CN117143019A - As beta 2 Pyrazole amide derivatives of allosteric antagonists of the adrenergic receptors - Google Patents

As beta 2 Pyrazole amide derivatives of allosteric antagonists of the adrenergic receptors Download PDF

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CN117143019A
CN117143019A CN202310937573.6A CN202310937573A CN117143019A CN 117143019 A CN117143019 A CN 117143019A CN 202310937573 A CN202310937573 A CN 202310937573A CN 117143019 A CN117143019 A CN 117143019A
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pyrazole
allosteric
beta
nmr
yield
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陈新
洪美龄
凌倩
李燕
张欣
李晴
郭雪
叶岢欣
钱明成
赵帅
侯亚男
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Changzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links

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

As beta 2 Pyrazole amide derivatives of allosteric antagonists of the adrenergic receptors
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and in particular relates to a pyrazole amide derivative and a preparation method thereof as beta 2 Use of modulators of allosteric adrenergic receptors.
Background
The G protein-coupled receptor (G-protein coupledreceptor, GPCR) superfamily consists of structurally similar proteins arranged in families (classes), which are the largest family of patent proteins in the human genome (more than 800) and are a class of membrane receptor proteins with seven transmembrane structures, also known as 7-helix-alpha helices transmembrane segment receptor (7 TM receptor), with very conserved spatial structures, the transmembrane domains consisting of lipid bilayers where 7 transmembrane alpha helices (TM 1-TM 7) repeatedly cross the cell membrane divide the receptor into an extracellular N-terminus, an intracellular C-terminus, 3 extracellular loops (EL 1-EL 3) and 3 intracellular loops (ICL 1-ICL 3).
GPCRs are found only in eukaryotes, and can be activated by extracellular molecules (including photosensitizing compounds, odors, pheromones, hormones and neurotransmitters) and elicit a cellular response, so they play a vital role in cellular signal transduction. In addition, GPCRs are involved in a variety of physiological processes such as regulation of vision, taste, smell, behavior and emotion, regulation of immune system activity and inflammation, autonomic nervous system transmission, cell density sensing, homeostasis regulation, and in the growth and metastasis of certain types of tumors, among others. GPCRs are a key drug target because they are involved in many disease-related signaling pathways, including mental, metabolic, immune, cardiovascular, inflammatory, sensory disorders, and cancer. Approximately 40% of all FDA approved drugs are GPCR targeted.
The exact size of the GPCR protein superfamily is not known, but it is predicted from genomic sequence analysis that the human genome encodes at least 831 different genes, approximately 4% of the entire protein-encoding genome. A number of classification schemes have been proposed for this family of proteins. a-F classification classifies GPCRs into 6 classes based on sequence homology and functional similarity: namely class A (Rhodopsin-like), class B (Secretin receptor family), class C (Metabotropic glutamate/pheomone), class D (Fungal mating pheromone receptors), class E (CyclicAMP receptors), class F (Frizzled/Smoothened). Recently, an alternative classification system called GRAFS (Glutamate, rhodopsin, adhesive, frizzled/Taste2, secretin) has been proposed for ridgesVertebro GPCRs, which correspond to class C, class a, class B2, class F and class B, respectively, in the classical class a-F classification. Despite the lack of sequence homology between classes, all GPCRs share a common structure and signaling mechanism. 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-a 19). In class a GPCRs, more than half of the members are expected to encode olfactory receptors. Beta-2 adrenergic receptor (Beta) 2 adrenoreceptor), also known as beta 2 AR, a member of the G protein-coupled receptor family, is widely expressed in vascular and bronchial smooth muscle. Beta 2 AR, when activated by endogenous agonists such as epinephrine, mediates cardiovascular function and pulmonary physiological processes, an important target for the treatment of vascular and respiratory diseases. In addition, it is also critical to overcome immunosuppression and to enhance the efficacy of immunotherapy. The allosteric (Allostery) concept was first proposed in 1961 by Jacques Monod, the institute of Pasteur, france,Jacob and Jean-Pierre Changeux have made a great breakthrough in structural biology in recent years, and the development of corresponding allosteric modulators for allosteric sites has become a great way of developing innovative drugs. Traditional drug development for GPCRs is directed primarily to the orthosteric binding site (i.e., the site of endogenous ligand binding of the receptor), which is highly conserved between the different subtypes, presenting a great challenge to the development of selective drugs. Allosteric modulators bind outside the normal ligand pocket of the receptor, and may have better subtype selectivity and lower toxicity than normal drugs targeting the active site due to relatively low conservation of binding sites; secondly, compared with an orthosteric agonist, the allosteric agonist is less likely to induce desensitization of proteins after being activated; in addition, the pharmacological effects of normal and allosteric modulators are based on different mechanisms of action, which makes it possible for allosteric modulators to overcome acquired resistance of normal drugs during treatment. Thus, the discovery of allosteric modulators provides a new concept for obtaining drugs with high selectivity.
201In 7 years, the laboratory, in cooperation with Duke's university scientist, reported a small molecule negative allosteric modulator compound 15 (Cmpd-15), which was the first β 2 An intracellular allosteric antagonist of the adrenergic receptor (Ahn S, et al Proc. Natl. Acad. Sci. U S A,2017,114:1708-1713;Liu X,et al.Nature,2017,548:480-484). However, cmpd-15 is a peptide compound, so that the water solubility is poor, the biological activity is low, the structure is relatively unstable, and the patentability of the compound can be possibly influenced. Therefore, the subject uses Cmpd-15 as a lead compound and adopts skeleton transition strategy, structure simplification and bioisostere concept to carry out drug design. The novel compounds synthesized were passed through a GloSensor TM cAMP Accumulation assay A biological activity screen of the classical signal pathway (G-protein signaling) (Binkowski BF et al ACS Chem biol.2011;6 (11): 1193-1197). It is expected to obtain a series of novel pyrazole derivatives with stable and simplified structure, novel skeleton, improved allosteric activity, improved water solubility and stable metabolism as beta 2 Allosteric modulators of AR.
Pyrazole nitrogen-containing heterocyclic compounds have broad-spectrum pharmacological properties and are very important core frameworks in drug design, so that in order to expand the structural types of the lead and expect to improve the bioactivity, selectivity, water solubility and stability of the drug, the peptide core structure of Cmpd-15 is replaced by a pyrazole framework (shown in the following figure), the (S) -2-amino-3- (3-bromophenyl) -N-methylpropanamide on the right is kept unchanged, and a series of pyrazole derivatives (Meng K et al, biorg. Med. Chem.2018, 26:2320-2330) are designed and synthesized.
Pyrazole derivative pair beta designed and synthesized in earlier stage of subject group 2 The G-protein signaling pathways of epinephrine all have differential antagonism, and can negatively regulate endogenous ligand isoprenaline(ISO) vs. beta 2 Adrenergic agonism. cAMP accumulation assay results indicate that most compounds are p 2 The allosteric antagonism of the adrenergic receptor is obviously better than the functional activity of the lead compound Cmpd-15, and the water solubility of the novel derivative compound is obviously improved compared with that of the Cmpd-15, thus being hopeful to be used as a Miao compound for treating the neovascular diseases (Chen Xin, et al, chinese patent publication No. CN 115745891A). However, as can be seen from the structural formula of the compound, phenylalanine containing chiral carbon atoms on the right side has larger steric hindrance and can influence the drug property.
Disclosure of Invention
Based on the problems pointed out in the background art, in order to simplify the structure of Cmpd-15 and obtain a compound with better allosteric antagonistic activity and water solubility, the invention designs to replace with simple substituted aniline, benzylamine or amine with nitrogen-oxygen heteroatom, etc. to synthesize new pyrazole compounds, and research the biological activity of the new derivatives to improve the patentability of the compounds with allosteric antagonistic activity.
The invention aims to provide pyrazole amide derivatives, which are used for developing brand new heterocyclic derivatives with stable chemical structure, high biological activity, good receptor subtype selectivity and good water solubility as beta 2 Allosteric modulators of AR provide new directions for the development of new drugs for cardiovascular, cerebrovascular, diabetes and cancer diseases.
The structural general formula of the pyrazole amide derivative is shown as formula I:
wherein R is 1 Any one of =h, cl;
R 2 ＝H,F,Cl,NO 2 ,CH 3 any one of them;
any one of the following.
The invention takes acetophenone with different substituents as raw material, and finally obtains a series of novel pyrazole amide derivatives through claisen condensation, cyclization, ester hydrolysis and amide coupling reaction.
TABLE 1 Structure of pyrazole amide derivatives
Synthetic route of pyrazole amide derivatives:
the specific synthesis method of the pyrazole amide derivative comprises the following steps:
the specific synthesis steps are as follows:
(1) Adding an ethanol solution of sodium ethoxide (EtONa: 20% w/w) into a round-bottom flask, adding absolute ethyl alcohol, protecting by nitrogen, cooling to 0 ℃ with an ice bath, completely dissolving the compound 1 with different substituents and diethyl oxalate with absolute ethyl alcohol, uniformly mixing, slowly dropwise adding into the flask, and continuously stirring. The reaction was continued for 30 minutes in ice bath, and after removing the ice bath, the reaction was continued at room temperature for 4 hours. After the reaction is monitored by TLC, the obtained pasty mixed solution is subjected to vacuum suction filtration, a filter cake is washed three times by using a proper amount of absolute ethyl alcohol, and the filter cake is dried to obtain a compound 2, wherein the molar ratio of sodium ethoxide to compound 1 to diethyl oxalate is 1:1:1.
(2) To the round bottom flask was added compound 2 and acetic acid, stirred, hydrazine hydrate (80%) was slowly added and heated to reflux and reacted for 3h. After the reaction was completed by TLC, heating was stopped to cool to room temperature, water was added, extraction was performed with ethyl acetate, and the organic phases were combined. The organic phase was washed with a saturated sodium bicarbonate solution, a saturated brine and dried over anhydrous sodium sulfate. Removing the solvent, purifying by silica gel column chromatography, eluting with an eluent [ V (petroleum ether): V (ethyl acetate) =5:1 ] to obtain the target compound 3; wherein the molar ratio of the compound 2 to the 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 1.5h. After the reaction is finished through TLC monitoring, stopping heating, cooling to room temperature, removing most of ethanol by rotary evaporation, adding L water into the obtained solution, slowly dropwise adding concentrated hydrochloric acid under the ice bath condition, adjusting the pH to 1-2, precipitating white solid, carrying out suction filtration, washing a filter cake with a proper amount of water, and recrystallizing the obtained crude product through an ethanol/water mixed solution to obtain a compound 4, wherein the mol ratio of the compound 3 to NaOH is 1:16, and the volume ratio of the ethanol to the NaOH solution is 1:1.
(4) Sequentially weighing compound 4, EDCl and HOBt, adding into a round bottom flask, adding DMF, stirring at room temperature, and adding compound H 2 N-R 3 After stirring at room temperature for 30 minutes, DIEA was added under ice bath conditions, and the reaction was continued under ice bath for 20 minutes and then allowed to react overnight at room temperature. The reaction was monitored by TLC to completion, a saturated ammonium chloride solution was added, and after thoroughly stirring and mixing, extracted with ethyl acetate, the organic phases were combined, washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. Removing solvent by rotary evaporation to obtain white solid crude product, recrystallizing with appropriate amount of ethyl acetate/ethanol/petroleum ether mixed solvent to obtain target compound 5, wherein the compounds 4, EDCl, HOBt, H 2 N-R 3 The molar ratio of DIEA is 1:1-1.1:1-1.2:3.
Novel derivatives of pyrazoles as beta 2 Allosteric antagonists of AR;
further, compound J in Table 1 1 、J 2 、J 12 、J 13 、J 15 、J 16 、J 17 、J 18 、J 19 、J 21 、J 22 、J 25 Has allosteric antagonistic activity and can be used as beta 2 Allosteric antagonists of AR.
The beneficial effects of the invention are as follows:
the novel derivatives of pyrazoles synthesized by the invention have beta in most parts 2 -AR allosteric antagonistic activity, compound J compared to the lead compound Cmpd-15 21 、J 22 、J 25 Beta pair 2 The allosteric antagonistic activity of the AR is significantly increased. The novel pyrazole derivative synthesized by the invention has the advantages of simple structure, simple synthesis route and easily available raw materials, and can realize industrial production. The novel pyrazole amide derivative can be used as beta 2 Allosteric antagonists of AR provide new directions for the development of new drugs for cardiovascular, cerebrovascular, diabetes and cancer diseases.
Description of the drawings:
FIG. 1 is J 25 Mediated ISO dose-response curve.
Detailed Description
Synthetic route of pyrazole amide derivatives:
example 1:
n-benzyl-5- (4-fluorophenyl) -1H-pyrazole-3-carboxamide J 1 Is prepared from
Step one: preparation of ethyl 4- (4-fluorophenyl) -2, 4-dioxobutyrate
To a 100mL round bottom flask was added sodium ethoxide in ethanol (3.4 g,10mmol, etONa:20% w/w), followed by 10mL absolute ethanol, nitrogen blanket, ice bath to 0deg.C, 4-fluoroacetophenone (10 mmol) and diethyl oxalate (1.46 g,10 mmol) were completely dissolved with 15mL absolute ethanol and mixed well, slowly added dropwise to the flask with constant stirring. The reaction was continued for 30 minutes in ice bath, and after removing the ice bath, the reaction was continued at room temperature for 4 hours. After the completion of the reaction by TLC monitoring, the obtained pasty mixed solution is subjected to vacuum filtration, a filter cake is washed three times by using a proper amount of absolute ethyl alcohol, and the filter cake is dried to obtain the ethyl 4- (4-fluorophenyl) -2, 4-dioxobutyrate as a reddish brown solid with the yield of 30%.
Step two: preparation of 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid ethyl ester
To a 50mL round bottom flask was added ethyl 4- (4-fluorophenyl) -2, 4-dioxabutyrate (2 mmol) and acetic acid 10mL, stirred, hydrazine hydrate (0.5 g,8mmol,4eq, 80%) was slowly added, heated to reflux and reacted for 3h. After the reaction was completed by TLC, the heating and cooling were stopped, cooled to room temperature, 20mL of water was added, extracted with ethyl acetate (3X 30 mL), and the organic phases were combined. The organic phase was washed with saturated sodium bicarbonate solution (2X 20 mL), once with saturated brine (20 mL), and dried over anhydrous sodium sulfate. The solvent was removed and purified by column chromatography on silica gel eluting with eluent [ V (petroleum ether): V (ethyl acetate) =5:1]The compound 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid ethyl ester was obtained as a pale yellow solid in 93% yield. 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 C NMR(75MHz,CDCl 3 )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).
Step three preparation of 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid
The compound 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid ethyl ester (1 mmol) was added to a 50mL round bottom flask, ethanol (4 mL) was added, stirred, 4mL of 4mol/L NaOH solution was added dropwise, heated to reflux, and reacted for 1.5H. After the reaction is finished through TLC monitoring, stopping heating, cooling to room temperature, removing most of ethanol by rotary evaporation, adding 10mL of water into the obtained solution, slowly dropwise adding concentrated hydrochloric acid under ice bath condition, adjusting pH to 1-2, precipitating white solid, carrying out suction filtration, washing a filter cake with a proper amount of water for 2 times, and recrystallizing the obtained crude product through an ethanol/water mixed solution to obtain the 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid as white solid with the yield of 91%.
Step four: 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,0.55mmol,1.1 eq), HOBt (74 mg,0.55mmol,1.1 eq) was weighed in sequence into a 25mL round bottom flask, 5mL DMF was added, stirring was carried out at room temperature, and after stirring at room temperature for 30 minutes, DIEA (194 mg,1.5mmol,3 eq) was added under ice bath conditions, and the reaction was continued under ice bath for 20 minutes until room temperature overnight. The reaction was completed by TLC, 20mL of a saturated ammonium chloride solution was added, and after thoroughly stirring and mixing, the mixture was extracted with ethyl acetate (3X 30 mL), and the organic phases were combined, washed with water (2X 20 mL) and saturated brine (20 mL), respectively, and dried over anhydrous sodium sulfate. The solvent is removed by rotary evaporation to obtain a white solid crude product, and the white solid crude product is recrystallized by a proper 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 the yield of 71%. 1 H NMR(300MHz,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＝6.1Hz,2H)；MS(ESI):m/z 296(M+1).。
Example 2:
5- (4-fluorophenyl) -N- (pyridine-2-methyl) -1H-pyrazole-3-carboxamide J 2 Is prepared from
Example 1 step four, benzyl amine was changed to pyridine-2-methylamine, and the other conditions were the same as in example 1. A white solid was obtained in 81% yield. Mp 205.5-206.4 ℃; 1 H NMR(300MHz,DMSO-d 6 ):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).。
example 3:
n- (3, 4-dimethoxybenzyl) -5- (4-fluorophenyl) -1H-pyrazole-3-carboxamide J 3 Is prepared from
Otherwise, the procedure of example 1, step four, was followed by changing benzylamine to 3, 4-dimethoxybenzylamine to give a white solid with a yield of 73%. Mp 205.1-206.1 ℃; 1 H NMR(300MHz,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＝5.9Hz,2H),3.74(s,3H),3.72(m,3H)；MS(ESI):m/z 356(M+1).。
example 4:
n- (2-chlorobenzyl) -5- (4-fluorophenyl) -1H-pyrazole-3-carboxamide J 4 Is prepared from
Otherwise the conditions were the same as in example 1, step four, the benzylamine was changed to 2-chlorobenzylamine to give a white solid with a yield of 74%. 1 H NMR(300MHz,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＝5.8Hz,2H)；MS(ESI):m/z 330(M+1).。
Example 5:
5- (4-fluorophenyl) -N- (3-nitrobenzyl) -1H-pyrazole-3-carboxamide J 5 Is prepared from
Other conditions were the same as in step four of example 1, changing benzylamine to 3-nitrobenzylamine, the product was pale yellow solid, and the yield was 82%. Mp 255.3-256.1 ℃; 1 H NMR(300MHz,DMSO-d 6 ):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).。
example 6:
n- (3-nitrobenzyl) -5- (p-tolyl) -1H-pyrazole-3-carboxamide J 6 Is prepared from
Step one: preparation of ethyl 2, 4-dione-4- (p-tolyl) butyrate
The procedure was as in step one of example 1, changing 4-fluoroacetophenone to 4-methylacetophenone, to give a pale yellow solid with 45% yield.
Step two: preparation of 5- (p-tolyl) -1H-pyrazole-3-carboxylic acid ethyl ester
The procedure was as in step two of example 1, substituting ethyl 4- (4-fluorophenyl) -2, 4-dioxobutyrate for ethyl 2, 4-dione-4- (p-tolyl) butyrate to give a bright yellow solid in 80% yield. 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 )δ161.2,147.9,138.6,129.6,127.5,125.7,105.0,61.1,21.4,14.2.MS(ESI):m/z 231[M+1]。
Step three: preparation of 5- (p-tolyl) -1H-pyrazole-3-carboxylic acid
The procedure is as in step three of example 1. 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid ethyl ester was changed to 5- (p-tolyl) -1H-pyrazole-3-carboxylic acid ethyl ester, to give a white solid in 93% yield. 1 H NMR(300MHz,DMSO-d 6 ):δ7.71(d,J＝8.1Hz,2H),7.24(d,J＝8.1Hz,2H),7.12(s,1H),2.31(s,3H).。
Step four: preparation of N- (3-nitrobenzyl) -5- (p-tolyl) -1H-pyrazole-3-carboxamide
The procedure was as in step four of example 1, substituting 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid with 5- (p-tolyl) -1H-pyrazole-3-carboxylic acid and substituting benzylamine with 3-nitrobenzylamine to give a white solid in 74% yield. Mp 262.3-263.1 ℃; 1 H NMR(300MHz,DMSO-d 6 ):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).。
example 7:
n- (pyridine-2-methylene) -5- (p-tolyl) -1H-pyrazole-3-carboxamide J 7 Is prepared from
The procedure was as in example 6, step four, changing 3-nitrobenzylamine to 2-pyridinemethylamine to give a white solid with a yield of 85%. 1 H NMR(300MHz,DMSO-d 6 ):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).。
Example 8:
n- (3, 4-Dimethoxybenzyl) -5- (p-tolyl) -1H-pyrazole-3-carboxamide J 8 Is prepared from
The procedure was as in example 6, step four, changing 3-nitrobenzylamine to 3, 4-dimethoxybenzylamine, to give a white solid with 77% yield. 1H NMR (300 MHz, DMSO-d 6): delta 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).
Example 9:
n-benzyl-5- (P-tolyl) -1H-pyrazole-3-carboxamide J 9 Is prepared from
Other conditions were the same as in example 6, step four, 3-nitrobenzylamine was changed to benzylamine, and 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).。
Example 10:
n- (2-chlorobenzyl) -5- (p-tolyl) -1H-pyrazole-3-carboxamide J 10 Is prepared from
Other conditions were the same as in example 6, step four, 3-nitrobenzylamine was changed to 2-chlorobenzylamine, and the product was a white solid with a yield of 73%. 1 H NMR(300MHz,DMSO-d 6 ):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).。
Example 11:
n-benzyl-5- (4-nitrophenyl) -1H-pyrazole-3-carboxamide J 11 Is prepared from
Step one: preparation of ethyl 4- (4-nitrobenzene) -2, 4-dioxobutyrate
The procedure is as in step one of example 1. 4-fluoro acetophenone was changed to 4-nitroacetophenone to give pale yellow solid powder with 49% yield. 1 H NMR(300MHz,CDCl 3 ):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).
Step two: preparation of 5- (4-nitrophenyl) -1H-pyrazole-3-carboxylic acid ethyl ester
The procedure is as in step two of example 1. Ethyl 4- (4-fluorophenyl) -2, 4-dioxobutyrate was exchanged for ethyl 4- (4-nitrobenzene) -2, 4-dioxobutyrate to give a yellow solid in 65% yield. 1 H NMR(300MHz,DMSO-d 6 ):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).
Step three: preparation of 5- (4-nitrophenyl) -1H-pyrazole-3-carboxylic acid
The procedure is as in step three of example 1. 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid ethyl ester is changed into 5- (4-nitrophenyl) -1H-pyrazole-3-carboxylic acid ethyl esterThe ester was obtained as a pale yellow solid in 68% yield. 1 H NMR(300MHz,DMSO-d 6 ):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).
Step four: preparation of N-benzyl-5- (4-nitrophenyl) -1H-pyrazole-3-carboxamide
Other conditions were the same as in step four of example 1, changing 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid to 5- (4-nitrophenyl) -1H-pyrazole-3-carboxylic acid, the product was a pale yellow solid, and the yield was 70%. Mp258.4-258.8 ℃; 1 H NMR(300MHz,DMSO-d 6 ):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).
example 12:
n- (2-morpholinoethyl) -5- (4-nitrophenyl) -1H-pyrazole-3-carboxamide J 12 Is prepared from
Otherwise the conditions were the same as in example 11, step four, the benzylamine was changed to 2-morpholinoethyl-1-amine, the product was a white solid with 29% yield. Mp 171.1-172.3 ℃; 1 H NMR(300MHz,DMSO-d 6 ):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).。
example 13:
n- (benzo [ d)][1,3]Dioxol-5-ylmethyl) -5- (4-nitrophenyl) -1H-pyrazole-3-carboxamide J 13 Is prepared from
Otherwise the conditions were the same as in example 11, step four, the benzylamine was changed to 3,4- (methylenedioxy) benzylamine and the product was a yellow solid in 26% yield. Mp 248.2-249.1 ℃; 1 H NMR(300MHz,DMSO-d 6 ):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-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).。
example 14:
5- (4-Nitrophenyl) -N- (pyridine-2-methyl) -1H-pyrazole-3-carboxamide J 14 Is prepared from
Other conditions were the same as in example 11, step four, changing benzylamine to pyridine-2-methylamine, the product was a pale yellow solid, yield 33%. Mp 258.8-259.6 ℃; 1 H NMR(300MHz,DMSO-d 6 ):δ14.08(s,1H),9.24(s,1H),8.54(s,1H),8.32(d,J＝8.2Hz,2H),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).。
example 15:
morpholin (5- (4-nitrophenyl) -1H-pyrazol-3-yl) methanone J 15 Is prepared from
Otherwise the conditions were the same as in example 11, step four, the benzylamine was changed to morpholine and the product was a white solid with a yield of 35%. Mp275.0-275.7 ℃; 1 H NMR(300MHz,DMSO-d 6 ):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).。
example 16:
n- (3, 4-Dimethoxybenzyl) -5- (4-nitrophenyl) -1H-pyrazole-3-carboxamide J 16 Is prepared from
Other conditions were the same as in example 11, step four, changing benzylamine to 3, 4-dimethoxyThe product was a pale yellow solid, 73% yield. Mp 196.4-197.2 ℃; 1 H NMR(300MHz,DMSO-d 6 ):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).。
example 17:
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n- (3-nitrobenzyl) -5- (4-nitrophenyl) -1H-pyrazole-3-carboxamide J 17 Is prepared from
Other conditions were the same as in example 11, step four, changing benzylamine to 3-nitrobenzylamine, the product was pale yellow solid, yield 80%. Mp 279.5-280.2 ℃; 1 HNMR(300MHz,DMSO-d 6 ):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).。
example 18:
5- (2, 4-dichlorophenyl) -N- (2-morpholinoethyl) -1H-pyrazole-3-carboxamide J 18 Is prepared from
Step one: preparation of ethyl 4- (2, 4-dichlorophenyl) -2, 4-dioxobutyrate
Other conditions were the same as in step one of example 1, except that 4-fluoroacetophenone was changed to 2, 4-dichloroacetophenone. Pale yellow solid powder was obtained in 40% yield. 1 H NMR(300MHz,DMSO-d 6 ):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).。
Step two: preparation of 5- (2, 4-dichlorophenyl) -1H-pyrazole-3-carboxylic acid ethyl ester
The procedure is as in step two of example 1. Ethyl 4- (4-fluorophenyl) -2, 4-dioxobutyrate was exchanged for ethyl 4- (2, 4-dichlorophenyl) -2, 4-dioxobutyrate to give a white solid in 70% yield. 1 H NMR(300MHz,CDCl 3 ):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).。
Step three: preparation of 5- (2, 4-dichlorophenyl) -1H-pyrazole-3-carboxylic acid
The procedure is as in step three of example 1. 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid ethyl ester was changed to 5- (2, 4-dichlorophenyl) -1H-pyrazole-3-carboxylic acid ethyl ester to give a white solid in 72% yield. 1 H NMR(300MHz,DMSO-d 6 ):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).。
Step four: preparation of 5- (2, 4-dichlorophenyl) -N- (2-morpholinoethyl) -1H-pyrazole-3-carboxamide
Other conditions were the same as in step four of example 1, 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid was changed to 5- (2, 4-dichlorophenyl) -1H-pyrazole-3-carboxylic acid, benzylamine was changed to 2-morpholinoethyl-1-amine, and the product was a white solid in 65% yield. Mp 249.3-250.1; 1 H NMR(300MHz,DMSO-d 6 ):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).。
example 19:
[5- (2, 4-dichlorophenyl) -1H-pyrazol-3-yl]-4-morpholinomethionone J 19 Is prepared from
Step one, two and three are the same as in example 18, step four is changed to morpholine, and other conditions are the same as in example 18, step four. The product was a white solid in 75% yield. Mp 204.3-205.5 ℃; 1 H NMR(300MHz,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).。
example 20:
5- (2, 4-dichlorophenyl) -N- (pyridine-2-methyl) -1H-pyrazole-3-carboxamide J 20 Is prepared from
Otherwise the conditions were the same as in example 18, step four, the benzylamine was changed to pyridine-2-methylamine, the product was a white solid with a yield of 58%. Mp 242.5-243.3 ℃; 1 H NMR(300MHz,DMSO-d 6 ):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-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).。
example 21:
(5- (2, 4-dichlorophenyl) -1H-pyrazol-3-yl) (piperidin-1-yl) methanone J 21 Is prepared from
Otherwise the conditions were the same as in example 18, step four, the benzylamine was changed to piperidine and the product was a white solid with a yield of 18%. 1 H NMR(400MHz,DMSO-d 6 )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).
Example 22:
n-cyclohexyl-5- (2, 4-dichlorophenyl) -1H-pyrazole-3-carboxamide J 22 Is prepared from
Otherwise the conditions were the same as in example 18, step four, the benzylamine was changed to cyclohexylamine, the product was a white solid with a yield of 74%. 1 H NMR(400MHz,DMSO-d 6 )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,1H),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).。
Example 23:
n- (3-bromophenyl) -3-phenyl-1H-pyrazole-5-carboxamide J 23 Is prepared from
Step one: preparation of ethyl 2, 4-dione-4-phenylbutyrate
The procedure is as in step one of example 1. The 4-fluoro acetophenone is changed into acetophenone to obtain the crude product of ethyl 2, 4-diketone-4-phenylbutyrate.
Step two: preparation of 5-phenyl-1H-pyrazole-3-carboxylic acid ethyl ester
The procedure is as in step two of example 1. Ethyl 4- (4-fluorophenyl) -2, 4-dioxobutyrate was exchanged for ethyl 2, 4-dione-4-phenylbutyrate to give a white solid with a yield of 80%. 1 H NMR(400MHz,CDCl 3 )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,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 three: preparation of 5-phenyl-1H-pyrazole-3-carboxylic acid
The procedure is as in step three of example 1. The ethyl 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylate was changed to ethyl 5-phenyl-1H-pyrazole-3-carboxylate to give a white solid in 90% yield. 1 H NMR(400MHz,CD 3 OD)δ7.79-7.72(m,2H),7.43(t,J＝7.5Hz,2H),7.40-7.31(m,1H),7.12(s,1H).
Step four: preparation of N- (3-bromophenyl) -3-phenyl-1H-pyrazole-5-carboxamide
Other conditions were the same as in step four of example 1, 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid was changed to 5-phenyl-1H-pyrazole-3-carboxylic acid, benzylamine was changed to m-bromoaniline, and 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)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/z 342(M+1).。
Example 24:
n- (3-bromobenzyl) -3-phenyl-1H-pyrazole-5-carboxamide J 24 Is prepared from
Step one, step two and step three are the same as in example 23, step four is to change benzylamine into m-bromobenzylamine, and other conditions are the same as in example 23, step four, and the product is white solid with a yield of 53%. 1 H NMR(400MHz,DMSO-d 6 )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).。
Example 25:
n-phenyl-3- (M-tolyl) -1H-pyrazole-5-carboxamide J 25 Is prepared from
Step one: preparation of ethyl 2, 4-dione-4- (m-tolyl) butyrate
The procedure is as in step one of example 1. The 4-fluoro acetophenone is changed into 3-methyl acetophenone, and the crude product of ethyl 2, 4-diketone-4- (m-tolyl) butyrate is obtained.
Step two: preparation of 5- (m-tolyl) -1H-pyrazole-3-carboxylic acid ethyl ester
The procedure is as in step two of example 1. Ethyl 4- (4-fluorophenyl) -2, 4-dioxobutyrate was exchanged for ethyl 2, 4-dione-4- (m-tolyl) butyrate, as a white solid, 76% yield. 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 three: preparation of 5- (m-tolyl) -1H-pyrazole-3-carboxylic acid
The procedure is as in step three of example 1. 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid ethyl ester was changed to 5- (m-tolyl) -1H-pyrazole-3-carboxylic acid ethyl ester, to give a white solid in 40% yield. 1 H NMR(400MHz,DMSO-d 6 )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).
Step four: preparation of N-phenyl-3- (M-tolyl) -1H-pyrazole-5-carboxamide
Other conditions were the same as in step four of example 1, 5- (4-fluorophenyl) -1H-pyrazole-3-carboxylic acid was changed to 5- (m-tolyl) -1H-pyrazole-3-carboxylic acid, and benzylamine was changed to aniline, and a white solid was obtained in 20% yield. 1 H NMR(400MHz,DMSO-d 6 )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).。
Biological Activity test
cAMP is a key signaling molecule for many G protein-coupled receptors. The accumulation level of cAMP was tested mainly using the GloSensor method, a bioluminescence-based cAMP biosensor (Promega). HEK293T cells in good condition were seeded in 6-well plates or 35MM cell culture dishes and placed at 37℃with 5% CO 2 Culturing in an incubator for 24 hours to enable cells to grow on the wall. Post-transfection of beta with transfection reagent 2 Adrenergic receptor plasmid and pGlosensor TM The-22 FcAMP plasmid is transferred into cells and placed in 5% CO at 37 DEG C 2 Culturing in an incubator for 24 hours to enable the target gene to be transcribed and expressed. The plasmid-transfected cells were then plated in 96-well plates at 37℃with 5% CO 2 The incubator cultures for 24 hours. Finally, the old culture solution of the 96-well plate is sucked and removed, the cells are washed once with the new culture solution, and then the culture solution containing GloSensor is added TM cAMP Reagent, serum and CO 2 Equilibrium culture of-independent medium at 37℃with 5% CO 2 The incubator is incubated for 1-2h or until a stable background signal is obtained. Dissolving Isoprenaline (ISO) in DMSO, sterilizing, filtering, and diluting with culture solution to different concentrationsGradient (DMSO concentration in formulated compound less than or equal to 0.1%). The formulated compounds were rapidly added to 96-well plates containing HEK293T cells to initiate dosing stimulation. The rapid rise in bioluminescence signal following addition of the compound was observed by a multifunctional microplate reader and the compound was added at a concentration of 50 μm immediately when the signal value increased to the point that no longer increased. Meanwhile, the bioluminescence signal is immediately collected by a multifunctional enzyme-labeled instrument, and the bioluminescence signal value rises along with the increase of the concentration of the compound. And finally, after finishing the data, processing the data by using GraphPad Prism8 software, and obtaining a dose-effect relation curve of the allosteric regulator by taking the concentration as an abscissa and the signal value as an ordinate.
TABLE 2 statistical table of functional Activity of novel pyrazole derivatives
Note that: wherein "+" means having the corresponding activity "-" means no corresponding activity
TABLE 3 comparison of the Activity of novel derivatives with allosteric antagonistic Activity with Cmpd-15
Note that: a values are expressed as blocking activity relative to Cmpd-15
Novel compound J in Glosensor cAMP accumulation assay test results 1 、J 2 、J 12 、J 13 、J 15 、J 16 、J 17 、J 18 、J 19 、J 21 、J 22 、J 25 All having beta 2 Antagonism of AR allosteric, the remaining novel compounds did not have allosteric antagonistic activity (see table 2). The above compound with allosteric antagonism has a higher activity than that of the lead compound Cmpd-15, J 19 The allosteric antagonistic activity of (C) is equivalent to that of Cmpd-15, J 21 、J 22 、J 25 The allosteric antagonistic activity of (C) is obviously improved compared with Cmpd-15, wherein the compound with the best activity is J 25 (see Table 3)。
As can be seen from Table 3, of all the novel pyrazole compounds, compound J 25 The allosteric antagonistic activity of (2) is preferably about 2.5 times that of Cmpd-15. When R is 2 The pyrazole amide compound which is a hydrogen atom or a 4-methyl substituted compound does not have allosteric antagonistic activity; when R is 2 The 4-bit fluorine substitution is adopted, the activity of the coupling benzylamine is better, if different substituted benzylamine is coupled, the coupling benzylamine is inactive, and in addition, the allosteric antagonistic activity of the coupling 2-pyridine methylamine is not obviously improved; when R is 2 In the case of 4-nitro substitution, most have allosteric antagonistic activity, but are not as active as Cmpd-15. When R is 1 ，R 2 Meanwhile, when chlorine is substituted, the activity of the product after coupling with morpholine, cyclohexylamine and piperidine is equivalent to that of the allosteric antagonist Cmpd-15, but the product after coupling with N-aminoethylmorpholine and 2-pyridine methylamine is inactive. In addition, when R 2 When the 3-methyl is substituted, the allosteric antagonistic activity is obviously improved.
Testing whether target Compounds could allosterically modulate beta by cAMP accumulation assay 2 The functional Activity of the AR endogenous ligand ISO, from the results in Table 1, is seen in J 1 、J 2 、J 12 、J 13 、J 15 、J 16 、J 17 、J 18 、J 19 、J 21 、J 22 、J 25 Are all beta 2 Negative allosteric modulation of AR, wherein at the same concentration (50. Mu.M) J 25 Is 2.5 times higher than the pharmacological activity of the lead compound (Cmpd-15). In addition, by adding J in a multiple relationship with the concentration 25 To further confirm whether the compound is beta 2 Negative allosteric modulators of AR. The results are shown in the appendix FIG. 1, when J 25 At concentrations up to 30. Mu.M, the ISO curve exhibits a large downward shift and reaches the maximum lower limit of the dose-response-modulating ISO functional activity, with a further increase in J 25 The ISO dose-response curve is hardly shifted down any more. This is illustrative J 25 The IC50 value of (C) may be between 15.0 mu M and 30.0 mu M, and the concentration curve shows a sudden drop, namely the newly synthesized pyrazole amide derivative J 25 Is beta with better activity 2 -AR negative allosteric antagonists or negative allosteric modulatorsAnd (3) an agent. This allosteric modulation phenomenon is consistent with the reported allosteric antagonistic modulation mechanism.
Taken together, these results indicate that the right (S) -2-amino-3- (3-bromophenyl) -N-methylpropanamide in Cmpd-15 structure is an important active moiety, and if the moiety is replaced by benzylamine or heteroatom-containing benzylamine, the antagonistic activity of most of the obtained compounds is not significantly improved; if the part is replaced by aniline, the allosteric antagonistic activity is obviously improved. Therefore, the structure of the aniline is simpler, and a compound with simpler structure and better biological activity can be obtained by replacing the right part of the Cmpd-15, so that the method has great significance in researching the pyrazole amide type structure antagonists.

Claims (3)

1. Beta-acting 2 -pyrazole amide derivatives of allosteric antagonists of the adrenergic receptors, characterized in that: the structural formula of the pyrazole amide derivative is shown as formula I:
wherein R is 1 Any one of =h, cl;
R 2 ＝H,F,Cl,NO 2 ,CH 3 any one of them;
any one of the following.
2. Beta-acting 2 -pyrazole amide derivatives of allosteric antagonists of the adrenergic receptors, characterized in that: the structural formula of the pyrazole amide derivative is shown as formula I:
wherein R is 1 Any one of =h, cl;
R 2 ＝F,Cl,NO 2 ,3-CH 3 any one of them;
any one of the following.
3. Use as beta according to claim 1 2 -use of pyrazole amide derivatives of allosteric antagonists of the adrenergic receptors, characterized in that: the pyrazole amide derivative is used for preparing beta 2 -use of an adrenoceptor allosteric antagonist in a medicament.
CN202310937573.6A 2023-07-28 2023-07-28 As beta 2 Pyrazole amide derivatives of allosteric antagonists of the adrenergic receptors Pending CN117143019A (en)

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