Camptothecin compound and use thereof

Abstract

The present invention relates to a compound represented by formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof, a pharmaceutical composition comprising same, and a use thereof in the prevention or treatment of cancers and / or tumors and diseases related thereto. The compound has good activity, and a lactone ring of the compound is more stable; in addition, the compound has a low efflux ratio and better safety, and has significant drug-forming potential.

Description

A camptothecin-type compound and its application Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to a novel camptothecin analogue, pharmaceutical compositions comprising the same, and their use in the preparation of pharmaceuticals. Background Technology

[0002] DNA topoisomerases are a class of enzymes found in the cell nucleus that catalyze the breaking and binding of DNA strands, thereby controlling the topological state of DNA. Topoisomerases participate in the regulation of supercoiled template structures. DNA topoisomerase I catalyzes changes in the topological isomerism of DNA replication by forming a brief single-strand cleavage-binding cycle. Camptothecin, a pentacyclic quinoline alkaloid originally isolated from the bark and stems of the Chinese camptotheca, is one of the important lead compounds in the development of anticancer drugs. Its structure is a planar pentacyclic system, including a quinoline ring AB, a pyrrole ring C, a pyridone ring D, and an α-hydroxylactone ring E, with the 20th chiral carbon in the S configuration serving as the active site of camptothecin. Camptothecin is a Topo I inhibitor; it forms a stable Topo I-DNA-CPT ternary complex by binding to the Topo I-DNA binary complex, inhibiting DNA rejoining, causing DNA damage, and ultimately leading to cell death. To address the drawbacks of camptothecin, such as poor solubility, stability, and high toxicity, camptothecin derivatives can be improved by introducing water-soluble groups or preparing prodrugs to increase water solubility and thus improve drug properties. Several camptothecin derivatives, such as topotecan and irinotecan, have already been approved for marketing and are used to treat solid tumors. Developed by Daiichi Sankyo, the irinotecan derivative Dxd is a highly effective TopoI inhibitor. As an effective payload for antibody-drug conjugates (ADCs), it selectively kills tumor cells through antibody targeting. Compared to traditional chemotherapy drugs, ADCs can more precisely kill tumor cells while reducing toxicity to normal tissues. Several ADC drugs using Dxd as the payload have been approved for marketing or entered clinical trials, demonstrating excellent efficacy and manageable safety in solid tumors such as breast cancer and non-small cell lung cancer. However, Dxd is a substrate for the transporters P-gp and BCRP, making it easily effluxed by tumor cells, reducing its efficacy. Meanwhile, Dxd exhibits poor sensitivity in killing gastrointestinal tumor cells, which may be associated with the development of interstitial lung disease. Therefore, developing novel camptothecin derivatives that are sensitive to the gastrointestinal tract and have low efflux as effective payloads for ADCs is of great significance and has promising application prospects.

[0003] Invention Overview

[0004] Through extensive research, the inventors have surprisingly discovered a camptothecin derivative, a prodrug toxin used in the preparation of ADC drugs. This camptothecin derivative exhibits good activity, a more stable lactone ring, low efflux rate, and better safety, demonstrating significant drug development potential.

[0005] One aspect of the present invention provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the compound has the structure of formula (I):

[0006] in,

[0007] X is selected from CR 3 R 4 , O, S or NR 5 ;

[0008] Ring A is selected from C 3-6 cycloalkyl or 3-6 membered heterocycloalkyl;

[0009] R 1 and R 2 Independently selected from halogens, amino (-NH2), hydroxyl (-OH), mercapto (-SH), CN, NO2, C 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Alkylthio; or

[0010] R 1 and R 2 C forms a straight chain 3-4 alkylene, wherein the C 3-4 One or two non-adjacent CH2 groups in an alkylene group are independently and optionally coated with O, S, or NR. 6 Substitute;

[0011] R 3 R 4 R 5 and R 6 Each is independently selected from H and C. 1-3 alkyl;

[0012] R is independently selected from halogen, hydroxyl, amino, mercapto, CN and NO2;

[0013] n is selected from 0, 1, 2, or 3;

[0014] m is selected from 1, 2, 3, or 4; and

[0015] key for or contain A mixture.

[0016] Another aspect of the invention provides a pharmaceutical composition comprising a preventive or therapeutically effective amount of the compound of the invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, and one or more pharmaceutically acceptable carriers.

[0017] Another aspect of the invention provides the use of the compounds of the invention or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, or pharmaceutical compositions of the invention in the preparation of medicaments for the prevention or treatment of cancer and / or tumors and related conditions.

[0018] Another aspect of the present invention provides compounds of the present invention or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, or pharmaceutical compositions of the present invention for the prevention or treatment of cancer and / or tumors and related conditions.

[0019] Another aspect of the present invention provides a method for preventing or treating cancer and / or tumors and related conditions, the method comprising administering to an individual in need an effective amount of the compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, or pharmaceutical composition of the present invention.

[0020] In another aspect, the present invention provides the use of the compounds of the present invention or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, or pharmaceutical compositions of the present invention in the preparation of ADC drugs.

[0021] Invention Details

[0022] definition

[0023] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to better explain the invention.

[0024] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0025] As used herein, the term "alkyl" is defined as a linear or branched saturated aliphatic hydrocarbon. In some embodiments, the alkyl group has 1 to 12, for example, 1 to 6 carbon atoms. For example, as used herein, the term "Cnalkyl" refers to a linear or branched group with 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or n-hexyl), optionally substituted with one or more (e.g., 1 to 3) suitable substituents such as halogens (in which case the group is referred to as "haloalkyl") (e.g., CH2F, CHF2, CF3, CCl3, C2F5, C2C15, CH2CF3, CH2Cl, or -CH2CH2CF3, etc.). The term "CMalkyl" refers to a linear or branched aliphatic hydrocarbon chain with 1 to 4 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl).

[0026] As used herein, the term "alkenyl" refers to a linear or branched monovalent hydrocarbon group containing a double bond; for example, "Cn-alkenyl" is an alkenyl group having 2-6 carbon atoms. The alkenyl group is, for example, vinyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, and 4-methyl-3-pentenyl. When the compounds of the present invention contain an alkenyl group, the compounds may exist in pure E (iso-side), pure Z (iso-side), or any mixture thereof.

[0027] As used herein, the term "alkynyl" refers to a monovalent hydrocarbon group containing one or more triple bonds; for example, "Cn-alkynyl" is an alkynyl group having 2 to 6 carbon atoms. Examples of alkynyl groups include ethynyl or propynyl.

[0028] As used herein, the term "cycloalkyl" refers to a saturated ("cycloalkyl") or unsaturated (i.e., having one or more double and / or triple bonds within the ring) monocyclic or polycyclic hydrocarbon ring having, for example, 3 to 10 (suitably 3 to 8, more preferably 3 to 6) cyclic carbon atoms, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclohexenyl, etc.

[0029] As used herein, the term "heterocyclic group" refers to a saturated ("heterocyclic alkyl") or partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) cyclic group having, for example, 3 to 10 (suitably 3 to 8, more preferably 3 to 6) ring atoms, wherein at least one ring atom is a heteroatom selected from N, O, S, and P, and the remaining ring atoms are C. For example, "3 to 10-membered heterocyclic group" is a saturated or partially unsaturated heterocyclic group having 2 to 9 (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) heteroatoms independently selected from N, O, and S. Examples of heterocyclic groups include, but are not limited to: ethylene oxide, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolinyl, pyrrolylyl, pyrrolidoneyl, imidazoalkyl, pyrazolyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, or trithianyl. The groups also encompass bicyclic systems, including spirocyclic, fused, or bridged systems (such as 8-azaspiro[4.5]decane, 3,9-diazaspiro[5.5]undecane, 2-azabicyclo[2.2.2]octane, etc.). The heterocyclic group may optionally be substituted with one or more (e.g., 1, 2, 3, or 4) suitable substituents.

[0030] As used herein, the term "aryl" refers to a monocyclic or polycyclic aromatic group with a conjugated electron system. For example, as used herein, the term "C WD "Aryl" refers to an aromatic group containing 6 to 10 carbon atoms, such as phenyl or naphthyl. The aryl group may optionally be replaced by one or more (such as 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, -NO2, Cw alkyl, etc.).

[0031] As used herein, the term "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic aromatic ring system. For example, "5-14 membered heteroaryl" refers to a system having 5, 6, 8, 9, 10, 11, 12, 13, or 14 ring atoms, particularly 1, or 2, or 3, or 4, or 5, or 6, or 9, or 10 carbon atoms, and containing at least one heteroatom that may be the same or different (the heteroatom being, for example, oxygen, nitrogen, or sulfur), and additionally, in each case, may be benzofused. Examples of "heteroaryl" include, but are not limited to: thienyl, furanyl, pyrroleyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiazolyl, etc., and their benzo[derivatives]; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., and their benzo[derivatives].

[0032] As used herein, the term “halogenated” or “halogenated” is defined as including F, Cl, Br, or I.

[0033] The term "substitution" refers to the selective replacement of one or more (e.g., one, two, three, or four) hydrogen atoms on a specified atom by a designated group, provided that the substitution does not exceed the normal valence of the specified atom in the present case and that the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound.

[0034] If a substituent is described as “optionally substituted,” then the substituent may be (1) unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted with one or more of the substituents in the list, then one or more hydrogens on the carbon (to the extent that any hydrogens are present) may be substituted individually and / or together with independently selected optional substituents. If the nitrogen of the substituent is described as being optionally substituted with one or more of the substituents in the list, then one or more hydrogens on the nitrogen (to the extent that any hydrogens are present) may each be substituted with independently selected optional substituents.

[0035] If a substituent is described as being “independently selected” from a group, then each substituent is selected independently of the others. Therefore, each substituent may be the same as or different from another (other) substituent.

[0036] As used herein, the term "one or more" means one or more under reasonable conditions, such as two, three, four, five, or ten.

[0037] Unless otherwise specified, as used herein, the connection point of a substituent may be derived from any suitable location of the substituent.

[0038] This invention also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the compounds of this invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the compounds of this invention include (but are not limited to) isotopes of hydrogen (e.g., deuterium). 2 H), tritium ( 3 H); carbon isotopes (e.g., H); n C 13 C and 14 C); isotopes of chlorine (e.g.) 36 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N);15 O、 17 O and 18 O); isotopes of phosphorus (e.g., O); phosphorus isotopes (e.g., O); 32 P); and isotopes of sulfur (e.g., ... 35 S). Certain isotope-labeled compounds of the present invention (e.g., those doped with radioactive isotopes) can be used in drug and / or substrate tissue distribution studies (e.g., analysis). Radioactive isotope tritium (i.e. 3 H) and carbon-14 (i.e. 14 C) It is particularly suitable for this purpose due to its ease of incorporation and detection. Using positron-emitting isotopes (e.g.) n C 18 F, 15 O and 13 N) substitution can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of the present invention can be prepared by methods similar to those described in the accompanying routes and / or examples and preparations, using a suitable isotopically labeled reagent instead of the previously used unlabeled reagent. Pharmaceutically acceptable solvates of the present invention include those in which the crystallization solvent can be isotopically substituted, for example, D. 2 O, acetone or DMSO-*.

[0039] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers).

[0040] Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It should be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0041] Solid lines may be used in this article. solid wedge Or virtual wedge The chemical bonds of the compounds of the present invention are depicted. Solid lines are used to depict bonds to asymmetric carbon atoms to indicate all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom. Solid or dashed wedges are used to depict bonds to asymmetric carbon atoms to indicate the presence of the indicated stereoisomers. When present in racemic mixtures, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0042] This invention encompasses all possible crystalline forms or polymorphs of the compounds of this invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion. It should also be understood that certain compounds of this invention may exist in a free form for therapeutic purposes, or, where appropriate, in their pharmaceutically acceptable derivative forms. In this invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, N-oxides, metabolites, or prodrugs, which, upon administration to a patient in need, can directly or indirectly provide the compounds of this invention or their metabolites or residues. Therefore, when referring to "compounds of this invention" herein, it is also intended to cover the various derivative forms of the compounds described above.

[0043] Pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts. Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts. A review of suitable salts can be found in Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0044] As used herein, the term "ester" means an ester derived from the various general formula compounds of this application, including physiologically hydrolyzable esters (the compounds of the invention that can be hydrolyzed under physiological conditions to release free acids or alcohols). The compounds of the invention may themselves also be esters.

[0045] The compounds of the present invention can exist as solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, can be stoichiometric or non-stoichiometric.

[0046] Those skilled in the art will understand that not all nitrogen-containing heterocycles can form oxides because nitrogen requires available lone pairs of electrons to be oxidized. Those skilled in the art will identify nitrogen-containing heterocycles that can form oxides. Those skilled in the art will also recognize that tertiary amines can form N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art, including the oxidation of heterocycles and tertiary amines with peroxyacids such as peracetic acid and m-chloroperoxybenzoic acid (MCPBA), hydrogen peroxide, alkyl peroxides such as tert-butyl peroxide, sodium perborate, and dioxiranes such as dimethyldioxirane. These methods for preparing W-oxides have been extensively described and reviewed in the literature, see, for example: T.L. Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp. 748-750; A.R. Katritzky and A.J. Boulton, Eds., Academic Press; and G.W. H. Heeseman and E.S. G. Wierstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp. 390-392, A.R. Katritzky and A.J. Boulton, Eds., Academic Press.

[0047] The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body when the compounds of this invention are administered. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds obtained by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

[0048] This invention further includes, within its scope, prodrugs of the compounds of the invention, which are certain derivatives of the compounds of the invention that may themselves have little or no pharmacological activity, which, when administered to or onto the body, can be converted, for example, by hydrolysis and cleavage into the compounds of the invention having the desired activity. Typically, such prodrugs are functional group derivatives of the compounds that are readily converted in vivo into the compounds with the desired therapeutic activity. Further information regarding the use of prodrugs can be found in “Pro-drugs as Novel Delivery Systems,” Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella). The prodrugs of the invention can be prepared, for example, by replacing suitable functional groups present in the compounds of the invention with certain portions known to those skilled in the art as “pro-moiety” (e.g., “Design of Prodmgs,” H. Bundgaard (Elsevier, 1985)).

[0049] This invention also covers compounds of the invention containing protecting groups. In any process of preparing the compounds of the invention, protection of sensitive or reactive groups on any relevant molecule may be necessary and / or desired, thereby forming a form of chemical protection for the compounds of the invention. This can be achieved by conventional protecting groups, for example, those described in T.W. Greene & P. ​​G. W. M. Uts, *Protective Groups in Organic Synthesis*, John Wiley & Sons, 1991, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art.

[0050] The term “about” means within ±10% of the stated value, preferably within ±5%, and more preferably within ±2%.

[0051] compound

[0052] One aspect of this disclosure provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein said compound has the structure of formula (I):

[0053] in,

[0054] X is selected from CR 3 R 4 , O, S or NR 5 ;

[0055] Ring A is selected from C 3-6cycloalkyl or 3-6 membered heterocycloalkyl;

[0056] R 1 and R 2 Independently selected from halogens, amino (-NH2), hydroxyl (-OH), mercapto (-SH), CN, NO2, C 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Alkylthio; or

[0057] R 1 and R 2 C forms a straight chain 3-4 alkylene, wherein the C 3-4 One or two non-adjacent CH2 groups in an alkylene group are independently and optionally coated with O, S, or NR. 6 Substitute;

[0058] R 3 R 4 R 5 and R 6 Each is independently selected from H and C. 1-3 alkyl;

[0059] R is independently selected from halogen, hydroxyl, amino, mercapto, CN and NO2;

[0060] n is selected from 0, 1, 2, or 3;

[0061] m is selected from 1, 2, 3, or 4; and

[0062] key for or contain A mixture.

[0063] In some implementation schemes, R 3 R 4 R 5 and R 6 Each is independently selected from H, methyl, ethyl, propyl, and isopropyl. In a preferred embodiment, R 3 R 4 R 5 and R 6 Each is independently represented by H.

[0064] In some embodiments, X is selected from CH2, O, S or NH, preferably CH2, O or S, more preferably CH2 or S.

[0065] In some implementation schemes, R 1 and R 2 Independently selected from halogens, -NH2, -OH, -SH, CN, C 1-3 Alkyl and C1-3 Alkoxy groups, preferably halogens, -NH2, -OH, C 1-3 Alkyl and C 1-3 Alkyl group.

[0066] In some implementation schemes, R 1 and R 2 C forms a straight chain 3-4 alkylene, wherein the C 3-4 One or two non-adjacent CH2 groups in the alkylene group are independently and optionally replaced by O, S, or NH. In some such embodiments, R 1 and R 2 The following sequences of ligatures are formed: -(CH2)3-, -(CH2)2-O-, -(CH2)2-S-, -(CH2)2-NH-, -O-(CH2)2-, -S-(CH2)2-, -NH-(CH2)2-, -OCH2O-, -SCH2S-, -OCH2S-, -SCH2O-, -OCH2NH-, -NHCH2O-, -SCH2NH-, -NHCH2S-, -CH2- O-CH2-, -CH2-S-CH2-, -CH2-NH-CH2-, -(CH2)4-, -O-(CH2)2-O-, -S-(CH2)2-S-, -O-(CH2)2- S-, -O-(CH2)2-S-, -NH-(CH2)2-O-, -O-(CH2)2-NH-, -NH-(CH2)2-S-, -S-(CH2)2-NH-, -NH-( CH2)2-NH-, -CH2-O-(CH2)2-, -CH2-S-(CH2)2-, -CH2-NH-(CH2)2-, -(CH2)2-O-CH2-, -(CH2) 2-S-CH2-, -(CH2)2-NH-CH2-, -O-CH2-O-CH2-, -O-CH2-S-CH2-, -S-CH2-S-CH2-, -O-CH2-NH- CH2-, -S-CH2-NH-CH2-, -NH-CH2-NH-CH2-, -CH2-O-CH2-O-, -CH2-O-CH2-S-, -CH2-S-CH2-S- , -CH2-O-CH2-NH-, -CH2-S-CH2-NH-, -CH2-NH-CH2-NH-, -CH2-NH-CH2-O-, or -CH2-NH-CH2-S-.

[0067] In some implementation schemes, R 1 and R 2 C forms a straight chain 3-4 alkylene, wherein the C 3-4One or two non-adjacent CH2 groups in the alkylene group are independently and optionally replaced by O or S. In some such embodiments, R 1 and R 2 Linkages form -(CH2)3-, -(CH2)2-O-, -(CH2)2-S-, -O-(CH2)2-, -S-(CH2)2-, -OCH2O-, -SCH2S-, -OCH2S-, -SCH2O-, -CH2-O-CH2-, -CH2-S-CH2-, -(CH2)4-, -O-(CH2)2-O-, -S-(CH2)2-S-, -O-(CH2)2-S -, -O-(CH2)2-S-, -CH2-O-(CH2)2-, -CH2-S-(CH2)2-, -(CH2)2-O-CH2-, -(CH2)2-S-CH2-, -O-CH 2-O-CH2-, -O-CH2-S-CH2-, -S-CH2-S-CH2-, -CH2-O-CH2-O-, -CH2-O-CH2-S-, or -CH2-S-CH2-S-.

[0068] In some implementation schemes, R 1 and R 2 C forms a straight chain 3-4 alkylene, wherein the C 3-4 One or two non-adjacent CH2 groups in the alkylene group are independently and optionally replaced by O. In some such embodiments, R 1 and R 2 The connections form -(CH2)3-, -(CH2)2-O-, -O-(CH2)2-, -OCH2O-, -CH2-O-CH2-, -(CH2)4-, -O-(CH2)2-O-, -CH2-O-(CH2)2-, -(CH2)2-O-CH2-, -O-CH2-O-CH2-, or -CH2-O-CH2-O-.

[0069] In some implementation schemes, R 1 and R 2 The linkage forms a straight-chain C3 alkylene group, wherein two non-adjacent CH2 groups in the C3 alkylene group are independently replaced by O (forming -OCH2O-, i.e., part).

[0070] In some embodiments, R is independently selected from halogen, hydroxyl, amino, mercapto and CN, with halogen, hydroxyl, amino and mercapto being preferred.

[0071] In some embodiments, this disclosure provides compounds of formula (I) described above, or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, or prodrugs thereof, wherein:

[0072] X is CH2 or S;

[0073] Ring A is independently selected as C 3-6 cycloalkyl or 3-6 membered heterocycloalkyl;

[0074] R 1 and R 2 Each is independently selected from halogens, -NH2, -OH, and C. 1-3 Alkyl or C 1-3 alkoxy; or

[0075] R 1 R 2 Composition with adjacent carbon

[0076] R is independently selected from halogen, hydroxyl, amino, and thiol groups;

[0077] n is selected from 0, 1, 2, or 3;

[0078] m is selected from 1, 2, 3, or 4;

[0079] key for Or a mixture containing it.

[0080] In some implementations, the C 3～6 The cycloalkyl group can be a monocyclic, spirocyclic, fused, or bridged ring. Preferably, the C 3～6 cycloalkyl groups are selected from C 3～6 Monocyclic cycloalkyl or C 5～6 Bridged cycloalkyl rings.

[0081] In some implementations, the C 3～6 The cycloalkyl group is selected from cyclopropane, cyclobutane, cyclopentane, cyclohexane, Preferred materials include cyclopropane, cyclobutane, cyclohexane, or...

[0082] In some embodiments, the 3-6 membered heterocyclic alkyl group may be ethylene oxide, aziridinyl, aziridine, oxacyclobutyl, tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, pyrrolidone, imidazoalkyl, pyrazolyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, or trithiaalkyl.

[0083] In some implementation schemes, R 1 Selected from halogens, -NH2, -OH, C1-3 Alkyl or C 1-3 Alkyl group.

[0084] In some implementation schemes, R 1 Selected from F, Cl, Br, I, -NH2, -OH, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, or propoxy. In some embodiments, R 1 Selected from methyl, methoxy, Cl, -OH or -NH 2。 In some implementation schemes, R 1 Selected from methyl, Cl, -OH or -NH 2。

[0085] In some implementation schemes, R 2 Selected from halogens, -NH2, -OH, C 1-3 Alkyl or C 1-3 Alkyl group.

[0086] In some implementation schemes, R 2 It is selected from F, Cl, Br, I, -NH2, -OH, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy or propoxy, preferably F.

[0087] In some embodiments, R is selected from halogens, hydroxyl groups, or mercapto groups. In some embodiments, R is selected from halogens or hydroxyl groups. In some embodiments, R is selected from F, Cl, Br, I, or OH, preferably F or OH. In some embodiments, R is selected from F, Cl, Br, or I, preferably F. In some embodiments, R is OH.

[0088] In some implementations, n is selected from 1 or 2, preferably 1.

[0089] In some implementations, m is selected from 1 or 2, preferably 1.

[0090] In some embodiments, this disclosure provides compounds of formula (I) as described above, wherein the compounds of formula (I) are compounds of formula (I-1):

[0091] in:

[0092] Ring A can be cyclopropane, cyclobutane, cyclopentane, or cyclohexane.

[0093] R 2 Preferred option: F;

[0094] R 1 Methyl group is preferred;

[0095] Other definitions are as described above.

[0096] In some embodiments, this disclosure provides compounds of formula (I) as described above, wherein the compounds of formula (I) are compounds of formula (I-1-1):

[0097] in:

[0098] R 1 Selected from methyl, Cl, -OH, -NH2 or methoxy;

[0099] R 2 Selected from F; or

[0100] R 1 R 2 Composition with adjacent carbon

[0101] R is selected from F; and

[0102] n is selected from 1;

[0103] Preferably, the compound of formula (I) is a compound of formula (IA):

[0104] In some embodiments, this disclosure provides compounds of formula (I) as described above, wherein the compounds of formula (I) are compounds of formula (I-1-2):

[0105] In equation (I-1-2),

[0106] R 1 Selected from methyl, Cl, -OH, -NH2 or methoxy;

[0107] R 2 Selected from F; or

[0108] R 1 R 2 Composition with adjacent carbon

[0109] R is selected from F; and

[0110] n is selected from 1.

[0111] In some embodiments, this disclosure provides compounds of formula (I) as described above, wherein the compounds of formula (I) are compounds of formula (I-1-3):

[0112] In equation (I-1-3),

[0113] R 1 Selected from methyl, Cl, -OH, -NH2 or methoxy;

[0114] R 2 Selected from F; or

[0115] R 1 R 2 Composition with adjacent carbon

[0116] R is selected from F; and

[0117] n is selected from 1.

[0118] In some embodiments, this disclosure provides compounds of formula (I) as described above, wherein the compounds of formula (I) are compounds of formula (I-1-4):

[0119] In equation (I-1-4),

[0120] R 1 Selected from C 1-3 Alkoxy;

[0121] R 2 Selected from F; and

[0122] n is selected from 0.

[0123] In some embodiments, this disclosure provides compounds of formula (I) as described above, wherein the compounds of formula (I) are compounds of formula (I-1-4):

[0124] In equation (I-1-4),

[0125] R 1 Selected from methyl, Cl, -OH, -NH2 or methoxy;

[0126] R 2 Selected from F; and

[0127] n is selected from 0.

[0128] In some embodiments, this disclosure provides compounds of formula (I) as described above, wherein the compounds of formula (I) are compounds of formula (I-1-5):

[0129] In equation (I-1-5),

[0130] R 1 Selected from methyl, Cl, -OH, -NH2 or methoxy;

[0131] R 2 Selected from F; or

[0132] R 1 R 2Composition with adjacent carbon

[0133] R is selected from F; and

[0134] n is selected from 1.

[0135] This invention covers compounds obtained by arbitrarily combining various embodiments.

[0136] In some embodiments, this disclosure provides compounds of formula (I) or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, or prodrugs thereof, wherein said compounds are selected from:

[0137] Preferably selected from:

[0138] Pharmaceutical compositions and treatment methods

[0139] In some embodiments, the present invention provides pharmaceutical compositions comprising a preventatively or therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug, and one or more pharmaceutically acceptable carriers. The pharmaceutical compositions are preferably solid, semi-solid, liquid, or gaseous formulations. In some embodiments, the pharmaceutical compositions may also comprise one or more other therapeutic agents. In preferred embodiments, the pharmaceutical compositions are preferably administered orally, intravenously, intraarterially, subcutaneously, intraperitoneally, intramuscularly, or transdermally.

[0140] In some embodiments, the present invention provides the use of the compounds of the present invention or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, or pharmaceutical compositions of the present invention in the preparation of medicaments for the prevention or treatment of cancer and / or tumors and related conditions.

[0141] In some embodiments, the present invention provides compounds of the present invention or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, or pharmaceutical compositions of the present invention for the prevention or treatment of cancer and / or tumors and related conditions.

[0142] In some embodiments, the present invention provides a method for preventing or treating cancer and / or tumors and related conditions, the method comprising administering to an individual in need an effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, or pharmaceutical composition of the present invention.

[0143] In some embodiments, the cancers and / or tumors and related conditions are preferably selected from: cancers occurring in the esophagus, stomach, intestines, rectum, mouth, pharynx, larynx, lungs, colon, breast, uterus, endometrium, ovary, prostate, testes, bladder, kidneys, liver, pancreas, bone, connective tissue, skin, eyes, brain and central nervous system, as well as thyroid cancer, leukemia, Hodgkin's disease, lymphoma and myeloma.

[0144] In another aspect, the present invention provides the use of the compounds of the present invention or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, or pharmaceutical compositions of the present invention in the preparation of ADC drugs.

[0145] In this invention, "pharmaceutically acceptable carrier" refers to a diluent, excipient, vehicle, or medium that is administered co-administered with a therapeutic agent and is suitable, to the extent of reasonable medical judgment, for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0146] Unless otherwise stated, as used herein, the term “treating” means to reverse, alleviate, or inhibit the progression of a disease or condition to which such term applies, or one or more symptoms of such a disease or condition, or to prevent such a disease or condition, or one or more symptoms of such a disease or condition.

[0147] As used herein, “individual” includes both human and non-human animals. Exemplary human individuals include human individuals suffering from a disease (such as the disease described herein) (referred to as patients) or normal individuals. In this invention, “non-human animals” includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.). Beneficial effects

[0148] The camptothecin derivative of this invention exhibits good activity, a more stable lactone ring, low efflux rate, and improved safety. It shows great potential as a prodrug toxin in the development of ADC drugs (e.g., drugs for treating gastrointestinal tumors). Detailed Implementation

[0149] Example

[0150] The present invention is further described below with reference to embodiments and experimental examples, but these embodiments are not intended to limit the scope of the invention.

[0151] The structure of the compound was confirmed by nuclear magnetic resonance spectroscopy (HNMR) or mass spectrometry (MS). The instrument used for HNMR spectroscopy was a Bruker 400MHz NMR spectrometer; the solvent used was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0152] The abbreviations in nuclear magnetic resonance spectroscopy have the following meanings: s: singlet; d: doublet; t: triplet; q: quartet; dd: double doublet; qd: quadruple doublet; ddd: double double doublet; ddt: double triplet; dddd: double double double doublet; m: multiplet; br: broad peak; / : coupling constant; Hz: Hertz.

[0153] Chemical shift (5) is given in parts per million (ppm).

[0154] The mass spectrometry (MS) instrument used was a SHIMADZU LCMS-2020.

[0155] Example 1: Preparation of compound 1a

[0156] Step 1: Synthesis of compound 1a

[0157] (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione methanesulfonate (500 mg, 0.94 mmol), 2-bromomethyl-1,1-difluorocyclopropane (322 mg, 1.88 mmol), and diisopropylethylamine (606 mg, 4.7 mmol) were dissolved in dimethyl sulfoxide (10 mL). The reaction was stirred at 100 °C for 16 hours. After the reaction, the compound was purified by HPLC (Waters Sunfire Prep C8 OBD 19*250mm 10µm, 0.1% FA aqueous solution-CH3CN, 254nm / 214nm, 20mL / min) to give compound 1a (30mg, yield: 6%). LCMS (ESI) m / z: 526.2 [M+H] + .

[0158] 1HNMR(400MHz,DMSO-d6)δ7.74(d,J=10.8Hz,1H),7.31(s,1H),6.52(s,1H),5.44–5.38(m,4H),4.29(s,1H),3.24–3.18(m,1H),3. 04–3.00(m,1H),2.88–2.83(m,2H),2.38(s,3H),2.27–2.22(m,1H),2.04–1.83(m,5H),1.60–1.56(m,2H),0.87(t,J=7.2Hz,3H).

[0159] Example 2: Preparation of compound 7a

[0160] Step 1: Synthesis of 3,3-difluorocyclobutane-1-carboxaldehyde

[0161] In a 25 mL single-necked flask, a solution of 3,3-difluorocyclobutane-1-methanol (1.5 g, 5.8 mmol, 1.0 equivalent) in dichloromethane (16 mL) was added to a mixture of pyridinium chlorochromate (2.82 g, 13.1 mmol, 1.6 equivalents). The reaction was stirred at room temperature for 1 hour. The reaction solution was diluted with ether (20 mL), filtered through a silica gel column, and the filtrate was concentrated under reduced pressure at low temperature to obtain crude 3,3-difluorocyclobutane-1-carboxaldehyde (700 mg). 1 H NMR (400MHz, DMSO-d6) δ9.69 (s, 1H), 4.14 (d, J = 6.0Hz, 1H), 2.50–2.32 (m, 4H).

[0162] Step 2: Synthesize compound 7a

[0163] In a 25 mL single-necked flask, triethylamine (9.5 mg, 0.09 mmol, 1.0 equivalent) was added to a mixture of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione methanesulfonate (50 mg, 0.09 mmol, 1.0 equivalent) and dimethyl sulfoxide (2 mL). After the reaction solution became clear, formic acid (22 mg, 0.47 mmol, 5.0 equivalent) and 3-fluorocyclobutane-1-carboxaldehyde (16 mg, 0.14 mmol, 1.5 equivalent) were added, and the mixture was stirred for 10 minutes. Sodium cyanoborohydride (8 mg, 0.14 mmol, 1.5 equivalent) was then added to the reaction solution. The reaction was stirred at room temperature for 2 hours. The reaction solution was filtered, and the filtrate was purified by high-performance liquid chromatography (HPLC) (Waters Sunfire Prep C8 OBD 19*250mm 10um, 0.1% FA aqueous solution-CH3CN, 254nm / 214nm, 20mL / min) to give (9S)-9-ethyl-5-fluoro-1-((3-fluorocyclobutyl)methyl)amino)-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione compound 7a (24 mg, 0.05 mmol, 49% yield), a white solid. LCMS (ESI) m / z: 540.3 [M+H]+;

[0164] 1 H NMR (400MHz, DMSO-d6) δ7.79(d,J=10.8Hz,1H),7.38(s,1H),5.48–5.32(m,4H),4.91(s,1H),3.37(s,1H),3.29–3.12(m,2H),3. 09–3.01(m,1H),2.74–2.59(m,3H),2.54(s,1H),2.47–2.30(m,5H),2.24–2.13(m,1H),1.92–1.77(m,2H),0.84(t,J=7.3Hz,2H).

[0165] Example 3: Preparation of compound 11a

[0166] Step 1: Synthesis of (3-fluorobicyclo[1.1.1]pentan-1-yl)methanol

[0167] Under ice bath conditions, a borane dimethyl sulfide complex (2.0 M, 38.5 mL, 77 mmol, 2.0 equivalent) was added to a tetrahydrofuran (50 mL) solution of 3-fluorobicyclo[1.1.1]pentane-1-carboxylic acid (5.0 g, 38.5 mmol, 1.0 equivalent). The reaction was stirred at room temperature for 16 hours. The reaction solution was quenched with methanol (100 mL), stirred at room temperature for 2 hours, and then concentrated under reduced pressure to obtain crude (3-fluorobicyclo[1.1.1]pentane-1-yl)methanol (4.0 g), which was used directly in the next step. 1 H NMR (400MHz, CDCl3) δ3.84 (s, 2H), 2.00 (s, 6H).

[0168] Step 2: Synthesis of (3-fluorobicyclo[1.1.1]pentan-1-yl)formaldehyde

[0169] (3-fluorobicyclo[1.1.1]pentan-1-yl)methanol (200 mg, 1.72 mmol, 1.0 equivalent) was dissolved in dichloromethane (5 mL), and Desmartin oxidant (1.1 g, 2.58 mmol, 1.5 equivalent) was added at 0 °C. After the addition was complete, the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain (3-fluorobicyclo[1.1.1]pentan-1-yl)formaldehyde (100 mg), which was used directly in the next step. 1 H NMR (400MHz, CDCl3) δ9.69 (d, J = 6.0 Hz, 1H), 2.30 (d, J = 2.4 Hz, 6H).

[0170] Step 3: Synthesize compound 11a

[0171] (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione methanesulfonate (50 mg, 0.094 mmol, 1.0 equivalent) and triethylamine (10 mg, 0.094 mmol, 1.0 equivalent) were added to dimethyl sulfoxide (2 mL) and stirred at room temperature for 10 minutes. Then, 3-fluorobicyclo[1.1.1]pentane-1-carboxaldehyde (16 mg, 0.141 mmol, 1.5 equivalent) and formic acid (22 mg, 0.470 mmol, 5 equivalent) were added sequentially to the reaction mixture. After the addition was complete, the mixture was stirred at room temperature for 30 minutes. Sodium cyanoborohydride (9 mg, 0.141 mmol, 1.5 equivalents) was added to the reaction solution. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was purified by preparative high performance liquid chromatography (Waters Sunfire Prep C8 OBD 19*250 mm 10 μm, 0.1% FA aqueous solution-CH3CN, 254 nm / 214 nm, 20 mL / min) to obtain the target compound 11a. 1 H NMR (400MHz, DMSO-d6) δ7.88(d,J=10.8Hz,1H),7.39(s,1H),5.54–5.39(m,4H),5.01(s,1H),3.52(d,J=12.8Hz,1H),3.28–3.11(m,2 H),3.09–3.01(m,1H),2.78–2.74(m,1H),2.56(s,2H),2.42(s,3H),2.12(d,J=2.4Hz,6H),1.94–1.85(m,2H),0.88(t,J=7.2Hz,3H).

[0172] Example 4: Preparation of compound 15a

[0173] Step 1: Synthesis of compound 15a

[0174] Triethylamine (9.52 mg, 0.09 mmol, 1 equivalent) was added to a solution of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione methanesulfonate (50 mg, 0.09 mmol, 1 equivalent) in dimethyl sulfoxide (1 mL). The mixture was stirred at room temperature for 15 minutes. 4,4-difluorocyclohexane-1-carboxaldehyde (16.72 mg, 0.112 mmol, 1.2 equivalent) and formic acid (21.65 mg, 0.047 mmol, 5 equivalent) were then added to the mixture. The reaction mixture was stirred at room temperature for 30 minutes under argon protection. Sodium cyanoborohydride (9 mg, 0.014 mmol, 1.5 equivalents) was added to the reaction solution. The reaction solution was stirred at room temperature for 2 hours under argon protection. The reaction solution was filtered and purified by preparative high-performance liquid chromatography (Waters Sunfire Prep C8 OBD 19*250mm 10µm, 0.1% FA aqueous solution-CH3CN, 254nm / 214nm, 20mL / min) to obtain the target compound 15a (26 mg, yield 48%). LCMS (ESI) m / z: 568.1 [M+H] + . 1 H NMR (400MHz, DMSO) δ7.88(s,1H),7.38(s,1H),5.54(d,J=19.4Hz,1H),5.46(s,2H),5.40(d,J=19.0Hz,1H),5.02(s,1H),3.17(d,J=42.2Hz, 4H),2.72(d,J=22.5Hz,1H),2.42(s,3H),2.18(s,1H),2.07–2.00(m,2H),1.94–1.75(m,7H),1.26(d,J=16.4Hz,4H),0.88(t,J=7.3Hz,3H).

[0175] Example 5: Preparation of compound 17a

[0176] Step 1: Synthesis of N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0177] Potassium tert-butoxide (16.1 g, 143 mmol, 2 equivalents) was added to anhydrous tetrahydrofuran (400 mL) and cooled to 0 °C. A solution of N-(3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (18 g, 71.64 mmol, 1 equivalent) in anhydrous tetrahydrofuran (100 mL) was slowly added dropwise to the reaction mixture. The reaction was stirred at room temperature for 0.5 h. Tert-butyl nitrite (14.8 g, 143 mmol, 2 equivalents) was slowly added dropwise to the reaction mixture at 0 °C. The reaction was stirred at room temperature for 16 h. The reaction mixture was poured into water (1000 mL) and extracted with ethyl acetate (400 mL x 2). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was slurried with diethyl ether to give the target compound 17-2,N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (15 g, 53.52 mmol, yield 75%), as a brown solid. LCMS (ESI) m / z: 281.1 [M+H] + .

[0178] Step 2: Synthesis of N-(7-amino-3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0179] (Z)-N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (6 g, 21.41 mmol, 1 equivalent) was dissolved in 150 mL of methanol, and 600 mg of 10% palladium on carbon and 15 mL of 3 M hydrochloric acid were added. The reaction was stirred at room temperature under hydrogen atmosphere for 2 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol, and the filtrate was concentrated under reduced pressure to give the target compound 17-3,N-(7-amino-3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (5 g, 18.8 mmol, yield 88%) as a black solid. LCMS (ESI) m / z: 267.1 [M+H] + .

[0180] Step 3: Synthesis of (9H-fluorene-9-yl)methyl(8-acetamido-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate

[0181] N-(7-amino-3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (5 g, 18.8 mmol, 1 equivalent) was dissolved in a mixture of 1,4-dioxane (200 mL) and water (50 mL). Sodium bicarbonate (6.31 g, 75.1 mmol, 4 equivalents) and 9-fluorenylmethyl-N-succinimide carbonate (7.6 g, 22.5 mmol, 1.2 equivalents) were slowly added to the reaction mixture at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into water (500 mL), extracted with ethyl acetate (200 mL), and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was subjected to high-performance liquid chromatography (HPLC) (acetonitrile, trifluoroacetic acid aqueous solution system) to obtain the target compound 17-4,(9H-fluorene-9-yl)methyl(8-acetamido-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate (4.3 g, 8.86 mmol, yield 47%), as a black solid. LCMS (ESI) m / z: 489.2 [M+H] + .

[0182] Step 4: Synthesis of (9H-fluorene-9-yl)methyl(8-amino-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate hydrochloride

[0183] (9H-fluorene-9-yl)methyl(8-acetamido-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate (1 g, 3.24 mmol, 1 equivalent) was dissolved in anhydrous methanol (10 mL), and 4M hydrochloric acid in methanol (10 mL) was added to the reaction solution. The reaction was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to give the target compound 17-5, (9H-fluorene-9-yl)methyl(8-amino-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate hydrochloride (1 g, 3.33 mmol, yield 101%), as a yellow solid. LCMS (ESI) m / z: 447.1 [M+H] + .

[0184] Step 5: Synthesis of (9H-fluorene-9-yl)methyl((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)carbamate

[0185] (9H-fluorene-9-yl)methyl(8-amino-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate hydrochloride (1 g, 2.07 mmol, 1 equivalent) was dissolved in anhydrous toluene solution (100 mL). (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f] nidene-3,6,10(4H)-trione (818 mg, 3.11 mmol, 1.5 equivalent) and p-toluenesulfonic acid (71 mg, 0.41 mmol, 0.2 equivalent) were added to the reaction solution. The reaction was stirred at 130 °C for 16 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (methanol:dichloromethane = 0-6%) to give the target compound 17-6, (9H-fluorene-9-yl)methyl ((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)carbamate (740 mg, 1.1 mmol, yield 53%), as a brown solid. LCMS (ESI) m / z: 674.2 [M+H] + .

[0186] Step 6: Synthesis of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione

[0187] (9H-fluorene-9-yl)methyl ((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)carbamate (700 mg, 1.04 mmol, 1 equivalent) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (152 mg, 2.08 mmol, 2 equivalents) was added. The reaction was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, the crude product was pulped with ethyl acetate, filtered, and the filter cake was dried to obtain (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione (380 mg, yield 81%), as a black solid.

[0188] Step 8: Synthesis of (1S)-9-ethyl-5-fluoro-1-(((3-fluorobicyclo[1.1.1]pent-1-yl)methyl)amino)-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione

[0189] Triethylamine (1.1 g, 11.1 mmol, 1.0 equivalent) was added to a dimethyl sulfoxide (4 mL) solution of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione (5.0 g, 0.4 mmol, 1.0 equivalent). After the reaction solution was clear, formic acid (2.5 g, 55.4 mmol, 5.0 equivalent) and 3-fluorobicyclo[1.1.1]pentane-1-carboxaldehyde (1.9 g, 16.6 mmol, 1.5 equivalent) were added. The reaction mixture was stirred at room temperature for 10 minutes, then sodium cyanoborohydride (1.0 g, 16.6 mmol, 1.5 equivalents) was added, and stirring continued at room temperature for 4 hours. The reaction mixture was filtered and purified by rapid silica gel column chromatography (dichloromethane:methanol = 50:1) to give the target compound 17 (1.6 g, 27% yield) as an off-white solid.

[0190] LCMS(ESI) m / z: 550.8 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ7.83(dd,J=12.4,4.9Hz,1H),7.33–7.26(m,1H),6.50( s,1H),5.45(dd,J=17.7,3.7Hz,3H),5.30(d,J=17.7Hz,1H),4.22(s,1H),3.94( d,J=5.4Hz,3H),3.24(d,J=17.5Hz,1H),3.14(d,J=12.3Hz,1H),3.03–2.88(m,2 H),2.25–2.09(m,2H),2.09–1.99(m,7H),1.94–1.84(m,2H),0.94–0.84(m,3H).

[0191] The racemic mixture of compound 17 was chirally resolved to give target compound 17a (retention time 1: 2.414 s; 900 mg, 15% yield, brown solid) and 17b (retention time 2: 4.134 s; 600 mg, 11% yield, brown solid).

[0192] Compound 17a: 1 H NMR (400MHz, DMSO-d6) δ7.83(d,J=12.4Hz,1H),7.29(s,1H),6.50(s,1H),5.48–5.27(m,3H),4.22(s,1H),3.94(s,3H),3. 27–3.22(m,1H),3.17–3.10(m,1H),3.02–2.94(m,2H),2.16(s,2H),2.02(s,7H),1.93–1.82(m,2H),0.88(t,J=7.2Hz,3H).

[0193] Compound 17b: 1 H NMR (400MHz, DMSO-d6) δ7.86(d,J=12.0Hz,1H),7.30(s,1H),6.50(s,1H),5.43(s,3H),5.33(d,J=19.1Hz,1H),4.25(s,1H),3.9 5(s,3H),3.28–3.23(m,1H),3.14(d,J=11.4Hz,1H),3.01(d,J=20.3Hz,2H),2.16(s,2H),2.02(s,7H),1.87(s,2H),0.88(s,3H).

[0194] Other compounds disclosed herein can be prepared by methods similar to those described in the above embodiments (with appropriate modifications if necessary).

[0195] Test Example 1: Cell Viability Test

[0196] The inhibitory activity of the drug compounds on the in vitro proliferation of SNU-5, MKN-45, SW480, LS1034, and AsPC-1 tumor cells was tested. After 6 days of culture, cell proliferation was detected using CGT reagent (CellTiter-Glo). The inhibitory activity was determined based on the IC50 value. 50 The in vitro activity of the compound was determined by measuring its value.

[0197] Note: DXd's sensitivity to killing tumor cells: SNU-5 (high), MKN-45 (medium), SW480 (medium), LS1034 (low) and AsPC-1 (low).

[0198] A. Materials and Equipment

[0199] 1. Reagents and consumables: SNU-5 (ATCC), MKN-45 (CTCC), SW480 (ATCC), LS1034 (ATCC), and AsPC-1 (ATCC); IMDM medium (Gibco), RPMI-1640 medium (Gibco), Leibovitz's L-15 medium (Gibco), trypsin (Gibco), fetal bovine serum (Vivacell), penicillin and streptomycin (Invitrogen), phenol blue (Biological Industries), DPBS (Biosera), DMSO (Sigma), Staurosporine 96 (Selleck), CellTiter-Glo (Promega), 96-well cell culture plates (Corning), 96-well V-plates (Corning)

[0200] 2. Instruments: Inverted microscope (OLYMPUS), CO2 incubator (ThermoFisher), centrifuge (Eppendorf), cell counter (Shanghai Meng Microbial Medical), multi-functional microplate reader (PerkinElmer).

[0201] B. Testing Methods

[0202] Experimental steps:

[0203] 1. Culture SNU-5, MKN-45, SW480, LS1034 and AsPC-1 in their respective cell cultures (see Table 1) until the confluence is about 70-90%.

[0204] 2. Passage the cells (cells should be passaged 2-15 after revival) and resuspend the cells in the corresponding complete culture medium (see Table 1). After cell counting (it is recommended that the cell viability be ≥90%), record the cell passage number and viability.

[0205] 3. Seed 90 μL of cells into each well of a 96-well plate and incubate overnight at 37°C.

[0206] 4. The following day, prepare the drug according to the preparation protocol in Table 2. Take out the 96-well plate and add 10 μL of 10x test drug, positive control staurosporine, or carrier control to each well. The total system volume is 100 μL.

[0207] 5. After mixing, place the cell culture plate in a 37°C, 5% CO2 or 37°C, 0% CO2 (SW480 cells) incubator and continue incubation for 6 days.

[0208] 6. After incubation, remove the cell culture plate and allow it to equilibrate to room temperature.

[0209] 7. Add 50 μL of CellTiter Glo reagent to each well, lyse at room temperature for 2 minutes, and then equilibrate for 10 minutes.

[0210] 8. Use an Envision microplate reader to read the plate and detect the luminescence value.

[0211] 9. Data Analysis: Using the four-parameter fitting formula in GraphPad software, an S-shaped curve of cell survival was plotted, and the IC50 of the drug on cell killing was calculated. 50 .

[0212] Table 1. Cell lines

[0213] Table 2. Medication Dispensing Plan

[0214] The in vitro inhibitory activity against tumor cell proliferation was tested according to the above experimental steps, and the results are shown in Table 3.

[0215] Table 3 *Indices of IC50 for Dxd cell killing in the same plate 50 value

[0216] Table 3 (continued) *IC50 for eczema-induced cell killing in the same plate. 50 value

[0217] Experimental results showed that compounds 1a, 7a, 11a, and 15a exhibited superior cytotoxic activity against all five tumor cell lines compared to Dxd. Specifically, compounds 1a, 7a, 11a, and 15a showed significantly better activity against Dxd in both Dxd and low-sensitivity cell lines. In the Dxd-sensitive tumor cell lines LS1034 and AsPc-1, compounds 1a, 7a, 11a, and 15a were comparable to or better than eczema.

[0218] Test Example 2: Caco-2 Cell Efflux Rate Assay

[0219] A. Materials and Equipment

[0220] Caco-2 cells (ATCC), Hanke balanced salt solution (HBSS), and non-essential amino acids (NEAA) were purchased from Thermo Fisher Scientific. HEPES, penicillin, streptomycin, and trypsin / EDTA were purchased from Solarbio. Fetal bovine serum (FBS) was purchased from AusGeneX. High-glucose DMEM medium was purchased from Hyclone. HTS-96-well Transwell plates and other sterile consumables were purchased from Corning. The Millicell electrical resistance measurement system was purchased from Millipore. Vision was purchased from Nexcelom Bioscience. The Infinite200PRO microplate reader was purchased from Tecan. The MTS2 / 4orbital shaker was purchased from IKA Labortechnik.

[0221] B. Testing Methods

[0222] 1. Cell culture and seeding plates

[0223] 1) Use high-glucose DMEM medium containing L-glutamine, with 10% fetal bovine serum, 0.1 mg / mL streptomycin, 100 units of penicillin, and 1× non-essential amino acids added for cell culture.

[0224] 2) Caco-2 cells were cultured in cell culture flasks. The incubator was set to 37°C, 5% CO2, and 95% relative humidity. When the cell confluence reached 70-90%, the cells could be used for Transwell inoculation.

[0225] 3) Before cell seeding, add 50 μL of cell culture medium to each well in the upper chamber of the Transwell and 25 mL of cell culture medium to the lower culture plate. After incubating the culture plate in a 37°C, 5% CO2 incubator for 1 hour, it can be used for cell seeding.

[0226] 4) After cell digestion, aspirate the cell suspension and transfer it to a round-bottom centrifuge tube, then centrifuge at 120g for 5 minutes.

[0227] 5) Resuspend the cells in culture medium to a final concentration of 6.86 × 10⁻⁶. 5 Cells / mL. Add 50 μL of cell suspension to each well of a 96-well Transwell plate, resulting in a final seeding density of 2.4 × 10⁶ cells / mL. 5 cells / cm 2 .

[0228] 6) Change the medium 48 hours after inoculation and culture for 14-18 days, changing the medium every other day.

[0229] 7) The process of changing the culture medium is as follows: Separate the Transwell chamber from the receiving plate, discard the culture medium in the receiving plate first, then discard the culture medium in the Transwell chamber, and finally add 75 μL of fresh culture medium to each chamber and 25 mL of fresh culture medium to the receiving plate.

[0230] 2. Evaluation of cell monolayer membrane integrity

[0231] 1) After 14-18 days of culture, Caco-2 should have completely merged and differentiated. At this point, it can be used for the breakthrough test.

[0232] 2) Measure the resistance of the single-layer film using a resistance meter (Millipore, USA) and record the resistance of each pore.

[0233] 3) After the measurement is completed, put the Transwell culture plate back into the incubator.

[0234] 4) Calculation of resistance value: Measure resistance value (ohms) × film area (cm²) 2 ) = TEER value (ohm·cm) 2 ),

[0235] If the TEER value is <230 ohms·cm 2 If the hole is not suitable for a penetration test, then the hole cannot be used.

[0236] 3. Solution preparation

[0237] 1) Prepare 1L HBSS (10mM HEPES, pH 7.4): Weigh 2.38g HEPES and 0.35g sodium bicarbonate, add 900mL of pure water to dissolve them, then add 100mL of 10×HBSS and stir well. Adjust the pH to 7.4 and finally filter.

[0238] 2) Prepare a high-concentration DMSO stock solution of the test substance and dilute it to 1 mM with DMSO. Then, dilute it accordingly with HBSS (10 mM HEPES, pH 7.4) to obtain a test concentration of 5 μM.

[0239] 3) Prepare high-concentration DMSO stock solutions of the control drugs digoxin and metoprolol, and dilute them to 2 mM stock solution with DMSO. Then, dilute accordingly with HBSS (10 mM HEPES, pH 7.4) to obtain a test concentration of 10 μM.

[0240] 4. Drug penetration test

[0241] 1) Remove the Transwell culture plate from the incubator. Rinse the cell monolayer twice with HBSS (10mM HEPES, pH 7.4) buffer and incubate at 37°C for 30 minutes.

[0242] 2) Determine the transport rate of the compound from the top to the base. Add 125 μL of the drug delivery solution to each well in the upper chamber (top), then transfer 50 μL of the sample to 200 μL of acetonitrile containing the internal standard as the sample delivered to the top of the Transwell plate at 0 minutes for detection. Add 235 μL of the receiver solution to each well in the lower chamber (base).

[0243] 3) Determine the transport rate of the compound from the base to the top. Add 75 μL of receiving end solution to each well in the upper chamber (top) and 285 μL of dosing end solution to each well in the lower chamber (base). Then transfer 50 μL of sample to 200 μL of acetonitrile containing internal standard as the 0-minute dosing sample from the base of the Transwell plate for detection.

[0244] 4) After merging the upper and lower transfer devices, incubate at 37°C for 2 hours.

[0245] 5) Transfer 50 μL of sample from the working solution preparation plate and add it to 200 μL of acetonitrile containing the internal standard as the 0-minute dosing sample for detection.

[0246] 6) After incubation, take 50 μL of sample from each well of the upper and lower chambers of the Transwell culture plate and add it to a new sample tube. Add 200 μL of acetonitrile containing the internal standard to the sample tube, vortex for 10 minutes, and then centrifuge at 3220g for 30 minutes. Take 150 μL of the supernatant, dilute it with an equal volume of water, and then perform LC-MS / MS analysis. All samples are prepared in duplicate.

[0247] 7) Evaluate the integrity of the cell monolayer after 2 hours of incubation using fluorescein (LY) leakage. Dilute the fluorescein stock solution to a final concentration of 100 μM using HBSS (10 mM HEPES, pH 7.4). Add 100 μL of fluorescein solution to each well of the upper Transwell plate and 300 μL of HBSS (10 mM HEPES, pH 7.4) to each well of the lower receiving plate. After incubation at 37°C for 30 minutes, aspirate 80 μL of solution from both the upper and lower layers of each well into a new 96-well plate. Measure fluorescence using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

[0248] 5. Data Analysis

[0249] All calculations were performed using Microsoft Excel. The apparent permeability coefficient (Papp, in cm / s) of the compound in Caco-2 cells was calculated using the following formula based on the specific concentrations at the receiving and administering ends:

[0250] in:

[0251] V A The volume of the receiving solution is 0.235 mL from the top to the base and 0.075 mL from the base to the top.

[0252] Area is the area of ​​the Transwell-96-well plate membrane (0.143 cm²). 2 );

[0253] time is the incubation time (unit: seconds);

[0254] [drug] receiver The drug concentration at the receiving end;

[0255] [drug]initial,donor is the initial drug concentration at the dosing end.

[0256] The efflux ratio is calculated using the following formula:

[0257] in:

[0258] P app(B-A) The apparent permeability coefficient is measured from the base to the tip.

[0259] P app(A-B) The apparent permeability coefficient is measured from the top to the base.

[0260] Recovery rate (Recovery%) is calculated using the following formula:

[0261] in:

[0262] V A The volume of the solution at the receiving end (unit: mL);

[0263] V D The volume of the solution at the administration end (unit: mL);

[0264] [drug] receiver The drug concentration at the receiving end;

[0265] [drug]initial,donor is the initial drug concentration at the dosing end.

[0266] [drug] donor This represents the drug concentration at the dosing end.

[0267] The integrity of the Caco-2 cell monolayer membrane was calculated using the following formula:

[0268] in:

[0269] I receiver This refers to the fluorescence density of the receiving aperture (0.3 mL).

[0270] I donor This refers to the fluorescence density of the drug delivery well (0.1 mL).

[0271] Integrity is expressed as LY Leakage percentage (%LY Leakage).

[0272] %LY Leakage < 1.5% indicates an intact monolayer cell membrane. For individual cases where %LY Leakage > 1.5%, if P... app The values ​​are similar to those of other parallel holes, and based on scientific judgment, the final data can be adopted.

[0273] The potential absorption and efflux of the test substance were investigated by measuring its permeability in the Caco-2 cell model, as shown in Table 4.

[0274] Table 4

[0275] Note: Digoxin is a positive reference.

[0276] Experimental results show that the efflux rates of compounds 1a, 11a, 17a, and 17b are significantly lower than those of Dxd.

[0277] Test Example 3: Hepatocyte Stability of the Compound

[0278] The in vitro metabolic stability of the test compounds in hepatocytes of different species was investigated. The concentration of the parent drug in the incubation system was determined by LC / MS / MS, and the intrinsic clearance rate of the test compounds in the hepatocyte system was calculated to assess their stability.

[0279] The test concentrations for the test compound and the positive control compound were 1 μM.

[0280] 1. Materials and Reagents

[0281] Hepatocytes were preserved in liquid nitrogen; details are shown in Table 5 below. Information on other reagents will be included in the experimental report.

[0282] Table 5

[0283] 2. Experimental Design

[0284] 2.1 Preparation of the compound working solution

[0285] High-concentration stock solutions of the test substance and the control drug verapamil powder were prepared using DMSO, and then diluted with DMSO to a 100 μM working solution before use. The final concentration of the test substance and verapamil was 1 μM.

[0286] 2.2 Preparation of hepatocytes

[0287] 1) Specific preparation information for hepatocyte resuscitation solution is shown in Table 6 below. Mix 49.5 mL of Williams' E medium and 0.5 mL of glutaMAX as the incubation solution. Preheat the hepatocyte resuscitation solution and incubation solution in a 37°C water bath for at least 15 minutes before use.

[0288] Table 6

[0289] 2) Take a tube of cryopreserved hepatocytes, ensuring that the hepatocytes remain frozen before thawing. Quickly place the hepatocytes in a 37°C water bath and gently shake until all ice crystals are dispersed. Spray with 70% ethanol and transfer to a biosafety cabinet.

[0290] 3) Pour the contents of the hepatocyte tubules into a centrifuge tube containing 50 mL of resuscitation medium and centrifuge at 100 g for 10 minutes. After centrifugation, aspirate the resuscitation medium and add sufficient incubation medium to obtain a cell density of approximately 1.0 × 10⁻⁶ cells / mL. 6 Cell suspension of cells per mL.

[0291] 4) Use AO / PI staining to count cells and determine viable cell density. Hepatocyte viability must be greater than 75%. Dilute cells with culture medium to a working cell density of 0.5 × 10⁻⁶. 6 live cells / mL.

[0292] 2.3 Test Methods

[0293] 1) Transfer 247.5 μL of live cell suspension to a 96-well deep-well plate and preheat the plate in an incubator for 10 minutes on a vortex. Perform double parallel incubation.

[0294] 2) Add 2.5 μL of 100 μM test substance or verapamil to each well to initiate the reaction, and then place the deep well plate back onto the incubator vortex.

[0295] 3) Place the incubation plate in an incubator. At 0, 15, 30, 60, 90, and 120 minutes, take 25 μL of the suspension and add it to a new 96-well plate containing 12 times its volume (300 μL) of acetonitrile as an internal standard (100 nM alprazolam, 200 nM caffeine, and 100 nM tolbutamide) as a quencher to terminate the reaction. Vortex the plate for 5 minutes, then centrifuge at 3220 rpm and 4°C for 45 minutes to precipitate the protein. Transfer 100 μL of the supernatant to a sample plate, add 100 μL of pure water, mix well, and proceed with UPLC-MS / MS analysis. All incubations should be performed in duplicate.

[0296] 3. Data Analysis

[0297] All calculations were performed using Microsoft Excel. Peak areas were detected by extracting ion spectra. The in vitro half-life (t) of the parent drug was determined by linearly fitting the natural logarithm of the elimination percentage of the parent drug to time. 1 / 2 ).

[0298] In vitro half-life (t) 1 / 2 ) Calculated by slope:

[0299] In vitro t 1 / 2 = -0.693 / k.

[0300] In vitro clearance rate (unit: μL / min / 10) 6 (Number of cells) is calculated using the following formula:

[0301] In vitro CL int =-kV / N

[0302] in:

[0303] V = Incubation volume per well (0.25 mL);

[0304] N = Number of cells per well (0.125 × 10⁻⁶) 6 (cells).

[0305] The in vitro scale-up clearance rate (mL / min / kg), predicted clearance rate (mL / min / kg), and liver extraction rate (ER) were calculated using the following formulas:

[0306] In vitro expanded clearance rate (unit: mL / min / kg):

[0307] In vitro expansion of CL int =(0.693 / t) 1 / 2 )×(1 / hepatocyte density(0.5×10 6 (cells / mL) × proportionality factor (Table 7).

[0308] Predicted Hepatic Clearance (unit: mL / min / kg):

[0309] Predicting liver CL H = (QH × extracorporeal expansion CL) int ×fub) / (QH+external expansion CL) int ×fub);

[0310] Liver extraction rate (ER) = In vitro expanded CL H / QH;

[0311] in:

[0312] QH is the hepatic blood flow rate (mL / min / kg) (Table 7).

[0313] fub represents the portion of the drug that is free in the plasma, assumed to be 1.

[0314] Table 7. Proportion factors for predicting clearance in vivo in humans, monkeys, mice, and rats. aProportion factor = (Hepatocyte density) × (Liver weight)

[0315] 4. Data Processing Rules

[0316] The data processing rules are summarized in Table 8.

[0317] Table 8. Data Processing Rules

[0318] Table 9: Half-life of compound 1a in hepatocytes of humans, monkeys, beagle dogs, rats, and mice.

[0319] Experimental results showed that compound 1a had a short half-life in human, monkey, beagle dog, rat and mouse hepatocytes, suggesting that compound 1a, as an ADC load, does not easily accumulate when it is effective.

[0320] In addition to those described herein, various modifications of the invention will be apparent to those skilled in the art based on the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. All references cited in this application (including all patents, patent applications, journal articles, books, and any other disclosures) are incorporated herein by reference in their entirety.

Claims

1. Compounds of formula (I): Or, a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug, in: X is selected from CR 3 R 4 , O, S or NR 5 ; Ring A is selected from C 3-6 cycloalkyl or 3-6 membered heterocycloalkyl; R 1 and R 2 are independently selected from the group consisting of halogen, amino (-NH2), hydroxy (-OH), thiol (-SH), CN, NO2, C 1-3 alkyl, C 1-3 alkoxy and C 1-3 alkylthio; or R 1 and R 2 form a straight chain C 3-4 alkylene, wherein 1 CH2, or 2 non-adjacent CH2in said C 3-4 alkylene is / are independently optionally replaced with O, S, or NR 6 ; R 3 , R 4 , R 5 and R 6 are each independently selected from the group consisting of H and C 1-3 alkyl; R is independently selected from halogen, hydroxyl, amino, mercapto, CN and NO2; n is selected from 0, 1, 2, or 3; m is selected from 1, 2, 3, or 4; For or comprising A mixture.

2. The compound of claim 1, wherein: (1) R 3 (2) R 4 (3) R 5 (4) R 6 each independently is selected from the group consisting of H, methyl, ethyl, propyl and isopropyl, preferably each independently is H; and / or (2) X is selected from CH2, O, S or NH, preferably CH2, O or S, more preferably CH2 or S; and / or (3) R 1 and R 2 are independently selected from the group consisting of halogen, -NH2, -OH, -SH, CN, C 1-3 alkyl and C 1-3 alkoxy, preferably halogen, -NH2, -OH, C 1-3 alkyl and C 1-3 alkoxy; or R 1 and R 2 form, together with the C atom to which they are attached, a straight-chain C 3-4 alkylene, wherein 1 CH2or 2 non-adjacent CH2in said C 3-4 alkylene is / are independently optionally replaced by O, S or NH, preferably 1 CH2or 2 non-adjacent CH2in said C 3-4 alkylene is / are independently optionally replaced by O or S, more preferably 1 CH2or 2 non-adjacent CH2in said C 3-4 alkylene is / are independently optionally replaced by O; further more preferably R 1 and R 2 form, together with the C atom to which they are attached, a straight-chain C3alkylene, wherein 2 non-adjacent CH2in said C3alkylene is / are independently replaced by O; and / or (4) R is independently selected from halogen, hydroxyl, amino, mercapto and CN, preferably halogen, hydroxyl, amino and mercapto.

3. The compound according to claim 1 or 2, wherein: X is CH2 or S; Ring A is independently C 3-6 cycloalkyl or 3-6 membered heterocycloalkyl; R 1 and R 2 are each independently selected from the group consisting of halogen, -NH2, -OH, C 1-3 alkyl or C 1-3 alkoxy; or R 1 , R 2 with the adjacent carbon R is independently selected from halogen, hydroxyl, amino, and thiol groups; n is selected from 0, 1, 2, or 3; m is selected from 1, 2, 3, or 4; key For Or a mixture containing it.

4. The compound of any one of claims 1-3, wherein The compound of formula (I) satisfies any of the following conditions: (1) said C 3～6 Cycloalkyl is monocyclic, spiro, fused or bridged; preferably, said C 3～6 Cycloalkyl is selected from C 3～6 Monocyclic cycloalkyl or C 5～6 Bridged cycloalkyl; (2) The 3-6 membered heterocyclic alkyl group is selected from ethylene oxide, aziridinyl, aziridine, oxadiazinyl, tetrahydrofuranyl, dioxadiazopentenyl, pyrrolyl, pyrrolidone, imidazoalkyl, pyrazolyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl or trithiaalkyl; (3) said R 1 selected from halogen, -NH2, -OH, C 1-3 alkyl or C 1-3 alkoxy; (4) said R 2 selected from halogen, -NH2, -OH, C 1-3 alkyl or C 1-3 alkoxy; (5) The R is selected from halogen, hydroxyl or mercapto, preferably F, Cl, Br, I or OH, more preferably F or OH; (6) The n is selected from 1 or 2; and (7) m is selected from 1 or 2.

5. The compound of any one of claims 1-4, wherein The compound of formula (I) satisfies any of the following conditions: (1) the C 3～6 cycloalkyl is selected from cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclopropane, cyclobutane, cyclohexane or (2) said R 1 is selected from F, CI, Br, I, -NH2, -OH, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy or propoxy, preferably methyl, methoxy, CI, -OH or -NH2, more preferably methyl, CI, -OH or -NH2; (3) said R 2 is selected from F, CI, Br, I, -NH2, -OH, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy or propoxy, preferably F; (4) The R mentioned is selected from F; (5) The n is selected from 1; and (6) m is selected from 1.

6. The compound according to any one of claims 1-5, characterized in that, The compound of formula (I) is a compound of formula (I-1): in: Ring A is cyclopropane, cyclobutane, cyclopentane, cyclohexane, said R 2 preferably F; and said R 1 preferably methyl; Preferably, the compound of formula (I) is a compound of formula (I-1-1): In the above formula (I-1-1), R 1 selected from methyl, Cl, -OH, -NH2, or methoxy; R 2 selected from F; or R 1 , R 2 with the adjacent carbon R is selected from F; and n is selected from 1; More preferably, the compound of formula (I-1-1) is a compound of formula (I-A): or, The compound of formula (I) is a compound of formula (I-1-2): In the above formula (I-1-2), R 1 selected from methyl, Cl, -OH, -NH2, or methoxy; R 2 selected from F; or R 1 , R 2 with the adjacent carbon R is selected from F; and n is selected from 1; or, The compound of formula (I) is a compound of formula (I-1-3): In the above formula (I-1-3), R 1 selected from methyl, Cl, -OH, -NH2, or methoxy; R 2 selected from F; or R 1 , R 2 with the adjacent carbon R is selected from F; and n is selected from 1; or, The compound of formula (I) is a compound of formula (I-1-4): In the above formula (I-1-4), R 1 selected from C 1-3 alkoxy; R 2 selected from F; and n is selected from 0; or, The compound of formula (I) is a compound of formula (I-1-4): In the above formula (I-1-4), R 1 selected from methyl, Cl, -OH, -NH2, or methoxy; R 2 selected from F; and n is selected from 0; Alternatively, the compound of formula (I) is a compound of formula (I-1-5): In the above formula (I-1-5), R 1 selected from methyl, Cl, -OH, -NH2, or methoxy; R 2 selected from F; or R 1 , R 2 with the adjacent carbon R is selected from F; and n is selected from 1.

7. The compound according to any one of claims 1-6, characterized in that, The compound of formula (I) is the following compound:

8. A pharmaceutical composition comprising a preventive or therapeutically effective amount of the compound as described in any one of claims 1-7 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, and one or more pharmaceutically acceptable carriers.

9. Use of any compound of claims 1-7 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, or pharmaceutical composition of claim 8 in the preparation of a medicament for the prevention or treatment of cancer and / or tumors and related conditions.

10. The compound of any one of claims 1-7 or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, or pharmaceutical composition of claim 8, for the prevention or treatment of cancer and / or tumors and related conditions.

11. A method for preventing or treating cancer and / or tumors and related conditions, the method comprising administering to an individual in need an effective amount of a compound as described in any one of claims 1-7 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, or a pharmaceutical composition as described in claim 8.

12. The method as described in claim 11, characterized in that, The cancers and / or tumors and related conditions are selected from: cancers occurring in the esophagus, stomach, intestines, rectum, mouth, pharynx, larynx, lungs, colon, breast, uterus, endometrium, ovary, prostate, testis, bladder, kidney, liver, pancreas, bone, connective tissue, skin, eye, brain and central nervous system, as well as thyroid cancer, leukemia, Hodgkin's disease, lymphoma and myeloma.

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