Improved conjugation linkers

Abstract

To provide: β-eliminative linkers suitable for the conjugation of small molecules, peptides and proteins; and compounds comprising the linkers.SOLUTION: The invention provides a hydrogel comprising a linker-drug (L-D) unit represented by the formula in the figure. [In the formula, n is an integer from 0 to 6; R1 is -CN or the like; R2 is H; each R4 independently represents C1-C3 alkyl or the like; Y is absent or -N(R6)CH2-, where R6 represents optionally substituted C1-C6 alkyl or the like; D is a drug; and the wavy line represents a point of connecting the linker-drug unit to a macromolecular carrier.]SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 830,280, filed April 5, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation by reference of sequence listing This application has been submitted with a Sequence Listing in electronic format, which is provided as a file entitled 670572002140SeqList.txt, created on April 3, 2020, and 2,112 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Technical Field The present disclosure relates generally to beta-elimination linkers, and compounds containing the linkers, suitable for conjugation to small molecules, peptides, oligonucleotides, and proteins. [Background technology]

[0004] Drug molecules are covalently attached to polymeric carriers to enhance pharmaceutical properties such as half-life, stability, solubility, tolerability, and safety. US Patents 8,680,315, 8,754,190, and 9,649,385 disclose drug conjugate systems with β-elimination linkers that allow drug release via a rate-controlled β-elimination mechanism. However, along with the released drug or cleaved crosslinks, the β-elimination process generates a linker residue attached to the polymeric carrier that contains an alkenyl group that can be activated for nucleophilic addition. As shown in Figure 1, potential nucleophiles for this addition under physiological conditions include thiols and amines, e.g., those abundant in proteins, or, more likely, amines released by cleaving crosslinks in hydrogels. Although amines would be expected to be protonated and unreactive at physiological pH, such aza-Michael additions have been surprisingly found to occur, at least in in vitro situations. Previously disclosed linkers (i.e., U.S. Pat. Nos. 8,680,315 and 8,754,190) have an alkyl group (R of formula (I) in U.S. Pat. No. 8,680,315) on the carbon bearing the leaving oxygen, as shown in FIG. 5 The addition of a 2-amino group (corresponding to a 2-amino group) has provided a means of remedying this undesired reaction. Improved linkers that can more effectively suppress the undesired aza-Michael addition are needed. Summary of the Invention

[0005] In one embodiment, the compound of formula (I) [ka] (I) [where n, R 1 , R 2 , R 4, X, and Z are as disclosed herein. In some embodiments, the linker is a beta-elimination linker. In some embodiments, the beta-elimination linker is suitable for conjugation of small molecule, peptide, and protein therapeutic agents.

[0006] In another embodiment, the compound of formula (II) [ka] (II) [where n, R 1 , R 2 , R 4 , Y, Z, and D are as disclosed herein. In some embodiments, a linker-drug of formula (II) is prepared by combining a linker of formula (I) with a drug, such as a small molecule, peptide, or protein therapeutic agent.

[0007] In yet another embodiment, the compound of formula (III) [ka] (III) [where n, q, R 1 , R 2 , R 4 , M, Y, Z * and D is as disclosed herein. In some embodiments, the conjugate of formula (III) is a conjugate of a drug D releasably linked to a polymeric carrier M via a linker of formula (I).

[0008] In yet another embodiment, the compound of formula (IV) [ka] (IV) [where n, r, R 1 , R 2 , R 4 , W, Z* , P 1 , and P 2 is as disclosed herein. In some embodiments, the compound of formula (IV) is a degradable crosslinked hydrogel. In some embodiments, the degradable crosslinked hydrogel comprises a residue of a linker of formula (I).

[0009] In yet another embodiment, methods for making and using the compounds of formula (I), (II), (III), and (IV) are provided. In another embodiment, pharmaceutical compositions are provided that include the conjugate of formula (III), or the hydrogel of formula (IV). [Brief description of the drawings]

[0010] [Figure 1] Figure 1 shows the cleavage of the carbamate linker disclosed in US Patent No. 9,649,385 in a conjugate. The intact conjugate 1 undergoes a pH-dependent beta-elimination reaction to cleave the linker and form the linker residue 2 along with a free amine 3. These products undergo a subsequent reversible aza-Michael reaction to form the relatively stable re-adduct 4. When R is an amine-containing drug or prodrug, the linker is a drug-releasing linker. When R is a second PEG prepolymer, the linker is part of a crosslinker in a PEG hydrogel.

[0011] [Diagram 2] Figure 2 shows the cleavage of the carbamate linker disclosed in U.S. Patent No. 9,649,385 in a conjugate where both R5 groups are alkyl. Steric hindrance by the alkyl groups at R5 is expected to slow the re-addition process.

[0012] [Diagram 3] FIG. 3 shows cleavage of a γ-disubstituted carbamate linker disclosed herein in a conjugate.

[0013] [Figure 4] Figure 4 shows the kinetics of aza-Michael addition of glycine to the linker vinyl sulfone at various pH values. Prism plots of glycine adduct concentration (mM) vs. time (h) for std, β-Me, and gem diMe linkers: A) 1.0 M glycine at pH 7.4; B) 1.0 M glycine at pH 8.4; C) 0.1 M glycine at pH 9.5. Tabulated data for each experiment, including kf, kr, and Keq, were calculated as described in the Methods section.

[0014] [Diagram 5] Figure 5 shows the kinetics of retro-aza-Michael elimination of glycine from β-N-glycylmethylsulfone. A) Schematic of the retro-aza-Michael reaction for each linker. B) Prism plot of glycine adduct concentration (μM) versus time (h). Std linker, kobs=0.0030h-1; β-Me linker, kobs=0.0050h-1; gem diMe linker, kobs=0.0060h-1. For each linker, calculated [Gly-adduct]equil≦1μM.

[0015] [Figure 6] FIG. 6 shows an exemplary structure of a hydrogel comprising crosslinks of formula (IV).

[0016] [Figure 7] 7 shows an exemplary structure of a drug-releasing hydrogel comprising a crosslinker of formula (IV). A linker-drug (LD) of formula (II) is attached to the hydrogel via the reactive group B.

[0017] [Figure 8]Figure 8 shows the plasma concentration of exenatide [N28Q] released from the exenatide-releasing hydrogel microsphere conjugate of Example 18. Rats (n=3) were subcutaneously injected with a suspension of the conjugate containing 9.2 μmol / kg of exenatide [N28Q] on day 0. Plasma samples were collected and analyzed by LC / MS. The conjugate provided continuous exposure of exenatide [N28Q] for at least 84 days after injection and showed a release t1 / 2 of 750 hours (31 days).

[0018] [Figure 9] FIG. 9 shows blood glucose measured in STZ-induced diabetic mice treated with the insulin lispro-releasing hydrogel microsphere conjugate of Example 17. Mice were treated with streptozotocin to induce diabetes, then subcutaneously injected with a suspension of the conjugate containing 1.2 μmol / kg (low dose, squares) or 4.8 μmol / kg (high dose, triangles) of insulin lispro on days 0 and 7. The control vehicle (circles) consisted of microspheres without peptide. Blood samples were taken and blood glucose was analyzed. The low dose suppressed blood glucose for one day, while the high dose suppressed blood glucose for five days after injection. The effect was repeated with a second dose.

[0019] [Figure 10] Figure 10 shows the body weight measured in STZ-induced diabetic mice treated with the insulin lispro-releasing hydrogel microsphere conjugate of Example 17. Mice were treated with streptozotocin to induce diabetes, then subcutaneously injected with a suspension of the conjugate containing 1.2 μmol / kg (low dose, squares) or 4.8 μmol / kg (high dose, triangles) of insulin lispro on days 0 and 7. The control vehicle (circles) consisted of microspheres without peptide. Both the low and high doses of the conjugate maintained the animal's body weight, whereas the control vehicle lost 17% over 6 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Compared to previously disclosed β-elimination linkers, it has been found that undesired aza-Michael additions can be suppressed much more effectively with the linkers disclosed herein, which incorporate a geminally substituted carbon adjacent to the carbon bearing the residual oxygen, i.e., at the gamma carbon, as shown in Figure 3. The resulting linkers of formula (I) disclosed herein provide conjugates that greatly suppress the rate of addition of nucleophiles to the linker residue, resulting in correspondingly much lower equilibrium constants.

[0021] definition In this specification, unless otherwise stated, the use of the terms "a", "an", etc. means one or more.

[0022] As used herein, unless otherwise indicated, the term "about" when used in connection with a numerical value contemplates within 15%, within 10%, within 5%, within 4%, within 3%, within 2%, within 1%, or within 0.5% of the numerical value.

[0023] The term "alkyl" includes linear, branched, or cyclic saturated hydrocarbon groups of 1 to 20, 1 to 12, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. In some embodiments, the alkyl is linear or branched. Examples of linear or branched alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. In some embodiments, the alkyl is cyclic. Examples of cyclic alkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, and the like.

[0024] The term "alkoxy" includes alkyl groups linked to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and the like.

[0025] The term "alkenyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon double bond and having 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0026] The term "alkynyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon triple bond and having 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0027] The term "aryl" includes aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. The term "heteroaryl" includes aromatic rings containing at least one N, O, or S atom and containing 3 to 15 carbons, preferably containing at least one N, O, or S atom and containing 3 to 7 carbons, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and the like.

[0028] In some instances, the alkenyl, alkynyl, aryl, or heteroaryl moiety may be attached to the remainder of the molecule via an alkyl bond. In these situations, the substituent is referred to as alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl to indicate that an alkylene moiety is between the alkenyl, alkynyl, aryl, or heteroaryl moiety and the molecule to which it is attached.

[0029] The term "halogen" or "halo" includes bromo, fluoro, chloro, and iodo.

[0030] The term "heterocycle" or "heterocyclyl" refers to a 3-15 membered aromatic or non-aromatic ring containing at least one N, O, or S atom. Examples include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as the exemplary groups provided above for the term "heteroaryl". In some embodiments, the heterocycle or heterocyclyl is non-aromatic. In some embodiments, the heterocycle or heterocyclyl is aromatic.

[0031] The term "polymer" refers to a molecule or residue of a molecule having a molecular weight of 5,000 to 1,000,000 daltons, preferably 10,000 to 500,000 daltons, more preferably 10,000 to 250,000 daltons. Polymers include, but are not limited to, proteins, including antibodies, antibody fragments, and enzymes; polypeptides, including poly(amino acids), such as poly(lysine) and poly(valine), and mixed sequence polypeptides; synthetic polymers, including poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(ethyleneimine) (PEI), and copolymers thereof; and polysaccharides, such as dextran. In some embodiments, the polymer contains at least one functional group suitable for conjugation, either naturally or after chemical conversion, such as an amine, carboxylic acid, alcohol, thiol, alkyne, azide, or maleimide group, as described above. In certain embodiments of the invention, the polymer is polyethylene glycol. The polyethylene glycol may be linear or branched, terminated at one end with a functional group suitable for conjugation and at the other end (or ends) with a capping group (e.g., methyl), or may comprise multiple arms, each arm terminated with a functional group suitable for conjugation. In a preferred embodiment of the invention, the polyethylene glycol is a linear, branched, or multi-armed polymer having an average molecular weight of 20,000-200,000 daltons, preferably 20,000-100,000 daltons, and most preferably about 40,000 daltons. Examples of such polyethylene glycols are known in the art and are commercially available, for example, from NOF Corporation (Tokyo, Japan).

[0032] The terms "protein" and "peptide" are used interchangeably regardless of chain length, and these terms further include peptidomimetics and pseudopeptides that contain linkages other than amide linkages, such as CH2NH2 linkages.

[0033] The terms "nucleic acid" and "oligonucleotide" are also used interchangeably regardless of chain length. Nucleic acid or oligonucleotide may be single-stranded or double-stranded, or may be DNA, RNA, or modified forms thereof with modified linkages such as phosphodiester, phosphoramidate, etc. For both proteins and nucleic acids useful as drugs in the present invention, these terms also include those with side chains not found in nature in the case of proteins and pseudopeptide bonds, and bases not found in nature in the case of nucleic acids and backbone variants such as peptide nucleic acids.

[0034] The term "small molecule" in the context of drugs is a term well understood in the art and is meant to include compounds other than proteins and nucleic acids that are synthesized or isolated from nature and generally do not resemble proteins or nucleic acids. Typically, they have a molecular weight <1,000, although no specific cutoff is recognized. Nevertheless, the term is well understood in the fields of pharmacology and medicine.

[0035] Unless otherwise specified, "optionally substituted" means that the group can be unsubstituted or substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituents, which can be the same or different. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, halogen, -CN, -OR, -CH, -CH- ... aa , -SR aa , -NR aa R bb , -NO2, -C=NH(OR aa ), -C(O)R aa , -OC(O)R aa , -C(O)OR aa , -C(O)NR aa R bb , -OC(O)NR aa R bb , -NR aa C(O)R bb , -NR aa C(O)ORbb , -S(O)R aa , -S(O)2R aa , -NR aa S(O)R bb , -C(O)NR aa S(O)R bb , -NR aa S(O)2R bb , -C(O)NR aa S(O)2R bb , -S(O)NR aa R bb , -S(O)NR aa R bb , -P(O)(OR aa )(OR bb ), heterocyclyl, heteroaryl, or aryl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl are each independently optionally selected from R cc may be substituted by, where R aa and R bb are each independently H, alkyl, alkenyl, alkynyl, heterocyclyl, heteroaryl, or aryl; R aa and R bb together with the nitrogen atom to which they are attached form a heterocyclyl, which is optionally substituted with alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, or -CN, where Each R cc is independently alkyl, alkenyl, alkynyl, halogen, heterocyclyl, heteroaryl, aryl, -CN, or -NO2.

[0036] Typically, the active form of the drug is released directly from the conjugates of the present invention, although in some cases it is possible to release the active drug in the form of its prodrug.

[0037] Linker In one aspect, the present invention relates to a compound of formula (I) [ka] (I) [In the formula, n is an integer from 0 to 6; R 1 and R 2 are independently an electron withdrawing group, alkyl, or H, where R 1 and R 2 at least one of is an electron withdrawing group; Each R 4 are independently C1-C3 alkyl, or two R 4 form a 3- to 6-membered ring together with the carbon atom to which they are attached; X is a leaving group; and Z is a functional group for connecting the linker to the polymeric carrier. A linker represented by the formula:

[0038] In some embodiments of the linker of Formula (I), n=1-6 and R 1 and R 2 are independently an electron withdrawing group, alkyl, or H, where R 1 and R 2 at least one of R is an electron-withdrawing group; 4 are independently C1-C3 alkyl or may be joined together to form a 3-6 membered ring; X is a halogen, an active ester such as N-succinimidyloxy, nitrophenoxy, or pentahalophenoxy, or imidazolyl, triazolyl, tetrazolyl, or N(R 6 ) CH2Cl, where R 6 is an optionally substituted C1-C6 alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl; and Z is a functional group for connecting the linker to the polymeric carrier.

[0039] In some embodiments, R 1 and R 2 The electron-withdrawing group is -CN; -NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR 3 , -SOR 3 , or -SO2R 3 , [Where, R 3 is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 8 , or -NR 8 2, where each R 8 are independently H or optionally substituted alkyl, or two R 8 the groups together with the nitrogen to which they are attached form a heterocycle; or S.R. 9 , [Where, R 9 is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl. It is.

[0040] In some embodiments, R 1 and R 2 The electron withdrawing group in R is -CN. 1 and R 2 The electron withdrawing group in R is -NO. 1 and R 2 The electron withdrawing group is an optionally substituted aryl containing 6 to 10 carbons. For example, in some embodiments, R 1 and R2 The electron withdrawing group in is optionally substituted phenyl, naphthyl, or anthracenyl. In some embodiments, R 1 and R 2 The electron withdrawing group is an optionally substituted heteroaryl containing 3 to 7 carbons and at least one N, O, or S atom. For example, in some embodiments, R 1 and R 2 The electron withdrawing group of is optionally substituted pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl. 1 and R 2 The electron withdrawing group is an optionally substituted alkenyl containing 2 to 20 carbon atoms. 1 and R 2 The electron withdrawing group is an optionally substituted alkynyl containing 2 to 20 carbon atoms. 1 and R 2 The electron-withdrawing group is -COR 3 , -SOR 3 , or -SO2R 3 where R 3 is H, optionally substituted alkyl containing 1 to 20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 8 or -NR 8 2, where each R 8 are independently H or optionally substituted alkyl containing 1 to 20 carbon atoms, or two R 8 The groups together with the nitrogen to which they are attached form a heterocycle. In some embodiments, R 1 and R 2 The electron-withdrawing group is -SR 9 where R 9is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl containing 1 to 20 carbon atoms.

[0041] In some embodiments of the linker of formula (I), R 1 and R 2 At least one of the following is -CN, -SOR 3 , or -SO2R 3 In some embodiments, R 1 and R 2 At least one of is -CN or -SO2R 3 In some embodiments, R 1 and R 2 At least one of the groups is -CN or -SO2R. 3 where R 3 is optionally substituted alkyl, optionally substituted aryl, or -NR 8 2. In some embodiments, R 1 and R 2 At least one of is -CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0042] In some embodiments of the linker of formula (I), each R 4 is independently C1-C3 alkyl. In some embodiments, R 4 are both methyl.

[0043] In some embodiments of the linker of Formula (I), n is an integer from 1 to 6. In some embodiments, n is an integer from 1 to 3. In some embodiments, n is an integer from 0 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0044] In some embodiments of the linker of formula (I), X is a halogen, an active ester (e.g., N-succinimidyloxy, nitrophenoxy, or pentahalophenoxy), an optionally substituted heteroaryl (e.g., imidazolyl, triazolyl, or tetrazolyl), or -N(R 6 ) CH2Cl, where R 6 is an optionally substituted C1-C6 alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl. In some embodiments, X is a halogen. In some embodiments, X is an active ester, such as succinimidyloxy. In some embodiments, X is -N(R 6 ) CH2Cl, where R 6 is optionally substituted aryl.

[0045] In the linker of formula (I), Z can be any functional group known in the art for conjugation. Such functional groups include, but are not limited to, for example, amine, aminooxy, ketone, aldehyde, maleimidyl, thiol, alcohol, azide, 1,2,4,5-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, cyclooctynyl, and protected variants thereof. In some embodiments, Z is a protected amine, a protected aminooxy, a ketone or a protected ketone, an aldehyde or a protected aldehyde, maleimidyl, a protected thiol, a protected alcohol, an azide, 1,2,4,5-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, or cyclooctynyl. In some embodiments, Z is an azide, a ketone, or a protected ketone.

[0046] In the description herein, it is understood that every description, variation, embodiment, or aspect of a moiety can be combined with every description, variation, embodiment, or aspect of any other moiety, just as if each and every combination of descriptions were specifically and individually set forth. For example, every description, variation, embodiment, or aspect provided herein with respect to n in formula (I) can be combined with R 1 , R 2 , R 4, X, and Z. It is also understood that all descriptions, variations, embodiments, or aspects of formulas such as formula (I), (II), (III), (IV), or (V) apply equally to other formulas described herein, where applicable, and all descriptions, variations, embodiments, or aspects are described equally as if they were described separately and individually for all formulas. For example, all descriptions, variations, embodiments, or aspects of formula (I) apply equally to any of the formulas described herein, such as formulas (II), (III), (IV), and (V), where applicable, and all descriptions, variations, embodiments, or aspects are described equally as if they were described separately and individually for all formulas.

[0047] Linker-drug In another embodiment, the compound of formula (II) [ka] (II) [where n, R 1 , R 2 , R 4 , X, and Z are as disclosed herein for formula (I); D is a drug; Y is absent when D is a drug attached through an amine, or Y is -N(R 6 )CH2-, where R 6 is an optionally substituted C1-C6 alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl. The compound is provided: In some embodiments, the compounds of formula (II) are linker-drugs prepared by combining a linker of formula (I) with a drug, such as a small molecule, peptide, or protein therapeutic.

[0048] In some embodiments of the compound of Formula (II), Y is absent. In some embodiments, Y is -N(R 6 )CH2-.

[0049] In some embodiments of the compound of formula (II), suitable drugs include, but are not limited to, small molecules, peptides, proteins, and nucleic acids. Suitable drugs include, but are not limited to, for example, antidiabetic drugs, growth promoters, antibacterial agents, including aminoglycosides, penicillins, cephalosporins, macrolides, and peptides, trimethoprim, piromidic acid, and sulfamethazine; analgesics and anti-inflammatory drugs, antiallergic and antiasthmatic drugs, antihypercholesterolemic drugs, beta-adrenergic blockers and antihypertensive drugs, antitumor drugs, and antiviral drugs.

[0050] Further examples of such drugs include alcohols, such as paclitaxel and analogs, epothilones and analogs, camptothecin and analogs, such as irinotecan, and nucleosides, such as 5-fluorouracil and capecitabine. In another embodiment, the drug is a peptide containing a serine residue. In another embodiment, the drug is a small molecule containing an arylol group; such drugs include, for example, SN-38, etilefrine, prenalterol, and estradiol. In another embodiment, the drug is a peptide containing a tyrosine residue. When the coupling is via S, the drug can be a small molecule containing a thiol group. Such drugs include, for example, penicillamine, captopril, and enalapril. The drug can be a small molecule containing a thioaryl or thioheteroaryl group; such drugs include, for example, 6-mercaptopurine. When coupling is through a non-basic N, the drug can be a small molecule or peptide containing a primary or secondary amide (such as a pyroglutamic acid residue, or other amides), or a sulfonamide, or a heteroaryl group such as an indole (e.g., tryptophan) or a purine, for example thyrotropin releasing hormone, bombesin, luteinizing hormone releasing hormone, follicle stimulating hormone releasing hormone, octreotide, 5-fluorouracil, allopurinol.

[0051] Examples of nucleic acid-based drugs include the sense and antisense strands of genes from animals, especially mammals. Such genes can be the subject of antisense DNA or RNA, or small interfering RNA, such as protein kinase C-alpha, BCL-2, ICAM-1, tumor necrosis factor alpha, etc., which are provided for the treatment of various diseases. Also included are CpG oligonucleotide agonists of Toll-like receptors. The nucleic acid can be directly linked to the linker or linked via modified groups on the nucleic acid, such as oligonucleotides that contain 5'-amine or 3'-amine modifications or amine-containing bases.

[0052] In some embodiments of the compound of formula (II), D is a peptide. Suitable peptides include, but are not limited to, octreotide (SEQ ID NO:5), exenatide and variants, including [N28Q]exenatide (SEQ ID NO:1), insulin lispro (A chain: SEQ ID NO:2; B chain: SEQ ID NO:3; disulfide bridges: A6-A11, A7-B7, A20-B19), or teduglutide ([Gly 2 ]GLP-2) (SEQ ID NO: 4), and sequence variants thereof. For example, both amidated and non-amidated forms of any of the sequences disclosed herein are contemplated. As another example, both L- and D-forms of any amino acid are contemplated. In some embodiments, octreotide is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (Cys2-Cys7 cyclic disulfide). SEQ ID NO: 1: HGEGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPS-NH2 ([N28Q]exenatide) SEQ ID NO: 2: GIVEQCCTSICSLYQLENYCN (A chain of insulin lispro) SEQ ID NO: 3: FVNQHLCGSHLVEALYLVCGERGFFYTKPT (B chain of insulin lispro) SEQ ID NO: 4: HGDGSFSDEMNTILDNLAARDFINWLIQTKITD (Teduglutide ([Gly 2 ]GLP-2) SEQ ID NO: 5: FCFWKTCT (Octreotide; Cys2-Cys7 cyclic disulfide)

[0053] Conjugates In another embodiment, the compound of formula (III) [ka] (III) [where n, R 1 , R 2 , R 4, D, and Y are as disclosed herein for Formula (I) or Formula (II); M is a polymeric carrier; q is an integer from 1 to 10 when M is a soluble polymer, or q is a number when M is an insoluble matrix; Z * indicates the attachment point to M. A conjugate represented by the formula: In some embodiments, the compound of formula (III) is a conjugate of a drug D releasably linked to a polymeric carrier M via a linker of formula (I). It is understood that when M is an insoluble matrix, multiple linker-drugs can be attached to M. For example, in some embodiments, M is a hydrogel of formula (IV) and P 1 and P 2 When both P are 4-arm polymers, one, two, three, or four linker-drugs may be attached to each P 1 -P 2 The linker-drug can be linked to the M unit. Thus, a desired number can be obtained by reacting the linker-drug with M in an appropriate ratio. In this way, an appropriate drug concentration can be obtained in the volume of the matrix.

[0054] In some embodiments of the conjugate of formula (III), the molecular carrier M is a soluble polymer and q is an integer between 1 and 10. In some embodiments, M is an insoluble matrix and q is a large number. In some embodiments, when M is an insoluble matrix, q is a large number such that an adequate drug concentration can be obtained in the volume of the matrix. Soluble polymers include, but are not limited to, polyethylene glycol or other synthetic polymers, dextran, antibodies, antibody fragments, albumin or other proteins of sufficient molecular size to inhibit the efficiency of renal filtration as understood in the art. In the case of polyethylene glycol, M can be single-chain, multi-chain, or multi-armed with an average molecular weight of 1,000 to 100,000 daltons, preferably 1,000 to 40,000 daltons. Insoluble matrices include, but are not limited to, hydrogels, implants, or surgical devices, which may be in bulk or in the form of microparticles or nanoparticles. In some embodiments, M is a soluble polymer. In some embodiments, M is an insoluble matrix. In some embodiments, M is a hydrogel of formula (IV) disclosed herein.

[0055] In some embodiments of the conjugate of formula (III), the molecular carrier M includes at least one functional group Z' associated with Z that allows for conjugation. For example, when Z is an amine, Z' is a carboxylic acid, an active ester, or an active carbonate, and Z * is an amide or carbamate. As another example, when Z is an azide, Z' is an alkynyl, bicyclononynyl, or cyclooctynyl, and Z * As another example, when Z is NH2O, Z' is a ketone or aldehyde and Z *As another example, when Z is SH, Z' is maleimide or halocarbonyl, and Z * Similarly, the roles of Z and Z' can be reversed to produce a conjugate of formula (III) in which Z is a thiosuccinimidyl or thioether. * In some embodiments, Z * includes an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

[0056] Hydrogels In another embodiment, the compound of formula (IV) [ka] (IV) [where n, R 1 , R 2 , R 4 , and Z * is as disclosed herein for formula (I), (II), or (III); W is not present or [ka] and wherein each of x, y, and z is independently an integer from 0 to 6; B is -NH2, -ONH2, a ketone, an aldehyde, -SH, -OH, -CO2H, a carboxamide group, or a group containing cyclooctyne or bicyclononyne; and C * is a carboxamide, a thioether, a thiosuccinimidyl, a triazole, or an oxime; and P 1 and P 2 are independently r-arm polymers having a molecular weight of 1 to 40 kDa, where r is an integer from 2 to 8. A hydrogel having the formula: (CH2)x is bonded to NH, C * , P 2It is understood that the hydrogel of formula (IV) is degradable.

[0057] In some embodiments of the hydrogel of formula (IV), P 1 and P 2 are synthetic polymers such as polyethylene glycol, dextran, hyaluronic acid, etc. Examples of the structure of such hydrogels are shown in FIG. 6. In these hydrogels, the polymer chains are crosslinked using a linker of formula (I) to form an insoluble three-dimensional matrix. The crosslinking is carried out by the groups R 1 and the group R 2 It slowly cleaves by non-hydrolytic beta-elimination at a rate governed by and eventually produces soluble polymer fragments. These hydrogels allow for linker-drug attachment in several ways. When W is present, a functional group B is introduced at each crosslink. As shown in FIG. 6, hydrogels can be formed with multiple Bs present. The group B can then be used directly for linker-drug attachment, where B is equivalent to the relevant group Z' above, or the group B can be derivatized to introduce the relevant group Z' for linking the linker-drug to a preformed hydrogel. This is advantageous when the hydrogel needs to be prepared ex vivo, for example when it is manufactured into a desired form such as a microsphere or a sheet of fixed dimensions. Alternatively, the group B can include the linker-drug of formula (II) when the hydrogel is manufactured, which is advantageous when it is gelled in situ by injecting a liquid mixture of the components prior to gel formation.

[0058] Pharmaceutical Compositions In another embodiment, a pharmaceutical composition is provided herein, comprising a polymeric carrier-drug conjugate or a pharma- ceutically acceptable salt thereof together with a pharma- ceutically acceptable buffer and / or excipient. The buffer is selected so that the stability of the linker is maintained during storage and upon reconstitution as necessary, typically having a pH of 2-7, preferably 2-6, more preferably 2-5. Acceptable buffers include acetic acid, citric acid, phosphoric acid, histidine, gluconic acid, aspartic acid, glutamic acid, lactic acid, tartaric acid, succinic acid, malic acid, fumaric acid, α-ketoglutaric acid, and the like. Excipients may include tonicity and osmolarity agents, such as sodium chloride; preservatives, such as citric acid or citrate salts, and parabens; antimicrobial agents, such as phenol and cresol; antioxidants, such as butylated hydroxytoluene, vitamin A, C, or E, cysteine, methionine, and the like; density adjusters, such as sucrose, polyols, hyaluronic acid, and carboxymethylcellulose. These formulations are described, for example, in "Remington's Pharmaceutical Science" AR Gennaro, ed., 17 th The pharmaceutical compositions may be prepared by conventional methods known to those skilled in the art, as described in the American Journal of Clinical Chemistry, 1985, Mack Publishing Company, Easton, PA, USA. The pharmaceutical compositions may be provided in liquid solution or suspension, or may be provided as solids, for example, by lyophilization of liquid compositions. Such lyophilized products may further comprise bulking agents for rapid and efficient reconstitution before use.

[0059] How to use In another aspect, the polymeric carrier-drug conjugates described herein and pharmaceutical compositions comprising the same can be used to treat or prevent disease or illness in individuals. In some embodiments, a method for treating disease or illness is provided, comprising administering the polymeric carrier-drug conjugates described herein or pharmaceutical compositions comprising the polymeric carrier-drug conjugates described herein to an individual in need thereof. The "individual" may be a human or an animal, such as a cat, dog, cow, rat, mouse, horse, rabbit, or other domestic animal.

[0060] Also provided is a composition comprising a polymeric carrier-drug conjugate as described herein for use in treating a disease or condition. Also provided herein is the use of a polymeric carrier-drug conjugate as described herein in the manufacture of a medicament for treating a disease or condition.

[0061] The applicable disease or illness that requires treatment will be recognized by those skilled in the art from the nature of the conjugate drug.For example, exenatide and insulin can be used to treat diabetes, octreotide can be used to treat acromegaly and various cancers, teduglutide can be used to treat short bowel syndrome, and SN-38 and TLR9 agonists can be used to treat cancer.Suitable routes of administration to humans and animals are envisioned by the present invention, including, for example, intravenous, intrathecal, intraocular, subcutaneous, intraarticular, intraperitoneal or other local injection, or by oral administration.

[0062] Preparation of linkers The linker of formula (I) can be prepared by several routes, as shown in the examples below. In one method, a geminal-dialkylcarbonyl compound (A) is converted to R 1 R 2 Condensate with CH2. [ka]

[0063] A suitable base is R 1 R 2 Those capable of deprotonating CH2, such as potassium tert-butoxide or tert-pentoxide, butyllithium, lithium diisopropylamide, NaH, and silazide bases, such as LiHMDS, NaHMDS, or KHMDS. 10 R can be H, C1-C6 alkoxy, or N(Me)OMe. 10 If R is H, alcohol (C) is produced directly. 10 is other than H, a ketone (B) is generated which is then reduced to an alcohol (C). Suitable reducing agents include borohydrides such as LiBH4 and NaBH4, although other reducing agents well known in the art may be used depending on the nature of the group Z. The alcohol (C) is then activated to generate the linker of formula (I). Typical activation conditions include conversion to a chloroformate (X=Cl) by the action of phosgene or a phosgene equivalent such as diphosgene or triphosgene; conversion to a succinimidyl carbonate (X=OSu) using N,N'-disuccinimidyl carbonate and 4-(dimethylamino)pyridine or by treating chloroformic acid with N-hydroxysuccinimide and pyridine; and conversion to an activated carbonate, for example by reaction with nitrophenyl chloroformate in the presence of a weak base such as pyridine.

[0064] X is -N(R 6 ) CH2Cl can be prepared as disclosed in U.S. Pat. No. 8,754,190.

[0065] Z can be a functional group known in the art for conjugation, such as amine, aminooxy, ketone, aldehyde, maleimidyl, thiol, alcohol, azide, 1,2,4,5-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, cyclooctynyl, and protected variants thereof. In some embodiments, Z is a protected amine, a protected aminooxy, a ketone or a protected ketone, an aldehyde or a protected aldehyde, maleimidyl, a protected thiol, a protected alcohol, an azide, 1,2,4,5-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, or cyclooctynyl. In some embodiments, Z is an azide, a ketone, or a protected ketone.

[0066] Linker-drug production The linker of formula (I) is reacted with a drug D to form a compound of formula (II) [ka] (II) A linker-drug can be produced, as shown in

[0067] Drugs suitable for use in the present invention include small molecules, peptides, proteins, and nucleic acids. For drugs containing a basic amine group, a linker of formula (I) where X is a halide or active ester is reacted with the drug in the presence of base in an organic solvent or in aqueous buffer to produce a linker-drug of formula (II). Such a basic amine may be part of the small molecule drug or may be the N-terminal amine or lysine e-amine of peptides and proteins. For drugs containing multiple basic amines, such as peptides and proteins, multiple linkers may be attached. For synthetic peptides, the linker can be attached at a specific position in the synthesis, for example, at the N-terminus by using a linker in the final coupling step, or using a temporary blocking group at an internal amino acid residue that can be selectively removed; then acylated with the linker, followed by global deprotection and purification of the linker-peptide. Suitable bases to facilitate attachment of the linker to the drug include tertiary amines, such as triethylamine or N,N-diisopropylethylamine, and guanidines, such as N,N-dimethylguanidine, and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, and others known in the art. When carried out in aqueous solution, the reaction is typically carried out at a pH value of 7-10.

[0068] X=N(R 6 The linker of formula (I), wherein R is CH2Cl, can be used to attach drugs through alcohols, phenols, thiols, thiophenols, imidazoles, and non-basic nitrogen atoms, similar to the methods disclosed in U.S. Pat. No. 8,754,190.

[0069] After preparation of the linker-drug, protecting groups on Z or the drug may be removed prior to conjugation using techniques well known in the art.

[0070] Conjugate Production Using the linker-drug of formula (II), the deprotected functional group Z is reacted with a cognate reactive group attached to the polymeric carrier M to form a linker-drug of formula (III): [ka] (III) A conjugate can be prepared,

[0071] Z can be a functional group known in the art for conjugation, such as amine, aminooxy, ketone, aldehyde, maleimidyl, thiol, alcohol, azide, 1,2,4,5-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, cyclooctynyl. The choice of attachment functional group will depend on the presence of other functional groups on the drug D, and will be apparent to one of skill in the art. M can be a water-soluble polymer, such as polyethylene glycol or other synthetic polymers, dextran, antibodies, antibody fragments, albumin or other proteins of sufficient molecular size to inhibit the efficiency of renal filtration as understood in the art. In the case of polyethylene glycol, M can be single-chain, multi-chain, or multi-armed, with an average molecular weight of 1,000-100,000 daltons, preferably 1,000-40,000 daltons. The polyethylene glycol contains at least one functional group Z' associated with Z that allows for conjugation. For example, when Z is an amine, Z' is a carboxylic acid, an activated ester, or an activated carbonate, and Z * In another example, when Z is an azide, Z' is an alkynyl, bicyclononynyl, or cyclooctynyl, and Z * As another example, when Z is NH2O, Z' is a ketone or aldehyde and Z * As another example, when Z is SH, Z' is maleimide or halocarbonyl, and Z* Similarly, the roles of Z and Z' can be reversed to produce a conjugate of formula (III) in which Z is a thiosuccinimidyl or thioether. * These conjugation reactions can be carried out under conditions known in the art, for example, where Z=azide and Z′=cyclooctyne, the conjugation can occur in any solvent in which both components exhibit suitable solubility, although aqueous solutions are known to exhibit more favorable reaction kinetics.

[0072] Similarly, M can be a water-insoluble matrix, for example a hydrogel, an implant, or a surgical device, which can be in bulk or in the form of microparticles or nanoparticles. In this case, M contains multiple groups Z as described above, allowing multiple linker-drugs to be attached. Although the matrix is ​​insoluble, reaction with a solution containing drug-linker is sufficient for conjugation to occur. For example, if the insoluble matrix is ​​a hydrogel, produced in bulk form or as microspheres or other particulate forms, the solution of linker-drug is mixed with the suspension of hydrogel for a sufficient time to allow linker-drug to penetrate the porous hydrogel matrix, and conjugation reaction occurs.

[0073] Hydrogel fabrication In some embodiments, M is a group represented by formula (IV): [ka] (IV) [In the formula, P 1 , P 2 , Z * , n, r, R 1 , R 2 , R 4 and W is as disclosed herein. It is a biodegradable hydrogel represented by the formula:

[0074] An example of such a hydrogel structure containing crosslinks of formula (IV) is shown in Figure 6. In these hydrogels, the polymer chains are crosslinked using a linker of formula (I) to form an insoluble three-dimensional matrix. The crosslinks are formed by the groups R 1 and the group R 2 The hydrogel slowly cleaves by non-hydrolytic beta-elimination at a rate governed by and eventually produces soluble polymer fragments. These hydrogels allow for linker-drug attachment in several ways. When W is present, a functional group B is introduced at each crosslink. As shown in FIG. 6, the hydrogel can be first formed to provide a hydrogel polymer with multiple Bs present, providing a degradable polymer that does not further include a linker-drug of formula (II). The group B can then be used directly for linker-drug attachment, if B is equivalent to the relevant group Z' described above, or the group B can be derivatized to introduce the relevant group Z' for attaching the linker-drug to a preformed hydrogel. This is advantageous when the hydrogel needs to be prepared ex vivo, for example when it is manufactured into a desired form such as a microsphere or a sheet of fixed dimensions. Alternatively, the group B can include the linker-drug of formula (II) when the hydrogel is manufactured, which is advantageous when gelling in situ by injecting a liquid mixture of the components prior to gel formation.

[0075] These hydrogels can be formed by mixing two multi-arm prepolymers, one having arms terminating in a group that includes the residue of a linker of formula (I) having a reactive end group Z, and the other having arms terminating in a group that includes the associated reactive end group Z'. In one embodiment, one prepolymer has formula (V): [ka] (V) wherein the groups are as defined above. and the other prepolymer has the formula P 1 -(Z')r. When mixed in a suitable solvent, typically at a pH between 2-7 when Z and Z' are azides / cyclooctynes, or at a pH between 6-9 when Z and Z' are active esters and amines, in an aqueous buffer, the Z and Z' groups react to form an insoluble hydrogel matrix containing crosslinks of formula (IV). This process can be carried out in the bulk phase or under conditions of emulsification in mixed organic / aqueous systems to form particulate suspensions such as microspheres suitable for injection.

[0076] Specific representative embodiments are provided below: EMBODIMENT 1 Formula (I) [ka] (I) [In the formula, n=1 to 6, R 1 and R 2 are both, independently, electron-withdrawing groups, or R 1 and R 2 one of which is an electron withdrawing group and the other is alkyl or H; Each R 4 are independently C1-C3 alkyl or may be joined together to form a 3- to 6-membered ring; Z is a group for attaching the linker to a carrier for conjugation; and X is a leaving group. , the linker. EMBODIMENT 2 The linker of embodiment 1, wherein X is halogen, N-succinimidyloxy, nitrophenoxy, pentahalophenoxy, imidazolyl, triazolyl, or tetrazolyl. EMBODIMENT 3 The linker of embodiment 1, wherein Z is a protected amine, a protected aminooxy, a ketone or a protected ketone, an aldehyde or a protected aldehyde, maleimidyl, a protected thiol, a protected alcohol, an azide, 1,2,4,5-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, or cyclooctynyl. EMBODIMENT 4 Formula (II) [ka] (II) wherein Y is absent when D is a drug attached through an amine, or Y is N(R 6 )CH2, where R 6 is optionally substituted C1-C6 alkyl, or optionally substituted aryl or heteroaryl. , denoted as linker-drug. EMBODIMENT 5 Formula (III) [ka] (III) [In the formula, M is a polymeric carrier, and Z * includes a carboxylic acid amide, an oxime, a 1,2,3-triazole, a thioether, a thiosuccinimide, or an ether, and q=1 to many. The conjugate is shown by: EMBODIMENT 6 Formula (IV) [ka] (IV) [In the formula, P 1 and P 2 is independently an r-arm polymer, where r=2 to 8, and W is absent or [ka] and where x, y, and z are each independently 0 to 6; B is an NH, ONH, ketone, aldehyde, SH, OH, COH, or carboxamide group; and C * is a carboxamide, a thioether, a thiosuccinimidyl, a triazole, or an oxime. The hydrogel is shown in FIG. EXAMPLES

[0077] The following examples are offered to illustrate, but not limit, the present disclosure. Example 1 Preparation of Linkers of Formula (I) Wherein Z=Azide [ka]

[0078] (1) 4-azido-1-cyano-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =CN,R 2 =H, R 4 = CH3, Z = N3, and X = succinimidyloxy) A 1M solution of potassium tert-butoxide in THF (3.5 mL, 3.5 mmol) was added to a solution of methyl 3-azido-2,2-dimethylpropionate (prepared according to Kim, Synthetic Communications; 300 mg, 1.9 mmol) and acetonitrile (0.365 mL, 7.0 mmol) in 7 mL of THF at -30°C. The mixture was stirred at -30°C for 30 min, then warmed to ambient temperature over 1 h and stirred for an additional 30 min. The mixture was cooled in ice and quenched by the addition of 6N HCl (0.62 mL, 3.7 mmol), then partitioned between EtOAc and water. The aqueous phase was extracted twice with EtOAc and the combined organics were washed with brine, dried over MgSO4, filtered and concentrated to give the crude ketone.

[0079] Sodium borohydride (33 mg, 0.88 mmol) was added to a solution of the crude ketone (300 mg, ca. 1.75 mmol) in 7 mL of methanol. The mixture was then stirred for 15 min, quenched by the addition of 6N HCl (0.7 mL), and partitioned between EtOAc and water. The aqueous phase was extracted twice with EtOAc, and the combined organics were washed with brine, dried over MgSO4, filtered, and concentrated to give the crude alcohol. Purification on SiO2 (20-40% EtOAc / hexanes) gave 4-azido-1-cyano-3,3-dimethyl-2-butanol (142 mg, 0.85 mmol). 1 H-NMR (CDCl3, 300 MHz) d 3.83-3.92 (m,1H), 3.43 (d, J=12.1 Hz,1H), 3.21 (d, J=12.1 Hz, 1H), 2.41-2.62 (m,3H), 0.97 (s,3H), 0.96 (s,3H).

[0080] Pyridine (136 μL, 1.7 mmol) was added dropwise to an ice-cold solution of 4-azido-1-cyano-3,3-dimethyl-2-butanol (142 mg, 0.85 mmol) and triphosgene (425 mg, 1.44 mmol) in 8 mL of THF. The resulting suspension was allowed to warm to ambient temperature and stirred for 15 min, then filtered and concentrated to give the crude chloroformate. This was dissolved in 8 mL of THF, cooled on ice, and treated with N-hydroxysuccinimide (291 mg, 2.5 mmol) and pyridine (204 μL, 2.53 mmol). The resulting suspension was allowed to warm to ambient temperature and stirred for 15 min, then partitioned between EtOAc and 5% KHSO4. The aqueous phase was extracted twice with EtOAc and the combined organics were washed with brine, dried over MgSO4, filtered and concentrated to give the crude succinimidyl carbonate. Purification on SiO2 (20-40% EtOAc / Hexanes) gave 4-azido-1-cyano-3,3-dimethyl-2-butyl succinimidyl carbonate (174 mg, 0.56 mmol). 1H-NMR (CDCl3, 300 MHz) d 5.03 (dd,J=7.0,5.1,1H), 3.27-3.41 (m,6H), 3.43 (d, J=12.1 Hz,1H), 3.21 (d, J=12.1 Hz, 1H), 2.41-2.62 (m,3H), 0.97 (s,3H), 0.96 (s,3H).

[0081] (2) 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2N(CH3)2, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). A 1.43 M solution of n-butyllithium in hexanes (70 mL, 100 mmol) was added to a stirred solution of N,N-dimethylmethanesulfonamide (12.33 g, 100 mmol) in 200 mL of anhydrous THF, kept at -50 °C under an inert atmosphere. The mixture was warmed to -20 °C for 1 h and then recooled to -50 °C before adding methyl 3-azido-2,2-dimethylpropionate (prepared according to Kim, Synthetic Communications; 7.70 g, 50 mmol). The mixture was warmed to +10 °C for 2 h and then quenched with 20 mL of 6N HCl. The mixture was diluted with methyl t-butyl ether (MTBE, 200 mL), washed twice with 100 mL of water and once with 100 mL of brine, dried over MgSO4, filtered, and concentrated to give 14.05 g of crude ketone product. Purification by chromatography on SiO2 (220 g) using a step gradient of 0, 20, 30, 40, and 50% EtOAc / hexanes afforded 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanone (10.65 g, 86%) as a crystalline solid.

[0082] The above ketone was dissolved in 200 mL of methanol, cooled on ice, and treated with sodium borohydride (0.96 g, 25 mmol) for 15 min, then quenched with 4 mL of 6 N HCl and concentrated. The resulting slurry was diluted with methyl t-butyl ether (MTBE, 200 mL), washed once with 100 mL of water and once with 100 mL of brine, dried over MgSO4, filtered, and concentrated to give 10.0 g of crystalline 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanol.

[0083] Pyridine (10.6 mL, 132 mmol) was added to a stirred mixture of N-hydroxysuccinimide (6.90 g, 60 mmol) and triphosgene (5.93 g, 20 mmol) in 250 mL of ice-cold dichloromethane in 10 min. The mixture was stirred for 15 min on ice and then warmed to ambient temperature in 30 min. A solution of 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanol (10.0 g, 40 mmol) in 20 mL of dichloromethane was added and the mixture was further stirred at ambient temperature for 1 h. After cooling on ice, the mixture was treated with 100 mL of water and the phases were separated. The organic phase was washed twice with water, once with 5% KHSO4, and once with brine, dried over MgSO4, filtered, and concentrated. The crude product was crystallized from 100 mL of 30% EtOAc / hexanes to give 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (11.1 g, 71%) as a white crystalline solid. (3) Additional compounds of formula (I) prepared according to these procedures include: 4-Azido-1-(methylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2CH3, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-((4-methylpiperidinyl)sulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2N(CH2CH2)2CHCH3, R 2 =H, R 4 = CH3, Z = N3, and X = succinimidyloxy). LC / MS was [M+H] + =446.15. 4-Azido-1-(phenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2Ph, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-(4-chlorophenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2PhCl, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-(4-morpholinosulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2N(CH2CH2)2O, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-(isopropylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2CH(CH3)2, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-((N-ethyl-N-methylamino)sulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2N(CH3)(CH2CH3), R 2 =H, R4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-((N,N-bis(2-methoxyethyl)aminosulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2N(CH2CH2OCH3)2, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-(4-methylphenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2PhCH3, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy).

[0084] Example 2 Preparation of the linker of formula (I) [ka] Another general method for preparing compounds of formula (I) is 1 =CN,R 2 =H, 2 R 4 =CH3, Z=N3, and X=N-succinimidyloxy.

[0085] (1) 5-azido-1-cyano-3,3-dimethyl-2-pentyl succinimidyl carbonate (wherein n=2, R 1 =CN,R 2 =H, R 4 = CH3, Z = N3, and X = succinimidyloxy) (a) Ethyl 4-chloro-2,2-dimethylbutanoate A heat gun dried 500 mL round bottom flask equipped with a stir bar, rubber septum, nitrogen inlet, and thermocouple probe was charged with iPr2NH (5.30 mL, 37.4 mmol, 1.1 equiv, final concentration 0.27 M) and THF (100 mL). With the reaction mixture cooled to 0 °C, a solution of nBuLi (1.28 M in hexanes, 27.8 mL, 35.7 mmol, 1.05 equiv, final concentration 0.26 M) was added dropwise via syringe at a rate (~10 min) such that the internal temperature did not exceed +10 °C. The reaction mixture was stirred at 0° C. for 15 min, cooled to −78° C., and a solution of ethyl isobutyrate (4.6 mL, 4.0 g, 34 mmol, 1.0 equiv, 0.24 M final concentration) in THF (5 mL) was added dropwise via syringe at a rate (~5 min) such that the internal temperature did not exceed −65° C. The reaction mixture was stirred at −78° C. for 45 min, then a solution of 1-bromo-2-chloroethane (2.8 mL, 34 mmol, 1.0 equiv, 0.24 M final concentration) in THF (5 mL) was added at a rate such that the internal temperature did not exceed −68° C. The reaction mixture was stirred at −78° C. for 15 min, warmed to 0° C., and stirred at 0° C. for 15 min. The reaction mixture was diluted with EtOAc (100 mL) and 5% KHSO4 (100 mL). The aqueous phase was separated and extracted with EtOAc (3×50 mL). The aqueous phase was separated and extracted with EtOAc (3×50 mL). The combined organic phases were washed with brine, dried over MgSO4, filtered, and concentrated from toluene (10 mL×2) to give 4.85 g (27 mmol, 79%) of the desired chloride as a pale yellow oil. 1 H NMR (CDCl3, 300 MHz) δ 4.14 (q, J=7.2 Hz, 2 H), 3.43 - 3.57 (m, 2 H), 1.94 - 2.19 (m, 2 H), 1.27 (t, J=7.1 Hz, 3 H), 1.22 (s, 6 H)

[0086] (b) Ethyl 4-azido-2,2-dimethylbutanoate [ka] A 100 mL round bottom flask equipped with a stir bar, rubber septum, and nitrogen inlet was charged with ethyl 4-chloro-2,2-dimethylbutanoate (2-1) (4.85 g, 27 mmol, 1.0 equiv, final concentration 0.54 M), DMSO (50 mL), and sodium azide (2.28 g, 35 mmol, 1.3 equiv, 0.70 M). The reaction mixture was stirred behind a blast shield at 70 °C for 18 h. The reaction mixture was cooled to ambient temperature and diluted with EtOAc (200 mL) and H2O (100 mL). The organic phase was separated, washed with H2O (3 x 100 mL) and brine (100 mL), dried over MgSO4, filtered, and concentrated. Purification by column chromatography (40 g silica gel cartridge; stepwise gradient elution: 0%, 5%, 10%, 20% EtOAc / hexanes) afforded 4.33 g (23.3 mmol, 87%) of the desired azide as a pale yellow oil. 1 H NMR (CDCl3, 300 MHz) δ 4.15 (q, J=7.1 Hz, 2 H), 3.22 - 3.35 (m, 2 H), 1.81 - 1.96 (m, 2 H), 1.27 (t, J=7.2 Hz, 3 H), 1.15 - 1.24 (m, 6 H)

[0087] (c) 5-Azido-1-cyano-3,3-dimethyl-2-pentanone [ka] A heat gun dried 100 mL round bottom flask fitted with a stir bar, rubber septum, nitrogen inlet, and thermocouple probe was charged with THF (20 mL) and iPr2NH (1.59 mL, 11.3 mmol, 2.1 equiv, final concentration 0.36 M). While the solution was cooled at 0 °C, a solution of nBuLi (1.28 M in hexanes, 8.64 mL, 10.8 mmol, 2.0 equiv, final concentration 0.34 M) was added dropwise (~5 min) at a rate such that the internal temperature did not exceed +10 °C, stirred at 0 °C for 10 min, and cooled to -78 °C. Acetonitrile (0.59 mL, 11.3 mmol, 2.1 equiv, final concentration 0.36 M) was added dropwise via syringe at a rate such that the internal temperature did not exceed -65 °C. The reaction mixture was stirred at -78 °C for 15 min, then a solution of ethyl 4-azido-2,2-dimethylbutanoate (1.0 g, 5.4 mmol, 1.0 equiv, final concentration 0.17 M) in THF (5 mL) was added via syringe such that the internal temperature did not exceed -65 °C (~3 min). The reaction mixture was stirred at -78 °C for 10 min, warmed to 0 °C, and stirred at 0 °C for 15 min. The reaction mixture was diluted with EtOAc (50 mL) and 5% KHSO4 (50 mL). The aqueous layer was separated and extracted with EtOAc (3 x 50 mL). The combined organic phase was washed with brine (50 mL), dried over MgSO4, filtered, and concentrated. Purification by column chromatography (40 g silica gel cartridge; stepwise gradient elution: 20%, 30%, 50% EtOAc / hexanes) afforded 537 mg (2.98 mmol, 55%) of the desired ketone as a pale yellow oil. 1 H NMR (CDCl3, 300 MHz) δ 3.66 (s, 2 H), 3.37 (t, J=6.7 Hz, 2 H), 1.86 (t, J=6.8 Hz, 2 H), 1.16 - 1.27 (m, 6 H)

[0088] (d) 5-azido-1-cyano-3,3-dimethyl-2-pentanol [ka] A 25 mL round bottom flask equipped with a stir bar, rubber septum, and nitrogen inlet was charged with 5-azido-1-cyano-3,3-dimethyl-2-pentanone (537 mg, 2.98 mmol, 1.0 equiv, 0.25 M final concentration) and MeOH (12 mL) and cooled to 0 °C. NaBH4 (56 mg, 1.49 mmol, 0.5 equiv, 0.13 M final concentration) was added in one portion as a solid. The reaction mixture was stirred at 0 °C for 30 min. The reaction mixture was diluted with EtOAc (50 mL) and 5% aqueous KHSO4 (50 mL). The aqueous phase was separated and extracted with EtOAc (3 x 50 mL). The combined organic phase was washed with brine (40 mL), dried over MgSO4, filtered, and concentrated. Purification by column chromatography (40 g silica gel cartridge; stepwise gradient elution: 20%, 30%, 40%, 50% EtOAc / hexanes) afforded 482 mg (2.64 mmol, 89%) of the desired alcohol as a pale yellow oil. 1 H NMR (CDCl3, 300 MHz) δ 3.76 (ddd, J=9.1, 5.4, 3.4 Hz, 1 H), 3.34 - 3.50 (m, 2 H), 2.38 - 2.64 (m, 3 H), 1.68 - 1.82 (m, 1 H), 1.50 (ddd, J=14.1, 7.4, 6.6 Hz, 1 H), 0.96 (s, 3 H), 0.94 (s, 3 H)

[0089] (e) 5-azido-1-cyano-3,3-dimethyl-2-pentyl succinimidyl carbonate [ka] A heat gun dried 50 mL round bottom flask equipped with a stir bar, rubber septum, and nitrogen inlet was charged with NHS (455 mg, 3.96 mmol, 1.5 equiv, 211 mM final concentration), DCM (17 mL), and triphosgene (392 mg, 1.32 mmol, 0.5 equiv, 70.4 mM final concentration) and cooled to 0° C. Pyridine (0.774 mL, 8.71 mmol, 3.3 equiv, 464 mM final concentration) was added dropwise via syringe while the reaction mixture was cooled at 0° C. The reaction mixture was allowed to warm to ambient temperature and stirred at ambient temperature for 30 minutes. A solution of 5-azido-1-cyano-3,3-dimethyl-2-pentanol (482 mg, 2.64 mmol, 1.0 equiv., final concentration 150 mM) in THF (1 mL) was added dropwise via syringe. The reaction mixture was stirred at ambient temperature for 1 h, cooled at 0° C., and quenched by addition of HO (10 mL). The reaction mixture was further diluted with EtOAc (50 mL) and HO (50 mL). The organic phase was separated, washed with water (50 mL), 5% aqueous KHSO4, brine (50 mL), dried over MgSO4, filtered, and concentrated. Purification by column chromatography (40 g silica gel cartridge; stepwise gradient elution: 25%, 30%, 35%, 40% acetone / hexanes) afforded 636 mg (1.96 mmol, 75% yield) of the desired activated linker as a white solid. 1 H NMR (CDCl3, 300 MHz) δ 4.90 - 4.99 (m, 1 H), 3.32 - 3.50 (m, 2 H), 2.84 - 2.88 (m, 4 H), 2.66 - 2.82 (m, 2 H), 1.58 - 1.80 (m, 2 H), 1.08 (s, 6 H).

[0090] (2) 6-azido-1-cyano-3,3-dimethyl-2-hexyl succinimidyl carbonate (wherein, n=3, R 1 =CN,R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). (a) Ethyl 5-chloro-2,2-dimethylpentanoate [ka] A heat gun dried 500 mL round bottom flask fitted with a stir bar, rubber septum, nitrogen inlet, and thermocouple probe was charged with iPr2NH (5.30 mL, 37.4 mmol, 1.1 equiv, final concentration 266 mM) and THF (100 mL). With the reaction mixture cooled to 0 °C, a solution of nBuLi (1.28 M in hexanes, 27.8 mL, 35.7 mmol, 1.05 equiv, final concentration 254 mM) was added dropwise via syringe at a rate (~10 min) such that the internal temperature did not exceed +10 °C. The reaction mixture was stirred at 0° C. for 15 min, cooled to −78° C., and a solution of ethyl isobutyrate (4.60 mL, 4.0 g, 34.0 mmol, 1.0 equiv, 242 mM final concentration) in THF (5 mL) was added dropwise via syringe at a rate (~5 min) such that the internal temperature did not exceed −65° C. The reaction mixture was stirred at −78° C. for 45 min, then a solution of 1-bromo-3-chloropropane (3.37 mL, 34.0 mmol, 1.0 equiv, 242 mM final concentration) in THF (5 mL) was added at a rate such that the internal temperature did not exceed −68° C. The reaction mixture was stirred at −78° C. for 15 min, warmed to 0° C., and stirred at 0° C. for 15 min. The reaction mixture was diluted with EtOAc (100 mL) and 5% KHSO4 (100 mL). The aqueous phase was separated and extracted with EtOAc (3×50 mL). The combined organic phase was washed with brine, dried over MgSO4, filtered, and concentrated from toluene (10 mL x 2) to give 6.17 g (32.0 mmol, 90%) of the desired chloride as a pale yellow oil. 1 H NMR (CDCl3, 300 MHz) δ 4.13 (q, J=7.2 Hz, 2 H), 3.49 - 3.56 (m, 2 H), 1.62 - 1.83 (m, 4 H), 1.26 (t, J=7.1 Hz, 3 H), 1.17 - 1.22 (m, 6 H)

[0091] (b) Ethyl 5-azido-2,2-dimethylpentanoate [ka] A 100 mL round bottom flask equipped with a stir bar, rubber septum, and nitrogen inlet was charged with ethyl 5-chloro-2,2-dimethylpentanoate (6.17 g, 32.0 mmol, 1.0 equiv., final concentration 533 mM), DMSO (60 mL), and sodium azide (2.7 g, 42 mmol, 1.3 equiv., 690 mM). The reaction mixture was stirred behind a blast shield at 70° C. for 18 h. The reaction mixture was cooled to ambient temperature and diluted with EtOAc (200 mL) and H2O (100 mL). The organic phase was separated, washed with H2O (3×100 mL) and brine (100 mL), dried over MgSO4, filtered, and concentrated. Purification by column chromatography (40 g silica gel cartridge; stepwise gradient elution: 0%, 5%, 10%, 20% EtOAc / hexanes) afforded 5.40 g (27.1 mmol, 85%) of the desired azide as a pale yellow oil. 1 H NMR (CDCl3, 300 MHz) δ 4.13 (q, J=7.0 Hz, 2 H), 3.26 (t, J=5.9 Hz, 2 H), 1.46 - 1.65 (m, 4 H), 1.26 (t, J=7.2 Hz, 3 H), 1.19 (s, 6 H).

[0092] (c) 6-Azido-1-cyano-3,3-dimethyl-2-hexanone [ka] A heat gun dried 100 mL round bottom flask fitted with a stir bar, rubber septum, nitrogen inlet, and thermocouple probe was charged with THF (40 mL) and iPr2NH (3.0 mL, 21 mmol, 2.1 equiv, final concentration 320 mM). While the solution was cooled at 0 °C, a solution of nBuLi (1.28 M in hexanes, 15.4 mL, 20.0 mmol, 2.0 equiv, final concentration 305 mM) was added dropwise (~5 min) at a rate such that the internal temperature did not exceed +10 °C, stirred at 0 °C for 10 min, and cooled to -78 °C. Acetonitrile (1.10 mL, 21.0 mmol, 2.1 equiv, final concentration 322 mM) was added dropwise via syringe at a rate such that the internal temperature did not exceed -65 °C. The reaction mixture was stirred at -78 °C for 15 min, then a solution of ethyl 5-azido-2,2-dimethylpentanoate (2.04 g, 10.0 mmol, 1.0 equiv, final concentration 153 mM) in THF (4 mL) was added via syringe such that the internal temperature did not exceed -65 °C (~3 min). The reaction mixture was stirred at -78 °C for 10 min, warmed to 0 °C, and stirred at 0 °C for 15 min. The reaction mixture was diluted with EtOAc (50 mL) and 5% KHSO4 (50 mL). The aqueous layer was separated and extracted with EtOAc (3 x 50 mL). The combined organic phase was washed with brine (50 mL), dried over MgSO4, filtered, and concentrated. Purification by column chromatography (40 g silica gel cartridge; stepwise gradient elution: 15%, 20%, 30%, 40% EtOAc / hexanes) afforded 1.18 g (6.07 mmol, 59%) of the desired ketone as a pale yellow oil. 1 H NMR (CDCl3, 300 MHz) δ 3.61 (d, J=0.4 Hz, 2 H), 3.32 (t, J=6.3 Hz, 2 H), 1.42 - 1.68 (m, 5 H), 1.17 - 1.24 (m, 6 H).

[0093] (d) 6-Azido-1-cyano-3,3-dimethyl-2-hexanol [ka] A 25 mL round bottom flask equipped with a stir bar, rubber septum, and nitrogen inlet was charged with 6-azido-1-cyano-3,3-dimethyl-2-hexanone (1.18 g, 6.08 mmol, 1.0 equiv, 243 mM final concentration) and MeOH (25 mL) and cooled to 0 °C. NaBH4 (114 mg, 3.04 mmol, 0.5 equiv, 122 mM final concentration) was added in one portion as a solid. The reaction mixture was stirred at 0 °C for 30 min. The reaction mixture was diluted with EtOAc (50 mL) and 5% aqueous KHSO4 (50 mL). The aqueous phase was separated and extracted with EtOAc (3 x 50 mL). The combined organic phase was washed with brine (40 mL), dried over MgSO4, filtered, and concentrated. Purification by column chromatography (40 g silica gel cartridge; stepwise gradient elution: 20%, 30%, 40%, 50% EtOAc / hexanes) afforded 1.1 g (5.61 mmol, 97%) of the desired linker alcohol as a pale yellow oil. 1 H NMR (CDCl3, 300 MHz) δ 3.68 - 3.79 (m, 1 H), 3.30 (t, J=6.6 Hz, 2 H), 2.39 - 2.60 (m, 2 H), 2.23 - 2.29 (m, 1 H), 1.20 - 1.68 (m, 4 H), 0.93 (s, 3H), 0.92 (s, 3H)

[0094] (e) 6-Azido-1-cyano-3,3-dimethyl-2-hexyl succinimidyl carbonate [ka] A heat gun dried 50 mL round bottom flask equipped with a stir bar, rubber septum, and nitrogen inlet was charged with NHS (440 mg, 3.83 mmol, 1.5 equiv, 217 mM final concentration), DCM (17 mL), and triphosgene (378 mg, 1.28 mmol, 0.5 equiv, 72 mM final concentration) and cooled to 0° C. Pyridine (0.68 mL, 8.4 mmol, 3.3 equiv, 48 mM final concentration) was added dropwise via syringe while the reaction mixture was cooled at 0° C. The reaction mixture was allowed to warm to ambient temperature and stirred at ambient temperature for 30 minutes. A solution of 6-azido-1-cyano-3,3-dimethyl-2-hexanol (500 mg, 2.55 mmol, 1.0 equiv, 144 mM final concentration) in THF (1 mL) was added dropwise via syringe. The reaction mixture was stirred at ambient temperature for 1 h, cooled at 0° C., and quenched by the addition of H2O (10 mL). The reaction mixture was further diluted with EtOAc (50 mL) and H2O (50 mL). The organic phase was separated, washed with water (50 mL), 5% aqueous KHSO4, brine (50 mL), dried over MgSO4, filtered, and concentrated. Purification by column chromatography (40 g silica gel cartridge; stepwise gradient elution: 25%, 30%, 35%, 40% acetone / hexanes) afforded 638 mg (1.89 mmol, 74% yield) of the desired activated linker as a white solid. 1 H NMR (CDCl3, 300 MHz) δ 4.93 (dd, J=7.1, 5.3 Hz, 1 H), 3.32 (s, 2 H), 2.86 (s, 4 H), 2.71 - 2.79 (m, 2 H), 1.29 - 1.72 (m, 4 H), 1.05 (s., 3 H), 1.04 (s., 3 H).

[0095] Example 3 Preparation of a linker of formula (I) where Z=protected ketone [ka] Succinimidyl 2,2-diethoxypropanoate Concentrated H2SO4 (0.5 mL) was added to an ice-cold mixture of pyruvic acid (8.8 g, 100 mmol) and triethyl orthoformate (40 mL, 240 mmol). The mixture was stirred on ice for 30 min, then diluted with CHCl2, washed twice with cold water followed by brine, dried over MgSO4, filtered and concentrated to give crude 2,2-diethoxypropanoic acid (11.16 g, 69 mmol). This was dissolved in 250 mL of CHCl2 and treated with N-hydroxysuccinimide (8.7 g, 76 mmol) followed by dicyclohexylcarbodiimide (15.6 g, 76 mmol) for 2 h. The thick white slurry was filtered to remove dicyclohexylurea and then passed through a pad of silica gel to remove most of the yellow color. The silica gel was rinsed with 1:1 EtOAc / hexanes and the combined eluents were concentrated. The residue was crystallized from warm 20% EtOAc / hexanes to give the first crop of succinimidyl ester (11.73 g, 45 mmol) as white crystals. Additional product was obtained by chromatography of the mother liquor (0-60% EtOAc / hexanes) on SiO2 followed by crystallization to give a total of 13.0 g of product (50% overall from pyruvic acid).

[0096] Ethyl 3-[(2,2-diethoxypropanoyl)amino]-2,2-dimethylpropanoate A solution of ethyl 3-amino-2,2-dimethylpropanoate (CombiBlocks; 1.82 g, 10 mmol) and succinimidyl 2,2-diethoxypropanoate (2.6 g, 10 mmol) in 10 mL of DMF was treated with N,N-diisopropylethylamine (3.5 mL, 20 mmol) at ambient temperature for 1 h. The mixture was diluted with EtOAc and washed successively with water, 5% KHSO4, saturated NaHCO3, and brine, then dried over MgSO4, filtered, and concentrated. Chromatography on SiO2 (0-70% MTBE / hexanes) afforded the product ester (2.48 g, 86%) as a colorless oil.

[0097] Conversion to succinimidyl carbonate was carried out following the procedures described for the other linkers above.

[0098] Example 4 X=N(R 6 ) Preparation of a linker of formula (I) where [ka] Step 1. 4-Azido-1-cyano-3,3-dimethyl-2-butyl 4-(N,N-diethylcarboxamido)phenylcarbamate Pyridine was added to a solution of 4-azido-1-cyano-3,3-dimethyl-2-butanol and triphosgene in THF. After 15 min, the mixture was filtered and concentrated. The residue was dissolved in CH2Cl2 and treated with N,N-diethyl 4-aminobenzamide and triethylamine. After 1 h, the mixture was diluted with CH2Cl2, washed with 5% KHSO4, water, and brine, then dried over MgSO4, filtered, and evaporated. The product crystallized.

[0099] Step 2. N-Chloromethyl 4-azido-1-cyano-3,3-dimethyl-2-butyl 4-(N,N-diethylcarboxamido)-phenylcarbamate A mixture of the carbamate from step 1 (1 mmol), paraformaldehyde (120 mg), chlorotrimethylsilane (0.5 mL), and 1,2-dichloroethane (4 mL) was sealed in a screw-cap vial and heated for 12 min at 50° C. After cooling, the mixture was concentrated and the residue was redissolved in 10 mL of MTBE, filtered, and reconcentrated to give the N-chloromethyl carbamate as a colorless oil.

[0100] Example 5 Preparation of Compounds of Formula (II) Wherein Z=Azide [ka] n=1, R 4 =CH3, Z=N3, and D is Phe 11 shows a method for preparing a compound of formula (II), which is octreotide linked via the alpha-amino group of

[0101] Boc-Octreotide A 58.4 mg / mL solution of t-butyl succinimidyl carbonate (385 μL) was added to a mixture of octreotide acetate (128 mg), and N,N-diisopropylethylamine (0.2 mL) in 2 mL of amine-free N,N-dimethylformamide. After 4 hours, HPLC analysis indicated the presence of 91.6% mono-Boc-octreotide, 3.0% di-Boc-octreotide, and 5.4% octreotide. UV spectrophotometric analysis indicated a total octreotide concentration of 43.5 mM. This solution was used without purification.

[0102] Common methods The compound of formula (I) was dissolved in amine-free DMF at 40 mM. An aliquot of Boc-octreotide from above (500 μL, 21.8 μmol total octreotide) was mixed with the solution of the compound of formula (I) (540 μL, 21.6 μmol) and kept at ambient temperature for 16 hours. The reaction was diluted with 5 mL of ice-cold 0.1 M acetic acid and the precipitated linker-peptide was collected by centrifugation. The pelleted material was dissolved in 4 mL of methanol and analyzed by preparative HPLC (C 18 The product was purified using a 20-80% MeCN / HO / 0.1% TFA buffer. After drying, the product was dissolved in 1 mL of ice-cold 95:5 trifluoroacetic acid / water to remove the Boc group, held for 10 min, and then precipitated by adding 10 mL of cold ether and dried.

[0103] Compounds of formula (II) which have been prepared according to this method are listed below. Nα-[(4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butoxy)carbonyl]octreotide (n=1, R 1 =SO2N(CH3)2, R 2 =H, R 4= CH3, Z = N3, D = octreotide attached via the α-amino group). Yield 23.6 mg (84%), LC-MS was [M+H] + =1295.75 (expected value 1295.6). Nα-[(4-azido-1-((N-ethyl-N-methylamino)sulfonyl)-3,3-dimethyl-2-butoxy)carbonyl]octreotide (n=1, R 1 =SO2N(CH3)(CH2CH3), R 2 =H, R 4 = CH3, Z = N3, D = octreotide attached via α-amino group). Yield 21.6 mg, LC-MS [M+H] + =1309.75 (expected value 1309.6). Nα-[(4-azido-1-((morpholinosulfonyl)-3,3-dimethyl-2-butoxy)carbonyl]octreotide (n=1, R 1 =SO2N(CH2CH2)2O, R 2 =H, R 4 = CH3, Z = N3, D = octreotide attached via the α-amino group). Yield 19.9 mg (84%), LC-MS was [M+H] + =1337.7 (expected value 1337.6). Nα-[(4-azido-1-((N,N-bis(2-methoxyethyl)aminosulfonyl)-3,3-dimethyl-2-butoxy)carbonyl]octreotide (n=1, R 1 =SO2N(CH2CH2OCH3)2, R 2 =H, R 4 = CH3, Z = N3, D = octreotide attached via α-amino group). Yield 35.4 mg, LC-MS [M+H] + =1383.8 (expected value 1383.7). Nα-[(4-azido-1-((isopropylsulfonyl)-3,3-dimethyl-2-butoxy)carbonyl]octreotide (n=1, R 1 =SO2CH(CH3)2, R 2 =H, R 4= CH3, Z = N3, D = octreotide attached via α-amino group). Yield 16.8 mg, LC-MS [M+H] + =1294.7 (expected value 1294.6). Nα-[(4-azido-1-(1-cyano-3,3-dimethyl-2-butoxy)carbonyl]octreotide (n=1, R 1 =CN,R 2 =H, R 4 =CH3, Z=N3, D=octreotide attached via the α-amino group). Nα-[(4-azido-1-(1-(methylsulfonyl)-3,3-dimethyl-2-butoxy)carbonyl]octreotide (n=1, R 1 =SO2CH3, R 2 =H, R 4 =CH3, Z=N3, D=octreotide attached via the α-amino group).

[0104] N α -Linker-[Gln 28 ]-Exenatide [ka] N α -{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-[Gln 28 ] Exenatide In a 25 mL fritted SPE column, the protected [Gln 28] Exenatide (Fmoc α-amine) was swollen in 10 mL DMF for 30 min at ambient temperature. The DMF was removed by syringe filtration using a F / F Luer adapter and a 12 mL syringe, and the swollen resin was treated with 5% 4-methylpiperidine in DMF (2 x 10 mL for 5 min each, then 2 x 10 mL for 20 min each). The Fmoc-deprotected resin was then washed with DMF (10 x 10 mL) and the supernatant removed by syringe filtration. The washed resin was suspended in 8.4 mL of DMF and treated with 3.6 mL of O-{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyl}-O'-succinimidyl carbonate (0.10 M in DMF, 0.36 mmol, 30 mM final concentration) and 4-methylmorpholine (40 μL, 0.36 mmol, 30 mM final concentration). The reaction mixture was agitated using an orbital shaker. After 20 h, the supernatant was removed by syringe filtration and the resin was washed sequentially with DMF (5 × 15 mL) and CHCl (5 × 15 mL). Kaiser test was negative for free amines in the intermediate linker-modified resin. The resin was then treated with 10 mL of pre-chilled (0° C.) 90:5:5 TFA:TIPS:H2O with gentle agitation on an orbital shaker. After 2 h, the resin was vacuum filtered and washed with TFA (2×1.5 mL). The filtrate was concentrated to ∼6 mL by rotary evaporation. The crude linker-peptide was precipitated by adding the TFA concentrate dropwise to 40 mL of −20° C. MTBE in a tared 50 mL Falcon tube. After incubation at −20° C. for 10 min, the crude linker-peptide suspension was pelleted by centrifugation (3000×g, 2 min, 4° C.) and the supernatant was decanted. The resulting pellet was suspended in 40 mL of −20° C. MTBE, vortexed to mix, centrifuged, and decanted as above. After drying under high vacuum, the pellet was isolated as an off-white solid (575 mg) and then dissolved in 8 mL of 5% AcOH (~70 mg / mL). After heating in a 50 °C water bath for 45 min, the solution was purified by preparative C18-HPLC to give 13 mL of the title compound (3.33 mM, 43 μmol, A 280(by HPLC) was obtained as an aqueous solution. Lyophilization gave 235 mg of a white solid. C18-HPLC purity (measured at 280 nm): 90.0% (RV=11.47 mL) M av Calculated value 4476.9; Measured value 4476

[0105] Example 6 Preparation of Compounds of Formula (II) Wherein Z=Ketone [ka] The preparation of compounds of formula (II) where Z=ketone is carried out by the steps of: 1 =SO2N(CH3)2, R 2 =H, each R 4 Illustrated by an example where =CH3, Z=NH-pyruvoyl, and D=Nα-linked octreotide.

[0106] A 250 mM solution of t-butyl succinimidyl carbonate in DMF (440 μL) was added to a mixture of octreotide acetate (128 mg) and N,N-diisopropylethylamine (0.2 mL) in 2 mL of amine-free DMF. After 1 h, HPLC analysis showed the presence of 90% mono-Boc-octreotide, 2.5% di-Boc-octreotide, and 7% octreotide. A 100 μL solution of 4-(2-diethoxypropionamido)-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (Example 3; 54 mg) in DMF was added. The mixture was kept at ambient temperature for 16 h, then diluted with EtOAc and washed with 5% KHSO4 and then with brine. After drying over Na2SO4, the mixture was filtered and concentrated to give the fully protected intermediate as a foam, which was dissolved in 1 mL of CH3CN and treated with 1 mL of 2N HCl at 50°C for 30 min. The solution was cooled to ambient temperature, diluted with 2 mL of water, and carefully added to 5 mL of 1M NaHCO3. The precipitated product was collected by centrifugation, washed with water and dichloromethane, and dissolved in 5 mL of methanol.

[0107] Example 7 Y=N(R 6 ) CH2, [ka] A solution of SN-38 (100 mg) in 10 mL of 1:1 DMF / THF was cooled on ice and treated dropwise with 1 M potassium tert-butoxide (0.26 mL, 1 equiv). The resulting orange suspension was stirred for 30 min, then a 1 mL THF solution of N-(chloromethyl)carbamate linker (Example 4; 1 mmol) was added. The orange color gradually faded and the suspension became clear. The mixture was quenched with 10% aqueous citric acid and then extracted with ethyl acetate. The organic extract was washed with water and brine, then dried over MgSO4, filtered and evaporated. Purification by chromatography on SiO2 using a gradient of 0-100% acetone in hexanes gave n=1, R 1 =CN,R 2 =H, each R 4 =Me, D=SN-38, Y=N(R 6 )CH2(where R 6 = 4-(N,N-diethylcarboxamidophenyl)), and Z = azide, to give a linker-drug of formula (II).

[0108] The corresponding compounds where Z = amine were prepared as follows: A THF solution of the compound where Z = azide was added to a mixture of 1 M trimethylphosphine in THF and acetic acid. After gas evolution ceased, water was added and the mixture was stirred for 1 h and then concentrated to dryness. The residue was partitioned between water and ethyl acetate and the aqueous phase was collected and dried. Final purification by preparative HPLC used a gradient of 0-100% acetonitrile / water / 0.1% TFA.

[0109] Example 8 M is a soluble PEG; Z * = 1,2,3-triazole, A mixture of 20 kDa 4-arm PEG-tetra(cyclooctyne) (prepolymer B, prepared according to Example 14 below) and the linker-drug of Example 5, where Z=azide, in acetonitrile was kept at 50° C. for 16 minutes. Dialysis (12 kDa SpectraPor2) against water followed by methanol afforded the soluble 4-arm PEG, where M is soluble and Z is soluble. * to obtain the purified conjugate of formula (III), which is a 1,2,3-triazole.

[0110] Example 9 M is a soluble PEG; Z * = Formation of a conjugate of formula (III) A 20 kDa 4-arm PEG-tetra(aminooxyacetamide) was prepared by reacting 20 kDa 4-arm PEG-tetraamine (100 mg, NOF America) with excess (Boc-aminooxy)acetic acid in the presence of HATU and N,N-diisopropylethylamine in DMF. After 1 h, the PEG was precipitated by slow addition to stirred MTBE, collected by centrifugation, and dried under vacuum. This was dissolved in 2 mL of 1:1 CHCl / CFCOH, held for 1 h, and evaporated to dryness. The residue was dissolved in 2 mL of THF and the product was precipitated by slow addition to stirred MTBE, collected by centrifugation, and dried under vacuum.

[0111] A 1:1 mixture of 20 kDa 4-arm PEG-tetra(aminooxyacetamide) and linker-drug where Z=ketone from Example 6 in DMSO / 0.1 M acetic acid was kept at 50° C. for 16 hours. Dialysis against water followed by methanol (12 kDa SpectraPor2) revealed that M was a soluble 4-arm PEG and Z was a soluble 4-arm PEG. * to obtain the purified conjugate of formula (III), which is an oxime.

[0112] Example 10 M is a soluble PEG; Z *Formation of a conjugate of formula (III), A THF mixture of 20 kDa 4-arm PEG-tetra(succinimidyl ester) (JenKem), N,N-diisopropylethylamine, and the linker-drug where Z=amine from Example 7 was stirred for 1 h. Dialysis against water followed by methanol (12 kDa SpectraPor2) revealed that M was a soluble 4-arm PEG and Z was a soluble 4-arm PEG. * To obtain the purified conjugate of formula (III), which is a carboxamide.

[0113] Example 11 Formation of the conjugate of formula (III) where M is an insoluble degradable PEG microsphere P 1 and P 2 Both are 10 kDa 4-arm PEGs, and Z * = 1,2,3-triazole, n = 1, each R 4 =CH3, R 1 =SO2N(CH3)2, R 2 =H, and W = CH((CH2)4NH2)C(=O)NH (i.e., x = 0, y = 4, z = 0, B = NH2, and C * A suspension (100 mL) of PEG microspheres of formula (IV), where Z' = carboxamide (prepared according to Example 14 below), was activated by reaction with 4-cyclooctynylsuccinimidyl carbonate and N,N-diisopropylethylamine in acetonitrile. The resulting hydrogel, containing multiple reactive groups Z' = cyclooctynyl, was then suspended in 50 mM acetate buffer, pH 5, and reacted with Z = azide, n = 1, each R 4 =CH3, R 1 =SO2N(CH3)2, R 2The microspheres were reacted with a solution of linker-drug of formula (II), where D=H and D=-HGEGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAP-NH2 (exenatide-[N28Q] (Example 5). After 48 hours at 50°C, the microspheres were washed extensively with acetate buffer to remove unconjugated peptide. Analysis showed that the packed microsphere slurry contained 2.1 µmol link peptide / mL of slurry.

[0114] Example 12 release kinetics Conjugates were dissolved at 0.25-2 mM in 100 mM buffer and kept at 37°C in a thermostated HPLC autosampler. Samples (5-10 μL) were periodically removed and analyzed by HPLC (Phenomenex Jupiter 5um 4.6×150 mm C 18 The columns were injected into a 100-well column (reverse phase) and eluted with a linear gradient of 0-100% MeCN / HO / 0.1% TFA at 1 mL / min. Peaks were detected at 280 nm (peptide) or 350 nm (dinitrophenyl-Lys) and combined to obtain the peak areas of the conjugate and released peptide. The extent of drug release was calculated as (area of ​​drug released) / [(area of ​​drug released)+(area of ​​conjugate)]. The α-amine-linked octreotide described in Example 5 above was conjugated to 20 kDa MeO-PEG-cyclooctyne as described in Example 8. The half-life of release at pH 7.4 was calculated from the results obtained at a given pH using the following formula: t 1 / 2 (pH7.4) = ln(2) 10 (X-7.4) / k obs [Table 1] Table 1. Release kinetics of α-linked octreotide from soluble PEG conjugates in borate buffer at various pH values ​​and temperatures. a t 1 / 2,37℃ = (t 1 / 2,38℃ )* 1.143 (see the Arrhenius equation in SI, Santi PNAS 2012). In all cases, n = 1, R 2 =H, each R 4 = Me, Z = N3, Y is absent, and D = Nα-linked octreotide.

[0115] Example 13 Kinetics of the Aza-Michael Reaction Kinetics of the forward reaction In each of three 1.5 mL glass HPLC vials, a 5 mM DMSO solution of either standard, β-methyl or gem dimethylvinylsulfone (0.1 mL, 0.5 μmol, final concentration 0.5 mM) was added to 0.9 mL of pre-heated glycine cleavage buffer A, B, or C. The vials were kept in a heated (37 °C) HPLC autosampler and the aza-Michael reaction was monitored periodically by C18-HPLC. Cleavage Buffer A: 1.1 M glycine (final concentration 1.0 M), 0.11 M HEPES, pH 7.4, @37°C. Cleavage Buffer B: 1.1 M Glycine (final concentration 1.0 M), 0.11 M Bicine, pH 8.4, @37°C. Cleavage buffer C: 0.11 M glycine (final concentration 0.10 M), pH 9.5, @37°C. K eq was calculated using the following formula: K eq = K eq app / [Gly] K eq app = [GA] eq / [VS] eq = stable value / (0.5mM-stable value) [Gly] = glycine concentration (M) [GA] = glycine adduct concentration (mM) [VS] = vinyl sulfone concentration (mM) Stable values ​​(mM) were determined using Prism.

[0116] The two unknowns, kf (association) and kr (dissociation), are the K eq app and k determined by Prism fitting obs It is calculated using the following two formulas: k obs = k f [Gly] + k r k f [Gly] / k r = K eq app

[0117] Kinetics of the reverse reaction The rate of the dissociative retro-aza-Michael reaction (Figure 2.3A) was measured directly from the isolated glycine adducts. Each purified glycine adduct was diluted into HEPES buffer at pH 7.4 at 37 °C without added glycine. The concentration of glycine adduct from the start was plotted against time and each curve was fitted to a first order decay with Prism (Figure 2.3B). For each reaction, the calculated infinity value was <1 μM, indicating that the reaction proceeded to completion, and therefore the k r is essentially k obs is equivalent to 0.7-3.8 mM DMSO solutions of either standard, β-methyl or gem dimethylsulfo-DIBO glycine adducts (11 nmol, final concentration 50 μM) were added to 0.2 mL of pre-warmed 0.1 M HEPES, pH 7.0, containing enough water to adjust the final volume to 0.22 mL. The reaction vials were kept in a heated (37 °C) HPLC autosampler and the retro-aza-Michael reaction was monitored periodically by C18-HPLC. The concentration (μM) of glycine adduct [GA] from the start was calculated using the following equation and plotted against time using Prism: [GA] = ga / (ga+vs)*50μM [GA] = glycine adduct concentration (μM) ga = integrated HPLC peak area of ​​glycine adduct (254 nm) vs = HPLC peak area accumulated for vinyl sulfone (254 nm) 50 μM = [GA]i and maximum possible value [VS] kr (dissociation) was determined by fitting the data to a first order decay of Prism.

[0118] Example 14 Fabrication of degradable PEG hydrogels [ka] The hydrogel of the present invention reacts to form a connecting functional group C * The hydrogel is prepared by polymerization of two prepolymers comprising groups C and C' to form: The prepolymer connection to one of C or C' further comprises a cleavable linker, which is introduced by reaction with a molecule of formula (I) to introduce a cleavable linker to each crosslink of the hydrogel.

[0119] In one embodiment, the first prepolymer comprises a 4-arm PEG, each arm of which is terminated with an adaptor unit bearing two mutually unreactive ("heterologous") functional groups B and C. B and C can initially exist in protected form, allowing selective chemical reactions in a subsequent step. In certain embodiments, the adaptor unit is a derivative of an amino acid, particularly lysine, cysteine, aspartic acid, or glutamic acid, including derivatives in which the alpha amine group has been converted to an azide, e.g., the monoester of 2-azidoglutaric acid. The adaptor unit comprises a connecting functional group A formed by condensation of a functional group A on each prepolymer arm with the associated functional group A' on the adaptor unit. * The second prepolymer comprises a 4-arm PEG, each arm terminated with a functional group C' having complementary reactivity to group C of the first prepolymer, and C and C' react to form C * When the polymer is formed, crosslinking between the two prepolymers occurs.

[0120] As an illustrative example, a first prepolymer was prepared as follows: H-Lys(Boc)-OH was acylated with a linker of formula (I) where Z=azide to give an adaptor unit where A=COOH, B=Boc-protected NH2, and C=azide. This was coupled to a 20 kDa 4-arm PEG-tetraamine and the Boc group was removed to give A * A first prepolymer was obtained in which B = amide, B = NH2, and C = azide, and a cleavable linker of formula (I) was incorporated into the bond between each arm and group C of the first prepolymer. The corresponding second prepolymer was prepared by acylation of a 20 kDa 4-arm PEG-tetraamine with 5-cyclooctynyl succinimidyl carbonate to obtain a second prepolymer with C' = cyclooctyne. When the first and second prepolymers were mixed, the reaction of the C = azide group with the C' = cyclooctyne group formed the corresponding triazole group, thereby crosslinking the two prepolymers into a three-dimensional network, where each crosslink contains a cleavable linker obtained by incorporation of a compound of formula (I), and where each node obtained by incorporation of the first prepolymer contains a residual functional group B = NH2 that can be derivatized for the addition of further linkers, drugs, fluorophores, metal chelators, etc.

[0121] A * Prepolymer A, where A = amide, B = amine, and C = azide. [ka] (1)N α -Boc-N ε -{4-Azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys-OH A solution of Boc-Lys-OH (2.96 g, 12.0 mmol) in 28 mL of HO was treated sequentially with 1 M aqueous NaOH (12.0 mL, 12.0 mmol), 1 M aqueous NaHCO3 (10.0 mL, 10.0 mmol), and a solution of O-{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyl}-O'-succinimidyl carbonate (3.91 g, 10.0 mmol, final concentration 0.1 M) in 50 mL of MeCN. After stirring at ambient temperature for 2 h, the reaction was judged complete by C18-HPLC (ELSD). The reaction was quenched with 30 mL of 1 M KHSO4 (aq). The mixture was partitioned between 500 mL of 1:1 EtOAc:HO. The aqueous phase was extracted with 100 mL of EtOAc. The combined organic phase was washed with H2O and brine (100 mL each), then dried over MgSO4, filtered, and concentrated by rotary evaporation to give the crude title compound (5.22 g, 9.99 mmol, 99.9% crude yield) as a white foam. C18-HPLC, purity determined by ELSD: 99.1% (RV = 9.29 mL). LC-MS (m / z): calculated 521.2; found 521.3 [MH] - . (2)N α -Boc-N ε -{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys-OSu Dicyclohexylcarbodiimide (60% in xylene, 2.6 M, 4.90 mL, 12.7 mmol) was dissolved in N α -Boc-N ε-{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys-OH (5.11 g, 9.79 mmol, final concentration 0.1 M) and N-hydroxysuccinimide (1.46 g, 12.7 mmol) were added to a solution of 98 mL of CHCl. ​​The reaction suspension was stirred at ambient temperature and monitored by C-HPLC (ELSD). After 2.5 h, the reaction mixture was filtered and the filtrate was loaded onto a SiliaSep120 g column. The product was eluted with a stepwise gradient of acetone in hexane (0%, 20%, 30%, 40%, 50%, 60%, 240 mL each). The fractions containing the purified product were combined and concentrated to give the title compound (4.95 g, 7.99 mmol, 81.6% yield) as a white foam. C18-HPLC, purity determined by ELSD: 99.7% (RV = 10.23 mL). LC-MS (m / z): calculated 520.2; found 520.2 [M+H-Boc] + .

[0122] (3)(N α -Boc-N ε -{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys)4-PEG 20kDa PEG 20kDa -(NH)4 (20.08 g, 0.9996 mmol, 3.998 mmol of NH2, final concentration 0.02 M NH2) was dissolved in 145 mL of MeCN. α -Boc-N εA solution of -{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys-OSu (2.976 g, 4.798 mmol) in 50 mL of MeCN was added. The reaction was stirred at ambient temperature and analyzed by C18-HPLC (ELSD). The starting material was converted to a single product peak via three slower eluting intermediate peaks. After 1 h, Ac2O (0.37 mL, 4.0 mmol) was added. The reaction mixture was stirred for an additional 30 min more and then concentrated to ~50 mL by rotary evaporation. The reaction concentrate was added to 400 mL of stirred MTBE. The mixture was stirred at ambient temperature for 30 min and then decanted. MTBE (400 mL) was added to the wet solid and the suspension was stirred for 5 min and decanted. The solid was transferred to a vacuum filter and washed / triturated with 3×100 mL MTBE. After drying on the filter for 10 min, the solid was transferred to a tared 250 mL HDPE packaging bottle. Residual volatiles were removed under high vacuum until a stable weight was reached to give the title compound (21.23 g, 0.9602 mmol, 96.1% yield) as a white solid. C18-HPLC, purity determined by ELSD: 89.1% (RV = 10.38 mL) with 10.6% impurity (RV = 10.08).

[0123] (4)(N ε -{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys)4-PEG 20kDa (N ε -{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys)4-PEG 20kDa(19.00 g, 0.8594 mmol, 3.438 mmol Boc, final concentration 0.02 M Boc) was dissolved in 86 mL of 1,4-dioxane. After stirring for 5 min to completely dissolve the PEG, 4 M HCl in dioxane (86 mL, 344 mmol HCl) was added. The reaction was stirred at ambient temperature and analyzed by C18-HPLC (ELSD). The starting material was converted to a single product peak via three faster eluting intermediate peaks. After 2 h, the reaction mixture was concentrated to .about.40 mL. To the concentrate was added THF (10 mL) and the solution was concentrated again to .about.40 mL. The viscous oil was poured into 400 mL of stirred Et2O. After stirring for 20 min at ambient temperature, the supernatant was decanted from the precipitate. The wet solid was transferred to a vacuum filter with 200 mL of Et2O and washed with Et2O (3 x 75 mL). The solid was allowed to dry on the filter for 10 min before being transferred to a tared 250 mL HDPE packaging bottle. Residual volatiles were removed under high vacuum overnight to give the title compound (17.52 g, 0.8019 mmol, 93.3% yield @ 4HCl) as a white solid. C18-HPLC, purity determined by ELSD: 99.2% (RV = 9.34 mL).

[0124] Prepolymer B, where C'=cyclooctynyl In a 4mL screw-top vial, PEG 20kDa-[NH2]4 (SunBritt PTE-200PA; 150 mg, 7.6 μmol PEG, 30.2 μmol NH2, 1.0 equiv, final amine concentration 20 mM), MeCN (1.5 mL), and iPr2NEt (7 μL, 40 μmol, 1.3 equiv, final concentration 27 mM) were charged. A solution of the active ester of cyclooctyne (39 μmol, 1.3 equiv, final concentration 27 mM) was added and the reaction mixture was stirred at ambient temperature. The reaction was monitored by C18-HPLC (20-80% B in 11 min) with ELSD. Upon completion, Ac2O (3 μL, 30 μmol, 1 equiv per starting NH2) was added to the reaction mixture and the mixture was stirred for 30 min. The reaction mixture was then concentrated to a thick oil and suspended in MTBE (20 mL). The resulting suspension was stirred vigorously for 10 min. The resulting solid was triturated three times with MTBE (20 mL) by vigorously mixing, pelleting in a centrifuge (2800 rpm, 4° C., 10 min), and removing the supernatant with a pipette. The resulting solid was dried under vacuum at ambient temperature within 30 min. A stock solution was prepared in 20 mM NaOAc (pH 5) with a target amine concentration of 20 mM. The cyclooctyne concentration was then confirmed by treatment with PEG7-N3 (2 eq.) and back titration of unreacted PEG7-N3 with DBCO-CO2H.

[0125] Macromonomers prepared using this technique include those in which the cyclooctyne group is MFCO, 5-hydroxycyclooctyne, 3-hydroxycyclooctyne, BCN, DIBO, 3-(carboxymethoxy)cyclooctyne, and 3-(2-hydroxyethoxy)cyclooctyne, prepared using MFCO pentafluorophenyl ester, 5-((4-nitrophenoxy-carbonyl)oxy)cyclooctyne, 3-(4-nitrophenoxycarbonyl)oxycyclooctyne, BCN hydroxysuccinimidyl carbonate, DIBO 4-nitrophenyl carbonate, 3-(carboxymethoxy)cyclooctyne succinimidyl ester, and 3-(hydroxyethoxy)cyclooctyne 4-nitrophenyl carbonate, respectively.

[0126] Fabrication of hydrogel microspheres Hydrogel microspheres were prepared and activated as described in Schneider et al. (2016) Bioconjugate Chemistry 27: 1210-15.

[0127] Example 15 Z=N3, n=1, R 1 = (4-methylphenyl)SO2, R 2 =H, each R 4 = Me, Y = absent, and D = Lys B28 The compound of formula (II) is insulin lispro linked via As an alternative to the preparation of compounds of formula (II) by solid phase peptide synthesis (see Example 5), compounds of formula (II) where D is a peptide can be formed by reaction of a preformed peptide with an activated linker of formula (I) under conditions where at least one amine group on the peptide is free for reaction. In cases where the peptide contains both an N-terminal alpha amine and one or more lysine epsilon amines, preferential attachment of the linker to the lysine epsilon amines can be obtained by carrying out the reaction in the presence of an excess of a tertiary amine at high pH or in an organic solvent.

[0128] One 10 mL vial of Humalog (100 U / mL) was adjusted to pH 5.4 using 0.1 N HCl, the resulting precipitate was collected by centrifugation, the pellet was washed with 2×15 mL ethanol, 1×15 mL methyl t-butyl ether (MTBE), and dried under vacuum. Dried insulin lispro (35 mg, 6 μmol) was dissolved in 3 mL dimethylformamide (DMF) and 30 μL (170 μmol) N,N-diisopropylethylamine (DIPEA). A 100 mM solution of 4-azido-3,3-dimethyl-1-(4-methylphenylsulfonyl)-2-butylsuccinimidyl carbonate in DMF (84 μL, 8.4 μmol) was added and the mixture was stirred at ambient temperature for 1 h. The mixture was evaporated to dryness under vacuum and the residue was dissolved in 10 mL of 3:1 water / acetonitrile / 0.1% trifluoroacetic acid. 21.2×150mm, Jupiter 5um 300A C 18 Purification by preparative HPLC using a reversed-phase column with a gradient of 30–50% acetonitrile / water / 0.1% TFA in 20 min at 15 mL / min afforded the B-chain Lys. 28 The pure azide linker-lispro was obtained with the linker attached via the amine of 1 = (4-methylphenyl)SO2, R 2 =H, each R 3 = Me, Y = absent, and D = Lys B28 (II), which is insulin lispro linked via

[0129] Similar linker-peptides of formula (II) include the peptide teduglutide, [Gly 2 ]GLP-2 was used to produce the compound.

[0130] Example 16 Insulin Lispro Releasing PEG Conjugate M = 20 kDa MeO-PEG, Z * = triazole, n=1, R 1 = (4-methylphenyl)SO2, R 2 =H, each R 4=Me, Y=absence, D=Lys B28 and q=1. A solution of 20 kDa methoxy-PEG-amine (BroadPharm, 100 mg, 5 μmol), DIPEA (3 μL, 17 μmol), and (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethylsuccinimidyl carbonate (BCN-OSu, Sigma, 2 mg, 7 μmol) in 1 mL of acetonitrile was stirred at ambient temperature for 1 h and then evaporated to dryness. The residue was dissolved in 1 mL of THF and the solution was added to 10 mL of MTBE with stirring. The precipitated PEG-cyclooctyne was collected, washed with MTBE, and dried under vacuum.

[0131] A mixture of azide-linker-insulin lispro (1.1 μmol) from Example 15 and PEG-cyclooctyne (20 mg, 1 μmol) in 1 mL of 1:1 isopropanol:citrate buffer (pH 4) was kept at ambient temperature for 4 hours and then dialyzed against water using a membrane with a cutoff of 12-14 kDa, followed by dialysis against methanol. The dialyzed product was evaporated to dryness to give MeO-PEG, Z = 20 kDa. * = triazole, n=1, R 1 = (4-methylphenyl)SO2, R 2 =H, Y=absent, D=Lys B28 and q=1.

[0132] When dissolved in 0.1 M borate buffer (pH 9.4, 37°C), the conjugate exhibited t 1 / 2 = 2.08 hours. This was achieved at pH 7.4 and 37°C with a t 1 / 2 =Estimated to be 208 hours.

[0133] R 1The related conjugate, where t = phenyl-SO2, was prepared similarly and dissolved in 0.1 M borate buffer (pH 9.4, 37 °C), at 1 / 2 = 0.8 hours. This was achieved at pH 7.4 and 37°C with a t 1 / 2 =Estimated to be 80 hours.

[0134] Example 17 Hydrogels containing releasable insulin lispro The azide-linker-insulin lispro of Example 15 was conjugated to the degradable PEG hydrogel of Example 14 to obtain a sustained release depot of insulin lispro. For the PEG hydrogel, prepolymer A was (Nα-Boc-Nε-{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys)4-PEG 10kDa and Prepolymer B is ((4-cyclooctynyloxy-carbonyl)amino)4-PEG 10kDa This means that P 1 and P 2 are both 10 kDa 4-arm poly(ethylene glycol)s, and Z * = triazole, n=1, R 1 = (N,N-dimethylamino)SO2, R 2 =H, each R 4 =CH3, and W=(CH2) x -CH[(CH2) y B]-(CH2) z C (where x=4, y=0, z=0, B=NH2) and C * = carboxamide, was obtained. The PEG hydrogel was formed as a microsphere as previously described by PCT Publication WO2019 / 152672 (herein incorporated by reference).

[0135] A loaded suspension of these hydrogel microspheres (B=NH2) in acetonitrile (3.5 g, containing 10.8 μmol NH2 by TNBS assay) was activated for linker-drug conjugation by reaction with BCN-OSu (16.2 μmol) and triethylamine (43.1 μmol) for 4.5 h. Acetic anhydride (10.8 μmol) was added to cap any unreacted amine groups, and after 2 h, the slurry was washed 5 times with 11 mL of acetonitrile, followed by 5 times with 11 mL of drug loading solvent (100 mM citrate in 1:1 iPrOH:HO, pH 3.0). The final loaded slurry was ~5.6 mL, containing 7.3 μmol of cyclooctyne (B=[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl]amino).

[0136] The mixture of activated hydrogel microspheres and azide-linker-insulin lispro in the drug loading solvent was gently mixed for 24 hours, and then the suspension was washed repeatedly with reaction buffer to remove unreacted azide-linker-insulin lispro and reaction by-products. The final microsphere preparation contained 1.5 μmol / mL insulin lispro, which was t 1 / 2 = Released at 350 hours.

[0137] Example 18 Releasable exenatide-containing hydrogel P 1 and P 2 Both are 10 kDa 4-arm poly(ethylene glycol), and Z * = triazole, n=1, R 1 = (N,N-dimethylamino)SO2, R 2 =H, each R 4 =CH3, and W=(CH2) x -CH[(CH2) y B]-(CH2) z C (where x=4, y=0, z=0, B=NH2) and C *The degradable hydrogel of Example 14, which is a = carboxamide, was activated by reaction with cyclooctynyl succinimidyl carbonate (5HCO-OSu) and then azide-linker-exenatide [N28Q] of Example 5 was attached. Amino microsphere slurry (1.2 mL) loaded in MeCN containing 10.3 μmol amine was combined with neat triethylamine (41.1 μmol) and 5HCO-NHS (15.4 μmol). The reaction was mixed end-over-end for 18 hours and the loading of amine was confirmed by a qualitative TNBS test (described in General Methods). Acetic anhydride was added (1 equivalent, 10.3 μmol) to cap the remaining free amine, and after 2 hours of reaction, the slurry was washed five times with 6 mL of acetonitrile. The final loaded slurry was 1.4 mL containing 10.3 μmol 5HCO. Two tared 10 mL syringes were loaded with ∼1 g of 5HCO-microsphere slurry in MeCN containing ∼9 μmol 5HCO. The slurry was washed 4 times with 6.5 mL of reaction solvent (100 mM citrate in 1:1 DMSO:HO, pH 3.0). N3-peptide was added to the loaded slurry at 1.2 equivalents relative to 5HCO (11 μmol N3) and incubated with stirring at 37° C. for 18 h.

[0138] The loaded microspheres were washed five times with 6.5 mL of reaction solvent (OD of the final wash). 280 The beads were washed three times with isotonic acetate buffer (10 mM sodium acetate, 143 mM NaCl, 0.05% polysorbate 20 (w / v), pH 5.0) and twice with isotonic acetate buffer containing 0.8% sodium carboxymethylcellulose. Payload concentration and loaded fraction were determined by solubilizing ∼50 μL of the loaded slurry (∼50 mg) in 9 volumes (∼450 μL) of 50 mM NaOH for 1 h at ambient temperature. Payload content was determined by the quantification of [Gln 28 ] Exenatide (E=5500M -1 cm -1) was measured by absorbance at 280 nm. PEG assays were performed on similar NaOH solubilized samples to measure the PEG concentration of each construct and compare to peptide concentration to determine fraction loading.

[0139] A sample of loaded microspheres (~50 mg) was placed in a 1.5 mL screw-top microcentrifuge tube and the release kinetics / degelation reaction was initiated by adding 19 volumes (0.95 mL) of 100 mM sodium borate buffer (pH 9.4) at 37 °C. Two reactions for each conjugate were initiated with an 18-h interval to capture as many time points as possible. Reactions were incubated with shaking in a water bath at 37 °C. At t = 0 and various time points, the microsphere slurry was pelleted at 20,000 x g, the visual presence or absence of a microsphere pellet was recorded, 20 μL of reaction supernatant was removed and quenched by adding 4 μL of 4 M acetic acid, and samples were stored at -20 °C. The concentration of exenatide [N28Q] in the reaction supernatant was determined by absorbance at 280 nm by Nanodrop UV-Vis. The state of the microspheres was also visually recorded at each time point (solid / present or solubilized / disappeared). 280 was plotted and fitted to a single exponential function to determine the release rate of each peptide. PEG assays were performed on the supernatant samples to measure the soluble PEG concentration at each time point and generate solubilization / reverse gelation curves. Assay of the microspheres showed a peptide content of 5.19 μmol / mL, corresponding to 95% loading of available B sites. At pH 9.4 and 37°C, these hydrogel microspheres had a t 1 / 2 = 17.5 hours, exenatide [N28Q] was released and dissolved with a degelation time of 32 hours.

[0140] Similar exenatide releasing hydrogels are prepared by replacing the cyclooctynyl succinimidyl carbonate used in the activation step with BCN-OSu, as shown in Example 17.

[0141] These exenatide-releasing hydrogel microspheres have the general formula shown in FIG. 7, where P 1 and P 2 are 4-arm poly(ethylene glycol), and Z * is a triazole, C * is a carboxamide, and B is * is a triazole and LD is the residue of a linker-drug of formula (II).

[0142] Example 19 Preparation of linker-oligonucleotides Phosphorothioate CpG oligonucleotide TLR9 receptor agonist conjugates were prepared as follows. [ka]

[0143] Process 1 N-[4-azido-3,3-dimethyl-1-(4-chlorophenyl)sulfonyl-2-butyloxycarbonyl]-Gly-OH A solution of glycine (18 mg, 0.24 mmol) in 0.56 mL of HO was treated sequentially with 1 M aqueous NaOH (0.24 mL, 0.24 mmol), 1 M aqueous NaHCO3 (0.20 mL, 0.20 mmol), and 4-azido-3,3-dimethyl-1-(4-chlorophenyl)sulfonyl-2-butylsuccinimidyl carbonate (92 mg, 0.20 mmol, final concentration 0.1 M) in 1.0 mL of MeCN. After stirring for 45 min at ambient temperature, the reaction was judged complete by C18-HPLC (ELSD). The reaction was quenched with 5 mL of 1 M aqueous KHSO4 and then partitioned with 20 mL of 1:1 EtOAc:HO. The aqueous phase was extracted with 5 mL of EtOAc. The combined organic phase was washed with H2O and brine (10 mL each), then dried over MgSO4, filtered, and concentrated by rotary evaporation to give the crude title compound (85 mg, 0.20 mmol, quantitative crude yield) as a cloudy film. C18-HPLC, purity determined by ELSD: 98.6% (RV=9.42 mL). nLC-MS (m / z): 35 Calculated Cl 417.1; Measured 417.0 [MH] - ; 37 Calculated Cl value: 419.1; measured value: 419.1 [MH] - .

[0144] Process 2 N-[4-azido-3,3-dimethyl-1-(4-chlorophenyl)sulfonyl-2-butyloxycarbonyl]-Gly-OSu Dicyclohexylcarbodiimide (60% in xylene, 2.6 M, 100 μL, 0.26 mmol) was added to a solution of N-[4-azido-3,3-dimethyl-1-(4-chlorophenyl)sulfonyl-2-butyloxycarbonyl]-Gly-OH (85 mg, 0.20 mmol, final concentration 0.1 M) and N-hydroxysuccinimide (30 mg, 0.26 mmol) in 1.9 mL of CHCl. ​​The reaction suspension was stirred at ambient temperature and monitored by C18-HPLC (ELSD). After 1 h, the reaction mixture was filtered through a cotton plug and the filtrate was loaded onto a SiliaSep4g column. The product was eluted with a stepwise gradient of acetone in hexane (0%, 20%, 30%, 40%, 50%, 60%, 70%; 25 mL each). Fractions containing the purified product were combined and concentrated to give the title compound (74 mg, 0.14 mmol, 70% yield) as a cloudy film, which was then dissolved in 1.4 mL of MeCN and stored at -20°C. C18-HPLC, purity determined by ELSD: 92.7% (RV=10.00 mL). The main impurity was a hydrolysis product (7.3%, @9.42 mL RV), which may have been formed during HPLC chromatography.

[0145] Process 3 3'-{N-[4-azido-3,3-dimethyl-1-(4-chlorophenyl)sulfonyl-2-butyloxycarbonyl]-Gly-aminoalkyl}-CpG-5' In a 15 mL Falcon tube, a solution of 0.78 mM CpG-3'-NH2 (900 μL, 0.70 μmol, final concentration 0.5 mM) in 0.11 M HEPES (pH 7.65, 22°C) was diluted with 340 μL of 0.11 M HEPES (final 100 mM HEPES, pH 7.4, 37°C). The solution was warmed in a 37°C water bath for 30 min and then treated with a solution of 100 mM N-[4-azido-3,3-dimethyl-1-(4-chlorophenyl)sulfonyl-2-butyloxycarbonyl]-Gly-OSu (140 μL, 14 μmol, final concentration 10 mM) in DMF. The reaction was kept at 37°C and periodically monitored by C18-HPLC. The starting material was converted to a single product peak at ∼90% within 30 min. The reaction was diluted to 10 mL with Milli-Q water and 2.5 mL of the solution was loaded onto each of four NAP-25 columns. The oligonucleotides were eluted from each column with 3.5 mL of Milli-Q water according to the manufacturer's protocol and the eluates were combined to obtain an A260 H2O A solution of 45 μM total oligonucleotide was obtained (14.0 mL, 0.63 μmol total oligo; 91% linker-oligo by C18-HPLC) as determined by [conc=0.65 / (290300)*100 / 5]. The oligo solution was then concentrated to 1.4 mL using two Amicon Ultra-4, 10 kDa spin filters and diluted with 10 mL of 10% ethanol at 20°C. H2O A solution containing 0.44 mM total oligonucleotide was obtained (1.4 mL, 0.62 μmol total oligo), as determined by [conc=0.64 / (290300)*1000 / 5]. C 18 The HPLC purity was determined at 260 nm: 90.9% (RV=5.98 mL). av , 10284 (calculated); measured value 10282 Da (ESI).

[0146] R 1 = methylsulfonyl, and R 1 = phenylsulfonyl. The corresponding linker-oligonucleotides were prepared similarly.

[0147] Example 20 Preparation of conjugated linker-oligonucleotides [ka] 3'-[4-branched-PEG 40kDa -BCN / N3-(GDM 4-ClPhSO2)-Gly-aminoalkyl]-CpG-5', 134BH32 In a 1.5 mL screw-cap Eppendorf tube, a solution of 3'-{N-[4-azido-3,3-dimethyl-1-(4-chlorophenyl)-sulfonyl-2-butyloxycarbonyl]-Gly-aminoalkyl}-CpG-5' (0.44 mM total oligo, 1.00 mL, 0.44 μmol total oligo) was diluted with 121 μL of 0.3 M MES buffer pH 6.0 (final buffer concentration 30 mM) and then diluted with 5 mM 4-arm PEG. 40kDa A solution of -BCN (88 μL, 0.44 μmol, final concentration 0.36 mM) in MeCN was added. The reaction was kept at ambient temperature and monitored by C18-HPLC. The starting material was converted to a single slower eluting product peak. After 18 h, the reaction mixture contained ~60% product. The reaction tube was placed in a heating block at 32 °C and stirred for 24 h, after which the reaction mixture contained ~67% product. The mixture was loaded onto a Phenomenex Jupiter C18 preparative column (150 × 21.2 mm) and the product was eluted with 20%–95% MeCN in 50 mM Et3N·HOAc (pH 7.0) over 20 min (8 mL / min). Fractions containing the purified product were combined and MeCN was removed by rotary evaporation to give a 260% yield. H2O An aqueous solution containing 14 μM total oligo (15 mL, 0.21 μmol) was obtained, as determined by [conc=0.80 / 290300) / 0.2 cm path]. The aqueous solution was further concentrated using two Amicon Ultra-15, 10 kDa spin filters, and the A H2O A solution containing 0.18 mM total oligonucleotide was obtained (1.4 mL, 0.25 μmol, 71% yield) as determined by [conc=0.26 / (290300)*1000 / 5]. C 18HPLC purity determined at 260 nm: 97.2% (RV=8.26 mL).

[0148] Example 21 Kinetics of oligonucleotide release from the conjugate In a 1.5 mL glass HPLC vial capped with multiple septa, 800 μL of 125 mM borate buffer (pH 9.0, @ 37°C) and 182 μL of HO were warmed in a 37°C autosampler for >30 min. 1 An aqueous solution of the conjugate of Example 19, where 3'-(4-chlorophenyl)sulfonyl (110 μM total oligo, 18 μL, total oligo final concentration 2 μM) was added to each and the cleavage reaction was periodically monitored by C18-HPLC. Product formation, CpG-3'-NH2 HPLC peak area (260 nm) as a fraction of the total area at 260 nm, was plotted against time to obtain the average t 1 / 2 = 1.2 hours, which was estimated to be 48 hours at pH 7.4.

[0149] All references disclosed herein are incorporated by reference in their entirety.

Claims

1. Formula (I) 【Chemistry 1】 (I) [In the formula, n is an integer from 0 to 6; R １ and R ２ are independently an electron withdrawing group, alkyl, or H, where R １ and R ２ at least one of is an electron withdrawing group; Each R ４ is independently １ -C ３ alkyl or two R ４ together with the carbon atoms to which they are attached form a 3- to 6-membered ring; X is a leaving group; and Z is a functional group for connecting the linker to the polymeric carrier. , the linker.

2. X is a halogen, an active ester, an optionally substituted heteroaryl, or -N(R ６ ) CH ２ Cl, where R ６ is an optionally substituted C １ -C ６ 2. The linker of claim 1 which is alkyl, optionally substituted aryl, or optionally substituted heteroaryl.

3. X is halogen, N-succinimidyloxy, nitrophenoxy, pentahalophenoxy, imidazolyl, triazolyl, tetrazolyl, or -N(R ６ ) CH ２ The linker of claim 1 or 2, wherein the linker is Cl.

4. 4. The linker of any one of claims 1 to 3, wherein Z is selected from the group consisting of amine, aminooxy, ketone, aldehyde, maleimidyl, thiol, alcohol, azide, 1,2,4,6-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, cyclooctynyl, and protected versions thereof.

5. R １ and R ２ The electron-withdrawing group is -CN; -NO ２ ; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR ３ , -SOR ３ , or -SO ２ R ３ , [where R ３ is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ８ or -NR ８ ２ where each R ８ are independently H or optionally substituted alkyl, or two R ８ the groups together with the nitrogen to which they are attached form a heterocycle; or SR ９ 、 [where R ９ is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl. The linker according to any one of claims 1 to 4,

6. R １ and R ２ At least one of the groups is -CN or -SO ２ R ３ The linker according to any one of claims 1 to 5,

7. Each R ４ But independently, C １ -C ３ The linker of any one of claims 1 to 6, which is alkyl.

8. Formula (II) 【Chemistry 2】 (II) [In the formula, n is an integer from 0 to 6; R １ and R ２ are independently an electron withdrawing group, alkyl, or H, where R １ and R ２ at least one of is an electron withdrawing group; Each R ４ is independently １ -C ３ alkyl or two R ４ together with the carbon atoms to which they are attached form a 3- to 6-membered ring; Z is a linker - a functional group for connecting the drug to the polymeric carrier; D is a drug; and Y is absent when D is a drug attached through an amine, or Y is -N(R ６ ) CH ２ -, where R ６ is an optionally substituted C １ -C ６ alkyl, or optionally substituted aryl or heteroaryl. The linker-drug is represented by the formula:

9. 9. The linker-drug of claim 8, wherein Z is selected from the group consisting of amine, aminooxy, ketone, aldehyde, maleimidyl, thiol, alcohol, azide, 1,2,4,6-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, cyclooctynyl, and protected versions thereof.

10. D is octreotide (SEQ ID NO: 5), [N28Q]exenatide (SEQ ID NO: 1), insulin lispro (A chain: SEQ ID NO: 2; B chain: SEQ ID NO: 3; disulfide bridges: A6-A11, A7-B7, A20-B19), and teduglutide ([Gly ２ 10. The linker-drug of claim 8 or 9, which is a peptide selected from the group consisting of: GLP-2) (SEQ ID NO: 4).

11. R １ and R ２ The electron-withdrawing group is -CN; -NO ２ ; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR ３ , -SOR ３ , or -SO ２ R ３ , [where R ３ is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ８ or -NR ８ ２ where each R ８ are independently H or optionally substituted alkyl, or two R ８ the groups together with the nitrogen to which they are attached form a heterocycle; or SR ９ 、 [where R ９ is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl. The linker-drug of any one of claims 8 to 10,

12. R １ and R ２ At least one of the groups is -CN or -SO ２ R ３ The linker-drug of any one of claims 8 to 11,

13. Each R ４ But independently, C １ -C ３ The linker-drug of any one of claims 8 to 12, which is alkyl.

14. Formula (III) 【Chemistry 3】 (III) [In the formula, n is an integer from 0 to 6; R １ and R ２ are independently an electron withdrawing group, alkyl, or H, where R １ and R ２ at least one of is an electron withdrawing group; Each R ４ is independently １ -C ３ alkyl or two R ４ together with the carbon atoms to which they are attached form a 3- to 6-membered ring; D is a drug; and Y is absent when D is a drug attached through an amine, or Y is -N(R ６ ) CH ２ -, where R ６ is an optionally substituted C １ -C ６ alkyl, or optionally substituted aryl or heteroaryl; M is a polymeric carrier; q is an integer from 1 to 10 when M is a soluble polymer, or q is a large number when M is an insoluble matrix; and Z ＊ indicates the attachment point to M. The conjugate is shown by:

15. The conjugate of claim 14, wherein M is a soluble polymer and q is an integer from 1 to 10.

16. 15. The conjugate of claim 14, wherein M is an insoluble matrix and q is a number.

17. Z ＊ The conjugate of any one of claims 14 to 16, wherein comprises a carboxylic acid amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

18. R １ and R ２ The electron-withdrawing group is -CN; -NO ２ ; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR ３ , -SOR ３ , or -SO ２ R ３ , [where R ３ is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ８ or -NR ８ ２ where each R ８ are independently H or optionally substituted alkyl, or two R ８ the groups together with the nitrogen to which they are attached form a heterocycle; or SR ９ 、 [where R ９ is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl. The conjugate according to any one of claims 14 to 17,

19. R １ and R ２ At least one of the groups is -CN or -SO ２ R ３ The conjugate according to any one of claims 14 to 18,

20. Each R ４ But independently, C １ -C ３ The conjugate of any one of claims 14 to 19, which is alkyl.

21. Formula (IV) 【Chemistry 4】 (IV) [In the formula, n is an integer from 0 to 6; R １ and R ２ are independently an electron withdrawing group, alkyl, or H, where R １ and R ２ at least one of is an electron withdrawing group; Each R ４ is independently １ -C ３ alkyl or two R ４ together with the carbon atoms to which they are attached form a 3- to 6-membered ring; W is not present or 【Chemistry 5】 and where each of x, y, and z is independently an integer from 0 to 6; B is -NH ２ , -ONH ２ , ketone, aldehyde, -SH, -OH, -CO ２ H, a carboxamide group, or a group containing cyclooctyne or bicyclononyne, and C ＊ is a carboxamide, a thioether, a thiosuccinimidyl, a triazole, or an oxime; and P １ and P ２ is independently an r-arm polymer, where r is an integer from 2 to 8. The hydrogel is shown in FIG.

22. P １ and P ２ The hydrogel of claim 21 , wherein both are 4-arm polymers.

23. R １ and R ２ The electron-withdrawing group is -CN; -NO ２ ; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR ３ , -SOR ３ , or -SO ２ R ３ , [where R ３ is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ８ or -NR ８ ２ where each R ８ are independently H or optionally substituted alkyl, or two R ８ the groups together with the nitrogen to which they are attached form a heterocycle; or SR ９ 、 [where R ９ is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

23. The hydrogel of claim 21 or 22, wherein

24. R １ and R ２ At least one of the groups is -CN or -SO ２ R ３ The hydrogel according to any one of claims 21 to 23,

25. Each R ４ But independently, C １ -C ３ The hydrogel of any one of claims 21 to 24, wherein the alkyl group is an alkyl group.

26. The hydrogel of any one of claims 21 to 25, further comprising a drug bound to the hydrogel via a linker-drug of any one of claims 8 to 13.

27. The drugs were octreotide (SEQ ID NO: 5), [N28Q]exenatide (SEQ ID NO: 1), insulin lispro (A chain: SEQ ID NO: 2; B chain: SEQ ID NO: 3; disulfide bridges: A6-A11, A7-B7, A20-B19), and teduglutide ([Gly ２ ]GLP-2) (SEQ ID NO: 4).

28. The process is as follows: contacting a drug with a linker according to any one of claims 1 to 7 under conditions in which a linker-drug is formed; and Optionally, isolating the linker-drug; A method for producing a linker-drug according to any one of claims 8 to 13, comprising:

29. The group Z reacts with the group Z′ to form a remaining bond functional group Z ＊ 21. A method of making a conjugate according to any one of claims 14 to 20, comprising reacting a linker-drug according to any one of claims 8 to 13 with a polymeric carrier comprising an associated reactive group Z' under conditions to form

30. The process is as follows: (a) a multi-arm polymer comprising a linker of formula (V), each arm of which is terminated with a linker of any one of claims 1 to 7 comprising a reactive functional group Z; 【Chemistry 6】 (V) providing a first prepolymer having the formula: (b) providing a second prepolymer comprising a multi-arm polymer, each arm terminating in an associated reactive functional group Z′ that reacts with Z; (c) Z and Z′ react to form a remaining bond functional group Z ＊ mixing the two polymers under conditions to form (d) isolating the resulting hydrogel; A method for producing the hydrogel of any one of claims 21 to 25, comprising:

31. A method of making a drug releasing degradable hydrogel conjugate, comprising reacting the hydrogel of any one of claims 21 to 25 with the linker-drug of any one of claims 8 to 13, wherein the functional group B on the hydrogel reacts with the group Z on the linker-drug to form a conjugate.

32. Step (a) further comprises: reacting a first prepolymer of formula (V) with a linker-drug according to any one of claims 8 to 13 under conditions where the Z group of the linker-drug is attached to the first prepolymer via reaction with group B; 31. The method of claim 30, comprising:

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