CN113200938A

CN113200938A - Benzisothiazole hypoxia inducible factor 2 agonist compound or pharmaceutically acceptable salt thereof, preparation method and application

Info

Publication number: CN113200938A
Application number: CN202110551171.3A
Authority: CN
Inventors: 张晓进; 余砚成; 刘思萌; 余权威; 吴晨阳
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-05-20
Filing date: 2021-05-20
Publication date: 2021-08-03
Anticipated expiration: 2041-05-20
Also published as: WO2022242462A1; CN113200938B

Abstract

The invention discloses a benzisothiazole hypoxia inducible factor 2 agonist compound or pharmaceutically acceptable salt thereof, wherein the compound can stimulate the transcriptional activity of hypoxia inducible factor 2 and enhance the generation and secretion of erythropoietin so as to promote the generation of red blood cells; the benzisothiazole hypoxia inducible factor 2 agonist compound or the pharmaceutically acceptable salt thereof prepared by the invention can be combined with prolyl hydroxylase inhibitor to play a synergistic role in improving HIF-2 transcription activity, and can be used for treating hypoxia inducible factor 2 related diseases, such as ischemic diseases and the like.

Description

Benzisothiazole hypoxia inducible factor 2 agonist compound or pharmaceutically acceptable salt thereof, preparation method and application

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a benzisothiazole hypoxia inducible factor 2 agonist compound or pharmaceutically acceptable salt thereof, and also relates to a preparation method and application of the compound.

Background

Anemia is a common disease that manifests as an insufficient level of red blood cells in the blood to supply oxygen transport in the body. 2-3X 10 is required for adults¹³The red blood cells are used for ensuring oxygen transmission, but the service life of the red blood cells is only 100-120 days. The erythropoiesis process takes place mainly in the bone marrow, and hemoglobin, the main constituent of erythrocytes, consists of globin and heme. In erythropoiesis, Erythropoietin (EPO) is the most critical hormone in erythropoiesis, while ferrous ions are the important raw material of heme, and the levels of both are critical for erythropoiesis.

Hypoxia Inducible Factor (HIF) is a class of transcription factor that responds to hypoxia, and its major 3 subtypes are HIF-1, HIF-2 and HIF-3, respectively. HIF is a heterodimeric protein composed of two subunits of hypoxia-inducible factor α (HIF- α) and stably expressed aromatic hydrocarbon receptor nuclear translocator (ARNT). Under normoxic conditions, certain proline residues in the oxygen-sensitive HIF- α subunit (HIF-1 α: Pro402 and Pro 564; HIF-2 α: Pro405 and Pro 531; HIF-3 α: Pro492) are hydroxylated by Prolyl Hydroxylase (PHD). Hydroxylated HIF- α is recognized by von Hippel-Lindau (VHL), which recruits E3 ubiquitin ligase complex to degrade HIF- α through the proteasome pathway. In a hypoxic environment, since the hydroxylation modification process of PHD requires oxygen to participate, the reduction of oxygen concentration will result in the inhibition of HIF-alpha hydroxylation modification by PHD. VHL has difficulty recognizing non-hydroxylated HIF- α, which leads to increased HIF- α levels in the cell, which dimerizes with ARNT to form HIF protein that enters the nucleus to activate transcriptional functions and thereby relieve hypoxia. Compared with HIF-1 alpha and HIF-3 alpha, HIF-2 alpha is an ideal target for treating related diseases due to its specific expression in kidney cells. When the body is lack of oxygen, HIF-2 transcription activates its downstream genes, induces various physiological processes (such as promoting erythropoiesis, enhancing oxygen metabolism, promoting angiogenesis, etc.) to promote the body to adapt to the lack of oxygen.

EPO production in adults occurs primarily in the kidney, which is directly regulated by HIF-2 (nat. rev. nephrol.11,394-410 (2015)). Increasing HIF-2 transcriptional activity can directly increase EPO synthesis and secretion in kidney cells. In addition, HIF-2 can also regulate proteins such as duodenal cytochrome b reductase (DCYTB), divalent metal transporter 1(DMT1), and ferroportin (FPN1) to promote the uptake of iron in food and the transport of iron in vivo, thereby increasing the ferrous ion content in vivo (j.biol.chem.286,4090-4097 (2011)).

Currently, the small molecules that can increase HIF-2 activity are the best known PHD inhibitors, which can inhibit PHD hydroxylation-modifying activity to prevent HIF- α degradation, ultimately increasing HIF levels in cells (j.med.chem.63,10045-10060 (2020)). In recent years 5 PHD inhibitors have been approved on the market for the treatment of renal anemia, namely, Rosxadustat (FG-4592), Vadavista (Vadadustat, AKB-6548), Dadostat (Daprodustat, GSK1278863), Ennaxsat (Enarodustat, JTZ-951), and Moristal (Molidustat, BAY 85-3934). However, vatacostat exhibited cardiovascular toxicity in later clinical trials. PHD inhibitors are poorly selective in stabilizing HIF- α, which can simultaneously elevate the levels of three HIF subtypes, detrimental to the specific activation of HIF-2.

Recently, two HIF-2 alpha direct agonists (nat. chem. biol.15, 367-376 (2019)) were discovered, respectively

The mechanism of the HIF-2 alpha agonist is different from that of a PHD inhibitor, the HIF-2 alpha agonist can be specifically combined with the PASB structure domain of HIF-2 alpha, the side chain conformation of an amino acid residue Tyr281 in a cavity is changed, the side chain conformation of the amino acid residue Tyr281 is promoted to form a hydrogen bond with Tyr456 in an ARNT subunit, the dimerization of the HIF-2 alpha subunit and the ARNT subunit is enhanced, and the HIF-2 content in cells is finally improved. The cavity exists only in HIF-2 alpha, and similar cavities do not exist in PASB domains of other HIF-alpha subtypes, so that HIF-2 alpha agonists can specifically activate HIF-2 pathways, however, the currently reported HIF-2 alpha agonists have extremely weak activity in HIF-2 agonism and cannot achieve expected effects when being used.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problem that the activity of the prior HIF-2 alpha agonist is weak when the HIF-2 is excited, the invention provides a benzisothiazole hypoxia inducible factor 2 agonist compound or pharmaceutically acceptable salt thereof; also provides a preparation method and application of the compound.

The technical scheme is as follows: the invention discloses a benzisothiazole hypoxia inducible factor 2 agonist compound or pharmaceutically acceptable salt thereof, which has a general formula as shown in formula I:

wherein X represents-S-or

L represents-NH-or

R¹And R²Each independently represents hydrogen, halogen radical, C₁-C₄Halogen-substituted alkyl;

preferably, when L is

When Ar represents a five-membered aromatic heterocycle or

R³Represents a substitution mode which at least contains one non-hydrogen substituent and has no chlorine substitution at the 4 position, wherein the substituents are respectively and independently halogen and C₁-C₄Alkyl of (C)₁-C₄Alkoxy group of (C)₁-C₄Halogen-substituted alkyl;

preferably, when L is-NH-, Ar represents

Wherein R is⁴And R⁵Each independently represents halogen or C₁-C₄Alkyl of (C)₁-C₄Alkoxy group of (C)₁-C₄Halogen-substituted alkyl.

Preferably, the salts include pharmaceutically acceptable metal salts such as sodium, potassium, lithium, calcium, magnesium, aluminum and zinc salts; carbonates and bicarbonates of pharmaceutically acceptable metal cations such as sodium, potassium, lithium, calcium, magnesium, aluminum and zinc; pharmaceutically acceptable organic primary, secondary and tertiary amines including aliphatic, aromatic, aliphatic and hydroxyalkylamines such as methylamine, ethylamine, 2-hydroxyethylamine, diethylamine, triethylamine, ethylenediamine, ethanolamine, diethanol.

The invention also discloses a preparation method of the compound,

when L is-NH-and X is S in the general formula (I), the preparation method of the compound comprises the following steps:

wherein R is¹、R²Ar is as defined above.

And carrying out substitution reaction on the compound 2 and arylamine to obtain a compound 3. The reaction temperature is preferably 20-60 ℃, and the reaction solvent can be selected from acetone, acetonitrile, N-dimethylformamide, tetrahydrofuran, toluene and the like. Alkali such as sodium hydroxide, potassium hydroxide, sodium hydride, potassium hydride, etc. should also be added in the reaction.

And carrying out a ring closing reaction of the compound 3 with the participation of elemental sulfur to obtain a compound of the general formula I. The reaction temperature is preferably 120-200 ℃, the reaction solvent is preferably a mixed solution of dimethyl sulfoxide, toluene, N-methylpyrrolidone, N-dimethylformamide and the like, and alkali such as potassium carbonate, sodium carbonate, cesium carbonate, potassium phosphate, potassium dihydrogen phosphate and the like is also added in the reaction.

When L in the formula (I) is

When X is S, the preparation method of the compound comprises the following steps:

wherein R is¹、R²Ar is as defined above.

Condensing the compound 4 with substituted carboxylic acid to obtain the compound shown in the general formula (I). The reaction temperature is 20-60 ℃. The solvent used in the reaction can be selected from dichloromethane, trichloromethane, acetone, acetonitrile, dioxane, etc. Organic or inorganic base such as potassium carbonate, sodium carbonate, cesium carbonate, triethylamine, pyridine, etc. may be added during the reaction. Condensing agents such as Dicyclohexylcarbodiimide (DCC), Diisopropylcarbodiimide (DIC), 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), benzotriazol-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU), and the like, may also be added to the reaction.

When X in the formula (I) is

The preparation method of the compound comprises the following steps:

wherein R is¹、R²Ar and L are as defined above.

And (3) carrying out oxidation reaction on the compound 5 to obtain the compound shown in the general formula (I). The reaction temperature is preferably-78 to-10 ℃, the reaction solvent is preferably dichloromethane, trichloromethane, tetrahydrofuran, sulfuric acid and the like, and an oxidant such as m-chloroperoxybenzoic acid, sulfuric acid, nitric acid and the like is also added in the reaction.

The invention also discloses the application of the benzisothiazole hypoxia inducible factor 2 agonist compound or pharmaceutically acceptable salt thereof in preparing a medicament for treating ischemic diseases.

Furthermore, the ischemic diseases comprise anemia, stroke and myocardial ischemia caused by ischemia, and the clinical dosage of the compound of the invention is 0.0l mg-1000 mg/day.

Further, the use of a prolyl hydroxylase inhibitor in combination with a prolyl hydroxylase inhibitor for the manufacture of a medicament for the treatment of ischemic diseases, said prolyl hydroxylase inhibitor comprising roxasistat (Roxadustat, FG-4592)

Vandastat (AKB-6548)

Daprodustat (Daprodustat, GSK1278863)

Ennasstat (Enarodustat JTZ-951)

Molisstat (Molidustat, BAY 85-3934)

The invention designs and synthesizes a novel benzisothiazole HIF-2 alpha agonist, as shown in figure 1, the invention analyzes by a molecular docking methodThe binding mode of HIF-2 alpha agonist M1002 and HIF-2 with better activity, the-SO of M1002₂The oxygen atom in the N-structure can form hydrogen bond promotion combination with Tyr307 in HIF-2 alpha-PASB structure domain as hydrogen bond acceptor, the hydrogen bond is an important combination characteristic of M1002 and protein, but only one side oxygen atom can form hydrogen bond with Tyr307, and the prompt is that-SO is removed₂The oxygen atom on N can reduce the solvation of the small molecule to finally strengthen the combination of the small molecule and the protein, and after the oxygen is removed, the sulfur and nitrogen atoms on the isothiazole ring can also form a hydrogen bond with Tyr 307; the invention removes-SO in benzisothiazole ring₂The special structural characteristics brought by one or two oxygen atoms in the N-structure can effectively activate HIF-2 signal channels, thereby promoting the transcriptional activation of HIF-2 downstream genes.

When the benzisothiazole HIF-2 alpha agonist or the pharmaceutically acceptable salt thereof and the PHD inhibitor are used together, the dosage of the benzisothiazole HIF-2 alpha agonist and the PHD inhibitor is 0.01mg-1000 mg/day respectively and can also deviate from the range according to the condition or dosage form; the compound and the PHD inhibitor may be administered separately, simultaneously or prepared as a complex for simultaneous administration. In vitro and in vivo pharmacodynamic experiments, the compound can be combined with a PHD inhibitor to play a synergistic role in promoting HIF-2 activation. PHD inhibitors and HIF-2 α agonists have different mechanisms in increasing HIF-2 transcriptional activity: PHD inhibitors prevent HIF-2 α degradation in cells by inhibiting the hydroxylation modifying activity of PHD on HIF-2 α, ultimately increasing HIF-2 complex levels to enhance HIF-2 transcriptional activity; HIF-2 alpha agonists enhance HIF-2 alpha and ARNT subunit dimerization by binding to the HIF-2 alpha-PASB domain, ultimately increasing HIF-2 levels to enhance HIF-2 transcriptional activity. The combination of the two can not only stabilize HIF-2 alpha level, but also improve the dimerization capacity of HIF-2 alpha and ARNT, and synergistically improve the HIF-2 complex content.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages.

1. The benzisothiazole hypoxia inducible factor 2 agonist compound prepared by the invention has good HIF-2 agonistic activity at the cellular level, and representative compounds 2 and 18 can respectively promote the HIF-2 transcriptional activity to 349% and 359%, which is far superior to the optimal transcriptional activity 141% of M1002 reported in the prior art;

2. the HIF-2 alpha agonist and the PHD inhibitor are combined to activate an HIF-2 pathway, compared with the single use of the PHD inhibitor, the benzisothiazole derivative and the PHD inhibitor are combined to improve the EPO level in the blood of a mouse more effectively and avoid the defect of subtype selectivity of the PHD inhibitor; the compound has certain application prospect in ischemic diseases such as renal anemia, cerebral arterial thrombosis and the like.

Drawings

FIG. 1: molecular docking analysis of the binding pattern of HIF-2 alpha agonist M1002 to HIF-2;

FIG. 2: luciferase reporter gene assays test the synergistic effect of compound 2 and PHD inhibitors on HIF-2 transcriptional activity;

FIG. 3: effect of Compound 2 on plasma EPO of mice when administered in combination with the PHD inhibitor AKB-6548.

Detailed Description

The technical scheme of the invention is further explained by combining the attached drawings.

Example 1

N- (3, 5-dibromophenyl) benzo [ d ] isothiazol-3-amine (1)

(1) Preparation of 2-chloro-N- (3, 5-dibromophenyl) benzamide

Sodium hydride (31.3mg,1.2mmol) and 3, 5-dibromoaniline (276.0mg,1.1mmol) were added to a solution of o-chlorobenzonitrile (138.9mg,1mmol) in dry dimethylsulfoxide (5mL) under ice-bath conditions. After stirring the reaction for 0.5 hour in ice bath, the reaction was gradually returned to room temperature and kept at room temperature for 24 hours. After completion of the reaction, the reaction mixture was quenched by adding water (5 mL). The reaction solution was adjusted to pH 3.0 using dilute hydrochloric acid to protonate the desired product and dissolve it in the aqueous phase, which was washed with dichloromethane (3X 10mL) to remove impurities from the organic phase. The aqueous phase was adjusted to pH 7.0 using saturated sodium bicarbonate solution and extracted with dichloromethane (3X 10 mL). And combining organic phases, drying the organic phases by using anhydrous sodium sulfate, and evaporating the solvent under reduced pressure to obtain a brown oily crude product, namely a product, wherein the brown oily crude product is directly used for the next reaction without further purification.

(2) Synthesis of N- (3, 5-dibromophenyl) benzo [ d ] isothiazol-3-amine

To a mixed solution of 2-chloro-N- (3, 5-dibromophenyl) benzamide (388.5mg,1mmol) in dimethylsulfoxide and toluene (5mL, v: v ═ 1:1) were added elemental sulfur (143.9mg,4.5mmol) and tripotassium phosphate (318.4mg,1.5 mmol). The reaction was carried out at 160 ℃ for 24 hours under nitrogen protection. After completion of the reaction, the reaction mixture was quenched by adding water (10mL), the elemental sulfur was removed by suction filtration, and the filtrate was extracted with ethyl acetate (15 mL. times.3) and washed with saturated brine (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent and purified by column chromatography (petroleum ether: ethyl acetate: 10:1) to give the title compound as a white solid (107.7mg, 28.2%). mp 165.3-166.2 ℃.¹H-NMR(400MHz,DMSO-d₆)δ9.92(s,1H),8.43(d,J＝8.1Hz,1H),8.24(d,J＝1.7Hz,2H),8.10(d,J＝8.1Hz,1H),7.67–7.51(m,2H),7.36(d,J＝1.8Hz,1H).¹³C-NMR(101MHz,DMSO-d₆)δ154.68,150.31,144.28,129.12,127.60,125.78,125.10,123.02,122.85,121.15,119.12,40.60,40.39,40.19,39.98,39.77,39.56,39.35.HRMS(ESI):calcd.for C₁₃H₈Br₂N₂S[M+H]⁺＝382.8848,found 382.8854.t_R＝10.564min,HPLC purity:96.78％。

Example 2

N- (3, 5-Dimethoxyphenyl) benzo [ d ] isothiazol-3-amine (2)

(1) Preparation of 2-chloro-N- (3, 5-dimethoxyphenyl) benzamide

To a solution of o-chlorobenzonitrile (1g,7.3mmol) in dry N, N-dimethylformamide (5mL) under ice bath conditions were added 3, 5-dimethoxyaniline (1.23g,8.0mmol) and sodium hydride (0.524g,22.0 mmol). The reaction was carried out at room temperature for 1 hour. After completion of the reaction, the reaction mixture was quenched by adding ice water (25 mL). The reaction solution was adjusted to pH 3.0 with dilute hydrochloric acid. The aqueous phase was washed with dichloromethane (3X 10mL) to remove impurities from the organic phase. The aqueous phase was adjusted to pH 7.0 with saturated sodium bicarbonate solution and extracted with dichloromethane (3X 10 mL). The organic phases were combined and the solvent was evaporated under reduced pressure to give a red oily product. The reaction was carried out without further purification.

(2) Preparation of N- (3, 5-dimethoxyphenyl) benzo [ d ] isothiazol-3-amine

2-chloro-N- (3, 5-dimethoxyphenyl) benzamide (700mg,2.4mmol) was dissolved in a mixed solution of dimethyl sulfoxide and toluene (12mL, v: v ═ 1:1), and precipitated sulfur (2.15g,8.0mmol) and tripotassium phosphate (0.71g,3.6mmol) were added in this order. The reaction was heated at 135 ℃ for 36 hours under nitrogen blanket. After the reaction, the reaction solution was filtered with suction and the filtrate was retained. The toluene in the filtrate was distilled off under reduced pressure, and water (25mL) was added to the residual liquid. The mixture was extracted with ethyl acetate (3X 25mL) and washed with saturated brine (15 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate: 10:1) to give the desired product as a white solid (427.9mg, 62.1%). mp 144.2-144.9 ℃.¹H-NMR(400MHz,DMSO-d₆)δ9.62(s,1H),8.51(d,J＝8.1Hz,1H),8.08(d,J＝7.9Hz,1H),7.58(dt,J＝26.8,7.2Hz,2H),7.30(s,2H),6.19(s,1H),3.79(s,6H).¹³C-NMR(101MHz,DMSO-d₆)δ161.10,155.48,150.13,143.27,128.87,127.87,124.81,123.10,121.00,96.67,93.88,55.52.HRMS(ESI):calcd.for C₁₅H₁₄N₂O₂S[M+H]⁺＝287.0849,found 287.0854.t_R＝6.014min,HPLC purity:98.74％。

Example 3

N- (3, 5-dimethylphenyl) benzo [ d ] isothiazol-3-amine (3)

(1) Preparation of 2-chloro-N- (3, 5-dimethylphenyl) benzamide

Sodium hydride (31.3mg,1.2mmol) and 3, 5-dimethylaniline (133.3mg,1.1mmol) were added to a solution of o-chlorobenzonitrile (138.9mg,1mmol) in dry dimethylsulfoxide (5mL) under ice-bath conditions. The reaction solution was allowed to react for 0.5 hour under ice-bath conditions, then returned to room temperature, and allowed to react for 24 hours while being maintained at room temperature. After completion of the reaction, the reaction mixture was quenched by adding water (5 mL). The reaction solution was adjusted to pH 3.0 with dilute hydrochloric acid. The aqueous phase was washed with dichloromethane (3X 10mL) to remove impurities from the organic phase. The aqueous phase was adjusted to pH 7.0 with saturated sodium bicarbonate solution and extracted with dichloromethane (3X 10 mL). The organic phases were combined and the solvent was evaporated under reduced pressure to give a tan oil crude which was used directly in the next reaction without further purification.

(2) Synthesis of N- (3, 5-dimethylphenyl) benzo [ d ] isothiazol-3-amine

To a mixed solution of 2-chloro-N- (3, 5-dimethylphenyl) benzamide (258.7mg,1mmol) in dimethylsulfoxide and toluene (5mL, v: v ═ 1:1) were added elemental sulfur (143.9mg,4.5mmol) and tripotassium phosphate (318.4mg,1.5mmol) in this order. The reaction solution was reacted at 160 ℃ for 24 hours under nitrogen protection. After completion of the reaction, the reaction mixture was quenched by adding water (10 mL). The reaction mixture was suction-filtered to remove sulfur, and the filtrate was extracted with ethyl acetate (15 mL. times.3) and washed with saturated brine (10 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate: 10:1 to 5:1) to give the title compound as a white solid (143.6mg, 56.5%). mp 123.0-123.8 ℃.¹H-NMR(300MHz,DMSO-d₆)δ9.44(s,1H),8.48(d,J＝8.2Hz,1H),8.06(d,J＝8.1Hz,1H),7.64–7.57(m,3H),7.51(t,J＝7.2Hz,1H),6.63(s,1H),2.28(s,6H).¹³C-NMR(75MHz,DMSO-d₆)δ155.63,150.14,141.59,138.03,128.79,127.96,124.76,123.36,123.20,120.96,115.94,40.79,40.51,40.23,39.96,39.68,39.40,39.12,21.77.HRMS(ESI):calcd.for C₁₅H₁₄N₂S[M+H]⁺＝255.0950,found 255.0953.t_R＝5.119min,HPLCpurity:99.17％。

Example 4

N- (3, 5-difluorophenyl) benzo [ d ] isothiazol-3-amine (4)

(1) Preparation of 2-chloro-N- (3, 5-difluorophenyl) benzamide

Sodium hydride (31.3mg,1.2mmol) and 3, 5-difluoroaniline (142.0mg,1.1mmol) were added to a dry solution of o-chlorobenzonitrile (138.9mg,1mmol) in dimethylsulfoxide (5mL) under ice-bath conditions. Under the condition of ice bath, the room temperature is naturally recovered after 0.5 hour of reaction, and the room temperature is kept for reaction for 24 hours. The reaction was quenched by addition of water (5 mL). Then, the pH of the reaction solution is adjusted to 3.0 by using dilute hydrochloric acid, so that the target product is protonated and dissolved in a water phase. The aqueous phase was washed with dichloromethane (3X 10mL) to remove impurities from the organic phase. The aqueous phase was adjusted to pH 7.0 with saturated sodium bicarbonate solution and extracted with dichloromethane (3X 10 mL). The organic phases were combined and the solvent was evaporated under reduced pressure to give a tan crude oil which was used directly in the next reaction without further purification.

(2) Synthesis of N- (3, 5-difluorophenyl) benzo [ d ] isothiazol-3-amine

To a mixed solution of 2-chloro-N- (3, 5-difluorophenyl) benzamide (266.0mg,1mmol) in dimethyl sulfoxide and toluene (5mL, v: v ═ 1:1) were added elemental sulfur (143.9mg,4.5mmol) and tripotassium phosphate (318.4mg,1.5mmol) in that order. The reaction was carried out at 160 ℃ for 24 hours under nitrogen protection. After completion of the reaction, the reaction mixture was quenched by adding water (10 mL). The elemental sulfur was removed by suction filtration, and the filtrate was extracted with ethyl acetate (15 mL. times.3) and washed with saturated brine (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent and purified by column chromatography (petroleum ether: ethyl acetate 15:1) to give the title compound as a white solid (123.4mg, 47.1%). mp 159.2-160.4 ℃.¹H-NMR(300MHz,DMSO-d₆)δ10.03(s,1H),8.45(d,J＝8.0Hz,1H),8.10(d,J＝8.1Hz,1H),7.71(dd,J＝10.5,2.2Hz,2H),7.67–7.51(m,2H),6.77(tt,J＝9.3,2.3Hz,1H).¹³C-NMR(75MHz,DMSO-d₆)δ164.84,164.63,161.63,161.42,154.88,150.31,144.34,144.15,143.96,129.08,127.57,125.03,123.04,121.13,100.93,100.54,96.82,96.47,96.12,40.79,40.51,40.24,39.96,39.68,39.40,39.13.HRMS(ESI):calcd.for C₁₃H₈F₂N₂S[M+H]⁺＝263.0449,found263.0451.t_R＝5.613min,HPLC purity:99.05％。

Example 5

N- (3, 5-dichlorophenyl) benzo [ d ] isothiazol-3-amine (5)

(1) Preparation of 2-chloro-N- (3, 5-dichlorophenyl) benzamide

Sodium hydride (31.3mg,1.2mmol) and 3, 5-dichloroaniline (176.0mg,1.1mmol) were added to a solution of o-chlorobenzonitrile (138.9mg,1mmol) in dry dimethylsulfoxide (5mL) under ice-bath conditions. After the reaction was carried out for 0.5 hour in an ice bath, the temperature was gradually returned to room temperature, and the reaction was maintained at room temperature for 24 hours. After completion of the reaction, water (5mL) was added to the reaction mixture to quench the reaction. The reaction solution was adjusted to pH 3.0 using dilute hydrochloric acid to protonate the desired product and dissolve it in the aqueous phase, which was washed with dichloromethane (3X 10mL) to remove impurities from the organic phase. The aqueous phase was adjusted to pH 7.0 using saturated sodium bicarbonate solution and extracted with dichloromethane (3X 10 mL). And combining organic phases, drying the organic phases by using anhydrous sodium sulfate, and evaporating the solvent under reduced pressure to obtain a target product which is a tan oily crude product, namely the product, wherein the target product is directly used for the next reaction without further purification.

(2) Synthesis of N- (3, 5-dichlorophenyl) benzo [ d ] isothiazol-3-amine

To a mixed solution of 2-chloro-N- (3, 5-dichlorophenyl) benzamide (299.6mg,1mmol) in dimethylsulfoxide and toluene (5mL, v: v ═ 1:1) were added elemental sulfur (143.9mg,4.5mmol) and tripotassium phosphate (318.4mg,1.5 mmol). The reaction was carried out at 160 ℃ for 24 hours under nitrogen protection. After the reaction, water (10mL) was added to the reaction solution to quench, the solution was filtered under suction to remove elemental sulfur, and the filtrate was extracted with ethyl acetate (15 mL. times.3) and washed with saturated brine (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and purified by column chromatography (petroleum ether: ethyl acetate: 10:1 to 5:1) to give the target compound as a white solid (150.5mg, 51.2%). mp 175.4-176.3 ℃.¹H-NMR(300MHz,DMSO-d₆)δ9.93(s,1H),8.41(d,J＝8.1Hz,1H),8.12–8.00(m,3H),7.56(dt,J＝25.7,7.2Hz,2H),7.08(s,1H).¹³C-NMR(75MHz,DMSO-d₆)δ155.24,150.83,144.44,135.03,129.57,128.12,125.55,123.54,121.60,120.98,116.45,41.33,41.05,40.77,40.49,40.21,39.94,39.66.HRMS(ESI):calcd.for C₁₃H₈Cl₂N₂S[M+H]⁺＝294.9858,found 294.9853.t_R＝2.218min,HPLC purity:100％。

Example 6

3- ((3, 5-dimethylphenyl) amino) benzo [ d ] isothiazole 1-oxide (6)

At-70 deg.C in N- (3, 5-dimethylphenyl) benzo [ d ]]To a solution of isothiazol-3-amine (100mg,0.39mmol,1.0eq) in dichloromethane (5mL) was slowly added dropwise a solution of m-chloroperoxybenzoic acid (87.89mg,0.43mmol,1.1eq) in dichloromethane (10 mL). After the dropwise addition, the reaction was returned to room temperature, and the reaction was stirred for 0.5 hour. After completion of the reaction, the reaction mixture was adjusted to neutral by adding a saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (3X 15 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent and purified by column chromatography (petroleum ether: ethyl acetate: 3:1) to give the title compound as a white solid (42.5mg, 40.3%). mp 236.7-237.6 ℃.¹H-NMR(400MHz,DMSO-d₆)δ10.25(s,1H),8.48(s,1H),8.10(d,J＝7.7Hz,1H),7.81(t,J＝11.5Hz,2H),7.64(s,2H),6.85(s,1H),2.32(s,6H).HRMS(ESI):calcd.for C₁₅H₁₄N₂OS[M+H]⁺＝271.0900,found 271.0899.t_R＝6.377min,HPLC purity:100％。

Example 7

N- (3, 5-bis (trifluoromethyl) phenyl) benzo [ d ] isothiazol-3-amine (7)

(1) Preparation of 2-chloro-N- (3, 5-bistrifluoromethylphenyl) benzamide

Sodium hydride (31.3mg,1.2mmol) and 3, 5-bistrifluoromethylaniline (252.0mg,1.1mmol) were added to a solution of o-chlorobenzonitrile (138.9mg,1mmol) in dry dimethylsulfoxide (5mL) under ice-bath conditions. After the reaction was carried out for 0.5 hour in an ice bath, the temperature was gradually returned to room temperature, and the reaction was carried out for 24 hours while maintaining the room temperature. After completion of the reaction, the reaction mixture was quenched by adding water (5 mL). The reaction solution was adjusted to pH 3.0 using dilute hydrochloric acid to protonate the desired product and dissolve it in the aqueous phase, which was washed with dichloromethane (3X 10mL) to remove impurities from the organic phase. The aqueous phase was adjusted to pH 7.0 using saturated sodium bicarbonate solution and extracted with dichloromethane (3X 10 mL). And combining organic phases, drying the organic phases by using anhydrous sodium sulfate, and evaporating the solvent under reduced pressure to obtain a brown oily crude product, namely a product, wherein the brown oily crude product is directly used for the next reaction without further purification.

(2) Synthesis of N- (3, 5-bis (trifluoromethyl) phenyl) benzo [ d ] isothiazol-3-amine

To a mixed solution of 2-chloro-N- (3, 5-bistrifluoromethylphenyl) benzamide (366.7mg,1mmol) in dimethylsulfoxide and toluene (5mL, v: v ═ 1:1) were added elemental sulfur (143.9mg,4.5mmol) and tripotassium phosphate (318.4mg,1.5 mmol). The reaction was heated in an oil bath at 160 ℃ under nitrogen with stirring and refluxing for 24 hours. After completion of the reaction, the reaction mixture was quenched by adding water (10 mL). The elemental sulfur was removed by suction filtration, and the filtrate was extracted with ethyl acetate (15 mL. times.3) and washed with saturated brine (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent and purified by column chromatography (petroleum ether: ethyl acetate: 10:1 to 5:1) to give the title compound as a white solid (193.3mg, 53.4%). mp 163.4-164.8.3 ℃.¹H-NMR(300MHz,DMSO-d₆)δ10.36(s,1H),8.74–8.68(m,2H),8.50(dt,J＝8.1,1.0Hz,1H),8.17(dt,J＝8.1,1.0Hz,1H),7.72–7.58(m,3H).¹³C-NMR(75MHz,DMSO-d₆)δ154.74,150.33,143.39,131.82,131.39,130.96,130.53,129.18,127.50,125.76,125.16,122.99,122.14,121.17,117.37,117.33,113.81,40.78,40.51,40.23,39.95,39.67,39.39,39.11.HRMS(ESI):calcd.for C₁₅H₈F₆N₂S[M+H]⁺＝363.0385,found 363.0390.t_R＝3.225min,HPLC purity:97.53％。

Example 8

3- ((3, 5-dibromophenyl) amino) benzo [ d ] isothiazole 1-oxide (8)

At-30 deg.C in N- (3, 5-dibromophenyl) benzo [ d]To a solution of isothiazol-3-amine (100mg) in concentrated sulfuric acid (2mL) was slowly added a cold mixed solution of concentrated sulfuric acid (225. mu.L) and concentrated nitric acid (225. mu.L). After the dropwise addition, the reaction solution was immediately poured into ice water to quench. The reaction mixture was adjusted to neutral pH by slowly adding saturated aqueous sodium carbonate solution, and extracted with ethyl acetate (3X 20mL) and washed with saturated brine (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent and purified by column chromatography (petroleum ether: ethyl acetate: 3:1) to give the title compound as a white solid (58.1mg, 51.2%). mp 216.2-217.4 ℃.¹H-NMR(300MHz,DMSO-d₆)δ11.09(s,1H),8.13(d,J＝7.4Hz,1H),8.05–7.81(m,4H),7.69(s,1H).HRMS(ESI):calcd.for C₁₃H₈Br₂N₂OS[M+Na]⁺＝467.8447,found 467.8452.t_R＝4.211min,HPLC purity:100％。

Example 9

3- ((3, 5-bis (trifluoromethyl) phenyl) amino) benzo [ d ] isothiazole 1-oxide (9)

At-30 deg.C in the compound N- (3, 5-bis-trifluoromethylphenyl) benzo [ d]To a solution of isothiazol-3-amine (100mg) in concentrated sulfuric acid (2mL) was slowly added a cold mixed solution of concentrated sulfuric acid (225. mu.L) and concentrated nitric acid (225. mu.L). After the dropwise addition, the reaction solution was immediately poured into ice water to quench. The pH was adjusted to neutral by adding a saturated aqueous sodium carbonate solution, and the mixture was extracted with ethyl acetate (3X 20mL) and washed with saturated brine (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and purified by column chromatography (petroleum ether: ethyl acetate: 5:1) to give the title compound as a white solid (56.0mg, 53.6%). mp 269.4-270.5 ℃.¹H-NMR(400MHz,DMSO-d₆)δ10.89(s,1H),8.74(s,2H),8.43(d,J＝7.6Hz,1H),8.16(d,J＝7.4Hz,1H),7.94–7.80(m,3H).HRMS(ESI):calcd.for C₁₅H₈F₆N₂OS[M+H]⁺＝379.0344,found 379.0334.t_R＝12.833min,HPLC purity:100％。

Example 10

3- ((3, 5-Dimethoxyphenyl) amino) benzo [ d ] isothiazole 1-oxide (10)

At-70 deg.C in N- (3, 5-dimethoxyphenyl) benzo [ d]A solution of m-chloroperoxybenzoic acid (39.0mg,0.21mmol,1.1eq) in dichloromethane (5mL) was slowly added dropwise to a solution of isothiazol-3-amine (57.2mg,0.2mmol,1.0eq) in dichloromethane (5 mL). After the dropwise addition, the reaction solution was naturally returned to room temperature, and stirred for reaction for 0.5 hour. After completion of the reaction, the reaction mixture was adjusted to neutral by adding a saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (3X 15 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent and purified by column chromatography (petroleum ether: ethyl acetate: 3:1) to give the title compound as a white solid (45.1mg, 74.6%). mp 235.5-235.6 ℃.¹H-NMR(300MHz,DMSO-d₆)δ10.28(s,1H),8.48(d,J＝7.3Hz,1H),8.11(d,J＝7.0Hz,1H),7.93–7.75(m,2H),7.29(s,2H),6.37(s,1H),3.79(s,6H)。

Example 11

N- (benzo [ d ] isothiazol-3-yl) furan-2-carboxamide (11)

1) Preparation of 1- (2-chlorophenyl) ethane-1-imine

Lithium bis (trimethylsilyl) amide (1M in THF,60mL,0.06mol) was slowly added dropwise to a solution of 2-chlorobenzonitrile (5g,0.036mol) in diethyl ether (60mL) under ice-bath conditions. After the dropwise addition, the reaction solution was gradually returned to room temperature, and the reaction was maintained at room temperature for 12 hours. After completion of the reaction, the reaction mixture was slowly poured into ice water (200mL) and quenched, and the suspension was extracted with ethyl acetate (3X 100mL) and washed with saturated brine (50 mL). The combined organic phases were dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure to give a red oily product which was directly subjected to the next reaction without further purification.

2) Preparation of benzo [ d ] isothiazol-3-amines

To a mixed solution of 1- (2-chlorophenyl) ethane-1-imine (278.3mg,1.8mmol) in dimethyl sulfoxide and toluene (10ml. v: v ═ 1:1) were added elemental sulfur (256mg,8mmol), and K₃PO₄(764.1mg,3.6 mmol). The reaction was carried out at 135 ℃ for 36 hours under nitrogen protection. After the reaction is finished, the sulfur is removed by pumping and filtering, and the toluene is removed by vacuum distillation from the filtrate. Water (20mL) was added to the residue, which was extracted with ethyl acetate (3X 20mL) and washed with saturated brine (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate 5:1) to give the desired product as a brown solid (247.1mg, 91.5%). mp deg.C.¹H-NMR(300MHz,DMSO-d₆)δ8.10(dt,J＝8.0,1.0Hz,1H),7.92(dt,J＝8.1,0.9Hz,1H),7.51(ddd,J＝8.1,7.0,1.2Hz,1H),7.38(ddd,J＝8.0,7.0,1.0Hz,1H),6.81(s,2H)。

3) Preparation of N- (benzo [ d ] isothiazol-3-yl) furan-2-carboxamide

In benzo [ d ]]To a solution of isothiazol-3-amine (100mg,0.67mmol) in dichloromethane (5mL) was added furan-2-carboxylic acid (75.1mg,0.67mmol), 1-hydroxybenzotriazole (135.1mg,1mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (191.7mg,1mol) and triethylamine (202.3mg,2mmol) in that order. The reaction was carried out at room temperature for 12 hours. After completion of the reaction, the reaction mixture was suction-filtered, and the filtrate was extracted with dichloromethane (3X 20mL) and washed with saturated brine (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent and purified by column chromatography (petroleum ether: ethyl acetate: 10:1) to give the desired product as a white solid (146.3mg, 75.6%). mp 256.1-257.0 ℃.¹H-NMR(300MHz,DMSO-d₆)δ11.16(s,1H),8.19(d,J＝8.2Hz,1H),8.00(d,J＝7.6Hz,2H),7.63(s,1H),7.56–7.42(m,2H),6.75(d,J＝4.1Hz,1H).¹³C-NMR(75MHz,DMSO-d₆)δ157.20,153.26,152.25,147.03,147.01,130.01,128.73,125.31,121.26,116.47,112.75.HRMS(ESI):calcd.for C₁₂H₈N₂O₂S[M+Na]⁺＝267.0199,found.267.0202t_R＝2.660min,HPLC purity:99.21％。

Example 12

N- (benzo [ d ] isothiazol-3-yl) -1H-pyrrole-2-carboxamide (12)

In benzo [ d ]]To a solution of isothiazol-3-amine (100mg,0.67mmol) in methylene chloride (5mL) were added pyrrole-2-carboxylic acid (75.0mg,0.67mmol), 1-hydroxybenzotriazole (135.1mg,1mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (191.7mg,1mol) and triethylamine (202.3mg,2mmol) in that order. The reaction was carried out at room temperature for 12 hours. After completion of the reaction, the reaction mixture was suction-filtered, and the filtrate was extracted with dichloromethane (3X 20mL) and washed with saturated brine (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent and purified by column chromatography (petroleum ether: ethyl acetate: 5:1) to give the desired product as a white solid (131.1mg, 81.1%). Mp>300℃.¹H-NMR(400MHz,DMSO-d₆)δ11.44(s,1H),9.32(s,1H),8.99(d,J＝2.5Hz,1H),8.87(t,J＝2.0Hz,1H),8.22(d,J＝8.2Hz,1H),8.06(d,J＝8.2Hz,1H),7.66(s,1H),7.52(s,1H).HRMS(ESI):calcd.for C₁₂H₁₀N₃OS[M+Na]⁺＝267.0359,found.279.0323t_R＝8.900min,HPLC purity:96.85％。

Example 13

N- (benzo [ d ] isothiazol-3-yl) thiophene-2-carboxamide (13)

Following the procedure of example 12, substituting thiophene-2-carboxylic acid for pyrrole-2-carboxylic acid, the desired product was obtained as a white solid (136.0mg, 78.1%). mp 276.4-177.2 ℃.¹H-NMR(300MHz,DMSO-d₆)δ11.27(s,1H),8.23–8.11(m,2H),8.02–7.91(m,2H),7.65(d,J＝1.1Hz,1H),7.51(dd,J＝8.1,1.1Hz,1H),7.32–7.25(m,1H).HRMS(ESI):calcd.for C₁₂H₉N₂OS₂[M+H]⁺＝261.0151,found.261.0148.t_R＝15.167min,HPLC purity:97.93％。

Example 14

N- (benzo [ d ] isothiazol-3-yl) -2-fluorobenzamide (14)

Following the procedure of example 12, substituting 2-fluorobenzoic acid for pyrrole-2-carboxylic acid, the title compound was obtained as a white solid (118.3mg, 65.8%). mp 231.1-231.9 ℃.¹H-NMR(400MHz,DMSO-d₆)δ11.26(s,1H),8.20(d,J＝8.2Hz,1H),8.11(d,J＝8.2Hz,1H),7.78(td,J＝7.4,1.8Hz,1H),7.67–7.61(m,2H),7.55–7.51(m,1H),7.41–7.35(m,2H).¹³C-NMR(100MHz,DMSO-d₆)δ164.03,160.94,158.46,153.02,152.06,133.61,130.71,130.68,129.53,128.82,125.40,125.10,125.07,124.72,121.30,116.81,116.59.HRMS(ESI):calcd.for C₁₄H₁₀FN₂OS[M+H]⁺＝273.0492,found.273.0492 t_R＝7.180min,HPLC purity:96.29％。

Example 15

N- (benzo [ d ] isothiazol-3-yl) -3-fluorobenzamide (15)

Following the procedure of example 12, substituting 3-fluorobenzoic acid for pyrrole-2-carboxylic acid, the desired product was obtained as a white solid (129.3mg, 70.8%). Mp>300℃.¹H-NMR(400MHz,DMSO-d₆)δ11.29(s,1H),8.21(d,J＝8.2Hz,1H),8.01(d,J＝8.2Hz,1H),7.93(d,J＝7.7Hz,1H),7.87(d,J＝9.8Hz,1H),7.65(t,J＝7.7Hz,2H),7.56–7.47(m,2H).HRMS(ESI):calcd.for C₁₄H₁₀FN₂OS[M+H]⁺＝273.0492,found.273.0496t_R＝7.156min,HPLC purity:100％。

Example 16

N- (benzo [ d ] isothiazol-3-yl) -4-fluorobenzamide (16)

Following the procedure of example 12, substituting 4-fluorobenzoic acid for pyrrole-2-carboxylic acid, the desired product was obtained as a white solid (141.1mg, 77.4%). mp 278.1-279.1 ℃.¹H-NMR(300MHz,DMSO-d₆)δ11.23(s,1H),8.21(dt,J＝8.2,0.9Hz,1H),8.15(dd,J＝8.9,5.5Hz,2H),7.99(dt,J＝8.2,1.0Hz,1H),7.64(ddd,J＝8.2,7.0,1.1Hz,1H),7.49(ddd,J＝8.1,6.9,1.0Hz,1H),7.42(t,J＝8.9Hz,2H).HRMS(ESI):calcd.for C₁₄H₁₀FN₂OS[M+H]⁺＝273.0492,found.273.0497 t_R＝3.301min,HPLC purity:97.31％。

Example 17

N- (benzo [ d ] isothiazol-3-yl) -2-methoxybenzamide (17)

The procedure of example 12 was followed, substituting 2-methoxybenzoic acid for pyrrole-2-carboxylic acid, to give the desired product as a white solid (127.6mg, 67.8%). mp 161.5-162.1 deg.C.¹H-NMR(300MHz,DMSO-d₆)δ10.84(s,1H),8.18(s,0H),8.07(s,0H),7.77(d,J＝7.6Hz,1H),7.65(t,J＝7.5Hz,1H),7.55(dt,J＝14.8,7.9Hz,2H),7.23(d,J＝8.4Hz,1H),7.11(t,J＝7.5Hz,1H),3.92(s,3H).HRMS(ESI):calcd.for C₁₅H₁₃N₂O₂S[M+H]⁺＝285.0692 found.285.0693.t_R＝3.581min,HPLC purity:98.36％。

Example 18

N- (benzo [ d ] isothiazol-3-yl) -4-bromobenzamide (18)

Following the procedure of example 12, pyridine was replaced with 4-bromobenzoic acidPyrrole-2-carboxylic acid, to give the desired product as a white solid (128.6mg, 58.1%). mp 277.1-279.3 ℃.¹H-NMR(300MHz,DMSO-d₆)δ11.30(s,1H),8.21(d,J＝8.3Hz,1H),8.06–7.95(m,3H),7.80(d,J＝8.6Hz,2H),7.64(s,1H),7.50(s,1H).¹³C-NMR(75MHz,DMSO-d₆)δ165.78,153.87,152.20,132.92,132.04,130.74,129.96,128.73,126.63,125.30,125.28,121.30.HRMS(ESI):calcd.for C₁₄H₁₀BrN₂OS[M+H]⁺＝332.9692,found.332.9688t_R＝4.632min,HPLC purity:97.77％。

Example 19

N- (benzo [ d ] isothiazol-3-yl) -4-bromo-2-fluorobenzamide (19)

Following the procedure of example 12, substituting 2-fluoro-4-bromobenzoic acid for furan-2-carboxylic acid, the desired product was obtained as a pale yellow solid (184.9mg, 78.6%). mp 192.3-193.1 deg.C.¹H-NMR(300MHz,DMSO-d₆)δ11.35(s,1H),8.20(d,J＝8.1Hz,1H),8.12(d,J＝8.1Hz,1H),7.86–7.70(m,2H),7.63(q,J＝9.1,8.2Hz,2H),7.52(t,J＝7.6Hz,1H).HRMS(ESI):calcd.for C₁₄H₈BrFN₂OS[M+H]⁺＝350.9598found.350.9596.t_R＝4.172min,HPLC purity:97.49％。

Example 20

2-fluoro-N- (6-fluorobenzo [ d ] isothiazol-3-yl) benzamide (20)

Following the procedure of example 11, substituting 2-chloro-4-fluorobenzonitrile for 2-chlorobenzonitrile and 2-fluorobenzoic acid for furan-2-carboxylic acid, the title product was obtained as a pale yellow solid (149.6mg, 76.9%). mp 202.1-203.1 deg.C.¹H-NMR(300MHz,DMSO-d₆)δ11.31(s,1H),8.20–8.05(m,2H),7.78(td,J＝7.4,1.8Hz,1H),7.70–7.57(m,1H),7.49–7.38(m,1H),7.44–7.31(m,2H).HRMS(ESI):calcd.for C₁₄H₉F₂N₂OS[M+H]⁺＝291.0398found.291.0398.t_R＝3.573min,HPLC purity:100％。

Example 21

4-bromo-N- (5- (trifluoromethyl) benzo [ d ] isothiazol-3-yl) benzamide (21)

Following the procedure of example 11, substituting 2-chloro-4-trifluoromethylbenzonitrile for 2-chlorobenzonitrile and p-bromobenzoic acid for furan-2-carboxylic acid, the desired product was obtained as a yellow solid (232.5mg, 86.5%). mp170.9-171 ℃.¹H-NMR(400MHz,DMSO-d₆)δ11.45(s,1H),8.47(d,J＝9.3Hz,2H),8.01(d,J＝8.2Hz,2H),7.98–7.90(m,1H),7.79(d,J＝8.2Hz,2H).HRMS(ESI):calcd.for C₁₅H₈BrF₃N₂OS[M+H]⁺＝402.2087 found.402.2085.t_R＝5.234min,HPLC purity:96.65％。

Example 22

N- (6-chlorobenzo [ d ] isothiazol-3-yl) -2-fluorobenzamide (22)

Following the procedure of example 12, substituting 2, 4-dichlorobenzonitrile for 2-chlorobenzonitrile and 2-fluorobenzoic acid for furan-2-carboxylic acid, the title product was obtained as a yellow solid (166.9mg, 81.7%). mp 171-171.1 ℃.¹H-NMR(300MHz,DMSO-d₆)δ11.34(s,1H),8.42–8.33(m,1H),8.12(dd,J＝8.7,0.6Hz,1H),7.78(td,J＝7.4,1.8Hz,1H),7.71–7.53(m,2H),7.46–7.31(m,2H).HRMS(ESI):calcd.for C₁₄H₈ClFN₂OSNa[M+Na]⁺＝328.9922,found.328.9929.t_R＝4.923min,HPLC purity:97.32％。

Example 23

N- (benzo [ d ] isothiazol-3-yl) -4-bromo-2-methylbenzamide (23)

Following the procedure of example 12, substituting 2-methyl-4-bromobenzoic acid for furan-2-carboxylic acid, the desired product was obtained as a white solid (204.5mg, 87.9%) mp 236-238.7 ℃.¹H-NMR(400MHz,DMSO-d₆)δ11.19(s,1H),8.20(dt,J＝8.3,0.9Hz,1H),8.12(dt,J＝8.2,1.0Hz,1H),7.64(ddd,J＝8.1,6.9,1.1Hz,1H),7.61(dt,J＝1.6,0.8Hz,1H),7.56–7.53(m,2H),7.54–7.49(m,1H),2.46(s,3H).HRMS(ESI):calcd.for C₁₅H₁₁BrN₂OS[M+H]⁺＝346.9868,found.346.9869.t_R＝7.325min,HPLC purity:100％。

Example 24

N- (6-Bromobenzo [ d ] isothiazol-3-yl) -2-fluorobenzamide (24)

Following the procedure of example 11, substituting 2-chloro-4-bromobenzonitrile for 2-chlorobenzonitrile and 2-fluorobenzoic acid for furan-2-carboxylic acid, the title product was obtained as a white solid (184.8mg, 76.8%). mp 166.5-167.1 ℃.¹H-NMR(400MHz,DMSO-d₆)δ11.33(s,1H),8.54(d,J＝1.7Hz,1H),8.05(d,J＝8.7Hz,1H),7.77(td,J＝7.4,1.8Hz,1H),7.70(dd,J＝8.7,1.8Hz,1H),7.66–7.60(m,1H),7.40–7.33(m,2H).HRMS(ESI):calcd.for C₁₄H₇BrFN₂OSNa[M+Na]⁺＝372.9417,found.372.9421.t_R＝5.613min,HPLC purity:99.18％。

Example 25

N- (5-chlorobenzo [ d ] isothiazol-3-yl) -2-fluorobenzamide (25)

The procedure is as in example 11, using 2, 5-dichlorobenzeneReplacing 2-chlorobenzonitrile with formonitrile and furan-2-carboxylic acid with 2-fluorobenzoic acid gave the title product as a white solid (157.8mg, 76.8%). Mp>300℃.¹H-NMR(300MHz,DMSO-d₆)δ11.33(s,1H),8.33–8.18(m,2H),7.80(d,J＝1.9Hz,1H),7.69(dd,J＝8.7,2.0Hz,2H),7.44–7.33(m,2H).HRMS(ESI):calcd.for C₁₄H₉ClFN₂OS[M+H]⁺＝307.0103,found.307.0109.t_R＝5.017min,HPLC purity:100.0％。

Example 26

N- (5- (trifluoromethyl) benzo [ d ] isothiazol-3-yl) benzamide (26)

Following the procedure of example 11, substituting 2-chloro-5- (trifluoromethyl) benzonitrile for 2-chlorobenzonitrile and p-bromobenzoic acid for furan-2-carboxylic acid, the title product was obtained as a yellow solid (211.9mg, 78.9%). mp 261.6-263.6 ℃.¹H-NMR(300MHz,DMSO-d₆)δ11.40(s,1H),8.52–8.43(m,2H),8.12–8.04(m,2H),7.96(s,1H),7.63–7.55(m,2H).HRMS(ESI):calcd.for C₁₅H₉BrF₃N₂OS[M+H]⁺＝400.9566,found.400.9568.t_R＝4.672min,HPLC purity:96.48％。

Example 27

2-fluoro-N- (5- (trifluoromethyl) benzo [ d ] isothiazol-3-yl) benzamide (27)

Following the procedure of example 11, substituting 2-chloro-5- (trifluoromethyl) benzonitrile for 2-benzonitrile and 2-fluorobenzoic acid for furan-2-carboxylic acid, the title product was obtained as a white solid (175.1mg, 76.8%). m.p.>300℃.¹H-NMR(300MHz,DMSO-d₆)δ11.48(s,1H),8.62(s,1H),8.48(d,J＝8.6Hz,1H),7.96(dd,J＝8.5,1.7Hz,1H),7.80(d,J＝1.9Hz,1H),7.70–7.60(m,1H),7.39(d,J＝6.9Hz,2H).HRMS(ESI):calcd.for C₁₅H₉F₄N₂OS[M+H]⁺＝341.0366,found.341.0368.t_R＝5.613min,HPLC purity:99.06％。

Example 28

N- (5, 6-Difluorobenzo [ d ] isothiazol-3-yl) benzamide (28)

Following the procedure of example 11, substituting 3, 4-difluoro-5-chloro-benzonitrile for 2-chlorobenzonitrile and benzoic acid for furan-2-carboxylic acid, the title product was obtained as a white solid (150.0mg, 77.1%). Mp>300℃.HRMS(ESI):calcd.for C₁₄H₈BrF₂N₂OS[M+H]⁺＝368.9503,found.368.9506.t_R＝7.324min,HPLC purity:98.1％。

Example 29

N- (benzo [ d ] isothiazol-3-yl) -4-bromo-2-methoxybenzamide (29)

Following the procedure of example 12, substituting 2-methoxy-4-bromobenzoic acid for pyrrole-2-carboxylic acid, the desired product was obtained as a white solid (162.8mg, 66.9%). Mp>300℃.¹H-NMR(400MHz,DMSO-d₆)δ10.87(s,1H),8.19(s,1H),8.07(s,1H),7.69–7.27(m,5H),3.91(s,3H).HRMS(ESI):calcd.for C₁₅H₁₁BrN₂O₂S[M+H]⁺＝362.9797,found.362.9798.t_R＝5.532min,HPLC purity:98.46％。

Example 30

N- (benzo [ d ] isothiazol-3-yl) -4-bromo-2, 6-difluorobenzamide (30)

Method according to example 12Method, substituting 2, 5-difluoro-4-bromobenzoic acid for pyrrole-2-carboxylic acid gave the desired product as a white solid (120.2mg, 48.6%). Mp>300℃.¹H-NMR(400MHz,Chloroform-d)δ9.12(s,1H),8.16(d,J＝8.2Hz,1H),7.90(dd,J＝8.2,0.9Hz,1H),7.58(d,J＝7.0Hz,1H),7.50(t,J＝7.1Hz,1H),7.25(d,J＝7.4Hz,2H).HRMS(ESI):calcd.for C₁₄H₇BrF₂N₂OS[M+H]⁺＝368.9503,found.368.9505.t_R＝8.485min,HPLC purity:96.53％。

The following are some of the pharmacodynamic experimental data for the compounds of the invention:

(1) luciferase reporter assay test Compounds for HIF-2 agonistic Activity

The experimental method comprises the following steps: a commercial virus (CLS-007L, Qiagen) containing hypoxia inducible element and luciferase gene sequences was transfected into 786-O cells to obtain stably transfected 786-O-HRE tool cells (J.Med.chem.61,9691-9721 (2018)). 786-O-HRE cells were seeded evenly in 96-well white plates. When the cells adhere to the wall, a drug-added group is added with a dimethyl sulfoxide (DMSO) solution of a corresponding compound, and a blank control group is added with DMSO with a corresponding concentration. At 37 deg.C, 5% CO₂After 24 hours incubation in an incubator under culture conditions, 10 μ L of luciferase substrate (RG055S, Beyotime) was added to the whiteboard according to the kit instructions and the luminescence was immediately detected. And comparing the luminous value of the drug adding hole with that of the blank control group to obtain the agonistic effect E of the compound. Meanwhile, Graphpad Prism 8 is used for carrying out nonlinear regression analysis on different concentrations of luminescence values of the compound to calculate EC₅₀The value is obtained.

E is equal to the luminous value of the drug adding group/the luminous value of the blank control group multiplied by 100 percent

TABLE 1 agonist Effect of partial Compounds of the invention on HIF-2 transcriptional Activity at 20. mu.M concentration

The data in Table 1 show the activation effect of some compounds of the invention on HIF-2 transcriptional activity at a concentration of 20. mu.M. The activity data show that the compounds in the invention have HIF-2 agonistic activity to a certain degree and are superior to the positive compound M1002(141.6 +/-8.2). The HIF-2 agonistic activity of some partial compounds is obviously better than that of the positive drug compound M1002, such as compounds 2,4, 8, 10, 14, 16 and 18.

The activity data of the compounds of the invention indicate that the two S ═ O double bonds of the sulfonamide structure in the isothiazole ring of the compound are not pharmacophores for HIF-2 agonistic activity and that the S ═ O double bonds can be removed to give a new parent structure. The sulfur atom of the isothiazole ring of the compound of the present invention contains only one S ═ O double bond or does not contain S ═ O double bond substitution, and the compound activity is generally superior to that of the positive compound M1002.

At the same time, some compounds of the invention activate HIF-2 transcriptional activity at low concentrations, which is the EC in luciferase reporter gene assays₅₀The data are shown in table 2. Wherein, compounds 2, 18, 19, 21 are effective at nanomolar concentrations for the activation of HIF-2. While the positive compound M1002 showed an abnormal decrease in the high concentration fluorescence values in the multi-concentration luciferase reporter assay, probably due to its cytotoxicity against 786-O-HRE cells. Abnormal fluorescence values leading to their EC₅₀The values are not fit and are less active than the compounds of the invention.

TABLE 2 EC in luciferase reporter gene assay for partial compounds of the invention₅₀

(2) Cell counting kit-8 (CCK-8) method for testing cytotoxicity of compound on kidney epithelial cell line HEK293

To initially evaluate the safety of the compounds of the present invention, the cytotoxicity of the renal epithelial cell line HEK293 was tested by the CCK-8 method.

The experimental method comprises the following steps: HEK293 cells were seeded in 96-well plates and after cell attachment, the dosing groups added DMSO solutions of the respective compounds to ensure a final concentration of 2, 20 μ M of each compound. The blank control group was added with the highest concentration of DMSO in the dosing group. And 3 multiple holes are arranged at the same time. After 24 hours of treatment, Cell Counting Kit-8(C0037, Beyotime) was added to each well according to Kit instructions. The 96-well plate was incubated at 37 ℃ for 2 hours in the dark, and absorbance at a wavelength of 450nm was measured for each well using a microplate reader. And calculating the ratio of the absorbance value of the administration group to the absorbance of the blank control group to determine the inhibition rate I, wherein the cytotoxicity is determined when the value I is higher than 10%.

I ═ 100% (1-absorbance in dosing/absorbance in blank) ×

The following are the cytotoxicity experimental data for a portion of the compounds (table 3):

the experiment results tested by the CCK-8 method show that the compound has no obvious cytotoxicity under the condition of high concentration of 20 mu M. Whereas the positive compound M1002 exhibited cytotoxicity at a concentration of 2 μ M. The compound of the present invention is superior to the positive compound M1002 in safety at the cellular level.

TABLE 3 cytotoxicity of partial compounds of the invention against HEK293 cells

(3) Luciferase reporter gene assays test synergistic effects of compound and PHD inhibitor

In addition, the present invention tests the synergistic effect of a portion of the compounds in combination with a PHD inhibitor on the enhancement of HIF-2 activity. At the cellular level, the compounds and PHD inhibitors were tested for synergy using a luciferase reporter assay. HIF-2 is not hydroxylated by PHD for degradation due to the depletion of VHL protein in 786-O cells. Therefore, the invention selects the human kidney epithelial cell line HEK293 cell line as a tool for research. The following are experimental methods: will contain hypoxia inducible elements and luciferase gene sequencesVirus (CLS-007L, Qiagen) was transfected into HEK293 cells, resulting in stably transfected HEK293-HRE tool cells. HEK293-HRE cells were plated evenly in 96-well plates. When cells adhere to the wall, a DMSO solution of the compound with corresponding concentration is added into the combined administration group to ensure that the final concentration is 0.2, 2 and 20 mu M, and the DMSO solution of the PHD inhibitor with corresponding concentration to ensure that the final concentration is 2 mu M; only adding DMSO solution of PHD inhibitor with corresponding concentration into PHD inhibitor control group to ensure that final concentration is 0.2, 2, 20 μ M; the blank control group was added with the highest concentration of DMSO in the dosing group. Three multiple holes are arranged at the same time. At 37 ℃ and 5% CO₂After incubation for 24 hours in a cell incubator under culture conditions, luciferase substrate (RG055S, Beyotime) was added to a 96-well white plate according to the instructions and its luminescence was immediately detected using a microplate reader. Calculating the effect value R of the combination drug, and defining that the R values of the compound of the invention at three concentrations of 0.2, 2 and 20 mu M are all more than 100 percent indicates that the two have synergistic effect in promoting HIF-2 transcriptional activity.

The luminescence value of the combined administration group/(the luminescence value of the single administration group + the luminescence value of the 2 mu M PHD inhibitor group) × 100%

TABLE 4 Experimental data for the use of compounds of some of the examples of the invention in combination with PHD inhibitors

The partial compounds of the present invention exhibit a synergistic effect in increasing HIF-2 transcriptional activity in combination with a PHD inhibitor, wherein ND means not tested. Experimental results indicate that the compound of the invention can show synergistic effect in promoting HIF-2 transcriptional activity by combining with PHD inhibitor. Meanwhile, the invention discloses a luciferase reporter gene experiment result graph (shown in figure 2) formed by combining the compound 2 and a PHD inhibitor. The results show that the compound 2 and the PHD inhibitor have good synergistic effect on the activation of HIF-2 in cells, and the combined effect is obviously better than that of a single administration group.

(4) Study of in vivo plasma EPO-elevating Activity

The experimental method comprises the following steps: 18-20 grams of C56BL/6 mice were selected and randomized into 8 groups: a blank control group, a low-dose single-administration group of compound 2, a medium-dose single-administration group of compound 2, a high-dose single-administration group of compound 2, a PHD inhibitor single-administration group, a PHD inhibitor and low-dose compound 2 combination group, a PHD inhibitor and medium-dose compound 2 combination group, a PHD inhibitor and high-dose compound 2 combination group, and 5 mice per group. The administration group is respectively administered with compound of corresponding concentration by intragastric administration, and the blank control group is administered with normal saline of the same volume as the administration group by intragastric administration. After 4-6 hours, each group of mice was bled by retroorbital venous plexus bleeding, and blood samples were centrifuged at 6000r/min for 5min to obtain plasma in the upper layer. According to the instruction of the kit, the content of EPO in the plasma of each group of mice is detected by using a murine EPO quantitative ELISA detection kit (ab119593, Abcam).

The invention discloses an experimental result of the combination of a compound 2 and a PHD inhibitor AKB-6548 (figure 3). The results show that at the animal level, the compound 2 and the PHD inhibitor AKB-6548 show good synergistic effect on the increase of EPO in C57BL/6 mice. The plasma EPO of mice in the combination group was significantly higher than that of AKB-6548 alone and the effect was compound 2 dose-dependent. The combination of the PHD inhibitor and the compound of the invention provides a unique advantage in rapidly increasing EPO levels over the PHD inhibitor alone or the compound of the invention alone.

The benzisothiazole derivative has good biological activity at cellular level and animal level. The compound can stimulate the transcriptional activity of hypoxia inducible factor 2 and enhance the generation and secretion of erythropoietin, thereby promoting the generation of red blood cells; the benzisothiazole hypoxia inducible factor 2 agonist compound or the pharmaceutically acceptable salt thereof prepared by the invention can be combined with prolyl hydroxylase inhibitor to play a synergistic role in improving HIF-2 transcription activity, and can be used for treating hypoxia inducible factor 2 related diseases, such as ischemic diseases and the like. Compared with a single PHD inhibitor, the combination of the two can quickly improve the EPO level in the blood of the mouse, and has a certain application prospect in ischemic diseases such as renal anemia, cerebral arterial thrombosis and the like.

Claims

1. A benzisothiazole hypoxia inducible factor 2 agonist compound or a pharmaceutically acceptable salt thereof is characterized in that the general formula of the compound is as shown in formula I:

wherein X represents-S-or

L represents-NH-or

when L is

When Ar represents a five-membered aromatic heterocycle or

when L is-NH-, Ar represents

Wherein R is⁴And R⁵Each independently represents halogen、C₁-C₄Alkyl of (C)₁-C₄Alkoxy group of (C)₁-C₄Halogen-substituted alkyl.

2. The benzisothiazole hypoxia inducible factor 2 agonist compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the salt comprises a pharmaceutically acceptable metal salt such as sodium, potassium, lithium, calcium, magnesium, aluminum or zinc salt; carbonates or bicarbonates of pharmaceutically acceptable metal cations such as sodium, potassium, lithium, calcium, magnesium, aluminum and zinc; pharmaceutically acceptable organic primary, secondary and tertiary amines including aliphatic, aromatic, aliphatic or hydroxyalkylamines such as methylamine, ethylamine, 2-hydroxyethylamine, diethylamine, triethylamine, ethylenediamine, ethanolamine or diethanol.

3. A process for preparing a compound of claim 1 or a pharmaceutically acceptable salt thereof,

when L in the formula (I) is

when X in the formula (I) is

The preparation method of the compound comprises the following steps:

4. the use of the benzisothiazole hypoxia inducible factor 2 agonist compound or the pharmaceutically acceptable salt thereof according to claim 1 in the preparation of drugs for treating ischemic diseases.

5. The use according to claim 4, wherein the ischemic disease comprises ischemia-induced anemia, stroke, and myocardial ischemia.

6. The use of claim 4, further comprising the use of a prolyl hydroxylase inhibitor in combination for the manufacture of a medicament for the treatment of ischemic diseases.

7. The use of claim 4, wherein the prolyl hydroxylase inhibitor comprises rosxastat, vatinostat, dadostat, ennalastat and morristat.