Toxin derivative for preparing drug conjugate

Abstract

A toxin derivative represented by formula (I) or a stereoisomer and pharmaceutically acceptable salt thereof, and a use thereof for preparing a drug conjugate. A drug conjugate and a use thereof. The toxin derivative is linked to an auristatin toxin by adjusting the structure of a linker to form a novel linker-effector small molecule, achieving excellent biological activity and efficient endocytosis when used for preparing a drug conjugate, and facilitating the expansion of the design, production and application of auristatin conjugates. X-L 1-A-NH(CH 2CH 2O) n-L 2-D (formula (I))

Description

Toxin derivatives for preparing drug conjugates TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a toxin derivative for preparing drug conjugates and use thereof, and further relates to a drug conjugate and use thereof. BACKGROUND

[0002] Drug conjugates with targeted delivery function are a new type of emerging treatment method as drug research and development develops towards precision treatment. Drug conjugates are composed of targeting molecules and effector molecules through conjugation. The targeting molecules selectively bind to cells or tissues related to diseases, and release effector molecules with therapeutic effects in a targeted manner. The targeting molecules can be antibodies, antibody fragments, ligands, polypeptides, small molecules, or viruses such as virus-like particles. At present, antibody-drug conjugates (ADC) with antibodies as targeting molecules have become the mainstream of the development of the next generation of precision treatment drugs, and have broad prospects in the treatment of diseases such as cancer and autoimmune diseases. ADC is usually composed of four parts: 1) an antibody with targeting function, which is used to recognize target cells and bring effector small molecule drugs (usually chemical drugs) to the cell surface or inside; 2) a cytotoxic or other effector small molecule, which plays a role in killing cells or regulating cell function; 3) a linker connecting the antibody and the drug, which plays a connecting and supporting role; and 4) a linker connecting the antibody and the drug, which has a special active group.

[0003] At present, more research is conducted on antibodies and effector small molecules. However, in ADC, the linker and the linker also play a very important role. The linker can be divided into non-cleavable linkers and cleavable linkers in terms of performance. The non-cleavable linker has a stable chemical bond composition and is more stable than the cleavable linker. Its mechanism is based on the endocytosis of the antibody-antigen complex of ADC, and then the ADC is degraded by lysosomes to release the effective load containing the linker, thereby killing tumor cells. Therefore, the advantage of the non-cleavable linker is that the ADC using this type of linker is less likely to release drugs in the blood circulation, has less off-target toxicity, and is safer. In addition, the ADC using the non-cleavable linker can change the chemical properties of the small molecule drug through the linker, enhance its metabolic properties or pharmacological effects, because the released effective load has a complete linker.

[0004] Among the marketed and clinically developed ADCs, the types of ADCs using non-cleavable linkers are relatively few. The relatively mature ones include thioether linkers and amide linkers, etc. Kadcyla of Roche is connected to DM1 through a stable thioether linker.

[0005] By rational design of the linker, the optimization of ADC molecules can be achieved, for example, the physicochemical properties, pharmacokinetic characteristics of the ADC can be optimized, the pharmacodynamic characteristics and toxicity characteristics of the ADC can be improved, or a high DAR value design can be achieved, further realizing the introduction of different kinds of effector small molecules, etc.

[0006] The coupling mode of the antibody and the effector small molecule also directly affects the DAR value and the uniformity of the ADC product, further affecting the physicochemical properties, biological activity, and in vivo therapeutic effect and toxicity of the ADC. Site-specific coupling of the ADC is one of the research hotspots that are currently concerned, and through genetic engineering, non-natural amino acids (UAA) with specific active groups can be introduced into the antibody molecule, thereby effectively controlling the DAR value and product uniformity of the ADC. SUMMARY

[0007] In order to make up for the deficiencies in the prior art, one object of the present application is to provide a toxin derivative, which is very suitable for preparing a drug conjugate, such as an antibody-drug conjugate (ADC), by adjusting the structure of the linker, so as to achieve excellent biological activity and endocytosis effect.

[0008] Another object of the present application is to provide a drug conjugate and use thereof.

[0009] The first aspect of the present application provides a compound as shown in formula (I) or a stereoisomer, a pharmaceutically acceptable salt thereof, X---L1---A---NH(CH2CH2O) n ---L2---D Formula (I)

[0010] X represents a protected or unprotected reactive group;

[0011] L1, L2 each independently represents a C1-C6 straight chain or branched chain alkylene group;

[0012] A represents a peptide formed by 1-4 amino acids selected from glycine and / or alanine;

[0013] D represents an auristatin toxin;

[0014] n represents 1, 2 or 3.

[0015] For the drug conjugate, the structure and physicochemical properties of the linker have a very great influence on the stability of the drug, drug metabolism, drug efficacy, etc., for example, it can affect the coupling efficiency and DAR value, the transmembrane property of the effector small molecule and the stability of the conjugate, the pharmacokinetic characteristics of the effector small molecule, and the like, for example, the structural residues of the linker on the effector small molecule can affect the effect response of the effector small molecule, and for example, various physicochemical properties of the linker can also affect the production process and drugability of the conjugate.

[0016] The compound of formula (I) provided by the present application forms a novel linker structure with amino acid residues and PEG units in specific number, specific type and specific connection order, and the conjugate formed by connecting the targeting molecule (such as an antibody) and the auristatin toxin has excellent biological activity and high efficient endocytosis effect.

[0017] In the compound of formula (I) provided by the present application, X can represent any active reactive group commonly used in the art, which can react with the reactive group on the targeting molecule (such as an antibody) (which can be the group contained in the targeting molecule itself, or the group carried by the targeting molecule after modification) to form the targeting molecule-drug conjugate. X can be an unprotected group or a group protected by a protecting group commonly used in the art, and the corresponding protecting group can be removed after reaction.

[0018] In some embodiments, X can represent the following group protected by a protecting group or unprotected: hydroxylamine group, amino group, hydroxyl group, sulfhydryl group, halogen atom, azido group, carbonyl group, dicarbonyl group, ester group or cycloalkyne group. In some preferred embodiments, X represents a hydroxylamine group, i.e. "NH2-O-".

[0019] In some embodiments, L1 and L2 can each independently represent a C1-C3 straight chain alkylene group, for example, methylene (-CH2-), ethylene (-CH2-CH2-), propylene (-CH2-CH2-CH2-). In some preferred embodiments, L1 and L2 can each independently represent methylene or ethylene.

[0020] In some embodiments, A can represent a dipeptide of "-glycine-glycine-", a dipeptide of "-glycine-alanine-", a dipeptide of "-alanine-alanine-", or a tripeptide of "-glycine-glycine-glycine-". In some preferred embodiments, the structure of A can be: wherein "1" represents the connection point with L1, and "2" represents the connection point at the other end.

[0021] In some embodiments, D can represent any of the Auristatins toxins commonly known in the art, which is an artificial synthetic derivative of the natural product dolastatin 10, belongs to the anti-mitotic agent, by acting on the β-subunit of α-β tubulin dimer, blocking tubulin polymerization, causing cell cycle arrest and apoptosis. Auristatins toxins include but not limited to MMAE, MMAF, MMAD, etc. In some preferred embodiments, D can be represented as MMAE or MMAF as shown below, the secondary amine group of the end group is removed by hydrogen and connected to other parts, so D can also be referred to as the residue of MMAE or MMAF after removing the hydrogen of the end group secondary amine group.

[0022] In some most preferred embodiments, the compound can be one of the following:

[0023] The second aspect of the present application provides the use of the compound or its stereoisomer, pharmaceutically acceptable salt of any of the above technical solutions for preparing a drug conjugate (such as an antibody-drug conjugate).

[0024] The compound or its stereoisomer, pharmaceutically acceptable salt of any of the above technical solutions provided by the present application can be coupled with any of the targeting molecules commonly known in the art, such as antibodies, antibody fragments, ligands, polypeptides, targeting proteins, etc. by any of the coupling methods commonly known in the art to form a conjugate. In some preferred embodiments, the antibody can be an anti-HER2 monoclonal antibody, such as trastuzumab.

[0025] The third aspect of the present application provides a drug conjugate prepared by coupling the compound or its stereoisomer, pharmaceutically acceptable salt of any of the above technical solutions with a targeting molecule through the reactive group (i.e. X).

[0026] In the antibody-drug conjugate provided by the present application, the compound or its stereoisomer, pharmaceutically acceptable salt of any of the above technical solutions can be coupled with any of the targeting molecules commonly known in the art, such as antibodies, polypeptides, targeting proteins, etc. by any of the coupling methods commonly known in the art to form a drug conjugate. In some preferred embodiments, the antibody can be an anti-HER2 monoclonal antibody, such as trastuzumab.

[0027] In some embodiments, the targeting molecule is a trastuzumab with a non-natural amino acid containing an end group carbonyl group, and the reactive group is a hydroxylamine group, and the drug conjugate is prepared by reacting the end group carbonyl group with the hydroxylamine group to form an oxime bond. The oxime bond formed by the reaction of carbonyl group and hydroxylamine group is as follows:

[0028] In some embodiments, the unnatural amino acid can be those described in Chinese patents ZL 202111019771.1 and ZL 202110865364.6. In some preferred embodiments, the unnatural amino acid is a compound having one of the following structures:

[0029] In some embodiments, the unnatural amino acid can be introduced into the specified site of the target molecule by a codon expansion technique (e.g., codon expansion technique implemented in E. coli), so as to realize subsequent site-specific coupling. In some preferred embodiments, the amino acid sequences of the heavy chain and the light chain of the trastuzumab used are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, wherein an unnatural amino acid is introduced at position 142 of the heavy chain.

[0030] The fourth aspect of the present application provides the use of the drug conjugate of any of the above technical solutions as a drug (i.e., for treatment).

[0031] The fifth aspect of the present application provides the use of the drug conjugate of any of the above technical solutions in the preparation of a drug for treating cancer.

[0032] The sixth aspect of the present application provides a method for preventing and / or treating cancer, comprising administering a therapeutically effective dose of the drug conjugate of any of the above technical solutions to a patient in need thereof.

[0033] In some embodiments, the cancer can be gastric cancer or breast cancer.

[0034] In some embodiments, the drug conjugate can be used alone or in combination with other antitumor drug(s).

[0035] The toxin derivative provided by the present application connects auristatin toxin by adjusting the structure of the linker, forming a new linker-effector small molecule derivative, which can achieve very excellent biological activity and efficient endocytosis when used for preparing drug conjugates, and is conducive to expanding the design, production and application of auristatin conjugates, and has very important economic and social value. BRIEF DESCRIPTION OF DRAWINGS

[0036] FIG. 1 is a map of the expression plasmid pCDNA3.1-Trastuzumab-UAG142 in Example 3. DETAILED DESCRIPTION

[0037] In the present application, "C1-Cn" includes C1-C2,..., C1-Cn. For example, the "C1-C6" group means that the moiety has 1 to 6 carbon atoms, i.e. the group contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.

[0038] The compounds or conjugates of the present application can contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds or conjugates of the present application, including but not limited to, diastereomeric, enantiomeric, atropisomeric and geometric (conformational) isomeric forms, and mixtures thereof such as racemates, are within the scope of the present application. Unless otherwise stated, structures depicted herein are also meant to include all isomeric (such as diastereomeric, enantiomeric, atropisomeric and geometric (conformational) isomeric forms) and tautomeric forms of the structure; for example, structures depicted as single structural isomers are also meant to include their tautomeric forms. For example, structures depicted as single structural isomers are also meant to include their tautomeric forms. Thus, individual stereo isomers as well as enantiomeric, diastereomeric, atropisomeric, and geometric (conformational) mixtures of the compounds or conjugates of the present application are within the scope of the application. Unless otherwise stated, structures depicted herein are also meant to include all isomeric (such as diastereomeric, enantiomeric, atropisomeric and geometric (conformational) isomeric forms) and tautomeric forms of the structure; for example, structures depicted as single structural isomers are also meant to include their tautomeric forms. For example, structures depicted as single structural isomers are also meant to include their tautomeric forms. Thus, individual stereo isomers as well as enantiomeric, diastereomeric, atropisomeric, and geometric (conformational) mixtures of the compounds or conjugates of the present application are within the scope of the application.

[0039] The term "pharmaceutically acceptable salt" as used herein alone or in combination means a salt of a compound or conjugate of the present application which retains the biological effectiveness and properties of the free acids or free bases and which is obtained by reaction of a free acid with a non-toxic inorganic or organic base or of a free base with a non-toxic inorganic or organic acid. Standard procedures as known in the art can be used for obtaining such salts. Suitable salts are listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977).

[0040] The technical solutions of the present application are further described below in combination with specific examples.

[0041] The non-natural amino acids NBGK, NPAK, and NBOK used in the embodiments of the present application were prepared according to Chinese Patent ZL 202111019771.1, and the comparative toxins P4AE and P4AF were prepared according to Chinese Patent ZL 201810957127.0, and their structures are as follows:

[0042] The toxin MMAE (monomethyl auristatin E) used in the embodiments was purchased from Nanjing Lianning Biopharmaceutical Co., Ltd., batch number: LN217-41-1, MMAF-OtBu (MMAF: monomethyl auristatin F) was purchased from Lianning (Suzhou) Biopharmaceutical Co., Ltd., batch number: LN507-38, and other raw materials or reagents were commercially available unless otherwise specified.

[0043] In the embodiments of the present application, the eluent ratios are all volume ratios, and the percentages referred to are mass percentages unless otherwise specified.

[0044] Preparation of compound GPAE in Example 1

[0045] The structural formula of GPAE is as follows:

[0046] The reaction process is as follows:

[0047] The preparation process includes the following steps:

[0048] First, the glycine linker part (1-6) is prepared, then the PEG2-MMAE (2-3) part is prepared, and then the two parts 1-6 and 2-3 are condensed to obtain the target compound GPAE.

[0049] a) In a reaction flask, glycine methyl ester hydrochloride (1.67 g, 10 mmol) was dissolved in a DCM / water (50 / 20 mL) mixed solution, the container was placed in an ice bath, K2CO3 (4.15 g, 30 mmol) and bromoacetyl bromide (2.42 g, 12 mmol) were added, and after stirring vigorously at room temperature for 18 hours, the mixture was extracted with DCM three times, the organic phases were combined and washed with water and brine, and the organic phase was concentrated and recrystallized (PE:EA = 10:1), and white crystals 1-1 (1.70 g, yield 68%) were obtained by filtration.

[0050] b) The product 1-1 (1.67 g, 6.6 mmol) from previous step was taken in a reaction flask, solvent DCM (20 mL) was added, the vessel was placed in an ice bath, trifluoroacetic acid (5 mL) was added, stirred at room temperature for 18 h, TLC indicated the completion of the reaction, the mixture was evaporated on a water bath rotary evaporator, most of the solvent was removed, water (30 mL) was added, and the product 1-2 was obtained by lyophilization and was used as such.

[0051] c) In a reaction flask, N-(2-bromoethyl)phthalimide (6.0 g, 24 mmol), tert-butyl N- hydroxy carbamate (4.8 g, 36 mmol) and K2CO3(8.3 g, 60 mmol) were added, solvent DMF (60 mL) was added, stirred at room temperature for 18 h, TLC indicated the completion of the reaction, the mixture was diluted with water, extracted with EA three times, the organic phase was combined, washed with water three times, dried over anhydrous Na2SO4, concentrated, column chromatography (eluent PE:EA = 10:1 and 2:1), afforded 1-3 (3.45 g, yield 47%) as a white solid.

[0052] d) In a reaction flask, the product 1-3 (0.5 g, 1.64 mmol) from previous step was dissolved in ethanol (20 mL), hydrazine hydrate (80%, 410 μL, 6.56 mmol) was added, the vessel was refluxed at 80 °C for 18 h, TLC indicated the completion of the reaction, the white precipitate was allowed to settle upon cooling, filtered, the filter cake was washed with ethanol, the filtrate was concentrated and column chromatography (eluent DCM:MeOH = 10:1) was performed to obtain 1-4 (165 mg, yield 57%) as a yellow oil (LCMS: m / z (ESI+) calcd: 176.12; Found: 177.10 [M+H] + ; 218.15 [M+MeCN+H] + ).

[0053] e) In a reaction flask, the product 1-4 (3.67 g, 20.8 mmol) from previous step, the product 1-2 (3.4 g, 17.3 mmol) prepared in step b) and solvent methanol (60 mL) were added, DIPEA (15 mL, 87 mmol) was added, stirred at 70 °C for 18 h, concentrated, slurry in EA, filtered to obtain 1-5 (5.56 g, used as such without purification) as a white solid.

[0054] f) In a reaction flask, dissolve the crude 1-5 (504 mg) from the previous step in water (5 mL), add triethylamine (0.72 mL, 5.2 mmol), a solution of Boc anhydride (0.94 g, 4.3 mmol) in dioxane (5 mL), and stir at room temperature for 18 hours. Concentrate to remove most of the dioxane, extract with DCM three times, combine the organic phases, and concentrate. Purify by column chromatography (eluting with DCM:MeOH = 10:1 and 5:1) to give 1-6 as a white solid (287 mg; LCMS: m / z (ESI+) calcd: 391.20, Found: 392.15 [M+H] + ; 414.15 [M+Na] + ).

[0055] g) In a reaction flask, add the substrate 2-0 (100 mg, 0.4 mmol, purchased from Bide Pharmatech, Cat. No. BD261696), dissolve in DMSO (3 mL), and add IBX (2-iodoxybenzoic acid, 224 mg, 0.8 mmol). Stir at room temperature for 18 hours. TLC monitoring (2,4-dinitrophenylhydrazine coloration) shows that the reaction is complete. Wash the mixture with water, extract with EA three times, dry over anhydrous Na2SO4, and concentrate. Purify by column chromatography (eluting with DCM:MeOH = 20:1) to give the product 2-1 (50 mg, yield 50.5%).

[0056] h) In a reaction flask, dissolve the product 2-1 (50 mg, 0.2 mmol) from the previous step and MMAE (57 mg, 0.08 mmol) in dichloroethane (5 mL). Place the vessel in an ice bath, add STAB (sodium triacetoxyborohydride, 51 mg, 0.24 mmol), and stir at room temperature for 18 hours. TLC monitoring (phosphomolybdic acid coloration) shows that the reaction is complete. Wash the mixture with water, extract with DCM three times, dry over anhydrous Na2SO4, and concentrate. Purify by column chromatography (eluting with DCM:MeOH = 20:1) to give 2-2 as a colorless oil (79 mg, yield 99%; LCMS: m / z (ESI+) calcd: 948.65; Found: 475.65 [M / 2+H] + , 949.80 [M+H] + , 971.80 [M+Na] + ).

[0057] i) In a reaction flask, dissolve the product 2-2 (79 mg, 0.08 mmol) of the previous step in DCM (2 mL), then add trifluoroacetic acid (1 mL), stir at room temperature for 3 hours, monitor the reaction completion by TLC (molybdenum phosporic acid coloration), concentrate the mixture at room temperature, add DCM, repeat the above operation 3 times to remove excess trifluoroacetic acid, finally add water and lyophilize to obtain the product 2-3 (67.9 mg, yield 99%).

[0058] j) In a reaction flask, add the product 1-6 (50 mg, 0.13 mmol) and solvent DCM (5 mL), then add HATU (60.8 mg, 0.16 mmol), DIPEA (57 μL, 0.32 mmol), stir at room temperature for 30 minutes, then add the product 2-3 (67.9 mg, 0.08 mmol) of the previous step, stir at room temperature for 18 hours, monitor the reaction completion by TLC (molybdenum phosporic acid coloration), wash the mixture with water, extract with DCM 3 times, dry with anhydrous Na2S04, concentrate, and purify by column chromatography (eluent DCM:MeOH = 15:1) to obtain the white solid 2-4 (63 mg; LCMS: m / z (ESI+) calcd: 1221.78, Found: 612.25 [M / 2+H] + ).

[0059] k) In a reaction flask, dissolve the product 2-4 (63 mg) of the previous step in dioxane (10 mL), slowly add hydrochloric acid (5 mL), stir at room temperature for 3 hours, monitor the reaction completion by TLC, concentrate the mixture at room temperature to remove part of the solvent, add water and lyophilize to obtain the white solid final product GPAE (25.6 mg, yield 48.6%).

[0060] LCMS: m / z (ESI+) calcd: 1021.68, Found: 512.20 [M / 2+H] + , 1046.40 [M+Na] + .

[0061] 1H-NMR (400 MHz, heavy water): δ 7.41 - 7.13 (m, 5H), 4.69 - 4.53 (m, 2H), 4.41 - 4.32 (m, 3H), 4.31 - 4.10 (m, 2H), 4.09 - 3.99 (m, 1H), 3.97 (s, 2H), 3.91 (s, 3H), 3.87 - 3.72 (m, 3H), 3.69 - 3.50 (m, 8H), 3.48 - 3.41 (m, 3H), 3.38 - 3.32 (m, 3H), 3.28 (d, J = 3.7 Hz, 3H), 3.22 (d, J = 5.9 Hz, 4H), 3.16 (s, 2H), 3.08 (s, 2H), 2.92 (d, J = 6.8 Hz, 4H), 2.69 - 2.60 (m, 1H), 2.54 - 2.31 (m, 3H), 2.30 - 2.18 (m, 2H), 2.09 - 1.92 (m, 2H), 1.88 - 1.67 (m, 2H), 1.63 - 1.37 (m, 2H), 1.20 (dd, J = 21.6, 6.5 Hz, 5H), 1.07 (d, J = 6.7 Hz, 1H), 1.05 - 0.67 (m, 23H).

[0062] Preparation of compound GPAF of Example 2

[0063] The structural formula of GPAF is as follows:

[0064] The reaction process is as follows:

[0065] The preparation process comprises the following steps:

[0066] The preparation of compounds 1-6 is the same as that of Example 1.

[0067] a) In a reaction flask, dissolve the substrate 3-0 (1.0 g, 6.7 mmol) and Fmoc-OSu (3.4 g, 10.0 mmol) in dioxane (15 mL), add sodium bicarbonate (1.7 g, 20.1 mmol) and water (10 mL), and react at room temperature for 24 hours. After removing part of the dioxane by concentration, extract with EA for 3 times, dry the combined organic phase with anhydrous Na2S04, and concentrate. Column chromatography (eluent PE:EA = 1:3) gives colorless oil 3-1 (2.2 g, yield 88%).

[0068] b) In a reaction flask, add the product 3-1 (150 mg, 0.41 mmol) from the previous step, dissolve in DMSO (5 mL), then add 2-iodoxybenzoic acid (IBX, 280 mg, 0.82 mmol), stir at room temperature for 18 hours, TLC monitor (2,4-dinitrophenylhydrazine coloration) reaction complete, the mixture is first washed with water, EA extraction 3 times, dried over anhydrous Na2S04, concentrated, column chromatography (eluent DCM:MeOH = 20:1) to get colorless oil 3-2 (103 mg, yield 68%; LCMS: m / z (ESI+) calcd: 370.15; Found: 424.15 [M+MeOH+Na] + ).

[0069] c) In a reaction flask, add the product 3-2 (74 mg, 0.2 mmol) from the previous step and MMAF-OtBu (60 mg, 0.076 mmol) dissolved in dichloroethane (5 mL), the container is placed in an ice bath, after adding sodium triacetoxyborohydride (STAB, 48 mg, 0.228 mmol) stir at room temperature for 18 hours, TLC monitor (phosphomolybdic acid coloration) reaction complete, the mixture is first washed with water, extracted with DCM 3 times, dried over anhydrous Na2S04, concentrated, column chromatography (eluent DCM:MeOH = 20:1) to get colorless oil crude 3-3, which is directly used in the next step.

[0070] d) In a reaction flask, dissolve the product 3-3 from the previous step in DCM (3 mL), then add diethylamine (1 mL), stir at room temperature for 18 hours, TLC monitor (phosphomolybdic acid coloration) reaction complete, the mixture is first washed with water, extracted with DCM 3 times, dried over anhydrous Na2S04, concentrated, column chromatography (eluent DCM:MeOH = 15:1) to get white solid 3-4 (40 mg, two-step yield 22%; LCMS: m / z (ESI+) calcd: 918.64; Found: 460.30; [M / 2+H] + ; 919.75 [M+H] + ; 941.70 [M+Na] + ).

[0071] e) In a reaction flask, product 1-6 (40 mg, 0.04 mmol) and solvent DCM (5 mL) were added, followed by HATU (34 mg, 0.09 mmol), DIPEA (22 μL, 0.12 mmol), stirring at room temperature for 15 minutes, then product 3-4 (35 mg, 0.09 mmol) from the previous step was added, stirring at room temperature for 18 hours, TLC monitoring (molybdenum phosphoric acid coloration) showed that the reaction was complete. The mixture was washed with water first, extracted with DCM three times, dried over anhydrous Na2S04, and concentrated, and column chromatography (eluent DCM:MeOH = 30:1) gave white solid 3-5 (36 mg, yield 70%).

[0072] f) In a reaction flask, product 3-5 (91 mg, 0.07 mmol) from the previous step was dissolved in DCM (4 mL), then trifluoroacetic acid (2 mL) was added, stirring at room temperature for 3 hours, TLC monitoring (molybdenum phosphoric acid coloration) showed that the reaction was complete, the mixture was concentrated at room temperature, then DCM was added, the above operation was repeated three times to remove excess trifluoroacetic acid, and finally water was added to freeze-dry the final product GPAF (69.3 mg, yield 95.7%).

[0073] LCMS: m / z (ESI+) calcd: 1035.66, Found: 518.95 [M / 2+H] + ,1037.10 [M+H] + .

[0074] 1 H-NMR (400 MHz, heavy water): δ 7.23-7.16 (m, 5H), 4.70-4.52 (m, 2H), 4.42-4.31 (m, 3H), 4.33-4.08 (m, 2H), 4.09-3.99 (m, 1H), 3.97 (s, 2H), 3.91 (s, 3H), 3.87-3.72 (m, 3H), 3.68-3.49 (m, 8H), 3.48-3.41 (m, 3H), 3.38-3.32 (m, 3H), 3.28 (d, J = 3.7 Hz, 3H), 3.22 (d, J = 5.9 Hz, 4H), 3.17 (s, 2H), 3.08 (s, 2H), 2.92 (d, J = 6.8 Hz, 4H), 2.69-2.60 (m, 1H), 2.53-2.30 (m, 3H), 2.23 (dd, J = 16.1, 10.2 Hz, 2H), 2.09-1.92 (m, 2H), 1.88-1.67 (m, 3H), 1.63-1.37 (m, 2H), 1.20-1.14 (m, 2H), 1.07 (d, J = 6.7 Hz, 1H), 1.06-0.67 (m, 23H).

[0075] Example 3 Preparation of antibody-drug conjugate

[0076] The anti-HER2 monoclonal antibody containing unnatural amino acids was expressed in eukaryotic expression system using unnatural amino acids NBGK, NPAK, NBOK, and drug conjugate was prepared.

[0077] (1) Obtaining of helper plasmid

[0078] The helper plasmid pCMV-MbPylRS was purchased from the plasmid depository organization addgene (item # 91706), which encodes the aminoacyl tRNA synthetase specifically recognizing pyrrolysine-derived unnatural amino acids in mammalian cells and the corresponding tRNA (recognizing the amber codon UAG).

[0079] (2) Construction of anti-HER2 antibody (trastuzumab) expression vector containing amber codon in the internal gene reading frame

[0080] The heavy chain and light chain DNA (the corresponding amino acid sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively) encoding trastuzumab were synthesized by full gene synthesis and subcloned into the eukaryotic expression vector pCDNA3.1+, and then the obtained expression vector was subjected to point mutation to obtain the expression plasmid pCDNA3.1-Trastuzumab-UAG142 in which the 142th amino acid codon in the heavy chain reading frame was mutated to amber codon, the map of which is shown in Figure 1, and the complete sequence is shown in SEQ ID NO: 3.

[0081] The heavy chain amino acid sequence of trastuzumab (SEQ ID NO: 1) is as follows:

[0082] The light chain amino acid sequence of trastuzumab (SEQ ID NO: 2) is as follows:

[0083] The gene sequence of the expression plasmid pCDNA3.1-Trastuzumab-UAG142 (SEQ ID NO: 3) is as follows:

[0084] (3) Insertion of unnatural amino acid

[0085] Using suspension-acclimated HEK293 cells, 0.3 x 10 5 / mL density was inoculated into Wayne293 TMQuacell Biotechnology, Cat. No. A21501), using 1 L of a shake flask, filling the liquid volume of 240 mL, under the condition of 120 rpm, 5% CO2, 80% humidity, the cells were cultured, when the cell density reached about 1 x 10 6

[0086] (4) Purification

[0087] The cell culture supernatant was purified by HiTrap Protein A, 1 mL pre-packed column, and the elution buffer was 100 mmol / L glycine, 200 mmol / L acetate, pH 3.5, to obtain the purified trastuzumab inserted with unnatural amino acids. The trastuzumab inserted with NBGK, NPAK, and NBOK was named trastuzumab-NBGK, trastuzumab-NPAK, and trastuzumab-NBOK, respectively.

[0088] (5) Coupling

[0089] The synthetic route is shown as follows (taking the coupling reaction of trastuzumab-NBGK and GPAE as an example):

[0090] ​The toxins (GPAE, GPAF, P4AE, P4AF) containing aminoxy end groups were coupled with the purified trastuzumab-NBGK, trastuzumab-NPAK, and trastuzumab-NBOK inserted with unnatural amino acids (wherein the direction from R1 to R2 is the N-terminal to C-terminal direction of the amino acid sequence) by an oximation reaction to obtain the antibody-toxin conjugates: trastuzumab-NBGK-GPAE (trastuzumab-NBGK modified by toxin GPAE), trastuzumab-NBGK-GPAF (trastuzumab-NBGK modified by toxin GPAF), trastuzumab-NBGK-P4AE (trastuzumab-NBGK modified by toxin P4AE), trastuzumab-NBGK-P4AF (trastuzumab-NBGK modified by toxin P4AF), trastuzumab-NPAK-GPAE (trastuzumab-NPAK modified by toxin GPAE), trastuzumab-NPAK-GPAF (trastuzumab-NPAK modified by toxin GPAF), trastuzumab-NPAK-P4AE (trastuzumab-NPAK modified by toxin P4AE), trastuzumab-NPAK-P4AF (trastuzumab-NPAK modified by toxin P4AF), trastuzumab-NBOK-GPAE (trastuzumab-NBOK modified by toxin GPAE), trastuzumab-NBOK-GPAF (trastuzumab-NBOK modified by toxin GPAF), trastuzumab-NBOK-P4AE (trastuzumab-NBOK modified by toxin P4AE), and trastuzumab-NBOK-P4AF (trastuzumab-NBOK modified by toxin P4AF).

[0091] The specific operation of the coupling process is as follows: the purified trastuzumab inserted with unnatural amino acids and GPAE (or GPAF or P4AE or P4AF) are mixed at a molar ratio of 1:12, then 10M acetic acid is used to adjust the pH to 4.0, and the mixture is shaken on a shaker (25°C, 200 rpm). After 48 hours, sampling is performed, and the reaction of trastuzumab and toxin is detected by HIC-HPLC based on the principle of hydrophobic chromatography. The result is shown in the form of drug-to-antibody ratio (DAR value), and the results are shown in Table 1.

[0092] The antibody-toxin conjugate after coupling is subjected to liquid exchange by a 50 kDa ultrafiltration centrifuge tube to remove unreacted toxin raw materials, and the buffer is replaced with 20 mM histidine buffer (pH 6.5).

[0093] The HIC-HPLC analysis conditions are as follows:

[0094] Mobile phase A (2 M ammonium sulfate, 75 mM K2HPO4, pH 7.2±0.2);

[0095] Mobile phase B (75 mM K2HPO4, 25% isopropanol, pH 7.2±0.2).

[0096] The peak area of coupling 1 toxin (DRUG1), the peak area of coupling 2 toxins (DRUG2), and the peak area of coupling 3 toxins (DRUG3) were calculated by area normalization method, and the DAR value was calculated. The DAR value calculation formula: DAR value = (DRUG1+DRUG2x2+DRUG3x3) / (DRUG1+DRUG2+DRUG3).

[0097] Table 1 DAR value of ADC sample

[0098] The results show that GPAE, GPAF, P4AE and P4AF are all combined to the non-natural amino acids NBGK, NPAK or NBOK of trastuzumab in the form of site-specific coupling, and the DAR value after GPAE coupling is higher than that of P4AE, and the DAR value after GPAF coupling is higher than that of P4AF. Since one non-natural amino acid for coupling reaction is inserted into each heavy chain of trastuzumab, ideally, each monoclonal antibody molecule can couple 2 molecules of toxin, and the ideal DAR value is 2.

[0099] Example 4 Activity analysis

[0100] The human gastric cancer cell NCI-N87 (ATCC, item number: CRL-5822) cell line was used to detect the inhibitory effect of the ADC sample on cell proliferation.

[0101] The specific process is as follows: NCI-N87 cells are cultured in RPMI-1640 Medium (Gibco, A10491-01) containing 10% fetal bovine serum (Gibco, 10099-141C) at 37°C, 5% carbon dioxide, and the cells are cultured to an adequate amount, the cell density is adjusted to an appropriate cell density with cell culture solution, cell plating is performed so that 4000 cells are added to each well, 90 μL / well, the solvent control wells are added with culture medium without cells, and the blank control wells are added with cells without drugs. The cell culture plate is incubated in a CO2 incubator (37±1°C, 5±0.5% CO2) overnight. All samples (trastuzumab-NBGK-GPAE, trastuzumab-NBGK-GPAF, trastuzumab-NBGK-P4AE, trastuzumab-NBGK-P4AF, trastuzumab-NPAK-GPAE, trastuzumab-NPAK-GPAF, trastuzumab-NPAK-P4AE, trastuzumab-NPAK-P4AF, trastuzumab-NBOK-GPAE, trastuzumab-NBOK-GPAF, trastuzumab-NBOK-P4AE, trastuzumab-NBOK-P4AF, trastuzumab-NBGK, trastuzumab-NPAK, trastuzumab-NBOK) are diluted from 100 nM to 0.05 nM in a gradient, a total of 9 concentrations, and 2 replicate wells for each dilution. The diluted samples are transferred to the culture plate with plated NCI-N87 cells, 10 μL per well, 10 μL of culture medium is added to the solvent control wells and blank control wells, and incubated at 37°C, 5% carbon dioxide for 96 h±2 h. After adding 50 μL of Promega CellTiter-Glo detection reagent (Promega, G9242) per well, shaking for 2 min, and placing at room temperature for 10 min, the plate is read. The inhibition rate (Growth inhibition) of the test sample is calculated using the following formula: Inhibition rate (%) = (1-(luminescence signal value 化合物 – luminescence signal value blank ) / (luminescence signal value control – luminescence signal value blank )) x 100%. The inhibition rates of different concentrations of compounds are calculated in Excel, and the IC50 is calculated using GraphPad Prism7 software. The results are shown in Table 2.

[0102] Table 2 Cell proliferation inhibition activity of ADC and antibody samples

[0103] The results show that the IC50 value of the GPAE, GPAF, P4AE or P4AF modified trastuzumab containing unnatural amino acids is only a few percent of that of the trastuzumab containing only unnatural amino acids, indicating that the former successfully connects the small molecule toxin. The tumor killing effect (inhibition rate) of the GPAE or GPAF modified trastuzumab containing unnatural amino acids is significantly improved compared with the P4AE or P4AF modified trastuzumab containing unnatural amino acids.

[0104] Example 5 Analysis of endocytic clearance efficiency in cells

[0105] The NCI-N87 cell line was used to detect the endocytic clearance efficiency of the ADC sample in cells.

[0106] The specific process is as follows:

[0107] (1) After observing the NCI-N87 cells under a microscope (Olmpus), place them in a biological safety cabinet, discard the original culture medium, rinse with 6 mL of PBS (Biyun Tian, C0221A) and discard, add 2 mL of 0.25% trypsin (Gibco, 25200072) and digest for 2 min. Add 6 mL of RPMI-1640 medium (Gibco, A10491-01) containing 10% FBS (Gibco, 10099-141C) to terminate digestion, centrifuge and discard the supernatant, add 1 mL of medium and mix, then take 100 μL of cell suspension to a sterile 1.5 mL centrifuge tube for counting.

[0108] (2) Add 500 μL of the resuspended cell suspension after digestion to each well of a 12-well plate, 2×10 5 cells / well, and place the culture plate of NCI-N87 in a 5% CO2, 37°C incubator overnight.

[0109] (3) Take out the 12-well plate, discard the original culture medium, add 300 μL of PBS to each well, wash twice, and discard the washing PBS.

[0110] (4) Add 300 μL of 10 μg / mL FITC-labeled (Thermo, F6434) test sample solution to each well, and place the 12-well plate in a 4°C environment for 60 min. Discard the test sample solution, add 300 μL of PBS to each well, wash twice, and discard the washing PBS.

[0111] (5) Take the cells in one well, trypsinize and transfer into flow tube (Corning, 352054), centrifuge, add 300 μL PBS per tube, wash twice, add 300 μL 4% paraformaldehyde (Biolegend, 420801) to fix for 20 min, centrifuge to discard paraformaldehyde, add 300 μL PBS per well, wash twice, discard the PBS, take this sample as the 0 h time point sample. During the paraformaldehyde fixation, add 500 μL complete medium to each well of the 12-well plate, incubate in a 37°C incubator for a certain period of time (0.5, 1, 2, 4, 6, 8, 16, 24 h), take out, wash twice with PBS, trypsinize and transfer into flow tubes, add 300 μL PBS per tube, wash twice, centrifuge to discard PBS, add 300 μL 4% paraformaldehyde to fix for 20 min, discard the fixing solution, add 300 μL PBS per tube, wash twice, centrifuge to discard the PBS. Add 300 μL FITC-labeled goat anti-human immunoglobulin kappa chain secondary antibody (FITC-Goat Anti-Human Kappa Antibody, 1 μg / 100 μL, SouthernBiotech, 2061-02) to each well, incubate at 4°C in the dark for 60 min. Discard the FITC-Goat Anti-Human Kappa Antibody solution, add 300 μL PBS per well, wash twice, and discard the PBS. After all the time point samples are collected, add 200 μL FACS buffer (PBS+2% FBS) per tube, and detect the mean fluorescence intensity (MFI) on a flow cytometer (Thermo NxT). The endocytosis efficiency of the sample to be tested is calculated using the following formula: endocytosis efficiency % = (MFI 0h –MFI 时间点 ) / (MFI 0h –MFI negtive control ))*100%. The signal value of the secondary antibody background group is the negative control.

[0112] Ideally, after the ADC binds to the tumor-related target, the ADC-receptor complex is internalized in a fast and efficient manner. Studying whether there is a difference in the endocytosis rate of the ADC has a significant impact on the final optimization of the intracellular transport of the targeted therapeutic drug ADC. The results, as shown in Tables 3-1 and 3-2, show that the GPAE and GPAF modified trastuzumab containing unnatural amino acids have an endocytosis efficiency about 14% to 35% higher than the P4AE and P4AF modified trastuzumab containing unnatural amino acids.

[0113] Table 3-1 Endocytosis effect of some ADC samples in cells (1)

[0114] Table 3-2 Intracellular uptake effect of some ADC samples (2)

[0115] As can be seen from Examples 4 and 5, the GPAE and GPAF prepared in the present application have different hydrophobicity and spatial structure relative to the existing auristatin P4AE and P4AF, thereby significantly improving the biological activity and intracellular uptake effect of the formed ADC.

[0116] Unless specifically defined otherwise, the terms used in the present application are understood to have the meanings commonly understood by those skilled in the art.

[0117] The embodiments described in the present application are for illustrative purposes only and are not intended to limit the scope of protection of the present application, and various other alternatives, changes and improvements can be made by those skilled in the art within the scope of the present application, and therefore the present application is not limited to the above-described embodiments, but is limited only by the claims.

Claims

1. A compound of Formula (I) or a stereoisomer, a pharmaceutically acceptable salt thereof, X— L1— A— NH(CH2CH2O) — L2— D Formula (I) n — L2— D Formula (I) wherein X represents a protected or unprotected reactive group; L1, L2 each independently represents a C1-C6 straight chain or branched chain alkylene; A represents a peptide formed by 1-4 amino acids selected from glycine and / or alanine; D represents an auristatin toxin; n represents 1, 2 or 3.

2. The compound according to claim 1 or a stereoisomer, a pharmaceutically acceptable salt thereof, wherein, Said X represents a protected or unprotected hydroxylamine group, amino group, hydroxyl group, thiol group, halogen atom, azido group, carbonyl group, dicarbonyl group, ester group or cycloalkyne group.

3. The compound according to claim 1 or 2, or a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, Said L1, L2 each independently represents a C1-C3 straight chain alkylene, preferably methylene or ethylene.

4. The compound according to any one of claims 1-3, or a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, Said A represents a dipeptide of -glycine-glycine-, a dipeptide of -glycine-alanine-, a dipeptide of -alanine-alanine- or a tripeptide of -glycine-glycine-glycine-.

5. The compound according to any one of claims 1-4, or a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, Said D represents an auristatin toxin MMAE, MMAF or MMAD.

6. The compound according to any one of claims 1-5, or a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, The compound is one of the following:

7. Use of the compound of any one of claims 1-6 or a stereoisomer, a pharmaceutically acceptable salt thereof for preparing a drug conjugate.

8. A drug conjugate prepared from the compound of any one of claims 1-6 or a stereoisomer, a pharmaceutically acceptable salt thereof by conjugation reaction of the reactive group with a targeting molecule; Preferably, the targeting molecule is an anti-HER2 monoclonal antibody, more preferably trastuzumab.

9. The drug conjugate of claim 8, wherein, Said targeting molecule is trastuzumab with site-specific insertion of a non-natural amino acid containing a terminal carbonyl group, and said reactive group is a hydroxylamine group, and the drug conjugate is prepared by reaction of the terminal carbonyl group with the hydroxylamine group to form an oxime bond; Preferably, the unnatural amino acid is a compound having one of the following structures:

10. Use of the drug conjugate of claim 8 or 9 in the preparation of a medicament for treating cancer. Preferably, the cancer is gastric cancer or breast cancer.

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