An indole-2-one derivative, its antibody-drug conjugate and its application

Abstract

The present invention discloses an indole-2-one derivative, an antibody-drug conjugate thereof and an application thereof. The compound of formula I disclosed in the present invention has an anti-tumor effect, and the present invention discloses an antibody-drug conjugate containing the compound, which can achieve tumor enrichment, eliminate or reduce the toxic and side effects caused by the indole-2-one derivative acting on non-disease tissues, and improve the therapeutic effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an indole-2-one derivative, an antibody-drug conjugate thereof and applications thereof. Background Art

[0002] The 3-disubstituted indole-2-one derivatives have been reported to be used for tumor treatment (e.g., patent documents US2004 / 0242563A1, WO2005 / 07107A1, WO2005 / 080335, WO2005 / 097107, WO2008071387, WO2008129075A1, WO 2010109008A1 and journal document J Med Chem, 2010, 53, 7140-7145). These studies have not confirmed the anti-cancer mechanism of such 3-disubstituted indole-2-one derivatives. The report shows that the 3-disubstituted indole-2-one derivatives BPHI or ErSO can kill ERα-positive tumor cells resistant to traditional therapies by binding to the allosteric position of ERα. The binding sites of BPHI and ErSO to ER are different from those of tamoxifen and fulvestrant (US2020190029A1, Sci Transl Med , 2021, 13,eabf1383(DOI: 10.1126 / scitranslmed.abf1383)), BPHI and ErSO bind to ER and induce persistent and lethal hyperactivation of the anticipatory pathway of activation of the unfolded protein response (hyperactivation of the anticipatory pathway of activation of the unfolded protein response, David J Shapiro, Front. Endocrinol., 2018, 9, 1-7).

[0003]

[0004] Although many studies have shown that 3-disubstituted indole-2-one derivatives have good anti-tumor activity, there is still no related molecule successfully used in tumor treatment.

[0005] Therefore, it is necessary to find indole-2-one derivative drugs with better tumor treatment effects. Summary of the invention

[0006] In order to solve the above problems, the present invention provides for the first time a compound of formula I or a pharmaceutically acceptable salt thereof, and an antibody-drug conjugate thereof, wherein the compound of formula I and the antibody-drug conjugate thereof have excellent anti-tumor activity, especially have excellent inhibitory activity against the MCF7 cell line.

[0007] Solutions used to solve the problem:

[0008] In a first aspect, the present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0009] .

[0010] In a second aspect, the present invention provides a compound as shown in Formula II or a pharmaceutically acceptable salt thereof:

[0011] .

[0012] In a third aspect, the present invention provides an antibody drug conjugate as shown in Formula III:

[0013] ;

[0014] in,

[0015] Tb antibody or antigen-binding fragment thereof;

[0016] q is an integer or decimal between 0 and 20, and q is not 0.

[0017] Preferably, q is any integer from 1 to 16.

[0018] Preferably, the Tb is an anti-Trop 2 antibody or an antigen-binding fragment thereof.

[0019] Preferably, the Tb is gosartumomab.

[0020] Preferably, the structure of the antibody drug conjugate represented by formula III is as shown in formula IV:

[0021] ;

[0022] Wherein, q is any integer from 1 to 12.

[0023] In a fourth aspect, the present invention provides a pharmaceutical composition, comprising a compound as shown in Formula I as described above or a pharmaceutically acceptable salt thereof, or a compound as shown in Formula II as described above or a pharmaceutically acceptable salt thereof, or an antibody-drug conjugate as shown in Formula III as described above.

[0024] Preferably, the composition further comprises a pharmaceutically acceptable excipient.

[0025] In a fifth aspect, the present invention provides the use of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, the compound as shown in Formula II or a pharmaceutically acceptable salt thereof, the antibody-drug conjugate as shown in Formula III as described above, or the pharmaceutical composition as described above in the preparation of a drug for treating and / or preventing diseases associated with abnormal cell activity.

[0026] Preferably, the disease associated with abnormal cell activity is selected from tumors and / or cancer.

[0027] Preferably, the tumor and / or cancer is selected from one or more of breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer and cervical cancer.

[0028] Preferably, the tumor and / or cancer is breast cancer.

[0029] Effects of the invention:

[0030] The compound of formula I provided by the present invention has excellent anti-tumor effect; at the same time, the ADC molecule with the compound of formula I as the effective load provided by the present invention, on the one hand, enriches the effective load in tumor tissues / cells, thereby improving the drug efficacy; on the other hand, reduces the drug distribution in non-target tissues, reduces toxicity, and further improves the therapeutic effect. DETAILED DESCRIPTION

[0031] In order to make the technical solutions and beneficial effects of the present invention more clearly understood, the following is a detailed description by listing specific embodiments. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0032] Terminology explanation:

[0033] In the present invention, unless otherwise specified, the scientific and technical terms used in the present invention have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used in the present invention are all conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0034] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent.

[0035] The term "pharmaceutical excipients" refers to all substances contained in pharmaceutical preparations other than active pharmaceutical ingredients, which are generally divided into two categories: excipients and additives. For details, please refer to the "Pharmacopoeia of the People's Republic of China (2020 Edition)".

[0036] The term "treating" refers to removing the cause or alleviating the symptoms.

[0037] The term "prevention" refers to reducing the risk of developing a disease.

[0038] The term "patient" refers to any animal, usually a mammal, such as a human, that needs to be treated or prevented. Mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc.

[0039] The term "therapeutically effective amount" refers to an amount administered to a patient that is sufficient to effectively treat a disease. The therapeutically effective amount will vary depending on the type of compound, the type of disease, the severity of the disease, the age of the patient, etc., but can be adjusted by those skilled in the art as appropriate.

[0040] The term "antibody-drug conjugate" refers to a substance obtained by connecting a bioactive compound fragment (drug molecule) to an antibody or its antigen-binding fragment. In some embodiments of the present invention, the bioactive compound fragment is connected to the targeting portion through a linker. The linker can be broken in a specific environment (e.g., a low pH environment in the cell) or under a specific action (e.g., the action of a lysosomal protease), thereby separating the bioactive compound fragment from the targeting portion or the antibody or its antigen-binding fragment. In some embodiments of the present invention, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present invention, the bioactive compound fragment is directly connected to the targeting portion or the antibody or its antigen-binding fragment through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.

[0041] The term "antibody" is interpreted in its broadest sense, including intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed by at least two intact antibodies, as long as they have the desired biological activity. In the present invention, "antibody" and "immunoglobulin" can be used interchangeably.

[0042] The term "monoclonal antibody" refers to an antibody that comes from a group of substantially homogeneous antibodies, i.e., the antibodies that make up the group are identical except for a small number of natural mutations that may exist. Monoclonal antibodies have high specificity for one determinant (epitope) of an antigen, while polyclonal antibodies, in contrast, contain different antibodies for different determinants (epitopes). In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" here indicates that the antibody is characterized by being from a substantially homogeneous group of antibodies and should not be understood as requiring production by a special method.

[0043] In some embodiments of the present invention, monoclonal antibodies also specifically include chimeric antibodies, that is, a portion of the heavy chain and / or light chain is identical or homologous to a certain type, class or subclass of antibody, and the remaining portion is identical or homologous to another type, class or subclass of antibody, as long as they have the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81: 6851-6855). Chimeric antibodies that can be used in the present invention include primatized antibodies, which contain variable region antigen binding sequences from non-human primates (e.g., ancient monkeys, orangutans, etc.) and human constant region sequences.

[0044] The term "antibody fragment" refers to a portion of an antibody, preferably the antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region fragments, diabodies, linear antibodies and single-chain antibody molecules.

[0045] The term "bispecific antibody", also known as "bifunctional antibody conjugate", refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a coupling arm. The conjugate retains the activity of each antibody and thus has bifunctionality and bispecificity.

[0046] The term "multispecific antibody" includes, for example, trispecific antibodies, which are antibodies with three different antigen-binding specificities, and tetraspecific antibodies, which are antibodies with four different antigen-binding specificities.

[0047] The term "complete antibody" refers to an antibody comprising an antigen-binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2 and CH3). The constant region may be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. A complete antibody is preferably a complete antibody having one or more effector functions.

[0048] The term "probody" is a modified antibody, including an antibody or an antibody fragment, which can specifically bind to its target and can be coupled to a masking group, wherein the masking group refers to a cleavage constant for the binding ability of the antibody or antibody fragment to its target that is at least 100 times, or 1000 times, or 10,000 times greater than the cleavage constant for the binding ability of the antibody or antibody fragment to its target without the coupled masking group.

[0049] In the present disclosure, a "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody containing a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which the hypervariable region residues of a human recipient immunoglobulin are replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibody) having the desired specificity, affinity, and function. In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Moreover, the humanized antibody may also contain residues that are not present in the recipient antibody or the donor antibody. These modifications are intended to further optimize the performance of the antibody. Humanized antibodies generally contain at least one, usually two, variable regions in which all or almost all of the hypervariable loops correspond to those of non-human immunoglobulins, while the FRs are completely or almost completely sequences of human immunoglobulins. Humanized antibodies may also contain at least a portion of an immunoglobulin constant region (Fc, usually human immunoglobulin Fc). For details, see, e.g., Jones et al., 1986, Nature , 321: 522-525; Riechmann et al., 1988, Nature , 332: 323-329; and Presta, 1992, Curr Op Struct Bwl 2: 593-596.

[0050] Intact antibodies can be divided into different "classes" based on the amino acid sequence of the constant region of their heavy chains. The five main classes are IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of the different classes of antibodies are called α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0051] In the present invention, although the amino acids in the antibody are L-amino acids in most cases, it is not limited thereto. In some embodiments, the antibody peptide chain may include one or more D-amino acids. Peptides containing D-amino acids are more stable and less prone to degradation in the oral cavity, intestinal tract or plasma than peptides containing only L-amino acids.

[0052] The monoclonal antibodies used in the present invention can be produced by many methods. For example, the monoclonal antibodies used in the present invention can be obtained by the hybridoma method using cells of many species (including mice, hamsters, rats and humans) (see, for example, Kohler et al., 1975, Nature, 256: 495), or by recombinant DNA technology (see, for example, US 4,816,567), or isolated from phage antibody libraries (see, for example, Clackson et al., 1991, Nature , 352: 624-628; and Marks et al., 1991, Journal of Molecular Biology , 222: 581-597).

[0053] In some embodiments of the present invention, the anti-Trop 2 antibody is RS7 described in U.S. Patent No. 7,517,964 (i.e., Sacituzumab of the present invention) and hRS7 described in US2012 / 0237518 (i.e., Sacituzumab of the present disclosure). The anti-Trop 2 antibody that can be used in the present invention can also be obtained by screening the vector design, construction, and antibody library for constructing display antibodies disclosed in CN103476941A, or by G-MAB of Sorrento Therapeutics, Inc. ® The library was screened.

[0054] In the present invention, Trop 2 or TROP2 refers to human trophoblast cell-surface antigens 2, also known as TACSTD2, M1S1, GA733-1, EGP-1, which is a cell surface receptor expressed by many human tumor cells (such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer). In some embodiments, the compounds or conjugates of the present invention can inhibit or kill cells expressing TROP2 receptors, such as breast cancer cells, colorectal cancer cells, lung cancer cells, pancreatic cancer cells, ovarian cancer cells, prostate cancer cells or cervical cancer cells.

[0055] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0056] In a first aspect, the present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0057] .

[0058] In a second aspect, the present invention provides a compound as shown in Formula II or a pharmaceutically acceptable salt thereof:

[0059] .

[0060] In a third aspect, the present invention provides an antibody drug conjugate as shown in Formula III:

[0061] ;

[0062] in,

[0063] Tb is an antibody or an antigen-binding fragment thereof;

[0064] q is an integer or decimal between 0 and 20, and q is not 0.

[0065] In certain embodiments, the Tb is an anti-Trop 2 antibody or an antigen-binding fragment thereof.

[0066] In certain embodiments, q is any integer from 1 to 16.

[0067] In some embodiments, q is any integer from 1 to 12, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0068] In certain embodiments, the Tb is gosartuzumab.

[0069] In certain embodiments, the structural formula of the drug conjugate of formula III is as shown in formula IV:

[0070] ;

[0071] Wherein, q is any integer from 1 to 12.

[0072] The antibody-drug conjugate of the present invention can achieve tumor enrichment, eliminate or reduce the toxic and side effects caused by the indole-2-one derivatives acting on non-disease tissues, and improve the therapeutic effect.

[0073] In a fourth aspect, the present invention provides a pharmaceutical composition, comprising a compound as shown in Formula I as described above or a pharmaceutically acceptable salt thereof, or a compound as shown in Formula II as described above or a pharmaceutically acceptable salt thereof, or an antibody-drug conjugate as shown in Formula III as described above.

[0074] In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0075] In a fifth aspect, the present invention provides the use of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, the compound as shown in Formula II or a pharmaceutically acceptable salt thereof, the antibody-drug conjugate as shown in Formula III as described above, or the pharmaceutical composition as described above in the preparation of a drug for treating and / or preventing diseases associated with abnormal cell activity.

[0076] In certain embodiments, the disease associated with abnormal cell activity is selected from tumors and / or cancer.

[0077] In certain embodiments, the tumor and / or cancer is a tumor and / or cancer associated with abnormal expression of Trop2.

[0078] In certain embodiments, the tumor and / or cancer is selected from one or more of breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer and cervical cancer.

[0079] In certain embodiments, the tumor and / or cancer is breast cancer.

[0080] The method of the present invention is described below by specific examples. It should be understood that these examples are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited by the scope of the following examples; the implementation conditions used in the examples can be further adjusted according to specific requirements, and the unspecified implementation conditions are usually the conditions in conventional experiments or selected according to the product instructions. The reagents and raw materials used in the present invention are all commercially available.

[0081] The structures of the compounds described in the following examples were confirmed by nuclear magnetic resonance (1H NMR) or mass spectrometry (MS).

[0082] In the following embodiments 1 H NMR was measured using a Bruker instrument (400 MHz), and chemical shifts were expressed in ppm. The measurement solvents were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6); the internal standard substance was tetramethylsilane (TMS). 1H NMR notation: s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartetdoublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz. The d value is expressed in ppm.

[0083] The mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0084] TLC: Thin layer chromatography. The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The specifications of the silica gel plate used in thin layer chromatography (TLC) are 0.2mm~0.3mm, and the specifications of the thin layer chromatography separation and purification products are 0.4mm~0.5mm.

[0085] Column chromatography generally uses Yantai Huanghai Silica Gel 200-300 mesh silica gel as the carrier.

[0086] DAR value determination method:

[0087] Sample treatment: Take 50 μg of ADC sample, dilute it to 0.5 mg / mL with ultrapure water, then add 1 μL of 1M DTT, mix well, centrifuge and take the supernatant for sampling.

[0088] The instruments, chromatographic conditions and mass spectrometry parameters used in this method are shown in Tables 1, 2 and 3.

[0089] Table 1

[0090]

[0091] Table 2

[0092]

[0093] Table 3

[0094]

[0095] In the following examples, unless otherwise specified, all temperatures are in degrees Celsius. Unless otherwise specified, various starting materials and reagents are commercially available or synthesized according to known methods. Commercially available materials and reagents are used directly without further purification. Unless otherwise specified, commercial manufacturers include but are not limited to Sinopharm Group, J&K Technology Co., Ltd., TCI (Shanghai) Chemical Industry Development Co., Ltd., Shanghai Bid Pharmaceutical Technology Co., Ltd. and Shanghai Myrel Chemical Technology Co., Ltd.

[0096] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0097] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0098] The reaction progress in the embodiment is monitored by thin layer chromatography (TLC), and the developing solvent used in the reaction, the eluent system of column chromatography or the developing solvent system of thin layer chromatography used for purifying the compound includes: A: petroleum ether and ethyl acetate system; B: dichloromethane and methanol system; C: n-hexane: ethyl acetate; wherein the volume ratio of the solvent varies according to the polarity of the compound, and a small amount of acidic or alkaline reagent, such as acetic acid or triethylamine, can also be added for adjustment.

[0099] Example 1 Synthesis of drug-linker conjugate

[0100] (1) Synthesis of INT1

[0101]

[0102] Step 1:

[0103] Compound PL-1-1 (100 mg, 0.36 mmol, synthesis method reference patent: WO2005097107) and phenol (103.1 mg, 1.08 mmol) were dissolved in 1,2-dichloroethane (2 mL), and then p-toluenesulfonic acid (347.2 mg, 1.80 mmol) was added, and the temperature was raised to 90 °C and stirred for 2 h; LCMS monitored the reaction; the reaction solution was cooled to room temperature, and a white solid precipitated, which was filtered and concentrated under reduced pressure. The crude product was purified by column chromatography (EA: PE = 0-40%) to obtain the target compound PL-1 (95 mg).

[0104] LCMS (ESI) [M+H] + =352.31.

[0105] 1 H NMR (400 MHz, DMSO- d 6) δ 11.42 (s, 1H), 9.47 (s, 1H), 7.13-7.11 (m,2H), 7.03-7.00 (m, 4H), 6.94-6.92 (m, 2H), 6.71-6.68 (m, 2H), 2.26 (s, 3H).

[0106] Step 2:

[0107] Compound PL-1 (100 mg, 0.28 mmol) and compound INT1-1 (78.48 mg, 0.20 mmol, purchased from Nanjing Yaoshi Technology Co., Ltd., product number: PBUB813) were added to DMF (2 mL), and then cesium carbonate (136.84 mg, 0.42 mmol) was added. Under nitrogen protection, the reaction was stirred at room temperature for 1 h; LCMS monitored the reaction; ethyl acetate (20 mL) was added to the reaction solution for dilution, the organic phase was washed with water (5 mL×2), and then concentrated under reduced pressure, and the crude product was purified by column chromatography (EA:PE=0-50%) to obtain the target compound INT1-2 (72 mg).

[0108] LCMS (ESI) [M+H] + = 572.37.

[0109] 1 H NMR (400 MHz, MeOD) δ 7.39-7.25 (m, 5H), 7.15-6.84 (m, 9H), 6.75 (d, J = 8.4 Hz, 1H), 5.09 (s, 2H), 4.30 (s, 2H), 3.75 (s, 2H), 2.30 (s, 3H).

[0110] Step 3:

[0111] Compound INT1-2 (20 mg, 0.035 mmol) was dissolved in MeOH (2 mL), and 10% Pd / C (20 mg) was added under nitrogen protection. The mixture was replaced with hydrogen three times, and then stirred at room temperature for 1 h under a hydrogen atmosphere. The reaction was monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT1 (15 mg), which was directly used in the next step.

[0112] LCMS (ESI) [M+H] + = 438.30.

[0113] (2) Synthesis of DL-1

[0114]

[0115] Step 1:

[0116] Compound INT1 (15 mg, 0.034 mmol) and compound DL-1-1 (20 mg, 0.034 mmol, synthesis method reference patent: WO2022170971) were dissolved in DMF (2 mL), and then DMTMM (9.41 mg, 0.034 mmol) was added and stirred for 1 h under nitrogen protection; the reaction was monitored by LCMS; the reaction solution was directly purified by preparative HPLC (CH3CN:H2O containing 0.1% FA = 5%-90%) to obtain the target compound DL-1 (10.3 mg).

[0117] LCMS (ESI) [M+H] + = 999.73.

[0118] 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.11 (s, 2H), 8.62 (t, J = 4.0 Hz, 1H),8.22 (s, 1H), 8.15-8.12 (m, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.86-7.83 (m, 1H),7.13-7.01 (m, 6H), 6.96-6.92 (m, 2H), 6.71-6.68 (m, 2H), 5.29-5.18 (m, 2H),4.24-4.20 (m, 2H), 3.71-3.69 (m, 2H), 3.41 (s, 3H), 2.55-2.51 (m, 2H), 2.40-2.26 (m, 11H), 2.01-1.95 (m, 1H), 1.88-1.72 (m, 2H), 1.72-1.45 (m, 2H), 1.40-1.24 (m, 9H), 0.90-0.79 (m, 12H).

[0119] Example 2 Synthesis of Antibody Drug Conjugate (ADC)

[0120] Preparation of Trop2-ADC-001

[0121] Take 1.0mL Trop2 antibody sacituzumab (gosartuzumab, 13.4 mg / mL, antibody preparation method reference patent: WO2003074566), dilute with 0.01 mL 20mM PB (containing 100mM disodium edetate solution, pH 7.6), adjust the pH to 7.48 with 0.5M Na2HPO4 solution, add 20 mM TCEP (tris (2-carboxyethyl) phosphine, 0.0249 ml, 0.498µmol) solution, mix well, and let stand at room temperature for 90 min. Then add dimethyl sulfoxide (0.1806 mL) solution containing DL-1 (1.135 mg, 12 times the molar amount of antibody substance), mix well, let stand at room temperature for 2h, and then use a centrifugal ultrafiltration tube (Merck, Amicon Ultra-15) to replace the liquid, and the buffer is replaced with a 20 mM His-Hcl buffer solution with pH 5.9. The conjugated product of DL-1 and Trop2 antibody sacituzumab (gosartuzumab) Trop2-ADC-001 was obtained (the structural formula is shown below, and its information is shown in Table 4). The DAR value determined by mass spectrometry was 6.4.

[0122]

[0123] Table 4

[0124]

[0125] In Table 4, LC represents antibody light chain; HC represents antibody heavy chain; LC+DAR1 represents a drug-linker conjugate comprising a light chain conjugate; HC+DAR1 represents a drug-linker conjugate comprising a heavy chain conjugate; LC+DAR2 represents a drug-linker conjugate comprising a light chain conjugate; HC+DAR2 represents a drug-linker conjugate comprising a heavy chain conjugate; HC+DAR3 represents a drug-linker conjugate comprising a heavy chain conjugate; HC+DAR4 represents a drug-linker conjugate comprising a heavy chain conjugate.

[0126] Example 3 Biological Activity Test of Compounds

[0127] Test of the inhibitory activity of compounds on MCF7 cell proliferation

[0128] According to the conventional method, MCF7 cells (sourced from ATCC, Catalog No. HTB-22) were revived and cultured in DMEM complete medium (sourced from Damas life, Catalog No. C8013) containing 10% FBS (sourced from Gibco, Catalog No. A5669701) and 10μg / mL insulin (sourced from Biyuntian, Catalog No. P3376), and plated in a 96-well flat-bottom plate at a density of 5000 cells / well and 142.5μL / well, and cultured in a 37°C incubator overnight. The next day, the test compound was diluted with DMSO to 400 times (400×) of the final concentration, and then diluted to 20 times (20×) of the final concentration with serum-free culture medium, so that the highest final concentration was 1000 nM, and 9 concentration points were diluted 4 times. 7.5 μL of 20× test dilution was added to each well. After culturing at 37°C for 4 days, 50 μL of Cell titer glo substrate (from ADamas life, catalog number RA-GL11-A) was added to each well, and the fluorescence value was detected after incubation for 10 minutes. The test results are shown in Table 5.

[0129] Table 5

[0130]

[0131] The test results show that the compounds disclosed in the present invention have a strong cell-killing effect in MCF7 tumor cells.

[0132] Example 4 Bioactivity Testing of Antibody Drug Conjugates (ADCs)

[0133] Test of ADC's inhibitory activity on MCF7 cell proliferation

[0134] According to the conventional method, MCF7 cells (sourced from ATCC, catalog number HTB-22) were revived and cultured in DMEM complete medium (sourced from Damas life, catalog number C8013) containing 10% FBS (sourced from Cornning, catalog number 35-081-CV) and 10μg / mL insulin (sourced from YEASEN, catalog number 40112ES60), and 5000 cells / well and 100μL / well were plated in a 96-well flat-bottom plate and cultured in a 37°C incubator overnight. The next day, the antibody drug conjugate was diluted with complete medium (the highest concentration was 2μM) and 4-fold dilution was performed for 10 points. 100μL of the diluted antibody drug conjugate was added to the cell culture wells. After culturing at 37°C for 4 days, 50μL Cell titer glo substrate (sourced from ADamas life, catalog number RA-GL11-A) was added to each well, and the fluorescence value was detected after incubation for 5 minutes. The test results are shown in Table 6.

[0135] Table 6

[0136]

[0137] The test results show that the antibody drug conjugate disclosed in the present invention has a strong cell killing effect in MCF7 tumor cells.

[0138] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A compound as shown in formula I or a pharmaceutically acceptable salt thereof: 。 2. A compound as shown in formula II or a pharmaceutically acceptable salt thereof: 。 3. An antibody-drug conjugate as shown in formula III: ； in, Tb is an antibody or an antigen-binding fragment thereof; q is an integer or decimal between 0 and 20, and q is not 0.

4. The antibody-drug conjugate according to claim 3, characterized in that: The Tb is an anti-Trop 2 antibody or an antigen-binding fragment thereof; And / or, q is any integer from 1 to 16.

5. The antibody-drug conjugate according to claim 4, characterized in that: The Tb is gosartuzumab.

6. The antibody-drug conjugate according to claim 5, characterized in that: The structural formula of the antibody drug conjugate shown in Formula III is shown in Formula IV: ； Wherein, q is any integer from 1 to 12.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound as shown in formula I according to claim 1 or a pharmaceutically acceptable salt thereof, or the compound as shown in formula II according to claim 2 or a pharmaceutically acceptable salt thereof, or the antibody drug conjugate as shown in formula III according to any one of claims 3 to 6.

8. Use of the compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof, the compound of formula II according to claim 2 or a pharmaceutically acceptable salt thereof, the antibody-drug conjugate of formula III according to any one of claims 3 to 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating and / or preventing a disease associated with abnormal cell activity.

9. The use according to claim 8, characterized in that The disease associated with abnormal cell activity is selected from tumors.

10. The use according to claim 9, characterized in that The tumor is selected from one or more of breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer and cervical cancer.

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