Prodrug based on enzyme catalysis and chemical conversion

Abstract

Provided are a prodrug based on enzyme catalysis and chemical conversion, a pharmaceutical composition comprising the prodrug, a preparation method therefor, and use thereof.

Description

A prodrug based on enzyme catalysis and chemical transformation

[0001] This application is based on and claims priority to the following applications: CN application number 202510125088.8 (filed January 26, 2025), CN application number 202510173572.8 (filed February 17, 2025), CN application number 202511119969.5 (filed August 11, 2025), and CN application number 202610092071.1 (filed January 22, 2026). The disclosures of the aforementioned applications are incorporated herein by reference in their entirety. Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to prodrugs based on enzyme catalysis and chemical transformation, as well as pharmaceutical compositions containing such prodrugs and their applications. Background Technology

[0003] Some drugs have a short duration of action and low bioavailability after administration due to poor absorption or metabolic effects, making it difficult for them to exert their effects. Furthermore, the rapid release of the drug during administration may cause toxic side effects.

[0004] A prodrug is a therapeutic agent that is virtually inactive on its own but predictably releases an active drug in the body through enzymatic and / or non-enzymatic conversion to exert its therapeutic effect. Prodrug design can alter the physicochemical properties of a drug, improve its pharmacokinetic processes in the body, prolong its duration of action, increase its bioavailability, and reduce its toxic side effects.

[0005] Designing prodrugs can, to some extent, overcome the side effects of high-dose injections. This involves allowing the drug to enter the body in its inactive form, and then slowly releasing its active form under specific conditions, thus achieving a stable drug effect. Currently, the main technology used for long-acting protein and peptide drugs is prodrug-based sustained-release technology. This involves administering the drug as a predrug, which then slowly releases its active form within the body. This reduces the frequency of administration, resulting in more stable blood glucose control and fewer and safer side effects.

[0006] Currently, common prodrugs are mainly divided into two categories: carrier prodrugs and biological prodrugs. Carrier prodrugs refer to compounds inactive or with low activity in vitro, formed by the linkage of an active drug and a carrier, which release the active drug in vivo through enzymatic or non-enzymatic transformation to exert their pharmacological effects. Biological prodrugs are new compounds obtained by molecularly modifying an active compound. This new compound serves as a substrate for metabolic enzymes, and the active metabolites produced through enzymatic metabolism are the intended active molecules.

[0007] However, these existing prodrug technologies mainly target small molecule drugs, with fewer prodrug technologies designed for protein and peptide drugs. Currently, the most commonly used technology for peptide drugs is carrier prodrug technology, such as polyethylene glycol modification technology. This type of technology mainly relies on chemical synthesis, which is complex. The coupling process requires precise control and optimization, and the additional materials and process steps required for the coupling process increase the production cost of the drug.

[0008] Diketopiperazine (DKP) is a cyclic molecule composed of two α-amino acids linked by peptide bonds; these two α-amino acids are called DKP dipeptides. DKP dipeptides with free α-amino groups can undergo specific chemical or enzymatic reactions in vivo or under simulated in vivo environments (such as suitable pH, temperature, and enzyme presence) to cyclize and form DKP, releasing biologically active drug molecules. Prodrugs that release active drugs through this mechanism are called DKP-based prodrugs, which can release active drugs under specific physiological conditions, improving drug targeting and bioavailability.

[0009] Because DKP-based prodrugs release the active drug through chemical transformation, improper storage and transportation conditions can lead to DKP dipeptide cyclization. For example, prolonged exposure to high temperature, high humidity, or light may accelerate the cyclization reaction of DKP dipeptides. Simultaneously, other components in the drug formulation may interact with the DKP dipeptide, resulting in its cyclization. Certain steps in the formulation process, such as mixing, homogenization, and drying, may also affect the stability of DKP dipeptides. This poses challenges to the manufacturing and formulation development of DKP-based prodrugs. Furthermore, once DKP-based prodrugs begin to cleave in vivo, their chemical transformation rate cannot be slowed down, making it difficult to control the drug release rate.

[0010] Therefore, designing prodrugs that are stable in vitro and have a controllable release rate in vivo will provide value for disease treatment. Summary of the Invention

[0011] prodrug

[0012] A first aspect of the present invention provides a prodrug or a pharmaceutically acceptable salt thereof, wherein the prodrug comprises the structure shown in formula (F-1).

[0013] in,

[0014] A1 and A2 are each an α-amino acid;

[0015] B is an α-amino acid or an α-amino acid modified with an azide group;

[0016] Each C is an N-alkylated α-amino acid;

[0017] n is an integer between 0 and 10;

[0018] m is an integer between 0 and 10;

[0019] The condition is that m and n are not both 0;

[0020] Dipeptide A1-A2 can be cleaved by DPP4 enzyme to be removed from the prodrug;

[0021] A1, A2, B, and C are connected by amide bonds.

[0022] In some embodiments, dipeptide A1-A2 can be cleaved by the DPP4 enzyme. The ability of dipeptide A1-A2 to be cleaved by the DPP4 enzyme means that dipeptide A1-A2 can be cleaved by the DPP4 enzyme at the C-terminus of amino acid A2, thereby being removed from the prodrug.

[0023] In some embodiments, when there are n dipeptides A1-A2, the first dipeptide A1-A2 at the N-terminus is first removed from the prodrug by DPP4 enzyme cleavage, and the remaining n-1 dipeptides A1-A2 are then removed from the prodrug by DPP4 enzyme cleavage in sequence.

[0024] In some embodiments, the dipeptide BC is cyclized to form DKP, which is then removed from the prodrug.

[0025] In some embodiments, when there are m dipeptides BC, the first N-terminal dipeptide BC is first cyclized to form DKP and removed from the prodrug, and the remaining m-1 dipeptides BC are sequentially cyclized to form DKP and removed from the prodrug.

[0026] In some implementations, the C terminal of A1 is connected to the N terminal of A2.

[0027] In some implementations, A1 is the N-terminus of the prodrug.

[0028] In some implementations, B is the N-terminus of the prodrug.

[0029] In some implementations, A2 is independently selected from Pro, Ala, Gly, Ser, Hyp (hydroxyproline), ΔHyp (dehydroproline), Thr, Val, and Leu.

[0030] In some implementations, A2 is independently selected from Pro, Ala, Gly, and Ser.

[0031] In some implementations, A2 is independently selected from Pro and Ala.

[0032] In some implementations, A1 is independently selected from protein amino acids and non-protein amino acids.

[0033] In some implementations, A1 is independently selected from Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Cys and non-protein amino acids (e.g., Nle, Nva).

[0034] In some implementations, A1 is independently selected from Lys, Gly, His, Tyr, Ser, and Arg.

[0035] In some implementations, A1 is independently selected from Lys, Gly, His, and Tyr.

[0036] In some implementations, A1 is independently selected from Lys, Gly, and His.

[0037] In some embodiments, dipeptides A1-A2 are each independently selected from Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, Nle-Pro, Nva-Pro, Gly-Ala, His-Ala, Lys-Ala, Met-Ala, Phe-Ala, Pro-Ala, Ser-Ala, Thr-Ala, Trp-Ala, Tyr-Ala, Val-Ala, Ala-Ala, Arg- Ala, Asn-Ala, Asp-Ala, Gln-Ala, Glu-Ala, Ile-Ala, Leu-Ala, Cys-Ala, Nle-Ala, Nva-Ala, Tyr-Ala, Tyr-Gly, His-Ser.

[0038] In some embodiments, dipeptides A1-A2 are each independently selected from Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, Nle- Pro, Nva-Pro, Gly-Ala, His-Ala, Lys-Ala, Met-Ala, Phe-Ala, Pro-Ala, Ser-Ala, Thr-Ala, Trp-Ala, Tyr-Ala, Va l-Ala, Ala-Ala, Arg-Ala, Asn-Ala, Asp-Ala, Gln-Ala, Glu-Ala, Ile-Ala, Leu-Ala, Cys-Ala, Nle-Ala and Nva-Ala.

[0039] In some implementations, (A1-A2)n is selected from Gly-Pro-Lys-Pro, Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, Nle-Pro, Nva-Pro, Gly-Ala, His-Ala, Tyr-Ala, Tyr-Gly, and His-Ser.

[0040] In some embodiments, dipeptides A1-A2 are each independently selected from Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, Nle-Pro, Nva-Pro, and His-Ala.

[0041] In some implementations, dipeptides A1-A2 are each independently selected from His-Ala, Gly-Pro, and Lys-Pro.

[0042] In some implementations, (A1-A2)n is selected from Gly-Pro-Lys-Pro, Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, Nle-Pro, Nva-Pro, Gly-Ala, and His-Ala.

[0043] In some embodiments, B is independently an L-type α-amino acid, a D-type α-amino acid, an azide-modified L-type α-amino acid, or an azide-modified D-type α-amino acid.

[0044] In some embodiments, B is independently selected from Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Cys, Nle, Nva, ε-azido-Lys, D-Lys, and ε-azido-D-Lys.

[0045] In some embodiments, B is independently selected from Lys, ε-azido-Lys, D-Lys, ε-azido-D-Lys, Gly, Ser, Phe, Thr, Ala, Arg, Leu, and Nva.

[0046] In some embodiments, B is independently selected from Lys, ε-azido-Lys, D-Lys, ε-azido-D-Lys, Gly, and Ser.

[0047] In some implementations, C is each independently an α-N-methylated α-amino acid.

[0048] In some embodiments, C is independently selected from N-methyl-Gly, N-methyl-Asp, N-methyl-Ser, N-methyl-Val, N-methyl-Phe, N-methyl-Leu, N-methyl-Tyr, α-N-methyl-Lys, N-methyl-Ile, N-methyl-Ala, N-methyl-Nle, N-methyl-Nva, and N-methyl-Glu.

[0049] In some implementations, dipeptides like As shown,

[0050] in,

[0051] R1 and R4 are each independently selected from H and C. 1-6 alkyl,

[0052] R2, R3, R5, and R6 are each independently selected from H and C. 1-6 Alkyl groups and 4-7 membered heterocyclic groups, wherein the C 1-6 The alkyl group may be optionally substituted by one or more substituents selected from OH, NH2, N3, SH, COOH, S-CH3, C(=O)NH2, NHC(=NH)NH2, NHC(=O)NH2, phenyl, indole, and hydroxyl groups of the phenyl group.

[0053] In some implementations, R1, R3, and R6 are H.

[0054] In some implementations, R2 is selected from H and C. 1-4 Alkyl, the C 1-4 The alkyl group may be optionally substituted by one or more substituents selected from OH, NH2, N3, SH, COOH, S-CH3, C(=O)NH2, NHC(=NH)NH2, NHC(=O)NH2, phenyl, indole, and hydroxyl groups of the phenyl group.

[0055] In some implementations, R2 is selected from H and C. 1-4 Alkyl, the C 1-4 The alkyl group may be optionally substituted by one or more substituents selected from OH, NH2, N3, SH, COOH, S-CH3, C(=O)NH2, NHC(=NH)NH2 and NHC(=O)NH2.

[0056] In some implementations, R2 is selected from H and C. 1-4 Alkyl groups (e.g., CH3, CH2CH2CH3, CH2CH(CH3)2), CH2Ph, CH2OH, CH(OH)CH3, CH2CH2CH2NHC(=NH)NH2, (CH2)4NH2 and (CH2)4N3.

[0057] In some embodiments, R2 is selected from H, CH2OH, (CH2)4NH2 and (CH2)4N3.

[0058] In some implementations, R4 is C 1-4 alkyl.

[0059] In some implementations, R4 is selected from CH3, CH2CH3 and CH(CH3)2.

[0060] In some implementations, R4 is CH3.

[0061] In some implementations, R5 is selected from H and C. 1-6Alkyl, the C 1-6 The alkyl group may be optionally substituted by one or more substituents selected from OH, NH2, N3, SH, COOH, S-CH3, C(=O)NH2, NHC(=NH)NH2, NHC(=O)NH2, phenyl, indole, and hydroxyl groups of the phenyl group.

[0062] In some implementations, R5 is selected from H and C. 1-4 Alkyl, the C 1-4 The alkyl group may be optionally substituted by one or more substituents selected from OH, NH2, N3, SH, COOH, S-CH3, C(=O)NH2, NHC(=NH)NH2, NHC(=O)NH2, phenyl, indole, and hydroxyl groups of the phenyl group.

[0063] In some implementations, R5 is selected from H, C 1-4 Alkyl groups, CH2Ph, CH2PhOH, CH2OH, CH2COOH, CH2CH2COOH and (CH2)4NH2.

[0064] In some implementations, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0065] An α-amino acid that can be cleaved by the DPP4 enzyme is introduced at the N-terminus of the active drug or DKP dipeptide to form a stable prodrug in vitro. This prodrug releases the active drug only after the N-terminal α-amino acid is cleaved by the enzyme in vivo, or it initiates intramolecular cyclization to form a DKP ring and simultaneously releases the active drug. Furthermore, by selecting the α-amino acid and DKP dipeptide that can be cleaved by the DPP4 enzyme, the drug release rate in vivo can be controlled.

[0066] In some implementations, n is 1, 2, 3, 4, or 5; m is 0, 1, or 2;

[0067] In some implementations, n is 1, 2, or 3; m is 0, 1, or 2;

[0068] In some implementations, n is 1 or 2; m is 0, 1 or 2.

[0069] In some implementations, n is 1; m is 0 or 1.

[0070] In some embodiments, the structure shown in equation (F-1) is selected from the structure shown in equation (F-1-1) or equation (F-1-2):

[0071] Wherein, A1, A2, B and C are as defined in any embodiment of the present invention.

[0072] In some embodiments, the structure shown in equation (F-1) is selected from the structure shown in equation (F-1-3) or equation (F-1-4):

[0073] in,

[0074] In equation (F-1-3), each A1 may be the same or different, and each A2 may be the same or different;

[0075] In equation (F-1-4), each B may be the same or different, and each C may be the same or different;

[0076] A1, A2, B, and C are as defined in any embodiment of the present invention.

[0077] In some implementations, the structure shown in equation (F-1) is selected from the structure shown in equation (F-1-5):

[0078] in,

[0079] n1 is 1, 2, 3, 4 or 5; preferably, n is 1, 2 or 3;

[0080] In equation (F-1-5), each A1 may be the same or different, and each A2 may be the same or different;

[0081] A1, A2, B, and C are as defined in any embodiment of the present invention.

[0082] The prodrug of the present invention or its pharmaceutically acceptable salt further comprises drug P.

[0083] In some embodiments, the drug P is an active drug.

[0084] In some embodiments, the active pharmaceutical ingredient is selected from peptide drugs, protein drugs, nucleic acid drugs, and small molecule drugs.

[0085] In some embodiments, the polypeptide drug is selected from glucagon-like peptide-1 (GLP-1), exendin-2, exendin-3, exendin-4, atrial factor lina (ANF), ghrellin, vasopressin, growth hormone-releasing hormone (GHRH), RC-3095, somatostatin, corticotropin, vilcapeptide, scutellarin, PCK-3145, and Phe-His-Ser-Cys-Asn (PHSCN, SEQ ID NO). NO:41), Insulin-like growth factor 1 (IGF1), B-type linalide, Peptide YY (PYY), Interferon, Platelet-Reactive Ingredient, Angiopoietin, Calcitonin, Gonadotropin-Releasing Hormone, Cetrolec, Ganirec, Hirudin, Glucagon, Anti-TNF-α, Fibroblast Growth Factor, Granulocyte Colony-Stimulating Factor, Obinepitide, Parathyroid Hormone (PTH) and its analogues (e.g., sequences with 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions, deletions or additions or any combination thereof compared to PTH), Leuprorelin, Sermorelin, and Propranolol. Lamorrelin, Nesiritide, Rotigaptide, Silenjitide, MBP-8298, AL-108, Enfuviride, Carfilzomib, Milvastatin, Thymofasin, 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 (e.g., leucine enkephalin and methionine enkephalin), Antimicrobial peptides, [des-Ala20,Gln34]-dermal antimicrobial peptide, anionic antimicrobial peptides associated with surface cleaning substances, Apidaecin IA, Apidaecin IB, Acetyl-Adhesin (1025-1044)amide, Theromacin (49-63), Percicanam (MSI-78), Indolicidin, Apelin-15 (63-77), CFP10 (71-85), inhibitors of anthrax-related lethal factors (LF), bovine antimicrobial peptides, 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), Humanin(HN)sHNG, [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), atebandin, aviptadin, R otigaptide (ZP123, GAP486), silengitide (EMD-121924, RGD peptide), A1buBNP, BN-054, angiotensin 11, MBP-8298, peptidylleucine arginine, ziconopeptide, AL-208, AL-108, carbeticon, coliven, ADNF-14, VIP (intestinal vascular cleansing peptide), thymosin, bacitracin, brevicin, 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, 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, ganirilin, 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), dynorphin A and cyclic peptides (e.g., romidepsin, voclosporin, ziconotide, linaclotide, plecanatide, plecanatide, lanreotide, vasopressin, terlipressin). The following are listed: parathyroid hormone (PTHrP), steroid hormone (SPH) 2, parathyroid hormone-associated protein (SPH) 2, parathyroid hormone-associated protein-1 (SDF-1), brain natriuretic peptide (BNP), neuropeptide Y (NPY), tyrosinase (PYY), and glucose-dependent insulinotropic peptide (GIP).

[0086] In some embodiments, the protein drug is selected from 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, insulin, erythropoietin, gonadotropin, parathyroid hormone), and cytokines (e.g., interleukins, interferons, colony-stimulating factors).

[0087] In some embodiments, the nucleic acid drug is selected from antisense nucleic acids (e.g., Fomivirsen, Mipomersen, Eteplirsen, Nusinersen, Inotersen, Volanesoresn, Golodirsen), small interfering RNAs (e.g., Onpattro, Givlaari), microRNAs, small activating RNAs, messenger RNAs, and aptamers.

[0088] In some embodiments, the small molecule drug is selected from antibiotics such as penicillin (e.g., penicillin, penicillin V), fluoroquinolones (e.g., ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, gemifloxacin), cephalosporins (e.g., cefixime, cefbufen, ceftoranil, cefdinir, cefpodoxime proxetil), aminoglycosides (e.g., isapamicin, amikacin, etimicin, netilimicin), macrolides (e.g., erythromycin, azithromycin, clarithromycin), tetracyclines (e.g., doxycycline, minocycline, tigecycline, omacycline, eracycline), sulfonamides, and sensitizers (e.g., trimethoprim, sulfonamides). Pyrimidines, sulfamethoxazole, sulfamethoxazole); urinary tract 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, mequinidine, promethazine, mizolastine, ebastine, loratadine, terfenadine, fexofenadine, desloratadine, cetirizine); antiviral drugs (e.g., oseltamivir, mabaloxavir, erteiravir, lenapavir, capeiravir, entecavir, nematamide / ritonavir, ribavirin, interferon). Arbidol, zanamivir, peramivir, amantadine, acyclovir, ganciclovir, valacyclovir, daclatasvir, asunaprevir, asunaprevir, adefovir dipivoxil, lamivudine, telbivudine, azvudine; antihypertensive drugs (e.g., amlodipine, felodipine, cilnidipine, nifedipine, nifedipine, 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, glimepiride, 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, oxytocin, adecyl bromide, glycopyrronium bromide, tiotropium bromide, umemet, glycopyrronium (glycopyrronium bromide), refenapyridine, roflumilast, erdosteine, carbocysteine);Antitumor drugs (e.g., methotrexate, pemetrexed, fluorouracil, 6-mercaptopurine, hydroxyurea, gemcitabine, cytarabine, doxorubicin, epirubicin, rubitine, irinotecan, topotecan, etoposide, taxanes, vinca alkaloids, eribulin, EGFR inhibitors (e.g., gefitinib, erlotinib, icotinib, afatinib, osimertinib), ALK inhibitors (e.g., crizotinib, alectinib, ceritinib, lorlatinib), MET inhibitors (e.g., cevotinib, carmatinib), RET inhibitors (e.g., pralatinib, LOX) O-292), NTRK inhibitors (e.g., larotrectinib, entrectinib), BRAF inhibitors (e.g., dabrafenib, vemurafenib, encorafenib), MEK inhibitors (e.g., trametinib, binimetinib), HER2 inhibitors (e.g., neratinib, tacardinib), CDK4 / 6 inhibitors (e.g., palbociclib, abeciclib), PARP inhibitors (e.g., olaparib, niraparib), anti-angiogenic multikinase inhibitors (e.g., nilotinib, apatinib, sunitinib, fruquintinib, pazopanib, abcitabine). Inhibitors include: imatinib, vandetanib, cabozantinib; mTOR inhibitors (e.g., everolimus); HDAC inhibitors (e.g., chidamide); ABL-BCR inhibitors (e.g., imatinib, dasatinib, nilotinib); PDGFR / C-KIT inhibitors (e.g., imatinib, nilotinib, avatinib); BTK inhibitors (e.g., ibrutinib, zanubrutinib, zanubrutinib); protease inhibitors (e.g., bortezomib, ixazomib); JAK inhibitors (e.g., ruxolitinib); selective nuclear transporter inhibitors (e.g., selinexor); and PI3K inhibitors. Inhibitors (e.g., apelisib), FGFR2 inhibitors (e.g., pemigatinib), IDH1 inhibitors (e.g., 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).

[0089] In some embodiments, the prodrug is as shown in formula (I).

[0090] in,

[0091] P represents the drug;

[0092] A1, A2, B, C, P, m, and n are as defined in any embodiment of the present invention;

[0093] C is linked to the amino group of P by an amide bond formed through its α-carboxyl group.

[0094] In some embodiments, the prodrug is as shown in formula (I-1) or formula (I-2).

[0095] Wherein, A1, A2, B, C and P are as defined in any embodiment of the present invention.

[0096] In some embodiments, the prodrug is as shown in formula (I-3) or formula (I-4).

[0097] in,

[0098] In equation (I-3), each A1 may be the same or different, and each A2 may be the same or different;

[0099] In equation (I-4), each B may be the same or different, and each C may be the same or different;

[0100] A1, A2, B, C, and P are as defined in any embodiment of the present invention.

[0101] In some embodiments, the prodrug is as shown in formula (I-5).

[0102] in,

[0103] n1 is 1, 2, 3, 4 or 5; preferably, n is 1, 2 or 3;

[0104] In equation (I-5), each A1 may be the same or different, and each A2 may be the same or different;

[0105] A1, A2, and P are as defined in any embodiment of the present invention.

[0106] In some embodiments, the prodrug is as shown in formula (I-5).

[0107] in,

[0108] A1-A2 is a dipeptide sequence that can be recognized and cleaved by the DPP4 enzyme;

[0109] n1 represents the number of A1-A2, where n1 is an integer from 1 to 10;

[0110] P represents a polypeptide molecule, which requires an intact N-terminus to be active, and the fusion of the dipeptide sequence at the N-terminus will cause the polypeptide molecule to be completely or partially inactivated.

[0111] In some embodiments, the second position of the dipeptide sequence is an amino acid residue selected from the group consisting of proline, alanine, hydroxyproline, dehydroproline, glycine, threonine, valine, leucine, and serine; more preferably, the dipeptide sequence is selected from the group consisting of HA, YA, YG, YP, HS, SP, GP, and RP; even more preferably, the dipeptide sequence is HA, YA, or YG.

[0112] In some embodiments, n1 is an integer from 1 to 5; more preferably, n1 is 1, 2 or 3; preferably, when n1 ≥ 2, the dipeptide sequences are the same or different.

[0113] In some embodiments, the prodrug is a fragment as shown in formula (I-5); wherein n1 is an integer from 1 to 10; preferably, n1 is an integer from 1 to 5; more preferably, n1 is 1, 2 or 3; in formula (I-5), each A1 may be the same or different, and each A2 may be the same or different; A1, A2 and P are as defined in any embodiment of the present invention.

[0114] In some embodiments, the polypeptide molecule is a polypeptide molecule obtained by biologically or chemically modifying an original polypeptide sequence, wherein the original polypeptide sequence includes the amino acid sequence of a ligand molecule of a GPCR; more preferably, the original polypeptide sequence includes one or more combinations of amino acid sequences selected from the group consisting of: glucagon-like peptide-1 (GLP-1), parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), GLP-2, stromal cell-derived factor-1 (SDF-1), brain natriuretic peptide (BNP), neuropeptide Y (NPY), tyrosinase (PYY), and glucose-dependent insulinotropic peptide (GIP).

[0115] In some embodiments, the biomodification involves fusing the original polypeptide sequence with a functional fragment to extend the half-life of the original polypeptide sequence; preferably, the biomodification involves fusing the functional fragment to the C-terminus of the original polypeptide sequence; more preferably, the biomodification involves linking the functional fragment to the C-terminus of the original polypeptide sequence via a linker sequence.

[0116] In some embodiments, the functional fragment is selected from the group consisting of: Fc domains or variants thereof, albumin, antibodies or antibody fragments (preferably VHH), and combinations thereof; preferably, the Fc domain is an Fc domain derived from an IgG antibody (e.g., IgG1, IgG2, IgG3, or IgG4), and the variant of the Fc domain has mutations of F234A / L235A, M252Y / S254T / T256E, M428L / N434S, T307A / E380A / N434A, M428L / N434S, V308P, H285D / T307Q / A378V, or L309D / Q311H / N434S compared to the wild-type Fc domain of an IgG-derived antibody; more preferably, the amino acid sequence of the Fc domain is as shown in any of SEQ ID NO:138-140.

[0117] In some embodiments, the adapter sequence is a flexible polypeptide sequence; preferably, the adapter sequence comprises (G4S). q , where q is a positive integer greater than or equal to 1, for example, an integer from 1 to 8 (preferably 3 or 4); more preferably, the amino acid sequence of the linker sequence is as shown in SEQ ID NO:141;

[0118] In some embodiments, the chemical modification is selected from fatty acid chain modification, polyethylene glycol modification, and combinations thereof; preferably, the chemical modification includes introducing a C-16, C-18, or C-20 fatty acid chain into the amino acid side chain of the therapeutic peptide, or polyethylene glycol modification.

[0119] In some embodiments, the polypeptide molecule is a GLP-1-Fc fusion protein or a PTH-Fc fusion protein, the dipeptide sequence is HA, and two or three HA sequences are fused to the N-terminus of the polypeptide molecule.

[0120] In some embodiments, the amino acid sequence of the GLP-1-Fc fusion protein is shown in SEQ ID NO:119, and the amino acid sequence of the PTH-Fc fusion protein is shown in SEQ ID NO:136.

[0121] In some embodiments, the amino acid sequence of the peptide molecule after fusing at least one dipeptide sequence to the N-terminus is as shown in any of SEQ ID NO:120-135 and 137.

[0122] In some embodiments, the method further includes the following step: selecting different dipeptide sequences to have different sustained-release effects based on in vitro enzymatic digestion rates and / or in vivo sustained-release results.

[0123] Furthermore, chemical modification helps prolong the duration of drug action and improve bioavailability. Long-acting macromolecules such as albumin or macromolecular carriers, such as the Fc portion of IgG, can slow down enzymatic degradation and renal filtration. Modifying the drug with fatty acids or fatty diacids allows the fatty acids or fatty diacids to form non-covalent bonds with albumin in vivo, or directly modifying the drug with the Fc portion of IgG, thereby slowing down enzymatic degradation and renal filtration, and prolonging the drug's plasma half-life. In some embodiments of the present invention, the prodrug is modified by introducing fatty acids, fatty diacids, or macromolecular carriers to prolong the drug's duration of action and increase its plasma half-life.

[0124] In some embodiments, the prodrug is as shown in formula (II).

[0125] in,

[0126] A1, A2, B, C, P, m, and n are as defined in any embodiment of the present invention;

[0127] C is linked to the amino group of P through an amide bond formed by its α-carboxyl group;

[0128] L stands for connector;

[0129] D is selected from antibody, Fc fragment, C 10-30 Fatty acids, C 10-30 Fatty acid, polyethylene glycol, polycarboxybetaine (pCB) and any combination thereof.

[0130] In some implementations, -OH, -SH, or -NH2 in P is replaced by L, or -N3 in P is replaced by L.

[0131] In some implementations, L is selected from a connector sequence, a key, or... NH, O, S, C (=O), C 1-6 Alkylenes and any combination thereof.

[0132] In some embodiments, the adapter sequence is a flexible polypeptide sequence.

[0133] In some implementations, the connector sequence includes (G4S). q , where q is a positive integer greater than or equal to 1, for example, an integer from 1 to 8 (for example, 3 or 4).

[0134] In some embodiments, the amino acid sequence of the linker sequence is as shown in SEQ ID NO:141.

[0135] In some implementations, L is selected from NH, O, S, C (=O), C 1-6 Alkylenes and any combination thereof.

[0136] In some implementations, L is selected from t can be 1, 2, 3, 4 or 5.

[0137] In some implementations, L is selected from t can be 1, 2, 3, 4 or 5.

[0138] In some implementations, L is selected from

[0139] In some implementations, L is selected from

[0140] In some embodiments, D is selected from H, Fc domain or variants thereof, albumin, antibody or antibody fragment (e.g., IgG, IgM, IgA, IgD, IgE, VHH), C 14-24 Fatty acids (e.g., C) 14 Fatty acids, C 16 Fatty acids, C 18 Fatty acids, C 20 Fatty acids, C 22 fatty acids), C 14-24 Fatty acids (e.g., C40) 14 Fatty acid, C 16 Fatty acid, C 18 Fatty acid, C 20 Fatty acid, C 22 Fatty acid), polyethylene glycol, pCB and any combination thereof.

[0141] In some implementations, the Fc domain is derived from the Fc domain of an IgG antibody, and the variant of the Fc domain has mutations of F234A / L235A, M252Y / S254T / T256E, M428L / N434S, T307A / E380A / N434A, M428L / N434S, V308P, H285D / T307Q / A378V, or L309D / Q311H / N434S compared to the wild-type Fc domain of the IgG antibody.

[0142] In some embodiments, the amino acid sequence of the Fc domain is as shown in any of SEQ ID NO:138-140;

[0143] In some implementations, D is selected from H, antibodies (e.g., IgG, IgM, IgA, IgD, IgE, VHH), C14-24 Fatty acids (e.g., C) 14 Fatty acids, C 16 Fatty acids, C 18 Fatty acids, C 20 Fatty acids, C 22 fatty acids), C 14-24 Fatty acids (e.g., C40) 14 Fatty acid, C 16 Fatty acid, C 18 Fatty acid, C 20 Fatty acid, C 22 Fatty acid), polyethylene glycol, pCB and any combination thereof.

[0144] In some implementations, D is selected from -(D1). p -(D2) q -D3,

[0145] in,

[0146] D1 is -C(=O)CH2(OCH2CH2)2NH-,

[0147] D2 is selected from Glu and Asp.

[0148] D3 is selected from C 14-24 Fatty diacids,

[0149] p is an integer between 0 and 24.

[0150] q is 1 or 2,

[0151] D1, D2, and D3 are connected by amide bonds, and the C (=O) end of D1 is connected to the L phase.

[0152] In some implementations, p is an integer between 0 and 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0153] In some implementations, D2 is The C (=O) terminal is connected to the -NH- phase of D1.

[0154] In some implementations, D3 is selected from -C(O)(CH2). 14 COOH, -C(O)(CH2) 15 COOH, -C(O)(CH2) 16 COOH, -C(O)(CH2) 17 COOH, -C(O)(CH2) 18 COOH, -C(O)(CH2) 19 COOH and -C(O)(CH2) 20 COOH.

[0155] In some implementations, D is selected from:

[0156] In some implementations, D is selected from:

[0157] In some embodiments, the prodrug is selected from the structure shown in formula (II-1) or formula (II-2):

[0158] Wherein, A1, A2, B, C, P, L and D are defined as in any embodiment of the present invention.

[0159] In some embodiments, the prodrug is selected from the structure shown in formula (II-3) or formula (II-4):

[0160] in,

[0161] In equation (II-3), each A1 may be the same or different, and each A2 may be the same or different;

[0162] In equation (II-4), each B may be the same or different, and each C may be the same or different;

[0163] A1, A2, B, C, P, L, and D are as defined in any embodiment of the present invention.

[0164] In some embodiments, the prodrug is selected from the structure shown in formula (II-5):

[0165] in,

[0166] n1 is 1, 2, 3, 4 or 5; preferably, n is 1, 2 or 3;

[0167] In equation (II-5), each A1 may be the same or different, and each A2 may be the same or different;

[0168] A1, A2, P, L, and D are as defined in any embodiment of the present invention.

[0169] In some embodiments, the prodrug is as shown in any one of SEQ ID NOs:21, 25-26, 30-37, 48-55, 58-106, 108-118.

[0170] In some embodiments, the prodrug is as shown in any one of SEQ ID NOs:15-20, 27-29, SEQ ID NOs:120-135 and 137.

[0171] In some embodiments, the prodrug is as shown in any one of SEQ ID NOs:15-20, 27-29.

[0172] In some embodiments, the prodrug is as shown in any one of SEQ ID NOs:2-14, 38-39, 56-57.

[0173] In some embodiments, the prodrug is as shown in any one of SEQ ID NOs:42-43.

[0174] In some embodiments, the prodrug is as shown in any one of SEQ ID NOs:44-47.

[0175] A second aspect of the invention provides a prodrug or a pharmaceutically acceptable salt thereof, wherein the prodrug comprises the structure shown in formula (F-2).

[0176] in,

[0177] A1, A2, B, C, m, n, L, and D are defined as in any embodiment of the present invention.

[0178] The -OH, -SH, or -NH2 in A1, A2, B, or C is replaced by LD, or the -N3 in A1, A2, B, or C is replaced by LD.

[0179] In some embodiments, the structure shown in equation (F-2) is selected from the structure shown in equation (F-2-1).

[0180] in,

[0181] A1, A2, B, C, m, n, L, and D are defined as in any embodiment of the present invention.

[0182] In some embodiments, dipeptides A1-A2 are each independently selected from Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, Nle- Pro, Nva-Pro, Gly-Ala, His-Ala, Lys-Ala, Met-Ala, Phe-Ala, Pro-Ala, Ser-Ala, Thr-Ala, Trp-Ala, Tyr-Ala, Va l-Ala, Ala-Ala, Arg-Ala, Asn-Ala, Asp-Ala, Gln-Ala, Glu-Ala, Ile-Ala, Leu-Ala, Cys-Ala, Nle-Ala and Nva-Ala.

[0183] In some embodiments, dipeptides A1-A2 are each independently selected from Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, Nle-Pro, Nva-Pro, and His-Ala.

[0184] In some implementations, like As shown,

[0185] in,

[0186] R1, R3, R4, R5, and R6 are defined as in any embodiment of the present invention.

[0187] R7 is selected from -C 1-4 Alkylene-LD, -C 1-4 Alkylene-oxy(O)-LD, -C 1-4 alkylene-nitrogen (NH)-LD and -C 1-4 Alkylene-sulfur (S)-LD, L, and D are as defined in any embodiment of the present invention.

[0188] In some implementations, R7 is selected from:

[0189] In some implementations, R7 is selected from:

[0190] In some embodiments, the prodrug further includes drug P, as defined in any embodiment of the present invention.

[0191] In some embodiments, the prodrug is as shown in formula (III).

[0192] in,

[0193] A1, A2, B, C, P, m, n, L, and D are as defined in any embodiment of the present invention;

[0194] C is linked to the amino group of P through an amide bond formed by its α-carboxyl group;

[0195] The -OH, -SH, or -NH2 in A1, A2, B, or C is replaced by LD, or the -N3 in A1, A2, B, or C is replaced by LD.

[0196] In some embodiments, the prodrug is selected from the structure shown in formula (III-1):

[0197] in,

[0198] A1, A2, B, C, P, m, n, L, and D are defined as in any embodiment of the present invention.

[0199] In some embodiments, the prodrug is selected from the structure shown in formula (III-1-1):

[0200] in,

[0201] A1, A2, B, C, P, L, and D are as defined in any embodiment of the present invention.

[0202] In some embodiments, the prodrug is selected from the structure shown in formula (III-1-2):

[0203] in,

[0204] Each A1 may be the same or different; each A2 may be the same or different.

[0205] A1, A2, B, C, P, L, and D are as defined in any embodiment of the present invention.

[0206] In some embodiments, the prodrug is as shown in SEQ ID NO: 22.

[0207] In some embodiments, the prodrug is as shown in SEQ ID NO: 23 or 24.

[0208] intermediate

[0209] A third aspect of the invention provides an intermediate of the prodrug described in the first aspect of the invention or a pharmaceutically acceptable salt thereof, wherein the intermediate is shown in formula (M-1).

[0210] Wherein, A1, A2, B, C, m and n are defined as in any embodiment of the present invention.

[0211] The fourth aspect of the invention provides the use of the intermediate described in the third aspect of the invention in the preparation of the prodrug described in the first aspect of the invention or a pharmaceutically acceptable salt thereof.

[0212] The fifth aspect of the invention provides an intermediate of the prodrug described in the second aspect of the invention or a pharmaceutically acceptable salt thereof, wherein the intermediate is shown in formula (M-2).

[0213] Wherein, A1, A2, B, C, m, n, L and D are defined as in any embodiment of the present invention.

[0214] The sixth aspect of the invention provides the use of the intermediate described in the fifth aspect of the invention in the preparation of the prodrug described in the second aspect of the invention or a pharmaceutically acceptable salt thereof.

[0215] Nucleic acid molecules

[0216] A seventh aspect of the present invention provides an isolated nucleic acid molecule comprising: (a) a polynucleotide molecule encoding a polypeptide drug molecule; and / or (b) its complementary sequence.

[0217] Nucleic acid constructs

[0218] The eighth aspect of the present invention provides a nucleic acid construct containing the polynucleotide molecule or its complementary sequence as described in the seventh aspect of the present invention.

[0219] In some embodiments, the nucleic acid construct is an expression cassette and also includes, for example, a promoter and a transcription termination sequence.

[0220] Recombinant vector

[0221] A ninth aspect of the present invention provides a recombinant vector containing the nucleic acid molecule described in the seventh aspect of the present invention or the nucleic acid construct described in the eighth aspect of the present invention.

[0222] In some implementations, the recombinant vector is a recombinant cloning vector or a recombinant expression vector.

[0223] host cells

[0224] The tenth aspect of the present invention provides a host cell containing the nucleic acid molecule described in the seventh aspect of the present invention, the nucleic acid construct described in the eighth aspect of the present invention, or the recombinant vector described in the ninth aspect of the present invention, and / or expressing a polypeptide drug molecule.

[0225] In some embodiments, the host cell is selected from Escherichia coli cells, insect cells, yeast cells, and mammalian cells.

[0226] In some embodiments, the host cell is an Escherichia coli cell, such as BL21(DE3).

[0227] Pharmaceutical Composition

[0228] The eleventh aspect of the present invention provides a pharmaceutical composition comprising at least one prodrug or a pharmaceutically acceptable salt thereof as described in the first or second aspect of the present invention, and one or more pharmaceutically acceptable carriers and / or excipients.

[0229] use

[0230] The twelfth aspect of the invention provides the use of the prodrug or a pharmaceutically acceptable salt thereof described in the first or second aspect of the invention, or the pharmaceutical composition described in the seventh aspect of the invention, in the preparation of a medicament for treating and / or preventing a disease or condition or reducing the severity of said disease or condition.

[0231] In some embodiments, the disease or condition is selected from hypoparathyroidism, osteoporosis, osteopenia, diabetes, diabetic nephropathy, chronic kidney disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cardiovascular disease (e.g., atherosclerosis, hypertension, heart failure), neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease), mental disorders (e.g., schizophrenia, depression), immune system diseases (e.g., autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus), allergic diseases (e.g., allergic rhinitis, asthma, urticaria), infectious diseases (e.g., influenza, pneumonia, tuberculosis)), pain and cancer (e.g., breast cancer, lung cancer, stomach cancer, colorectal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, or melanoma), obesity, hypoglycemia, bone metabolic diseases, hypoparathyroidism, hypocalcemia, Crohn's disease, neurodegenerative diseases, or anorexia nervosa, and any combination thereof.

[0232] The thirteenth aspect of the present invention provides the use of the prodrug or a pharmaceutically acceptable salt thereof described in the first or second aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention, in the preparation of a sustained-release polypeptide drug.

[0233] Methods to enhance drug sustained-release capability

[0234] The thirteenth aspect of the present invention provides a method for enhancing the sustained-release capability of a drug, comprising: fusing at least one fragment of formula (F-1); and causing the drug to be wholly or partially inactivated when the drug is fused with the fragment.

[0235] In some embodiments, the drug is selected from drugs mentioned in the first aspect of the invention.

[0236] In some embodiments, the drug is a polypeptide drug.

[0237] In some embodiments, the polypeptide drug is a PTH polypeptide analog or a GLP-1 polypeptide analog.

[0238] The fourteenth aspect of the present invention provides a method for enhancing the sustained-release capability of a polypeptide drug, comprising fusing at least one dipeptide sequence to the N-terminus of a polypeptide molecule, the dipeptide sequence being cleavable by a DPP-4 enzyme; wherein the polypeptide molecule requires an intact N-terminus to be active, and the fusing of the dipeptide sequence to the N-terminus causes complete or partial inactivation of the polypeptide molecule.

[0239] In some embodiments, the second position of the dipeptide sequence is an amino acid residue selected from the group consisting of proline, alanine, hydroxyproline, dehydroproline, glycine, threonine, valine, leucine, and serine; more preferably, the dipeptide sequence is selected from the group consisting of HA, YA, YG, YP, HS, SP, GP, and RP; even more preferably, the dipeptide sequence is HA, YA, or YG.

[0240] In some embodiments, n1 dipeptide sequences are fused to the N-terminus of the polypeptide molecule, where n1 is an integer from 1 to 10; preferably, n1 is an integer from 1 to 5; more preferably, n1 is 1, 2 or 3; preferably, when n1 ≥ 2, the dipeptide sequences are the same or different.

[0241] In some embodiments, the dipeptide sequence is a fragment as shown in formula (F-1-5); wherein n1 is an integer from 1 to 10; preferably, n1 is an integer from 1 to 5; more preferably, n1 is 1, 2 or 3; in formula (F-1-5), each A1 may be the same or different, and each A2 may be the same or different; A1 and A2 are as defined in any embodiment of the present invention.

[0242] In some embodiments, the polypeptide molecule is a polypeptide molecule obtained by biologically or chemically modifying an original polypeptide sequence, wherein the original polypeptide sequence includes the amino acid sequence of a ligand molecule of a GPCR; more preferably, the original polypeptide sequence includes one or more combinations of amino acid sequences selected from the group consisting of: glucagon-like peptide-1 (GLP-1), parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), GLP-2, stromal cell-derived factor-1 (SDF-1), brain natriuretic peptide (BNP), neuropeptide Y (NPY), tyrosinase (PYY), and glucose-dependent insulinotropic peptide (GIP).

[0243] In some embodiments, the biomodification involves fusing the original polypeptide sequence with a functional fragment to extend the half-life of the original polypeptide sequence; preferably, the biomodification involves fusing the functional fragment to the C-terminus of the original polypeptide sequence; more preferably, the biomodification involves linking the functional fragment to the C-terminus of the original polypeptide sequence via a linker sequence.

[0244] In some embodiments, the functional fragment is selected from the group consisting of: Fc domains or variants thereof, albumin, antibodies or antibody fragments (preferably VHH), and combinations thereof; preferably, the Fc domain is an Fc domain derived from an IgG antibody (e.g., IgG1, IgG2, IgG3, or IgG4), and the variant of the Fc domain has mutations of F234A / L235A, M252Y / S254T / T256E, M428L / N434S, T307A / E380A / N434A, M428L / N434S, V308P, H285D / T307Q / A378V, or L309D / Q311H / N434S compared to the wild-type Fc domain of an IgG-derived antibody; more preferably, the amino acid sequence of the Fc domain is as shown in any of SEQ ID NO:138-140.

[0245] In some embodiments, the adapter sequence is a flexible polypeptide sequence; preferably, the adapter sequence comprises (G4S). q , where q is a positive integer greater than or equal to 1, for example, an integer from 1 to 8 (preferably 3 or 4); more preferably, the amino acid sequence of the linker sequence is as shown in SEQ ID NO:141;

[0246] In some embodiments, the chemical modification is selected from fatty acid chain modification, polyethylene glycol modification, and combinations thereof; preferably, the chemical modification includes introducing a C-16, C-18, or C-20 fatty acid chain into the amino acid side chain of the therapeutic peptide, or polyethylene glycol modification.

[0247] In some embodiments, the polypeptide molecule is a GLP-1-Fc fusion protein or a PTH-Fc fusion protein, the dipeptide sequence is HA, and two or three HA sequences are fused to the N-terminus of the polypeptide molecule.

[0248] In some embodiments, the amino acid sequence of the GLP-1-Fc fusion protein is shown in SEQ ID NO:119, and the amino acid sequence of the PTH-Fc fusion protein is shown in SEQ ID NO:136.

[0249] In some embodiments, the amino acid sequence of the peptide molecule after fusing at least one dipeptide sequence to the N-terminus is as shown in any of SEQ ID NO:120-135 and 137.

[0250] In some embodiments, the method further includes the following step: selecting different dipeptide sequences to have different sustained-release effects based on in vitro enzymatic digestion rates and / or in vivo sustained-release results.

[0251] Terminology Definition

[0252] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this application, definitions and explanations of relevant terms are provided below.

[0253] When the terms “for example,” “such as,” or variations thereof are used in this document, these terms will not be considered restrictive terms but will be interpreted as meaning “including but not limited to” or “not limited to.”

[0254] When this document uses the terms “including,” “contains,” or variations thereof, it also provides the meanings of the terms “consisting of” and “substantially composed of.”

[0255] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0256] As used herein, the term “and / or” should be considered as a specific disclosure of each of two or more specified features or elements, and any combination of two or more features or elements. Therefore, the term “and / or” as used in phrases, such as “A and / or B” herein, is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).

[0257] As used herein, the term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0258] As used herein, the term "prodrug" refers to a compound that releases a parent drug in vivo through enzymatic and / or non-enzymatic means, such as chemical transformation. In some embodiments, the starting prodrug releases an intermediate prodrug (e.g., a DKP-based prodrug) via enzymatic means, which further releases the parent drug via a non-enzymatic process. As used herein, the terms "parent drug" or "active drug" refer to a pharmacologically active compound released from a prodrug through conversion. As used herein, the term "conversion" refers to a process in which a prodrug is converted via enzymatic and / or non-enzymatic means, resulting in the release of the parent drug or an intermediate prodrug (e.g., a DKP-based prodrug). Further, "enzymatic cleavage conversion" refers to the process in which a prodrug is converted via enzymatic means to release the parent drug or an intermediate prodrug (e.g., a DKP-based prodrug), and "autocleavage conversion" refers to the process in which a prodrug is converted via a non-enzymatic means, such as chemical transformation, to release the parent drug. In this paper, the peptide that is cleaved from the prodrug by an enzyme, thereby releasing the parent drug or intermediate prodrug (e.g., a DKP-based prodrug), is called an "enzyme-cleaved peptide," and the dipeptide that forms DKP and releases the active drug is called a "DKP dipeptide." The rate of conversion can be measured by the "conversion half-life," which is the time required for the prodrug concentration to be halved due to conversion.

[0259] As used herein, the term "amino acid" refers to any amino acid, both proteinogenic and non-proteinogenic. As used herein, the term "proteinogenic amino acid" refers to the 20 standard amino acids encoded by the human genetic code: Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, and Cys. As used herein, the term "non-proteinogenic amino acid" refers to any amino acid that does not qualify as a proteinogenic amino acid. Generally, amino acid residues as used herein (e.g., in the context of a polypeptide sequence) can be represented by their full name, their single-letter code, and / or their three-letter code. These three methods are completely equivalent and interchangeable; for example, alanine can be represented by A or Ala. In the following text, unless otherwise stated, each amino acid of the present invention's optical isomers should be understood to refer to the L-isomer. Examples of non-protein amino acids include, but are not limited to: 2-aminoisobutyric acid (Aib), norleucine (Nle), norvaline (Nva), and the D-isomers of protein amino acids. The single-letter code for the D-configuration of a protein amino acid is the lowercase letter of its L-configuration code; for example, the lowercase letter k represents the D-configuration of lysine. ε-azido-Lys or K(ε-N3) represents lysine with an azido group modified at the ε-position, with the structural formula...

[0260] As used herein, the term "polypeptide" or "polypeptide sequence" refers to a compound comprising a series of two or more amino acids linked together by amide bonds. When the amino acids in a polypeptide are α-amino acids, the amide bonds are formed by the linkage of the α-carboxyl group of one amino acid to the α-amino group of another amino acid.

[0261] As used herein, for a polypeptide or polypeptide sequence, the term "N-terminus" refers to the end having a free amino group (-NH2), and for a single amino acid, the term "N-terminus" refers to the end having an α-amino group, which can be either a free amino group or a non-free amino group, such as the NH form that has been dehydrogenated and attached to the α-carboxyl group of another amino acid.

[0262] As used herein, for a polypeptide or polypeptide sequence, the term “C-terminus” refers to the end having a free carboxyl group (-COOH), and for a single amino acid, the term “C-terminus” refers to the end having an α-carboxyl group, which can be a free carboxyl group or a non-free carboxyl group, such as a C (=O) form that is attached to the α-amino group of another amino acid without a hydroxyl group.

[0263] As used herein, the term "dipeptidyl peptidase-4 (DPP4)," also known as T-cell antigen CD26, is a typical serine-type protease in the proline oligopeptidase family. It inactivates many bioactive peptides by removing the N-terminal (N-terminal) dipeptide. DPP4 is expressed as a cell surface protein, but it also detaches from the membrane and functions as a soluble protein in plasma. DPP4 is widely expressed in many tissues, including endothelial cells in multiple vascular beds, making the enzyme readily available to peptide substrates circulating through the intestines, liver, lungs, and kidneys. DPP4 is a multifunctional protein that, in addition to its catalytic activity, functions as a binding protein and ligand for various extracellular molecules. Numerous studies have elucidated its diverse functional characteristics in immunology, inflammation, glucose homeostasis, oncology, and neuroendocrinology.

[0264] DPP4 is known to cleave dozens of natural peptides, including various chemokines, neuropeptides, and regulatory peptides, such as glucagon-like peptide-1 (GLP-1), GLP-2, stromal cell-derived factor-1 (SDF-1), brain natriuretic peptide (BNP), neuropeptide Y (NPY), tyrosinase Y (PYY), and glucose-dependent insulinotropic peptide (GIP). DPP4 recognizes peptide substrates containing a proline or alanine residue at the second N-terminus, but it can also be hydroxyproline, dehydroproline, glycine, threonine, valine, serine, or leucine, with different amino acids exhibiting different cleavage rates. DPP4 cleaves the peptide bond after the second N-terminus, thereby inactivating the peptide and / or generating new bioactive peptides, thus regulating various biological processes.

[0265] As used herein, the term "analyte" refers to a polypeptide whose sequence has one or more amino acid alterations compared to a reference amino acid sequence. These amino acid alterations may include amino acid additions, deletions, and / or substitutions. Amino acid substitutions, deletions, and / or additions may also be referred to as "mutations."

[0266] As used herein, the term "substitution" refers to the replacement of a hydrogen atom in a group by a corresponding substituent, for example, replacing SH in -CH2-SH with LD to obtain -CH2-SLD. It should be understood that substituents are only present in their possible chemical positions. As used herein, the term "substitution" refers to the replacement of the group itself by a corresponding group, for example, replacing -N3 in -(CH2)4-N3 with LD to obtain -(CH2)4-LD. The terms "substitution" or "substitution" are used only to indicate the manner of connection and do not represent a method of preparation.

[0267] As used herein, the term "fatty acid" refers to a carboxylic acid having an aliphatic hydrocarbon chain (straight or branched), wherein the aliphatic hydrocarbon chain may be saturated or unsaturated. For example, "C 10-24"Fatty acid" refers to a substance having 10 to 24 carbon atoms, with one of the carbon atoms forming a carboxyl group. Examples of fatty acids include, but are not limited to, myristic acid, palmitic acid, stearic acid, and eicosanoic acid.

[0268] As used herein, the term "fatty diacid" refers to a dicarboxylic acid having an aliphatic hydrocarbon chain (straight or branched), wherein the aliphatic hydrocarbon chain may be saturated or unsaturated. For example, "C 10-24 "Aliphatic diacids" refer to diacids with 10 to 24 carbon atoms, two of which form a carboxyl group. Examples of aliphatic diacids include, but are not limited to, adipic acid, octanoic acid, sebacic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, and tetradecanoic acid.

[0269] As used herein, the term "alkyl" refers to a straight-chain or branched monovalent saturated hydrocarbon group, such as "C 1-6 "Alkyl" refers to an alkyl group having 1 to 6 carbon atoms, such as 1, 2, 3, 4, 5, or 6 carbon atoms; "C" 1-4 "Alkyl" refers to a compound having 1 to 4 carbon atoms, such as 1, 2, 3, or 4 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, and n-butyl.

[0270] As used herein, the term "alkylene" refers to a straight-chain or branched divalent saturated hydrocarbon group, such as "C 1-6 "Alkylene" refers to a compound having 1 to 6 carbon atoms, such as 1, 2, 3, 4, 5, or 6 carbon atoms; "C" 1-4 "Alkyl" refers to a compound having 1 to 4 carbon atoms, such as 1, 2, 3, or 4 carbon atoms. Examples of alkyl groups include, but are not limited to, methylene, ethylene, and propylene.

[0271] In this document, unless otherwise stated, when one or more variables are defined as defined in one or more embodiments, combinations of those one or more variables may also be covered as defined in this or these embodiments. For example, for equation (I), A1, A2, B, C, P, m, and n are defined as defined in any embodiment of the present invention, representing the variables themselves and the structural units obtained by combining these variables. For example, A1-A2 may also be defined as defined in any embodiment of the present invention.

[0272] In this paper, when a general formula contains multiple groups with the same designation, these groups are independent of each other and may be the same or different. For example, in formula (I-3), each A1 may be the same or different, each A2 may be the same or different, and each A1-A2 may be the same or different.

[0273] As used herein, the term "antibody" is used in the broadest sense to encompass a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The constant domains do not directly participate in antibody-antigen binding but exhibit 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. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0274] As used herein, the term "nanobody (VHH)" has the meaning commonly understood by those skilled in the art, referring to an antibody fragment consisting 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 may 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.

[0275] As used herein, the terms "Fc fragment" or "Fc domain" refer to an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody, or an antibody fragment formed by the second and third constant regions of the second heavy chain of an antibody, or an antibody fragment formed by the combination of the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain via disulfide bonds. Antibody Fc fragments have various functions but do not participate in antigen binding.

[0276] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delaying agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Stabilizers have the meaning commonly understood by those skilled in the art for stabilizing the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate).

[0277] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the bioavailability and properties of a compound or conjugate, and is biologically or otherwise suitable for use in pharmaceuticals. In many cases, the prodrugs disclosed herein can form acid and / or base salts in the presence of amino and / or carboxyl or similar groups. Pharmaceutically acceptable acid addition salts can consist of inorganic and organic acids. Inorganic acids that can be derivatized to form salts include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids that can be derivatized to form salts include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Pharmaceutically acceptable base addition salts can consist of inorganic and organic bases. Inorganic bases that can be derived to form salts include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum; particularly preferred are ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases that can be derived to form salts include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc., specifically, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Many such salts are known in the art, such as those described in WO87 / 05297 by Johnston et al., published on September 11, 1987 (which is incorporated herein by reference in its entirety).

[0278] In cases where the compound name used in this article differs from the chemical structural formula, the chemical structural formula shall prevail.

[0279] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this application, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0280] As used herein, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) suffers from hepatitis B, particularly chronic hepatitis B.

[0281] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for treating a disease means an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0282] As used herein, the term "enhanced drug release capability" refers to reducing the instantaneous blood concentration of a drug upon injection, allowing for a slow release of the drug within the body, thereby reducing potential drug toxicity. In some cases, enhanced drug release capability can also prolong the drug's half-life in the body.

[0283] In one specific embodiment, the present invention first screens for suitable DPP4 cleavage sites on the target peptide, and then fuses the DPP4 cleavage sites (such as different repeat sequences of HA) to the N-terminus of the target peptide. The fusion molecule is obtained through recombinant expression technology, thus yielding an inactive prodrug. After the prodrug enters the body, it undergoes enzymatic cleavage by the DPP4 enzyme in the blood, gradually releasing the active molecule and thus exerting a more stable and effective therapeutic effect.

[0284] Regarding the improvement of drug properties, this invention can prolong the duration of action while reducing the side effects caused by high-dose injections, thus achieving a stable drug effect. For example, high-dose injections of GLP-1-related drugs can cause hypoglycemia, and high-dose injections of PTH-related drugs can cause hypercalcemia. However, the prodrug designed based on the method of this invention can avoid similar side effects. Beneficial effects

[0285] 1. The prodrug provided by this invention is stable in vitro and is not easily affected by external factors during production, formulation development, storage and transportation.

[0286] 2. The prodrug provided by this invention stably releases the active drug in vivo and has a long half-life, which can be used to develop long-acting sustained-release drugs, reduce the frequency of administration, and improve patient compliance; in addition, it can effectively eliminate adverse reactions caused by peak activity at the time of administration and increase the therapeutic index of the active drug.

[0287] 3. By selecting a combination of enzyme-cleaved peptides and DKP dipeptides, this invention can regulate the release time of the active drug, enabling the active drug to take effect at the target site at the appropriate time.

[0288] 4. Compared to the current main technologies for long-acting peptide drugs in this field, it has the following expected advantages:

[0289] (1) Easy to operate. Long-acting formulations such as sustained-release microspheres are complex to operate and have high process requirements. Various chemical modifications also require additional chemical reactions and have high requirements for reaction conditions. The sustained-release technology based on DPP4 in this invention only requires the addition of a few amino acids during peptide synthesis, and basically no additional operations are required.

[0290] (2) Low cost. Compared with some chemical modification and formulation technologies, the DPP4-based sustained-release technology of this invention is very simple to operate and can directly obtain the target product through recombinant expression, thus the cost is relatively low.

[0291] (3) Versatility. Compared to the sequence restrictions imposed by some modification methods, the DPP4-based sustained-release technology of this invention can theoretically be applied to all peptides that require an intact N-terminus (no more or less amino acids) to produce activity. This includes many natural peptides, such as GLP-1, GLP-2, SDF-1, parathyroid hormone (PTH), etc., as well as many marketed or investigational peptide drugs, such as GLP-1 analogs like smegglutide and dulaglutide (GLP-1-Fc), PTH analogs like teriparatide, etc.

[0292] (4) Adjustability. Compared to the fixed long-acting performance of some other methods, this invention, based on the enzymatic cleavage efficiency of DPP-4 enzyme on different substrates, allows for the selection and combination of different cleavage sites and repetitions to achieve an adjustable sustained-release effect, resulting in a stable and slow release of active molecules. This reduces the frequency of drug administration while also reducing drug side effects.

[0293] (5) It can be used in conjunction with other technologies that extend the half-life. For example, in one embodiment of the present invention, it is used in conjunction with Fc fusion proteins (such as GLP-1-Fc and PTH-Fc) to achieve a sustained release effect while extending the molecular half-life. Attached Figure Description

[0294] Figure 1. Hydrolysis of PTH polypeptide conjugate SEQ ID NO:19 in PBS buffer and human plasma;

[0295] Figure 2. Hydrolysis of PTH polypeptide conjugates SEQ ID NOs:31, 33, 35, and 37 in human plasma;

[0296] Figure 3. In vitro cAMP activity results of PTH peptide conjugate SEQ ID NO:23;

[0297] Figure 4. In vitro cAMP activity results of PTH polypeptide conjugate SEQ ID NO:26 (human serum);

[0298] Figure 5. In vitro cAMP activity results of PTH polypeptide conjugate SEQ ID NO:26 (rat serum);

[0299] Figure 6. Effect of PTH polypeptide conjugate SEQ ID NO:26 on serum calcium in PTx rats;

[0300] Figure 7. Effect of PTH polypeptide conjugate SEQ ID NO:26 on serum phosphorus in PTx rats;

[0301] Figure 8. Changes in serum calcium after a single dose of (HA)3-PTH-Fc fusion protein SEQ ID NO:137 and PTH-Fc fusion protein SEQ ID NO:136.

[0302] The relevant sequences used in this application are shown in Table 1 below.

[0303] Table 1. Sequence Information Note: The meanings of Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, and Z13 are shown in Table 2 below:

[0304] Table 2 Meanings of Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, and Z13

[0305] In this application, the symbol “{}” is used to represent an amino acid as a whole, and the “()” in the symbol “{}” is used to represent the modifying group of the amino acid represented by a single letter or three letters within the symbol “{}”. For example, {(N-Me)G} represents N-methylated Gly, {K(ε-N3)} represents Lys modified by an azide group at the ε position, that is, Lys in which the amino group of the side chain is replaced by an azide group, and {C(β-S-Z2)} represents Lys modified by -S-NHS-CH2CH2NH-2OEG-γGlu-CO(CH2) at the β position. 18 Cys modified with COOH, i.e., the thiol group of the side chain is replaced by -NHS-CH2CH2NH-2OEG-γGlu-CO(CH2). 18Cys substituted with COOH; Meanwhile, when the modifying group in "()" consists of a position number and a certain group, it indicates that the position is occupied by that group. For example, in {K(ε-Z4)}, (ε-Z4) consists of the position number ε and the group Z4, indicating that the ε position is occupied by the Z4 group. {K(ε-Z4)} represents Lys with the Z4 group at the ε position, i.e., Lys where the amino group in the side chain is replaced by the Z4 group. {K(ε-NH-Z7)} represents Lys with the -NH-Z7 group at the ε position, i.e., Lys where the amino group in the side chain is replaced by the -NH-Z7 group. In some embodiments, the single-letter or three-letter amino acid in the symbol "{}" is connected to a specific chemical group to represent the modified amino acid obtained after the side chain group of the single-letter or three-letter amino acid is replaced by that chemical group. For example, and Both represent SEQ ID NO:16. and

[0306] All of these represent SEQ ID NO:26. It should be noted that the terms "substitute" or "replace" used above refer only to the connection method and do not represent the preparation method.

[0307] The sequence description lists each mutation site based on the reference amino acid sequence.

[0308] The following provides examples of sequence descriptions of the prodrugs of the present invention comprising an enzyme-cleaved peptide, a DKP dipeptide, and a PTH peptide analog as a parent drug. For example, the sequence of SEQ ID NO:26 is described as PTH(1-34),G-3,P-2,K-1,(N-Me)D0,Nle8,Nle18,Aib25,K(ε-Azide-DBCO-CH2CH2NH-2OEG-γGlu-CO(CH2)). 18 COOH)34. In this prodrug, the enzyme-cleaved peptide contains Gly and Pro linked by an amide bond, the DKP dipeptide contains Lys and N-methylated Asp linked by an amide bond, and the parent drug is a PTH peptide analog that, compared to the reference amino acid sequence (PTH(1-34), SEQ ID NO:1), has Nle at position 8, Nle at position 18, Aib at position 25, and the ε position of the side chain at position 34 is Azide-DBCO-CH2CH2NH-2OEG-γGlu-CO(CH2). 18 In the COOH-modified Lys-DKP dipeptide, the N-methylated Asp is linked to the N-terminus of the PTH peptide analog via an amide bond, and the Pro in the enzyme-cleaved peptide is linked to the N-terminal amino group of the DKP dipeptide via an amide bond. The complete structure of this compound is shown above.

[0309] The following provides an example sequence description of a prodrug of the present invention comprising an enzyme-cleaved peptide, a DKP dipeptide, and a GLP-1 peptide analog as a parent drug. For example, the sequence of SEQ ID NO:70 is described as (GLP-1)7-37,F3,P4,A5,(N-Me)Ser6,Aib8,K(ε-Z4)26. In this prodrug, the enzyme-cleaved peptide comprises Phe and Pro linked by an amide bond, the DKP dipeptide comprises Ala and N-methylated Ser linked by an amide bond, and the parent drug is a GLP-1 peptide analog that, compared to the reference amino acid sequence ((GLP-1)7-37, SEQ ID NO:107), has Aib at position 8, a side chain at position 26, and Lys at position Z4, where the specific structure of Z4 is shown in the table above. In the DKP dipeptide, the N-methylated Ser is linked to the N-terminus of the GLP-1 peptide analog via an amide bond, and the Pro in the enzyme-cleaved peptide is linked to the N-terminal amino group of the DKP dipeptide via an amide bond. Detailed Implementation

[0310] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without specified manufacturers are all commercially available conventional products. The PBS buffer used in the following examples was purchased from Sangon Biotech, catalog number: E607009-0500.

[0311] General purification methods

[0312] Preparative HPLC purification method (1): In C18 (10 μm particle size, Purification was performed on a column with a pore size of 0.1% (mobile phase A: H2O (0.1% TFA), mobile phase B: CH3CN (0.1% TFA), linear gradient: 35 to 50% B, flow rate: 20 mL / min, total time: within 40 minutes), and detection was performed at a wavelength of 220 nm.

[0313] Preparative HPLC purification method (2): Reverse column chromatography purification was performed on an XBridge C18 (10 μm, 19 x 250 mm) column (mobile phase A: H2O (0.1% TFA), mobile phase B: CH3CN (0.1% TFA), elution gradient: 20% B to 80%, flow rate: 35 mL / min, total time: 22 min), using dual wavelength detection at 254 nm and 214 nm.

[0314] Example 1: Determination of DKP dipeptide cleavage rate

[0315] The conversion half-life of DKP-based prodrugs obtained by linking various dipeptides to the parent model peptide via amide bonds was determined using a parathyroid hormone (PTH) analog (NH2-X1VX2EIQLMHNLGKHLNSMERVEWL{Aib}KKLQDVHNF-COOH; SEQ ID NO:40) as the parent model peptide.

[0316] On a peptide synthesizer, a parent model peptide is assembled sequentially from the C-terminus to the N-terminus using the standard FMOC chemical synthesis method, and amino acid C and amino acid B are sequentially added to the parent model peptide binding resin to synthesize a DKP-based prodrug. The sequence of the prodrug is shown in SEQ ID NO:2 to SEQ ID NO:14, and the prodrug is obtained by preparative HPLC purification method (1).

[0317] A reaction mixture was prepared by dissolving the prodrugs shown in SEQ ID NO:2 to SEQ ID NO:14 in PBS buffer at a concentration of 1 mg / mL and maintaining the solution at 37°C. Aliquots were periodically removed for LC-MS and HPLC analysis. The rate of cleavage from the prodrug to the parent model peptide 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.

[0318] The remaining prodrug area (A) is obtained by integrating the peak. s ) and the released maternal model peptide area (A P ), calculate the reaction percentage according to the following formula: %reaction = A P / (A P +A s )*100, T 1 / 2 =50% reaction. The results are shown in Table 3.

[0319] Table 3: Peptide Information and Conversion Half-Life

[0320] Example 2: Synthesis of (S)-25-carboxy-1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-4,13,22,27-tetraoxo-6,9,15,18-tetraoxa-3,12,21,26-tetraazahexacosane-46-acid (compound 2)

[0321] 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (compound 2-2, 86.34 mg, 0.489 mmol, 1.05 eq) and (S)-22-carboxy-1-((2,5-dioxopyrrolidine-1-yl)oxy)-1,10,19,24-tetraoxo-3,6,12,15-tetraoxa-9,18,23-triazatetrazole-43-acid (compound 2-1, 400 mg, 0.466 mmol, 1.0 eq) were dissolved in dichloromethane (10 mL). N,N-diisopropylethylamine (DIEA) (95 μL, 0.547 mmol, 1.2 eq) was added with stirring at room temperature, and the mixture was stirred overnight at room temperature. LCMS monitoring showed that the starting materials were completely consumed. The solvent was removed by concentration under reduced pressure, and the solution was purified by reversed-phase column chromatography (C18, mobile phase A: H2O, mobile phase B: MeOH, elution gradient: 0% B to 100% B) to give (S)-25-carboxy-1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-4,13,22,27-tetraoxo-6,9,15,18-tetraoxa-3,12,21,26-tetraazahexacosane-46-acid (2,260 mg of compound, yield: 63%).

[0322] LCMS(ES+)m / z:884.5(M+H).

[0323] Example 3: Synthesis of (S)-25-carboxy-1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-4,13,22,27-tetraoxo-6,9,15,18-tetraoxa-3,12,21,26-tetraazapentacosane-44-acid (compound 3)

[0324] 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (compound 2-2, 221.52 mg, 1.2 mmol, 1.0 eq) and (S)-22-carboxy-1-((2,5-dioxopyrrolidine-1-yl)oxy)-1,10,19,24-tetraoxo-3,6,12,15-tetraoxa-9,18,23-triazahexahexadecane-41-acid (compound 3-1, 1 g, 1.2 mmol, 1.0 eq) were dissolved in dichloromethane (10 mL). N,N-diisopropylethylamine (DIEA) (95 μL, 0.547 mmol, 1.2 eq) was added with stirring at room temperature, and the mixture was stirred for 14 hours at room temperature. The solvent was removed by concentration under reduced pressure, and the solution was purified by reversed-phase column chromatography (C18, mobile phase A: H2O, mobile phase B: MeOH, elution gradient: 0% B to 100% B) to give (S)-25-carboxy-1-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-4,13,22,27-tetraoxo-6,9,15,18-tetraoxa-3,12,21,26-tetraazapentacosane-44-acid (compound 3,520 mg, yield: 50.5%).

[0325] LCMS(ES+)m / z:856.36(M).

[0326] Example 4: Synthesis of 19-[[(1S)-3-[[2-[2-[2-[2-[(3-(2-azatricyclic[10.4.0.0^(4,9)]hexadecano-1(12),4(9),5,7,13,15-hexene-10-yn-2-yl)-3-oxopropyl]carbamoyl]methoxyethoxy)ethyl]carbamoylmethoxy)ethoxy]ethylcarbamoyl)-1-carboxypropyl]carbamoylnonadecanoic acid (compound 4)

[0327] (S)-22-carboxy-1-((2,5-dioxopyrrolidone-1-yl)oxy)-1,10,19,24-tetraoxo-3,6,12,15-tetraoxa-9,18,23-triazatetrazole-43-acid (compound 2-1, 5.5 g, 6.403 mmol, 1.00 eq) was dissolved in acetonitrile (72 mL), and 3-amino-1-(2-azatricyclo

[10] ) was added to the reaction solution. [4.0.0^(4,9)] Hexadecane-1(12),4(9),5,7,13,15-hexaden-10-yn-2-yl)propane-1-one (compound 4-1, 1.77 g, 6.403 mmol, 1.00 eq.) was added in portions with stirring at room temperature, followed by the addition of N,N-diisopropylethylamine (DIEA) (1.66 g, 12.806 mmol, 2 eq). The mixture was stirred overnight at room temperature. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, the solvent was removed by concentration under reduced pressure. 0.1 N HCl (50 mL) and ethyl acetate (75 mL) were added to the residue to separate the organic phase. The aqueous phase was extracted three times with ethyl acetate (75 mL). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by normal-phase column chromatography (silica gel, mobile phase A: DCM, mobile phase B: MeOH, elution gradient: 7% B), 19-[[(1S)-3-[[2-[2-[2-[2-[(3-(2-azatricyclo[10.4.0.0^(4,9)]hexadecano-1(12),4(9),5,7,13,15-hexaden-10-yn-2-yl)-3-oxopropyl]carbamoyl]methoxyethoxy)ethyl]carbamoylmethoxy)ethoxy]ethylcarbamoyl)-1-carboxypropyl]carbamoylnonadecanoic acid (compound 4, 3.0331 g, yield: 43.09%).

[0328] LCMS(ES+)m / z:1020.6(M+H).

[0329] Example 5: Synthesis of 17-[[(1S)-3-[[2-[2-[[2-[2-[3-(2-azatricyclic[10.4.0.0^(4,9)]hexadecane-1(12),4(9),5,7,13,15-hexene-10-yn-2-yl)-3-oxopropyl]carbamoyl]methoxyethoxy]ethyl]carbamoylmethoxy]ethoxy]ethylcarbamoyl]-1-carboxypropyl]carbamoyl]heptadecanoic acid (compound 5)

[0330] (S)-22-carboxy-1-((2,5-dioxopyrrolidone-1-yl)oxy)-1,10,19,24-tetraoxo-3,6,12,15-tetraoxa-9,18,23-triazahexadecane-41-acid (compound 3-1, 7.10 g, 8.54 mmol, 1.00 eq) and 3-amino-1-(2-azatricyclo[10.4.0.0^(4,9)]) Hexadecane-1(12),4(9),5,7,13,15-hexaden-10-yn-2-yl)propane-1-one (compound 4-1, 2.48 g, 8.97 mmol, 1.05 eq.) was dissolved in acetonitrile (72 mL). N,N-diisopropylethylamine (DIEA) (2.21 g, 17.09 mmol, 2.00 eq.) was added with stirring at room temperature, and the mixture was stirred overnight at room temperature. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, the solvent was removed by concentration under reduced pressure. 0.1 N HCl (400 mL) and ethyl acetate (400 mL) were added to the residue to separate the organic phase. The aqueous phase was extracted twice with ethyl acetate (200 mL). The combined organic layers were washed with saturated brine (300 mL) and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated under reduced pressure. Purified by normal-phase column chromatography (silica gel, mobile phase A: DCM, mobile phase B: n-hexane, elution gradient: A:B = 1:3), 17-[[(1S)-3-[[2-[2-[2-[2-[3-(2-azatricyclo[10.4.0.0^(4,9)]hexadecane-1(12),4(9),5,7,13,15-hexene-10-yn-2-yl)-3-oxopropyl]carbamoyl]methoxyethoxy]ethyl]carbamoylmethoxy]ethoxy]ethylcarbamoyl]-1-carboxypropyl]carbamoyl]heptadecanoic acid (compound 5, 6.2972 g, yield: 64.4%).

[0331] LCMS(ES+)m / z:992.55(M+H).

[0332] 1H NMR: (400MHz, DMSO-d6, ppm) δ12.25(s,2H),8.03(d,J=7.7Hz,1H),7.88(t,J=5.6Hz,1H),7.65–7.59(m,3H),7.53–7.43(m,4 H),7.42–7.29(m,3H),5.04(d,J=14.0Hz,1H),4.13(td,J=8.5,5.1Hz,1H),3.86(d,J=6.1Hz,2H),3.73(d,J=14.0Hz,1H),3.5 4(hd,J=5.0,2.4Hz,5H),3.48–3.43(m,3H),3.43–3.35(m,4H),3.25(q,J=5.9Hz,2H),3.18(q,J=5.6Hz,3H),3.07–2.96(m,1H ), 2.44(dd,J=8.4,6.5Hz,1H),2.22–2.02(m,6H),1.95-1.82(m,2H),1.75–1.71(m,1H),1.47(t,J=7.1Hz,4H),1.23(s,24H).

[0333] Example 6: Synthesis of PTH polypeptide conjugate 6 (SEQ ID NO:16)

[0334] Step 1: Synthesis of polypeptide compound 6-1 (SEQ ID NO:15)

[0335] PTH peptide analogs were assembled on 2-chlorotriphenylmethyl chloride resin (CTC resin) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-five peptides were assembled sequentially from the C-terminus to the N-terminus using standard FMOC chemical synthesis, with proline (Pro) and glycine (Gly) continuously added to the thirty-five peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:15. The peptide compound 6-1 (SEQ ID NO:15) was obtained by preparative HPLC purification (1).

[0336] LCMS(ES+)m / z:4304.5(M).

[0337] Step 2: Synthesis of polypeptide conjugate 6 (SEQ ID NO:16)

[0338] The polypeptide compound 6-1 (SEQ ID NO:15, 10 mg, 2.3234 μmol) was dissolved in deionized water (32 μL) and acetonitrile (96 μL), and then a dimethylformamide solution (20 mg / mL, 143.7 μL) of compound 2 (2.87 mg, 3.2528 μmol) was added, and the mixture was stirred for 18 hours. After the reaction was completed, the polypeptide conjugate 6 (SEQ ID NO:16, 11.2 mg, 2.930 μmol, 93%) was isolated by preparative HPLC purification method (2) after LCMS monitoring.

[0339] LCMS(ES+)m / z:5184.6666(M).

[0340] Example 7: Synthesis of PTH polypeptide conjugate 7 (SEQ ID NO:17)

[0341] The polypeptide compound 6-1 (SEQ ID NO:15, 10 mg, 2.3234 μmol) was dissolved in deionized water (32 μL) and acetonitrile (96 μL), and then a dimethylformamide solution (20 mg / mL, 139.2 μL) of compound 3 (2.78 mg, 3.2528 μmol) was added, and the mixture was stirred for 18 hours. After the reaction was completed, the polypeptide conjugate 7 (SEQ ID NO:17, 11.5 mg, 2.2324 μmol, 96%) was isolated by preparative HPLC purification method (2).

[0342] LCMS(ES+)m / z:5156.6392(M).

[0343] Example 8: Synthesis of PTH polypeptide conjugate 8 (SEQ ID NO:19)

[0344] Step 1: Synthesis of polypeptide compound 8-1 (SEQ ID NO:18)

[0345] PTH peptide analogs were assembled on 2-chlorotriphenylmethyl chloride resin (CTC resin) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-four peptides were assembled sequentially from the C-terminus to the N-terminus using standard FMOC chemical synthesis, and proline (Pro) and glycine (Gly) were successively added to the thirty-four peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:18. The peptide compound 8-1 (SEQ ID NO:18) was obtained by preparative HPLC purification method (1).

[0346] LCMS(ES+)m / z:4226.8(M).

[0347] Step 2: Synthesis of polypeptide conjugate 8 (SEQ ID NO:19)

[0348] The polypeptide compound 8-1 (SEQ ID NO:18, 4 mg, 0.9463 μmol) was dissolved in deionized water (0.05% trifluoroacetic acid, 86 μL) and acetonitrile (0.05% trifluoroacetic acid, 258 μL), and then a dimethylformamide solution (20 mg / mL, 55.4 μL) of compound 4 (1.11 mg, 1.0883 μmol) was added, and the mixture was stirred for 21 hours. After the reaction was completed, the polypeptide conjugate 8 (SEQ ID NO:19, 3 mg, 0.5718 μmol, 60%) was separated by preparative HPLC purification method (2).

[0349] LCMS(ES+)m / z:5243.7450(M).

[0350] Example 9: Synthesis of PTH polypeptide conjugate 9 (SEQ ID NO:20)

[0351] The polypeptide compound 8-1 (SEQ ID NO:18, 4 mg, 0.9463 μmol) was dissolved in deionized water (0.05% trifluoroacetic acid, 86 μL) and acetonitrile (0.05% trifluoroacetic acid, 258 μL), and then a dimethylformamide solution (20 mg / mL, 54 μL) of compound 5 (1.08 mg, 1.0883 μmol) was added, and the mixture was stirred for 21 hours. After the reaction was completed, the polypeptide conjugate 9 (SEQ ID NO:20, 4.1 mg, 0.7857 μmol, 83%) was separated by preparative HPLC purification method (2).

[0352] LCMS(ES+)m / z:5215.7200(M).

[0353] Example 10: Synthesis of PTH polypeptide conjugate 10 (SEQ ID NO:22)

[0354] Step 1: Synthesis of polypeptide compound 10-1 (SEQ ID NO:21)

[0355] PTH peptide analogs were assembled on 2-chlorotriphenylmethyl chloride resin (CTC resin) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-four peptides were assembled sequentially from the C-terminus to the N-terminus using standard FMOC chemical synthesis, with sarcosine ((N-Me)Gly), ε-azido-lysine (Lys(ε-N3)), alanine (Ala), and histidine (His) being added sequentially to the thirty-four peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:21. The peptide compound 10-1 (SEQ ID NO:21) was obtained by preparative HPLC purification (1); LCMS (ES+) m / z: 4473.36 (M).

[0356] Step 2: Synthesis of polypeptide conjugate 10 (SEQ ID NO:22)

[0357] The polypeptide compound 10-1 (SEQ ID NO:21, 2.2 mg, 0.4914 μmol) was dissolved in PBS buffer (1.1 mL), and then a dimethylformamide solution (20 mg / mL, 55.4 μL) of compound 4 (0.692 mg, 0.6782 μmol) was added. The mixture was stirred for 16 hours. After the reaction was completed, the polypeptide conjugate 10 (SEQ ID NO:22, 2.4 mg, 0.4366 μmol, 89%) was purified by preparative HPLC method (2).

[0358] LCMS(ES+)m / z:5492.94(M).

[0359] Example 11: Synthesis of PTH polypeptide conjugate 11 (SEQ ID NO:23)

[0360] The polypeptide compound 11-1 (SEQ ID NO: 2, 4 mg, 0.9364 μmol) was dissolved in deionized water (0.05% trifluoroacetic acid, 66 μL) and acetonitrile (0.05% trifluoroacetic acid, 200 μL), and then a dimethylformamide solution (20 mg / mL, 57.3 μL) of compound 4 (1.14 mg, 1.1236 μmol) was added, and the mixture was stirred for 7 hours. After the reaction was completed, the polypeptide conjugate 11 (SEQ ID NO: 23, 4.1 mg, 0.7748 μmol, 83%) was separated by preparative HPLC purification method (2).

[0361] LCMS(ES+)m / z:5288.82(M).

[0362] Example 12: Synthesis of PTH polypeptide conjugate 12 (SEQ ID NO:24)

[0363] The polypeptide compound 12-1 (SEQ ID NO: 3, 4 mg, 0.924 μmol) was dissolved in deionized water (0.05% trifluoroacetic acid, 330 μL) and acetonitrile (0.05% trifluoroacetic acid, 1000 μL), and then a dimethylformamide solution (20 mg / mL, 56.5 μL) of compound 4 (1.13 mg, 1.1085 μmol) was added, and the mixture was stirred for 16 hours. After the reaction was completed, the polypeptide conjugate 12 (SEQ ID NO: 24, 4.3 mg, 0.8038 μmol, 87%) was separated by preparative HPLC purification method (2).

[0364] LCMS(ES+)m / z:5346.81(M).

[0365] Example 13: Synthesis of PTH polypeptide conjugate 13 (SEQ ID NO:26)

[0366] Step 1: Synthesis of polypeptide compound 13-1 (SEQ ID NO:25)

[0367] PTH peptide analogs were assembled on 2-chlorotriphenylmethyl chloride resin (CTC resin) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-four peptides were assembled sequentially from the C-terminus to the N-terminus using standard FMOC chemical synthesis, and N-methyl-aspartic acid (N-Me-Asp), lysine (Lys), proline (Pro), and glycine (Gly) were successively added to the thirty-four peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:25. The peptide compound 13-1 (SEQ ID NO:25) was obtained by preparative HPLC purification method (1).

[0368] LCMS(ES+)m / z:4429.0(M).

[0369] Step 2: Synthesis of polypeptide conjugate 13 (SEQ ID NO:26)

[0370] The polypeptide compound 13-1 (SEQ ID NO:25, 10 mg, 2.2578 μmol) was dissolved in deionized water (0.05% trifluoroacetic acid, 108 μL) and acetonitrile (0.05% trifluoroacetic acid, 324 μL), and then a dimethylformamide solution (20 mg / mL, 132.3 μL) of compound 4 (2.647 mg, 2.5965 μmol) was added, and the mixture was stirred for 21 hours. After the reaction was monitored by LCMS, the polypeptide conjugate 13 (SEQ ID NO:26, 11.5 mg, 2.1106 μmol, 93%) was isolated by preparative HPLC purification method (2).

[0371] LCMS(ES+)m / z:5446.0240(M).

[0372] Example 14: Synthesis of PTH conjugate 14 (SEQ ID NO:27)

[0373] The polypeptide compound 6-1 (SEQ ID NO: 15, 4.9 mg, 1.1384 μmol) was dissolved in deionized water (32.6 μL) and acetonitrile (33.2 μL), and then an acetonitrile solution (11 mg / mL, 32 μL) of 1-(5-aminopentyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate (CAS: 222159-87-7, 0.352 mg, 1.1990 μmol) was added. The mixture was stirred at 30 °C for 2 hours. After the reaction was completed, the polypeptide conjugate 14 (SEQ ID NO: 27, 4.7 mg, 1.047 μmol, 92%) was separated by preparative HPLC purification method (2).

[0374] LCMS(ES+)m / z:4485.3(M).

[0375] Example 15: Synthesis of PTH conjugate 15 (SEQ ID NO:29)

[0376] Step 1: Synthesis of polypeptide compound 15-1 (SEQ ID NO:28)

[0377] PTH peptide analogs were assembled on 2-chlorotriphenylmethyl chloride resin (CTC resin) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-five peptides were assembled sequentially from the C-terminus to the N-terminus using standard FMOC chemical synthesis, with proline (Pro) and lysine (Lys) continuously added to the thirty-five peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:28. The peptide compound 15-1 (SEQ ID NO:28) was obtained by preparative HPLC purification (1).

[0378] LCMS(ES+)m / z:4375.2(M).

[0379] Step 2: Synthesis of polypeptide conjugate 15 (SEQ ID NO:29)

[0380] The polypeptide compound 15-1 (SEQ ID NO:28, 2.459 mg, 0.56 μmol) was dissolved in deionized water (16.3 μL) and acetonitrile (15.5 μL), and then an acetonitrile solution (11 mg / mL, 17.1 μL) of 1-(5-aminopentyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate (CAS: 222159-87-7, 0.19 mg, 0.644 μmol) was added. The mixture was stirred at 30 °C for 2 hours. After the reaction was monitored by LCMS, the polypeptide conjugate 15 (SEQ ID NO:29, 2.4 mg, 0.5266 μmol, 94%) was purified by preparative HPLC (2).

[0381] LCMS(ES+)m / z:4554.39(M).

[0382] Example 16: Synthesis of PTH conjugate 16 (SEQ ID NO:31)

[0383] Step 1: Synthesis of polypeptide compound 16-1 (SEQ ID NO:30)

[0384] PTH peptide analogs were assembled on 2-chlorotriphenylmethyl chloride resin (CTC resin) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-four peptides were assembled sequentially from the C-terminus to the N-terminus using standard FMOC chemical synthesis, and N-methyl-aspartic acid (N-Me-Asp), lysine (Lys), proline (Pro) and lysine (Lys) were successively added to the thirty-four peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:30. The peptide compound 16-1 (SEQ ID NO:30) was obtained by preparative HPLC purification method (1).

[0385] LCMS(ES+)m / z:4500.0(M).

[0386] Step 2: Synthesis of polypeptide conjugate 16 (SEQ ID NO:31)

[0387] The polypeptide compound 16-1 (SEQ ID NO:30, 10 mg, 2.2222 μmol) was dissolved in deionized water (0.05% trifluoroacetic acid, 216 μL) and acetonitrile (0.05% trifluoroacetic acid, 648 μL), and then a dimethylformamide solution (20 mg / mL, 130.2 μL) of compound 4 (2.60 mg, 2.5556 μmol) was added, and the mixture was stirred for 4 hours. After the reaction was completed, the polypeptide conjugate 16 (SEQ ID NO:31, 10.2 mg, 1.848 μmol, 83%) was separated by preparative HPLC purification method (2).

[0388] LCMS(ES+)m / z:5517.1116(M).

[0389] Example 17: Synthesis of PTH conjugate 17 (SEQ ID NO:33)

[0390] Step 1: Synthesis of polypeptide compound 17-1 (SEQ ID NO:32)

[0391] PTH peptide analogs were assembled on 2-chlorotriphenylmethyl chloride resin (CTC resin) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-four peptides were assembled sequentially from the C-terminus to the N-terminus using standard FMOC chemical synthesis, and N-methyl-aspartic acid (N-Me-Asp), lysine (Lys), alanine (Ala), and histidine (His) were successively added to the thirty-four peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:32. The peptide compound 17-1 (SEQ ID NO:32) was obtained by preparative HPLC purification method (1).

[0392] LCMS(ES+)m / z:4483.0(M).

[0393] Step 2: Synthesis of polypeptide conjugate 17 (SEQ ID NO:33)

[0394] Polypeptide compound 17-1 (SEQ ID NO:32, 10 mg, 2.2306 μmol) was dissolved in deionized water (0.05% trifluoroacetic acid, 216 μL) and acetonitrile (0.05% trifluoroacetic acid, 648 μL), and then a dimethylformamide solution (20 mg / mL, 132.3 μL) of compound 4 (2.62 mg, 2.5652 μmol) was added, and the mixture was stirred for 4 hours. After the reaction was completed, the polypeptide conjugate 17 (SEQ ID NO:33, 10.2 mg, 1.8536 μmol, 83%) was isolated by preparative HPLC purification method (2).

[0395] LCMS(ES+)m / z:5500.0445(M).

[0396] Example 18: Synthesis of PTH conjugate 18 (SEQ ID NO:35)

[0397] Step 1: Synthesis of polypeptide compound 18-1 (SEQ ID NO:34)

[0398] PTH peptide analogs were assembled on 2-chlorotriphenylmethyl chloride resin (CTC resin) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-four peptides were assembled sequentially from the C-terminus to the N-terminus using standard FMOC chemical synthesis, and N-methyl-glycine (N-Me-Gly), serine (Ser), proline (Pro) and glycine (Gly) were successively added to the thirty-four peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:34. The peptide compound 18-1 (SEQ ID NO:34) was obtained by preparative HPLC purification method (1).

[0399] LCMS(ES+)m / z:4329.5(M).

[0400] Step 2: Synthesis of polypeptide conjugate 18 (SEQ ID NO:35)

[0401] The polypeptide compound 18-1 (SEQ ID NO:34, 10 mg, 2.3096 μmol) was dissolved in deionized water (0.05% trifluoroacetic acid, 216 μL) and acetonitrile (0.05% trifluoroacetic acid, 648 μL), and then a dimethylformamide solution (20 mg / mL, 135.4 μL) of compound 4 (2.7 mg, 2.6560 μmol) was added, and the mixture was stirred for 4 hours. After the reaction was completed, the polypeptide conjugate 18 (SEQ ID NO:35, 9.1 mg, 1.7011 μmol, 74%) was separated by preparative HPLC purification method (2).

[0402] LCMS(ES+)m / z:5346.9509(M).

[0403] Example 19: Synthesis of PTH conjugate 19 (SEQ ID NO:37)

[0404] Step 1: Synthesis of polypeptide compound 19-1 (SEQ ID NO:36)

[0405] PTH peptide analogs were assembled on 2-chlorotriphenylmethyl chloride resin (CTC resin) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-four peptides were assembled sequentially from the C-terminus to the N-terminus using standard FMOC chemical synthesis, with N-methyl-glycine (N-Me-Gly), D-lysine (D-Lys), proline (Pro), and glycine (Gly) being added sequentially to the thirty-four peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:36. The peptide compound 19-1 (SEQ ID NO:36) was obtained by preparative HPLC purification (1).

[0406] LCMS(ES+)m / z:4370.5(M).

[0407] Step 2: Synthesis of polypeptide conjugate 19 (SEQ ID NO:37)

[0408] The polypeptide compound 19-1 (SEQ ID NO:36, 10 mg, 2.2878 μmol) was dissolved in deionized water (0.05% trifluoroacetic acid, 216 μL) and acetonitrile (0.05% trifluoroacetic acid, 648 μL), and then a dimethylformamide solution (20 mg / mL, 134 μL) of compound 4 (2.68 mg, 2.631 μmol) was added, and the mixture was stirred for 18 hours. After the reaction was completed, the polypeptide conjugate 19 (SEQ ID NO:37, 10.4 mg, 1.9293 μmol, 84%) was separated by preparative HPLC purification method (2).

[0409] LCMS(ES+)m / z:5388.0110(M).

[0410] Example 20: Synthesis of PTH conjugate 20 (SEQ ID NO:39)

[0411] Step 1: Synthesis of polypeptide compound 20-1 (SEQ ID NO:38)

[0412] PTH peptide analogs were assembled on 2-chlorotriphenylmethyl chloride resin (CTC resin) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-four peptides were assembled sequentially from the C-terminus to the N-terminus using standard FMOC chemical synthesis, and N-methyl-aspartic acid (N-Me-Asp) and lysine (Lys) were successively added to the thirty-four peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:38. The peptide compound 20-1 (SEQ ID NO:38) was obtained by preparative HPLC purification method (1).

[0413] LCMS(ES+)m / z:4274.0(M).

[0414] Step 2: Synthesis of polypeptide conjugate 20 (SEQ ID NO:39)

[0415] Polypeptide compound 20-1 (SEQ ID NO:38, 10 mg, 2.3392 μmol) was dissolved in deionized water (0.05% trifluoroacetic acid, 216 μL) and acetonitrile (0.05% trifluoroacetic acid, 648 μL), and then a dimethylformamide solution (20 mg / mL, 137.2 μL) of compound 4 (2.74 mg, 2.6902 μmol) was added, and the mixture was stirred for 18 hours. After the reaction was completed, the polypeptide conjugate 20 (SEQ ID NO:39, 10.4 mg, 1.9643 μmol, 84%) was obtained by preparative HPLC purification method (2) monitored by LCMS.

[0416] LCMS(ES+)m / z:5291.9361(M).

[0417] Example 21: Synthesis of PTH conjugate 21 (SEQ ID NO:43)

[0418] Step 1: Synthesis of polypeptide compound 21-1 (SEQ ID NO:42)

[0419] The polypeptide compound 21-1 (SEQ ID NO:42) was synthesized and purified according to the first step of Example 20;

[0420] LCMS(ES+)m / z:4654.5(M).

[0421] Step 2: Synthesis of polypeptide conjugate 21 (SEQ ID NO:43)

[0422] Referring to the second step of Example 20, polypeptide compound 20-1 (SEQ ID NO:38) was replaced with polypeptide compound 21-1 (SEQ ID NO:42, 5 mg, 1.0742 μmol), and polypeptide conjugate 21 (SEQ ID NO:43, 4.8 mg, 0.8458 μmol) was synthesized and purified.

[0423] LCMS(ES+)m / z:5671.2503(M).

[0424] Example 22: Synthesis of PTH conjugate 22 (SEQ ID NO:45)

[0425] Step 1: Synthesis of polypeptide compound 22-1 (SEQ ID NO:44)

[0426] The polypeptide compound 22-1 (SEQ ID NO:44) was synthesized and purified according to the first step of Example 20;

[0427] LCMS(ES+)m / z:4613.5(M).

[0428] Step 2: Synthesis of polypeptide conjugate 22 (SEQ ID NO:45)

[0429] Referring to the second step of Example 20, polypeptide compound 20-1 (SEQ ID NO:38) was replaced with polypeptide compound 22-1 (SEQ ID NO:44, 5 mg, 1.0742 μmol), and polypeptide conjugate 22 (SEQ ID NO:45, 4.8 mg, 0.8458 μmol) was synthesized and purified.

[0430] LCMS(ES+)m / z:5630.1987(M).

[0431] Example 23: Synthesis of PTH conjugate 23 (SEQ ID NO:47)

[0432] Step 1: Synthesis of polypeptide compound 23-1 (SEQ ID NO:46)

[0433] The polypeptide compound 23-1 (SEQ ID NO:46) was synthesized and purified according to the first step of Example 20;

[0434] LCMS(ES+)m / z:4669.5(M).

[0435] Step 2: Synthesis of polypeptide conjugate 23 (SEQ ID NO:47)

[0436] Referring to the second step of Example 20, polypeptide compound 20-1 (SEQ ID NO:38) was replaced with polypeptide compound 23-1 (SEQ ID NO:46, 4 mg, 0.8566 μmol), and polypeptide conjugate 23 (SEQ ID NO:47, 3.7 mg, 0.6504 μmol) was synthesized and purified.

[0437] LCMS(ES+)m / z:5686.3054(M).

[0438] Example 24: Synthesis of PTH conjugate 24 (SEQ ID NO:57)

[0439] Step 1: Synthesis of polypeptide compound 24-1 (SEQ ID NO:56)

[0440] The polypeptide compound 24-1 (SEQ ID NO:56) was synthesized and purified according to the first step of Example 20;

[0441] LCMS(ES+)m / z:4288.5(M).

[0442] Step 2: Synthesis of polypeptide conjugate 24 (SEQ ID NO:57)

[0443] Referring to the second step of Example 20, polypeptide compound 20-1 (SEQ ID NO:38) was replaced with polypeptide compound 24-1 (SEQ ID NO:56, 5 mg, 1.1658 μmol), and polypeptide conjugate 24 (SEQ ID NO:57, 4.7 mg, 0.8854 μmol) was synthesized and purified.

[0444] LCMS(ES+)m / z:5306.0538(M).

[0445] Example 25: Synthesis of PTH peptide analogs and PTH conjugates

[0446] Following the method of Example 20, PTH polypeptide analogs or PTH conjugates as shown in SEQ ID NOs:48-55, 58-65 were prepared, and the LCMS (ES+) detection results are shown in Table 4 below.

[0447] Table 4 shows the LCMS (ES+) detection results of PTH peptide analogs or PTH conjugates shown in SEQ ID NOs: 48-55 and 58-65.

[0448] Example 26: Synthesis of PTH conjugate 26 (SEQ ID NO: 66)

[0449] Step 1: Synthesis of polypeptide conjugate 26 (SEQ ID NO:66)

[0450] PTH peptide analogs were assembled on Rink amide resin using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling protocol. Thirty-four peptides were assembled sequentially from C-terminus to N-terminus using standard FMOC chemical synthesis, with N-methylglutamic acid (N-Me-Glu), glycine (Gly), proline (Pro), and glycine (Gly) successively added to the thirty-four peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO: 112. The resin was treated with 5% hydrazine hydrate for 30 min to remove the isoprene (ivDde) ​​protecting group, followed by washing five times with DMF. Solid-phase peptide synthesis (SPPS) was then performed using the same protocol as in standard FMOC chemical synthesis, with the substituent Z7 linked according to the method described in Mol. Pharmaceuticals 2021, 18, 3260-3271.

[0451] After the side chain synthesis was completed, the resin was washed with DCM and dried with methanol (MeOH), then treated with 92.5:5:2.5 (v / v / v) TFA / water / TIS for 2-3 hours, followed by precipitation with methyl tert-butyl ether. The precipitate was separated by centrifugation, washed with methyl tert-butyl ether, dissolved in a solvent (2:1 water / MeCN), and allowed to stand until all unstable adducts decomposed. The polypeptide conjugate 26 (SEQ ID NO: 66) was then purified by preparative HPLC method (1); LCMS (ES+) m / z: 5175.0 (M).

[0452] Example 27: Synthesis of PTH conjugate 27 (SEQ ID NO: 67)

[0453] Step 1: Synthesis of polypeptide conjugate 27 (SEQ ID NO:67)

[0454] Referring to Example 26, the peptide sequence shown in SEQ ID NO:113 was synthesized, and substituent Z8 was linked according to the method described in J.Med.Chem.2015,58,7370-7380, and the peptide conjugate 27 (SEQ ID NO:67) was purified.

[0455] LCMS(ES+)m / z:5151.2(M).

[0456] Example 28: Synthesis of PTH conjugate 28 (SEQ ID NO: 68)

[0457] Step 1: Synthesis of polypeptide conjugate 28 (SEQ ID NO:68)

[0458] Referring to Example 26, the peptide sequence shown in SEQ ID NO:114 was synthesized, and substituent Z9 was linked according to the method described in J.Med.Chem.2015,58,7370-7380, and the peptide conjugate 28 (SEQ ID NO:68) was purified.

[0459] LCMS(ES+)m / z:4989.6(M).

[0460] Example 29: Synthesis of GLP-1 conjugate 29 (SEQ ID NO:70)

[0461] Step 1: Synthesis of polypeptide compound 29-1 (SEQ ID NO:69)

[0462] GLP-1 peptide analogs were assembled on 2-chlorotriphenylmethyl chloride resin (CTC resin) using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-one peptides were assembled sequentially from the C-terminus to the N-terminus using standard FMOC chemical synthesis, with N-methyl-serine (N-Me-Ser), alanine (Ala), proline (Pro), and phenylalanine (Phe) successively added to the thirty-one peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:69. The peptide compound 29-1 (SEQ ID NO:69) was obtained by preparative HPLC purification (1).

[0463] LCMS(ES+)m / z:3840.4(M).

[0464] Step 2: Synthesis of polypeptide conjugate 29 (SEQ ID NO:70)

[0465] The polypeptide compound 29-1 (SEQ ID NO: 69, 5 mg, 1.3020 μmol) was dissolved in deionized water (106 μL) and acetonitrile (318 μL), and then a dimethylformamide solution (20 mg / mL, 76.3 μL) of compound 4 (1.52 mg, 1.4973 μmol) was added, and the mixture was stirred for 2 hours. After the reaction was completed, the polypeptide conjugate 29 (SEQ ID NO: 70, 5 mg, 1.0288 μmol, 79%) was separated by preparative HPLC purification method (2).

[0466] LCMS(ES+)m / z:4857.5811(M).

[0467] Example 30: Synthesis of GLP-1 peptide analogs and GLP-1 conjugates

[0468] Following the method of Example 20, GLP-1 polypeptide analogs or GLP-1 conjugates as shown in SEQ ID NOs: 71-106 were prepared, and the LCMS (ES+) detection results are shown in Table 5 below.

[0469] Table 5. LCMS (ES+) detection results of GLP-1 peptide analogs or GLP-1 conjugates shown in SEQ ID NOs: 71–106

[0470] Example 31: Synthesis of GLP-1 conjugate 31 (SEQ ID NO: 108)

[0471] Step 1: Synthesis of polypeptide conjugate 31 (SEQ ID NO:108)

[0472] GLP-1 peptide analogs were assembled on Rink amide resin using a peptide synthesizer and the Fmoc / Oxyma / DIC coupling scheme. Thirty-one peptides were assembled sequentially from C-terminus to N-terminus using standard FMOC chemical synthesis, with N-methylglutamic acid ((N-Me)Glu), glycine (Gly), proline (Pro), and glycine (Gly) successively added to the thirty-one peptide-binding resin to synthesize the peptide sequence shown in SEQ ID NO:115. The resin was treated with 5% hydrazine hydrate for 30 min to remove the ivDde protecting group, followed by washing five times with DMF. SPPS was then performed using the same scheme as in standard FMOC chemical synthesis, with the substituent Z10 linked according to the method described in US12280124B2.

[0473] After the side chain synthesis was completed, the resin was washed with DCM and dried with methanol (MeOH), then treated with 92.5:5:2.5 (v / v / v) TFA / water / TIS for 2-3 hours, followed by precipitation with methyl tert-butyl ether. The precipitate was separated by centrifugation, washed with methyl tert-butyl ether, dissolved in a solvent (2:1 water / MeCN), and allowed to stand until all unstable adducts decomposed. The polypeptide conjugate 31 (SEQ ID NO: 108) was then purified by preparative HPLC method (1); LCMS (ES+) m / z: 4609.2 (M).

[0474] Example 32: Synthesis of GLP-1 conjugate 32 (SEQ ID NO: 109)

[0475] Step 1: Synthesis of polypeptide conjugate 32 (SEQ ID NO:109)

[0476] Referring to Example 31, the peptide sequence shown in SEQ ID NO:116 was synthesized, and the substituent Z11 was linked according to the method described in J.Med.Chem.2015,58,7370-7380, and the peptide conjugate 32 (SEQ ID NO:109) was purified.

[0477] LCMS(ES+)m / z:4550.8(M).

[0478] Example 33: Synthesis of GLP-1 conjugate 33 (SEQ ID NO: 110)

[0479] Step 1: Synthesis of polypeptide conjugate 33 (SEQ ID NO:110)

[0480] Referring to Example 31, the peptide sequence shown in SEQ ID NO:117 was synthesized, and the substituent Z12 was linked according to the method described in US20230203121A1, and the peptide conjugate 33 (SEQ ID NO:110) was purified.

[0481] LCMS(ES+)m / z:4390.0(M).

[0482] Example 34: Synthesis of GLP-1 conjugate 34 (SEQ ID NO: 111)

[0483] Step 1: Synthesis of polypeptide conjugate 34 (SEQ ID NO:111)

[0484] Referring to Example 31, the peptide sequence shown in SEQ ID NO:118 was synthesized, and substituent Z13 was linked according to the method described in US12280124B2, and the peptide conjugate 34 (SEQ ID NO:111) was purified.

[0485] LCMS(ES+)m / z:4553.2(M).

[0486] Example 35: Plasmid construction, cell transfection, and protein expression

[0487] 1.1 Expression of GLP-1-Fc and PTH-Fc with different types of dipeptides fused to the N-terminus, and GLP-1-Fc and PTH-Fc without fused dipeptides.

[0488] GLP-1-Fc sequences, PTH-Fc sequences, and GLP-1-Fc and PTH-Fc sequences of unfused dipeptides fused to the N-terminus (see Table 1) were synthesized by Baiying Biotechnology after codon optimization based on the protein sequence and ligated into a protein expression plasmid (pcDNA3.1, Baiying Biotechnology). Sequence accuracy was verified by sequencing. The concentration of the obtained expression plasmid was determined by centrifugation, and the different components of the plasmid were mixed in an appropriate ratio.

[0489] Take an appropriate amount of cells (CHOK1, ECACC), centrifuge to remove the supernatant, add an appropriate amount of electroporation buffer (prepared by Baiying Biotechnology) to the cells, mix well, and then add plasmids. After thorough mixing, add the suspension to an electroporation tube, place the tube in an electroporator (Celetrix, model SLT) for electroporation. After electroporation, aliquot the cells from the electroporation tube into shake flasks containing culture medium and incubate statically for 40 min. After incubation, place the shake flasks in a 37°C, 110 rpm, 8% CO2 incubator. After 24 h, add sodium butyrate and continue culturing for 6 to 7 days.

[0490] 1.2 Purification of Fusion Protein

[0491] The fusion protein was purified using a Protein A affinity chromatography column (in-house developed by Baiying Biotechnology), and the protein purity was then determined by SDS-PAGE and SEC-HPLC.

[0492] The SDS-PAGE used pre-cast protein gels (made in-house by Baiying Biotechnology) with gradients of 4%-18%. The SC-HPLC used an LC-20AT high-performance liquid chromatograph and a gel chromatography column (manufacturer: Saifen SRT-C).

[0493] Test Example 1: Evaluation of Self-Cleavage Conversion Half-Life (in PBS)

[0494] This test was performed to evaluate the conversion half-life of the active peptide released from the DKP dipeptide by DKP-based prodrug cleavage.

[0495] 1.1. Procedure for incubating samples in PBS buffer

[0496] A reaction mixture was prepared by dissolving the prodrug in PBS buffer and adjusting the pH to 7.4. The resulting solution was incubated at 37°C. At designed time points (0.5 h, 1 h, 2 h, 3 h, 8 h), aliquots were periodically removed for LC-MS and HPLC analysis. 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 parent peptide were quantified using Peak Simple Chromatography software.

[0497] The remaining prodrug area (A) is obtained by integrating the peak. s) and the surface area of ​​the released active peptide (A P ), calculate the reaction percentage according to the following formula: %reaction = A P / (A P +A s )*100, T 1 / 2 =50% reaction.

[0498] The results are shown in Table 6.

[0499] Table 6. Self-cleavage conversion half-life of the prodrug in PBS buffer.

[0500] The results in Table 6 show that the DKP dipeptide at the N-terminus of the prodrug cyclizes in PBS buffer to form DKP, releasing the active polypeptide.

[0501] Test Example 2: Evaluation of the half-life of enzyme cleavage transformation (in PBS with added DPP4 enzyme)

[0502] 1.1. Procedure for incubating samples in PBS buffer

[0503] See Test Example 1.

[0504] 1.2. Procedure for incubating samples in PBS buffer (with added DPP4 enzyme)

[0505] A reaction mixture was prepared by dissolving the prodrug in PBS buffer and adjusting the pH to 7.4, resulting in a concentration of 0.67 mg / mL. 500 μL of this solution was taken, and 1 μL of human DPP4 solution (400 μg / mL, Human DPPIV / CD26 Protein, His Tag, active dimer (active enzyme, MALS verified), ACRO) was added. The resulting solution was incubated at 37 °C. At designed time points (0.5 h, 1 h, 2 h, 3 h, 8 h), aliquots were periodically removed, and 50 μL of the sample was taken as the test sample. 5 μL of ZnCl2 (1 μM) was added, mixed, transferred to a vial, and analyzed by LC-MS and HPLC. The cleavage rate was qualitatively monitored by LC-MS and quantitatively studied by HPLC. The retention time and relative peak area of ​​the prodrug and the parent model peptide were quantified using Peak Simple Chromatography software.

[0506] The remaining prodrug area (A) is obtained by integrating the peak. s ) and the surface area of ​​the released active peptide (A P ) or the intermediate prodrug area (A) of the active polypeptide with DKP dipeptide linked to the N-terminus. s’ ), calculate the reaction percentage according to the following formula: %reaction = A P / (AP +A s )*100, T 1 / 2 =50% reaction; % reaction = A s’ / (A s’ +A s )*100, T 1 / 2 =50% reaction.

[0507] The results are shown in Table 7.

[0508] Table 7. Conversion half-life of the prodrug in PBS buffer and PBS buffer with added DPP4 enzyme.

[0509] The results in Table 7 show that the prodrugs with introduced enzyme-cleaved peptides are stable in PBS. When DPP4 enzyme is added, the dipeptides His-Ala, Gly-Pro and Lys-Pro at the N-terminus of the prodrug are removed by DPP4 enzyme, releasing the active peptide or DKP-based prodrug.

[0510] Test Example 3: Assessment of Conversion Half-Life in Plasma

[0511] 1.1. Procedure for incubating samples in ddH2O

[0512] A reaction mixture was prepared by dissolving the prodrug in ddH₂O and adjusting the pH to 7.4. The resulting solution was incubated at 37°C. At designed time points (0.5 h, 1 h, 2 h, 3 h, 8 h), aliquots were periodically removed for LC-MS and HPLC analysis. 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 parent peptide were quantified using Peak Simple Chromatography software.

[0513] The remaining prodrug area (A) is obtained by integrating the peak. s ) and the surface area of ​​the released active peptide (A P ), calculate the reaction percentage according to the following formula: %reaction = A P / (A P +A s )*100, T 1 / 2 =50% reaction.

[0514] 1.2. Procedure for incubating samples in human plasma

[0515] A reaction mixture was prepared by dissolving the prodrug in ddH₂O and adjusting the pH to 7.4. The resulting solution was then mixed thoroughly with human plasma. The solution was incubated at 37°C. At designed time points (0.5h, 1h, 2h, 3h, 8h), aliquots were periodically transferred, and 30 μL of the sample was added to 150 μL of pre-cooled 90% methanol at -20°C. The mixture was incubated at 4°C for 30 min, centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was transferred to a vial for LC-MS and HPLC analysis. 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.

[0516] The remaining prodrug area (A) is obtained by integrating the peak. s ) and the surface area of ​​the released active peptide (A P ), calculate the reaction percentage according to the following formula: %reaction = A P / (A P +A s )*100, T 1 / 2 =50% reaction.

[0517] The results are shown in Tables 8 and 9, Figure 1 and Figure 2.

[0518] Table 8. Conversion half-life of prodrug in ddH2O and plasma.

[0519] The results in Table 8 and Figure 1 indicate that the prodrug with the enzyme-cleaved peptide is stable in ddH2O, and its N-terminal dipeptide Gly-Pro can be removed from human plasma to release the active polypeptide.

[0520] Table 9. Conversion half-life of the prodrug in ddH2O and plasma. Note: m is an integer from 1 to 10.

[0521] Table 9 and Figure 2 show that the prodrugs containing the enzyme-cleaved peptides and one or more DKP dipeptides are stable in ddH2O. The N-terminal enzyme-cleaved peptides Gly-Pro, Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, Nle-Pro, Nva-Pro, Gly-Pro-Lys-Pro, Gly-Ala, and His-Ala can be removed from human plasma, thereby spontaneously initiating the formation of DKP from the DKP dipeptide and releasing the active polypeptide. Furthermore, the release time of the active drug varies depending on the combination of different enzyme-cleaved peptides and DKP dipeptides.

[0522] Test Example 4: In vitro cAMP activity detection

[0523] The test sample was dissolved in ddH2O and the pH was adjusted to 7.4. It was then mixed with human serum or rat serum and incubated at 37°C. At the same time, the test sample and PTH(1-34) (as shown in SEQ ID NO:1) peptide were dissolved in PBS buffer. The in vitro cAMP activity of the sample was detected using a cAMP kit (Cisbio:62AM4PEC).

[0524] Prepare test buffer 1 (0.5% BSA in DMEM) and test buffer 2 (0.5 mM IBMX in test buffer 1) according to the kit instructions. Add 5 μL of each concentration of sample diluted with test buffer 1 (SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 39) to a 384-well plate. Then add 5 μL of Saos-2 cell suspension (human osteoblast cells, Henan Applied Microbiology Engineering Research Center, BNCC338485) diluted with test buffer 2 to the 384-well plate and incubate the 384-well plate in a cell culture incubator for 30 minutes. After incubation, add 5 μL of 1X cAMP-d2 and 5 μL of 1X Anti-cAMP-Cryptate successively, incubate at room temperature for 1 hour, and then incubate in PerkinElmer. Data were read from the 2105 (665nm and 620nm) display and analyzed using GraphPad Prism 10.2.3 software. Experimental results are shown in Tables 10-12 and Figures 3-5.

[0525] Table 10 Results of in vitro cAMP bioactivity of SEQ ID NO:23

[0526] The results in Table 10 and Figure 3 indicate that the PTH prodrug (PTH polypeptide conjugate 11) shown in SEQ ID NO:23 is active in PBS buffer, and its N-terminal DKP dipeptide will cyclize to form DKP in PBS buffer, releasing the active PTH polypeptide. In human plasma, the PTH prodrug shown in SEQ ID NO:23 will also cyclize to form DKP. The results show that the active PTH polypeptide can be stably released for at least 14 days, thus maintaining cAMP activity.

[0527] Table 11 Results of in vitro cAMP bioactivity of SEQ ID NO:39

[0528] Table 12 Results of in vitro cAMP bioactivity of SEQ ID NO:26

[0529] Tables 11 and 12, and Figures 4 and 5 show that the PTH prodrug (PTH polypeptide conjugate 20) shown in SEQ ID NO:39 is active in PBS, mainly because the DKP dipeptide is unstable and cyclizes to form DKP, thus releasing the active PTH polypeptide conjugate in PBS. However, the PTH prodrug (PTH polypeptide conjugate 13) shown in SEQ ID NO:26 is inactive in PBS. The DKP dipeptide is cleaved by the enzyme and remains stable without initiation. However, in human or rat plasma, the enzyme-cleaved peptide of SEQ ID NO:26 is cleaved by the DPP4 enzyme in the plasma, exposing the DKP dipeptide, which cyclizes to form DKP, and can stably release the active PTH polypeptide for at least 7 days, thereby maintaining cAMP activity.

[0530] Test Example 5: Pharmacological Evaluation of PTx Rats

[0531] Rats (sex: female; strain: Sprague Dawley; average weight: 400-450g; age: 9-10 weeks; diet: standard food) were randomly divided into a drug administration group and a sham operation group. The drug administration group underwent parathyroidectomy (PTx) to establish a PTx rat model. The sham operation group underwent surgery without parathyroidectomy.

[0532] The test sample was dissolved in ddH2O and the pH was adjusted to 7.4.

[0533] After successful modeling, PTx rats in the drug-treated group (n=6) were subcutaneously injected with the PTH prodrug (PTH polypeptide conjugate 13, dose 40 nmol / kg, Q3D*3) shown in SEQ ID NO:26; rats in the sham-operated group (n=6) were subcutaneously injected with PBS buffer (PBS-Sham, QD). Blood samples were collected from rats in the drug-treated group immediately before injection and at 24 and 72 hours after administration, while blood samples were collected from rats in the sham-operated group immediately before injection and on days 1, 2, 4, 5, 6, 7, and 9 after injection. Blood was rotated (2000 rcf, RT, 10 min) and serum was collected and stored at -80°C. Calcium and phosphorus concentrations were determined according to the manufacturer's instructions using commercially available kits (purchased from Sangon Biotech, catalog numbers: D799341-0050 and D799343-0050, respectively).

[0534] The results are shown in Figures 6 and 7. The results indicate that PTH prodrug has the effect of increasing serum calcium levels and decreasing serum phosphorus levels in PTx rats.

[0535] Test Example 6: DPP4 digestion at different sites and repetitive sequences

[0536] This experiment was used to test the enzymatic digestion results of DPP4 on different sites and different fusion proteins in vitro.

[0537] 6.1 Enzyme digestion

[0538] At 37℃, equal amounts of DPP4 enzyme (name: Human DPPIV / CD26 Protein, brand: ACRO; catalog number DP4-H5221; batch number 81-83UF1-XW) were added to GLP-1-Fc capsules with various N-terminal tags. Samples were taken at 0h, 1h, 2h, 4h, and 8h, and 10uM Zn was added. 2+ To terminate.

[0539] 6.2 Mass Spectrometry Analysis

[0540] The reaction system from step 6.1 was subjected to mass spectrometry analysis. 50 μL of each sample was reduced with 2 μL of 500 mM DTT and incubated at 37 °C for 1 h. The resulting solution was then transferred to vials for mass spectrometry analysis. Liquid chromatography (LC) was performed using a BioResolve RP column (Waters, 450 Å, 2.7 μm, catalog number: 186008946) and a Thermo Orbitrap Exploris 240 mass spectrometer for molecular weight determination. Detailed experimental parameters are shown in the table below. Results were processed using Thermo BioPharma Finder 5.0 software. A cation source was used for mass spectrometry.

[0541] The release of active molecules was determined by analyzing the molecular weight of the samples. The results are shown in Table 13 below.

[0542] Table 13 shows the enzymatic digestion results of DPP4 on different fusion proteins.

[0543] As shown in Table 13, DPP4 can cleave fusion proteins containing HA, YA, YG, YP, HS, SP, GP, RP and their respective repeat sequences. The cleavage rate varies for different sequences, and the cleavage rate also varies for different repeat lengths of the same sequence.

[0544] Different enzyme digestion rates can affect the subsequent release of molecules. If the digestion rate is too fast, the active drug will be released too quickly, failing to achieve a sustained-release effect; if the digestion rate is too slow, the stability of the molecule will be limited by its own half-life, and the active drug may be cleared before it can be released.

[0545] For GLP-1-Fc, sequences with relatively fast digestion rates, such as YA, SP, YP, GP, RP, and HA, can achieve different sustained-release effects through repetition of multiple sequences. The length of the repetition sequence can be selected based on the half-life of the peptide drug. In this invention, sequences with a moderate digestion rate need to be selected for further validation.

[0546] In the embodiment where the sequence is fused to the N-terminus of GLP-1-Fc, the digestion rate of HA and its repeating sequences is relatively stable. GLP-1-Fc carrying the repeating HA site exhibits multiple digestion states during digestion, and the digestion rate gradients of different digestion states are relatively stable. Therefore, HA was selected as an example site for further validation in subsequent in vivo pharmacokinetic and pharmacodynamic studies.

[0547] In contrast, sequences like YG and HS, which have slower cleavage rates, are less suitable for drugs with shorter half-lives, such as GLP-1-Fc and PTH-Fc. However, data suggests that these dipeptide sequences are more suitable for drugs that are relatively stable and have longer half-lives.

[0548] Test Case 7: (HA) n -GLP-1-Fc fusion molecule single-dose p-value assay

[0549] This experiment was used to test the levels of DPP4 pairs (HA) in mouse serum. n Tag resection capability, measured (HA). n PK of the -GLP-1-Fc fusion molecule.

[0550] 7.1 Animal Preparation

[0551] Twenty-four male mice were used, with six mice per group. Animals were weighed and their weight recorded before administration. All animals had free access to food and water before administration. A single subcutaneous injection of 0.6 mg / kg was administered. Blood samples were collected at different time points after administration (before administration (0h), and 4h, 8h, 16h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h after administration). Three animals were collected at each time point, with blood samples collected alternately according to the experimental design.

[0552] 7.2 Sample Analysis: Detection of GLP-1-Fc content in serum

[0553] The detection was performed using a sandwich ELISA method. The antibody specifically recognizing the N-terminus of GLP-1 (GLP-1 Monoclonal Antibody, Biotin, Invitrogen: Catalog No. ABS 033-10B-005) was used for coating, along with rabbit anti-human IgG4 antibody (Abcam, Catalog No. ab238320) and anti-rabbit secondary antibody (BETHYL, Catalog No. A120-211P).

[0554] 7.3PK Parameter Analysis

[0555] Using a non-room model, (Version 8.2, Pharsight, Mountain View, CA) Analyzed blood drug concentration-time data.

[0556] PK parameters include (if data allows) but are not limited to peak concentration (C0). max Peak time (T) max Eliminate half-life (T) 1 / 2 ), area under the serum concentration-time curve (AUC), mean residence time (MRT), etc. The meanings and calculation formulas for each PK parameter are shown in Table 14 below:

[0557] Table 14 Pharmacokinetic Parameters

[0558] The sequences of GLP-1-Fc fusion proteins with different lengths of HA sequences fused to the N-terminus are shown in Table 1, and the PK results of the DPP4 restriction site are shown in Table 15 below.

[0559] Table 15. PK results of GLP-1-Fc fusion proteins with N-terminal fused HA sequences of different lengths via the DPP4 restriction site.

[0560] As shown in Table 15, GLP-1-Fc (derived from recombinant expression by Baiying Biotechnology), as can be seen from the PK results, has a peak plasma concentration C after entering the body.max The concentration was 421 ng / ml. Since GLP-1 drugs lower blood sugar, excessively high blood concentrations can easily trigger a series of side effects, including hypoglycemia.

[0561] The plasma concentration of (HA)2-GLP-1-Fc was 320 ng / ml, while the peak plasma concentration of the active molecule of (HA)3-GLP-1-Fc was 283 ng / ml. PK results showed that, compared with GLP-1-Fc, (HA)2-GLP-1-Fc and (HA)3-GLP-1-Fc significantly reduced the peak plasma concentration of the active molecule, and also had a longer duration of action and longer efficacy.

[0562] From the above results, it can be concluded that (HA) n The tag can be cleaved by DPP4 in mouse serum, releasing active GLP-1-Fc; compared to untagged GLP-1-Fc, (HA) n -GLP-1-Fc showed more stable blood drug concentrations.

[0563] Compared to (HA)2-GLP-1-Fc, (HA)3-GLP-1-Fc has different T values. 1 / 2 AUC (0-t) AUC (0-∞) They are quite similar; both (HA)2-GLP-1-Fc and (HA)3-GLP-1-Fc showed lower peak plasma concentrations (C0) compared to GLP-1-Fc. max The results suggest that the introduction of HA can improve the stability of GLP-1 blood drug levels. The MRT(0-t) and MRT(0-∞) values ​​show that the introduction of HA increases the retention time of GLP-1 in vivo. In summary, the introduction of the N-terminal DPP4 cleavage site allows for a more stable and prolonged release of the active molecule, thereby maintaining stable blood glucose levels and preventing hypoglycemia during drug use.

[0564] Test Example 8: Pharmacodynamic Study of PTH-Fc Fusion Protein After Single Dosage

[0565] The main physiological function of PTH in the body is to regulate the balance of calcium and phosphorus and maintain serum calcium homeostasis. Clinically, it can be used to treat patients with hypoparathyroidism to maintain physiological serum calcium levels. The normal physiological range of serum calcium is 8.6-10.3 mg / dL. In patients with hypoparathyroidism, serum calcium levels are lower than physiological levels, requiring medication to raise them. In this experiment, wild-type rats were used to observe the pharmacodynamic effects of PTH-Fc fusion protein SEQ ID NO:136 and (HA)3-PTH-Fc fusion protein SEQ ID NO:137 on increasing serum calcium levels. This experiment mainly assesses the stability of the drug's efficacy by observing the magnitude and stability of the increase in serum calcium levels after administration.

[0566] Twelve male SD rats aged 6-8 weeks were randomly divided into four groups. A single subcutaneous injection of 40 nmol / kg was administered. Blood samples were collected at different time points after administration, centrifuged, and the supernatant was used to detect serum total calcium content using a Sysmex BX4000 blood biochemistry analyzer. Sampling points: before administration (0 hours), and after administration: 3 hours, 10 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 144 hours, and 168 hours. Changes in serum calcium after a single administration are shown in Table 16.

[0567] Table 16 Changes in serum calcium after a single dose

[0568] As shown in Table 16 and Figure 8, PTH-Fc and (HA)3-PTH-Fc can exert their normal efficacy at the same dosage. The highest serum calcium concentration after PTH-Fc administration was 12.53 mg / dL. Since the physiological function of PTH in the body is mainly to maintain calcium and phosphorus homeostasis, the physiological concentration of serum calcium in wild-type rats is 9.2-13 mg / dL. Exceeding the physiological range can easily lead to hypercalcemia.

[0569] In comparison, the highest serum calcium concentration of (HA)3-PTH-Fc was 11.56, indicating a decrease in peak serum calcium concentration. Therefore, at the same dose and during the same dosing period, the fluctuation in serum calcium levels of the (HA)3-PTH-Fc molecule was more stable, suggesting that the N-terminal HA fusion sequence can help the active molecule exert its efficacy more steadily, maintaining physiological serum calcium levels while avoiding hypercalcemia.

[0570] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A prodrug or a pharmaceutically acceptable salt thereof, wherein, The prodrug comprises a structure represented by Formula (F-1), in, A1 and A2 are each an α-amino acid; B is an α-amino acid or an α-amino acid modified with an azide group; Each C is an N-alkylated α-amino acid; n is an integer between 0 and 10; m is an integer between 0 and 10; The condition is that m and n are not both 0; Dipeptide A1-A2 can be cleaved by DPP4 enzyme to be removed from the prodrug; A1, A2, B, and C are connected by amide bonds.

2. The prodrug of claim 1 or a pharmaceutically acceptable salt thereof, wherein, A2 is independently selected from Pro, Ala, Gly, Ser, Hyp, ΔHyp, Thr, Val, and Leu; Preferably, A2 is independently selected from Pro, Ala, Gly, and Ser; Preferably, A2 is independently selected from Pro and Ala; Preferably, each A1 is independently selected from protein amino acids and non-protein amino acids; Preferably, each of A1 is independently selected from Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Cys and non-protein amino acids (e.g., Nle, Nva); Preferably, A1 is independently selected from Lys, Gly, His, Tyr, Ser, and Arg; Preferably, A1 is independently selected from Lys, Gly, His, and Tyr; Preferably, A1 is independently selected from Lys, Gly, and His; Preferably, dipeptides A1-A2 are each independently selected from Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, Nle-Pro, Nva-Pro, Gly -Ala, His-Ala, Lys-Ala, Met-Ala, Phe-Ala, Pro-Ala, Ser-Ala, Thr-Ala, Trp-Ala, Tyr-Ala, Val-Ala, Ala-Ala, Arg-A la, Asn-Ala, Asp-Ala, Gln-Ala, Glu-Ala, Ile-Ala, Leu-Ala, Cys-Ala, Nle-Ala, Nva-Ala, Tyr-Ala, Tyr-Gly, His-Ser; Preferably, dipeptides A1-A2 are each independently selected from Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, and Nle-Pro. , Nva-Pro, Gly-Ala, His-Ala, Lys-Ala, Met-Ala, Phe-Ala, Pro-Ala, Ser-Ala, Thr-Ala, Trp-Ala, Tyr-Ala, Val -Ala, Ala-Ala, Arg-Ala, Asn-Ala, Asp-Ala, Gln-Ala, Glu-Ala, Ile-Ala, Leu-Ala, Cys-Ala, Nle-Ala and Nva-Ala; Preferably, (A1-A2)n is selected from Gly-Pro-Lys-Pro, Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-P ro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, Nle-Pro, Nva-Pro, Gly-Ala, His-Ala, Tyr-Ala, Tyr-Gly, His-Ser; Preferably, (A1-A2)n is selected from Gly-Pro-Lys-Pro, Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, Nle-Pro, Nva-Pro, Gly-Ala, and His-Ala.

3. The prodrug or a pharmaceutically acceptable salt thereof as described in claim 1 or 2, wherein, B can be an L-type α-amino acid, a D-type α-amino acid, an L-type α-amino acid modified with an azide group, or a D-type α-amino acid modified with an azide group; Preferably, B is independently selected from Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Cys, Nle, Nva, ε-azido-Lys, D-Lys, and ε-azido-D-Lys; Preferably, B is independently selected from Lys, ε-azido-Lys, D-Lys, ε-azido-D-Lys, Gly, Ser, Phe, Thr, Ala, Arg, Leu, and Nva.

4. The prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein, C is an α-N-methylated α-amino acid; Preferably, C is independently selected from N-methyl-Gly, N-methyl-Asp, N-methyl-Ser, N-methyl-Val, N-methyl-Phe, N-methyl-Leu, N-methyl-Tyr, α-N-methyl-Lys, N-methyl-Ile, N-methyl-Ala, N-methyl-Nle, N-methyl-Nva and N-methyl-Glu.

5. The prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein, Dipeptides As As shown, where, R1 and R4 are each independently selected from H and C. 1-6 alkyl, R2, R3, R5, and R6 are each independently selected from H and C. 1-6 Alkyl groups and 4-7 membered heterocyclic groups, wherein the C 1-6 The alkyl group may be optionally substituted by one or more substituents of a phenyl group selected from OH, NH2, N3, SH, COOH, S-CH3, C(=O)NH2, NHC(=NH)NH2, NHC(=O)NH2, phenyl, indole, and hydroxyl groups; Preferably, R1, R3, and R6 are H; Preferably, R2 is selected from H and C. 1-4 Alkyl, the C 1-4 The alkyl group may be optionally substituted by one or more substituents of a phenyl group selected from OH, NH2, N3, SH, COOH, S-CH3, C(=O)NH2, NHC(=NH)NH2, NHC(=O)NH2, phenyl, indole, and hydroxyl groups; Preferably, R2 is selected from H and C. 1-4 Alkyl groups (e.g., CH3, CH2CH2CH3, CH2CH(CH3)2), CH2Ph, CH2OH, CH(OH)CH3, CH2CH2CH2NHC(=NH)NH2, (CH2)4NH2 and (CH2)4N3; Preferably, R4 is C 1-4 alkyl; Preferably, R4 is selected from CH3, CH2CH3 and CH(CH3)2; Preferably, R4 is CH3; Preferably, R5 is selected from H and C. 1-6 Alkyl, the C 1-6 The alkyl group may be optionally substituted by one or more substituents of a phenyl group selected from OH, NH2, N3, SH, COOH, S-CH3, C(=O)NH2, NHC(=NH)NH2, NHC(=O)NH2, phenyl, indole, and hydroxyl groups; Preferably, R5 is selected from H and C. 1-4 Alkyl, the C 1-4 The alkyl group may be optionally substituted by one or more substituents of a phenyl group selected from OH, NH2, N3, SH, COOH, S-CH3, C(=O)NH2, NHC(=NH)NH2, NHC(=O)NH2, phenyl, indole, and hydroxyl groups; Preferably, R5 is selected from H and C. 1-4 Alkyl groups, CH2Ph, CH2PhOH, CH2OH, CH2COOH, CH2CH2COOH and (CH2)4NH2.

6. The prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, n is 1, 2, 3, 4 or 5; m is 0, 1 or 2; Preferably, n is 1, 2, or 3; m is 0, 1, or 2; Preferably, n is 1 or 2; m is 0, 1 or 2.

7. The prodrug or pharmaceutically acceptable salt thereof of any one of claims 1-6, wherein, The structure shown in equation (F-1) is selected from the structures shown in equations (F-1-1), (F-1-2), (F-1-3), (F-1-4), or (F-1-5): in, n1 is 1, 2, 3, 4 or 5; preferably, n is 1, 2 or 3; In equations (F-1-3) and (F-1-5), each A1 may be the same or different, and each A2 may be the same or different; In equation (F-1-4), each B may be the same or different, and each C may be the same or different; A1, A2, B, and C are as defined in any one of claims 1-5.

8. The prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, wherein, The prodrug also includes drug P; Preferably, the drug P is an active drug; Preferably, the active pharmaceutical ingredient is selected from polypeptide drugs, protein drugs, nucleic acid drugs, and small molecule drugs; Preferably, the polypeptide drug is selected from 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), insulin-like growth factor 1 (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, and granulocyte colony-stimulating factor. Obinepitide, parathyroid hormone (PTH) and its analogues (e.g., sequences with substitutions, deletions, additions, or any combination thereof of 1, 2, 3, 4, 5, 6, or 7 amino acids compared to PTH), leuprorelin, sermorelin, pramorelin, nesiritide, rotigaptide, silengitide, MBP-8298, AL-108, enfuviride, carfilzomib, mirvastatin, 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 (e.g., leucine enkephalin and methionine enkephalin), dermatostatin, [des-Ala20,Gln34]-dermal antimicrobial peptide, anionic antimicrobial peptides associated with surface cleaning substances, Apidaecin IA, Apidaecin IB, Acetyl-Adhesin (1025-1044)amide, Theromacin (49-63), Percicanam (MSI-78), Indolicidin, Apelin-15 (63-77), CFP10 (71-85), inhibitors of anthrax-related lethal factors (LF), bovine antimicrobial peptides, 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), Humanin(HN)sHNG, [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), atebandin, aviptadin, R otigaptide (ZP123, GAP486), silengitide (EMD-121924, RGD peptide), A1buBNP, BN-054, angiotensin 11, MBP-8298, peptidylleucine arginine, ziconopeptide, AL-208, AL-108, carbeticon, coliven, ADNF-14, VIP (intestinal vascular cleansing peptide), thymosin, bacitracin, brevicin, 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, 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, ganirilin, 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), dynorphin A and cyclic peptides (e.g., romidepsin, voclosporin, ziconotide, linaclotide, plecanatide, pesireotide, lanreotide, vasopressin, terlipopressin). (Resistin), Bremlanotide, Setmelanotide, Daptomycin, Telavancin, Dalbavancin, Oritavancin, Caspofungin, Micafungin, Anidulafungin), Parathyroid hormone-related protein (PTHrP), GLP-2, Stromal cell-derived factor-1 (SDF-1), Brain natriuretic peptide (BNP), Neuropeptide Y (NPY), Pyropeptide (PYY), and Glucose-dependent insulinotropic peptide (GIP); Preferably, the protein drug is selected from 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, insulin, erythropoietin, gonadotropin, parathyroid hormone), and cytokines (e.g., interleukin, interferon, colony-stimulating factor). Preferably, the nucleic acid drug is selected from antisense nucleic acids (e.g., Fomivirsen, Mipomersen, Eteplirsen, Nusinersen, Inotersen, Volanesoresn, Golodirsen), small interfering RNA (e.g., Onpattro, Givlaari), microRNA, small activating RNA, messenger RNA, and aptamers; Preferably, the small molecule drug is selected from antibiotics such as penicillin (e.g., penicillin, penicillin V), fluoroquinolones (e.g., ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, gemifloxacin), cephalosporins (e.g., cefixime, cefbufen, ceftoranil, cefdinir, cefpodoxime proxetil), aminoglycosides (e.g., isapamicin, amikacin, etimicin, netilimicin), macrolides (e.g., erythromycin, azithromycin, clarithromycin), tetracyclines (e.g., doxycycline, minocycline, tigecycline, omalicycline, eracycline), sulfonamides and sensitizers (e.g., trimethoprim, sulfadiazine ... Aminomethoxazole, sulfamethoxazole); urinary tract 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, mequinidine, promethazine, mizolastine, ebastine, loratadine, terfenadine, fexofenadine, desloratadine, cetirizine); antiviral drugs (e.g., oseltamivir, mabaloxavir, erteiravir, lenapavir, capeiravir, entecavir, nematamide / ritonavir, ribavirin, interferon, arbidol). Zanamivir, Peramivir, Amantadine, Acyclovir, Ganciclovir, Valacyclovir, Daclatasvir, Asunaprevir, Adefovir Dipivoxil, Lamivudine, Telbivudine, Azvudine; Antihypertensive drugs (e.g., Amlodipine, Felodipine, Cisnidipine, Nifedipine, Nifedipine, Enalapril, Fosinopril, Lisinopril, Perindopril, Midapril, Losartan, Valsartan, Irbesartan, Telmisartan, Olmesartan, Hydrochlorothiazide, Indapamide, Metoprolol, Bisoprolol, Atenolol, Labetolol, Propranolol, Prazosin, Terazosin, Doxazosin); Diabetes medications (e.g., Glibenclamide, Glycol ... Lecithin, glipizide, glimepiride, 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, umemetium bromide, glycopyrronium (glycopyrronium bromide), refenapyridine, roflumilast, erdosteine, carbocysteine);Antitumor drugs (e.g., methotrexate, pemetrexed, fluorouracil, 6-mercaptopurine, hydroxyurea, gemcitabine, cytarabine, doxorubicin, epirubicin, rubitine, irinotecan, topotecan, etoposide, taxanes, vinca alkaloids, eribulin, EGFR inhibitors (e.g., gefitinib, erlotinib, icotinib, afatinib, osimertinib), ALK inhibitors (e.g., crizotinib, alectinib, ceritinib, lorlatinib), MET inhibitors (e.g., cevotinib, carmatinib), RET inhibitors (e.g., pralatinib, LOX) O-292), NTRK inhibitors (e.g., larotrectinib, entrectinib), BRAF inhibitors (e.g., dabrafenib, vemurafenib, encorafenib), MEK inhibitors (e.g., trametinib, binimetinib), HER2 inhibitors (e.g., neratinib, tacardinib), CDK4 / 6 inhibitors (e.g., palbociclib, abeciclib), PARP inhibitors (e.g., olaparib, niraparib), anti-angiogenic multikinase inhibitors (e.g., nilotinib, apatinib, sunitinib, fruquintinib, pazopanib, abcitabine). Inhibitors include: imatinib, vandetanib, cabozantinib; mTOR inhibitors (e.g., everolimus); HDAC inhibitors (e.g., chidamide); ABL-BCR inhibitors (e.g., imatinib, dasatinib, nilotinib); PDGFR / C-KIT inhibitors (e.g., imatinib, nilotinib, avatinib); BTK inhibitors (e.g., ibrutinib, zanubrutinib, zanubrutinib); protease inhibitors (e.g., bortezomib, ixazomib); JAK inhibitors (e.g., ruxolitinib); selective nuclear transporter inhibitors (e.g., selinexor); and PI3K inhibitors. Inhibitors (e.g., apelisib), FGFR2 inhibitors (e.g., pemigatinib), IDH1 inhibitors (e.g., 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).

9. The prodrug or pharmaceutically acceptable salt thereof of any one of claims 1-8, wherein, The prodrug is represented by formula (I), in, P represents the drug; A1, A2, B, C, P, m, and n are as defined in any one of claims 1-8; C is linked to the amino group of P by an amide bond formed through its α-carboxyl group.

10. The prodrug or pharmaceutically acceptable salt thereof of any one of claims 1-9, wherein, said prodrug is of formula (I-1), (I-2), (I-3), (I-4) or (I-5), in, n1 is 1, 2, 3, 4 or 5; preferably, n is 1, 2 or 3; In equations (I-3) and (I-5), each A1 may be the same or different, and each A2 may be the same or different; In equation (I-4), each B may be the same or different, and each C may be the same or different; A1, A2, B, C, and P are as defined in any one of claims 1-9.

11. The prodrug or pharmaceutically acceptable salt thereof of any one of claims 1-8, wherein, The prodrug is represented by formula (II), in, A1, A2, B, C, P, m, and n are as defined in any one of claims 1-8; C is linked to the amino group of P through an amide bond formed by its α-carboxyl group; L stands for connector; D is selected from antibody, Fc fragment, C 10-30 Fatty acids, C 10-30 Fatty acid, polyethylene glycol, pCB and any combination thereof.

12. The prodrug of claim 11 or a pharmaceutically acceptable salt thereof, wherein, L is selected from a linker sequence, a bond, or NH, O, S, C (=O), C 1-6 Alkylenes and any combination thereof; Preferably, the adapter sequence is a flexible polypeptide sequence; more preferably, the adapter sequence comprises (G4S). q , where q is a positive integer greater than or equal to 1, for example, an integer from 1 to 8 (for example, 3 or 4); more preferably, the amino acid sequence of the linker sequence is as shown in SEQ ID NO:141; Preferably, L is selected from NH, O, S, C(=O), C 1-6 alkylene and any combination thereof; Preferably, L is selected from t is 1, 2, 3, 4, or 5; Preferably, L is selected from Preferably, -OH, -SH, or -NH2 in P is replaced by L, or -N3 in P is replaced by L.

13. The prodrug or a pharmaceutically acceptable salt thereof as claimed in claim 11 or 12, wherein, D is selected from H, Fc domain or variants thereof, albumin, antibody or antibody fragment (e.g., IgG, IgM, IgA, IgD, IgE, VHH), C 14-24 Fatty acids (e.g., C) 14 Fatty acids, C 16 Fatty acids, C 18 Fatty acids, C 20 Fatty acids, C 22 fatty acids), C 14-24 Fatty acids (e.g., C40) 14 Fatty acid, C 16 Fatty acid, C 18 Fatty acid, C 20 Fatty acid, C 22 Fatty acid), polyethylene glycol, pCB and any combination thereof; Preferably, the Fc domain is an Fc domain derived from an IgG antibody, and the variant of the Fc domain has mutations of F234A / L235A, M252Y / S254T / T256E, M428L / N434S, T307A / E380A / N434A, M428L / N434S, V308P, H285D / T307Q / A378V, or L309D / Q311H / N434S compared to the wild-type Fc domain of the IgG antibody; more preferably, the amino acid sequence of the Fc domain is as shown in any one of SEQ ID NO:138-140; Preferably, D is selected from H, antibodies (e.g., IgG, IgM, IgA, IgD, IgE, VHH), and C. 14-24 Fatty acids (e.g., C) 14 Fatty acids, C 16 Fatty acids, C 18 Fatty acids, C 20 Fatty acids, C 22 fatty acids), C 14-24 Fatty acids (e.g., C40) 14 Fatty acid, C 16 Fatty acid, C 18 Fatty acid, C 20 Fatty acid, C 22 Fatty acid), polyethylene glycol, pCB and any combination thereof; Preferably, D is selected from -(D1) p -(D2) q -D3, wherein, D1 is -C(=O)CH2(OCH2CH2)2NH-, D2 is selected from Glu and Asp. D3 is selected from C 14-24 fatty diacids, p is an integer between 0 and 24. q is 1 or 2, D1, D2 and D3 are connected by amide bonds, and the C (=O) end of D1 is connected to the L phase; Preferably, p is an integer between 0 and 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Preferably, D2 is The C (=O) terminal is connected to the -NH- phase of D1; Preferably, D3 is selected from -C(O)(CH2). 14 COOH, -C(O)(CH2) 15 COOH, -C(O)(CH2) 16 COOH, -C(O)(CH2) 17 COOH, -C(O)(CH2) 18 COOH, -C(O)(CH2) 19 COOH and -C(O)(CH2) 20 COOH; Preferably, D is selected from:

14. The prodrug or pharmaceutically acceptable salt thereof of any one of claims 11-13, wherein, The prodrug is selected from the group consisting of a structure represented by Formula (II-1), Formula (II-2), Formula (II-3), or Formula (II-4): in, n1 is 1, 2, 3, 4 or 5; preferably, n is 1, 2 or 3; In equations (II-3) and (II-5), each A1 may be the same or different, and each A2 may be the same or different; In equation (II-4), each B may be the same or different, and each C may be the same or different; A1, A2, B, C, P, L and D are as defined in any one of claims 1-13; Preferably, Selected from sequences such as SEQ ID NO:119 or SEQ ID NO:

136.

15. The prodrug or pharmaceutically acceptable salt thereof of any one of claims 1-14, wherein, The prodrug 1) As shown in any one of SEQ ID NOs: 21, 25-26, 30-37, 48-55, 58-106, 108-118; or 2) As shown in any one of SEQ ID NOs:15-20, 27-29, SEQ ID NOs:120-135 and 137; or 3) As shown in any one of SEQ ID NOs: 2-14, 38-39, 56-57; or 4) As shown in any one of SEQ ID NOs:42-43; or 5) As shown in any one of SEQ ID NOs:44-47.

16. A prodrug or a pharmaceutically acceptable salt thereof, wherein, The prodrug comprises a structure represented by Formula (F-2), in, A1, A2, B, C, m, and n are defined as in any one of claims 1-6, and L and D are defined as in any one of claims 11-13; The -OH, -SH, or -NH2 in A1, A2, B, or C is replaced by LD, or the -N3 in A1, A2, B, or C is replaced by LD.

17. The prodrug of claim 16, or a pharmaceutically acceptable salt thereof, wherein, the structure represented by formula (F-2) is selected from the group consisting of a structure represented by formula (F-2-1), in, A1, A2, B, C, m, n, L, and D are as defined in claim 16; Preferably, dipeptides A1-A2 are each independently selected from Lys-Pro, Met-Pro, Phe-Pro, Pro-Pro, Ser-Pro, Thr-Pro, Trp-Pro, Tyr-Pro, Val-Pro, Ala-Pro, Arg-Pro, Asn-Pro, Asp-Pro, Gln-Pro, Glu-Pro, Gly-Pro, His-Pro, Ile-Pro, Leu-Pro, Cys-Pro, and Nle-Pro. , Nva-Pro, Gly-Ala, His-Ala, Lys-Ala, Met-Ala, Phe-Ala, Pro-Ala, Ser-Ala, Thr-Ala, Trp-Ala, Tyr-Ala, Val -Ala, Ala-Ala, Arg-Ala, Asn-Ala, Asp-Ala, Gln-Ala, Glu-Ala, Ile-Ala, Leu-Ala, Cys-Ala, Nle-Ala and Nva-Ala; Preferably, As As shown, in, R1, R3, R4, R5, and R6 are as defined in claim 5. R7is selected from C 1-4 alkylene-L-D, C 1-4 alkylene-oxygen(O)-L-D, C 1-4 alkylene-nitrogen(NH)-L-D, and C 1-4 alkylene-sulfur(S)-L-D, L and D are as defined in any of claims 11-13; Preferably, R7 is selected from:

18. The prodrug of claim 16 or 17, or a pharmaceutically acceptable salt thereof, wherein, The prodrug further includes drug P, as defined in claim 8.

19. The prodrug or pharmaceutically acceptable salt thereof of any one of claims 16-18, wherein, The prodrug is represented by formula (III), in, A1, A2, B, C, P, m, and n are defined as in any one of claims 1-8, and L and D are defined as in any one of claims 11-13; C is linked to the amino group of P through an amide bond formed by its α-carboxyl group; The -OH, -SH, or -NH2 in A1, A2, B, or C is replaced by LD, or the -N3 in A1, A2, B, or C is replaced by LD.

20. The prodrug of claim 19, or a pharmaceutically acceptable salt thereof, wherein, said prodrug is selected from the group consisting of structures represented by formula (III-1): in, A1, A2, B, C, P, m, n, L, and D are as defined in claim 19; Preferably, the prodrug is selected from the group consisting of formula (III-1-1) or formula (III-1-2): in, In equation (III-1-2), each A1 may be the same or different, and each A2 may be the same or different; A1, A2, B, C, P, L and D are as defined in claim 19.

21. The prodrug or pharmaceutically acceptable salt thereof of any one of claims 16-20, wherein, The prodrug: 1) As shown in SEQ ID NO: 22; or 2) As shown in SEQ ID NO: 23 or 24.

22. A polypeptide drug molecule, wherein, The sequence of the polypeptide drug molecule from N-terminus to C-terminus is shown as formula (I-5): in, A1-A2 is a dipeptide sequence that can be recognized and cleaved by the DPP4 enzyme; n1 represents the number of A1-A2, where n1 is an integer from 1 to 10; P represents a polypeptide molecule, which requires an intact N-terminus to be active, and the fusion of the dipeptide sequence at the N-terminus will cause the polypeptide molecule to be completely or partially inactivated.

23. The polypeptide drug molecule of claim 22, wherein, The second position of the dipeptide sequence is an amino acid residue selected from the group consisting of: proline, alanine, hydroxyproline, dehydroproline, glycine, threonine, valine, leucine, and serine. Preferably, the dipeptide sequence is selected from the group consisting of: HA, YA, YG, YP, HS, SP, GP, and RP; Preferably, the dipeptide sequence is HA, YA, or YG; Preferably, n1 is an integer from 1 to 5; more preferably, n1 is 1, 2 or 3; preferably, when n1 ≥ 2, the dipeptide sequences are the same or different. Preferably, the polypeptide molecule is a polypeptide molecule obtained by biologically or chemically modifying the original polypeptide sequence, wherein the original polypeptide sequence includes the amino acid sequence of the ligand molecule of GPCR; more preferably, the original polypeptide sequence includes one or more combinations of amino acid sequences selected from the group consisting of: glucagon-like peptide-1 (GLP-1), parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), GLP-2, stromal cell-derived factor-1 (SDF-1), brain natriuretic peptide (BNP), neuropeptide Y (NPY), tyrosine peptide (PYY), and glucose-dependent insulinotropic peptide (GIP). Preferably, the biomodification involves fusing the original polypeptide sequence with a functional fragment to extend the half-life of the original polypeptide sequence; more preferably, the biomodification involves fusing the functional fragment to the C-terminus of the original polypeptide sequence; even more preferably, the biomodification involves linking the functional fragment to the C-terminus of the original polypeptide sequence via a linker sequence. Preferably, the functional fragment is selected from the group consisting of: an Fc domain or a variant thereof, albumin, an antibody or antibody fragment (preferably VHH), and combinations thereof; preferably, the Fc domain is an Fc domain derived from an IgG antibody (e.g., IgG1, IgG2, IgG3, or IgG4), and the variant of the Fc domain has mutations of F234A / L235A, M252Y / S254T / T256E, M428L / N434S, T307A / E380A / N434A, M428L / N434S, V308P, H285D / T307Q / A378V, or L309D / Q311H / N434S compared to the wild-type Fc domain of an IgG-derived antibody; more preferably, the amino acid sequence of the Fc domain is as shown in any of SEQ ID NO:138-140; Preferably, the linker sequence is a flexible polypeptide sequence; preferably, the linker sequence comprises (G4S) q wherein q is a positive integer greater than or equal to 1, for example an integer from 1 to 8 (preferably 3 or 4); more preferably, the amino acid sequence of the linker sequence is set forth in SEQ ID NO: 141 ; Preferably, the chemical modification is selected from: fatty acid chain modification, polyethylene glycol modification, and combinations thereof; more preferably, the chemical modification includes introducing a C-16, C-18 or C-20 fatty acid chain into the amino acid side chain of the therapeutic peptide, or polyethylene glycol modification. Preferably, the polypeptide molecule is a GLP-1-Fc fusion protein or a PTH-Fc fusion protein, the dipeptide sequence is HA, and two or three HA sequences are fused to the N-terminus of the polypeptide molecule; preferably, the amino acid sequence of the GLP-1-Fc fusion protein is as shown in SEQ ID NO:119, and the amino acid sequence of the PTH-Fc fusion protein is as shown in SEQ ID NO:

136.

24. An intermediate for the prodrug or pharmaceutically acceptable salt thereof of any one of claims 1-23, wherein, The intermediate is represented by formula (M-1) Wherein, A1, A2, B, C, m and n are as defined in any one of claims 1-23.

25. Use of the intermediate of claim 24 in the preparation of the prodrug of any one of claims 1-23 or a pharmaceutically acceptable salt thereof.

26. An intermediate for the prodrug or pharmaceutically acceptable salt thereof of claim 24 or 25, wherein, The intermediate is represented by formula (M-2) Wherein, A1, A2, B, C, m and n are defined as in any one of claims 1-6, and L and D are defined as in any one of claims 11-13.

27. Use of the intermediate according to any one of claims 24-26 in the preparation of the prodrug according to any one of claims 1-23 or a pharmaceutically acceptable salt thereof.

28. A pharmaceutical composition comprising at least one prodrug as described in any one of claims 1-23 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers and / or excipients.

29. The use of the prodrug of any one of claims 1-23 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 28, in the preparation of a medicament for treating and / or preventing a disease or condition or reducing the severity of said disease or condition; Preferably, the disease or condition is selected from hypoparathyroidism, osteoporosis, osteopenia, diabetes, diabetic nephropathy, chronic kidney disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cardiovascular diseases (e.g., atherosclerosis, hypertension, heart failure), neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease), mental disorders (e.g., schizophrenia, depression), immune system diseases (e.g., autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus), allergic diseases (e.g., allergic rhinitis, asthma, urticaria), infectious diseases (e.g., influenza, pneumonia, tuberculosis)), pain and cancer (e.g., breast cancer, lung cancer, stomach cancer, colorectal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, melanoma), obesity, hypoglycemia, bone metabolic diseases, hypoparathyroidism, hypocalcemia, Crohn's disease, neurodegenerative diseases, or anorexia nervosa and any combination thereof.

30. Use of the prodrug or a pharmaceutically acceptable salt thereof as described in any one of claims 1-23, or the pharmaceutical composition of claim 28, in the preparation of a sustained-release polypeptide drug.

31. An isolated nucleic acid molecule, wherein, The nucleic acid molecules include: (a) a polynucleotide molecule encoding a polypeptide drug molecule; and / or, (b) Its complementary sequence.

32. A nucleic acid construct comprising, in 5' to 3' order: Contains the polynucleotide molecule of claim 31 or its complementary sequence; Preferably, the nucleic acid construct is an expression cassette and further includes, for example, a promoter and a transcription termination sequence.

33. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 31 or the nucleic acid construct as described in claim 32; Preferably, the recombinant vector is a recombinant cloning vector or a recombinant expression vector.

34. A host cell, characterized in that, The host cell contains the nucleic acid molecule as described in claim 31, the nucleic acid construct as described in claim 32, or the recombinant vector as described in claim 33, and / or expresses the polypeptide drug molecule as described in claim 1; Preferably, the host cell is selected from Escherichia coli cells, insect cells, yeast cells, and mammalian cells; Preferably, the host cell is an Escherichia coli cell, such as BL21(DE3).

35. A method of enhancing the ability of a drug to be released, comprising, The method comprises: fusing at least one fragment represented by formula (F-1): in, A1, A2, B, C, m, and n have the definitions described in any one of claims 1-23; and When the drug is fused with the fragment, it will cause complete or partial inactivation.

36. A method of enhancing the ability of a drug to be released, comprising, The method comprises fusing at least one fragment represented by formula (F-2) at the N-terminus of the polypeptide molecule: in, A1, A2, B, C, m, n, L, and D are as defined in claim 16; and When the drug is fused with the fragment, it will cause complete or partial inactivation.

37. The method of claim 35 or 36, wherein, The drug is selected from the drugs described in claim 8; Preferably, the drug is a polypeptide drug; More preferably, the polypeptide drug is a PTH polypeptide analog or a GLP-1 polypeptide analog.

38. A method of enhancing the slow release ability of a polypeptide drug, comprising, The method includes fusing at least one dipeptide sequence to the N-terminus of a polypeptide molecule, the dipeptide sequence being cleaved by the DPP4 enzyme; wherein the polypeptide molecule requires an intact N-terminus to be active, and the fusing of the dipeptide sequence to the N-terminus causes complete or partial inactivation of the polypeptide molecule.

39. The method of claim 38, wherein, The second position of the dipeptide sequence is an amino acid residue selected from the group consisting of: proline, alanine, hydroxyproline, dehydroproline, glycine, threonine, valine, leucine, and serine. Preferably, the dipeptide sequence is selected from the group consisting of: HA, YA, YG, YP, HS, SP, GP, and RP; Preferably, the dipeptide sequence is HA, YA, or YG; Preferably, n dipeptide sequences are fused to the N-terminus of the polypeptide molecule, where n is an integer from 1 to 10; more preferably, n is an integer from 1 to 5; even more preferably, n is 1, 2, or 3; preferably, when n ≥ 2, the dipeptide sequences are the same or different. Preferably, the polypeptide molecule is a polypeptide molecule obtained by biologically or chemically modifying the original polypeptide sequence, wherein the original polypeptide sequence includes the amino acid sequence of the ligand molecule of GPCR; more preferably, the original polypeptide sequence includes one or more combinations of amino acid sequences selected from the group consisting of: glucagon-like peptide-1 (GLP-1), parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), GLP-2, stromal cell-derived factor-1 (SDF-1), brain natriuretic peptide (BNP), neuropeptide Y (NPY), tyrosine peptide (PYY), and glucose-dependent insulinotropic peptide (GIP). Preferably, the biomodification involves fusing the original polypeptide sequence with a functional fragment to extend the half-life of the original polypeptide sequence; more preferably, the biomodification involves fusing the functional fragment to the C-terminus of the original polypeptide sequence; even more preferably, the biomodification involves linking the functional fragment to the C-terminus of the original polypeptide sequence via a linker sequence. Preferably, the functional fragment is selected from the group consisting of: an Fc domain or a variant thereof, albumin, an antibody or antibody fragment (preferably VHH), and combinations thereof; preferably, the Fc domain is an Fc domain derived from an IgG antibody (e.g., IgG1, IgG2, IgG3, or IgG4), and the variant of the Fc domain has mutations of F234A / L235A, M252Y / S254T / T256E, M428L / N434S, T307A / E380A / N434A, M428L / N434S, V308P, H285D / T307Q / A378V, or L309D / Q311H / N434S compared to the wild-type Fc domain of an IgG-derived antibody; more preferably, the amino acid sequence of the Fc domain is as shown in any of SEQ ID NO:138-140; Preferably, the linker sequence is a flexible polypeptide sequence; preferably, the linker sequence comprises (G4S) q wherein q is a positive integer greater than or equal to 1, for example an integer from 1 to 8 (preferably 3 or 4); more preferably, the amino acid sequence of the linker sequence is set forth in SEQ ID NO: 141 ; Preferably, the chemical modification is selected from: fatty acid chain modification, polyethylene glycol modification, and combinations thereof; more preferably, the chemical modification includes introducing a C-16, C-18 or C-20 fatty acid chain into the amino acid side chain of the therapeutic peptide, or polyethylene glycol modification. Preferably, the polypeptide molecule is a GLP-1-Fc fusion protein or a PTH-Fc fusion protein, the dipeptide sequence is HA, and two or three HA sequences are fused to the N-terminus of the polypeptide molecule; preferably, the amino acid sequence of the GLP-1-Fc fusion protein is as shown in SEQ ID NO:119, and the amino acid sequence of the PTH-Fc fusion protein is as shown in SEQ ID NO:

136. Preferably, the amino acid sequence of the polypeptide molecule after fusing at least one dipeptide sequence to the N-terminus is as shown in any one of SEQ ID NO:120-135 and 137.

40. The method of any one of claims 35 to 39, wherein, The method further includes the following steps: selecting different dipeptide sequences to achieve different sustained-release effects based on in vitro enzymatic digestion rates and / or in vivo sustained-release results.

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