WO2017162204A1 - Manufacturing method of benzofuran analog and intermediate and crystalline form of same - Google Patents

Abstract

The present invention discloses a manufacturing method of a benzofuran analog and an intermediate and crystalline form of same.

Description

Preparation method of benzofuran analog, intermediate and crystal form thereof Technical field

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

Background technique

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 therapies are 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 currently urgently needed.

HCV is a single-stranded positive-strand RNA virus. It belongs to the 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.

Summary of the invention

The invention provides a preparation method of the compound 1,

It contains the following steps,

among them,

R is selected from Me, Et,

B is selected from alkali _{TMPMgCl·LiCl, TMP 2 Mg · 2LiCl,} i-PrMgCl, i-PrMgCl·LiCl, LDA, n-butyllithium;

The cyanation reagent is selected from the group consisting of TsCN,

The molar ratio of compound 8 to base B is selected from 1:1 to 3;

The molar ratio of the compound 8 to the cyanation reagent is selected from 1:1 to 3;

The solvent B is selected from a single ether solvent or a mixed solvent of an ether solvent and 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 aspects 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, a mixed solvent of ethylene glycol dimethyl ether and toluene.

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 cyanating agent is selected from the group consisting of 1:1.2 to 2.

In some embodiments of the invention, the molar ratio of the above compound 8 to the cyanating agent 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,

among them,

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,

among them,

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 the group consisting of: dichloromethane, acetonitrile, DMF;

Optionally, compound 12 and compound 19 are reacted under catalyst catalysis, and the catalyst is selected from DMAP;

The molar ratio of the compound 12 to the 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;

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,

among them,

The base C is selected from the group consisting of K ₂ CO ₃ , Na ₂ CO ₃ , Cs ₂ CO ₃ ;

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,

among them,

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;

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 the group consisting of no or PPh ₃ , tricyclohexylphosphine, tributylphosphine.

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,

among them,

The base E is selected from the group consisting of LiOH, NaOH, KOH, Na ₂ CO ₃ , K ₂ CO ₃ ;

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 analytical data of the XRPD pattern 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 crystal form of the compound 1 described above has a differential scanning calorimetry curve having a starting point of an 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 pattern as shown in FIG.

In some aspects 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 the crystal form A, which comprises adding any one form of the 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 and 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.

Definition and description:

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 skilled in the art. Well-known equivalents, 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-tetramethylpiperidine); TMP ₂ Mg·2LiCl represents two (2,2',6,6' - tetramethylpiperidine) magnesium dichloride; DPP-Cl represents diphenylphosphoryl chloride; i-PrMgCl represents isopropyl magnesium chloride; LDA represents lithium diisopropylamide; TsCN represents 4-toluenesulfonyl nitrile; HATU stands for 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-di Methylaminopropyl)-3-ethylcarbodiimide hydrochloride; DCC stands for dicyclohexylcarbodiimide; DIC stands for N,N-diisopropylcarbodiimide; PyBOP stands for benzohexafluorophosphate Triazol-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} tetrakis (triphenylphosphine) palladium Representative; Pd (dppf) Cl ₂ representative of 1,1'-bis (diphenylphosphino) ferrocene Palladium chloride; PPh ₃ triphenylphosphine Representative; Pd (OAc) ₂ palladium acetate _{Representative; Pd (PPh 3) 2 Cl} 2 representative of dichlorobis (triphenylphosphine) palladium (II); DIEA representative of N, N- Diisopropylethylamine.

Compound by hand or

Software naming, commercially available compounds using the supplier catalog name.

X-ray powder diffractometer (XRPD) method of the present invention

Instrument model: Brooke D8advance X-ray diffractometer;

Test method: about 10-20 mg sample is used for XRPD detection;

The detailed XRPD parameters are as follows:

Light pipe: Cu, kα,

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

Divergence slit: 0.60mm;

Detector slit: 10.50mm;

Anti-scattering slit: 7.10mm;

Scanning range: 4-40deg;

Step diameter: 0.02 deg;

Step size: 0.12 seconds;

Sample pan speed: 15 rpm.

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 .

Thermal Gravimetric Analyzer (TGA) method of the present invention

Instrument model: TA Q5000IR thermogravimetric analyzer

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

Technical effect:

The compound A provided by the invention has stable crystal form, good solubility and good wettability, and has 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.

DRAWINGS

Figure 1 is an XRPD spectrum of Cu-Kα radiation of Form A.

Figure 2 is a DSC spectrum of Form A.

Figure 3 is a TGA spectrum of Form A.

detailed description

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.

Example 1 Preparation of Compound 11-1

Synthetic route 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-hydrazinyl borate (1.497 g, 5.896 mmol), bis (1,5-cyclooctadiene) was added in sequence. Di-μ-methoxydiazide (I) (0.216 g, 0.32 mmol), 4,4'-di-tert-butyl-2,2'-bipyridine (0.144 g, 0.536 mmol), the reaction was protected under nitrogen The reaction was refluxed for 2 hours. 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 reaction mixture was warmed to 50 ° C for 20 hours. After cooling, the reaction mixture was evaporated to dryness mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj 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 mixture was reacted at 160 ° C for 4 hours, after cooling Ethyl acetate (50 mL) was added, the organic phase was washed with brine (20 mL), dried over anhydrous sodium sulfate 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 dissolved in water (1000 mL) at 25-30 ° C over one hour. 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 mL). The organic layer was combined, washed with EtOAc EtOAc EtOAcjHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH MS (ESI) m / z: 250 [M+H] ⁺ . ^{1 H NMR (400MHz, CHLOROFORM-} d) δ7.37-7.36 (m, 5H), 5.18 (s, 2H), 4.29-4.28 (m, 2H), 3.83-3.82 (m, 2H), 3.76 (t, J = 5.2 Hz, 2H), 3.53 (t, J = 5.2 Hz, 2H).

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 a crude material 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 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 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 with stirring. 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 (400MHz, CHLOROFORM-} d) δ7.39-7.32 (m, 5H), 5.88 (br, 1H), 5.13 (s, 2H), 4.35 (t, J = 5.2Hz, 2H), 3.69 (t , J = 5.2 Hz, 2H), 3.64 (t, J = 4.8 Hz, 4H), 2.81-2.80 (m, 4H). 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) Used directly in the next step. Yield: 35.5 g. MS (ESI) m / z: 306 [M+H] ⁺ . ^{1 H NMR (400MHz, CHLOROFORM-} d) δ7.36-7.31 (m, 5H), 5.12 (s, 2H), 4.33 (t, J = 7.2Hz, 2H), 3.65-3.59 (m, 6H), 3.0 -2.85 (m, 4H).

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

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, MeOH-MeOH (2L), then EtOAc (EtOAc (EtOAc) 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 in the next step of synthesis. MS (ESI) m / z: 337 [M + H] +, 339 [M + H + 2] +. _{^{1 H NMR (400MHz, CDCl 3}} ) 8.02 (s, 1H), 7.81 (s, 1H), 7.52 (s, 1H), 4.46 (q, J = 7.19Hz, 2H), 1.44 (t, J = 7.03Hz , 3H).

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), tricyclohexylphosphine (20.80) g, 74.16 mmol) was 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), and the organic phase was dried over sodium sulfate and filtered to give a crude product. The title compound 8-1 (brown-black oil, 210 g, yield: 93.99%). MS (ESI) m / z: 299 [M+H] ⁺ . _{^{1 H NMR (400MHz, CDCl 3}} ) 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 a magnesium salt of magnesium dichloride (2,2,6,6-tetramethylpiperidine) was added under a nitrogen atmosphere at -40 °C. (1.5M, 893.33mL), stirred at -40 ° C for 2 hours, then added 4-toluenesulfonyl nitrile (182.27 g, 1.01 mol) in anhydrous tetrahydrofuran (500 mL), at -40 ° C to -10 ° C conditions 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, filtered, and the filtrate was evaporated. mjjjjjjjj 90 g, yield: 41.52%). ^{1 H NMR (400MHz, MeOD)} δ7.76 (s, 1H), 7.66 (s, 1H), 4.53 (q, J = 7.2Hz, 2H), 2.21 ~ 2.17 (m, 1H), 1.46 (t, J = 7.2 Hz 3H), 1.14 to 1.11 (m, 2H), 0.86 to 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. The mixture was stirred at 25 ° C for 2 hours, and the starting material was completely disappeared by TLC (PE: EA = 30:1). The solvent tetrahydrofuran was evaporated under reduced pressure, and the aqueous phase was adjusted to pH 4 to 5 with 2N HCl. Ethyl acetate (2.0 L*3) was evaporated. 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 give a pale-yellow solid, which was evaporated 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 to 2.20 (m, 1H), 1.35 to 1.13 (m, 2H), 0.87 to 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 EtOAc (EtOAc, EtOAc, EtOAc, EtOAc. ). The reaction solution was stirred at 25 ° C for 1.5 hours. LCMS showed complete reaction of the starting material. After the completion of the reaction, 120 mL of water was added, and the precipitate was filtered, and the mixture was evaporated, mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj MS (ESI) m / z: 449 [M+H] ⁺ . ^{1 H NMR (400MHz, CHLOROFORM-} d) δ7.62 (s, 1H), 7.50 (s, 1H), 4.36 (t, J = 8.0Hz, 2H), 3.97 (m, 4H), 3.67 (t, J = 8.0 Hz, 2H), 3.16-3.13 (m, 4H), 2.12-2.06 (m, 1H), 1.15-1.11 (m, 2H), 0.81 - 0.79 (m, 2H).

Example 4 Crystalline Screening Experiment of Compound 1

A suitable amount of Compound 1 was placed in a 50 mL vial, and an appropriate amount of acetonitrile, tetrahydrofuran, acetone, toluene, dioxane, ethyl acetate, isopropanol, acetone + water and acetonitrile + water were added thereto, and heated to complete dissolution. 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

Serial number	Solvent used	Weight (mg)	Solvent amount (mL)	Heating temperature	XRPD
1	Acetonitrile	500	20	80 ° C	A crystal form
2	Tetrahydrofuran	500	25	70 ° C	A crystal form
3	acetone	500	25	56°C	A crystal form
4	Toluene	200	30	110 ° C	A crystal form
5	Dioxane	300	30	101 ° C	A crystal form
6	Ethyl acetate	200	25	80 ° C	A crystal form
7	Isopropanol	100	25	85 ° C	A crystal form
8	Acetone + water	500	30+1.5	60 ° C	A crystal form
9	Acetonitrile + water	540	15+0.75	85 ° C	A crystal form

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 organic solvent and water is shown in Table 3.

Table 3 Approximate Solubility of APIs in Organic Solvents and Water

Serial number	Solvent	Form A (mg)	Solubility (mg/ml)
1	Methanol	2.29	<0.46
2	Ethanol	2.32	<0.46
3	acetone	2.29	~1.15
4	Isopropanol	2.28	<0.46
5	Acetonitrile	2.58	~1.03
6	Ethyl acetate	2.39	~0.53
7	Dichloromethane	2.54	~7.47
8	water	2.15	<0.43

Experimental in vitro cell viability test

Purpose:

With HCV genotype 1a (HCV-1a) and 1b (HCV-1b) stably transfected replicon (replicon) Cell ₅₀ measured values of the anti-HCV compound EC ₅₀ and CC. 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, 10T1280I and K1846T adaptive mutations.

Background introduction:

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 proteins and fluorescein The enzyme can be stably expressed in cells. The level of replication of the HCV replicon 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) culture 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).

CO2 incubator, Thermo 240 I.

Multidrop automatic dispenser, Thermo.

POD 810 Plate Assembler fully automated microplate pretreatment system, Labcyte.

EnVision Multilabel Plate Readers Microplate Spectrophotometer, Perkine Elmer.

Experimental steps 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. It was digested at 37 ° C in a 5% CO ₂ incubator.

3) Each T150 flask was suspended with 10-15 mL of the culture solution, and 0.1 mL of the cells were diluted with a trypan blue solution and counted twice.

4) Dilute the cells with culture medium to 8 × 10 ⁴ /mL, and add the diluted cells to a compound-containing 96-well plate (Greiner, Cat. #655090) (100 μL/well, using an automatic liquid separator (Thermo Scientific). 8000cells/hole). The cells were cultured at 37 ° C for 3 days 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 to detect the signal using 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 >700nm) with chemiluminescence detection system Envison; analyze the anti-HCV replicon inhibitory activity of the compound according to luciferase data And used to calculate the EC ₅₀ value.

c) Data processing and analysis:

Using GraphPad Prism software percent inhibition (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/CC50 test results of HCV replicon cells

Compound	EC50(1b)	EC50(1a)	CC50
Compound 1	A	A	>3000

Note: A (0.1nM ~ 100nM).

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

Claims (15)

1. a method for preparing compound 1,

   

   It contains the following steps,

   

   among them,

   R is selected from Me, Et,

   

   The base B is selected from the group consisting of TMPMgCl·LiCl, TMP ₂ Mg·2LiCl, i-PrMgCl, i-PrMgCl·LiCl, LDA, n-butyl lithium;

   The cyanation reagent is selected from the group consisting of TsCN,

   

   The molar ratio of compound 8 to base B is selected from 1:1 to 3;

   The molar ratio of the compound 8 to the cyanation reagent is selected from 1:1 to 3;

   The solvent B is selected from a single ether solvent or a mixed solvent of an ether solvent and toluene.
2. The process 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, ethylene glycol dimethyl ether; 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.
3. The production 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;

   The molar ratio of the compound 8 to the cyanating agent is selected from the group consisting of 1:1.2 to 2, preferably from 1:1.3 to 1.6.
4. The production method according to claim 1, wherein the compound 11 is produced by the following steps,

   

   among them,

   X is selected from the group consisting of F, Cl, Br, and I.
5. The preparation method according to claim 1, further comprising the following steps,

   

   among them,

   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 the group consisting of: dichloromethane, acetonitrile, DMF;

   Optionally, compound 12 and compound 19 are reacted under catalyst catalysis, and the catalyst is selected from DMAP;

   The molar ratio of the compound 12 to the 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;

   The reaction temperature is selected from 10 ° C to 40 ° C.
6. The preparation method according to claim 1, further comprising the following reaction step,

   

   

   among them,

   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;

   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 a mixed solvent of toluene and water, and 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 ;

   The ligand is selected from the group consisting of no or PPh ₃ , tricyclohexylphosphine, tributylphosphine;

   The base E is selected from the group consisting of LiOH, NaOH, KOH, Na ₂ CO ₃ , K ₂ CO ₃ ;

   The solvent E is selected from the group consisting of tetrahydrofuran, 1,4-dioxane, and acetonitrile.
7. As a compound of the formula:

   
8. 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 °.
9. The crystal form of Compound A according to claim 8, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2 theta 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°.
10. The crystalline form A of Compound 1 according to claim 9, the XRPD pattern of which is shown in FIG.
11. The crystal form A 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.
12. The crystal form of Form A according to claim 11, wherein the DSC pattern is as shown in FIG.
13. The crystal form of A 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 a weight loss of 0.2094±0.2% at 232.5±3° C., 233.0±3. The weight loss at °C is 0.3501±0.2%.
14. The crystal form of Form A according to claim 13, wherein the TGA pattern is as shown in FIG.
15. A method for preparing a crystalline form according to any one of claims 8 to 10, which comprises adding any one of the compounds of Form 1 to a solvent for recrystallization or beating,

    among them,

    The recrystallization solvent or beating solvent is selected from the group consisting of methanol, ethanol, isopropanol, dioxane, acetonitrile, tetrahydrofuran, acetone, toluene, or selected from the group consisting of methanol, ethanol, isopropanol, dioxane, acetonitrile, tetrahydrofuran. A mixed solvent of any solvent and water in acetone.

Images