Sustained release delivery system comprising traceless adapter

Abstract

Described herein are drug delivery systems, pharmaceutically acceptable salts thereof, for delivering bioactive agents comprising a primary or secondary amine, or a ring nitrogen atom of an aza-heteroaryl ring; a drug delivery agent associated therewith; a pharmaceutical composition comprising the drug delivery system; and the use of the drug delivery system as sustained release therapy.

Description

[0001] This application is a divisional application of Chinese patent application 201880026014.7, whose application date is April 19, 2018 and whose invention name is “Sustained release delivery system comprising a traceless joint”. Technical Field

[0002] Described herein are drug delivery systems for delivering biologically active agents containing a primary or secondary amine, or a ring nitrogen atom of an azaheteroaryl ring, pharmaceutically acceptable salts thereof; drug delivery agents related thereto; pharmaceutical compositions containing the drug delivery systems; and the use of the drug delivery systems as sustained release treatments. Background Art

[0003] The regulation of the physicochemical or pharmacokinetic properties of a drug in vivo may be affected by the conjugation of the drug to a carrier. In particular, the conjugation of a drug to a carrier is often used to increase the therapeutic duration of action, reduce the maximum concentration of the drug after administration, or deliver the drug locally to a desired tissue or compartment, or a combination of these purposes. Typically, carriers in drug delivery systems are (a) used in a non-covalent manner, where the drug is physicochemically formulated into a solvent-carrier mixture, or (b) connected by covalent attachment of a carrier agent to a functional group present in the drug.

[0004] Non-covalent methods require efficient drug encapsulation to prevent uncontrolled burst release of the drug, which may occur upon initial administration of the carrier-drug system or during carrier degradation after administration to a subject. Inhibition of diffusion of unbound water-soluble drug molecules requires strong van der Waals contacts, typically mediated by hydrophobic moieties, hydrogen bonding, or electrostatic binding mediated by charged moieties. Many conformationally sensitive drugs, such as proteins, peptides, or antibodies, become dysfunctional during the encapsulation process and / or during subsequent storage of the encapsulated drug.

[0005] Alternatively, the drug can be covalently conjugated to the carrier via a stable linker or via a reversible linker moiety (from which the drug is released). If the drug is stably linked to the carrier, such a conjugate needs to exhibit sufficient residual activity to have a pharmaceutical effect, and the conjugate is always in an active form.

[0006] If the drug is conjugated to the carrier via a cleavable linker, such conjugates are often referred to as carrier-linked drugs. This approach can be applied to bioactive molecules of various types and sizes from low molecular weight organic molecules, natural products, antibodies and their analogs, proteins, peptides, etc. An important consideration for carrier-linked drugs is the mechanism for releasing the drug from the carrier. The release mechanism can be enzymatic, pH-dependent, or by autonomous hydrolysis. Typically, drug release is easily controlled and difficult to maintain for long periods of time.

[0007] There continues to be a need for new drug delivery systems suitable for sustained release of biologically active moieties in therapeutic applications.Described herein are drug delivery systems that provide sustained release of biologically active moieties for therapeutically relevant applications. Summary of the invention

[0008] Described herein are drug delivery systems comprising a biologically active molecule, a carrier, and a traceless linker that provides a means for attaching the biologically active molecule to the carrier, pharmaceutical compositions comprising the drug delivery systems, and the use of the drug delivery systems as sustained release therapeutics.

[0009] One embodiment described herein is a drug delivery system comprising DR represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

[0010]

[0011] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0012] R 1 is hydrogen or C1-C4 alkyl;

[0013] R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0014] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0015] a is 0, 1, 2, 3, or 4;

[0016] R 3 is hydrogen or C1-C4 alkyl;

[0017] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0018] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0019] R 12 is hydrogen or C1-C4 alkyl;

[0020] R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0021] R 13 is C1-C4 alkyl; or

[0022] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0023] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0024] R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0025] R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0026] b is an integer from 1 to 10;

[0027] Z is CH-LA, CH-A, NLA or NA;

[0028] L is an optionally substituted divalent linker;

[0029] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0030] R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and

[0031] R 11 It is a carrier.

[0032] In one aspect, D comprises a protein, a peptide, a polypeptide, a nucleic acid, an oligonucleotide or polynucleotide, a carbohydrate, an oligosaccharide or polysaccharide, or a small molecule, each of which comprises at least one primary amine, a secondary amine, or a ring nitrogen atom comprising an azaheteroaryl ring, and the small molecule has a molecular weight between 100 g / mol and about 2000 g / mol.

[0033] In another aspect, D comprises a protein, a peptide, a nucleic acid, a Fab, a scFv, a monoclonal antibody, or a small molecule. In another aspect, D comprises brolucizumab (D1) (SEQ ID NO: 4), D2 (SEQ ID NO: 5), D3 (SEQ ID NO: 6), D4 (SEQ ID NO: 7), NO:7), abciximab, adalimumab, aflibercept, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab, canakinumab, certolizumab pegol, cetuximab, daclizumab, daratumumab, denosumab, eculizumab, efalizumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lambalizumab, muromonab-CD3, natalizumab, nivolumab, ofatumumab, omalizumab, palivizumab, panitumumab, pegpleranib, pembrolizumab, ranibizumab, rituximab, tocilizumab, tositumomab, trastuzumab, ustekinumab, or vedolizumab or a combination thereof. In another aspect, D comprises one or more biologically active peptides, including but not limited to C-type natriuretic peptide (CNP), atrial natriuretic peptide (ANP), exenatide-4, insulin, adrenocorticotropic hormone, adrenomedullin, adropin, ω-funnel web spider toxin, agouti protein-related protein, one or more angiotensin, apelin 12, apelin 13, apelin 36, or derivatives thereof, bradykinin, calcitonin, cocaine and amphetamine regulated transcript (CART), corticotropin releasing factor, CRF, α-defensin, β-defensin, δ-sleep-inducing peptide (DSIP), elastase-specific inhibitor (Elafin), endokinin, endomorphin, endorphin, endothelin, exenatide, fibronectin active fragment, galanin, galanin-like peptide, big gastrin, gastrin I, gastrin-related peptide, gastric inhibitory polypeptide, gastrin-releasing peptide, ghrelin, desacyl ghrelin, glucagon, glucagon-like peptide, growth hormone-releasing factor, Chilean bird spider (Grammostolaspatulata)GsMTx-4, guangxitoxin-1E, guanylin, hepatic bactericidal peptide 1, hepatic bactericidal peptide / LEAP-1, histamine 5, human growth hormone, humanin, tiger bird spider toxin, Iberiotoxin, emperor scorpion toxin (imperatoxin A), insulin-like growth factor, pituitary hormone, IRL1620, connecting peptide, short-skinned scorpion toxin, kisspeptin, kurtoxin, lipolysis hormone, lipolysis hormone, hepatocyte growth factor, liver-expressed antimicrobial peptide 2, luteinizing hormone-releasing hormone, lyseni n), LL-37, Mager toxin, mast degranulation peptide, mast cell degranulation peptide, melanin concentrating hormone, melanocyte stimulating hormone, MSH release inhibitory factor, midkine, mollusk cardiac excitatory neuropeptide, morphine tolerance peptide, motilin, muscarinic toxin, neuroendocrine regulatory peptide-2, neurokinin A, neurokinin B, neuromedin, neurostatin-13 (neuronostatin-13), neuropeptide, neurotensin, neurotoxin NSTX-3, pain stabilizer, pain sensitive peptide, obesity inhibitor, opioid peptides (enkephalin, endorphin, BAM-12P, casomorphin, dynorphin, endomorphin, neoendorphin, pain sensitive peptide), orexin-A / -B, orphan peptide, osteocalcin (osteocalci) , oxytocin, pancreatic inhibin, parathyroid hormone, parathyroid hormone-related protein, peptide 234, peptide histidine-methionine, peptide T, peptide YY, frog peptide, pituitary adenylate cyclase-activating polypeptide, platelet factor-4, plectasin, pleiotrophin, preadrenomedullin, prolactin-releasing peptide, psalmotoxin 1, purotoxin-1, pyroglutamylated RFamide peptide, renin, RFamide-related peptide-1, RFamide-related peptide-3, salusin-α / β, sarafotoxin, schizophrenia-related peptide, scyll atoxin), secretin, secrelaxant, serum thymic factor, sodium potassium ATPase inhibitor-1, somatostatin, carpet anemone toxin, substance K, apnopressin, apnopressin-related peptide, substance P, tachykinin, tarantula SNX-482, tarantula ProTx-I, tarantula ProTx-II, tertiapin, tityustoxin Ka, thyrotropin-releasing hormone, phagostimulin, uroguanylin, uroguanylin isomer A or B, urocortin, urocortin II, urotensin II, urotensin II-related peptide, vasoactive intestinal peptide, vasopressin, oxytocin, viral replication inhibitory peptide, xenin, or a combination thereof.

[0034] In another aspect, D is a small molecule having a molecular weight between about 100 g / mol and about 2000 g / mol or less, which is linked to R through a primary amine, a secondary amine, or a ring nitrogen atom of an azaheteroaryl ring.

[0035] In another aspect, the small molecule comprises 5-(5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; 5-(6,7-dihydro-5H-pyrrolo[3,4d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; N -(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6-((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide; (S)-5-(6-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yloxy)-N-(5-(1-methylcyclopropyl)-1H-pyrazol-3-yl)-1H-indole-1-carboxamide, or a combination thereof.

[0036] In another aspect, R 1 is hydrogen or methyl, R 1a is hydrogen or methyl, or CR 1 R 1a , combined to form a cyclopropane-1,1-diyl group. In another aspect, R 3 and R 3a In another aspect, X is O. In another aspect, Y is C(O)R 4 , and R 4 is C1-C6 alkyl, C3-C6 cycloalkyl or C1-C2 alkoxy C1-C2 alkyl. 4 In another aspect, Y is SiR 5 R 6 R 7 ; R 5 and R 6 are each methyl, ethyl, propyl or isopropyl; and R 7 is C1-C4 alkyl, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, 2-ethoxyethoxy, 2-isopropoxy-ethoxy, tetrahydropyranyloxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein b is 2, 3 or 4.

[0037] In another aspect, Z is CH-LA, CH-A, NLA or NA;

[0038] L is an optionally substituted divalent linker Q-[Sp-Q] h -Q;

[0039] Q is independently selected at each occurrence from a bond, O, C(O), N(H), N(C1-C4 alkyl), C(O)NH, C(O)N(C1-C4 alkyl), N(H)C(O), N(C1-C4 alkyl)C(O), N(H)C(O)O, N(C1-C4 alkyl)C(O)O, OC(O)N(H), OC(O)N(C1-C4 alkyl), N(H)C(O)N(H), N(C1-C4 alkyl)C(O)N(H), N(H)C(O)N(C1-C4 alkyl), N(C1-C4 alkyl)C( C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O), OC(O)O, S, S(O)2, N(H)S(O)2, N(C1-C4 alkyl)S(O)2, S(O)2N(H), S(O)2N(C1-C4 alkyl), C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O)C1-C2 alkyl, 1,2,3-triazole, OP(O)2, P(O)2O, C1-C4 alkyl-P(O)2-O, or OP(O)2-C 1-4 alkyl;

[0040] Sp is independently selected at each occurrence from optionally substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, [WO] g 、C1-C8 alkyl-[OW] g , [OW] g -O-C1-C8 alkyl, C1-C8C alkyl-[OW] g -O-C1-C8 alkyl, oligopeptide;

[0041] h is an integer between 1 and 20;

[0042] g is a weighted average between about 2 and about 50;

[0043] W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom;

[0044] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)OC1-C8 alkyl, C(O)N(H)C1-C8 alkyl, R 10 or R11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0045] R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and

[0046] R 11 It is a carrier.

[0047] In another aspect, Z is NR 9 ; R 9 is C(O)-(CH2) n -QA or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA; n is an integer from 1 to 8; and A is R 10 or R 11 In another aspect, R 10 is an azido group, an alkynyl group, a substituted or unsubstituted C7-C 12 Cycloalkynyl, substituted or unsubstituted C7-C 12 Heterocycloalkynyl, substituted or unsubstituted C7-C 12 In another aspect, R is cycloalkenyl, norbornyl, substituted or unsubstituted vinylcarboxyl, substituted or unsubstituted vinylsulfonyl, substituted or unsubstituted C2-C8 alkenyl, amino, thiol, substituted or unsubstituted C1-C8 carboxyl, substituted or unsubstituted C1-C8 carbonyl, -O-NH2, hydrazide, maleimide, α-halogenated carbonyl, furan, substituted or unsubstituted tetrazinyl, lysine, glutamine, cyclodextrin or adamantyl. 10 Contains a reactive functional group suitable for coupling Formula (I) to a support. In another aspect, R 11 In another aspect, R 11 In another aspect, R 11 In another aspect, R 11The invention comprises a polymer, a cross-linked polymer, or a hydrogel comprising one or more of hyaluronic acid, polyethylene glycol, polypropylene glycol, polyethylene oxide, polypropylene oxide, polyglutamate, polylysine, polysialic acid, polyvinyl alcohol, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl pyrrolidone, polyoxazoline, polyiminocarbonate, polyamino acid, hydrophilic polyester, polyamide, polyurethane, polyurea, dextran, agarose, xylan, mannan, carrageenan, alginate, gelatin, collagen, albumin, cellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl starch, chitosan, nucleic acid, derivatives thereof, copolymers thereof, or a combination thereof. In another aspect, R 11 A cross-linked hydrogel comprising hyaluronic acid, polyethylene glycol, a cross-linked hydrogel of hyaluronic acid, a cross-linked hydrogel of polyethylene glycol, or a combination thereof. 11 In another aspect, R 11 The invention relates to a hydrogel comprising cross-linked hyaluronic acid or cross-linked polyethylene glycol. In another aspect, the hyaluronic acid or polyethylene glycol is functionalized with at least one functional group comprising an azido group, an alkynyl group, a substituted or unsubstituted C7-C 12 Cycloalkynyl, substituted or unsubstituted C7-C 12 Heterocycloalkynyl, C7-C 12 Cycloalkenyl, norbornyl, vinylcarboxyl, vinylsulfonyl, C2-C8 alkenyl, amino, thiol, C1-C8 carboxyl, C1-C8 carbonyl, -O-NH2, carbohydrazide, maleimide, α-halocarbonyl, furan, substituted or unsubstituted tetrazinyl, lysine, glutamine, cyclodextrin, adamantyl, or a combination thereof. In another aspect, R 11 A hydrogel comprising cross-linked hyaluronic acid, wherein the hyaluronic acid comprises a cross-linked hyaluronic acid selected from -NH(W1)(O(W1)) d -V, wherein W1 is a C2-C4 alkyl-1,2-diyl group in which a hydrogen, methyl or ethyl side chain may be present on any main chain carbon atom; d is a number average with an average value of 0 to 500; and V is a C7-C4 alkyl group containing an azido group, an alkynyl group, a substituted or unsubstituted C7-C 12 Cycloalkynyl, substituted or unsubstituted C7-C 12 Heterocycloalkynyl, C7-C 12 In one aspect, V is an azide.

[0048] Another embodiment described herein is a method for preparing a cross-linked carrier formulation, the method comprising: (a) making a carrier molecule R 11 (b) preparing a reactive cross-linking agent; and (c) reacting the functionalized carrier molecule with the reactive cross-linking agent to form a cross-linked carrier by incubating at a temperature of about 4°C to about 60°C for about 0.5 hours to about 48 hours. 11 In another aspect, R 11 The invention relates to a hydrogel comprising one or more cross-linked polymers. On the one hand, the carrier molecule comprises hyaluronic acid or polyethylene glycol. On the other hand, the carrier molecule is functionalized by azide, sulfhydryl, amine, aminooxy (O-NH2) or aldehyde moiety to provide reactive functional groups for cross-linking. On the other hand, the preparation of reactive cross-linking agent comprises polyethylene glycol and 1-((tert-butoxycarbonyl)amino)cyclopropane-1-carboxylic acid, 3-((tert-butoxycarbonyl)amino)propionic acid, 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid, 2-(1-((tert-butoxycarbonyl)amino)methyl)cyclopropyl)acetic acid, 2-methyl-3-((tert-butoxycarbonyl)amino)propionic acid or 7-((tert-butoxycarbonyl)amino)heptanoic acid, and functionalized polyethylene glycol ester is deprotected. In another aspect, the preparation of the reactive cross-linking agent further comprises introducing at least two bicyclo[6.1.0]non-4-yn-9-yl)methyl groups after deprotection of the functionalized polyethylene glycol ester. In another aspect, the preparation of the reactive cross-linking agent further comprises reacting the deprotected functionalized polyethylene glycol ester with a suitable reagent or reagents to introduce terminal reactive groups (e.g., alkynyl, cycloalkynyl, heterocycloalkynyl, carboxylic acid or activated carboxylic acid, aldehyde or ketone, amino or aminooxy moiety) to provide reactive functional groups for cross-linking with carrier molecules.

[0049] Another embodiment described herein is a cross-linked hydrogel obtainable using the methods described herein. In one aspect, the carrier molecule is functionalized with an azide.

[0050] Another embodiment described herein is a method for preparing a drug adduct comprising a traceless linker R coupled to a biologically active agent D, the method comprising: (a) providing a biologically active agent D comprising a primary amine, a secondary amine, or a ring nitrogen atom of an azaheteroaryl ring; (b) reacting the biologically active agent with a traceless linker R having an activated carbonyl functional group; and (c) purifying the drug adduct from the reagent. In one aspect, R is as defined above in formula (I).

[0051] Another embodiment described herein is a drug adduct obtainable using the methods described herein.

[0052] Another embodiment described herein is a method for preparing a drug delivery system, the method comprising: (a) preparing a carrier molecule R 11 , where R 11 is a cross-linked hydrogel, and then the step includes a method for preparing the hydrogel; the carrier molecule can be optionally purified at this stage; (b) separately conjugating a traceless linker R to a biologically active agent D comprising a primary amine, a secondary amine or a ring nitrogen atom of an azaheteroaryl ring to form a traceless linker-D adduct; the traceless linker-D adduct can be optionally purified at this stage, (c) conjugating the carrier molecule R 11 conjugating with a traceless linker-D adduct; and (d) purifying the drug delivery system from the reagents.

[0053] Another embodiment described herein is a drug delivery system obtainable using the methods described herein.

[0054] Another embodiment described herein is a method for treating an eye disorder, such as macular degeneration (i.e., wet age-related macular degeneration), comprising administering to a subject in need thereof a drug delivery system comprising a conjugate DR represented by formula (I), or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring, the biologically active moiety comprising brocizumab (SEQ ID NO:4), aflibercept, peprazib, ranibizumab, 5-(5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; 5-(6,7-dihydro-5H-pyrrolo[3,4d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; N-(1-methyl -1H-indole-1-carboxamide; (S)-5-(6-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yloxy)-N-(5-(1-methylcyclopropyl)-1H-pyrazol-3-yl)-1H-indole-1-carboxamide, or a combination thereof; R is a linker suitable for releasing the biologically active moiety D:

[0055]

[0056] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0057] R 1 is hydrogen or C1-C4 alkyl;

[0058] R 1ais hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0059] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0060] a is 0, 1, 2, 3, or 4;

[0061] R 3 is hydrogen or C1-C4 alkyl;

[0062] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0063] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0064] R 12 is hydrogen or C1-C4 alkyl;

[0065] R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0066] R 13 is C1-C4 alkyl; or

[0067] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0068] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0069] R 5 and R6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0070] R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0071] b is an integer from 1 to 10;

[0072] Z is CHR 8 or NR 9 ;

[0073] R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA;

[0074] q is 1, 2, or 3;

[0075] g and p each independently have a weighted average length between about 2 and about 50;

[0076] m is 1 or 0;

[0077] W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom;

[0078] Q is a bond, O, N(H) or N(C1-C4 alkyl);

[0079] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0080] R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and

[0081] R 11 It is a carrier.

[0082] Another embodiment described herein is a method for treating a musculoskeletal disorder, the method comprising administering to a subject in need thereof a drug delivery system comprising a conjugate DR represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring, the biologically active moiety comprising D2 (SEQ ID NO: 4), D3 (SEQ ID NO: 5), other insulin-like growth factors, or a combination thereof; and R is a linker suitable for releasing the biologically active moiety D:

[0083]

[0084] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0085] R 1 is hydrogen or C1-C4 alkyl;

[0086] R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0087] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0088] a is 0, 1, 2, 3, or 4;

[0089] R 3 is hydrogen or C1-C4 alkyl;

[0090] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0091] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0092] R 12 is hydrogen or C1-C4 alkyl;

[0093] R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0094] R 13 is C1-C4 alkyl; or

[0095] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0096] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0097] R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0098] R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0099] b is an integer from 1 to 10;

[0100] Z is for CHR 8 or NR 9 ;

[0101] R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA;

[0102] q is 1, 2, or 3;

[0103] g and p each independently have a weighted average length between about 2 and about 50;

[0104] m is 1 or 0;

[0105] W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom;

[0106] Q is a bond, O, N(H) or N(C1-C4 alkyl);

[0107] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0108] R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and

[0109] R 11 It is a carrier.

[0110] Another embodiment described herein is a method for treating a cardiac disorder, the method comprising administering to a subject in need thereof a drug delivery system comprising a conjugate DR represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring, the biologically active moiety comprising relaxin or SEQ ID NO: 7; R is a linker suitable for releasing the biologically active moiety D:

[0111]

[0112] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0113] R 1 is hydrogen or C1-C4 alkyl;

[0114] R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0115] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0116] a is 0, 1, 2, 3, or 4;

[0117] R 3 is hydrogen or C1-C4 alkyl;

[0118] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0119] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0120] R 12 is hydrogen or C1-C4 alkyl;

[0121] R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0122] R 13 is C1-C4 alkyl; or

[0123] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0124] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0125] R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0126] R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) bO-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0127] b is an integer from 1 to 10;

[0128] Z is CHR 8 or NR 9 ;

[0129] R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA;

[0130] q is 1, 2, or 3;

[0131] g and p each independently have a weighted average length between about 2 and about 50;

[0132] m is 1 or 0;

[0133] W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom;

[0134] Q is a bond, O, N(H) or N(C1-C4 alkyl);

[0135] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0136] R 10 is a reactive functional group suitable for coupling Formula I to a support; and

[0137] R 11 It is a carrier.

[0138] Another embodiment described herein is a method for treating a disease or disorder comprising administering to a subject in need thereof a drug delivery system comprising a conjugate DR represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

[0139]

[0140] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0141] R 1 is hydrogen or C1-C4 alkyl;

[0142] R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0143] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0144] a is 0, 1, 2, 3, or 4;

[0145] R 3 is hydrogen or C1-C4 alkyl;

[0146] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0147] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0148] R 12 is hydrogen or C1-C4 alkyl;

[0149] R 12a is hydrogen or C1-C4 alkyl, or CR12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0150] R 13 is C1-C4 alkyl; or

[0151] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0152] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0153] R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0154] R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0155] b is an integer from 1 to 10;

[0156] Z is CHR 8 or NR 9 ;

[0157] R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA;

[0158] q is 1, 2, or 3;

[0159] g and p each independently have a weighted average length between about 2 and about 50;

[0160] m is 1 or 0;

[0161] W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom;

[0162] Q is a bond, O, N(H) or N(C1-C4 alkyl);

[0163] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0164] R 10 is a reactive functional group suitable for coupling Formula I to a support; and

[0165] R 11 It is a carrier.

[0166] Another embodiment described herein is a method for extending the half-life of a biologically active moiety D comprising at least one primary amine, secondary amine, or ring nitrogen atom of an azaheteroaryl ring, the method comprising attaching D to R, represented by formula (I), wherein R is a linker suitable for releasing the biologically active moiety D:

[0167]

[0168] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0169] R 1 is hydrogen or C1-C4 alkyl;

[0170] R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0171] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0172] a is 0, 1, 2, 3, or 4;

[0173] R 3 is hydrogen or C1-C4 alkyl;

[0174] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3aCombine to form a C3-C6 cycloalkane-1,1-diyl;

[0175] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0176] R 12 is hydrogen or C1-C4 alkyl;

[0177] R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0178] R 13 is C1-C4 alkyl; or

[0179] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0180] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0181] R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0182] R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0183] b is an integer from 1 to 10;

[0184] Z is for CHR 8 or NR 9 ;

[0185] R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA;

[0186] q is 1, 2, or 3;

[0187] g and p each independently have a weighted average length between about 2 and about 50;

[0188] m is 1 or 0;

[0189] W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom;

[0190] Q is a bond, O, N(H) or N(C1-C4 alkyl);

[0191] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0192] R 10 is a reactive functional group suitable for coupling Formula I to a support; and

[0193] R 11 It is a carrier.

[0194] Another embodiment described herein is a drug delivery system comprising formula (III):

[0195]

[0196] Wherein brocuzumab comprises D1, SEQ ID NO: 4; R 4 is CH3-, CH3-O-CH2-, CH3CH2- or (CH3)2CH-; and the carrier comprises hyaluronic acid, polyethylene glycol, a hydrogel thereof, a cross-linked hydrogel thereof, or a combination thereof.

[0197] Another embodiment described herein is a drug delivery system comprising formula (IV):

[0198]

[0199] wherein D2 comprises SEQ ID NO:5, D3 comprises SEQ ID NO:6; R 4 is CH3-, CH3-O-CH2-, CH3CH2- or (CH3)2CH-; and the carrier comprises hyaluronic acid, polyethylene glycol, a hydrogel thereof, a cross-linked hydrogel thereof, or a combination thereof.

[0200] Another embodiment described herein includes DRR 11 The drug delivery system of claim 1, wherein R comprises a traceless linker coupled to a bioactive agent D; D comprises a bioactive moiety comprising at least one primary amine, secondary amine, or ring nitrogen atom comprising an azaheteroaryl ring; and R 11 Comprising a plurality of hyaluronic acid monomers, each of which comprises a random series of unfunctionalized hyaluronic acid monomers, hyaluronic acid monomers functionalized by one or more cross-linked hyaluronic acid monomers and one or more drug adducts DR. In one aspect, D comprises brocuzumab (SEQ ID NO: 4), D2 (SEQ ID NO: 5), D3 (SEQ ID NO: 6), D4 (SEQ ID NO: 7), celecoxib, aflibercept, peprazib, ranibizumab, 5-(5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; 5-(6,7-dihydro-5H-pyrrolo[3,4d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; ]-amide; N-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6-((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide; (S)-5-(6-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yloxy)-N-(5-(1-methylcyclopropyl)-1H-pyrazol-3-yl)-1H-indole-1-carboxamide, or a combination thereof. In one aspect, the traceless linker R is as described herein.

[0201] Another embodiment described herein is a drug delivery system comprising DR represented by formula (XXIV) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

[0202]

[0203] Another embodiment described herein is a drug delivery system comprising DR represented by formula (XXV) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

[0204]

[0205] Another embodiment described herein is a drug delivery system comprising DR represented by formula (XXVI) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

[0206]

[0207] Another embodiment described herein is a drug delivery system comprising DR represented by formula (XXVII) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

[0208]

[0209] Another embodiment described herein is a drug delivery system comprising DR represented by formula (XXVIII) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

[0210]

[0211] Another embodiment described herein is a drug delivery system comprising DR represented by formula (XXIX) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

[0212]

[0213] Another embodiment described herein is a drug delivery system represented by formula (XXX) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R 4 is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl or methoxymethyl:

[0214]

[0215] Another embodiment described herein is a drug delivery system comprising DR represented by formula (XXXI) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and

[0216] R is a linker suitable for releasing the biologically active moiety D:

[0217]

[0218] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0219] Y is C(O)R 4 ;

[0220] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0221] Z is NLA;

[0222] L is C(O)CH2CH2NH;

[0223] A is for R 11 ;

[0224] R 11 is a hydrogel derived from cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH2CH2O)3CH2CH2N3, and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[3-(((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)propanoate]. In one aspect, D is brocuzumab, and R 4 In another aspect, D is brolucizumab, and R 4 It's CH(CH3)CH3.

[0225] Another embodiment described herein is a drug delivery system represented by formula (XXXII) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R 4 is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl or methoxymethyl:

[0226]

[0227] In one aspect, D is brolucizumab, and R 4 In another aspect, D is brolucizumab, and R 4 It's CH(CH3)CH3.

[0228] Another embodiment described herein is a drug delivery system comprising DR represented by Formula XXXIII, or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and

[0229] R is a linker suitable for releasing the biologically active moiety D:

[0230]

[0231] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0232] Y is C(O)R 4 ;

[0233] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0234] Z is NLA;

[0235] L is C(O)CH2CH2NH;

[0236] A is for R 11 ;

[0237] R 11 A hydrogel derived from cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH2CH2O)3CH2CH2N3, and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[1-((((1'R,8'S,9's)-bicyclo[6.1.0]non-4'-yn-9'-yl)methoxy)carbonyl)amino-cyclopropane-1-carboxylate].

[0238] In one aspect, D is brolucizumab, and R 4 In another aspect, D is brolucizumab, and R 4 It's CH(CH3)CH3.

[0239] Another embodiment described herein is a drug delivery system represented by formula (XXXIV) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R 4 is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl or methoxymethyl:

[0240]

[0241] In one aspect, D is brolucizumab, and R 4 In another aspect, D is brolucizumab, and R 4 It's CH(CH3)CH3.

[0242] Another embodiment described herein is a drug delivery system comprising DR represented by Formula XXXV, or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and

[0243] R is a linker suitable for releasing the biologically active moiety D:

[0244]

[0245] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0246] Y is C(O)R 4 ;

[0247] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0248] Z is NLA;

[0249] L is C(O)CH2CH2NH;

[0250] A is for R 11 ;

[0251] R 11 A hydrogel derived from cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH2CH2O)3CH2CH2N3, and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[1-((((1'R,8'S,9's)-bicyclo[6.1.0]non-4'-yn-9'-yl)methoxy)carbonyl)piperidine-4-carboxylate].

[0252] In one aspect, D is brolucizumab, and R 4 In another aspect, D is brolucizumab, and R 4 It's CH(CH3)CH3.

[0253] Another embodiment described herein is a drug delivery system represented by formula (XXXVI) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R 4 is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl or methoxymethyl:

[0254]

[0255] In one aspect, D is brolucizumab, and R 4 In another aspect, D is brolucizumab, and R 4 It's CH(CH3)CH3.

[0256] Another embodiment described herein is a drug delivery system comprising DR represented by Formula XXXVII, or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and

[0257] R is a linker suitable for releasing the biologically active moiety D:

[0258]

[0259] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0260] Y is C(O)R 4 ;

[0261] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0262] Z is NLA;

[0263] L is C(O)CH2CH2NH;

[0264] A is for R 11 ;

[0265] R 11A hydrogel derived from cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH2CH2O)3CH2CH2N3, and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[7-(((((1'R,8'S,9's)-bicyclo[6.1.0]non-4'-yn-9'-yl)methoxy)carbonyl)amino)heptanoate].

[0266] In one aspect, D is brolucizumab, and R 4 In another aspect, D is brolucizumab, and R 4 It's CH(CH3)CH3.

[0267] Another embodiment described herein is a drug delivery system represented by formula (XXXVIII), or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R 4 is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl or methoxymethyl:

[0268]

[0269] In one aspect, D is brolucizumab, and R 4 In another aspect, D is brolucizumab, and R 4 It's CH(CH3)CH3.

[0270] Another embodiment described herein is a drug delivery system comprising DR represented by formula (XXXIX) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and

[0271] R is a linker suitable for releasing the biologically active moiety D:

[0272]

[0273] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0274] Y is C(O)R 4 ;

[0275] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0276] Z is NLA;

[0277] L is C(O)CH2CH2NH;

[0278] A is for R 11 ;

[0279] R 11 is a hydrogel derived from cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH2CH2O)3CH2CH2N3, and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[2-methyl-3-(((((1'R,8'S,9's)-bicyclo[6.1.0]non-4'-yn-9'-yl)methoxy)carbonyl)amino)propanoate]. In one aspect, D is brocizumab, and R 4 In another aspect, D is brolucizumab, and R 4 It's CH(CH3)CH3.

[0280] Another embodiment described herein is a carrier system or a pharmaceutically acceptable salt thereof, represented by the following formula:

[0281]

[0282] in

[0283] L is an optionally substituted divalent linker Q-[Sp-Q] h -Q;

[0284] Q is independently selected at each occurrence from a bond, O, C(O), N(H), N(C1-C4 alkyl), C(O)NH, C(O)N(C1-C4 alkyl), N(H)C(O), N(C1-C4 alkyl)C(O), N(H)C(O)O, N(C1-C4 alkyl)C(O)O, OC(O)N(H), OC(O)N(C1-C4 alkyl), N(H)C(O)N(H), N(C1-C4 alkyl)C(O)N(H), N(H)C(O)N(C1-C4 alkyl), N(C1-C4 alkyl)C( C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O), OC(O)O, S, S(O)2, N(H)S(O)2, N(C1-C4 alkyl)S(O)2, S(O)2N(H), S(O)2N(C1-C4 alkyl), C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O)C1-C2 alkyl, 1,2,3-triazole, OP(O)2, P(O)2O, C1-C4 alkyl-P(O)2-O, or OP(O)2-C 1-4 alkyl;

[0285] Sp is independently selected at each occurrence from optionally substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, [WO] g 、C1-C8 alkyl-[OW] g , [OW] g -O-C1-C8 alkyl, C1-C8C alkyl-[OW] g -O-C1-C8 alkyl or oligopeptide;

[0286] h is an integer between 1 and 20;

[0287] g is a weighted average between about 2 and about 50;

[0288] W is C2-C4 alkyl-1,2-diyl, in which hydrogen, methyl or ethyl side chains may be present on any backbone carbon atom; and

[0289] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy. BRIEF DESCRIPTION OF THE DRAWINGS

[0290] Figure 1 A schematic diagram of a drug delivery system is shown, which includes a drug conjugated to a carrier via a traceless linker, and the drug after release. The reaction of the trigger generates a nucleophilic functional group that cleaves the amide bond connecting the drug to the drug delivery system. In this non-limiting example, the drug is connected via a primary amine group.

[0291] Figure 2 A partial chemical representation of a hydrogel-drug conjugate is shown, where the hydrogel is composed of hyaluronic acid cross-linked with PEG, and the drug is conjugated to the hydrogel via a traceless linker with a trigger Y. Figure 2 In the specification and throughout the specification, polymer molecules (e.g., hyaluronic acid) are represented by [P] n , wherein P represents a monomer repeating unit, and n represents the average number of repeating units of a monomer. For any given polymer group, the number of repeating units is randomly distributed. In addition, the relative connectivity of the independent molecules of the hydrogel or drug delivery system described herein is random within the group. The structural description of the hydrogel or drug delivery system represents a single potential structural unit in two dimensions, while these complexes are three-dimensional with many structural subunits.

[0292] Figure 3AAn exemplary reaction of a traceless linker with an azide-functionalized hyaluronic acid polymer is shown.

[0293] Figure 3B An exemplary reaction of a traceless linker with an azide-functionalized hydrogel is shown.

[0294] Figure 4A (Illustrative Example 8.1) shows an example of in vitro release of brocuzumab from a hydrogel conjugate.

[0295] Figure 4B The comparative in vitro release of brocuzumab from hydrogel conjugates C1a, C2a, C3a and C4a is shown.

[0296] Figure 5A (Illustrative Example 8.2) shows the in vitro release of D2 from the hydrogel conjugates, comparing the release from traceless linkers L1, L2 and L5 into PBS buffer.

[0297] Figure 5B The in vitro release of D2 from the hydrogel conjugates is shown, comparing the release from traceless linkers L1, L2 and L5 into bovine synovial fluid (BSF).

[0298] Figure 6 (Illustrative Example 8.3) shows the in vitro release of D4 from a traceless linker, illustrating the loss of the parent, the appearance and subsequent disappearance of the intermediate and the release of D4.

[0299] FIG. 7 (Illustrative Example 9.1) shows data from in vivo testing of brolucizumab-hydrogel at two different dose levels. Fig. 7A Fluorescein leakage data from the rabbit VEGF challenge model are shown. Figure 7B Shown in Fig. 7A Levels of brolucizumab D1 were measured in the vitreous humor of the same rabbits.

[0300] Figure 8 (Illustrative Example 9.3) shows the in vivo release of D4 from the hydrogel conjugate.

[0301] Fig. 9 (Illustrative Example 8.2) The activity of D2 released from the hydrogel in vitro into bovine synovial fluid (sampled daily) was shown. DETAILED DESCRIPTION

[0302] Described herein are drug delivery systems for delivering biologically active agents containing a primary or secondary amine, or a ring nitrogen atom of an azaheteroaryl ring, pharmaceutically acceptable salts thereof; drug delivery agents related thereto; pharmaceutical compositions containing the drug delivery systems; and the use of the drug delivery systems as sustained release treatments.

[0303] As used herein, the term "alkyl" refers to a straight, branched or cyclic carbon chain. Unless otherwise specified, one or more hydrogen atoms of an alkyl carbon may be replaced by a substituent. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc. When two parts of a molecule are connected by an alkyl group, it is also referred to as an alkylene group. As used herein, "low alkyl" is a subset of alkyl, is preferred in some embodiments, and refers to a straight or branched hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of low alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc. Unless otherwise indicated, the term "alkyl" or "lower alkyl" is intended to include substituted and unsubstituted alkyl or lower alkyl groups, and these groups may be substituted with additional organic and / or inorganic groups, including but not limited to groups selected from the group consisting of halogen (e.g., to form haloalkyl), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl, hydroxy, alkoxy (thereby producing polyalkoxy groups such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, thiol, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro, cyanoalkyl-S(O), m 、Haloalkyl-S(O) m、 Alkenyl-S(O)n, Alkynyl-S(O) m、 Cycloalkyl-S(O) m、 Cycloalkylalkyl-S(O) m 、Aryl-S(O) m 、Arylalkyl-S(O) m 、Heterocyclic-S(O) m , or heterocycloalkyl-S(O) m , where m=0, 1, 2 or 3.

[0304] As used herein, the term "alkenyl" individually or as part of another group refers to a straight or branched hydrocarbon containing from 1 to 10, 20 or 30 or more carbon atoms (or 1 to 4 carbon atoms in lower alkenyl), the hydrocarbon comprising 1 to 4, 5 or 6 or more double bonds in the normal chain. Representative examples of alkenyl include but are not limited to vinyl, 2-propenyl, 3-butene, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, etc. Unless otherwise indicated, the term "alkenyl" or "lower alkenyl" is intended to include substituted and unsubstituted alkenyl or lower alkenyl, and these groups can be substituted by the groups described above for alkyl and lower alkyl.

[0305] As used herein, the term "alkynyl" alone or as part of another group refers to a straight or branched hydrocarbon containing 1 to 10, 20, 30, or 40 or more carbon atoms (or 1 to 4 carbon atoms in lower alkynyl), the hydrocarbon containing 1, 2, or 3 or more triple bonds in the normal chain. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, etc. Unless otherwise indicated, the term "alkynyl" or "lower alkynyl" is intended to include substituted and unsubstituted alkynyl or lower alkynyl, and these groups may be substituted with the same groups associated with the above-mentioned alkyl and lower alkyl.

[0306] As used herein, the term "cycloalkynyl" refers to a hydrocarbon ring system having between 6 and 16 carbon atoms and 1, 2 or 3 fused or bridged rings containing at least 1 or more triple bonds in the ring structure.

[0307] As used herein, the term "heterocycloalkynyl" refers to a cyclic hydrocarbon ring system having: between 6 and 16 carbon atoms; 1, 2, 3 or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur; and 1, 2 or 3 rings that are fused or bridged, said rings containing at least 1 or more triple bonds in the ring structure.

[0308] As used herein, the term "cycloalkyl" alone or as part of another group refers to a saturated or partially unsaturated cycloalkyl containing 3, 4, 5, 6, 7 or 8 carbons (carbons therein can be replaced in a heterocyclic group as described below). Representative examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. These rings can be optionally substituted by other substituents (e.g., halogen or low alkyl) as described herein. Unless otherwise indicated, the term "cycloalkyl" is general and is intended to include heterocyclic groups as described below.

[0309] As used herein, the term "heterocycle" or "heterocyclyl", alone or as part of another group, refers to an aliphatic (e.g., a fully or partially saturated heterocycle) or aromatic (e.g., heteroaryl) monocyclic or bicyclic ring system. Examples of monocyclic ring systems are exemplified by any 3 to 8 membered ring containing 1, 2, 3 or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur. The 5-membered ring has from 0 to 2 double bonds, and the 6-membered ring has from 0 to 3 double bonds. Representative examples of monocyclic ring systems include, but are not limited to, azetidine, aziridine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazolidine, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to an aryl group as described herein, a cycloalkyl group as described herein, or another monocyclic ring system as described herein. Representative examples of bicyclic ring systems include, but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxane, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like. These rings include quaternized derivatives thereof and may be optionally substituted with additional organic and / or inorganic groups, including but not limited to groups selected from the group consisting of halogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl, hydroxy, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, thiol, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro, cyano, alkyl-S(O) m Haloalkyl-S(O) m 、Alkenyl-S(O) m , Alkynyl-S(O) m 、Cycloalkyl-S(O) m、Cycloalkylalkyl-S(O) m 、Aryl-S(O) m 、Arylalkyl-S(O) m、 Heterocyclic-S(O) m 、Heterocycloalkyl-S(O) m , where m=0, 1, 2 or 3.

[0310] As used herein, the term "heteroaryl" is as described with respect to the heterocycle above.

[0311] As used herein, the terms "cycloalkylalkyl," "cycloalkylalkenyl," and "cycloalkylalkynyl," by themselves or as part of another group, refer to a cycloalkyl group as described herein appended to the parent molecular moiety through an alkyl, alkenyl or alkynyl group, respectively, as described herein.

[0312] As used herein, the term "alkoxy" alone or as part of another group refers to an alkyl or lower alkyl group as described herein (and thus includes substituted forms, such as polyalkoxy) attached to the parent molecular moiety through an oxy group -O-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like. In some aspects, when part of a more complex molecule, the alkoxy group comprises an alkoxy substituent attached to the alkyl or lower alkyl group via an ether bond.

[0313] As used herein, the term "halo" or "halogen" refers to any suitable halogen, including -F, -Cl, -Br or -I.

[0314] As used herein, the term "acyl" by itself or as part of another group refers to a -C(O)R' group, where R' is any suitable substituent (e.g., aryl, alkyl, alkenyl, alkynyl, cycloalkyl) or other suitable substituents as described herein.

[0315] As used herein, the term "alkylthio" alone or as part of another group refers to an alkyl group as described herein attached to the parent molecular moiety through a thiol moiety as described herein. Representative examples of alkylthio include, but are not limited to, methylthio, ethylthio, tert-butylthio, hexylthio, and the like.

[0316] As used herein, the terms "amide," "amido," or "amidyl" as used herein alone or as part of another group refer to -C(O)NR a R b Group, where R a and R band R is independently any suitable substituent (eg, alkyl, hydrogen, cycloalkyl, alkenyl, alkynyl, or aryl).

[0317] As used herein, the term "ester" by itself or as part of another group refers to a -C(O)OR' group, where R' is any suitable substituent (eg, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl).

[0318] As used herein, the term "ether" by itself or as part of another group refers to a -COR' group, where R' is any suitable substituent (eg, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl).

[0319] As used herein, the term "sulfone" by itself or as part of another group refers to a -S(O)(O)R' group, where R' is any suitable substituent (eg, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl).

[0320] As used herein, the term "sulfonamide" alone or as part of another group refers to -S(O)2NR a R b Group, where R a and R b and R is independently any suitable substituent (eg, H, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl).

[0321] As used herein, the term "carboxy" refers to the group -C(O)OH.

[0322] As used herein, the term "hydroxy" refers to the group -OH.

[0323] As used herein, the term "amino" refers to the group -NH2.

[0324] As used herein, vinyl refers to the group -CH2CH2.

[0325] As used herein, the term "sulfonate" refers to the group -S(O)(O)OR', where R' is any suitable substituent (eg, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl).

[0326] As used herein, the term "sulfonyl" refers to the group -S(O)(O)R', where R' is any suitable substituent (eg, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl).

[0327] As used herein, thiol refers to the group -SH.

[0328] As used herein, oxyamine or aminooxy refers to the group -ONH2.

[0329] As used herein, "hydrazide" or "hydrazide group" refers to the group -C(O)-NH-NH2.

[0330] As used herein, "maleimide or maleimide group" refers to a cyclic compound having the molecular formula C2H2(C(O))2NH or a group -N(C(O))2C2H2 having at least one CC double bond.

[0331] As used herein, furan refers to a five-membered aromatic ring having four carbon atoms and one oxygen.

[0332] As used herein, "tetrazine" or "tetrazine group" refers to a six-membered aromatic ring having the molecular formula C2H2N4 containing four nitrogen atoms or the radical -C2HN4.

[0333] As used herein, the term "azide," "azidyl" or "azido" refers to a -N3 group.

[0334] As used herein, the term "BCN" refers to a bicyclo[6.1.0]non-4-yn-9-yl)methyl group, wherein the exocyclic methylene group can have an exo or endo orientation relative to the bicyclic ring, as shown:

[0335]

[0336] As used herein, throughout the specification, most structures are depicted without stereochemistry for convenience and, therefore, represent all possible stereoisomers.

[0337] With particular regard to the structure of the triazole product of the reaction between BCN and an azido compound, as used herein, throughout the specification, for convenience, the N-attached substituents on the triazole ring are shown at a single regiochemical position. One of ordinary skill in the art will recognize that the reaction of the BCN alkyne with the azido compound will result in a stereoisomeric mixture of products having N-attached substituents at the 1- and 3-positions of the triazole, as shown below:

[0338]

[0339] As used herein, "reactive functional group" refers to a functional group suitable for use in an orthogonal coupling reaction. Suitable reactive functional groups are those that readily undergo orthogonal reactions. Exemplary and non-limiting orthogonal chemical reactions include the functional groups shown in Table 1.

[0340] As used herein, "trigger" refers to a functional group present in the drug-traceless linker adduct DR that is capable of undergoing a chemical reaction to generate a new functional group. The presence of the new functional group significantly reduces the stability of the bond between the drug and the traceless linker counterpart and leads to an increased likelihood of drug release.

[0341] The term "number average molecular weight" or "M n " refers to the statistical average molecular weight of all molecules in a sample, expressed in g / mol. The number average molecular weight can be determined by techniques known in the art, such as gel permeation chromatography (wherein M can be calculated based on the following known standards: n , these standards are based on online detection systems such as refractive index, UV or other detectors), viscometry, mass spectrometry, or colligative methods (e.g., vapor pressure osmometry, end group determination or proton NMR). The number average molecular weight is defined by the following equation,

[0342]

[0343] Among them, M i is the molecular weight of the molecule, and N i is the number of molecules of that molecular weight.

[0344] The term "weight average molecular weight" or "M w ” refers to the statistical average molecular weight of all molecules (taking into account the weight of each molecule in determining its contribution to the molecular weight average), expressed in g / mol. The higher the molecular weight of a given molecule, the greater its contribution to the M w The greater the contribution of the weight average molecular weight, the greater the contribution of the weight average molecular weight. The weight average molecular weight can be calculated by techniques known in the art that are sensitive to molecular size, such as static light scattering, small angle neutron scattering, X-ray scattering, and sedimentation velocity. The weight average molecular weight is defined by the following equation,

[0345]

[0346] Among them'M i ' is the molecular weight of the molecule, and 'N i ' is the number of molecules of that molecular weight.

[0347] The term "viscosity average molecular weight" or "M v ” refers to the statistical average molecular weight of all molecules (taking into account the weight of each molecule in determining its contribution to the molecular weight average) expressed in g / mol. Large molecules or polymers have higher viscosities than smaller molecules. The viscosity average molecular weight is defined by the following equation,

[0348]

[0349] Among them'M i ' is the molecular weight of the molecule, and 'N i' is the number of molecules of the molecular weight, and a is a constant determined by the molecule, solvent, and temperature. Flexible polymer molecules have a values ​​of 0.5≤a≤0.8. Semi-flexible polymer molecules have a≥0.8. The viscosity average molecular weight can be determined by intrinsic viscosity experiments or size exclusion chromatography.

[0350] As used herein, a "drug" comprises one or more biologically active moieties comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring. The biologically active moiety can be a small molecule, a macromolecule, such as a protein, a peptide, or a nucleic acid, or a combination thereof. Specifically with respect to the traceless linker R, the drug or biologically active moiety comprises a "D" in the DR representation. The terms or phrases "drug", "biologically active molecule", "biologically active moiety", "biologically active agent", "active agent" refer to any substance that can affect any physical or biochemical properties of an organism, including but not limited to viruses, bacteria, fungi, plants, animals and humans. In particular, drugs or biologically active molecules include any substance intended for diagnosis, cure, relief, treatment or prevention of human or other animal diseases, or otherwise enhancing the physical and mental health of humans or animals. As used herein, several drugs are referred to as D1-D4 throughout. D1 comprises brocizumab (SEQ ID NO: 4). D2 comprises (SEQ ID NO: 5). D3 comprises (SEQ ID NO: 6). D4 comprises (SEQ ID NO: 7).

[0351] As used herein, "a biologically active moiety comprising at least one primary amine, secondary amine, or ring nitrogen atom containing an azaheteroaryl ring" refers to the free biologically active moiety prior to attachment to a traceless linker or the free biologically active moiety "DH" produced after cleavage from a traceless linker adduct "DR". In some aspects, the drug adduct DR can be biologically active.

[0352] As used herein, "free form" of a drug or biologically active moiety refers to the drug in its unmodified, pharmacologically active form, e.g., before attachment to a traceless linker or after release from a traceless linker in a drug delivery system.

[0353] As used herein, a "traceless linker", R, is a linker represented by formula (I) suitable for releasing a biologically active moiety D comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring:

[0354]

[0355] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0356] R 1 is hydrogen or C1-C4 alkyl;

[0357] R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0358] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0359] a is 0, 1, 2, 3, or 4;

[0360] R 3 is hydrogen or C1-C4 alkyl;

[0361] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0362] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0363] R 12 is hydrogen or C1-C4 alkyl;

[0364] R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0365] R 13 is C1-C4 alkyl; or

[0366] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0367] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0368] R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0369] R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0370] b is an integer from 1 to 10;

[0371] Z is CH-LA, CH-A, NLA or NA;

[0372] L is an optionally substituted divalent linker;

[0373] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0374] R 10 is a reactive functional group suitable for coupling Formula I to a support; and

[0375] R 11 It is a carrier.

[0376] In another embodiment, R is a linker represented by formula (I) suitable for releasing a biologically active moiety D comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring:

[0377]

[0378] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0379] R 1 is hydrogen or C1-C4 alkyl;

[0380] R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0381] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0382] a is 0, 1, 2, 3, or 4;

[0383] R 3 is hydrogen or C1-C4 alkyl;

[0384] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0385] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0386] R 12 is hydrogen or C1-C4 alkyl;

[0387] R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0388] R 13 is C1-C4 alkyl; or

[0389] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0390] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0391] R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0392] R 7is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0393] b is an integer from 1 to 10;

[0394] Z is CH-LA, CH-A, NLA or NA;

[0395] L is an optionally substituted divalent linker Q-[Sp-Q] h -Q;

[0396] Q is independently selected at each occurrence from a bond, O, C(O), N(H), N(C1-C4 alkyl), C(O)NH, C(O)N(C1-C4 alkyl), N(H)C(O), N(C1-C4 alkyl)C(O), N(H)C(O)O, N(C1-C4 alkyl)C(O)O, OC(O)N(H), OC(O)N(C1-C4 alkyl), N(H)C(O)N(H), N(C1-C4 alkyl)C(O)N(H), N(H)C(O)N(C1-C4 alkyl), N(C1-C4 alkyl)C( C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O), OC(O)O, S, S(O)2, N(H)S(O)2, N(C1-C4 alkyl)S(O)2, S(O)2N(H), S(O)2N(C1-C4 alkyl), C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O)C1-C2 alkyl, 1,2,3-triazole, OP(O)2, P(O)2O, C1-C4 alkyl-P(O)2-O, or OP(O)2-C 1-4 alkyl;

[0397] Sp is independently selected at each occurrence from optionally substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, [WO] g 、C1-C8 alkyl-[OW] g , [OW] g -O-C1-C8 alkyl, C1-C8C alkyl-[OW] g -O-C1-C8 alkyl, oligopeptide;

[0398] h is an integer between 1 and 20;

[0399] g is a weighted average between about 2 and about 50;

[0400] W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom;

[0401] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)OC1-C8 alkyl, C(O)N(H)C1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0402] R 10 is a reactive functional group suitable for coupling Formula I to a support; and

[0403] R 11 It is a carrier.

[0404] In another embodiment, R is a linker represented by formula (I) suitable for releasing a biologically active moiety D comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring:

[0405]

[0406] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0407] R 1 is hydrogen or C1-C4 alkyl;

[0408] R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0409] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0410] a is 0, 1, 2, 3, or 4;

[0411] R 3 is hydrogen or C1-C4 alkyl;

[0412] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3aCombine to form a C3-C6 cycloalkane-1,1-diyl;

[0413] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0414] R 12 is hydrogen or C1-C4 alkyl;

[0415] R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0416] R 13 is C1-C4 alkyl; or

[0417] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0418] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0419] R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0420] R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0421] b is an integer from 1 to 10;

[0422] Z is for CHR 8 or NR 9 ;

[0423] R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA;

[0424] q is independently 1, 2, or 3 at each occurrence;

[0425] g and p each independently have a weighted average length between about 2 and about 50;

[0426] m is 1 or 0;

[0427] W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom;

[0428] Q is a bond, O, N(H) or N(C1-C4 alkyl);

[0429] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0430] R 10 is a reactive functional group suitable for coupling Formula I to a support; and

[0431] R 11 It is a carrier.

[0432] In one aspect, the traceless linker is a linker suitable for sustained release of a biologically active moiety. In one aspect, "R" is a traceless linker in the representation DR, wherein "D" refers to a drug comprising a biologically active moiety comprising at least one primary amine, secondary amine, or ring nitrogen atom of an azaheteroaryl ring.

[0433] As used herein, a "drug adduct" is a drug D linked to a traceless linker R. With specific reference to the traceless linker R, the drug adduct comprises a "DR".

[0434] As used herein, a "carrier" is a soluble polymer, a biopolymer, or a cross-linked polymer or biopolymer. The carrier comprises a protein, a nucleic acid, a carbohydrate, or polyethylene glycol. In one aspect, the carrier or carriers are cross-linked intermolecularly, intramolecularly, or a combination thereof. In one aspect, the cross-linked carrier comprises a hydrogel. With specific reference to the traceless linker R, the carrier comprises R 11 In one aspect, Z is linked to a carrier, typically a polymer or a hydrogel. The carrier is attached to a traceless linker R directly or via a non-cleavable spacer. As non-limiting examples, the carrier may comprise polyethylene glycol, a hyaluronic acid polymer, or cross-linked hyaluronic acid or polyethylene glycol capable of forming a hydrogel.

[0435] As used herein, "polymer" refers to a molecule consisting of repeating structural units (monomers) connected by chemical bonds in a linear, cyclic, branched or dendritic manner or a combination thereof, which can be of synthetic origin, or of biological origin, or a combination of the two. Typically, a polymer has an average molecular weight of at least 1 kDa. A copolymer is a polymer consisting of at least two chemically different monomers. Typically, a polymer consists of molecules with a molecular weight distribution. One way to describe the molecular weight distribution of a polymer is the average molecular weight. Typically, a polymer consists of molecules with a distribution of degrees of polymerization. One way to describe the distribution of the degree of polymerization of a polymer is the average degree of polymerization.

[0436] As described herein and depicted in the structures herein, a polymer molecule (e.g., polyethylene glycol or hyaluronic acid) is represented as [P] n , wherein P represents a monomer repeat unit, and n represents the average degree of polymerization of the monomers in the polymer. It will be appreciated by those skilled in the art that if the difference in n is about 10% or less, the two polymer molecules depicted with the same repeat unit P but different n are considered to be equivalent. For the hyaluronic acid copolymers described herein and depicted in the structure, the distribution of monomers in the polymer is uncertain and assumed to be random. In addition, the relative connectivity of the independent molecules of the hydrogel or drug delivery system described herein is random within the group. The structural description of the hydrogel or drug delivery system represents a single potential structural unit in two dimensions, while these complexes are three-dimensional with many structural subunits.

[0437] As described herein, for the traceless linker-drug adduct RD, the exact position of amide bond formation is unclear. This situation occurs when D contains more than one primary or secondary amine capable of forming an amide bond with a linker R (e.g., a protein) or a ring nitrogen atom containing an azaheteroaryl ring. For convenience, the position of the unclear amide bond formation in the adduct is depicted herein as follows:

[0438]

[0439] As used herein, "hydrogel" refers to a three-dimensional, hydrophilic or amphiphilic polymer network capable of absorbing large amounts of water. The network is composed of homopolymers or copolymers and is insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglement) crosslinks. Crosslinking provides the network structure and physical integrity. Hydrogels exhibit thermodynamic compatibility with water, allowing them to swell in aqueous media. The chains of the network are connected in such a way that pores are present and water-soluble solutes having a size smaller than the pores can diffuse in and out of the network.

[0440] As used herein, "linker" or "non-bioactive linker" refers to a linker that does not produce any pharmacological effect. In some embodiments, the linker comprises a bivalent or multivalent organic linker that is compatible with biological systems. "Bivalent" means having a reactive group at each end of the polymer that is suitable for attachment to a traceless linker or drug D. "Multivalent" means having a reactive group at each end of the polymer that is suitable for attachment to a traceless linker or drug D, with additional reactive moieties scattered along the linker molecule.

[0441] As used herein, a "drug delivery system" comprises a drug adduct DRR 11 A vector connected to the 11 The drug delivery system as described herein is a molecular conjugate comprising: one or more drugs D; one or more traceless linkers R; and one or more carriers R 11 In some embodiments, there may be multiple different drug species conjugated to the drug delivery system. For example, two different drugs (eg, D1 and D2) may be conjugated in a single drug delivery system.

[0442] As used herein, the phrase "water-insoluble" refers to a swellable three-dimensional cross-linked molecular network that forms a hydrogel. If the hydrogel is suspended in a large excess of water or an aqueous buffer of physiological osmotic pressure concentration, the hydrosol may absorb a large amount of water, for example, up to 10 times on a weight / weight basis, and thus be swellable, but retain the physical stability and shape of the gel after the excess water is removed. Such a shape can have any geometric shape, and it should be understood that such a single hydrogel object should be considered as a single molecule composed of multiple components, wherein each component is connected to each other by chemical bonds.

[0443] As used herein, "sustained release" or "sustained release rate" refers to a situation in which the interval between subsequent doses of each drug delivery system required to achieve a desired therapeutic effect is extended. A drug administered in daily doses can, for example, be converted into a sustained release form with a one-week or even longer time interval between two administrations.

[0444] As used herein, "functional group" refers to a group of atoms within a molecule that exhibits a specific chemical activity. Examples are amide, amine, alcohol, carbonyl, carboxylic acid, thiol.

[0445] As used herein, "protecting group" refers to a moiety that temporarily protects a functional group of a molecule during synthesis to obtain chemical selectivity in a subsequent chemical reaction. Protecting groups for alcohols are, for example, benzyl and trityl; protecting groups for amines are, for example, tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl and benzyl; and examples of protecting groups for thiols are 2,4,6-trimethoxybenzyl, phenylthiomethyl, acetamidomethyl, p-methoxybenzyloxycarbonyl, tert-butylthio, triphenylmethyl, 3-nitro-2-pyridylthio, 4-methyltrityl.

[0446] As used herein, "protected functional group" means a functional group that is protected by one or more protecting groups.

[0447] As used herein, "PBS" refers to phosphate buffered saline.

[0448] As used herein, the term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the biologically active agent and are generally not biologically or otherwise undesirable. In many cases, biologically active agents are capable of forming acid and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto.

[0449] Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids, for example, acetate, aspartate, benzoate, benzenesulfonate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlorophyllinate, citrate, edisylate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, dodecylsulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthoate, naphthylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, polygalacturonate, propionate, stearate, succinate, subsalicylate, tartrate, toluenesulfonate and trifluoroacetate.

[0450] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0451] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like.

[0452] Pharmaceutically acceptable base addition salts can be formed with inorganic bases and organic bases.

[0453] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table. In certain embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.

[0454] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; basic ion exchange resins, etc. Certain organic amines include isopropylamine, benzathine, choline salts, diethanolamine, diethylamine, lysine, meglumine, piperazine, or tromethamine.

[0455] The pharmaceutically acceptable salts can be synthesized from a parent compound, a basic or acidic moiety, by conventional chemical methods.

[0456] Typically, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of a suitable base (e.g., hydroxides of Na, Ca, Mg or K, carbonates, bicarbonates, etc.), or by reacting the free base forms of these compounds with a stoichiometric amount of a suitable acid. Such reactions are typically carried out in water or an organic solvent or in a mixture of the two. Typically, where feasible, it is desirable to use a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile. Lists of additional suitable salts can be found, for example, in “Remington's Pharmaceutical Sciences”, 18th ed., Mack Publishing Company, Easton, Pa., (1990) and in “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

[0457] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc. and the like and combinations thereof, as will be known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329). Except for conventional carriers that are incompatible with the active ingredient, other conventional carriers may be used in therapeutic or pharmaceutical compositions.

[0458] The term "therapeutically effective amount" of a biologically active agent refers to an amount of the biologically active agent that will elicit a biological or medical response in a subject, such as improvement of symptoms, alleviation of a condition, slowing or delaying disease progression, or preventing a disease.

[0459] In another non-limiting embodiment, the term "therapeutically effective amount" refers to the following amount of a bioactive agent, which, when applied to a cell, or a tissue, or a non-cellular biological material or medium, is effective in at least partially treating a disease or disorder. As will be appreciated by those of ordinary skill in the art, the absolute amount of an effective specific agent can vary according to such factors as a desired biological endpoint, an agent to be delivered, a target tissue, etc. Those skilled in the art will appreciate that a "therapeutically effective amount" can be administered in a single dose or can be achieved by multiple doses. For example, in the case of an agent for the treatment of heart failure, an effective amount can be an amount sufficient to cause clinical improvement in the patient, such as increasing exercise tolerance / ability, increasing blood pressure, reducing fluid retention and / or improving the results of quantitative tests of cardiac function (e.g., ejection fraction, exercise capacity (failure time), etc.).

[0460] As used herein, the term "subject" refers to an animal. Typically, the animal is a mammal. Subjects also refer to, for example, primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human.

[0461] As used herein, the terms "inhibit," "inhibition," or "inhibiting" refer to the reduction or suppression of a given condition, symptom or disorder, or a disease, or a significant decrease in the baseline activity of a biological activity or process.

[0462] As used herein, the term "treat, treating or treatment" of any disease or disorder refers to, in one embodiment, alleviating a disease or disorder (i.e., slowing down or stagnating or reducing the development of a disease or at least one of its clinical symptoms). In another embodiment, "treat, treating or treatment" refers to alleviating or alleviating at least one physical parameter, including those that cannot be discerned by the patient. In yet another embodiment, "treat, treating or treatment" refers to regulating a disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In yet another embodiment, "treat, treating or treatment" refers to preventing or delaying the onset or development or progression of the disease or disorder. As used herein, the term "prevent, preventing and prevention" refers to the prevention of the recurrence, onset or development of one or more symptoms of a disorder of a subject caused by administration of therapy (e.g., a therapeutic agent) or a combination of administration of therapy (e.g., a combination of therapeutic agents).

[0463] As used herein, a subject is "in need of" or "in need of" a treatment if the subject would benefit biologically, medically, or in quality of life from such treatment.

[0464] As used herein, the terms “a”, “an”, “the” and similar terms used herein (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0465] As used herein, the term "about" refers to any value, including whole and fractional components, within a variation of up to ±10% of the value modified by the term "about."

[0466] The term "or" may be a conjunction or a disjunctive conjunction such that "or" encompasses "and / or".

[0467] One embodiment described herein is a drug adduct DR comprising a drug and a traceless linker, wherein R is a linker represented by formula (I) suitable for releasing a biologically active moiety D comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring:

[0468]

[0469] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0470] R1 is hydrogen or C1-C4 alkyl;

[0471] R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0472] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0473] a is 0, 1, 2, 3, or 4;

[0474] R 3 is hydrogen or C1-C4 alkyl;

[0475] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0476] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0477] R 12 is hydrogen or C1-C4 alkyl;

[0478] R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0479] R 13 is C1-C4 alkyl; or

[0480] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0481] R 4is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0482] R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0483] R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0484] b is an integer from 1 to 10;

[0485] Z is CH-LA, CH-A, NLA or NA;

[0486] L is an optionally substituted divalent linker;

[0487] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0488] R 10 is a reactive functional group suitable for coupling Formula I to a support; and

[0489] R 11 It is a carrier.

[0490] Another embodiment described herein is a drug adduct DR comprising a drug and a traceless linker, wherein R is a linker represented by formula (I) suitable for releasing a biologically active moiety D comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring:

[0491]

[0492] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0493] R 1 is hydrogen or C1-C4 alkyl;

[0494] R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0495] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0496] a is 0, 1, 2, 3, or 4;

[0497] R 3 is hydrogen or C1-C4 alkyl;

[0498] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0499] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0500] R 12 is hydrogen or C1-C4 alkyl;

[0501] R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0502] R 13 is C1-C4 alkyl; or

[0503] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0504] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0505] R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0506] R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0507] b is an integer from 1 to 10;

[0508] Z is CH-LA, CH-A, NLA or NA;

[0509] L is an optionally substituted divalent linker Q-[Sp-Q] h -Q;

[0510] Q is independently selected at each occurrence from a bond, O, C(O), N(H), N(C1-C4 alkyl), C(O)NH, C(O)N(C1-C4 alkyl), N(H)C(O), N(C1-C4 alkyl)C(O), N(H)C(O)O, N(C1-C4 alkyl)C(O)O, OC(O)N(H), OC(O)N(C1-C4 alkyl), N(H)C(O)N(H), N(C1-C4 alkyl)C(O)N(H), N(H)C(O)N(C1-C4 alkyl), N(C1-C4 alkyl)C( C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O), OC(O)O, S, S(O)2, N(H)S(O)2, N(C1-C4 alkyl)S(O)2, S(O)2N(H), S(O)2N(C1-C4 alkyl), C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O)C1-C2 alkyl, 1,2,3-triazole, OP(O)2, P(O)2O, C1-C4 alkyl-P(O)2-O, or OP(O)2-C 1-4 alkyl;

[0511] Sp is independently selected at each occurrence from optionally substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, [WO] g 、C1-C8 alkyl-[OW] g, [OW] g -O-C1-C8 alkyl, C1-C8C alkyl-[OW] g -O-C1-C8 alkyl, oligopeptide;

[0512] h is an integer between 1 and 20;

[0513] g is a weighted average between about 2 and about 50;

[0514] W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom;

[0515] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)OC1-C8 alkyl, C(O)N(H)C1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0516] R 10 is a reactive functional group suitable for coupling Formula I to a support; and

[0517] R 11 It is a carrier.

[0518] Another embodiment described herein is a drug adduct DR comprising a drug and a traceless linker, wherein R is a linker represented by formula (I) suitable for releasing a biologically active moiety D comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring:

[0519]

[0520] wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring;

[0521] R 1 is hydrogen or C1-C4 alkyl;

[0522] R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0523] R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group;

[0524] a is 0, 1, 2, 3, or 4;

[0525] R 3 is hydrogen or C1-C4 alkyl;

[0526] R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0527] Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ;

[0528] R 12 is hydrogen or C1-C4 alkyl;

[0529] R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl;

[0530] R 13 is C1-C4 alkyl; or

[0531] CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers;

[0532] R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy;

[0533] R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl;

[0534] R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) bO-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups;

[0535] b is an integer from 1 to 10;

[0536] Z is CHR 8 or NR 9 ;

[0537] R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA;

[0538] q is independently 1, 2, or 3 at each occurrence;

[0539] g and p each independently have a weighted average length between about 2 and about 50;

[0540] m is 1 or 0;

[0541] W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom;

[0542] Q is a bond, O, N(H) or N(C1-C4 alkyl);

[0543] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0544] R 10 is a reactive functional group suitable for coupling Formula I to a support; and

[0545] R 11 It is a carrier.

[0546] In some aspects, R of formula (I) 1 -R 8 has the following meanings. In one aspect, R 1 is hydrogen or methyl. In another aspect, R 1ais hydrogen or methyl. In another aspect, R 1 and R 1a In another aspect, R 2 In another aspect, the two R 2 The groups combine to form a fused or spiro C3-C6 cycloalkyl group. In another aspect, R 3 is hydrogen or methyl. In another aspect, R 3a is hydrogen or methyl. In another aspect, R 3 and R 3a In another aspect, Y is C(O)R 4 , and R 4 is C1-C6 alkyl, C1-C2 alkoxy, C1-C2 alkyl or C1-C6 alkoxy. 4 In another aspect, Y is SiR 5 R 6 OR 7 ; and R 5 and R 6 are each methyl, ethyl, propyl or isopropyl; and R 7 is ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, ethoxyethyl, ethoxyisopropyl, tetrahydropyranyl or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein b is 2, 3 or 4.

[0547] In some aspects, R of formula (I) 8 and R 9 has the following meanings. In one aspect, R 9 In another aspect, R 9 In another aspect, R 9 is C(O)C5 alkyl. 9 is C(O)C2 alkyl. 9 is C(O)C1 alkyl. 9 In another aspect, R 9 is C(O)(CH2CH2O) b CH2CH2, wherein b is 1, 2, 3 or 4. In another aspect, R 9 In another aspect, R 9 In another aspect, R9 is a C1 alkylamide. 9 It is a C2 alkylamide group.

[0548] In some aspects, Z is CH-LA, CH-A, NLA, or NA;

[0549] L is an optionally substituted divalent linker;

[0550] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0551] R 10 is a reactive functional group suitable for coupling Formula I to a support; and

[0552] R 11 It is a carrier.

[0553] In some aspects, Z is CH-LA, CH-A, NLA, or NA;

[0554] L is an optionally substituted divalent linker Q-[Sp-Q] h -Q;

[0555] Q is independently selected at each occurrence from a bond, O, C(O), N(H), N(C1-C4 alkyl), C(O)NH, C(O)N(C1-C4 alkyl), N(H)C(O), N(C1-C4 alkyl)C(O), N(H)C(O)O, N(C1-C4 alkyl)C(O)O, OC(O)N(H), OC(O)N(C1-C4 alkyl), N(H)C(O)N(H), N(C1-C4 alkyl)C(O)N(H), N(H)C(O)N(C1-C4 alkyl), N(C1-C4 alkyl)C( C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O), OC(O)O, S, S(O)2, N(H)S(O)2, N(C1-C4 alkyl)S(O)2, S(O)2N(H), S(O)2N(C1-C4 alkyl), C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O)C1-C2 alkyl, 1,2,3-triazole, OP(O)2, P(O)2O, C1-C4 alkyl-P(O)2-O, or OP(O)2-C 1-4 alkyl;

[0556] Sp is independently selected at each occurrence from optionally substituted C1-C20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, [WO] g 、C1-C8 alkyl-[OW] g , [OW] g -O-C1-C8 alkyl, C1-C8C alkyl-[OW] g -O-C1-C8 alkyl, oligopeptide;

[0557] h is an integer between 1 and 20;

[0558] g is a weighted average between about 2 and about 50;

[0559] W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom;

[0560] A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)OC1-C8 alkyl, C(O)N(H)C1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace;

[0561] R 10 is a reactive functional group suitable for coupling Formula I to a support; and

[0562] R 11 It is a carrier.

[0563] In certain aspects, g and h are from 1 to about 25 or from 1 to about 10.

[0564] In some aspects, Z of formula (I) is NR 9 In one aspect, R 9 Further R 10 In another aspect, R 9 Further R 11 replace.

[0565] According to formula (I), R 10 Is a reactive functional group suitable for orthogonal coupling reactions. Suitable reactive functional groups are those that are prone to orthogonal reactions. Exemplary and non-limiting orthogonal chemical reactions include the functional groups shown in Table 1. For a given row in Table 1, functional group X (left column) is suitable for coupling reactions with functional group Y (right column). The coupling reaction can be a covalent bond or an intermolecular complex. In most embodiments, the coupling reaction produces a covalent bond. In other reactions, such as the reaction of adamantane with cyclodextrin, the coupling is a non-covalent molecular association. In one aspect, the functional group is selected from azido, alkynyl, substituted or unsubstituted C6-C12 Cycloalkynyl, C6-C 12 Heterocycloalkynyl, C6-C 12 In one aspect, the functional group is a substituted C6-C8 alkylene group, a substituted C1 ... 12 In one aspect, the functional group is cycloalkynyl, wherein the substitution comprises a fused cyclopropyl group. In another aspect, the functional group is bicyclo[6.1.0]non-4-yn-9-yl. In another aspect, the functional group is azido.

[0566]

[0567]

[0568]

[0569]

[0570] In some embodiments, the traceless linker R comprises formula (II):

[0571]

[0572] Among them, R 9 and D are as defined above in formula (I), and T1 comprises a substituted or unsubstituted C2-C 10 Esters, substituted or unsubstituted C3-C 10 Silyl ethers (which contain one or more heteroatoms selected from nitrogen or oxygen).

[0573] In some aspects, T1 is -CR 1 R 1a OY, which comprises a trigger portion, and comprises one of the following structures in Table 2.

[0574]

[0575]

[0576] In some embodiments, the traceless linker R according to Formula I or II comprises any one of the following structures in Table 3.

[0577]

[0578]

[0579]

[0580] In another embodiment, the traceless linker R of formula (I) or formula (II) is conjugated to a drug or a biologically active moiety. In one aspect, the conjugated drug traceless linker comprises any one of the following structures in Table 4.

[0581]

[0582]

[0583]

[0584] Synthesis of traceless linker R

[0585] Another embodiment is a method for making a traceless linker R of formula (I), comprising any of the following steps: (A) reacting the N4 nitrogen of a piperazine compound containing a nucleophilic group at the C2 position with a functionalized acyl linker compound to form an N4-acylpiperazine; (B) carboxymethylating the N1 nitrogen of the piperazine ring of the N4-acylpiperazine, wherein the carboxyl group is covalently attached to a suitable protecting group; (C) reacting the nucleophilic group of the N4-acylpiperazine with a trigger compound as described herein; (D) if necessary, complicating the functionalized acyl linker on N4 to include a functional group suitable for attaching the traceless linker to a carrier compound. In one aspect, the nucleophilic group at the C2 position of the piperazine compound comprises a hydroxyl group. In another aspect, the nucleophilic group at the C2 position of the piperazine compound is a primary alcohol. In another aspect, the primary alcohol present in the piperazine compound of step (A) or (D) is conjugated to a suitable protecting group or a trigger group. In another aspect, the protecting group or triggering group comprises an ester, a silyl ether, an acetal, a carbamate, a carbonate or a compound containing disiloxane. In one aspect, the N4 carboxymethyl ester protecting group is deprotected to form a carboxylic acid suitable for forming an amide bond of a bioactive agent comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring. In another aspect, in the carboxymethylation step, the carboxyl group of the carboxymethylating agent is covalently attached via an amide bond to a bioactive agent comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring. In one aspect, the functional group of step A is suitable for attaching a traceless linker to a carrier compound.

[0586] Another embodiment is a method for making a traceless linker R of formula (I), comprising any of the following steps: (A) carboxymethylating the N1 nitrogen of a piperazine compound containing a nucleophilic group at the C2 position, wherein the carboxyl group is covalently attached to a suitable protecting group; (B) reacting the N4 nitrogen of the piperazine compound with a functionalized alkyl linker to form an N4-alkylpiperazine; (C) reacting the nucleophilic group of the N4-alkylpiperazine with a trigger compound described herein; (D) if necessary, complicating the functionalized alkyl linker on N4 to include a functional group suitable for attaching the traceless linker to a carrier compound. In one aspect, the reactive group on the piperazine compound comprises a hydroxyl group. In another aspect, the nucleophilic group at the C2 position of the piperazine compound is a primary alcohol. In another aspect, the primary alcohol present on the piperazine compound of step (A) or (D) is conjugated to a suitable protecting group or trigger group. In another aspect, the trigger compound comprises an ester, a silyl ether, an acetal, a carbamate, a carbonate or a compound containing disiloxane. In one aspect, the N4 carboxymethyl ester protecting group is deprotected to form a carboxylic acid suitable for forming an amide bond of a bioactive agent comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring. In another aspect, in the carboxymethylation step, the carboxyl group of the carboxymethylating agent is covalently attached via an amide bond to a bioactive agent comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring. In one aspect, the functional group of step A is suitable for attaching a traceless linker to a carrier compound.

[0587] Another embodiment is a method for making a traceless linker R of formula (I), the method comprising any of the following steps: (A) introducing a functionalized linker at the C4 position of a pyridine compound containing a nucleophilic group at the C2 position via a cross-coupling or substitution reaction; (B) hydrogenating a 4-substituted pyridine compound to form a 4-substituted piperidine compound; (C) carboxylmethylating the N1 nitrogen of the 4-substituted piperidine compound, wherein the carboxyl group is covalently attached to a suitable protecting group; (D) reacting the nucleophilic group of the 4-substituted piperidine compound with a trigger compound described herein; (E) if necessary, complicating the functionalized alkyl linker on C4 to include a functional group suitable for attaching the traceless linker to the carrier compound. In one aspect, the nucleophilic group on the pyridine or piperidine compound comprises a hydroxyl group. In another aspect, the nucleophilic group at the C2 position of the pyridine or piperidine compound is a primary alcohol. In another aspect, the trigger compound comprises an ester, a silyl ether, an acetal, a carbamate, a carbonate, or a disiloxane-containing compound. In one aspect, the N4 carboxymethyl ester protecting group is deprotected to form a carboxylic acid suitable for forming an amide bond to a biologically active agent comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring. In another aspect, in the carboxymethylation step, the carboxyl group of the carboxymethylating agent is covalently attached to a biologically active agent comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring via an amide bond. In one aspect, the functional group of step A is suitable for attaching a traceless linker to a carrier compound.

[0588] Suitable blocking groups are the following parts, which are reversibly connected to reactive functional groups or chemical functional groups, thereby making it impossible to react with, for example, other chemical functional groups. Exemplary and non-limiting amino blocking groups include fluorenyl methylene oxygen group (FMOC), tert-butyloxycarbonyl (BOC), carboxyl benzyl (Cbz) etc. Exemplary and non-limiting alcohol blocking groups include tert-butyl ether, propyl ether, benzyl ether, tert-butyl dimethyl silyl ether (TBDMS) etc. Deprotection groups can be added or removed as needed in the whole building-up process, to close and expose specific parts.

[0589] In one embodiment, the method for making a traceless joint is according to reaction schemes 1-3. The following schemes are general and non-limiting schemes for making a traceless joint. As shown in the examples herein, some synthetic pathways do not particularly conform to these general schemes. In another aspect, the method for making a traceless joint is according to the reaction scheme provided in Example 1.

[0590]

[0591] Wherein one or more of the steps shown above may be performed in a different sequential order or omitted depending on the reagents used;

[0592] X is Cl, O-NHS, O(C=O)-R 2a or an activated group of X-OH, and the reaction involves standard peptide coupling reagents such as 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU);

[0593] R 2a is a C1-C8 alkyl or aryl group;

[0594] P4 is a suitable protecting group or H;

[0595] L is an optionally substituted divalent linker;

[0596] P1 and P2 are protecting groups which may be the same;

[0597] Y is a suitable trigger provided in Table 2; and

[0598] FG comprises one or more suitable functional groups that can be conjugated to a carrier as described herein.

[0599]

[0600] Wherein one or more of the steps shown above may be performed in a different sequential order or omitted depending on the reagents used;

[0601] Wherein X2 is an activating group, such as Cl, Br, I or trifluoromethanesulfonate;

[0602] Y, L, P1, P2, P4 and FG are as described in Scheme 1;

[0603] P3 is a protecting group which may be the same as P1 and P2.

[0604]

[0605] wherein one or more of the steps shown above may be performed in a different sequential order or omitted depending on the reagents used; and

[0606] L, P1, P2, P3, Y and FG are as described in Scheme 2.

[0607] As described herein, the adduct DR of formula (I) or formula (II) is prepared by reacting the carboxylic acid form of the traceless linker R with the amino group of the biologically active agent containing at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring to form an amide bond. This method is described in Scheme 4. A suitable carboxylic acid activating agent is used to promote the amide bond forming reaction. As an example, the carboxylic acid can be converted to an amino-reactive form by the action of disuccinimidyl carbonate to form an NHS ester; then, the NHS ester of the traceless linker R is reacted with the amino-containing drug D to form the adduct DR.

[0608] In Scheme 4, L, Y and FG are as described in Schemes 1-3; D is a biologically active agent containing at least one primary or secondary amine or a ring nitrogen atom containing an azaheteroaryl ring.

[0609]

[0610] One embodiment described herein is a traceless linker R of formula (I) or formula (II) attached to a carrier composition via a linker having one or more functional groups. In one aspect, the carrier composition comprises a traceless linker R as described herein. 11 In one aspect, R 11 Containing polymers, biopolymers, or R 8 or R 9 In one aspect, the carrier composition is a hydrogel. In one aspect, the hydrogel composition comprises hyaluronic acid, polyethylene glycol, polypropylene glycol, polyethylene oxide, polyglutamate, polylysine, polysialic acid, polyvinyl alcohol, polyacrylate, polymethacrylate, polyacrylate / polymethacrylate copolymer, polyacrylamide, polymethacrylamide, polyvinyl pyrrolidone, polyoxazoline, polyiminocarbonate, polyamino acid, hydrophilic polyester, polyamide, polyurethane, polyurea, dextran, agarose, xylan, mannan, carrageenan, alginate, gelatin, collagen, albumin, cellulose, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, hydroxyethyl starch, chitosan, nucleic acid, derivatives thereof, copolymers thereof, or combinations thereof. In another aspect, R 11 In one aspect, the hydrogel comprises hyaluronic acid with polyethylene glycol. In another aspect, R 11 A cross-linked hydrogel comprising hyaluronic acid or polyethylene glycol. In one aspect, in the case of drug delivery systems used in the eye or synovial joints, R 11 Contains hyaluronic acid or cross-linked hyaluronic acid.

[0611] As described in this article, R 11The carrier composition is cross-linked to connect multiple molecules together and promote hydrogel formation. Cross-linking can be accomplished using any method known in the art (see, e.g., Liu et al., Chem. Commun. 51, (2015)). In one embodiment described herein, hyaluronic acid or polyethylene glycol is functionalized with one or more functional groups shown in Table 1 to provide reactive functional groups for cross-linking. In another aspect, hyaluronic acid or polyethylene glycol is functionalized with a functional group selected from an azido group, an alkynyl group, a substituted or unsubstituted C7-C 12 Cycloalkynyl, C7-C 12 Cycloalkenyl, substituted or unsubstituted C7-C 12 Heterocycloalkynyl, vinylcarboxyl, vinylsulfonyl, C2-C8 alkenyl, amino, thiol, C1-C8 carboxyl, C1-C8 carbonyl, oxyamine, carbohydrazide, maleimide, α-halocarbonyl, furan, substituted or unsubstituted tetrazinyl, lysine, glutamine, cyclodextrin and adamantyl. In another aspect, the hyaluronic acid is functionalized by an azido group.

[0612] The degree of functionalization can determine the porosity of the hydrogel. In one aspect, about 5% to about 50% of the carrier polymer is functionalized, including all integers within the specified range. In one aspect, the carrier polymer is functionalized by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or even higher.

[0613] In one aspect, the carrier is hyaluronic acid. In another aspect, hyaluronic acid is reacted with 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium-4-ium chloride (CAS No. 3945-69-5) and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethylamine (CAS No. 134179-38-7) to form azide functionalized hyaluronic acid ([HA-N3]). In one aspect, the reaction is:

[0615]

[0616] Experimental conditions are described in Example 3. In one aspect, about 5% to about 50% of the hyaluronic acid is functionalized, including all integers within the specified range. In one aspect, the hyaluronic acid is functionalized by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% or even higher.

[0617] In another embodiment, polyethylene glycol is functionalized with various agents to form cross-linkers for linking the functionalized hyaluronic acid monomers as discussed above.

[0618] Synthesis of 2kDa 2-arm PEG-BCN crosslinker

[0619] In one aspect, as shown, M n About 2 kDa polyethylene glycol diamine hydrochloride was reacted.

[0620] The reaction conditions are described in Example 4.

[0621]

[0622] Synthesis of 10 kDa 4-arm PEG-BCN crosslinker

[0623] In another aspect, as shown, M n The reaction was carried out with 10 kDa 4-arm polyethylene glycol amine hydrochloride (pentaerythritol core, JenKem Technology). The reaction conditions are described in Example 4.

[0624]

[0625] Synthesis of 2 kDa 2-arm β-alanine PEG-BCN crosslinker

[0626] In another aspect, as shown, M n About 2 kDa polyethylene glycol and 3-((tert-butoxycarbonyl)amino)propionic acid were reacted. The reaction conditions are described in Example 4.

[0627]

[0628] Synthesis of 2 kDa 2-arm aminocyclopropanecarboxylic acid PEG-BCN crosslinker

[0629] In another aspect, as shown, M n About 2 kDa polyethylene glycol and 1-((tert-butoxycarbonyl)amino)cyclopropane-1-carboxylic acid were reacted. The reaction conditions are described in Example 4.

[0630]

[0631] Another embodiment described herein is one or more cross-linking agents. In one aspect, the cross-linking agent comprises Formula V:

[0632]

[0633] In another aspect, the cross-linking agent comprises Formula VI:

[0634]

[0635] In another aspect, the cross-linking agent comprises Formula VIa:

[0636]

[0637] In another aspect, the cross-linking agent comprises Formula VIb:

[0638]

[0639] In another aspect, the cross-linking agent comprises Formula VIc:

[0640]

[0641] In another aspect, the cross-linking agent comprises Formula VII:

[0642]

[0643] In another aspect, the cross-linking agent comprises Formula VIII:

[0644]

[0645] In another aspect, the cross-linking agent comprises Formula IX:

[0646]

[0647] In another embodiment described herein, R 11 A functionalized carrier is included, which has been reacted with one or more cross-linking agents to form a cross-linked carrier. In one embodiment, the cross-linked carrier forms a hydrogel. On the one hand, the cross-linked carrier is hyaluronic acid, which has been functionalized and cross-linked with one or more polyethylene glycol cross-linking agents as described herein to form a hydrogel.

[0648] In one embodiment, a cross-linked carrier antibody comprising hyaluronic acid can be prepared by appropriately reacting a functionalized polymer as follows:

[0649]

[0650] Although not limited to any particular example and description, in one embodiment, as determined by viscometry, the sodium salt of hyaluronate is labeled by the supplier Lifecore Biomedical (HA200K, Chaska, MN) as having a nominal average molecular weight that varies from batch to batch in the range of 151 kDa-300 kDa. For the purpose of illustration, a hyaluronate sodium salt molecule with an assumed nominal average molecular weight of 200 kDa will consist of an average of about 500 monomer units. In this structure and the following structures, unmodified monomer units are defined as "m"; monomer units modified by azido groups are defined as "n"; azido monomer units conjugated to cross-linked molecules are defined as "p"; and azido monomer units conjugated to traceless linker-drug adducts are defined as "q". For a polymer chain comprising ≈500 monomer units, (n+m+p+q≈500). If the modification percentage of the sodium hyaluronate molecule is 25%, then m = 75% and (n + p + q) = 25%.

[0651] Similarly, the PEG unit present in one embodiment of the cross-linker is derived from a starting PEG described as having a nominal average molecular weight of 2 kDa, and will be composed of approximately 44 repeating PEG monomer units (depicted as "w" in these depicted structures).

[0652] In the following formula, in one embodiment, for a nominal average molecular weight of about 200 kDa as determined by viscometry, the sum of the unmodified disaccharide repeating units (m) of hyaluronic acid plus the modified disaccharide repeating units (n+p+q) of hyaluronic acid in a random distribution (=m+n+p+q) can contain about 500 units. This applies to formulas X to XXII.

[0653] In one aspect, the cross-linked carrier comprises Formula X:

[0654]

[0655] In another aspect, the cross-linked carrier comprises Formula XI:

[0656]

[0657] In another aspect, the cross-linked carrier comprises Formula XII:

[0658]

[0659] In another aspect, the cross-linked carrier comprises Formula XIIa:

[0660]

[0661] In another aspect, the cross-linked carrier comprises Formula XIIb:

[0662]

[0663] In another aspect, the cross-linked carrier comprises Formula XIIc:

[0664]

[0665] In another aspect, the cross-linked carrier comprises Formula XIII:

[0666]

[0667] Another embodiment described herein is a drug adduct DR, wherein D is a biologically active moiety comprising at least one primary amine, secondary amine, or ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety. In one aspect, D comprises a protein, a nucleic acid, a carbohydrate, a peptide, a nucleotide, an oligosaccharide, or a small molecule, each of which has at least one primary or secondary amine, and the small molecule has a molecular weight between about 100 g / mol and about 2000 g / mol.

[0668] In one embodiment described herein, D comprises a low molecular weight biologically active molecule. In one aspect, D comprises a biologically active molecule containing at least one primary amine, secondary amine, or ring nitrogen atom containing an azaheteroaryl ring and having a molecular weight of about 100 g / mol to about 2000 g / mol, including all integers within the specified range. In one aspect, D comprises a biologically active molecule comprising at least one primary amine, secondary amine, or ring nitrogen atom comprising an azaheteroaryl ring and having a molecular weight of about 100 g / mol, about 150 g / mol, about 200 g / mol, about 250 g / mol, about 300 g / mol, about 350 g / mol, about 400 g / mol, about 450 g / mol, about 500 g / mol, about 550 g / mol, about 600 g / mol, about 650 g / mol, about 700 g / mol, about 750 g / mol, about 800 g / mol, about 850 g / mol, about 900 g / mol, about 950 g / mol 1, about 1000 g / mol, about 1050 g / mol, about 1100 g / mol, about 1150 g / mol, about 1200 g / mol, about 1250 g / mol, about 1300 g / mol, about 1350 g / mol, about 1400 g / mol, about 1450 g / mol, about 1500 g / mol, about 1550 g / mol, about 1600 g / mol, about 1650 g / mol, about 1700 g / mol, about 1750 g / mol, about 1800 g / mol, about 1850 g / mol, about 1900 g / mol, about 1950 g / mol, or about 2000 g / mol.

[0669] In another embodiment, D comprises a biologically active molecule containing at least one primary amine, secondary amine, or ring nitrogen atom containing an azaheteroaryl ring and having a molecular weight of about 100 g / mol to about 2000 g / mol, which modulates at least one biologically relevant target in a therapeutically beneficial manner. In one aspect, D comprises a central nervous system-active agent, an anti-infective agent, an anti-allergic agent, an immunomodulator, an anti-obesity agent, an anticoagulant, an antidiabetic agent, an antineoplastic agent, an antibacterial agent, an antifungal agent, an analgesic agent, a contraceptive, an anti-inflammatory agent, an anti-angiogenic agent, an anti-glaucoma agent, a regenerative agent, a steroid, a vasodilator, a vasoconstrictor, or a cardiovascular drug.In one aspect, D comprises acarbose, alapropylate, alendronate, amantadine, amikacin, amineptine, aminoglutethimide, amisulpride, amlodipine, amitosaline, amoxapine, amoxicillin, amphetamine, amphetamine, amphetamine B, ampicillin, amprenavir, aminone, anilidine, apraclonidine, apramycin, articaine, atenolol, atomoxetine, avizafone, baclofen, benazepril, benserazide, benzocaine, betaxolol, bleomycin, bromfenac, bromofaromine, carvedilol, norpseudoephedrine, cathinone, amosulbutamide, cephalexin, clinafloxacin, ciprofloxacin, deferoxamine, delavirdine daunorubicin, desipramine, daunorubicin, dexmethylphenidate, dexmethylphenidate, diaminophenyl sulfone, dizocilpine, dopamine, dobutamine, dorzolamide, adriamycin, duloxetine, eflornithine, enalapril, epinephrine, epirubicin, ergoline, ertapenem, esmolol, enoxacin, ethambutol, fenfluramine, fenoldopam, fenoterol, fingolimod, flecamide, trifluramine, fosamprenavir, frovatriptan, furosemide, fluoxetine, gabapentin, gatifloxacin, gemifloxacin, gentamicin, grepafloxacin, sea Cocaine, hydralazine, hydrochlorothiazide, ifenac spray, idarubicin, imiquimod, mecamylamine, isoproterenol, isradipine, kanamycin A, ketamine, labetalol, lamivudine, levobunolol, levodopa, levothyroxine, lisinopril, lomefloxacin, loracarbef, maprotiline, mefloquine, melphalan, memantine, meropenem, mesalamine, mescaline, methyldopa, methylenedioxymethamphetamine, metoprolol, milnacipran, mitoxantrone, moxifloxacin, norepinephrine, norfloxacin, nortriptyline, neomycin B, nystatin, oseltamivir, pamidronate, paroxetine, pazufloxacin, pemetrexed Trixetine, perindopril, phenmetrazine, phenelzine, pregabalin, procaine, pseudoephedrine, protriptyline, reboxetine, ritodrine, sarubicin, salbutamol, serotonin, sertraline, sitagliptin, sotalol, spectinomycin, sulfadiazine, sulfamerazine, sertraline, spectinomycin, sulfamethoxazole, tacrine, tamsulosin, terbutaline, timolol, tirofiban, tobramycin, tocamide, tofloxacin, trandolapril, tranexamic acid, tranylcypromine, trimetrexate, trovafloxacin, valacyclovir, valganciclovir, vancomycin, puromycin, viloxazine, and zalcitabine.

[0670] In another embodiment, D comprises one or more bioactive peptides, including but not limited to C-type natriuretic peptide (CNP), atrial natriuretic peptide (ANP), exenatide-4, insulin, adrenocorticotropic hormone, adrenomedullin, atropine, ω-funnel web spider toxin, agouti protein-related protein, one or more angiotensin, apelin 12, apelin 13, apelin 36, or derivatives thereof, bradykinin, calcitonin and amphetamine-regulated transcript (CART), corticotropin releasing factor (CRF) , α-defensin, β-defensin, δ-sleep-inducing peptide (DSIP), elastase-specific inhibitor (Elafin), endokinin, endomorphin, endorphin, endothelin, exenatide, fibronectin active fragment, galanin, galanin-like peptide, big gastrin, gastrin I, gastrin-related peptide, gastric inhibitory polypeptide, gastrin-releasing peptide, ghrelin, desacyl ghrelin, glucagon, glucagon-like peptide, growth hormone-releasing factor, Chilean bird spider (Grammostolaspatulata)GsMTx-4, guangxitoxin-1E, guanylin, hepatic bactericidal peptide 1, hepatic bactericidal peptide / LEAP-1, histamine 5, human growth hormone, humanin, tiger bird spider toxin, Iberiotoxin, emperor scorpion toxin (imperatoxin A), insulin-like growth factor, pituitary hormone, IRL1620, connecting peptide, short-skinned scorpion toxin, kisspeptin, kurtoxin, lipolysis hormone, lipolysis hormone, hepatocyte growth factor, liver-expressed antimicrobial Phytokines 2, luteinizing hormone-releasing hormone, lysenin, LL-37, Mager toxin, mast degranulation peptide, mast cell degranulation peptide, melanin-concentrating hormone, melanocyte-stimulating hormone, MSH release inhibitory factor, midkine, mollusk cardiac excitatory neuropeptide, morphine tolerance peptide, motilin, muscarinic toxin, neuroendocrine regulatory peptide-2, neurokinin A, neurokinin B, neuromedin, neurostatin-13, neuropeptide, neurotensin, neurotoxin NSTX-3, pain stabilizer, pain-sensitive peptide, obesity inhibitor, opioid peptides (enkephalin, endophrenia, BAM-12P, casomorphin, dynorphin, endomorphin, neoendophrenia, pain-sensitive peptide ), orexin-A / -B, orphanin, osteocalcin, oxytocin, pancreatic inhibin, parathyroid hormone, parathyroid hormone-related protein, peptide 234, peptide histidine-methionine, peptide T, peptide YY, frog peptide, pituitary adenylate cyclase-activating polypeptide, platelet factor-4, plectasin, pleiotrophin, preadrenomedullin, prolactin-releasing peptide, psalmotoxin 1, purotoxin-1, pyroglutamylated RFamide peptide, renin, RFamide-related peptide-1, RFamide-related peptide-3, sarucin-α / β, viperin , schizophrenia-related peptide, blue crab toxin, secretin, relaxin, serum thymic factor, sodium potassium ATPase inhibitor-1, somatostatin, carpet anemone toxin, substance K, apnopressin, apnopressin-related peptide, substance P, tachykinin, tarantula SNX-482, tarantula ProTx-I, tarantula ProTx-II, tropolamine, neodymium scorpion toxin Ka, thyrotropin-releasing hormone, phagocytosis, uroguanylin, uroguanylin isomer A or B, urocortin, urocortin II, uropressin II, uropressin II-related peptide, vasoactive intestinal peptide, vasopressin, oxytocin, viral replication inhibitory peptide, clawed frog peptide, or a combination thereof.

[0671] In another aspect, D comprises ACTH, adenosine deaminase, β-galactosidase, alpha-1 antitrypsin (AAT), alpha-1 proteinase inhibitor (API), alteplase, amylin (amylin, pramlintide), anistreplase, ancroserine protease, antibodies (monoclonal or polyclonal,and fragments or fusions thereof), antithrombin III, antitrypsin, aprotinin, asparaginase, atosiban, biphalin, bivalirudin, bone morphogenetic protein, bovine pancreatic trypsin inhibitor (BPTI), cadherin fragments, calcitonin (salmon), collagenase, complement C1 esterase inhibitor, conotoxin, cytokine receptor fragments, DNA enzyme, dynorphin A, endorphin, enfuvirtide, enkephalin, erythropoietin, exenatide, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, fibrinolytic enzyme, fibroblast growth factor FGF, growth hormone releasing peptide 2 (GHRP2), fusion protein, follicle stimulating hormone, gramicidin, ghrelin, desaccharide-ghrelin, granulocyte colony stimulating factor (G-CSF), galactosidase, glucagon, glucagon-like peptide, glucocerebrosidase, granulocyte macrophage colony stimulating factor (GM-CSF), human heat shock protein (HSP), phospholipase-activating protein (PLAP), human chorionic gonadotropin (hCG), hemoglobin, hepatitis B vaccine, hirudin, human serine protease inhibitor, hyaluronidase, iduronidase, immunoglobulin, influenza vaccine, white Cytokines (1α, 1β, 2, 3, 4, 6, 10, 11, 12, 13, 21), IL-1 receptor antagonist (rhIL-1ra), insulin, insulin-like growth factor, insulin-like growth factor binding protein (rhIGFBP), interferon (α2a, α2b, α2c, β1a, β1b, γ1a, γ1b), intracellular adhesion molecules, keratinocyte growth factor (KGF), P-selectin glycoprotein ligand (PSGL), transforming growth factor, lactase, leptin, leuprorelin, levothyroxine, luteinizing hormone, Lyme vaccine, natriuretic peptides (ANP, BNP, CNP and fragments), neuropeptide Y, pancreatic lipase, pancreatic polypeptide, papain, parathyroid hormone, PDGF, pepsin, peptide YY, platelet activating factor acetylhydrolase (PAF-AH), prolactin, protein C, thymosin, octreotide, secretin, sermorelin, soluble tumor necrosis factor receptor (TNFR), superoxide dismutase (SOD), somatotropin (growth hormone), somatostatin, streptokinase, sucrase, terlipressin, tetanus toxin fragment, galactosidase, thrombin, thymosin, thyroid stimulating hormone, thyrotropin, tumor necrosis factor (TNF), TNF receptor-IgG Fc, tissue plasminogen activator (tPA), TSH, urodilator, urate oxidase, urokinase, vaccine, vascular endothelial growth factor (VEGF), vasoactive intestinal peptide, vasopressin, ziconotide, lectin, ricin, or a combination thereof. ,

[0672] In another embodiment, D comprises abciximab, adalimumab, aflibercept, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab, canakinumab, certolizumab pegol, cetuximab, daclizumab, daratumumab, denosumab, eculizumab, efalizumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lambalizumab, muromonab-CD3, natalizumab, nivolumab, ofatumumab, omalizumab, palivizumab, panitumumab, peprazib, pembrolizumab, ranibizumab, rituximab, tocilizumab, tositumomab, trastuzumab, ustekinumab, or vedolizumab or a derivative or combination thereof.

[0673] In another embodiment, D comprises abatacept, albumin, alefacept, alpha-1 proteinase inhibitor, alpha-galactosidase A, alpha-L-iduronidase, botulinum toxin A, botulinum toxin B, darbepoietin, denileukin, dornase alpha, collagenase, complement protein, erythropoietin, etanercept, factor VIII, fibrin, fibrinogen, filgrastim, follicle-stimulating hormone, glucocerebrosidase, granulocyte colony stimulating factor (G-CSF), sargramostim, human growth hormone, human chorionic gonadotropin, hyaluronidase, insulin, insulin-like growth factor, insulin-like growth factor 1 (IGF-1), interferon (IF), luteinizing hormone, oprelvekin, pramlintide, mecasermin, N-acetylgalactosamine-4-sulfatase (galactosamine), tissue plasminogen activator (TPA), a protein-based vaccine, or a combination thereof.

[0674] In another embodiment, D comprises one or more of a protein, a peptide, an antibody, a humanized antibody, an antibody fragment, Fab, scFv, a nucleic acid, a carbohydrate, a small molecule, or a combination thereof.

[0675] In another embodiment, D comprises one or more of a nucleotide, an oligonucleotide, a polynucleotide, a nucleic acid, including but not limited to DNA, RNA, PNA, siRNA, miRNA, mRNA, tRNA, rRNA, or a combination thereof.

[0676] In one embodiment, D comprises brocuzumab (SEQ ID NO:4), D2 (SEQ ID NO:5), D3 (SEQ ID NO:6), or D4 (SEQ ID NO:7), ranibizumab, bevacizumab, 5-(5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; 5-(6,7-dihydro-5H-pyrrolo[3,4d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; N-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6-((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide; (S)-5-(6-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yloxy)-N-(5-(1-methylcyclopropyl)-1H-pyrazol-3-yl)-1H-indole-1-carboxamide, or a combination thereof.

[0677] Another embodiment described herein is a drug delivery system DRR comprising a carrier-traceless linker bioactive agent conjugate 11 , wherein D comprises a biologically active moiety as described herein that contains at least one primary amine, secondary amine, or ring nitrogen atom containing an azaheteroaryl ring, and R comprises a 11 attached as described herein without trace joint, the R 11 comprising a carrier polymer or hydrogel as described herein. In one aspect, R 11In one aspect, R comprises a non-traceable linker as described herein. In another aspect, D comprises a protein, a peptide, an antibody Fab, Fc, scFv, a nucleic acid, a siRNA, a miRNA, an mRNA, a carbohydrate, a peptide nucleic acid (PNA), an organic small molecule comprising at least one primary amine, a secondary amine or a ring nitrogen atom of an azaheteroaryl ring, or a combination thereof. In one aspect, D comprises brocuzumab (SEQ ID NO: 4), D2 (SEQ ID NO: 5), D3 (SEQ ID NO: 6), or D4 (SEQ ID NO: 7). NO:7), ranibizumab, bevacizumab, 5-(5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; 5-(6,7-dihydro-5H-pyrrolo[3,4d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; N-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6-((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide; (S)-5-(6-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yloxy)-N-(5-(1-methylcyclopropyl)-1H-pyrazol-3-yl)-1H-indole-1-carboxamide, or a combination thereof.

[0678] In another embodiment, DRR 11 Comprising Formula XIV, wherein "drug" can be one or more of the following: brocuzumab (SEQ ID NO: 4), D2 (SEQ ID NO: 5), D3 (SEQ ID NO: 6), or D4 (SEQ ID NO: 7), ranibizumab, bevacizumab, 5-(5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; 5-(6,7-dihydro-5H-pyrrolo[3,4d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; N-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6-((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide; (S)-5-(6-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yloxy)-N-(5-(1-methylcyclopropyl)-1H-pyrazol-3-yl)-1H-indole-1-carboxamide, or a combination thereof.

[0679] In some embodiments, DRR 11 Any of the species shown in Formulae XIV-XXIII are included.

[0680] In one embodiment, the drug or bioactive agent D comprises polypeptide D2 (SEQ ID NO: 5) or a pegylated form thereof, such as, for example, D3 (SEQ ID NO: 6). D2 and D3 polypeptides and derivatives thereof can be prepared as described in International Patent Application Publication No. WO2007 / 146689, which is incorporated herein by reference for such teachings.

[0681] In another embodiment, the drug or bioactive agent D comprises brocuzumab (SEQ ID NO: 4). Brocuzumab is a single chain variable fragment (scFv) comprising a VH region (SEQ ID NO: 1), a VL region (SEQ ID NO: 2), and a linker region (SEQ ID NO: 3). Brocuzumab and its derivatives can be prepared as described in International Patent Application Publication No. WO 2009 / 155724, which is incorporated herein by reference for such teachings. Methionine derived from the start codon is present in the VL of brocuzumab. In some cases, methionine can be cleaved post-translationally before being assembled into a drug delivery system as described herein.

[0682] In another embodiment, the drug or bioactive agent D comprises D4 (SEQ ID NO: 7). D4 is a 13 amino acid peptide comprising the sequence pE-RPRLCHKGP-Nle-CF-OH (SEQ ID NO: 7), with C6 and C 12 wherein pE represents pyroglutamic acid and Nle represents norleucine. D4 and its derivatives can be prepared as described in International Patent Application Publication No. WO 2013 / 111110, which is incorporated herein by reference for such teachings.

[0683] In another embodiment, the drug or bioactive agent D comprises apelin 12, apelin 13, apelin 36, or a derivative thereof. Apelin and its derivatives are disclosed in WO 2013 / 111110, WO 2014 / 081702, WO 2015 / 013168, WO 2015 / 013165, or WO 2015 / 013167, each of which is incorporated herein by reference for such teachings.

[0684] In another embodiment, the drug or bioactive agent D comprises one or more anti-vascular endothelial growth factor drugs, such as bevacizumab or ranibizumab. Bevacizumab is a recombinant human monoclonal IgG1 antibody that binds to human vascular endothelial growth factor (VEGF) and inhibits its biological activity in in vitro and in vivo assay systems. Bevacizumab contains a human framework region and a complementary determining region of a mouse antibody that binds to VEGF. The antibody has a molecular weight of approximately 149 kDa. Bevacizumab is produced in a Chinese hamster ovary expression system in a nutrient medium. Ranibizumab is a recombinant humanized IgG1κ isotype monoclonal antibody fragment designed for intraocular use. Ranibizumab binds to human vascular endothelial growth factor A (VEGF-A) and inhibits its biological activity. Ranibizumab, which lacks an Fc region, has a molecular weight of approximately 48 kDa and is produced by an E. coli expression system in a nutrient medium. Bevacizumab and ranibizumab are described in International Patent Application Publication Nos. WO 1994 / 004679 and WO 1998 / 45331, respectively, both of which are incorporated herein by reference for such teachings.

[0685] In another embodiment, the drug or bioactive agent D comprises 5-(5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; 5-(6,7-dihydro-5H-pyrrolo[3,4d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide. -amide; N-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6-((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide; or (S)-5-(6-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yloxy)-N-(5-(1-methylcyclopropyl)-1H-pyrazol-3-yl)-1H-indole-1-carboxamide. The synthesis of these molecules is described in International Patent Application Publication No. WO 2010 / 066684, which is incorporated herein by reference for such teachings.

[0686] Another embodiment described herein is a method for assembling a drug delivery system; the method may include one of the following steps in the following order:

[0687] (a) Preparation of carrier molecule R 11 , where R 11 is a cross-linked hydrogel, and the step then includes a method for preparing the hydrogel; the carrier molecule may optionally be purified at this stage;

[0688] (b) separately conjugating a traceless linker R to a biologically active agent D comprising a primary amine, a secondary amine, or a ring nitrogen atom of an azaheteroaryl ring, thereby forming a traceless linker-D adduct; the traceless linker-D adduct may optionally be purified at this stage,

[0689] (c) The carrier molecule R 11 conjugation with a traceless linker-D adduct; and

[0690] (d) Purification of the drug delivery system from the reagents. This method is shown in Scheme 5A below.

[0691] Another embodiment described herein is a method for assembling a drug delivery system, the method comprising the steps of:

[0692] (a) Preparation of carrier molecule R 11 , where R 11 is a cross-linked hydrogel, and the step then includes a method for preparing the hydrogel; the carrier molecule may optionally be purified at this stage;

[0693] (b) The traceless linker R and the carrier molecule R 11 conjugation, thereby forming a carrier molecule-traceless linker adduct, which can optionally be purified at this stage;

[0694] (c) conjugating a biologically active agent D comprising a primary amine, a secondary amine, or a ring nitrogen atom of an azaheteroaryl ring to a carrier molecule-traceless linker adduct; and

[0695] (d) Purification of the drug delivery system from the reagents. This method is shown in Scheme 5B below.

[0696] Another embodiment described herein is a method for assembling a drug delivery system, the method comprising the steps of:

[0697] (a) Preparation of non-crosslinked carrier molecules R 11 , the carrier molecule may optionally be purified at this stage;

[0698] (b) separately conjugating a traceless linker R to a biologically active agent D comprising a primary amine, a secondary amine, or a ring nitrogen atom of an azaheteroaryl ring, thereby forming a traceless linker-D adduct; the traceless linker-D adduct may optionally be purified at this stage;

[0699] (c) The carrier molecule R 11 conjugation with a traceless linker-D adduct, which can optionally be purified at this stage;

[0700] (d) By combining the non-crosslinked carrier molecule-traceless linker-bioactive agent R 11-RD is incubated with a suitable cross-linking agent to form a hydrogel to prepare a cross-linked hydrogel; and

[0701] (e) Purification of the drug delivery system from the reagents. This method is shown in Scheme 5C below.

[0702] Another embodiment described herein is a method for assembling a drug delivery system, the method comprising the steps of:

[0703] (a) Preparation of non-crosslinked carrier molecules R 11 , the carrier molecule may optionally be purified at this stage;

[0704] (b) The traceless linker R and the carrier molecule R 11 conjugation, thereby forming a carrier molecule-traceless linker adduct, which can optionally be purified at this stage; and

[0705] (c) conjugating a biologically active agent D comprising a primary amine, a secondary amine, or a ring nitrogen atom of an azaheteroaryl ring to a carrier molecule-traceless linker adduct;

[0706] (d) By combining the non-crosslinked carrier molecule-traceless linker-bioactive agent R 11 -RD is incubated with a suitable cross-linking agent to form a hydrogel to prepare a cross-linked hydrogel; and

[0707] (e) Purification of the drug delivery system from the reagents. This method is shown in Scheme 5D below.

[0708] Another embodiment described herein is a method for assembling a drug delivery system, the method comprising the steps of:

[0709] (a) Preparation of non-crosslinked carrier molecules R 11 , the carrier molecule may optionally be purified at this stage;

[0710] (b) The traceless linker R and the carrier molecule R 11 conjugation, thereby forming a carrier molecule-traceless linker adduct, which can optionally be purified at this stage;

[0711] (c) By combining the non-cross-linked carrier molecule-traceless linker-adduct R 11 -R is incubated with a suitable cross-linking agent to form a hydrogel to prepare a cross-linked hydrogel, which can be optionally purified at this stage;

[0712] (d) conjugating a biologically active agent D comprising a primary amine, a secondary amine, or a ring nitrogen atom of an azaheteroaryl ring to the cross-linked carrier molecule-traceless linker adduct, which conjugate may optionally be purified at this stage; and

[0713] (e) Purification of the drug delivery system from the reagents. This method is shown in Scheme 5E below.

[0714] Scheme 5A-E. Scheme 5 shows five methods, AE, for assembling a drug delivery system as described herein.

[0715] legend:

[0716]

[0717] In one embodiment, the drug delivery system may comprise formula (XIV):

[0718]

[0719] wherein L2 represents a spacer group that may be specific for a particular cross-linker, and "drug" refers to a biologically active molecule containing at least one primary amine, secondary amine, or ring nitrogen atom of an azaheteroaryl ring that modulates at least one biologically relevant target in a therapeutically beneficial manner.

[0720] In one embodiment, spacer L2 comprises any species shown in Table 5.

[0721]

[0722]

[0723] In another embodiment, the drug delivery system may comprise formula (XV), wherein the drug is brolucizumab:

[0724]

[0725] In another embodiment, the drug delivery system may comprise formula (XVI), wherein the drug is brolucizumab:

[0726]

[0727] In another embodiment, the drug delivery system may comprise formula (XVII), wherein the drug is brolucizumab:

[0728]

[0729] In another embodiment, the drug delivery system may comprise formula (XVIII), wherein the drug is brolucizumab:

[0730]

[0731] In another embodiment, the drug delivery system may comprise formula (XVIIIa), wherein the drug is brolucizumab:

[0732]

[0733] In another embodiment, the drug delivery system may comprise formula (XVIIIb), wherein the drug is brolucizumab:

[0734]

[0735] In another embodiment, the drug delivery system may comprise formula (XVIIIc), wherein the drug is brolucizumab:

[0736]

[0737] In another embodiment, the drug delivery system may comprise formula (XVIIId), wherein the drug is brolucizumab:

[0738]

[0739] In another embodiment, the drug delivery system may comprise formula (XVIIIe), wherein the drug is brolucizumab:

[0740]

[0741] In another embodiment, the drug delivery system may comprise formula (XVIIIf), wherein the drug is brolucizumab:

[0742]

[0743] In another embodiment, the drug delivery system may comprise formula (XIX), wherein the drug is D2:

[0744]

[0745] In another embodiment, the drug delivery system may comprise formula (XX), wherein the drug is D2:

[0746]

[0747] In another embodiment, the drug delivery system may comprise formula (XXI), wherein the drug is D2:

[0748]

[0749] In another embodiment, the drug delivery system may comprise formula (XXII), wherein the drug is D2:

[0750]

[0751] In another embodiment, the drug delivery system may comprise formula (XXIII), wherein the drug is D4:

[0752]

[0753] Another embodiment described herein is a method for treating a disease or disorder using a drug delivery system as described herein. Another embodiment described herein relates to a drug delivery system comprising DR represented by formula (I) or a pharmaceutically acceptable salt thereof, used as a medicine. Another embodiment is a drug delivery system comprising DR represented by formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a drug for treating cardiac disorders, musculoskeletal disorders, and eye disorders, the eye disorders including but not limited to wet age-related macular degeneration, macular edema, retinal branch vein occlusion, central retinal vein occlusion, glaucoma, hereditary hemorrhagic telangiectasia (HHT), proliferative diabetic retinopathy, uveitis, paroxysmal nocturnal hemoglobinuria (PNH), geographic atrophy, dry age-related macular degeneration, neovascularization, diabetic macular edema or mid-term age-related macular degeneration. On the one hand, the pharmaceutical composition of the drug delivery system as described herein can be administered to a subject in need thereof by injection. On the one hand, it can be administered by injection or surgical implantation.

[0754] One embodiment described herein is a method for treating an eye disorder including but not limited to wet age-related macular degeneration, macular edema, branch retinal vein occlusion, central retinal vein occlusion, glaucoma, hereditary hemorrhagic telangiectasia (HHT), proliferative diabetic retinopathy, uveitis, paroxysmal nocturnal hemoglobinuria (PNH), geographic atrophy, dry age-related macular degeneration, neovascularization, diabetic macular edema or intermediate age-related macular degeneration, the method comprising administering a drug delivery system DR as described herein, wherein D comprises ranibizumab, bevacizumab, brolucizumab (SEQ ID NO:4), 5-(5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; 5-(6,7-dihydro-5H-pyrrolo[3,4d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; N-(1- methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6-((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide; (S)-5-(6-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yloxy)-N-(5-(1-methylcyclopropyl)-1H-pyrazol-3-yl)-1H-indole-1-carboxamide, or a combination thereof. In one aspect, the composition is a solution or suspension injected into the eye. In one aspect, the injection is an intraocular injection (e.g., an intravitreal injection). In another aspect, the composition is a gel or semisolid composition implanted into the eye using surgery or by injection.

[0755] Another embodiment described herein is a method for treating musculoskeletal disorders including but not limited to cartilage regeneration, tendon healing, wound healing, or bulbospinal muscular atrophy, the method comprising administering a drug delivery system DR as described herein, wherein D comprises D2 (SEQ ID NO: 5), a pegylated form of D2 such as D3 (SEQ ID NO: 6), or a combination thereof.

[0756] Another embodiment described herein relates to a drug delivery system comprising DR represented by formula (I) or a pharmaceutically acceptable salt thereof (wherein D comprises D2 (SEQ ID NO: 5), a pegylated form of D2 such as D3 (SEQ ID NO: 6), or a combination thereof) for use in the treatment of cardiac disorders, musculoskeletal disorders, or ocular disorders, including but not limited to wet age-related macular degeneration, macular edema, branch retinal vein occlusion, central retinal vein occlusion, glaucoma, hereditary hemorrhagic telangiectasia (HHT), proliferative diabetic retinopathy, uveitis, paroxysmal nocturnal hemoglobinuria (PNH), geographic atrophy, dry age-related macular degeneration, neovascularization, diabetic macular edema, or intermediate age-related macular degeneration. In one aspect, the composition is a solution or suspension that is injected into a joint. In another aspect, the composition is a gel or semisolid composition that is implanted into a joint using surgery or by macroaperture injection. In another aspect, the composition is in the form of particles that are injected into or near a joint. In another aspect, the composition is implanted in or near a joint as a biodegradable mesh or gauze that is ultimately absorbed or processed in situ. In another aspect, the composition is impregnated into sutures, staples, plates, meshes, or similar articles that are used during surgery to reconnect or repair tendons, ligaments, cartilage, bones, or other joint components after trauma or disease.

[0757] Another embodiment described herein is a method for treating the following cardiac disorders, such as heart failure, acute compensated heart failure (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular disease, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including gestational diabetes), obesity, peripheral arterial disease, cerebrovascular accident, transient ischemic attack, traumatic brain injury, amyotrophic lateral sclerosis, burns (including sunburn) or pre-eclampsia, the method comprising administering a drug delivery system DR as described herein, wherein D comprises D4 (SEQ ID NO: 7). On the one hand, the composition is a solution or suspension delivered intravenously, intra-arterially, subcutaneously, intramuscularly or intraperitoneally. In another aspect, the composition is a gel or semisolid composition implanted using surgical methods or large-aperture injections. In another aspect, the composition is a gel or semisolid composition applied topically or directly to a skin wound.

[0758] In one embodiment, the drug delivery system as described herein releases the bioactive agent at a specific release rate. On the one hand, the release rate can be adjusted or regulated by the "trigger" component of the traceless joint R. Without being bound by any theory, it is believed that the reaction of the trigger under physiological conditions produces a nucleophilic functional group, such as the hydroxyl functional group of the amide bond that connects the drug to the drug delivery system in an intramolecular manner. In one embodiment, the reaction of the trigger is a hydrolysis reaction that causes the formation of the hydroxyl functional group. Without being bound by any theory, it is believed that the steric hindrance of the trigger part is related to the slower reaction of the trigger, and the presence and proximity of the electron withdrawing group are related to the faster reaction of the trigger.

[0759] In one embodiment, the half-life of release of the biologically active portion is about 0.5 hours, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 or more than 4 years. In one aspect, the biologically active portion release half-life is about 2.5 days, about 4.5 days, about 7 days, about 10 days, about 11 days, about 12 days, about 14 days, about 15 days, about 21 days, about 28 days, about 30 days, about 31 days, about 32 days, about 40 days, about 58 days, about 60 days, about 65 days, about 70 days, about 80 days, about 125 days, about 165 days, about 380 days, about 940 days, or even longer.

[0760] In one embodiment, the half-life of the traceless linker trigger ester hydrolysis is about 0.5 hours, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 or more than 4 years. In one aspect, the traceless linker trigger ester hydrolysis half-life is about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 4 days, about 5 days, about 10 days, about 12 days, about 15 days, about 20 days, about 30 days, about 32 days, about 35 days, about 40 days, about 55 days, about 60 days, about 90 days, about 120 days, about 150 days, about 180 days, about 200 days, about 300 days, about 400 days, or even longer.

[0761] In another embodiment, the drug delivery system RR 11 In releasing drug D (e.g., DRR 11 →RR 11 In some embodiments, the drug delivery system is eliminated from a tissue, organ or compartment to which it is administered with a half-life of about 0.5 hours, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 or more than 4 years.

[0762] Other embodiments described herein are drug delivery systems DRR comprising the drug delivery system described herein 11 In one aspect, the pharmaceutical composition is suitable for injection or implantation in a subject in need thereof.

[0763] Pharmaceutical compositions suitable for administration by injection or implantation include sterile aqueous solutions, suspensions, or dispersions and sterile powders or lyophilisates for the extemporaneous preparation of sterile injectable solutions or dispersion.

[0764] For intravenous administration, suitable carriers include phosphate buffered saline (PBS), normal saline, Ringer's solution or water for injection. In all cases, the composition should be sterile and must have a fluidity that reaches the degree of easy injection. Preferred pharmaceutical formulations should be stable under manufacturing and storage conditions and must be preserved against microbial contamination such as bacteria and fungi. Generally speaking, the relevant carrier can be a solvent or dispersion medium comprising the following substances: for example, water, buffer, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and mixtures suitable therefor. Suitable fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the required particle size in the case of a dispersion, and by using a surfactant. Preventing the effect of microorganisms can be achieved by different antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents will preferably be included in the composition, such as sugars, polyols such as mannitol, amino acids, sorbitol, sodium chloride or a combination thereof. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0765] Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fat emulsions or suspensions. The compositions may be sterilized and / or contain adjuvants (e.g., preservatives, stabilizers, wetting agents or emulsifiers, solution promoters, salts and / or buffers for regulating osmotic pressure). In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulation or coating methods, respectively, and contain about 0.1%-75%, or about 1%-50% of active ingredients.

[0766] As required, sterile injectable solutions or suspensions can be prepared by: the desired amount of drug delivery system is incorporated into a suitable solvent together with a combination of one or more ingredients, followed by filtration sterilization. Typically, solutions or suspensions are prepared by incorporating the active compound into a sterile vehicle (such as sterile PBS) and any excipient. With regard to the sterile powder for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient and any other desired ingredients from its previous sterile filtered solution.

[0767] Transmucosal or transdermal administration is also possible. Suitable compositions for transdermal administration include an effective amount of a bioactive agent and a suitable carrier. Carriers suitable for transdermal delivery include absorbable pharmacologically acceptable solvents that help pass through the recipient's skin. For example, a transdermal device is in the form of a bandage that includes a back member, a reservoir containing the compound optionally with a carrier, a rate-controlling barrier for delivering the compound to the host's skin at a controlled and predetermined rate over an extended period of time, and a device for fixing the device to the skin.

[0768] Compositions suitable for topical application (e.g., to the skin, eyes or joints) include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like. Such topical delivery systems will be particularly suitable for skin applications. It is therefore particularly suitable for local uses, including cosmetics, formulations well known in the art. These may contain solubilizers, stabilizers, tension enhancers, buffers or preservatives.

[0769] As used herein, topical administration may also involve inhalation or intranasal administration. It may be conveniently delivered in the form of a dry powder (alone, as a mixture, e.g., a dry blend with lactose, or mixed component particles, e.g., mixed with phospholipids) from a dry powder inhaler with or without a suitable propellant, or in the form of an aerosol spray from a pressurized container, pump, spray, nebulizer or atomizer.

[0770] Also described herein are pharmaceutical compositions and dosage forms comprising one or more agents that reduce the rate of decomposition of the compositions described herein as active ingredients. Such agents (referred to herein as "stabilizers") include, but are not limited to, antioxidants (such as ascorbic acid), pH buffers, or salt buffers, etc.

[0771] The pharmaceutical composition of effective amount used will for example depend on treatment situation and object.It will be appreciated by those skilled in the art that therefore suitable dosage level for treatment will partly change according to the following: the therapeutic agent incorporated into the drug delivery system, the indication using the drug delivery system, the route of administration and the patient's physique (body weight, body surface area or organ size) and / or condition (age and general health status).Therefore, clinicians can titrate dosage and change the route of administration to obtain the best therapeutic effect.

[0772] The frequency of administration will depend on the pharmacokinetic parameters of the therapeutic agent incorporated into the drug delivery system used. Typically, the clinician will use the composition until the dosage for the desired effect is reached. Therefore, the composition can be used in a single dose, over time with two or more doses (may contain or may not contain the same amount of the desired molecule), or via an implant device or catheter continuous infusion. Further refinement of appropriate doses is routinely performed by those of ordinary skill in the art, and within the scope of their routinely performed tasks. Appropriate doses can be determined via the use of appropriate dose-response data.

[0773] The drug delivery system can be prepared into a solution or suspension of microparticles. On the one hand, the carrier is formed into microparticles, which can be administered to the target tissue by a standard syringe, for example, by subcutaneous, intravitreal, intraarticular, intratendon or intramuscular administration. Such particles can have an average particle size distribution between 1 μm and 5000 μm. Other aspects include biodegradable gauze, mesh or sutures impregnated with a drug delivery system as described herein.

[0774] In certain embodiments, the microparticles of drug delivery systems as described herein can be produced by emulsion polymerization, photolithography, spinning, molding, spray drying, grinding, extrusion, mechanical crushing or similar methods known in the art. In one embodiment, drug delivery systems as described herein, carrier polymers or hydrogels can be divided into microparticles by extruding via a net or sieve. On the one hand, extrusion can be repeated many times and / or continuously passed through smaller meshes to obtain a desired particle distribution size.

[0775] In one embodiment, based on laser diffraction measurement particle size, when suspended in isotonic aqueous formulation buffer, the drug delivery system has an average particle size distribution between 1 μm and 5000 μm. In some aspects, when suspended in isotonic buffer, the drug delivery system has an average particle size distribution between 10 μm and 1000 μm. In another aspect, when suspended in isotonic aqueous buffer, the drug delivery system has an average particle size distribution between 50 μm and 500 μm. In another aspect, when suspended in isotonic aqueous buffer, the drug delivery system has an average particle size distribution between 100 μm and 300 μm. In another aspect, when suspended in isotonic aqueous buffer, the drug delivery system has an average particle size distribution between 200 μm and 300 μm. In some embodiments, the average particle size distribution includes about 10 μm, about 50 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 750 μm, about 1000 μm, about 1500 μm, about 2000 μm, about 2500 μm or about 5000 μm.

[0776] Particle size can be determined using standard techniques known to those of ordinary skill in the art. Exemplary techniques that can be used to measure the particle size distribution of drug delivery system particles can include laser diffraction analysis, light scattering (e.g., dynamic light scattering), microscopic particle image analysis, elutriation, or aerosol mass spectrometry. Samples of drug delivery system particles can be measured as dry samples or wet samples. Following the recommended operating procedures, any commercially available instrument for measuring particle size can be used according to the manufacturer's instructions, including from Cilas; Brookhaven Instruments Corporation; Malvern Instruments; Horiba Scientific; or instruments of Wyatt.

[0777] Particle sizes measured using the techniques described herein can be expressed as derived diameters with normal or non-normal distributions in the case of mean, median (e.g., mass median diameter), and particle size mode. Particle size distributions can be expressed as diameter number distributions, surface area distributions, or particle volume distributions. The mean of a particle size distribution can be calculated and expressed in a variety of ways, such as volume mean diameter (D[4,3] or d[4,3]). 43 ), average surface area diameter (D[3,2] or d 32 ) or mean particle size (D[1,0] or d 10 ). Because particle size distribution values ​​can vary depending on the measurement method and the way the distribution is represented, comparisons of different average particle size distributions must be calculated by the same method in order to make an accurate comparison. For example, a sample having a measured and calculated volume average diameter must be compared to a second sample having a measured and calculated volume average diameter, ideally measured under the same conditions using the same measuring instrument. Therefore, the specific particle size distributions described herein are not intended to be limited to any one type of method for measuring or calculating particle size distributions (e.g., diameter number distributions, surface area distributions, or particle volume distributions), but rather are indicative of particle size values ​​and distributions thereof for each method of measuring particle diameters described herein.

[0778] In one embodiment, the drug delivery system can be administered by injection via the following needle: less than 0.6mm inner diameter (e.g., 20 gauge) needle, preferably less than 0.3mm inner diameter (e.g., 25 gauge) needle, more preferably less than 0.25mm inner diameter (e.g., 27 gauge) needle, even more preferably less than 0.2mm inner diameter (e.g., 28 gauge) needle, and most preferably less than 0.16mm inner diameter (e.g., 30 gauge) needle. In one embodiment, when the drug delivery system is administered intraocularly or intravitreally, it is preferably less than 0.16mm inner diameter (e.g., 30-34 gauge) needle. For example, when injecting a drug delivery system with a particle size distribution of 100 μm to 300 μm, a 20 gauge needle may be the best for delivery. However, since the particle morphology is flexible, a needle size narrower than the drug delivery system particle size can be successfully used.

[0779] The phrase and term "can be administered by injection", "injectable" or "syringability" refers to a combination of factors, such as a certain force applied to the plunger of a syringe containing a drug delivery system described herein, which drug delivery system swells in a liquid at a certain concentration (w / v) and at a certain temperature, a needle of a given inner diameter connected to the outlet of such syringe; and the time required to expel a certain volume of the drug delivery system from the syringe through the needle.

[0780] In one embodiment, the injectability test is performed on the drug delivery system suspended in PBS or saline to a concentration of about 0.1% to about 20% (w / v), including all integers within the specified percentage range.

[0781] Therefore, drug delivery system shows the beneficial effect of controlling the release rate with respect to the drug DH released. Preferably, sustained release rate is obtained. Sustained release means that compared with the administration of drugs in the absence of drug delivery system, the administration interval of each drug delivery system described herein is extended. For example, based on the drug usually administered once a day or several times, drug delivery system provides at least three days, at least one week, at least one month, several months or several years of effective level of treatment.

[0782] Another embodiment described herein is a pharmaceutical composition of the drug delivery system described herein. The pharmaceutical composition may include one or more excipients, such as:

[0783] (i) Buffers: Physiologically tolerated buffers that maintain the pH in the desired range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulfate, nitrate, chloride, pyruvate. Antacids such as Mg(OH)2 or ZnCO3 may also be used. The buffering capacity may be adjusted to match the conditions most sensitive to pH stability.

[0784] (ii) Isotonicity modifiers: minimize pain caused by cell damage due to osmotic pressure differences at the injection depot. Glycerol and sodium chloride are examples. Effective concentrations can be determined by osmometry using an assumed serum osmotic pressure of 285 mOsmol / kg-315 mOsmol / kg.

[0785] (iii) Preservatives and / or antimicrobial agents: Multiple-dose parenteral formulations may require the addition of preservatives in sufficient concentrations to minimize the risk of infection to subjects following injection, and regulatory requirements have been established. Typical preservatives include m-cresol, phenol, methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosol, sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and benzalkonium chloride.

[0786] (iv) Stabilizers: Stabilization is achieved by enhancing the stability of the protein, by destabilizing the denatured state, or by direct binding of the excipient to the protein. Stabilizers can be amino acids such as alanine, arginine, aspartic acid, glycine, histidine, lysine, proline; sugars such as glucose, sucrose, trehalose; polyols such as glycerol, mannitol, sorbitol; salts such as potassium phosphate, sodium sulfate; chelating agents such as EDTA, hexaphosphate; ligands such as divalent metal ions (zinc, calcium, etc.); other salts or organic molecules (such as phenol derivatives). In addition, oligomers or polymers such as cyclodextrins, dextran, dendrimers, polyethylene glycol, polyvinyl pyrrolidone, protamine or human serum albumin can be used.

[0787] (v) Anti-adsorption agents: Ionic or nonionic surfactants or other proteins or soluble polymers are mainly used to competitively coat or adsorb to the inner surface of the composition container, such as poloxamer (Pluronic F-68), PEG dodecyl ether (Brij 35), polysorbate 20 and 80, dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA, and gelatin. The concentration and type of excipients selected depend on the effects to be avoided, but usually a monolayer of surfactant is formed at the interface only above the CMC value.

[0788] (vi) Freeze-drying or cryoprotectants: During freeze-drying or spray-drying, excipients may counteract the destabilizing effects caused by hydrogen bond breakage and water removal. For this purpose, sugars and polyols can be used, but corresponding positive effects have also been observed for surfactants, amino acids, non-aqueous solvents and other peptides. Trehalose is particularly effective in reducing aggregation caused by moisture and also improves thermal stability that may be caused by exposure of protein hydrophobic groups to water. Mannitol and sucrose can also be used as separate freeze-drying / cryoprotectants, or in combination with each other where higher proportions of mannitol or sucrose are known to enhance the physical stability of the freeze-dried cake. Mannitol can also be combined with trehalose. Trehalose can also be combined with sorbitol, or sorbitol can be used as the only protective agent. Starch or starch derivatives can also be used.

[0789] (vii) Oxidative protectants: Antioxidants such as ascorbic acid, ectoine, methionine, glutathione, monothioglycerol, morin, polyethyleneimine (PEI), propyl gallate, vitamin E, chelating agents (such as citric acid, EDTA, hexaphosphate, thioglycolic acid).

[0790] (viii) Viscosifiers or viscosity enhancers: delay the settling of particles in vials and syringes and serve to facilitate mixing and resuspension of particles and make the suspension easier to inject (ie, less force on the syringe plunger). Suitable viscosity enhancers or viscosity enhancers are, for example, carbomer viscosity enhancers such as Carbopol 940, Carbopol Ultrez 10; cellulose derivatives such as hydroxypropylmethylcellulose (hydroxypropyl methylcellulose, HPMC) or diethylaminoethylcellulose (DEAE or DEAE-C); colloidal magnesium silicate (Veegum) or sodium silicate; hydroxyapatite gel; tricalcium phosphate gel; xanthan gum; carrageenan (such as Satiagum UTC 30); aliphatic poly(hydroxy acids) such as poly(D,L- or L-lactic acid) (PLA) and poly(glycolic acid) (PGA) and copolymers thereof (PLGA); terpolymers of D,L-lactide, glycolide and caprolactone; poloxamers; triblocks of hydrophilic poly(oxyethylene) blocks and hydrophobic poly(oxypropylene) blocks (to form triblocks of poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) (e.g. Pluronic. TM)); polyetherester copolymers, such as polyethylene terephthalate / polybutylene terephthalate copolymers; sucrose acetate isobutyrate (SAIB); dextran or its derivatives; combinations of dextran and PEG; polydimethylsiloxane; collagen; chitosan; polyvinyl alcohol (PVA) and derivatives; polyalkylimides; poly(acrylamide-co-diallyldimethylammonium (DADMA)); polyvinylpyrrolidone (PVP); glycosaminoglycans (GAGs), such as dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin, heparan sulfate; hyaluronic acid; ABA triblock or AB block copolymers composed of a hydrophobic A block (e.g., polylactide (PLA) or poly(lactide-co-glycolide) (PLGA)) and a hydrophilic B block (e.g., polyethylene glycol (PEG) or polyvinylpyrrolidone);. Such block copolymers, as well as the above-mentioned poloxamers, may exhibit opposite thermogelation behaviors (fluid state at room temperature to facilitate administration, and gel state above the sol-gel transition temperature at body temperature after injection).

[0791] (ix) Diffusants: Change the permeability of connective tissue by hydrolyzing components of the extracellular matrix in the intercellular space (such as, but not limited to, hyaluronic acid, a polysaccharide found in the intercellular spaces of connective tissue). Dispersants (such as, but not limited to, hyaluronidase) temporarily reduce the viscosity of the extracellular matrix and promote the diffusion of injected drugs.

[0792] (x) Other adjuvants: such as wetting agents, viscosity modifiers, antibiotics, hyaluronidase. Acids and bases (such as hydrochloric acid and sodium hydroxide) are necessary adjuvants for adjusting pH during the manufacturing process.

[0793] The drug delivery system may be provided as a liquid, a suspension or as a dry composition.

[0794] In one embodiment, the drug delivery system is a dry composition. Suitable methods for drying are, for example, spray drying and lyophilization (freeze drying). On the one hand, the drug delivery system is dried by lyophilization.

[0795] In one embodiment, the drug delivery system is fully administered with compositions, to provide the bioactive agent of therapeutically effective amount in a single application for at least 12 hours. On the one hand, the single use of the drug delivery system is enough to continue about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years or even longer.

[0796] In one embodiment, the drug delivery system is provided as a single dose, meaning that the container in which the drug delivery system is provided contains one drug dose.

[0797] In another embodiment, the composition is provided as a multi-dose composition, meaning that the composition contains more than one therapeutic dose. Preferably, the multi-dose composition contains at least 2 doses. Such a multi-dose drug delivery system can be used for different subjects in need thereof, or intended for one subject, wherein the remaining dose is stored until needed after the first dose is administered.

[0798] In another embodiment, the drug delivery system is contained in one or more containers. For liquid or suspension compositions, the container is preferably a single chamber syringe. For dry compositions, preferably, the container is a dual chamber syringe. The dry composition is provided in a first chamber of the dual chamber syringe, and the reconstitution solution is provided in a second chamber of the dual chamber syringe.

[0799] Before the dry drug delivery system is applied to a subject in need thereof, the dry composition is reconstituted. Reconstruction can be carried out in a container (e.g., a vial, a syringe, a dual-chamber syringe, an ampoule, and a cartridge) providing the dry drug delivery system. Reconstruction is completed by adding a predetermined amount of reconstitution solution to the dry composition. The reconstitution solution is a sterile liquid, such as phosphate-buffered saline, isotonic saline, water for injection, or other buffer, which may contain other excipients, such as preservatives and / or antimicrobials, such as benzyl alcohol and cresol. Preferably, the reconstitution solution is sterile phosphate-buffered saline (PBS) or physiological saline. Alternatively, the reconstitution solution is sterile water for injection.

[0800] Another embodiment is a method for preparing a reconstructed composition comprising a therapeutically effective amount of a drug delivery system, and optionally one or more pharmaceutically acceptable excipients, the method comprising the step of contacting the composition with a volume of a reconstructed vehicle. The reconstructed drug delivery system can then be administered by injection or other routes.

[0801] Another embodiment is a reconstituted composition comprising a therapeutically effective amount of a drug delivery system, a reconstitution vehicle, and optionally one or more pharmaceutically acceptable excipients.

[0802] Another embodiment is a pre-filled syringe comprising a solution or suspension, the solution or suspension comprising a therapeutically effective amount of a drug delivery system, and optionally one or more pharmaceutically acceptable excipients. On the one hand, the syringe is filled with a drug delivery system as described herein between about 0.01mL and about 5mL. On the one hand, the syringe is filled with about 0.05mL and about 5mL, about 1mL and about 2mL, about 0.1mL and about 0.15mL, about 0.1mL and about 0.5mL, about 0.15mL and about 0.175mL or about 0.5 to about 5mL. In one embodiment, the syringe is filled with 0.165mL of a drug delivery system as described herein. In some aspects, the syringe is filled with about 0.01mL, about 0.02mL, about 0.03mL, about 0.04mL, about 0.05mL, about 0.06mL, about 0.07mL, about 0.08mL, about 0.09mL, about 0.1mL, about 0.2mL, about 0.3mL, about 0.4mL, about 0.5mL, about 0.6mL, about 0.7mL, about 0.8mL, about 0.9mL, about 1mL, about 1.2mL, about 1.5mL, about 1.75mL, about 2mL, about 2.5mL, about 3mL, about 4mL or about 5mL of drug delivery system as described herein. Considering the waste caused by " dead angle " in syringe and needle, the dosage of syringe filling is usually more than the required dosage applied to the patient. When the doctor starts the syringe, there can also be a waste of a predetermined amount, so as to prepare for the patient to inject.

[0803] In one embodiment, depending on the injection route of the drug delivery system as described herein, the syringe is filled with a dose volume (i.e., the volume of drug intended for delivery to the patient) between about 0.01 mL and about 5 mL (e.g., between about 0.01 mL and about 0.1 mL, between about 0.1 mL and about 0.5 mL, between about 0.2 mL and about 2 mL, between about 0.5 mL and about 5 mL, or between about 1 mL and about 5 mL). In one embodiment intended for intravitreal injection, as described herein, the syringe is filled with a drug delivery system solution or suspension with a dose volume between about 0.01 mL and about 0.1 mL, wherein the drug concentration is 1 mg / mL to 40 mg / mL. In one embodiment intended for intra-articular injection, as described herein, the syringe is filled with a drug delivery system solution or suspension with a dose volume between about 0.05 mL and about 5.0 mL, wherein the drug concentration is 1 mg / mL to 40 mg / mL. In one embodiment intended for subcutaneous injection, as described herein, the syringe is filled with a drug delivery system solution or suspension having a dose volume of between about 0.1 mL and about 5.0 mL, wherein the drug concentration is 0.1 mg / mL to 40 mg / mL. In embodiments intended for injection by other routes, as described herein, the syringe is filled with a drug delivery system solution or suspension having a dose volume of between about 0.01 mL and about 5.0 mL, wherein the drug concentration is 0.1 mg / mL to 40 mg / mL. In some aspects, a syringe is filled with about 0.01 mL, about 0.02 mL, about 0.03 mL, about 0.04 mL, about 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08 mL, about 0.09 mL, about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1 mL, about 1.2 mL, about 1.5 mL, about 1.75 mL, about 2 mL, about 2.5 mL, about 3 mL, about 4 mL, or about 5 mL of a drug delivery system as described herein for delivery to a patient in need thereof.

[0804] Since the syringe contains a drug solution, the outlet can be reversibly sealed to maintain the sterility of the drug. The seal can be achieved by sealing means known in the art, such as a luer lock or a tamper-evident seal.

[0805] Another embodiment is a medicine box comprising one or more pre-filled syringes, and the one or more pre-filled syringes include a solution or suspension of one or more drug delivery systems as described herein. In one embodiment, such a medicine box includes a pre-filled syringe, and the syringe is included in a drug delivery system as described herein in a blister package or a sealed sleeve. The inside of the blister package or sleeve can be sterile. On the one hand, the pre-filled syringe as described herein can be placed in this blister package or sleeve before sterilizing (e.g., terminal sterilization).

[0806] Such a medicine box may further include one or more needles for administering the drug delivery system described herein. When the drug delivery system is to be administered intravitreally, a 30 gauge × 1 / 2-inch needle is generally used, although a 31 gauge or 32 gauge needle can be used. For intravitreal administration, a 33 gauge or 34 gauge needle can be used interchangeably. Such medicine boxes may further include instructions for use, drug labels, contraindications, warnings, or other relevant information. One embodiment described herein is a carton or package containing one or more pre-filled syringes, which are contained in a blister pack. One or more drug delivery systems as described herein, needles, and optional instructions for administration, drug labels, contraindications, warnings, or other relevant information.

[0807] The syringe can be sterilized using a terminal sterilization process, and such a process can use known processes such as ethylene oxide or hydrogen peroxide (H2O2) sterilization processes. The needle used with the syringe can be sterilized by the same method as the medicine box described herein. On the one hand, the packaging is exposed to a sterilized gas until the outside of the syringe is sterile. After such a process, the outer surface of the syringe can remain sterile (when in a blister pack) for 6 months, 9 months, 12 months, 15 months, 18 months, 24 months or longer. Therefore, in one embodiment, the pre-filled syringe as described herein (in its blister pack) can have a shelf life of up to 6 months, 9 months, 12 months, 15 months, 18 months, 24 months or even longer. In one embodiment, after 18 months of storage, less than one in a million syringes have detectable microorganisms on the outside of the syringe. On the one hand, a sterile assurance level of at least 10 -6 The prefilled syringes are sterilized with ethylene oxide. In another aspect, the prefilled syringes are sterilized with a sterility assurance level of at least 10 -6The prefilled syringe is sterilized with hydrogen peroxide. A significant amount of sterilizing gas should not be allowed to enter the variable volume chamber of the syringe. As used herein, the term "significant amount" refers to the amount of gas that will cause an unacceptable change in the drug delivery system solution or suspension in the variable volume chamber. In one embodiment, the sterilization process causes alkylation of the drug delivery system to be ≤10% (preferably ≤5%, ≤3%, ≤1%). In one embodiment, the prefilled syringe has been sterilized with ethylene oxide, but the outer surface of the syringe has ≤1ppm, preferably ≤0.2ppm of ethylene oxide residual. In one embodiment, the prefilled syringe has been sterilized with hydrogen peroxide, but the outer surface of the syringe has ≤1ppm, preferably ≤0.2ppm of hydrogen peroxide residual. In another embodiment, the prefilled syringe has been sterilized with ethylene oxide, and the total ethylene oxide residual found on the outside of the syringe and the inside of the blister package is ≤0.1mg. In another embodiment, the pre-filled syringe has been sterilized using hydrogen peroxide and the total hydrogen peroxide residual found on the outside of the syringe and the inside of the blister pack is ≤ 0.1 mg.

[0808] Another aspect is a kit. For liquid and suspension compositions, and when the drug delivery device is only a hypodermic syringe, the drug box may include a syringe, a needle and a container containing a drug delivery system composition for use with a syringe. In the case of a dry composition, the container may have a chamber containing a dry drug delivery system composition, and a second chamber containing a reconstitution solution. In one embodiment, the injection device is an adapted hypodermic syringe, so that a separate container with a drug delivery system composition can be engaged with the injection device so that during use, the liquid or suspension in the container or the reconstructed dry composition is in fluid communication with the outlet of the injection device. The example of a drug delivery device includes, but is not limited to, a hypodermic syringe and a pen-type syringe device. A particularly preferred injection device is a pen-type syringe, in which case the container is a cartridge, preferably a disposable cartridge.

[0809] Another embodiment comprises a kit comprising a needle and a container containing a drug delivery system composition and optionally further containing a reconstitution solution, the container being adapted for use with the needle. In one aspect, the container is a prefilled syringe. In another aspect, the container is a dual chamber syringe.

[0810] Another embodiment is a cartridge comprising a composition of the drug delivery system as described above, the cartridge being used with a pen injector device.The cartridge may contain a single dose or a multi-dose drug delivery system.

[0811] In another embodiment, the drug delivery system solution or suspension comprises a drug delivery system and one or more excipients, and also comprises other bioactive agents in its free form or as a drug or in combination with other drug delivery systems (e.g., a pegylated drug or a drug connected with a hydrogel). On the one hand, this other one or more bioactive agents are free form drugs or a second drug delivery system.

[0812] In another embodiment, one or more drug delivery systems are administered simultaneously, wherein each drug delivery system has a separate or related biological activity.

[0813] In alternative embodiments, the drug delivery system is combined with a second biologically active compound in a manner such that the drug delivery system is first administered to a subject in need thereof, followed by administration of the second compound. Alternatively, the drug delivery system composition is administered to the same subject in need thereof after another compound has been administered to the subject.

[0814] Another embodiment is a drug delivery system comprising DR represented by formula (I) or a pharmaceutically acceptable salt thereof, for use as a medicament.

[0815] Another embodiment is a drug delivery system comprising DR represented by formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of musculoskeletal disorders.

[0816] Another embodiment is the use of a drug delivery system comprising DR represented by formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating musculoskeletal disorders.

[0817] Another embodiment is a drug delivery system or pharmaceutical composition for use in a method of treating or preventing a disease or disorder that can be treated by the biologically active moiety released from the drug delivery system.

[0818] Another embodiment is a method for manufacturing a solution or suspension composition of a drug delivery system. In one embodiment, such a composition is prepared by:

[0819] (i) mixing the drug delivery system with one or more excipients;

[0820] (ii) transferring an amount of the liquid or suspension composition equivalent to a single dose or multiple doses into a suitable container; and

[0821] (iii) sealing the container.

[0822] Another embodiment is a method for manufacturing a dry composition of a drug delivery system. In one embodiment, such a composition is prepared by:

[0823] (i) mixing the drug delivery system with one or more excipients;

[0824] (ii) transferring an amount equivalent to a single dose or multiple doses into a suitable container;

[0825] (iii) drying the composition in said container; and

[0826] (iv) sealing the container.

[0827] Suitable containers are vials, syringes, dual chamber syringes, ampoules and cartridges.

[0828] Another embodiment is a method for synthesizing a drug delivery system as described above or its pharmaceutically acceptable salt. The precursor of a drug delivery system or a drug delivery system can be prepared by known methods, or according to a reaction sequence as described below. The starting material used in the preparation (synthesis) of a drug delivery system or its precursor is known or commercially available, or can be prepared by known methods or as described below.

[0829] It will be apparent to those of ordinary skill in the relevant art that appropriate modifications and adaptations may be made to the compositions, methods, and administrations described herein without departing from the scope of any embodiment or aspect thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any specific embodiment. All of the various embodiments, aspects, and selections disclosed herein may be combined in any and all variations or repetitions. The scope of the compositions, formulations, methods, and processes described herein includes all actual or potential combinations of the embodiments, aspects, selections, examples, and preferences described herein.

[0830] Examples

[0831] abbreviation

[0832] Ac Acetyl ACN Acetonitrile AcOH Acetic acid Ac2O Acetic anhydride aq. water-based atm atmosphere Boc, BOC tert-butyl carboxyl Boc-anhydride di-tert-butyl dicarbonate (Boc)2O, (BOC)2O di-tert-butyl dicarbonate br. Width

[0833] BSA bovine serum albumin BuOH Butanol CAD charged aerosol detector calcd. Calculated Cat, cat catalytic CBZ, Cbz benzyloxycarbonyl Cu(OTf)2 Copper(II) trifluoromethanesulfonate d Doublet dd double double peak DCM Dichloromethane DIAD Diisopropyl azodicarboxylate DIPEA, DIEA N,N-diisopropylethylamine DMAP 4,4-dimethylaminopyridine DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide DOSY-NMR One-dimensional diffusion order NMRECL Electrochemiluminescence

[0834] Elem.Anal. Elemental analysis

[0835] ELSD Evaporative Light Scattering Detector

[0836] EDC·HCl N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride ESI Electrospray ionization

[0837] EtOAc, AcOEt Ethyl acetate

[0838] Et Ethyl

[0839] EtOH

[0840] FCC Flash Column Chromatography

[0841] FITC Fluorescein isothiocyanate

[0842] g

[0843] G Specifications

[0844] h hour

[0845] HATU 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium methylammonium hexafluorophosphate

[0846] HC HPLC conditions

[0847] HPLC High Performance Liquid Chromatography

[0848] IPA 2-Propanol

[0849] IR infrared spectroscopy

[0850] i or iso

[0851] IVT, ivt intravitreal K2CO3 Potassium carbonate

[0852] kD, kDa kilodaltons

[0853] L Liter

[0854] LCMS liquid chromatography-mass spectrometry

[0855] M mole / liter

[0856] MHz Megahertz

[0857] m multiplet

[0858] Me Methyl

[0859] MeCN Acetonitrile

[0860] MeOH Methanol

[0861] MES 2-(N-morpholino)ethanesulfonic acid

[0862] mg milligram

[0863] μg microgram

[0864] mM millimole / liter

[0865] mm Millimeters

[0866] min

[0867] mL milliliters

[0868] mmol millimole

[0869] μL microliter

[0870] μmol micromole

[0871] MOPS 3-(N-morpholino)propanesulfonic acid

[0872] MS

[0873] MsCl Methanesulfonyl chloride

[0874] MsOH Methanesulfonic acid

[0875] MWCO Molecular weight cutoff

[0876] m / z mass-to-charge ratio

[0877] N Positive

[0878] NA Not available

[0879] NaBH4 Sodium borohydride

[0880] NaBH3CN Sodium cyanoborohydride

[0881] Na(AcO)3BH Sodium triacetoxyborohydride

[0882] ng

[0883] NH4Cl Ammonium chloride

[0884] NHS N-Hydroxysuccinimide

[0885] nM nanomoles / liter

[0886] NMR Nuclear Magnetic Resonance

[0887] OMe Methoxy

[0888] PBS Phosphate buffered saline

[0889] 1× PBS Phosphate buffered saline, typically about 10 mM PO4 3-

[0890] Pd / C Palladium Carbon

[0891] Pd(dppf)Cl2 1,1′-Bis(diphenylphosphino)ferrocene-palladium(II) chloride

[0892] Pd(dppf)Cl2·CH2Cl2 adduct 1,1′-bis(diphenylphosphino)ferrocene-palladium(II) chloride dichloromethane complex

[0894] Pd(PPh3)4Tetrakis(triphenylphosphine)palladium(0)

[0895] Ph Phenyl

[0896] ppm parts per million

[0897] psi pounds per square inch

[0898] rac racemic

[0899] rcf relative centrifugal force

[0900] RP Invert

[0901] rt, RT room temperature

[0902] s Single peak

[0903] sat. saturated

[0904] SDS-page Sodium dodecyl sulfate polyacrylamide gel electrophoresis

[0905] SEC Size Exclusion Chromatography

[0906] SFC Supercritical Fluid Chromatography

[0907] t Triple Peak

[0908] t-Bu, tBu tert-butyl

[0909] t 1 / 2 half life

[0910] t r or ret.time retention time

[0911] TBAF Tetra-n-butylammonium fluoride

[0912] TBSCl, TBDMSCl tert-Butyldimethylsilyl chloride

[0913] TEA, Et3N, NEt3 triethylamine

[0914] tert- uncle

[0915] TFA Trifluoroacetic acid

[0916] Tf2O Trifluoromethanesulfonic anhydride

[0917] THF Tetrahydrofuran

[0918] TLC Thin Layer Chromatography

[0919] TMS Trimethylsilyl

[0920] TMSOTf Trimethylsilyl trifluoromethanesulfonate Tris(hydroxymethyl)aminomethane

[0921] Triton X-100 tert-Octylphenoxypolyethoxyethanol (CAS 9002-93-1) Ts p-Toluenesulfonyl

[0922] TsOH p-Toluenesulfonic acid

[0923] Tween 20 Polysorbate 20, Polyoxyethylene (20) Sorbitan Monolaurate

[0924] UPLC Ultra-Performance Liquid Chromatography

[0925] UV

[0926] VEGF Vascular endothelial growth factor

[0927] v / v volume / volume

[0928] w / v weight / volume

[0929] w / w weight / weight

[0930] The following drugs described in these examples are referred to by the following abbreviations:

[0931] D1: brolucizumab (SEQ ID NO: 4);

[0932] D2: (SEQ ID NO: 5);

[0933] D3: (SEQ ID NO: 6); and

[0934] D4: (SEQ ID NO: 7).

[0935] method

[0936] Place the following

[0937]

[0938] The synthesis of ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate used in some examples is described in AM Jawalekar, et al.; Molecules, 2013, 18, 7346-7363.

[0939] The following

[0940]

[0941] The synthesis of ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(4-nitrophenyl)carbonate used in some examples is described in J. Dommerholt, et al.; Angew. Chem. Int. Ed. [Angewandte Chemie International Edition] 2010, 49, 9422-9425.

[0942] All other intermediates and reagents not specifically described as a synthesis herein are commercially available and were used as stated.

[0943] LCMS method

[0944] Method 1

[0945] Column SunFire C18 3.5μm 3.0×30mm; column temperature 40℃; flow rate 2.0mL / min; stop time 2.20min; pH 2.2; eluent A1 0.05% TFA in water; eluent B1 acetonitrile; gradient time (min) / %A (eluent A1):%B (eluent B1); 0.00 / 95:5; 1.70 / 5:95; 2.00 / 5:95; 2.10 / 95:5.

[0946] Method 2

[0947] Column AcQuity UPLC BEH C18 1.7 μm 2.1×30 mm; column temperature 50°C; flow rate 1.0 mL / min; stop time 2.00 min; pH 2.6; eluent A1 0.1% formic acid in water; eluent B1 0.1% formic acid in acetonitrile; gradient time (min) / % A (eluent A1): % B (eluent B1)

[0948] 0.00 / 98:2; 0.10 / 98:2; 1.50 / 2:98; 1.80 / 2:98; 1.90 / 98:2; 2.00 / 98:2.

[0949] Method 3

[0950] Column: Kinetex 2.6 μm C18 100A (100×4.6 mm); mobile phase A (0.1% formic acid in water), B (acetonitrile); gradient (time (min) / % B): 0 / 5, 1 / 30, 3 / 95, 4 / 95, 4.1 / 5, 6 / 5.

[0951] Method 4

[0952] Column: AcQuity UPLC BEH C18 1.7μm 2.1×30mm; column temperature 50°C; flow rate 1.0mL / min; stop time: 2.00min; pH 2.6; eluent A1 0.1% formic acid in water; eluent B1 0.1% formic acid in acetonitrile; gradient time (min) / %A(eluent A1):%B(eluent B1); 0.00 / 98:2; 0.10 / 98:2; 1.50 / 2:98; 1.80 / 2:98; 1.90 / 98:2; 2.00 / 98:2.

[0953] Method 5

[0954] Column: SunFire C18 3.5μm 3.0×30mm; column temperature 40℃; flow rate 2.0mL / min; stop time 2.20min; pH 2.2; eluent A1 5mM ammonium hydroxide in water; eluent B1 acetonitrile; gradient time (min) / %A (eluent A1):%B (eluent B1); 0.00 / 95:5; 1.70 / 5:95; 2.00 / 5:95; 2.10 / 95:5.

[0955] Method 6

[0956] Column: Kinetex 2.6 μm C18 100A, (100×4.60 mm); Gradient / (time (min) / % B) 0 / 5, 1 / 30, 3 / 95, 4 / 95, 4.1 / 5, 6 / 5; Mobile phase: 0.1% formic acid (A) / acetonitrile (B) in water; Flow rate: 1.4 mL / min; Column temperature: 40°C.

[0957] Method 7

[0958] Column: Synergi 2.5 μm MAX-RP 100A Mercury (100×4.6 mm); mobile phase A (0.1% formic acid in water), B (acetonitrile); gradient (time (min) / % B): 0 / 30, 0.5 / 30, 1.5 / 95, 2.4 / 95, 2.5 / 30, 3.0 / 30.

[0959] Method 8

[0960] Column: Kinetex 2.6 μm C18 100A (100×4.6 mm); Mobile phase A (0.1% formic acid in water), B (acetonitrile); Gradient (time (min) / % B): 0 / 50, 1 / 70, 2 / 100, 4 / 100, 4.1 / 50, 6 / 50. ESI-MS data were recorded on an Acquity G2 Xevo-QTOF-MS. Positive ion mass spectra were deconvoluted using the MaxEnt 1 program in the MassLynx software package.

[0961] Method 9

[0962] Column: Proswift Monolith (4.6x50 mm); Mobile phase A (0.1% formic acid in water), B (0.1% formic acid in acetonitrile); Gradient (time (min) / % B): 0 / 2, 0.7 / 2, 2 / 98, 2.1 / 98, 2.3 / 2, 3.3 / 2. ESI-MS data were recorded on an AcquityG2 Xevo-QTOF-MS. Positive ion mass spectra were deconvoluted using the MaxEnt 1 program in the MassLynx software package. (M+H) was recorded. + The deconvoluted m / z.

[0963] Example 1

[0964] No trace joint

[0965] This example describes the synthesis of a variety of traceless linkers capable of conjugation to amine-containing drugs as well as to carriers.

[0966]

[0967]

[0968]

[0969]

[0970] Common intermediates in the synthesis of traceless linkers: L-INT-1c

[0971]

[0972] L-INT-1b.1-(N-Cbz-β-alanyl)-3-(hydroxymethyl)piperazine

[0973]

[0974] To a 0°C solution of commercially available N-Cbz-β-alanine (SM-1, 2.5 g, 11.2 mmol) in tetrahydrofuran (30 mL) was added triethylamine (1.13 g, 11.2 mmol) and ethyl chloroformate (1.21 g, 11.2 mmol). The solution was stirred at 0°C for 2 hours to provide in situ formation of N-Cbz-β-alanine ethyl carbonic anhydride (L-INT-1a). The reaction mixture was added to a solution of 2-(hydroxymethyl)piperazine (SM-2, 1.95 g, 16.8 mmol) and triethylamine (1.13 g, 11.2 mmol) in water (30 mL), and the resulting mixture was stirred at room temperature for 12 hours. The solution was made basic with sodium carbonate solution (2M, 1 mL). Brine (2 mL) was added, and the aqueous phase was concentrated to give a crude solid. Purification by column chromatography (basic alumina, eluting with 3%-15% methanol: dichloromethane) provided L-INT-1b. MS (ESI+) m / z 322.2 (M+H).

[0975] L-INT-1c.1-(Boc)-2-(Hydroxymethyl)-4-(N-Cbz-β-alanyl)piperazine

[0976]

[0977] To a 0°C solution of L-INT-1b (1 g, 3.1 mmol) in dichloromethane (20 mL) was added triethylamine (0.504 g, 4.98 mmol) and di-tert-butyl dicarbonate (611 mg, 2.8 mmol). The solution was stirred at room temperature for 2 hours. The reaction mixture was treated with water and extracted with dichloromethane. The organic layer was dried (sodium sulfate) and evaporated. Purification by flash chromatography (SiO2, eluted with 2:98 methanol:dichloromethane) provided L-INT-1c. MS (ESI+) m / z 422.2 (M+H).

[0978] Linker intermediate L-INT-1.

[0979]

[0980] L-INT-1d.1-(Boc)-2-(2'-methoxyacetoxymethyl)-4-(N-Cbz-β-alanyl)piperazine

[0981]

[0982] To a 0°C solution of L-INT-1c (1 g, 2.37 mmol) in dichloromethane (15 mL) was added triethylamine (0.60 g, 5.93 mmol). Methoxyacetyl chloride (307 mg, 2.85 mmol) was then added dropwise and the solution was stirred at room temperature for 12 hours. The reaction mixture was treated with water and extracted with dichloromethane. The organic layer was dried (sodium sulfate) and evaporated. Purification by flash chromatography (SiO2 with 1%-2% methanol: dichloromethane) provided L-INT-1d. MS (ESI+) m / z 494.2 (M+H).

[0983] L-INT-1e.1-(N-Cbz-β-alanyl)-3-(2'-methoxyacetoxymethyl)piperazine, trifluoroacetate

[0984]

[0985] To a 0°C solution of L-INT-1d (1 g, 2.02 mmol) in dichloromethane (26 mL) was added trifluoroacetic acid (7 mL). The solution was stirred at room temperature for 3 hours. The reaction mixture was concentrated, washed with pentane, and dried under high vacuum to provide the trifluoroacetate salt L-INT-1e. MS (ESI+) m / z 394 (M+H).

[0986] L-INT-1.2-(2'-methoxyacetoxymethyl)-4-(N-Cbz-β-alanyl)-1-piperazineacetic acid tert-butyl ester

[0987]

[0988] To a 0°C mixture of L-INT-1e (1.0 g, 2.54 mmol) and potassium carbonate (1.05 g, 7.63 mmol) in acetonitrile (25 mL) was added tert-butyl bromoacetate (0.744 g, 3.82 mmol) dropwise. The solution was stirred at room temperature for 24 hours. The reaction mixture was treated with water and extracted with ethyl acetate. The organic layer was dried (sodium sulfate) and evaporated. Purification by flash chromatography (SiO2 with 1%-2% methanol: dichloromethane) provided L-INT-1 as a colorless sticky solid. LCMS (Method 8): Retention time = 3.43 min; MS (ESI+) m / z 508.3 (M+H). 1H NMR(400MHz, CDCl3)7.35-7.28(m,5H),5.58-5.52(m,1H),5.08(s,2H),4.38-4.22(m,1H),4.20- 4.08(m,1H),4.10-4.00(m,3H),4.00-3.90(m,1H),3.70-3.60(m,1H),3.60-3.54(m,1H),3.50-3 .49(d,J=4,4H),3.45(s,3H),3.42-3.38(m,1H),3.33(s,1H),3.32-3.29(m,1H),3.28-3.20(m,1 H),3.18-3.10(m,1H),3.10-3.00(m,1H),2.86-2.72(m,2H),2.53-2.51(t,J=4,2H),1.46(s,9H).

[0989] The species shown in Table 7 were prepared using a method similar to that used to synthesize L-INT-1.

[0990]

[0991]

[0992] L-INT-5.2-(1'-Methylcyclopropylcarbonyloxymethyl)-4-(N-Cbz-β-alanyl)-1-piperazineacetic acid tert-butyl ester

[0993]

[0994] L-INT-5a.1-(Boc)-2-(1'-methylcyclopropylcarbonyloxymethyl)-4-(N-Cbz-β-alanyl)piperazine

[0995]

[0996] To a solution of L-INT-1c (1.0 g, 2.37 mmol), 1-methylcyclopropanecarboxylic acid (0.475 g, 4.75 mmol) and 2-chloro-1-methyl-pyridinium iodide (1.21 g, 4.75 mmol) in toluene (20 mL) was added triethylamine (5.0 mL) and the resulting reaction was stirred at 110 °C for 24 hours. The reaction mixture was treated with water and extracted with ethyl acetate. The organic phase was washed with brine, dried (sodium sulfate) and evaporated. Purification by flash chromatography (SiO2 with 1%-2% methanol: dichloromethane) provided L-INT-5a. MS (ESI+) m / z 504.2 (M+H).

[0997] L-INT-5.2-(1'-Methylcyclopropylcarbonyloxymethyl)-4-(N-Cbz-β-alanyl)-1-piperazineacetic acid tert-butyl ester

[0998] L-INT-5 was prepared from L-INT-5a in two steps according to the general method described for the synthesis of L-INT-1e and L-INT-1. L-INT-5: LCMS (Method 8): Retention time = 3.84 min; MS (ESI+) m / z 518.3 1 H NMR(400MHz, CDCl3)7.35-7.30(m,5H),5.5(s 1H),5.08(s,2H),4.28-4.12(m,2H),4.06-3.88(m,2H),3.67-3.49(m,3H),3.41-3.28(m,3H),3.26-3.17(m,1H),3.12-3.05(m,1 H),3.03-2.90(m,1H),2.78-2.74(m,2H),2.53-2.50(t,J=8,2H),1.45(s,9H),1.29(s,3H),1.25-1.20(m,2H),0.73-0.68(m,2H).

[0999] Chiral separation of L-INT-6 and L-INT-7. tert-butyl 2-(acetoxymethyl)-4-(N-Cbz-β-alanyl)-1-piperazineacetate

[1000]

[1001] The individual enantiomers of L-INT-2 were purified by preparative chiral HPLC of L-INT-2 (Phenomenex Lux Amylose-2 column, 5 μm, 250 mm×21.2 mm), separated by eluting with a mobile phase of 20 mL / min with the following combination (isocratic): 55% n-hexane, 45% 70:30 ethanol:isopropanol (containing 0.1% diethylamine). The fractions containing the product were evaporated, dissolved in chloroform, washed with water, dried (sodium sulfate), and evaporated to provide stereoisomers L-INT-6 and L-INT-7.

[1002] L-INT-6: LCMS (Method 8): Retention time = 1.93 min; MS (ESI+) m / z 478.3 (M+H). Chiral HPLC (Method as follows): Retention time = 8.018 min; 98.3% purity).

[1003] L-INT-7: LCMS (Method 8): Retention time = 1.92 min; MS (ESI+) m / z 478.3 (M+H). Chiral HPLC (Method as follows): Retention time = 9.694 min; 94.4% purity).

[1004] Chiral HPLC method: column: Phenol Lux Amylose-2, 5 μm, 250 mm×4.60 mm; column temperature 25°C; flow rate 1.0 mL / min; mobile phase: A = hexane, B = ethanol:methanol 1:1; elution: isocratic 30:70 A:B.

[1005] No trace connector; L1-L7

[1006]

[1007] L-2a. tert-Butyl 2-(acetoxymethyl)-4-(β-alanyl)-1-piperazineacetate

[1008]

[1009] L-INT-2 (185.5 mg, 0.39 mmol) was dissolved in methanol (12 mL) and subjected to flow hydrogenolysis (Pd-C cartridge, 5 atm H2, 35°C, 1 hour at 1 mL / min flow rate). The solution was evaporated to provide the title compound L-2a. LCMS (Method 1): Retention time = 0.72 min; MS (ESI+) m / z 344.1 (M+H).

[1010] L-2b: 2-(Acetoxymethyl)-4-(β-alanyl)-1-piperazineacetic acid, bis(trifluoroacetate)

[1011]

[1012] L-2a (123.2 mg, 0.36 mmol) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (1 mL). The reaction was stirred at room temperature overnight and then evaporated to provide the crude title material (L-2b). LCMS (Method 1): Retention time = 0.10 min; MS (ESI+) m / z 288.0 (M+H).

[1013] L2.2-(Acetoxymethyl)-4-(N-(((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxycarbonyl)-β-alanyl)-1-piperazineacetic acid

[1014]

[1015] Crude L-2b (163 mg, 0.57 mmol) was taken up in dichloromethane and acetonitrile to provide a slightly turbid mixture. Triethylamine (0.40 mL, 2.84 mmol) and ((1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (182 mg, 0.63 mmol) were added to produce a solution which was evaporated after 0.5 h. The crude material was dissolved in 6 mL of 3:1 acetonitrile:water and purified by HPLC (column: Waters XBridge BEH 5 μm 19×150 mm; method: 15%-30% acetonitrile in water 10 min gradient (10 mM ammonium hydroxide), 30 mL / min). Fractions were evaporated, flash frozen and lyophilized to provide L2. LCMS (Method 1): Retention time = 0.97 min; MS (ESI+) m / z 464.1 (M+H). 1 H-NMR (CD3OD, ppm) (sample contained traces of residual triethylamine): 4.17 (m, 4H); 4.00 (m, 1H), 3.75 (m, 1H), 3.5-3.32 (m, 5H), 3.06 (m, 1H), 2.91 (m, 1H), 2.79 (m, 1H), 2.60 (t, 2H), 2.19 (m, 6H), 2.05 (m, 3H), 1.59 (m, 2H), 1.38 (m, 1H), 0.96 (m, 2H).

[1016] The species shown in Table 8 were prepared using a method similar to that used to synthesize L2.

[1017]

[1018]

[1019]

[1020]

[1021] Traceless linker L8.2-(2'-methoxyacetoxymethyl)-4-(6'-azidohexanoyl)-1-piperazineacetic acid

[1022]

[1023] L-8a. 1-(Boc)-2-(Hydroxymethyl)-4-(6'-azidohexanoyl)piperazine

[1024]

[1025] 6-Azidohexanoic acid (743 mg, 4.73 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.0 g, 5.22 mmol) were weighed into a round bottom flask and dissolved in dichloromethane (20 mL). After 5 minutes, solid tert-butyl 2-(hydroxymethyl)piperazine-1-carboxylate (0.98 g, 4.53 mmol) and diisopropylethylamine (1.0 mL, 5.73 mmol) were added and the solution was stored at room temperature overnight. The next day, the reaction mixture was diluted with 100 mL of dichloromethane and washed with 1M HCl (2×50 mL), 1M NaOH (1×50 mL) and brine (1×50 mL). The organic phase was concentrated to provide L-8a. MS (ESI+) m / z 356.3 (M+H).

[1026] L-8b. 1-(Boc)-2-(2'-methoxyacetoxymethyl)-4-(6"-azidohexanoyl)piperazine

[1027]

[1028] In a round-bottom flask, L-8a (959 mg, 2.70 mmol) was dissolved in dichloromethane (12 mL). A stirring bar and diisopropylethylamine (1 mL, 5.73 mmol) were added, followed by 2-methoxyacetyl chloride (0.32 mL, 3.51 mmol), and the flask was capped. The reaction was stirred at room temperature. After 6 hours, 100 μL of 2-methoxyacetyl chloride was added. After 30 minutes, the reaction mixture was diluted with 90 mL of ethyl acetate and washed with 1 M HCl (2 × 25 mL) and brine (1 × 20 mL). The organic phase was concentrated using a rotary evaporator. The product was purified by flash column chromatography on silica with an ethyl acetate: heptane gradient. The fractions containing the product were combined and concentrated to provide L-8b. MS (ESI+) m / z = 428.3 (M + H).

[1029] L-8c. 1-(6'-azidohexanoyl)-3-(2"-methoxyacetoxymethyl)piperazine, trifluoroacetate

[1030]

[1031] L-8b (846 mg, 1.979 mmol) was dissolved in trifluoroacetic acid (10 mL, 130 mmol) and dichloromethane (10 mL). The solution was stirred at room temperature. After 1 hour, the solvent was removed using a rotary evaporator, and the product was dried under vacuum to provide L-8c. MS (ESI+) m / z 328.2 (M+H).

[1032] L-8d. tert-Butyl 2-(2'-methoxyacetoxymethyl)-4-(6"-azidohexanoyl)-1-piperazineacetate

[1033]

[1034] In a glass vial with a stirring bar, L-8c (439 mg, 0.994 mmol) was dissolved in dimethylformamide (5 mL). Tert-butyl bromoacetate (0.220 mL, 1.491 mmol) and diisopropylethylamine (0.868 mL, 4.97 mmol) were added and the vial was capped. The reaction was stirred at 50 ° C overnight. The next day, the reaction mixture was removed from the heat source and stored at -20 ° C until the next day. Purification. The reaction mixture was purified by flash column chromatography on silica with a heptane: ethyl acetate gradient without extractive post-treatment. The fractions containing the product were combined and concentrated to provide L-8d. MS (ESI-) m / z = 486.5 (M + formate). L8.2-(2'-methoxyacetoxymethyl)-4-(6"-azidohexanoyl)-1-piperazineacetic acid

[1035]

[1036] L-8d (150 mg, 0.317 mmol) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (5 mL), and the reaction was stirred at room temperature overnight. The next day, the solution was concentrated using a rotary evaporator, and the residue was redissolved in 4 mL of acetonitrile. The solution was filtered and purified by preparative reverse phase HPLC with mass oriented fraction collection (method below). The fractions containing the product were combined, frozen, and lyophilized to provide L8. MS (ESI+) m / z=386.5 (M+H). Preparative HPLC conditions: Waters Sunfire C18; particle size: 5 μm; column size: 30×50 mm; eluent / gradient: 10% CH3CN / H2O / 0.7 min, 10%-30% CH3CN / H2O / 3.5 min, 30%-95% CH3CN / H2O 0.5 min (CH3CN and H2O containing 0.1% TFA); flow rate: 75 mL / min; column temperature: room temperature; total m / z: +385.

[1037] No trace connector: L9-L11

[1038]

[1039] L-9a.2-(Propanyloxymethyl)-4-(N-Cbz-β-alanyl)-1-piperazineacetic acid, trifluoroacetate

[1040]

[1041] L-INT-3 (250 mg, 0.51 mmol) was dissolved in dichloromethane (5 mL) at 0°C, then treated with trifluoroacetic acid (2.5 mL) and stirred at room temperature under argon for 6 hours. The reaction was evaporated, washed with pentane, and dried to provide crude L-9a as a trifluoroacetate salt. LCMS (Method 5): Retention time = 0.262 min; MS (ESI+) m / z 436.2 (M+H). L-9b. 2-(Propanyloxymethyl)-4-(β-alanyl)-1-piperazineacetic acid

[1042]

[1043] L-9a (250 mg, 0.57 mmol) was dissolved in ethanol (5 mL), and 10% palladium on carbon (25 mg) was added thereto. The solution was stirred under a hydrogen balloon for 6 hours, and then Filter and wash with ethanol. The combined filtrates were evaporated to provide compound L-9b. LCMS (Method 5): Retention time = 0.774 min; MS (ESI+) m / z 302.2 (M+H).

[1044] L9.

[1045]

[1046] L-9b (100 mg, 0.33 mmol) and 5-azidopentanoic acid (57 mg, 0.40 mmol) in ethyl acetate (5 mL) were treated with diisopropylethylamine (107 mg, 0.83 mmol) and propylphosphonic anhydride (50% solution in ethyl acetate, 158 mg, 0.50 mmol) at 0°C. The reaction was stirred at room temperature under argon for 16 hours, then quenched with water and evaporated. Initial flash chromatography (SiO2, 10% methanol: dichloromethane) followed by preparative HPLC (column: zorbax C-18 4.6×150 mm; mobile phase A=: methanol (1:1) 0.01% TFA in water, mobile phase B=acetonitrile: methanol (1:1); time=0 min: 30% B; 1 min: 70% B; 6 min: 100% B; 1 mL / min) to provide L9. LCMS (Method 5): Retention time = 0.18 min; MS (ESI+) m / z 427.1 (M+H). 1H-NMR (CD3OD, ppm) (all assignments are provisional) 4.23-4.17 (m, 2H), 4.16-3.98 (m, 1H), 3.78-3.62 (m, 1H), 3.54 (s, 2H), 3.46-3.43 (t, J=4, 3H), 3.26-3.20 (m, 1H), 3.18-3.05 (m, 1H), 3.03-2.76 (m, 2H), 2.64-2.61 (t, J=8, 2H), 2.43-2.36 (m, 2H), 2.24-2.21 (t, J=8, 2H), 1.75-1.58 (m, 4H), 1.15-1.12 (t, J=8, 3H).

[1047] Using a method similar to that used to synthesize L-9, the species shown in Table 9 were prepared:

[1048]

[1049]

[1050]

[1051] No-mark connectors: L12 and L13

[1052]

[1053] L-12a.1-(Boc)-2-(Hydroxymethyl)-4-(N-maleyl-β-alanyl)piperazine

[1054]

[1055] Tert-butyl 2-(hydroxymethyl)piperazine-1-carboxylate (200 mg, 0.93 mmol) was dissolved in dichloromethane (10 mL) at 0 ° C. Diisopropylethylamine (359 mg, 2.78 mmol) and HATU (422 mg, 1.11 mmol) were added, and the solution was stirred at 0 ° C for 15 minutes. 3-Maleimidopropionic acid (187 mg, 1.11 mmol) was added, and the reaction was then stirred at room temperature under argon for 12 hours. The reaction was diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate and concentrated. Purification by flash chromatography (neutral alumina, 2% methanol: ethyl acetate) provided the title material L-12a. LCMS (method 6): retention time = 2.87 min.

[1056] L-12b. 1-(Boc)-2-(Acetoxymethyl)-4-(N-maleoyl-β-alanyl)piperazine

[1057]

[1058] To L-12a (300 mg, 0.82 mmol) and triethylamine (330 mg, 3.27 mmol) in dichloromethane (4 mL) was added acetyl chloride (125 mg, 1.64 mmol) dropwise at 0°C under argon. The reaction was stirred at room temperature for 3 hours, then treated with water and extracted with dichloromethane. The organic layer was dried (sodium sulfate), concentrated, and purified by flash chromatography (neutral alumina, 2% methanol:ethyl acetate) to provide the title material L-12b. LCMS (Method 6): Retention time = 3.2 min.

[1059] L-12c.1-(N-Maleyl-β-alanyl)-3-(acetoxymethyl)piperazine, trifluoroacetate

[1060]

[1061] L-12b (200 mg, 0.49 mmol) was dissolved in dichloromethane (5 mL) at 0°C, then treated dropwise with trifluoroacetic acid (0.4 mL) and stirred for 3 hours at room temperature under argon. The reaction was evaporated, washed with pentane, and dried to provide the crude title material L-12c. LCMS (Method 6): Retention time = 1.709 min; MS (ESI+) m / z 310 (M+H).

[1062] L12. 2-(Acetoxymethyl)-4-(N-maleyl-β-alanyl)-1-piperazineacetic acid tert-butyl ester

[1063]

[1064] To L-12c (140 mg, 0.45 mmol) and potassium carbonate (156 mg, 1.13 mmol) in acetonitrile (5 mL) at 0°C was added tert-butyl bromoacetate (100 mg, 0.54 mmol) dropwise. The reaction was heated to 10°C-15°C and then allowed to stir at room temperature under argon for 12 hours. The reaction was diluted with water and extracted with ethyl acetate. The organic layer was dried (sodium sulfate), concentrated, and purified by preparative HPLC (column: zorbax eclipse XDB C18, 21.2×150 mm, 5 μm; mobile phase A=water, mobile phase B=acetonitrile; time=0 min: 30% B; 2 min: 40% B; 10 min: 60% B) to provide L12. LCMS (Method 6): retention time=3.08 min; MS (ESI+) m / z 424 (M+H). 1H-NMR(CDCl3,ppm)6.69(s,2H),4.25-4.14(m,2H),4.09-3.96(m,2H),3.88-3.83(t,J=9,2H),3.65-3.54(m,1H),3.46-3.33( m,2H),3.31-3.17(m,2H),3.05-2.98(m,2H),2.84-2.75(m,2H),2.68-2.63(t,J=9,2H),2.09-2.07(d,J=6,3H),1.45(s,9H).

[1065] L13 was prepared using a method similar to that used for the synthesis of L12:

[1066] L13. 2-(2'-methoxyacetoxymethyl)-4-(N-maleyl-β-alanyl)-1-piperazineacetic acid tert-butyl ester (1-(2-(tert-butoxy)-2-oxoethyl)-4-(3-(maleimido)propionyl)piperazin-2-yl)methyl 2-methoxyacetate

[1067]

[1068] L13. LCMS (Method 6): Retention time = 3.04 min; MS (ESI+) m / z 454.5 (M+H). 1 H-NMR (CDCl3, ppm) 6.70 (s, 2H), 4.40-4.24 (m, 1H), 4.20-4.10 (m, 1H), 4.07-4.60 (d, J = 4, 2H), 4. 02-3.94(m,1H),3.87-3.83(t,J=8,2H),3.70-3.60(m,1H),3.68-3.60(m,1H),3.58-3.51(m,1H) ,3.45(s,3H),3.41-3.39(d,J=8,1H),3.36-3.32(s,1H),3.32-3.28(s,1H),3.18-3.10(m,1H),3 .04-2.97(m,1H),2.83-2.81(t,J=4,1H),2.79-2.75(m,1H),2.67-2.64(t,J=8,2H),1.46(s,9H).

[1069] No trace connector; L14-L16

[1070]

[1071] L14a. 1-(Boc)-2-(Hydroxymethyl)-4-(3'-(2"-(2"'-(2""-((N-Cbz)-amino)ethoxy)ethoxy)ethoxy)propanoyl)piperazine

[1072]

[1073] To tert-butyl 2-(hydroxymethyl)piperazine-1-carboxylate (1.0 g, 4.63 mmol) dissolved in dichloromethane (20 mL) at 0°C was added diisopropylethylamine (1.79 g, 13.9 mmol) and HATU (2.10 g, 5.55 mmol). The solution was stirred at 0°C for 15 minutes. 3-(2'-(2"-(2"'-((N-Cbz)-amino)ethoxy)ethoxy)ethoxy)propanoic acid (1.64 g, 4.63 mmol) was added and the reaction was stirred at room temperature under argon for 16 hours. The reaction was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate and concentrated. Purification by flash chromatography (SiO2, 1%-4% methanol:dichloromethane) provided the title material L-14a. LCMS (Method 5): Retention time = 1.27 min; MS (ESI+) m / z 554.3 (M+H). L-14b. 1-(Boc)-2-(acetoxymethyl)-4-(3'-(2"-(2"'-(2""-((N-Cbz)-amino)ethoxy)ethoxy)ethoxy)propanoyl)piperazine

[1074]

[1075] To L-14a (500 mg, 0.90 mmol) and triethylamine (228 mg, 2.26 mmol) dissolved in dichloromethane (20 mL) was added acetyl chloride (83 mg, 1.08 mmol) dropwise at 0°C. The reaction was stirred at room temperature under argon for 16 hours. The reaction was diluted with water and extracted with dichloromethane. The combined organic layers were dried over sodium sulfate and concentrated, then purified by flash chromatography (SiO2, 1%-4% methanol: dichloromethane) to provide the title material L-14b. LCMS (Method 7): Retention time = 1.54 min; MS (ESI+) m / z 595.8 (M+H).

[1076] L-14c. 1-(3'-(2"-(2"'-(2""-((N-Cbz)-amino)ethoxy)ethoxy)ethoxy)propanoyl)-3-(acetoxymethyl)piperazine, trifluoroacetate

[1077]

[1078] L-14b (500 mg, 0.84 mmol) was dissolved in dichloromethane (10 mL) at 0° C., then treated dropwise with trifluoroacetic acid (3 mL) and stirred at room temperature under argon for 3 h. The reaction was evaporated, washed with pentane, and dried to provide the crude title material L-14c, which was used in the next reaction without further purification or analysis.

[1079] L14. tert-Butyl 2-(acetoxymethyl)-4-(3'-(2"-(2"'-(2""-((N-Cbz)-amino)ethoxy)ethoxy)ethoxy)propanoyl)-1-piperazineacetate

[1080]

[1081] To L-14c (500 mg, 1.0 mmol) and potassium carbonate (418 mg, 3.03 mmol) in acetonitrile (10 mL) was added tert-butyl bromoacetate (295 mg, 1.51 mmol) dropwise at 0°C. The reaction was stirred at room temperature under argon for 20 hours. The reaction was diluted with water and extracted with ethyl acetate. The organic layer was dried (sodium sulfate), concentrated and purified by initial flash chromatography (SiO2, 1%-5% methanol: dichloromethane) followed by preparative HPLC (column: zorbax eclipse XDB C18, 4.6×150 mm, 5 μm; mobile phase A=0.01% TFA in water, mobile phase B=acetonitrile: methanol (1:1), 1 mL / min; time=0 min: 30% B; 1 min: 70% B; 6 min: 100% B) to provide L14. LCMS (Method 5): Retention time = 3.43 min; MS (ESI+) m / z 610.35 (M+H). 1 H-NMR(CDCl3,ppm)7.36-7.31(m,5H),5.49(s,1H),5.09(s,2H),4.26-4.16(m,1H),4.07-3.98(m,1H),3.78-3.74(t,J=6,2 H),3.60-3.53(m,11H),3.39-3.32(m,4H),2.84-2.72(m,2H),2.61-2.57(t,J=3,2H),2.08-2.06(d,J=6,2H),1.45(s,9H).

[1082] Using a method similar to that used to synthesize L14, the species shown in Table 10 were prepared:

[1083]

[1084]

[1085]

[1086]

[1087]

[1088]

[1089]

[1090] Example 2

[1091] Traceless adducts of linkers with biologically active moieties

[1092] This example describes the synthesis of a variety of traceless linker-drug adducts that can also be conjugated to a carrier.

[1093]

[1094]

[1095]

[1096] Synthesis of adducts

[1097] Bioactive Moieties and Traceless Linkers:

[1098] Acylation of L4-NHS. 2-(isobutyryloxymethyl)-4-(N-(((1R',8'S,9's)-bicyclo[6.1.0]non-4'-yn-9'-yl)methoxycarbonyl)-β-alanyl)-1-piperazineacetic acid N-hydroxysuccinimide ester

[1099]

[1100] A suspension of L4 (22 mg, 0.045 mmol) in dichloromethane (5 mL) was treated with triethylamine (0.0062 mL, 0.045 mmol), dimethylaminopyridine (0.55 mg, 0.0045 mmol) and N,N'-disuccinimidyl carbonate (10.9 mg, 0.043 mmol) and stirred at room temperature for 2 hours, then evaporated and used immediately without further purification. LCMS (Method 1): Retention time = 1.33 min; MS (ESI+) m / z 589.6 (M+H).

[1101] The species shown in Table 13 were prepared using a method similar to that used to synthesize L4-NHS.

[1102]

[1103]

[1104] L4-Brucizumab (L4D1b)

[1105]

[1106] A 60 mg / mL solution of brocuzumab (10 mL, 0.023 mmol) in buffer (composed of 20 mM trisodium citrate dihydrate, 125 mM sodium chloride, 0.01 mg / mL Tween 20 at pH 6.25) was diluted with 1×PBS (10 mL, pH 7.4) to provide a 30 mg / mL solution, which was then slowly treated with 38 mM L4-NHS in DMSO (0.65 mL, 0.023 mmol), vortexed, and kept at room temperature for 1 hour. Purification by ultracentrifugation (Vivaspin concentrator, 10 kDa MWCO) and washing with 1×PBS (4 times) to remove residual small molecule impurities produced a mixture of L4D1 and unmodified brocuzumab (D1). MS (Method 9) deconvoluted m / z 26312 (brocuzumab, M+H); deconvoluted m / z 26786 (L4D1, M+H, expected MW=MW(brocuzumab)+473).

[1107] An important analytical parameter is to determine the ratio of (unmodified brolucizumab, n=0):(monoacylated brolucizumab, n=1):(polyacylated brolucizumab, n>1). Comparison of the relative peak heights of the various species in the mass spectrum allows an estimate of the ratio of products to be produced, but this ratio may not be accurate because the various products may not have similar mole / liter response factors in the MS analysis. In this example: MS analysis showed that the product ratio [(n=0):(n=1):(n=2)] was approximately 5:1:0 (Method 9).

[1108] Another method for estimating the ratio of reaction products is to utilize SEC (size exclusion chromatography) or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of the derivatized reaction products; derivatization is required to achieve separation of the reaction products.

[1109] L4D1b derivatization reaction

[1110]

[1111]

[1112] An aliquot of the reaction solution from a 30 mg / mL L4D1b reaction mixture (0.005 mL, 0.0057 μmol) was washed with M n The cells were treated with approximately 5 kDa methoxy-PEG-azide (Sigma-Aldrich, 689475, 10 μL of a 100 mg / mL solution in 1×PBS, 0.20 μmol), vortexed, and then shaken at 37° C. for 45 minutes.

[1113] Analysis of L4D1b derivatives

[1114] SEC: (column: Agilent AdvanceBioSEC 300A 2.7 μm 4.6×150 mm; mobile phase: 150 mM potassium phosphate in water containing 5% isopropanol (isocratic); fast method: 0.2 mL / min for 13 min; slow method: 0.1 mL / min for 25 min; detection: 210 nm). The ratio determined for the L4D1b derivatives was (unmodified brolucizumab, n=0, retention time=12.74 min (slow method)): (mono-acylated brolucizumab, n=1, retention time=11.26 min (slow method)): (di-acylated brolucizumab, n=2, retention time=10.36 min (slow method)) = [(n=0): (n=1): (n=2)] = 81:18:1.

[1115] SDS-Page: (MOPS running buffer; 4-12% Bis-Tris Gels; Coomassie blue stain; Bio-Rad ChemiDoc imager; Image Lab5.2.1 analysis software): The ratio determined by the L4D1b derivative is (unmodified brolucizumab, n=0): (mono-acylated brolucizumab, n=1): (poly-acylated brolucizumab, n=2) = [(n=0): (n=1): (n=2)] about 67:33: trace.

[1116] Using a method similar to that used to synthesize L4D1b, the adducts shown in Tables 14 and 15 were prepared. The reaction conditions used to prepare the adducts are listed in Table 14; the characteristics of these adducts are described in Table 15.

[1117]

[1118]

[1119]

[1120]

[1121] L8D4

[1122]

[1123] L8 (43.4 mg, 0.113 mmol) was dissolved in dimethylformamide (2.55 mL), followed by EDC·HCl (21.2 mg, 0.111 mmol) and N-hydroxyphthalimide (18.6 mg, 0.114 mmol). The solution was stirred at room temperature overnight (about 16 hours). To the solution was added D4 (35 mg, 0.02 mmol) dissolved in 1.5 mL of dimethylformamide and 1.5 mL of 100 mM phosphate buffer (pH 6.5). After 2 hours, the reaction was diluted with 4 mL of acetonitrile, filtered, and purified by preparative reverse phase HPLC with mass oriented fraction collection (method below). The fractions containing the product were combined, frozen, and lyophilized to provide L8D4. MS (ESI+) m / z=952.5 (M+2H) 2+ .

[1124] Preparative HPLC conditions: Waters XSelect CSH OBD; particle size: 5 μm; column size: 19×150 mm; eluent / gradient: 24% CH3CN / H2O / 1.65 min, 24%-34% CH3CN / H2O / 8.35 min (CH3CN and H2O containing 0.1% TFA); flow rate: 30 mL / min; column temperature: room temperature; total m / z: +951.7.

[1125] Example 3

[1126] Functionalization of hyaluronic acid

[1127] This example describes the synthesis of functionalized hyaluronic acid, which can be a carrier by itself or can also be reacted with a cross-linking moiety to form a hydrogel.

[1128] Synthesis of hyaluronic acid intermediate [HA-N3]:

[1129]

[1130] Hyaluronic acid sodium salt is a linear polymer composed of the repeating dimer units of glucuronic acid and N-acetylgalactosamine, wherein the molecular weight of the repeating unit is 401.3Da. In this example, the mole number of the hyaluronic acid reported refers to the mole number of the repeating unit, and relative to the mole number of the hyaluronic acid repeating unit, the equivalent of the reagent used in the reaction with hyaluronic acid is reported. The average molecular weight of the polymer determines the average number / polymer chain of the repeating unit. As determined by viscometry, the hyaluronic acid sodium salt labeled as having a nominal average molecular weight of 200kDa by supplier Lifecore Biomedical (HA200K, Chaska, Minnesota (Chaska, MN)) can vary from batch to batch in the range of 151kDa-300kDa. In this example, this molecule of the hyaluronic acid sodium salt with the nominal average molecular weight of 200kDa assumed to be assuming will have an average length of about 500 monomer units.

[1131] Synthesis of [HA-N3]-23%

[1132] A solution of hyaluronic acid, sodium salt (nominal average molecular weight 200 kD; 250.1 mg, 0.623 mmol; Lifecore Biomedical, LLC; product number HA200K) was completely dissolved in 25 mL of MES buffer (50 mM, pH 5.5). 4-(4',6'-dimethoxy-1',3',5'-triazine-2'-yl)-4-methylmorpholinium-4-ium chloride (DMTMM, 295 mg, 1.07 mmol, 1.71 equivalents) was added to the solution, followed by 11-azido-3,6,9-trioxaundecane-1-amine (N3-PEG3-NH2, 196 mg, 0.90 mmol) after 5 minutes. The reaction was stirred overnight, then diluted with 25 mL of 0.25 M NaCl solution and purified by tangential flow filtration.

[1133] Tangential flow filtration was performed using a 30 kDa MWCO Vivaflow-50R hydrosart cartridge from Sartorius, eluting with 400 mL of 0.25 M NaCl solution followed by 400 mL of water.

[1134] The product was flash frozen and lyophilized to provide the title material [HA-N3]-23%.

[1135] 1 H NMR (400MHz, D2O) δ4.45 (bs, 2H), 4.0-3.1 (m, 15.5H), 1.95 (s, 3H).

[1136] DOSY-NMR. A Bruker AVANCE III 400 MHz (for 1 H) instrument, using a stimulated echo with a perturbation gradient pulse sequence (stegp1s1d), collect one-dimensional diffusion ordered NMR spectra (DOSY). The diffusion time and diffusion gradient time were set to 50ms and 4ms, respectively. Two spectra were collected, where the gradient strength (gpz6) was set to 2% and 95%. Comparison of the two spectra showed no difference except for the solvent peak, indicating that there were no small molecule impurities in the polymer.

[1137] Elemental analysis: C: 40.05%; H: 5.67%; N: 5.70%.

[1138] The degree of substitution of [HA-N3] is defined as the % repeat unit in which the carboxylate moiety has undergone reaction to give the amide shown. Elemental analysis is used to determine the degree of substitution. The [%C / %N] ratio determined by elemental analysis of the purified sample is entered into the following formula to provide the degree of substitution. Where y = [(%C) / (%N)]), then:

[1139]

[1140]

[1141] In this example, the degree of substitution (DS) of [HA-N3] is 23%.

[1142] The 200 kDa hyaluronic acid functionalized with 23% azide linker was labeled as [HA-N3]-23%.

[1143] In the remaining examples, 200 kDa hyaluronic acid functionalized with X% of an azide linker was labeled as [HA-N3]-X%.

[1144] Use similar method for synthesizing [HA-N3]-23% to prepare and characterize the species in Table 16. We find that the degree of substitution achieved depends on the given raw material of the DMTMM reagent used, and may be heterogeneous. Generally, for a given raw material of DMTMM, the degree of substitution increases as the equivalent number of the DMTMM used increases, and the degree of substitution decreases as the equivalent number of the DMTMM used decreases. Some [HA-N3] intermediates are purified ([HA-N3]-23%b, [HA-N3]-37%) by dialysis instead of tangential flow filtration. In these cases, the crude reaction mixture is filled into a regenerated cellulose dialysis membrane (MWCO 1-25kD) and dialyzed for 1-3 days with 0.25M-1M NaCl, wherein the dialysis solution is replaced several times, and then after dialysis with deionized water for 1-3 days, the dialysis solution is also replaced many times. After completion, the sample is taken out from the dialysis tubing, quickly frozen and lyophilized.

[1145]

[1146]

[1147] Example 4

[1148] Preparation of PEG polymers, XL-1-XL-10

[1149] This example describes the synthesis of a crosslinker that can react with other functionalized polymers to form a hydrogel.

[1150] PEG-based cross-linked polymers

[1151] General structure1.

[1152]

[1153] General structure2.

[1154]

[1155]

[1156]

[1157] Structure of XL-8:

[1158]

[1159] MW of starting PEG: M n About 10,000 kDa.

[1160] Synthesis of PEG-based cross-linked polymers

[1161] The degree of substitution is an important parameter for PEG-derivatized cross-linkers and is defined as the percentage of the PEG terminal groups that are substituted with the functional group one wishes to introduce. 1 The degree of substitution was determined by H-NMR, comparing the integral of the methylene group specific for the PEG terminal group with the integral of the proton specific for the introduced functional group. For XL-1-XL-6, the methylene group specific for the PEG moiety was an ester or carbamate methylene [-(C=O)-OC H 2-], comparing the integral of the methylene group to the protons in the bicyclo[6.1.0]non-4-yne moiety. Degree of Substitution = ((by 1 H-NMR, molar number of functional groups introduced) / (by 1 H-NMR, molar number of PEG end groups))×100.

[1162] XL-1.PEG(2000)-bis-[3-(((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)propanoate]

[1163]

[1164] XL-1a.PEG(2000)-bis-[3-((tert-butoxycarbonyl)amino)propionate]

[1165]

[1166] 3-((tert-Butyloxycarbonyl)amino)propanoic acid (1.892 g, 10.00 mmol) and M n About 2 kDa PEG (5 g, 2.500 mmol) was dissolved in 50 mL of dichloromethane. Dimethylaminopyridine (0.153 g, 1.250 mmol) and EDC·HCl (1.922 g, 10.03 mmol) were added and the reaction mixture was stirred at room temperature overnight. The crude product was purified by flash column chromatography on silica with a 0-15% dichloromethane: methanol gradient. The fractions containing the product were combined and decompressed to dryness to provide XL-1a. 1 H NMR (400 MHz, methanol-d4) δ 4.23 (m, 4H), 3.70 (m, 4H), 3.63 (br s, 167H), 2.52 (m, 4H), 1.43 (br s, 18H). XL-1b. PEG (2000)-bis-[3-(amino) propionate], bis-trifluoroacetic acid

[1167]

[1168] XL-1a (600 mg, 0.261 mmol) was dissolved in dichloromethane (6 mL). Trifluoroacetic acid (2 mL) was added and the reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure. The crude product was triturated twice with Et2O and then dried under vacuum to provide XL-1b. 1 H NMR (400 MHz, methanol-d4) δ 4.33 (m, 4H), 3.73 (m, 4H), 3.64 (brs, 183H), 2.80 (m, 4H).

[1169] XL-1-1.PEG(2000)-bis-[3-(((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)propanoate]

[1170]

[1171] XL-1b (200 mg, 0.086 mmol) was dissolved in acetonitrile (3 mL). Triethylamine (0.599 mL, 4.30 mmol) was added, followed by ((1R, 8S, 9s)-bicyclo [6.1.0] non-4-yn-9-yl) methyl (2,5-dioxopyrrolidin-1-yl) carbonate (200 mg, 0.688 mmol), and the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was directly purified by preparative reverse phase HPLC with ELSD triggered fraction collection (method below). The fractions containing the product were combined, frozen and lyophilized to provide XL-1-1. For storage purposes, XL-1-1 was kept in a freezer as acetonitrile, DMSO or methanol solution. Analytical HPLC-CAD (method below): retention time = 11.93 min. 1 H NMR (400MHz, methanol-d4) δ4.23(m,4H),4.14(m,4H),3.63(br s,175H),2.54(m,4H),2.22(m,12H),1.60(m,4H),1.37(m,2H),0.94(m,4H).

[1172] Preparative HPLC conditions: Waters XBridge C18; particle size: 5 μm; column size: 19×250 mm; eluent / gradient: 5% CH3CN / H2O / 0.5 min, 5-95% CH3CN / H2O / 12.5 min, 95% CH3CN / H2O / 3 min; flow rate: 30 mL / min; column temperature: room temperature.

[1173] Analytical HPLC-CAD conditions: Waters XBridge BEH300 C18; particle size: 3.5 μm; column size: 4.6×100 mm; eluent / gradient: 2% CH 3 CN / H 2 O / 0.5 min, 2%-98% CH 3 CN / H 2 O / 17.5 min (CH 3 CN containing 0.05% TFA and H 2 O containing 0.1% TFA); flow rate: 1 mL / min; column temperature: 50° C.

[1174] Analytical HPLC-CAD conditions (Method 10): Waters ACQUITY BEH C18; particle size: 1.7 μm; pore size: Column size: 2.1×50 mm; eluent / gradient: 5% CH 3 CN / H 2 O / 1.2 min, 5%-95% CH 3 CN / H 2 O / 1.8 min (CH 3 CN containing 0.1% TFA and H 2 O containing 0.1% TFA); flow rate: 1 mL / min; column temperature: 45° C.

[1175] Alternatively, the reaction mixture was purified directly by flash chromatography (SiO2, 0-10% methanol: dichloroethane) to provide a less pure batch of XL-1-2.

[1176] Using a method similar to that used to synthesize XL-1 (except XL-1-2), the species in Table 18 were prepared:

[1177]

[1178]

[1179]

[1180]

[1181] Intermediates used in the synthesis of PEG-based cross-linked polymers:

[1182]

[1183]

[1184]

[1185] XL-5.PEG(2000)-bis-[3-(((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methylcarbamoyl]

[1186]

[1187] Mn About 2kDa PEG diamine hydrochloride (JenKem Technology, 300mg, 0.148mmol) was dissolved in acetonitrile (3mL). Triethylamine (0.413mL, 2.96mmol) was added, followed by ((1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (345mg, 1.184mmol), and the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was directly purified by preparative reverse phase HPLC with ELSD triggered fraction collection (method below). The fractions containing the product were combined, frozen and lyophilized to provide XL-5. For storage purposes, XL-5 was kept in a freezer as acetonitrile, DMSO or methanol solution. Analytical HPLC-CAD (method below): retention time = 11.85min. 1 H NMR (400MHz, methanol-d4) δ4.14(m,4H),3.63(br s,186H),3.54(m,4H),2.22(m,12H),1.61(m,4H),1.38(m,2H),0.94(m,4H).

[1188] Preparative HPLC conditions: Waters XBridge C18; particle size: 5 μm; column size: 19×250 mm; eluent / gradient: 5% CH3CN / H2O / 0.5 min, 5-95% CH3CN / H2O / 12.5 min, 95% CH3CN / H2O / 3 min; flow rate: 30 mL / min; column temperature: room temperature.

[1189] Analytical HPLC-CAD conditions: Waters XBridge BEH300 C18; particle size: 3.5 μm; column size: 4.6×100 mm; eluent / gradient: 2% CH 3 CN / H 2 O / 0.5 min, 2%-98% CH 3 CN / H 2 O / 17.5 min (CH 3 CN containing 0.05% TFA and H 2 O containing 0.1% TFA); flow rate: 1 mL / min; column temperature: 50°C.

[1190] XL-6.PEG(10000)-Tetrakis-[3-(((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methylcarbamoyl]

[1191]

[1192] M n4-arm PEG amine hydrochloride (pentaerythritol core, JenKem Technology, 500 mg, 0.05 mmol) with a 10 kDa reaction mixture was dissolved in dichloromethane (15 mL). Triethylamine (0.554 mL, 4.00 mmol) was added followed by ((1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (175 mg, 0.600 mmol) and the reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by dialysis against methanol using a 2 kDa MWCO Spectra / Por 6 regenerated cellulose dialysis membrane (Spectrum, Inc.). For analysis and use in further reactions, XL-6 was isolated by concentration using a rotary evaporator. For storage purposes, the compound was stored as a methanol solution at room temperature. 1 H NMR (400 MHz, methanol-d4) δ 4.14 (m, 8H), 3.63 (br s, 995H), 2.22 (m, 24H), 1.61 (m, 8H), 1.38 (m, 4H), 0.94 (m, 8H).

[1193] XL-8.

[1194]

[1195] M n 8-arm PEG amine hydrochloride (hexaglycerol core, JenKem Technology, 800 mg, 0.08 mmol) of about 10 kDa was dissolved in dichloromethane (5 mL). Triethylamine (0.887 mL, 6.40 mmol) was added, followed by ((1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (807 mg, 2.56 mmol), and the reaction mixture was stirred at room temperature for 5 hours. Using a rotary evaporator, the reaction mixture was concentrated, then dissolved in methanol, and purified by dialysis through methanol using a 2 kDa MWCO Spectra / Por 6 regenerated cellulose dialysis membrane (Spectrum, Inc.). For analysis and use in further reactions, XL-8 was isolated by concentration using a rotary evaporator, but for storage purposes, XL-8 was stored as a methanol solution at room temperature. 1 H NMR (400 MHz, methanol-d4) δ 4.14 (m, 16H), 3.63 (br s, 935H), 2.23 (m, 48H), 1.61 (m, 16H), 1.38 (m, 8H), 0.95 (m, 16H).

[1196] Example 5

[1197] This example describes the synthesis of a hydrogel prepared by reacting a suitably functionalized polymer with a cross-linking agent.

[1198] Synthesis of hyaluronic acid hydrogel: H1a

[1199]

[1200]

[1201] [HA-N3]-23%b (60 mg, 136 μmol, degree of substitution = 23%) was dissolved in 3.8 mL of sterile 1×PBS buffer, pH 7.4 in a 5 mL microcentrifuge tube at room temperature for 30 min. The molecular weight of the unsubstituted carboxylic acid sodium salt repeating dimer unit is 401.3 Da. The MW of the azidated repeating dimer unit is 579.6 Da. The average MW of the dimer unit in the sodium salt form of [HA-N3]-23%b is 442.3 Da = ((401.3×0.77)+(579.6×0.23)). Using the average MW of the sodium salt dimer unit, the total mole of the repeating dimer unit is 136 μmol, and the mole number of the azidated repeating dimer unit is 31 μmol.

[1202] To this solution was added a 50 mg / mL solution of XL-1-1 (0.215 mL, 4.30 μmol reagent assuming a MW of 2500 Da, 8.60 μmol reactive functionality). This produced a 1.5% w / v solution of [HA-N3]-23%b, and in which 6.3% of the [HA-N3]-22%b repeating units were predicted to be cross-linked by XL-1-1] ((8.60 μmol [XL-1-1 - reactive functionality] / 136 μmol [HA units) × 100 = 6.3%).

[1203] The mixture was vortexed briefly to mix and then dispensed into two 3 mL syringes, which were capped and kept at 37°C overnight to provide H1a. Visual inspection (reverse test) indicated successful gelation.

[1204] Preparation of hyaluronic acid-based hydrogel particles

[1205] H1a (2 mL) was forced through a 100 mesh stainless steel screen disk into a 5 mL syringe to produce coarse gel particles. 1 mL of 1×PBS was added to the syringe and then vortexed to mix. The syringe was kept at room temperature for 6 hours to allow the hydrogel to swell. This produced a mixture with 1.0% about [HA-N3]-23%b. The swollen coarse gel particles of H1a were forced through a 200 mesh stainless steel screen disk 20 times to produce fine gel particles as the final product.

[1206] Similar to H1a, hyaluronic acid-based hydrogels (and corresponding hydrogel particles) were prepared as listed in Table 20. The hydrogels were defined by the [HA-N3] component used and its concentration in the cross-linking reaction, as well as the PEG cross-linker used, and the degree of cross-linking expected to form with the backbone polymer (% cross-linking).

[1207]

[1208] Example 6

[1209] Synthesis of hydrogel-drug conjugates

[1210] This example describes the synthesis of a hydrogel-drug conjugate in which the drug is conjugated to a carrier via a traceless linker.

[1211] When preparing samples for in vivo administration, all manipulations on uncapped materials or solutions were performed under sterile conditions in a laminar flow hood. All consumables used previously were unopened and labeled “sterile”.

[1212] C4b(H1a-L4D1)

[1213]

[1214]

[1215] A suspension of hydrogel particles of H1a (2.5 mL, 10 mg / mL, 23 mM total dimer repeat units) was treated with a solution of L4D1b (14.5 mL of a 42 mg / mL solution in 1×PBS), vortexed and kept overnight at 37°C. The reaction tube was centrifuged (1000 rcf, 5 minutes) and the supernatant was removed from the tube using a needle and syringe. Fresh 1×PBS (10-15 mL) was added to the tube and the tube was oscillated to resuspend the gel. Centrifugation, removal of the supernatant, and dilution with fresh buffer were repeated. Washing was continued until no brocizumab was detected in the supernatant (using a Nanodrop spectrophotometer to detect absorbance at 280 nm, a total of approximately 65 mL of PBS wash). The remaining hydrogel precipitate [C4b] was transferred to a syringe for storage and distribution.

[1216] The key description of a hydrogel-drug conjugate is drug loading / hydrogel volume (=drug loading). When the drug is a protein, there are several ways to determine the loading.

[1217] The protein loading of the hydrogel was determined by forced release of the conjugated protein from a known volume of hydrogel followed by quantification of the released protein. In this example, 37.3 μg of [C4b] was weighed into a reaction tube (equivalent to 37.3 μL of sample assuming a density of about 1 g / mL for the hydrogel). The sample was treated with 1 M Tris·HCl buffer, pH 9.5 (1 mL), vortexed and shaken at 37°C for 48 hours (or until the measured concentration reached steady state). In this example, the concentration of the released drug (brocetizumab) was measured (Nanodrop spectrophotometer) as 0.48 mg / mL, corresponding to 13.35 mg brocetizumab / mL of the hydrogel in [C4b] ((0.048 mg / mL / 0.0373 mL) × (1.0373 mL total volume) = 13.35 mg / mL). The analysis was performed in triplicate. Prior to dilution, the average measured protein load for [C4b] was 13.9 mg brocuzumab / mL hydrogel.

[1218] An alternative method to determine protein loading relies on differential calculation. In this method, the measured (Nanodrop spectrophotometer) brocuzumab recovered in the hydrogel washes is subtracted from the known total added protein (e.g., brocuzumab + L4D1). The difference gives the brocuzumab loading based on the total hydrogel volume.

[1219] The hydrogels in Table 21 were prepared using a method similar to that used to synthesize C4b.

[1220]

[1221]

[1222] The hydrogel-drug conjugate can be diluted with 1×PBS to obtain the desired final drug concentration for administration. The amount of 1×PBS to be added can be calculated by the following equation: (PBS dilution (mL)) = [(total drug (mg) / desired drug load (mg / mL)] - (initial volume of conjugate (mL)).

[1223] Conjugate C4b prepared as described above (2 mL hydrogel conjugate, 13.9 mg / mL brolucizumab, total brolucizumab = 27.8 mg) was diluted by adding 1×PBS (0.8 mL). The syringe was capped and shaken vigorously to homogenize. This resulted in a total sample volume of 2.8 mL with protein loading = 27.8 mg brolucizumab / 2.8 mL = 9.93 mg / mL brolucizumab.

[1224] Conjugate C4b was dispensed for dosing in a 0.5 mL insulin syringe with an attached 30G needle by removing the plunger and backfilling with the desired volume of C4b.

[1225] The hydrogel-drug conjugates were stored at 4 °C.

[1226] Synthesis of Hydrogel Drug Conjugates: C13

[1227]

[1228] The linker-drug adduct L8D4 (trifluoroacetate, 19.9 mg, 8.86 μmol) was treated with a solution of XL-8 (0.157 mL of a 200 mg / mL solution in DMSO), shaken, and kept for 80 minutes, during which time HPLC analysis showed complete conjugation of L8D4 to XL-8. To this solution was added a solution of XL-7 (0.154 mL of a 200 mg / mL solution in DMSO). The reaction was shaken and kept at room temperature for 4 hours.

[1229] The block gel is forced to pass through a 100 mesh stainless steel mesh disk into a 3 mL syringe to produce coarse gel particles. 2 mL of DMSO is added to the syringe and vortexed to mix. The syringe is kept at room temperature for 1 h to allow the gel to swell. The swollen coarse gel particles are forced to pass through a 200 mesh stainless steel mesh disk 20 times to produce fine gel particles.

[1230] The fine gel particle suspension (in the syringe with cap) was centrifuged at 2000rcf for 5 minutes to precipitate the gel. Using a needle and syringe, the supernatant was removed, and the precipitate was washed with cold 1 × PBS, pH 6.5 to remove unconjugated species from the gel. The suspension was centrifuged at 2000rcf for 5 minutes to precipitate the gel. Using a needle and syringe, the supernatant was removed from the syringe, fresh 1 × PBS (pH 6.5) was added to fill the syringe, and the syringe with cap was vibrated to resuspend the gel. Centrifugation, removal of the supernatant and dilution with fresh buffer were repeated four more times. After final centrifugation and removal of buffer, the endotoxin in the test supernatant was tested, and the washed gel precipitate was diluted to a final cumulative volume of 1.4mL with 1 × PBS (pH 6.5). The diluted particle suspension was vortexed to mix, and the product, C13, was stored at 4 ° C.

[1231] The concentration of D4 in C13 (assuming 100% conjugation) was 10.3 mg / mL.

[1232] Example 7

[1233] This example describes the preparation of a series of model traceless linker-drug conjugates, where the model "drug" is a low molecular weight amine-containing compound, most commonly p-methoxybenzylamine. Compounds M1-M27 were not conjugated to polymers, hydrogels, or other carriers. In vitro release studies of low molecular weight amine-containing compounds from Compounds M1-M27 allowed for the evaluation of a variety of different release triggers.

[1234]

[1235]

[1236]

[1237]

[1238]

[1239]

[1240] Synthesis of model compounds:

[1241] M1. (4-(3'-(((benzyloxy)carbonyl)amino)propanoyl)-1-(2"-((4"'-methoxybenzyl)amino)-2"-oxoethyl)piperazin-2-yl)methylpropanoate

[1242]

[1243] M1. (4-(3'-(((benzyloxy)carbonyl)amino)propanoyl)-1-(2"-((4"'-methoxybenzyl)amino)-2"-oxoethyl)piperazin-2-yl)methylpropanoate

[1244]

[1245] A solution of L-INT-3 (61.7 mg, 0.13 mmol) in dichloromethane (3 mL) was treated with trifluoroacetic acid (1 mL) and stirred at room temperature overnight before evaporation. The crude intermediate was dissolved in dichloromethane (3 mL) and treated with diisopropylethylamine (0.066 mL, 0.38 mmol), HATU (57.3 mg, 0.15 mmol) and p-methoxybenzylamine (0.017 mL, 0.13 mmol), stirred for 1 hour, then evaporated. Purification by flash chromatography (SiO2, 0-15% methanol: dichloromethane) provided M1. LCMS (Method 1): Retention time = 1.16 min; MS (ESI+) m / z 555.2 (M+H). 1 H-NMR(400MHz, CDCl3)7.25(m,5H),7.12(m,2H),6.80(m,2H),5.43(m,1H),5.01(m,2H),4.33(d,2H),4.02( tdt,2H),3.73(s,3H),3.55-3.2(m,7H),3.09(m,1H),2.72(m,2H),2.41(dq,3H),2.18(m,2H),1.04(dt,3H).

[1246] M2. (4-(3'-(((benzyloxy)carbonyl)amino)propanoyl)-1-(2"-((4"'-methoxybenzyl)amino)-2"-oxoethyl)piperazin-2-yl)methyl acetate

[1247]

[1248] M2 was prepared from intermediate (L-INT-2) using a method similar to that used for the synthesis of M1. LCMS (Method 1): Retention time = 1.10 min: MS (ESI+) m / z 541.1 (M+H). NMR (400 MHz, CDCl3) 7.35 (m, 5H), 7.20 (m, 2H), 6.88 (m, 2H), 5.53 (m, 1H), 5.09 (m, 2H), 4.41 (d, 2H), 4.10 (tdt, 2H), 3.81 (s, 3H), 3.55-3.2 (m, 7H), 3.17 (dd, 1H), 2.79 (m, 2H), 2.49 (dq, 3H), 2.0 (m, 3H).

[1249] M3. (4-(3'-(((benzyloxy)carbonyl)amino)propanoyl)-1-(2"-((4"'-methoxybenzyl)amino)-2"-oxoethyl)piperazin-2-yl)methyl methoxyacetate

[1250]

[1251] M3 was prepared from intermediate (L-INT-1) using a method similar to that used for the synthesis of M1. LCMS (Method 1): Retention time = 1.11 min MS (ESI+) m / z 571.2 (M+H). NMR (400 MHz, CDCl3) 7.37 (m, 5H), 7.21 (m, 2H), 6.90 (m, 2H), 5.49 (m, 1H), 5.10 (m, 2H), 4.42 (d, 2H), 4.23 (tdd, 2H), 3.83 (s, 3H), 3.55-3.2 (m, 9H), 3.20 (dd, 1H), 2.83 (d, 2H), 2.52 (m, 3H).

[1252] M4. (4-(3'-(((benzyloxy)carbonyl)amino)propanoyl)-1-(2"-((4"'-methoxybenzyl)(methyl)amino)-2"-oxoethyl)piperazin-2-yl)methyl 2-methoxyacetate

[1253]

[1254] M4 was prepared from intermediate (L-INT-1) using a method similar to that used for the synthesis of M1, except that p-methoxybenzylamine was replaced with N-methyl-p-methoxybenzylamine. LCMS (Method 1): Retention time = 1.04 min MS (ESI+) m / z 585.2 (M+H). NMR (400 MHz, CDCl3) 7.35 (m, 5H), 7.20 (m, 2H), 6.80 (m, 2H), 5.43 (m, 1H), 5.13 (m, 2H), 4.7-3.9 (m, 7H), 3.82 (m, 3H), 3.8-3.2 (m, 10H), 2.8 (m, 3H), 2.7 (m, 3H), 2.6 (m, 2H).

[1255] M5. (4-(3'-(((benzyloxy)carbonyl)amino)propanoyl)-1-(2"-((4"'-methoxyphenyl)amino)-2"-oxoethyl)piperazin-2-yl)methyl 2""-methoxyacetate

[1256]

[1257] M5 was prepared from intermediate (L-INT-1) using a method similar to that used for the synthesis of M1, except that p-methoxybenzylamine was replaced with 4-methoxyaniline. LCMS (Method 1): Retention time = 1.14 min MS (ESI+) m / z 557.1 (M+H). NMR (400 MHz, CDCl3) 8.7 (br s, 1H), 7.4 (d, 2H), 7.37 (m, 5H), 6.8 (m, 2H), 5.5 (m, 1H), 5.1 (m, 2H), 4.3 (m, 2H), 4.0 (m, 2H), 3.8 (s, 4H), 3.7-3.2 (m, 11H), 3.2 (m, 1H), 2.8 (m, 2H), 2.5 (m, 2H), 2.18 (m, 3H).

[1258] M6. (4-Acetyl-1-(2-((4-methoxybenzyl)amino)-2-oxoethyl)piperazin-2-yl)methyl acetate

[1259]

[1260] M6a.1-N-Acetyl-3-hydroxymethylpiperazine

[1261]

[1262] A solution of 2-(hydroxymethyl)piperazine (2 g, 17.2 mmol) in water (25 mL) was treated dropwise with triethylamine (3.5 mL, 25 mmol) at 0°C under argon, stirred for 10 minutes, and then treated dropwise with a solution of acetic anhydride (1.64 mL, 17.3 mmol) in water (15 mL). The reaction was stirred at room temperature for 2 hours, then treated with 2M sodium carbonate (10 mL) and saturated sodium chloride (20 mL). The aqueous solution was evaporated, and the residue was purified by flash chromatography (basic alumina, 3% methanol: dichloromethane) to provide M6a. MS (ESI+) m / z 159.0 (M+H).

[1263] M6b. tert-Butyl 4-acetyl-2-(hydroxymethyl)piperazine-1-carboxylate

[1264]

[1265] A solution of M6a (1.4 g, 8.84 mmol) in dichloromethane (20 mL) was treated dropwise with triethylamine (1.78 g, 17.6 mmol) at 0°C under argon, stirred for 10 minutes, and then treated dropwise with di-tert-butyl dicarbonate (2.12 g, 9.73 mmol). The reaction was stirred at room temperature for 12 hours, then diluted with dichloromethane, washed with brine, dried over sodium sulfate, and evaporated. Purification by flash chromatography (basic alumina, 3% methanol: dichloromethane) provided M6b. MS (ESI+) m / z 259.0 (M+H).

[1266] M6c. tert-Butyl 2-(acetoxymethyl)-4-acetylpiperazine-1-carboxylate

[1267]

[1268] A solution of M6b (0.20 g, 0.77 mmol) in dichloromethane (5 mL) was treated dropwise with triethylamine (0.20 g, 1.94 mmol) at 0°C under argon, stirred for 10 minutes, and then treated dropwise with acetyl chloride (0.072 g, 0.93 mmol). The reaction was stirred at room temperature for 4 hours, then diluted with DCM, washed with brine, dried over sodium sulfate and evaporated. Purification by flash chromatography (basic alumina, 3% methanol: dichloromethane) provided M6c. MS (ESI+) m / z 301.1 (M+H).

[1269] M6. (4-Acetyl-1-(2'-((4"-methoxybenzyl)amino)-2'-oxoethyl)piperazin-2-yl)methyl acetate

[1270]

[1271] At 0°C, under argon, a solution of M6c (0.15 g, 0.49 mmol) in dichloromethane (5 mL) was treated dropwise with trifluoroacetic acid (2.0 mL) and stirred at room temperature for 4 hours, then evaporated. The material was dissolved in acetonitrile (10 mL) and kept at 0°C, under argon, then treated with potassium carbonate (0.414 g, 2.99 mmol) and 2-bromo-N-(4-methoxybenzyl)acetamide (0.232 g, 0.89 mmol). The reaction was stirred at room temperature for 12 hours, then evaporated. The crude mixture was extracted with dichloromethane, the extract was washed with brine, dried over sodium sulfate and evaporated. Purification by flash chromatography (basic alumina, 5% methanol: dichloromethane) followed by preparative HPLC (column: Zorbax Eclipse XDB C18 (150×21.5 mm*5 mm); mobile phase: A=water, B=acetonitrile; gradient: 0-2 min: 10% B; 2-8 min: 20% B; 8 min to end: 40% B;) provided M6. LCMS (Method 8): retention time=2.67 min; MS (ESI+) m / z 378.2 (M+H).

[1272] M7. Methyl 4-acetyl-1-(2'-((4"-methoxybenzyl)amino)-2'-oxoethyl)piperazine-2-carboxylate

[1273]

[1274] M7a. Methyl 4-acetylpiperazine-2-carboxylate, trifluoroacetate

[1275]

[1276] A solution of 1-(tert-butyl) 2-methylpiperazine-1,2-dicarboxylate (0.20 g, 0.82 mmol) and triethylamine (0.33 g, 3.27 mmol) in dichloromethane (4 mL) was treated dropwise with acetyl chloride (0.125 mg, 25 mmol) at 0°C under argon and then stirred at room temperature for 2 hours. The reaction was diluted with water, extracted with dichloromethane, and the organic phase was dried over sodium sulfate and evaporated. Purification by flash chromatography (basic alumina, 2% methanol:ethyl acetate) provided the intermediate product: MS (ESI+) m / z 287 (M+H). The material was dissolved in dichloromethane (5 mL) at 0°C and treated dropwise with trifluoroacetic acid and then stirred at room temperature for 3 hours. The reaction mixture was evaporated, washed with pentane and dried to provide M7a as a trifluoroacetate salt, which was used without further purification. MS (ESI+) m / z 187 (M+H).

[1277] M7b. Methyl 4-acetyl-1-(2'-(benzyloxy)-2'-oxoethyl)piperazine-2-carboxylate

[1278]

[1279] At 0°C, under argon, a mixture of M7a (0.16 g, 0.86 mmol) and potassium carbonate (0.30 mg, 2.15 mmol) in acetonitrile (5 mL) was treated dropwise with benzyl bromoacetate (0.234 mg, 1.03 mmol), allowed to slowly reach room temperature, and then stirred at room temperature for 12 hours. The reaction was diluted with water, extracted with ethyl acetate, and the organic phase was dried over sodium sulfate and evaporated. Purification by flash chromatography (neutral alumina, 1% methanol:ethyl acetate) provided M7b. MS (ESI+) m / z 335 (M+H).

[1280] M7c.2-(4'-Acetyl-2'-(methoxycarbonyl)piperazin-1'-yl)acetic acid

[1281]

[1282] To a solution of M7b (0.285 g, 0.85 mmol) was added 10% palladium on carbon (0.02 g) and then stirred under hydrogen balloon pressure at room temperature for 12 hours. The reaction was filtered through celite, washed with methanol, dried over sodium sulfate and evaporated. M7c was used without further purification. MS (ESI+) m / z 245 (M+H).

[1283] M7. Methyl 4-acetyl-1-(2'-((4"-methoxybenzyl)amino)-2'-oxoethyl)piperazine-2-carboxylate

[1284]

[1285] A solution of M7c (0.21 g, 0.86 mmol) in dichloromethane (5 mL) at 0°C under argon was treated with diisopropylethylamine (0.275 g, 2.14 mmol) and HATU (0.39 g, 1.03 mmol), then 15 minutes later with 4-methoxybenzylamine (0.117 g, 0.86 mmol). The reaction was stirred at room temperature for 12 hours, then treated with water and extracted with dichloromethane, the extracts were washed with brine, dried over sodium sulfate and evaporated. Purification by preparative HPLC (column: XBridge (150×19 mm); mobile phase: A=(0.01% TFA in water), B=acetonitrile; gradient: 0-2 min: 20% B; 2-8 min: 20% B; 8 min to end: 80% B) provided M7. LCMS (Method 3): Retention time = 2.85 min; MS (ESI+) m / z 364 (M+H).

[1286] M8. (4-Acetyl-1-(2'-((4"-methoxybenzyl)amino)-2'-oxoethyl)piperazin-2-yl)methyl methyl carbonate

[1287]

[1288] M8a (4-acetyl-1-(tert-butoxycarboxy)piperazin-2-yl)methyl methyl carbonate

[1289]

[1290] A solution of M6b (0.30 g, 1.16 mmol) in dichloromethane (5 mL) was treated dropwise with triethylamine (0.20 g, 1.94 mmol) at 0°C under argon, stirred for 10 minutes, and then treated dropwise with methyl chloroformate (0.54 g, 5.8 mmol). The reaction was stirred at room temperature for 72 hours, then diluted with dichloromethane, washed with brine, dried over sodium sulfate, and evaporated. Purification by flash chromatography (basic alumina, 3% methanol: dichloromethane) provided M8a. MS (ESI+) m / z 317.1 (M+H).

[1291] M8. (4-Acetyl-1-(2'-((4"-methoxybenzyl)amino)-2'-oxoethyl)piperazin-2-yl)methyl methyl carbonate

[1292]

[1293] At 0°C, under argon, a solution of M8a (0.09 g, 0.28 mmol) in dichloromethane (5 mL) was treated dropwise with trifluoroacetic acid (2.0 mL) and stirred at room temperature for 4 hours, then evaporated. The material was dissolved in acetonitrile (10 mL) and kept at 0°C, under argon, then treated with potassium carbonate (0.23 g, 1.66 mmol) and 2-bromo-N-(4-methoxybenzyl)acetamide (0.128 g, 0.49 mmol). The reaction was stirred at room temperature for 12 hours, then evaporated. The crude mixture was extracted with dichloromethane, the extract was washed with brine, dried over sodium sulfate and evaporated. Purification by flash chromatography (basic alumina, 5% methanol: dichloromethane) followed by preparative HPLC (column: Zorbax Eclipse XDB C18 (150×21.5 mm*5 mm); mobile phase: A=water, B=acetonitrile; gradient: 0-2 min: 10% B; 2-10 min: 20% B; 10 min to end: 50% B;) provided M8. LCMS (Method 7): retention time=0.24 min; MS (ESI+) m / z 394.2 (M+H).

[1294] M9. 1-(4'-Acetyl-1'-(2"-((4"'-methoxybenzyl)amino)-2"-oxoethyl)piperazin-2'-yl)methyl N-methylcarbamate

[1295]

[1296] M9a. 1-(4'-Acetyl-1'-(tert-butoxycarbonyl)piperazin-2'-yl)methyl N-methylcarbamate

[1297]

[1298] A solution of M6b (0.30 g, 1.16 mmol) in dichloromethane (5 mL) was treated dropwise with triethylamine (0.30 mg, 2.90 mmol) at 0 °C under argon and stirred for 10 minutes. Methylcarbamoyl chloride (0.11 g, 1.27 mmol) was added and the reaction was stirred at room temperature for 72 hours. The reaction was diluted with dichloromethane, washed with brine, dried over sodium sulfate and evaporated. Purification by flash chromatography (basic alumina, 3% methanol:ethyl acetate) provided M9a. MS (ESI+) m / z 316.1 (M+H).

[1299] M9. 1-(4'-Acetyl-1'-(2"-((4"'-methoxybenzyl)amino)-2"-oxoethyl)piperazin-2'-yl)methyl N-methylcarbamate

[1300]

[1301] A solution of M9a (0.12 g, 0.38 mmol) in dichloromethane (5 mL) at 0°C under argon was treated dropwise with trifluoroacetic acid (2.0 mL) and stirred at room temperature for 4 hours, then evaporated and used without further purification. MS (ESI+) m / z 216.1 (M+H). The material was dissolved in acetonitrile (10 mL) and kept at 0°C under argon, then treated with potassium carbonate (0.385 g, 2.78 mmol) and 2-bromo-N-(4-methoxybenzyl)acetamide (0.215 g, 0.83 mmol). The reaction was stirred at room temperature for 12 hours, then evaporated. The crude mixture was extracted with dichloromethane, the extracts were washed with brine, dried over sodium sulfate and evaporated. Flash chromatography (basic alumina, 5% methanol: dichloromethane) followed by preparative HPLC (column: Zorbax Eclipse XDB C18 (150×21.5 mm*5 mm); mobile phase: A=water, B=acetonitrile; gradient: 0-2 min: 10% B; 2-8 min: 20% B; 8 min to end: 50% B) provided M9. LCMS (Method 3): retention time=2.61 min; MS (ESI+) m / z 393.2 (M+H).

[1302] M10. 2-(4'-acetyl-2'-(((tetrahydro-2"H-pyran-2"-yl)oxy)methyl)piperazin-1'-yl)-N-(4"'-methoxybenzyl)acetamide

[1303]

[1304] M10a. 2-(4'-Acetyl-2'-(hydroxymethyl)piperazin-1'-yl)-N-(4"-methoxybenzyl)acetamide

[1305]

[1306] A solution of M6a (1.0 g, 6.32 mmol) in acetonitrile (20 mL) was maintained at 0°C under argon and then treated with potassium carbonate (2.62 g, 19.0 mmol) and 2-bromo-N-(4'-methoxybenzyl)acetamide (1.4 g, 5.68 mmol). The reaction was stirred at room temperature for 16 hours and then treated with water, extracted with ethyl acetate, and the combined organic layers were dried over sodium sulfate and evaporated. Purification by flash chromatography (SiO2, 1%-2% methanol: dichloromethane) provided M10a. LCMS (Method 8): Retention time = 2.61 min; MS (ESI+) m / z 336.3 (M+H).

[1307] M10. 2-(4'-acetyl-2'-(((tetrahydro-2"H-pyran-2"-yl)oxy)methyl)piperazin-1'-yl)-N-(4"'-methoxybenzyl)acetamide

[1308]

[1309] A solution of M10a (0.10 g, 0.30 mmol) in dichloromethane (5 mL) was treated with 3,4-dihydro-2H-pyran (0.05 g, 0.60 mmol) at 0°C under argon, followed by the addition of p-toluenesulfonic acid (0.030 g, 0.89 mmol) in portions, and the reaction was stirred at room temperature for 4 hours. The reaction was treated with saturated aqueous sodium bicarbonate and extracted with dichloromethane, the extracts were washed with brine, dried over sodium sulfate and evaporated. Purification by flash chromatography (SiO2, 1%-2% methanol: dichloromethane) provided M10. LCMS (Method 8): Retention time = 2.78 min; MS (ESI+) m / z 420.3 (M+H).

[1310] M11. 2-(4'-Acetyl-2'-(((tert-butyldimethylsilyl)oxy)methyl)piperazin-1'-yl)-N-(4"'-methoxybenzyl)acetamide

[1311]

[1312] M11. 2-(4'-Acetyl-2'-(((tert-butyldimethylsilyl)oxy)methyl)piperazin-1'-yl)-N-(4"'-methoxybenzyl)acetamide

[1313]

[1314] A solution of M10a (0.10 g, 0.30 mmol) in dichloromethane (5 mL) was treated with imidazole (0.061 g, 0.89 mmol) at 0°C under argon, followed by dropwise addition of tert-butyldimethylsilyl chloride (0.054 g, 0.36 mmol), and the reaction was stirred at room temperature for 2 hours. The reaction was treated with saturated aqueous ammonium chloride and extracted with dichloromethane, the extracts were washed with brine, dried over sodium sulfate and evaporated. Purification by preparative HPLC (column: Zorbax Eclipse XDB C18 (150×21.5 mm*5 mm); mobile phase: A=water, B=acetonitrile; gradient: 0-2 min: 30% B; 2-8 min: 40% B; 8 min to end: 90% B;) provided M11. LCMS (Method 8): retention time = 3.48 min; MS (ESI+) m / z 450.3 (M+H).

[1315] M17. 1-(4'-Acetyl-1'-(2"-oxo-2"-(pyridin-2"'-ylamino)ethyl)piperazin-2'-yl)methyl acetate

[1316]

[1317] M17a 1-(4'-Acetylpiperazin-2'-yl)methyl acetate, trifluoroacetate

[1318]

[1319] To a solution of tert-butyl 2-(hydroxymethyl)piperazine-1-carboxylate (0.70 g, 3.23 mmol) in DMF (7 mL) at 0°C under argon was added diisopropylethylamine (1.65 mL, 2.76 mmol), 4-dimethylaminopyridine (0.007 g) and acetyl chloride (0.57 mL, 8.09 mmol) and stirred at room temperature for 16 hours. The reaction was treated with water and extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over sodium sulfate and evaporated. Purification by flash chromatography (basic alumina, 50% ethyl acetate: hexanes) provided an intermediate compound that was used without further purification. MS (ESI+) m / z 323 (M+Na). The material was dissolved in dichloromethane (2 mL) at 0°C and treated with trifluoroacetic acid (1 mL) and stirred at room temperature for 16 hours, then evaporated. The residue was washed with pentane and dried under high vacuum to afford M17a as the trifluoroacetate salt which was used without further purification. MS (ESI+) m / z 201 (M+H).

[1320] M17b. Benzyl 2-(2'-(acetoxymethyl)-4'-acetylpiperazin-1'-yl) acetate

[1321]

[1322] Under argon, a solution of M17a (0.75 g, 3.73 mmol) in acetonitrile (8 mL) was treated with potassium carbonate (1.54 g, 11.9 mmol) and benzyl 2-bromoacetate (0.59 mL, 3.73 mmol) and stirred at room temperature for 16 hours. The reaction was concentrated and purified by flash chromatography (basic alumina, 1% methanol: dichloromethane) to provide M17b. MS (ESI+) m / z 349 (M+H).

[1323] M17c. 2-(2'-(Acetoxymethyl)-4'-acetylpiperazin-1'-yl)acetic acid

[1324]

[1325] To a solution of M17b (0.30 g, 0.86 mmol) in ethanol (6 mL) was added 5% palladium on carbon (0.060 g), and the reaction was stirred under hydrogen balloon pressure for 16 hours. The reaction was concentrated and purified by preparative HPLC (column: Zorbax EclipseXDB C18 (150×21.5 mm*5 mm); mobile phase: A=water, B=acetonitrile; gradient: 0-2 minutes: 10% B; 2-10 minutes: 20% B; 10 minutes to the end: 40% B) to provide M17c. MS (ESI+) m / z 259 (M+H).

[1326] M17. 1-(4'-Acetyl-1'-(2"-oxo-2"-(pyridin-2"'-ylamino)ethyl)piperazin-2'-yl)methyl acetate

[1327]

[1328] To a solution of M17c (0.070 g, 0.27 mmol) in dichloromethane (1 mL) was added diisopropylethylamine (0.039 mL, 0.81 mmol), HATU (0.35 g, 0.90 mmol) and 2-aminopyridine (0.028 g, 0.29 mmol) under argon and stirred at room temperature for 16 hours. The reaction was treated with water and extracted with dichloromethane. The combined organic layers were washed with brine and evaporated. Purification by preparative HPLC (column: Zorbax Eclipse XDB C18 (150×21.5 mm*5 mm); mobile phase: A=water, B=acetonitrile; gradient: 0-2 min: 10% B; 2-6 min: 20% B; 6 min to end: 70% B) provided M17. LCMS (Method 3): retention time=2.35 min; MS (ESI+) m / z 335.2 (M+H).

[1329] M18. 1-(4'-methyl-1'-(2"-oxo-2"-(pyridin-2"'-ylamino)ethyl)piperazin-2'-yl)methyl acetate

[1330]

[1331] M18a. 3-(((tert-Butyldimethylsilyl)oxy)methyl)-1-methylpiperazine

[1332]

[1333] To a solution of (4-methylpiperazin-2-yl)methanol (0.60 g, 4.6 mmol) in dichloromethane (7 mL) was added triethylamine (1.08 mL, 7.82 mmol), 4-dimethylaminopyridine (0.01 g) and then a solution of tert-butyldimethylsilyl chloride (1 g, 6.91 mmol) in dichloromethane (20 mL) dropwise under argon and stirred at room temperature for 16 hours. Additional triethylamine (0.6 eq) and tert-butyldimethylsilyl chloride (0.6 eq) were added to the solution and the solution was stirred at room temperature for 2 hours. The reaction was treated with water and extracted with dichloromethane. The combined organic layers were dried over sodium sulfate and evaporated. Purification by flash chromatography (basic alumina, 1% methanol: dichloromethane) provided M18a. MS (ESI+) m / z 245 (M+H).

[1334] M18c. Benzyl 2-(2'-(acetoxymethyl)-4'-methylpiperazin-1'-yl) acetate

[1335]

[1336] Under argon, a solution of M18a (1.2 g, 4.91 mmol) in acetonitrile (12 mL) was treated with potassium carbonate (1.69 g, 12.3 mmol) and benzyl 2-bromoacetate (0.7 mL, 4.42 mmol) and stirred at room temperature for 16 hours. The reaction was treated with water and extracted with ethyl acetate. The combined organic layers were washed with water, dried over sodium sulfate and concentrated. Purification by flash chromatography (basic alumina, 1% methanol: dichloromethane) provided intermediate M18b. To a solution of M18b (0.49 g, 1.25 mmol) in degassed (with argon) dichloromethane (12 mL) was added copper (II) trifluoromethanesulfonate (0.45 g, 1.25 mmol). The mixture was degassed again (10 minutes), followed by the addition of acetic anhydride (0.31 g, 3.0 mmol), degassed a third time (10 min), and stirred at room temperature under argon for 48 hours. The reaction was treated with saturated aqueous sodium bicarbonate and extracted with dichloromethane. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated. Purification by flash chromatography (neutral alumina, 0.25% methanol: dichloromethane) provided M18c. MS (ESI+) m / z 321 (M+H)

[1337] M18d. 2-(2'-(Acetoxymethyl)-4'-methylpiperazin-1'-yl)acetic acid

[1338]

[1339] To a solution of M18c (0.225 g, 0.70 mmol) in ethanol (5 mL) was added 5% palladium on carbon (0.045 g) and the reaction was stirred under hydrogen balloon pressure for 5 hours. The reaction was filtered through celite, the filtrate was dried over sodium sulfate and concentrated to provide M18d, which was used without further purification. MS (ESI+) m / z 231.2 (M+H)

[1340] M18. 1-(4'-methyl-1'-(2"-oxo-2"-(pyridin-2"'-ylamino)ethyl)piperazin-2'-yl)methyl acetate

[1341]

[1342] To a solution of M18d (0.060 g, 0.26 mmol) in dichloromethane (5 mL) at 0°C under argon was added diisopropylethylamine (0.10 g, 0.78 mmol) and HATU (0.146 g, 0.39 mmol). The solution was stirred for 10 minutes, then 2-aminopyridine (0.029 g, 0.31 mmol) was added. The reaction was stirred at room temperature for 16 hours, then treated with water and extracted with dichloromethane. The combined organic layers were washed with brine, dried over sodium sulfate and evaporated. Purification by flash chromatography (SiO2, 10%-12% methanol: dichloromethane) followed by preparative HPLC (column: XBridge (150×19 mm); mobile phase: A=(0.1% TFA in water), B=(CAN:methanol); gradient: 0-2 min: 10% B; 2-8 min: 20% B; 8 min to end: 50% B) provided M18. LCMS (Method 8): retention time = 2.12 min; MS (ESI+) m / z 307.1 (M+H).

[1343] M19. 1-(4'-ethyl-1'-(2"-oxo-2"-(pyridin-2"'-ylamino)ethyl)piperidin-2'-yl)methyl acetate

[1344]

[1345]

[1346] M19b.1-(4'-Ethylpiperidin-2'-yl)methanol

[1347]

[1348] To a solution of 1-(4'-bromopyridin-2'-yl)methanol (2 g, 10.6 mmol) in degassed toluene (20 mL) was added tributyl vinyl tin (4.44 g, 16.0 mmol) and PdCl2(PPh3)2 (0.746 g, 1.06 mmol) followed by a second 10 minute degassing. The reaction was stirred at 50°C under argon for 18 hours and then treated with additional portions of butyl vinyl tin (0.5 eq) and PdCl2(PPh3)2 (0.05 eq) followed by stirring at 80°C for 18 hours. The reaction was evaporated. Purification by flash chromatography (SiO2, 30% ethyl acetate: hexanes) provided intermediate M19a. A mixture of M19a (1.4 g, 10.4 mmol), platinum oxide (0.941 g, 4.15 mmol) and acetic acid (0.622 g, 10.4 mmol) in methanol (54 mL) and water (36 mL) was placed in a Parr shaker under 80 psi hydrogen pressure for 36 hours. The reaction was stirred for 2 hours. Filtered and concentrated. The residue was treated with water and extracted with ethyl acetate. The aqueous layer was concentrated to provide M19b, which was used without further purification. MS (ESI+) m / z 144 (M+H).

[1349] M19c. 2-(((tert-Butyldimethylsilyl)oxy)methyl)-4-ethylpiperidine

[1350]

[1351] To a solution of M19b (0.4 g, 2.79 mmol) in dichloromethane (8 mL) was added triethylamine (0.85 g, 8.4 mmol), 4-dimethylaminopyridine (0.01 g), and then a solution of tert-butyldimethylsilyl chloride (1.26 g, 8.4 mmol) in dichloromethane (4 mL) was added dropwise and stirred at room temperature for 16 hours. The reaction was treated with water and extracted with dichloromethane. The combined organic layers were dried over sodium sulfate and evaporated. Purification by flash chromatography (neutral alumina, 10% ethyl acetate: hexanes) provided M19c. MS (ESI+) m / z 258.6 (M+H). M19e. Benzyl 2-(2'-(acetoxymethyl)-4'-ethylpiperidin-1'-yl)acetate

[1352]

[1353] A solution of M19c (0.50 g, 1.94 mmol) and potassium carbonate (0.67 g, 4.86 mmol) in acetonitrile (10 mL) was treated dropwise with benzyl 2-bromoacetate (0.53 g, 2.33 mmol) at 0°C under argon and stirred at room temperature for 6 hours. The reaction was treated with saturated aqueous ammonium chloride and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated. Purification by flash chromatography (SiO2, 10% ethyl acetate: hexane) provided intermediate M19d. To a solution of M19d (0.30 g, 0.74 mmol) in degassed (with argon) dichloromethane (12 mL) was added copper (II) trifluoromethanesulfonate (0.267 g, 0.74 mmol). The mixture was degassed again (10 minutes), followed by the addition of acetic anhydride (0.18 g, 1.78 mmol), degassed a third time (10 minutes), and stirred at room temperature under argon for 24 hours. The reaction was treated with saturated aqueous sodium bicarbonate and extracted with dichloromethane. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated. Purification by flash chromatography (SiO2, 15% ethyl acetate: hexanes) provided M19e. MS (ESI+) m / z 334.0 (M+H).

[1354] M19f.2-(2'-(Acetoxymethyl)-4'-ethylpiperidin-1'-yl)acetic acid

[1355]

[1356] To a solution of M19e (0.40 g, 1.2 mmol) in ethanol (8 mL) was added 5% palladium on carbon (0.08 g) and the reaction was stirred under hydrogen balloon pressure for 5 h. Filtered. The filtrate was dried over sodium sulfate and concentrated to provide M19f, which was used without further purification. MS (ESI+) m / z 244.1 (M+H).

[1357] M19. 1-(4'-ethyl-1'-(2"-oxo-2"-(pyridin-2"'-ylamino)ethyl)piperidin-2"-yl)methyl acetate

[1358]

[1359] To a solution of M19f (0.10 g, 0.41 mmol) in dichloromethane (5 mL) at 0°C under argon was added diisopropylethylamine (0.16 g, 1.23 mmol) and HATU (0.187 g, 0.39 mmol). The solution was stirred for 15 minutes, then 2-aminopyridine (0.046 g, 0.49 mmol) was added. The reaction was stirred at room temperature for 16 hours, then treated with water and extracted with dichloromethane. The combined organic layers were washed with brine, dried over sodium sulfate and evaporated. Purification by flash chromatography (SiO2, 25%-30% ethyl acetate:hexanes) provided M19. LCMS (Method 8): Retention time = 2.58 min; MS (ESI+) m / z 320.5 (M+H).

[1360] M20. Benzyl N-(3-(3'-(((diethyl(isopropoxy)silyl)oxy)methyl)-4'-(2"-((4"'-methoxybenzyl)amino)-2"-oxoethyl)piperazin-1'-yl)-3-oxopropyl)carbamate

[1361]

[1362] M20a. Benzyl (3-(3-(hydroxymethyl)-4-(2-((4-methoxybenzyl)amino)-2-oxoethyl)piperazin-1-yl)-3-oxopropyl)carbamate

[1363]

[1364] L-INT-1b (342 mg, 1.065 mmol) was dissolved in acetonitrile (5 mL) and cooled to 0 ° C. The reaction was treated with potassium carbonate (402 mg, 2.91 mmol) and 2-bromo-N-(4-methoxybenzyl)acetamide (250 mg, 0.969 mmol) and stirred at room temperature overnight. The reaction mixture was treated with water and extracted with ethyl acetate. The organic phase was washed with brine, dried (sodium sulfate) and evaporated. Purification by flash chromatography (SiO2, eluted with 0-10% methanol: dichloromethane) provided M20a. LCMS (Method 1): Retention time = 0.91 min; MS (ESI+) m / z 499.4 (M+H).

[1365] M20b. Benzyl N-(3-(3'-(((chlorodiethylsilyl)oxy)methyl)-4'-(2"-((4"'-methoxybenzyl)amino)-2"-oxoethyl)piperazin-1'-yl)-3-oxopropyl)carbamate

[1366]

[1367] M20a (40 mg, 0.080 mmol) and imidazole (273 mg, 4.01 mmol) were dissolved in tetrahydrofuran (2 mL) at room temperature and added dropwise to a solution of dichlorodiethylsilane (0.120 mL, 0.802 mmol) in dimethylformamide (1 mL) and tetrahydrofuran (1 mL) at about 50° C. over several minutes. The resulting solution was removed from the cold bath and stirred at room temperature for 1 hour to provide M20b, which was used without further purification. LCMS (Method 5): Retention time = 1.13 min; MS (ESI+) m / z 601.3 (M+H of hydrolyzed M20b).

[1368] M20. Benzyl N-(3-(3'-(((diethyl(isopropoxy)silyl)oxy)methyl)-4'-(2"-((4"'-methoxybenzyl)amino)-2"-oxoethyl)piperazin-1-yl)-3-oxopropyl)carbamate

[1369]

[1370] At room temperature, 2-propanol (0.061 mL, 0.802 mmol) was added dropwise to a solution of the crude M20b prepared above in DMF / THF. The solution was stirred at room temperature for 2 hours and then concentrated. Purification was performed by preparative HPLC (column: Waters XBridge 5 μm 30×50 mm; method: 3.5 minute gradient, 75 mL / min, 45% to 70% acetonitrile / H2O (containing 10 mM NH4OH)) to yield fractions, which were concentrated, flash frozen and lyophilized to provide M20. LCMS (method 5): retention time = 1.35 min; MS (ESI+) m / z 643.3 (M+H). 1 H-NMR (CD3OD, ppm): δ: 7.25-7.39 (m, 5H), 7.22 (d, J = 8.7Hz, 2H), 6.85-6.90 (m, 2H), 5 .06(d,J=1.3Hz,2H),4.27-4.42(m,2H),4.08-4.12(m,1H),3.70-3.84(m,5H),3.61- 3.66(m,1H),3.44-3.55(m,1H),3.34-3.43(m,4H),3.03-3.19(m,2H),2.74-2.85(m, 1H), 2.39-2.66 (m, 4H), 1.15 (d, J = 6.0Hz, 6H), 0.92-0.98 (m, 6H), 0.52-0.63 (m, 4H).

[1371] The species in Table 23 were prepared using a method similar to that used to synthesize M20:

[1372]

[1373]

[1374]

[1375]

[1376]

[1377]

[1378]

[1379] Example 8

[1380] In vitro drug release studies

[1381] 8-1. Release of brolucizumab from hydrogel-drug conjugates

[1382]

[1383] wherein Y is selected from Table 2 above.

[1384] like Figure 4A As shown, release of brocizumab from hydrogel conjugates C1a, C2a, C3a and C4a occurred. In this study, a series of hydrogel-brocizumab-conjugates were maintained in 1× PBS at 37°C to evaluate drug release after a traceless linker cleavage reaction. These reactions were sampled at various time points and analyzed to determine the concentration of released brocizumab in the supernatant.

[1385] Each hydrogel (approximately 50 μL; exact amount determined by weighing, assuming hydrogel density = 1 g / mL) was added to Inserts (Corning, Inc; polycarbonate membrane, 10 μm thickness, 6.5 mm diameter, 0.4 μm pore size). The wells of the plate were filled with 1 mL of 1×PBS and the inserts containing the hydrogels were placed in the wells. Three wells were prepared for each hydrogel sample. The plate was securely capped and maintained at 37° C. in a humid environment.

[1386] Samples of the collection buffer were removed from the wells at various time points up to 180 days. At each time point and for each well, The inserts were removed from the wells and the entire volume of the collection buffer was transferred to a clean tube and weighed, then the time point samples were removed from the collection buffer (0.10 mL) and stored at 4°C until final analysis. The collection buffer was supplemented with 1× PBS to return the collection buffer volume to 1 mL, which was then returned to the wells. The inserts were then returned to their wells and the plate was returned to the incubator.

[1387] After the study was completed, the protein was measured using a protein assay kit (DC TM Protein Assay Kit II, Bio-Rad 5000112) was used to measure the protein concentration of the time point samples (representing the protein concentration in each well at that time point), where a standard curve was generated from brocizumab. Each time point sample was analyzed in duplicate, and the results were averaged. The total amount of protein in a given well at a given time point was calculated by multiplying the protein concentration at that time point by the measured volume (assuming a density of 1 g / mL). The amount of protein removed from a given well at a given time point was calculated by multiplying the protein concentration at that time point by the volume removed (0.10 mL). The cumulative brocizumab released from each hydrogel into each well until that time point was calculated by adding the total amount of brocizumab in the well at each time point to the cumulative amount of brocizumab removed from the well at each time point before that time point. Until a given time point, the cumulative percentage of protein released from conjugates C1a, C2a, C3a and C4a is shown in Figure 4B The cumulative percentage of brolucizumab released was calculated by dividing the cumulative brolucizumab released at a given time point by the total brolucizumab present in the initial hydrogel sample and multiplying by 100. The data were normalized by setting the measured amount of released drug that had reached a plateau value as 100%. Figure 4B It is evident that the release of brocuzumab from the carrier lasts for more than several months and that the release rate is affected by the structure of the traceless linker used in the conjugate.

[1388] At time point t, the concentration of protein in the well is:

[1389] [P](t)

[1390] At time point t, the total volume in the well is:

[1391] V(t)

[1392] Total protein in the well at time point t:

[1393] P 孔 (t)=[P](t)×V(t)

[1394] At time point t, the protein removed from the well is:

[1395] R(t)=[P](t)×0.1mL

[1396] Cumulative protein released from the hydrogel until time point t:

[1397]

[1398] 8-2. Release of D2 from hydrogel-drug conjugate

[1399] At 6.5mm In vitro release assays were performed in Transwell inserts (8 μm pore size, Corning). 100 μL aliquots of drug-linked HA hydrogels were added to each Transwell insert along with 100 μL bovine synovial fluid (BSF, from Bioreclamation IVT, supplemented with the following antimicrobials: 100 U / mL penicillin, 100 μg / mL streptomycin (Life Technologies), and 1% amphotericin B (Life Technologies)) or 100 μL PBS. The total initial volume of gel and release medium in the insert was 200 μL. 900 μL of BSF (plus antimicrobial) or PBS was pipetted into the wells of the Transwell plate, the inserts containing the hydrogels were placed in the wells, and the plate was incubated in a humidified incubator at 37°C, 5% CO2 (two inserts were prepared for each hydrogel sample). At each time point, the Transwell inserts were transferred to new wells filled with 1000 μL of fresh BSF (plus antimicrobial) or PBS, and the contents of the old wells were removed and stored at -80°C until assayed.

[1400] Determine the quantification of the D2 released by sandwich ELISA. This scheme is similar to the scheme described in the human IGF-1 DuoSet Elisa development system (R&D Systems), but has the following modifications: for the preparation of the plate, the plate of 96-well format (Costar) is coated with the capture antibody (Novartis) diluted in BupH carbonate-bicarbonate buffer Pack (Thermoscientific), and incubated overnight at 4°C. Except BSA (Sigma), washing buffer KPL (Kirkegaard & Perry Laboratory) and blocking solution KPL milk diluent / blocking agent (Kirkegaard & Perry Laboratory), all other reagents are from R&D Systems. The detection antibody was incubated for 1.5 hours. During incubation with PBS or BSF, the cumulative amount of protein released from hydrogel C10, C11 and C12 is shown in Figure 5. The amount of protein released in the wells at a given time point was calculated by multiplying the protein concentration in the release medium (BSF or PBS) at that time point by the total volume of the release medium (1.1 mL). The cumulative protein released from each hydrogel until a given time point was calculated by adding the amount of protein released at that time point (average from two wells for each hydrogel) to the cumulative amount of protein released at each time point before that time point. As is apparent from Figure 5, the release of D2 from the carrier lasted for more than several weeks, and the release rate was affected by the structure of the traceless linker used in the conjugate.

[1401] D2 is a drug that causes IGF1 receptor phosphorylation in NIH3T3 cells that overexpress the human IGF-1 receptor. When NIH3T3 cells that overexpress the human IGF-1 receptor are incubated with drug-containing release media from various time points (collected as described above), the biological activi...

Claims

1. A drug delivery system comprising DR represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D: wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring; R 1 is hydrogen or C1-C4 alkyl; R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The group and the one or more carbon atoms to which it is attached combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group; a is 0, 1, 2, 3, or 4; R 3 is hydrogen or C1-C4 alkyl; R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl; Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ; R 12 is hydrogen or C1-C4 alkyl; R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 13 is C1-C4 alkyl; or R 12 and R 13 With C(R 12a ) and O are combined to form a 5-, 6- or 7-membered cyclic ether; R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy; R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl; R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups; b is an integer from 1 to 10; Z is CH-LA, CH-A, NLA or NA; L is an optionally substituted divalent linker; A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace; R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and R 11 It is a carrier.

2. drug delivery system as claimed in claim 1 or pharmaceutically acceptable salt, wherein D comprises protein, peptide, polypeptide, nucleic acid, oligonucleotide or polynucleotide, carbohydrate, oligosaccharide or polysaccharide or small molecule, it each comprises at least one primary amine, secondary amine or comprises the ring nitrogen atom of azaheteroaryl ring, and this small molecule has the molecular weight between 100g / mol and about 2000g / mol.

3. The drug delivery system or pharmaceutically acceptable salt of claim 1 or 2, wherein D comprises a protein, a peptide, a nucleic acid, a Fab, a scFv, a monoclonal antibody or a small molecule.

4. The drug delivery system or pharmaceutically acceptable salt of any one of claims 1 to 3, wherein D comprises brocuzumab (SEQ ID NO: 4), D2 (SEQ ID NO: 5), D3 (SEQ ID NO: 6), D4 (SEQ ID NO:7), abciximab, adalimumab, aflibercept, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab, canakinumab, certolizumab pegol, cetuximab, daclizumab, daratumumab, denosumab, eculizumab, efalizumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lambalizumab, muromonab-CD3, natalizumab, nivolumab, ofatumumab, omalizumab, palivizumab, panitumumab, peprazib, pembrolizumab, ranibizumab, rituximab, tocilizumab, tositumomab, trastuzumab, ustekinumab, or vedolizumab or a combination thereof.

5. The drug delivery system or pharmaceutically acceptable salt of any one of claims 1 to 3, wherein D comprises one or more bioactive peptides, including but not limited to C-type natriuretic peptide (CNP), atrial natriuretic peptide (ANP), exenatide-4, insulin, adrenocorticotropic hormone, adrenomedullin, atropine, ω-funnel web spider toxin, agouti protein-related protein, one or more angiotensin, apelin 12, apelin 13, apelin 36, or a derivative thereof, bradykinin, calcitonin and amphetamine-regulated transcript (CART), corticotropin releasing factor (CRF), α-defensin, β-defensin, δ-sleep-inducing peptide ( DSIP), Elafin, Endokinin, Endomorphin, Endophytin, Endothelin, Exenatide, Fibronectin active fragment, Galanin, Galanin-like peptide, Gastrin, Gastrin I, Gastrin-related peptide, Gastric inhibitory polypeptide, Gastrin-releasing peptide, Ghrelin, Desacyl ghrelin, Glucagon, Glucagon-like peptide, Growth hormone releasing factor, Chilean bird spider GsMTx-4, Guangxi toxin-1E, Guanylin, Hepatic bactericidal peptide 1, Hepatic bactericidal peptide / LEAP-1, Histamine 5, Human growth hormone, Human peptide, Tiger bird spider toxin, Iberian scorpion toxin, Emperor scorpion toxin A, Insulin-like growth factor, Pituitin, IRL1620, connecting peptide, short-skinned scorpion toxin, base silk peptide, Kut toxin, lipolysis, lipolysis, hepatocyte growth factor, liver expressed antimicrobial peptide 2, luteinizing hormone releasing hormone, lysin, LL-37, Marg toxin, mast degranulation peptide, mast cell degranulation peptide, melanin concentrating hormone, melanocyte stimulating hormone, MSH release inhibitory factor, midkine, mollusk cardiac excitatory neuropeptide, morphine tolerance peptide, motilin, muscarinic toxin, neuroendocrine regulatory peptide-2, neuro Kinin A, neurokinin B, neuromedin, neurostatin-13, neuropeptide, neurotensin, neurotoxin NSTX-3, nociceptin, nociceptin, obestatin, opioid peptides (enkephalin, endorphin, BAM-12P, casomorphin, dynorphin, endomorphin, neoendorphin, nociceptin), orexin-A / -B, orphan peptide, osteocalcin, oxytocin, pancreatic inhibin, parathyroid hormone, parathyroid hormone-related protein, peptide 234, peptide histidine-methionine, peptide T, peptide YY, bubble frog Peptide, pituitary adenylate cyclase activating polypeptide, platelet factor-4, mycoplasmin, pleiotrophic factor, proadrenomedullin, prolactin-releasing peptide, wolf spider toxin 1, prototoxin-1, pyroglutamylated RFamide peptide, renin, RFamide-related peptide-1, RFamide-related peptide-3, sarusin-α / β, viper toxin, schizophrenia-related peptide, blue crab toxin, secretin, relaxin, serum thymic factor, sodium potassium ATPase inhibitor-1, somatostatin, carpet anemone toxin, substance K, apnopressin, apnopressin-related peptide, substance P, tachykinin, tarantula SNX-482, tarantula ProTx-I, tarantula ProTx-II, tropeptide, neodymium scorpion toxin Ka, thyrotropin-releasing hormone, phagocytosis, uroguanylin, uroguanylin isomer A or B, urocortin, urocortin II, uropressin II, uropressin II-related peptide, vasoactive intestinal peptide, vasopressin, oxytocin, viral replication inhibitory peptide, clawed toad peptide, or a combination thereof.

6. as the compound described in any one in claim 1 to 3, wherein D is to have molecular weight between about 100g / mol to about 2000g / mol or smaller small molecule, and it is connected with R through the ring nitrogen atom of primary amine, secondary amine or aza-heteroaryl ring.

7. The drug delivery system or pharmaceutically acceptable salt of claim 6, wherein the small molecule comprises 5-(5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; 5-(6,7-dihydro-5H-pyrrolo[3,4d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide. [00136] Indole-3-yl]-amide; N-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6-((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide; (S)-5-(6-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yloxy)-N-(5-(1-methylcyclopropyl)-1H-pyrazol-3-yl)-1H-indole-1-carboxamide, or a combination thereof.

8. The drug delivery system or pharmaceutically acceptable salt according to any one of claims 1 to 7, wherein R 1 is hydrogen or methyl, R 1a is hydrogen or methyl, or CR 1 R 1a The combination forms a cyclopropane-1,1-diyl group.

9. The drug delivery system or pharmaceutically acceptable salt of any one of claims 1 to 8, wherein variable a is 0.

10. The drug delivery system or pharmaceutically acceptable salt according to any one of claims 1 to 9, wherein R 3 and R 3a Each is hydrogen.

11. The drug delivery system or pharmaceutically acceptable salt of any one of claims 1 to 10, wherein Y is C(O)R 4 , and R 4 It is a C1-C6 alkyl group, a C3-C6 cycloalkyl group or a C1-C2 alkoxy C1-C2 alkyl group.

12. The drug delivery system or pharmaceutically acceptable salt of claim 11, wherein R 4 It is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl or methoxymethyl.

13. The drug delivery system or pharmaceutically acceptable salt of any one of claims 1 to 10, wherein Y is SiR 5 R 6 R 7 ; R 5 and R 6 are each methyl, ethyl, propyl or isopropyl; and R 7 is C1-C4 alkyl, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, 2-ethoxyethoxy, 2-isopropoxy-ethoxy, tetrahydropyranyloxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein b is 2, 3 or 4.

14. The drug delivery system or pharmaceutically acceptable salt of any one of claims 1 to 13, wherein Z is CH-LA, CH-A, NLA or NA; L is an optionally substituted divalent linker Q-[Sp-Q] h -Q; Q is independently selected at each occurrence from a bond, O, C(O), N(H), N(C1-C4 alkyl), C(O)NH, C(O)N(C1-C4 alkyl), N(H)C(O), N(C1-C4 alkyl)C(O), N(H)C(O)O, N(C1-C4 alkyl)C(O)O, OC(O)N(H), OC(O)N(C1-C4 alkyl), N(H)C(O)N(H), N(C1-C4 alkyl)C(O)N(H), N(H)C(O)N(C1-C4 alkyl), N(C1-C4 alkyl)C( C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O), OC(O)O, S, S(O)2, N(H)S(O)2, N(C1-C4 alkyl)S(O)2, S(O)2N(H), S(O)2N(C1-C4 alkyl), C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O)C1-C2 alkyl, 1,2,3-triazole, OP(O)2, P(O)2O, C1-C4 alkyl-P(O)2-O, or OP(O)2-C 1-4 alkyl; Sp is independently selected at each occurrence from optionally substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, [WO] g 、C1-C8 alkyl-[OW] g , [OW] g -O-C1-C8 alkyl, C1-C8C alkyl-[OW] g -O-C1-C8 alkyl or oligopeptide; h is an integer between 1 and 20; g is a weighted average between about 2 and about 50; W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom; A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)OC1-C8 alkyl, C(O)N(H)C1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace; R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and R 11 It is a carrier.

15. The drug delivery system or pharmaceutically acceptable salt of any one of claims 1 to 13, wherein Z is CHR 8 or NR 9 ; R 8 and R 9 Each independently selected from hydrogen, C1-C8 alkyl, C(O)-(CH2) n -QA, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA; q is independently 1, 2, or 3 at each occurrence; g and p each independently have a weighted average length between about 2 and about 50; m is 1 or 0; W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom; Q is a bond, O, N(H) or N(C1-C4 alkyl); A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace; R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and R 11 It is a carrier.

16. The drug delivery system or pharmaceutically acceptable salt of claim 15, wherein Z is NR 9 ; R 9 is C(O)-(CH2) n -QA or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA; n is an integer from 1 to 8; and A is for R 10 or R 11 .

17. The drug delivery system or pharmaceutically acceptable salt according to any one of claims 1 to 16, wherein R 10 is an azido group, an alkynyl group, a substituted or unsubstituted C7-C 12 Cycloalkynyl, substituted or unsubstituted C7-C 12 Heterocycloalkynyl, substituted or unsubstituted C7-C 12 cycloalkenyl, norbornyl, substituted or unsubstituted vinylcarboxyl, substituted or unsubstituted vinylsulfonyl, substituted or unsubstituted C2-C8 alkenyl, amino, thiol, substituted or unsubstituted C1-C8 carboxyl, substituted or unsubstituted C1-C8 carbonyl, -O-NH2, hydrazide, maleimide, α-halogenated carbonyl, furan, substituted or unsubstituted tetrazinyl, lysine, glutamine, cyclodextrin or adamantyl.

18. The drug delivery system or pharmaceutically acceptable salt according to any one of claims 1 to 17, wherein R 10 Contains a reactive functional group suitable for coupling the drug delivery system having formula (I) or a pharmaceutically acceptable salt to a carrier.

19. The drug delivery system or pharmaceutically acceptable salt according to any one of claims 1 to 16, wherein R 11 Is biodegradable.

20. The drug delivery system or pharmaceutically acceptable salt of any one of claims 1 to 16 or 19, wherein R 11 Contains polymers or cross-linked polymers.

21. The drug delivery system or pharmaceutically acceptable salt of claim 19 or 20, wherein R 11 A hydrogel is included that includes one or more cross-linked polymers.

22. The drug delivery system or pharmaceutically acceptable salt of any one of claims 1 to 16 and 19 to 21, wherein R 11 The invention relates to a polymer, a cross-linked polymer, or a hydrogel, wherein the polymer, the cross-linked polymer, or the hydrogel comprises one or more of hyaluronic acid, polyethylene glycol, polypropylene glycol, polyethylene oxide, polypropylene oxide, polyglutamate, polylysine, polysialic acid, polyvinyl alcohol, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl pyrrolidone, polyoxazoline, polyiminocarbonate, polyamino acid, hydrophilic polyester, polyamide, polyurethane, polyurea, dextran, agarose, xylan, mannan, carrageenan, alginate, gelatin, collagen, albumin, cellulose, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, hydroxyethyl starch, chitosan, nucleic acid, derivatives thereof, copolymers thereof, or combinations thereof.

23. A drug delivery system or a pharmaceutically acceptable salt as described in any one of claims 1 to 16 and 19 to 22, wherein R 11 Contains hyaluronic acid, polyethylene glycol, a cross-linked hydrogel of hyaluronic acid, a cross-linked hydrogel of polyethylene glycol, or a combination thereof.

24. A drug delivery system or a pharmaceutically acceptable salt as described in any one of claims 1 to 16 and 19 to 23, wherein R 11 Contains hyaluronic acid or polyethylene glycol.

25. A drug delivery system or a pharmaceutically acceptable salt as described in any one of claims 1 to 16 and 19 to 23, wherein R 11 Contains a hydrogel comprising cross-linked hyaluronic acid or cross-linked polyethylene glycol.

26. drug delivery system or pharmaceutically acceptable salt as claimed in claim 24 or 25, wherein the hyaluronic acid or polyethylene glycol is functionalized by at least one functional group comprising an azido, an alkynyl, a substituted or unsubstituted C7-C 12 Cycloalkynyl, substituted or unsubstituted C7-C 12 Heterocycloalkynyl, C7-C 12 Cycloalkenyl, norbornyl, vinylcarboxyl, vinylsulfonyl, C2-C8 alkenyl, amino, thiol, C1-C8 carboxyl, C1-C8 carbonyl, -O-NH2, carbohydrazide, maleimide, α-halocarbonyl, furan, substituted or unsubstituted tetrazinyl, lysine, glutamine, cyclodextrin, adamantyl or a combination thereof.

27. A drug delivery system or a pharmaceutically acceptable salt as described in any one of claims 1 to 16 and 19 to 26, wherein R 11 A hydrogel comprising cross-linked hyaluronic acid, wherein the hyaluronic acid comprises a cross-linked hyaluronic acid selected from -NH(W1)(O(W1)) d - at least one side chain of V, wherein W1 is a C2-C4 alkyl-1,2-diyl group, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom; d is the number mean with an average value ranging from 0 to 500; and V is an azido group, an alkynyl group, a substituted or unsubstituted C7-C 12 Cycloalkynyl, substituted or unsubstituted C7-C 12 Heterocycloalkynyl, C7-C 12 Suitable functional groups include cycloalkenyl, norbornyl, vinylcarboxyl, vinylsulfonyl, C2-C8 alkenyl, amino, thiol, C1-C8 carboxyl, C1-C8 carbonyl, -O-NH2, carbohydrazide, maleimide, α-halocarbonyl, furan, substituted or unsubstituted tetrazinyl, lysine, glutamine, cyclodextrin or adamantyl.

28. The drug delivery system or pharmaceutically acceptable salt of claim 27, wherein V is an azide.

29. A method for preparing a cross-linked carrier formulation, the method comprising: (a) Make the carrier molecule R 11 Functionalization; (b) preparing a reactive cross-linking agent; as well as (c) reacting the functionalized carrier molecule with the reactive cross-linking agent to form a cross-linked carrier by incubating at a temperature of about 4° C. to about 60° C. for about 0.5 hour to about 48 hours.

30. The method of claim 29, wherein the carrier molecule comprises hyaluronic acid or polyethylene glycol.

31. The method of claim 30, wherein the carrier molecule is functionalized with an azide, thiol, amine, aminooxy (O-NH2) or aldehyde moiety to provide reactive functional groups for cross-linking.

32. The method of any one of claims 29 to 31, wherein the preparation of the reactive cross-linking agent comprises reacting polyethylene glycol with 1-((tert-butoxycarbonyl)amino)cyclopropane-1-carboxylic acid, 3-((tert-butoxycarbonyl)amino)propionic acid, 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid, 2-(1-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)acetic acid, 2-methyl-3-((tert-butoxycarbonyl)amino)propionic acid or 7-((tert-butoxycarbonyl)amino)heptanoic acid, and deprotecting the functionalized polyethylene glycol ester.

33. The method of claim 32, wherein the preparation of the reactive cross-linking agent further comprises introducing at least two bicyclo[6.1.0]non-4-yn-9-yl)methyl groups after deprotection of the functionalized polyethylene glycol ester.

34. A cross-linked hydrogel obtainable using the method of any one of claims 29 to 33.

35. The cross-linked hydrogel of claim 34, wherein the carrier molecule is functionalized with an azide.

36. A method for preparing a drug adduct comprising a traceless linker R coupled to a bioactive agent D, the method comprising: (a) providing a biologically active agent D comprising a primary amine, a secondary amine or a ring nitrogen atom of an azaheteroaryl ring; (b) reacting the bioactive agent with a traceless linker R having an activated carbonyl functional group; and (c) purifying the drug adduct from the reagent.

37. A drug adduct obtainable by the method of claim 36.

38. A method for preparing a drug delivery system, the method comprising: (a) Preparation of carrier molecule R 11 , where R 11 is a cross-linked hydrogel; optionally, step (a) may further comprise purifying the cross-linked hydrogel carrier molecule R 11 ; (b) separately conjugating a traceless linker R to a biologically active agent D comprising a primary amine, a secondary amine, or a ring nitrogen atom of an azaheteroaryl ring, thereby forming a traceless linker-D adduct; step (b) may optionally further comprise purifying the traceless linker-D adduct, (c) The carrier molecule R 11 conjugating with the traceless linker-D adduct; and (d) purifying the drug delivery system from the reagent.

39. A drug delivery system prepared using the method of claim 38.

40. A method for treating macular degeneration, the method comprising administering to a subject in need thereof a drug delivery system comprising a conjugate DR represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring, the biologically active moiety comprising brocuzumab (SEQ ID NO: 4), aflibercept, peprazib, ranibizumab, 5-(5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; 5-(6,7-dihydro-5H-pyrrolo[3,4d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; N-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6-((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide; (S)-5-(6-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yloxy)-N-(5-(1-methylcyclopropyl)-1H-pyrazol-3-yl)-1H-indole-1-carboxamide, or a combination thereof; R is a linker suitable for releasing the biologically active moiety D: wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring; R 1 is hydrogen or C1-C4 alkyl; R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group; a is 0, 1, 2, 3, or 4; R 3 is hydrogen or C1-C4 alkyl; R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl; Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ; R 12 is hydrogen or C1-C4 alkyl; R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 13 is C1-C4 alkyl; or CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers; R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with hydroxy, amino, C1-C4 alkoxy or mono- and di-C1-C4 alkylamino; R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl; R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups; b is an integer from 1 to 10; Z is for CHR 8 or NR 9 ; R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA; q is independently 1, 2, or 3 at each occurrence; g and p each independently have a weighted average length between about 2 and about 50; m is 1 or 0; W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom; Q is a bond, O, N(H) or N(C1-C4 alkyl); A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace; R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and R 11 It is a carrier.

41. A method for treating a musculoskeletal disorder, the method comprising administering to a subject in need thereof a drug delivery system comprising a conjugate DR represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring, the biologically active moiety comprising D2 (SEQ ID NO: 4), D3 (SEQ ID NO: 5), other insulin-like growth factors, or a combination thereof; R is a linker suitable for releasing the biologically active moiety D: wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring; R 1 is hydrogen or C1-C4 alkyl; R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group; a is 0, 1, 2, 3, or 4; R 3 is hydrogen or C1-C4 alkyl; R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl; Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ; R 12 is hydrogen or C1-C4 alkyl; R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 13 is C1-C4 alkyl; or CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers; R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with hydroxy, amino, C1-C4 alkoxy or mono- and di-C1-C4 alkylamino; R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl; R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups; b is an integer from 1 to 10; Z is for CHR 8 or NR 9 ; R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA; q is independently 1, 2, or 3 at each occurrence; g and p each independently have a weighted average length between about 2 and about 50; m is 1 or 0; W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom; Q is a bond, O, N(H) or N(C1-C4 alkyl); A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace; R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and R 11 It is a carrier.

42. A method for treating a cardiac disorder, the method comprising administering to a subject in need thereof a drug delivery system comprising a conjugate DR represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring, the biologically active moiety comprising relaxin or SEQ ID NO: 7; R is a linker suitable for releasing the biologically active moiety D: wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring; R 1 is hydrogen or C1-C4 alkyl; R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group; a is 0, 1, 2, 3, or 4; R 3 is hydrogen or C1-C4 alkyl; R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl; Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ; R 12 is hydrogen or C1-C4 alkyl; R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 13 is C1-C4 alkyl; or CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers; R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with hydroxy, amino, C1-C4 alkoxy or mono- and di-C1-C4 alkylamino; R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl; R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups; b is an integer from 1 to 10; Z is for CHR 8 or NR 9 ; R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA; q is independently 1, 2, or 3 at each occurrence; g and p each independently have a weighted average length between about 2 and about 50; m is 1 or 0; W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom; Q is a bond, O, N(H) or N(C1-C4 alkyl); A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace; R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and R 11 It is a carrier.

43. A method for treating a disease or disorder, the method comprising administering to a subject in need thereof a drug delivery system comprising a conjugate DR represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D: wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring; R 1 is hydrogen or C1-C4 alkyl; R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group; a is 0, 1, 2, 3, or 4; R 3 is hydrogen or C1-C4 alkyl; R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl; Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ; R 12 is hydrogen or C1-C4 alkyl; R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 13 is C1-C4 alkyl; or CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers; R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with hydroxy, amino, C1-C4 alkoxy or mono- and di-C1-C4 alkylamino; R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl; R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups; b is an integer from 1 to 10; Z is for CHR 8 or NR 9 ; R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA; q is independently 1, 2, or 3 at each occurrence; g and p each independently have a weighted average length between about 2 and about 50; m is 1 or 0; W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom; Q is a bond, O, N(H) or N(C1-C4 alkyl); A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace; R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and R 11 It is a carrier.

44. A method for extending the half-life of a biologically active moiety D comprising at least one primary amine, secondary amine, or ring nitrogen atom of an azaheteroaryl ring, the method comprising attaching D to R, represented by formula (I), wherein R is a linker suitable for releasing the biologically active moiety D: wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring; R 1 is hydrogen or C1-C4 alkyl; R 1a is hydrogen or C1-C4 alkyl, or CR 1 R 1a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 2 are independently selected at each occurrence from C1-C4 alkyl or oxo, or two R 2 The groups combine to form a fused C3-C6 cycloalkyl or spiro C3-C6 cycloalkane-1,1-diyl group; a is 0, 1, 2, 3, or 4; R 3 is hydrogen or C1-C4 alkyl; R 3a is hydrogen, C1-C4 alkyl, or CR 3 R 3a Combine to form a C3-C6 cycloalkane-1,1-diyl; Y is C(O)R 4 、C(O)OR 4 、C(O)NHR 4 、C(O)NR 5 R 6 、SiR 5 R 6 R 7 or CR 12 R 12a OR 13 ; R 12 is hydrogen or C1-C4 alkyl; R 12a is hydrogen or C1-C4 alkyl, or CR 12 R 12a Combine to form a C3-C6 cycloalkane-1,1-diyl; R 13 is C1-C4 alkyl; or CHR 12 OR 13 Combine to form 5-, 6- or 7-membered cyclic ethers; R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with hydroxy, amino, C1-C4 alkoxy or mono- and di-C1-C4 alkylamino; R 5 and R 6 Each independently selected from C1-C4 alkyl and C3-C6 cycloalkyl; R 7 is C1-C8 alkyl, C3-C7 cycloalkyl, C1-C8 alkoxy, C3-C7 cycloalkoxy, heterocycloalkoxy or -(OCHR 3 CH2) b O-C1-C4 alkyl, wherein the heterocycloalkoxy is a 4- to 7-membered saturated heterocycle having one ring heteroatom selected from N, O and S, and is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups; b is an integer from 1 to 10; Z is for CHR 8 or NR 9 ; R 8 and R 9 Each is independently selected from hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl or -C(O)(CH2) q [OW] g (NHC(O)) m (CH2) q [OW] p -QA, wherein the alkyl group is optionally substituted with 0 or 1 QA; q is independently 1, 2, or 3 at each occurrence; g and p each independently have a weighted average length between about 2 and about 50; m is 1 or 0; W is a C2-C4 alkyl-1,2-diyl radical, in which hydrogen, methyl or ethyl side chains may be present on any main chain carbon atom; Q is a bond, O, N(H) or N(C1-C4 alkyl); A is hydrogen, C1-C8 alkyl, C(O)C1-C8 alkyl, C(O)N(H)C1-C8 alkyl, C(O)OC1-C8 alkyl, R 10 or R 11 , wherein the alkyl group is optionally replaced by 0 or 1 R 10 replace; R 10 is a reactive functional group suitable for coupling the compound of formula (I) to a carrier; and R 11 It is a carrier.

45. A drug delivery system comprising formula (III): Wherein brocuzumab comprises SEQ ID NO:4; R 4 is CH3-, CH3-O-CH2-, CH3CH2- or (CH3)2CH-; and the carrier comprises hyaluronic acid, polyethylene glycol, a hydrogel thereof, a cross-linked hydrogel thereof, or a combination thereof.

46. ​​A drug delivery system comprising formula (IV): wherein D2 comprises SEQ ID NO:5, D3 comprises SEQ ID NO:6; R 4 is CH3-, CH3-O-CH2-, CH3CH2- or (CH3)2CH-; and the carrier comprises hyaluronic acid, polyethylene glycol, a hydrogel thereof, a cross-linked hydrogel thereof, or a combination thereof.

47. A method comprising DRR 11 The drug delivery system of claim 1, wherein R comprises a traceless linker coupled to a bioactive agent D; D comprises a bioactive moiety comprising at least one primary amine, secondary amine, or ring nitrogen atom comprising an azaheteroaryl ring; and R 11 A plurality of hyaluronic acid polymers are included, each of which comprises a random series of unfunctionalized D-glucuronic acid monomers, one or more D-glucuronic acid monomers functionalized with a cross-linking agent, or one or more D-glucuronic acid monomers functionalized with a drug adduct DR.

48. The method of claim 44, wherein D comprises brolucizumab D1 (SEQ ID NO: 4), D2 (SEQ ID NO: 5), D3 (SEQ ID NO: 6), D4 (SEQ ID NO: 7), celecoxib, aflibercept, peprazib, ranibizumab, 5-(5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide; 5-(6,7-dihydro-5H-pyrrolo[3,4d]pyrimidin-4-yloxy)-indole-1-carboxylic acid [5-(1-methyl-cyclopropyl)-2H-pyrazol-3-yl]-amide ]-amide; N-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6-((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide; (S)-5-(6-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yloxy)-N-(5-(1-methylcyclopropyl)-1H-pyrazol-3-yl)-1H-indole-1-carboxamide, or a combination thereof.

49. A drug delivery system comprising DR represented by formula (XXIV) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

50. A drug delivery system comprising DR represented by formula (XXV) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

51. A drug delivery system comprising DR represented by formula (XXVI) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

52. A drug delivery system comprising DR represented by formula (XXVII) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

53. A drug delivery system comprising DR represented by formula (XXVIII) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

54. A drug delivery system comprising DR represented by formula (XXIX) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D:

55. A drug delivery system represented by formula (XXX) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R 4 is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl or methoxymethyl:

56. A drug delivery system comprising DR represented by formula (XXXI) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D: wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring; Y is C(O)R 4 ; R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy; Z is NLA; L is C(O)CH2CH2NH; A is for R 11 ; R 11 A hydrogel derived from cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH2CH2O)3CH2CH2N3, and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[3-(((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)propanoate].

57. A drug delivery system represented by formula (XXXII) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R 4 is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl or methoxymethyl:

58. A drug delivery system comprising DR represented by formula XXXIII, or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D: wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring; Y is C(O)R 4 ; R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy; Z is NLA; L is C(O)CH2CH2NH; A is for R 11 ; R 11 A hydrogel derived from cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH2CH2O)3CH2CH2N3, and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[1-((((1'R,8'S,9's)-bicyclo[6.1.0]non-4'-yn-9'-yl)methoxy)carbonyl)amino-cyclopropane-1-carboxylate].

59. A drug delivery system represented by formula (XXXIV) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R 4 is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl or methoxymethyl:

60. A drug delivery system comprising DR represented by formula XXXV, or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D: wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring; Y is C(O)R 4 ; R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy; Z is NLA; L is C(O)CH2CH2NH; A is for R 11 ; R 11 A hydrogel derived from cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH2CH2O)3CH2CH2N3, and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[1-((((1'R,8'S,9's)-bicyclo[6.1.0]non-4'-yn-9'-yl)methoxy)carbonyl)piperidine-4-carboxylate].

61. A drug delivery system represented by formula (XXXVI) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R 4 is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl or methoxymethyl:

62. A drug delivery system comprising DR represented by formula XXXVII, or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D: wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring; Y is C(O)R 4 ; R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy; Z is NLA; L is C(O)CH2CH2NH; A is for R 11 ; R 11 A hydrogel derived from cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH2CH2O)3CH2CH2N3, and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[7-(((((1'R,8'S,9's)-bicyclo[6.1.0]non-4'-yn-9'-yl)methoxy)carbonyl)amino)heptanoate].

63. A drug delivery system represented by formula (XXXVIII) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom comprising an azaheteroaryl ring; and R 4 is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl or methoxymethyl:

64. A drug delivery system comprising DR represented by formula (XXXIX) or a pharmaceutically acceptable salt thereof, wherein D is a biologically active moiety comprising at least one primary or secondary amine or a ring nitrogen atom of an azaheteroaryl ring; and R is a linker suitable for releasing the biologically active moiety D: wherein the dashed line represents the ring nitrogen atom attached to the primary amine, secondary amine or azaheteroaryl ring; Y is C(O)R 4 ; R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy; Z is NLA; L is C(O)CH2CH2NH; A is for R 11 ; R 11 A hydrogel derived from cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH2CH2O)3CH2CH2N3, and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[2-methyl-3-(((((1'R,8'S,9's)-bicyclo[6.1.0]non-4'-yn-9'-yl)methoxy)carbonyl)amino)propanoate].

65. The drug delivery system of any one of claims 55 to 64, wherein D is brocuzumab, and R 4 It is CH2CH3.

66. The drug delivery system of any one of claims 55 to 64, wherein D is brocuzumab, and R 4 It's CH(CH3)CH3.

67. A carrier composition or a pharmaceutically acceptable salt thereof, represented by the following formula: in L is an optionally substituted divalent linker Q-[Sp-Q] h -Q; Q is independently selected at each occurrence from a bond, O, C(O), N(H), N(C1-C4 alkyl), C(O)NH, C(O)N(C1-C4 alkyl), N(H)C(O), N(C1-C4 alkyl)C(O), N(H)C(O)O, N(C1-C4 alkyl)C(O)O, OC(O)N(H), OC(O)N(C1-C4 alkyl), N(H)C(O)N(H), N(C1-C4 alkyl)C(O)N(H), N(H)C(O)N(C1-C4 alkyl), N(C1-C4 alkyl)C( C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O), OC(O)O, S, S(O)2, N(H)S(O)2, N(C1-C4 alkyl)S(O)2, S(O)2N(H), S(O)2N(C1-C4 alkyl), C1-C2 alkyl-C(O)N(H), N(H)C(O)C1-C2 alkyl, C1-C2 alkyl-C(O)O, OC(O)C1-C2 alkyl, 1,2,3-triazole, OP(O)2, P(O)2O, C1-C4 alkyl-P(O)2-O, or OP(O)2-C 1-4 alkyl; Sp is independently selected at each occurrence from optionally substituted C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, [WO] g 、C1-C8 alkyl-[OW] g , [OW] g -O-C1-C8 alkyl, C1-C8C alkyl-[OW] g -O-C1-C8 alkyl or oligopeptide; h is an integer between 1 and 20; g is a weighted average between about 2 and about 50; W is a C2-C4 alkyl-1,2-diyl radical in which hydrogen, methyl or ethyl side chains may be present on any backbone carbon atom; and R 4 is C1-C8 alkyl or C3-C7 cycloalkyl, wherein the cycloalkyl is optionally substituted with 0, 1 or 2 independently selected C1-C4 alkyl groups, and wherein the alkyl is optionally substituted with C1-C4 alkoxy.

68. The carrier composition of claim 67, having one of the following formulas:

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