Detailed Description
In the compounds of the present invention, when any variable (e.g., R, etc.) occurs more than one time in any constituent, its definition in each occurrence is independent of its definition in every other occurrence. Also, combinations of substituents and variables are permissible only if such combinations result in stable compounds. The line drawn from a substituent into the ring system indicates that the indicated bond can be attached to any ring atom that can be substituted. If the ring system is polycyclic, it means that such a bond is only attached to any suitable carbon atom of the adjacent ring. It is to be understood that substituents and substitution patterns on the compounds of the present invention may be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by those skilled in the art and by the methods set forth below from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these groups may be on the same carbon atom or on different carbon atoms, so long as the structure is stable.
The term "alkyl" as used herein is intended to include both branched and straight chain saturated aliphatic hydrocarbon radicals having the specified number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a straight or branched chain arrangement. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl.
The term "alkoxy" refers to a group in which the alkyl group is directly attached to the oxygen, i.e., a group having the structure-O-alkyl, such as-OCH3、-OCH2CH3、-OCH2CH2CH3、-O-CH2CH(CH3)2、-OCH2CH2CH2CH3、-O-CH(CH3)2And the like.
The term "cycloalkyl" or "carbocycle" refers to a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
The term "heterocyclyl" or "heterocycle" is a saturated or partially unsaturated monocyclic or polycyclic cyclic substituent in which one or more ring atoms are selected from N, O or a heteroatom of s (o) m (where m is an integer from 0 to 2), the remaining ring atoms being carbon, for example: morpholinyl, piperidinyl, tetrahydropyrrolyl, pyrrolidinyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, tetrahydrofuranyl, tetrahydrothienyl, and the like, and N-oxides thereof, and the attachment of the heterocyclic substituent may be through a carbon atom or through a heteroatom.
The term "heteroaryl" refers to an aromatic ring containing 1 or more heteroatoms selected from O, N or S, and heteroaryl groups within the scope of the present invention include, but are not limited to: quinolyl, pyrazolyl, pyrrolyl, thienyl, furyl, pyridyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, benzofuryl, benzothienyl, benzoxazole, indolyl, etc.; "heteroaryl" is also understood to include any N-oxide derivative of a nitrogen-containing heteroaryl group.
The term "substituted" refers to the replacement of a hydrogen radical in a particular structure with a radical of a specified substituent.
As understood by those skilled in the art, "halo" or "halo" as used herein means chloro, fluoro, bromo, and iodo.
Unless otherwise defined, alkyl, cycloalkyl, aryl, heteroaryl, and heterocycloalkyl substituents may be unsubstituted or substituted. For example, C1-C6 alkyl may be substituted with one, two or three substituents selected from OH, halogen, alkoxy, dialkylamino or heterocyclyl such as morpholinyl, piperidinyl and the like.
The invention includes the free form of a compound of formula (I), formula (II), formula (IV), formula (V), formula (VI) or formula (VII), as well as pharmaceutically acceptable salts and stereoisomers thereof. Pharmaceutically acceptable salts of the invention can be synthesized from compounds of the invention containing a basic or acidic moiety by conventional chemical methods. In general, salts of basic compounds are prepared by ion exchange chromatography or by reaction of the free base with a stoichiometric amount or excess of an inorganic or organic acid in the form of the desired salt in an appropriate solvent or combination of solvents. Similarly, salts of acidic compounds are formed by reaction with suitable inorganic or organic bases.
Thus, pharmaceutically acceptable salts of the compounds of the present invention include the conventional non-toxic salts of the compounds of the present invention formed by the reaction of a basic compound of the present invention and an inorganic or organic acid. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, as well as those prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxy-monobenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethane disulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, and the like.
If the compounds of the invention are acidic, suitable "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic and organic bases, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Particularly preferred are ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases including salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N' -dibenzylethylenediamine, diethylamine, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, histidine, hydroxycobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, piperdine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
Berg et al, "Pharmaceutical Salts," j.pharm.sci.' 1977: 66: 1-19 describe in more detail the preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts.
Since acidic moieties such as carboxyl groups deprotonated in a compound under physiological conditions may be anionic and such charge may then be balanced out by a protonated or alkylated basic moiety such as a quaternary nitrogen atom bearing a cation internally, it should be noted that the compounds of the present invention are potential internal salts or zwitterions.
In one embodiment, the invention provides a method of treating diseases and/or conditions associated with GLP-1R downstream signaling pathways in humans and other mammals using compounds having a structure represented by formula (I), formula (II), formula (IV), formula (V), formula (VI) or formula (VII), and pharmaceutically acceptable salts or stereoisomers thereof.
Wherein the disease and/or symptom associated with the downstream signaling pathway of GLP-1R may be selected from, but not limited to: type 1 diabetes (T1DM), type 2 diabetes (T2DM), gestational diabetes and other specific types of diabetes (idiopathic T1D, early-onset T2DM, adult-onset diabetes of young adults, juvenile-onset atypical diabetes, malnutrition-related diabetes, latent autoimmune diabetes adult, etc.), diabetic retinopathy, diabetic cerebrovascular disease, diabetic neuropathy, insulin resistance, hyperglycemia, diabetic nephropathy, hypertension, cataracts, osteoporosis, hyperuricemia, and various infections resulting from diabetes, obesity, metabolic syndrome, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), fibrosis, heart disease, stroke, cirrhosis, liver cancer, metabolic acidosis, ketosis, cardiovascular discomfort, epilepsy, and atherosclerosis, Parkinson's disease, Alzheimer's disease, etc.
Combined medication: the compounds of formula (I), formula (II), formula (IV), formula (V), formula (VI) or formula (VII) may be combined with other agents known to treat or ameliorate similar conditions. When administered in combination, the original drug is administered in a manner and dosage which remains the same, while the compound of formula (I), formula (II), formula (IV), formula (V), formula (VI) or formula (VII) is administered simultaneously or subsequently. When the compound of formula (I), formula (II), formula (IV), formula (V), formula (VI) or formula (VII) is administered simultaneously with one or more other drugs, it is preferred to use a pharmaceutical composition containing one or more known drugs together with the compound of formula (I), formula (II), formula (IV), formula (V), formula (VI) or formula (VII). The combination also includes administration of a compound of formula (I), formula (II), formula (IV), formula (V), formula (VI) or formula (VII) in overlapping time periods with one or more other known agents. When a compound of formula (I), formula (II), formula (IV), formula (V), formula (VI) or formula (VII) is administered in combination with one or more other drugs, the dose of the compound of formula (I), formula (II), formula (IV), formula (V), formula (VI) or formula (VII) or known drug may be lower than when they are administered alone.
Drugs or active ingredients that can be combined pharmaceutically with a compound of formula (I), formula (II), formula (IV), formula (V), formula (VI) or formula (VII) include, but are not limited to, the following drugs for the treatment of diabetes: biguanides (e.g., metformin), sulfonylureas (acetohexamide, chlorpropamide, tolbutamide, tolazamide, amisulbutamide, glyburide, gliclazide, glipizide, gliquidone, glibornuride, glisoxepid and glimepiride), thiazolidinediones (e.g., troglitazone, rosiglitazone, pioglitazone, ciglitazone), meglitinides (e.g., nateglinide, repaglinide, mitiglinide), dipeptidyl peptidase 4 inhibitors (sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, rigagliptin and tiagliptin), sodium-glucose transporter 2 inhibitors (canagliflozin, dapagliflozin, rigagliflozin, glutagliflozin and troglifloxagliflozin), sodium-glucose transporter 1/2 inhibitors, GPR40 agonists, GIPR agonists, GPR agonists, GIPR agonists, and meglumine, A GIP/GLP-1 dual receptor agonist, an alpha glucosidase inhibitor, insulin or an insulin analog.
Drugs or active ingredients that may be combined pharmaceutically with a compound of formula (I), formula (II), formula (IV), formula (V), formula (VI) or formula (VII) include, but are not limited to, the following drugs for the treatment of obesity: peptide YY or its analogs, neuropeptide Y receptor type 2 agonists, melanocortin receptor 4 agonists, amylin, GIPR agonists, phosphodiesterases, AMP-activated protein kinases, neuropeptide Y5 receptor antagonists, GPR40 agonists, GIP/GLP-1 dual receptor agonists, naltrexone/bupropion, lorcaserin, phentermine/topiramate, orlistat, liraglutide.
Drugs or active ingredients that may be combined pharmaceutically with a compound of formula (I), formula (I), formula (III), formula (IV), formula (V), formula (VI) or formula (VII) include, but are not limited to, the following drugs for treating NASH: farnesoid X receptor agonists, PPAR α/δ agonists, fibroblast growth factor 19/21 analogs, thyroid hormone receptor β agonists, sodium glucose cotransporter (SGLT) -1/2 inhibitors, acetyl coa carboxylase inhibitors, chemokine receptor-2/5 inhibitors, anti-apoptotic signal-regulating kinase 1 inhibitors, ATP-binding transporter 1 agonists, 5-lipoxygenase inhibitors, vascular adhesion protein 1 inhibitors.
The synthesis method comprises the following steps: the compounds of the invention can be prepared using the methods in the following synthetic schemes (schemes 1-10) in addition to standard methods known in the literature or exemplified in experimental procedures. The compounds and methods of synthesis described in the present invention can be better understood in conjunction with the synthetic schemes described below. The synthetic schemes describe the methods that can be used to prepare the compounds of the present invention, and the methods are described as illustrative schemes for illustrative purposes only and do not limit the scope of the present invention.
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Scheme 5
Scheme 6
Scheme 7
Scheme 8
Scheme 9
Scheme 10
The following are specific examples.
Example 1: preparation of 4- (6-chloropyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester (intermediate 0104-1) (prepared according to scheme one route)
Step 1a preparation of 1- (tert-butyl) 4-methyl 4- (6-chloropyridin-2-yl) piperidine-1, 4-dicarboxylate (Compound 0102-1): to a solution of methyl N-tert-butoxycarbonyl-4-piperidinecarboxylate (3.00 g, 12.33 mmol, 1.0 eq) in tetrahydrofuran (40 ml) was added dropwise a solution of lithium bis (trimethylsilyl) amide in tetrahydrofuran (1.0 mol/l, 25 ml, 24.66 mmol, 2.0 eq) under nitrogen. The mixture was stirred at 0 ℃ for 1.5 h, then a solution of 2, 6-dichloropyridine (2.00 g, 13.51 mmol, 1.1 eq) in tetrahydrofuran (5 ml) was added dropwise. The mixture was warmed to room temperature and stirred for 2.5 hours. The reaction was quenched with saturated aqueous ammonium chloride (10 ml) and separated with water (70 ml) and ethyl acetate (160 ml). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product of 1- (tert-butyl) 4-methyl 4- (6-chloropyridin-2-yl) piperidine-1, 4-dicarboxylate (4.49 g, yield: 103%). The product was used in the next step without further purification.
Step 1b preparation of 1- (tert-Butoxycarbonyl) -4- (6-chloropyridin-2-yl) piperidine-4-carboxylic acid (compound 0103-1): the crude product 1- (tert-butyl) 4-methyl 4-was dissolved in methanol (12 ml) at 43 ℃ (6-chloropyridin-2-yl) piperidine-1, 4-dicarboxylate (0102-1) (4.49g), and 4 mol/l aqueous sodium hydroxide solution (9 ml) was added dropwise over 20 minutes. The mixture was warmed to 50 ℃ and stirred for 35 minutes. The mixture was cooled to room temperature and the pH was adjusted to 6 mol/L aqueous hydrochloric acid (6 ml) in an ice-water bath
2, after which a solid precipitate forms. The slurry was diluted with water (10 ml) and stirred for 40 min, then the solid was collected by filtration, washed with water and dried under vacuum to give 1- (tert-butoxycarbonyl) -4- (6-chloropyridin-2-yl) piperidine-4-carboxylic acid (3.85 g, yield: 89.0%) as a white solid. Lcms (esi): 341(M + H)
+.
Step 1c preparation of tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (Compound 0104-1): a solution of 1- (tert-butoxycarbonyl) -4- (2-yl 6-chloropyridine) -piperidine-4-carboxylic acid (0103-1) (3.85 g, 11.32 mmol) in 1, 2-dichloroethane (30 ml) was heated to 82 ℃ and stirred overnight under nitrogen. After cooling to room temperature, the solvent was removed under reduced pressure. The crude product was purified by column chromatography (PE/EA ═ 10/1) to give tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (1.61g, yield: 48.0%) as a white solid. Lcms (esi): m/z 297(M + H) +.
Example 2: preparation of methyl (S) -2- (chloromethyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b ] pyridine-5-carboxylate (intermediate 0116-1) (prepared according to scheme one route)
Step 2a preparation of 6-chloro-5-nitropyridine-2-carboxylic acid (compound 0106-1) to a solution of 2-chloro-6-methyl-3-nitropyridine (0105-1) (5.0 g, 29.1 mmol, 1.0 eq) in concentrated sulfuric acid (20 ml) at room temperature chromium trioxide (8.6 g, 87.3 mmol, 3.0 eq) was added in portions. The mixture was stirred at 60 ℃ overnight. After cooling to room temperature, the mixture was poured into ice water, and then the solid was filtered and dried in vacuo to give 6-chloro-5-nitropyridine-2-carboxylic acid (4.15 g, yield: 71%) as a gray solid. Lcms (esi): m/z 201(M-H)-.
And step 2 b: preparation of methyl 6-chloro-5-nitropyridine-2-carboxylate (Compound 0107-1) to a mixture of 6-chloro-5-nitropyridine-2-carboxylic acid (0106-1) (4.0 g, 19.8 mmol, 1.0 eq) and N, N-dimethylformamide (2 drops) in dichloromethane (60 mL) was added oxalyl chloride (5.0 g, 39.6 mmol, 2.0 eq) under nitrogen at 0 ℃. The mixture was stirred at room temperature for 1 hour. Methanol (4 ml) was added and the mixture was stirred at room temperature for 10 minutes. Water was added and the mixture was partitioned. The organic layer was concentrated and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate: 20/1 to 5/1) to give methyl 6-chloro-5-nitropyridine-2-carboxylate (4.2 g, yield: 97.7%) as a white solid. Lcms (esi): 217(M + H) +.
And step 2 c: preparation of (S) -2- ((benzyloxy) methyl) oxetane (Compound 0109) to a mixture of potassium tert-butoxide (13.6 g, 122.0 mmol, 2.0 equiv.) in tert-butanol (180 mL) was added trimethyl sulfoxide iodide (26.8 g, 122.0 mmol, 2.0 equiv.) under nitrogen at room temperature. The mixture was stirred at 60 ℃ for 30 minutes. (S) -2- ((benzyloxy) methyl) oxirane (0108) (10.0 g, 61.0 mmol, 1.0 eq) was added and the mixture was stirred at 80 ℃ for 2 h. After cooling to room temperature, the mixture was filtered, and the filtrate was washed with water and concentrated. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 20/1 to 10/1) to give (S) -2- ((benzyloxy) methyl) oxetane (4.1 g, yield: 38%) as a colorless oily liquid.
Step 2 d: preparation of (S) -2-hydroxymethyl oxetane (Compound 0110) A solution of (S) -2- ((benzyloxy) methyl) oxetane (0109) (9.2 g, 51.7 mmol, 1.0 eq) and Pd/C (1.8 g, 10% by mass) in methanol (100 ml) was stirred overnight at room temperature under hydrogen. The mixture was filtered, and the filtrate was concentrated in vacuo to give (S) -2-hydroxymethyloxetane (5.0 g, yield: 111%) as a colorless liquid.
Step 2 e: preparation of (S) -oxetan-2-ylmethyl methanesulfonate (Compound 0111) Methanesulfonyl chloride (9.1 g, 79.5 mmol, 1.4 equiv.) is added dropwise to a mixture of (S) -2-hydroxymethyloxetane (0110) (5.0 g, 56.8 mmol, 1.0 equiv.) and triethylamine (217.2 g, 170.4 mmol, 3.0 equiv.) in dichloromethane (80 ml) under nitrogen at 0 ℃. The mixture was stirred at room temperature for 3 hours. The mixture was washed with water and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 5/1 to 1/1) to give (S) -oxetan-2-ylmethyl methanesulfonate as a colorless oily liquid (5.8 g, yield: 61.7%).
Step 2 f: preparation of (S) -oxetan-2-ylmethylamine (Compound 0113) A solution of (S) -oxetan-2-ylmethyl methanesulfonate (0111) (5.8 g, 35 mmol, 1.0 eq), sodium azide (2.7 g, 42 mmol, 1.2 eq) and potassium iodide (1.2 g, 7.0 mmol, 0.2 eq) in N, N-dimethylformamide (80 mL) was stirred at 60 ℃ overnight. After cooling to room temperature, methyl tert-butyl ether was added and the mixture was washed with water. The organic layer was concentrated, and the residue was dissolved in tetrahydrofuran and Pd/C (600 mg, 10% by mass) was added. The mixture was stirred at room temperature under hydrogen overnight. The mixture was filtered and the filtrate was concentrated in vacuo to give (S) -oxetan-2-ylmethylamine as a colorless oil, which was used in the next step without further purification.
Step 2 g: preparation of methyl (S) -5-nitro-6- ((oxetan-2-ylmethyl) amino) pyridine-2-carboxylate (Compound 0114-1) A mixture of methyl 6-chloro-5-nitropyridine-2-carboxylate (0107-1) (1.2 g, 5.6 mmol, 1.0 eq), N, N-diisopropylethylamine (2.2 g, 16.8 mmol, 3.0 eq) and (S) -oxetan-2-ylmethylamine (0113) (1.0 g, 11.2 mmol, 2.0 eq) in N, N-dimethylformamide (30 ml) was stirred at room temperature overnight. Ethyl acetate and water were added to the mixture, and the mixture was separated, and the organic layer was washed with saturated brine and concentrated. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 8/1 to 2/1) to give methyl (S) -5-nitro-6- ((oxetan-2-ylmethyl) amino) pyridine-2-carboxylate (1.0 g, yield: 80.6%) as a yellow solid. Lcms (esi): m/z 268(M + H)+.
Step 2 h: preparation of methyl (S) -5-amino-6- ((oxetan-2-ylmethyl) amino) pyridine-2-carboxylate (Compound 0115-1) A mixture of methyl (S) -5-nitro-6- ((oxetan-2-ylmethyl) amino) pyridine-2-carboxylate (0114-1) (1.0 g, 3.7 mmol, 1.0 eq) and Pd/C (100 mg, 10% by mass) in methanol (30 ml) was stirred at room temperature overnight under hydrogen. The mixture was filtered and the filtrate was concentrated in vacuo to give methyl (S) -5-amino-6- ((oxetan-2-ylmethyl) amino) pyridine-2-carboxylate (800 mg, yield: 90.1%) as a yellow solid. Lcms (esi): 238(M + H) +.
Step 2 i: (S) -2- (chloromethyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]Preparation of methyl pyridine-5-carboxylate (Compound 0116-1) A mixture of methyl (S) -5-amino-6- ((oxetan-2-ylmethyl) amino) pyridine-2-carboxylate (0115-1) (800 mg, 3.4 mmol, 1.0 eq) and 2-chloroacetic anhydride (872 mg, 5.1 mmol, 1.5 eq) in tetrahydrofuran (50 ml) was stirred at 70 ℃ overnight under nitrogen protection. After cooling to room temperature, an aqueous sodium carbonate solution and acetic acid were addedEthyl ester, and separating the mixture. The organic layer was washed with saturated brine and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluent: dichloromethane/methanol 200/1 to 100/1) to give (S) -2- (chloromethyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b ] as a yellow solid]Pyridine-5-carboxylic acid methyl ester (1.0 g, yield: 100%). Lcms (esi): 296(M + H)+.
Example 3: preparation of (S) -methyl 2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylate (intermediate 0116-3) (prepared according to scheme one route)
Step 3a preparation of methyl (S) -4-nitro-3- ((oxetan-2-ylmethyl) amino) benzoate (Compound 0114-3): a mixture (60 ml) of methyl 3-fluoro-4-nitrobenzoate (0107-3) (6.23 g, 31.30 mmol, 1.0 eq), (S) -oxetan-2-ylmethylamine (0113) (3.00 g, 34.44 mmol, 1.1 eq) and potassium carbonate (8.97 g, 64.87 mmol, 2.0 eq) in N, N-dimethylformamide was stirred at room temperature for 3 hours under nitrogen. The reaction was quenched with water (90 ml) and extracted with ethyl acetate (80 ml). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate 5/1) to give methyl (S) -4-nitro-3- ((oxetan-2-ylmethyl) amino) benzoate (6.61 g, yield: 79.4%) as a white solid. Lcms (esi): 267(M + H) +.
And step 3 b: preparation of methyl (S) -4-amino-3- ((oxetan-2-ylmethyl) amino) benzoate (Compound 0115-3) A mixture of methyl (S) -4-nitro-3- ((oxetan-2-ylmethyl) amino) benzoate (0114-3) (6.61 g, 24.84 mmol, 1.0 eq) and palladium on carbon (661 mg, 10% by mass) in tetrahydrofuran (80 ml) was stirred at room temperature under hydrogen for 4 hours. The reaction was filtered and the filtrate was concentrated in vacuo to give methyl (S) -4-amino-3- ((oxetan-2-ylmethyl) amino) benzoate as a pale solid (5.88 g, yield: 100.2%). Lcms (esi): 237(M + H)+.
And step 3 c: (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (formula)Preparation of compound 0116-3): a solution of (S) -methyl 4-amino-3- ((oxetan-2-ylmethyl) amino) benzoate (0115-3) (5.88 g, 24.89 mmol, 1.0 eq), 2-chloro-1, 1, 1-trimethoxyethane (4.62 g, 29.86 mmol, 1.2 eq) and p-toluenesulfonic acid monohydrate (514 mg, 2.99 mmol, 0.12 eq) in tetrahydrofuran (60 ml) was heated to 45 ℃ and stirred under dry conditions overnight. After cooling to room temperature, the reaction was diluted with water (40 ml) and with ethyl acetate (60 ml). The crude product was purified by column chromatography (dichloromethane/methanol ═ 50/1) to give (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid ]Imidazole-6-carboxylic acid methyl ester (6.54 g, yield: 74.5%). Lcms (esi): 295(M + H) with M/z+.
Example 4: preparation of (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b ] pyridine-5-carboxylic acid (Compound 1) (preparation according to scheme two line)
Step 4 a: preparation of 7- (hydroxymethyl) benzofuran-4-carbonitrile (compound 0201-1):
a mixture of 5-bromo-2-methylphenol (2.0 g, 10.7 mmol, 1.0 eq), 2-bromo-1, 1-diethoxyethane (2.5 g, 12.8 mmol, 1.2 eq), potassium carbonate (3.0 g, 21.4 mmol, 2.0 eq) and potassium iodide (0.2 g, 10% by mass) in N, N-dimethylformamide was stirred at 120 ℃ overnight under a nitrogen blanket. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product of 4-bromo-2- (2, 2-diethoxyethoxy) -1-methylbenzene (2.88 g, yield: 88.9%). The product was used in the next step without further purification.
4-bromo-2- (2, 2-diethoxyethoxy) -1-methylbenzene (2.9 g, 9.55 mmol, 1.0 eq) obtained above and polyphosphoric acid (8.0 g, 23) were combined under nitrogen blanket. 75 mmol, 2.5 equivalents) of dichloroethane was refluxed overnight. After cooling to room temperature, the reaction was quenched with water (40 ml) and extracted with ethyl acetate (50 ml). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether) to give 4-bromo-7-methylbenzofuran (1.61 g, yield: 80.5%) as a yellow oil. Lcms (esi): m/z 211(M + H)+.
A mixture of 4-bromo-7-methylbenzofuran (800 mg, 3.8 mmol, 1.0 equiv.) obtained above and cuprous cyanide (1.7 g, 19.0 mmol, 5.0 equiv.) in N, N-dimethylformamide (15 ml) was stirred at 120 ℃ overnight under nitrogen blanket. After cooling to room temperature, the reaction solution was filtered through celite and washed with ethyl acetate (15 ml). The filtrate was diluted with water (30 ml) and extracted with ethyl acetate (30 ml). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to give 7-methylbenzofuran-4-carbonitrile as a white solid (305 mg, yield: 51.3%). Lcms (esi): m/z 158(M + H) +.
A mixture of 7-methylbenzofuran-4-carbonitrile (305 mg, 1.94 mmol, 1.0 equiv.) obtained above, N-bromosuccinimide (416 mg, 2.33 mmol, 1.2 equiv.) and azobisisobutyronitrile (65 mg, 0.39 mmol, 0.2 equiv.) in dichloroethane (10 ml) was stirred at 72 ℃ overnight under a nitrogen blanket. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was diluted with water (20 ml) and extracted with petroleum ether/ethyl acetate 2/1(30 ml). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to give 7- (bromomethyl) benzofuran-4-carbonitrile as a white solid (377 mg, yield: 82.0%). Lcms (esi): 236(M + H) is defined as M/z+.
A mixture (10 ml) of 7- (bromomethyl) benzofuran-4-carbonitrile obtained above (326 mg, 1.38 mmol, 1.0 equiv.) and potassium acetate (1.35 g, 13.76 mmol, 10.0 equiv.) in N, N-dimethylformamide was stirred at room temperature for 3 hours. The reaction was quenched with water (20 ml) and extracted with ethyl acetate (30 ml). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product of acetic acid (4-cyanobenzofuran-7-yl) methyl ester (190 mg, yield: 64.2%). The product was used in the next step without further purification.
To a solution of the above-obtained (4-cyanobenzofuran-7-yl) methyl acetate (168 mg, 0.78 mmol, 1.0 eq) in tetrahydrofuran (5 ml) was added a solution of sodium methoxide in methanol (5.4 mol/l, 282 mg, 1.56 mmol, 2.0 eq). The mixture was stirred at room temperature for 1 hour. The reaction solution was adjusted to a pH of about 6 with 1 mol/l hydrochloric acid, and then a solid precipitate was formed. The slurry was diluted with water (15 ml) and extracted with ethyl acetate (20 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (eluent: petroleum ether/ethyl acetate-4/1) to give 7- (hydroxymethyl) benzofuran-4-carbonitrile (135 mg, yield: 100.0%) as a yellow solid. Lcms (esi): m/z 174(M + H)+.
And 4 b: preparation of tert-butyl 4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0202-1): a mixture of tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (0104-1) (125 mg, 0.44 mmol, 1.0 eq), 7- (hydroxymethyl) benzofuran-4-carbonitrile (0201-1) (106 mg, 0.61 mmol, 1.4 eq), cesium carbonate (400 mg, 1.22 mmol, 2.0 eq), 2-dicyclohexylphosphonium-2 ',6' -diisopropoxy-1, 1' -biphenyl (58 mg, 0.12 mmol, 0.2 eq) and tris (dibenzylideneacetone) dipalladium (0) (57 mg, 0.06 mmol, 0.1 eq) in toluene (10 ml) was refluxed at 125 ℃ overnight under nitrogen protection. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate (15 ml). The filtrate was diluted with water (20 ml) and extracted with ethyl acetate (20 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to give 4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine as a white solid Tert-butyl (165 mg, yield: 86.8%) 1-carboxylate. Lcms (esi): m/z 434(M + H)+.
And 4 c: preparation of 7- (((6- (piperidin-4-yl) pyridinyl-2-yl) oxy) methyl) benzofuran-4-carbonitrile p-toluenesulfonate (compound 0203-1): a mixture of tert-butyl 4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-1) (165 mg, 0.38 mmol, 1.0 eq) and p-toluenesulfonic acid monohydrate (197 mg, 1.14 mmol, 3.0 eq) in ethyl acetate (15 ml) was stirred at 60 ℃ overnight. After cooling to room temperature, the reaction solution was filtered, and the filter cake was washed with ethyl acetate and dried in vacuo to give 7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzofuran-4-carbonitrile p-toluenesulfonate as a white solid (150 mg, yield: 90.9%). LCMS (ESI): m/z 343(M + H)+.
And 4 d: (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]Preparation of pyridine-5-carboxylic acid methyl ester (Compound 0204-1) 7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzofuran-4-carbonitrile p-toluenesulfonate (0203-1) (150 mg, 0.29 mmol, 1.2 equiv), (S) -2- (chloromethyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4, 5-b) ]A solution of methyl pyridine-5-carboxylate (0116-1) (72 mg, 0.24 mmol, 1.0 equiv.) and potassium carbonate (115 mg, 0.83 mmol, 4.0 equiv.) in acetonitrile (5 ml) was stirred at 60 ℃ for 4 hours. After cooling to room temperature, water was added and extraction was performed with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain white solid (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxyl) pyridine-2-yl) piperidine-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4, 5-b)]Pyridine-5-carboxylic acid methyl ester (132 mg, yield: 75.9%). Lcms (esi): 593(M + H)+.
And 4 e: (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4, 5-b)]Preparation of pyridine-5-carboxylic acid (Compound 1) by reacting (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]A mixture of pyridine-5-carboxylic acid methyl ester (0204-1) (132 mg, 0.22 mmol, 1.0 eq) and lithium hydroxide monohydrate (11 mg, 0.25 mmol, 1.1 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. After the mixture was cooled to room temperature, the pH was adjusted to about 6 with 1 mol/l hydrochloric acid, and then a solid precipitate was formed. The slurry was diluted with water (12 ml), stirred for 4 hours, and the solid was collected by filtration. The solid was washed with water and then dried under vacuum. The solid was dissolved in dichloromethane/methanol 1/2(5 ml), then filtered, the filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was dried in vacuo to give (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b ] as a white solid ]Pyridine-5-carboxylic acid (97 mg, yield: 75.2%). Lcms (esi): m/z 579(M + H)+.1H NMR(500MHz,DMSO)δ13.04(s,1H),8.33(d,J=2.2Hz,1H),8.14(d,J=8.2Hz,1H),7.99(d,J=8.2Hz,1H),7.77(d,J=7.7Hz,1H),7.68–7.61(m,1H),7.55(d,J=7.8Hz,1H),7.19(d,J=2.2Hz,1H),6.87(d,J=7.3Hz,1H),6.72(d,J=8.1Hz,1H),5.72(s,2H),5.24–5.15(m,1H),4.84(dd,J=14.6,6.4Hz,1H),4.72(dd,J=14.6,4.3Hz,1H),4.53–4.46(m,1H),4.37(dt,J=8.9,6.0Hz,1H),3.94(dd,J=32.0,13.7Hz,2H),2.97–2.87(m,2H),2.73–2.63(m,1H),2.56(ddd,J=16.8,8.5,4.7Hz,1H),2.47(d,J=7.1Hz,1H),2.22(tt,J=16.1,8.1Hz,2H),1.73(d,J=10.1Hz,4H).
Example 5: preparation of (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b ] pyridine-5-carboxylic acid (Compound 2) (preparation according to scheme three-line)
Step 5 a: 6-chloro-3 ', 6' -dihydro- [2,4' -bipyridine]Preparation of tert-butyl (Compound 0302-2) 1'(2' H) -carboxylate 2-bromo-6-chloropyridine (0301-2) (2.00 g, 10.39 mmol, 1.0 eq.), N-tert-butoxycarbonyl-1, 2,5, 6-tetrahydropyridine-4-boronic acid pinacol ester (3.21 g, 10.39 mmol, 1.0 eq.) under nitrogen blanketAmount), sodium carbonate (2.20 g, 20.78 mmol, 2.0 equivalents) and [1,1' -bis (diphenylphosphino) ferrocene]A mixture of palladium (II) dichloride (761 mg, 1.04 mmol, 0.1 eq) in dioxane/water 10/1(30 ml) was stirred at 90 ℃ overnight. After cooling to room temperature, the reaction solution was filtered through celite, and washed with ethyl acetate (30 ml). The filtrate was diluted with water (30 ml) and extracted with ethyl acetate (20 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to give 6-chloro-3 ', 6' -dihydro- [2,4' -bipyridine as a white solid ]-1'(2' H) -carboxylic acid tert-butyl ester (2.51 g, yield: 81.9%). Lcms (esi): 295(M + H) with M/z+.
And step 5 b: 6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine]-preparation of 1'(2' H) -carboxylic acid tert-butyl ester (Compound 0303-2): under the protection of nitrogen, 6-chloro-3 ',6' -dihydro- [2,4' -bipyridine]A mixture of tert-butyl (0302-2) (491 mg, 1.67 mmol, 1.2 eq), 7- (hydroxymethyl) benzofuran-4-carbonitrile (0201-1) (241 mg, 1.39 mmol, 1.0 eq), cesium carbonate (906 mg, 2.78 mmol, 2.0 eq), 2-dicyclohexylphosphonium-2 ',6' -diisopropoxy-1, 1' -biphenyl (130 mg, 0.28 mmol, 0.2 eq) and tris (dibenzylideneacetone) dipalladium (0) (127 mg, 0.14 mmol, 0.1 eq) in toluene (10 ml) at 125 ℃ is refluxed overnight. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate (15 ml). The filtrate was diluted with water (20 ml) and extracted with ethyl acetate (20 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to give 6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine as a white solid ]-1'(2' H) -carboxylic acid tert-butyl ester (462 mg, yield: 76.9%). Lcms (esi): m/z 432(M + H)+.
And step 5 c: 7- (((1', 2', 3', 6' -tetrahydro- [2,4' -bipyridine)]]Preparation of (E) -6-yl) oxy) methyl) benzofuran-4-carbonitrile hydrochloride (compound 0304-2): reacting 6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine ]]Tert-butyl (0303-2) (462 mg, 1.07 mmol, 1.0 equiv.) of (E) -1'(2' H) -carboxylate was dissolved in dioxane hydrochloride solution (4 mol/L, 5 mL), and the reaction was stirred at room temperature overnight. The reaction solution was then filtered, and the filter cake was washed with ethyl acetate and dried under vacuum to give 7- (((1', 2', 3', 6' -tetrahydro- [2,4' -bipyridine) as a white solid]]-6-yl) oxy) methyl) benzofuran-4-carbonitrile hydrochloride (400 mg, yield: 100.0%). Lcms (esi): 332(M + H) M/z+.
And step 5 d: (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]Preparation of pyridine-5-carboxylic acid methyl ester (Compound 0305-2) 7- (((1', 2', 3', 6' -tetrahydro- [2,4' -bipyridine)]]-6-yl) oxy) methyl) benzofuran-4-carbonitrile hydrochloride (0304-2) (200 mg, 0.54 mmol, 1.0 eq), (S) -2- (chloromethyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b ]A mixture of pyridine-5-carboxylic acid methyl ester (0116-1) (160 mg, 0.54 mmol, 1.0 equiv.) and potassium carbonate (298 mg, 2.16 mmol, 4.0 equiv.) in acetonitrile (5 ml) was stirred at 60 ℃ for 4 hours. The reaction mixture was cooled to room temperature, and then water was added thereto, followed by extraction with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain white solid (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxyl) -3', 6' -dihydro- [2,4' -bipyridine)]-1'(2' H) -yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]Pyridine-5-carboxylic acid methyl ester (212 mg, yield: 66.7%). Lcms (esi): 590(M + H) M/z+.
And step 5 e: (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]Preparation of pyridine-5-carboxylic acid (Compound 2) from (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]Pyridine-5-carboxylic acid methyl ester (0305-2) (70 mg, 0.12 mmol, 1.0 eq.) and lithium hydroxide monohydrate (25 mg, 0.48 mmol) 4.0 equiv.) of acetonitrile/water 5/1(3 ml) was stirred at 40 ℃ overnight. After the mixture was cooled to room temperature, the pH was adjusted to about 6 with 1 mol/l hydrochloric acid, and then a solid precipitate was formed. The slurry was diluted with water (12 ml) and stirred for 4 hours. The solid was then collected by filtration, washed with water and then dried under vacuum. The solid was dissolved in dichloromethane/methanol 1/2(5 ml) and filtered. The filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Vacuum drying to obtain white solid (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine)]-1'(2' H) -yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]Pyridine-5-carboxylic acid (47 mg, yield: 69.1%). Lcms (esi): m/z 576(M + H)+.1H NMR(500MHz,DMSO)δ8.32(s,1H),8.05(d,J=8.0Hz,1H),7.96(d,J=8.0Hz,1H),7.78(d,J=7.5Hz,1H),7.69(t,J=7.7Hz,1H),7.54(d,J=7.6Hz,1H),7.20(s,1H),7.07(d,J=7.2Hz,1H),6.77(d,J=8.1Hz,1H),6.63(s,1H),5.74(s,2H),4.99(s,1H),4.83(dd,J=14.3,7.4Hz,1H),4.65(d,J=14.0Hz,1H),4.42(d,J=6.9Hz,1H),4.33(d,J=7.2Hz,1H),4.16(d,J=13.5Hz,1H),3.91(d,J=13.4Hz,1H),3.20(s,2H),2.71(s,2H),2.51(s,1H),2.44(s,2H),2.29(s,1H).
Example 6: preparation of (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 3) (preparation according to scheme two line)
Step 6 a: (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-3) 7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzofuran-4-carbonitrile p-toluenesulfonate (0203-1) (105 mg, 0.24 mmol, 1.2 equiv.), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] d]A mixture of methyl imidazole-6-carboxylate (0116-3) (60 mg, 0.20 mmol, 1.0 equiv.) and potassium carbonate (111 mg, 0.81 mmol, 4.0 equiv.) in acetonitrile (5 ml) was stirred at 60 ℃ for 4 h. The reaction mixture was cooled to room temperature, and then water was added thereto, followed by extraction with dichloromethane. For organic phaseDried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain white solid (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxyl) pyridine-2-yl) piperidine-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (78 mg, yield: 65.5%). Lcms (esi): m/z 592(M + H)+.
And 6 b: (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 3) from (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]A mixture of imidazole-6-carboxylic acid methyl ester (0204-3) (78 mg, 0.13 mmol, 1.0 eq) and lithium hydroxide monohydrate (11 mg, 0.26 mmol, 2.0 eq) in acetonitrile/water 5/1(3 ml) was stirred at 40 ℃ overnight. After the mixture was cooled to room temperature, the pH was adjusted to about 6 with 1 mol/l hydrochloric acid, and then a solid precipitate was formed. The slurry was diluted with water (12 ml), stirred for 4 hours, then the solid was collected by filtration, washed with water and then dried under vacuum. The solid was dissolved in dichloromethane/methanol 1/2(5 ml) and filtered. The filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was dried in vacuo to give (S) -2- ((4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid]Imidazole-6-carboxylic acid (54 mg, yield: 71.1%). Lcms (esi): m/z 578(M + H)+.1H NMR(500MHz,DMSO)δ12.65(s,1H),8.28(d,J=22.1Hz,2H),7.80(dd,J=28.6,7.5Hz,2H),7.64(d,J=7.0Hz,2H),7.54(d,J=7.4Hz,1H),7.18(s,1H),6.87(d,J=6.9Hz,1H),6.72(d,J=7.9Hz,1H),5.72(s,2H),5.10(s,1H),4.76(s,1H),4.64(d,J=14.4Hz,1H),4.45(d,J=5.8Hz,1H),4.36(s,1H),3.95(d,J=12.5Hz,1H),3.79(d,J=11.9Hz,1H),2.97(d,J=8.7Hz,1H),2.84(s,1H),2.68(s,1H),2.57(s,1H),2.42(s,1H),2.21(d,J=29.3Hz,2H),1.72(d,J=12.1Hz,4H).
Example 7: preparation of (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 4) (preparation according to scheme three-line)
Step 7 a: (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine ]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0305-4) from 7- (((1', 2', 3', 6' -tetrahydro- [2,4' -bipyridine)]]-6-yl) oxy) methyl) benzofuran-4-carbonitrile hydrochloride (0304-2) (100 mg, 0.27 mmol, 1.0 eq), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]]A mixture of methyl imidazole-6-carboxylate (0116-3) (80 mg, 0.27 mmol, 1.0 eq) and potassium carbonate (149 mg, 1.08 mmol, 3.0 eq) in acetonitrile (5 ml) was stirred at 60 ℃ for 4 h. The reaction mixture was cooled to room temperature, and water was added thereto, followed by extraction with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain white solid (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxyl) -3', 6' -dihydro- [2,4' -bipyridine)]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (97 mg, yield: 60.9%). Lcms (esi): 590(M + H) M/z+.
And 7 b: (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Preparation of imidazole-6-carboxylic acid (Compound 4) (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine)]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0305-4) (97 mg, 0.16 mmol, 1.0 eq) and lithium hydroxide monohydrate (25 mg, 0.48 mmol, 3.0 eq) in acetonitrile/water 5/1(3 ml) was stirred at 40 ℃ overnight. After the mixture was cooled to room temperature, the pH was adjusted to about 6 with 1 mol/l hydrochloric acid, and then a solid precipitate was formed. The slurry was diluted with water (12 ml), stirred for 4 hours, then the solid was collected by filtration, washed with water and then dried under vacuum. Dissolve the solid in dichloromethane/methanol1/2(5 ml), then filtered, the filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Vacuum drying to obtain white solid (S) -2- ((6- ((4-cyanobenzofuran-7-yl) methoxy) -3', 6' -dihydro- [2,4' -bipyridine)]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (68 mg, yield: 76.6%). Lcms (esi): m/z 576(M + H)+.1H NMR(500MHz,DMSO)δ12.67(s,1H),8.32(s,1H),8.25(s,1H),7.79(dd,J=18.0,8.0Hz,2H),7.74–7.61(m,2H),7.55(d,J=7.7Hz,1H),7.21(s,1H),7.08(d,J=7.4Hz,1H),6.78(d,J=8.1Hz,1H),6.62(s,1H),5.74(s,2H),5.05(d,J=5.3Hz,1H),4.79(dd,J=15.2,7.3Hz,1H),4.64(d,J=13.4Hz,1H),4.46(dd,J=13.8,7.3Hz,1H),4.35(dd,J=14.6,5.9Hz,1H),4.05(d,J=13.5Hz,1H),3.91(d,J=13.5Hz,1H),3.22–3.15(m,2H),2.77–2.60(m,3H),2.46–2.31(m,3H).
Example 8: preparation of (S) -2- ((4- (6- ((4-cyano-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 5) (preparation according to scheme two line)
Step 8 a: preparation of 7- (hydroxymethyl) -2-methylbenzofuran-4-carbonitrile (Compound 0201-5):
a mixture of methyl 4-bromo-2-hydroxybenzoate (1.0 g, 4.33 mmol, 1.0 eq), 3-bromopropyne (618 mg, 5.19 mmol, 1.2 eq), and potassium carbonate (896 mg, 6.49 mmol, 1.5 eq) in N, N-dimethylformamide was stirred at 50 deg.C for 5 h under nitrogen. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure. The residue was dissolved with N, N-diethylaniline (5 ml). Cesium fluoride (855 mg, 5.63 mmol, 1.3 equiv.) was added to the mixture. The mixture was stirred at 220 ℃ for 3 hours. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfateAnd (5) drying. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-10/1) to give methyl 4-bromo-2-methylbenzofuran-7-carboxylate (563 mg, yield: 48.53%) as a yellow solid. Lcms (esi): m/z 269(M + H)+.
A solution of methyl 4-bromo-2-methylbenzofuran-7-carboxylate (500 mg, 1.86 mmol, 1.0 eq), palladium tetratriphenylphosphine (215 mg, 0.186 mmol, 0.1 eq) and zinc cyanide (326 mg, 2.79 mmol, 1.5 eq) in N, N-dimethylformamide (5 ml) was stirred at 90 ℃ overnight under nitrogen. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-10/1) to give methyl 4-cyano-2-methylbenzofuran-7-carboxylate (290 mg, yield: 72.68%) as a yellow solid. Lcms (esi): 216(M + H) +.
Methyl 4-cyano-2-methylbenzofuran-7-carboxylate (290 mg, 1.348 mmol, 1.0 equiv) obtained above was added to a tetrahydrofuran mixture of lithium aluminum hydride (62 mg, 1.618 mmol, 1.2 equiv) under nitrogen and ice bath. The mixture was stirred for 1 hour under ice bath. The reaction was quenched with water. The mixture was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-2/1) to give 7- (hydroxymethyl) -2-methylbenzofuran-4-carbonitrile as a yellow solid (248 mg, yield: 98.41%). Lcms (esi): 188(M + H) M/z+.
And step 8 b: preparation of tert-butyl 4- (6- ((4-cyano-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0202-5): 4- (6-Chloropyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester (0104-1) (220 mg, 0.74 mmol, 1.0 eq), 7- (hydroxymethyl) -2-methylbenzofuran-4-carbonitrile (0201-5) (166 mg, 0.88 mmol, 1.2 eq), cesium carbonate (481 mg, 1.48 mmol, 2.0 eq), 2-dicyclohexylphosphonium-2 ',6' -diisopropoxy-1, 1' -biphenyl (138 mg, 0 h) under nitrogen protection296 mmol, 0.4 eq) and tris (dibenzylideneacetone) dipalladium (0) (135 mg, 0.148 mmol, 0.2 eq) in toluene (10 ml) were stirred at 125 ℃ overnight. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-10/1) to give tert-butyl 4- (6- ((4-cyano-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (237 mg, yield: 71.60%) as a yellow oil. Lcms (esi): 448(M + H) +.
And step 8 c: preparation of 2-methyl-7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzofuran-4-carbonitrile hydrochloride (compound 0203-5): tert-butyl 4- (6- ((4-cyano-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-5) (248 mg, 0.55 mmol, 1.0 eq) and a solution of dioxane (4 ml) of hydrochloric acid (4M, 1 ml) in dioxane (4 ml) was stirred at room temperature overnight. The mixture was concentrated under reduced pressure to give 2-methyl-7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzofuran-4-carbonitrile hydrochloride (105 mg, crude) which was used in the next step without further purification. Lcms (esi): m/z 348(M + H)+.
And step 8 d: (S) -2- ((4- (6- ((4-cyano-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-5) 2-methyl-7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzofuran-4-carbonitrile hydrochloride (0203-5) (98 mg, 0.255 mmol, 1.5 equiv), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]]A solution of methyl imidazole-6-carboxylate (0116-3) (50 mg, 0.17 mmol, 1.0 eq) and potassium carbonate (140 mg, 1.01 mmol, 4.0 eq) in acetonitrile (5 ml) was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, and water was added thereto, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain yellow solid (S) -2- ((4- (6- ((4-cyano-2-methylbenzene) Furan-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (70 mg, yield: 68.03%). Lcms (esi): m/z 606(M + H)+.
Step 8 e: (S) -2- ((4- (6- ((4-cyano-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 5) (S) -2- ((4- (6- ((4-cyano-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0204-5) (27.6 mg, 0.045 mmol, 1.0 eq) and lithium hydroxide monohydrate (4 mg, 0.09 mmol, 2.0 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l hydrochloric acid, and a solid precipitate was formed. The mixture was filtered. The residue was washed with water. The residue was purified by thick preparative thin layer silica gel chromatography (eluent: dichloromethane/methanol-10/1) to give (S) -2- ((4- (6- ((4-cyano-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] c ]Imidazole-6-carboxylic acid (21.6 mg, yield: 80.12%). Lcms (esi): m/z 592(M + H)+.1H NMR(500MHz,DMSO)δ12.69(s,1H),8.26(s,1H),7.81(dd,J=8.4,1.3Hz,1H),7.73–7.59(m,3H),7.44(d,J=7.8Hz,1H),6.87(d,J=7.3Hz,1H),6.83(d,J=1.0Hz,1H),6.72(d,J=8.1Hz,1H),5.68(s,2H),5.10(ddd,J=14.5,7.2,2.8Hz,1H),4.77(dd,J=15.2,7.1Hz,1H),4.64(dd,J=15.2,2.6Hz,1H),4.46(dt,J=13.8,7.0Hz,1H),4.36(dt,J=9.0,5.9Hz,1H),3.93(d,J=13.6Hz,1H),3.77(d,J=13.5Hz,1H),2.97(d,J=11.1Hz,1H),2.84(d,J=11.2Hz,1H),2.74–2.65(m,1H),2.59–2.54(m,1H),2.48(d,J=0.8Hz,3H),2.43(ddd,J=11.1,9.0,5.6Hz,1H),2.19(dt,J=32.3,10.5Hz,2H),1.80–1.60(m,4H).
Example 9: (S) -2-((4-(6-((2-chloro-4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 6)Preparation (preparation according to scheme two circuit)
Step 9 a: preparation of tert-butyl 4- (6- ((2-chloro-4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0202-6): lithium diisopropylamide (0.42 ml, 2 mol/l tetrahydrofuran, 0.843 mmol, 2 eq) was added dropwise to a solution of tert-butyl 4- (6- ((4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-1) (180 mg, 0.416 mmol, 1 eq) in 10 ml tetrahydrofuran under nitrogen and at-70 ℃. The mixture was stirred at-70 ℃ for 1 hour. A solution of hexachloroethane (98.5 mg, 0.416 mmol, 1 eq) in tetrahydrofuran (1 ml) was added to the mixture. The mixture was stirred at-70 ℃ for 1 hour. The reaction was quenched with ammonium chloride. The mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over sodium sulfate. The mixture was concentrated under reduced pressure. The residue was purified by preparative silica gel thin layer chromatography (eluent: petroleum ether/ethyl acetate 5/1) to give 4- (6) as a clear oil -Tert-butyl ((2-chloro-4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (54 mg, yield: 27.74%). Lcms (esi): m/z 468(M + H)+.
And step 9 b: 2-chloro-7-Preparation of ((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzofuran-4-carbonitrile 4-methylbenzenesulfonate (compound 0203-6): to a solution of tert-butyl 4- (6- ((2-chloro-4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-6) (54 mg, 0.115 mmol, 1.0 eq) in ethyl acetate (10 ml) was added p-toluenesulfonic acid monohydrate (59 mg, 0.346 mmol, 3.0 eq). The mixture was stirred at 60 ℃ overnight. The mixture is decompressed and concentrated to obtain a crude product 2-chloro-7-(((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzofuran-4-carbonitrile 4-methylbenzenesulfonate (93 mg, crude). The product was used in the next step without further purification.
And step 9 c: (S) -2- ((4- (6- ((2-chloro-4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-6) 2-chloro-7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methylYl) benzofuran-4-carbonitrile 4-methylbenzenesulfonate (0203-6) (93 mg, 0.173 mmol, 1.5 equivalents), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] c ]A mixture of methyl imidazole-6-carboxylate (0116-3) (34 mg, 0.115 mmol, 1.0 eq) and potassium carbonate (63.6 mg, 0.461 mmol, 4.0 eq) in acetonitrile (10 ml) was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, and water was added thereto, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain white solid (S) -2- ((4- (6- ((2-chloro-4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (60 mg, yield: 83.34%). Lcms (esi): 626(M + H) M/z+.
And step 9 d: (S) -2-((4-(6-((2-chloro-4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 6) from (S) -2- ((4- (6- ((2-chloro-4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0204-6) (60 mg, 0.096 mmol, 1.0 eq) and lithium hydroxide monohydrate (12 mg, 0.288 mmol, 3 eq) in tetrahydrofuran/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l dilute sulfuric acid, after which a solid precipitate formed. The mixture was filtered. The residue was purified by preparative silica gel thin layer chromatography (eluent: dichloromethane/methanol 10/1) to give (S) -2 as a white solid -((4-(6-((2-chloro-4-cyanobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (32.7 mg, yield: 55.66%). Lcms (esi): m/z 612(M + H)+Melting point: 125-130 ℃;1H NMR(500MHz,DMSO)δ12.70(s,1H),8.27(d,J=0.7Hz,1H),7.87–7.76(m,2H),7.70–7.60(m,2H),7.55(d,J=7.9Hz,1H),7.36(s,1H),6.87(d,J=7.3Hz,1H),6.73(d,J=8.1Hz,1H),5.68(s,2H),5.10(qd,J=7.2,2.8Hz,1H),4.78(dd,J=15.2,7.2Hz,1H),4.65(dd,J=15.2,2.7Hz,1H),4.47(dt,J=13.8,7.0Hz,1H),4.36(dt,J=9.0,5.9Hz,1H),3.93(d,J=13.5Hz,1H),3.78(d,J=13.5Hz,1H),2.96(d,J=10.8Hz,1H),2.84(d,J=11.0Hz,1H),2.74–2.65(m,1H),2.59–2.53(m,1H),2.43(ddd,J=11.1,8.9,5.6Hz,1H),2.18(dt,J=29.7,11.0Hz,2H),1.77–1.54(m,4H).
example 10: preparation of (S) -2- ((4- (6- ((4-cyanobenzo [ b ] thiophen-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 8) (prepared according to scheme two line)
Step 10 a: preparation of 7- (hydroxymethyl) benzo [ b ] thiophene-4-carbonitrile (compound 0201-8):
methyl 2-amino-4-bromobenzoate (2.3 g, 10.0 mmol, 1.0 eq) was added to dilute hydrochloric acid (2 ml of concentrated hydrochloric acid and 8 ml of water). The mixture was stirred at 80 ℃ for 15 minutes and then cooled to 0 ℃. Sodium nitrite (828 mg, 12 mmol, 1.2 eq) was dissolved in 8 ml water and added dropwise to the mixture above and the mixture was stirred at 0 ℃ for 2 h. Potassium ethyl xanthate (2.72 g, 17 mmol, 1.7 eq) was added to 8 ml of water and stirred at 65 ℃. The mixture of diazonium salts obtained above was added to the mixture over 20 minutes and the mixture was stirred at 65 ℃ for 2 hours. Dichloromethane was added for extraction, and the mixture was washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was added to a solution of potassium hydroxide (5.6 g, 100 mmol, 10.0 eq) in 10 ml of ethanol and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure and water was added. The pH of the aqueous phase was adjusted to 1 by adding concentrated HCl. Ethyl acetate was added for extraction, and the mixture was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure to give a mixture of 3-bromopropyne (619 mg, 5.2 mmol, 1.3 equiv.) and potassium carbonate (884 mg, 6.4 mmol, 1.6 equiv.) in N, N-dimethylformamide and stirred at 50 ℃ for 3 hours . After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give 4-bromo-2-mercaptobenzoic acid as a red oil (2.19 g, crude) which was used in the next step without further purification. MS (ES)-):m/z=231(M-H)-.
The 4-bromo-2-mercaptobenzoic acid obtained above (2.19 g, 9.48 mmol, 1.0 eq) was dissolved in 50 ml of methanol and thionyl chloride (3.38 g, 28.44 mmol, 3.0 eq) was added dropwise at 0 ℃. The mixture was stirred at 75 ℃ for 3 hours. The mixture was concentrated under reduced pressure, and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give methyl 4-bromo-2-mercaptobenzoate (2.23 g, crude) as a yellow oil which was used in the next step without further purification. MS (ES)-):m/z=245(M-H)-。
A mixture of methyl 4-bromo-2-mercaptobenzoate (2.23 g, 9.03 mmol, 1.0 eq), 2-bromo-1, 1-diethoxyethane (2.13 g, 10.84 mmol, 1.2 eq) and potassium carbonate (2.49 g, 18.03 mmol, 2.0 eq) obtained above in 35 ml of N, N-dimethylformamide was stirred at 70 ℃ for 3 hours. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine and dried over anhydrous sodium sulfate. The organic phase was concentrated in vacuo and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate: 30/1 to 10/1) to give methyl 4-bromo-2- ((2, 2-diethoxyethyl) thio) benzoate (1.46 g, yield: 44.5%) as a yellow oil.
A toluene mixture of methyl 4-bromo-2- ((2, 2-diethoxyethyl) thio) benzoate (1.26 g, 3.47 mmol, 1.0 eq.) obtained above and 1.29 g of polyphosphoric acid was stirred at 120 ℃ overnight. After cooling to room temperature, water was added, the mixture was acidified with ethyl acetate and the named mixture by adding concentrated hydrochloric acid, and the mixture was extracted by adding a mixed solvent of ethyl acetate and petroleum ether. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1 to 50/1) to give methyl 4-bromobenzo [ b ] thiophene-7-carboxylate (849 mg, yield: 90.6%) as a red solid.
4-bromo-benzo [ b ] obtained above]Thiophene-7-carboxylic acid methyl ester (800 mg, 2.96 mol, 1.0 eq) was dissolved in 25 ml of ethanol and the mixture was stirred at 45 ℃. Sodium borohydride (1.14 g, 30 mmol, 10 eq) was added and the mixture was stirred overnight. The mixture was concentrated under reduced pressure, and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure and the residue is purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 20/1 to 5/1) to give (4-bromobenzo [ b ] as a yellow solid ]Thiophen-7-yl) methanol (470 mg, yield: 65.6%). Lcms (esi): 225(M + H-H) M/z2O)+.
Under the protection of nitrogen, the (4-bromobenzo [ b ] obtained above is reacted]Thien-7-yl) methanol (440 mg, 1.82 mmol, 1.0 equiv) and cuprous cyanide (1.63 g, 18.2 mmol, 10.0 equiv) in a mixture of N-methylpyrrolidone (15 ml) was stirred at 190 ℃ for 8 h. After cooling to room temperature, the mixture was diluted with ethyl acetate. The mixture was filtered through celite, and the filtrate was washed with saturated brine, and then dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure and the residue is purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate: 10/1 to 4/1) to give 7- (hydroxymethyl) benzo [ b]Thiophene-4-carbonitrile (125 mg, yield: 36.3%). Lcms (esi): m/z 172(M + H-H)2O)+.
Step 10 b: 4- (6- ((4-cyanobenzo [ b ]]Preparation of thiophen-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester (compound 0202-8): 4- (6-Chloropyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester (0104-1) (138 mg, 0.46 mmol, 1.0 eq), 7- (hydroxymethyl) benzo [ b ] under nitrogen]A mixture of thiophene-4-carbonitrile (0201-8) (105 mg, 0.56 mmol, 1.2 equivalents), cesium carbonate (378 mg, 1.16 mmol, 2.5 equivalents), 2-biscyclohexylphosphine-2 ',6' -diisopropoxybiphenyl (33 mg, 0.07 mmol, 0.15 equivalents), and tris (dibenzylideneacetone) dipalladium (0) (38 mg, 0.042 mmol, 0.09 equivalents) in toluene (50 ml) was refluxed at 125 ℃ overnight. After cooling to room temperature, the reaction mixture is cooled The reaction solution was filtered through celite and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1 to 6/1) to give 4- (6- ((4-cyanobenzo [ b ] b) as a yellow solid]Thiophen-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester (192 mg, yield: 92.3%). Lcms (esi): m/z 450(M + H)+.
Step 10 c: 7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzo [ b]Preparation of thiophene-4-carbonitrile hydrochloride (Compound 0203-8): to 4- (6- ((4-cyanobenzo [ b ]]Thien-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester (0202-8) (192 mg, 0.43 mmol, 1.0 eq) to a mixture of 5 ml dioxane was added 1 ml of a 4M dioxane solution of hydrogen chloride. The mixture was stirred at room temperature overnight. The mixture was concentrated to dryness under reduced pressure to give 7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzo [ b ] a white solid]Thiophene-4-carbonitrile hydrochloride (240 mg, crude). Lcms (esi): 350(M + H) M/z+.
Step 10 d: (S) -2- ((4- (6- ((4-cyanobenzo [ b ]]Thien-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Preparation of imidazole-6-carboxylic acid methyl ester (compound 0204-8): reacting 7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzo [ b]Thiophene-4-carbonitrile hydrochloride (0203-8) (240 mg, 0.43 mmol, 2.5 equivalents) was added to 5 ml of N-methylpyrrolidinone, followed by N, N-diisopropylethylamine (175 mg, 1.36 mmol, 8.0 equivalents). The mixture was stirred at room temperature for 5 minutes. Adding (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]]Imidazole-6-carboxylic acid methyl ester (0116-3) (50 mg, 0.17 mmol, 1.0 equiv) and the mixture is stirred at 60 ℃ overnight. The mixture was diluted with ethyl acetate, washed with water and saturated brine. The organic phase was concentrated under reduced pressure and the residue was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol ═ 20/1) to give (S) -2- ((4- (6- ((4-cyanobenzo [ b) as a yellow solid]Thien-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (89 mg, yield: 86.1%). M/z 608(M + H)+.
Step 10 e: (S) -2- ((4- (6- ((4-cyanobenzo [ b ]]Thien-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Preparation of imidazole-6-carboxylic acid (Compound 8) (S) -2- ((4- (6- ((4-cyanobenzo [ b)]Thien-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0204-8) (80 mg, 0.13 mmol, 1.0 equiv.) and lithium hydroxide monohydrate (44 mg, 1.05 mmol, 8.0 equiv.) in a mixed solvent of 5 ml acetonitrile and 1 ml water was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. Dilute sulfuric acid was added to adjust the pH of the residue to 6, ethyl acetate was added for extraction, and the mixture was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol ═ 10/1) to give (S) -2- ((4- (6- ((4-cyanobenzo [ b) a yellow solid]Thien-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (49 mg, yield: 62.0%). Lcms (esi): m/z 594(M + H)+Melting point: 124-126 ℃.1H NMR(500MHz,DMSO)δ12.73(s,1H),8.28(s,1H),8.12(d,J=5.5Hz,1H),7.94(d,J=7.6Hz,1H),7.81(dd,J=8.4,1.0Hz,1H),7.71–7.55(m,4H),6.88(d,J=7.3Hz,1H),6.75(d,J=8.2Hz,1H),5.71(s,2H),5.10(qd,J=7.2,2.8Hz,1H),4.79(dd,J=15.2,7.1Hz,1H),4.66(dd,J=15.2,2.4Hz,1H),4.46(dd,J=13.7,7.6Hz,1H),4.36(dt,J=9.0,5.9Hz,1H),3.94(d,J=13.6Hz,1H),3.80(s,1H),2.95(d,J=10.7Hz,1H),2.83(d,J=10.8Hz,1H),2.75–2.65(m,1H),2.56(t,J=11.5Hz,1H),2.47–2.38(m,1H),2.28–2.10(m,2H),1.75–1.55(m,4H).
Example 11: preparation of (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 12) (preparation according to scheme two line)
Step 11 a: preparation of (4-chlorobenzofuran-7-yl) methanol (Compound 0201-12):
a mixture of 5-chloro-2-methylphenol (2.0 g, 14.03 mmol, 1.0 eq), 2-bromo-1, 1-diethoxyethane (3.31 g, 16.83 mmol, 1.2 eq), and potassium carbonate (2.9 g, 21.04 mmol, 1.5 eq) in N, N-dimethylformamide was stirred at 120 ℃ overnight under nitrogen. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated in vacuo to give 4-chloro-2- (2, 2-diethoxyethoxy) -1-methylbenzene (4 g, crude). The product was used in the next step without further purification.
A mixture of 4-chloro-2- (2, 2-diethoxyethoxy) -1-methylbenzene (4.18 g, 14.03 mmol, 1.0 eq) and polyphosphoric acid (7.09 g, 21.04 mmol, 1.5 eq) obtained above was refluxed overnight under nitrogen blanket. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether) to give 4-chloro-7-methylbenzofuran (1.98 g, yield: 84.98%) as a clear oil.
A solution of 4-chloro-7-methylbenzofuran (1.98 g, 11.93 mmol, 1.0 eq.) obtained above, N-bromosuccinimide (2.54 g, 14.31 mmol, 1.2 eq.) and azobisisobutyronitrile (391 mg, 2.38 mmol, 0.2 eq.) in dichloroethane (10 ml) was stirred at 72 ℃ overnight under a nitrogen blanket. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether) to give 7- (bromomethyl) -4-chlorobenzofuran (2.54 g, yield: 87.28%) as a yellow oil.
A mixture (20 ml) of the above-obtained 7- (bromomethyl) -4-chlorobenzofuran (2.54 g, 10.37 mmol, 1.0 equivalent) and potassium acetate (10.16 g, 103.7 mmol, 10.0 equivalent) in N, N-dimethylformamide was stirred at room temperature for 3 hours. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give (4-chlorobenzofuran-7-yl) methyl acetate (2.29 g, crude) which was used in the next step without further purification.
To a solution of (4-chlorobenzofuran-7-yl) methyl acetate (2.29 g, 10.22 mmol, 1.0 eq) obtained above in tetrahydrofuran (10 ml) was added a solution of sodium methoxide in methanol (5.4 mol/l, 3.8 ml, 20.44 mmol, 2.0 eq). The mixture was stirred at room temperature for 1 hour. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate-200/1-4/1) to give (4-chlorobenzofuran-7-yl) methanol (1.3 g, yield: 69.89%) as a yellow solid. Lcms (esi): m/z 183(M + H)+.
Step 11 b: preparation of tert-butyl 4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate tert-butyl ester tert-butyl (compound 0202-12): a mixture of tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (0104-1) (160 mg, 0.548 mmol, 1 eq), (4-chlorobenzofuran-7-yl) methanol (0201-12) (120 mg, 0.66 mmol, 1.2 eq), cesium carbonate (356 mg, 1.096 mmol, 2.0 eq), 4, 5-bis diphenylphosphino-9, 9-dimethylxanthene (76 mg, 0.131 mmol, 0.2 eq), and tris (dibenzylideneacetone) dipalladium (0) (60 mg, 0.065 mmol, 0.1 eq) in toluene (10 ml) was stirred at 125 ℃ overnight under nitrogen protection. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-10/1) to give 4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester as a yellow oil (235 mg, yield: 98.74%). Lcms (esi): m/z 443(M + H) +.
Step 11 c: 2- ((4-chlorobenzofuran-7-yl)) Preparation of methoxy) -6- (piperidin-4-yl) pyridine hydrochloride (compound 0203-12): to a mixture of tert-butyl 4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-12) (235 mg, 0.53 mmol, 1.0 eq) and hydrogen chloride in dioxane (4M, 1 ml) was added dioxane (2 ml) and the mixture was stirred at room temperature overnight. The mixture was filtered. The residue was washed with dioxane. The residue was dried to give 2- ((4-chlorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine hydrochloride (142 mg, yield: 70.64%) as a yellow solid lcms (esi): m/z 343(M + H)+.
Step 11 d: (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-12) 2- ((4-chlorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine hydrochloride (0203-12) (142 mg, 0.374 mmol, 1.5 equiv), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A solution of methyl imidazole-6-carboxylate (0116-3) (73 mg, 0.249 mmol, 1.0 eq) and N, N-diisopropylethylamine (161 mg, 1.248 mmol, 4.0 eq) in N-methylpyrrolidone (5 ml) was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, and water was added thereto, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain yellow solid (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxyl) pyridine-2-yl) piperidine-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Imidazole-6-carboxylic acid methyl ester (115 mg, yield: 77.18%). Lcms (esi): 601(M + H) M/z+.
Step 11 e: (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 12) (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d [, c]Imidazole-6-carboxylic acid methyl ester (0204-12) (115 mg, 0.19 mmol, 1.0 eq) and lithium hydroxide monohydrate (16 mg, 0.38 mmol)2.0 eq) of acetonitrile/water 5/1(6 ml) mixture was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l hydrochloric acid, and a solid precipitate was formed. The mixture was filtered. The residue was washed with water. The mixture was slurried with methanol to give (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid]Imidazole-6-carboxylic acid (88 mg, yield: 78.57%). Lcms (esi): m/z 587(M + H)+.1H NMR(500MHz,DMSO)δ12.74(s,1H),8.29(s,1H),8.15(d,J=2.2Hz,1H),7.82(s,1H),7.75–7.60(m,2H),7.44(d,J=7.9Hz,1H),7.34(d,J=8.0Hz,1H),7.03(d,J=2.1Hz,1H),6.88(d,J=7.1Hz,1H),6.69(d,J=7.9Hz,1H),5.63(s,2H),5.10(d,J=5.6Hz,1H),4.80(dd,J=15.3,7.2Hz,1H),4.72–4.63(m,1H),4.46(dd,J=13.7,7.4Hz,1H),4.36(dt,J=9.0,5.9Hz,1H),3.95(s,1H),3.78(d,J=10.6Hz,1H),2.93(d,J=71.7Hz,2H),2.76–2.56(m,2H),2.42(s,1H),2.34–2.03(m,2H),1.80(s,4H).
Example 12: preparation of (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b ] pyridine-5-carboxylic acid (Compound 14) (preparation according to scheme two line)
Step 12 a: (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]Preparation of pyridine-5-carboxylic acid methyl ester (Compound 0204-14) 2- ((4-chlorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine hydrochloride (0203-12) (139 mg, 0.366 mmol, 1.5 equiv), (S) -2- (chloromethyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4, 5-b)]A solution of methyl pyridine-5-carboxylate (0116-1) (72 mg, 0.244 mmol, 1.0 eq) and N, N-diisopropylethylamine (126 mg, 0.976 mmol, 4.0 eq) in N-methylpyrrolidone (5 ml) was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, water was added thereto, and ethyl acetate was added for extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to give (S) -2- ((4- (6-((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]Pyridine-5-carboxylic acid methyl ester (70 mg, yield: 47.62%). Lcms (esi): 602(M + H) is defined as M/z+.
Step 12 b: (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b ]Preparation of pyridine-5-carboxylic acid (Compound 14) from (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]A mixture of pyridine-5-carboxylic acid methyl ester (0204-14) (70 mg, 0.116 mmol, 1.0 eq) and lithium hydroxide monohydrate (10 mg, 0.232 mmol, 2.0 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l hydrochloric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water. The residue was purified by preparative silica gel thin layer chromatography on preparative plates (eluent: dichloromethane/methanol-10/1) to give (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4, 5-b) as a yellow solid]Pyridine-5-carboxylic acid (30 mg, yield: 44.12%). Lcms (esi): m/z 588(M + H)+.1H NMR(500MHz,DMSO)δ12.99(s,1H),8.39–7.93(m,3H),7.61(d,J=6.7Hz,1H),7.41(s,1H),7.32(d,J=6.9Hz,1H),7.01(s,1H),6.87(s,1H),6.67(d,J=7.7Hz,1H),5.61(s,2H),5.29(d,J=93.9Hz,1H),4.80(d,J=53.8Hz,2H),4.42(d,J=50.8Hz,2H),3.89(d,J=105.4Hz,2H),2.97(s,2H),2.75–2.51(m,3H),2.31(d,J=53.9Hz,2H),1.77(d,J=15.7Hz,4H).
Example 13: preparation of (S) -2- ((4- (6- ((2, 4-dichlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 15) (prepared according to scheme two line)
Step 13 a: preparation of tert-butyl 4- (6- ((2, 4-dichlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0202-15): under nitrogen protection and at-70 ℃,4- (6- ((4-chlorobenzo)Furan-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester (0202-12) (200 mg, 0.452 mmol, 1 eq) was added to a solution of lithium diisopropylamide (0.45 ml, 2 mol/l tetrahydrofuran, 0.905 mmol, 2 eq) in 10 ml tetrahydrofuran. The mixture was stirred at-70 ℃ for 1 hour. To the mixture was added hexachloroethane (107 mg, 0.452 mmol, 1 eq) in tetrahydrofuran (1 ml). The mixture was stirred at-70 ℃ for 1 hour. The reaction was quenched with ammonium chloride. The mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over sodium sulfate. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-10/1) to give tert-butyl 4- (6- ((2, 4-dichlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (180 mg, yield: 83.72%) as a clear oil. Lcms (esi): m/z 477(M + H)+.
Step 13 b: preparation of 2- ((2, 4-dichlorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (compound 0203-15): a solution of tert-butyl 4- (6- ((2, 4-dichlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-15) (180 mg, 0.377 mmol, 1.0 eq) and p-toluenesulfonic acid (195 mg, 1.132 mmol, 3.0 eq) in ethyl acetate (15 ml) was stirred at 60 ℃ overnight. The mixture was cooled to room temperature. The mixture was filtered. The residue was washed with ethyl acetate. The residue was dried to give 2- ((2, 4-dichlorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (253 mg, crude) as a white solid lcms (esi): m/z 377(M + H) +.
Step 13 c: (S) -2- ((4- (6- ((2, 4-dichlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-15) 2- ((2, 4-Dichlorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (0203-15) (206 mg, 0.377 mmol, 1.2 equiv), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] c]Imidazole-6-carboxylic acid methyl ester (0116-3) (92 mg, 0.314 mmol, 1.0 eq) and potassium carbonate (173 mg, 1.256 mmol, 4.0 eq) in acetonitrile (5 ml)) The solution was stirred at 60 ℃ for 16 hours. The reaction mixture was cooled to room temperature, water was added thereto, and ethyl acetate was added for extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative silica gel thin layer chromatography (eluent: ethyl acetate) to give (S) -2- ((4- (6- ((2, 4-dichlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid]Imidazole-6-carboxylic acid methyl ester (180 mg, yield: 90.9%). Lcms (esi): m/z 635(M + H)+.
Step 13 d: (S) -2- ((4- (6- ((2, 4-Dichlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Preparation of imidazole-6-carboxylic acid (Compound 15) from (S) -2- ((4- (6- ((2, 4-dichlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0204-15) (64 mg, 0.1 mmol, 1.0 eq) and lithium hydroxide monohydrate (13 mg, 0.3 mmol, 3.0 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l sulfuric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water. The residue was purified by preparative silica gel thin layer chromatography (eluent: dichloromethane/methanol 10/1) to give (S) -2- ((4- (6- ((2, 4-dichlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid]Imidazole-6-carboxylic acid (55 mg, yield: 88.71%). Lcms (esi): m/z 621(M + H)+.1H NMR(500MHz,DMSO)δ12.67(s,1H),8.26(s,1H),7.80(d,J=8.2Hz,1H),7.63(dd,J=12.7,8.1Hz,2H),7.44(d,J=8.0Hz,1H),7.37(d,J=8.1Hz,1H),7.15(s,1H),6.86(d,J=7.2Hz,1H),6.68(d,J=8.1Hz,1H),5.59(s,2H),5.11(d,J=5.0Hz,1H),4.79(dd,J=15.2,7.0Hz,1H),4.65(d,J=14.0Hz,1H),4.46(dd,J=13.5,7.1Hz,1H),4.41–4.31(m,1H),3.94(d,J=13.5Hz,1H),3.78(d,J=13.5Hz,1H),2.99(d,J=10.4Hz,1H),2.85(d,J=10.7Hz,1H),2.76–2.66(m,1H),2.58(s,1H),2.45(d,J=8.9Hz,1H),2.20(dt,J=21.7,10.4Hz,2H),1.83–1.64(m,4H).
Example 14: preparation of (S) -2- ((4- (6- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 16) (preparation according to scheme two line)
Step 14 a: preparation of tert-butyl 4- (6- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0202-16): lithium diisopropylamide (1.13 ml, 2 mol/l tetrahydrofuran, 2.26 mmol, 2 eq) was added to a solution of tert-butyl 4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-12) (500 mg, 1.13 mmol, 1 eq) in 10 ml tetrahydrofuran under nitrogen protection at-70 ℃. The mixture was stirred at-70 ℃ for 1 hour. To the mixture was added N-fluorobisbenzenesulfonamide (356 mg, 1.13 mmol, 1 eq) in tetrahydrofuran (1 ml). The mixture was stirred at-70 ℃ for 1 hour. The reaction was quenched with ammonium chloride. The mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over sodium sulfate. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-10/1) to give tert-butyl 4- (6- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (200 mg, yield: 38.38%) as a clear oil. Lcms (esi): m/z 461(M + H)+.
Step 14 b: preparation of 2- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (compound 0203-16): a solution of tert-butyl 4- (6- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-16) (200 mg, 0.43 mmol, 1.0 eq) and p-toluenesulfonic acid (224 mg, 1.3 mmol, 3.0 eq) in ethyl acetate (15 ml) was stirred at 60 ℃ overnight. The mixture was cooled to room temperature. The mixture was filtered. The residue was washed with ethyl acetate. The residue was dried to give 2- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (208 mg, crude) as a white solid lcms (esi): m/z 361(M + H) +.
Step 14 c: (S) -2- ((4- (6- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl)1- (Oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-16) 2- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (0203-16) (104 mg, 0.203 mmol, 1.2 equiv), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] c]A solution of methyl imidazole-6-carboxylate (0116-3) (50 mg, 0.169 mmol, 1.0 eq) and potassium carbonate (93 mg, 0.676 mmol, 4.0 eq) in acetonitrile (5 ml) was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, and water was added thereto, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative silica gel thin layer chromatography (eluent: ethyl acetate) to give (S) -2- ((4- (6- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid]Imidazole-6-carboxylic acid methyl ester (76 mg, yield: 72.38%). Lcms (esi): m/z 619(M + H)+.
Step 14 d: (S) -2- ((4- (6- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Preparation of imidazole-6-carboxylic acid (Compound 16) (S) -2- ((4- (6- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0204-16) (76 mg, 0.123 mmol, 1.0 eq) and lithium hydroxide monohydrate (15 mg, 0.369 mmol, 3.0 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l sulfuric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water. The residue was purified by preparative silica gel thin layer chromatography on plate (eluent: dichloromethane/methanol-10/1) to give (S) -2- ((4- (6- ((4-chloro-2-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] e as a yellow solid]Imidazole-6-carboxylic acid (54 mg, yield: 72.97%). Lcms (esi): m/z 605(M + H)+.1H NMR(500MHz,DMSO)δ12.64(s,1H),8.26(d,J=0.8Hz,1H),7.80(dd,J=8.4,1.5Hz,1H),7.68–7.57(m,2H),7.42(d,J=8.2Hz,1H),7.38(d,J=8.2Hz,1H),6.86(d,J=7.3Hz,1H),6.68(d,J=8.2Hz,1H),6.49(d,J=6.4Hz,1H),5.56(s,2H),5.11(qd,J=7.2,2.7Hz,1H),4.79(dd,J=15.2,7.2Hz,1H),4.65(dd,J=15.2,2.7Hz,1H),4.47(dt,J=13.7,7.0Hz,1H),4.37(dt,J=9.0,5.9Hz,1H),4.00–3.89(m,1H),3.78(d,J=13.5Hz,1H),2.99(d,J=11.2Hz,1H),2.85(d,J=11.3Hz,1H),2.74–2.66(m,1H),2.62–2.54(m,1H),2.43(ddd,J=11.1,8.9,5.6Hz,1H),2.28–2.11(m,2H),1.84–1.60(m,4H).
Example 15: preparation of (S) -2- ((4- (6- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 17) (preparation according to scheme two line)
Step 15 a: preparation of (4-chloro-2-methylbenzofuran-7-yl) methanol (compound 0201-17):
a mixture of methyl 4-chloro-2-hydroxybenzoate (1.87 g, 10.0 mmol, 1.0 eq), 3-bromopropyne (1.42 g, 12 mmol, 1.2 eq), and potassium carbonate (2.1 g, 15 mmol, 1.5 eq) in N, N-dimethylformamide was stirred at 50 ℃ for 5 hours under nitrogen. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was dissolved with N, N-diethylaniline (15 ml). Cesium fluoride (2.67 g, 17.63 mmol, 1.3 eq) was added to the mixture. The mixture was stirred at 220 ℃ for 3 hours. After cooling to room temperature, the reaction was quenched with water, and the pH of the residue was adjusted to 6 with 2 mol/l hydrochloric acid and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate-10/1) to give 4-chloro-2-methylbenzofuran-7-carboxylic acid methyl ester (1.34 g, yield: 59.56%) as a yellow solid. Lcms (esi): m/z 225(M + H) +.
A solution of methyl 4-chloro-2-methylbenzofuran-7-carboxylate (1.34 g, 5.98 mmol, 1.0 eq) obtained above and sodium borohydride (4.55 g, 119.64 mmol, 20 eq) in methanol (13 ml) was stirred at room temperature for 3 hours under a nitrogen blanket. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate-10/1) to give (4-chloro-2-methylbenzofuran-7-yl) methanol (1.1 g, yield: 93.86%) as a yellow solid. Lcms (esi): 196(M + H) is equal to M/z+.
Step 15 b: preparation of tert-butyl 4- (6- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0202-17): under nitrogen protection, a mixture of tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (0104-1) (137 mg, 0.46 mol, 1.0 eq), (4-chloro-2-methylbenzofuran-7-yl) methanol (0201-17) (100 mg, 0.51 mmol, 1.1 eq), cesium carbonate (333 mg, 1.02 mmol, 2.0 eq), 4, 5-bis-diphenylphosphino-9, 9-dimethylxanthene (59 mg, 0.05 mmol, 0.1 eq), and tris (dibenzylideneacetone) dipalladium (0) (47 mg, 0.148 mmol, 0.2 eq) in toluene (10 ml) was refluxed at 125 ℃ overnight. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to give 4- (6- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester as a yellow oil (145 mg, yield: 69.13%). Lcms (esi): 456(M + H) M/z +.
And step 15 c: preparation of 2- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (compound 0203-17): a solution of tert-butyl 4- (6- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-17) (145 mg, 0.31 mmol, 1.0 eq) and p-toluenesulfonic acid (159 mg, 0.93 mmol, 3.0 eq) in ethyl acetate (10 ml) was stirred at 60 ℃ overnight. The mixture is washed with water and the pH of the residue is adjustedThe ester was adjusted to 8 with 2 mol/l sodium hydroxide solution and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 2- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (92 mg, crude) which was used in the next step without further purification. Lcms (esi): 357(M + H) is the ratio of M/z+.
Step 15 d: (S) -2- ((4- (6- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-17) 2- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (0203-17) (90 mg, 0.24 mmol, 1.2 equiv), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] c ]A solution of methyl imidazole-6-carboxylate (0116-3) (60 mg, 0.20 mmol, 1.0 eq) and potassium carbonate (110 mg, 0.80 mmol, 4.0 eq) in acetonitrile (5 ml) was stirred at 60 ℃ for 16 h. After cooling to room temperature, the mixture was diluted with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative silica gel thin layer chromatography (eluent: ethyl acetate/petroleum ether: 2/1) to give (S) -2- ((4- (6- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid]Imidazole-6-carboxylic acid methyl ester (100 mg, yield: 81.30%). Lcms (esi): m/z 615(M + H)+.
Step 15 e: (S) -2- ((4- (6- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 17) (S) -2- ((4- (6- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0204-17) (100 mg, 0.16 mmol, 1.0 eq) and lithium hydroxide monohydrate (21 mg, 0.49 mmol, 3.0 eq) in acetonitrile/water 5/1(12 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue is 1 mol/l Hydrochloric acid was adjusted to 6, after which a solid precipitate formed. The mixture was filtered and the residue was washed with water. The residue was purified by thick preparative thin layer silica gel chromatography (eluent: dichloromethane/methanol-10/1) to give (S) -2- ((4- (6- ((4-chloro-2-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] c]Imidazole-6-carboxylic acid (56 mg, yield: 58.24%). Lcms (esi): 601(M + H) M/z+.1H NMR(500MHz,DMSO)δ12.67(s,1H),8.27(s,1H),7.80(s,1H),7.64(d,J=7.2Hz,2H),7.33(d,J=7.8Hz,1H),7.26(d,J=8.0Hz,1H),6.87(d,J=7.0Hz,1H),6.67(d,J=13.6Hz,2H),5.59(s,2H),5.10(s,1H),4.78(s,1H),4.66(d,J=14.2Hz,1H),4.46(dd,J=13.7,7.5Hz,1H),4.36(dt,J=8.9,5.9Hz,1H),3.94(s,1H),3.79(s,1H),2.99(s,1H),2.86(s,1H),2.70(dt,J=16.1,8.0Hz,1H),2.62(d,J=18.8Hz,1H),2.40(d,J=42.1Hz,4H),2.21(d,J=33.5Hz,2H),1.76(s,4H).
Example 16: preparation of (S) -2- ((4- (6- (4-chloro-2-methylbenzofuran-7-carboxamido) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 20) (prepared according to scheme two line)
Step 16 a: preparation of tert-butyl 4- (6- (4-chloro-2-methylbenzofuran-7-carboxamido) pyridin-2-yl) piperidine-1-carboxylate (compound 0202-20):
to a solution of 4-chloro-2-methylbenzofuran-7-carboxylic acid methyl ester (885 mg, 3.95 mmol, 1 eq) in 10 ml of tetrahydrofuran was added a 2N sodium hydroxide solution (5 ml). The mixture was stirred at 40 ℃ for 16 hours. The reaction was cooled to room temperature. The pH of the mixture was adjusted to 1 with 2N hydrochloric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water. The residue was dried to give 4-chloro-2-methylbenzofuran-7-carboxylic acid (700 mg, yield: 84.03%) as a yellow solid. Lcms (esi): m/z 211(M + H) +.
6-Bromopyridin-2-amine (519 mg, 3 mmol) was reacted under nitrogen blanket1.0 eq), a mixture of tert-butyl 4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 6-dihydropyridine-1 (2H) -carboxylate (1113 mg, 3.6 mmol, 1.2 eq), sodium carbonate (636 mg, 6 mmol, 2.0 eq) and bis-triphenylphosphine palladium dichloride (210 mg, 0.3 mmol, 0.1 eq) in toluene/ethanol/water (10/5/5 ml) was stirred at 110 ℃ for 16H. After cooling to room temperature, the reaction solution was extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over sodium sulfate. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-2/1) to give 6-amino-3 ',6' -dihydro- [2,4' -bipyridine as a yellow oil]-1'(2' H) -carboxylic acid tert-butyl ester (600 mg, yield: 72.46%). Lcms (esi): m/z 276(M + H)+.
Reacting 6-amino-3 ',6' -dihydro- [2,4' -bipyridine]A solution of-1 '(2' H) -carboxylic acid tert-butyl ester (600 mg, 2.18 mmol, 1.0 eq) and palladium on carbon (60 mg, Wt% ═ 10%) in methanol (15 ml) was stirred at room temperature for 5 hours. The mixture was filtered. The residue was washed with methanol. The residue was concentrated under reduced pressure to give 4- (6-aminopyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester (553 mg, crude) as a white solid lcms (esi): 278(M + H) M/z +.
Oxalyl chloride (181 mg, 1.428 mmol, 3 equivalents) was added dropwise to a solution of 4-chloro-2-methylbenzofuran-7-carboxylic acid (100 mg, 0.476 mmol, 1 equivalent) in dichloromethane/tetrahydrofuran/N, N-dimethylformamide (10/5/0.01 ml) under nitrogen and ice bath. The mixture was stirred at room temperature for 5 hours. The mixture was concentrated under reduced pressure. The residue was dissolved in 5 ml of dichloromethane. To the solution was added tert-butyl 4- (6-aminopyridin-2-yl) piperidine-1-carboxylate (131 mg, 0.476 mmol, 1 eq) and triethylamine (144 mg, 1.428 mmol, 3 eq). The mixture was stirred at room temperature for 3 hours. The reaction was quenched with water. The mixture was extracted with dichloromethane. The organic phase was washed with saturated brine and dried over sodium sulfate. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-2/1) to give tert-butyl 4- (6- (4-chloro-2-methylbenzofuran-7-carboxamido) pyridin-2-yl) piperidine-1-carboxylate (143 mg,yield: 64.41%). Lcms (esi): 470(M + H) M/z+.
Step 16b preparation of 4-chloro-2-methyl-N- (6- (piperidin-4-yl) pyridin-2-yl) benzofuran-7-carboxamide 4-methylbenzenesulfonate (compound 0203-20): a solution of tert-butyl 4- (6- (4-chloro-2-methylbenzofuran-7-carboxamido) pyridin-2-yl) piperidine-1-carboxylate (compound 0202-20) (143 mg, 0.305 mmol, 1.0 eq) and p-toluenesulfonic acid (158 mg, 0.915 mmol, 3.0 eq) in ethyl acetate (5 ml) was stirred at 60 ℃ overnight. The mixture was cooled to room temperature. The mixture was filtered. The residue was washed with ethyl acetate. The residue was dried to give 4-chloro-2-methyl-N- (6- (piperidin-4-yl) pyridin-2-yl) benzofuran-7-carboxamide 4-methylbenzenesulfonate (200 mg, crude) as a white solid lcms (esi): m/z 370(M + H) +.
Step 16c (S) -2- ((4- (6- (4-chloro-2-methylbenzofuran-7-carboxamido) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-20) 4-chloro-2-methyl-N- (6- (piperidin-4-yl) pyridin-2-yl) benzofuran-7-carboxamide 4-methylbenzenesulfonate (0203-20) (106 mg, 0.203 mmol, 1.2 equivalents), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] d]A solution of methyl imidazole-6-carboxylate (0116-3) (50 mg, 0.169 mmol, 1.0 eq) and potassium carbonate (93 mg, 0.676 mmol, 4.0 eq) in acetonitrile (5 ml) was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, and water was added thereto, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative silica gel thin layer chromatography on a plate (eluent: ethyl acetate) to give (S) -2- ((4- (6- (4-chloro-2-methylbenzofuran-7-formylamino) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid]Imidazole-6-carboxylic acid methyl ester (70 mg, yield: 66.03%). Lcms (esi): 628(M + H) M/z+.
Step 16d (S) -2- ((4- (6- (4-chloro-2-methylbenzofuran-7-carboxamido) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Preparation of imidazole-6-carboxylic acid (Compound 20) preparation of (S) -2- ((4- (6- (4-chloro-2-methylbenzene)Furan-7-carboxamido) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0204-20) (70 mg, 0.11 mmol, 1.0 eq) and lithium hydroxide monohydrate (14 mg, 0.33 mmol, 3.0 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l sulfuric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water. The residue was purified by preparative silica gel thin layer chromatography on plate (eluent: dichloromethane/methanol-10/1) to give (S) -2- ((4- (6- (4-chloro-2-methylbenzofuran-7-carboxamido) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] c]Imidazole-6-carboxylic acid (48 mg, yield: 70.03%). Lcms (esi): m/z 614(M + H)+.1H NMR(500MHz,DMSO)δ12.57(s,1H),10.33(s,1H),8.26(d,J=0.8Hz,1H),8.04(d,J=8.2Hz,1H),7.79(dd,J=8.7,7.2Hz,2H),7.72(d,J=8.2Hz,1H),7.64(d,J=8.4Hz,1H),7.42(d,J=8.2Hz,1H),7.09(d,J=7.5Hz,1H),6.79(d,J=1.0Hz,1H),5.09(ddd,J=14.4,7.2,2.8Hz,1H),4.82(dd,J=15.2,7.2Hz,1H),4.67(dd,J=15.2,2.7Hz,1H),4.49(dd,J=13.6,7.7Hz,1H),4.37(dt,J=9.0,5.9Hz,1H),3.95(d,J=13.5Hz,1H),3.81(d,J=13.5Hz,1H),3.01(d,J=11.2Hz,1H),2.89(d,J=11.2Hz,1H),2.78–2.62(m,2H),2.55(s,3H),2.42(ddd,J=15.9,11.1,7.0Hz,1H),2.23(dt,J=29.5,10.7Hz,2H),1.86(t,J=13.5Hz,2H),1.81–1.67(m,2H).
Example 17: preparation of 2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (2-methoxyethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (compound 23) (prepared according to scheme one and two routes)
Step 17 a: preparation of methyl 3- ((2-methoxyethyl) amino) -4-nitrobenzoate (compound 0118-23) to a mixture of methyl 3-fluoro-4-nitrobenzoate (0107-23) (1.0 g, 5.02 mmol, 1.0 eq) and N, N-diisopropylethylamine (1.34 ml, 7.53 mmol, 1.5 eq) in tetrahydrofuran (15 ml) was added 2-methoxyethylamine (0.45 g, 6.03 mmol, 1.2 eq). The mixture was heated to 55 ℃ and reacted overnight. The solvent was removed under reduced pressure. Residue ofDiluted with water (30 ml). The aqueous layer was extracted with ethyl acetate (30 ml × 4). The combined organic layers were washed with saturated brine (30 ml. times.1), dried over anhydrous sodium sulfate and concentrated to give methyl 3- ((2-methoxyethyl) amino) -4-nitrobenzoate as a yellow solid (1.17 g, yield: 91%). Lcms (esi): m/z 255[ M +1 ]]+(ii) a TLC: rf0.5 (petroleum ether: ethyl acetate ═ 5: 1).
Step 17 b: preparation of methyl 4-amino-3- ((2-methoxyethyl) amino) benzoate (Compound 0119-23) to a mixture of methyl 3- ((2-methoxyethyl) amino) -4-nitrobenzoate (0118-23) (1.17 g, 4.61 mmol, 1.0 eq) in methanol (30 mL) was added palladium on charcoal (0.22 g). The mixture was stirred at room temperature under hydrogen balloon pressure for 3.5 hours. The mixture was filtered. The filtrate was concentrated under reduced pressure to give methyl 4-amino-3- ((2-methoxyethyl) amino) benzoate (1.03 g, yield: 100%) as a white solid. Lcms (esi): m/z 225[ M +1 ] ]+(ii) a TLC: rf0.3 (petroleum ether: ethyl acetate ═ 5: 1).
And step 17 c: 2- (chloromethyl) -1- (2-methoxyethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (compound 0120-23) to a mixture of methyl 4-amino-3- ((2-methoxyethyl) amino) benzoate (0119-23) (1.03 g, 4.60 mmol, 1.0 equiv.) and p-toluenesulfonic acid monohydrate (17.5 mg, 0.09 mmol, 0.02 equiv.) in tetrahydrofuran (20 ml) was added 2-chloro-1, 1, 1-trimethoxyethane (0.78 g, 5.06 mmol, 1.1 equiv.). The mixture was heated to 45 ℃ and reacted overnight. The solvent was removed under reduced pressure. The residue is subjected to column chromatography on silica gel (petroleum ether: ethyl acetate 5:1) to give 2- (chloromethyl) -1- (2-methoxyethyl) -1H-benzo [ d ] a white solid]Imidazole-6-carboxylic acid methyl ester (1.25 g, yield: 93%). Lcms (esi): m/z 283[ M +1 ]]+(ii) a TLC: rf0.5 (petroleum ether: ethyl acetate ═ 2: 1).
Step 17 d: 2-((4-(6-((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (2-methoxyethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0205-23) methyl ester to 2- ((4-chlorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine p-toluenesulfonate (0203-12) (90 mg, 0.18 mmol, 1.1 equiv.) and 2 - (chloromethyl) -1- (2-methoxyethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (0120-23) (45 mg, 0.16 mmol, 1.0 eq) to a mixture of acetonitrile (6 ml) was added potassium carbonate (66 mg, 0.48 mmol, 3.0 eq). The mixture was heated to 60 ℃ and reacted overnight. The solvent was removed under reduced pressure. The residue was purified by preparative thin layer chromatography (petroleum ether: ethyl acetate 1:1) to give 2 as a white solid-((4-(6-((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (2-methoxyethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (69 mg, yield: 74%). Lcms (esi): m/z 589[ M +1 ]]+(ii) a TLC: rf0.5 (petroleum ether: ethyl acetate ═ 1: 1).
Step 17 e: 2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (2-methoxyethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 23): general 2-((4-(6-((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (2-methoxyethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (0205-23) (69 mg, 0.12 mmol, 1.0 equiv.) to a mixture of tetrahydrofuran (6 ml) and water (3 ml) was added lithium hydroxide monohydrate (15 mg, 0.36 mmol, 3.0 equiv). The mixture was heated to 40 ℃ and reacted overnight. The mixture was diluted with water (15 ml). A 1N diluted hydrochloric acid solution was added to adjust pH 5, and then the aqueous layer was extracted with ethyl acetate (20 ml × 3). The combined organic layers were washed with saturated brine (20 ml × 1), dried over anhydrous sodium sulfate and concentrated. The residue was purified by preparative thin layer chromatography (dichloromethane: methanol ═ 10: 1) to give 2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (2-methoxyethyl) -1H-benzo [ d ] e as a white solid ]Imidazole-6-carboxylic acid (52 mg, yield: 78%). LCMS (ESI): m/z 575[ M +1 ]]+(ii) a TLC: rf0.5 (dichloromethane: methanol ═ 10: 1); melting point: 123-126 ℃.1H NMR(500MHz,DMSO)δ12.75(s,1H),8.24–8.11(m,2H),7.81(d,J=8.2Hz,1H),7.62(t,J=7.8Hz,2H),7.42(d,J=8.0Hz,1H),7.33(d,J=8.0Hz,1H),7.02(d,J=2.1Hz,1H),6.86(d,J=7.3Hz,1H),6.67(d,J=8.2Hz,1H),5.61(s,2H),4.60(s,2H),3.84(s,2H),3.75(t,J=5.0Hz,2H),3.21(s,3H),2.92(d,J=10.8Hz,2H),2.61(dd,J=19.2,7.7Hz,1H),2.21(t,J=10.7Hz,2H),1.75(dd,J=33.6,10.7Hz,4H).
Example 18: preparation of (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- ((tetrahydrofuran-2-yl) methyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 24) (prepared according to scheme one and two routes)
Step 18 a: preparation of methyl (S) -4-nitro-3- ((tetrahydrofuran-2-yl) methyl) amino) benzoate (compound 0118-24): a solution of methyl 3-fluoro-4-nitrobenzoate (0107-23) (894.8 mg, 4.4937 mmol, 1.0 eq), (S) - (tetrahydrofuran-2-yl) methylamine (500 mg, 4.9431 mmol, 1.1 eq) and potassium carbonate (1242 mg, 8.987 mmol, 2.0 eq) in a mixture of N, N-dimethylformamide (10 ml) was stirred at ambient temperature for 3 hours under nitrogen. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 5/1) to give methyl (S) -4-nitro-3- ((tetrahydrofuran-2-yl) methyl) amino) benzoate (1120 mg, yield: 88.96%) as a yellow solid. LCMS (ESI): m/z 281(M + H) +.
Step 18 b: preparation of methyl (S) -4-amino-3- (((tetrahydrofuran-2-yl) methyl) amino) benzoate (compound 0119-24): a tetrahydrofuran mixture (13 ml) of methyl (S) -4-nitro-3- ((tetrahydrofuran-2-yl) methyl) amino) benzoate (0118-24) (1120 mg, 3.996 mmol, 1.0 eq) and palladium on carbon (112 mg, 10% by mass) under hydrogen was stirred at room temperature overnight. The reaction was filtered, and the filtrate was concentrated under reduced pressure to give methyl (S) -4-amino-3- (((tetrahydrofuran-2-yl) methyl) amino) benzoate as a yellow oil (1090 mg, 109% yield). The product was used in the next step without further purification.
Step 18 c: ((S) -2- (chloromethyl) -1- ((tetrahydrofuran-2-yl) methyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (compound 0120-24): under nitrogen, (S) -4-amino-3- (((tetrahydrofuran-2-yl) methyl) amino) benzoic acid methyl ester (0119-24) (1090 mg, 4.35 mmol, 1.0 eq.), 2-chloro-A solution of 1,1, 1-trimethoxyethane (807 mg, 5.55 mmol, 1.2 units) and p-toluenesulfonic acid monohydrate (89.889 mg, 0.522 mmol, 0.12 units) in tetrahydrofuran (10 ml) was heated to 45 ℃ and stirred overnight. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. Concentrating the organic phase under reduced pressure to obtain (S) -2- (chloromethyl) -1- ((tetrahydrofuran-2-yl) methyl) -1H-benzo [ d ]Methyl imidazole-6-carboxylate (609.5 mg, yield: 45.44%) was a yellow solid. Lcms (esi): m/z 309(M + H)+.
Step 18 d: (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- ((tetrahydrofuran-2-yl) methyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0205-24): a mixture of 2- ((4-chlorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (compound 0203-12) (96.82 mg, 0.1948 mmol, 1.2 equiv.) and potassium carbonate (89.5896 mg, 0.6492 mmol, 4.0 equiv.) in acetonitrile (5 ml) was stirred at 60 ℃ to pH 7-8, after which (S) -2- (chloromethyl) -1- ((tetrahydrofuran-2-yl) methyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (0120-24) (50 mg, 0.1623 mmol, 1.0 eq) was added to the mixture and stirred overnight. After cooling to room temperature, the reaction solution was quenched with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain yellow solid compound (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxyl) pyridine-2-yl) piperidine-1-yl) methyl) -1- ((tetrahydrofuran-2-yl) methyl) -1H-benzo [ d ]Imidazole-6-carboxylic acid methyl ester (79.8 mg, yield 79.94). Lcms (esi): m/z 616(M + H)+.
Step 18 e: (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- ((tetrahydrofuran-2-yl) methyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 24): (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- ((tetrahydrofuran-2-yl) methyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (0205-24) (79.8 mg, 0.1298 mmol, 1.0mAmount) and lithium hydroxide monohydrate (10.98 mg, 0.1298 mmol, 10 parts per volume) in acetonitrile/water 5/1(6 ml) were stirred at 40 ℃ overnight. The mixture was then cooled to room temperature and the acid-base temperature was adjusted to about 6 by the addition of 1.0M H2SO4 before a solid precipitate formed. The mixture was filtered. The residue was washed with water. The mixture was slurried with methanol to give (S) -2- (4- (6- (4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- ((tetrahydrofuran-2-yl) methyl) -1H-benzo [ d ] as a yellow solid]Imidazole-6-carboxylic acid (49.8 mg, yield 63.86%). lcms (esi): 602(M + H) is defined as M/z+.1H NMR(500MHz,DMSO)δ12.72(s,1H),8.21(s,1H),8.14(d,J=2.2Hz,1H),7.81(d,J=8.4Hz,1H),7.62(dd,J=14.0,6.4Hz,2H),7.42(d,J=8.0Hz,1H),7.33(d,J=8.0Hz,1H),7.02(d,J=2.2Hz,1H),6.86(d,J=7.3Hz,1H),6.67(d,J=8.2Hz,1H),5.61(s,2H),4.55(d,J=13.6Hz,1H),4.44(dd,J=14.6,8.1Hz,1H),4.32–4.23(m,1H),3.99(d,J=13.6Hz,1H),3.77(dt,J=22.8,10.4Hz,2H),3.60(dd,J=14.0,7.3Hz,1H),3.01(d,J=11.2Hz,1H),2.85(d,J=10.8Hz,1H),2.61(td,J=11.2,5.6Hz,1H),2.27(t,J=10.7Hz,1H),2.17(t,J=10.6Hz,1H),2.11–2.01(m,1H),1.76(dd,J=15.5,8.4Hz,5H),1.71–1.61(m,2H).
Example 19: preparation of 2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- ((1-ethyl-1H-imidazol-5-yl) methyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (compound 25) (prepared according to scheme one and two routes)
Step 19 a: preparation of methyl 4-amino-3- (((1-ethyl-1H-imidazol-5-yl) methyl) amino) benzoate (compound 0119-25):
a solution of 1-ethyl-1H-imidazole-5-carboxylic acid (500 mg, 3.6 mmol, 1.0 eq) in thionyl chloride (5 ml) was refluxed for two hours. After cooling to room temperature, the solvent was spun off in vacuo and the residue was dissolved in (6 ml) tetrahydrofuran and 0.4 mol/l aminodioxane solution (1 ml) was added dropwise at 0 ℃. After the addition was complete, the mixture was stirred for 30 minutes. Potassium carbonate (1 g) was added,the mixture was stirred for 30 minutes. The mixture was filtered, and the filtrate was concentrated in vacuo to give 1-ethyl-1H-imidazole-5-carboxamide (500 mg, yield: 100%) as a white solid. Lcms (esi): 140(M-H) M/z+.
To a solution of 1-ethyl-1H-imidazole-5-carboxamide (500 mg, 3.6 mmol, 1.0 eq) in tetrahydrofuran (10 ml) was added a solution of borane dimethylsulfide (10 mol/l) (0.72 ml, 7.2 mmol, 2.0 eq). The mixture was stirred at room temperature for 3 hours. Methanol (2 ml) was added and the mixture was stirred for 30 minutes. The mixture was concentrated in vacuo, 5 mol/l of methanol hydrochloride (5 ml) was added, and the mixture was stirred for 10 minutes. The mixture was concentrated in vacuo to give (1-ethyl-1H-imidazol-5-) methylamine hydrochloride (550 mg, yield: 94.8%) as a white solid. Lcms (esi): m/z 126(M + H) +.
A mixture of methyl 3-fluoro-4-nitrobenzoate (367 mg, 1.85 mmol, 1.0 eq), potassium carbonate (511 g, 3.70 mmol, 2.0 eq) and (1-ethyl-1H-imidazole-5-) methylamine hydrochloride (300 mg, 1.85 mmol, 1.0 eq) in N-methylpyrrolidone (10 ml) was stirred at room temperature overnight. Ethyl acetate and water were added to the mixture, and the mixture was separated, and the organic layer was washed with saturated brine and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane/methanol 100/1 to 30/1) to give methyl 3- (((1-ethyl-1H-imidazol-5-yl) methyl) amino) -4-nitrobenzoate (340 mg, yield: 60.5%) as a yellow solid. Lcms (esi): m/z 305(M + H)+.
A mixture of methyl 3- (((1-ethyl-1H-imidazol-5-yl) methyl) amino) -4-nitrobenzoate (340 mg, 1.1 mmol, 1.0 eq) and Pd/C (34 mg, 10% mass) in methanol (6 ml) was stirred at room temperature under hydrogen overnight. The mixture was filtered, and the filtrate was concentrated in vacuo to give methyl 4-amino-3- (((1-ethyl-1H-imidazol-5-yl) methyl) amino) benzoate as a white solid (300 mg, yield: 98.0%). Lcms (esi): 275(M + H) M/z+.
Step 19 b: preparation of methyl 2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- ((1-ethyl-1H-imidazol-5-yl) methyl) -1H-benzo [ d ] imidazole-6-carboxylate (compound 0205-25):
2- ((4-chlorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (0203-12) (231 mg, 0.45 mmol, 1.5 equiv.), ethyl 2-bromoacetate (50 mg, 0.30 mmol, 1.0 equiv.) and potassium carbonate (165 mg, 1.20 mmol, 4.0 equiv.) were stirred in a solution of N, N-dimethylformamide (10 mL) at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, and water was added thereto, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 20/1-1/1) to give ethyl 2- (4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) acetate as a white solid (120 mg, yield: 93.24%). Lcms (esi): m/z 429(M + H)+.
To a solution of ethyl 2- (4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) acetate (120 mg, 0.28 mmol, 1.0 eq) in methanol/tetrahydrofuran-2/1 (9 ml) was added an aqueous solution of sodium hydroxide (2 mol/l, 5 ml). The mixture was stirred at room temperature for 3 hours. The pH of the mixture was adjusted to 7 with 1 mol/l dilute hydrochloric acid, after which a solid precipitate formed. The mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give a crude product of 2- (4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) acetic acid (120 mg, crude). The product was used in the next step without further purification.
To a solution of 2- (4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) acetic acid (120 mg, 0.30 mmol, 1.0 eq) in N, N-dimethylformamide (10 ml) was added methyl 4-amino-3- (((1-ethyl-1H-imidazol-5-yl) methyl) amino) benzoate (0119-25) (73 mg, 0.27 mmol, 0.9 eq) and N, N-diisopropylethylamine (109 mg, 0.84 mmol, 2.8 eq). 2- (7-Azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (136.8 mg, 0.36 mmol, 1.2 equivalents) was added to the reaction at the end. The mixture was stirred at room temperature overnight. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate.The organic phase was concentrated under reduced pressure and the residue was purified by preparative silica gel thin layer chromatography (eluent: dichloromethane/methanol-15/1) to give methyl 4- (2- (4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) acetamido) -3- (((((1-ethyl-1H-imidazole) -5-yl) methyl) amino) benzoate as a yellow oil (50 mg, yield: 28.19%). Lcms (esi): 657(M + H) M/z+.
Methyl 4- (2- (4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) acetamido) -3- ((((1-ethyl-1H-imidazol) -5-yl) methyl) amino) benzoate was stirred in a solution of acetic acid (10 ml) at 55 ℃ for 16H. After cooling to room temperature, the solvent was spin dried under reduced pressure. The residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. Concentrating the organic phase under reduced pressure, and purifying the residue with silica gel thin layer chromatography preparative plate (eluent: dichloromethane/methanol-10/1) to obtain yellow solid methyl 2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxyl) pyridine-2-yl) piperidine-1-yl) methyl) -1- ((1-ethyl-1H-imidazole-5-yl) methyl) -1H-benzo [ d ]Imidazole-6-carboxylic acid methyl ester (34 mg, yield: 69.96%). Lcms (esi): m/z 639(M + H)+.
Step 19 c: 2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- ((1-ethyl-1H-imidazol-5-yl) methyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 25) methyl 2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- ((1-ethyl-1H-imidazol-5-yl) methyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0205-25) (34 mg, 0.053 mmol, 1.0 eq) and lithium hydroxide monohydrate (6.7 mg, 0.16 mmol, 3 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l dilute sulfuric acid, after which a solid precipitate formed. The mixture was filtered. The residue was purified by preparative silica gel thin layer chromatography on preparative plates (eluent: dichloromethane/methanol-10/1) to give 2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- ((1-ethyl-1H-imidazol-5-yl) methyl) -1H-benzo [ d ] o]Imidazole-6-carboxylic acid (18.3 mg,yield: 55.25%). Lcms (esi): 625(M + H) M/z +.1H NMR(500MHz,DMSO)δ12.69(s,1H),8.15(d,J=1.9Hz,1H),8.06(s,1H),7.81(d,J=8.3Hz,1H),7.76–7.55(m,3H),7.42(d,J=8.0Hz,1H),7.34(d,J=7.9Hz,1H),7.03(d,J=1.9Hz,1H),6.81(d,J=7.2Hz,1H),6.67(t,J=7.1Hz,1H),6.46(s,1H),5.73(s,2H),5.61(s,2H),3.98(q,J=7.2Hz,2H),3.81(s,2H),2.87(d,J=10.8Hz,2H),2.53(d,J=11.4Hz,1H),2.15(t,J=11.1Hz,2H),1.72(d,J=11.8Hz,2H),1.60–1.42(m,2H),1.14(t,J=7.2Hz,3H).
Example 20: preparation of (S) -2- ((4- (6- ((4-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 28) (prepared according to scheme two route)
Step 20 a: preparation of (4-fluorobenzofuran-7-yl) methanol (compound 0201-28):
a mixture of 5-fluoro-2-methylphenol (3.0 g, 23.8 mmol, 1.0 eq), 2-bromo-1, 1-diethoxyethane (5.63 g, 28.5 mmol, 1.2 eq), potassium carbonate (4.92 g, 35.7 mmol, 1.5 eq) and potassium iodide (0.3 g, 10% by mass) in N, N-dimethylformamide was stirred at 120 ℃ overnight under a nitrogen blanket. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product, 2- (2, 2-diethoxyethoxy) -4-fluoro-1-methylbenzene (5.3 g, yield: 92.0%). The product was used in the next step without further purification.
A mixture of 2- (2, 2-diethoxyethoxy) -4-fluoro-1-methylbenzene (1.0 g, 4.13 mmol, 1.0 eq.) obtained above and polyphosphoric acid (3.5 g, 10.3 mmol, 2.5 eq.) in 1, 2-dichloroethane (20 mL) was refluxed overnight under a nitrogen blanket. After cooling to room temperature, the reaction was quenched with water (40 ml) and extracted with ethyl acetate (50 ml). The organic layer was washed with saturated brine and with anhydrous sulfur Drying sodium salt, and concentrating under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether) to give 4-fluoro-7-methylbenzofuran (300 mg, yield: 49.0%) as a yellow oil. Lcms (esi): m/z 151(M + H)+.
A mixture of 4-fluoro-7-methylbenzofuran (300 mg, 2.00 mmol, 1.0 equiv), N-bromosuccinimide (415 mg, 2.40 mmol, 1.2 equiv) and azobisisobutyronitrile (66 mg, 0.4 mmol, 0.2 equiv) in 1, 2-dichloroethane (10 ml) was stirred at 72 ℃ overnight under a nitrogen blanket. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was diluted with water (20 ml) and extracted with petroleum ether/ethyl acetate 2/1(30 ml). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to give 7- (bromomethyl) -4-fluorobenzofuran (400 mg, yield: 87.3%) as a white solid. Lcms (esi): 229(M + H)+.
A mixture of the above-obtained 7- (bromomethyl) -4-fluorobenzofuran (400 mg, 1.73 mmol, 1.0 equivalent) and potassium acetate (1.67 g, 17.3 mmol, 10.0 equivalent) in N, N-dimethylformamide (15 ml) was stirred at room temperature for 3 hours. The reaction was quenched with water (20 ml) and extracted with ethyl acetate (30 ml). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product of (4-fluorobenzofuran-7-yl) methyl acetate (500 mg of crude product). The product was used in the next step without further purification.
To a solution of (4-fluorobenzofuran-7-yl) methyl acetate (500 mg, 2.42 mmol, 1.0 eq) obtained above in tetrahydrofuran (10 ml) was added a solution of sodium methoxide in methanol (5.4 mol/l, 0.7 ml, 3.64 mmol, 1.5 eq). The mixture was stirred at room temperature for 1 hour. The reaction was adjusted to a pH of about 6 with 1 mol/l hydrochloric acid and a solid precipitate was formed. The slurry was diluted with water (15 ml) and extracted with ethyl acetate (20 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (eluent: petroleum ether/ethyl acetate 4/1) to give a yellow solidThe bulk (4-fluorobenzofuran-7-yl) methanol (195 mg, yield: 67.9%). Lcms (esi): 167(M + H)+.
Step 20 b: 4- (6-Preparation of tert-butyl ((4-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0202-28): a mixture of tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (0104-1) (195 mg, 0.66 mmol, 1.0 eq), (4-fluorobenzofuran-7-yl) methanol (0201-28) (132 mg, 0.79 mmol, 1.2 eq), cesium carbonate (430 mg, 1.32 mmol, 2.0 eq), 2-dicyclohexylphosphonium-2 ',6' -diisopropoxy-1, 1' -biphenyl (61 mg, 0.132 mmol, 0.2 eq) and tris (dibenzylideneacetone) dipalladium (0) (55 mg, 0.06 mmol, 0.1 eq) in toluene (10 ml) was refluxed at 120 ℃ overnight under nitrogen protection. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate (15 ml). The filtrate was diluted with water (20 ml) and extracted with ethyl acetate (20 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to give 4- (6) as a white solid -Tert-butyl ((4-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (230 mg, yield: 82.1%). Lcms (esi): m/z 427(M + H)+.
And step 20 c: preparation of 2- ((4-fluorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine hydrochloride (compound 0203-28): will be 4- (6)-Tert-butyl ((4-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-28) (110 mg, 0.258 mmol, 1.0 eq) was added to a solution of dioxane (15 ml) hydrochloride and stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure to give a white solid 2-((4-fluorobenzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine hydrochloride (84 mg crude). M/z 327(M + H)+.
And step 20 d: (S) -2- ((4- (6)-((4-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-28) 2- ((4-fluorobenzofuran-7-yl) methoxy) -6-(piperidin-4-yl) pyridine hydrochloride (0203-28) (84 mg, 0.257 mmol, 1.2 equiv), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of methyl imidazole-6-carboxylate (compound 0116-3) (60 mg, 0.21 mmol, 1.0 eq) and N, N-diisopropylethylamine (0.5 ml) in N-methylpyrrolidone (5 ml) was stirred at 60 ℃ for 4 h. The reaction mixture was cooled to room temperature, and water was added thereto, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain white solid (S) -2 -((4-(6-((4-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (99 mg, yield: 80.4%). Lcms (esi): 585(M + H) with M/z+.
Step 20 e: (S) -2- ((4- (6- ((4-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 28) by reacting (S) -2-((4- (6- ((4-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0204-28) (99 mg, 0.184 mmol, 1.0 eq) and lithium hydroxide monohydrate (24 mg, 0.553 mmol, 3.0 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. After the mixture was cooled to room temperature, the pH was adjusted to about 6 with 1 mol/l hydrochloric acid, and then a solid precipitate was formed. The slurry was diluted with water (12 ml), stirred for 4 hours, and the solid was collected by filtration. The solid was washed with water and then dried under vacuum. The solid was dissolved in dichloromethane/methanol 1/2(5 ml), then filtered, the filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was dried in vacuo to give (S) -2- ((4- (6- ((4-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid ]Imidazole-6-carboxylic acid (42 mg, yield: 40.0%). Lcms (esi): 571(M + H) M/z+Melting point: 131 to 143 ℃;1H NMR(500MHz,DMSO)δ12.71(s,1H),8.27(s,1H),8.08(s,1H),7.80(d,J=8.3Hz,1H),7.70–7.56(m,2H),7.44(dd,J=7.8,5.5Hz,1H),7.08(dd,J=8.9,5.5Hz,2H),6.86(d,J=7.2Hz,1H),6.66(d,J=8.1Hz,1H),5.60(s,2H),5.11(d,J=5.2Hz,1H),4.79(dd,J=15.1,7.1Hz,1H),4.66(d,J=13.7Hz,1H),4.46(dd,J=13.6,7.2Hz,1H),4.37(dd,J=14.3,5.8Hz,1H),3.95(d,J=13.5Hz,1H),3.78(d,J=13.5Hz,1H),3.00(d,J=10.5Hz,1H),2.86(d,J=10.7Hz,1H),2.70(dt,J=16.0,7.8Hz,1H),2.61(t,J=11.5Hz,1H),2.44(t,J=13.0Hz,1H),2.22(dt,J=21.6,10.6Hz,2H),1.86–1.65(m,4H).
example 21: preparation of (S) -2- ((4- (6- ((4-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 30) (preparation according to scheme two line)
Step 21 a: preparation of (4-methylbenzofuran-7-yl) methanol (Compound 0201-30):
a mixture of methyl 2-hydroxy-4-methylbenzoate (831 mg, 5.0 mmol, 1.0 eq), 2-bromo-1, 1-diethoxyethane (1.48 g, 7.5 mmol, 1.5 eq) and potassium carbonate (1.73 g, 12.5 mmol, 2.5 eq) in N, N-dimethylformamide was stirred at 120 ℃ overnight. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give methyl 2- (2, 2-diethoxyethoxy) -4-methylbenzoate as a yellow oil (4 g, crude) which was used in the next step without further purification.
A mixture of methyl 2- (2, 2-diethoxyethoxy) -4-methylbenzoate (1.72 g, 5.0 mmol, 1.0 eq.) obtained above and polyphosphoric acid (300 mg, 0.88 mmol, 0.18 eq.) in toluene was refluxed overnight. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate: 50/1) to give methyl 4-methylbenzofuran-7-carboxylate (496 mg, yield: 52.2%) as a colorless oil.
Methyl 4-methylbenzofuran-7-carboxylate (430 mg, 2.26 mmol, 1.0 equiv.) obtained above was dissolved in anhydrous tetrahydrofuran under nitrogen protection and the mixture was cooled to 0 ℃. Lithium aluminum hydride (129 mg, 3.39 mmol, 1.5 eq) was added and the mixture was stirred for 1.5 hours. 0.13 ml of water, 0.13 ml of a 15% aqueous sodium hydroxide solution and 0.39 ml of water are added in this order at 0 ℃. The mixture was stirred for 15 minutes. 1.6 g of anhydrous sodium sulfate was added, and the mixture was stirred at room temperature for 0.5 hour. The mixture was filtered under reduced pressure. The filtrate was concentrated under reduced pressure to give (4-methylbenzofuran-7-yl) methanol (391 mg, crude) as a yellow solid. Lcms (esi): 145(M + H-H) with M/z2O)+.
Step 21 b: (S) -2- ((4- (6-chloropyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-20): to a mixture of 4- (6-chloropyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester (0104-1) (200 mg, 0.67 mmol, 1.0 eq) in 4 ml dioxane was added 1.5 ml dioxane solution of 4M hydrogen chloride. The mixture was stirred at room temperature overnight. The mixture was concentrated to dryness under reduced pressure. The residue was added to 5 ml of N-methylpyrrolidone, and N, N-diisopropylethylamine (432 mg, 3.35 mmol, 5.0 eq.) was added. The mixture was stirred at room temperature for 5 minutes. Adding (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Imidazole-6-carboxylic acid methyl ester (compound 0116-3) (180 mg, 0.61 mmol, 0.9 eq) and the mixture was stirred at 60 ℃ overnight. The mixture was diluted with ethyl acetate, washed with water and saturated brine. The organic phase was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 5/1 to 1/1) to give (S) -2- ((4- (6-chloropyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a yellow solid]Imidazole-6-carboxylic acid methyl ester (245 mg, yield: 80.3%). Lcms (esi): 455(M + H) is defined as M/z+Under nitrogen protection, (S) -2- ((4- (6-chloropyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] obtained above]A mixture of methyl imidazole-6-carboxylate (120 mg, 0.264 mmol, 1.0 eq), (4-methylbenzofuran-7-yl) methanol (0201-30) (52 mg, 0.317 mmol, 1.2 eq), cesium carbonate (215 mg, 0.66 mmol, 2.5 eq), 2-bicyclohexylphosphine-2 ',6' -diisopropoxybiphenyl (12 mg, 0.026 mmol, 0.1 eq), and tris (dibenzylideneacetone) dipalladium (0) (60 mg, 0.013 mmol, 0.05 eq) in toluene (20 ml) was refluxed at 125 ℃ overnight. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol ═ 33/1) to give (S) -2- ((4- (6- ((4-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a yellow solid ]Imidazole-6-carboxylic acid methyl ester (68 mg, yield: 44.4%). Lcms (esi): m/z 581(M + H)+.
Step 21 c: (S) -2- ((4- (6- ((4-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 30) (S) -2- ((4- (6- ((4-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d [ -d]A mixture of imidazole-6-carboxylic acid methyl ester (0204-30) (68 mg, 0.12 mmol, 1.0 equiv.) and lithium hydroxide monohydrate (10 mg, 0.23 mmol, 2.0 equiv.) in a mixed solvent of 5 ml acetonitrile and 1 ml water was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with dilute sulfuric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water. The mixture was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol ═ 15/1) to give (S) -2- ((4- (6- ((4-methylbenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a yellow solid]Imidazole-6-carboxylic acid (45 mg, yield: 67.1%). Lcms (esi): m/z 567(M + H) +Melting point: 105 to 107 ℃.1H NMR(500MHz,DMSO)δ12.70(s,1H),8.28(s,1H),7.98(d,J=2.2Hz,1H),7.81(d,J=8.1Hz,1H),7.73–7.56(m,2H),7.31(d,J=7.4Hz,1H),7.03(t,J=4.5Hz,2H),6.86(d,J=7.2Hz,1H),6.65(d,J=8.1Hz,1H),5.59(s,2H),5.11(d,J=5.2Hz,1H),4.79(dd,J=15.3,7.2Hz,1H),4.66(dd,J=15.2,2.6Hz,1H),4.46(dd,J=13.7,7.6Hz,1H),4.36(dt,J=9.0,5.9Hz,1H),3.96(s,1H),3.79(s,1H),3.03(s,1H),2.87(s,1H),2.76–2.56(m,2H),2.45(d,J=16.4Hz,4H),2.20(dd,J=29.0,21.6Hz,2H),1.79(d,J=30.1Hz,4H).
Example 22: preparation of (S) -2- ((4- (6- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 31) (preparation according to scheme two line)
Step 22 a: preparation of (2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methanol (compound 0201-31):
a mixture of 2-bromo-5- (trifluoromethyl) phenol (964 mg, 4.0 mmol, 1.0 eq), 3-bromopropyne (619 mg, 5.2 mmol, 1.3 eq) and potassium carbonate (884 mg, 6.4 mmol, 1.6 eq) in N, N-dimethylformamide was stirred at 50 ℃ for 3 hours. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give 1-bromo-2- (prop-2-yn-1-yloxy) -4- (trifluoromethyl) benzene (920 mg, yield: 82.1%) which was used in the next step without further purification.
A mixture of 1-bromo-2- (prop-2-yn-1-yloxy) -4- (trifluoromethyl) benzene (920 mg, 3.3 mmol, 1.0 eq) obtained above and cesium fluoride (752 mg, 4.95 mmol, 14.5 eq) in N, N-diethylaniline was stirred at 220 ℃ for 5 hours under nitrogen. After cooling to room temperature, the mixture was acidified by adding concentrated hydrochloric acid, and extracted with a solvent mixture of ethyl acetate and petroleum ether. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether) to give 7-bromo-2-methyl-4- (trifluoromethyl) benzofuran (650 mg, yield: 70.6%).
A mixture of 7-bromo-2-methyl-4- (trifluoromethyl) benzofuran (580 mg, 2.08 mmol, 1.0 equivalent) obtained above, 1, 3-bis (diphenylphosphino) propane (172 mg, 0.42 mmol, 0.2 equivalent), palladium acetate (95 mg, 0.42 mmol, 0.2 equivalent), and triethylamine (2.1 g, 20.8 mmol, 10.0 equivalent) in a mixed solvent of 25 ml of methanol and 5 ml of N, N-dimethylformamide was added to the pressurized reaction tank. The reactor was charged with 0.3 mpa carbon monoxide and stirred at 80 ℃ overnight. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 50/1 to 20/1) to give methyl 2-methyl-4- (trifluoromethyl) benzofuran-7-carboxylate (521 mg, yield: 97.1%) as a yellow solid.
Methyl 2-methyl-4- (trifluoromethyl) benzofuran-7-carboxylate (521 mg, 2.0 mol, 1.0 eq) obtained above was dissolved in anhydrous tetrahydrofuran under nitrogen protection and the mixture was cooled to 0 ℃. Lithium aluminum hydride (116 mg, 3.0 mmol, 1.5 equivalents) was added and the mixture was stirred for 1.5 hours. 0.12 ml of water, 0.12 ml of a 15% aqueous sodium hydroxide solution and 0.36 ml of water were added in this order at 0 ℃. The mixture was stirred for 15 minutes. 1.5 g of anhydrous sodium sulfate was added, and the mixture was stirred at room temperature for 0.5 hour. The mixture was filtered under reduced pressure. The filtrate was concentrated under reduced pressure to give (2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methanol (507 mg, crude) as a yellow oil. Lcms (esi): 175(M + H-H) in M/z 2O)+.
Step 22 b: preparation of tert-butyl 4- (6- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0202-31): under nitrogen protection, tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (0104-1) (150 mg, 0.51 mmol, 1.0 eq), (2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methanol (0201-31) (140 mg, 0.61 mmol, 1.2 eq), cesium carbonate (416 mg, 1.28 mmol, 2.5 eq), 2-dicyclohexylphosphine-2 ',6' -diisopropoxybiphenyl (36 mg )0.077 mmol, 0.15 equiv) and tris (dibenzylideneacetone) dipalladium (0) (42 mg, 0.046 mmol, 0.09 equiv) in toluene (50 ml) were refluxed at 125 ℃ overnight. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on preparative silica gel (eluent: petroleum ether/ethyl acetate 20/1 to 6/1) to give tert-butyl 4- (6- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (220 mg, yield: 88.0%) as a yellow solid. Lcms (esi): 491(M + H)+.
Step 22 c: preparation of 2- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine hydrochloride (compound 0203-31): to a mixture of tert-butyl 4- (6- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-31) (220 mg, 0.45 mmol, 1.0 eq) in 5 ml dioxane was added 1 ml of a 4M hydrogen chloride in dioxane solution. The mixture was stirred at room temperature overnight. The mixture was concentrated to dryness under reduced pressure to give 2- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine hydrochloride (250 mg, crude) as a white solid. Lcms (esi): m/z 391(M + H) +.
Step 22 d: (S) -2- ((4- (6- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-31): 2- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) -6- (piperidin-4-yl) pyridine hydrochloride (0203-21) (250 mg, 0.45 mmol, 2.6 equivalents) was added to 10 mL of N-methylpyrrolidinone, followed by N, N-diisopropylethylamine (171 mg, 1.25 mmol, 7.3 equivalents). The mixture was stirred at room temperature for 5 minutes. Adding (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (0116-3) (50 mg, 0.17 mmol, 1.0 equiv) and the mixture is stirred at 60 ℃ overnight. The mixture was diluted with ethyl acetate, washed with water and saturated brine. Concentrating the organic phase under reduced pressure, and purifying the residue by preparative thin-layer chromatography (eluent: stone)Oleyl ether/ethyl acetate ═ 2/1) to give (S) -2- ((4- (6- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a yellow solid]Imidazole-6-carboxylic acid methyl ester (74 mg, yield: 67.2%). M/z 649(M + H) +.
Step 22 e: (S) -2- ((4- (6- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 31) (S) -2- ((4- (6- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d [ -d]A mixture of imidazole-6-carboxylic acid methyl ester (0204-31) (74 mg, 0.11 mmol, 1.0 equiv.) and lithium hydroxide monohydrate (12 mg, 0.8 mmol, 2.5 equiv.) in a mixed solvent of 5 ml acetonitrile and 1 ml water was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. Dilute sulfuric acid was added to adjust the pH of the residue to 6, ethyl acetate was added for extraction, and the mixture was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure and the residue was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol-10/1) to give (S) -2- ((4- (6- ((2-methyl-4- (trifluoromethyl) benzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (55 mg, yield: 75.3%). Lcms (esi): m/z 635(M + H) +Melting point: 126-128 ℃.1H NMR(500MHz,DMSO)δ12.72(s,1H),8.27(s,1H),7.81(dd,J=8.4,1.4Hz,1H),7.64(t,J=7.8Hz,2H),7.54(d,J=7.9Hz,1H),7.47(d,J=7.8Hz,1H),6.87(d,J=7.3Hz,1H),6.78–6.67(m,2H),5.68(s,2H),5.10(qd,J=7.2,2.8Hz,1H),4.78(dd,J=15.2,7.2Hz,1H),4.64(dd,J=15.2,2.6Hz,1H),4.45(dt,J=13.9,7.0Hz,1H),4.35(dt,J=9.0,5.9Hz,1H),3.95(d,J=13.6Hz,1H),3.78(d,J=13.6Hz,1H),2.98(d,J=10.6Hz,1H),2.85(d,J=10.9Hz,1H),2.73–2.64(m,1H),2.62–2.54(m,1H),2.49–2.39(m,4H),2.21(dt,J=21.7,10.6Hz,2H),1.81–1.62(m,4H).
Example 23: preparation of (S) -2- ((4- (6- ((5-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 32) (four-way preparation according to scheme)
Step 23 a: preparation of (5-fluorobenzofuran-6-yl) methanol (compound 0401-32):
to a solution of methyl 2-fluoro-5-hydroxybenzoate (2.0 g, 11.76 mmol, 1.0 eq) in acetic acid (20 ml) under nitrogen was added N-iodosuccinimide (2.9 g, 12.94 mmol, 1.1 eq) and the mixture was stirred at room temperature overnight. The reaction solution was poured into water and stirred for 30 minutes. The mixture was filtered, and the filter cake was washed with water and dried under reduced pressure to give methyl 2-fluoro-5-hydroxy-4-iodobenzoate (2.7 g, yield: 77.59%) as a white solid. Lcms (esi): m/z 297(M + H) +.
To a solution of methyl 2-fluoro-5-hydroxy-4-iodobenzoate (2.7 g, 9.12 mmol, 1.0 eq), triethylamine (2.76 g, 27.36 mmol, 3.0 eq), cuprous iodide (26 mg, 0.137 mmol, 0.02 eq) and bis (triphenylphosphine) palladium dichloride (192 mg, 0.274 mmol, 0.03 eq) in tetrahydrofuran (20 ml) obtained above was added trimethylethynylsilane (1.79 g, 18.24 mmol, 2.0 eq) under nitrogen protection and the mixture was stirred at 70 ℃ overnight. After cooling to room temperature, the solvent was removed under reduced pressure and methanol (20 ml), diisopropylethylamine (3.5 g, 27.36 mmol, 3.0 eq) and cuprous iodide (1.7 g, 9.12 mmol, 1.0 eq) were added. The mixture was refluxed for 2 hours. After cooling to room temperature, the mixture was poured into water. A PE/EA solution of 2/1 was added to the mixture to conduct extraction, and the organic layer was concentrated under reduced pressure. Tetrahydrofuran (20 ml) and tetrabutylammonium fluoride trihydrate (1.19 g, 4.56 mmol, 0.5 eq) were added and the mixture refluxed for 1 hour. After cooling to room temperature, the mixture was washed with water and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1 to 20/1) to give methyl 5-fluorobenzofuran-6-carboxylate (1 g, yield: 55.87%) as a yellow solid. Lcms (esi): m/z=195(M+H)+.
Lithium aluminum hydride (94.3 mg, 2.48 mmol, 2 equiv.) was added to a solution of methyl 5-fluorobenzofuran-6-carboxylate (241 mg, 1.24 mmol, 1.0 equiv.) in tetrahydrofuran (20 ml) under nitrogen. The mixture was stirred at room temperature for 3 hours. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 20/1 to 5/1) to give (5-fluorobenzofuran-6-yl) methanol (200 mg, yield: 97.08%) as a yellow solid. Lcms (esi): 167(M + H)+.
Step 23 b: preparation of tert-butyl 4- (6- ((5-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0402-32): under nitrogen, a mixture of tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (0104-1) (297 mg, 1.0 mmol, 1.0 eq), (5-fluorobenzofuran-6-yl) methanol (0401-32(200 mg, 1.2 mmol, 1.2 eq), cesium carbonate (652 mg, 2.0 mmol, 2.0 eq), 2-dicyclohexylphosphonium-2 ',6' -diisopropoxy-1, 1' -biphenyl (46.7 mg, 0.1 mmol, 0.1 eq) and tris (dibenzylideneacetone) dipalladium (0) (45.8 mg, 0.05 mmol, 0.05 eq) in toluene (10 ml) was refluxed overnight at 125 ℃ 20/1-5/1) to give tert-butyl 4- (6- ((5-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate as a yellow oil (250 mg, yield: 58.55%). Lcms (esi): m/z 427(M + H) +.
Step 23 c: preparation of 2- ((5-fluorobenzofuran-6-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (compound 0403-32): to a solution of tert-butyl 4- (6- ((5-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0402-32) (250 mg, 0.587 mmol, 1.0 eq) in ethyl acetate (10 ml) was added p-toluenesulfonic acid monohydrate (303 mg, 1.76 mmol, 3 eq). The mixture is in 6Stir at 0 ℃ overnight. The mixture was filtered. The residue was washed with ethyl acetate. The residue was dried to give 2- ((5-fluorobenzofuran-6-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate as a white solid (254 mg, yield: 86.99%). lcms (esi): m/z 327(M + H)+.
Step 23 d: (S) -2- ((4- (6- ((5-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of methyl imidazole-6-carboxylate (Compound 0404-32) 2- ((5-Fluorobenzofuran-6-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (0403-32) (203 mg, 0.407 mmol, 1.5 equivalents), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] (S-O-R-O-N-methyl ester)]A solution of methyl imidazole-6-carboxylate (0116-3) (80 mg, 0.27 mmol, 1.0 eq) and potassium carbonate (149 mg, 1.08 mmol, 4.0 eq) in acetonitrile (10 ml) was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, and water was added thereto, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain white oily substance (S) -2- ((4- (6- ((5-fluorobenzofuran-6-yl) methoxyl) pyridine-2-yl) piperidine-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Imidazole-6-carboxylic acid methyl ester (100 mg, yield: 63.29%). Lcms (esi): 585(M + H) with M/z+.
Step 23 e: (S) -2- ((4- (6- ((5-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 32) (S) -2- ((4- (6- ((5-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d [, e]A mixture of imidazole-6-carboxylic acid methyl ester (0404-32) (100 mg, 0.17 mmol, 1.0 eq) and lithium hydroxide monohydrate (11 mg, 0.26 mmol, 1.5 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l hydrochloric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water and dried under reduced pressure to give (S) -2- ((4- (6- ((5-fluorobenzofuran) as a white solid-6-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (70 mg, yield: 72.16%). Lcms (esi): 571(M + H) M/z+.1H NMR(500MHz,DMSO)δ8.25(s,1H),8.01(d,J=2.2Hz,1H),7.80(dd,J=8.4,1.4Hz,1H),7.76(d,J=5.7Hz,1H),7.62(dd,J=12.2,4.8Hz,2H),7.49(d,J=9.9Hz,1H),6.94(dd,J=2.1,0.8Hz,1H),6.86(d,J=7.3Hz,1H),6.67(d,J=8.1Hz,1H),5.46(s,2H),5.11(qd,J=7.3,2.8Hz,1H),4.80(dd,J=15.2,7.2Hz,1H),4.66(dd,J=15.2,2.6Hz,1H),4.46(dt,J=13.9,7.0Hz,1H),4.37(dt,J=9.0,5.9Hz,1H),3.95(d,J=13.5Hz,1H),3.78(d,J=13.5Hz,1H),3.00(d,J=11.2Hz,1H),2.86(d,J=11.3Hz,1H),2.74–2.66(m,1H),2.62(tt,J=11.4,4.0Hz,1H),2.48–2.40(m,1H),2.29–2.22(m,1H),2.22–2.14(m,1H),1.85–1.69(m,4H).
Example 24: preparation of (S) -2- ((4- (6- ((6-fluorobenzofuran-5-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b ] pyridine-5-carboxylic acid (compound 33) (prepared according to the five-line scheme)
Step 24 a: preparation of (6-fluorobenzofuran-5-yl) methanol (compound 0501-33):
to a solution of 2-fluoro-4-hydroxybenzaldehyde (5.0 g, 35.7 mmol, 1.0 eq) in concentrated sulfuric acid (30 ml) was added N-iodosuccinimide (8.84 g, 39.3 mmol, 1.1 eq) and the mixture was stirred at room temperature for 3 hours. The reaction solution was poured into ice water and stirred for 1 hour. The mixture was filtered and the filter cake was washed with water and dried under reduced pressure to give a crude product of 2-fluoro-4-hydroxy-5-iodobenzaldehyde (5.6 g, crude) which was used in the next step without further purification.
2-fluoro-4-hydroxy-5-iodobenzaldehyde (5.6 g, 20.8 mmol, 1.0 eq), triethylamine (6.3 g, 62.4 mmol, 3.0 eq), cuprous iodide (59.3 mg, 0.31 mmol, 0.02 eq) and bis (triphenylphosphine) palladium dichloride (438 mg) obtained as described above under nitrogen0.62 mmol, 0.03 equiv) of tetrahydrofuran (50 ml) was added trimethylethynylsilane (4.08 g, 41.6 mmol, 2.0 equiv) and the mixture was stirred at 70 ℃ overnight. After cooling to room temperature, the solvent was removed under reduced pressure and methanol (150 ml), diisopropylethylamine (8.06 g, 62.4 mmol, 3.0 eq) and cuprous iodide (4.0 g, 20.8 mmol, 1.0 eq) were added. The mixture was refluxed for 2 hours. After cooling to room temperature, the mixture was poured into water. A PE/EA solution of 2/1 was added to the mixture to conduct extraction, and the organic layer was concentrated under reduced pressure. Tetrahydrofuran (50 ml) and tetrabutylammonium fluoride trihydrate (2.7 g, 10.4 mmol, 0.5 eq) were added and the mixture refluxed for 1 hour. After cooling to room temperature, the mixture was washed with water and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1 to 20/1) to give 6-fluorobenzofuran-5-carbaldehyde (2.1 g, yield: 61.19%) as a yellow solid. Lcms (esi): m/z 165(M + H) +.
To a solution of 6-fluorobenzofuran-5-carbaldehyde (1 g, 6.10 mmol, 1.0 eq) in methanol (25 ml) was added sodium borohydride (277 mg, 7.32 mmol, 1.2 eq). The mixture was stirred at room temperature for 1 hour. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give (6-fluorobenzofuran-5-yl) methanol (947 mg, yield: 93.58%) as a yellow solid. Lcms (esi): 167(M + H)+.
Step 24 b: 4- (6-Preparation of tert-butyl ((6-fluorobenzofuran-5-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0502-33): under nitrogen, a mixture of tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (0104-1) (350 mg, 1.18 mmol, 1.0 eq), (6-fluorobenzofuran-5-yl) methanol (0501-33) (235 mg, 1.416 mmol, 1.2 eq), cesium carbonate (769 mg, 2.36 mmol, 2.0 eq), 2-dicyclohexylphosphonium-2 ',6' -diisopropoxy-1, 1' -biphenyl (55 mg, 0.118 mmol, 0.1 eq), and tris (dibenzylideneacetone) dipalladium (0) (54 mg, 0.059 mmol, 0.05 eq) in toluene (10 ml) was refluxed at 125 ℃ overnight. After cooling to room temperature, the reaction mixture was passed through a celite The soil was filtered and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1-5/1) to give 4- (6) as a yellow oil-Tert-butyl ((6-fluorobenzofuran-5-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (450 mg, yield: 89.64%). Lcms (esi): m/z 427(M + H)+.
Step 24 c: preparation of 2- ((6-fluorobenzofuran-5-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (compound 0503-33): to a solution of tert-butyl 4- (6- ((6-fluorobenzofuran-5-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0502-33) (450 mg, 1.06 mmol, 1.0 eq) in ethyl acetate (10 ml) was added p-toluenesulfonic acid monohydrate (546 mg, 3.17 mmol, 3 eq). The mixture was stirred at 60 ℃ overnight. The mixture was filtered. The residue was washed with ethyl acetate. The residue was dried to give 2- ((6-fluorobenzofuran-5-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate as a white solid (510 mg, yield: 96.59%). lcms (esi): m/z 327(M + H)+.
Step 24 d: (S) -2- ((4- (6- ((6-fluorobenzofuran-5-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b ]Preparation of pyridine-5-carboxylic acid methyl ester (Compound 0504-33) 2- ((6-fluorobenzofuran-5-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (0503-33) (127 mg, 0.255 mmol, 1.5 equiv), (S) -2- (chloromethyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4, 5-b)]A solution of methyl pyridine-5-carboxylate (0116-1) (50 mg, 0.17 mmol, 1.0 equiv.) and potassium carbonate (94 mg, 0.68 mmol, 4.0 equiv.) in acetonitrile (10 ml) was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, water was added thereto, and ethyl acetate was added for extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain white solid (S) -2- ((4- (6- ((6-fluorobenzofuran-5-yl) methoxyl) pyridine-2-yl) piperidine-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4, 5-b)]Pyridine-5-carboxylic acid methyl ester (66 mg, yield: 66.37%). Lcms (esi): m/z 586(M + H)+.
Step 24 e: (S) -2- ((4- (6- ((6-fluorobenzofuran-5-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b]Preparation of pyridine-5-carboxylic acid (Compound 33) (S) -2- ((4- (6- ((6-fluorobenzofuran-5-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4,5-b ]Pyridine-5-carboxylic acid methyl ester) 0504-33) (66 mg, 0.11 mmol, 1.0 eq) and lithium hydroxide monohydrate (5 mg, 0.12 mmol, 1.1 eq) in acetonitrile/water 5/1(6 ml) were stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l hydrochloric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water and dried under reduced pressure to give (S) -2- ((4- (6- ((6-fluorobenzofuran-5-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4, 5-b) as a white solid]Pyridine-5-carboxylic acid (40 mg, yield: 63.49%). Lcms (esi): m/z 572(M + H)+.1H NMR(500MHz,DMSO)δ8.04(d,J=8.1Hz,1H),7.97(dd,J=14.1,5.2Hz,2H),7.81(d,J=7.3Hz,1H),7.66–7.53(m,2H),6.97–6.91(m,1H),6.86(d,J=7.3Hz,1H),6.65(d,J=8.2Hz,1H),5.44(s,2H),5.03(d,J=4.6Hz,1H),4.87(dd,J=14.7,7.4Hz,1H),4.69(dd,J=14.7,3.0Hz,1H),4.41(dd,J=14.0,7.1Hz,1H),4.32(dt,J=12.2,6.1Hz,1H),4.05(d,J=13.6Hz,1H),3.82(d,J=13.6Hz,1H),3.00(d,J=11.2Hz,1H),2.87(d,J=11.2Hz,1H),2.61(td,J=11.0,5.5Hz,1H),2.50(s,1H),2.37–2.16(m,3H),1.88–1.66(m,4H).
Example 25: preparation of (S) -2- ((4- (6- ((7-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 44) (six-way preparation according to scheme)
Step 25 a: preparation of (7-fluorobenzofuran-6-yl) methanol (Compound 0601-44)
A mixture of 2-fluoro-3-methylphenol (2.5 g, 19.84 mmol, 1.0 eq), 2-bromo-1, 1-diethoxyethane (4.69 g, 23.81 mmol, 1.2 eq), potassium carbonate (4.11 g, 21.4 mmol, 1.5 eq) in N, N-dimethylformamide (50 ml) was stirred at 120 ℃ overnight under nitrogen. After cooling to room temperature, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by a silica gel column (petroleum ether/ethyl acetate ═ 10/1 to 5/1) to give 1- (2, 2-diethoxyethoxy) -2-fluoro-3-methylbenzene (4.78 g, yield: 99.6%) as a yellow oily product.
To a solution of polyphosphoric acid (15.89 g, 47.00 mmol, 2.5 eq) in toluene (100 ml) under nitrogen blanket was added 1- (2, 2-diethoxyethoxy) -2-fluoro-3-methylbenzene (4.55 g, 18.80 mmol, 1.0 eq) and the mixture was stirred at 120 ℃ overnight. After cooling to room temperature, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether) to give 7-fluoro-6-methylbenzofuran (2.08 g, yield: 70.2%) as a yellow oily product.
A solution of 7-fluoro-6-methylbenzofuran (2.0 g, 13.33 mmol, 1.0 equiv.), N-bromosuccinimide (2.85 g, 15.99 mmol, 1.2 equiv.), and azobisisobutyronitrile (437 mg, 2.67 mmol, 0.2 equiv.) in 1, 2-dichloroethane (25 mL) was stirred at 72 ℃ overnight under nitrogen. The reaction solution was diluted with water and extracted with ethyl acetate, and the organic phase was dried and concentrated under reduced pressure. The residue was purified by a silica gel column (eluent: petroleum ether) to give 6- (bromomethyl) -7-fluorobenzofuran (2.13 g, yield: 69.8%) as a white solid product.
A mixture of 6- (bromomethyl) -7-fluorobenzofuran (2.03 g, 8.86 mmol, 1.0 eq) and potassium acetate (8.7 g, 88.6 mmol, 10.0 eq) in N, N-dimethylformamide (40 ml) was stirred at room temperature for 3 hours. The reaction solution was diluted with water and extracted with ethyl acetate, and the organic phase was dried and concentrated under reduced pressure. The residue was used directly in the next step without further purification.
To a solution of acetic acid (7-fluorobenzofuran-6-yl) methyl ester (1.87 g, 8.99 mmol, 1.0 eq) in tetrahydrofuran was added a solution of sodium methoxide in methanol (5.4 mol/l, 3.33 ml, 17.98 mmol, 2.0 eq) under nitrogen, and the mixture was stirred at room temperature for 1.0 h. The reaction solution was diluted with water and extracted with ethyl acetate, and the organic phase was dried and concentrated under reduced pressure. The residue was purified by a silica gel column (petroleum ether/ethyl acetate ═ 15/1 to 5/1) to give the product (7-fluorobenzofuran-6-yl) methanol (1.2 g, yield: 80.4%) as a yellow solid.
Step 25 b: preparation of tert-butyl 4- (6- ((7-fluorophenylfuran-6-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (Compound 0602-44) under nitrogen protection, tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (0104-1) (178.8 mg, 0.602 mmol, 1.0 eq), (7-fluorophenylfuran-6-yl) methanol (0601-44) (120 mg, 0.722 mmol, 1.2 eq), cesium carbonate (391 mg, 1.2 mmol, 2.0 eq), 2-dicyclohexylphosphonium-2 ',6' -diisopropoxy-1, 1' -biphenyl (56 mg, 0.12 mmol, 0.2 eq) and tris (dibenzylideneacetone) dipalladium (0) (55 mg, 0.06 mmol, 0.1 eq) of toluene (10 ml) was stirred at 120 ℃ overnight. The reaction solution was diluted with water and extracted with ethyl acetate, and the organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column (petroleum ether/ethyl acetate ═ 20/1 to 10/1) to give the product tert-butyl 4- (6- ((7-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (254 mg, yield: 98.8%) as a yellow solid. Lcms (esi): m/z 427[ M +1] +.
Step 25 c: preparation of 2- ((7-fluorobenzofuran-6-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (compound 0603-44): to a solution of tert-butyl 4- (6- ((7-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0602-44) (254 mg, 0.596 mmol, 1.0 eq) in ethyl acetate (10 ml) was added p-toluenesulfonic acid monohydrate (206 mg, 1.19 mmol, 2.0 eq) and the mixture was stirred at 60 ℃ overnight. The reaction was concentrated under reduced pressure, and the residue was used in the next step without further purification. Lcms (esi): m/z 327(M + H)+.
Step 25 d: (S) -2- ((4- (6- ((7-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidine-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of methyl imidazole-6-carboxylate (Compound 0604-44) 2- ((7-fluorobenzofuran-6-yl) methoxy) -6- (piperidin-4-yl) pyridine 4-methylbenzenesulfonate (0603-44) (72 mg, 0.22 mmol, 1.3 equivalents), (S) -2- (chloromethyl) -3- (oxetan-2-ylmethyl) -1H-benzo [ d]A solution of methyl imidazole-6-carboxylate (0116-3) (50 mg, 0.169 mmol, 1.0 eq) and potassium carbonate (93.5 mg, 0.678 mmol, 4.0 eq) in acetonitrile (10 ml) was stirred at 60 ℃ overnight. The reaction solution was diluted with water and extracted with ethyl acetate, and the organic phase was dried and concentrated under reduced pressure. The residue was purified by preparative thin layer chromatography (dichloromethane/methanol ═ 16/1) to give the product (S) -2- ((4- (6- ((7-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] ne as a white solid ]Imidazole-6-carboxylic acid methyl ester (89 mg, yield: 90.3%). Lcms (esi): 584(M + H)+.
Step 25 e: (S) -2- ((4- (6- ((7-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 44) (S) -2- ((4- (6- ((7-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d [ -d]A mixture of imidazole-6-carboxylic acid methyl ester (0704-44) (89 mg, 0.147 mmol, 1.0 eq) and lithium hydroxide monohydrate (16.3 mg, 0.388 mmol, 2.5 eq) in acetonitrile/water 5/1(12 ml) was stirred at 40 ℃ overnight. The reaction solution was adjusted to pH 6 with 2 mol/l hydrochloric acid, extracted with ethyl acetate, and the resulting organic phase was dried and concentrated under reduced pressure to give (S) -2- ((4- (6- ((7-fluorobenzofuran-6-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] ne]Imidazole-6-carboxylic acid (76 mg, yield: 87.7%). Lcms (esi): 571(M + H) M/z+.1H NMR(500MHz,DMSO)δ12.88(s,1H),8.36(s,1H),8.10(d,J=1.6Hz,1H),7.89(d,J=8.3Hz,1H),7.79(d,J=8.2Hz,1H),7.68(t,J=7.7Hz,1H),7.53–7.41(m,2H),7.12–7.04(m,1H),6.92(d,J=5.6Hz,1H),6.72(d,J=8.2Hz,1H),5.54(s,2H),5.05(d,J=5.9Hz,1H),4.84(dd,J=15.4,6.9Hz,2H),4.70(d,J=14.6Hz,1H),4.49(dd,J=13.5,7.2Hz,1H),4.35(dd,J=14.7,5.9Hz,1H),3.81(s,2H),3.35(s,3H),2.96(s,1H),2.71(dt,J=16.3,8.0Hz,1H),2.35(d,J=7.8Hz,1H),2.29–2.01(m,4H).
Example 26: preparation of (S) -2- ((6- ((4-cyano-3-methylbenzofuran-7-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridinyl ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 46) (preparation according to scheme three line)
Step 26 a: preparation of 7- (hydroxymethyl) -3-methylbenzofuran-4-carbonitrile (Compound 0201-46):
a mixture of methyl 4-bromo-2-hydroxybenzoate (1.0 g, 4.33 mmol, 1.0 eq), 1-bromopropan-2-one (711 mg, 5.19 mmol, 1.2 eq), and potassium carbonate (896 mg, 6.49 mmol, 1.5 eq) in N, N-dimethylformamide was stirred at room temperature for 1 hour under nitrogen. The reaction was quenched with water and a solid precipitated. The mixture was filtered. The residue was washed with water and dried to give a crude product of methyl 4-bromo-2- (2-oxopropoxy) benzoate (1 g, crude). The product was used in the next step without further purification. Lcms (esi): 287(M + H)+.
A mixture of methyl 4-bromo-2- (2-oxopropoxy) benzoate (1 g, 3.48 mmol, 1.0 eq.) and polyphosphoric acid (1.76 g, 5.23 mmol, 1.5 eq.) in dichloroethane was refluxed overnight under nitrogen blanket. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether) to give methyl 4-bromo-3-methylbenzofuran-7-carboxylate (357 mg, yield: 38.26%) as a yellow solid. Lcms (esi): m/z 269(M + H) +.
Methyl 4-bromo-3-methylbenzofuran-7-carboxylate (320 mg, 1.19 mmol, 1.0 eq), palladium tetrakistriphenylphosphine (137 mg, 0.1) under nitrogen blanket2 mmol, 0.1 equiv) and zinc cyanide (208 mg, 1.78 mmol, 1.5 equiv) were stirred in N, N-dimethylformamide (5 ml) at 90 ℃ overnight. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-10/1) to give methyl 4-cyano-3-methylbenzofuran-7-carboxylate (242 mg, yield: 95.01%) as a yellow solid. Lcms (esi): m/z 216(M + H)+.
To a mixture of lithium aluminum tetrahydride (55 mg, 1.44 mmol, 1.2 eq) in tetrahydrofuran under nitrogen and ice bath was added methyl 4-cyano-3-methylbenzofuran-7-carboxylate (259 mg, 1.2 mmol, 1.0 eq). The mixture was stirred for 1 hour under ice bath. The reaction was quenched with water. The mixture was dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-2/1) to give 7- (hydroxymethyl) -3-methylbenzofuran-4-carbonitrile as a yellow solid (135 mg, yield: 60%). Lcms (esi): 188(M + H) M/z +.
Step 26 b: 6- ((4-cyano-3-methylbenzofuran-7-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine]Preparation of 1'(2' H) -carboxylic acid tert-butyl ester (Compound 0303-46): under the protection of nitrogen, 6-chloro-3 ',6' -dihydro- [2,4' -bipyridine]A mixture of tert-butyl (0302-2) (174 mg, 0.59 mmol, 1.2 eq), 7- (hydroxymethyl) -3-methylbenzofuran-4-carbonitrile (0201-46) (92 mg, 0.492 mmol, 1.0 eq), cesium carbonate (320 mg, 0.984 mmol, 2.0 eq), 2-dicyclohexylphosphorus-2 ',6' -diisopropoxy-1, 1' -biphenyl (92 mg, 0.197 mmol, 0.4 eq), and tris (dibenzylideneacetone) dipalladium (0) (89 mg, 0.098 mmol, 0.2 eq) in toluene (10 ml) at 125 ℃ under reflux overnight. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-10/1) to give 6- ((4-cyano-3-methylbenzofuran-7-yl) methoxy group as a yellow oil) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -carboxylic acid tert-butyl ester (123 mg, yield: 56.16%). Lcms (esi): m/z 446(M + H) +.
Step 26 c: 3-methyl-7- (((1', 2', 3',6' -tetrahydro- [2,4' -bipyridine)]]Preparation of (E) -6-yl) oxy) methyl) benzofuran-4-carbonitrile hydrochloride (compound 0304-46): reacting 6- ((4-cyano-3-methylbenzofuran-7-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -carboxylic acid tert-butyl ester (0303-46) (123 mg, 0.276 mmol, 1.0 equiv.) and a dioxane (4 mL) solution of hydrochloric acid in dioxane (4M, 1 mL) was stirred at room temperature overnight. The mixture was concentrated under reduced pressure to give crude 3-methyl-7- (((1', 2', 3',6' -tetrahydro- [2,4' -bipyridine)]]-6-yl) oxy) methyl) benzofuran-4-carbonitrile hydrochloride (105 mg, crude). The product was used in the next step without further purification. Lcms (esi): 346(M + H)+.
Step 26 d: (S) -2- ((6- ((4-cyano-3-methylbenzofuran-7-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of methyl imidazole-6-carboxylate (Compound 0305-46) 3-methyl-7- (((1', 2', 3',6' -tetrahydro- [2,4' -bipyridine)]]-6-yl) oxy) methyl) benzofuran-4-carbonitrile hydrochloride (0304-46) (105 mg, 0.276 mmol, 1.5 equivalents), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] ]A solution of methyl imidazole-6-carboxylate (0116-3) (54 mg, 0.184 mmol, 1.0 eq) and N, N-diisopropylethylamine (95 mg, 0.736 mmol, 4.0 eq) in N-methylpyrrolidone (5 ml) was stirred at 60 ℃ for 16 h. After cooling to room temperature, water was added, and ethyl acetate was added for extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain yellow solid (S) -2- ((6- ((4-cyano-3-methylbenzofuran-7-yl) methoxyl) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (80 mg, yield: 72.26%). Lcms (esi): 604(M + H)+.
Step 26 e: (S) -2- ((6- ((4-cyano-3-methylbenzofuran-7-yl) methoxy) -3'6 '-dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 46) from (S) -2- ((6- ((4-cyano-3-methylbenzofuran-7-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0305-46) (80 mg, 0.13 mmol, 1.0 eq) and lithium hydroxide monohydrate (11 mg, 0.26 mmol, 2.0 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l hydrochloric acid, and a solid precipitate was formed. The mixture was filtered. The residue was washed with water. The mixture was slurried with methanol to give (S) -2- ((6- ((4-cyano-3-methylbenzofuran-7-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine) as a yellow solid ]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (66 mg, yield: 78.14%). Lcms (esi): 590(M + H) M/z+.1H NMR(500MHz,DMSO)δ8.25(s,1H),8.05(s,1H),7.82(d,J=8.4Hz,1H),7.76–7.60(m,3H),7.50(d,J=7.8Hz,1H),7.06(d,J=7.5Hz,1H),6.76(d,J=8.2Hz,1H),6.61(s,1H),5.70(s,2H),5.05(qd,J=7.2,2.8Hz,1H),4.78(dd,J=15.2,7.2Hz,1H),4.63(d,J=13.0Hz,1H),4.46(dd,J=13.7,7.5Hz,1H),4.35(dt,J=8.9,5.9Hz,1H),4.05(d,J=13.6Hz,1H),3.91(d,J=13.5Hz,1H),3.19(d,J=12.6Hz,2H),2.65(ddd,J=17.7,14.8,12.1Hz,3H),2.45–2.31(m,6H).
Example 27: preparation of (S) -2- ((4- (6- ((7-cyanobenzofuran-4-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 47) (preparation according to scheme seven line)
Step 27 a: preparation of 4- (hydroxymethyl) benzofuran-7-carbonitrile (Compounds 0701-47):
4-methylbenzofuran-7-carbonitrile (960 mg, 6.11 mmol, 1.0 eq.) N-bromosuccinimide under Nitrogen blanketingA solution of (1300 mg, 7.33 mmol, 1.2 eq) and azobisisobutyronitrile (200 mg, 1.22 mmol, 0.2 eq) in dichloroethane (10 ml) was stirred at 72 ℃ overnight. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 200/1-10/1) to give 4- (bromomethyl) benzofuran-7-carbonitrile (968 mg, yield: 67.22%) as a white solid. Lcms (esi): 236(M + H) M/z +.
A mixture of 4- (bromomethyl) benzofuran-7-carbonitrile (968 mg, 4.1 mmol, 1.0 eq) and potassium acetate (4 g, 41 mmol, 10.0 eq) in N, N-dimethylformamide (10 ml) was stirred at room temperature for 3 h. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give a crude product of (7-cyanobenzofuran-4-yl) methyl acetate (766 mg, crude). The product was used in the next step without further purification.
To a solution of (7-cyanobenzofuran-4-yl) methyl acetate (766 mg, 3.56 mmol, 1.0 eq) in tetrahydrofuran (5 ml) was added a solution of sodium methoxide in methanol (5.4 mol/l, 1.32 ml, 7.12 mmol, 2.0 eq). The mixture was stirred at room temperature for 1 hour. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 200/1-2/1) to give 4- (hydroxymethyl) benzofuran-7-carbonitrile (968 mg, yield: 66.23%) as a yellow solid. Lcms (esi): m/z-174 (M + H) +.
Step 27 b: preparation of tert-butyl 4- (6- ((7-cyanobenzofuran-4-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0702-47): 4- (6-Chloropyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester (0104-1) (178 mg, 0.6 mmol, 1.2 eq), 4- (hydroxymethyl) benzofuran-7-carbonitrile (0701-47(87 mg, 0.5 mmol, 1.0 eq), cesium carbonate (325 mg), under nitrogen protectionA mixture of 1 mmol, 2.0 equivalents), 2-dicyclohexylphosphonium-2 ',6' -diisopropoxy-1, 1' -biphenyl (93 mg, 0.2 mmol, 0.4 equivalents) and tris (dibenzylideneacetone) dipalladium (0) (91 mg, 0.1 mmol, 0.2 equivalents) in toluene (10 ml) was stirred at 125 ℃ overnight. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-10/1) to give 4- (6- ((7-cyanobenzofuran-4-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester as a yellow oil (94 mg, yield: 43.32%). Lcms (esi): m/z 434(M + H)+.
Step 27 c: preparation of 4- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzofuran-7-carbonitrile hydrochloride (compound 0703-47): a mixture of tert-butyl 4- (6- ((7-cyanobenzofuran-4-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0702-47) (94 mg, 0.217 mmol, 1.0 eq) and dioxane (4 ml) as a solution of hydrochloric acid in dioxane (4M, 1 ml) was stirred at room temperature overnight. The mixture was concentrated under reduced pressure to give crude 4- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzofuran-7-carbonitrile hydrochloride (100 mg, crude). Lcms (esi): m/z 334(M + H) +.
Step 27 d: (S) -2- ((4- (6- ((7-cyanobenzofuran-4-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of methyl imidazole-6-carboxylate (Compound 0704-47) 4- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) benzofuran-7-carbonitrile hydrochloride (0703-47) (80 mg, 0.217 mmol, 1.3 equiv), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ], (S)]A solution of methyl imidazole-6-carboxylate (0116-3) (50 mg, 0.17 mmol, 1.0 equiv.) and N, N-diisopropylethylamine (88 mg, 0.68 mmol, 4.0 equiv.) in N-methylpyrrolidone (5 ml) was stirred at 60 deg.C for 16 h. After cooling to room temperature, water was added, and ethyl acetate was added for extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain yellow(S) -2- ((4- (6- ((7-cyanobenzofuran-4-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a colored solid]Imidazole-6-carboxylic acid methyl ester (40 mg, yield: 40%). Lcms (esi): m/z 592(M + H)+.
Step 27 e: (S) -2- ((4- (6- ((7-cyanobenzofuran-4-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Preparation of imidazole-6-carboxylic acid (Compound 47) (S) -2- ((4- (6- ((7-cyanobenzofuran-4-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d [, e]A mixture of methyl imidazole-6-carboxylate (0704-47) (40 mg, 0.068 mmol, 1.0 eq) and lithium hydroxide monohydrate (6 mg, 0.136 mmol, 2.0 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l hydrochloric acid, and a solid precipitate was formed. The mixture was filtered. The residue was washed with water. The mixture was slurried with methanol and filtered to give (S) -2- ((4- (6- ((7-cyanobenzofuran-4-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid]Imidazole-6-carboxylic acid (33 mg, yield: 84.61%). Lcms (esi): m/z 578(M + H)+.1HNMR(500MHz,DMSO)δ8.26(d,J=1.0Hz,1H),8.22(d,J=2.2Hz,1H),7.84–7.77(m,2H),7.64(dd,J=8.0,6.9Hz,2H),7.47(d,J=7.8Hz,1H),7.28(d,J=2.2Hz,1H),6.87(d,J=7.3Hz,1H),6.73(d,J=8.1Hz,1H),5.69(s,2H),5.10(ddd,J=14.5,7.2,2.8Hz,1H),4.78(dd,J=15.2,7.2Hz,1H),4.65(dd,J=15.2,2.8Hz,1H),4.46(td,J=7.8,5.9Hz,1H),4.36(dt,J=9.0,5.9Hz,1H),3.93(d,J=13.5Hz,1H),3.77(d,J=13.5Hz,1H),2.96(d,J=11.4Hz,1H),2.83(d,J=11.3Hz,1H),2.73–2.64(m,1H),2.56(tt,J=11.8,3.7Hz,1H),2.46–2.38(m,1H),2.18(ddd,J=31.6,11.8,9.6Hz,2H),1.74(t,J=13.5Hz,2H),1.70–1.56(m,2H).
Example 28: preparation of (S) -2- ((4- (6- ((4-cyanonaphthalen-1-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 49) (prepared according to scheme eight line)
Step 28 a: preparation of 4- (hydroxymethyl) -1-naphthalenecarbonitrile (Compounds 0801-49):
A mixed solution of 1-bromo-4-methylnaphthalene (1.5 g, 6.78 mmol, 1.0 equiv.) and cuprous cyanide (1.2 g, 13.57 mmol, 2 equiv.) in N, N-dimethylformamide (20 mL) was stirred at 120 ℃ overnight under nitrogen. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The mixture was filtered through celite and the solid was washed with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 15/1-10/1) to give 4-methyl-1-naphthonitrile (889 mg, yield: 78.53%) as a yellow solid. Lcms (esi): m/z equals 168(M + H) +
A solution of 4-methyl-1-naphthacenitrile (400 mg, 2.4 mmol, 1.0 eq), N-bromosuccinimide (511 mg, 2.87 mmol, 1.2 eq), and azobisisobutyronitrile (78.6 mg, 0.479 mmol, 0.2 eq) in dichloroethane (10 ml) was stirred at 72 ℃ overnight under nitrogen. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give a crude product of 4- (bromomethyl) -1-naphthonitrile (678 mg, crude). The product was used in the next step without further purification.
A mixture of 4- (bromomethyl) -1-naphthalenecarbonitrile (678 mg, 2.76 mmol, 1.0 eq) and potassium acetate (2.7 g, 27.6 mmol, 10.0 eq) in N, N-dimethylformamide (10 ml) was stirred at room temperature for 3 h. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give a crude product of (4-cyanonaphthalen-1-yl) methyl acetate (823 mg, crude). The product was used in the next step without further purification.
In methyl (4-cyanonaphthalen-1-yl) acetate (823 mg)3.65 mmol, 1.0 eq) of tetrahydrofuran (5 ml) was added a solution of sodium methoxide in methanol (5.4 mol/l, 1.35 ml, 7.31 mmol, 2.0 eq). The mixture was stirred at room temperature for 1 hour. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 20/1-2/1) to give 4- (hydroxymethyl) -1-naphthonitrile (300 mg, yield: 44.91%) as a yellow solid. Lcms (esi): 184(M + H) M/z+.
Step 28 b: preparation of tert-butyl 4- (6- ((4-cyanonaphthalen-1-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0802-49): a mixture of tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (0104-1) (202 mg, 0.68 mmol, 1 eq), 4- (hydroxymethyl) -1-naphthacenitrile (0801-49) (150 mg, 0.82 mmol, 1.2 eq), cesium carbonate (443.36 mg, 1.36 mmol, 2.0 eq), 2-dicyclohexylphosphonium-2 ',6' -diisopropoxy-1, 1' -biphenyl (32 mg, 0.068 mmol, 0.1 eq) and tris (dibenzylideneacetone) dipalladium (0) (31 mg, 0.034 mmol, 0.05 eq) in toluene (10 ml) was stirred at 125 ℃ under nitrogen protection overnight. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 100/1-8/1) to give 4- (6- ((4-cyanonaphthalen-1-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester as a yellow oil (272 mg, yield: 90.37%). Lcms (esi): m/z 444(M + H) +.
Step 28 c: preparation of 4- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) -1-naphthacenitrile hydrochloride (compound 0803-49): tert-butyl 4- (6- ((4-cyanonaphthalen-1-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0802-49) (272 mg, 0.614 mmol, 1.0 eq) and a dioxane solution of hydrochloric acid (4M, 5 ml) were stirred at room temperature overnight. The mixture was filtered. The residue was washed with dioxane. The residue was dried to give 4- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) -1-naphthacenitrile hydrochloride as a yellow solid (215 mg, yield: 92.28%)(ESI):m/z=344(M+H)+.
Step 28 d: (S) -2- (((4- (6- ((4-cyanonaphthalen-1-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0804-49): 4-(((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl) -1-naphthacenitrile hydrochloride salt (0803-49) (96 mg, 0.254 mmol, 1.5 eq), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A solution of methyl imidazole-6-carboxylate (0116-3) (50 mg, 0.17 mmol, 1.0 equiv.) and potassium carbonate (93.5 mg, 0.678 mmol, 4.0 equiv.) in acetonitrile (5 ml) was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, and water was added thereto, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain white solid (S) -2- (((4- (6- ((4-cyanonaphthalen-1-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]Imidazole-6-carboxylic acid methyl ester (50 mg, yield: 49.02%). Lcms (esi): 602(M + H) is defined as M/z+.
Step 28 e: (S) -2- ((4- (6- ((4-cyanonaphthalen-1-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 49) (S) -2- (((4- (6- ((4-cyanonaphthalen-1-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d [ -d]A mixture of methyl imidazole-6-carboxylate (0804-49) (50 mg, 0.083 mmol, 1.0 eq) and lithium hydroxide monohydrate (7 mg, 0.17 mmol, 2.0 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l hydrochloric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water and dried under reduced pressure to give (S) -2- ((4- (6- ((4-cyanonaphthalen-1-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid]Imidazole-6-carboxylic acid (35 mg, yield: 71.75%). Lcms (esi): m/z 588(M + H)+.1HNMR(500MHz,DMSO)δ12.68(s,1H),8.30(d,J=8.3Hz,2H),8.17(t,J=7.0Hz,2H),7.81(ddd,J=15.3,14.3,7.0Hz,4H),7.67(d,J=7.1Hz,2H),6.91(d,J=6.7Hz,1H),6.74(d,J=7.8Hz,1H),5.93(s,2H),5.07(d,J=5.1Hz,1H),4.78(dd,J=15.1,6.7Hz,1H),4.64(d,J=14.8Hz,1H),4.44(d,J=6.3Hz,1H),4.33(dt,J=9.0,5.9Hz,1H),3.94(s,1H),3.77(s,1H),2.97(s,1H),2.85(s,1H),2.74–2.55(m,2H),2.44–2.31(m,1H),2.18(s,2H),1.91(d,J=102.8Hz,4H).
Example 29: preparation of (S) -2- ((4- (6- ((4-cyano-6-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 53) (preparation according to scheme two line)
Step 29 a: preparation of 6-fluoro-7- (hydroxymethyl) benzofuran-4-carbonitrile (compound 0201-53):
to a solution of methyl 4-bromo-2, 6-difluorobenzoate (2.5 g, 9.96 mmol, 1.0 eq) in methanol was added a solution of sodium methoxide in methanol (5.4 mol/l, 5.5 ml, 29.88 mmol, 3.0 eq) under nitrogen, and the mixture was stirred at room temperature for 16.0 h. The reaction mixture was diluted with water, adjusted to pH 5 with 2 mol/l hydrochloric acid and extracted with ethyl acetate, the organic phase was dried and concentrated under reduced pressure. The residue was purified by a silica gel column (petroleum ether/ethyl acetate ═ 10/1 to 5/1) to give methyl 4-bromo-2-fluoro-6-methoxybenzoate (2.5 g, yield: 96.2%) as a colorless oily product.
Boron tribromide (3.4 g, 13.69 mmol, 1.5 equivalents) was added to a solution of methyl 4-bromo-2-fluoro-6-methoxybenzoate (2.4 g, 9.12 mmol, 1.0 equivalent) in dichloromethane (120 ml) under nitrogen, and the mixture was stirred at 0 ℃ for 1.5 hours. The reaction was quenched with methanol, diluted with water and extracted with dichloromethane, the organic phase dried and concentrated under reduced pressure. The residue was purified by a silica gel column (developing solvent: dichloromethane) to give methyl 4-bromo-2-fluoro-6-hydroxybenzoate (2.03 g, yield: 89.4%) as a white solid product. Lcms (esi): m/z 247[ M +1 ═ M ]-。
A mixture of methyl 4-bromo-2-fluoro-6-hydroxybenzoate (1.5 g, 6.02 mmol, 1.0 eq), 2-bromo-1, 1-diethoxyethane (1.42 g, 7.23 mmol, 1.2 eq), potassium carbonate (1.25 g, 9.03 mmol, 1.5 eq) in N, N-dimethylformamide (15 ml) was stirred at 120 ℃ under nitrogen atmosphere overnight. After cooling to room temperature, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by a silica gel column (petroleum ether/ethyl acetate ═ 10/1 to 8/1) to give 4-bromo-2- (2, 2-diethoxyethoxy) -6-fluorobenzoate as a yellow oily product (2.15 g, yield: 98.2%).
To a solution of polyphosphoric acid (4.98 g, 14.73 mmol, 2.5 eq) in toluene (80 ml) under nitrogen blanket was added 4-bromo-2- (2, 2-diethoxyethoxy) -6-fluorobenzoate (2.15 g, 5 mmol, 1.0 eq) and the mixture was stirred at 120 ℃ overnight. After cooling to room temperature, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether) to give methyl 4-bromo-6-fluorobenzofuran-7-carboxylate (1.34 g, yield: 83.4%) as a yellow oily product. Lcms (esi): 273[ M +1 ] M/z ]+。
To a solution of methyl 4-bromo-6-fluorobenzofuran-7-carboxylate (950 mg, 3.48 mmol, 1.0 eq) in methanol (30 ml) at 0 ℃ under nitrogen atmosphere was added sodium borohydride (2.6 g, 69.60 mmol, 20.0 eq) and the mixture was stirred at room temperature for 3.0 h. The reaction was quenched with water and extracted with ethyl acetate, and the organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column (eluent: petroleum ether/ethyl acetate 10/1 to 2/1) to give the product (4-bromo-6-fluorobenzofuran-7-yl) methanol (850 mg, yield: 99.6%) as a yellow solid.
To a solution of (4-bromo-6-fluorobenzofuran-7-yl) methanol (630 mg, 2.57 mmol, 1.0 eq) in N-methylpyrrolidone (20 ml) under nitrogen blanket was added cuprous cyanide (2.3 g, 25.71 mmol, 10.0 eq) and stirred at 190 ℃ for 8 h. After cooling to room temperature, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1 to 2/1) to give 6-fluoro-7- (hydroxymethyl) benzofuran-4-carbonitrile (92 mg, yield: 18.8%) as a white solid.
Step 29 b: preparation of tert-butyl 4- (6- ((4-cyano-6-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (compound 0202-53): under nitrogen, a mixture of tert-butyl 4- (6-chloropyridin-2-yl) piperidine-1-carboxylate (0104-1) (143 mg, 0.482 mmol, 1.0 eq), 6-fluoro-7- (hydroxymethyl) benzofuran-4-carbonitrile (0201-53) (92 mg, 0.482 mmol, 1.0 eq), cesium carbonate (314 mg, 0.964 mmol, 2.0 eq), 2-dicyclohexylphosphonium-2 ',6' -diisopropoxy-1, 1' -biphenyl (45 mg, 0.0964 mmol, 0.2 eq), and tris (dibenzylideneacetone) dipalladium (0) (44 mg, 0.0482 mmol, 0.1 eq) in toluene (10 ml) was stirred at 120 ℃ overnight. The reaction solution was diluted with water and extracted with ethyl acetate, and the organic phase was dried and concentrated under reduced pressure. The residue was purified by a silica gel column (petroleum ether/ethyl acetate ═ 10/1 to 5/1) to give tert-butyl 4- (6- ((4-cyano-6-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (180 mg, yield: 82.6%) as a yellow solid product. Lcms (esi): 452[ M +1] +, M/z.
Step 29 c: 6-fluoro-7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl]Preparation of benzofuran-4-carbonitrile hydrochloride (Compound 0203-53): to a solution of tert-butyl 4- (6- (((4-cyano-6-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidine-1-carboxylate (0202-53) (180 mg, 0.40 mmol, 1.0 eq) in dioxane (4 ml) was added a solution of hydrochloric acid-dioxane (4 mol solution, 1 ml), the mixture was stirred at room temperature overnight, the reaction was concentrated under reduced pressure, and the residue was used in the next step without further purification, lcms (esi) M/z 352(M + H)+.
Step 29 d: (S) -2- (((4- (6- ((4-cyano-6-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 0204-53) 6-fluoro-7- (((6- (piperidin-4-yl) pyridin-2-yl) oxy) methyl]Benzofuran-4-carbonitrile hydrochloride (0203-53) (77 mg, 0.22)0 mmol, 1.3 equivalents), (S) -2- (chloromethyl) -3- (oxetan-2-ylmethyl) -1H-benzo [ d]A solution of methyl imidazole-6-carboxylate (0116-3) (50 mg, 0.169 mmol, 1.0 eq) and potassium carbonate (93.5 mg, 0.678 mmol, 4.0 eq) in acetonitrile (10 ml) was stirred at 60 ℃ overnight. The reaction solution was diluted with water and extracted with ethyl acetate, and the organic phase was dried and concentrated under reduced pressure. The residue was purified by preparative thin layer chromatography (dichloromethane/methanol ═ 16/1) to give the product (S) -2- ((4- (6- ((4-cyano-6-fluorobenzofuran-7-yl) methoxy) pyridinyl-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d-oxetan-2-ylmethyl) -1H-benzo [ d ] c ]Imidazole-6-carboxylic acid methyl ester (55 mg, yield: 53.4%). Lcms (esi): m/z 610(M + H)+.
Step 29 e: (S) -2- ((4- (6- ((4-cyano-6-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 53) (S) -2- ((4- (6- ((4-cyano-6-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0204-53) (55 mg, 0.09 mmol, 1.0 eq) and lithium hydroxide monohydrate (5.7 mg, 0.135 mmol, 1.5 eq) in acetonitrile/water 5/1(6 ml) was stirred at 42 ℃ overnight. The reaction solution was adjusted to pH 6 with 2 mol/l hydrochloric acid, extracted with ethyl acetate, and the resulting organic phase was dried and concentrated under reduced pressure to give (S) -2- ((4- (6- ((4-cyano-6-fluorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperidin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] a white solid]Imidazole-6-carboxylic acid (39 mg, yield: 72.8%). Lcms (esi): m/z 596(M + H)+.1H NMR(500MHz,DMSO)δ12.71(s,1H),8.35(d,J=2.1Hz,1H),8.26(s,1H),7.89(d,J=10.0Hz,1H),7.81(d,J=8.4Hz,1H),7.62(dd,J=16.8,8.9Hz,2H),7.20(d,J=2.1Hz,1H),6.87(d,J=7.3Hz,1H),6.66(d,J=8.2Hz,1H),5.69(s,2H),5.12(dd,J=7.1,2.5Hz,1H),4.80(dd,J=15.1,7.1Hz,1H),4.67(d,J=12.9Hz,1H),4.47(dd,J=13.7,7.6Hz,1H),4.37(dt,J=9.0,5.9Hz,1H),3.95(d,J=13.6Hz,1H),3.80(d,J=13.6Hz,1H),2.99(d,J=10.9Hz,1H),2.86(d,J=11.0Hz,1H),2.76–2.68(m,1H),2.56(t,J=11.5Hz,1H),2.44(dd,J=18.8,8.1Hz,1H),2.20(dt,J=21.1,10.4Hz,2H),1.78–1.59(m,4H).
Example 30: preparation of (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 54) (prepared according to scheme nine route)
Step 30 a: preparation of 2-bromo-6- ((4-chlorobenzofuran-7-yl) methoxy) pyridine (compounds 0901-54) to a mixture of (4-chlorobenzofuran-7-yl) methanol (0201-12) (230 mg, 1.26 mmol, 1.0 eq) in tetrahydrofuran under nitrogen protection was added potassium hexamethyldisilazide (1 mol/l, 3.78 mmol, 3.0eq) at 10-15 deg.C, and the mixture was stirred at 15 deg.C for 1 hour. Dissolving 2-bromo-6-fluoropyridine in tetrahydrofuran, and dropwise adding the solution into a reaction system. The mixture was stirred at 15 ℃ for 1 hour. The mixture was quenched with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate-10/1) to give 2-bromo-6- ((4-chlorobenzofuran-7-yl) methoxy) pyridine (140 mg, yield: 32.87%) as a yellow solid. Lcms (esi): 338(M + H) M/z+.
Step 30 b: preparation of tert-butyl 4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazine-1-carboxylate (compound 0902-54): a mixture of 2-bromo-6- ((4-chlorobenzofuran-7-yl) methoxy) pyridine (0901-54) (150 mg, 0.44 mmol, 1.0 eq), 1-tert-butoxycarbonylpiperazine (92 mg, 0.49 mmol, 1.1 eq), cesium carbonate (290 mg, 0.89 mmol, 2.0 eq), 1 '-binaphthyl-2, 2' -bis-diphenylphosphine (31 mg, 0.05 mmol, 0.1 eq), and tris (dibenzylideneacetone) dipalladium (0) (28 mg, 0.03 mmol, 0.05 eq) in toluene (15 ml) was stirred at 125 ℃ under nitrogen overnight. After cooling to room temperature, the reaction was filtered through celite and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to purify the crude product, thereby obtaining tert-butyl 4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazine-1-carboxylate (71 mg, product yield) as a yellow oily liquid Rate: 36.43%). Lcms (esi): m/z 443(M + H)+.
Step 30 c: preparation of 1- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazine hydrochloride (compound 0903-54): tert-butyl 4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazine-1-carboxylate (0902-54) (71 mg, 0.16 mmol, 1.0 eq) and a solution of ethyl acetate in hydrochloric acid (2M, 4 ml) in methanol (4 ml) were stirred at 40 ℃ for 2 hours. The mixture was concentrated under reduced pressure to give crude 1- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazine hydrochloride (55 mg, crude). Lcms (esi): m/z 343(M + H)+.
Step 30 d: (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of methyl imidazole-6-carboxylate (Compound 0904-54) 1- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazine hydrochloride (0903-54) (55 mg, 0.16 mmol, 1.2 eq), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d-]A solution of methyl imidazole-6-carboxylate (0116-3) (40 mg, 0.13 mmol, 1.0 eq) and N, N-diisopropylethylamine (1 ml) in N-methylpyrrolidone (10 ml) was stirred at 60 ℃ overnight. The reaction mixture was cooled to room temperature, water was added thereto, and ethyl acetate was added thereto for extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative silica gel thin layer chromatography using a preparative plate (eluent: ethyl acetate: petroleum ether ═ 3/1) to give (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] c ]Imidazole-6-carboxylic acid methyl ester (85 mg, yield: 100%). Lcms (esi): m/z 603(M + H)+.
Step 30 e: (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 54) (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d []Imidazole-6-carboxylic acid methyl ester (0904-58) (85 mg)0.14 mmol, 1.0 eq) and lithium hydroxide monohydrate (18 mg, 0.43 mmol, 3.0 eq) in acetonitrile/water 5/1(12 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The mixture was washed with water and the pH of the residue was adjusted to 6 with 1 mol/l hydrochloric acid. The residue was extracted with ethyl acetate and concentrated under reduced pressure. The crude product was purified by preparative thin layer chromatography on silica gel (eluent: dichloromethane: methanol ═ 10/1) to give (S) -2- ((4- (6- ((4-chlorobenzofuran-7-yl) methoxy) pyridin-2-yl) piperazin-1-yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] c]Imidazole-6-carboxylic acid (71 mg, yield: 86.25%). Lcms (esi): m/z 588(M + H) +.1H NMR(500MHz,DMSO)δ8.14(d,J=2.2Hz,1H),8.09(s,1H),7.81(d,J=8.3Hz,1H),7.49–7.42(m,2H),7.38(d,J=8.0Hz,1H),7.33(d,J=8.0Hz,1H),7.03(d,J=2.2Hz,1H),6.31(d,J=8.1Hz,1H),6.09(d,J=7.8Hz,1H),5.55(s,2H),5.15–5.08(m,1H),4.71(dd,J=15.2,7.0Hz,1H),4.59(dd,J=15.3,3.1Hz,1H),4.49(dd,J=13.3,8.0Hz,1H),4.37(dt,J=9.0,5.9Hz,1H),3.92(d,J=13.5Hz,1H),3.77(d,J=13.4Hz,1H),3.43(dd,J=12.0,7.3Hz,4H),3.29–3.24(m,4H),2.73–2.65(m,1H),2.47–2.41(m,1H).
Example 31: preparation of (S) -2- ((6- (2- (4-chlorobenzofuran-7-yl) ethoxy) -3',6' -dihydro- [2,4' -bipyridinyl ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 55) (prepared according to scheme three-line)
Step 31 a: preparation of 2- (4-chlorobenzofuran-7-yl) ethan-1-ol (Compound 0201-55):
to a solution of 7- (bromomethyl) -4-chlorobenzofuran (1 g, 4.08 mmol, 1.0 eq) and potassium carbonate (1.12 g, 8.16 mmol, 2 eq) in tetrahydrofuran (10 ml) under nitrogen was added trimethylsilyl cyanide (808 mg, 8.16 mmol, 2 eq). The mixture was stirred at 90 ℃ for 4 hours. The reaction was cooled to room temperature. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 50/1-10/1) to give 2- (4-chlorobenzofuran-7-yl) acetonitrile (880 mg, crude) as a yellow oil. Lcms (esi): m/z 192(M + H) +.
To a solution of 2- (4-chlorobenzofuran-7-yl) acetonitrile (880 mg, 6.24 mmol, 1.0 eq) in ethanol/water (10/10 ml) was added potassium hydroxide (1.74 g, 31.2 mmol, 5 eq). The mixture was stirred at 90 ℃ for 16 hours. The reaction was cooled to room temperature. The pH of the mixture was adjusted to 1 with 2N hydrochloric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water and dried to give 2- (4-chlorobenzofuran-7-yl) acetic acid (510 mg, yield: 38.75%) as a yellow solid. Lcms (esi): m/z 211(M + H) +.
Borane (0.64 ml, 10 mol/l dimethyl sulfide, 6.375 mmol, 2.5 eq) is added dropwise to a solution of 2- (4-chlorobenzofuran-7-yl) acetic acid (510 mg, 2.43 mmol, 1.0 eq) in tetrahydrofuran (10 ml) under nitrogen at a temperature between 0 and 10 ℃. The mixture was stirred at room temperature for 1 hour. The reaction was quenched with methanol and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 20/1-5/1) to give 2- (4-chlorobenzofuran-7-yl) ethan-1-ol as a clear oil (397 mg, yield: 41.18%). Lcms (esi): m/z 197(M + H) +.
Step 31 b: 6- ((2- (4-chlorobenzofuran-7-yl) ethoxy) -3',6' -dihydro- [2,4' -bipyridine]Preparation of 1'(2' H) -carboxylic acid tert-butyl ester (Compound 0303-55): potassium hexamethyldisilazide (3.5 ml, 1 mol/l tetrahydrofuran, 3.448 mmol, 2 equiv.) was added to a solution of 2- (4-chlorobenzofuran-7-yl) ethan-1-ol (0201-55) (338 mg, 1.724 mmol, 1.0 equiv.) in tetrahydrofuran (20 ml) at 0 to 10 ℃ under nitrogen. The mixture was stirred at 10 ℃ for 45 minutes. 2-bromo-6-fluoropyridine (0301-2) (303 mg, 1.724 mmol, 1 eq.) was added in portions to the reaction mixture at 10 ℃ and the mixture was at room temperature Stirring was continued for 1 hour. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 200/1-20/1) to give 2-bromo-6- (2- (4-chlorobenzofuran-7-yl) ethoxy) pyridine (383 mg, yield: 63.09%) as a yellow solid. Lcms (esi): 352(M + H) M/z+Under nitrogen protection, the mixture of 2-bromo-6- (2- (4-chlorobenzofuran-7-yl) ethoxy) pyridine (272 mg, 0.77 mmol, 1.05 eq), 4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (226 mg, 0.73 mmol, 1 eq), sodium carbonate (155 mg, 1.46 mmol, 2.0 eq), bis (triphenylphosphine) palladium dichloride (51 mg, 0.073 mmol, 0.1 eq) in N, N-dimethylformamide/water-10/1 (11 ml) obtained above was stirred at 92 ℃ overnight. After cooling to room temperature, water was added, and ethyl acetate was added for extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 20/1-10/1) to give 6- ((2- (4-chlorobenzofuran-7-yl) ethoxy) -3',6' -dihydro- [2,4' -bipyridine) as a clear oil ]-1'(2' H) -carboxylic acid tert-butyl ester (123 mg, yield: 37.05%). Lcms (esi): 455(M + H) is defined as M/z+.
Step 31 c: preparation of 6- ((2- (4-chlorobenzofuran-7-yl) ethoxy) -1',2',3',6' -tetrahydro-2, 4 '-bipyridine 4-methylbenzenesulfonate (Compound 0304-55) Or 6- ((2- (4-chlorobenzofuran-7-yl) ethoxy) -3',6 '-dihydro- [2,4' -bipyridine]-1'(2' H) -carboxylic acid tert-butyl ester (0303-55) (100 mg, 0.22 mmol, 1.0 equiv.) in ethyl acetate (5 mL) was added p-toluenesulfonic acid monohydrate (114 mg, 0.66 mmol, 3 equiv.). The mixture was stirred at 60 ℃ overnight. The mixture was cooled to room temperature. The mixture was concentrated under reduced pressure. The residue was used directly in the next step (103 mg, crude) without purification. Lcms (esi): 355(M + H) M/z+.
Step 31 d: (S) -2- ((6- (2- (4-chlorobenzofuran-7-yl) ethoxy) -3',6' -dihydro- [2,4' -bipyridine)]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (formula)Preparation of Compound 0305-55) 6- ((2- (4-chlorobenzofuran-7-yl) ethoxy) -1',2',3',6' -tetrahydro-2, 4' -bipyridine 4-methylbenzenesulfonate (0304-55) (103 mg, 0.203 mmol, 1.2 equiv), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ]A solution of methyl imidazole-6-carboxylate (0116-3) (50 mg, 0.17 mmol, 1.0 eq) and potassium carbonate (94 mg, 0.68 mmol, 4.0 eq) in acetonitrile (10 ml) was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, water was added thereto, and ethyl acetate was added for extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying the residue with silica gel thin layer chromatography preparative plate (eluent: ethyl acetate) to obtain yellow oily substance (S) -2- ((6- (2- (4-chlorobenzofuran-7-yl) ethoxy) -3',6' -dihydro- [2,4' -bipyridine)]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (69 mg, yield: 66.34%). Lcms (esi): 613(M + H) M/z+.
Step 31 e: (S) -2- ((6- (2- (4-chlorobenzofuran-7-yl) ethoxy) -3',6' -dihydro- [2,4' -bipyridine)]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 55) (S) -2- ((6- (2- (4-chlorobenzofuran-7-yl) ethoxy) -3',6' -dihydro- [2,4' -bipyridine)]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (0604-55) (69 mg, 0.11 mmol, 1.0 eq) and lithium hydroxide monohydrate (14 mg, 0.33 mmol, 3 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The pH of the residue was adjusted to 6 with 1 mol/l hydrochloric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water. The residue was purified by preparative silica gel thin layer chromatography on preparative plates (eluent: dichloromethane/methanol-10/1) to give (S) -2- ((6- (2- (4-chlorobenzofuran-7-yl) ethoxy) -3',6' -dihydro- [2,4' -bipyridine) as a yellow solid ]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (27 mg, yield: 40.29%). Lcms (esi): 599(M + H)+.1H NMR(500MHz,DMSO)δ12.72(s,1H),8.25(s,1H),8.08(d,J=2.2Hz,1H),7.81(dd,J=8.4,1.4Hz,1H),7.70–7.60(m,2H),7.25(q,J=8.0Hz,2H),7.03(d,J=7.4Hz,1H),6.98(d,J=2.2Hz,1H),6.70(s,1H),6.59(d,J=8.2Hz,1H),5.06(ddd,J=14.4,7.3,2.8Hz,1H),4.79(dd,J=15.2,7.3Hz,1H),4.70–4.55(m,3H),4.46(dd,J=13.6,7.7Hz,1H),4.35(dt,J=9.0,5.9Hz,1H),4.07(d,J=13.6Hz,1H),3.92(d,J=13.5Hz,1H),3.27–3.21(m,2H),2.80–2.69(m,2H),2.65(ddd,J=16.2,8.7,5.4Hz,1H),2.51(s,2H),2.44–2.34(m,1H).
Example 32: preparation of 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] [1,3] dioxolan-4-yl) piperidin-1-yl) methyl) -1- ((((S) -oxetan-2-yl) methyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 56) (prepared according to scheme ten lines)
Step 32 a: preparation of tert-butyl 4- (2, 3-dihydroxyphenyl) piperidine-1-carboxylate (Compounds 1003-56):
to a mixture of 3-bromocatechol (1.2 g, 6.35 mmol, 1.0 equiv.) and N, N-diisopropylethylamine (2.82 ml, 15.87 mmol, 2.5 equiv.) in dichloromethane (20 ml) was added 2- (trimethylsilyl) ethoxymethyl chloride (2.65 g, 15.87 mmol, 2.5 equiv.) dropwise at 0 ℃. The mixture was warmed to room temperature and stirred for 2 hours. The solvent was removed under reduced pressure. The residue was subjected to column chromatography on silica gel (petroleum ether: ethyl acetate 30:1) to give a colorless oil ((((((3-bromo-1, 2-phenylene) bis (oxy)) bis (methylene)) bis (oxy)) bis (ethane-2, 1-diyl)) bis (trimethylsilane) (2.27 g, yield: 79%). TLC: rf0.5 (petroleum ether: ethyl acetate ═ 30: 1).
To ((((((3-bromo-1, 2-phenylene) bis (oxy)) bis (methylene) bis (oxy)) bis (ethane-2, 1-diyl)) bis (trimethylsilane) (2.27 g, 5.04 mmol, 1.0 eq), [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride (221 mg, 0.302 mmol, 0.06 equiv.) and sodium carbonate (1.60 g, 15.12 mmol, 3.0 equiv.) to a mixture of dioxane (30 ml) and water (7.5 ml) was added 4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -3, 6-dihydropyridine-1 (2H)) -carboxylic acid tert-butyl ester (1.87 g, 6.05 mmol, 1.2 equiv.). The mixture was heated to 85 ℃ under nitrogen and stirred for 16 hours. The solvent was removed under reduced pressure. Column chromatography of the residue on silica gel (petroleum ether: ethyl acetate 30: 1) gave 4- (2, 3-bis ((2- (trimethylsilyl) ethoxy) methoxy) phenyl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester as a pale yellow oil (2.37 g, yield: 85%). Lcms (esi): m/z 552[ M +1 ]]+(ii) a TLC: rf0.5 (petroleum ether: ethyl acetate ═ 10:1).
To a mixture of tert-butyl 4- (2, 3-bis ((2- (trimethylsilyl) ethoxy) methoxy) phenyl) -3, 6-dihydropyridine-1 (2H) -carboxylate (1.0 g, 1.81 mmol, 1.0 eq) in methanol (15 ml) was added palladium on carbon (0.2 g). The mixture was stirred under hydrogen balloon pressure at room temperature overnight. The mixture was filtered. The filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography on silica gel (petroleum ether: ethyl acetate 30: 1) to give tert-butyl 4- (2, 3-bis ((2- (trimethylsilyl) ethoxy) methoxy) phenyl) piperidine-1-carboxylate (0.48 g, yield: 48%) as a white solid. Lcms (esi): m/z554[ M +1 ] ]+(ii) a TLC: rf0.5 (petroleum ether: ethyl acetate: 10: 1).
A mixture of tert-butyl 4- (2, 3-bis ((2- (trimethylsilyl) ethoxy) methoxy) phenyl) piperidine-1-carboxylate (1.1 g, 1.99 mmol, 1.0 eq) in methanolic hydrogen chloride solution (4M solution, 15 ml) was stirred at room temperature overnight. The solvent was removed under reduced pressure and dried under vacuum to give 4- (2, 3-dihydroxyphenyl) piperidine hydrochloride (601 mg, crude) as a white solid. Lcms (esi): m/z 194[ M +1 ]]+(ii) a TLC: rf0.3 (dichloromethane: methanol ═ 10: 1).
To a mixture of 4- (2, 3-dihydroxyphenyl) piperidine hydrochloride (454 mg, 1.99 mmol, 1.0 eq) and sodium bicarbonate (502 mg, 5.97 mmol, 3.0 eq) in methanol (6 ml) was added di-tert-butyl dicarbonate (456 mg, 2.09 mmol, 1.05 eq). The mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: methanol 30: 1) to give tert-butyl 4- (2, 3-dihydroxyphenyl) piperidine-1-carboxylate (583 mg, yield: 100%) as a gray solid. Lcms (esi): m/z 294[ M +1 ]]+(ii) a TLC: rf0.5 (dichloromethane: methanol ═ 10: 1).
Step 32 b: 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d) ][1,3]Preparation of Dioxolan-4-yl) piperidine-1-carboxylic acid tert-butyl ester (Compound 1004-56) to a mixture of tert-butyl 4- (2, 3-dihydroxyphenyl) piperidine-1-carboxylate (1003-56) (0.25 g, 0.85 mmol, 1.0 eq), triruthenium dodecacarbonyl (27 mg, 0.043 mmol, 0.05 eq) and sodium bicarbonate (72 mg, 0.85 mmol, 1.0 eq) in toluene (10 mL) was added 4-chloro-7-ethynylbenzofuran (Compound 1101-57) (150 mg, 0.85 mmol, 1.0 eq). The mixture was heated to 120 ℃ under nitrogen and stirred overnight. The solvent was removed under reduced pressure. The residue was subjected to column chromatography on silica gel (petroleum ether: ethyl acetate 30: 1) to give 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] as a pale yellow oil][1,3]Dioxolan-4-yl) piperidine-1-carboxylic acid tert-butyl ester (70 mg, yield: 18%). Lcms (esi): m/z 470[ M +1 ]]+(ii) a TLC: rf0.5 (petroleum ether: ethyl acetate ═ 10: 1).
Step 32 c: 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Preparation of Dioxolan-4-yl) piperidine hydrochloride (Compounds 1005-56) 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d][1,3]A mixture of tert-butyl dioxolan-4-yl) piperidine-1-carboxylate (1004-56) (70 mg, 0.15 mmol, 1.0 eq) in hydrogen chloride dioxane solution (4M solution, 1.5 mL) was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure and dried under vacuum to give 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] as a white solid ][1,3]Dioxolan-4-yl) piperidine hydrochloride (61 mg, crude). Lcms (esi): m/z 370[ M +1 ]]+(ii) a TLC: rf0.3 (dichloromethane: methanol ═ 10: 1).
Step 32 d: 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compounds 1006-56) by reacting 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) piperidine hydrochloride (1005-56) (61 mg, 0.15 mmol, 1.0 eq.) and (S) -2- (chloromethyl) -1- (oxetane-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (0116-3) (44 mg, 0.15 mmol, 1.0 eq) to a mixture of acetonitrile (5 ml) was added potassium carbonate (51 mg, 0.37 mmol, 2.5 eq). The mixture was heated at 60 ℃ overnight. The solvent was removed under reduced pressure. The residue was subjected to column chromatography on silica gel (petroleum ether: ethyl acetate 3: 1) to give 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] as a white solid][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (81 mg, yield: 87%). Lcms (esi): m/z 628[ M +1 ] ]+(ii) a TLC: rf0.5 (petroleum ether: ethyl acetate ═ 1: 1).
Step 32 e: 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 56) by reacting 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (1006-56) (81 mg, 0.13 mmol, 1.0 equiv.) to a mixture of tetrahydrofuran (5 ml) and water (2 ml) was added lithium hydroxide monohydrate (16 mg, 0.39 mmol, 3.0 equiv.). The mixture was heated at 40 ℃ overnight. The mixture was diluted with water (15 ml). A 1N diluted hydrochloric acid solution was added to adjust pH 5 and then the aqueous layer was extracted with ethyl acetate (15 ml × 3). The combined organic layers were washed with saturated brine (15 ml × 1), dried over anhydrous sodium sulfate and concentrated. The residue was purified by preparative thin layer chromatography (ethyl acetate: methanol ═ 10: 1) to give 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] as a white solid][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d ]Imidazole-6-carboxylic acid (53 mg, yield: 66%). Lcms (esi): m/z 614[ M +1]+(ii) a TLC: rf0.5 (ethyl acetate: methanol ═ 10: 1); melting point: 165-167 ℃.1H NMR(500MHz,DMSO)δ12.70(s,1H),8.27(d,J=2.1Hz,1H),8.21(d,J=6.3Hz,1H),7.80(d,J=8.5Hz,1H),7.64(d,J=8.4Hz,1H),7.44(dd,J=8.1,2.9Hz,1H),7.37(d,J=8.1Hz,1H),7.06(s,1H),6.75(dd,J=16.6,4.4Hz,3H),5.17–5.03(m,1H),4.78(dd,J=15.1,7.2Hz,1H),4.65(d,J=15.7Hz,1H),4.44(dt,J=13.0,7.6Hz,1H),4.41–4.32(m,1H),3.96(dd,J=13.5,3.6Hz,1H),3.78(d,J=13.5Hz,1H),3.01(s,1H),2.86(d,J=10.5Hz,1H),2.67(dd,J=23.9,16.3Hz,2H),2.44(s,1H),2.26(s,1H),2.16(s,4H),1.82–1.67(m,4H).
Example 33: preparation of 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] [1,3] dioxolan-4-yl) -3, 6-dihydropyridin-1 (2H) -yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 57) (prepared according to scheme ten line)
Step 33 a: preparation of 4-chloro-7-ethynylbenzofuran (compound 1001-57):
a solution of 2-bromo-5-chlorophenol (5.0 g, 24.1 mmol, 1.0 eq), 2-bromo-1, 1-diethoxyethane (5.7 g, 28.9 mmol, 1.2 eq) and potassium carbonate (6.7 g, 48.2 mmol, 2.0 eq) in N, N-dimethylformamide (70 mL) was stirred at 120 ℃ overnight. After cooling to room temperature, the mixture was poured into water, ethyl acetate was added thereto for extraction, and the organic layer was washed with saturated brine and concentrated. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 200/1 to 100/1) to give 1-bromo-4-chloro-2- (2, 2-diethoxyethoxy) benzene as a yellow oil (8.0 g, yield: 102%).
A solution of 1-bromo-4-chloro-2- (2, 2-diethoxyethoxy) benzene (8.0 g, 19.8 mmol, 1.0 eq.) and polyphosphoric acid (12.5 g, 37.1 mmol, 1.5 eq.) in dichloroethane (150 mL) was stirred at 72 deg.C overnight under nitrogen. After cooling to room temperature, water was added and the mixture was stirred for 30 minutes. After the liquid separation, the organic layer was washed with saturated brine and concentrated. The residue was purified by silica gel column chromatography (eluent: petroleum ether) to give 7-bromo-4-chlorobenzofuran (4.2 g, yield: 73.7%) as a colorless liquid.
A mixture of 7-bromo-4-chlorobenzofuran (2.0 g, 8.6 mmol, 1.0 eq), palladium (303 mg, 0.43 mmol, 0.05 eq), cuprous iodide (41 mg, 0.215 mmol, 0.025 eq), trimethylsilylacetylene (4.2 g, 43.0 mmol, 5.0 eq) and triethylamine (15 ml) in tetrahydrofuran (20 ml) was stirred at 90 ℃ overnight under nitrogen. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated. The residue was dissolved in ethyl acetate, potassium carbonate (1.0 g) was added, and the mixture was stirred at room temperature for 2 hours. Water was added and the mixture was partitioned. The organic layer was concentrated, and the residue was purified by silica gel column chromatography (eluent: petroleum ether) to give 4-chloro-7-ethynylbenzofuran (1.4 g, yield: 92.1%) as a yellow solid.
Step 33 b: preparation of 4-bromo-2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] [1,3] dioxolane (compound 1002-57) A mixture of 4-chloro-7-ethynylbenzofuran (1001-57) (1.3 g, 7.4 mmol, 1.0 eq.), 3-bromo-1, 2-benzenediol (1.5 g, 8.1 mmol, 1.1 eq.), sodium bicarbonate (622 mg, 7.4 mmol, 1.0 eq.) and ruthenium dodecacarbonyl (236 mg, 0.37 mmol, 0.05 eq.) in toluene (50 ml) was stirred at 120 ℃ overnight under nitrogen. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: petroleum ether) to give 4-bromo-2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] [1,3] dioxolane (0.66 g, yield: 24.4%) as a yellow solid.
Step 33 c: 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Preparation of Dioxolan-4-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (Compound 1004-57) 4-bromo-2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] under Nitrogen protection][1,3]Dioxolane (1002-57) (660 mg, 1.8 mmol, 1.0 eq), tert-butyl 4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -3, 6-dihydropyridine-1 (2H) -carboxylate (612 g, 1.98 mmol, 1.1 eq), [1,1' -bis (diphenylphosphino) ferrocene]A dioxane/water (10/1 ml) mixture of palladium dichloride (132 mg, 0.18 mmol, 0.1 eq) and sodium carbonate (572 mg, 5.4 mmol, 3.0 eq) was stirred at 90 ℃ overnight. After cooling to room temperature, water and ethyl acetate were added. The mixture was separated and the organic layer was concentrated in vacuo. The residue was purified by silica gel column chromatography (washing)The agent removing method comprises the following steps: petroleum ether/ethyl acetate 200/1 to 30/1) to yield 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] as a yellow solid][1,3]Dioxolan-4-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (680 mg, yield: 80.5%). Lcms (esi): m/z 468(M + H)+.
Step 33 d: preparation of 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] [1,3] dioxolan-4-yl) -1,2,3, 6-tetrahydropyridine hydrochloride (Compound 1005-57) A4 molar solution of 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] [1,3] dioxolan-4-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (1004-57) (100 mg, 0.21 mmol, 1.0 eq) in dioxane (4 mL) hydrochloride was stirred at room temperature overnight. The solvent was spun off in vacuo to give 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] [1,3] dioxolan-4-yl) -1,2,3, 6-tetrahydropyridine hydrochloride as a yellow solid which was used in the next step without further purification.
Step 33 e: 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) -3, 6-dihydropyridin-1 (2H) -yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compounds 1006-57) by reacting 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) -1,2,3, 6-tetrahydropyridine hydrochloride (1005-57) (80 g, 0.2 mmol, 1.0 eq), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A solution of methyl imidazole-6-carboxylate (0116-3) (35 g, 0.12 mmol, 0.6 equiv.) and potassium carbonate (138 mg, 1.0 mmol, 5.0 equiv.) in acetonitrile (6 ml) was stirred at 60 ℃ overnight. After cooling to room temperature, ethyl acetate and water were added, and the mixture was partitioned. The organic layer was concentrated and the residue was purified by silica gel thin layer chromatography preparative plate (eluent: ethyl acetate/methanol-30/1) to give 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] as a white solid][1,3]Dioxolan-4-yl) -3, 6-dihydropyridin-1 (2H) -yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (55 mg, yield: 44.4%). Lcms (esi): 626(M + H) M/z+.
Step 33 f: 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-) Methylbenzo [ d ]][1,3]Dioxolan-4-yl) -3, 6-dihydropyridin-1 (2H) -yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 57) 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) -3, 6-dihydropyridin-1 (2H) -yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]A mixture of methyl imidazole-6-carboxylate (1006-57) (55 mg, 0.09 mmol, 1.0 eq) and lithium hydroxide monohydrate (19 mg, 0.45 mmol, 5.0 eq) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. After cooling to room temperature, water was added and 1.0 mol/l hydrochloric acid was added to adjust the pH to 6-7. Dichloromethane was added for extraction and the organic layer was concentrated in vacuo. The residue was purified by preparative silica gel thin layer chromatography on preparative plates (eluent: dichloromethane/methanol-12/1) to give 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] as a white solid][1,3]Dioxolan-4-yl) -3, 6-dihydropyridin-1 (2H) -yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (40 mg, yield: 74.1%). Lcms (esi): m/z 612(M + H)+.1H NMR(500MHz,DMSO)δ12.53(s,1H),8.25(s,1H),8.21(d,J=2.2Hz,1H),7.82(d,J=8.4Hz,1H),7.65(d,J=8.1Hz,1H),7.43(d,J=8.1Hz,1H),7.36(d,J=8.1Hz,1H),7.06(d,J=2.2Hz,1H),6.90–6.79(m,3H),6.39(d,J=3.5Hz,1H),5.11–5.02(m,1H),4.83–4.74(m,1H),4.64(d,J=13.1Hz,1H),4.49–4.40(m,1H),4.34(tt,J=11.8,5.9Hz,1H),4.06(d,J=13.7Hz,1H),3.92(d,J=13.6Hz,1H),3.25–3.19(m,2H),2.75(s,2H),2.65(dd,J=17.6,8.4Hz,1H),2.54(s,1H),2.47–2.35(m,2H),2.15(s,3H).
Example 34: 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] [1,3] dioxolan-4-yl) piperidin-1-yl) methyl) -3- (((S) -oxetan-2-yl) methyl) -3H-imidazo [4,5-b ] pyridine-5-carboxylic acid (Compound 58) (prepared according to scheme ten lines)
Step 34 a: 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -3- (((S) -oxetan-2-yl) methyl) -3H-imidazo [4,5-b]Preparation of pyridine-5-carboxylic acid methyl ester (Compounds 1006-58) by reacting 4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) piperidine hydrochloride (1005-56) (33 mg, 0.08 mmol, 1.0 equiv.) and (S) -2- (chloromethyl) -3- (oxetan-2-ylmethyl) -3H-imidazo [4, 5-b-]Pyridine-5-carboxylic acid methyl ester (0116-1) (24 mg, 0.08 mmol, 1.0 eq) to a mixture of acetonitrile (4 ml) was added potassium carbonate (28 mg, 0.20 mmol, 2.5 eq). The mixture was heated at 60 ℃ overnight. The solvent was removed under reduced pressure. The residue was purified by preparative thin layer chromatography (ethyl acetate: methanol 60: 1) to give 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d ] as a pale yellow solid][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -3- (((S) -oxetan-2-yl) methyl) -3H-imidazo [4,5-b]Pyridine-5-carboxylic acid methyl ester (29 mg, yield: 57%). Lcms (esi): m/z 629[ M +1 ]]+(ii) a TLC: rf0.5 (dichloromethane: methanol ═ 30: 1).
Step 34 b: 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d) ][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -3- (((S) -oxetan-2-yl) methyl) -3H-imidazo [4,5-b]Preparation of pyridine-5-carboxylic acid (Compound 58) by reacting 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -3- (((S) -oxetan-2-yl) methyl) -3H-imidazo [4,5-b]Pyridine-5-carboxylic acid methyl ester (1006-58) (29 mg, 0.05 mmol, 1.0 equiv.) to a mixture of tetrahydrofuran (4 ml) and water (2 ml) was added lithium hydroxide monohydrate (6 mg, 0.15 mmol, 3.0 equiv). The mixture was heated at 40 ℃ overnight. The mixture was diluted with water (15 ml). A 1N diluted hydrochloric acid solution was added to adjust pH 5 and then the aqueous layer was extracted with ethyl acetate (20 ml × 3). The combined organic layers were washed with saturated brine (20 ml × 1), dried over anhydrous sodium sulfate and concentrated. The residue was purified by preparative thin layer chromatography (dichloromethane: methanol ═ 6: 1) to give 2- ((4- (2- (4-chlorobenzofuran-7-yl) -2-methylbenzo [ d) as a white solid][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -3- (((S) -oxetan-2-yl) methyl) -3H-imidazo [4,5-b]Pyridine-5-carboxylic acid (18 mg, yield: 64%). Lcms (esi): m/z 615[ M +1 ]+(ii) a TLC: rf0.5 (dichloromethane: methanol ═ 6: 1); melting point: 129 ℃ and 132 ℃.1HNMR(500MHz,DMSO)δ8.22(s,1H),8.08(s,1H),7.99(d,J=7.5Hz,1H),7.44(d,J=8.0Hz,1H),7.37(d,J=8.0Hz,1H),7.06(s,1H),6.76(d,J=11.1Hz,3H),5.19(s,1H),4.82(s,1H),4.72(s,1H),4.45(s,1H),4.36(s,1H),3.98(s,1H),3.87(d,J=13.9Hz,1H),2.99(s,1H),2.90(s,1H),2.67(s,2H),2.52(s,1H),2.24(dd,J=26.5,14.2Hz,2H),2.17(d,J=12.3Hz,3H),1.87–1.63(m,4H).
Example 35: preparation of 2- ((4- (2- (4-cyanobenzofuran-7-yl) -2-methylbenzo [ d ] [1,3] dioxolan-4-yl) piperidin-1-yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 61) (preparation according to scheme ten lines)
Step 35 a: preparation of 7-ethynylbenzofuran-4-carbonitrile (compound 1001-61):
a mixture of 7- (hydroxymethyl) benzofuran-4-carbonitrile (441 mg, 2.55 mmol, 1.0 eq) and manganese dioxide (3.33 g, 38.26 mmol, 15.0 eq) in dichloromethane (10 ml) was stirred at room temperature overnight. The reaction solution was filtered through celite and washed with dichloromethane (15 ml). And concentrating the filtrate under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to give 7-formylbenzofuran-4-carbonitrile (288 mg, yield: 65.7%) as a white solid. Lcms (esi): m/z 172(M + H)+.
A mixture of 7-formylbenzofuran-4-carbonitrile (251 mg, 1.47 mmol, 1.0 eq), (1-diazo-2-oxopropyl) phosphonic acid dimethyl ester (338 mg, 1.76 mmol, 1.2 eq) and potassium carbonate (405 mg, 2.94 mmol, 2.0 eq) in methanol (6 ml) was stirred overnight under nitrogen atmosphere. The reaction was quenched with water and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to give 7-ethynylbenzofuran-4-carbonitrile (228 mg, yield: 92.3%) as a white solid. Lcms (esi): m/z 168(M + H) +.
Step (ii) of35 b: 4- (2- (4-cyanobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Preparation of Dioxolan-4-yl) piperidine-1-carboxylic acid tert-butyl ester (Compounds 1004-61): a mixture of 7-ethynylbenzofuran-4-carbonitrile (1001-61) (228 mg, 1.37 mmol, 1.0 eq), tert-butyl 4- (2, 3-dihydroxyphenyl) piperidine-1-carboxylate (1003-56) (442 mg, 1.51 mmol, 1.1 eq), sodium bicarbonate (115 mg, 1.37 mmol, 1.0 eq) and triruthenium dodecacarbonyl (44 mg, 0.07 mmol, 0.05 eq) in toluene (6 ml) was refluxed at 120 ℃ overnight under nitrogen. After cooling to room temperature, the reaction solution was filtered through celite, and washed with ethyl acetate (12 ml). The filtrate was diluted with water (15 ml) and extracted with ethyl acetate (10 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate 10/1) to give 4- (2- (4-cyanobenzofuran-7-yl) -2-methylbenzo [ d ] as a white solid][1,3]Dioxolan-4-yl) piperidine-1-carboxylic acid tert-butyl ester (80 mg, yield: 12.7%). Lcms (esi): m/z 461(M + H)+.
Step 35 c: 7- (2-methyl-4- (piperidin-4-yl) benzo [ d ][1,3]Preparation of Dioxolan-2-yl) benzofuran-4-carbonitrile hydrochloride (Compounds 1005-61): to 4- (2- (4-cyanobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) piperidine-1-carboxylic acid tert-butyl ester (1004-61) (80 mg, 0.17 mmol, 1.0 equiv.) in dioxane (2 ml) was added a dioxane solution of hydrogen chloride (4.0 mol/l, 4 ml) and the mixture was stirred at room temperature overnight. Removing the solvent under reduced pressure to obtain a crude product of 7- (2-methyl-4- (piperidin-4-yl) benzo [ d][1,3]Dioxolan-2-yl) benzofuran-4-carbonitrile hydrochloride. The product was used in the next step without further purification. Lcms (esi): m/z 361(M + H)+.
Step 35 d: 2- ((4- (2- (4-cyanobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid methyl ester (Compound 1006-61) by reacting 7- (2-methyl-4- (piperidin-4-yl) benzo [ d][1,3]Dioxolan-2-yl) benzofuran-4-carbonitrile hydrochloride (1005-61) (80 mg, assumed to be 0.17 mmol1.0 equivalent), (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of methyl imidazole-6-carboxylate (0116-3) (52 mg, 0.17 mmol, 1.0 equiv.) and potassium carbonate (96 mg, 0.70 mmol, 4.0 equiv.) in acetonitrile (5 ml) was stirred at 60 ℃ for 4 h. The reaction mixture was cooled to room temperature, and water was added thereto, followed by extraction with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative silica gel thin layer chromatography (eluent: petroleum ether/ethyl acetate 1/10) to give 2- ((4- (2- (4-cyanobenzofuran-7-yl) -2-methylbenzo [ d ] as a white solid ][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (50 mg, yield: 46.7%). Lcms (esi): m/z 619(M + H)+.
Step 35 e: 2- ((4- (2- (4-cyanobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Preparation of imidazole-6-carboxylic acid (Compound 61) 2- ((4- (2- (4-cyanobenzofuran-7-yl) -2-methylbenzo [ d)][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]A mixture of imidazole-6-carboxylic acid methyl ester (1006-61) (50 mg, 0.08 mmol, 1.0 eq) and lithium hydroxide monohydrate (7 mg, 0.16 mmol, 2.0 eq) in acetonitrile/water 5/1(3 ml) was stirred at 40 ℃ overnight. After the mixture was cooled to room temperature, the pH was adjusted to about 6 with 1 mol/l hydrochloric acid, and then a solid precipitate was formed. The slurry was diluted with water (12 ml), stirred for 4 hours, and the solid was collected by filtration. The solid was washed with water and then dried under vacuum. The solid was dissolved in dichloromethane/methanol 1/2(5 ml), then filtered, the filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was dried in vacuo to give 2- ((4- (2- (4-cyanobenzofuran-7-yl) -2-methylbenzo [ d) as a white solid ][1,3]Dioxolan-4-yl) piperidin-1-yl) methyl) -1- (((S) -oxetan-2-yl) methyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (40 mg, yield: 81.6%). Lcms (esi): m/z 605(M + H)+.1H NMR(500MHz,DMSO)δ12.72(s,1H),8.38(dd,J=7.9,2.2Hz,1H),8.27(d,J=3.0Hz,1H),7.82(t,J=7.9Hz,2H),7.64(d,J=8.3Hz,1H),7.59(dd,J=7.9,2.9Hz,1H),7.24(t,J=2.1Hz,1H),6.79(dd,J=6.6,3.4Hz,2H),6.77–6.73(m,1H),5.12(tt,J=12.1,3.6Hz,1H),4.78(dd,J=14.6,6.6Hz,1H),4.65(dd,J=12.5,4.7Hz,1H),4.50–4.42(m,1H),4.37(td,J=12.2,6.1Hz,1H),3.96(dd,J=13.5,4.7Hz,1H),3.79(d,J=13.6Hz,1H),3.02(d,J=9.2Hz,1H),2.87(d,J=8.9Hz,1H),2.68(dt,J=18.9,6.5Hz,2H),2.53(d,J=8.0Hz,1H),2.48–2.42(m,1H),2.25(d,J=10.9Hz,1H),1.74(dd,J=29.5,15.3Hz,4H).
Example 36 biological Activity assay
The control compound used in the present invention was PF-06882961, having the following structure:
furthermore, the present invention also synthesized the following 4 control compounds:
the synthesis method comprises the following steps:
preparation of (S) -2- ((6- (benzo [ d ] [1,3] dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 24067)
Step 1-1: preparation of 2- (benzo [ d ] [1,3] dioxolan-5-ylmethoxy) -6-bromopyridine:
under the protection of nitrogen, (benzo [ d ] is][1,3]Dioxolan-5-yl) methanol (304 mg, 2.0 mmol, 1.0 eq) was added to anhydrous tetrahydrofuran (10 ml) and cooled to 0 ℃. 1M solution of potassium hexamethyldisilazide in tetrahydrofuran (4.0 mL, 4.0 mmol, 2.0 equiv.) was added dropwise to the mixture, with stirring at 0 deg.CStirring for one hour. A solution of 2-bromo-6-fluoropyridine (388 mg, 2.2 mmol, 1.1 eq) in tetrahydrofuran was added dropwise to the mixture and stirred at room temperature for one hour. Quenched by addition of aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, and dried over anhydrous sodium sulfate. Concentrating under reduced pressure, and purifying with silica gel column chromatography (eluent: petroleum ether/ethyl acetate 20/1) to obtain white solid 2- (benzo [ d) ][1,3]Dioxolan-5-ylmethoxy) -6-bromopyridine (600 mg, yield: 97.4%). Lcms (esi): m/z 308(M + H)+.
Step 1-2: preparation of tert-butyl 6- (benzo [ d ] [1,3] dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -carboxylate:
under the protection of nitrogen, 2- (benzo [ d ]][1,3]Dioxolan-5-ylmethoxy) -6-bromopyridine (600 mg, 1.95 mmol, 1.0 equivalent), tert-butyl 4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 6-dihydropyridine-1 (2H) -carboxylate (723 mg, 2.34 mmol, 1.2 equivalents), dichlorobis (triphenylphosphine) palladium (69 mg, 0.098 mmol, 0.05 equivalent), and sodium carbonate (414 mg, 3.9 mmol, 2.0 equivalents) were added to a mixed solvent of toluene (20 ml), ethanol (10 ml), and water (10 ml), and the reaction was stirred at 110 ℃ for two hours. Extracting with ethyl acetate, washing with water and saturated brine, drying over anhydrous sodium sulfate, concentrating under reduced pressure, and purifying with silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1-3/1) to obtain anhydrous oily substance 6- (benzo [ d ]][1,3]Dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -carboxylic acid tert-butyl ester (815 mg, yield: 96%). Lcms (esi): m/z 411(M + H) +。
Step 1-3: preparation of methyl (S) -2- ((6- (benzo [ d ] [1,3] dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridinyl ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylate:
at room temperature, reacting the 6- (benzo [ d ]][1,3]Dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine]Tert-butyl (1 '(2' H) -carboxylate (200 mg, 0.49 mmol, 1.8 eq.) was dissolved in 5 ml dioxane, 1 ml of 4M dioxane solution of hydrogen chloride was added and the mixture was stirred at room temperature for 50 minutes. The mixture was concentrated to dryness under reduced pressure, and the residue was added to 5 ml of N-methylpyrrolidone. 1 ml of diisopropylethylamine and (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] was added]Imidazole-6-carboxylic acid methyl ester (80 mg, 0.27 mmol, 1.0 equiv.) the mixture was stirred at 60 ℃ overnight. Adding ethyl acetate, washing with water and saturated salt water, drying over anhydrous sodium sulfate, concentrating the organic phase under reduced pressure, and purifying by preparative thin layer chromatography (developing solvent: petroleum ether/ethyl acetate 1/1) to give (S) -2- ((6- (benzo [ d ] b) as a yellow solid][1,3]Dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine ]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (46 mg, yield: 29.8%). Lcms (esi): m/z 569(M + H)+。
Step 1-4 preparation of (S) -2- ((6- (benzo [ d ] [1,3] dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridinyl ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (Compound 24067):
reacting (S) -2- ((6- (benzo [ d ])][1,3]Dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Methyl imidazole-6-carboxylate (46 mg, 0.081 mmol, 1.0 equiv.) and lithium hydroxide monohydrate (11 mg, 0.243 mmol, 3.0 equiv.) were added to a mixed solvent of 5 ml of acetonitrile and 1 ml of water, and the mixture was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and,and concentrated under reduced pressure. Dilute sulfuric acid was added to adjust the pH of the residue to 6, ethyl acetate was added for extraction, and the mixture was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol ═ 15/1) to give (S) -2- ((6- (benzo [ d ] b) a yellow solid][1,3]Dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine ]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (22 mg, yield: 48.8%). Lcms (esi): m/z 555(M + H)+Melting point: 144 to 146 ℃.1H NMR(500MHz,DMSO)δ12.71(s,1H),8.26(s,1H),7.81(d,J=8.4Hz,1H),7.65(dd,J=8.0,5.8Hz,2H),7.06(d,J=7.5Hz,1H),7.00(s,1H),6.93(d,J=8.0Hz,1H),6.87(d,J=7.9Hz,1H),6.75(s,1H),6.68(d,J=8.2Hz,1H),5.99(s,2H),5.26(s,2H),5.06(dt,J=7.2,4.8Hz,1H),4.80(dd,J=15.2,7.2Hz,1H),4.66(dd,J=15.2,2.3Hz,1H),4.47(dd,J=13.7,7.6Hz,1H),4.36(dt,J=8.9,5.9Hz,1H),4.07(d,J=13.5Hz,1H),3.93(d,J=13.5Hz,1H),3.21(d,J=17.6Hz,2H),2.81–2.71(m,2H),2.66(dt,J=16.3,7.7Hz,1H),2.54(s,2H),2.44–2.34(m,1H).
Preparation of (S) -2- ((6- (2, 2-difluorobenzo [ d ] [1,3] dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (compound 24068)
Step 2-1: preparation of 2-bromo-6- (2, 2-difluorobenzo [ d ] [1,3] dioxolan-5-ylmethoxy) pyridine:
under the protection of nitrogen, 2-difluorobenzo [ d ]][1,3]Dioxolan-5-ylmethanol (376 mg, 2.0 mmol, 1.0 eq.) was added to anhydrous tetrahydrofuran (10 mL) and cooled to 0 ℃. 1M solution of hexamethyldisilazane in tetrahydrofuran (4.0 ml, 4.0 mmol, 2.0 equiv.) was added dropwise to the mixture, and stirred at 0 ℃ for one hour. 2-bromo-6-fluoropyridine (388 mg, 2.2 mmol, 1.1 eq) in tetrahydrofuran was added dropwise to the mixture, inStirred at room temperature for one hour. Quenched by addition of aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, and dried over anhydrous sodium sulfate. Concentrating under reduced pressure, and purifying with silica gel column chromatography (eluent: petroleum ether/ethyl acetate 20/1) to obtain colorless solid 2-bromo-6- (2, 2-difluorobenzo [ d ] ][1,3]Dioxolan-5-ylmethoxy) pyridine (626 mg, yield: 90.9%). Lcms (esi): 344(M + H) is given M/z+.
Step 2-2: preparation of tert-butyl 6- (2, 2-difluorobenzo [ d ] [1,3] dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -carboxylate:
under the protection of nitrogen, 2-bromo-6- (2, 2-difluorobenzo [ d ]][1,3]Dioxolan-5-ylmethoxy) pyridine (626 mg, 1.82 mmol, 1.0 equivalent), tert-butyl 4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 6-dihydropyridine-1 (2H) -carboxylate (675 mg, 2.18 mmol, 1.2 equivalents), bis (triphenylphosphine) palladium dichloride (64 mg, 0.09 mmol, 0.05 equivalent), and sodium carbonate (386 mg, 3.64 mmol, 2.0 equivalents) were added to a mixed solvent of toluene (20 ml), ethanol (10 ml), and water (10 ml), and the reaction was stirred at 110 ℃ for two hours. Extracting with ethyl acetate, washing with water and saturated brine, drying over anhydrous sodium sulfate, concentrating the organic phase under reduced pressure, and purifying with silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1-3/1) to give 6- (2, 2-difluorobenzo [ d ] a yellow solid][1,3]Dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -carboxylic acid tert-butyl ester (800 mg, yield: 98.4%). Lcms (esi): m/z 447(M + H) +。
Step 2-3: preparation of methyl (S) -2- ((6- (2, 2-difluorobenzo [ d ] [1,3] dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylate:
at room temperature, 6- (2, 2-difluorobenzo [ d ]][1,3]Dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine]Tert-butyl (1 '(2' H) -carboxylate (300 mg, 0.67 mmol, 3.3 eq.) was dissolved in 5 ml dioxane, 1 ml of 4M dioxane solution of hydrogen chloride was added and the mixture was stirred at room temperature for 60 minutes. The mixture was concentrated to dryness under reduced pressure, and the residue was added to 5 ml of N-methylpyrrolidone. 1 ml of diisopropylethylamine and (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] was added]Imidazole-6-carboxylic acid methyl ester (60 mg, 0.20 mmol, 1.0 eq) and the mixture was stirred at 60 ℃ overnight. Adding ethyl acetate, washing with water and saturated salt water, drying with anhydrous sodium sulfate, concentrating the organic phase under reduced pressure, and purifying by preparative thin layer chromatography (developing solvent: ethyl acetate) to obtain (S) -2- ((6- (2, 2-difluorobenzo [ d ]) as yellow oil][1,3]Dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine ]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (57 mg, yield: 46.3%). Lcms (esi): m/z 605(M + H)+。
Step 2-4: preparation of (S) -2- ((6- (2, 2-difluorobenzo [ d ] [1,3] dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (compound 24068):
reacting (S) -2- ((6- (2, 2-difluorobenzo [ d ]][1,3]Dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Methyl imidazole-6-carboxylate (57 mg, 0.094 mmol, 1.0 equiv.) and lithium hydroxide monohydrate (12 mg, 0.284 mmol, 3.0 equiv.) were added to a mixed solvent of 5 ml of acetonitrile and 1 ml of water, and the mixture was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. Adjusting the residue by adding dilute sulfuric acidAfter the pH of the mixture was adjusted to 6, ethyl acetate was added thereto and the mixture was extracted, followed by washing with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol ═ 12/1) to give (S) -2- ((6- (2, 2-difluorobenzo [ d ] as a yellow solid ][1,3]Dioxolan-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (36 mg, yield: 64.8%). Lcms (esi): 591(M + H) M/z+Melting point: 102-104 ℃.1H NMR(500MHz,DMSO)δ12.69(s,1H),8.26(s,1H),7.81(d,J=8.4Hz,1H),7.67(dd,J=12.3,7.9Hz,2H),7.49(s,1H),7.38(d,J=8.2Hz,1H),7.30(d,J=8.2Hz,1H),7.07(d,J=7.4Hz,1H),6.78–6.68(m,2H),5.36(s,2H),5.07(d,J=7.1Hz,1H),4.80(dd,J=15.1,7.2Hz,1H),4.65(d,J=14.8Hz,1H),4.46(dd,J=13.9,6.9Hz,1H),4.36(dd,J=14.2,6.2Hz,1H),4.07(d,J=13.5Hz,1H),3.93(d,J=13.5Hz,1H),3.21(d,J=17.1Hz,2H),2.75(s,2H),2.66(dd,J=17.6,7.9Hz,1H),2.52(s,2H),2.39(dd,J=17.6,8.5Hz,1H).
Preparation of (S) -2- ((6- ((1-methyl-1H-benzo [ d ] imidazol-6-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (compound 24069)
Step 3-1: preparation of (1-methyl-1H-benzo [ d ] imidazol-6-yl) methanol:
under the protection of nitrogen, 1-methyl-1H-benzo [ d]Imidazole-6-carboxylic acid (300 mg, 1.7 mmol, 1.0 eq) was added to anhydrous tetrahydrofuran (10 ml) and cooled to 0 ℃. Lithium aluminum hydride (260 mg, 6.8 mmol, 4.0 equiv) was added to the mixture and stirred at room temperature for three hours. Dropwise adding 0.26 ml of water, 0.26 ml of 15% sodium hydroxide aqueous solution and 0.78 ml of water in sequence under ice bath, heating to room temperature, adding anhydrous sodium sulfate, stirring, suction filtering, concentrating the filtrate under reduced pressure, and purifying by silica gel column chromatography (eluent: dichloromethane/methanol-20/1) to obtain white solid (1-methyl-1H-Benzo [ d ] carbonyl ]Imidazol-6-yl) methanol (126 mg, yield: 45.6%). Lcms (esi): 163(M + H) M/z+.
Step 3-2: preparation of 6- (((6-bromopyridin-2-yl) oxy) methyl) -1-methyl-1H-benzo [ d ] imidazole:
under the protection of nitrogen, (1-methyl-1H-benzo [ d)]Imidazol-6-yl) methanol (159 mg, 1.0 mmol, 1.0 eq) was added to anhydrous tetrahydrofuran (10 ml) and cooled to 0 ℃. 1M solution of hexamethyldisilazane in tetrahydrofuran (2.0 ml, 2.0 mmol, 2.0 equiv.) was added dropwise to the mixture, and stirred at 0 ℃ for one hour. A solution of 2-bromo-6-fluoropyridine (194 mg, 1.1 mmol, 1.1 eq) in tetrahydrofuran was added dropwise to the mixture and stirred at room temperature for one hour. Quenched by addition of aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure and purified by column chromatography on silica gel (eluent: dichloromethane/methanol 20/1) to give 6- (((6-bromopyridin-2-yl) oxy) methyl) -1-methyl-1H-benzo [ d ] as a white solid]Imidazole (288 mg, yield: 91.1%). Lcms (esi): m/z 318(M + H)+.
Step 3-3: preparation of tert-butyl 6- (1-methyl-1H-benzo [ d ] imidazol-6-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -carboxylate:
Under the protection of nitrogen, 6- (((6-bromopyridin-2-yl) oxy) methyl) -1-methyl-1H-benzo [ d]Imidazole (288 mg, 0.91 mmol, 1.0 eq), tert-butyl 4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 6-dihydropyridine-1 (2H) -carboxylate (337 mg, 1.09 mmol, 1.2 eq), dichlorobis (triphenylphosphine) palladium (32 mg, 0.046 mmol, 0.05 eq) and sodium carbonate (193 mg, 1.82 mmol, 2.0 eq) were added to toluene(10 ml), ethanol (5 ml) and water (5 ml) in a mixed solvent, and the reaction was stirred at 110 ℃ for two hours. Extraction with ethyl acetate, washing with water and saturated brine, drying over anhydrous sodium sulfate, concentrating under reduced pressure the organic phase, and purification by column chromatography on silica gel (eluent: dichloromethane/methanol-33/1) to give 6- (1-methyl-1H-benzo [ d ] a yellow solid]Imidazol-6-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -carboxylic acid tert-butyl ester (374 mg, yield: 97.3%). Lcms (esi): 421(M + H) M/z+。
Step 3-4: preparation of methyl (S) -2- ((6- (1-methyl-1H-benzo [ d ] imidazol-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylate:
At room temperature, the 6- (1-methyl-1H-benzo [ d)]Imidazol-6-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine]Tert-butyl (1 '(2' H) -carboxylate (187 mg, 0.45 mmol, 2.25 eq) was dissolved in 5 ml dioxane, 1 ml dioxane solution of 4M hydrogen chloride was added and the mixture was stirred at room temperature for 60 min. The mixture was concentrated to dryness under reduced pressure, and the residue was added to 5 ml of N-methylpyrrolidone. 1 ml of diisopropylethylamine and (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] was added]Imidazole-6-carboxylic acid methyl ester (60 mg, 0.20 mmol, 1.0 eq) and the mixture was stirred at 60 ℃ overnight. Ethyl acetate was added, washed with water and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by preparative thin layer chromatography (developing solvent: dichloromethane/methanol-15/1) to give (S) -2- ((6- (1-methyl-1H-benzo [ d ] b) as a yellow oil]Imidazol-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (67 mg, yield: 57.8%). Lcms (esi): m/z 579(M + H)+。
Step 3-5 preparation of (S) -2- ((6- ((1-methyl-1H-benzo [ d ] imidazol-6-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridinyl ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (compound 24069):
Reacting (S) -2- ((6- (1-methyl-1H-benzo [ d ]]Imidazol-5-ylmethoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Methyl imidazole-6-carboxylate (67 mg, 0.116 mmol, 1.0 equiv.) and lithium hydroxide monohydrate (19 mg, 0.46 mmol, 4.0 equiv.) were added to a mixed solvent of 5 ml acetonitrile and 1 ml water, and the mixture was stirred at 40 ℃ overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. Dilute sulfuric acid was added to adjust the pH of the residue to 6, ethyl acetate was added for extraction, and the mixture was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol ═ 12/1) to give (S) -2- ((6- ((1-methyl-1H-benzo [ d) a yellow solid]Imidazol-6-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (41 mg, yield: 62.5%). Lcms (esi): 565(M + H)+Melting point: 135-137 ℃.1H NMR(500MHz,DMSO)δ12.61(s,1H),8.26(s,1H),8.17(s,1H),7.82(dd,J=8.4,1.5Hz,1H),7.70–7.64(m,3H),7.62(d,J=8.2Hz,1H),7.31(dd,J=8.3,1.3Hz,1H),7.06(d,J=7.4Hz,1H),6.78(s,1H),6.71(d,J=8.2Hz,1H),5.49(s,2H),5.07(ddd,J=14.5,7.3,2.8Hz,1H),4.80(dd,J=15.3,7.3Hz,1H),4.66(dd,J=15.2,2.7Hz,1H),4.47(dt,J=14.0,7.1Hz,1H),4.36(dt,J=9.0,5.9Hz,1H),4.08(d,J=13.5Hz,1H),3.97–3.90(m,1H),3.81(s,3H),3.26(d,J=12.6Hz,2H),2.80–2.74(m,2H),2.70–2.62(m,1H),2.60–2.53(m,2H),2.45–2.38(m,1H).
Preparation of (S) -2- ((6- ((2, 3-dihydrobenzo [ b ] [1, 4] dioxin-6-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (compound 24070) step 4-1: preparation of 6-chloro-3 ',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -carboxylic acid tert-butyl ester:
2-bromo-6-chloropyridine (1.0 g, 5.20 mmol, 1.0 eq), 4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (1.6 g, 5.20 mmol, 1.0 eq), 1,1' -bis (diphenylphosphino) ferrocene were reacted under nitrogen protection]Palladium dichloride and sodium carbonate were added to a mixed solvent (11 ml) of dioxane and water (10:1), and stirred overnight at 90 ℃. The reaction was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. Concentrating the organic phase under reduced pressure, and purifying the residue by silica gel column chromatography (eluent: petroleum ether/ethyl acetate: 10/1) to obtain 6-chloro-3 ',6' -dihydro- [2,4' -bipyridine as a yellow solid]-1'(2' H) -carboxylic acid tert-butyl ester (3.26 g, yield: 214%). Lcms (esi): 295(M + H) with M/z+.
Step 4-2: preparation of 6-chloro-1 ',2',3',6' -tetrahydro-2, 4' -bipyridine p-toluenesulfonate salt:
a mixture (50 ml) of 6-chloro-3 ',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -carboxylic acid tert-butyl ester (2.94 mg, 9.99 mmol, 1.0 eq) and p-toluenesulfonic acid (4.3 g, 24.97 mmol, 2.5 mmol) in ethyl acetate was stirred at 60 ℃ for 30 min. The reaction was filtered and the filter cake afforded 6-chloro-1 ',2',3',6' -tetrahydro-2, 4' -bipyridine p-toluenesulfonate as a white solid (232 mg, 12% yield). The product was used in the next step without further purification.
Step 4-3: preparation of methyl (S) -2- ((6-chloro-3 ',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylate:
a mixture of 6-chloro-1 ',2',3',6' -tetrahydro-2, 4' -bipyridine p-toluenesulfonate (232 mg, 0.634 mmol, 1.2 eq) and potassium carbonate (291 mg, 2.11 mmol, 4.0 equiv) in acetonitrile (10 ml) was heated to 60 ℃ until the pH reached 7-8, and then (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] (S) -2- (chloromethyl) -1- (oxetan-2-ylmethyl) -1]Methyl imidazole-6-carboxylate (150 mg, 0.528 mmol, 1.0 mono amount) was added to the mixture and stirred overnight. After cooling to room temperature, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. Concentrating the organic phase under reduced pressure to obtain (S) -2- ((6-chloro-3 ',6' -dihydro- [2,4' -bipyridine)]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Methyl imidazole-6-carboxylate (188.5 mg, yield: 81.74%) was a yellow solid. Lcms (esi): m/z 453.9(M + H)+.
Step 4-4: preparation of methyl (S) -2- ((6- ((2, 3-dihydrobenzo [ b ] [1, 4] dioxin-6-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylate:
Mixing (S) -2- ((6-chloro-3 ',6' -dihydro- [2,4' -bipyridine)]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (188.5 mg, 0.417 mmol, 1.0 eq), (2, 3-dihydrobenzo [ b ]][1,4]A mixture of dioxin-6-yl) methanol (83.1 mg, 0.500 mmol, 1.2 equiv.), tris (dibenzylideneacetone) dipalladium (19.23 mg, 0.021 mmol, 0.05 equiv.), 2-dicyclohexylphosphine-2 ',6' -diisopropoxybiphenyl (19.46 mg, 0.0417 mmol, 0.1 equiv.) and cesium carbonate (382.86 mg, 1.175 mmol, 2.5 equiv.) in toluene (70 ml) was stirred at 120 ℃ overnight. After cooling to room temperature, the reaction was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by preparative thin layer chromatography on silica gel (eluent): petroleum ether/ethyl acetate 1/2 to obtain yellow solid compound (S) -2- ((6- ((2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid methyl ester (20 mg, 8.44% yield). Lcms (esi): m/z 583.6(M + H)+.
And 4-5: preparation of (S) -2- ((6- ((2, 3-dihydrobenzo [ b ] [1, 4] dioxin-6-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d ] imidazole-6-carboxylic acid (compound 24070):
Reacting (S) -2- ((6- ((2, 3-dihydrobenzo [ b ]][1,4]Dioxin-6-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]A mixture of methyl imidazole-6-carboxylate (20 mg, 0.034 mmol, 1.0 eq) and lithium hydroxide monohydrate (2.85 mg, 0.068 mmol, 2.0 parts) in acetonitrile/water 5/1(6 ml) was stirred at 40 ℃ overnight. The mixture was then cooled to room temperature and the pH was adjusted to about 6 by the addition of 1.0M sulfuric acid, after which a solid precipitate formed. The mixture was filtered. The residue was washed with water. The mixture was slurried with methanol to give (S) -2- ((6- ((2, 3-dihydrobenzo [ b ] b) as a yellow solid][1,4]Dioxin-6-yl) methoxy) -3',6' -dihydro- [2,4' -bipyridine]-1'(2' H) -yl) methyl) -1- (oxetan-2-ylmethyl) -1H-benzo [ d]Imidazole-6-carboxylic acid (18 mg, 92.4% yield). Lcms (esi): m/z 569(M + H)+.1H NMR(500MHz,DMSO)δ12.67(s,1H),8.26(s,1H),7.81(dd,J=8.4,1.2Hz,1H),7.65(t,J=8.2Hz,2H),7.05(d,J=7.5Hz,1H),6.92(d,J=1.8Hz,1H),6.89(dd,J=8.2,1.9Hz,1H),6.81(d,J=8.2Hz,1H),6.74(s,1H),6.67(d,J=8.2Hz,1H),5.24(s,2H),5.07(qd,J=7.2,2.8Hz,1H),4.80(dd,J=15.2,7.3Hz,1H),4.65(dd,J=15.2,2.6Hz,1H),4.47(dd,J=13.6,7.7Hz,1H),4.36(dt,J=8.9,5.9Hz,1H),4.20(s,4H),4.07(d,J=13.5Hz,1H),3.93(d,J=13.5Hz,1H),3.23(s,2H),2.79–2.72(m,2H),2.66(ddd,J=8.8,8.2,5.5Hz,1H),2.54(s,2H),2.40(ddd,J=16.1,11.2,7.1Hz,1H).
Measurement of cyclic adenosine monophosphate (cAMP) level in hGLP-1R 293Ta cell strain
1. Experimental Material
2. Principle of experiment
The cyclic adenosine monophosphate assay kit of Cisbio is a competitive immunoassay method and aims to measure the accumulation of cyclic adenosine monophosphate in cells. The principle is based on
Technology, native cyclic adenosine monophosphate or unlabeled cyclic adenosine monophosphate produced by cells (standard curve) competes with d 2-labeled cyclic adenosine monophosphate (red acceptor) for binding to monoclonal cyclic adenosine monophosphate Europium Cryptate-labeled antibody (Europium donor). The specific signal is inversely proportional to the concentration of cyclic adenosine monophosphate in the standard or sample.
For all other HTRF analyses, the calculation of the fluorescence ratio (665nm/620nm) eliminates any possible photophysical interference, which means that the analysis is not affected by the experimental medium conditions (e.g. medium, serum, biotin, colored compounds, etc.).
3. Preparation of the experiment
1) Constructing a hGLP-1R 293Ta stable transfer cell strain:
a) the hGLP-1R full-length sequence was synthesized by Suzhou Jinzhi Biotechnology Ltd and ligated to the lentiviral vector pLEX-MCS.
b) Transfecting 293Ta cells with a lentivirus plasmid pLEX-MCS connected with an hGLP-1R sequence to generate lentiviruses respectively;
c) the 293Ta cell is infected by lentivirus, and is screened by puromycin to obtain the 293Ta stable cell line containing the hGLP-1R sequence.
2) Preparation of reagents:
a) preparing cyclic adenosine monophosphate detection reagent (the reagent needs to be placed at room temperature for 30min)
b) 3-isobutyl-1-methylxanthine: 3-isobutyl-1-methylxanthine (500mM) was diluted to 50mM in DMSO and dispensed into 20ul tubes and stored at 4 ℃. 20ul plus 2ml stimulation Buffer 1 diluted to 0.5mM was used.
4. Cell experiments
1) Compound dilution 2mM stock solution was diluted to 200nM (40X) in DMSO, 2ul was diluted to 10nM (2X) with 38ul of stimulating Buffer 1 (containing 3-isobutyl-1-methylxanthine 0.5mM) and the final concentration of the reaction was 5 nM. The samples were diluted 5-fold in sequence with stimulation Buffer 1 (containing 0.5mM 3-isobutyl-1-methylxanthine) for 8 concentration points.
2) The hGLP-1R 293Ta cells were harvested by digestion centrifugation, resuspended cells were blown up by adding DMEM medium (containing 10% FBS), and counted using a Scepter automated cell counter (Millipore # PHCC 00000). Adjusting the number of cells to 1X106cells/ml, 96-well microwell plates were loaded with 5ul per well (i.e., 5000 cells per well).
3) To 96 wells 5ul of diluted 8 concentration gradient 2X compound was added, and to one well 5ul of stimulating Buffer 1 (containing 0.5mM 3-isobutyl-1-methylxanthine) was added as a control. Cover the flask and incubate at 37 ℃ for 30 min.
4) 50ul of cyclic adenosine monophosphate-d 2 and 50ul of anti-cyclic adenosine monophosphate Eu-Cryptate are respectively added into 200ul of lysis & detection Buffer 1 to prepare working solution. Adding 5ul of adenosine cyclophosphate-d 2 working solution into each hole of a 96-hole plate, and then adding 5ul of Anti-adenosine cyclophosphate Eu-Cryptate working solution. Mixing, sealing 96-well plate with sealing plate, and incubating at room temperature for 2 hr.
5) The plate-sealing membrane was removed and the HTRF signal was read using a diken INFINITE F NANO + microplate reader. The Ratio of acceptor and donor excitation signals for each single well was calculated using the formula Ratio Signal 665nm/Signal 620nm 10000. Data were processed with GraphPad Prism software to calculate EC50 values by sigmoidal dose-response curve fitting, with HTRF signal ratios and corresponding compound concentrations. The test results are shown in table 1.
TABLE 1 determination of the Effect of Compounds on the cellular Cyclic adenosine monophosphate levels of hGLP-1R 293Ta
As can be seen from the above table, the compounds of the invention can activate cAMP levels in hGLP-1R 293Ta cells and are more active than the control compounds PF-06882961, 24067, 24068, 24069 and 24070.
Second, Pharmacokinetic (PK) experiments
1. Experimental method
Male SD rats, weighing 180-. The test compound was dissolved in 30% sulfobutyl-beta-cyclodextrin (SBE-beta-CD) and administered by single gavage at 20 mg/kg. Blood was collected from the tail end fracture at 15 minutes, 30 minutes, and 1, 2, 4, 6, 8, and 24 hours after administration, about 0.3ml per time point, placed in a centrifuge tube containing K2-EDTA, centrifuged (2000g, 10 minutes, 4 ℃) to collect plasma, and stored in an ultra-low temperature refrigerator at-70 ℃ to-80 ℃. A50. mu.L sample of plasma was vortexed with 135. mu.L acetonitrile (containing an internal standard of 0.5. mu.g/mL) for protein precipitation, centrifuged, and the supernatant was analyzed by LC-MS/MS.
2. Results of the experiment
After the benzimidazole or azabenzimidazole-6-carboxylic acid compounds provided by the invention are orally administered to rats, the compounds are well absorbed and the blood exposure is high, and the results are shown in figures 1 and 2 and table 2. T of benzimidazole or azabenzimidazole-6-carboxylic acid compound of the present invention max0.5-2.67 hours, CmaxIs 180-2553.33ng/ml, AUC0-24h934.28-9583.02ng/ml x h. CmaxRefers to the maximum blood concentration, T1/2Is half-life, AUC0-24Means the area under the 0-24 hour time-concentration curve, AUC0-infRefers to the area under the 0-Inf time-concentration curve.
TABLE 2 pharmacokinetic parameters of gavage administration (20mg/kg) in rats
Third, pharmacodynamics experiment
1. Experimental methods
Genetically engineered hGLP1R mice, male, 7 weeks, were purchased from Jiangsu Gene biotechnology, Inc., of Baiosai, Inc. The experimental animals are all raised in a horizontal flow independent ventilation cage, the temperature is 20-26 ℃, the relative humidity is 40-70% RH, the ventilation frequency is 15-30 times/hour, the air cleanliness is 7 levels, and the day and night light and shade alternating time is 12h/12 h; continuously supplying complete pellet feed (maintenance feed for big and small mice in the medical experimental animal center of Guangdong province) for the cobalt 60 radiation sterilized mice, and allowing unlimited free intake; drinking tap water (used after high-pressure steam sterilization) is supplied with water continuously and is freely taken. The rearing cage is a transparent polyetherimide cage box (Suzhou Echolin purifying equipment limited, horizontal flow mouse cage box) without pathogenic microorganisms; the padding is corncob (used after high-pressure steam sterilization in Guangdong province medical experimental animal center), 2-5 animals are placed in each cage, and IACUC approval numbers, experiment starting time, subject responsible persons, experimenters, animal sources, groups, animal numbers and the like are marked on cage cards. The animal use method of this trial was approved by the Guangzhou Bifibrate medical technology Co., IACUC.
hGLP1R mouse oral glucose tolerance test: animals are fasted overnight without water, the animals are orally administered with a single dose of a solvent or a corresponding compound, the blood sugar (equivalent to the blood sugar value of-60 min) is measured before the solvent or the compound is administered, the blood sugar (2g/kg) is orally administered after the solvent or the compound is administered for 60 minutes, the blood sugar (equivalent to the blood sugar value of 0 min) is measured before the glucose is administered, and the blood sugar is measured 15min, 30min, 60min, 90min and 120min after the glucose is administered. The vehicle was 40% PEG400+ 60% MCT (0.5% MC/0.1% Tween80) in water, and the compound dose was 0.3mg/kg (n 2-4) and 1mg/kg (n 2-4), respectively.
2. Results of the experiment
As shown in fig. 3 and 4, when compound 12 and compound 16 were orally administered with PF-06882961 at the same dose (1mg/kg) in a single dose, compound 12 and PF-06882961 showed comparable glucose lowering effect in terms of blood glucose curve and blood glucose AUC (0-2 h); the compound 16 has better blood sugar reducing effect than PF-06882961.
As shown in FIG. 5 and FIG. 6, compound 6 and compound 16, together with PF-06882961, showed slightly better hypoglycemic effect than PF-06882961 in the blood glucose curve and blood glucose AUC (0-2h) when administered orally at the same dose (0.3mg/kg) in a single dose; the blood sugar reducing effect of the compound 16 is obviously better than that of PF-06882961.
As shown in fig. 7 and 8, compound 3, compound 16, and compound 58 showed the best hypoglycemic effect of compound 16 in terms of the glycemic profile and glycemic AUC (0-2h) when administered orally in a single dose at the same dose (0.3 mg/kg).
The technical features of the above-mentioned embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the following embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the combinations should be considered as the scope of the present description.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.