TW202300148A - Preparation method of camptothecin derivatives - Google Patents

Abstract

The present disclosure relates to a preparation method of camptothecin derivatives, and specifically relates to a preparation method of a compound shown as formula (II) including a step of reaction of a compound shown as formula (III) and a compound shown as formula (IV), wherein definition of each functional group is as described in the specification. The method is high-yield and reaction conditions thereof is mild, and is suitable for commercial manufacture.

Description

The preparation method of camptothecin derivative

The disclosure belongs to the field of medicine, and relates to a preparation method of camptothecin derivatives.

This application claims the priority of the Chinese patent application 202110286025.2 with the filing date of 2021/3/17. This application cites the full text of the above-mentioned Chinese patent application.

Antibody drug conjugate (ADC) connects a monoclonal antibody or antibody fragment with a biologically active cytotoxin through a stable chemical linker compound, making full use of the antibody's specificity and ability to bind to surface antigens on normal cells and tumor cells The high efficiency of cytotoxin avoids defects such as low curative effect of the former and excessive toxicity of the latter. This also means that, compared with traditional chemotherapy drugs in the past, antibody drug complexes can precisely bind tumor cells and reduce the impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329–332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

There are several types of cytotoxic small molecules used in antibody-drug complexes, one of which is camptothecin derivatives, which have anti-tumor effects by inhibiting topoisomerase I. It is reported that the camptothecin derivative exitecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl- 1H,12H-Benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione) applied to antibody The literature on drug complex (ADC) includes WO2014057687 and so on. Among them, Exitecan is used in a large amount as the toxin part and is expensive.

WO2020063676, WO2020063673, CN105829346A, CN111689980A, CN112125915A, etc. disclose a series of exixitecan analogs and preparation methods thereof.

The purpose of the present disclosure is to provide a new preparation method of camptothecin derivatives.

其中， R選自氫原子或羧基保護基； R ₁、R ₂各自獨立地選自氫原子、C _1-6烷基、3-6元環烷基、6-10元芳基或5-10元雜芳基，其中所述的烷基、環烷基、芳基、雜芳基任選被一個或多個選自C ₁-C ₆烷基、鹵素、羥基、胺基、氧代、3-6元環烷基、6-10元芳基或C ₁-C ₆烷氧基的取代基所取代， 或者，R ₁和R ₂與其相連接的碳原子一起形成任選被一個或多個選自C ₁-C ₆烷基、鹵素、羥基、胺基、氧代或C ₁-C ₆烷氧基的取代基所取代的3-6元環烷基； R ₃、R ₄、R ₅各自獨立地選自氫原子或C _1-6烷基； n為2至8的整數； m為0至4的整數。 One aspect of the present disclosure provides a method for preparing a compound represented by formula (II), comprising the step of reacting a compound represented by formula (III) with a compound represented by formula (IV),

Wherein, R is selected from hydrogen atom or carboxyl protecting group; R ₁ and R ₂ are each independently selected from hydrogen atom, C _1-6 alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl or 5-10 Elementary heteroaryl, wherein said alkyl, cycloalkyl, aryl, and heteroaryl are optionally replaced by one or more selected from C ₁ -C ₆ alkyl, halogen, hydroxyl, amino, oxo, 3 - Substituents of 6-membered cycloalkyl, 6-10 membered aryl or C ₁ -C ₆ alkoxyl, or, R ₁ and R ₂ are formed together with the carbon atom to which they are connected, optionally by one or more A 3-6 membered cycloalkyl group substituted by a substituent selected from C ₁ -C ₆ alkyl, halogen, hydroxyl, amino, oxo or C ₁ -C ₆ alkoxy; R ₃ , R ₄ , R ₅ each independently selected from a hydrogen atom or a C _1-6 alkyl group; n is an integer from 2 to 8; m is an integer from 0 to 4.

In certain embodiments, the carboxyl protecting group is selected from methyl, substituted methyls, ethyl, 2-substituted ethyls, allyl, tertiary butyl, alkoxyalkyls, Alkoxyalkoxyalkyls, 2,6-dialkylphenyls, benzyls, substituted benzyls, silanes or stannyls; The substituted methyl group is selected from 9-fluorenylmethyl, triisopropylsilylmethyl, cyclopropylmethyl, diphenylmethyl or triphenylmethyl; The 2-substituted ethyl group is selected from 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(p-toluenesulfonyl)ethyl or 2-cyano Ethyl; The alkoxyalkyl group is selected from methoxymethyl, benzyloxymethyl or triisopropylsilyloxymethyl; The alkoxyalkoxyalkyl group is selected from methoxyethoxymethyl; The 2,6-dialkylphenyls are selected from 2,6-dimethylphenyl, 2,6-diisopropylphenyl or 2,6-two tertiary butyl-4-methoxy phenyl; The substituted benzyl group is selected from p-methylbenzyl, 2,4-dimethoxybenzyl, 2,6-dimethoxybenzyl, p-nitrobenzyl or o-nitrobenzyl; The silyl group is selected from trimethylsilyl, triethylsilyl, triisopropylsilyl or phenyldimethylsilyl; The stannyl group is selected from trimethylstannyl; Preferably methyl, allyl, tertiary butyl, benzyl, 2,4-dimethoxybenzyl, p-methylbenzyl, pentafluorophenyl or methoxyethoxymethyl, more preferably 2, 4-Dimethoxybenzyl.

The reaction described is a condensation reaction, and the condensing agent that forms an amide bond includes but is not limited to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N , N '-di Cyclohexylcarbodiimide, N , N '-diisopropylcarbodiimide, O-benzotriazole- N , N , N ', N '-tetramethyluronium tetrafluoroborate, 1-Hydroxybenzotriazole, 1-Hydroxy-7-azobenzotriazole, O-benzotriazole- N , N , N ', N '-tetramethyluronium hexafluorophosphate, 2- (7-Azobenzotriazole) -N , N , N ', N '-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino) Phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 4-(4,6-dimethoxytrimethoxy)-4-methylmorphine.

The above-mentioned condensation reaction is preferably carried out under alkaline conditions, and the base is selected from organic bases or inorganic bases, and the organic bases are preferably triethylamine, diethylamine, N , N -diisopropylethylamine (diisopropyl ethylamine), pyridine, sodium hexamethyldisilazide, n-butyllithium, potassium tertiary butoxide or tetrabutylammonium bromide, and the inorganic base is selected from lithium hydroxide, sodium hydroxide, Potassium hydroxide, sodium hydride, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, or cesium carbonate.

In addition, the reaction can also be carried out by using an acyl halide method in which an acyl halide is first prepared by using an acyl halide such as thionyl chloride or oxalyl chloride and a carboxylic acid in the presence of a base.

The solvent used in the reaction can be a conventional solvent, such as water, dimethylformamide, 1-methyl-2-pyrrolidone, tetrahydrofuran, methyltetrahydrofuran, dioxane, toluene, xylene, dimethylsulfoxide , diethyl ether, isopropyl ether, methyl tertiary butyl ether, acetonitrile, propionitrile, C ₁ -C ₆ alkyl alcohol, acetone, ethyl acetate, dichloromethane, chloroform one or more.

In certain embodiments, _R is selected from a hydrogen atom, a C _1-6 alkyl group, a 3-6 membered cycloalkyl group, a 6-10 membered aryl group or a 5-10 membered heteroaryl group, wherein the alkyl group , cycloalkyl, aryl, and heteroaryl are optionally replaced by one or more members selected from C ₁ -C ₆ alkyl, halogen, hydroxyl, amino, oxo, 3-6-membered cycloalkyl, 6-10-membered Aryl or C ₁ -C ₆ alkoxy substituents, R ₂ is selected from hydrogen atom or C ₁ optionally substituted by one or more substituents selected from halogen, hydroxyl, amino, oxo -C ₆ alkyl, alternatively, R ₁ and R ₂ are formed together with the carbon atom to which they are attached, and are optionally replaced by one or more selected from C ₁ -C ₆ alkyl, halogen, hydroxyl, amino, oxo or C ₁ A 3-6 membered cycloalkyl group substituted by a substituent of -C ₆ alkoxy group.

In certain embodiments, R ₁ is selected from C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkyl substituted by 3-6 membered cycloalkyl, C substituted by 6-10 membered aryl _1-6 alkyl, 3-6-membered cycloalkyl, 6-10-membered aryl or 5-10-membered heteroaryl, R ₂ is a hydrogen atom, or R ₁ and R ₂ together form 3 -6-membered cycloalkyl; preferably R ₁ is selected from C _1-6 alkyl, C _1-6 haloalkyl, 3-6 membered cycloalkyl, R ₂ is a hydrogen atom, or R ₁ and R ₂ are connected to it The carbon atoms are taken together to form a 3-6 membered cycloalkyl group.

In certain embodiments, R ₃ , R ₄ , and R ₅ are all hydrogen atoms.

。 In certain embodiments, R is a carboxyl protecting group, and the method further includes the following step of deprotecting the group,

.

其中，R選自甲基、烯丙基、三級丁基、苄基、2,4-二甲氧基苄基、對甲基苄基、五氟代苯基或甲氧乙氧甲基，優選2,4-二甲氧基苄基。 In some embodiments, the method includes

Wherein, R is selected from methyl, allyl, tertiary butyl, benzyl, 2,4-dimethoxybenzyl, p-methylbenzyl, pentafluorophenyl or methoxyethoxymethyl, Preference is given to 2,4-dimethoxybenzyl.

。 In some embodiments, the method also includes

.

， 其中，R為羧基保護基，R ₁、R ₂、R ₃、R ₄、R ₅、m、n如前所述。 The present disclosure also provides a preparation method of the compound shown in formula (II-1), comprising the step of decarboxylation protecting group of the compound shown in formula (II),

, wherein, R is a carboxyl protecting group, and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , m, and n are as described above.

In some embodiments, the step of decarboxylation protecting group is carried out in the presence of acid, which includes common inorganic and organic acids, including but not limited to hydrochloric acid, acetic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, oxalic acid, citric acid acid, benzenesulfonic acid, substituted benzenesulfonic acid, benzoic acid, substituted benzoic acid, maleic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, dichloroacetic acid, difluoroacetic acid, etc. Substituted benzenesulfonic acid or benzoic acid means that benzenesulfonic acid or benzoic acid is selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, halogen, cyano, One or more substituents in hydroxyl and amino groups are substituted.

In some embodiments, the molar ratio of the compound represented by the formula (II) to the acid can be 1:1-1:100, preferably 1:1-1:30, more preferably 1:1-1: 10.

The solvent used in the reaction can be a conventional solvent, such as water, dimethylformamide, 1-methyl-2-pyrrolidone, tetrahydrofuran, methyltetrahydrofuran, dioxane, toluene, xylene, dimethylsulfoxide , diethyl ether, isopropyl ether, methyl tertiary butyl ether, acetonitrile, propionitrile, C ₁ -C ₆ alkyl alcohol, acetone, ethyl acetate, dichloromethane, chloroform one or more.

其中，R ₆選自氫原子、氘原子、C _1-6烷基、6-10元芳基或5-10元雜芳基，其中所述的烷基、芳基、雜芳基任選被一個或多個選自C ₁-C ₆烷基、鹵素、羥基、胺基和氧代的取代基所取代，優選R ₆選自氫原子、C _1-6烷基，鹵代C _1-6烷基或羥基C _1-6烷基，更優選氫原子； R為羧基保護基，R ₁、R ₂、R ₃、R ₄、R ₅、m、n如前所述。 The present disclosure also provides a preparation method of camptothecin derivatives, including the step of reacting the compound shown in formula (II-1) with the compound shown in formula (V) to prepare the compound shown in formula (I), and also including the step of preparing the compound shown in formula (II) ) the step of deprotecting the compound shown in the compound shown in the formula ((II-1),

Wherein, R is selected from a hydrogen atom, a deuterium atom, a C _1-6 alkyl group, a 6-10 membered aryl _group or a 5-10 membered heteroaryl group, wherein the alkyl group, aryl group, and heteroaryl group are optionally replaced by One or more substituents selected from C ₁ -C ₆ alkyl, halogen, hydroxyl, amino and oxo are substituted, preferably R ₆ is selected from hydrogen atom, C _1-6 alkyl, halogenated C _1-6 Alkyl or hydroxy C _1-6 alkyl, more preferably a hydrogen atom; R is a carboxyl protecting group, and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , m, and n are as described above.

The reaction of the compound represented by the formula (II-1) with the compound represented by the formula (V) is a condensation reaction, and the condensing agent is as described above. The solvent used in the reaction can be a conventional solvent, such as water, dimethylformamide, 1-methyl-2-pyrrolidone, tetrahydrofuran, methyltetrahydrofuran, dioxane, toluene, xylene, dimethylsulfoxide , diethyl ether, isopropyl ether, methyl tertiary butyl ether, acetonitrile, propionitrile, C ₁ -C ₆ alkyl alcohol, acetone, ethyl acetate, dichloromethane, chloroform, preferably dimethyl Formamide, 1-methyl-2-pyrrolidone, methanol, ethanol, dichloromethane, chloroform, dichloromethane/methanol, dichloromethane/ethanol, etc. In addition, the reaction can also be carried out by using an acyl halide method in which an acyl halide is first prepared by using an acyl halide such as thionyl chloride or oxalyl chloride and a carboxylic acid in the presence of a base.

In some embodiments, the method further includes the method for preparing the compound represented by formula (II) described in the present disclosure.

其中，R選自甲基、烯丙基、三級丁基、苄基、2,4-二甲氧基苄基、對甲基苄基、五氟代苯基或甲氧乙氧甲基，優選2,4-二甲氧基苄基。 In some embodiments, the method includes

Wherein, R is selected from methyl, allyl, tertiary butyl, benzyl, 2,4-dimethoxybenzyl, p-methylbenzyl, pentafluorophenyl or methoxyethoxymethyl, Preference is given to 2,4-dimethoxybenzyl.

， 其中，R、R ₁、R ₂、R ₃、R ₄、R ₅、m、n如前所述。 The present disclosure also provides a compound shown in formula (II):

, wherein, R, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , m, n are as described above.

。 The present disclosure also provides a compound represented by formula (IIb),

.

。 The present disclosure also provides a compound represented by formula (IIa-1),

.

其中Ab為抗體或抗原結合片段，k為1至20（包括1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意兩數值之間任意數值）， R ₁、R ₂、R ₃、R ₄、R ₅、R ₆、m、n如前所述。 The present disclosure also provides a method for preparing an antibody-drug complex, comprising: the step of preparing the compound represented by formula (I) described in the present disclosure, and after reduction of Ab, coupling reaction with the compound represented by formula (I) to obtain The step of the antibody-drug complex shown in formula (X),

Wherein Ab is an antibody or an antigen-binding fragment, k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any value between any two values), R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , m, n are as described above.

The reducing agent is preferably TCEP, and in particular, preferably reduces disulfide bonds on antibodies.

In certain embodiments, the antibody is selected from chimeric antibodies, humanized antibodies or fully human antibodies; preferably monoclonal antibodies.

In some embodiments, wherein said antibody or antigen-binding fragment thereof is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti- HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, Anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, or anti-Mesothelin antibody or an antigen-binding fragment thereof.

In certain embodiments, the antibody or antigen-binding fragment thereof is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumab, or an antigen-binding fragment thereof .

In certain embodiments, k is 2-8, preferably 5-9. Non-limiting examples include 3, 4, 5, 6, 7.2, 7.5, 8, 8.5, 9.

。 In some embodiments, the method includes

.

The antibody-drug complexes described in the present disclosure can be prepared by referring to known methods, for example, WO2020063676 discloses a series of preparation methods of antibody-drug complexes, which are incorporated herein in full.

The preparation method of camptothecin derivatives described in the present disclosure has high reaction yield and lower cost, and is more suitable for industrial production. The side chain reaction conditions are mild, effectively avoiding the impurities generated by the decomposition of the side chain. The synthesis method of the existing technology is likely to cause the side chain to be broken and decomposed during the preparation process to generate impurities (such as impurity 1, etc.), affecting product quality.

The "alkyl" in the present disclosure is preferably a C ₁ -C ₆ alkyl.

The "alkenyl" in the present disclosure is preferably a C ₂ -C ₆ alkenyl.

The "alkynyl" in the present disclosure is preferably a C ₂ -C ₆ alkynyl.

The "alkylene" in the present disclosure is preferably a C ₁ -C ₆ alkylene.

The "alkenylene group" described in the present disclosure is preferably a C ₂ -C ₆ alkenylene group.

The "alkynylene group" described in the present disclosure is preferably a C ₂ -C ₆ alkynylene group.

The "alkoxy" in the present disclosure is preferably C ₁ -C ₆ alkoxy.

The "alkylsulfide group" described in the present disclosure is preferably a C ₁ -C ₆ alkylsulfide group.

The "cycloalkyl" in the present disclosure is preferably 3 to 12 membered, more preferably 3 to 6 membered cycloalkyl.

The "fused cycloalkyl group" described in the present disclosure is preferably 6 to 14 membered, more preferably 7 to 10 membered fused cycloalkyl group.

The "heterocyclic group" described in the present disclosure is preferably a 3- to 12-membered heterocyclic group, more preferably a 3- to 6-membered heterocyclic group.

The "fused heterocyclic group" described in the present disclosure is preferably 6 to 14 membered, more preferably 7 to 10 membered condensed heterocyclic group.

The "aryl group" described in the present disclosure is preferably a 6- to 14-membered aryl group, more preferably a 6- to 10-membered aryl group.

The "heteroaryl" in the present disclosure is preferably 5 to 12 membered, more preferably 5 to 10 membered heteroaryl.

Unless stated to the contrary, the terms used in the specification and claims for invention have the following meanings.

The term "antibody-drug complex" refers to the linking of a ligand with a biologically active drug through a stable linker unit. In this disclosure, "antibody drug conjugate" (antibody drug conjugate, ADC) refers to linking a monoclonal antibody or antibody fragment with a biologically active glucocorticoid through a stable linker unit. Wherein the antibody or antibody fragment can be combined with the glucocorticoid molecule containing the linker through a specific group (such as an interchain disulfide bond).

The term "drug loading" refers to the average amount of drug carried by each antibody-drug complex molecule in the population of antibody-drug complexes, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The range of drug loading can be 1-20, preferably 1-10 glucocorticoids (D) linked to each antibody (Ab). In the embodiments of the present disclosure, the drug loading is expressed as k, which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean of any two values in between. Preferably 1-10, more preferably 1-8, or 2-8, or 2-7, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or 4-5 mean value. The average amount of drug per ADC molecule after conjugation can be characterized by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assay, capillary electrophoresis analysis (CE-SDS) and HPLC.

The term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are µ chain, δ chain, γ chain, α chain, and ε chain. The same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa chains or lambda chains by difference in the constant region. Each of the five Ig classes can have either a kappa chain or a lambda chain.

The sequence of about 110 amino acids near the N-terminal of the heavy and light chains of the antibody varies greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminal are relatively stable and are the constant region. The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. The three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.

The term "murine antibody" in this disclosure refers to an antibody prepared using a mouse according to the knowledge and skill in the art. In preparation, test subjects are injected with the specified antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.

The term "chimeric antibody" is an antibody that fuses the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then select and clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody according to the need, and then clone the variable region gene of the mouse. The region gene is connected with the human constant region gene to form a chimeric gene and then inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.

The term "humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), refers to the antibody variable region framework grafted with mouse CDR sequences to humans, that is, different types of human germline antibodies Antibodies generated in the framework sequences. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, Found in E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid decreased immunogenicity and decreased activity, minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequence to maintain activity. The humanized antibody of the present disclosure also includes the humanized antibody after affinity maturation of CDR by phage display. Further descriptions of methods involving the use of mouse antibodies in humanization include, for example, Queen et al., Proc., Natl. 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].

The term "fully human antibody", "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", the variable region and constant region of the antibody are all human, and the immunogen sex and side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The present disclosure is a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), gene transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc. .

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of full-length antibodies can be utilized to perform the antigen-binding function of the antibody. Examples of binding fragments encompassed by "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') ₂ fragments comprising (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally via A synthetic linker is a combination of two or more separate CDRs joined. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, recombinant methods can be used to link them via a synthetic linker, making it possible to produce a single protein in which the VL and VH regions pair to form a monovalent molecule chain (referred to as single-chain Fv (scFv); see, eg, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those of ordinary skill in the art to which the invention pertains, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibody can be of a different isotype, eg, an IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibody.

Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating an IgG antibody molecule with papain protease (cleaving the amino acid residue at position 224 of the H chain), wherein the H chain N About half of the end sides and the entire L chain are held together by disulfide bonds.

F(ab')2 is an antibody fragment having a molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions connected at the hinge position obtained by pepsin digestion of the lower portion of the two disulfide bonds in the IgG hinge region .

Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.

In addition, the Fab' fragment can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryote expression vector or a eukaryote expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.

The term "single-chain antibody", "single-chain Fv" or "scFv" is meant to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker molecules. Such scFv molecules may have the general structure: _NH2 -VL-linker-VH-COOH or _NH2 -VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA90: 6444-6448 ). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001 ), Cancer Immunol.

The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDRs applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and to CDR1, CDR2, L3 of the heavy chain variable domain. CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3). Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. Amino acid sequence boundaries for CDRs can be determined using any of a variety of well-known schemes, including "Sequence Boundaries, Package Numbering Conventions" (see Kabat et al. (1991), "991) and see "Which to determine which to determine proteins of immunological in, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "," hesda numbering convention (see Al-Lazikani et al., (1997) JMB 273:927-948) and ImMunoGenTics (IMGT ) numbering convention (see Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)) and the like. For example, for the classical format, following the Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) ; CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Following the Chothia rule, the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1 ), 50-52 (LCDR2) and 91-96 (LCDR3). Defined by combining the CDRs of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acids in human VL Residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) constitute. Following the IMGT rules, the numbering of CDR amino acid residues in VH is approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the numbering of CDR amino acid residues in VL is approximately 27 -32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3). Following the IMGT rules, the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.

The term "antibody framework" refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen-binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

The terms "specifically bind", "selectively bind", "selectively bind" and "specifically bind" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, antibodies bind with an affinity (KD) of less than about 10 ^"7M , eg, about less than 10 ^"8M , 10 ^"9M or ^10" 10M or less.

The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plastid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or can integrate into the genome of the host cell after introduction, thereby following The host genome replicates together (eg, non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as Cold Spring Harbor's Antibody Laboratory Technique Guide, Chapters 5-8 and Chapter 15. Antigen-binding fragments can also be prepared by conventional methods. In the antibody or antigen-binding fragment described in the invention, one or more human FR regions are added to the non-human CDR region by genetic engineering methods. The human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from Immunoglobulin Journal, 2001ISBN012441351 get.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant or animal cells. Bacteria that are readily transformed include members of the enterobacteriaceae such as strains of Escherichia coli or Salmonella; the Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.

Antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Positive colonies are expanded in serum-free media in bioreactors for antibody production. The culture fluid from which the antibody has been secreted can be purified by conventional techniques. For example, use an A or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. The bound antibody was then eluted by the pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.

Amino acid sequence "identity" means that the amino acid sequences are aligned and gaps are introduced as necessary to achieve the maximum percent sequence identity, and that any conservative substitutions are not considered part of the sequence identity, the first sequence with The percentage of amino acid residues that are identical to the amino acid residues in the second sequence. For purposes of determining percent amino acid sequence identity, alignment can be accomplished in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 Or Megalign (DNASTAR) software. Those skilled in the art to which this invention pertains can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a linear or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, second-butyl, n-pentyl, 1,1-dimethylpropyl , 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl- 2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1 ,3-Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl , 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-di Methylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl , 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4 -Ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3 -Ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary butyl, di Butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl Base, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2- Dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methyl Amylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, said substituents being preferably one or more of the following groups independently selected from alkyl radical, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkane Oxy group, heterocycloalkoxy group, cycloalkylthio group, heterocycloalkylthio group, oxo group, carboxyl group or carboxylate group.

The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably containing 3 to 12 carbon atoms, more preferably containing 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Base, cyclooctyl, etc.; polycyclic cycloalkyl includes spiro ring, fused ring and bridged ring cycloalkyl. "Carbocycle" refers to the ring system in a cycloalkyl group.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) _m (where m is an integer from 0 to 2), excluding ring portions of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. Preferably it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably it contains 3 to 6 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidine Piperyl, piperyl, morpholinyl, thiomorpholinyl, homopiperyl, etc., preferably piperidinyl and pyrrolidinyl. Polycyclic heterocyclyls include spiro, fused and bridged heterocyclyls. "Heterocycle" refers to a ring system in a heterocyclyl group.

和

； 芳基可以是取代的或非取代的，當被取代時，取代基優選為一個或多個以下基團，其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基、羧基或羧酸酯基，優選苯基。 The term "aryl" refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) group, preferably 6 to 10 membered, having a conjugated π-electron system, such as benzene base and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring bonded to the parent structure is the aryl ring. "Aromatic ring" refers to a ring system in an aryl group. Non-limiting examples of aryl groups include:

and

; aryl may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio , alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, Heterocycloalkylthio, carboxyl or carboxylate, preferably phenyl.

和

。 The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 12 membered, such as imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyridyl group, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring bonded to the parent structure is a heteroaryl ring. "Heteroaryl" refers to a ring system in a heteroaryl group. Non-limiting examples of heteroaryl include:

and

.

Heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio group, heterocycloalkylthio group, carboxyl group or carboxylate group.

"Carboxyl protecting group" is a suitable group known in the art for carboxyl protection, see the literature ("Protective Groups in Organic Synthesis", 5 ^Th Ed. TW Greene & PGM Wuts) in the carboxyl protecting group, as For example, the carboxyl protecting group can be a substituted or unsubstituted C _1-10 straight chain or branched chain alkyl, a substituted or unsubstituted C _2-10 straight chain or branched alkenyl or alkynyl, a substituted Or unsubstituted C _3-8 cyclic alkyl, substituted or unsubstituted C _5-10 aryl or heteroaryl, or (C _1-8 alkyl or aryl) ₃ silyl, etc.

"Amino-protecting groups" are suitable groups known in the art for the protection of amine groups, see Amine Protection in the literature ("Protective Groups in Organic Synthesis", 5 ^Th . Ed. TW Greene & PGM Wuts) Group, preferably, the amino protecting group can be (C _1-10 alkyl or aryl) acyl group, for example: formyl, acetyl, benzoyl etc.; can be (C _{1 -6} alkyl or C _6-10 aryl) sulfonyl; also can be (C _1-6 alkoxy or C _6-10 aryloxy) carbonyl, for example: Boc or Cbz; can also be substituted or Unsubstituted alkyl such as trityl (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).

"Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, a "heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes cases where the heterocycle group is substituted with an alkyl group and cases where the heterocycle group is not substituted with an alkyl group .

”並未指定構型，即如果化學結構中存在構型異構，鍵“

”可以為“

”或“

”，或者同時包含“

”和“

”兩種構型。本公開所述化合物的化學結構中，鍵“

”並未指定構型，即可以為Z構型或E構型，或者同時包含兩種構型。 In the chemical structures of the compounds described in this disclosure, the bond "

"No configuration is specified, i.e. if there is configurational isomerism in the chemical structure, the bond"

"can be"

"or"

, or both "

"and"

"Two configurations. In the chemical structures of the compounds described in this disclosure, the bond"

" does not specify the configuration, it can be the Z configuration or the E configuration, or both configurations.

The present disclosure will be explained in detail below in conjunction with specific examples, so that those skilled in the art to which the present invention pertains can more fully understand that the specific examples of the present disclosure are only used to illustrate the technical solutions of the present disclosure, and do not limit the present disclosure in any way.

Compound structures were determined by nuclear magnetic resonance (NMR) or/and mass spectroscopy (MS). NMR shifts (δ) are given in units of 10 ^-6 (ppm). The determination of NMR was carried out with a Bruker AVANCE-400 nuclear magnetic instrument, and the determination solvents were deuterated dimethyl sulfide (DMSO- d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD), and the internal standard was four Methylsilane (TMS).

MS was determined with a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC e2695-2489 high pressure liquid chromatography were used for high performance liquid chromatography (HPLC) analysis.

Chiral HPLC analysis and determination using Agilent 1260 DAD high performance liquid chromatography.

High-performance liquid phase preparation uses Waters 2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson-281 preparative chromatographs.

Chiral preparation A Shimadzu LC-20AP preparative chromatograph was used.

The CombiFlash rapid preparation instrument uses Combiflash Rf200 (TELEDYNE ISCO).

The HPLC detection conditions for impurity 1 are: detection chromatographic column is Sunfire C18 3.5µm 4.6mm*75mm, mobile phase: TFA/MeCN/H ₂ O, detection wavelength: 214 nm. Retention time of impurity 1: 6.96 min.

TLC silica gel plates use Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications of the silica gel plates used in thin-layer chromatography (TLC) are 0.15 mm to 0.2 mm, and the specifications of TLC separation and purification products are 0.4 mm. ~0.5 mm.

Silica gel column chromatography generally uses Yantai Huanghai silica gel 200~300 mesh silica gel as the carrier. Kinase average inhibition rate and IC ₅₀ value were measured with a NovoStar microplate reader (BMG, Germany).

The known starting materials of the present disclosure can be adopted or synthesized according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc), Darui Chemicals and other companies.

Unless otherwise specified in the examples, the reactions can all be carried out under an argon atmosphere or a nitrogen atmosphere.

The argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a volume of about 1 L.

The hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

The pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and Qinglan QL-500 hydrogen generator or HC2-SS hydrogenator.

The hydrogenation reaction is usually vacuumized and filled with hydrogen, and the operation is repeated 3 times.

For the microwave reaction, a CEM Discover-S 908860 microwave reactor was used.

Unless otherwise specified in the examples, the solution refers to an aqueous solution.

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

將化合物IVc (800 mg，1.39 mmol，根據WO2020063676公開的方法製備)溶於二氯甲烷(4 mL)，加入二乙胺(2 mL)，在氬氣保護下室溫攪拌至反應結束；10℃下，減壓濃縮除去有機溶劑，殘餘物中加入正己烷(10 mL)，振盪後靜置，倒去上層清液，重複三次，最後減壓濃縮至幹，得到化合物IVb粗品。將上述粗品溶於DMF (10 mL)，加入IIIa (658 mg，1.39 mmol，根據WO2020063676公開的方法製備)，DMTMM (462 mg，1.67 mmol)，室溫攪拌至反應結束，在反應液中加入水(60 mL)淬滅，乙酸乙酯萃取，合併有機相，依次用水(30 mL×2)，飽和氯化鈉溶液洗滌(30 mL×2)，無水硫酸鈉乾燥，過濾，減壓濃縮，所得殘餘物柱層析（矽膠預先用含0.2%TEA的DCM溶液鹼化，濕法上樣，流動相為甲醇和含0.05%TEA的DCM溶液，0~100:1），濃縮，殘餘物加入乙醚(10 mL)，充分振盪後靜置，倒去上層清液；重複兩次，減壓濃縮至幹，得到化合物IIb (875 mg，收率77.9%，HPLC純度98%)。 ¹H NMR (500 MHz, DMSO- d ₆) δ 8.53 (t, J= 6.8 Hz, 1H), 8.28 (d, J= 6.0 Hz, 1H), 8.11 (d, J= 8.0 Hz, 1H), 8.08 (d, J= 6.0 Hz, 1H), 8.01 (d, J= 5.9 Hz, 1H), 7.25 (m,  5H), 7.19 (d, J= 6.8 Hz, 1H), 6.99 (s, 2H), 6.58 (s, 1H), 6.51 (d, J= 8.3 Hz, 1H), 5.10 (d, J= 12.0 Hz, 1H), 5.01 (d, J= 12.0 Hz, 1H), 4.63 – 4.48 (m, 3H), 3.78 (s, 3H), 3.76 (s, 3H), 3.70 (m, 5H), 3.59 (m, 1H), 3.37 (t, J= 7.2 Hz, 2H), 3.06 (m, 1H), 2.80 (m, 1H), 2.11 (t, J= 7.5 Hz, 2H), 1.47 (m, 4H), 1.26 – 1.14 (m, 3H), 1.01 (m, 1H), 0.45 (d, J= 8.4 Hz, 2H), 0.38 – 0.31 (m, 2H). MS: m/z 829.3 [M+Na] ⁺. Example 1

Dissolve compound IVc (800 mg, 1.39 mmol, prepared according to the method disclosed in WO2020063676) in dichloromethane (4 mL), add diethylamine (2 mL), and stir at room temperature under argon until the reaction is complete; 10°C The residue was concentrated under reduced pressure to remove the organic solvent, and n-hexane (10 mL) was added to the residue, shaken and left to stand, and the supernatant liquid was poured off. This was repeated three times, and finally concentrated to dryness under reduced pressure to obtain the crude compound IVb. Dissolve the above crude product in DMF (10 mL), add IIIa (658 mg, 1.39 mmol, prepared according to the method disclosed in WO2020063676), DMTMM (462 mg, 1.67 mmol), stir at room temperature until the reaction is complete, add water to the reaction solution (60 mL), extracted with ethyl acetate, combined organic phases, washed with water (30 mL×2), saturated sodium chloride solution (30 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain Residue column chromatography (silica gel was pre-basified with DCM solution containing 0.2% TEA, wet loading, mobile phase was methanol and DCM solution containing 0.05% TEA, 0~100:1), concentrated, and the residue was added to diethyl ether (10 mL), fully shaken and left to stand, poured off the supernatant; repeated twice, concentrated to dryness under reduced pressure to obtain compound IIb (875 mg, yield 77.9%, HPLC purity 98%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 8.53 (t, J = 6.8 Hz, 1H), 8.28 (d, J = 6.0 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 6.0 Hz, 1H), 8.01 (d, J = 5.9 Hz, 1H), 7.25 (m, 5H), 7.19 (d, J = 6.8 Hz, 1H), 6.99 (s, 2H), 6.58 (s, 1H), 6.51 (d, J = 8.3 Hz, 1H), 5.10 (d, J = 12.0 Hz, 1H), 5.01 (d, J = 12.0 Hz, 1H), 4.63 – 4.48 (m, 3H) , 3.78 (s, 3H), 3.76 (s, 3H), 3.70 (m, 5H), 3.59 (m, 1H), 3.37 (t, J = 7.2 Hz, 2H), 3.06 (m, 1H), 2.80 ( m, 1H), 2.11 (t, J = 7.5 Hz, 2H), 1.47 (m, 4H), 1.26 – 1.14 (m, 3H), 1.01 (m, 1H), 0.45 (d, J = 8.4 Hz, 2H ), 0.38 – 0.31 (m, 2H). MS: m/z 829.3 [M+Na] ⁺ .

在反應瓶中加入化合物IIb (3.46 g，4.29 mmol)，反應在冰浴下降溫至0~5℃，然後加入三乙基矽烷(1.49 g，12.81 mmol，2 mL)和140 mL二氯乙酸的DCM溶液(2%，v/v)，保溫攪拌至反應結束。在反應液中緩慢滴加乙醚(420 mL)，加畢，混合物在室溫下打漿攪拌30分鐘，過濾；濾餅用乙醚(120 mL)在室溫繼續打漿30分鐘，過濾；濾餅繼續用乙醚(120 mL)室溫打漿30分鐘，過濾，減壓乾燥，得到目標化合物IIa-1 (2.84 g，收率100%，HPLC純度92.05%，其中雜質1為0.98%)。 ¹H NMR (500 MHz, DMSO- d ₆) δ 12.56 (s, 1H), 8.51 (t, J= 6.9 Hz, 1H), 8.28 (t, J= 6.0 Hz, 1H), 8.11 (d, J= 8.1 Hz, 1H), 8.09 – 8.04 (m, 1H), 8.00 (t, J= 5.9 Hz, 1H), 7.23 (m, 4H), 7.19 (d, J= 6.8 Hz, 1H), 7.00 (s, 2H), 4.62 – 4.49 (m, 3H), 3.79 – 3.66 (m, 4H), 3.60 (m, 2H), 3.45(d, J= 7.6 Hz, 1H), 3.41 – 3.38 (m, 1H), 3.08 (m, 1H), 2.80 (dd, J= 13.8, 9.8 Hz, 1H), 2.11 (t, J= 7.4 Hz, 2H), 1.48 (m, 4H), 1.26 – 1.15 (m, 3H), 1.03 (q, J= 6.5 Hz, 1H), 0.46 (m, 2H), 0.36 (m, 2H). MS: m/z 679.0 [M+Na] ⁺. Example 2

Compound IIb (3.46 g, 4.29 mmol) was added to the reaction flask, and the reaction was cooled to 0~5°C in an ice bath, then triethylsilane (1.49 g, 12.81 mmol, 2 mL) and 140 mL of dichloroacetic acid were added. DCM solution (2%, v/v), insulated and stirred until the end of the reaction. Diethyl ether (420 mL) was slowly added dropwise to the reaction solution. After the addition was complete, the mixture was beaten and stirred at room temperature for 30 minutes, and filtered; the filter cake was continuously beaten with ether (120 mL) for 30 minutes at room temperature, and filtered; Diethyl ether (120 mL) was beaten at room temperature for 30 minutes, filtered, and dried under reduced pressure to obtain the target compound IIa-1 (2.84 g, yield 100%, HPLC purity 92.05%, of which impurity 1 was 0.98%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 12.56 (s, 1H), 8.51 (t, J = 6.9 Hz, 1H), 8.28 (t, J = 6.0 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 8.09 – 8.04 (m, 1H), 8.00 (t, J = 5.9 Hz, 1H), 7.23 (m, 4H), 7.19 (d, J = 6.8 Hz, 1H), 7.00 (s, 2H), 4.62 – 4.49 (m, 3H), 3.79 – 3.66 (m, 4H), 3.60 (m, 2H), 3.45(d, J = 7.6 Hz, 1H), 3.41 – 3.38 (m, 1H), 3.08 (m, 1H), 2.80 (dd, J = 13.8, 9.8 Hz, 1H), 2.11 (t, J = 7.4 Hz, 2H), 1.48 (m, 4H), 1.26 – 1.15 (m, 3H), 1.03 ( q, J = 6.5 Hz, 1H), 0.46 (m, 2H), 0.36 (m, 2H). MS: m/z 679.0 [M+Na] ⁺ .

在反應瓶中加入鹽酸依喜替康 (1.45 g，3.07 mmol)和DMF (44 mL)，反應在冰浴下降至0~5℃，然後加入三乙胺(466 mg，4.61 mmol，642µL)，反應在冰浴保護下攪拌，將化合物IIa-1 (2.82 g，4.29 mmol)溶於DMF (12 mL)後，加入至上述反應液中，再加入DMTMM (1.27 g，4.59 mmol)，自然升至室溫攪拌至反應結束。在反應液中加入DCM-MeOH溶液(DCM:MeOH=10：1，200 mL)，水(180 mL)，混合物在室溫攪拌，靜置分層；水相用DCM-MeOH溶液(DCM:MeOH=10：1)萃取，合併有機相，有機相用水洗，無水硫酸鈉乾燥，過濾，減壓濃縮，所得殘餘物柱層析(combiflash，展開劑：DCM:MeOH=10:1)，得到較純的目標產物化合物Ia  (1.08 g，收率33%，純度97.02%)和部分粗品，粗品再次柱層析得到較純的目標產物化合物Ia  (813 mg，收率25%，純度94.62%)。 Example 3

Exitecan hydrochloride (1.45 g, 3.07 mmol) and DMF (44 mL) were added into the reaction flask, and the reaction was cooled to 0-5 °C in an ice bath, then triethylamine (466 mg, 4.61 mmol, 642 µL) was added, The reaction was stirred under the protection of an ice bath. After dissolving Compound IIa-1 (2.82 g, 4.29 mmol) in DMF (12 mL), it was added to the above reaction solution, and then DMTMM (1.27 g, 4.59 mmol) was added and naturally rose to Stir at room temperature until the reaction is complete. DCM-MeOH solution (DCM:MeOH=10:1, 200 mL) and water (180 mL) were added to the reaction solution, the mixture was stirred at room temperature, and the layers were left to stand; =10:1) extraction, combined organic phase, organic phase washed with water, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, the resulting residue column chromatography (combiflash, developing solvent: DCM:MeOH=10:1), obtained a relatively Pure target product compound Ia (1.08 g, yield 33%, purity 97.02%) and part of the crude product, the crude product was re-column chromatography to obtain relatively pure target product compound Ia (813 mg, yield 25%, purity 94.62%).

Since this disclosure has been described in terms of specific embodiments thereof, certain modifications and equivalents will be apparent to those skilled in the art to which this invention pertains and are intended to be within the scope of this disclosure.

none

Claims (23)

A method for preparing the compound shown in formula (II), comprising the step of reacting the compound shown in formula (III) with the compound shown in formula (IV),

Wherein, R is selected from hydrogen atom or carboxyl protecting group; R ₁ and R ₂ are each independently selected from hydrogen atom, C _1-6 alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl or 5-10 Elementary heteroaryl, wherein said alkyl, cycloalkyl, aryl, and heteroaryl are optionally replaced by one or more selected from C ₁ -C ₆ alkyl, halogen, hydroxyl, amino, oxo, 3 - Substituents of 6-membered cycloalkyl, 6-10 membered aryl or C ₁ -C ₆ alkoxyl, or, R ₁ and R ₂ are formed together with the carbon atom to which they are connected, optionally by one or more A 3-6 membered cycloalkyl group substituted by a substituent selected from C ₁ -C ₆ alkyl, halogen, hydroxyl, amino, oxo or C ₁ -C ₆ alkoxy; R ₃ , R ₄ , R ₅ each independently selected from a hydrogen atom or a C _1-6 alkyl group; n is an integer from 2 to 8; m is an integer from 0 to 4. According to the preparation method described in claim 1, wherein the carboxyl protecting group is selected from methyl, substituted methyl, ethyl, 2-substituted ethyl, allyl, tertiary butyl, alkoxy Alkyl, alkoxyalkoxyalkyl, 2,6-dialkylphenyl, benzyl, substituted benzyl, silane or stannyl, preferably methyl, allyl , tertiary butyl, benzyl, 2,4-dimethoxybenzyl, p-methylbenzyl, pentafluorophenyl or methoxyethoxymethyl, more preferably 2,4-dimethoxybenzyl base. According to the preparation method described in Claim 1 or 2, the condensing agent used in the reaction is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N , N '- Dicyclohexylcarbodiimide, N , N '-diisopropylcarbodiimide, O-benzotriazole- N , N , N ', N '-tetramethyluronium tetrafluoroborate , 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole- N , N , N ', N '-tetramethyluronium hexafluorophosphate, 2 -(7-Azobenzotriazole) -N , N , N ', N '-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytri(dimethylamino )phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 4-(4,6-dimethoxytrimethoxy)-4-methylmorphine one or more of. According to the preparation method described in any one of claims 1 to 3, wherein the reaction is carried out under alkaline conditions, the base is selected from organic bases or inorganic bases, and the organic bases are preferably triethylamine and diethylamine , N , N -Diisopropylethylamine (Diisopropylethylamine), Pyridine, Sodium Hexamethyldisilazide, n-BuLi, Potassium Butoxide or Tetrabutylammonium Bromide , the inorganic base is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium hydride, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate or cesium carbonate. According to the preparation method described in any one of claims 1 to 4, wherein, _R is selected from a hydrogen atom, a C _1-6 alkyl group, a 3-6 membered cycloalkyl group, a 6-10 membered aryl group or a 5-10 membered Heteroaryl, wherein said alkyl, cycloalkyl, aryl, and heteroaryl are optionally replaced by one or more selected from C ₁ -C ₆ alkyl, halogen, hydroxyl, amino, oxo, 3- Substituted by a 6-membered cycloalkyl group, a 6-10-membered aryl group or a C ₁ -C ₆ alkoxy group, R ₂ is selected from a hydrogen atom or is optionally selected from one or more halogen, hydroxyl, amino, A C ₁ -C ₆ alkyl group substituted by an oxo substituent, or, R ₁ and R ₂ are formed together with the carbon atom to which they are connected, and are optionally replaced by one or more selected from C ₁ -C ₆ alkyl, halogen, 3-6 membered cycloalkyl substituted by hydroxyl, amino, oxo or C ₁ -C ₆ alkoxy substituents; preferably R ₁ is selected from C _1-6 alkyl, C _1-6 haloalkyl, 3 -6-membered cycloalkyl substituted C _1-6 alkyl, 6-10 membered aryl substituted C _1-6 alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl or 5-10 membered hetero Aryl, R ₂ is a hydrogen atom, or R ₁ and R ₂ together form a 3-6-membered cycloalkyl group with the carbon atom it is connected to; more preferably R ₁ is selected from C _1-6 alkyl, C _1-6 haloalkyl , 3-6-membered cycloalkyl group, R ₂ is a hydrogen atom, or R ₁ and R ₂ form a 3-6-membered cycloalkyl group together with the carbon atoms they are connected to. The preparation method according to any one of claims 1-5, wherein R ₃ , R ₄ , and R ₅ are all hydrogen atoms. According to the preparation method described in any one of claims 1 to 6, wherein R is a carboxyl protecting group, the method also includes the step of deprotecting the compound shown in formula (II-1),

. According to the preparation method described in any one of claims 1-7, wherein the method comprises

Wherein, R is selected from methyl, allyl, tertiary butyl, benzyl, 2,4-dimethoxybenzyl, p-methylbenzyl, pentafluorophenyl or methoxyethoxymethyl, Preference is given to 2,4-dimethoxybenzyl. According to the preparation method described in claim 8, wherein said method also includes

. A preparation method of camptothecin derivatives, comprising the step of reacting the compound shown in the formula (II-1) with the compound shown in the formula (V) to prepare the compound shown in the formula (I), and also including removing the compound shown in the formula (II) Protecting group The step of preparing the compound shown in formula ((II-1), wherein, R is _selected from hydrogen atom, deuterium atom, C _1-6 alkyl, 6-10 yuan of aryl or 5-10 yuan of heteroaryl, The alkyl, aryl, and heteroaryl groups mentioned therein are optionally substituted by one or more substituents selected from C ₁ -C ₆ alkyl, halogen, hydroxyl, amino, and oxo, preferably R ₆ is selected from Hydrogen atom, C _1-6 alkyl, halogenated C _1-6 alkyl or hydroxy C _1-6 alkyl, more preferably hydrogen atom; R is a carboxyl protecting group, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , m, n are as described in Claim 1,

. According to the preparation method described in Claim 10, the method further includes the preparation method of the compound represented by formula (II) described in any one of Claims 1-9. According to the preparation method described in claim 10 or 11, the method comprises

Wherein, R is selected from methyl, allyl, tertiary butyl, benzyl, 2,4-dimethoxybenzyl, p-methylbenzyl, pentafluorophenyl or methoxyethoxymethyl, Preference is given to 2,4-dimethoxybenzyl. A method for preparing an antibody-drug complex, comprising the steps of the method for preparing a camptothecin derivative described in any one of claim items 10 to 12, and after Ab reduction, a coupling reaction with a compound represented by formula (I), The step of obtaining the antibody-drug complex shown in formula (X),

Where Ab is an antibody or an antigen-binding fragment, k is 1 to 20, and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , m, and n are as described in claim 1. According to the preparation method described in claim 13, wherein the antibody is selected from chimeric antibodies, humanized antibodies or fully human antibodies; preferably monoclonal antibodies. According to the preparation method described in claim item 13 or 14, wherein said antibody or antigen-binding fragment thereof is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 ( ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody , anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 Antibodies or anti-Mesothelin antibodies or antigen-binding fragments thereof. According to the preparation method described in any one of claims 13-15, wherein the antibody or antigen-binding fragment thereof is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 , Glematumamab, or an antigen-binding fragment thereof. The preparation method according to any one of claims 13-16, wherein k is 2-8, preferably 5-9. The preparation method according to any one of claims 13-17, wherein the method comprises

. A preparation method of the compound shown in formula (II-1), comprising the step of decarboxylation protecting group of the compound shown in formula (II),

, wherein, R is a carboxyl protecting group, and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , m, and n are as described in Claim 1. According to the preparation method described in claim item 19, wherein the step of decarboxylation protecting group is carried out in the presence of an acid, the acid is selected from hydrochloric acid, acetic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, oxalic acid, citric acid, benzenesulfonic acid, substituted One or more of benzenesulfonic acid, benzoic acid, substituted benzoic acid, maleic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, dichloroacetic acid, difluoroacetic acid. A compound shown in formula (II):

, wherein, R, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , m, n are as described in Claim 1. According to the compound described in claim item 21, it is a compound shown in formula (IIb),

. According to the compound described in claim 21, it is a compound shown in formula (IIa-1),

.