Use of a drug in the treatment of a neoplastic disease

Abstract

The present invention relates to the use of a medicament for the treatment of a tumor disease. In particular, the present invention provides the use of a bioactive agent conjugate of formula (I) for the treatment of a tumor disease, in particular preferably a non-resectable locally advanced or metastatic solid tumor refractory to existing standard treatment, 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 tumor.

Description

[0001] This application is a divisional application of Chinese National Application No. 202280025989.4, which entered the Chinese national phase on September 28, 2023, and is entitled "Application of Drugs in the Treatment of Tumor Diseases". Technical Field

[0002] This application relates to the use of drugs in the treatment of diseases associated with abnormal cell activity, including but not limited to oncological diseases, particularly refractory and unresectable locally advanced or metastatic solid tumors that are resistant to existing standard of treatment, such as tumors that have failed and / or relapsed after first-line chemotherapy, tumors that have failed and / or relapsed after radiotherapy, and / or tumors that have failed and / or relapsed after targeted therapy. Background Technology

[0003] Cancer is a major burden on global public health. In the United States, cancer remains the second leading cause of death after cardiovascular disease; in China, the number of cancer cases is also on the rise. In 2019, there were approximately 4.4 million new cases of malignant tumors and approximately 2.624 million deaths from cancer in China. The continuous increase in the number of cancer cases and deaths will lead to an expansion of the overall market size for cancer treatment.

[0004] Chemotherapy is one of the main treatments for cancer, but traditional chemotherapy drugs lack tumor-specific recognition capabilities, easily damaging normal cells and causing serious adverse reactions in patients. To improve the survival rate of cancer patients, there is an urgent need for innovation in treatment methods to keep pace with advancements in detection and diagnosis. Despite significant progress in many indications, mortality rates for some of the most difficult-to-treat solid tumors have not improved significantly since the 1970s, and more effective treatments with fewer side effects are still needed. Molecularly targeted drugs are an important direction in current drug design.

[0005] Monoclonal antibody drugs have advantages such as strong targeting, high specificity, and a low incidence of serious adverse reactions, but their large molecular weight limits their efficacy as monotherapy. Antibody-drug conjugates (ADCs) are a class of drugs that use chemical linkers to conjugate monoclonal antibodies to varying numbers of small-molecule cytotoxic molecules (effect molecules). After entering the body, ADC molecules can bind to antigens on the surface of target cells through the guidance of monoclonal antibodies, entering the target cells. Once inside the cells, ADC molecules release effector molecules through chemical and / or enzymatic reactions, thereby eliminating the target cells. ADC drugs combine the advantages of strong targeting of monoclonal antibodies and the high activity of small-molecule toxins, reducing the toxic side effects of small-molecule cytotoxic molecules while improving drug efficacy.

[0006] Currently, several ADC drugs are available globally, covering indications such as leukemia, lymphoma, and breast cancer. However, some ADC drugs still have issues with pharmacokinetic properties and safety, potentially leading to serious adverse reactions in patients. Furthermore, the efficacy rates for treating some metastatic, recurrent, and / or refractory cancers still need further improvement. Therefore, there is still a need to develop ADC drugs for the treatment of these metastatic, recurrent, and / or refractory cancers, maximizing their effectiveness and minimizing their toxicity to meet the medication needs of cancer patients. Summary of the Invention

[0007] This invention provides the use of the bioactive conjugate of formula (I) in the preparation of medicaments for treating tumor diseases;

[0008] {D-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]} γ -A

[0009] Formula (I)

[0010] in,

[0011] L1 is Each of R1 and R2 is independently hydrogen (e.g., protium or deuterium), halogen, carboxylic acid group, sulfonic acid group, cyano group, or C. 1-6 Alkyl, Halogenated C 1-6 alkyl and cyano substituted C 1-6 Alkyl groups (e.g., -CH2CN), C 1-6 Alkoxy, C 2-10 alkenyl or C 2-10 Alkyne group; Z1 is an amino acid or a peptide composed of 2 to 10 amino acids; x1 and x2 are each independently 0, 1, 2, 3, 4, 5 or 6; and L1 is connected to D at position 1 and L2 at position 2.

[0012] L2 is Where y1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and L2 is connected to L1 at position 1 and L2 is connected to L3 at position 2.

[0013] L3 is a 5- to 12-membered heterocyclic aromatic ring;

[0014] L4 is Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 etyne group, and C 3-8 Cycloalkylene; R3 is selected from H and C 1-6Alkyl group; Z3 is absent or is C 1-6 Alkylene; or, R3 and Z3 together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic group; α is 0, 1, 2, 3, 4, 5 or 6, and L4 is attached to E at position 2 and to L3 at position 1;

[0015] E is In this system, each R4 is independently hydrogen (e.g., protium or deuterium), β is 0, 1 or 2, and E is connected to A at position 2 (e.g., to a thiol group on A) and to L4 at position 1.

[0016] m1, m2 and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0017] D represents a bioactive molecular fragment;

[0018] γ refers to {D-[L1-(L2]} which is linked to A via a thioether bond. m1 -(L3) m2 -(L4) m3 The number of the part γ is selected from an integer between 1 and 10; preferably, γ is selected from an integer between 3 and 8 (e.g., 3, 4, 5, 6, 7 or 8).

[0019] A is a monoclonal antibody against Trop-2 or its antigen-binding fragment.

[0020] In some implementations, the tumor is an unresectable locally advanced or metastatic solid tumor that has failed standard treatment, or for which there is no standard treatment option, or for which standard treatment is not currently applicable. In some implementations, the standard treatment refers to the standard treatment regimen recommended by the NCCN guidelines and CSCO guidelines for the tumor.

[0021] In some embodiments, the tumor is a tumor that has failed and / or relapsed after first-line chemotherapy. In other embodiments, the first-line chemotherapy drug refers to the first-line chemotherapy drug recommended by the NCCN guidelines and CSCO guidelines for the tumor.

[0022] In some embodiments, the tumor is a failed and / or recurrent tumor that has undergone radiotherapy. In other embodiments, the radiotherapy refers to a radiotherapy regimen recommended by the NCCN and CSCO guidelines for the tumor.

[0023] In some embodiments, the tumor is a tumor that has failed to respond to targeted therapy or immunotherapy and / or has recurred. In other embodiments, the targeted therapy or immunotherapy refers to the targeted therapy or immunotherapy recommended by the NCCN guidelines and CSCO guidelines for the treatment of the tumor.

[0024] In some embodiments, the tumor disease includes, but is not limited to, breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial carcinoma, esophageal cancer, liver cancer, colorectal cancer, cervical cancer, endometrial cancer, pancreatic cancer, bladder cancer, or brain tumor; preferably breast cancer (e.g., triple-negative breast cancer or Her2-positive breast cancer), ovarian cancer (e.g., ovarian epithelial carcinoma), gastric cancer, lung cancer, pancreatic cancer, bladder cancer, or urothelial carcinoma; more preferably, the tumor disease is triple-negative breast cancer, Her2-positive breast cancer, ovarian cancer, gastric cancer, lung cancer, or pancreatic cancer; even more preferably, the tumor disease is triple-negative breast cancer, Her2-positive breast cancer, ovarian cancer, or gastric cancer.

[0025] In some implementations, the tumor is breast cancer.

[0026] In some implementations, the breast cancer includes, but is not limited to, the following types: Luminal A, Luminal B, Her-2 positive, and triple negative.

[0027] In some implementations, the tumor disease is triple-negative breast cancer.

[0028] In some implementations, the tumor is Her2-positive breast cancer.

[0029] In some implementations, the tumor disease is ovarian cancer.

[0030] In some implementations, the ovarian cancer includes, but is not limited to, the following types: platinum-sensitive and platinum-resistant.

[0031] In some implementations, the tumor disease is gastric cancer.

[0032] In some implementations, the gastric cancer includes, but is not limited to, the following types: adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, and neuroendocrine tumor.

[0033] In some implementations, the tumor disease is pancreatic cancer.

[0034] In some implementations, the pancreatic cancer includes, but is not limited to, the following types: epithelial tumors, exocrine tumors, borderline tumors, ductal adenocarcinomas, endocrine tumors, mature teratomas, mesenchymal tumors, malignant lymphomas, and secondary tumors.

[0035] In some implementations, the tumor is bladder cancer.

[0036] In some implementations, the bladder cancer includes, but is not limited to, the following types: urothelial (transitional cell) carcinoma, squamous cell carcinoma, and adenocarcinoma.

[0037] In some implementations, the tumor is urothelial carcinoma.

[0038] In some implementations, the urothelial carcinoma includes, but is not limited to, the following types: basal-like, luminal-like, and wild-type.

[0039] In some implementations, the tumor is lung cancer.

[0040] In some implementations, the lung cancer includes, but is not limited to, the following types: small cell lung cancer and non-small cell lung cancer.

[0041] In some embodiments, the coupling has the following structure:

[0042] L1 is selected from , , , , and Furthermore, position 1 of L1 is connected to D, and position 2 of L1 is connected to L2.

[0043] L2 is Where y1 is 3, 4, 5, 6, 7, 8, 9 or 10; and L2 is connected to L1 at position 1 and L2 is connected to L3 at position 2.

[0044] L3 is selected from 5-6 membered heteroaromatic rings, such as pyrazole or triazole;

[0045] L4 is Z2 is selected from C 1-3 Alkylene; R3 is H; Z3 is selected from C 1-3 Alkylene; α is 0, and L4 is connected to E at position 2 and to L3 at position 1;

[0046] E is In this system, each R4 is independently hydrogen (e.g., protium or deuterium), β is 0, 1 or 2, and E is connected to A at position 2 (e.g., to a thiol group on A) and to L4 at position 1.

[0047] m1, m2 and m3 are all 1;

[0048] The bioactive molecules are selected from and Preferably, the bioactive molecule is linked to position 1 of L1 via its own hydroxyl group.

[0049] γ is selected from an integer between 3 and 8 (e.g., 3, 4, 5, 6, 7 or 8);

[0050] A represents Sacituzumab or its antigen-binding fragment.

[0051] In some embodiments, the coupling has the following structure:

[0052] L1 is selected from , , , , and Furthermore, position 1 of L1 is connected to D, and position 2 of L1 is connected to L2.

[0053] L2 is Where y1 is 3, 4, 5, 6, 7, 8, 9 or 10; and L2 is connected to L1 at position 1 and L2 is connected to L3 at position 2.

[0054] L3 is selected from 5-6 membered heteroaromatic rings, such as pyrazole or triazole;

[0055] L4 is Z2 is selected from C 1-3 Alkylene; R3 is H; Z3 is selected from C 1-3 Alkylene; α is 1, and L4 is connected to E at position 2 and to L3 at position 1;

[0056] E is In this system, each R4 is independently hydrogen (e.g., protium or deuterium), β is 0, 1 or 2, and E is connected to A at position 2 (e.g., to a thiol group on A) and to L4 at position 1.

[0057] m1, m2 and m3 are all 1;

[0058] The bioactive molecules are selected from and Preferably, the bioactive molecule is linked to position 1 of L1 via its own hydroxyl group.

[0059] γ is selected from an integer between 3 and 8 (e.g., 3, 4, 5, 6, 7 or 8);

[0060] A represents Sacituzumab or its antigen-binding fragment.

[0061] In some implementation schemes, D is selected from... and .

[0062] In some technical solutions, the coupling element is coupling element A with the structure shown in the following formula:

[0063]

[0064] Wherein, γ is an integer from 1 to 10; preferably, γ is selected from an integer between 5 and 8.

[0065] In some embodiments, the DAR value of the coupling is 1 to 12; preferably 1 to 10; more preferably 5 to 8; for example, 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.

[0066] In another aspect, the present invention provides a treatment method for a tumor disease, the method comprising the step of administering to an individual in need a therapeutically effective amount of a bioactive conjugate of formula (I) as described above and / or a pharmaceutical composition comprising a bioactive conjugate of formula (I).

[0067] In some implementations, the tumor is an unresectable locally advanced or metastatic solid tumor that has failed standard treatment, or for which there is no standard treatment option, or for which standard treatment is not currently applicable. In some implementations, the standard treatment refers to the standard treatment regimen recommended by the NCCN guidelines and CSCO guidelines for the tumor.

[0068] In some embodiments, the tumor is a tumor that has failed and / or relapsed after first-line chemotherapy. In other embodiments, the first-line chemotherapy drug refers to the first-line chemotherapy drug recommended by the NCCN guidelines and CSCO guidelines for the tumor.

[0069] In some embodiments, the tumor is a failed and / or recurrent tumor that has undergone radiotherapy. In other embodiments, the radiotherapy refers to a radiotherapy regimen recommended by the NCCN and CSCO guidelines for the tumor.

[0070] In some embodiments, the tumor is a tumor that has failed to respond to targeted therapy or immunotherapy and / or has recurred. In other embodiments, the targeted therapy or immunotherapy refers to the targeted therapy or immunotherapy recommended by the NCCN guidelines and CSCO guidelines for the treatment of the tumor.

[0071] In some embodiments, the tumor disease includes, but is not limited to, breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial carcinoma, esophageal cancer, liver cancer, colorectal cancer, cervical cancer, endometrial cancer, pancreatic cancer, bladder cancer, or brain tumor; preferably breast cancer (e.g., triple-negative breast cancer or Her2-positive breast cancer), ovarian cancer (e.g., ovarian epithelial carcinoma), gastric cancer, lung cancer, pancreatic cancer, bladder cancer, or urothelial carcinoma; more preferably, the tumor disease is triple-negative breast cancer, Her2-positive breast cancer, ovarian cancer, gastric cancer, lung cancer, or pancreatic cancer; even more preferably, the tumor disease is triple-negative breast cancer, Her2-positive breast cancer, ovarian cancer, or gastric cancer.

[0072] In some implementations, the tumor is breast cancer.

[0073] In some implementations, the breast cancer includes, but is not limited to, the following types: Luminal A, Luminal B, Her-2 positive, and triple negative.

[0074] In some implementations, the tumor disease is triple-negative breast cancer.

[0075] In some implementations, the tumor is Her2-positive breast cancer.

[0076] In some implementations, the tumor disease is ovarian cancer.

[0077] In some implementations, the ovarian cancer includes, but is not limited to, the following types: platinum-sensitive and platinum-resistant.

[0078] In some implementations, the tumor disease is gastric cancer.

[0079] In some implementations, the gastric cancer includes, but is not limited to, the following types: adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, and neuroendocrine tumor.

[0080] In some implementations, the tumor disease is pancreatic cancer.

[0081] In some implementations, the pancreatic cancer includes, but is not limited to, the following types: epithelial tumors, exocrine tumors, borderline tumors, ductal adenocarcinomas, endocrine tumors, mature teratomas, mesenchymal tumors, malignant lymphomas, and secondary tumors.

[0082] In some implementations, the tumor is bladder cancer.

[0083] In some implementations, the bladder cancer includes, but is not limited to, the following types: urothelial (transitional cell) carcinoma, squamous cell carcinoma, and adenocarcinoma.

[0084] In some implementations, the tumor is urothelial carcinoma.

[0085] In some implementations, the urothelial carcinoma includes, but is not limited to, the following types: basal-like, luminal-like, and wild-type.

[0086] In some implementations, the tumor is lung cancer.

[0087] In some embodiments, the 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 the bioactive ingredient conjugate and a pharmaceutically acceptable carrier and / or excipient.

[0088] In some embodiments, the bioactive conjugate or the pharmaceutical composition is administered once every 7 to 35 days, preferably once every 7 to 28 days, for example, once every 7, 14, 21, 28, or 35 days.

[0089] In some embodiments, the administration routes of the conjugate or pharmaceutical composition include, but are not limited to, oral, transdermal, rectal, mucosal, intramuscular, intramedullary, intravenous, or intraperitoneal injection, with intravenous injection being preferred.

[0090] In some embodiments, the dosage of the bioactive conjugate administered per dose is from 1 mg / kg to 30 mg / kg based on the patient's body weight; preferably from 1 mg / kg to 20 mg / kg; more preferably from 2 mg / kg to 12 mg / kg; further preferably from 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.

[0091] In some embodiments, the administration regimen of the bioactive conjugate is divided into one or more administration phases (e.g., one phase, two phases, three phases or four phases), and the administration period and dosage of each phase are independently selected from the administration period or dosage described above.

[0092] In some embodiments, the use or method of the present invention causes tumor elimination or reduction in size.

[0093] In some embodiments, the use or method of the present invention causes a reduction in tumor volume of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, or at least 40%.

[0094] definition

[0095] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to better explain the invention.

[0096] Drug-antibody conjugate ratio (DAR) refers to the average loading of a small molecule toxin drug in a conjugate. While there is a precise value for the ratio of the small molecule toxin drug portion to the antibody portion for a specific conjugate molecule, it should be understood that when used to describe samples containing many molecules, this value refers to an average value calculated based on the percentage of different specific conjugate molecules. This average loading is referred to herein as the average conjugate ratio or "DAR".

[0097] The NCCN guidelines are clinical practice guidelines for various malignant tumors published by the National Comprehensive Cancer Network (NCCN).

[0098] The CSCO guidelines refer to the clinical diagnosis and treatment guidelines for various malignant tumors published by the Chinese Society of Clinical Oncology (CSCO).

[0099] The objective response rate (ORR) refers to the proportion of patients whose tumors shrink to a certain extent and remain so for a certain period of time, including cases of complete remission (CR) and partial remission (PR). The response evaluation criteria in solid tumors version 1.1 (RECIST 1.1 criteria) are used to assess objective response. Subjects must have measurable tumor lesions at baseline. The efficacy evaluation criteria are based on RECIST 1.1 and are categorized as complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD).

[0100] Disease progression (PD): The minimum sum of the diameters of all target lesions measured throughout the study is used as a reference, with a relative increase of at least 20% in the sum of diameters (or the baseline value if the baseline measurement is the minimum); in addition, the absolute value of the sum of diameters must increase by at least 5 mm (the appearance of one or more new lesions is also considered disease progression).

[0101] Stable disease (SD): The degree of reduction in target lesions does not reach the PR level, nor does the degree of increase reach the PD level; it falls between the two. The minimum value of the sum of diameters can be used as a reference in studies.

[0102] Partial response (PR): The sum of target lesions is reduced by at least 30% compared to baseline.

[0103] Complete remission (CR): All target lesions disappear and the short diameter of all pathological lymph nodes (including target nodules and non-target nodules) must be reduced to <10 mm.

[0104] Dose-limiting toxicities (DLT): Drug toxicity that is the main reason for limiting further increases in drug dosage.

[0105] Adverse events (AEs) refer to any adverse medical events that occur after a patient or clinical research subject has received a drug, but are not necessarily causally related to the treatment.

[0106] Treatment Emergent Adverse Event (TEAE): Any adverse event that occurs or worsens during or after the first dose. Attached Figure Description

[0107] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0108] Figure 1 This shows that conjugate A is related to the marketed drug Trodelvy. TM The stability of in vitro plasma. Detailed Implementation

[0109] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0110] Example 1. 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7] indo[1,2-b]quinoline-4-yl) carbonate

[0111]

[0112] Step 1: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-yneamide

[0113] Proprynne-1-amine (189 mg, 3.4 mmol) and compound 3-4 (800 mg, 2.83 mmol) were dissolved in dichloromethane (10 mL) at 25°C. N,N-diisopropylethylamine (738 mg, 5.67 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (1.63 g, 4.25 mmol) were added sequentially, and the mixture was stirred for 2 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by rapid silica gel column chromatography (ethyl acetate / petroleum ether = 3 / 1) to give the title compound, 700 mg. ESI-MS (m / z): 306.1 [M+H]+.

[0114] 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-nonazo-3,9-diazapentadecanoylamino)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-2H-pyrano[2,3-b]-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl) carbonate

[0115] Under nitrogen protection at 25°C, T-030 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL), cooled to 0°C, and a solution of 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL) was added. Then, a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL) was slowly added dropwise. After the addition was complete, the mixture was stirred at 0°C for 20 min, and the reaction solution was purged with nitrogen for 20 min. A solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonazo-6-azatriacetamyl)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyphenyl)diphenylmethyl)amino)acetamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added. After addition, the mixture was stirred at 0°C for 1 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography to give the title compound, 500 mg. ESI-MS (m / z): 1597.5 [M+H]+.

[0116] Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzazido[1,2-b]quinoline-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-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-diazapentadecanoamide)benzyl)carbonate

[0117] Compound 33-1 (14 mg, 0.05 mmol) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL) at room temperature, and cuprous bromide (11 mg, 0.08 mmol) was added. The mixture was stirred for 1 h. The solution was purified by preparative high-performance liquid chromatography (HPLC) to give the title compound, 30 mg. ESI-MS (m / z): 815.9[(M-273) / 2+H]+.

[0118] Step 4: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7] indo[1,2-b]quinoline-4-yl) carbonate (compound IM-1)

[0119] Compound 33-2 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL) and reacted with trifluoroacetic acid (0.2 mL) at room temperature for 30 min. The solution was purified by preparative high-performance liquid chromatography (Method C) to give 20.0 mg of the trifluoroacetate salt of the title compound. Its structure is characterized as follows:

[0120] 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.10 (s, 2H), 8.38 (t, J =5.56 Hz, 1H), 8.32 (d, J = 8.40 Hz, 1H), 8.22 – 8.20 (m, 2H), 8.09 (t, J =5.68 Hz, 1H), 7.91–7.87 (m, 2H), 7.82–7.78 (m, 1H),7.69 (brs, 3H), 7.61 (d, J= 8.56 Hz, 2H), 7.32 (d, J = 8.56 Hz, 2H), 7.06 (s, 1H), 5.56 (d, J = 16.96Hz, 1H), 5.51 (d, J = 16.96 Hz, 1H), 5.47 (d, J = 19.28 Hz, 1H), 5.42 (d, J =19.28 Hz, 1H), 5.14 (d, J = 12.20 Hz, 1H), 5.07 (d, J = 12.16 Hz, 1H), 4.48(t, J = 5.24 Hz, 2H), 4.46 – 4.43 (m, 1H), 4.29 (d, J = 5.60 Hz, 2H), 4.08 –3.95 (m, 5H), 3.79 (t, J = 5.28 Hz, 2H), 3.51 – 3.43 (m, 32H), 3.40 (s, 3H),3.39 – 3.35 (m, 2H), 3.30 – 3.26 (m, 2H), 3.00 (s, 3H), 2.82 – 2.74 (m, 2H),2.56 (t, J = 7.08 Hz, 2H), 2.29 (t, J = 7.36 Hz, 2H), 2.23 – 2.13 (m, 2H),1.82 (p, J = 7.24 Hz, 2H), 1.78 – 1.63 (m, 2H), 1.61 – 1.49 (m, 2H), 1.42 –1.27 (m, 2H), 1.15 (d, J = 6.80 Hz, 3H) , 1.13 (d, J = 6.76 Hz, 3H) , 0.90(t, J = 7.32 Hz, 3H). ESI-MS (m / z): 816.0[M / 2 + H] + 。 The temperature is -19.55° (c = 1.000 g / 100 mL, CH3CN).

[0121] Example 2. Preparation of Coupling A

[0122] Take 0.3 mL of Sacituzumab antibody (anti-Trop-2, 33.5 mg / mL), dilute with 0.25 mL of a solution containing 20 mM PB, 150 mM NaCl, and 20 mM sodium edetate (pH 7.6), then add 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) and mix well. Adjust the pH to 7.4 with 1 M Na₂HPO₄ solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution and mix well. Incubate at room temperature for 30 min. Add 10 times the amount of IM-1 trifluoroacetate dissolved in dimethyl sulfoxide to the above solution system, mix well, and incubate at room temperature for 2 h. Finally, add 6.1 μL of 100 mM cysteine ​​to terminate the reaction. Finally, replace the buffer with PBS buffer solution at pH 6.5 using a G-25 gel column to obtain the product of IM-1 conjugated with Sacituzumab antibody, named conjugate A.

[0123]

[0124] The molecular weight of conjugate A was analyzed using LCMS. 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 conjugated with different numbers of toxins. It was determined that each antibody molecule in conjugate A was conjugated with 1 to 10 toxins (i.e., γ was 1 to 10). The average conjugation ratio (DAR) was calculated to be approximately 6.9 based on the percentage of conjugate molecules conjugated with different numbers of toxins.

[0125] Referring to Example 2, conjugate A samples with DAR values ​​ranging from 6 to 8 (e.g., 7.3 or 7.4) were prepared in batches and then subjected to the following non-clinical and clinical studies.

[0126] Experimental Example 1. Detection of the inhibitory effect of the conjugate on the proliferation of pancreatic cancer cell lines

[0127] Using CellTiter-Glo ®The effect of conjugate A on the proliferation of BxPC-3 cells (pancreatic cancer cell line, derived from ATCC, TROP2 positive cells) was investigated using a chemiluminescent cell viability assay (CTG method). On day 1, BxPC-3 cells in the exponential growth phase were collected, and the cell suspension concentration was adjusted with culture medium and added to 96-well cell culture plates to a final cell concentration of 2000 cells / well. Cells were incubated overnight at 37°C with 5% CO2. On day 2, conjugate A (final concentration 0.152–1000 nM) was serially diluted 1:3 to a 10-fold working solution. 10 µl of the corresponding 10-fold working solution was added to each well, with three replicates for each drug concentration. Cells were then incubated at 37°C with 5% CO2 for 72 hours. On day 5 of the experiment, 50 µl (1 / 2 culture volume) of pre-melted and equilibrated CTG solution to room temperature was added to each well. The mixture was shaken for 2 minutes using a microplate shaker and left to stand at room temperature for 20 minutes before the fluorescence signal was measured using an Envision 2104 plate reader.

[0128] The test results showed that conjugate A significantly inhibited the proliferation of TROP2-positive BxPC-3 cells, with an IC50 value of [missing information]. 50 The effective concentration was 14.3 nM, and the maximum inhibition rate was 95.3%. This indicates that conjugate A can inhibit the proliferation of TROP2-positive pancreatic cancer cells, suggesting that it has a therapeutic effect on pancreatic cancer.

[0129] Experimental Example 2. Detecting the effects of conjugates on cardiovascular and respiratory function.

[0130] Crab-eating macaques were divided into groups of 10 (half male and half female) and administered conjugate A intravenously at doses of 25 mg / kg, 50 mg / kg, and 75 mg / kg, respectively, once every 2 weeks for a total of 4 administrations. Electrocardiograms and respiratory rates in lead II were detected using a large animal non-invasive physiological signal telemetry system, and arterial blood pressure was measured using a non-invasive sphygmomanometer to evaluate the effects of conjugate A on the cardiovascular and respiratory functions of the crab-eating macaques.

[0131] The results showed that no arrhythmias were observed in lead II electrocardiograms of monkeys in all groups treated with conjugate A before the first administration, 2-3 hours after administration, 24-25 hours after administration, 72-73 hours after administration, 7 days after administration, approximately 2-3 hours after the last administration, and before the end of the recovery period. No significant abnormalities were observed in lead II electrocardiogram parameters such as heart rate, RR interval, P wave duration, PR interval, QRS duration, QT interval, corrected QT interval, systolic blood pressure, diastolic blood pressure, mean arterial pressure, and respiratory rate. These findings indicate that conjugate A has no effect on the cardiovascular and respiratory systems of cynomolgus monkeys and demonstrates good clinical safety.

[0132] Experiment Example 3. Detecting the toxic metabolic behavior of conjugates in animals.

[0133] Crab-eating macaques were divided into four groups according to different dosages: a control group, a 25 mg / kg dosage group, a 50 mg / kg dosage group, and a 75 mg / kg dosage group, with five males and five females in each group. At dosage levels of 25 mg / kg and 50 mg / kg, the macaques were intravenously injected with conjugate A four times every two weeks. Blood samples were collected from each group of monkeys before the first and last administration, immediately after administration (0-1 minute after administration), at 4 h, 24 h, 48 h, 96 h, and 168 h, one hour before the second and third administrations, immediately after administration (0-1 minute after administration), and 336 h after the last administration. In the 75 mg / kg dosage group, blood samples were also collected at 4 h, 24 h, 48 h, 96 h, and 168 h after the third administration to detect the toxicokinetics of the conjugate and its released toxin molecules in vivo.

[0134] The table below shows the peak plasma concentrations (C0) of conjugate A and the toxin molecules released by conjugate A in male and female monkeys after administration in the manner described above. max The results showed that the highest harmless non-toxic dose (HNSTD) of conjugate A was 50 mg / kg. After the last administration of this dose, the exposure levels of toxin molecules in female and male monkeys were 3.85 h*μg / mL and 5.86 h*μg / mL, respectively, and the exposure levels of conjugate A in female and male monkeys were 45.8 h*mg / mL and 64.2 h*mg / mL, respectively.

[0135]

[0136] The experimental results show that the exposure levels of ADC and toxins, or C, were different in each group of animals after the first and last administration. max The data are all quite similar, indicating that conjugate A has no significant toxicity accumulation after continuous intravenous administration and is well tolerated in animals, showing good prospects for clinical use.

[0137] Experimental Example 4. In vitro metabolic stability of coupled substances

[0138] This experiment first involved coupling compound A and Trodelvy TM Working solutions of 3.4 mg / mL were prepared using physiological saline. Conjugate A and Trodelvy were then added. TM Working solution was added to blank human plasma to obtain human plasma samples with a concentration of 0.05 mg / ml. The plasma samples were incubated at 37 °C, and the release of toxin molecules was measured at 1 h, 3 h, 24 h, 48 h, 72 h, and 144 h. Conjugate A was compared with the marketed drug Trodelvy. TM Differences in in vitro plasma stability. Results are attached. Figure 1 As shown.

[0139] Instruction manual attached Figure 1 The results showed that with increasing incubation time, the amount of free toxins generated in human plasma increased. After 24 hours of incubation, the levels of conjugate A and the marketed ADC drug Trodelvy in human plasma increased. TM The percentages of toxin molecule release were 28.5% and 93.4%, respectively; after 48 hours of incubation, the levels of conjugate A and the marketed ADC drug Trodelvy in human plasma were... TM The percentages of toxin molecules released were 44.6% and 109.1%, respectively; after 72 hours of incubation, the levels of conjugate A and the marketed ADC drug Trodelvy in human plasma were... TM The percentages of toxin molecules released were 53.7% and 100.6%, respectively; after 144 hours of incubation, the levels of conjugate A and the marketed ADC drug Trodelvy in human plasma were... TM The percentages of released toxin molecules were 65.3% and 104.8%, respectively.

[0140] The results showed that the release rate of toxin molecules from human plasma by conjugate A was significantly lower than that of Trodelvy. TM This indicates that conjugate A is relatively stable in human plasma, with low toxin release, effectively achieving the goal of targeting tumor sites and re-releasing toxins. Simultaneously, it is compatible with the marketed drug Trodelvy. TM In comparison, conjugate A can reduce the risk of serious adverse reactions caused by the rapid and excessive release of toxins into the plasma.

[0141] Trial Example 1. Phase I Clinical Study

[0142] I. Test Plan

[0143] Conjugate A was used to treat patients with histologically confirmed epithelial-derived malignancies. All patients had unresectable locally advanced or metastatic solid tumors that had failed standard therapy, had no standard treatment regimen, or were not currently eligible for standard therapy. Based on patient weight, five dose levels (2, 4, 6, 9, and 12 mg / kg) were designed, administered intravenously every two weeks in 28-day cycles until disease progression or intolerable toxicity. The BLRM method was used to assess toxicity in all planned dose groups and some intermediate dose groups. Dosage escalation decisions were made jointly by the sponsor and investigators based on model analysis.

[0144] II. Safety Results

[0145] During treatment, no Grade 3 or higher "Treatment Emergent Adverse Events (TEAEs)" occurred in the 2 mg / kg dose group; Grade 3 TEAEs occurred in the 4 mg / kg dose group, including oral mucositis and anemia; Grade 3 or higher TEAEs occurred in the 6 mg / kg dose group, including decreased neutrophil count and decreased white blood cell count; the above Grade 3 or higher TEAEs could be resolved after symptomatic treatment, and continued medication was possible, indicating that conjugate A has good clinical safety.

[0146] III. Validity Results

[0147] The treatment outcomes for each indication are as follows:

[0148] 1. Triple-negative breast cancer (TNBC)

[0149] Among all patients with triple-negative breast cancer, some experience partial remission.

[0150] In a patient who had previously received neoadjuvant chemotherapy with doxorubicin / cyclophosphamide / paclitaxel, undergone left mastectomy, and received left thoracic radiotherapy, and subsequently experienced disease progression, conjugate A was administered intravenously at a dose of 4 mg / kg based on patient weight, following the aforementioned dosing cycle. Partial disease remission was observed after 18 weeks, lasting for 6 weeks. In multiple efficacy assessments, the total target lesion volume decreased by up to 40%. No serious adverse events occurred in the patient.

[0151] 2. Ovarian cancer

[0152] Among all ovarian cancer patients, some experience partial remission.

[0153] In a patient with ovarian cancer who had failed multiple lines of therapy, including hysterectomy, ovarian tumor resection, carboplatin / paclitaxel, rucapranib, and carboplatin / docetaxel / bevacizumab, conjugate A was administered intravenously at a dose of 4 mg / kg based on patient weight, following the aforementioned dosing cycle. Partial disease remission was observed after 21 weeks and lasted for 15 weeks. Multiple efficacy assessments showed a 64.8% reduction in total target lesion volume, with one target lesion completely disappearing. No serious adverse events occurred in the patient.

[0154] 3. HER2-positive breast cancer

[0155] In a HER2-positive breast cancer patient who had previously undergone modified radical mastectomy, epirubicin / paclitaxel, HER2 monoclonal antibody / docetaxel, or capecitabine treatment and had failed, conjugate A was administered intravenously at a dose of 6 mg / kg based on patient weight, following the aforementioned dosing cycle. After 8 weeks, partial remission was observed. Multiple efficacy assessments showed a reduction in total target lesion volume of up to 49.6%.

[0156] 4. Stomach cancer

[0157] In a gastric cancer patient who had previously failed total gastrectomy, paclitaxel / tegafur, anlotinib / tegafur, anlotinib / capecitabine, oxaliplatin / fluorouracil, and oxaliplatin / raltitrexed, conjugate A was administered intravenously at a dose of 4 mg / kg based on patient weight, following the aforementioned dosing cycle. After 15 weeks, partial disease remission was observed and has persisted for 10 weeks; the patient is still benefiting. Multiple efficacy assessments showed a 62.8% reduction in the total volume of target lesions, with one target lesion disappearing completely. No serious adverse events occurred in the patient.

[0158] 5. Pancreatic cancer

[0159] In a pancreatic cancer patient who had failed multiple lines of therapy, including distal pancreatic resection, radiotherapy, gemcitabine / capecitabine, gemcitabine / irinotecan / fluorouracil, gemcitabine / nab-paclitaxel / oxaliplatin / fluorouracil, and nivolumab / cabiralizumab, conjugate A was administered intravenously at a dose of 4 mg / kg based on the patient's body weight, following the aforementioned dosing cycle. After 7 weeks, disease stability was observed, which lasted for 30.3 weeks. After multiple efficacy assessments, the total volume of the target lesion decreased by 8.8%, and the patient did not experience any serious adverse events.

[0160] In summary, the Phase I clinical trial demonstrated that conjugate A showed promising preliminary efficacy in at least the aforementioned unresectable metastatic TNBC, ovarian cancer, HER2-positive breast cancer, gastric cancer, and pancreatic cancer that had failed standard treatment, without inducing serious toxicities that could hinder clinical use. These results demonstrate that conjugate A has good safety and efficacy in treating cancer, and conjugate A from different batches with DAR values ​​ranging from 6 to 8 (e.g., DAR values ​​of 7.3 or 7.4) exhibited essentially consistent efficacy and safety profiles.

[0161] In addition to those described herein, various modifications to the invention will be apparent to those skilled in the art based on the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. All references cited in this application (including all patents, patent applications, journal articles, books, and any other disclosures) are incorporated herein by reference in their entirety.

Claims

1. Use of a bioactive conjugate or a pharmaceutical composition comprising a bioactive conjugate in the preparation of a medicament for treating tumor diseases, wherein the bioactive conjugate has the following structure: in, γ is an integer from 1 to 10; The tumor disease is triple-negative breast cancer or Her-2 positive breast cancer; The triple-negative breast cancer mentioned refers to triple-negative breast cancer that has progressed after chemotherapy with doxorubicin, cyclophosphamide and paclitaxel, surgery and radiotherapy; The Her-2 positive breast cancer referred to is HER2 positive breast cancer that has not responded to surgery, epirubicin, paclitaxel, HER2 monoclonal antibody, docetaxel, and capecitabine.

2. Use of a bioactive conjugate or a pharmaceutical composition comprising a bioactive conjugate in the preparation of a medicament for treating tumor diseases, wherein the bioactive conjugate has the following structure: in, γ is an integer from 1 to 10; The tumor disease is platinum-resistant ovarian cancer.

3. Use of a bioactive conjugate or a pharmaceutical composition comprising a bioactive conjugate in the preparation of a medicament for treating tumor diseases, wherein the bioactive conjugate has the following structure: in, γ is an integer from 1 to 10; The tumor disease is ovarian cancer that has failed surgery and treatment with carboplatin, paclitaxel, rucapranib, docetaxel, and bevacizumab.

4. Use of a bioactive conjugate or a pharmaceutical composition comprising a bioactive conjugate in the preparation of a medicament for treating tumor diseases, wherein the bioactive conjugate has the following structure: in, γ is an integer from 1 to 10; The tumor disease referred to is gastric cancer that has not responded to treatment with paclitaxel, tegafur, anlotinib, capecitabine, oxaliplatin, fluorouracil, and raltitrexed after gastrectomy.

5. Use of a bioactive conjugate or a pharmaceutical composition comprising a bioactive conjugate in the preparation of a medicament for treating tumor diseases, wherein the bioactive conjugate has the following structure: in, γ is an integer from 1 to 10; The tumor disease referred to is pancreatic cancer that has failed surgery, radiotherapy, and treatment with gemcitabine, capecitabine, irinotecan, fluorouracil, albumin-bound paclitaxel, oxaliplatin, nivolumab, and cabirazumab.

6. In the use according to any one of claims 1-5, γ is selected from an integer between 3 and 8 in the bioactive conjugate.

7. The use according to any one of claims 1-5, wherein the DAR value of the bioactive conjugate is 1 to 10.

8. In the use according to claim 7, the DAR value of the bioactive conjugate is 5 to 8.

9. The use according to claim 8, wherein the DAR value of the bioactive conjugate is 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.

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