A substituted 2H-benzopyran-3-carboxanilide compound and its preparation method and application

Abstract

The present invention discloses a substituted 2H-benzopyran-3-formylanilide compound, a preparation method, and an application thereof. The structure of the substituted 2H-benzopyran-3-formylanilide compound is shown in Formula I. #imgabs0# The substituted 2H-benzopyran-3-formylanilide compound has the potential to be developed into an anti-radiation drug.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a substituted 2H-benzopyran-3-carboxanilide compound, a preparation method thereof, and application thereof in preventing and treating radiation damage. Background Art

[0002] Radiation protection drugs, namely radiation damage prevention drugs and / or radiation damage treatment drugs, can directly counteract multi-system damage caused by radiation, effectively alleviate the symptoms of acute radiation sickness, and win precious time for subsequent comprehensive treatment.

[0003] Aminothiol compounds are among the earliest small-molecule radioprotectants developed. A representative example is amifostine (WR2721), chemically known as 2-(3-aminopropylamine)-ethyl phosphorothioate. It was once used as an anti-radiation drug, exerting its efficacy after being metabolized in the body and removing the phosphate. The drug exhibited strong anti-radiation activity in animal models, but was abandoned because its radiation protection dose was close to a toxic dose. It was approved for marketing by the US FDA after its dosage was adjusted to mitigate the toxic side effects of radiotherapy in cancer patients. Currently, small-molecule anti-radiation drugs in the US are undergoing clinical development, including 5-AED (5-androstenediol), BIO 300 (genistein), and Ex-RAD. Summary of the Invention

[0004] The purpose of the present invention is to address the technical defects in the prior art.

[0005] In a first aspect, a substituted 2H-benzopyran-3-carboxanilide compound is provided, the structure of which is Formula I:

[0006]

[0007] Wherein, R1 is selected from H, halogen, alkoxy; R2, R3, R4, R5 are independently selected from any one of H, halogen, alkyl, alkoxy, and NO2.

[0008] The alkyl group is methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl; preferably methyl, ethyl or tert-butyl;

[0009] The alkoxy group is methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy or tert-butoxy; preferably methoxy, ethoxy or tert-butoxy.

[0010] R1 is selected from halogen, alkoxy; R2, R4, R5 are independently selected from any one of H, halogen, alkyl; R3 is selected from H, halogen, alkyl, alkoxy, NO2.

[0011] R1 is selected from H; R2 is selected from halogen; R3 is selected from H, halogen, alkyl, alkoxy, NO2; R4 and R5 are independently selected from any one of H, halogen, and alkyl.

[0012] R1, R2 are selected from H; R3 is selected from halogen; R4 is selected from H, alkyl; R5 is selected from H, halogen, alkyl, alkoxy;

[0013] Preferably, R1 and R2 are selected from H; R3 is selected from halogen; R4 is selected from H; R5 is selected from H, halogen, alkyl; or

[0014] Preferably, R1 and R2 are selected from H; R3 is selected from halogen; R4 is selected from alkyl; R5 is selected from H, halogen, and alkoxy.

[0015] R1, R2, and R3 are selected from H; and R4 and R5 are selected from halogen.

[0016] Includes any of the following:

[0017] N-(4-fluoro-2-methoxyphenyl)-2H-chromen-3-carboxamide;

[0018] N-(2-Fluoro-4-chlorophenyl)-2H-chromen-3-carboxamide;

[0019] N-(2,3,4-trifluorophenyl)-2H-benzopyran-3-carboxamide;

[0020] N-(4'-fluoro-2'-methylphenyl-)-2H-benzopyran-3-carboxamide;

[0021] N-(2',4'-difluorophenyl)-2H-benzopyran-3-carboxamide;

[0022] N-(2,4,5-trifluorophenyl)-2H-chromen-3-carboxamide;

[0023] N-(2-methylphenyl)-2H-benzopyran-3-carboxamide;

[0024] N-(2,4-dimethylphenyl)-2H-benzopyran-3-carboxamide;

[0025] N-(4-chlorophenyl)-2H-benzopyran-3-carboxamide;

[0026] N-(3-Fluoro-4-chlorophenyl)-2H-benzopyran-3-carboxamide;

[0027] N-(4-bromophenyl)-2H-chromen-3-carboxamide;

[0028] N-(3,5-dimethylphenyl)-2H-benzopyran-3-carboxamide;

[0029] N-(2,4-dimethoxyphenyl)-2H-chromen-3-carboxamide;

[0030] N-(2-Fluoro-4-nitrophenyl)-2H-benzopyran-3-carboxamide;

[0031] N-(3-chloro-2-methylphenyl)-2H-benzopyran-3-carboxamide;

[0032] N-(4-chloro-3-methylphenyl)-2H-benzopyran-3-carboxamide;

[0033] N-(4-Fluorophenyl)-2H-chromen-3-carboxamide;

[0034] N-(2,3-dimethylphenyl)-2H-benzopyran-3-carboxamide;

[0035] N-(2,4-dichlorophenyl)-2H-benzopyran-3-carboxamide;

[0036] N-(4-bromophenyl)-7-methoxy-2H-chromen-3-carboxamide;

[0037] N-(4-Fluorophenyl)-7-methoxy-2H-chromen-3-carboxamide;

[0038] N-(2,4-difluorophenyl)-7-methoxy-2H-chromen-3-carboxamide;

[0039] N-(4-bromophenyl)-7-bromo-2H-benzopyran-3-carboxamide;

[0040] N-(4-Fluorophenyl)-7-bromo-2H-chromen-3-carboxamide;

[0041] N-(2,4-Difluorophenyl)-7-bromo-2H-chromen-3-carboxamide.

[0042] In a second aspect, the present invention provides a composition comprising the above-mentioned substituted 2H-benzopyran-3-carboxanilide compound, its geometric isomers, its pharmaceutically acceptable salts, its hydrates or its solvates, and a pharmaceutically acceptable carrier or excipient.

[0043] In a third aspect, the present invention provides a method for preparing the above-mentioned substituted 2H-benzopyran-3-carboxanilide compounds, comprising the following steps:

[0044] Step (1): heating the compound of formula II and thionyl chloride under reflux until the compound of formula II is completely converted, as shown in the reaction formula 1; evaporating under reduced pressure to dryness; and dissolving the residue in an appropriate solvent to prepare reaction solution A;

[0045]

[0046] Step (2): dissolving the compound of formula III in a suitable solvent and adding an organic base to prepare reaction solution B;

[0047]

[0048] Step (3): Place the reaction solution B in an ice bath and stir, add the reaction solution A dropwise, and then react at room temperature until completion to obtain the compound of formula I;

[0049] The solvent is preferably dichloromethane, tetrahydrofuran, N,N-dimethylformamide and pyridine; the organic base is preferably 4-dimethylaminopyridine, triethylamine, pyridine and N-methylmorpholine.

[0050] In a fourth aspect, the present invention provides use of the above-mentioned substituted 2H-benzopyran-3-carboxanilide compounds or the above-mentioned compositions in the preparation of drugs for preventing and / or treating radiation damage (especially ionizing radiation damage).

[0051] The compounds with radiation prevention and treatment activity provided by the present invention have the characteristics of high efficiency and low toxicity, both prevention and treatment, stable quality, easy administration, and effectiveness both orally and by injection. They can effectively prolong the survival of irradiated mice and provide new ideas and directions for the development of efficient and safe radiation protection drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1-Figure 3 Shown is the compound D-18 1 H NMR spectrum, 13 C NMR and MS spectra.

[0053] Figure 4 Shown is the 30-day survival rate change curve of mice in the animal experiment. DETAILED DESCRIPTION

[0054] the term

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

[0056] Unless otherwise indicated, the present invention employs conventional methods such as mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the skill of the art. Unless specific definitions are provided, the nomenclature and laboratory procedures and techniques associated with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those known to those skilled in the art. In general, the aforementioned techniques and steps can be implemented by conventional methods well known in the art and described in various general and more specific literature, which are cited and discussed in this specification.

[0057] The term "alkyl" refers to an aliphatic hydrocarbon group, which can be a branched or straight chain alkyl group. Depending on the structure, the alkyl group can be a monovalent group or a divalent group (i.e., an alkylidene group). In the present invention, the alkyl group is preferably an alkyl group having 1 to 8 carbon atoms, more preferably a "low alkyl group" having 1 to 6 carbon atoms, and even more preferably an alkyl group having 1 to 4 carbon atoms. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, amyl, hexyl, etc. It should be understood that "alkyl" mentioned herein includes all possible configurations and conformations of the alkyl group, for example, "propyl" mentioned herein includes n-propyl and isopropyl, "butyl" includes n-butyl, isobutyl and tert-butyl, and "pentyl" includes n-pentyl, isopentyl, neopentyl, tert-pentyl, and penta-3-yl, etc.

[0058] The term "alkoxy" refers to an -O-alkyl group, wherein alkyl is as defined herein. Typical alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like.

[0059] The term "cycloalkyl" refers to a monocyclic or polycyclic radical containing only carbon and hydrogen. Cycloalkyl includes a group having 3-12 ring atoms. According to structure, cycloalkyl can be a monovalent group or a divalent group (e.g., cycloalkylidene). In the present invention, cycloalkyl is preferably a cycloalkyl having 3-8 carbon atoms, more preferably a "low cycloalkyl" having 3-6 carbon atoms. The example of cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl and adamantyl.

[0060] The term "aromatic" refers to a planar ring having a delocalized π electron system and containing 4n+2 π electrons, where n is an integer. The aromatic ring can be composed of five, six, seven, eight, nine, or more than nine atoms. The aromatic group can be optionally substituted. The term "aromatic" includes carbocyclic aromatic groups (e.g., phenyl) and heterocyclic aromatic (or "heteroaryl" or "heteroaromatic") groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings that share adjacent pairs of carbon atoms) groups.

[0061] As used herein, the term "aryl" refers to an aromatic ring in which each of the atoms forming the ring is a carbon atom. The aryl ring can be composed of five, six, seven, eight, nine, or more than nine atoms. An aryl group can be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, phenanthrenyl, anthracenyl, fluorenyl, and indenyl. Depending on the structure, an aryl group can be a monovalent group or a divalent group (i.e., an arylene group).

[0062] The term "aryloxy" refers to an -O-aryl group, wherein aryl is as defined herein.

[0063] The term "heteroaryl" refers to an aromatic group that includes one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. The N-containing "heteroaryl" moiety refers to an aromatic group in which at least one skeletal atom on the ring is a nitrogen atom. Depending on the structure, a heteroaryl group can be a monovalent group or a divalent group (i.e., a heteroarylidene group). Examples of heteroaryl groups include, but are not limited to, pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolyl, isoquinolyl, indolyl, benzimidazolyl, benzofuranyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, isoindole, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, naphthyridinyl, and furopyridinyl.

[0064] As used herein, the term "heteroalkyl" refers to an alkyl group as defined herein where one or more of the backbone chain atoms is a heteroatom, such as oxygen, nitrogen, sulfur, silicon, phosphorus, or a combination thereof. The heteroatom(s) may be located at any position within the heteroalkyl group or at the position where the heteroalkyl group is attached to the rest of the molecule.

[0065] As used herein, the term "heterocycloalkyl" or "heterocyclyl" refers to a non-aromatic ring in which one or more of the atoms forming the ring are heteroatoms selected from nitrogen, oxygen, and sulfur. The heterocycloalkyl ring may be composed of three, four, five, six, seven, eight, nine, or more than nine atoms. The heterocycloalkyl ring may be optionally substituted. Examples of heterocycloalkyls include, but are not limited to, lactams, lactones, cyclic imines, cyclic thioimides, cyclic carbamates, tetrahydrothiopyran, 4H-pyran, tetrahydropyran, piperidine, 1,3-dioxin, 1,3-dioxane, 1,4-dioxin, 1,4-dioxane, piperazine, 1,3-oxathiinane, 1,4-oxathiinane, 1,4-oxathiinane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbital Acid, dioxopiperazine, hydantoin, dihydrouracil, morpholine, trioxane, hexahydro-1,3,5-triazine, tetrahydrothiophene, tetrahydrofuran, pyrroline, pyrrolidine, imidazolidine, pyrrolidone, pyrazoline, pyrazolidine, imidazolidine, imidazolidine, 1,3-dioxole, 1,3-dioxolane, 1,3-dithiole, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine and 1,3-oxathiolane. Depending on the structure, the heterocycloalkyl group can be a monovalent group or a divalent group (i.e., a heterocycloalkylene group).

[0066] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0067] The terms "haloalkyl," "haloalkoxy," and "haloheteroalkyl" include structures of alkyl, alkoxy, or heteroalkyl groups in which at least one hydrogen atom is replaced by a halogen atom. In certain embodiments, if two or more hydrogen atoms are replaced by halogen atoms, the halogen atoms may be the same or different from one another.

[0068] The term "amino" refers to a -NH2 group.

[0069] The term "hydroxy" refers to an -OH group.

[0070] The term "cyano" refers to a -CN group.

[0071] The term "ester group" refers to a chemical moiety having the formula -COOR, where R is selected from alkyl, cycloalkyl, aryl, heteroaryl (attached through a ring carbon), and heterocyclyl (attached through a ring carbon).

[0072] The term "amido" or "amido" refers to a -NR-CO-R' group, wherein R and R' are each independently hydrogen or alkyl.

[0073] The term "aminoacyl" or "aminoacyl" refers to a -CO-NH2 group.

[0074] The term "alkylaminoacyl" or "alkylaminoacyl" refers to a -CO-NH-R group where R is alkyl as defined herein.

[0075] The term "optionally" refers to one or more events described later that may or may not occur, and includes both events that occur and events that do not occur. The term "optionally substituted" or "substituted" refers to that the group mentioned can be substituted by one or more additional groups, each of which is independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, hydroxyl, alkoxy, cyano, halogen, amide, nitro, haloalkyl, amino, methylsulfonyl, alkylcarbonyl, alkoxycarbonyl, heteroarylalkyl, heterocycloalkylalkyl, aminoacyl, amino protecting group, etc. Among them, the amino protecting group is preferably selected from pivaloyl, tert-butyloxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, benzyl, p-methoxybenzyl, allyloxycarbonyl, and trifluoroacetyl, etc.

[0076] The compounds provided by the present invention are substituted 2H-benzopyran-3-carboxanilide compounds, and their structure is Formula I:

[0077]

[0078] Wherein, R1 is selected from H, halogen, alkoxy; R2, R3, R4, R5 are independently selected from any one of H, halogen, alkyl, alkoxy, and NO2.

[0079] For example: R1 is selected from any one of H, F, Cl, Br, and OCH3; R2, R3, R4, and R5 are independently selected from any one of H, F, Cl, Br, CH3, OCH3, and NO2.

[0080] The above substituted 2H-benzopyran-3-carboxanilide compounds also include geometric isomers of the compound of formula I, pharmaceutically acceptable salts thereof, hydrates or solvates thereof, and pharmaceutical compositions of pharmaceutically acceptable carriers or excipients. Among them: isomers or hydrates, such as optical isomers or racemic compounds; pharmaceutically acceptable salts can be sulfates, pyrosulfates, hydrogen sulfates, sulfites, hydrogen sulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, hydrochlorides, bromides, iodides, acetates, propionates, octanoates, acrylates, formates, isobutyrates, heptanoates, decanoates, propiolates, oxalates, malonates, succinates, suberates of the compound of formula I. , sebacate, fumarate, maleate, 2-butyne-1,4-dioate, 3-cyclohexyne-2,5-dioate, benzoate, chlorobenzoate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hippurate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, glutamate, arginine, lysine, etc., preferably hydrochloride and phosphate.

[0081] The substituted 2H-benzopyran-3-carboxanilide compounds provided by the present invention can be administered orally or parenterally. Oral medications can be tablets, pills, granules, capsules, coatings, oral liquids, emulsions, and powders; parenteral medications can be injections, suppositories, or other suitable forms. The excipients used to make the above dosage forms are all commonly used adjuvants, such as the adjuvants used in solid dosage forms such as tablets, capsules, and coatings include starch, gelatin, gum arabic, silica, and polyethylene glycol; the solvents used in liquid dosage forms include water, ethanol, propylene glycol, and vegetable oils (such as corn oil, peanut oil, olive oil, etc.). Other adjuvants, such as surfactants, lubricants, disintegrants, preservatives, flavoring agents, pigments, etc., are also commonly used adjuvants.

[0082] Compounds of formula (I) can be synthesized using standard synthetic techniques known to those skilled in the art or using methods known in the art in combination with the methods described herein. In addition, the solvents, temperatures and other reaction conditions given herein can be varied according to the skill in the art. As further guidance, the following synthetic methods can also be utilized.

[0083] The reactions can be used sequentially to provide the compounds described herein; or they can be used to synthesize fragments that are subsequently added by methods described herein and / or known in the art.

[0084] The compounds can be synthesized using methods similar to those described below by using appropriate optional starting materials. The starting materials for synthesizing the compounds described herein can be synthesized or can be obtained from commercial sources. The compounds described herein and other related compounds with different substituents can be synthesized using techniques and raw materials known to those skilled in the art. The general method for preparing the compounds disclosed herein can be derived from reactions known in the art, and the reactions can be modified by reagents and conditions deemed appropriate by those skilled in the art to introduce various parts into the molecules provided herein.

[0085] If desired, the reaction products can be isolated and purified using conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography, etc. These products can be characterized using conventional methods, including physical constants and spectral data.

[0086] The present invention also provides a method for preparing the compound of formula I. The synthesis steps of the substituted 2H-benzopyran-3-carboxanilide compound are as follows:

[0087] Step (1): Heat the compound of formula II and thionyl chloride under reflux until the compound of formula II is completely converted and then evaporate to dryness under reduced pressure. The reaction formula is shown in Formula 1. Dissolve the residue in an appropriate solvent to prepare reaction solution A;

[0088]

[0089] Step (2): dissolving the compound of formula III in a suitable solvent and adding an organic base to prepare reaction solution B;

[0090]

[0091] Step (3): Place the reaction solution B in an ice bath and stir, add the reaction solution A dropwise, and then react at room temperature until completion to obtain the compound shown in formula I.

[0092] The solvent is preferably dichloromethane, tetrahydrofuran, N,N-dimethylformamide and pyridine; the organic base is preferably 4-dimethylaminopyridine, triethylamine, pyridine and N-methylmorpholine.

[0093] The substituted 2H-benzopyran-3-carboxanilide compounds having the structure of Formula I provided by the present invention can be used to prevent and / or treat radiation damage, and may be suitable for preventing and / or treating damage to the body caused by radiation, including early symptoms such as dizziness, fatigue, and loss of appetite caused by different radiation doses in humans or animals, DNA damage, digestive tract damage, hematopoietic system damage, and brain damage.

[0094] The following is a more detailed description of the present invention with reference to specific examples, and further elaboration of the present invention. However, these examples are by no means intended to limit the present invention.

[0095] The following specific examples illustrate the preparation and properties of the compounds of the present invention, which are not exhaustive. The reagents used in the examples were all purchased from the market, the nuclear magnetic resonance spectra were measured using a Bruker 600M superconducting nuclear magnetic spectrometer, the ESI mass spectra were measured using an Agilent 1260-G6230A mass spectrometer, and the high-resolution mass spectra were measured using a Bruker 9.4T ultra-high performance hybrid tandem Fourier transform ion cyclotron resonance mass spectrometer (Q-FT-ICR-MS).

[0096] Example 1: Synthesis of N-(4-fluoro-2-methoxyphenyl)-2H-benzopyran-3-carboxamide (D-1)

[0097] The synthesis of 2H-benzopyran-3-carboxylic acid is shown in Formula 2:

[0098]

[0099] 2.444 g (20.0 mmol) of salicylaldehyde, 1.274 g (24.0 mmol) of acrylonitrile, and 0.269 g (2.4 mmol) of triethylenediamine were placed in a 35 mL microwave reaction bottle. The reaction bottle was placed in a CEM Discovery SP microwave synthesizer. The reaction was carried out at 140°C in the microwave for 30 min. After cooling, the mixture was removed and an appropriate amount of dichloromethane was added. The solution was transferred to a 100 mL round-bottom flask and evaporated to dryness under reduced pressure. 40 mL of 5 M sodium hydroxide solution was added to the flask and refluxed with stirring for 3 h. The mixture was cooled to room temperature, acidified with 5 M hydrochloric acid, extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and evaporated to dryness to obtain the crude product. Column chromatography gave 2.445 g of a white solid with a yield of 69.4% and an mp of 116-117°C.

[0100] The synthesis of N-(4-fluoro-2-methoxyphenyl)-2H-benzopyran-3-carboxamide is shown in Formula 3:

[0101]

[0102] To a 25 mL round-bottom flask, add 0.529 g (3.0 mmol) of 2H-benzopyran-3-carboxylic acid and 6 mL of thionyl chloride. Heat under reflux for 1.5 hours and evaporate to dryness under reduced pressure. Dissolve the mixture in 10 mL of dry tetrahydrofuran to prepare Reaction Solution A. Dissolve 0.353 g (2.5 mmol) of 4-fluoro-2-methoxyaniline in 5 mL of dry tetrahydrofuran and add 0.42 mL of triethylamine (3.0 mmol) to prepare Reaction Solution B. While stirring in an ice bath, slowly add Reaction Solution A to Reaction Solution B. Stir the reaction at room temperature for 3 hours, then dilute with 100 mL of water. precipitate a solid, filter, wash with water, and air-dry to obtain the crude product. Column chromatography yields 0.660 g of a pale yellow solid (88.2% yield). mp 149-151°C. 1H NMR (600MHz, DMSO-d6) δ9.30 (s, 1H), 7.56 (d, J = 8.7Hz, 1H), 7.48 (s, 1H), 7.26 (t, J = 7.9Hz, 2H), 7.04 -6.96(m,2H),6.88(d,J=7.9Hz,1H),6.77(td,J=8.6,2.7Hz,1H),4.96(d,J=1.0Hz,2H),3.84(s,3H). 13 C NMR (151MHz, DMSO-d6) δ163.67 (s), 160.61 (d, J = 241.7Hz), 154.70 (s), 153.82 ( d,J=10.5Hz),131.72(s),129.10(s),128.42(s),126.95(s),126.63(d,J=10.1 Hz),123.04(d,J=3.0Hz),122.33(s),121.62(s),116.18(s),106.51(d,J=22.2 Hz), 100.42 (d, J = 26.8Hz), 64.80 (s), 56.63 (s). MS (ESI-TOF) m / z: 300.10 (M+H) + ,322.08(M+Na) + .

[0103] Example 2: Synthesis of N-(2-fluoro-4-chlorophenyl)-2H-benzopyran-3-carboxamide (D-2)

[0104] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 2-fluoro-4-chloroaniline, 0.635 g of a white powder was obtained, with a yield of 83.7%. mp 127-128°C. 1 H NMR(600MHz,DMSO-d6)δ9.99(s,1H),7.61(s,1H),7.52(d,J=3.9Hz,2H),7.29(dd,J= 7.6, 2.6Hz, 3H), 6.99 (d, J = 0.9Hz, 1H), 6.89 (d, J = 8.0Hz, 1H), 4.97 (d, J = 1.2Hz, 2H). 13C NMR (151MHz, DMSO-d6) δ163.84(s),155.73(d,J=250.66Hz),154.80(s),131.99(s),130.21(d,J=9.6Hz),129.50(s),129.24(s),128.07(s),1 26.25(s),125.15(d,J=12.08),125.02(d,J=3.02Hz),122.41(s),121. 48(s),117.04(s),116.25(s),64.70(s).MS(ESI-TOF)m / z: 304.05(M+H) + ,326.03(M+Na) + .

[0105] Example 3: Synthesis of N-(2,3,4-trifluorophenyl)-2H-benzopyran-3-carboxamide (D-3)

[0106] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 2,3,4-trifluoroaniline, 0.650 g of a white solid was obtained, with a yield of 85.2%. mp 128-129°C. 1 H NMR (600MHz, DMSO-d6) δ10.14(s,1H),7.52(s,1H),7.35(d,J=5.3Hz,2H),7.29(dd,J=6.6,4.9Hz,2 H),7.00(d,J=1.0Hz,1H),6.89(d,J=8.0Hz,1H),4.97(d,J=1.2Hz,2H).HRMS(ESI)m / z:Calculated for C 16 H 11 F3NO2 + ([M+H] + )306.0736,Found 306.0737.

[0107] Example 4: Synthesis of N-(4-fluoro-2-methylphenyl)-2H-benzopyran-3-carboxamide (D-4)

[0108] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 4-fluoro-2-methylaniline, 0.612 g of a white solid was obtained, with a yield of 86.4%. mp 147-149°C. 1H NMR (600MHz, DMSO-d6) δ9.67(s,1H),7.47(s,1H),7.28(dd,J=7.7,3.4Hz,3H),7.13(dd,J =9.7, 2.8Hz, 1H), 6.99 (s, 2H), 6.89 (t, J = 9.3Hz, 1H), 4.98 (d, J = 1.1Hz, 2H), 2.21 (s, 3H). 13 C NMR(151MHz,DMSO-d6)δ163.67(s),160.61(d,J=241.6Hz),154.70(s),153.82(d,J =10.5Hz),131.72(s),129.10(s),128.42(s),126.95(s),126.63(d,J=10.1Hz),12 3.04(d,J=3.0Hz),122.33(s),121.62(s),116.18(s),106.52(d,J=22.65Hz),100. 43(d,J=25.67Hz),100.34(s),64.80(s),56.63(s).MS(ESI-TOF)m / z: 284.11(M+H) + ,306.09(M+Na) + .

[0109] Example 5: Synthesis of N-(2,4-difluorophenyl)-2H-benzopyran-3-carboxamide (D-5)

[0110] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 2,4-difluoroaniline, 0.564 g of a white powder was obtained, with a yield of 78.6%. mp 123-125°C. 1 H NMR(600MHz,DMSO-d6)δ9.93(s,1H),7.54(td,J=8.9,6.2Hz,1H),7.51(s,1H),7.35(ddd,J=10.9,9.2,2.8Hz,1H),7.30-7.26(m,2H ),7.11(ddd,J=8.3,2.9,1.4Hz,1H),6.99(d,J=0.9Hz,1H),6.89(d,J=8.0Hz,1H),4.97(d,J=1.2Hz,2H).HRMS(ESI)m / z:Calculated for C 16 H 12 F2NO2 + ([M+H] + )288.0831,Found288.0830.

[0111] Example 6: Synthesis of N-(2,4,5-trifluorophenyl)-2H-benzopyran-3-carboxamide (D-6)

[0112] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 2,4,5-trifluoroaniline, 0.640 g of a white solid was obtained, with a yield of 83.9%. mp 149-151°C. 1 H NMR(600MHz,DMSO-d6)δ10.04(s,1H),7.77-7.63(m,2H),7.53(s,1H),7.32-7.26(m,2H),7 .03-6.97(m,1H),6.89(d,J=8.0Hz,1H),4.97(d,J=1.0Hz,2H).HRMS(ESI)m / z:Calculated for C 16 H 11 F3NO2 + ([M+H] + )306.0736,Found306.0736.

[0113] Example 7: Synthesis of N-(2-methylphenyl)-2H-benzopyran-3-carboxamide (D-7)

[0114] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 2-methylaniline, 0.561 g of a white powder was obtained, with a yield of 84.6%. mp 118-120°C. 1 H NMR(600MHz,DMSO-d6)δ9.65(s,1H),7.48(s,1H),7.33-7.22(m,4H),7.20(s,1H),7.16( d,J=7.3Hz,1H),6.99(t,J=7.4Hz,1H),6.88(d,J=8.0Hz,1H),4.99(s,2H),2.22(s,3H). 13 C NMR(151MHz,DMSO-d6)δ163.72(s),154.72(s),136.41(s),134.03(s),131.67(s),130.80(s),129.05(s),128.29(s),127.17 (s),126.83(s),126.43(d,J=7.8Hz),122.34(s),121.68(s),116.19(s),64.93(s),18.38(s).MS(ESI-TOF)m / z: 266.12(M+H) + ,288.10(M+Na) + .

[0115] Example 8: Synthesis of N-(2,4-dimethylphenyl)-2H-benzopyran-3-carboxamide (D-8)

[0116] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 2,4-dimethylaniline, 0.609 g of a white solid was obtained, with a yield of 87.2%. mp 112-114°C. 1 H NMR(600MHz,DMSO-d6)δ9.58(s,1H),7.45(s,1H),7.28-7.24(m,2H),7.14(d,J=7.9Hz,1H),7 .06(s,1H),7.01-6.96(m,2H),6.88(d,J=8.0Hz,1H),4.97(s,2H),2.27(s,3H),2.17(s,3H). 13 C NMR(151MHz,DMSO-d6)δ163.72(s),154.69(s),135.51(s),133.82(d,J=13.1Hz),131.62(s),131.33(s),129.01(s),128.10(s),12 7.24(s),126.98(s),126.79(s),122.33(s),121.71(s),116.18(s),64.94(s),21.00(s),18.30(s).MS(ESI-TOF)m / z: 280.13(M+H) + ,302.11(M+Na) + .

[0117] Example 9: Synthesis of N-(4-chlorophenyl)-2H-benzopyran-3-carboxamide (D-9)

[0118] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 4-chloroaniline, 0.61g of yellow solid was obtained in an 86.3% yield. mp 161-163°C. 1 H NMR(600MHz,DMSO-d6)δ10.18(s,1H),7.78-7.72(m,2H),7.48(s,1H),7.40(d,J=8.8Hz,2 H),7.32-7.25(m,2H),6.99(t,J=7.4Hz,1H),6.88(d,J=8.0Hz,1H),4.98(d,J=1.2Hz,2H). 13C NMR(151MHz,DMSO-d6)δ163.91(s),154.74(s),138.31(s),131.86(s),129.14(s),129.04(s),128.68(s),12 7.64(s),127.14(s),122.40(s),122.04(s),121.52(s),116.24(s),64.78(s).MS(ESI-TOF)m / z:286.06(M+H) + ,308.04(M+Na) + .

[0119] Example 10: Synthesis of N-(3-fluoro-4-chlorophenyl)-2H-benzopyran-3-carboxamide (D-11)

[0120] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 3-fluoro-4-chloroaniline, 0.670 g of a yellow solid was obtained, with a yield of 88.2%. mp 131-133°C. 1 H NMR (600MHz, DMSO-d6) δ10.23(s,1H),8.07-7.97(m,1H),7.71-7.63(m,1H),7.48(s,1H),7.41(dd,J=11 .4,6.7Hz,1H),7.28(dd,J=15.2,7.6Hz,2H),7.00(t,J=7.4Hz,1H),6.89(d,J=8.0Hz,1H),4.98(s,2H). 13 C NMR(151MHz,DMSO-d6)δ163.91(s),154.76(s),153.76(d,J=243.11Hz),136.60(s),131.93(s),129.16(s),128.89(s),126.89(s),122.40 (s),121.90(s),121.43(s),120.75(s),119.49(d,J=18.2Hz),117.32(d,J=19.63Hz)116.25(s),64.71(s).MS(ESI-TOF)m / z:304.05(M+H) + ,326.04(M+Na) + .

[0121] Example 11: Synthesis of N-(4-bromophenyl)-2H-benzopyran-3-carboxamide (D-12)

[0122] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 4-bromoaniline, 0.687 g of a white solid was obtained, with a yield of 83.2%. mp 173-175°C. 1 H NMR (600MHz, DMSO-d6) δ10.18 (s, 1H), 7.72-7.67 (m, 2H), 7.52 (dd, J = 6.8, 2.1Hz, 2H), 7. 48(s,1H),7.29(dd,J=9.3,7.9Hz,2H),6.99(s,1H),6.88(d,J=8.1Hz,1H),4.98(s,2H). 13 C NMR (151MHz, DMSO-d6) δ161.63 (s), 152.48 (s), 136.48 (s), 129.61 (d, J = 12.6Hz), 126.86 (s), 126.44 ( s),124.86(s),120.12(d,J=6.0Hz),119.24(s),113.97(s),62.52(s).MS(ESI-TOF)m / z:330.01(M+H) + ,353.99( 81 M+Na) + .

[0123] Example 12: Synthesis of N-(3,5-dimethylphenyl)-2H-benzopyran-3-carboxamide (D-13)

[0124] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 3,5-dimethylaniline, 0.591 g of a white powder was obtained, with a yield of 84.6%. mp 143-144°C. 1 H NMR(600MHz,DMSO-d6)δ9.88(s,1H),7.46(s,1H),7.33(s,2H),7.26(dd,J=12.5,4.7Hz,2H) ,7.01-6.97(m,1H),6.88(d,J=7.8Hz,1H),6.73(s,1H),4.97(d,J=1.3Hz,2H),2.25(s,6H). 13C NMR(151MHz,DMSO-d6)δ163.65(s),154.68(s),139.13(s),138.01(s),131.71(s),129.04(s),128.19(s),127.43( s),125.60(s),122.36(s),121.63(s),118.35(s),116.20(s),64.85(s),21.59(s).MS(ESI-TOF)m / z: 280.13(M+H) + ,302.12(M+Na) + .

[0125] Example 13: Synthesis of N-(2,4-dimethoxyphenyl)-2H-benzopyran-3-carboxamide (D-14)

[0126] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 2,4-dimethoxyaniline, 0.676 g of a white solid was obtained, with a yield of 86.9%. mp 147-149°C. 1 H NMR (600MHz, DMSO-d6) δ9.16 (s, 1H), 7.49 (s, 1H), 7.45 (d, J = 3.1Hz, 1H), 7.31-7.24 (m, 2H), 6.99 (ddd, J = 8.4, 6.3 ,2.6Hz,2H),6.88(d,J=8.1Hz,1H),6.71(dd,J=8.9,3.1Hz,1H),4.98(d,J=1.2Hz,2H),3.80(s,3H),3.70(s,3H). 13 C NMR(151MHz,DMSO-d6)δ163.46(s),154.72(s),153.30(s),145.41(s),131.77(s),129.20(s),128.43(s),127.75(s),127.05(s),1 22.30(s),121.59(s),116.17(s),112.44(s),110.35(s),109.82(s),64.77(s),56.66(s),55.82(s).MS(ESI-TOF)m / z: 312.12(M+H) + ,334.10(M+Na) + .

[0127] Example 14: Synthesis of N-(2-fluoro-4-nitrophenyl)-2H-benzopyran-3-carboxamide (D-16)

[0128] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 2-fluoro-4-nitroaniline, 0.563 g of a yellow powder was obtained, with a yield of 71.6%. mp 136-138°C. 1 H NMR(600MHz,DMSO-d6)δ10.12(s,1H),7.96(dd,J=7.7,1.7Hz,1H),7.85(s,1H),7.82-7.78(m,1H),7.54(t,J =7.5Hz,1H),7.46-7.42(m,2H),7.37-7.33(m,1H),7.02(t,J=7.4Hz,1H),6.93(d,J=8.2Hz,1H),5.09(s,2H). 13 C NMR(151MHz,DMSO-d6)δ190.42(s),162.86(s),155.25(s),151.29(s),136.19(s),135.86(s),133.22(s),131. 46(s),130.18(s),128.43(s),127.30(s),124.32(s),122.53(s),121.31(d,J=72.7Hz),116.39(s),64.29(s).

[0129] Example 15: Synthesis of N-(3-chloro-2-methylphenyl)-2H-benzopyran-3-carboxamide (D-17)

[0130] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 3-chloro-2-methylaniline, 0.619 g of a white solid was obtained, with a yield of 82.6%. mp 162-164°C. 1 H NMR (600MHz, DMSO-d6) δ9.92 (s, 1H), 7.50 (s, 1H), 7.35 (d, J = 7.7Hz, 1H), 7.30-7.22 (m ,4H),6.99(t,J=7.4Hz,1H),6.89(d,J=8.0Hz,1H),4.99(d,J=2.5Hz,2H),2.23(s,3H). 13C NMR(151MHz,DMSO-d6)δ163.94(s),154.76(s),138.09(s),134.27(s),132.54(s),131.80(s),129.12(s),128.75(s),127.34( s),127.18(s),126.79(s),126.12(s),122.37(s),121.59(s),116.22(s),64.85(s),15.84(s).MS(ESI-TOF)m / z: 300.09(M+H) + ,322.07(M+Na) + .

[0131] Example 16: Synthesis of N-(4-chloro-3-methylphenyl)-2H-benzopyran-3-carboxamide (D-18)

[0132] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 4-chloro-3-methylaniline, 0.648 g of a white solid was obtained, with a yield of 86.5%. mp 161-163°C. 1 H NMR (600MHz, DMSO-d6) δ10.09(s,1H),7.70(d,J=2.4Hz,1H),7.57(d,J=8.7Hz,1H),7.47(s,1H),7.36(d,J=8. 6Hz,1H),7.27(dd,J=13.4,7.6Hz,2H),6.99(t,J=7.4Hz,1H),6.88(d,J=8.0Hz,1H),4.98(s,2H),2.31(s,3H). 13 CNMR(151MHz,DMSO-d6)δ163.82(s),154.73(s),138.23(s),135.94(s),131.84(s),129.40(s),129.12(s),128.61(s),127.97 (s),127.15(s),122.94(s),122.39(s),121.53(s),119.69(s),116.24(s),64.78(s),20.35(s).MS(ESI-TOF)m / z: 300.09(M+H) + ,322.07(M+Na) + .

[0133] Example 17: Synthesis of N-(4-fluorophenyl)-2H-benzopyran-3-carboxamide (D-19)

[0134] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 4-fluoroaniline, 0.561 g of a white solid was obtained, with a yield of 83.4%. mp 132-134°C. 1 H NMR (600MHz, DMSO-d6) δ10.11(s,1H),7.72(dd,J=8.5,5.1Hz,2H),7.46(s,1H),7.28(dd,J=16.1,7. 6Hz, 2H), 7.18 (t, J = 8.9Hz, 2H), 6.99 (t, J = 7.8Hz, 1H), 6.88 (d, J = 8.1Hz, 1H), 4.98 (d, J = 1.0Hz, 2H). 13 C NMR (151MHz, DMSO-d6) δ163.74(s),158.69(d,J=239.9Hz),154.71(s),135.67(d,J=2.2Hz),131.78(s),129.08(s),128.40(s),12 7.27(s),122.39(d,J=2.6Hz),122.35(s),121.56(s),116.23(s),115.70(d,J=22.2Hz),64.82(s).MS(ESI-TOF)m / z: 270.10(M+H) + ,292.08(M+Na) + .

[0135] Example 18: Synthesis of N-(2,3-dimethylphenyl)-2H-benzopyran-3-carboxamide (D-20)

[0136] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 2,3-dimethylaniline, 0.566 g of a white solid was obtained, with a yield of 81.1%. mp 186-188°C. 1 H NMR (600MHz, DMSO-d6) δ9.71 (s, 1H), 7.47 (s, 1H), 7.27 (dd, J = 13.5, 7.5Hz, 2H), 7.07 (d, J = 3.0Hz, 3H), 6.98 (t, J = 7.4Hz, 1H), 6.88 (d, J = 8.0Hz, 1H), 4.98 (d, J = 1.2Hz, 2H), 2.27 (s, 3H), 2.08 (s, 3H). 13C NMR(151MHz,DMSO-d6)δ163.84(s),154.70(s),137.48(s),136.23(s),133.08(s),131.63(s),129.02(s),128.15(s),127.98(s),1 27.24(s),125.71(s),124.96(s),122.33(s),121.71(s),116.18(s),64.95(s),20.60(s),14.73(s).MS(ESI-TOF)m / z: 280.13(M+H) + ,302.12(M+Na) + .

[0137] Example 19: Synthesis of N-(2,4-dichlorophenyl)-2H-benzopyran-3-carboxamide (D-21)

[0138] Following the procedure of Example 1, except that 4-fluoro-2-methoxyaniline was replaced with 2,4-dichloroaniline, 0.656 g of a yellow solid was obtained, with a yield of 81.9%. mp 140-142°C. 13 C NMR(151MHz,DMSO-d6)δ163.86(s),154.81(s),137.21(s),132.43(s),132.02(s),129.39(s),129.27(s),128.48(s),12 8.39(s),128.27(s),127.26(s),126.31(s),122.43(s),121.46(s),116.27(s),64.70(s).MS(ESI-TOF)m / z: 320.03(M+H) + ,342.01(M+Na) + .

[0139] Example 20: Synthesis of N-(4-bromophenyl)-7-methoxy-2H-benzopyran-3-carboxamide (D-22)

[0140] The synthesis of 7-methoxy-2H-benzopyran-3-carboxylic acid is shown in Formula 4:

[0141]

[0142] 3.043 g (20.0 mmol) of 4-methoxysalicylaldehyde, 1.274 g (24.0 mmol) of acrylonitrile, and 0.269 g (2.4 mmol) of triethylenediamine were placed in a 35 mL microwave reaction vial. The reaction vial was placed in a CEM Discovery SP microwave synthesizer. The reaction was carried out at 140°C in the microwave for 30 min. After cooling, the solution was removed from the flask and an appropriate amount of dichloromethane was added. The solution was transferred to a 100 mL round-bottom flask and evaporated to dryness under reduced pressure. 40 mL of 5 M sodium hydroxide solution was added to the flask and the mixture was refluxed with stirring for 3 h. The mixture was cooled to room temperature, acidified with 5 M hydrochloric acid, extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and evaporated to dryness to obtain the crude product. Column chromatography yielded 3.011 g of a white solid (73.0% yield), mp 127-129°C.

[0143] The synthesis of N-(4-bromophenyl)-7-methoxy-2H-benzopyran-3-carboxamide is shown in Formula 5:

[0144]

[0145] To a 25 mL round-bottom flask, add 0.619 g (3.0 mmol) of 7-methoxy-2H-benzopyran-3-carboxylic acid and 6 mL of thionyl chloride. Heat under reflux for 1.5 hours and evaporate to dryness under reduced pressure. Dissolve in 10 mL of dry tetrahydrofuran to prepare reaction solution A. Dissolve 0.430 g (2.5 mmol) of 4-bromoaniline in 5 mL of dry tetrahydrofuran and add 0.42 mL of triethylamine (3.0 mmol) to prepare reaction solution B. While stirring in an ice bath, slowly add reaction solution A to reaction solution B. Stir the reaction at room temperature for 3 hours, then dilute with 100 mL of water. precipitate a solid, filter, wash with water, and air-dry to obtain the crude product. Column chromatography yields 0.73 g of a white solid (82.1% yield). mp 175-177°C. 1 H NMR (600MHz, DMSO-d6) δ10.08(s,1H),7.68(d,J=8.8Hz,2H),7.51(d,J=8.7Hz,2H),7.46(s,1H),7.2 2(d,J=8.3Hz,1H),6.58(d,J=8.4Hz,1H),6.49(d,J=2.3Hz,1H),4.95(d,J=0.9Hz,2H),3.77(s,3H). 13C NMR(151MHz,DMSO-d6)δ161.85(s),160.37(s),154.13(s),136.68(s),129.69(s),128.00(s),126.78(s),121.46 (s),120.12(s),113.30(s),112.32(s),106.45(s),99.67(s),62.68(s),53.70(s).MS(ESI-TOF)m / z: 360.03(M+H) + ,382.01(M+Na) + .

[0146] Example 21: Synthesis of N-(4-fluorophenyl)-7-methoxy-2H-benzopyran-3-carboxamide (D-23)

[0147] Following the procedure of Example 20, except that 4-bromoaniline was replaced with 4-fluoroaniline, 0.578 g of a white powder was obtained, with a yield of 77%. mp 140-142°C. 1 H NMR(600MHz,DMSO-d6)δ10.01(s,1H),7.74-7.68(m,2H),7.45(s,1H),7.22(d,J=8.4Hz,1H), 7.17(t,J=8.9Hz,2H),6.59(s,1H),6.49(d,J=2.4Hz,1H),4.95(d,J=1.0Hz,2H),3.77(s,3H). 13 C NMR (151MHz, DMSO-d6) δ162.45(s),157.64(d,J=240.4Hz),154.37(s),134.48(d,J=2.2Hz),129.35(s),126.60(s),126.37(s),12 4.23(s),122.68(s),121.30(d,J=7.6Hz),119.75(s),118.07(s),114.63(d,J=22.3Hz),64.14(s).MS(ESI-TOF)m / z: 300.11(M+H) + ,322.09(M+Na) + .

[0148] Example 22: Synthesis of N-(2,4-difluorophenyl)-7-methoxy-2H-benzopyran-3-carboxamide (D-24)

[0149] Following the procedure of Example 20, except that 4-bromoaniline was replaced with 2,4-difluoroaniline, 0.589 g of a yellow powder was obtained, with a yield of 74.2%. mp 138-139°C.1 H NMR (600MHz, CDCl3) δ8.28(d,J=8.8Hz,1H),7.61(s,1H),7.12-7.06(m,2H),6.90(t,J=5.0Hz,2H),6.52(d d,J=8.4,2.4Hz,1H),6.45(d,J=2.4Hz,1H),5.06(d,J=1.1Hz,2H),3.81(s,3H).HRMS(ESI)m / z: Calculated for C 17 H 14 F2NO3 + ([M+H] + )318.0936, Found 318.0935.

[0150] Example 23: Synthesis of N-(4-bromophenyl)-7-bromo-2H-benzopyran-3-carboxamide (D-25)

[0151] The synthesis of 7-bromo-2H-benzopyran-3-carboxylic acid is shown in Formula 6:

[0152]

[0153] 4.020 g (20.0 mmol) of 4-bromosalicylaldehyde, 1.274 g (24.0 mmol) of acrylonitrile, and 0.269 g (2.4 mmol) of triethylenediamine were placed in a 35 mL microwave reaction bottle. The reaction bottle was placed in a CEM Discovery SP microwave synthesizer. The reaction was carried out at 140°C in the microwave for 30 min. After cooling, the mixture was removed and an appropriate amount of dichloromethane was added. The solution was transferred to a 100 mL round-bottom flask and evaporated to dryness under reduced pressure. 40 mL of 5 M sodium hydroxide solution was added to the flask and refluxed with stirring for 3 h. The mixture was cooled to room temperature, acidified with 5 M hydrochloric acid, extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and evaporated to dryness to obtain the crude product. Column chromatography gave 3.933 g of an off-white solid with a yield of 77.1% and an mp of 118-120°C.

[0154] The synthesis of N-(4-bromophenyl)-7-bromo-2H-benzopyran-3-carboxamide is shown in Formula 7:

[0155]

[0156] To a 25 mL round-bottom flask, add 0.765 g (3.0 mmol) of 7-bromo-2H-benzopyran-3-carboxylic acid and 6 mL of thionyl chloride. Heat under reflux for 1.5 hours and evaporate to dryness under reduced pressure. Dissolve the mixture in 10 mL of dry tetrahydrofuran to prepare Reaction Solution A. Dissolve 0.430 g (2.5 mmol) of 4-bromoaniline in 5 mL of dry tetrahydrofuran and add 0.42 mL of triethylamine (3.0 mmol) to prepare Reaction Solution B. Slowly add Reaction Solution A to Reaction Solution B while stirring in an ice bath. Stir the reaction at room temperature for 3 hours, then dilute with 100 mL of water. precipitate a solid, filter, wash with water, and air-dry to obtain the crude product. Column chromatography yields 0.851 g of a yellow solid (83.2% yield). mp 197-199°C. 1 H NMR (600MHz, DMSO-d6) δ10.20 (s, 1H), 7.70-7.66 (m, 2H), 7.52 (d, J = 8.8Hz, 2H), 7.45 (s, 1H) ,7.25(d,J=8.1Hz,1H),7.19(d,J=1.8Hz,1H),7.13(d,J=1.3Hz,1H),5.01(d,J=1.3Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ163.72(s),155.48(s),138.64(s),131.96(s),130.50(s),127.77(s),127.56(s),1 25.35(s),123.88(s),122.42(s),120.79(s),119.19(s),115.81(s),65.19(s).MS(ESI-TOF)m / z: 409.92[M( 79 Br 81 Br)+H] + .

[0157] Example 24: Synthesis of N-(4-fluorophenyl)-7-bromo-2H-benzopyran-3-carboxamide (D-26)

[0158] Following the procedure of Example 23, except that 4-bromoaniline was replaced with 4-fluoroaniline, 0.707 g of a white solid was obtained, with a yield of 81.2%. mp 183-185°C. 1 H NMR(600MHz,DMSO-d6)δ10.14(s,1H),7.71(dd,J=9.2,5.0Hz,2H),7.45-7.42(m,1H), 7.25(d,J=8.1Hz,1H),7.21-7.16(m,3H),7.13(d,J=1.8Hz,1H),5.01(d,J=1.4Hz,2H). 13C NMR (151MHz, DMSO-d6) δ162.45(s),157.64(d,J=240.4Hz),154.37(s),134.48(d,J=2.2Hz),129.35(s),126.60(s),126.37(s),12 4.23(s),122.68(s),121.30(d,J=7.6Hz),119.75(s),118.07(s),114.63(d,J=22.3Hz),64.14(s).MS(ESI-TOF)m / z: 348.00(M+H) + ,369.99(M+Na) + .

[0159] Example 25: Synthesis of N-(2,4-difluorophenyl)-7-bromo-2H-benzopyran-3-carboxamide (D-27)

[0160] Following the procedure of Example 23, except that 4-bromoaniline was replaced with 2,4-difluoroaniline, 0.652 g of a yellow powder was obtained, with a yield of 71.2%. mp 144-145°C. 1 H NMR(600MHz,DMSO-d6)δ9.96(s,1H),7.54(d,J=6.2Hz,1H),7.49(s,1H),7.35(s,1H),7.25(d,J=8.1Hz ,1H),7.19(dd,J=8.0,1.4Hz,1H),7.16-7.06(m,2H),5.00(d,J=1.3Hz,2H).HRMS(ESI)m / z:Calculated for C 16 H 12 BrF2NO2 + ([M+H] + )365.9936, Found 364.9936.

[0161] The compounds of the above embodiments all have 1 H NMR spectrum, 13 C NMR and MS patterns were used to confirm the structure of the compound. Due to space limitations, compound D-18 was used as an example. 1 H NMR spectrum, 13 C NMR and MS spectra are shown in Figure 1-Figure 3 .

[0162] The structural formulas of the target compounds of Examples 1-25 are shown in Table 1.

[0163] Table 1 Structures of target compounds of Examples 1-25

[0164]

[0165]

[0166]

[0167] Experiment 1: In vitro anti-radiation activity evaluation experiment

[0168] The compounds listed in Table 1 were evaluated for their anti-radiation activity. Compounds were treated with the compounds before irradiation of cultured cells in vitro. Cell proliferation was then assessed to assess the protective effects of the compounds on the irradiated cells. X-rays were applied to the cells using an RS2000 biological irradiator (Rad Source Technologies Inc.).

[0169] The compounds listed in Table 1 were prepared into stock solutions with concentrations of 1.0 mM and 2.0 mM respectively using DMSO and diluted with culture medium before use.

[0170] Logarithmically growing AHH-1 cells (human peripheral blood B lymphocytes, commercially available) were taken and gently pipetted to uniformly suspend the cells. The cells were counted using a cell counter and the cell density was adjusted to 1.5 × 10 cells / mL using RPMI-1640 medium (Gibco) containing 10% fetal bovine serum. 5 Cells were seeded into a 96-well plate at a density of 15,000 cells / mL (100 μL per well). The outermost wells of the 96-well plate were filled with 200 μL of purified water. A radiation control group (cells irradiated but not treated with the compound), a test compound group (cells irradiated and treated with the compounds listed in Table 1), a negative irradiation group (normal cell culture without irradiation), and a blank group (no cells, no compound, 100 μL of culture medium per well) were set up, with triplicate wells per group. After incubation of the 96-well plate in a 5% CO2 incubator for 4-6 hours, 50 μL of the corresponding compound stock solution diluted in culture medium was added to the test compound group (to achieve the desired final concentration). 50 μL of DMSO diluted in culture medium (containing the same DMSO concentration as the test compound group) was added to the negative irradiation and blank groups. After 24 hours in a 5% CO2 incubator, the cells were removed and placed in an RS2000 biological irradiator for 9.0 Gy irradiation. Continue incubating in a 5% CO2 incubator for 24 hours. Prepare a CCK-8 solution diluted with culture medium (the volume ratio of culture medium to CCK-8 stock solution (included in the kit) is 38:12). Add 50 μL of CCK-8 solution to each well and incubate in a 5% CO2 incubator for 2 hours. Measure the absorbance (OD) at 450 nm using a microplate reader to calculate the survival rate.

[0171] Survival rate (%) = (OD value of the test compound group - OD value of the blank group) / (OD value of the negative control group - OD value of the blank group) × 100%

[0172] Survival rate was expressed as mean ± standard deviation The survival rate data between groups were compared using SPSS 13.0 software for one-way analysis of variance, with P < 0.05 indicating statistically significant differences. Taking the compounds listed in Tables 2 to 4 as examples, the experimental results are shown in Tables 2 to 4.

[0173] Table 2 Radiation protection of compounds against 9.0 Gy irradiated AHH-1 cells (I)

[0174]

[0175] The experimental results in Table 2 show that at 20 μM, D-4, D-5, D-6, D-11, D-12, D-18, and D-19 can significantly increase the survival rate of irradiated cells compared to the radiation control group, showing significant anti-radiation activity. The inventors analyzed that the substituents on the aminophenyl ring of 2H-benzopyran-3-carboxanilide compounds have a significant influence on the activity, and halogen substitutions such as fluorine substitution, bromine substitution, etc. at the para position (R3) of the aminophenyl ring are beneficial to the anti-radiation activity; halogen substitution at the para position (R3) of the aminophenyl ring and alkyl or halogen substitution at the meta position (R4) of the aminophenyl ring are beneficial to the anti-radiation activity; halogen substitution at the ortho position (R5) or meta position (R2 or R4) of the aminophenyl ring are beneficial to the anti-radiation activity; halogen or alkoxy substitutions on the benzopyranyl group also show good anti-radiation activity.

[0176] Compounds D-4, D-5, D-6, D-11, D-12, D-18 and D-19 that can significantly improve cell survival in the experiment in Table 2 and subsequently synthesized compounds D-22, D-23, D-24, D-25, D-26 and D-27 were selected to evaluate their effects on cell survival at different concentrations. The final concentrations were set to 20 μM, 40 μM and 60 μM, the irradiation dose was 9.0 Gy, the experimental steps were the same as before, and the experimental results are shown in Tables 3 and 4 (Tables 2-4 show the results of three batches of experiments).

[0177] Table 3 Radiation protection of compounds against 9.0 Gy irradiated AHH-1 cells (II)

[0178]

[0179]

[0180] Table 4 Radiation protection of compounds against 9.0 Gy irradiated AHH-1 cells (III)

[0181]

[0182] The experimental results in Tables 3 and 4 show that the compounds can significantly improve the survival of irradiated cells compared with the radiation control group in the range of 20-60 μM. The protective effect of most compounds on irradiated AHH-1 cells increases with increasing drug concentration. Compound D-19 and compound D-23 have basically the highest cell survival rate values ​​at all three concentrations.

[0183] Experiment 2: Cytotoxicity evaluation experiment

[0184] The compounds listed in Table 1 were selected to evaluate their toxicity to AHH-1 cells in the range of 20-80 μM.

[0185] Take logarithmically growing AHH-1 cells, gently pipette to make the cells uniformly suspended, count them using a cell counter, and adjust the cell density to 1.5×10 5 Cells were seeded into a 96-well plate at 100 μL per well, i.e., 15,000 cells per well. The outermost wells of the 96-well plate were filled with 200 μL of purified water. A negative control group and a test compound group were set up. After 3 hours of cell culture, 50 μL of culture medium containing DMSO was added to the negative control group (prepared at a ratio of 6 μL DMSO dispersed in 194 μL culture medium), and 50 μL of the prepared 60 μM, 120 μM, 180 μM, and 240 μM test compound solutions were added to the test compound group (preparation process is the same as experiment 1), with the final concentrations of the compounds being 20 μM, 40 μM, 60 μM, and 80 μM, respectively. Three replicates were set up for each group. Another 96-well plate was used to set up a blank group, and 150 μL of culture medium containing DMSO was added to each well (the ratio is the same as before).

[0186] The 96-well plates seeded with cells and the blank 96-well plates were placed in a 37°C, 5% carbon dioxide (CO2) incubator for 24 hours. A certain amount of CCK-8 solution diluted with culture medium was prepared according to the instructions of the CCK-8 kit (the volume ratio of culture medium to CCK-8 stock solution (provided in the kit) was 38:12), and 50 μL of CCK-8 solution was added to each well. Incubate in a 5% carbon dioxide incubator for 3 hours, and measure the absorbance (OD) value at 450 nm using a microplate reader.

[0187] The cell survival rate was calculated by the following formula: survival rate (%) = (OD value of the test compound - OD value of the blank group) / (OD value of the negative control group - OD value of the blank group) × 100%, and the cell survival rate was expressed as the mean ± standard deviation. It is shown that one-way ANOVA was used for the comparison between groups of survival rate data with SPSS 13.0 software, and P < 0.05 indicates significant statistical significance. Taking D-5, D-18, D-19, D-22, D-23, D-24, D-25, D-26 and D-27 as examples, the experimental results are shown in Table 5.

[0188] Table 5 Effects of compounds on the proliferation of AHH-1 cells

[0189]

[0190] The experimental results show that within the range of 20 - 80 μM, the proliferation of AHH-1 cells is inhibited with the increase of the compound concentration. When the compound concentration is 20 - 60 μM, the compounds in Table 5 have little effect on the proliferation of AHH-1 cells.

[0191] Experiment 3: 30-day survival experiment of irradiated mice

[0192] The compounds listed in Table 1 were selected to carry out the 30-day survival experiment of irradiated mice. The positive drugs were Ex-RAD and nilestriol. Adult male C57 / BL mice were bred by Beijing Speifo Biotechnology Co., Ltd. The body weight of the mice was 20 - 22 g, and the experimental animal license number was: SCXK(Beijing)2019 - 0010. The mice were housed in a SPF-level laboratory, with 5 mice in each cage, fed with a special feed prepared for mice, and given free access to water. The temperature in the animal laboratory was maintained at 25 °C, and the relative humidity was 40% - 70%. The daily sunshine was 12 hours. The solvent was physiological saline containing 20 wt% hydroxypropyl-β-cyclodextrin (HPCD). Ex-RAD was formulated into a solution with a concentration of 30 mg / mL with the solvent; nilestriol was formulated into a solution with a concentration of 0.5 mg / mL with the solvent; the compounds were formulated into a suspension with a concentration of 20 mg / mL with the solvent.

[0193] Taking compounds D-19 and D-23 as examples, the experimental settings included a radiation control group (given physiological saline containing 20% HPCD), a nilestriol group at 5 mg / kg and an Ex-RAD group at 300 mg / kg (positive control group), a 200 mg / kg D-19 group and a 200 mg / kg D-23 group. The radiation control group, D-19 group, D-23 group and Ex-RAD group were administered by intraperitoneal injection, once at 24 hours before irradiation and once at 15 minutes before irradiation, 0.2 mL per mouse each time. The nilestriol group was administered by gavage, once at 24 hours before irradiation, 0.2 mL per mouse each time. The irradiation ray was 60The mice were irradiated with whole-body Go gamma rays at a dose of 8.6 Gy at a dose rate of 55.69 cGy / min. Survival was observed for 30 days, with the day of irradiation designated as day 0. Mice were weighed 1, 4, 7, 10, 14, 18, 22, and 30 days after irradiation, and weight changes were recorded over the 30-day period.

[0194] The mouse body weight and survival rate data were statistically analyzed using GraphPad Prism 5 software. The differences between the groups were analyzed by one-way analysis of variance, and P < 0.05 indicated that the differences were statistically significant. The results are shown in Tables 6 and 7.

[0195] Table 6 30-day survival experiment of mice

[0196] Group Number of mice before irradiation Number of mice at 30 days Mouse survival rate Radiation control group 10 0 0％ Nylestriol group 10 5 50％ Ex-RAD group 10 2 20％ D-19 Group 10 7 70％ D-23 Group 10 2 20％

[0197] Depend on Figure 1 As shown in Table 6, after whole-body irradiation of 8.6 Gy, mice in the radiation control group rapidly died within 10 days (combined with Table 7). The 30-day survival rate of mice in the positive drug Ex-RAD group was 20%, the nilestriol group was 50%, the D-23 group was 20%, and the D-19 group was 70%. Statistical analysis of the survival curves of mice with compound D-19 compared to the radiation control group, using GraphPad, revealed a statistically significant difference (p=0.0027), indicating that compound D-19 significantly increased the survival rate of irradiated mice and exhibited a certain anti-radiation effect.

[0198] Table 7 Weight changes of irradiated mice over 30 days

[0199]

[0200] As shown in Table 7, the weight changes of irradiated mice over 30 days after irradiation, the weight of mice in each group continued to decrease from 0 to 10 days after irradiation. The weight loss in the Ex-RAD group continued until the 18th day, and the weight loss in the compound D-19 and D-23 groups continued to the 7th and 10th days, respectively. After that, the weight of mice in each treatment group began to recover, and by the 22nd day, it had basically returned to the pre-irradiation level. Among them, the weight of mice in the D-19 group increased significantly more than that in the Ex-RAD group from the 10th day to the 30th day after irradiation. This result shows that D-19 has a good radiation protection effect at a dose of 200 mg / Kg, and can effectively improve the survival rate and recovery rate of mice exposed to lethal doses of radiation.

[0201] Experiment 4: Effects of Compounds on the Blood Count of Irradiated Mice

[0202] D-19 mice were selected for a 30-day blood count change experiment after irradiation, and Ex-RAD and nialestradiol were selected as positive agents. Adult male C57 / BL mice were bred by Beijing Sibeifu Biotechnology Co., Ltd., weighing 18-20 g. The experimental animal license number is SCXK (Beijing) 2019-0010. Mice were housed in an SPF-grade laboratory, with 5 mice per cage. They were fed a diet specially formulated for mice and had free access to water. The animal laboratory temperature was maintained at 25°C, the relative humidity was between 40% and 70%, and there was 12 hours of sunlight per day. The solvent was normal saline containing 20 wt% hydroxypropyl-β-cyclodextrin (HPCD). Ex-RAD was prepared into a solution with a concentration of 30 mg / mL using the solvent; nialestradiol was prepared into a solution with a concentration of 0.5 mg / mL using the solvent; and D-19 was prepared into a suspension with a concentration of 30 mg / mL using the solvent.

[0203] The experiment set up a radiation control group (administered with normal saline containing 20% ​​HPCD), a 5mg / kg nialestradiol group, a 300mg / kg Ex-Rad group (positive drug group), and a 300mg / kg D-19 group. The radiation control group, the D-19 group, and the Ex-Rad group were administered with intraperitoneal injection, once 24 hours and 15 minutes before irradiation, each time 0.2mL / animal. The nialestradiol group was administered with oral gavage, once 24 hours before irradiation, each time 0.2mL / animal. The irradiation dose was 60 Whole-body irradiation was performed using Go gamma irradiation, with a dose of 6.0 Gy and a dose rate of 72.81 cGy / min. (Appropriate irradiation conditions were selected to ensure survival of mice in the irradiated control group during the experimental period.) With the day of irradiation designated as day 0, tail vein blood samples were collected from mice before irradiation and on days 1, 4, 7, 10, 14, 18, 22, and 30 after irradiation to measure peripheral blood white blood cell counts, red blood cell counts, hemoglobin levels, and platelet counts. The experimental results are shown in Tables 8-11.

[0204] Table 8 Leukocyte counts of mice 30 days after irradiation

[0205]

[0206] The data in Table 8 show that the white blood cell counts of mice in all groups decreased significantly on days 1 and 4 after irradiation. On days 7, 10, 14, and 18 after irradiation, the white blood cell counts of mice in the radiation control, Ex-RAD, and nylestriol groups did not change significantly. However, the white blood cell count in the D-19 group continued to increase, and the magnitude of the increase was significantly greater than that in the radiation control and Ex-RAD groups from day 7 to day 22 after irradiation. On day 30 after irradiation, the white blood cell count of mice in all groups increased significantly, but the differences were not statistically significant.

[0207] Table 9 Red blood cell content of mice 30 days after irradiation

[0208]

[0209]

[0210] The data in Table 9 show that the red blood cell count of mice in each group decreased significantly after irradiation, and then began to recover on days 18, 22, and 30 after irradiation. The D-19 group showed the most improvement, with red blood cell counts significantly higher than those in the irradiated and Ex-RAD groups on days 1, 4, 7, 10, 14, 18, and 22. The rate of recovery in the D-19 group was significantly higher than in the other groups.

[0211] Table 10 Hemoglobin content in mice 30 days after irradiation

[0212]

[0213] The data in Table 10 show that the hemoglobin levels of mice in the radiation control and Ex-RAD groups decreased rapidly on days 1, 4, 7, 10, and 14 after irradiation, and began to rise again on day 18. The hemoglobin levels of mice in the D-19 and nilestriol groups began to rise again on day 10, and the hemoglobin levels on days 10, 14, and 18 were significantly higher than those in the radiation control group.

[0214] Table 11 Platelet counts of mice 30 days after irradiation

[0215]

[0216] The data in Table 11 show that platelet counts in all groups of mice decreased significantly on days 1, 4, and 7 after irradiation. Platelet counts in the irradiation control and Ex-RAD groups continued to decrease for 10 days after irradiation. Platelet counts in the nilestriol group were significantly higher than those in the irradiation control group on days 10, 14, and 18. Platelet counts in the D-19 group were significantly higher than those in the irradiation control and Ex-RAD groups on days 10 and 14.

[0217] Analysis of the white blood cell count, red blood cell count, hemoglobin content, and platelet count of irradiated mice 30 days after irradiation revealed that all indicators experienced a period of decline and recovery after irradiation, returning to pre-irradiation levels by day 30. This suggests that 6.0 Gy whole-body irradiation damaged the hematopoietic system of mice within 30 days of irradiation, but was not fatal. Compared with the irradiation control group, the nialestradiol and D-19 groups significantly improved these four indicators, with the compound D-19 group showing particularly significant improvement. This suggests that it promotes the recovery of the hematopoietic system in irradiated mice and has the potential to be developed as an anti-radiation drug.

[0218] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A substituted 2H-benzopyran-3-carboxanilide compound, characterized in that: Its structure is Formula I: Wherein, R1 is selected from halogen and alkoxy; R2, R4, and R5 are independently selected from any one of H and halogen; R3 is selected from halogen; and the alkoxy is methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, or tert-butoxy.

2. The substituted 2H-benzopyran-3-carboxanilide compound according to claim 1, characterized in that: The alkoxy group is selected from methoxy, ethoxy or tert-butoxy.

3. The substituted 2H-benzopyran-3-carboxanilide compound according to claim 1 or 2, characterized in that: Includes any of the following: N-(4-bromophenyl)-7-methoxy-2H-chromen-3-carboxamide; N-(4-Fluorophenyl)-7-methoxy-2H-chromen-3-carboxamide; N-(2,4-difluorophenyl)-7-methoxy-2H-chromen-3-carboxamide; N-(4-bromophenyl)-7-bromo-2H-benzopyran-3-carboxamide; N-(4-Fluorophenyl)-7-bromo-2H-chromen-3-carboxamide; N-(2,4-Difluorophenyl)-7-bromo-2H-chromen-3-carboxamide.

4. A composition comprising the substituted 2H-benzopyran-3-carboxanilide compound according to any one of claims 1 to 3, its geometric isomers, its pharmaceutically acceptable salts, its hydrates or its solvates, and a pharmaceutically acceptable carrier or excipient.

5. A method for preparing the substituted 2H-benzopyran-3-carboxanilide compound according to any one of claims 1 to 3, characterized in that: Here are the steps: Step (1): heating the compound of formula II and thionyl chloride under reflux until the compound of formula II is completely converted, as shown in the reaction formula 1; evaporating under reduced pressure to dryness; and dissolving the residue in an appropriate solvent to prepare reaction solution A; Step (2): dissolving the compound of formula III in a suitable solvent and adding an organic base to prepare a reaction solution B; Step (3): Place the reaction solution B in an ice bath and stir, add the reaction solution A dropwise, and then react at room temperature until completion to obtain the compound shown in formula I.

6. The method according to claim 5, characterized in that The solvent is selected from one or more of dichloromethane, tetrahydrofuran, N,N-dimethylformamide and pyridine.

7. The method according to claim 5 or 6, characterized in that: The organic base is selected from one or more of 4-dimethylaminopyridine, triethylamine, pyridine and N-methylmorpholine.

8. Use of the substituted 2H-benzopyran-3-carboxanilide compound according to any one of claims 1 to 3 or the composition according to claim 4 in the preparation of a drug for preventing and / or treating radiation damage.

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