A toxin derivative for preparing drug conjugates

Abstract

The present invention provides a toxin derivative as shown in formula (I) or its stereoisomer, a pharmaceutically acceptable salt and its use for preparing a drug conjugate. The present invention also provides a drug conjugate and its use. The toxin derivative provided by the present invention connects to auristatin toxins by adjusting the structure of the linker to form a new linker-effect small molecule, which can achieve very excellent biological activity and efficient cellular endocytosis effect when used to prepare drug conjugates, which is conducive to expanding the design, production and application of auristatin conjugates and has very important economic and social value. X——L1——A——NH(CH2CH2O) n ——L2——D. Formula (I)

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a toxin derivative for preparing a drug conjugate and a use thereof, and also to a drug conjugate and a use thereof. Background Art

[0002] Drug conjugates with targeted delivery function are an emerging type of treatment method that has developed as drug research and development moves towards precision treatment. Drug conjugates are composed of targeting molecules coupled to effector molecules. The targeting molecules selectively bind to disease-related cells or tissues and release effector molecules with therapeutic effects in a targeted manner. Targeting molecules can be antibodies, antibody fragments, ligands, peptides, small molecules, or virus-like particles. At present, antibody-drug conjugates (ADCs) with antibodies as targeting molecules have become the mainstream of the development of next-generation precision therapeutic drugs, and have broad prospects in the treatment of diseases such as cancer and autoimmune diseases. ADCs are usually composed of four parts: 1) targeted antibodies, which are used to identify target cells and bring effector small molecule drugs (usually chemical drugs) to the cell surface or inside; 2) small molecules with cytotoxicity or other effects, which play a role in killing cells or regulating cell functions; 3) linkers that connect antibodies and drugs, which play a role in connection and support; 4) linkers that couple linkers to antibodies, which have special active groups.

[0003] At present, there are many studies on antibodies and effector small molecules. However, in ADC, linkers and adapters also play a very important role. Linkers can be divided into non-cleavable linkers and cleavable linkers based on their performance. Non-cleavable linkers have a stable chemical bond composition and are more stable than cleavable linkers. Their mechanism of action is based on the antibody-antigen complex of ADC being endocytosed by cells, and then ADC is degraded through lysosomes to release the effective load containing the linker, which plays the effect of killing tumor cells. Therefore, the advantage of non-cleavable linkers is that ADCs using this type of linker are not easy to release drugs in the blood circulation, have low off-target toxicity, and are safer. In addition, ADCs using non-cleavable linkers can change the chemical properties of small molecule drugs through linkers to enhance their metabolic properties or efficacy because the released effective load carries a complete linker.

[0004] There are relatively few ADCs that are already on the market or in clinical development that use non-cleavable linkers. The more mature ones include thioether linkers, amide linkers, etc. For example, Roche's Kadcyla connects DM1 to the antibody via a stable thioether linker.

[0005] Through the rational design of linkers, the optimization of ADC molecules can be achieved. For example, the physical and chemical properties and pharmacokinetic properties of ADC can be optimized, the pharmacodynamic properties and toxicity properties of ADC can be improved, or a high DAR value design can be achieved, and further the introduction of different types of effector small molecules can be realized, etc.

[0006] The coupling mode of antibodies and effector small molecules also directly affects the DAR value and the uniformity of ADC products, further affecting their physicochemical properties, biological activity, in vivo efficacy and toxicity. Site-specific coupling of ADC is one of the research hotspots that has attracted much attention. Through genetic engineering, unnatural amino acids (UAAs) with specific active groups can be introduced into antibody molecules, thereby achieving effective control of the DAR value and product uniformity of ADC. Summary of the invention

[0007] In order to make up for the deficiencies in the prior art, an object of the present invention is to provide a toxin derivative, which is very suitable for preparing drug conjugates, such as antibody-drug conjugates (ADCs), by adjusting the structure of the linker, and can achieve excellent biological activity and cellular endocytosis effects.

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

[0009] The first aspect of the present invention provides a compound as shown in formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof,

[0010] XL1ANH(CH2CH2O) n L2D

[0011] Formula (I)

[0012] Wherein, X represents a reactive group protected or unprotected by a protecting group;

[0013] L1 and L2 each independently represent a C1-C6 straight or branched alkylene group;

[0014] A represents a peptide formed by 1 to 4 amino acids selected from glycine and / or alanine;

[0015] D indicates auristatin toxin;

[0016] n represents 1, 2 or 3.

[0017] For drug conjugates, the structure and physicochemical properties of the linker have a very large impact on the stability, metabolism, and efficacy of the drug. For example, it can affect the conjugation efficiency and DAR value, the transmembrane properties of the effector small molecule and the stability of the conjugate, the pharmacokinetic properties of the effector small molecule, etc. For another example, the structural residues of the linker on the effector small molecule can affect the effector response of the effector small molecule. For another example, the various physicochemical properties of the linker can also affect the production process and drugability of the conjugate.

[0018] In the compound represented by formula (I) provided by the present invention, a new linker structure is formed by amino acid residues and PEG units of a 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 efficient cellular endocytosis effect.

[0019] In the compound represented by formula (I) provided by the present invention, X can represent any active reactive group commonly used in the art, which can react with a reactive group on a targeting molecule (such as an antibody) (which can be a group contained in the targeting molecule itself or a group carried by the targeting molecule after modification), thereby forming a 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 during the reaction.

[0020] In some embodiments, the X can represent the following groups protected or unprotected by a protecting group: hydroxylamine, amino, hydroxyl, sulfhydryl, halogen atom, azido, carbonyl, dicarbonyl, ester or cycloalkyne. In some preferred embodiments, the X represents a hydroxylamine group, i.e., "NH2-O-".

[0021] In some embodiments, L1 and L2 may 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 may each independently represent a methylene group or an ethylene group.

[0022] In some embodiments, A may 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 may be: Among them, "1" represents the connection point with L1, and "2" represents the connection point at the other end.

[0023] In some embodiments, the D may represent any common auristatins toxin in the art, which is a synthetic derivative of the natural product dolastatin 10, an anti-mitotic agent, which blocks tubulin polymerization by acting on the β-subunit of the α-β tubulin dimer, promoting cell cycle arrest and apoptosis. Auristatins include but are not limited to MMAE, MMAF, MMAD, etc. In some preferred embodiments, the D may be represented as MMAE or MMAF as shown below, wherein the secondary amine group of the terminal group is dehydrogenated and connected to other parts, so D may also be referred to as the residue after MMAE or MMAF removes the hydrogen of the terminal secondary amine group.

[0024]

[0025] In some most preferred embodiments, the compound may be one of the following:

[0026]

[0027] The second aspect of the present invention provides the use of the compound described in any one of the above technical solutions or its stereoisomers, and pharmaceutically acceptable salts for preparing drug conjugates (such as antibody-drug conjugates).

[0028] The compound or its stereoisomer or pharmaceutically acceptable salt described in any one of the above technical solutions provided by the present invention can be coupled with any common antibody, antibody fragment, ligand, polypeptide, targeting protein or other targeting molecule in the art through any common coupling method in the art to form a conjugate. In some preferred embodiments, the antibody can be an anti-HER2 monoclonal antibody, such as trastuzumab.

[0029] The third aspect of the present invention provides a drug conjugate, which is prepared by coupling the compound described in any one of the above technical solutions or its stereoisomers, pharmaceutically acceptable salts through the reactive group (ie, X) with a targeting molecule.

[0030] In the antibody-drug conjugate provided by the present invention, the compound or its stereoisomer or pharmaceutically acceptable salt described in any one of the above technical solutions can be coupled with any antibody, polypeptide, targeting protein or other targeting molecule commonly used in the art through the reactive group by any coupling method commonly used in the art to form a drug conjugate. In some preferred embodiments, the antibody can be an anti-HER2 monoclonal antibody, such as trastuzumab.

[0031] In some embodiments, the targeting molecule is trastuzumab with a non-natural amino acid containing a terminal carbonyl group inserted at a fixed point, the reactive group is a hydroxylamine group, and the drug conjugate is prepared by reacting the terminal carbonyl group with the hydroxylamine group to form an oxime bond, and the oxime bond formed by the reaction of the carbonyl group with the hydroxylamine group is as follows:

[0032]

[0033] In some embodiments, the non-natural amino acid may be those described in Chinese Patents ZL 202111019771.1 and ZL202110865364.6. In some preferred embodiments, the non-natural amino acid is a compound having one of the following structures:

[0034]

[0035]

[0036] In some embodiments, the non-natural amino acid can be introduced into the designated site of the targeting molecule by codon expansion technology (e.g., codon expansion technology is implemented in Escherichia coli), thereby achieving subsequent site-specific coupling. In some preferred embodiments, the amino acid sequences of the heavy chain and light chain of trastuzumab used are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, wherein the non-natural amino acid is introduced at position 142 of the heavy chain.

[0037] The fourth aspect of the present invention provides use of the drug conjugate described in any one of the above technical solutions in the preparation of a drug for treating cancer.

[0038] In some embodiments, the cancer may be gastric cancer or breast cancer.

[0039] In some embodiments, the drug conjugate can be used alone or in combination with one or more other anti-tumor drugs.

[0040] The toxin derivatives provided by the present invention are connected to auristatins by adjusting the structure of the linker to form a completely new linker-effector small molecule derivative, which can achieve very excellent biological activity and efficient cellular endocytosis effect when used to prepare drug conjugates, is conducive to expanding the design, production and application of auristatins conjugates, and has very important economic and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the map of the expression plasmid pCDNA3.1-Trastuzumab-UAG142 in Example 3. DETAILED DESCRIPTION

[0042] In the present invention, "C1~Cn" includes C1~C2,...C1~Cn. For example, the "C1~C6" group means that the part has 1 to 6 carbon atoms, that is, the group contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.

[0043] The compounds or conjugates of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds or conjugates of the present invention, including but not limited to diastereomers, enantiomers, hindered isomers and geometric (conformation) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention. Unless otherwise indicated, the structure described in the present invention also includes all isomers of the structure (e.g., diastereomers, enantiomers, steric isomers and geometric (conformation) isomer forms; for example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, steric isomers of biphenyl structures (see "Basic Organic Chemistry" (Second Edition), Volume 1, Xing Qiyi et al., p104-105); PAC, 1996, 68, 2193. (Basic terminology ofstereochemistry (IUPAC Recommendations 1996, on page 2201)), (Z) and (E) conformational isomers. Therefore, single stereoisomers of the compounds or conjugates of the present invention as well as enantiomeric mixtures, diastereomeric mixtures, steric isomers and geometric (conformation) isomer mixtures are all within the scope of the present invention.

[0044] The term "pharmaceutically acceptable salt" used herein, alone or in combination, refers to a compound or conjugate of the present invention that retains the biological effectiveness and properties of a free acid or free base, and the free acid is obtained by reacting with a non-toxic inorganic base or organic base, and the free base is obtained by reacting with a non-toxic inorganic acid or organic acid. Standard procedures well known in the art can be used to obtain. Suitable salts are listed in Remingtong's Pharmaceutical Scicences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977).

[0045] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments.

[0046] The non-natural amino acids NBGK, NPAK, and NBOK used in the embodiments of the present invention were prepared with reference to Chinese Patent ZL202111019771.1, and the comparative toxins P4AE and P4AF used were prepared with reference to Chinese Patent ZL 201810957127.0, and their structures are as follows:

[0047]

[0048] The toxin MMAE (monomethyl auristatin E) used in the examples 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.

[0049] In the embodiments of the present invention, all the ratios of eluent involved are volume ratios, and all the percentages involved are mass percentages unless otherwise specified.

[0050] Example 1 Preparation of Compound GPAE

[0051] The structural formula of GPAE is shown below:

[0052]

[0053] The reaction process is shown below:

[0054]

[0055] The preparation process includes the following steps:

[0056] First, the glycine linker part (1-6) is prepared, and then the PEG2-MMAE (2-3) part is prepared. Then, the two parts 1-6 and 2-3 are condensed to obtain the target compound GPAE.

[0057] a) In a reaction flask, glycine methyl ester hydrochloride (1.67 g, 10 mmol) was dissolved in a mixed solution of DCM / water (50 / 20 mL), 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 vigorous stirring at room temperature for 18 hours, the mixture was extracted 3 times with DCM, the organic phases were combined and washed with water and brine (brine), the organic phase was concentrated and recrystallized (PE: EA = 10:1), and filtered to obtain white crystals 1-1 (1.70 g, yield 68%).

[0058] b) The product 1-1 (1.67 g, 6.6 mmol) of the previous step was placed in a reaction flask, and solvent DCM (20 mL) was added. The container was placed in an ice bath, trifluoroacetic acid (5 mL) was added, and stirred at room temperature for 18 hours. The reaction was complete when detected by TLC. The mixture was evaporated in a water bath to remove most of the solvent. After adding 30 mL of water, the product 1-2 was freeze-dried and used for standby.

[0059] c) In a reaction flask, N-(2-bromoethyl)phthalimide (6.0 g, 24 mmol), tert-butyl N-hydroxycarbamate (4.8 g, 36 mmol) and K2CO3 (8.3 g, 60 mmol) were added, and solvent DMF (60 mL) was added. The mixture was stirred at room temperature for 18 hours and monitored by TLC. An appropriate amount of water was added to the mixture, and the mixture was extracted with EA three times. After the organic phases were combined, they were washed with water three times, dried over anhydrous Na2SO4, and concentrated. Column chromatography (eluent PE:EA = 10:1 and 2:1) was performed to obtain a white solid 1-3 (3.45 g, yield 47%).

[0060] d) In a reaction flask, the product 1-3 (0.5 g, 1.64 mmol) of the previous step was dissolved in ethanol (20 mL), and hydrazine hydrate (80%, 410 μL, 6.56 mmol) was added. The container was refluxed at 80 degrees for 18 hours. The reaction was complete by TLC detection. The white precipitate was precipitated by cooling and standing. It was removed by filtration. The filter cake was washed with ethanol. The filtrate was concentrated and subjected to column chromatography (eluent DCM: MeOH = 10: 1) to obtain a yellow oil 1-4 (165 mg, yield 57%, LCMS: m / z (ESI+) calcd: 176.12; Found: 177.10 [M+H] + ; 218.15[M+MeCN+H] + ).

[0061] e) In a reaction flask, the product 1-4 (3.67 g, 20.8 mmol) of the previous step, the product 1-2 (3.4 g, 17.3 mmol) prepared in step b) and the solvent methanol (60 mL) were added, and then DIPEA (15 mL, 87 mmol) was added. After stirring at 70 degrees for 18 hours, it was directly concentrated, slurried with EA, and filtered to obtain a white solid 1-5 (5.56 g, not purified, directly used in the next step).

[0062] f) Based on a 50% yield, the crude product 1-5 (504 mg) of the previous step was dissolved in water (5 mL), and a solution of triethylamine (0.72 mL, 5.2 mmol) and Boc anhydride (0.94 g, 4.3 mmol) in dioxane (5 mL) was added, and the mixture was stirred at room temperature for 18 hours. The mixture was first concentrated to remove most of the dioxane, and extracted with DCM for 3 times. The organic phases were combined and concentrated, and column chromatography (eluent DCM: MeOH = 10: 1 and 5: 1) was performed to obtain a white solid 1-6 (287 mg; LCMS: m / z (ESI+) calcd: 391.20, Found: 392.15 [M+H] + ; 414.15[M+Na] + ).

[0063] g) In a reaction flask, add substrate 2-0 (100 mg, 0.4 mmol, purchased from Bidex Pharmaceuticals, catalog number: BD261696), dissolve it in DMSO (3 mL), add 2-iodobenzoic acid (IBX, 224 mg, 0.8 mmol), stir at room temperature for 18 hours, monitor the reaction by TLC (2,4-dinitrophenylhydrazine color development), and the mixture is washed with water, extracted with EA three times, dried over anhydrous Na2SO4, and concentrated. Column chromatography (eluent DCM: MeOH = 20:1) gives the product 2-1 (50 mg, yield 50.5%).

[0064] h) In a reaction flask, the product 2-1 (50 mg, 0.2 mmol) and MMAE (57 mg, 0.08 mmol) of the previous step were dissolved in dichloroethane (5 mL), the container was placed in an ice bath, sodium triacetoxyborohydride (STAB, 51 mg, 0.24 mmol) was added and stirred at room temperature for 18 hours. TLC monitoring (phosphomolybdic acid color development) showed that the reaction was complete. The mixture was first washed with water, extracted with DCM three times, dried over anhydrous Na2SO4 and concentrated. Column chromatography (eluent DCM: MeOH = 20: 1) gave a colorless oil 2-2 (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] + ).

[0065] i) In a reaction flask, the product 2-2 (79 mg, 0.08 mmol) of the previous step was dissolved in DCM (2 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 3 hours. The reaction was completed by TLC monitoring (phosphomolybdic acid color development). The mixture was concentrated at room temperature, and DCM was added. The above operation was repeated 3 times to remove excess trifluoroacetic acid. Finally, water was added and lyophilized to obtain the product 2-3 (67.9 mg, yield 99%).

[0066] j) In a reaction flask, product 1-6 (50 mg, 0.13 mmol) and solvent DCM (5 mL) were added, followed by HATU (60.8 mg, 0.16 mmol) and DIPEA (57 μL, 0.32 mmol), and the mixture was stirred at room temperature for 30 minutes. The product 2-3 (67.9 mg, 0.08 mmol) from the previous step was added, and the mixture was stirred at room temperature for 18 hours. The reaction was complete as monitored by TLC (phosphomolybdic acid colorimetric). The mixture was washed with water, extracted 3 times with DCM, dried over anhydrous Na2SO4, and concentrated. Column chromatography (eluent DCM: MeOH = 15: 1) gave a white solid 2-4 (63 mg; LCMS: m / z (ESI+) calcd: 1221.78, Found: 612.25 [M / 2+H] + ).

[0067] k) In a reaction flask, the product 2-4 (63 mg) of the previous step was dissolved in dioxane (10 mL), hydrochloric acid (5 mL) was slowly added, and the mixture was stirred at room temperature for 3 hours. The reaction was completed after monitoring by TLC. The mixture was concentrated at room temperature to remove part of the solvent, and then lyophilized with water to obtain the final product GPAE (25.6 mg, yield 48.6%) as a white solid.

[0068] LCMS: m / z(ESI+)calcd: 1021.68, Found: 512.20[M / 2+H] + ,1046.40[M+Na] + .

[0069] 1H-NMR (400 MHz, heavy water): δ7.41–7.13 (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=5.9Hz,4H),3.16(s,2H),3.08(s,2H),2.92(d,J=6.8Hz,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=21.6,6.5Hz,5H),1.07(d,J=6.7Hz,1H),1.05–0.67(m,23H).

[0070] Example 2 Preparation of compound GPAF

[0071] The structural formula of GPAF is shown below:

[0072]

[0073] The reaction process is shown below:

[0074]

[0075] The preparation process includes the following steps:

[0076] The preparation of compound 1-6 is the same as that of Example 1.

[0077] a) In a reaction flask, substrate 3-0 (1.0 g, 6.7 mmol) and Fmoc-OSu (3.4 g, 10.0 mmol) were dissolved in dioxane (15 mL), and sodium bicarbonate (1.7 g, 20.1 mmol) and water (10 mL) were added. After reacting at room temperature for 24 hours, the dioxane was partially removed by concentration, and the mixture was extracted with EA three times. The organic phases were combined, dried over anhydrous Na2SO4, and concentrated. Column chromatography (eluent PE:EA = 1:3) was performed to obtain a colorless oil 3-1 (2.2 g, yield 88%).

[0078] b) Add the product 3-1 (150 mg, 0.41 mmol) from the previous step to a reaction flask, dissolve it in DMSO (5 mL), add 2-iodobenzoic acid (IBX, 280 mg, 0.82 mmol), stir at room temperature for 18 hours, and monitor the reaction by TLC (2,4-dinitrophenylhydrazine color development). The reaction is complete. The mixture is first washed with water, extracted with EA three times, dried over anhydrous Na2SO4, concentrated, and then concentrated by column chromatography (eluent DCM: MeOH = 20: 1) to obtain a colorless oil 3-2 (103 mg, yield 68%; LCMS: m / z (ESI+) calcd: 370.15; Found: 424.15 [M+MeOH+Na] + ).

[0079] c) In a reaction flask, the product 3-2 from the previous step (74 mg, 0.2 mmol) and MMAF-OtBu (60 mg, 0.076 mmol) were added and dissolved in dichloroethane (5 mL). The container was placed in an ice bath, and sodium triacetoxyborohydride (STAB, 48 mg, 0.228 mmol) was added and stirred at room temperature for 18 hours. The reaction was completed by TLC monitoring (phosphomolybdic acid color development). The mixture was first washed with water, extracted 3 times with DCM, dried over anhydrous Na2SO4 and concentrated. Column chromatography (eluent DCM: MeOH = 20:1) was used to obtain a colorless oily crude product 3-3, which was directly used in the next step.

[0080] d) In a reaction flask, the product 3-3 of the previous step was dissolved in DCM (3 mL), and diethylamine (1 mL) was added. The mixture was stirred at room temperature for 18 hours. The reaction was completed by TLC monitoring (phosphomolybdic acid color development). The mixture was first washed with water, extracted with DCM three times, dried over anhydrous Na2SO4 and concentrated. Column chromatography (eluent DCM: MeOH = 15: 1) gave a 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] + ).

[0081] e) Add product 1-6 (40 mg, 0.04 mmol) and solvent DCM (5 mL) to a reaction flask, then add HATU (34 mg, 0.09 mmol) and DIPEA (22 μL, 0.12 mmol), stir at room temperature for 15 minutes, then add product 3-4 (35 mg, 0.09 mmol) from the previous step, stir at room temperature for 18 hours, and monitor the reaction by TLC (phosphomolybdic acid color development). The mixture is washed with water, extracted with DCM three times, dried with anhydrous Na2SO4, concentrated, and column chromatography (eluent DCM: MeOH = 30: 1) to obtain a white solid 3-5 (36 mg, yield 70%).

[0082] f) In a reaction flask, the product 3-5 (91 mg, 0.07 mmol) of the previous step was dissolved in DCM (4 mL), and trifluoroacetic acid (2 mL) was added. The mixture was stirred at room temperature for 3 hours. The reaction was completed by TLC monitoring (phosphomolybdic acid color development). The mixture was concentrated at room temperature, and DCM was added. The above operation was repeated 3 times to remove excess trifluoroacetic acid. Finally, water was added and lyophilized to obtain the final product GPAF (69.3 mg, yield 95.7%).

[0083] LCMS: m / z(ESI+)calcd: 1035.66, Found: 518.95[M / 2+H] + ,1037.10[M+H] + .

[0084] 1 H-NMR (400 MHz, heavy water): δ7.23–7.16 (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=5.9Hz,4H),3.17(s,2H),3.08(s,2H),2.92(d,J=6.8Hz,4H),2.69–2.60(m,1H),2.53–2.30(m,3H),2.23(dd,J=16.1,10.2H z,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=6.7Hz,1H),1.06–0.67(m,23H).

[0085] Example 3 Preparation of Antibody-Drug Conjugates

[0086] Unnatural amino acids NBGK, NPAK, and NBOK were used to express anti-HER2 monoclonal antibodies containing unnatural amino acids in a eukaryotic expression system, and drug conjugates were prepared.

[0087] (1) Obtaining auxiliary plasmids

[0088] The auxiliary plasmid pCMV-MbPylRS was purchased from the plasmid collection organization addgene (catalog number #91706). The plasmid encodes the aminoacyl tRNA synthetase and the corresponding tRNA (recognizing the amber codon UAG) that specifically recognizes the unnatural amino acid derived from pyrrolysine in mammalian cells.

[0089] (2) Construction of an anti-HER2 antibody (trastuzumab) expression vector containing an amber codon within the gene reading frame

[0090] The heavy chain and light chain DNA encoding trastuzumab (the corresponding amino acid sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively) were synthesized by whole gene synthesis and subcloned into the eukaryotic expression vector pCDNA3.1+. The obtained expression vector was then subjected to point mutation to obtain the expression plasmid pCDNA3.1-Trastuzumab-UAG142 in which the amino acid codon at position 142 of the heavy chain reading frame was mutated to an amber codon. The map is shown in Figure 1 The complete sequence is shown in SEQ ID NO:3.

[0091] The heavy chain amino acid sequence of trastuzumab (SEQ ID NO: 1) is as follows:

[0092]

[0093]

[0094] The amino acid sequence of the light chain of trastuzumab (SEQ ID NO: 2) is as follows:

[0095]

[0096] The gene sequence of the expression plasmid pCDNA3.1-Trastuzumab-UAG142 (SEQ ID NO: 3) is as follows:

[0097]

[0098]

[0099]

[0100]

[0101] (3) Insertion of unnatural amino acids

[0102] Use suspension-cultured HEK293 cells at 0.3 × 10 5 / mL density inoculated into Wayne293 TM Culture medium (Quacell Biotechnology, catalog number: A21501) was used in a 1L shake flask with a volume of 240 mL. The culture was shaken at 120 rpm, 5% CO2, and 80% humidity. When the cell density reached about 1×10 6 / mL, extract the auxiliary plasmid pCMV-MbPylRS and expression plasmid pCDNA3.1-Trastuzumab-UAG142 described in steps (1) and (2), remove endotoxin and set aside. Use 120 μg of expression plasmid and auxiliary plasmid for transfection in each shake flask. Add the plasmid to a centrifuge tube and dilute it to 7.2 mL with 1× PBS buffer. Then add 720 μg of PEI transfection reagent (polyethyleneimine) to another centrifuge tube, and then add 1× PBS buffer was diluted to 7.2 mL, and after mixing, the two centrifuge tubes were allowed to stand for 5 minutes each. Then, the liquids in the two centrifuge tubes were gently mixed and allowed to stand for 10 minutes. A total of 14.4 mL was slowly added dropwise to the 240 mL cell suspension in a 1 L shake flask. During the addition process, the mixture was gently shaken continuously. After shaking culture at 120 rpm, 5% CO2 and 80% humidity for 4 h, a non-natural amino acid with a final concentration of 1 mM was added. The mixture was continued to be shaken at 120 rpm, 5% CO2 and 80% humidity for 5 days. The cell culture supernatant was harvested for antibody purification.

[0103] (4) Purification

[0104] The cell culture supernatant was purified using 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 were named trastuzumab-NBGK, trastuzumab-NPAK, and trastuzumab-NBOK, respectively.

[0105] (5) Coupling

[0106] The synthetic route is as follows (taking the coupling reaction of trastuzumab-NBGK and GPAE as an example):

[0107]

[0108] The toxins containing aminooxy terminal groups (GPAE, GPAF, P4AE, P4AF) were site-specifically coupled with the purified trastuzumab-NBGK, trastuzumab-NPAK, and trastuzumab-NBOK (wherein the direction from R1 to R2 is from the N-terminus to the C-terminus of the amino acid sequence) inserted with non-natural amino acids by oximation reaction to obtain monoclonal antibody-toxin conjugates: trastuzumab-NBGK-GPAE (trastuzumab-NBGK modified with toxin GPAE), trastuzumab-NBGK-GPAF (trastuzumab-NBGK modified with toxin GPAF), trastuzumab-NBGK-P4AE (trastuzumab-NBGK modified with toxin P4AE), trastuzumab-NBGK-P4AF (trastuzumab-NBGK modified with toxin P4AF), and trastuzumab-NBGK-NPAK. PAK-GPAE (trastuzumab-NPAK modified with toxin GPAE), trastuzumab-NPAK-GPAF (trastuzumab-NPAK modified with toxin GPAF), trastuzumab-NPAK-P4AE (trastuzumab-NPAK modified with toxin P4AE), trastuzumab-NPAK-P4AF (trastuzumab-NPAK modified with toxin P4AF), trastuzumab-NBOK-GPAE (trastuzumab-NBOK modified with toxin GPAE), trastuzumab-NBOK-GPAF (trastuzumab-NBOK modified with toxin GPAF), trastuzumab-NBOK-P4AE (trastuzumab-NBOK modified with toxin P4AE), trastuzumab-NBOK-P4AF (trastuzumab-NBOK modified with toxin P4AF).

[0109] The specific operation of the coupling process is as follows: the purified trastuzumab inserted with a non-natural amino acid is mixed and dissolved with GPAE (or GPAF or P4AE or P4AF) at a molar ratio of 1:12, and then the pH is adjusted to 4.0 with 10M acetic acid, and the mixture is shaken on a shaker (25°C, 200rpm). After 48 hours, samples are taken and the reaction between trastuzumab and toxin is detected by HPLC (HIC-HPLC) based on the principle of hydrophobic chromatography. The results are presented in the form of toxin-monoclonal antibody coupling ratio (drug-to-antibody ratio, DAR value). The results are shown in Table 1.

[0110] The coupled monoclonal antibody-toxin conjugate was replaced with a 50 kDa ultrafiltration centrifuge tube to remove unreacted toxin raw materials, and the replacement buffer was 20 mM histidine buffer (pH 6.5).

[0111] The HIC-HPLC analysis conditions are as follows:

[0112] Mobile phase A (2 M ammonium sulfate, 75 mM K2HPO4, pH 7.2 ± 0.2);

[0113] Mobile phase B (75 mM K2HPO4, 25% isopropanol, pH 7.2±0.2).

[0114]

[0115] The peak area of ​​1 toxin coupled (DRUG1), 2 toxins coupled (DRUG2), and 3 toxins coupled (DRUG3) were calculated by area normalization method to calculate the DAR value. The DAR value calculation formula is: DAR value = (DRUG1 + DRUG2 × 2 + DRUG3 × 3) / (DRUG1 + DRUG2 + DRUG3).

[0116] Table 1 DAR values ​​of ADC samples

[0117]

[0118]

[0119] The results showed that GPAE, GPAF, P4AE and P4AF were all conjugated to the unnatural amino acids NBGK, NPAK or NBOK of trastuzumab in the form of site-specific conjugation, and the DAR value of GPAE after conjugation was higher than that of P4AE, and the DAR value of GPAF after conjugation was higher than that of P4AF. Since each heavy chain of trastuzumab has an unnatural amino acid inserted for conjugation reaction, ideally each monoclonal antibody molecule can be conjugated to 2 molecules of toxin, and the ideal DAR value is 2.

[0120] Example 4 Activity Analysis

[0121] The human gastric cancer cell line NCI-N87 (ATCC, catalog number: CRL-5822) was used to detect the inhibitory effect of ADC samples on cell proliferation.

[0122] The specific process is as follows: NCI-N87 cells were cultured with RPMI-1640 Medium (Gibco, A10491-01) containing 10% fetal bovine serum (Gibco, 10099-141C) at 37°C and 5% carbon dioxide until sufficient volume was achieved, the cell density was adjusted to an appropriate cell density using cell culture fluid, and the cells were plated so that 4000 cells were added to each well, 90 μL / well, medium without cells was added to the vehicle control wells, and only cells without drugs were added to the blank control wells. The cell culture plates were placed in a CO2 incubator (37±1°C, 5±0.5% CO2) and incubated 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) were serially diluted from 100 nM to 0.05 nM, for a total of 9 concentrations, with 2 replicates for each dilution. The diluted samples were transferred to the culture plate seeded with NCI-N87 cells, 10 μL per well, 10 μL culture medium was added to the solvent control well and the blank control well, and cultured at 37°C and 5% carbon dioxide for 96 h ± 2 h. Then, 50 μL Promega CellTiter-Glo detection reagent (Promega, G9242) was added to each well. After shaking for 2 min, the plate was placed at room temperature for 10 min before reading. The inhibition rate (Growth inhibition) of the test sample was calculated using the following formula: Inhibition rate (%) = (1-(luminescence signal value 化合物 – Luminescence signal value blank ) / (luminescence signal value c ontrol – Luminescence signal value blank )) × 100%. The inhibition rate of different concentrations of compounds was calculated in Excel, and then IC50 was calculated using GraphPad Prism7 software. The results are shown in Table 2.

[0123] Table 2 Cell proliferation inhibitory activity of ADC and antibody samples

[0124]

[0125]

[0126] The results showed that the IC50 value of trastuzumab containing unnatural amino acids modified by GPAE, GPAF, P4AE or P4AF was only a few hundredths of that of trastuzumab containing only unnatural amino acids, indicating that the former successfully linked to the toxin small molecule. The tumor killing effect (inhibition rate) of trastuzumab containing unnatural amino acids modified by GPAE or GPAF was significantly improved compared with trastuzumab containing unnatural amino acids modified by P4AE or P4AF.

[0127] Example 5 Analysis of endocytic clearance efficiency in cells

[0128] The NCI-N87 cell line was used to detect the endocytic clearance efficiency of ADC samples in cells.

[0129] The specific process is as follows:

[0130] (1) After observing NCI-N87 cells under a microscope (Olmpus), place them in a biosafety cabinet, discard the original culture medium, add 6 mL PBS (Biyuntian, C0221A) for rinsing and discard, add 2 mL 0.25% trypsin (Gibco, 25200072) for digestion for 2 min. Add 6 mL RPMI-1640 culture medium (Gibco, A10491-01) containing 10% FBS (Gibco, 10099-141C) to terminate digestion, centrifuge and discard the supernatant, add 1 mL culture medium and mix well, take 100 μL of cell suspension in a sterile 1.5 mL centrifuge tube for counting.

[0131] (2) Add 500 μL of digested and resuspended cell suspension to each well of a 12-well plate. 5 The culture plate containing NCI-N87 cells was placed in a 5% CO2, 37°C incubator for overnight culture.

[0132] (3) Take out the 12-well plate, discard the original culture medium, add 300 μL PBS to each well, wash twice, and discard the washing PBS.

[0133] (4) Add 300 μL of 10 μg / mL FITC-labeled (Thermo, F6434) sample solution to each well, place the 12-well plate at 4°C for 60 min, discard the sample solution, add 300 μL PBS to each well, wash twice, and discard the PBS.

[0134] (5) Take cells from one well, digest them with trypsin and transfer them to a flow tube (Corning, 352054). After centrifugation, add 300 μL PBS to each tube, wash twice, add 300 μL 4% paraformaldehyde (Biolegend, 420801) to fix for 20 min, centrifuge to discard formaldehyde, add 300 μL PBS to each well, wash twice, and discard the washing PBS. This sample is the sample at time 0 h. During the formaldehyde fixation period, add 500 μL complete medium to each well of the 12-well plate and place it in a 37°C incubator for a certain period of time (0.5, 1, 2, 4, 6, 8, 16, 24 h), take it out, wash twice with PBS, digest it with trypsin and transfer it to a flow tube. Add 300 μL PBS to each tube, wash twice, centrifuge to discard PBS, and then add 300 μL 4% paraformaldehyde to fix for 20 min. Discard the fixative, add 300 μL PBS to each tube, wash twice, centrifuge to discard the washing PBS. 300 μL of FITC-labeled goat anti-human immunoglobulin Kappa chain secondary antibody (FITC-Goat Anti-Human Kappa antibody, 1 μg / 100 μL, SouthernBiotech, 2061-02) was added to each well and incubated at 4°C in the dark for 60 min. The FITC-Goat Anti-Human Kappa antibody solution was discarded, 300 μL of PBS was added to each well, and the cells were washed twice, and the washing PBS was discarded. After the cells at all time points were collected, 200 μL of FACS buffer (PBS + 2% FBS) was added to each tube and the cells were analyzed on a flow cytometer (Thermo The mean fluorescence intensity (MFI) was detected on NxT, and the endocytosis efficiency of the sample to be tested was calculated using the following formula: endocytosis efficiency % = (MFI 0h –MFI 时间点 ) / (MFI 0h –MFI negtive control ))*100%. The secondary antibody background signal value is the negative control.

[0135] Ideally, after ADC binds to tumor-related targets, the ADC-receptor complex is internalized in a rapid and efficient manner. Studying whether there is a difference in ADC endocytosis rate has a significant impact on the ultimate optimization of intracellular transport of targeted therapeutic drug ADC. The results are shown in Tables 3-1 and 3-2. The results show that the endocytosis efficiency of GPAE and GPAF-modified trastuzumab containing unnatural amino acids is about 14% to 35% higher than that of P4AE and P4AF-modified trastuzumab containing unnatural amino acids.

[0136] Table 3-1 Cell endocytosis effects of some ADC samples (1)

[0137]

[0138]

[0139] Table 3-2 Cell endocytosis effects of some ADC samples (2)

[0140]

[0141] It can be seen from Examples 4 and 5 that, compared with the existing auristatin toxins P4AE and P4AF, the GPAE and GPAF prepared by the present invention have different hydrophobicity and spatial structure, thereby significantly improving the biological activity and cellular endocytosis effect of the formed ADC.

[0142] Unless otherwise defined, the terms used in the present invention have the meanings commonly understood by those skilled in the art.

[0143] The embodiments described in the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various other substitutions, changes and improvements within the scope of the present invention. Therefore, the present invention is not limited to the above embodiments, but is only limited by the claims.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, X-L1-A-NH(CH2CH2O) n -L2-D Formula (I) in, X represents a reactive group, and the reactive group is -ONH2; L1 and L2 each independently represent a C1-C6 straight or branched alkylene group; A represents a peptide formed by 1 to 4 glycine residues; D represents auristatin toxins MMAE, MMAF or MMAD; n represents 1, 2, or 3.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein The above-mentioned L1 and L2 each independently represent a C1-C3 straight-chain alkylene group.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein The L1 and L2 each independently represent a methylene group or an ethylene group.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein The A represents a dipeptide of -glycine-glycine- or a tripeptide of -glycine-glycine-glycine-.

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein: The compound is one of the following:

6. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof for preparing a drug conjugate.

7. A drug conjugate, which is prepared by reacting the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof with a targeting molecule through the reactive group; wherein: The targeting molecule is trastuzumab with a non-natural amino acid containing a terminal acetyl group inserted at a fixed point, and the drug conjugate is prepared by reacting the carbonyl group in the terminal acetyl group with -ONH2 to form an oxime bond; The non-natural amino acid is a compound having one of the following structures:

8. Use of the drug conjugate according to claim 7 in the preparation of a drug for treating cancer, wherein the cancer is gastric cancer.

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