WO2022028154A1 - 作为p2x3受体拮抗剂的n-甲酰胺基吡唑啉类衍生物及应用 - Google Patents

作为p2x3受体拮抗剂的n-甲酰胺基吡唑啉类衍生物及应用 Download PDF

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WO2022028154A1
WO2022028154A1 PCT/CN2021/103402 CN2021103402W WO2022028154A1 WO 2022028154 A1 WO2022028154 A1 WO 2022028154A1 CN 2021103402 W CN2021103402 W CN 2021103402W WO 2022028154 A1 WO2022028154 A1 WO 2022028154A1
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ethyl
dihydro
pyrazole
carboxamide
methylphenyl
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PCT/CN2021/103402
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French (fr)
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程云锋
朱心强
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杭州维坦医药科技有限公司
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Priority to JP2022548136A priority Critical patent/JP7385852B2/ja
Priority to EP21854166.2A priority patent/EP4079725A4/en
Priority to US17/909,429 priority patent/US20230357218A1/en
Publication of WO2022028154A1 publication Critical patent/WO2022028154A1/zh

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Definitions

  • the invention belongs to the field of medicine, and relates to an N-formamidopyrazoline derivative as a ligand-gated non-selective cation channel receptor subtype P2X3 inhibitor, and its preparation for treating diseases mediated by P2X3 application in medicine.
  • P2X3 is a ligand-gated non-selective cation channel receptor subtype, belonging to the ionotropic P2X receptor in the P2 class of purinergic receptors, originally cloned in mammals in 1995 (Chen C et al., Nature , 1995, 428; Lewis, C et al., Nature, 1995, 432), so far, there are 7 subtypes of P2X receptors (P2X1 ⁇ P2X7) cloned in mammals. Each P2X receptor molecule consists of intracellular N-terminal and C-terminal and two transmembrane domains. Although there are differences in subtypes and species of P2X receptors, there is no obvious difference in the basic structure of P2X receptors.
  • P2X3 is homologous. Trimers, P2X1/2, P2X2/3 are heterotrimers (Jacobson et al., Neuropharmacology, 2016, 31). P2X3 receptors are widely distributed in the body, mainly expressed in peripheral sensory neurons related to nociceptive information, and play an important mediating role in the generation and transmission of nociceptive information.
  • P2X3 is involved in a variety of physiological and pathological responses, including inflammatory pain, neuropathic pain, cancer pain and other pathological pain, cough, hypertension, bladder urination, etc.
  • up-regulation of P2X3 receptor expression can lead to the formation of pain sensitivity, which is involved in pain signaling, and knockout of P2X3 receptor can alleviate pain-related behaviors in mice (Cockayne DA et al., Nature, 2000, 1011).
  • Inhibition of P2X3 receptors can improve the reflex function of spontaneous cardiac baroreceptors and help inhibit sympathetic nerves in rats, thereby exerting antihypertensive effects (Pijacks W et al., Nature Medicine, 2016, 1151).
  • P2X3 receptors are correlated with the urination frequency of mice, and the deletion of P2X3 receptors will reduce the urination frequency of mice (Gao XF et al., Nature Communication, 2015, 7650).
  • the ATP/P2X3 signaling pathway in human skin is associated with chronic pruritus, so P2X3 receptor inhibitors can also be used for the treatment of chronic pruritus (Chauret, N. et al., 49th Annual ESDR Meeting, 2019).
  • the P2X3 inhibitor with the novel compound structure of the present invention has unexpected and favorable properties in terms of selectivity, pharmacokinetics and pharmacodynamics, etc., and can greatly expand the research and development of P2X3 targeted drugs, which constitutes a basis of the present invention.
  • the present invention provides a novel N-formamidopyrazoline derivative as a ligand-gated non-selective cation channel receptor subtype P2X3 inhibitor.
  • the invention also discloses the application of the above N-formamidopyrazoline derivatives.
  • N-formamidopyrazoline derivative having the compound structure represented by the general formula (I) or its enantiomer, diastereomer, epimer and racemate , or a pharmaceutically acceptable salt thereof:
  • Ar is selected from substituted or unsubstituted phenyl, substituted or unsubstituted five-membered heteroaromatic ring containing 1-3 atoms selected from O, N and S, or substituted or unsubstituted six-membered heteroaryl containing 1-2 N atoms Aromatic ring group; when Ar is selected from the ortho-position double-substituted phenyl group, the two ortho-position substituent groups are independent of each other or form a ring;
  • R 1 is selected from substituted or unsubstituted phenyl or substituted or unsubstituted pyridyl
  • R 2 is selected from methyl, ethyl, isopropyl, cyclopropyl;
  • R 3 , R 4 are each independently selected from H, C1-C5 alkyl, C1-C5 alkoxycarbonyl, substituted or unsubstituted C1-C5 acyl; or R 3 and R 4 together with the N to which they are both connected together to form a substituted or unsubstituted C5-C6 heterocycloalkyl or C5-C6 heterocycloalkyl ketone or C5-C6 heteroaryl; the carbon atoms on the heterocycloalkyl or heterocycloalkyl ketone ring may further Replaced by one or more O, N.
  • Said Ar is selected from the following substituted phenyl, substituted five- or six-membered heteroaromatic groups:
  • the R 5 , R 6 are the same or different or deleted, and are independently selected from H, halogen, nitro, cyano, methyl, trifluoromethyl, trifluoromethoxy, methoxy, and dioxymethylene , C1-C3 straight-chain or branched alkoxy groups, and substituted on any ring carbon atom; when selecting the dioxymethylene group, the R 5 and R 6 are the oxygen atoms therein, and the connected methylene base ring;
  • R7 is selected from methyl, ethyl, isopropyl, cyclopropyl.
  • the halogen includes F, Cl, Br.
  • the C1-C5 alkyl group includes methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, etc.;
  • the C1-C5 alkoxycarbonyl group includes methyl, ethyl, propyl, etc. , isopropyl, butyl, tert-butyl, etc. linked oxycarbonyl.
  • the C1-C5 acyl groups include acyl groups connected by methyl, ethyl, propyl, isopropyl, butyl, tert-butyl and the like.
  • the C1-C3 linear or branched alkoxy includes methoxy, ethoxy, propoxy or isopropyloxy and the like.
  • the substituent on the phenyl or pyridyl ring is selected from halogen, C1-C3 straight or branched chain alkyl (including methyl, ethyl, propyl, isopropyl, etc.), C1-C3 linear or branched alkoxy, trifluoromethyl, trifluoromethoxy.
  • R 3 and R 4 are each independently selected from H, methyl, ethyl, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, tert-butoxycarbonyl, formyl, acetyl, N,N - dialkylacetyl; or R3 , R4 together with the N to which they both are attached to form a substituted or unsubstituted morpholinone, substituted or unsubstituted morpholino, substituted or unsubstituted piperidine base, substituted or unsubstituted piperazinyl, substituted or unsubstituted piperazinone, substituted or unsubstituted pyrrolidone, substituted or unsubstituted oxazolidinone, substituted or unsubstituted imidazolidinone, substituted or unsubstituted imidazole group, substituted or unsubstituted pyrazoly
  • Substituents are selected from C1-C3 straight or branched chain alkyl, formyl, acetyl, C1-C5 straight or branched alkoxy (including methyl, ethyl, propyl, isopropyl, butyl, tert-butyl attached oxy substituent).
  • it has the structure represented by the general formula (Ia), (Ib), (Ic) or (Id) or its enantiomer, diastereomer, racemate, or its pharmacy Acceptable salt on:
  • the Ar is selected from the group consisting of phenyl-p-trifluoromethylphenyl, dimethoxyphenyl, dioxolane phenyl, p-cyanophenyl, chlorophenyl, methoxypyridyl, tri- Fluoromethylpyridyl, chloro-substituted pyridyl, difluoropyridyl, trifluoromethoxypyridyl, trifluoromethylpyrazinyl, methylpyrazinyl, chloropyrazinyl, methoxypyridazinyl , trifluoromethylpyridazinyl, chloropyridazinyl, trifluoromethylpyrimidinyl, methylpyrimidinyl, chloropyrimidinyl, methyl-1,2,4-oxadiazolyl, methyl-1 ,3,4-oxadiazolyl; R 1 is selected from p-methylphenyl, p-me
  • preferred compounds include but are not limited to:
  • Another object of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising at least one active ingredient and one or more pharmaceutically acceptable carriers or excipients
  • the active ingredient may be N-formamidopyrazoline derivatives of the present invention and their pharmaceutically acceptable salts, isomers, enantiomers, diastereomers and racemates of the compounds
  • Described salt selects pharmaceutically acceptable inorganic acid salt, organic acid salt for use
  • Described inorganic acid salt includes the salt formed with hydrohalic acid, nitric acid, carbonic acid, sulfuric acid, phosphoric acid
  • Described organic acid salt includes and malic acid, L-malic acid, D-malic acid, citric acid, fumaric acid, oxalic acid, lactic acid, camphorsulfonic acid, L-camphorsulfonic acid, D-camphorsulfonic acid, The salt formed by p-toluenesulfonic acid, methanesulfonic acid and benzoic
  • the carriers or excipients include conventional fillers in the pharmaceutical field, diluents, wetting agents, lubricants, binders, disintegrants, absorption enhancers, surfactants, adsorption carriers, antioxidants, emulsifiers, metals Chelating agents, pH adjusters, etc., if necessary, flavoring agents, sweeteners, etc. can also be added.
  • the medicine of the present invention can be made into various forms such as tablets, capsules, inhalants, emulsions, suspensions, gels, powders, granules, oral liquids and injections. method of preparation.
  • the present invention also provides the use of any one of the compounds of the general formula (I), and pharmaceutically acceptable salts thereof, used alone and/or in combination with other drugs in the preparation of P2X3 inhibitors, especially in the preparation of P2X3 inhibitors.
  • the diseases mediated by P2X3 include respiratory diseases, such as chronic obstructive pulmonary disease (COPD), asthma, acute/subacute and chronic cough, bronchospasm; pain caused by various reasons, such as surgical pain, inflammation Sexual pain, cancer pain, bladder pain, endometriosis pain, diabetic neuralgia, traumatic pain, toothache, migraine, and pain associated with irritable bowel syndrome; skin disorders such as chronic pruritus.
  • COPD chronic obstructive pulmonary disease
  • the title molecule was prepared by condensation of the corresponding substituted N-formyl chloride pyrazoline with substituted arylmethylamine or substituted heteroarylmethylamine under basic conditions.
  • the obtained target molecule is a mixture of epimers, which can be separated by column chromatography gradient to obtain S,R- Isomers and R,R-isomers
  • the obtained target molecule can be separated by column chromatography gradient to obtain S, S-isomer and R ,
  • the preparation of intermediate substituted N-formyl chloride-based pyrazoline can use the corresponding amino ketone as raw material, react with formaldehyde under alkaline conditions to generate ketene, and then react with hydrazine to obtain cyclic compound pyrazoline, and pyrazoline is further reacted with Triphosgene reaction to prepare substituted N-formyl chloride pyrazoline;
  • R-substituted arylmethylamine, R-substituted heteroarylmethylamine, or S-substituted arylmethylamine, S-substituted heteroarylmethylamine can use corresponding aromatic aldehyde as raw material, and S-tert-butylsulfinic acid can be used as raw material.
  • the amide or R-tert-butyl sulfinamide is reacted to obtain imine, which is then added with the corresponding Grignard reagent or alkyl lithium under low temperature conditions, and hydrolyzed to obtain R-substituted arylmethylamine or S-substituted heteroaryl methylamine.
  • the present invention confirms through experiments that the compounds of the present invention can selectively inhibit the activity of P2X3 channels, and can be used in medicines for treating diseases mediated by P2X3.
  • the inhibitors provided by the present invention also include pharmaceutical compositions of the compounds.
  • Figure 1 shows the chemical structure (A), 3D simulation (B), and 1 HNMR (C) of the title compound 1 .
  • FIG. 2 is the NOESY spectrum of the title compound 1.
  • Fig. 3 shows the chemical structure (A), 3D simulation diagram (B), and 1 HNMR (C) of compound 1a.
  • Figure 4 is the NOESY spectrum (D) of compound 1a.
  • Figure 5 is the inhibitory effect of the title compound on ATP/citrate-induced cough in guinea pigs (intraperitoneal injection).
  • Figure 6 is the inhibitory effect of the title compound on ATP/citrate-induced cough in guinea pigs (oral administration).
  • Figure 7 shows the percentages of drinking water and quinine water after administration of experimental animals in each title group.
  • Figure 8 is the residual percent degradation of title compound 4 in artificial gastrointestinal fluid.
  • Figure 9 shows the residual percent degradation of title compound 22 in artificial gastrointestinal fluid.
  • Triphosgene (1.30 g, 4.32 mmol) and dichloromethane (20 mL) were added to a dry reaction flask, and the temperature was lowered to -10 °C under N 2 protection, and compound A-1c (2.0 g, 8.64 mmol), pyridine (2.04 g, 25.80 mmol) and dichloromethane (20 mL) were mixed, and the mixture was added dropwise to the reaction flask at -10 ° C. After the addition was completed, the reaction was stirred at room temperature for 10 min, and diluted hydrochloric acid was added to quench the reaction. The organic phase and the aqueous phase were separated.
  • the organic layer was separated, the aqueous phase was extracted with dichloromethane (10 mL ⁇ 2), the organic phases were combined, the organic phases were washed with saturated brine, dried over anhydrous Na 2 SO 4 , filtered, and the filtrate was concentrated to dryness under reduced pressure.
  • Triphosgene (1.55g, 5.14mmol) and anhydrous dichloromethane (25mL) were added to a dry reaction flask, protected by N, cooled to -8°C to -10°C, and compound A-3a (2.5g, 10.28 mmol) and pyridine (2.65 g, 22.54 mmol) were dissolved in dichloromethane (25 mL), and added dropwise to the reaction flask at -10 °C. After the dropwise addition was completed, the mixture was stirred at room temperature for 10 min, and diluted hydrochloric acid was added to quench the reaction.
  • the intermediate A-17 listed in Table 1.1 uses 4-methoxy- ⁇ -bromoacetophenone instead of 4-methyl- ⁇ -bromoacetophenone as the raw material, according to the same method for the preparation of compound A-4 synthesized.
  • the intermediates B-7 to B-22 listed in Table 1.2 are the corresponding substituted heteroaromatic aldehydes (B-7: 2-methylpyrimidine-5-carbaldehyde; B-8: 2-chloropyrimidine-5-carbaldehyde; B-9, B-21: 6-trifluoromethylpyridine-3-carbaldehyde; B-10: 6-methoxypyridine-3-carbaldehyde; B-11: 6-trifluoromethoxypyridine-3- formaldehyde; B-12: 5,6-difluoropyridine-3-carbaldehyde; B-13: 6-chloropyridine-3-carbaldehyde; B-14: 5-trifluoromethylpyridine-2-carbaldehyde; B-15 : 5-methoxypyridine-2-carbaldehyde; B-16: 5-trifluoromethylpiperazine-2-carbaldehyde; B-17: 5-methylpiperazine-2-carbaldehyde; B-18: 6- Trifluoromethylpyri
  • Synthesis steps Compound A-1 (1.5g, 5.1mmol) and Compound B-21 (0.95g, 5mmol) were added to dichloromethane (10mL), and then N,N-diisopropylethylamine (DIPEA) was added. , 2.0 g, 12.2 mmol), stirred at room temperature overnight, added hydrochloric acid (0.5N, 10 mL) to quench the reaction, and stirred for 10 min.
  • DIPEA N,N-diisopropylethylamine
  • HEK293 cell line The cells used HEK293 cell line.
  • HEK293 cells were cultured in a 37°C incubator with 5% CO 2 . Passaging was performed when the cells had grown to 80%-90% density. Discard the medium in the original 60mm petri dish, wash the cells 3 times with sterilized PBS solution, and then dry the solution. Add 1 mL of 0.25% trypsin (Gibco) to digest for 10 s, blot dry the trypsin and add 3 mL of cell culture medium to resuspend the cells.
  • Gibco trypsin
  • this mixed solution was slowly added to an equal volume of 2 ⁇ HBS solution (NaCl 140mM, Na 2 HPO 4 1.5mM, HEPES 50mM, adjusted to pH 6.96), and mixed with a pipette tip while adding, all the After adding, gently blow 8-10 times. After standing for 5 min, add it to a small dish, shake the small dish slightly to mix the solution evenly, and place it in a cell incubator. Change the medium after 8–10 h. Electrophysiological experiments were performed 24-48 h after transfection.
  • 2 ⁇ HBS solution NaCl 140mM, Na 2 HPO 4 1.5mM, HEPES 50mM, adjusted to pH 6.96
  • Electrophysiological recording electrodes were obtained by two-step drawing using an electrode drawing machine (PP-10, Narishige).
  • a high potassium salt solution containing nystatin K 2 SO 4 75 mM, KCl 120 mM, MgSO 4 5 mM, HEPES 10 mM, adjusted to pH 7.2 with Tris-base, and used after filtration
  • nystatin K 2 SO 4 75 mM, KCl 120 mM, MgSO 4 5 mM, HEPES 10 mM, adjusted to pH 7.2 with Tris-base, and used after filtration
  • This experiment uses an Axon200B amplifier, Digidata 1440A digital-to-analog converter, and Clampfit in voltage-clamp mode to record data.
  • the clamping voltage is -60mv
  • the sampling frequency is 10kHz
  • the filtering is performed under the condition of 2kHz.
  • the concentration of P2X3 receptor agonist ATP was 1 ⁇ M, and the interval between two ATP administrations was 10 min. All test compounds, including reference substance AF-219, were pre-administered for 2 min before co-administration with ATP.
  • the inhibitory activity of the title compound on human PAR-1 was determined by calcium ion flux fluorescence method (FLIPR method). For its method, see the literature report of Zhong et al. [Zhong et al. J Biomol Struct Dyn, 2017, 35(13):2853].
  • the HEK293/Ga15 recombinant cell line stably expressing human PAR-1 was cultured in DMEM (Thermo Fisher Scientific, Shanghai) medium (containing 10% calf serum, 800 ⁇ g/mL G418)).
  • the incubator conditions were 37 °C and air humidity with 5% CO2 .
  • the cell assay plate was placed in a FLIPR instrument (Molecular Device) and 10 ⁇ L of compound (3x final concentration) from the compound plate was added to the corresponding wells of the cell assay plate. After 10 minutes, 10 ⁇ L of TFLLR-NH2 (TFA) (final concentration of 10 ⁇ M) was added to each well to stimulate the generation of intracellular calcium flux signals.
  • TFLLR-NH2 TFLLR-NH2
  • Analytical data was collected using the FLIPR program.
  • the inhibitory (or agonistic) activity of the compounds at each concentration was assessed as peak fluorescence.
  • the IC50 values of the compounds were calculated using EXCEL and PRISM programs.
  • the compounds were administered by intravenous injection (3 mg/kg), intraperitoneal injection (10 mg/kg) or gavage (30 mg/kg) at different doses.
  • blood was collected into EDTA blood sample collection tubes. Plasma was isolated by centrifugation and stored at -20°C pending analysis.
  • Standard curve and quality control sample preparation and processing Dilute the compound stock solution with 50% methanol water into standard working solution containing each compound concentration of 20-10000ng/mL, and 60, 600, 6000ng/mL quality control working solution. Take 47.5 ⁇ L of blank rat plasma and add 2.50 ⁇ L of standard curve working solution and quality control working solution, respectively, to prepare standard curve with concentrations of 1.00-500.00ng/mL of each compound and concentrations of 3.00, 30.00 and 300.00ng/mL 200 ⁇ L of acetonitrile (containing internal standard verapamil 5ng/mL) were added to the quality control samples, vortexed for 3 min, centrifuged at 20,000 rcf at 4°C for 15 min, and the supernatant was collected for LC-MS/MS analysis.
  • acetonitrile containing internal standard verapamil 5ng/mL
  • Compound blood sample processing take 5 ⁇ L of plasma sample, add 45 ⁇ L of blank rat plasma, add 200 ⁇ L of acetonitrile (including internal standard verapamil 5ng/mL), vortex for 3 min, centrifuge at 20000rcf for 15 min at 4°C, and take the supernatant LC-MS/MS analysis was performed (column: ACQUITY BEH C18 2.1 x 50 mm 1.7 ⁇ m; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; flow rate: 0.35 mL/min).
  • the guinea pig cough model was induced by ATP/citrate to observe the inhibitory effect of compounds on guinea pig cough by intraperitoneal injection and gavage [Carceau and Chauret (2019) Pulmonary Pharmacology&Therapeutics 56:56–62].
  • Guinea pig screening Put the guinea pigs in a plexiglass box of about 25cm ⁇ 10cm ⁇ 8cm (length, width, height), spray 20% citric acid solution for 1 minute, and record the animal’s cough within 10 minutes with a multi-channel physiological signal acquisition and processing system According to the number of coughs of the guinea pigs, the guinea pigs that did not cough were eliminated, and finally 35 guinea pigs of each sex were screened out, and the guinea pigs were grouped by stratified random method for formal experiment.
  • Formal test 20 minutes after intraperitoneal injection of guinea pigs in each group, or 45 minutes after intragastric administration, guinea pigs in each group were placed in a plexiglass box of about 25cm ⁇ 10cm ⁇ 8cm (length, width, height). The 10 ⁇ M ATP solution was continuously sprayed into the box with a pressure nebulizer for 2 min (spraying flow rate 0.6 ml/min). Then, 20% citric acid solution was sprayed for 1 min, and the number of coughs of animals within 10 min was recorded with a multi-channel physiological signal acquisition and processing system.
  • the number of coughs was recorded with Excel software. GraphPad Prism 8 software was used for graphing, and SPSS 18 software was used for statistical analysis. The results are shown in Figures 5 and 6. The number of coughs and the incubation period in each group were tested for homogeneity of variance. If the variance is equal (P>0.05), then do one-way ANOVA, if there is significant difference (P ⁇ 0.05), then do Dunnett test between each dose group and the control group; otherwise, the test ends. If the variance is unequal (P ⁇ 0.05), a non-parametric test (Kruskal-Wallis H test, ie KWH test) is performed. If there is a statistical difference in the KWH test (P ⁇ 0.05), the difference between each dose group and the control group is determined. Do the Mann-Whitney U test, otherwise the test ends.
  • KWH test KWH test
  • Animals and groupings 40 male SD rats, about 300 grams per rat. The animals were randomly divided into 4 groups with 10 animals in each group, and the weights of each group were similar, and they were kept in single cages.
  • Data statistics Count the drinking amount of quinine bitter water, tap water and total drinking water, as well as the percentage of quinine water in total drinking water, compare the drinking amount of animal bitter water in each group, and compare the differences between groups by variance analysis. whether the difference is significant.
  • LC-MS/MS method wherein: the liquid phase method adopts gradient elution method.
  • Mass spectrometry conditions ESI source, positive ion, multiple reaction monitoring (MRM) mode was used for mass spectrometry analysis.
  • liver microparticle incubation sample Take the liver microparticle incubation sample, add 400 ⁇ L of acetonitrile solution containing the internal standard (loratadine 1 ng/mL) to stop the reaction, vortex at 2500 rpm to mix well, centrifuge at 20,000 rcf for 10 min at 4°C, and take the supernatant into a clean lined tube.
  • the content of the prototype drug was detected by LC-MS/MS.
  • Administration group stock solution Weigh appropriate amount of compound 4 and compound 22 powder respectively, dissolve with dimethyl sulfoxide (DMSO), prepare 10mM administration group stock solution, and store in -20°C refrigerator;
  • DMSO dimethyl sulfoxide
  • Testosterone stock solution Weigh an appropriate amount of testosterone, dissolve it with dimethyl sulfoxide (DMSO), prepare a 10mM testosterone stock solution, and store it in a -20°C refrigerator;
  • DMSO dimethyl sulfoxide
  • Working solution of administration group Pipette appropriate amount of stock solution of compound 4 and compound 22 administration group, and prepare 500 ⁇ M working solution with methanol;
  • Testosterone working solution Pipette an appropriate amount of testosterone stock solution and prepare a 500 ⁇ M working solution with methanol.
  • Metabolic rate test Take human liver microsomes, thaw and thaw on ice, and shake gently. Liver microsomes were pipetted and added to 100 mM phosphate buffer, and then the working solution of compound 4 (or compound 22) administration group was added to make the microsome concentration 0.625 mg/mL and the compound 4 or compound 22 concentration 1.25 ⁇ M. Pipette 80 ⁇ L of the above solution, pre-incubate in a 37°C water bath for 5 min, add 20 ⁇ L of 5mM NADPH solution, and incubate in a 37°C water bath.
  • the total volume of each metabolic incubation system sample is 100 ⁇ L, and the incubation system includes liver microsomal protein at a final concentration of 0.5 mg/mL, 1 ⁇ M of compound 4 (or compound 22) and 1 mM of NADPH.
  • the reaction was terminated with 4 times the volume of acetonitrile (containing the internal standard loratadine 1 ng/mL), vortexed to mix, and centrifuged at 4°C at 20,000 rcf for 10 min. The supernatant was subjected to LC-MS/MS detection. Each incubation sample was run in triplicate.
  • the positive control was the metabolic sample of the microsomes of the corresponding species to the positive drug testosterone (1 ⁇ M).
  • the reaction was terminated with 4 times the volume of acetonitrile (including internal standard loratadine 1 ng/mL), and the incubation time points were 0, 30, and 60 min.
  • the Analyst1.6.3 software was used for data acquisition. After optimizing the integration parameters, the target chromatographic peak was automatically integrated, and individual chromatographic peaks were not allowed to be integrated or manually integrated, and the retention time and peak area were calculated. The data was collected by Analyst 1.6.3, and the retention time and peak area were obtained after integration processing. The peak area ratio of the analyte to the internal standard was calculated, and then secondary processing was used in Microsoft Office Excel to calculate the half-life t 1/2 and its clearance rate CLint. The percent test compound remaining (% Control) is calculated from the ratio of the concentrations in the non-zero time point sample to the zero time sample. Ln (% Control) was plotted against incubation time and a linear fit was performed.
  • LC-MS/MS method wherein: the liquid phase method adopts gradient elution method.
  • Mass spectrometry conditions ESI source, positive ion, multiple reaction monitoring (MRM) mode was used for mass spectrometry analysis.
  • Working solution of administration group Pipette appropriate amount of stock solution of compound 4 and compound 22 administration group and prepare 50 ⁇ M working solution with methanol;
  • Blank artificial gastric juice take 3.28 mL of dilute hydrochloric acid, add 160 mL of water, shake well to fully dissolve, adjust the pH value to 1.3, add water and dilute to 200 mL, which is blank artificial gastric juice.
  • Blank artificial intestinal juice Weigh 1.36 g of potassium dihydrogen phosphate, add 100 mL of water to dissolve it, adjust the pH to 6.8 with 0.1 mol/L sodium hydroxide solution, and add water to make up to 200 mL, which is blank artificial intestinal juice.
  • Artificial intestinal juice Weigh 1.36 g of potassium dihydrogen phosphate, add 100 mL of water to dissolve it, adjust the pH to 6.8 with 0.1 mol/L sodium hydroxide solution, and add 2 g of trypsin to dissolve in an appropriate amount of water, and mix the two solutions. , add water to volume to 200mL, which is blank artificial intestinal juice.
  • MassLynx V4.2 software was used for data acquisition. After optimizing the integration parameters, the target chromatographic peak was automatically integrated, and individual chromatographic peaks were not allowed to be integrated or manually integrated, and the retention time and peak area were calculated. The data was collected by MassLynx V4.2, the retention time and peak area were obtained after integration processing, the peak area ratio of analyte to internal standard was calculated, and then secondary processing was used in Microsoft Office Excel to calculate the average residual percentage.

Abstract

一种N-甲酰胺基吡唑啉类衍生物，具有通式（I）的化合物或其对映异构体、非对映异构体、差向异构体、外消旋体，或其在药学上可接受的盐。所述化合物是配体门控非选择性阳离子通道受体亚型P2X3的拮抗剂，可用治疗或预防由P2X3受体介导的各类疾病。

Description

作为P2X3受体拮抗剂的N-甲酰胺基吡唑啉类衍生物及应用 技术领域
本发明属于医药领域，涉及一种作为配体门控非选择性阳离子通道受体亚型P2X3抑制剂的N-甲酰胺基吡唑啉类衍生物，及其在制备治疗由P2X3介导的疾病药物中的应用。
背景技术
P2X3是一种配体门控非选择性阳离子通道受体亚型，归属于嘌呤能受体P2大类中的离子型P2X受体，最初于1995年在哺乳动物中克隆(Chen C等，Nature,1995，428；Lewis,C等，Nature,1995,432),至今，在哺乳动物中已克隆的P2X受体亚型有7种(P2X1～P2X7)。每个P2X受体分子由细胞内的N-端和C-端及两个跨膜结构区域组成。虽然P2X受体存在亚型及种属差异性，但是组成P2X受体的基本结构并没有明显差异,都是由3个同源或异源的亚基构成的三聚体,如P2X3为同源三聚体,P2X1/2、P2X2/3为异源三聚体(Jacobson等，Neuropharmacology，2016，31)。P2X3受体在体内分布广泛，主要表达在与伤害性信息有关的周围感觉神经元，在伤害信息的产生、传递中起着重要的介导作用。当机体受到伤害或神经损伤后释放大量ATP，与P2X3受体结合而导致受体的跨膜区域产生构象变化，从而激活P2X3受体，引起大量Ca 2+内流，细胞内钙浓度增加激活蛋白激酶A、蛋白激酶C等的磷酸化，促进谷氨酸的释放和NMDA受体等的进一步激活，最终导致中枢神经敏感化。
P2X3参与多种生理病理反应，包括炎症性疼痛、神经病理性疼痛、癌性疼痛等病理性疼痛,咳嗽高血压、膀胱排尿等。比如，P2X3受体表达上调可导致痛敏形成，参与疼痛的信号传递，P2X3受体敲除后，可缓解小鼠疼痛相关行为(Cockayne DA等，Nature,2000,1011)。P2X3受体的抑制作用可以改善自发性心脏压力感受器的反射功能，有助于抑制大鼠的交感神经，从而发挥抗高血压作用(Pijacks W等，Nature Medicine,2016,1151)。P2X3受体与小鼠的排尿频率具有相关性，P2X3受体的缺失，小鼠的排尿频率也会随之降低(Gao XF等，Nature Communication，2015，7650)。人体皮肤中的ATP/P2X3信号通道与慢性瘙痒相关，所以P2X3受体抑制剂亦可用于慢性瘙痒的治疗(Chauret,N.等，49th Annual ESDR Meeting，2019)。
另有研究表明，P2X3表达于豚鼠C—fibres迷走神经中，经其激动剂ATP激活后，可刺激呼吸道中的感觉神经并引发咳嗽(Abdulqawi R等，Eur Respir J,2013)。研究发现，咳嗽高敏综合征(Cough Hypersensitivity Syndrome)，即慢性咳嗽，特别是难治性慢性咳嗽，为呼吸系统最常见的疾病之一，病因包括多种呼吸系统疾病、环境、吸烟和药物过敏等。该类疾病严重影响患者的健康与生活质量，并受环境恶化和人口老龄化等因素的影响呈逐年递增趋势，但现有药物的治疗选择却甚少。因此，需对咳嗽机 制进行研究以揭开新的治疗靶点，并开发新的治疗方法以改善患者生活质量。近年临床数据显示，P2X3受体抑制剂AF-219在降低慢性咳嗽患者的咳嗽频率与严重程度上效果显著(Abdulqawi R等，Eur Respir J,2013,42；Abdulqawi R等，Lancet,2015,1198；Garceau D等，Pulmonary Pharmacology&Therapeutics,2019,56；Muccino D等，Pulmonary Pharmacology&Therapeutics,2019,75)。
Figure PCTCN2021103402-appb-000001
但与此同时，临床试验亦表明，AF-219通过影响舌头的味觉，可导致治疗对象的味觉紊乱(Abdulqawi等,Lancet 2015)。这种副作用归因于P2X2/3通道(P2X2和P2X3亚型的异源三聚体)的阻塞(Cockayne,D.A.等，J.Physiol.2005,621；Pulmonary Pharmacology&Therapeutics,2019)。因此，开发具有P2X3亚型受体选择性的拮抗剂可解决此类慢性病治疗期间的病人依从性不足的问题。本发明所述的具有新型化合物结构的P2X3抑制剂在选择性、药代动力学和药效学等方面具有出人意料且有利的性质，可以很大程度上拓展P2X3靶向药物的研发，这构成了本发明的基础。
发明内容
本发明提供了一种新型的作为配体门控非选择性阳离子通道受体亚型P2X3抑制剂的N-甲酰胺基吡唑啉类衍生物。
本发明同时公开了上述N-甲酰胺基吡唑啉类衍生物的应用。
一种N-甲酰胺基吡唑啉类衍生物，具有通式(I)所示的化合物结构或其对映异构体、非对映异构体、差向异构体、外消旋体，或其在药学上可接受的盐:
Figure PCTCN2021103402-appb-000002
其中：
Ar选自取代或无取代苯基、包含1-3个选自O、N和S原子的取代或无取代五元杂芳环基或包含1-2个N原子的取代或无取代六元杂芳环基；当Ar选自邻位双取代苯基时，两个邻位取代基相互独立或者成环；
R 1选自取代或未取代苯基或取代或未取代吡啶基；R 2选自甲基、乙基、异丙基、环丙基；
R 3，R 4分别各自独立的选自H、C1～C5烷基、C1～C5烷氧羰基、取代或未取代的C1～C5酰基；或R 3、R 4连同它们两者所连接的N一起形成取代或未取代的C5～C6杂 环烷基或C5～C6杂环烷基酮或C5～C6杂芳基；所述杂环烷基或杂环烷基酮环上的碳原子可进一步被一个或多个O、N取代。
所述Ar选自下列取代苯基、取代的五元或六元杂芳环基：
Figure PCTCN2021103402-appb-000003
所述R 5，R 6相同或不同或缺失，分别独立选自H、卤素、硝基、氰基、甲基、三氟甲基、三氟甲氧基、甲氧基、二氧亚甲基、C1-C3直链或支链烷氧基，且在任意环碳原子上取代；选择所述二氧亚甲基时，所述R 5，R 6为其中的氧原子，与相连的亚甲基成环；
R 7选自甲基、乙基、异丙基、环丙基。
所述卤素包括F、Cl、Br。
本发明中，所述C1～C5烷基包括甲基、乙基、丙基、异丙基、丁基、叔丁基等；所述C1～C5烷氧羰基包括甲基、乙基、丙基、异丙基、丁基、叔丁基等连接的氧羰基。所述C1～C5酰基包括甲基、乙基、丙基、异丙基、丁基、叔丁基等连接的酰基。所述C1-C3直链或支链烷氧基包括甲氧基、乙氧基、丙氧基或者异丙基氧基等。
作为优选，所述R 1中，苯基或吡啶基环上的取代基选自卤素、C1-C3直链或支链烷基(包括甲基、乙基、丙基、异丙基等)、C1-C3直链或支链烷氧基、三氟甲基、三氟甲氧基。
作为优选，R 3，R 4分别各自独立的选自H、甲基、乙基、甲氧羰基、乙氧羰基、丙氧羰基、异丙氧羰基、叔丁氧羰基、甲酰基、乙酰基、N,N-二烷基乙酰基；或R 3、R 4连同它们这两者所连接的N一起形成取代或未取代吗啉酮基、取代或未取代吗啉基、取代或未取代哌啶基、取代或未取代哌嗪基、取代或未取代哌嗪酮基、取代或未取代吡咯烷酮基、取代或未取代恶唑烷酮基、取代或未取代咪唑烷酮基、取代或未取代咪唑基、取代或未取代吡唑基、1,2,3-三氮唑基、1,2,4-三氮唑基。
作为进一步优选，所述吗啉酮基、吗啉基、哌啶基、哌嗪基、哌嗪酮基、吡咯烷酮基、恶唑烷酮基、咪唑烷酮基、咪唑基、吡唑基上的取代基选自C1-C3直链或支链烷基、甲酰基、乙酰基、C1-C5直链或支链烷氧基(包括甲基、乙基、丙基、异丙基、丁基、叔丁基连接的氧基取代基)。
除非另有说明，本文所述被任选取代的成分可以在任何化学上可能的位置被取代。
作为优选，其具有通式(Ia)、(Ib)、(Ic)或(Id)所示的结构或其对映异构体、非对映异构体、外消旋体、或其在药学上可接受的盐：
Figure PCTCN2021103402-appb-000004
作为优选，所述Ar选自苯基对三氟甲基苯基、二甲氧基苯基、二氧五环苯基、对氰基苯基、氯代苯基、甲氧基吡啶基、三氟甲基吡啶基、氯取代吡啶基、二氟吡啶基、三氟甲氧基吡啶基、三氟甲基吡嗪基、甲基吡嗪基、氯代吡嗪基、甲氧基哒嗪基、三氟甲基哒嗪基、氯代哒嗪基、三氟甲基嘧啶基、甲基嘧啶基、氯代嘧啶基、甲基-1,2,4-恶二唑基、甲基-1,3,4-恶二唑基；R 1选自对甲基苯基、对甲氧基苯基、对氟苯基、对氯苯基、甲基吡啶基；R 2选自甲基、乙基；R 3,R 4分别各自独立的选自H、甲基、乙基、甲氧羰基、乙氧羰基、乙酰基、N,N-二烷基乙酰基；或R 3、R 4连同它们这两者所连接的N一起形成吗啉酮基、哌嗪酮基、N-甲基哌嗪酮基、甲基哌嗪酮基、吡咯烷酮基、恶唑烷酮基、咪唑烷酮基、乙酰基咪唑烷酮基、吗啉基、哌嗪基、N-乙酰基哌嗪基、吡唑基。
更具体地，本发明通式(I)化合物中，优选的化合物包括但不限于：
Figure PCTCN2021103402-appb-000005
Figure PCTCN2021103402-appb-000006
Figure PCTCN2021103402-appb-000007
Figure PCTCN2021103402-appb-000008
Figure PCTCN2021103402-appb-000009
Figure PCTCN2021103402-appb-000010
Figure PCTCN2021103402-appb-000011
Figure PCTCN2021103402-appb-000012
Figure PCTCN2021103402-appb-000013
Figure PCTCN2021103402-appb-000014
Figure PCTCN2021103402-appb-000015
Figure PCTCN2021103402-appb-000016
Figure PCTCN2021103402-appb-000017
Figure PCTCN2021103402-appb-000018
Figure PCTCN2021103402-appb-000019
本发明的另一个目的是提供一种药物组合物，所述药物组合物包含至少一种活性组分以及一种或多种药学上可接受的载体或赋形剂，所述的活性组分可以是本发明的N-甲酰胺基吡唑啉类衍生物及其在药学上可接受的盐、所述化合物的异构体、对映异构体、非对映异构体、外消旋体中的任意一种或任意多种；所述的盐选用药用上接受的无机酸盐、有机酸盐；所述的无机酸盐包括与氢卤酸，硝酸，碳酸，硫酸，磷酸形成的盐；所述的有机酸盐包括与苹果酸、L-苹果酸、D-苹果酸、枸橼酸、富马酸、草酸、乳酸、樟脑磺酸、L-樟脑磺酸、D-樟脑磺酸、对甲苯磺酸、甲磺酸、苯甲酸形成的盐；所述氢卤酸选用氢氟酸、氢溴酸、氢碘酸、盐酸。
所述载体或赋形剂包括药学领域的常规填充剂，稀释剂，湿润剂，润滑剂，粘合剂，崩解剂，吸收促进剂，表面活性剂，吸附载体，抗氧化剂、乳化剂、金属螯合剂、pH调节剂等，必要时还可以加入香味剂，甜味剂等。本发明药物可以制成片剂、胶囊、吸入剂、乳剂、混悬剂、凝胶剂、粉剂、颗粒剂、口服液及注射剂等多种形式，上述各剂型的药物均可以按照药学领域的常规方法制备。
本发明还提供通式(I)所述的任一项化合物、及其药学上可接受的盐单独和/或与其他药物联合使用在制备P2X3抑制剂中的应用，特别是在制备治疗由P2X3介导的疾病中的应用。所述的由P2X3介导的疾病包括呼吸系统疾病，如慢性阻塞性肺疾病(COPD)、哮喘、急性/亚急性及慢性咳嗽、支气管痉挛；由多种原因造成的疼痛，如手术疼痛、炎性疼痛、癌性疼痛、膀胱疼痛、子宫内膜异位症疼痛、糖尿病性神经痛、创伤性疼痛、牙痛、偏头痛、及与肠易激综合征有关的疼痛；皮肤疾病如慢性瘙痒。
本发明具有通式(I)的化合物的制备可以通过以下步骤实现：
相应的取代N-甲酰氯基吡唑啉在碱性条件下与取代芳甲胺或取代杂芳甲胺缩合制得标题分子。其中，当选用手性分子R-取代芳甲胺或R-取代杂芳甲胺为合成子时，所得目标分子为差向异构体混合物，其可经柱层析梯度分离获得S,R-异构体和R,R-异构体，所用洗脱剂为石油醚：乙酸乙酯＝3:1～1:3(v/v)或甲醇：乙酸乙酯＝1:3～1:6(v/v)；当选用手性分子S-取代芳甲胺或S-取代杂芳甲胺为合成子时，所得目标分子可经柱层析梯度分离获得S,S-异构体和R,S-异构体，所用洗脱剂为石油醚：乙酸乙酯＝4:1～1:3(v/v)或甲醇：乙酸乙酯＝1:3～1:6(v/v)；标题化合物的立体构型分别经COSY相关谱、 NOSEY谱确证。
Figure PCTCN2021103402-appb-000020
其中:
中间体取代N-甲酰氯基吡唑啉的制备可以相应的氨基酮为原料，在碱性条件下与甲醛反应生成烯酮，再与肼反应得环合物吡唑啉，吡唑啉进而与三光气反应制得取代N-甲酰氯基吡唑啉；
Figure PCTCN2021103402-appb-000021
中间体R-取代芳甲胺、R-取代杂芳甲胺，或S-取代芳甲胺、S-取代杂芳甲胺的制备可以相应的芳醛为原料，与S-叔丁基亚磺酰胺或R-叔丁基亚磺酰胺反应制得亚胺，进而与相应的格氏试剂或烷基锂在低温条件下经加成，水解制得R-取代芳甲胺或S-取代杂芳甲胺。
Figure PCTCN2021103402-appb-000022
本发明通过实验证实，本发明中的化合物可选择性抑制P2X3通道活性，可应用于治疗由P2X3介导的疾病的药物中。本发明提供的抑制剂还包括该化合物的药物组合物。
附图说明
图1为标题化合物1的化学结构(A)、3D模拟图(B)、 1HNMR(C)。
图2为标题化合物1的的NOESY谱。
图3为化合物1a的化学结构(A)、3D模拟图(B)、 1HNMR(C)。
图4为化合物1a的NOESY谱(D)。
图5为标题化合物对ATP/枸橼酸诱导的豚鼠咳嗽的抑制作用(腹腔注射)。
图6为标题化合物对ATP/枸橼酸诱导的豚鼠咳嗽的抑制作用(口服给药)。
图7为标题各组实验动物给药后的饮水及饮用奎宁水的百分比情况。
图8为标题化合物4在人工胃肠液中的降解剩余百分率。
图9为标题化合物22在人工胃肠液中的降解剩余百分率。
具体实施方式
本发明结合实施例作进一步的说明，以下实施例仅是具体地说明本发明，而不是以任何方式限制本发明。此外，关于本实施例中使用的原料、中间体、试剂等，若没有特别记载，则是在本领域按照通常实施的方法制备得到、或者是可通过商业购买得到。
一、主要中间体的制备方法
1.中间体N-甲酰氯基吡唑啉衍生物(A)的制备
1).4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑-1-甲酰氯(A-1)的合成
Figure PCTCN2021103402-appb-000023
步骤1.N-甲基-N-(2-氧代-2-(4-甲基苯基)乙基)乙酰胺(A-1a)的合成
将30％甲胺醇溶液(7.29g，70.41mmol)加入至乙腈(20mL)中，N 2保护下，降温至-15℃～-10℃，慢慢滴加4-甲基-α-溴代苯乙酮(5g，23.47mmol)的乙腈(40mL)溶液，滴加完毕，维持-15℃～-10℃搅拌20min。将反应液升温至-5℃～0℃，滴加冰水(60mL)，搅拌5min，加入乙酸乙酯(30mL)，搅拌5min，静置分层，水相用乙酸乙酯萃取(20mL×2)，合并有机相，用水洗涤两次并用无水Na 2SO 4干燥，过滤，向滤液中加入三乙胺(3.5g，35.2mmol)，N 2保护下降至-10℃～-5℃，滴加乙酰氯(3.68g，46.94mmol)，继续搅拌10～20min，加水(80mL)，室温搅拌10min，水相用乙酸乙酯萃取(30mL×2)，合并有机相，无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱层析分离(PE:EA＝3:1,v/v)，得化合物A-1a，淡黄色固体，收率＝41.5％；ESI-MS:m/z＝206[M+1] +。
步骤2.N-甲基-N-(3-氧代-3-(4-甲基苯基)丙基-1-烯-2-基)乙酰胺(A-1b)的合成
将化合物A-1a(2.0g，9.76mmol)，37％甲醛溶液(2.38g，29.28mmol)和哌啶(0.41g，4.88mmol)加入至THF(10mL)中，加热至70～75℃搅拌5h后，再加入37％甲醛溶液(2.38g，29.28mmol)和哌啶(0.41g，4.88mmol)，搅拌过夜，TLC监 控至无原料，降至室温，过滤，滤液减压浓缩至干，残留物经硅胶柱层析分离(PE:EA＝2:1,v/v),得化合物A-1b，收率67.0％； 1H NMR(500MHz,DMSO-d 6):δ7.77(d,J＝7.3Hz,2H),7.35(d,J＝7.4Hz,2H),5.70(d,J＝2.3Hz,1H),5.28(d,J＝2.2Hz,1H),3.22(s,3H),2.42(s,3H),1.95(s,3H)；ESI-MS:m/z＝218[M+1] +。
步骤3.4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑-1-甲酰氯(A-1)的合成
将化合物A-1b(3.0g，13.8mmol)、水合肼(1.38g，27.6mmol)、EtOH(15mL)加入至反应瓶中，N 2保护，加热至80℃搅拌3h，反应液降至室温，减压蒸去乙醇，加入水(10mL)，搅拌，用乙酸乙酯(15mL×2)萃取，有机相用无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，得化合物A-1c粗品，未经进一步纯化，直接用于下一步反应。
将三光气(1.30g，4.32mmol)、二氯甲烷(20mL)加入至干燥反应瓶中，N 2保护降温至-10℃，另将化合物A-1c(2.0g，8.64mmol)、吡啶(2.04g，25.80mmol)与二氯甲烷(20mL)混合，并于-10℃将混合液滴加至反应瓶中，加毕，室温搅拌10min，加入稀盐酸淬灭反应，分出有机相，水相用二氯甲烷萃取(10mL×2)，合并有机相，用饱和食盐水洗涤，无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱层析分离(PE:EA＝3:1～1:3,v/v),得化合物A-1，淡黄色固体，二步收率46％； 1H NMR(500MHz,CDCl 3)：δ7.86(d,J＝8.0Hz,2H),7.26(d,J＝8.0Hz,2H),5.20-5.11(m,1H),4.02-3.87(m,2H),3.27(s,3H),2.42(s,3H),2.32(s,3H)；ESI-MS:m/z＝294[M+1] +。
表1.1中所列中间体A-6是使用4-氟-α-溴代苯乙酮代替4-甲基-α-溴代苯乙酮为原料，按制备化合物A-1同样的方法合成得到。
2).4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑-1-甲酰氯(A-2)的合成
Figure PCTCN2021103402-appb-000024
步骤1.4-(3-氧代-3-(4-甲基苯基)丙基-1-烯-2-基)吗啉-3-酮(A-2a)的合成
将4-(2-氧代-2-(4-甲基苯基)乙基)吗啉-3-酮(10.0g，42.9mmol)、多聚甲醛(3.8g，128.7mmol)和哌啶(1.8g，21.5mmol)加入至THF(100mL)中，在70～75℃下搅拌5h后，再加入多聚甲醛(3.8g，128.7mmol)和哌啶(1.8g，21.5mmol)，搅拌过夜，TLC监控至无原料，降至室温，过滤，滤液减压浓缩至干，残留物经硅胶柱层析分离(PE:EA＝3:1,v/v),得化合物A-2a，收率88.0％； 1HNMR(500MHz,DMSO-d 6)：δ7.67(d,J＝7.5Hz,2H),7.55(d,J＝7.5Hz,2H),5.70(d,J＝2.1Hz,1H),5.24(d,J＝2.1Hz,1H),4.21(s,2H),3.56(m,2H),3.37(m,2H),2.41(s,3H)；ESI-MS:m/z＝246[M+1] +。
步骤2.4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑-1-甲酰氯(A-2)的合 成
将化合物A-2a(5.0g，20.4mmol)、水合肼(2.05g，40.75mmol)加入至EtOH(25mL)中，N 2保护下在80～82℃搅拌3h，TLC监控至无原料，降至室温，减压蒸去体系中乙醇，加入水，搅拌，用乙酸乙酯萃取，有机相用无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，得化合物A-2b粗品，未经进一步纯化，直接用于下一步反应。
将三光气(1.71g，5.76mmol)、二氯甲烷(30mL)加入至干燥反应瓶中，N 2保护下降至-10℃～-5℃，将化合物A-2b(3.0g，11.55mmol)、吡啶(2.7g，34.2mmol)加入至二氯甲烷(30mL)中，并于-10℃～-5℃滴加至反应瓶中，滴加完毕，于室温下搅拌10min，加入稀盐酸淬灭反应，分出有机层，水相用二氯甲烷萃取(10mL×2)，合并有机相，有机相用饱和食盐水洗涤,用无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，经硅胶柱层析分离(PE:EA＝4:1～1:1,v/v),得中间体化合物A-2,淡黄色固体,二步收率56％； 1H NMR(500MHz,CDCl 3):δ7.72(d,J＝8.0Hz,2H),7.25(d,J＝8.0Hz,2H),6.61-6.57(m,1H),4.29-4.17(m,2H),4.03-3.94(m,2H),3.78-3.67(m,2H),3.21-2.93(m,2H),2.39(s,3H)；ESI-MS:m/z＝322[M+1] +。
表1.1中所列化合物A-7～A-14是使用相应的取代吗啉酮或取代吗啉或取代哌嗪或取代哌嗪酮(A-7：4-(2-氧代-2-(4-氟苯基)乙基)吗啉-3-酮；A-8：4-(2-氧代-2-(4-氯苯基)乙基)吗啉-3-酮；A-9：4-(2-氧代-2-(4-甲氧基苯基)乙基)吗啉-3-酮；A-10：4-(2-氧代-2-(4-氟苯基)乙基)吗啉；A-11：4-乙酰基-1-(2-氧代-2-(4-甲基苯基)乙基)哌嗪；A-12：4-甲基-1-(2-氧代-2-(4-甲基苯基)乙基)哌嗪；A-13：4-甲基-1-(2-氧代-2-(4-甲基苯基)乙基)哌嗪-2-酮；A-14：4-(2-氧代-2-(4-甲基苯基)乙基)吗啉)替代4-(2-氧代-2-(4-甲基苯基)乙基)吗啉-3-酮为原料，按制备化合物A-2同样的方法合成得到。
3).4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑-1-甲酰氯(A-3)的合成
Figure PCTCN2021103402-appb-000025
步骤1.1-(3-(4-甲基苯基)-4,5-二氢-1H-吡唑-4-基)吡咯烷-2-酮(A-3a)的合成
将1-(2-氧代-2-(4-甲基苯基)乙基)吡咯烷-2-酮(5.0g,23.01mmol)和37％甲醛溶液(2.70mL，33.2mmol)溶于二氧六环(50mL)中，滴加哌啶(2.83g,33.2mmol)，加毕，在105℃下搅拌6h，降至室温，减压蒸去二氧六环。向体系中加入45mL乙酸乙酯，50mL饱和食盐水，搅拌5min，静置分层，水相用乙酸乙酯(20mL)萃取，合并有机相，无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，得黄色油状物。将上述黄色油状物溶于乙醇(120mL)中，加入水合肼(4.16g,66.5mmol)，混合物于70℃～80℃加热搅拌反应3hr，冷至室温，减压蒸除去溶剂，残留物中加THF(30mL)和水(10mL),室温搅拌，过滤，滤饼用少量THF洗二次，固体物真空干燥，得化合物A-3a,收率65％； 1H NMR(500MHz,DMSO-d 6):δ9.15(brs,1H),7.70(d,J＝7.4Hz,2H),7.30(d,J＝7.4Hz,2H),4.51(m,1H),3.30-3.04(m,4H),2.42(s,3H),2.24(m,2H),1.95(m,2H)；ESI-MS:m/z＝ 244[M+1] +。
步骤2.4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑-1-甲酰氯(A-3)的合成
将三光气(1.55g，5.14mmol)、无水二氯甲烷(25mL)加入至干燥反应瓶中，N 2保护，降温至-8℃～-10℃，另将化合物A-3a(2.5g，10.28mmol)、吡啶(2.65g，22.54mmol)溶于二氯甲烷(25mL)中，并于-10℃滴加至反应瓶中，滴加毕，室温搅拌10min，加入稀盐酸淬灭反应，分出有机相，水相用二氯甲烷萃取(10mL×2)，合并有机相，用饱和食盐水洗涤，无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱层析分离(PE:EA＝4:1～1:2,v/v),得化合物A-3，淡黄色固体，收率66％； 1HNMR(500MHz,CDCl 3)：δ7.78(d,J＝8.0Hz,2H),7.21(d,J＝8.0Hz,2H),5.10-4.86(m,1H),3.65-3.54(m,2H),3.29-3.20(m,2H),2.42(s,3H),2.35-2.27(m,2H),1.99-1.91(m,2H)；ESI-MS:m/z＝306[M+1] +。
表1.1中所列化合物A-15,A-16,A-18～A-21是使用相应的原料(A-15：1-(2-氧代-2-(4-氟苯基)乙基)吡咯烷-2-酮；A-16：3-(2-氧代-2-(4-甲基苯基)乙基)恶唑烷-2-酮；A-18：3-(2-氧代-2-(4-氯苯基)乙基)恶唑烷-2-酮；A-19：1-(2-氧代-2-(4-甲基苯基)乙基)-3-甲基咪唑烷-2-酮；A-20：1-(2-氧代-2-(4-甲基苯基)乙基)-3-乙酰基咪唑烷-2-酮；A-21：1-(2-氧代-2-(5-甲基吡啶-2-基)乙基)吡咯烷-2-酮)替代1-(2-氧代-2-(4-甲基苯基)乙基吡咯烷-2-酮)，按制备化合物A-3同样的方法合成得到。
4).4-(N-甲基-N-叔丁氧羰基胺基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑-1-甲酰氯(A-4)的合成
Figure PCTCN2021103402-appb-000026
步骤1.N-甲基-N-(2-氧代-2-(4-甲基苯基)乙基)氨基甲酸叔丁酯(A-4a)的合成
将30％甲胺醇溶液(11.5mL，70.41mmol)、乙腈(20mL)加入至反应瓶中，N 2保护下降温至-15℃～-10℃，慢慢滴加4-甲基-α-溴代苯乙酮(5g，23.47mmol)的乙腈(40mL)溶液,滴加完毕，保温搅拌20min。将反应体系升温至-5℃～0℃，滴加冰水(60mL)，搅拌5min，加入乙酸乙酯(30mL)，搅拌5min，静置分层，水相用乙酸乙酯萃取(20mL×2)，合并有机相，有机相用水洗涤两次，无水Na 2SO 4干燥，过滤，向滤液中加入三乙胺(4.8mL，35.2mmol)，N 2保护下降至-10℃～-5℃，滴加(Boc) 2O(7.68g，35.21mmol)，加毕，室温搅拌30min，加入水(80mL)，室温下继续搅拌10min，静置分层，水相用乙酸乙酯萃取(20mL×2)，合并有机相，用无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱层析分离(PE:EA＝3:1～1:1,v/v)，得化合物A-4a,淡黄色固体,收率63.9％；ESI-MS:m/z＝264[M+1] +。
步骤2.N-甲基-N-(3-氧代-3-(4-甲基苯基)丙基-1-烯-2-基)氨基甲酸叔丁酯(A-4b)的合成
将化合物A-4a(3.5g，13.29mmol)，37％甲醛溶液(3.0mL，39.87mmol)和哌啶(0.6mL，6.65mmol)加入至二氧六环(30mL)中，在105℃下搅拌6h，TLC监控至无原料，降至室温，减压蒸去二氧六环。向体系中加入15mL乙酸乙酯，30mL饱和食盐水，搅拌5min，静置分层，水相用乙酸乙酯(15mL)萃取一次，合并有机相，用无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，即得化合物A-4b粗品，黄色油状，收率65％，无需进一步精制，可直接用于下一步反应。
步骤3.4-(N-甲基-N-叔丁氧羰基胺基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑(A-4c)的合成
将化合物A-4b(5.12g,18.6mmol)、水合肼(1.0mL，19.98mmol)加入至乙醇(40mL)中，N 2保护，加热至85℃搅拌3h，降至室温，减压蒸去乙醇，残留物中加入水(10mL)，搅拌，用乙酸乙酯萃取(2×20mL)，合并有机相，用无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，得化合物A-4c粗品，黄色油状物，收率68％，无需进一步精制，可直接用于下一步反应。
步骤4.4-(N-甲基-N-叔丁氧羰基胺基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑-1-甲酰氯(A-4)的合成
将三光气(1.97g，6.65mmol)、干燥二氯甲烷(30mL)加入至三口瓶中，N 2保护下降至-10℃，将含化合物A-4c(6.07g，21.0mmol)、吡啶(3.4mL，39.87mmol)的二氯甲烷(30mL)慢慢滴加至上述三口瓶中，加毕，于室温搅拌10min，加水淬灭反应，分离有机层，水相用二氯甲烷萃取(2×20mL)，合并有机相，用饱和食盐水洗涤，无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱层析分离(PE:EA＝3:1～1:1,v/v),得中间体A-4，淡黄色固体，收率76％； 1HNMR(500MHz,CDCl 3):δ7.82(d,J＝7.8Hz,2H),7.31(d,J＝8.0Hz,2H),4.68(m,1H),3.60＝3.47(m,2H),3.27(s,3H),2.41(s,3H),1.41(s,9H)；ESI-MS:m/z＝352[M+1] +。
表1.1中所列中间体A-17是使用4-甲氧基-α-溴代苯乙酮代替4-甲基-α-溴代苯乙酮为原料，按制备化合物A-4同样的方法合成得到。
5).4-(吡唑-1-基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑-1-甲酰氯(A-5)的合成
Figure PCTCN2021103402-appb-000027
步骤1.2-(1H-吡唑-1-基)-1-(4-甲基苯基)丙-2-烯-1-酮(A-5a)的合成
将2-(吡唑-1-基)-1-(4-甲基苯基)乙基-1-酮(10.02g,50mmol)和甲醇20mL分别加入反应瓶中，搅拌下加入37％甲醛水溶液(20mL,250mmol),哌啶(2.5g,30mmol)及冰醋酸2.8mL，室温搅拌反应约5hr，用TLC检测反应终点，反应毕，用乙酸乙酯(100mL×2)提取，乙酸乙酯层用饱和食盐水洗涤，无水Na 2SO 4干燥，过滤，滤液减压回收乙酸乙酯，得化合物A-5a，淡黄色油状物，收率66％，不经进一步纯化，直接用于下步反应。
步骤2.4-(吡唑-1-基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑(A-5b)的合成
将化合物A-5a(6.5g,30mmol)溶于乙醇70mL中，搅拌下加入水合肼(3g,60mmol)，加热至45℃～50℃反应40min，减压蒸除乙醇，残留物用二氯甲烷(50mL×2)提取，饱和食盐水洗涤，无水Na 2SO 4干燥，过滤，残留物用乙酸乙酯/石油醚重结晶，得化合物A-5b，类白色固体，收率62％；ESI-MS:m/z＝227[M+1] +。
步骤3.4-(吡唑-1-基)-3-(4-甲基苯基)-4,5-二氢-1H-吡唑-1-甲酰氯(A-5)的合成
将三光气(2.75g，9.09mmol)、无水二氯甲烷(45mL)加入至干燥反应瓶中，N 2保护降温至-15℃～-10℃，另将化合物A-5b(4.0g，18.2mmol)、吡啶(4.7g，39.9mmol)溶于二氯甲烷(45mL)中，并于-15℃～-10℃滴加至反应瓶中，滴加毕，室温搅拌10min，加入稀盐酸淬灭反应，分出有机相，水相用二氯甲烷萃取(20mL×2)，合并有机相，用饱和食盐水洗涤，无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱分离(PE:EA＝3:1～1.5:1,v/v),得-化合物A-5,淡黄色固体，收率56％； 1HNMR(500MHz,CDCl 3):δ7.83(d,J＝7.5Hz,1H),7.68(d,J＝8.0Hz,2H),7.30(d，J＝7.5Hz,1H),7.21(d,J＝8.0Hz,2H),6.40-6.33(m,1H),6.25-6.22(m,1H),3.81-3.56(m,2H),2.43(s,3H)；ESI-MS:m/z＝289[M+1] +。
表1.1中间体A-6～A-21的结构与质谱数据
Figure PCTCN2021103402-appb-000028
Figure PCTCN2021103402-appb-000029
2.中间体手性-α-取代-芳基/杂芳基甲胺衍生物(B)的制备
1).(R)-1-(4-(三氟甲基)苯基)乙烷-1-胺(B-1)的合成
Figure PCTCN2021103402-appb-000030
步骤1.(E)-2-甲基-N-(4-(三氟甲基)苯亚甲基)丙基-2-亚磺酰胺(B-1a)的合成
将4-三氟甲基苯甲醛(5.0g，28.7mmol)、S-叔丁基亚磺酰胺(3.80g，31.7mmol)、无水硫酸铜(9.16g，57.4mmol)加入至二氯甲烷(70mL)中，50℃保温搅拌18～20h，TLC监控至无原料，降至室温，过滤,滤液减压浓缩，残留物经硅胶柱层析(PE:EA＝5:1～1:1,v/v)分离，得化合物B-1a，白色固体，收率76.6％。ESI-MS:m/z＝278[M+1] +。
步骤2.N-((R)-1-(4-(三氟甲基)苯基)乙基)-2-甲基-丙基-2-亚磺酰胺(B-1b)的合成
将化合物B-1a(5.0g，18.03mmol)、甲苯(80mL)加入至三口瓶中，N 2保护下降温至-70℃，将甲基锂(1.6M乙醚溶液10.2mL，16.3mmol)缓慢滴加至三口瓶中，并维持-78℃搅拌反应1h，加入饱和氯化铵(50mL)淬灭反应并室温搅拌10min，静置，分层，水相用二氯甲烷萃取三次，合并有机相，有机相用无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱层析(PE:EA＝3:1～1:3,v/v)分离，得化合物B-1b,白色固体,收率52.2％。ESI-MS:m/z＝294[M+1] +。
步骤3.(R)-1-(4-(三氟甲基)苯基)乙烷-1-胺(B-1)的合成
将化合物B-1b(2.0g，6.8mmol)、甲醇(30mL)加入至100mL反应瓶中，室温下滴加4.0M HCl的二氧六环液6mL(24mmol)，继续室温搅拌1h，压减回收溶剂，残留物中加入10ml碳酸钾水溶液，搅拌10min后，用二氯甲烷-甲醇混合液(10:1)萃取四次，减压回收溶剂，得化合物B-1,淡黄色油状物,收率71.3％。ee值：99％(HPLC,Chiralpak AD-3柱,检测条件：已烷/异丙醇＝93/7,流速＝0.8mL/min,uv-vis检测器)； 1HNMR(400MHz,CDCl 3):δ8.77(brs,2H),7.54(m,2H),7.21(m,2H),4.01(m,1H),1.27(d,J＝6.8Hz,3H)；ESI-MS:m/z＝190[M+1] +。
表1.2中所列中间体B-2～B-5是使用相应的取代芳醛(B-2：4-氯苯甲醛；B-3：3，4-二甲氧基苯甲醛；B-4：4-氰基苯甲醛；B-5：3,4-亚甲基二氧苯甲醛)为原料代替4-三氟甲基苯甲醛，按制备化合物B-1同样的方法合成得到。
2).(R)-1-(2-(三氟甲基)嘧啶-5-基)乙烷-1-胺(B-6)的合成
Figure PCTCN2021103402-appb-000031
步骤1.(E)-2-甲基-N-((2-(三氟甲基)嘧啶-5-基)亚甲基)丙基-2-亚磺酰胺(B-6a)的合成
将2-三氟甲基嘧啶-5-甲醛(5g，28.4mmol)、S-叔丁基亚磺酰胺(3.79g，31.3mmol)、无水硫酸铜(9.06g，56.8mmol)加入至二氯甲烷(70mL)中，50℃保温搅拌18～20h，TLC监控至无原料，降至室温，过滤，滤液减压浓缩，残留物经硅胶柱层析(PE:EA＝5:1～1:1,v/v)分离，得化合物B-6a,白色固体，收率70％。ESI-MS:m/z＝280[M+1] +。
步骤2.N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-2-甲基-丙基-2-亚磺酰胺(B-6b)的合成
将化合物B-6a(4.0g，14.3mmol)、干燥二氯甲烷(80mL)加入至三口瓶中，N 2保护下降温至-78℃，将甲基溴化镁(3.0M的四氢呋喃溶液10.5mL，31.5mmol)缓慢滴加至 三口瓶中，并维持-78℃～-70℃搅拌反应2.5h,然后升温至-40℃继续搅拌反应2h后，加入饱和氯化铵(50mL)淬灭反应，室温搅拌10min，静置，分层，水相用二氯甲烷萃取三次，合并有机相，有机相用无水Na 2SO 4干燥，过滤,滤液减压浓缩至干，残留物经硅胶柱层析(PE:EA＝3:1～1:3,v/v)分离，得化合物B-16b,白色固体，收率45％。ESI-MS:m/z＝296[M+1] +。
步骤3.(R)-1-(2-(三氟甲基)嘧啶-5-基)乙烷-1-胺(B-6)的合成
将化合物B-6b(1.2g，4.06mmol)、甲醇(20mL)加入至100mL反应瓶中，室温下将HCl(4.0M的二氧六环溶液；5mL，20mmol)滴加至反应瓶中，室温搅拌1h，TLC检测原料消失。减压回收溶剂，加入10mL碳酸钾水溶液，搅拌10min后，用二氯甲烷:MeOH(10:1)萃取四次，减压回收溶剂，得化合物B-6,淡黄色油状物，收率80％。ee值：99％(HPLC,Chiralpak AD-3柱，检测条件：已烷/异丙醇＝93/7,流速＝0.8mL/min,uv-vis检测器)； 1HNMR(500MHz,CDCl 3):δ8.85(s,2H),8.77(brs,1H),4.91-4.85(m,1H),1.67(d,J＝7.0Hz,3H)；ESI-MS:m/z＝192[M+1] +。
表1.2中所列中间体B-7～B-22是使用相应的取代杂芳醛(B-7:2-甲基嘧啶-5-甲醛；B-8:2-氯嘧啶-5-甲醛；B-9、B-21:6-三氟甲基吡啶-3-甲醛；B-10:6-甲氧基吡啶-3-甲醛；B-11:6-三氟甲氧基吡啶-3-甲醛；B-12:5,6-二氟吡啶-3-甲醛；B-13:6-氯吡啶-3-甲醛；B-14:5-三氟甲基吡啶-2-甲醛；B-15:5-甲氧基吡啶-2-甲醛；B-16:5-三氟甲基哌嗪-2-甲醛；B-17:5-甲基哌嗪-2-甲醛；B-18:6-三氟甲基哒嗪-3-甲醛；B-19:6-氯哒嗪-3-甲醛；B-20:2-三氟甲基嘧啶-5-甲醛；B-22:6-甲氧基哒嗪-3-甲醛)为原料替代2-三氟甲基嘧啶-5-甲醛，按制备化合物B-6同样的方法合成得到。其中，中间体B-20,B-21的制备采用R-叔丁基亚磺酰胺为手性源试剂。
表1.2中间体B-2～B-22的结构与质谱数据
Figure PCTCN2021103402-appb-000032
Figure PCTCN2021103402-appb-000033
二、标题化合物的制备实例
制备实施例1.(S)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(化合物1)和差向异构体1a的合成
Figure PCTCN2021103402-appb-000034
合成步骤：将化合物A-2(1.05g，3.26mmol)、化合物B-9(0.62g，3.26mmol)加入至二氯甲烷(10mL)中，再加入N，N-二异丙基乙胺(DIPEA,1.26g，9.75mmol)，室温搅拌过夜，加入0.5N盐酸(10mL)搅拌10min。静置分层，分出有机相，水相用二氯甲烷(10mL×3)萃取，合并有机相，用无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱层析梯度分离(PE:EA＝3:1～1:3，v/v)得标题化合物1，收率32％和它的R,R-差向异构体1a，收率29％。
标题化合物1与其差向异构体1a的结构和 1H-NMR核磁谱见图1和图3，标题化合物1与其差向异构体1a的立体结构解析(图2，图4)：
1)鉴于标题化合物1的合成中，中间体B-9为R-型手性合成子，而另一合成子 A-2为消旋体，且标题化合物1与1a的质谱数据均为ESI-MS:m/z＝476[M+H] +，同时，二者氢质子数相同，故互为差向异构体；
2)根据标题化合物1 1H-NMR(400MHz,CDCl 3)的氢质子化学位移及 1H- 1HCOSY谱对标题化合物1氢质子各信号进行归属：
δ8.74(s,1H,H-16),7.86(d,J＝7.9Hz,1H,H-17),7.64(d,J＝8.0Hz,1H,H-18),7.62(d,J＝8.0Hz,2H,H-5H-6),7.21(d,J＝7.9Hz,2H,H-3H-4),6.60(dd,J＝11.0,3.7Hz,1H,H-9),6.34(d,J＝7.0Hz,1H,H-19),5.14(brs,1H,H-15),4.23-4.13(m,2H,H-13),4.00(t,J＝12.0Hz,1H,H-10a)，3.96(dd,J＝12.9,3.7Hz,1H,H-10b)，3.78(m,1H,H-12a),3.67(m,1H,H-12b),3.22(m,1H,H-11a),2.95(m,1H,H-11b),2.37(s,3H,H-1),1.64(d,J＝7.0Hz,3H,H-14)；
化合物1a氢谱各信号从低场到高场的归属顺序与标题化合物1一致。
3)根据化合物1及1a的化学结构结合三维模拟图可知，甲基苯环/吡唑啉环/碳酰胺三部分均分别基本处于同平面结构。当C-9位构型为S时，吗啉酮环与C-14位甲基同侧，NOSEY谱中显示C-14位甲基氢质子与11，13位氢质子有相关，由此确定标题化合物1结构中C-9位构型为S-型。反之，当C-9位构型为R-型时，吗啉酮环与C-14位甲基异侧，NOSEY谱中显示C-14位甲基氢质子与C-11，C-12，C-13位氢质子均无相关，由此确定化合物1a结构中C-9位构型为R-型。
制备实施例2.(S)-4-(4-甲基-2-氧代哌嗪-1-基)-3-(4-甲基苯基)-N-((R)-1-(4-氯苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(化合物2)和差向异构体2a的合成
Figure PCTCN2021103402-appb-000035
合成步骤：将化合物B-2(500mg,2.63mmol)加入至二氯甲烷(5mL)中，冷却至0℃，N 2保护下加入三光气(370mg,1.25mmol)，Et 3N(660mg,6.55mmol)，升至室温搅拌1h。将反应液倒入水中，用二氯甲烷萃取(10mL×2)，有机相用无水Na 2SO 4干燥，过滤，减压回收溶剂，得异氰酸酯粗品，未经进一步纯化，直接用于下步反应。
将化合物1-(3-(4-甲基苯基)-4,5-二氢-1H-吡唑-4-基)-4-甲基哌嗪-2-酮(500mg,1.80mmol)，异氰酸酯粗品(410mg,2.27mmol)，K 2CO 3加入到丙酮(10mL)中，N 2保护，室温搅拌2h，TLC显示原料消失。将反应液倒入二氯甲烷中，水洗，饱和食盐 水洗。有机相用无水Na 2SO 4干燥，过滤，滤液减压回收溶剂，残留物经硅胶柱层析梯度分离(MeOH:EA＝1:4,v/v)，得标题化合物2，白色固体，收率35％，和它的R,R-差向异构体2a，收率25％。
标题化合物2: 1H NMR(400MHz,CDCl 3):δ7.65(d,J＝7.4Hz,2H),7.36-7.28(m,4H),7.24(d,J＝7.8Hz,2H),6.68-6.60(m,1H),6.29(d,J＝8.0Hz,1H),5.08(dd,J＝14.3,7.4Hz,1H),4.12-3.95(m,2H),3.27-3.01(m,4H),2.83-2.47(m,2H),2.41(s,3H),2.26(s,3H),1.60(d,J＝6.9Hz,3H)；ESI-MS:m/z＝454[M+H] +。
差向异构体2a: 1H NMR(400MHz,CDCl 3):δ7.70(d,J＝7.8Hz,2H),7.46-7.42(m,4H),7.22(d,J＝7.8Hz,2H),6.66-6.58(m,1H),6.32(d,J＝8.0Hz,1H),5.06(dd,J＝14.0,7.0Hz,1H),4.11-3.94(m,2H),3.30-3.05(m,4H),2.86-2.42(m,2H),2.42(s,3H),2.28(s,3H),1.62(d,J＝7.2Hz,3H)；ESI-MS:m/z＝454[M+H] +。
制备实施3.(S)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(化合物3)和差向异构体3a的合成
Figure PCTCN2021103402-appb-000036
合成步骤：将化合物A-2(1.05g，3.26mmol)、化合物B-6(0.62g，3.25mmol)加入至二氯甲烷(10mL)中，再加入N,N-二异丙基乙胺(DIPEA,1.26g，9.75mmol)，室温搅拌过夜，加入0.5N盐酸10mL)搅拌10min。静置分层，分出有机相，水相用二氯甲烷(10mL×3)萃取，合并有机相，用无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱层析梯度分离(PE:EA＝2:1～1:3,v/v)得标题化合物3，收率37％，和它的R,R-差向异构体3a，收率30％。
标题化合物3: 1H NMR(500MHz,CDCl 3):δ8.91(s,2H),7.62(d,J＝8.2Hz,2H),7.23(d,J＝8.0Hz,2H),6.62(dd,J＝11.1,4.1Hz,1H),6.34(d,J＝7.0Hz,1H),5.20–5.12(m,1H),4.20(m,2H),4.05(dd,J＝13.0,11.1Hz,1H),3.96(dd,J＝13.0,4.1Hz,1H),3.80(m,1H),3.68(m,1H),3.23(m,1H),2.94(m,1H),2.39(s,3H),1.69(d,J＝7.1Hz,3H)；ESI-MS:m/z＝477[M+1] +。
差向异构体3a: 1H NMR(500MHz,CDCl 3):δ8.88(s,2H),7.70(d,J＝8.0Hz,2H),7.25(d,J＝8.0Hz,2H),6.60(dd,J＝11.0,4.0Hz,1H),6.40(brs,1H),5.22–5.13(m,1H),4.22(m,2H),4.02(dd,J＝12.2,11.0Hz,1H),3.94(dd,J＝12.0,4.0Hz,1H),3.78(m,1H),3.65(m,1H),3.21(m,1H),2.92(m,1H),2.36(s,3H),1.65(d,J＝7.5Hz,3H)；ESI-MS:m/z＝477[M+1] +。
制备实施4.(S)-4-(N-甲基-N-乙氧羰基胺基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(化合物4)的合成
Figure PCTCN2021103402-appb-000037
步骤1.(S)-4-(N-甲基-N-叔丁氧羰基胺基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(4-a)和差向异构体4c的合成
将化合物A-4(1.0g，2.84mmol)，化合物B-6(0.52g，2.70mmol)加入至二氯甲烷(10mL)中，再加入N,N-二异丙基乙胺(DIPEA，1.0g，7.75mmol)，室温搅拌过夜，加入盐酸(0.5N，10mL)并搅拌10min，静置分层，水相用二氯甲烷萃取(10mL×3)，合并有机相，有机相用无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱层析递度分离(PE:EA＝2:1～1:3,v/v)，得化合物4-a，收率40.7％；ESI-MS:m/z＝507[M+1] +。同时得化合物4-b,收率35％；ESI-MS:m/z＝507[M+1] +。
步骤2.(S)-4-(N-甲基-N-乙氧羰基胺基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(化合物4)和差向异构体4c的合成
将化合物4-a(0.73g,1.44mmol)加入至二氯甲烷(10mL)中，加入三氟乙酸(0.3mL,4.32mmol)，室温搅拌1h，减压蒸去过量三氟乙酸，向反应瓶中加入二氯甲烷(10mL)，三乙胺(0.8mL,5.76mmol)，将反应体系降温至-10～-5℃，并在此温度下向反应中滴加氯甲酸乙酯(0.2mL,1.73mmol)，加毕，升至室温搅拌1h，加水淬灭反应，分出有机层，水层用乙酸乙酯萃取(10mL×3)，合併有机相，用无水Na 2SO 4干燥,过滤，滤液减压浓缩至干，残留物经硅胶柱层析梯度分离(PE:EA＝2:1～1:3,v/v)，得标题化合物4，收率56％； 1H NMR(400MHz,CDCl 3):δ8.97(d,J＝1.7Hz,2H),7.71(d,J＝7.4Hz,2H),7.26(d,J＝7.9Hz,2H),6.39(d,J＝6.3Hz,1H),5.24–5.15(m,1H),4.22(q,J＝7.0Hz,2H),4.11–4.02(m,1H),3.94(td,J＝12.6,4.6Hz,1H),2.64(d,J＝16.1Hz,1H),2.43(s,3H),1.72(d,J＝7.5Hz,3H),1.65(s,3H),1.30(t,J＝6.7Hz,3H)；ESI-MS:m/z＝479[M+1] +。
以化合物4b为原料，按制备标题化合物4同样的方法合成得到差向异构体4c,收率47％； 1H NMR(400MHz,CDCl 3):δ8.92(s,2H),7.73(d,J＝7.6Hz,2H),7.28(d,J＝7.4Hz,2H),6.44(br,1H),5.20–5.08(m,1H),4.58-4.51(m,1H),4.25(q,J＝7.8Hz,2H),3.88(td,J＝12.8,5.0Hz,1H),2.64(d,J＝16.1Hz,1H),2.43(s,3H),1.74(d,J＝7.6Hz,3H),1.67(s,3H),1.35(t,J＝7.6Hz,3H)；ESI-MS:m/z＝479[M+1] +。
制备实施例5～13,18,20～25
制备实施例5～13,18,20～25中标题化合物5～13,18,20～25和它们的R,R-差向异构体是使用相应的中间体A与中间体B为原料，按制备标题化合物1同样的方法合成得到(表2)。
Figure PCTCN2021103402-appb-000038
制备实施例14,17,19,37～39
制备实施例14,17,19,37～39中标题化合物14,17,19,37～39和它们的R,R-差向异构体是使用相应的中间体A与中间体B为原料，按制备标题化合物2同样的方法合成得到(表2)。
Figure PCTCN2021103402-appb-000039
制备实施例26～36,40～44
制备实施例26～36,40～44中标题化合物26～36,40～44和它们的R,R-差向异构体是使用相应的中间体A与中间体B为原料，按制备标题化合物3同样的方法合成得 到(表2)。
Figure PCTCN2021103402-appb-000040
制备实施例15,16
制备实施例15,16中标题化合物15,16和它们的R,R-差向异构体是使用相应的中间体A(同时化合物15制备时：利用ClCOCH 2N(CH 3) 2代替实施例4中ClCOOEt；化合物16制备时：利用ClCOOMe代替实施例4中ClCOOEt)与中间体B为原料，按制备标题化合物4同样的方法合成得到(表2)。
Figure PCTCN2021103402-appb-000041
表2.标题化合物5～44的核磁及质谱数据
Figure PCTCN2021103402-appb-000042
Figure PCTCN2021103402-appb-000043
Figure PCTCN2021103402-appb-000044
Figure PCTCN2021103402-appb-000045
Figure PCTCN2021103402-appb-000046
Figure PCTCN2021103402-appb-000047
Figure PCTCN2021103402-appb-000048
Figure PCTCN2021103402-appb-000049
Figure PCTCN2021103402-appb-000050
Figure PCTCN2021103402-appb-000051
Figure PCTCN2021103402-appb-000052
Figure PCTCN2021103402-appb-000053
Figure PCTCN2021103402-appb-000054
Figure PCTCN2021103402-appb-000055
Figure PCTCN2021103402-appb-000056
Figure PCTCN2021103402-appb-000057
Figure PCTCN2021103402-appb-000058
Figure PCTCN2021103402-appb-000059
Figure PCTCN2021103402-appb-000060
Figure PCTCN2021103402-appb-000061
Figure PCTCN2021103402-appb-000062
制备实施例45.(S)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(3-甲基-1,2,4-恶二唑-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(化合物45)以及差向异构体45a的合成
1).(R)-1-(3-甲基-1,2,4-恶二唑-5-基)乙胺(B-22)的合成
Figure PCTCN2021103402-appb-000063
步骤1.(R)-(1-(3-甲基-1,2,4-恶二唑-5-基)乙基)氨基甲酸叔丁酯(中间体B-22a)的合成
将N-叔丁氧羰基-D-丙氨酸(10.0g,53.0mmol)溶于DMF(40mL)中，再分别加入三乙胺(22mL),N-羟基乙脒盐酸盐(4.6g,41.8mmol)和正丙基磷酸酐20mL(50％DMF溶液),搅拌下加热至100℃～110℃反应3.5h，冷至室温，加水淬灭反应，用乙酸乙酯提取(300mL×3)，合併的有机层，用饱和食盐水洗3次，无水Na 2SO 4干燥，减压浓缩，残留物用硅胶柱层析(PE:EA＝3:1,v/v)分离，得中间体化合物B-22a,收率75％；ESI-MS:m/z＝228[M+1] +。
步骤2.(R)-1-(3-甲基-1,2,4-恶二唑-5-基)乙胺(B-22)的合成
将化合物B-22a5.0g中溶于乙醇(35mL)中，冷至0℃，慢慢滴加盐酸甲醇溶液(50mL)，加毕，室温搅拌过夜，反应液减压浓缩，得化合物B-22盐酸盐，类白色固体，收率95％；ESI-MS:m/z＝128[M+1] +。
2).(S)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(3-甲基-1,2,4-恶二唑-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(化合物45)的合成
Figure PCTCN2021103402-appb-000064
将化合物A-3(1.0g，3.27mmol)、化合物B-22(0.53g，3.27mmol)加入至二氯甲烷(10mL)中，再加入N,N-二异丙基乙胺(DIPEA,1.27g，9.81mmol)，室温搅拌过夜，加入0.5N盐酸(10mL)搅拌10min。静置分层，分出有机相，水相用二氯甲烷(10mL×3)萃取，合并有机相，用无水Na 2SO 4干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱层析梯度分离(PE:EA＝2:1～1:3,v/v),得标题化合物45,收率35％。 1HNMR(500MHz,CDCl 3):δ7.65(d,J＝8.1Hz,2H),7.22(d,J＝8.0Hz,2H),6.45(d,J＝7.2Hz,1H),6.04(dd,J＝11.2,4.0Hz,1H),5.10(m,1H),3.99(dd,J＝12.0,11.0Hz,1H),3.87(dd,J＝12.0,3.8Hz,1H),3.24-3.21(m,1H),2.90-2.82(m,1H),2.39(s,3H),2.27(s,3H),2.32-2.23(m,1H),2.05-1.78(m,3H),1.66(d,J＝7.1Hz,3H)；ESI-MS:m/z＝397[M+1] +。
差向异构体45a,收率32％。 1HNMR(400MHz,CDCl 3):δ7.62(d,J＝7.8Hz,2H),7.21(d,J＝7.6Hz,2H),6.59(dd,J＝11.0,4.0Hz,1H),6.48(brs,1H),5.12(m,1H),3.99(dd,J＝11.0,4.0Hz,1H),3.86(dd,J＝11.0,3.8Hz,1H),3.36-3.29(m,2H),2.43(s,3H),2.34(s,3H),2.32-2.23(m,2H),2.05-1.78(m,2H),1.65(d,J＝7.2Hz,3H)；ESI-MS:m/z＝397[M+1] +。
制备实施例46.(S)-4-(3-氧代吗啉基)-3-(4-甲基苯基)-N-((R)-1-(3-甲基-1,2,4-恶二唑-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(化合物46)和差向异构体46a的合成
Figure PCTCN2021103402-appb-000065
合成步骤参考制备实施例45.步骤3.只是用化合物A-7和化合物B-22反应制备标题化合物46； 1HNMR(500MHz,CDCl 3):δ7.78(m,2H),7.35(m,2H),6.62(dd,J＝11.0,3.8Hz,1H),6.55(d,J＝7.2Hz,1H),5.13-5.08(m,1H),4.24-4.10(m,2H),4.06(t,J＝12.0Hz,1H),3.89(dd,J＝12.5,3.8Hz,1H),3.80-3.70(m,1H),3.67-3.58(m,1H),3.20-3.10(m,1H),2.90-2.82(m,1H),2.35(s,3H),1.58(d,J＝7.2Hz,3H)；ESI-MS:m/z＝417[M+1] +。
差向异构体46a； 1HNMR(400MHz,CDCl 3):δ7.80(m,2H),7.37(m,2H),6.65(m,1H),6.60(dd,J＝11.0,4.0Hz,1H),5.12(m,1H),4.23-4.13(m,2H),4.02(t,J＝11.0Hz,1H),3.92(dd,J＝11.6,3.6Hz,1H),3.79(m,1H),3.65(m,1H),3.22-3.13(m,1H),2.93(m,1H),2.37(s,3H),1.63(d,J＝7.0Hz,3H)；ESI-MS:m/z＝417[M+1] +。
制备实施例47.(S)-4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((S)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(化合物47)和差向异构体47a的合成
Figure PCTCN2021103402-appb-000066
合成步骤:将化合物A-1(1.5g，5.1mmol)、化合物B-21(0.95g，5mmol)加入至二氯甲烷(10mL)中，再加入N,N-二异丙基乙胺(DIPEA,2.0g，12.2mmol)，在室温下搅拌过夜，加入盐酸(0.5N，10mL)淬灭反应，搅拌10min。静置，分层，水相用二氯甲烷(10mL)萃取三次，合并有机相，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩至干，残留物经硅胶柱层析梯度分离(PE:EA＝2:1～1:3,v/v)，得粗品，粗品经硅胶板制备层析分离，得标题化合物47，收率25％和它的R,S-差向异构体47a，收率22％。
标题化合物47: 1H NMR(400MHz,CDCl 3):δ8.79(s,1H),7.90(d,J＝7.0Hz,1H),7.68(d,J＝8.2Hz,1H),7.62(d,J＝8.0Hz,2H),7.21(d,J＝8.0Hz,2H),6.62(dd,J＝11.2,4.0Hz,1H),6.34(d,J＝6.9Hz,1H),5.20–5.10(m,1H),4.05(t,J＝12.2Hz,1H),3.83(dd,J＝12.8,4.2Hz,1H),2.67(s,3H),2.38(s,3H),2.07(s,3H),1.63(d,J＝6.8Hz,3H)；ESI-MS:m/z＝448[M+1] +。
差向异构体47a: 1H NMR(400MHz,CDCl 3):δ8.75(s,1H),7.87(d,J＝8.1Hz,1H),7.66–7.60(m,3H),7.21(d,J＝8.0Hz,2H),6.61(dd,J＝11.3,4.3Hz,1H),6.34(d,J＝7.4Hz,1H),5.16(t,J＝7.2Hz,1H),4.06–3.98(m,1H),3.86(dd,J＝12.8,4.4Hz,1H),2.71(s,3H),2.38(s,3H),2.08(s,3H),1.64(d,J＝7.1Hz,3H)。
三、生物学评估
1.电生理法测定标题化合物对P2X的抑制活性
化合物对于P2X受体的IC 50检测实验是采取穿孔膜片钳完成[Wang et al.(2018)Proc Natl AcadSci USA 115(19):4939–4944]。所有配置溶液所需化合物均从sigma公司购买。
1)细胞培养
细胞使用HEK293细胞系。HEK293细胞放至37℃的含5％CO 2的培养箱中进行培养。在细胞生长至80％－90％密度时，进行传代。弃置原60mm培养皿中的培养基，使用灭菌过的PBS溶液清洗细胞3次后吸干溶液。加入1mL 0.25％胰酶(Gibco)消化10s，吸干胰酶后加入3mL细胞培养基重悬细胞。取0.5mL重悬液加到新的培养皿中，并将总的培养液补至5mL，轻轻晃动培养皿使细胞分布均匀后放至37℃的含5％CO 2的培养箱中进行培养。
2)质粒转染
首先在35mm小培养皿中铺好处理过的玻片，加入100μL重悬细胞，再将培养基总量补至2mL。采取钙转的方法将目的基因转入HEK293细胞系。对需转染的小皿在转染前1h换液，加入2mL新鲜细胞培养液。取1.5mL EP管，加入200μL的0.25M的CaCl 2溶液，加入3μg所需转染质粒，混合均匀成DNA–CaCl 2溶液。再将此混合溶液缓慢加入等体积的2×HBS溶液(NaCl 140mM,Na 2HPO 4 1.5mM,HEPES 50mM，调节pH至6.96)中，在加入的过程中边加边使用枪头进行混合，全部加完后，轻柔吹打8－10次。静置5min后加入小皿中，轻微晃动小皿使溶液混合均匀后放置细胞培养箱中。8–10h后更换培养基。转染24-48h后进行电生理实验。
3)电生理实验
使用电极拉制仪(PP-10，Narishige)，通过两步法拉制得到电生理记录电极，电极中注入內液后，入水电阻为3–5MΩ。实验时，使用含制霉菌素的高钾盐內液(K 2SO 4 75mM,KCl 120mM,MgSO 4 5mM,HEPES 10mM，使用Tris-base调节pH至7.2，过滤后使用)作为电极內液，当电极封接上细胞之后，不需要进一步吸破细胞膜，而是通过制霉菌素对细胞膜的穿孔作用，使其在细胞膜上钻孔，形成孔道。本实验使用Axon200B放大器，Digidata 1440A数模转换器，在电压钳模式下使用Clampfit记录数据。钳制电压为-60mv，采样频率为10kHz,在2kHz条件下进行滤波。
P2X3受体激动剂ATP的使用浓度为1μM，两次ATP给药间隔时间为10min，所有测试化合物包括对照品AF-219先预给2min后，再与ATP进行共给。
表3.1标题化合物对人源P2X3的抑制活性(电生理法)
Figure PCTCN2021103402-appb-000067
Figure PCTCN2021103402-appb-000068
*AF-219购自MedChemExpress(CAS No.1015787-98-0)
表3.2化合物对大鼠和豚鼠P2X3的抑制活性(电生理法)
Figure PCTCN2021103402-appb-000069
表3.3标题化合物对人源P2X2/3的抑制活性(电生理法)
Figure PCTCN2021103402-appb-000070
表3.4标题化合物对人源P2X4的抑制活性(电生理法)
Figure PCTCN2021103402-appb-000071
4)结论：由上述检测结果可知，标题化合物对人源、鼠源P2X3有较强的抑制活性，对人源P2X2/3的抑制活性明显偏弱，而对P2X4无抑制作用，说明本发明得到的化合物对P2X3有良好选择性。
2.标题化合物对PAR-1(血小板蛋白酶激活受体)的抑制活性测定
标题化合物对人源PAR-1的抑制活性采用钙离子通量荧光法(FLIPR法)测定。其方法参见Zhong等人的文献报道[Zhong et al.J BiomolStructDyn,2017,35(13):2853]。
1)细胞培养
稳定表达人源PAR-1的HEK293/Ga15重组细胞株(HD Biosciences,Shanghai,China)在DMEM(Thermo Fisher Scientific,上海)培养液(含有10％小牛血清，800μg/mL G418))中培养。培养箱条件为37℃和含5％CO 2的空气湿度。
2)化合物分析板准备
第一天，将4E+5cells/mL浓度的PAR-1细胞每孔20μL播种在384孔分析板(Corning 3712)中，将细胞分析板以300rpm的速度离心1min，然后在37℃、含5％CO 2的培养箱中培养24h。第二天,倾除每孔的培养液，然后每孔加入20μL的1x loading dye(2μM Fluo-8 AM in 1x HBSS with 20mM HEPES,1mM Probenecid,0.025％Pluronic F-127 assay buffer)。将细胞分析板在室温培养1h。
3)样品准备
将化合物溶于100％DMSO做成10mM的贮备液，在-20℃贮存。在使用当天，将化合物贮备液在室温下解冻。将每个化合物以30μM为起始浓度，用DMEM培养液进行11个浓度点的1:3系列稀释。每个化合物做双份稀释供测试。阳性对照化合物采用SCH79797,以30μM为起始浓度进行同样稀释。
4)FLIPR测定
将细胞分析板置于FLIPR仪器(Molecular Device)内，将化合物板中的10μL化合物(3x最终浓度)加到细胞分析板上的对应孔位。10分钟后，再在每孔加入10μL的TFLLR-NH2(TFA)(终浓度为10μM)，以刺激产生细胞内的钙通量信号。在激发波长470/495nm，发射波长515/575nm下连续监测Ca ++依赖性萤光信号，以分析化合物的抑制活性。
5)数据分析
用FLIPR程序采集分析数据。以荧光峰值评估化合物在各浓度下的抑制(或激动)活性。用EXCEL和PRISM程序计算化合物的IC 50值。
表3.5标题化合物对PAR-1的抑制活性(电生理法)
Figure PCTCN2021103402-appb-000072
6)结论：在上述实验条件下，标题化合物对PAR-1无抑制作用。
3.标题化合物大鼠体内药代动力学实验
1)给药方式
采用SD大鼠，雌雄各半,化合物按不同剂量采用静脉注射(3mg/kg)、腹腔注射(10mg/kg)或灌胃给药(30mg/kg)。在给药后的不同时间点，采集血液到EDTA血样收集管中。通过离心分离血浆并在-20℃下保存，等待分析。
2)血样处理和LC-MS/MS分析
标准曲线和质控样本配制处理：取化合物储备液用50％甲醇水稀释成含各化合物浓度分别为20-10000ng/mL的标准工作液，60、600、6000ng/mL的质控工作液。分别取47.5μL空白大鼠血浆中加入2.50μL的标准曲线工作液和质控工作液，配置成含各化合物浓度为1.00-500.00ng/mL的标曲和浓度为3.00、30.00和300.00ng/mL的质控样本，分别加入200μL的乙腈(含内标维拉帕米5ng/mL)，涡旋振荡3min后，20000rcf，4℃离心15min，取上清液进行LC-MS/MS分析。
化合物血样处理：取血浆样品5μL，加入空白大鼠血浆45μL，加入200μL的乙腈(含内标维拉帕米5ng/mL)，涡旋振荡3min后，20000rcf，4℃离心15min，取 上清液进行LC-MS/MS分析(色谱柱：ACQUITY
Figure PCTCN2021103402-appb-000073
BEH C18 2.1x50mm 1.7μm；流动相A：0.1％甲酸水；流动相B：乙腈；流速：0.35mL/min)。
表3.6标题化合物的主要药代动力学参数
Figure PCTCN2021103402-appb-000074
3)结论：由上述实验结果可知，标题化合物有良好的口服药代动力学性质和口服生物利用度。
4.ATP/枸橼酸诱导的豚鼠咳嗽实验
采用ATP/枸橼酸诱导豚鼠咳嗽模型，观察化合物经腹腔注射和灌胃给药对豚鼠咳嗽的抑制作用[Carceau and Chauret(2019)Pulmonary Pharmacology&Therapeutics 56:56–62]。
1)实验动物
采用普通级SD豚鼠，雄性动物体重：244～320g；雌性动物体重：227～331g。豚鼠饲养于塑料笼盒中，自由摄食，自由饮水，笼盒内铺有玉米芯垫料，雌雄分笼饲养，每笼不10只。控制温度20～26℃，相对湿度为40％～70％。采用自动光照，每12h明暗交替，8:00a.m.开灯，8:00p.m.关灯。
2)动物分组和给药
根据性别及初筛时枸橼酸诱导咳嗽次数，采用分层随机法分组，每组10只。化合物按不同剂量采用腹腔注射或灌胃给药。阳性对照化合物采用AF-219。空白溶媒为5％DMSO加5％Solutol HS 0.9％氯化钠溶液。
3)实验方法
豚鼠筛选：将豚鼠置于约25cm×10cm×8cm(长、宽、高)的有机玻璃盒内，喷入20％枸橼酸溶液1min，用多道生理信号采集处理系统记录10min内动物的咳嗽次数，根据豚鼠的咳嗽次数，淘汰未发生咳嗽的豚鼠，最终筛选出雌雄各35只豚鼠，分层随机法分组，进行正式试验。
4)正式试验：各组豚鼠腹腔注射给药后20min，或灌胃给药后45min，将各组豚鼠置于约25cm×10cm×8cm(长、宽、高)的有机玻璃盒内。用压力雾化器向盒内持续喷入10μM的ATP溶液2min(喷入流速0.6ml/min)。再喷入20％枸橼酸溶液1min，用多道生理信号采集处理系统记录10min内动物的咳嗽次数。
5)数据统计
用Excel软件录入咳嗽次数。采用GraphPad Prism 8软件作图，SPSS 18软件进行统计分析。结果见图5和图6，将各组咳嗽次数和潜伏期时间分别作方差齐性检验。若方差齐(P>0.05)则做单因素方差分析，有显著性差别(P≤0.05)，则各剂量组与对照组间做Dunnett检验；否则检验结束。若方差不齐(P≤0.05)，则进行非参数检验(Kruskal-Wallis H检验，即K-W H检验)，如K-W H检验有统计学差异(P≤0.05)，则各剂量组与对照组间做Mann-Whitney U检验,否则检验结束。
6)结论：在上述实验条件下，标题化合物经腹腔注射或灌胃给药均可明显降低实验动物的咳嗽频率。
5.大鼠味觉试验
实验目的：观察标题化合物对SD大鼠味觉的影响。
1)实验方法
1.1)动物及分组：雄性SD大鼠40只，300克/只左右。将动物随机分为4组，每组10只，各组体重相近，单笼饲养。
1.2)饮水习惯训练：各组动物每天上午8.30和下午16.30分别给正常饮水30分钟，其余时间禁水，持续5天。
1.3)给药：实验前一天晚上禁水，次日上午按以下剂量腹腔注射给予以下药物：
(1)溶剂(5％DMSO/5％Solutol HS/0.9％氯化钠溶液)10mL/kg
(2)Compound 22 10mg/kg(体积10mL/kg)
(3)Compound 4 10mg/kg(体积10mL/kg)
(4)AF-21910mg/kg(体积10mL/kg)
1.4)饮水量测量：注射后将动物放回原来的笼子，溶剂、Compound 22、Compound 4、AF-219的注射时间分别在各种药物的Tmax区间，每个笼子同时放入一瓶正常饮用水，一瓶含0.3mM盐酸奎宁(Quinie)的饮水，所有动物饲养笼中两瓶水放置的左右位置一致。让动物自由饮水15分钟后，分别测量两瓶水的饮用量，精确到0.1mL。
1.5)数据统计：分别统计奎宁苦味水、自来水的饮用量和总饮水量，以及奎宁水占总饮水量的百分比，比较各组动物苦味水的饮用量，用方差分析比较各组之间的差异有无显著性。
2)实验结果
各给药组SD大鼠总饮水量与溶剂对照组比较均无显著性差异(P>0.05)，而阳性对照AF-219组SD大鼠苦味水的饮用量有明显增加，表明在本试验条件下受试化合物对大鼠味觉没有明显影响(图7、表3.7)。
表3.7各组实验动物的饮水量
Figure PCTCN2021103402-appb-000075
Figure PCTCN2021103402-appb-000076
6.肝微粒体酶稳定性实验
1)检测方法
LC-MS/MS法,其中:液相方法采用梯度洗脱法。色谱柱：ACQUITY
Figure PCTCN2021103402-appb-000077
BEH C18(2.1mm×50mm，1.7μm)；流速：0.35mL/min；进样体积：1μL(YS001)、3μL(睾酮)；柱温：40℃；自动进样器温度：4℃；流动相组成：流动相A：0.1％甲酸水，流动相B：乙腈。质谱条件采用ESI源，正离子、多反应监测(MRM)模式进行质谱分析。
2)样品处理
取肝微粒孵育样品，加含有内标(氯雷他定1ng/mL)的乙腈溶液400μL终止反应，2500rpm涡旋混匀，4℃，20000rcf离心10min，取上清液至干净内衬管中，LC-MS/MS检测原型药物含量。
3)溶液配制
储备液的配制
给药组储备液：分别称取化合物4及化合物22粉末适量，用二甲基亚砜(DMSO)溶解，配制成10mM的给药组储备液，存于-20℃冰箱中；
睾酮储备液：称取睾酮适量，用二甲基亚砜(DMSO)溶解，配制成10mM的睾酮储备液，存于-20℃冰箱中；
内标储备液：称取氯雷他定适量，用甲醇溶解，配制成1.0mg/mL的氯雷他定储备液，存于-20℃冰箱中。
工作液的配制
给药组工作液：移取化合物4及化合物22给药组储备液适量，用甲醇配制成500μM的工作液；
睾酮工作液：移取睾酮储备液适量，用甲醇配制成500μM的工作液。
4)试验内容
代谢速率试验：取人肝微粒体，于冰上解冻融化，轻轻摇匀。移取肝微粒体加至100mM磷酸缓冲液中，再分别加入化合物4(或化合物22)给药组工作液，使微粒体浓度为0.625mg/mL，化合物4或化合物22浓度为1.25μM。移取上述溶液80μL，于37℃水浴中预孵育5min后，加入5mM的NADPH溶液20μL，37℃水浴孵育。每个代谢孵育体系样本总体积100μL，孵育体系包括终浓度为0.5mg/mL的肝微粒体蛋白、1μM的化合物4(或化合物22)和1mM的NADPH。反应在进行0、10、30、60、90min后，以4倍体积乙腈(含内标氯雷他定1ng/mL)终止反应，涡旋混匀，4℃，20000rcf下高速离心10min，取上清液进行LC-MS/MS检测。每一孵育样品平行试验三份。阳性对照为相应种属微粒体对阳性药物睾酮(1μM)的代谢样本，以4倍体积乙腈(含内标氯雷他定1ng/mL)终止反应，孵育时间点为0、30、60min。
5)数据处理
采用Analyst1.6.3软件进行数据采集，在优化积分参数后，对目标色谱峰自动积分，不允许对个别色谱峰单独积分或手动积分，计算得出保留时间和峰面积。由Analyst1.6.3采集数据，经积分处理后得到保留时间、峰面积，计算分析物与内标的峰面积比，然后使用Microsoft Office Excel二次处理，计算半衰期t 1/2其清除率CLint。受试化合物剩余百分比(％Control)由非零时间点样品与零时刻样品中浓度之比计算出。将Ln(％Control)对孵育时间作图并进行线性拟合。计算受试化合物清除常数(ke，1/min)、清除半衰期(t 1/2，min)以及体外内在清除率(CLint，μL/(mg·min))。t 1/2＝0.693/ke，CLint＝ke/Cprotein(注：ke为斜率，Cprotein为蛋白浓度。)
Figure PCTCN2021103402-appb-000078
6)结论：在上述实验条件下，标题化合物对肝微粒体酶有较好稳定性。
7.模拟胃液、模拟肠液稳定性实验
1)检测方法
LC-MS/MS法,其中:液相方法采用梯度洗脱法。色谱柱：ACQUITY
Figure PCTCN2021103402-appb-000079
BEH C18(2.1mm×50mm，1.7μm)；流速：0.35mL/min；进样体积：1μL；柱温：40℃；自动进样器温度：10℃；流动相组成：流动相A：0.1％甲酸水，流动相B：乙腈。质谱条件采用ESI源，正离子、多反应监测(MRM)模式进行质谱分析。
2)样品处理
取模拟胃液、模拟肠液样品，加含有内标(氯雷他定5ng/mL)的乙腈溶液1200μL，2500rpm涡旋混匀，4℃，20000rcf离心10min，取上清液至干净内衬管中，LC-MS/MS检测原型药物含量。
3)溶液配制
工作液的配制
给药组工作液：移取化合物4及化合物22给药组储备液适量，用甲醇配制成50μM的工作液；
内标工作液：移取氯雷他定储备液适量，用乙腈配制成5ng/mL的氯雷他定工作液
模拟胃液、模拟肠液的配制
空白人工胃液：取稀盐酸3.28mL，加水160mL，摇匀使其充分溶解后，调节pH值至1.3，加水稀释定容至200mL，即为空白人工胃液。
人工胃液：取稀盐酸3.28mL，加水160mL与胃蛋白酶2g，摇匀使其充分溶解后，调节pH值至1.3，加水稀释定容至200mL，即为人工胃液。
空白人工肠液：称取磷酸二氢钾1.36g，加水100mL，使其溶解，用0.1mol/L氢氧化钠溶液调节pH值至6.8，加水定容至200mL，即为空白人工肠液。
人工肠液：称取磷酸二氢钾1.36g，加水100mL，使其溶解，用0.1mol/L氢氧化钠溶液调节pH值至6.8，另外称取2g胰蛋白酶加适量水溶解，将两液混合后，加水定容至200mL，即为空白人工肠液。
4)试验内容
取模拟胃液、模拟肠液，轻轻摇匀。移取模拟胃液、模拟肠液，分别加入化合物4(或化合物22)工作液，化合物4(或化合物22)浓度为1.25μM。移取上述溶液300μL，于37℃水浴中预孵育，于0、0.5、1、1.5、2、3、5h取样，以4倍体积乙腈(含内标氯雷他定5ng/mL)，涡旋混匀，4℃，20000rcf下高速离心10min，取上清液进行LC-MS/MS检测。每一孵育样品平行试验三份。
5)数据处理
采用MassLynx V4.2软件进行数据采集，在优化积分参数后，对目标色谱峰自动积分，不允许对个别色谱峰单独积分或手动积分，计算得出保留时间和峰面积。由MassLynx V4.2采集数据，经积分处理后得到保留时间、峰面积，计算分析物与内标的峰面积比，然后使用Microsoft Office Excel二次处理，计算平均剩余百分率。
6)实验结果
在上述实验条件下，化合物4及化合物22在人工胃肠液中较稳定(图8,图9)。

Claims (14)

  1. 一种N-甲酰胺基吡唑啉类衍生物，其特征在于，为具有通式(I)所示结构的化合物或其对映异构体、非对映异构体、差向异构体、外消旋体，或其在药学上可接受的盐:
    Figure PCTCN2021103402-appb-100001
    其中：
    Ar选自取代或无取代苯基、包含1-3个选自O、N和S原子的取代或无取代五元杂芳环基或包含1-2个N原子的取代或无取代六元杂芳环基；当Ar选自邻位双取代苯基时，两个邻位取代基相互独立或者成环；
    R 1选自取代或未取代苯基或取代或未取代吡啶基；R 2选自甲基、乙基、异丙基、环丙基；
    R 3，R 4分别各自独立的选自H、C1～C5烷基、C1～C5烷氧羰基、取代或未取代的C1～C5酰基；或R 3、R 4连同它们两者所连接的N一起形成取代或未取代的C5～C6杂环烷基或C5～C6杂环烷基酮或C5～C6杂芳基；所述杂环烷基或杂环烷基酮环上的碳原子可进一步被一个或多个O、N取代。
  2. 根据权利要求1所述的N-甲酰胺基吡唑啉类衍生物，其特征在于，所述Ar选自下列取代苯基、取代的五元或六元杂芳环基：
    Figure PCTCN2021103402-appb-100002
    所述R 5,R 6相同或不同，分别独立的选自H、卤素、硝基、氰基、甲基、三氟甲基、三氟甲氧基、甲氧基、二氧亚甲基、C1-C3直链或支链烷氧基，且在任意环碳原子上取代；选择所述二氧亚甲基时，所述R 5,R 6为其中的氧原子，与相连的亚甲基成环；
    R 7选自甲基、乙基、异丙基、环丙基。
  3. 根据权利要求1所述的N-甲酰胺基吡唑啉类衍生物，其特征在于，所述R 1中， 苯基或吡啶基环上的取代基选自卤素、C1-C3直链或支链烷基、C1-C3直链或支链烷氧基、三氟甲基、三氟甲氧基。
  4. 根据权利要求1所述的N-甲酰胺基吡唑啉类衍生物，其特征在于，R 3，R 4分别各自独立的选自H、甲基、乙基、甲氧羰基、乙氧羰基、丙氧羰基、异丙氧羰基、叔丁氧羰基、甲酰基、乙酰基、N,N-二烷基乙酰基；或R 3、R 4连同它们这两者所连接的N一起形成取代或未取代吗啉酮基、取代或未取代吗啉基、取代或未取代哌啶基、取代或未取代哌嗪基、取代或未取代哌嗪酮基、取代或未取代吡咯烷酮基、取代或未取代恶唑烷酮基、取代或未取代咪唑烷酮基、取代或未取代咪唑基、取代或未取代吡唑基、1,2,3-三氮唑基、1,2,4-三氮唑基。
  5. 根据权利要求4所述的N-甲酰胺基吡唑啉类衍生物，其特征在于，所述吗啉酮基、吗啉基、哌啶基、哌嗪基、哌嗪酮基、吡咯烷酮基、恶唑烷酮基、咪唑烷酮基、咪唑基、吡唑基上的取代基选自C1-C3直链或支链烷基、甲酰基、乙酰基、C1-C5直链或支链烷氧基。
  6. 根据权利要求1～5任一项所述的N-甲酰胺基吡唑啉类衍生物，其特征在于，所述通式(I)的化合物为具有通式(Ia)、(Ib)、(Ic)或(Id)所示的结构：
    Figure PCTCN2021103402-appb-100003
  7. 根据权利要求6所述的N-甲酰胺基吡唑啉类衍生物，其特征在于，所述Ar选自苯基、对三氟甲基苯基、二甲氧基苯基、二氧五环苯基、对氰基苯基、氯代苯基、甲氧基吡啶基、三氟甲基吡啶基、氯代吡啶基、二氟吡啶基、三氟甲基氧基吡啶基、三氟甲基吡嗪基、甲基吡嗪基、氯代吡嗪基、甲氧基哒嗪基、三氟甲基哒嗪基、氯代哒嗪基、三氟甲基嘧啶基、甲基嘧啶基、氯代嘧啶基、甲基-1,2,4-恶二唑基、甲基-1,3,4-恶二唑基；
    R 1选自对甲基苯基、对甲氧基苯基、对氟苯基、对氯苯基、甲基吡啶基；
    R 2选自甲基、乙基；R 3,R 4分别各自独立的选自H、甲基、乙基、甲氧羰基、乙氧羰基、乙酰基、N,N-二烷基乙酰基；或R 3、R 4连同它们两者所连接的N一起形成吗啉酮基、哌嗪酮基、N-甲基哌嗪酮基、甲基哌嗪酮基、吡咯烷酮基、恶唑烷酮基、 咪唑烷酮基、乙酰基咪唑烷酮基、吗啉基、哌嗪基、N-乙酰基哌嗪基、吡唑基。
  8. 根据权利要求1所述的N-甲酰胺基吡唑啉类衍生物，其特征在于，所述通式(I)的化合物为如下化合物中的至少一种：
    (S)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(1)
    (R)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(1a)(S)-4-(4-甲基-2-氧代哌嗪-1-基)-3-(4-甲基苯基)-N-((R)-1-(4-氯苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(2)
    (R)-4-(4-甲基-2-氧代哌嗪-1-基)-3-(4-甲基苯基)-N-((R)-1-(4-氯苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(2a)
    (S)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(3)
    (R)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(3a)
    (S)-4-(N-甲基-N-乙氧羰基胺基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(4)
    (R)-4-(N-甲基-N-乙氧羰基胺基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(4c)
    (S)-4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((R)-1-(4-(三氟甲基)苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(5)
    (R)-4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((R)-1-(4-(三氟甲基)苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(5a)
    (S)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(3,4-二甲氧基苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(6)
    (R)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(3,4-二甲氧基苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(6a)
    (S)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(3,4-(亚甲基二氧)苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(7)
    (R)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(3,4-(亚甲基二氧)苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(7a)
    (S)-4-(3-乙酰基-2-氧代咪唑烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(4-腈基苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(8)
    (R)-4-(3-乙酰基-2-氧代咪唑烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(4-腈基苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(8a)
    (S)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(5-甲氧基吡啶-2-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(9)
    (R)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(5-甲氧基吡啶-2-基)乙基)-4,5- 二氢-1H-吡唑-1-甲酰胺(9a)
    (S)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(5-(三氟甲基)吡啶-2-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(10)
    (R)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(5-(三氟甲基)吡啶-2-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(10a)
    (S)-4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((R)-1-(6-氯吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(11)
    (R)-4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((R)-1-(6-氯吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(11a)
    (S)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(5,6-二氟吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(12)
    (R)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(5,6-二氟吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(12a)(S)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲氧基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(13)
    (R)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲氧基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(13a)
    (S)-4-(吗啉-4-基)-3-(4-氟苯基)-N-((R)-1-(6-甲氧基吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(14)
    (R)-4-(吗啉-4-基)-3-(4-氟苯基)-N-((R)-1-(6-甲氧基吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(14a)
    (S)-4-(2-(二甲胺基)-N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(15)
    (R)-4-(2-(二甲胺基)-N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(15a)
    (S)-4-(N-甲基-N-甲氧羰基胺基)-3-(4-甲氧基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(16)
    (R)-4-(N-甲基-N-甲氧羰基胺基)-3-(4-甲氧基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(16a)
    (S)-4-(4-甲基-2-氧代哌嗪-1-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(17)
    (R)-4-(4-甲基-2-氧代哌嗪-1-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(17a)
    (S)-4-(3-乙酰基-2-氧代咪唑烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(18)
    (R)-4-(3-乙酰基-2-氧代咪唑烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(18a)
    (S)-4-(4-乙酰哌嗪-1-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙 基)-4,5-二氢-1H-吡唑-1-甲酰胺(19)
    (R)-4-(4-乙酰哌嗪-1-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(19a)
    (S)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(4-(三氟甲基)苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(20)
    (R)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(4-(三氟甲基)苯基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(20a)
    (S)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(21)
    (R)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(21a)
    (S)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(22)
    (R)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(22a)
    (S)-4-(2-氧代恶唑烷-3-基)-3-(5-甲基吡啶-2-基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(23)
    (R)-4-(2-氧代恶唑烷-3-基)-3-(5-甲基吡啶-2-基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(23a)
    (S)-4-(3-氧代吗啉-4-基)-3-(4-氟苯基)-N-((R)-1-(2-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(24)
    (R)-4-(3-氧代吗啉-4-基)-3-(4-氟苯基)-N-((R)-1-(2-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(24a)
    (S)-4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(25)
    (R)-4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(25a)
    (S)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(5-(三氟甲基)吡嗪-2-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(26)
    (R)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(5-(三氟甲基)吡嗪-2-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(26a)
    (S)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(5-甲基吡嗪-2-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(27)
    (R)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(5-甲基吡嗪-2-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(27a)
    (S)-4-(2-氧代吡咯烷-1-基)-3-(4-氟苯基)-N-((R)-1-(5-甲基吡嗪-2-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(28)
    (R)-4-(2-氧代吡咯烷-1-基)-3-(4-氟苯基)-N-((R)-1-(5-甲基吡嗪-2-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(28a)
    (S)-4-(2-氧代恶唑烷-3-基)-3-(4-氯苯基)-N-((R)-1-(6-(甲氧基)哒嗪-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(29)
    (R)-4-(2-氧代恶唑烷-3-基)-3-(4-氯苯基)-N-((R)-1-(6-(甲氧基)哒嗪-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(29a)
    (S)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)哒嗪-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(30)
    (R)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(6-(三氟甲基)哒嗪-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(30a)
    (S)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(6-氯哒嗪-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(31)
    (R)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(6-氯哒嗪-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(31a)
    (S)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(32)
    (R)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(32a)
    (S)-4-(3-氧代吗啉-4-基)-3-(4-氟苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(33)
    (R)-4-(3-氧代吗啉-4-基)-3-(4-氟苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(33a)
    (S)-4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(34)
    (R)-4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(34a)
    (S)-4-(3-氧代吗啉-4-基)-3-(4-氯苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(35)
    (R)-4-(3-氧代吗啉-4-基)-3-(4-氯苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(35a)
    (S)-4-(3-氧代吗啉-4-基)-3-(4-甲氧基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(36)
    (R)-4-(3-氧代吗啉-4-基)-3-(4-甲氧基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(36a)
    (S)-4-(4-乙酰哌嗪-1-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(37)
    (R)-4-(4-乙酰哌嗪-1-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙 基)-4,5-二氢-1H-吡唑-1-甲酰胺(37a)
    (S)-4-(4-甲基哌嗪-1-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(38)
    (R)-4-(4-甲基哌嗪-1-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(38a)
    (S)-4-(吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(39)
    (R)-4-(吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(39a)
    (S)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(40)
    (R)-4-(2-氧代恶唑烷-3-基)-3-(4-甲基苯基)-N-((R)-1-(2-(三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(40a)
    (S)-4-(3-甲基-2-氧代咪唑烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(2-三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(41)
    (R)-4-(3-甲基-2-氧代咪唑烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(2-三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(41a)
    (S)-4-(吡唑-1-基)-3-(4-甲基苯基)-N-((R)-1-(2-三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(42)
    (R)-4-(吡唑-1-基)-3-(4-甲基苯基)-N-((R)-1-(2-三氟甲基)嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(42a)
    (S)-4-(2-氧代恶唑烷-3-基)-3-(4-氯苯基)-N-((R)-1-(2-甲基嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(43)
    (R)-4-(2-氧代恶唑烷-3-基)-3-(4-氯苯基)-N-((R)-1-(2-甲基嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(43a)
    (S)-4-(2-氧代吡咯烷-1-基)-3-(4-氟苯基)-N-((R)-1-(2-氯嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(44)
    (R)-4-(2-氧代吡咯烷-1-基)-3-(4-氟苯基)-N-((R)-1-(2-氯嘧啶-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(44a)
    (S)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(3-甲基-1,2,4-恶二唑-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(45)
    (R)-4-(2-氧代吡咯烷-1-基)-3-(4-甲基苯基)-N-((R)-1-(3-甲基-1,2,4-恶二唑-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(45a)
    (S)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(3-甲基-1,2,4-恶二唑-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(46)
    (R)-4-(3-氧代吗啉-4-基)-3-(4-甲基苯基)-N-((R)-1-(3-甲基-1,2,4-恶二唑-5-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(46a)(S)-4-(N-甲基乙酰胺基)-3-(4-甲基苯 基)-N-((S)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(47)
    (R)-4-(N-甲基乙酰胺基)-3-(4-甲基苯基)-N-((S)-1-(6-(三氟甲基)吡啶-3-基)乙基)-4,5-二氢-1H-吡唑-1-甲酰胺(47a)。
  9. 根据权利要求1所述的N-甲酰胺基吡唑啉类衍生物，其特征在于，所述在药学上可接受的盐选用药用上接受的无机酸盐、有机酸盐；所述的无机酸盐包括与氢卤酸，硝酸，碳酸，硫酸或磷酸形成的盐；所述的有机酸盐包括与苹果酸、L-苹果酸、D-苹果酸、枸橼酸、富马酸、草酸、乳酸、樟脑磺酸、L-樟脑磺酸、D-樟脑磺酸、对甲苯磺酸、甲磺酸、苯甲酸形成的盐。
  10. 一种权利要求1～9任一项所述N-甲酰胺基吡唑啉类衍生物单独或与其他药物联合使用在制备治疗由P2X3介导的疾病的药物中的应用。
  11. 根据权利要求9所述的应用，其特征在于，所述的疾病包括呼吸系统疾病、多种原因造成的疼痛、皮肤疾病；所述呼吸系统疾病包括慢性阻塞性肺疾病、哮喘、急性、亚急性及慢性咳嗽、支气管痉挛；所述疼痛包括手术疼痛、炎性疼痛、癌性疼痛、膀胱疼痛、子宫内膜异位症疼痛、糖尿病性神经痛、创伤性疼痛、牙痛、偏头痛、及与肠易激综合症有关的疼痛；所述皮肤疾病包括慢性瘙痒。
  12. 一种药物组合物，其特征在于，包括权利要求1～9任一项所述N-甲酰胺基吡唑啉类衍生物以及一种或多种药学上可接受的载体或赋形剂。
  13. 根据权利要求12所述的药物组合物，其特征在于，所述载体或赋形剂包括填充剂，稀释剂，湿润剂，润滑剂，粘合剂，崩解剂，吸收促进剂，表面活性剂，吸附载体，抗氧化剂、乳化剂、金属螯合剂、pH调节剂，香味剂，甜味剂中的一种或多种。
  14. 一种由权利要求12所述药物组合物制成的药物制剂，其特征在于，包括片剂、胶囊、吸入剂、乳剂、混悬剂、凝胶剂、粉剂、颗粒剂、口服液及注射剂。
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