Prodrug and drug having controllable drug release rate

Abstract

The present invention relates to a prodrug and drug having a controllable drug release rate. Specifically provided are a prodrug or a stereoisomer, deuterated substance, solvate, polymorph or pharmaceutically acceptable salt thereof. The prodrug contains a drug and a linker, wherein the drug is linked to the linker, and the linker contains a structure as represented by formula (1). The prodrug has a long half-life, does not require an enzymatic or redox environment, can controllably and spontaneously release the drug under physiological conditions, and exhibits a high drug delivery efficiency. The released drug exhibits a complete activity and is not affected by the linker or a macromolecular carrier.

Description

A prodrug and drug with a controllable drug release rate

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to CN application No. 202411918109.3 filed on December 24, 2024, and CN application No. 202510763971.X filed on June 9, 2025, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to the field of biomedicine, and more specifically to a prodrug and a drug having a controllable drug release rate. Background Technology

[0004] Many drugs and drug candidates suffer from short durations of action, resulting in ineffective or non-functional outcomes, due to unfavorable pharmacokinetic properties. For example, therapeutically valuable peptides and proteins often have serum half-lives of only minutes to hours due to metabolic instability and renal clearance. Similarly, therapeutically valuable nucleic acids such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) suffer from short half-lives and low delivery efficiency due to poor stability and cell impermeability.

[0005] One effective strategy to address this issue is conjugation with macromolecular carriers (such as polyethylene glycol (PEG), the Fc moiety of IgG, serum albumin, and other long-acting macromolecules) or with fatty acid carriers. Macromolecular carriers can slow down enzyme degradation and renal filtration, thereby prolonging the plasma half-life of the attached drug. To date, the use of PEG as a macromolecular carrier has been very successful. PEG is non-toxic and non-immunogenic, and its plasma half-life is size-dependent. Conjugation of drugs with PEG molecules below 40 kDa has been successfully applied to peptides, nucleic acids, and small molecule drugs, achieving half-lives of up to 7 days in humans. Fatty acids can form non-covalent bonds with human serum albumin (HSA), slowing down enzyme degradation and thus prolonging the plasma half-life of the drug.

[0006] All commercially available PEGylated protein conjugates are permanently PEGylated, and this stable conjugate has limitations. The attachment of large PEG molecules often reduces drug activity, requiring higher concentrations of conjugate to achieve the desired biological activity. Another limiting factor is that permanent PEGylation is generally unsuitable for small molecule drugs because larger carriers often prevent drug binding to the target and cell penetration.

[0007] Therefore, current research focuses on using conjugated compounds as drug delivery carriers. In this scenario, the drug attaches to a macromolecular carrier (usually PEG) via a linker. Under physiological conditions, the linker is cleaved by enzymes or chemicals, releasing the active drug. A key advantage of this type of conjugated compound is that the released drug retains its full activity, unaffected by the macromolecular carrier.

[0008] These types of macromolecules and drug conjugates all have potential drawbacks. Some require hydrolysis by serum proteases or esterases, while others require a reducing environment to cleave disulfide bonds. The released prodrug can spontaneously split into the active drug and small, potentially toxic alkylating agents.

[0009] Therefore, designing macromolecular-drug conjugates that do not require enzymes or redox environments and have controllable spontaneous release under physiological conditions will provide valuable therapeutic tools for disease treatment. Summary of the Invention

[0010] To address the problems existing in the prior art, the inventors of this application, through experimental research, provide a prodrug and drug-macromolecule conjugate capable of controlling and prolonging the release of drugs in vitro and in vivo. This prodrug and drug-macromolecule conjugate have a long half-life, do not require enzymes or redox environments, can controllably and spontaneously release drugs under physiological conditions, exhibit high drug delivery efficiency, and the released drug possesses complete activity, unaffected by linkers or macromolecular carriers.

[0011] Specifically, in a first aspect of the invention, the invention provides a prodrug or its stereoisomer, deuterated derivative, solvate, polymorph, or pharmaceutically acceptable salt thereof, said prodrug comprising a drug and a linker, said drug being linked to said linker, said linker comprising the structure shown in formula (1).

[0012] in:

[0013] n is an integer selected from 0 to 10, preferably an integer selected from 1 to 5, and more preferably 1, 2 or 3;

[0014] m is an integer selected from 0 to 10, preferably an integer selected from 1 to 5, and more preferably 1;

[0015] X is selected from CH2, O, and S, with CH2 and O being preferred.

[0016] Furthermore, when X is CH2, m and n satisfy conditions a)-d):

[0017] a) When m is 0, n is neither 1 nor 2.

[0018] b) When m is 2, n is not 1.

[0019] c) When n is 1, m is not 0 or 2.

[0020] d) When n is 2, m is not 0;

[0021] R 1 R 2 Each is independently selected from H, C1-C12 alkyl, electron-withdrawing group, C6-C14 aryl, 5-14 heteroaryl, and R 1 and R 2 In, at least one is an electron-withdrawing group; or, R 1 R 2 Together with the carbon atoms they are connected to, they form C6-C14 aryl or 5-14 heteroaryl groups; the C6-C14 aryl or the 5-14 heteroaryl group is optionally replaced by one or more electron-withdrawing groups and / or electron-donating groups;

[0022] R 3 It is N3;

[0023] Y does not exist or is selected from Preferably, the amino terminus is connected to the remaining portion of the general formula;

[0024] X1, X2, X3, and X4 are each independently selected from H and electron-withdrawing groups;

[0025] y is an integer selected from 0 to 10.

[0026] In some implementation schemes, R 1 R 2 Each is independently selected from H, -COR, -CO2R, -SOR, -SO2R, -CN, -NO2, -CF3, -CHF2, and R 1 and R 2 Not both H; or, R 1 R 2 Together with the carbon atoms bonded to them, they form

[0027] Each R is independently selected from H, C1-C12 alkyl, C1-C12 alkoxy, -NR a R b C6-C14 aryl or 5-14 heteroaryl, R a R b Each is independently selected from H and C1-C12 alkyl groups;

[0028] G is selected from direct bond, C (=O), SO, SO2, CZ2 or CZ2CZ2, where each Z is independently selected from H, Cl, F.

[0029] In some implementation schemes, R1 R 2 Each is independently selected from H, -SOR, -SO2R, and -CN, and R 1 and R 2 Not both H; or, R 1 R 2 Together with the carbon atoms bonded to them, they form

[0030] In some implementation schemes, R 1 R 2 Each is independently selected from H, -SO2R, and -CN, and R 1 and R 2 Not both H; or, R 1 R 2 Together with the carbon atoms bonded to them, they form

[0031] In some implementation schemes, R 1 R 2 In this context, one of them is H, and the other is selected from -SO2R or -CN; or, R 1 R 2 Together with the carbon atoms bonded to them, they form

[0032] In some embodiments, each R is independently selected from H, C1-C12 alkyl, -NR a R b R a R b Each is independently selected from H and C1-C12 alkyl groups.

[0033] In some embodiments, each R is independently selected from H, C1-C6 alkyl, -NR a R b R a R b Each is independently selected from H and C1-C6 alkyl groups.

[0034] In some embodiments, each R is independently selected from C1-C6 alkyl groups and -NR groups. a R b R a R b Each is independently selected from C1-C6 alkyl groups.

[0035] In some embodiments, each R is independently selected from methyl, isopropyl,

[0036] In some implementations, G is a direct key.

[0037] In some implementation schemes, As a whole, selected from

[0038] In some implementations, Y is absent or selected from Preferably, the amino terminus is connected to the remaining portion of the general formula.

[0039] In some implementation schemes, X1 and X2 are each independently selected from H, Cl, F, CN, NO2, CF3, and CHF2.

[0040] In some implementation schemes, either X1 or X2 is H, and the other is selected from H, Cl, F, CN, NO2, CF3, or CHF2.

[0041] In some implementation schemes, X1 and X2 are each independently selected from H, Cl, and F.

[0042] In some implementations, either X1 or X2 (e.g., X2) is H, and the other (e.g., X1) is selected from H, Cl, or F.

[0043] In some implementations, either X1 or X2 (e.g., X2) is H, and the other (e.g., X1) is H or Cl.

[0044] In some implementations, y is an integer selected from 0 to 10, preferably an integer selected from 1 to 5, and more preferably 1, 4 or 5.

[0045] In some implementations, Y is absent or selected from Preferably, the amino terminus is connected to the remaining portion of the general formula.

[0046] In some embodiments, the connector includes the structure shown in equation (1-1).

[0047] in:

[0048] n is an integer selected from 0 to 10, preferably an integer selected from 1 to 5, and more preferably 1, 2 or 3;

[0049] m is an integer selected from 0 to 10, preferably an integer selected from 1 to 5, and more preferably 1;

[0050] R 1 R 2 As defined in any of the aforementioned technical solutions;

[0051] R 3 As defined in any of the aforementioned technical solutions;

[0052] Y is as defined in any of the aforementioned technical solutions.

[0053] In some embodiments, the connector includes the structure shown in equations (1-2).

[0054] in:

[0055] n is an integer selected from 0 to 10, preferably an integer selected from 1 to 5, and more preferably 1, 2 or 3;

[0056] m is an integer selected from 0 to 10, preferably an integer selected from 1 to 5, and more preferably 1;

[0057] R 1 R 2 As defined in any of the aforementioned technical solutions;

[0058] R 3 As defined in any of the aforementioned technical solutions;

[0059] Y' is selected from X1, X2, X3, and X4 are each independently selected from H and electron-withdrawing groups; preferably, the amino terminus is connected to the remaining part of the general formula structure;

[0060] Preferably, Y' is selected from Preferably, the amino terminus is connected to the remaining portion of the general formula;

[0061] X1 and X2 are each independently selected from H, Cl, F, CN, NO2, CF3, and CHF2;

[0062] Preferably, in X1 and X2, either one is H, and the other is selected from H, Cl, F, CN, NO2, CF3, CHF2;

[0063] Preferably, X1 and X2 are each independently selected from H, Cl, and F;

[0064] More preferably, of X1 and X2, one (e.g., X2) is H, and the other (e.g., X1) is selected from H, Cl, and F;

[0065] Most preferably, in X1 and X2, either one (e.g., X2) is H, and the other (e.g., X1) is H or Cl;

[0066] y is an integer selected from 0 to 10, preferably an integer selected from 1 to 5, and more preferably 1, 4 or 5;

[0067] Preferably, Y' is selected from Preferably, the amino terminus is connected to the remaining portion of the general formula.

[0068] In some implementations, the connector comprises a structure selected from the following:

[0069] In some embodiments, the prodrug has the structure shown in formula (2).

[0070] in:

[0071] n, m, X, R 1 R 2 R 3 Y is as defined in any of the aforementioned technical solutions;

[0072] D represents a drug, preferably a peptide drug, protein drug, nucleic acid drug, or small molecule drug.

[0073] In some embodiments, the peptide drug includes, but is not limited to, glucagon-like peptide-1 (GLP-1), exendin-2, exendin-3, exendin-4, atrial factor (ANF), ghrellin, vasopressin, growth hormone-releasing hormone (GHRH), RC-3095, somatostatin, corticotropin, vilcapeptide, scutellarin, PCK-3145, Phe-His-Ser-Cys-Asn (PHSCN), IGF-1, B-type linapeptide, peptide YY (PYY), interferon, thrombopoietin, angiopoietin, calcitonin, gonadotropin-releasing hormone, cetrorexone, ganirexone, hirudin, glucagon, anti-TNF-α, fibroblast growth factor, granulocyte colony-stimulating factor, and onipeptide. (obinepitide), pituitary thyroid hormone (PTH), leuprorelin, sermorelin, pramorelin, nesiritide, rotigaptide, silengitide, MBP-8298, AL-108, enfuviride, carfilzomib, mirtamicin, thymosin, daptomycin, HLF1-I, lactoferrin, delmitide, glutathione, T-cell epitope PR1, protease-3-peptide 1-11, B-cell epitope P3, luteinizing hormone-releasing hormone (LHRH), substance P, neurokinin A, neurokinin B, CCK-8, enkephalins (including leucine enkephalin and methionine enkephalin), skin antimicrobial peptides, [des-Ala20,Gln34]-skin antimicrobial peptides, anionic antimicrobial peptides related to surface cleaning substances, apidaecin IA, apidaecin IB, OV-2, 1025,Ethylene adhesion peptide (1025-1044) amide, Theromacin (49-63), Percicanam (MSI-78), Indolicidin, Apelin-15 (63-77), CFP10 (71-85), anthrax-associated lethal factor (LF) inhibitor, bovine antimicrobial peptide, hepatitis C virus NS3 protease inhibitor 2, hepatitis C virus NS3 protease inhibitor 3, hepatitis C virus NS3 protease inhibitor 4, NS4A-NS4B hepatitis C virus (NS3 protease inhibitor 1), HIV-1, HIV-2 protease matrix, anti-Flt1 peptide, Bak-BH3, BaxBH3 peptide (55-74) (wild type), Bid BH3-r8, CTT (gelatinase inhibitor), E75 (Her-2 / neu) (369-377), GRP78-binding chimeric peptide motif, p53 (17-26), EGFR2 / KDR antagonist, Colivelin AGA-(C8R)HNGl 7 (Humanin derivative), activity-dependent neurotrophic factor (ADNF), β-secretase inhibitor 1, β-secretase inhibitor 2, ch[β]amyloid (30-16), Humanun(HN)sHNG, [Gly14]-HN, [Gly14]-Humanin, angiotensin-converting enzyme inhibitor (BPP), renin inhibitor II1, annexin 1 (ANXA-1, Ac2-12), anti-inflammatory peptide 1, anti-inflammatory peptide 2, anti-inflammatory apelin 12. [D-Phe12,Leu14]-frog dermatin, tentacledopeptide (acid) (penetrating protein), tentacledopeptide precursor (CT), wasp venom, sulfated [Thr28,Nle3 1]-cholecystokinin (25-33), pain-sensitive peptide (1-13) (amide), fibrinolysis inhibitor, γ-fibrinogen (377-395), Xenin, obesity suppressant (human), [Hisl,Lys6]-GHRP (GHRP-6), [Ala5,[β]-Ala8]-Neurokinin A (4-10), Neurotransmitter B, Neurotransmitter C, Neurotransmitter N, Activity-dependent Neurotrophic Factor (ADNF-14), Acetalin 1 (Opioid Receptor Antagonist 1), Acetalin 2 (Opioid Receptor Antagonist 2), Acetalin 3 (Opioid Receptor Antagonist 3), ACTH (1-39) (human), ACTH (7-38) (human), Frog Skin Antihypertensive Peptide, Fat Mobilizing Hormone (Locust), Myristylated ADP-ribosylated Factor 6, myr-ARF6 (2-13), PAMP (1-20) (Adrenal Myelin Proton (1-20) human), AGRP (25-5) 1) Amylin (8-37) (human), Angiotensin 1 (human), Angiotensin II (human), Apstatin (aminopeptidase P inhibitor), Brevinin-1, Xenopus 1, RL-37, LL-37 (antimicrobial peptide) (human), Silkworm antimicrobial peptide A, Antioxidant peptide A, Antioxidant peptide B, L-carnosine, BcI 9-2, NPVF, Neuropeptide AF (hNPAF) (human), Bax BH3 peptide (55-74), bFGF inhibitory peptide, bFGF inhibitory peptide II, Calidin, [Des-Argl O]-HOE 140, Caspase 1 inhibitor II, Caspase 1 inhibitor VIII, Smac N7 protein (MEKI-derived peptide inhibitor 1), hBD-1 ([β]defensin-1) (human), hBD-3 ([β]defensin-3) (human), hBD-4 ([β]defensin-4) (human), HNP-1 (human defensin neutrophil peptide 1), HNP-2 (human defensin neutrophil peptide-2 dynorphin A (1-17)), endorphin-1, [β]-endorphin (human, porcine), endothelin 2 (human), fibrinogen binding inhibitory peptide, Cyclo (-GRGDSP), TP508 (thrombin-derived peptide), growth hormone neuropeptide (human), GIP (human), gastric Gastrointestinal releasing peptide (human), gastrin-1 (human), Ghrelin (human), PDGF-BB peptide, [D-Lys3]-GHRP-6, HCV core protein (1–20), a3B1 integrin peptide fragment (325) (amide), laminin thymolpentin (amide), melanocyte-stimulating factor (MPF), VA-[β]-MSH, lipolysis hormone γ (derived from melatonin), atrial linapeptide (1–28) (human), angiotensin-releasing peptide (1–27), [Ala5,B-Ala8]-neurokine A (4–10), neurotransmitter L (NKA), Ac-(Leu28,31)-Neuropeptide Y (24-26), Alitec, Brain Neuropeptide II, [D-tyr11]-Neurohypertensive Peptide, 1KKy NEMO Binding Region (NBD) Inhibitory Peptide, PTD-p50 (NLS) Inhibitory Peptide, Alitec A (Bovine, Human, Mouse, Rat), Alitec B (Human), Aquaporin-2 (254-267) (Human Trypsin) (37-52), Pancreatic Polypeptide (Human), Neuropeptide, Peptide YY (3-36) (Human), Hydroxymethyl-Phytochelatide 2, PACAP (1-27) (Amide, Human, Bovine, Rat), Prolactin-Releasing Peptide (1-31) (Human), Salusin-α, Salusin-β, Saponified Protein C22, Secretin (Human), L-Selective Protein, Endokinin A / B, Endokinin C (Human), Endokinin D (human), thrombin receptor (42-48) agonist (human), LSKL (thrombin-sensitive inhibitor), thyrotropin-releasing hormone (TRH), p55-TNFR fragment, urotenzien II (human), VIP (human, pig, rat), VIP antagonist, cyclophosphamide, exenatide, ZPI0 (AVE00I00), pramlintide, AC162352 (PYY) (3-36), PYY, onnipidide, glucagon, GRP, glucone (GHRP6), leuprorelin, histamine, oxytocin, atosiban (RWJ22164), sermorelin, nesiritide, bivalirudin (Hirulog), aniband, aviptadin, rotigapti de(ZP123,GAP486), silengitide (EMD-121924,RGD peptide), A1buBNP, BN-054, angiotensin 11, MBP-8298, peptidylleucine arginine, ziconopeptide, AL-208, AL-108, Carbeticon, tripeptide, SAL, Coliven, Humanin, ADNF-14, VIP (intestinal vascular cleansing peptide), thymosin, bacitracin, brevicatin, pecidogenam (MSI-78), PI13, PAC-113, SCV-07, HLF1-I1 (lactoferrin), DAPTA, TRI-1144, Tritrpticin, Antiflammin 2, Gattex (Teduglutide,ALX-0600), Stimuvax (L-BLP25), Chrysalin (TP508), Melanonan II, Spantide II, succinate, sicalite, pentagastrin, secretin, endostatin, E-selectin, HER2, PDGF, thrombin-sensitive peptide, uPA(1), uPA(2), VEGF, VEGF(2), thymopentin-3, XXLRR, β-amyloid microfibrillogenin, endorphin-2, TIP 39 (segmented funnel-shaped neuropeptide), PACAP(1-38) (amide, human, bovine, rat), TGFB activating peptide, insulin sensitizing factor (ISF402), transforming growth factor carnosine (TGF-B1), frog skin release factor, IELLQAR (8-branch) MAPS), tigaposide PK3145, goserelin, abaric, cetrorelin, ganirilix, degarelix (by prazoline), barusiban (FE 200440), pramorelin, oxytocin, eptifibatide, netamiftide (INN-00835), daptomycin, spantide II, delmitide (RDP-58), AL-209, enfuviride, IDR-I, hexapeptide-6, insulin A chain, lanreotide, hexapeptide-3, insulin B chain, glargine insulin A chain, glargine insulin B chain, insulin-LisPro B-chain analogs, insulin-aspart B-chain analogs, insulin-glucosine insulin B-chain analogs, insulin-detemir insulin B-chain analogs, somatostatin tumor suppressor analogs, trypsin (37-52), vasoactive intestinal peptide fragments (KKYL-NH2), and dynorphin A, cyclic peptides (e.g., Romidepsin, Voclosporin, Ziconotide, Linaclotide, Plecanatide, Pasireotide, Lanreotide, Vasopressin, Terlipressin, Bremeranotide, Setmelanotide, Daptomycin, Telavancin, Dalbavancin, Oritavancin, Caspofungin, Micafungin, Anidulafungin).

[0074] In some embodiments, the protein drug includes, but is not limited to, enzymes (e.g., asparaginase, sacrosidase, pegvaliase, laronidase, glucosidase, β-glucocerebrosidase), coagulation factors (e.g., coagulation factor VIII, coagulation factor IX, coagulation factor XIII), protein hormones (e.g., growth hormone GH, insulin, erythropoietin, gonadotropin, parathyroid hormone), and cytokines (e.g., interleukins, interferons, colony-stimulating factors).

[0075] In some embodiments, the nucleic acid drug includes, but is not limited to, antisense nucleic acids (ASO) (e.g., Fomivirsen, Mipomersen, Eteplirsen, Nusinersen, inotersen, Volanesoresn, Golodirsen), small interfering RNA (siRNA) (e.g., Onpattro, Givlaari), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), and aptamers.

[0076] In some embodiments, the small molecule drug includes, but is not limited to, antibiotics including penicillins (e.g., penicillin, penicillin V), fluoroquinolones (e.g., ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, gemifloxacin), cephalosporins (e.g., cefixime, cefbufen, ceftriaxone, cefdinir, cefpodoxime proxetil), aminoglycosides (e.g., isapamicin, amikacin, etimicin, netilmicin), macrolides (e.g., erythromycin, azithromycin, clarithromycin), and tetracyclines (e.g., doxycycline, minocycline). Tigecycline, omalicycline, iracycline); sulfonamides and sensitizers (e.g., trimethoprim, sulfadiazine, sulfamethoxazole, sulfamethoxazole); urinary preparations (e.g., nitrofurantoin, fosfomycin); analgesics and anti-inflammatory drugs (e.g., aspirin, ibuprofen, indomethacin, nalbuprofen, acetaminophen, lidocaine, procaine, tetracaine); antihistamines (e.g., chlorpheniramine, triprolidine, diphenhydramine, triphenylphenamine, mequidine, promethazine, mizolastine, ebastine, loratadine, terfenadine, fexofenadine, desloratadine, cetirizine). Antiviral drugs (e.g., oseltamivir, mabaloxavir, erteiravir, lenapavir, capeiravir, entecavir, nematvir / ritonavir, ribavirin, interferon, arbidol, zanamivir, peramivir, amantadine, acyclovir, ganciclovir, valacyclovir, daclatasvir, asunaprevir, adefovir dipivoxil, lamivudine, telbivudine, azvudine); antihypertensive drugs (e.g., amlodipine, felodipine, cilnidipine, nifedipine, enalapril, fosinopril, lisinopril, perindopril). Midapril, losartan, valsartan, irbesartan, telmisartan, olmesartan, hydrochlorothiazide, indapamide, metoprolol, bisoprolol, atenolol, labetalol, propranolol, prazosin, terazosin, doxazosin; diabetes medications (e.g., glibenclamide, gliclazide, glipizide, glimepiride, repaglinide, nateglinide, acarbose, voglibose, miglitol, pioglitazone, rosiglitazone, sitagliptin, linagliptin, alogliptin, saxagliptin, vildagliptin, dapagliflozin, empagliflozin, canagliflozin);Chronic obstructive pulmonary disease (COPD) medications (e.g., fenoterol, levosalbutamol, salbutamol, terbutaline, afortrol, formoterol, indacaterol, olodaterol, salmeterol, ipratropium bromide, oxytropium bromide, adecyl bromide, glycopyrronium bromide, tiotropium bromide, umemet, glycopyrronium (glycopyrronium bromide), refenapyridine, roflumilast, erdocysteine, carbocysteine) and antitumor drugs (e.g., methotrexate, pemetrexed, fluorouracil, 6-mercaptopurine, hydroxyurea, gemcitabine, cytarabine, doxorubicin, epirubicin, lurbinectedin, irinotecan, topotecan, etoposide, taxanes) Vincristine alkaloids, eribulin, EGFR inhibitors: gefitinib, erlotinib, icotinib, afatinib, osimertinib, ALK inhibitors: crizotinib, alectinib, ceritinib, lorlatinib, MET inhibitors: cevotinib, carmatinib, RET inhibitors: prallatinib, LOXO-292, NTRK inhibitors: larotrectinib, entrectinib, BRAF inhibitors: dabrafenib, vemurafenib, encorafenib, MEK inhibitors: trametinib, binimetinib, HER2 inhibitors: neratinib, tacotinib, CDK4 / 6 inhibitors: Palbociclib, Abeciclib; PARP inhibitors: Olaparib, Niraparib; Anti-angiogenic multikinase inhibitors: Nilotinib, Apatinib, Sunitinib, Fruquintinib, Pazopanib, Axitinib, Vandetanib, Cabozantinib; mTOR inhibitors: Everolimus; HDAC inhibitors: Chidamide; ABL-BCR inhibitors: Imatinib, Dasatinib, Nilotinib; PDGFR, C-KIT inhibitors: Imatinib, Nilotinib, Avatinib; BTK inhibitors: Ibrutinib, Zanubrutinib, Zanubrutinib; Protease inhibitors: Bortezomib, Ixazomib; JAK inhibitors: Ruco TINIBs (selective nuclear transporter inhibitors: selinexor, PI3K inhibitors: alpelisib, FGFR2 inhibitors: pemigatinib, IDH1 inhibitors: ivosidenib); psoriasis medications (e.g., apremilast, deuterocelexitinib); antipsychotics (e.g., risperidone, quetiapine, chlorpromazine, perphenazine, clozapine, olanzapine, sulpiride); antidepressants (e.g., fluoxetine, paroxetine, sertraline, citalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, vortioxetine, mirtazapine).

[0077] In some embodiments, the prodrug has the structure shown in formula (2-1).

[0078] in:

[0079] n, m, R 1 R 2 R 3Y is as defined in any of the aforementioned technical solutions;

[0080] D is as defined in any of the aforementioned technical solutions.

[0081] In some embodiments, the prodrug has the structure shown in formula (2-2).

[0082] in:

[0083] n, m, X, R 1 R 2 R 3 Y' is as defined in any of the aforementioned technical solutions;

[0084] D is as defined in any of the aforementioned technical solutions.

[0085] In some embodiments, the prodrug has a structure selected from the following:

[0086] Each D is independently defined as described in any of the aforementioned technical solutions.

[0087] In one aspect, the present invention provides a nanobody or antigen-binding fragment thereof capable of specifically binding to serum albumin, comprising:

[0088] (a) CDR1 or a variant thereof contained in the heavy chain variable region (VHH) as shown in SEQ ID NO:30, CDR2 or a variant thereof contained in the heavy chain variable region (VHH) as shown in SEQ ID NO:30, and CDR3 or a variant thereof contained in the heavy chain variable region (VHH) as shown in SEQ ID NO:30; or,

[0089] (b) CDR1 or a variant thereof contained in the heavy chain variable region (VHH) as shown in SEQ ID NO:34, CDR2 or a variant thereof contained in the heavy chain variable region (VHH) as shown in SEQ ID NO:34, and CDR3 or a variant thereof contained in the heavy chain variable region (VHH) as shown in SEQ ID NO:34.

[0090] The variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0091] In some embodiments, the nanobody or its antigen-binding fragment comprises:

[0092] (a) CDR1, CDR2, and CDR3 contained in the heavy chain variable region (VHH) as shown in SEQ ID NO:30; or,

[0093] (b) CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VHH) as shown in SEQ ID NO:34.

[0094] In some embodiments, the nanobody or its antigen-binding fragment comprises:

[0095] (a) CDR1 as shown in SEQ ID NO:31, CDR2 as shown in SEQ ID NO:32, and CDR3 as shown in SEQ ID NO:33; or,

[0096] (b) CDR1 as shown in SEQ ID NO:35, CDR2 as shown in SEQ ID NO:36, and CDR3 as shown in SEQ ID NO:37.

[0097] In some implementations, the three CDRs contained in the VHH are defined by the IMGT numbering system.

[0098] In some embodiments, the nanobody or its antigen-binding fragment comprises a heavy chain framework region sequence of an immunoglobulin (e.g., a heavy chain framework region sequence of a human or camel immunoglobulin).

[0099] In some embodiments, the nanobody or its antigen-binding fragment comprises the heavy chain framework region of a human immunoglobulin.

[0100] In some embodiments, the nanobody or its antigen-binding fragment comprises a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline gene; preferably, the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) reversion mutations from human residues to camel residues.

[0101] In some embodiments, the nanobody or its antigen-binding fragment comprises an amino acid sequence selected from the following:

[0102] (i) A sequence as shown in either SEQ ID NO:30 or 34;

[0103] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in any one of SEQ ID NO: 30 or 34; or

[0104] (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in any one of SEQ ID NO: 30 or 34;

[0105] Preferably, the substitution is a conservative substitution.

[0106] In a second aspect, the present invention provides a drug-macromolecule conjugate or its stereoisomers, deuterated derivatives, solvates, polymorphs, or pharmaceutically acceptable salts thereof, said drug-macromolecule conjugate comprising a drug, a macromolecule, and a linking unit, said drug being linked to said macromolecule via said linking unit, said linking unit comprising the structure shown in formula (3).

[0107] in:

[0108] n, m, X, R 1 R 2 Y is as defined in any of the technical solutions in the first aspect.

[0109] In some embodiments, the connecting unit includes the structure shown in equation (3-1).

[0110] in:

[0111] n, m, R 1 R 2 Y is as defined in any of the technical solutions in the first aspect.

[0112] In some embodiments, the connecting unit includes the structure shown in equation (3-2).

[0113] in:

[0114] n, m, X, R 1 R 2 Y' is as defined in any of the technical solutions of the first aspect.

[0115] In some embodiments, the connection unit includes a structure selected from the following:

[0116] In some embodiments, the drug-macromolecule conjugate comprises the structure shown in formula (4).

[0117] in:

[0118] n, m, X, R 1 R 2 Y is as defined in any of the technical solutions of the first aspect;

[0119] D is as defined in any of the technical solutions in the first aspect.

[0120] In some embodiments, the drug-macromolecule conjugate comprises the structure shown in formula (4-1).

[0121] in:

[0122] n, m, R 1 R 2 Y is as defined in any of the technical solutions of the first aspect;

[0123] D is as defined in any of the technical solutions in the first aspect.

[0124] In some embodiments, the drug-macromolecule conjugate comprises the structure shown in formula (4-2).

[0125] in:

[0126] n, m, X, R 1 R 2 Y' is as defined in any of the technical solutions of the first aspect.

[0127] D is as defined in any of the technical solutions in the first aspect.

[0128] In some embodiments, the drug-macromolecule conjugate comprises a structure selected from the following:

[0129] Each D is independently defined as in any of the technical solutions of the first aspect.

[0130] In some embodiments, the drug-macromolecule conjugate has the structure shown in formula (5).

[0131] in:

[0132] n, m, X, R 1 R 2 Y is as defined in any of the technical solutions of the first aspect;

[0133] D is as defined in any of the technical solutions in the first aspect;

[0134] P represents a group that contains macromolecules;

[0135] Preferably, the macromolecule is an antibody (e.g., IgG, IgM, IgA, IgD or IgE) or its antigen-binding fragment, a fatty acid (e.g., C16, C18, C20 fatty acid), PEG, pCB or any conjugation thereof.

[0136] In some embodiments, the macromolecule is a nanobody or its antigen-binding fragment.

[0137] In some embodiments, the macromolecule is a nanobody that specifically binds to serum albumin or an antigen-binding fragment thereof.

[0138] In some embodiments, the nanobody that specifically binds to serum albumin or its antigen-binding fragment is as described above.

[0139] In some embodiments, the drug-macromolecule conjugate has the structure shown in formula (5-1).

[0140] in:

[0141] n, m, R 1 R 2 Y is as defined in any of the technical solutions of the first aspect;

[0142] D is as defined in any of the technical solutions in the first aspect;

[0143] P is as defined in any of the aforementioned technical solutions.

[0144] In some embodiments, the drug-macromolecule conjugate has the structure shown in formula (5-2).

[0145] in:

[0146] n, m, X, R 1 R 2 Y' is as defined in any of the technical solutions of the first aspect;

[0147] D is as defined in any of the technical solutions in the first aspect;

[0148] P is as defined in any of the aforementioned technical solutions.

[0149] In some embodiments, the drug-macromolecule conjugate has a structure selected from the following:

[0150] Wherein, each D is independently defined as in any of the technical solutions of the first aspect, and each P is independently defined as in any of the aforementioned technical solutions.

[0151] In a third aspect, the present invention provides a prodrug intermediate or its stereoisomer, deuterated derivative, solvate, polymorph, or pharmaceutically acceptable salt thereof, said prodrug intermediate having the structure shown in formula (6).

[0152] in:

[0153] n, m, X, R 1 R 2 R 3 As defined in any of the technical solutions in the first aspect;

[0154] Y1 is selected from X1, X2, X3, and X4 are each independently selected from H and electron-withdrawing groups;

[0155] Preferably, Y1 is selected from

[0156] X1 and X2 are each independently selected from H, Cl, F, CN, NO2, CF3, and CHF2;

[0157] Preferably, in X1 and X2, either one is H, and the other is selected from H, Cl, F, CN, NO2, CF3, CHF2;

[0158] Preferably, X1 and X2 are each independently selected from H, Cl, and F;

[0159] More preferably, of X1 and X2, one (e.g., X2) is H, and the other (e.g., X1) is selected from H, Cl, and F;

[0160] Most preferably, in X1 and X2, either one (e.g., X2) is H, and the other (e.g., X1) is H or Cl;

[0161] y is an integer selected from 0 to 10, preferably an integer selected from 1 to 5, and more preferably 1, 4 or 5;

[0162] Most preferably, Y1 is selected from

[0163] In some embodiments, the prodrug intermediate has the structure shown in formula (6-1).

[0164] in:

[0165] n, m, R1 R 2 R 3 As defined in any of the technical solutions in the first aspect;

[0166] Y1 is defined as in any of the aforementioned technical solutions.

[0167] In some embodiments, the prodrug intermediate has the structure shown in formula (6-2).

[0168] in:

[0169] n, m, X, R 1 R 2 R 3 As defined in any of the technical solutions in the first aspect;

[0170] Y1' is selected from X1, X2, X3, and X4 are each independently selected from H and electron-withdrawing groups;

[0171] Preferably, Y1' is selected from

[0172] X1 and X2 are each independently selected from H, Cl, F, CN, NO2, CF3, and CHF2;

[0173] Preferably, in X1 and X2, either one is H, and the other is selected from H, Cl, F, CN, NO2, CF3, CHF2;

[0174] Preferably, X1 and X2 are each independently selected from H, Cl, and F;

[0175] More preferably, of X1 and X2, one (e.g., X2) is H, and the other (e.g., X1) is selected from H, Cl, and F;

[0176] Most preferably, in X1 and X2, either one (e.g., X2) is H, and the other (e.g., X1) is H or Cl;

[0177] y is an integer selected from 0 to 10, preferably an integer selected from 1 to 5, and more preferably 1, 4 or 5;

[0178] Most preferably, Y1' is selected from

[0179] In some embodiments, the prodrug intermediate has a structure selected from the following:

[0180] In a fourth aspect, the present invention provides a prodrug intermediate or its stereoisomer, deuterated derivative, solvate, polymorph, or pharmaceutically acceptable salt thereof, said prodrug intermediate comprising the structure shown in formula (7).

[0181] in:

[0182] n, m, X, R 1 R 2 R 3 As defined in any of the technical solutions in the first aspect.

[0183] In some embodiments, the prodrug intermediate comprises a structure selected from the following:

[0184] In some embodiments, the prodrug intermediate has the structure shown in formula (8).

[0185] in:

[0186] n, m, X, R 1 R 2 R 3 As defined in any of the technical solutions in the first aspect;

[0187] Q is

[0188] In some embodiments, the prodrug intermediate has a structure selected from the following:

[0189] In a fifth aspect of the invention, the invention provides a pharmaceutical composition comprising a prodrug or its stereoisomer, deuterated derivative, solvate, polymorph, or pharmaceutically acceptable salt as described in any of the technical solutions of the first aspect; or a drug-macromolecule conjugate or its stereoisomer, deuterated derivative, solvate, polymorph, or pharmaceutically acceptable salt as described in any of the technical solutions of the second aspect; or a prodrug intermediate or its stereoisomer, deuterated derivative, solvate, polymorph, or pharmaceutically acceptable salt as described in any of the technical solutions of the third or fourth aspect.

[0190] In some embodiments, the composition further comprises pharmaceutically acceptable excipients.

[0191] In some embodiments, the composition further comprises one or more additional therapeutic agents.

[0192] In a sixth aspect of the invention, the invention provides the use of a prodrug or its stereoisomer, deuterated derivative, solvate, polymorph or pharmaceutically acceptable salt as described in any of the technical solutions of the first aspect, or a drug-macromolecule conjugate or its stereoisomer, deuterated derivative, solvate, polymorph or pharmaceutically acceptable salt as described in any of the technical solutions of the second aspect, or a prodrug intermediate or its stereoisomer, deuterated derivative, solvate, polymorph or pharmaceutically acceptable salt as described in any of the technical solutions of the third or fourth aspect, or a pharmaceutical composition as described in any of the technical solutions of the fifth aspect, in the preparation of a medicament for treating and / or preventing diseases.

[0193] In a seventh aspect of the invention, the invention provides a prodrug or its stereoisomer, deuterated derivative, solvate, polymorph or pharmaceutically acceptable salt as described in any of the technical solutions of the first aspect; or a drug-macromolecule conjugate or its stereoisomer, deuterated derivative, solvate, polymorph or pharmaceutically acceptable salt as described in any of the technical solutions of the second aspect; or a prodrug intermediate or its stereoisomer, deuterated derivative, solvate, polymorph or pharmaceutically acceptable salt as described in any of the technical solutions of the third or fourth aspect; or a pharmaceutical composition as described in any of the technical solutions of the fifth aspect, for the treatment and / or prevention of disease.

[0194] In an eighth aspect of the invention, the invention provides a method for treating and / or preventing disease, comprising: administering to a subject in need an effective amount of a prodrug or its stereoisomer, deuterated form, solvate, polymorph, or pharmaceutically acceptable salt as described in any of the first aspects; or a drug-macromolecule conjugate or its stereoisomer, deuterated form, solvate, polymorph, or pharmaceutically acceptable salt as described in any of the second aspects; or a prodrug intermediate or its stereoisomer, deuterated form, solvate, polymorph, or pharmaceutically acceptable salt as described in any of the third or fourth aspects; or a pharmaceutical composition as described in any of the fifth aspects.

[0195] In some embodiments, the disease is the disease treated by the prodrug or the active drug in the drug-macromolecule conjugate. Beneficial effects

[0196] 1. This invention provides prodrugs and drug-macromolecule conjugates capable of controlling and prolonging the in vitro and in vivo release of drugs. The inventors of this application have discovered that the release rate can be controlled by increasing the hydrophilicity of long carbon chains, selecting specific substituents on the triggering group, and different substituents on 4-(hydroxymethyl)aniline. The compounds provided by this invention are prodrugs with tunable release rates, or drug-macromolecule conjugates exhibiting tunable controlled release rates.

[0197] 2. The prodrugs and drug-macromolecule conjugates provided by the present invention have long half-lives, do not require enzymes or redox environments, and can release drugs spontaneously and controllably under physiological conditions (for example, as shown in Figures 1 and 2). They have high drug delivery efficiency, and the released drugs have complete activity and are not affected by linkers or macromolecular carriers.

[0198] Terminology Definition

[0199] It should be understood that the terminology used here is intended to describe specific implementation schemes and is not intended to be restrictive.

[0200] Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and combined with each other. Such combinations and combinations of group definitions and compound structures should be understood as being within the scope of this application specification and / or claims.

[0201] In this invention, unless otherwise explicitly stated, the descriptive phrase “...each independently selected” used throughout this document can mean either that the specific options expressed by the same or different symbols in different groups do not affect each other, or that the specific options expressed by the same or different symbols in the same group do not affect each other.

[0202] The substituents in the compounds of this invention are disclosed according to the type or range of groups. In particular, this invention includes every independent sub-combination of the members of these types and ranges. For example, the term "C1-C6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl groups.

[0203] In this invention, the term "C1-C12 alkyl" refers to an alkyl group having 1 to 12 carbon atoms, preferably "C1-C6 alkyl". The term "C1-C6 alkyl" refers to an alkyl group having 1 to 6 carbon atoms, preferably "C1-C4 alkyl", more preferably "C1-C3 alkyl", and most preferably "C1-C2 alkyl". Examples of "C1-C6 alkyl" include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl). Examples of "C1-C4 alkyl" include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), and butyl (e.g., n-butyl, isobutyl, tert-butyl). Examples of "C1-C3 alkyl" include methyl, ethyl, and propyl (e.g., n-propyl, isopropyl). Examples of "C1-C2 alkyl" include methyl and ethyl.

[0204] In this invention, the term "C1-C12 alkoxy" refers to any of the above-mentioned C1-C12 alkyl groups that are attached to the rest of the molecule by an oxygen atom (-O-), examples of which include methoxy, ethoxy, isopropoxy, etc.

[0205] In this invention, the term "heteroatom" refers to N, O, or S.

[0206] In this invention, the term "substituted" means that any one or more hydrogen atoms on a specified atom or group are selectively replaced by a specified group, provided that the replacement does not exceed the normal valence state of the specified atom.

[0207] In this invention, the term "aryl" refers to an all-carbon monocyclic or fused polycyclic group with a conjugated π-electron system that may be optionally substituted. For example, "C6-C14 aryl" refers to a monocyclic, bicyclic, or tricyclic aryl group having 6 to 14 carbon atoms, preferably "C9-C14 aryl". Non-limiting examples of "aryl" include phenyl, naphthyl, etc.

[0208] In this invention, the term "heteroaryl" refers to an aromatic monocyclic, bicyclic, tricyclic, or more cyclic group, optionally substituted with 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered heteroatom (N, O, or S) in at least one ring. The heteroatom-containing ring optionally also has one, two, or three heteroatoms selected from N, O, or S, and is a bicyclic, tricyclic, or more cyclic heteroaryl group. It is required that the bicyclic, tricyclic, or multicyclic group constitutes an aromatic system, preferably a "9-14-membered heteroaryl group". Non-limiting examples of the term "heteroaryl" include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, thiazolyl, pyrroleyl, phenyl-pyrroleyl, furanyl, phenyl-furanyl, oxazolyl, isoxazolyl, pyrazolyl, thiophenyl, benzofuranyl, benzothiophenyl, benzimidazolyl, indazoleyl, quinolinyl, isoquinolinyl, etc.

[0209] In this invention, "optionally replaced" means that "replacement" may but does not have to occur, and this description includes situations where it occurs or does not occur.

[0210] In this invention, the terms "electron-donating group" and "electron-withdrawing group" are used interchangeably. In this invention, if the electron-donating ability of a substituent is stronger than that of hydrogen, it is called an electron-donating group; if the electron-withdrawing ability of a substituent is stronger than that of hydrogen, it is called an electron-withdrawing group.

[0211] In this invention, the term "direct bond" refers to a bond in which groups on either side are directly connected, for example, R 1 R 2 Together with the carbon atoms bonded to them, they form When G is a direct bond, it represents that R 1 R 2 Together with the carbon atoms bonded to them, they form

[0212] In this invention, the term "prodrug" refers to a derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide an active compound. Prodrugs become active compounds only after undergoing this reaction under biological conditions, or they do not have or only have low activity in their unreacted forms.

[0213] In this invention, the term "stereoisomer" refers to compounds with the same molecular formula but different spatial arrangements of atoms or groups of atoms. Stereoisomers are a special class of isomers whose differences in molecular structure are mainly reflected in the spatial positions of atoms or groups of atoms. Stereoisomers include enantiomers and diastereomers. Enantiomers are two molecules that are mirror images of each other but cannot be superimposed, and they have opposite optical rotations. Diastereomers include cis-trans isomers (also called geometric isomers) and conformational isomers, etc. In this invention, when specifically designated by chemical name as (R)- or (S)- isomers, it should be understood that the predominant configuration is (R)- or (S)-, respectively. Any asymmetric carbon atom can exist in (R)-, (S)-, or (R, S)- configurations, preferably in (R)- or (S)- configurations.

[0214] In this invention, the terms "solvent" or "solvent compound" are used interchangeably and refer to a compound existing in combination with a solvent molecule. This combination may include a stoichiometric amount of a solvent, such as a monohydrate or dihydrate, or may include any amount of water; similarly, methanol or ethanol may form an "alcohol," which may be stoichiometric or non-stoichiometric. The term "solvent compound" as used herein refers to a solid form, i.e., a compound in solution of a solvent that, while solvated, is not a solvate compound as used herein.

[0215] In this invention, the term "polymorph" refers to the different crystal structures formed when the compounds of this invention, due to various factors affecting their crystallization, undergo changes in intramolecular or intermolecular bonding, resulting in different arrangements of molecules or atoms in the crystal lattice space. The compounds of this invention can exist in one crystal structure or multiple crystal structures, i.e., they possess "polymorphism." The compounds of this invention can exist in different crystal forms.

[0216] In this invention, the term "pharmaceutically acceptable salt" refers to (i) a salt formed by an acidic functional group (e.g., -COOH) present in the compounds provided by this invention and a suitable inorganic or organic cation (base), including but not limited to, alkali metal salts such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts such as calcium salts, magnesium salts, etc.; other metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts such as ammonium salts; organic base salts such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenylethylamine salts, piperazine salts, tetramethylamine salts, and tris(hydroxymethyl)aminomethane salts. (ii) The salts formed by the basic functional group (e.g., -NH2) present in the compounds provided by the present invention and suitable inorganic or organic anions (acids), including but not limited to, hydrohalides such as hydrofluoric acid, hydrochloride, hydrobromide, hydroiodide, etc.; inorganic acid salts such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkyl sulfonates such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; aryl sulfonates such as benzenesulfonates, p-benzenesulfonates, etc.; organic acid salts such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts such as glycine salts, trimethylglycine salts, arginine salts, ornithine salts, glutamate salts, aspartate salts, etc.

[0217] In this invention, the term "deuterated product" refers to a molecule or group obtained by replacing one or more hydrogen atoms in a molecule or group with deuterium atoms.

[0218] The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable excipients, including but not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycerol, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin, etc.

[0219] The pharmaceutical composition of the present invention can be prepared in various forms according to different routes of administration. For example, the pharmaceutical composition can be administered in any of the following ways: orally, by spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, and non-enteric administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intrasternal, and intracranial injection or infusion, or via an external implantation device. Oral or intravenous administration is preferred.

[0220] The compounds described in this invention may optionally be used in combination with one or more other active ingredients, and the dosage and ratio of each ingredient may be adjusted by those skilled in the art according to the specific symptoms, the patient's condition, and clinical needs.

[0221] In this invention, "treatment" generally refers to achieving the desired pharmacological and / or physiological effect. This effect may be preventative based on the complete or partial prevention of the disease or its symptoms; and / or therapeutic based on the partial or complete stabilization or cure of the disease and / or side effects resulting from the disease. As used herein, "treatment" encompasses any treatment of a patient's disease, including: (a) prevention of disease or symptoms occurring in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppression of the symptoms of the disease, i.e., prevention of its progression; or (c) relief of the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

[0222] In this invention, the term "subject" refers to a vertebrate. In some embodiments, a vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In some embodiments, a mammal refers to a non-human. In some embodiments, a mammal refers to a human.

[0223] In this invention, the term "effective amount" refers to the amount that effectively achieves the desired therapeutic or preventative effect at the necessary dose and time. The "therapeutic effective amount" of the substance / molecule of this invention may vary depending on factors such as an individual's disease state, age, sex, weight, and the ability of the substance / molecule to elicit the desired response in the individual. Therapeutic effective amount also encompasses the amount in which the beneficial therapeutic effect of the substance / molecule outweighs any toxic or harmful consequences. "Preventative effective amount" refers to the amount that effectively achieves the desired preventative effect at the necessary dose and time. Typically, but not necessarily, the preventative effective amount will be lower than the therapeutic effective amount because the preventative dose is administered to the subject before the onset of the disease or in the early stages of the disease. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; shrink the tumor volume; inhibit (i.e., slow down, preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down, preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent.

[0224] In this invention, the term "antibody" is used in the broadest sense to include various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule may consist of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). The antibody light chain may be classified as κ (kappa) and λ (lambda) light chains. The heavy chain may be classified as μ, δ, γ, α, or ε, and the isotypes of the antibody are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of a single CL domain. While not directly involved in antibody-antigen binding, the constant domain exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The distribution of amino acids in different regions or domains can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0225] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0226] In this invention, the terms "nanobody" and "single-domain antibody (sdAb)" have the meanings commonly understood by those skilled in the art and are used interchangeably. They refer to antibody fragments composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region, VHH), typically derived from the variable region of a heavy chain antibody (e.g., a camel antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity-determining regions, having a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to lack one or both of those framework regions, as long as they substantially maintain antigen binding and specificity.

[0227] In this invention, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0228] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined using the IMGT, Kabat, or Chothia numbering system. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined using the IMGT numbering system.

[0229] In this invention, the term "framework region" or "FR" residue refers to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0230] In this invention, the term "antigen-binding fragment" of an antibody refers to a molecule other than the intact antibody, which includes a portion of the intact antibody that binds to the antigen bound to the intact antibody. For example, an antigen-binding fragment may be a polypeptide fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound to the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as an "antigen-binding moiety." See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. An antigen-binding fragment contains at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0231] In this invention, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) or half-maximal effective concentration (EC50) of the interaction. 50 )express.

[0232] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the “binding rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The values ​​of KD, kon, and kdis can be measured using any effective method. In some embodiments, the dissociation constant can be measured using bioluminescent interferometry (e.g., the ForteBio Octet method). Alternatively, surface plasmon resonance techniques (e.g., Biacore) or Kinexa can be used to measure the dissociation constant.

[0233] In this invention, the term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be achieved using, for example, methods readily available through computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0234] In this invention, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0235] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0236] In this invention, the terms "macromolecule" and "macromolecule carrier" are used interchangeably. A macromolecule is essentially non-cytotoxic, non-hormonal, or non-cell signaling active, but can be conjugated with an active drug molecule via a linker for loading the active drug molecule in systemic circulation and providing a reservoir for gradual release over time. Examples of macromolecules include, but are not limited to, antibodies (e.g., IgG, IgM, IgA, IgD, or IgE) or their antigen-binding fragments, fatty acids (e.g., C16, C18, C20 fatty acids), PEG, or pCB. In some embodiments, the macromolecule contains at least one functional group suitable for conjugation after natural or chemical transformation, such as amine, carboxylic acid, alcohol, thiol, alkyne, azide, or maleimide groups. Thus, in some embodiments, the macromolecule can be any conjugation structure of the foregoing examples; for example, the macromolecule can be a PEG-antibody conjugate, a fatty acid-antibody conjugate, a fatty acid-PEG conjugate, etc. In some embodiments of the invention, the macromolecule is polyethylene glycol. Polyethylene glycol can be linear or branched, with at least one end being a functional group suitable for coupling, or it can comprise multiple arms, each arm ending in a functional group suitable for coupling. Examples of such polyethylene glycols are known in the art and are commercially available.

[0237] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0238] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0239] Figure 1: An example diagram of drug-macromolecule conjugate drug release of the present invention.

[0240] Figure 2: An example diagram of drug-macromolecule conjugate drug release of the present invention.

[0241] Figure 3: cAMP results of compound C7 in human serum from Saos-2 cells.

[0242] Figure 4A: Results of serum calcium level detection in PTx rats using compound C7.

[0243] Figure 4B: Results of serum phosphorus level detection of compound C7 in PTx rats.

[0244] Figure 5: Detection results of PTH prodrug peptide conjugate releasing PTH analogs in PBS. Detailed Implementation

[0245] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0246] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0247] Example 1: General preparation methods A, B, and C

[0248] (1) A general method for preparing chloroformate and N-hydroxysuccinimide carbonate as shown in Formula II:

[0249] Pyridine (2 eq) was slowly added dropwise to a solution of compound I (1.0 eq) and triphosgene (1.5 eq) in anhydrous tetrahydrofuran (7.87 mL / mmol), and the mixture was stirred at room temperature for 30–60 minutes. The reaction mixture was filtered under vacuum, and the filter cake was washed with ethyl acetate. The combined filtrates were concentrated under reduced pressure using a rotary evaporator to obtain a crude oily chloroformate product, which could be used directly in the next step without further purification.

[0250] Preparation of N-hydroxysuccinimide carbonate: Crude chloroformate was dissolved in anhydrous tetrahydrofuran (7.87 mL / mmol), and pyridine (3.0 eq) and N-hydroxysuccinimide (3.0 eq) were added at room temperature. After reacting for 30 minutes, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The solution was washed successively with 0.1 M HCl, water, saturated sodium bicarbonate aqueous solution, water, 0.1 M HCl, and saturated sodium chloride aqueous solution, then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude N-hydroxysuccinimide carbonate as shown in Formula II. This product can be used directly in the next step without further purification.

[0251] (2) General preparation method B for N-[4-(hydroxymethyl)phenyl]carbamate and (4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate as shown in Formula III:

[0252] The previously prepared N-hydroxysuccinimide carbonate (1.0 eq) and 4-aminobenzyl alcohol (2.0 eq) were dissolved in tetrahydrofuran (3.5 mL / mmol) and stirred at room temperature for 14 hours. The reaction solution was concentrated under reduced pressure, and the residue was further dissolved in dichloromethane, filtered, and the filter cake was washed with dichloromethane. The filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to obtain N-[4-(hydroxymethyl)phenyl]carbamate, a red oily substance.

[0253] Preparation of (4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate: N-[4-(hydroxymethyl)phenyl]carbamate was dissolved in anhydrous dichloromethane (3.2 mL / mmol) / acetonitrile (3.2 mL / mmol). The reaction mixture was placed in an ice bath, and N,N′-disuccinimidyl carbonate (1.5 eq) and 4-dimethylaminopyridine (0.5 eq) were added sequentially. The mixture was stirred at room temperature for 3 hours, and then concentrated under vacuum using a rotary evaporator. The residue was diluted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude (4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate, which was used directly in the next step without further purification.

[0254] (3) A general method for preparing N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate and (3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate as shown in Formula III:

[0255] The previously prepared N-hydroxysuccinimide carbonate (1.0 eq) and 2-chloro-4-aminobenzyl alcohol (2.0 eq) were dissolved in acetonitrile (4.5 mL / mmol) and stirred at room temperature for 5 minutes. Then, an aqueous solution of sodium bicarbonate (6.0 eq) (4.5 mL / mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate, washed successively with water, 1N HCl, and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate.

[0256] Preparation of (3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate: N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate was dissolved in anhydrous dichloromethane (3.2 mL / mmol) / acetonitrile (3.2 mL / mmol). The reaction was carried out in an ice bath. N,N′-disuccinimidyl carbonate (2.0 eq) and 4-dimethylaminopyridine (0.5 eq) were added sequentially to the reaction solution, and the mixture was stirred in an ice bath for 3 hours. The reaction solution was diluted with ethyl acetate, washed sequentially with 0.1N HCl aqueous solution and brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude (3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate, which was used directly in the next step without further purification.

[0257] Example 2: General Preparation Method D

[0258] Equation IV shows N 2 -((2-((5-azidopentyl)oxy)ethoxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine or N as shown in formula V 2 -(((4-(((2-((5-azidopentyl)oxy)ethoxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine or N 2 -(((4-(((2-(((5-azidopentyl)oxy)ethoxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 General preparation method for -(2,4-dinitrophenyl)-L-lysine D:

[0259] N ε-(2,4-dinitrophenyl)-L-lysine hydrochloride (1.1 eq) was dissolved in water (5 mL / mmol). An aqueous solution of sodium bicarbonate (2.0-4.0 eq) (3-6 mL / mmol) was added to the reaction solution. Under stirring at room temperature, an acetonitrile solution (7.2 mL / mmol) of N-hydroxysuccinimide carbonate (1.0 eq) of Formula II, or an acetonitrile solution (7.2 mL / mmol) of (3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (1.0 eq) of Formula III, or an acetonitrile solution (7.2 mL / mmol) of (4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (1.0 eq) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed as monitored by LCMS, the solid was filtered off, and the filtrate was directly purified by reversed-phase column chromatography to obtain N as shown in Formula IV. 2 -((2-((5-azidopentyl)oxy)ethoxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine or N as shown in formula V 2 -(((4-(((2-((5-azidopentyl)oxy)ethoxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine or N 2 -(((4-(((2-(((5-azidopentyl)oxy)ethoxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine.

[0260] Example 3: Preparation of Intermediate 3

[0261] Step 1: Synthesis of 5-azidopentan-1-ol (compound 3-2):

[0262] 5-Chloropentan-1-ol (compound 3-1, 3.5 g, 28.5 mmol, 1.0 eq) was dissolved in water (100 mL), and sodium azide (5.0 g, 76.9 mmol, 2.7 eq) was added to the reaction solution. The reaction solution was stirred at 110 °C for 16 hours, and the reaction was monitored by LCMS until completion. The reaction was brought to room temperature, diluted with water (100 mL), and then extracted three times with dichloromethane (30 mL). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 5-azidopentan-1-ol (compound 3-2, 3.3 g, yield: 89%), which was used directly in the next step.

[0263] LCMS(ES+)m / z:130.1(M+H).

[0264] Step 2: Synthesis of 5-azidopentanal (compound 3):

[0265] 5-Azidopentan-1-ol (compound 3-2, 3.3 g, 25.5 mmol, 1.0 eq), TEMPO (0.08 g, 0.5 mmol, 0.01 eq), and sodium bicarbonate (2.57 g, 30.6 mmol, 1.2 eq) were dissolved in a mixed solvent of dichloromethane (100 mL) and water (10 mL). Trichloroisocyanuric acid (2.19 g, 9.4 mmol, 0.37 eq) was added in portions under vigorous stirring at room temperature, and the reaction was allowed to proceed for 30 minutes at room temperature. The reaction was monitored by LCMS until completion. The reaction solution was filtered through diatomaceous earth to separate the organic phase. The solution was washed with saturated NaHCO3 aqueous solution (50 mL) and saturated brine (50 mL), then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was diluted with anhydrous THF (30 mL) and then concentrated under reduced pressure to approximately 10 mL of solvent to obtain 5-azidopentanal (compound 3, 2.9 g, yield 89%), which was used directly in the next step.

[0266] LCMS(ES+)m / z:100.0(M–N2+H).

[0267] Example A1

[0268] Step 1: Synthesis of 6-azido-1-methanesulfonyl-2-hexanol (compounds 4-3):

[0269] Dimethyl sulfone (compound 4-1, 2.0 g, 21.2 mmol, 1.0 eq) was dissolved in anhydrous tetrahydrofuran (60 mL). At -60 °C, n-BuLi (9.3 mL, 23.3 mmol, 1.1 eq, 2.5 M in hexane) was slowly added dropwise. After the addition was complete, the reaction system temperature was slowly raised to 0 °C. After 30 minutes, the reaction system was returned to -60 °C, and a solution of 5-azido-1-pentanal (compound 3, 3 g, 17.3 mmol, 1.0 eq) in anhydrous tetrahydrofuran (10 mL) was slowly added dropwise. After the addition was complete, the mixture was stirred at -60 °C for 15 minutes. LC-MS showed the reaction was complete. The reaction solution was slowly heated, and when it became clear, saturated ammonium chloride aqueous solution (5 mL) was added. The temperature was then raised to room temperature (approximately 15 minutes). The reaction solution was diluted with ethyl acetate (70 mL), washed successively with H₂O (100 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by normal-phase column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 50% B) to give 6-azido-1-methanesulfonyl-2-hexanol (compound 4-3, 2.6 g, yield: 55%).

[0270] LCMS(ES+)m / z:244.1(M+Na).

[0271] Step 2: Synthesis of 1-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}oxy)pyrrolidine-2,5-dione (compound 4-4):

[0272] 6-Azide-1-methanesulfonyl-2-hexanol (compound 4-3, 2.8 g, 12.7 mmol, 1.0 eq) was treated according to general preparation method A to obtain 1-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}oxy)pyrrolidine-2,5-dione (compound 4-4, 4.2 g, yield: 91%).

[0273] LCMS(ES+)m / z:385.1(M+Na).

[0274] 1 H NMR (400MHz, DMSO-d6) δ5.31-5.21(m,1H),3.78(dd,J=14.8,8.8Hz,1H),3.60(dd,J=15.2,2.0Hz,1H),3 .33(t,J=6.8Hz,2H),3.04(s,3H),2.81(s,4H),1.86-1.72(m,2H),1.58-1.50(m,2H),1.44-1.32(m,2H).

[0275] Step 3: Synthesis of 6-azido-1-methanesulfonylhexane-2-ylN-[4-(hydroxymethyl)phenyl]carbamate (compounds 4-5)

[0276] 1-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}oxy)pyrrolidine-2,5-dione (compound 4-4, 4.2 g, 11.6 mmol, 1.0 eq.) was treated according to general preparation method B to obtain 6-azido-1-methanesulfonylhexane-2-yl N-[4-(hydroxymethyl)phenyl]carbamate (compound 4-5, 4.6 g, yield: 98%).

[0277] LCMS(ES+)m / z:393.1(M+Na).

[0278] Step 4: Synthesis of [4-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino)phenyl]methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compounds 4-6)

[0279] 6-Azide-1-methanesulfonylhexane-2-yl N-[4-(hydroxymethyl)phenyl]carbamate (compound 4-5, 2.9 g, 7.8 mmol, 1.0 eq) was treated according to general preparation method B to obtain [4-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino)phenyl]methyl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 4-6, 3.6 g, yield: 89%).

[0280] LCMS(ES+)m / z: 534.2(M+Na).

[0281] 1 H NMR(400MHz, DMSO-d6)δ9.88(s,1H),7.52(d,J=8.8Hz,2H),7.39(d,J=8.8Hz,2H),5.31(s,2H),5.27-5.23(m,1H),3.5 5-3.49(m,2H),3.36-3.30(m,2H),3.05(s,3H),2.80(s,4H),1.76-1.70(m,2H),1.59-1.52(m,2H),1.43-1.38(m,2H).

[0282] Step 5: Synthesis of (2S)-2-[({[4-({[(6-azido-1-methanesulfonylhex-2-yl)oxy]carbonyl}amino)phenyl]methoxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 4)

[0283] [4-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino)phenyl]methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 4-6, 40 mg, 0.08 mmol, 1.0 eq) was treated according to general preparation method D to obtain (2S)-2-[({[4-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino)phenyl]methoxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 4, 13.9 mg, yield: 25%).

[0284] LCMS(ES+)m / z: 731.2(M+Na).

[0285] 1H NMR (400MHz, DMSO-d6) δ9.77(s,1H),8.89(d,J=5.6Hz,1H),8.86(d,J=2.8Hz,1H),8.24(dd ,J=9.2,2.8Hz,1H),7.54(d,J=8.0Hz,1H),7.46(d,J=8.4Hz,2H),7.27(d,J=8.4Hz,2H),7.2 1(d,J=9.6Hz,1H),5.28-5.19(m,1H),4.94(s,2H),3.97-3.89(m,1H),3.54-3.46(m,4H),3 .32(t,J=6.8Hz,2H),3.04(s,3H),1.77-1.67(m,3H),1.65-1.50(m,5H),1.45-1.34(m,4H).

[0286] Example A2

[0287] Step 1: Synthesis of 6-azido-1-methanesulfonylhexane-2-ylN-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 5-1)

[0288] 1-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}oxy)pyrrolidine-2,5-dione (compound 4-4, 120 mg, 0.33 mmol, 1.0 eq) was treated according to general preparation method C to obtain 6-azido-1-methanesulfonylhexane-2-yl N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 5-1, 120 mg, yield: 89%).

[0289] LCMS(ES+)m / z:374.0(M+Na)

[0290] Step 2: Synthesis of [4-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino)-2-chlorophenyl]methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 5-2)

[0291] 6-Azide-1-methanesulfonylhexane-2-yl N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 5-1, 120 mg, 0.3 mmol, 1.0 eq) was treated according to general preparation method C to obtain [4-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino)-2-chlorophenyl]methyl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 5-2, 150 mg, yield: 93%).

[0292] LCMS(ES+)m / z:568.0(M+Na).

[0293] Step 3: Synthesis of (2S)-2-[({[4-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino]-2-chlorophenyl]methoxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 5)

[0294] [4-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino)-2-chlorophenyl]methyl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 5-2, 75 mg, 0.14 mmol, 1.0 eq) was treated according to general preparation method D to obtain (2S)-2-[({[4-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino]-2-chlorophenyl]methoxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 5, 44.5 mg, yield: 43.6%).

[0295] LCMS(ES+)m / z: 765.1(M+Na)

[0296] 1 H NMR (400MHz, DMSO-d6) δ12.48(s,1H),10.00(s,1H),8.89(t,J=6.0Hz,1H),8.86(d,J =2.8Hz,1H),8.24(dd,J=9.6,2.8Hz,1H),7.71-7.57(m,2H),7.40(s,2H),7.21(d,J=1 0.0Hz,1H),5.30-5.20(m,1H),5.02(s,2H),3.98-3.89(m,1H),3.56-3.42(m,4H),3.3 7-3.29(m,2H),3.04(s,3H),1.80-1.69(m,3H),1.66-1.51(m,5H),1.47-1.36(m,4H).

[0297] Example A3

[0298] Step 1: Synthesis of (2S)-2-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino)-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 6)

[0299] 1-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}oxy)pyrrolidine-2,5-dione (compound 4-4, 30 mg, 0.07 mmol, 1.0 eq) was treated according to general preparation method D to obtain (2S)-2-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino)-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 6, yield: 35.6%).

[0300] Compound 6 was separated under the following conditions: Waters MS-triggered Prep-LC, WELCH Xtimate C18, eluent A: H2O (0.1% FA), eluent B: ACN, gradient: 50% ACN to 60% ACN, 10 min, to obtain compound 6-P1 (first peak, 6.3 mg, retention time: 8.4 min) and compound 6-P2 (last peak: 10.2 mg, retention time: 9.1 min).

[0301] Compound 6-P1: LCMS (ES+) m / z: 560.2 (M+H).

[0302] Compound 6-P1: 1 H NMR (400MHz, DMSO-d6) δ8.90(d,J=6.0Hz,1H),8.86(d,J=2.8Hz,1H),8.25(dd,J=9.6,2.8Hz,1H),7.57 (d,J=8.4Hz,1H),7.22(d,J=9.6Hz,1H),5.13-5.03(m,1H),3.97-3.89(m,1H),3.50-3.45(m,3H),3.39 -3.38(m,1H),3.30-3.28(m,2H),2.98(s,3H),1.77-1.58(m,6H),1.55-1.47(m,2H),1.45-1.31(m,4H).

[0303] Compound 6-P2: LCMS (ES+) m / z: 560.2 (M+H).

[0304] Compound 6-P2: 1H NMR (400MHz, DMSO-d6) δ8.89(t,J=6.0Hz,1H),8.87(d,J=2.8Hz,1H),8.25(dd,J =9.6,2.8Hz,1H),7.58(d,J=8.0Hz,1H),7.22(d,J=9.6Hz,1H),5.10-5.00(m,1H ),3.99-3.88(m,1H),3.48(dd,J=14.8,8.0Hz,3H),3.31-3.27(m,3H),2.99(s,3 H),1.80-1.57(m,6H),1.54-1.46(m,2H),1.46-1.38(m,2H),1.38-1.29(m,2H).

[0305] Example A4

[0306] Step 1: Synthesis of ethyl 2-(2-azidoethoxy)acetate (compound 7-3)

[0307] Under nitrogen protection, NaH (1.01 g, 25.26 mmol, 1.1 eq, 60% in oil) was dissolved in tetrahydrofuran (10 mL). At 0 °C, a tetrahydrofuran (10 mL) solution of azide ethanol (compound 7-2, 2 g, 22.96 mmol, 1.0 eq) was added dropwise to the reaction mixture. The mixture was gradually brought to room temperature and stirred for 1 hour. Ethyl bromoacetate (compound 7-1, 4.6 g, 27.56 mmol, 1.2 eq) was slowly added to the reaction mixture at 0 °C, and the mixture was gradually brought to room temperature and stirred for 1 hour. The reaction was monitored by TLC until it ended. The reaction was quenched with water (5 mL) at 0 °C, and then extracted twice with DCM (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by normal column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 5% B) to give ethyl 2-(2-azidoethoxy)acetate (compound 7-3, 3.1 g, yield: 78%).

[0308] LCMS(ES+)m / z:196.01(M+Na).

[0309] Step 2: Synthesis of 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-one (compound 7-4)

[0310] Dimethyl sulfone (compound 4-1, 2.44 g, 26 mmol, 1.5 eq) was dissolved in anhydrous tetrahydrofuran (30 mL). At -65 °C, n-BuLi (10.4 mL, 26 mmol, 1.5 eq, 2.5 M in hexane) was slowly added dropwise to the reaction mixture. After the addition was complete, the temperature of the reaction system was slowly increased to -10 °C (over 1 h). After 1 hour, at -65 °C, a solution of 2-(2-azidoethoxy)ethyl acetate (compound 7-3, 3 g, 17.3 mmol, 1.0 eq) in anhydrous tetrahydrofuran (10 mL) was slowly added dropwise. After the addition was complete, the mixture was stirred at -65 °C for another 1 hour. TLC showed that the dimethyl sulfone reacted completely. The reaction solution was then heated to room temperature, and the reaction was quenched by adding 10 mL of saturated ammonium chloride aqueous solution. The reaction solution was extracted five times with 50 mL of ethyl acetate. The organic phases were combined, washed with 200 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by normal-phase column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 40% B) to give 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-one (compound 7-4, 2.1 g, yield: 55%).

[0311] LCMS(ES+)m / z:193.96(M–N2+H).

[0312] Step 3: Synthesis of 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-ol (compound 7-5)

[0313] 1-(2-Azideethoxy)-3-(methanesulfonyl)prop-2-one (compound 7-4, 2.1 g, 9.5 mmol, 1.0 eq) was dissolved in a mixture of dichloromethane (15 mL) and methanol (15 mL). NaBH4 (542 mg, 14.3 mmol, 1.5 eq) was added in portions to the reaction mixture at 0 °C, and the mixture was slowly heated to room temperature with stirring for 1 hour. After 1 hour, the reaction was monitored by LCMS to ensure complete reaction. The reaction mixture was concentrated under reduced pressure and purified by normal-phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 1% B to 5% B) to give 1-(2-Azideethoxy)-3-(methanesulfonyl)prop-2-ol (compound 7-5, 2.1 g, yield: 94%).

[0314] LCMS(ES+)m / z:195.97(M–N2+H).

[0315] 1H NMR(400MHz,DMSO-d6)δ5.48(d,J=5.6Hz,1H),4.13–4.00(m,1H),3.62(dd,J=5.4,4.4Hz,2H ),3.50–3.36(m,4H),3.25(dd,J=14.7,9.2Hz,1H),3.13–3.05(m,1H),3.00(d,J=1.0Hz,3H).

[0316] Step 4: Synthesis of 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(2,5-dioxopyrrolidone-1-yl) carbonate (compounds 7-6)

[0317] 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-ol (compound 7-5, 223 mg, 1.0 mmol, 1.0 eq) was treated according to general preparation method A to give 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 7-6, 238 mg, yield: 65.4%).

[0318] LCMS(ES+)m / z:336.99(M–N2+H).

[0319] 1 H NMR (400MHz, DMSO-d 6) δ5.45–5.36 (m, 1H), 3.84–3.58 (m, 6H), 3.44 (t, J = 4.9Hz, 2H), 3.09 (s, 3H), 2.83 (s, 4H).

[0320] Step 5: Synthesis of 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(4-(hydroxymethyl)phenyl)carbamate (compound 7-7)

[0321] 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(2,5-dioxopyrrolidone-1-yl) carbonate (compound 7-6, 130 mg, 0.36 mmol, 1.0 eq) was treated according to general preparation method B to obtain 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(4-(hydroxymethyl)phenyl)carbamate (compound 7-7, 124 mg, yield: 93%).

[0322] LCMS(ES+) m / z: 354.98 (M–H2O).

[0323] Step 6: Synthesis of 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compounds 7-8)

[0324] 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(4-(hydroxymethyl)phenyl)carbamate (compound 7-7, 124 mg, 0.33 mmol, 1.0 eq) was treated according to general preparation method B to obtain 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(4-(((((2,5-dioxopyrrolidine-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 7-8, 167 mg, crude product), which was directly used in the next step.

[0325] LCMS(ES+)m / z:536.06(M+Na).

[0326] Step 7: N 2 -(((4-((((1-(2-azidoethoxy)-3-cyanopropane-2-yl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 7)

[0327] 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compounds 7-8, 167 mg, crude) was treated according to general preparation method D to finally obtain N. 2 -(((4-((((1-(2-azidoethoxy)-3-cyanopropane-2-yl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 7,61.2 mg, yield: 26%).

[0328] LCMS(ES+)m / z:733.23(M+Na).

[0329] 1H NMR (400MHz, DMSO-d6) δ9.88(s,1H),8.91(d,J=5.9Hz,1H),8.88(d,J=2.7Hz,1H),8.26( dd,J=9.7,2.8Hz,1H),7.54–7.46(m,3H),7.29(d,J=8.4Hz,2H),7.23(d,J=9.7Hz,1H),5 .44–5.32(m,1H),4.96(s,2H),4.00–3.86(m,1H),3.75–3.70(m,3H),3.66–3.61(m,1H), 3.59–3.47(m,4H),3.45–3.41(m,2H),3.09(s,3H),1.74–1.56(m,4H),1.49–1.40(m,2H).

[0330] Example A5

[0331] Step 1: Synthesis of 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 8-1)

[0332] 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(2,5-dioxopyrrolidine-1-yl) carbonate (compounds 7-6, 146 mg, 0.4 mmol, 1.0 eq) was treated according to general preparation method C to obtain 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 8-1, 140 mg, yield: 86%), which was used directly in the next step.

[0333] LCMS(ES+) m / z: 387.99 (M–H2O).

[0334] Step 2: Synthesis of 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 8-2)

[0335] 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 8-1, 170 mg, 0.42 mmol, 1.0 eq) was treated according to general preparation method C to obtain 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(3-chloro-4-(((((2,5-dioxopyrrolidine-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 8-2, 229 mg, crude product), which was directly used in the next step.

[0336] LCMS(ES+)m / z:569.98(M+Na).

[0337] Step 3: N 2 -(((4-((((1-(2-azidoethoxy)-3-cyanopropane-2-yl)oxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 8)

[0338] 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 8-2, 229 mg, crude) was treated according to general preparation method D to obtain N 2 -(((4-((((1-(2-azidoethoxy)-3-cyanopropane-2-yl)oxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 8, 114.5 mg, yield: 36.8%).

[0339] LCMS(ES+)m / z: 767.19 (M+Na).

[0340] 1 H NMR (400MHz, DMSO-d6) δ10.10(s,1H),8.90(d,J=6.0Hz,1H),8.88(d,J=2.8Hz,1H),8. 26(dd,J=9.6,2.8Hz,1H),7.68(s,1H),7.61(d,J=7.9Hz,1H),7.42(s,2H),7.23(d,J= 9.7Hz,1H),5.46–5.35(m,1H),5.03(s,2H),3.99–3.90(m,1H),3.77–3.69(m,3H),3.6 6–3.55(m,3H),3.54–3.45(m,4H),3.09(s,3H),1.81–1.56(m,4H),1.53–1.37(m,2H).

[0341] Example A6

[0342] Step 1: N 2 -(((1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 9)

[0343] 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(2,5-dioxopyrrolidone-1-yl) carbonate (compound 7-6, 100 mg, 0.27 mmol, 1.0 eq) was treated according to general preparation method D to obtain N. 2 -(((1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 9,33.5 mg, yield: 22.4%).

[0344] LCMS(ES+)m / z:562.01(M+H).

[0345] 1 H NMR (400MHz, DMSO-d6) δ8.90(d,J=5.9Hz,1H),8.88(d,J=2.6Hz,1H),8.27(dt,J=9.6,2.2Hz,1H),7.67–7.57(m,1H),7.24(dd,J=9.7,1.8Hz ,1H),5.21–5.15(m,1H),3.97–3.88(m,1H),3.70–3.58(m,4H),3.55– 3.47(m,3H),3.44–3.40(m,3H),1.77–1.58(m,4H),1.51–1.37(m,2H).

[0346] Example A7

[0347] Step 1: Synthesis of 2-(2-(2-azido)ethoxy)acetaldehyde (compound 10-2)

[0348] At 25°C, 2-(2-(2-azido)ethoxy)ethane-1-ol (compound 10-1, 500 mg, 2.85 mmol, 1.0 eq), TEMPO (4.45 mg, 0.03 mmol, 0.01 eq), and sodium bicarbonate solid (287.7 mg, 3.42 mmol, 1.2 eq) were added sequentially to a two-phase mixture of dichloromethane (10 mL) and water (1 mL). While stirring, trichloroisocyanuric acid (245.4 mg, 1.06 mmol, 0.37 eq) was added in portions. After the addition was complete, the reaction was continued at room temperature for 30 minutes. After the reaction was completed as monitored by TLC, the reaction solution was filtered with diatomaceous earth, the filter cake was washed with dichloromethane (10 mL * 2), the organic phase was washed successively with saturated sodium bicarbonate aqueous solution (20 mL) and saturated sodium chloride aqueous solution (20 mL), dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 30% B) to give a colorless oily substance 2-(2-(2-azido)ethoxy)acetaldehyde (compound 10-2, 251 mg, yield: 51%).

[0349] 1 H NMR (400MHz, Chloroform-d) δ9.75 (s, 1H), 4.20 (s, 2H), 3.80–3.67 (m, 6H), 3.42 (t, J = 5.0Hz, 2H).

[0350] Step 2: Synthesis of 1-(2-(2-azidoethoxy)ethoxy-3-(methanesulfonyl)prop-2-ol (compound 10-3)

[0351] Under nitrogen protection at -65°C, dimethyl sulfone (compound 4-1, 136 mg, 1.45 mmol, 1.0 eq) was dissolved in ultra-dry tetrahydrofuran (5 mL). Butyllithium (0.64 mL, 1.59 mmol, 1.1 eq) was slowly added dropwise to the reaction solution, maintaining the internal temperature below -60°C. After the addition was complete, stirring was continued at -65°C for 20 minutes. After 20 minutes, the reaction system temperature was slowly increased (30 minutes, from -65°C to -10°C). The reaction system temperature was then lowered again to -65°C, and a tetrahydrofuran (2 mL) solution of 2-(2-(2-azidooxy)ethoxy)acetaldehyde (compound 10-2, 250 mg, 1.45 mmol, 1.0 eq) was slowly added dropwise, maintaining the internal temperature below -60°C. After the addition was complete, stirring was continued at -65°C for 30 minutes. The reaction was confirmed by LCMS. The reaction solution was heated to 0℃, and the reaction was quenched by slowly adding saturated ammonium chloride aqueous solution (5 mL). After the addition was complete, ethyl acetate (20 mL * 2 times) was added for extraction. The combined organic phases were washed with saturated sodium chloride aqueous solution (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 80% B) to obtain 1-(2-(2-azidoethoxy)ethoxy-3-(methanesulfonyl)prop-2-ol (compound 10-3, 130 mg, yield: 34%).

[0352] 1 H NMR (400MHz, DMSO-d6) δ5.42(s,1H),4.14–3.98(m,1H),3.62–3.59(m,2H),3.56(s,4H),3.47–3.29(m,6H),3.00(s,3H).

[0353] Step 3: Synthesis of 1-(2-(2-(-2-azidoethoxy)ethoxy)-3-(methanesulfonyl)propyl-2-yl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 10-4)

[0354] 1-(2-(2-azidoethoxy)ethoxy-3-(methanesulfonyl)prop-2-ol (compound 10-3, 80 mg, 0.3 mmol, 1.0 eq) was treated according to general preparation method A to give 1-(2-(2-(-2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-yl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 10-4, 70 mg, yield: 57%).

[0355] 1H NMR (400MHz, Chloroform-d) δ5.49-5.40(m,1H),3.93-3.80(m,2H),3.75-3.63(m,8H),3.43-3.39(m,2H),3.05(s,3H),2.87(s,4H).

[0356] Step 4: N 2 -(((1-(2-(2-azidoethoxy)ethoxy)-3-(methanesulfonyl)propyl-2-yl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 10)

[0357] 1-(2-(2-(-2-azidoethoxy)ethoxy)-3-(methanesulfonyl)propyl-2-yl(2,5-dioxopyrrolidone-1-yl) carbonate (compound 10⁻⁴, 21 mg, 0.05 mmol, 1.0 eq) was treated according to general preparation formula D to obtain N 2 -(((1-(2-(2-azidoethoxy)ethoxy)-3-(methanesulfonyl)propyl-2-yl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 10, 12 mg, yield: 38%).

[0358] 1 H NMR(400MHz,Chloroform-d)δ9.12(d,J=2.6Hz,1H),8.57(d,J=5.7Hz,1H),8.27(dd,J=9.5,2.7Hz,1H),6.95(dd,J=9.6,7.0Hz,1H),5.39(d d,J=11.6,5.5Hz,1H),4.45–4.20(m,1H),3.73–3.63(m,8H),3.50–3.3 5(m,6H),3.00(d,J=5.5Hz,3H),2.06–1.71(m,4H),1.66–1.49(m,2H).

[0359] Example A8

[0360] Step 1: Synthesis of ethyl 2-(2-(2-azido)ethoxy)ethyl acetate (compound 11-1)

[0361] Under nitrogen protection at 0°C, NaH (127 mg, 3.2 mmol, 1.1 eq, 60% in oil) was dissolved in anhydrous tetrahydrofuran. A solution of 2-(2-azidoethoxy)ethoxyethanol-1-ol (compound 10-1, 500 mg, 2.89 mmol, 1 eq) in anhydrous tetrahydrofuran (5 mL) was slowly added dropwise to the reaction mixture. The reaction system was raised to room temperature and stirred for 1 hour. After 1 hour, the reaction system was cooled to 0°C, and then ethyl 2-bromoacetate (compound 7-1, 579 mg, 3.5 mmol, 1.2 eq) was slowly added, with stirring continued for 30 minutes. The reaction was monitored by TLC until complete. The reaction was quenched by adding water (3 mL) at 0 °C. The mixture was extracted with DCM (10 mL * 2). The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 50% B to 65% B) to give ethyl 2-(2-(2-azido)ethoxy)acetyl (compound 11-1, 450 mg, yield: 60%).

[0362] 1 H NMR (400MHz, Chloroform-d) δ4.21(q,J=7.1Hz,2H),4.14(s,2H),3.76–3.66(m,10H),3.38(t,J=5.1Hz,2H),1.28(t,J=7.1Hz,3H).

[0363] Step 2: Synthesis of 1-(2-(2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-one (compound 11-2)

[0364] Under nitrogen protection, dimethyl sulfone (compound 4-1, 243 mg, 2.58 mmol, 1.5 eq) was dissolved in ultra-dry tetrahydrofuran (10 mL). Butyllithium (1 mL, 2.58 mmol, 1.5 eq) was slowly added dropwise at -65 °C. After the addition was complete, the reaction temperature was slowly increased (from -65 °C to -10 °C over 1 hour). After 1 hour, the reaction temperature was lowered again to -65 °C, and a tetrahydrofuran (5 mL) solution of ethyl 2-(2-(2-azido)ethoxy)acetyl (compound 11-1, 450 mg, 1.72 mmol, 1 eq) was slowly added dropwise. The mixture was stirred at -65 °C for another 1 hour. TLC monitoring showed no dimethyl sulfone remaining. The reaction solution was heated to -10°C, and the reaction was quenched by slowly adding saturated ammonium chloride solution (3 mL). After heating to 25°C, ethyl acetate (10 mL) and water (5 mL) were added, followed by extraction with DCM:MeOH (10:1, 20 mL * 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (60 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 50% B to 65% B) to obtain 1-(2-(2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-one (compound 11-2, 305 mg, yield: 57%).

[0365] 1 H NMR (400MHz, Chloroform-d) δ4.27 (s, 2H), 4.24 (s, 2H), 3.75 -3.65 (m, 10H), 3.39 (t, J = 5.0Hz, 2H), 3.07 (s, 3H).

[0366] Step 3: Synthesis of 1-(2-(2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-ol (compound 11-3)

[0367] At 0 °C, 1-(2-(2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-one (compound 11-2, 300 mg, 0.97 mmol, 1 eq) was dissolved in a mixed solution of methanol (10 mL) and dichloromethane (1 mL). Then, sodium borohydride (55 mg, 1.46 mmol, 1.5 eq) was slowly added. After the addition was complete, the reaction system temperature was raised to room temperature and the reaction was continued for 30 minutes. After the reaction was monitored by TLC until it was complete, the reaction solution was concentrated under reduced pressure to obtain the crude product, which was purified by normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 50% B to 75% B) to obtain 1-(2-(2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-ol (compound 11-3, 250 mg, yield: 83%).

[0368] 1 H NMR (400MHz, Chloroform-d) δ 4.44–4.35 (m, 1H), 3.73–3.50 (m, 12H), 3.40 (t, J = 5.0Hz, 2H), 3.23 (dd, J = 15.0, 9.3Hz, 2H), 3.05 (s, 4H).

[0369] Step 4: Synthesis of 1-(2-(2-(-2-azidoethoxy)ethoxy)-3-(methanesulfonyl)propyl-2-yl(2,5-dioxopyrrolidine-1-yl) carbonate (compounds 11-4)

[0370] 1-(2-(2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-ol (compound 11-3, 100 mg, 0.32 mmol, 1.0 eq) was treated according to general preparation method A to give 1-(2-(2-(-2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-yl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 11-4, 120 mg, yield: 82%).

[0371] 1 H NMR (400MHz, Chloroform-d) δ 5.47–5.38 (m, 1H), 3.91 -3.80 (m, 2H), 3.78 -3.63 (m, 12H), 3.42 (t, J = 5.0Hz, 2H), 3.05 (s, 3H), 2.87 (s, 4H).

[0372] Step 5: N 2 -(13-azido-3-((methanesulfonyl)methyl)-2,5,8,11-tetraoxatridecanoyl)-N 6Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 11)

[0373] 1-(2-(2-(-2-azidoethoxy)ethoxy)-3-(methanesulfonyl)propyl-2-yl(2,5-dioxopyrrolidone-1-yl) carbonate (compound 11-4, 80 mg, 0.18 mmol, 1 eq) was treated according to general preparation method D to obtain N. 2 -(13-azido-3-((methanesulfonyl)methyl)-2,5,8,11-tetraoxatridecanoyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 11,89.3 mg, yield: 78%).

[0374] 1 H NMR(400MHz,Chloroform-d)δ9.11(d,J=2.6Hz,1H),8.62–8.51(m,1H),8.27(dd,J=9.5,2.6Hz,1H),6.99–6.89(m,1H),5.38(dd,J=16.2,10 .5Hz,1H),4.45–4.28(m,1H),3.75–3.62(m,12H),3.49–3.36(m,6H), 3.00(s,3H),2.07–1.90(m,1H),1.91–1.70(m,3H),1.66–1.50(m,2H).

[0375] Example A9

[0376] Step 1: Synthesis of 6-azido-1-(propane-2-sulfonyl)-2-hexanol (compound 12-3)

[0377] Under nitrogen protection, at -60°C, 9.7 mL (24.3 mmol, 1.1 eq, 2.5 M in hexane) of n-butyllithium was slowly added dropwise to a 50 mL solution of anhydrous tetrahydrofuran containing 2-methanesulfonylpropane (compound 12-1, 2.7 g, 22.1 mmol, 1.0 eq). The reaction mixture was slowly heated to 0°C over 30 minutes. The reaction mixture was then cooled to -60°C, and a 20 mL solution of anhydrous tetrahydrofuran containing 5-azidopentanal (compound 3, 3.09 g, 24.3 mmol, 1.1 eq) was slowly added dropwise. After stirring for 15 minutes, the reaction mixture was heated to 0°C over 10 minutes. After the reaction was complete as monitored by LCMS, a 5 mL solution of saturated ammonium chloride was added to quench the reaction. The reaction solution was diluted with ethyl acetate (100 mL), washed successively with water (100 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by normal-phase column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 30% B) to give 6-azido-1-(propane-2-sulfonyl)-2-hexanol (compound 12-3, 3.77 g, yield: 68%).

[0378] LCMS(ES+)m / z:272.1(M+Na).

[0379] Step 2: Synthesis of 1-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)oxy]pyrrolidine-2,5-dione (compound 12-4)

[0380] 6-Azide-1-(propane-2-sulfonyl)-2-hexanol (compound 12-3, 3.77 g, 15.1 mmol, 1.0 eq) was treated according to general preparation method A to give 1-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)oxy]pyrrolidine-2,5-dione (compound 12-4, 5.4 g, yield: 91%).

[0381] LCMS(ES+)m / z:413.1(M+Na).

[0382] 1H NMR (400MHz, DMSO-d6) δ5.30-5.21(m,1H),3.73(dd,J=14.8,8.8Hz,1H),3.53(dd,J=14.8,3.2Hz,1H),3.33(t,J=6.8Hz,2H),3 .25(dt,J=13.6,6.8Hz,1H),2.80(s,4H),1.86-1.76(m,2H),1.60-1.50(m,2H),1.46-1.34(m,2H),1.26(dd,J=6.8,3.6Hz,6H).

[0383] Step 3: Synthesis of 6-azido-1-(propane-2-sulfonyl)hex-2-yl-N-[4-(hydroxymethyl)phenyl]carbamate (compounds 12-5)

[0384] 1-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)oxy]pyrrolidine-2,5-dione (compound 12-4, 5.2 g, 13.3 mmol, 1.0 eq) was treated according to general preparation method B to obtain 6-azido-1-(propane-2-sulfonyl)hexane-2-yl-N-[4-(hydroxymethyl)phenyl]carbamate (compound 12-5, 4.47 g, yield: 84%).

[0385] LCMS(ES+)m / z:421.1(M+Na).

[0386] Step 4: Synthesis of {4-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)amino]phenyl}methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 12-6)

[0387] 6-Azide-1-(propane-2-sulfonyl)hex-2-N-[4-(hydroxymethyl)phenyl]carbamate (compound 12-5, 1.5 g, 3.8 mmol, 1.0 eq) was treated according to general preparation method B to obtain {4-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)amino]phenyl}methyl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 12-6, 1.5 g, yield: 73%).

[0388] LCMS(ES+)m / z: 562.2(M+Na).

[0389] 1H NMR(400MHz, DMSO-d6)δ9.88(s,1H),7.52(d,J=8.8Hz,2H),7.39(d,J=8.8Hz,2H),5.31(s,2H),5.27-5.23(m,1H),3.5 5-3.49(m,2H),3.36-3.30(m,2H),3.05(s,3H),2.80(s,4H),1.76-1.70(m,2H),1.59-1.52(m,2H),1.43-1.38(m,2H).

[0390] Step 5: Synthesis of (2S)-2-{[({4-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl]amino]phenyl}methoxy)carbonyl]amino}-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 12)

[0391] {4-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl]amino]phenyl}methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 12-6, 59 mg, 0.11 mmol, 1.0 eq) was treated according to general preparation method D to obtain (2S)-2-{({4-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl]amino]phenyl}methoxy)carbonyl]amino}-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 12, 14.2 mg, yield: 17%).

[0392] LCMS(ES+)m / z:759.2(M+Na).

[0393] 1H NMR (400MHz, DMSO-d6) δ9.75(s,1H),8.89(t,J=6.0Hz,1H),8.86(d,J=2.8Hz,1H),8.24(dd,J=9.6,2.8Hz,1H ),7.54(d,J=8.4Hz,1H),7.46(d,J=8.0Hz,2H),7.27(d,J=8.4Hz,2H),7.21(d,J=10.0Hz,1H),5.29-5.21(m,1 H),4.94(s,2H),3.98-3.87(m,1H),3.53-3.43(m,3H),3.42(d,J=4.8Hz,1H),3.39-3.35(m,1H),3.31-3.24( m,2H),1.82-1.67(m,3H),1.65-1.49(m,5H),1.45-1.35(m,4H),1.26(d,J=7.2Hz,3H),1.24(d,J=7.2Hz,3H).

[0394] Example A10

[0395] Step 1: Synthesis of (2S)-2-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 13)

[0396] 1-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)oxy]pyrrolidine-2,5-dione (compound 12-4, 80 mg, 0.2 mmol, 1.0 eq) was treated according to general preparation method D to obtain (2S)-2-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 13, 54.2 mg, yield: 45%).

[0397] LCMS(ES+)m / z:588.2(M+H).

[0398] 1H NMR(400MHz, DMSO-d6)δ12.50(s,1H),8.87(t,J=5.6Hz,1H),8.85(d,J=2.8Hz, 1H),8.24(dd,J=9.6,2.8Hz,1H),7.50(d,J=8.0Hz,1H),7.20(dd,J=10.0,2.0Hz ,1H),5.14-5.02(m,1H),3.96-3.85(m,1H),3.50-3.40(m,3H),3.29-3.20(m,4 H),1.74-1.56(m,6H),1.55-1.46(m,2H),1.43-1.29(m,4H),1.25-1.17(m,6H).

[0399] Example A11

[0400] Step 1: Synthesis of 6-azido-1-(propane-2-sulfonyl)hex-2-yl-N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 14-1)

[0401] 1-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)oxy]pyrrolidine-2,5-dione (compound 12-4, 100 mg, 0.26 mmol, 1.0 eq) was treated according to general preparation method C to obtain 6-azido-1-(propane-2-sulfonyl)hexane-2-yl-N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 14-1, 110 mg, yield: 94%).

[0402] LCMS(ES+)m / z:455.1(M+Na).

[0403] Step 2: Synthesis of {4-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)amino]-2-chlorophenyl}methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 14-2)

[0404] 6-Azide-1-(propane-2-sulfonyl)hex-2-yl-N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 14-1, 110 mg, 0.25 mmol, 1.0 eq) was treated according to general preparation method C to obtain {4-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)amino]-2-chlorophenyl}methyl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 14-2, 130 mg, yield: 89%), which was used directly in the next step without purification.

[0405] LCMS(ES+)m / z: 596.4 (M+Na).

[0406] Step 3: Preparation of (2S)-2-{[({4-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)amino]-2-chlorophenyl}methoxy)carbonyl]amino}-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 14)

[0407] {4-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)amino]-2-chlorophenyl}methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 14-2, 65 mg, 0.11 mmol, 1.0 eq) was treated according to general preparation method D to obtain (2S)-2-{({4-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)amino]-2-chlorophenyl}methoxy)carbonyl]amino}-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 14, 43.4 mg, yield: 50%).

[0408] LCMS(ES+)m / z:793.1(M+Na).

[0409] 1 H NMR (400MHz, DMSO-d6) δ12.54(s,1H),9.97(s,1H),8.88(t,J=6.0Hz,1H),8.86(d,J=2. 8Hz,1H),8.24(dd,J=9.6,2.4Hz,1H),7.69-7.55(m,2H),7.43-7.33(m,2H),7.21(d,J=9 .6Hz,1H),5.31-5.23(m,1H),5.01(s,2H),3.96-3.87(m,1H),3.54-3.37(m,5H),3.32-3 .25(m,2H),1.81-1.69(m,3H),1.66-1.51(m,5H),1.46-1.36(m,4H),1.28-1.22(m,6H).

[0410] Example A12

[0411] Step 1: Synthesis of 7-azido-3-hydroxyheptanonitrile (compound 15-3)

[0412] Under nitrogen protection, at -60°C, 10.7 mL (26.8 mmol, 1.1 eq, 2.5 M in hexane) of n-butyllithium was slowly added dropwise to a 50 mL solution of anhydrous tetrahydrofuran containing acetonitrile (compound 15-1, 1.0 g, 24.4 mmol, 1.0 eq). After addition, the reaction mixture was slowly heated to 0°C over 30 minutes. The reaction mixture was then cooled again to -60°C, and a 20 mL solution of anhydrous tetrahydrofuran containing 5-azidopentanal (compound 3, 3.1 g, 24.4 mmol, 1.0 eq) was slowly added dropwise. After stirring for 15 minutes, the reaction mixture was heated to 0°C over 10 minutes. After the reaction was completed as monitored by TLC, a 10 mL solution of saturated ammonium chloride was added to quench the reaction. The reaction solution was diluted with ethyl acetate (100 mL), washed successively with water (100 mL * 2) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by normal-phase column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 15% B) to give 7-azido-3-hydroxyheptanonitrile (compound 15-3, 1.8 g, yield: 44%).

[0413] LCMS(ES+)m / z:191.1(M+Na).

[0414] Step 2: Synthesis of 7-azido-3-({[(2,5-dioxopyrrolidone-1-yl)oxy]carbonyl}oxy)heptanilonitrile (compound 15-4)

[0415] 7-Azide-3-hydroxyheptanenitrile (compound 15-3, 1.1 g, 6.5 mmol, 1.0 eq) was treated according to general preparation method A to give 7-azido-3-({[(2,5-dioxopyrrolidone-1-yl)oxy]carbonyl}oxy)heptanenitrile (compound 15-4, 1.3 g, yield: 65%).

[0416] LCMS(ES+)m / z:332.1(M+Na).

[0417] Step 3: Synthesis of 6-azido-1-cyanohexane-2-yl-N-[4-(hydroxymethyl)phenyl]carbamate (compounds 15-5)

[0418] 7-Azide-3-({[(2,5-dioxopyrrolidone-1-yl)oxy]carbonyl}oxy)heptanilate (compound 15-4, 200 mg, 0.65 mmol, 1.0 eq) was treated according to general preparation method B to obtain 6-azido-1-cyanohexane-2-yl-N-[4-(hydroxymethyl)phenyl]carbamate (compound 15-5, 147 mg, yield: 96%).

[0419] LCMS(ES+)m / z:340.2(M+Na).

[0420] Step 4: Synthesis of [4-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino)phenyl]methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compounds 15-6)

[0421] 6-Azide-1-cyanohexane-2-yl-N-[4-(hydroxymethyl)phenyl]carbamate (compound 15-5, 220 mg, 0.69 mmol, 1.0 eq) was treated according to general preparation method B to obtain [4-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino)phenyl]methyl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 15-6, 280 mg, yield: 88%).

[0422] LCMS(ES+)m / z:481.0(M+Na).

[0423] Step 5: Synthesis of (2S)-2-[({[4-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino]phenyl]methoxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 15)

[0424] [4-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino)phenyl]methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 15-6, 70 mg, 0.15 mmol, 1.0 eq) was treated according to general preparation method D to obtain (2S)-2-[({[4-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino]phenyl]methoxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 15, 21.8 mg, yield: 22%).

[0425] LCMS(ES+)m / z: 678.0 (M+Na).

[0426] 1H NMR (400MHz, DMSO-d6) δ12.51(s,1H),9.87(s,1H),8.89(t,J=5.6Hz,1H),8.86( d,J=2.8Hz,1H),8.24(dd,J=9.6,2.8Hz,1H),7.56-7.41(m,3H),7.29-7.18(m,3 H),4.98-4.90(m,3H),3.96-3.88(m,1H),3.47(q,J=6.4Hz,2H),3.36-3.34(m,1 H),3.31-3.28(m,1H),3.03-2.88(m,2H),1.74-1.54(m,8H),1.45-1.33(m,4H).

[0427] Example A13

[0428] Step 1: Synthesis of (2S)-2-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino)-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 16)

[0429] 7-Azide-3-({[(2,5-dioxopyrrolidone-1-yl)oxy]carbonyl}oxy)heptanilic acid (compound 15-4, 50 mg, 0.16 mmol, 1.0 eq) was treated according to general preparation method D to obtain (2S)-2-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino)-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 16, 50.9 mg, yield: 62%).

[0430] LCMS(ES+)m / z:507.2(M+H).

[0431] 1 H NMR(400MHz,DMSO-d6)δ12.55(s,1H),8.93-8.79(m,2H),8.25(dd,J=9.6,2.8Hz,1 H),7.63(dd,J=10.8,8.0Hz,1H),7.22(d,J=9.6Hz,1H),4.84-4.73(m,1H),3.97-3. 86(m,1H),3.48(q,J=6.8Hz,2H),3.32-3.28(m,2H),2.90-2.77(m,2H),1.79-1.70( m,1H),1.69-1.57(m,5H),1.56-1.48(m,2H),1.48-1.38(m,2H),1.38-1.27(m,2H).

[0432] Example A14

[0433] Step 1: Synthesis of 6-azido-1-cyanohexane-2-yl-N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 17-1)

[0434] 7-Azide-3-({[(2,5-dioxopyrrolidone-1-yl)oxy]carbonyl}oxy)heptanilonitrile (compound 15-4, 100 mg, 0.32 mmol, 1.0 eq) was treated according to general preparation method C to obtain 6-azido-1-cyanohexane-2-yl-N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 17-1, 110 mg, yield: 94%).

[0435] LCMS(ES+)m / z:374.0(M+Na).

[0436] Step 2: Synthesis of [4-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino)-2-chlorophenyl]methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 17-2)

[0437] 6-Azide-1-cyanohexane-2-yl-N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 17-1, 100 mg, 0.28 mmol, 1.0 eq) was treated according to general preparation method C to give [4-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino)-2-chlorophenyl]methyl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 17-2, 110 mg, yield: 79%).

[0438] LCMS(ES+)m / z: 514.9 (M+Na).

[0439] Step 3: Synthesis of (2S)-2-[({[4-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino]-2-chlorophenyl]methoxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 17)

[0440] [4-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino)-2-chlorophenyl]methyl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 17-2, 55 mg, 0.11 mmol, 1.0 eq) was treated according to general preparation method D to give (2S)-2-[({[4-({[(6-azido-1-cyanohexane-2-yl)oxy]carbonyl}amino]-2-chlorophenyl]methoxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 17, 30.8 mg, yield: 40%).

[0441] LCMS(ES+)m / z: 711.9 (M+Na).

[0442] 1 H NMR (400MHz, DMSO-d6) δ12.51(s,1H),10.10(s,1H),8.89(t,J=6.0Hz,1H),8.86(d,J=2.8 Hz,1H),8.24(dd,J=9.6,2.4Hz,1H),7.66(s,1H),7.61(d,J=7.6Hz,1H),7.41(s,2H),7.2 1(d,J=9.6Hz,1H),5.02(s,2H),4.98-4.92(m,1H),3.96-3.89(m,1H),3.47(q,J=6.8Hz,2 H),3.37-3.34(m,2H),2.98(qd,J=16.8,4.8Hz,2H),1.77-1.53(m,8H),1.46-1.34(m,4H).

[0443] Example A15

[0444] Step 1: Synthesis of 4-(2-azidoethoxy)-3-oxobutyronitrile (compound 18-1)

[0445] Under nitrogen protection at -65°C, 1.4 mL of n-butyllithium (3.5 mmol, 1.2 eq, 2.5 M in hexane) was added dropwise to anhydrous tetrahydrofuran (5 mL) solution. At -65°C, a tetrahydrofuran solution of acetonitrile (compound 15-1, 178 mg, 4.3 mmol, 1.5 eq) (2 mL) was added dropwise to the reaction solution. After stirring at this temperature for 1 hour, a tetrahydrofuran solution of ethyl 2-(2-azidoethoxy)acetate (compound 7-3, 500 mg, 2.9 mmol, 1 eq) (3 mL) was slowly added dropwise to the reaction solution. Stirring was continued at -65°C for 1 hour. TLC showed the reaction was complete. The reaction solution was poured into ice water (20 mL), and the pH was adjusted to neutral with 0.1 N hydrochloric acid. Then, it was extracted three times with EA (30 mL), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain 4-(2-azidoethoxy)-3-oxobutyronitrile (compound 18-1, 380 mg, crude product), which was directly used for the next step.

[0446] Step 2: Synthesis of 4-(2-azidoethoxy)-3-hydroxybutyronitrile (compound 18-2)

[0447] The 4-(2-azidoethoxy)-3-oxobutyronitrile (compound 18-1, 380 mg, crude) obtained in the previous step was dissolved in MeOH (3 mL). Sodium borohydride (165 mg, 4.3 mmol, 1.5 eq) was added in portions at 0 °C, and the reaction was brought back to room temperature and stirred for 30 min. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure and purified by normal phase column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 30% B) to finally obtain 4-(2-azidoethoxy)-3-hydroxybutyronitrile (compound 18-2, 56 mg, two-step yield: 11%).

[0448] 1 H NMR (400MHz, DMSO-d6) δ5.55 (d, J=5.2Hz, 1H), 3.95–3.87 (m, 1H), 3.70–3.62 (m, 2H), 3.48 (dd, J= 10.0,5.3Hz,1H),3.45–3.37(m,3H),2.69(dd,J=16.8,4.4Hz,1H),2.57(dd,J=16.8,6.6Hz,1H).

[0449] Step 3: Synthesis of 1-(2-azidoethoxy)-3-cyano-2-propyl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 18-3)

[0450] 4-(2-azidoethoxy)-3-hydroxybutyronitrile (compound 18-2, 36 mg, 0.21 mmol, 1 eq) was treated according to general preparation method A to obtain 1-(2-azidoethoxy)-3-cyano-2-propyl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 18-3, 42 mg, yield: 63.8%), which was used directly in the next step.

[0451] LCMS(ES+)m / z:334.00(M+Na).

[0452] Step 4: Synthesis of 1-(2-azidoethoxy)-3-cyano-2-propyl(4-(hydroxymethyl)phenyl)carbamate (compound 18-4)

[0453] 1-(2-azidoethoxy)-3-cyano-2-propyl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 18-3, 90 mg, 0.29 mmol, 1 eq) was treated according to general preparation method B to give 1-(2-azidoethoxy)-3-cyano-2-propyl(4-(hydroxymethyl)phenyl)carbamate (compound 18-4, 72 mg, yield: 78%).

[0454] LCMS(ES+)m / z:342.05(M+Na).

[0455] Step 5: Synthesis of 1-(2-azidoethoxy)-3-cyano-2-propyl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 18-5)

[0456] 1-(2-Azideethoxy)-3-cyano-2-propyl(4-(hydroxymethyl)phenyl)carbamate (compound 18-4, 70 mg, 0.22 mmol, 1 eq) was treated according to general preparation method B to obtain 1-(2-Azideethoxy)-3-cyano-2-propyl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 18-5, 88 mg, crude product), which was directly used in the next step.

[0457] LCMS(ES+)m / z:483.17(M+Na).

[0458] Step 6: N 2 -(((4-((((1-(2-azidoethoxy)-3-cyanopropane-2-yl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 18)

[0459] 1-(2-azidoethoxy)-3-cyano-2-propyl(4-((((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 18-5, 88 mg, crude) was treated according to general synthetic method D to obtain N. 2 -(((4-((((1-(2-azidoethoxy)-3-cyanopropane-2-yl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 18, 30.8 mg, yield: 25%).

[0460] 1 H NMR (400MHz, DMSO-d6) δ9.95 (s, 1H), 8.94–8.85 (m, 2H), 8.26 (dd, J = 9.6, 2.8Hz, 1H), 7.56 ( d,J=8.0Hz,1H),7.49(d,J=8.2Hz,2H),7.30(d,J=8.3Hz,2H),7.23(d,J=9.6Hz,1H),5.19– 5.11(m,1H),4.96(s,2H),3.99–3.91(m,1H),3.74–3.64(m,4H),3.54–3.47(m,4H),3.03(d d,J=17.2,4.9Hz,1H),2.95(dd,J=17.3,6.5Hz,1H),1.79–1.58(m,4H),1.49–1.42(m,2H).

[0461] Example A16

[0462] Step 1: N 2 -(((1-(2-azidoethoxy)-3-cyanopropane-2-yl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 19)

[0463] 1-(2-azidoethoxy)-3-cyano-2-propyl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 18-3, 42 mg, 0.14 mmol, 1.0 eq) was treated according to general preparation method D to obtain N. 2 -(((1-(2-azidoethoxy)-3-cyanopropane-2-yl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 19,20 mg, yield: 29%).

[0464] LCMS(ES+)m / z:509.29(M+H).

[0465] 1 H NMR(400MHz, DMSO-d6)δ8.92–8.87(m,2H),8.27(dt,J=9.7,2.4Hz,1H),7.67(s,1H),7.24(d,J=9.7Hz,1H), 5.01–4.92(m,1H),3.90–3.93(m,1H),3.71–3.46(m,8H),2.96–2.82(m,2H),1.80–1.59(m,4H),1.44(s,2H).

[0466] Example A17

[0467] Step 1: Synthesis of 1-(2-azidoethoxy)-3-cyano-2-propyl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 20-1)

[0468] 1-(2-azidoethoxy)-3-cyano-2-propyl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 18-3, 73 mg, 0.24 mmol, 1 eq) was treated according to general preparation method C to obtain 1-(2-azidoethoxy)-3-cyano-2-propyl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 20-1, 75 mg, yield: 90%), which was used directly in the next step.

[0469] LCMS(ES+)m / z:336.03(M+Na).

[0470] Step 2: Synthesis of 1-(2-azidoethoxy)-3-cyano-2-propyl(3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 20-2)

[0471] 1-(2-Azideethoxy)-3-cyano-2-propyl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 20-1, 75 mg, 0.21 mmol, 1 eq) was treated according to general preparation method C to obtain 1-(2-Azideethoxy)-3-cyano-2-propyl(3-chloro-4-(((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 20-2, 100 mg, crude product), which was directly used in the next step.

[0472] LCMS(ES+)m / z:517.14(M+Na).

[0473] Step 3: N 2-(((4-((((1-(2-azidoethoxy)-3-cyanopropane-2-yl)oxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 20)

[0474] 1-(2-azidoethoxy)-3-cyano-2-propyl(3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 20-2, 90 mg, crude) was treated according to general preparation method D to obtain N. 2 -(((4-((((1-(2-azidoethoxy)-3-cyanopropane-2-yl)oxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 20, 34.2 mg, yield: 27%).

[0475] 1 H NMR(400MHz,DMSO-d6)δ10.18(s,1H),8.94–8.85(m,2H),8.26(dd,J=9.6,2.8Hz, 1H),7.71–7.60(m,2H),7.43(s,2H),7.23(d,J=9.7Hz,1H),5.20–5.11(m,1H),5.0 4(s,2H),4.00–3.91(m,1H),3.76–3.63(m,4H),3.54–3.44(m,4H),3.04(dd,J=17. 3,4.9Hz,1H),2.96(dd,J=17.3,6.4Hz,1H),1.82–1.58(m,4H),1.52–1.37(m,2H).

[0476] Example A18

[0477] Step 1: Synthesis of 6-azido-2-hydroxy-N,N-dimethylhexane-1-sulfonamide (compound 21-3)

[0478] Under nitrogen protection, at -60°C, 3.2 mL (8.0 mmol, 1.1 eq, 2.5 M in hexane) of n-butyllithium was slowly added dropwise to a 50 mL solution of anhydrous tetrahydrofuran containing N,N-dimethylmethanesulfonamide (compound 21-1, 0.9 g, 7.3 mmol, 1.0 eq). After addition, the reaction mixture was slowly heated to 0°C over 30 minutes. The reaction mixture was then cooled to -60°C, and a 20 mL solution of anhydrous tetrahydrofuran containing 5-azidopentanal (compound 3, 1.02 g, 8.0 mmol, 1.1 eq) was slowly added dropwise. After stirring for 15 minutes, the reaction mixture was heated to 0°C over 10 minutes. After the reaction was monitored by LCMS, saturated ammonium chloride solution (5 mL) was added to quench the reaction. The reaction solution was diluted with ethyl acetate (100 mL), washed successively with water (100 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by normal phase column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 40% B) to give 6-azido-2-hydroxy-N,N-dimethylhexane-1-sulfonamide (compound 21-3, 1.15 g, yield: 63%).

[0479] LCMS(ES+)m / z:273.1(M+Na).

[0480] Step 2: Synthesis of 6-azido-2-({[(2,5-dioxopyrrolidone-1-yl)oxy]carbonyl}oxy)-N,N-dimethylhexane-1-sulfonamide (compound 21-4)

[0481] 6-Azide-2-hydroxy-N,N-dimethylhexane-1-sulfonamide (compound 21-3, 1.15 g, 4.6 mmol, 1.0 eq) was treated according to general preparation method A to obtain 6-azido-2-({[(2,5-dioxopyrrolidone-1-yl)oxy]carbonyl}oxy)-N,N-dimethylhexane-1-sulfonamide (compound 21-4, 1.73 g, yield: 96%).

[0482] LCMS(ES+)m / z:414.1(M+Na).

[0483] 1 H NMR (400MHz, DMSO-d6) δ5.20-5.11(m,1H),3.64(dd,J=14.8,8.4Hz,1H),3.49(dd,J=14.8,3.2Hz,1H),3 .32(d,J=6.8Hz,2H),2.80(s,4H),2.77(s,6H),1.85-1.72(m,2H),1.60-1.50(m,2H),1.45-1.33(m,2H).

[0484] Step 3: Synthesis of 6-azido-1-(N,N-dimethylaminosulfonyl)hex-2-yl(4-(hydroxymethyl)phenyl)carbamate (compounds 21-5)

[0485] 6-Azide-2-({[(2,5-dioxopyrrolidone-1-yl)oxy]carbonyl}oxy)-N,N-dimethylhexane-1-sulfonamide (compound 21-4, 150 mg, 0.38 mmol, 1.0 eq) was treated according to general preparation method B to obtain 6-azido-1-(N,N-dimethylaminosulfonyl)hex-2-yl(4-(hydroxymethyl)phenyl)carbamate (compound 21-5, 147 mg, yield: 96%).

[0486] LCMS(ES+)m / z:422.1(M+Na).

[0487] Step 4: Synthesis of 6-azido-1-(N,N-dimethylaminosulfonyl)hex-2-yl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compounds 21-6)

[0488] 6-Azide-1-(N,N-dimethylaminosulfonyl)hex-2-yl(4-(hydroxymethyl)phenyl)carbamate (compound 21-5, 140 mg, 0.35 mmol, 1.0 eq) was prepared according to general preparation method B to give 6-azido-1-(N,N-dimethylaminosulfonyl)hex-2-yl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 21-6, 193 mg, yield: 97%).

[0489] LCMS(ES+)m / z:563.1(M+Na).

[0490] 1 H NMR (400MHz, DMSO-d6) δ9.85(s,1H),7.53(d,J=8.5Hz,2H),7.38(d,J=8.6Hz,2H),5.31(s,2H),5.20–5.15(m,1H),3.37(dd,J =5.9,2.9Hz,2H),3.34(s,2H),2.80(s,4H),2.78(s,6H),1.81–1.70(m,2H),1.55(dd,J=10.5,6.8Hz,2H),1.45–1.39(m,2H).

[0491] Step 5: N 2-(((4-((((6-azido-1-(N,N-dimethylaminosulfonyl)hex-2-yl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 21)

[0492] 6-Azide-1-(N,N-dimethylaminosulfonyl)hex-2-yl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 21-6, 60 mg, 0.11 mmol, 1.0 eq) was treated according to general preparation method D to obtain N 2 -(((4-((((6-azido-1-(N,N-dimethylaminosulfonyl)hex-2-yl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 21, 13.6 mg, yield: 17%).

[0493] LCMS(ES+)m / z: 760.2(M+Na).

[0494] 1 H NMR (400MHz, DMSO) δ12.46(s,1H),9.74(s,1H),8.89(t,J=6.0Hz,1H),8.86(d,J=2.8Hz,1H),8.24(d d,J=9.6,2.8Hz,1H),7.53(d,J=8.0Hz,1H),7.46(d,J=8.4Hz,2H),7.27(d,J=8.8Hz,2H),7.21(d,J=1 0.0Hz,1H),5.22-5.11(m,1H),4.93(s,2H),3.97-3.87(m,1H),3.47(dd,J=13.2,6.4Hz,2H),3.37-3 .35(m,2H),3.31-3.30(m,2H),2.78(s,6H),1.82-1.67(m,3H),1.66-1.50(m,5H),1.46-1.35(m,4H).

[0495] Example A19

[0496] Step 1: Synthesis of (2S)-2-[({[6-azido-1-(dimethylaminosulfonyl)hex-2-yl]oxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 22)

[0497] 6-Azide-2-({[(2,5-dioxopyrrolidone-1-yl)oxy]carbonyl}oxy)-N,N-dimethylhexane-1-sulfonamide (compound 21-4, 70 mg, 0.18 mmol, 1.0 eq) was treated according to general preparation method D to obtain (2S)-2-[({[6-azido-1-(dimethylaminosulfonyl)hex-2-yl]oxy}carbonyl)amino]-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 22, 65.2 mg, yield: 62%).

[0498] LCMS(ES+)m / z: 611.2(M+Na).

[0499] 1 H NMR (400MHz, DMSO-d6) δ12.51(s,1H),8.89(t,J=5.2Hz,1H),8.86(dd,J=2.8,0.8Hz,1H),8.25(dd,J=9.6,2.4Hz,1H),7.51(dd,J=16.8,8.0Hz,1H),7 .22(dd,J=9.6,1.6Hz,1H),5.07-4.93(m,1H),3.99-3.88(m,1H),3.53-3.4 2(m,2H),3.32-3.27(m,4H),2.76(s,3H),2.74(s,3H),1.74-1.28(m,12H).

[0500] Example A20

[0501] Step 1: Synthesis of 6-azido-1-(dimethylaminosulfonyl)hex-2-yl-N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 23-1)

[0502] 6-Azide-2-({[(2,5-dioxopyrrolidone-1-yl)oxy]carbonyl}oxy)-N,N-dimethylhexane-1-sulfonamide (compound 21-4, 120 mg, 0.31 mmol, 1.0 eq) was treated according to general preparation method C to obtain 6-azido-1-(dimethylaminosulfonyl)hex-2-yl-N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 23-1, 120 mg, yield: 90%).

[0503] LCMS(ES+)m / z:456.1(M+Na).

[0504] Step 2: Synthesis of {4-[({[6-azido-1-(dimethylaminosulfonyl)hexane-2-yl]oxy}carbonyl)amino]-2-chlorophenyl}methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 23-2)

[0505] 6-Azide-1-(dimethylaminosulfonyl)hex-2-yl-N-[3-chloro-4-(hydroxymethyl)phenyl]carbamate (compound 23-1, 120 mg, 0.28 mmol, 1.0 eq) was treated according to general preparation method C to obtain {4-[({[6-azido-1-(dimethylaminosulfonyl)hexan-2-yl]oxy}carbonyl)amino]-2-chlorophenyl}methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 23-2, 150 mg, yield: 94%).

[0506] LCMS(ES+)m / z:597.1(M+Na).

[0507] Step 3: Synthesis of (2S)-2-{[({4-[({[6-azido-1-(dimethylaminosulfonyl)hexane-2-yl]oxy}carbonyl)amino]-2-chlorophenyl}methoxy)carbonyl]amino}-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 23)

[0508] {4-[({[6-azido-1-(dimethylaminosulfonyl)hexan-2-yl]oxy}carbonyl)amino]-2-chlorophenyl}methyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 23-2, 75 mg, 0.13 mmol, 1.0 eq) was treated according to general preparation method D to obtain (2S)-2-{({4-[({[6-azido-1-(dimethylaminosulfonyl)hexan-2-yl]oxy}carbonyl)amino]-2-chlorophenyl}methoxy)carbonyl]amino}-6-[(2,4-dinitrophenyl)amino]hexanoic acid (compound 23, 45.7 mg, yield: 45%).

[0509] LCMS(ES+)m / z:794.1(M+Na).

[0510] 1H NMR (400MHz, DMSO-d6) δ12.52(s,1H),9.97(s,1H),8.89(t,J=6.0Hz,1H),8.86(d,J =2.8Hz,1H),8.24(dd,J=9.6,2.4Hz,1H),7.66(s,1H),7.62(d,J=8.0Hz,1H),7.40(s ,2H),7.21(d,J=9.6Hz,1H),5.23-5.15(m,1H),5.01(s,2H),3.97-3.88(m,1H),3.51 -3.42(m,2H),3.39-3.30(m,4H),2.78(s,6H),1.80-1.48(m,8H),1.47-1.35(m,4H).

[0511] Example A21

[0512] Step 1: Synthesis of S-(1-(5-((((4-((((6-azido-1-(isopropylsulfonyl)hex-2-yl)oxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)amino)pentyl)-2,5-dioxopyrrolidone-3-yl)-L-cysteine ​​(compound 24)

[0513] [4-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}amino)-2-chlorophenyl]methyl(2,5-dioxopyrrolidone-1-yl) carbonate (compound 5-2, 23.4 mg, 0.042 mmol, 1.0 eq)) was dissolved in anhydrous acetonitrile (0.5 mL). An anhydrous acetonitrile solution (677 μL) of 1-(5-aminopentyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate (compound 24-1, 9.5 mg, 0.032 mmol, 0.75 eq) and N-methylmorpholine (3 μL, 0.032 mmol, 0.75 eq) was added to the reaction solution, and the mixture was stirred at room temperature for 2.5 hours. The reaction was monitored by TLC until it ended. An aqueous solution (0.33 mL) of cysteine ​​hydrochloride (compound 24-3, 5 mg, 0.032 mmol, 0.75 eq) was added to the reaction mixture, and stirring was continued for 76 hours. The reaction was monitored by LCMS until it ended, yielding compound S-(1-(5-((((4-((((6-azido-1-(isopropylsulfonyl)hex-2-yl)oxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)amino)pentyl)-2,5-dioxopyrrolidone-3-yl)-L-cysteine ​​(compound 24).

[0514] LCMS(ES+)m / z:734.2(M).

[0515] Example A22

[0516] Step 1: Synthesis of S-(1-(2-((((6-azido-1-(isopropylsulfonyl)hexane-2-yl)oxy)carbonyl)amino)ethyl)-2,5-dioxopyrrolidone-3-yl)-L-cysteine ​​(compound 25)

[0517] 1-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)oxy]pyrrolidine-2,5-dione (compound 12-4, 20 mg, 0.05 mmol, 1.0 eq) was dissolved in anhydrous N,N-dimethylformamide (0.5 mL). Anhydrous N,N-dimethylformamide solution (0.5 mL) of N-(2-aminoethyl)maleimide hydrochloride (compound 25-1, 9.7 mg, 0.055 mmol, 1.1 eq) and N-methylmorpholine (6 μl, 0.055 mmol, 1.1 eq) was added to the reaction solution, and the mixture was stirred at room temperature for 2.5 hours. After TLC monitoring showed the reaction was complete, anhydrous N,N-dimethylformamide solution (0.58 mL) of cysteine ​​hydrochloride (compound 24-3, 8.7 mg, 0.055 mmol, 1.1 eq) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. After LCMS monitoring showed the reaction was complete, 0.1 N hydrochloric acid aqueous solution (10 mL) was added to adjust the pH to 2, and the mixture was extracted with ethyl acetate (2 mL). The extract was washed successively with water (5 mL) and saturated sodium chloride aqueous solution (2 mL). The aqueous phases were combined to give compound S-(1-(2-((((6-azido-1-(isopropylsulfonyl)hexane-2-yl)oxy)carbonyl)amino)ethyl)-2,5-dioxopyrrolidine-3-yl)-L-cysteine ​​(compound 25).

[0518] LCMS(ES+)m / z:537.1(M+H).

[0519] Example A23

[0520] Step 1: Synthesis of S-(6-((1-((6-azido-1-(isopropylsulfonyl)hexane-2-yl)oxy)vinyl)amino)hexyl)-2,5-dioxopyrrolidone-3-yl)-L-cysteine ​​(compound 26)

[0521] 1-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)oxy]pyrrolidine-2,5-dione (compound 12-4, 20 mg, 0.05 mmol, 1.0 eq) was dissolved in anhydrous acetonitrile (0.5 mL). A solution of 1-(6-aminopentyl)-1H-pyrrolidine-2,5-dione 2,2,2-trifluoroacetate (compound 24-1, 17.1 mg, 0.055 mmol, 1.1 eq) and N-methylmorpholine (6 μL, 0.055 mmol, 1.1 eq) in anhydrous acetonitrile (0.5 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete as monitored by TLC, an aqueous solution of cysteine ​​hydrochloride (compound 24-3, 8.7 mg, 0.055 mmol, 1.1 eq) (0.58 mL) was added to the reaction solution, and the mixture was stirred for another 16 hours. TLC monitoring showed that the reaction contained residual 6-azido-1-(isopropylsulfonyl)hex-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound 26-1). An aqueous solution (0.21 mL) of cysteine ​​hydrochloride (compound 24-3, 3.2 mg, 0.02 mmol, 0.4 eq) was added, and stirring continued for 20 hours. LCMS monitoring indicated the reaction was complete, yielding compound S-(6-((1-((6-azido-1-(isopropylsulfonyl)hexane-2-yl)oxy)vinyl)amino)hexyl)-2,5-dioxopyrrolo-3-yl)-L-cysteine ​​(compound 26).

[0522] LCMS(ES+)m / z:593.2(M+H).

[0523] Example A24

[0524] Synthesis of S-(1-(6-((1-((6-azido-1-(isopropylsulfonyl)hexane-2-yl)oxy)vinyl)amino)hexyl)-2,5-dioxopyrrolidone-3-yl)cysteine ​​(compound 27):

[0525] 1-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)oxy]pyrrolidine-2,5-dione (compound 12-4, 20 mg, 0.05 mmol, 1.0 eq) was dissolved in anhydrous acetonitrile (0.5 mL). A solution of 1-(6-aminopentyl)-1H-pyrrolidine-2,5-dione 2,2,2-trifluoroacetate (17.1 mg, 0.055 mmol, 1.1 eq.) and N-methylmorpholine (6 μL, 0.055 mmol, 1.1 eq) in anhydrous acetonitrile (0.5 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete as monitored by TLC, an aqueous solution of cysteine ​​hydrochloride (8.7 mg, 0.055 mmol, 1.1 eq) (0.58 mL) was added, and the mixture was stirred for another 16 hours. TLC monitoring showed that the reaction contained residual 6-azido-1-(isopropylsulfonyl)hex-2-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexyl)carbamate (compound 27-2). Cysteine ​​hydrochloride (3.2 mg, 0.02 mmol, 0.4 eq) in aqueous solution (0.21 mL) was added, and stirring continued for 20 hours. LCMS monitoring indicated the reaction was complete, yielding compound S-(1-(6-((1-((6-azido-1-(isopropylsulfonyl)hexane-2-yl)oxy)vinyl)amino)hexyl)-2,5-dioxopyrrolo-3-yl)cysteine ​​(compound 27).

[0526] LCMS(ES+)m / z:593.2(M+H).

[0527] Example A25

[0528] Step 1: Synthesis of 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-one (compound 30-1)

[0529] Under nitrogen protection at -65°C, 1.02 mL (2.56 mmol, 1.3 eq., 2.5 M in hexane) was added dropwise to a 5 mL solution of anhydrous tetrahydrofuran containing 2-methanesulfonylpropane (compound 12-1, 361 mg, 2.95 mmol, 1.5 eq.). The mixture was then gradually heated to 0°C and stirred for 1 hour. Subsequently, a 2 mL solution of tetrahydrofuran containing 2-(2-azidoethoxy)ethyl acetate (compound 7-3, 375 mg, 2.17 mmol, 1 eq.) was slowly added dropwise to the reaction mixture, and stirring was continued at -65°C for another 1 hour. TLC showed the reaction was complete. The reaction solution was gradually heated to -10°C, and the reaction was quenched by adding 20 mL of saturated ammonium chloride aqueous solution at this temperature. Then, ethyl acetate (20 mL) was added for extraction three times. The organic phase was collected, washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under vacuum. The solution was purified by normal column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 25% B) to obtain 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-one (compound 30-1,435 mg, yield: 90%).

[0530] LCMS(ES+)m / z:222.03(M-N2+H).

[0531] Step 2: Synthesis of 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-ol (compound 30-2)

[0532] 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-one (compound 30-1, 435 mg, 1.74 mmol, 1 eq) was dissolved in MeOH (5 mL). NaBH4 (100 mg, 2.61 mmol, 1.5 eq) was added in portions at 0 °C, and the mixture was then brought to room temperature and stirred for 30 min. The reaction was monitored by LCMS until complete. The solvent was evaporated to dryness, and the crude product was dissolved in dichloromethane (10 mL), washed with H2O (10 mL), and the aqueous phase was extracted with ethyl acetate (20 mL * 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness, finally yielding 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-ol (compound 30-2, 391 mg, yield: 89%).

[0533] LCMS(ES+)m / z:274.06(M+Na).

[0534] Step 3: Synthesis of (2,5-dioxopyrrolidone-1-yl)carbonate 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-ester (compound 30-3)

[0535] 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-ol (compound 30-2, 391 mg, 1.56 mmol, 1 eq) was treated according to general preparation method A to give (2,5-dioxopyrrolidone-1-yl)carbonate 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-ester (compound 30-3, 363 mg, yield: 59%).

[0536] LCMS(ES+)m / z:415.06(M+Na).

[0537] Step 4: Synthesis of 1-(2-azidoethoxy)-3-(isopropylsulfonyl)propyl-2-yl(4-(hydroxymethyl)phenyl)carbamate (compound 30-4)

[0538] (2,5-Dioxopyrrolidone-1-yl)carbonate 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-ester (compound 30-3, 76 mg, 0.194 mmol, 1 eq) was treated according to general preparation method B to give 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-yl(4-(hydroxymethyl)phenyl)carbamate (compound 30-4, 72 mg, yield: 93%).

[0539] LCMS(ES+)m / z:423.14(M+Na).

[0540] Step 5: Synthesis of 1-(2-azidoethoxy)-3-(isopropylsulfonyl)propyl-2-yl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 30-5)

[0541] 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-yl(4-(hydroxymethyl)phenyl)carbamate (compound 30-4, 72 mg, 0.18 mmol, 1 eq) was treated according to general preparation method B to give 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-yl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 30-5, 97 mg, yield: 94%).

[0542] LCMS(ES+)m / z:564.13(M+Na).

[0543] Step 6: N 2 -(((4-((((1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-yl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 30)

[0544] 1-(2-Azideethoxy)-3-(isopropylsulfonyl)propyl-2-yl(4-(((((2,5-dioxopyrrolid-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 30-5, 97 mg, 0.179 mmol, 1 eq) was treated according to general synthetic method D to give N 2 -(((4-((((1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-yl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 30, 53 mg, yield: 40%).

[0545] LCMS(ES+)m / z: 761.27 (M+Na).

[0546] 1 H NMR (400MHz, DMSO-d6) δ9.85 (s, 1H), 8.93–8.86 (m, 2H), 8.26 (dd, J = 9.6, 2.8Hz, 1H), 7. 53(d,J=8.0Hz,1H),7.49(d,J=8.2Hz,2H),7.30(d,2H),7.23(d,J=9.7Hz,1H),5.44–5.3 7(m,1H),4.96(s,2H),3.99–3.92(m,1H),3.77–3.70(m,3H),3.67–3.61(m,1H),3.54–3 .47(m,4H),3.45–3.41(m,3H),1.81–1.57(m,5H),1.50–1.40(m,2H),1.30–1.26(m,6H).

[0547] Example A26

[0548] Step 1: N 2 Synthesis of -(((1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-yl)oxy)carbonyl)-N6-(2,4-dinitrophenyl)-L-lysine (compound 31)

[0549] (2,5-Dioxopyrrolidone-1-yl)carbonate 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-ester (compound 30-3.54 mg, 0.138 mmol, 1 eq) was treated according to general preparation method D to give N 2-(((1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-yl)oxy)carbonyl)-N6-(2,4-dinitrophenyl)-L-lysine (Compound 31, 28.8 mg, yield: 36%).

[0550] LCMS(ES+)m / z:590.15(M+H).

[0551] 1 H NMR(400MHz, DMSO-d6)δ12.63(s,1H),8.94–8.84(m,2H),8.27(dd,J=9.6,2.8Hz,1H),7.68–7.54(m,1H),7.24(dd,J=9.7,2.5Hz,1H),5.27–5.1 7(m,1H),4.00–3.89(m,1H),3.74–3.55(m,4H),3.54–3.38(m,6H),3.30 –3.24(m,1H),1.83–1.55(m,4H),1.54–1.37(m,2H),1.28–1.23(m,6H).

[0552] Example A27

[0553] Step 1: Synthesis of 1-(2-azidoethoxy)-3-(isopropylsulfonyl)propyl-2-yl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 32-1)

[0554] (2,5-Dioxopyrrolidone-1-yl)carbonate 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-ester (compound 30-3,72 mg, 0.183 mmol, 1 eq) was treated according to general preparation method C to give 1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-yl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 32-1,76 mg, yield: 95%).

[0555] LCMS(ES+)m / z:457.12(M+Na).

[0556] Step 2: Synthesis of 1-(2-azidoethoxy)-3-(isopropylsulfonyl)propyl-2-yl(3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 32-2)

[0557] 1-(2-azidoethoxy)-3-(isopropylsulfonyl)propyl-2-yl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 32-1, 76 mg, 0.175 mmol, 1 eq) was treated according to general preparation method C to give 1-(2-azidoethoxy)-3-(isopropylsulfonyl)propyl-2-yl(3-chloro-4-(((((2,5-dioxopyrrolidine-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 32-2, 98 mg, yield: 97%).

[0558] LCMS(ES+)m / z:597.98(M+Na).

[0559] Step 3: N 2 -(((4-((((1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-yl)oxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 32)

[0560] 1-(2-azidoethoxy)-3-(isopropylsulfonyl)propyl-2-yl(3-chloro-4-(((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 32-2, 98 mg, 0.17 mmol, 1 eq) was treated according to general preparation method D to give N 2 -(((4-((((1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-yl)oxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 32, 16.3 mg, yield: 12%).

[0561] LCMS(ES+)m / z:795.30(M+Na).

[0562] 1H NMR(400MHz,DMSO-d6)δ10.07(s,1H),8.94–8.86(m,2H),8.26(dd,J=9.6,2.8Hz, 1H),7.68(s,1H),7.61(s,1H),7.43(s,2H),7.23(d,J=9.7Hz,1H),5.45–5.38(m, 1H),5.04(s,2H),3.97–3.91(m,1H),3.73(dd,J=11.3,4.0Hz,3H),3.67–3.62(m, 1H),3.54–3.40(m,7H),1.79–1.61(m,4H),1.49–1.41(m,3H),1.29–1.27(m,6H).

[0563] Example A28

[0564] Step 1: Synthesis of 3-(2-azidoethoxy)-N,N-dimethyl-2-oxoprop-1-sulfonamide (compound 34-1)

[0565] Under nitrogen protection at -65°C, 0.955 mL (2.39 mmol, 1.3 eq., 2.5 M in hexane) was added dropwise to a 5 mL solution of anhydrous tetrahydrofuran containing N,N-dimethylmethanesulfonamide (compound 21-1, 373 mg, 3.03 mmol, 1.5 eq.). The mixture was then gradually heated to 0°C and stirred for 1 hour. Subsequently, a 2 mL solution of tetrahydrofuran containing ethyl 2-(2-azidoethoxy)acetate (compound 7-3, 350 mg, 2.02 mmol, 1 eq.) was slowly added dropwise to the reaction mixture, and stirring was continued at -65°C for 1 hour. TLC showed the reaction was complete. The reaction solution was gradually heated to -10°C, and the reaction was quenched by adding 20 mL of saturated ammonium chloride aqueous solution at this temperature. Then, ethyl acetate (20 mL) was added for extraction three times. The organic phase was collected, washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under vacuum. The solution was purified by normal column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 25% B) to give 3-(2-azidoethoxy)-N,N-dimethyl-2-oxoprop-1-sulfonamide (compound 34-1, 460 mg, yield: 87%).

[0566] LCMS(ES+)m / z:273.07(M+Na).

[0567] Step 2: Synthesis of 3-(2-azidoethoxy)-2-hydroxy-N,N-dimethylpropane-1-sulfonamide (compound 34-2)

[0568] 3-(2-azidoethoxy)-N,N-dimethyl-2-oxopropane-1-sulfonamide (compound 34-1, 430 mg, 1.72 mmol, 1 eq) was dissolved in MeOH (5 mL). NaBH4 (98 mg, 2.58 mmol, 1.5 eq) was added in portions at 0 °C, and the mixture was then brought to room temperature and stirred for 30 min. The reaction was monitored by LCMS until complete. The solvent was evaporated to dryness, and the crude product was dissolved in ethyl acetate (10 mL), washed with H2O (10 mL), and the aqueous phase was extracted with ethyl acetate (20 mL * 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness, finally yielding 3-(2-azidoethoxy)-2-hydroxy-N,N-dimethylpropane-1-sulfonamide (compound 34-2, 370 mg, yield: 85%).

[0569] LCMS(ES+)m / z:275.09(M+Na).

[0570] Step 3: Synthesis of 1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)propyl-2-yl(2,5-dioxopyrrolidone-1-yl) carbonate (compound 34-3)

[0571] 3-(2-azidoethoxy)-2-hydroxy-N,N-dimethylpropane-1-sulfonamide (compound 34-2, 370 mg, 1.47 mmol, 1 eq) was treated according to general preparation method A to give 1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)propyl-2-yl(2,5-dioxopyrrolidone-1-yl) carbonate (compound 34-3, 400 mg, yield: 69%).

[0572] LCMS(ES+)m / z:415.06(M+Na).

[0573] Step 4: Synthesis of 1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)propyl-2-yl(4-(hydroxymethyl)phenyl)carbamate (compounds 34-4)

[0574] 1-(2-Azideethoxy)-3-(N,N-dimethylaminosulfonyl)propyl-2-yl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 34-3, 77 mg, 0.195 mmol, 1 eq) was treated according to general preparation method B to give 1-(2-Azideethoxy)-3-(isopropylsulfonyl)propyl-2-yl(4-(hydroxymethyl)phenyl)carbamate (compound 34-4, 72 mg, yield: 93%).

[0575] LCMS(ES+)m / z:424.13(M+Na).

[0576] Step 5: Synthesis of 1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)prop-2-yl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compounds 34-5)

[0577] 1-(2-azidoethoxy)-3-(isopropylsulfonyl)propyl-2-yl(4-(hydroxymethyl)phenyl)carbamate (compound 34-4, 72 mg, 0.18 mmol, 1 eq) was treated according to general preparation method B to obtain 1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)propyl-2-yl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 34-5, 114 mg, crude).

[0578] LCMS(ES+)m / z: 565.03 (M+Na).

[0579] Step 6: N 2 -(((4-((((1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)prop-2-yl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 34)

[0580] 1-(2-Azideethoxy)-3-(N,N-dimethylaminosulfonyl)prop-2-yl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 34-5, 114 mg, crude) was treated according to general synthetic method D, N 2 (((4-((((1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)prop-2-yl)oxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 34, 23.7 mg, yield: 17.8%).

[0581] LCMS(ES+)m / z: 762.27 (M+Na).

[0582] 1H NMR (400MHz, DMSO-d6) δ12.64(s,1H),9.87(s,1H),8.91(t,J=7.7Hz,1H),8.88(d,J=2. 5Hz,1H),8.26(dd,J=7.7,2.8Hz,1H),7.49(d,J=7.7Hz,2H),7.29(d,J=7.7Hz,2H),7.2 3(d,J=10.0Hz,2H),5.34–5.31(m,1H),4.95(s,2H),3.91(s,1H),3.73–3.61(m,4H),3. 51–3.47(m,2H),3.46–3.41(m,4H),2.80(s,6H),1.77–1.58(m,4H),1.46–1.40(m,2H).

[0583] Example A29

[0584] Step 1: N 2 -(((1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)prop-2-yl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 35)

[0585] 1-(2-Azideethoxy)-3-(N,N-dimethylaminosulfonyl)propyl-2-yl(2,5-dioxopyrrolidone-1-yl) carbonate (compound 34-3,54 mg, 0.137 mmol, 1 eq) was treated according to general preparation method D to obtain N 2 -(((1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)prop-2-yl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 35,54 mg, yield: 67%).

[0586] LCMS(ES+)m / z:591.15(M+H).

[0587] 1H NMR (400MHz, DMSO-d6) δ8.93–8.84(m,2H),8.27(dt,J=9.6,2.2Hz,1H),7.59(d,J=7.9Hz,1H),7.24(dd,J=9.7,2.0Hz,1H),5.20–5 .07(m,1H),4.00–3.87(m,1H),3.73–3.59(m,4H),3.54–3.38(m,6H),2.78(d,J=6.9Hz,6H),1.84–1.56(m,4H),1.56–1.36(m,2H).

[0588] Example A30

[0589] Step 1: Synthesis of 1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)propyl-2-yl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 38-1)

[0590] 1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)propyl-2-yl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 34-3, 77 mg, 0.195 mmol, 1 eq) was treated according to general preparation method C to give 1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)propyl-2-yl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 38-1, 75 mg, yield: 88%).

[0591] LCMS(ES+)m / z:458.11(M+Na).

[0592] Step 2: Synthesis of 1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)prop-2-yl(3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 38-2)

[0593] 1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)prop-2-yl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 38-1, 75 mg, 0.17 mmol, 1 eq) was treated according to general preparation method C to obtain 1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)prop-2-yl(3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 38-2, 86 mg, crude).

[0594] LCMS(ES+)m / z:597.98(M+Na).

[0595] Step 3: N 2 -(((4-((((1-(2-azidoethoxy)-3-(N,N-dimethylaminosulfonyl)prop-2-yl)oxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 38)

[0596] 1-(2-Azideethoxy)-3-(N,N-dimethylaminosulfonyl)prop-2-yl(3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 38-2,86 mg, crude) was treated according to general preparation method D to obtain N 2 -(((4-((((1-(2-azidoethoxy)-3-(isopropylsulfonyl)prop-2-yl)oxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 38, 17.9 mg, yield: 13.6%).

[0597] LCMS(ES+)m / z:796.25(M+Na).

[0598] 1H NMR (400MHz, DMSO-d6) δ10.08(s,1H),8.91(t,J=7.7Hz,1H),8.88(d,J=2.5Hz,1H),8.2 6(dd,J=7.7,2.8Hz,1H),7.68(d,J=2.5Hz,1H),7.43(s,2H),7.24(d,J=10.0Hz,2H),5.3 6–5.33(m,1H),5.03(s,2H),3.91–3.85(m,1H),3.74–3.61(m,4H),3.49–3.47(m,2H),3 .46–3.41(m,4H),2.81(s,6H),1.67–1.60(m,2H),1.46–1.40(m,2H),1.27–1.25(m,2H).

[0599] Example A31

[0600] Step 1: Synthesis of 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)acetone (compound 129-2)

[0601] Under nitrogen protection at -65°C, 0.9 mL (2.25 mmol, 1.3 eq., 2.5 M in hexane) of n-butyllithium was added dropwise to a 5 mL solution of anhydrous tetrahydrofuran containing fluorene (compound 129-1, 432 mg, 2.6 mmol, 1.5 eq.). The mixture was then gradually heated to 0°C and stirred for 1 hour. Subsequently, a 2 mL solution of tetrahydrofuran containing ethyl 2-(2-azidoethoxy)acetate (compound 7-3, 300 mg, 1.73 mmol, 1 eq.) was slowly added dropwise to the reaction mixture, and stirring was continued at -65°C for 1 hour. TLC showed the reaction was complete. The reaction solution was gradually heated to -10°C, and the reaction was quenched by adding 20 mL of saturated ammonium chloride aqueous solution at this temperature. Then, ethyl acetate (20 mL) was added for extraction three times. The organic phase was collected, washed with 60 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under vacuum. The solution was purified by normal column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 6% B) to give 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)acetone (compound 129-2, 291 mg, yield: 57%).

[0602] LCMS(ES+)m / z:266.11(M–N2+H).

[0603] Step 2: Synthesis of 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)ethanol (compound 129-3)

[0604] 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)acetone (compound 129-2, 291 mg, 1.17 mmol, 1 eq) was dissolved in MeOH (5 mL). NaBH4 (68 mg, 1.76 mmol, 1.5 eq) was added in portions at 0 °C, and the mixture was then brought back to room temperature and stirred for 30 min. The reaction was monitored by TLC until complete. The solvent was evaporated to dryness, and the crude product was dissolved in ethyl acetate (10 mL), washed with H2O (10 mL), and the aqueous phase was extracted with ethyl acetate (20 mL * 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness, finally yielding 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)ethanol (compound 129-3, 283 mg, yield: 96%).

[0605] LCMS(ES+)m / z:268.14(M–N2+H).

[0606] 1H NMR (400MHz, DMSO-d6) δ7.87(d,J=7.6,1.6Hz,2H),7.74(d,J=7.4Hz,1H),7.64(d,J=7.5Hz,1H),7.43–7.28(m,4H),5.26(d,J=4.6Hz,1H), 4.43–4.36(m,1H),4.19(d,J=4.5Hz,1H),3.55–3.39(m,2H),3.32–3.27(m,2H),3.22(dd,J=10.2,7.0Hz,1H),3.03(dd,J=10.2,3.9Hz,1H).

[0607] Step 3: Synthesis of 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)ethyl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 129-4)

[0608] 2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethanol (compound 129-3, 200 mg, 0.677 mmol, 1 eq) was treated according to general preparation method A to give 2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethyl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 129-4, 264 mg, yield: 89%).

[0609] LCMS(ES+)m / z:409.16(M–N2+H).

[0610] Step 4: Synthesis of 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)ethyl(4-(hydroxymethyl)phenyl)carbamate (compound 129-5)

[0611] 2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethyl(2,5-dioxopyrrolidine-1-yl)carbonate (compound 129-4, 85 mg, 0.195 mmol, 1 eq) and p-aminobenzyl alcohol (48 mg, 0.390 mmol, 2 eq) were dissolved in anhydrous tetrahydrofuran (3 mL). The reaction was carried out in an oil bath at 50 °C for 2 h. The reaction was monitored by LCMS until complete. EA (10 mL) was added to the reaction solution, followed by washing with 1N HCl (20 mL). The organic phase was washed with saturated brine (20 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain 2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethyl(4-(hydroxymethyl)phenyl)carbamate (compound 129-5, 86 mg, yield: 98%).

[0612] LCMS(ES+)m / z:467.18(M+Na).

[0613] Step 5: Synthesis of 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)ethyl(4-(((((2,5-dioxopyrrolidine-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 129-6)

[0614] 2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethyl(4-(hydroxymethyl)phenyl)carbamate (compound 129-5, 86 mg, 0.193 mmol, 1 eq) was treated according to general preparation method B to give 2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethyl(4-(((((2,5-dioxopyrrolidine-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 129-6, 97 mg, yield: 86%).

[0615] LCMS(ES+)m / z:608.14(M+Na).

[0616] Step 6: N 2 -(((4-(((2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethoxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 129)

[0617] 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)ethyl(4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 129-6, 97 mg, 0.166 mmol, 1 eq) was treated according to general synthetic method D, N 2 -(((4-(((2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethoxy)carbonyl)amino)benzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 129,48 mg, yield: 31%).

[0618] LCMS(ES+)m / z:805.31(M+Na).

[0619] 1H NMR (400MHz, DMSO-d6) δ12.57(s,1H),9.91(s,1H),8.90(t,J=7.7Hz,1H),8.88(d,J=2.5Hz,1H),8.26(dd,J=7.7,2.8H z,1H),7.94(dd,J=7.7,2.8Hz,2H),7.81(d,J=7.7Hz,1H),7.71(d,J=10.0Hz,1H),7.51–7.47(m,3H),7.44(d,J=7.7Hz ,2H),7.40–7.36(m,2H),7.29(d,J=7.7Hz,2H),7.23(d,J=10.0Hz,1H),5.55–5.51(m,1H),4.96(s,2H),4.44(d,J=10. 0Hz,1H),3.97–3.91(m,1H),3.55–3.42(m,5H),3.32–3.28(m,1H),1.78–1.59(m,5H),1.46–1.41(m,2H),1.26(s,1H).

[0620] Example A32

[0621] Step 1: N 2 -((2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)ethoxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 130)

[0622] 2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethyl(2,5-dioxopyrrolidone-1-yl)carbonate (compound 129-4, 54 mg, 0.137 mmol, 1 eq) was treated according to general preparation method D to obtain N 2 -((2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)ethoxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 130, 68.9 mg, yield: 59%).

[0623] LCMS(ES+)m / z: 656.20 (M+Na).

[0624] 1H NMR(400MHz, DMSO-d6)δ9.01–8.70(m,2H),8.30–8.09(m,1H),7.90(dd,J=13.8,7.5Hz,2H),7.86–7.57(m,3H),7.50–7.13(m,5H),5 .40–5.25(m,1H),4.35–4.19(m,1H),4.08–3.93(m,1H),3.54–3.40(m,4H),3.30–3.20(m,3H),1.89–1.59(m,4H),1.54–1.41(m,2H).

[0625] Example A33

[0626] Step 1: Synthesis of 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)ethyl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 131-1)

[0627] 2-Chloro-4-aminobenzyl alcohol (41 mg, 0.26 mmol, 2 eq) and 2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethyl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 129-4, 57 mg, 0.13 mmol, 1 eq) were dissolved in acetonitrile (2 mL). With stirring at room temperature, an aqueous solution of sodium bicarbonate (66 mg, 0.7 mmol, 6 eq) (1 mL) was added. The reaction was carried out in an oil bath at 50 °C for 2 h. LC-MS monitoring confirmed complete reaction. Ice water (5 mL) was added to the reaction solution, followed by the addition of 1 N... Extracted with HCl (5 mL), ethyl acetate (10 mL), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)ethyl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 131-1, 60 mg, yield: 96%).

[0628] LCMS(ES+)m / z:501.15(M+Na).

[0629] Step 2: Synthesis of 2-(2-azidoethoxy)-1-(9H-fluorene-9-yl)ethyl(3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 131-2)

[0630] 2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethyl(3-chloro-4-(hydroxymethyl)phenyl)carbamate (compound 131-1, mg, mmol, 1 eq) was treated according to general preparation method C to obtain 2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethyl(3-chloro-4-(((((2,5-dioxopyrrolidine-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 131-2, 80 mg, crude product).

[0631] LCMS(ES+)m / z: 642.15(M+Na).

[0632] Step 3: N 2 -(((4-(((2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethoxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 Synthesis of -(2,4-dinitrophenyl)-L-lysine (compound 131)

[0633] 2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethyl(3-chloro-4-(((((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 131-2, 80 mg, crude) was treated according to general preparation method D to obtain N 2 -(((4-(((2-(2-azidoethoxy)-1-(9H-fluoren-9-yl)ethoxy)carbonyl)amino)-2-chlorobenzyl)oxy)carbonyl)-N 6 -(2,4-dinitrophenyl)-L-lysine (compound 131, 33.4 mg, yield: 32%).

[0634] LCMS(ES+)m / z:839.27(M+Na).

[0635] 1H NMR(400MHz,DMSO-d6)δ10.11(s,1H),8.94–8.84(m,2H),8.25(dd,J=9.7,2.8Hz,1H),7.93 (dd,J=7.5,3.9Hz,2H),7.79(d,J=7.4Hz,1H),7.70(d,J=7.6Hz,2H),7.55(s,1H),7.50–7.3 3(m,6H),7.23(d,J=9.6Hz,1H),5.61–5.51(m,1H),5.03(s,2H),4.45(d,J=5.1Hz,1H),4.0 1–3.87(m,1H),3.58–3.39(m,6H),3.32–3.23(m,3H),1.80–1.56(m,4H),1.50–1.38(m,2H).

[0636] Example B1

[0637] Step 1: Synthesis of the polypeptide conjugate (compound B1, SEQ ID NO:19)

[0638] Peptides were assembled on 2-Chlorotrityl chloride resin using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. An octapeptide (NH2-H{Aib}EKQSWR-COOH; SEQ ID NO:18) was synthesized using standard FMOC chemistry. 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(2,5-dioxopyrrolidone-1-yl) carbonate (compounds 7-6) was added to the octapeptide-binding resin to generate a peptide conjugate (compound B1; SEQ ID NO:19). The peptide conjugate (compound B1; SEQ ID NO:19) was purified by preparative HPLC using standard methods; LCMS (ES+) m / z: 1304.5 (M).

[0639] Example B2

[0640] Step 1: Synthesis of the polypeptide conjugate (compound B2, SEQ ID NO:21)

[0641] PTH peptides were assembled on 2-Chlorotrityl chloride resin (CTC resin, compound B2-1) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. The hexapeptide (NH2-GPSVSEIQLMHNLGKHLNSM ERVEWLRKK(Dde)LQDVHN F-COOH) was synthesized using standard FMOC chemistry. The Dde protecting group was removed by reacting the peptide resin with hydrazine reagent (2% N2H4 in DMF) to obtain the peptide (SEQ ID NO:20). Then, 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(2,5-dioxopyrrolidine-1-yl) carbonate (compounds 7-6) was added to the hexapeptide-binding resin to generate the peptide sequence of the peptide conjugate. The protecting groups on the resin and peptide branches were removed by standard methods. The peptide conjugate (compound B2; SEQ ID NO:21) was obtained by preparative HPLC purification; LCMS (ES+) m / z: 4520.4 (M).

[0642] Example C1

[0643] Step 1: Synthesis of 1-(2-(2-azidoethoxy)ethoxy)-3-(methanesulfonyl)propyl-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound C1-1)

[0644] 1-(2-(2-(-2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-yl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 10-4, 20 mg, 0.049 mmol, 1.0 eq) was dissolved in anhydrous acetonitrile (389 μL), and then 1-(5-aminopentyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate (compound 24-1, 8.7 mg, 0.0294 mmol, 0.0 eq) was added to the reaction solution. A solution of 0.39 mL of anhydrous acetonitrile containing 6 eq) and N-methylmorpholine (3.25 μL, 0.0294 mmol, 0.6 eq) was stirred at 25 °C for 4.5 h. After the reaction was completed as monitored by LCMS, 1-(2-(2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound C1-1) was obtained. The reaction solution was aliquoted and used directly in the next step.

[0645] LCMS(ES+)m / z:476.16(M+H).

[0646] Step 2: Synthesis of the polypeptide conjugate (compound C1-3, SEQ ID NO:2)

[0647] The peptide sequence of the peptide (HA{AIB}V{AIB}EIQLMHNLGKHLNSMERVEWL{AIB}KKLQDVHNFC) was synthesized using standard FMOC chemistry, and the peptide (compound C1-2, SEQ ID NO:1) was purified by reverse-phase HPLC using standard methods. The peptide (compound C1-2, SEQ ID NO:1, 5 mg, 1.1484 μmol, 1.0 eq) was dissolved in deionized water (33.1 μL) and anhydrous acetonitrile (21.2 μL), and then a solution (45.7 μL) of 1-(2-(2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound C1-1, 0.819 mg, 1.7225 μmol, 1.5 eq) in acetonitrile was added, and the mixture was stirred for 16 hours. After the reaction was monitored by LCMS, the polypeptide conjugate (compound C1-3, SEQ ID NO: 2, 2.8 mg) was prepared and isolated.

[0648] LCMS(ES+)m / z:4826.45(M).

[0649] Step 3: Synthesis of the polypeptide conjugate (compound C1, SEQ ID NO:3)

[0650] The polypeptide conjugate (compound C1-3, SEQ ID NO: 2, 2.8 mg, 0.58014 μmol, 1.0 eq) was dissolved in deionized water (0.05% trifluoroacetic acid, 61 μL) and anhydrous acetonitrile (0.05% trifluoroacetic acid, 183 μL). Then, a DMF solution (36 μL) of fatty acid (compound C1-4, 0.72 mg, 0.725 μmol, 20 mg / ml) was added and stirred for 2 hours to prepare and isolate the polypeptide conjugate (compound C1, SEQ ID NO: 3, 1.7 mg). LCMS (ES+) m / z: 5818.0407 (M).

[0651] Example C2

[0652] Step 1: Synthesis of 1-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-3-(methanesulfonyl)propyl-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound C2-1)

[0653] 1-(2-(2-(-2-azidoethoxy)ethoxy)-3-(methanesulfonyl)prop-2-yl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 11-4, 20 mg, 0.044 mmol, 1.0 eq) was dissolved in anhydrous acetonitrile (380 μL), and then 1-(5-aminopentyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate (compound 24-1, 8.5 mg, 0.0287 mmol, 0.65 eq) was added to the reaction solution. A mixture of anhydrous acetonitrile (0.381 mL) and N-methylmorpholine (3.3 μL, 0.0287 mmol, 0.65 eq) was stirred at 25 °C for 4.5 h. After the reaction was completed as monitored by LCMS, 1-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-3-(methanesulfonyl)prop-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound C2-1) was obtained. The reaction solution was dispensed and used directly in the next step.

[0654] LCMS(ES+)m / z:520.21(M+H).

[0655] Step 2: Synthesis of the polypeptide conjugate (compound C2-2, SEQ ID NO:4)

[0656] The polypeptide (compound C1-2, SEQ ID NO: 1, 5 mg, 1.1484 μmol, 1.0 eq) was dissolved in deionized water (33.1 μL) and anhydrous acetonitrile (21.2 μL). Then, an anhydrous acetonitrile solution (45.7 μL) of 1-(2-(2-azidoethoxy)ethoxy)ethoxy)-3-(methanesulfonyl)prop-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound C2-1, 0.894 mg, 1.7225 μmol, 1.5 eq) was added to the reaction solution, and the mixture was stirred for 16 hours. After the reaction was completed as monitored by LCMS, the polypeptide conjugate (compound C2-2, SEQ ID NO: 4, 2.6 mg) was prepared and isolated.

[0657] LCMS(ES+)m / z:4870.47(M).

[0658] Step 3: Synthesis of the polypeptide conjugate (compound C2, SEQ ID NO:5)

[0659] The polypeptide conjugate (compound C2-2, SEQ ID NO:4, 2.6 mg, 0.53383 μmol, 1.0 eq) was dissolved in deionized water (0.05% trifluoroacetic acid, 56 μL) and anhydrous acetonitrile (0.05% trifluoroacetic acid, 168 μL). Then, a DMF solution (33 μL) of fatty acid (compound C1-4, 0.66 mg, 0.66729 μmol, 20 mg / ml) was added and stirred for 2 hours to prepare and isolate the polypeptide conjugate (compound C2, SEQ ID NO:5, 2.1 mg). LCMS (ES+) m / z: 5862.0635 (M).

[0660] Example C3

[0661] Step 1: Synthesis of 6-azido-1-(isopropylsulfonyl)hex-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound 26-1):

[0662] 1-[({[6-azido-1-(propane-2-sulfonyl)hexane-2-yl]oxy}carbonyl)oxy]pyrrolidine-2,5-dione (compound 12-4, 20 mg, 0.05 mmol, 1.0 eq) was dissolved in anhydrous acetonitrile (0.5 mL). A mixture of 1-(6-aminopentyl)-1H-pyrrolidine-2,5-dione 2,2,2-trifluoroacetate (compound 24-1, 11.1 mg, 0.0375 mmol, 0.75 eq) and N-methylmorpholine (4 μL, 0.0375 mmol, 0.75 eq) in anhydrous acetonitrile (0.5 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 2.5 hours. The reaction was monitored by LCMS until it ended, yielding 6-azido-1-(isopropylsulfonyl)hex-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound 26-1). The reaction solution was dispensed and used in the next step.

[0663] LCMS(ES+)m / z:458.2(M+H).

[0664] Step 2: Synthesis of the polypeptide conjugate (compound C3-2, SEQ ID NO:7)

[0665] The peptide sequence of the polypeptide ({AIB}V{AIB}EIQLMHNLGKHLNSMERVEWL{AIB}KKLQDVHNFC) was synthesized using standard FMOC chemistry, and the polypeptide (compound C3-1, SEQ ID NO:6) was purified by reverse-phase HPLC using standard methods. The polypeptide (compound C3-1, SEQ ID NO:6, 5 mg, 1.1484 μmol, 1.0 eq) was dissolved in deionized water (33.1 μL) and anhydrous acetonitrile (21.2 μL), and then an acetonitrile solution (45.7 μL) of 6-azido-1-(isopropylsulfonyl)hex-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound 26-1, 0.819 mg, 1.7225 μmol, 1.5 eq) was added, and the mixture was stirred for 16 hours. After the reaction was monitored by LCMS, the polypeptide conjugate (compound C3-2, SEQ ID NO: 7, 2 mg) was prepared and isolated.

[0666] LCMS(ES+)m / z:4826.45(M).

[0667] Step 3: Synthesis of the polypeptide conjugate (compound C3, SEQ ID NO:8)

[0668] The polypeptide conjugate (compound C3-2, SEQ ID NO:7, 2 mg, 0.435 μmol, 1.0 eq) was dissolved in deionized water (0.2% trifluoroacetic acid, 261 μL) and anhydrous acetonitrile (21.2 μL). Then, a DMF solution (29.5 μL) of fatty acid (compound C3-3, 0.59 mg, 0.578 μmol, 1.33 eq, 20 mg / ml) was added. The mixture was stirred for 16 hours to prepare and isolate the polypeptide conjugate (compound C3, SEQ ID NO:8, 1.6 mg).

[0669] LCMS(ES+)m / z:5619.94(M).

[0670] Example C4

[0671] Step 1: Synthesis of 6-azido-1-(methanesulfonyl)hex-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound C4-1):

[0672] 1-({[(6-azido-1-methanesulfonylhexane-2-yl)oxy]carbonyl}oxy)pyrrolidine-2,5-dione (compound 4-4, 20 mg, 0.055 mmol, 1.0 eq) was dissolved in anhydrous acetonitrile (0.546 mL). A mixture of 1-(6-aminopentyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate (compound 24-1, 12.2 mg, 0.04125 mmol, 0.75 eq) and N-methylmorpholine (4.57 μL, 0.04125 mmol, 0.75 eq) in anhydrous acetonitrile (0.546 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 2.5 hours. The reaction was monitored by LCMS until it ended, yielding 6-azido-1-(methanesulfonyl)hex-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound C4-1). The reaction solution was dispensed and used in the next step.

[0673] LCMS(ES+)m / z:430.1(M+H).

[0674] Step 2: Synthesis of the polypeptide conjugate (compound C4-2, SEQ ID NO:9)

[0675] The polypeptide (compound C1-2, SEQ ID NO: 1, 5 mg, 1.1484 μmol, 1.0 eq) was dissolved in deionized water (33.3 μL) and anhydrous acetonitrile (21.8 μL), and then an acetonitrile solution of compound C4-1 (0.740 mg, 1.7225 μmol, 1.5 eq) (44.8 μL) was added. The mixture was stirred for 16 hours. After the reaction was completed by LCMS monitoring, the polypeptide conjugate (compound C4-2, SEQ ID NO: 9, 3.27 mg) was prepared and isolated.

[0676] LCMS(ES+)m / z:4780.42(M).

[0677] Step 3: Synthesis of the polypeptide conjugate (compound C4, SEQ ID NO:10)

[0678] The polypeptide conjugate (compound C4-2, SEQ ID NO: 9, 3.3 mg, 0.711 μmol, 1.0 eq) was dissolved in deionized water (0.05% trifluoroacetic acid, 74 μL) and anhydrous acetonitrile (0.05% trifluoroacetic acid, 222 μL). Then, a DMF solution (44 μL) of fatty acid (compound C1-4, 0.88 mg, 0.889 μmol, 1.33 eq, 20 mg / ml) was added, and the mixture was stirred for 2 hours to prepare and isolate the polypeptide conjugate (compound C4, SEQ ID NO: 10, 3 mg).

[0679] LCMS(ES+)m / z:5771.9942(M).

[0680] Example C5

[0681] Step 1: Synthesis of 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound C5-1):

[0682] 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(2,5-dioxopyrrolidine-1-yl) carbonate (compound 7-6, 20 mg, 0.055 mmol, 1.0 eq) was dissolved in anhydrous acetonitrile (0.563 mL). A mixture of 1-(6-aminopentyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate (compound 24-1, 12.5 mg, 0.0424 mmol, 0.77 eq) and N-methylmorpholine (4.7 μL, 0.0424 mmol, 0.77 eq) in anhydrous acetonitrile (0.563 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 2.5 hours. The reaction was monitored by LCMS until it ended, yielding 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamate (compound C5-1). The reaction solution was dispensed and used in the next step.

[0683] LCMS(ES+)m / z:432.14(M+H).

[0684] Step 2: Synthesis of the polypeptide conjugate (compound C5-2, SEQ ID NO:11)

[0685] The polypeptide (compound C1-2, SEQ ID NO: 1, 5 mg, 1.1484 μmol, 1.0 eq) was dissolved in deionized water (33 μL) and anhydrous acetonitrile (21.1 μL), and then an acetonitrile solution (45.9 μL) of compound C5-1 (0.743 mg, 1.7225 μol, 1.5 eq.) was added. The mixture was stirred for 18 hours. After the reaction was completed as monitored by LCMS, the polypeptide conjugate PHPL14-MC5-CS-A4_2PEG (compound C5-2, SEQ ID NO: 11, 3.65 mg) was prepared and isolated.

[0686] LCMS(ES+)m / z:4782.35(M).

[0687] Step 3: Synthesis of the polypeptide conjugate (compound C5, SEQ ID NO:12)

[0688] The polypeptide conjugate PHPL14-MC5-CS-A4_2PEG (compound C5-2, SEQ ID NO:11, 7.3 mg, 1.5258 μmol, 1.0 eq) was dissolved in deionized water (0.05% trifluoroacetic acid, 160 μL) and anhydrous acetonitrile (0.05% trifluoroacetic acid, 480 μL). Then, a DMF solution (87 μL) of fatty acid (compound C1-4, 1.74 mg, 1.7546 μmol, 1.15 eq, 20 mg / mL) was added. The mixture was stirred for 18 hours to prepare and isolate the polypeptide conjugate (compound C5, SEQ ID NO:12, 6.6 mg).

[0689] LCMS(ES+)m / z:5774.0117(M).

[0690] Example C6

[0691] Step 1: Synthesis of 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(3-chloro-4-((((5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamoyl)oxy)methyl)phenyl)carbamate (compound C6-1):

[0692] 1-(2-azidoethoxy)-3-(methanesulfonyl)prop-2-yl(3-chloro-4-(((((2,5-dioxopyrrolidine-1-yl)oxy)carbonyl)oxy)methyl)phenyl)carbamate (compound 8-2, 20 mg, 0.0366 mmol, 1.0 eq.) was dissolved in anhydrous acetonitrile (0.4 mL). A mixture of 1-(6-aminopentyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate (compound 24-1, 8 mg, 0.0270 mmol, 0.74 eq.) and N-methylmorpholine (3 μL, 0.0270 mmol, 0.74 eq.) in anhydrous acetonitrile (0.4 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 2.5 hours. The reaction was monitored by LCMS until it ended, yielding 1-(2-azidoethoxy)-3-(methanesulfonyl)propyl-2-yl(3-chloro-4-((((5-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)pentyl)carbamoyl)oxy)methyl)phenyl)carbamate (compound C6-1). The reaction solution was dispensed and used in the next step.

[0693] LCMS(ES+)m / z: 637.12(M+Na).

[0694] Step 2: Synthesis of the polypeptide conjugate (compound C6-2, SEQ ID NO:13)

[0695] The polypeptide (compound C1-2, SEQ ID NO: 1, 5 mg, 1.1484 μmol, 1.0 eq) was dissolved in deionized water (33 μL) and anhydrous acetonitrile (21.1 μL), and then an acetonitrile solution (45.9 μL) of compound C6-1 (1.059 mg, 17.225 μol, 1.5 eq.) was added. The mixture was stirred for 17 hours. After the reaction was completed as monitored by LCMS, the polypeptide conjugate (compound C6-2, SEQ ID NO: 13, 5.2 mg) was prepared and isolated.

[0696] LCMS(ES+)m / z:4966.35(M).

[0697] Step 3: Synthesis of the polypeptide conjugate (compound C6, SEQ ID NO:14)

[0698] The polypeptide conjugate PHPL14-MC5-CS-A(2Cl)_2PEG (compound C6-2, SEQ ID NO:13, 5.2 mg, 1.0467 μmol, 1.0 eq) was dissolved in deionized water (0.05% trifluoroacetic acid, 115 μL) and anhydrous acetonitrile (0.05% trifluoroacetic acid, 345 μL). Then, a DMF solution (87 μL) of fatty acid (compound C1-4, 1.19 mg, 1.2037 μmol, 1.15 eq, 20 mg / mL) was added. The mixture was stirred for 18 hours to prepare and isolate the polypeptide conjugate (compound C6, SEQ ID NO:14, 3.9 mg).

[0699] LCMS(ES+)m / z:5958.0024(M).

[0700] Example C7

[0701] Step 1: Synthesis of the polypeptide conjugate (compound C7-2, SEQ ID NO:16)

[0702] The peptide sequence (SEQ ID NO: 15) of the polypeptide (GPSVSEIQLMHNLGKHLNSMERVEWL{AIB}KKLQDVHNFC) was synthesized using standard FMOC chemistry and purified by reversed-phase HPLC to obtain the polypeptide (compound C7-1, SEQ ID NO: 15). The polypeptide (compound C7-1, SEQ ID NO: 15, 9.8 mg, 2.277 μmol, 1.0 eq) was dissolved in deionized water (65.2 μL) and anhydrous acetonitrile (40 μL), and then a solution of compound C4-1 (1.466 mg, 3.415 μmol, 1.5 eq) in acetonitrile (90.4 μL) was added. The mixture was stirred for 16 hours. After the reaction was monitored by LCMS, the polypeptide conjugate (compound C7-2, SEQ ID NO: 16, 6.3 mg) was prepared and isolated.

[0703] LCMS(ES+)m / z:4732.4(M).

[0704] Step 2: Synthesis of the polypeptide conjugate (compound C7, SEQ ID NO:17)

[0705] The polypeptide conjugate (compound C7-2, SEQ ID NO: 16, 2.8 mg, 0.5916 μmol, 1.0 eq) was dissolved in deionized water (0.05% trifluoroacetic acid, 100 μL) and anhydrous acetonitrile (0.05% trifluoroacetic acid, 300 μL). Then, a DMF solution (34.7 μL) of fatty acid (compound C3-3, 0.69 mg, 0.68 μmol, 20 mg / ml) was added, and the mixture was stirred for 2 hours to prepare and isolate the polypeptide conjugate (compound C7, SEQ ID NO: 17, 2.7 mg); LCMS (ES+) m / z: 5749.93 (M).

[0706] Example C8

[0707] Step 1: Synthesis of the polypeptide conjugate (compound C8, SEQ ID NO:22)

[0708] The polypeptide conjugate (compound B1, 15 mg, 11.5 μmol, 1.3 eq) was dissolved in deionized water (0.5 mL) and anhydrous acetonitrile (0.3 mL), shaken, and centrifuged (repeated twice). Then, an acetonitrile solution of DBCO-PEG_5K (compound C8-1, 44 mg, 8.8 μmol, 1.0 eq) in 1 mL was added to the reaction solution, shaken, and centrifuged (repeated twice). The reaction was allowed to proceed for 24 h.

[0709] The polypeptide conjugate (compound C8; SEQ ID NO:22) was obtained by concentrating and replacing the buffer (ddH2O:ACN = 4:1) using a 3KD ultrafiltration tube.

[0710] Example D1: Peptide-Macromolecule Conjugate

[0711] Step 1: Immunoassay of Anti-HSAVHH-cystin protein sequence

[0712] Alpacas and camels were immunized with a mixture of human serum albumin (HSA), cynomolgus serum albumin (CSA), and rat serum albumin (RSA) in equal mass ratios. Peripheral blood was collected after four immunizations to construct an immune library of alpacas and camels.

[0713] 50 ml of fresh peripheral blood that has reached the required immunization titer was collected, PBMCs were isolated, total RNA was extracted, and total cDNA was synthesized by reverse transcription. Two rounds of PCR were performed using the reverse transcription products as templates: In the first round of PCR, primers were designed for the vector signal peptide and the constant region of the antibody heavy chain, amplifying two bands of approximately 1000 bp and 750 bp, respectively. The 750 bp band was recovered by gel electrophoresis and used as the template for the second round of PCR. In the second round of PCR, primers were designed for the FR1 and FR4 regions of the antibody heavy chain, and restriction enzyme sites were introduced into the phage vector. After restriction enzyme digestion and agarose gel recovery, the second round PCR products were loaded into the digested phage vector and electroporated into TG1 *E. coli* to complete the construction of the immunoglobulin.

[0714] The immunological library was packaged into a phage library. Biotinylated HSA (HSA-biotin) or RSA (RSA-biotin) was used as the antigen for liquid chromatography panning. The bacterial culture after the first and second rounds of panning was diluted, plated, and incubated overnight. Single clones were picked from the plate, packaged with helper phage M13KO7, and the phage supernatant was used for ELISA screening. Positive clones were sequenced to obtain the anti-serum albumin sequence.

[0715] The variable region sequences of anti-serum albumin selected by immunological library sequencing were humanized using a CDR transplantation strategy and partial reversion point mutations were performed. Cystin was added to the end of the humanized variable region for expression in mammalian cells, followed by affinity purification. The protein nomenclature and sequence information are shown in Table A below, while the sequence information of the anti-serum albumin nanobody is shown in Table B below.

[0716] Table A: Antiserum albumin VHH-cystin protein sequence

[0717] Table B: Sequences of anti-serum albumin nanobodies (CDRs are divided according to IMGT encoding method)

[0718] Step 2: Synthesis of the macromolecular conjugate (compound D1-3, SEQ ID NO:24)

[0719] The nanobody (VHH-C, compound D1-1, SEQ ID NO: 23, 4 mg, 0.2888 μmol, 1.0 eq, 2 mg / ml) was dissolved in PBS buffer (2 mL). Tris(2-carboxyethyl)phosphonic hydrochloride (TCEP-HCl, TCI, IUZ8A-OS, 0.745 mg, 2.599 μmol, 9 eq, 10 mg / ml) in PBS buffer (74.5 μL) was added, and the mixture was stirred for 3 hours. Then, N-[19-(11,12-dihydrodibenzo[b,f]azacyclooctyne-5(6H)-yl)-15-oxo-3,6,9,12-tetraoxa-16-azanonadecan-1-yl]-2,5-dihydro-2,5-dioxo-1H-pyrrole-1-propionamide (Dibenzocyclooctyne-PEG4) was added. A DMSO solution (155.9 μL) containing maleimide, BroadPharm, compound D1-2, B121-129, 1.5589 mg, 2.3104 μmol, 8 eq, 10 mg / ml was stirred for 20 hours. The reaction mixture was purified by 10 KD Amicon to obtain the product (compound D1-3, SEQ ID NO: 24, 3.992 mg, 0.2748 μmol, 3.992 mg / ml), which was dissolved in PBS buffer (pH = 6.1). LCMS (ES+) m / z: 14523.44 (M).

[0720] Step 3: Synthesis of the polypeptide-macromolecule conjugate (compound D1, SEQ ID NO:25)

[0721] The macromolecular conjugate (compound D1-3, SEQ ID NO:24, 0.383 mg, 0.0264 μmol, 1.0 eq) was dissolved in PBS buffer (pH = 6.1) (174 μL), and then a DMSO solution (25.2 μL) of the peptide conjugate (compound C4-2, SEQ ID NO:9, 0.252 mg, 0.05273 μmol, 2 eq, 10 mg / ml) was added. The mixture was stirred for 19 hours, and the reaction mixture was purified by 10 KD Amicon to obtain the peptide-macromolecular conjugate (compound D1, SEQ ID NO:25, 3.992 mg, 2.748 μmol, 3.992 mg / ml) dissolved in PBS buffer (pH = 6.1), LCMS (ES+) m / z: 19307.1367 (M).

[0722] Example D2: Polypeptide-Macromolecule Conjugate

[0723] Step 1: Synthesis of the macromolecular conjugate (compound D2-2, SEQ ID NO:27)

[0724] The nanobody (VHH-GGGGS-C, compound D2-1, SEQ ID NO:26, 2.5 mg, 0.1769 μmol, 1.0 eq, 2 mg / ml) was dissolved in PBS buffer (1.25 mL), and then PBS buffer containing tris(2-carboxyethyl)phosphonic hydrochloride (TCEP-HCl, TCI, IUZ8A-OS, 0.507 mg, 1.769 μmol, 10 eq, 10 mg / ml) (50.7 μL) was added. The mixture was stirred for 3 hours, and then N-[19-(11,12-dihydrodibenzo[b,f]azacyclooctyne-5(6H)-yl)-15-oxo-3,6,9,12-tetraoxa-16-azanonadecan-1-yl]-2,5-dihydro-2,5-dioxo-1H-pyrrole-1-propionamide (Dibenzocyclooctyne-PEG4) was added. A DMSO solution (95.5 μL) containing maleimide (BroadPharm, compound D1-2, B121-129, 0.955 mg, 1.415 μmol, 8 eq, 10 mg / ml) was stirred for 20 hours. The reaction mixture was purified by 10 KD Amicon to obtain the product (compound D2-2, SEQ ID NO: 27, 1.882 mg, 0.1271 μmol, 2.353 mg / ml), which was dissolved in PBS buffer (pH = 6.1). LCMS (ES+) m / z: 14802.91 (M).

[0725] Step 2: Synthesis of the polypeptide-macromolecule conjugate (compound D2, SEQ ID NO:29)

[0726] The polypeptide (compound C7-1, SEQ ID NO: 15, 30 mg, 6.97 μmol) was prepared into a polypeptide conjugate (compound D2-3, SEQ ID NO: 28, 24.6 mg, 5.196 μmol) according to the method in Example C5, with LCMS (ES+) m / z: 4732.3995 (M).

[0727] The macromolecular conjugate (compound D2-2, SEQ ID NO:27, 1.53 mg, 0.1033 μmol, 2.353 mg / ml) was dissolved in PBS buffer (pH = 6.1) (650 μL), and then a DMSO solution (100 μL) of the peptide conjugate (compound D2-3, SEQ ID NO:28, 2.4 mg, 0.5069 μmol, 4.9 eq, 0.024 mg / ml) was added. The mixture was stirred for 19 hours, and the reaction mixture was purified by 10 KD Amicon to obtain the peptide-macromolecular conjugate (compound D2, SEQ ID NO:29, 1.32 mg, 0.06756 μmol, 5.5 mg / ml) dissolved in PBS buffer (pH = 6.1), LCMS (ES+) m / z: 19538.5859 (M).

[0728] In addition, Tables C and D are provided below to more clearly illustrate sequences 1-29 above.

[0729] Table C: Detailed Description of Sequences 1-29

[0730] Note: Each X in Table C is only used to describe the structure of each variable in each polypeptide chain in that table, and does not interfere with the definitions of the variables in the general formula above.

[0731] Table D: Specific structures of each group in Table C

[0732] Biological evaluation

[0733] Test Example 1

[0734] Test Name: General Method for Determining the Release Rate of a Drug from a Compound of Formula IV or V

[0735] A mother liquor of the compound is prepared by dissolving the compound in a suitable water-miscible solvent such as DMSO or acetonitrile. An appropriate volume of this mother liquor is then diluted to a buffer solution, optionally containing an internal standard for HPLC analysis, such as sodium benzoate, to obtain a clear solution, which is maintained at a set temperature. Poorly soluble compounds may require the addition of a co-solvent such as DMSO. Periodically, aliquots are removed and immediately analyzed by HPLC or the reaction is quenched by adding 1M acetic acid (AcOH) solution and stored for subsequent analysis. The peak areas of the remaining compound of formula IV or V and (if possible) the drug itself are then determined and compared with the peak area of ​​the internal standard.

[0736] As an example, N is determined as follows: ε The rate at which -(2,4-dinitrophenyl)-L-lysine, "H-Lys(DNP)-OH", is released from each compound.

[0737] A reaction mixture containing 0.1 M buffer and approximately 170 μM of the starting compound was prepared and maintained at 37 °C. Periodically aliquots were removed and the reaction was quenched by adding 1 M acetic acid (AcOH) solution and injected into a Varian Polaris 3 μm C18-A reversed-phase HPLC column (150 x 4.6 mm) for analysis. The column was equilibrated with a 50:50 water / methanol solution (each containing 0.5% acetic acid) at a flow rate of 0.8 mL / min. The compound was eluted from the column using a gradient of 100% acetonitrile + 1% trifluoroacetic acid (TFA) and detected by absorbance at 254 nm. The area (A) of the remaining starting material was obtained by integrating the peaks. s ) and the released H-Lys(DNP)-OH(A P The area of ​​(A) P ), calculate the reaction percentage according to the following formula.

[0738] % reaction = A P / (A P +A s Multiply by 100, then calculate the release rate, and the half-life is calculated as T. 1 / 2 =50% reaction.

[0739] The results generated in these experiments are shown in Table 1, presenting the present disclosure as a model for drug release, N ε The half-life of the release of -(2,4-dinitrophenyl)-L-lysine (H-Lys(DNP)-OH) was determined under HEPES, pH 8.4, and 37°C (accelerated conditions).

[0740] Table 1: Cleavage half-life of drugs from compounds of formula IV or V

[0741] Experimental conclusions

[0742] Obviously, the hydrophilicity of long carbon chains, R 1 and R 2 The properties of its substituents and the different substituents on 4-(hydroxymethyl)aniline provide a wide range of release rates.

[0743] Test Example 2

[0744] In vitro biological cell assay design: cAMP activity detection

[0745] Two buffers were prepared according to the instructions: assay buffer 1 (0.5% BSA in DMEM) and assay buffer 2 (0.5mM IBMX in assay buffer 1). 5 μL of assay buffer 1 diluted samples of various concentrations (compound C7, SEQ ID NO:17, compound D1 (SEQ ID NO:25), compound D2 (SEQ ID NO:29), as examples) were added to a 384-well plate. 5 μL of assay buffer 2 diluted cell suspension (Saos-2 cells) was added to the 384-well plate, and the plate was incubated in a cell culture incubator for 30 minutes. 5 μL of 1X cAMP-d2 and 5 μL of 1X Anti-cAMP-Cryptate (Cisbio:62AM4PEC) were added sequentially, and the plate was incubated at room temperature for 1 hour. The cells were then transferred to PerkinElmer. Data were read at 2105 nm (665 nm and 620 nm) and analyzed using GraphPad Prism 10.2.3 software. The results from these experiments are presented in Figure 3 and Table 2, showing the cAMP activity of the PTH peptide conjugates disclosed herein. This demonstrates that the controllable peptide conjugates of PTH prodrugs (such as SEQ ID NO:17, SEQ ID NO:25, and SEQ ID NO:29) can stably release active PTH and maintain cAMP activity in human plasma for up to 7–14 days.

[0746] Table 2: Drug Release and Activity Assay

[0747] Test Example 3

[0748] Pharmacological evaluation of PTx rats:

[0749] The solvent (PBS-sham, QD) and compound C7 (SEQ ID NO:17 (as an example), at doses of 40 nmol / kg, Q3D*3 and 30 nmol / kg, Q2D*3) were subcutaneously injected into two groups of six rats (sex; female; strain; Sprague Dawley; mean body weight; 400–450 g; age: 9–10 weeks; diet: standard chow food)). Blood was collected from each group of rats immediately before injection and at 24, 48, and 96 hours later. Blood was rotated and serum was collected and stored at -80°C. Calcium and phosphorus concentrations were determined using a commercially available assay (cAMP Gs Dynamic kit, Cisbio; catalog number: 62AM4PEC) according to the manufacturer's instructions. The results are shown in Figures 4A and 4B. This demonstrates the efficacy of the PTH prodrug-controlled peptide conjugate in increasing serum calcium and decreasing serum phosphorus levels in PTx rats.

[0750] Test Example 4

[0751] Assessment of cleavage half-life on PTH peptide conjugates (in PBS):

[0752] A reaction mixture was prepared by dissolving the PTH prodrug peptide or a PTH prodrug peptide conjugate in PBS buffer and adjusting the pH to 7.4. The resulting solution was incubated at 37°C. At designed time points, aliquots were periodically removed and injected into LC-MS and HPLC. The cleavage rate was qualitatively monitored by LC-MS and quantitatively studied by HPLC. The retention times and relative peak areas of the prodrug and the parent model peptide were quantified using Peak Simple Chromatography software.

[0753] The integral of the peak yields the area (A) of the remaining starting material. s ) and the area of ​​the released peptide (A P ), calculate the reaction percentage according to the following formula.

[0754] % reaction = A P / (A P +A s Multiply by 100, then calculate the release rate, and the half-life is calculated as T. 1 / 2 =50% reaction.

[0755] The results of these experiments are shown in Table 3 and Figure 5. The cleavage half-lives of various embodiments of the PTH peptide conjugates of this disclosure are presented. As a model for drug release, the PTH multi-release half-life was determined in PBS, pH 7.4, at 37°C. Clearly, the hydrophilicity of long carbon chains, R... 1 and R 2The properties of its substituents, and the substituents on 4-(hydroxymethyl)aniline, provide a wide range of release rates.

[0756] Table 3: Cleavage Half-Life of PTH Peptide Conjugates

[0757] The embodiments of the technical solution of the present invention have been described above by way of example. It should be understood that the protection scope of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the protection scope of the claims of this application.

Claims

1. A prodrug or its stereoisomer, deuterated derivative, solvate, polymorph, or pharmaceutically acceptable salt thereof, said prodrug comprising a drug and a linker, said drug being linked to said linker, said linker comprising the structure shown in formula (1). wherein: n is an integer selected from 0 to 10, preferably from 1 to 5, more preferably 1, 2 or 3; m is an integer selected from 0 to 10, preferably from 1 to 5, and more preferably 1; X is selected from CH2, O, S, preferably from CH2, O; and, when X is CH2, m and n satisfy conditions a) to d): a) when m is 0, n is not 1 or 2, b) when m is 2, n is not 1, c) when n is 1, m is not 0 or 2, d) when n is 2, m is not 0; R 1 , R 2 are each independently selected from the group consisting of H, C1-C12 alkyl, an electron- withdrawing group, C6-C14 aryl, 5-14 membered heteroaryl, and, R 1 and R 2 , at least one of R 1 , R 2 and the carbon atom to which they are both attached, form a C6-C14 aryl or a 5-14 membered heteroaryl; said C6-C14 aryl or said 5-14 membered heteroaryl is optionally substituted with 1 or more electron- withdrawing groups and / or electron-donating groups; R 3 is N3; Y is absent or selected from the group consisting of preferably, the amino end is attached to the remainder of the general structure; X1, X2, X3, X4are each independently selected from H, an electron withdrawing group; y is an integer selected from 0 to 10.

2. The prodrug or stereoisomer, deuterated metabolite, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 1, wherein, R 1 , R 2 each independently is selected from the group consisting of H, -COR, -CO2R, -SOR, -SO2R, -CN, -NO2, -CF3, -CHF2, and R 1 and R 2 are not simultaneously H; or R 1 , R 2 and the carbon atom to which they are jointly attached form a Preferably, R 1 , R 2 are each independently selected from the group consisting of H, -SOR, -SO2R, -CN, and R 1 and R 2 are not simultaneously H; or R 1 , R 2 and the carbon atom to which they are both attached form a More preferably, R 1 , R 2 are each independently selected from the group consisting of H, -SO2R, -CN, and R 1 and R 2 are not simultaneously H; or R 1 , R 2 and the carbon atom to which they are both attached together form Most preferably, R 1 , R 2 , R 1 , R 2 and the carbon atom to which they are both attached together form each R is independently selected from H, C1-C12 alkyl, C1-C12 alkoxy, -NR a R b , C6-C14 aryl or 5-14 membered heteroaryl, R a , R b each is independently selected from H, C1-C12 alkyl; Preferably, each R is independently selected from H, C1-C12 alkyl, -NR a R b , R a , R b each independently selected from H, C1-C12 alkyl; More preferably, each R is independently selected from H, C1-C6alkyl, -NR a R b , R a , R b each is independently selected from H, C1-C6alkyl; Further preferably, each R is independently selected from C1-C6 alkyl, -NR a R b , R a , R b each is independently selected from C1-C6 alkyl; Most preferably, each R is independently selected from methyl, isopropyl, G is selected from a direct bond, C(=O), SO, SO2, CZ2or CZ2CZ2, wherein each Z is independently selected from H, Cl, F; preferably, G is a direct bond; Most preferably, As a whole, selected from 3. The prodrug or stereoisomer, deuterated metabolite, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 1 or 2, wherein, Y is absent or selected from Preferably, the amino terminus is attached to the remainder of the general structure; X1, X2are each independently selected from H, Cl, F, CN, NO2, CF3, CHF2; preferably, of X1, X2, one is H and the other is selected from H, Cl, F, CN, NO2, CF3, CHF2. preferably, X1, X2are each independently selected from H, Cl, F; more preferably, of X1, X2, one (e.g. X2) is H and the other (e.g. X1) is selected from H, Cl, F; most preferably, of X1, X2, one (e.g. X2) is H, and the other (e.g. X1) is H, Cl; y is an integer selected from 0 to 10, preferably from 1 to 5, further preferably 1, 4 or 5; Most preferably, Y is absent or selected from preferably, the amino end is attached to the remainder of the general structure.

4. The prodrug or stereoisomer, deuterated metabolite, solvate, polymorph, or pharmaceutically acceptable salt thereof of any one of claims 1-3, wherein, the linker comprises a structure represented by Formula (1-1), wherein: n is an integer selected from 0 to 10, preferably from 1-5, more preferably 1, 2 or 3; m is an integer selected from O to 10, preferably from 1 to 5, and more preferably 1; R 1 , R 2 as defined in any one of claims 1 to 3; R 3 as defined in any one of claims 1 to 3; Y is as defined in any one of claims 1 to 3.

5. The prodrug or stereoisomer, deuterated metabolite, solvate, polymorph, or pharmaceutically acceptable salt thereof of any one of claims 1-3, wherein, the linker comprises a structure represented by Formula (1-2), wherein: n is an integer selected from 0 to 10, preferably from 0 to 5, more preferably 1, 2 or 3; m is an integer from 0 to 10, preferably from 1 to 5, and more preferably 0; R 1 , R 2 as defined in any one of claims 1 to 3; R 3 as defined in any one of claims 1 to 3; Y' is selected from X1, X2, X3, X4are each independently selected from H, and electron withdrawing group; preferably, the amino end is attached to the remainder of the general structure; Preferably, Y' is selected from preferably, the amino end is attached to the remainder of the general structure; X1and X2are each independently selected from H, Cl, F, CN, NO2, CF, CHF2; preferably, of X1, X2, one is H and the oth er is selected from H, Cl, F, CN, NO2, CF3, CHF ; preferably, X1, X2are each independently selected from H, Cl, F; preferably, of X1, X2, one (e.g. X2) is H and t he other (e.g. X1) is selected from H, Cl, F; most preferably of X1, X2, one (e.g. X2) is H, and t he other (e.g. X1) is H, Cl; y is an integer selected from O to 10, preferably from 1 to 5, further preferably 1, 4, or 5; Most preferably, Y' is selected from preferably, the amino end is attached to the remainder of the general structure; 6. The prodrug or stereoisomer, deuterated metabolite, solvate, polymorph, or pharmaceutically acceptable salt thereof of any one of claims 1-5, wherein, The linker comprises a structure selected from:

7. The prodrug or stereoisomer, deuterated metabolite, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 1, wherein, The prodrug has a structure represented by Formula (2), wherein: n, m, X, R 1 , R 2 , R 3 , Y is as defined in any one of claims 1 to 3; D is a drug, preferably a peptide drug, a protein drug, a nucleic acid drug, or a small molecule drug; Preferably, the peptide drug includes, but is not limited to, glucagon-like peptide 1 (GLP-1), exendin-2, exendin-3, exendin-4, atrial natriuretic factor (ANF), ghrellin, vasopressin, growth hormone releasing hormone (GHRH), RC-3095, somatostatin, corticotropin, velacapeptin, bombesin, PCK-3145, Phe-His-Ser-Cys-Asn (PHSCN), IGF1, B-type natriuretic peptide, peptide YY (PYY), interferon, thrombocytin, angiogenin, calcitonin, gonadotropin-releasing hormone, cetrorelix, ganirelix, leechin, glucagon, anti-TNF-α, fibroblast growth factor, granulocyte colony stimulating factor, obinepitide, pituitary thyroid hormone (PTH), leuprolide, sermorelin, pramlidomide, nesiritide, rotigaptide, cilengitide, MBP-8298, AL-108, enfuvirtide, caifizomib, mivazerol, thymalfasin, daptomycin, HLF1-I, lactoferrin, Delmitide, glutathione, T-cell epitope PR1, proteinase-3-peptide 1-11, B-cell epitope P3, luteinizing hormone-releasing hormone (LHRH), substance P, neurokinin A, neurokinin B, CCK-8, enkephalins (including leucine enkephalin and methionine enkephalin), dermcidin, [des-Ala20, Gln34]-dermcidin, anionic antimicrobial peptides associated with surface cleanliness, Apidaecin IA, Apidaecin IB, OV-2, 1025,Ethanolamine adhesion peptide (1025-1044) amide, Theromacin (49-63), Pexiganan (MSI-78), Indolicidin, Apelin-15 (63-77), CFP10 (71-85), Inhibitor of lethal factor (LF) associated with anthrax, Bovine antibacterial peptide, Hepatitis C virus NS3 protease inhibitor 2, Hepatitis C virus NS3 protease inhibitor 3, Hepatitis C virus NS3 protease inhibitor 4, NS4A-NS4B Hepatitis C virus (NS3 protease inhibitor 1), HIV-1, HIV-2 protease substrate, Anti-Fltl peptide, Bak-BH3, Bax BH3 peptide (55-74) (wild type), Bid BH3-r8, CTT (gelatinase inhibitor), E75 (Her-2 / neu) (369-377), GRP78 binding chimeric peptide motif, p53 (17-26), EGFR2 / KDR antagonist, Colivelin AGA-(C8R)HNGl 7 (Humanin derivative), Activity-dependent neurotrophic factor (ADNF), Beta secretase inhibitor 1, Beta secretase inhibitor 2, ch[β] amyloid (30-16), Humanun (HN) sHNG, [Glyl4]-HN, [Glyl4]-Humanin, Angiotensin converting enzyme inhibitor (BPP), Renin inhibitor II 1, Annexin 1 (ANXA-1, Ac2-12), Anti-inflammatory peptide 1, Anti-inflammatory peptide 2, Anti-inflammatory Apelin 12, [D-Phe12,Leul4]-Bombesin, Antennapedia peptide (acid) (penetratin), Antennapedia peptide leader peptide (CT), Bombina naja venom peptide, Sulfated [Thr28,Nle31]-Cholecystokinin (25-33), Allatostatin (1-13) (amide), Antistasin, Gamma fibrinogen (377-395), Xenin, Leptin (human), [Hisl,Lysl]-GHRP (GHRP-6), [Ala5,[β]-Ala8]-neurokinin A (4-10), neuromedin B, neuromedin C, neuromedin N, activity-dependent neurotrophic factor (ADNF-14), Acetalin 1 (opioid receptor antagonist 1), Acetalin 2 (opioid receptor antagonist 2), Acetalin 3 (opioid receptor antagonist 3), ACTH (1-39) (human), ACTH (7-38) (human), bombesin, fat mobilizing hormone (locusta migratoria), eicosylated ADP-ribosylation factor 6, myr-ARF6 (2-13), PAMP (1-20) (pro- adrenomedullin (1-20) human), AGRP (25-51), Amylin (8-37) (human), Angiotensin 1 (human), Angiotensin II (human), Apstatin (aminopeptidase P inhibitor), Brevinin-1, Ranatuerin 1, RL-37, LL-37 (antimicrobial peptide) (human), Cecropin A, Antioxidant Peptide A, Antioxidant Peptide B, L-carnosine, Bcl 9-2, NPVF, Neuropeptide AF (hNPAF) (human), Bax BH3 peptide (55-74), bFGF inhibitory peptide, bFGF inhibitory peptide II, caramidin, [Des-Argl O]-HOE 140, Caspase 1 inhibitor II, Caspase 1 inhibitor VIII, Smac N7 protein (MEKI-derived peptide inhibitor 1), hBD-1 ([beta]-defensin-1) (human), hBD-3 ([beta]-defensin-3) (human), hBD-4 ([beta]-defensin-4) (human), HNP-1 (human defensin neutrophil peptide 1), HNP-2 (human defensin neutrophil peptide-2 strong enkephalin A (1-17)), endorphin-1, [beta]-endorphin (human, pig), endothelin 2 (human), fibrinogen binding inhibitory peptide, Cyclo(-GRGDSP), TP508 (thrombin-derived peptide), growth hormone-releasing neuropeptide (human), GIP (human), gastrin-releasing peptide (human), Gastrin-1 (human), Ghrelin (human), PDGF-BB peptide, [D-Lys3]-GHRP-6, HCV core protein (1-20), a3B1 integrin peptide fragment (325) (amide), laminin thymosin alpha 1 (amide), melanocyte-stimulating hormone potentiating factor (MPF), VA-[beta]-MSH, lipolytic hormone y (derived from alpha melanocyte-stimulating hormone), atrial natriuretic peptide (1-28) (human), vasonatrin peptide (1-27), [Ala5, B-Ala8]-neurokinin A (4-10), neuromedin L (NKA), Ac-(Leu28,31) - Neuropeptide Y (24-26), Allatostatins, Brain-Neuropeptide II, [D-tyr11] - Neurotensin, 1 KKy NEMO Binding Domain (NBD) Inhibitory Peptide, PTD-p50 (NLS) Inhibitory Peptide, Orexin A (bovine, human, mouse, rat), Orexin B (human), Aquaporin-2 (254-267) (human pancreatic secretin) (37-52), Pancreatic Polypeptide (human), Neuropeptide, Peptide YY (3-36) (human), Hydroxymethyl-phytochelatin 2, PACAP (1-27) (amide, human, bovine, rat), Prolactin-Releasing Peptide (1-31) (human), Salusin-alpha, Salusin-beta, Saposin C22, Secretin (human), L-selectin, Endokinin A / B, Endokinin C (human), Endokinin D (human), Thrombin Receptor (42-48) Agonist (human), LSKL (inhibitor of Thrombin Sensitizers), Thyrotropin-Releasing Hormone (TRH), P55-TNFR Fragment, Urotensin II (human), VIP (human, pig, rat), VIP Antagonist, Toxins, Exenatide, ZP10 (AVE00100), Pramlintide, AC162352 (PYY) (3-36), PYY, Oxyntomodulin, Glucagon, GRP, Gherlin (GHRP6), Leuprolide, Histrelin, Oxytocin, Atosiban (RWJ22164), Semorelin, Nesiritide, Bivalirudin (Hirulog), Etorphine, Aviptadin, Rotigaptide (ZP123, GAP486), Celanidip (EMD-121924, RGD Peptide), A1buBNP, BN-054, Angiotensin 11, MBP-8298, Peptide Leucine Arginine, Ziconotide, AL-208, AL-108, Carbeticon, Tripeptide, SAL, Coliven, Humanin, ADNF-14, VIP (Vasoactive Intestinal Peptide), Thymalfasin, Bacitracin, Short Bacitracin, Pexiganan (MSI-78), PI13, PAC-113, SCV-07, HLF1-I1 (Lactoferrin), DAPTA, TRI-1144, Tritrpticin, Antiflammin 2, Gattex (Teduglutide,ALX-0600), Stimuvax (L-BLP25), Chrysalin (TP508), Melanonan II, Spantide II, Chrypstin, Syn-B, Pentagastrin, Secretin, Endostatin peptide, E-selectin, HER2, PDGF, Thrombospondin, uPA (1), uPA (2), VEGF, VEGF (2), Thymosin-beta-3, XXLRR, beta-amyloid fibrillogenesis inhibitor, Endomorphin-2, TIP 39 (Phrelin), PACAP (1-38) (amide, human, bovine, rat), TGFB-activating peptide, Insulin sensitizing factor (ISF402), Transforming growth factor neuropeptide (TGF-B1), Bombesin releasing factor, IELLQAR (8-branch MAPS), Teglaseran PK3145, Goserelin, Abarelix, Cetrorelix, Ganirelix, Degarelix (from Prolin), Barusiban (FE 200440), Pramlintide, Orvastatin, Eptifibatide, Netamiftide (INN-00835), Daptomycin, Spantide II, Delmitide (RDP-58), AL-209, Enfuvirtide, IDR-I, Hexapeptide-6, Insulin A chain, Lanreotide, Hexa[rho]eptide-3, Insulin-B chain, Insulin- Lispro A-chain analogue, Insulin-Aspart B-chain analogue, Insulin-Glulisine B-chain analogue, Insulin-Detemir B-chain analogue, Somatostatin tumour-inhibiting analogue, Chymase (37-52), Vasoactive intestinal peptide fragment (KKYL-NH2), and Dynorphin A, cyclic peptides (e.g., Romidepsin, Voclosporin, Ziconotide, Linaclotide, Plecanatide, Pasireotide, Lanreotide, Vasopressin, Terlipressin, Bremelanotide, Setmelanotide, Daptomycin, Telavancin, Dalbavancin, Oritavancin, Caspofungin, Micafungin, Anidulafungin), Preferably, the protein drugs include but are not limited to enzymes (e.g., asparaginase, sacrosidase, pegvaliase, laronidase, glucosidase, beta-glucocerebrosidase), blood coagulation factors (e.g., coagulation factor VIII, coagulation factor IX, coagulation factor XIII), protein hormones (e.g., growth hormone GH, insulin, erythropoietin, gonadotropin, parathyroid hormone), cytokines (e.g., interleukins, interferons, colony-stimulating factors); Preferably, the nucleic acid drugs include but are not limited to antisense nucleic acids (ASO) (e.g., Fomivirsen, Mipomersen, Eteplirsen, Nusinersen, inotersen, Volanesoresn, Golodirsen), small interfering RNA (siRNA) (e.g., Onpattro, Givlaari), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer; Preferably, the small molecule drugs include, but are not limited to, antibiotics including penicillins (e.g., penicillin, penicillin V), fluoroquinolones (e.g., ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, gemifloxacin), cephalosporins (e.g., cefixime, ceftibuten, cefditoren pivoxil, cefdinir, cefpodoxime proxetil), aminoglycosides (e.g., isepamicin, amikacin, etimicin, netilmicin), macrolides (e.g., erythromycin, azithromycin, clarithromycin), tetracyclines (e.g., doxycycline, minocycline, tigecycline, omadacycline, eravacycline), sulfonamides and sensitizers (e.g., trimethoprim, sulfadiazine, sulfamethoxazole, sulfamethoxazole), urinary preparations (e.g., nitrofurantoin, fosfomycin); analgesic anti-inflammatory drugs (e.g., aspirin, ibuprofen, indomethacin, nabumetone, acetaminophen, lidocaine, procaine, tetracaine); antiallergics (e.g., chlorpheniramine, triprolidine, diphenhydramine, tripelennamine, methapyrilene, promethazine, azelastine, ebastine, loratadine, terfenadine, fexofenadine, desloratadine, cetirizine); antiviral drugs (e.g., oseltamivir, maraviroc, elvitegravir, ledipasvir, cobicistat, entecavir, nirmatrelvir / ritonavir, ribavirin, interferon, arbidol, zanamivir, peramivir, amantadine, acyclovir, ganciclovir, valacyclovir, dalitiravir, asunaprevir, asunaprevir, adefovir dipivoxil, lamivudine, telbivudine, azvudine); antihypertensive drugs (e.g., amlodipine, felodipine, cinaldipine, nifedipine, nitrendipine, enalapril, fosinopril, lisinopril, perindopril, imidapril, losartan, valsartan, irbesartan, telmisartan, olmesartan, hydrochlorothiazide, indapamide, metoprolol, bisoprolol, atenolol, labetalol, propranolol, prazosin, terazosin, doxazosin); diabetes drugs (e.g., glibenclamide, gliclazide, glipizide, gliclazide, glimepiride, repaglinide, nateglinide, acarbose, voglibose, miglitol, pioglitazone, rosiglitazone, sitagliptin, linagliptin, anagliptin, saxagliptin, vildagliptin, remogliflozin, emapamil);Chronic obstructive pulmonary disease drugs (e.g., fenoterol, levalbuterol, salbutamol, terbutaline, arformoterol, formoterol, indacaterol, olodaterol, salmeterol, ipratropium bromide, oxitropium bromide, aclidinium bromide, glycopyrronium bromide, tiotropium bromide, umclidinium bromide, glycopyrronium (glycopyrronium bromide), rufinacain, roflumilast, erdosteine, carbocysteine), antitumor drugs (e.g., methotrexate, pemetrexed, fluorouracil, 6-mercaptopurine, hydroxyurea, gemcitabine, cytarabine, doxorubicin, epirubicin, Lurbinectedin, irinotecan, topotecan, etoposide, taxoids, vinca alkaloids, eribulin, EGFR inhibitors: gefitinib, erlotinib, icotinib, afatinib, osimertinib, ALK inhibitors: crizotinib, alectinib, ceritinib, loratinib, MET inhibitors: savolitinib, capmatinib, RET inhibitors: pralsetinib, LOXO-292, NTRK inhibitors: larotrectinib, entrectinib, BRAF inhibitors: dabrafenib, vemurafenib, encorafenib, MEK inhibitors: trametinib, binimetinib, HER2 inhibitors: neratinib, tucatinib, CDK4 / 6 inhibitors: ribociclib, abemaciclib, PARP inhibitors: olaparib, niraparib, anti-angiogenic multi-kinase inhibitors: nilotinib, apatinib, sunitinib, vandetanib, pazopanib, axitinib, vandetanib, cabozantinib, mTOR inhibitors: everolimus, HDAC inhibitors: chidamide, ABL-BCR inhibitors: imatinib, dasatinib, nilotinib, PDGFR, C-KIT inhibitors: imatinib, nilotinib, avatinib, BTK inhibitors: ibrutinib, zanubratinib, zanabrutinib, proteasome inhibitors: bortezomib, ixazomib, JAK inhibitors: ruxolitinib, selective nuclear transport protein inhibitors: selinexor, PI3K inhibitors: alpelisib, FGFR2 inhibitors: pemigatinib, IDH1 inhibitors: ivosidenib); psoriasis drugs (e.g., apremilast, deucravacitinib); antipsychotic drugs (e.g., risperidone, quetiapine, chlorpromazine, perphenazine, clozapine, olanzapine, sulpiride); antidepressants (e.g., fluoxetine, paroxetine, sertraline, citalopram, venlafaxine, duloxetine, milnacipran, bupropion, agomelatine, fluvoxamine, mirtazapine.

8. The prodrug or stereoisomer, deuterated metabolite, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 7, wherein, The prodrug has a structure represented by Formula (2-1), wherein: n, m, R 1 , R 2 , R 3 , Y is as defined in claim 4; D is as defined in claim 7.

9. The prodrug or stereoisomer, deuterated metabolite, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 7, wherein, said prodrug has a structure according to Formula (2-2), wherein: n, m, X, R 1 , R 2 , R 3 , Y' is as defined in claim 5; D is as defined in claim 7.

10. The prodrug or stereoisomer, deuterated metabolite, solvate, polymorph, or pharmaceutically acceptable salt thereof of any one of claims 7-9, wherein, The prodrug has a structure selected from the group consisting of: wherein each D is independently as defined in claim 7.

11. A drug-macromolecule conjugate or its stereoisomers, deuterated derivatives, solvates, polymorphs, or pharmaceutically acceptable salts thereof, said drug-macromolecule conjugate comprising a drug, a macromolecule, and a linking unit, said drug being linked to said macromolecule via said linking unit, said linking unit comprising the structure shown in formula (3). wherein: n, m, X, R 1 , R 2 , Y are as defined in any one of claims 1 to 3.

12. The drug-macromolecule conjugate or a stereoisomer, deuteride, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 11, wherein, The connection unit comprises a structure represented by formula (3-1), wherein: n, m, R 1 , R 2 , Y are as defined in claim 4.

13. The drug-macromolecule conjugate or a stereoisomer, deuterated form, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 11, wherein, The connection unit includes a structure represented by formula (3-2), wherein: n, m, X, R 1 R 2 Y' is as defined in claim 5.

14. The drug-macromolecule conjugate or a stereoisomer, deuterated analog, solvate, polymorph, or pharmaceutically acceptable salt thereof of any one of claims 11-13, wherein, The connecting unit comprises a structure selected from the group consisting of:

15. The drug-macromolecule conjugate or a stereoisomer, deuterated form, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 11, wherein, The drug-macromolecule conjugate comprises a structure represented by formula (4), wherein: n, m, X, R 1 R 2 Y is as defined in any one of claims 1-3; D is as defined in claim 7.

16. The drug-macromolecule conjugate or a stereoisomer, deuterated analog, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 15, wherein, The drug-macromolecule conjugate comprises a structure represented by formula (4-1), wherein: n, m, R 1 , R 2 , Y are as defined in claim 4; D is as defined claim 7.

17. The drug-macromolecule conjugate or a stereoisomer, deuterated analog, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 15, wherein, The drug-macromolecule conjugate comprises a structure represented by formula (4-2), wherein: n, m, X, R 1 , R 2 , Y' is as defined in claim 5, D is as defined in claim 7.

18. The drug-macromolecule conjugate or a stereoisomer, deuterated analog, solvate, polymorph, or pharmaceutically acceptable salt thereof of any one of claims 15-17, wherein, The drug-macromolecule conjugate comprises a structure selected from the group consisting of wherein each of D is independently as defined in claim 7.

19. The drug-macromolecule conjugate or a stereoisomer, deuterated analog, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 11, wherein, The drug-macromolecule conjugate has a structure shown in formula (5), wherein: n, m, X, R 1 R 2 Y is as defined in any one of claims 1-3; D is as defined in claim 7; P is a macromolecule-containing group; Preferably, the macromolecule is an antibody (e.g., IgG, IgM, IgA, IgD or IgE) or an antigen-binding fragment thereof, a fatty acid (e.g., C16, C18, C20 fatty acid), PEG, pCB or any coupling structure thereof.

20. The drug-macromolecule conjugate or a stereoisomer, deuterated form, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 19, wherein, The drug-macromolecule conjugate has a structure represented by Formula (5-1), wherein: n, m, R 1 , R 2 , Y are as defined in claim 4; D is as defined in claim 7; P is as defined in claim 19.

21. The drug-macromolecule conjugate or a stereoisomer, deuterated form, solvate, polymorph, or pharmaceutically acceptable salt thereof of claim 19, wherein, The drug-macromolecule conjugate has a structure represented by Formula (5-2), wherein: n, m, X, R 1 R 2 Y' is as defined in claim 5; D is as defined in claim 7; P is as defined in claim19.

22. The drug-macromolecule conjugate or a stereoisomer, deuterated analog, solvate, polymorph, or pharmaceutically acceptable salt thereof of any one of claims 19-21, wherein, The drug-macromolecule conjugate has a structure selected from the group consisting of wherein each of D is independently as defined in claim 7, and each of P is independently as defined in claim 19.

23. A prodrug intermediate having the structure of Formula (6), Formula (6) or a stereoisomer, deuterated form, solvate, polymorph, or pharmaceutically acceptable salt thereof. wherein: n, m, X, R 1 , R 2 , R 3 as defined in any one of claims 1 to 3; Y1is selected from the group consisting of X1, X2, X3, X4are each independently selected from H, an electron-withdrawing group; Preferably, Y1is selected from X1, X2are each independently selected from H, Cl, F, CN, NO2, CF3, CHF2; Preferably, among X1, X2, one is H and the other is selected from H, Cl, F, CN, NO2, CF3, CHF2. Preferably, X1, X2are each independently selected from H, Cl, F; More preferably, among X1, X2, one (e.g., X2) is H and the other (e.g., X1) is selected from H, Cl, F; Most preferably, among X1, X2, one (e.g., X2) is H, and the other (e.g., X1) is H, Cl. y is an integer selected from 0-10, preferably an integer selected from 1-5, more preferably 1, 4 or 5; Most preferably, Y1is selected from 24. The prodrug intermediate of claim 23, or a stereoisomer, deuterated analog, solvate, polymorph, or pharmaceutically acceptable salt thereof, wherein, The prodrug intermediate has a structure represented by Formula (6-1), wherein: n, m, R 1 , R 2 , R 3 as defined in claim 4; Y1is as defined in claim 23.

25. The prodrug intermediate of claim 23, or a stereoisomer, deuterated analog, solvate, polymorph, or pharmaceutically acceptable salt thereof, wherein, The prodrug intermediate has a structure represented by formula (6-2), wherein: n, m, X, R 1 , R 2 , R 3 as defined in claim 5; Y1' is selected from X1, X2, X3, X4are each independently selected from H, an electron- withdrawing group; Preferably, Y1' is selected from X1, X2are each independently selected from H, CI, F, CN, NO2, CF3, CHF2; Preferably, of X1, X2, any one is H, and the other is selected from H, CI, F, CN, NO2, CF3, CHF2. Preferably, X1, X2are each independently selected from H, CI, F. More preferably, of X1, X2, any one (such as X2) is H, and the other (such as X1) is selected from H, CI, F. Most preferably, of X1, X2, any one (such as X2) is H and the other (such as X1) is H, CI. y is an integer selected from 0-10, preferably an integer selected from 1- 5, more preferably 1, 4 or 5; Most preferably, Y1' is selected from 26. The prodrug intermediate of any one of claims 23-25, or a stereoisomer, deuterated analog, solvate, polymorph, or pharmaceutically acceptable salt thereof, wherein, The prodrug intermediate has a structure selected from the group consisting of:

27. A prodrug intermediate comprising a structure represented by Formula (7), Formula (7), or a stereoisomer, deuterated form, solvate, polymorph, or pharmaceutically acceptable salt thereof. wherein: n, m, X, R 1 , R 2 , R 3 as defined in any one of claims 1 to 3.

28. The prodrug intermediate of claim 27, or a stereoisomer, deuterated derivative, solvate, polymorph, or pharmaceutically acceptable salt thereof, wherein, The prodrug intermediate comprises a structure selected from:

29. The prodrug intermediate of claim 27, or a stereoisomer, deuterated derivative, solvate, polymorph, or pharmaceutically acceptable salt thereof, wherein, The prodrug intermediate has a structure shown in formula (8), wherein: n, m, X, R 1 , R 2 , R 3 as defined in any one of claims 1 to 3; Q is 30. The prodrug intermediate of claim 29, or a stereoisomer, deuterated derivative, solvate, polymorph, or pharmaceutically acceptable salt thereof, wherein, The prodrug intermediate has a structure selected from the group consisting of:

31. A pharmaceutical composition comprising a prodrug of any one of claims 1-10, or a stereoisomer, deuterated analog, solvate, polymorph, or pharmaceutically acceptable salt thereof, or a drug-macromolecule conjugate of any one of claims 11-22, or a stereoisomer, deuterated analog, solvate, polymorph, pharmaceutically acceptable salt thereof, or a prodrug intermediate of any one of claims 23-30, or a stereoisomer, deuterated analog, solvate, polymorph, a pharmaceutically acceptable salt thereof. Preferably, the composition further comprises a pharmaceutically acceptable excipient. Preferably, the composition further comprises one or more additional therapeutic agents.

32. Use of a prodrug of any one of claims 1-10, or a stereoisomer, deut erated analog, solvate, polymorph, or pharmaceutically acceptable salt thereof, or of a drug-macromolecule conjugate of any one of claims 11-22, or of a stereoisomer, deuterated analog, solvate, polymorph, pharmaceuti cally acceptable salt thereof, or of a prodrug intermediate of any one of claims 23-30, or of a stereoisomer, deuterated analog, solvate, polymorph a pharmaceutically acceptable salt thereof, or of a pharmaceutical composition of claim 31, for the manufacture of a medicament for the treatment and / or prevention of a disease.

33. A Nanobody or antigen-binding fragment thereof capable of specifically binding to serum albumin, comprising: (a) a CDR1 as contained in a heavy chain variable region (VHH) as set forth in SEQ ID NO: 30 or a variant thereof, a CDR2 as contained in a heavy chain variable region (VHH) as set forth in SEQ ID NO : 30 or a variant thereof, and a CDR3 as contained in a heavy chain variable region (VHH) as set forth in SEQ ID NO 30 or a variant thereof; or (b) a CDR1 as contained in a heavy chain variable region (VHH) as set fort in SEQ ID NO: 34 or a variant thereof, a CDR2 as contained in a heavy chain variable region (VH H) as set forth in SEQ ID NO: 34 or a variant thereof, and a CDR3 as contained in a heavy chain variable region ( VHH) as set forth in SEQ ID NO: 34 or a variant thereof. wherein, The variant has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably the substitution is a conservative substitution; Preferably, the Nanobody or antigen-binding fragment thereof comprises: (a) a CDR1, CDR2 and CDR3 contained within a heavy chain variable region (VHH) as depicted in SEQ ID NO: 30; or, (b) a CDR1, CDR2 and CDR3 contained within a heavy chain variable Preferably, the Nanobody or antigen-binding fragment thereof comprises: (a) a CDRl as depicted in SEQ ID NO: 31, a CDR2 as depicted in SEQ ID NO: 32, and a CDR3 as depicted in SEQ ID NO: 33; or, (b) a CDRl as depicted in SEQ ID NO: 35, a CDR2 as depicted in SEQ ID NO: 36, and a CDR3 as depicted in SEQ ID NO:

37.

34. The nanobody or antigen-binding fragment thereof of claim 33, wherein, The Nanobody or antigen-binding fragment thereof comprises a heavy chain framework region sequence of an immunoglobulin (e.g. a human or a camelid immunoglobulin heavy chain framework region sequence); Preferably, the Nanobody or antigen-binding fragment thereof comprises a heavy chain framework region of a human immunoglobulin; Preferably, the Nanobody or antigen-binding fragment thereof comprises a heavy chain framework region comprised within an amino acid sequence encoded by a human heavy chain germline gene; preferably the heavy chain framework region optionally comprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) backmutations from a human residue to a camelid residue.

35. The Nanobody or antigen-binding fragment thereof of any one of claims 33-34, which comprises an amino acid sequence selected from the group consisting of: (i) a sequence as depicted in any one of SEQ ID NOs: 30, 34; (ii) a sequence having one or several (e.g. 1, 2, 3, 4 or 5) amino acid substitutions, deletions or additions compared to the sequence as depicted in any one of SEQ ID NOs: 30, 34; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence as depicted in any one of SEQ ID NOs: 30, 34; Preferably, the substitution is a conservative substitution.

Images