Use of the medicament in the treatment of tumor diseases

Abstract

The present invention relates to the use of medicaments in the treatment of tumor disease.In particular, the present invention provides the use of the bioactive conjugate represented by formula (I) in the treatment of tumor disease.Tumor disease refers in particular and preferably to unresectable locally advanced or metastatic solid tumors that are refractory to existing treatment standards, including (but not limited to) breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, esophageal cancer, liver cancer, colorectal cancer, cervical cancer, endometrial cancer, pancreatic cancer and brain cancer.

Description

[Technical field]

[0001] This application is based on and claims priority to CN Application No. 202110482012.2, filed on April 30, 2021, the disclosure of which is incorporated by reference in its entirety into this specification.

[0002] Technical Field The present application relates to the use of the medicament in the treatment of tumor diseases, in particular diseases associated with abnormal cellular activity, including unresectable locally advanced or metastatic solid tumors that are refractory to existing standards of treatment, such as tumors that have failed and / or recurred after first-line chemotherapy, tumors that have failed and / or recurred after radiation therapy and / or tumors that have failed and / or recurred after targeted therapy. [Background technology]

[0003] Cancer (hereafter referred to as cancer) is a major burden on global public health. In the United States, cancer remains the second leading cause of death after cardiovascular disease. The number of cancer cases is also increasing in China. In 2019, the number of new cases of malignant tumors in China was approximately 4.4 million, and the number of deaths was approximately 2.624 million. The increase in the number of cancer patients and deaths leads to an expansion of the overall scale of commercial activities in oncology treatment.

[0004] Chemotherapy is one of the main means of cancer treatment, but traditional chemotherapy is not specific in tumor recognition, which easily damages normal cells and causes serious adverse reactions in patients. To improve the survival rate of cancer patients, innovation in treatment methods is urgently needed to match advances in detection and diagnosis. Although great progress has been made in many indications, the mortality rate of some of the most intractable solid tumors has not improved significantly since the 1970s, and more effective treatments with fewer side effects are still needed. Molecularly targeted drugs are currently an important trend in drug design.

[0005] Monoclonal antibody drugs have the advantages of strong targeting, high specificity, and low incidence of serious adverse reactions, but their molecular weight is large, and their therapeutic effect is limited when used alone. ADC (antibody-drug conjugate) is a drug in which a monoclonal antibody is coupled to various numbers of small molecule cytotoxins (effector molecules) through a chemical linker. After entering the body, the ADC molecule binds to antigens on the surface of target cells through the induction of monoclonal antibodies and enters the target cells. The ADC molecule that enters the cells releases the effector molecules through chemical and / or enzymatic action to achieve the purpose of destroying the target cells. ADC drugs combine the advantages of strong targeting of monoclonal antibodies and high activity of small molecule toxins, which can not only reduce the toxic effects and side effects of small molecule cytotoxins, but also improve the efficacy of drugs.

[0006] Currently, various ADC drugs are commercially available worldwide, and their indications include leukemia, lymphoma, breast cancer, etc. However, some problems still exist in the pharmacokinetics and safety of some ADC drugs, which may cause serious adverse reactions in patients after use. In addition, their response rates against some metastatic, recurrent and / or refractory cancers still need to be further improved. Therefore, there is still a need to develop the use or treatment methods of ADC drugs for treating these metastatic, recurrent and / or refractory cancers to maximize their efficacy and minimize their toxicity, and meet the drug needs of cancer patients. Summary of the Invention

[0007] General The present invention relates to the use of a compound of formula (I) in the manufacture of a medicament for treating a tumor disease. [ka] [In the formula, L1 is [ka] where R1 and R2 are each independently hydrogen (e.g., protium or deuterium), halogen, carboxylate, sulfonate, cyano, C 1-6 Alkyl, halogenated C 1-6 Alkyl, cyano-substituted C 1-6 Alkyl (e.g., -CHCN), C 1-6 Alkoxy, C 2-10 Alkenyl or C 2-10 alkynyl; Z1 is an amino acid or a peptide consisting of 2 to 10 amino acids; x1 and x2 are each independently 0, 1, 2, 3, 4, 5, or 6; position 1 of L1 is bonded to D and position 2 of L1 is bonded to L2; L2 is [ka] where y1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; position 1 of L2 is attached to L1 and position 2 of L2 is attached to L3; L3 is a 5- to 12-membered heteroaromatic ring; L4 is [ka] where Z2 is C 1-6 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylene and C 3-8 cycloalkylene; R3 is selected from H and C 1-6 alkyl; Z3 is absent or C 1-6 alkylene; or R3 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclyl; α is 0, 1, 2, 3, 4, 5 or 6, position 2 of L4 is bonded to E and position 1 of L4 is bonded to L3; E is [ka] where each R4 is independently hydrogen (e.g., protium or deuterium), β is 0, 1, or 2, position 2 of E is attached to A (e.g., at a sulfhydryl on A) and position 1 of E is attached to L4; m1, m2 and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; D is a biologically active molecular fragment; γ is the moiety {D-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E] is selected from integers from 1 to 10; preferably, γ is selected from integers from 3 to 8 (e.g., 3, 4, 5, 6, 7, or 8); and A is a monoclonal antibody or an antigen-binding fragment thereof against Trop-2. The present invention provides the use of a biologically active conjugate represented by the formula:

[0008] In some embodiments, the tumor disease is an unresectable locally advanced or metastatic solid tumor that has failed standard treatment or has no standard treatment regimen or is not currently suitable for standard treatment. In some embodiments, standard treatment refers to the standard treatment regimen for tumor disease recommended by NCCN guidelines and CSCO guidelines for diagnosis and treatment.

[0009] In some embodiments, the tumor disease is a tumor that has failed and / or recurred after first-line chemotherapy. In some embodiments, first-line chemotherapy refers to the first-line chemotherapy for tumor disease recommended by NCCN guidelines and CSCO guidelines for diagnosis and treatment.

[0010] In some embodiments, the tumor disease is a tumor that has failed treatment and / or recurred after radiation therapy. In some embodiments, radiation therapy refers to the radiation therapy regimen for tumor disease recommended by NCCN guidelines and CSCO guidelines for diagnosis and treatment.

[0011] In some embodiments, the tumor disease is a tumor that has failed and / or recurred after targeted drug or immunotherapy. In some embodiments, targeted drug or immunotherapy refers to targeted drug or immunotherapy for tumor disease recommended by NCCN guidelines and CSCO guidelines for diagnosis and treatment.

[0012] In some embodiments, the tumor disease includes (but is not limited to) breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, esophageal cancer, liver cancer, colorectal cancer, cervical cancer, endometrial cancer, pancreatic cancer, bladder cancer or brain cancer, preferably includes breast cancer (e.g., triple-negative breast cancer or Her2-positive breast cancer), ovarian cancer (e.g., ovarian epithelial cancer), gastric cancer, lung cancer, pancreatic cancer, bladder cancer or urothelial cancer, more preferably, the tumor disease is triple-negative breast cancer, Her2-positive breast cancer, ovarian cancer, gastric cancer, lung cancer or pancreatic cancer, and even more preferably, the tumor disease is triple-negative breast cancer, Her2-positive breast cancer, ovarian cancer or gastric cancer.

[0013] In some embodiments, the neoplastic disease is breast cancer.

[0014] In some embodiments, breast cancer includes, but is not limited to, the following types: Luminal type A, Luminal type B, Her-2 positive type, and Triple negative type.

[0015] In some embodiments, the neoplastic disease is triple-negative breast cancer.

[0016] In some embodiments, the neoplastic disease is Her2-positive breast cancer.

[0017] In some embodiments, the neoplastic disease is ovarian cancer.

[0018] In some embodiments, ovarian cancer includes, but is not limited to, the following types: platinum-sensitive and platinum-resistant types.

[0019] In some embodiments, the neoplastic disease is gastric cancer.

[0020] In some embodiments, gastric cancer includes, but is not limited to, the following types: adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, and neuroendocrine tumors.

[0021] In some embodiments, the neoplastic disease is pancreatic cancer.

[0022] In some embodiments, pancreatic cancer includes, but is not limited to, the following types: epithelial tumors, exocrine tumors, borderline tumors, tubular adenocarcinoma, endocrine tumors, mature teratomas, mesenchymal tumors, malignant lymphomas, and secondary tumors.

[0023] In some embodiments, the neoplastic disease is bladder cancer.

[0024] In some embodiments, bladder cancer includes, but is not limited to, the following types: urothelial (transitional cell) carcinoma, squamous cell carcinoma, and adenocarcinoma.

[0025] In some embodiments, the neoplastic disease is urothelial carcinoma.

[0026] In some embodiments, urothelial carcinoma includes, but is not limited to, the following types: basal, luminal and wild-type urothelial carcinoma.

[0027] In some embodiments, the neoplastic disease is lung cancer.

[0028] In some embodiments, lung cancer includes, but is not limited to, the following types: small cell lung cancer and non-small cell lung cancer.

[0029] In some embodiments, the conjugate has the structure: L1 is [ka] wherein position 1 of L1 is attached to D and position 2 of L1 is attached to L2; L2 is [ka] where y1 is 3, 4, 5, 6, 7, 8, 9, or 10, position 1 of L2 is attached to L1, and position 2 of L2 is attached to L3; L3 is selected from a 5-6 membered heteroaromatic ring, such as pyrazole or triazole; L4 is [ka] where Z2 is C 1-3 alkylene, R3 is H, and Z3 is C 1-3 alkylene, α is 0, position 2 of L4 is bonded to E, and position 1 of L4 is bonded to L3; E is [ka] where each R4 is independently hydrogen (e.g., protium or deuterium), β is 0, 1, or 2, position 2 of E is attached to A (e.g., at a sulfhydryl on A) and position 1 of E is attached to L4; m1, m2 and m3 are all 1; Bioactive molecules are [ka] Preferably, the biologically active molecule is attached to position 1 of L1 via its hydroxyl; γ is selected from an integer from 3 to 8 (e.g., 3, 4, 5, 6, 7, or 8); A is sacituzumab or an antigen-binding fragment thereof.

[0030] In some embodiments, the conjugate has the structure: L1 is [ka] wherein position 1 of L1 is bound to D and position 2 of L1 is bound to L2; L2 is [ka] wherein y1 is 3, 4, 5, 6, 7, 8, 9, or 10, position 1 of L2 is attached to L1, and position 2 of L2 is attached to L3; L3 is selected from a 5-6 membered heteroaromatic ring, such as pyrazole or triazole; L4 is [ka] where Z2 is C 1-3 alkylene, R3 is H, and Z3 is C 1-3 alkylene, α is 1, position 2 of L4 is bonded to E, and position 1 of L4 is bonded to L3; E is [ka] where each R4 is independently hydrogen (e.g., protium or deuterium), β is 0, 1 or 2, position 2 of E is attached to A (e.g., at a sulfhydryl on A) and position 1 of E is attached to L4; m1, m2, and m3 are all 1; Bioactive molecules are [ka] Preferably, the biologically active molecule is attached to position 1 of L1 via its hydroxyl; γ is selected from an integer from 3 to 8 (e.g., 3, 4, 5, 6, 7, or 8); and A is sacituzumab or an antigen-binding fragment thereof.

[0031] In some embodiments, D is [ka] is selected from.

[0032] In some technical solutions, the conjugate has the following formula: [ka] (In the formula, γ is an integer of 1 to 10, and preferably, γ is selected from integers of 5 to 8.) and conjugate A having a structure represented by the following formula:

[0033] In some embodiments, the conjugate has a DAR value of 1 to 12, preferably 1 to 10, and more preferably the DAR value is 5 to 8. For example, the DAR values ​​are 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.

[0034] In another aspect, the present invention provides a method for treating a tumor disease comprising administering a therapeutically effective amount of the above-mentioned biologically active conjugate of formula (I) and / or a pharmaceutical composition comprising a biologically active conjugate of formula (I) to an individual in need thereof.

[0035] In some embodiments, the tumor disease is an unresectable locally advanced or metastatic solid tumor that has failed standard treatment or has no standard treatment regimen or is not currently suitable for standard treatment. In some embodiments, standard treatment refers to the standard treatment regimen for tumor disease recommended by NCCN guidelines and CSCO guidelines for diagnosis and treatment.

[0036] In some embodiments, the tumor disease is a tumor that has failed and / or recurred after first-line chemotherapy. In some embodiments, first-line chemotherapy refers to the first-line chemotherapy for tumor disease recommended by NCCN guidelines and CSCO guidelines for diagnosis and treatment.

[0037] In some embodiments, the tumor disease is a tumor that has failed treatment and / or recurred after radiation therapy. In some embodiments, radiation therapy refers to the radiation therapy regimen for tumor disease recommended by NCCN guidelines and CSCO guidelines for diagnosis and treatment.

[0038] In some embodiments, the tumor disease is a tumor that has failed and / or recurred after targeted drug or immunotherapy. In some embodiments, targeted drug or immunotherapy refers to targeted drug or immunotherapy for tumor disease recommended by NCCN guidelines and CSCO guidelines for diagnosis and treatment.

[0039] In some embodiments, the tumor disease includes (but is not limited to) breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, esophageal cancer, liver cancer, colorectal cancer, cervical cancer, endometrial cancer, pancreatic cancer, bladder cancer or brain cancer, preferably includes breast cancer (e.g., triple-negative breast cancer or Her2-positive breast cancer), ovarian cancer (e.g., ovarian epithelial cancer), gastric cancer, lung cancer, pancreatic cancer, bladder cancer or urothelial cancer, more preferably, the tumor disease is triple-negative breast cancer, Her2-positive breast cancer, ovarian cancer, gastric cancer, lung cancer or pancreatic cancer, and even more preferably, the tumor disease is triple-negative breast cancer, Her2-positive breast cancer, ovarian cancer or gastric cancer.

[0040] In some embodiments, the neoplastic disease is breast cancer.

[0041] In some embodiments, breast cancer includes, but is not limited to, the following types: Luminal type A, Luminal type B, Her-2 positive type, and Triple negative type.

[0042] In some embodiments, the neoplastic disease is triple-negative breast cancer.

[0043] In some embodiments, the neoplastic disease is Her2-positive breast cancer.

[0044] In some embodiments, the neoplastic disease is ovarian cancer.

[0045] In some embodiments, ovarian cancer includes, but is not limited to, the following types: platinum-sensitive and platinum-resistant types.

[0046] In some embodiments, the neoplastic disease is gastric cancer.

[0047] In some embodiments, gastric cancer includes, but is not limited to, the following types: adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, and neuroendocrine tumors.

[0048] In some embodiments, the neoplastic disease is pancreatic cancer.

[0049] In some embodiments, pancreatic cancer includes, but is not limited to, the following types: epithelial tumors, exocrine tumors, borderline tumors, tubular adenocarcinoma, endocrine tumors, mature teratomas, mesenchymal tumors, malignant lymphomas, and secondary tumors.

[0050] In some embodiments, the neoplastic disease is bladder cancer.

[0051] In some embodiments, bladder cancer includes, but is not limited to, the following types: urothelial (transitional cell) carcinoma, squamous cell carcinoma, and adenocarcinoma.

[0052] In some embodiments, the neoplastic disease is urothelial carcinoma.

[0053] In some embodiments, urothelial carcinoma includes, but is not limited to, the following types: basal, luminal and wild-type urothelial carcinoma.

[0054] In some embodiments, the neoplastic disease is lung cancer.

[0055] In some embodiments, lung cancer includes, but is not limited to, the following types: small cell lung cancer and non-small cell lung cancer. In some embodiments, the pharmaceutical composition comprises a bioactive conjugate and a pharma- ceutically acceptable carrier and / or excipient.

[0056] In some embodiments, the bioactive conjugate or pharmaceutical composition is administered once every 7 to 35 days, preferably once every 7 to 28 days, such as once every 7, 14, 21, 28 or 35 days.

[0057] In some embodiments, the route of administration of the conjugate or pharmaceutical composition includes, but is not limited to, oral administration, transdermal injection, rectal administration, mucosal administration, intramuscular injection, intramedullary injection, intravenous injection, or intraperitoneal injection, preferably intravenous injection.

[0058] In some embodiments, the dose of the bioactive conjugate per administration based on the patient's weight is 1 mg / kg to 30 mg / kg, preferably 1 mg / kg to 20 mg / kg, more preferably 2 mg / kg to 12 mg / kg, even more preferably 2 to 5 mg / kg, 4 to 7 mg / kg, 6 to 9 mg / kg, 8 to 11 mg / kg, 10 to 13 mg / kg, or 12 to 15 mg / kg, for example, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 11 mg / kg, or 12 mg / kg.

[0059] In some embodiments, the administration regimen of the biologically active conjugate is divided into one or more administration phases (e.g., 1, 2, 3, or 4 phases), with the administration cycles and doses in each phase being independently selected from the administration cycles or doses described above.

[0060] In some embodiments, the uses or methods of the invention result in tumor elimination or volume reduction.

[0061] In some embodiments, the uses or methods of the invention result in a tumor volume reduction of at least 5%, at least 10%, at least 15%, at least 20%, at least 30% or at least 40%.

[0062] definition Unless otherwise specified below, all scientific and technical terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. References to technology used herein are intended to be references to technology commonly understood in the art, including technical modifications or equivalent technical substitutions that are obvious to those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are provided to better explain the present invention.

[0063] Drug-to-antibody ratio (DAR) refers to the average loading of the small molecule toxin drug by the antibody in the conjugate. For a particular conjugate molecule, the binding ratio of the small molecule toxin drug moiety to the antibody moiety has an exact value, but when used to describe a sample containing a large number of molecules, this value is calculated according to the percentage of the various particular conjugate molecules, and this average loading is referred to herein as the average coupling ratio or "DAR."

[0064] NCCN Guidelines refer to clinical practice guidelines for various malignancies published by the National Comprehensive Cancer Network.

[0065] CSCO Guidelines for Diagnosis and Treatment refers to the guidelines for clinical diagnosis and treatment of various malignant tumors issued by the Chinese Society of Clinical Oncology (CSCO).

[0066] Objective response rate (ORR) refers to the proportion of patients whose tumor shrinks to a certain extent and maintains that extent over a certain period of time (including CR and PR cases). Objective tumor response is evaluated by Response Evaluation Criteria in Solid Tumors Version 1.1 (RECIST 1.1). Subjects must have measurable tumor lesions at baseline. According to the RECIST 1.1 criteria, efficacy evaluation criteria are divided into complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD).

[0067] Progressive Disease (PD): A relative increase in the sum of diameters of at least 20% relative to the smallest sum of all diameters of measured target lesions (if the baseline measurement is smallest, then baseline is considered the reference); and an absolute increase in the sum of diameters of at least 5 mm (appearance of one or more new lesions is also considered progressive disease).

[0068] Stable disease (SD): The shrinkage of the target lesions does not reach PR, the increase does not reach PD levels, the symptoms are intermediate between them, and the minimum sum of diameters can be used as the criterion in the study.

[0069] Partial Response (PR): At least a 30% reduction in the sum of target lesions compared to baseline.

[0070] Complete response (CR): All target lesions must disappear and the short axis of all pathological lymph nodes (including target and non-target nodes) must shrink to less than 10 mm.

[0071] Dose-limiting toxicity (DLT): Toxic and side effects of a drug that are the primary reason for limiting continued increases in drug dose.

[0072] Adverse Event (AE): means an untoward medical occurrence that occurs after a patient or clinical research subject receives a drug, but which does not necessarily have a causal relationship to the treatment.

[0073] Treatment-Emergent Adverse Event (TEAE): means any AE that occurs or worsens at the time of or after the first dose. [Brief description of the drawings]

[0074] [Figure 1] The drawings described in this application are used to provide further understanding of the present invention and constitute a part of this application. The exemplary examples of the present invention and their description are used to explain the present invention and do not unduly limit the present invention. In the drawings, Figure 1 shows the in vitro plasma stability of conjugate A and the commercially available drug Trodelvy™.

[0075] Detailed Description In the following, the technical solutions in the embodiments of the present invention are clearly and completely described with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part, not all, of the embodiments of the present invention. The following description of at least one exemplary embodiment is merely exemplary in nature, and is not intended to limit the present invention, its application or use in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are included in the scope of protection of the present invention.

[0076] Example 1. 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamino)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate [ka]

[0077] Step 1: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(propane-2-alkyn-1-yl)hexa-5-alkynamide At 25°C, prop-2-yn-1-amine (189mg, 3.4mmol) and compound 3-4 (800mg, 2.83mmol) were dissolved in dichloromethane (10mL), N,N-diisopropylethylamine (738mg, 5.67mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.63g, 4.25mmol) were added in sequence, and the mixture was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by fast silica gel column (ethyl acetate / petroleum ether=3 / 1) to give the title compound (700mg). ESI-MS (m / z): 306.1 [M+H]+.

[0078] Step 2: Synthesis of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontane amino)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-2H-pyrano[2,3-b]-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-4-yl)carbonate Under nitrogen protection at 25°C, T-030 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0°C, then 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL) was added, followed by slow dropwise addition of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL). The mixture was then stirred at 0°C for 20 minutes, and the reaction mixture was bubbled with nitrogen for 20 minutes. (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacetamidyl)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyphenyl)diphenylmethyl)amino)acetamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added. The mixture was then reacted with stirring for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC to give the title compound (500 mg). ESI-MS (m / z): 1597.5 [M+H]+.

[0079] Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontaneamino)benzyl)carbonate] At room temperature, compound 33-1 (14 mg, 0.05 mmol) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), copper(I) bromide (11 mg, 0.08 mmol) was added, and the mixture was stirred and reacted for 1 hour. The system was purified by preparative HPLC to obtain the title compound (30 mg). ESI-MS (m / z): 815.9 [(M-273) / 2+H]+.

[0080] Step 4: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamino)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound IM-1) Compound 33-2 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL), trifluoroacetic acid (0.2 mL) was added to the reaction solution, and the mixture was reacted at room temperature for 30 minutes. After purification by preparative high performance liquid chromatography (method C), the trifluoroacetate of the title compound was obtained (20.0 mg). Its structure is characterized as follows:

[0081] [Table 1]

[0082] Example 2. Preparation of Conjugate A 0.3 mL of sacituzumab antibody (anti-Trop-2, 33.5 mg / mL) was taken and diluted with 0.25 mL of a solution containing 20 mM PB, 150 mM NaCl and 20 mM sodium edetate (pH 7.6), then 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) was added, and the mixture was mixed well. The pH of the reaction mixture was adjusted to 7.4 with 1 M Na2HPO4 solution, 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added, the mixture was mixed well, and left at room temperature for 30 minutes. 10 times molar IM-1 trifluoroacetate dissolved in dimethyl sulfoxide was added to the solution system, the system was mixed well, and left at room temperature for 2 hours. Then 6.1 μl of 100 mM cysteine ​​was added to stop the reaction. Finally, the buffer solution was replaced with PBS buffer solution at pH 6.5 using a G-25 gel column to obtain the product of IM-1 coupled to sacituzumab antibody, which was named conjugate A. [ka]

[0083] The molecular weight of conjugate A was analyzed by LCMS, and the measured molecular weights of the light and heavy chains of conjugate A were correlated with the theoretical molecular weights of the light and heavy chains coupled to different numbers of toxins. Each antibody molecule in conjugate A was found to be coupled to 1-10 toxins (i.e., γ was 1-10). The average coupling ratio (DAR) was then calculated to be about 6.9 according to the respective proportions of the conjugate molecules coupled to different numbers of toxins.

[0084] Referring to Example 2, conjugate A samples having DAR values ​​in the range of 6 to 8 (e.g., 7.3 or 7.4) were produced in batches and subjected to the following non-clinical and clinical studies.

[0085] Experimental Example 1. Detection of the inhibitory effect of the conjugate on the proliferation of pancreatic cancer cell lines The effect of conjugate A on the proliferation of BxPC-3 cells (pancreatic cancer cell line from ATCC, TROP2 positive cells) was detected by Celititer-Glo® chemiluminescent cell viability assay (i.e., CTG). On the first day of the experiment, BxPC-3 cells in logarithmic growth phase were harvested, the concentration of the cell suspension was adjusted with culture medium, and the suspension was added to a 96-well cell culture plate to a final cell concentration of 2000 cells / well. The cells were cultured overnight at 37°C in a 5% CO2 incubator. On the second day of the experiment, conjugate A (final concentration 0.152-1000 nM) was diluted in a 1:3 gradient into a 10-fold diluted working solution, and 10 μl of the corresponding 10-fold diluted working solution was added to each well (three duplicate wells for each drug concentration). After the addition of the drugs, the cells were cultured at 37°C in a 5% CO2 incubator for 72 hours. On the fifth day of the experiment, 50 μl (1 / 2 the culture volume) of premelted and room temperature equilibrated CTG solution was added to each well, and the mixture was mixed well for 2 min on a microplate shaker. After standing at room temperature for 20 min, the fluorescence signal values ​​were measured using an Envision 2104 plate reader.

[0086] The results showed that conjugate A had an IC of 14.3 nM. 50 and significantly inhibited the proliferation of TROP2-positive cells BxPC-3, with a maximum inhibition rate of 95.3%, indicating that conjugate A can inhibit the proliferation of TROP2-positive pancreatic cancer cells, suggesting that it has a therapeutic effect on pancreatic cancer.

[0087] Experimental Example 2. Detection of the effects of the conjugate on cardiovascular and respiratory functions Cynomolgus monkeys were divided into groups of 10 (half male and half female). Conjugate A was intravenously injected once every 2 weeks for a total of 4 times at doses of 25 mg / kg, 50 mg / kg, and 75 mg / kg, respectively. To evaluate the effect of Conjugate A on the cardiovascular and respiratory functions of cynomolgus monkeys, lead II ECG and respiratory frequency were detected by a large animal non-invasive physiological signal telemetry system, and arterial blood pressure was measured by a non-invasive sphygmomanometer.

[0088] The results showed that for monkeys in each group receiving Conjugate A before the first dose, 2-3 hours after the first dose, 24-25 hours, 72-73 hours, 7 days after, about 2-3 hours after the last dose, and before the end of the recovery period, no arrhythmias were observed in lead II ECG, and no significant abnormalities were observed in systolic blood pressure, diastolic blood pressure, mean arterial pressure, and respiratory rate in heart rate, RR interval, P wave duration, PR interval, QRS wave duration, QT interval, corrected QT interval, and other parameters of lead II ECG. These results indicate that Conjugate A does not affect the cardiovascular and respiratory systems of cynomolgus monkeys and has good prospects for clinical drug safety.

[0089] Experimental Example 3. Detection of toxic metabolic behavior of conjugates in animals Cynomolgus monkeys were divided into four groups according to different doses: control group, 25 mg / kg dose group, 50 mg / kg dose group, and 75 mg / kg dose group, each group consisting of 5 males and 5 females. Cynomolgus monkeys were intravenously injected with conjugate A at the dose levels of 25 mg / kg and 50 mg / kg once every 2 weeks for four consecutive injections. For each group of monkeys, blood samples were taken before and immediately after the first and last dose (0-1 min after the end of the dose), as well as 4 hours, 24 hours, 48 ​​hours, 96 hours, and 168 hours, before and immediately after the second and third dose (0-1 min after the end of the dose), and 336 hours after the last dose. For monkeys in the 75 mg / kg dose group, blood samples were further collected at 4, 24, 48, 96 and 168 hours after the third dose to detect the toxicokinetic behavior of the conjugate and the toxin molecules released thereby in vivo.

[0090] The following table shows the peak plasma concentrations (C) of conjugate A and the toxin molecules released by conjugate A in male and female monkeys following administration in the manner described above. max The results show that the highest non-severe toxicity dose (HNSTD) of conjugate A was 50 mg / kg, and after the last administration at this dose, the exposure to the toxin molecule in female and male monkeys was 3.85 h, respectively. * μg / mL and 5.86h * and exposure to conjugate A in female and male monkeys was 45.8 h, respectively. * mg / mL and 64.2h * This indicates that the concentration was mg / mL.

[0091] [Table 2]

[0092] Experimental results showed that exposure to ADC and toxins or C in all animal groups maxIt can be seen that the data was close after the first and last doses, indicating that conjugate A showed no obvious toxicity accumulation after continuous intravenous administration and was well tolerated in animals, and therefore it has good prospects for clinical drug safety.

[0093] Experimental Example 4. Metabolic stability of the conjugate in vitro In this experiment, conjugate A and Trodelvy™ were first formulated into a 3.4 mg / mL working solution, respectively, in saline, and then conjugate A and Trodelvy™ working solutions were spiked into human blank plasma, respectively, to obtain 0.05 mg / mL human plasma samples. Plasma samples were incubated at 37° C., and the release of toxin molecules was measured at 1 hour, 3 hours, 24 hours, 48 ​​hours, 72 hours, and 144 hours. Conjugate A and the commercially available drug Trodelvy™ were compared for differences in plasma stability in vitro. The results are shown in FIG. 1.

[0094] As shown in FIG. 1 herein, the yield of free toxin in human plasma increased with increasing incubation time; after 24 hours of incubation, the release rates of the toxin molecules of conjugate A and the commercially available ADC drug Trodelvy™ in human plasma were 28.5% and 93.4%, respectively; after 48 hours of incubation, the release rates of the toxin molecules of conjugate A and the commercially available ADC drug Trodelvy™ in human plasma were 44.6% and 109.1%, respectively; after 72 hours of incubation, the release rates of the toxin molecules of conjugate A and the commercially available ADC drug Trodelvy™ in human plasma were 53.7% and 100.6%, respectively; and after 144 hours of incubation, the release rates of the toxin molecules of conjugate A and the commercially available ADC drug Trodelvy™ in human plasma were 65.3% and 104.8%, respectively.

[0095] The results show that the release rate of the toxin molecules of conjugate A in human plasma is significantly lower than that of Trodelvy™, indicating that conjugate A is relatively stable in human plasma, releases less toxin in plasma, and effectively achieves the goal of releasing the toxin after reaching the target tumor site. Furthermore, compared with the commercially available drug Trodelvy™, conjugate A may reduce the risk of severe side effects caused by the rapid and excessive release of toxin in plasma.

[0096] Clinical Trial Example 1: Phase I Clinical Trial I. Protocol Patients with malignant tumors of epithelial origin diagnosed by histological examination were treated with conjugate A. All patients had unresectable locally advanced or metastatic solid tumors that had failed standard treatment or had no standard treatment regimen or were not currently suitable for standard treatment. Five dose levels were designed based on the patient's weight, 2, 4, 6, 9 and 12 mg / kg, which were administered intravenously once every 2 weeks for 28 days in each cycle, until disease progression or intolerable toxic reactions occurred in each cycle. Toxicity of all planned dose groups and several intermediate dose groups was evaluated by BLRM. Dose escalation decisions were made jointly by the sponsor and investigator based on model analysis.

[0097] II. Safety Results During treatment, no "treatment-emergent adverse events (hereinafter referred to as TEAEs)" of grade 3 or higher occurred in the 2 mg / kg dose group. Grade 3 TEAEs occurred in the 4 mg / kg dose group, including oral mucositis, anemia, etc. Grade 3 TEAEs occurred in the 6 mg / kg dose group, including decreased neutrophil count, decreased white blood cell count, etc. The above grade 3 TEAEs were reversible after symptomatic treatment, and the drug could be administered continuously, indicating that conjugate A has good clinical safety.

[0098] III. Efficacy Results Treatment outcomes for various indications are as follows:

[0099] 1. Triple-negative breast cancer (TNBC) In all patients with triple-negative breast cancer, some patients had a partial response.

[0100] A patient with progressive disease after prior adriamycin / cyclophosphamide / paclitaxel neoadjuvant chemotherapy, left mastectomy and left thoracic radiotherapy was given intravenous injections of conjugate A at a dose of 4 mg / kg based on the patient's weight, according to the above administration cycle. After 18 weeks, a partial response was observed, which lasted for 6 weeks. At several efficacy evaluations, the total volume of the target lesions could be reduced by 40%. The patient did not show any serious adverse events.

[0101] 2. Ovarian cancer In all patients with ovarian cancer, some patients showed partial responses.

[0102] Patients with ovarian cancer who had failed prior hysterectomy, oophorectomy, carboplatin / paclitaxel, rucaparib, and carboplatin / docetaxel / bevacizumab treatment were intravenously injected with conjugate A at a dose of 4 mg / kg based on the patient's weight according to the above administration cycle. After 21 weeks, a partial response was observed, which lasted for 15 weeks. In several efficacy evaluations, the total volume of the target lesions could be reduced by 64.8%, and one of the target lesions completely disappeared. The patient did not show any serious adverse events.

[0103] 3.HER2 positive breast cancer Patients with HER2-positive breast cancer who had failed prior modified radical mastectomy, epirubicin / paclitaxel, HER2 monoclonal antibody / docetaxel and capecitabine treatment were given intravenous injections of conjugate A at a dose of 6 mg / kg based on the patient's weight according to the above administration cycle. After 8 weeks, partial responses were observed. At several efficacy evaluations, the total volume of the target lesions could be reduced by 49.6%.

[0104] 4. Stomach cancer Patients with gastric cancer who had failed treatment with prior total gastrectomy, paclitaxel / tegafur, anlotinib / tegafur, anlotinib / capecitabine, oxaliplatin / fluorouracil and oxaliplatin / raltitrexed were intravenously injected with conjugate A at a dose of 4mg / kg based on the patient's weight according to the above administration cycle. After 15 weeks, partial response was observed, which lasted for 10 weeks and the patient is still benefiting. In several efficacy evaluations, the total volume of the target lesions could be reduced by 62.8%, and one of the target lesions completely disappeared. No serious adverse events occurred in the patient.

[0105] 5. Pancreatic cancer Patients with pancreatic cancer who had failed prior pancreatic cancer distal resection, radiation therapy, gemcitabine / capecitabine, gemcitabine / irinotecan / fluorouracil, gemcitabine / albumin paclitaxel / oxaliplatin / fluorouracil, and nivolumab / caviralizumab multi-line therapy were intravenously injected with conjugate A at a dose of 4mg / kg based on the patient's weight according to the above administration cycle, and stable disease was observed after 7 weeks, which lasted for 30.3 weeks. In several efficacy evaluations, the total volume of the target lesions could be reduced by 8.8%, and the patient did not experience any serious adverse events.

[0106] In summary, the phase I clinical trial showed that conjugate A has good efficacy at least for the above-mentioned unresectable metastatic TNBC, ovarian cancer, HER2-positive breast cancer, gastric cancer, and pancreatic cancer that have failed standard treatment, without inducing severe toxicity that may prevent clinical use. These results prove that conjugate A has good safety and efficacy in the treatment of tumor diseases, and that conjugate A with DARs ranging from 6 to 8 (e.g., DAR7.3 or DAR7.4) manufactured in various batches has basically consistent efficacy and safety.

[0107] Various modifications of the present invention other than those described herein will be apparent to those skilled in the art from the above description. Such modifications are intended to be included within the scope of the appended claims. All references cited in this application (including all patents, patent applications, journal articles, books, and any other publications) are incorporated herein by reference in their entirety.

Claims

1. In the manufacture of a medicament for treating a tumor disease, 【Chemistry 1】 [In the formula, L １ teeth 【Chemistry 2】 where R １ and R ２ are each independently hydrogen, halogen, carboxylate, sulfonate, cyano, C １－６ Alkyl, halogenated C １－６ Alkyl, cyano-substituted C １－６ Alkyl, C １－６ Alkoxy, C ２－１０ Alkenyl or C ２－１０ alkynyl; Z １ is an amino acid or a peptide of 2 to 10 amino acids; x １ and x ２ are each independently 0, 1, 2, 3, 4, 5 or 6; L １ Position 1 of L is bonded to D １ Position 2 of L ２ binds to; L ２ teeth 【Chemistry 3】 where y1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; L ２ Position 1 of L １ Binds to L ２ Position 2 of L ３ binds to; L ３ is a 5-12 membered heteroaromatic ring; L ４ teeth 【Chemistry 4】 where Z ２ is C １－６ Alkylene, C ２－１０ Alkenylene, C ２－１０ Alkynylene and C ３－８ cycloalkylene; R ３ is H and C １－６ alkyl; Z ３ does not exist, or C １－６ alkylene; or R ３ and Z ３ together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclyl; α is 0, 1, 2, 3, 4, 5, or 6; L ４ Position 2 of L is bonded to E; ４ Position 1 of L ３ binds to; E is 【Chemistry 5】 where each R ４ are independently hydrogen, β is 0, 1 or 2, position 2 of E is bonded to A, and position 1 of E is L ４ is bound to; m １ , m ２ and m ３ are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; D is a biologically active molecular fragment; γ is selected from an integer from 1 to 10; and A is a monoclonal antibody against Trop-2 or an antigen-binding fragment thereof. The use of a bioactive conjugate represented by the formula:

2. 2. The use according to claim 1, wherein the tumor disease is an unresectable locally advanced or metastatic solid tumor that has failed standard treatment or that has no standard treatment regimen or is currently unsuitable for standard treatment.

3. 3. The use according to claim 2, wherein the tumor disease is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, esophageal cancer, liver cancer, colorectal cancer, cervical cancer, endometrial cancer, pancreatic cancer, bladder cancer or brain cancer.

4. The conjugate has the following structure: L １ but 【Chemistry 6】 is selected from L １ Position 1 of L is bonded to D １ Position 2 of L ２ Binds to L ２ but 【Chemistry 7】 where y １ is 3, 4, 5, 6, 7, 8, 9 or 10; L ２ Position 1 of L １ is bonded to L ２ Position 2 of L ３ is bound to L ３ is selected from 5- to 6-membered heteroaromatic rings; L ４ but 【Chemistry 8】 where Z ２ is C １－３ alkylene; R ３ is H, and Z ３ is C １－３ alkylene, α is 1, and L ４ Position 2 of L is bonded to E; ４ Position 1 of L ３ is bound to E 【Chemistry 9】 where each R ４ are independently hydrogen, β is 0, 1 or 2, position 2 of E is bonded to A, and position 1 of E is L ４ is bound to m １ , m ２ and m ３ are all 1, Bioactive molecules 【Chemistry 10】 is selected from γ is selected from an integer from 3 to 8; 2. The use according to claim 1, wherein A is sacituzumab or an antigen-binding fragment thereof.

5. The structure of the conjugate is 【Chemistry 11】 (wherein γ is an integer from 1 to 10) The use according to claim 1,

6. A pharmaceutical composition for the treatment of a tumor disease comprising a biologically active conjugate according to claim 1 and a pharma- ceutically acceptable carrier and / or excipient.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is administered once every 7 to 35 days.

8. 7. The pharmaceutical composition according to claim 6, wherein the route of administration of the pharmaceutical composition includes oral administration, transdermal injection, rectal administration, mucosal administration, intramuscular injection, intramedullary injection, intravenous injection or intraperitoneal injection.

9. The pharmaceutical composition according to claim 6, wherein each dose of the biologically active conjugate is 1 mg / kg to 30 mg / kg based on the patient's body weight.

10. The pharmaceutical composition according to claim 6, wherein the administration of the biologically active conjugate is divided into one or more administration steps, the administration cycle in each step being as described in claim 7, and / or the administration dose in each step being as described in claim 9.

11. 2. The use of the biologically active conjugate of claim 1, wherein position 2 of E is linked to A via a sulfhydryl on A.

12. 2. The use of the biologically active conjugate according to claim 1, wherein γ is selected from the integers 3 to 8.

13. The use according to claim 3, wherein the breast cancer is selected from luminal type A, luminal type B, Her-2 positive type and triple negative type breast cancer.

14. The use according to claim 3, wherein the ovarian cancer is selected from platinum-sensitive ovarian cancer and platinum-resistant ovarian cancer.

15. 4. The use according to claim 3, wherein the gastric cancer is selected from gastric adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma and neuroendocrine tumor.

16. The use according to claim 3, wherein the pancreatic cancer is selected from pancreatic epithelial tumors, pancreatic exocrine tumors, pancreatic borderline tumors, pancreatic tubular adenocarcinomas, pancreatic endocrine tumors, pancreatic mature teratomas, pancreatic mesenchymal tumors, pancreatic malignant lymphomas and pancreatic secondary tumors.

17. The use according to claim 3, wherein the bladder cancer is selected from urothelial (transitional cell) carcinoma, bladder squamous cell carcinoma and bladder adenocarcinoma.

18. 18. The use according to claim 17, wherein the urothelial carcinoma is selected from basal-type, luminal-type and wild-type urothelial carcinoma.

19. The use according to claim 3, wherein the lung cancer is selected from small cell lung cancer and non-small cell lung cancer.

20. The use according to claim 3, wherein the tumor disease is endometrial cancer.

21. 6. The use according to claim 5, wherein γ is selected from the integers from 5 to 8.

22. 7. The pharmaceutical composition of claim 6, wherein the composition has a drug-to-antibody ratio (DAR) value of 1-12.

23. The pharmaceutical composition according to claim 22, having a DAR value of 1 to 10.

24. The pharmaceutical composition according to claim 23, having a DAR value of 5 to 8.

25. 25. The pharmaceutical composition of claim 24, having a DAR value of 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.

0.

26. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is administered once every 7 to 28 days.

27. 27. The pharmaceutical composition of claim 26, wherein the pharmaceutical composition is administered once every 7, 14, 21, 28 or 35 days.

28. The pharmaceutical composition according to claim 8, wherein the route of administration is intravenous injection.

29. 10. The pharmaceutical composition of claim 9, wherein each dose of the biologically active conjugate is 1 mg / kg to 20 mg / kg based on the patient's body weight.

30. 30. The pharmaceutical composition of claim 29, wherein each dose of the biologically active conjugate is from 2 mg / kg to 12 mg / kg based on the patient's body weight.

31. 31. The pharmaceutical composition of claim 30, wherein each dose of the biologically active conjugate is 2-5 mg / kg, 4-7 mg / kg, 6-9 mg / kg, 8-11 mg / kg, 10-13 mg / kg or 12-15 mg / kg based on the patient's weight.

Images