CN117143019A - Pyrazole amide derivatives used as β2-adrenergic receptor allosteric antagonists - Google Patents

Abstract

The invention belongs to the field of pharmaceutical chemistry, and in particular relates to a beta-cell inhibitor ₂ Pyrazole amide derivatives of allosteric antagonists of the adrenergic receptors. The invention takes acetophenone with different substituents as raw material, and finally obtains a series of novel pyrazole derivatives through claisen condensation, cyclization, hydrolysis and amide coupling, the structure of the novel pyrazole derivatives is shown as formula I, wherein R is shown as the specification ₁ Is a hydrogen atom, a chlorine atom; r is R ₂ Is a hydrogen atom, a halogen atom, a nitro group or a methyl group; r is R ₃ Is that All the synthesized compounds were screened for the functional activity of G-protein dependent signaling pathway, and these novel derivatives were found to be useful as beta ₂ Allosteric antagonistic modulators of adrenergic receptors.

Description

Pyrazole amide derivatives as β2-adrenaline receptor allosteric antagonists

Technical Field

The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a pyrazole amide derivative and its application as a _β2 adrenergic receptor allosteric antagonist modulator.

Background Art

The G-protein coupled receptor (GPCR) superfamily is composed of structurally similar proteins arranged into families (categories). It is the largest druggable protein family in the human genome (more than 800). Because it is a type of membrane receptor protein with seven transmembrane structures, it is also called seven-α helices transmembrane protein receptor (7TM receptor), which has a very conserved spatial structure. The transmembrane domain composed of these seven transmembrane α helices (TM1~TM7) repeatedly passes through the lipid bilayer of the cell membrane, dividing the receptor into an extracellular N-terminus, an intracellular C-terminus, three extracellular loops (EL1~EL3) and three intracellular loops (ICL1~ICL3).

GPCRs exist only in eukaryotes and can be activated by extracellular molecules (including photosensitive compounds, odors, pheromones, hormones, and neurotransmitters) and cause cellular responses, so they play a vital role in cell signal transduction. In addition, GPCRs are also involved in a variety of physiological processes such as vision, taste, smell, behavior and emotional regulation, regulation of immune system activity and inflammation, autonomic nervous system transmission, cell density sensing, homeostasis regulation, and participation in the growth and metastasis of certain types of tumors. Because GPCRs are involved in many disease-related signaling pathways, including mental, metabolic, immune, cardiovascular, inflammatory, sensory disorders, and cancer diseases, it is a key drug target. About 40% of all FDA-approved drugs target GPCRs.

The exact size of the GPCR protein superfamily is unknown, but based on genome sequence analysis, it is predicted that the human genome has at least 831 different genes encoding it, which is about 4% of the entire protein-coding genome. Many classification schemes have been proposed for this protein family. The AF classification divides GPCRs into six categories based on sequence homology and functional similarity: Class A (Rhodopsin-like), Class B (Secretin receptor family), Class C (Metabotropicglutamate/pheromone), Class D (Fungal mating pheromone receptors), Class E (CyclicAMPreceptors), and Class F (Frizzled/Smoothened). Recently, an alternative classification system called GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, Secretin) has been proposed for vertebrate GPCRs, which correspond to Class C, Class A, Class B2, Class F, and Class B in the classic AF classification, respectively. Despite the lack of sequence homology between the classes, all GPCRs have a common structure and signal transduction mechanism. By far the largest class is class A, which accounts for nearly 85% of GPCR genes. Class A GPCRs are further subdivided into 19 subgroups (A1-A19). Among class A GPCRs, more than half of the members are expected to encode olfactory receptors. Beta-2 adrenergic receptor (β ₂ adrenoreceptor), also known as β ₂ AR, is a star member of the G protein-coupled receptor family and is widely expressed in blood vessels and bronchial smooth muscle. After being activated by endogenous agonists such as adrenaline, _{β 2} AR mediates cardiovascular function and pulmonary physiological processes and is an important target for the treatment of vascular and respiratory diseases. In addition, it is also crucial for overcoming immunosuppression and improving the efficacy of immunotherapy. The concept of allostery was first proposed in 1961 by Jacques Monod and Jacob and Jean-Pierre Changeux, due to the major breakthroughs in structural biology in recent years, the development of corresponding allosteric modulators targeting allosteric sites has become a breakthrough for the development of innovative drugs. Traditional drug development for GPCRs is mainly aimed at the orthosteric binding site (that is, the position where the endogenous ligand of the receptor binds). The high conservation of this site between different subtypes poses a great challenge to the development of selective drugs. Allosteric modulators bind outside the orthosteric ligand pocket of the receptor. Due to the relatively low conservation of their binding position, they may have better subtype selectivity and lower toxicity than orthosteric drugs targeting active sites; secondly, in terms of efficacy, allosteric agonists are less likely to induce desensitization of proteins after activation than orthosteric agonists; in addition, orthosteric modulators and allosteric modulators exert their efficacy based on different mechanisms of action, which makes it possible for allosteric modulators to overcome the acquired resistance of orthosteric drugs during treatment. Therefore, the discovery of allosteric modulators provides a new idea for obtaining highly selective drugs.

In 2017, our laboratory collaborated with scientists from Duke University in the United States to report a small molecule negative allosteric modulator compound 15 (Cmpd-15), which is the first intracellular allosteric antagonist of β ₂ -adrenaline receptor (Ahn S, et al. Proc. Natl. Acad. Sci. USA, 2017, 114: 1708-1713; Liu X, et al. Nature, 2017, 548: 480-484). However, since Cmpd-15 is a peptide compound with poor water solubility and low biological activity, its structure is relatively unstable, which may affect its drugability. Therefore, this project uses Cmpd-15 as the lead compound and uses the skeleton transition strategy, structural simplification and bioisostere concept for drug design. The synthesized new compounds were screened for biological activity in the classical signaling pathway (G-protein signaling) by GloSensor ^TM cAMPAccumulation assay (Binkowski BF et al. ACS Chem Biol. 2011; 6(11): 1193-1197). It is expected to obtain a series of new pyrazole derivatives with stable and simplified structures, novel skeletons, enhanced allosteric activity, improved water solubility and metabolic stability as allosteric modulators of β ₂ -AR.

Pyrazole nitrogen-containing heterocyclic compounds have a broad spectrum of pharmacological properties and are very important core skeletons in drug design. Based on this, in order to expand the structural types of lead compounds and to improve the biological activity, selectivity, water solubility and stability of drugs, the peptide core structure of Cmpd-15 was replaced with a pyrazole skeleton (as shown in the figure below), while the (S)-2-amino-3-(3-bromophenyl)-N-methylpropionamide on the right was kept unchanged, and a series of pyrazole derivatives were designed and synthesized (Meng K et al, Bioorg. Med. Chem. 2018, 26: 2320-2330).

The pyrazole derivatives designed and synthesized by our research group in the early stage all have allosteric antagonism on the G-protein signaling pathway of β ₂ adrenaline, and can negatively regulate the agonism of endogenous ligand isoproterenol (ISO) on β ₂ adrenaline. The results of cAMP accumulation experiments show that the allosteric antagonism of most compounds on β ₂ adrenaline receptors is significantly better than the functional activity of its lead compound Cmpd-15, and the water solubility of the new derivative compounds is significantly improved compared with Cmpd-15, and it is expected to be used as a lead compound for the treatment of new vascular diseases (Chen Xin, et al., China Invention Patent Publication No.: CN115745891A). However, it can be seen from the structural formula of this type of compound that the phenylalanine containing a chiral carbon atom on the right side has a large steric hindrance, which may affect the drugability.

Summary of the invention

Based on the problems pointed out in the background technology, in order to simplify the structure of Cmpd-15 and obtain compounds with allosteric antagonistic activity and better water solubility, the present invention designs to use simple substituted aniline, benzylamine or amines with nitrogen and oxygen heteroatoms to synthesize new pyrazole compounds, and studies the biological activity of the new derivatives to improve the drugability of compounds with allosteric antagonistic activity.

The purpose of the present invention is to provide a class of pyrazole amide derivatives, so as to develop new heterocyclic derivatives with stable chemical structure, high biological activity, receptor subtype selectivity and good water solubility as allosteric modulators of β ₂ -AR, and to provide a new direction for the research and development of new drugs for cardiovascular and cerebrovascular diseases, diabetes and cancer diseases.

The general structural formula of pyrazole amide derivatives is shown in Formula I:

Wherein, R ₁ = any one of H and Cl;

R ₂ = any one of H, F, Cl, NO ₂ , CH ₃ ;

Any one of .

The invention uses acetophenone with different substituents as raw materials, and finally obtains a series of new pyrazole amide derivatives through Claisen condensation, cyclization, ester hydrolysis and amide coupling reactions.

Table 1 Structures of pyrazole amide derivatives

Synthesis route of pyrazole amide derivatives:

The specific synthesis steps of pyrazole amide derivatives are as follows:

The specific synthesis steps are:

(1) Add an ethanol solution of sodium ethoxide (EtONa: 20% w/w) to a round-bottom flask, then add anhydrous ethanol, protect with nitrogen, ice bath to 0°C, use anhydrous ethanol to completely dissolve compound 1 with different substituents and diethyl oxalate, mix evenly, slowly add dropwise to the flask, and stir continuously. Continue to react for 30 minutes under ice bath conditions, then remove the ice bath and react at room temperature for 4 hours. After the reaction is completed as monitored by TLC, the resulting paste-like mixed solution is filtered under reduced pressure, and the filter cake is washed three times with an appropriate amount of anhydrous ethanol. After the filter cake is dried, compound 2 is obtained, wherein the molar ratio of sodium ethoxide, compound 1, and diethyl oxalate is 1:1:1.

(2) Add compound 2 to a round-bottom flask, add acetic acid, stir, then slowly add hydrazine hydrate (80%), heat to reflux, and react for 3 hours. After the reaction is completed as monitored by TLC, stop heating and cool to room temperature, add water, extract with ethyl acetate, and combine the organic phases. The organic phase is washed with saturated sodium bicarbonate solution, washed with saturated brine, and dried over anhydrous sodium sulfate. Remove the solvent, purify by silica gel column chromatography, eluent [V (petroleum ether): V (ethyl acetate) = 5:1], and obtain the target compound 3; wherein the molar ratio of compound 2 to hydrazine hydrate is 1:4.

(3) Compound 3 was added to a round-bottom flask, ethanol was added, stirred, NaOH solution was added dropwise, heated to reflux, and reacted for 1.5 hours. After the reaction was completed as monitored by TLC, heating was stopped, the mixture was cooled to room temperature, most of the ethanol was removed by rotary evaporation, and L of water was added to the resulting solution. Concentrated hydrochloric acid was slowly added dropwise under ice bath conditions to adjust the pH to 1-2. A white solid precipitated, which was filtered by suction. The filter cake was washed with an appropriate amount of water. The resulting crude product was recrystallized from a mixed solution of ethanol/water to obtain compound 4, wherein the molar ratio of compound 3 to NaOH was 1:16, and the volume ratio of ethanol to NaOH solution was 1:1.

(4) Compound 4, EDCl, and HOBt were weighed in sequence and added to a round-bottom flask. DMF was added and stirred at room temperature. Compound H ₂ NR ₃ was then added. After stirring at room temperature for 30 minutes, DIEA was added under ice bath conditions. Stirring was continued under ice bath conditions for 20 minutes and then reacted at room temperature overnight. After the reaction was complete as monitored by TLC, a saturated ammonium chloride solution was added. After sufficient stirring and mixing, the mixture was extracted with ethyl acetate. The organic phases were combined, washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain a white solid crude product, which was recrystallized from an appropriate amount of a mixed solvent of ethyl acetate/ethanol/petroleum ether to obtain the target compound 5, wherein the molar ratio of compound 4, EDCl, HOBt, H ₂ NR ₃ , and DIEA was 1:1 to 1.1:1 to 1.1:1 to 1.2:3.

New derivatives of pyrazoles can act as allosteric antagonists of β ₂ -AR;

Furthermore, compounds J ₁ , J ₂ , J ₁₂ , J ₁₃ , J _{15 ,} J ₁₆ , J 17 , J ₁₈ , J ₁₉ , J ₂₁ , J ₂₂ , and J ₂₅ in Table 1 have allosteric antagonist activity and can be used as allosteric antagonists of β ₂ -AR.

The beneficial effects of the present invention are:

Most of the new pyrazole derivatives synthesized by the present invention have β ₂ -AR allosteric antagonist activity. Compared with the lead compound Cmpd-15, the allosteric antagonist activity of compounds J ₂₁ , J ₂₂ and J ₂₅ on β ₂ -AR is significantly improved. The new pyrazole derivatives synthesized by the present invention have a simple structure, a simple synthesis route, and readily available raw materials, and can be industrially produced. The new pyrazole amide derivatives of the present invention can be used as β ₂ -AR allosteric antagonists, providing a new direction for the research and development of new drugs for cardiovascular and cerebrovascular diseases, diabetes and cancer diseases.

Description of the drawings:

FIG1 is a graph showing the dose-response curve of ISO mediated by _J25 .

DETAILED DESCRIPTION

Synthesis route of pyrazole amide derivatives:

Embodiment 1:

Preparation of N-benzyl-5-(4-fluorophenyl)-1H-pyrazole-3-carboxamide J ₁

Step 1: Preparation of ethyl 4-(4-fluorophenyl)-2,4-dioxobutyrate

Add an ethanol solution of sodium ethoxide (3.4 g, 10 mmol, EtONa: 20% w/w) to a 100 mL round-bottom flask, then add 10 mL of anhydrous ethanol, protect with nitrogen, ice bath to 0 ° C, use 15 mL of anhydrous ethanol to completely dissolve 4-fluoroacetophenone (10 mmol) and diethyl oxalate (1.46 g, 10 mmol), mix evenly, slowly drop into the flask, and stir continuously. Continue to react for 30 minutes under ice bath conditions, then remove the ice bath and react at room temperature for 4 hours. After the reaction is completed by TLC monitoring, the resulting paste-like mixed solution is filtered under reduced pressure, and the filter cake is washed three times with an appropriate amount of anhydrous ethanol. After the filter cake is dried, 4-(4-fluorophenyl)-2,4-dioxobutyric acid ethyl ester is obtained as a reddish brown solid with a yield of 30%.

Step 2: Preparation of ethyl 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylate

Add 4-(4-fluorophenyl)-2,4-dioxobutyric acid ethyl ester (2mmol) to a 50mL round-bottom flask, add 10mL of acetic acid, stir, then slowly add hydrazine hydrate (0.5g, 8mmol, 4eq, 80%), heat to reflux, and react for 3h. After the reaction is completed by TLC monitoring, stop heating and cool to room temperature, add 20mL of water, extract with ethyl acetate (3×30mL), and combine the organic phases. The organic phase is washed with saturated sodium bicarbonate solution (2×20mL), washed once with 20mL of saturated brine, and dried over anhydrous sodium sulfate. Remove the solvent and purify by silica gel column chromatography, eluent [V (petroleum ether): V (ethyl acetate) = 5:1] to obtain compound 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid ethyl ester as a light yellow solid with a yield of 93%. ¹ H NMR (300MHz, DMSO-d ₆ ): δ12.06 (br, 1H), 7.71-7.78 (m, 2H), 7.01-7.15 (m, 3H), 4.30-4.43 (m, 2H), 1.31-1.42 (m, 3H). ¹³ CNMR (75MHz, CDCl ₃ ) δ 164.6, 161. 3,160.9,147.7,139.9,127.6,127.5,126.8,116.1,115.8,105.1,61.3,14.2.MS(ESI):m/z 235(M+1).

Step 3: Preparation of 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid

Compound 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid ethyl ester (1mmol) was added to a 50mL round-bottom flask, ethanol (4mL) was added, stirred, 4mL of 4mol/L NaOH solution was added dropwise, heated to reflux, and reacted for 1.5h. After the reaction was completed by TLC monitoring, heating was stopped, cooled to room temperature, most of the ethanol was removed by rotary evaporation, 10mL of water was added to the resulting solution, concentrated hydrochloric acid was slowly added dropwise under ice bath conditions, pH was adjusted to 1-2, a white solid was precipitated, suction filtered, the filter cake was washed twice with an appropriate amount of water, and the resulting crude product was recrystallized from a mixed solution of ethanol/water to obtain 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid as a white solid with a yield of 91%.

Step 4: Preparation of N-benzyl-5-(4-fluorophenyl)-1H-pyrazole-3-carboxamide

The compound 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid (129 mg, 0.5 mmol), EDCl (105 mg, 0.55 mmol, 1.1 eq), and HOBt (74 mg, 0.55 mmol, 1.1 eq) were weighed in sequence and added to a 25 mL round-bottom flask, and 5 mL of DMF was added. The mixture was stirred at room temperature, and then benzylamine (0.6 mmol, 1.2 eq) was added. After stirring at room temperature for 30 minutes, DIEA (194 mg, 1.5 mmol, 3 eq) was added under ice bath conditions, and the mixture was stirred for 20 minutes under ice bath conditions and then reacted overnight at room temperature. The reaction was complete as monitored by TLC, and 20 mL of saturated ammonium chloride solution was added. After sufficient stirring and mixing, the mixture was extracted with ethyl acetate (3×30 mL), and the organic phases were combined, washed with water (2×20 mL) and saturated brine (20 mL), respectively, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain a white solid crude product, which was recrystallized from an appropriate amount of ethyl acetate/ethanol/petroleum ether mixed solvent to obtain the target compound N-benzyl-5-(4-fluorophenyl)-1H-pyrazole-3-carboxamide as a white solid with a yield of 71%. ¹ H NMR (300 MHz, DMSO-d ₆ ): δ13.69 (s, 1H), 8.81 (br, 1H), 7.82-7.86 (m, 2H), 7.14-7.34 (m, 8H), 5.47 (d, J=6.1 Hz, 2H); MS (ESI): m/z 296 (M+1).

Embodiment 2:

Preparation of 5-(4-fluorophenyl)-N-(pyridine-2-methyl)-1H-pyrazole-3-carboxamide J ₂

In step 4 of Example 1, benzylamine was replaced with pyridine-2-methylamine, and the other conditions were the same as those of Example 1. A white solid was obtained with a yield of 81%. Mp 205.5-206.4°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ13.73 (br, 1H), 8.88 (br, 1H), 8.52 (dd, J＝4.8, 0.8 Hz, 1H), 7.74-7.88 (m, 3H), 7.18-7.35 (m, 5H), 4.58 (d, J＝6.0 Hz, 2H); MS (ESI): m/z 297 (M+1).

Embodiment 3:

Preparation of N-(3,4-dimethoxybenzyl)-5-(4-fluorophenyl)-1H-pyrazole-3-carboxamide J ₃

Other conditions were the same as those in Example 1, except that in step 4, benzylamine was replaced with 3,4-dimethoxybenzylamine to obtain a white solid with a yield of 73%. Mp 205.1-206.1°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ13.67 (s, 1H), 8.69 (br, 1H), 7.81-7.86 (m, 2H), 7.28-7.33 (m, 2H), 7.13 (s, 1H), 6.84-6.97 (m, 3H), 4.40 (d, J=5.9 Hz, 2H), 3.74 (s, 3H), 3.72 (m, 3H); MS (ESI): m/z 356 (M+1).

Embodiment 4:

Preparation of N-(2-chlorobenzyl)-5-(4-fluorophenyl)-1H-pyrazole-3-carboxamide J ₄

Other conditions were the same as those in Example 1, except that in step 4, benzylamine was replaced with 2-chlorobenzylamine to obtain a white solid with a yield of 74%. ¹ HNMR (300 MHz, DMSO-d ₆ ): δ13.73 (s, 1H), 9.81 (br, 1H), 7.83-7.87 (m, 2H), 7.13-7.47 (m, 7H), 4.53 (d, J=5.8 Hz, 2H); MS (ESI): m/z 330 (M+1).

Embodiment 5:

Preparation of 5-(4-fluorophenyl)-N-(3-nitrobenzyl)-1H-pyrazole-3-carboxamide J ₅

Other conditions were the same as those in step 4 of Example 1, except that benzylamine was replaced with 3-nitrobenzylamine. The product was a light yellow solid with a yield of 82%. Mp 255.3-256.1°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ13.66 (br, 1H), 9.10 (s, 1H), 8.20 (s, 1H), 8.12 (d, J＝9.4 Hz, 1H), 7.79-7.87 (m, 3H), 7.64 (t, J＝8.0 Hz, 1H), 7.31 (t, J＝8.9 Hz, 2H), 7.15 (s, 1H), 4.58 (d, J＝6.2 Hz, 2H); MS (ESI): m/z 341 (M+1).

Embodiment 6:

Preparation of N-(3-nitrobenzyl)-5-(p-tolyl)-1H-pyrazole-3-carboxamide J ₆

Step 1: Preparation of ethyl 2,4-diketo-4-(p-tolyl)butyrate

The method is the same as step 1 of Example 1, except that 4-fluoroacetophenone is replaced by 4-methylacetophenone to obtain a light yellow solid with a yield of 45%.

Step 2: Preparation of ethyl 5-(p-tolyl)-1H-pyrazole-3-carboxylate

The method is the same as step 2 of Example 1, except that 4-(4-fluorophenyl)-2,4-dioxobutyric acid ethyl ester is replaced with 2,4-diketo-4-(p-tolyl)butyric acid ethyl ester to obtain a bright yellow solid with a yield of 80%. ¹ H NMR (300MHz, CDCl ₃ ): δ11.95 (br, 1H), 7.61 (d, J = 8.0Hz, 2H), 7.21 (d, J = 8.0Hz, 2H), 7.02 (s, 1H), 4.31 (q, J = 7.1Hz, 2H), 2.37 (s, 3H), 1.32 (t, J = 7.1Hz, 3H) . ¹³ C NMR (75MHz, CDCl ₃ ) δ 161.2, 147.9, 138.6, 129.6, 127.5, 125.7, 105.0, 61.1, 21.4, 14.2. MS (ESI): m/z 231 [M+1].

Step 3: Preparation of 5-(p-tolyl)-1H-pyrazole-3-carboxylic acid

The method is the same as step 3 of Example 1. 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid ethyl ester is replaced by 5-(p-tolyl)-1H-pyrazole-3-carboxylic acid ethyl ester to obtain a white solid with a yield of 93%. ¹ H NMR (300 MHz, DMSO-d ₆ ): δ7.71 (d, J=8.1 Hz, 2H), 7.24 (d, J=8.1 Hz, 2H), 7.12 (s, 1H), 2.31 (s, 3H).

Step 4: Preparation of N-(3-nitrobenzyl)-5-(p-tolyl)-1H-pyrazole-3-carboxamide

The method is the same as step 4 of Example 1, except that 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid is replaced by 5-(p-tolyl)-1H-pyrazole-3-carboxylic acid, and benzylamine is replaced by 3-nitrobenzylamine to obtain a white solid with a yield of 74%. Mp 262.3-263.1°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ13.69 (br, 1H), 9.03 (br, 1H), 8.19 (s, 1H), 8.11 (d, J＝8.1 Hz, 1H), 7.60-7.80 (m, 4H), 7.26 (d, J＝7.9 Hz, 2H), 7.06 (s, 1H), 4.57 (d, J＝5.9 Hz, 2H), 2.32 (s, 3H); MS (ESI): m/z 337 (M+1).

Embodiment 7:

Preparation of N-(pyridine-2-methylene)-5-(p-tolyl)-1H-pyrazole-3-carboxamide J ₇

The method is the same as that of Example 6, except that in step 4, 3-nitrobenzylamine is replaced by 2-pyridinemethylamine to obtain a white solid with a yield of 85%. ¹ H NMR (300 MHz, DMSO-d ₆ ): δ13.65 (br, 1H), 9.11 (br, 1H), 8.51 (d, J=6.1 Hz, 1H), 7.77 (td, J=7.7, 1.7 Hz, 1H), 7.68 (d, J=8.1 Hz, 2H), 7.25-7.33 (m, 4H), 7.06 (s, 1H), 4.56 (d, J=6.0 Hz, 2H), 2.33 (s, 3H); MS (ESI): m/z 293 (M+1).

Embodiment 8:

Preparation of N-(3,4-dimethoxybenzyl)-5-(p-tolyl)-1H-pyrazole-3-carboxamide J ₈

The method is the same as that of Example 6, except that 3-nitrobenzylamine is replaced with 3,4-dimethoxybenzylamine in step 4 to obtain a white solid with a yield of 77%. 1H NMR (300MHz, DMSO-d6): δ13.60 (s, 1H), 8.65 (br, 1H), 7.67 (d, 8.1Hz, 2H), 7.26 (d, 8, 1Hz, 2H), 6.82-7.03 (m, 4H), 4.38 (d, 5.9Hz, 2H), 3.73 (s, 3H), 3.71 (s, 3H), 2.32 (s, 3H); MS (ESI): m/z 352 (M+1).

Embodiment 9:

Preparation of N-benzyl-5-(p-tolyl)-1H-pyrazole-3-carboxamide J ₉

Other conditions were the same as those in Example 6. In step 4, 3-nitrobenzylamine was replaced with benzylamine. The product was a white solid with a yield of 75%. ¹ H NMR (300MHz, DMSO-d ₆ ): δ13.62 (s, 1H), 8.77 (br, 1H), 7.68 (d, J = 8.0Hz, 2H), 7.25-7.33 (m, 7H), 7.04 (s, 1H), 4.46 (d, J = 5.5Hz, 2H), 2.32 (s, 3H); ¹³ C NMR (75MHz, DMSO-d ₆ ): δ162.27,148.12,144.01,140.33,138.44,130.03,128.71,127.71,127.16,126.46,125.64,102.73,42.41,21.27; MS (ESI): m/z 292( M+1).

Embodiment 10:

Preparation of N-(2-chlorobenzyl)-5-(p-tolyl)-1H-pyrazole-3-carboxamide J ₁₀

Other conditions were the same as those in Example 6, except that in step 4, 3-nitrobenzylamine was replaced with 2-chlorobenzylamine, and the product was a white solid with a yield of 73%. ¹ H NMR (300 MHz, DMSO-d ₆ ): δ13.66 (br, 1H), 8.78 (br, 1H), 7.69 (d, J＝8.1 Hz, 2H), 7.26-7.46 (m, 6H), 7.06 (s, 1H), 4.52 (d, J＝5.9 Hz, 2H), 2.32 (s, 3H); MS (ESI): m/z 326 (M+1).

Embodiment 11:

Preparation of N-benzyl-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide J ₁₁

Step 1: Preparation of ethyl 4-(4-nitrobenzene)-2,4-dioxobutanoate

The method is the same as step 1 of Example 1. 4-Fluoroacetophenone is replaced by 4-nitroacetophenone to obtain a light yellow solid powder with a yield of 49%. ¹ H NMR (300 MHz, CDCl ₃ ): δ8.36 (d, J＝9.2 Hz, 2H), 8.17 (d, J＝9.2 Hz, 2H), 7.11 (s, 1H), 4.43 (q, J＝7.3 Hz, 2H), 1.43 (t, J＝7.3 Hz, 3H).

Step 2: Preparation of ethyl 5-(4-nitrophenyl)-1H-pyrazole-3-carboxylate

The method is the same as step 2 of Example 1. Replace 4-(4-fluorophenyl)-2,4-dioxobutyric acid ethyl ester with 4-(4-nitrobenzene)-2,4-dioxobutyric acid ethyl ester to obtain a yellow solid with a yield of 65%. ¹ H NMR (300 MHz, DMSO-d ₆ ): δ14.40 (s, 1H), 4.28 (d, J＝7.6 Hz, 2H), 8.16 (d, J＝7.6 Hz, 2H), 7.54 (s, 1H), 4.36 (q, J＝7.1 Hz, 2H), 1.34 (t, J＝7.1 Hz, 3H).

Step 3: Preparation of 5-(4-nitrophenyl)-1H-pyrazole-3-carboxylic acid

The method is the same as step 3 of Example 1. 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid ethyl ester is replaced by 5-(4-nitrophenyl)-1H-pyrazole-3-carboxylic acid ethyl ester to obtain a light yellow solid with a yield of 68%. ¹ H NMR (300 MHz, DMSO-d ₆ ): δ14.21 (s, 1H), 13.65 (s, 1H), 8.29 (d, J＝8.9 Hz, 2H), 8.15 (d, J＝8.9 Hz, 2H), 7.47 (s, 1H).

Step 4: Preparation of N-benzyl-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide

Other conditions were the same as those in step 4 of Example 1, except that 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid was replaced with 5-(4-nitrophenyl)-1H-pyrazole-3-carboxylic acid. The product was a light yellow solid with a yield of 70%. Mp 258.4-258.8°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ 9.04 (s, 1H), 8.32 (d, J = 8.9 Hz, 2H), 8.06 (d, J = 8.9 Hz, 2H), 7.44 (s, 1H), 7.24-7.35 (m, 5H), 4.49 (d, J = 6.1 Hz, 2H); MS (ESI): m/z 323 (M+1).

Embodiment 12:

Preparation of N-(2-morpholinoethyl)-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide J ₁₂

Other conditions were the same as those in Example 11, except that in step 4, benzylamine was replaced with 2-morpholinoethyl-1-amine, and the product was a white solid with a yield of 29%. Mp 171.1-172.3°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ14.00 (s, 1H), 8.32 (d, J=8.8 Hz, 2H), 8.06 (d, J=8.8 Hz, 2H), 7.37 (s, 1H), 3.58 (t, J=4.5 Hz, 4H), 3.39-3.43 (m, 4H), 2.41-2.49 (m, 4H); MS (ESI): m/z 346 (M+1).

Embodiment 13:

Preparation of N-(Benzo[d][1,3]dioxol-5-ylmethyl)-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide J ₁₃

Other conditions were the same as those in Example 11. In step 4, benzylamine was replaced with 3,4-(methylenedioxy)benzylamine. The product was a yellow solid with a yield of 26%. Mp 248.2-249.1°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ13.98 (s, 1H), 9.27 (t, J＝5.9 Hz, 1H), 8.85 (s, 1H), 7.27-7.90 (m, 6H), 7.06 (s, 1H), 4.58 (s, 2H); ¹³ C NMR (75 MHz, DMSO-d ₆ ): δ162.10,159.16,149.40,147.59,139.66,137.28,134.56,132.62,132.13,130.39,128.29,127.40,122.72,121.68,107.03,44.60; MS (ESI): m/z 367(M+1).

Embodiment 14:

Preparation of 5-(4-nitrophenyl)-N-(pyridine-2-methyl)-1H-pyrazole-3-carboxamide J ₁₄

Other conditions were the same as those in Example 11, except that in step 4, benzylamine was replaced with pyridine-2-methylamine, and the product was a light yellow solid with a yield of 33%. Mp 258.8-259.6°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ14.08 (s, 1H), 9.24 (s, 1H), 8.54 (s, 1H), 8.32 (d, J＝8.2 Hz, 2H), 8.08 (s, 2H), 7.78 (t, J＝7.1 Hz, 1H), 7.29-7.38 (m, 3H), 4.60 (s, 2H); MS (ESI): m/z 324 (M+1).

Embodiment 15:

Preparation of Morpholino (5-(4-nitrophenyl)-1H-pyrazol-3-yl)methanone J ₁₅

Other conditions were the same as those in Example 11, except that benzylamine was replaced by morpholine in step 4, and the product was a white solid with a yield of 35%. Mp 275.0-275.7°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ 13.98 (s, 1H), 8.31 (d, J = 8.6 Hz, 2H), 8.11 (d, J = 8.6 Hz, 2H), 7.32 (s, 1H), 3.90 (s, 1H), 3.66 (s, 7H); MS (ESI): m/z 303 (M+1).

Embodiment 16:

Preparation of N-(3,4-dimethoxybenzyl)-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide J ₁₆

Other conditions were the same as those in Example 11, except that in step 4, benzylamine was replaced with 3,4-dimethoxybenzylamine, and the product was a light yellow solid with a yield of 73%. Mp 196.4-197.2°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ8.96 (br, 1H), 8.32 (d, J＝8.8 Hz, 2H), 8.06 (d, J＝8.8 Hz, 2H), 6.84-6.98 (m, 3H), 4.41 (d, J＝5.9 Hz, 2H), 3.75 (s, 3H), 3.73 (s, 3H); MS (ESI): m/z 383 (M+1).

Embodiment 17:

Preparation of N-(3-nitrobenzyl)-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide J ₁₇

Other conditions were the same as those in Example 11, except that in step 4, benzylamine was replaced with 3-nitrobenzylamine, and the product was a light yellow solid with a yield of 80%. Mp 279.5-280.2°C; ¹ HNMR (300 MHz, DMSO-d ₆ ): δ9.20 (br, 1H), 8.33 (d, 8.9 Hz, 2H), 8.22 (s, 1H), 8.14 (d, 8.1 Hz, 1H), 8.08 (d, 8.9 Hz, 2H), 7.66 (t, 7.9 Hz, 1H), 4.61 (d, 5.9 Hz, 2H); MS (ESI): m/z 368 (M+1).

Embodiment 18:

Preparation of 5-(2,4-dichlorophenyl)-N-(2-morpholinoethyl)-1H-pyrazole-3-carboxamide J ₁₈

Step 1: Preparation of ethyl 4-(2,4-dichlorophenyl)-2,4-dioxobutanoate

Other conditions were the same as those in step 1 of Example 1, except that 4-fluoroacetophenone was replaced with 2,4-dichloroacetophenone. A light yellow solid powder was obtained with a yield of 40%. ¹ H NMR (300 MHz, DMSO-d ₆ ): δ7.34-7.52 (m, 3H), 5.66 (br, 1H), 4.71 (s, 1H), 4.07 (q, J=7.1 Hz, 2H), 1.20 (t, J=7.1 Hz, 3H).

Step 2: Preparation of ethyl 5-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylate

The method is the same as step 2 of Example 1. Replace 4-(4-fluorophenyl)-2,4-dioxobutyric acid ethyl ester with 4-(2,4-dichlorophenyl)-2,4-dioxobutyric acid ethyl ester to obtain a white solid with a yield of 70%. ¹ H NMR (300 MHz, CDCl ₃ ): δ11.92 (br, 1H), 7.69 (d, J＝8.4 Hz, 1H), 7.49 (d, J＝2.1 Hz, 1H), 7.27-7.32 (m, 2H), 7.41 (q, J＝7.1 Hz, 2H), 1.40 (t, J＝7.1 Hz, 3H).

Step 3: Preparation of 5-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylic acid

The method is the same as step 3 of Example 1. 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid ethyl ester is replaced by 5-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylic acid ethyl ester to obtain a white solid with a yield of 72%. ¹ H NMR (300 MHz, DMSO-d ₆ ): δ13.76 (br, 2H), 7.81 (d, J＝8.3 Hz, 1H), 7.75 (s, 1H), 7.53 (d, J＝8.3 Hz, 1H), 7.18 (s, 1H).

Step 4: Preparation of 5-(2,4-dichlorophenyl)-N-(2-morpholinoethyl)-1H-pyrazole-3-carboxamide

Other conditions were the same as those in step 4 of Example 1, except that 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid was replaced by 5-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylic acid, and benzylamine was replaced by 2-morpholinoethyl-1-amine. The product was a white solid with a yield of 65%. Mp 249.3-250.1; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ 13.84 (s, 1H), 8.53 (s, 1H), 7.77 (s, 2H), 7.55 (d, J=7.8 Hz, 1H), 7.40 (s, 1H), 3.58 (t, J=4.5 Hz, 4H), 3.37-3.42 (m, 4H), 2.42-2.49 (m, 4H); MS (ESI): m/z 369 (M+1).

Embodiment 19:

Preparation of [5-(2,4-dichlorophenyl)-1H-pyrazol-3-yl]-4-morpholinone J ₁₉

Steps 1, 2 and 3 are the same as those in Example 18, and in step 4, benzylamine is replaced by morpholine. Other conditions are the same as those in step 4 of Example 18. The product is a white solid with a yield of 75%. Mp 204.3-205.5°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ13.89 (s, 1H), 7.50-7.82 (m, 3H), 7.01 (s, 1H), 4.02 (s, 1H), 3.65 (s, 7H); MS (ESI): m/z 326 (M+1).

Embodiment 20:

Preparation of 5-(2,4-dichlorophenyl)-N-(pyridine-2-methyl)-1H-pyrazole-3-carboxamide J ₂₀

Other conditions were the same as those in Example 18. In step 4, benzylamine was replaced with pyridine-2-methylamine. The product was a white solid with a yield of 58%. Mp 242.5-243.3°C; ¹ H NMR (300 MHz, DMSO-d ₆ ): δ9.27 (t, J=5.9 Hz, 1H), 8.52 (d, J=4.3 Hz, 1H), 7.70-7.90 (m, 3H), 7.50-7.62 (m, 1H), 7.27-7.38 (m, 2H), 7.06 (s, 1H), 4.58 (s, 2H); ¹³ C NMR (75 MHz, DMSO-d ₆ ): δ162.10,159.01,149.40,147.59,139.66,137.28,134.56,132.62,132.13,130.39,128.29,127.40,122.72,121.68,107.03,44.60; MS (ESI): m/z 347(M+1).

Embodiment 21:

Preparation of (5-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)(piperidin-1-yl)methanone J ₂₁

Other conditions were the same as those in Example 18. In step 4, benzylamine was replaced with piperidine. The product was a white solid with a yield of 18%. ¹ HNMR (400 MHz, DMSO-d ₆ ) δ 7.76–7.73 (m, 2H), 7.54 (dd, J=8.4, 2.2 Hz, 1H), 6.92 (s, 1H), 3.74 (s, 2H), 3.60 (d, J=6.7 Hz, 2H), 1.66–1.61 (m, 2H), 1.56–1.50 (m, 4H). MS (ESI): m/z 324 (M+1).

Embodiment 22:

Preparation of N-cyclohexyl-5-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxamide J ₂₂

Other conditions were the same as those in Example 18, except that in step 4, benzylamine was replaced with cyclohexylamine, and the product was a white solid with a yield of 74%. ¹ HNMR (400 MHz, DMSO-d ₆ ) δ 13.79 (s, 1H), 8.21 (s, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 2.2 Hz, 1H), 7.52 (dd, J = 8.5, 2.2 Hz, 1H), 7.31 (s, 1H), 3.78–3.71 (m, 1H), 1.83–1.79 (m, 2H), 1.73–1.71 (m, 2H), 1.60 (d, J = 12.8 Hz, 1H), 1.36–1.25 (m, 4H), 1.16–1.07 (m, 1H). MS (ESI): m/z 338 (M+1).

Embodiment 23:

Preparation of N-(3-bromophenyl)-3-phenyl-1H-pyrazole-5-carboxamide J ₂₃

Step 1: Preparation of ethyl 2,4-diketo-4-phenylbutyrate

The method is the same as step 1 of Example 1. 4-Fluoroacetophenone is replaced by acetophenone to obtain a crude product of ethyl 2,4-diketo-4-phenylbutyrate.

Step 2: Preparation of ethyl 5-phenyl-1H-pyrazole-3-carboxylate

The method is the same as step 2 of Example 1. Replace 4-(4-fluorophenyl)-2,4-dioxobutyric acid ethyl ester with 2,4-diketo-4-phenylbutyric acid ethyl ester to obtain a white solid with a yield of 80%. ¹ H NMR (400 MHz, CDCl ₃ ) δ7.73-7.67 (m, 2H), 7.40-7.29 (m, 3H), 6.97 (s, 1H), 4.17 (q, J＝7.1 Hz, 2H), 1.19 (t, J＝7.1 Hz, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ161.2, 147.7, 140.7, 130.2, 129.0, 128.6, 125.8, 105.2, 61.1, 14.1, 1.1. MS (ESI): m/z 217 (M+1).

Step 3: Preparation of 5-phenyl-1H-pyrazole-3-carboxylic acid

The method is the same as step 3 of Example 1. 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid ethyl ester is replaced by 5-phenyl-1H-pyrazole-3-carboxylic acid ethyl ester to obtain a white solid with a yield of 90%. ¹ H NMR (400 MHz, CD ₃ OD) δ 7.79-7.72 (m, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.40-7.31 (m, 1H), 7.12 (s, 1H).

Step 4: Preparation of N-(3-bromophenyl)-3-phenyl-1H-pyrazole-5-carboxamide

The other conditions were the same as those in step 4 of Example 1, except that 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid was replaced by 5-phenyl-1H-pyrazole-3-carboxylic acid, and benzylamine was replaced by m-bromoaniline. The product was a white solid with a yield of 53%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ13.86 (s, 1H), 10.33 (s, 1H), 8.18 (s, 1H), 7.85 (d, J = 7.6Hz, 3H), 7.49 (t, J = 7.6Hz, 2H), 7.41–7.24 (m, 4H). ¹³ C NMR (101MHz, DMSO) δ1 60.6,147.6,144.0,140.6,130.6,129.1,128.9,128.7,128.6,126.0,125.5,125.1,122.5,121.4,119.0,103.4ppm.MS(ESI):m/z342(M+1).

Embodiment 24:

Preparation of N-(3-bromobenzyl)-3-phenyl-1H-pyrazole-5-carboxamide J ₂₄

Step ^{1, step 2 and step 3 are the same as those in Example 23. In step 4, benzylamine is replaced by m-bromobenzylamine. Other conditions are the same as those in step 4 of Example 23. The product is a white solid with a yield of 53%. 1} H NMR (400 MHz, DMSO-d ₆ ) δ 8.95 (s, 1H), 7.77–7.75 (m, 2H), 7.48–7.39 (m, 4H), 7.35–7.29 (m, 2H), 7.28–7.24 (m, 1H), 7.11 (s, 1H), 4.42 (s, 2H). MS (ESI): m/z 356 (M+1).

Embodiment 25:

Preparation of N-phenyl-3-(M-tolyl)-1H-pyrazole-5-carboxamide J ₂₅

Step 1: Preparation of ethyl 2,4-diketo-4-(m-tolyl)butyrate

The method is the same as step 1 of Example 1. 4-Fluoroacetophenone is replaced by 3-methylacetophenone to obtain crude product 2,4-diketo-4-(m-tolyl)butyric acid ethyl ester.

Step 2: Preparation of ethyl 5-(m-tolyl)-1H-pyrazole-3-carboxylate

The method is the same as step 2 of Example 1. Replace 4-(4-fluorophenyl)-2,4-dioxobutyric acid ethyl ester with 2,4-diketo-4-(m-tolyl)butyric acid ethyl ester, white solid, yield 76%. ¹ HNMR (300MHz, CDCl ₃ ) δ7.54–7.50(m,2H),7.27(t,J＝7.6Hz,1H),7.15–7.13(m,1H),7.02(s,1H),4.25(q,J＝7.1Hz,2H),2.35(s,3H),1.26(t,J＝7.1Hz,3H ). ¹³ C NMR (75MHz, CDCl ₃ ) δ161.2,148.1,140.5,138.6,130.2,129.4,128.9,126.5,122.9,105.2,61.1,21.5,14.2.MS(ESI): m/z 231(M+1).

Step 3: Preparation of 5-(m-tolyl)-1H-pyrazole-3-carboxylic acid

The method is the same as step 3 of Example 1. 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid ethyl ester is replaced by 5-(m-tolyl)-1H-pyrazole-3-carboxylic acid ethyl ester to obtain a white solid with a yield of 40%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.68 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.16 (d, J = 7.1 Hz, 2H), 2.35 (s, 3H).

Step 4: Preparation of N-phenyl-3-(M-tolyl)-1H-pyrazole-5-carboxamide

Other conditions were the same as those in step 4 of Example 1, except that 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid was replaced by 5-(m-tolyl)-1H-pyrazole-3-carboxylic acid, and benzylamine was replaced by aniline. The product was a white solid with a yield of 20%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 13.79 (s, 1H), 10.14 (s, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.69 (s, 1H), 7.64 (d, J=7.8 Hz, 1H), 7.39-7.34 (m, 1H), 7.20 (d, J=7.6 Hz, 1H), 7.13 (t, J=7.4 Hz, 1H), 2.38 (s, 1H). MS (ESI): m/z 278 (M+1).

Biological activity test

cAMP is a key signaling molecule for many G protein-coupled receptors. The accumulation level of cAMP is mainly tested using the GloSensor method, a bioluminescent cAMP biosensor (Promega). HEK293T cells in good condition are seeded in 6-well plates or 35MM cell culture dishes and placed in a 37°C, 5% CO ₂ incubator for 24 hours to allow the cells to adhere to the wall and grow. Then, the β ₂ adrenergic receptor plasmid and pGloSensor ^TM -22FcAMP plasmid are transferred into the cells using a transfection reagent and placed in a 37°C, 5% CO ₂ incubator for 24 hours to allow the target gene to be transcribed and expressed. Then, the cells transfected with the plasmid are evenly seeded in a 96-well plate and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. Finally, discard the old culture medium in the 96-well plate, wash the cells with new culture medium, and then add the balanced culture medium containing GloSensor ^TM cAMP Reagent, serum and CO ₂ -independent medium and culture at 37°C, 5% CO ₂ incubator for 1-2 hours or until a stable background signal is obtained. Dissolve isoproterenol (ISO) with DMSO, sterilize and filter it, and dilute it with culture medium to different concentration gradients (DMSO concentration in the prepared compound is less than or equal to 0.1%). Quickly add the prepared compound to the 96-well plate containing HEK293T cells to start drug administration stimulation. The rapid rise of bioluminescent signal after adding the compound can be observed by a multifunctional microplate reader. When the signal value rises to the peak and no longer increases, the compound with a concentration of 50μM is immediately added. At the same time, the bioluminescent signal is immediately collected with a multifunctional microplate reader, and the bioluminescent signal value increases with the increase of compound concentration. Finally, the data is sorted and processed with GraphPadPrism8 software to obtain the dose-effect curve of the allosteric modulator with concentration as the horizontal axis and signal value as the vertical axis.

Table 2 Statistics of functional activities of new pyrazole derivatives

Note: "+" indicates corresponding activity and "-" indicates no corresponding activity

Table 3 Comparison of the activity of the new derivatives with allosteric antagonistic activity and Cmpd-15

Note: ^aThe values are expressed relative to the blocking activity of Cmpd-15

In the Glosensor cAMP accumulation test results, new compounds J ₁ , J ₂ , J ₁₂ , J ₁₃ , J 15, J ₁₆ , J ₁₇ , J ₁₈ , J ₁₉ , J ₂₁ , J ₂₂ , and _{J 25 all} have β ₂ AR allosteric antagonism, and the remaining new compounds have no allosteric antagonism activity (see Table 2). Compared with the lead compound Cmpd-15, the allosteric antagonism activity of the above _- mentioned compounds with allosteric antagonism is comparable to that of Cmpd-15, and the allosteric antagonism activity of J ₂₁ , J ₂₂ , and J ₂₅ is significantly improved compared with that of Cmpd-15, among which J ₂₅ has the best activity (see Table 3).

As can be seen from Table 3, among all the new pyrazole compounds, compound J ₂₅ has the best allosteric antagonistic activity, which is about 2.5 times that of Cmpd-15. When R ₂ is a hydrogen atom or methyl substituted at the 4th position, the pyrazole amide compounds do not have allosteric antagonistic activity; when R ₂ is fluorine substituted at the 4th position, the coupling activity is better when coupled with benzylamine, but there is no activity if it is coupled with benzylamines with different substitutions. In addition, the allosteric antagonistic activity is not significantly improved when coupled with 2-pyridinemethylamine; when R ₂ is nitro substituted at the 4th position, most of them have allosteric antagonistic activity, but the activity is not as good as Cmpd-15. When R ₁ and R ₂ are chlorine substituted at the same time, the product after coupling with morpholine, cyclohexylamine, and piperidine has the same activity as the allosteric antagonist Cmpd-15, but it is inactive when coupled with N-aminoethylmorpholine and 2-pyridinemethylamine. In addition, when R ₂ is methyl substituted at the 3rd position, its allosteric antagonistic activity is significantly improved.

The cAMP accumulation test was used to test whether the target compound could allosterically regulate the functional activity of the endogenous ligand ISO of β ₂ AR. From the results in Table 1, it can be seen that among the new compounds, J ₁ , J ₂ , J ₁₂ , J ₁₃ , J ₁₅ , J ₁₆ , J ₁₇ , J ₁₈ , J ₁₉ , J ₂₁ , J ₂₂ , and J ₂₅ are all negative allosteric regulators of β ₂ AR, among which the pharmacological activity of J ₂₅ at the same concentration (50 μM) is 2.5 times that of the lead compound (Cmpd-15). In addition, by adding J ₂₅ with a multiple relationship in concentration, it is further confirmed whether this type of compound is a negative allosteric regulator of β ₂ -AR. The results are shown in Appendix Figure 1. When the concentration of J ₂₅ increased to 30 μM, the curve of ISO showed a significant downward shift and reached the maximum downward shift limit of the dose-effect regulation of ISO functional activity. Increasing the concentration of J ₂₅ further almost no longer caused the dose-effect curve of ISO to shift downward. This indicates that the IC50 value of J ₂₅ may be between 15.0 μM and 30.0 μM, which is manifested as a sudden drop in the concentration curve. That is, the newly synthesized pyrazole amide derivative J ₂₅ is a relatively active β ₂ -AR negative allosteric antagonist or negative allosteric modulator. This allosteric regulation phenomenon is consistent with the reported allosteric antagonistic regulation mechanism.

In summary, these results show that the right side (S)-2-amino-3-(3-bromophenyl)-N-methylpropionamide in the Cmpd-15 structure is an important active part. If this part is replaced with benzylamine or benzylamine containing heteroatoms, the antagonistic activity of most of the obtained compounds will not be significantly improved; if this part is replaced with aniline, the allosteric antagonistic activity is significantly improved. It can be seen that the structure of aniline is simpler, and replacing the right side of Cmpd-15 can also obtain compounds with simpler structures but better biological activities, which is of great significance to the study of pyrazole amide class allosteric antagonists.

Claims (3)

1. A pyrazole amide derivative used as a β ₂ -adrenergic receptor allosteric antagonist, characterized in that: the structural formula of the pyrazole amide derivative is as shown in Formula I: Among them, R ₁ = any one of H and Cl; R ₂ = any one of H, F, Cl, NO ₂ and CH ₃ ; any of them. 2. A pyrazole amide derivative used as a β ₂ -adrenergic receptor allosteric antagonist, characterized in that: the structural formula of the pyrazole amide derivative is as shown in Formula I: Among them, R ₁ = any one of H and Cl; R ₂ =Any one of F, Cl, NO ₂ and 3-CH ₃ ; any of them. 3. Application of a pyrazole amide derivative as a β ₂ -adrenergic receptor allosteric antagonist as claimed in claim 1, characterized in that: the pyrazole amide derivative is used in the preparation of β ₂ - Application of adrenergic receptor allosteric antagonist drugs.