Oxindole derivative, preparation method thereof and application in TRPA1 inhibitor

Abstract

The invention provides an oxoindole derivative, a preparation method thereof and application of the oxoindole derivative in the direction of a TRPA1 inhibitor. The structural general formula of the oxoindole derivative is shown in the formula I, the compound ZQMT1-26 provided by the invention does not influence the cell activity in the administration range, has high TRPA1 inhibitor activity, reduces the number of times of licking a C57BL / 6J mouse after the mouse is smeared with a TRPA1 agonist, improves the capacity of contact cold foot pain, reduces the total time of abnormal behaviors in an abdominal pain experiment, remarkably improves the MDT (mechanical sensation threshold) of the mouse and the like, and has good clinical application prospects. In addition, the compound ZQMT10 and the TRPA1 have strong binding capacity. In conclusion, the compound ZQMT1-26 can be used for treating neuropathic pain, inflammatory pain and the like. Especially, the compound ZQMT1-26 can block pain feeling from the root of pain generation caused by cold, and the compound plays an important role in preparation of drugs for preventing or treating pain caused by frostbite. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of organic compound synthesis and pharmaceutical application, and specifically relates to an oxindole derivative and a preparation method thereof and application thereof in the direction of TRPA1 inhibitors. Background Art

[0002] Transient receptor potential ankyrin 1 (TRPA1) is a non-selective calcium channel and a member of the TRP ion channel family that functions as a sensor for a range of physical and chemical stimuli. Functional TRPA1 channels are tetramers formed by four identical subunits, each consisting of six transmembrane helices forming a central pore and including a large intracellular domain of 14 N-terminal ankyrin repeats. TRPA1 acts as a sensor of pain and inflammation and is widely expressed in sensory neurons throughout the body. Specifically, TRPA1 is activated by endogenous ligands such as proinflammatory cytokines and exogenous stimuli including allyl isothiocyanate, allicin, and cinnamaldehyde, which mediate channel switching by covalently modifying the cysteine-rich region of the N-terminal ankyrin repeats. Upon channel activation, increased calcium flux leads to the release of neurotransmitters, eliciting a pain response.

[0003] Studies have shown that cold and low-temperature environments are closely related to the occurrence and development of a variety of pain diseases. Long-term exposure of the body to cold and humid environments can cause local or systemic tissue damage and cause pain, such as chest pain, abdominal pain, and muscle and joint pain. At present, there are four main hypotheses about how low temperature causes or aggravates pain. Among them, the 2021 Nobel Prize in Physiology or Medicine winner proposed that low temperature can increase the excitability of TRP channels, thereby causing or aggravating pain, and reported the theory of mechanically gated ion channels that mediate cation influx in mammals. Harmful low temperature stimulation directly activates TRPA1 channels, leading to the generation of neuronal action potentials, thereby inducing pain and inflammation. Blocking TRPA1 channels can effectively reduce the hyperalgesia and abnormal pain reactions induced by harmful stimuli.

[0004] In the past few years, TRPA1 has been considered an attractive and promising drug target for the treatment of neuropathic pain because several rodent knockout studies have linked TRPA1 to pain signaling and because it can be activated by low temperature (0-20°C), mechanical stimulation, etc., and is involved in a variety of physiological and pathological processes such as cold perception and pain. Inhibiting the switch of TRPA1 channels can effectively reduce pain caused by cold, but few TRPA1 inhibitors have passed clinical trials.

[0005] Nowadays, the analgesics commonly used in clinical practice are still non-steroidal analgesics and opioid central analgesics. They are unable to block the pain perception from the root cause of the pain caused by cold. Therefore, the development of a clear and effective anti-cold pain drug is an urgent problem that needs to be solved. Summary of the invention

[0006] Purpose of the invention: The present invention proposes an oxindole derivative and a preparation method thereof and its application in the direction of TRPA1 inhibitors. Its purpose is to provide an oxindole derivative or a pharmaceutically acceptable salt thereof having the ability to inhibit TRPA1 and a preparation method of the oxindole derivative, and also points out the application of the oxindole derivative in drugs for treating cold injuries.

[0007] Technical solution:

[0008] In a first aspect, the present invention provides an oxindole compound or a pharmaceutically acceptable salt thereof, wherein the compound has a general structural formula as shown in Formula I below:

[0009]

[0010] Wherein: R1 is H or a carbonyl group, R2 is a substituent at the end of the side chain of the parent core, and R2 is selected from the following structures:

[0011]

[0012] The second aspect of the present invention provides an oxindole compound represented by general formula I or a pharmaceutically acceptable salt thereof, selected from:

[0013]

[0014]

[0015] The third aspect of the present invention provides a method for preparing the oxygen indole compounds, comprising the steps of: dissolving a carboxylic acid compound in DMF, adding a condensing agent, and then adding DIPEA, reacting at room temperature for 15-60 minutes, adding an amino compound after the reaction, extracting and purifying to obtain compound ZQMT1-26.

[0016] Preferably, the carboxylic acid-containing compound is one of 2-oxoindoline-6-carboxylic acid, 2-oxoindoline-5-carboxylic acid, 2-indole-7-carboxylic acid, 2-indole-4-carboxylic acid, and 2,3-dioxoindoline-7-carboxylic acid.

[0017] Preferably, the condensing agent is one or more of EDCI, HOBT and TBTU.

[0018] Preferably, the amino compound is one of p-methoxybenzylamine, 4-fluorobenzylamine, 4-methylbenzylamine, p-bromobenzylamine, p-trifluoromethylbenzylamine, 4-chlorobenzylamine and p-trifluoromethoxybenzylamine.

[0019] The fourth aspect of the present invention provides a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0020] Furthermore, the compound is added with one or more pharmaceutically acceptable excipients to prepare a preparation, and the dosage form of the preparation includes tablets, granules, powders, capsules, soft capsules, solutions, injections, mixtures, suspensions and the like.

[0021] The fifth aspect of the present invention provides the use of the compound in the preparation of a drug for treating pain, including neuralgia, inflammatory pain, and the like.

[0022] The sixth aspect of the present invention proposes the use of the compound in the preparation of a drug for treating pain caused by frostbite.

[0023] The seventh aspect of the present invention provides the use of the compound in preparing a TRPA1 inhibitor.

[0024] Beneficial effects:

[0025] The present invention proposes an oxindole derivative and a preparation method thereof and an application thereof in the direction of TRPA1 inhibitors, wherein the oxindole derivative has a novel structure, and a substituent group is introduced at the 4, 5, 6 or 7 positions with oxindole as the parent nucleus. The developed compound ZQMT1-26 does not affect cell viability within the administration range by performing MTT cytotoxicity experiments, calcium influx experiments, foot licking experiments, in vivo acute toxicity tests, cold plate experiments, abdominal pain experiments, von Fery tests and thermal migration experiments in DRG cells, primary DRG cells, and HEK-293T cells overexpressing TRPA1, and has high anti-TRPA1 inhibitor activity. The compound reduces the number of foot licking of C57BL / 6J mice, improves the ability of contact cold foot pain, reduces the total time of abnormal behavior in the abdominal pain experiment, significantly improves the mechanical perception threshold MDT of mice, and the compound ZQMT10 has a good affinity with TRPA1. In summary, the compound ZQMT1-26 can treat neuralgia, inflammatory pain, and the like. In particular, compound ZQMT1-26 can block pain perception from the root of pain caused by cold, and this type of compound plays an important role in the preparation of drugs for preventing or treating pain caused by frostbite. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the preparation method of compound ZQMT1-26;

[0027] Figure 2 The toxicity data of compound ZQMT1-26 at different concentrations on DRG cells measured by MTT;

[0028] Figure 3 This is a graph showing the results of a calcium ion influx experiment of compound ZQMT1-26 in DRG cells;

[0029] Figure 4 This is the data diagram of the toxicity of the compound to primary DRG cells measured by MTT;

[0030] Figure 5 This is the result of immunofluorescence experiment of primary DRG cells;

[0031] Figure 6 This is a graph showing the results of a calcium influx experiment of compound ZQMT1-26 in primary DRG cells;

[0032] Figure 7 This is the toxicity data of compound ZQMT1-26 on HEK-293T cells overexpressing TRPA1 measured by MTT;

[0033] Figure 8 This is a graph showing the results of calcium influx experiments of compounds in HEK-293T cells overexpressing TRPA1;

[0034] Fig. 9 This is the result of the mouse licking foot experiment;

[0035] Fig.10 The results of acute toxicity experiment in mice are shown in Figure A, which shows the weight change of mice in 14 days, B shows the organ index, and C shows the HE staining of the main organs;

[0036] Fig.11 This is the result of the cold plate experiment on the seventh day;

[0037] Fig.12 This is the result of the cold plate experiment on the fourteenth day;

[0038] Fig.13 This is a graph showing the results of an abdominal pain experiment in mice. In the graph, A is the total duration of abnormal abdominal pain behaviors in mice, B is the first appearance time of abnormal abdominal pain behaviors in mice, and C is HE staining of the colon after the end of the experiment.

[0039] Fig.14 This is the result of measuring the mechanical receptive field of C57BL / 6J mice on day 7 (without CFA-induced stimulation);

[0040] Fig.15 This is the result of measuring the mechanical receptive field of C57BL / 6J mice on day 14 (without CFA-induced stimulation);

[0041] Fig.16This is the result of the change of mechanoreceptive field of mice within 24 hours after CFA induction;

[0042] Fig.17 The results of the thermal migration experiment of ZQMT10 in primary DRG cells are shown in Figure A and B, respectively, which show the CETSA analysis of primary DRG expression after ZQMT10 treatment. DETAILED DESCRIPTION

[0043] The present invention is described below by means of specific examples, but the present invention is not limited to these examples.

[0044] The structure of the compound was determined by nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HR-MS). NMR was measured using a Bruker AVANCE-300 / 600 nuclear magnetic resonance spectrometer, the solvent for the measurement was DMSO-d6, and the internal standard was TMS. Column chromatography used 200-300 mesh silica gel (produced by Qingdao Ocean Chemical Plant).

[0045] Example 1 N-(4-bromobenzyl)-2-oxoindoline-6-carboxamide (Compound ZQMT1)

[0046]

[0047] like Figure 1 As shown, 200 mg (1.5 equivalents) of 2-oxoindoline-6-carboxylic acid was added to a round-bottom flask (50 ml) containing N, N-dimethylformamide (DMF) (8 ml), and 362.48 mg (1.5 equivalents) of O-(benzotriazole-1-yl)-N, N, N′, N′-tetramethyluronium tetrafluoroborate (TBTU) and 291.82 mg (3 equivalents) of N, N-diisopropylethylamine (DIPEA) were added, and stirred at room temperature. After 20 minutes, 140.03 mg (1 equivalent) of p-bromobenzylamine was added. After 4 hours, the mixture was extracted with water and ethyl acetate. The ethyl acetate layer was concentrated and purified by flash column chromatography to obtain 77.94 mg of a white product with a yield of 30%. 1 H NMR(400MHz, DMSO-d6)δ10.55(s,1H),9.03(t,J=6.0Hz,1H),7.54–7.50(m,2H),7 .48(dd,J=7.7,1.7Hz,1H),7.35–7.19(m,4H),4.42(d,J=5.9Hz,2H),3.53(s,2H). 13C NMR (101MHz, DMSO) δ176.8,166.6,144.4,139.7,134.2,131.6,129.9,129.9,124.5,120.8,120.2,108.3,42.5,36.3.HR-ESI-MS:367.0052[M+NA] + ,(calcd for C 16 H 13 N2O2NaBr,367.0058).

[0048] Example 2 N-(4-chlorobenzyl)-2-oxoindoline-6-carboxamide (Compound ZQMT2)

[0049]

[0050] like Figure 1 As shown, p-bromobenzylamine replaced p-chlorobenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 1 to obtain compound ZQMT2. 88 mg of white product, yield 38.9%. 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 9.03 (t, J = 6.0 Hz, 1H), 7.48 (dd, J = 7.6, 1.6 Hz, 1H), 7.42-7.36 (m, 2H), 7.32 (d, J = 8.5 Hz, 2H), 7.31-7.25 (m, 2H), 4.44 (d, J = 5.9 Hz, 2H), 3.53 (s, 2H). 13C NMR(101MHz,DMSO)δ176.8,166.6,144.4,139.3,129.9,129.6,128.7,124.5,120.8,108.3,42.5,36.3.HR-ESI-MS:323.0564[M+Na]+,(calcd for C 16 H 13 N2O2NaCl,323.0563).

[0051] Example 3 N-(4-fluorobenzyl)-2-oxoindoline-6-carboxamide (Compound ZQMT3)

[0052]

[0053] like Figure 1As shown, 200 mg (1.5 equivalents) of 2-oxoindoline-6-carboxylic acid was added to a round-bottom flask (50 ml) containing DMF, 291.82 mg (3 equivalents) of DIPEA was added, and the mixture was stirred at room temperature. 291.82 mg (3 equivalents) of DIPEA, 152.55 mg (1.5 equivalents) of 1-hydroxybenzotriazole monohydrate (HOBT) and 216.42 mg (1.5 equivalents) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) were added, and the mixture was stirred at room temperature. After 20 min, 94.19 mg (1 equivalent) of 4-fluorobenzylamine was added. After 6 h, the mixture was extracted with water and ethyl acetate. The ethyl acetate layer was concentrated and purified by flash column chromatography to obtain 79 mg of a yellow product with a yield of 36.9%. 1 H NMR (400MHz, DMSO-d6) δ10.54(s,1H),9.00(t,J=6.0Hz,1H),7.48(dd,J=7.7,1.6Hz,1H),7 .39–7.31(m,2H),7.31–7.24(m,2H),7.20–7.08(m,2H),4.43(d,J=5.9Hz,2H),3.53(s,2H). 13 C NMR (101MHz, DMSO) δ176.8,166.6,162.8,160.4,144.3,134.3,129.7,129.6,12 4.5,120.8,115.6,115.3,108.3,56.5,42.4,36.3.HR-ESI-MS:307.0862[M+Na] + ,(calcd for C 16 H 13 N2O2NaF,307.0859).

[0054] Example 4 N-(4-methylbenzyl)-2-oxoindoline-6-carboxamide (Compound ZQMT4)

[0055]

[0056] like Figure 1As shown, p-bromobenzylamine replaced p-methylbenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 1 to obtain compound ZQMT4. 67 mg of pink-white product, yield 25.4%. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 8.95 (t, J = 6.0 Hz, 1H), 7.48 (dd, J = 7.7, 1.6 Hz, 1H), 7.34-7.24 (m, 2H), 7.19 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 8.0 Hz, 2H), 4.41 (d, J = 5.9 Hz, 2H), 3.52 (d, J = 1 .1Hz,2H),2.27(s,3H).13CNMR(101MHz,DMSO)δ176.8,166.5,144.3,137.2,136.2,134.5,129 .7,129.3,127.7,124.5,120.8,108.4,42.8,36.3,21.1.HR-ESI-MS:303.1109[M+Na]+,(calcd for C 17 H 16 N2O2Na,303.1109).

[0057] Example 5 N-(4-methoxybenzyl)-2-oxoindoline-6-carboxamide (Compound ZQMT5)

[0058]

[0059] like Figure 1 As shown, p-bromobenzylamine replaced p-methoxybenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 1 to obtain compound ZQMT5. 276 mg of yellow product, yield 46.2%. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 8.93 (t, J = 6.0 Hz, 1H), 7.47 (dd, J = 7.7, 1.6 Hz, 1H), 7.32-7.07 (m, 4H), 6.99-6.79 (m, 2H), 4.38 (d, J = 5.9 Hz, 2H), 3.72 (s, 3H), 3.52 (s, 2H). 13C NMR(101MHz,DMSO)δ176.8,166.4,158.6,144.3,134.5,132.2,129.0,124.5,120.8,114.1,108.3,55.5,42.5,36.3.HR-ESI-MS:319.1060[M+Na]+,(calcd for C 17 H 16 N2O3Na,319.1059).

[0060] Example 6 2-Oxo-N-(4-(trifluoromethoxy)benzyl)indole-6-carboxamide (Compound ZQMT6)

[0061]

[0062] like Figure 1 As shown, p-bromobenzylamine replaced p-trifluoromethoxybenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 1 to obtain compound ZQMT6. 86 mg of white product, yield 29.1%. 1 H NMR (400MHz, DMSO-d6) δ10.55(s,1H),9.06(t,J=6.0Hz,1H),7.50(dd,J=7.7,1.6 Hz,1H),7.46–7.40(m,2H),7.34–7.26(m,4H),4.48(d,J=5.9Hz,2H),3.53(s,2H). 13 C NMR (101MHz, DMSO) δ176.8,166.6,144.4,139.7,134.2,129.9,129.5,124.5,121.4,120.8,108.3,42.5,36.3.HR-ESI-MS:373.0768[M+Na] + ,(calcd for C 17 H 13 N2O3NaF3,373.0776).

[0063] Example 7 2-Oxo-N-(4-(trifluoromethyl)benzyl)indole-6-carboxamide (Compound ZQMT7)

[0064]

[0065] like Figure 1 As shown, 4-fluorobenzylamine replaced trifluoromethylbenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 3 to obtain compound ZQMT7. 120 mg of light yellow product, yield 47.7%. 1 H NMR (400MHz, DMSO-d6) δ9.74 (s, 1H), 8.29 (t, J = 6.0Hz, 1H), 6.88 (d, J = 8.1Hz, 2H),6.80–6.58(m,3H),6.58–6.35(m,2H),3.72(d,J=5.8Hz,2H),2.72(s,2H). 13C NMR(101MHz,DMSO)δ176.8,166.7,145.1,144.4,134.1,130.0,128.3,125.7,12 5.7,125.6,125.6,124.6,120.8,108.3,42.8,36.3.HR-ESI-MS:357.0822[M+Na] + ,(calcd for C 17 H 13 N2O2NaF3,357.0827).

[0066] Example 8 N-(4-bromobenzyl)-2-oxoindoline-5-carboxamide (Compound ZQMT8)

[0067]

[0068] like Figure 1 As shown, 2-oxoindoline-6-carboxylic acid was replaced by 2-oxoindoline-5-carboxylic acid, and other conditions remained unchanged. The synthesis method was the same as in Example 1 to obtain compound ZQMT8. 59 mg of white product, yield 22.7%. 1 H NMR (400MHz, DMSO-d6) δ10.62(s,1H),8.90(t,J=6.0Hz,1H),7.77(d,J=7.6Hz,2H),7.59 –7.43(m,2H),7.33–7.20(m,2H),6.94–6.77(m,1H),4.41(d,J=5.9Hz,2H),3.53(s,2H). 13 C NMR (101MHz, DMSO) δ177.1,166.5,147.0,139.9,131.6,129.9,128.0,127.6,126.2,123.9,120.1,109.0,42.5,36.1.HR-ESI-MS:367.0058[M+Na] + ,(calcd for C 16 H 13 N2O2NaBr,367.0058).

[0069] Example 9 N-(4-chlorobenzyl)-2-oxoindoline-5-carboxamide (Compound ZQMT9)

[0070]

[0071] like Figure 1 As shown, p-bromobenzylamine was replaced by p-chlorobenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 8 to obtain compound ZQMT9. 79 mg of white product was obtained with a yield of 27.9%.1 H NMR (400MHz, DMSO-d6) δ10.63(s,1H),8.90(t,J=6.0Hz,1H),7.77(d,J=7.7Hz,2H),7.37(d,J =8.5Hz,2H),7.32(d,J=8.5Hz,2H),6.86(d,J=8.0Hz,1H),4.43(d,J=5.9Hz,2H),3.53(s,2H). 13 C NMR (101MHz, DMSO) δ177.1,166.5,147.0,139.5,131.7,129.5,128.7,128.0,127.6,126.2,123.9,109.0,42.4,36.1.HR-ESI-MS:323.0563[M+Na] + ,(calcd for C 16 H 13 N2O2NaCl,323.0563).

[0072] Example 10 N-(4-fluorobenzyl)-2-oxoindoline-5-carboxamide (Compound ZQMT10)

[0073]

[0074] like Figure 1 As shown, p-bromobenzylamine was replaced by p-fluorobenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 8 to obtain compound ZQMT10. 89 mg of white product was obtained with a yield of 33.3%. 1 H NMR (400MHz, DMSO-d6) δ10.62(s,1H),8.88(t,J=6.0Hz,1H),7.77(d,J=7.6Hz,2H),7.39 –7.23(m,2H),7.22–7.03(m,2H),6.99–6.75(m,1H),4.43(d,J=5.9Hz,2H),3.53(s,2H). 13 C NMR (101 MHz, DMSO)δ

[0075] 177.1,166.5,162.8,160.4,147.0,136.6,136.6,129.7,129.6,128.0,127.7 ,126.2,124.0,115.5,115.3,109.0,42.4,36.1.HR-ESI-MS:307.0862[M+Na] + ,(calcd forC 16 H 13N2O2FNa,307.0859).

[0076] Example 11 N-(4-methylbenzyl)-2-oxoindoline-5-carboxamide (Compound ZQMT11)

[0077]

[0078] like Figure 1 As shown, p-bromobenzylamine was replaced with p-methylbenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 8 to obtain compound ZQMT11. 85 mg of a pinkish white product was obtained, with a yield of 32.3%. 1 H NMR (400MHz, DMSO-d6) δ10.61(s,1H),8.82(t,J=6.0Hz,1H),7.77(d,J=7.3Hz,2H),7.18(d,J=8. 0Hz, 2H), 7.14–7.09 (m, 2H), 6.88–6.82 (m, 1H), 4.40 (d, J = 5.9Hz, 2H), 3.53 (s, 2H), 2.26 (s, 3H). 13 C NMR(101MHz,DMSO)δ177.1,166.4,146.9,137.4,136.1,129.2,129.1,128.0,12 7.9,127.6,126.1,123.9,108.9,42.8,36.1,21.1.HR-ESI-MS:303.1113[M+Na] + ,(calcd for C 17 H 16 N2O2Na,303.1109).

[0079] Example 12 N-(4-methoxybenzyl)-2-oxoindoline-5-carboxamide (Compound ZQMT12)

[0080]

[0081] like Figure 1 As shown, p-bromobenzylamine was replaced with p-methoxybenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 8 to obtain compound ZQMT12. 77 mg of pink product was obtained with a yield of 27.7%. 1 H NMR (400MHz, DMSO-d6) δ10.61(s,1H),8.80(t,J=6.0Hz,1H),7.76(d,J=7.4Hz,2H),7.28–7.15(m,2H),6.95–

[0082] 6.78(m,3H),4.38(d,J=5.9Hz,2H),3.72(s,3H),3.53(s,2H). 13 C NMR(101MHz,DMSO)δ177.1,166.3,158.6,146.9,132.4,129.0,128.0,128.0 ,126.1,124.0,114.1,108.9,55.5,42.5,36.1.HR-ESI-MS:319.1057[M+Na] + ,(calcd for C 17 H 16 N2O3Na,319.1059).

[0083] Example 13 2-Oxo-N-(4-(trifluoromethoxy)benzyl)indoline-5-carboxamide (Compound ZQMT13)

[0084]

[0085] like Figure 1 As shown, p-bromobenzylamine was replaced with p-trifluoromethoxybenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 8 to obtain compound ZQMT13. 127 mg of yellow-white product was obtained, with a yield of 38.6%. 1 H NMR (400MHz, DMSO-d6) δ10.63(s,1H),8.92(s,1H),7.78(d,J=7.7Hz,2H),7.47–7.3 9(m,2H),7.35–7.26(m,2H),6.93–6.75(m,1H),4.47(d,J=5.9Hz,2H),3.54(s,2H). 13 C NMR (101MHz, DMSO) δ177.1,166.5,147.6,147.0,139.9,129.5,128.0,127.6,126.2,124.0,121.4,109.0,42.4,36.1.HR-ESI-MS:373.0771[M+Na] + ,(calcd for C 17 H 13 N2O3NaF3,373.0776).

[0086] Example 14 2-Oxo-N-(4-(trifluoromethyl)benzyl)indoline-5-carboxamide (Compound ZQMT14)

[0087]

[0088] like Figure 1As shown, p-bromobenzylamine was replaced with p-trifluoromethylbenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 8 to obtain compound ZQMT14. 76 mg of yellow product was obtained with a yield of 30.3%. 1 H NMR (400MHz, DMSO-d6) δ10.63(s,1H),8.98(t,J=6.0Hz,1H),7.78(d,J=7.7Hz,2H),7.69(d,J=8.0 Hz,2H),7.51(d,J=8.0Hz,2H),6.90–6.84(m,1H),4.53(d,J=5.8Hz,2H),3.54(s,2H),3.32(s,3H). 13 C NMR(101MHz,DMSO)δ177.1,166.6,147.1,145.3,128.3,128.0,127.7,127.5 ,126.2,125.6,125.6,124.0,109.0,42.8,36.1.HR-ESI-MS:357.0822[M+Na] + ,(calcd for C 17 H 13 N2O2NaF3,357.0827).

[0089] Example 15 N-(4-bromobenzyl)-2-oxoindoline-4-carboxamide (Compound ZQMT15)

[0090]

[0091] like Figure 1 As shown, 2-oxoindoline-6-carboxylic acid was replaced by 2-indolone-4-carboxylic acid, and other conditions remained unchanged. The synthesis method was the same as in Example 1 to obtain compound ZQMT15. 78 mg of yellow product was obtained with a yield of 30.11%. 1 H NMR(400MHz,DMSO-d6)δ10.49(s,1H),8.88(t,J=6.0Hz,1H),7.66–7.41(m,2H),7 .41–7.19(m,4H),6.94(dd,J=7.6,1.1Hz,1H),4.41(d,J=5.9Hz,2H),3.66(s,2H). 13 C NMR (101MHz, DMSO) δ176.8,166.8,145.0,139.5,131.6,129.9,128.1,125.9,120.2,120.1,111.8,42.4,37.1.HR-ESI-MS:367.0061[M+Na] + ,(calcd for C 16H 13 N2O2NaBr,367.0058).

[0092] Example 16 N-(4-chlorobenzyl)-2-oxoindoline-4-carboxamide (Compound ZQMT16)

[0093]

[0094] like Figure 1 As shown, p-bromobenzylamine was replaced by p-chlorobenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 15 to obtain compound ZQMT16. 69 mg of light yellow product was obtained, with a yield of 20.35%. 1 H NMR (400MHz, DMSO-d6) δ10.49(s,1H),8.88(t,J=6.0Hz,1H),7.50–7.15(m,6H),6.94(dd,J=7.6,1.1Hz,1H),4.43(d,J=5.9Hz,2H),3.66(s,2H). 13 C NMR (101MHz, DMSO) δ176.8,166.8,145.0,139.1,131.8,131.3,129.6,128.7,128.1,125.9,120.1,111.8,42.3,37.1.HR-ESI-MS:323.0559[M+Na] + ,(calcd forC 16 H 13 N2O2NaCl,323.0563).

[0095] Example 17 N-(4-fluorobenzyl)-2-oxoindoline-4-carboxamide (Compound ZQMT17)

[0096]

[0097] like Figure 1 As shown, p-bromobenzylamine was replaced with p-fluorobenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 15 to obtain compound ZQMT17. 156 mg of pink product was obtained, with a yield of 73%. 1 H NMR (400MHz, DMSO-d6) δ10.47(d,J=13.5Hz,1H),8.97–8.75(m,1H),7.39–7.33(m,2H),7.32(d,J=1.2Hz,1H ),7.27(t,J=7.8Hz,1H),7.19–7.11(m,2H),6.94(dd,J=7.7,1.1Hz,1H),4.43(d,J=5.9Hz,2H),3.66(s,2H). 13CNMR(101MHz,DMSO)δ176.8,166.8,145.0,136.2,136.2,129.8,129.7,129.6,128.1,111.8,42.2,40.7,40.6,37.1.HR-ESI-MS:307.0859[M+Na] + ,(calcd for C 16 H 13 N2O2FNa,307.0859).

[0098] Example 18 N-(4-methylbenzyl)-2-oxoindoline-4-carboxamide (Compound ZQMT18)

[0099]

[0100] like Figure 1 As shown, p-bromobenzylamine was replaced with p-methylbenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 15 to obtain compound ZQMT18. 126 mg of yellow product was obtained with a yield of 47.9%. 1 H NMR (400MHz, DMSO-d6) δ10.48(s,1H),8.81(t,J=6.0Hz,1H),7.32(dd,J=8.0,1.2Hz,1H),7.26(t,J=7.8Hz,1H),7.21 (d,J=8.0Hz,2H),7.13(d,J=7.9Hz,2H),6.93(dd,J=7.6,1.1Hz,1H),4.40(d,J=6.0Hz,2H),3.65(s,2H),2.27(s,3H). 13 C NMR (101MHz, DMSO) δ176.84,166.71,144.93,137.01,136.22,131.55,129.30,128.06,127.67,125.82,120.10,111.67,42.64,37.12,21.14.

[0101] HR-ESI-MS:303.1111[M+Na] + ,(calcd for C 17 H 16 N2O2Na,303.1109).

[0102] Example 19 N-(4-methoxybenzyl)-2-oxoindoline-4-carboxamide (Compound ZQMT19)

[0103]

[0104] like Figure 1 As shown, p-bromobenzylamine was replaced with p-methoxybenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 15 to obtain compound ZQMT19. 174 mg of yellow product was obtained with a yield of 62.6%. 1 H NMR (400MHz, DMSO-d6) δ10.48(s,1H),8.79(t,J=6.0Hz,1H),7.33–7.20(m,4H),6.95–6.84(m,3H),4.38(d,J=5.9Hz,2H),3.73(s,3H),3.65(s,2H). 13 C NMR (101MHz, DMSO) δ176.8,166.7,158.7,144.9,132.0,131.6,129.0,128.1 ,125.8,120.1,114.2,111.7,55.5,42.3,37.1.HR-ESI-MS:319.1063[M+Na] + ,(calcd for C 17 H 16 N2O3Na,319.1059).

[0105] Example 20 2-Oxo-N-(4-(trifluoromethoxy)benzyl)indoline-4-carboxamide (Compound ZQMT20)

[0106]

[0107] like Figure 1 As shown, p-bromobenzylamine was replaced with p-trifluoromethoxybenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 15 to obtain compound ZQMT20. 63 mg of white product was obtained with a yield of 24%. 1 H NMR (400MHz, DMSO-d6) δ10.49(s,1H),8.90(t,J=6.0Hz,1H),7.55–7.40(m,2H),7.33(ddd,J=7.7,4. 2,1.9Hz,3H),7.27(t,J=7.8Hz,1H),6.95(dd,J=7.6,1.1Hz,1H),4.47(d,J=5.9Hz,2H),3.67(s,2H). 13 C NMR (101MHz, DMSO) δ176.8,166.9,147.6,145.0,139.6,131.3,129.5,128.1,125.9,121.4,120.1,111.8,42.3,37.1.HR-ESI-MS:373.0775[M+Na] + ,(calcd for C 17H 13 N2O3NaF3,373.0776).

[0108] Example 21 2-Oxo-N-(4-(trifluoromethyl)benzyl)indoline-4-carboxamide (Compound ZQMT21)

[0109]

[0110] like Figure 1 As shown, p-bromobenzylamine was replaced with p-trifluoromethylbenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 15 to obtain compound ZQMT21. 124 mg of white product was obtained, with a yield of 39.5%. 1 H NMR (400MHz, DMSO-d6) δ10.50(s,1H),8.96(t,J=6.0Hz,1H),7.70(d,J=8.1Hz,2H),7.54(d,J=8.0Hz,2H),7.35 (dd,J=8.0,1.1Hz,1H),7.29(d,J=7.7Hz,1H),6.95(dd,J=7.6,1.0Hz,1H),4.54(d,J=5.9Hz,2H),3.67(s,2H). 13 C NMR(101MHz,DMSO)δ176.8,166.9,145.0,145.0,131.2,128.3,128.1,128.1,126.0 ,125.7,125.7,125.7,125.6,120.1,111.9,42.6,37.1.HR-ESI-MS:357.0821[M+Na] + ,(calcd for C 17 H 13 N2O2NaF3,357.0827).

[0111] Example 22 N-(4-chlorobenzyl)-2-oxoindoline-7-carboxamide (Compound ZQMT22)

[0112]

[0113] like Figure 1 As shown, 2-oxoindoline-6-carboxylic acid was replaced with 2-indolone-7-carboxylic acid, and other conditions remained unchanged. The synthesis method was the same as in Example 2 to obtain compound ZQMT22. 179 mg of light yellow product was obtained, with a yield of 52.8%. 1H NMR (400MHz, DMSO-d6) δ9.87 (s, 1H), 9.15 (t, J = 6.0Hz, 1H), 7.71 (dd, J = 8.2, 1.1Hz, 1H ),7.46–7.23(m,5H),7.03(dd,J=8.1,7.3Hz,1H),4.47(d,J=5.9Hz,2H),3.53(s,2H). 13 C NMR (101MHz, DMSO) δ176.2,166.5,144.3,139.0,131.8,129.6,128.7,127.8,127.7,125.7,121.3,114.9,42.2,35.3.HR-ESI-MS:323.0559[M+Na] + ,(calcd for C 16 H 13 N2O2NaCl,323.0563).

[0114] Example 23 2-Oxo-N-(4-(trifluoromethoxy)benzyl)indole-7-carboxamide (Compound ZQMT23)

[0115]

[0116] like Figure 1 As shown, p-chlorobenzylamine was replaced with p-trifluoromethoxybenzylamine, and other conditions remained unchanged. The synthesis method was the same as Example 22 to obtain compound ZQMT23. 77 mg of white product was obtained with a yield of 23.4%. 1 H NMR(400MHz, DMSO-d6)δ9.88(s,1H),9.17(t,J=6.0Hz,1H),7.72(dd,J=8.1,1.1Hz,1H),7.52–7 .40(m,2H),7.41–7.24(m,3H),7.03(dd,J=8.1,7.3Hz,1H),4.51(d,J=5.9Hz,2H),3.53(s,2H). 13 C NMR (101MHz, DMSO) δ176.2,166.5,144.3,139.5,129.5,127.8,127.7,125.7,121.5,121.3,114.9,42.2,35.3.HR-ESI-MS:373.0775[M+Na] + ,(calcd for C 17 H 13 N2O3NaF3,373.0776).

[0117] Example 24 N-(4-methylbenzyl)-2,3-dioxoindoline-7-carboxamide (Compound ZQMT24)

[0118]

[0119] like Figure 1 As shown, 2-oxoindoline-6-carboxylic acid was replaced with 2,3-dioxoindoline-7-carboxylic acid, and other conditions remained unchanged. The synthesis method was the same as in Example 4 to obtain compound ZQMT24. 96 mg of white product was obtained with a yield of 47%. 1 H NMR (400MHz, DMSO-d6) δ10.60(s,1H),9.22(t,J=6.0Hz,1H),8.01(dd,J=8.1,1.2Hz,1H),7.66(dd ,J=7.4,1.2Hz,1H),7.23(d,J=7.7Hz,2H),7.18–7.09(m,3H),4.45(d,J=5.8Hz,2H),2.28(s,3H). 13 C NMR (101MHz, DMSO) δ183.9,165.2,159.4,150.4,136.6,136.4,135.7,129.4,12 9.3,127.8,127.5,122.6,119.5,117.7,42.7,21.1.HR-ESI-MS:317.0896[M+Na] + ,(calcd forC 17 H 14 N2O3Na,317.0902).

[0120] Example 25 N-(4-methoxybenzyl)-2,3-dioxoindoline-7-carboxamide (Compound ZQMT25)

[0121]

[0122] like Figure 1 As shown, p-methylbenzylamine was replaced with p-methoxybenzylamine, and other conditions remained unchanged. The synthesis method was the same as Example 24 to obtain compound ZQMT25. 97 mg of light yellow product, yield 30%. 1 H NMR (400MHz, DMSO-d6) δ10.61(s,1H),9.19(s,1H),8.00(dd,J=8.0,1.3Hz,1H),7.65(s,1H),7.2 7(d,J=8.6Hz,2H),7.15(t,J=7.7Hz,1H),6.95–6.86(m,2H),4.42(d,J=5.8Hz,2H),3.73(s,3H). 13C NMR (101MHz, DMSO) δ183.9,165.2,159.8,158.8,150.4,135.7,131.6,129.2,12 7.4,122.6,119.5,117.8,114.3,114.2,55.5,42.4.HR-ESI-MS:333.0846[M+Na] + ,(calcd for C 17 H 14 N2O4Na,333.0851).

[0123] Example 26 2,3-Dioxo-N-(4-(trifluoromethoxy)benzyl)indole-7-carboxamide (Compound ZQMT26)

[0124]

[0125] like Figure 1 As shown, p-methylbenzylamine was replaced with p-trifluoromethoxybenzylamine, and other conditions remained unchanged. The synthesis method was the same as Example 24 to obtain compound ZQMT26. 95 mg of white product was obtained with a yield of 31%. 1 H NMR (400MHz, DMSO-d6) δ10.62(s,1H),9.30(t,J=5.9Hz,1H),8.02(dd,J=8.1,1.3Hz,1H),7.67(dd,J=7.4 ,1.2Hz,1H),7.49–7.44(m,2H),7.33(dt,J=7.6,1.0Hz,2H),7.16(t,J=7.7Hz,1H),4.52(d,J=5.8Hz,2H). 13 C NMR (101MHz, DMSO) δ183.9,165.4,159.4,150.4,147.7,139.2,135.7,129.7, 129.5,127.6,122.6,121.5,119.5,117.6,42.3.HR-ESI-MS:387.0571[M+Na] + ,(calcd forC 17 H 11 N2O4NaF3,387.0569).

[0126] Example 27: Evaluation of DRG Cytotoxicity

[0127] The DRG cytotoxicity of the compounds was evaluated by MTT assay. The cells were seeded in 96-well plates (8000 cells / well, 100 μL cell suspension / well) and allowed to adhere overnight. The cells were then exposed to different concentrations (100 μM, 33.3 μM, 11.1 μM, 3.7 μM, 1.23 μM, 0.411 μM) of compound ZQMT1-26 for 24 h (100 μL of different concentrations of drug / well) cell suspension / well. MTT solution (20 μL, 0.5 mg / mL) was added to each well, and the cells were incubated with MTT at 37 °C for 4 h. After aspirating the supernatant, DMSO (150 μL) was added and the absorbance was read at 490 nm. All assays were repeated three times in parallel. Figure 2 As shown, there was no significant difference between the maximum concentration of most compounds and the blank group, and the administration concentration within this range did not affect cell viability.

[0128] Example 28: Primary screening of activity by calcium influx assay using DRG cells

[0129] The purpose is to detect whether the compound ZQMT1-26 has an inhibitory effect on TRPA1 channels. TRPA1 channels are cation channels that mainly transmit Ca 2+ The binding of the inhibitor to the TRPA1 channel causes the channel to close, allowing Ca 2+ The smaller the change in intracellular calcium ion fluorescence before and after the addition of the agonist, the stronger the inhibitory effect.

[0130] Cells were plated one day in advance, and the old culture medium was removed on the next day. The cells were washed twice with PBS, and the calcium ion probe Flou 4 was added and incubated for 30 min. The calcium ion probe Flou 4 was removed and the cells were washed twice with PBS. The compound was added to each well at a final concentration of 10 μM and incubated in a 37°C incubator for 60 min. The cells were first detected using a fluorescence microplate reader and the fluorescence intensity value was output. Then, 100 μl of the agonist AITC (final concentration 200 μM / well) was added to each well for stimulation and the fluorescence intensity value was output again using a microplate reader.

[0131] Fluorescence intensity change rate = data processing is fluorescence intensity after AITC stimulation / fluorescence intensity before AITC stimulation.

[0132] Depend on Figure 3 It can be seen that at the same concentration (10 μM), compared with the blank group, HC-030031, compounds ZQMT-7, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 33, 23, 24, 25, and 26 have significant differences, indicating that these combined compounds have an inhibitory effect on inhibiting TRPA1 activation caused by AITC in DRG cells.

[0133] Example 29: Extracting primary DRG cells and measuring the inhibitory effects of different compounds by calcium influx fluorescence

[0134] 1. Extract primary DRG cells;

[0135] 2. The MTT assay was used to evaluate whether compound ZQMT1-26 has primary DRG cytotoxicity. Cells were seeded in 96-well plates (8000 cells / well, 100 μL cell suspension / well) and allowed to adhere overnight. The cells were then exposed to compounds at a concentration of 10 μM (100 μL / well) for 24 h. MTT solution (20 μL, 0.5 mg / mL) was added to each well, and the cells were incubated with MTT at 37°C in the dark for 4 h. After aspirating the supernatant, DMSO (150 μL) was added and the absorbance was read at 490 nm. All measurements were repeated three times in parallel. Figure 4 As shown, most of the compounds had no significant difference compared with the blank group, and this administration concentration did not affect cell viability.

[0136] 3. Immunofluorescence Experiment

[0137] Immunofluorescence experiments were used to determine the purity of the extracted primary DRG cells and the number of TRPA1 channels contained in the primary DRG cells.

[0138] (1) Take primary DRG cells in the logarithmic growth phase, inoculate them in a 6-well plate, make cell slides, and incubate them in a 37° incubator overnight;

[0139] (2) Discard the liquid in the wells, wash three times with PBS, fix the cells with 4% paraformaldehyde for 20 min, discard the paraformaldehyde, and wash three times with PBS;

[0140] (3) 0.5% TritonX was used for permeabilization for 10 min. After permeabilization, the liquid in the well was discarded and the well was washed three times with PBS.

[0141] (4) Block the cells with 10% goat serum for 30 min and discard the blocking solution;

[0142] (5) Add TRPA1 primary antibody and incubate overnight;

[0143] (6) The next day, recover the primary antibody after rewarming, and then wash three times with PBS;

[0144] (7) Add fluorescent secondary antibody, incubate at room temperature in the dark for 3 h, recover the secondary antibody, and wash three times with PBS;

[0145] (8) DAPI staining solution was used to counterstain the cell nucleus for 30 min, the liquid in the wells was discarded, and the wells were washed three times with PBS;

[0146] (9) Add anti-fluorescence quenching blocking solution to the slide, discard the water from the slide, gently invert it and place it on the slide, dry it at room temperature away from light, observe it under a microscope, use two kinds of light (green light and blue light) to take two pictures for one field of view; use Image J software to merge them together for observation, such as Figure 5 shown.

[0147] By calculating the fluorescence intensity, the following conclusions were drawn: 1. The cell purity of the extracted primary DRG cells was 95.3% (average fluorescence intensity of green fluorescence / average fluorescence intensity of blue fluorescence); 2. The primary DRG cells contained 91.2% TRPA1 channels (average fluorescence intensity of green fluorescence / average fluorescence intensity of blue fluorescence).

[0148] 4. Calcium ion fluorescence

[0149] Cells were plated one day in advance, and the old culture medium was removed on the second day. The cells were washed twice with PBS, and the calcium ion probe Flou 4 was added and incubated for 30 min. The calcium ion probe Flou 4 was removed and the cells were washed twice with PBS. A compound with a final concentration of 10 μM was added to each well and incubated in a 37°C incubator for 60 min. The cells were first detected using a fluorescence microplate reader and the fluorescence intensity value was output. Then, 100 μL of the agonist AITC (final concentration 200 μM / well) was added to each well for stimulation and the fluorescence intensity value was output again using a microplate reader.

[0150] Fluorescence intensity change rate = fluorescence intensity after AITC stimulation / fluorescence intensity before AITC stimulation.

[0151] Depend on Figure 6 It can be seen from the experimental results that at the same concentration (10 μM), compared with the blank group, HC-030031, compounds ZQMT3, 4, 5, 9, 10, 14, 15, 17, 19, and 20 have significant differences, indicating that these compounds have an inhibitory effect on inhibiting AITC-induced TRPA1 activation in primary DRG cells.

[0152] Example 30: Calcium influx assay using transfected HEK293T-TRPA1 cells (specifically expressing TRPA1) to determine the inhibitory effects of different compounds

[0153] The purpose is to detect the inhibitory effect of the combined compound ZQMT1-26. TRPA1 channel is a cation channel that mainly passes calcium ions. The binding of inhibitors to TRPA1 channels will cause the channels to close. If the change in intracellular calcium ion fluorescence before and after the addition of agonists is smaller, it means that the inhibitory effect is stronger.

[0154] 1. The cytotoxicity of compound ZQMT1-26 to transfected HEK293T-TRPA1 cells was evaluated by MTT assay. Cells were seeded in 96-well plates (8000 cells / well, 100 μL cell suspension / well) and allowed to adhere overnight. The cells were then exposed to compounds at a concentration of 10 μM (100 μL of different drugs / well) for 24 h. MTT solution (20 μL, 0.5 mg / mL) was added to each well, and the cells were incubated with MTT at 37 °C in the dark for 4 h. After aspirating the supernatant, DMSO (150 μL) was added and the absorbance was read at 490 nm. All measurements were repeated three times in parallel. Figure 7 As shown, most of the compounds had no significant difference compared with the blank group, and this administration concentration did not affect cell viability, so the subsequent experiments used a concentration of 10 μM.

[0155] 2. Calcium ion fluorescence experiment

[0156] The purpose is to detect whether the compound ZQMT1-26 has an inhibitory effect on TRPA1 channels. TRPA1 channels are cation channels that mainly transmit Ca 2+ The binding of the inhibitor to the TRPA1 channel causes the channel to close, allowing Ca 2+ The smaller the change in intracellular calcium ion fluorescence before and after the addition of the agonist, the stronger the inhibitory effect.

[0157] Cells were plated one day in advance, and the old culture medium was removed on the next day. The cells were washed twice with PBS, and the calcium ion probe Flou 4 was added and incubated for 30 minutes. The calcium ion probe Flou 4 was removed and the cells were washed twice with PBS. The compound with a final concentration of 10 μM was added to each well and incubated in a 37°C incubator for 60 minutes. The cells were first detected using a fluorescence microplate reader and the fluorescence intensity value was output. Then, 100 μl of the agonist AITC (final concentration 200 μM / well) was added to each well for stimulation and then the cells were detected again using a microplate reader and the fluorescence intensity value was output.

[0158] Fluorescence intensity change rate = fluorescence intensity after AITC stimulation / fluorescence intensity before AITC stimulation.

[0159] Depend on Figure 8 It can be seen that at the same concentration (10 μM), compared with the blank group, HC-030031, compounds 1, 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 15, 17, 22, 23, 25, and 26 have significant differences, indicating that these combined compounds have an inhibitory effect on inhibiting TRPA1 activation caused by AITC in transfected HEK293T-TRPA1 cells.

[0160] Example 31: Foot licking experiment

[0161] The goal is to determine whether the compound can reduce or inhibit the TRPA1 stimulatory effect caused by AITC. AITC is a selective agonist of TRPA1. Activating TRPA1 can cause adverse reactions such as itching in mice, which can cause foot licking and other phenomena. Inhibiting TRPA1 can reduce the frequency of such adverse reactions in mice or even prevent them from occurring.

[0162] The top ten compounds with good activity in the cell calcium influx test were selected for the paw licking test. The compounds were prepared (stock solution 10 mM), and 50 μL of the stock solution was added to 5 mL of normal saline for dilution, with a final concentration of 0.1 mM.

[0163] Healthy C57BL / 6J mice were randomly divided into blank group and drug-treated group with 10 mice in each group.

[0164] Blank group: physiological saline was first applied to the soles of the feet, and the agonist AITC was applied after the feet were dry for 3 minutes;

[0165] Drug-treated group: Apply the compound to the sole of the foot first, and then apply the agonist AITC after the foot is dry for 3 minutes;

[0166] A person who is unaware of the experiment is selected to observe and record the number of times the mice lick their paws and abnormal behaviors within 5 minutes.

[0167] like Fig. 9 As shown, all compounds reduced the number of paw licking in C57BL / 6J mice, but abnormal behaviors such as itching still occurred. After the application of compound ZQMT-10, C57BL / 6J mice had almost no abnormal behaviors such as itching, and the number of paw licking was the least, so the screened compound ZQMT-10 was the optimal compound.

[0168] The following experiments use compound ZQMT-10 as an example for illustration, and other compounds can still achieve similar technical effects as compound ZQMT-10.

[0169] Example 32: In vivo acute toxicity test

[0170] The purpose of the in vivo acute toxicity test is to determine whether the compound is toxic to animals. The up-down method was used to determine whether the compound ZQMT10 was toxic to mice. The test was performed using a single administration method.

[0171] The sodium carboxymethyl cellulose used to dissolve the compound was purchased from Tianjin Damao Chemical Reagent Factory.

[0172] C57BL / 6J mice, male, weighing 20±2g, were provided by Liaoning Changsheng Biotechnology Co., Ltd., and standard feed and bedding were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The temperature of the feeding room was 22±2℃, the relative humidity of the environment was 50%-60%, and the experimental environment was alternating between light and dark for 12h. Mice were free to take food and water, and the experiment was carried out after 7 days of adaptive feeding. All experimental operation procedures were in accordance with the "Regulations on the Administration of Experimental Animals (Revised in 2017)".

[0173] Five C57BL / 6J mice were fasted for 6 hours before administration and administered by gavage at a concentration of 2000 mg / kg (600 mg of the compound dissolved in 2 mL of sodium carboxymethyl cellulose, 400 μL was administered by gavage to each mouse). After fasting for 4 hours after administration, normal feeding was resumed. The mice were weighed and their weights and adverse reactions were recorded every day.

[0174] The body weight was recorded after 14 days. If there was no death or adverse reaction, it means that the compound was calculated as LD50. 50 >2000mg / kg.

[0175] On the fourteenth day, blood was drawn from the eyeballs of C57BL / 6J mice and the mice were killed. The heart, liver, spleen, lungs, and kidneys were removed for organ index statistics and HE staining was performed to observe whether the compounds had an effect on the pathological level of the internal organs.

[0176] like Fig.10 As shown, there was no significant fluctuation in the body weight of mice within 14 days, and there was no significant difference in organ index compared with the blank group. HE staining also showed that there was no pathological change in the heart, liver, spleen, lung and kidney of the drug group and the blank group.

[0177] Example 33: Cold Plate Experiment

[0178] The objective was to determine the effects of compounds on cold allodynia.

[0179] Grouping: Healthy C57BL / 6J mice were randomly divided into high, medium and low dose groups (50, 100, 200 mg / kg), a positive drug group (HC-030031—100 mg / kg), a blank group and a cold exposure blank group, with 12 mice in each group.

[0180] The mice were gavaged continuously for 14 days. Before the cold plate test, the C57BL / 6J mice were placed in a cold plate compartment at room temperature for 1 hour to adapt. 30 minutes after the last administration, the mice were placed on a -5°C cold plate. The number of licking feet, prolonged foot retraction and foot lifting within 5 minutes, and the time of the first occurrence of the above behaviors were recorded. The continuous behavior lasting 1 second was recorded as 1. The cold plate test was performed on C57BL / 6J mice on the 7th and 14th days of administration.

[0181] On the 7th day, the cold plate test Fig.11As shown in the figure, the cold plate test measured after 7 days of drug administration showed that the total number of licking and lifting of the paw, the total time and the time of the first abnormal behavior were significantly lower than those of the blank group. There were significant differences in all doses between the drug group and the positive drug group compared with the blank group, and the effect of the compound ZQMT10 at the drug concentration of 50mg / kg, 100mg / kg and 200mg / kg was higher than that of the positive drug group of HC-030031 at the drug concentration of 100mg / kg. However, except for the time of the first occurrence of harmful behavior, the other two indicators did not show dose dependence. As shown in the cold plate test on the 14th day, Fig.12 As shown in the figure, from the data measured after 14 days of administration, the compound ZQMT10 significantly improved the efficacy of the compound ZQMT10 after 14 days of administration compared with the compound ZQMT10 after 7 days of administration. The cold plate test showed that the total number of licking and lifting the foot, the total time and the time of the first abnormal behavior were significantly lower than those of the blank group. After 14 days of administration, all doses except the first time of nociceptive reaction in the 50mg / kg administration group and the positive drug group showed significant differences, and the three groups of measurement data showed dose dependence. The pharmacodynamic effects of the compound ZQMT10 at the concentrations of 50mg / kg, 100mg / kg and 200mg / kg were also higher than those of the HC-030031 positive drug group at the concentration of 100mg / kg. The ability of the compound ZQMT10 to improve contact cold foot pain increased with the increase of drug concentration. These results show that the compound ZQMT10 has a good effect in resisting pain caused by cold.

[0182] Example 34: Abdominal pain experiment

[0183] The aim is to determine the inhibitory effect of compounds on TRPA1 selective agonist (AITC)

[0184] Grouping: Healthy C57BL / 6J mice were randomly divided into high, medium and low dose groups (50, 100, 200 mg / kg), a positive drug group (HC-030031—100 mg / kg), a blank group and a cold exposure blank group, with 12 mice in each group.

[0185] The mice were gavaged continuously for 14 days. 30 minutes after the last day of gavage, 0.75% mustard oil (AITC) was used for enema (2.5 μL / g). The blank group was given an equal amount of saline for enema. A small amount of vaseline was applied to the perianal area of ​​the mice before enema to prevent the mustard oil from irritating the perianal area and causing discomfort. The enema depth was about 4 cm from the anal sphincter. After enema, the mice were kept upside down for 1 minute to prevent the leakage of mustard oil. The abdominal pain behaviors such as licking the abdomen, abdominal contraction and inward retraction, abdominal stretching, abdominal pressing the floor, body twisting, hind limb extension, etc. were observed within 10 minutes, and the time of the first occurrence of the above behaviors was observed. After the experiment, the mice were euthanized and the colon tissues were taken for HE staining.

[0186] like Fig.13As shown, oral administration of C57BL / 6J mice at different doses reduced the total time of abnormal behavior in the abdominal pain experiment compared with the model group, and delayed the time of the first appearance of abnormal behavior. Both reductions were dose-dependent. The drug efficacy was the best when the drug group was 200 mg / kg, but the total time of abnormal behavior in abdominal pain did not exceed that of the positive drug HC-030031 group with a drug concentration of 100 mg / kg. Pathological sections showed that the colon tissue morphology of the control group was normal. However, in the model group, there were obvious necrotic epithelial cells and intestinal gland cells in the mucosal layer of colon tissue, with darker nuclear staining; there were a large number of dilated intestinal glands in the lamina propria; there was edema and loosely arranged connective tissue in the submucosal layer. In the low-dose group, there were a small number of necrotic epithelial cells in the mucosal layer of the colon, local intestinal glands in the lamina propria were dilated, glandular cells were flat, there was a small amount of eosinophilic substances in the glandular cavity, and there was mild connective tissue hyperplasia around, and focal lymphocyte infiltration. The colon tissue morphology of the medium-dose group and high-dose group of compound ZQMT10 was normal. In the HC-030031 group, the morphology and structure of colonic tissue were normal, with slight focal lymphocyte infiltration; a small amount of hydrated degenerated muscle fibers could be seen in the muscle layer, indicating that compound ZQMT10 can improve the abdominal pain caused by mustard oil enema.

[0187] Example 35: von Fery test

[0188] The aim was to assess the mechanical nociceptive threshold (impact on tactile allodynia).

[0189] Grouping: Healthy C57BL / 6J mice were randomly divided into high, medium and low dose groups (50, 100, 200 mg / kg), a positive drug group (HC-030031—100 mg / kg), a blank group and a cold exposure blank group, with 12 mice in each group.

[0190] Gavage was continued for 14 days, and 20 μL CFA was injected into the sole of the foot 30 minutes after the last day of gavage. The threshold of painful mechanical stimulation was measured with von Frey filaments (stent). Mice were placed in acrylic cages on a wired grid floor for 15 minutes. The inoculated hind paws were then touched with a series of fibers with logarithmic increasing hardness (0.008-4g) until they slightly bent. A positive reaction was characterized by the sudden removal of the touched paw. The mechanical nociception threshold was calculated using the up-and-down method.

[0191] like Fig.14As shown in the figure, the mechanoreceptive threshold MDT of mice measured after continuous administration was significantly higher than that of the blank group. There were significant differences among the four administration groups except for the low-dose group with a concentration of 50 mg / kg on the 7th day, and there was a dose-dependency. With the increase of the administration concentration, the mechanoreceptive threshold MDT of mice increased. The drug efficacy was the best when the administration concentration was 200 mg / kg. However, at the same administration concentration (100 mg / kg), the mechanoreceptive threshold MDT of mice in the administration group was better than that of compound ZQMT10 after 7 days of administration compared with the positive drug group. Fig.15 As shown in the figure, at the same administration concentration (100 mg / kg), the administration group was superior to the positive drug group at 14 days of administration, indicating that the compound ZQMT-10 increased the mechanical perception threshold of mice.

[0192] On the 14th day of administration, 20 μL of CFA was injected into the sole of the foot 30 minutes after oral gavage. The threshold of painful mechanical stimulation caused by persistent inflammation was measured using von Frey filaments (scaffolds), and the changes in the mechanical perception threshold of C57BL / 6J mice were measured 0.5 h, 1 h, 3 h, 6 h, and 24 h after injection.

[0193] The results are as follows Fig.16 As shown in the figure, it can be seen that during the CFA-induced persistent inflammation, plantar injection of CFA caused long-term inflammatory abnormal pain in mice, such as a sharp drop in the nociceptive threshold. The threshold of pain mechanical stimulation of mice at 0.5h, 1h, 3h, and 6h was lower than that of the blank group, but higher than that of the model group. 24 hours after plantar injection of CFA, the compound ZQMT10 at a concentration of 200mg / kg and the positive drug HC-030031 at a concentration of 100mg / kg reversed the CFA-induced persistent inflammation and increased the level of mechanical receptive field.

[0194] Example 36: Thermal migration experiment

[0195] To elucidate the binding ability of ZQMT-10 to the TRPA1 target, we used the cell-based thermal shift assay (CETSA) to evaluate the affinity of the compound to the target, which is good at quantifying the binding efficiency of drugs to targets in cells. Fig.17 As shown, the compound ZQMT10 at a concentration of 10 μM has a stronger ability to bind to TRPA1 than DMSO, which indicates that the compound ZQMT10 can bind to TRPA1 well.

[0196] In summary, compound ZQMT1-26 can treat neuralgia, inflammatory pain, etc. In particular, compound ZQMT1-26 can block pain perception from the source of pain caused by cold, and this type of compound plays an important role in the preparation of drugs for preventing or treating pain caused by frostbite.

Claims

1. An oxindole compound or a pharmaceutically acceptable salt thereof, wherein the compound has the general structural formula shown in Formula I below: in: R1 is H or a carbonyl group, R2 is a substituent at the end of the side chain of the parent core, and R2 is selected from the following structures:

2. An oxindole compound of formula I or a pharmaceutically acceptable salt thereof, selected from:

3. A method for preparing an oxindole compound as claimed in claim 1 or 2, characterized in that: The steps are: The carboxylic acid compound is dissolved in DMF, a condensing agent is added, and then DIPEA is added, and the reaction is carried out at room temperature for 15-60 minutes. After the reaction, an amino compound is added, and the compound ZQMT1-26 is obtained after extraction and purification.

4. The preparation method according to claim 3, characterized in that: The carboxylic acid-containing compound is one of 2-oxoindoline-6-carboxylic acid, 2-oxoindoline-5-carboxylic acid, 2-indole-7-carboxylic acid, 2-indole-4-carboxylic acid, and 2,3-dioxoindoline-7-carboxylic acid; and the condensing agent is one or more of EDCI, HOBT, and TBTU.

5. The preparation method according to claim 3, characterized in that: The amino compound is one of p-methoxybenzylamine, 4-fluorobenzylamine, 4-methylbenzylamine, p-bromobenzylamine, p-trifluoromethylbenzylamine, 4-chlorobenzylamine and p-trifluoromethoxybenzylamine.

6. A pharmaceutical composition, characterized in that Contains the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

7. The pharmaceutical composition according to claim 6, characterized in that A preparation prepared by adding one or more pharmaceutically acceptable excipients to the compound as claimed in claim 1 or 2.

8. Use of the compound according to claim 1 or 2 in the preparation of a drug for treating pain.

9. Use of the compound as claimed in claim 1 or 2 in the preparation of a medicament for treating pain caused by frostbite.

10. Use of the compound according to claim 1 or 2 in the preparation of a TRPA1 inhibitor.

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