WO2017097253A1

WO2017097253A1 - Deuterium-modified elbasvir derivative, drug composition containing same, and use thereof

Info

Publication number: WO2017097253A1
Application number: PCT/CN2016/109181
Authority: WO
Inventors: 张寅生; 敖汪伟
Original assignee: 正大天晴药业集团股份有限公司
Priority date: 2015-12-10
Filing date: 2016-12-09
Publication date: 2017-06-15
Also published as: CN108290904A; CN108290904B

Abstract

The present invention relates to the field of medicine, and in particular to a deuterium-modified elbasvir derivative, a drug composition containing same, and a use thereof in the preparation of a drug for treating hepatitis C virus infection. The deuterium-modified elbasvir derivative of the present invention has more excellent properties in one or more assays (e.g., activity assay, pharmacokinetics assay, and liver microsome metabolism stability assay), and is expected to lower the clinical dose, thereby reducing the treatment costs for patients.

Description

Deuterium-modified ebasvir derivative, pharmaceutical composition containing the same, and use thereof

Technical field

The present invention belongs to the field of medicine, and in particular relates to a hydrazine-modified elbasvir derivative, a pharmaceutical composition containing the same, and use thereof for the preparation of a medicament for treating hepatitis C virus infection.

Background technique

Many current drugs suffer from poor absorption, distribution, metabolism, and/or excretion (ADME) characteristics, which hampers their wider use. Poor ADME properties are also a major cause of failure of drug candidates in clinical trials. One potentially attractive strategy for improving the metabolic properties of drugs is to modify the sputum. In this method, an attempt is made to slow CYP-mediated drug metabolism by replacing one or more hydrogen atoms with deuterium atoms. Helium is a safe, stable, non-radiative hydrogen isotope. Tantalum and carbon form stronger bonds than hydrogen. In selected examples, an increase in bond strength given by sputum can have a positive impact on the ADME properties of the drug, creating the potential for improved drug efficacy, safety, and/or tolerance. At the same time, because the size and shape of the crucible is substantially the same as that of hydrogen, the replacement of hydrogen with deuterium does not affect the biochemical efficacy and selectivity of the drug compared to the original chemical containing only hydrogen.

The effect of hydrazine substitution on metabolic rate has been reported in some drugs (see, for example, Blake, MI et al, J Pharm Sci, 1975, 64: 367-91; Foster, AB, Adv Drug Res, 1985, 14: 1- 40; Kushner, DJ et al, Can J Physiol Pharmacol, 1999, 79-88; Fisher, MB et al, Curr Opin Drug Discov Devel, 2006, 9: 101-09), the results are variable and unpredictable. For some compounds, deuteration causes a reduction in metabolic clearance in the body. For other compounds, no changes were made to metabolism. For some compounds, increased metabolic clearance has been demonstrated. The variability of sputum effects has also led experts to question or abandon the idea that 氘 modification is a viable drug design strategy to inhibit harmful metabolism (see Foster page 35 and Fisher page 101).

Even when a ruthenium atom is bound to a known site of a metabolite, the effect of oxime modification on the metabolic properties of the drug is unpredictable. Only if the deuterated drugs are actually prepared and tested can the metabolic rate be determined and how it differs from the non-deuterated counterparts. See, for example, Fukuto et al. (J. Med. Chem., 1991, 34, 2871-76). Many drugs have multiple sites where metabolic reactions may occur. The degree of deuteration that is required for hydrazine substitution and the extent to which the effects of metabolism are visible, if any, is different for each drug.

Merck's full oral cocktail, Grazoprevir/elbasvir (100 mg/50 mg), is a once-a-day compound monolith consisting of grazoprevir (an NS3/4A protease inhibitor) and elbasvir (an NS5A polymerase inhibitor). The FDA granted two new breakthrough drug (BTD) qualifications in 2015, namely: (1) BTD status for treating genotype 4 chronic hepatitis C virus (HCV GT4) infection; (2) treatment with end BTD status of genotype 1HCV (HCV GT1) infected with hemodialysis treatment for end stage renal disease.

Despite the beneficial activity of elbasvir, there is a need to further develop new compounds that have inhibitory activity against hepatitis C virus NS5A and can be used to prevent or treat hepatitis C virus infection.

Summary of the invention

In a first aspect of the invention, there is provided a compound of formula 、, or a pharmaceutically acceptable salt thereof:

Wherein R ₁ or R ₂ are each independently selected from CH ₃ , CH ₂ D, CHD ₂ or CD ₃ , and R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ or R ₉ are each independently selected from H (hydrogen) or D (氘), provided that R ₁ to R ₉ contain at least one deuterium atom.

In some embodiments of the invention, R ₁ , R _{2 are} both CH ₃ or CD ₃ . In one embodiment of the invention, R ₁ and R _{2 are} both CH ₃ . In another embodiment of the invention, R ₁ and R _{2 are} both CD ₃ .

In some embodiments of the invention, R ₅ , R ₆ , R ₇ , R ₈ , R _{9 are} both H or D. In one embodiment of the invention, R ₅ , R ₆ , R ₇ , R ₈ , R _{9 are} both H. In another embodiment of the invention, R ₅ , R ₆ , R ₇ , R ₈ , R _{9 are} both D.

In some preferred embodiments of the invention, examples of compounds of formula 如下 are as follows:

化合物Compound	R₁ R ₁	R₂ R ₂	R₃ R ₃	R₄ R ₄	R₅ R ₅	R₆ R ₆	R₇ R ₇	R₈ R ₈	R₉ R ₉
11	CD₃ CD ₃	CD₃ CD ₃	HH	HH	HH	HH	HH	HH	HH
22	CD₃ CD ₃	CD₃ CD ₃	DD	HH	HH	HH	HH	HH	HH
33	CD₃ CD ₃	CD₃ CD ₃	HH	DD	HH	HH	HH	HH	HH
44	CD₃ CD ₃	CD₃ CD ₃	DD	HH	DD	DD	DD	DD	DD
55	CD₃ CD ₃	CD₃ CD ₃	DD	DD	HH	HH	HH	HH	HH
66	CD₃ CD ₃	CD₃ CD ₃	DD	DD	DD	DD	DD	DD	DD
77	CH₃ CH ₃	CH₃ CH ₃	DD	HH	HH	HH	HH	HH	HH
88	CH₃ CH ₃	CH₃ CH ₃	DD	DD	HH	HH	HH	HH	HH
99	CH₃ CH ₃	CH₃ CH ₃	DD	HH	DD	DD	DD	DD	DD
1010	CH₃ CH ₃	CH₃ CH ₃	DD	DD	DD	DD	DD	DD	DD
1111	CH₃ CH ₃	CH₃ CH ₃	HH	DD	HH	HH	HH	HH	HH

In a further preferred embodiment of the invention, examples of compounds of the formula 如下 are as follows:

The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness of the free acids and bases of a particular compound without biologically adverse effects. Examples of pharmaceutically acceptable salts include, but are not limited to, (1) acid addition salts, and salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, nitric acid, phosphoric acid, etc.; or with organic acids such as malic acid, rich Horse acid, maleic acid, benzoic acid, phenylacetic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, glycolic acid, cinnamic acid, pyruvic acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid a salt formed by acrylic acid, mandelic acid or the like; or (2) a base addition salt, a salt formed with an alkali metal such as lithium, sodium, potassium or the like; a salt formed with an alkaline earth metal such as calcium, magnesium, or the like; and an organic base such as ammonium a salt formed by choline, diethanolamine, lysine, ethylenediamine, tert-butylamine, tert-octylamine, tris(hydroxymethyl)aminomethane, N-methylglucamine, triethanolamine, dehydroabietylamine or the like.

Any atom of the compound to be labeled and synthesized in the present invention may represent any stable isotope of the atom unless otherwise specified. Unless otherwise stated, when a position in the structure is defined as H, hydrogen (H-1), the position contains only naturally occurring isotope amounts. Similarly, unless specifically stated, when a position in the structure is defined as D, ie, helium (H-2), the isotope content at that position is at least 3340 times greater than the naturally occurring isotope amount (0.015%) (ie, at least 50.1) % 氘 isotope).

The deuteration rate of the compound synthesized by the present invention refers to the ratio of the isotopic content of the labeled synthesis to the naturally occurring isotope amount. The deuterated atom of each of the specified deuterium atoms of the compound synthesized in the present invention may be at least 3500 times (52.5%), at least 4000 times (60%), at least 4500 times (67.5%), at least 5000 times. (75%), at least 5500 times (82.5%), at least 6000 times (90%), at least 6333.3 times (95%), at least 6466.7 times (97%), at least 6566.7 times (98.5%), At least 6600 times (99%), at least 6633.3 times (99.5%).

Herein, any atom not designated as 氘 exists in its natural isotopic abundance.

The compounds of the present invention can be prepared by the method disclosed in WO2010111483 (for example, the method disclosed in Example 189b (optional procedure), and using deuterated reagents (for example, CD ₃ OD, D6-benzaldehyde, Heavy water, etc.) in place of the corresponding non-deuterated reagents.

A further aspect of the invention provides a pharmaceutical composition comprising a compound of formula 或其 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

A further aspect of the invention provides a pharmaceutical composition according to formula I, or a pharmaceutically acceptable salt thereof, comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of hepatitis C Use in drugs infected with the virus.

A further aspect of the invention provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof for use in preventing or treating a hepatitis C virus infection.

A further aspect of the invention provides a method of preventing or treating a hepatitis C virus infection, the method comprising administering a compound of formula I, or a pharmaceutically acceptable salt thereof, a compound of formula I or A pharmaceutical composition of a pharmaceutically acceptable salt thereof is administered to a patient in need thereof.

The hepatitis C virus includes various genotypes thereof and a plurality of gene subtypes, such as 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a, 6a and the like.

Compared with elbasvir, compounds of formula I have superior properties in one or more assays (eg, activity assays, pharmacokinetic assays, liver microsomal metabolic stability assays, etc.) and are expected to reduce clinical Use doses to reduce treatment costs for more patients.

The terms used in this article have the following meanings:

The term "DMA" refers to N,N-dimethylacetamide.

The term "B(Pin) ₂ " refers to a pinacol borate.

The term "Pd(dppf)Cl ₂ " refers to [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride.

The term "HOBt" refers to 1-hydroxybenzotriazole.

detailed description

The preparation method of the compound of the present invention as shown in the formula is more specifically described below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention may also be conveniently prepared by combining various synthetic methods described in the specification or known in the art, and such combinations can be readily manipulated by those skilled in the art to which the present invention pertains.

Preparation of Compound 1 of Example 1

Step (1) Preparation of 3-bromophenyl-2-(2,5-dibromophenyl)acetate

50 g of 2,5-dibromophenylacetic acid and 600 mL of dichloromethane were added to a 1000 mL three-necked flask. After the solution was dissolved, 0.62 g of DMF was added, and 28.3 g of oxalyl chloride was added dropwise at room temperature. After the addition, the reaction was carried out for 1 hour at room temperature, and then the reaction solution was concentrated. To a volume of about 250 mL, a 2,5-dibromophenylacetyl chloride solution was obtained.

To another 1000 mL three-necked bottle, 31.5 g of m-bromophenol, 265 mL of dichloromethane, and 36.6 g of 2,6-lutidine were added dropwise, keeping the temperature below 25 ° C. After cooling to 0-5 ° C, the above prepared 2,5-dibromophenylacetyl chloride solution was added dropwise, and the reaction was carried out at 0-5 ° C for 1 hour after the addition. After the reaction was completed, it was quenched with 265 mL of 1N HCl. The organic phase was washed with 265 mL of water, then the organic phase was concentrated and acetonitrile was added, and concentrated to give a volume of about 150 mL of acetonitrile solution, 180 mL of water was added dropwise, stirred for 1 hour, filtered, and the filter cake was used with 160 mL. The mixture was washed with acetonitrile / water (1:1), and dried in vacuo at 50 ° C to give 69.7 g of pale yellow solid, compound 1-a, yield 90.6%.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 7.54 (d, J = 3.0 Hz, 1H), 7.49 (d, J = 10.6 Hz, 1H), 7.39 (dd, J = 10.0, 2.4 Hz, 1H) , 7.35-7.32 (m, 2H), 7.25 (d, J = 10.1 Hz, 1H), 7.10 (dd, J = 10.6, 1.6 Hz, 1H), 4.00 (s, 2H).

¹³ C-NMR (CDCl ₃ , 300 MHz): δ 167.9, 151.0, 135.5, 134.5, 134.3, 132.4, 130.5, 129.3, 124.9, 123.8, 122.4, 121.4, 120.4, 41.5.

HRMS (M-H) m/z: 444.8.

Step (2) Preparation of 1-(4-bromo-2-hydroxyphenyl)-2-(2,5-dibromophenyl)ethanone

To a 1000 mL three-necked bottle, 7.9 g of methanesulfonic anhydride and 436 g of methanesulfonic acid were sequentially added, and the mixture was heated to 90 ° C under N ₂ for 1 hour, and then cooled to 65 ° C. 68 g of compound 1-a was added thereto, and reacted at 65 ° C for 24 hours, followed by cooling to At room temperature, 572 mL of isopropanol/water (3:1) was added dropwise. After the addition, the mixture was stirred for 20 min and then filtered. The filter cake was washed with 215 mL of isopropyl alcohol/water (1:1), and the obtained solid was dried at 60 ° C for 12 hours to obtain 58.8. g gray solid, compound 1-b, yield 86.5%.

^{1 H-NMR (300MHz, DMSO} -d6): δ11.60 (s, 1H), 7.87 (d, J = 8.5Hz, 1H), 7.67 (d, J = 2.4Hz, 1H), 7.59 (d, J =8.7 Hz, 1H), 7.45 (dd, J = 8.7, 2.4 Hz, 1H), 7.26 (d, J = 1.9 Hz, 1H), 7.20 (dd, J = 8.4, 1.8 Hz, 1H), 4.59 (s) , 2H).

¹³ C-NMR (300 MHz, DMSO-d6): δ 199.3, 160.7, 138.4, 135.5, 134.5, 132.6, 132.1, 129.2, 124.5, 123.0, 121.5, 120.9, 120.8, 47.9.

HRMS (M-H) m/z: 444.8079.

Step (3) Preparation of 5-bromo-2-(2-(2,5-dibromophenyl)-1-iminoethyl)phenol

160 mL of 7 M methanolic ammonia and 28 g of compound 1-b were added to a 500 mL single-mouth flask, and the mixture was reacted at room temperature for 24 hours, and then filtered, and the obtained solid was dried at 40 ° C for 8 hours to obtain 23.8 g of a yellow solid, Compound 1-c, yield 85%.

¹ H-NMR (CDCl ₃ , 300MHz): δ 9.97, (s, 1H), 7.56 (d, J = 9.1 Hz, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.42 (d, J) =2.4 Hz, 1H), 7.40 (d, J = 2.4 Hz, 1H), 7.22 (d, J = 2.0 Hz, 1H), 7.02 (dd, J = 8.6, 2.0 Hz, 1H), 4.22 (s, 2H) ).

¹³ C-NMR (CDCl ₃ , 300 MHz): δ 175.9, 164.2, 135.3, 135.1, 134.9, 133.0, 128.8, 127.7, 124.4, 122.1, 122.0, 121.2, 116.9, 42.6.

HRMS (M+H) m/z: 445.8385.

Step (4) Preparation of (R)-2-(1-amino-2-(2,5-dibromophenol)ethyl)-5-bromophenol

To a 350 mL pressure-resistant tube, 130 mL of dichloromethane, 11.5 g of compound 1-c, 3.56 g of ammonium formate, and 47.7 mg of (R,R)Teth-TsDPEN-RuCl (Alfa Aesar) were added, and reacted at 70 ° C for 24 hours. Then, it was neutralized to pH=7.5 with 10% NaHCO ₃ solution, and the organic phase was washed with 2×20 mL of water, concentrated, and then added with about 100 ml of acetonitrile, and concentrated to obtain about 60 mL of acetonitrile solution, stirred at room temperature for 1 hour, filtered, and dried in vacuo to give 10.43 g. Gray solid, compound 1-d, yield 90.3%.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 7.46 (d, J = 8.4 Hz, 1H), 7.27 (m, 1H), 7.22 (d, J = 2.4 Hz, 1H), 7.06 (d, J = 1.9 Hz, 1H), 6.87 (dd, J = 8.1, 2.0 Hz, 1H), 6.70 (d, J = 8.1 Hz, 1H), 4.45 (dd, J = 8.8, 5.7 Hz, 1H), 3.15 (m, 2H).

¹³ C-NMR (CDCl ₃ , 300 MHz): δ 158.7, 139.1, 134.5, 134.4, 131.8, 129.0, 124.8, 123.5, 122.2, 122.1, 121.5, 120.6, 55.5, 42.8.

HRMS (M+H) m/z:447.8542.

Step (5) Preparation of (R)-5-bromo-2-(5-bromoindoline-2-yl)phenol

21.35 g of compound 1-d, 452 mg of cuprous iodide, 30.9 g of cesium carbonate were successively added to a 250 mL three-necked flask, and reacted at 45 ° C for 1 hour under nitrogen atmosphere. Then, 427 mL of ethyl acetate and 132 mL of a saturated ammonium chloride solution were added, and the pH was adjusted to 7.5 with 1 N HCl. The aqueous phase was extracted twice with 2×50 mL of ethyl acetate. The organic phases were combined and then were taken from <RTI ID=0.0> The organic phase was concentrated to about 120 mL of acetonitrile solution, 120 mL of water was added dropwise, filtered, and dried in vacuo to give 13.13 g of a white solid, compound 1-e, yield 75%.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 9.58 (s, 1H), 7.31 (s, 1H), 7.25 (d, J = 8.3 Hz, 1H), 7.10 (d, J = 2.0 Hz, 1H) , 6.98 (dd, J = 8.0, 2.0 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 4.94 (m, 1H), 4.40 (s, 1H), 3.30 (t, J = 8.6 Hz, 1H), 3.10 (dd, J = 15.7, 12.4 Hz, 1H).

¹³ C-NMR (CDCl ₃ , 300 MHz): δ 157.6, 147.4, 132.8, 130.5, 129.4, 127.8, 123.4, 122.7, 122.4, 120.9, 111.8, 113.3, 65.1, 38.0.

HRMS (M+H) m/z: 367.9269.

Step (6) (S, 12aR)-3,10-Dibromo-6-phenyl-12,12a-dihydro-6H-benzoyl[5,6][1,3]oxazepine

Preparation of [3,4-a]吲哚

6 g of compound 1-e, 30 mL of acetonitrile, 2.41 g of benzaldehyde, and 93 mg of trifluoroacetic acid were successively added to a 100 mL three-necked flask, and reacted at 35 ° C for 3 hours under nitrogen atmosphere. Then, 2.75 mL of 5% NaHCO ₃ solution and 12 mL of water were added dropwise, and the filter cake was washed successively with 18 mL of acetonitrile/water (2:1) and 12 mL of water, and dried under vacuum to obtain 6.66 g of a gray solid, compound 1-f. The rate is 89.6%.

^{_{1 H-NMR (CDCl 3,}} 300MHz): δ7.58 (m, 2H), 7.40-7.36 (m, 4H), 7.23 (s, 1H), 7.08 (d, J = 2.0Hz, 1H), 6.98 ( Dd, J = 8.2, 2.0 Hz, 1H), 6.82 (m, 2H), 6.76 (s, 1H), 4.71 (d, J = 8.9 Hz, 1H), 3.52 (dd, J = 15.7, 8.9 Hz, 1H) ), 3.12 (d, J = 15.7 Hz, 1H).

¹³ C-NMR (CDCl ₃ , 300 MHz): δ 153.2, 148.0, 137.6, 131.4, 130.4, 128.8, 128.5, 128.4, 127.8, 126.8, 124.4, 123.8, 121.2, 120.3, 112.6, 110.9, 83.1, 55.5, 36.0.

HRMS (M+H) m/z: 455.9577.

Step (7) (S)-3,10-Dibromo-6-phenyl-6H-benzoyl[5,6][1,3]

Preparation of [3,4-a]吲哚

To a 250 mL three-necked bottle, 6.66 g of compound 1-f, 67 mL of DMA, 3.67 g of NaHCO ₃ were added in sequence, and cooled to 10 ° C. 4.4 g of potassium permanganate was added in portions, and 16.7 mL of water was added dropwise to keep the temperature below 15 °C, reacted at 10 ° C overnight after the addition. 133 mL of ethyl acetate was added, and 67 mL of an aqueous NaHSO ₃ solution was added dropwise, and the mixture was allowed to stand for separation. The aqueous phase was extracted again with 67 mL of ethyl acetate. The organic phase was combined and washed three times with 3×33 mL of 10% NaCl solution, and the obtained organic phase was concentrated. 67 mL of isopropanol solution, further added 67 mL of water, stirred overnight, filtered, and the filter cake was washed with 25 mL of isopropanol/water (1:1), and dried under vacuum to obtain 5.62 g of a solid, ie, compound 1-g, yield 84.8%. .

¹ H-NMR (CDCl ₃ , 300 MHz): δ 7.81 (d, J = 1.8 Hz, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.37-7.28 (m, 3H), 7.21-7. m, 3H), 7.11-7.08 (m, 3H), 6.85 (s, 1H), 6.70 (d, J = 8.8 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 4.94 (m, 1H), 4.40 (s, 1H), 3.30 (t, J = 8.6 Hz, 1H), 3.10 (dd, J = 15.7, 12.4 Hz, 1H).

¹³ C-NMR (CDCl ₃ , 300 MHz): δ 150.0, 136.2, 133.9, 132.1, 130.7, 123.0, 126.8, 125.6, 125.1, 123.4, 122.4, 121.3, 117.0, 114.2, 111.2, 96.8, 84.5.

HRMS (M+H) m/z: 453.9485.

Step (8) (2S, 2'S)-di-tert-butyl 2,2'-(5,5'-((S)-6-phenyl-6H-benzoyl [5,6][1,3] Oxygen and nitrogen

Preparation of [3,4-a]indole-3,10-diyl)bis(1H-imidazol-5,2-diyl))bis(pyrrolidine-1-carboxylate)

60 mL of 1,4-dioxane, 2.5 g of compound 1-g, 3.03 g of B(Pin) ₂ , 2.12 g of potassium acetate, 200 mg of Pd(dppf)Cl ₂ , and 110 ° C under nitrogen atmosphere were added to a 250 mL three-necked flask. After 3 hours of reaction, the mixture was cooled and cooled, and then 40 mL of 1,4-dioxane and 10 mL of water, 4.11 g of compound 1-h, 1.6 g of Pd(dppf)Cl ₂ , 4.5 g of potassium carbonate were added, and the reaction was carried out at 110 ° C under nitrogen atmosphere overnight. . The reaction solution was subjected to post-purification and purified by column chromatography (EA: EtOAc: EtOAc: EtOAc)

¹ H-NMR (DMSO-d6, 300MHz): δ 11.89 (m, 2H), 7.99 (s, 1H), 7.79 (d, 1H), 7.71 (s, 1H), 7.54-7.46 (m, 3H) , 7.42-7.37 (m, 2H), 7.30-7.27 (m, 4H), 7.08 (s, 1H), 4.82 (m, 2H), 3.55 (br s, 2H), 3.37 (m, 2H), 2. 2.10 (m, 2H), 2.08-1.83 (m, 6H), 1.41-1.16 (m, 18H).

¹³ C-NMR (DMSO-d6, 300 MHz): δ 170.8, 154.6, 153.8, 149.4, 139.1, 138.3, 134.6, 129.6, 129.3, 129.1, 126.8, 124.5, 120.3, 119.4, 116.1, 113.4, 97.3, 79.0, 78.7, 60.2, 55.7, 47.0, 46.8, 33.8, 28.7, 28.4, 24.3, 23.6, 21.2, 14.6.

HRMS (M+H) m/z: 768.33.

Step (9) 6-Phenyl-3,10-bis(2-((S)-pyrrolidin-2-yl)-1H-imidazol-5-yl)-6H-benzoyl [5,6][ 1,3] oxygen and nitrogen

Preparation of [3,4-a]hydrazine hydrochloride

2.7 g of compound 1-i, 15 mL of methanol, 2.8 g of concentrated hydrochloric acid were successively added to a 250 mL single-necked bottle, and reacted at 45 ° C for 4 hours, cooled to room temperature, 45 mL of acetonitrile was added dropwise, stirred at room temperature overnight, filtered, and dried under vacuum to give 2.18 g of solid. , that is, compound 1-j, the yield was 87.2%.

¹ H-NMR (DMSO-d6, 300 MHz): δ 8.31 (d, J = 0.7 Hz, 1H), 8.21 (s, 1H), 8.19 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H) ), 7.88 (s, 1H), 7.72 (m, 3H), 7.47 (d, J = 8.8 Hz, 1H), 7.32 (m, 4H), 7.02 (m, 1H), 5.16 (t, J = 8.0 Hz) , 1H), 5.06 (t, J = 8.0 Hz, 1H), 3.52-3.37 (m, 4H), 2.57-2.47 (m, 5H), 2.24 - 2.17 (m, 2H), 2.08-1.91 (m, 2H) ).

¹³ C-NMR (DMSO-d6, 300 MHz): δ 149.7, 142.7, 141.5, 137.4, 135.7, 135.2, 132.3, 130.0, 129.3, 129.1, 126.8, 125.7, 121.0, 120.6, 120.2, 118.5, 118.0, 117.4, 115.5, 115.0, 111.5, 98.9, 83.6, 53.1, 52.6, 46.0, 45.9, 30.0, 29.9, 24.6, 24.5.

HRMS (M+H) m/z: 568.2780.

Step (10) Preparation of (S)-2-((tritylmethoxycarbonyl)amino)-3-methylbutyric acid

To a 250 mL single-mouth bottle, 9.94 g of triphosgene, 60 mL of chloroform were added, and the mixture was cooled to 0 ° C in an ice salt bath. Then, 7.2 g of CD ₃ OD was added, and 10.16 g of a solution of triethylamine in chloroform (20 mL) was added dropwise. After reacting for 2 hours at room temperature, it was washed with 3×25 mL of ice water, dried over anhydrous magnesium sulfate, and then evaporated to dryness to give 5.

Add 54.3 mL of water, 2.17 g of sodium hydroxide, 2.88 g of sodium carbonate, and add 6.36 g of L-valine to a 100 mL three-necked flask, cool down to 0 ° C, and add the above 5.72 g of trimethyl chloroformate dropwise. After the completion of the reaction at room temperature for 3 hours. It was extracted three times with 3×25 mL of diethyl ether. The aqueous phase was cooled to 0° C., and the mixture was adjusted to pH 1-2 with concentrated hydrochloric acid, and then extracted three times with 3×80 mL of dichloromethane, dried over anhydrous magnesium sulfate and concentrated to dryness , that is, compound 1-k, the yield was 40.8%.

¹ H-NMR (DMSO-d6, 300 MHz): δ 12.51 (s, 1H, ValCOOH), 7.29 (s, 1H, ValNH), 3.82 to 3.87 (m, 1H, ValCH), 2.00 to 2.04 (m, 1H) , ValCH(CH ₃ ) ₃ ), 0.87 (d, 6H, CH(CH ₃ ) ₂ ).

¹³ C-NMR (DMSO-d6, 300 MHz): δ 134.70, 113.55, 7.28, 6.62, 5.63, 3.12.

HRMS (M+H) m/z: 177.0923.

Step (11) Di(trimethylene) ((2S,2'S)-((2S,2'S)-2,2'-(5,5'-((S)-6-phenyl-6H-Benzyl) Acyl [5,6][1,3]oxazepine

[3,4-a]indole-3,10-diyl)bis(1H-imidazole-5,2-diyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl Preparation of 1-butanone-2,1-diyl)dicarboxylate

500 mg of compound 1-j, 6 mL of acetonitrile were sequentially added to a 50 mL two-necked flask, cooled to 5 ° C, and 274 mg of compound 1-k, 496 mg of N-methylmorpholine, 47.3 mg of HOBt, 295 mg of EDC·HCl were added, and the reaction was carried out at room temperature after the addition. overnight. 8 mL of ethyl acetate was added thereto, washed with 4 mL of 2M NH ₄ Cl, 3 mL of 10 wt% NaHCO ₃ , 3 mL of 10 wt% Na ₂ CO ₃ , 3 mL of water, dried with anhydrous magnesium sulfate, and concentrated. MeOH: DCM = 3: 100) afforded 428 mg (yield: mp.

^{1 H-NMR (DMSO-d6,300MHz} ): δ8.13 (s, 1H), 8.10 (s, 1H), 8.07 (d, 1H), 7.99 (s, 1H), 7.89 (s, 1H), 7.56 -7.54 (m, 3H), 7.52 (d, 1H), 7.35 (s, 1H), 7.31-7.27 (m, 5H), 6.97 (m, 2H), 5.16 (t, 1H), 5.12 (t, 1H) ), 4.15-4.11 (m, 2H), 3.91-3.80 (m, 4H), 2.45-2.36 (m, 2H), 2.02-1.98 (m, 8H), 0.85 (d, 3H), 0.82 (d, 3H) ), 0.80 (d, 3H), 0.77 (d, 3H).

¹³ C-NMR (DMSO-d6, 300 MHz): δ 170.8, 157.3, 149.9, 149.4, 138.7, 138.1, 136.9, 134.4, 131.9, 130.1, 129.6, 129.3, 129.1, 126.8, 124.6, 120.2, 119.3, 116.1, 115.9, 113.8, 113.5, 111.4, 110.3, 97.1, 58.5, 58.4, 55.4, 54.7, 51.9, 47.3, 31.4, 30.3, 24.7, 24.6, 19.5, 19.4, 19.1, 19.0.

HRMS (M+H) m/z: 888.4388.

Preparation of Compound 9 of Example 2

Step (1) (S, 12aR)-3,10-Dibromo-6-phenyl-12,12a-dihydro-6D-pentabenzoyl [5,6][1,3]oxazepine

Preparation of [3,4-a]吲哚

To a 100 mL three-necked flask were successively added 2.64 g of compound 1-e, 13 mL of acetonitrile, 1.12 g of D6-benzaldehyde (Aldrich), 40.8 mg of trifluoroacetic acid, and reacted at 35 ° C for 3 hours under nitrogen atmosphere. Then, 1.2 mL of a 5% NaHCO ₃ solution and 5.3 mL of water were successively added dropwise, and the mixture was filtered, and the filter cake was washed with 8 mL of acetonitrile/water (2:1), and dried under vacuum to give 2.85 g of a gray solid, compound 9-a, yield. 86%.

¹ H-NMR (CDCl ₃ , 300 MHz): δ 7.28 (m, 1H), 7.23 (s, 1H), 7.08 (d, J = 2.0 Hz, 1H), 6.98 (dd, J = 8.2, 2.0 Hz, 1H), 6.82 (m, 2H), 4.71 (d, J = 8.9 Hz, 1H), 3.52 (dd, J = 15.7, 8.9 Hz, 1H), 3.12 (d, J = 15.7 Hz, 1H).

HRMS (M+H) m/z: 461.9970.

Step (2) (S)-3,10-Dibromo-6-phenyl-6D-pentabenzoyl [5,6][1,3]oxazepine

Preparation of [3,4-a]吲哚

To a 100 mL three-necked bottle, 2.85 g of compound 9-a, 29 mL of DMA, 1.55 g of NaHCO ₃ were added in sequence, and the mixture was cooled to 10 ° C, and 1.86 g of potassium permanganate was added in portions, and 7.2 mL of water was added dropwise to keep the temperature below 15 °C, reacted at 10 ° C overnight after the addition. Was added 57mL of ethyl acetate was added dropwise 29mL NaHSO ₃ solution, allowed to stand separated and the aqueous phase was extracted with 30mL ethyl acetate and the combined organic phases were washed three times with a 3 × 15mL 10% NaCl solution, the organic phase was concentrated to give about 29 mL of isopropanol solution, 29 mL of water was added dropwise, stirred overnight, and filtered. The filter cake was washed with 12 mL of isopropanol/water (1:1), and dried under vacuum to give 2.08 g of solid, compound 9-b, yield 73.3%. .

¹ H-NMR (CDCl ₃ , 300 MHz): δ 7.81 (d, J = 1.8 Hz, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.25-7.16 (m, 3H), 6.85 (s, 1H), 6.70 (d, J = 8.8 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 4.94 (m, 1H), 4.40 (s, 1H), 3.30 (t, J = 8.6 Hz, 1H), 3.10 (dd, J = 15.7, 12.4 Hz, 1H).

HRMS (M+H) m/z: 459.9814.

Step (3) (2S, 2'S)-di-tert-butyl 2,2'-(5,5'-((S)-6-phenyl-6D-pentabenzoyl [5,6][1, 3] Oxygen and nitrogen

To a 250 mL three-necked bottle, 55 mL of 1,4-dioxane, 2.0 g of compound 9-b, 2.39 g of B(Pin) ₂ , 1.68 g of potassium acetate, 125.3 mg of Pd(dppf)Cl ₂ , and 110 under nitrogen were added. After reacting for 3 hours at °C, the mixture was cooled and cooled, and then 32 mL of 1,4-dioxane and 8 mL of water, 3.25 g of compound 1-h, 1.25 g of Pd(dppf)Cl ₂ , 3.54 g of potassium carbonate and 110 ° C under nitrogen atmosphere were added. overnight. The reaction mixture was subjected to post-purification and purified by column chromatography (EA: EtOAc: EtOAc: EtOAc: EtOAc:

^{1 H-NMR (DMSO-d6,300MHz} ): δ11.89 (m, 2H), 7.79 (d, 1H), 7.71 (s, 1H), 7.54-7.46 (m, 3H), 7.42-7.37 (m, 2H), 7.08 (s, 1H), 4.82 (m, 2H), 3.55 (br s, 2H), 3.37 (m, 2H), 2.32-2.10 (m, 2H), 2.08-1.83 (m, 6H), 1.41-1.16 (m, 18H).

HRMS (M+H) m/z: 774.4075.

Step (4) 6-Phenyl-3,10-bis(2-((S)-pyrrolidin-2-yl)-1H-imidazol-5-yl)-6D-pentabenzoyl [5,6 ][1,3]Oxygen and nitrogen

Preparation of [3,4-a]hydrazine hydrochloride

To a 50 mL single-mouth bottle, 2.0 g of compound 9-c, 11 mL of methanol, and 2.0 g of concentrated hydrochloric acid were successively added, and the mixture was reacted at 45 ° C for 4 hours, cooled to room temperature, 33 mL of acetonitrile was added dropwise, stirred at room temperature overnight, filtered, and dried in vacuo to give 1.7 g of solid. , compound 9-d, yield 91.4%.

¹ H-NMR (DMSO-d6, 300 MHz): δ 8.31 (d, J = 0.7 Hz, 1H), 8.19 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.88 (s, 1H) ), 7.72 (m, 3H), 7.47 (d, J = 8.8 Hz, 1H), 5.16 (t, J = 8.0 Hz, 1H), 5.06 (t, J = 8.0 Hz, 1H), 3.52-3.37 (m) , 4H), 2.57-2.47 (m, 5H), 2.24 - 2.17 (m, 2H), 2.08 - 1.91 (m, 2H).

HRMS (M+H) m/z: 574.3203.

Step (5) Dimethyl ((2S,2'S)-((2S,2'S)-2,2'-(5,5'-((S)-6-phenyl-6D-pentabenzoyl) [ 5,6][1,3] oxazepine

720 mg of compound 9-d, 8 mL of acetonitrile were sequentially added to a 50 mL two-necked flask, cooled to 5 ° C, and 385 mg of N-Moc-L-proline, 708 mg of N-methylmorpholine, 67.6 mg of HOBt, 421.7 mg of EDC·HCl were added. After the addition, the reaction was carried out at room temperature overnight. 12 mL of ethyl acetate was added thereto, washed with 6 mL of 2M NH ₄ Cl, 4.5 mL of 10 wt% NaHCO ₃ , 4.5 mL of 10 wt% Na ₂ CO ₃ , 4.5 mL of water, dried with anhydrous magnesium sulfate, and concentrated. Chromatography (MeOH: DCM = 3: 100) afforded 686 mg of y.

¹ H-NMR (DMSO-d6, 300MHz): δ 8.10 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.99 (s, 1H), 7.89 (s, 1H), 7.56-7.54 (m, 3H), 7.52 (d, J = 8.7 Hz, 1H), 7.35 (s, 1H), 6.97 (m, 2H), 5.16 (t, J = 7.3 Hz, 1H), 5.12 (t, J = 7.3 Hz, 1H), 4.15-4.11 (m, 2H), 3.91-3.80 (m, 4H), 3.55 (s, 3H), 3.54 (s, 3H), 2.45-2.36 (m, 2H), 2.02-1.98 (m, 8H), 0.85 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H), 0.80 (d, J = 6.8 Hz, 3H), 0.77 (d, J = 6.8 Hz) , 3H).

HRMS (M+H) m/z: 888.4613.

Preparation of Compound 7 of Example 3

Step (1) Preparation of D1-benzaldehyde

150 mL of diethyl ether, 37.92 g of anhydrous stannous chloride, 62.8 g of acetyl chloride were added dropwise to a 500 mL three-necked flask, cooled to -10 ° C, and 15.02 g of heavy water was added dropwise at -10 ° C, and then 10.3 g of benzonitrile was added. After completion of the reaction at room temperature overnight, the mixture was filtered, and then filtered, and then evaporated to dryness to give a white solid. </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt;

Step (2) (S, 12aR)-3,10-Dibromo-6-phenyl-12,12a-dihydro-6D-benzoyl[5,6][1,3]oxazepine

Preparation of [3,4-a]吲哚

To a 100 mL three-necked flask, 7.38 g of compound 1-e, 30 mL of acetonitrile, 3 g of D1-benzaldehyde, and 114 mg of trifluoroacetic acid were successively added, and reacted at 35 ° C for 3 hours under nitrogen atmosphere. Then, 3.3 mL of 5% NaHCO ₃ solution and 15 mL of water were successively added dropwise, and the mixture was filtered, and the filter cake was washed successively with 22.5 mL of acetonitrile/water (2:1), and dried under vacuum to obtain 7.69 g of a gray solid, compound 7-a, yield. 84%.

¹ H-NMR (CDCl ₃ , 500 MHz): δ 7.55 (m, 2H), 7.54-7.25 (m, 4H), 7.19 (d, 1H), 7.06 (dd, 1H), 6.97-6.82 (m, 1H) ), 6.81-6.78 (m, 2H), 4.68 (d, 1H), 4.69 (dd, 1H), 3.52 (m, 1H), 3.11 (d, 1H).

^{_{13 C-NMR (CDCl 3,}} 500MHz): δ153.4,148.2,137.8,131.6,130.6,129.0,128.7,128.0,127.0,124.6,124.0,121.4,120.5,112.8,110.0,83.1,55.7,36.2.

HRMS (M+H) m/z: 456.9898.

Step (3) (S)-3,10-Dibromo-6-phenyl-6D-benzoyl[5,6][1,3]oxazepine

Preparation of [3,4-a]吲哚

7.6 g of compound 7-a, 76 mL of DMA, 4.18 g of NaHCO ₃ were sequentially added to a 500 mL three-necked flask, cooled to 10 ° C, 5.01 g of potassium permanganate was added in portions, and 19 mL of water was added dropwise to keep the system temperature below 15 ° C. After the addition, the reaction was carried out at 10 ° C overnight. 152 mL of ethyl acetate was added, and 76 mL of aq. NaHSO ₃ was added dropwise, and the mixture was allowed to stand for separation. The aqueous phase was extracted with ethyl acetate (80 mL). The organic phase was combined, washed three times with 3×40 mL of 10% NaCl solution, washed, concentrated, and added. 120 ml of isopropanol, concentrated to obtain about 76 mL of isopropanol solution, and then added dropwise with 76 mL of water, stirred overnight, filtered, and the filter cake was washed with 32 mL of isopropanol/water (1:1), and dried under vacuum to give 6.387 g of a solid. 7-b, yield 84.4%.

¹ H-NMR (CDCl ₃ , 500 MHz): δ 7.78 (d, 1H), 7.51 (d, 1H), 7.34-7.29 (m, 3H), 7.29-7.28 (m, 3H), 7.06 (d, 2H) ), 6.83 (s, 1H), 6.70-6.68 (d, 1H).

¹³ C-NMR (CDCl ₃ , 500 MHz): δ 150.2, 136.3, 134.1, 132.3, 130.9, 130.1, 129.1, 127.0, 126.6, 125.7, 125.2, 123.6, 122.6, 121.5, 117.2, 114.3, 111.3, 97.0.

HRMS (M+H) m/z: 454.9.

Step (4) (2S, 2'S)-di-tert-butyl 2,2'-(5,5'-((S)-6-phenyl-6D-benzoyl [5,6][1,3] Oxygen and nitrogen

Add 80 mL of 1,4-dioxane, 3 g of compound 7-b, 3.63 g of B(Pin) ₂ , 2.54 g of potassium acetate, 190 mg of Pd(dppf)Cl ₂ to a 250 mL three-necked flask, and react at 110 ° C under nitrogen. After 3 hours, the mixture was cooled and cooled, and then 48 mL of 1,4-dioxane and 12 mL of water, 4.93 g of compound 1-h, 1.9 g of Pd(dppf)Cl ₂ and 5.37 g of potassium carbonate were added, and the reaction was carried out at 110 ° C under nitrogen atmosphere overnight. The reaction mixture was subjected to post-purification and purified by column chromatography (EtOAc: EtOAc: EtOAc: EtOAc:

¹ H-NMR (DMSO-d6, 500 MHz): δ 11.95 (m, 2H), 7.78 (d, 1H), 7.78 (s, 1H), 7.54-7.46 (m, 3H), 7.42-7.37 (m, 2H), 7.30-7.27 (m, 4H), 7.08 (s, 1H), 4.82 (m, 2H), 3.55 (br s, 2H), 3.37 (m, 2H), 2.32-2.10 (m, 2H), 2.08-1.83 (m, 6H), 1.41-1.16 (m, 18H).

¹³ C-NMR (DMSO-d6, 500 MHz): δ 170.8, 154.6, 153.8, 149.4, 139.1, 138.3, 134.6, 129.6, 129.3, 129.1, 126.8, 124.5, 120.3, 119.4, 116.1, 113.4, 97.3, 79.0, 78.7, 60.2, 55.7, 47.0, 46.8, 33.8, 28.7, 28.4, 24.3, 23.6, 21.2, 14.6.

HRMS (M+H) m/z: 76.93926.

Step (5) 6-Phenyl-3,10-bis(2-((S)-pyrrolidin-2-yl)-1H-imidazol-5-yl)-6D-benzoyl [5,6][ 1,3] oxygen and nitrogen

Preparation of [3,4-a]hydrazine hydrochloride

To a 50 mL single-mouth bottle, 1.7 g of compound 7-c, 8.5 mL of methanol, and 1.3 g of concentrated hydrochloric acid were successively added, and the mixture was reacted at 45 ° C for 4 hours, cooled to room temperature, 50 mL of acetonitrile was added dropwise, stirred at room temperature overnight, filtered, and dried under vacuum to give 1.35 g. Solid, compound 7-d, yield 85.5%.

^{1 H-NMR (DMSO-d6,500MHz} ): δ8.31 (d, 1H), 8.18 (m, 2H), 8.05 (d, 1H), 7.70 (m, 3H), 7.48 (d, 1H), 7.32 (m, 4H), 7.02 (m, 2H), 5.15 (t, 1H), 5.04 (t, 1H), 3.39-3.37 (m, 4H), 2.57-2.47 (m, 5H), 2.24 - 2.17 (m , 2H), 2.08-1.91 (m, 2H)

¹³ C-NMR (DMSO-d6, 500 MHz): δ 149.7, 142.7, 141.5, 137.4, 135.7, 135.2, 132.3, 130.0, 129.3, 129.1, 126.8, 125.7, 121.0, 120.6, 120.2, 118.5, 118.0, 117.4, 115.5, 115.0, 111.5, 98.9, 83.6, 53.1, 52.6, 46.0, 45.9, 30.0, 29.9, 24.6, 24.5.

HRMS (M+H) m/z: 569.2869.

Step (6) Dimethyl ((2S,2'S)-((2S,2'S)-2,2'-(5,5'-((S)-6-phenyl-6D-benzoyl [5, 6][1,3] oxazepine

Add 1000 mg of compound 7-d, 10 mL of acetonitrile to a 50 mL two-necked bottle, cool to 5 ° C, add 539 mg of N-Moc-L-proline, 992 mg of N-methylmorpholine, 95 mg of HOBt, 590 mg of EDC·HCl, plus After the completion of the reaction at room temperature overnight. 17 mL of ethyl acetate was added thereto, washed with 8.3 mL of 2M NH ₄ Cl, 6.3 mL of 10 wt% NaHCO ₃ , 6.3 mL of 10 wt% Na ₂ CO ₃ , 6.3 mL of water, and dried over anhydrous magnesium sulfate. Column chromatography (MeOH: DCM = 3: 100) gave 1.02 mg of yellow solid, compound 7 as a yield of 82.4%, purity 96.7%, 99%.

¹ H-NMR (DMSO-d6, 500MHz): δ 8.10 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.99 (s, 1H), 7.89 (s, 1H), 7.56-7.54 (m, 3H), 7.52 (d, J = 8.7 Hz, 1H), 7.35 (s, 1H), 7.31-7.27 (m, 5H), 6.97 (m, 2H), 5.16 (t, J = 7.3 Hz, 1H), 5.12 (t, J = 7.3 Hz, 1H), 4.15-4.11 (m, 2H), 3.91-3.80 (m, 4H), 3.55 (s, 3H), 3.54 (s, 3H), 2.45-2.36 (m, 2H), 2.02-1.98 (m, 8H), 0.85 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H), 0.80 (d, J = 6.8 Hz, 3H) , 0.77 (d, J = 6.8 Hz, 3H).

¹³ C-NMR (DMSO-d6, 500 MHz): δ 170.8, 157.3, 149.9, 149.4, 138.7, 138.1, 136.9, 134.4, 131.9, 130.1, 129.6, 129.3, 129.1, 126.8, 124.6, 120.2, 119.3, 116.1, 115.9, 113.8, 113.5, 111.4, 110.3, 97.1, 58.5, 58.4, 55.4, 54.7, 51.9, 47.3, 31.4, 30.3, 24.7, 24.6, 19.5, 19.4, 19.1, 19.0.

HRMS (M+H) m/z: 883.4388.

Effect Example 1 Evaluation of in vitro anti-hepatic activity

experimental method:

The test compound was diluted 1:3 in 8 series points, double duplicate wells, and added to a 96-well plate. The HCV GT-1a and HCV GT-1b replicon cells were separately suspended in a culture solution containing 10% FBS, and seeded at a density of 8000 cells per well into a 96-well plate containing the test compound. The cells were cultured for 3 days at 5% CO ₂ at 37 °C. The cell viability assay reagent was added, and the fluorescence signal (RFU, cell viability raw data) was detected 1 hour later. Subsequently, the luciferase luminescent substrate Bright-Glo was added to each well of the cells, and after 5 minutes, the chemiluminescent signal value RLU was detected using a chemiluminescence detection system. The raw data RFU and RLU were analyzed to calculate the percentage of viability of HCV GT-1a and HCV GT-1b replicon cells and the percentage of inhibitory activity of the test compound against HCV GT-1a and HCV GT-1b genotype replicons. GraphPad Prism software application inhibitory activity and cell viability percentages nonlinear fitting, EC ₅₀ compound calculated and CC _50.

Experimental results:

The CC _{50 of} inhibiting cell viability in Elbasvir, Compound 1, and Compound 9 was greater than 0.5 nM, and the EC ₅₀ for HCV GT-1a genotype replicon inhibitory activity was 0.004, 0.005, 0.004 nM, respectively (see Table 1), for HCV GT. -1b genotype replicon inhibition activity of EC ₅₀ were 0.005,0.005,0.003nM (see Table 2).

Table 1 Compound in vitro cytotoxicity and anti-hepatitis C virus activity (HCV GT-1a)

Table 2 In vitro cytotoxicity and anti-hepatitis C virus activity of compounds (HCV GT-1b)

Effect Example 2 Evaluation of in vitro human liver microsome stability

In 300 μL of the final incubation system, 30 μL of human liver microsomes (protein concentration: 0.15 mg/mL), 30 μL of NADPH + MgCl ₂ , 3 μL of substrate, and 237 μL of PBS buffer were contained. Among them, the ratio of the organic solvent (acetonitrile) was 1%. Make 2 parts in parallel, 0.3 mL each. For each sample, first prepare a total volume of 270 μL of substrate and enzyme mixture, and NADPH pre-incubated at 37 ° C for 5 min, then add 30 μL of NADPH + MgCl ₂ mixed, respectively, at 0, 5, 15, 40 μL was taken out at 30, 60, and 120 min, and the reaction was terminated with 300 μL of ice-acetonitrile containing an internal standard. In addition, three blanks (KB) were each made up to 300 μL, respectively, KB1: no NADPH; KB2: no substrate; KB3: no enzyme.

Aspirate 40 μL of the incubation sample, add 300 μL of ice-acetonitrile precipitate containing internal standard, vortex for 5 min, and centrifuge (13,000 rpm, 4 ° C) for 10 min. 80 μL of the supernatant was pipetted into the sample plate, 80 μL of a 50% aqueous acetonitrile solution was added, and the mixture was mixed, and 0.5 μL of the injection was analyzed.

The Ln values of the percentage of test compound remaining at each time point were plotted against time, and t _1/2 and CL _{int were} calculated by the corresponding formulas.

Slope = -K _mic

Note: V/M=1/protein concentration

Table 3 shows experimental data of the metabolic stability of human liver microsomes in vitro for test compounds.

Table 3 Test compound in vitro human liver microsome metabolic stability

The results showed that the in vitro human liver microsome metabolic stability of the compound 1 and the compound 9 of the present invention was superior to that of the elbasvir.

Effect Example 3 Pharmacokinetic Evaluation in Rats

SD rats, weighing 200-220 g, were randomly divided into 2 groups after 3-5 days, and 3 rats in each group were given the test compound elbasvir and compound 7 at a dose of 10 mg/kg.

The test animals (SD rats) were fasted for 12 h before administration, and given food for 4 h after administration. Drinking water was free before and after the experiment and during the experiment.

After intragastric administration, 0.10 mL of blood was taken from the eyelids at 0 min, 15 min, 30 min, 1 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 24 h, EDTA-K2 was anticoagulated, and the blood sample was immediately placed in ice for 30 min. The plasma was separated by centrifugation at 4 ° C, 4000 rpm, and 10 min. Immediately after collection of all plasma, the samples were stored at -20 ° C.

Pipette 40 μL of plasma sample and standard sample, add 200 μL of acetonitrile solution containing internal standard, mix by shaking for 5 min, centrifuge at 13000 rpm for 10 min, take 80 μL of supernatant to the sample plate, add 80 μL of 50% acetonitrile aqueous solution, mix, and absorb 1 μL for LC. - MS/MS measurement (liquid phase conditions: column: ACQUITY UPLC BEH C18 (1.7 μm 2.1 × 50 mm); aqueous phase: 0.1% aqueous formic acid, organic phase: methanol solution; flow rate: 0.3 ml/min, gradient conditions: water The ratio of phase 0-0.5min: 90%; 0.5-0.8min: 90% ~ 40%; 0.8-1.2min: 40% ~ 5%; 1.2-2.0min: 5%; 2.0-2.2min: 5% ~ 90 %; 2.2-3.0min: 90%. Mass spectrometry conditions: LC-MS/MS (Waters TQ-S) detection, ion source: ESI+, cone voltage: 85V, capillary voltage: 3.2kV, ion source temperature: 150°C , desolvation gas temperature: 400 ° C, desolvation gas flow rate: 800 L / hr. Elbasvir selective reaction detection of ions [M + H] m / z 882.5 → 656.42, selective reaction of compound 7 detection of ions [M + H] m/z 883.43→657.37. The internal standard diazepam selective reaction detection ion [M+H]m/z285.06→154.04), and the chromatogram was recorded.

The oral bioavailability of the compounds of the invention was assessed by pharmacokinetic experiments in rats.

Table 4 Pharmacokinetic parameters of test compounds

Claims

A compound of the formula: or a pharmaceutically acceptable salt thereof:

Wherein R 1 or R 2 are each independently selected from CH 3 , CH 2 D, CHD 2 or CD 3 , and R 3 , R 4 , R 5 , R 6 , R 7 , R 8 or R 9 are independently selected from H Or D, provided that R 1 to R 9 contain at least one deuterium atom.
A compound according to claim 1, wherein R 1 and R 2 are simultaneously CH 3 or CD 3 .
A compound according to claim 1, wherein R 5 , R 6 , R 7 , R 8 and R 9 are simultaneously H or D.

A compound according to claim 1, wherein the compound of the formula:

Compound R ₁ R ₂ R ₃ R ₄ R ₅ R ₆ R ₇ R ₈ R ₉ 1 CD ₃ CD ₃ H H H H H H H 2 CD ₃ CD ₃ D H H H H H H 3 CD ₃ CD ₃ H D H H H H H 4 CD ₃ CD ₃ D H D D D D D 5 CD ₃ CD ₃ D D H H H H H 6 CD ₃ CD ₃ D D D D D D D 7 CH ₃ CH ₃ D H H H H H H 8 CH ₃ CH ₃ D D H H H H H 9 CH ₃ CH ₃ D H D D D D D 10 CH ₃ CH ₃ D D D D D D D 11 CH ₃ CH ₃ H D H H H H H

A compound according to claim 1, wherein the compound of the formula:
A pharmaceutical composition comprising the compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 6, for the preparation of a medicament for preventing or treating hepatitis C virus infection.
The use according to claim 7, wherein the hepatitis C virus is selected from the group consisting of 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a or 6a.
A pharmaceutical composition comprising the compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of a hepatitis C virus infection.
The pharmaceutical composition according to claim 9, wherein the hepatitis C virus is selected from the group consisting of 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a or 6a.
A method for preventing or treating a hepatitis C virus infection, wherein the method comprises the compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a compound of formula I or A pharmaceutical composition of a pharmaceutically acceptable salt is administered to a patient in need thereof.
The method of claim 11 wherein said hepatitis C virus is selected from the group consisting of: 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a or 6a.