A substituted pyridine derivative and its application

Abstract

The present invention discloses a substituted pyridine derivative and its application, belonging to the technical field of pharmaceutical chemistry. The substituted pyridine derivative is a compound represented by general formula I or a pharmaceutically acceptable salt thereof. The substituted pyridine derivative of the present invention can be used as an ALKBH5 small molecule inhibitor, has obvious inhibitory activity on ALKBH5, inhibits RNA demethylase at the protease level, modifies and regulates m 6 A level can be used to prepare drugs for treating diseases related to ALKBH5 dysfunction.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to a substituted pyridine derivative and application thereof. Background Art

[0002] The dynamic modification of RNA plays a vital role in many physiological processes of living organisms, such as cell cycle regulation, cell proliferation and differentiation, and is an important research topic in the field of epigenetic regulation. 6 -methyladenosine (N 6 -methyladenosine, m 6 A) is the most common and conservative modification of messenger RNA (mRNA) and is also one of the hot topics of current research. ALKBH5 is an mRNA demethylase that can regulate m 6 A level. Studies have shown that ALKBH5 is abnormally expressed in a variety of cancers, such as ovarian cancer, endometrial cancer, cervical cancer, breast cancer, acute myeloid leukemia and gastric cancer.

[0003] Therefore, the use of small molecule inhibitors to inhibit the enzymatic activity of ALKBH5 can be used to treat indications related to ALKBHI5 protein dysfunction. Currently, there are no reports of highly active ALKBH5 small molecule inhibitors. Summary of the invention

[0004] Purpose of the invention: Based on the lead compounds of substituted pyridine, a series of derivatives were designed and synthesized, and a preliminary structure-activity relationship discussion was conducted. Finally, a new type of ALKBH5 small molecule inhibitor with excellent activity was obtained, which can significantly inhibit the action of ALKBH5 on m 6 The demethylation activity of A is expected to become a promising anti-tumor drug candidate.

[0005] One of the purposes of the present invention is to provide a compound as shown in general formula I or a pharmaceutically acceptable salt thereof:

[0006]

[0007] in,

[0008] R 1 Selected from -COOR a 、-CONR b R c , -CO-NH-OH, cyano, hydroxyl, thiol, halogen, nitro, amino, methoxy, trifluoromethyl, where R a , R b , R cEach independently represents hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, phenyl or substituted phenyl, the substituent of the substituted phenyl is halogen, C1-C4 alkyl, C1-C4 haloalkyl, cyano, hydroxyl, mercapto, halogen, nitro, amino, methoxy or trifluoromethyl;

[0009] R 2 Selected from H, F, C1-C4 alkyl or C1-C4 haloalkyl;

[0010] L 1 , L 2 Selected from carbonyl, sulfonyl, amino, methylene,

[0011] Ring A is selected from substituted phenyl, substituted or unsubstituted naphthyl, including

[0012] Where R d is substituted or unsubstituted phenyl.

[0013] In certain preferred embodiments, R d When it is a substituted phenyl group, the substituent is selected from fluorine, nitro, trifluoromethyl or methoxy.

[0014] In certain preferred embodiments, R 1 Selected from COOH, COOCH3 or CO-NH-OH.

[0015] In certain preferred embodiments, R 2 Selected from H or CH3.

[0016] In certain preferred embodiments, L 1 , L 2 is selected from carbonyl, sulfonyl or methylene.

[0017] In some preferred embodiments, the pharmaceutically acceptable salts include acid addition salts formed by the compound of formula I and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid; and also include basic salts formed by the compound of formula I and inorganic bases: sodium salt, potassium salt, lithium salt, calcium salt, zinc salt, magnesium salt, meglumine salt, tromethamine salt, diethylamine salt or ethanolamine salt.

[0018] The compounds of the general formula I of the present invention are preferably the following compounds:

[0019]

[0020]

[0021] The above-mentioned compounds of general formula I of the present invention may also exist in the form of their salts, which are converted into compounds of general formula I in vivo and have the same pharmacological effects as the compounds of general formula I. For example, within the scope of the present invention, the compounds of the present invention are converted into pharmaceutically acceptable salt forms according to processes known in the art, and they are used in the form of salts.

[0022] In certain embodiments, the compounds according to Formula I may contain acidic or basic functional groups sufficient to form salts. Representative salts include pharmaceutically acceptable inorganic bases, organic bases.

[0023] Another object of the present invention is to provide a pharmaceutical composition, which comprises a compound of formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0024] The pharmaceutical composition of the present invention can be administered in various known ways, such as orally, by injection, or by inhalation spray. The pharmaceutical composition of the present invention can be administered alone or in combination with other drugs. The oral composition can be in any orally acceptable dosage form, including but not limited to tablets, capsules, suspensions and solutions. Commonly used pharmaceutically acceptable carriers or excipients include diluents, surfactants, lubricants, antioxidants, adhesives, colorants, emulsifiers, etc. Sterile injectable compositions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents that can be used include water, sodium chloride solution, etc.

[0025] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The dosage level selected depends on a variety of factors, including the activity of the specific compound of the present invention or its salt used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0026] Another object of the present invention is to provide a use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease associated with ALKBH5 protein dysfunction. The disease associated with ALKBH5 protein dysfunction is glioblastoma, ovarian cancer, endometrial cancer, cervical cancer, breast cancer, acute myeloid leukemia, pituitary adenoma, oral squamous cell carcinoma, esophageal squamous cell carcinoma, gastric cancer, hepatitis B virus-related hepatocellular carcinoma, and intrahepatic bile duct carcinoma.

[0027] Beneficial effects: The compounds of general formula I prepared by the present invention and their pharmaceutically acceptable salts are a class of effective ALKBH5 small molecule inhibitors, which have obvious inhibitory activity against ALKBH5, inhibit RNA demethylase at the protease level, modify and regulate m 6 A level, can be used to treat diseases related to ALKBH5, such as ovarian cancer, endometrial cancer, cervical cancer, breast cancer and other cancers. Therefore, the above compounds can be used to prepare drugs for treating clinical diseases related to ALKBH5. DETAILED DESCRIPTION

[0028] The preparation methods of the compounds of general formula I of the present invention are described below in conjunction with specific examples, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily performed by those skilled in the art to which the present invention belongs.

[0029] The starting materials, reaction reagents, etc. used in the specific embodiments of the present invention are all commercially available. The present invention can be prepared into a salt form by a commonly used salt-forming method in the art, for example: at room temperature, the compound is dissolved in hydrochloric acid ethanol for reaction to generate a hydrochloride; or benzenesulfonic acid is added thereto for reaction to generate a benzenesulfonate; or at room temperature, the compound is dissolved in a methanol solution of sodium hydroxide for reaction to generate a sodium salt; or under reflux conditions, ethanolamine is added to an acetone solution thereof for reaction to generate an ethanolamine salt.

[0030] The experimental methods in the examples of the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by raw material or product manufacturers. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0031] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). 1 H NMR and 13 C NMR spectra were measured by Bruker AV-300 NMR spectrometer, the solvent was deuterated dimethyl sulfoxide (DMSO-d6), and the internal standard was tetramethylsilane (TMS). The mass spectrum was measured by Agilent's 1946A-MSD mass spectrometer (ESI-MS). The chemical reaction was detected by 0.25 mm GF254 thin layer chromatography silica gel plate and observed by ZF7 triple UV analyzer.

[0032] In the examples, unless otherwise specified, the reaction is carried out under air atmosphere. In the examples, unless otherwise specified, the reaction temperature is room temperature of 20°C to 30°C.

[0033] Example 1: Preparation of 2-(((3-propenylaminophenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 1) 1

[0035] Step 1: Synthesis of compound 1-2

[0036] Compound 1-1 (3 g, 19.85 mmol) was dissolved in 20 mL of pyridine, and SeO2 (3.3 g, 29.77 mmol) was added. The reaction was allowed to proceed at 120 °C for 2 h. After the reaction was completed by TLC monitoring, most of the pyridine was removed by rotary evaporation, 10 ml of water was added, and the residue was removed by filtration. The aqueous phase was adjusted to pH = 5 using 2M HCl, and a large amount of solid precipitated. Compound 1-2 was obtained by filtration as a white-grey solid with a yield of 55.63%. m / z (ESI-MS): 180.1 [MH] - .

[0037] Step 2: Synthesis of compounds 1-4

[0038] Compound 1-2 (1.00 g, 5.52 mmol), compound 1-3 (1.67 g, 8.28 mmol) and NMI (1.59 ml, 8.28 mmol) were dissolved in 30 mL of anhydrous acetonitrile, reacted at room temperature for 15 min, and then tetramethyl chlorouronium hexafluorophosphate (1.59 g, 8.28 mmol) was added, and the reaction was continued at room temperature overnight. The reaction was monitored by TLC, and the solvent was removed by rotary evaporation. Compound 1-4 was purified by silica gel column chromatography (DCM: MeOH = 100: 1) as a white solid with a yield of 75.87%. m / z (ESI-MS): 366.3 [M+H] + .

[0039] Step 3: Synthesis of compounds 1-5

[0040] Compound 1-4 (1.50 g, 4.11 mmol) was dissolved in 30 mL of methanol, and Pd / C (10%) was added in batches. The reaction system was stirred at room temperature overnight under a hydrogen atmosphere. The reaction was monitored by TLC and filtered with the aid of diatomaceous earth. The crude product after the filtrate was concentrated was purified by silica gel column chromatography (DCM: MeOH = 80: 1) to obtain compound 1-5 as a white solid with a yield of 71.17%. m / z (ESI-MS): 336.3 [M+H] + .

[0041] Step 4: Synthesis of compounds 1-6

[0042] Compound 1-5 (900 mg, 2.68 mmol) was dissolved in a mixture of MeOH, H2O and THF (1:1:1), and LiOH (193 mg, 8.05 mmol) was added. The reaction was allowed to react at room temperature for 5 h. TLC monitored the reaction completion, and the solvent was removed by rotary evaporation. An appropriate amount of water was added, and 2M HCl was adjusted to pH = 3. A white precipitate was precipitated and filtered to obtain compound 1-6 as a white solid with a yield of 92.77%. m / z (ESI-MS): 320.0 [MH] - .

[0043] Step 5: Synthesis of Compound 1

[0044] Under argon atmosphere, compound 1-5 (100 mg, 0.311 mmol) was dissolved in 5 mL of anhydrous dichloromethane, cooled to 0 ° C, and compound 1-7 (30 μl, 0.373 mmol) was added dropwise. The reaction mixture was then stirred at room temperature overnight. TLC monitored the completion of the reaction, and the crude product was washed with water 3 times, dried over anhydrous sodium sulfate, and concentrated to obtain compound 1 as a white solid with a yield of 53.93%. 1 H NMR (300MHz, DMSO-d6) δ13.19(s,1H),10.58(s,1H),8.90(d,J=4.9Hz,1H),8.41(t,J=2.0Hz,1H),8.34-8.28(m,1H),8.07(dd,J=4. 9,1.7Hz,1H),7.99(dd,J=8.2,1.9Hz,1H),7.78-7.67(m,1H),7.59(t,J=8.0Hz,1H),6.55-6.23(m,2H),5.82(dd,J=9.9,2.2Hz,1H).

[0045] Example 2: Preparation of 2-(((2-propenylaminophenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 2)

[0046]

[0047] Referring to the preparation method of Example 1, compound 2 was synthesized according to steps 2-5 in Example 1, except that compound 1-3 was replaced by compound 2-1 in step 2 of this example.

[0048] Compound 2-2 was a white solid with a yield of 70.34%. m / z (ESI-MS): 366.3 [M+H] + .

[0049] Compound 2-3 was a white solid with a yield of 68.29%. m / z (ESI-MS): 336.3 [M+H] + .

[0050] Compound 2-4 was a white solid with a yield of 90.30%. m / z (ESI-MS): 320.0 [MH] -.

[0051] Compound 2 was a white solid with a yield of 50.21%. 1 H NMR (300MHz, DMSO-d6) δ9.90 (s, 1H), 8.91 (d, J = 4.9Hz, 1H), 8.32 (d, J = 8.3Hz, 2H), 8.19-8.00 (m, 2H), 7.71 (t, J = 7.9Hz ,1H),7.39(t,J=7.7Hz,1H),6.54(dd,J=17.0,10.2Hz,1H),6.30(dd,J=17.0,1.7Hz,1H),5.89(dd,J=10.3,1.7Hz,1H).

[0052] Example 3: Preparation of 2-(((3-(2-chloroacetylamino)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 3)

[0053]

[0054] The preparation method of Reference Example 1 is different in that compound 1-7 is replaced by compound chloroacetyl chloride in step 5.

[0055] Compound 3 was a white solid, and the yield was 56.97%. 1 H NMR (300MHz, DMSO-d6) δ10.76(s,1H),8.91(d,J=4.9Hz,1H),8.36(t,J=2.0Hz,1H),8.30(s,1H),8.10 (dd,J=5.0,1.7Hz,1H),7.95-7.84(m,1H),7.76(d,J=7.8Hz,1H),7.62(t,J=8.0Hz,1H),4.31(s,2H).

[0056] Example 4: Preparation of 2-(((2-(2-chloroacetylamino)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 4)

[0057]

[0058] The preparation method of reference example 1 was used, except that compound 2-4 was used to replace compound 1-6, and compound chloroacetyl chloride was used to replace compound 1-7 in step 5. Compound 4 was obtained as a white solid with a yield of 53.28%. 1H NMR (300MHz, DMSO-d6) δ10.06(s,1H),8.92(d,J=5.0Hz,1H),8.32(s,1H),8.15-8.06(m,3H),7.81-7.64(m,1H),7.50-7.37(m,1H),4.49(s,2H).

[0059] Example 5: Preparation of 2-(((3-((4,6-dichloro-1,3,5-triazin-2-yl)amino)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 5)

[0060]

[0061] Compound 1-5 (100 mg, 0.311 mmol) and triethylamine (43 μl, 0.311 mmol) were dissolved in 10 mL of acetone, and 5 mL of cyanuric chloride acetone solution (57 mg, 0.311 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 h. A large amount of white precipitate was precipitated, filtered, and the filter cake was washed with a small amount of acetone to obtain compound 5 as a white solid with a yield of 53.41%. 1 H NMR(300MHz,DMSO-d6)δ11.49(s,1H),8.92(dt,J=5.0,1.3Hz,1H),8.29(m,2H),8.10(dt ,J=5.0,1.7Hz,1H),8.01-7.90(m,1H),7.88-7.75(m,1H),7.67(dt,J=16.0,8.0Hz,1H).

[0062] Example 6: Preparation of 2-(((2-((4,6-dichloro-1,3,5-triazin-2-yl)amino)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 6)

[0063]

[0064] The synthesis steps were the same as those in Example 5, except that compound 2-4 was used in place of compound 1-5. Compound 6 was obtained as a white solid with a yield of 47.97%. 1 H NMR (300MHz, DMSO-d6) δ10.29(s,1H),8.80(d,J=1.5Hz,1H),8.68(d,J=7.4Hz,1H),8.04(dd,J=7.4,1.5Hz,1H),7 .83(dd,J=7.5,1.5Hz,1H), 7.62(td,J=7.5,1.6Hz,1H), 7.47(dd,J=7.6,1.6Hz,1H), 7.37(td,J=7.5,1.6Hz,1H).

[0065] Example 7: Preparation of methyl 2-(((3-formyl-4-hydroxyphenyl)sulfonyl)carbamoyl)isonicotinate (Compound 7)

[0066]

[0067] Step 1: Synthesis of compound 7-2

[0068] Add 10 mL of chlorosulfonic acid to a 100 mL reaction bottle and pre-cool in an ice bath for 30 min. Dissolve compound 7-1 (2 g, 14.69 mmol) in 2 mL of dichloromethane and slowly drop it into chlorosulfonic acid. Continue to react in an ice bath for 30 min, then return to room temperature and react overnight. After monitoring the reaction by TLC, slowly drop the reaction solution into ice water to quench, extract with EA 3 times, wash with saturated brine, dry with anhydrous sodium sulfate, evaporate under reduced pressure to remove the solvent, and purify by silica gel column chromatography (PE: EA = 10: 1) to obtain compound 7-2 as a white solid with a yield of 60.92%. m / z (ESI-MS): 235.6 [M+H] + .

[0069] Step 2: Synthesis of compound 7-3

[0070] Compound 7-2 (2 g, 8.52 mmol) was dissolved in 30 mL of anhydrous 1,4-dioxane, and 100 mL of 0.4 M ammonia in 1,4-dioxane was added dropwise, and the reaction was allowed to proceed overnight at room temperature. TLC monitored the completion of the reaction, and appropriate amount of water was added to the reaction solution, 2 M HCl was used to adjust pH = 4, and the aqueous phase was extracted with DCM for 3 times. The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by evaporation under reduced pressure. Compound 7-3 was purified by silica gel column chromatography (PE: EA = 2: 1) as a white solid with a yield of 82.86%. m / z (ESI-MS): 216.2 [M + H] + .

[0071] Step 3: Synthesis of compound 7-4

[0072] Compound 7-3 (1.5 g, 6.97 mmol) was dissolved in 30 mL of anhydrous dichloromethane, and 10 mL of BBr3 (2.0 M in methylene chloride) was slowly added dropwise at -20 °C. After reacting at -20 °C for 30 min, the mixture was returned to room temperature and reacted overnight. After TLC monitoring, the reaction was completed, and methanol was slowly added dropwise to quench the boron tribromide. The solvent was removed by evaporation under reduced pressure, and compound 7-4 was purified by silica gel column chromatography (pure DCM) as a white solid with a yield of 71.31%. m / z (ESI-MS): 202.2 [M+H] + .

[0073] Step 4: Synthesis of compound 7

[0074] Referring to the preparation method of Example 1, the synthesis steps are the same as step 2 of Example 1, except that compound 7-4 is used to replace compound 1-3. Compound 7 is a white solid, and the yield is 67.20%. 1 H NMR (300MHz, DMSO-d6) δ12.10(s,1H),10.49(s,1H),9.13(d,J=5.1Hz,1H),8.56-8.41(m,2H),8.43-8.22(m,2H),7.40(d,J=8.8Hz,1H),3.93(s,3H).

[0075] Example 8: Preparation of 2-(((3-formyl-4-hydroxyphenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 8)

[0076]

[0077] Referring to the preparation method of Example 1, the synthesis steps are the same as step 4 of Example 1, except that compound 7 is used to replace compound 1-5. Compound 8 is obtained as a white solid with a yield of 88.39%. 1 H NMR (300MHz, DMSO-d6) δ12.13(s,1H),10.52(s,1H),9.10(d,J=5.1Hz,1H),8.58-8.43(m,2H),8.43-8.22(m,2H),7.41(d,J=8.8Hz,1H).

[0078] Example 9: N 2 -(3-formyl-4-hydroxyphenyl)sulfonyl)-N 4 Preparation of -hydroxypyridine-2,4-diamide (Compound 9)

[0079]

[0080] Potassium hydroxide (11.2 g, 200 mmol) was dissolved in 30 mL of anhydrous methanol and added dropwise to a methanol solution of hydroxylamine hydrochloride (9.34 g, 134.4 mmol) under ice bath conditions. The reaction was stirred for 1 h. The precipitate was removed by filtration, and the filtrate was collected to obtain a fresh hydroxylamine solution for further reaction. Compound 7 (100 mg, 0.274 mmol) was dissolved in a fresh hydroxylamine solution under ice bath conditions. The reaction mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC to terminate, and 2 M HCl was used for neutralization, precipitation was separated, suction filtered, and washed with a small amount of water to obtain compound 9 as a light yellow solid with a yield of 21.33%. 1H NMR (300MHz, DMSO-d6) δ11.60(s,1H),10.97(d,J=4.9Hz,1H),10.17(s,1H),8.96(d,J=4.9Hz,1H),8.85-8.73 (m, 2H), 8.22 (d, J = 1.6Hz, 1H), 7.95 (dd, J = 7.4, 1.5Hz, 1H), 7.88 (dd, J = 7.6, 1.5Hz, 1H), 7.03 (d, J = 7.5Hz, 1H).

[0081] Example 10: Preparation of 2-((3-formyl-4-hydroxybenzyl)carbamoyl)isonicotinic acid (Compound 10)

[0082]

[0083] Step 1: Synthesis of compound 10-2

[0084] Compound 10-1 (2 g, 16.24 mmol) was dissolved in 40 mL THF, 40 mL NaHCO3 aqueous solution (4.09 g, 48.72 mmol) was added, (Boc)2O (4.10 mL, 17.86 mmol) was added dropwise, and the reaction was carried out at room temperature for 4 h. TLC monitored the reaction to be complete. The reaction solution was extracted with EA three times, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to remove the solvent to obtain 3.3 g of pink oily liquid, which was directly used for the next step without further purification. m / z (ESI-MS): 224.3 [M+H] + .

[0085] Step 2: Synthesis of compound 10-3

[0086] Compound 10-2 (3 g, 13.44 mmol), paraformaldehyde (2.42 mg, 80.62 mmol), triethylamine (7.47 mL, 16.12 mmol) were dissolved in 30 mL of acetonitrile, and magnesium chloride (1.92 g, 20.15 mmol) was added in batches. After all the addition was completed, the temperature was raised and refluxed for 12 h. TLC monitoring showed that the reaction was complete. The reaction solution was extracted with EA and water, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and purified by silica gel column chromatography (PE: EA = 8: 1) to obtain compound 10-3 as a white solid with a yield of 30.44%. m / z (ESI-MS): 252.3 [M + H] + .

[0087] Step 3: Synthesis of compound 10-4

[0088] Compound 10-3 (800 mg, 3.18 mmol) was dissolved in 30 mL of hydrogen chloride-ethyl acetate solution and reacted at room temperature overnight. A large amount of white solid precipitated, which was filtered to obtain compound 10-4 as a white solid with a yield of 89.71%. m / z (ESI-MS): 188.6 [M+H] + .

[0089] Step 4: Synthesis of compound 10-5

[0090] Referring to the preparation method of Example 1, the synthesis steps are the same as step 2 of Example 1, except that compound 10-4 is used to replace compound 1-3. Compound 10-5 is obtained as a white solid with a yield of 73.67%. m / z (ESI-MS): 315.3 [M+H] + .

[0091] Step 5: Synthesis of compound 10

[0092] Referring to the preparation method of Example 1, the synthesis steps are the same as step 4 of Example 1, except that compound 10-5 is used to replace compound 1-5. Compound 10 is obtained as a white solid with a yield of 85.60%. 1 H NMR (300MHz, DMSO-d6) δ11.31(s,1H),10.18(s,1H),9.02-8.72(m,2H),8.30(d,J=1.5Hz,1H),8.09(dd,J =7.4, 1.5Hz, 1H), 7.79 (q, J = 1.4Hz, 1H), 7.25 (m, 1H), 6.88 (d, J = 7.5Hz, 1H), 4.49 (dt, J = 10.1, 1.0Hz, 2H).

[0093] Example 11: Preparation of 2-((3-formyl-4-hydroxybenzoyl)carbamoyl)isonicotinic acid (Compound 11)

[0094]

[0095] Step 1: Synthesis of compound 11-2

[0096] Compound 11-1 (500 mg, 3.40 mmol) and potassium carbonate (939 mg, 12.33 mmol) were dissolved in 3 mL DMSO, and 2 mL 30% H2O2 aqueous solution was added under ice bath conditions, and the reaction was allowed to proceed at room temperature for 10 min. The reaction was monitored by TLC, and a large amount of white precipitate was precipitated by adding water. Compound 11-2 was obtained as a white solid by suction filtration, with a yield of 80.36%. m / z (ESI-MS): 166.1 [M+H] + .

[0097] Step 2: Synthesis of compound 11-3

[0098] Referring to the preparation method of Example 1, the synthesis steps are the same as step 2 of Example 1, except that compound 11-2 is used to replace compound 1-3. Compound 11-3 is obtained as a white solid with a yield of 65.08%. m / z (ESI-MS): 329.3 [M+H] + .

[0099] Step 3: Synthesis of compound 11

[0100] Referring to the preparation method of Example 1, the synthesis steps are the same as step 4 of Example 1, except that compound 11-3 is used to replace compound 1-5. Compound 11 is obtained as a white solid with a yield of 87.21%. 1 H NMR(300MHz,DMSO-d6)δ12.81(s,1H),12.28(s,1H),11.46(s,1H),10.17(s,1H),8.85-8.75(m,2H),8 .27(d,J=1.4Hz,1H), 8.10(dd,J=7.6,1.5Hz,1H), 8.00(dd,J=7.6,1.5Hz,1H), 7.00(d,J=7.4Hz,1H).

[0101] Example 12: Preparation of 2-(((3-formyl-4-hydroxyphenyl)sulfonyl)(methyl)carbamoyl)isonicotinic acid (Compound 12)

[0102]

[0103] Step 1: Synthesis of compound 12-1a

[0104] Compound 1-1 (1 g, 7.29 mmol), benzyl bromide (1.5 g, 8.75 mmol) and K2CO3 (2 g, 14.58 mmol) were dissolved in 20 mL DMF and reacted at room temperature for 4 h. TLC monitored the reaction to be complete. The reaction solution was poured into 100 mL of water, extracted with EA 3 times, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and purified by silica gel column chromatography (PE) to obtain compound 12-1a, which was a colorless oily liquid with a yield of 87.44%. m / z (ESI-MS): 228.3 [M+H] + .

[0105] Step 2: Synthesis of compound 12-2a

[0106] Compound 12-1a (1.44 g, 6.34 mmol) was dissolved in 20 mL of pyridine, and SeO2 (1.48, 13.31 mmol) was added. The reaction was carried out at 120°C for 2 h. TLC monitoring showed that the reaction was complete and post-processing was performed. Most of the pyridine was removed by evaporation under reduced pressure. 10 mL of water was added and the mixture was filtered. The filtrate was adjusted to pH = 7 with saturated sodium bicarbonate, and a precipitate was precipitated and filtered to obtain compound 12-2a as a yellow solid with a yield of 68.08%. m / z (ESI-MS): 256.2 [MH]-.

[0107] Step 3: Synthesis of compound 12-1b

[0108] Methylamine hydrochloride (575 mg, 8.52 mmol) was dissolved in 20 mL of anhydrous dichloromethane, TEA (1.78 mL, 12.78 mmol) and DMAP (52 mg, 0.426 mmol) were added, and stirred at room temperature for 30 min. A dichloromethane solution of compound 7-2 (1 g, 4.26 mmol) was slowly added dropwise to the reaction system, and the reaction was allowed to proceed for 4 h at room temperature. TLC monitored the reaction to be complete. Water and DCM were used for extraction, the organic phases were combined, washed with citric acid, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and purified by silica gel column chromatography (PE: EA = 30: 1) to obtain compound 12-1b as a white solid with a yield of 71.22%. m / z (ESI-MS): 230.2 [M+H] + .

[0109] Step 4: Synthesis of compound 12-2b

[0110] Reference Example 7 preparation method, the synthesis steps are the same as Example 7 step 3, the difference is that the compound 12-2b is used to replace the compound 7-3. Compound 12-2b is a white solid, the yield is 59.61%. m / z (ESI-MS): 216.2 [M + H] + .

[0111] Step 5: Synthesis of compound 12-3

[0112] Referring to the preparation method of Example 1, the synthesis steps are the same as step 2 of Example 1, except that compound 12-2a is used to replace compound 1-2, and compound 12-2b is used to replace compound 1-3. Compound 12-3 is a white solid with a yield of 60.43%. m / z (ESI-MS): 455.4 [M+H] + .

[0113] Step 6: Synthesis of compound 12

[0114] Compound 12-3 (100 mg, 0.220 mmol) was dissolved in 4 mL of anhydrous dichloromethane, and 4 mL of BF3 in ether was slowly added dropwise, and stirred at room temperature overnight. TLC monitored the reaction to be complete. The reaction solution was neutralized with saturated NH4Cl aqueous solution, extracted with DCM, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to remove the solvent. Compound 12 was purified by silica gel column chromatography (DCM; MeOH = 20:1) as a white solid with a yield of 30.77%. 1 H NMR (300MHz, DMSO-d6) δ11.60(s,1H),10.17(s,1H),8.85(d,J=7.4Hz,1H),8.36(d,J=1.6Hz,1H),8.16(d, J=1.5Hz,1H),8.08(dd,J=7.6,1.4Hz,1H),7.80(dd,J=7.5,1.5Hz,1H),7.04(d,J=7.5Hz,1H),2.97(s,3H).

[0115] Example 13: Preparation of 2-(((3-formyl-4-hydroxyphenyl)sulfonamide)methyl)isonicotinic acid (Compound 13)

[0116] Step 1: Synthesis of compound 13-2

[0117] Compound 13-1 (2 g, 12.33 mmol) was dissolved in 30 mL of methanol, and (Boc)2O (2.84 mL, 12.33 mmol) and NiCl2·6H2O (300 mg, 1.23 μmol) were added at 0°C. After the reaction was stirred at 0°C for 5 min, NaBH4 (2.8 g, 73.98 mmol) was added in batches and reacted at room temperature for 24 h. TLC monitored the reaction to be complete. The solvent was evaporated under reduced pressure, extracted with water and EA, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and purified by silica gel column chromatography (PE:EA=3:1) to obtain compound 13-2, which was a yellow oily liquid with a yield of 24.36%. m / z (ESI-MS): 267.3 [M+H] + .

[0118] Step 2: Synthesis of compound 13-3

[0119] Compound 2 (800 mg, 3.00 mmol) was dissolved in 20 mL of methanol, and 20 mL of HCl-EA was added. The mixture was reacted at room temperature overnight. The precipitate was filtered to obtain compound 13-3 as a white solid with a yield of 41.77%. m / z (ESI-MS): 203.6 [M+H] + .

[0120] Step 3: Synthesis of compound 13-4

[0121] Reference Example 12 preparation method, the synthesis steps are the same as Example 12 step 3, the difference is that the compound 13-3 is used to replace methylamine hydrochloride. Compound 13-4 is a white solid, the yield is 69.76%. m / z (ESI-MS): 365.4 [M + H] + .

[0122] Step 4: Synthesis of compound 13-5

[0123] Referring to the preparation method of Example 7, the synthesis steps are the same as step 3 of Example 7, except that compound 13-4 is used to replace compound 7-3. Compound 13-5 is a white solid with a yield of 53.11%. m / z (ESI-MS): 351.3 [M+H] + .

[0124] Step 5: Synthesis of compound 13

[0125] Referring to the preparation method of Example 1, the synthesis steps are the same as step 4 of Example 1, except that compound 13-5 is used to replace compound 1-5. Compound 13 is obtained as a white solid with a yield of 89.23%. 1 H NMR (300MHz, DMSO-d6) δ11.50(s,1H),10.14(s,1H),8.65(d,J=7.5Hz,1H),8.19(d,J=1.5Hz,1H),7.99(t,J=11.9Hz,1H),7. 94(d,J=1.5Hz,1H), 7.83(dd,J=7.5,1.6Hz,1H), 7.74(dd,J=7.4,1.5Hz,1H), 7.03(d,J=7.5Hz,1H), 4.22(d,J=11.9Hz,2H).

[0126] Example 14: Preparation of 2-(((4-dihydroxyboryl-3-formylphenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 14)

[0127]

[0128] Step 1: Synthesis of compound 14-1

[0129] Compound 7 (1.0 g, 2.74 mmol) and potassium carbonate (1.52 g, 10.98 mmol) were dissolved in 5 mL of anhydrous DMF, stirred for 5 min under ice bath, and then CF3SO2Cl (1.17 mL, 10.98 mmol) was added and reacted at room temperature for 4 h. TLC monitored the reaction to be complete. The reaction solution was poured into 50 mL of water, extracted with EA 3 times, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to remove the solvent to obtain compound 14-1, which was a yellow oily liquid and was directly used for the next step without further purification. m / z (ESI-MS): 497.4 [M+H] + .

[0130] Step 2: Synthesis of compound 14-2

[0131] Compound 14-1 (500 mg, 1.01 mmol), B2pin2 (307 mg, 1.21 mmol), Pd(dppf)2·CH2Cl2 (7.68 mg, 10.07 μmol) and potassium acetate (297 mg, 3.02 mmol) were dissolved in 6 mL of 1,4-dioxane solution in a 25 mL sealed tube and reacted at 100 °C for 7 h. TLC monitored the reaction to be complete. The solvent was evaporated, extracted with EA and water, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and purified by silica gel column chromatography (pure DCM) to obtain compound 14-2 as a white solid with a yield of 11.03%. m / z (ESI-MS): 393.1 [M+H] + .

[0132] Step 3: Synthesis of compound 14

[0133] Referring to the preparation method of Example 1, the synthesis steps are the same as step 4 of Example 1, except that compound 14-2 is used to replace compound 1-5. Compound 14 is a white solid with a yield of 73.29%. 1 H NMR(300MHz,DMSO-d6)δ10.02(s,1H),8.85-8.77(m,2H),8.51(s,2H),8.39(d,J=1.4H z, 1H), 8.08 (dd, J = 7.4, 1.5 Hz, 1H), 7.97 ( dd, J = 7.5, 1.4 Hz, 1H), 7.87 ( d, J = 7.5 Hz, 1H).

[0134] Example 15: Preparation of 2-(((3-acetyl-4-hydroxyphenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 15)

[0135]

[0136] Compounds 15-2 to 15 were synthesized according to the steps of Example 8, except that compound 15-1 was used to replace compound 7-1. Compound 15-2 was obtained as a white solid with a yield of 78.04%. m / z (ESI-MS): 249.7 [M+H] + .

[0137] Compound 15-3 is a white-grey solid with a yield of 69.87%. m / z (ESI-MS): 230.2 [M+H] + .

[0138] Compound 15-4 was a white solid with a yield of 53.29%. m / z (ESI-MS): 216.2 [M+H] + .

[0139] Compound 15-5 was a white solid with a yield of 63.22%. m / z (ESI-MS): 379.3 [M+H] + .

[0140] Compound 15 was a white solid with a yield of 92.03%. 1 H NMR(300MHz,DMSO-d6)δ12.23(s,1H),8.92(d,J=4.9Hz,1H),8.43(d,J=2.5Hz,1H), 8.35-8.25(m,1H),8.12(dt,J=6.8,2.3Hz,2H),7.21(d,J=8.8Hz,1H),2.67(s,3H).

[0141] Example 16: Preparation of 2-(((3-acetyl-4-dihydroxyborylphenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 16)

[0142]

[0143] Compounds 16-1 to 16 were synthesized according to the steps of Example 14, except that compound 15-5 was used to replace compound 7. Compound 16-1 was obtained as a yellow oily liquid. m / z (ESI-MS): 511.4 [M+H] + .

[0144] Compound 16-2 was a white solid with a yield of 18.37%. m / z (ESI-MS): 407.2 [M+H] + .

[0145] Compound 16 was a white solid with a yield of 83.05%. 1H NMR(300MHz,DMSO-d6)δ8.84-8.70(m,2H),8.57(s,2H),8.39(d,J=1.5Hz,1H),8.06(d d, J=7.4, 1.5Hz, 1H), 7.91 (dd, J=7.4, 1.5Hz, 1H), 7.85 (d, J=7.5Hz, 1H), 2.71 (s, 3H).

[0146] Example 17: Preparation of methyl 2-((naphthalene-1-sulfonyl)carbamoyl)isonicotinate (Compound 17)

[0147]

[0148] Step 1: Synthesis of compound 17-2

[0149] Reference Example 7 preparation method, the synthesis steps are the same as Example 7 step 2, the difference is that compound 17-1 is used to replace compound 7-2. Compound 17-2 is a white solid, the yield is 83.31%. m / z (ESI-MS): 208.2 [M + H] + .

[0150] Step 2: Synthesis of compound 17

[0151] Referring to the preparation method of Example 1, the synthesis steps are the same as step 2 of Example 1, except that compound 17-2 is used to replace compound 1-3. Compound 17 is obtained as a white solid with a yield of 68.54%. 1 H NMR (300MHz, DMSO-d6) δ8.91(d,J=5.1Hz,1H),8.85(d,J=8.3Hz,1H),8.42(s,1H),8.33(d,J=7.3Hz ,1H),8.18(d,J=8.2Hz,1H),8.08-8.01(m,1H),8.01-7.92(m,1H),7.72-7.53(m,3H),3.93(s,3H).

[0152] Example 18: Preparation of 2-((naphthalene-1-ylsulfonyl)carbamoyl)isonicotinic acid (Compound 18)

[0153]

[0154] Referring to the preparation method of Example 1, the synthesis steps were the same as step 4 of Example 1, except that compound 17 was used to replace compound 1-5. Compound 18 was obtained as a white solid with a yield of 87.32%. 1H NMR (300MHz, DMSO-d6) δ8.91(d,J=5.1Hz,1H),8.85(d,J=8.3Hz,1H),8.42(s,1H),8.33(d,J =7.3Hz,1H),8.18(d,J=8.2Hz,1H),8.10-8.01(m,1H),8.01-7.91(m,1H),7.73-7.52(m,3H).

[0155] Example 19: N 4 -Hydroxy-N 2 Preparation of -(naphthalene-1-ylsulfonyl)pyridine-2,4-diamide (Compound 19)

[0156]

[0157] Compound 19 was synthesized according to the procedure of Example 9, except that Compound 17 was used instead of Compound 7.

[0158] Compound 19 was a light yellow solid, and the yield was 20.78%. 1 H NMR (300MHz, DMSO-d6) δ8.97-8.87(m,1H),8.66(s,1H),8.34(d,J=4.8Hz,2H),8.25-8.17(m,1H),8.05-7.91(m,2H),7.65-7.48(m,4H).

[0159] Example 20: Preparation of 2-((naphthalen-1-ylmethyl)carbamoyl)isonicotinic acid (Compound 20)

[0160]

[0161] Step 1: Synthesis of compound 20-2

[0162] Referring to the preparation method of Example 1, the synthesis steps are the same as step 2 of Example 1, except that compound 20-1 is used to replace compound 1-3. Compound 20-2 is a white solid with a yield of 78.26%. m / z (ESI-MS): 321.3 [M+H] + .

[0163] Step 2: Synthesis of compound 20

[0164] Referring to the preparation method of Example 1, the synthesis steps are the same as step 4 of Example 1, except that compound 20-2 is used to replace compound 1-5. Compound 20 is obtained as a white solid with a yield of 91.07%. 1H NMR (300MHz, DMSO-d6) δ9.26(t,J=6.2Hz,1H),8.65(d,J=4.9Hz,1H),8.30-8.22(m,1H),8.12-8.01(m,1H),7.83(dd, J=4.9,1.7Hz,1H),7.76(dd,J=7.4,2.0Hz,1H),7.65(dd,J=7.4,2.1Hz,1H),7.45-7.23(m,4H),4.81(d,J=6.2Hz,2H).

[0165] Example 21: Preparation of 2-((naphthalene-1-sulfonamido)methyl)isonicotinic acid (Compound 21)

[0166]

[0167] Step 1: Synthesis of compound 21-1

[0168] Referring to the preparation method of Example 12, the synthesis steps are the same as step 3 of Example 12, except that methylamine hydrochloride is replaced by compound 13-3, and compound 17-1 is replaced by compound 7-2. Compound 21-1 is obtained as a white solid with a yield of 79.52%. m / z (ESI-MS): 357.4 [M+H] + .

[0169] Step 2: Synthesis of compound 21

[0170] Referring to the preparation method of Example 1, the synthesis steps were the same as step 4 of Example 1, except that compound 21-1 was used to replace compound 1-5. Compound 21 was obtained as a white solid with a yield of 89.71%. 1 H NMR (300MHz, DMSO-d6) δ8.66(d,J=7.5Hz,1H),8.15-7.98(m,4H),7.94(d,J=1.5Hz,1H),7.84(dd,J=7.6,1.6 Hz,1H),7.81-7.70(m,2H),7.60(td,J=7.6,1.4Hz,1H),7.43(td,J=7.5,1.5Hz,1H),4.22(d,J=11.9Hz,2H).

[0171] Example 22: Preparation of 2-((1-naphthylamino)methyl)isonicotinic acid (Compound 22)

[0172]

[0173] Step 1: Synthesis of compound 22-2

[0174] Referring to the preparation method of Example 1, the synthesis steps are the same as step 2 of Example 1, except that compound 13-3 is used to replace compound 1-3, and compound 22-1 is used to replace compound 1-2. Compound 22-2 is obtained as a white solid with a yield of 78.26%. m / z (ESI-MS): 321.3 [M+H] + .

[0175] Step 2: Synthesis of compound 22

[0176] Referring to the preparation method of Example 1, the synthesis steps are the same as step 4 of Example 1, except that compound 22-2 is used to replace compound 1-5. Compound 22 is obtained as a white solid with a yield of 89.90%. 1 H NMR (300MHz, DMSO-d6) δ13.70(s,1H),9.27(t,J=6.0Hz,1H),8.77(d,J=5.0Hz,1H),8.28(dt,J=8.5,3.0Hz,1H),8.07(d,J =8.2Hz,1H),8.04-7.98(m,1H),7.93(s,1H),7.73(ddd,J=12.1,6.0,1.5Hz,2H),7.66-7.55(m,3H),4.74(d,J=6.0Hz,2H).

[0177] Example 23: Preparation of methyl 2-(((3-formyl-4-hydroxynaphthalen-1-yl)sulfonyl)carbamoyl)isonicotinate (Compound 23)

[0178]

[0179] Step 1: Synthesis of compound 23-2

[0180] Reference Example 10 preparation method, the synthesis steps are the same as Example 10 step 2, the difference is that compound 23-1 is used to replace compound 10-2. Compound 23-2 is a yellow solid, the yield is 60.21%. m / z (ESI-MS): 173.2 [M + H] + .

[0181] Step 2: Synthesis of compound 23-3

[0182] Compound 23-2 (5 g, 29.04 mmol) and potassium carbonate (6.09 g, 43.56 mmol) were dissolved in 10 mL DMF, iodomethane (2.71 mL, 43.56 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. TLC monitored the reaction completion, water was added and stirred for 30 min, EA was extracted 3 times, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and purified by silica gel column chromatography (PE: EA = 100: 1) to obtain compound 23-3 as a yellow solid with a yield of 88.01%. m / z (ESI-MS): 187.2 [M+H] + .

[0183] Step 3: Synthesis of Compound 23-4 to Compound 23

[0184] Referring to the preparation method of Example 7, compounds 23-4 to 23 were synthesized according to steps 1-4 of Example 7, except that compound 23-3 was used to replace compound 7-1.

[0185] Compound 23-4 was a white solid with a yield of 50.30%. m / z (ESI-MS): 285.7 [M+H] + .

[0186] Compound 23-5 was a white solid with a yield of 68.21%. m / z (ESI-MS): 266.3 [M+H] + .

[0187] Compound 23-6 was a white solid with a yield of 70.35%. m / z (ESI-MS): 252.2 [M+H] + .

[0188] Compound 23 was a white solid, and the yield was 58.32%. 1 H NMR (300MHz, DMSO-d6) δ10.38(s,1H),8.91(d,J=4.9Hz,1H),8.72(d,J=8.5Hz,1H),8.65(s,1H),8.48(d,J=8. 3Hz, 1H), 8.23 ​​(s, 1H), 8.08 (td, J = 4.9, 1.7Hz, 1H), 7.82 (t, J = 7.8Hz, 1H), 7.65 (t, J = 7.6Hz, 1H), 3.89 (s, 3H).

[0189] Example 24: Preparation of 2-(((3-formyl-4-hydroxynaphthalen-1-yl)sulfonyl)carbamoyl)isonicotinic acid (Compound 24)

[0190]

[0191] Referring to the preparation method of Example 1, the synthesis steps were the same as step 4 of Example 1, except that compound 23 was used to replace compound 1-5. Compound 24 was obtained as a white solid with a yield of 83.05%. 1 H NMR(300MHz,DMSO-d6)δ10.43(s,1H),8.97-8.86(m,1H),8.77(dd,J=12.3,8.6Hz,1H),8.73-8.49(m ,1H),8.47-8.36(m,1H),8.21(dt,J=2.3,1.2Hz,1H),8.07(dd,J=4.9,1.6Hz,1H),7.95-7.60(m,2H).

[0192] Example 25: N 2 -(3-formyl-4-hydroxynaphthalen-1-yl)sulfonyl)-N 4 Preparation of -hydroxypyridine-2,4-diamide (Compound 25)

[0193]

[0194] Compound 19 was synthesized according to the procedure of Example 9, except that compound 23 was used instead of compound 7.

[0195] Compound 25 was a light yellow solid, and the yield was 19.37%. 1 H NMR (300MHz, DMSO-d6) δ12.09(s,1H),10.81(d,J=4.9Hz,1H),10.19(s,1H),8.96(d,J=4.9Hz,1H),8.85–8.75(m,2H),8.41(dd d,J=15.6,7.4,1.5Hz,2H),8.25(s,1H),7.87(dd,J=7.4,1.5Hz,1H),7.58(td,J=7.5,1.6Hz,1H),7.45(td,J=7.4,1.6Hz,1H).

[0196] Example 26: Preparation of 2-(((4-dihydroxyboryl-3-formylnaphthalen-1-yl)sulfonyl)carbamoyl)isonicotinic acid (Compound 26)

[0197]

[0198] Referring to the preparation method of Example 14, compounds 26-1 to 26 were synthesized according to steps 1-3 of Example 14, except that compound 23 was used to replace compound 7.

[0199] Compound 26-1 is a yellow oily liquid. m / z (ESI-MS): 547.4 [M+H]+ .

[0200] Compound 26-2 was a white solid with a yield of 17.39%. m / z (ESI-MS): 443.2 [M+H] + .

[0201] Compound 26 was a white solid with a yield of 87.52%. 1 H NMR (300MHz, DMSO-d6) δ10.00 (s, 1H), 8.80 (d, J = 1.5Hz, 1H), 8.78-8.67 (m, 2H), 8.47-8.36 ( m, 4H), 8.08 (dd, J = 7.4, 1.5 Hz, 1H), 7.57 (td, J = 7.5, 1.6 Hz, 1H), 7.47 (td, J = 7.4, 1.6 Hz, 1H).

[0202] Example 27: Preparation of methyl 2-(((3-acetyl-4-hydroxynaphthalen-1-yl)sulfonyl)carbamoyl)isonicotinate (Compound 27)

[0203]

[0204] Referring to the preparation method of Example 23, compounds 27-2 to 27 were synthesized according to steps 2-6 of Example 23, except that compound 27-1 was used to replace compound 23-2.

[0205] Compound 27-2 was a white solid with a yield of 89.33%. m / z (ESI-MS): 201.2 [M+H] + .

[0206] Compound 27-3 was a white solid with a yield of 70.21%. m / z (ESI-MS): 299.7 [M+H] + .

[0207] Compound 27-4 was a white solid with a yield of 73.68%. m / z (ESI-MS): 280.3 [M+H] + .

[0208] Compound 27-5 was a white solid with a yield of 70.21%. m / z (ESI-MS): 266.3 [M+H] + .

[0209] Compound 27 was a white solid, and the yield was 79.98%. 1H NMR (300MHz, DMSO-d6) δ14.57(s,1H),8.96-8.89(m,1H),8.80(d,J=8.6Hz,1H),8.74(s,1H),8.51-8.43(m,1H),8.24-8 .19(m,1H),8.08(dd,J=5.0,1.7Hz,1H),7.90(ddd,J=8.5,7.0,1.4Hz,1H),7.78-7.69(m,1H),3.88(s,3H),2.84(s,3H).

[0210] Example 28: Preparation of 2-(((3-acetyl-4-hydroxynaphthalen-1-yl)sulfonyl)carbamoyl)isonicotinic acid (Compound 28)

[0211]

[0212] Referring to the preparation method of Example 1, the synthesis steps were the same as step 4 of Example 1, except that compound 27 was used to replace compound 1-5. Compound 28 was obtained as a white solid with a yield of 89.77%. 1 H NMR (300MHz, DMSO-d6) δ14.59(s,1H),8.91(dd,J=4.9,0.9Hz,1H),8.81(d,J=8.6Hz,1H),8.75(s,1H),8.48(dd,J=8.4,1.4Hz,1H) ,8.26-8.17(m,1H),8.08(dd,J=4.9,1.7Hz,1H),7.91(ddd,J=8.5,7.0,1.4Hz,1H),7.74(ddd,J=8.2,7.0,1.1Hz,1H),2.85(s,3H).

[0213] Example 29: N 2 -((3-acetyl-4-hydroxynaphthalen-1-yl)sulfonyl)-N 4 Preparation of -hydroxypyridine-2,4-diamide (Compound 29)

[0214]

[0215] Compound 29 was synthesized according to the procedure of Example 9, except that Compound 27 was used instead of Compound 7.

[0216] Compound 29 was a light yellow solid, and the yield was 24.96%. 1H NMR (300MHz, DMSO-d6) δ8.78(d,J=8.4Hz,1H),8.65(d,J=5.0Hz,1H),8.36(s,1H),8.31(d,J =8.2Hz,1H),8.25(s,1H),7.68(d,J=5.2Hz,1H),7.55(dq,J=15.0,7.1Hz,2H),2.44(s,3H).

[0217] Example 30: Preparation of 2-(((3-acetyl-4-dihydroxyboronaphthalene-1-yl)sulfonyl)carbamoyl)isonicotinic acid (Compound 30)

[0218]

[0219] Referring to the preparation method of Example 14, compounds 30-1 to 30 were synthesized according to steps 1-3 of Example 14, except that compound 27 was used instead of compound 7.

[0220] Compound 30-1 is a yellow oily liquid. m / z (ESI-MS): 561.5 [M+H] + .

[0221] Compound 30-2 was a white solid with a yield of 21.75%. m / z (ESI-MS): 457.2 [M+H] + .

[0222] Compound 30 was a white solid with a yield of 89.21%. 1 H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.70(dd,J=7.4,2.2Hz,2H),8.52(d,J=10. 3Hz, 3H), 8.39-8.30 (m, 1H), 8.08 (dd, J = 7.4, 1.5Hz, 1H), 7.57-7.38 (m, 2H), 2.75 (s, 3H).

[0223] Example 31: Preparation of 2-(((3-((4-nitrophenoxy)carbonyl)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 31)

[0224]

[0225] Step 1: Synthesis of compound 31-3

[0226] Compound 31-1 (500 mg, 2.49 mmol) was dissolved in 20 mL of anhydrous dichloromethane, and EDCI (715 mg, 3.73 mmol), DMAP (455 mg, 3.73 mmol) and TEA (1.04 mL, 7.46 mmol) were added in sequence. After activation at 0°C for 30 min, compound 31-2 (415 mg, 2.98 mmol) was added and reacted at room temperature overnight. TLC monitored the reaction to be complete. Extracted with DCM and water, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (PE: EA = 20: 1) to obtain compound 31-3 as a yellow solid with a yield of 69.81%. m / z (ESI-MS): 323.3 [M+H] + .

[0227] Step 2: Synthesis of compound 31-4

[0228] Referring to the preparation method of Example 1, the synthesis steps are the same as step 2 of Example 1, except that compound 31-3 is used to replace compound 1-3. Compound 31-4 is a yellow solid with a yield of 67.72%. m / z (ESI-MS): 562.5 [M+H] + .

[0229] Step 3: Synthesis of compound 31

[0230] Referring to the preparation method of Example 12, the synthesis steps are the same as step 6 of Example 12, except that compound 31-4 is used to replace compound 12-3. Compound 31 is obtained as a yellow solid with a yield of 33.07%. 1 H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.67(d,J=7.4Hz,1H),8.44(t,J=1.5Hz,1H),8.29-8.20(m,2H),8.16(dt ,J=7.5,1.5Hz,1H),8.06(dd,J=7.4,1.5Hz,1H),7.97(dt,J=7.5,1.5Hz,1H),7.61(t,J=7.5Hz,1H),7.37-7.27(m,2H).

[0231] Example 32: Preparation of 2-(((3-((4-trifluoromethylphenoxy)carbonyl)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 32)

[0232]

[0233] Referring to the preparation method of Example 31, compounds 32-2 to 32 were synthesized according to steps 1-3 of Example 31, except that compound 32-1 was used to replace compound 31-2.

[0234] Compound 32-2 was a white solid with a yield of 71.66%. m / z (ESI-MS): 346.3 [M+H] + .

[0235] Compound 32-3 was a white solid with a yield of 59.06%. m / z (ESI-MS): 585.5 [M+H] + .

[0236] Compound 32 was a white solid, and the yield was 28.09%. 1 H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.65(d,J=7.6Hz,1H),8.41(t,J=1.5Hz,1H),8.14(dt,J=7.4 ,1.5Hz,1H),8.06(dd,J=7.4,1.5Hz,1H),7.93(dt,J=7.6,1.5Hz,1H),7.71-7.56(m,3H),7.29-7.19(m,2H).

[0237] Example 33: Preparation of 2-(((3-((4-fluorophenoxy)carbonyl)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 33)

[0238]

[0239] Referring to the preparation method of Example 31, compounds 33-2 to 33 were synthesized according to steps 1-3 of Example 31, except that compound 33-1 was used to replace compound 31-2.

[0240] Compound 33-2 was a white solid with a yield of 69.43%. m / z (ESI-MS): 296.3 [M+H] + .

[0241] Compound 33-3 was a white solid with a yield of 56.89%. m / z (ESI-MS): 535.5 [M+H] + .

[0242] Compound 33 was a white solid, and the yield was 29.01%. 1H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.70(d,J=7.5Hz,1H),8.46(t,J=1.5Hz,1H),8.15(dt,J=7.4,1.5Hz,1H),8.08 (dd,J=7.4,1.5Hz,1H),7.96(dt,J=7.4,1.5Hz,1H),7.65-7.54(m,1H),7.16(ddt,J=6.6,5.1,1.5Hz,2H),7.14-7.02(m,2H).

[0243] Example 34: Preparation of 2-(((3-((4-methoxyphenoxy)carbonyl)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 34)

[0244]

[0245] Referring to the preparation method of Example 31, compounds 34-2 to 34 were synthesized according to steps 1-3 of Example 31, except that compound 34-1 was used to replace compound 31-2.

[0246] Compound 34-2 was a white solid with a yield of 71.01%. m / z (ESI-MS): 308.3 [M+H] + .

[0247] Compound 34-3 was a white solid with a yield of 51.77%. m / z (ESI-MS): 547.5 [M+H] + .

[0248] Compound 34 was a white solid with a yield of 30.10%. 1 H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.70(d,J=7.5Hz,1H),8.46(t,J=1.5Hz,1H),8.17(dt,J=7.5,1.5Hz,1H),8.06 (dd,J=7.4,1.5Hz,1H),7.95(dt,J=7.6,1.5Hz,1H),7.59(t,J=7.5Hz,1H),7.18-7.08(m,2H),6.90-6.81(m,2H),3.77(s,3H).

[0249] Example 35: Preparation of 2-(((2-((4-nitrophenoxy)carbonyl)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 35)

[0250]

[0251] Referring to the preparation method of Example 31, compounds 35-2 to 35 were synthesized according to steps 1-3 of Example 31, except that compound 35-1 was used instead of compound 31-1.

[0252] Compound 35-2 was a yellow solid with a yield of 68.32%. m / z (ESI-MS): 323.3 [M+H] + .

[0253] Compound 35-3 was a yellow solid with a yield of 53.87%. m / z (ESI-MS): 562.5 [M+H] + .

[0254] Compound 35 was a yellow solid, and the yield was 29.01%. 1 H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.67(d,J=7.4Hz,1H),8.31-8.22(m,2H),8.14(dd,J=7.4,1.5Hz,1 H), 8.05 (ddd, J=7.4, 4.6, 1.6Hz, 2H), 7.68 (td, J=7.5, 1.6Hz, 1H), 7.55 (td, J=7.4, 1.6Hz, 1H), 7.33-7.23 (m, 2H).

[0255] Example 36: Preparation of 2-(((2-((4-trifluoromethylphenoxy)carbonyl)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 36)

[0256]

[0257] Referring to the preparation method of Example 31, compounds 36-1 to 36 were synthesized according to steps 1-3 of Example 31, except that compound 35-1 was used to replace compound 31-1, and compound 32-1 was used to replace compound 31-2.

[0258] Compound 36-1 was a white solid with a yield of 68.32%. m / z (ESI-MS): 346.3 [M+H] + .

[0259] Compound 36-2 was a white solid with a yield of 53.87%. m / z (ESI-MS): 585.5 [M+H] + .

[0260] Compound 36 was a white solid, and the yield was 29.01%. 1H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.65(d,J=7.5Hz,1H),8.15(dd,J=7.4,1.6Hz ,1H),8.05(dt,J=7.4,1.6Hz,2H),7.73-7.62(m,3H),7.67-7.52(m,1H),7.26-7.16(m,2H).

[0261] Example 37: 2-(((2-((4-fluorophenoxy)carbonyl)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 37)

[0262]

[0263] Referring to the preparation method of Example 31, compounds 37-1 to 37 were synthesized according to steps 1-3 of Example 31, except that compound 35-1 was used to replace compound 31-1, and compound 33-1 was used to replace compound 31-2.

[0264] Compound 37-1 was a white solid with a yield of 61.06%. m / z (ESI-MS): 296.3 [M+H] + .

[0265] Compound 37-2 was a white solid with a yield of 57.33%. m / z (ESI-MS): 535.5 [M+H] + .

[0266] Compound 37 was a white solid, and the yield was 28.91%. 1 H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.70(d,J=7.5Hz,1H),8.11(ddd,J=16.4,7.5,1.5Hz,2H),7.98(dd, J=7.5,1.6Hz,1H),7.74(td,J=7.5,1.5Hz,1H),7.56(td,J=7.4,1.5Hz,1H),7.19(ddt,J=6.6,4.9,1.4Hz,2H),7.14 -7.03(m,2H).

[0267] Example 38: Preparation of 2-(((2-((4-methoxyphenoxy)carbonyl)phenyl)sulfonyl)carbamoyl)isonicotinic acid (Compound 38)

[0268]

[0269] Referring to the preparation method of Example 31, compounds 38-1 to 38 were synthesized according to steps 1-3 of Example 31, except that compound 35-1 was used to replace compound 31-1, and compound 34-1 was used to replace compound 31-2.

[0270] Compound 38-1 was a white solid with a yield of 63.08%. m / z (ESI-MS): 308.3 [M+H] + .

[0271] Compound 38-2 was a white solid with a yield of 55.01%. m / z (ESI-MS): 547.5 [M+H] + .

[0272] Compound 38 was a white solid, and the yield was 26.33%. 1 H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.70(d,J=7.5Hz,1H),8.14(dd,J=7.5,1.6Hz,1H),8.03(ddd,J=14.1,7.4 ,1.5Hz,2H),7.74(td,J=7.4,1.5Hz,1H),7.55(td,J=7.4,1.5Hz,1H),7.17-7.08(m,2H),6.90-6.81(m,2H),3.77(s,3H).

[0273] Example 39: Preparation of 2-(((4-methoxy-3-((4-nitrophenoxy)carbonyl)naphthalen-1-yl)sulfonyl)carbamoyl)isonicotinic acid (Compound 39)

[0274]

[0275] Step 1: Synthesis of compound 39-2

[0276] Reference Example 7 preparation method, the synthesis steps are the same as Example 7 step 1, the difference is that compound 39-1 is used to replace compound 7-1. Compound 39-2 is a white solid, the yield is 73.05%. m / z (ESI-MS): 299.7 [MH] - .

[0277] Step 2: Synthesis of compound 39-3

[0278] Referring to the preparation method of Example 7, the synthesis steps are the same as step 2 of Example 7, except that compound 39-2 is used to replace compound 7-2. Compound 39-3 is a white solid with a yield of 73.05%. m / z (ESI-MS): 280.3 [MH] -Step 3: Synthesis of Compound 39-4 to Compound 39

[0279] Referring to the preparation method of Example 31, compounds 39-4 to 39 were synthesized according to steps 1-3 of Example 31, except that compound 39-3 was used to replace compound 31-1.

[0280] Compound 39-4 was a yellow solid with a yield of 62.78%. m / z (ESI-MS): 403.4 [M+H] + .

[0281] Compound 39-5 is a yellow solid with a yield of 53.90%. m / z (ESI-MS): 642.6 [M+H] + .

[0282] Compound 39 was a yellow solid with a yield of 27.74%. 1 H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.66(d,J=7.6Hz,1H),8.55-8.43(m,2H),8.34(d,J=1.3Hz,1H),8.32(d,J=1.3Hz,1H),8.24(s,1H) ,8.05(dd,J=7.4,1.4Hz,1H),7.61(td,J=7.5,1.6Hz,1H),7.48(td,J=7. 5,1.6Hz,1H),7.34(d,J=1.3Hz,1H),7.31(d,J=1.3Hz,1H),3.81(s,3H). Example 40: Preparation of 2-(((4-methoxy-3-((4-trifluoromethylphenoxy)carbonyl)naphthalen-1-yl)sulfonyl)carbamoyl)isonicotinic acid (Compound 40)

[0283]

[0284] Referring to the preparation method of Example 31, compounds 40-1 to 40 were synthesized according to steps 1-3 of Example 31, except that compound 39-3 was used to replace compound 31-1, and compound 32-1 was used to replace compound 31-2.

[0285] Compound 40-1 was a white solid with a yield of 63.20%. m / z (ESI-MS): 426.4 [M+H] + .

[0286] Compound 40-2 was a white solid with a yield of 56.81%. m / z (ESI-MS): 665.6 [M+H] + .

[0287] Compound 40 was a white solid with a yield of 25.32%. 1 H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.66(d,J=7.6Hz,1H),8.49(ddd,J=7.2,2.6,1.5Hz,2H),8.22(s,1H),8.05(dd,J=7.4,1.4Hz ,1H),7.76-7.63(m,2H),7.58(td,J=7.5,1.6Hz,1H),7.48(td,J=7.5,1.6Hz,1H),7.20(d,J=1.3Hz,1H),7.18(d,J=1.3Hz,1H),3.82(s,3H).

[0288] Example 41: Preparation of 2-(((4-methoxy-3-((4-fluorophenoxy)carbonyl)naphthalen-1-yl)sulfonyl)carbamoyl)isonicotinic acid (Compound 41)

[0289]

[0290] Referring to the preparation method of Example 31, compounds 41-1 to 41 were synthesized according to steps 1-3 of Example 31, except that compound 39-3 was used to replace compound 31-1, and compound 33-1 was used to replace compound 31-2.

[0291] Compound 41-1 was a white solid with a yield of 58.91%. m / z (ESI-MS): 376.4 [M+H] + .

[0292] Compound 41-2 was a white solid with a yield of 60.02%. m / z (ESI-MS): 615.6 [M+H] + .

[0293] Compound 41 was a white solid with a yield of 27.65%. 1 H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.65(d,J=7.6Hz,1H),8.54(dd,J=7.4,1.6Hz,1H),8.48(dd,J=7.4,1.6Hz,1H),8.21(s,1H),8.06( dd,J=7.4,1.4Hz,1H),7.60(td,J=7.4,1.6Hz,1H),7.48(td,J=7.4,1.6H z,1H),7.18(ddt,J=6.6,5.1,1.4Hz,2H),7.14–7.04(m,2H),3.83(s,3H).

[0294] Example 42: Preparation of 2-(((4-methoxy-3-((4-methoxyphenoxy)carbonyl)naphthalen-1-yl)sulfonyl)carbamoyl)isonicotinic acid (Compound 42)

[0295]

[0296] Referring to the preparation method of Example 31, compounds 42-1 to 42 were synthesized according to steps 1-3 of Example 31, except that compound 39-3 was used to replace compound 31-1, and compound 34-1 was used to replace compound 31-2.

[0297] Compound 42-1 was a white solid with a yield of 64.83%. m / z (ESI-MS): 388.4 [M+H] + .

[0298] Compound 42-2 was a white solid with a yield of 54.71%. m / z (ESI-MS): 627.6 [M+H] + .

[0299] Compound 42 was a white solid with a yield of 28.60%. 1 H NMR (300MHz, DMSO-d6) δ8.80(d,J=1.5Hz,1H),8.66(d,J=7.6Hz,1H),8.53(dd,J=7.4,1.5Hz,1H),8.48(dd,J=7.4,1.6Hz,1H),8.22(s,1H),8.05 (dd,J=7.4,1.5Hz,1H),7.60(td,J=7.4,1.6Hz,1H),7.48(td,J=7.5,1.6 Hz,1H),7.15-7.07(m,2H),6.89-6.79(m,2H),3.81(s,3H),3.77(s,3H).

[0300] Example 43: Preparation of Sodium 2-((Naphthalen-1-ylmethyl)carbamoyl)isonicotinate (Compound 43)

[0301]

[0302] Compound 20 (100 mg, 0.326 mmol) was dissolved in 10 mL of methanol, and 326 μL of 1 M NaOH-methanol solution was accurately added using a pipette, which immediately turned into a colorless clear solution. After 20 min, the methanol was removed by concentration under reduced pressure and dried to obtain compound 43 as a white solid with a yield of 83.21%. 1H NMR (300MHz, DMSO-d6) δ8.94-8.82(m,2H),8.78(d,J=1.5Hz,1H),8.21-8.09(m,1H),8.05(dd,J=7.5,1.6Hz,1H),7.87 (ddt,J=8.4,7.5,1.5Hz,2H),7.60-7.46(m,2H),7.46-7.36(m,1H),7.34(dd,J=7.5,1.7Hz,1H),4.83(d,J=9.9Hz,2H).

[0303] Example 44: Preparation of 2-((naphthalen-1-ylmethyl)carbamoyl)isonicotinoic acid hydrochloride (Compound 44)

[0304]

[0305] Compound 20 (100 mg, 0.326 mmol) was dissolved in 10 mL of hydrochloric acid-ethanol solution and reacted at room temperature for 4 h. The ethanol was removed by concentration under reduced pressure, and an appropriate amount of ethyl acetate was added and filtered to obtain compound 44 as a white solid with a yield of 76.08%. 1 H NMR (300MHz, DMSO-d6) δ8.92(t,J=9.9Hz,1H),8.81(d,J=7.5Hz,1H),8.52(s,1H),8.30(d,J=1.5Hz,1H),8.17-8.05(m,2H), 7.86(ddt,J=10.7,7.1,1.5Hz,2H),7.60-7.46(m,2H),7.45-7.34(m,1H),7.33(dd,J=7.5,1.7Hz,1H),4.79(d,J=9.9Hz,2H).

[0306] Example 45: Expression and purification of ALKBH5 protein

[0307] ALKBH5 gene plasmid was purchased from GenScript Biotech.

[0308] Experimental steps:

[0309] The recombinant plasmid was used to transfect the E. coli BL21 (DE3) strain, and the cells were revived in sterile LB medium at 37°C. A single clone was picked and added to 10 mL LB liquid medium (containing 50 μg / mL kanamycin, Amp), cultured at 37°C with shaking (220 rpm) overnight, transferred to 1 L LB liquid medium (containing 50 μg / mL kanamycin, Amp), cultured at 37°C with shaking (220 rpm) for 6-8 hours until OD600 was 0.6-0.8, cooled to 12°C, induced by adding 1 mM IPTG (Merck) for 16 hours (180 rpm), and then harvested and stored at -80°C for later use.

[0310] 4g of the above-mentioned bacterial sludge was added to 40mL of bacterial lysis solution, and PMSF (Biyuntian) was added after mixing. Ultrasonic lysis was performed for 40 minutes. The lysed mixture was centrifuged at low temperature and high speed (10000rpm, 20min, 4°C), and the supernatant was filtered (0.4μm microporous filter membrane). It was separated and purified by AKTA pure 25 (GE Healthcare, Life Sciences) using His column (balance solution: 20mM pH 8.0 Tris-HCl, 300mM NaCl, 10mM imidazole; eluent: 20mM pH 8.0 Tris-HCl, 300mM NaCl, 500mM imidazole), 10% SDS-PAGE was used to confirm the molecular weight and purity of the band, and dialyzed overnight (20mM pH 8.0 Tris-HCl, 300mM NaCl). The obtained protein was stored at -80°C for future use after the concentration was determined by BCA.

[0311] Example 46: Determination of the inhibitory activity of compounds against ALKBH5 based on fluorescence polarization (FP)

[0312] The present invention constructs a fluorescent molecular probe based on a segment of ssDNA that binds to ALKBH5 as a method for studying the competition of such compounds for the binding of ALKBH5 to ssDNA, and determines the inhibition rate of such compounds under different concentration conditions, and then calculates the IC 50 value.

[0313] The equipment and reagents used are as follows:

[0314] The instrument used in this experiment was SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices). The protein used was ALKBH5 (protein expressed and purified by the laboratory, sequence 66-292). The probe used was fluorescently labeled m 6 A-ssDNA(5′-ATTGTCA(dm 6A) CAGCAGA-FAM-3′, synthesized by GenScript Biotech. All test compounds were prepared into 10 mM stock solution dissolved in DMSO. The 384-well black plate used in the experiment was produced by Corning.

[0315] Experimental steps:

[0316] The final test volume was 60 μL, and 20 μL of compounds of different concentrations (each compound was diluted twice with 10 to 14 gradients, with an initial concentration of 100 μM), 20 μL of ALKBH5 protein (300 nM, final concentration 100 nM), and 20 μL of fluorescent probe (30 nM, final concentration 10 nM) were added to the wells in the order of 20 μL of compounds of different concentrations (each compound was diluted twice with 10 to 14 gradients, with an initial concentration of 100 μM), 20 μL of ALKBH5 protein (30 nM, final concentration 10 nM), and 20 μL of fluorescent probe (30 nM, final concentration 10 nM). A blank control (40 μL of pH = 7.5 Tris-HCl buffer + 20 μL of 10 nM fluorescent probe) and a negative control (20 μL of pH = 7.5 Tris-HCl buffer + 20 μL of ALKBH5 protein + 20 μL of 10 nM fluorescent probe) were set for each experiment. After adding the sample, cover the 384-well plate with tin foil and incubate it on a shaker at room temperature for 1 hour. Use SpectraMax Paradigm Multi-Mode Microplate Reader to read the fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 535 nm to calculate the mP value. After calculating the inhibition rate using the following formula, GraphPad Prism5.0 was used to calculate the IC 50 value.

[0317] Inhibition rate = (mP value of compound group - mP value of blank group) / (mP value of negative control group - mP value of blank group) × 100%

[0318] The experimental results are shown in Table 1. The compound MD-9 reported in the literature (Chem Asian J 2018, 13 (19), 2854-2867.) was used as the lead compound, and its activity data was used as the control. The structural formula of MD-9 is

[0319]

[0320] Table 1 IC values ​​of the compounds of the present invention for ALKBH5 protein 50 value

[0321]

[0322]

[0323] From the results in Table 1, it can be seen that the compounds of the present invention have significant inhibitory activity against ALKBH5, and can be used as small molecule inhibitors of ALKBH5 protein to inhibit RNA demethylase at the protease level.

[0324] In summary, the substituted pyridine compounds provided by the present invention have significant inhibitory activity against ALKBH5 and are effective ALKBH5 inhibitors. Therefore, the drug containing the above compound as an active ingredient can be used to prepare a drug for treating clinical diseases related to ALKBH5.

[0325] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A substituted pyridine compound or a pharmaceutically acceptable salt thereof, characterized in that: Selected from:

2. A pharmaceutical composition, characterized in that: The invention comprises the compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

3. Use of the compound according to claim 1 in the preparation of a medicament for treating diseases associated with ALKBH5 protein dysfunction.

4. The use according to claim 3, characterized in that The diseases associated with ALKBH5 protein dysfunction are glioblastoma, ovarian cancer, endometrial cancer, cervical cancer, breast cancer, acute myeloid leukemia, pituitary adenoma, oral squamous cell carcinoma, esophageal squamous cell carcinoma, gastric cancer, hepatitis B virus-related hepatocellular carcinoma and intrahepatic bile duct carcinoma.

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