An oxindole derivative and its preparation method and application in TRPA1 inhibitors

Abstract

The present invention proposes a kind of oxindole derivative and its preparation method and application in the direction of TRPA1 inhibitor, and the general structural formula is shown in Formula I below;Compound ZQMT1-26 proposed in the present invention does not affect cell viability within the scope of administration, has high TRPA1 inhibitor activity, and reduces the number of licking feet of C57BL / 6J mice after TRPA1 agonist smearing, improves the ability of contact cold foot pain, reduces the total time of abnormal behavior in abdominal pain experiment, significantly improves the mechanical perception threshold MDT of mice, and compound ZQMT10 has strong binding ability with TRPA1. In summary, compound ZQMT1-26 can treat neuralgia, inflammatory pain, etc. In particular, compound ZQMT1-26 can block pain perception from the pain source caused by cold, and such compounds play an important role in preparing pain drugs for preventing or treating frostbite.

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 as a TRPA1 inhibitor. 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 acts as a sensor for a range of physical and chemical stimuli. Functional TRPA1 channels are tetramers formed by four identical subunits, each composed 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 stimulating the cysteine-rich region of the N-terminal ankyrin repeats through covalent modification. Upon channel activation, increased calcium flux leads to the release of neurotransmitters, inducing 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. Prolonged exposure of the body to cold and humid environments can cause local or systemic tissue damage, thereby causing 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 winner in Physiology or Medicine 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. The stimulation of harmful low temperatures 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] Over the past few years, several rodent knockout studies have linked TRPA1 to pain signaling. Because it can be activated by low temperatures (0-20°C) and mechanical stimulation, and is involved in a variety of physiological and pathological processes such as cold perception and pain, TRPA1 has been considered an attractive and highly promising drug target for the treatment of neuropathic pain. Inhibiting the opening and closing of TRPA1 channels can effectively reduce cold-induced pain, but few TRPA1 inhibitors have passed clinical trials.

[0005] Nowadays, the analgesics commonly used in clinical practice are still two major categories: 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, a preparation method thereof, and its application in the field 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. It 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 the general structural formula shown in Formula I below:

[0009]

[0010] Wherein: R1 is H or carbonyl, R2 is a substituent on 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 above-mentioned oxindole 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, and 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 forms of the preparation include 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 provides the use of the compound in the preparation of a medicament for treating pain caused by frostbite.

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

[0024] Beneficial effects:

[0025] The present invention proposes an oxindole derivative, its preparation method, and its application as a TRPA1 inhibitor. The oxindole derivative has a novel structure, with substituents introduced at positions 4, 5, 6, or 7 using an oxindole nucleus. Compound ZQMT1-26, developed by performing MTT cytotoxicity assays, calcium influx assays, paw licking assays, in vivo acute toxicity tests, cold plate assays, abdominal pain assays, von Fery assays, and thermal migration assays in DRG cells, primary DRG cells, and HEK-293T cells overexpressing TRPA1, exhibited high anti-TRPA1 inhibitory activity without affecting cell viability within the dosing range. Furthermore, the compound reduced paw licking frequency in C57BL / 6J mice, improved contact cold paw pain, decreased the total duration of abnormal behavior in an abdominal pain assay, and significantly increased the mechanoreceptor threshold (MDT) in mice. Furthermore, compound ZQMT10 exhibits excellent affinity for TRPA1. In summary, compound ZQMT1-26 is capable of treating neuropathic pain, inflammatory pain, and the like. In particular, compound ZQMT1-26 can block pain perception at the root of cold-induced pain. 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 This is 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 influx experiment of compound ZQMT1-26 in DRG cells;

[0029] Figure 4 This is the data 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 with 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 in HEK-293T cells overexpressing TRPA1;

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

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

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

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

[0038] Figure 13 Figure 1 is the result of the abdominal pain experiment in mice. A is the total time of abnormal abdominal pain behavior in mice, B is the time when abnormal behavior first appeared in the abdominal pain experiment in mice, and C is the HE staining of the colon after the end of the experiment.

[0039] Figure 14 The graph shows the results of the mechanical receptive field of C57BL / 6J mice measured on day 7 (without CFA-induced stimulation);

[0040] Figure 15 The graph shows the results of the mechanical receptive field of C57BL / 6J mice measured on day 14 (without CFA-induced stimulation);

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

[0042] Figure 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 cells after treatment with ZQMT10. DETAILED DESCRIPTION

[0043] The present invention will be described below with reference to specific examples, but the present invention is not limited to these examples.

[0044] The structures of the compounds were confirmed by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HR-MS). NMR measurements were performed using a Bruker AVANCE-300 / 600 NMR spectrometer in DMSO-d6 solvent with TMS as the internal standard. Column chromatography was performed using 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-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and 291.82 mg (3 equivalents) of N,N-diisopropylethylamine (DIPEA) were added. The mixture was stirred at room temperature, and 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 give 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 was substituted for p-chlorobenzylamine, and other conditions remained unchanged. Compound ZQMT2 was synthesized as in Example 1. 88 mg of the white product was obtained in a 38.9% yield. 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, and 291.82 mg (3 equivalents) of DIPEA was added. 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. The mixture was stirred at room temperature. After 20 minutes, 94.19 mg (1 equivalent) of 4-fluorobenzylamine was added. After 6 hours, the mixture was extracted with water and ethyl acetate. The ethyl acetate layer was concentrated and purified by flash column chromatography to give 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 was substituted for p-methylbenzylamine, and other conditions remained unchanged. The synthesis method was the same as in Example 1 to obtain compound ZQMT4. 67 mg of the white product was obtained with a yield of 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 was substituted for p-methoxybenzylamine, and other conditions remained unchanged. Compound ZQMT5 was synthesized as in Example 1. 276 mg of the yellow product was obtained in a 46.2% yield. 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 was substituted for 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 was obtained with a yield of 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 was substituted for p-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 was obtained with a yield of 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 with 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 was obtained with a yield of 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 with 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 with 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 the 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 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 with 2-indolone-4-carboxylic acid. Other conditions remained unchanged and 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 with 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. Other conditions remained unchanged and 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 was obtained, with a yield of 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 compound 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 24h (100 μL different concentration 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 4h. 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 2 As shown in the figure, the maximum concentrations of most compounds had no significant difference compared with the blank group, and the administration concentrations 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 test whether the compound ZQMT1-26 has an inhibitory effect on TRPA1 channel, which is a cation channel that mainly 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 second 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 was added to each well at a final concentration of 10 μM 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 fluorescence intensity value was detected again using a microplate reader.

[0131] Fluorescence intensity change rate = data processed as 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 the inhibitory effect of inhibiting TRPA1 activation caused by AITC in DRG cells.

[0133] Example 29: Extracting primary DRG cells and measuring the inhibitory effects of different compounds using 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 hours. 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 hours. 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) Primary DRG cells in the logarithmic growth phase were seeded in 6-well plates to prepare cell slides, and incubated in a 37°C incubator overnight;

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

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

[0141] (4) Block the cells with 10% goat serum for 30 minutes 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) Counterstain the cell nucleus with DAPI for 30 min, discard the liquid in the well, and wash three times with PBS;

[0146] (9) Add anti-fluorescence quenching blocking solution on the slide, discard the water from the slide, gently invert it on the slide, dry it at room temperature away from light, observe it under a microscope, use two kinds of light (green light, 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] Through fluorescence intensity calculation, 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 minutes. 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 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 fluorescence intensity value was detected 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 From the experimental results, it can be seen that at the same concentration (10 μM), compared with the blank group, HC-030031 and compounds ZQMT3, 4, 5, 9, 10, 14, 15, 17, 19, and 20 have significant differences, indicating that these compounds have the inhibitory effect of inhibiting TRPA1 activation caused by AITC 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 the inhibitor to the TRPA1 channel will cause the channel to close. The smaller the change in intracellular calcium ion fluorescence before and after the addition of the agonist, the stronger the inhibitory effect.

[0154] 1. The cytotoxicity of compound ZQMT1-26 to transfected HEK293T-TRPA1 cells 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 compounds at a concentration of 10 μM (100 μL different drugs / well) for 24 hours. 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 hours. 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. 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 test whether the compound ZQMT1-26 has an inhibitory effect on TRPA1 channel, which is a cation channel that mainly 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 second 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 was added to each well at a final concentration of 10 μM 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 fluorescence intensity value was detected again using a microplate reader.

[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 the inhibitory effect of inhibiting TRPA1 activation caused by AITC in transfected HEK293T-TRPA1 cells.

[0160] Example 31: Foot licking experiment

[0161] The goal was to determine whether compounds could reduce or inhibit AITC-induced TRPA1 stimulation. AITC is a selective agonist of TRPA1. Activating TRPA1 can cause adverse reactions in mice, such as itching, which can lead to paw licking. Inhibiting TRPA1 can reduce or even prevent these adverse reactions in mice.

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

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

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

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

[0166] A person who was unaware of the experiment was selected to observe and record the number of times the mice licked their feet and any abnormal behaviors within 5 minutes.

[0167] like Figure 9 As shown, all compounds reduced the number of paw licking in C57BL / 6J mice, but abnormal behaviors such as itching still occurred. After applying compound ZQMT-10, C57BL / 6J mice almost did not experience 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. Other compounds can still achieve similar technical effects as compound ZQMT-10.

[0169] Example 32: In vivo acute toxicity test

[0170] The purpose of in vivo acute toxicity testing is to determine whether a compound is toxic to animals. Compound ZQMT10 was tested for toxicity to mice using the up-down method. A single dose was used.

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

[0172] Male C57BL / 6J mice, weighing 20 ± 2 g, were provided by Liaoning Changsheng Biotechnology Co., Ltd. Standard feed and bedding were purchased from the same company. The housing temperature was 22 ± 2°C, the relative humidity was 50%–60%, and the experimental environment was a 12-hour light-dark cycle. Mice had free access to food and water, and experiments were conducted after acclimating for 7 days. All experimental procedures complied with the Regulations on the Administration of Laboratory Animals (revised in 2017).

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

[0174] Record the body weight after 14 days. If there is no death or adverse reaction of mice, it means that the compound is LD50. 50 >2000mg / kg.

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

[0176] like Figure 10 As shown, there was no significant fluctuation in the body weight of the mice over the 14-day period, and no significant differences in organ indexes were observed compared to the blank group. HE staining also showed no pathological changes in the heart, liver, spleen, lung, and kidney in either the drug-treated or 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] C57BL / 6J mice were gavaged for 14 consecutive days. Before the cold plate test, C57BL / 6J mice were placed in a cold plate compartment at room temperature for 1 hour. Thirty minutes after the last dose, the mice were placed on a -5°C cold plate. The number of paw licking, prolonged paw withdrawal, and paw lifting events within 5 minutes, as well as the time of the first occurrence of these behaviors, were recorded. A continuous behavior lasting 1 second was scored as 1. The cold plate test was performed on C57BL / 6J mice on days 7 and 14 of dosing.

[0181] On the 7th day, the cold plate experiment was as follows Figure 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. The effect of compound ZQMT10 at the drug concentration of 50 mg / kg, 100 mg / kg and 200 mg / kg was higher than that of the HC-030031 positive drug group at the drug concentration of 100 mg / 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 figure, the cold plate test on the 14th day showed Figure 12 As shown, data from 14 days of dosing indicate that compound ZQMT10 significantly improved its overall efficacy compared to 7 days of dosing. The cold plate test revealed that the total number of paw licking and lifting, total duration, and time to first onset of abnormal behavior were significantly reduced compared to the blank control group. At 14 days of dosing, all doses showed significant differences, except for the time to first onset of nociceptive reactions in the 50 mg / kg group and the positive drug group. Data from all three groups demonstrated dose-dependency. Compound ZQMT10 at concentrations of 50 mg / kg, 100 mg / kg, and 200 mg / kg all demonstrated greater efficacy than the 100 mg / kg HC-030031 positive drug group. Compound ZQMT10's ability to alleviate contact cold foot allergy increased with increasing drug concentration. These results demonstrate that compound ZQMT10 has a potent anti-cold pain effect.

[0182] Example 34: Abdominal pain experiment

[0183] The purpose 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. Thirty minutes after the final day of gavage, 0.75% mustard oil (AITC) was administered enema (2.5 μL / g). A control group received an equal volume of saline enema. Before enema, a small amount of petroleum jelly was applied to the perianal area of ​​the mice to prevent irritation caused by the mustard oil. The enema was administered approximately 4 cm from the anal sphincter. After enema, the mice were held in an inverted position for 1 minute to prevent leakage of the mustard oil. The mice were observed for any abdominal pain behaviors within 10 minutes, including licking the abdomen, abdominal contraction and inward retraction, abdominal stretching, pressing the abdomen against the floor, torso twisting, and hind limb extension. The time of the first occurrence of these behaviors was also observed. At the end of the experiment, the mice were euthanized, and colon tissue was collected for HE staining.

[0186] like Figure 13As shown, oral administration of different doses to C57BL / 6J mice reduced the total duration of abnormal behaviors during the abdominal pain test and delayed the onset of abnormal behaviors compared to the model group. Both reductions were dose-dependent, with the 200 mg / kg dose producing the most effective treatment. However, the total duration of abnormal behaviors did not exceed that of the 100 mg / kg dose of the active drug HC-030031. Pathological sections revealed normal colonic morphology in the control group. However, in the model group, colonic tissue showed marked necrotic epithelial cells and intestinal glandular cells with darker nuclear staining in the mucosal layer, numerous dilated intestinal glands in the lamina propria, and edema and loosely arranged connective tissue in the submucosal layer. In the low-dose group, a small number of necrotic epithelial cells were present in the mucosal layer, and localized intestinal glandular dilation in the lamina propria was accompanied by flattened glandular cells. A small amount of eosinophilic material was present in the glandular lumen, with mild connective tissue hyperplasia and focal lymphocytic infiltration. Colonic morphology was normal in the medium- and high-dose groups of the compound ZQMT10. In the HC-030031 group, colonic tissue morphology and structure were normal, with mild focal lymphocytic infiltration; a small amount of hydrated, degenerated muscle fibers were observed in the muscle layer, indicating that compound ZQMT10 can alleviate the abdominal pain induced by mustard oil enema.

[0187] Example 35: von Fery test

[0188] The aim was to assess mechanical nociception 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] The mice were gavaged continuously for 14 days. Thirty minutes after the last day of gavage, 20 μL of CFA was injected into the sole of the foot. The threshold for painful mechanical stimulation was measured using von Frey filaments (stents). Mice were placed in acrylic cages with a wired grid floor for 15 minutes. The inoculated hind paws were then contacted with a series of fibers with logarithmic increasing hardness (0.008-4g) until they slightly flexed. A positive reaction was characterized by the abrupt removal of the touched paw. The mechanical nociception threshold was calculated using the up-and-down method.

[0191] like Figure 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 drug-treated groups except for the low-dose group with a concentration of 50 mg / kg on the 7th day. The difference was dose-dependent. As the drug concentration increased, the mechanoreceptive threshold (MDT) of mice increased. The drug effect was best when the drug concentration was 200 mg / kg. However, at the same drug concentration (100 mg / kg), the drug-treated group was better than the positive drug group in improving the mechanoreceptive threshold (MDT) of mice after 7 days of drug administration than compound ZQMT10. Figure 15 As shown in the figure, at the same dosage concentration (100 mg / kg), the ZQMT-10 group was superior to the positive drug group at 14 days after 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 gavage. The threshold of painful mechanical stimulation caused by persistent inflammation was measured using von Frey filaments (stents). 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 Figure 16 As shown in the figure, during the CFA-induced persistent inflammation, intraplantar injection of CFA caused long-term inflammatory allodynia in mice, as evidenced by a sharp drop in the nociceptive threshold. At 0.5, 1, 3, and 6 hours, the thresholds for mechanical pain in mice were lower than those in the blank group, but higher than those in the model group. Twenty-four hours after intraplantar injection of CFA, administration of the compound ZQMT10 at a concentration of 200 mg / kg and the active drug HC-030031 at a concentration of 100 mg / kg reversed the CFA-induced persistent inflammation and increased the level of mechanoreceptive field.

[0194] Example 36: Thermal migration experiment

[0195] To elucidate the binding ability of ZQMT-10 to the TRPA1 target, we used the cellular 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. Figure 17 As shown in the figure, the compound ZQMT10 at a concentration of 10 μM has a stronger binding ability 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, and the like. In particular, compound ZQMT1-26 can block pain perception at the root of cold-induced pain, making it an important compound for the preparation of drugs to prevent or treat pain caused by frostbite.

Claims

1. An oxindole compound or a pharmaceutically acceptable salt thereof, wherein the compound structure is selected from: 。 2. A pharmaceutical composition, characterized in that Contains the compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

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

4. Use of an oxindole compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating pain; The oxindole compound is: 。 5. The use according to claim 4, characterized in that The compound is used in preparing medicine for treating pain caused by frostbite.

6. The use according to claim 4, characterized in that Application of the compound in preparing TRPA1 inhibitors.

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