CN113214222B - N-(arylsulfonyl)-indole-2-carboxamide FBPase inhibitors and uses thereof - Google Patents

Abstract

The invention discloses a 1-methyl-7-chloro-4- ((methoxyl aryl) amino) -N- (aryl sulfonyl) -indole-2-formamide FBPase inhibitor with a new structure, a preparation method, a pharmaceutical composition and application thereof. In particular, the invention relates to an N-acyl sulfonamide FBPase inhibitor shown in a general formula I and a general formula II and salts thereof, a preparation method thereof, a composition containing one or more compounds, application of the compounds in preparing the FBPase inhibitor or a medicine for treating diseases related to the FBPase, and application in preparing a medicine for preventing and/or treating diabetes.

Description

N-(arylsulfonyl)-indole-2-carboxamide FBPase inhibitors and uses thereof

Technical Field

The present invention discloses a novel structure of 1-methyl-7-chloro-4-((methoxyaromatic heteroyl)amino)-N-(arylsulfonyl)-indole-2-carboxamide FBPase inhibitor, and its preparation method, pharmaceutical composition and use. Specifically, the present invention relates to N-acylsulfonamide FBPase inhibitors and salts thereof represented by general formula I and general formula II, preparation methods thereof, compositions containing one or more such compounds, and use of such compounds in preparing FBPase inhibitors or drugs for treating diseases related to FBPase, and in preparing drugs for preventing and/or treating diabetes.

Background Art

Diabetes is a chronic metabolic disease regulated by multiple genes, which is mainly manifested by persistent high blood sugar and diabetes. Continuous high blood sugar can lead to many complications, such as retinal, kidney, nervous system diseases and vascular complications (New. Engl. J. Med., 2010, 362: 1090-1101). Hyperglycemia patients are often accompanied by hyperlipidemia. The number of diabetes patients in China ranks first in the world. According to the latest survey on "Prevalence and Control of Adult Diabetes in China", the prevalence of diabetes in adults aged 18 and above in China has reached 11.6%, and the prevalence of prediabetes has reached 50.1%.

Diabetes is divided into insulin-dependent (type I) and non-insulin-dependent (type II), of which type II diabetes accounts for 90% to 95% of the total number of diabetic patients. Biological studies have shown that the main pathological basis for diabetes is insufficient insulin secretion, insulin resistance and increased hepatic glucose production. Most of the anti-diabetic drugs currently used in clinical practice increase insulin secretion (such as sulfonylureas and glinides insulin secretagogues) or treat diabetes by avoiding peripheral insulin resistance (such as thiazolidinedione insulin sensitizers).

Studies have shown that increased endogenous glucose production is the main cause of elevated fasting blood glucose in diabetic patients. Endogenous glucose mainly comes from the liver, and gluconeogenesis and glycogenolysis are the two main pathways for producing glucose in the liver. The gluconeogenesis pathway is one of the main causes of elevated blood glucose in patients with type 2 diabetes. Therefore, regulating the gluconeogenesis pathway and reducing the production of endogenous glucose has become a new strategy for developing anti-type 2 diabetes drugs with new mechanisms of action.

Gluconeogenesis is the process of converting three-carbon precursors such as glycerol and lactate into glucose under the catalysis of multiple enzymes. In the process of gluconeogenesis, fructose 1,6-bisphosphatase, fructose 6-phosphatase and pyruvate carboxylase play a key role in gluconeogenesis; among them, fructose 1,6-bisphosphatase (FBPase) catalyzes the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate, which is one of the rate-controlling steps in the gluconeogenesis pathway; therefore, fructose-1,6-bisphosphatase is a potential target for anti-diabetic drugs with a new mechanism of action. Fructose-1,6-bisphosphatase inhibitors are potential anti-diabetic drugs with a new mechanism of action.

In 2003, Pfizer Pharmaceuticals obtained indole carboxylic acid compounds through high-throughput screening, and the IC ₅₀ value of FBPase inhibition activity was at the micromolar level (Bioorg. Med. Chem ^. Lett., 2003, 13: 2055-2058). In 2006, von Geldern et al. reported benzoxazole-2-benzenesulfonamide compounds, and the IC ₅₀ value of FBPase inhibition activity was at the ^10-6-10-7 molar level (Bioorg. Med. Chem. Lett., 2006, 16: 1811-1815). In 2010, Roche Pharmaceuticals reported thiazole-substituted sulfonylurea FBPase inhibitors discovered through high-throughput screening, and the IC ₅₀ value of its inhibitory activity was at ^{the 10-7-10-8} molar level (Bioorg. Med. Chem. Lett., 2010, 20: 594-599). From 2007 to 2010, Metbasis Pharmaceuticals reported the use of structure-based drug molecule design strategies to discover AMP analogs and search for FBPase inhibitors. After continuous structural optimization, they obtained benzimidazole FBPase inhibitors (J.Am.Chem.Soc., 2007, 129: 15480-15490; J.Med.Chem., 2010, 53: 441-451) and thiazole FBPase inhibitors (J.Am.Chem.Soc., 2007, 129: 15491-15502; J.Med.Chem., 2011, 54: 153-165). MB07803 is a phosphorodiamidate prodrug of thiazole compounds and is currently in Phase II clinical trials (US, 225259A1[P]. 2007-09-27.). In 2012-2014, He et al. found that the inhibitory activity of oxadiazole compounds on FBP enzyme was _4.51 μM after optimization through high-throughput screening (Heterocycles, 2012, 85: 2693-2712; Eur. J. Med. Chem, 2014, 83: 15-25). In 2019, Hang et al. used virtual screening based on pharmacophores and found that the inhibitory activity of furanthiophene compounds on FBP enzyme was 5.3 μM (J. Mol. Grap. Model. 2019, 86: 142-148).

Although many structural types of FBPase inhibitors have been reported, so far, only one FBPase inhibitor (MB07083) is in Phase II clinical research in the form of phosphonic acid diamide prodrug. This compound is a second-generation FBPase inhibitor developed by Metabasis. The first-generation FBPase inhibitor CS-917 (also in the form of phosphonic acid diamide prodrug) was terminated in clinical research in 2008 due to the toxicity of its metabolites. Therefore, it is of great clinical significance to find new and efficient FBPase inhibitors with reasonable physicochemical properties and druggability.

The present invention is based on the N-acylsulfonamide inhibitors obtained in the previous period (patent application number: 2016101058649), and the 4-substituent of the indole ring, the 2-sulfonamide substituent, and the salt of the acylsulfonamide are structurally modified to obtain a FBPase inhibitor with high activity, and/or reasonable physicochemical properties (such as improved solubility), and/or drugability. The present invention designs and synthesizes a new structure of 1-methyl-7-chloro-4-((methoxyaromatic heteroyl)amino)-N-(arylsulfonyl)-indole-2-carboxamide and its salt FBPase inhibitors, which lays a structural foundation for obtaining FBPase inhibitors with good pharmacokinetic properties and can be orally administered. The present invention aims to find a new oral and effective anti-diabetic drug with strong FBPase inhibitory activity and good drugability.

Summary of the invention

The technical problem solved by the present invention is to provide N-acylsulfonamide FBPase inhibitors shown in formula I, I-A and formula II, II-A, their preparation methods, compositions containing one or more of such compounds, and the use of such compounds in the preparation of FBPase inhibitors or drugs for treating diseases related to FBPase, and the use of such compounds in the preparation of drugs for preventing and/or treating diabetes.

In order to solve the technical problem of the present invention, the present invention provides the following technical solutions:

The first aspect of the technical solution of the present invention is to provide N-acylsulfonamide derivatives as shown in general formula I, IA and formula II, IIA:

In Formula I,

A, B, D, E are independently selected from CR or N; R is selected from H, F, Cl, CH ₃ , OCH ₃ ;

Ar ₁ is selected from the following groups or structural fragments:

Phenyl, substituted phenyl, unsubstituted nitrogen-containing six-membered aromatic heterocycle (including pyridine, pyrimidine, pyridazine, pyrazine), substituted nitrogen-containing six-membered aromatic heterocycle (six-membered aromatic heterocycle includes pyridine, pyrimidine, pyridazine, pyrazine);

wherein the substituent is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclopropylmethylene, cyclobutyl, halogen-substituted C1-4 straight or branched chain alkyl (including CF ₃ , CH ₂ F, CHF ₂ , CH ₂ CF ₃ , CF ₂ CH ₃ , CH ₂ CH ₂ CF ₃ ), F, Cl, Br, NO ₂ , CN, methylenedioxy, ORs ₁ , NRs ₂ Rt ₁ , CONRs ₃ Rt ₂ ;

wherein Rs ₁ , Rs ₂ , Rs ₃ , Rt ₁ , and Rt ₂ are independently selected from H, C1-4 straight or branched chain alkyl (including methyl, ethyl, propyl, isopropyl, butyl, isobutyl), halogen-substituted C1-4 straight or branched chain alkyl (including CF ₃ , CH ₂ F, CHF ₂ , CH ₂ CF ₃ , CF ₂ CH ₃ , CH ₂ CH ₂ CF ₃ ), cyclopropyl, cyclopropylmethylene, and cyclobutyl;

The benzene ring and the nitrogen-containing six-membered aromatic heterocyclic ring may be monosubstituted or polysubstituted;

The six-membered aromatic heterocyclic ring may contain one nitrogen atom or multiple nitrogen atoms;

The halogen includes F, Cl and Br.

In I-A,

A, B, D, E are independently selected from CR or N; R is selected from H, F, Cl, CH ₃ , OCH ₃ ;

_M1 is independently selected from different alkali metal (lithium, sodium, potassium, cesium) or alkaline earth metal salts (calcium, magnesium, barium).

Ar ₁ is selected from the following groups or structural fragments:

Phenyl, substituted phenyl, unsubstituted nitrogen-containing six-membered aromatic heterocycle (including pyridine, pyrimidine, pyridazine, pyrazine), substituted nitrogen-containing six-membered aromatic heterocycle (six-membered aromatic heterocycle includes pyridine, pyrimidine, pyridazine, pyrazine);

wherein the substituent is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclopropylmethylene, cyclobutyl, halogen-substituted C1-4 straight or branched chain alkyl (including CF ₃ , CH ₂ F, CHF ₂ , CH ₂ CF ₃ , CF ₂ CH ₃ , CH ₂ CH ₂ CF ₃ ), F, Cl, Br, NO ₂ , CN, methylenedioxy, ORs ₁ , NRs ₂ Rt ₁ , CONRs ₃ Rt ₂ ;

wherein Rs ₁ , Rs ₂ , Rs ₃ , Rt ₁ , and Rt ₂ are independently selected from H, C1-4 straight or branched chain alkyl (including methyl, ethyl, propyl, isopropyl, butyl, isobutyl), halogen-substituted C1-4 straight or branched chain alkyl (including CF ₃ , CH ₂ F, CHF ₂ , CH ₂ CF ₃ , CF ₂ CH ₃ , CH ₂ CH ₂ CF ₃ ), cyclopropyl, cyclopropylmethylene, and cyclobutyl;

The benzene ring and the nitrogen-containing six-membered aromatic heterocyclic ring may be monosubstituted or polysubstituted;

The six-membered aromatic heterocyclic ring may contain one nitrogen atom or multiple nitrogen atoms;

The halogen includes F, Cl and Br.

In Formula II,

A', B', D', E' are independently selected from CR' or N; R' is selected from H, F, Cl, CH ₃ , OCH ₃ ;

Ar ₂ is selected from the following groups or structural fragments:

Phenyl, substituted phenyl, unsubstituted nitrogen-containing six-membered aromatic heterocycle (including pyridine, pyrimidine, pyridazine, pyrazine), substituted nitrogen-containing six-membered aromatic heterocycle (six-membered aromatic heterocycle includes pyridine, pyrimidine, pyridazine, pyrazine);

wherein the substituent is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclopropylmethylene, cyclobutyl, halogen-substituted C1-4 straight or branched chain alkyl (including CF ₃ , CH ₂ F, CHF ₂ , CH ₂ CF ₃ , CF ₂ CH ₃ , CH ₂ CH ₂ CF ₃ ), F, Cl, Br, NO ₂ , CN, methylenedioxy, ORs ₁ , NRs ₂ Rt ₁ , CONRs ₃ Rt ₂ ;

wherein Rs ₁ , Rs ₂ , Rs ₃ , Rt ₁ , and Rt ₂ are independently selected from H, C1-4 straight or branched chain alkyl (including methyl, ethyl, propyl, isopropyl, butyl, isobutyl), halogen-substituted C1-4 straight or branched chain alkyl (including CF ₃ , CH ₂ F, CHF ₂ , CH ₂ CF ₃ , CF ₂ CH ₃ , CH ₂ CH ₂ CF ₃ ), cyclopropyl, cyclopropylmethylene, and cyclobutyl;

The benzene ring and the nitrogen-containing six-membered aromatic heterocyclic ring may be monosubstituted or polysubstituted;

The six-membered aromatic heterocyclic ring may contain one nitrogen atom or multiple nitrogen atoms;

The halogen includes F, Cl and Br.

In Formula II-A,

A', B', D', E' are independently selected from CR' or N; R' is selected from H, F, Cl, CH ₃ , OCH ₃ ;

_M2 is independently selected from different alkali metals (lithium, sodium, potassium, cesium) or alkaline earth metal salts (calcium, magnesium, barium).

Ar ₂ is selected from the following groups or structural fragments:

Phenyl, substituted phenyl, unsubstituted nitrogen-containing six-membered aromatic heterocycle (including pyridine, pyrimidine, pyridazine, pyrazine), substituted nitrogen-containing six-membered aromatic heterocycle (six-membered aromatic heterocycle includes pyridine, pyrimidine, pyridazine, pyrazine);

wherein the substituent is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclopropylmethylene, cyclobutyl, halogen-substituted C1-4 straight or branched chain alkyl (including CF ₃ , CH ₂ F, CHF ₂ , CH ₂ CF ₃ , CF ₂ CH ₃ , CH ₂ CH ₂ CF ₃ ), F, Cl, Br, NO ₂ , CN, methylenedioxy, ORs ₁ , NRs ₂ Rt ₁ , CONRs ₃ Rt ₂ ;

wherein Rs ₁ , Rs ₂ , Rs ₃ , Rt ₁ , and Rt ₂ are independently selected from H, C1-4 straight or branched chain alkyl (including methyl, ethyl, propyl, isopropyl, butyl, isobutyl), halogen-substituted C1-4 straight or branched chain alkyl (including CF ₃ , CH ₂ F, CHF ₂ , CH ₂ CF ₃ , CF ₂ CH ₃ , CH ₂ CH ₂ CF ₃ ), cyclopropyl, cyclopropylmethylene, and cyclobutyl;

The benzene ring and the nitrogen-containing six-membered aromatic heterocyclic ring may be monosubstituted or polysubstituted;

The six-membered aromatic heterocyclic ring may contain one nitrogen atom or multiple nitrogen atoms;

The halogen includes F, Cl and Br.

To achieve the purpose of the present invention, preferred compounds include but are not limited to the following compounds:

The second aspect of the technical solution of the present invention is to provide a method for preparing the compound described in the first aspect, and the technical solution adopted comprises the following steps: in the presence of sodium ethoxide and ethyl trifluoroacetate, 2-bromo-5-chloro-benzaldehyde reacts with ethyl azidoacetate to prepare compound 1, using o-dichlorobenzene as solvent, compound 1 undergoes a ring-closing reaction at 180°C to obtain 4-bromo-7-chloro-1-H-indole-2-carboxylic acid ethyl ester, and then undergoes methylation at the indole-1-position under the condition of cesium carbonate to obtain 4-bromo-7-chloro-1-methyl-indole-2-carboxylic acid ethyl ester; subsequently, compound 3 undergoes a coupling reaction with a substituted aromatic amine under palladium catalysis to obtain compounds 4 and 4', and after a hydrolysis reaction, the corresponding compounds 5 and 5' with a carboxyl group at the 2-position are obtained. Compounds 5 and 5' undergo a condensation reaction with an aromatic sulfonamide to obtain compounds of general structural formulas I and II, respectively, and finally undergo an acid-base reaction with different bases to obtain N-acylsulfonamide salt compounds I-A and II-A.

Reagents and reaction conditions: (a) ethyl azidoacetate, ethyl trifluoroacetate, sodium, ethanol, -15°C to 0°C; (b) o-dichlorobenzene, 180°C; (c) iodomethane, cesium carbonate, DMF, rt; (d) Ar ₁ -NH ₂ , Pd ₂ (dba) ₃ , Xantphos, sodium carbonate, toluene, water, 100°C; (d) sodium hydroxide, THF, ethanol, water, rt; (e) Ar ₂ -S(O) ₂ -NH ₂ , HATU, DMAP, Et ₃ N, rt; (g) alkali metal or alkaline earth metal base, water, rt.

The third aspect of the technical solution of the present invention is to provide a pharmaceutical composition, which comprises the compound as described in the first aspect of the technical solution of the present invention and a commonly used pharmaceutical carrier.

The present invention also provides a pharmaceutical composition with the compound of the present invention as an active ingredient, the composition comprising at least one compound of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition is selected from tablets, capsules, pills, injections, sustained-release preparations, controlled-release preparations or various microparticle delivery systems. The pharmaceutical composition can be prepared according to methods well known in the art. The compound of the present invention can be combined with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants to prepare any dosage form suitable for human or animal use. The content of the compound of the present invention in its pharmaceutical composition is usually 0.1-95% by weight.

The compound of the present invention or the pharmaceutical composition containing it can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

The dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w types, w/o types and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.

The compound of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

These preparations are prepared according to methods well known to those skilled in the art. The adjuvants used to make tablets, capsules, and coatings are conventional adjuvants, such as starch, gelatin, gum arabic, silica, polyethylene glycol, and the solvents used in liquid dosage forms are, for example, water, ethanol, propylene glycol, and vegetable oils such as corn oil, peanut oil, olive oil, etc. The preparations containing the compounds of the present invention may also contain other adjuvants, such as surfactants, lubricants, disintegrants, preservatives, flavoring agents, pigments, etc.

In order to prepare the compound of the present invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent may be water, ethanol, isopropanol, etc.; the binder may be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, etc.; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

In order to prepare the dosing unit into a capsule, the active ingredient compound of the present invention can be mixed with a diluent and a glidant, and the mixture can be directly placed in a hard capsule or a soft capsule. The active ingredient compound of the present invention can also be first prepared into granules or pellets with a diluent, a binder, and a disintegrant, and then placed in a hard capsule or a soft capsule. The diluents, binders, wetting agents, disintegrants, and glidants used to prepare tablets of the compound of the present invention can also be used to prepare capsules of the compound of the present invention.

To prepare the compound of the present invention into an injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent and an appropriate amount of a solubilizer, a cosolvent, a pH adjuster, and an osmotic pressure regulator commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If a lyophilized powder injection is prepared, mannitol, glucose, etc. can also be added as a support agent.

Furthermore, if necessary, colorants, preservatives, perfumes, flavoring agents or other additives may be added to the pharmaceutical preparations.

To achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.

The dosage of the pharmaceutical composition of the compound of the present invention can vary widely according to the nature and severity of the disease to be prevented or treated, the individual conditions of the patient or animal, the route of administration and the dosage form. Generally speaking, the daily suitable dosage range of the compound of the present invention is 0.01-500 mg/Kg body weight, preferably 0.1-300 mg/Kg body weight. The above dosage can be administered in one dosage unit or divided into several dosage units, depending on the doctor's clinical experience and the dosage regimen including the use of other treatment means.

The compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

The fourth aspect of the technical solution of the present invention provides the use of the compound described in the first aspect of the present invention in the preparation of FBPase inhibitors and in the preparation of drugs for preventing and\or treating diseases and conditions related to FBPase. The application is characterized in that the diseases and conditions related to FBPase are selected from diabetes, chronic complications of diabetes, hyperlipidemia and obesity. The diabetes is selected from type I diabetes and type II diabetes; the chronic complications of diabetes are selected from retinal, kidney, nervous system lesions and vascular complications, local ischemic heart disease or atherosclerosis. The hyperlipidemia includes high glycerides and high cholesterol esters.

DETAILED DESCRIPTION

The invention will be further described below with reference to the embodiments, but the scope of the invention is not limited thereto.

The structure of the compound is determined by nuclear magnetic resonance (NMR) or high resolution mass spectrometry (HRMS). NMR is measured using Varian mercury 300 or Varian mercury 400, the measuring solvent is CDCl ₃ , DMSO-d ₆ , acetone-d ₆ , CD ₃ OD, the internal standard is TMS, and the chemical shift is given in ppm. Ms is measured using a Thermo Exactive Plus mass spectrometer. mp is the melting point given in °C, and the temperature is not corrected. Silica gel column chromatography generally uses 200-300 mesh silica gel as the carrier.

The reagents used in the experiments were chemically pure or analytically pure. The solvents used were all analytically pure. The anhydrous solvents used were obtained from the solvent purification system produced by INNOVATIVE TECHNOLOGY in the United States. Other solvents were not treated unless otherwise specified.

List of abbreviations:

CDCl ₃ : deuterated chloroform DMSO-d ₆ : deuterated dimethyl sulfoxide

HOBt: 1-hydroxybenzotriazole Pd(OAc) ₂ : palladium acetate

DCM: dichloromethane NCS: N-chlorosuccinimide

DMF: N,N-dimethylformamide DMAP: 4-dimethylaminopyridine

EA: ethyl acetate Et ₃ N: triethylamine

HATU: 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

XantPhos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

min: minute; h: hour

P/E: petroleum ether/ethyl acetate; D/M: dichloromethane/methanol

Preparation of intermediates:

7-Chloro-4-bromo-1-methyl-indole-2-carboxylic acid ethyl ester

a). Ethyl 2-azido-3-(5-chloro-2-bromophenyl)acrylate

Sodium metal (8.35 g, 363.2 mmol) was dissolved in anhydrous ethanol (200 mL). After the sodium metal was completely dissolved, the reaction solution was cooled to -15°C. 2-Bromo-5-chlorobenzaldehyde (50 g, 227 mmol), ethyl azidoacetate (44 g, 341 mmol) and ethyl trifluoroacetate (TFAE, 51.5 g, 363.2 mmol) were added to the reaction solution in batches. After reacting at -15°C for 1 hour, the temperature was raised to -5°C for 1 hour, and then the temperature was raised to 0°C for 5 hours. The temperature was raised to room temperature for overnight reaction. The reaction solution was poured into saturated NH ₄ Cl aqueous solution and extracted with EA (300 mL×3). The organic layers were combined, washed with saturated sodium chloride solution (100 mL×2), dried over anhydrous magnesium sulfate, concentrated, column chromatographed (DCM:PE＝1:1), concentrated, and recrystallized with PE:EA＝20:1. The first batch of product 36.5 g was obtained with a yield of 48.9%. ¹ H-NMR (400HMz, CDCl ₃ ) δ (ppm): 8.13 (s, 1H), 7.53 (d, J = 8.4Hz, 1H), 7.15 (s, 2H), 4.40 (q, J = 7.2Hz, 2H), 1.42 (t, J = 7.2Hz, 3H).

b). 7-Chloro-4-bromo-1H-indole-2-carboxylic acid ethyl ester

The compound 2-azido-3-(5-chloro-2-bromophenyl) ethyl acrylate (15 g) was placed in a reaction bottle, added to o-dichlorobenzene (15 mL), heated to 180°C for reaction, and the reaction was stopped after 1 hour. The solid was cooled, and solid was precipitated. The filter cake was washed with petroleum ether to obtain 7.2 g of white solid with a yield of 52.7%; mp: 154-155°C. ¹ H-NMR (400HMz, CDCl ₃ )δ(ppm):9.11(brs,1H),7.27(d,J＝8.4Hz,2H),7.18(d,J＝8.0Hz,1H),4.44(q,J＝7.2Hz,2H),1.44(t,J＝7.2Hz,3H).

c). 1-Methyl-7-chloro-4-bromo-indole-2-carboxylic acid ethyl ester

The compound ethyl 7-chloro-4-bromo-1H-indole-2-carboxylate (26.2 g, 86.24 mmol) was placed in a reaction bottle, and DMF (300 mL) was added. Cesium carbonate (55.9 g, 172.58 mmol) and iodomethane (24.7 g, 132.58 mmol) were added and stirred at room temperature. The reaction was stopped after 1 h, and the reaction solution was poured into ice water. Solid precipitated, which was filtered with suction, and the filter cake was washed with water to obtain 26 g of an off-white solid with a yield of 94.8%. ¹ H-NMR (400HMz, CDCl ₃ ) δ (ppm): 7.32 (s, 1H), 7.19 (d, J = 8.0Hz, 1H), 7.13 (d, J = 8.0Hz, 1H), 4.46 (s, 3H), 4.39 (q, J = 7.2Hz, 2H), 1.43 (t, J = 7.2Hz, 3H).

Example 1: 1-methyl-4-((6-methoxypyridin-2-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

a). 1-Methyl-4-((6-methoxypyridin-2-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester

Under argon protection, the compound 1-methyl-7-chloro-4-bromo-indole-2-carboxylic acid ethyl ester (350mg, 1.113mmol) was dissolved in toluene (30mL), Pd ₂ (dba) ₃ (255mg, 0.28mmoll) and Xantphos (325mg, 0.56mmol) were added, and then Na ₂ CO ₃ (353mg, 3.341mmol) was dissolved in (7.5mL) water and added to the reaction solution, 2-amino-6-methoxypyridine (413mg, 3.339mmol) was added to the reaction bottle, and refluxed for 20h. Filter, add ethyl acetate to the filtrate, wash with dilute hydrochloric acid, wash with saturated sodium bicarbonate solution, wash with saturated sodium chloride solution, dry with anhydrous magnesium sulfate, and column chromatography (E:P=1:30) to obtain 300mg of light yellow solid with a yield of 75%. ¹ H-NMR (400MHz, CDCl ₃ ) δ (ppm): 7.43 (t, J = 7.5Hz, 1H), 7.37 (d, J = 8.0Hz, 1H), 7.32 (s, 1H), 7.22 (d, J = 7.6Hz, 1H), 6.66 (brs, 1H), 6.44 (d, J = 7.6Hz, 1H), 6.24 (d,J＝7.6Hz,1H),4.47(s,3H),4.37(q,J＝7.2Hz,2H),3.93(s,3H),1.41(t,J＝7.2Hz,3H).

b). 1-Methyl-4-((6-methoxypyridin-2-yl)amino)-7-chloro-indole-2-carboxylic acid

The compound 1-methyl-4-((6-methoxypyridin-2-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester (265 mg, 0.738 mmol) was dissolved in a mixture of THF (7.5 mL) and EtOH (7.5 mL), and NaOH (88.5 mg, 2.214 mmol) was dissolved in 2.5 mL of water and added to the reaction solution. The mixture was reacted at 40° C. for 2 h, concentrated, and a small amount of water was added. The mixture was extracted with ether, and the pH of the aqueous layer was adjusted to 2-3 with dilute hydrochloric acid. A solid precipitated and was filtered. The filter cake was washed with water to obtain 210 mg of a light yellow solid with a yield of 86%. ¹ H-NMR (500MHz, DMSO-d ₆ ) δ (ppm): 13.04 (s, 1H), 8.93 (s, 1H), 8.04 (d, J = 7.2Hz, 1H), 7.85 (s, 1H), 7.53 (t, J = 5.6Hz, 1H), 7.25 (d, J = 7.6Hz, 1H), 6.71 (d, J ＝6.0Hz,1H),6.22(d,J＝6.4Hz,1H),4.37(s,3H),3.86(s,3H).

c). 1-methyl-4-((6-methoxypyridin-2-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

Under Ar protection, 1-methyl-4-((6-methoxypyridin-2-yl)amino)-7-chloro-indole-2-carboxylic acid (85 mg, 0.26 mmol) was dissolved in dry DCM, and HATU (176 mg, 0.46 mmol), DMAP (16 mg, 0.14 mmol) and TEA (78 mg, 0.77 mmol) were added in sequence. After stirring evenly, p-methoxybenzenesulfonamide (87 mg, 0.46 mmol) was added. The reaction was stopped after reacting at 35 degrees for 30 minutes. After evaporating the solvent, the mixture was washed with dilute hydrochloric acid, water and brine in sequence. The EA layers were combined and recrystallized to obtain 82 mg of a light yellow-green solid with a yield of 64%. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ (ppm): 12.58 (s, 1H), 9.00 (s, 1H), 7.97-7.95 (d, J = 10.4Hz, 1H), 7.84 (s, 1H), 7.53 (m, 1H), 7.27-7.25 (d, J = 8.4Hz, 1H), 7.19 -7.17(d,J＝8.4Hz,1H),6.73-6.71(d,J＝8.4Hz,1H),6.26-6.24(d,J＝8.4Hz,1H),4.12(s,3H),3.86(s,3H),3.83(s,3H).

Example 2 1-Methyl-4-((6-methoxypyridin-2-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide sodium salt

Under Ar protection, compound 1-methyl-4-((6-methoxypyridin-2-yl)amino)-7-chloro-indole-2-carboxylic acid (2 g, 6.04 mmol) was dissolved in DCM (40 mL), and HATU (4.59 g, 12.08 mmol), DMAP (737 mg, 6.04 mmol) and TEA (2.61 mL, 18.12 mmol) and p-methoxybenzenesulfonamide (2.265 mg, 12.08 mmol) were added. The mixture was stirred at room temperature for about 1 h, and DCM (50 mL) was added. The mixture was washed with 1N hydrochloric acid solution, saturated sodium bicarbonate solution, and water. Solid precipitated and filtered. The filter cake was washed with water and DCM to obtain 1.2 g of khaki solid. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ (ppm): 8.84 (s, 1H), 7.96 (d, J = 8.4Hz, 1H), 7.70-7.81 (m, 2H), 7.48 (t, J = 4.0Hz, 2H), 7.05 (d, J = 8.4Hz, 1H), 6.91-6.95 (m, 2H), 6.72(d,J＝8.0Hz,1H),6.16(d,J＝7.6Hz,1H),4.30(s,3H),3.85(s,3H),3.78(s,3H).

Example 3 1-Methyl-4-((4-methoxypyridin-2-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

a). 1-Methyl-4-((4-methoxypyridin-2-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester

Under argon protection, the compound 1-methyl-7-chloro-4-bromo-indole-2-carboxylic acid ethyl ester (133 mg, 0.42 mmol) was dissolved in toluene (20 mL), and Pd ₂ (dba) ₃ (40 mg, 0.04 mmol), Xantphos (50 mg, 0.09 mmol) and Na ₂ CO ₃ (135 mg, 1.26 mmol, 3 mL H ₂ O) were added in sequence. After stirring evenly, 4-methoxy-2-aminopyridine (156 mg, 1.26 mmol) was added, and the reaction was refluxed for 9 h. The raw material completely disappeared. The mixture was cooled to room temperature, concentrated, and EA (20 mL) was added to dissolve the residue. The residue was washed with water and salt, and 115 mg of a light yellow solid was obtained by column chromatography with a yield of 76%. ¹ H-NMR (400MHz, CDCl ₃ ) δ (ppm): 8.05 (s, 3H), 7.32 (s, 1H), 7.24-7.22 (m, 2H), 7.02 (br, 1H), 6.39 (s, 2H), 4.47 (s, 3H), 4.36 (m, 2H), 3.77 (s, 3H), 1.39 (t, J＝8.0Hz,3H).

b). 1-Methyl-4-((4-methoxypyridin-2-yl)amino)-7-chloro-indole-2-carboxylic acid

The compound 1-methyl-4-((4-methoxypyridin-2-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester (100 mg, 0.28 mmol) was dissolved in THF/EtOH (1/2, v/v, 12 mL), and NaOH solution (56 mg, 1.39 mmol, 2 mL H ₂ O) was added. The reaction was allowed to react at room temperature overnight. After stopping the reaction, the solvent was evaporated, a small amount of water was added, and 1 M hydrochloric acid was used to adjust the pH to 2. The precipitated solid was filtered and dried to obtain 89 mg of a light yellow-green solid with a yield of 96%. ¹ H-NMR(400MHz,DMSO-d ₆ )δ(ppm):13.36(s,1H),10.68(s,1H),7.94(d,J＝6.4Hz,1H),7.44-7.28(m,3H),6.71(d,J＝7.6Hz,2H),4.40(s,3H),3.92(s,3H).

c). 1-Methyl-4-((4-methoxypyridin-2-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

Under Ar protection, 1-methyl-4-((4-methoxypyridin-2-yl)amino)-7-chloro-indole-2-carboxylic acid (100 mg, 0.30 mmol) was dissolved in dry DCM, and HATU (207 mg, 0.54 mmol), DMAP (20 mg, 0.15 mmol) and TEA (92 mg, 0.91 mmol) were added in sequence. After stirring evenly, p-methoxybenzenesulfonamide (102 mg, 0.54 mmol) was added. The reaction was stopped after reacting at 35 degrees for 30 min. The solvent was evaporated and washed with dilute hydrochloric acid, water and brine in sequence. The EA layers were combined and separated by column to obtain a slightly yellow solid (84 mg, 55%). ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 12.54 (s, 1H), 9.04 (s, 1H), 8.00 (d, J = 6.0Hz, 1H), 7.94 (dd, J ₁ = 8.4Hz, J ₂ = 4.0Hz, 3H), 7.74 (s, 1H), 7.24 (d, J = 8.4Hz, 1H ),7.18-7.11(m,2H),6.67(d,J＝2.4Hz,1H),6.50(dd,J ₁ ＝6.0,J ₁ ＝2.4Hz,1H),4.14(s,3H),3.86(s,3H),3.82(s,3H).HRMS(ESI):m/z,calcd.for C ₂₃ H ₂₀ N ₄ O ₅ ClS[MH] ^- :499.08,found499.06

Example 4 1-Methyl-4-((5-methoxypyridin-3-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

a). 1-Methyl-4-((5-methoxypyridin-3-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester

Under argon protection, the compound 1-methyl-7-chloro-4-bromo-indole-2-carboxylic acid ethyl ester (195 mg, 0.62 mmol) was dissolved in toluene (20 mL), and Pd ₂ (dba) ₃ (58 mg, 0.06 mmol), Xantphos (72 mg, 0.12 mmol) and Na ₂ CO ₃ (192 mg, 1.85 mmol, 3 mL H ₂ O) were added in sequence. After stirring evenly, 5-methoxy-3-aminopyridine (230 mg, 1.85 mmol) was added, and the reaction was refluxed for 8 h. The raw material completely disappeared. The mixture was cooled to room temperature, concentrated, and EA (20 mL) was added to dissolve the residue. The residue was washed with water and salt, and 133 mg of a light yellow solid was obtained by column chromatography with a yield of 60%. ¹ H-NMR (400MHz, CDCl ₃ ) δ (ppm): 8.07 (s, 1H), 7.91 (s, 1H), 7.27 (s, 1H), 7.18 (d, J = 8.0Hz, 1H), 6.95 (s, 1H), 6.85 (d, J = 8.0Hz, 1H), 6.06 (s, 1H), 4.47 (s, 3H), 4.40-4.32 (m, 2H), 3.82 (s, 3H), 1.39 (t, J = 4.0Hz, 3H).

b). 1-Methyl-4-((5-methoxypyridin-3-yl)amino)-7-chloro-indole-2-carboxylic acid

The compound 1-methyl-4-((5-methoxypyridin-3-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester (100 mg, 0.28 mmol) was dissolved in THF/EtOH (1/2, v/v, 12 mL), and NaOH solution (56 mg, 1.39 mmol, 2 mL H ₂ O) was added. The reaction was allowed to react at room temperature overnight. After stopping the reaction, the solvent was evaporated, a small amount of water was added, and 1 M hydrochloric acid was used to adjust the pH to 2. The precipitated solid was filtered and dried to obtain 90 mg of a light yellow-green solid with a yield of 97%.

¹ H-NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.17 (s, 1H), 8.97 (m, 1H), 8.12 (s, 1H), 7.96 (s, 1H), 7.53 (s, 1H) ),7.34(s,1H),7.25(d,J＝8.4Hz,1H),6.98(d,J＝8.4Hz,1H),4.37(s,3H),3.85(s,3H).

c). 1-Methyl-4-((5-methoxypyridin-3-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

Under Ar protection, 1-methyl-4-((5-methoxypyridin-3-yl)amino)-7-chloro-indole-2-carboxylic acid (75 mg, 0.23 mmol) was dissolved in dry DCM, and HATU (155 mg, 0.41 mmol), DMAP (14 mg, 0.11 mmol) and TEA (70 mg, 0.68 mmol) were added in sequence. After stirring evenly, p-methoxybenzenesulfonamide (76 mg, 0.41 mmol) was added. The reaction was stopped after reacting at 35 degrees for 50 min. After evaporating the solvent, the mixture was washed with dilute hydrochloric acid, water and brine in sequence. The EA layers were combined and separated by column to obtain a slightly yellow solid (60 mg, 52%). ¹ H-NMR (400MHz, DMSO-d ₆ ) δ (ppm): 11.96 (s, 1H), 8.59 (s, 1H), 8.07 (s, 1H), 7.94 (d, J = 8.4Hz, 2H), 7.85 (s, 1H), 7.63 (s, 1H), 7.19 (m, 3H), 7.08 (s, 1H), 6.90(d,J＝10.4Hz,1H),4.14(s,3H),3.86(s,3H),3.79(s,3H).

Example 5 1-methyl-4-((6-methoxypyridin-3-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

a). 1-Methyl-4-((6-methoxypyridin-3-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester

Under argon protection, the compound 1-methyl-7-chloro-4-bromo-indole-2-carboxylic acid ethyl ester (190 mg, 0.60 mmol) was dissolved in toluene (20 mL), and Pd ₂ (dba) ₃ (55 mg, 0.06 mmol), Xantphos (70 mg, 0.12 mmol) and Na ₂ CO ₃ (191 mg, 1.80 mmol, 3 mL H ₂ O) were added in sequence. After stirring evenly, 2-methoxy-5-aminopyridine (224 mg, 1.80 mmol) was added, and the reaction was refluxed for 7 h until the raw material completely disappeared. The mixture was cooled to room temperature, concentrated, and EA (20 mL) was added to dissolve the residue, washed with water, washed with salt, and subjected to column chromatography to obtain 130 mg of a light yellow solid with a yield of 59%. ¹ H-NMR (400MHz, CDCl ₃ ) δ (ppm): 8.09 (s, 1H), 7.52 (d, J = 8.4Hz, 1H), 7.31 (s, 1H), 7.09 (d, J = 7.6Hz, 1H), 6.77 (d, J = 10.0Hz, 1H), 6.47 (s, 1H), 5.90 (s, 1H) ,4.46(s,3H),4.36(m,2H),3.97(s,3H),1.40(t,J=7.2Hz,3H).

b). 1-Methyl-4-((6-methoxypyridin-3-yl)amino)-7-chloro-indole-2-carboxylic acid

1-Methyl-4-((6-methoxypyridin-3-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester (110 mg, 0.31 mmol) was dissolved in THF/EtOH (1/2, v/v, 12 mL), and NaOH solution (62 mg, 1.53 mmol, 2 mL H ₂ O) was added. The reaction was allowed to react overnight at room temperature. After stopping the reaction, the solvent was evaporated, a small amount of water was added, and the pH was adjusted to 2 with 1 M hydrochloric acid. The precipitated solid was filtered and dried to obtain 98 mg of a light yellow-green solid with a yield of 97%. ¹ H-NMR(400MHz,DMSO-d ₆ )δ(ppm):8.07(s,1H),7.65(s,2H),7.09(d,J＝8.4Hz,1H),6.87(d,J＝8.4Hz,1H),6.48(d,J＝8.8Hz,1H),4.34(s,3H),3.85(s,3H).

c). 1-methyl-4-((6-methoxypyridin-3-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

Under Ar protection, 1-methyl-4-((6-methoxypyridin-3-yl)amino)-7-chloro-indole-2-carboxylic acid (76 mg, 0.23 mmol) was dissolved in dry DCM, and HATU (158 mg, 0.41 mmol), DMAP (14 mg, 0.11 mmol) and TEA (70 mg, 0.69 mmol) were added in sequence. After stirring evenly, p-methoxybenzenesulfonamide (78 mg, 0.41 mmol) was added. The reaction was stopped after reacting at 35 degrees for 1.0 h. The solvent was evaporated and washed with dilute hydrochloric acid, water and brine in sequence. The EA layers were combined and separated by column to obtain a slightly yellow solid (61 mg, 53%). ¹ H-NMR (400MHz, DMSO-d ₆ ) δ (ppm): 12.50 (s, 1H), 8.24 (s, 1H), 8.06 (s, 1H), 7.95 (d, J = 8.8Hz, 2H), 7.66 (s, 1H), 7.59 (dd, J ₁ = 8.4Hz, J ₂ = 2.4Hz, 1H), 7.18 (d,J＝8.8Hz,2H),7.10(d,J＝8.4Hz,1H),6.83(d,J＝8.4Hz,1H),6.48(d,J＝8.4Hz,1H),4.10(s,3H),3.87(s,3H),3.84(s,3H).HRMS(ESI):m/z,calcd.for C ₂₃ H ₂₀ N ₄ O ₅ ClS[MH] ^- :499.08,found499.06.

Example 6 1-Methyl-4-((2-methoxypyrimidin-6-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

a). 1-Methyl-4-((2-methoxypyrimidin-6-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester

Under argon protection, the compound 1-methyl-7-chloro-4-bromo-indole-2-carboxylic acid ethyl ester (750mg, 2.39mmol) was dissolved in toluene (40mL), Pd ₂ (dba) ₃ (547mg, 0.6mmol) and Xantphos (694mg, 1.2mmol) were added, and then Na ₂ CO ₃ (847mg, 7.99mmol) was dissolved in (10mL) water and added to the reaction solution, and 2-methoxy-4-aminopyrimidine (1g, 7.99mmol) was added to the reaction bottle, and refluxed for 20h. Filter, add ethyl acetate to the filtrate, wash with dilute hydrochloric acid, wash with saturated sodium bicarbonate solution, wash with saturated sodium chloride solution, dry with anhydrous magnesium sulfate, and obtain 720mg of off-white solid by column chromatography with a yield of 83%. ¹ H-NMR (400MHz, CDCl ₃ ) δ (ppm): 8.09 (d, J = 4.4Hz, 1H), 7.25-7.28 (m, 3H), 7.07 (s, 1H), 6.33 (s, 1H), 4.48 (s, 3H), 4.37 (q, J = 6.4Hz, 2H), 3.97 (s, 3H), 1.39 (t,J＝6.4Hz,3H).

b). 1-Methyl-4-((2-methoxypyrimidin-6-yl)amino)-7-chloro-indole-2-carboxylic acid

The compound 1-methyl-4-((2-methoxypyrimidin-6-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester (650 mg, 1.805 mmol) was dissolved in a mixture of THF (10 mL) and EtOH (10 mL), and NaOH (217 mg, 5.42 mmol) was dissolved in 3.5 mL of water and added to the reaction solution. The mixture was reacted at 40° C. for 2 h, concentrated, and a small amount of water was added. The mixture was extracted with ether, and the pH of the aqueous layer was adjusted to 2-3 with dilute hydrochloric acid. Solid precipitated and was filtered. The filter cake was washed with water to obtain 600 mg of a light yellow solid with a yield of 99%. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ (ppm): 10.82 (s, 1H), 8.17 (dd, J ₁ = 8.4Hz, J ₂ = 1.2Hz, 1H), 7.78 (t, J = 8.0Hz, 1H), 7.69 (s, 1H), 7.38 (dd, J ₁ = 8.4Hz, J ₂ = 1.2Hz ,1H),6.94(d,J=8.4Hz,1H),4.39(s,3H),3.96(s,3H).

c). 1-methyl-4-((2-methoxypyrimidin-6-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

Under Ar protection, 1-methyl-4-((2-methoxypyrimidin-6-yl)amino)-7-chloro-indole-2-carboxylic acid (75 mg, 0.23 mmol) was dissolved in dry DCM, and HATU (156 mg, 0.41 mmol), DMAP (15 mg, 0.12 mmol) and TEA (70 mg, 0.69 mmol) were added in sequence. After stirring evenly, p-methoxybenzenesulfonamide (77 mg, 0.41 mmol) was added. The reaction was stopped after reacting at 35 degrees for 30 min. After evaporating the solvent, the mixture was washed with dilute hydrochloric acid, water and brine in sequence. The EA layers were combined and separated by column to obtain a slightly yellow solid (63 mg, 54%). ¹ H-NMR (400MHz, DMSO-d ₆ ) δ (ppm): 12.63 (s, 1H), 9.60 (s, 1H), 8.15 (s, 1H), 7.93 (d, J = 8.8Hz, 2H), 7.82 (d, J = 8.4Hz, 1H), 7.72 (s, 1H), 7.32 (d, J = 8.4Hz, 1 H),7.15(d,J＝8.7Hz,2H),6.66(s,1H),4.16(s,3H),3.85(s,3H),3.83(s,3H).

Example 7 1-Methyl-4-((2-methoxypyrimidin-6-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide sodium salt

Under Ar protection, 1-methyl-4-((2-methoxypyrimidin-6-yl)amino)-7-chloro-indole-2-carboxylic acid (498 mg, 1.5 mmol) was dissolved in DCM (30 mL), and HATU (1.14 g, 3 mmol), DMAP (91.5 mg, 0.75 mmol) and TEA (0.65 mL, 4.5 mmol) and p-methoxybenzenesulfonamide (563 mg, 3 mmol) were added. The mixture was stirred at room temperature for about 1 h, and DCM (25 mL) was added. The mixture was washed with 1N hydrochloric acid solution, saturated sodium bicarbonate solution, and water. Solid precipitated and filtered. The filter cake was washed with water and DCM to obtain 470 mg of khaki solid with a yield of 62%. ^1. 93(d,J＝8.8Hz, _2H ),6.73(d,J＝6.0Hz,1H),4.31(s,3H),3.85(s,3H),3.78(s,3H).

Example 8 1-methyl-4-((4-methoxypyrimidin-6-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

a). 1-Methyl-4-((4-methoxypyrimidin-6-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester

Under argon protection, the product 1-methyl-7-chloro-4-bromo-indole-2-carboxylic acid ethyl ester (158 mg, 0.50 mmol) was dissolved in toluene (20 mL), and Pd ₂ (dba) ₃ (46 mg, 0.05 mmol), Xantphos (58 mg, 0.10 mmol) and Na ₂ CO ₃ (160 mg, 1.50 mmol, 3 mL H ₂ O) were added in sequence. After stirring evenly, 6-methoxy-4-aminopyrimidine (188 mg, 1.50 mmol) was added, and the reaction was refluxed for 10 h. The raw material completely disappeared. The mixture was cooled to room temperature, concentrated, and EA (20 mL) was added to dissolve the residue. The residue was washed with water and salt, and separated by column chromatography (PE/EA=10/1-5/1) to obtain 108 mg of light yellow solid with a yield of 60%. ¹ H-NMR (400MHz, CDCl ₃ ) δ (ppm): 8.39 (s, 1H), 7.30 (s, 1H), 7.10 (d, J = 8.4Hz, 1H), 6.03 (s, 1H), 4.48 (s, 3H), 4.36 (d, J = 6.8Hz, 2H), 3.92 (s, 3H), 1.41 (d, J = 8.0Hz,3H).

b). 1-Methyl-4-((4-methoxypyrimidin-6-yl)amino)-7-chloro-indole-2-carboxylic acid

1-Methyl-4-((4-methoxypyrimidin-6-yl)amino)-7-chloro-indole-2-carboxylic acid ethyl ester (95 mg, 0.26 mmol) was dissolved in THF/EtOH (1/2, v/v, 12 mL), and NaOH solution (53 mg, 1.32 mmol, 2 mL H ₂ O) was added. The reaction was allowed to react overnight at room temperature. After stopping the reaction, the solvent was evaporated, a small amount of water was added, and 1 M hydrochloric acid was used to adjust the pH to 2. The precipitated solid was filtered and dried to obtain 85 mg of a light yellow-green solid with a yield of 96%. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ (ppm): 9.45 (s, 1H), 8.43 (s, 1H), 7.82 (d, J = 8.4Hz, 1H), 7.66 (s, 1H), 7.29 (d, J = 8.4Hz, 1H), 6.38 (s, 1H), 4.37 (s, 3H), 3.87 (s, 3H).

c). 1-methyl-4-((4-methoxypyrimidin-6-yl)amino)-7-chloro-N-(4-methoxybenzenesulfonyl)-indole-2-carboxamide

Under Ar protection, 1-methyl-4-((4-methoxypyrimidin-6-yl)amino)-7-chloro-indole-2-carboxylic acid (70 mg, 0.21 mmol) was dissolved in dry DCM, and HATU (145 mg, 0.38 mmol), DMAP (14 mg, 0.11 mmol) and TEA (64 mg, 0.63 mmol) were added in sequence. After stirring evenly, p-methoxybenzenesulfonamide (72 mg, 0.38 mmol) was added. The reaction was stopped after reacting at 35 degrees for 1.5 hours. After evaporating the solvent, the mixture was washed with dilute hydrochloric acid, water and brine in sequence. The EA layers were combined and separated by column to obtain a slightly yellow solid (72 mg, 67%). ¹ H-NMR (400MHz, DMSO-d ₆ )δ(ppm):11.95(s,1H),9.38(s,1H),8.40(s,1H),7.96(m,2H),7.69(m,2H),7.18(m,1H),7.12(m,2H),6.26(s,1H),4.14(s,3H ),3.86(s,6H).

Pharmacological experiments:

Experimental Example 1: Evaluation of the inhibitory activity of compounds on FBP enzyme

Experimental methods and results:

1. Recombinant expression of human liver FBPase

The constructed human liver FBPase gene plasmid PETα8a-FBP was transformed into competent bacteria BL21 (DE3) Rosetta cells (Novagen, Inc.), and 100ul of competent bacteria containing the plasmid was applied to LB solid culture plates containing kanamycin (30ug/mL), and cultured in an incubator at 37°C for 12-14h until obvious single colonies grew. Pick a single colony from the solid culture medium, inoculate it into LB liquid culture medium containing kanamycin (30ug/ml), and expand and culture it at 37°C and 200rpm for 12h. IPTG was added to the expanded culture solution (so that its final concentration in the bacterial solution was 0.1mM), and cultured in a shaker at 16°C and 200rpm for 24h to induce protein expression. Then the bacterial solution was centrifuged at 4°C and 5500rpm for 5min, the supernatant was discarded, and the precipitate (bacteria) was added to FBPaseTris-Buffer to resuspend the bacteria. The cells were disrupted by ultrasound, and then centrifuged at 4°C, 12,000 rpm for 1 min. The supernatant was collected, the enzyme activity was determined, and the aliquots were stored at -80°C for later use.

2. Determination of FBP enzyme activity

FBPase activity assay principle: Utilize the coupled reaction of FBPase with phosphoglucose isomerase and glucose-6-phosphate dehydrogenase, and use a spectrophotometer to detect the concentration of the reaction product to reflect the activity of the enzyme. The reaction consumes NADP ⁺ to generate NADPH, and the absorbance change at 340nm directly reflects the activity of the enzyme. First, add FBPase Tris-Buffer (100mM Tris, 2mM MgCl ₂ , 0.1mM EDTA, pH7.5), inhibitor (final concentration 0.08uM, 0.4uM, 2uM or 10uM, all reaction wells are replaced by FBPase Tris-Buffer) and FBPase (final concentration 0.72unit) to the reaction well. After incubation at 37℃ for 10min, add the reaction substrate mixture (final concentration 0.2mM NADP ⁺ , 0.01units phosphoglucose isomerase, 0.01units glucose-6-phosphate dehydrogenase and 0.2mM FBP), and the reaction begins. Dynamically monitor the absorbance of the reaction product (NADPH). Take the absorbance of the entire reaction well as 100% to calculate the inhibition rate of the inhibitor reaction well, that is, the inhibition strength of the enzyme activity. Obtain the inhibition curve through the concentration of the inhibitor and the activity of the enzyme.

Table 1. FBPase inhibitory activity of compounds

Example (IC ₅₀ M) Example (IC ₅₀ M) 1 8.30×10 ^-8 2 1.50×10 ^-8 3 4.78×10 ^-8 4 1.81×10 ^-8 5 4.49×10 ^-8 6 3.05× ^10-8 7 9.70×10 ^-8 8 2.71×10 ^-8

Experimental Example 2: Animal in vivo pharmacodynamics experiment

Experimental methods and results:

1. Experimental Methods

Normal male ICR mice were fasted overnight, and blood samples were taken at zero o'clock the next morning (-120min). Afterwards, Example Compound 2 (Example 2, 150mg/kg) or positive drug metformin (Met, 150mg/kg) was administered by gavage, and the blank control group was replaced with water. After 2h of administration, blood samples were collected after administration (0min), and sodium pyruvate (2g/kg), a gluconeogenic substrate, was orally administered by gavage, and blood samples of mice were collected 30min after gavage of sodium pyruvate (30min). Glucose concentration in blood samples was measured by GOD method. Compare whether the difference in blood sugar between 0min and 30min in each group is statistically different from that in the normal control group, and determine the hypoglycemic activity of the compound.

2. Experimental results

The results are shown in Table 2. The compound of Example 2 can significantly inhibit the 30-min rise in blood glucose in the sodium pyruvate tolerance test of normal ICR mice, and its effect is better than that of metformin at an equal dose.

Table 2. Effect of single oral administration in Example 2 on oral sodium pyruvate tolerance test in ICR mice

The data are shown as mean ± SD, *P < 0.05, ***P < 0.001, compared with the control group.

Experimental Example 3: Pharmacokinetic Experiment Methods and Results:

1. Experimental Methods

1. Sample Collection

Six SD rats were randomly divided into two groups, an intravenous injection group of Example 2 and an oral administration group, with 3 rats in each group. After the rats were intravenously injected with the compound of Example 2 (3 mg/kg), blood was collected from the orbital venous plexus at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, and 72 h, respectively. After the rats were orally administered with the compound of Example 2 (30 mg/kg), blood was collected from the orbital venous plexus at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, and 72 h, respectively. The blood was collected in heparin-anticoagulated EP tubes and placed on ice. After centrifugation at 8000 r/min×5 min, the plasma was separated and used for later use.

2. Preparation of standard curve

Standard curve of the compound in Example 2: Take 50 μl of blank plasma from SD rats, add 480 μl of acetonitrile, and then add 10 μl of methanol solution of the compound in Example 2 at different concentrations to make the final concentrations of 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 50000, 10000, 20000, 50000 ng/ml, mix well, and high-speed centrifuge twice at 14000 r/min×5 min. Take 5 μl of the supernatant for HPLC-MS/MS analysis.

3. Sample Processing

Take 20 μl of plasma, add 200 μl of acetonitrile containing internal standard to precipitate protein, centrifuge twice at 14000 r/min×5 min, and take 5 μl of the supernatant for HPLC-MS/MS analysis.

2. Experimental results

Plasma pharmacokinetic parameters

After oral and intravenous administration of the compound of Example 2 to rats, the plasma pharmacokinetics were analyzed using WinNonLin software and fitted with a non-compartmental model. The results are shown in Table 3.

Table 3 Plasma pharmacokinetic parameters of rats after oral and intravenous injection of the compound of Example 2

Experimental Example 4: In vitro inhibition of cytochrome P450 (CYP)

1. Experimental methods

1. Human liver microsome incubation test

The experiment was conducted in three groups, namely, control group (no inhibitor), positive inhibitor group, and test compound inhibition group (10 μM), with three parallel samples in each group. The inhibitor was added to human liver microsomes and incubated with probe substrates of each subtype, and the metabolites generated by the probe substrate were measured and compared with the control group to reflect the degree of inhibition of the inhibitor on the isozyme.

The liver microsome incubation system includes human liver microsome protein (0.5 mg/mL), NADPH generating system, Tris-HCl buffer (50 mM, pH=7.4), substrate and inhibitor, and the total reaction volume is 200 μL (organic solvent content 1%). The concentration and incubation time of CYP1A2, 2D6, 2C9, 2C19, 3A4, and 2E1 substrates are phenacetin 50 μM/30 min, dextromethorphan 5 μM/10 min, diclofenac sodium 20 μM/10 min, mephenytoin 40 μM/30 min, midazolam 5 μM/5 min, and chlorzoxazone 80 μM/20 min. The final concentrations of positive inhibitors of CYP1A2, 2D6, 2C9, 2C19, 3A4, and 2E1 isozymes were 10 μM furafylline, 2 μM quinidine, 5 μM sulfaphenazole, 5 μM ticlopidine, 1 μM ketoconazole, and 50 μM diethyl disulfide ammonium formate. After the reaction was completed, 400 μL of glacial acetonitrile containing internal standard was added to terminate the reaction, mixed, centrifuged twice at 14000 rpm × 5 min, and 5 μL of the supernatant was taken for LC-MS/MS analysis to determine the content of the probe substrate metabolite.

2. Experimental results

The metabolic inhibition rates of positive inhibitors on CYP1A2, 2C19, 2C9, 2D6, 3A4, and 2E1 probe substrates were 61.23%, 93.52%, 76.81%, 89.77%, 93.82%, and 80.37%, respectively, and they had obvious inhibitory effects on the substrates of the six isozymes.

The inhibitory activity of the test compound on each subtype was weaker than that of the positive control inhibitor, and had good safety. The results are shown in Table 4.

Table 4 In vitro inhibition of compounds (10 μM) on human liver microsome CYP450 isozymes

Claims (15)

1. A compound as shown in general formula I or a pharmaceutically acceptable salt thereof, In Formula I, A, B, D, and E are independently selected from CR or N; at least one atom among A, B, D, and E is N; R is selected from H, F, Cl, CH ₃ , and OCH ₃ ; Ar ₁ is selected from the following groups or structural fragments: Phenyl, substituted phenyl, unsubstituted nitrogen-containing six-membered aromatic heterocycle, substituted nitrogen-containing six-membered aromatic heterocycle; wherein the substituent is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclopropylmethylene, cyclobutyl, halogen-substituted C1-4 straight or branched alkyl, F, Cl, Br, NO ₂ , CN, methylenedioxy, ORs ₁ , NRs ₂ Rt ₁ , CONRs ₃ Rt ₂ ; wherein Rs ₁ , Rs ₂ , Rs ₃ , Rt ₁ , and Rt ₂ are independently selected from H, C1-4 straight or branched alkyl, halogen-substituted C1-4 straight or branched alkyl, cyclopropyl, cyclopropylmethylene, and cyclobutyl; The phenyl group and the nitrogen-containing six-membered aromatic heterocyclic ring are mono- or poly-substituted; The six-membered aromatic heterocyclic ring contains one nitrogen atom or multiple nitrogen atoms; The halogen is selected from F, Cl, and Br. 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that the compound is as shown in general formula I-A: In I-A, A, B, D, and E are independently selected from CR or N; at least one atom among A, B, D, and E is N; R is selected from H, F, Cl, CH ₃ , and OCH ₃ ; _M1 is independently selected from different alkali metal or alkaline earth metal salts; Ar ₁ is selected from the following groups or structural fragments: Phenyl, substituted phenyl, unsubstituted nitrogen-containing six-membered aromatic heterocycle, substituted nitrogen-containing six-membered aromatic heterocycle; wherein the substituent is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclopropylmethylene, cyclobutyl, halogen-substituted C1-4 straight or branched alkyl, F, Cl, Br, NO ₂ , CN, methylenedioxy, ORs ₁ , NRs ₂ Rt ₁ , CONRs ₃ Rt ₂ ; wherein Rs ₁ , Rs ₂ , Rs ₃ , Rt ₁ , and Rt ₂ are independently selected from H, C1-4 straight or branched alkyl, halogen-substituted C1-4 straight or branched alkyl, cyclopropyl, cyclopropylmethylene, and cyclobutyl; The phenyl group and the nitrogen-containing six-membered aromatic heterocyclic ring are mono- or poly-substituted; The six-membered aromatic heterocyclic ring contains one nitrogen atom or multiple nitrogen atoms; The halogen is selected from F, Cl, and Br. 3. The compound or pharmaceutically acceptable salt thereof according to claim 2, characterized in that said _M1 is selected from lithium, sodium, potassium, cesium, calcium, magnesium and barium. 4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, characterized in that The fragment is selected from the following structural fragments: R ₁ is selected from F, Cl, CH ₃ , OCH ₃ ; In the substituted nitrogen-containing six-membered aromatic heterocycle or the unsubstituted nitrogen-containing six-membered aromatic heterocycle, the nitrogen-containing six-membered aromatic heterocycle is selected from pyridine, pyrimidine, pyridazine, and pyrazine; The halogen-substituted C1-4 straight or branched alkyl group is independently selected from CF ₃ , CH ₂ F, CHF ₂ , CH ₂ CF ₃ , CF ₂ CH ₃ , CH ₂ CH ₂ CF ₃ ; The C1-4 straight or branched alkyl group is selected from methyl, ethyl, propyl, isopropyl, butyl and isobutyl. 5. A compound as shown in general formula II or a pharmaceutically acceptable salt thereof, In Formula II, A', B', D', E' are independently selected from CR' or N; at least one atom among A', B', D', E' is N; R' is selected from H, F, Cl, CH ₃ , OCH ₃ ; Ar ₂ is selected from the following groups or structural fragments: Phenyl, substituted phenyl, unsubstituted nitrogen-containing six-membered aromatic heterocycle, substituted nitrogen-containing six-membered aromatic heterocycle; wherein the substituent is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclopropylmethylene, cyclobutyl, halogen-substituted C1-4 straight or branched alkyl, F, Cl, Br, NO ₂ , CN, methylenedioxy, ORs ₁ , NRs ₂ Rt ₁ , CONRs ₃ Rt ₂ ; wherein Rs ₁ , Rs ₂ , Rs ₃ , Rt ₁ , and Rt ₂ are independently selected from H, C1-4 straight or branched alkyl, halogen-substituted C1-4 straight or branched alkyl, cyclopropyl, cyclopropylmethylene, and cyclobutyl; The phenyl group and the nitrogen-containing six-membered aromatic heterocyclic ring are mono- or poly-substituted; The six-membered aromatic heterocyclic ring contains one nitrogen atom or multiple nitrogen atoms; The halogen is selected from F, Cl, and Br. 6. The compound or pharmaceutically acceptable salt thereof according to claim 5, characterized in that the compound is as shown in general formula II-A: In Formula II-A, A', B', D', E' are independently selected from CR' or N; at least one atom among A', B', D', E' is N; R' is selected from H, F, Cl, CH ₃ , OCH ₃ ; _M2 is independently selected from different alkali metal or alkaline earth metal salts; Ar ₂ is selected from the following groups or structural fragments: Phenyl, substituted phenyl, unsubstituted nitrogen-containing six-membered aromatic heterocycle, substituted nitrogen-containing six-membered aromatic heterocycle; wherein the substituent is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclopropylmethylene, cyclobutyl, halogen-substituted C1-4 straight or branched alkyl, F, Cl, Br, NO ₂ , CN, methylenedioxy, ORs ₁ , NRs ₂ Rt ₁ , CONRs ₃ Rt ₂ ; wherein Rs ₁ , Rs ₂ , Rs ₃ , Rt ₁ , and Rt ₂ are independently selected from H, C1-4 straight or branched alkyl, halogen-substituted C1-4 straight or branched alkyl, cyclopropyl, cyclopropylmethylene, and cyclobutyl; The phenyl group and the nitrogen-containing six-membered aromatic heterocyclic ring are mono- or poly-substituted; The six-membered aromatic heterocyclic ring contains one nitrogen atom or multiple nitrogen atoms; The halogen is selected from F, Cl, and Br. 7. The compound or pharmaceutically acceptable salt thereof according to claim 6, characterized in that said _M2 is selected from lithium, sodium, potassium, cesium, calcium, magnesium, and barium. 8. The compound according to any one of claims 5 to 7 or a pharmaceutically acceptable salt thereof, characterized in that The fragment is selected from the following structural fragments: R ₂ is selected from F, Cl, CH ₃ , OCH ₃ ; In the substituted nitrogen-containing six-membered aromatic heterocycle or the unsubstituted nitrogen-containing six-membered aromatic heterocycle, the nitrogen-containing six-membered aromatic heterocycle is selected from pyridine, pyrimidine, pyridazine, and pyrazine; The halogen-substituted C1-4 straight or branched alkyl group is independently selected from CF ₃ , CH ₂ F, CHF ₂ , CH ₂ CF ₃ , CF ₂ CH ₃ , CH ₂ CH ₂ CF ₃ ; The C1-4 straight or branched alkyl group is selected from methyl, ethyl, propyl, isopropyl, butyl and isobutyl. 9. The compound or pharmaceutically acceptable salt thereof according to claim 1 or 5, characterized in that the compound is selected from: 10. A method for preparing a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, comprising the steps of: (1) 2-bromo-5-chloro-benzaldehyde is reacted with sodium and ethyl azidoacetate in the presence of sodium ethoxide and ethyl trifluoroacetate to prepare compound 1; (2) Using o-dichlorobenzene as solvent, compound 1 undergoes a ring-closing reaction at 180°C to obtain ethyl 7-chloro-4-bromo-1H-indole-2-carboxylate; (3) methylation at the indole-1-position occurs under the condition of cesium carbonate to obtain 4-bromo-7-chloro-1-methyl-indole-2-carboxylic acid ethyl ester; (4) Compound 3 undergoes a coupling reaction with a substituted aromatic amine under palladium catalysis to obtain compounds 4 and 4'; (5) Compounds 4 and 4' are hydrolyzed to obtain corresponding compounds 5 and 5' having a carboxyl group at the 2-position; (6) Compounds 5 and 5' undergo condensation reaction with arylsulfonamide to obtain compounds of general structural formulas I and II; (7) Finally, react with different bases to obtain salts of N-acylsulfonamide compounds IA and II-A; wherein A, B, D, E, A', B', D', E', Ar ₁ , Ar ₂ , M ₁ , and M ₂ are as defined in any one of claims 1 to 9. 11. A pharmaceutical composition, characterized in that it comprises an effective dose of a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof and a pharmacodynamically acceptable carrier. 12. Use of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof in the preparation of a FBPase inhibitor. 13. Use of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and/or treating a disease associated with FBPase. 14. The use according to claim 13, characterized in that the disease associated with FBPase is selected from diabetes, diabetic complications, hyperlipidemia and obesity. 15. The use according to claim 14, characterized in that the diabetes is selected from type I diabetes and type II diabetes; the diabetic complications are selected from retinal, kidney, nervous system lesions and vascular complications, local ischemic heart disease or atherosclerosis; and the hyperlipidemia includes high triglycerides and high cholesterol esters.