Pyrazole amide derivatives as beta2-adrenergic receptor allosteric modulators

Abstract

The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a pyrazole amide derivative used as a beta2-adrenergic receptor allosteric modulator. The present application takes phenylethanone with different substituents as raw material, and performs Claisen condensation, ring formation, hydrolysis and amide coupling, so as to finally obtain a series of new pyrazole derivatives, and the structure is shown as formula I, wherein R1 is a hydrogen atom or a chlorine atom; R2 is a hydrogen atom, a halogen atom, a nitro group or a methyl group; and R3 is a methyl group. The functional activity screening of G protein-dependent signal pathways is performed on all synthesized compounds, and it is found that the new derivatives can be used as a beta2-adrenergic receptor allosteric modulator.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and specifically relates to a pyrazolamide derivative and its application as an allosteric antagonist modulator of β2-adrenergic receptor. Background Technology

[0002] The G protein coupled receptor (GPCR) superfamily is composed of structurally similar proteins arranged in a family (class). It is the largest family of drug-producing proteins in the human genome (more than 800). Because it is a type of membrane receptor protein with a seven-transmembrane structure, it is also called the seven-alpha helices transmembrane segment receptor (7TM receptor). It has a very conserved spatial structure. These seven transmembrane alpha helices (TM1 to TM7) repeatedly pass through the transmembrane domain composed of the lipid bilayer of the cell membrane, dividing the receptor into an extracellular N-terminus, an intracellular C-terminus, three extracellular loops (EL1 to EL3), and three intracellular loops (ICL1 to ICL3).

[0003] GPCRs, found only in eukaryotes, can be activated by extracellular molecules (including photosensitive compounds, odorants, pheromones, hormones, and neurotransmitters) and elicit cellular responses, thus playing a crucial role in cell signal transduction. Furthermore, GPCRs are involved in various physiological processes such as visual, gustatory, olfactory, behavioral, and mood regulation; regulation of immune system activity and inflammation; autonomic nervous system transmission; cell density sensing; homeostasis regulation; and involvement in the growth and metastasis of certain types of tumors. Because GPCRs are involved in many disease-related signaling pathways, including those affecting mental health, metabolism, immunity, cardiovascular health, inflammation, sensory disorders, and cancer, they are a key drug target. Approximately 40% of all FDA-approved drugs target GPCRs.

[0004] The exact size of the GPCR protein superfamily is unknown, but genome sequence analysis predicts that it is encoded by at least 831 different genes in the human genome, representing approximately 4% of the entire protein-coding genome. Many classification schemes have been proposed for this protein family. The AF classification divides GPCRs into six classes based on sequence homology and functional similarity: class A (Rhodopsin-like), class B (Secretin receptor family), class C (Metabotropic glutamate / pheromone), class D (Fungal mating pheromone receptors), class E (Cyclic AMP receptors), and class F (Frizzled / Smoothened). Recently, an alternative classification system called GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled / Taste2, Secretin) has been proposed for vertebrate GPCRs, corresponding to classes C, A, B2, F, and B in the classic AF classification. Despite the lack of sequence homology between classes, all GPCRs share common structures and signal transduction mechanisms. The largest class to date is class A, accounting for nearly 85% of GPCR genes. Class A GPCRs are further subdivided into 19 subgroups (A1-A19). It is estimated that more than half of the members in class A GPCRs encode olfactory receptors. The Beta-2 adrenergic receptor (β2AR), also known as β2-adrenergic receptor, is a star member of the G protein-coupled receptor family, widely expressed in vascular and bronchial smooth muscle. β2AR, activated by endogenous agonists such as adrenaline, mediates cardiovascular function and pulmonary physiological processes, making it an important target for the treatment of vascular and respiratory diseases. Furthermore, it is crucial for overcoming immunosuppression and improving the efficacy of immunotherapy. The concept of allostery was first proposed in 1961 by Jacques Monod of the Pasteur Institute in France. Due to significant breakthroughs in structural biology in recent years, Jacob and Jean-Pierre Changeux have found that developing allosteric modulators targeting allosteric sites has become a breakthrough in innovative drug development. Traditional drug development for GPCRs primarily targets the orthogonal binding site (i.e., the location where the receptor's endogenous ligand binds). The high conservation of this site across different subtypes poses a significant challenge to developing selective drugs. Allosteric modulators bind outside the orthogonal ligand pocket of the receptor. Because their binding site is less conserved, they may exhibit better subtype selectivity and lower toxicity compared to orthogonal drugs targeting the active site. Secondly, in terms of efficacy, allosteric agonists are less likely to induce protein desensitization after activation compared to orthogonal agonists. Furthermore, orthogonal and allosteric modulators exert their effects through different mechanisms of action, making it possible for allosteric modulators to overcome acquired resistance to orthogonal drugs during treatment. Therefore, the discovery of allosteric modulators provides a new approach to obtaining highly selective drugs.

[0005] In 2017, our laboratory collaborated with scientists at Duke University to report a small-molecule negative allosteric modulator compound 15 (Cmpd-15), which is the first intracellular allosteric antagonist of the β2-adrenergic receptor (Ahn S, et al. Proc. Natl. Acad. Sci. USA, 2017, 114:1708-1713; Liu X, et al. Nature, 2017, 548:480-484). However, Cmpd-15 is a peptide compound with poor water solubility and low biological activity, and its structure is relatively unstable, which may affect its druggability. Therefore, this project uses Cmpd-15 as a lead compound and employs skeletal transition strategies, structural simplification, and bioelectronic isosterism concepts for drug design. The synthesized new compound was analyzed using GloSensor... TM The cAMPAccumulation assay was used to screen for the bioactivity of classical signaling pathways (G-protein signaling) (Binkowski BF et al. ACS Chem Biol. 2011; 6(11):1193-1197). The aim was to obtain a series of novel pyrazole derivatives with stable and simplified structures, novel skeletons, enhanced allosteric activity, improved water solubility, and metabolic stability as allosteric regulators of β2-AR.

[0006]

[0007] Pyrazole nitrogen-containing heterocyclic compounds possess broad-spectrum pharmacological properties and are a crucial core framework in drug design. Accordingly, in order to expand the structural types of lead compounds and to improve the bioactivity, 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 keeping the (S)-2-amino-3-(3-bromophenyl)-N-methylpropionamide on the right side unchanged, and a series of pyrazole derivatives were designed and synthesized (Meng K et al, Bioorg. Med. Chem. 2018, 26: 2320-2330).

[0008]

[0009] Our research group previously designed and synthesized pyrazole derivatives that allosterically antagonized the G-protein signaling pathway of β2-adrenergic receptors and negatively regulated the agonistic effect of the endogenous ligand isoproterenol (ISO) on β2-adrenergic receptors. cAMP accumulation experiments showed that most compounds exhibited significantly better allosteric antagonism against β2-adrenergic receptors than their lead compound, Cmpd-15, and the newly derived compounds showed significantly improved water solubility compared to Cmpd-15, suggesting their potential as precursor compounds for treating new vascular diseases (Chen Xin, et al., Chinese Invention Patent Publication No.: CN115745891A). However, the structural formulas of these compounds show that the phenylalanine containing a chiral carbon atom on the right side has significant steric hindrance, which may affect their drug-likeness. Summary of the Invention

[0010] Based on the problems pointed out in the background art, in order to simplify the structure of Cmpd-15 and obtain compounds with better allosteric antagonistic activity and water solubility, this invention designs to use simple substitutions such as aniline, benzylamine or amines with nitrogen and oxygen heteroatoms to synthesize new pyrazole compounds, and studies the bioactivity of the new derivatives to improve the drug-likeness of compounds with allosteric antagonistic activity.

[0011]

[0012] The purpose of this invention is to provide a class of pyrazole amide derivatives to develop novel heterocyclic derivatives with stable chemical structure, high biological activity, receptor subtype selectivity and good water solubility, as allosteric modulators of β2-AR, providing a new direction for the development of new drugs for cardiovascular and cerebrovascular diseases, diabetes and cancer.

[0013] The general structural formula of pyrazole amide derivatives is shown in Formula I:

[0014]

[0015] Wherein, R1 = any one of H and Cl;

[0016] R2 = any one of H, F, Cl, NO2, CH3;

[0017] Any one of them.

[0018] This invention uses acetophenone with different substituents as raw materials, and through Claisen condensation, cyclization, ester hydrolysis and amide coupling reaction, finally obtains a series of new pyrazole amide derivatives.

[0019] Table 1. Structure of pyrazole amide derivatives

[0020]

[0021]

[0022] Synthetic routes for pyrazole amide derivatives:

[0023]

[0024] The specific steps for synthesizing pyrazole amide derivatives are as follows:

[0025] The specific synthesis steps are as follows:

[0026] (1) Add an ethanol solution of sodium ethoxide (EtONa: 20% w / w) to a round-bottom flask, then add anhydrous ethanol. Under nitrogen protection, heat to 0°C in an ice bath. Dissolve compound 1 with different substituents and diethyl oxalate completely in anhydrous ethanol and mix thoroughly. Slowly add the mixture dropwise to the flask while stirring continuously. Continue the reaction under ice bath conditions for 30 minutes, then remove the ice bath and react at room temperature for 4 hours. After the reaction is complete as monitored by TLC, filter the resulting paste-like mixture under reduced pressure and wash the filter cake three times with an appropriate amount of anhydrous ethanol. After drying the filter cake, compound 2 is obtained, in which the molar ratio of sodium ethoxide, compound 1, and diethyl oxalate is 1:1:1.

[0027] (2) Compound 2 was added to a round-bottom flask, along with acetic acid. The mixture was stirred, and then hydrazine hydrate (80%) was slowly added. The mixture was heated to reflux and reacted for 3 hours. After the reaction was completed by TLC monitoring, heating was stopped, and the mixture was cooled to room temperature. Water was added, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phases were washed with saturated sodium bicarbonate solution, then with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed, and the mixture was purified by silica gel column chromatography using a 5:1 eluent to obtain the target compound 3. The molar ratio of compound 2 to hydrazine hydrate was 1:4.

[0028] (3) Compound 3 was added to a round-bottom flask, ethanol was added, the mixture was stirred, NaOH solution was added dropwise, and the mixture was heated to reflux for 1.5 h. After the reaction was completed by TLC monitoring, the heating was stopped, the mixture was cooled to room temperature, most of the ethanol was removed by rotary evaporation, L of water was added to the resulting solution, and concentrated hydrochloric acid was slowly added dropwise under ice bath conditions to adjust the pH to 1-2. A white solid precipitated out, which was filtered, and the filter cake was washed with an appropriate amount of water. The crude product was recrystallized from the ethanol / water mixture 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.

[0029] (4) Compound 4, EDCl, and HOBt were weighed sequentially and added to a round-bottom flask. DMF was added, and the mixture was stirred at room temperature. Then, compound H2N-R3 was added, and the mixture was stirred at room temperature for 30 minutes. DIEA was added under ice bath conditions, and the mixture was stirred under ice bath conditions for another 20 minutes before reacting overnight at room temperature. The reaction was monitored by TLC until complete. A saturated ammonium chloride solution was added, and the mixture was stirred thoroughly. The mixture was extracted with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain a white solid crude product. The product was recrystallized from an appropriate amount of ethyl acetate / ethanol / petroleum ether mixed solvent to obtain the target compound 5. The molar ratio of compound 4, EDCl, HOBt, H2N-R3, and DIEA was 1:1 to 1.1:1 to 1.1:1 to 1.2:3.

[0030] Novel pyrazole derivatives can serve as allosteric antagonists of β2-AR;

[0031] Furthermore, compounds J1, J2, and J in Table 1 12 J 13 J 15 J 16 J 17 J 18 J 19 J 21 J 22 J 25 It has allosteric antagonistic activity and can be used as an allosteric antagonist of β2-AR.

[0032] The beneficial effects of this invention are as follows:

[0033] Most of the novel pyrazole derivatives synthesized in this invention possess β2-AR allosteric antagonistic activity. Compared with the lead compound Cmpd-15, compound J... 21 J 22 J 25The allosteric antagonistic activity against β2-AR is significantly enhanced. The novel pyrazole derivatives synthesized in this invention have simple structures, a straightforward synthetic route, readily available raw materials, and can be industrially produced. These novel pyrazole amide derivatives can serve as allosteric antagonists against β2-AR, providing a new direction for the development of new drugs for cardiovascular, cerebrovascular, diabetic, and cancer diseases. Attached Figure Description

[0034] Figure 1 For J 25 mediated ISO dose-response curve. Detailed Implementation

[0035] Synthetic routes for pyrazole amide derivatives:

[0036]

[0037] Example 1:

[0038]

[0039] Preparation of N-benzyl-5-(4-fluorophenyl)-1H-pyrazole-3-carboxamide J1

[0040] Step 1: Preparation of ethyl 4-(4-fluorophenyl)-2,4-dioxobutyrate

[0041] An ethanol solution of sodium ethoxide (3.4 g, 10 mmol, EtONa: 20% w / w) was added to a 100 mL round-bottom flask, followed by 10 mL of anhydrous ethanol. The flask was kept under nitrogen protection and heated to 0 °C on an ice bath. 4-fluoroacetophenone (10 mmol) and diethyl oxalate (1.46 g, 10 mmol) were completely dissolved in 15 mL of anhydrous ethanol and mixed thoroughly. This solution was then slowly added dropwise to the flask with constant stirring. The reaction was continued on an ice bath for 30 minutes, then the ice bath was removed, and the reaction was allowed to proceed at room temperature for 4 hours. After the reaction was completed as monitored by TLC, the resulting paste-like mixture was filtered under reduced pressure. The filter cake was washed three times with an appropriate amount of anhydrous ethanol. After drying, ethyl 4-(4-fluorophenyl)-2,4-dioxobutyrate was obtained as a reddish-brown solid, with a yield of 30%.

[0042] Step 2: Preparation of ethyl 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylate

[0043] To a 50 mL round-bottom flask, add 2 mmol of ethyl 4-(4-fluorophenyl)-2,4-dioxobutyrate and 10 mL of acetic acid. Stir, then slowly add 0.5 g, 8 mmol, 4 eq, 80% hydrazine hydrate. Heat to reflux and react for 3 h. After the reaction is complete as monitored by TLC, stop heating and cool to room temperature. Add 20 mL of water and extract with ethyl acetate (3 × 30 mL). Combine the organic phases. Wash the organic phase with saturated sodium bicarbonate solution (2 × 20 mL), then wash once with 20 mL of saturated brine, and dry with anhydrous sodium sulfate. Remove the solvent and purify by silica gel column chromatography with eluent [V(petroleum ether):V(ethyl acetate) = 5:1] to give ethyl 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid as a pale yellow solid, yield 93%. 1 H NMR (300MHz, DMSO-d6): 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, CDCl3)δ164.6,161.3,160.9,147.7,139.9,127.6,127.5,126.8,116.1,115.8,105.1,61.3,14.2.MS(ESI):m / z 235(M+1).

[0044] Step 3: Preparation of 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid

[0045] Ethyl 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid (1 mmol) was added to a 50 mL round-bottom flask, followed by 4 mL of ethanol. The mixture was stirred, and 4 mL of 4 mol / L NaOH solution was added dropwise. The mixture was heated to reflux and reacted for 1.5 h. After the reaction was completed by TLC monitoring, heating was stopped, and the mixture was cooled to room temperature. Most of the ethanol was removed by rotary evaporation. 10 mL of water was added to the resulting solution, and concentrated hydrochloric acid was slowly added dropwise under ice bath conditions to adjust the pH to 1-2. A white solid precipitated out. The solid was filtered, and the filter cake was washed twice with an appropriate amount of water. The crude product was recrystallized from an ethanol / water mixture to give 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid as a white solid, with a yield of 91%.

[0046] Step 4: Preparation of N-benzyl-5-(4-fluorophenyl)-1H-pyrazole-3-carboxamide

[0047] 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 sequentially and added to a 25 mL round-bottom flask. 5 mL of DMF was added, and the mixture was stirred at room temperature. Then, benzylamine (0.6 mmol, 1.2 eq) was added, and the mixture was stirred 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 another 20 minutes under ice bath conditions until the reaction proceeded overnight at room temperature. The reaction was monitored by TLC until complete. 20 mL of saturated ammonium chloride solution was added, and the mixture was stirred thoroughly. The mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were 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. The crude product 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 (300MHz, DMSO-d6): δ13.69 (s, 1H), 8.81 (br, 1H), 7.82-7.86 (m, 2H), 7.14-7.34 (m, 8H), 5.47 (d, J = 6.1Hz, 2H); MS (ESI): m / z 296 (M+1).

[0048] Example 2:

[0049]

[0050] Preparation of 5-(4-fluorophenyl)-N-(pyridin-2-methyl)-1H-pyrazole-3-carboxamide J2

[0051] In step four of Example 1, benzylamine was replaced with pyridine-2-methylamine, while other conditions remained the same as in Example 1. A white solid was obtained, with a yield of 81%. Mp 205.5-206.4℃; 1 H NMR (300MHz, DMSO-d6): δ13.73(br,1H),8.88(br,1H),8.52(dd,J=4.8,0.8Hz, 1H),7.74-7.88(m,3H),7.18-7.35(m,5H),4.58(d,J=6.0Hz,2H); MS(ESI):m / z 297(M+1).

[0052] Example 3:

[0053]

[0054] Preparation of N-(3,4-dimethoxybenzyl)-5-(4-fluorophenyl)-1H-pyrazole-3-carboxamide J3

[0055] Other conditions were the same as in Example 1, except in step four, benzylamine was replaced with 3,4-dimethoxybenzylamine, yielding a white solid with a yield of 73%. Mp 205.1-206.1℃; 1 H NMR (300MHz, DMSO-d6): 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=5.9Hz,2H),3.74(s,3H),3.72(m,3H);MS(ESI):m / z 356(M+1).

[0056] Example 4:

[0057]

[0058] Preparation of N-(2-chlorobenzyl)-5-(4-fluorophenyl)-1H-pyrazole-3-carboxamide J4

[0059] Other conditions are the same as in Example 1. In step four, benzylamine is replaced with 2-chlorobenzylamine to obtain a white solid with a yield of 74%. 1 HNMR (300MHz, DMSO-d6): δ13.73 (s, 1H), 9.81 (br, 1H), 7.83-7.87 (m, 2H), 7.13-7.47 (m, 7H), 4.53 (d, J = 5.8Hz, 2H); MS (ESI): m / z 330 (M+1).

[0060] Example 5:

[0061]

[0062] Preparation of 5-(4-fluorophenyl)-N-(3-nitrobenzyl)-1H-pyrazole-3-carboxamide J5

[0063] Other conditions were the same as in step four 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℃; 1H NMR (300MHz, DMSO-d6): δ13.66(br,1H),9.10(s,1H),8.20(s,1H),8.12(d,J=9.4Hz,1H),7.79-7.87( m,3H),7.64(t,J=8.0Hz,1H),7.31(t,J=8.9Hz,2H),7.15(s,1H),4.58(d,J=6.2Hz,2H); MS(ESI):m / z 341(M+1).

[0064] Example 6:

[0065]

[0066] Preparation of N-(3-nitrobenzyl)-5-(p-tolyl)-1H-pyrazole-3-carboxamide J6

[0067] Step 1: Preparation of ethyl 2,4-dione-4-(p-tolyl)butyrate

[0068] The method is the same as step one of Example 1, except that 4-fluoroacetophenone is replaced with 4-methylacetophenone, resulting in a light yellow solid with a yield of 45%.

[0069] Step 2: Preparation of ethyl 5-(p-Tolyl)-1H-pyrazole-3-carboxylate

[0070] The method is the same as step two of Example 1, except that ethyl 4-(4-fluorophenyl)-2,4-dioxobutyrate is replaced with ethyl 2,4-dione-4-(p-tolyl)butyrate, to obtain a bright yellow solid with a yield of 80%. 1 H NMR (300MHz, CDCl3): 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, CDCl3) 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].

[0071] Step 3: Preparation of 5-(p-Tolyl)-1H-pyrazole-3-carboxylic acid

[0072] The method is the same as step three of Example 1. Ethyl 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylate was replaced with ethyl 5-(p-tolyl)-1H-pyrazole-3-carboxylate, yielding a white solid with a yield of 93%. 1H NMR (300MHz, DMSO-d6): δ7.71(d,J=8.1Hz,2H),7.24(d,J=8.1Hz,2H),7.12(s,1H),2.31(s,3H).

[0073] Step 4: Preparation of N-(3-nitrobenzyl)-5-(p-tolyl)-1H-pyrazole-3-carboxamide

[0074] The method is the same as step four of Example 1, except that 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid is replaced with 5-(p-tolyl)-1H-pyrazole-3-carboxylic acid, and benzylamine is replaced with 3-nitrobenzylamine, yielding a white solid with a yield of 74%. Mp 262.3-263.1℃; 1 H NMR (300MHz, DMSO-d6): δ13.69(br,1H),9.03(br,1H),8.19(s,1H),8.11(d,J=8.1Hz,1H),7.60- 7.80(m,4H),7.26(d,J=7.9Hz,2H),7.06(s,1H),4.57(d,J=5.9Hz,2H),2.32(s,3H); MS(ESI):m / z 337(M+1).

[0075] Example 7:

[0076]

[0077] Preparation of N-(pyridin-2-methylene)-5-(p-tolyl)-1H-pyrazole-3-carboxamide J7

[0078] The method is the same as in Example 6, except that in step four, 3-nitrobenzylamine is replaced with 2-pyridinemethylamine to obtain a white solid with a yield of 85%. 1 H NMR (300MHz, DMSO-d6): δ13.65(br,1H),9.11(br,1H),8.51(d,J=6.1Hz,1H),7.77(td,J=7.7,1.7Hz,1H) ,7.68(d,J=8.1Hz,2H),7.25-7.33(m,4H),7.06(s,1H),4.56(d,J=6.0Hz,2H),2.33(s,3H); MS(ESI):m / z 293(M+1).

[0079] Example 8:

[0080]

[0081] Preparation of N-(3,4-dimethoxybenzyl)-5-(p-tolyl)-1H-pyrazole-3-carboxamide J8

[0082] The method was the same as in Example 6, except that in step four, 3-nitrobenzylamine was replaced with 3,4-dimethoxybenzylamine, yielding a white solid with a yield of 77%. ¹H NMR (300MHz, DMSO-d6): δ 13.60 (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).

[0083] Example 9:

[0084]

[0085] Preparation of N-benzyl-5-(P-tolyl)-1H-pyrazole-3-carboxamide J9

[0086] Other conditions are the same as in Example 6. In step four, 3-nitrobenzylamine is replaced with benzylamine. The product is a white solid with a yield of 75%. 1 H NMR (300MHz, DMSO-d6): 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-d6): δ162.27,148.12,144.01,140.33,138.44,130.03,1 28.71,127.71,127.16,126.46,125.64,102.73,42.41,21.27; MS(ESI):m / z 292(M+1).

[0087] Example 10:

[0088]

[0089] N-(2-Chlorobenzyl)-5-(p-Tolyl)-1H-Pyrazole-3-carboxamide J 10 Preparation

[0090] Other conditions are the same as in Example 6. In step four, 3-nitrobenzylamine is replaced with 2-chlorobenzylamine. The product is a white solid with a yield of 73%. 1H NMR (300MHz, DMSO-d6): δ13.66(br,1H),8.78(br,1H),7.69(d,J=8.1Hz,2H),7 .26-7.46(m,6H),7.06(s,1H),4.52(d,J=5.9Hz,2H),2.32(s,3H);MS(ESI):m / z 326(M+1).

[0091] Example 11:

[0092]

[0093] N-Benzyl-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide J 11 Preparation

[0094] Step 1: Preparation of ethyl 4-(4-nitrobenzene)-2,4-dioxobutyrate

[0095] The method is the same as step one in Example 1. 4-Fluoroacetophenone was replaced with 4-nitroacetophenone, yielding a light yellow solid powder with a yield of 49%. 1 H NMR (300MHz, CDCl3): δ8.36 (d, J = 9.2Hz, 2H), 8.17 (d, J = 9.2Hz, 2H), 7.11 (s, 1H), 4.43 (q, J = 7.3Hz, 2H), 1.43 (t, J = 7.3Hz, 3H).

[0096] Step 2: Preparation of ethyl 5-(4-nitrophenyl)-1H-pyrazole-3-carboxylate

[0097] The method is the same as step two of Example 1. Ethyl 4-(4-fluorophenyl)-2,4-dioxobutyrate was replaced with ethyl 4-(4-nitrobenzene)-2,4-dioxobutyrate, yielding a yellow solid with a yield of 65%. 1 H NMR (300MHz, DMSO-d6): δ14.40 (s, 1H), 4.28 (d, J = 7.6Hz, 2H), 8.16 (d, J = 7.6Hz, 2H), 7.54 (s, 1H), 4.36 (q, J = 7.1Hz, 2H), 1.34 (t, J = 7.1Hz, 3H).

[0098] Step 3: Preparation of 5-(4-nitrophenyl)-1H-pyrazole-3-carboxylic acid

[0099] The method is the same as step three of Example 1. Ethyl 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylate was replaced with ethyl 5-(4-nitrophenyl)-1H-pyrazole-3-carboxylate, yielding a pale yellow solid with a yield of 68%. 1H NMR (300MHz, DMSO-d6): δ14.21(s,1H),13.65(s,1H),8.29(d,J=8.9Hz,2H),8.15(d,J=8.9Hz,2H),7.47(s,1H).

[0100] Step 4: Preparation of N-benzyl-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide

[0101] Other conditions were the same as in step four 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 pale yellow solid with a yield of 70%. Mp 258.4-258.8℃; 1 H NMR (300MHz, DMSO-d6): δ9.04 (s, 1H), 8.32 (d, J = 8.9Hz, 2H), 8.06 (d, J = 8.9Hz, 2H), 7.44 (s, 1H), 7.24-7.35 (m, 5H), 4.49 (d, J = 6.1Hz, 2H); MS (ESI): m / z 323(M+1).

[0102] Example 12:

[0103]

[0104] N-(2-morpholinoethyl)-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide J 12 Preparation

[0105] Other conditions were the same as in Example 11, except in step four, benzylamine was replaced with 2-morpholinoethyl-1-amine. The product was a white solid with a yield of 29%. Mp 171.1-172.3℃; 1 H NMR (300MHz, DMSO-d6): δ14.00(s,1H),8.32(d,J=8.8Hz,2H),8.06(d,J=8.8Hz,2H),7 .37(s,1H),3.58(t,J=4.5Hz,4H),3.39-3.43(m,4H),2.41-2.49(m,4H); MS(ESI):m / z 346(M+1).

[0106] Example 13:

[0107]

[0108] N-(benzo[d][1,3]dioxo-5-ylmethyl)-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide J 13 Preparation

[0109] Other conditions were the same as in Example 11, except in step four, benzylamine was replaced with 3,4-(methylenedioxy)benzylamine. The product was a yellow solid with a yield of 26%. Mp 248.2-249.1℃; 1 H NMR (300MHz, DMSO-d6): δ13.98(s,1H),9.27(t,J=5.9Hz,1H),8.85(s,1H),7.27-7.90(m,6H),7.06(s,1H),4.58(s,2H); 13 C NMR (75MHz, DMSO-d6): δ162.10,159.16,149.40,147.59,139.66,137.28,134.56,13 2.62,132.13,130.39,128.29,127.40,122.72,121.68,107.03,44.60; MS(ESI):m / z 367(M+1).

[0110] Example 14:

[0111]

[0112] 5-(4-nitrophenyl)-N-(pyridine-2-methyl)-1H-pyrazole-3-carboxamide J 14 Preparation

[0113] Other conditions were the same as in Example 11, except that in step four, benzylamine was replaced with pyridine-2-methylamine. The product was a light yellow solid with a yield of 33%. Mp 258.8-259.6℃; 1 H NMR (300MHz, DMSO-d6): δ14.08(s,1H),9.24(s,1H),8.54(s,1H),8.32(d,J=8.2Hz,2 H),8.08(s,2H),7.78(t,J=7.1Hz,1H),7.29-7.38(m,3H),4.60(s,2H); MS(ESI):m / z 324(M+1).

[0114] Example 15:

[0115]

[0116] Morpholine (5-(4-nitrophenyl)-1H-pyrazol-3-yl) methyl ketone J 15 Preparation

[0117] Other conditions were the same as in Example 11, except that in step four, benzylamine was replaced with morpholine. The product was a white solid with a yield of 35%. Mp 275.0-275.7℃; 1H NMR (300MHz, DMSO-d6): δ13.98 (s, 1H), 8.31 (d, J = 8.6Hz, 2H), 8.11 (d, J = 8.6Hz, 2H), 7.32 (s, 1H), 3.90 (s, 1H), 3.66 (s, 7H); MS (ESI): m / z 303(M+1).

[0118] Example 16:

[0119]

[0120] N-(3,4-Dimethoxybenzyl)-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide J 16 Preparation

[0121] Other conditions were the same as in Example 11, except in step four, benzylamine was replaced with 3,4-dimethoxybenzylamine. The product was a light yellow solid with a yield of 73%. Mp 196.4-197.2℃; 1 H NMR (300MHz, DMSO-d6): δ8.96(br,1H),8.32(d,J=8.8Hz,2H),8.06(d,J=8.8Hz,2H ),6.84-6.98(m,3H),4.41(d,J=5.9Hz,2H),3.75(s,3H),3.73(s,3H); MS(ESI):m / z 383(M+1).

[0122] Example 17:

[0123]

[0124] N-(3-nitrobenzyl)-5-(4-nitrophenyl)-1H-pyrazole-3-carboxamide J 17 Preparation

[0125] Other conditions were the same as in Example 11, except in step four, benzylamine was replaced with 3-nitrobenzylamine. The product was a light yellow solid with a yield of 80%. Mp 279.5-280.2℃; 1 HNMR(300MHz,DMSO-d6): δ9.20(br,1H),8.33(d,8.9Hz,2H),8.22(s,1H),8.14(d,8 .1Hz,1H),8.08(d,8.9Hz,2H),7.66(t,7.9Hz,1H),4.61(d,5.9Hz,2H); MS(ESI):m / z 368(M+1).

[0126] Example 18:

[0127]

[0128] 5-(2,4-Dichlorophenyl)-N-(2-morpholinylethyl)-1H-pyrazole-3-carboxamide J 18 Preparation

[0129] Step 1: Preparation of ethyl 4-(2,4-dichlorophenyl)-2,4-dioxobutyrate

[0130] Other conditions were the same as in step one 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 (300MHz, DMSO-d6): δ7.34-7.52 (m, 3H), 5.66 (br, 1H), 4.71 (s, 1H), 4.07 (q, J = 7.1Hz, 2H), 1.20 (t, J = 7.1Hz, 3H).

[0131] Step 2: Preparation of ethyl 5-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylate

[0132] The method is the same as step two of Example 1. Ethyl 4-(4-fluorophenyl)-2,4-dioxobutyrate was replaced with ethyl 4-(2,4-dichlorophenyl)-2,4-dioxobutyrate, yielding a white solid with a yield of 70%. 1 H NMR (300MHz, CDCl3): δ11.92(br,1H),7.69(d,J=8.4Hz,1H),7.49(d,J=2.1Hz,1H),7.27-7.32(m,2H),7.41(q,J=7.1Hz,2H),1.40(t,J=7.1Hz,3H).

[0133] Step 3: Preparation of 5-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylic acid

[0134] The method is the same as step three of Example 1. Ethyl 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylate was replaced with ethyl 5-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylate, yielding a white solid with a yield of 72%. 1 H NMR (300MHz, DMSO-d6): δ13.76(br,2H),7.81(d,J=8.3Hz,1H),7.75(s,1H),7.53(d,J=8.3Hz,1H),7.18(s,1H).

[0135] Step 4: Preparation of 5-(2,4-dichlorophenyl)-N-(2-morpholinylethyl)-1H-pyrazole-3-carboxamide

[0136] Other conditions were the same as in step four of Example 1, except that 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid was replaced with 5-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylic acid, and benzylamine was replaced with 2-morpholinoethyl-1-amine. The product was a white solid with a yield of 65%. Mp 249.3-250.1; 1 H NMR (300MHz, DMSO-d6): δ13.84(s,1H),8.53(s,1H),7.77(s,2H),7.55(d,J=7.8Hz,1H) ,7.40(s,1H),3.58(t,J=4.5Hz,4H),3.37-3.42(m,4H),2.42-2.49(m,4H); MS(ESI):m / z 369(M+1).

[0137] Example 19:

[0138]

[0139] [5-(2,4-dichlorophenyl)-1H-pyrazol-3-yl]-4-morpholinone J 19 Preparation

[0140] Steps one, two, and three are the same as in Example 18. In step four, benzylamine is replaced with morpholine, and other conditions are the same as in step four of Example 18. The product is a white solid with a yield of 75%. Mp 204.3-205.5℃; 1 H NMR (300MHz, DMSO-d6): 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).

[0141] Example 20:

[0142]

[0143] 5-(2,4-Dichlorophenyl)-N-(pyridin-2-methyl)-1H-pyrazole-3-carboxamide J 20 Preparation

[0144] Other conditions were the same as in Example 18, except that in step four, benzylamine was replaced with pyridine-2-methylamine. The product was a white solid with a yield of 58%. Mp 242.5-243.3℃; 1H NMR (300MHz, DMSO-d6): δ9.27(t,J=5.9Hz,1H),8.52(d,J=4.3Hz,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 (75MHz, DMSO-d6): δ162.10,159.01,149.40,147.59,139.66,137.28,134.56,13 2.62,132.13,130.39,128.29,127.40,122.72,121.68,107.03,44.60; MS(ESI):m / z 347(M+1).

[0145] Example 21:

[0146]

[0147] (5-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)(piperidin-1-yl)methyl ketone J 21 Preparation

[0148] Other conditions are the same as in Example 18. In step four, benzylamine is replaced with piperidine. The product is a white solid with a yield of 18%. 1 HNMR(400MHz,DMSO-d6)δ7.76–7.73(m,2H),7.54(dd,J=8.4,2.2Hz,1H),6.92(s,1H), 3.74(s,2H),3.60(d,J=6.7Hz,2H),1.66–1.61(m,2H),1.56–1.50(m,4H).MS(ESI):m / z 324(M+1).

[0149] Example 22:

[0150]

[0151] N-Cyclohexyl-5-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxamide J 22 Preparation

[0152] Other conditions are the same as in Example 18. In step four, benzylamine is replaced with cyclohexylamine. The product is a white solid with a yield of 74%. 1HNMR (400MHz, DMSO-d6) δ13.79(s,1H),8.21(s,1H),7.80(d,J=8.5Hz,1H),7.74(d,J=2.2Hz,1H),7.52(dd,J=8.5,2.2Hz,1H),7.31(s,1 H),3.78–3.71(m,1H),1.83–1.79(m,2H),1.73–1.71(m,2H),1.60(d,J=12.8Hz,1H),1.36–1.25(m,4H),1.16–1.07(m,1H).MS(ESI):m / z 338(M+1).

[0153] Example 23:

[0154]

[0155] N-(3-Bromophenyl)-3-phenyl-1H-pyrazole-5-carboxamide J 23 Preparation

[0156] Step 1: Preparation of ethyl 2,4-dione-4-phenylbutyrate

[0157] The method is the same as step one in Example 1. 4-Fluoroacetophenone is replaced with acetophenone to obtain the crude product ethyl 2,4-dione-4-phenylbutyrate.

[0158] Step 2: Preparation of ethyl 5-phenyl-1H-pyrazole-3-carboxylate

[0159] The method is the same as step two of Example 1. Ethyl 4-(4-fluorophenyl)-2,4-dioxobutyrate was replaced with ethyl 2,4-dione-4-phenylbutyrate, yielding a white solid with a yield of 80%. 1 H NMR (400MHz, CDCl3) δ7.73-7.67(m,2H),7.40-7.29(m,3H),6.97(s,1H),4.17(q,J=7.1Hz,2H),1.19(t,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) 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).

[0160] Step 3: Preparation of 5-phenyl-1H-pyrazole-3-carboxylic acid

[0161] The method is the same as step three of Example 1. Ethyl 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylate was replaced with ethyl 5-phenyl-1H-pyrazole-3-carboxylate, yielding a white solid with a yield of 90%. 1 H NMR (400MHz, CD3OD) δ7.79-7.72(m,2H),7.43(t,J=7.5Hz,2H),7.40-7.31(m,1H),7.12(s,1H).

[0162] Step 4: Preparation of N-(3-bromophenyl)-3-phenyl-1H-pyrazole-5-carboxamide

[0163] Other conditions are the same as in step four of Example 1, except that 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid is replaced with 5-phenyl-1H-pyrazole-3-carboxylic acid, and benzylamine is replaced with m-bromoaniline. The product is a white solid with a yield of 53%. 1 H NMR (400MHz, DMSO-d6) 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) δ160.6,147.6,144.0,140.6,130.6,129.1,128.9,128.7,128 .6,126.0,125.5,125.1,122.5,121.4,119.0,103.4ppm.MS(ESI):m / z342(M+1)..

[0164] Example 24:

[0165]

[0166] N-(3-Bromobenzyl)-3-phenyl-1H-pyrazole-5-carboxamide J 24 Preparation

[0167] Steps one, two, and three are the same as in Example 23. In step four, benzylamine is replaced with m-bromobenzylamine. Other conditions are the same as in step four of Example 23. The product is a white solid with a yield of 53%. 1 H NMR(400MHz,DMSO-d6)δ8.95(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).

[0168] Example 25:

[0169]

[0170] N-Phenyl-3-(M-Tolyl)-1H-Pyrazole-5-carboxamide J 25 Preparation

[0171] Step 1: Preparation of ethyl 2,4-dione-4-(m-tolyl)butyrate

[0172] The method is the same as step one in Example 1. 4-Fluoroacetophenone is replaced with 3-methylacetophenone to obtain the crude product ethyl 2,4-dione-4-(m-tolyl)butyrate.

[0173] Step 2: Preparation of ethyl 5-(m-Tolyl)-1H-pyrazole-3-carboxylate

[0174] The method is the same as step two of Example 1. Ethyl 4-(4-fluorophenyl)-2,4-dioxobutyrate was replaced with ethyl 2,4-dione-4-(m-tolyl)butyrate, resulting in a white solid with a yield of 76%. 1 HNMR(300MHz, CDCl3)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, CDCl3) 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).

[0175] Step 3: Preparation of 5-(m-Tolyl)-1H-pyrazole-3-carboxylic acid

[0176] The method is the same as step three of Example 1. Ethyl 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylate was replaced with ethyl 5-(m-tolyl)-1H-pyrazole-3-carboxylate, yielding a white solid with a yield of 40%. 1 H NMR (400MHz, DMSO-d6) δ7.68(s,1H),7.63(d,J=7.8Hz,1H),7.32(t,J=7.6Hz,1H),7.16(d,J=7.1Hz,2H),2.35(s,3H).

[0177] Step 4: Preparation of N-phenyl-3-(M-tolyl)-1H-pyrazole-5-carboxamide

[0178] Other conditions are the same as in step four of Example 1, except that 5-(4-fluorophenyl)-1H-pyrazole-3-carboxylic acid is replaced with 5-(m-tolyl)-1H-pyrazole-3-carboxylic acid, and benzylamine is replaced with aniline. The product is a white solid with a yield of 20%. 1 H NMR (400MHz, DMSO-d6) δ13.79(s,1H),10.14(s,1H),7.83(d,J=8.0Hz,1H),7.69(s,1H),7.64(d,J=7 .8Hz,1H),7.39–7.34(m,1H),7.20(d,J=7.6Hz,1H),7.13(t,J=7.4Hz,1H),2.38(s,1H).MS(ESI):m / z 278(M+1).

[0179] Bioactivity test

[0180] cAMP is a key signaling molecule in many G protein-coupled receptors. The accumulation level of cAMP is mainly measured using the GloSensor method, a bioluminescent cAMP biosensor (Promega). Healthy HEK293T cells were seeded in 6-well plates or 35 mm cell culture dishes and incubated at 37°C with 5% CO2 for 24 h to allow cell adhesion. Then, the β2-adrenergic receptor plasmid and pGloSensor were transfected using transfection reagents. TM The -22FcAMP plasmid was transfected into cells and incubated at 37°C with 5% CO2 for 24 hours to allow for the transcription and expression of the target gene. The transfected cells were then seeded evenly into 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. Finally, the old culture medium in the 96-well plates was discarded, the cells were washed once with fresh culture medium, and then GloSensor was added. TMThe cAMP Reagent, serum, and CO2-independent medium were incubated at 37°C in a 5% CO2 incubator for 1-2 hours or until a stable background signal was obtained. Isoproterenol (ISO) was dissolved in DMSO, sterilized, filtered, and used as the stock solution for the compound, then diluted with culture medium to different concentration gradients (the DMSO concentration in the prepared compound was less than or equal to 0.1%). The prepared compound was rapidly added to 96-well plates containing HEK293T cells to begin stimulation. A rapid rise in the bioluminescent signal after the addition of the compound was observed using a multi-mode microplate reader. When the signal value reached its peak and stopped increasing, a 50 μM concentration of the compound was immediately added. Simultaneously, the bioluminescent signal was collected using the multi-mode microplate reader; the bioluminescent signal value increased with increasing compound concentration. Finally, the data were processed using GraphPadPrism8 software, with concentration on the x-axis and signal value on the y-axis to obtain a dose-response curve for the allosteric modulator.

[0181] Table 2. Statistical table of the functional activities of new pyrazole derivatives

[0182]

[0183] Note: "+" indicates the presence of the corresponding activity, and "-" indicates the absence of the corresponding activity.

[0184] Table 3. Comparison of the activities of the new allosteric antagonistic derivatives with Cmpd-15.

[0185]

[0186] Note: a The numerical values ​​are expressed as blocking activity relative to Cmpd-15.

[0187] In the Glosensor cAMP cumulative assay results, the new compounds J1, J2, and J... 12 J 13 J 15 J 16 J 17 J 18 J 19 J 21 J 22 J 25 All of them exhibited β2AR allosteric antagonistic activity, while the remaining new compounds did not (see Table 2). Compared with the lead compound Cmpd-15, the compounds with allosteric antagonistic activity showed J 19 Its allosteric antagonistic activity is comparable to that of Cmpd-15, J 21 J 22 J 25The allosteric antagonistic activity of J was significantly improved compared to Cmpd-15, with the most active compound being J. 25 (See Table 3).

[0188] As can be seen from Table 3, among all the new pyrazole compounds, compound J... 25 The allosteric antagonistic activity of this compound is the best, approximately 2.5 times that of Cmpd-15. Pyrazolamide compounds with R2 being hydrogen-atom substituted or substituted at the 4-position do not exhibit allosteric antagonistic activity. When R2 is fluorine-substituted at the 4-position, coupling with benzylamine shows better activity; coupling with benzylamines of different substitutions shows no activity. Furthermore, coupling with 2-pyridinemethylamine does not significantly improve its allosteric antagonistic activity. When R2 is nitro-substituted at the 4-position, most compounds exhibit allosteric antagonistic activity, but less than that of Cmpd-15. When both R1 and R2 are chlorinated, the products coupled with morpholine, cyclohexylamine, and piperidine have activity comparable to the allosteric antagonist Cmpd-15, but coupling with N-aminoethylmorpholine and 2-pyridinemethylamine shows no activity. Additionally, when R2 is methyl-substituted at the 3-position, its allosteric antagonistic activity is significantly improved.

[0189] The cAMP accumulation assay was used to test whether the target compounds could allosterically modulate the functional activity of the β2AR endogenous ligand ISO. As shown in Table 1, among the new compounds, J1, J2, and J... 12 J 13 J 15 J 16 J 17 J 18 J 19 J 21 J 22 J 25 Both are negative allosteric regulation of β2AR, with the highest concentration (50 μM) J 25 The pharmacological activity was 2.5 times that of the lead compound (Cmpd-15). Furthermore, by adding J in a concentration-dependent manner... 25 To further confirm whether this class of compounds are negative allosteric modulators of β2-AR. Results are shown in the appendix. Figure 1 As shown, when J 25 When the concentration increased to 30 μM, the ISO curve showed a significant downward shift, reaching the lower limit of the maximum downward shift in dose-dependent ISO functional activity. Further increases in J... 25 The concentration of J almost no longer shifts the dose-response curve of ISO downwards. This indicates that J 25 The IC50 value may be between 15.0 μM and 30.0 μM, and it will show a sharp drop in concentration on the concentration curve, i.e., the newly synthesized pyrazole amide derivative J 25 It is a relatively active β2-AR negative allosteric antagonist or negative allosteric modulator. This allosteric modulation phenomenon is consistent with the reported allosteric antagonistic regulatory mechanisms.

[0190] In summary, these results indicate that the (S)-2-amino-3-(3-bromophenyl)-N-methylpropionamide on the right side of the Cmpd-15 structure is the key active moiety. Replacing this moiety with benzylamine or benzylamine containing heteroatoms does not significantly improve the antagonistic activity of most of the resulting compounds; however, replacing this moiety with aniline significantly enhances the allosteric antagonistic activity. This demonstrates that aniline has a simpler structure, and replacing the right-side portion of Cmpd-15 can yield compounds with simpler structures but better biological activity, which is of great significance for the research of pyrazolamide allosteric antagonists.

Claims

1. A pyrazolamide derivative used as an allosteric antagonist of the β2-adrenergic receptor, characterized in that: The structural formula of the pyrazole amide derivative is as follows: 、 、 、 、 、 、 、 。 2. Use of a pyrazole carboxamide derivative as claimed in claim 1 as a β2-adrenergic receptor allosteric modulator, characterized in that: The pyrazole amide derivative is used for preparing β2-adrenergic receptor allosteric agonist drugs.

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