Polypeptide coupling medicine as well as preparation method and application thereof

Abstract

The invention belongs to the field of medical chemistry and relates to a polypeptide coupling medicine as well as a preparation method and application thereof. In particular to a polypeptide coupling medicine as shown in a general formula (I) and application thereof, P comprises polypeptide as shown in a formula (II), L is selected from a non-cleavable connexon or a cleavable connexon, and Dug is an anti-tumor agent. The polypeptide coupling drug provided by the invention can be specifically combined with GRPr with high affinity, so that tumor cells are efficiently killed, and a novel anti-tumor targeting drug is provided for the field of tumor treatment.

Description

Technical Field

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

[0002] Cancer, also known as malignant tumor, is one of the important causes of human death, characterized by the uncontrolled growth and spread of abnormal cells in the body. Cancer cells can invade nearby tissues and organs and can spread to other parts of the body through the blood or lymphatic system, forming new tumors in the process. There are many types of cancer, which can have different effects on the human body, but ultimately will damage human health. Treatment options for cancer include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, etc.

[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 treatment method. However, due to the lack of early specific symptoms and the short cancer course and rapid progression, surgery alone cannot achieve the desired effect; another main clinical treatment method for pancreatic cancer is chemotherapy-based treatment, and some receive combined treatment of radiotherapy and chemotherapy. However, most cases of pancreatic cancer are either insensitive to chemotherapy or develop drug resistance during the treatment process, and the 5-year survival rate of patients is relatively low.

[0004] Polypeptide conjugate drug (PDC) is an emerging targeted therapy method that can improve the penetrability and selectivity of tumors. PDC mainly consists of three parts - polypeptide, linker, and cytotoxic payload.

[0005] Gastrin-releasing peptide receptor (GRPr) is a member of the bombesin G protein-coupled receptor family. Recent studies have shown that abnormal expression of GRPr is associated with various cancers, including prostate cancer, renal cancer, lung cancer, pancreatic cancer, breast cancer, and colorectal cancer, indicating that GRPr can be used as a biomarker for cancer.

[0006] PDCs are a research hotspot that has received extensive attention in tumor treatment. For example, the PDC molecules and their applications disclosed in Chinese patents CN114728089A and CN101965358B. However, their applications in PC treatment are currently less. The literature reports PDC molecules with good water solubility and tumor targeting: RGD-HSA-GEM / CURNPs (ANovel Delivery SystemofRGD-HSA Loaded GEM / CURNanoparticles for the Treatment ofPancreatic CancerTherapy), which mainly inhibits the growth of PC by inhibiting DNA synthesis.

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

[0008] The purpose of the present invention is to provide a polypeptide-conjugated drug and its preparation method and use. The polypeptide has the characteristics of high selectivity, strong specificity, small molecular weight, easy availability, no immunogenicity, safety and reliability, etc. The polypeptide-conjugated drug can significantly enhance the therapeutic effect of tumor drugs.

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

[0010] P-L-Drug

[0011] (I)

[0012] Wherein,

[0013] The P contains the polypeptide represented by the formula (II)

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

[0015] (II)

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

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

[0018] X 3Selected 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-form or L-form;

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

[0022] Drug is an anti-tumor agent.

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

[0024] Preferably, the derivatives of said amino acids are 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 (citrulline), 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] More preferably, the derivatives of said amino acids are 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), 3-Me-Phe (3-methylphenylalanine).

[0026] In some specific embodiments, 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 derivatives of said amino acids;

[0030] X4 Selected from Ala, Leu, Phe, Val, Trp, Tyr or derivatives of said amino acids.

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

[0032] In some specific embodiments, the 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 polypeptides represented by general formulas (I) to (IV) are modified or unmodified. The modification forms include but are not limited to acetylation, carboxylation, alkylation, acylation, carbamylation.

[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, the linker L comprises an uncleavable linker or a cleavable linker. The uncleavable 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 group, a dipeptide linker, a tripeptide linker, a tetrapeptide linker, a peptidomimetic linker, a β-glucuronidase-cleavable linker, a β-galactosidase-cleavable linker, a phosphatase cleavage-based linker, a pH-sensitive linker, a sulfatase-cleavable linker, 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, a valine-alanine (Val-Ala) dipeptide linker.

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

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

[0080] In some specific embodiments, L is selected from non-cleavable linkers or cleavable linkers. The non-cleavable linkers are selected from PEG linkers, linkers with thioether groups, linkers with oxime groups or combinations thereof; the cleavable linkers are selected from linkers with disulfide groups, valine-citrulline (Val-Cit) dipeptide linker, phenylalanine-lysine (Phe-Lys) dipeptide linker, valine-alanine (Val-Ala) dipeptide linker, β-glucuronidase-cleavable linker, β-galactosidase-cleavable linker, phosphatase cleavage-based linker, pH-sensitive linker, sulfatase-cleavable linker or combinations thereof.

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

[0082] In some specific embodiments, L comprises the following structure or combinations thereof,

[0083]

[0084]

[0085] m is independently selected from integers from 1 to 24.

[0086] Preferably, m is independently selected from integers from 1 to 8.

[0087] Preferably, m is independently selected from integers from 1 to 4.

[0088] In some specific embodiments, the L structure further comprises a PAB structure, and the PAB structure is

[0089] In some specific embodiments, the L structure further comprises a PABC spacer, and the PABC spacer structure is The carbonyl group in the structure is connected to the Drug moiety.

[0090] In some specific embodiments, the L structure further contains a β-Ala spacer group.

[0091] In some specific embodiments, the L structure further contains a [Sar]n spacer group, where n is an integer selected from 1 to 15.

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

[0093] In some specific embodiments, the L structure further contains a β-Ala-[Sar]n spacer group, where n is an integer selected from 1 to 15, and the carboxyl group in [Sar]n is connected to the P part by an amide bond.

[0094] In some specific embodiments, the L structure further contains a β-Ala-[Sar]n spacer group, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0095] In some specific embodiments, L is selected from the following structures or combinations thereof,

[0096]

[0097] wherein m is independently an integer selected from 1 to 24.

[0098] Preferably, m is independently an integer selected from 1 to 8.

[0099] Preferably, m is independently an integer selected from 1 to 4.

[0100] In some specific embodiments, L is selected from the following structures or combinations thereof,

[0101] m is independently an integer selected from 1 to 4.

[0102] In some specific embodiments, L is selected from the following structures or combinations thereof, m is independently an integer selected from 1 to 4.

[0103] In some specific embodiments, L is

[0104] In some specific embodiments, L is selected from the following structures or combinations thereof, m is independently an integer selected from 1 to 4.

[0105] In some specific embodiments, L is

[0106] In some specific embodiments, L is selected from the following structures or combinations thereof, m is independently selected from integers of 1 to 4.

[0107] In some specific embodiments, L is

[0108] In some specific embodiments, L is

[0109]

[0110] In some specific embodiments, the Drug is selected from microtubule-disrupting drugs and DNA-damaging drugs.

[0111] In some specific embodiments, the Drug is selected from dolastatin and its auristatin derivatives (MMAE, MMAF, MMAD), maytansine and maytansinoid derivatives (DM1, DM2, DM3, DM4), Tubulysins, cryptophycins, kinesins, gemcitabine, pyrrolo[2,1-c][1,4]benzodiazepines, duocarmycins, camptothecin and camptothecin derivatives (irinotecan, topotecan), calicheamicin, amanitin, paclitaxel, vinblastine, vincristine, etoposide, doxorubicin, cyclophosphamide, docetaxel, methotrexate, cisplatin, cytarabine, melphalan and chlorambucil or combinations thereof.

[0112] Preferably, the Drug is selected from MMAE, MMAF, PTX, gemcitabine, Dxd (Deruxtecan).

[0113] More preferably, the Drug is selected from MMAE, gemcitabine, Dxd.

[0114] The polypeptide conjugate drug of the present invention is selected from the following structures,

[0115]

[0116]

[0117]

[0118] On the other hand, the present invention provides an application of the above-mentioned polypeptide conjugate drug in the preparation of a cancer targeted therapeutic drug.

[0119] In some specific embodiments, the application is an application of the polypeptide conjugate drug in the preparation of a cancer targeted therapeutic drug for GRPR-positive.

[0120] Furthermore, the GRPR-positive cancers are 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, uterine cancer, and further preferably, the GRPR-positive cancer is pancreatic cancer.

[0121] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that play a role similar to that of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that have been subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (e.g., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics are chemical compounds whose structure is different from the general chemical structure of amino acids but play a role similar to that of naturally occurring amino acids.

[0122] The amino acid sequences of the present invention contain the standard single-letter or three-letter codes of twenty natural amino acids.

[0123] When the listed linking groups do not specify their linking directions, the linking directions are arbitrary.

[0124] 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, the embodiments formed by their combination with other chemical synthesis methods, and equivalent replacement methods 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

[0125] Figure 1 It is the liquid-phase detection diagram of Compound 1;

[0126] Figure 2 It is the mass spectrometry detection diagram of Compound 1;

[0127] Figure 3 It is the liquid-phase detection diagram of Compound 2;

[0128] Figure 4 It is the mass spectrometry detection diagram of Compound 2;

[0129] Figure 5 It is the liquid-phase detection diagram of Compound 3;

[0130] Figure 6 It is the mass spectrometry detection diagram of Compound 3;

[0131] Figure 7 It is the liquid-phase detection chart of Compound 4;

[0132] Figure 8 It is the mass spectrometry detection chart of Compound 4;

[0133] Figure 9 It is the liquid-phase detection chart of Compound 5;

[0134] Figure 10 It is the mass spectrometry detection chart of Compound 5;

[0135] Figure 11 It is the liquid-phase detection chart of Compound 6;

[0136] Figure 12 It is the mass spectrometry detection chart of Compound 6;

[0137] Figure 13 It is the liquid-phase detection chart of Compound 7;

[0138] Figure 14 It is the mass spectrometry detection chart of Compound 7;

[0139] Figure 15 It is the liquid-phase detection chart of Compound 8;

[0140] Figure 16 It is the mass spectrometry detection chart of Compound 8;

[0141] Figure 17 It is the liquid-phase detection chart of Compound 9;

[0142] Figure 18 It is the mass spectrometry detection chart of Compound 9;

[0143] Figure 19 It is the liquid-phase detection chart of Compound 10;

[0144] Figure 20 It is the mass spectrometry detection chart of Compound 10;

[0145] Figure 21 It is the liquid-phase detection chart of Compound 12;

[0146] Figure 22 It is the mass spectrometry detection chart of Compound 12;

[0147] Figure 23 It is the statistical chart of the killing effect IC 50 value of the small molecule compound in Example 12 on different cells;

[0148] Figure 24 It is the imaging picture of the internalization of the positive polypeptide of the present invention causing GRPR receptor in Example 13;

[0149] Figure 25Images of GRPR receptor endocytosis induced by some compounds (Compound 1 - Compound 7) of the present invention in Example 13;

[0150] Figure 26 Images of GRPR receptor endocytosis induced by some compounds (Compound 8, Compound 9, Compound 10, Compound 12) of the present invention in Example 13;

[0151] Figure 27 IC of the killing effect of the polypeptide - conjugated drug of the present invention on different cells in Example 14 50 value statistical chart; wherein, Figure A shows the IC of the killing effect of MMAE on different cells 50 statistical results; Figure B shows the IC of the killing effect of gemcitabine on different cells 50 statistical results; Figure C shows the IC of the killing effect of Dxd on different cells 50 statistical results; Figure D shows the IC of the killing effect of Compound 4 on different cells 50 statistical results; Figures E - N respectively show the IC of the killing effect of Compounds 1, 2, 3, 5, 6, 7, 8, 9, 10, 12 on different cells 50 statistical results;

[0152] Figure 28 Mouse plasma stability results of some compounds (Compound 1 - Compound 5) of the present invention in Example 15;

[0153] Figure 29 Mouse plasma stability results of some compounds (Compound 8, Compound 9, Compound 10, Compound 12) of the present invention in Example 15;

[0154] Figure 30 Human plasma stability results of some compounds (Compound 4 - Compound 7) of the present invention in Example 16;

[0155] Figure 31 Human plasma stability results of some compounds (Compound 1 - Compound 5) of the present invention in Example 16;

[0156] Figure 32 Human plasma stability results of some compounds (Compound 8, Compound 9, Compound 10, Compound 12) of the present invention in Example 16;

[0157] Figure 33 Tissue distribution results of Compound 4 in Hs766T pancreatic tumor - bearing mice in Example 17;

[0158] Figure 34 Tissue distribution results of MMAE released by Compound 4 in Hs766T tumor - bearing mice in Example 17;

[0159] Figure 35 Tumor relative proliferation rate of Compound 4 and MMAE in the HPAF-II pancreatic tumor model in Example 18. Detailed implementation manners

[0160] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0161] The polypeptide compounds and their derivatives provided by the present disclosure are synthesized by a solid-phase synthesis method to synthesize their linear precursors, and the synthesis carrier is RinkAmide-AM Resin. During the synthesis process, first, the RinkAmide-AM Resin is fully swollen in N,N-dimethylformamide (DMF), and then the solid-phase carrier and the activated amino acid derivatives are repeatedly subjected to the operations of condensation → washing → deprotection of Fmoc → washing → the next round of amino acid condensation to reach the length of the polypeptide chain to be synthesized. After that, a mixed solution of trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) is reacted with the resin to cleave the polypeptide from the solid-phase carrier, and then the solid crude product of the linear precursor is obtained by precipitation with cold methyl tert-butyl ether. The crude linear precursor after cleavage is purified and separated by a C-18 reversed-phase preparative chromatographic column using a system of acetonitrile / water with 0.1% trifluoroacetic acid to obtain a polypeptide with a higher purity. The polypeptide is conjugated with the MMAE conjugate in the liquid phase, and after the reaction, the pure product of the polypeptide and its derivative is obtained by purification and separation by a C-18 reversed-phase preparative chromatographic column using a system of acetonitrile / water with 0.1% trifluoroacetic acid. Or during the synthesis process, first, the RinkAmide-AM Resin is fully swollen in N,N-dimethylformamide (DMF), and then the solid-phase carrier and the activated amino acid derivatives are repeatedly subjected to the operations of condensation → washing → deprotection of Fmoc → washing → the next round of amino acid condensation to reach the length of the polypeptide chain to be synthesized. The gemcitabine conjugate is conjugated in the solid phase. After that, a mixed solution of trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) is reacted with the resin to cleave the polypeptide from the solid-phase carrier, and then the solid crude product of the linear precursor is obtained by precipitation with cold methyl tert-butyl ether. The crude linear precursor after cleavage is purified and separated by a C-18 reversed-phase preparative chromatographic column using a system of acetonitrile / water with 0.1% trifluoroacetic acid to obtain the pure product of the polypeptide and its derivative.

[0162] Experimental reagents

[0163]

[0164]

[0165]

[0166] Example 1. Preparation of Compound 1

[0167]

[0168] Step 1: Synthesis of the linear precursor peptide chain

[0169] The linear precursor peptide chain of Compound 1:

[0170] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Leu-Nle.

[0171] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swollen in DMF for 1 h. Then the synthesis was carried out in the order from the carboxyl terminus to the amino terminus according to the linear precursor sequence. Each coupling cycle was carried out as follows:

[0172] · 20% piperidine / DMF (20% v / v, 10 mL) was used for Fmoc-deprotection twice, 8 min each time.

[0173] · The resin was rinsed with DMF 6 - 8 times until neutral pH.

[0174] · 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) were dissolved in DMF, added to the resin and reacted at room temperature for 1 h.

[0175] · The resin was rinsed with DMF 4 - 6 times before the next amino acid coupling.

[0176] After the synthesis of the linear polypeptide, the resin was rinsed with DMF 5 times.

[0177] Step 2: Cleavage of the linear precursor peptide chain

[0178] Freshly prepared cleavage cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in Step 1, and the reaction was shaken at room temperature for 2 h. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain the crude product. The crude product was washed with MTBE 3 times and dried in vacuo.

[0179] Step 3: Purification and preparation of the pure polypeptide

[0180] The crude polypeptide was dissolved in 20% aqueous acetonitrile solution, filtered through a 0.45 μm membrane, and separated by a reverse-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). Among them, the chromatographic column was a BR-C18 (Sepax) reverse-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 20 - 50% acetonitrile in 40 min. The fractions related to the product were collected, and after the purity was identified by HPLC, the fractions with >75% purity were combined and freeze-dried to obtain the pure polypeptide.

[0181] Step 4: Synthesize MMAE conjugate 1

[0182]

[0183] Step 4.1 Synthesize compound 1-2

[0184]

[0185] Step 4.1.1 Swell 840 mg (1 mmol) of 2-Chlorotrityl chloride resin in 15 mL of DCM for 1 h. Weigh Fmoc-Cit-OH (3 mmol) and diisopropylethylamine (DIEA, 6 mmol), dissolve them in 10 mL of DCM, add them to the resin, and react at room temperature for 2 h. After the reaction is completed, add the blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) and block at room temperature for 10 min. The blocked resin is washed 5 times with DCM and 5 times with DMF.

[0186] Step 4.1.2 Add the deprotection solution (10 mL) of 4-methylpiperidine: DMF (20:80, v:v) to the resin obtained in Step 4.1.1 and shake to react. The deprotection time is 25 min, divided into two times, the first time is 5 min, and the second time is 20 min. After the reaction, wash with DMF 6 - 8 times.

[0187] Weigh Fmoc-Val-OH (5 mmol), HATU (2.85 mmol), and DIPEA (6 mmol), dissolve them sufficiently in 10 mL of DMF, and then add them to the resin obtained in Step 4.1.2 and shake to react at room temperature for 2 h. After the reaction, drain the solution and wash 5 times with DMF.

[0188] Step 4.1.4 Add the deprotection solution (10 mL) of 4-methylpiperidine:DMF (20:80, v:v) to the resin obtained in Step 4.1.3, and carry out an oscillating reaction. The deprotection time is 25 min, divided into two times, the first time is 5 min and the second time is 20 min. Rinse the resin with DMF 6 - 8 times until the pH value is neutral to obtain Compound 1-2.

[0189] Step 4.2 Synthesis of Compound 1-4

[0190]

[0191] Step 4.2.1 Weigh monomethyl succinate (3 mmol), HATU (2.85 mmol), and DIPEA (6 mmol), dissolve them fully in 10 mL of DMF, and then add them to the resin obtained in Step 4.1.4. Carry out an oscillating reaction at room temperature for 2 h. After the reaction, drain the solution, wash it 5 times with DMF and 5 times with DCM to obtain Compound 1-4.

[0192] Step 4.3 Synthesis of Compound 1-5

[0193]

[0194] Step 4.3.1 Add the cleavage buffer (15 mL) of hexafluoroisopropanol:DCM (20:80, v:v) to the resin obtained in Step 4.2.1, carry out an oscillating reaction for 1 h, and repeat it twice. After each reaction, filter and collect the filtrate. Use a rotary evaporator to concentrate the collected filtrate under reduced pressure. After removing the solvent, dry it under vacuum to obtain Compound 1-5.

[0195] Step 4.4 Synthesis of Compound 1-6

[0196]

[0197] Step 4.4.1 Dissolve Compound 1-5 (1 eq) in a mixed solution of appropriate amount of DCM (10 mL) and MeOH (5 mL), add EEDQ (2 eq) and p-aminobenzyl alcohol (1.3 eq), and carry out a stirring reaction at room temperature for 16 h under nitrogen protection. Monitor the reaction result by LC-MS. After the reaction is completed, use a rotary evaporator to concentrate the reaction solution under reduced pressure to remove the solvent and obtain a solid crude product.

[0198] Step 4.4.2 The purification adopts flash column chromatography. Dissolve the crude product obtained in Step 4.4.1 with a small amount of DCM, load it into a 40 g flash silica gel column. The A / B phase solvents are DCM and MeOH respectively, the gradient is set to 0 - 15% MeOH in 30 min, and the flow rate is set to 40 mL / min. After purification, concentrate the collected fractions by using a rotary evaporator under reduced pressure to remove the solvent, and then a pale yellow solid Compound 1-6 can be obtained.

[0199] Step 4.5 Synthesis of Compound 1-7

[0200]

[0201] Step 4.5.1 Dissolve Compound 1-6 (1 eq) in an appropriate amount of DMF (10 mL), slowly add DIPEA (5 eq) and bis(p-nitrophenyl) carbonate (4 eq), and stir the reaction at room temperature for 1 hour under nitrogen protection. Monitor the reaction result by LC-MS and prepare for purification after the reaction ends.

[0202] Step 4.5.2 Purification is carried out by HPLC method. Dilute the crude product obtained in Step 4.5.1 according to the volume ratio of stock solution: acetonitrile: pure water = 1:1:0.1. The A / B phase solvents are pure water and pure acetonitrile respectively. Among them, the chromatographic column is a BR-C18 (Sepax) reversed-phase chromatographic column, the gradient is set to 40%-70% acetonitrile in 40 min, the flow rate is set to 15 mL / min, and the target fraction is collected after purification and freeze-dried to obtain the white solid Compound 1-7.

[0203] Step 4.6 Synthesis of Compound 1-9

[0204]

[0205] Step 4.6.1 Dissolve Compound 1-7 (1 eq) in an appropriate amount of DMF (1 g / 10 mL), slowly add HOBt (1.3 eq), DIPEA (3 eq) and MMAE (0.9 eq), and stir the reaction at room temperature for 16 hours under nitrogen protection. Monitor the reaction result by LC-MS and prepare for purification after the reaction ends.

[0206] Step 4.6.2 Purification is carried out by HPLC method. Inject the crude product obtained in Step 4.6.1 directly without dilution. The A / B phase solvents are pure water and pure acetonitrile respectively. Among them, the chromatographic column is a BR-C18 (Sepax) reversed-phase chromatographic column, the gradient is set to 40%-70% acetonitrile in 40 min, the flow rate is set to 15 mL / min, and the target fraction is collected after purification and freeze-dried to obtain the white solid Compound 1-9.

[0207] Step 4.7 Synthesis of Compound 1-10

[0208]

[0209] Step 4.7.1 Dissolve Compound 1-9 (1 eq) in a mixed solution of an appropriate amount of THF (3 mL) and H2O (3 mL), and then slowly add LiOH.H 2O (5 eq), under nitrogen protection, stir the reaction at room temperature for 1 hour. Monitor the reaction result by LC-MS. After the reaction is completed, concentrate THF under reduced pressure using a rotary evaporator and then adjust the pH to 7 with glacial acetic acid.

[0210] Step 4.7.2 Purification is carried out by HPLC method. Dissolve the crude product obtained in Step 4.7.1 with pure water. The A / B phase solvents are pure water and pure acetonitrile respectively. First, rinse with 10% acetonitrile for 40 min, and then set for purification with 100% acetonitrile. Among them, the chromatographic column is a BR-C18 (Sepax) reverse-phase chromatographic column, the flow rate is set at 15 mL / min, and the target fraction is collected after purification and freeze-dried to obtain the white solid compound 1-10.

[0211] Step 4.8 Synthesis of compound 1-12

[0212]

[0213] Step 4.8.1 Dissolve compound 1-10 (1 eq) in a mixed solution 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). Under nitrogen protection, stir the reaction at room temperature for 3 hours. Monitor the reaction result by LC-MS. After the reaction is completed, concentrate DCM under reduced pressure using a rotary evaporator and prepare for purification.

[0214] Step 4.8.2 Purification is carried out by HPLC method. Dilute the crude product obtained in Step 4.8.1 according to the volume ratio of stock solution: acetonitrile: pure water = 1:0.5:0.5. The A / B phase solvents are pure water and pure acetonitrile respectively. Among them, the chromatographic column is a BR-C18 (Sepax) reverse-phase chromatographic column, the gradient is set at 30-80% acetonitrile in 40 min, the flow rate is set at 15 mL / min, and the target fraction is collected after purification and freeze-dried to obtain the white solid compound 1-12 (i.e., MMAE conjugate 1).

[0215] Step 5: Conjugation of polypeptide with MMAE conjugate 1

[0216] Dissolve 40 mg of the pure polypeptide obtained in Step 3 in 4 mL of DMF, slowly add DIPEA (3 eq) and MMAE conjugate 1 (1 eq) obtained in Step 4. Under nitrogen protection, stir the reaction at room temperature for 16 hours. Monitor the reaction result by LC-MS. After the reaction is completed, directly carry out preparative purification.

[0217] Step 6: Preparation and purification of the target product

[0218] The crude product obtained in step 5 was diluted according to the volume ratio of stock solution: acetonitrile: pure water = 1:1:1, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were 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 chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 5-90% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions > 95% were combined and freeze-dried to obtain the pure target product.

[0219] Step 7: Detection and Characterization Methods

[0220] The purity of the target product in step 6 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry and the N-terminal MMAE conjugate 1 of the polypeptide compound was tested. The test results are as follows Figure 1 、 Figure 2 As shown.

[0221] Example 2. Preparation of Compound 2

[0222]

[0223] Step 1: Synthesis of linear precursor peptide chain

[0224] Linear precursor peptide chain of compound 2:

[0225] DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Leu-Nle.

[0226] The steps for synthesizing the linear precursor peptide chain are the same as those in step 1 of Example 1.

[0227] Step 2: Linear Precursor Peptide Chain Cleavage

[0228] Add freshly prepared cutting cocktail (10 mL) 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 is completed, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain a crude product. Wash the crude product 3 times with MTBE and dry it in a vacuum.

[0229] Step 3: Purification and preparation of pure peptides

[0230] The crude polypeptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were 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 chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 20-50% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >75% were combined and freeze-dried to obtain the pure polypeptide.

[0231] Step 4: Synthesis of MMAE conjugate 2

[0232]

[0233] Step 4.1 Synthesis of compound 2-2

[0234]

[0235] Step 4.1.1 Fully swell 840mg (1mmol) of 2-Chlorotrityl chloride resin in 15mL DCM for 1h. Weigh Fmoc-AEEA-OH (3mmol) and diisopropylethylamine (DIEA, 4mmol) and dissolve in 10mL DCM and add to the resin, react at room temperature for 2h. After the reaction is completed, add blocking solution (10mL) DCM: methanol: DIEA (85:10:5, v:v:v) at room temperature for 10min for blocking. The blocked resin is washed 5 times with DCM and 5 times with DMF.

[0236] Step 4.1.2 Add deprotection solution (10 mL) 4-methylpiperidine:DMF (20:80, v:v) to the resin obtained in step 4.1.1 and shake for reaction. The deprotection time is 25 min, divided into two times, the first time is 5 min, and the second time is 20 min. After the reaction, rinse the resin with DMF 6-8 times to a neutral pH value to obtain compound 2-2.

[0237] Step 4.2 Synthesis of compound 2-4

[0238]

[0239] Step 4.2.1 Weigh monomethyl succinate (3 mmol), HATU (2.85 mmol), and DIPEA (6 mmol) and fully dissolve them in 10 mL DMF, then add them to the resin obtained in step 4.1.4 and shake at room temperature for 2 h. After the reaction, drain the solution, wash with DMF 5 times, and wash with DCM 5 times to obtain compound 2-4.

[0240] Step 4.3 Synthesis of Compound 2-5

[0241]

[0242] Step 4.3.1 Add cleavage buffer (15 mL) hexafluoroisopropanol:DCM (20:80, v:v) to the resin obtained in Step 4.2.1, shake the reaction for 1 h, and 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.

[0243] Step 4.4 Synthesis of Compound 2-7

[0244]

[0245] Step 4.4.1 Dissolve Compound 2-5 (1 eq) in an appropriate amount of DMF (1 g / 10 mL), slowly add HOBt (1.3 eq), DIPEA (3 eq) and MMAE (0.9 eq), and stir the reaction at room temperature for 16 h under nitrogen protection. Monitor the reaction result by LC-MS. After the reaction is completed, prepare for purification.

[0246] Step 4.4.2 Purification is carried out by HPLC. Inject the crude product obtained in Step 4.4.1 directly without dilution. The A / B phase solvents are pure water and pure acetonitrile respectively. Among them, the chromatographic column is a BR-C18 (Sepax) reversed-phase chromatographic column. The gradient is set to 40%-70% acetonitrile in 40 min, and the flow rate is set to 15 mL / min. After purification, collect the target fraction and freeze-dry to obtain the white solid Compound 2-7.

[0247] Step 4.5 Synthesis of Compound 2-8

[0248]

[0249] Step 4.5.1 Dissolve Compound 2-7 (1 eq) in a mixed solution of an appropriate amount of THF (3 mL) and H2O (3 mL), and then slowly add LiOH·H2O (5 eq). Stir the reaction at room temperature for 1 h under nitrogen protection. Monitor the reaction result by LC-MS. After the reaction is completed, concentrate THF by rotary evaporation under reduced pressure and adjust the pH = 7 using glacial acetic acid.

[0250] Step 4.5.2 Purification is carried out by HPLC. Dissolve the crude product obtained in Step 4.5.1 in pure water. The A / B phase solvents are pure water and pure acetonitrile respectively. First, rinse with 10% acetonitrile for 40 min, and then set for purification with 100% acetonitrile. Among them, the chromatographic column is a BR-C18 (Sepax) reversed-phase chromatographic column, and the flow rate is set to 15 mL / min. After purification, collect the target fraction and freeze-dry to obtain the white solid Compound 2-8.

[0251] Step 4.6 Synthesis of Compound 2-10

[0252]

[0253] Step 4.6.1 Dissolve Compound 2-8 (1 eq) in a mixed solution 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), and stir the reaction at room temperature for 3 hours under nitrogen protection. Monitor the reaction result by LC-MS. After the reaction is completed, concentrate DCM under reduced pressure using a rotary evaporator and prepare for purification.

[0254] Step 4.6.2 Purification is carried out by HPLC method. Dilute the crude product obtained in Step 4.6.1 according to the volume ratio of stock solution: acetonitrile: pure water = 1:0.5:0.5. The A / B phase solvents are pure water and pure acetonitrile respectively. Among them, the chromatographic column is a BR-C18 (Sepax) reversed-phase chromatographic column, the gradient is set to 30-80% acetonitrile in 40 min, and the flow rate is set to 15 mL / min. After purification, collect the target fraction and freeze-dry to obtain the white solid Compound 2-10 (i.e., MMAE conjugate 2).

[0255] Step 5: Peptide Conjugation with MMAE Conjugate 2

[0256] Dissolve 40 mg of the pure peptide obtained in Step 3 in 4 mL of DMF, slowly add DIPEA (3 eq) and MMAE conjugate 1 (1 eq) obtained in Step 4, and stir the reaction at room temperature for 16 hours under nitrogen protection. Monitor the reaction result by LC-MS. After the reaction is completed, directly carry out preparative purification.

[0257] Step 6: Preparation and Purification of the Target Product

[0258] Dilute the crude product obtained in Step 5 according to the volume ratio of stock solution: acetonitrile: pure water = 1:1:1, filter it through a 0.45 μm membrane, and separate it using a reversed-phase high-performance liquid chromatography system. The buffer solutions are A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). Among them, the chromatographic column is a BR-C18 (Sepax) reversed-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph is set to 230 nm, the flow rate is 15 mL / min, and the gradient is 35-65% acetonitrile in 40 min. Collect the fractions related to the product, combine the fractions with a purity > 95% after HPLC identification, and freeze-dry to obtain the pure target product.

[0259] Step 7: Detection and Characterization Methods

[0260] The pure target product from Step 6 was analyzed by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry to determine the purity and the N-terminal MMAE conjugate 2 of the polypeptide compound, and the test results are as Figure 3 , Figure 4 shown.

[0261] Example 3. Preparation of Compound 3

[0262]

[0263] Step 1: Synthesis of the linear precursor peptide chain

[0264] The linear precursor peptide chain of Compound 3:

[0265] DSer-DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle.

[0266] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swollen in DMF for 1 h. Then the linear precursor sequence was synthesized in the order from the carboxyl terminus to the amino terminus. Each coupling cycle was carried out as follows:

[0267] · 20% piperidine / DMF (20% v / v, 10 mL) was used for Fmoc-deprotection twice, 8 min each time.

[0268] · The resin was rinsed with DMF 6 - 8 times until neutral pH.

[0269] · 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) were dissolved in DMF, added to the resin and reacted at room temperature for 1 h.

[0270] · The resin was rinsed with DMF 4 - 6 times before the next amino acid coupling.

[0271] After the synthesis of the linear polypeptide, the resin was rinsed with DMF 5 times.

[0272] Step 2: Cleavage of the linear precursor peptide chain

[0273] Freshly prepared cleavage cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in Step 1, and the reaction was shaken at room temperature for 2 h. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid and combined with the reaction solution. The crude product was precipitated with 4 volumes of cold MTBE. The crude product was washed with MTBE 3 times and dried in vacuo.

[0274] Step 3: Purification and preparation of pure peptides

[0275] After the crude polypeptide was dissolved in 20% acetonitrile aqueous solution, it was filtered through a 0.45um membrane and separated using a reversed-phase high-performance liquid chromatography system. The buffers were 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 chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 20-50% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >75% were combined and freeze-dried to obtain the pure polypeptide.

[0276] Step 4: Peptide coupling MMAE conjugate 1

[0277] 40 mg of the pure peptide obtained in step 3 was dissolved in 4 mL of DMF, and DIPEA (3 eq) and MMAE conjugate 1 (1 eq) were slowly added. Under nitrogen protection, the mixture was stirred at room temperature for 16 hours. The reaction results were monitored by LC-MS, and the preparation and purification were directly carried out after the reaction was completed.

[0278] Step 5: Preparation and purification of target product

[0279] The crude product obtained in step 4 was diluted according to the volume ratio of stock solution: acetonitrile: pure water = 1:1:1, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase chromatographic column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 40-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions > 95% were combined and freeze-dried to obtain the pure target product.

[0280] Step 6: Detection and Characterization Methods

[0281] The purity of the target product of step 5 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry and the N-terminal MMAE conjugate 1 of the polypeptide compound was tested. The test results are as follows Figure 5 、 Figure 6 As shown.

[0282] Example 4. Preparation of Compound 4

[0283]

[0284] Step 1: Synthesis of linear precursor peptide chain

[0285] ​Linear precursor peptide chain of Compound 4: DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle.

[0286] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swollen in DMF for 1 h. Then the linear precursor sequence was synthesized in the order from the carboxyl terminus to the amino terminus. Each coupling cycle was carried out as follows:

[0287] · 20% piperidine / DMF (20% v / v, 10 mL) was used for Fmoc-deprotection twice, 8 min each time.

[0288] · The resin was rinsed with DMF 6 - 8 times until neutral pH.

[0289] · 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) were dissolved in DMF, added to the resin and reacted at room temperature for 1 h.

[0290] · The resin was rinsed with DMF 4 - 6 times before the next amino acid coupling.

[0291] After the synthesis of the linear polypeptide, the resin was rinsed with DMF 5 times.

[0292] Step 2: Cleavage of the linear precursor peptide chain

[0293] Freshly prepared cleavage cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in Step 1, and the reaction was shaken at room temperature for 2 h. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid and combined with the reaction solution. The crude product was precipitated with 4 volumes of cold MTBE. The crude product was washed 3 times with MTBE and dried in vacuo.

[0294] Step 3: Purification and preparation of the pure polypeptide

[0295] The crude polypeptide was dissolved in 20% aqueous acetonitrile solution, filtered through a 0.45 μm membrane and separated by a reverse-phase high performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). Among them, the chromatographic column was a BR-C18 (Sepax) reverse-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 20 - 50% acetonitrile in 40 min. The relevant fractions of the product were collected, and the fractions with >75% purity were combined after HPLC identification, freeze-dried to obtain the pure polypeptide.

[0296] Step 4: Peptide coupling MMAE conjugate 1

[0297] 40 mg of the pure peptide obtained in step 3 was dissolved in 4 mL of DMF, and DIPEA (3 eq) and MMAE conjugate 1 (1 eq) were slowly added. Under nitrogen protection, the mixture was stirred at room temperature for 16 hours. The reaction results were monitored by LC-MS, and the preparation and purification were directly carried out after the reaction was completed.

[0298] Step 5: Preparation and purification of target product

[0299] The crude product obtained in step 4 was diluted according to the volume ratio of stock solution: acetonitrile: pure water = 1:1:1, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were 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 chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 45-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions > 95% were combined and freeze-dried to obtain the pure target product.

[0300] Step 6: Detection and Characterization Methods

[0301] The purity of the target product of step 5 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry and the N-terminal MMAE conjugate 1 of the polypeptide compound was tested. The test results are as follows Figure 7 、 Figure 8 As shown.

[0302] Example 5. Preparation of Compound 5

[0303]

[0304] Step 1: Synthesis of linear precursor peptide chain

[0305] The linear precursor peptide chain of compound 5: DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle.

[0306] The steps for synthesizing the linear precursor peptide chain are the same as those in step 1 of Example 4.

[0307] Step 2: Linear Precursor Peptide Chain Cleavage

[0308] Add freshly prepared cutting cocktail (10 mL) 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 is completed, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain a crude product. Wash the crude product 3 times with MTBE and dry it in a vacuum.

[0309] Step 3: Purification and preparation of pure peptides

[0310] After the crude polypeptide was dissolved in 20% acetonitrile aqueous solution, it was filtered through a 0.45um membrane and separated using a reversed-phase high-performance liquid chromatography system. The buffers were 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 chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 20-50% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >75% were combined and freeze-dried to obtain the pure polypeptide.

[0311] Step 4: Peptide coupling MMAE conjugate 2

[0312] 40 mg of the pure peptide obtained in step 3 was dissolved in 4 mL of DMF, and DIPEA (3 eq) and MMAE conjugate 2 (1 eq) were slowly added. Under nitrogen protection, the mixture was stirred at room temperature for 16 hours. The reaction results were monitored by LC-MS, and the preparation and purification were carried out directly after the reaction was completed.

[0313] Step 5: Preparation and purification of target product

[0314] The crude product obtained in step 4 was diluted according to the volume ratio of stock solution: acetonitrile: pure water = 1:1:1, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase chromatographic column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 40-65% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions > 95% were combined and freeze-dried to obtain the pure target product.

[0315] Step 6: Detection and Characterization Methods

[0316] The purity of the target product of step 5 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry and the N-terminal MMAE conjugate 2 of the polypeptide compound was tested. The test results are as follows Figure 9 、 Figure 10as shown

[0317] Example 6. Preparation of Compound 6

[0318]

[0319] Step 1: Synthesis of the linear precursor peptide chain

[0320] The linear precursor peptide chain of Compound 6: DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle.

[0321] The synthesis steps of the linear precursor peptide chain are the same as those in Step 1 of Example 4.

[0322] Step 2 Synthesis of Gemcitabine Conjugate 1

[0323]

[0324] Step 2.1 Synthesis of Compound 3-2

[0325]

[0326] Step 2.1.1 Dissolve di-tert-butyl dicarbonate (DBDC) (44 mg, 0.2 mmol) in a mixed solution of dioxane (4 mL) and water (1 mL), slowly add Compound 3-1, namely gemcitabine (500 mg, 0.2 mmol), and 800 mg of sodium carbonate. Under nitrogen protection, stir the reaction at room temperature for 48 h. After the reaction is completed, add 2 mL of water to the reaction solution, and then add 2 - 30 mL of ethyl acetate for extraction. The obtained organic phase is washed with 5 mL of water and 5 mL of saturated brine, and then dried over anhydrous sodium sulfate and filtered. The reaction solution is concentrated under reduced pressure using a rotary evaporator to remove the solvent, and a crude solid is obtained.

[0327] Step 2.1.2 Purification is carried out by flash column chromatography. Dissolve the crude product obtained in Step 2.1.1 in a small amount of DCM, load it into a 40 g flash silica gel column, the B-phase solvent is CH2Cl2 - acetone - EtOH 1:1:0.02, the gradient is set to 100% B in 30 min, and the flow rate is set to 40 mL / min. After purification, the collected fractions are concentrated under reduced pressure using a rotary evaporator to remove the solvent, and then the solid Compound 3-2 can be obtained.

[0328] Step 2.2 Synthesis of Compound 3-3

[0329]

[0330] Step 2.2.1 Dissolve compound 3-2 (73 mg, 0.2 mmol) in 8 mL of dioxane, add di-tert-butyl dicarbonate (DBDC) (436 mg, 2 mmol), and react with shaking at 37 °C for 70 h. After the reaction, concentrate the reaction solution by rotary evaporation under reduced pressure to remove the solvent and obtain a crude solid.

[0331] Step 2.2.2 Purification is carried out by flash column chromatography. Dissolve the crude product obtained in Step 2.2.1 in a small amount of DCM, load it onto a 40 g flash silica gel column, and the solvents for phases A / B are DCM and acetone respectively. The gradient is set to 10-20% acetone in 30 min, and the flow rate is set to 40 mL / min. After purification, concentrate the collected fractions by rotary evaporation under reduced pressure to remove the solvent, and then compound 3-3 can be obtained.

[0332] Step 2.3 Synthesis of compound 3-4

[0333]

[0334] 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, and add DIPEA (1.13 mL, 6.47 mmol). Stir the reaction at room temperature for 16 h. After the reaction, concentrate the reaction solution by rotary evaporation under reduced pressure to remove the solvent and obtain a crude solid.

[0335] Step 2.3.2 Purification is carried out by HPLC. Dissolve the crude product obtained in Step 4.5.1 in pure water. The solvents for phases A / B are pure water and pure acetonitrile respectively. Among them, the chromatographic column is a BR-C18 (Sepax) reversed-phase chromatographic column, the gradient is set to 10-95%, and the flow rate is set to 15 mL / min. After purification, collect the target fractions and freeze-dry them to obtain the white solid compound 3-4, namely gemcitabine conjugate 1.

[0336] Step 3: Solid-phase coupling of gemcitabine conjugate 1

[0337] 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, add them to the resin obtained in Step 1, and react with shaking at room temperature for 16 h. After the reaction, wash the resin with DMF 5 times and with DCM 5 times. Dry the resin in vacuo.

[0338] Step 4: Cleavage of the linear precursor peptide chain

[0339] Add freshly prepared cutting cocktail (10 mL) 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 is completed, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain a crude product. Wash the crude product 3 times with MTBE and dry it in a vacuum.

[0340] Step 5: Preparation and purification of peptides

[0341] After the crude polypeptide was dissolved in 50% acetonitrile aqueous solution, it was filtered through a 0.45um membrane and separated using a reversed-phase high-performance liquid chromatography system. The buffers were 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 chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 25-45% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide.

[0342] Step 6: Detection and Characterization Methods

[0343] The purity of the peptide obtained in step 5 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry, and the N-terminus of the peptide compound was coupled to gemcitabine conjugate 1. The test results are as follows Figure 11 、 Figure 12 As shown.

[0344] Example 7. Preparation of Compound 7

[0345]

[0346] Step 1: Synthesis of linear precursor peptide chain

[0347] The linear precursor peptide chain of compound 7: AEEA-DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle.

[0348] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swelled in DMF for 1 hour. Then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0349] ·20% piperidine / DMF (20% v / v, 10 mL) was used for Fmoc-deprotection twice, each time for 8 min.

[0350] · Rinse the resin 6-8 times with DMF until the pH is neutral.

[0351] Dissolve 1.0mmol Fmoc-AA, 1.0mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol 4-methylmorpholine (NMM) in DMF, add the resin and react at room temperature for 1h.

[0352] · Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0353] After the linear peptide synthesis, rinse the resin with DMF 5 times.

[0354] Step 2: Solid phase coupling of gemcitabine conjugate 1

[0355] Dissolve 0.4mmol gemcitabine conjugate, 1,1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol 4-methylmorpholine (NMM) in DMF, add to the resin obtained in step 1, and shake at room temperature for 16h. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM. Drain the resin in a vacuum.

[0356] Step 3: Linear Precursor Peptide Chain Cleavage

[0357] Add freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 3 and shake the reaction at room temperature for 2 hours. After the reaction is completed, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain a crude product. Wash the crude product 3 times with MTBE and dry it in a vacuum.

[0358] Step 4: Preparation and purification of peptides

[0359] After the crude polypeptide was dissolved in 50% acetonitrile aqueous solution, it was filtered through a 0.45um membrane and separated using a reversed-phase high-performance liquid chromatography system. The buffers were 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 chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 25-55% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide.

[0360] Step 5: Detection and Characterization Methods

[0361] The purity of the peptide obtained in step 4 was determined by analytical HPLC and LC / MS and the N-terminus of the peptide compound was coupled to gemcitabine conjugate 1. The test results were as follows:​​Figure 13 , Figure 14 as shown

[0362] Example 8. Synthesis of Compound 8

[0363]

[0364] Step 1: Synthesis of the linear precursor peptide chain

[0365] The linear precursor peptide chain of Compound 8:

[0366] βAla-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle

[0367] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swollen in DMF for 1 h. Then the linear precursor sequence was synthesized in the order from the carboxyl terminus to the amino terminus. Each coupling cycle was carried out as follows:

[0368] · 20% piperidine / DMF (20% v / v, 10 mL) was used for Fmoc-deprotection twice, 8 min each time.

[0369] · The resin was rinsed with DMF 6 - 8 times until neutral pH.

[0370] · 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) were dissolved in DMF, added to the resin and reacted at room temperature for 1 h.

[0371] · The resin was rinsed with DMF 4 - 6 times before the next amino acid coupling.

[0372] After the synthesis of the linear polypeptide, the resin was rinsed with DMF 5 times.

[0373] Step 2: Cleavage of the linear precursor peptide chain

[0374] Freshly prepared cleavage cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in Step 1, and the reaction was shaken at room temperature for 2 h. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid and combined with the reaction solution. The crude product was precipitated with 4 volumes of cold MTBE. The crude product was washed with MTBE 3 times and dried in vacuo.

[0375] Step 3: Purification and preparation of the pure polypeptide

[0376] After the crude polypeptide was dissolved in 20% acetonitrile aqueous solution, it was filtered through a 0.45um membrane and separated using a reversed-phase high-performance liquid chromatography system. The buffers were 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 chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 25-40% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >75% were combined and freeze-dried to obtain the pure polypeptide.

[0377] Step 4: Peptide coupling MMAE conjugate 1

[0378] 40 mg of the pure peptide obtained in step 3 was dissolved in 4 mL of DMF, and DIPEA (3 eq) and MMAE conjugate 1 (1 eq) were slowly added. Under nitrogen protection, the mixture was stirred at room temperature for 16 hours. The reaction results were monitored by LC-MS, and the preparation and purification were directly carried out after the reaction was completed.

[0379] Step 5: Preparation and purification of target product

[0380] The crude product obtained in step 4 was diluted according to the volume ratio of stock solution: acetonitrile: pure water = 1:1:1, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were 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 chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 35-55% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions > 95% were combined and freeze-dried to obtain the pure target product.

[0381] Step 6: Detection and Characterization Methods

[0382] The purity of the target product of step 5 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry and the N-terminal MMAE conjugate 1 of the polypeptide compound was tested. The test results are as follows Figure 15 、 Figure 16 As shown.

[0383] Example 9. Synthesis of Compound 9

[0384]

[0385] Step 1: Synthesis of linear precursor peptide chain

[0386] Linear precursor peptide chain of compound 9:

[0387] βAla-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle

[0388] The synthesis steps of the linear precursor peptide chain are the same as those in Step 1 of Example 8.

[0389] Step 2: Cleavage of the linear precursor peptide chain

[0390] Add freshly prepared cleavage cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) to the resin obtained in Step 1, and react with shaking 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 with 4 volumes of cold MTBE to obtain the crude product. Wash the crude product with MTBE 3 times and dry it in vacuo.

[0391] Step 3: Purification and preparation of the pure polypeptide

[0392] Dissolve the crude polypeptide in 20% aqueous acetonitrile solution, filter it through a 0.45 um membrane, and then separate it using a reverse-phase high-performance liquid chromatography system. The buffers are A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). Among them, the chromatographic column is a BR-C18 (Sepax) reverse-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph is set at 230 nm, the flow rate is 15 mL / min, and the gradient is 25 - 40% acetonitrile in 40 min. Collect the relevant fractions of the product, combine the fractions with a purity > 75% after HPLC identification, and lyophilize to obtain the pure polypeptide.

[0393] Step 4: Synthesis of MMAE conjugate 3

[0394]

[0395] Step 4.1 Synthesis of compound 4-1

[0396]

[0397] 8.4 g (10 mmol) of 2-Chlorotrityl chloride resin was fully swollen in 150 mL of DCM for 1 h. 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 the reaction was shaken for 2 h. After the reaction was completed, it was washed 3 times with DCM. 100 mL of blocking solution (85% DCM + 10% methanol + 5% DIPEA) was added, and the reaction was carried out for 10 min. After the reaction was completed, it was washed 5 times with DCM and 5 times with DMF. An appropriate amount of 20% 4-methylpiperidine / 80% DMF was added to the above resin and shaken on a shaker. The deprotection time was 25 min, divided into two times, the first time was 5 min, and the second time was 20 min. A small amount of resin was taken out for ninhydrin detection. If the ninhydrin detection was positive, the next step was carried out; otherwise, this step was repeated for 10 min, and the resin was rinsed 6 - 8 times with DMF until the pH value was neutral. The prepared amino acid solution (5 eq Fmoc-Val-OH, 2.85 eq HATU, 6 eq DIPEA dissolved in 100 mL of DMF) was added to the resin, and the reaction was shaken for 2 h. A small amount of resin was taken out for ninhydrin detection. If the ninhydrin detection was negative, the next step was carried out; otherwise, this step was repeated for one hour. After the reaction was completed, it was washed 3 times with DMF. The deprotection and coupling steps were repeated, and Fmoc-Glu(otBu)-OH was coupled in turn. After the reaction was completed, it was washed 3 times with DMF. Subsequently, the resin was cleaved with a 30% hexafluoroisopropanol / 70% DCM solution, and the precipitate was precipitated with tert-butyl methyl ether. The precipitate was dried in vacuo to obtain compound 4-1.

[0398] Step 4.2 Synthesis of compound 4-2

[0399]

[0400]

[0401] Compound 4-1 (2.12 g, 1.0 eq) was dissolved in DMF (5 mL), EEDQ (1.54 g, 2.0 eq) and (4-aminophenyl) methanol (0.77 g, 2.0 eq) were added, and the mixture was stirred at 20 °C for 16 h. The reaction result was monitored by LC-MS. After the reaction was completed, it was purified by HPLC and freeze-dried to obtain 1.12 g of a white solid.

[0402] Step 4.3 Synthesis of compound 4-3

[0403]

[0404] 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 reaction was carried out at room temperature for 3 hours. The reaction result was monitored by LC-MS. After the reaction was completed, it was purified by HPLC and freeze-dried to obtain white solid compound 4-3.

[0405] Step 4.4 Synthesis of compound 4-4

[0406]

[0407] Compound 4-3 (328 mg, 1 eq) was dissolved in an appropriate amount of DMF (1 g / 10 mL), and HOBt (0.2 eq, 9 mg), pyridine (8 eq, 181 mg) and MMAE (0.85 eq, 210 mg) were slowly added. The reaction was carried out at room temperature for 16 hours. The reaction result was monitored by LC-MS. After the reaction was completed, 4-methylpiperidine (0.8 eq) was added and the reaction continued at room temperature for 30 minutes. A small amount of the reaction solution was taken for LCMS monitoring. After the reaction was completed, it was purified. The purification was carried out by HPLC, and after purification, it was freeze-dried to obtain white solid compound 4-4.

[0408] Step 4.5 Synthesis of compound 4-5

[0409]

[0410] Compound 4-4 (304 mg, 1 eq) was dissolved in 3 mL of DMF solution, and glutaric anhydride (34 mg, 2 eq) and N,N-diisopropylethylamine (DIEA) (48 mg, 2 eq) were added. The reaction solution was stirred at room temperature for 3 h. The reaction result was monitored by LC-MS. After the reaction was completed, it was purified. The purification was carried out by HPLC, and the obtained crude product was purified and then freeze-dried to obtain white solid compound 4-5.

[0411] Step 4.6 Synthesis of compound 4-6

[0412]

[0413] Compound 4-5 (1 eq, 231 mg) was dissolved in a mixed solution of DMF (6 mL) and DCM (2 mL), and EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (3 eq, 93 mg) and NHS (N-hydroxysuccinimide) (3 eq, 56 mg) were slowly added. The reaction was carried out at room temperature for 3 hours; the reaction result was monitored by LC-MS. After the reaction was completed, DCM was concentrated under reduced pressure using a rotary evaporator and then purified. In this experiment, the purification was carried out by HPLC, and after purification, it was freeze-dried to obtain white solid compound 4-6.

[0414] Step 4.7 Synthesis of compound 4-7

[0415]

[0416] Compound 4-6 (188 mg, 1 eq) was dissolved in DCM (19 mL), TFA (1.9 mL) was slowly added, and the reaction was allowed to proceed at room temperature for 30 minutes; the reaction results were monitored by LC-MS. After the reaction was completed, DCM was concentrated under reduced pressure using a rotary evaporator and then purified. This experiment used HPLC to purify the compound, and after purification, freeze-dried to obtain a white solid compound 4-7 (i.e., MMAE conjugate 3).

[0417] Step 5: Peptide coupling MMAE conjugate 3

[0418] 18 mg of the pure peptide obtained in step 3 was dissolved in 2 mL of DMF, and DIPEA (30 eq, 35 mg) and MMAE conjugate 3 (13 mg, 1 eq) obtained in step 4 were slowly added. The mixture was stirred at room temperature for 16 hours under nitrogen protection. The reaction results were monitored by LC-MS, and the preparation and purification were directly carried out after the reaction was completed.

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

[0420] The crude product obtained in step 5 was diluted according to the volume ratio of stock solution: acetonitrile: pure water = 1:1:1, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were 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 chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure target product.

[0421] Step 7: Detection and Characterization Methods

[0422] The purity of the target product of step 6 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry and the N-terminal MMAE conjugate 3 of the polypeptide compound was tested. The test results are as follows Figure 17 、 Figure 18 As shown.

[0423] Example 10. Synthesis of Compound 10

[0424]

[0425] Step 1: Synthesis of linear precursor peptide chain

[0426] Linear precursor peptide chain of compound 10: ​

[0427] βAla-Sar-Sar-Sar-Sar-DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle。

[0428] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swollen in DMF for 1 h. Then the synthesis was carried out in the order from the carboxyl terminus to the amino terminus according to the linear precursor sequence. Each coupling cycle was carried out as follows:

[0429] · 20% piperidine / DMF (20% v / v, 10 mL) was used for Fmoc-deprotection twice, 8 min each time.

[0430] · The resin was rinsed with DMF 6 - 8 times until neutral pH.

[0431] · 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) were dissolved in DMF, added to the resin and reacted at room temperature for 1 h.

[0432] · The resin was rinsed with DMF 4 - 6 times before the next amino acid coupling.

[0433] After the synthesis of the linear polypeptide, the resin was rinsed with DMF 5 times.

[0434] Step 2: Cleavage of the linear precursor peptide chain

[0435] Freshly prepared cleavage cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in Step 1, and the reaction was shaken at room temperature for 2 h. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid and combined with the reaction solution. The crude product was precipitated with 4 volumes of cold MTBE. The crude product was washed 3 times with MTBE and dried in vacuo.

[0436] Step 3: Purification and preparation of the pure polypeptide

[0437] The crude polypeptide was dissolved in 20% aqueous acetonitrile solution, filtered through a 0.45 um membrane and separated by a reverse-phase high performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). Among them, the chromatographic column was a BR-C18 (Sepax) reverse-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 25 - 45% acetonitrile in 40 min. The relevant fractions of the product were collected. After the purity was identified by HPLC, the fractions with > 75% purity were combined and lyophilized to obtain the pure polypeptide.

[0438] Step 4: Peptide coupling MMAE conjugate 3

[0439] 18 mg of the pure peptide obtained in step 3 was dissolved in 2 mL of DMF, and DIPEA (30 eq, 35 mg) and MMAE conjugate 3 (13 mg, 1 eq) were slowly added. The mixture was stirred at room temperature for 16 hours under nitrogen protection. The reaction results were monitored by LC-MS, and the preparation and purification were directly carried out after the reaction was completed.

[0440] Step 5: Preparation and purification of target product

[0441] The crude product obtained in step 4 was diluted according to the volume ratio of stock solution: acetonitrile: pure water = 1:1:1, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase chromatographic column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 5-90% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions > 95% were combined and freeze-dried to obtain the pure target product.

[0442] Step 6: Detection and Characterization Methods

[0443] The purity of the target product of step 5 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry and the N-terminal MMAE conjugate 3 of the polypeptide compound was tested. The test results are as follows Figure 19 、 Figure 20 As shown.

[0444] Example 11. Synthesis of Compound 12

[0445]

[0446] Step 1: Synthesis of linear precursor peptide chain

[0447] The linear precursor peptide chain of compound 12: DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle.

[0448] The steps for synthesizing the linear precursor peptide chain are the same as those in step 1 of Example 4.

[0449] Step 2: Linear Precursor Peptide Chain Cleavage

[0450] Add freshly prepared cleavage cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) to the resin obtained in Step 1, and shake the reaction 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 with 4 volumes of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it in vacuo.

[0451] Step 3: Purification and preparation of pure polypeptide

[0452] Dissolve the crude polypeptide in 20% aqueous acetonitrile solution, filter it through a 0.45 μm membrane, and separate it using a reverse-phase high-performance liquid chromatography system. The buffers are A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). Among them, the chromatographic column is a BR-C18 (Sepax) reverse-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph is set at 230 nm, the flow rate is 15 mL / min, and the gradient is 20 - 50% acetonitrile in 40 min. Collect the relevant fractions of the product, combine the fractions with a purity > 75% after HPLC identification, and lyophilize to obtain pure polypeptide.

[0453] Step 4 Synthesis of Dxd conjugate 1

[0454]

[0455] Step 4.1 Synthesis of compound 5-1 (Fmoc-GGFGG-OH)

[0456]

[0457] 25.2 g (30 mmol) of 2-Chlorotrityl chloride resin was fully swollen in 400 mL of DCM for 1 h. After the resin was swollen, the DCM was drained. ① The prepared amino acid solution (3 eq Fmoc-G-OH, 6 eq DIPEA) was added, and the reaction was shaken for 2 h. After the reaction was completed, it was washed 3 times with DCM. 300 mL of blocking solution (85% DCM + 10% methanol + 5% DIPEA) was added, and the reaction was carried out for 10 min. After the reaction was completed, it was washed 5 times with DCM and 5 times with DMF. An appropriate amount of 20% 4-methylpiperidine / 80% DMF was added to the above resin and shaken on a shaker. The deprotection time was 25 min, divided into two times, the first time was 5 min, and the second time was 20 min. A small amount of resin was taken out for ninhydrin detection. If the ninhydrin detection was positive, the next step was carried out. Otherwise, this step was repeated for 10 min, and the resin was rinsed 6 - 8 times with DMF until the pH value was neutral; ② The prepared amino acid solution (3 eq Fmoc-G-OH, 2.85 eq HATU, 6 eq DIPEA) was added to the resin, and the above steps were repeated; ③ The prepared amino acid solution (3 eq Fmoc-F-OH, 2.85 eq HATU, 6 eq DIPEA) was added to the resin and the above steps were repeated; ④ The prepared amino acid solution (3 eq Fmoc-G-OH, 2.85 eq HATU, 6 eq DIPEA) was added to the resin and the above steps were repeated; ⑤ The prepared amino acid solution (3 eq Fmoc-G-OH, 2.85 eq HATU, 6 eq DIPEA) was added to the resin and the above steps were repeated; Finally, after the reaction was completed, it was washed 3 times with DMF; Subsequently, the resin was cleaved with a 30% hexafluoroisopropanol / 70% DCM solution, and the precipitate was precipitated with tert-butyl methyl ether. The precipitate was dried in vacuo to obtain the white solid compound Fmoc-GGFGG-OH.

[0458] Step 4.2 Synthesis of Compound 5-2

[0459]

[0460] Compound 5-1 (4 g, 6.50 mmol), Pd(OAc) 4 / acetic acid solution (8.64 g), 120 mL of THF and 40 mL of toluene were added to a 50 mL round-bottomed two-necked flask. Under nitrogen protection, the flask was placed in an ice-water bath and stirred for 10 minutes, then pyridine (1.3 mL, 16.8 mmol) was slowly added, and then the temperature was raised to 50 °C and the reaction was carried out for about 3 h. After the reaction was complete, the reaction solution was concentrated in vacuo and purified by column chromatography to obtain a pale yellow solid compound 5-2.

[0461] Step 4.3 Synthesis of Compound 5-3

[0462]

[0463] Into a 25 mL round-bottom flask, add 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. React at 0 °C for about 3 h under nitrogen protection. After monitoring the reaction to completion by LCMS, concentrate the reaction solution in vacuo and purify by column chromatography to obtain the white solid compound 5-3.

[0464] Step 4.4 Synthesis of compound 5-4

[0465]

[0466] Into a 100 mL round-bottom flask, add compound 5-3 (1.06 g, 1.44 mmol), Pd / C (389 mg), 25 mL of ethanol and 15 mL of ethyl acetate. After displacing hydrogen three times, react at room temperature for 2 h. After monitoring the reaction to completion by TLC, filter the reaction solution and concentrate the filtrate in vacuo to obtain 800 mg of the white solid compound 5-4.

[0467] Step 4.5 Synthesis of compound 5-5

[0468]

[0469] Into a 25 mL round-bottom flask, add compound DXd-002-4 (800 mg, 1.24 mmol), irinotecan mesylate (478 mg, 0.9 mmol), HOBt (165 mg, 1.22 mmol), EDCI (250.8 mg, 1.3 mmol), 5 mL of DMSO and 4 mL of THF. Under nitrogen protection, add triethylamine (121 mg, 1.20 mmol) dropwise and react at room temperature for 4 h. After monitoring the reaction to completion by LCMS, subject it to preparative purification. Concentrate the preparative solution in vacuo and lyophilize to obtain 560 mg of the gray solid compound DXd-002-5.

[0470] Step 4.6 Synthesis of compound 5-6

[0471]

[0472] Into a 25 mL round-bottom flask, add compound 5-5 (560 mg, 0.50 mmol) and 12 mL of THF. Under nitrogen protection, add DBU (80 mg, 0.52 mmol) dropwise and react at room temperature for 3 h. After monitoring the reaction to completion by TLC, concentrate the reaction solution in vacuo and recrystallize with methyl tert-butyl ether to obtain 394 mg of the gray solid compound 5-6.

[0473] Step 4.7 Synthesis of compound 5-7

[0474]

[0475] Compound 5-6 (210 mg, 0.25 mmol), glutaric anhydride (57 mg, 0.5 mmol), and 2 mL DMF were added to a 10 mL round-bottom flask, and DIEA (80 mg, 0.62 mmol) was added dropwise under nitrogen protection, and the reaction was carried out at room temperature for 3 h. After the reaction was completed by TLC monitoring, EDCI (206 mg, 1.07 mmol) and NHS (123 mg, 1.01 mmol) were added to the reaction solution and the reaction was continued for 8 h. After the reaction was completed by LCMS monitoring, the solution was sent for preparation and purification, and the preparation solution was vacuum concentrated and freeze-dried to obtain a white solid compound 5-7 (i.e., Dxd conjugate 1).

[0476] Step 5: Peptide coupling Dxd conjugate 1

[0477] Take 23.5 mg of the pure peptide obtained in step 3 and dissolve it in 2 ml of DMF, slowly add DIPEA (30 eq, 35 mg) and Dxd conjugate 1 (20 mg, 1 eq) obtained in step 4, and stir at room temperature for 3 hours under nitrogen protection. LC-MS monitors the reaction results, and the preparation and purification are carried out directly after the reaction is completed.

[0478] Step 6: Preparation and purification of target product

[0479] The crude product obtained in step 5 was diluted according to the volume ratio of stock solution: acetonitrile: pure water = 1:1:1, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were 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 chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure target product.

[0480] Step 7: Detection and Characterization Methods

[0481] The purity of the target product of step 6 was determined by analytical HPLC and LC / MS and the N-terminal Dxd conjugate 1 of the peptide compound was detected as shown in Figure 21 、 Figure 22 As shown.

[0482] Example 12: Test of small molecule compounds killing tumor cells (screening cell experiment)

[0483] 1. Materials and Methods

[0484] 1.1 Experimental materials:

[0485] ​​Small molecule compounds: Monomethyl aunistatin E (MMAE, GLPBIO), Gemcitabine (gemcitabine, GLPBIO), Monomethylauristatin F (MMAF, MCE), PTX (paclitaxel, Medicinal Chemistry Department), 5-Fluorouracil (5-Fu, MCE).

[0486] Experimental cells: SW1990 (Procell), CFPAC-1 (Procell), HPAF-II (BNCC), BxPC-3 (Procell), PANC-1 (Procell), Capan-1 (Procell), Capan-2 (Procell), HUTU-80 (Procell), Hs 766T (Nanjing Kebai Biotechnology).

[0487] 1.2 Reagents and consumables related to the experiment: 96-well transparent cell culture plates (Agilent); 96-well black clear-bottom plates (Jing'an); 1×DPBS

[0488] (self-made); 96-well transparent cell culture plates (Corning); Cell Counting Kit-8 (GLPBIO).

[0489] 1.3 Experimental instruments: Cytation5 multi-functional microplate reader (BioTek).

[0490] 2. Experimental methods:

[0491] (1) Cell culture:

[0492] SW1990 and PANC-1 cells are cultured and grown in the medium (DMEM, 10% FBS, 1% double antibody (penicillin-streptomycin mixture)). When the cell growth density reaches 80-90% of the culture flask, first rinse the cells with DPBS, and then digest the cells with 0.25% trypsin (containing 0.5 mM EDTA); then add the corresponding growth medium to terminate the digestion, collect the cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 3 min, and remove the supernatant medium; then add 3-5 mL of fresh growth medium to resuspend the cells, and passage them at a ratio of 1:3 to 1:8, and place them in a 37°C, 5% CO2 incubator for culture. Change the medium or passage every 2-3 days after passage.

[0493] Hs766T cells were cultured and grown in a medium (DMEM, 15% FBS, 1% double antibody (penicillin-streptomycin mixture)). 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); then the corresponding growth medium was added to terminate the digestion. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 min, and the supernatant medium was removed; then 3 - 5 mL of fresh growth medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:3 to 1:8, and placed in an incubator at 37°C with 5% CO 2 and cultured. After passage, the medium was changed or the cells were passaged every 2 - 3 days.

[0494] CFPAC-1 and Capan-1 cells were cultured and grown in a medium (IMDM, 10% FBS, 1% double antibody (penicillin-streptomycin mixture)). 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); then the complete medium was added to terminate the digestion. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 min, and the supernatant medium was removed; then 3 - 5 mL of fresh growth medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:3 to 1:8, and placed in an incubator at 37°C with 5% CO 2 and cultured. After passage, the medium was changed or the cells were passaged every 2 - 3 days.

[0495] HPAF-II and HUTU-80 cells were cultured and grown in a medium (MEM, 10% FBS, 1% double antibody (penicillin-streptomycin mixture)). 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); then the complete medium was added to terminate the digestion. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 min, and the supernatant medium was removed; then 3 - 5 mL of fresh growth medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:3 to 1:8, and placed in an incubator at 37°C with 5% CO 2 and cultured. After passage, the medium was changed or the cells were passaged every 2 - 3 days.

[0496] BxPC-3 cells were cultured and grown in a medium (RPMI-1640, 10% FBS, 1% double antibody (penicillin-streptomycin mixture)). 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); then the corresponding growth medium was added to terminate the digestion. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 min, and the supernatant medium was removed; then 3 - 5 mL of fresh growth medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:3 to 1:8, and placed in an incubator at 37°C with 5% CO2 Cultured in an incubator. Change the medium or passage the cells every 2 - 3 days after passage.

[0497] Capan - 2 cells were cultured and grown in a medium (McCoy's 5A, 10% FBS, 1% double antibody (penicillin - streptomycin mixture)). 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); then the corresponding growth medium was added to terminate the digestion. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 min, and the supernatant medium was removed; then 3 - 5 mL of fresh growth medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:3 to 1:8, and placed in an incubator at 37°C with 5% CO 2 Cultured in an incubator. Change the medium or passage the cells every 2 - 3 days after passage.

[0498] (2) Experimental procedures

[0499] Amplify all the above cells by passage to the required cell number respectively. After digesting and resuspending the cells, incubate them in a 96-well cell culture plate at a cell density of 6000 cells / well and 100 μL / well of cell resuspension for 1 day for experimental detection. First, dilute the above small molecule compounds to 2× concentration using complete medium according to the following cell usage concentrations: The final concentration of MMAF is 30 μM on CFPAC-1, Capan-1, Capan-2 and SW1990 cells, and 10 μM on HPAF-II, PANC-1, Bx-PC3 and HUTU-80 cells, and dilute them in 7 concentrations with a 10-fold gradient; The final concentration of gemcitabine is 10 μM on HPAF-II, PANC-1, Bx-PC3, Capan-1, Capan-2 and HUTU-80 cells, 1 μM on CFPAC-1 cells, 30 μM on SW1990 cells, and 60 μM on Hs766T cells, and dilute them in 7 concentrations with a 5-fold gradient; The final concentration of MMAE is 10 nM on PANC-1, Bx-PC3, CFPAC-1 and HUTU-80 cells, 1 μM on Capan-1 and Capan-2 cells, 10 μM on SW1990 cells, and 400 nM on HPAF-II and Hs766T cells, and dilute them in 7 concentrations with a 5-fold gradient; The final concentration of PTX is 100 nM on HPAF-II, PANC-1, Bx-PC3, CFPAC-1 and HUTU-80 cells, 1 μM on Capan-1 and Capan-2 cells, 10 μM on SW1990 cells, and 400 nM on Hs766T cells, and dilute them in 7 concentrations with a 5-fold gradient; The final concentration of 5-Fluorouracil is 30 μM on HPAF-II, PANC-1, Bx-PC3, CFPAC-1, Capan-1 and Capan-2 cells, and 78.75 μM on SW1990 and HUTU-80 cells, and dilute them in 7 concentrations with a 10-fold gradient.

[0500] Then remove 100 μL of the supernatant from the cell plate, and add 100 μL of each of the above-diluted small molecule compounds, make 3 replicates, and place them in an incubator at 37 °C and 5% CO 2 Incubate for 72 h. Finally, add 10 μL of CCK8 cell viability detection reagent, place it in an incubator at 37 °C and 5% CO 2 Incubate for 1 h, and use a Cytation5 multifunctional microplate reader to detect the OD 450 value.

[0501] The experimental results are as Figure 15, as shown in Table 1.

[0502] Table 1 Statistical table of the killing effects of small molecule compounds on different cells, IC 50 value:

[0503]

[0504] It can be seen from the experimental results 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 has no obvious killing effect on other cells. According to the above table, three tumor cells, HPAF-II, Hs766T, and HUTU-80, with obvious cell killing curves and positive GRPR expression shown in the previous data, were selected as the cells for the subsequent verification experiment. The small molecule compound MMAE with strong cell killing effect and gemcitabine, a small molecule compound reported in the literature for the treatment of pancreatic cancer, were selected as the combined drug molecules for the subsequent development of PDC drugs and used as the naked drug control.

[0505] Example 13: Test for GRPR receptor endocytosis on HEK293-GRPR-GFP cells

[0506] 1. Materials and methods

[0507] 1.1 Experimental materials

[0508] PDC drug (self-made synthetic polypeptide combined drug): the polypeptide conjugate drug of the present invention.

[0509] Positive polypeptide (self-made synthetic or outsourced polypeptide): 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.

[0510] 1.2 Cells and related reagents and consumables

[0511] HEK293-GRPR-GFP cells (obtained by transfection and screening); G418 (Aladdin, 11811031 / ant-gn-5 / G110917-5); FBS (Hyclone, SV30208.02); trypsin (Gibco, 27250-018). 96-well black bottom transparent cell culture plate (Agilent), fixing solution (Beyotime); DAPI (Beyotime); 1×DPBS (self-made).

[0512] 2. Experimental method:

[0513] (1) Cell preparation: HEK293 - GRPR - GFP cells were cultured and grown in a 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); then the corresponding growth medium was added to terminate the digestion. The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 min, and the supernatant medium was removed; then 3 - 5 mL of fresh growth medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:3 - 1:8 and placed in an incubator at 37°C and 5% CO 2 2. The culture was carried out in an incubator. The medium was changed or the cells were passaged every 2 - 3 days after passage.

[0514] (2) Experimental procedure: The PDC drug and the positive control polypeptide sample were diluted to 3 μM (1×) with the basal medium and then serially diluted 3 - fold, with a total of 4 concentrations. 100 μL of the medium was aspirated from the cell plate, and then 100 μL of the diluted PDC drug and the positive control polypeptide were added, and the mixture was placed in an incubator at 37°C and 5% CO 2 for 0.5 h. 100 μL of the sample solution was aspirated, and 100 μL of 1×DPBS was added to wash the cells, and this step was repeated 3 - 5 times. The 1×DPBS was aspirated, 60 μL of the fixing solution was added, and the mixture was incubated in a refrigerator at 4°C for 0.5 h. Then the cells were washed 1 - 2 times with 1×DPBS. The 1×DPBS was aspirated, 50 μL of the DAPI nuclear staining solution was added, and the mixture was placed in an incubator at 37°C and 5% CO 2 for 0.5 h. Then the cells were washed 1 - 2 times with 1×DPBS, and photographed using a Cytition10 confocal imaging system.

[0515] The endocytosis imaging pictures of the experimental results are as Figures 24 - 26 shown. The experimental results show that all the polypeptide - conjugated drugs of the present invention have obvious endocytosis, proving that these polypeptide - conjugated drugs can target GRPR and enter cells.

[0516] Example 14: Cell killing test

[0517] 1. Materials and methods

[0518] 1.1 Experimental materials:

[0519] PDC drug (self - made synthetic polypeptide - conjugated drug): The polypeptide - conjugated drug of the present invention.

[0520] Small molecule compounds: Monomethyl aunistatin E (MMAE, GLPBIO), Gemcitabine (GLPBIO).

[0521] 1.2 Experimental cells and related reagents: HEK293-GRPR-GFP cells (obtained by transfection and screening), HEK293 (Saibakang), HPAF-II (Beina Bio), HUTU-80 (Punosai), Hs 766T (Nanjing Kebai Bio); 96-well black clear-bottom cell culture plates (Agilent), 96-well transparent cell culture plates (Corning); 1×DPBS (self-made), Cell Counting Kit-8 (GLPBIO).

[0522] 1. Experimental methods:

[0523] Digest, resuspend and dilute HEK293-GRPR-GFP, HEK293, HPAF-II and HUTU-80 cells, and plate them at a cell density of 3000 cells per well; digest, resuspend and dilute Hs766T cells and plate them at a cell density of 6000 cells per well. Add them to 96-well cell culture plates with a volume of 100 μL each, and place them in an incubator at 37 °C and 5% CO 2 for 1 day.

[0524] Dilute the PDC drug to 30 μM (3×) with complete medium, and then serially dilute it 8-fold; dilute MMAE to 1.2 μM (3×) with complete medium, and then serially dilute it 5-fold; dilute gemcitabine to 30 μM (3×) with complete medium, and then serially dilute it 8-fold.

[0525] Directly add 50 μL of the PDC drug and control small molecule compounds into the cell plates, with 3 replicates for each concentration, and place them in an incubator at 37 °C and 5% CO 2 for 72 h. Add 15 μL of CCK8 cell viability detection reagent, and place it in an incubator at 37 °C and 5% CO 2 for 1 h, and then use a Cytation5 multifunctional microplate reader to detect the OD 450 value. The experimental results of the killing effect of the PDC drug on different cells are as Figure 27 shown. The IC 50 value statistical tables of the killing effect of each polypeptide conjugate drug on different cells are shown in Table 2. The test results show that the polypeptide conjugate drugs of the present invention have a killing effect on different cells. Compounds 4 and 5 using MMAE as the conjugated small molecule have significantly better tumor cell killing effects than the polypeptide conjugate drugs conjugated with gemcitabine, and the cell killing activity of Compound 4 is better than that of Compound 5.

[0526] Table 2 Statistical table of IC 50 values of the killing effect of polypeptide conjugate drugs on different cells

[0527]

[0528] Example 15: Mouse plasma (heparin sodium) stability

[0529] 1. Materials and Methods

[0530] 1.1 Experimental materials:

[0531] PDC drug (self-made synthetic peptide conjugate drug): peptide conjugate drug of the present invention.

[0532] Small molecule compound: Monomethyl aunistatin E (MMAE, GLPBIO)

[0533] Experimental related reagents: positive control (nenitride, 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).

[0534] Experimental instruments: equilibrium dialysis device HTD (Shanghai Meixing Gaode); triple quadrupole liquid chromatography mass spectrometer LCMS-8060NX (Shimadzu).

[0535] 2. Experimental methods:

[0536] Dissolve nenitide in DMSO to 1mM, dissolve TAT in DMSO to 1mM, and dissolve the peptide to be tested in DMSO to 1mM, and place at -20℃ for use. Plasma thawing: Take out plasma (sample number * 2.1)mL from the -80℃ refrigerator and quickly thaw it in a 37℃ water bath. Prepare MIX (mixture): Take 693μL of plasma and add it to a 1.5mL EP tube, prepare 3 parallel samples at each time point, and prepare 3 tubes of MIX. Add 7μL of the sample to be tested to each tube, so that the final concentration is a detectable concentration or an in vivo drug concentration of 10μM. Shake on a vortex oscillator, and incubate after each aliquot of 100μL according to the time gradient. Incubation: Incubate in a 37℃ water bath at six time points: 0min, 15min, 30min, 60min, 90min, and 120min. Stop the reaction: After incubation, add 4 times the volume of 0.1% formic acid in methanol precipitation agent. Mixing: Oscillate on a vortex oscillator for 30 seconds. Centrifugation: 4°C, 15000r / min for 10 minutes. Take the supernatant, transfer it to a small injection tube, and send it to LC-MS / MS for analysis.

[0537] 1 Experimental results: ​​

[0538] The vertical axis is the original drug remaining rate (%), and the horizontal axis is the time point line graph. It can be seen that the degradation trend of the sample in the in vitro plasma changes with time, and the result of the sample stability is obtained.

[0539]

[0540] Use GraphPad Prism 8 software according to nonlinear regression One phase decay to obtain the half-life T of the compound in plasma 1 / 2 .

[0541] Experimental results are as follows Figure 28 、 29 , as shown in Table 3, the half-life of compound 5>120min, compound 2>120min, compound 9>120min, compound 10>120min, compound 12>120min, compound 3 is 43.01min, compound 1 is 19.87min, compound 4 is 16.51min, and compound 8 is 10.76min.

[0542] Table 3 Mouse plasma (heparin sodium) stability

[0543]

[0544] Example 16: Human Plasma (Heparin Sodium) Stability

[0545] 1. Materials and Methods

[0546] 1.1 Experimental materials:

[0547] PDC drug (self-made synthetic peptide conjugate drug): peptide conjugate drug of the present invention.

[0548] Small molecule compound: Monomethyl aunistatin E (MMAE, GLPBIO)

[0549] 1.2 Experimental related reagents: positive control (nenitride, 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).

[0550] ​​1.3 Experimental instruments: Equilibrium dialysis device HTD (Shanghai Meixing Gaode); Triple quadrupole liquid chromatography-mass spectrometry LCMS-8060NX (Shimadzu).

[0551] 2. Experimental methods:

[0552] Dissolve nainipeptide in DMSO to 1 mM, dissolve TAT in DMSO to 1 mM, dissolve the polypeptide to be tested in DMSO to 1 mM, and place them at -20 °C for later use. Plasma thawing: Take out human plasma (sample number * 2.1) mL from the -80 °C refrigerator and quickly thaw it in a 37 °C water bath. Prepare MIX (mixture): Add 693 μL of plasma into a 1.5 mL EP tube, with 3 parallel samples at each time point, and prepare 3 tubes of MIX. Then add 7 μL of the sample to be tested to each tube to make the final concentration 10 μM, which is the detectable concentration or the in vivo drug concentration. Shake on a vortex oscillator for 30 s, and aliquot 100 μL according to the time gradient and incubate. Incubation: Incubate in a 37 °C water bath at six time points: 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min. Terminate the reaction: After incubation, add a methanol precipitant containing 4 times the volume of 0.1% formic acid. Mixing: Shake on a vortex oscillator for 30 s. Centrifugation: Centrifuge at 4 °C and 15000 r / min for 10 min. Take the supernatant, transfer it to an injection vial, and send it to LC-MS / MS for analysis.

[0553] 3. Experimental results: A dot-line graph with the ordinate as the remaining rate of the original drug (%) and the abscissa as time. It can be seen that there is a change trend of the sample degrading in vitro plasma over time, and the results of sample stability are obtained.

[0554]

[0555] Using GraphPad Prism 8 software, according to nonlinear regression One phase decay, the half-life T of the compound in plasma was obtained 1 / 2 .

[0556] The experimental results are shown in Table 4, Figures 30 - 32 as follows. The half-lives are 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 is 55.54 min, and Compound 6 is 9.438 min.

[0557] Table 4 Stability in human plasma (sodium heparin)

[0558]

[0559]

[0560] Example 17: Tissue Distribution Study in HS766T Pancreatic Tumor-bearing Mice

[0561] 1. Materials and Methods

[0562] 1.1 PDC drug (self-made synthetic polypeptide conjugate): The polypeptide conjugate drug of the present invention.

[0563] 1.2 Related reagents and consumables: 1 mL insulin syringe, 1.5 mL EDTA anticoagulant tube, 0.3 mL EP tube, EDTA, protease inhibitor, methanol, Monomethyl auristatin E (MMAE).

[0564] 1.3 Experimental animals: 12 male Balb / c nu mice, 6 - 7 weeks old, purchased from Hunan An Sheng Mei Pharmaceutical Research Institute Co., Ltd.

[0565] 2. Experimental Methods

[0566] Balb / c male nude mice were raised for 6 - 7 weeks under standard conditions. Human pancreatic cancer cells Hs766T were suspended in serum-free medium and mixed with Matrigel at a ratio of 1:1. Then, 1*10 6 cells were inoculated subaxillarily into each mouse. When the tumor volume reached 100 - 200 mm 3 , the mice were randomly divided into 4 groups, with 3 mice in each group. 8 μL of EDTA and protease inhibitor were respectively added to the EDTA anticoagulant tube, and compound 4 (2 mg / kg) was injected into the tail vein of the mice according to their body weight. After the administration, the mice were anesthetized at 0.5 h, 1 h, 2 h, and 4 h, and about 200 μL of blood was collected from the ophthalmic plexus vein of the mice. After collecting the blood with EDTA, it was immediately centrifuged, and 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, perfusion was immediately carried out, and then the liver, kidney, pancreas, and muscle tissues were taken, washed with normal saline, blotted dry with filter paper, stored on dry ice, and then transferred to a -80°C refrigerator.

[0567] A protease inhibitor mixture containing 2% and 0.5 M EDTA (pH = 8.0) were added to the mouse tissues, and then a PBS solution containing 50% methanol and 1.0% formic acid with 5 times the volume was added. The tissues were placed in a grinder to grind and homogenize, and then vortexed for 3 min. After homogenization, 50 μL of the sample was taken into an EP tube, 50 μL of the diluent was added, and then 200 μL of the precipitant (0.1% formic acid methanol solution) was added, and vortexed for 3 min. Then, it was centrifuged at 13000 rpm for 10 min, and the supernatant was taken for injection.

[0568] 3. Experimental Results

[0569] The distribution of compound 4 in the tissues of Hs766T pancreatic tumor-bearing mice was determined by LC-MS, and the results are as follows Figure 33 shown. The content of compound 4 in the tissues of tumor-bearing mice decreased with time. Among them, the content in plasma was the highest, followed by the liver and kidneys, and the distribution in the pancreas, tumor and muscle was less. In addition, the distribution of MMAE released by compound 4 in various tissues of tumor-bearing mice was further determined, as follows Figure 34 shown. The concentration of MMAE in the plasma and kidneys of mice decreased with time, increased first and then decreased in the liver of mice, and reached the peak concentration at 1 h. The content of MMAE in the muscle, tumor and pancreas of mice increased with time.

[0570] Example 18: Pharmacodynamic study of HPAF-II pancreatic tumor-bearing mice

[0571] 1. Materials and methods

[0572] 1.1 PDC drug (self-made synthetic polypeptide conjugate drug): the polypeptide conjugate drug of the present invention.

[0573] 1.2 Related reagents and consumables: 1 mL insulin syringe, 0.5 mL EP tube, Monomethyl auristatin E (MMAE), DMSO, Solutol HS-15.

[0574] 1.3 Experimental animals: 30 male Balb / c nu mice, 6-7 weeks old, purchased from Hunan Anshengmei Pharmaceutical Research Institute Co., Ltd.

[0575] 2. Experimental methods

[0576] Male Balb / C nu mice were raised for 6-7 weeks under standard conditions. Human pancreatic cancer cells HPAF-II were suspended in serum-free medium, and 1×10 6 tumor cells were inoculated into each mouse. When the tumor volume reached 100-150 mm 3 , the mice were randomly divided into 6 groups, with 5 mice in each group. They were respectively injected with compound 4 (0.61 mg / kg), compound 4 (1.22 mg / kg), compound 4 (1.83 mg / kg), Vehicle (2% DMSO + 2% Solutol HS-15 + 96% normal saline) via the tail vein. The administration volume was 10 mL / kg, the administration cycle was once every 4 days, and the administration was continued for 4 times. The experiment was terminated 4 days after the last administration. After the experiment, the animals were sacrificed by cervical dislocation, and the tumor volume (tumor volume (V) = long diameter × short diameter^2 / 2), RTV (relative tumor volume = Vt / V0) and other indexes of each group of animals were compared by the t-test method.

[0577] 3. Experimental Results

[0578] During the experimental observation, the body weights of the mice in the MMAE (0.18 mg / kg), Compound 4 (0.61 mg / kg), Compound 4 (1.22 mg / kg), and Vehicle groups were basically maintained within the range tolerable for animal toxic and side reactions during the 4 - time administration. The body weight of the mice in the Compound 4 (1.83 mg / kg) group showed a gradually decreasing trend. As shown in Table 5, with the increase of the administration time, compared with the Vehicle group, the other groups all showed a trend of tumor proliferation inhibition, and compared with MMAE at the same dose, Compound 4 had a better anti - tumor effect than MMAE. At the same time, as can be seen from Figure 35 it that with the increase of the administration dose, the anti - pancreatic tumor HPAF - II proliferation effect of Compound 4 became more obvious, and the tumor volume showed a regression trend. The experimental endpoint data showed that under the conditions of 1.22 mg / kg and 1.83 mg / kg, after the first administration, the relative tumor growth rates were 55.82% and 34.63% respectively, and after the end of the administration, the relative tumor proliferation rates were 13.44% and 3.90% respectively. This proves that under medium - dose 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.

[0579] Table 5 Growth Inhibitory Effects of Compound 4 and MMAE on Pancreatic Cancer Tumor Models in Tumor - bearing Mice at Different Doses

[0580]

[0581]

[0582] The above - mentioned embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above - mentioned embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope 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 represented by formula (IV) DSer-DTyr-Gln-Trp-Ala-X 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: The P is selected from the following polypeptides; DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle (SEQ ID No. 1), DSer-DTyr-Gln-Trp-Ala-Leu-βAla-His-Leu-Nle (SEQ ID 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.

Images