Novel camptothecin derivatives, compositions containing same and uses thereof

Abstract

The present invention provides camptothecin derivatives, compositions containing the same, and uses thereof. Specifically, the present invention provides a compound of general formula (1) and a method for preparing the same, as well as the use of the compound of general formula (1) and its optical isomers, crystalline forms, and pharmaceutically acceptable salts in the preparation of a medicament for treating cancer. [Formula 1] TIFF2023552610000112.tif53168

Description

[Technical Field]

[0001] This application claims priority to Chinese Patent Application No. 2020114637044, filed on December 11, 2020, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to the field of medicinal chemistry, and in particular to novel camptothecin derivatives, compositions containing same and uses thereof. [Background technology]

[0003] DNA topoisomerases exist in the cell nucleus and use DNA as a substrate. They are involved in cell replication, transcription, and mitosis. The main role of topoisomerases is to disrupt the supercoil structure of DNA. Topoisomerases are classified as topoisomerase I (TopoI) and topoisomerase II (TopoII). When topoisomerases are inhibited, large amounts of broken DNA accumulate in tumor cells, inducing tumor cell death. DNA topoisomerase I inhibitors, including camptothecin and its derivatives, are clinically used to treat malignant tumors.

[0004] Camptothecin, first isolated from Camptotheca acuminata Decne (family Nyssaceae), has relatively strong cytotoxicity and exhibits excellent therapeutic effects against malignant tumors, such as gastrointestinal tumors (gastric cancer, colon cancer, and rectal cancer), liver cancer, breast cancer, bladder cancer, and leukemia. Camptothecin has major drawbacks, including relatively poor solubility and stability, and high toxicity, limiting its clinical use. The water solubility of camptothecin derivatives can be increased by introducing water-soluble groups or preparing prodrugs, thereby improving their druggability. Numerous camptothecin derivatives with significantly improved solubility, such as topotecan and its carbamate prodrug, irinotecan, are commercially available.

[0005] Camptothecin derivatives are used not only as chemotherapy drugs for tumor treatment but also as small molecule toxins (payloads) attached to antibodies in antibody-drug conjugates (ADCs). ADCs combine an antibody with a small molecule toxin. These ADCs combine the specificity of an antibody for binding to tumor cell surface antigens and the high activity of a cytotoxic drug for inhibiting and killing tumor cells. Compared with conventional chemotherapy drugs, ADCs can more precisely kill tumor cells, thereby reducing the impact on normal cells. In recent years, ADCs using camptothecin derivatives as small molecule toxins have been actively developed. DS-8201a (trastuzumab deruxtecan) is the first ADC developed and approved for marketing in Japan by Daiichi Sankyo Co., Ltd. This ADC uses the camptothecin derivative deruxtecan as the small molecule toxin and a self-cleaving GGF-G tetrapeptide as a linker that is hydrolyzable by cathepsin B.

[0006] [ka]

[0007] However, ADCs using camptothecin derivatives as small molecule toxins typically require a relatively large drug-to-antibody ratio (DAR), are difficult to manufacture, and are prone to instability. Therefore, novel camptothecin derivatives with higher activity have the potential for broad application as antitumor drugs or small molecule toxins for ADCs. The novel camptothecin derivatives provided by the present invention have significantly improved cellular activity compared to known compounds such as deruxtecan, and are of great significance for the development of novel antitumor drugs and ADCs.

[0008] (Summary of the Invention) The present invention provides a camptothecin derivative compound of general formula (1), or an optical isomer, crystalline form, pharmaceutically acceptable salt, hydrate, or solvate thereof. [ka] Here, in general formula (1), m is an integer of 0, 1, or 2; X is -O-, -S-, -S(O)-, -S(O2)-, and -N(R 4 )-selected from R 1 and R 2 are independently H, halogen, OH, C 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Haloalkyl, C 3~6 cycloalkyl, NH, NO, and CN, or R 1 and R 2 together with the phenyl ring attached thereto, by cyclization to give [ka] Forming R 3 is C 1~6 Alkyl, C 2~6 Alkenyl, C 1~3 Alkoxy-substituted C 1~3 Alkyl, and C 1~6 haloalkyl; R 4 is H, C 1~6 Alkyl and C 1~6 haloalkyl; R 5 is H, C 1~6 Alkyl and C 3~6 cycloalkyl; R 6 and R 7 are independently H, C 1~6 Alkyl, C 1~6 Haloalkyl, and C 3~6 cycloalkyl; or R 6 and R 7 together with the carbon atoms attached to them, form C 3~6 cycloalkyl or 4- to 7-membered heterocycloalkyl; or R 6 and R 5 are linked to form a 5- to 7-membered lactam ring, and R7 is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and C 3~6 cycloalkyl; R 8 OH and NR 9 R 10 Selected from R 9 and R 10 are independently H, C 1~6 Alkyl and C 3~6 cycloalkyl; or R 9 and R 10 together with the N atom to which they are attached form, upon cyclization, a 4- to 7-membered heterocycloalkyl, said 4- to 7-membered heterocycloalkyl being unsubstituted or 1~6 It is substituted with 1 to 3 groups selected from alkyl, halogen, OH, CN, and NH2.

[0009] In another preferred embodiment, in formula (1), R 8 is OH.

[0010] In another preferred embodiment, in formula (1), R 1 and R 2 are independently selected from H, halogen, OH, Me, Et, OMe, OEt, CF, NH, NO, and CN; R 1 and R 2 are independently preferably H, F, Cl, Me, Et, OMe, OEt or CF; R 1 and R 2 are independently more preferably H, F, Me, Et or OMe; R 1 and R 2 are independently more preferably F or Me; or R 1 and R 2 together with the phenyl ring attached thereto, by cyclization to give [ka] Form.

[0011] In another preferred embodiment, in formula (1), R 3 Me, Et, [ka] Selected from;R 3 is preferably Et, [ka] and;R 3 is more preferably Et.

[0012] In another preferred embodiment, in formula (1), X is selected from —O—, —S—, —S(O)—, —S(O2)—, —N(H)—, and —N(Me)—; X is preferably —O—, —S—, —S(O)—, —S(O2)—, or —N(Me)—; X is more preferably —S—.

[0013] In another preferred embodiment, in formula (1), R 5 are H, Me, Et, and [ka] Selected from;R 5 is preferably H or Me, and R 5 is more preferably H.

[0014] In another preferred embodiment, in formula (1), R 6 and R 7 are independently H, Me, Et, CHF2, CF3, CH2CF3, [ka] Selected from;R 6 and R 7 are independently preferably H, Me, CF3, [ka] and;R 6 and R7 are independently, more preferably, H, or [ka] and;R 6 and R 7 are independently more preferably H; R 6 and R 7 are independently, more preferably [ka] is.

[0015] In another preferred embodiment, in formula (1), [ka] teeth, [ka] is selected from, preferably [ka] and more preferably, [ka] is.

[0016] In another preferred embodiment, in formula (1), R 8 is OH, and [ka] teeth, [ka] is selected from, preferably [ka] and more preferably, [ka] is.

[0017] In some embodiments of the present invention, a camptothecin derivative compound or a pharmaceutically acceptable salt thereof is provided, wherein said compound has the following structure: [ka] I have one of the following: TIFF2023552610000022.tif153168.

[0018] In some embodiments of the present invention, a camptothecin derivative compound or a pharmaceutically acceptable salt thereof is provided, wherein said compound has the following structure: [ka] It has one of the following.

[0019] In some embodiments of the present invention, the compound of general formula (1), or an isomer or pharmaceutically acceptable salt thereof, has the following structure: [ka] I have one of the following: TIFF2023552610000025.tif248168.

[0020] In some embodiments of the present invention, the compound of general formula (1), or an isomer or pharmaceutically acceptable salt thereof, has the following structure: [ka] It has one of the following: TIFF2023552610000027.tif229168TIFF2023552610000028.tif38168.

[0021] In some embodiments of the invention, the invention provides an antibody-drug conjugate, wherein the antibody-drug conjugate has the following structure: [ka] TIFF2023552610000030.tif173168TIFF2023552610000031.tif168168, wherein Ab represents a monoclonal antibody, preferably an anti-her2 antibody, more preferably trastuzumab, and n is 2 to 8, preferably 4 to 8, more preferably 7 to 8, for example 7.2 or 7.3.

[0022] An object of the present invention is to provide the use of a compound of the present invention, or an optical isomer, a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof, as a small molecule toxin in the preparation of an antibody drug conjugate (ADC).

[0023] Another object of the present invention is to provide a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier and, as an active ingredient, a compound of the present invention or an optical isomer thereof, or a pharmaceutically acceptable inorganic or organic salt thereof.

[0024] Yet another object of the present invention is to provide the use of a compound of the present invention, or an optical isomer thereof, or a pharmaceutically acceptable inorganic or organic salt thereof, or said pharmaceutical composition, in the preparation of a medicament for treating tumors and related diseases.

[0025] Yet another object of the present invention is to provide an antibody-drug conjugate (ADC), wherein the antibody-drug conjugate comprises an antibody, a small molecule toxin, and a linker, wherein the small molecule toxin is a compound of the present invention, and the linker connects the antibody and the small molecule toxin via a covalent bond.

[0026] It is to be understood that both the foregoing general description and the following detailed description of the invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

[0027] (Compound synthesis) Methods for preparing the compounds of general formula (1) disclosed in the present specification are specifically described below, but these specific methods do not limit the present invention in any way.

[0028] The compounds of formula (1) above can be synthesized using standard synthetic techniques, well-known techniques, in combination with the methods described herein. Furthermore, the solvents, temperatures, and other reaction conditions described herein may vary. Starting materials for the synthesis of the compounds can be obtained synthetically or commercially. The compounds described herein and other related compounds with different substituents are described in March, ADVANCED ORGANIC CHEMISTRY, 4 th Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY, 4 th Ed., Vols. A and B (Plenum 2000, 2001), and Green and Wuts, Protective Groups in Organic Synthesis, 3 rd Ed., (Wiley 1999). General methods for preparing compounds can be modified by using appropriate reagents and conditions to introduce various groups into the formulas provided herein.

[0029] In one embodiment, the compounds described herein are prepared according to methods well known in the art. However, the conditions of the methods, such as reactants, solvents, bases, amounts of compounds used, reaction temperatures, and reaction times, are not limited to those described below. In addition, the compounds of the present invention can be easily prepared by any combination of various synthetic methods described herein or known in the art, and such combinations can be easily determined by those skilled in the art to which the present invention pertains. In one embodiment, the present invention also provides a method for preparing a compound of general formula (1), which is prepared using the following general reaction scheme 1.

[0030] General Reaction Scheme 1 [ka] [ka] In the formula, R 1 , R 2 , R 3 , R 5 , R 6 , R 7 , R 8 and X is as defined above.

[0031] Compound A1 is used as the starting material and undergoes a nucleophilic substitution reaction to give compound A2, which is then nitro-reduced with iron powder and hydrochloric acid and then hydrolyzed to give compound A3, which is then subjected to a Friedel-Crafts acylation reaction to give compound A4, which is then acetylated to give compound A5, which is then subjected to nitration, reduction, and acetylation to give compound A6, and the acetyl group on the aniline in compound A6 is removed to give intermediate compound A7.

[0032] Compound B1 is used as the starting material and is brominated to give compound B2, which is reacted with a chiral amino acid to give compound B3, which is subjected to an affinity substitution reaction to give compound B4, which is cyano-reduced to give compound B5, which is diazotized and subjected to a nucleophilic reaction to give compound B6, which is finally cyclized to give intermediate compound B7.

[0033] Intermediate compound A7 and intermediate compound B7 are cyclized to give intermediate compound B8, which is then deacetylated to give compound B9, which is finally amino-modified or functional group converted to give the target compound of the present invention.

[0034] Further forms of the compound As used herein, "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not cause a compound to lose its biological activity or properties, is relatively non-toxic, e.g., does not cause undesired biological effects or adverse interactions with any of its components when the substance is administered to an individual.

[0035] The term " pharmaceutically acceptable salt " refers to the form of a compound that does not cause significant irritation to the organism when administered with a drug or does not eliminate the biological activity and properties of the compound.In certain embodiments, pharmaceutically acceptable salts are obtained by reacting the compound of general formula (1) with an acid, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, and nitric acid, organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, trifluoroacetic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid, acidic amino acids such as aspartic acid and glutamic acid.

[0036] It should be understood that pharmaceutically acceptable salts include solvent addition forms or crystalline forms, particularly solvates or polymorphs. Solvates contain stoichiometric or non-stoichiometric amounts of solvent and are selectively formed upon crystallization with pharmaceutically acceptable solvents, such as water and ethanol. Hydrates are formed when the solvent is water, and alcoholates are formed when the solvent is ethanol. Solvates of compounds of general formula (1) are conveniently prepared or formed according to the methods described herein. For example, hydrates of compounds of general formula (1) are conveniently prepared by recrystallization from a water / organic solvent mixture, where the organic solvent used includes, but is not limited to, tetrahydrofuran, acetone, ethanol, or methanol. Furthermore, the compounds referred to herein can exist in both unsolvated and solvated forms. In general, solvated forms are considered equivalent to unsolvated forms for the purposes of the compounds and methods provided herein.

[0037] In other specific examples, compounds of general formula (1) are prepared in different forms, including, but not limited to, amorphous, crushed, and nanoparticle forms. Furthermore, compounds of formula (1) may include crystalline forms and may also be polymorphs. Polymorphs contain different lattice arrangements of the same elements of a compound. Polymorphs typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystalline forms, optical properties, electrical properties, stability, and solubility. Various factors, such as recrystallization solvents, crystallization rates, and storage temperatures, may result in the predominance of a single crystal.

[0038] In another embodiment, the compounds of general formula (1) may have chiral centers and / or axial asymmetry and therefore may exist in the form of racemates, racemic mixtures, single enantiomers, diastereomeric compounds, single diastereomers, and cis-trans isomers. Each chiral center or axial asymmetry independently produces two optical isomers, and all possible optical isomers, diastereomeric mixtures, and pure or partially pure compounds are included within the scope of the present invention. The present invention is meant to include all such isomers of these compounds.

[0039] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may contain tritium ( 3 H), iodine-125( 125 I) and C-14( 14 The compounds may be labeled with a radioactive isotope such as C. As another example, deuterium can be used to replace a hydrogen atom to form a deuterated compound, and the bond formed between deuterium and carbon is stronger than the bond formed between ordinary hydrogen and carbon. Compared to non-deuterated drugs, deuterated drugs generally have advantages such as reduced toxicity and side effects, increased drug stability, improved therapeutic efficacy, and prolonged in vivo half-life. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0040] Explanation of terms Unless otherwise specified, the terms used in this specification and claims are defined as follows. It should be noted that in this specification and the appended claims, the singular forms "a" and "an" include the plural reference unless the context clearly dictates otherwise. Conventional methods such as mass spectrometry, nuclear magnetic resonance spectroscopy, HPLC, protein chemistry, biochemistry, recombinant DNA technology and pharmacology are used unless otherwise specified. As used herein, "or" or "and" means "and / or" unless otherwise specified.

[0041] Unless otherwise specified, "alkyl" refers to saturated aliphatic hydrocarbon groups, including straight-chain and branched groups, having 1 to 6 carbon atoms. Lower alkyls having 1 to 4 carbon atoms are preferred, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, or tert-butyl. As used herein, "alkyl" includes unsubstituted and substituted alkyls, particularly alkyls substituted with one or more halogens. Preferred alkyls are CH3, CH3CH2, CF3, CHF2, CF3CH2, CF3(CH3)CH, i Pr, n Pr, i Bu, n Bu and t Bu.

[0042] Unless otherwise specified, "alkylene" refers to a divalent alkyl as defined above. Examples of alkylene include, but are not limited to, methylene and ethylene.

[0043] Unless otherwise specified, "alkenyl" refers to an unsaturated aliphatic hydrocarbon group containing a carbon-carbon double bond, and includes straight-chain or branched groups containing 1 to 14 carbon atoms. Lower alkenyl containing 1 to 4 carbon atoms, such as vinyl, 1-propenyl, 1-butenyl, or 2-methylpropenyl, is preferred.

[0044] Unless otherwise specified, "alkynyl" refers to an unsaturated aliphatic hydrocarbon group containing a carbon-carbon triple bond, and includes straight-chain and branched groups containing 1 to 14 carbon atoms. Lower alkynyl groups containing 1 to 4 carbon atoms, such as ethynyl, 1-propynyl, or 1-butynyl, are preferred.

[0045] Unless otherwise specified, "cycloalkyl" refers to a non-aromatic hydrocarbon ring system (monocyclic, bicyclic, or polycyclic); partially unsaturated cycloalkyls may be referred to as "cycloalkenyls" if the carbocyclic ring contains at least one double bond, or "cycloalkynyls" if the carbocyclic ring contains at least one triple bond. Cycloalkyls can include monocyclic or polycyclic groups, as well as spirocycles (e.g., having 2, 3, or 4 fused rings). In some embodiments, cycloalkyls are monocyclic. In some embodiments, cycloalkyls are monocyclic or bicyclic. Ring carbon atoms of cycloalkyls can be optionally oxidized to form oxo or sulfido groups. Cycloalkyls further include cycloalkylene. In some embodiments, cycloalkyls contain zero, one, or two double bonds. In some embodiments, cycloalkyls contain one or two double bonds (partially unsaturated cycloalkyls). In some embodiments, cycloalkyls can be fused with aryls, heteroaryls, cycloalkyls, and heterocycloalkyls. In some embodiments, cycloalkyls can be fused with aryls, cycloalkyls, and heterocycloalkyls. In some embodiments, cycloalkyls can be fused with aryls and heterocycloalkyls. In some embodiments, cycloalkyls can be fused with aryls and cycloalkyls. Examples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norcamphanyl, norpinanyl, norcarnyl, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, and the like.

[0046] Unless otherwise specified, "alkoxy" refers to an alkyl group attached to the remainder of the molecule via an ether oxygen atom. Representative alkoxy groups are those having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, and tert-butoxy. As used herein, "alkoxy" includes unsubstituted and substituted alkoxy, particularly alkoxy substituted with one or more halogens. Preferred alkoxy are OCH3, OCF3, CHF2O, CF3CHO, i- PrO, n- PrO, i- BuO, n- BuO and t- BuO.

[0047] Unless otherwise specified, "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group. For example, a monocyclic aryl ring may be fused with one or more carbocyclic aromatic groups. Examples of aryl include, but are not limited to, phenyl, naphthyl, and phenanthryl.

[0048] Unless otherwise specified, "heterocycloalkyl" refers to a non-aromatic ring or ring system having at least one heteroatom ring member independently selected from boron, phosphorus, nitrogen, sulfur, oxygen, and phosphorus, and which may optionally contain one or more alkenylene moieties as part of the ring structure. Partially unsaturated heterocycloalkyls may be referred to as "heterocycloalkenyls" when the heterocycloalkyl contains at least one double bond, and "heterocycloalkynyls" when the heterocycloalkyl contains at least one triple bond. Heterocycloalkyls may include monocyclic, bicyclic, spirocyclic, or polycyclic systems (e.g., having two fused or bridged rings). In some embodiments, heterocycloalkyls are monocyclic groups having 1, 2, or 3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. The ring carbon atoms and heteroatoms of a heterocycloalkyl may be optionally oxidized to form oxo or sulfide groups or other oxidized bonds (e.g., C(O), S(O), C(S), or S(O), N-oxide, etc.), or the nitrogen atom may be quaternized. A heterocycloalkyl may be bonded through a ring carbon atom or a ring heteroatom. In some embodiments, a heterocycloalkyl contains 0 to 3 double bonds. In some embodiments, a heterocycloalkyl contains 0 to 2 double bonds. Also included within the definition of heterocycloalkyl are moieties having one or more aromatic rings fused (i.e., sharing a bond) to the heterocycloalkyl ring, e.g., benzo derivatives such as piperidine, morpholine, azepine, and thienyl. A heterocycloalkyl containing a fused aromatic ring may be bonded through any ring atom, including a ring atom of the fused aromatic ring.Examples of heterocycloalkyl include azetidinyl, azepinyl, dihydrobenzofuryl, dihydrofuryl, dihydropyranyl, N-morpholinyl, 3-oxa-9-azaspiro[5.5]undecyl, 1-oxa-8-azaspiro[4.5]decyl, piperidinyl, piperazinyl, oxopiperazinyl, pyranyl, pyrrolidinyl, quininyl, tetrahydrofuryl, tetrahydropyranyl, 1,2,3,4-tetrahydroquinolinyl, tropanyl, 4,5,6,7-tetrahydrothiazolo[5,4-c]pyridinyl, 4,5,6,7-tetrahydro-1H-imidazo[4, 5-c]pyridine, N-methylpiperidinyl, tetrahydroimidazolyl, pyrazolidinyl, butyrolactam, valerolactam, imidazolidinonyl, hydantoinyl, dioxolanyl, phthalimidyl, pyrimidine-2,4(1H,3H)-dione, 1,4-dioxanyl, morpholinyl, thiomorpholinyl, thiomorpholinyl-S-oxide, thiomorpholinyl-S,S-oxide, piperazinyl, pyranyl, pyridonyl, 3-pyrrolinyl, thiopyranyl, pyronyl, tetrahydrothienyl, 2-azaspiro[3.3]heptanyl, indolinyl, [ka] These include, but are not limited to:

[0049] Unless otherwise specified, "halogen" (or halo) refers to fluorine, chlorine, bromine, or iodine. The term "halo" (or "halogenated") before a group name indicates that the group is partially or fully halogenated, i.e., substituted by F, Cl, Br, or I, preferably F or Cl, in any combination.

[0050] "Optional" or "optionally" means that the subsequently described event or circumstance may occur, but does not necessarily occur, and the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0051] The substituent "-O-CH2-O-" means that the two oxygen atoms in the substituent are bonded to two adjacent carbon atoms in a heterocycloalkyl, aryl, or heteroaryl, for example: [ka]

[0052] When the number of a linker group is 0, such as -(CH2)0-, it means that the linker group is a single bond.

[0053] When one of the variables is selected from a chemical bond, it means that the two groups linked by this variable are directly linked. For example, when L in XLY represents a chemical bond, it actually means that the structure is XY.

[0054] Unless otherwise indicated, the absolute configuration of the stereocenter is represented by a solid wedge bond. [ka] and wedge-shaped dashed bond [ka] The relative configuration of the stereocenters is represented by a straight solid bond. [ka] and straight dashed bond [ka] It is expressed as:

[0055] Wavy line [ka] is a solid wedge connection [ka] or wedge-shaped dashed bond [ka] or a wavy line [ka] is a straight solid line connection [ka] or straight dashed bond [ka] Represents.

[0056] Unless otherwise indicated, single or double bonds are [ka] It is expressed by:

[0057] Specific pharmaceutical and medical terms As used herein, the term "acceptable" means that the formulation or active ingredient does not have excessively deleterious effects on the health of the general subject being treated.

[0058] As used herein, the terms "treatment," "course of treatment," or "treatment" include alleviating, inhibiting, or ameliorating a symptom or condition of a disease, inhibiting the development of a complication, improving or preventing the underlying metabolic syndrome, inhibiting the development of a disease or condition (e.g., controlling the progression of a disease or condition), alleviating a disease or symptom, relieving a disease or symptom, alleviating a complication caused by a disease or condition, or preventing or treating a symptom caused by a disease or condition. As used herein, a compound or pharmaceutical composition, when administered, can ameliorate a disease, symptom, or condition, and in particular, can improve the severity, delay the onset, slow the progression, or shorten the duration of a disease. Fixed or temporary administration, or continuous or intermittent administration, can result from or relate to administration.

[0059] The term "active ingredient" refers to a compound of formula (1) and pharmaceutically acceptable inorganic or organic salts of a compound of formula (1). The compounds of the present invention may contain one or more asymmetric centers (chiral centers or axial asymmetry) and therefore may exist in the form of racemates, racemic mixtures, single enantiomers, diastereomeric compounds, and single diastereomers. The asymmetric centers that may be present depend on the properties of the various substituents on the molecule. Each of these asymmetric centers independently generates two optical isomers, and all possible optical isomers, diastereomeric mixtures, and pure or partially pure compounds are within the scope of the present invention. The present invention is meant to include all such isomeric forms of these compounds.

[0060] As used herein, terms such as "compound," "composition," "drug," or "medicine or pharmaceutical agent" are used interchangeably and refer to any compound or composition that, when administered to an individual (human or animal), is capable of eliciting a desired pharmacological and / or physiological response through local and / or systemic action.

[0061] The terms "administered, administering, or administration" as used herein refer to direct administration of a compound or composition, or the administration of a prodrug, derivative, analog, etc. of an active compound.

[0062] While the numerical ranges and parameters defining the broad scope of the present invention are approximations, the relevant values ​​set forth in specific embodiments are set forth herein as precisely as possible. However, any numerical value inherently contains standard deviations necessarily resulting from certain testing methods. Herein, the term "about" generally means that the actual numerical value is within a particular numerical value or range ±10%, 5%, 1%, or 0.5%. Alternatively, the term "about" indicates that the actual numerical value falls within an acceptable standard error of the mean, as would be understood by one of ordinary skill in the art. Except in experimental examples or unless otherwise indicated, all ranges, amounts, values, and percentages used herein (e.g., to describe amounts of materials, lengths of time, temperatures, operating conditions, proportions of amounts, etc.) are understood to be modified by the term "about." Accordingly, unless otherwise indicated, all numerical parameters set forth in this specification and the appended claims are approximations that may vary, if desired. At the very least, these numerical parameters should be construed as representing significant digits or as derived using conventional rounding rules.

[0063] Scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, unless otherwise defined herein. Furthermore, as used herein, singular nouns include their plural forms, and plural nouns as used herein also include their singular forms, unless contradictory to the context.

[0064] therapeutic use The present invention provides methods of treating diseases, including but not limited to cancer, using the compounds, antibody-drug conjugates or pharmaceutical compositions of the present invention.

[0065] In some embodiments, methods of treating cancer are provided, comprising administering to an individual in need thereof an effective amount of a pharmaceutical composition of any of the aforementioned compounds and antibody-drug conjugates. In other embodiments, the cancer is a blood cancer or solid tumor, including, but not limited to, leukemia, breast cancer, lung cancer, pancreatic cancer, colon cancer, bladder cancer, brain cancer, urothelial cancer, prostate cancer, liver cancer, ovarian cancer, head and neck cancer, gastric cancer, mesothelioma, or all cancer metastases.

[0066] Route of administration The compounds of the present invention and their pharmaceutically acceptable salts can be prepared into various formulations containing a safe and effective amount of the compounds of the present invention or their pharmaceutically acceptable salts and a pharmaceutically acceptable excipient or carrier, where "safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the compound is determined according to the age, condition, course of treatment, and other specific conditions of the subject to be treated.

[0067] A "pharmaceutically acceptable excipient or carrier" refers to one or more compatible solid or liquid fillers or gel substances that must be suitable for human use and have sufficient purity and low toxicity. "Compatible" here means that the components of the composition can be intermixed with the compounds of the present invention without significantly reducing the pharmaceutical efficacy of the compounds. Examples of pharmaceutically acceptable excipients or carriers include cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, or cellulose acetate), gelatin, talc, solid lubricants (e.g., stearic acid or magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween®), wetting agents (e.g., sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0068] The compounds of the present invention can be administered orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously) or topically.

[0069] Solid dosage forms for oral administration include capsules, tablets, pills, pulvises, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or the following ingredients: (a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retardants such as paraffin; (f) absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glycerol monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, and sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, said dosage forms may further comprise buffering agents.

[0070] Solid dosage forms such as tablets, sugar-coated tablets, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials known in the art.These may contain opacifying agents, and the active compound or compounds in such compositions can be delayed in certain parts of the digestive tract.The examples of embedding components that can be used include polymeric materials and wax-based materials.If necessary, the active compound can be formed into microcapsules with one or more of the above-mentioned excipients.

[0071] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0072] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0073] Suspensions may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methylate, and agar-agar, or mixtures of these substances.

[0074] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0075] Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, inhalants, etc. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier, and any preservatives, buffers, or propellants that may be required, as required.

[0076] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds. When the pharmaceutical composition of the present invention is used, a safe and effective amount of the compound of the present invention is administered to the mammal (e.g., human) to be treated, and the dosage is a pharmaceutically effective dosage. For a human weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 50 to 1000 mg. The specific dosage is determined taking into consideration factors such as the route of administration and the patient's health condition, which are well known to those skilled in the art.

[0077] The above-described features of the present invention or the features described in the embodiments can be arbitrarily combined. All features disclosed herein can be used in any composition, and various features disclosed herein can be replaced with any alternative features that serve the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely generic examples of equivalent or similar features. [Brief explanation of the drawings]

[0078] [Figure 1] 1 shows the results of antitumor activity in mice of Example 11 according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0079] (Detailed explanation) Various specific aspects, features and advantages of the above compounds, methods and pharmaceutical compositions will be described in detail below, which will make the content of the present invention clearer. It should be understood that the following detailed description and examples describe specific embodiments for reference. After reading the description of the present invention, those skilled in the art can make various changes or modifications to the present invention, and such equivalents also fall within the scope of the present invention as defined herein.

[0080] In all examples, 1H-NMR spectra were recorded on a Varian Mercury 400 nuclear magnetic resonance spectrometer, and chemical shifts were expressed in δ (ppm). Unless otherwise specified, 200–300 mesh silica gel was used for separation, and the ratio of eluents was by volume.

[0081] In the present invention, the following abbreviations are used: room temperature (RT, rt); aqueous solution (aq.); petroleum ether (PE); ethyl acetate (EA); dichloromethane (DCM); 1,4-dioxane (dioxane); methanol (MeOH); methyl tert-butyl ether (MTBE); ethanol (EtOH); tetrahydrofuran (THF); dimethylformamide (DMF); N-methylpyrrolidone (NMP); dimethyl sulfoxide (DMSO); triethylamine (TEA); diisopropylethylamine (DIPEA); 4-dimethylaminopyridine (DMAP); carbon tetrachloride (CCl4); palladium on carbon (Pd / C); Eaton's reagent (7.7% by weight of phosphorus pentoxide in methanesulfonic acid); iron powder (Fe); zinc powder (Zn); Raney nickel (Ranyi Ni); acetyl chloride (AcCl); acetic acid (AcOH); acetic anhydride (AcO); m-chloroperbenzoic acid (m-CPBA); butyl nitrite (n-BuNO); sodium nitrite (NaNO); sodium hydride (NaH); magnesium sulfate (MgSO); N-bromosuccinimide (NBS); p-toluenesulfonic acid monohydrate (TsOH.HO); sodium carbonate (NaCO); potassium carbonate (KCO); equivalent (eq); gram / milligram (g / mg); mole / millimole (mol / mmol); liter / milliliter (L / mL); minute (min(s)); time (h, hr, hrs); nitrogen (N); nuclear magnetic resonance (NMR); liquid crystal mass spectrometry (LC-MS); thin layer chromatography (TLC); preparative liquid chromatography (pre-HPLC). [Example]

[0082] Preparation Example 1: Synthesis of N-(5-amino-7-fluoro-8-methyl-4-oxothiochroman-3-yl)acetamide (A7-a)

[0083] [ka]

[0084] Step 1: Synthesis of methyl 3-((3-fluoro-2-methyl-5-nitrophenyl)thio)propanoate [ka]

[0085] A 50 mL three-neck flask was charged with A1-a (1.73 g, 10 mmol, 1 equiv.), methyl 3-mercaptopropanoate (1.8 g, 15 mmol, 1.5 equiv.), and NMP (10 mL). After dissolution, potassium carbonate (2 g, 15 mmol, 1.5 equiv.) was added and the mixture was stirred at 60 °C for 8 hours under an argon atmosphere. After cooling to room temperature, water (30 mL) was added to dilute the mixture. The precipitated solid was filtered and washed with water. The filter cake was separated by column chromatography (PE / EA = 1 / 12-1 / 7) to obtain a yellow solid A2-a (1.55 g, 56.8% yield). LC-MS: 274.2 [M+H] + .

[0086] Step 2: Synthesis of 3-((3-fluoro-2-methyl-5-aminophenyl)thio)propionic acid [ka]

[0087] A 250 mL three-neck flask was charged with A2-a (1.55 g, 5.67 mmol, 1 equiv.), iron powder (1.27 g, 22.69 mmol, 4 equiv.), ethanol (80 mL), and aqueous ammonium chloride solution (2.8 M, 28 mL, 5 equiv.). The mixture was stirred at 90 °C for 16 h under an argon atmosphere. After cooling to room temperature, the mixture was filtered through Celite and washed with ethanol. The filtrate was concentrated, and the crude product was diluted with water (30 mL) and extracted with EA (30 mL × 2). The organic phase was washed with saturated brine, dried over sodium sulfate, and concentrated to give the crude product (1.5 g, quantitative yield). LC-MS: 244.3 [M+H] + .

[0088] The crude product (1.5 g, 5.67 mmol, 1 equivalent) and 1,4-dioxane (15 mL) were added to a 50 mL three-neck flask. After dissolution, concentrated hydrochloric acid (37%, 10 mL) was added and the mixture was stirred at 65°C for 4 hours under an argon atmosphere. After cooling to room temperature, a 3N aqueous solution of sodium carbonate was added to adjust the pH to 5. The mixture was extracted with EA (30 mL x 2), and the organic phase was washed with saturated brine, dried over sodium sulfate, and concentrated. The crude product was slurried (EA / PE = 1 / 5) to give a white solid A3-a (985 mg, 75.8% yield over two steps). LC-MS: 228.2 [M H ] - .

[0089] Step 3: Synthesis of 5-amino-7-fluoro-8-methylthiochroman-4-one [ka]

[0090] A 50 mL three-neck flask was charged with A3-a (985 mg, 4.3 mmol, 1 equivalent) and Eaton's reagent (15 mL), and the mixture was stirred at 60°C for 1 hour under an argon atmosphere. After cooling to room temperature, the reaction mixture was poured into ice water, and the pH was adjusted to 8 by adding 3N aqueous sodium carbonate solution. The mixture was extracted with EA (30 mL x 2), and the organic phase was washed with saturated brine, dried over sodium sulfate, and concentrated to give crude product A4-a (1.05 g, quantitative yield). LC-MS: 212.3 [M+H]+ .

[0091] Step 4: Synthesis of N-(7-fluoro-8-methyl-4-oxothiochroman-5-yl)acetamide [ka]

[0092] A 50 mL three-neck flask was charged with A4-a (1.05 g, 4.3 mmol, 1 equiv.), DMAP (52.5 mg, 0.43 mmol, 0.1 equiv.), DCM (15 mL), acetyl chloride (674 mg, 8.6 mmol, 2 equiv.), and triethylamine (869 mg, 8.6 mmol, 2 equiv.) in an ice bath. The mixture was allowed to warm to room temperature and then stirred for 1 h until the starting material was completely consumed. The mixture was quenched with water (20 mL) and then separated. The aqueous phase was extracted with DCM (20 mL × 2), and the combined organic phases were washed with saturated brine, dried, and concentrated. The residue was subjected to column chromatography (EA / PE = 1 / 1) to give a yellow solid A5-a (910 mg, 89% yield for two steps). LC-MS: 254.3 [M+H] + .

[0093] Step 5: Synthesis of N,N'-(7-fluoro-8-methyl-4-oxothiochroman-3,5-diyl)diacetamide [ka]

[0094] Potassium tert-butoxide (191 mg, 1.7 mmol, 1.1 equiv.), anhydrous THF (5 mL), A5-a (375 mg, 1.48 mmol, 1 equiv.), and butyl nitrite (190 mg, 1.85 mmol, 1.25 equiv.) were sequentially added to a 50 mL three-neck flask at -20 °C. The mixture was heated to 5 °C and stirred for 2 h until the starting material was completely consumed. MTBE (15 mL) was added to the mixture, filtered, and the solid was dissolved in acetic acid (5 mL). Zinc powder (200 mg, 3.1 mmol, 2.1 equiv.) was added and the mixture was stirred at room temperature for 5 min. Acetic anhydride (1 mL) was added and the mixture was stirred for 2 h. The system was washed with MeOH / DCM (3 / 30 mL), concentrated, and the crude product was separated by column chromatography (EA / DCM = 1 / 10 to 1 / 5) to give a light brown solid A6-a (175 mg, 41%). LC-MS: 311.1 [M+H] + .

[0095] Step 6: Synthesis of N-(5-amino-7-fluoro-8-methyl-4-oxothiochroman-3-yl)acetamide [ka]

[0096] A 50 mL three-neck flask was charged with A6-a (175 mg, 0.56 mmol, 1 equivalent) and methanol / 1,4-dioxane (4 / 8 mL). After dissolution, concentrated hydrochloric acid (37%, 4 mL) was added and the mixture was stirred at 40°C for 2 hours under an argon atmosphere. After cooling to room temperature, 3N aqueous sodium carbonate solution was added to adjust the pH to 8. The mixture was filtered, and the solid was dried to give A7-a (137 mg, 91% yield). LC-MS: 269.2 [M+H] + .

[0097] Similar to the synthesis of A7-a, the intermediates shown in the table below are obtained.

[0098] [Table 1] TIFF2023552610000055.tif117168

[0099] Preparation Example 2: Synthesis of (S)-4-(2-fluoroethyl)-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (B7-a) [ka]

[0100] Step 1: Synthesis of ethyl 2-bromo-2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolan]-7-yl)acetate [ka]

[0101] B1-a (3 g, 9.9 mmol, 1 equiv.) was dissolved in DMF (75 mL), NBS (1.5 g, 12 mmol, 1.2 equiv.) and m-CPBA (170 mg, 1 mmol, 0.1 equiv.) were added, and the mixture was stirred overnight at room temperature, then poured into 500 mL of ice water and filtered. The solid was washed with water and dried to give a gray solid B2-a (3.8 g, equivalent yield). LC-MS: 383.2 [M+H] + .

[0102] Step 2: Synthesis of 1-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolan]-7-yl)-2-ethoxy-2-oxoethyltosyl-D-prolinate [ka]

[0103] B2-a (1 g, 2.6 mmol, 1 equiv.), sodium tosyl-D-prophosphate (1 g, 3.9 mmol, 1.5 equiv.), and K2CO3 (362 mg, 2.6 mmol, 1 equiv.) were dissolved in DMF (20 mL). The mixture was stirred at 65 °C under an argon atmosphere for 2 h until the starting material was completely consumed. The mixture was diluted with water (100 mL) and extracted with EA (100 mL × 3). The combined organic phase was washed twice with water, washed with saturated brine, dried, and concentrated. The residue was separated by column chromatography (EA / DCM = 1 / 6) to give white solid B3-a (1.1 g, 74% yield). LC-MS: 572.2 [M+H] + .

[0104] Step 3: Synthesis of (S)-2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolan-7-yl)-1-ethoxy-4-fluoro-1-oxobutan-2-yl tosyl-D-prolinate [ka]

[0105] B3-a (1 g, 1.7 mmol, 1 equiv.) was dissolved in DMF (20 mL), and NaH (101 mg, 60%, 2.5 mmol, 1.5 equiv.) was added in an ice bath. The mixture was allowed to warm to room temperature and stirred for 1 hour. 2-Fluoroiodoethane (1.5 g, 8.6 mmol, 5 equiv.) was added in an ice bath, and the mixture was allowed to warm to room temperature and stirred overnight. After the reaction was complete, the mixture was poured into ice water (100 mL) and extracted with EA (100 mL x 3). The combined organic phase was washed twice with water and saturated brine, dried, and concentrated. The residue was separated by column chromatography (EA / DCM = 1 / 6) to give 743 mg of crude product. This was subjected to pre-HPLC to give white solid B4-a (150 mg, 70% dehydrogenase, 15% yield). LC-MS: 618.2 [M+H] + .

[0106] Step 4: Synthesis of (S)-2-(6-(acetamidomethyl)-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolan]-7-yl)-1-ethoxy-4-fluoro-1-oxobutan-2-yl tosyl-D-prolinate [ka]

[0107] Raney nickel (600 mg, 50% water content) was added to a 50 mL three-neck flask, and the system was washed three times with HOAc. Under an argon atmosphere, a solution of B4-a (150 mg, 0.24 mmol, 1 equiv.) in AcO / HOAc (4 / 1 mL) was added, purged with hydrogen three times, and reacted at 65 °C for 3 hours and then filtered. The solid was washed with AcOH, and the filtrate was concentrated and separated by column chromatography (MeOH / DCM = 1 / 20) to give a colorless oily solution B5-a (130 mg, 83% yield). LC-MS: 664.2 [M+H] + .

[0108] Step 5: Synthesis of (S)-2-(6-(acetoxymethyl)-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2'-[1,3]dioxolan]-7-yl)-1-ethoxy-4-fluoro-1-oxobutan-2-yl tosyl-D-prolinate [ka]

[0109] B5-a (130 mg, 0.2 mmol, 1 equiv.) was dissolved in AcO / HOAc (3 / 1 mL), and NaNO (68 mg, 1 mmol, 5 equiv.) was added in an ice bath. The system was returned to room temperature and stirred for 1 hour. After the reaction was completed, the system was filtered, the solid was washed with AcOH, the filtrate was concentrated, CCl (15 mL) was added, and the mixture was stirred under reflux overnight. The system was washed with water and saturated brine, dried, and concentrated. The residue was separated by column chromatography (MeOH / DCM = 1 / 20) to obtain 90 mg of a colorless oily solution. This was subjected to pre-HPLC to obtain white solid B6-a (90 mg, 69% yield). LC-MS: 665.2 [M+H] + .

[0110] Step 6: Synthesis of (S)-4-(2-fluoroethyl)-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione [ka]

[0111] B6-a (90 mg, 0.14 mmol, 1 equiv.) was dissolved in EtOH (3 mL), and 1 N aqueous sodium carbonate solution (1 mL) was added. The mixture was stirred at room temperature for 1 hour until the starting material was completely consumed, then concentrated at room temperature and lyophilized. The crude product was dissolved in 85% aqueous TFA solution (5 mL) and stirred at 85°C for 1 hour. After the reaction was completed, the mixture was concentrated, and the crude product was separated by pre-HPLC to give white solid B7-a (7 mg, 17% yield, 68% de). LC-MS: 282.2 [M+H]+.

[0112] Similar to the synthesis of B7-a, the intermediates shown in the table below are obtained.

[0113] [Table 2]

[0114] Example 1: Synthesis of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,9,12,15-tetrahydro-13H-pyrano[3',4':6,7]indolizino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione (Compound 1) [ka]

[0115] Step 1: Synthesis of N-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[3',4':6,7]indolizino[1,2-b]thiopyrano[4,3,2-de]quinolin-1-yl)acetamide [ka]

[0116] A 50 mL three-neck flask was charged with A7-a (108 mg, 0.4 mmol, 1 equiv.), B7-b (156 mg, 0.6 mmol, 1.5 equiv.), p-toluenesulfonic acid monohydrate (45 mg, 0.24 mmol, 0.6 equiv.), anhydrous magnesium sulfate (1 g), and acetic acid (10 mL). The mixture was stirred at 105 °C under an argon atmosphere for 24 hours until the starting material was completely consumed. The mixture was filtered, and the filter cake was washed with EA and concentrated. The crude product was separated by column chromatography (MeOH / DCM = 1 / 40-1 / 20) to give a light brown solid B8-a (131 mg, 67%). LC-MS: 496.2 [M+H] + .

[0117] Step 2: Synthesis of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,9,12,15-tetrahydro-13H-pyrano[3',4':6,7]indolizino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione [ka]

[0118] B8-a (131 mg, 0.26 mmol, 1 equivalent) and 1,4-dioxane (5 mL) were added to a 50 mL three-neck flask. After dissolution, concentrated hydrochloric acid (37%, 5 mL) was added and the mixture was stirred at 80°C under an argon atmosphere for 24 hours. The mixture was cooled to room temperature and then concentrated. The crude product was slurried with ACN / EA (1 / 1) to give a brown solid, 1 (hydrochloride salt) (107 mg, 84.6% yield). LC-MS: 454.2 [M+H] + .

[0119] Similar to the synthesis of compound 1, the compounds shown in the table below are obtained.

[0120] [Table 3] TIFF2023552610000068.tif231168TIFF2023552610000069.tif42168

[0121] Example 2: Chiral separation of compound 1 Compound 1 is a mixture of a pair of diastereomers, and two diastereoisomers of compound 1, 1-1 and 1-2, can be obtained by employing methods of separation and purification by salification, recrystallization, or pre-HPLC. [ka]

[0122] Chromatography conditions: Preparative liquid chromatograph equipped with Shimadzu LC-20AP; chromatography column: Waters SunFire Prep C18 OBD (50 × 150 mm, 5 μm); mobile phase: acetonitrile-0.5‰ trifluoroacetic acid aqueous solution = 66:34; flow rate: 48.0 mL / min; detection wavelength: 254 nm; injection volume: 3000 μm.

[0123] The experimental procedure is as follows: An appropriate amount of 1 was taken and adjusted to a constant volume using 50% acetonitrile aqueous solution. A test sample solution with a concentration of 25 mg / mL was prepared. This test sample solution was taken and placed in a preparative liquid chromatograph for detection according to the chromatography conditions of the present invention, and data was recorded.

[0124] As a result, the solution was separated by pre-HPLC to give 1-1 (33 mg) and 1-2 (31 mg). The retention times of these two components were 5.419 minutes and 7.614 minutes, respectively, and their purities were 99.38% and 99.21%, respectively.

[0125] Similar to the chiral separation method for compound 1, the compounds shown in the table below can be obtained.

[0126] [Table 4] TIFF2023552610000072.tif193168

[0127] Example 3: Synthesis of Compound 32-1 and Compound 32-2 [ka]

[0128] The long-retention component 1-2 (98 mg, 0.2 mmol, 1 equiv.), 2-hydroxyacetic acid (18.4 mg, 0.24 mmol, 1.2 equiv.), anhydrous DCM (5 mL), HATU (84 mg, 0.3 mmol, 1.5 equiv.), and DIPEA (90.3 mg, 0.7 mmol, 3.5 equiv.) were sequentially added to a 50 mL three-neck flask under ice bath conditions. After maintaining the temperature for 0.5 h, the system was diluted with water (5 mL) and then extracted with DCM (5 mL x 2). The organic phase was washed with saturated brine, dried, and concentrated. The residue was subjected to column chromatography (MeOH / DCM = 1 / 40) to give compound 32-2 (75 mg, 73% yield).

[0129] 1H NMR (400 MHz, DMSO-d6) δ: 8.21 (dd, J = 13.3, 8.5 Hz, 1H), 7.78 (d, J = 10.6 Hz, 1H), 7.31 (d, J = 1.2 Hz, 1H), 6.53 (s, 1H), 5.89-5.79 (m, 1H), 5.56-5.49 (m, 1H), 5.43 (s, 2H), 5.38 (s, 1H), 5.31 (d, J = 4.8 Hz, 1H), 3.90 (d, J = 4.7 Hz, 2H), 3.50 -3.35 (m, 3H), 2.44 (s, 3H), 1.91-1.80 (m, 2H), 0.87 (t, J = 6.5 Hz, 3H), LC-MS: 512.2 [M+H] + .

[0130] Similar to the synthesis of compound 32-2, the diastereoisomer 32-1 of compound 32-2 can be obtained by using another component 1-1 with a shorter retention time.

[0131] Similar to the synthesis of compound 32-1 and compound 32-2, by using different intermediates, the compounds shown in the table below can be obtained.

[0132] [Table 5] TIFF2023552610000075.tif237168TIFF2023552610000076.tif236168TIFF2023552610 000077.tif222168TIFF2023552610000078.tif247168TIFF2023552610000079.tif81168

[0133] Example 4: Preparation of antibody drug conjugate (ADC-1) [ka]

[0134] Step 1: Preparation of compound LD-1 [ka]

[0135] L-1 (76 mg, 0.12 mmol, 1.0 equiv.), the long-retention component compound 1-2 (55 mg, 0.12 mmol, 1.0 equiv.), NMI (50.6 mg, 0.62 mmol, 5.0 equiv.), and DMF (2 mL) were added to a 50 mL single-neck flask. The mixture was stirred vigorously and then cooled to 0 °C. TCFH (41.5 mg, 0.15 mmol, 1.2 equiv.) was added to the reaction mixture, which was stirred for 30 min and then analyzed by LC-MS. After completion of the reaction, the reaction mixture was purified by reverse-phase C18 column chromatography (MeCN / water = 0-60%). The target fraction was lyophilized to obtain a yellow solid, LD-1 (80 mg, 61.5% yield).

[0136] 1H-NMR (400 MHz, DMSO-d6) δ: 8.56 (t, J = 6.4 Hz, 1H), 8.30 (dd, J = 14.0, 8.3 Hz, 2H), 8.11 (d, J = 7.9 Hz, 1H), 8.06 (t, J = 5.7 Hz, 1H), 7.99 (t, J = 5.7 Hz, 1H), 7.76 (d, J = 10.7 Hz, 1H), 7.31 (d, J = 2.8 Hz, 1H), 7.27-7.12 (m, 5H), 6.98 (s, 2H), 6.54 (d, J = 1.8 Hz, 1H), 5.84-5.83 (m, 1H), 5.49-5.31 (m, 3H), 5.27-5.21 (m, 1H), 4.66-4.53 (m, 2H), 4.48-4.41 (m, 1H), 3.98 (s, 2H), 3.75-3.54 (m, 6H), 3.42 (s, 2H), 3.36-3.35 (m, 2H), 3.04-2.98 (m, 1H), 2.80-2.74 (m, 1H), 2.42 (s, 3H), 2.08 (t, J = 7.4, 2H), 1.93-1.79 (m, 2H), 1.50-1.41 (dd, J = 13.2, 5.9 Hz, 4H), 1.25-1.12 (m, 2H), 0.88-0.84 (m, 3H), LC-MS: 1052.1 [M+H] + , 1050.1 [MH] - .

[0137] Step 2: Preparation of ADC-1

[0138] The prepared tris(2-carboxyethyl)phosphine aqueous solution (10 mM, 0.082 mL) was added to a PBS buffer solution of the antibody trastuzumab (0.05 PBS buffer, pH 6.5, 2.5 mL, 9.96 mg / mL, 0.168 nmol) at 37°C, and the system was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL.

[0139] Compound LD-1 (2.02 nmol) was dissolved in DMSO (0.10 mL) and added to 2.0 mL of the above solution. The system was placed in a water bath shaker and shaken at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer, pH 6.5, containing 0.001 M EDTA) to obtain ADC in PBS buffer (5.0 mg / mL, 1.1 mL). This was frozen and stored at 4°C. Average value calculated by UV-HPLC: n = 7.2.

[0140] Example 5: Preparation of antibody drug conjugate (ADC-2) [ka]

[0141] Step 1: Preparation of compound LD-2 [ka]

[0142] L-1 (76 mg, 0.12 mmol, 1.0 equiv.), the short-retention component compound 1-1 (55 mg, 0.12 mmol, 1.0 equiv.), NMI (50.6 mg, 0.62 mmol, 5.0 equiv.), and DMF (2 mL) were added to a 50 mL single-neck flask. The mixture was stirred vigorously and then cooled to 0 °C. TCFH (41.5 mg, 0.15 mmol, 1.2 equiv.) was added to the reaction mixture, which was stirred for 30 min and then analyzed by LC-MS. After completion of the reaction, the reaction mixture was purified by reverse-phase C18 column chromatography (MeCN / water = 0-60%). The fraction containing the target substance was lyophilized to obtain LD-2 (80 mg, 61.5% yield) as a yellow solid.

[0143] 1 H-NMR (400 MHz, DMSO-d6) 1H-NMR (400 MHz, DMSO-d6) δ: 8.56 (t, J = 6.4 Hz, 1H), 8.30 (dd, J = 14.0, 8.3 Hz, 2H), 8.11 (d, J = 7.9 Hz, 1H), 8.06 (t, J = 5.7 Hz, 1H), 7.99 (t, J = 5.7 Hz, 1H), 7.76 (d, J = 10.7 Hz, 1H), 7.31 (d, J = 2.8 Hz, 1H), 7.30-7.16 (m, 5H), 6.98 (s, 2H), 6.54 (d, J = 1.8 Hz, 1H), 5.90-5.86 (m, 1H), 5.46-5.30 (m, 3H), 5.26-5.21 (m, 1H), 4.68-4.53 (m, 2H), 4.46-4.41 (m, 1H), 3.98 (s, 2H), 3.75-3.54 (m, 6H), 3.42 (s, 2H), 3.36-3.35 (m, 2H), 3.04-2.98 (m, 1H), 2.80-2.74 (m, 1H), 2.42 (s, 3H), 2.08 (t, J = 7.4, 2H), 1.93-1.79 (m, 2H), 1.50-1.41 (dd, J = 13.2, 5.9 Hz, 4H), 1.25-1.12 (m, 2H), 0.88-0.84 (m, 3H), LC-MS: 1052.1 [M+H] + , 1050.1 [MH] - .

[0144] Step 2: Preparation of ADC-2 The prepared tris(2-carboxyethyl)phosphine aqueous solution (10 mM, 0.082 mL) was added to a PBS buffer solution of the antibody trastuzumab (0.05 PBS buffer, pH 6.5, 2.5 mL, 9.96 mg / mL, 0.168 nmol) at 37°C, and the system was placed in a water bath shaker and shaken at 37°C for 3 hours to react, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL.

[0145] Compound LD-2 (2.02 nmol) was dissolved in DMSO (0.10 mL) and added to 2.0 mL of the above solution. The system was placed in a water bath shaker and shaken at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer, pH 6.5, containing 0.001 M EDTA) to obtain ADC in PBS buffer (5.0 mg / mL, 1.1 mL). This was frozen and stored at 4°C. Average value calculated by UV-HPLC: n = 7.2.

[0146] Example 6: Preparation of antibody drug conjugates (ADC-3 and ADC-4) [ka]

[0147] Step 1: Preparation of Compound LD-3 and Compound LD-4 [ka]

[0148] L-2 (80 mg, 0.12 mmol, 1.0 equiv.), the short-retention component compound 1-1 (55 mg, 0.12 mmol, 1.0 equiv.), NMI (50.6 mg, 0.62 mmol, 5.0 equiv.), and DMF (2 mL) were added to a 50 mL single-neck flask. The mixture was stirred vigorously and then cooled to 0 °C. TCFH (41.5 mg, 0.15 mmol, 1.2 equiv.) was added to the reaction mixture, which was stirred for 30 minutes and then analyzed by LC-MS. After completion of the reaction, the reaction mixture was purified by reverse-phase C18 column chromatography to obtain two fractions: a short-retention fraction (LD-3) and a long-retention fraction (LD-4). The fraction containing the target substance was lyophilized to obtain yellow solids, LD-3 (20 mg) and LD-4 (25 mg). LC-MS: 1092.4 [M+H] + .

[0149] Chromatography conditions: Thermo Fisher Semi-Preparative Liquid Chromatograph U3000; Chromatography column: Welch Ultimate XB-Phenyl; Mobile phase: acetonitrile containing 0.1% formic acid - 0.1% formic acid aqueous solution = 50:50; Flow rate: 30.0 mL / min; Detection wavelength: 370 nm; Injection volume: 100 μL.

[0150] The experimental procedure was as follows: A suitable amount of the mixture of LD-3 and LD-4 was taken and dissolved in DMF. A test sample solution with a concentration of 10 mg / mL was prepared. The test sample solution was taken and placed in a preparative liquid chromatograph for detection according to the chromatographic conditions of the present invention, and data was recorded. Multiple injections were performed.

[0151] As a result, LD-3 and LD-4 were separated by pre-HPLC, and the retention times of both components were 5.29 minutes and 5.87 minutes, respectively, and their purities were 99.38% and 99.21%, respectively.

[0152] Step 2: Preparation of ADC-3 and ADC-4 The prepared aqueous tris(2-carboxyethyl)phosphine solution (10 mM, 0.082 mL) was added to the antibody trastuzumab in PBS buffer (0.05 M PBS buffer, pH 6.5; 2.5 mL, 9.96 mg / mL, 0.168 nmol) at 37°C. The system was placed in a water bath shaker and shaken at 37°C for 3 hours to react, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL. Two aliquots were prepared in parallel.

[0153] Compound LD-3 (2.0 nmol) was dissolved in DMSO (0.10 mL) and added to 2.0 mL of the above solution. The system was placed in a water bath shaker and shaken at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer, pH 6.5, containing 0.001 M EDTA) to obtain ADC in PBS buffer (5.0 mg / mL, 1 mL). This was frozen and stored at 4°C. Average value calculated by UV-HPLC: n = 7.3.

[0154] ADC-4 was similarly prepared using compound LD-4 (n=7.3).

[0155] Example 7: Preparation of antibody drug conjugates (ADC-5 and ADC-6) [ka]

[0156] Step 1: Preparation of Compound LD-5 and Compound LD-6 [ka]

[0157] L-2 (80 mg, 0.12 mmol, 1.0 equiv.), the long-retention component compound 1-2 (55 mg, 0.12 mmol, 1.0 equiv.), NMI (50.6 mg, 0.62 mmol, 5.0 equiv.), and DMF (2 mL) were added to a 50 mL single-neck flask. The mixture was stirred vigorously and then cooled to 0 °C. TCFH (41.5 mg, 0.15 mmol, 1.2 equiv.) was added to the reaction mixture, which was stirred for 30 minutes and then analyzed by LC-MS. After completion of the reaction, the reaction mixture was purified by reverse-phase C18 column chromatography to obtain two fractions: a short-retention fraction designated LD-5 and a long-retention fraction designated LD-6. The fraction containing the target substance was lyophilized to obtain yellow solids, LD-5 (20 mg) and LD-6 (23 mg). LC-MS: 1092.4 [M+H] + .

[0158] Chromatography conditions: Thermo Fisher Semi-Preparative Liquid Chromatograph U3000; Chromatography column: Welch Ultimate XB-Phenyl; Mobile phase: acetonitrile containing 0.1% formic acid - 0.1% formic acid aqueous solution = 50:50; Flow rate: 30.0 mL / min; Detection wavelength: 370 nm; Injection volume: 100 μL.

[0159] The experimental procedure is as follows: An appropriate amount of the mixture of LD-5 and LD-6 was taken and dissolved in DMF. A test sample solution with a concentration of 10 mg / mL was prepared. The test sample solution was taken and placed in a preparative liquid chromatograph for detection according to the chromatographic conditions of the present invention, and data was recorded. Multiple injections were performed.

[0160] As a result, LD-5 and LD-6 were separated by pre-HPLC, and the retention times of both components were 6.04 and 6.48 minutes, respectively, and their purities were 98.58% and 99.13%, respectively.

[0161] Step 2: Preparation of ADC-5 and ADC-6 The prepared aqueous tris(2-carboxyethyl)phosphine solution (10 mM, 0.082 mL) was added to a PBS buffer solution of the antibody trastuzumab (0.05% PBS buffer, pH 6.5; 2.5 mL, 9.96 mg / mL, 0.168 nmol) at 37°C. The system was placed in a water bath shaker and shaken at 37°C for 3 hours to react, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL. Two aliquots were prepared in parallel.

[0162] Compound LD-5 (2.0 nmol) was dissolved in DMSO (0.10 mL) and added to 2.0 mL of the above solution. The system was placed in a water bath shaker and shaken at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer, pH 6.5, containing 0.001 M EDTA) to obtain ADC in PBS buffer (5.0 mg / mL, 1.1 mL). This was frozen and stored at 4°C. Average value calculated by UV-HPLC: n = 7.3.

[0163] ADC-6 was prepared in a similar manner using compound LD-6 (n=7.3).

[0164] Example 8: Other ADCs Other compounds similar to LD-1, LD-2, LD-3, LD-4, LD-5, or LD-6 (the camptothecin derivatives of the present application are small molecule toxins) can be prepared in a similar manner. LD-1, LD-2, LD-3, LD-4, LD-5, or LD-6, and similar compounds, can be further combined with the antibody trastuzumab or other similar antibodies to prepare antibody-drug conjugates containing the camptothecin derivatives of the present application as small molecule toxins.

[0165] Example 9: Assay of antiproliferative activity against SK-BR-3 cells The activity of the antibody-drug conjugates of the present invention can be measured by assaying the in vitro antiproliferative activity of camptothecin derivatives as small molecule toxins against SK-BR-3 cells.

[0166] SK-BR-3 cells were seeded in a 384-well plate (Fisher 142762) at 3,000 cells per well. The next day, serially diluted compounds were added, and 72 hours later, intracellular ATP content was measured using CellTiter-Lumi (Beyotime C0068XL). Cell proliferation was assessed, and the relative IC of compounds on cell proliferation was calculated. 50 The screening results are shown in Table 6.

[0167] [Table 6]

[0168] Compared with commercially available camptothecin drugs, exatecan, deruxtecan, and topotecan, the compounds of the present invention have potent in vitro antiproliferative activity against SK-BR-3 cells, particularly when X in general formula (1) is S or O. For example, compounds 1-2 are twice as active as exatecan, and compound 32 is three times more active than deruxtecan. In particular, compounds of general formula (1) having a group that is easy to bind, such as OH or NH, in the side chain and having potent cellular activity are suitable for use as small molecule toxins in ADCs.

[0169] Example 10: In vitro antitumor activity of antibody-drug conjugates of the present invention SK-BR-3 cells, which highly express HER2, were selected as the cell line for in vitro activity detection in experiments and used to evaluate the dose-effect relationship of the antibody-drug conjugate (ADC) of the present invention on cell killing. The plating density of each type of cell was initially selected to be 1500-2000 cells / well, and cell cytotoxicity assays were performed after 12 hours. ADC was added at a starting concentration of 10 nM and serially diluted 3-10 times to obtain the final concentration. The killing effect was observed for 144 hours. Chemiluminescent staining was performed using the CellTiter-Glo@Luminescent Cell Viability Assay, and the IC was calculated after reading the fluorescence data. 50 was calculated.

[0170] Activity tests showed that all ADCs exhibited a certain degree of antitumor activity, and the activity of some ADCs was higher than that of DS-8201a.

[0171] [Table 7]

[0172] Activity tests showed that all ADCs exhibited a certain degree of antitumor activity, and the activity of some ADCs was higher than that of DS-8201a.

[0173] Example 11: In vivo antitumor activity of antibody-drug conjugates of the present invention Human gastric cancer cells (NCI-N87) dissolved in 100 μL of PBS solution were subcutaneously injected into the right side of the neck or back of 6- to 8-week-old female Balb / c nude mice. The average tumor volume was approximately 150-200 mm. 3At this point, 32 nude mice were randomly divided into four groups based on tumor size, with eight mice per group. They were administered intravenously via tail vein. Group 01 was the blank control group, Group 02 was the DS-8201a (4.5 mg / kg) group, Group 03 was the ADC-1 (4.5 mg / kg) group, and Group 04 was the ADC-2 (4.5 mg / kg) group. Animal weights and tumor volumes were measured twice weekly, and animal survival was monitored throughout the experiment. The specific results of tumor volume changes in each group are shown in Figure 1.

[0174] As can be seen from Figure 1, both ADC samples of the invention exhibited in vivo antitumor activity comparable to that of DS-8201a.

[0175] Although specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that these embodiments are merely exemplary and that many changes or modifications can be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A camptothecin derivative compound of general formula (1) or an optical isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate, or a solvate thereof. 【Chemistry 1】 (wherein, in general formula (1), m is an integer of 0, 1, or 2; X is selected from —O— and —S—; R1 is F; R ２ is H, halogen, OH, Me, Et, OMe, OEt, CF3, NH ２ , NO ２ , and CN; R ３ is Me, Et, and the group of the following formula: 【Chemistry 2】 is selected from R ４ is H, C １～６ Alkyl and C １～６ haloalkyl; A partial structure in general formula (1) of the following formula: 【Transformation 3】 teeth, Based on the formula below 【Chemistry 4】 is selected from

2. The compound has the structure 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 2. The camptothecin derivative compound of formula (1) according to claim 1, wherein the compound is one of the following compounds:

3. The compound has the structure 【Chemistry 6-1】 【Chemistry 6-2】 【Transformation 6-3】 2. The camptothecin derivative compound of formula (1) according to claim 1, wherein the compound is one of the following compounds:

4. The compound has the structure 【Chemistry 7-1】 【Chemistry 7-2】 or an optical isomer thereof, a crystalline form, a pharmaceutically acceptable salt, a hydrate, or a solvate thereof, wherein the compound is one of the following:

5. The compound has the structure 【Transformation 8】 or an optical isomer, crystalline form, pharmaceutically acceptable salt, hydrate, or solvate thereof.

6. a pharmaceutically acceptable excipient or carrier; A pharmaceutical composition comprising, as an active ingredient, the camptothecin derivative compound according to any one of claims 1 to 5, or an optical isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate, or a solvate thereof.

7. An antibody-drug conjugate comprising: The structure of the formula below 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 is one of the compounds wherein Ab represents an anti-her2 antibody and n is 2 to 8.

8. The antibody-drug conjugate of claim 7, wherein Ab represents trastuzumab and n is 4 to 8.

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