Difluoromethoxy phenyl compound and medical application thereof

Abstract

The invention provides a difluoromethoxy phenyl compound as shown in formula (I), or pharmaceutically acceptable salts, solvates, hydrates, prodrugs, stereoisomers, polymorphs, eutectic crystals or isotope markers of the difluoromethoxy phenyl compound, and application of the difluoromethoxy phenyl compound and the pharmaceutically acceptable salts, solvates, hydrates, prodrugs, stereoisomers, polymorphs, eutectic crystals or isotope markers in medicine, and the difluoromethoxy phenyl compound and the pharmaceutically acceptable salts, solvates, hydrates, prodrugs, stereoisomers, polymorphs, eutectic crystals or isotope markers can be used for preparing anti-tumor drugs # imgabs0 #.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a difluoromethoxyphenyl compound and use thereof in preparing a medicine for preventing and / or treating tumors. Background Art

[0002] Malignant tumors are the most serious type of disease that endangers human health. Their incidence rate is second only to cardiovascular and cerebrovascular diseases, making them the second largest "killer" of human health, and their mortality rate even exceeds that of cardiovascular and cerebrovascular diseases, ranking first among all diseases. Therefore, finding and developing new drugs that can treat tumors is one of the major issues currently facing us.

[0003] The PDE4 target has been developed for the treatment of a variety of inflammatory diseases, including respiratory diseases (such as chronic obstructive pulmonary disease, asthma, etc.), various skin diseases (such as psoriasis, atopic dermatitis, etc.), and immune system diseases (such as systemic lupus erythematosus, rheumatoid arthritis, etc.). Common PDE4 inhibitors include: aprenomide for moderate to severe psoriasis and rheumatoid arthritis, crisaborole for systemic / topical use for mild to moderate psoriasis, roflumilast for adjuvant treatment of chronic obstructive pulmonary disease (COPD), etc. At present, PDE4 inhibitors have become the first-line oral drugs for the treatment of psoriasis and some other chronic inflammatory diseases, and their safe and efficient efficacy has been widely recognized.

[0004] Patent PCT / CN2024 / 122614 records a difluoromethoxyphenyl PDE4 inhibitor, which has good anti-inflammatory effects and can be used to treat psoriasis, lung damage, chronic obstructive pulmonary disease and other diseases. Summary of the invention

[0005] The inventors of the present invention obtained a series of difluoromethoxyphenyl compounds when synthesizing the PDE4 inhibitors described in patent PCT / CN2024 / 122614, and surprisingly found that they have good anti-tumor activity (such as melanoma, skin cancer, etc.), and are expected to play a potential in tumor treatment and expand the number and types of anti-tumor drugs. Based on this, the present invention is specially proposed.

[0006] The first aspect of the present invention provides a difluoromethoxyphenyl compound as shown in formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof,

[0007]

[0008] Among them, R 1represents hydrogen or C1-C6 alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from halogen, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy or C3-C8 cycloalkyl;

[0009] R 2 Represents -C(O)R 3 , a 5- to 12-membered heterocyclic group or a 5- to 12-membered heteroaryl group, wherein the heterocyclic group or the heteroaryl group contains 1 to 3 heteroatoms selected from N, O, and S as ring atoms, and the heterocyclic group or the heteroaryl group is optionally selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -OR 4 、-C(O)OR 4 、-C(O)R 4 、-C(O)NHR 4 、-NHC(O)R 4 is substituted by one or more substituents;

[0010] R 3 represents hydrogen, hydroxyl, C1-C6 alkyl or C1-C6 alkoxy;

[0011] R 4 are the same or different and each independently represents hydrogen, C1-C6 alkyl, 5- to 12-membered heterocyclic group or 5- to 12-membered heteroaryl, wherein the heterocyclic group or heteroaryl group contains 1 to 3 heteroatoms selected from N, O and S as ring atoms.

[0012] In some embodiments according to the present invention, R 1 represents hydrogen, C1-C4 alkyl substituted by halogen or C3-C6 cycloalkyl. 1 represents hydrogen, C1-C4 alkyl substituted by F or cyclopropyl. In some more preferred embodiments, R 1 Represents hydrogen,

[0013] In some embodiments according to the present invention, R 2 express Among them, R 3 represents hydrogen or hydroxyl.

[0014] In some embodiments according to the present invention, R 2 express

[0015] Ra stands for -C(O)OR 4 or -C(O)NHR 4 ;

[0016] R 4 are the same or different and each independently represents hydrogen, C1-C6 alkyl or 5-6 membered heteroaryl containing 1-3 heteroatoms selected from N, O and S as ring atoms;

[0017] Rb represents hydrogen or C1-C6 alkyl;

[0018] m represents 1, 2, or 3.

[0019] In some preferred embodiments, R 2 express

[0020] Ra stands for -C(O)OR 4 or -C(O)NHR 4 ;

[0021] R 4 are the same or different and each independently represents hydrogen, C1-C4 alkyl or 5-membered heteroaryl containing 1 N and 1 S as ring atoms;

[0022] Rb represents hydrogen or a C1-C4 alkyl group.

[0023] In some more preferred embodiments, R 2 express

[0024] R 4 represents a C1-C4 alkyl group (including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, etc.), for example, it may represent a methyl group.

[0025] In some most preferred embodiments according to the present invention, the difluoromethoxyphenyl compound is selected from the following compounds:

[0026] .

[0028] The second aspect of the present invention provides a difluoromethoxyphenyl compound as shown in formula (II), or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof,

[0029]

[0030] Among them, R 1 , R 4 Each is independently defined as in any one of the above technical solutions.

[0031] The third aspect of the present invention provides a method for preparing a difluoromethoxyphenyl compound as shown in formula (II) as described in any one of the above technical schemes, wherein 3,4-dihydroxybenzaldehyde as shown in formula (II-1) is used as a starting material to first form a benzaldehyde intermediate as shown in formula (II-2), and then react with 2,3-diaminobenzoic acid ester as shown in formula (II-3) to obtain,

[0032]

[0033] Among them, R 1 , R 4 Each is independently defined as in any one of the above technical solutions.

[0034] A fourth aspect of the present invention provides a pharmaceutical composition comprising:

[0035] (1) The difluoromethoxyphenyl compound described in any one of the above technical solutions, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof;

[0036] (2) optionally one or more other pharmaceutically active ingredients; and

[0037] (3) Pharmaceutically acceptable carriers and / or excipients.

[0038] A fifth aspect of the present invention provides the use of the difluoromethoxyphenyl compound described in any one of the above technical schemes, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof, or the pharmaceutical composition described in any one of the above technical schemes in the preparation of a medicament for preventing and / or treating tumors.

[0039] In some most preferred embodiments according to the present invention, the tumor is melanoma or skin cancer. DETAILED DESCRIPTION

[0040] the term

[0041] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

[0042] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to." Unless otherwise specified, "comprising" includes "consisting of.

[0043] The term "and / or" used herein, alone or in combination, such as "X and / or Y," should be understood to mean "X and Y" or "X or Y" and should be used to provide clear support for both meanings or either meaning.

[0044] "C1-Cn" used herein includes C1-C2, C1-C3, ... C1-Cn. For example, the "C1-C6" group refers to a group having 1 to 6 carbon atoms in the portion, that is, the group contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms. Therefore, for example, "C1-C4 alkyl" refers to an alkyl group containing 1 to 4 carbon atoms, that is, the alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. The numerical ranges herein, such as "1-6", "1-4", etc., refer to each integer in the given range.

[0045] The term "alkyl" used herein alone or in combination refers to an optionally substituted straight chain or optionally substituted branched saturated aliphatic hydrocarbon. The "alkyl" herein preferably has 1 to 6 carbon atoms, for example, 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1-3 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, and the like. When a group defined herein, such as "alkyl", appears in a numerical range, for example, "C1-C6 alkyl" refers to an alkyl group that can be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, and the alkyl group herein also includes the case where no numerical range is specified. The alkyl group can be substituted or unsubstituted.

[0046] As used herein, "alkyl" refers to an alkyl group attached to another group, for example, alkoxy, haloalkyl, alkyl in haloalkoxy, and has the same definition as when used alone.

[0047] The term "haloalkyl" used herein alone or in combination refers to one or more, or even all of the hydrogen in the alkyl group being replaced by halogen, C1-C6 haloalkyl includes, for example, C1-C3 haloalkyl or C1-C4 haloalkyl, and non-limiting examples of haloalkyl include trifluoromethyl, trichloromethyl, etc. Haloalkyl can be substituted or unsubstituted.

[0048] The term "alkoxy" as used herein, alone or in combination, refers to an alkyl group as defined above attached to the parent molecular moiety through an oxygen atom, C1-C6 alkoxy includes, for example, C1-C3 alkoxy or C1-C4 alkoxy, and non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, etc. Alkoxy can be substituted or unsubstituted.

[0049] The term "haloalkoxy" used herein alone or in combination refers to one or more, or even all of the hydrogen in the alkoxy group being replaced by halogen, and C1-C6 haloalkoxy includes, for example, C1-C3 haloalkoxy or C1-C4 haloalkoxy, and non-limiting examples of haloalkoxy include trifluoromethoxy, trichloromethoxy, etc. Haloalkoxy may be substituted or unsubstituted.

[0050] The term "cycloalkyl" used herein alone or in combination refers to an optionally substituted non-aromatic saturated carbocyclic ring, which may include a monocyclic ring (having one ring), a bicyclic ring (having two rings) or a polycyclic ring (having more than two rings), and the ring types include fused rings, bridged rings and spiro rings. Preferably, the cycloalkyl group may have 3 to 8 ring-forming carbon atoms, for example, 3 to 6 ring-forming carbon atoms. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. The cycloalkyl group may be substituted or unsubstituted.

[0051] The term "heterocyclyl" used herein alone or in combination refers to an optionally substituted non-aromatic 5 to 12-membered monocyclic, bicyclic or polycyclic ring, the ring types include fused rings, bridged rings and spirocyclic rings, and the ring atoms may contain 1 to 3 atoms selected from nitrogen, oxygen and / or sulfur. Preferably, the heterocyclyl may have 5 to 8 ring atoms, for example, 5 to 6 ring atoms. Non-limiting examples of heterocyclyl include, but are not limited to, morpholinyl, oxetane, thiomorpholinyl, tetrahydrofuranyl, tetrahydropyranyl, 1,1-dioxo-thiomorpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo[3.2.1]octyl, piperazinyl, hexahydropyrimidine, etc. The heterocyclyl may be substituted or unsubstituted.

[0052] The term "heteroaryl" used herein alone or in combination refers to an optionally substituted aromatic 5- to 12-membered monocyclic, bicyclic or polycyclic ring, the ring types of which include fused rings, bridged rings and spiro rings, and the ring atoms thereof may contain 1 to 3 atoms selected from nitrogen, oxygen and / or sulfur. Preferably, the heteroaryl may have 5 to 8 ring atoms, for example, 5 to 6 ring atoms. Non-limiting examples of heteroaryl groups include, but are not limited to, furanyl, pyridinyl, 2-oxo-1,2-dihydropyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, benzodioxolyl, benzothienyl, benzimidazolyl, indolyl, isoindolyl, 1,3-dioxo-isoindolyl, quinolyl, indazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, isothiazolyl, 1H-1,2,4-triazolyl, 4H-1,2,4 The heteroaryl group may be substituted or unsubstituted.

[0053] The term "halogen" as used herein, alone or in combination, refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0054] The term "hydroxy," as used herein, alone or in combination, refers to -OH.

[0055] The term "pharmaceutically acceptable" as used herein, alone or in combination, refers to a substance that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, i.e., the substance can be administered to a subject without causing an adverse biological response or interacting in an adverse manner with any components contained in the composition.

[0056] The term "pharmaceutically acceptable salt" used herein, alone or in combination, refers to a compound of the present invention that retains the biological effectiveness and properties of a free acid or free base, and the free acid is obtained by reacting with a non-toxic inorganic base or organic base, and the free base is obtained by reacting with a non-toxic inorganic acid or organic acid. Standard procedures well known in the art can be used to obtain. Suitable salts are listed in Remingtong's Pharmaceutical Scicences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977).

[0057] The term "solvate" as used herein, alone or in combination, refers to a physical aggregate of a compound of the invention and one or more solvent molecules formed by solvation, which includes varying degrees of ionic and covalent bonding, such as hydrogen bonding. "Hydrate" is a physical aggregate formed by the reaction of water (H 2 O) The molecule is a solvate of a solvent.

[0058] The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers, hindered isomers and geometric (conformation) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention. Unless otherwise indicated, the structure described in the present invention also includes all isomers of the structure (e.g., diastereomers, enantiomers, steric isomers and geometric (conformation) isomer forms; for example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, steric isomers of biphenyl structures (see "Basic Organic Chemistry" (Second Edition), Volume 1, Xing Qiyi et al., p104-105); PAC, 1996, 68, 2193. (Basic terminology of stereochemistry (IUPAC Recommendations 1996, on page 2201)), (Z) and (E) conformational isomers. Therefore, single stereoisomers of the compounds of the present invention as well as enantiomeric mixtures, diastereomeric mixtures, steric isomers and geometric (conformation) isomer mixtures are all within the scope of the present invention.

[0059] The term "prodrug" used herein alone or in combination refers to a compound of the present invention that can be converted into a biologically active compound through in vivo metabolism. The prodrug of the present invention is prepared by modifying the amino or carboxyl group in the compound of the present invention, and the modification can be removed by conventional operations or in vivo to obtain the parent compound. When the prodrug of the present invention is administered to a mammalian subject, the prodrug is cleaved to form a free amino or carboxyl group.

[0060] The term "cocrystal" used herein alone or in combination refers to a crystal formed by the active pharmaceutical ingredient (API) and the cocrystal former (CCF) under the action of hydrogen bonds or other non-covalent bonds, wherein the pure state of the API and the CCF are solid at room temperature, and there is a fixed stoichiometric ratio between the components. Cocrystal is a multi-component crystal, including both binary eutectics formed between two neutral solids and multi-component eutectics formed between neutral solids and salts or solvates.

[0061] The carbon, hydrogen, oxygen, sulfur, nitrogen, F, Cl, Br, I, etc. involved in the compounds of the present invention include their isotopes. The carbon, hydrogen, oxygen, sulfur or nitrogen involved in the compounds of the present invention are optionally further replaced by one or more of their corresponding isotopes, wherein the carbon isotopes include 12 C. 13 C and 14 C, hydrogen isotopes include protium (H), deuterium (D, also called heavy hydrogen), tritium (T, also called super tritium), oxygen isotopes include 16 O. 17 O and 18 O, sulfur isotopes include 32 S. 33 S. 34 S and 36 S, isotopes of nitrogen include 14 N and 15 N, fluorine isotopes include 17 F and 19 F, isotopes of chlorine include 35 Cl and 37 Isotopes of Cl and bromine include 79 Br and 81 Br.

[0062] The term "pharmaceutical composition" as used herein, alone or in combination, refers to a biologically active compound optionally mixed with at least one pharmaceutically acceptable chemical component, wherein the pharmaceutically acceptable chemical component includes but is not limited to carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, excipients, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoaming agents, inclusion agents, humectants, absorbents, flocculants and deflocculating agents, filter aids, release retardants, etc.

[0063] The term "subject" or "patient" used herein, alone or in combination, refers to an animal, including a human patient in need of treatment. In certain aspects, this article can also be applied to any mammal or other animal in need of such immune-targeted therapy in veterinary practice, including but not limited to non-human primates, canines, felines, pigs, horses, and any other animals.

[0064] The term "treat" and other similar synonyms used herein, alone or in combination, include alleviating, alleviating or ameliorating symptoms of a disease or condition, preventing other symptoms, ameliorating or preventing potential metabolic causes of symptoms, inhibiting a disease or condition, such as preventing the development of a disease or condition, alleviating a disease or condition, making a disease or condition better, alleviating symptoms caused by a disease or condition, or stopping symptoms of a disease or condition, and in addition, the term includes the purpose of prevention. The term also includes obtaining a therapeutic effect and / or a preventive effect.

[0065] The term "room temperature" or "normal temperature" as used herein, alone or in combination, refers to 25±5°C.

[0066] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments.

[0067] Unless otherwise specified, the raw materials or reagents used in the examples of the present invention are commercially available products.

[0068] The percentages used in the embodiments of the present invention are all by mass unless otherwise specified.

[0069] Example 1 Synthesis of Compound 2

[0070]

[0071] 3 g of compound 3,4-dihydroxybenzaldehyde (21.72 mmol, 1.0 eq.) and 6.91 g of sodium carbonate (65.16 mmol, 3.0 eq.) were weighed and dissolved in 30 mL of N,N-dimethylformamide. Finally, 3.44 g of ethyl difluorochloroacetate (21.72 mmol, 1.0 eq.) was added. After reacting at 80° C. for 8 h, TLC was used to detect that the raw material reaction was complete. Water was added to quench the reaction, and ethyl acetate was extracted 3 times. The organic phase was washed with a saturated sodium chloride solution. After the organic phase was concentrated, 1.4 g of a white solid product, namely compound 2, was obtained by flash column chromatography (petroleum ether / ethyl acetate elution system). The yield was 34%.

[0072] 1 H NMR (500 MHz, CDCl 3 )δ9.90(s,1H),7.53(d,J=2.0Hz,1H),7.44(dd,J=8.5,2.0Hz,1H),7.26(d,J=8.5Hz,1H),6.66(t,J=73.5Hz,1H).

[0073] ESI-HRMS m / z: calculated value is C 8 H 6 O 3 F 2 Na + [M+Na] + ,211.0177; the measured value is 211.0166.

[0074] Example 2 Synthesis of Compound 3

[0075]

[0076] Weigh 2.1 g of compound 2 (11.16 mmol, 1.0 eq.), add 3.09 g of potassium carbonate (22.32 mmol, 2.0 eq.), then add 20 mL of N,N-dimethylformamide, and finally add 2.26 g of bromomethylcyclopropane (16.74 mmol, 1.5 eq), react at 80 ° C for 8 h, detect the complete reaction of the raw materials by TLC, add water to quench the reaction, extract 3 times with ethyl acetate, wash the organic phase with saturated sodium chloride solution, concentrate the organic phase, and obtain 2.3 g of yellow oily product, namely compound 3, with a yield of 85%.

[0077] 1 H NMR (500 MHz, DMSO-d 6)δ9.94(s,1H),7.59–7.54(m,2H),7.39(d,J=8.0Hz,1H),7.28(t,J=73.5Hz,1H) ,3.98(d,J=7.0Hz,2H),1.30–1.21(m,1H),0.61–0.55(m,2H),0.39–0.33(m,2H).

[0078] ESI-MS m / z: calculated value is C 12 H 13 O 3 F 2 + [M+H] + ,243.1; the measured value is 243.1.

[0079] Example 3 Synthesis of Compound IA

[0080]

[0081] Compound 2 (10 g, 1 eq.) and aminosulfonic acid (1.5 eq.) were stirred evenly in 100 mL of acetonitrile aqueous solution (volume ratio = 1:1), cooled to 0°C, and sodium chlorite (1.5 eq.) was added in 3 batches with an interval of 10 min between each batch. After the addition, the temperature was slowly raised to room temperature and reacted overnight. The acetonitrile solution was concentrated under reduced pressure, and appropriate amounts of ethyl acetate and water were added to the residue, separated and extracted. The organic phase was washed twice with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 7.5 g of a white solid product, namely compound IA, with a yield of 69.1%.

[0082] MS m / z: calculated as C 12 H 13 O 3 + [MH] - ,203.0; the measured value is 203.0.

[0083] Example 4 Synthesis of Compound IB

[0084]

[0085] Compound 3 (12.38 mmol, 3.0 g) was weighed, sodium hydroxide (99 mmol, 4 g) was added, and then 30 mL of a mixed solution of methanol and water (volume ratio of 10:1) was added, and finally 30% hydrogen peroxide (37.14 mmol, 4.2 g) was added. After reacting at 50 ° C for 2 h, TLC was used to detect that the raw material reaction was complete, and an aqueous solution was added to quench the reaction. The reaction was adjusted to acidity with dilute hydrochloric acid, and a large amount of solid precipitated. It was then filtered and dried in vacuo to obtain 2.4 g of the product, namely compound IB, with a yield of 75%.

[0086] 1 H NMR (500 MHz, DMSO-d 6 )δ7.60–7.54(m,2H),7.27(d,J=8.0Hz,1H),7.21(t,J=74.0Hz,1H),3.94( d,J=7.0Hz,2H),1.29–1.23(m,1H),0.61–0.54(m,2H),0.38–0.33(m,2H).

[0087] ESI-HRMS m / z: calculated value is C 12 H 12 F 2 O 4 + [M+H] + ,281.0596; the measured value is 281.1688.

[0088] Example 5 Synthesis of Compound IC

[0089]

[0090] Referring to the synthesis method of Example 1, the amount of ethyl difluorochloroacetate was doubled to obtain a yellow oily product, namely, Compound IC, with a yield of 49%.

[0091] 1 H NMR (400 MHz, CDCl 3 )δ9.91(s,1H),7.75–7.73(m,2H),7.39(d,J=8.8Hz,1H),6.64(t,J=72.8Hz,1H),6.59(t,J=72.8Hz,1H).

[0092] ESI-HRMS m / z: calculated value is C 9 H 7 F 4 O 3 + [M+H] + ,239.0326; the measured value is 239.0464.

[0093] Example 6 Synthesis of Compound ID

[0094]

[0095] Compound IC (25 g, 1.0 eq.) and methyl 2,3-diaminobenzoate (1.0 eq.) were dissolved in 30 mL of N,N-dimethylformamide solution, sodium metabisulfite (1.2 eq.) was added, and the mixture was stirred at 80° C. overnight. TLC detected that the reaction of the raw materials was complete, and aqueous solution was added to quench the reaction. The mixture was extracted with ethyl acetate three times, and the organic phase was washed with a saturated sodium chloride solution. After the organic phase was concentrated, a yellow solid product, namely compound ID, was obtained by flash column chromatography (petroleum ether / ethyl acetate elution system) with a yield of 49%.

[0096] 1 H NMR (400 MHz, CDCl 3 )δ10.75(br,1H),8.04(d,J=2.0Hz,1H),8.01(d,J=8.0Hz,1H),7.93–7.91(m,2H),7.41(d,J=8 .4Hz,1H),7.32(t,J=8.0Hz,1H),6.65(t,J=73.2Hz,1H),6.A5(t,J=72.8Hz,1H),4.03(s,3H).

[0097] ESI-HRMS m / z: calculated value is C 17 H 13 F 4 N 2 O 4 + [M+H] + ,385.0806; the measured value is 385.0801.

[0098] Example 7 Synthesis of Compound IE

[0099]

[0100] Compound ID (16.5 g, 1.0 eq.) and sodium hydroxide (5.0 eq.) were dissolved in 200 mL of ethanol-water solution (ethanol: water = 5:1, volume ratio), and the reaction was stirred at room temperature for 5 h. TLC detected that the raw material reaction was complete. Ethanol was removed by rotary evaporation, an appropriate amount of water was added, the pH was adjusted to 7 with dilute hydrochloric acid, filtered, and the solid was washed with small amounts of water several times. The solid was dried to obtain 13.2 g of a yellow solid product, namely compound IE, with a yield of 83%.

[0101] 1 H NMR (500 MHz, DMSO-d 6)δ12.57(br,1H),12.52(br,1H),8.33(s,1H),8.30(d,J=8.5Hz,1H),7.95(d,J=8 .0Hz,1H),7.84(dd,J=7.5,1.0Hz,1H),7.52(d,J=8.5Hz,1H),7.49–7.19(m,3H).

[0102] ESI-HRMS m / z: calculated value is C 16 H 11 F 4 N 2 O 4 + [M+H] + ,371.0649; the measured value is 371.0652.

[0103] Example 8 Synthesis of Compound IF

[0104]

[0105] Compound IE (8.1 g, 1.0 eq.) was dissolved in 20 mL of N,N-dimethylformamide solution, HATU (1.0 eq.) and DIPEA (3.0 eq.) were added, and the mixture was stirred at room temperature for 20 min. 2-aminothiazole (1.1 eq.) was added, and the mixture was reacted at room temperature for 6 h. TLC monitoring was performed. When the reaction of the raw material was complete, water was added to quench the mixture, and the mixture was extracted with ethyl acetate-water (volume ratio of 1:1). The mixture was washed once with a saturated sodium chloride solution, and the ethyl acetate layer was dried over anhydrous sodium sulfate. The solution was concentrated and purified by column chromatography (petroleum ether / ethyl acetate elution system) to obtain a solid product. The solid product was dissolved in 100 mL of tetrahydrofuran, heated to 60 ° C and stirred to completely dissolve it. 10 g of activated carbon was added and stirred at room temperature for 10 min. The activated carbon was removed by suction, the tetrahydrofuran was spin-dried and 100 mL of ethanol was added. The mixture was stirred at room temperature for 30 min, the solid was suction-filtered, and the solid was dried to obtain 6.2 g of a solid product, namely compound IF.

[0106] 1 H NMR (400 MHz, DMSO-d 6 )δ13.87(br,1H),13.40(br,1H),8.26–8.18(m,2H),8.04(d,J=7.6Hz,1H),7.91( d,J=8.0Hz,1H),7.68(d,J=8.8Hz,1H),7.61(d,J=2.0Hz,1H),7.55–7.13(m,4H). 13 C NMR (100 MHz, DMSO-d 6)δ1A5.4,157.6,150.9,144.1,141.9,138.2,135.5,129.7,126.6,125.4,123.6,123 .4,121.3,120.2,119.4,117.1,116.6(t,J=259.1Hz),116.4(t,J=258.5Hz),114.4.

[0107] ESI-HRMS m / z: calculated value is C 19 H 13 F 4 N 4 O 3 S + [M+H] + ,453.0639; the measured value is 453.0614.

[0108] Example 9 Tumor cell inhibition effect experiment

[0109] 1. Experimental Purpose and Principle

[0110] Experimental purpose: CCK-8 method was used to detect the inhibitory effect of the compounds of the present invention on tumor cell proliferation.

[0111] Experimental principle: CCK-8 method is a method based on WST-8 and widely used for rapid and highly sensitive detection of cell proliferation and cytotoxicity. WST-8 is a compound similar to MTT. In the presence of an electron coupling agent, it can be reduced by some dehydrogenases in the mitochondria to generate orange-yellow formazan. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of color is linearly related to the number of cells. The absorbance value (OD value) at 450nM is detected by an enzyme-linked immunosorbent assay, and the number of living cells can be reflected according to the absorbance value. Within a certain range, the smaller the OD value, the weaker the cell activity and the better the drug's inhibitory effect on proliferation.

[0112] 2. Basic information of reagents

[0113]

[0114] 3. Reagent Preparation

[0115] 1. RPMI-1640 complete medium

[0116] When in use, add 50 mL of fetal bovine serum to 450 mL of RPMI-1640 culture medium to form a complete culture medium, which can be used for cell culture.

[0117] 2. Compound preparation

[0118] Take the autoclaved EP tube to weigh the compound, add the corresponding amount of DMSO to the EP tube to make the liquid form a 100mM mother solution, and dilute it to 100mM, 50mM, 25mM, 12.5mM, 6.25mM, 3.125mM respectively. When using, dilute it 1000 times with the corresponding amount of culture medium to prepare a drug-containing culture medium with a concentration of 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM.

[0119] IV. Experimental Procedure

[0120] (1) Mouse skin melanoma cells B16-F10 and human skin keratinocytes HaCaT in the logarithmic growth phase were digested and the cell number concentration was adjusted to 5×10 4 100 μL / well was inoculated into a 96-well plate and incubated at 37°C with 5% CO 2 Culture in a cell culture incubator overnight until the cells adhere.

[0121] (2) The original culture medium was aspirated and different concentrations of drug-containing culture medium were added to each group. The gradient concentrations of each compound were 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM, respectively. Each treatment was repeated 3 times. 0.1% DMSO was used as the negative control group, and no cells and compounds were used as the blank control. The cells were cultured in the cell culture incubator for 72 h.

[0122] (3) Add 10 μL of CCK-8 solution to each well and incubate in a 37°C incubator for 1-2 h.

[0123] (4) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0124] (5) Calculate the cell growth inhibition rate according to the following formula:

[0125] Inhibition rate = [(As-Ab) / (Ac-Ab)] × 100%

[0126] As: absorbance of the experimental well (including cells, CCK-8, compounds)

[0127] Ac: Absorbance of control well (containing cells, CCK-8, no compound)

[0128] Ab: absorbance of blank well (without cells and compounds, with CCK-8)

[0129] V. Experimental Results

[0130] The experimental results show that the compound of the present invention has certain inhibitory activity on mouse skin melanoma cells B16-F10 and human skin keratinocytes HaCaT.

[0131] In particular, compound ID showed a half-maximal inhibitory concentration (IC 50 value) is 16.99 μM, indicating that it has good melanoma cell inhibitory activity and can effectively inhibit the proliferation of tumor cells, so it has good application potential in the treatment of melanoma.

[0132] Unless otherwise defined, the terms used in the present invention have the meanings commonly understood by those skilled in the art.

[0133] The embodiments described in the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various other substitutions, changes and improvements within the scope of the present invention. Therefore, the present invention is not limited to the above embodiments, but is only limited by the claims.

Claims

1. A difluoromethoxyphenyl compound as shown in formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof, in, R1 represents hydrogen or C1-C6 alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy or C3-C8 cycloalkyl; R2 represents -C(O)R3, a 5- to 12-membered heterocyclic group or a 5- to 12-membered heteroaryl group, wherein the heterocyclic group or heteroaryl group contains 1 to 3 heteroatoms selected from N, O, and S as ring atoms, and the heterocyclic group or heteroaryl group may be optionally substituted by one or more substituents selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -OR4, -C(O)OR4, -C(O)R4, -C(O)NHR4, and -NHC(O)R4; R3 represents hydrogen, hydroxyl, C1-C6 alkyl or C1-C6 alkoxy; R4 are the same or different and each independently represents hydrogen, C1-C6 alkyl, 5-12 membered heterocyclic group or 5-12 membered heteroaryl group, wherein the heterocyclic group or heteroaryl group contains 1 to 3 heteroatoms selected from N, O and S as ring atoms.

2. The difluoromethoxyphenyl compound according to claim 1, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof, wherein: R1 represents hydrogen, C1-C4 alkyl substituted by halogen or C3-C6 cycloalkyl; Preferably, R1 represents hydrogen, C1-C4 alkyl substituted by F or cyclopropyl; More preferably, R1 represents hydrogen, 3. The difluoromethoxyphenyl compound according to claim 1 or 2, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof, wherein: R2 means R3 represents hydrogen or hydroxy.

4. The difluoromethoxyphenyl compound according to claim 1 or 2, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof, wherein: R2 means Ra represents -C(O)OR4 or -C(O)NHR4; R4 are the same or different and each independently represents hydrogen, C1-C6 alkyl or 5-6 membered heteroaryl, wherein the heteroaryl contains 1-3 heteroatoms selected from N, O and S as ring atoms; Rb represents hydrogen or C1-C6 alkyl; m means 1, 2 or 3; Preferably, R2 represents Ra represents -C(O)OR4 or -C(O)NHR4; R4 are the same or different and each independently represents hydrogen, C1-C4 alkyl or 5-membered heteroaryl containing 1 N and 1 S as ring atoms; Rb represents hydrogen or C1-C4 alkyl; More preferably, R2 represents R4 represents a C1-C4 alkyl group, and preferably represents a methyl group.

5. The difluoromethoxyphenyl compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof, wherein: The difluoromethoxyphenyl compound is selected from the following compounds:

6. A difluoromethoxyphenyl compound as shown in formula (II), or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof, in, R1 and R4 are each independently defined as in any one of claims 1 to 5.

7. The method for preparing the difluoromethoxyphenyl compound of formula (II) according to claim 6, wherein: Using 3,4-dihydroxybenzaldehyde as shown in formula (II-1) as the starting material, firstly forming a benzaldehyde intermediate as shown in formula (II-2), and then reacting with 2,3-diaminobenzoate as shown in formula (II-3) to obtain: Wherein, R1 and R4 are as defined in claim 6.

8. A pharmaceutical composition comprising: (1) The difluoromethoxyphenyl compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof; (2) optionally one or more other active pharmaceutical ingredients; as well as (3) Pharmaceutically acceptable carriers and / or excipients.

9. Use of the difluoromethoxyphenyl compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or isotope-labeled substance thereof, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for preventing and / or treating tumors.

10. The use according to claim 9, wherein The tumor is melanoma or skin cancer.

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