CN111423377B - 5-amino-1H-pyrazole derivative, preparation method and medical application - Google Patents

5-amino-1H-pyrazole derivative, preparation method and medical application Download PDF

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CN111423377B
CN111423377B CN202010396597.1A CN202010396597A CN111423377B CN 111423377 B CN111423377 B CN 111423377B CN 202010396597 A CN202010396597 A CN 202010396597A CN 111423377 B CN111423377 B CN 111423377B
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江程
胡庆华
陆冉
周梦泽
刘春晓
马思楠
李白杨
孟子博
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Abstract

The invention discloses a 5-amino-1H-pyrazole derivative shown as a general formula (I) or a pharmaceutically acceptable salt thereof, a preparation method thereof and a compound serving as P2Y14Use of a receptor inhibitor. Compared with the prior art, the invention discloses a pair P2Y145-amino-1H-pyrazole derivatives with receptor inhibiting effect and pharmaceutically acceptable salts thereof, and the compounds are proved to be P2Y through pharmacological experiments14The receptor has obvious inhibiting effect, and can be particularly used as a medicament for treating inflammatory diseases.

Description

5-amino-1H-pyrazole derivative, preparation method and medical application
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to novel 5-amino-1 (H) -pyrazole derivatives, a pharmaceutical composition containing the same, a preparation method thereof, and a therapeutic agent of the derivatives, particularly P2Y14The medical use of receptor antagonists.
Background
Cell signal transduction refers to a process in which extracellular factors are combined with receptors (membrane receptors or nuclear receptors), and obtained information is enhanced, differentiated, integrated and transmitted to a downstream sensor to trigger a series of biochemical reactions and protein-protein interaction in cells until genes required by cell physiological reactions begin to express and various biological effects are formed. G protein-coupled receptors are currently the largest family of membrane surface receptors that are involved in a variety of signaling processes.
G protein-coupled receptors (GPCRs) are classified into three classes according to the type of downstream-coupled G protein: gsProtein, GiProtein, GqProteins and G12/13Four proteins, each mediating different signaling. G protein-coupled receptors are currently found only in eukaryotes. Ligands that can bind to G protein-coupled receptors include neurotransmitters, pheromones, polypeptides, and small molecule compounds, among others. G protein-coupled receptors are involved in the development of many diseases and are therefore important drug targets. It is reported statistically that about 30% of the drugs currently on the market target G protein-coupled receptors.
Gq/GiThere are many subfamilies of coupled receptors, including: (1) purine Receptor family (Purinergic Receptor), members of P1, P2; (2) members of the Adenosine Receptor family (Adenosine Receptor) are a1, A2A, A2B and A3. The purine receptor family plays an important role in regulating myocardial oxygen consumption, coronary blood flow, anti-inflammation, vascular reactivity, apoptosis, cytokine secretion and the like.
P2Subfamilies can be further divided into 5 subtypes according to tissue distribution and pharmacological characteristics: P2X, P2Y, P2Z, P2U and P2T. Wherein P2X and P2Z belong to ion channel type receptors; P2Y, P2UAnd P2T belong to G protein-coupled receptors.
The P2Y receptor family of G protein-coupled receptors has been reported to contain 8 subtypes (P2Y1, 2, 4, 6, 11, 12, 13, 14), which are widely distributed in various cells and tissues and have low homology among subtypes, so that different subtypes have high selectivity for ligands. Wherein the P2Y1, 2, 4, 6 receptor binds GqAnd activating the PLC path; P2Y12, 13, 14 receptor binding GiInhibiting adenylate cyclase activity; P2Y4Receptor coupling Gq/GiTwo G proteins; P2Y11Coupling Gq/GsTwo G proteins. The P2Y receptor mediates a series of biological effects such as immunoregulation, platelet aggregation, smooth muscle cell proliferation and the like.
P2Y14Receptors are mainly present in the heart, placenta, adipose tissue, gastrointestinal tract and peripheral immune cells, and can improve hypersensitivity of microglia and fluidity of neutrophils; increase mast cell release mediators and renal leap cell inflammation, and inhibit astrocytes from releasing interstitial metalloprotease and tumor necrosis factor in the central nervous system. Recent studies have shown that the activity of the enzyme is shown at P2Y14In receptor knockout mice, P2Y14Antagonism of the receptor has potential therapeutic effects on diabetes. UDP and UDP-glu have also been reported to activate P2Y as ligands14The receptor has a great relationship with diseases such as inflammation, asthma and the like.
For P2Y at present14Receptor inhibitor studies have reported only 3 structural classes of compounds (pyrimidopiperides, 2-naphthoates and 3-substituted benzoic acids), but all have also been in preclinical studies. The 2-naphthoic acid inhibitor has the highest activity and selectivity, but the currently reported 2-naphthoic acid inhibitor has the defects of poor solubility, low oral bioavailability, great difficulty in synthesis and purification and the like, and brings great difficulty for further discussion of structure-activity relationship and biological evaluation. Thus finding a new structure type of P2Y14The receptor antagonist solves the problems of poor drug forming property and the like of the 2-naphthoic acid inhibitor, and is found to be P2Y with strong activity and good selectivity14Novel strategies for receptor inhibitors.
Disclosure of Invention
The invention aims to provide a novel structure with P2Y145-amino-pyrazole derivatives having receptor antagonistic action and pharmaceutically acceptable salts thereof.
It is another object of the present invention to provide a process for producing the above 5-amino-1H-pyrazole derivative.
It is still another object of the present invention to provide a use of the above 5-amino-1H-pyrazole derivative for treating inflammatory diseases.
5-amino-1H-pyrazole derivatives of general formula (i), and their enantiomers, diastereomers, tautomers, N-oxides, solvates, formulations and pharmaceutically acceptable salts:
Figure BDA0002487824760000021
wherein R is1Is selected from CnH2n-COOR5、CnH2n-CONHR5、CnH2n-CN and tetrazole, wherein n is selected from 0, 1 and 2;
R2selected from H, F, CN, C1-3-alkyl, CF3；
R3Is CmH2mR6、COCmH2mR6、CONHCmH2mR6、SOCmH2mR6Or SO2CmH2mR6Wherein m is selected from 0, 1 and 2;
R4is C5-10Monocyclic aryl or bicyclic aryl, C containing 1-4 heteroatoms selected from N, O or S5-14Monocyclic or bicyclic heteroaryl, C3-10A cycloalkyl or heterocycloalkyl radical, C1-10Alkyl radical, C1-10-an alkoxy group,
wherein said aryl or heteroaryl is independently optionally substituted with 1 to 5 substituents selected from: NO2、CN、COH、COCH3、OH、NH2、F、Cl、Br、I、C1-6-alkyl, halo-C1-6Alkyl radical, C1-6-alkoxy, C3-10A cycloalkyl or heterocycloalkyl radical, C0-6alkylene-C3-10-cycloalkyl, C0-6Alkylene- (5-or 6-membered heteroaryl), C0-6-alkylene-NR7R8、C0-6alkylene-COR7、C0-6-alkylene-CONR7R8、C0-6alkylene-SO2R7、C0-6alkylene-SO3R7、C0-6alkylene-SO2NR7R8，
Wherein said alkyl, alkoxy, cycloalkyl and heterocycloalkyl are unsubstituted or independently substituted with 1 to 6 substituents selected from the group consisting of: F. OH, NH2、C1-3Alkyl radical, C1-3-alkoxy, C0-4-alkylene-NR7R8、C0-4-alkylene-OR8、C0-4-alkylene-CONR7R8、C0-4alkylene-COR8、C0-4alkylene-SO2R8、C0-4alkylene-SO2NR7R8、C0-4alkylene-SO3R8，
Or wherein two adjacent substituents form a 3-8 membered saturated or unsaturated ring containing carbon atoms and optionally containing 1-3 heteroatoms selected from N, O or S;
R5is H, C1-6Alkyl, wherein alkyl is unsubstituted or substituted with 1 to 6 substituents selected from the group consisting of: F. OH, O-C1-3-an alkyl group;
R6is C5-10Monocyclic aryl or bicyclic aryl, C containing 1-4 heteroatoms selected from N, O or S5-14Monocyclic or bicyclic heteroaryl, C3-10A cycloalkyl or heterocycloalkyl radical, C1-10Alkyl radical, C1-10-an alkoxy group;
wherein said aryl or heteroaryl is independently optionally substituted with 1 to 5 substituents selected from the group consisting of: F. cl, Br, I,C1-6Alkyl radical, C1-6Alkoxy, NO2、CN、COH、COCH3、OH、NH2(ii) a Said alkyl, alkoxy, cycloalkyl and heterocycloalkyl are unsubstituted or independently substituted with 1 to 6 substituents selected from the group consisting of: F. OH, C1-3Alkyl radical, C1-3-an alkoxy group;
R7is H;
R8selected from H, C1-10Alkyl radical, C3-10-cycloalkyl, C3-10-heterocycloalkyl radical, C1-10alkylene-C3-10-cycloalkyl radical, C1-10alkylene-C3-10-heterocycloalkyl radical, C1-10-alkylene- (5-membered heteroaryl), wherein alkyl, alkylene, cycloalkyl, heterocycloalkyl and heteroaryl are unsubstituted or independently substituted with 1 to 7 substituents selected from the group consisting of: OR (OR)9、C1-6Alkyl, CO2R9、CONR9R10、NR9COR9、C3-10-a heterocycloalkyl group;
or R7And R8Together with the nitrogen to which they are attached form a 3-8 membered ring containing carbon atoms and optionally 1 or 2 heteroatoms selected from N, O or S, wherein the ring is unsubstituted or substituted with 1-4 substituents selected from: c0-6alkylene-CO2R9、C1-6Alkyl radical, C3-8-a heterocycloalkyl group;
R9selected from H, C1-6-an alkyl group, wherein the alkyl group is unsubstituted or substituted with 1 to 6 substituents selected from the group consisting of: F. O-C1-3-an alkyl group;
R10is H.
Preferably, the compound of formula i:
R1is selected from CH2COOH, COOH or CONH2；
R2Selected from H or F;
R3is selected from CmH2mR6、COCmH2mR6、SOCmH2mR6Or SO2CmH2mR6Wherein m is selected from 0;
R6is C5-10Monocyclic aryl radical, C containing 1-4 heteroatoms selected from N, O or S5-14Monocyclic heteroaryl, C3-10A cycloalkyl or heterocycloalkyl group;
wherein said aryl or heteroaryl is independently optionally substituted with 1 to 5 substituents selected from the group consisting of: F. cl, Br, I, C1-6Alkyl radical, C1-6Alkoxy, NO2、CN、COH、COCH3、OH、NH2(ii) a Said cycloalkyl and heterocycloalkyl being unsubstituted or independently substituted with 1 to 6 substituents selected from the group consisting of: F. OH, C1-3Alkyl radical, C1-3-alkoxy groups.
R4Selected from:
Figure BDA0002487824760000041
wherein Ring is C5-7Monocyclic aryl radical, C containing 1-4 heteroatoms selected from N, O or S5-7A monocyclic heteroaryl group having a ring structure,
wherein R is11A substituent selected from: NO2、CN、OH、NH2、F、Cl、Br、I、C1-6-alkyl, halo-C1-6Alkyl radical, C1-6-alkoxy, halo-C1-6-alkoxy, C0-6-alkylene-NR7R8、C0-6alkylene-COR8、C0-6alkylene-CO2R8、C0-6-alkylene-CONR7R8、C0-6alkylene-SO3R8、C0-6alkylene-SO2NR7R8Or wherein two adjacent substituents form a 3-8 membered saturated or unsaturated ring containing carbon atoms and optionally containing 1-3 heteroatoms selected from N, O or S;
wherein R is7And R8As described above.
Further preferably, R4Selected from:
Figure BDA0002487824760000042
ring is phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, thienyl, furyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl;
R11selected from NO2、CN、OH、NH2、F、Cl、Br、I、C1-6-alkyl, halo-C1-6Alkyl radical, C1-6-alkoxy, halo-C1-6Alkoxy radical, COR8、CONR7R8、CO2R8、SO2NR7R8、SO3R8；
R7Is H;
R8selected from H, C1-6Alkyl radical, C3-6-cycloalkyl, CH2CONH2、CH2CONMe2、CH2CH2OH、CH2CH2Me、(CH2)3OH、(CH3)3OMe、(CH2)4OH、(CH2)4OMe、(CH2)5OH、(CH2)2COOH、(CH2)3COOH、(CH2)4COOH、(CH2)5COOH、(CH2)2CH(CH3)COOH、C(CH2OH)3、C(CH2OH)2CH3、CH2CH(CH3)OH、(CH2)2CF3、(CH2)3CF3、(CH2)4CF3
Figure BDA0002487824760000051
Most preferably, R4Selected from:
Figure BDA0002487824760000052
ring is phenyl, pyrimidinyl, thienyl, furyl;
R11selected from NO2、CN、F、Cl、C1-6-alkyl, halo-C1-6Alkyl radical, C1-6-alkoxy, halo-C1-6-alkoxy, CONR7R8、CO2R8、SO2NR7R8、SO3R8；
R7Is H;
R8selected from H, C1-6Alkyl radical, C3-6-cycloalkyl, CH2CONH2、CH2CONMe2、CH2CH2OH、CH2CH2OMe、(CH2)3OH、(CH3)3OMe、(CH2)4OH、(CH2)4OMe、(CH2)2COOH、(CH2)3COOH、(CH2)4COOH、(CH2)2CH(CH3)COOH、C(CH2OH)3、C(CH2OH)2CH3、CH2CH(CH3)OH、
Figure BDA0002487824760000053
As the most preferred embodiment of the present invention, said compound of formula I is selected from the following compounds:
Figure BDA0002487824760000061
it will be understood by those of ordinary skill in the art that where a list of alternative substituents includes members that are not available for substitution of a particular group due to their valency requirements or other reasons, reference to that list is intended to include only those members of the list that are suitable for substitution of that particular group, as per the knowledge of those of ordinary skill in the art. The same applies to the number of possible substituents on a group.
The compounds used in the present invention may be in the form of pharmaceutically acceptable salts or solvates. The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids. If the compounds of the invention contain one or more acidic or basic groups, the invention also encompasses the corresponding pharmaceutically or toxicologically acceptable salts thereof, in particular the pharmaceutically usable salts thereof. Thus, according to the invention, the compounds of the invention which contain acidic groups can be used, for example, in the form of alkali metal salts, alkaline earth metal salts or ammonium salts. More specific examples of these salts include sodium salts, potassium salts, calcium salts, magnesium salts, or salts with ammonia or organic amines (e.g., ethylamine, ethanolamine, triethylamine, or amino acids). According to the invention, the compounds of the invention containing one or more basic groups (i.e. protonatable groups) can be used in the form of their addition salts with inorganic or organic acids. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, napadisylic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and acids known to those of ordinary skill in the art. If the compounds of the invention contain both acidic or basic groups in the molecule, the invention also includes inner salts or betaines (zwitterions) in addition to the salt forms mentioned. The various salts can be obtained by the usual methods known to the person skilled in the art, for example by reacting these substances with organic or inorganic acids or bases in solvents or dispersants or by anion exchange or cation exchange with other salts. The invention also encompasses all salts of the compounds of the invention which, owing to their low physiological compatibility, are not directly suitable for use in medicaments but which can be employed, for example, as intermediates in chemical reactions or for the preparation of pharmaceutically acceptable salts.
It is another object of the present invention to provide a process for the preparation of a compound of formula I, comprising the steps of:
(1) the compound 1 in the general formula is reduced by nitro to prepare a compound 2 in the general formula;
(2) the compound 2 in the general formula is subjected to condensation reaction with different carboxylic acid or sulfonic acid derivatives or substitution reaction with different hydrocarbyl derivatives to obtain a compound 3 in the general formula;
(3) carrying out substitution reaction and deprotection reaction on the compound 3 with different hydrocarbyl derivatives to obtain a compound 4 with a general formula, namely a compound shown in a general formula (I);
Figure BDA0002487824760000071
R1、R2、R3and R4The method of claim 1.
The invention also provides a pharmaceutical composition which contains the compound of the general formula (I) or pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials.
The invention finally provides the application of the compound of the general formula (I) or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing P2Y14The use of receptor inhibitors in medicine.
And the application of the compound of the general formula (I) or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing the medicine for treating P2Y14The use in the preparation of medicaments for treating inflammatory diseases related to receptors.
The compounds of the invention are described as P2Y14When the receptor inhibitor is used, the inhibitor can be used alone, can be matched with other medicines for simultaneous use, or can be prepared into a compound preparation together with other medicines for use, and the P2Y can be inhibited14The purpose of the receptor.
The pharmaceutically acceptable auxiliary materials refer to various conventional auxiliary materials required when preparing different dosage forms, such as diluents, adhesives, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorbing materials and the like, and the pharmaceutically acceptable auxiliary materials are prepared into any one of common oral preparations by a conventional preparation method, such as granules, powder, tablets, capsules, pills, oral liquid, decoction, dropping pills and the like.
Has the advantages that: compared with the prior art, the invention discloses a pair P2Y145-amino-1H-pyrazole derivatives with receptor inhibiting effect and pharmaceutically acceptable salts thereof, and the compounds are proved to be P2Y through pharmacological experiments14The receptor has obvious inhibiting effect, and can be particularly used as a medicament for treating inflammatory diseases.
Drawings
FIG. 1 shows THP-1 cells P2Y14Receptor protein relative expression, where data are mean ± sd (n ═ 4), and analysis of variance was performed using one-way anova (# represents P < 0.001 compared to normal, P < 0.05 compared to model control, P < 0.01 compared to model control, and P < 0.001 compared to model control).
FIG. 2 is the IL-1 β levels in the supernatant of THP-1 cell culture medium, where the data are mean. + -. standard deviation and one-way anova was used for the analysis of variance (## # represents P < 0.001 compared to normal, P < 0.05 compared to model control, and P < 0.001 compared to model control).
FIG. 3 shows the synovial tissue P2Y of 4 rats14Relative receptor protein expression, where the mean ± sd of the data was analyzed by one-way anova for variance (## # represents P < 0.001 compared to normal, P < 0.05 compared to model control, P < 0.001 compared to model control).
FIG. 4 is serum IL-1 β levels of 10 rats, wherein the mean values of the data. + -. standard deviation, were subjected to analysis of variance using one-way anova (# represents P < 0.001 compared to the normal group, # represents P < 0.05 compared to the model control group, and # represents P < 0.001 compared to the model control group).
Detailed Description
The present invention will be described in detail with reference to examples. In the present invention, the following examples are given for better illustration of the present invention and are not intended to limit the scope of the present invention.
Examples 1 and 2
The method comprises the following steps: 1- (4-fluorobenzyl)) -5-Nitro-1H-pyrazole-3-carboxylic acid ethyl ester (1a)
Figure BDA0002487824760000081
5-Nitro-1H-pyrazole-3-carboxylic acid ethyl ester (1.0g, 5.40mmol) was dissolved in N, N-dimethylformamide (20mL), and K was added2CO3(1.49g, 10.80mmol), the mixture was stirred at room temperature for 20min, then 4-fluorobenzyl bromide (1.23g, 6.48mmol) was added and stirred at room temperature for 6 h. After the reaction was completed, water was added and extraction was performed with ethyl acetate (3 times). The combined organic phases were washed with brine and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (5: 1 petroleum ether/ethyl acetate) gave 1.30g of a pale yellow solid in 82% yield.
Step two: 5-amino-1- (4-fluorobenzyl) -1H-pyrazole-3-carboxylic acid ethyl ester (1b)
Dissolving ethyl 1- (4-fluorobenzyl) -5-nitro-1H-pyrazole-3-carboxylate (2.0g, 6.82mmol) in a mixed solvent of ethanol/water (10/1) (30mL), adding NH4Iron powder (3.81g, 68.20mmol) was slowly added to Cl (3.65g, 68.20mmol) at 0 deg.C, and after the addition was complete, the reaction was allowed to warm to room temperature and stirred for 7 h. After the reaction is complete, NaHCO is added3The saturated solution was adjusted to pH 7 and extracted with dichloromethane (3 times). The combined organic phases were washed with brine and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (1: 1 petroleum ether/ethyl acetate) gave 1.52g of a white solid in 84% yield.
Step three: 1- (4-Fluorobenzyl) -5- (4-methylbenzamido) -1H-pyrazole-3-carboxylic acid ethyl ester (1)
Ethyl 5-amino-1- (4-fluorobenzyl) -1H-pyrazole-3-carboxylate (150mg, 0.569mmol) was dissolved in dichloromethane (3mL), and 4-methylbenzoic acid (118.31mg, 0.869mmol), 4-Dimethylaminopyridine (DMAP) (141.64mg, 1.16mmol) and Kate condensation agent (BOP) (384.22mg, 0.869mmol) were sequentially added thereto, followed by stirring at room temperature for 10 hours. After the reaction was completed, insoluble matter was removed by filtration, and the filtrate was extracted with ethyl acetate (3 times) by adding water to the filtrate, and combined withThe organic phase was washed with brine and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (5: 1 petroleum ether/ethyl acetate) gave 190mg of white solid in 72% yield.
Step four: 1- (4-Fluorobenzyl) -5- (4-methylbenzamido) -1H-pyrazole-3-carboxylic acid (2)
Ethyl 1- (4-fluorobenzyl) -5- (4-methylbenzamido) -1H-pyrazole-3-carboxylate (190mg,0.498mmol) was dissolved in methanol (1mL), 4mol/L LiOH solution (1mL) was added, and the reaction was stirred at room temperature for 5 hours after the addition. After completion of the reaction, 1N HCl was added to adjust the pH of the solution to 2, stirring was continued for 30min, and filtration was carried out to obtain 120mg of a white solid with a yield of 69%.1H NMR(400MHz,DMSO-d6)δ7.86–7.78(m,2H),7.34(dddd,J＝7.2,6.2,2.1,1.0Hz,4H),7.22–7.12(m,2H),6.52(s,1H),5.60(t,J＝1.0Hz,2H),2.39(d,J＝0.8Hz,3H)。
Example 3
The following compounds were prepared analogously as in example 1 and example 2:
Figure BDA0002487824760000091
Figure BDA0002487824760000101
Figure BDA0002487824760000111
example 4 and example 5
The method comprises the following steps: 5- (Cyclopropanecarboxamido) -1- (4-fluorobenzyl) -1H-pyrazole-3-carboxylic acid ethyl ester (4)
Figure BDA0002487824760000112
Reacting 5-amino-1- (4-fluorobenzyl) -1H-pyridineOxazole-3-carboxylic acid ethyl ester (1b) (150mg, 0.569mmol) was dissolved in dichloromethane (2mL) solvent, triethylamine (115mg, 1.138mmol) was added, cyclopropanecarbonyl chloride (89.19mg, 0.854mmol) was added slowly at 0 deg.C, and after the addition was complete, the reaction was allowed to move to room temperature for 3 h. After the reaction was completed, water was added, extraction was performed with ethyl acetate (3 times), and the organic phases were combined, washed with saturated brine, and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) gave 130mg of a white solid in 69% yield.1H NMR(400MHz,DMSO-d6)δ6.76(s,1H),4.20(q,J＝7.0Hz,2H),1.41(p,J＝6.4Hz,1H),1.28(t,J＝7.0Hz,3H),1.07–0.88(m,4H)。
Step two: 5- (Cyclopropanecarboxamido) -1- (4-fluorobenzyl) -1H-pyrazole-3-carboxylic acid (5)
5- (cyclopropanecarboxamido) -1- (4-fluorobenzyl) -1H-pyrazole-3-carboxylic acid ethyl ester (130mg,0.393mmol) was dissolved in methanol (1mL), and 4mol/L LiOH solution (1mL) was added thereto, followed by stirring at room temperature for 5 hours. After completion of the reaction, 1N HCl was added to adjust the pH of the solution to 2, stirring was continued for 30min, and filtration was carried out to obtain 95mg of a white solid with a yield of 80%.
Example 6
The following compounds were prepared analogously as in example 4 and example 5:
Figure BDA0002487824760000121
Figure BDA0002487824760000131
example 7 and example 8
Figure BDA0002487824760000132
The method comprises the following steps: 1- (4-Fluorobenzyl) -5- ((4-methylphenyl) sulfonamido) -1H-pyrazole-3-carboxylic acid ethyl ester (7)
Ethyl 5-amino-1- (4-fluorobenzyl) -1H-pyrazole-3-carboxylate (1b) (150mg, 0.569mmol) was dissolved in tetrahydrofuran (3mL) solvent, triethylamine (115mg, 11.138mmol) and p-methylbenzenesulfonyl chloride (108mg, 0.569mmol) were added, and after completion of the addition, the mixture was stirred at room temperature overnight. After completion of the reaction, the solvent was removed under reduced pressure, the crude product was added with water, extracted with dichloromethane (3 times), the organic phases were combined, washed with saturated brine, anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) gave 180mg of a white solid in 71% yield.1H NMR(400MHz,DMSO-d6)δ7.34(ddt,J＝8.5,1.6,0.8Hz,2H),7.14–7.06(m,2H),6.72(s,1H),4.20(q,J＝7.0Hz,2H),2.37(d,J＝0.6Hz,3H),1.28(t,J＝7.0Hz,3H)。
Step two: 1- (4-Fluorobenzyl) -5- ((4-methylphenyl) sulfonamido) -1H-pyrazole-3-carboxylic acid (8)
Ethyl 1- (4-fluorobenzyl) -5- ((4-methylphenyl) sulfonamide) -1H-pyrazole-3-carboxylate (150mg,0.336mmol) was dissolved in methanol (1mL) solvent, and 4mol/L LiOH solution (1mL) was added thereto, after which the reaction was stirred at room temperature for 5 hours. After completion of the reaction, 1N HCl was added to adjust the pH of the solution to 2, stirring was continued for 30min, and filtration was carried out to obtain 115mg of a white solid with a yield of 82%.
Example 9
The following compounds were prepared analogously as in example 7 and example 8:
Figure BDA0002487824760000133
Figure BDA0002487824760000141
examples 10 and 11
The method comprises the following steps: 1- (4-Fluorobenzyl) -5- ((4-methylbenzyl) amino) -1H-pyrazole-3-carboxylic acid ethyl ester (10)
Figure BDA0002487824760000142
1- (4-Fluorobenzyl) -5-amino-1H-pyrazole-3-carboxylic acid ethyl ester (150mg, 0.569mmol) was dissolved in N, N-dimethylformamide (2mL), K was added2CO3(121mg, 0.854mmol), the mixture was stirred at room temperature for 20min, then 4-methylbenzyl bromide (126mg, 0.682mmol) was added and stirred at room temperature for 6 h. After the reaction was completed, water was added and extraction was performed with ethyl acetate (3 times). The combined organic phases were washed with brine and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (5: 1 petroleum ether/ethyl acetate) gave 175mg of a pale yellow solid in 84% yield.1H NMR(400MHz,DMSO-d6)δ7.92(t,J＝5.5Hz,1H),7.24–7.10(m,4H),5.75(s,1H),4.68(dt,J＝5.5,0.9Hz,2H),4.20(q,J＝7.0Hz,2H),2.25(d,J＝0.7Hz,3H),1.28(t,J＝7.0Hz,3H)。
Step two: 1- (4-Fluorobenzyl) -5- ((4-methylbenzyl) amino) -1H-pyrazole-3-carboxylic acid (11)
Ethyl 1- (4-fluorobenzyl) -5- ((4-methylbenzyl) amino) -1H-pyrazole-3-carboxylate (150mg,0.409mmol) was dissolved in methanol (1mL), 4mol/L LiOH solution (1mL) was added, and after the addition, the reaction was stirred at room temperature for 5 hours. After completion of the reaction, 1N HCl was added to adjust the pH of the solution to 2, stirring was continued for 30min, and filtration was carried out to obtain 120mg of a white solid with a yield of 86%.
Example 12
The following compounds were prepared analogously as in example 10 and example 11:
Figure BDA0002487824760000143
Figure BDA0002487824760000151
example 13 and example 14
Figure BDA0002487824760000152
The method comprises the following steps: 5- (4-Methylbenzoylamino) -1H-pyrazole-3-carboxylic acid ethyl ester (13a)
Ethyl 5-amino-1H-pyrazole-3-carboxylate (200mg, 1.29mmol) was dissolved in dichloromethane (5mL) solvent, and 4-methylbenzoic acid (263mg, 1.935mmol), 4-Dimethylaminopyridine (DMAP) (315mg, 2.58mmol) and Cartesian condensing agent (BOP) (855mg, 1.935mmol) were added in this order, and after completion of the addition, the reaction was stirred at room temperature for 10 hours. After the reaction was completed, insoluble matter was removed by filtration, water was added to the filtrate, extraction was performed with ethyl acetate (3 times), and the organic phases were combined, washed with saturated brine and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (5: 1 petroleum ether/ethyl acetate) gave 150mg of a white solid in 43% yield.1H NMR(400MHz,DMSO-d6)δ7.86–7.78(m,2H),7.37–7.25(m,2H),4.20(q,J＝7.0Hz,2H),2.39(d,J＝0.6Hz,3H),1.28(t,J＝7.0Hz,3H)。
Step two: 5- (4-methylbenzamido) -1- (4- (trifluoromethyl) benzyl) -1H-pyrazole-3-carboxylic acid ethyl ester (13)
5- (4-methylbenzamido) -1H-pyrazole-3-carboxylic acid ethyl ester (150mg, 0.549mmol) is dissolved in N, N-dimethylformamide (2.5mL), NaH (60% oil dispersion, 24mg, 0.604mmol) is slowly added at 0 ℃, stirring is carried out at 0 ℃ for 20min after the addition is finished, 4-fluorobenzyl bromide (155mg, 0.823mmol) is added, and after the addition is finished, the mixture is moved to room temperature for reaction overnight. After the reaction was completed, water was added, extraction was performed with ethyl acetate (3 times), and the organic phases were combined, washed with saturated brine, and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) gave 165mg of a white solid in 79% yield.
Step three: 5- (4-methylbenzamido) -1- (4- (trifluoromethyl) benzyl) -1H-pyrazole-3-carboxylic acid (14)
Ethyl 5- (4-methylbenzamido) -1- (4- (trifluoromethyl) benzyl) -1H-pyrazole-3-carboxylate (150mg,0.394mmol) was dissolved in methanol (1mL), 4mol/L LiOH solution (1mL) was added, and the reaction was stirred at room temperature for 5 hours after the addition. After completion of the reaction, 1N HCl was added to adjust the pH of the solution to 2, stirring was continued for 30min, and filtration was carried out to obtain 110mg of a white solid with a yield of 80%.
Example 15
The following compounds were prepared analogously as in example 13 and example 14:
Figure BDA0002487824760000161
Figure BDA0002487824760000171
example 16, example 17 and example 18
Figure BDA0002487824760000172
The method comprises the following steps: 4- ((3- (ethoxycarbonyl) -5- (4-methylbenzamido) -1H-pyrazol-1-yl) methyl) benzyl Tert-butyl ester (16)
Ethyl 5- (4-methylbenzamido) -1H-pyrazole-3-carboxylate (150mg, 0.549mmol) was dissolved in N, N-dimethylformamide (2.5mL), NaH (60% oil dispersion, 24mg, 0.604mmol) was slowly added at 0 ℃ and after the addition was completed, the mixture was stirred at 0 ℃ for 20min, and then tert-butyl 4-bromomethylbenzoate (223mg, 0.823mmol) was added and the mixture was allowed to stand at room temperature for overnight reaction. After the reaction was completed, water was added, extraction was performed with ethyl acetate (3 times), and the organic phases were combined, washed with saturated brine, and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) gave 215mg of white solid in 81% yield.
Step two: 4- ((3- (ethoxycarbonyl) -5- (4-methylbenzamido) -1H-pyrazol-1-yl) methyl) benzyl Acid (17)
Tert-butyl 4- ((3- (ethoxycarbonyl) -5- (4-methylbenzamido) -1H-pyrazol-1-yl) methyl) benzoate (215mg, 0.46mmol) was dissolved in trifluoroacetic acid (4mL) solvent, and the reaction was stirred at room temperature for 3H. After completion of the reaction, TFA was removed by concentration under reduced pressure to give a pale yellow oil. Adding water, extracting with dichloromethane (3 times), mixing organic phases, washing with saturated brine, anhydrous Na2SO4And (5) drying. The solvent was dried by rotary evaporation under reduced pressure to give 168mg of a pale yellow solid, yield 89%.
Step three: 4- ((3-carboxy-5- (4-methylbenzamido) -1H-pyrazol-1-yl) methyl) benzoic acid (18)
4- ((3- (ethoxycarbonyl) -5- (4-methylbenzamido) -1H-pyrazol-1-yl) methyl) benzoic acid (200mg, 0.385mmol) was dissolved in a methanol (1mL) solvent, and a 4mol/L LiOH solution (1mL) was added thereto, and after the addition, the reaction was stirred at room temperature for 5 hours. After completion of the reaction, 1N HCl was added to adjust the pH of the solution to 2, stirring was continued for 30min, and filtration was carried out to obtain 147mg of a white solid with a yield of 78%.1H NMR(400MHz,DMSO-d6)δ8.17–8.09(m,2H),7.86–7.78(m,2H),7.64(dt,J＝8.3,1.1Hz,2H),7.37–7.29(m,2H),6.83(s,1H),5.54(t,J＝1.0Hz,2H),2.39(d,J＝0.8Hz,3H)。
Example 19
The following examples were prepared similarly as in example 17 and optionally example 18:
Figure BDA0002487824760000181
Figure BDA0002487824760000191
examples 20 and 21
Figure BDA0002487824760000192
The method comprises the following steps: 5- (4-methylbenzamido) -1- (4- (3- (tetrahydro-2H-pyran-4-yl) propionyl) benzyl) - 1H-pyrazole-3-carboxylic acid ethyl ester (20)
4- ((3- (ethoxycarbonyl) -5- (4-methylbenzamido) -1H-pyrazol-1-yl) methyl) benzoic acid (200mg, 0.385mmol) was dissolved in a solvent of anhydrous dichloromethane (5mL), and 1-hydroxybenzotriazole hydrate (71mg, 0.462mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (89mg, 0.462mmol) were added in this order at 0 ℃. ) And reacting for 10min under the protection of nitrogen. (tetrahydro-2H-pyran-4-yl) methylamine (44mg, 0.385mmol) and diethylisopropylamine (1.21mmol) were then dissolved in anhydrous dichloromethane (2.5mL) and added to the reaction mixture. The mixture was cooled to room temperature and stirred under nitrogen for 4 h. After completion of the reaction, the reaction mixture was diluted with dichloromethane and successively diluted with 1N HCl and saturated NaHCO3The aqueous solution and the saturated brine solution were washed, and the organic phase was washed with anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (petroleum ether/ethyl acetate 8:1) gave 163mg of white solid in 68% yield.
Step two: 5- (4-methylbenzamido) -1- (4- (3- (tetrahydro-2H-pyran-4-yl) propionyl) benzyl) - 1H-pyrazole-3-carboxylic acid (21)
Ethyl 5- (4-methylbenzamido) -1- (4- (3- (tetrahydro-2H-pyran-4-yl) propionyl) benzyl) -1H-pyrazole-3-carboxylate (163mg, 0.262mmol) was dissolved in methanol (1mL) solvent, 4mol/L LiOH solution (1mL) was added, and after the addition, the reaction was stirred at room temperature for 5H. After completion of the reaction, 1N HCl was added to adjust the pH of the solution to 2, stirring was continued for 30min, and filtration was carried out to obtain 122mg of a white solid with a yield of 81%.
Example 22
The following compounds were prepared analogously as in example 20 and example 21:
Figure BDA0002487824760000201
example 23 and example 24
Figure BDA0002487824760000202
The method comprises the following steps: 5-amino-1- (thien-2-ylmethyl) -1H-pyrazole-3-carboxylic acid ethyl ester (23a)
Sulfuric acid was slowly added to a solution of thiophene-2-methylhydrazinium hydrochloride (1.0g, 6.58mmol) in water at 0 ℃. The solution was added dropwise to a suspension of 3-cyano-2-oxo-propionic acid ethyl ester sodium salt (1.08g, 6.58mmol), acetonitrile and sodium ethoxide in dichloromethane (30mL) at room temperature. After stirring the biphasic mixture for 12h, the conversion to the corresponding hydrazone was completed. The phases were separated and extracted with dichloromethane (3 times). The combined organic phases were washed with brine and anhydrous Na2SO4And (5) drying. Concentration by evaporation, the residue dissolved in ethanol (25mL), refluxed for 16h, cyclization was complete and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate 1:2) to give 0.87g of a white solid in 53% yield.
Step two: 5- (4-Methylbenzoylamino) -1- (thien-2-ylmethyl) -1H-pyrazole-3-carboxylic acid ethyl ester (23)
Ethyl 5-amino-1- (thien-2-ylmethyl) -1H-pyrazole-3-carboxylate (200mg, 0.796mmol) was dissolved in dichloromethane (3mL), 4-methylbenzoic acid (162mg, 1.19mmol), 4-Dimethylaminopyridine (DMAP) (194mg, 1.59mmol) and Cartesian condensing agent (BOP) (529mg, 1.19mmol) were sequentially added, and after completion of the addition, the reaction was stirred at room temperature for 10 hours. After the reaction was completed, insoluble matter was removed by filtration, water was added to the filtrate, extraction was performed with ethyl acetate (3 times), and the organic phases were combined, washed with saturated brine and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (5: 1 petroleum ether/ethyl acetate) gave 200mg of a white solid in 66% yield.
Step three: 5- (4-methylbenzamido) -1- (thiophen-2-ylmethyl) -1H-pyrazole-3-carboxylic acid (24)
5- (4-methylbenzamido) -1- (thiophene-2-ylmethyl) -1H-pyrazole-3-carboxylic acid ethyl ester (200mg, 0.518mmol) was dissolved in methanol (1mL), 4mol/L LiOH solution (1mL) was added, and after the addition, the reaction was stirred at room temperature for 5 hours. After completion of the reaction, 1N HCl was added to adjust the pH of the solution to 2, stirring was continued for 30min, and filtration was carried out to obtain 170mg of a white solid with a yield of 84%.1H NMR(400MHz,DMSO-d6)δ7.86–7.78(m,2H),7.33(dq,J＝8.5,0.7Hz,2H),7.25(dd,J＝4.2,3.1Hz,1H),7.01–6.91(m,2H),6.52(s,1H),5.81(s,2H),2.39(t,J＝0.7Hz,3H)。
Example 25
The following compounds were prepared analogously as in example 23 and example 24:
Figure BDA0002487824760000211
Figure BDA0002487824760000221
example 26 and example 27
Figure BDA0002487824760000222
The method comprises the following steps: 5-amino-4-fluoro-1H-pyrazole-3-carboxylic acid ethyl ester (26a)
Will be provided with5-amino-1H-pyrazole-3-carboxylic acid ethyl ester(1.0g, 5.91mmol) was dissolved in acetonitrile (20mL), and to this solution was added a fluorinating reagent (Selectfluor) (2.8g, 7.97mmol), and stirred at room temperature overnight. The resultant solid was filtered, the filtrate was concentrated, and purified by silica gel column chromatography (PE/EA ═ 1:1) to obtain 430mg of the compound as a yellow solid in 42% yield.
Step two: 4-fluoro-5- (4-methylbenzamido) -1H-pyrazole-3-carboxylic acid ethyl ester (26b)
5-amino-4-fluoro-1H-pyrazole-3-carboxylic acid ethyl ester (500mg, 2.89mmol) was dissolved inTo dichloromethane (3mL) was added 4-methylbenzoic acid (590mg, 4.33mmol), 4-Dimethylaminopyridine (DMAP) (706mg, 5.78mmol), and Cartesian condensing agent (BOP) (191mg, 4.33mmol) in this order, and after completion of the addition, the reaction was stirred at room temperature for 10 hours. After the reaction was completed, insoluble matter was removed by filtration, water was added to the filtrate, extraction was performed with ethyl acetate (3 times), and the organic phases were combined, washed with saturated brine and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (5: 1 petroleum ether/ethyl acetate) gave 460mg of a white solid in 51% yield.
Step three: 4-fluoro-1- (4-fluorobenzyl) -5- (4-methylbenzamido) -1H-pyrazole-3-carboxylic acid ethyl ester (26)
4-fluoro-5- (4-methylbenzamido) -1H-pyrazole-3-carboxylic acid ethyl ester (200mg, 0.651mmol) is dissolved in N, N-dimethylformamide (3mL), NaH (60% oil dispersion, 29mg, 0.716mmol) is slowly added at 0 ℃, after the addition is completed, stirring is performed at 0 ℃ for 20min, p-fluorobenzyl bromide (184mg, 0.976mmol) is then added, and the mixture is transferred to room temperature for reaction overnight after the addition is completed. After the reaction was completed, water was added, extraction was performed with ethyl acetate (3 times), and the organic phases were combined, washed with saturated brine, and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) gave 195mg of a white solid in 75% yield.
Step four: 4-fluoro-1- (4-fluorobenzyl) -5- (4-methylbenzamido) -1H-pyrazole-3-carboxylic acid (27)
Ethyl 4-fluoro-1- (4-fluorobenzyl) -5- (4-methylbenzamido) -1H-pyrazole-3-carboxylate (150mg, 0.376mmol) was dissolved in methanol (1mL), and 4mol/L LiOH solution (1mL) was added thereto, followed by stirring at room temperature for 5 hours. After completion of the reaction, 1N HCl was added to adjust the pH of the solution to 2, stirring was continued for 30min, and filtration was carried out to obtain 115mg of a white solid with a yield of 83%.
Example 28
The following compounds were prepared analogously as in example 26 and example 27:
Figure BDA0002487824760000231
examples 29 and 30
Figure BDA0002487824760000232
The method comprises the following steps: ethyl 2- (5-amino-1H-pyrazol-3-yl) acetate (29a)
Dissolving 2- (5-amino-1H-pyrazol-3-yl) acetic acid (5g, 35.46mmol) in ethanol (25mL), cooling to 0 ℃, and dropwise adding SOCl at 0 DEG C2(15mL), after the dropwise addition, the reaction mixture was allowed to warm to room temperature and stirred for 2 h. After the reaction is complete NaHCO is slowly added3Saturated solution (100mL), stirring for 30min, extracting with ethyl acetate (3 times), combining organic phases, washing with saturated brine, anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) gave about 4.8g of a white solid in 80% yield.
Step two: 2- (5- (4-methylbenzamido) -1H-pyrazol-3-yl) acetic acid ethyl ester (29b)
Ethyl 2- (5-amino-1H-pyrazol-3-yl) acetate (500mg, 2.95mmol) was dissolved in methylene chloride (8mL) solvent, and 4-methylbenzoic acid (604mg, 4.44mmol), 4-Dimethylaminopyridine (DMAP) (722mg, 5.91mmol) and Kate condensing agent (BOP) (1.96g, 4.44mmol) were sequentially added thereto, and after completion of the addition, the reaction was stirred at room temperature for 10 hours. After the reaction was completed, insoluble matter was removed by filtration, water was added to the filtrate, extraction was performed with ethyl acetate (3 times), and the organic phases were combined, washed with saturated brine and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (5: 1 petroleum ether/ethyl acetate) gave 0.55g of a white solid in 65% yield.
Step three: ethyl 2- (1- (4-fluorobenzyl) -5- (4-methylbenzamido) -1H-pyrazol-3-yl) acetate (29)
Ethyl 2- (5- (4-methylbenzamido) -1H-pyrazol-3-yl) acetate (200mg, 0.696mmol) was dissolved in N, N-dimethylformamide (5mL) solventNaH (60% oil dispersion, 30mg, 0.766mmol) was slowly added at 0 deg.C, after the addition was complete, stirring was carried out at 0 deg.C for 20min, p-fluorobenzyl bromide (197mg, 1.04mmol) was then added, and after the addition was complete, the reaction was allowed to proceed at room temperature overnight. After the reaction was completed, water was added, extraction was performed with ethyl acetate (3 times), and the organic phases were combined, washed with saturated brine, and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) gave 230mg of a white solid in 83% yield.
Step four: 2- (1- (4-fluorobenzyl) -5- (4-methylbenzamido) -1H-pyrazol-3-yl) acetic acid (30)
Ethyl 1- (4-fluorobenzyl) -5- (4-methylbenzamido) -1H-pyrazole-3-carboxylate (150mg, 0.380mmol) was dissolved in methanol (1mL), 4mol/L LiOH solution (1mL) was added, and the reaction was stirred at room temperature for 5 hours after the addition. After completion of the reaction, 1N HCl was added to adjust the pH of the solution to 2, stirring was continued for 30min, and filtration was carried out to obtain 120mg of a white solid with a yield of 86%.
Example 31
The following compounds were prepared analogously as in example 29 and example 30:
Figure BDA0002487824760000241
Figure BDA0002487824760000251
example 32
Figure BDA0002487824760000252
The method comprises the following steps: 5- (4-methylbenzamido) -1H-pyrazole-3-carboxamide (32a)
P-methylbenzoic acid (1.3g, 9.52mmol) was dissolved in ethyl acetate (25mL) solvent, and N, N' -carbonyldiimidazole (1.5g, 9.52mmol) was added at room temperatureStirring for 1H, adding 5-amino-1H-pyrazole-3-carboxamide (1.0g, 7.93mmol), transferring to 80 deg.C oil bath, heating and stirring for reaction overnight, precipitating a large amount of white solid after reaction is complete, filtering, washing filter cake with diethyl ether, and drying to obtain white solid 2.1g with yield of 92%.1H NMR(400MHz,DMSO-d6)δ7.96(s,2H),7.86–7.78(m,2H),7.33(dq,J＝8.5,0.7Hz,2H),6.60(s,1H),2.39(t,J＝0.7Hz,3H).
Step two: 5- (4-methylbenzamido) -1H-pyrazole-3-carboxamide (32)
Ethyl 2- (5- (4-methylbenzamido) -1H-pyrazol-3-yl) acetate (200mg, 0.819mmol) was dissolved in N, N-dimethylformamide (5mL), NaH (60% oil dispersion, 36mg, 0.90mmol) was slowly added at 0 ℃, after the addition was completed, stirring was performed at 0 ℃ for 20min, p-fluorobenzyl bromide (232mg, 1.23mmol) was then added, and the mixture was allowed to stand at room temperature for overnight reaction. After the reaction was completed, water was added, extraction was performed with ethyl acetate (3 times), and the organic phases were combined, washed with saturated brine, and anhydrous Na2SO4And (5) drying. Purification by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) gave 187mg of white solid in 65% yield.
Example 33
The following compounds were prepared analogously as in example 32:
Figure BDA0002487824760000261
the pharmacological experiments and results of the partial compounds of formula i are as follows:
the experimental method comprises the following steps:
stable rotation P2Y14The recipient HEK293 cell line was cultured in DMEM medium (containing 10% fetal calf serum, 100U/ml penicillin and 100. mu.g/ml streptomycin), inoculated to a culture plate before experiment, and changed to serum-free medium at an inoculation density of 1X 105Cell/well, cell at 37 ℃ 95% O2、5％CO2Culturing under humidity condition. Addition of IBMX inhibited PDEs activity to ensure cAMP at a higher level. Using the AC agonist Forskolin (30 μ M) stimulates cellular cAMP production, and different concentrations of test compounds (0.01, 0.1, 1, 10, 100nm) were pre-added, with PPTN as a positive control. Then 1. mu.M of P2Y was added14Receptor agonist UDPG, cAMP Glo 4h laterTMThe Assay kit (PROMEGA co. ltd, usa) detects the amount of intracellular cAMP. Calculation of IC from the inhibition of cAMP content50The value is obtained.
Pharmacological research experiment method for inhibiting urate crystal-induced macrophage inflammatory reaction by test compound
Human THP-1 cells were cultured in RPMI-1640 medium (containing 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin) and inoculated onto culture plates at a density of 1X 10 before the experiment5Cell/well, cell at 37 ℃ 95% O2、5％CO2Culturing under humidity condition. Before the experiment, 100ng/ml PMA is added into each hole and incubated for 24h to induce the THP-1 cells to differentiate into macrophages. The test compounds (2.5, 5, 10. mu.M), PPTN (5. mu.M) and dexamethasone (5. mu.M) were added to the medium beforehand for intervention, and urate crystals (MSU) were added to the cells at a final concentration of 500. mu.g/ml after 1h, after 6h the following indices were determined:
western Blot method for detecting P2Y in cells14Protein expression of the receptor;
and detecting the level of IL-1 beta in the cell culture medium supernatant according to an ELISA kit (Shenzhen Xinbo Sheng) method.
Pharmacological experimental research method for therapeutic effect of tested compound on acute gouty arthritis at whole animal level
Male clean grade SD rats, 200 + -20 g in weight, free water diet, 12h daily lighting, ambient temperature 25 + -2 deg.C. Animals were divided into several groups: the normal control group, the model control group and the administration group (tested compound, PPTN and dexamethasone) adopt a one-time joint cavity injection MSU to induce the acute gouty arthritis model, and the normal control group and the normal administration group adopt the same amount of physiological saline to be injected into the joint cavity. Test compounds (5, 10, 20mg/kg), PPTN (10mg/kg) and dexamethasone (10mg/kg) were administered by intra-articular injection to each administration group. Detecting the joint circumference of the rat by adopting a line binding method, selecting 0h, 2h, 4h, 8h, 12h and 24h at the time point of measurement, taking blood from the retrobulbar venous plexus of the rat after 24h, centrifuging for 5min under the condition of 10000 Xg centrifugation, taking serum, and storing at 4 ℃ for later use. Then, the neck is cut off to kill the animal, joint synovial tissue is rapidly divided on an ice bench, and the following indexes are detected:
detection of P2Y in synovial tissue by Western Blot method14Protein expression of the receptor.
And (3) detecting the IL-1 beta level in serum and synovial tissue according to an ELISA kit (Shenzhen Xinbo Sheng) method.
The experimental results are as follows:
table 1 partial compound at cellular level vs. P2Y14IC of R50The value:
Figure BDA0002487824760000271
table 2: effect (cm) of the composition on the circumference of the joints of rats with acute gouty arthritis
Figure BDA0002487824760000272
Figure BDA0002487824760000281
The data in Table 2 are the mean values. + -. standard deviation of the joint circumference of 10 rats at different time points, and the analysis of variance was performed by one-way anova (# represents P < 0.01 in comparison with the normal group, # represents P < 0.001 in comparison with the normal group, # represents P < 0.05 in comparison with the model control group, # represents P < 0.01 in comparison with the model control group, # represents P < 0.001 in comparison with the model control group). As can be seen from Table 2: MSU causes the joint swelling of rats, and the obvious difference appears in 4h, 8h, 12h and 24h, which indicates that the modeling is successful; the tested compositions with different dosages can relieve the increase of the joint circumference of the rat caused by MSU to different degrees, and show significant difference compared with a model control group; PPTN and dexamethasone also show expected effects, which shows that the experimental results are true and reliable.
As can be seen from fig. 1: MSU causes THP-1 cell P2The Y14 receptor protein expression is obviously increased, which indicates that the modeling is successful; different doses of the test composition can reduce P to different degrees2Y14 receptor protein expression shows significant difference compared with a model control group; the PPTN also shows expected effect, which shows that the experimental result is real and credible. Dexamethasone was not shown to be P2Y14Modulation of receptor expression.
As can be seen from fig. 2: MSU causes the IL-1 beta level in the THP-1 cell culture medium supernatant to be obviously increased, which indicates that the molding is successful; the test composition with different dosages can reduce the IL-1 beta level in the cell culture medium supernatant to different degrees, and shows significant difference compared with a model control group; PPTN and dexamethasone also show expected effects, which shows that the experimental results are true and reliable.
As can be seen from fig. 3: MSU causes synovial membrane P2Y in rats14The expression of the receptor protein is obviously increased, which indicates that the modeling is successful; different doses of the test composition can down-regulate synovial membrane P2Y to different degrees14The expression of receptor protein shows significant difference compared with a model control group; the PPTN also shows expected effect, which shows that the experimental result is real and credible. Dexamethasone was not shown to be P2Y14Modulation of receptor expression.
As can be seen from fig. 4: MSU causes the IL-1 beta level of the serum of the rat to be obviously increased, which prompts the success of modeling; the tested compositions with different dosages can reduce the level of the IL-1 beta in the serum to different degrees, and show significant difference compared with a model control group; PPTN and dexamethasone also show expected effects, which shows that the experimental results are true and reliable.

Claims (5)

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof:
Figure FDA0002994274300000011
the compound of the formula I is selected from the following compounds:
Figure FDA0002994274300000012
2. a process for the preparation of a compound of formula i as claimed in claim 1, comprising the steps of:
(1) the compound 1 in the general formula is reduced by nitro to prepare a compound 2 in the general formula;
(2) the compound 2 in the general formula is subjected to condensation reaction with different carboxylic acid or sulfonic acid derivatives or substitution reaction with different hydrocarbyl derivatives to obtain a compound 3 in the general formula;
(3) carrying out substitution reaction and deprotection reaction on the compound 3 with different hydrocarbyl derivatives to obtain a compound 4 with a general formula, namely a compound shown in a general formula (I);
Figure FDA0002994274300000021
R1、R2、R3and R4The method of claim 1.
3. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.
4. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 3 in the preparation of P2Y14The use of receptor inhibitors in medicine.
5. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 3 in the preparation of a medicament for the treatment of P2Y14The use in the preparation of medicaments for treating inflammatory diseases related to receptors.
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