TW201734011A - Preparation method of benzofuryl analogue and intermediate and crystal form thereof - Google Patents

Abstract

The present invention discloses a method of benzofuryl analogue and intermediate and crystal form thereof.

Description

Preparation method of benzofuran analog, intermediate and crystal form thereof

The present invention relates to a process for the preparation of benzofuran analogs, as well as intermediates and crystal forms thereof.

HCV is one of the major human pathogens, with an estimated 170 million chronic HCV infections worldwide, five times the number of human immunodeficiency virus type 1 infections. People with chronic HCV infection develop severe progressive liver disease, including cirrhosis and hepatocellular carcinoma. Therefore, chronic HCV infection is the leading cause of death in patients worldwide due to liver disease.

Currently, standard chronic HCV infection therapy is a combination of alpha-interferon and ribavirin with one of the direct-acting antiviral (DAA) drugs approved in the last two years. Although the curative effect is significantly improved compared with the previous combination of α-interferon and ribavirin, it is still ineffective for some patients with chronic HCV infection, and the virus can produce drug resistance. In addition, α-interferon and ribavirin have obvious side effects. Therefore, new and effective drugs for the treatment of chronic HCV infection are urgently needed.

HCV is a single-stranded positive-strand RNA virus. It belongs to a separate genus of the Flaviviridae family. All members of the Flaviviridae are enveloped virions containing a positive-stranded RNA genome that encodes all known virus-specific proteins by translation of a single uninterrupted open reading frame (ORF).

The nucleotides of the HCV genome and the encoded amino acid sequence are quite heterogeneous. At least 6 major genotypes and more than 50 sub-genotypes have been identified. The main genotypes of HCV are distributed globally. Although a large number of genotypes have been studied for pathogenesis and therapeutic effects, the clinical importance of HCV genetic heterogeneity remains unclear.

The invention provides a preparation method of the compound 1, It contains the following steps, Where R is selected from Me, Et, The base B is selected from the group consisting of TMPMgCl•LiCl, TMP2Mg•2LiCl, i-PrMgCl, i-PrMgCl•LiCl, LDA, n-butyllithium; the cyanation reagent is selected from TsCN, , The molar ratio of the compound 8 to the base B is selected from 1:1 to 3; the molar ratio of the compound 8 to the cyanating agent is selected from 1:1 to 3; the solvent B is selected from a single ether solvent or an ether solvent and A mixed solvent of toluene.

In some embodiments of the invention, the solvent B is selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, tert-butyl ether, isopropyl ether, methyl tert-butyl ether, and ethylene glycol dimethyl ether.

In some embodiments of the invention, the solvent B is selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, tert-butyl ether, isopropyl ether, methyl tert-butyl ether, ethylene glycol dimethyl ether and toluene. Solvent.

In some embodiments of the invention, the molar ratio of the above compound 8 to base B is selected from the group consisting of 1:1.2 to 2.5.

In some embodiments of the invention, the molar ratio of the above compound 8 to the cyanation reagent is selected from 1:1.2 to 2.

In some embodiments of the invention, the molar ratio of the above compound 8 to the cyanation reagent is selected from the group consisting of 1:1.3 to 1.6.

In some embodiments of the invention, the above compound 11 is prepared by the following steps, Wherein X is selected from the group consisting of F, Cl, Br and I.

In some aspects of the present invention, the above preparation method further includes the following steps, Wherein the condensing agent is selected from the group consisting of: HATU, HBTU, CDI, HOBT, EDCI, DCC, DIC, PyBOP, DPP-Cl; the base A is selected from the group consisting of: K ₂ CO ₃ , Na ₂ CO ₃ , NaHCO ₃ , NaOH, KOH, TEA, DIPEA, pyridine; solvent A is selected from: dichloromethane, acetonitrile, DMF; optionally, compound 12 and compound 19 are reacted under catalyst catalysis, the catalyst is selected from DMAP; the molar ratio of compound 12 to condensing agent is selected From 1:1 to 3; the molar ratio of compound 12 to compound 19 is selected from 1:1 to 1.5; and the reaction temperature is selected from 10 ° C to 40 ° C.

In some aspects of the present invention, the above preparation method further comprises the following reaction steps, Wherein the base C is selected from the group consisting of K ₂ CO ₃ , Na ₂ CO ₃ , and Cs ₂ CO ₃ ; and the solvent C is selected from the group consisting of DMF and CH ₃ CN.

In some aspects of the present invention, the above preparation method further comprises the following reaction steps, Wherein the base D is selected from the group consisting of K ₃ PO ₄ , K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ ; the solvent D is selected from the group consisting of THF, DMF, toluene, xylene, 1,4-dioxane, water a single solvent or a mixed solvent of two solvents; the catalyst is selected from the group consisting of Pd ₂ (dba) ₃ , Pd (dppf) Cl ₂ , Pd(PPh ₃ ) ₄ , Pd(OAc) ₂ ; the ligand is selected from none or PPh ₃ , three Cyclohexyl hydrazine, tributyl hydrazine.

In some aspects of the invention, the solvent D is selected from the group consisting of a mixed solvent of toluene and water, and the volume ratio of toluene to water is selected from 1 to 3:1.

In some aspects of the present invention, the above preparation method further comprises the following reaction steps, Wherein the base E is selected from the group consisting of LiOH, NaOH, KOH, Na ₂ CO ₃ , and K ₂ CO ₃ ; and the solvent E is selected from the group consisting of tetrahydrofuran, 1,4-dioxane, and acetonitrile.

In some aspects of the invention, the method includes the following reaction steps, The invention also provides a compound of the formula: as an intermediate for the preparation of compound 1: , , , , , .

The present invention also provides Form A of Compound 1, the X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2 theta angles: 10.58 ± 0.2 °, 20.41 ± 0.2 °, 21.98 ± 0.2 °.

In some aspects of the present invention, the X crystal form of the above Compound 1 has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 5.34±0.2°, 10.58±0.2°, 12.70±0.2°, 20.41±0.2°. , 20.74 ± 0.2 °, 21.16 ± 0.2 °, 21.98 ± 0.2 °, 24.55 ± 0.2 °.

In some aspects of the invention, the X-form of Form A of Compound 1 above is shown in Figure 1. In some aspects of the present invention, the X-form analysis data of the A crystal form of the above Compound 1 is shown in Table-1. Table-1 XRPD pattern analysis data of Form A of Compound 1

In some aspects of the invention, the A crystalline form of Compound 1 above has a differential scanning calorimetry curve having a starting point of endothermic peak at 212.07 °C ± 3 °C.

In some aspects of the invention, the A crystal form of the above compound 1 has a DSC spectrum as shown in FIG.

In some embodiments of the present invention, the A crystal form of the above compound 1 has a thermogravimetric analysis curve having a weight loss of 0.3107±0.2% at 120.0±3° C. and a weight loss of 0.2094±0.2% at 232.5±3° C., 233.0±3° C. The weight loss was 0.3501±0.2%.

In some aspects of the invention, the T crystal of Form A of Compound 1 above is shown in Figure 3.

The invention also provides a preparation method of Form A, which comprises adding any one form of Compound 1 to a solvent for recrystallization or beating, wherein the recrystallization solvent or the beating solvent is selected from the group consisting of methanol, ethanol, isopropanol, Dioxane, acetonitrile, tetrahydrofuran, acetone, toluene.

In some aspects of the present invention, the preparation A crystal recrystallization or beating solvent is selected from the group consisting of methanol, ethanol, isopropanol, dioxane, acetonitrile, tetrahydrofuran, acetone, and a mixed solvent of water and water.

Another object of the present invention is to provide a use of Form A in the preparation of a medicament for the treatment of a disease associated with HCV.

Definitions and Descriptions: Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered undefined or unclear without a particular definition, but should be understood in the ordinary sense. When a trade name appears in this document, it is intended to refer to its corresponding commodity or its active ingredient.

The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, combinations thereof with other chemical synthesis methods, and those well known to those skilled in the art. Equivalent alternatives, preferred embodiments include, but are not limited to, embodiments of the invention.

The chemical reaction of a particular embodiment of the invention is carried out in a suitable solvent which is suitable for the chemical changes of the invention and the reagents and materials required thereof. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthetic steps or reaction schemes based on the prior embodiments.

An important consideration in any synthetic route planning in the art is the selection of a suitable protecting group for a reactive functional group, such as an amino group in the present invention. For trained practitioners, Greene and Wuts (Protective Groups In Organic Synthesis, Wiley and Sons, 1991) is the authority in this regard. All references cited in the present invention are incorporated by reference in their entirety.

The invention is specifically described by the following examples, which are not intended to limit the invention.

The invention adopts the following abbreviations: TMPMgCl•LiCl represents lithium dichloride (2,2,6,6-tetramethylacridine); TMP2Mg•2LiCl represents two (2,2',6,6'-four Methyl acridine) magnesium dichloride; DPP-Cl stands for diphenylphosphonium chloride; i-PrMgCl stands for isopropyl magnesium chloride; LDA stands for lithium diisopropylamide; TsCN stands for 4-toluenesulfononitrile; Represents 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HBTU stands for benzotriazole-N,N,N',N '-Tetramethylurea hexafluorophosphate; CDI stands for N,N'-carbonyldiimidazole; DMAP stands for 4-dimethylaminopyridine; HOBT stands for 1-hydroxybenzotriazole; EDCI stands for 1-(3-dimethyl Aminopropyl)-3-ethylcarbodiimide hydrochloride; DCC stands for dicyclohexylcarbodiimide; DIC stands for N,N-diisopropylcarbodiimide; PyBOP stands for benzotrifluorophosphate Zin-1-yl-oxytripyrrolidinylphosphine; DMF stands for N,N-dimethylformamide; DIPEA stands for diisopropylethylamine; TEA stands for triethylamine; THF stands for tetrahydrofuran; Pd ₂ ( dba) ₃ representative of tris (dibenzylideneacetone) dipalladium; Pd (PPh _{3) 4} Representative phosphonium tetrakistriphenylphosphine palladium; Pd (dppf) Cl ₂ representative of 1,1'-bis (diphenylphosphino) bis Ferric chloride, palladium; PPh ₃ Representative triphenylphosphonium; Pd (OAc) ₂ palladium acetate _{Representative; Pd (PPh 3) 2 Cl} 2 dichlorobis Representative (triphenylphosphonium) palladium (II); DIEA Representative N, N - Diisopropylethylamine.

Compounds are named by hand or by ChemDraw® software, and commercial compounds are listed under the supplier's catalogue.

Inventive powder X-ray diffraction (X-ray powder diffractometer, XRPD) method.

Instrument model: Brooke D8 advance X-ray diffractometer.

Test method: Approximately 10-20 mg samples were used for XRPD testing.

The detailed XRPD parameters are as follows.

Light pipe: Cu, kα, (λ=1.54056Ǻ).

Light pipe voltage: 40 kV, light pipe current: 40 mA.

Divergence slit: 0.60 mm.

Detector slit: 10.50 mm.

Anti-scatter slit: 7.10 mm.

Scan range: 4-40 deg.

Step size: 0.02 deg.

Step size: 0.12 seconds.

Sample tray speed: 15 rpm.

The differential scanning Calorimeter (DSC) method of the present invention.

Instrument model: TA Q2000 Differential Scanning Calorimeter

Test method: The sample (~ 1 mg) was placed in a DSC aluminum pan for testing, and the sample was heated from 25 ° C to 300 ° C at a heating rate of 10 ° C / min under 50 mL / min N ₂ .

The method of the invention is a Thermo Gravimetric Analyzer (TGA) method.

Instrument model: TA Q5000IR thermogravimetric analyzer.

Test method: Samples (2 ~ 5 mg) were placed in a TGA platinum pot for testing. The sample was heated from room temperature to 300 °C at a heating rate of 10 °C/min under 25 mL/min N ₂ .

Technical Effects: The compound A provided by the present invention has stable crystal form, good solubility, good wettability, and good promising prospects.

The process for synthesizing compound 1 and its intermediates provided by the invention has the advantages that the raw materials are cheap and easy to obtain, and the disadvantages of the reagents used are large, the reaction conditions are harsh, the separation and purification are difficult, and the industrialization is difficult.

The compound 11 was prepared by a one-step reaction from the compound 8, and the post-treatment was simple, and the reaction yield was high.

For a better understanding of the content of the present invention, the following detailed description is made in conjunction with the specific embodiments, but the specific embodiments are not limited to the content of the present invention.

Preparation of Compound 11-1 of Example 1 : Synthesis Scheme 1.

Step 1: Synthesis of Compound 9-1.

Compound 8-1 (1.6 g, 5.36 mmol) was dissolved in n-hexane (30 mL) then bis-pinacol borate (1.497 g, 5.896 mmol), bis (1,5-cyclooctadiene) Di-[mu]-methoxydioxan (I) (0.216 g, 0.32 mmol), 4,4'-di-tert-butyl-2,2'-bipyridine (0.144 g, 0.536 mmol), The reaction was refluxed for 2 hours under a nitrogen atmosphere. The reaction mixture was dried with EtOAc (EtOAc:EtOAc:EtOAc) MS (ESI) m / z: 343 [M-8+H] ⁺ .

Step 2: Synthesis of Compound 10-1.

Compound 9-1 (0.30 g, 0.707 mmol) was dissolved in methanol (8 mL), then copper bromide (0.316 g, 1.41 mmol) was added and the mixture was warmed to 50 ° C for 20 hours. After cooling, the reaction mixture was evaporated to dryness mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj MS (ESI) m / z: 377 [M + H] +, 379 [M + H + 2] +.

Step 3: Synthesis of Compound 11-1.

Compound 10-1 (0.160 g, 0.424 mmol) was dissolved in nitrogen, nitrogen-dimethylformamide (3 mL), then CuCN (0.152 g, 1.7 mmol) was added, and the reaction was reacted at 160 ° C for 4 hours. After cooling, ethyl acetate (50 mL) was added, and the organic phase was washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. Petroleum ether = 1/3) The title compound 11-1 was obtained (yellow solid, 0.040 g, yield: 29%). MS (ESI) m/z: 324 [M+H] ⁺ .

Example 2 Preparation of Compound 19.

synthetic route: .

Step 1: Synthesis of Compound 14.

Compound 13 (500 g, 2.27 mol) was dissolved in concentrated hydrochloric acid (1250 mL) and water (1250 mL), then sodium nitrite (469.89 g, 6.81 mol) was added dropwise at 25-30 ° C for one hour. 1000 mL) solution. The reaction was stirred at 25 ° C for 1 hour. TLC (petroleum ether: ethyl acetate = 1:1) showed the reaction was completed. The reaction was poured into ethyl acetate (2 L). The organic phase was combined, washed with EtOAc EtOAc EtOAc m. MS (ESI) m/z: 250 [M+H] ⁺ . ¹ H NMR (400 MHz, CHLOROFORM-d) δ 7.37-7.36 (m, 5 H), 5.18 (s, 2 H), 4.29-4.28 (m, 2 H), 3.83-3.82 (m, 2 H), 3.76 (t, J = 5.2 Hz, 2 H), 3.53 (t, J = 5.2 Hz, 2 H).

Step 2: Synthesis of Compound 15.

Compound 14 (1.00 kg, 4.01 mol) was dissolved in methanol (10 L), then ammonium chloride (1.50 kg, 28.08 mol) was added, and zinc powder (1.05 kg, 16.05 mol) was added in portions over one hour. The reaction solution was heated to 55 ° C for 2 hours. TLC (petroleum ether: ethyl acetate = 4:1) showed complete conversion of starting material. The reaction was filtered through EtOAc (EtOAc)EtOAc. The filtrate was concentrated to give crude product 15 (yellow oil, 950 g). MS (ESI) m/z: 236 [M+H] ⁺ .

Step 3: Synthesis of Compound 17.

Compound 16 (415.64 g, 2.91 mol) and diisopropylethylamine (626.21 g, 4.85 mol) were dissolved in dichloromethane (10 L), then the compound 15 was slowly added dropwise at -65 °C over one hour. (570 g, 2.42 mol) was dissolved in 2 L of dichloromethane. The reaction was reacted at -65 °C for 2 hours. LCMS showed the reaction was completed. The reaction mixture was washed with water (4 L×2), EtOAc (EtOAc (EtOAc) . The crude product 17 (600 g, 1.76 mol) was dissolved in dichloromethane (1.2 L) and petroleum ether (1.2 L) was slowly added dropwise over one hour. The precipitate was collected by filtration to give crude product 17 (500 g). The white solid product was again dissolved in dichloromethane (1 L) and petroleum ether (1 L) was slowly added dropwise over one hour. The precipitate was collected by filtration to give the title compound 17 (white solid, 300 g, crystallization yield: 49.87%, reaction, total crystal yield: 42.50%). Further, the recrystallized mother liquor was combined and concentrated to give a crude product (300 g, purity: 60%) which was further purified by repeating the above recrystallization. ¹ H NMR (400 MHz, CHLOROFORM-d) δ 7.39-7.32 (m, 5 H), 5.88 (br, 1 H), 5.13 (s, 2 H), 4.35 (t, J = 5.2 Hz, 2 H) , 3.69 (t, J = 5.2 Hz, 2 H), 3.64 (t, J = 4.8 Hz, 4 H), 2.81-2.80 (m, 4 H). MS (ESI) m/z: 342 [M+H] ⁺ .

Step 4: Synthesis of Compound 18.

Compound 17 (40 g, 117.03 mmol) was dissolved in acetonitrile (400 mL) then EtOAc (EtOAc (EtOAc) LCMS showed the reaction was complete. The reaction solution was filtered off with cesium carbonate, washed with acetonitrile (100 mL*3), and the filtrate was concentrated and dried. The residue was dissolved in ethyl acetate (1 L), EtOAc (EtOAc (EtOAc) The crude solid was used directly in the next reaction. Yield: 35.5 g. MS (ESI) m/z: 306 [M+H] ⁺ . ¹ H NMR (400 MHz, CHLOROFORM-d) δ 7.36-7.31 (m, 5 H), 5.12 (s, 2 H), 4.33 (t, J = 7.2 Hz, 2 H), 3.65-3.59 (m, 6 H), 3.0-2.85 (m, 4 H).

Step 5: Synthesis of compound 19.

Compound 18 (34.5 g, 112.99 mmol) was dissolved in methanol (700 mL), then Pd/C (3.45 g) was added and stirred at room temperature (25 ° C) under hydrogen (50 Psi) for 18 hours. The reaction is complete. The Pd/C was filtered, and the filter cake was washed with methanol (100 mL x 2), and the filtrate was concentrated to dryness, and the crude compound (19.0 g) was used directly in the next step. ¹ H NMR (400 MHz, CHLOROFORM-d) δ 4.32 (t, J = 8 Hz, 2 H), 3.64 (t, J = 8 Hz, 2 H), 3.01-2.93 (m, 8 H).

Example 3 Preparation of Compound 1.

Synthetic route 2: .

Step 1: Synthesis of Compound 6-1.

Compound 4 (250 g, 929.3 mmol) was dissolved in nitrogen, nitrogen-dimethylformamide (2 L), then potassium carbonate (256.88 g, 1.86 mol) was added, and ethyl bromoacetate (310.39 g, 1.86) was slowly added. Mm). The reaction was reacted at 25 ° C for 1 hour and then heated to 90-95 ° C and stirred for 11 hours. The reaction solution turned brown, and the reaction was completed by LCMS. After cooling to 25 ° C, the mixture was filtered. The filtrate was poured into 6 L of cold water, stirred for 2 hours, filtered, and dried in air to give the title compound 6-1 (yellow solid, yield: 69.27 % ) Used directly for the next step of synthesis. MS (ESI) m/z: 337 [M+H] ⁺ , 339 [M+H+2] ⁺ . ¹ H NMR (400 MHz, CDCl3) 8.02 (s, 1 H), 7.81 (s, 1 H), 7.52 (s, 1 H), 4.46 (q, J=7.19 Hz, 2 H), 1.44 (t, J = 7.03 Hz, 3 H).

Step 2: Synthesis of Compound 8-1.

Compound 6-1 (250 g, 741.64 mmol), cyclopropylboronic acid (76.45 g, 889.97 mmol), potassium phosphate (393.57 g, 1.85 mol), palladium acetate (15.00 g, 66.81 mmol), tricyclohexyl hydrazine ( 20.80 g, 74.16 mmol) Dissolved in toluene (2 L) and water (500 mL), then ventilated, and then heated to 100 ° C under nitrogen for 12 hours. HPLC showed the reaction to be complete. After cooling, the layers were separated, and the aqueous phase was extracted with ethyl acetate (1 L×2). The organic phase was washed with brine (500 mL×2). Eluent, ethyl acetate/petroleum ether = 1/20) mp. MS (ESI) m/z: 299 [M+H] ⁺ . ¹ H NMR (400 MHz, CDCl3) 7.54 (s, 1H), 7.49 (s, 1H), 7.42 (s, 1H), 4.47-4.42 (m, 2H), 2.07-2.02 (m, 1H), 1.44- 1.41 (m, 3H), 1.06-1.02 (m, 2H), 0.77-0.73 (m, 2H).

Step 3: Synthesis of Compound 11-1.

Compound 8-1 (200.00 g, 670.56 mmol) was dissolved in anhydrous tetrahydrofuran (2.0 L), and magnesium dichloride (2,2,6,6-tetramethylacridine) lithium salt solution was added under a nitrogen atmosphere at -40 °C. (1.5 M, 893.33 mL), stirred at -40 °C for 2 hours, then added 4-toluenesulfononitrile (182.27 g, 1.01 mol) in anhydrous tetrahydrofuran (500 mL) at -40 ° C to -10 ° C Stirring was continued for 1 hour. TLC (petroleum ether / ethyl acetate = 20/1) showed product. The reaction was quenched with 120 mL of water. Ethyl acetate (5 L) was added to the above mixture and the filtrate was evaporated. EtOAcjjjjjjjjj 90 g, yield: 41.52%). ¹ H NMR (400 MHz, MeOD) δ 7.76 (s, 1H), 7.66 (s, 1H), 4.53 (q, J = 7.2 Hz, 2H), 2.21~2.17 (m, 1H), 1.46 (t, J = 7.2 Hz 3H), 1.14~1.11 (m, 2H), 0.86~0.84 (m, 2H).

Step 4: Synthesis of Compound 12.

Compound 11-1 (405.00 g, 1.25 mol) was dissolved in tetrahydrofuran (3.00 L), then lithium hydroxide hydrate (105.14 g, 2.51 mol, 2.00 eq) and water (1.25 L) were added to the above solution. After stirring at 25 ° C for 2 hours, TLC (PE: EA = 30:1) was used to detect complete disappearance of the starting material. The solvent tetrahydrofuran was removed by rotary distillation under reduced pressure, and the aqueous phase was adjusted to pH 4 to 5 with 2N HCl. Ethyl acetate (2.0 L*3) was extracted with EtOAc (EtOAc)EtOAc. The mixed solvent (petroleum ether: ethyl acetate = 30:1, 600 mL) was added to the above crude product, and the mixture was stirred and beaten, and then the funnel was filtered to obtain a pale yellow solid, which was dried under a rotary distillation to give a pale yellow pure compound 12 ( 300.00 g, yield: 79.67% yield). MS (ESI) m/z: 296 (M+H) ⁺ . ¹ H NMR (400 MHz, MeOD) δ 7.75 (s, 1H), 7.65 (s, 1H), 2.23~2.20 (m, 1H), 1.35~1.13 (m, 2H), 0.87~0. 86 (m, 2H).

Step 5: Synthesis of Compound 1.

Compound 19 (5.0 g, 29.21 mmol) was dissolved in DMF (80 mL) then compound 12 (8.0 g, 27.10 mmol), HATU (12.36 g, 32.52 mmol), DIEA (10.51 g, 81.30) Mm). The reaction solution was stirred at 25 ° C for 1.5 hours. LCMS showed the starting material was completely reacted. After the completion of the reaction, 120 mL of water was added, and the precipitate was filtered. The solid was added to methanol (120 mL) and stirred for 15 minutes, filtered, and dried in vacuo to give the title compound as a white solid (7.4 g, yield: 60.89%). MS (ESI) m/z: 449 [M+H] ⁺ . ¹ H NMR (400 MHz, CHLOROFORM-d) δ 7.62 (s, 1H), 7.50 (s, 1H), 4.36 (t, J = 8.0 Hz, 2 H), 3.97 (m, 4H), 3.67 (t, J = 8.0 Hz, 2 H), 3.16-3.13 (m, 4 H), 2.12-2.06 (m, 1 H), 1.15-1.11 (m, 2 H), 0.81-0.79 (m, 2 H).

Example 4 Crystalline screening experiments for Compound 1.

Take appropriate amount of compound 1 into a 50 mL vial, add appropriate amount of acetonitrile, tetrahydrofuran, acetone, toluene, dioxane, ethyl acetate, isopropanol, acetone + water and acetonitrile + water, and heat until completely dissolved. The mixture was slowly cooled to 0 ° C, and the solid obtained by filtration was subjected to XRPD, and the XRPD results are shown in Table 2. Table 2 Compound 1 crystal form screening

Example 5 A crystal form solubility test of Compound 1.

Weigh about 2 mg of Form A into a 7 mL vial and add appropriate amounts of methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, water and ethyl acetate, and mix well. Continue adding the appropriate amount of solvent until the compound is completely dissolved. The solubility of the obtained drug substance in an organic solvent and water is shown in Table 3. Table 3 Approximate Solubility of APIs in Organic Solvents and Water

Experimental Example In vitro cell viability assay.

Purpose: with HCV genotype 1a (HCV-1a) and 1b (HCV-1b) ₅₀ measured values stably transfected compound anti-HCV replicon EC ₅₀ and CC (replicon) cells. The genotype 1a replicon is derived from the H77 clone and contains K1691R, K2040R and S2204I adaptive mutations. The genotype 1b replicon is derived from the Con1 clone and contains E1202G, 10 T1280I and K1846T adaptive mutations.

BACKGROUND: The HCV 1a (HCV-1a) and 1b (HCV-1b) genotype subgenomic replication subsystems contain the relevant HCV gene subtype non-structural protein genes, the G418 resistance gene NEO and the luciferase gene, making HCV-related Protein and luciferase are stably expressed in cells. The level of replication of HCV replicons can be determined by detecting the level of expression of the luciferase gene. Therefore, this system serves as a model for screening the activity of anti-HCV compounds in vitro.

Experimental Materials.

HCV replicon cell lines: HCV-1a and HCV-1b cells.

Cell culture medium: DMEM (Invitrogen, Cat. # 11960077) medium, plus 10% fetal bovine serum (FBS, Sigma, Cat. # 12003C) and 1% double antibody (penicillin 5000 IU/mL, streptomycin 10 mg / mL, Hyclone, Cat.# SV30010).

Trypsin (Invitrogen, Cat. # 25200072).

PBS (Invitrogen, Cat. # 10010023).

Trypan Blue (Invitrogen, Cat. # 15250061).

Cell Titer-fluor (Promega, Cat.# G6082).

Bright-Glo (Promega, Cat. # E2650).

CO ₂ incubator, Thermo 240 I.

Multidrop automatic dispenser, Thermo.

POD 810 Plate Assembler Fully Automatic Microplate Pretreatment System, Labcyte.

EnVision Multilabel Plate Readers Microplate spectrophotometer, Perkine Elmer.

Experimental procedures and methods: a) Compound solution preparation, dilution and loading: The compound powder was dissolved in 100% DMSO. The compound was then diluted 6 points by 5 fold. Echo liquid handler 5 was added to the cell plate. The final concentration of DMSO was 0.5%. Each compound is doubled. b) Cell culture (HCV-1a or HCV-1b replicon cells): 1) Aspirate the culture supernatant of the cell culture and wash the cells with 10 mL of PBS. 2) Add the pre-warmed trypsin to the washed cell culture flask, and rotate the culture flask to evenly cover the bottom of the flask. Digested in a 37 ° C, 5% CO ₂ incubator. 3) Each T150 flask was suspended with 10-15 mL of culture medium, and 0.1 mL of the cells were diluted with trypan blue solution and diluted twice. 4) Dilute the cells to 8 × 10 ⁴ /mL with the culture solution, and add the diluted cells to a compound-containing 96-well plate (Greiner, Cat. # 655090) (100 L/well, using an automatic liquid separator (Thermo Scientific). 8000 cells/hole). Incubate for 3 days at 37 ° C in a 5% CO ₂ incubator. Cell control wells: no compound, only 0.5% DMSO. 5) Add the chemiluminescent substrate Cell Titer-fluor to the cell wells and incubate for 30 minutes. The signal is detected by the chemiluminescence detection system Envison (Ex at 405 nm and read at 515 nm). Analysis of compounds on HCV replication data Effects The light emitting sub-cellular activity, and value calculation for ₅₀ CC. 6) Then add luciferase luminescent substrate Bright-Glo, incubate for 5 minutes, detect the luciferase activity (length >700 nm) with chemiluminescence detection system Envison; analyze the anti-HCV replication of the compound according to luciferase data The subsuppressive activity was used together to calculate the EC ₅₀ value. c) data processing and analysis: The percent inhibition GraphPad Prism software (inh%) non-linear data fitting analysis to give ₅₀ or EC ₅₀ value CC. The experimental results are shown in Table 4. Table 4 EC50 replicon cell EC ₅₀ /CC ₅₀ test results Note: A (0.1 nM ~ 100 nM).

Conclusion: Compound 1 has excellent anti-hepatitis C virus activity in vitro.

Figure 1 is an XRPD spectrum of Cu-Kα radiation of Form A; Figure 2 is a DSC spectrum of Form A of Figure A; and Figure 3 is a TGA spectrum of Form A of Form A.

Claims (15)

a method for preparing compound 1, It contains the following steps, Where R is selected from Me, Et, The base B is selected from the group consisting of TMPMgCl•LiCl, TMP2Mg•2LiCl, i-PrMgCl, i-PrMgCl•LiCl, LDA, n-butyllithium; the cyanation reagent is selected from TsCN, , ; the molar ratio of the compound 8 to the base B is selected from 1:1 to 3; the molar ratio of the compound 8 to the cyanating agent is selected from 1:1 to 3; and the solvent B is selected from a single ether solvent or an ether solvent. A mixed solvent with toluene. The preparation method according to claim 1, wherein the solvent B is selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, tert-butyl ether, isopropyl ether, methyl tert-butyl ether, and ethylene glycol dimethyl ether. Or a mixed solvent selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, tert-butyl ether, isopropyl ether, methyl tert-butyl ether, ethylene glycol dimethyl ether and toluene. The preparation method according to claim 1, wherein the molar ratio of the compound 8 to the base B is selected from 1:1.2 to 2.5; or the molar ratio of the compound 8 to the cyanating agent is selected from 1:1.2 to 2, preferably From 1:1.3 to 1.6. The production method according to claim 1, wherein the compound 11 is produced by the following steps, Wherein X is selected from the group consisting of F, Cl, Br and I. The preparation method according to claim 1, which further comprises the following steps, Wherein the condensing agent is selected from the group consisting of: HATU, HBTU, CDI, HOBT, EDCI, DCC, DIC, PyBOP, DPP-Cl; the base A is selected from the group consisting of: K ₂ CO ₃ , Na ₂ CO ₃ , NaHCO ₃ , NaOH, KOH, TEA , DIPEA, pyridine; solvent A is selected from: dichloromethane, acetonitrile, DMF; optionally, compound 12 and compound 19 are reacted under catalyst catalysis, the catalyst is selected from DMAP; the molar ratio of compound 12 to the condensing agent is selected from 1:1~3; The molar ratio of the compound 12 to the compound 19 is selected from 1:1 to 1.5; and the reaction temperature is selected from 10 ° C to 40 ° C. The preparation method according to claim 1, which further comprises the following reaction step, ;or ;or ;or Wherein the base C is selected from the group consisting of K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ ; the solvent C is selected from the group consisting of DMF and CH ₃ CN; and the base D is selected from the group consisting of K ₃ PO ₄ , K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ ; The solvent D is selected from the group consisting of THF, DMF, toluene, xylene, 1,4-dioxane, a single solvent of water or a mixed solvent of two solvents; specifically, the solvent D is selected from the group consisting of toluene and water. a mixed solvent, the volume ratio of toluene to water is selected from 1 to 3:1; the catalyst is selected from the group consisting of Pd ₂ (dba) ₃ , Pd (dppf) Cl ₂ , Pd(PPh ₃ ) ₄ , Pd(OAc) ₂ ; From no or PPh ₃ , tricyclohexyl hydrazine, tributyl hydrazine; base E is selected from the group consisting of LiOH, NaOH, KOH, Na ₂ CO ₃ , K ₂ CO ₃ ; and solvent E is selected from the group consisting of tetrahydrofuran, 1,4-dioxane Ring, acetonitrile. As a compound of the formula: , , , , , . The Form A of Compound 1 has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2 theta angles: 10.58 ± 0.2 °, 20.41 ± 0.2 °, 21.98 ± 0.2 °. The X crystal form of Compound 1 according to claim 8 has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 5.34±0.2°, 10.58±0.2°, 12.70±0.2°, 20.41±0.2°, 20.74. ±0.2°, 21.16±0.2°, 21.98±0.2°, 24.55±0.2°. The crystal form of Compound A of Compound 1 as claimed in claim 9 has an XRPD pattern as shown in FIG. The A crystal form according to any one of claims 8 to 10, wherein the differential scanning calorimetry curve has a starting point of an endothermic peak at 212.07 ° C ± 3 ° C. The A crystal form as described in claim 11 has a DSC spectrum as shown in Fig. 2. The A crystal form according to any one of claims 8 to 10, wherein the thermogravimetric analysis curve has a weight loss of 0.3107±0.2% at 120.0±3° C., and the weight loss at 0.22.5±3° C. is 0.2094±0.2%, 233.0±3. The weight loss at °C is 0.3501±0.2%. The TGA pattern of the A crystal form described in claim 13 is shown in Fig. 3. The method for preparing a crystalline form according to any one of claims 8 to 10, which comprises the step of adding a compound 1 of any one form to a solvent for recrystallization or beating, wherein the recrystallization solvent or the beating solvent is selected from the group consisting of methanol and ethanol. , isopropanol, dioxane, acetonitrile, tetrahydrofuran, acetone, toluene, or a mixed solvent selected from the group consisting of methanol, ethanol, isopropanol, dioxane, acetonitrile, tetrahydrofuran, acetone .