Polypeptide-conjugated drug, method for preparing same, and use thereof

Abstract

Provided are a polypeptide-conjugated drug, a method for preparing same, and use thereof. Specifically, the present invention relates to a polypeptide-conjugated drug as represented by general formula (I) and use thereof, wherein P comprises a polypeptide of formula (II), L is selected from a non-cleavable linker or a cleavable linker, and Drug is an antitumor agent. The polypeptide-conjugated drug can specifically bind to GRPr with high affinity and thereby efficiently kill tumor cells, thus providing a targeted antitumor therapy for the field of tumor treatment. P-L-Drug (I) Ser-Tyr-Gln-X1-Ala-X2-βAla-X3-X4-Nle (II)

Description

A polypeptide-coupled drug and its preparation method and use Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a polypeptide-coupled drug, a preparation method thereof, and use of the polypeptide-coupled drug in drugs for treating tumors or other diseases. Background Art

[0002] Cancer, also known as malignant tumors, is a leading cause of death and is characterized by the uncontrolled growth and spread of abnormal cells within the body. Cancer cells can invade nearby tissues and organs and spread through the bloodstream or lymphatic system to other parts of the body, forming new tumors in the process. There are many types of cancer, each with different effects on the body, but all ultimately cause damage to human health. Treatment options for cancer include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy.

[0003] Pancreatic cancer (PC) is one of the most common malignant tumors, characterized by high malignancy, insidious onset, and poor prognosis. Currently, surgery is the only curative treatment. However, due to the lack of specific early symptoms and the short course and rapid progression of the cancer, surgery alone cannot achieve the desired results. Another major clinical treatment for pancreatic cancer is chemotherapy, with some patients receiving combined radiotherapy and chemotherapy. However, the majority of pancreatic cancer cases are either insensitive to chemotherapy or develop drug resistance during treatment, resulting in a low 5-year survival rate.

[0004] Peptide-drug conjugates (PDCs) are an emerging targeted therapy that can improve tumor penetration and selectivity. PDCs are primarily composed of three parts: a peptide, a linker, and a cytotoxic payload.

[0005] The gastrin-releasing peptide receptor (GRPr) is a member of the bombesin G protein-coupled receptor family. Recent studies have shown that abnormal GRPr expression is associated with a variety of cancers, including prostate cancer, kidney cancer, lung cancer, pancreatic cancer, breast cancer, and colorectal cancer, suggesting that GRPr may serve as a cancer biomarker.

[0006] PDCs (polysaccharide-derived cytokines) (PDCs) are a research hotspot garnering widespread attention in tumor treatment, as exemplified by the PDC molecules and their applications disclosed in Chinese patents CN114728089A and CN101965358B. However, their application in pancreatic cancer (PC) treatment is currently limited. A novel delivery system of RGD-HSA-GEM / CURNPs (RGD-HSA-loaded GEM / CUR Nanoparticles for the Treatment of Pancreatic Cancer Therapy) has been reported, demonstrating excellent water solubility and tumor targeting. These molecules inhibit PC growth primarily by inhibiting DNA synthesis.

[0007] PDCs have high selectivity and specificity, and have good therapeutic effects. As a new type of targeted drug delivery system, it is gradually entering the stage of targeted therapy for various cancers and is expected to provide new hope for the clinical treatment of PC patients. Summary of the Invention

[0008] The present invention aims to provide a peptide-drug conjugate, its preparation method, and use. The peptide-drug conjugate exhibits high selectivity, strong specificity, low molecular weight, ease of availability, lack of immunogenicity, and safety and reliability. The peptide-drug conjugate can significantly enhance the therapeutic efficacy of tumor drugs.

[0009] The first aspect of the present invention is to provide a polypeptide-conjugated drug represented by general formula (I) or a pharmaceutically acceptable salt thereof.

[0010] PL-Drug

[0011] (I)

[0012] in,

[0013] The P comprises a polypeptide represented by formula (II)

[0014] Ser-Tyr-Gln-X 1 -Ala-X 2 -βAla-X 3 -X 4 -Nle

[0015] (II)

[0016] X 1 Selected from Ala, Trp, Tyr, Phe or derivatives of said amino acids;

[0017] X 2 Selected from Ala, Leu, Val, Ile or derivatives of said amino acids;

[0018] X3 Selected from His, Lys, Arg or derivatives of said amino acids;

[0019] X 4 Selected from Ala, Leu, Phe, Val, Trp, Tyr or derivatives of said amino acids;

[0020] The configuration of each amino acid in the sequence represented by the general formula (II) is independently selected from D-type or L-type;

[0021] L is selected from a non-cleavable linker or a cleavable linker;

[0022] Drug is an antitumor agent.

[0023] In the polypeptide-coupled drug of the present invention, the N-terminus of P is connected to L.

[0024] Preferably, the amino acid derivative is selected from Cha (3-cyclohexylalanine), Dap (diaminopropionic acid), 1-Nal (1-naphthylalanine), 2-Nal (2-naphthylalanine), Aib (2-aminoisobutyric acid), Abu (2-aminobutyric acid), Nva (norvaline), Dab (2,4-diaminobutyric acid), Har (homoarginine), Cit (guanidine), HomoLeu (homoleucine), (Nle norleucine), 4-Cl-Phe (4-chlorophenylalanine), 3-Cl-Phe (3-chlorophenylalanine), 4-Me-Phe (4-methylphenylalanine), 3-Me-Phe (3-methylphenylalanine).

[0025] Further preferably, the amino acid derivative is selected from Cha (3-cyclohexylalanine), 1-Nal (1-naphthylalanine), 2-Nal (2-naphthylalanine), Abu (2-aminobutyric acid), Nva (norvaline), 4-Cl-Phe (4-chlorophenylalanine), 3-Cl-Phe (3-chlorophenylalanine), 4-Me-Phe (4-methylphenylalanine), and 3-Me-Phe (3-methylphenylalanine).

[0026] In some specific embodiments, wherein in formula (II),

[0027] X 1 Selected from Ala, Trp, Tyr, Phe or derivatives of said amino acids;

[0028] X 2 Selected from Ala, Leu, Val or derivatives of said amino acids;

[0029] X 3 Selected from His or a derivative of said amino acid;

[0030] X 4 Selected from Ala, Leu, Phe, Val, Trp, Tyr or derivatives of said amino acids.

[0031] In some specific embodiments, the configurations of Ser and Tyr in formula (II) are each independently D-type.

[0032] In some specific embodiments, wherein said P comprises a polypeptide represented by formula (III)

[0033] DSer-DTyr-Gln-X 1 -Ala-X 2 -βAla-X 3 -X 4 -Nle

[0034] (III)

[0035] X 1 Selected from Ala, Trp;

[0036] X 2 Selected from Leu, Val;

[0037] X 3 Selected from His, DHis;

[0038] X 4 Selected from Cha, Leu, Phe, Nva;

[0039] In some specific embodiments, P comprises a polypeptide represented by formula (IV)

[0040] DSer-DTyr-Gln-Trp-Ala-X 2 -βAla-His-X 4 -Nle

[0041] (IV)

[0042] X 2 Selected from Leu, Val;

[0043] X 4 Selected from Cha, Leu, Phe, Nva;

[0044] In some specific embodiments, the C-terminus and / or N-terminus of the amino acid sequence of the polypeptide represented by general formula (I) to (IV) is modified or unmodified. Modifications include but are not limited to acetylation, carboxylation, alkylation, acylation, and carbamoylation.

[0045] In some specific embodiments, P is selected from any one of the following polypeptides:

[0046] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle(SEQ ID No.1)、

[0047] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Leu-Nle(SEQ ID No.2)、

[0048] DSer-DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle(SEQ ID No.3)、

[0049] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(Ethylamide)(SEQ ID No.4)、

[0050] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(Butylamide)(SEQ ID No.5)、

[0051] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(Methyl ester)(SEQ ID No.6)、

[0052] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(Ethyl ester)(SEQ ID No.7)、

[0053] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-D-His-Phe-Nle(SEQ ID No.8)、

[0054] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-D-Phe-Nle(SEQ ID No.9)、

[0055] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-D-Leu-Nle(SEQ ID No.10)、

[0056] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-4-Cl-Phe-Nle(SEQ ID No.11)、

[0057] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-4-Me-phe-Nle(SEQ ID No.12)、

[0058] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-3-Cl--Phe-Nle(SEQ ID No.13)、

[0059] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-D-His-D-Phe-Nle(SEQ ID No.14)、

[0060] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Nva-Nle(SEQ ID No.15)、

[0061] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Abu-Nle(SEQ ID No.16)、

[0062] DSer-DTrp-Gln-Trp-Ala-Val-βAla-His-Phe-Nle(SEQ ID No.17)、

[0063] DSer-DTyr-Gln-Tyr-Ala-Val-βAla-His-Phe-Nle(SEQ ID No.18)、

[0064] DSer-DTyr-Gln-Phe-Ala-Val-βAla-His-Phe-Nle(SEQ ID No.19)、

[0065] DSer-DTyr-Gln-1-Nal-Ala-Val-βAla-His-Phe-Nle(SEQ ID No.20)、

[0066] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(SEQ ID No.21)、

[0067] DSer-DTyr-Gln-Trp-Ala-Val-βAla-Lys-Phe-Nle(SEQ ID No.22)、

[0068] DSer-DTyr-Gln-Trp-Ala-Val-βAla-Arg-Phe-Nle(SEQ ID No.23)、

[0069] DSer-DTyr-Gln-Trp-Ala-Val-βAla-His-Trp-Nle (SEQ ID No. 24),

[0070] DSer-DTyr-Gln-Trp-Ala-Val-βAla-His-Tyr-Nle (SEQ ID No. 25),

[0071] DSer-DTyr-Gln-Trp-Ala-Val-βAla-His-1-Nal-Nle (SEQ ID No. 26).

[0072] In some specific embodiments, P is selected from any one of the following polypeptides:

[0073] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle (SEQ ID No. 1),

[0074] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Leu-Nle (SEQ ID No. 2),

[0075] DSer-DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle (SEQ ID No. 3).

[0076] In some specific embodiments, L comprises a non-cleavable linker or a cleavable linker, the non-cleavable linker is selected from a PEG linker, a linker with a thioether group, a linker with an oxime group, or a combination thereof; the cleavable linker is selected from a linker with a disulfide bond group, a dipeptide linker, a tripeptide linker, a tetrapeptide linker, a peptidomimetic linker, a linker cleaved by β-glucuronidase, a linker cleaved by β-galactosidase, a linker based on phosphatase cleavage, a pH-sensitive linker, a linker cleaved by sulfatase, or a combination thereof.

[0077] In some specific embodiments, the dipeptide linker is selected from a valine-citrulline (Val-Cit) dipeptide linker, a phenylalanine-lysine (Phe-Lys) dipeptide linker, and a valine-alanine (Val-Ala) dipeptide linker.

[0078] In some specific embodiments, the tripeptide linker is selected from a glutamic acid-valine-citrulline (Glu-Val-Cit) tripeptide linker and an alanine-valine-citrulline (Ala-Val-Cit) tripeptide linker.

[0079] In some specific embodiments, the tetrapeptide linker is selected from a glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) tetrapeptide linker and an aspartic acid-glutamic acid-valine-aspartic acid (Asp-Glu-Val-Asp) tetrapeptide linker.

[0080] In some specific embodiments, L is selected from a non-cleavable linker or a cleavable linker, the non-cleavable linker is selected from a PEG linker, a linker with a thioether group, a linker with an oxime group, or a combination thereof; the cleavable linker is selected from a linker with a disulfide bond group, a valine-citrulline (Val-Cit) dipeptide linker, a phenylalanine-lysine (Phe-Lys) dipeptide linker, a valine-alanine (Val-Ala) dipeptide linker, a β-glucuronidase-cleaved linker, a β-galactosidase-cleaved linker, a phosphatase-cleaved linker, a pH-sensitive linker, a sulfatase-cleaved linker, or a combination thereof.

[0081] In some specific embodiments, L comprises a non-cleavable linker or a cleavable linker, the non-cleavable linker is selected from a PEG linker; the cleavable linker is selected from a linker with a disulfide bond group, a valine-citrulline (Val-Cit) dipeptide linker, a phenylalanine-lysine (Phe-Lys) dipeptide linker, a valine-alanine (Val-Ala) dipeptide linker, a glutamic acid-valine-citrulline (Glu-Val-Cit) tripeptide linker, and a glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) tetrapeptide linker.

[0082] In some specific embodiments, wherein L comprises the following structure or a combination thereof,

[0083] The m is independently selected from integers of 1-24.

[0084] Preferably, m is independently selected from integers of 1-8.

[0085] Preferably, m is independently selected from integers of 1-4.

[0086] In some specific embodiments, the L structure further comprises a PAB structure, wherein the PAB structure is

[0087] In some specific embodiments, the L structure further comprises a PABC spacer, wherein the PABC spacer structure is In the structure, the carbonyl group is connected to the Drug portion.

[0088] In some specific embodiments, the L structure further comprises a β-Ala spacer.

[0089] In some specific embodiments, the L structure further comprises a [Sar]n spacer, where n is an integer selected from 1 to 15.

[0090] In some specific embodiments, the L structure further comprises a [Sar]n spacer, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0091] In some specific embodiments, the L structure further comprises a β-Ala-[Sar]n spacer, where n is an integer selected from 1 to 15, and the carboxyl group in the [Sar]n is connected to the P portion via an amide bond.

[0092] In some specific embodiments, the L structure further comprises a β-Ala-[Sar]n spacer, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0093] In some specific embodiments, wherein L is selected from the following structures or combinations thereof, The m is independently selected from integers of 1-24.

[0094] Preferably, m is independently selected from integers of 1-8.

[0095] Preferably, m is independently selected from integers of 1-4.

[0096] In some specific embodiments, L is selected from the following structures or combinations thereof,

[0097] m is independently selected from integers of 1-4.

[0098] In some specific embodiments, L is selected from the following structures or combinations thereof, m is independently selected from integers of 1-4.

[0099] In some specific embodiments, L is

[0100] In some specific embodiments, L is selected from the following structures or combinations thereof, m is independently selected from integers of 1-4.

[0101] In some specific embodiments, L is

[0102] In some specific embodiments, L is selected from the following structures or combinations thereof, m is independently selected from integers of 1-4.

[0103] In some specific embodiments, L is

[0104] In some specific embodiments, L is

[0105] In some specific embodiments, the drug is selected from microtubule-damaging drugs and DNA-damaging drugs.

[0106] In some specific embodiments, the drug is selected from dolastatin and its auristatins derivatives (MMAE, MMAF, MMAD), maytansine and maytansinoids (DM1, DM2, DM3, DM4), Tubulysins, cryptocolistin, spindle kinesin, gemcitabine, pyrrolo[2,1-c][1,4]benzodiazepine, dukamycin, camptothecin and camptothecin derivatives (irinotecan, topotecan), calicheamicin, amatoxin, paclitaxel, vinblastine, vincristine, etoposide, doxorubicin, cyclophosphamide, docetaxel, methotrexate, cisplatin, cytarabine, phenylalanine mustard and chlorambucil, or a combination thereof.

[0107] Preferably, the drug is selected from MMAE, MMAF, PTX, gemcitabine, and Dxd (Deruxtecan).

[0108] Further preferably, the drug is selected from MMAE, gemcitabine, and Dxd.

[0109] The polypeptide-coupled drug of the present invention is selected from the following structures:

[0110] In another aspect, the present invention provides a use of the above-mentioned polypeptide-coupled drug in the preparation of a cancer targeted therapeutic drug.

[0111] In some specific embodiments, the application is the use of a polypeptide-coupled drug in the preparation of a GRPR-positive cancer targeted therapeutic drug.

[0112] Furthermore, the GRPR-positive cancer is selected from at least one of prostate cancer, breast cancer, colon cancer, pancreatic cancer, renal cell carcinoma, small cell lung cancer, head and neck cancer, ovarian cancer, and uterine cancer. More preferably, the GRPR-positive cancer is pancreatic cancer.

[0113] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure (e.g., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group) as naturally occurring amino acids, for example, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds whose structure is different from the general amino acid chemical structure, but that function similarly to naturally occurring amino acids.

[0114] The amino acid sequences of the present invention contain the standard one-letter or three-letter codes for the twenty naturally occurring amino acids.

[0115] When the listed linking groups do not specify their linking direction, their linking direction is arbitrary.

[0116] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 is a liquid phase detection diagram of compound 1;

[0118] Figure 2 is a mass spectrometry diagram of compound 1;

[0119] Figure 3 is a liquid phase detection diagram of compound 2;

[0120] Figure 4 is a mass spectrometry diagram of compound 2;

[0121] Figure 5 is a liquid phase detection diagram of compound 3;

[0122] Figure 6 is a mass spectrometry diagram of compound 3;

[0123] Figure 7 is a liquid phase detection diagram of compound 4;

[0124] Figure 8 is a mass spectrometry diagram of compound 4;

[0125] Figure 9 is a liquid phase detection diagram of compound 5;

[0126] Figure 10 is a mass spectrometry diagram of compound 5;

[0127] Figure 11 is a liquid phase detection diagram of compound 6;

[0128] Figure 12 is a mass spectrometry diagram of compound 6;

[0129] Figure 13 is a liquid phase detection diagram of compound 7;

[0130] Figure 14 is a mass spectrum detection diagram of compound 7;

[0131] Figure 15 is a liquid phase detection diagram of compound 8;

[0132] Figure 16 is a mass spectrometry diagram of compound 8;

[0133] Figure 17 is a liquid phase detection diagram of compound 9;

[0134] Figure 18 is a mass spectrometry diagram of compound 9;

[0135] Figure 19 is a liquid phase detection diagram of compound 10;

[0136] Figure 20 is a mass spectrum detection diagram of compound 10;

[0137] Figure 21 is a liquid phase detection diagram of compound 12;

[0138] Figure 22 is a mass spectrum detection diagram of compound 12;

[0139] Figure 23 shows the killing effect of the small molecule compound of Example 12 on different cells IC 50 Value statistics chart;

[0140] Figure 24 is an image of GRPR receptor endocytosis induced by the positive polypeptide of the present invention in Example 13;

[0141] Figure 25 is an image showing GRPR receptor endocytosis induced by some compounds of the present invention (Compounds 1 to 7) in Example 13;

[0142] Figure 26 is an image showing GRPR receptor endocytosis induced by some compounds of the present invention (Compound 8, Compound 9, Compound 10, Compound 12) in Example 13;

[0143] Figure 27 shows the killing effect IC of the peptide-drug conjugate of the present invention on different cells in Example 14. 50 Statistical graph of values; Figure A shows the IC value of MMAE on different cell killing effects 50 Statistical results; Figure B shows the IC value of gemcitabine on different cell lines 50 Statistical results; Figure C shows the IC of Dxd's killing effect on different cells 50 Statistical results; Figure D shows the IC of compound 4 against different cells 50 Statistical results; Figures E to N respectively show the IC values ​​of compounds 1, 2, 3, 5, 6, 7, 8, 9, 10, and 12 on different cell lines. 50Statistical results;

[0144] Figure 28 shows the mouse plasma stability results of some compounds (Compounds 1 to 5) of the present invention in Example 15;

[0145] Figure 29 shows the mouse plasma stability results of some compounds of the present invention (Compound 8, Compound 9, Compound 10, Compound 12) in Example 15;

[0146] Figure 30 shows the human plasma stability results of some compounds (Compounds 4 to 7) of the present invention in Example 16;

[0147] Figure 31 shows the human plasma stability results of some compounds (Compounds 1 to 5) of the present invention in Example 16;

[0148] Figure 32 shows the human plasma stability results of some compounds of the present invention (Compound 8, Compound 9, Compound 10, Compound 12) according to Example 16;

[0149] FIG33 shows the tissue distribution results of Compound 4 of Example 17 in Hs766T pancreatic tumor-bearing mice;

[0150] FIG34 shows the tissue distribution results of MMAE released by Compound 4 in Example 17 in Hs766T tumor-bearing mice;

[0151] FIG35 shows the relative tumor growth rate of Example 18 Compound 4 and MMAE in the HPAF-II pancreatic tumor model. DETAILED DESCRIPTION

[0152] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0153] The polypeptide compounds and derivatives provided herein are synthesized using a solid-phase synthesis method to synthesize their linear precursors, using Rink Amide-AM Resin as a synthetic support. During the synthesis process, the Rink Amide-AM Resin is first fully swollen in N,N-dimethylformamide (DMF). The solid support is then subjected to repeated condensation with an activated amino acid derivative, followed by washing, Fmoc deprotection, washing, and the next round of amino acid condensation to achieve the desired polypeptide chain length. The polypeptide is then cleaved from the solid support by reacting with the resin using a mixed solution of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v). The crude solid product of the linear precursor is then precipitated with frozen methyl tert-butyl ether to obtain a solid product. The crude linear precursor after cleavage was purified and separated by a C-18 reverse phase preparative chromatography column using a 0.1% trifluoroacetic acid acetonitrile / water system to obtain a polypeptide with high purity. The polypeptide was coupled with an MMAE conjugate in the liquid phase, and after the reaction, it was purified and separated by a C-18 reverse phase preparative chromatography column using a 0.1% trifluoroacetic acid acetonitrile / water system to obtain pure polypeptides and their derivatives. Alternatively, during the synthesis process, Rink Amide-AM Resin is first fully swollen in N,N-dimethylformamide (DMF). The solid support is then subjected to repeated condensation with an activated amino acid derivative, followed by washing, Fmoc deprotection, washing, and the next round of amino acid condensation to achieve the desired polypeptide chain length. The gemcitabine conjugate is then coupled to the solid phase. The peptide is then cleaved from the solid support by reacting with a mixture of trifluoroacetic acid, water, triisopropylsilane, and thioanisole (90:2.5:2.5:5, v:v:v:v). The peptide is then precipitated with chilled methyl tert-butyl ether to yield a crude solid of the linear precursor. The cleaved crude linear precursor is then purified using a C-18 reverse-phase preparative chromatography column in acetonitrile / water with 0.1% trifluoroacetic acid to yield pure peptides and their derivatives.

[0154] Experimental reagents

[0155] Example 1. Preparation of Compound 1

[0156] Step 1: Synthesis of linear precursor peptide chain

[0157] Linear precursor peptide chain of compound 1:

[0158] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Leu-Nle.

[0159] 294 mg (0.2 mmol) of Rink Amide-AM Resin was fully swelled in DMF for 1 hour. The linear precursor sequence was then synthesized from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0160] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0161] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0162] Dissolve 1.0 mmol of Fmoc-AA, 1.0 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0163] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0164] After the linear peptide synthesis, the resin was washed with DMF five times.

[0165] Step 2: Cleavage of the linear precursor peptide chain

[0166] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0167] Step 3: Purification and preparation of pure peptide

[0168] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 20-50% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 75% were combined and lyophilized to obtain the pure peptide.

[0169] Step 4: Synthesis of MMAE conjugate 1

[0170] Step 4.1 Synthesis of compound 1-2

[0171] Step 4.1.1: Fully swell 840 mg (1 mmol) of 2-Chlorotrityl chloride resin in 15 mL of DCM for 1 h. Dissolve 3 mmol of Fmoc-Cit-OH and 6 mmol of diisopropylethylamine (DIEA) in 10 mL of DCM and add to the resin. Allow to react at room temperature for 2 h. After completion, add 10 mL of blocking solution (DCM:methanol:DIEA (85:10:5, v:v:v)) at room temperature for 10 min. Wash the blocked resin five times with DCM and five times with DMF.

[0172] Step 4.1.2: Add 10 mL of the deprotection solution (4-methylpiperidine:DMF) (20:80, v:v) to the resin obtained in step 4.1.1 and shake. Deprotection time is 25 minutes, divided into two steps: the first step is 5 minutes, and the second step is 20 minutes. After the reaction, wash with DMF 6-8 times.

[0173] Step 4.1.3: Weigh Fmoc-Val-OH (5 mmol), HATU (2.85 mmol), and DIPEA (6 mmol) and dissolve thoroughly in 10 mL of DMF. Add to the resin obtained in Step 4.1.2 and shake at room temperature for 2 h. Drain the solution and wash with DMF five times.

[0174] Step 4.1.4: Add 10 mL of the deprotection solution (4-methylpiperidine:DMF) (20:80, v:v) to the resin obtained in step 4.1.3 and shake the mixture. Deprotection time is 25 minutes, divided into two steps: the first step is 5 minutes, and the second step is 20 minutes. Rinse the resin with DMF 6-8 times until the pH is neutral to obtain compound 1-2.

[0175] Step 4.2 Synthesis of Compound 1-4

[0176] Step 4.2.1: Weigh monomethyl succinate (3 mmol), HATU (2.85 mmol), and DIPEA (6 mmol) and thoroughly dissolve them in 10 mL of DMF. Add the mixture to the resin obtained in Step 4.1.4 and shake at room temperature for 2 h. Drain the solution and wash the mixture five times with DMF and five times with DCM to yield compound 1-4.

[0177] Step 4.3 Synthesis of Compound 1-5

[0178] In step 4.3.1, add lysis buffer (15 mL) and hexafluoroisopropanol:DCM (20:80, v:v) to the resin obtained in step 4.2.1. Shake and react for 1 h. Repeat twice. After each reaction, filter and collect the filtrate. Concentrate the collected filtrate under reduced pressure using a rotary evaporator. After removing the solvent, dry under vacuum to obtain compound 1-5.

[0179] Step 4.4 Synthesis of Compound 1-6

[0180] Step 4.4.1: Dissolve compound 1-5 (1 eq) in a mixture of DCM (10 mL) and MeOH (5 mL). Add EEDQ (2 eq) and p-aminobenzyl alcohol (1.3 eq). Stir the mixture at room temperature under nitrogen for 16 hours. Monitor the reaction using LC-MS. After completion, concentrate the reaction mixture on a rotary evaporator to remove the solvent and obtain a crude solid.

[0181] Purification in step 4.4.2 was performed using flash column chromatography. The crude product from step 4.4.1 was dissolved in a small amount of DCM and loaded onto a 40 g flash silica gel column. The A / B solvents were DCM and MeOH, respectively, with a gradient of 0-15% MeOH over 30 min at a flow rate of 40 mL / min. After purification, the collected fractions were concentrated under reduced pressure on a rotary evaporator to remove the solvent, yielding Compound 1-6 as a pale yellow solid.

[0182] Step 4.5 Synthesis of Compound 1-7

[0183] Step 4.5.1: Dissolve compound 1-6 (1 eq) in DMF (10 mL). Slowly add DIPEA (5 eq) and di(p-nitrobenzene) carbonate (4 eq). Stir at room temperature under nitrogen for 1 hour. Monitor the reaction by LC-MS. After completion, prepare for purification.

[0184] Step 4.5.2 was purified by HPLC. The crude product obtained in step 4.5.1 was diluted according to the volume ratio of stock solution: acetonitrile: pure water = 1:1:0.1. The A / B phase solvents were pure water and pure acetonitrile, respectively. The chromatographic column was a BR-C18 (Saifen) reverse phase column, the gradient was set to 40%-70% acetonitrile in 40 min, and the flow rate was set to 15 mL / min. After purification, the target fractions were collected and lyophilized to obtain white solid compound 1-7.

[0185] Step 4.6 Synthesis of compound 1-9

[0186] Step 4.6.1: Dissolve compound 1-7 (1 eq) in DMF (1 g / 10 mL). Slowly add HOBt (1.3 eq), DIPEA (3 eq), and MMAE (0.9 eq). Stir at room temperature under nitrogen for 16 hours. Monitor the reaction by LC-MS. After completion, prepare for purification.

[0187] Purification in step 4.6.2 was performed by HPLC. The crude product obtained in step 4.6.1 was injected directly without dilution. The A / B solvents were pure water and pure acetonitrile, respectively. The chromatographic column was a BR-C18 (Saifen) reversed-phase column, with a gradient of 40% to 70% acetonitrile in 40 min at a flow rate of 15 mL / min. After purification, the target fractions were collected and lyophilized to obtain Compound 1-9 as a white solid.

[0188] Step 4.7 Synthesis of Compound 1-10

[0189] Step 4.7.1: Dissolve compound 1-9 (1 eq) in a mixture of THF (3 mL) and H₂O (3 mL). Slowly add LiOH.H₂O (5 eq). Stir at room temperature under nitrogen for 1 hour. Monitor the reaction by LC-MS. After completion, concentrate the THF using a rotary evaporator under reduced pressure and adjust the pH to 7 using glacial acetic acid.

[0190] Step 4.7.2 was purified by HPLC. The crude product obtained in step 4.7.1 was dissolved in pure water. The A / B solvents were pure water and pure acetonitrile, respectively. The product was first rinsed with 10% acetonitrile for 40 minutes, and then purified with 100% acetonitrile. The chromatographic column was a BR-C18 (Saifen) reverse-phase column at a flow rate of 15 mL / min. After purification, the target fractions were collected and lyophilized to obtain Compound 1-10 as a white solid.

[0191] Step 4.8 Synthesis of Compound 1-12

[0192] Step 4.8.1: Dissolve compound 1-10 (1 eq) in a mixture of DMF (6 mL) and DCM (2 mL). Slowly add EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (3 eq) and NHS (N-hydroxysuccinimide) (3 eq). Stir at room temperature under nitrogen for 3 hours. Monitor the reaction by LC-MS. After completion, concentrate the DCM on a rotary evaporator under reduced pressure for purification.

[0193] Step 4.8.2 was purified by HPLC. The crude product obtained in step 4.8.1 was diluted to a volume ratio of 1:0.5:0.5 stock solution:acetonitrile:purified water, with the A / B solvents being purified water and acetonitrile, respectively. The chromatographic column was a BR-C18 (Saifen) reversed-phase column with a gradient of 30-80% acetonitrile in 40 min at a flow rate of 15 mL / min. After purification, the target fractions were collected and lyophilized to obtain Compound 1-12 (i.e., MMAE conjugate 1) as a white solid.

[0194] Step 5: Peptide coupling to MMAE conjugate 1

[0195] Dissolve 40 mg of the purified peptide obtained in Step 3 in 4 mL of DMF. Slowly add 3 eq of DIPEA and 1 eq of the MMAE conjugate 1 obtained in Step 4. Stir at room temperature under nitrogen for 16 hours. Monitor the reaction by LC-MS. Purification is then performed immediately after completion of the reaction.

[0196] Step 6: Preparation and purification of target product

[0197] The crude product obtained in step 5 was diluted to a volume ratio of 1:1:1 stock solution: acetonitrile: purified water. After filtering through a 0.45 μm membrane, it was separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 5-90% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions with >95% purity were combined and lyophilized to obtain the pure desired product.

[0198] Step 7: Detection and Characterization Methods

[0199] The purity of the target product of step 6 and the N-terminal MMAE conjugate 1 of the polypeptide compound were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry. The detection results are shown in Figures 1 and 2.

[0200] Example 2. Preparation of Compound 2

[0201] Step 1: Synthesis of linear precursor peptide chain

[0202] The linear precursor peptide chain of compound 2:

[0203] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Leu-Nle.

[0204] The steps for synthesizing the linear precursor peptide chain are the same as step 1 in Example 1.

[0205] Step 2: Cleavage of the linear precursor peptide chain

[0206] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0207] Step 3: Purification and preparation of pure peptide

[0208] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 20-50% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 75% were combined and lyophilized to obtain the pure peptide.

[0209] Step 4: Synthesis of MMAE conjugate 2

[0210] Step 4.1 Synthesis of compound 2-2

[0211] Step 4.1.1: Fully swell 840 mg (1 mmol) of 2-Chlorotrityl chloride resin in 15 mL of DCM for 1 h. Weigh 3 mmol of Fmoc-AEEA-OH and 4 mmol of diisopropylethylamine (DIEA) in 10 mL of DCM and add to the resin. Allow to react at room temperature for 2 h. After completion, add 10 mL of blocking solution (DCM:methanol:DIEA (85:10:5, v:v:v)) at room temperature for 10 min. Wash the blocked resin five times with DCM and five times with DMF.

[0212] Step 4.1.2: Add 10 mL of the deprotection solution (4-methylpiperidine:DMF) (20:80, v:v) to the resin obtained in step 4.1.1 and shake the reaction. Deprotection time is 25 minutes, divided into two steps: the first step is 5 minutes, and the second step is 20 minutes. After the reaction, rinse the resin 6-8 times with DMF to a neutral pH to obtain compound 2-2.

[0213] Step 4.2 Synthesis of compound 2-4

[0214] Step 4.2.1: Weigh monomethyl succinate (3 mmol), HATU (2.85 mmol), and DIPEA (6 mmol) and thoroughly dissolve them in 10 mL of DMF. Add the mixture to the resin obtained in Step 4.1.4 and shake at room temperature for 2 h. Drain the solution and wash the mixture five times with DMF and five times with DCM to yield compound 2-4.

[0215] Step 4.3 Synthesis of Compound 2-5

[0216] In step 4.3.1, add lysis buffer (15 mL) and hexafluoroisopropanol:DCM (20:80, v:v) to the resin obtained in step 4.2.1. Shake and react for 1 h. Repeat twice. After each reaction, filter and collect the filtrate. Concentrate the collected filtrate under reduced pressure using a rotary evaporator. After removing the solvent, dry under vacuum to obtain compound 2-5.

[0217] Step 4.4 Synthesis of compound 2-7

[0218] Step 4.4.1: Dissolve compound 2-5 (1 eq) in DMF (1 g / 10 mL). Slowly add HOBt (1.3 eq), DIPEA (3 eq), and MMAE (0.9 eq). Stir at room temperature under nitrogen for 16 hours. Monitor the reaction by LC-MS. After completion, prepare for purification.

[0219] In step 4.4.2, purification was performed by HPLC. The crude product obtained in step 4.4.1 was injected directly without dilution. The A / B solvents were pure water and pure acetonitrile, respectively. The chromatographic column was a BR-C18 (Saifen) reversed-phase column, with a gradient of 40% to 70% acetonitrile in 40 min at a flow rate of 15 mL / min. After purification, the target fractions were collected and lyophilized to obtain compound 2-7 as a white solid.

[0220] Step 4.5 Synthesis of Compound 2-8

[0221] Step 4.5.1: Dissolve compound 2-7 (1 eq) in a mixture of THF (3 mL) and H₂O (3 mL). Slowly add LiOH.H₂O (5 eq). Stir at room temperature under nitrogen for 1 hour. Monitor the reaction by LC-MS. After completion, concentrate the THF using a rotary evaporator under reduced pressure and adjust the pH to 7 using glacial acetic acid.

[0222] Step 4.5.2 was purified by HPLC. The crude product obtained in step 4.5.1 was dissolved in pure water. The A / B solvents were pure water and pure acetonitrile, respectively. The product was first rinsed with 10% acetonitrile for 40 minutes, and then purified with 100% acetonitrile. The chromatographic column was a BR-C18 (Saifen) reverse-phase column at a flow rate of 15 mL / min. After purification, the target fractions were collected and lyophilized to obtain compound 2-8 as a white solid.

[0223] Step 4.6 Synthesis of compound 2-10

[0224] Step 4.6.1: Dissolve compound 2-8 (1 eq) in a mixture of DMF (6 mL) and DCM (2 mL). Slowly add EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (3 eq) and NHS (N-hydroxysuccinimide) (3 eq). Stir at room temperature under nitrogen for 3 hours. Monitor the reaction by LC-MS. After completion, concentrate the DCM on a rotary evaporator under reduced pressure for purification.

[0225] Step 4.6.2 was purified by HPLC. The crude product obtained in step 4.6.1 was diluted to a volume ratio of 1:0.5:0.5 stock solution:acetonitrile:purified water, with the A / B phase solvents being purified water and acetonitrile, respectively. The chromatographic column was a BR-C18 (Saifen) reversed-phase column with a gradient of 30-80% acetonitrile in 40 min at a flow rate of 15 mL / min. After purification, the target fractions were collected and lyophilized to obtain compound 2-10 (i.e., MMAE conjugate 2) as a white solid.

[0226] Step 5: Peptide coupling to MMAE conjugate 2

[0227] Dissolve 40 mg of the purified peptide obtained in Step 3 in 4 mL of DMF. Slowly add 3 eq of DIPEA and 1 eq of the MMAE conjugate 1 obtained in Step 4. Stir at room temperature under nitrogen for 16 hours. Monitor the reaction by LC-MS. Purification is then performed immediately after completion of the reaction.

[0228] Step 6: Preparation and purification of target product

[0229] The crude product obtained in step 5 was diluted to a volume ratio of 1:1:1 stock solution: acetonitrile: purified water. After filtering through a 0.45 μm membrane, it was separated using a reverse-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reverse-phase column. During the purification process, the chromatograph detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 35-65% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions with >95% purity were combined and lyophilized to obtain the pure desired product.

[0230] Step 7: Detection and Characterization Methods

[0231] The purity of the target product of step 6 and the N-terminal MMAE conjugate 2 of the polypeptide compound were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry. The detection results are shown in Figures 3 and 4.

[0232] Example 3. Preparation of Compound 3

[0233] Step 1: Synthesis of linear precursor peptide chains

[0234] Linear precursor peptide chain of compound 3:

[0235] DSer-DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle.

[0236] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swelled in DMF for 1 hour. The linear precursor sequence was then synthesized from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0237] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0238] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0239] Dissolve 1.0 mmol of Fmoc-AA, 1.0 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0240] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0241] After the linear peptide synthesis, the resin was washed with DMF five times.

[0242] Step 2: Cleavage of the linear precursor peptide chain

[0243] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0244] Step 3: Purification and preparation of pure peptide

[0245] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 20-50% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 75% were combined and lyophilized to obtain the pure peptide.

[0246] Step 4: Peptide coupling to MMAE conjugate 1

[0247] Dissolve 40 mg of the purified peptide obtained in Step 3 in 4 mL of DMF. Slowly add DIPEA (3 eq) and MMAE conjugate 1 (1 eq). Stir at room temperature under nitrogen for 16 hours. Monitor the reaction by LC-MS. Purification is then performed immediately after completion of the reaction.

[0248] Step 5: Preparation and purification of target product

[0249] The crude product obtained in step 4 was diluted to a volume ratio of 1:1:1 stock solution: acetonitrile: purified water. After filtering through a 0.45 μm membrane, it was separated using a reverse-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reverse-phase column. During the purification process, the chromatograph detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 40-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions with >95% purity were combined and lyophilized to obtain the pure desired product.

[0250] Step 6: Detection and Characterization Methods

[0251] The purity of the target product of step 5 and the N-terminal MMAE conjugate 1 of the polypeptide compound were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry. The detection results are shown in Figures 5 and 6.

[0252] Example 4. Preparation of Compound 4

[0253] Step 1: Synthesis of linear precursor peptide chains

[0254] The linear precursor peptide chain of compound 4: DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle.

[0255] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swelled in DMF for 1 hour. The linear precursor sequence was then synthesized from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0256] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0257] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0258] Dissolve 1.0 mmol of Fmoc-AA, 1.0 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0259] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0260] After the linear peptide synthesis, the resin was washed with DMF five times.

[0261] Step 2: Cleavage of the linear precursor peptide chain

[0262] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0263] Step 3: Purification and preparation of pure peptide

[0264] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 20-50% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 75% were combined and lyophilized to obtain the pure peptide.

[0265] Step 4: Peptide coupling to MMAE conjugate 1

[0266] Dissolve 40 mg of the purified peptide obtained in Step 3 in 4 mL of DMF. Slowly add DIPEA (3 eq) and MMAE conjugate 1 (1 eq). Stir at room temperature under nitrogen for 16 hours. Monitor the reaction by LC-MS. Purification is then performed immediately after completion of the reaction.

[0267] Step 5: Preparation and purification of target product

[0268] The crude product obtained in step 4 was diluted to a volume ratio of 1:1:1 stock solution: acetonitrile: purified water. After filtering through a 0.45 μm membrane, it was separated using a reversed-phase HPLC system using buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 45-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions with >95% purity were combined and lyophilized to obtain the pure desired product.

[0269] Step 6: Detection and Characterization Methods

[0270] The purity of the target product of step 5 and the N-terminal MMAE conjugate 1 of the polypeptide compound were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry. The detection results are shown in Figures 7 and 8.

[0271] Example 5. Preparation of Compound 5

[0272] Step 1: Synthesis of linear precursor peptide chains

[0273] The linear precursor peptide chain of compound 5: DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle.

[0274] The steps for synthesizing the linear precursor peptide chain are the same as those in step 1 of Example 4.

[0275] Step 2: Cleavage of the linear precursor peptide chain

[0276] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0277] Step 3: Purification and preparation of pure peptide

[0278] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 20-50% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 75% were combined and lyophilized to obtain the pure peptide.

[0279] Step 4: Peptide coupling to MMAE conjugate 2

[0280] Dissolve 40 mg of the purified peptide obtained in Step 3 in 4 mL of DMF. Slowly add DIPEA (3 eq) and MMAE conjugate 2 (1 eq). Stir at room temperature under nitrogen for 16 hours. Monitor the reaction by LC-MS. Purification is then performed immediately after completion of the reaction.

[0281] Step 5: Preparation and purification of target product

[0282] The crude product obtained in step 4 was diluted to a volume ratio of 1:1:1 stock solution: acetonitrile: purified water. After filtering through a 0.45 μm membrane, it was separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 40-65% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions with >95% purity were combined and lyophilized to obtain the pure desired product.

[0283] Step 6: Detection and Characterization Methods

[0284] The purity of the target product of step 5 and the N-terminal MMAE conjugate 2 of the polypeptide compound were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry. The detection results are shown in Figures 9 and 10.

[0285] Example 6. Preparation of Compound 6

[0286] Step 1: Synthesis of linear precursor peptide chain

[0287] The linear precursor peptide chain of compound 6: DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle.

[0288] The steps for synthesizing the linear precursor peptide chain are the same as those in step 1 of Example 4.

[0289] Step 2 Synthesis of Gemcitabine Conjugate 1

[0290] Step 2.1 Synthesis of compound 3-2

[0291] Step 2.1.1: Dissolve DBDC (di-tert-butyl dicarbonate) (44 mg, 0.2 mmol) in a mixture of dioxane (4 mL) and water (1 mL). Slowly add compound 3-1, gemcitabine (500 mg, 0.2 mmol), and 800 mg of sodium carbonate. Stir the mixture at room temperature under nitrogen for 48 hours. After the reaction, add 2 mL of water to the reaction solution, followed by extraction with 2-30 mL of ethyl acetate. The organic phase obtained by extraction is washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered. The reaction solution is concentrated under reduced pressure using a rotary evaporator to remove the solvent to obtain a crude solid.

[0292] Step 2.1.2: Purify by flash column chromatography. Dissolve the crude product from step 2.1.1 in a small amount of DCM and load onto a 40 g flash silica gel column. Phase B solvent: CH2Cl2-acetone-EtOH 1:1:0.02, with a gradient of 100% B in 30 min and a flow rate of 40 mL / min. After purification, the collected fractions were concentrated under reduced pressure on a rotary evaporator to remove the solvent, yielding solid compound 3-2.

[0293] Step 2.2 Synthesis of compound 3-3

[0294] Step 2.2.1: Dissolve compound 3-2 (73 mg, 0.2 mmol) in 8 mL of dioxane, add DBDC (di-tert-butyl dicarbonate) (436 mg, 2 mmol), and shake at 37°C for 70 h. After completion of the reaction, concentrate the reaction mixture under reduced pressure using a rotary evaporator to remove the solvent to obtain a crude solid.

[0295] Step 2.2.2: Purification was performed using flash column chromatography. The crude product from step 2.2.1 was dissolved in a small amount of DCM and loaded onto a 40 g flash silica gel column. The A / B solvents were DCM and acetone, respectively. The gradient was set to 10-20% acetone over 30 minutes at a flow rate of 40 mL / min. After purification, the collected fractions were concentrated under reduced pressure on a rotary evaporator to remove the solvent, yielding compound 3-3.

[0296] Step 2.3 Synthesis of compound 3-4

[0297] Step 2.3.1: Dissolve compound 3-3 (300 mg, 0.65 mmol) and succinic anhydride (518 mg, 5.18 mmol) in 20 mL of DCM, add DIPEA (1.13 mL, 6.47 mmol), and stir at room temperature for 16 h. After completion of the reaction, concentrate the reaction mixture under reduced pressure using a rotary evaporator to remove the solvent to obtain a crude solid.

[0298] Step 2.3.2 was purified using HPLC. The crude product obtained in step 4.5.1 was dissolved in pure water. The A / B solvents were pure water and pure acetonitrile, respectively. The chromatographic column was a BR-C18 (Saifen) reverse-phase column with a gradient setting of 10-95% and a flow rate of 15 mL / min. After purification, the target fraction was collected and lyophilized to obtain compound 3-4, gemcitabine conjugate 1, as a white solid.

[0299] Step 3: Solid-phase coupling of gemcitabine conjugate 1

[0300] Dissolve 0.4 mmol of gemcitabine conjugate, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol of 4-methylmorpholine (NMM) in DMF and add to the resin obtained in Step 1. Shake and react at room temperature for 16 hours. After the reaction, rinse the resin five times with DMF and five times with DCM. Drain the resin in vacuo.

[0301] Step 4: Cleavage of the linear precursor peptide chain

[0302] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 3 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0303] Step 5: Peptide purification and preparation

[0304] The crude peptide was dissolved in 50% acetonitrile-water solution, filtered through a 0.45 μm membrane, and separated using a reversed-phase high-performance liquid chromatography system with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 25-45% acetonitrile in 40 minutes. The product-related fractions were collected, and after HPLC analysis of purity, the fractions with >95% purity were combined and lyophilized to obtain the pure peptide.

[0305] Step 6: Detection and Characterization Methods

[0306] The purity of the pure polypeptide obtained in step 5 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry, and the N-terminus of the polypeptide compound was coupled to gemcitabine conjugate 1. The detection results are shown in Figures 11 and 12.

[0307] Example 7. Preparation of Compound 7

[0308] Step 1: Synthesis of linear precursor peptide chain

[0309] The linear precursor peptide chain of compound 7: AEEA-DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle.

[0310] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swelled in DMF for 1 hour. The linear precursor sequence was then synthesized from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0311] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0312] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0313] Dissolve 1.0 mmol of Fmoc-AA, 1.0 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0314] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0315] After the linear peptide synthesis, the resin was washed with DMF five times.

[0316] Step 2: Solid-phase coupling of gemcitabine conjugate 1

[0317] Dissolve 0.4 mmol of gemcitabine conjugate, 1.1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol of 4-methylmorpholine (NMM) in DMF and add to the resin obtained in Step 1. Shake and react at room temperature for 16 hours. After the reaction, rinse the resin five times with DMF and five times with DCM. Drain the resin in vacuo.

[0318] Step 3: Cleavage of the linear precursor peptide chain

[0319] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 3 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0320] Step 4: Peptide purification and preparation

[0321] The crude peptide was dissolved in 50% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 25-55% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0322] Step 5: Detection and Characterization Methods

[0323] The purity of the pure polypeptide obtained in step 4 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry, and the N-terminus of the polypeptide compound was coupled to gemcitabine conjugate 1. The detection results are shown in Figures 13 and 14.

[0324] Example 8. Synthesis of Compound 8

[0325] Step 1: Synthesis of linear precursor peptide chain

[0326] The linear precursor peptide chain of compound 8:

[0327] βAla-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle

[0328] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swelled in DMF for 1 hour. The linear precursor sequence was then synthesized from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0329] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0330] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0331] Dissolve 1.0 mmol of Fmoc-AA, 1.0 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0332] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0333] After the linear peptide synthesis, the resin was washed with DMF five times.

[0334] Step 2: Cleavage of the linear precursor peptide chain

[0335] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0336] Step 3: Purification and preparation of pure peptide

[0337] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase high-performance liquid chromatography system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 25-40% acetonitrile in 40 minutes. The product fractions were collected, and after HPLC analysis of purity, the fractions with >75% purity were combined and lyophilized to obtain the pure peptide.

[0338] Step 4: Peptide coupling to MMAE conjugate 1

[0339] Dissolve 40 mg of the purified peptide obtained in Step 3 in 4 mL of DMF. Slowly add DIPEA (3 eq) and MMAE conjugate 1 (1 eq). Stir at room temperature under nitrogen for 16 hours. Monitor the reaction by LC-MS. Purification is then performed immediately after completion of the reaction.

[0340] Step 5: Preparation and purification of target product

[0341] The crude product obtained in step 4 was diluted to a volume ratio of 1:1:1 stock solution: acetonitrile: purified water. After filtering through a 0.45 μm membrane, it was separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 35-55% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions with >95% purity were combined and lyophilized to obtain the pure desired product.

[0342] Step 6: Detection and Characterization Methods

[0343] The purity of the target product of step 5 and the N-terminal MMAE conjugate 1 of the polypeptide compound were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry. The detection results are shown in Figures 15 and 16.

[0344] Example 9. Synthesis of Compound 9

[0345] Step 1: Synthesis of linear precursor peptide chain

[0346] The linear precursor peptide chain of compound 9:

[0347] βAla-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle

[0348] The steps for synthesizing the linear precursor peptide chain are the same as those in step 1 of Example 8.

[0349] Step 2: Cleavage of the linear precursor peptide chain

[0350] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0351] Step 3: Purification and preparation of pure peptide

[0352] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase high-performance liquid chromatography system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 25-40% acetonitrile in 40 minutes. The product fractions were collected, and after HPLC analysis of purity, the fractions with >75% purity were combined and lyophilized to obtain the pure peptide.

[0353] Step 4: Synthesis of MMAE conjugate 3

[0354] Step 4.1 Synthesis of compound 4-1

[0355] 8.4 g (10 mmol) of 2-Chlorotrityl chloride resin was fully swelled in 150 mL of DCM for 1 hour. After the resin was swollen, the DCM was drained and the prepared amino acid solution (3 eq Fmoc-Cit-OH, 6 eq DIPEA dissolved in 100 mL of DCM) was added and shaken for 2 hours. After the reaction was complete, the resin was washed three times with DCM. 100 mL of blocking solution (85% DCM + 10% methanol + 5% DIPEA) was added and allowed to react for 10 minutes. After the reaction was complete, the resin was washed five times with DCM and five times with DMF. An appropriate amount of 20% 4-methylpiperidine / 80% DMF was added to the resin and shaken on a shaker. Deprotection was performed for 25 minutes, divided into two steps: the first step was 5 minutes, and the second step was 20 minutes. A small amount of resin was removed for ninhydrin testing. If the ninhydrin test was positive, the next step was performed. Otherwise, this step was repeated for 10 minutes. The resin was rinsed with DMF 6-8 times until the pH was neutral. The prepared amino acid solution (5 eq Fmoc-Val-OH, 2.85 eq HATU, 6 eq DIPEA dissolved in 100 mL DMF) was added to the resin and shaken for 2 hours. A small amount of resin was removed for ninhydrin testing. If the ninhydrin test was negative, the next step was performed. Otherwise, this step was repeated for one hour. After the reaction was completed, the solution was washed with DMF three times. The deprotection and coupling steps were repeated, and Fmoc-Glu(otBu)-OH was coupled in sequence. After the reaction was completed, the solution was washed with DMF three times. The resin was then cleaved with a 30% hexafluoroisopropanol / 70% DCM solution, and the precipitate was precipitated with tert-butyl methyl ether. The precipitate was dried under vacuum to yield compound 4-1.

[0356] Step 4.2 Synthesis of compound 4-2

[0357] Compound 4-1 (2.12 g, 1.0 eq) was dissolved in DMF (5 mL), and EEDQ (1.54 g, 2.0 eq) and (4-aminophenyl)methanol (0.77 g, 2.0 eq) were added. The mixture was stirred at 20°C for 16 hours. The reaction was monitored by LC-MS. After completion of the reaction, the mixture was purified by HPLC and lyophilized to obtain 1.12 g of a white solid.

[0358] Step 4.3 Synthesis of compound 4-3

[0359] Compound 2-2 (393 mg, 1.0 eq) was dissolved in DMF (5 mL), and DIEA (322 mg, 5.0 eq) and bis(4-nitrophenyl) carbonate (608 mg, 4.0 eq) were added. The mixture was allowed to react at room temperature for 3 hours. The reaction was monitored by LC-MS. After completion of the reaction, the mixture was purified by HPLC and lyophilized to obtain compound 4-3 as a white solid.

[0360] Step 4.4 Synthesis of compound 4-4

[0361] Compound 4-3 (328 mg, 1 eq) was dissolved in an appropriate amount of DMF (1 g / 10 mL). HOBt (0.2 eq, 9 mg), pyridine (8 eq, 181 mg), and MMAE (0.85 eq, 210 mg) were slowly added and allowed to react at room temperature for 16 hours. LC-MS was used to monitor the reaction. After completion, 4-methylpiperidine (0.8 eq) was added and the reaction continued at room temperature for 30 minutes. A trace amount of the reaction solution was collected for LCMS monitoring. After completion of the reaction, purification was performed. Purification was performed by HPLC and lyophilization was performed to obtain compound 4-4 as a white solid.

[0362] Step 4.5 Synthesis of compound 4-5

[0363] Compound 4-4 (304 mg, 1 eq) was dissolved in 3 mL of DMF, and glutaric anhydride (34 mg, 2 eq) and N,N-diisopropylethylamine (DIEA) (48 mg, 2 eq) were added. The reaction mixture was stirred at room temperature for 3 h. LC-MS was used to monitor the reaction. After completion of the reaction, purification was performed. HPLC was used to purify the crude product, which was then lyophilized to yield compound 4-5 as a white solid.

[0364] Step 4.6 Synthesis of compound 4-6

[0365] Compound 4-5 (1 eq, 231 mg) was dissolved in a mixture of DMF (6 mL) and DCM (2 mL). EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (3 eq, 93 mg) and NHS (N-hydroxysuccinimide) (3 eq, 56 mg) were slowly added and allowed to react at room temperature for 3 hours. The reaction was monitored by LC-MS. After completion of the reaction, the DCM was concentrated under reduced pressure using a rotary evaporator and then purified. HPLC was used for purification in this experiment, and after purification, lyophilization was performed to obtain compound 4-6 as a white solid.

[0366] Step 4.7 Synthesis of compound 4-7

[0367] Compound 4-6 (188 mg, 1 eq) was dissolved in DCM (19 mL), and TFA (1.9 mL) was slowly added. The reaction was allowed to react at room temperature for 30 minutes. The reaction was monitored by LC-MS. After completion of the reaction, the DCM was concentrated under reduced pressure using a rotary evaporator and then purified. This experiment was purified using HPLC, and after purification, lyophilization was performed to obtain compound 4-7 (i.e., MMAE conjugate 3) as a white solid.

[0368] Step 5: Peptide coupling to MMAE conjugate 3

[0369] 18 mg of the purified peptide obtained in step 3 was dissolved in 2 mL of DMF. DIPEA (30 eq, 35 mg) and MMAE conjugate 3 (13 mg, 1 eq) obtained in step 4 were slowly added. The reaction was stirred at room temperature under nitrogen for 16 hours. The reaction was monitored by LC-MS. After completion of the reaction, the product was directly purified.

[0370] Step 6: Preparation and purification of target product

[0371] The crude product obtained in step 5 was diluted to a volume ratio of 1:1:1 stock solution: acetonitrile: purified water. After filtering through a 0.45 μm membrane, it was separated using a reverse-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reverse-phase column. During the purification process, the chromatograph detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions with >95% purity were combined and lyophilized to obtain the pure desired product.

[0372] Step 7: Detection and Characterization Methods

[0373] The purity of the target product of step 6 and the N-terminal MMAE conjugate 3 of the polypeptide compound were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry. The detection results are shown in Figures 17 and 18.

[0374] Example 10. Synthesis of Compound 10

[0375] Step 1: Synthesis of linear precursor peptide chains

[0376] The linear precursor peptide chain of compound 10:

[0377] βAla-Sar-Sar-Sar-Sar-DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle.

[0378] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swelled in DMF for 1 hour. The linear precursor sequence was then synthesized from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0379] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0380] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0381] Dissolve 1.0 mmol of Fmoc-AA, 1.0 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0382] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0383] After the linear peptide synthesis, the resin was washed with DMF five times.

[0384] Step 2: Cleavage of the linear precursor peptide chain

[0385] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0386] Step 3: Purification and preparation of pure peptide

[0387] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 25-45% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 75% were combined and lyophilized to obtain the pure peptide.

[0388] Step 4: Peptide coupling to MMAE conjugate 3

[0389] Dissolve 18 mg of the purified peptide obtained in Step 3 in 2 mL of DMF. Slowly add DIPEA (30 eq, 35 mg) and MMAE conjugate 3 (13 mg, 1 eq). Stir at room temperature under nitrogen for 16 hours. Monitor the reaction by LC-MS. Purification is then performed immediately after completion of the reaction.

[0390] Step 5: Preparation and purification of target product

[0391] The crude product obtained in step 4 was diluted to a volume ratio of 1:1:1 stock solution: acetonitrile: purified water. After filtration through a 0.45 μm membrane, the product was separated using a reversed-phase HPLC system using buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the chromatograph detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 5-90% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions with >95% purity were combined and lyophilized to obtain the pure desired product.

[0392] Step 6: Detection and Characterization Methods

[0393] The purity of the target product of step 5 and the N-terminal MMAE conjugate 3 of the polypeptide compound were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry. The detection results are shown in Figures 19 and 20.

[0394] Example 11. Synthesis of Compound 12

[0395] Step 1: Synthesis of linear precursor peptide chains

[0396] The linear precursor peptide chain of compound 12: DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle.

[0397] The steps for synthesizing the linear precursor peptide chain are the same as those in step 1 of Example 4.

[0398] Step 2: Cleavage of the linear precursor peptide chain

[0399] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0400] Step 3: Purification and preparation of pure peptide

[0401] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 20-50% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 75% were combined and lyophilized to obtain the pure peptide.

[0402] Step 4 Synthesis of Dxd conjugate 1

[0403] Step 4.1 Synthesis of compound 5-1 (Fmoc-GGFGG-OH)

[0404] Fully swell 25.2 g (30 mmol) of 2-Chlorotrityl chloride resin in 400 mL of DCM for 1 hour. After the resin has swelled, drain the DCM. ① Add the prepared amino acid solution (3 eq Fmoc-G-OH, 6 eq DIPEA) and shake for 2 hours. After the reaction is complete, wash the resin three times with DCM. Add 300 mL of blocking solution (85% DCM + 10% methanol + 5% DIPEA) and let it react for 10 minutes. After the reaction is complete, wash the resin five times with DCM and five times with DMF. Add an appropriate amount of 20% 4-methylpiperidine / 80% DMF to the resin and shake on a shaker. Deprotection takes 25 minutes, divided into two steps: the first step is 5 minutes, and the second step is 20 minutes. Take out a small amount of resin for ninhydrin test. If the ninhydrin test is positive, proceed to the next step. Otherwise, repeat this step for 10 minutes and rinse the resin with DMF 6-8 times until the pH value is neutral. ② Add the prepared amino acid solution (3eq Fmoc-G-OH, 2.85eq HATU, 6eq DIPEA) to the resin and repeat the above steps; ③ Add the prepared amino acid solution (3eq Fmoc-F-OH, 2.85eq HATU, 6eq DIPEA) to the resin and repeat the above steps; ④ Add the prepared amino acid solution (3eq Fmoc-G-OH, 2.85eq HATU, 6eq DIPEA) to the resin and repeat the above steps; ⑤ Add the prepared amino acid solution (3eq Fmoc-G-OH, 2.85eq HATU, 6eq DIPEA) to the resin and repeat the above steps. After the reaction was completed, the resin was washed with DMF three times; the resin was then cut with a 30% hexafluoroisopropanol / 70% DCM solution, and the precipitate was precipitated with tert-butyl methyl ether. The precipitate was vacuum dried to obtain a white solid compound Fmoc-GGFGG-OH.

[0405] Step 4.2 Synthesis of compound 5-2

[0406] A 50 mL round-bottomed, double-necked flask was charged with compound 5-1 (4 g, 6.50 mmol), a Pd(OAc)4 / acetic acid solution (8.64 g), 120 mL of THF, and 40 mL of toluene. Under nitrogen, the flask was placed in an ice-water bath and stirred for 10 minutes. Pyridine (1.3 mL, 16.8 mmol) was then slowly added, and the temperature was raised to 50°C for approximately 3 hours. After completion of the reaction, the reaction mixture was concentrated in vacuo and purified by column chromatography to afford compound 5-2 as a pale yellow solid.

[0407] Step 4.3 Synthesis of compound 5-3

[0408] To a 25 mL round-bottom flask, compound 5-2 (1 g, 1.59 mmol), benzyl glycolate (264 mg, 1.59 mmol), tris(pentafluorophenyl)borane (163 mg, 0.32 mmol), and 30 mL of DME were added and reacted at 0°C under nitrogen for approximately 3 h. After completion of the reaction as monitored by LCMS, the reaction solution was concentrated in vacuo and purified by column chromatography to afford compound 5-3 as a white solid.

[0409] Step 4.4 Synthesis of compound 5-4

[0410] A 100 mL round-bottom flask was charged with compound 5-3 (1.06 g, 1.44 mmol), Pd / C (389 mg), 25 mL of ethanol, and 15 mL of ethyl acetate. The atmosphere was replaced with hydrogen three times and the mixture was reacted at room temperature for 2 h. After completion of the reaction as monitored by TLC, the reaction mixture was filtered and the filtrate was concentrated in vacuo to yield 800 mg of compound 5-4 as a white solid.

[0411] Step 4.5 Synthesis of compound 5-5

[0412] To a 25 mL round-bottom flask, compound DXd-002-4 (800 mg, 1.24 mmol), isotecan mesylate (478 mg, 0.9 mmol), HOBt (165 mg, 1.22 mmol), EDCI (250.8 mg, 1.3 mmol), 5 mL DMSO, and 4 mL THF were added dropwise under nitrogen. Triethylamine (121 mg, 1.20 mmol) was added and allowed to react at room temperature for 4 h. After completion of the reaction, the mixture was purified by preparative purification. The preparative solution was concentrated in vacuo and lyophilized to yield 560 mg of compound DXd-002-5 as a gray solid.

[0413] Step 4.6 Synthesis of compound 5-6

[0414] Compound 5-5 (560 mg, 0.50 mmol) and 12 mL of THF were added to a 25 mL round-bottom flask. DBU (80 mg, 0.52 mmol) was added dropwise under nitrogen and allowed to react at room temperature for 3 h. After completion of the reaction as monitored by TLC, the reaction solution was concentrated in vacuo and recrystallized from methyl tert-butyl ether to obtain 394 mg of compound 5-6 as a gray solid.

[0415] Step 4.7 Synthesis of compound 5-7

[0416] To a 10 mL round-bottom flask, compound 5-6 (210 mg, 0.25 mmol), glutaric anhydride (57 mg, 0.5 mmol), and 2 mL of DMF were added. DIEA (80 mg, 0.62 mmol) was added dropwise under nitrogen and allowed to react at room temperature for 3 h. After TLC monitoring of the reaction completion, EDCI (206 mg, 1.07 mmol) and NHS (123 mg, 1.01 mmol) were added to the reaction solution and the reaction continued for 8 h. After LCMS monitoring of the reaction completion, the solution was sent for preparative purification. The preparative solution was concentrated in vacuo and lyophilized to obtain compound 5-7 (i.e., Dxd conjugate 1) as a white solid.

[0417] Step 5: Peptide coupling to Dxd conjugate 1

[0418] Dissolve 23.5 mg of the purified peptide obtained in Step 3 in 2 mL of DMF. Slowly add DIPEA (30 eq, 35 mg) and Dxd conjugate 1 (20 mg, 1 eq) obtained in Step 4. Stir at room temperature under nitrogen for 3 hours. Monitor the reaction by LC-MS. Purification is then carried out immediately after completion of the reaction.

[0419] Step 6: Preparation and purification of target product

[0420] The crude product obtained in step 5 was diluted to a volume ratio of 1:1:1 stock solution: acetonitrile: purified water. After filtering through a 0.45 μm membrane, it was separated using a reverse-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reverse-phase column. During the purification process, the chromatograph detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions with >95% purity were combined and lyophilized to obtain the pure desired product.

[0421] Step 7: Detection and Characterization Methods

[0422] The purity of the target product of step 6 and the N-terminal Dxd conjugate 1 of the polypeptide compound were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry. The detection results are shown in Figures 21 and 22.

[0423] Example 12: Test of small molecule compounds killing tumor cells (screening cell experiment)

[0424] 1. Materials and Methods

[0425] 1.1 Experimental Materials:

[0426] Small molecule compounds: Monomethyl aunistatin E (MMAE, GLPBIO), Gemcitabine (Gemcitabine, GLPBIO), Monomethylauristatin F (MMAF, MCE), PTX (paclitaxel, Pharmaceutical Chemistry Department), 5-Fluorouracil (5-Fu, MCE).

[0427] Experimental cells: SW1990 (Punocai), CFPAC-1 (Punocai), HPAF-II (Beina Biotech), BxPC-3 (Punocai), PANC-1 (Punocai), Capan-1 (Punocai), Capan-2 (Punocai), HUTU-80 (Punocai), and Hs 766T (Nanjing Kebai Biotech).

[0428] 1.2 Experimental reagents and consumables: 96-well clear cell plate (Agilent); 96-well black transparent bottom plate (Jingan); 1× DPBS

[0429] (self-made); 96-well clear cell plate (Corning); Cell Counting Kit-8 (GLPBIO).

[0430] 1.3 Experimental instrument: Cytation5 multifunctional microplate reader (BioTek).

[0431] 2. Experimental methods:

[0432] (1) Cell culture:

[0433] SW1990 and PANC-1 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, 10% FBS, 1% double-antibody (penicillin-streptomycin mixture)). When the cell density reached 80-90% of the flask, cells were rinsed with DPBS and then digested with 0.25% trypsin (containing 0.5 mM EDTA). Digestion was terminated by adding the corresponding growth medium. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. The supernatant was removed and the cells were resuspended in 3-5 mL of fresh growth medium. Cells were passaged at a ratio of 1:3 to 1:8 and cultured in a 37°C, 5% CO2 incubator. After passage, the medium was changed or the cells were passaged every 2-3 days.

[0434] Hs766T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, 15% FBS, 1% double-antibody (penicillin-streptomycin mixture)). When the cell density reached 80-90% of the flask, cells were rinsed with DPBS and then digested with 0.25% trypsin (containing 0.5 mM EDTA). Digestion was terminated by adding the appropriate growth medium. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. The supernatant was removed and the cells were resuspended in 3-5 mL of fresh growth medium. Cells were passaged at a ratio of 1:3 to 1:8 and cultured in a 37°C, 5% CO2 incubator. After passage, the medium was changed or the cells were passaged every 2-3 days.

[0435] CFPAC-1 and Capan-1 cells were cultured in IMDM, 10% FBS, and 1% double-streptomycin (penicillin-streptomycin mixture). When the cell density reached 80-90% of the flask, cells were rinsed with DPBS and then digested with 0.25% trypsin (containing 0.5 mM EDTA). Complete culture medium was added to terminate the digestion. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. The supernatant was removed and the cells were resuspended in 3-5 mL of fresh growth medium. Cells were passaged at a ratio of 1:3 to 1:8 and cultured in a 37°C, 5% CO2 incubator. After passage, the medium was changed or the cells were passaged every 2-3 days.

[0436] HPAF-II and HUTU-80 cells were cultured and grown in MEM (MEM, 10% FBS, 1% double-antibody (penicillin-streptomycin mixture)). When the cell density reached 80-90% of the flask, the cells were rinsed with DPBS and then digested with 0.25% trypsin (containing 0.5 mM EDTA). Complete culture medium was added to terminate the digestion, and the cell suspension was collected into a centrifuge tube. Centrifuged at 1000 rpm for 3 minutes, the supernatant removed, and the cells were resuspended in 3-5 mL of fresh growth medium. The cells were passaged at a ratio of 1:3 to 1:8 and cultured in a 37°C, 5% CO2 incubator. After passage, the medium was changed or the cells were passaged every 2-3 days.

[0437] BxPC-3 cells were cultured in RPMI-1640, 10% FBS, and 1% double-streptomycin (penicillin-streptomycin mixture). When the cells reached 80-90% of the flask density, they were rinsed with DPBS and then digested with 0.25% trypsin (containing 0.5 mM EDTA). The digestion was terminated by adding the appropriate growth medium. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. The supernatant was removed and the cells were resuspended in 3-5 mL of fresh growth medium. The cells were passaged at a ratio of 1:3 to 1:8 and cultured in a 37°C, 5% CO2 incubator. The medium was changed or the cells were passaged every 2-3 days after passage.

[0438] Capan-2 cells were cultured in McCoy's 5A medium (10% FBS, 1% double-streptomycin mixture). When the cells reached 80-90% of the flask density, they were rinsed with DPBS and then digested with 0.25% trypsin (containing 0.5 mM EDTA). The digestion was terminated by adding the appropriate growth medium. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. The supernatant was removed and the cells were resuspended in 3-5 mL of fresh growth medium. The cells were passaged at a ratio of 1:3 to 1:8 and cultured in a 37°C, 5% CO2 incubator. The medium was changed or the cells were passaged every 2-3 days after passage.

[0439] (2) Experimental steps

[0440] All the above cells were passaged and expanded to the required cell number. After digestion and resuspending, the cells were incubated in a 96-well cell culture plate at a cell density of 6000 cells / well and 100 μL / well cell resuspension solution for 1 day before use in experimental detection. First, the above small molecule compounds were diluted to 2× concentrations using complete culture medium according to the following cell use concentrations: MMAF was used at a final concentration of 30 μM on CFPAC-1, Capan-1, Capan-2 and SW1990 cells, and at a final concentration of 10 μM on HPAF-Ⅱ, PANC-1, Bx-PC3 and HUTU-80 cells, all with a 10-fold serial dilution of 7 concentrations; gemcitabine was used at a final concentration of 10 μM on HPAF-Ⅱ, PANC-1, Bx-PC3, Capan-1, Capan-2 and HUTU-80 cells, at a final concentration of 1 μM on CFPAC-1 cells, at a final concentration of 30 μM on SW1990 cells, and at a final concentration of 60 μM on Hs766T cells, all with a 5-fold serial dilution of 7 concentrations; MMAE was used at a final concentration of 10 nM on PANC-1, Bx-PC3, CFPAC-1 and HUTU-80 cells, and at a final concentration of 10 nM on Capan-1 and The final concentration of PTX was 1 μM on Capan-2 cells, 10 μM on SW1990 cells, and 400 nM on HPAF-Ⅱ and Hs766T cells, all diluted 5-fold into 7 concentrations; the final concentration of PTX was 100 nM on HPAF-Ⅱ, PANC-1, Bx-PC3, CFPAC-1 and HUTU-80 cells, 1 μM on Capan-1 and Capan-2 cells, and 1 μM on SW1990 cells. The final concentration used on 1990 cells was 10 μM, and the final concentration used on Hs766T cells was 400 nM, both of which were diluted 5-fold into 7 concentrations; the final concentration of 5-Fluorouracil used on HPAF-Ⅱ, PANC-1, Bx-PC3, CFPAC-1, Capan-1 and Capan-2 cells was 30 μM, and the final concentration used on SW1990 and HUTU-80 cells was 78.75 μM, both of which were diluted 10-fold into 7 concentrations.

[0441] Then, 100 μL of the supernatant was removed from the cell plate, and 100 μL of each of the diluted small molecule compounds was added to the wells in triplicate. The wells were placed in a 37°C, 5% CO2 incubator and incubated for 72 hours. Finally, 10 μL of CCK8 cell activity detection reagent was added, the wells were placed in a 37°C, 5% CO2 incubator, incubated for 1 hour, and the OD was measured using a Cytation5 multifunctional microplate reader. 450 value.

[0442] The experimental results are shown in Figure 15 and Table 1.

[0443] Table 1 IC50 of small molecule compounds against different cells 50 Value statistics table:

[0444] The experimental results show that MMAE is the small molecule compound with the best cell-killing effect. 5-Fu has a significant killing effect on Capan-1 cells, but no significant killing effect on other cells. As shown in the table above, three tumor cell lines, HPAF-Ⅱ, Hs766T, and HUTU-80, with clear cell-killing curves and preliminary data demonstrating GRPR-positive expression, were selected for use in subsequent validation experiments. MMAE, a small molecule compound with strong cell-killing activity, and gemcitabine, a small molecule compound reported in the literature for the treatment of pancreatic cancer, were selected as co-drug molecules for subsequent PDC drug development and used as bare drug controls.

[0445] Example 13: GRPR receptor endocytosis test in HEK293-GRPR-GFP cells

[0446] 1. Materials and Methods

[0447] 1.1 Experimental Materials

[0448] PDC drug (self-made synthetic polypeptide conjugate drug): polypeptide conjugate drug of the present invention.

[0449] Positive polypeptides (self-synthesized or outsourced): SEQ ID No.1, SEQ ID No.1-Cy5, SEQ ID No.2, SEQ ID No.2-Cy5, SEQ ID No.3, SEQ ID No.1-Cy5.

[0450] 1.2 Cells and Related Reagents and Consumables

[0451] HEK293-GRPR-GFP cells (obtained by transfection and selection); G418 (Aladdin, 11811031 / ant-gn-5 / G110917-5); FBS (Hyclone, SV30208.02); Trypsin (Gibco, 27250-018). 96-well black clear-bottom cell plates (Agilent), fixative (Beyotime); DAPI (Beyotime); 1× DPBS (homemade).

[0452] 2. Experimental methods:

[0453] (1) Cell preparation: HEK293-GRPR-GFP cells were cultured and grown in culture medium (DMEM, 10% FBS, 1% double-antibody (penicillin-streptomycin mixture), 400 μg / mL G418). When the cell growth density reached 80-90% of the culture flask, the cells were first rinsed with DPBS and then digested with 0.25% trypsin (containing 0.5 mM EDTA). The corresponding growth medium was then added to terminate the digestion. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. The supernatant medium was removed. 3-5 mL of fresh growth medium was then added to resuspend the cells. The cells were passaged at a ratio of 1:3 to 1:8 and cultured in a 37°C, 5% CO2 incubator. After passage, the medium was changed or passaged every 2-3 days.

[0454] (2) Experimental procedure: PDC drug and positive control peptide samples were diluted to 3 μM (1×) using basal culture medium, and then diluted 3-fold to a total of 4 concentrations. 100 μL of culture medium was aspirated from the cell plate, and 100 μL of diluted PDC drug and positive control peptide were added, and the cells were incubated in a 37°C, 5% CO2 incubator for 0.5 h. 100 μL of sample solution was aspirated, and 100 μL of 1× DPBS was added to wash the cells, and this step was repeated 3-5 times. 1× DPBS was aspirated, 60 μL of fixative solution was added, and the cells were incubated in a 4°C refrigerator for 0.5 h, and then the cells were washed 1-2 times with 1× DPBS. 1× DPBS was aspirated, 50 μL of DAPI nuclear staining solution was added, and the cells were incubated in a 37°C, 5% CO2 incubator for 0.5 h, and then the cells were washed 1-2 times with 1× DPBS, and photographed using a Cytition10 confocal imaging system.

[0455] The experimental results are shown in the endocytosis imaging images in Figures 24 to 26. The experimental results show that all the peptide-drug conjugates of the present invention have obvious endocytosis, proving that these peptide-drug conjugates can target GRPR and enter cells.

[0456] Example 14: Cell Killing Test

[0457] 1. Materials and Methods

[0458] 1.1 Experimental Materials:

[0459] PDC drug (self-made synthetic polypeptide conjugate drug): polypeptide conjugate drug of the present invention.

[0460] Small molecule compounds: Monomethyl aunistatin E (MMAE, GLPBIO), Gemcitabine (Gemcitabine, GLPBIO).

[0461] 1.2 Experimental cells and related reagents: HEK293-GRPR-GFP cells (obtained by transfection and screening), HEK293 (Cyborg), HPAF-II (Beina Biotech), HUTU-80 (Punosai), Hs 766T (Nanjing Kebai Biotech); 96-well black transparent bottom cell plates (Agilent), 96-well clear cell plates (Corning); 1× DPBS (homemade), Cell Counting Kit-8 (GLPBIO).

[0462] 1. Experimental methods:

[0463] HEK293-GRPR-GFP, HEK293, HPAF-II, and HUTU-80 cells were digested, resuspended, and diluted before plating at a cell density of 3,000 cells per well. Hs766T cells were digested, resuspended, and diluted before plating at a cell density of 6,000 cells per well. All cells were added to a 96-well cell culture plate at a volume of 100 μL and cultured in a 37°C, 5% CO2 incubator for 1 day.

[0464] PDC drugs were diluted to 30 μM (3×) using complete culture medium, and then diluted 3-fold to 8 concentrations; MMAE was diluted to 1.2 μM (3×) using complete culture medium, and then diluted 5-fold to 8 concentrations; gemcitabine was diluted to 30 μM (3×) using complete culture medium, and then diluted 3-fold to 8 concentrations.

[0465] Add 50 μL of PDC drug and control small molecule compound directly to the cell plate, with 3 replicates for each concentration, and incubate in a 37°C, 5% CO2 incubator for 72 hours. Add 15 μL of CCK8 cell activity detection reagent, incubate in a 37°C, 5% CO2 incubator for 1 hour, and then use a Cytation5 multifunctional microplate reader to measure OD. 450 The experimental results of the killing effect of PDC drugs on different cells are shown in Figure 27. The killing effect IC values ​​of each peptide-coupled drug on different cells are shown in Figure 27. 50 The statistical table of values ​​is shown in Table 2. The test results show that the peptide-drug conjugates of the present invention have killing effects on different cells. Compounds 4 and 5 using MMAE as the co-drug small molecule have significantly better tumor cell killing effects than the peptide-drug conjugates with gemcitabine, among which the cell killing activity of compound 4 is better than that of compound 5.

[0466] Table 2 IC of peptide-drug conjugates against different cells 50 Value Statistics Table

[0467] Example 15: Mouse Plasma (Heparin Sodium) Stability

[0468] 1. Materials and Methods

[0469] 1.1 Experimental Materials:

[0470] PDC drug (self-made synthetic polypeptide conjugate drug): polypeptide conjugate drug of the present invention.

[0471] Small molecule compound: Monomethyl aunistatin E (MMAE, GLPBIO)

[0472] Experimental related reagents: positive control (nenitide, Tat-NR2B9C / NA-1) (purchased from Nanjing GenScript Biotechnology Co., Ltd.); negative control (TAT, {d-Tyr}G{d-Arg}{d-Lys}{d-Lys}{d-Arg}{d-Arg}{d-Gln}{d-Arg}{d-Arg}{d-Arg}), purchased from Nanjing GenScript Biotechnology Co., Ltd.); methanol (purchased from Sigma); formic acid (purchased from Aladdin); DMSO (dimethyl sulfoxide) (purchased from Aladdin); mouse plasma (Slack) (self-collected).

[0473] Experimental instruments: equilibrium dialysis device HTD (Shanghai Meixing Gaode); triple quadrupole liquid chromatography mass spectrometer LCMS-8060NX (Shimadzu).

[0474] 2. Experimental methods:

[0475] Dissolve nenitide in DMSO to 1 mM, dissolve TAT in DMSO to 1 mM, and dissolve the peptide to be tested in DMSO to 1 mM. Store at -20°C until ready to use. Thaw plasma: Remove (number of samples * 2.1) mL of plasma from a -80°C freezer and rapidly thaw in a 37°C water bath. Prepare the MIX: Add 693 μL of plasma to a 1.5 mL EP tube. Prepare three replicates for each time point, and prepare three MIX tubes. Add 7 μL of the test sample to each tube to a final concentration of 10 μM, which is detectable or an in vivo drug concentration. Vortex the mixture and aliquot 100 μL of the sample into a time-series aliquots. Incubate the mixture in a 37°C water bath for six time points: 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min. Terminate the reaction: After incubation, add 4 volumes of 0.1% formic acid in methanol as a precipitant. Mix: Vortex on a vortex shaker for 30 seconds. Centrifuge: 4°C, 15,000 rpm for 10 minutes. Transfer the supernatant to a vial for analysis by LC-MS / MS.

[0476] 1 Experimental results:

[0477] The dot-line graph with the original drug remaining rate (%) on the ordinate and time on the abscissa shows the degradation trend of the sample in plasma in vitro over time, providing the results of the sample stability.

[0478] GraphPad Prism 8 software was used to calculate the half-life of the compound in plasma according to the nonlinear regression of One phase decay. 1 / 2 .

[0479] The experimental results are shown in Figures 28 and 29 and Table 3. The half-life of compound 5 is >120 min, compound 2 is >120 min, compound 9 is >120 min, compound 10 is >120 min, compound 12 is >120 min, compound 3 is 43.01 min, compound 1 is 19.87 min, compound 4 is 16.51 min, and compound 8 is 10.76 min.

[0480] Table 3 Mouse plasma (heparin sodium) stability

[0481] Example 16: Human Plasma (Heparin Sodium) Stability

[0482] 1. Materials and Methods

[0483] 1.1 Experimental Materials:

[0484] PDC drug (self-made synthetic polypeptide conjugate drug): polypeptide conjugate drug of the present invention.

[0485] Small molecule compound: Monomethyl aunistatin E (MMAE, GLPBIO)

[0486] 1.2 Experimental reagents: positive control (nenitide, Tat-NR2B9C / NA-1) (purchased from Nanjing GenScript Biotechnology Co., Ltd.); negative control (TAT, {d-Tyr}G{d-Arg}{d-Lys}{d-Lys}{d-Arg}{d-Arg}{d-Gln}{d-Arg}{d-Arg}{d-Arg}), purchased from Nanjing GenScript Biotechnology Co., Ltd.); methanol (purchased from Sigma); formic acid (purchased from Aladdin); DMSO (dimethyl sulfoxide) (purchased from Aladdin); human plasma (Shanghai Xuanya).

[0487] 1.3 Experimental instruments: equilibrium dialysis device HTD (Shanghai Meixing Gaode); triple quadrupole liquid chromatography mass spectrometer LCMS-8060NX (Shimadzu).

[0488] 2. Experimental methods:

[0489] Dissolve nenitide in DMSO to 1 mM, dissolve TAT in DMSO to 1 mM, and dissolve the peptide to be tested in DMSO to 1 mM. Store at -20°C until ready for use. Thaw plasma: Remove (number of samples * 2.1) mL of human plasma from a -80°C freezer and rapidly thaw in a 37°C water bath. Prepare the MIX: Add 693 μL of plasma to a 1.5 mL EP tube. Prepare three replicates for each time point, and prepare three MIX tubes. Add 7 μL of the test sample to each tube to a final concentration of 10 μM, which is detectable or an in vivo drug concentration. Vortex on a vortex for 30 seconds. Aliquot 100 μL of the sample into a time-series aliquots and incubate. Incubate in a 37°C water bath for six time points: 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min. Terminate the reaction: After incubation, add 4 volumes of 0.1% formic acid in methanol as a precipitant. Mix: Vortex on a vortex shaker for 30 seconds. Centrifuge: 4°C, 15,000 rpm for 10 minutes. Transfer the supernatant to a vial for analysis by LC-MS / MS.

[0490] 3. Experimental results: A dot-line graph with the original drug remaining rate (%) on the ordinate and time on the abscissa shows the degradation trend of the sample in plasma over time, providing the stability of the sample.

[0491] GraphPad Prism 8 software was used to calculate the half-life of the compound in plasma according to the nonlinear regression of One phase decay. 1 / 2 .

[0492] The experimental results are shown in Table 4 and Figures 30 to 32. The half-lives of MMAE>120 min, compound 1>120 min, compound 4>120 min, compound 5>120 min, compound 2>120 min, compound 3>120 min, compound 8>120 min, compound 12>120 min, compound 10>120 min, compound 9>120 min, compound 7 was 55.54 min, and compound 6 was 9.438 min.

[0493] Table 4 Human plasma (heparin sodium) stability

[0494] Example 17: Tissue distribution study in HS766T pancreatic tumor-bearing mice

[0495] 1. Materials and Methods

[0496] 1.1PDC drug (self-made synthetic polypeptide conjugate drug): polypeptide conjugate drug of the present invention.

[0497] 1.2 Related reagents and consumables: 1mL insulin needle, 1.5mL EDTA anticoagulant tube, 0.3mL EP tube, EDTA, protease inhibitors, methanol, Monomethyl auristatin E (MMAE).

[0498] 1.3 Experimental Animals: Twelve male Balb / c nu mice, 6-7 weeks old, were purchased from Hunan Anshengmei Pharmaceutical Research Institute Co., Ltd.

[0499] 2. Experimental Methods

[0500] Balb / c male nude mice were raised under standard conditions for 6-7 weeks. Human pancreatic cancer cells Hs766T were suspended in serum-free medium and mixed with matrigel at a ratio of 1:1. Each mouse was inoculated under the armpit with 1*10 6 When the tumor volume reaches 100-200mm 3 At the same time, mice were randomly divided into 4 groups, 3 in each group. 8 μL EDTA and protease inhibitors were added to EDTA anticoagulant tubes respectively, and compound 4 (2 mg / kg) was injected into the tail vein of mice according to their weight. After the administration, the mice were anesthetized at 0.5h, 1h, 2h, and 4h, and about 200 μL of blood was collected from the eye plexus vein of the mice. The blood was collected with EDTA and immediately centrifuged. The supernatant was taken and stored on dry ice and then transferred to a -80°C refrigerator. After the blood of the mice was drained, it was immediately perfused. Subsequently, the liver, kidney, pancreas, and muscle tissue were washed with normal saline, dried with filter paper, stored on dry ice, and then transferred to a -80°C refrigerator.

[0501] Mouse tissue was treated with a 2% protease inhibitor cocktail and 0.5M EDTA (pH 8.0). Five volumes of PBS containing 50% methanol and 1.0% formic acid were then added. The tissue was homogenized in a grinder and vortexed for 3 minutes. After homogenization, 50 μL of sample was transferred to an EP tube, 50 μL of diluent was added, and 200 μL of precipitant (0.1% formic acid in methanol) was added. The tube was vortexed for 3 minutes. The tube was then centrifuged at 13,000 rpm for 10 minutes, and the supernatant was sampled.

[0502] 3. Experimental Results

[0503] The distribution of compound 4 in the tissues of Hs766T pancreatic tumor-bearing mice was determined using LC-MS. The results, shown in Figure 33, show that the content of compound 4 in tumor-bearing mouse tissues decreased over time. The highest concentration was found in plasma, followed by the liver and kidneys, with lesser concentrations in the pancreas, tumors, and muscle. Furthermore, the distribution of MMAE released by compound 4 in various tissues of tumor-bearing mice was further determined. As shown in Figure 34, MMAE concentrations in mouse plasma and kidneys decreased over time, while in the liver they initially increased and then decreased, reaching a peak concentration at 1 hour. MMAE levels in mouse muscle, tumors, and pancreas increased over time.

[0504] Example 18: Study on the efficacy of HPAF-II in pancreatic tumor-bearing mice

[0505] 1. Materials and Methods

[0506] 1.1PDC drug (self-made synthetic polypeptide conjugate drug): polypeptide conjugate drug of the present invention.

[0507] 1.2 Related reagents and consumables: 1mL insulin needle, 0.5mL EP tube, Monomethyl auristatin E (MMAE), DMSO, Solutol HS-15.

[0508] 1.3 Experimental animals: 30 Balb / c nu mice, male, 6-7 weeks old, purchased from Hunan Anshengmei Pharmaceutical Research Institute Co., Ltd.

[0509] 2. Experimental Methods

[0510] Male Balb / C nu mice, 6-7 weeks old, were raised under standard conditions. Human pancreatic cancer cells HPAF-II were suspended in serum-free medium and inoculated into each mouse at a rate of 1*10 6 When the tumor volume reaches 100-150mm 3 Mice were randomly divided into 6 groups, 5 mice per group, and injected via the tail vein with compound 4 (0.61 mg / kg), compound 4 (1.22 mg / kg), compound 4 (1.83 mg / kg), and vehicle (2% DMSO + 2% Solutol HS-15 + 96% saline). The dosage volume was 10 mL / kg, and the administration cycle was 4 days / 1, with four consecutive doses. The experiment was terminated 4 days after the last dose. After the experiment, the animals were sacrificed by cervical dissection, and the tumor volume (tumor volume (V) = major diameter * minor diameter^2 / 2) and RTV (relative tumor volume = Vt / V0) of the animals in each group were compared using the t-test.

[0511] 3. Experimental Results

[0512] During the experimental observation process, the body weight of mice in the MMAE (0.18 mg / kg), compound 4 (0.61 mg / kg), compound 4 (1.22 mg / kg) and vehicle groups was basically maintained within the range that the animal toxicity and side effects could be tolerated during the four doses. The body weight of mice in the compound 4 (1.83 mg / kg) group showed a gradual downward trend. As shown in Table 5, as the dosing time increased, compared with the vehicle group, the other groups all showed a trend of tumor proliferation inhibition, and the anti-tumor effect of compound 4 was better than that of MMAE at the same dose. At the same time, it can be seen from Figure 35 that as the dose increased, the anti-pancreatic tumor HPAF-II proliferation effect of compound 4 became more obvious, and the tumor volume showed a trend of regression. The experimental endpoint data showed that under the conditions of 1.22 mg / kg and 1.83 mg / kg, after the first dose, the relative tumor growth rates were 55.82% and 34.63%, respectively. After the end of the dose, the relative tumor growth rates were 13.44% and 3.90%, respectively. This proves that under medium and high dose conditions, compound 4 can completely inhibit the growth of pancreatic tumors, indicating that compound 4 has excellent in vivo anti-pancreatic tumor effects.

[0513] Table 5 Inhibitory effects of compound 4 and MMAE on the growth of pancreatic cancer tumor model in tumor-bearing mice at different doses

[0514] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A polypeptide-conjugated drug represented by general formula (I) or a pharmaceutically acceptable salt thereof, PL-Drug Formula (I) It is characterized in that The P comprises a polypeptide represented by formula (II) Ser-Tyr-Gln-X 1 -No-X 2 -βAla-X 3 -X 4 -I (II) X 1 Selected from Ala, Trp, Tyr, Phe or derivatives of said amino acids; X 2 Selected from Ala, Leu, Val, Ile or derivatives of said amino acids; X 3 Selected from His, Lys, Arg or derivatives of said amino acids; X 4 Selected from Ala, Leu, Phe, Val, Trp, Tyr or derivatives of said amino acids; The configuration of each amino acid in the sequence represented by the general formula (II) is independently selected from D-type or L-type; L is selected from a non-cleavable linker or a cleavable linker; Drug is an anti-tumor agent.

2. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The P comprises a polypeptide represented by formula (III) DSer-DTyr-Gln-X 1 -No-X 2 -βAla-X 3 -X 4 -I (III) X 1 Selected from Ala, Trp; X 2 Selected from Leu, Val; X 3 Selected from His, DHis; X 4 Selected from Cha, Leu, Phe, Nva; L is selected from a non-cleavable linker or a cleavable linker; Drug is an anti-tumor agent.

3. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The P comprises a polypeptide DSer-DTyr-Gln-Trp-Ala-X represented by formula (IV) 2 -βAla-His-X 4 -Nle (IV) X 2 Selected from Leu, Val; X 4 Selected from Cha, Leu, Phe, Nva; 4. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that: The C-terminus and / or N-terminus of the amino acid sequence of the polypeptide represented by the general formula (II) to (IV) is modified or unmodified.

5. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle(SEQ ID No.1)、 DSer-DTyr-Gln-Trp-Ala-Leu-Leu-Ala-Leu-His ID (SEQ ID No.1) No.2)、 DSer-DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle(SEQ ID No.3)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(Ethylamide)(SEQ ID No.4)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(Butylamide)(SEQ ID No.5)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(Methyl ester)(SEQ ID No.6)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(Ethyl ester)(SEQ ID No.7)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-D-His-Phe-Nle(SEQ ID No.8)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-D-Phe-Nle(SEQ ID No.9)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-D-Leu-Nle(SEQ ID No.10)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-4-chloro-Phe-Nle(SEQ ID No.11)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-4-methyl-phe-Nle(SEQ ID No.12)、. DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-3-chloro-Phe-Nle(SEQ ID No.13)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-D-His-D-phe-Nle(SEQ ID No.14) DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Nva-Nle(SEQ ID no.15)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-abu-Nle(SEQ ID No.16)、 DSer-DTrp-Gln-Trp-Ala-Val-βAla-His-Phe-Nle(SEQ ID No.17)、 DSer-DTyr-Gln-Tyr-Ala-Val-βAla-His-Phe-Nle(SEQ ID No.18)、 DSer-DTyr-Gln-Phe-Ala-Val-βAla-His-Phe-Nle(SEQ ID No.19)、 DSer-DTyr-Gln-1-Nal-Ala-Val-βAla-His-Phe-Nle(SEQ ID No.20)、 DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(SEQ ID No.21)、 DSer-DTyr-Gln-Trp-Ala-Val-βAla-Lys-Phe-Nle(SEQ ID No.22)、 DSer-DTyr-Gln-Trp-Ala-Val-βAla-Arg-Phe-Nle(SEQ ID No.23)、 DSer-DTyr-Gln-Trp-Ala-Val-βAla-His-Trp-Nle(SEQ ID No.24)、 DSer-DTyr-Gln-Trp-Ala-Val-βAla-His-Tyr-Nle(SEQ ID No.25)、 DSer-DTyr-Gln-Trp-Ala-Val-βAla-His-1-Nal-Nle(SEQ ID No.26)。.

6. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The L comprises a non-cleavable linker or a cleavable linker; the non-cleavable linker is selected from a PEG linker, a linker with a thioether group, a linker with an oxime group, or a combination thereof; the cleavable linker is selected from a linker with a disulfide bond group, a dipeptide linker, a tripeptide linker, a tetrapeptide linker, a peptidomimetic linker, a linker cleaved by β-glucuronidase, a linker cleaved by β-galactosidase, a linker based on phosphatase cleavage, a pH-sensitive linker, a linker cleaved by sulfatase, or a combination thereof.

7. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: The L comprises the following structure or a combination thereof, The m is independently selected from integers of 1-24.

8. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to claim 6 or 7, characterized in that: The linker structure also includes a PAB structure.

9. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to any one of claims 6 to 8, characterized in that: The linker structure also includes a β-Ala spacer and / or a [Sar]n spacer, wherein n is selected from an integer of 1-15.

10. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to any one of claims 6 to 9, characterized in that: The L is selected from the following structures, 11. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The drug is selected from microtubule-destroying drugs and DNA-damaging drugs.

12. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to claim 11, characterized in that: The drug is selected from dolastatin and its auristatin derivatives auristatins (MMAE, MMAF, MMAD), maytansine and maytansinoids (DM1, DM2, DM3, DM4), tubulysins, cryptocolistin, spindle kinesin, gemcitabine, pyrrolo[2,1-c][1,4]benzodiazepine, dukamycin, camptothecin and camptothecin derivatives, calicheamicin, amatoxin, paclitaxel, vinblastine, vincristine, etoposide, doxorubicin, cyclophosphamide, docetaxel, methotrexate, cisplatin, cytarabine, phenylalanine mustard and chlorambucil or a combination thereof.

13. The polypeptide-conjugated drug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The polypeptide-conjugated drug is selected from the following structures, 14. Use of the polypeptide-coupled drug according to any one of claims 1 to 13 in the preparation of cancer targeted therapeutic drugs.

15. The use according to claim 14, characterized in that: The application is the use of polypeptide-coupled drugs in the preparation of GRPR-positive cancer targeted therapeutic drugs; the GRPR-positive cancer is selected from at least one of prostate cancer, breast cancer, colon cancer, pancreatic cancer, renal cell carcinoma, small cell lung cancer, head and neck cancer, ovarian cancer, and uterine cancer.

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