Use of a heterocyclic compound for reducing adverse reactions caused by chemotherapeutic drugs

Abstract

The present invention relates to the use of heterocyclic compounds for reducing the adverse reactions caused by chemotherapeutic drugs, and particularly relates to the use of the compound shown in formula (1) or its pharmaceutically acceptable form in the preparation of a drug for reducing the adverse reactions caused by chemotherapeutic drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceuticals and relates to the use of heterocyclic compounds in the preparation of drugs for reducing adverse reactions caused by chemotherapeutic drugs. Background Art

[0002] The most fundamental and broad-spectrum methods in cancer treatment are chemotherapy and radiotherapy, also simply referred to as chemoradiotherapy. Both will damage normal cells in the patient's body, especially cells in tissues with relatively rapid turnover, thereby causing various adverse reactions (so-called side effects) of chemoradiotherapy, seriously affecting the quality of life of cancer patients. Some patients have a low tolerance to the adverse reactions caused by chemoradiotherapy and have to reduce the treatment dose of chemoradiotherapy or adjust the treatment cycle, which will greatly affect the treatment effect of cancer to a large extent.

[0003] The intestine is the organ with the fastest cell turnover rate in the human body. A large number of rapidly proliferating stem cells and progenitor cells in it are sensitive to chemoradiotherapy. Therefore, intestinal injury is one of the main side effects of chemoradiotherapy. Relevant research shows that among cancer patients receiving chemotherapy or radiotherapy, about 40% - 60% will have digestive tract adverse reactions, and among patients receiving pre-chemotherapy before stem cell transplantation surgery, this proportion will almost reach 100%. However, so far, no approved auxiliary drug can specifically reduce the intestinal adverse reactions in chemoradiotherapy. Therefore, it is of great significance to find new drugs for reducing adverse reactions caused by chemoradiotherapy. Summary of the Invention

[0004] The inventors of the present invention unexpectedly found through a large number of experiments that the heterocyclic compound represented by formula (1) or its pharmaceutically acceptable form has the use of reducing adverse reactions caused by chemotherapeutic drugs, thereby completing the present invention.

[0005] In a first aspect, the present invention provides the use of the compound represented by formula (1) or its pharmaceutically acceptable form in the preparation of a drug for reducing adverse reactions caused by chemotherapeutic drugs, and the structure of the compound represented by formula (1) is as follows:

[0006]

[0007] Wherein:

[0008] R 1 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, 3 - 8-membered heteroalkyl, C 6-10 aryl or 5 - 10-membered heteroaryl, and R 1 is optionally substituted by one or more R a substituents,

[0009] The R aSelected from hydrogen, hydroxyl, halogen, nitro, cyano, C 6-10 aryl, 5- to 10-membered heteroaryl, C 1-6 alkyl, -OC 1-6 alkyl, C 2-6 alkenyl, -OC 2-6 alkenyl, C 2-6 alkynyl, -OC 2-6 alkynyl, C 3-6 cycloalkyl, -C 1-6 alkylene-C 3-6 cycloalkyl, -OC 3-6 cycloalkyl, -OC 1-6 alkylene-C 3-6 cycloalkyl, -C 1-6 alkylene-C 6-10 aryl, -OC 6-10 aryl, -OC 1-6 alkylene-C 6-10 aryl, CHO, -(CO)R b , -O(CO)R b , -O(CO)OR b , -C 1-6 alkylene-OR b , -OC 2-6 alkylene-OR b , -C 1-6 alkylene-(CO)R b , -OC 1-6 alkylene-(CO)R b , -CO2R b , -C 1-6 alkylene-CO2R b , -OC 1-6 alkylene-CO2R b , -NR b R c , -C 1-6 alkylene-NR b R c , -OC 2-6 alkylene-NR b R c , -C 1-6 alkylene-(CO)NR b R c , -OC 1-6 alkylene-(CO)NR b R c , -NR b (CO)R c , -C 1-6 alkylene-NR b (CO)R c , -OC2-6 Alkylene-NR b (CO)R c 、-NR b (CO)NR b R c 、-C 1-6 Alkylene-NR b (CO)NR b R c 、-SR b 、-C 1-6 Alkylene-SR b 、-OC 2-6 Alkylene-SR b 、-(SO)R b 、-C 1-6 Alkylene-(SO)R b 、-OC 2-6 Alkylene-(SO)R b 、-SO2R b 、-C 1-6 Alkylene-SO2R b 、-OC 2-6 Alkylene-SO2R b 、-(SO2)NR b R c 、-C 1-6 Alkylene-(SO2)NR b R c 、-OC 1-6 Alkylene-(SO2)NR b R c 、-NR b (SO2)R c 、-C 1-6 Alkylene-NR b (SO2)R c 、-OC 2-6 Alkylene-NR b (SO2)R c 、-NR b (SO2)NR b R c 、-C 1-6 Alkylene-NR b (SO2)NR b R c 、-OC 2-6 Alkylene-NR b (SO2)NR b R c 、-(CO)NR b R c 、-O(CO)NR b R c 、-NRb OR c 、 -NR b (CO)OR c 、 -C 1-6 alkylene - NR b (CO)OR c or - OC 2-6 alkylene - NR b (CO)OR c , wherein the aryl, heteroaryl, alkyl, alkenyl, alkynyl, cycloalkyl, alkylene are optionally substituted by one or more substituents selected from halogen, hydroxy, cyano, nitro, C 1-6 alkyl, - OC 1-6 alkyl or C 3-6 cycloalkyl,

[0010] R 2 is selected from hydrogen, hydroxy, halogen, nitro, cyano, C 6-10 aryl, 5 - 10 - membered heteroaryl, C 1-6 alkyl, - OC 1-6 alkyl, C 2-6 alkenyl, - OC 2-6 alkenyl, C 2-6 alkynyl, - OC 2-6 alkynyl, C 3-6 cycloalkyl, - C 1-6 alkylene - C 3-6 cycloalkyl, - OC 3-6 cycloalkyl, - OC 1-6 alkylene - C 3-6 cycloalkyl, - C 1-6 alkylene - C 6-10 aryl, - OC 6-10 aryl, - OC 1-6 alkylene - C 6-10 aryl, CHO, -(CO)R b 、 - O(CO)R b 、 - O(CO)OR b 、 - C 1-6 alkylene - OR b 、 - OC 2-6 alkylene - OR b 、 - C 1-6 alkylene - (CO)R b 、 - OC 1-6 alkylene - (CO)R b 、 - CO2R b 、 - C 1-6 alkylene - CO2R b 、 - OC 1-6 alkylene - CO2R b 、 - NR b Rc ,-C 1-6 alkylene - NR b R c ,-OC 2-6 alkylene - NR b R c ,-C 1-6 alkylene-(CO)NR b R c ,-OC 1-6 alkylene-(CO)NR b R c ,-NR b (CO)R c ,-C 1-6 alkylene - NR b (CO)R c ,-OC 2-6 alkylene - NR b (CO)R c ,-NR b (CO)NR b R c ,-C 1-6 alkylene - NR b (CO)NR b R c ,-SR b ,-C 1-6 alkylene - SR b ,-OC 2-6 alkylene - SR b ,-(SO)R b ,-C 1-6 alkylene-(SO)R b ,-OC 2-6 alkylene-(SO)R b ,-SO2R b ,-C 1-6 alkylene - SO2R b ,-OC 2-6 alkylene - SO2R b ,-(SO2)NR b R c ,-C 1-6 alkylene-(SO2)NR b R c ,-OC 2-6 alkylene-(SO2)NR b R c ,-NR b (SO2)R c ,-C 1-6 alkylene - NR b (SO2)R c ,-OC2-6 Alkylene-NR b (SO2)R c 、-NR b (SO2)NR b R c , -C 1-6 Alkylene-NR b (SO2)NR b R c 、-OC 2-6 Alkylene-NR b (SO2)NR b R c 、-(CO)NR b R c 、-O(CO)NR b R c 、-NR b OR c 、-NR b (CO)OR c , -C 1-6 Alkylene-NR b (CO)OR c or -OC 2-6 Alkylene-NR b (CO)OR c The aryl, heteroaryl, alkyl, alkenyl, alkynyl, cycloalkyl, alkylene group is optionally substituted by one or more selected from halogen, hydroxyl, cyano, nitro, C 1-6 Alkyl, -OC 1-6 Alkyl or C 3-6 Substituents of the cycloalkyl group are substituted,

[0011] R b and R c are independently selected from hydrogen, C 1-6 Alkyl, -C 1-6 Alkylene-C 6-10 Aryl, -C 1-6 Alkylene-(5-10 membered heteroaryl), C 3-7 Cycloalkyl or C 6-10 Aryl,

[0012] R 3 Select from -OR 7 or -NR 7 R 8 ,

[0013] R 7 and R 8 independently selected from hydrogen, hydroxyl, C 1-6 Alkyl, -OC 1-6 Alkyl, C 2-6 Alkenyl, C 2-6Alkynyl, -C 1-6 Alkylene-C 6-10 Aryl, -C 1-6 Alkylene-(5-10 membered heteroaryl), -C 1-6 Alkylene-(3-8 membered heterocycloalkyl), C 3-7 Cycloalkyl or C 6-10 Aryl, wherein the aryl, heteroaryl, alkyl, alkenyl, alkynyl, cycloalkyl, alkylene are optionally substituted by one or more substituents selected from halogen, hydroxy, cyano, nitro, C 1-6 Alkyl, -OC 1-6 Alkyl or C 3-6 Cycloalkyl,

[0014] Or R 7 And R 8 Together with the N atom to which it is attached form a 3-8 membered heterocycloalkyl or a 5-10 membered heteroaryl,

[0015] X is selected from O or NR d , R d Is selected from hydrogen, C 1-6 Alkyl, -C 1-6 Alkylene-C 6-10 Aryl, -C 1-6 Alkylene-(5-10 membered heteroaryl), C 3-7 Cycloalkyl or C 6-10 Aryl;

[0016] The pharmaceutically acceptable forms are selected from pharmaceutically acceptable salts, esters, stereoisomers, tautomers, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs.

[0017] In some embodiments, in the compound of formula (1), R 1 Is selected from C 1-6 Alkyl, C 6-10 Aryl or 5-10 membered heteroaryl; preferably, R 1 Is selected from pyridyl, methyl, phenyl, thienyl, benzothienyl, furyl, benzofuryl, pyrrolyl or thiazolyl; more preferably, R 1 Is selected from -CH3,

[0018] In some embodiments, the compound of formula (1) is the compound of formula (2) below:

[0019]

[0020] Wherein:

[0021] R 4 , R5 and R 6 are independently selected from hydrogen, hydroxy, halogen, nitro, cyano, C 6-10 aryl, 5- to 10-membered heteroaryl, C 1-6 alkyl, -OC 1-6 alkyl, C 2-6 alkenyl, -OC 2-6 alkenyl, C 2-6 alkynyl, -OC 2-6 alkynyl, C 3-6 cycloalkyl, -C 1-6 alkylene-C 3-6 cycloalkyl, -OC 3-6 cycloalkyl, -OC 1-6 alkylene-C 3-6 cycloalkyl, -C 1-6 alkylene-C 6-10 aryl, -OC 6-10 aryl, -OC 1-6 alkylene-C 6-10 aryl, CHO, -(CO)R b 、-O(CO)R b 、-O(CO)OR b 、-C 1-6 alkylene-OR b 、-OC 2-6 alkylene-OR b 、-C 1-6 alkylene-(CO)R b 、-OC 1-6 alkylene-(CO)R b 、-CO2R b 、-C 1-6 alkylene-CO2R b or -OC 1-6 alkylene-CO2R b ,wherein the aryl, heteroaryl, alkyl, alkenyl, alkynyl, cycloalkyl, alkylene are optionally substituted by one or more substituents selected from halogen, hydroxy, cyano, nitro, C 1-6 alkyl, -OC 1-6 alkyl or C 3-6 cycloalkyl,

[0022] Y is selected from S, O or NH,

[0023] X, R b 、R 2 、R 7 and R 8 are as defined in formula (1).

[0024] In some embodiments, the compound represented by formula (2) is the compound represented by formula (3):

[0025]

[0026] Wherein, R 4 and R 5 and R 6 and R 2 and R 7 and R 8 are defined as in formula (2).

[0027] In some embodiments, in the compounds represented by formula (2) or formula (3), R 4 and R 5 and R 6 are independently selected from hydrogen, hydroxyl, halogen, nitro, cyano, C 6-10 aryl, 5- to 10-membered heteroaryl, C 1-6 alkyl, -OC 1-6 alkyl, C 2-6 alkenyl, -OC 2-6 alkenyl, C 2-6 alkynyl, -OC 2-6 alkynyl, C 3-6 cycloalkyl, -OC 3-6 cycloalkyl or -OC 6-10 aryl, and the aryl, heteroaryl, alkyl, alkenyl, alkynyl, cycloalkyl are optionally substituted with one or more substituents selected from halogen, hydroxyl, cyano, nitro or C 1-6 alkyl;

[0028] Preferably, R 4 and R 5 and R 6 are independently selected from hydrogen, hydroxyl, halogen, nitro, cyano, C 6-10 aryl or C 1-6 alkyl; More preferably, R 4 and R 5 and R 6 are independently selected from hydrogen, hydroxyl, halogen, nitro, cyano, phenyl, methyl or n-butyl.

[0029] In some embodiments, in the compounds represented by formula (1), formula (2) or formula (3), R 2 is selected from hydrogen, hydroxyl, halogen, nitro, cyano, C 6-10 aryl, 5- to 10-membered heteroaryl, C 1-6 alkyl, -OC 1-6 alkyl, C 2-6 alkenyl, -OC 2-6 alkenyl, C 2-6 alkynyl, -OC 2-6 alkynyl, C 3-6 cycloalkyl, -OC 3-6Cycloalkyl, -OC 6-10 aryl or CHO; preferably, R 2 is selected from hydrogen, hydroxy, halogen, nitro, cyano or methyl.

[0030] In some embodiments, in the compounds represented by formula (1), formula (2) or formula (3), R 7 and R 8 are independently selected from hydrogen, hydroxy, C 1-6 alkyl, -OC 1-6 alkyl, -C 1-6 alkylene-(5-10 membered heteroaryl), -C 1-6 alkylene-(3-8 membered heterocycloalkyl), C 3-7 cycloalkyl or C 6-10 aryl, and the aryl, heteroaryl, alkyl, cycloalkyl, alkylene are optionally substituted with one or more substituents selected from halogen, hydroxy, cyano, nitro or C 1-6 alkyl, or R 7 and R 8 together with the N atom to which they are attached form a 3-8 membered heterocycloalkyl,

[0031] preferably, R 7 and R 8 are independently selected from hydrogen, hydroxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tert-butyl, methyl, n-butyl, n-propyl, methoxy, ethoxy,

[0032] In some embodiments, the compounds represented by formula (1) are selected from the following compounds:

[0033]

[0034]

[0035] In a second aspect, the present invention provides the use of the compounds represented by formula (1) to formula (3) or their pharmaceutically acceptable forms in the preparation of a drug for reducing the adverse reactions caused by chemotherapeutic drugs, wherein the adverse reactions are intestinal adverse reactions.

[0036] In a third aspect, the present invention provides a method for reducing the adverse reactions caused by chemotherapeutic drugs, which comprises the following steps: administering an effective amount of the compounds represented by formula (1) to formula (3) or their pharmaceutically acceptable forms to an individual in need thereof.

[0037] The present invention is not limited to the specific embodiments described herein; it should also be understood that the terms used herein are for descriptive purposes only and not for limiting specific embodiments.

[0038] General Terms and Definitions

[0039] Unless otherwise defined, the meanings of the terms used herein are the same as those commonly understood by those skilled in the art. The techniques used herein are intended to refer to the techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to those skilled in the art. Although the following terms are easily understood by those skilled in the art, they are still described below for better explanation of the present invention.

[0040] The terms "comprising", "including", "having" or "involving" and other variant forms thereof used herein refer to an inclusive or open set concept and do not exclude other unenumerated elements or method steps. Those skilled in the art should understand that the above terms such as "comprising" cover the meaning of "consisting of".

[0041] The term "one or more" or a similar expression "at least one" means, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0042] When the lower and upper limits of a numerical range are disclosed, any numerical value or any sub-range falling within that range is specifically disclosed. In particular, each numerical range of parameters disclosed herein (for example, in the form of "about a to b", or equivalently "approximately a to b", or equivalently "about a - b") should be understood to cover each numerical value and sub-range therein. For example, "C 1-6 " should be understood to cover any sub-range and each point value therein, such as C 2-5 、C 3-4 、C 1-2 、C 1-3 、C 1-4 、C 1-5 etc., and C1, C2, C3, C4, C5, C6, etc. Again, for example, "3 - 10 yuan" should be understood to cover any sub-range and each point value therein, such as 3 - 4 yuan, 3 - 5 yuan, 3 - 6 yuan, 3 - 7 yuan, 3 - 8 yuan, 3 - 9 yuan, 4 - 5 yuan, 4 - 6 yuan, 4 - 7 yuan, 4 - 8 yuan, 5 - 7 yuan, 5 - 8 yuan, 6 - 7 yuan, etc., and 3, 4, 5, 6, 7, 8, 9, 10 yuan, etc.

[0043] When used alone or in combination with other groups herein, the term "alkyl" refers to a straight-chain or branched-chain saturated aliphatic hydrocarbon group. For example, the term "C 1-6"Alkyl" refers to a saturated straight-chain or branched-chain hydrocarbon group having 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6 carbon atoms). For example, "C 1-6 "Alkyl" can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or n-hexyl, etc.

[0044] As used herein, either alone or in combination with other groups, the term "alkylene" refers to a saturated straight-chain or branched-chain divalent hydrocarbon group. For example, the term "C 1-6 "Alkylene" as used herein refers to a saturated straight-chain or branched-chain divalent hydrocarbon group having 1 to 6 carbon atoms, such as methylene, ethylene, propylene, or butylene, etc.

[0045] As used herein, either alone or in combination with other groups, the term "cycloalkyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g., fused-ring, bridged-ring or spiro-ring) non-aromatic hydrocarbon group. For example, the term "C 3-6 "Cycloalkyl" as used in the present invention refers to a cycloalkyl having 3 to 6 carbon atoms. For example, cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or bicyclo[2.2.1]heptyl, etc.

[0046] As used herein, either alone or in combination with other groups, the term "heterocycloalkyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g., fused-ring, bridged-ring or spiro-ring) non-aromatic group, the ring atoms of which are composed of carbon atoms and at least one heteroatom selected from N, O, and S. If the valence bond requirements are met, the heterocyclic group can be connected to the rest of the molecule through any one of the ring atoms. For example, the term "3-8 membered heterocycloalkyl" as used in the present invention refers to a heterocyclic group having 3 to 8 ring atoms. For example, the heterocyclic group can be oxiranyl, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuryl, dioxolanyl, pyrrolidinyl, pyrrolidinone, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithianyl, or trithianyl.

[0047] As used herein, either alone or in combination with other groups, the term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated π electron system. For example, the term "C 6-10 "Aryl" as used in the present invention refers to an aryl having 6 to 10 carbon atoms. For example, aryl can be phenyl, naphthyl, anthryl, phenanthryl, acenaphthylenyl, azulenyl, fluorenyl, indenyl, pyrenyl, etc.

[0048] As used herein, alone or in combination with other groups, the term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated π-electron system, the ring atoms of which are composed of carbon atoms and at least one heteroatom selected from N, O, and S. If the valence requirements are met, the heteroaryl can be attached to the rest of the molecule through any one of the ring atoms. For example, the term "5- to 10-membered heteroaryl" as used in the present invention refers to a heteroaryl having 5 to 10 ring atoms. For example, the heteroaryl can be thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and their benzo derivatives, pyrrolopyridyl, pyrrolopyrazinyl, pyrazolopyridyl, imidazolopyridyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, etc.

[0049] As used herein, alone or in combination with other groups, the term "alkenyl" refers to a straight-chain or branched-chain aliphatic hydrocarbon group having one or more carbon-carbon double bonds. For example, the term "C 2-6 alkenyl" as used herein refers to an alkenyl having 2 to 6 carbon atoms and one, two or three (preferably one) carbon-carbon double bonds (such as 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, 4-methyl-3-pentenyl, etc.).

[0050] As used herein, alone or in combination with other groups, the term "alkynyl" refers to a straight-chain or branched-chain aliphatic hydrocarbon group having one or more carbon-carbon triple bonds. For example, the term "C 2-6 alkynyl" as used herein refers to an alkynyl having 2 to 6 carbon atoms and one, two or three (preferably one) carbon-carbon triple bonds (such as ethynyl, 1-propynyl, 2-propynyl, 2-butynyl, 3-butynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, etc.).

[0051] As used herein, alone or in combination with other groups, the term "halo" or "halogen" group means F, Cl, Br or I.

[0052] As used herein, alone or in combination with other groups, the term "hydroxy" refers to -OH.

[0053] As used herein, alone or in combination with other groups, the term "cyano" refers to -CN.

[0054] As used herein, alone or in combination with other groups, the term "nitro" refers to -NO2.

[0055] As used herein, either alone or in combination with other groups, the term "amino" refers to -NH2.

[0056] As used herein, either alone or in combination with other groups, the term "oxo" refers to =O.

[0057] As used herein, the term "each independently" or "independently" means that at least two groups (or moieties) having the same or similar range of values present in a structure can have the same or different meanings in a particular situation. For example, if substituents X and Y are each independently hydrogen, halogen, hydroxy, -CN, alkyl or aryl, then when substituent X is hydrogen, substituent Y can be either hydrogen or halogen, hydroxy, -CN, alkyl or aryl; similarly, when substituent Y is hydrogen, substituent X can be either hydrogen or halogen, hydroxy, -CN, alkyl or aryl.

[0058] The term "substituted" and its other variant forms as used herein mean that one or more (such as 1, 2, 3 or 4) atoms or groups of atoms (such as a hydrogen atom) on the specified atom are replaced by other equivalents, provided that the normal valence of the specified atom or group of atoms in the current situation is not exceeded and a stable compound can be formed. If an atom or group of atoms is described as "optionally substituted by...", it can either be substituted or unsubstituted. Unless otherwise specified, the attachment site of a substituent can be from any suitable position of the substituent. When the connecting bond in a substituent is shown as a chemical bond passing between two interconnected atoms in a ring system, it means that the substituent can be attached to any ring-forming atom in the ring system.

[0059] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is substantially non-toxic to an organism. Pharmaceutically acceptable salts generally include (but are not limited to) salts formed by reacting a compound of the present invention with a pharmaceutically acceptable inorganic acid / organic acid / acidic amino acid or inorganic base / organic base / basic amino acid, and such salts are also referred to as acid addition salts or base addition salts. A review of suitable salts can be found, for example, in Jusiak, Soczewinski, et al., Remington’s Pharmaceutical Sciences [M], Mack Publishing Company, 2005 and Stahl, Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use [M], Wiley-VCH, 2002. Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0060] The term "pharmaceutically acceptable ester" refers to an ester that is substantially non-toxic to living organisms and hydrolyzes in vivo to the compound of the present invention or its salt. Additionally, the compounds of the present invention themselves may also be esters.

[0061] The term "isomer" refers to compounds that have the same molecular weight due to having the same number and type of atoms, but differ in the spatial arrangement or configuration of the atoms.

[0062] The term "stereoisomer" (or "optical isomer") refers to stable isomers that have a perpendicular plane of asymmetry due to having at least one chiral element (including chiral centers, chiral axes, chiral planes, etc.), and thus can rotate plane-polarized light. Due to the presence of asymmetric centers and other chemical structures in the compounds of the present invention that may lead to stereoisomerism, the present invention also includes these stereoisomers and their mixtures. Since the compounds of the present invention (or their pharmaceutically acceptable salts) may contain asymmetric carbon atoms, they can exist in the form of a single stereoisomer, a racemate, a mixture of enantiomers and diastereomers. The term "enantiomer" refers to a pair of stereoisomers that are non-superimposable mirror images of each other. The term "diastereomer" or "diastereoisomer" refers to optical isomers that are not mirror images of each other. The term "racemic mixture" or "racemate" refers to a mixture containing equal amounts of a single enantiomer (i.e., an equimolar mixture of two R and S enantiomers). The term "non-racemic mixture" refers to a mixture containing unequal amounts of a single enantiomer. Unless otherwise indicated, all stereoisomeric forms of the compounds of the present invention are within the scope of the present invention.

[0063] The term "tautomer" (or "tautomeric form") refers to structural isomers that have different energies and can be interconverted through a low energy barrier. If tautomerism is possible (e.g., in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (or proton-transfer tautomers) include (but are not limited to) interconversions that occur through proton migration, such as keto-enol isomerization, imine-enamine isomerization, amide-imidol isomerization, nitroso-oxime isomerization, etc. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.

[0064] The term "solvate" refers to a substance formed by the compound of the present invention (or its pharmaceutically acceptable salt) binding to at least one solvent molecule through non-covalent intermolecular forces. The compounds of the present invention can exist in the form of solvates, which contain polar solvents as elements of the crystal lattice structure. The amount of the polar solvent can exist in a stoichiometric or non-stoichiometric ratio.

[0065] The term "isotope label" refers to a derivative compound formed by replacing a specific atom in the compound of the present invention with its isotope atom. Unless otherwise indicated, the compounds of the present invention include various isotopes of H, C, N, O, F, P, S, Cl, such as 2 H(D), 3 H(T), 13 C, 14 C, 13 N, 15 N, 17 O, 18 O, 18 F, 31 P, 32 P, 34 S, 35 S, 36 S, 37 Cl and 125 I. For example, 12 C can be replaced by 13 C or 14 C; 1 H can be replaced by 2 H(D, deuterium) or 3 H(T, tritium); 16 O can be replaced by 18 O, etc.

[0066] Those skilled in the art will understand that since nitrogen requires available lone pairs of electrons to be oxidized to an oxide, not all nitrogen-containing heterocycles are capable of forming N-oxides. Those skilled in the art will identify nitrogen-containing heterocycles that can form N-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 and include oxidizing heterocycles and tertiary amines with peroxyacids such as peracetic acid and meta-chloroperoxybenzoic acid (mCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate, and dioxiranes such as dimethyldioxirane. These methods for preparing N-oxides have been widely described and reviewed in the literature, see, for example: T.L. Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp748-750 (A.R. Katritzky and A.J. Boulton, Eds., Academic Press); and G.W.H. Cheeseman and E.S.G. Werstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp 390-392 (A.R. Katritzky and A.J. Boulton, Eds., Academic Press).

[0067] The term "metabolite" refers to a derivative compound formed after the metabolism of the compound of the present invention, such as those produced through oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis and other reactions. For further information on metabolism, see Goodman and Gilman's: The Pharmacological Basis of Therapeutics [M], McGraw-Hill International Editions, 1996. The present invention encompasses all possible metabolite forms of the compound of the present invention, i.e., substances formed in the body of an individual to whom the compound of the present invention is administered. The metabolites of a compound can be identified by well-known techniques in the art, and their activities can be characterized through tests.

[0068] The term "prodrug" refers to a derivative compound that can directly or indirectly provide the compound of the present invention after being administered to an individual. Particularly preferred derivative compounds or prodrugs are those that can improve the bioavailability of the compound of the present invention (e.g., are more readily absorbed into the blood) when administered to an individual, or compounds that facilitate the delivery of the parent compound to the site of action (e.g., the lymphatic system). Unless otherwise indicated, all prodrug forms of the compound of the present invention are within the scope of the present invention, and various prodrug forms are known in the art, for example, see T. Higuchi, V. Stella, Pro-drugs as Novel Drug Delivery Systems [J], American Chemical Society, Vol. 14, 1975. In addition, the present invention also encompasses compounds of the present invention containing protecting groups. During any process for preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive groups or reactive groups on any relevant molecule, thereby forming chemically protected forms of the compounds of the present invention. This can be achieved through conventional protecting groups, such as those described in T.W. Greene, P.G.M. Wuts, Protective Groups in Organic Synthesis [M], John Wiley & Sons, 2006. These protecting groups can be removed at appropriate subsequent stages using methods known in the art.

[0069] As used herein, the term "effective amount" refers to the amount of an active ingredient that will, to some extent, achieve the desired effect after administration, such as alleviating one or more symptoms of the disorder being treated or preventing the occurrence of the disorder or its symptoms.

[0070] As used herein, "individual" includes human or non-human animals. Exemplary human individuals include human individuals suffering from a disease (such as the diseases described herein) (referred to as patients) or normal individuals. In the present invention, "non-human animals" include all vertebrates, such as non-mammals (such as birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals and / or domesticated animals (such as sheep, dogs, cats, cows, pigs, etc.).

[0071] The compounds of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations by combining with suitable pharmaceutically acceptable excipients, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols, etc.

[0072] Typical routes of administration of the compounds of the present invention or their pharmaceutically acceptable forms include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

[0073] In some embodiments, the preparation is in oral form. For oral administration, the preparation can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present invention to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, suspensions, etc. for oral administration to patients.

[0074] Solid oral preparations can be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally milling the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain the core of a tablet or dragee. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants or flavoring agents, etc.

[0075] The compounds of the present invention can also be administered in the form of sterile injectables, including sterile aqueous or oily suspensions, or sterile aqueous or oily solutions. Among them, the carriers that can be used include, but are not limited to: water, Ringer's solution and isotonic sodium chloride solution. In addition, sterilized non-volatile oils can also be used as solvents or suspension media, such as monoglycerides or diglycerides.

[0076] The compounds of the present invention are also suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.

[0077] The dosing regimen can be adjusted to provide the optimal desired response. For example, when administered in the form of an injection, a single bolus injection, an infusion bolus, and / or a continuous infusion can be administered. For example, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the treatment situation. It should be noted that the dose value can vary depending on the type and severity of the condition to be alleviated, and can include a single or multiple doses. Generally, the dose of treatment varies depending on considerations such as: the age, sex, and general health of the patient to be treated; the frequency of treatment and the nature of the desired effect; the degree of tissue damage; the duration of the symptoms; and other variables that can be adjusted by the individual physician. It is further understood that for any specific individual, the specific dosing regimen should be adjusted over time based on individual needs and the professional judgment of the person administering the pharmaceutical composition or supervising the administration of the composition. The dosage and dosing regimen of the pharmaceutical composition can be readily determined by a person of ordinary skill in the clinical art. For example, the compounds of the present invention can be administered once every 4 days to once every 3 days in divided doses, and the dosage can be, for example, 0.01 to 1000 mg / dose. The required dose can be administered one or more times to achieve the desired result. The compounds according to the present invention can also be provided in unit dose form. Also, for all methods of administration of the general formula compounds described herein, the dose administered per day is 0.01 mg / kg to 50 mg / kg body weight, preferably 0.03 mg / kg to 30 mg / kg body weight, more preferably 0.05 mg / kg to 20 mg / kg body weight, in the form of a single or separate dose.

[0078] Chemotherapeutic drugs include, for example, alkylating agents (nimustine, carmustine, cyclophosphamide, temozolomide, etc.), antimetabolites (5-fluorouracil, gemcitabine, methotrexate, etc.), antitumor antibiotics (doxorubicin, epirubicin, mitomycin, etc.), plant-derived anticancer drugs (paclitaxel, vinblastine, etoposide, irinotecan, etc.), antitumor hormones (tamoxifen, megestrol acetate, etc.), cisplatin, etc.

[0079] Adverse reactions caused by chemotherapeutic drugs are, for example, intestinal adverse reactions, myelosuppression, reduced immunity, organ weakness, and inflammation. Intestinal adverse reactions are, for example, loss of appetite, nausea and vomiting, oral ulcers, abdominal pain, and diarrhea.

[0080] The compounds of the present invention can be used alone, or can be used in combination with other known drugs for alleviating the adverse reactions caused by chemotherapeutic drugs.

[0081] The compounds of the present invention can be used before the use of chemotherapeutic drugs, used simultaneously with chemotherapeutic drugs, or used after the use of chemotherapeutic drugs. Description of the Drawings

[0082] Figure 1: Protection Experiment of ISX against 5-Fluorouracil-induced Injury of Mouse Small Intestine Organoids - Immunofluorescence Staining Experiment

[0083] Figure 2 : Protection Experiment of ISX against 5-Fluorouracil-induced Injury of Mouse Small Intestine Organoids - Bright-Field and Live / Dead Cell Double Staining Experiment

[0084] Figure 3 : Statistical Results of Protection Experiment of ISX against 5-Fluorouracil-induced Injury of Mouse Small Intestine Organoids - Bright-Field and Live / Dead Cell Double Staining Experiment

[0085] Figure 4 : Protection Experiment of ISX against Etoposide-induced Injury of Mouse Small Intestine Organoids - Immunofluorescence Staining Experiment

[0086] Figure 5 : Protection Experiment of ISX against Etoposide-induced Injury of Mouse Small Intestine Organoids - Western Experiment

[0087] Figure 6 : Protection Experiment of ISX against Etoposide-induced Injury of Mouse Small Intestine Organoids - Bright-Field and Live / Dead Cell Double Staining Experiment

[0088] Figure 7 : Statistical Results of Protection Experiment of ISX against Etoposide-induced Injury of Mouse Small Intestine Organoids - Bright-Field and Live / Dead Cell Double Staining Experiment

[0089] Figure 8 : Protection Experiment of ISX on Human Colonic Organoids - Bright-Field Experiment

[0090] Figure 9 : Statistical Analysis Results of Protection Experiment of ISX on Human Colonic Organoids

[0091] Figure 10 : Protection Experiment of ISX on Human Colonic Organoids - Live / Dead Cell Double Staining Experiment

[0092] Figure 11 : Protection Effect of ISX on Mouse Intestine - TUNEL Experiment

[0093] Figure 12 : Statistical Results of Protection Effect of ISX on Mouse Intestine - TUNEL Experiment

[0094] Figure 13 : Comparative Experiment on the Protection Effects of PD332991, CHIR99021 and ISX on Intestinal Organoids Detailed Implementation Modes

[0095] Experimental Materials and Animals

[0096] Animals: Healthy 6 - 8 - week - old C57BL / 6 mice, weighing 20 - 23 g, purchased from Beijing Biocytogen Co., Ltd. and housed in the SPF - level experimental animal center of Tsinghua University.

[0097] Materials and Reagents:

[0098] ISX (selleck s7914),

[0099] PD0332991, CHIR99021, 5 - fluorouracil, and etoposide (Selleck, S1579, S1263, S1209, S1225).

[0100] Human organ culture medium (STEMdiff intestinal organoid kit, STEMCELL, catalog number 05140), mouse organoid culture medium (intestiCult organoid growth medium, STEMCELL, 06005).

[0101] Cleaved caspase - 3 antibody (CST, catalog number 9661S), HRP - conjugated secondary antibodies (Thermo Fisher, catalog numbers 31430 and 31460 respectively), β - Tubulin antibody (Three - arrow Biotech, catalog number KM9003T), fluorescent secondary antibody Goat anti - Rabbit IgG(H + L)Cross - Adsorbed Secondary Antibody, Alexa Fluor 568 (Invitrogen, catalog number A - 11011).

[0102] Live / Dead Cell Double Staining Kit (Calcein - AM / PI) (BioLebio Co., Ltd., catalog number HR0444), One - Step TUNEL Apoptosis Detection Kit (Red Fluorescence) (Beyotime Biotechnology, catalog number C1089).

[0103] PBS (Zhongke Maichen, product number CC008.1), Matrigel (Corning, product number 254230), cellstripping solution (cellrecovery solution, Corning, 354253), cell lysis buffer (Beyotime Biotechnology, product number P0013), DAPI (Beyotime Biotechnology, C1002), Triton X-100 (sigma, product number 9036-19-5), BSA (Albumin, Zhongke Maichen, LS000290), Tween-20 (sigma, 9005-64-5), immunofluorescence buffer (IF buffer): Triton X-100 (0.1%, diluted with PBS) + Tween-20 (0.05%, diluted with PBS), anti-quenching agent (ProLong Gold anti-fade mounting medium, Invitrogen, product number P10144), Proteinase K (Beyotime Biotechnology, product number ST532), ECL (Pierce ECLPlus Western Blotting Substrate, product number 32132), Tween 80 (Merk, STS0204), CMC-Na (selleck, s6703).

[0104] Polyacrylamide (PAGE) gel: The formulations of PAGE separating gels and stacking gels with different concentrations are shown in the following table:

[0105] Formulation of PAGE separating gels with different concentrations (lower layer gels)

[0106]

[0107] When preparing, add the first 5 components in the order shown in the table. After confirming the correct volume and setting up the gel casting plate, add TEMED (Sigma, T7024) to the separating gel, mix well, and pour the solution into the gap of the gel plate until it is 1 cm from the upper edge (generally, for a 0.75 mm thick gel, 3.2 - 3.4 mL of solution needs to be poured). Add 70% ethanol to flatten the liquid surface. Let it stand at room temperature for 30 minutes to 1 hour to allow the gel to solidify completely. It can be placed near a fluorescent lamp tube to accelerate solidification. It should be noted that when the room temperature is relatively low, white crystals may precipitate from the 10% SDS solution. In this case, the SDS solution should be placed in a 37 - 60 °C water bath to redissolve the SDS crystals until the solution returns to a uniform and clear state before continuing to prepare the PAGE gel.

[0108] Formulation of PAGE stacking gels with different volumes (upper layer gels)

[0109] Components 2 mL 4 mL 6 mL 8 mL 10 mL 20 M H2O 1.4 mL 2.7 mL 4.1 mL 5.5 mL 6.8 mL 30% AB 330 μL 670 μL 1 mL 1.3 mL 1.7 mL Tris·HCl, pH 6.8 250 μL 500 μL 750 μL 1 mL 1.25 mL 10% SDS 20 μL 40 μL 60 μL 80 μL 100 μL 10% APS 20 μL 40 μL 60 μL 80 μL 100 μL TEMED 2 μL 4 μL 6 μL 8 μL 10 μL

[0110] After the separating gel has solidified, suck out the ethanol completely with a vacuum pump. Add TEMED to the stacking gel solution, mix well, and immediately pour it into the gap of the gel plate until it is full, without leaving any bubbles. Carefully insert the gel comb (choose a 10-well or 15-well comb according to needs). Let it stand at room temperature for another 20 - 30 minutes until it is completely solidified. At this time, the gel can be used immediately for electrophoresis or stored at 4°C (to be used within 3 days). It is recommended to use freshly prepared gel on the same day.

[0111] When preparing the gradient gel, first pour the high-concentration PAGE separating gel solution, and then immediately and carefully pour the low-concentration PAGE separating gel solution evenly until it reaches 1 cm below the upper edge of the short glass plate. Flatten it with 70% ethanol. The subsequent steps are the same as those for preparing a single-concentration PAGE.

[0112] 70% ethanol: Dilute absolute ethanol to a volume concentration of 70% with deionized water, and use it to flatten the liquid surface when preparing the PAGE separating gel.

[0113] 10% ammonium persulfate solution (10% APS): Weigh 5 g of ammonium persulfate powder (Sigma, A9164), dissolve it in 50 mL of deionized water, aliquot it into 1.5 mL tubes (300 - 500 μL per tube), and store at -20°C.

[0114] 10× SDS running buffer: 151 g of Tris base, 470 g of glycine, 250 g of SDS, make up to 5 L with deionized water, and store at room temperature.

[0115] 1× SDS running buffer (1 L): 100 mL of 10× SDS running buffer, 900 mL of deionized water.

[0116] 10× transfer buffer: 291.4 g of Tris base, 146.5 g of glycine, make up to 5 L with deionized water, and store at room temperature.

[0117] 1× transfer buffer (1 L): 100 mL of 10× transfer buffer, 200 mL of methanol, 700 mL of distilled water.

[0118] 20× TBS (5 L): Dissolve 800 g of NaCl, 20 g of KCl, and 300 g of Tris base in deionized water, adjust the pH to 7.4 with concentrated hydrochloric acid, and make up the volume, then store at room temperature.

[0119] 1× TBST (1 L): Add 50 mL of 20× TBS to a 1 L reagent bottle. When the volume is close to 1 L after adding deionized water, add 500 μL of Tween-20 (AMRESCO), and then make up the volume with deionized water.

[0120] Blocking solution (5% milk, TBST): Weigh 2.5 g of skim milk powder, dissolve it in 45 mL of 1×TBST, and make up the volume to 50 mL. Prepare it freshly before use.

[0121] Primary antibody dilution solution (5% BSA, TBST): Weigh 2.5 g of bovine serum albumin (BSA, Maichen), dissolve it in 45 mL of 1×TBST, add 150 μL of 10% sodium azide aqueous solution, and make up the volume to 50 mL. Store it at 4°C.

[0122] Experimental Operations

[0123] 1. Isolation of crypts and organoid culture

[0124] Select mice with appropriate genotypes and genetic backgrounds according to experimental requirements. Generally, 6 - 8 - week - old C57BL / 6 mice are selected as experimental materials to ensure the survival efficiency of organoids. Before the experiment, make the following preparations: pre - cool the centrifuge to 4°C; put the pipette tips in the refrigerator for pre - cooling; pre - heat the multi - well cell culture plate in the cell culture incubator; pre - cool PBS and 5 mM EDTA (prepared with PBS) on ice.

[0125] (1) After sacrificing the mouse by cervical dislocation, place it ventral - side up in a 10 - cm cell culture plate, and disinfect and moisten its abdomen with 75% medical alcohol.

[0126] (2) Cut open the abdominal cavity with scissors and take out the small intestine (pay attention to removing the adipose tissue on the outer wall of the intestine during the separation process). Cut off the first half of the small intestine and transfer it to a cell culture dish containing pre - cooled PBS.

[0127] (3) Cut open the intestine with surgical scissors, hold the small intestine tissue with forceps and rinse it back and forth in PBS several times, then discard the PBS. Rinse it again with fresh PBS until the intestinal contents are completely washed away.

[0128] (4) Cut the small intestine tissue into small pieces about 4 mm on each side, and transfer them to a cell culture dish pre - added with pre - cooled 5 mM EDTA, and let it stand at 4°C for 30 minutes.

[0129] (5) Transfer the small intestine tissue to a 50 - mL centrifuge tube with forceps, add 10 mL of pre - cooled PBS, and shake it vigorously up and down 10 times (at this time, the intestinal villi will fall off).

[0130] (6) Clamp the small intestine tissue back into the 50 - mL centrifuge tube with forceps, add 10 mL of pre - cooled PBS, shake it vigorously up and down 10 times again, and pour the liquid in the tube into the cell culture dish.

[0131] (7) Repeat the above operation once. At this time, the villous part of the small intestine tissue has basically fallen off, showing a white and semi - transparent thin sheet.

[0132] (8) Use tweezers to put it back into the 5 mM EDTA solution and let it stand at 4°C for 30 minutes.

[0133] (9) Transfer the small intestinal tissue to a 50 ml centrifuge tube, add 10 ml of pre-cooled PBS, gently shake up and down 10 times, and pour the liquid in the tube into the cell culture dish.

[0134] (10) Use forceps to clamp the small intestinal tissue back into a 50-ml centrifuge tube, add 10 ml of pre-cooled PBS, and shake vigorously up and down 10 times (at this time, a large number of small intestinal crypts will fall into the PBS). Pass the PBS containing the crypts through a 70-μm sieve and collect it in a new 50-ml centrifuge tube (operate on ice).

[0135] (11) Repeat the above steps 2 times.

[0136] (12) Centrifuge the collected crypts at 300 × g for 2 min, discard the supernatant, place on ice, add 1 ml of pre-chilled PBS to resuspend the crypts, and count them.

[0137] (13) Take an appropriate amount of the crypt suspension and dilute it to 100 crypts per μL. Pipette an equal volume of Matrigel and mix it evenly (operate on ice to prevent Matrigel from solidifying).

[0138] (14) Drop the mixed Matrigel into a preheated multi-well cell culture plate and spread it as flat as possible (but avoid touching the edge of the well). Add 20 μl of Matrigel to each well of the 24-well plate.

[0139] (15) Place the cell culture plate in a 37°C incubator and let it sit for 10 to 15 minutes. At this time, you can preheat the intestinal organoid culture medium.

[0140] (16) After Matrigel is fully solidified, add the preheated culture medium into the multi-well cell culture plate along the well wall. Add 500 μL to each well of the 24-well plate. Add the same volume of PBS to the remaining wells to prevent uneven evaporation of the liquid.

[0141] (17) The cells were transferred to a 37°C cell culture incubator containing 5% carbon dioxide for culture. The growth of the organoids was observed on the second day, and the culture medium was replaced every other day.

[0142] 2. Passaging of Organoids

[0143] (1) Use a 1 ml pipette tip (with a small cut using scissors) to scrape off the Matrigel wrapped around the organoids and transfer it to a 15 ml centrifuge tube. Then change to a normal 1 ml pipette tip and blow vigorously until the organoids are broken up.

[0144] (2) Centrifuge at 300×g for 1 minute, discard the supernatant, add 10 mL of PBS to wash once, centrifuge at 200×g for 2 minutes, and discard the supernatant.

[0145] (3) Resuspend the precipitated organoid fragments with an appropriate amount of PBS and place on ice. Take an appropriate volume of the suspension and mix well with an equal volume of Matrigel.

[0146] (4) Drop the Matrigel onto a pre-warmed multi-well cell culture plate and flatten it as much as possible. Place it in the cell culture incubator. After the Matrigel has fully solidified, add the culture medium. The passage ratio is generally 1:4 to 1:6.

[0147] 3. Organoid immunofluorescence

[0148] (1) Carefully transfer the Matrigel coating the organoids to a 1.5 mL centrifuge tube using a 1 mL pipette tip, centrifuge at 500×g for 1 minute, and discard the supernatant.

[0149] (2) Add the de-gel solution (cell recovery solution) and let it stand on ice for 40 minutes.

[0150] (3) Carefully aspirate the de-gel solution, wash once with PBS, add 4% paraformaldehyde, and fix overnight at 4°C.

[0151] (4) Carefully aspirate the paraformaldehyde, wash twice with PBS (let the organoids settle naturally on ice).

[0152] (5) Aspirate the PBS, add the permeabilization solution (Triton X-100, 1%, diluted with PBS), and permeabilize at room temperature for 20 minutes.

[0153] (6) Aspirate the permeabilization solution, add the blocking solution (Triton X-100 (0.1%, diluted with PBS) + BSA (3%, diluted with PBS)), and block at room temperature for 1 hour.

[0154] (7) Aspirate the blocking solution, add the primary antibody diluted (1:1000) with the blocking solution, and let it stand overnight at 4°C.

[0155] (8) Carefully aspirate the primary antibody into a centrifuge tube (it can be recycled).

[0156] (9) Add the immunofluorescence buffer (IF buffer) to the tube containing the organoids, flick to mix well, wash at 500 revolutions per minute at room temperature for 5 minutes, let it stand for 2 minutes until the organoids precipitate to the bottom of the tube, and carefully aspirate the liquid. Repeat 3 times, and aspirate as dry as possible for the last time.

[0157] (10) Dilute the corresponding fluorescent secondary antibody (1:500) with the blocking solution and add it to the centrifuge tube containing the organoids, incubate in the dark at room temperature for 45 minutes.

[0158] (11) Carefully aspirate the secondary antibody, and add DAPI (1 μg / mL) diluted with immunofluorescence buffer to stain the nuclei for 10 minutes.

[0159] (12) Wash 3 times with immunofluorescence buffer (same as step 9)

[0160] (13) Paste the spacer onto the glass slide, transfer the organoids to the holes of the spacer using a 200 μL pipette tip with a small cut, and aspirate the remaining liquid.

[0161] (14) Add 12 μL of anti - quenching agent to the holes of the spacer.

[0162] (15) Cover with a coverslip and seal the edges with nail polish.

[0163] (16) Incubate at room temperature in the dark and well - ventilated for 1 - 2 hours. After the nail polish has fully solidified, observation and photography can be carried out, or store at 4 °C in the dark.

[0164] 4. Calcein - AM / PI Live / Dead Cell Double Staining Assay

[0165] Calcein - AM can penetrate the cell membrane, and the esterase in live cells can remove its AM group to produce Calcein (calcein) and emit fluorescence. PI (Propidium Iodide) cannot pass through the intact cell membrane. Only when the cell membrane is damaged and its permeability changes can PI enter the cell and bind to DNA to emit fluorescence. Therefore, Calcein - AM / PI can be used to label live and dead cells.

[0166] (1) Prepare a 10 mM stock solution of Calcein - AM by dissolving it in DMSO, aliquot and store at - 20 °C in the dark. Prepare a 1 mM stock solution of PI by dissolving it in PBS, aliquot and store at - 20 °C in the dark, and can be stored at 4 °C in the short term.

[0167] (2) Dilute the Calcein - AM stock solution with PBS (dilute 20 - fold). Add the diluted Calcein - AM and PI stock solutions to the culture medium respectively and mix well (final concentrations are 0.5 μM and 1 μM respectively) to prepare the staining working solution, and heat in a 37 °C water bath in the dark for 5 minutes.

[0168] (3) Replace the culture medium with the staining working solution and incubate in a 37 °C incubator for 20 minutes.

[0169] (4) Observe the staining under a fluorescence microscope and take pictures.

[0170] 5. Western (Immunoblotting Assay)

[0171] (1) Electrophoresis: Initially use a constant voltage mode with a voltage of 80 volts. After the sample enters the separating gel, the voltage can be increased (up to 160 volts) until electrophoresis is completed.

[0172] (2) Transfer: Generally, wet transfer is used. Soak the PVDF membrane in methanol, then wash it once with deionized water and once with transfer buffer. Carefully cover the entire polyacrylamide gel, cover it with filter paper, expel air bubbles, fasten the clip, and place it in the transfer tank. Add transfer buffer (select the appropriate methanol ratio according to the size of the protein to be detected), and transfer at a constant current of 300 mA for 1 - 3 hours. At this time, the blocking solution can be prepared (5% skim milk powder prepared with TBST solution).

[0173] (3) Blocking: After transfer, place the PVDF membrane in a box containing the blocking solution and incubate it on a shaker at room temperature for 1 hour.

[0174] (4) Primary antibody incubation: After blocking, wash off the blocking solution with TBST, cut the PVDF membrane, add the primary antibody diluted in an appropriate ratio (select the appropriate antibody dilution buffer for different antibodies), and incubate it on a shaker at 4°C overnight.

[0175] (5) After the primary antibody incubation is completed, wash the PVDF membrane 3 times with TBST on a shaker at room temperature, 10 minutes each time.

[0176] (6) Add the secondary antibody diluted in an appropriate ratio (generally dilute the secondary antibody with TBST, dilute special antibodies with the blocking solution), and incubate it on a shaker at room temperature for 1 hour.

[0177] (7) After the secondary antibody incubation is completed, wash the PVDF membrane 3 times with TBST on a shaker at room temperature, 5 - 10 minutes each time.

[0178] (8) Drop an appropriate amount of ECL on the PVDF membrane for chemiluminescence detection, and expose it with X-ray film in a darkroom, or use an imager to take pictures.

[0179] 6. TUNLE experiment:

[0180] (1) Dewax the paraffin sections of mouse small intestine tissue in xylene for 5 - 10 minutes. Replace with fresh xylene and dewax for another 5 - 10 minutes.

[0181] (2) Dewax with absolute ethanol for 5 minutes; 90% ethanol for 2 minutes; 70% ethanol for 2 minutes; distilled water for 2 minutes.

[0182] (3) Drop 20 μg / ml proteinase K without DNase and act at 20 - 37°C for 15 - 30 minutes.

[0183] (4) Wash 3 times with PBS or HBSS. Note: In this step, proteinase K must be washed clean, otherwise it will seriously interfere with the subsequent labeling reaction.

[0184] (5) Incubate at room temperature for 20 minutes in 3% hydrogen peroxide solution prepared with PBS to inactivate endogenous peroxidase in the sections, and then wash 3 times with PBS or HBSS.

[0185] (6) Prepare the TUNEL detection solution: Mix 5 μl of TdT enzyme with 45 μl of fluorescent labeling solution.

[0186] (7) Wash 2 times with PBS or HBSS.

[0187] (8) Add 50 μl of TUNEL detection solution to the sample and incubate at 37 °C in the dark for 60 minutes.

[0188] (9) Wash 3 times with PBS or HBSS.

[0189] (10) Observe and photograph under a fluorescence microscope after mounting the slides.

[0190] The compounds in the examples of the present invention (such as ISX, 5-fluorouracil, etoposide, PD332991, CHIR99021) were all tested in the form of solutions dissolved in DMSO, and the concentrations of the compounds in the examples were all the final concentrations after being added to the organoid medium.

[0191] Example 1: Protection Experiment of ISX against 5-FU-Induced Small Intestinal Organoid Injury in Mice - Immunofluorescence Staining Experiment Experiment

[0192] Take the small intestine tissues of 6-8-week-old mice and perform organoid culture. After the culture is stable, passage and continue to culture for 3-4 days, and then add the compounds as Figure 1 shown. Divide them into 4 groups: a control group with only DMSO added (DMSO group), a group with only the chemotherapeutic drug 5-fluorouracil (5-FU, 50 μM) added (5-FU group), a group with only ISX (25 μM) added (ISX group), and a group with both 5-FU (50 μM) and ISX (25 μM) added (5-FU+ISX group). Fix the organoids after 4 hours of treatment, perform an immunofluorescence staining experiment with a primary antibody against cleaved caspase-3, and observe and record the results.

[0193] As Figure 1 shown, the DAPI signal represents the cell nucleus, and the signal of cleaved caspase-3 represents dead cells. It can be seen that compared with the DMSO group, the number of dead cells in the 5-FU group increased significantly, while compared with the 5-FU group, the number of dead cells in the 5-FU+ISX group decreased significantly, and the ISX group did not have a significant effect on the signal of cleaved caspase-3. The above experiments show that ISX can reduce the damage of the chemotherapeutic drug 5-fluorouracil to intestinal tissues.

[0194] Example 2: Protection Experiment of ISX against 5-FU-Induced Small Intestinal Organoid Injury in Mice - Bright-Field and Live / Dead Cell Double Staining Experiment Experiment

[0195] Take the small intestinal tissues of 6-8-week-old mice and perform organoid culture. After the culture is stable, passage is carried out and the culture is continued for 2-3 days. Then, add the compound shown in Figure 2 to the culture medium and divide it into 3 groups: the control group with DMSO alone (DMSO group), the group with the chemotherapeutic drug 5-FU (50 μM) alone (5-FU group), and the group with both 5-FU (50 μM) and ISX (25 μM) added simultaneously (5-FU + ISX group). After 8 hours of treatment, terminate the treatment with all compounds, wash 3 times with PBS, replace the culture medium with fresh medium, continue the culture for 4 days, and then perform a live / dead cell double staining experiment, and observe and photograph under a microscope for recording.

[0196] As shown in Figure 2 , it can be observed in the bright field image that compared with the DMSO group, the biomass in the 5-FU group is significantly reduced, and compared with the 5-FU group, the biomass in the 5-FU + ISX group is significantly increased. In the fluorescence image, the signal generated by PI represents dead cells, and the signal generated by Calcein-AM represents live cells. It can be observed that the number of live cells in the 5-FU + ISX group is significantly more than that in the 5-FU group. Statistical analysis is performed on the number of surviving crypt structures in each organoid. As shown in Figure 3 , there is a significant difference between the 5-FU + ISX group and the 5-FU group (p < 0.01). The above experiments show that ISX can reduce the damage of the chemotherapeutic drug 5-fluorouracil to mouse small intestinal organoids.

[0197] Example 3: Protection Experiment of ISX against Etoposide-Induced Small Intestinal Organoid Injury in Mice - Immunofluorescence Staining Experiment Experiment

[0198] Take the small intestinal tissues of 6-8-week-old mice and perform organoid culture. After the culture is stable, passage is carried out and the culture is continued for 3-4 days. Then, add the compound shown in Figure 4 to the culture medium and divide it into 3 groups: the control group with DMSO alone (DMSO group), the group with the chemotherapeutic drug etoposide (Eto, 50 μM) alone (Eto group), and the group with both etoposide (Eto, 50 μM) and ISX (25 μM) added simultaneously (Eto + ISX group). After 4 hours of treatment, fix the organoids and perform immunofluorescence staining, and observe and photograph under a microscope for recording.

[0199] As shown in Figure 4 , the DAPI signal represents the cell nucleus, and the signal of cleaved caspase-3 represents apoptotic cells. It can be seen that ISX can significantly reduce the death of mouse small intestinal organoid cells caused by etoposide treatment.

[0200] Example 4: Protection Experiment of ISX against Etoposide-Induced Small Intestinal Organoid Injury in Mice - Western Experiment

[0201] Take the small intestine tissues of 6 - 8 - week - old mice and perform organoid culture. After the culture is stable, passage is carried out and the culture is continued for 2 - 3 days. Then, add the compounds as shown in Figure 5 to the culture medium. Divide them into 4 groups: the control group with only DMSO added (DMSO group), the group with only ISX (25 μM) added (ISX group), the group with only the chemotherapeutic drug etoposide (Eto, 50 μM) added (Eto group), and the group with both etoposide (Eto, 50 μM) and ISX (25 μM) added (Eto + ISX group). After treatment for 4 hours, collect the samples of the 4 groups and perform western experiments.

[0202] As shown in Figure 5 , ISX can significantly reduce the death of mouse small intestine organoid cells caused by etoposide treatment.

[0203] Example 5: Protection Experiment of ISX against Etoposide-Induced Small Intestinal Organoid Injury in Mice - Bright-Field and Live Cell / Dead Cell Double Staining Experiment Experiment

[0204] Take the small intestine tissues of 6 - 8 - week - old mice and perform organoid culture. After the culture is stable, passage is carried out and the culture is continued for 2 - 3 days. Then, add the compounds as shown in Figure 6 to the culture medium. Divide them into 4 groups: the control group with only DMSO added (DMSO group), the group with only ISX (25 μM) added (ISX group), the group with only the chemotherapeutic drug etoposide (Eto, 50 μM) added (Eto group), and the group with both etoposide (Eto, 50 μM) and ISX (25 μM) added (Eto + ISX group). After 8 - hour treatment, terminate the treatment of all compounds, wash 3 times with PBS, change to a new culture medium, continue the culture for 4 days, and then perform a live / dead cell double - staining experiment. Observe and photograph under a microscope.

[0205] As shown in Figure 6 , in the bright - field image, it can be observed that compared with the DMSO group, the biomass in the Eto group is significantly reduced, while compared with the Eto group, the biomass in the Eto + ISX group is significantly increased. In the fluorescence image, the signal generated by PI represents dead cells, and the signal generated by Calcein - AM represents live cells. It can be observed that the number of live cells in the Eto + ISX group is significantly more than that in the Eto group. Perform statistical analysis on the number of surviving crypt structures in each organoid. As shown in Figure 7 , there is a significant difference between the Eto + ISX group and the Eto group (p < 0.01). The above experiments show that ISX can significantly reduce the death of mouse small intestine organoid cells caused by etoposide treatment.

[0206] Example 6: Protection Experiment of ISX on Human Colonic Organoids - Bright-Field and Live Cell / Dead Cell Double Staining Experiment

[0207] Colon tissue was isolated from normal human tissues and subjected to organoid culture. After stable culture, subculture was performed and continued to be cultured for 3 - 5 days. The formation of crypt structures could be observed. Then, the compounds shown as follows were added to the culture medium, divided into 4 groups: a control group with only DMSO added (DMSO group), a group with only the chemotherapeutic drug 5 - fluorouracil (5 - FU, 20 uM) added (5 - FU group), a group with only ISX (10 uM) added (ISX group), and a group with both 5 - FU (50 uM) and ISX (25 uM) added (5 - FU + ISX group). After 48 - hour treatment, the morphology of organoids in different treatment groups was observed and photographed under a microscope, and the number of crypts in each organoid among different treatment groups was statistically analyzed ( Figure 8 、 Figure 8 、 Figure 9 ).

[0208] As shown in Figure 8 and Figure 9 , the crypt structures of organoids in the DMSO group were intact, while the number of crypt structures in the 5 - FU group was significantly reduced. There was a significant difference between the DMSO group and the 5 - FU group (p < 0.01). There was no obvious difference between the 5 - FU + ISX group and the DMSO group, and there was no significant difference between the 5 - FU + ISX group and the DMSO group (p = 0.29). There was no significant difference between the ISX group and the DMSO group (p = 0.9). The number of crypts in the 5 - FU + ISX group was significantly increased compared with the 5 - FU group, and there was a significant difference between the 5 - FU + ISX group and the 5 - FU group (p < 0.01).

[0209] Continue the culture. By the fifth day, all treatment groups were subjected to double staining of live / dead cells respectively, and the results were observed and photographed under a fluorescence microscope. The signal generated by PI in the fluorescence image represents dead cells, and the signal generated by Calcein - AM represents live cells. As shown in Figure 10 , compared with the DMSO group, the number of dead cells in the 5 - FU group was significantly increased, while there was no obvious difference between the 5 - FU + ISX group and the DMSO group. The above experiments indicate that ISX can reduce the damage of the chemotherapeutic drug 5 - fluorouracil to human colon organoids.

[0210] Example 7: Protective Effect of ISX on Mouse Intestine - TUNEL Experiment

[0211] Wild-type B6 mice at 6-8 weeks of age were randomly divided into two groups. One group was intraperitoneally injected with the chemotherapeutic drug etoposide (20 mg / kg), followed by oral gavage of the solubilizer (5% DMSO + 0.5% Tween 80 + 0.5% CMC-Na) (5 mL / kg). The other group was intraperitoneally injected with the chemotherapeutic drug etoposide (20 mg / kg), followed by oral gavage of ISX (20 mg / kg) dissolved in the solubilizer. Three hours later, the mice were sacrificed and the intestinal tissues were fixed, and then TUNEL staining was performed.

[0212] As Figure 11 shown, the TUNEL signal in the group gavaged with ISX was significantly lower than that in the group gavaged with the solubilizer. Statistical analysis was performed on the number of TUNEL signals in each crypt, and there was a significant difference between the two groups (p < 0.01) ( Figure 12 ). The above experiments indicate that ISX can reduce the damage of the chemotherapeutic drug etoposide to the mouse intestine.

[0213] Example 8: Comparative Experiment on the Protective Effects of PD332991, CHIR99021 and ISX on Intestinal Organoids

[0214] PD0332991 is a CDK4 / 6 inhibitor, which has been reported to reduce the killing effect of chemotherapeutic drugs on normal cells. CHIR99021 is an inhibitor of GSK3β, which has been reported to reduce the damage of chemotherapeutic drugs to the intestine. In this example, PD0332991 and CHIR99021 were used as control compounds to evaluate the protective effect of the compounds of the present application on intestinal organoids.

[0215] Small intestinal tissues of 6-8-week-old mice were taken and intestinal organoids were cultured. After the culture was stable, subculture was performed and the culture was continued for 2-3 days. Then, the compounds shown in Figure 13 were added to the culture medium, and they were divided into 5 groups: a control group with DMSO alone (DMSO group), a group with 5-FU (25 μM) alone (5-FU group), a group with 5-FU (25 μM) and ISX (25 μM) simultaneously (5-FU + ISX group), a group with 5-FU (25 μM) and PD0332991 (100 nM) simultaneously (5-FU + PD group), and a group with 5-FU (25 μM) and CHIR99021 (10 μM) simultaneously (5-FU + CHIR group). Treatments were carried out. After 8 hours, all compound treatments were terminated, and the cells were washed 3 times with PBS, and new culture medium was replaced. After continuing to culture for 4 days, a live / dead cell double staining experiment was performed, and observations and photographs were recorded under a microscope.

[0216] As Figure 13 shown, ISX can significantly reduce the death of mouse small intestinal organoid cells caused by 5-FU treatment, while PD332991 and CHIR99021 cannot protect the small intestinal organoids.

Claims

1. Use of the compound shown in formula (2) or a pharmaceutically acceptable salt thereof in the preparation of a drug for reducing intestinal adverse reactions caused by chemotherapeutic drugs, wherein the structure of the compound shown in formula (2) is as follows: Wherein: R 4 , R 5 and R 6 are independently selected from hydrogen, halogen or C 1-6 alkyl; Y is selected from S, O or NH; X is selected from O or NR d , R d is hydrogen; R 2 selected from hydrogen, a halogen or C 1-6 alkyl R 7 and R 8 are independently selected from hydrogen or C 3-7 cycloalkyl, provided that R 7 and R 8 are not the same.

2. The use according to claim 1, wherein, R 4 、R 5 and R 6 are independently selected from hydrogen, halogen or methyl; R 2 selected from hydrogen, a halogen or a methyl group; R 7 and R 8 are independently selected from hydrogen, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, provided that R 7 and R 8 are not the same.

3. The use according to claim 1 or 2, wherein, The compound represented by the formula (2) is the compound represented by the formula (3): wherein, R 4 、R 5 、R 6 、R 2 、R 7 and R 8 are as defined in claim 1 or 2.

4. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for reducing intestinal adverse reactions caused by chemotherapeutic drugs, wherein the compound is selected from:

Images