Preparation method for camptothecin derivative and intermediate thereof

Abstract

The present invention relates to a preparation method for a camptothecin derivative and an intermediate thereof. Specifically, the present invention relates to a preparation method for a compound represented by formula (V) or a salt thereof, comprising a step of preparing a compound represented by formula (II) or a salt thereof from a compound represented by formula (I) or a salt thereof, a step of preparing a compound represented by formula (III) or a salt thereof from the compound represented by formula (II) or the salt thereof, a step of preparing a compound represented by formula (IV) or a salt thereof from the compound represented by formula (III) or the salt thereof, and a step of preparing the compound represented by formula (V) or the salt thereof from the compound represented by formula (IV) or the salt thereof.

Description

A method for preparing camptothecin derivatives and their intermediates Technical Field

[0001] This disclosure pertains to the pharmaceutical field and specifically relates to a method for preparing a camptothecin derivative and its intermediates. Background Technology

[0002] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds. This fully leverages the specificity of antibodies in binding to antigens on the surface of both normal and tumor cells, as well as the high efficiency of cytotoxins, while avoiding the lower efficacy of the former and the excessive toxicity of the latter. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells.

[0003] Several classes of small molecules with cytotoxicity are used in antibody-drug conjugates, one of which is camptothecin derivatives, which have antitumor effects by inhibiting topoisomerase I. Reports on the application of the camptothecin derivative eczemacon (formula (C), 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) in antibody-drug conjugates (ADCs) include WO2014057687, WO2020063676, WO2020063673, CN112125915A, and CN115925796A.

[0004] EP0495432B1 discloses the icherotecan compound and its preparation method, wherein the compound of formula (D) is an important reaction intermediate. CN1104409C discloses an improved preparation method. Summary of the Invention

[0005] Researchers discovered that during the synthesis of the compound shown in route (D) of CN1104409C, a new byproduct I-1 was generated during the oxidation of compound IV-1 to compound V-1. This byproduct affected the quality of the important intermediate D, as well as the production efficiency of the intermediate and even the final product. This disclosure provides a method for reusing the byproduct as an intermediate in the synthesis of camptothecin derivatives, and also provides a synthetic route for the byproduct.

[0006] This disclosure provides, in one aspect, the compound of formula (I) or a salt thereof,

[0007] R1 is selected from halogens; R2 is selected from amino protecting groups.

[0008] This disclosure also provides compounds of formula (II) or salts thereof.

[0009] Wherein, R1 is selected from halogens; R2 is selected from amino protecting groups; R3 is selected from C1-C6 alkyl groups, wherein the alkyl group is optionally replaced by one or more substituents selected from 3-6 membered cycloalkyl groups and 6-10 membered aryl groups.

[0010] This disclosure also provides compounds of formula (III) or salts thereof.

[0011] R1 is selected from halogens; R2 is selected from amino protecting groups.

[0012] In some embodiments, R2 is selected from acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, neopentanoyl, formyl, benzoyl, phthaloyl, 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, and benzyloxycarbonyl. In some embodiments, R2 is selected from acetyl.

[0013] In some implementations, R1 is selected from F.

[0014] In some embodiments, the compound represented by formula (I) or a salt thereof is selected from...

[0015] In some embodiments, the compound represented by formula (II) or a salt thereof is selected from...

[0016] In some embodiments, the compound represented by formula (III) or a salt thereof is selected from...

[0017] This disclosure also provides a method for preparing a compound of formula (V) or a salt thereof, comprising the steps of preparing a compound of formula (II) or a salt thereof from a compound of formula (I), preparing a compound of formula (III) or a salt thereof from a compound of formula (II), preparing a compound of formula (IV) or a salt thereof from a compound of formula (III), and preparing a compound of formula (V) or a salt thereof from a compound of formula (IV).

[0018] Wherein, R1 is selected from halogens; R2 is selected from amino protecting groups; R3 is selected from C1-C6 alkyl groups, wherein the alkyl group is optionally replaced by one or more substituents selected from 3-6 membered cycloalkyl groups and 6-10 membered aryl groups.

[0019] In some embodiments, R1 is selected from halogens; R2 is selected from amino protecting groups; and R3 is selected from C1-C6 alkyl groups, wherein the alkyl group is optionally substituted by one or more substituents selected from 6-10 aryl groups.

[0020] In some embodiments, R1 is selected from halogens; R2 is selected from amino protecting groups; and R3 is selected from C1-C6 alkyl and benzyl groups.

[0021] In some embodiments, the preparation method involves preparing the compound of formula (II) or its salt by an O-alkylation reaction of a carboxylic acid.

[0022] In some embodiments, the alkylating agent used in the preparation method is selected from iodomethane, iodoethane, bromomethane, bromoethane, benzyl bromide, and benzyl chloride. In some embodiments, the alkylating agent used in the preparation method is selected from iodoethane.

[0023] In some embodiments, the alkylating agent used in the alkylation reaction is selected from iodomethane, iodoethane, bromomethane, bromoethane, benzyl bromide, and benzyl chloride. In some embodiments, the alkylating agent used in the alkylation reaction is selected from iodoethane.

[0024] In some embodiments, in the preparation method, the compound of formula (II) or its salt is prepared by a reduction reaction to the compound of formula (III) or its salt.

[0025] In some embodiments, the reducing agent used in the preparation method is selected from diisobutylaluminum hydride (DIABL-H), lithium aluminum hydride (LiAlH4), lithium borohydride (LiBH4), sodium borohydride (NaBH4), potassium borohydride (KBH4), sodium / ethanol (Na / EtOH), trisec-butyllithium borohydride (L-selectride), red aluminum, and sodium borohydride / zinc chloride (NaBH4 / ZnCl2). In some embodiments, the reducing agent used in the preparation method is selected from DIABL-H.

[0026] In some embodiments, the reducing agent used in the step of preparing the compound of formula (III) or its salt by reduction reaction is selected from diisobutylaluminum hydride (DIABL-H), lithium aluminum hydride (LiAlH4), lithium borohydride (LiBH4), sodium borohydride (NaBH4), potassium borohydride (KBH4), sodium / ethanol (Na / EtOH), trisec-butyllithium borohydride (L-selectride), red aluminum, sodium borohydride / zinc chloride (NaBH4 / ZnCl2).

[0027] In some embodiments, the reducing agent used in the step of preparing the compound of formula (III) or its salt by a reduction reaction is selected from DIABL-H.

[0028] In some embodiments, the preparation method involves preparing the compound of formula (III) or its salt by reduction dehydroxylation.

[0029] In some embodiments, the reducing agent used in the preparation method is selected from triethylsilane (Et3SiH), triphenylphosphine / 1,2-diiodoethane (Ph3P / ICH2CH2I), hydrogen / hydrogen iodide (H2 / HI), and sodium iodide / formic acid (NaI / HCOOH). In some embodiments, the reducing agent used in the preparation method is selected from Et3SiH.

[0030] In some embodiments, the reducing agent used in the step of preparing the compound of formula (IV) or its salt by reduction dehydroxylation is selected from triethylsilane (Et3SiH), triphenylphosphine / 1,2-diiodoethane (Ph3P / ICH2CH2I), hydrogen / hydrogen iodide (H2 / HI), sodium iodide / formic acid (NaI / HCOOH).

[0031] In some embodiments, the reducing agent used in the step of preparing the compound of formula (III) or a salt thereof by reduction dehydroxylation to the compound of formula (IV) or a salt thereof is selected from Et3SiH. In some embodiments, in the preparation method, the compound of formula (IV) or a salt thereof is prepared by oxidation to the compound of formula (V) or a salt thereof.

[0032] In some embodiments, the oxidant used in the preparation method is selected from potassium permanganate (KMnO4), cerium ammonium nitrate (CAN), and selenium dioxide (SeO2). In some embodiments, the oxidant used in the preparation method is selected from KMnO4.

[0033] In some embodiments, the oxidant used in the oxidation reaction is selected from potassium permanganate (KMnO4), cerium ammonium nitrate (CAN), and selenium dioxide (SeO2). In some embodiments, the oxidant used in the oxidation reaction is selected from KMnO4.

[0034] In some embodiments, R2 is selected from acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, neopentanoyl, formyl, benzoyl, phthaloyl, 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, and benzyloxycarbonyl. In some embodiments, R2 is selected from acetyl.

[0035] In some implementations, R1 is selected from F.

[0036] In some embodiments, the compound represented by formula (V) or a salt thereof is selected from the compound represented by formula (V-1) or a salt thereof, and the preparation method includes:

[0037] This disclosure also provides a method for preparing the compound of formula (D) or a salt thereof, including the method for preparing the compound of formula (V) or a salt thereof as described in this disclosure.

[0038] This disclosure also provides a method for preparing a compound of formula (C) or a salt thereof, comprising the method for preparing a compound of formula (V) or a salt thereof as described in this disclosure, and / or the method for preparing a compound of formula (D) or a salt thereof as described in this disclosure.

[0039] This disclosure also provides a method for preparing a compound of formula (B) or a salt thereof, comprising at least one of the methods for preparing a compound of formula (V) or a salt thereof, the method for preparing a compound of formula (D) or a salt thereof, and the method for preparing a compound of formula (C) or a salt thereof.

[0040] in,

[0041] L1 is selected from -(C(R) 11 (R) 12 )) n -(CH2) m - 3-6 saturated cycloalkyl and 3-6 saturated heterocycloalkyl, wherein each of the 3-6 saturated cycloalkyl and 3-6 saturated heterocycloalkyl is independently optionally substituted by one or more substituents selected from halogen, hydroxyl, amino and haloalkyl;

[0042] R 11 R 12 Each is independently selected from hydrogen atoms, C1-C6 alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl, and 5-10 membered heteroaryl, wherein the alkyl, cycloalkyl, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino, oxo, 3-6 membered cycloalkyl, 6-10 membered aryl, and C1-C6 alkoxy groups, or R 11 R 12 Together with the carbon atom connected thereto, they form a 3-6 membered cycloalkyl group which may be optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino, oxo and C1-C6 alkoxy groups;

[0043] R 13 Selected from hydrogen atom, deuterium atom, C 1-6Alkyl, 6-10 aryl and 5-10 heteroaryl, wherein the alkyl, aryl and heteroaryl groups are optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino and oxo groups;

[0044] m is 0 or 1, preferably 0; n is 1, 2, 3 or 4, preferably 1;

[0045] p is an integer from 2 to 8.

[0046] In some implementation schemes, R 11 R 12 Each is independently selected from hydrogen atoms, C1-C6 alkyl groups, 3-6 membered cycloalkyl groups, 6-10 membered aryl groups, and 5-10 membered heteroaryl groups, wherein the alkyl, cycloalkyl, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from C1-C6 alkyl groups, halogens, hydroxyl groups, amino groups, and oxo groups, or R 11 R 12 Together with the carbon atom attached thereto, they form a 3-6 membered cycloalkyl group, optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino, oxo, and C1-C6 alkoxy groups.

[0047] In some implementation schemes, R 11 R 12 Each is independently selected from hydrogen atoms, C1-C6 alkyl groups, and 3-6 membered cycloalkyl groups, wherein the alkyl and cycloalkyl groups are optionally substituted by one or more substituents selected from C1-C6 alkyl groups, halogens, hydroxyl groups, amino groups, and oxo groups, or R 11 R 12 Together with the carbon atoms attached to it, they form 3-6 membered cycloalkyl groups.

[0048] In some implementation schemes, R 13 The group is selected from hydrogen atoms, deuterium atoms, and C1-C6 alkyl groups, wherein the alkyl group is optionally substituted by one or more substituents selected from C1-C6 alkyl groups, halogens, hydroxyl groups, amino groups, and oxo groups.

[0049] In some embodiments, the compound represented by formula (B) is selected from,

[0050] This disclosure also provides a method for preparing the antibody-drug conjugate of formula (A), comprising: the step of preparing the compound of formula (B) or a salt thereof as described in this disclosure, and the step of reducing Ab and then coupling it with the compound of formula (B) to obtain the antibody-drug conjugate of formula (A).

[0051] Where Ab is the antibody or 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), L1, R 13 As mentioned before, p.

[0052] In some implementations, the reducing agent may be TCEP, for example, reducing disulfide bonds on an antibody.

[0053] In some embodiments, the antibody is selected from chimeric antibodies, humanized antibodies, or fully human antibodies; for example, monoclonal antibodies.

[0054] In some embodiments, the antibody or its antigen-binding fragment 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 its antigen-binding fragment.

[0055] In some embodiments, the antibody or its antigen-binding fragment is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumamab, or its antigen-binding fragment.

[0056] In some implementations, k is 2 to 8, preferably 5 to 9. Non-limiting embodiments include 3, 4, 5, 6, 7.2, 7.5, 8, 8.5, and 9.

[0057] The compound shown in formula (B) or its salt, and the compound shown in formula (A) can be prepared by referring to the methods disclosed in patents or applications such as CN104755494B, CN111228511A, CN112125915A, CN112512591A, CN115197088A, CN115197234A, CN115925796A, and CN117015549A, which are cited in full here.

[0058] In some embodiments, the preparation method of this disclosure optionally includes a purification step, which includes one or more of column chromatography, solvent slurrying, and recrystallization.

[0059] The salts of the compounds described in this disclosure can be inorganic acid salts and organic acid salts. Inorganic acid salts can be hydrochlorides, sulfates, phosphates, hydrobroms, trifluoroacetates, etc., and organic acids can be formates, acetates, sulfonates, substituted alkyl sulfonates, succinates, maleates, tartrates, citrates, lactates, oxalates, gluconates, fumarates, malonates, malates, etc.

[0060] This disclosure also provides a method for preparing a compound of formula (I) or a salt thereof, comprising the step of preparing a compound of formula (I) or a salt thereof from a compound of formula (IA), wherein the compound of formula (IB) or a salt thereof is prepared by one or more steps:

[0061] R1 is selected from halogens; R2 is selected from amino protecting groups.

[0062] In some embodiments, the compound represented by formula (I) or a salt thereof is selected from the compound represented by formula (I-1) or a salt thereof, and the preparation method includes:

[0063] In some embodiments, the compound represented by formula (I) or a salt thereof is selected from the compound represented by formula (I-1) or a salt thereof, and the preparation method includes:

[0064] In some embodiments, the method for preparing the compound or its salt represented by formula (I) above includes:

[0065] In some embodiments, the compound represented by formula (I) or a salt thereof is selected from the compound represented by formula (I-1) or a salt thereof, and the preparation method includes:

[0066] This disclosure also provides compounds of formula (IB) or salts thereof.

[0067] R1 is selected from halogens; R2 is selected from amino protecting groups.

[0068] This disclosure also provides compounds of formula (IC) or salts thereof.

[0069] R1 is selected from halogens.

[0070] This disclosure also provides compounds of formula (ID) or salts thereof.

[0071] R1 is selected from halogens.

[0072] In some embodiments, the compound represented by formula (IB) or a salt thereof is selected from,

[0073] In some embodiments, the compound represented by formula (IC) or a salt thereof is selected from,

[0074] In some embodiments, the compound represented by formula (ID) or a salt thereof is selected from,

[0075] This disclosure also provides the use of the compound of formula (I) or a salt thereof, the compound of formula (II) or a salt thereof, and the compound of formula (III) or a salt thereof as intermediates for the synthesis of camptothecin derivatives.

[0076] In some embodiments, the camptothecin derivative is selected from the compound shown in formula (C) or a salt thereof, the compound shown in formula (B) or a salt thereof, and the antibody-drug conjugate shown in formula (A).

[0077] In some embodiments, the compound represented by formula (I) or a salt thereof is selected from the compound represented by formula (I-1) or a salt thereof. In some embodiments, the compound represented by formula (II) or a salt thereof is selected from the compound represented by formula (II-1) or a salt thereof. In some embodiments, the compound represented by formula (III) or a salt thereof is selected from the compound represented by formula (III-1) or a salt thereof.

[0078] This disclosure also provides the use of the compound of formula (I) or its salt as an impurity reference for camptothecin derivatives or intermediates thereof.

[0079] In some embodiments, the camptothecin derivative or intermediate thereof is selected from the compound of formula (V) or its salt, the compound of formula (D) or its salt, the compound of formula (C) or its salt, the compound of formula (B) or its salt, and the antibody-drug conjugate of formula (A).

[0080] In some embodiments, the compound of formula (I) or a salt thereof is selected from the compound of formula (I-1) or a salt thereof.

[0081] This disclosure also provides a pharmaceutical composition comprising a camptothecin derivative or an intermediate thereof, and a compound of formula (I) or a salt thereof.

[0082] In some embodiments, the content of the compound represented by formula (I) or its salt in the composition is less than 1%. In some embodiments, the content of the compound represented by formula (I) or its salt in the composition is less than 0.8%, less than 0.5%, less than 0.3%, less than 0.10%, or less than 0.05%. In some embodiments, the content of the compound represented by formula (I) or its salt may be 0.50%, 0.49%, 0.48%, 0.47%, 0.46%, 0.45%, 0.44%, 0.43%, 0.42%, 0.41%, 0.40%, 0.39%, 0.38%, 0.37%, 0.36%, 0.35%, 0.34%, 0.33%, 0.32%, 0.31%, 0.30%, 0.29%, 0.28%, etc. 0.27%, 0.26%, 0.25%, 0.24%, 0.23%, 0.22%, 0.21%, 0.20%, 0.19%, 0.18%, 0.17%, 0.16%, 0.15%, 0.14%, 0.13%, 0.12%, 0.11%, 0.10%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% or lower.

[0083] In some embodiments, the content of the compound represented by formula (I-1) or its salt in the composition is less than 1%. In some embodiments, the content of the compound represented by formula (I-1) or its salt in the composition is less than 0.8%, less than 0.5%, less than 0.3%, less than 0.10%, or less than 0.05%. In some embodiments, the content of the compound represented by formula (I-1) or its salt may be 0.50%, 0.49%, 0.48%, 0.47%, 0.46%, 0.45%, 0.44%, 0.43%, 0.42%, 0.41%, 0.40%, 0.39%, 0.38%, 0.37%, 0.36%, 0.35%, 0.34%, 0.33%, 0.32%, 0.31%, 0.30%, 0.29%, or 0.28%. 0.27%, 0.26%, 0.25%, 0.24%, 0.23%, 0.22%, 0.21%, 0.20%, 0.19%, 0.18%, 0.17%, 0.16%, 0.15%, 0.14%, 0.13%, 0.12%, 0.11%, 0.10%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% or lower.

[0084] This disclosure provides a method for recovering and reusing byproducts generated during the synthesis of camptothecin derivatives. Byproducts, including compound (I) or its salts, found during the synthesis of the important intermediate compound (D), negatively impact the quality and production efficiency of camptothecin derivatives and their intermediates, increasing costs during process scale-up. This disclosure employs a novel route to resynthesize the important intermediate compound (D) using the compound (I) or its salts, thereby reducing costs and providing a new approach. Furthermore, this disclosure provides a synthetic route for the compound (I) or its salts, yielding a reference standard, thus facilitating quality control of camptothecin derivatives and their intermediates.

[0085] Terminology Explanation:

[0086] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0087] In the preparation method described in this disclosure, the reactions connected by "→" all refer to one-step reactions that yield the product.

[0088] The term "antibody-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker. In this disclosure, "antibody-drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active glucocorticoid via a stable linker. The antibody or antibody fragment may bind to a glucocorticoid molecule containing a linker via specific groups therein (e.g., interchain disulfide bonds).

[0089] The term "drug loading" refers to the average amount of drug carried by each antibody-drug conjugate molecule in a population of antibody-drug conjugates, and can also be expressed as the ratio of drug amount to antibody amount. The drug loading range can be 1-20, preferably 1-10, glucocorticoids (D) linked to each antibody (Ab). In embodiments of this disclosure, the drug loading is represented by k, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the average of any two values. 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 the average of 4-5. The average amount of drug per ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, CE-SDS (monoclonal antibody size variant assay), and HPLC characterization.

[0090] The term "antibody" encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies can refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, different subclasses can be distinguished; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ chains or λ chains based on differences in their constant regions. Each of the five types of Ig can have either a κ chain or a λ chain.

[0091] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0092] The antibodies disclosed herein include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

[0093] The term "mouse antibody" in this disclosure refers to antibodies prepared using mice in accordance with the knowledge and skills in the art. Preparation involves injecting a test subject with a specific antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics.

[0094] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can reduce the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies must first be established. Then, the variable region gene is cloned from the murine hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0095] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., a human germline antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse or reversion mutations to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR. Further literature describing methods that can be used to participate in humanization includes, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and Winter et al. [Jones et al., Nature, 321, 522 (1986), Riechmann et al., Nature, 332, 323-327 (1988), Verhoeyen et al., Science, 239, 1534 (1988)].

[0096] The term "fully human antibody," also known as a "fully human monoclonal antibody," refers to an antibody whose variable and constant regions are both human-derived, eliminating immunogenicity and toxicity. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. This disclosure pertains to fully human monoclonal antibodies. Related technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0097] The term “antigen-binding fragment” refers to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in “antigen-binding fragments” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bridges on hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of the antibody; (v) single-domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) separate complementarity-determining regions (CDRs) or (vii) combinations of two or more separate CDRs optionally linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, 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 included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0098] Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by treating IgG antibody molecules with the protease papain (which cleaves the amino acid residue at position 224 of the H chain). Approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0099] F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000, possessing antigen-binding activity, and containing two Fab regions connected at the hinge position, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

[0100] Fab' is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2.

[0101] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote to express the Fab'.

[0102] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have a 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 variants with 1–4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444–6448). Other connectors that may be used in this 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, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0103] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable domain and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable domain. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, in the classic format, following 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); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 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).Combining the CDR definitions from Kabat and Chothia, the CDR is composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). Following IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGap Align procedure.

[0104] The term "antibody framework" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0105] The term “epitope” or “antigenic determinant” refers to the site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)).

[0106] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example: approximately less than 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (KD) binding.

[0107] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0108] 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 "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

[0109] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to a non-human CDR region. Human FR germline sequences can be obtained by comparing with the IMGT Human Antibody Variable Region Germline Gene Database and MOE software, from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr, or from the journal Immunoglobulins, 2001 ISBN012441351.

[0110] 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. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, 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 NSO cells.

[0111] The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0112] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence, after aligning the amino acid sequences and, where necessary, introducing gaps to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0113] "Amino protecting group" is a group known in the art that can be used to protect an amino group, see amino protecting groups in the literature (Protective Groups in Organic Synthesis, 5th Ed. TW Greene & P. ​​GMWuts). Examples include, but are not limited to, urethane protecting groups such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)-ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Al loc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups such as formyl, acetyl, trichloroacetyl, benzoyl, and nitrophenylacetyl; sulfonamide protecting groups such as 2-nitrobenzenesulfonyl; and imine and cyclic imine protecting groups such as phthalimide and dithiosuccinyl.

[0114] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms (C1-C2). 12 Alkyl groups). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-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-Dimethylpentyl, 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 their various branched isomers, etc. More preferably, alkyl groups (C1 to C6 alkyl groups) containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-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, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. 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, oxo, carboxyl, or carboxylic acid ester group.

[0115] The term "alkenyl" refers to an alkyl group in which the molecule contains at least one carbon-carbon double bond, wherein the alkyl group is defined as described above and has 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) carbon atoms (C2-C). 12 Alkenyl group. The alkenyl group is preferably an alkenyl group having 2 to 6 carbon atoms (C2-C6 alkenyl). Non-limiting examples include vinyl, propenyl, isopropenyl, butenyl, etc. The alkenyl group can be optionally 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 carboxylic acid ester group.

[0116] The term "alkynyl" refers to an alkyl group in a molecule that contains at least one carbon-carbon triple bond, wherein the alkyl group is defined as described above and has 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) carbon atoms (C2-C). 12 The alkynyl group is preferably an alkynyl group having 2 to 6 carbon atoms (C2-C6 alkynyl). Non-limiting examples include: ethynyl, propynyl, butynyl, pentyynyl, hexynyl, etc. The alkynyl group can be optionally 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 carboxylic acid ester group.

[0117] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally 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 carboxylic acid ester group.

[0118] The term "alkathio" refers to -S- (alkyl) and -S- (unsubstituted cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkathio groups include: methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, and cyclohexylthio. Alkathio groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkathio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkathio, and heterocycloalkathio.

[0119] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups. "Carbocyclic" refers to the ring system within the cycloalkyl group.

[0120] The term "spirocycloalkyl" refers to a polycyclic group consisting of 5 to 20 quintile rings sharing a single carbon atom (called a spiro atom), which may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6 to 14 quintiles, more preferably 7 to 10 quintiles. Spirocycloalkyl groups are classified into monospirocycloalkyl, bispirocycloalkyl, or polyspirocycloalkyl groups based on the number of shared spiro atoms between the rings, with monospirocycloalkyl and bispirocycloalkyl groups being preferred. More preferably, it is a 4-quintile, 4-quintile, 4-quintile, 5-quintile, or 5-quintile / 6-quintile monospirocycloalkyl group. "Spirocarbon ring" refers to the ring system within the spirocycloalkyl group. Non-limiting examples of spirocycloalkyl groups include:

[0121] The term "fused-ring alkyl" refers to a 5- to 20-membered polycyclic carbon group in which each ring shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a fully conjugated π-electron system. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused-ring alkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl. "Fused-carbon ring" refers to the ring system within a fused-ring alkyl group. Non-limiting examples of fused-ring alkyl groups include:

[0122] The term "bridged cycloalkyl" refers to a 5- to 20-membered polycyclic carbon group in which any two rings share two non-directly bonded carbon atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl groups include:

[0123] The cycloalkyl ring may be fused to an aryl, heteroaryl, or heterocycloalkyl ring, wherein the ring connected to the parent structure is a cycloalkyl group, and non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, etc. The cycloalkyl group may be optionally substituted or unsubstituted; 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, oxo, carboxyl, or carboxylic acid ester group.

[0124] The terms "heterocyclic alkyl" or "heterocyclic group" refer to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m The ring consists of heteroatoms (where m is an integer from 0 to 2), but excludes ring portions of -OO-, -OS-, or -SS-, with 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 heterocyclic alkyl or heterocyclic groups include pyrrolidinyl, imidazoalkyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazoyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl or pyrrolidinyl. Polycyclic heterocyclic alkyl or heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic alkyl or heterocyclic groups. "Heterocyclic" refers to the ring system within the heterocyclic alkyl or heterocyclic group.

[0125] The term "spiroheteroalkyl" or "spiroheterocyclic" refers to a polycyclic heterocyclic group consisting of 5 to 20 member monocyclic rings sharing a single atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O). m(where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6 to 14 quinary, more preferably 7 to 10 quinary. Spirohedroalkyl or spirochetal groups are classified into monospiroalkyl or spirochetal groups, bispiroalkyl or spirochetal groups, or polyspiroalkyl or spirochetal groups based on the number of shared spiroatoms between rings, preferably monospiroalkyl or spirochetal groups and bispiroalkyl or spirochetal groups. More preferably, it is 4-quinary / 4-quinary, 4-quinary / 5-quinary, 4-quinary / 6-quinary, 5-quinary / 5-quinary, or 5-quinary / 6-quinary monospiroalkyl or spirochetal groups. "Spirohedrone" refers to the ring system in a spirochetal alkyl or spirochetal group. Non-limiting examples of spirochetal alkyl or spirochetal groups include:

[0126] The term "fused heterocyclic alkyl" or "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with the other rings in the system. One or more rings may contain one or more double bonds, but no ring has a fully conjugated π-electron system, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. Preferably, they are 6 to 14 members, more preferably 7 to 10 members. Depending on the number of rings, they can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic alkyl or fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic alkyl or fused heterocyclic groups. "Fused heterocycle" refers to the ring system in fused heterocyclic alkyl or fused heterocyclic groups. Non-limiting examples of fused heterocyclic alkyl or fused heterocyclic groups include:

[0127] The term "bridged heterocyclic alkyl" or "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two atoms that are not directly connected. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system, and one or more ring atoms are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. Preferably, it consists of 6 to 14 rings, more preferably 7 to 10 rings. Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic alkyl or bridged heterocyclic groups, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic alkyl or bridged heterocyclic groups include:

[0128] The heterocyclic alkyl or heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic alkyl or heterocyclic group, and non-limiting examples include:

[0129] wait.

[0130] Heterocyclic alkyl or heterocyclic groups may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkylthio, heterocyclic alkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0131] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring. "Aromatic ring" refers to the ring system within the aryl group. Non-limiting examples of aryl groups include:

[0132] The aryl group can be substituted or unsubstituted. 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 carboxylic acid ester group, preferably phenyl.

[0133] The term "fused-ring aryl" can refer to an unsaturated aromatic fused-ring structure containing 8-14 ring atoms, formed by two or more ring structures sharing two adjacent atoms. Preferably, it contains 8-12 ring atoms. Examples include fully unsaturated fused-ring aryl groups such as naphthalene and phenanthrene, as well as partially saturated fused-ring aryl groups such as benzo[3-8] saturated monocyclic cycloalkyl groups and benzo[3-8] partially saturated monocyclic cycloalkyl groups. "Fused aromatic ring" refers to the ring system within the fused-ring aryl group. Specific examples of fused-ring aryl groups include 2,3-dihydro-1H-indenyl, 1H-indenyl, 1,2,3,4-tetrahydronaphthyl, and 1,4-dihydronaphthyl.

[0134] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 12-membered, such as imidazolyl, furanyl, thiophenel, thiazolyl, pyrazolyl, oxazolyl, pyrrololyl, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl, or thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. "Heteroaryl ring" refers to the ring system within the heteroaryl group. Non-limiting examples of heteroaryl groups include:

[0135] The heteroaryl group can be optionally substituted or unsubstituted. 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 carboxylic acid ester group.

[0136] The term "fused aryl" can refer to an unsaturated aromatic fused ring structure containing 5-14 ring atoms (including at least one heteroatom) formed by two or more ring structures sharing two adjacent atoms. The carbon, nitrogen, and sulfur atoms can be substituted with oxygen. Preferably, it includes "5-12 fused aryl", "7-12 fused aryl", "9-12 fused aryl", etc., such as benzofuranyl, benzoisofuranyl, benzothiopheneyl, indole, isoindole, benzoxazolyl, benzoimidazolyl, indazole, benzotriazolyl, quinolinyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolinyl, acridinel, phenanthridinel, benzopyridinyl, phthalazinyl, quinazolinyl, quinoxalinyl, quinoxalinyl, phenoxalinyl, phenazinyl, pteridinel, purinel, naphthidyl, phenazine, phenothiazine, etc. "Dense aromatic rings" refers to the ring system in dense aromatic groups.

[0137] The fused heteroaryl group can be optionally substituted or unsubstituted. 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 carboxylic acid ester group.

[0138] The term "hydroxyl group" refers to the -OH group.

[0139] The term "oxo" refers to an =O group. For example, a carbon atom is connected to an oxygen atom by a double bond, forming a ketone or aldehyde group.

[0140] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0141] The term "amino" refers to -NH2.

[0142] The term "cyano" refers to -CN.

[0143] The term "nitro" refers to -NO2.

[0144] The term "carboxyl group" refers to -C(O)OH.

[0145] The term "aldehyde group" refers to -CHO.

[0146] The term "carboxylic acid ester group" refers to -C(O)O (alkyl) or -C(O)O (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0147] In the chemical structure of the compounds described in this disclosure, the bonds... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include Two configurations. In the chemical structure of the compounds described in this disclosure, the bonds... No configuration was specified, i.e., key The configuration can be E-type or Z-type, or it can contain both E-type and Z-type configurations.

[0148] "Optional" or "optional" means that the event or situation subsequently described may, but does not have to, occur; the description includes the possibility or possibility that the event or situation may or may not occur. For example, "optionally halogenated or cyano-substituted C..." 1-6 "Alkyl" means that halogens or cyano groups may or may not be present. This description includes cases where alkyl groups are substituted by halogens or cyano groups and cases where alkyl groups are not substituted by halogens or cyano groups.

[0149] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0150] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs, along with other chemical components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

[0151] "Pharmaceutical excipients" include, but are not limited to, any adjuvant, carrier, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the U.S. Food and Drug Administration (FDA) for use in humans or livestock.

[0152] The term "effective amount" or "effective therapeutic amount" as used in this disclosure includes an amount sufficient to improve or prevent symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. Detailed Implementation

[0153] The following detailed explanation of this disclosure will be provided with specific examples to enable those skilled in the art to have a more comprehensive understanding of this disclosure. The specific examples are only used to illustrate the technical solutions of this disclosure and do not limit this disclosure in any way.

[0154] The abbreviations used in the following experiments have the following meanings: PE represents petroleum ether; EA represents ethyl acetate; THF represents tetrahydrofuran; PCC represents pyridinium chlorochromate; DMF represents N,N-dimethylformamide.

[0155] Example 1

[0156] Add acetone (300 mL) to a flask, and while stirring at room temperature, add IV-1 (50 g, 0.226 mol), followed by sodium bicarbonate (22.7 g, 0.271 mol). Maintain the internal temperature at 15–30 °C while stirring. Add potassium permanganate (150 g, 0.949 mol) in batches over 1.5–2 hours. After the addition is complete, continue stirring in a water bath at 20–30 °C for 1.5–2.5 hours. Quench the reaction mixture with 5% sodium bisulfite solution until the purple-red color of the potassium permanganate disappears. Filter, and slurry the filter cake with acetone (600 mL) for 15–20 minutes. Filter again, combine the filtrates, and concentrate the filtrate to remove most of the acetone. Add 5 L of water for crystallization over 0.5 hours. Filter. Dissolve the resulting filter cake in 800 mL of dichloromethane and wash with water (500 mL × 2), allowing the layers to separate. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. 300 mL of ethyl acetate was added and heated to dissolve the filtrate. Then, 600 mL of n-heptane was added, and the mixture was cooled to crystallize for 0.5–1 hours. The crystals were filtered and dried to obtain compound V-1 (33 g, containing 0.44% I-1).

[0157] Example 2

[0158] Step 1: Preparation of compound ethyl 4-acetamido-2-fluoro-5,6,7,8-tetrahydronaphthalene-1-carboxylate (II-1)

[0159] Compound I-1 (60 mg, 257.2209 μmol) and sodium carbonate (54.5249 mg, 514.4419 μmol) were added to DMF (5 mL), and iodoethane (60.1762 mg, 385.8314 μmol) was added dropwise. The reaction mixture was reacted at room temperature for 2 hours. The reaction mixture was purified by reverse-phase chromatography (column: YMC-Triart Prep C18-S12nm ID50*250 mm, 7 μm; mobile phase 1: 0.2% trifluoroacetic acid aqueous solution; mobile phase 2: acetonitrile) to give compound II-1 (60 mg, yield: 83.52%).

[0160] 1 H NMR (400MHz, DMSO) δ9.26(s,1H),7.37(t,J=16.60Hz,1H),4.28(q,J=7.00Hz,2H),2.65(t,J= 5.28Hz,2H),2.54(d,J=5.80Hz,2H),2.06(s,3H),1.74–1.60(m,4H),1.26(d,J=7.20Hz,3H).

[0161] Step 2: Preparation of compound N-(3-fluoro-4-(hydroxymethyl)-5,6,7,8-tetrahydronaphth-1-yl)acetamide (III-1)

[0162] Compound II-1 (60 mg, 214.82 μmol) was added to THF (5 mL), followed by dropwise addition of diisobutylaluminum hydride (1 M, 429.64 μmol, 429.64 μL). The reaction mixture was reacted at room temperature for 2 hours. The reaction mixture was purified by reverse-phase chromatography (column: YMC-Triart Prep C18-S12nm ID50*250 mm, 7 μm; mobile phase 1: 0.2% trifluoroacetic acid aqueous solution; mobile phase 2: acetonitrile) to give compound III-1 (50 mg, yield: 98.10%).

[0163] 1 H NMR (400MHz, DMSO) δ9.15 (s, 1H), 7.14 (d, J = 11.60Hz, 1H), 4.42 (d, J = 1.80Hz, 2H), 2.81 (d, J = 5.60Hz, 2H), 2.51 (s, 2H), 2.02 (s, 3H), 1.66 (d, J = 2.7Hz, 4H).

[0164] Step 3: Preparation of compound N-(3-fluoro-4-methyl-5,6,7,8-tetrahydronaphth-1-yl)acetamide (IV-1)

[0165] Compound III-1 (50 mg, 210.73 μmol) was added to trifluoroacetic acid (1 mL), followed by the dropwise addition of triethylsilane (122.51 mg, 1.05 mmol). The reaction mixture was allowed to react at room temperature for 1 hour. The reaction mixture was then purified by reverse-phase chromatography (column: YMC-Triart Prep C18-S12nm ID50*250 mm, 7 μm; mobile phase 1: 0.2% trifluoroacetic acid aqueous solution; mobile phase 2: acetonitrile) to give compound IV-1 (35 mg, yield: 75.06%).

[0166] 1 H NMR (400MHz, DMSO) δ9.14 (s, 1H), 7.08 (d, J = 11.40Hz, 1H), 2.57–2.47 (m, 4H), 2.01 (s, 6H), 1.72–1.59 (m, 4H).

[0167] Step 4: Preparation of compound N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (V-1)

[0168] Compound IV-1 (5 g, 22.6 mmol) was dissolved in acetone (60 mL), and sodium bicarbonate (2.27 g, 27.1 mmol) was added with stirring at room temperature. Potassium permanganate (15 g, 94.9 mmol) was then added in portions, and the reaction was carried out at 20-30 °C with stirring for 2 h. The reaction was quenched with 5% sodium bisulfite solution, concentrated under reduced pressure, and the aqueous phase was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1) to give compound V-1 (3 g, yield: 56.4%).

[0169] Example 3

[0170] Compound D was prepared using the method disclosed in patent application CN1104409C, with compound V-1 as the raw material.

[0171] Example 4

[0172] Step 1: Preparation of compound N-(5-bromo-4-(bromomethyl)-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (2)

[0173] N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (5 g, 21 mmol, compound 1) was added to carbon tetrachloride (50 mL), followed by the addition of N-bromosuccinimide (15 g, 85 mmol) and benzoyl peroxide (515 mg, 2.1 mmol) in portions. The reaction mixture was reacted at 60 °C for 3 hours. Ethyl acetate (200 mL) and water (100 mL) were added to the reaction mixture, and the mixture was separated. The aqueous phase was extracted with ethyl acetate (100 mL × 2), and the organic phases were combined. The organic phase was washed with saturated sodium sulfite solution (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-30%) to give compound 2 (7.5 g, yield: 89.78%).

[0174] Step 2: Preparation of compound N-(5-bromo-3-fluoro-4-(hydroxymethyl)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (3)

[0175] Compound 2 (7.5 g, 19.0817 mmol) was added to water (10 mL) and acetone (70 mL), followed by silver oxide (2.43 g, 10.50 mmol). The reaction mixture was allowed to react overnight at room temperature. Ethyl acetate (100 mL) and water (30 mL) were added to the reaction mixture. The mixture was separated, and the aqueous phase was extracted with ethyl acetate (100 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-50%) to give compound 3 (3.5 g, yield: 55.56%).

[0176] 1H NMR (400MHz, DMSO) δ12.08(s,1H),8.38(d,J=13.20Hz,1H),6.15(s,1H),4.68(d,J=12.40Hz,1H),4.51(dd,J =12.40,2.7Hz,1H),2.99(ddd,J=18.40,12.7,5.7Hz,1H),2.78–2.68(m,1H),2.56–2.39(m,2H),2.15(s,3H).

[0177] Step 3: Preparation of compound N-(5-bromo-3-fluoro-4-formyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (4)

[0178] Compound 3 (3.5 g, 10.6013 mmol) was added to dichloromethane (50 mL), followed by PCC (4.5703 g, 21.2025 mmol) and diatomaceous earth (3.5 g, 58.3333 mmol). The reaction mixture was reacted at room temperature for 2 hours. Ethyl acetate (100 mL) and water (100 mL) were added to the reaction mixture. The mixture was separated, and the aqueous phase was extracted with ethyl acetate (100 mL × 2). The organic phases were combined, washed with saturated sodium sulfite solution (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (PE:EA = 0-50%) to give compound 4 (3.0 g, yield: 86.24%).

[0179] 1H NMR (400MHz, DMSO) δ12.27(s,1H),10.31(s,1H),8.47(d,J=14.90Hz,1H),6.72(s,1H),2.98(ddd,J=18. 6,13.00,5.60Hz,1H),2.74(dd,J=18.40,4.2Hz,1H),2.58–2.47(m,1H),2.43–2.32(m,1H),2.20(s,3H).

[0180] Step 4: Preparation of compound N-(3-fluoro-4-formyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (5)

[0181] Compound 4 (3.0 g, 9.14 mmol) was added to EA (30 mL) and THF (30 mL), and palladium on carbon (1.0 g, 8.23 ​​mmol) was added at room temperature. The reaction solution was purged three times under nitrogen, and hydrogen gas was bubbled through the reaction solution. The reaction solution was allowed to react overnight at room temperature. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain crude compound 5 (2.6 g).

[0182] 1 H NMR (600MHz, DMSO) δ12.47 (s, 1H), 10.27 (s, 1H), 8.36 (d, J = 15.00Hz, 1H), 3.31 -3.30(m,2H),2.69–2.63(m,2H),2.19(s,3H),1.95(dt,J=12.60,6.20Hz,2H).

[0183] Step 5: Preparation of compound 4-acetamido-2-fluoro-5-oxo-5,6,7,8-tetrahydronaphthalene-1-carboxylic acid (IA-1)

[0184] Compound 5 (2.6 g, 10.43 mmol) was added to water (10 mL) and acetone (40 mL), followed by potassium permanganate (1.97 g, 12.51 mmol). The reaction mixture was reacted at room temperature for 15 hours. The reaction mixture was purified by reverse-phase chromatography (column: YMC-Triart Prep C18-S12nm ID50*250 mm, 7 μm; mobile phase 1: 0.2% formic acid aqueous solution; mobile phase 2: acetonitrile), and then lyophilized to give compound IA-1 (1.8 g, yield: 65.05%).

[0185] 1 H NMR (400MHz, DMSO) δ12.23(s,1H),8.27(d,J=12.8Hz,1H),2.91(t,J=6.0Hz,2 H),2.64(t,J=6.6Hz,2H),2.14(d,J=3.6Hz,3H),1.93(dt,J=12.6,6.4Hz,2H).

[0186] Step 6: Preparation of compound 4-acetamido-2-fluoro-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carboxylic acid (IB-1)

[0187] Compound IA-1 was added to methanol (15 mL), and sodium borohydride (192.56 mg, 5.08 mmol) was added at 0 °C. The reaction mixture was reacted at room temperature for 1 hour. The reaction mixture was purified by reverse-phase chromatography (column: YMC-Triart Prep C18-S12nm ID50*250 mm, 7 μm; mobile phase 1: 0.2% formic acid aqueous solution; mobile phase 2: acetonitrile) to give compound IB-1 (0.8 g, yield: 88.21%).

[0188] Step 7: Preparation of compounds 4-acetamido-2-fluoro-5,6,7,8-tetrahydronaphthalene-1-carboxylic acid (I-1), 4-amino-2-fluoro-5,6,7,8-tetrahydronaphthalene-1-carboxylic acid (ID-1) and 4-amino-2-fluoro-5-hydroxy-5,6,7,8-tetrahydronaphthalene-1-carboxylic acid (IC-1).

[0189] Compound IB-1 (0.8 g, 2.9934 mmol) was added to trifluoroacetic acid (10 mL), and triethylsilane (3.48 g, 29.93 mmol, 10 mL) was added dropwise to the reaction solution at room temperature. The reaction solution was reacted at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure and purified by reverse-phase chromatography (column: YMC-Triart Prep C18-S12nm ID50*250 mm, 7 μm, mobile phase 1: 0.2% formic acid aqueous solution; mobile phase 2: acetonitrile) to give compounds I-1 (30 mg, yield: 3.98%), compound ID-1 (50 mg, yield: 7.98%), and compound IC-1 (50 mg, yield: 7.41%).

[0190] Example 5

[0191] Step 1: Synthesis of compound 4-amino-2-fluoro-5,6,7,8-tetrahydronaphthalene-1-carboxylic acid (ID-1)

[0192] Compound IC-1 (0.1 g, 444.0186 μmol) was added to trifluoroacetic acid (3 mL), and triethylsilane (3 mL) was added dropwise to the reaction solution at room temperature. The reaction solution was reacted at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure and purified by reverse-phase chromatography (column: YMC-Triart Prep C18-S12nm ID50*250 mm, 7 μm, mobile phase 1: 0.2% trifluoroacetic acid aqueous solution; mobile phase 2: acetonitrile) to give compound ID-1 (50 mg, yield: 53.82%).

[0193] Step 2: Synthesis of compound 4-acetamido-2-fluoro-5,6,7,8-tetrahydronaphthalene-1-carboxylic acid (I-1)

[0194] Compound ID-1 (100 mg, 477.97 μmol) was added to dichloromethane (10 mL). Acetic anhydride (97.59 mg, 955.94 μmol) and triethylamine (96.73 mg, 955.94 μmol) were added dropwise to the reaction solution at room temperature, and the reaction was carried out for 15 hours at room temperature. The reaction solution was concentrated under reduced pressure and purified by reverse-phase preparative chromatography (column: YMC-Triart Prep C18-S12nm ID50*250 mm, 7 μm; mobile phase 1: 0.2% formic acid aqueous solution; mobile phase 2: acetonitrile) to obtain compound I-1 (90 mg, yield: 74.94%).

[0195] 1H NMR (400MHz, DMSO) δ13.33 (s, 1H), 9.20 (s, 1H), 7.31 (d, J = 11.60Hz, 1H), 2.6 8(t,J=5.28Hz,2H),2.54(d,J=5.80Hz,2H),2.05(s,3H),1.74–1.60(m,4H).

[0196] Since this disclosure has been described in accordance with its specific implementation, certain modifications and equivalent variations will be apparent to those skilled in the art and are included within the scope of this disclosure.

Claims

1. The compound shown in formula (I) or a salt thereof, in, R1 is selected from halogens; R2 is selected from amino protecting groups; The compound of preferred formula (I) or its salt is selected from, 2. The compound shown in formula (II) or its salt, in, R1 is selected from halogens; R2 is selected from amino protecting groups; R3 is selected from C1-C6 alkyl groups, wherein the alkyl group is optionally substituted by one or more substituents selected from 3-6 membered cycloalkyl groups and 6-10 membered aryl groups; The compound of preferred formula (II) or its salt is selected from, 3. The compound shown in formula (III) or its salt, in, R1 is selected from halogens; R2 is selected from amino protecting groups; The compound of preferred formula (III) or its salt is selected from, 4. A method for preparing a compound of formula (V) or a salt thereof, comprising the steps of preparing a compound of formula (II) or a salt thereof from a compound of formula (I), preparing a compound of formula (III) or a salt thereof from a compound of formula (II), preparing a compound of formula (IV) or a salt thereof from a compound of formula (III), and preparing a compound of formula (V) or a salt thereof from a compound of formula (IV). in, R1 is selected from halogens; R2 is selected from amino protecting groups; R3 is selected from C1-C6 alkyl groups, wherein the alkyl group is optionally replaced by one or more substituents selected from 3-6 membered cycloalkyl groups and 6-10 membered aryl groups.

5. In the preparation method according to claim 4, R2 is selected from acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, neopentanoyl, formyl, benzoyl, phthaloyl, 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, and benzyloxycarbonyl; R2 is preferably selected from acetyl groups.

6. The preparation method according to claim 4 or 5, wherein the compound of formula (V) or a salt thereof is selected from the compound of formula (V-1) or a salt thereof, and the preparation method comprises:

7. A method for preparing a compound of formula (D) or a salt thereof, comprising the method for preparing a compound of formula (V) or a salt thereof as described in any one of claims 4-6.

8. A method for preparing a compound of formula (C) or a salt thereof, comprising the method for preparing a compound of formula (V) or a salt thereof as described in any one of claims 4-6, and / or the method for preparing a compound of formula (D) or a salt thereof as described in claim 7.

9. A method for preparing a compound of formula (B) or a salt thereof, comprising at least one of the methods for preparing a compound of formula (V) or a salt thereof according to any one of claims 4-6, the method for preparing a compound of formula (D) or a salt thereof according to claim 7, and the method for preparing a compound of formula (C) or a salt thereof according to claim 8. in, L1 is selected from -(C(R) 11 (R) 12 )) n -(CH2) m - 3-6 saturated cycloalkyl and 3-6 saturated heterocycloalkyl, wherein each of the 3-6 saturated cycloalkyl and 3-6 saturated heterocycloalkyl is independently optionally substituted by one or more substituents selected from halogen, hydroxyl, amino and haloalkyl; R 11 R 12 Each is independently selected from hydrogen atoms, C1-C6 alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl, and 5-10 membered heteroaryl, wherein the alkyl, cycloalkyl, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino, oxo, 3-6 membered cycloalkyl, 6-10 membered aryl, and C1-C6 alkoxy groups, or R 11 R 12 Together with the carbon atom connected thereto, they form a 3-6 membered cycloalkyl group which may be optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino, oxo and C1-C6 alkoxy groups; R 13 Selected from hydrogen atom, deuterium atom, C 1-6 Alkyl, 6-10 aryl and 5-10 heteroaryl, wherein the alkyl, aryl and heteroaryl groups are optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino and oxo groups; m is 0 or 1, preferably 0; n is 1, 2, 3 or 4, preferably 1; p is an integer from 2 to 8.

10. The preparation method according to claim 9, wherein the compound shown in formula (B) is selected from, 11. A method for preparing an antibody-drug conjugate of formula (A), comprising the steps of preparing the compound of formula (B) or a salt thereof as described in any one of claims 9-10, and the step of reducing Ab and then coupling it with the compound of formula (B) to obtain the antibody-drug conjugate of formula (A). Where Ab is an antibody or antigen-binding fragment, k is 1 to 20, and L1, R 13 And p as described in claim 9.

12. A method for preparing a compound of formula (I) or a salt thereof, comprising the step of preparing a compound of formula (IB) from a compound of formula (IA), wherein the compound of formula (IB) is prepared into a compound of formula (I) or a salt thereof through one or more steps: in, R1 is selected from halogens; R2 is selected from amino protecting groups.

13. Use of the compound of formula (I) according to claim 1 or a salt thereof as an impurity reference for camptothecin derivatives or intermediates thereof.

14. The use according to claim 13, wherein the camptothecin derivative or intermediate thereof is selected from the compound of formula (V) or its salt, the compound of formula (D) or its salt, the compound of formula (C) or its salt, the compound of formula (B) or its salt, and the antibody-drug conjugate of formula (A).

15. A pharmaceutical composition comprising a camptothecin derivative or an intermediate thereof, and a compound of formula (I) or a salt thereof; The preferred composition contains less than 1% of the compound of formula (I) or its salt. More preferably, the content of the compound of formula (I) or its salt in the composition is less than 0.8%, less than 0.5%, less than 0.3%, less than 0.10%, or less than 0.05%.

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