CD44-targeted multi-arm conjugate

Abstract

The present invention discloses a multi-branched drug conjugate that can specifically target CD44. This type of compound specifically binds to CD44 and can target tumor cells and tissues that highly express CD44, so that the concentration of the conjugate in the target tissue is higher, its clinical treatment effect is improved, and toxicity is reduced. The compound of the present invention is suitable for treating all tumors that highly express CD44, including but not limited to gastric cancer, pancreatic cancer, small cell lung cancer, colon cancer, breast cancer, lung adenocarcinoma, liver cancer, nasopharyngeal carcinoma, malignant glioma, lymphoma, kidney cancer, ovarian cancer, head and neck cancer, squamous cell carcinoma, etc.

Description

Technical Field

[0001] The present invention generally relates to a multi-arm targeting conjugate, and in particular, to a multi-arm conjugate of a CD44 targeting molecule and an anticancer agent that act synergistically. Background Art

[0002] The morbidity and mortality rates of malignant tumors have shown a clear upward trend in recent years, posing a serious threat to human health.

[0003] CD44 is a group of membrane-integrated glycoproteins with a wide distribution and diverse molecular forms, including extracellular regions, transmembrane regions, and membrane-proximal regions. As a type I transmembrane glycoprotein, CD44 first binds to hyaluronic acid, triggering intracellular signaling pathways, which in turn leads to a series of cell shedding, metastasis, and invasion reactions. As a member of the cell adhesion molecule family, CD44 plays an important role in regulating the adhesion and binding between cells and between cells and the extracellular matrix, and is very important for maintaining the integrity of tissue structure. CD44 can also transmit intracellular signals and regulate the occurrence and activity of tumor cells, such as breast cancer and colorectal cancer. Currently, a large number of studies believe that CD44 is closely related to the occurrence, development, infiltration, and metastasis of tumors. The expression of CD44 often appears in the early stages of tumor occurrence, and as the tumor develops, the difference becomes more significant. For example, CD44 is expressed in some precancerous lesions.

[0004] "CD44 targeting polypeptide" refers to a polypeptide that can bind to CD44 in a targeted manner. Typical "CD44 targeting polypeptides" include polypeptides having the following structures: Ac-KPSSPPEE-NH2 and Ac-NASAPPEE-NH2. The above two polypeptides have sequence homology with part of the linking domain of CD44 and can bind to CD44 in a targeted manner.

[0005] Among them, the polypeptide with the structure of Ac-KPSSPPEE-NH2 is also called A6 polypeptide. The structure of A6 is as follows:

[0006]

[0007] The peptide structure of Ac-NASAPPEE-NH2 is as follows:

[0008]

[0009] WO2005028539, WO2010019233, WO2011063156, and WO2011063158 disclose a drug nktr-102 in phase III clinical trials. The drug is mainly used for metastatic breast cancer and is developed by Nektar Therapeutics. The drug is a water-soluble multi-branched polymer drug precursor to increase drug loading, and its structure is as follows:

[0010]

[0011] The compound is connected to irinotecan using multi-arm PEG to improve water solubility and drug loading, and reduce side effects without changing the anti-cancer effect. However, the drug still has disadvantages, such as poor targeting, inability to act on specific cancer cells, and while killing cancer cells, it also affects the performance of normal cells, resulting in a relatively high incidence of adverse reactions.

[0012] Chinese patent application CN 108727583A discloses a multi-arm polymer targeted anticancer conjugate, which not only exhibits high loading capacity but also has targeting properties, making the conjugate have a higher concentration in the target tissue. However, due to the abnormal structure, function and metabolism of malignant tumor cells, cell division and proliferation are endless and disorderly, making them very difficult to eliminate, requiring drug developers to continuously develop anticancer drugs with better efficacy. Summary of the invention

[0013] The present invention discloses a multi-branched drug conjugate with targeting, wherein the conjugate has three or more branches and can be represented by the following formula:

[0014]

[0015] R is an organic center, POLY is a polymer, L is a multivalent linker, T is a targeting molecule, D is an active agent, and q is any integer between 3-8.

[0016] Organic Center, "R"

[0017] In formula (I), "R" is an organic core group of 1-100 atoms. Preferably, R contains 3-50 atoms, and more preferably, R contains about 3-30 atoms. R can be a core composed entirely of carbon atoms, and can optionally contain one or more heteroatoms, such as O, S, N, P, etc., depending on the specific central molecule used. R can be linear, branched or cyclic, emitting at least 3 independent polymer branches. In formula (I), "q" corresponds to the number of polymer branches emanating from "R".

[0018] In some specific embodiments, "R" has three polymer branches, and "R" is preferably:

[0019]

[0020] In some specific embodiments, "R" has four polymer branches, and "R" is preferably:

[0021]

[0022] In some specific embodiments, "R" has six polymer branches, and "R" is preferably:

[0023]

[0024] In some specific embodiments, "R" has eight polymer branches, and "R" is preferably:

[0025]

[0026] Polymer, “POLY”

[0027] In the structural formula (I), "POLY" is a polymer, preferably a water-soluble polymer, any water-soluble polymer can be used to form the conjugate of the present invention, and the polymer referred to in the present invention can be of any geometric shape or form. Representative polymers include but are not limited to: polyethylene glycol, polypropylene glycol, poly (vinyl pyrrolidone), poly (hydroxyalkyl methacrylic amine), poly (hydroxyalkyl methacrylate), poly (sugar), poly (α-hydroxy acid), poly (acrylic acid), poly (vinyl acetate), polyphosphazene, polyoxazoline, poly (N-acryloylmorpholine), etc.

[0028] In typical compounds, "POLY" is polyethylene glycol, which can be in any geometric shape or form, including straight chain, branched chain, forked chain, etc. Preferably, the preferred "POLY" of the present invention is linear polyethylene glycol, and the typical structure is:

[0029] Represents the connection of atoms, and the oxygen atom marked with "&" is the atom connected to the organic center "R". The value range of k is about 5 to 500, preferably 50 to 200, and preferably 113. The average molecular weight is about 1k to 60kDa. Those skilled in the art should know that in the field of polymers, k represents the degree of polymerization of the polymer, that is, the average number of repeating units contained in the polymer macromolecular chain, which depends on the molecular weight of the polymer. For example, when k is 113, it means that the average value is 113.

[0030] r is any integer between 1 and 10. More preferably, "POLY" of the present invention is:

[0031]

[0032] POLY of the present invention can also be:

[0033]

[0034] wait.

[0035] In a preferred embodiment of the present invention, "POLY" is a linear polyethylene glycol linker, that is, the conjugate of the present invention is preferably a compound of the following types:

[0036]

[0037] In the present invention, the multivalent linker L is preferably:

[0038]

[0039] The symbol "*" here represents the connection point between the multivalent linker L and the targeting molecule T, "#" represents the connection point between the multivalent linker L and the active agent D, and "%" represents the connection point between the multivalent linker L and POLY. Wherein, m is any integer between 1-20, and l and n are any integers between 1-10. Most preferably, l is 5, m is 3, and n is 4, that is, the preferred multivalent linker L of the present invention is

[0040]

[0041] D is a camptothecin drug as shown below:

[0042]

[0043] R1-R5 are independently selected from the following groups: hydrogen, halogen, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, alkynyl, cycloalkyl, hydroxyl, cyano, nitro, azido, amide, hydrazine, amino, substituted amino, hydroxycarbonyl, alkoxycarbonyl, alkoxycarbonyloxy, carbamoyloxy, arylsulfonyloxy, alkylsulfonyloxy; R6 is H or OR8, R8 is alkyl, alkenyl, cycloalkyl, haloalkyl or hydroxyalkyl; R7 is hydroxyl, amino, or thiol. Preferably, D is irinotecan or SN38.

[0044] Among them, the structure of irinotecan is as follows:

[0045]

[0046] The structure of SN38 is as follows:

[0047]

[0048] T is a targeting molecule, which is a "CD44 targeting polypeptide" that can bind to CD44. Typical "CD44 targeting polypeptides" include polypeptides having the following sequence:

[0049] Ac-KPSSPPEE-NH2; Ac-NASAPPEE-NH2; QETWFQNGWQGKNP;

[0050] KEKWFENEWQGKNP;KEQWFGNRWHEGYR;

[0051] QIRQQPRDPPTETLELEVSPDPAS.

[0052] Also included are substitution variants, addition variants and chemically modified derivatives of the above polypeptides.

[0053] T can also be a polypeptide disclosed in WO99 / 05263, i.e., a polypeptide having the following sequence or a substitution variant, addition variant, and chemically modified derivative thereof: Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu (KPSSPPEE), Asn-Ala-Ser-Ala-Pro-Pro-Glu-Glu (NASAPPEE). In addition, T can also be a FKBP-L polypeptide disclosed in WO2007141533.

[0054] The targeting molecule T may also be a monoclonal antibody or its natural ligand that can bind to CD44. Natural ligands that can bind to CD44 include but are not limited to hyaluronic acid (HA), osteopontin (OPN), serglycin, collagen, fibronectin, laminin, chondroitin sulfate (CSC), heparan sulfate (HS), ankyrin, galectin-3, L-selectin, P-selectin, C-type lectin, and addressin, etc.

[0055] The preferred targeting molecules T of the present invention are Ac-KPSSPPEE-NH2 and Ac-NASAPPEE-NH2. The two polypeptides are connected to the multivalent linker L via the blocked NH2 end.

[0056] Preferred compounds of the present invention are:

[0057]

[0058] In compound a, D is irinotecan and T is A6, i.e. Ac-KPSSPPEE-NH2.

[0059]

[0060] In compound b, D is irinotecan and T is Ac-NASAPPEE-NH2.

[0061]

[0062] In compound c, D is SN38 and T is A6, i.e. Ac-KPSSPPEE-NH2

[0063]

[0064] In compound d, D is SN38 and T is Ac-NASAPPEE-NH2.

[0065] The present invention is a multi-arm polymer-modified targeted anticancer conjugate, wherein the water-soluble polymer modification can enhance the water solubility of the conjugate, thereby increasing the drug loading; the targeting molecule increases the targeting property, so that the concentration of the conjugate in the target tissue is higher; L is an arbitrary connecting linker, which first connects the targeting molecule with the anticancer drug, and then connects the "targeting molecule, anticancer drug and polymer arm, so that the entire conjugate forms an organic whole. The conjugate of the present invention is a typical drug precursor. Through hydrolysis or enzymolysis, the active agent D is released, separated from the parent body, and exerts physiological activity. Compared with unconjugated drugs, the conjugate of the present invention can show a stronger effect and is more enriched in the tissues of the human body or other animals.

[0066] The pharmaceutically acceptable salts of the conjugates of the present invention include inorganic salts and organic salts. Typical salts include nitrates, sulfates, phosphates, hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, formates, lactates, benzoates, acetates, trifluoroacetates, dichloroacetates, trichloroacetates, mixed chlorofluoroacetates, citrates, oxalates, sulfonates, methanesulfonates, trifluoromethanesulfonates, heptanesulfonates, etc. Among them, hydrochlorides and acetates are preferred. Salts can be formed by conventional means in the field of pharmaceutical chemistry.

[0067] Typical hydrochlorides of compound a, compound b, compound c and compound d are hydrochlorides in which each branch chain is respectively bound to one or two molecules of hydrochloric acid, and the preferred conjugate is a hydrochloride of four or eight molecules.

[0068] Typical acetate salts of compound a, compound b, compound c and compound d are acetate salts in which each branch chain is respectively bonded with one or two molecules of acetic acid, and the preferred conjugate is four or eight molecules of acetate salt.

[0069] The compound of the present invention specifically binds to CD44 and can target tumor cells and tissues that highly express CD44, so that the concentration of the conjugate in the target tissue is higher, thereby improving its clinical therapeutic effect and reducing toxicity.

[0070] The compounds of the present invention are suitable for treating all tumors with high expression of CD44, including but not limited to gastric cancer, pancreatic cancer, small cell lung cancer, colon cancer, breast cancer, lung adenocarcinoma, liver cancer, nasopharyngeal carcinoma, malignant glioma, lymphoma, kidney cancer, ovarian cancer, head and neck cancer, squamous cell carcinoma, etc. Specific embodiments

[0071] The present invention will be described in detail below. However, the present invention may be embodied in many different forms, and it should not be limited to the embodiments described herein, and the purpose of providing these embodiments is to make the disclosed content more complete and comprehensive. The reagents and raw materials used, except for those providing the preparation method, are commercially available. 4armPEG20K-SCM was purchased from Beijing Jiankai Technology Co., Ltd. with a molecular weight of approximately 20kDa. Unless otherwise defined, all scientific and technological terms in this article have the same meaning as those commonly understood by personnel in the technical field to which the subject matter of the claims belongs.

[0072] The structure of 4arm-PEG20K-SCM is as follows:

[0073]

[0074] Glossary

[0075]

[0076] Example 1

[0077]

[0078] 1. Preparation of BP103a

[0079] To a 250 mL three-necked flask, add 18.20 g of compound BP103a05 and 100 ml of EA, stir to dissolve, then cool to 0°C, add 150 ml of HCl / EA (3.5 M), keep warm at 0°C, monitor the completion of the reaction by TLC, concentrate the reaction solution to dryness, add methyl tert-butyl ether to make a pulp, filter, wash the filter cake with TBME, and dry in vacuo to obtain 10.40 g of white solid BP103a.

[0080] 2. Preparation of BP103a07

[0081] To a 100 mL flask, 3.00 g BP103a (1.0 eq), 4.02 g (1.0 eq) BP102c07, 40 ml DCM, and 4.0 ml DIEA (2.0 eq) were added. The mixture was stirred at room temperature. The reaction was monitored by TLC. The organic solvent was evaporated and column chromatography was performed to obtain 6.4 g of an oily substance BP103a075.26 g.

[0082] 3. Preparation of BP103a08

[0083] To a 200 mL three-necked flask, add 9.00 g BP103a07 (1.0 eq), 3.96 g HOSU (1.53 eq), 90 ml DCM, and 6.60 g EDC·HCl (1.53 eq). React at room temperature for 2 h. After the reaction is completed under TLC monitoring, dilute with DCM, wash twice with 50 mmol / L potassium dihydrogen phosphate aqueous solution, wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate to obtain 5.93 g BP103a08 as a colorless oil.

[0084] 4. Preparation of BP103a30

[0085] To a 200 mL flask, 2.93 g of NH2-Lys(Boc)-OH·HCl (1.0 eq), 60 ml of water, and 2.00 g of NaHCO3 (2.0 eq) were added dropwise with stirring, and a solution of 5.91 g of BP103a08 (1.0 eq) dissolved in 60 ml of DME was added, and 60 ml of THF was added. The mixture was stirred overnight, and the reaction was monitored by TLC. The organic solvent was evaporated, and the pH was adjusted to 3 with dilute hydrochloric acid. The mixture was extracted with EA, dried over anhydrous sodium sulfate, and concentrated to obtain 4.50 g of a colorless oil BP103a30. 5. Preparation of BP103a31

[0086] To a 100 mL three-necked flask, 5.00 g BP103a30 (1.0 eq), 1.40 g HOSU (1.53 eq), 50 ml DCM, and 2.33 g EDC·HCl (1.53 eq) were added. The mixture was reacted at room temperature for 2 h. After the reaction was completed under TLC monitoring, the mixture was diluted with DCM, washed twice with 50 mmol / L potassium dihydrogen phosphate aqueous solution, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 4.89 g BP103a31 as a colorless oil.

[0087] Example 2 Synthesis of intermediate Ac-KPSSPPEEC-NH2

[0088]

[0089] Ac-KPSSPPEEC-NH2 was synthesized by the Fmoc solid phase synthesis method well known to professionals, using Rink-amide Resin, 20% piperidine / DMF to remove Fmoc, HOBT / DIC as a coupling reagent, DMF as a reaction solvent, and ninhydrin detection method to monitor the reaction. The following protected amino acids were connected to the resin in sequence: Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro ... moc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, and acetylated with acetic anhydride and pyridine, then washed with DMF, methanol, and DCM, and dried, and a cleavage reagent (TFA: thioanisole: phenol: TIS = 85:5:5:5) was added, and after reacting for 2 hours, it was precipitated with ice TBME and centrifuged to obtain the crude product of Ac-KPSSPPEEC-NH2, which was prepared and purified by HPLC and then freeze-dried to obtain the pure product.

[0090] Example 3 Synthesis of intermediate Ac-NASAPPEEC-NH2

[0091]

[0092] Ac-NASAPPEEC-NH2 was synthesized by the Fmoc solid phase synthesis method well known to professionals in the art. Rink-amide resin was used to remove Fmoc using 20% ​​piperidine / DMF. HOBT / DIC was used as a coupling reagent and DMF was used as a reaction solvent. The reaction was monitored by the ninhydrin detection method. The following protected amino acids were connected to the resin in sequence: Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-O H, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, and acetylated with acetic anhydride and pyridine, then washed with DMF, methanol, and DCM, and dried, and a cleavage reagent (TFA: thioanisole: phenol: TIS = 85:5:5:5) was added, and after reacting for 2 hours, it was precipitated with ice TBME and centrifuged to obtain the crude Ac-NASAPPEEC-NH2, which was prepared and purified by HPLC and then freeze-dried to obtain the pure product.

[0093] Example 4

[0094]

[0095] 1. Preparation of BP143b02

[0096] To a 250 mL round-bottom flask, 3.50 g of irinotecan hydrochloride trihydrate (1.0 eq) and 52.5 ml of DMF were added, and the mixture was heated to 60° C. to dissolve. After 5-10 min, DMF was evaporated under reduced pressure, and 300 ml of n-heptane was added and distilled under reduced pressure. The mixture was repeated three times. After being spin-dried, 105 ml of DCM, 1.08 g of Boc-Gly-OH (1.2 eq), and 63 mg of DMAP (0.1 eq) were added dropwise, and a solution of 1.59 g of DCC (1.5 eq) dissolved in 10 ml of DCM was added. The mixture was reacted at 20° C. for 4 hours. After the reaction was completed under TLC monitoring, the mixture was filtered, and 120 ml of IPA was added when the mixture was concentrated to a remaining volume of 25%. 75% of the solvent was evaporated, and 150 ml of n-heptane was added. The mixture was stirred at room temperature for 1 hour, filtered, washed twice with n-heptane, and dried to obtain 4.02 g of BP143b02 as a light yellow solid.

[0097] 2. Preparation of BP143b03

[0098] To a 100mL three-necked flask, add 4.02g BP143b02 and 50ml DCM, stir to dissolve, then add 11.6ml TFA dropwise, react at room temperature for 2h, add 150ml acetonitrile after the reaction is completed under TLC monitoring, distill 120ml of the solvent under reduced pressure and pour into 320ml TBME solution, stir for 30min, filter, and wash the filter cake with TBME to obtain 4.00g of light yellow solid BP143b03.

[0099] 3. Preparation of BP143b04

[0100] To a 200 mL three-necked flask, 3.69 g BP143b03, 100 ml DCM, 3.21 g (1.05 eq) BP103a30, 2.7 ml DIEA (3.0 eq), and 1.2 ml DEPC (1.5 eq) were added. The mixture was reacted at room temperature for 1 h. After the reaction was completed under TLC monitoring, the mixture was diluted with DCM, washed twice with water, once with saturated brine, dried, concentrated, purified by HPLC, and lyophilized to obtain 1.85 g of light yellow solid BP143b04.

[0101] 4. Preparation of BP143b05

[0102] 1.20 g BP143b04 and 10 ml 10% TFA / DCM were added to a 50 mL round-bottom flask and reacted at room temperature. After the reaction was completed under TLC monitoring, the mixture was poured into TBME, centrifuged and dried to obtain 210 mg of a light yellow solid compound BP143b05.

[0103] 5. Preparation of BP143b06

[0104] To a 10 mL round-bottom flask, 51 mg BP143b05 (4.0 eq), 2 ml DCM, 11 ul TEA (8.0 eq), and 201 mg 4armPEG20K-SCM (1.0 eq) were added. After reacting at room temperature overnight, the mixture was concentrated, added to TBME, centrifuged, purified by HPLC, and freeze-dried to obtain 85 mg of a yellow-green solid compound BP143b06.

[0105] Example 5

[0106]

[0107]

[0108] 1. Preparation of BP149a01

[0109] To a 100-mL three-necked flask, 10.00 g of compound SN38 (1.0 eq) and 110 ml of DMSO were added. After dissolving, 5.56 g of (Boc)2O (1.0 eq) and 9.82 g of DIEA (3.0 eq) were added. After reacting for 2 hours, the reaction was completed by monitoring by TLC. The reaction solution was poured into purified water, extracted with ethyl acetate three times, and the organic phases were combined, washed with purified water, washed with saturated brine, and dried over anhydrous sodium sulfate to obtain an off-white solid. Methyl tert-butyl ether was slurried and filtered. After drying, 7.95 g of compound BP149a01 as an off-white solid was obtained.

[0110] 2. Preparation of BP149a02

[0111] To a 250 mL round-bottom flask, 7.95 g of compound BP149a01 (1.0 eq), 2.83 g of Boc-Gly-OH (1.0 eq), 80 ml of dichloromethane, and 390 mg of DMAP (0.2 eq) were added dropwise, and a solution of 5.00 g of DCC (1.5 eq) dissolved in 20 ml of DCM was added dropwise. The mixture was reacted at room temperature for 1 hour. After the reaction was completed by monitoring by TLC, the mixture was filtered and concentrated until no liquid was evaporated. 30 ml of IPA was added, and 200 ml of n-heptane was added after the mixture was dissolved. The mixture was stirred for 30 minutes, filtered, washed twice with n-heptane, and dried to obtain 8.73 g of compound BP149a02 as a light yellow solid.

[0112] 3. Preparation of BP149a03

[0113] To a 100 mL three-necked flask, add 8.73 g of compound BP149a02 and 40 ml of DCM, stir to dissolve, then cool to 0°C, add 40 ml of TFA dropwise, react at room temperature for 2 h, monitor the reaction by TLC, pour the reaction solution into 800 ml of TBME solution, stir for 30 min, filter, wash the filter cake with TBME, and dry in vacuo to obtain 4.95 g of light yellow solid BP149a03.

[0114] 4. Preparation of BP149b01

[0115] To a 200 mL three-necked flask, 2.3 g of compound BP149a03, 45 ml of DCM, 3.196 g (1.05 eq) of compound BP103a31, and 1.75 ml of TEA (3.0 eq) were added, and the mixture was reacted at room temperature for 4 h. After the reaction was completed under TLC monitoring, the mixture was diluted with DCM, washed once with dilute hydrochloric acid, washed twice with water, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was prepared and purified by HPLC, extracted with DCM, dried over anhydrous sodium sulfate, and concentrated to obtain 2.20 g of a light yellow solid BP149b01.

[0116] 5. Preparation of BP149b02

[0117] 2.0 g of compound BP149b01 and 40 ml of 10% TFA / DCM were added to a 200 mL three-necked flask and reacted at room temperature. After the reaction was completed under TLC monitoring, the mixture was poured into TBME, centrifuged and dried to obtain 1.75 g of light yellow solid BP149b02.

[0118] 6. Preparation of BP149b03

[0119] To a 200 mL three-necked flask, 4.08 g of compound BP149b02 (5.0 eq), 100 ml of DMF, 1.96 g of DIEA (20 eq), and 16.05 g of 4armPEG20K-SCM (1.0 eq) were added. After reacting at room temperature for 2 hours, the mixture was poured into 1 L of methyl tert-butyl ether, stirred for 30 minutes, filtered, and dried to obtain 20.50 g of a crude off-white solid. The crude product was purified by HPLC and freeze-dried to obtain 12.36 g of pure BP149b03.

[0120] Example 6

[0121]

[0122] Under room temperature, 2.00 g (1.0 eq) BP143b06 was added to a 100 ml three-necked reaction bottle, and 35 ml of PBS solution with pH = 7.0 was stirred to dissolve. 0.45 g (5.0 eq) of targeting peptide 1 (Ac-KPSSPPEEC-NH2) was dissolved in 30 ml of purified water, and the pH was adjusted to 6.5 with sodium bicarbonate solution and then added to the reaction bottle. After 0.5 hours, HPLC detected that the reaction was complete, and the compound a was prepared and purified by HPLC and freeze-dried to obtain 1.65 g of pure product.

[0123] The molecular weight detected by MALDI-TOF was 28810.73.

[0124] Example 7

[0125]

[0126] Under room temperature, 2.00 g (1.0 eq) BP143b06 was added to a 100 ml three-necked reaction bottle, and 35 ml of PBS solution with pH = 7.0 was stirred to dissolve. 0.38 g (5.0 eq) of targeting peptide 2 (Ac-NASAPPEEC-NH2) was dissolved in 30 ml of purified water, and the pH was adjusted to 6.5 with sodium bicarbonate solution and then added to the reaction bottle. After 0.5 hours, HPLC detected that the reaction was complete. The compound b was prepared and purified by HPLC and freeze-dried to obtain 1.42 g of pure product.

[0127] The molecular weight detected by MALDI-TOF was 28931.52.

[0128] Example 8

[0129]

[0130] Under room temperature, 2.00 g (1.0 eq) BP149b03 was added to a 100 ml three-necked reaction bottle, and 35 ml of PBS solution with pH = 7.0 was stirred to dissolve. 0.46 g (5.0 eq) of targeting peptide 1 (Ac-KPSSPPEEC-NH2) was dissolved in 30 ml of purified water, and the pH was adjusted to 6.5 with sodium bicarbonate solution and then added to the reaction bottle. After 0.5 hours, HPLC detected that the reaction was complete. The compound was prepared and purified by HPLC and freeze-dried to obtain 1.45 g of pure compound c.

[0131] The molecular weight detected by MALDI-TOF was 28191.63.

[0132] Example 9

[0133]

[0134] Under room temperature, 2.00 g (1.0 eq) BP149b03 was added to a 100 ml three-necked reaction bottle, and 35 ml of PBS solution with pH = 7.0 was stirred to dissolve. 0.39 g (5.0 eq) of targeting peptide 2 (Ac-NASAPPEEC-NH2) was dissolved in 30 ml of purified water, and the pH was adjusted to 6.5 with sodium bicarbonate solution and then added to the reaction bottle. After 0.5 hour, HPLC detected that the reaction was complete. The compound d was prepared and purified by HPLC and freeze-dried to obtain 1.53 g of pure product.

[0135] The molecular weight detected by MALDI-TOF was 27982.32.

[0136] Example 10 Synthesis of biotin-labeled targeting peptide 3

[0137]

[0138] The synthesis of biotin-labeled targeting peptide 3 was carried out by solid phase synthesis of Fmoc method well known to professionals in the art, using Rink-amide Resin, 20% piperidine / DMF to remove Fmoc, HOBT / DIC as coupling reagent, DMF as reaction solvent, and ninhydrin detection method to monitor the reaction, and the following protected amino acids were connected to the resin in sequence: Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu) ... moc-Pro-OH, Fmoc-Lys(Dde)-OH, remove Fmoc protection, use acetic anhydride and pyridine for acetylation, add DMF solution of hydrazine hydrate to remove Dde protecting group, connect Fmoc-PEG3-OH and biotin in sequence, then wash with DMF, methanol and DCM, and dry, add cleavage reagent (TFA: thioanisole: phenol: EDT: water = 82.5:5:5:2.5:5), react for 2 hours, precipitate with ice TBME, centrifuge to obtain crude product, prepare and purify by HPLC, and then freeze-dry to obtain pure product.

[0139] Example 11 Synthesis of biotin-labeled targeting peptide 4

[0140]

[0141] The biotin-labeled targeting peptide 4 was synthesized by the Fmoc solid phase method well known to professionals in the art, using Rink-amide Resin, 20% piperidine / DMF to remove Fmoc, HOBT / DIC as a coupling reagent, DMF as a reaction solvent, and ninhydrin detection method to monitor the reaction. The following protected amino acids were connected to the resin in sequence: Fmoc-Cys(Trt)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu )-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, remove Fmoc, then acetylate with acetic anhydride and pyridine, add DMF solution of hydrazine hydrate to remove Dde protecting group, connect Fmoc-PEG3-OH and Biotin in sequence, then wash with DMF, methanol and DCM, and dry, add cleavage reagent (TFA: thioanisole: phenol: EDT: water = 82.5:5:5:2.5:5), react for 2 hours, precipitate with ice TBME, centrifuge to obtain a crude product, prepare and purify by HPLC, and then freeze-dry to obtain a pure product.

[0142] Example 12 Preparation of Compound a-Biotin Conjugate

[0143]

[0144] Under room temperature, 2.00 g (1.0 eq) BP143b06 was added to a 100 ml three-necked reaction bottle, and 35 ml of PBS solution with pH = 7.0 was stirred to dissolve, 0.57 g (5.0 eq) of biotin-labeled targeting peptide 3 was dissolved in 30 ml of purified water, and the pH was adjusted to 6.5 with sodium bicarbonate solution and then added to the reaction bottle. After 0.5 hours, HPLC detected that the reaction was complete, and HPLC preparation, purification, and freeze-drying were performed to obtain 1.24 g of pure product. The molecular weight detected by MALDI-TOF was 30325.51.

[0145] Example 13 Preparation of Compound b-Biotin Conjugate

[0146]

[0147] Under room temperature, 2.00 g (1.0 eq) BP143b06 was added to a 100 ml three-necked reaction bottle, and 35 ml of PBS solution with pH = 7.0 was stirred to dissolve, 0.60 g (5.0 eq) of biotin-labeled targeting peptide 4 was dissolved in 30 ml of purified water, and the pH was adjusted to 6.5 with sodium bicarbonate solution and then added to the reaction bottle. After 0.5 hours, HPLC detected that the reaction was complete, and HPLC preparation, purification, and freeze-drying were performed to obtain 1.33 g of pure product. The molecular weight detected by MALDI-TOF was 30596.56.

[0148] Example 14 Preparation of Compound C-Biotin Conjugate

[0149]

[0150] Under room temperature, 2.00 g (1.0 eq) BP149b03 was added to a 100 ml three-necked reaction bottle, and 35 ml of PBS solution with pH = 7.0 was stirred to dissolve, 0.55 g (5.0 eq) of biotin-labeled targeting peptide 3 was dissolved in 30 ml of purified water, and the pH was adjusted to 6.5 with sodium bicarbonate solution and then added to the reaction bottle. After 0.5 hours, HPLC detected that the reaction was complete, and HPLC preparation, purification, and freeze-drying were performed to obtain 1.26 g of pure product. The molecular weight detected by MALDI-TOF was 30223.25.

[0151] Example 15 Preparation of Compound d-Biotin Conjugate

[0152]

[0153] Under room temperature, 2.00 g (1.0 eq) BP149b03 was added to a 100 ml three-necked reaction bottle, and 35 ml of PBS solution with pH = 7.0 was stirred to dissolve, 0.58 g (5.0 eq) of biotin-labeled targeting peptide 4 was dissolved in 30 ml of purified water, and the pH was adjusted to 6.5 with sodium bicarbonate solution and then added to the reaction bottle. After 0.5 hours, HPLC detected that the reaction was complete, and HPLC preparation, purification, and freeze-drying were performed to obtain 1.33 g of pure product. The molecular weight detected by MALDI-TOF was 30491.18.

[0154] Example 16 The compounds of the present invention specifically bind to CD44

[0155] 1. Materials and Methods

[0156] 1.1 Conjugates of the compounds of the present invention labeled with biotin

[0157] The structures and preparation methods of compound a-biotin conjugate, compound b-biotin conjugate, compound c-biotin conjugate and compound d-biotin conjugate are shown in Examples 10-15, and they are respectively referred to as conjugate a, conjugate b, conjugate c and conjugate d in the experiments.

[0158] 1.2 Cell culture

[0159] SKOV3 cells were cultured in McCoy's 5A medium supplemented with 10% FBS, 1000 U / mL penicillin, 100 μg / mL streptomycin, and 4 mmol / L l-glutamine.

[0160] 1.3 Binding of conjugates to SKOV3 cells

[0161] 5mmol / L bis(sulfosuccinimide) suberate was added to conjugate a, conjugate b, conjugate c, and conjugate d (dissolved in iced PBS to a concentration of 100μmol / L) to bind to SKOV3 cells. TritonX-100 cell lysis buffer was added, the cells were lysed and centrifuged, and transferred to polyvinylidene fluoride (PVDF) membranes for immunoblot analysis. The PVDF membrane was incubated with streptavidin conjugated with horseradish peroxidase (HRP-SA) to detect proteins cross-linked with the conjugates. CD44 was detected by anti-CD44 monoclonal antibody DF1485 and peroxidase-labeled donkey anti-mouse immunoglobulin blot staining. Detection was performed using freshly prepared chemiluminescent reagents and Hypermax ECL film exposure.

[0162] 1.4 Immunoprecipitation of conjugate-labeled proteins

[0163] SKOV3 cells cross-linked with the conjugate were lysed by Triton X-100, pre-cleaned by incubation with protein A agarose for 2 hours at room temperature, and 100 μg of the washed lysate was incubated with 20 μL of protein A agarose supplemented with 2 μg of DF1485 (antibody against Cd44) or mouse IgG1 overnight at room temperature. After washing five times with lysis buffer, the immunoprecipitated proteins were eluted from the protein A-agarose after boiling in SDS-PAGE loading buffer. The eluted material and 5 μg of the original material were loaded on SDS-PAGE, transferred to PVDF membrane, and stained with HRP-SA.

[0164] 2. Results

[0165] SKOV3 cells were bound to the conjugate. Lysates prepared from these cells were immunoprecipitated with DF1485. Eluted proteins and starting lysates were subjected to PAGE / blotting and stained with HRP-SA to detect the biotin tag and DF1485 to detect CD44.

[0166] When the reaction system includes the starting lysate, the conjugate, and HRP-SA, many nonspecific bands appear in the range of 0-250 kDa; when the reaction system includes the starting lysate and HRP-SA, there are no bands in the range of 0-250 kDa, indicating that the protein labeled with the conjugate is specifically stained with HRP-SA. When the reaction system includes the immunoprecipitated protein, the conjugate, and HRP-SA, a specific band appears at 85 kDa; when the reaction system includes the immunoprecipitated protein and HRP-SA, there are no bands in the range of 0-250 kDa, indicating that DF1485 specifically precipitates the protein labeled with the conjugate of about 85 kDa. Compared with the starting lysate, the staining degree of this band in the immunoprecipitated protein is more obvious, indicating that the protein has been greatly enriched by anti-CD44 immunoprecipitation.

[0167] When the reaction system includes the starting lysate, the conjugate and DF1485, a specific band appears at 85 kDa; when the reaction system includes the starting lysate and DF1485, a specific band appears at 85 kDa. When the reaction system includes the immunoprecipitated protein, the conjugate and DF1485, a specific band appears at 85 kDa; when the reaction system includes the immunoprecipitated protein and DF1485, a specific band appears at 85 kDa. This indicates that the 85 kDa band is CD44, and CD44 is present in all samples.

[0168] The above results indicate that the compound of the present invention binds specifically to CD44 and can target tumor cells and tissues that highly express CD44, thereby increasing the concentration of the conjugate in the target tissue, improving its clinical therapeutic effect and reducing toxicity.

[0169] In Examples 17-21, all animal experiments were performed in strict compliance with animal use and management regulations. The calculation of tumor-related parameters was based on the China CFDA "Technical Guidelines for Non-clinical Studies of Cytotoxic Anti-tumor Drugs" and the China SFDA "Technical Guidelines for Non-clinical Studies of Cytotoxic Anti-tumor Drugs" (November 2006). %T / C≤40% and P<0.05 after statistical analysis were considered effective. The lower the relative tumor proliferation rate, the better the tumor inhibition effect.

[0170] The tumor volume (TV) was calculated as follows: TV (mm 3 )=l×w 2 / 2

[0171] Wherein, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

[0172] The relative tumor volume (RTV) is calculated as follows: RTV = TV t / TV initial

[0173] Among them, TV initial TV is the tumor volume measured at the time of group administration; t It is the tumor volume at each measurement during the drug administration period.

[0174] The relative tumor proliferation rate (%T / C) was calculated as follows: %T / C = 100% × (RTV T / RTV C )

[0175] Among them, RTV T represents the treatment group RTV; RTV C represents the solvent control group RTV.

[0176] The calculation formula of tumor growth inhibition rate TGI (%) is: TGI = 100% × [1-(TV t(T) -TV initial(T) ) / (TV t(C) -TV initial(C) )]

[0177] Among them, TV t(T) TV represents the tumor volume of each treatment group; initial(T) TV represents the tumor volume of the treatment group at the time of group administration; t(C) TV represents the tumor volume of the vehicle control group at each measurement; initial(C) It represents the tumor volume of the solvent control group at the time of group administration.

[0178] The formula for calculating the animal weight loss rate is: Animal weight loss rate = 100% × (BW initial -BW t ) / BW initial

[0179] Among them, BW t Indicates the animal body weight measured at each time during the dosing period; BW initial Indicates the body weight of animals at the time of grouping and dosing.

[0180] The calculation formula of tumor weight inhibition rate IR (%) is: IR (%) = 100% × (W C -W T ) / W C

[0181] Among them, WC W represents the tumor weight of the control group; T Indicates tumor weight in treatment group.

[0182] The experimental data were calculated and statistically processed using Microsoft Office Excel 2007. Unless otherwise specified, the data were expressed as mean ± standard error (Mean ± SE), and the t-test was used for comparison between groups. P < 0.05 was considered significant.

[0183] Experimental animals: Female BALB / c nude mice (number: 100; age: 6-8 weeks) were fed normally for about 1 week. Mice with good physical signs after veterinary examination were selected for this experiment. Before grouping, the tail base of the animal was marked with a marker pen. After grouping, each animal was marked by ear cut.

[0184] Preparation of compound M, compound a and compound b for administration: Before each administration, accurately weigh each compound, add 2.5 mL of normal saline, and vortex to completely dissolve the drug. The effective concentration of irinotecan in the solution is 4.0 mg·mL-1.

[0185] Preparation of compound c and compound d for administration: Before each administration, accurately weigh each compound, add 2.5 mL of normal saline, and vortex to completely dissolve the drug. The effective concentration of SN38 in the solution is 4.0 mg·mL-1.

[0186] Random grouping: After the animals were inoculated with tumor cells, when the tumor volume reached 100-300 mm3, the animals were divided into 6 groups by random block method, with 6 mice in each group, to ensure that the tumor volume and mouse weight were uniform among the groups. The difference between the mean tumor volume of each group and the mean tumor volume of all experimental animals did not exceed ±10%.

[0187] After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal body weight was weighed and recorded.

[0188] The animal administration of Examples 17-20 was as follows: the first administration started on the day of grouping. Group 1 was the solvent control group, which was injected with normal saline via the tail vein. Groups 2-6 were injected with the test samples Compound M, Compound a, Compound b, Compound c, and Compound d, respectively.

[0189] After the experiment, the animals were euthanized (CO2) after being weighed and the tumor diameter was measured. The tumor tissues were removed and weighed, and the RTV and %T / C were calculated.

[0190] The control test compound M involved in the embodiment is disclosed in Chinese patent application CN108727583A, and its structure is as follows:

[0191]

[0192] Example 17 Antitumor efficacy of the compounds of the present invention on human breast cancer MDA-MB-231 xenograft tumors in nude mice

[0193] Test samples: Compound M, Compound a, Compound b, Compound c, Compound d.

[0194] Reagents: DMEM culture medium, fetal bovine serum (FBS), trypsin, blue-chain double antibody, normal saline.

[0195] Transplanted tumor cell line: human breast cancer MDA-MB-231, from the cell bank of Type Culture Collection Committee of Chinese Academy of Sciences (CAS, frozen in liquid nitrogen in this laboratory).

[0196] Cell culture: MDA-MB-231 cells were cultured in DMEM medium (DMEM, USA) containing 10% fetal bovine serum (FBS) (GIBCO, USA) in a 37°C incubator containing 5% CO2.

[0197] Animal model preparation: The nude mouse subcutaneous tumor transplantation model was established by cell inoculation method. Tumor cells in the logarithmic growth phase were collected, counted, and resuspended in 1× PBS. The cell suspension concentration was adjusted to 1×10 7 / ml. A 1ml syringe (gauge 4 needle) was used to inoculate tumor cells subcutaneously on the right back of nude mice, 1×10 6 / mice.

[0198] Dosage frequency and dosage: Each group was given once a week for a total of three times, QW×3, and the dosage was 6 mg·kg -1 (as irinotecan or SN38).

[0199] The results are shown in Table 1:

[0200] Table 1 Tumor proliferation rate T / C (%)

[0201]

[0202] *Compared with the RTV of blank solvent and compound M group, P<0.05

[0203] #Compared with the %T / C of the blank solvent and compound M groups, P<0.05

[0204] The experimental results show that the compound of the present invention has a good inhibitory effect on human breast cancer MDA-MB-231 nude mouse transplanted tumors and is superior to compound M.

[0205] Example 18 The inhibitory effect of the compounds of the present invention on the in vivo growth of tumors in nude mice transplanted tumor models of human gastric cancer NCI-N87 cell line.

[0206] Test samples: Compound M, Compound a, Compound b, Compound c, Compound d.

[0207] Reagents: RPMI-1640 culture medium, fetal bovine serum (FBS), trypsin, blue-chain double antibody, normal saline.

[0208] Transplanted tumor cell line: Human gastric cancer cell line NCI-N87, from the cell bank of Type Culture Collection Committee of Chinese Academy of Sciences (CAS, frozen in liquid nitrogen in this laboratory).

[0209] Cell culture: NCI-N87 cells were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum at 5% CO2 and 37°C. Cells were digested and passaged with 0.25% trypsin. Depending on cell growth, cells were passaged 1 to 2 times per week at a ratio of 1:2 to 1:6.

[0210] Animal model preparation: NCI-N87 cells in the logarithmic growth phase were collected, counted, and resuspended in serum-free RPMI-1640 medium. The cell concentration was adjusted to 5×10 7 cells / mL; pipette the cells to disperse them evenly and then put them into a 50mL centrifuge tube, which was placed in an ice box; use a 1mL syringe to draw up the cell suspension and inject it into the subcutaneous part of the right axilla of the nude mice. Each animal was inoculated with 100μL (5×10 6 The NCI-N87 nude mouse xenograft tumor model was established.

[0211] Dosage frequency and dosage: Each group was given once a week for a total of four times, QW×4, and the dosage was 20 mg·kg -1 .

[0212] The results are shown in Table 2:

[0213] Table 2 Tumor proliferation rate T / C (%)

[0214]

[0215] *Compared with the RTV of blank solvent and compound M group, P<0.05

[0216] #Compared with the %T / C of the blank solvent and compound M groups, P<0.05

[0217] The experimental results show that the compound of the present invention has a good inhibitory effect on the tumor growth of the nude mouse transplanted tumor model of human gastric cancer NCI-N87 cell line, and is superior to compound M.

[0218] Example 19 In vivo drug efficacy evaluation in HT-29 nude mouse transplanted tumor model

[0219] Test samples: Compound M, Compound a, Compound b, Compound c, Compound d.

[0220] Reagents: McCoy's 5A culture medium, fetal bovine serum (FBS), trypsin, blue-chain double antibody, water for injection, normal saline, lactic acid, sorbitol.

[0221] Transplanted tumor cell line: human colon cancer cell HT-29, from the cell bank of Type Culture Collection Committee of Chinese Academy of Sciences (CAS, frozen in liquid nitrogen in this laboratory).

[0222] Cell culture: HT-29 cells were routinely cultured in McCoy's 5A medium containing 10% fetal bovine serum at 5% CO2 and 37°C. The cells were digested and passaged with 0.25% trypsin. Depending on the cell growth, the cells were passaged 2 to 3 times per week at a passage ratio of 1:4 to 1:6.

[0223] Animal model preparation: HT-29 cells in the logarithmic growth phase were collected, counted, and resuspended in serum-free McCoy's 5A medium. The cell concentration was adjusted to 4×10 7 cells / mL; pipette the cells to disperse them evenly and then put them into a 50mL centrifuge tube, which was placed in an ice box; use a 1mL syringe to draw up the cell suspension and inject it into the subcutaneous part of the right axilla of the nude mice. Each animal was inoculated with 100μL (4×10 6 The HT-29 nude mouse xenograft tumor model was established.

[0224] Dosage frequency and dosage: Each group was given once a week for a total of four times, QW×4, and the dosage was 20 mg·kg -1 .

[0225] The results are shown in Table 3:

[0226] Table 3 Tumor proliferation rate T / C (%)

[0227]

[0228] *Compared with the RTV of blank solvent and compound M group, P<0.05

[0229] #Compared with the %T / C of the blank solvent and compound M groups, P<0.05

[0230] The experimental results show that the compound of the present invention has a good inhibitory effect on the growth of human colon cancer HT-29 nude mouse transplanted tumor model in vivo, and is superior to compound M.

[0231] Example 20 Inhibitory Effect on Human Pancreatic Cancer MIA Paca-2 Nude Mouse Xenograft Model

[0232] Test samples: Compound M, Compound a, Compound b, Compound c, Compound d.

[0233] Transplanted tumor cell line: Human pancreatic cancer MIA Paca-2 was purchased from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai.

[0234] Cell culture: MIA Paca-2 cells were cultured in DMEM containing 10% fetal bovine serum (FBS) (GIBCO, USA) and 2.5% HS. The cells were cultured in a 5% CO2 incubator at 37°C.

[0235] Animal model preparation: The human pancreatic cancer MIA Paca-2 nude mouse subcutaneous transplant tumor model was established by cell inoculation method: tumor cells in the logarithmic growth phase were collected, counted, and resuspended in 1× PBS. The cell suspension concentration was adjusted to 3×10 7 / ml. A 1ml syringe (gauge 4 needle) was used to inoculate tumor cells subcutaneously on the right back of nude mice, 3×10 6 / 0.1ml / mouse.

[0236] Dosage frequency and dosage: Each group was given once a week for a total of three times, QW×3, and the dosage was 6 mg·kg -1 .

[0237] The results are shown in Table 4:

[0238] Table 4 Tumor proliferation rate T / C (%)

[0239]

[0240] *Compared with the RTV of blank solvent and compound M group, P<0.05

[0241] #Compared with the %T / C of the blank solvent and compound M groups, P<0.05

[0242] The experimental results show that the compound of the present invention has a good inhibitory effect on human pancreatic cancer MIA Paca-2 nude mouse transplanted tumors and is superior to compound M.

[0243] Example 21 Inhibitory effect on human small cell lung cancer cell NCI-H446 nude mouse xenograft model

[0244] Test samples: irinotecan, nktr-102, compound a, compound b, compound c, and compound d.

[0245] Irinotecan (API) was purchased. The preparation method of nktr-102 is as follows:

[0246] BP143b03 (829 mg, 4.5 eq) in Example 4 was added to a 250 mL reaction bottle, and DCM (50 mL) and triethylamine (221 mg, 9.0 eq) were added. After dissolving, 4ARM-PEG20K-SCM (5.00 g, 1.0 eq) was added to the reaction bottle. After HPLC monitoring showed no obvious progress in the reaction, about 20 mL of DCM was distilled off under reduced pressure, and the solution was poured into 300 mL of TBME and stirred for precipitation. Filtering gave 5.4 g of crude product, which was prepared and purified by HPLC, desalted, adjusted to pH 5-6 with dilute hydrochloric acid, and freeze-dried to give 2.71 g of light green powder nktr-102.

[0247] Preparation of irinotecan dosing preparation: Weigh 12.0 mg of irinotecan, add 0.15 mL of 1% lactic acid, vortex to completely dissolve the drug, then add 2.85 mL of 1% sorbitol aqueous solution, vortex to mix evenly, the ratio of 1% lactic acid to 1% sorbitol aqueous solution in the solution is about 5:95 (v / v). The effective concentration of irinotecan in the solution is 4.0 mg·mL -1 .

[0248] Preparation of nktr-102 dosing preparation: Before each administration, accurately weigh 101.5 mg of nktr-102, add 2.5 mL of normal saline, and vortex to completely dissolve the drug. The effective concentration of irinotecan in the solution is 4.0 mg mL -1 .

[0249] Transplanted tumor cell line: Human small cell lung cancer cell line NCI-H446 was obtained from the cell bank of Type Culture Collection Committee of Chinese Academy of Sciences.

[0250] Cell culture: NCI-H446 cells were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum under 5% CO2, 37°C, and saturated humidity. Depending on cell growth, cells were digested and passaged 2 to 4 times a week with a passage ratio of 1:3 to 1:4 using 0.25% trypsin.

[0251] Animal model preparation: The nude mouse subcutaneous tumor transplantation model was established by cell inoculation method: NCI-H446 cells in the logarithmic growth phase were collected, counted and resuspended in 50% RPMI-1640 basal medium and 50% Matrigel, and the cell concentration was adjusted to 4×107 cells / mL; the cells were blown with a pipette to disperse them evenly and then placed in a 50-mL centrifuge tube, which was placed in an ice box; the cell suspension was drawn up with a suitable syringe and injected into the subcutaneous part of the right axilla of the nude mouse. Each animal was inoculated with 200 μL (8×10 6 Cells / mouse) were used to establish the NCI-H446 nude mouse xenograft tumor model.

[0252] Animal administration: The first administration started on the day of grouping. Group 1 was the solvent control group, which was given normal saline by tail vein injection. Groups 2-7 were given the test samples irinotecan, nktr102, compound a, compound b, compound c, and compound d by tail vein injection, respectively. The above groups were administered once a week for a total of three times, QW×3, and the dosage was 6 mg·kg -1 .

[0253] The results are shown in Table 5:

[0254] Table 5 Tumor proliferation rate T / C (%)

[0255]

[0256]

[0257] *Compared with RTV of blank solvent, irinotecan, and nktr102 groups, P<0.05

[0258] #Compared with the %T / C of blank solvent, irinotecan, and nktr102 groups, P<0.05

[0259] The experimental results show that the compound of the present invention has a good inhibitory effect on human small cell lung cancer cell NCI-H446 nude mouse transplanted tumors, and is superior to irinotecan and nktr102.

Claims

1. A multi-branched drug conjugate having the following structural formula (I) or a pharmaceutically acceptable salt thereof: R is an organic center, POLY is a polymer, L is a multivalent linker, T is a targeting molecule, D is an active agent, and q is any integer between 3 and 8, wherein, D is a camptothecin drug as shown below: Wherein, R1-R5 are independently selected from the following groups: hydrogen, halogen, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, alkynyl, cycloalkyl, hydroxyl, cyano, nitro, azido, amide, hydrazine, amine, substituted amine, hydroxycarbonyl, alkoxycarbonyl, alkoxycarbonyloxy, carbamoyloxy, arylsulfonyloxy, alkylsulfonyloxy; R6 is H or OR8, R8 is alkyl, alkenyl, cycloalkyl, haloalkyl or hydroxyalkyl; R7 is hydroxyl; T is a CD44 targeting polypeptide, wherein the CD44 targeting polypeptide is Ac-KPSSPPEE-NH2 or Ac-NASAPPEE-NH2; POLY is polyethylene glycol; R is: The multivalent linker L is: The symbol "*" represents the connection point between the multivalent linker L and the targeting molecule T, "#" represents the connection point between the multivalent linker L and the active agent D, and "%" represents the connection point between the multivalent linker L and POLY, wherein m is any integer between 1 and 20, and l and n are any integers between 1 and 10, respectively.

2. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: POLY is: k is 5 to 500, and r is any integer between 1 and 10.

3. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: k is 50 to 200.

4. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: k is 113.

5. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The multi-branched drug conjugate is:

6. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 5, characterized in that: l is 5, m is 3, and n is 4.

7. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: D is irinotecan or SN38.

8. A multi-branched drug conjugate or a pharmaceutically acceptable salt thereof, which is:

9. The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to claim 8, characterized in that: The multi-branched drug conjugate or a pharmaceutically acceptable salt thereof is: Each branch of compound a, compound b, compound c and compound d is respectively bound to one or two molecules of hydrochloride of hydrochloric acid and each branch is respectively bound to one or two molecules of acetate of acetic acid.

10. Use of the multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 in the preparation of a medicament for treating gastric cancer, pancreatic cancer, small cell lung cancer, colon cancer, breast cancer, or malignant glioma.

11. A pharmaceutically acceptable composition comprising the multi-branched drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, and a pharmaceutically acceptable excipient.

12. Use of the composition according to claim 11 in the preparation of a medicament for treating gastric cancer, pancreatic cancer, small cell lung cancer, colon cancer, breast cancer, and malignant glioma.

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