Use of pyrazole derivatives as β2 - adrenergic receptor antagonists

Abstract

The present invention belongs to the field of medicinal chemistry, and specifically discloses the application of pyrazole derivatives as allosteric antagonists of β2-adrenergic receptors. A series of novel pyrazole derivatives are obtained in the present invention, and the structure is shown in the following formula, wherein R1 is a hydrogen atom, a halogen atom, a nitro group, a methyl group, a methoxy group, etc.; R2 is a hydrogen atom, a hydrogen halogen atom; R3 is a hydrogen atom, a halogen atom. In the present invention, the cumulative experiment of GloSensor cAMP is used to test the functional activity of the target compound on β2AR in the G protein-dependent signaling pathway and to clarify whether the new compound can allosterically regulate the functional activity of the endogenous ligand isoproterenol (ISO) of β2AR. The pharmacological results show that most of the synthesized pyrazole derivatives have good antagonistic activity against β2AR and can negatively allosterically regulate the functional activity of ISO.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and relates to pyrazole derivatives, their preparation methods, and pharmaceutical compositions containing them. Functional activity screening of a series of synthesized compounds was carried out on the G protein-dependent signaling pathway and other applications. Background Art

[0002] G-protein-coupled receptors (GPCRs) are a class of membrane protein receptors with a seven-transmembrane helix structure, and are also the largest family of cell signal transduction receptors in the human body. More than 800 of its members encode 4.1%-4.3% of the human genome (British Journal of Pharmacology, 2019, 176: S21-S141). They are expressed in various tissues and organs of the human body and are involved in regulating processes such as the growth, development, signal perception, and metabolism of the body, affecting almost all life activities. GPCRs play a very important regulatory role in a variety of physiological activities and pathological processes. Abnormalities in its function may directly or indirectly affect the health of the body, leading to the occurrence of some major diseases such as heart disease, diabetes, Parkinson's disease, cardiovascular disease, cancer, inflammation, and asthma. The importance of GPCRs makes them the most successful and most concerned drug target protein family. Among the drugs approved by the FDA, drugs targeting GPCRs account for 34% of the approved drugs (Cell 2018, 172(1-2): 41-+). Therefore, studying the structure of GPCRs and their signaling pathways is of great significance for the drug design and development of metabolic diseases, neuro-related diseases, immune diseases, especially high-incidence diseases such as cardiovascular diseases and cancer.

[0003] β2-Adrenergic receptor (β2AR) is a typical G-protein-coupled receptor and has long been used as a model system for studying the regulatory mechanism of GPCRs, playing an important role in understanding the pathology of cardiovascular diseases and asthma. Previously, we first reported the first intracellular allosteric antagonist of β2-adrenergic receptor, Cmpd-15. However, due to the poor solubility and low bioactivity of Cmpd-15, the relative instability of its polypeptide structure will affect its drugability. Therefore, we adopted a scaffold migration strategy to replace the amide of the left fragment of the original compound Cmpd-15 with a nitrogen-containing heterocycle (see Formula I), which may make the compound have better solubility and pharmacological activity, and obtain a new class of structurally stable heterocyclic scaffolds. Functional activity tests were carried out on all synthesized compounds through the G protein-dependent signaling pathway (GloSensor TM cAMP Assay Promega), and some derivatives with allosteric antagonistic activity and solubility superior to the lead compound Cmpd-15 were screened out.

[0004] Summary of the Invention

[0005] The present invention uses acetophenones with different substituents as starting materials, and through Claisen condensation reaction, cyclization reaction, ester hydrolysis reaction and amide coupling reaction, a series of new pyrazole derivatives are finally obtained (see Formula II). The purpose of the present invention is to prepare new pyrazole derivatives, derivatives without substituents on the N of pyrazole, so as to develop new heterocyclic derivatives with stable chemical structure, novel skeleton, high biological activity, good subtype selectivity of receptors and improved water solubility as allosteric antagonists of β2-AR, providing a solid foundation for the development of new drugs for diseases such as cardiovascular diseases, asthma and cancer.

[0006]

[0007] Table 1-1 Structural Formulas of Novel Pyrazole Derivatives

[0008]

[0009]

[0010] The synthesis route of pyrazole ring derivatives is as follows:

[0011]

[0012] The specific synthesis method steps of pyrazole derivatives are as follows:

[0013] The specific synthesis steps are as follows:

[0014] (1) Under ice bath, dissolve substituted acetophenone 1 and diethyl oxalate in a solvent, add freshly prepared sodium ethoxide, and continue stirring until it becomes room temperature to form β-ketoester compound 2. The solvent is anhydrous ethanol; the molar ratio of acetophenone 1:diethyl oxalate:sodium ethoxide is 1:1.2:3; stir for 30 min under ice bath and stir for 10 h at room temperature.

[0015] (2) Under ice bath, dissolve compound 2 in a solvent, add hydrazine monohydrate, and continue stirring until it becomes room temperature. Close the ring to form pyrazole ring structure compound 3. The solvent is acetic acid; the molar ratio of compound 2:hydrazine monohydrate is 1:1.2; stir for 10 min under ice bath and stir for 8 h at room temperature.

[0016] ) Dissolve compound 3 in a solvent, add an alkali solution, react at room temperature, add an acid to adjust the pH to form compound 4. The solvent is methanol; the alkali solution is 4N sodium hydroxide solution; stir at room temperature for 10 h.

[0017] (4) Dissolve compound 4 and the activator in a solvent, add (S)-2-amino-3-(3-bromophenyl)-N-methylpropanamide (compound A; Bioorganic & Medicinal Chemistry, 2018, 26: 2320-2330) under an ice bath, then add the acid-binding agent and the amide coupling agent and continue stirring until room temperature is reached to obtain the pyrazole amide derivative 5. The solvent is N,N-dimethylformamide; the activator is 1-hydroxy-7-azabenzotriazole (HOAT); the acid-binding agent is N-methylmorpholine (NMM); the amide coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI); the molar ratio of compound 5:HOAT:compound A:NMM:EDCI is 1:1.2:1:0.7:1.2; react under an ice bath for 1.5 h and react at room temperature for 12 h.

[0018] The beneficial effects of the present invention are as follows:

[0019] The beneficial effects of the present invention are mainly manifested in the following aspects. First, the allosteric antagonistic activity of the newly synthesized derivative against the β2-AR target is increased by 1 to 3 times compared with the lead compound Cmpd-15; second, the solubility of the new derivative is increased by 10-100 times compared with the lead compound Cmpd-15; finally, the skeleton of the newly synthesized derivative is novel, and its structure is greatly simplified compared with the lead compound Cmpd-15, optimizing the synthesis route and reducing the synthesis difficulty. The target compound can be obtained starting from economically available raw materials, which may greatly improve the economic benefits of this type of compound. Description of the Drawings

[0020] Figure 1 It is a partial allosteric antagonistic curve diagram mediated by pyrazole derivatives;

[0021] Figure 2 It is a dose-response curve diagram of ISO mediated by pyrazole derivatives L1, L4, and L14. Detailed Embodiments

[0022] Preparation of pyrazole derivatives:

[0023] Example 1:

[0024] Preparation of (S)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(2-fluorophenyl)-1H-pyrazole-5-carboxamide L1.

[0025] Step 1: Preparation of ethyl 4-(2-fluorophenyl)-2,4-dioxobutyrate

[0026] Under an ice bath, 2-fluoroacetophenone (1 g, 7.2 mmol) and diethyl oxalate (0.7 mL, 10.3 mmol) were dissolved in sodium ethoxide (10 mL, 20% mass content, 0.01 mmol), and stirred at room temperature for 10 h. Under an ice bath, the pH was adjusted to 2 - 3 with 4N hydrochloric acid, extracted with ethyl acetate, and the organic phase was washed with saturated brine, concentrated to obtain the crude product, and directly proceed to the next step.

[0027] Step 2: Preparation of ethyl 3-(2-fluorophenyl)-1H-pyrazole-5-carboxylate

[0028] The crude product from Step 1 was dissolved in 10 mL of acetic acid, and hydrazine monohydrate (0.3 mL, 4.0 mmol) was added under an ice bath, and stirred at room temperature for 8 h. Extracted with ethyl acetate, and the organic phase was washed with saturated brine, concentrated and column chromatographed (petroleum ether:ethyl acetate = 3:1) to obtain 750 mg of a milky white solid compound 3, with a total two-step yield of 80%.

[0029] Step 3: Preparation of 3-(2-fluorophenyl)-1H-pyrazole-5-carboxylic acid

[0030] Ethyl 3-(2-fluorophenyl)-1H-pyrazole-5-carboxylate (500 mg, 2.1 mmol) was dissolved in 12 mL of methanol, 4 mL of 4N sodium hydroxide solution was added, and stirred at room temperature for 10 h. Under ice bath conditions, 4N hydrochloric acid was added to adjust the pH to 4, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried and concentrated to obtain 396 mg of a white solid compound 5, with a yield of 90%.

[0031] Step 4: Preparation of (S)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(2-fluorophenyl)-1H-pyrazole-5-carboxamide

[0032] 3-(2-fluorophenyl)-1H-pyrazole-5-carboxylic acid (150 mg, 0.73 mmol) and HOAT (119 mg, 0.87 mmol) were dissolved in 10 mL of DMF. After stirring for 10 min, (S)-2-amino-3-(3-bromophenyl)-N-methylpropanamide (187 mg, 0.73 mmol) was added. Under an ice bath, N-methylmorpholine (0.057 mL, 0.51 mmol) was added and stirred for 10 min, then EDCI (167 mg, 0.87 mmol) was added, and stirred at 0 °C for 1 h, and reacted at room temperature for 12 h. Extracted with ethyl acetate, and the organic phase was washed with saturated brine, concentrated and column chromatographed (petroleum ether:ethyl acetate = 1:1) to obtain 103 mg of a white solid product, with a yield of 32%. 1HNMR(400MHz, DMSO-d6): δ 13.76 (s, 1H), 8.23 - 7.80 (m, 3H), 7.51 - 7.17 (m, 8H), 4.66 (s, 1H), 3.00 (d, J = 52.8Hz, 2H), 2.62 (d, J = 4.5Hz, 3H). 13 C NMR(100MHz, DMSO-d6): δ 171.1, 132.0, 130.2, 129.2, 128.3, 121.4, 25.7. HRMS(ESI, m / z): Calcd. for C 20 H 18 BrFN4O2 [M+Na] + 467.0489, found: 467.0494.

[0033] Example 2:

[0034] (S)-N-(3-(3-Bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(3-fluorophenyl)-1H-pyrazole-5-carboxamide L2 Preparation

[0035] The preparation method is the same as that of Example 1, except that 3-fluoroacetophenone is used instead of 2-fluoroacetophenone in Step 1, and a white solid is obtained with a yield of 35%. 1 H NMR(400MHz, DMSO-d6): δ 13.84 - 13.63 (m, 1H), 8.17 - 8.02 (m, 2H), 7.70 - 7.59 (m, 2H), 7.57 - 7.45 (m, 3H), 7.41 - 7.28 (m, 2H), 7.27 - 7.11 (m, 3H), 4.73 - 4.60 (m, 1H), 3.15 - 2.87 (m, 2H), 2.62 (d, J = 4.5Hz, 3H).

[0036] Example 3:

[0037] (S)-N-(3-(3-Bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(4-fluorophenyl)-1H-pyrazole-5-carboxamide L3 Preparation

[0038] The preparation method is the same as that of Example 1, except that 4-fluoroacetophenone is used instead of 2-fluoroacetophenone in Step 1, and a white solid is obtained with a yield of 35%. 11H NMR (400 MHz, DMSO-d6): δ 13.62 (d, J = 48.1 Hz, 1H), 8.17 - 7.98 (m, 2H), 7.82 (s, 2H), 7.51 (d, J = 30.0 Hz, 1H), 7.39 - 7.17 (m, 6H), 4.66 (s, 1H), 3.18 - 2.90 (m, 2H), 2.62 (d, J = 4.5 Hz, 3H). 13 13C NMR (100 MHz, DMSO-d6): δ 171.0, 162.3, 161.2, 147.3, 140.9, 132.0, 130.2, 129.2, 128.3, 127.6, 127.0, 121.4, 116.2, 115.9, 115.6, 102.8, 53.5, 37.3, 35.8, 30.8, 25.6.

[0039] Example 4:

[0040] (S)-N-(3-(3-Bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(2-chlorophenyl)-1H-pyrazole-5-carboxamide L4 Preparation

[0041] The preparation method is the same as that of Example 1, except that 2-chloroacetophenone is used instead of 2-fluoroacetophenone in Step 1, and a white solid is obtained with a yield of 35%. 1 1H NMR (400 MHz, DMSO-d6): δ 13.74 (d, J = 22.7 Hz, 1H), 8.26 - 7.75 (m, 2H), 7.68 - 7.59 (m, 1H), 7.58 - 7.43 (m, 3H), 7.42 - 7.25 (m, 3H), 7.21 (t, J = 7.9 Hz, 1H), 4.67 (s, 1H), 3.16 - 2.90 (m, 2H), 2.62 (d, J = 4.5 Hz, 3H). 13 13C NMR (100 MHz, DMSO-d6): δ 171.1, 132.0, 130.3, 129.2, 128.3, 121.4, 25.7.

[0042] Example 5:

[0043] (S)-N-(3-(3-Bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(3-chlorophenyl)-1H-pyrazole-5-carboxamide L5 Preparation

[0044] The preparation method is the same as that of Example 1, except that 3-chloroacetophenone is used instead of 2-fluoroacetophenone in Step 1, and a white solid is obtained with a yield of 37%. 1H NMR (400MHz, DMSO-d6): δ13.77(d,J=44.7Hz,1H),8.12-8.03(m,1H),7.95-7.70(m,2H),7.59-7.45(m,2H),7.45-7.29(m,3H),7.28 -7.12(m,3H),4.67(d,J=7.1Hz,1H),3.16-3.02(m,1H),2.94(q,J=13.3,12.9Hz,1H),2.68-2.42(m,3H).HRMS(ESI,m / z):Calcd.for C 20 H 18 BrClN4O2[M+Na] + 483.0194,found:483.0198.

[0045] Example 6:

[0046] Preparation of (S)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(4-chlorophenyl)-1H-pyrazole-5-carboxamide L6

[0047] The preparation method was the same as that of Example 1, except that 4-chloroacetophenone was used instead of 2-fluoroacetophenone in step 1 to obtain a white solid with a yield of 35%. 1 H NMR (400MHz, DMSO-d6): δ13.74(d,J=56.9Hz,1H),8.08(d,J=28.1Hz,2H),7.81(s,2 H),7.52(s,4H),7.40-7.16(m,4H),4.66(s,1H),3.07(s,2H),2.61(d,J=4.5Hz,3H). 13 C NMR (100MHz, DMSO-d6): δ171.0,162.3,140.9,132.0,130.2,129.2,129.0,128.3,126.9,121.4,79.2,37.2,35.8,30.8,25.6.HRMS(ESI,m / z):Calcd.forC 20 H 18 BrClN4O2[M+Na] + 483.0194,found:483.0196.

[0048] Example 7:

[0049] (S)-3-(3-Bromophenyl)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-1H-pyrazole-5-carboxamide L7 Preparation

[0050] The preparation method is the same as that of Example 1, except that 3-bromoacetophenone is used instead of 2-fluoroacetophenone in Step 1 to obtain a white solid with a yield of 35%. 1 H NMR(400MHz,DMSO-d6):δ13.80(d,J=49.0Hz,1H),8.16(dd,J1=29.5,J2=4.8Hz,1H),8.10-7.74(m,3H),7.58-7.46(m,2H),7.46-7.36(m,2H),7.33(d,J=11.0Hz,1H),7.28-7.13(m,2H),4.67(t,J=7.2Hz,1H),3.18-2.89(m,2H),2.62(d,J=4.5Hz,3H). 13 C NMR(100MHz,DMSO-d6):δ132.0,131.2,130.2,129.2,128.3,121.4.HRMS(ESI,m / z):Calcd.for C 20 H 18 Br2N4O2[M+Na] + 528.9668,found:528.9678.

[0051] Example 8:

[0052] (S)-3-(4-Bromophenyl)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-1H-pyrazole-5-carboxamide L8 Preparation

[0053] The preparation method is the same as that of Example 1, except that 4-bromoacetophenone is used instead of 2-fluoroacetophenone in Step 1 to obtain a white solid with a yield of 37%. 1 H NMR(400MHz,DMSO-d6):δ13.73(d,J=56.7Hz,1H),8.23-8.01(m,2H),7.73(d,J=9.8Hz,2H),7.66(d,J=10.2Hz,2H),7.52(d,J=32.8Hz,1H),7.35(d,J=8.2Hz,2H),7.27(d,J=23.8Hz,1H),7.20(t,J=7.7Hz,1H),4.66(s,1H),3.16–2.89(m,2H),2.62(d,J=4.6Hz,3H). 1313C NMR(100MHz,DMSO-d6):δ171.0,132.0,130.2,129.2,128.3,121.4,25.7.HRMS(ESI,m / z):Calcd.for C 20 H 18 Br2N4O2[M+Na]+528.9668,found:528.9675.

[0054] Example 9:

[0055] Preparation of (S)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(2-nitrophenyl)-1H-pyrazole-5-carboxamide L9

[0056] The preparation method was the same as that of Example 1, except that 2-nitroacetophenone was used instead of 2-fluoroacetophenone in Step 1, and a white solid was obtained with a yield of 37%. 1 1H NMR(400MHz,DMSO-d6):δ13.80(s,1H),8.51(d,J=281.5Hz,2H),7.80(d,J=42.5Hz,3H),7.57(d,J=24.4Hz,2H),7.38-7.34(m,1H),7.30(d,J=7.6Hz,1H),7.21(t,J=7.8Hz,2H),4.67-4.61(m,1H),3.14-2.86(m,2H),2.62(d,J=4.5Hz,3H).HRMS(ESI,m / z):Calcd.for C 20 H 18 BrN5O4[M+Na] + 494.0434,found:494.0434.

[0057] Example 10:

[0058] Preparation of (S)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(3-nitrophenyl)-1H-pyrazole-5-carboxamide L10

[0059] The preparation method was the same as that of Example 1, except that 3-nitroacetophenone was used instead of 2-fluoroacetophenone in Step 1, and a white solid was obtained with a yield of 32%. 11H NMR (400 MHz, DMSO-d6): δ 14.00 (d, J = 89.9 Hz, 1H), 8.93 - 8.50 (m, 2H), 8.31 - 8.09 (m, 3H), 7.77 - 7.73 (m, 1H), 7.65 - 7.46 (m, 2H), 7.35 (d, J = 8.3 Hz, 1H), 7.33 - 7.24 (m, 1H), 7.21 (t, J = 7.8 Hz, 1H), 4.67 (d, J = 8.0 Hz, 1H), 3.14 - 3.04 (m, 1H), 3.05 - 2.90 (m, 1H), 2.62 (d, J = 4.1 Hz, 3H).

[0060] Example 11:

[0061] Preparation of (S)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(3-hydroxyphenyl)-1H-pyrazole-5-carboxamide L11

[0062] The preparation method was the same as that of Example 1, except that 3-hydroxyacetophenone was used instead of 2-fluoroacetophenone in Step 1, and a white solid was obtained with a yield of 36%. 1 1H NMR (400 MHz, DMSO-d6): δ 13.60 (d, J = 53.3 Hz, 1H), 9.72 (s, 1H), 8.10 (d, J = 45.2 Hz, 1H), 7.50 (s, 1H), 7.40 - 7.33 (m, 1H), 7.32 - 7.25 (m, 2H), 7.23 - 7.18 (m, 4H), 6.86 (d, J = 61.1 Hz, 2H), 4.65 (q, J = 8.4, 7.8 Hz, 1H), 3.14 - 2.93 (m, 2H), 2.61 (d, J = 4.5 Hz, 3H).

[0063] Example 12:

[0064] Preparation of (S)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(4-hydroxyphenyl)-1H-pyrazole-5-carboxamide L12

[0065] The preparation method was the same as that of Example 1, except that 4-hydroxyacetophenone was used instead of 2-fluoroacetophenone in Step 1, and a white solid was obtained with a yield of 35%. 11H NMR (400 MHz, DMSO-d6) δ 13.50 (s, 1H), 9.82 (s, 1H), 8.07 (d, J = 58.8 Hz, 2H), 7.63 - 7.45 (m, 3H), 7.39 - 7.33 (m, 1H), 7.30 - 7.17 (m, 2H), 6.85 (d, J = 8.3 Hz, 2H), 4.69 - 4.63 (m, 1H), 3.13 - 2.95 (m, 2H), 2.61 (d, J = 4.5 Hz, 3H).

[0066] Example 13:

[0067] (S)-N-(3-(3-Bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(o-tolyl)-1H-pyrazole-5-carboxamide L13 Preparation

[0068] The preparation method is the same as that of Example 1, except that 2-methylacetophenone is used instead of 2-fluoroacetophenone in Step 1, and a white solid is obtained with a yield of 33%. HRMS (ESI, m / z): Calcd. for C 21 H 21 BrN4O2 [M+Na] + 463.0740, found: 463.0743.

[0069] Example 14:

[0070] (S)-N-(3-(3-Bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(m-tolyl)-1H-pyrazole-5-carboxamide L14 Preparation

[0071] The preparation method is the same as that of Example 1, except that 3-methylacetophenone is used instead of 2-fluoroacetophenone in Step 1, and a white solid is obtained with a yield of 35%. 1 1H NMR (400 MHz, DMSO-d6): δ 13.70 (s, 1H), 8.18 (s, 1H), 7.66 - 7.56 (m, 2H), 7.51 (s, 1H), 7.36 - 7.27 (m, 3H), 7.24 - 7.12 (m, 2H), 4.63 - 4.69 (m, 1H), 3.20 - 2.90 (m, 2H), 2.61 (d, J = 4.5 Hz, 3H), 2.35 (s, 3H). 13 13C NMR (75 MHz, DMSO-d6): δ 171.1, 141.0, 132.0, 130.3, 129.3, 128.9, 128.4, 125.8, 122.4, 121.4, 102.7, 37.2, 25.7, 21.1.

[0072] Example 15:

[0073] Preparation of (S)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-(p-tolyl)-1H-pyrazole-5-carboxamide L15

[0074] The preparation method is the same as that of Example 1, except that 4-methylacetophenone is used instead of 2-fluoroacetophenone in Step 1, and a white solid is obtained with a yield of 36%. 1 H NMR(400MHz,DMSO-d6):δ8.53(d,J=8.5Hz,1H),8.04(q,J=4.5Hz,1H),7.75-7.69(m,2H),7.56(t,J=1.8Hz,1H),7.37-7.29(m,2H),7.27-7.17(m,3H),4.63(ddd,J1=10.6,J2=8.4,J3=4.3Hz,1H),3.08(dd,J1=13.6,J2=4.3Hz,1H),3.00-2.93(m,1H),2.62(d,J=4.6Hz,3H),2.33(s,3H). 13 CNMR(100MHz,DMSO-d6):δ171.5,166.2,141.5,141.2,132.0,131.2,130.2,129.1,128.7,128.3,127.5,121.4,54.7,36.9,25.7,21.0.HRMS(ESI,m / z):Calcd.for C 21 H 21 BrN4O2[M+Na] + 463.0740,found:463.0745.

[0075] Example 16:

[0076] Preparation of (S)-N-(3-(3,5-difluorophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-phenyl-1H-pyrazole-5-carboxamide L16

[0077] The preparation method is the same as that of Example 1, except that acetophenone is used instead of 2-fluoroacetophenone in Step 1, and (S)-2-amino-3-(3,5-difluorophenyl)-N-methylpropanamide is used instead of (S)-2-amino-3-(3-bromophenyl)-N-methylpropanamide in Step 4, and a white solid is obtained with a yield of 37%. 11H NMR (400 MHz, MeOH-d4): δ 7.78 - 7.57 (m, 3H), 7.44 (t, J = 7.5 Hz, 2H), 7.36 (dd, J1 = 8.3, J2 = 6.3 Hz, 1H), 7.02 (s, 1H), 6.90 (h, J = 4.2 Hz, 2H), 6.77 (dd, J1 = 9.2, J2 = 2.4 Hz, 1H), 4.82 (dd, J1 = 8.6, J2 = 6.0 Hz, 1H), 3.23 (dd, J1 = 13.7, J2 = 6.0 Hz, 2H), 2.74 (d, J = 4.2 Hz, 3H). 13 13C NMR (100 MHz, MeOH-d4): δ 130.1, 126.6, 113.4, 113.1, 103.7, 103.070, 55.407, 26.507, 26.4.

[0078] Example 17:

[0079] (S)-N-(3-(3,5-Dichlorophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-phenyl-1H-pyrazole-5-carboxamide L17 Preparation

[0080] The preparation method is the same as that of Example 1, except that acetophenone is used instead of 2-fluoroacetophenone in Step 1, and (S)-2-amino-3-(3,5-dichlorophenyl)-N-methylpropanamide is used instead of (S)-2-amino-3-(3-bromophenyl)-N-methylpropanamide in Step 4, obtaining a white solid with a yield of 38%. 1 1H NMR (400 MHz, MeOH-d4): δ 7.76 - 7.57 (m, 3H), 7.43 (dd, J1 = 8.3, J2 = 6.7 Hz, 2H), 7.38 - 7.32 (m, 1H), 7.25 (s, 3H), 6.97 (d, J = 35.0 Hz, 1H), 4.80 (dd, J1 = 8.6, J2 = 6.0 Hz, 1H), 3.19 (dd, J1 = 13.7, J2 = 6.0 Hz, 1H), 3.03 (dd, J1 = 13.7, J2 = 8.6 Hz, 1H), 2.73 (s, 3H). 13 13C NMR (100 MHz, MeOH-d4): δ 173.3, 142.5, 135.9, 130.1, 129.1, 127.8, 126.6, 103.7, 55.4, 26.4.

[0081] Example 18:

[0082] Preparation of (S)-N-(3-(3,5-dibromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-phenyl-1H-pyrazole-5-carboxamide L18

[0083] The preparation method was the same as that of Example 1, except that acetophenone was used instead of 2-fluoroacetophenone in Step 1, and (S)-2-amino-3-(3,5-dibromophenyl)-N-methylpropanamide was used instead of (S)-2-amino-3-(3-bromophenyl)-N-methylpropanamide in Step 4, to obtain a white solid with a yield of 39%. 1 H NMR(400MHz,DMSO-d6):δ13.77-13.49(m,1H),8.10(dd,J1=18.1,J2=6.8Hz,2H),7.82-7.75(m,2H),7.63(d,J=1.9Hz,1H),7.59-7.50(m,2H),7.45(q,J=7.9Hz,2H),7.40-7.33(m,1H),4.66(dd,J1=9.0,J2=5.4Hz,1H),3.15-2.87(m,2H),2.63(d,J=4.5Hz,3H). 13 C NMR(100MHz,DMSO-d6):δ143.1,131.3,129.1,125.3,122.0,25.6.HRMS(ESI,m / z):Calcd.for C 20 H 18 Br2N4O2[M+Na] + 528.9668,found:528.9676.

[0084] Example 19:

[0085] Preparation of (S)-N-(3-(3-bromo-5-fluorophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-phenyl-1H-pyrazole-5-carboxamide L19

[0086] The preparation method was the same as that of Example 1, except that acetophenone was used instead of 2-fluoroacetophenone in Step 1, and (S)-2-amino-3-(3-bromo-5-fluorophenyl)-N-methylpropanamide was used instead of (S)-2-amino-3-(3-bromophenyl)-N-methylpropanamide in Step 4, to obtain a white solid with a yield of 35%. 11H NMR (400 MHz, DMSO-d6): δ 13.64 (d, J = 55.7 Hz, 1H), 8.09 (dd, J1 = 17.3, J2 = 6.8 Hz, 2H), 7.83 - 7.74 (m, 2H), 7.50 - 7.29 (m, 5H), 7.16 (d, J = 10.1 Hz, 1H), 4.69 (q, J = 8.5, 7.8 Hz, 1H), 3.19 - 2.86 (m, 2H), 2.63 (d, J = 4.5 Hz, 3H). 13 13C NMR (100 MHz, DMSO-d6): δ 129.1, 128.5, 125.3, 37.0, 25.7. HRMS (ESI, m / z): Calcd. for C 20 H 18 BrFN4O2 [M + Na] + 467.0489, found: 467.0489.

[0087] Example 20:

[0088] (S)-N-(3-(2-Bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-phenyl-1H-pyrazole-5-carboxamide L20 Preparation

[0089] The preparation method is the same as that of Example 1, except that acetophenone is used instead of 2-fluoroacetophenone in Step 1, and (S)-2-amino-3-(2-bromophenyl)-N-methylpropanamide is used instead of (S)-2-amino-3-(3-bromophenyl)-N-methylpropanamide in Step 4, to obtain a white solid with a yield of 37%. 1 1H NMR (400 MHz, CDCl3): δ 9.69 (d, J = 8.5 Hz, 1H), 7.70 (dd, J1 = 30.8, J2 = 7.7 Hz, 3H), 7.37 - 7.30 (m, 3H), 7.29 - 7.24 (m, 2H), 7.20 - 7.14 (m, 1H), 7.09 (t, J = 7.4 Hz, 1H), 6.96 (s, 1H), 6.91 (t, J = 7.7 Hz, 1H), 5.06 (q, J = 8.1 Hz, 1H), 3.27 - 3.16 (q, J = 11.6, 9.3 Hz, 2H), 2.80 - 2.70 (m, 3H). 13 13C NMR (100 MHz, CDCl3): δ 136.3, 133.1, 129.2, 128.9, 128.7, 125.6, 29.8, 26.6, 1.1.

[0090] Example 21:

[0091] Preparation of (S)-N-(3-(4-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-3-phenyl-1H-pyrazole-5-carboxamide L21

[0092] The preparation method was the same as that in Example 1, except that acetophenone was used instead of 2-fluoroacetophenone in Step 1, and (S)-2-amino-3-(4-bromophenyl)-N-methylpropanamide was used instead of (S)-2-amino-3-(3-bromophenyl)-N-methylpropanamide in Step 4, obtaining a white solid with a yield of 35%. 1 H NMR(400MHz,MeOH-d4):δ7.69(d,J=7.8Hz,2H),7.46-7.32(m,5H),7.19-7.15(m,2H),7.02(s,1H),4.79(dd,J1=8.2,J2=6.2Hz,1H),3.17(dd,J1=13.8,J2=6.2Hz,1H),3.02(dd,J1=13.7,J2=8.2Hz,1H),2.71(s,3H). 13 C NMR(100MHz,MeOH-d4):δ173.6,137.6,132.5,132.3,130.1,126.6,121.7,103.7,55.6,38.7,26.4.

[0093] Compound solubility experiment

[0094] Solubility is a property that refers to the ability of a substance to dissolve in a specific solvent. Solubility has a broader and deeper impact in the process of drug research and development, affecting the in vitro activity, in vivo activity, and pharmacokinetic behavior of candidate compounds. The experimental process is briefly described as follows. The compound was prepared into a stock solution of 10 uM, and then diluted into a 5 mM solution with purified water, phosphate buffer solution (PBS), and DMEM (Gibco) culture medium respectively. Compound Cmpd-15 was prepared into solutions of 5 mM, 500 uM, and 50 uM respectively. After vortexing at room temperature, it was left standing for 30 min, and the clarity of the solution was visually observed under sunlight. The experimental results in Table 1-2 show that except for a large amount of white particles precipitating out in L7, the solubility of the selected representative compounds was significantly improved. Compared with the lead compound Cmpd-15, their solubility was increased by 10 to 100 times.

[0095] Table 1-2 Statistical table of solubility results of novel pyrazole derivatives

[0096]

[0097] Biological activity screening

[0098] GloSensor TM cAMP Assay for measuring cAMP content:

[0099] cAMP is a key signaling molecule for many G protein-coupled receptors. The cumulative level of cAMP is mainly measured using GloSensor, a bioluminescence-based biosensor that can directly detect intracellular cAMP (Promega). The principle is that a cAMP binding domain is inserted at the N-terminus and C-terminus of firefly luciferase by genetic engineering technology, rendering the enzyme in an inactive state. When cAMP binds to the cAMP binding domain, the enzyme is activated, thereby oxidizing the substrate luciferin to produce bioluminescence. The cAMP accumulation experiment is used to test the functional activity of target compounds on β2AR and to clarify whether a new compound is a negative allosteric modulator (NAM) of β2AR. Briefly described as follows, HEK 293T cells are seeded into 6-well plates at 4×10 5 cells per well. The next day, β2AR and pGloSensor-22F cAMP plasmids are co-transfected into HEK 293T cells using FuGene transfection reagent (Promega). After 48 h, the transfected cells are washed with CO2-independent medium and then incubated with a balanced solution containing 2% v / v GloSensor cAMP reagent stock solution (dissolved in CO2-independent medium containing 10% FBS). After incubating at 37 °C for 1 h and then at room temperature for 1 h, the bioluminescence signal is detected using a multimode microplate reader until a steady-state baseline signal is obtained. Then, new derivatives and control compound Cmpd-15 at different concentration gradients are added to the cells, and after incubating at 37 °C for 30 min, positive control ISO (final concentration 1 nM - 100 μM) is added. The change in bioluminescence is read using a microplate reader.

[0100] [[ID=1l]]

[0101] Statistical Table of the Test Results of the Biological Activities of Novel Pyrazole Derivatives

[0102]

[0103]

[0104] Note: Among them, "+" indicates having the corresponding activity, and "-" indicates having no corresponding activity

[0105] The test results show (see Table 1-3) that the novel pyrazole derivatives have no agonist activity on β2-adrenergic receptor (β2AR), and have an antagonistic effect on β2-adrenergic receptor at high concentrations. The IC 50 curve is shown in Figure 1 .

[0106] Allosteric Antagonistic Activity Screening

[0107] Using the GloSensor cAMP accumulation assay, isoproterenol (ISO) at different concentration gradients was used as the positive control (final concentration 1 nM - 30 μM), and compound Cmpd-15 (final concentration 50 μM) was used as the reference control to compare the allosteric antagonistic activity of the novel pyrazole derivatives (final concentration 50 μM) with that of the lead compound Cmpd-15. The test results in Tables 1 - 4 showed that all the synthesized compounds had varying degrees of allosteric antagonistic effects on the β2-adrenergic receptor. Except for L12, L15, and L20, the allosteric antagonistic activity of the remaining compounds against β2AR was significantly better than that of the lead compound Cmpd-15, and the allosteric antagonistic activity of L6 was three times that of the lead compound.

[0108]

[0109] Table 1 - 4 Statistical Table of the Comparison Results of the Allosteric Activity of Novel Pyrazole Derivatives and the Activity of Cmpd-15

[0110]

[0111]

[0112] Note: a The value represents the blocking activity relative to Cmpd-15

[0113] Study on the Allosteric Antagonistic Mechanism

[0114] In addition, a further study on the allosteric antagonistic mechanism was carried out using the cAMP accumulation assay to test whether the target compounds could allosterically regulate the functional activity of the endogenous ligand ISO of β2AR to determine whether they were negative allosteric modulators (NAMs) of β2AR. Briefly, first, new compounds at multiple concentration levels (1 nM - 120 μM) were added to the cells. After incubation at 37 °C for 30 min, positive control ISO at different concentration gradients (final concentration 1 nM - 30 μM) was added to the cells, and whether the change in bioluminescence showed a concentration-dependent limited downward trend was tested. The specific experimental procedure was the same as the Glosensor cAMP accumulation assay described above. The experimental results showed that the target compounds represented by L1, L4, and L14 could negatively allosterically regulate the functional activity of the endogenous ligand ISO of β2AR (see Figure 2 ), that is, the pyrazole derivatives in the present invention are allosteric antagonists of the β2-adrenergic receptor.

Claims

1. Use of a pyrazole derivative in the preparation of a β2 - adrenergic receptor antagonist drug, characterized in that: The pyrazole derivative has a structure as shown in formula (I): 。 2. Use of the pyrazole derivative according to claim 1 in the preparation of a β2-adrenergic receptor antagonist drug, characterized in that: The structural formula of the pyrazole derivative is as follows: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。

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