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CN118754934B - A toxin derivative for preparing drug conjugates - Google Patents

A toxin derivative for preparing drug conjugates Download PDF

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Publication number
CN118754934B
CN118754934B CN202410723672.9A CN202410723672A CN118754934B CN 118754934 B CN118754934 B CN 118754934B CN 202410723672 A CN202410723672 A CN 202410723672A CN 118754934 B CN118754934 B CN 118754934B
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trastuzumab
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compound
pharmaceutically acceptable
acceptable salt
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CN118754934A (en
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霍鹏超
梁学军
夏钢
叶诚浩
杨金纬
王谦
丁文
刘利
曹锴
黄浩
靳婷
祝静静
应跃斌
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Zhejiang New Code Biomedical Co ltd
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract

本发明提供了一种如式(Ⅰ)所示的毒素衍生物或其立体异构体、药学上可接受的盐以及其用于制备药物偶联物的用途。本发明还提供了一种药物偶联物及其用途。本发明提供的毒素衍生物通过调整连接子的结构来连接奥瑞他汀类毒素，形成了全新的连接子‑效应小分子，用于制备药物偶联物时能够取得非常优异的生物活性和高效的细胞内吞效果，有利于扩大奥瑞他汀类偶联物的设计、生产和应用，具有十分重要的经济和社会价值，X——L1——A——NH(CH2CH2O)n——L2——D。式(Ⅰ)The present invention provides a toxin derivative or its stereoisomer as shown in formula (Ⅰ), a pharmaceutically acceptable salt thereof, and the use thereof 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 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——L 1 ——A——NH(CH 2 CH 2 O) n ——L 2 ——D. Formula (Ⅰ)

Description

Toxin derivative for preparing drug conjugate
Technical Field
The invention relates to the field of medicines, in particular to a toxin derivative for preparing a drug conjugate and application thereof, and further relates to a drug conjugate and application thereof.
Background
Drug conjugates with targeted delivery are an emerging class of therapeutic approaches that evolve with drug development toward precise therapies. The drug conjugate consists of targeting molecules which are selectively bound to cells or tissues related to diseases through coupling effector molecules, and the targeting molecules are used for directionally releasing the effector molecules with therapeutic effects. The targeting molecule may be an antibody, antibody fragment, ligand, polypeptide, small molecule, or, for example, a virus-like particle. At present, an antibody-drug conjugate (Antibody Drug Conjugate, ADC) using an antibody as a targeting molecule becomes the main stream of development of next-generation accurate therapeutic drugs, and has wide prospect in the treatment of diseases such as cancers, autoimmune diseases and the like. ADCs are typically composed of four parts, 1) an antibody with targeting properties for recognizing target cells and carrying effector small molecule drugs (usually chemical drugs) to the cell surface or interior, 2) small molecules with cytotoxicity or other effects to kill cells or regulate cell function, 3) a Linker (Linker) connecting the antibody and drug for connection and support, and 4) a Linker for coupling the Linker to the antibody with a specific active group.
Antibodies and effector small molecules are currently being studied more, however, in ADCs, linkers and linkers also play a very important role. Linkers can be categorized into non-cleavable linkers and cleavable linkers in terms of their properties. The non-cleavage type linker has stable chemical bond composition, is more stable than the cleavage type linker, has an action mechanism based on that an antibody-antigen complex of the ADC is endocytosed by cells, and then the ADC is degraded by lysosomes to release a payload containing the linker, thereby exerting the effect of killing tumor cells. Therefore, the non-cleavage type linker has the advantages that the ADC using the linker is not easy to release medicine in blood circulation, has low off-target toxicity and is safer. In addition, ADCs using non-cleaving linkers can alter the chemical properties of small molecule drugs by the linkers, enhancing their metabolic properties or potency, as the released payload carries the intact linkers.
In the market and clinical development, the types of ADCs using non-cleavable linkers are relatively few, and more mature are thioether linkers, amide linkers, etc., such as Roche's Kadcyla are used to attach DM1 to antibodies via stable thioether linkers.
Through reasonable design of the connector, the optimization of ADC molecules can be realized, for example, the physicochemical property, the pharmacokinetic property, the pharmacodynamic property and the toxicity property of the ADC can be optimized, or the design of high DAR value can be realized, and the introduction of different kinds of effect small molecules can be further realized.
The coupling mode of the antibody and the effector small molecule also directly influences the DAR value and the uniformity of the ADC product, and further influences the physicochemical property, the biological activity, the in-vivo curative effect and the toxicity of the antibody and the effector small molecule. Site-directed coupling of ADCs is one of the currently attracting attention research focus, and Unnatural Amino Acids (UAAs) with specific active groups can be introduced into antibody molecules through genetic engineering, so that effective control over DAR values and product uniformity of ADCs can be achieved.
Disclosure of Invention
To overcome the disadvantages of 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 (ADC), by adjusting the structure of the linker, and can achieve excellent bioactivity and endocytic effect.
It is another object of the present invention to provide a drug conjugate and its use.
In a first aspect, the present invention provides a compound of formula (I) or a stereoisomer, pharmaceutically acceptable salt thereof,
XL1ANH(CH2CH2O)nL2D
Formula (I)
Wherein X represents a reactive group protected or unprotected by a protecting group;
L 1、L2 each independently represents a C1 to C6 linear or branched alkylene group;
a represents a peptide formed by 1 to 4 amino acids selected from glycine and/or alanine;
D represents an auristatin toxoid;
n represents 1,2 or 3.
For drug conjugates, the structure and physicochemical properties of the linker have a very large influence on the stability of the drug, the metabolism of the drug, the efficacy, etc., for example, the coupling efficiency and DAR value, the transmembrane property of the effector small molecule, the stability of the conjugate, the pharmacokinetic properties of the effector small molecule, etc., and for example, the structural residue of the linker on the effector small molecule can influence the effector response of the effector small molecule, and for example, the various physicochemical properties of the linker can also influence the production process and the drug formation of the conjugate.
In the compound shown in the formula (I), a brand-new linker structure is formed by amino acid residues and PEG units with specific numbers, specific types and specific connection sequences, and the conjugate formed by connecting the targeting molecule (such as an antibody) and the auristatin toxoid has excellent bioactivity and efficient endocytosis effect.
In the compound shown in the formula (I), X can represent any active reactive group common in the field, and can react with a reactive group (can be a group contained in a targeting molecule or a group carried by the targeting molecule after modification) on the targeting molecule (such as an antibody) to form the targeting molecule-drug conjugate. X can be an unprotected group or a group protected by a protecting group common in the field, and the corresponding protecting group is removed during the reaction.
In some embodiments, X may represent a hydroxy amine group, an amino group, a hydroxy group, a mercapto group, a halogen atom, an azido group, a carbonyl group, a dicarbonyl group, an ester group, or a cycloalkynyl group, which may be protected with a protecting group or not. In some preferred embodiments, the X represents a hydroxylamine group, i.e., "NH 2 -O-".
In some embodiments, the L 1、L2 may each independently represent a C1-C3 linear alkylene group, e.g., methylene (-CH 2 -), ethylene (-CH 2-CH2 -), propylene (-CH 2-CH2-CH2 -). In some preferred embodiments, the L 1、L2 may each independently represent a methylene or ethylene group.
In some embodiments, the a may represent a dipeptide of "-glycine-", a dipeptide of "-glycine-alanine-", a dipeptide of "-alanine-", or a tripeptide of "-glycine-". In some preferred embodiments, the structure of a may be: where "1" indicates a connection point with L 1 and "2" indicates a connection point at the other end.
In some embodiments, the D may represent any of the auristatin (Auristatins) toxins common in the art, which are artificially synthesized derivatives of the natural product dolastatin dolastatin, which are antimitotics that promote cell cycle arrest and apoptosis by acting on the β -subunit of the α - β tubulin dimer to block tubulin polymerization. The auristatin toxoids include, but are not limited to MMAE, MMAF, MMAD and the like. In some preferred embodiments, D may be represented as MMAE or MMAF as shown below, with the secondary amine groups of the terminal groups dehydrogenized to other moieties, so D may also be referred to as residues of MMAE or MMAF after removal of the hydrogens of the terminal secondary amine groups.
In some most preferred embodiments, the compound may be one of the following:
a second aspect of the invention provides the use of a compound according to any one of the preceding claims, or a stereoisomer, pharmaceutically acceptable salt thereof, for the preparation of a drug conjugate (e.g. an antibody-drug conjugate).
The compound or stereoisomer and pharmaceutically acceptable salt thereof in any one of the technical schemes provided by the invention can be coupled with any targeting molecule such as an antibody, an antibody fragment, a ligand, a polypeptide, a targeting protein and the like which are common in the field through any coupling mode common in the field to form a conjugate. In some preferred embodiments, the antibody may be an anti-HER 2 mab, such as trastuzumab.
In a third aspect, the present invention provides a drug conjugate, which is prepared by coupling the compound according to any one of the above technical schemes or a stereoisomer or a pharmaceutically acceptable salt thereof with a targeting molecule through the reactive group (i.e. X).
In the antibody-drug conjugate provided by the invention, the compound or the stereoisomer and the pharmaceutically acceptable salt thereof in any one of the technical schemes can be coupled with any targeting molecule such as an antibody, a polypeptide, a targeting protein and the like which are common in the field through the reactive group to form the drug conjugate through any coupling mode common in the field. In some preferred embodiments, the antibody may be an anti-HER 2 mab, such as trastuzumab.
In some embodiments, the targeting molecule is trastuzumab with site-directed insertion of an unnatural amino acid containing a terminal carbonyl group, the reactive group is a hydroxylamine group, an oxime bond is formed by reacting the terminal carbonyl group with the hydroxylamine group, and thus a drug conjugate is prepared, and the oxime bond formed by reacting the carbonyl group with the hydroxylamine group is as follows:
In some embodiments, the unnatural amino acids 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 with one of the following structures:
In some embodiments, subsequent site-directed coupling can be achieved by introducing unnatural amino acids into a designated site of the targeting molecule through a codon extension technique (e.g., the codon extension technique is implemented in E.coli). In some preferred embodiments, the amino acid sequences of the heavy and light chains of 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.
A fourth aspect of the present invention provides the use of a drug conjugate according to any one of the preceding claims in the manufacture of a medicament for the treatment of cancer.
In some embodiments, the cancer may be gastric cancer or breast cancer.
In some embodiments, the drug conjugates may be used alone or in combination with other anti-tumor drug or drugs.
The toxin derivative provided by the invention is connected with the auristatin toxoid through adjusting the structure of the linker, so that a novel linker-effect small molecule derivative is formed, and when the toxin derivative is used for preparing a drug conjugate, the toxin derivative can obtain very excellent bioactivity and high-efficiency endocytosis effect, is beneficial to expanding the design, production and application of the auristatin conjugate, and has very important economic and social values.
Drawings
FIG. 1 is a map of the expression plasmid pCDNA3.1-Trastuzumab-UAG142 in example 3.
Detailed Description
In the present invention, "C1 to Cn" includes C1 to C2, C1 to Cn, for example, the "C1-C6" group means that the moiety has 1-6 carbon atoms, i.e., a group contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.
The compounds or conjugates of the invention may contain asymmetric or chiral centers and thus exist in different stereoisomers. It is contemplated that all stereoisomeric forms of the compounds or conjugates of the invention, including but not limited to diastereomers, enantiomers, sterically hindered isomers and geometric (conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the invention. Unless otherwise indicated, structures described herein also include all stereoisomers (e.g., diastereomers, enantiomers, sterically hindered isomers and geometric (conformational) isomeric forms of such structures, e.g., R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, sterically hindered isomers of biphenyl structures (see, basic organic chemistry (second edition) upper book, xing Jiyi et al ,p104-105);PAC,1996,68,2193.(Basic terminology of stereochemistry(IUPAC Recommendations 1996,on page 2201))、(Z) and (E) conformational isomers.
The term "pharmaceutically acceptable salt" as used herein, alone or in combination, refers to salts of the compounds or conjugates of the present invention that retain the biological effectiveness and properties of the free acid or free base by reaction with a non-toxic inorganic or organic base. Available using standard procedures well known in the art. 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).
The technical scheme of the invention is further described in detail below with reference to specific embodiments.
The unnatural amino acid NBGK, NPAK, NBOK used in the examples of the invention is prepared by referring to Chinese patent ZL 202111019771.1, and the comparison toxins P4AE and P4AF used are prepared by referring to Chinese patent ZL 201810957127.0, and the structure is as follows:
Toxin MMAE (monomethyl auristatin E) used in the examples was purchased from Nanjing Biopharmaceutical Co., ltd, lot number LN217-41-1, MMAF-OtBu (MMAF: monomethyl auristatin F) was purchased from BINING (Suzhou) Biopharmaceutical Co., ltd, lot number LN507-38, and other materials or reagents were commercially available unless otherwise specified.
In the embodiments of the invention, the eluent proportions are all volume proportions, and the percentages referred to are mass percentages unless otherwise specified.
Example 1 preparation of Compound GPAE
GPAE has the structural formula shown below:
the reaction process is shown in the following formula:
the preparation process comprises the following steps:
glycine linker moiety (1-6) is prepared, PEG2-MMAE (2-3) moiety is prepared, and then the two moieties 1-6 and 2-3 are condensed to obtain the target compound GPAE.
A) In a reaction flask, methyl glycine hydrochloride (1.67 g,10 mmol) was dissolved in a mixed solution of DCM/water (50/20 mL), the vessel was placed in an ice bath, K 2CO3 (4.15 g,30 mmol) and bromoacetyl bromide (2.42 g,12 mmol) were added, 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 phases concentrated and recrystallized (PE: EA=10:1), filtered to give white crystals 1-1 (1.70 g, yield 68%).
B) The product 1-1 (1.67 g,6.6 mmol) of the previous step was placed in a reaction flask, solvent DCM (20 mL) was added, the vessel was placed in an ice bath, trifluoroacetic acid (5 mL) was added, stirring was performed at room temperature for 18 hours, TLC detection was complete, the mixture was rotary evaporated with a water bath, most of the solvent was removed, water was added 30mL, and the product 1-2 was obtained by freeze drying for use.
C) In a reaction flask, N- (2-bromoethyl) phthalimide (6.0 g,24 mmol), tert-butyl N-hydroxycarbamate (4.8 g,36 mmol) and K 2CO3 (8.3 g,60 mmol) were added, solvent DMF (60 mL) was added, stirred at room temperature for 18 hours, TLC was monitored to completion, a suitable amount of water was added to the mixture, extracted 3 times with EA, the organic phases were combined, washed 3 times with water, dried over anhydrous Na 2SO4, concentrated, column chromatographed (eluent PE: EA=10:1 and 2:1) to give white solid 1-3 (3.45 g, yield 47%).
D) In a reaction flask, the product of the previous step 1-3 (0.5 g,1.64 mmol) was dissolved in ethanol (20 mL), hydrazine hydrate (80%, 410. Mu.L, 6.56 mmol) was added, the vessel refluxed for 18 hours at 80℃and TLC was complete to detect completion of the reaction, cooled down and left to stand to precipitate a white precipitate, filtered off, the filter cake was washed with ethanol, the filtrate was concentrated and column chromatographed (eluent DCM: meOH=10:1) to give 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]+).
E) In a reaction flask was added the product 1-4 from the previous step (3.67 g,20.8 mmol), the product 1-2 from step b) (3.4 g,17.3 mmol) and the solvent methanol (60 mL), followed by DIPEA (15 mL,87 mmol), stirred at 70℃under reflux for 18 hours, concentrated directly, slurried with EA and filtered to give a white solid 1-5 (5.56 g, without purification, used directly in the next step).
F) Calculated in 50% yield, crude 1-5 (504 mg) from the previous step was dissolved in water (5 mL), triethylamine (0.72 mL,5.2 mmol) and Boc anhydride (0.94 g,4.3 mmol) in dioxane (5 mL) were added, stirred at room temperature for 18 hours, concentrated first to remove most of the dioxane, extracted 3 times with DCM, the organic phases were combined and concentrated, and column chromatography (eluent DCM: meOH=10:1 and 5:1) afforded 1-6 (2877 mg; LCMS: m/z (ESI+) calcd:391.20, found:392.15[ M+H ] +;414.15[M+Na]+) as a white solid.
G) In a reaction flask, substrate 2-0 (100 mg,0.4mmol, available from Bifide. Under the trade name BD 261696), dissolved in DMSO (3 mL) and 2-iodoxybenzoic acid (IBX, 224mg,0.8 mmol) were added and stirred at room temperature for 18 hours, TLC monitoring (2, 4-dinitrophenylhydrazine development) was complete, the mixture was washed with water, EA extracted 3 times, dried over anhydrous Na 2SO4 and concentrated, column chromatography (eluent DCM: meOH=20:1) gave product 2-1 (50 mg, yield 50.5%).
H) In a reaction flask, the product of the previous step 2-1 (50 mg,0.2 mmol) and MMAE (57 mg,0.08 mmol) were dissolved in dichloroethane (5 mL), the vessel was placed in an ice bath and stirred at room temperature for 18 hours after adding sodium triacetoxyborohydride (STAB, 51mg,0.24 mmol), TLC monitoring (phosphomolybdic acid development) was complete, the mixture was washed with water, extracted 3 times with DCM, dried over anhydrous Na 2SO4 and concentrated, column chromatography (eluent DCM: meOH=20:1) gave 2-2 (79 mg, yield) as a colorless oil 99％;LCMS:m/z(ESI+)calcd:948.65;Found:475.65[M/2+H]+,949.80[M+H]+,971.80[M+Na]+).
I) In a reaction flask, the product of the previous step 2-2 (79 mg,0.08 mmol) was dissolved in DCM (2 mL), trifluoroacetic acid (1 mL) was added and stirred at room temperature for 3 hours, TLC monitoring (phosphomolybdic acid development) was complete, the mixture was concentrated at room temperature, DCM was added, the above procedure was repeated 3 times to remove excess trifluoroacetic acid, and finally water was added to freeze-dry to give the product 2-3 (67.9 mg, 99% yield).
J) In a reaction flask, product 1-6 (50 mg,0.13 mmol) and solvent DCM (5 mL) were added, then HATU (60.8 mg,0.16 mmol), DIPEA (57. Mu.L, 0.32 mmol) were added, stirred for 30 min at RT, then product 2-3 of the previous step (67.9 mg,0.08 mmol) was added, stirred at RT for 18 h, TLC monitored (phosphomolybdic acid development) was complete, the mixture was washed with water first, extracted 3 times with DCM, dried over anhydrous Na 2SO4, concentrated, column chromatography (eluent DCM: meOH=15:1) to give white solid 2-4 (63 mg; LCMS: m/z (ESI+) calcd:1221.78, found: 612.25M/2+H ] +).
K) In a reaction flask, the product of the previous step 2-4 (63 mg) was dissolved in dioxane (10 mL), hydrochloric acid (5 mL) was slowly added, stirring was performed at room temperature for 3 hours, TLC monitored for completion of the reaction, the mixture was concentrated at room temperature to remove a portion of the solvent, and water was added to freeze-dry to give the final product GPAE (25.6 mg, yield 48.6%) as a white solid.
LCMS:m/z(ESI+)calcd:1021.68,Found:512.20[M/2+H]+,1046.40[M+Na]+。
1 H-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＝3.7Hz,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).
Example 2 preparation of Compound GPAF
GPAF has the structural formula shown below:
the reaction process is shown in the following formula:
the preparation process comprises the following steps:
Wherein compounds 1-6 were prepared as in example 1.
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), sodium bicarbonate (1.7 g,20.1 mmol) and water (10 mL) were added, reacted at room temperature for 24 hours, concentrated to remove part of dioxane, extracted 3 times with EA, the organic phases were combined, dried over anhydrous Na 2SO4, concentrated, and column chromatographed (eluent PE: EA=1:3) to give colorless oil 3-1 (2.2 g, yield 88%).
B) In a reaction flask, add the product of the previous step 3-1 (150 mg,0.41 mmol), dissolve with DMSO (5 mL), add 2-iodoylbenzoic acid (IBX, 280mg,0.82 mmol), stir at room temperature for 18 hours, TLC monitor (2, 4-dinitrophenylhydrazine color development) reaction completion, wash the mixture with water first, EA extract 3 times, dry concentrate with anhydrous Na 2SO4, concentrate, column chromatography (eluent DCM: meOH=20:1) to give colorless oil 3-2 (103 mg, yield 68%; LCMS: m/z (ESI+) calcd:370.15; found:424.15[ M+MeOH+Na ] +).
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 vessel was placed in an ice bath and stirred at room temperature for 18 hours after adding sodium triacetoxyborohydride (STAB, 48mg,0.228 mmol), TLC monitoring (phosphomolybdic acid development) was complete, the mixture was washed with water first, extracted 3 times with DCM, dried over anhydrous Na 2SO4 and concentrated, column chromatography (eluent DCM: meOH=20:1) to give crude 3-3 as a colorless oil, which was used directly in the next step.
D) In a reaction flask, the product 3-3 of the previous step was dissolved in DCM (3 mL), diethylamine (1 mL) was added and stirred at room temperature for 18 hours, TLC was monitored (phosphomolybdic acid development) and the reaction was complete, the mixture was washed with water, extracted 3 times with DCM, dried over anhydrous Na 2SO4 and concentrated, column chromatography (eluent DCM: meOH=15:1) gave 3-4 as a white solid (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]+).
E) In the 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. Mu.L, 0.12 mmol), stirring at room temperature for 15 min, then product 3-4 of the previous step (35 mg,0.09 mmol) and stirring at room temperature for 18 h, TLC monitoring (phosphomolybdic acid development) reaction was complete. The mixture was washed with water, extracted 3 times with DCM, dried over anhydrous Na 2SO4, and concentrated, and column chromatographed (eluent DCM: meoh=30:1) to give 3-5 as a white solid (36 mg, yield 70%).
F) In a reaction flask, the product of the previous step 3-5 (91 mg,0.07 mmol) was dissolved in DCM (4 mL), trifluoroacetic acid (2 mL) was added and stirred at room temperature for 3 hours, TLC monitoring (phosphomolybdic acid development) was complete, the mixture was concentrated at room temperature, DCM was added, the above procedure was repeated 3 times to remove excess trifluoroacetic acid, and finally water was added and lyophilized to give the final product GPAF (69.3 mg, yield 95.7%).
LCMS:m/z(ESI+)calcd:1035.66,Found:518.95[M/2+H]+,1037.10[M+H]+。
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＝3.7Hz,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＝16.1,10.2Hz,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).
Example 3 preparation of antibody-drug conjugates
Unnatural amino acid NBGK, NPAK, NBOK is used to express unnatural amino acid-containing anti-HER 2 mab in eukaryotic expression systems and drug conjugates are prepared.
(1) Acquisition of helper plasmids
Helper plasmid pCMV-MbPylRS, which encodes an aminoacyl tRNA synthetase that specifically recognizes a pyrrolysine-derived unnatural amino acid and the corresponding tRNA (recognizes the amber codon UAG) in mammalian cells, is purchased from plasmid deposit tissue addgene (accession number # 91706).
(2) Construction of an expression vector for an anti-HER 2 antibody (trastuzumab) containing an amber codon within the Gene reading frame
Heavy chain and light chain DNA (corresponding amino acid sequences are SEQ ID NO:1 and SEQ ID NO: 2) of the coded Trastuzumab are synthesized through a total gene synthesis mode, subcloned into a eukaryotic expression vector pCDNA3.1+, and subjected to point mutation to obtain an expression plasmid pCDNA3.1-Trastuzumab-UAG142 with the amino acid codon at the 142 th position of a heavy chain reading frame mutated into an amber codon, wherein the map is shown in figure 1, and the complete sequence is shown in SEQ ID NO:3.
The heavy chain amino acid sequence of trastuzumab (SEQ ID NO: 1) is as follows:
the light chain amino acid sequence of trastuzumab (SEQ ID NO: 2) is as follows:
The gene sequence of the expression plasmid pCDNA3.1-Trastuzumab-UAG142 (SEQ ID NO: 3) is as follows:
(3) Insertion of unnatural amino acids
The suspension-conditioned HEK293 cells were used, inoculated into Wayne293 TM medium (Quacell Biotechnology, cat# A21501) at a density of 0.3X10: 5/mL, the culture was performed by shaking culture using a 1L shaking flask, the amount of liquid was 240mL, at 120rpm, 5% CO 2, 80% humidity, when the cell density reached about 1X 10 6/mL, transfection was performed, the helper plasmid pCMV-MbPylRS described in the steps (1) and (2) and the expression plasmid pCDNA3.1-Trastuzumab-UAG142 were extracted, and endotoxin was removed for later use, each 120. Mu.g of the expression plasmid and helper plasmid was used when each shaking flask was transfected, the plasmid was added into the centrifuge tube, diluted to 7.2mL with 1 XPBS buffer, 720. Mu.g of PEI transfection reagent (polyethylene imine ) was added into the other shaking flask, after the transfection was performed when the cell density reached about 1X 10:6242/mL, the two centrifuge tubes were subjected to 5 minutes, the two shaking tubes were gently mixed liquid was gently mixed with the shaking flask, and the shaking flask was further cooled to a concentration of 10.5 mL, and the shaking flask was continuously added for the culture was stopped at 120 h under the shaking flask of 120.5% CO buffer, and the medium was continuously stirred until the concentration of the shaking flask was not reached at 120.5% CO buffer, and the medium was continuously cooled until the medium was not reached, and the medium was cultured until the medium was cultured.
(4) Purification
Purifying the cell culture supernatant by using HiTrap Protein A and 1mL pre-packed column, wherein the elution buffer is 100mmol/L glycine, 200mmol/L acetate and the pH is 3.5, so as to obtain the purified trastuzumab inserted with unnatural amino acid. Trastuzumab defy orders inserted NBGK, NPAK, NBOK was trastuzumab-NBGK, trastuzumab-NPAK, and trastuzumab-NBOK, respectively.
(5) Coupling of
The synthetic route is shown below (taking trastuzumab-NBGK and GPAE coupling reactions as examples):
Respectively performing fixed-point coupling on toxins (4 AE and P4 AF) containing an ammoxidation terminal group and the purified trastuzumab- (trastuzumab-modified by the non-natural amino acid) with the amino acid sequence in the N-terminal to C-terminal direction to obtain a trastuzumab-toxin conjugate, wherein the trastuzumab- (trastuzumab-modified by the toxin), the trastuzumab-P4 AE (trastuzumab-modified by the toxin P4 AE), the trastuzumab-P4 AF (trastuzumab-modified by the toxin P4 AF), the trastuzumab- (trastuzumab-modified by the toxin), the trastuzumab- (trastuzumab-P4 AE), the trastuzumab-P4 AE (trastuzumab-modified by the toxin), and the trastuzumab-P4 AE (trastuzumab-modified by the toxin) with the amino acid sequence, trastuzumab-NBOK-P4 AF (trastuzumab-NBOK is modified with toxin P4 AF).
The coupling procedure was performed by mixing purified trastuzumab inserted with unnatural amino acid with GPAE (or GPAF or P4AE or P4 AF) at a molar ratio of 1:12, adjusting pH to 4.0 with 10M acetic acid, shaking on a shaker (25 ℃ C., 200 rpm), sampling for 48h, detecting trastuzumab reaction with toxin by HPLC (HIC-HPLC) using hydrophobic chromatography principle, and displaying the results as toxin-monoclonal antibody coupling ratio (DAR value) as shown in Table 1.
The conjugated mab-toxin conjugate was exchanged through a 50kDa ultrafiltration centrifuge tube to remove unreacted toxin starting material, and the displacement buffer was 20mM histidine buffer (pH 6.5).
The HIC-HPLC analysis conditions were as follows:
mobile phase a (2M ammonium sulfate, 75mM K 2HPO4, pH 7.2±0.2);
Mobile phase B (75 mM K 2HPO4, 25% isopropyl alcohol, pH 7.2±0.2).
The area normalization method calculates the peak area of 1 toxin coupled (DRUG 1), 2 toxins coupled (DRUG 2), and 3 toxins coupled (DRUG 3), and calculates the DAR value. The DAR value calculation formula: DAR value= (drug1+ drug2×2+drug3 x 3)/(drug1+drug2+drug3).
TABLE 1 DAR values for ADC samples
The results show that GPAE, GPAF, P AE and P4AF are both bound to the unnatural amino acids NBGK, NPAK or NBOK of trastuzumab in a site-directed coupled form, and the DAR value after GPAE coupling is higher than P4AE and the DAR value after GPAF coupling is higher than P4AF on different unnatural amino acids. Since each heavy chain of trastuzumab is inserted with 1 unnatural amino acid available for coupling reaction, ideally each mab molecule can be coupled to 2 molecules of toxin, with an ideal DAR value of 2.
EXAMPLE 4 Activity assay
The inhibition of cell proliferation by ADC samples was examined using human gastric cancer cell NCI-N87 (ATCC, cat# CRL-5822) cell line.
The method comprises culturing NCI-N87 cells in RPMI-1640Medium (Gibco, A10491-01) containing 10% fetal bovine serum (Gibco, 10099-141C) at 37deg.C, Cell density was adjusted to the appropriate cell density with cell culture media at 5% carbon dioxide to perform cell plating such that 4000 cells per well, 90 μl/well, medium without cells was added to vehicle control wells, cells alone without drug were added to blank control wells, and cell plates were allowed to stand in a CO 2 incubator (37±1 ℃,5±0.5% CO 2) overnight. all samples (trastuzumab-NBGK-GPAE, trastuzumab-NBGK-GPAF, trastuzumab-NBGK-P4 AE, trastuzumab-NBGK-P4 AF, trastuzumab-NPAK-GPAE, trastuzumab-NPAK-GPAF, trastuzumab-NPAK-P4 AE, trastuzumab-NPAK-P4 AF, trastuzumab-NBOK-GPAE, trastuzumab-NBOK-GPAF, trastuzumab-NBOK-P4 AE, trastuzumab-NBOK-P4 AF, trastuzumab-NBGK, trastuzumab-NPAK, trastuzumab-NBOK) was diluted from a 100nM gradient to 0.05nM at 9 total concentrations of 2 duplicate wells per dilution. the diluted samples were transferred to culture plates of NCI-N87 cells of the established plate, 10. Mu.L of each well, 10. Mu.L of medium was added to the vehicle control well and the blank control well, and after 96 h.+ -. 2h of incubation at 37 ℃ under 5% carbon dioxide, 50. Mu. L Promega CellTiter-Glo detection reagent (Promega, G9242) was added to each well, and after shaking for 2min, the plates were read after standing at room temperature for 10 min. The inhibition ratio (Growth inhibition) of the test sample was calculated by the following formula, i.e., inhibition ratio (%) = (1- (luminescence signal value Compounds of formula (I) -luminescence signal value blank)/(luminescence signal value c ontrol-luminescence signal value blank)) ×100%. inhibition rates of compounds at different concentrations were calculated in Excel and then IC50 was calculated using GRAPHPAD PRISM software. The results are shown in Table 2.
TABLE 2 cell proliferation inhibitory Activity of ADC and antibody samples
The results show that GPAE, GPAF, P AE or P4AF modified trastuzumab with unnatural amino acid had an IC50 value of only a few percent of the latter compared to trastuzumab with unnatural amino acid alone, indicating successful attachment of the toxin small molecule. Compared with P4AE or P4AF modified trastuzumab containing unnatural amino acid, GPAE or GPAF modified trastuzumab containing unnatural amino acid has obviously improved tumor killing effect (inhibition rate).
EXAMPLE 5 endocytic clearance efficiency analysis within cells
The endocytic clearance efficiency of ADC samples in cells was examined using NCI-N87 cell line.
The specific process is as follows:
(1) NCI-N87 cells were observed under a microscope (Olmpus), placed in a biosafety cabinet, the original medium was discarded, 6mL PBS (Biyun Tian, C0221A) was added for rinsing, the solution was discarded, and 2mL of 0.25% trypsin (Gibco, 25200072) was added for digestion for 2min. Digestion was terminated by adding 6mL of RPMI-1640 medium (Gibco, A10491-01) containing 10% FBS (Gibco, 10099-141C), centrifuging to discard the supernatant, adding 1mL of medium, mixing well and taking 100. Mu.L of cell suspension to count in a sterile 1.5mL centrifuge tube.
(2) Mu.L of the resuspended cell suspension after digestion was added to each well of the 12-well plate, 2X 10 5 cells/well, and the culture plates for NCI-N87 were placed in a 5% CO 2, 37℃incubator overnight.
(3) The 12-well plate was removed, the original medium was aspirated, 300. Mu.L of PBS was added to each well, washed twice, and the washed PBS was aspirated.
(4) 300. Mu.L of a 10. Mu.g/mL FITC-labeled (Thermo, F6434) sample solution to be tested was added to each well, the 12-well plate was allowed to bind at 4℃for 60min, 300. Mu.L of PBS was added to each well after the sample solution to be tested was pipetted off, washed twice, and the PBS was pipetted off.
(5) Cells from 1 well were trypsinized and transferred to a flow tube (Corning, 352054), centrifuged, 300 μl of PBS was added to each tube, washed twice, 300 μl of 4% paraformaldehyde (Biolegend, 420801) was added for 20min, centrifuged to remove formaldehyde, 300 μl of PBS was added to each well, washed twice, and washed PBS was removed by suction, and this sample was a 0h time point sample. During formaldehyde fixation, wells of the 12-well plate were incubated in 37℃incubator for a certain period of time after adding 500. Mu.L of complete medium (0.5, 1, 2, 4,6, 8, 16, 24 h), removed, washed twice with PBS, transferred to flow tubes after digestion with pancreatin, 300. Mu.L of PBS was added to each tube, washed twice, centrifuged to discard PBS, 300. Mu.L of 4% paraformaldehyde was added to fix for 20min, fixative was aspirated, 300. Mu.L of PBS was added to each tube, washed twice, and centrifuged to discard washed PBS. 300. Mu.L of FITC-labeled goat anti-Human immunoglobulin Kappa chain secondary antibody (FITC-Goat Anti-Human Kappa antibody, 1. Mu.g/100. Mu.L, southernBiotech, 2061-02) was added to each well and incubated at 4℃for 60min in the absence of light. FITC-Goat Anti-Human Kappa antibody solution was blotted, 300. Mu.L PBS was added to each well, washed twice, and the washed PBS was blotted. After cell collection at all time points was completed, 200 μl FACS buffer (pbs+2% FBS) was added to each tube, and the cells were collected on a flow cytometer (ThermoNxT) and calculating the endocytosis efficiency of the sample to be tested by using the following formula, wherein the endocytosis efficiency is%= (MFI 0h–MFI Time point )/(MFI0h–MFInegtive control)) and 100%. The secondary antibody background group signal value is the negative control.
Ideally, the ADC-receptor complex internalizes in a rapid and efficient manner after the ADC binds to a tumor-associated target. Research on whether there is a difference in the endocytic rate of the ADC has a significant impact on the final optimization of the targeted therapeutic ADC delivery within the cell. The results are shown in tables 3-1 and 3-2, and show that GPAE and GPAF modified trastuzumab containing unnatural amino acid are about 14% -35% higher in endocytosis efficiency than P4AE and P4AF modified trastuzumab containing unnatural amino acid.
TABLE 3-1 endocytic Effect of partial ADC samples (1)
TABLE 3-2 endocytic Effect of partial ADC samples (2)
As can be seen from examples 4 and 5, GPAE and GPAF prepared according to the present invention have different hydrophobicity and spatial structure relative to the existing auristatin toxoids P4AE and P4AF, thereby significantly improving the biological activity and endocytic effect of the formed ADC.
Unless otherwise defined, all terms used herein are intended to have the meanings commonly understood by those skilled in the art.
The described embodiments of the present invention are intended to be illustrative only and not to limit the scope of the invention, and various other alternatives, modifications, and improvements may be made by those skilled in the art within the scope of the invention, and therefore the invention is not limited to the above embodiments but only by the claims.

Claims (8)

1.一种如式(Ⅰ)所示的化合物或其药学上可接受的盐，1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, X-L1-A-NH(CH2CH2O)n-L2-DX-L1-A-NH(CH2CH2O) n -L2-D 式(Ⅰ)Formula (I) 其中，X表示反应性基团，所述反应性基团为-ONH2；Wherein, X represents a reactive group, and the reactive group is -ONH 2 ; L1、L2各自独立地表示C1～C6的直链或支链亚烷基；L 1 and L 2 each independently represent a C1-C6 straight-chain or branched alkylene group; A表示由1～4个甘氨酸所形成的肽；A represents a peptide formed by 1 to 4 glycine residues; D表示奥瑞他汀类毒素MMAE、MMAF或MMAD；D represents auristatin toxins MMAE, MMAF or MMAD; n表示1、2或3。n represents 1, 2 or 3. 2.根据权利要求1所述的化合物或其药学上可接受的盐，其中，所述L1、L2各自独立地表示C1～C3的直链亚烷基。2 . The compound or pharmaceutically acceptable salt thereof according to claim 1 , wherein L 1 and L 2 each independently represent a C1-C3 straight-chain alkylene group. 3.根据权利要求2所述的化合物或其药学上可接受的盐，其中，所述L1、L2各自独立地表示亚甲基或亚乙基。3 . The compound or pharmaceutically acceptable salt thereof according to claim 2 , wherein L 1 and L 2 each independently represent a methylene group or an ethylene group. 4.根据权利要求1所述的化合物或其药学上可接受的盐，其中，所述A表示-甘氨酸-甘氨酸-的二肽或-甘氨酸-甘氨酸-甘氨酸-的三肽。4 . The compound or pharmaceutically acceptable salt thereof according to claim 1 , wherein A represents a dipeptide of -glycine-glycine- or a tripeptide of -glycine-glycine-glycine-. 5.根据权利要求1-4任一项所述的化合物或其药学上可接受的盐，其中，所述化合物为以下之一：5. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the compound is one of the following: 6.权利要求1-5任一项所述的化合物或其药学上可接受的盐用于制备药物偶联物的用途。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. 7.一种药物偶联物，其由权利要求1-5任一项所述的化合物或其药学上可接受的盐通过所述反应性基团与靶向分子进行反应而制得；其中，所述靶向分子为定点插入含有端基乙酰基的非天然氨基酸的曲妥珠单抗，通过端基乙酰基中的羰基与-ONH2反应形成肟键由此制得药物偶联物；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; 所述非天然氨基酸为具有以下结构之一的化合物：The non-natural amino acid is a compound having one of the following structures: 8.权利要求7所述的药物偶联物在制备用于治疗癌症的药物中的用途，所述癌症为胃癌。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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