4-Arylthiophenecarboxylic acid compounds as ANO1 protein inhibitors

Abstract

The present invention discloses 4-arylthiophenecarboxylic acid compounds as ANO1 protein inhibitors, and the 4-arylthiophenecarboxylic acid compounds are shown as formula (I). In addition, the present invention also discloses a preparation method of the compounds and a pharmaceutical composition containing the same. The compounds have a pharmacological effect of significantly inhibiting the activity of ANO1 protein and have pharmacodynamic effects such as analgesia.

Description

Technical Field

[0001] The present invention relates to, but is not limited to, the field of pharmaceutical technology. Specifically, it relates to 4-arylthiophenecarboxylic acid compounds having an inhibitory effect on ANO1 (Anoctamin 1, or TMEM16A) protein, which can be used for the development of analgesic-related drugs related to ANO1 inhibition. Background Art

[0002] Pain is the fifth vital sign in addition to blood pressure, respiration, pulse, and body temperature. The importance of controlling a patient's pain is self-evident. It is of great significance to research and develop more efficient and less side-effect analgesic drugs.

[0003] In addition to typical central opioid receptor analgesics and non-steroidal antipyretics, there are few other types of analgesic drugs in clinical practice at present. And the above two have their own limitations in application. Central analgesic drugs have a wide range of adverse reactions including addiction and drug resistance. Peripheral antipyretics also have limitations. One is gastrointestinal adverse reactions, such as aspirin and paracetamol with poor selectivity for cyclooxygenase (COX); the other is cardiovascular adverse reactions caused by excessive selectivity, such as the COX-2 selective inhibitor rofecoxib; in addition, COX inhibitors all have an impact on the kidneys, mainly including sodium and potassium retention, and in severe cases, acute renal failure, interstitial nephritis, etc.

[0004] In addition to the application limitations of analgesic drugs themselves, there are also many types of pain in clinical practice for which there are no specific drugs. For example, some chronic pain and neuropathic pain are characterized by intense pain and long duration. In the treatment of these pains, the current two types of analgesic drugs far from meet the needs.

[0005] Calcium-activated chloride channel (CaCC), as an anion channel in the human body, is widely distributed in glandular epithelial cells with secretory functions such as the respiratory tract and digestive tract, and also exists in the retina, dorsal root ganglia, and smooth muscle cells, participating in various cell physiological activities, such as trans-epithelial transport of secreted proteins and salts, nerve signal conduction, action potential repolarization, smooth muscle contraction, etc. So far, the molecular basis of CaCC that has been discovered mainly includes CLCA (Chloride channel accessory), Tweety (hTTHY1 and hTTYH3), Bestrophins, and Anoctamins.

[0006] ANO1 (Anoctamin 1), also known as TMEM16A (Transmembrane protein 16A), is a membrane protein with an eight-transmembrane structure. ANO1 was initially found to be highly expressed specifically in some tumor tissues, such as gastrointestinal stromal tumors, oral squamous cell carcinomas, head and neck squamous cell carcinomas, etc., while being lowly expressed or not expressed in normal tissues of the same origin. In 2008, ANO1 protein was first confirmed as the molecular basis of CaCC and could exhibit complete calcium-activated chloride current characteristics in vitro.

[0007]

[0008] Currently known ANO1 protein inhibitors

[0009] Known ANO1-specific inhibitors mainly include the CaCCinh series, TMEM16Ainh-A series, MONNA series, Ani9 series, and some natural compounds. The above compounds generally have obvious inhibitory effects on the CaCC current in ANO1 stable cell lines, but in human tissue cells with CaCC electrophysiological characteristics, the inhibitory effects are inconsistent; in addition, the above inhibitors also have differences in the inhibitory effects on ANO1-related tumor proliferation. CaCCinh-A01 and the natural product Tannic Acid have obvious inhibitory effects, while the remaining inhibitors are ineffective.

[0010] ANO1 plays an important role in pain conduction. If its molecular basis as CaCC is inhibited, it will lead to a significant reduction in the efficiency of nerve signal conduction. In this way, theoretically, the conduction of pain signals by the corresponding afferent nerves can be effectively inhibited, achieving an analgesic effect. For example, ANO1 is highly expressed in dorsal root ganglion (DRG) cells related to pain. Blocking or knocking down ANO1 protein in DRG can significantly reduce the pain response of mice in pain models. In addition, in pain-related models induced by capsaicin, the pain behavior can be reduced by the ANO1 inhibitor TMEM16Ainh-A01.

[0011] The analgesic mechanism of ANO1 protein ANO1 inhibitors is different from that of classical analgesic drugs. From the currently known research reports, it mainly exerts its effect through the ion channel function of ANO1 protein. When nerve cells are in a resting state, the membrane potential shows a negative value, and the potential inside the cell membrane is lower than that outside the cell membrane; during the generation of nerve impulses, the membrane potential rises, and this process is mainly achieved through the influx of cations and the efflux of anions. ANO1, as a calcium-activated chloride channel, when inhibited, cannot pump anions out of the cell, making it difficult to achieve an increase in membrane potential and generate nerve impulses. Currently, many studies have confirmed this theory: after inhibiting or silencing ANO1, the sensitivity of experimental animals to pain stimuli decreases significantly. In the hot plate experiment, acupuncture experiment of physiological pain, and inflammatory pain induced by capsaicin, formalin, and carrageenan, ANO1 inhibitors all have obvious analgesic effects. However, due to the lack of highly efficient ANO1 protein inhibitor molecules, the above research is not yet complete. In particular, for the detailed mechanism of the analgesic process of ANO1 inhibitors and their relationship with upstream and downstream pathways, current research still needs to be further deepened.

[0012] In summary, ANO1 protein inhibitors can effectively inhibit the physiological function of calcium-activated chloride channels and play an important role in pain treatment; ANO1 protein, as the only reported peripheral anion channel analgesic target, has great novelty; in addition, ANO1 protein also has other functions and has great potential in the treatment of many diseases. Summary of the Invention

[0013] The following is an overview of the subject matter described in detail in this article. This overview is not intended to limit the scope of protection of this application.

[0014] The inventors have developed a series of 4-arylthiophenecarboxylic acid compounds, which have significant pharmacological effects of inhibiting ANO1 protein activity and calcium-activated chloride channels, and at the same time have pharmacodynamic effects of analgesia.

[0015] In a first aspect, the present invention provides a use of a 4-arylthiophenecarboxylic acid compound represented by the general formula (1), or a pharmaceutically acceptable stereoisomer, prodrug, salt, solvate or hydrate thereof, as an ANO1 protein inhibitor:

[0016]

[0017] Wherein, in formula (I), R is an optionally substituted pyrazinyl group, an optionally substituted pyridyl group, an optionally substituted quinolinyl group, a C1-C6 alkyl-substituted thiophene, or the following group:

[0018] Here, R1 is halogen, unsubstituted C1-C4 alkyl, halogenated C1-C4 alkyl, unsubstituted C1-C4 alkoxy, or halogenated C1-C4 alkoxy; R3 is hydrogen; R2, R4, and R5 are each independently hydrogen, halogen, unsubstituted C1-C4 alkyl, halogenated C1-C4 alkyl, unsubstituted C1-C4 alkoxy, or halogenated C1-C4 alkoxy.

[0019] In an embodiment of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein the halogen is fluorine, chlorine, bromine, or iodine.

[0020] In an embodiment of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein the unsubstituted C1-C4 alkyl is methyl, ethyl, n-propyl, isopropyl (2-propyl), n-butyl, isobutyl (2-methyl-propyl), sec-butyl (2-butyl), or tert-butyl. The halogenated C1-C4 alkyl refers to one or more hydrogens on the C1-C4 alkyl being replaced by a halogen atom, including but not limited to trifluoromethyl.

[0021] In an embodiment of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein the unsubstituted C1-C4 alkoxy is methoxy, ethoxy, n-propoxy, isopropoxy (2-propoxy), n-butoxy, isobutoxy (2-methyl-propoxy), sec-butyl (2-butoxy), or tert-butoxy. The halogenated C1-C4 alkoxy refers to one or more hydrogens on the C1-C4 alkoxy being replaced by a halogen atom, including but not limited to trifluoromethoxy.

[0022] In some embodiments of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein the optionally substituted pyrazinyl, optionally substituted pyridinyl, or optionally substituted quinolinyl in formula (I) respectively refer to unsubstituted pyrazinyl, unsubstituted pyridinyl, or unsubstituted quinolinyl, or pyrazinyl, pyridinyl, or quinolinyl substituted by a group selected from the following: C1-C4 alkyl, C1-C4 alkoxy, hydroxy, halogen, nitro, or cyano; optionally, the pyrazinyl is pyrazin-2-yl, the pyridinyl is pyridin-2-yl, pyridin-3-yl, or pyridin-4-yl, and the quinolinyl is quinolin-2-yl, quinolin-3-yl, quinolin-4-yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl, or quinolin-8-yl.

[0023] In some embodiments of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein R in formula (I) is a C1-C6 alkyl-substituted thiophene, and the C1-C6 alkyl-substituted thiophene can be 3-C1-C6 alkyl-thiophen-2-yl, 4-C1-C6 alkyl-thiophen-2-yl, or 5-C1-C6 alkyl-thiophen-2-yl, such as 3-methyl-thiophen-2-yl.

[0024] In some embodiments of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein in formula (I), R is the following group:

[0025] Here, R1 is halogen, unsubstituted C1-C4 alkyl, halogenated C1-C4 alkyl, unsubstituted C1-C4 alkoxy, or halogenated C1-C4 alkoxy; R3 is hydrogen; one of R2, R4, and R5 is halogen, unsubstituted C1-C4 alkyl, halogenated C1-C4 alkyl, unsubstituted C1-C4 alkoxy, or halogenated C1-C4 alkoxy, and the other two are hydrogen.

[0026] In some embodiments of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein in formula (I), R is the following group:

[0027] Here, R1 is fluorine, chlorine, bromine, iodine, methyl, ethyl, trifluoromethyl, methoxy, ethoxy, or trifluoromethoxy; R3 is hydrogen; one of R2, R4, and R5 is fluorine, chlorine, bromine, iodine, methyl, ethyl, trifluoromethyl, methoxy, ethoxy, or trifluoromethoxy, and the other two are hydrogen.

[0028] In some embodiments of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein in formula (I), R is the following group:

[0029] Here, R2, R3, R4, and R5 are all hydrogen, and R1 is fluorine, chlorine, bromine, iodine, methyl, ethyl, trifluoromethyl, methoxy, ethoxy, or trifluoromethoxy.

[0030] In some embodiments of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein in formula (I), R is the following group:

[0031] Here, R2, R3, and R4 are all hydrogen, and R1 and R5 are each independently fluorine, chlorine, bromine, iodine, methyl, ethyl, trifluoromethyl, methoxy, ethoxy, or trifluoromethoxy.

[0032] In some embodiments of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein in formula (I), R is the following group:

[0033] Here, R3, R4, and R5 are all hydrogen, and R1 and R2 are each independently fluorine, chlorine, bromine, iodine, methyl, ethyl, trifluoromethyl, methoxy, ethoxy, or trifluoromethoxy.

[0034] In some embodiments of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein R in formula (I) is the following groups:

[0035] Here, R2, R3, and R5 are all hydrogen, and R1 and R4 are each independently fluorine, chlorine, bromine, iodine, methyl, ethyl, trifluoromethyl, methoxy, ethoxy, or trifluoromethoxy.

[0036] In some preferred embodiments of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein R in formula (I) is pyrazin-2-yl, 3-methyl-thiophen-2-yl, pyridin-3-yl, pyridin-4-yl, quinolin-5-yl.

[0037] In some preferred embodiments of the present application, the 4-arylthiophenecarboxylic acid compounds provided by the present application, wherein R in formula (I) is: 2-trifluoromethoxyphenyl, 2,5-dichlorophenyl, 2,3-dichlorophenyl, 2,6-dichlorophenyl, 2-chloro-5-methoxyphenyl, 2-chloro-5-bromophenyl, 2-chloro-5-trifluoromethylphenyl, 2-chloro-5-iodophenyl, 2-chloro-5-methylphenyl, 2-bromo-5-chlorophenyl, 2-trifluoromethyl-5-chlorophenyl, 2-iodo-5-chlorophenyl, 2-fluoro-5-chlorophenyl, 2-chloro-5-fluorophenyl, 2-bromo-5-fluorophenyl, 2-iodo-5-fluorophenyl, 2,5-difluorophenyl, 2-fluoro-5-bromophenyl, or 2-fluoro-5-iodophenyl.

[0038] In particularly preferred embodiments of the present invention, the 4-arylthiophenecarboxylic acid compounds provided by the present invention, the compounds are selected from the following compounds:

[0039] 4-(4-chlorophenyl)-2-(pyrazine-2-carboxamido)thiophene-3-carboxylic acid;

[0040] 4-(4-chlorophenyl)-2-(2-(trifluoromethoxy)benzamido)thiophene-3-carboxylic acid;

[0041] 4-(4-chlorophenyl)-2-(2,5-dichlorobenzamido)thiophene-3-carboxylic acid;

[0042] 4-(4-chlorophenyl)-2-(3-methylthiophene-2-carboxamido)thiophene-3-carboxylic acid;

[0043] 4-(4-chlorophenyl)-2-(nicotinamido)thiophene-3-carboxylic acid;

[0044] 4-(4-chlorophenyl)-2-(isonicotinamido)thiophene-3-carboxylic acid;

[0045] 4-(4-chlorophenyl)-2-(quinoline-5-carboxamido)thiophene-3-carboxylic acid;

[0046] 4-(4-chlorophenyl)-2-(2,3-dichlorobenzamido)thiophene-3-carboxylic acid;

[0047] 4-(4-chlorophenyl)-2-(2,6-dichlorobenzamido)thiophene-3-carboxylic acid;

[0048] 2-(2-chloro-5-methoxybenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

[0049] 2-(5-bromo-2-chlorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

[0050] 2-(2-chloro-5-((difluoro-1-3-methyl)-12-fluorenyl)benzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

[0051] 2-(2-chloro-5-iodobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

[0052] 2-(2-chloro-5-methylbenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

[0053] 2-(2-bromo-5-chlorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

[0054] 2-(5-chloro-2-(trifluoromethyl)benzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

[0055] 2-(5-chloro-2-iodobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

[0056] 2-(5-chloro-2-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

[0057] 2-(2-chloro-5-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

[0058] 2-(2-bromo-5-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

[0059] 4-(4-chlorophenyl)-2-(5-fluoro-2-iodobenzamido)thiophene-3-carboxylic acid;

[0060] 4-(4-chlorophenyl)-2-(2,5-difluorobenzamido)thiophene-3-carboxylic acid;

[0061] 2-(5-bromo-2-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid; or

[0062] 4-(4-chlorophenyl)-2-(2-fluoro-5-iodobenzamido)thiophene-3-carboxylic acid;

[0063] or a pharmaceutically acceptable isomer, prodrug, salt, solvate, or hydrate thereof.

[0064] In an embodiment of the present invention, the pharmaceutically acceptable salt of the compound, for example, forms a non-toxic base addition salt with an inorganic base or an organic base, and examples include: triethylamine salt, sodium salt, potassium salt, or magnesium salt, etc.

[0065] In a second aspect, the present invention also provides a method for preparing the 4-arylthiophenecarboxylic acid compound as described above, and the preparation method includes the following steps:

[0066] (1) The acetophenone compound shown in formula (II) reacts with the compound shown in formula (III) to obtain the compound shown in formula (IV);

[0067]

[0068] (2) The compound shown in formula (IV) undergoes a condensation reaction with the acyl chloride shown in formula (V) to obtain the compound shown in formula (VI);

[0069]

[0070] (3) The compound shown in formula (VI) undergoes a hydrolysis reaction to obtain the compound shown in formula (I);

[0071]

[0072] Here, the substituent R6 in formulas (III), (IV), and (VI) is a C1-C6 alkyl or benzyl group, and the definition of the substituent R in formulas (V) and (VI) is as defined for the compound shown in formula (I) above.

[0073] In a third aspect, the present invention provides a pharmaceutical composition, and the pharmaceutical composition includes a pharmacologically effective amount of the 4-arylthiophenecarboxylic acid compound of the present invention or a pharmaceutically acceptable isomer, prodrug, salt, solvate, or hydrate thereof and a pharmaceutically acceptable carrier, and the carrier may include an excipient and / or a diluent. The compound is mixed with the carrier to obtain the pharmaceutical composition of the present invention. The pharmaceutical composition can be orally administered in the form of tablets, capsules, pills, powders, granules, powders, or syrups, or parenterally administered in the form of injections. The unit dose of the pharmaceutical composition is 0.1 mg to 1 g.

[0074] Fourthly, the present invention provides the use of the above-mentioned 4-arylthiophenecarboxylic acid compounds or their pharmaceutically acceptable isomers, prodrugs, salts, solvates, or hydrates, or their pharmaceutical compositions as ANO1 protein inhibitors. As an ANO1 protein inhibitor, the above-mentioned 4-arylthiophenecarboxylic acid compounds or their pharmaceutically acceptable isomers, prodrugs, salts, solvates, or hydrates, or their pharmaceutical compositions provided by the present invention can be used for the treatment of various diseases related to ANO1 protein, such as analgesia, including but not limited to analgesia for inflammatory pain or analgesia for bone cancer pain.

[0075] The beneficial effects of the present invention are as follows: The compounds of the present invention are a new type of compound and have ANO1 protein inhibitory activity, and can be used as ANO1 protein inhibitors, thus being able to be used for the treatment of various diseases related to ANO1 protein, such as analgesia; in addition, the compounds of the present invention provide advantages in drug use, such as in one or more aspects of their mechanism of action, binding, inhibitory activity, safety profile, solubility, or bioavailability.

[0076] It has been experimentally proven that the 4-arylthiophenecarboxylic acid compounds provided by the present invention show high affinity and high inhibitory activity against ANO1 protein in functional experiments. Among them, functional tests for ANO1 protein inhibitory activity were carried out using FRT cells stably expressing ANO1. In this experiment, the IC 50 of the compounds of the present invention is between 100 μM and 10 nM.

[0077] Other features and advantages of the present application will be described in the subsequent specification, and part of them will be obvious from the specification, or understood by implementing the present application. Other advantages of the present application can be achieved and obtained through the solutions described in the specification and the drawings. Description of the Drawings

[0078] The drawings are used to provide an understanding of the technical solutions of the present application, and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solutions of the present application, and do not constitute a limitation to the technical solutions of the present application.

[0079] Figure 1 Shows the whole-cell patch-clamp results of the inhibitory effects of Compounds 30 and 39 of the present invention on ANO1 protein at 30 μM;

[0080] Figure 2 Shows the IC 50 diagram of the inhibitory effect of Compound 39 of the present invention on the electrophysiological activity of ANO1 channels;

[0081] Figure 3 Shows the whole-cell patch-clamp results of the inhibitory effects of Compounds 30 and 39 of the present invention on ANO2 protein at 30 μM;

[0082] Figure 4 It represents the IC of the compound 39 of the present invention for inhibiting the electrophysiological activity of the ANO2 channel 50 Figure;

[0083] Figure 5 It represents the whole-cell patch-clamp results of the inhibitory effect of the compound 39 of the present invention on the hERG channel;

[0084] Figure 6 It represents the analgesic effect of the compound 39 of the present invention on formalin-induced inflammatory pain in mice;

[0085] Figure 7 It represents the analgesic effect of the compound 39 of the present invention on capsaicin-induced inflammatory pain in mice;

[0086] Figure 8 It represents the analgesic effect of the compound 39 of the present invention on complete Freund's adjuvant-induced inflammatory pain in rats;

[0087] Figure 9 It represents the analgesic effect of the compound 39 of the present invention on rat tibial bone cancer pain. Detailed implementation mode

[0088] The following examples are used to further illustrate the present invention and do not constitute any limitation to the present invention in any form.

[0089] In the present invention, the abbreviations are as follows:

[0090] ANO1 Anoctamin 1 protein

[0091] FRT cells Fisher rat thyroid follicular epithelial cells

[0092] ATP Adenosine triphosphate

[0093] CaCC Calcium-activated chloride channel

[0094] IC 50 Half inhibitory concentration

[0095] The nuclear magnetic resonance data was measured by a Bruker Avance III 400 nuclear magnetic resonance spectrometer with TMS (tertramethyl silance) as the internal standard; the nuclear magnetic resonance data was processed by mestReNova (Ver.6.1.0, mesrelab Research S.L.) software; the high-resolution mass spectrometry data (ESI-TOF) was measured by a Bruker Apex IV FTMS Fourier ion cyclotron transform mass spectrometer; the thin-layer chromatography silica gel plate (GF254, Shanghai Shangbang Industrial Co., Ltd.); the column chromatography silica gel (200-300 mesh, Shanghai Shangbang Industrial Co., Ltd.).

[0096] All solvents, raw materials and reagents are commercially available analytical reagents unless otherwise specified.

[0097] Synthesis method 1:

[0098]

[0099] Among them, the definitions of each substituent are shown in detail in the examples.

[0100] a1. Ethyl cyanoacetate (1.2 eq.), morpholine (2.2 eq.), acetic acid (1 eq.), ethanol, 70 °C, 3 hours, under argon protection;

[0101] a2. Sulfur powder (1.2 eq.), 70 °C, 48 hours, under argon protection;

[0102] b. Aroyl chloride is slowly added dropwise (1 eq.), dichloromethane, 0 °C; after the addition is complete, the reaction is carried out at room temperature for 8 hours;

[0103] c. NaOH (5 eq.), H2O, methanol, THF, reaction at room temperature for 8 hours.

[0104] Example 1

[0105] Compound 15

[0106]

[0107] Synthesis method:

[0108] Synthesized by "Synthesis Method 1". Take a 1 L round-bottom flask, add 18.55 g (120 mmol) of 4-chloroacetophenone, 16.29 g (144 mmol) of ethyl cyanoacetate, 7.12 g (120 mmol) of acetic acid, 23.00 g (264 mmol) of morpholine and 500 mL of ethanol. Stir at 70 °C under argon protection for 3 hours, add 4.91 g (144 mmol) of sulfur powder, and stir overnight under argon protection. After the reaction is completed, add 200 mL of dichloromethane to dilute the reaction solution, wash it four times with brine and dry it with anhydrous sodium sulfate, and separate it by column chromatography to obtain white powder ethyl 2-amino-4-(4-chlorophenyl)thiophene-3-carboxylate, with a yield of about 55%. Dissolve 0.15 g (1.2 mmol) of 2-pyrimidinecarboxylic acid in 10 mL of dichloromethane, add 0.1 mL of DMF and 0.26 mL (3.6 mmol) of thionyl chloride, reflux at 60 °C for 3 hours, then spin-dry the reaction solution and redissolve it with 10 mL of dichloromethane. Another 0.28 g (1 mmol) of ethyl 2-amino-4-(4-chlorophenyl)thiophene-3-carboxylate was dissolved in 10 mL of dichloromethane, and the obtained 1-naphthoyl chloride solution was added to the aminothiophene solution at 0 °C. After the addition was completed, the reaction was carried out at room temperature for 8 hours. After the reaction was completed, column chromatography separation was carried out to obtain white powder 2-(2-pyrimidinylamino)-4-(4-chlorophenyl)thiophene-3-carboxylate, with a yield of about 75%. Dissolve 0.06 g (0.15 mmol) of 2-(2-pyrimidinylamino)-4-(4-chlorophenyl)thiophene-3-carboxylate and 0.03 g (0.75 mmol) of NaOH in a mixed solvent of tetrahydrofuran / methanol / water, stir at 65 °C overnight, adjust the pH to neutral with acetic acid after the reaction is completed, and add water to precipitate a white solid compound 15 0.025 g (0.07 mmol). The total yield is 15%.

[0109] 1 H-NMR(400MHz,DMSO-d6)δ9.38(d,J=1.3Hz,1H),9.00(d,J=2.5Hz,1H),8.87(t,J=1.9Hz,1H),7.40(q,J=8.5Hz,4H),7.07(s,1H). 13 C-NMR(101MHz,DMSO-d6)δ166.42,160.92,149.14,148.31,144.56,144.36,143.07,139.04,136.43,132.28,131.28,127.91,117.28,114.00.HRMS(ESI-TOF + )C 16 H9N3O3SCl[M-H] - m / z theoretical value: 358.0055; measured value: 358.0053;

[0110] Example 2

[0111] Compound 16

[0112]

[0113] The synthesis method is the same as that of Compound 15, total yield: 21%.

[0114] 1 H-NMR(400MHz, DMSO-d6) δ8.03(dd, J = 7.7, 1.8Hz, 1H), 7.80(td, J = 7.9, 1.8Hz, 1H), 7.66–7.60(m, 2H), 7.39(q, J = 8.6Hz, 4H), 7.03(s, 1H). 13 C-NMR(101MHz, DMSO-d6) δ166.73, 161.35, 148.58, 145.69, 138.92, 136.53, 134.55, 132.27, 131.82, 131.29, 128.79, 127.89, 127.01, 122.44, 117.17, 113.67. HRMS(ESI-TOF + )C 19 H 10 NO4F3SCl[M-H] - Theoretical value of m / z: 439.9973; Measured value: 439.9971;

[0115] Example 3

[0116] Compound 17

[0117]

[0118] The synthesis method is the same as that of Compound 15, total yield: 25%.

[0119] 1 H-NMR(400MHz, DMSO-d6) δ7.91(t, J = 1.4Hz, 1H), 7.69(d, J = 1.7Hz, 2H), 7.43–7.35(m, 4H), 7.07(s, 1H). 13 C-NMR(101MHz, DMSO-d6) δ166.58, 162.09, 147.86, 138.85, 136.39, 135.46, 132.89, 132.80, 132.63, 132.30, 131.21, 130.16, 129.49, 127.96, 117.29, 114.49. HRMS(ESI-TOF + )C18 H9NO3SCl3[M-H] - Theoretical m / z value: 423.9369; Measured value: 423.9369;

[0120] Example 4

[0121] Compound 18

[0122]

[0123] The synthesis method is the same as that of Compound 15, with an overall yield of 27%.

[0124] 1 H-NMR (400 MHz, DMSO-d6) δ 12.06 (s, 1H), 7.85 (d, J = 4.9 Hz, 1H), 7.38 (q, J = 8.6 Hz, 4H), 7.14 (d, J = 4.9 Hz, 1H), 6.97 (s, 1H), 2.62 (s, 3H). 13 C-NMR (101 MHz, DMSO-d6) δ 167.34, 159.43, 149.69, 143.06, 138.74, 136.63, 133.44, 132.23, 131.33, 131.19, 130.03, 127.88, 116.82, 112.61, 16.14. HRMS (ESI-TOF + ) C 17 H 11 NO3S2Cl[M-H] - Theoretical m / z value: 375.987; Measured value: 375.9869;

[0125] Example 5

[0126] Compound 19

[0127]

[0128] The synthesis method is the same as that of Compound 15, with an overall yield of 32%.

[0129] 1 H-NMR (400 MHz, DMSO-d6) δ 9.12 (d, J = 2.3 Hz, 1H), 8.86 (d, J = 4.9 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.68 (dd, J = 8.0, 4.9 Hz, 1H), 7.44–7.36 (m, 4H), 7.03 (s, 1H). 13C-NMR(101MHz, DMSO-d6) δ 167.24, 162.42, 153.68, 149.23, 148.64, 136.50, 135.59, 132.27, 131.30, 128.50, 127.92, 124.65, 117.02, 113.75. HRMS(ESI-TOF + ) C 17 H 10 N2O3SCl [M-H] - Calculated m / z: 357.0099; Found: 357.0101;

[0130] Example 6

[0131] Compound 20

[0132]

[0133] The synthesis method is the same as that of Compound 15, with an overall yield of 19%.

[0134] 1 H-NMR(400MHz, DMSO-d6) δ 8.90–8.87 (m, 2H), 7.85–7.83 (m, 2H), 7.43–7.36 (m, 4H), 7.04 (s, 1H). 13 C-NMR(101MHz, DMSO-d6) δ 167.17, 162.35, 151.45, 149.09, 139.76, 138.96, 136.47, 132.28, 131.32, 127.91, 121.34, 117.21, 114.14. HRMS(ESI-TOF + ) C 17 H 12 N2O3SCl [M-H] - Calculated m / z: 359.0253; Found: 359.0257;

[0135] Example 7

[0136] Compound 22

[0137]

[0138] The synthesis method is the same as that of Compound 15, with an overall yield of 22%.

[0139] 1H-NMR(400MHz, DMSO-d6) δ 9.03 (dd, J = 4.2, 1.6 Hz, 1H), 8.89 (d, J = 8.9 Hz, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 7.1 Hz, 1H), 7.94 (t, J = 7.8 Hz, 1H), 7.69 (dd, J = 8.7, 4.1 Hz, 1H), 7.40 (q, J = 8.5 Hz, 4H), 7.05 (s, 1H). 13 C-NMR(101MHz, DMSO-d6) δ 167.08, 164.66, 151.79, 149.01, 148.20, 138.92, 136.61, 133.97, 133.58, 132.22, 132.09, 131.29, 129.26, 127.90, 127.12, 125.80, 123.15, 116.82. HRMS(ESI-TOF + )C 21 H 12 N2O3SCl [M-H] - m / z calculated: 407.0258; found: 407.0257;

[0140] Example 8

[0141] Compound 23

[0142]

[0143] The synthesis method is the same as that of Compound 15, with an overall yield of 18%.

[0144] 1 H-NMR(400MHz, DMSO-d6) δ 11.75 (s, 1H), 7.65–7.56 (m, 3H), 7.40 (q, J = 8.6 Hz, 4H), 7.10 (s, 1H). 13 C-NMR(101MHz, DMSO-d6) δ 166.64, 162.91, 147.89, 138.86, 136.52, 136.41, 133.35, 133.29, 132.31, 131.23, 129.48, 128.99, 128.79, 127.96, 117.25, 114.47, 40.62, 40.41, 40.20, 39.99, 39.79, 39.58, 39.37. HRMS(ESI-TOF + )C 18 H9NO3SCl3 [M-H] - m / z calculated: 423.937; found: 423.9369;

[0145] Example 9

[0146] Compound 24

[0147]

[0148] The synthesis method is the same as that of Compound 15, total yield: 15%.

[0149] 1 H-NMR(400MHz, DMSO-d6) δ 11.75(s, 1H), 7.65–7.56(m, 3H), 7.40(q, J = 8.6Hz, 4H), 7.10(s, 1H). 13 C-NMR(101MHz, DMSO-d6) δ 166.41, 161.61, 146.57, 138.74, 136.24, 134.64, 132.88, 132.35, 131.85, 131.12, 128.98, 128.03, 117.47, 115.36. HRMS(ESI-TOF + )C 18 H9NO3SCl3[M-H] - Theoretical value of m / z: 423.9374; Measured value: 423.9369;

[0150] Example 10

[0151] Compound 25

[0152]

[0153] The synthesis method is the same as that of Compound 15, total yield: 24%.

[0154] 1 H-NMR(400MHz, DMSO-d6) δ 11.96(s, 1H), 7.55(d, J = 8.9Hz, 1H), 7.42–7.34(m, 5H), 7.19(dd, J = 8.9, 3.1Hz, 1H), 7.04(s, 1H), 3.83(s, 3H). 13 C-NMR(101MHz, DMSO-d6) δ 166.79, 163.02, 158.76, 148.44, 138.88, 136.48, 134.38, 132.30, 132.00, 131.27, 127.93, 121.63, 119.17, 117.11, 115.63, 113.84, 56.32. HRMS(ESI-TOF + )C 19 H 12NO4SCl2[M-H] - Theoretical m / z value: 419.9871; Measured value: 419.9864;

[0155] Example 11

[0156] Compound 26

[0157]

[0158] The synthesis method is the same as that of Compound 15, with an overall yield of 21%.

[0159] 1 1H-NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H), 8.02 (d, J = 2.5 Hz, 1H), 7.81 (dd, J = 8.6, 2.5 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.43–7.34 (m, 4H), 7.06 (s, 1H). 13 13C-NMR (101 MHz, DMSO-d6) δ 166.63, 162.00, 147.90, 138.85, 136.40, 135.82, 135.69, 132.98, 132.85, 132.32, 131.22, 130.09, 127.97, 120.92, 117.30, 114.47. HRMS (ESI-TOF + ) C 18 H9NO3SCl2Br[M-H] - Theoretical m / z value: 467.8859; Measured value: 467.8864;

[0160] Example 12

[0161] Compound 27

[0162]

[0163] The synthesis method is the same as that of Compound 15, with an overall yield of 27%.

[0164] 1 1H-NMR (400 MHz, DMSO-d6) δ 11.99 (s, 1H), 8.20 (d, J = 2.3 Hz, 1H), 7.98 (dd, J = 8.4, 2.3 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.43–7.33 (m, 4H), 7.07 (s, 1H). 13C-NMR(101MHz, DMSO-d6) δ 166.60, 162.18, 147.76, 138.84, 136.36, 135.22, 134.90, 132.33, 132.15, 131.20, 128.95, 128.62, 127.98, 127.60, 127.56, 117.37, 114.64. HRMS(ESI-TOF + )C 19 H9NO3SCl2F3 [M-H] - Calculated m / z: 457.9628; Found: 457.9632;

[0165] Example 13

[0166] Compound 28

[0167]

[0168] The synthesis method is the same as that of Compound 15, with an overall yield of 21%.

[0169] 1 H-NMR(400MHz, DMSO-d6) δ 11.91(s, 1H), 8.13(d, J = 2.2Hz, 1H), 7.93(dd, J = 8.5, 2.2Hz, 1H), 7.43–7.34(m, 5H), 7.04(s, 1H). 13 C-NMR(101MHz, DMSO-d6) δ 166.66, 161.92, 147.97, 141.63, 138.82, 138.63, 136.39, 135.59, 132.75, 132.30, 131.21, 130.68, 127.96, 117.26, 114.32, 93.60. HRMS(ESI-TOF + )C 18 H9NO3SCl2I [M-H] - Calculated m / z: 515.8726; Found: 515.8725;

[0170] Example 14

[0171] Compound 29

[0172]

[0173] The synthesis method is the same as that of Compound 15, with an overall yield of 22%.

[0174] 1H-NMR(400MHz, DMSO-d6) δ 11.96 (s, 1H), 7.63 (d, J = 2.1Hz, 1H), 7.51 (d, J = 8.2Hz, 1H), 7.43–7.34 (m, 5H), 7.02 (s, 1H), 2.36 (s, 3H). 13 C-NMR(101MHz, DMSO-d6) δ 166.81, 163.34, 148.51, 138.84, 138.11, 136.48, 133.89, 133.28, 132.27, 131.25, 131.01, 130.71, 127.91, 127.52, 117.05, 113.71, 20.69. HRMS(ESI-TOF + )C 19 H 12 NO3SCl2[M - H] - m / z calculated: 403.9917; found: 403.9915;

[0175] Example 15

[0176] Compound 30

[0177]

[0178] The synthesis method is the same as that of Compound 15, total yield: 19%.

[0179] 1 H-NMR(400MHz, DMSO-d6) δ 11.81 (s, 1H), 7.86–7.79 (m, 2H), 7.58 (dd, J = 8.6, 2.6Hz, 1H), 7.43–7.34 (m, 4H), 7.05 (s, 1H). 13 C-NMR(101MHz, DMSO-d6) δ 166.64, 163.20, 147.86, 138.83, 138.10, 136.36, 135.61, 133.37, 132.80, 132.31, 131.20, 129.79, 127.96, 118.02, 117.25, 114.46. HRMS(ESI-TOF + )C 18 H9NO3SCl2Br[M - H] - m / z calculated: 467.8865; found: 467.8864;

[0180] Example 16

[0181] Compound 31

[0182]

[0183] The synthesis method is the same as that of Compound 15, with an overall yield of 17%.

[0184] 1 H-NMR (400 MHz, DMSO-d6) δ 11.81 (s, 1H), 8.03 (d, J = 2.1 Hz, 1H), 7.95 (d, J = 8.6 Hz, 1H), 7.90–7.86 (m, 1H), 7.44–7.40 (m, 2H), 7.39–7.35 (m, 2H), 7.08 (s, 1H). 13 C-NMR (101 MHz, DMSO-d6) δ 166.49, 163.06, 147.66, 138.77, 138.20, 136.34, 136.03, 132.31, 131.67, 131.18, 129.39, 129.34, 129.26, 127.99, 125.80, 125.48, 117.31, 114.87. HRMS (ESI-TOF + )C 19 H9NO3F3SCl2 [M-H] - Theoretical value of m / z: 457.9632; Measured value: 457.9632;

[0185] Example 17

[0186] Compound 34

[0187]

[0188] The synthesis method is the same as that of Compound 15, with an overall yield of 25%.

[0189] 1 H-NMR (400 MHz, DMSO-d6) δ 11.68 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 2.5 Hz, 1H), 7.42–7.39 (m, 3H), 7.36 (d, J = 8.6 Hz, 2H), 7.06 (s, 1H). 13 C-NMR (101 MHz, DMSO-d6) δ 166.70, 164.91, 148.07, 142.31, 141.91, 138.84, 136.39, 134.05, 132.58, 132.31, 131.22, 128.88, 127.97, 117.20, 114.34, 92.21. HRMS (ESI-TOF + )C 18 H9NO3SCl2I [M-H] -Theoretical m / z value: 515.8734; Measured value: 515.8725;

[0190] Example 18

[0191] Compound 35

[0192]

[0193] The synthesis method is the same as that of Compound 15, with an overall yield of 31%.

[0194] 1 1H-NMR (400 MHz, DMSO-d6) δ 12.46 (d, J = 10.0 Hz, 1H), 8.02 (td, J = 6.6, 2.8 Hz, 1H), 7.78 (dq, J = 8.8, 3.1 Hz, 1H), 7.53 (dd, J = 11.2, 8.8 Hz, 1H), 7.40–7.34 (m, 4H), 7.01 (s, 1H). 13 13C-NMR (101 MHz, DMSO-d6) δ 166.67, 158.85, 158.82, 148.58, 138.94, 136.51, 135.16, 135.07, 132.25, 131.28, 131.21, 129.83, 129.80, 127.87, 127.85, 121.51, 121.38, 119.58, 119.33, 117.46, 113.78. HRMS (ESI-TOF + ) C 18 C9H9NO3FSCl2 [M-H] - Theoretical m / z value: 407.9667; Measured value: 407.9664;

[0195] Example 19

[0196] Compound 36

[0197]

[0198] The synthesis method is the same as that of Compound 15, with an overall yield of 19%.

[0199] 1 1H-NMR (400 MHz, DMSO-d6) δ 11.96 (s, 1H), 7.72 (ddd, J = 15.8, 8.8, 4.0 Hz, 2H), 7.50 (td, J = 8.4, 3.1 Hz, 1H), 7.42–7.34 (m, 4H), 7.05 (s, 1H). 13C-NMR(101MHz, DMSO-d6) δ 166.66, 162.32, 162.10, 147.99, 138.86, 136.39, 135.40, 135.33, 133.00, 132.91, 132.32, 131.22, 127.94, 126.06, 120.45, 120.22, 117.78, 117.53, 117.28, 114.27. HRMS(ESI-TOF + ) C 18 H9NO3FSCl2 [M-H] - Calculated m / z: 407.967; Found: 407.9664;

[0200] Example 20

[0201] Compound 37

[0202]

[0203] Synthesized in the same way as Compound 15, total yield: 26%.

[0204] 1 H-NMR(400MHz, DMSO-d6) δ 11.79 (s, 1H), 7.84 (dd, J = 8.8, 5.0 Hz, 1H), 7.69 (dd, J = 8.6, 3.1 Hz, 1H), 7.45–7.34 (m, 5H), 7.06 (s, 1H). 13 C-NMR(101MHz, DMSO-d6) δ 166.67, 163.27, 160.39, 147.94, 138.84, 138.13, 136.37, 135.98, 135.90, 132.32, 131.22, 127.97, 120.42, 120.19, 117.54, 117.30, 117.25, 114.31, 114.24. HRMS(ESI-TOF + ) C 18 H9NO3SClBrF [M-H] - Calculated m / z: 451.9157; Found: 451.9159;

[0205] Example 21

[0206] Compound 38

[0207]

[0208] Synthesized in the same way as Compound 15, total yield: 28%.

[0209] 1H-NMR(400MHz, DMSO-d6) δ 11.71 (s, 1H), 8.02 (dd, J = 8.7, 5.3 Hz, 1H), 7.61 (dd, J = 8.9, 3.0 Hz, 1H), 7.42–7.34 (m, 4H), 7.24 (td, J = 8.6, 3.0 Hz, 1H), 7.05 (s, 1H). 13 C-NMR(101MHz, DMSO-d6) δ 166.74, 164.96, 163.65, 161.19, 148.16, 142.35, 142.25, 142.17, 138.85, 136.39, 132.32, 131.23, 127.95, 120.29, 120.08, 117.19, 116.80, 116.56, 114.19. HRMS(ESI-TOF + )C 18 H9NO3FSClI [M-H] - m / z theoretical value: 499.9028; measured value: 499.902;

[0210] Example 22

[0211] Compound 39

[0212]

[0213] The synthesis method is the same as that of Compound 15, with an overall yield of 32%.

[0214] 1 H-NMR(400MHz, DMSO-d6) δ 12.48 (d, J = 10.3 Hz, 1H), 7.86–7.80 (m, 1H), 7.59 (ddt, J = 15.3, 9.7, 4.6 Hz, 2H), 7.39 (q, J = 8.3 Hz, 4H), 7.03 (d, J = 3.2 Hz, 1H). 13 C-NMR(101MHz, DMSO-d6) δ 166.66, 159.00, 148.59, 138.97, 136.53, 132.26, 131.28, 127.88, 122.47, 119.27, 118.01, 117.74, 117.47, 113.77. HRMS(ESI-TOF + )C 18 H9NO3F2SCl [M-H] - m / z theoretical value: 391.9958; measured value: 391.996;

[0215] Example 23

[0216] Compound 40

[0217]

[0218] The synthesis method is the same as that of Compound 15, with an overall yield of 26%.

[0219] 1 H-NMR (400 MHz, DMSO-d6) δ 12.44 (d, J = 10.0 Hz, 1H), 8.14 (dd, J = 6.6, 2.7 Hz, 1H), 7.89 (ddd, J = 8.8, 4.3, 2.7 Hz, 1H), 7.47 (dd, J = 11.3, 8.8 Hz, 1H), 7.40–7.33 (m, 4H), 7.00 (s, 1H). 13 C-NMR (101 MHz, DMSO-d6) δ 166.67, 158.77, 158.37, 148.58, 138.93, 138.08, 137.99, 136.50, 134.14, 132.25, 131.27, 127.87, 121.81, 121.68, 119.89, 119.64, 117.55, 117.52, 117.46, 113.76. HRMS (ESI-TOF + )C 18 C9H9NO3FSClBr [M-H] - Theoretical value of m / z: 451.9170; Measured value: 451.9159;

[0220] Example 24

[0221] Compound 41

[0222]

[0223] The synthesis method is the same as that of Compound 15, with an overall yield of 28%.

[0224] 1 H-NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H), 8.30 (dd, J = 7.0, 2.4 Hz, 1H), 8.02 (ddd, J = 8.6, 4.7, 2.4 Hz, 1H), 7.41–7.34 (m, 4H), 7.33–7.28 (m, 1H), 6.99 (s, 1H). 13C-NMR(101MHz, DMSO-d6) δ 166.68, 159.11, 158.84, 148.77, 143.75, 140.03, 138.97, 136.60, 132.18, 131.29, 127.88, 127.84, 122.11, 121.99, 119.90, 119.65, 117.28, 114.00. HRMS(ESI-TOF + ) m / z C 18 H9NO3FSClI [M-H] - Theoretical m / z value: 499.9018; Measured value: 499.902.

[0225] Experimental Example

[0226] The inhibitory effect of 4-arylthiophenecarboxylic acid compounds on ANO1 protein in the present invention.

[0227] In the present invention, pharmacological and pharmacodynamic experiments were conducted on the pharmacological activity. The pharmacological experiments included the yellow fluorescent protein-iodide fluorescence quenching experiment and the whole-cell patch clamp experiment, which jointly demonstrated the inhibitory effect of 4-arylthiophenecarboxylic acid compounds on ANO1 protein in the present invention. The pharmacodynamic experiments included several analgesic experiments, namely: mouse formalin-induced pain experiment, mouse capsaicin-induced pain experiment, rat complete Freund's adjuvant-induced pain experiment, and rat tibial bone cancer pain experiment. They jointly demonstrated the analgesic effect of 4-arylthiophenecarboxylic acid compounds in the present invention.

[0228] 1. Yellow fluorescent protein-iodide fluorescence quenching experiment:

[0229] This method uses FRT cells co-transfected with ANO1 protein and yellow fluorescent protein (YFP). The yellow fluorescence of YFP can be quenched by iodide ions. When ATP is added to the extracellular fluid, calcium ions are released from the intracellular calcium store, increasing the calcium ion concentration, thereby activating the CaCC (ANO1) channel. The CaCC (ANO1) channel can transport extracellular iodide ions into the cell interior. Therefore, when no regulator is added, the YFP fluorescence in the cell will be quenched, and the fluorescence value will decrease significantly under the detection of an enzyme-linked immunosorbent assay (ELISA) reader. When a CaCC (ANO1) inhibitor is added, the rate of fluorescence value decrease will slow down; conversely, when an agonist is added, the rate of fluorescence value decrease will accelerate. By comparing the rate of fluorescence value decline, the activity of the regulator can be indirectly evaluated.

[0230] Activity data of some compounds provided in the present invention in the yellow fluorescent protein-iodide fluorescence quenching experiment:

[0231]

[0232]

[0233] The above data demonstrate the inhibitory effect of the 4-arylthiophenecarboxylic acid compounds provided by the present invention on ANO1 protein.

[0234] 2. Whole-cell patch clamp experiment:

[0235] The patch clamp technique is a technique that records the current passing through ion channels to reflect the activity of channel molecules. It is the "gold standard" for measuring ion channel activity. The whole-cell patch clamp uses a special hollow glass electrode to absorb and break the cell surface, forming a closed loop between the cell membrane and the electrode. The electrode can detect the ion transport current of the whole cell membrane. Calcium ions are added to the intracellular fluid through the hollow electrode to activate the ANO1 channel. At this time, a gradient voltage is applied to the electrode to detect the change in current. When the inhibitor is added to the extracellular fluid, the channel closes and the resistance increases. Under the same gradient voltage, the ion transport current of the inhibitor group is lower than that of the blank group without drug addition; the opposite is true for the agonist. This can evaluate the effect of regulators on ion channels.

[0236] The whole-cell patch clamp experiment results of some compounds provided by the present invention showing an inhibitory effect on ANO1 channels are as follows Figure 1 As shown, the blue line is the electrophysiological characteristic curve of calcium-activated chloride channel without drug addition, and the red line is the calcium-activated chloride channel current curve after adding 30 μM compound A01, Ani9, 30, 39. The IC of compound 39 provided in the present invention in the whole-cell patch clamp experiment 50 The activity data is 6.7 μM. Figure 2 shown.

[0237] The whole-cell patch clamp experiment results of some compounds provided in the present invention that have an inhibitory effect on ANO2 channels are as follows Figure 3 As shown, Figure 3 The left figure shows the electrophysiological characteristic curve of the calcium-activated rate channel under applied voltage when no drug is added, and the right figure shows the electrophysiological characteristic curve of the calcium-activated chloride channel under applied voltage after adding 30 μM compounds A01, Ani9, 30, 39; Figure 4 is the IC of compound 39 for inhibition of electrophysiological activity of ANO2 channel 50 picture.

[0238] Among them, A01 and Ani9 are reported ANO1 inhibitors used as positive references;

[0239] The inhibitory activity data of the compounds provided in the present invention on other analgesic channels in whole-cell patch clamp experiments at a concentration of 30 μM:

[0240]

[0241]

[0242] Experiments show that the 4-arylthiophenecarboxylic acid compounds provided by the present invention have an inhibitory effect on ANO1 protein and are selective for ANO1 / ANO2.

[0243] 3. Cytotoxicity detection of the compound on human embryonic kidney cells HEK293T

[0244] The present invention uses the CCK8 kit to detect the cytotoxicity of the 4-arylthiophenecarboxylic acid compounds provided by the present invention on human embryonic kidney cells HEK293T.

[0245] Cell Counting Kit 8, abbreviated as CCK8 kit, is a rapid and highly sensitive detection kit based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) and is widely used for the detection of cell proliferation and cytotoxicity. The working principle is as follows: In the presence of an electron coupling reagent, WST-8 can be reduced by dehydrogenases in mitochondria to produce a highly water-soluble orange-yellow formazan product. The intensity of the color is proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured at a wavelength of 450 nM using an enzyme-linked immunosorbent assay (ELISA) reader to indirectly reflect the number of viable cells.

[0246] Among them, adriamycin is an antitumor drug - cytotoxic drug and is used as a positive reference; CaCC inh -A01 is a reported ANO1 inhibitor and is used as a comparison:

[0247] Note: n = 3; *P < 0.05; **P < 0.01; ***P < 0.001; CaCC inh -A01 group compared with the non-CaCC inh -A01 group comparison;

[0248] Cytotoxicity data of the compounds provided by the present invention on HEK293T at concentrations of 100 μM and 30 μM:

[0249]

[0250] The above experiments show that at a concentration of 30 μM, the cell proliferation and inhibition rate of compound 39 provided by the present invention are reduced to less than 20%. The cytotoxicity of the compounds of the present invention may be independent of ANO1 inhibitory activity, which can reduce the influence of cytotoxicity on experimental results.

[0251] 4. Acute toxicity detection of the compounds of the present invention on mice

[0252] The present invention conducted an acute toxicity experiment on adult male C57BL / 6 mice via intragastric administration. Using the maximum dose method, under the premise of reasonable maximum administration concentration and administration volume, a single dose or multiple doses within 24 hours (the dose generally does not exceed 5 g / kg body weight) were administered at the maximum allowed dose, and the reactions of the animals were observed.

[0253] The experimental results showed that all adult C57BL / 6 mice (n = 8) intragastrically administered with compound 39 at a dose of 1 g / kg survived after administration. The acute toxicity reactions of the animals were not obvious, and there were no strong adverse reactions such as rapid breathing or convulsions. The willingness of the mice to move began to decline 15 minutes after administration, gradually returned to normal about 6 hours later, and there were no subsequent abnormal reactions. The acute toxicity experiment indicated that in mice, compound 39 provided by the present invention is very safe via intragastric administration at least at a dose of 1 g / kg.

[0254] 5. Detection of the cardiotoxicity of the compounds of the present invention on the hERG potassium channel

[0255] The patch clamp technique was used to detect the cardiotoxicity of the 4-arylthiophenecarboxylic acid compounds provided by the present invention on the rapidly activating delayed rectifier potassium channel (Ikr) encoded by the human ether-a-go-go related gene hERG.

[0256] The patch clamp experimental results of some compounds provided by the present invention on the hERG channel are as Figure 5 shown. The patch clamp was used to measure the inhibition rate of the compounds on the hERG channel current at concentrations of 30 μM, 10 μM, 3 μM, 1 μM, and 0.3 μM to plot and fit the IC 50 . Among them, cisapride was used as a positive reference. The above experiments showed that the hERG inhibition IC 50 of compound 39 provided by the present invention was greater than 30 μM, and almost no cardiotoxicity mediated by the hERG channel was produced.

[0257] 6. Evaluation of the analgesic effect of the compounds of the present invention in a formalin-induced inflammatory pain model in mice

[0258] The formalin-induced pain experiment is a common spontaneous pain model for evaluating peripheral analgesic drugs. After injecting formalin subcutaneously into the hind paw of a rat or mouse, obvious and persistent pain reactions begin to appear 10 minutes later, specifically manifested as the mouse retracting or shaking and licking and biting the injected foot, mainly used for some studies on pain mechanisms. The specific experimental steps are as follows:

[0259] Adult male C57BL / 6 mice were randomly and evenly divided into a control group and a drug administration group, and fasted for 8 hours without water restriction. They were placed in a formalin experiment automatic monitor at 3 - 4 mice per batch to adapt for 30 min. After adaptation, the drug solution was administered by gavage (the Vehicle group was given a solvent without the compound). 15 min later, a metal ring for monitoring was placed on the right hind limb of the mouse in the supine position, and then 20 μL of formalin solution was subcutaneously injected into the palm of the paw with a microsyringe. Immediately after injection, the mouse was placed in the formalin automatic monitor to record the number of foot licks for 1 h. Data processing was performed using Origin software and ANOVA.

[0260] The experimental results are as Figure 6 shown, where ibuprofen and pregabalin are first-line drugs for peripheral analgesia and are used as positive references:

[0261] Note: *P < 0.05; **P < 0.01; ***P < 0.001; NS P > 0.05; n = 7;

[0262] It has been experimentally proven that Compound 39 provided by the present invention has obvious analgesic effects at two doses of 40 mg / kg and 80 mg / kg, and the analgesic effect at the 80 mg / kg dose is better, which is comparable to the analgesic effects of the peripheral analgesic positive drugs ibuprofen and pregabalin at 40 mg / kg.

[0263] 7. Evaluation of the analgesic effect of the compound of the present invention on mice in a capsaicin-induced inflammatory pain model

[0264] Capsaicin is a ligand of transient receptor potential vanilloid subtype 1 (TRPV1), and can promote the release of various inflammatory substances by activating TRPV1, causing tissue inflammation. After injecting the capsaicin solution subcutaneously into the hind paw of a mouse, the mouse will show obvious pain responses within the next five minutes, mainly manifested as the mouse continuously licking the paw injected with capsaicin. The specific experimental steps are as follows:

[0265] Adult male C57BL / 6 mice were randomly and evenly divided into a control group and a drug administration group, and fasted for 8 hours without water restriction. They were placed on an isolated observation table at four mice per group to adapt for 30 min. After adaptation, the drug solution was administered by gavage. 0.5 h later, 20 μL of capsaicin solution was subcutaneously injected into the center of the right hind paw of the mouse in the supine position. Immediately after injection, the mouse was placed back on the observation table for a 5 - min video recording. The duration of foot licking by the mouse was statistically analyzed, and data processing was performed using Origin software and ANOVA.

[0266] The experimental results are as Figure 7 shown, where ibuprofen and pregabalin are first-line drugs for peripheral analgesia and are used as positive references; SB-705498 is a small molecule inhibitor targeting TRPV1 in clinical phase II and is used as a positive reference:

[0267] Note: *P < 0.05; **P < 0.01; ***P < 0.001; NS P > 0.05; n = 7;

[0268] It has been experimentally proven that Compound 39 provided by the present invention has an obvious analgesic effect on this model at a dose of 80 mg / kg, which is equivalent to the analgesic activity of the TRPV1 inhibitor at a dose of 40 mg / kg.

[0269] Evaluation of the analgesic effect of the compound of the present invention on rats in an inflammatory pain model induced by Freund's complete adjuvant (CFA)

[0270] Freund's complete adjuvant is one of the conventional models for studying chronic inflammatory pain in rodents. The components in CFA include inactivated viruses, which achieve the purpose of inflammation by causing antigen-antibody reactions in the body. After injecting the CFA solution subcutaneously into the hind paws of rats, the hind feet of the rats will gradually show swelling and hyperalgesia. This model is divided into an acute phase (within 24 hours) and a chronic phase (after 24 hours). As time goes by, rats will become more and more sensitive to non-noxious stimuli. The specific experimental steps are as follows:

[0271] Adult male Sprague Dawley rats were randomly and evenly divided into a control group and a drug administration group, and fasted for 8 hours without water deprivation. 100 μL of CFA solution was injected subcutaneously into the center of the right hind paw of the rats in the supine position (the control group was injected with normal saline). After 12 hours, the rats were placed on an isolated observation table in groups of 3 to adapt for 30 min. After the adaptation was completed, the drug solution was administered by gavage. After 0.5 hour, the pain threshold of the rats was measured using an electronic needle. After 7 days, the change in the pain threshold of the rats after gavage was measured in the same way. Data processing was performed using Origin software and ANOVA.

[0272] The experimental results are as Figure 8 shown, where ibuprofen and pregabalin are first-line drugs for peripheral analgesia and are used as positive references:

[0273] Note: *P < 0.05; **P < 0.01; ***P < 0.001; NS P > 0.05; n = 7;

[0274] It has been experimentally proven that Compound 39 provided by the present invention has obvious analgesic activity at two doses of 40 mg / kg and 80 mg / kg, which is equivalent to the analgesic effects of the peripheral analgesic positive drugs ibuprofen and pregabalin.

[0275] 9. Evaluation of the analgesic effect of the compound of the present invention on rats in a bone cancer pain model

[0276] Since the pain manifested by animals with tibial bone cancer pain is similar to that of clinical metastatic bone cancer pain, the tibial bone cancer pain model can be regarded as a metastatic bone cancer pain model. Cancer pain is a special type of inflammatory pain, and a large number of inflammatory factors are released around the tumor tissue, accompanied by the occurrence of pain behaviors. Injecting breast cancer cells into the bone marrow cavity of the rat tibia, after the tumor cells grow for ten days, the rats will show stable pain behaviors, that is, hyperalgesia. As the tumor grows, the cancer cells will gradually erode the bone mass and cortex of the rat tibia. Phenotypically, the leg at the rat's tibia will gradually swell and become more and more sensitive to non-noxious stimuli. The specific experimental steps are as follows:

[0277] Adult female Sprague Dawley rats were randomly and evenly divided into a control group and a drug administration group. The rats were intraperitoneally injected with 10% chloral hydrate. After the anesthesia effect appeared in the rats, they were fixed on the operating table. Wipe the knee joint with alcohol, make a 1-cm transverse incision at the knee joint to expose the knee triangle. Insert a 22G gauge needle into the knee triangle until there is a sense of falling through, then remove it, insert a micro syringe along the original path, and inject the cell solution. Slowly pull out the needle, seal the wound with bone wax, apply penicillin powder, suture, and disinfect the surgical wound with iodophor.

[0278] After ten days, observe the wound condition of the rats. Fast the rats for 8 hours without restricting water intake. Put them in separate observation tables in groups of 3 and let them adapt for 30 min. After the adaptation is completed, administer the drug solution by gavage. After 0.5 hour, use a Von Frey fiber instrument to measure the pain threshold of the rats. On the fourteenth day, measure the pain threshold of the rats after gavage in the same way. The data was processed using GraphPad Prism software, ANOVA.

[0279] The experimental results are as Figure 9 shown, where ibuprofen and gabapentin are first-line drugs for treating cancer pain and are used as positive references:

[0280] Note: *P < 0.05; **P < 0.01; ***P < 0.001; NS P > 0.05; n = 7;

[0281] It has been experimentally proven that Compound 39 provided by the present invention has significant analgesic activity against the bone cancer pain model at a dose of 80 mg / kg, and is stronger than the analgesic activities of ibuprofen and pregabalin.

[0282] The above experiments prove that the 4-arylthiophenecarboxylic acid compounds provided by the present invention have significant analgesic effects on inflammatory pain and bone cancer pain.

[0283] This application describes multiple embodiments, but the description is exemplary rather than restrictive, and it will be apparent to those of ordinary skill in the art that there can be more embodiments and implementations within the scope encompassed by the embodiments described in this application.

Claims

1. A 4-arylthiophenecarboxylic acid compound, selected from the following compounds: 4-(4-chlorophenyl)-2-(2,5-dichlorobenzamido)thiophene-3-carboxylic acid; 2-(2-bromo-5-chlorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid.

2. A pharmaceutical composition, the pharmaceutical composition comprising a pharmacologically effective amount of the compound according to claim 1 and a pharmaceutically acceptable carrier.

3. A 4-arylthiophenecarboxylic acid compound, the compound being: 4-(4-chlorophenyl)-2-(2,5-difluorobenzamido)thiophene-3-carboxylic acid.

4. A pharmaceutical composition, the pharmaceutical composition comprising a pharmacologically effective amount of the compound according to claim 3 and a pharmaceutically acceptable carrier.

5. Use of the compound according to claim 3 or the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating diseases related to ANO1 protein inhibitors, wherein, The treatment of diseases related to ANO1 protein inhibitors is for the analgesia of inflammatory pain or the analgesia of bone cancer pain.

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