CN108290904A - Elbasvir derivatives, the medical composition and its use containing the compound of deuterium modification - Google Patents

Abstract

The invention belongs to field of medicaments, and in particular to elbasvir derivatives, the pharmaceutical composition containing the compound and its purposes in the drug for preparing treatment hepatitis c virus infection of deuterium modification.The elbasvir derivatives of the deuterium modification of the present invention are measured in one or more（Such as determination of activity, pharmacokinetics measurement, hepatomicrosome metabolic stability measure etc.）In there is superior property, be expected to reduce clinical dosage, to reduce treatment cost to allow more patients to be benefited.

Description

Elbasvir derivative, the medical composition and its use containing the compound of deuterium modification Technical field

The invention belongs to field of medicaments, and in particular to elbasvir derivative, the pharmaceutical composition containing the compound and its purposes in the drug of preparation treatment hepatitis c virus infection of deuterium modification.

Background technique

Many current drugs are absorbed, are distributed, are metabolized and/or are drained the puzzlement of (ADME) feature difference, and which hinders them widely to apply.ADME characteristic difference is also the big reason that drug candidate fails in clinical test.A strategy for having potential attraction for improving drug metabolism characteristic is to carry out deuterium modification.In this approach, the drug metabolism for slowing down CYP mediation by substituting one or more hydrogen atoms with D-atom is attempted.Deuterium is safe and stable, non-radiative hydrogen isotope.Compared to hydrogen, deuterium and carbon form stronger key.In selected example, bond strength increase that deuterium is given can the ADME characteristic to drug bring positive influences, generate and improve the potentially possible of pharmaceutical efficacy, safety and/or tolerance.Meanwhile because the size and shape of deuterium is substantially identical as hydrogen, compared to the precursor chemicals for only including hydrogen, the biochemical activity and selectivity of drug will not influence with deuterium substitution hydrogen.

Deuterium replace influence to metabolic rate reported on some drugs (for example, with reference to 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 result is that variable and uncertain.For some compounds, deuterate causes internal metabolite clearance to reduce.For other compounds, metabolism is not changed.For some compounds, it has therefore proved that metabolite clearance increases.The changeability of deuterium effect, which also results in expert's query or abandoned deuterium, modifies the idea for inhibiting detrimental metabolic as feasible drug design strategies (referring at Foster page 35 and at Fisher page 101).

Even if deuterium modification is also uncertain to the effect of the metabolic characteristic of drug when D-atom is integrated to the known site of metabolin.It only actually prepares and has detected deuterated drug, just can determine that whether and how metabolic rate is different from non-deuterated counterpart.For example, see Fukuto et al. (J.Med.Chem., 1991,34,2871-76).Many drugs have multiple possible sites that metabolic response occurs.It needs the site of deuterium substitution and makes the visible necessary degree of deuterium of the influence to metabolism, if any, each drug is all different.

It is a kind of compound monolithic once a day that the hepatitis of Mo Shadong takes orally cocktail grazoprevir/elbasvir (100mg/50mg) entirely, is made of grazoprevir (a kind of NS3/4A protease inhibitors) and elbasvir (a kind of NS5A polymerase inhibitors).FDA was granted by 2 new breakthrough drug (BTD) qualifications in 2015, was respectively as follows: the BTD qualification of 4 chronic hepatitis C viral of (1) therapeutic gene type (HCV GT4) the infected；(2) treatment is carrying out the BTD qualification of genotype 1HCV (HCV GT1) the infected of hemodialysis with end-stage renal disease.

Although elbasvir has favorable activity, it is desired nonetheless to which further exploitation has inhibitory activity to hepatitis C virus NS 5 A, can be used for preventing or treating the noval chemical compound of hepatitis c virus infection.

Summary of the invention

In the first aspect of the invention, one kind is provided such as formula Ι compound represented or its pharmaceutically acceptable salt:

Wherein, R₁Or R₂Separately it is selected from CH₃、CH₂D、CHD₂Or CD₃, R₃、R₄、R₅、R₆、R₇、R₈Or R₉It is separately R selected from H (hydrogen) or D (deuterium), condition₁~R₉In contain at least one D-atom.

In some embodiments of the present invention, R₁、R₂It is simultaneously CH₃Or CD₃.In one embodiment of the invention, R₁、R₂It is simultaneously CH₃.In another embodiment of the present invention, R₁、R₂It is simultaneously CD₃。

In some embodiments of the present invention, R₅、R₆、R₇、R₈、R₉It is simultaneously H or D.In one embodiment of the invention, R₅、R₆、R₇、R₈、R₉It is simultaneously H.In another embodiment of the present invention, R₅、R₆、R₇、R₈、R₉It is simultaneously D.

In certain preferred embodiments of the invention, the example such as formula Ι compound represented is as follows:

Compound	R1	R2	R3	R4	R5	R6	R7	R8	R9
1	CD3	CD3	H	H	H	H	H	H	H
2	CD3	CD3	D	H	H	H	H	H	H
3	CD3	CD3	H	D	H	H	H	H	H
4	CD3	CD3	D	H	D	D	D	D	D
5	CD3	CD3	D	D	H	H	H	H	H
6	CD3	CD3	D	D	D	D	D	D	D
7	CH3	CH3	D	H	H	H	H	H	H
8	CH3	CH3	D	D	H	H	H	H	H
9	CH3	CH3	D	H	D	D	D	D	D
10	CH3	CH3	D	D	D	D	D	D	D
11	CH3	CH3	H	D	H	H	H	H	H

In further preferred embodiment of the present invention, the example such as formula Ι compound represented is as follows:

Term " pharmaceutically acceptable salt " refers to the salt for remaining the biological efficacy of the free bronsted lowry acids and bases bronsted lowry of specific compound without biology ill-effect.The example of pharmaceutically acceptable salt includes but is not limited to: (1) salt of the formation such as acid-addition salts and inorganic acid such as hydrochloric acid, sulfuric acid, hydrobromic acid, nitric acid, phosphoric acid；Or the salt with the formation such as organic acids such as malic acid, fumaric acid, maleic acid, benzoic acid, phenylacetic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, hydroxyacetic acid, cinnamic acid, pyruvic acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, acrylic acid, mandelic acid；Or the salt of the formation such as (2) base addition salts and alkali metal such as lithium, sodium, potassium；With the salt of the formation such as alkaline-earth metal such as calcium, magnesium；With the salt of the formation such as organic bases such as ammonium, choline, diethanol amine, lysine, ethylenediamine, tert-butylamine, t-octanylamine, three (methylol) aminomethanes, N- methyl glucose osamine, triethanolamine, dehydroabietylamine.

If any atom of the compound of institute's labelled synthesis does not specify in the present invention, the stable isotope of any one of the atom can be represented.Unless stated otherwise, when position a certain in structure is defined as H i.e. hydrogen (H-1), which contains only naturally occurring isotopic mass.Equally, unless stated otherwise, when position a certain in structure is defined as D i.e. deuterium (H-2), the position is at least 3340 times bigger than naturally occurring isotopic mass (0.015%) (at least having 50.1% deuterium isotope) containing isotopic mass.

The deuterated rate of the compound of institute's labelled synthesis refers to the isotopic content of labelled synthesis and the ratio of naturally occurring isotopic mass in the present invention. Each of compound of institute's labelled synthesis specifies the deuterated rate of D-atom that can be at least 3500 times (52.5%) in the present invention, 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 each atom for being not designated as deuterium is with the presence of its natural isotopic abundance.

The compound of the present invention can be prepared by the following method: i.e. the method (such as method disclosed in embodiment 189b (optional program)) with reference to disclosed in WO2010111483, and use deuterated reagent (such as CD₃OD, D6- benzaldehyde, heavy water etc.) replace corresponding non-deuterated reagent.

It is another aspect of the present invention to provide a kind of pharmaceutical compositions, and it includes such as formula Ι compound represented or its pharmaceutically acceptable salt and pharmaceutically acceptable excipient.

It is a further aspect of the present invention to provide the purposes of compound shown in formula I or its pharmaceutically acceptable salt, the pharmaceutical composition containing compound shown in formula I or its pharmaceutically acceptable salt in the drug of preparation prevention or treatment hepatitis c virus infection.

It is a further aspect of the present invention to provide the pharmaceutical compositions containing compound or its pharmaceutically acceptable salt shown in formula I for preventing or treating hepatitis c virus infection.

Another aspect of the invention is to provide a kind of method prevented or treat hepatitis c virus infection, and the method includes compound shown in formula I or its pharmaceutically acceptable salt, the pharmaceutical composition containing compound shown in formula I or its pharmaceutically acceptable salt are given to the patient of needs.

The hepatitis C virus includes its Multi-genotype and several genes hypotype, such as 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a, 6a etc..

Compared with elbasvir, compound shown in formula I has superior property in one or more measurements (such as determination of activity, pharmacokinetics measurement, hepatomicrosome metabolic stability measure etc.), it is expected to reduce clinical dosage, to reduce treatment cost to allow more patients to be benefited.

Term used herein has following meaning:

Term " DMA " refers to DMAC N,N' dimethyl acetamide.

Term " B (Pin)₂" refer to connection boric acid pinacol ester.

Term " Pd (dppf) Cl₂" refer to [bis- (diphenylphosphine) ferrocene of 1,1'-] palladium chloride.

Term " HOBt " refers to I-hydroxybenzotriazole.

Specific embodiment

The preparation method of the present invention such as formula Ι compound represented is described more particularly below, but these specific methods do not form any restrictions to the present invention.Various synthetic methods describing in the present specification or known in the art can also optionally be combined and are easily made by the compounds of this invention, and such combination can be easy to carry out operation by those skilled in the art in the invention.

The preparation of 1 compound 1 of embodiment

The preparation of step (1) 3- bromophenyl -2- (2,5- dibromo phenyl) acetic acid esters

50g 2,5- dibromo phenylacetic acid, 600mL methylene chloride are added into 1000mL there-necked flask, 0.62g DMF is added after dissolved clarification, room temperature is added dropwise 28.3g oxalyl chloride, reacts at room temperature 1 hour after adding, then reaction solution is concentrated into about 250mL volume, obtains 2,5- dibromobenzene chloride solution.

31.5g m -bromoacetophenone is sequentially added into another 1000mL there-necked flask, 265mL methylene chloride, 36.6g 2 is added dropwise again, 6- lutidines (2,6-lutidine) keeps temperature to be lower than 25 DEG C, 0-5 DEG C is cooled to after adding, above-mentioned 2, the 5- dibromobenzene chloride solution prepared is added dropwise again, adds latter 0-5 DEG C and reacts 1 hour.It is quenched after reaction with 265mL 1N HCl, organic phase is washed with 265mL again, then organic phase is concentrated to and is added acetonitrile, be concentrated to get the acetonitrile solution of about 150mL volume, 180mL water is added dropwise, stirring 1 hour, filtering, filter cake are washed with 160mL acetonitrile/water (1:1), obtain 69.7g faint yellow solid after 50 DEG C of vacuum drying, that is compound 1-a, yield 90.6%.

¹H-NMR(CDCl₃, 300MHz): δ 7.54 (d, J=3.0Hz, 1H), 7.49 (d, J=10.6Hz, 1H), 7.39 (dd, J=10.0,2.4Hz, 1H), 7.35-7.32 (m, 2H), 7.25 (d, J=10.1Hz, 1H), 7.10 (dd, J=10.6,1.6Hz, 1H), 4.00 (s, 2H).

¹³C-NMR(CDCl₃, 300MHz): δ 167.9,151.0,135.5,134.5,134.3,132.4,130.5,129.3,124.9,12 3.8,122.4, 121.4,120.4,41.5。

HRMS (M-H) m/z:444.8073.

The preparation of step (2) 1- (the bromo- 2- hydroxy phenyl of 4-) -2- (2,5- dibromo phenyl) ethyl ketone

7.9g methanesulfonic acid acid anhydride, 436g methanesulfonic acid, N are sequentially added into 1000mL there-necked flask₂65 DEG C are cooled to after being heated to 90 DEG C of reactions under protection 1 hour; 68g compound 1-a is added; 65 DEG C reaction 24 hours after be cooled to room temperature; it is added dropwise 572mL isopropanol/water (3:1), is filtered after stirring 20min after adding, filter cake is washed with 215mL isopropanol/water (1:1); the drying 12 hours of 60 DEG C of obtained solid; obtain 58.8g gray solid, i.e. compound 1-b, yield 86.5%.

¹H-NMR (300MHz, DMSO-d6): δ 11.60 (s, 1H), 7.87 (d, J=8.5Hz, 1H), 7.67 (d, J=2.4Hz, 1H), 7.59 (d, J=8.7Hz, 1H), 7.45 (dd, J=8.7,2.4Hz, 1H), 7.26 (d, J=1.9Hz, 1H), 7.20 (dd, J=8.4,1.8Hz, 1H), 4.59 (s, 2H).

¹³C-NMR (300MHz, DMSO-d6): δ 199.3,160.7,138.4,135.5,134.5,132.6,132.1,129.2,124.5,12 3.0,121.5,120.9,120.8,47.9.

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

The preparation of step (3) bromo- 2- of 5- (2- (2,5- dibromo phenyl) -1- iminoethyl) phenol

160mL 7M methanol ammonia, 28g compound 1-b are added into 500mL single port bottle, room temperature reaction is filtered after 24 hours, and the drying 8 hours of 40 DEG C of obtained solid obtains 23.8g yellow solid, i.e. compound 1-c, yield 85%.

¹H-NMR(CDCl₃, 300MHz): δ 9.97, (s, 1H), 7.56 (d, J=9.1Hz, 1H), 7.48 (d, J=8.6Hz, 1H), 7.42 (d, J=2.4Hz, 1H), 7.40 (d, J=2.4Hz, 1H), 7.22 (d, J=2.0Hz, 1H), 7.02 (dd, J=8.6,2.0Hz, 1H), 4.22 (s, 2H).

¹³C-NMR(CDCl₃, 300MHz): δ 175.9,164.2,135.3,135.1,134.9,133.0,128.8,127.7,124.4,12 2.1,122.0,121.2,116.9,42.6.

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

The preparation of step (4) (R) -2- (1- amino -2- (2,5- dibromophenol) ethyl) -5- bromophenol

130mL methylene chloride is added into 350mL pressure pipe, 11.5g compound 1-c, 3.56g ammonium formate, 47.7mg (R, R) Teth-TsDPEN-RuCl (AlfaAesar), 70 DEG C are reacted 24 hours.Then 10%NaHCO is used₃Solution is neutralized to pH=7.5, and organic phase is concentrated after being washed with 2 × 20mL, and about 100ml acetonitrile is added, is concentrated to get about 60mL acetonitrile solution, and after being stirred at room temperature 1 hour, filtering obtains 10.43g gray solid, i.e. compound 1-d, yield 90.3% after vacuum drying.

¹H-NMR(CDCl₃, 300MHz): δ 7.46 (d, J=8.4Hz, 1H), 7.27 (m, 1H), 7.22 (d, J=2.4Hz, 1H), (7.06 d, J=1.9Hz, 1H), 6.87 (dd, J=8.1,2.0Hz, 1H), 6.70 (d, J=8.1Hz, 1H), 4.45 (dd, J=8.8,5.7Hz, 1H), 3.15 (m, 2H).

¹³C-NMR(CDCl₃, 300MHz): δ 158.7,139.1,134.5,134.4,131.8,129.0,124.8,123.5,122.2,12 2.1,121.5,120.6,55.5,42.8.

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

The preparation of the bromo- 2- of step (5) (R) -5- (5--bromo indole quinoline -2- base) phenol

21.35g compound 1-d, 452mg cuprous iodide is sequentially added into 250mL there-necked flask, 30.9g cesium carbonate, lower 45 DEG C of nitrogen protection are reacted 1 hour.Then 427mL ethyl acetate and 132mL saturated ammonium chloride solution is added, pH=7.5 is adjusted with 1N HCl again, water phase is extracted twice with 2 × 50mL ethyl acetate again, merges organic phase, then is washed respectively with 88mL 10%NaCl, 88mL, organic phase is concentrated into about 120mL acetonitrile solution, 120mL water is added dropwise, filtering obtains 13.13g gray solid after vacuum drying, that is compound 1-e, yield 75%.

¹H-NMR(CDCl₃, 300MHz): δ 9.58 (s, 1H), 7.31 (s, 1H), 7.25 (d, J=8.3Hz, 1H), 7.10 (d, J=2.0Hz, 1H), 6.98 (dd, J=8.0,2.0Hz, 1H), 6.90 (d, J=8.0Hz, 1H), 6.73 (d, J=8.0Hz, 1H), 4.94 (m, 1H), 4.40 (s, 1H), 3.30 (t, J=8.6Hz, 1H), 3.10 (dd, J=15.7,12.4Hz, 1H).

¹³C-NMR(CDCl₃, 300MHz): δ 157.6,147.4,132.8,130.5,129.4,127.8,123.4,122.7,122.4,12 0.9,111.8,113.3,65.1,38.0.

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

Bromo- 6- phenyl -12,12a- dihydro -6H- benzoyl [5,6] [1,3] the oxygen azepine of step (6) (S, 12aR) -3,10- twoThe preparation of [3,4-a] indoles

6g compound 1-e, 30mL acetonitrile is sequentially added into 100mL there-necked flask, 2.41g benzaldehyde, 93mg trifluoroacetic acid, lower 35 DEG C of nitrogen protection are reacted 3 hours.Then 2.75mL 5%NaHCO is successively added dropwise₃Solution and 12mL water, filtering, filter cake successively use 18mL acetonitrile/water (2:1) and 12mL to wash, 6.66g gray solid, i.e. compound 1-f, yield 89.6% are obtained after vacuum drying.

¹H-NMR(CDCl₃, 300MHz): δ 7.58 (m, 2H), 7.40-7.36 (m, 4H), 7.23 (s, 1H), 7.08 (d, J=2.0Hz, 1H), 6.98 (dd, J=8.2,2.0Hz, 1H), 6.82 (m, 2H), 6.76 (s, 1H), 4.71 (d, J=8.9Hz, 1H), 3.52 (dd, J=15.7,8.9Hz, 1H), 3.12 (d, J=15.7Hz, 1H).

¹³C-NMR(CDCl₃, 300MHz): δ 153.2,148.0,137.6,131.4,130.4,128.8,128.5,128.4,127.8,12 6.8,124.4,123.8,121.2,120.3,112.6,110.9,83.1,55.5,36.0.

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

The bromo- 6- phenyl -6H- benzoyl [5,6] [1,3] of step (7) (S) -3,10- two is miscellaneousThe preparation of [3,4-a] indoles

6.66g compound 1-f, 67mL DMA, 3.67g NaHCO are sequentially added into 250mL there-necked flask₃, 10 DEG C are cooled to, 4.4g potassium permanganate is added portionwise, then 16.7mL water is added dropwise, system temperature is kept to be lower than 15 DEG C, 10 DEG C of reactions are stayed overnight after adding.133mL ethyl acetate is added, then 67mL NaHSO is added dropwise₃Aqueous solution, stratification, water phase uses 67mL ethyl acetate to extract again, merges organic phase, is washed three times with 3 × 33mL 10%NaCl solution, gained organic phase is concentrated to get about 67mL aqueous isopropanol, it is added dropwise after 67mL water is stirred overnight and filters again, filter cake is washed with 25mL isopropanol/water (1:1), and 5.62g solid is obtained after vacuum drying, that is compound 1-g, yield 84.8%.

¹H-NMR(CDCl₃, 300MHz): δ 7.81 (d, J=1.8Hz, 1H), 7.51 (d, J=8.1Hz, 1H), 7.37-7.28 (m, 3H), 7.21-7.17 (m, 3H), 7.11-7.08 (m, 3H), 6.85 (s, 1H), 6.70 (d, J=8.8Hz, 1H), 6.90 (d, J=8.0Hz, 1H), 6.73 (d, J=8.0Hz, 1H), 4.94 (m, 1H), 4.40 (s, 1H), 3.30 (t, J=8.6Hz, 1H), 3.10 (dd, J=15.7,12.4Hz, 1H).

¹³C-NMR(CDCl₃, 300MHz): δ 150.0,136.2,133.9,132.1,130.7,123.0,126.8,125.6,125.1,12 3.4,122.4,121.3,117.0,114.2,111.2,96.8,84.5.

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

Step (8) (2S, 2 ' S)-di-t-butyl 2,2 '-(5,5 '-((S) -6- phenyl -6H- benzoyl [5,6] [1,3] oxygen azepines[3,4-a] indoles -3,10- diyl) two (1H- imidazoles -5,2- diyls)) two (pyrrolidines -1- formic acid esters) preparation

60mL Isosorbide-5-Nitrae-dioxane, 2.5g compound 1-g, 3.03g B (Pin) are sequentially added into 250mL there-necked flask₂, 2.12g potassium acetate, 200mg Pd (dppf) Cl₂, nitrogen protection it is lower 110 DEG C reaction 3 hours after cool, add 40mL Isosorbide-5-Nitrae-dioxane and 10mL water, 4.11g compound 1-h, 1.6g Pd (dppf) Cl₂, 4.5g potassium carbonate, the lower 110 DEG C of reactions of nitrogen protection are overnight.The post-treated rear pillar chromatographic purifying (EA:PE=45:55) of reaction solution, obtains 2.74g yellow solid, i.e. compound 1-i, yield 66%.

¹H-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), 2.32-2.10 (m, 2H), 2.08-1.83 (m, 6H), 1.41-1.16 (m, 18H).

¹³C-NMR (DMSO-d6,300MHz): 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:768.3887.

Step (9) 6- phenyl -3,10- two (2- ((S)-pyrrolidin-2-yl) -1H- imidazoles -5- base) -6H- benzoyl [5,6] [1,3] oxygen azepineThe preparation of [3,4-a] indole hydrochloride

2.7g compound 1-i, 15mL methanol, 2.8g concentrated hydrochloric acid are sequentially added into 250mL single port bottle, 45 DEG C reaction 4 hours after be cooled to room temperature, be added dropwise 45mL acetonitrile, filtered after being stirred overnight at room temperature, 2.18g solid, i.e. compound 1-j, yield 87.2% are obtained after vacuum drying.

¹H-NMR (DMSO-d6,300MHz): δ 8.31 (d, J=0.7Hz, 1H), 8.21 (s, 1H), 8.19 (s, 1H), 8.05 (d, J=8.0Hz, 1H), 7.88 (s, 1H), 7.72 (m, 3H), 7.47 (d, J=8.8Hz, 1H), 7.32 (m, 4H), 7.02 (m, 1H), 5.16 (t, J=8.0Hz, 1H), 5.06 (t, J=8.0Hz, 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).

¹³C-NMR (DMSO-d6,300MHz): 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.

The preparation of step (10) (S) -2- ((three deuterium methoxycarbonyls) amino) -3 Methylbutanoic acid

9.94g triphosgene, 60mL chloroform are sequentially added into 250mL single port bottle, ice salt bath is cooled to 0 DEG C, adds 7.2g CD₃Chloroform (20mL) solution of 10.16g triethylamine is added dropwise in OD, adds and moves back to room temperature reaction 2 hours, it is washed with 3 × 25mL ice water, is concentrated and does after anhydrous magnesium sulfate is dry, obtain 5.72g colourless liquid, that is three deuterium methylchloroformates are directly used in and react in next step.

54.3mL water, 2.17g sodium hydroxide are added into 100mL there-necked flask, 2.88g sodium carbonate adds 6.36g Valine, cools to 0 DEG C, above-mentioned tri- deuterium methylchloroformate of 5.72g is added dropwise, reacts at room temperature 3 hours after adding.Three times with the extraction of 3 × 25mL ether, water phase is cooled to 0 DEG C, adjusts pH=1~2 with concentrated hydrochloric acid, and then three times with the extraction of 3 × 80mL methylene chloride, concentration is dry after anhydrous magnesium sulfate is dry, obtains 3.942g white solid, i.e. compound 1-k, yield 40.8%.

¹H-NMR (DMSO-d6,300MHz): δ 12.51 (s, 1H, ValCOOH), 7.29 (s, 1H, ValNH), 3.82~3.87 (m, 1H, ValCH), 2.00~2.04 (m, 1H, ValCH (CH₃)₃),0.87(d,6H,CH(CH₃)₂)。

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

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

Step (11) two (three deuterium methyl) ((2S, 2 ' S)-((2S, 2 ' S) -2,2 '-(5,5 '-((S) -6- phenyl -6H- benzoyl [5,6] [1,3] oxygen azepines[3,4-a] indoles-3,10- diyl) two (1H- imidazoles-5,2- diyls)) two (pyrrolidines-2,1- diyls)) two (3- methyl-1-butanone-2,1- diyls)) dicarboxylic acid esters preparation

500mg compound 1-j, 6mL acetonitrile is sequentially added into 50mL two-mouth bottle, is cooled to 5 DEG C, 274mg compound 1-k, 496mg N-methylmorpholine is added, and 47.3mg HOBt, 295mg EDCHCl is reacted at room temperature overnight after adding.8mL ethyl acetate is added thereto, uses 4mL 2M NH respectively₄Cl is washed, 3mL 10wt%NaHCO₃It washes, 3mL 10wt%Na₂CO₃It washes, 3mL washing, the dry organic phase of anhydrous magnesium sulfate, concentration rear pillar chromatography (MeOH:DCM=3:100) obtains 428mg yellow solid, i.e. compound 1, yield 68.8%, purity 96%, 99%de.

¹H-NMR (DMSO-d6,300MHz): δ 8.13 (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).

¹³C-NMR (DMSO-d6,300MHz): 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.

The preparation of 2 compound 9 of embodiment

Bromo- 6- phenyl -12,12a- dihydro -6D- five deuterium benzoyl [5,6] [1,3] the oxygen azepine of step (1) (S, 12aR) -3,10- twoThe preparation of [3,4-a] indoles

2.64g compound 1-e, 13mL acetonitrile is sequentially added into 100mL there-necked flask, 1.12g D6- benzaldehyde (Aldrich), 40.8mg trifluoroacetic acid, lower 35 DEG C of nitrogen protection are reacted 3 hours.Then 1.2mL 5%NaHCO is successively added dropwise₃Solution and 5.3mL water, filtering, filter cake are successively washed with 8mL acetonitrile/water (2:1), and 2.85g gray solid, i.e. compound 9-a, yield 86% are obtained after vacuum drying.

¹H-NMR(CDCl₃, 300MHz): δ 7.28 (m, 1H), 7.23 (s, 1H), 7.08 (d, J=2.0Hz, 1H), 6.98 (dd, J=8.2,2.0Hz, 1H), 6.82 (m, 2H), 4.71 (d, J=8.9Hz, 1H), 3.52 (dd, J=15.7,8.9Hz, 1H), 3.12 (d, J=15.7Hz, 1H).

¹³C-NMR(CDCl₃, 300MHz): δ 153.2,148.0,137.6,131.4,130.4,128.8,128.5,128.4,127.8,12 6.8,124.4,123.8,121.2,120.3,112.6,110.9,83.1,55.5,36.0.

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

Bromo- 6- phenyl -6D- five deuterium benzoyl [5,6] [1,3] the oxygen azepine of step (2) (S) -3,10- twoThe preparation of [3,4-a] indoles

2.85g compound 9-a, 29mL DMA, 1.55g NaHCO are sequentially added into 100mL there-necked flask₃, 10 DEG C are cooled to, in batches 1.86g potassium permanganate is added, then 7.2mL water is added dropwise, system temperature is kept to be lower than 15 DEG C, is reacted overnight for 10 DEG C after adding.57mL ethyl acetate is added, then 29mL NaHSO is added dropwise₃Aqueous solution, stratification, water phase uses 30mL ethyl acetate to extract again, merges organic phase, is washed three times with 3 × 15mL 10%NaCl solution, gained organic phase is concentrated to get about 29mL aqueous isopropanol, it is added dropwise after 29mL water is stirred overnight and filters again, filter cake is washed with 12mL isopropanol/water (1:1), and 2.08g solid is obtained after vacuum drying, that is compound 9-b, yield 73.3%.

¹H-NMR(CDCl₃, 300MHz): δ 7.81 (d, J=1.8Hz, 1H), 7.51 (d, J=8.1Hz, 1H), 7.25-7.16 (m, 3H), 6.85 (s, 1H), 6.70 (d, J=8.8Hz, 1H), 6.90 (d, J=8.0Hz, 1H), 6.73 (d, J=8.0Hz, 1H), 4.94 (m, 1H), 4.40 (s, 1H), 3.30 (t, J=8.6Hz, 1H), 3.10 (dd, J=15.7,12.4Hz, 1H).

¹³C-NMR(CDCl₃, 300MHz): δ 150.0,136.2,133.9,132.1,130.7,123.0,126.8,125.6,125.1,12 3.4,122.4,121.3,117.0,114.2,111.2,96.8,84.5.

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

Step (3) (2S, 2 ' S)-di-t-butyl 2,2 '-(5,5 '-((S) -6- phenyl -6D- five deuterium benzoyl [5,6] [1,3] oxygen azepines[3,4-a] indoles -3,10- diyl) two (1H- imidazoles -5,2- diyls)) two (pyrrolidines -1- formic acid esters) preparation

55mL Isosorbide-5-Nitrae-dioxane, 2.0g compound 9-b, 2.39g B (Pin) are sequentially added into 250mL there-necked flask₂, 1.68g potassium acetate, 125.3mg Pd (dppf) Cl₂, nitrogen protection it is lower 110 DEG C reaction 3 hours after cool, add 32mL Isosorbide-5-Nitrae-dioxane and 8mL water, 3.25g compound 1-h, 1.25g Pd (dppf) Cl₂, 3.54g potassium carbonate, the lower 110 DEG C of reactions of nitrogen protection are overnight.The post-treated rear pillar chromatographic purifying (EA:PE=45:55) of reaction solution, obtains 2.05g yellow solid, i.e. compound 9-c, yield 61.5%.

¹H-NMR (DMSO-d6,300MHz): δ 11.89 (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).

¹³C-NMR (DMSO-d6,300MHz): 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:774.4075.

Step (4) 6- phenyl -3,10- two (2- ((S)-pyrrolidin-2-yl) -1H- imidazoles -5- base) -6D- five deuterium benzoyl [5,6] [1,3] oxygen azepineThe preparation of [3,4-a] indole hydrochloride

Sequentially add 2.0g compound 9-c, 11mL methanol into 50mL single port bottle, 2.0g concentrated hydrochloric acid, 45 DEG C are cooled to room temperature after reaction 4 hours, and 33mL acetonitrile is added dropwise, filters after being stirred overnight at room temperature, 1.7g solid, i.e. compound 9-d, yield 91.4% is obtained after vacuum drying.

¹H-NMR (DMSO-d6,300MHz): δ 8.31 (d, J=0.7Hz, 1H), 8.19 (s, 1H), 8.05 (d, J=8.0Hz, 1H), 7.88 (s, 1H), 7.72 (m, 3H), 7.47 (d, J=8.8Hz, 1H), 5.16 (t, J=8.0Hz, 1H), 5.06 (t, J=8.0Hz, 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).

¹³C-NMR (DMSO-d6,300MHz): 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:574.3203.

Step (5) dimethyl ((2S, 2 ' S)-((2S, 2 ' S) -2,2 '-(5,5 '-((S) -6- phenyl -6D- five deuterium benzoyl [5,6] [1,3] oxygen azepines[3,4-a] indoles-3,10- diyl) two (1H- imidazoles-5,2- diyls)) two (pyrrolidines-2,1- diyls)) two (3- methyl-1-butanone-2,1- diyls)) dicarboxylic acid esters preparation

720mg compound 9-d, 8mL acetonitrile is sequentially added into 50mL two-mouth bottle, is cooled to 5 DEG C, 385mg N-Moc-L- valine, 708mg N-methylmorpholine is added, and 67.6mg HOBt, 421.7mg EDCHCl is reacted at room temperature overnight after adding.12mL ethyl acetate is added thereto, uses 6mL 2M NH respectively₄Cl is washed, 4.5mL 10wt%NaHCO₃It washes, 4.5mL 10wt%Na₂CO₃It washes, 4.5mL washing, the dry organic phase of anhydrous magnesium sulfate, concentration rear pillar chromatography (MeOH:DCM=3:100) obtains 686mg yellow solid, i.e. compound 9, yield 77.3%, purity 95.8%, 99%de.

¹H-NMR (DMSO-d6,300MHz): δ 8.10 (s, 1H), 8.07 (d, J=8.4Hz, 1H), 7.99 (s, 1H), 7.89 (s, 1H), 7.56-7.54 (m, 3H), 7.52 (d, J=8.7Hz, 1H), 7.35 (s, 1H), 6.97 (m, 2H), 5.16 (t, J=7.3Hz, 1H), 5.12 (t, J=7.3Hz, 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=6.8Hz, 3H), 0.82 (d, J=6.8Hz, 3H), 0.80 (d, J=6.8Hz, 3H), 0.77 (d, J=6.8Hz, 3H).

¹³C-NMR (DMSO-d6,300MHz): 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:888.4613.

The preparation of 3 compound 7 of embodiment

The preparation of step (1) D1- benzaldehyde

150mL ether is sequentially added into 500mL there-necked flask, 62.8g chloroacetic chloride is added dropwise in 37.92g anhydrous stannous chloride, -10 DEG C are cooled to, -10 DEG C of dropwise addition 15.02g heavy water add 10.3g benzonitrile, add room temperature reaction overnight, filtering, filter cake are dried in vacuo to obtain 22.45g white solid, make its hydrolysis with 250ml hot water, it is concentrated after ether extraction dry, obtain 7.2g liquid, yield 67.3%, purity 99%.

Bromo- 6- phenyl -12,12a- dihydro -6D- benzoyl [5,6] [1,3] the oxygen azepine of step (2) (S, 12aR) -3,10- twoThe preparation of [3,4-a] indoles

7.38g compound 1-e, 30mL acetonitrile is sequentially added into 100mL there-necked flask, 3g D1- benzaldehyde, 114mg trifluoroacetic acid, lower 35 DEG C of nitrogen protection are reacted 3 hours.Then 3.3mL 5%NaHCO is successively added dropwise₃Solution and 15mL water, filtering, filter cake are successively washed with 22.5mL acetonitrile/water (2:1), and 7.69g gray solid, i.e. compound 7-a, yield 84% are obtained after vacuum drying.

¹H-NMR(CDCl₃, 500MHz): 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).

¹³C-NMR(CDCl₃, 500MHz): δ 153.4,148.2,137.8,131.6,130.6,129.0,128.7,128.0,127.0,12 4.6,124.0,121.4,120.5,112.8,110.0,83.1,55.7,36.2.

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

Bromo- 6- phenyl -6D- benzoyl [5,6] [1,3] the oxygen azepine of step (3) (S) -3,10- twoThe preparation of [3,4-a] indoles

7.6g compound 7-a, 76mL DMA, 4.18g NaHCO are sequentially added into 500mL there-necked flask₃, 10 DEG C are cooled to, 5.01g potassium permanganate is added portionwise, then 19mL water is added dropwise, system temperature is kept to be lower than 15 DEG C, 10 DEG C of reactions are stayed overnight after adding.152mL ethyl acetate is added, then 76mL NaHSO is added dropwise₃Aqueous solution, stratification, water phase use 80mL ethyl acetate to extract again, merge organic phase, it is washed three times with 3 × 40mL 10%NaCl solution, is concentrated after washing, about 120ml isopropanol is added, it is concentrated to get about 76mL aqueous isopropanol, it is added dropwise after 76mL water is stirred overnight and filters again, filter cake is washed with 32mL isopropanol/water (1:1), and 6.387g solid is obtained after vacuum drying, that is compound 7-b, yield 84.4%.

¹H-NMR(CDCl₃, 500MHz): 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).

¹³C-NMR(CDCl₃, 500MHz): δ 150.2,136.3,134.1,132.3,130.9,130.1,129.1,127.0,126.6,12 5.7,125.2,123.6,122.6,121.5,117.2,114.3,111.3,97.0.

HRMS (M+H) m/z:454.9389.

Step (4) (2S, 2 ' S)-di-t-butyl 2,2 '-(5,5 '-((S) -6- phenyl -6D- benzoyl [5,6] [1,3] oxygen azepines[3,4-a] indoles -3,10- diyl) two (1H- imidazoles -5,2- diyls)) two (pyrrolidines -1- formic acid esters) preparation

80mL Isosorbide-5-Nitrae-dioxane, 3g compound 7-b, 3.63g B (Pin) are sequentially added into 250mL there-necked flask₂, 2.54g potassium acetate, 190mg Pd (dppf) Cl₂, nitrogen protection it is lower 110 DEG C reaction 3 hours after cool, add 48mL Isosorbide-5-Nitrae-dioxane and 12mL water, 4.93g compound 1-h, 1.9g Pd (dppf) Cl₂, 5.37g potassium carbonate, the lower 110 DEG C of reactions of nitrogen protection are overnight.The post-treated rear pillar chromatographic purifying (EA:PE=45:55) of reaction solution, obtains 3.22g yellow solid, i.e. compound 7-c, yield 64.3%.

¹H-NMR (DMSO-d6,500MHz): δ 11.95 (m, 2H), (7.98 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).

¹³C-NMR (DMSO-d6,500MHz): 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:769.3926.

Step (5) 6- phenyl -3,10- two (2- ((S)-pyrrolidin-2-yl) -1H- imidazoles -5- base) -6D- benzoyl [5,6] [1,3] oxygen azepineThe preparation of [3,4-a] indole hydrochloride

1.7g compound 7-c, 8.5mL methanol, 1.3g concentrated hydrochloric acid are sequentially added into 50mL single port bottle, 45 DEG C reaction 4 hours after be cooled to room temperature, be added dropwise 50mL acetonitrile, filtered after being stirred overnight at room temperature, 1.35g solid, i.e. compound 7-d, yield 85.5% are obtained after vacuum drying.

¹H-NMR (DMSO-d6,500MHz): δ 8.31 (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)

¹³C-NMR (DMSO-d6,500MHz): δ 149.7,142.7,141.5,137.4,135.7,135.2,132.3,130.0,129.3,12 9.1, 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] oxygen azepines[3,4-a] indoles-3,10- diyl) two (1H- imidazoles-5,2- diyls)) two (pyrrolidines-2,1- diyls)) two (3- methyl-1-butanone-2,1- diyls)) dicarboxylic acid esters preparation

1000mg compound 7-d, 10mL acetonitrile is sequentially added into 50mL two-mouth bottle, is cooled to 5 DEG C, 539mg N-Moc-L- valine, 992mg N-methylmorpholine is added, and 95mg HOBt, 590mg EDCHCl is reacted at room temperature overnight after adding.17mL ethyl acetate is added thereto, uses 8.3mL 2M NH respectively₄Cl is washed, 6.3mL 10wt%NaHCO₃It washes, 6.3mL 10wt%Na₂CO₃It washes, 6.3mL washing, the dry organic phase of anhydrous magnesium sulfate, concentration rear pillar chromatography (MeOH:DCM=3:100) obtains 1.02mg yellow solid, i.e. compound 7, yield 82.4%, purity 96.7%, 99%de.

¹H-NMR (DMSO-d6,500MHz): δ 8.10 (s, 1H), 8.07 (d, J=8.4Hz, 1H), 7.99 (s, 1H), 7.89 (s, 1H), 7.56-7.54 (m, 3H), 7.52 (d, J=8.7Hz, 1H), 7.35 (s, 1H), 7.31-7.27 (m, 5H), 6.97 (m, 2H), 5.16 (t, J=7.3Hz, 1H), 5.12 (t, J=7.3Hz, 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=6.8Hz, 3H), 0.82 (d, J=6.8Hz, 3H), 0.80 (d, J=6.8Hz, 3H), 0.77 (d, J=6.8Hz, 3H).

¹³C-NMR (DMSO-d6,500MHz): 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.

The external anti-hepatovirus activity rating of effect example 1

Experimental method:

Test compound carries out 1:3 and is serially diluted 8 concentration points, and duplicate hole is added in 96 orifice plates.HCV GT-1a and HCV GT-1b replicon cell is suspended in respectively in the culture solution containing 10%FBS, is inoculated into 96 orifice plates containing the test compound with the density of every 8000 cells in hole.CO of the cell 5%₂, cultivate 3 days under the conditions of 37 DEG C.Cell viability detection reagent is added, detects fluorescence signal (RFU, cell viability initial data) after 1 hour.Then, luciferase luminous substrate Bright-Glo is added into every hole cell, detects chemiluminescence signal value RLU with chemiluminescence detection system after five minutes.Initial data RFU and RLU are analyzed, the percent viability and test compound for calculating HCV GT-1a and HCV GT-1b replicon cell are to the inhibitory activity percentage of HCV GT-1a and HCV GT-1b genocopy.Nonlinear fitting is carried out to inhibitory activity and cell viability percentage data using GraphPad Prism software, calculates the EC of compound₅₀And CC₅₀。

Experimental result:

Elbasvir, compound 1, compound 9 inhibit the CC of cell viability in vitro₅₀It is all larger than 0.5nM, to the EC of the sub- inhibitory activity of HCV GT-1a genocopy₅₀Respectively 0.004,0.005,0.004nM (being shown in Table 1), to the EC of the sub- inhibitory activity of HCV GT-1b genocopy₅₀Respectively 0.005,0.005,0.003nM (being shown in Table 2).

1 Compound ira vitro cytotoxicity of table and anti-hepatitis C virus are active (HCV GT-1a)

2 Compound ira vitro cytotoxicity of table and anti-hepatitis C virus are active (HCV GT-1b)

The external people's hepatomicrosome estimation of stability of effect example 2

It is incubated in system in the final temperature of 300 μ L, contains 30 μ L people hepatomicrosomes (protein concentration: 0.15mg/mL), 30 μ L NADPH+MgCl₂, 3 μ L substrates, 237 μ L PBS buffer solution.Wherein, the ratio of organic solvent (acetonitrile) is 1%.2 parts are done in parallel, every part of 0.3mL.For every part of sample, 30 μ L NADPH+MgCl are added with NADPH respectively after 37 DEG C of pre-temperatures incubate 5min in the mixing liquid for first preparing substrate and enzyme that total volume is 270 μ L₂Mixing, takes out 40 μ L respectively at 0,5,15,30,60,120min, terminates reaction with the 300 μ L of ice acetonitrile of containing the internal standard.In addition, being 3 parts of each 300 μ L of blank (KB) respectively, NADPH respectively KB1: is not added；KB2: substrate is not added；KB3: not enzyme.

It draws 40 μ L temperature and incubates sample, the ice acetonitrile precipitation of 300 μ L containing the internal standards is added, be vortexed after concussion 5min, be centrifuged (13000rpm, 4 DEG C) 10min.80 μ L of Aspirate supernatant is added 80 μ L, 50% acetonitrile solution, mixes, sample introduction is analyzed by 0.5 μ L to sample introduction plate.

It is mapped by the Ln value of each time point residue test compound percentage and time, t is calculated by respective formula_1/2And CL_int。

Slope=- K_mic

Note: V/M=1/ protein concentration

Table 3 shows the test data of the external people's hepatomicrosome metabolic stability of test compound.

The external people's hepatomicrosome metabolic stability of 3 test compound of table

The result shows that external people's hepatomicrosome metabolic stability of the compound of the present invention 1 and compound 9 is more excellent compared with elbasvir.

Pharmacokinetic Evaluation in 3 rat of effect example

SD rat, 200~220g of weight are randomly divided into 2 groups, every group 3, give test compound elbasvir, the compound 7 of 10mg/kg dosage respectively after adapting to 3-5 days.

Fasting 12h before animal subject (SD rat) is administered, 4h is to food, experiment front and back and equal free water in experimentation after administration.

After gastric infusion, in 0min, 15min, 30min, 1h, 3h, 4h, 5h, 6h, 8h, 10h, for 24 hours in eye socket blood 0.10mL or so is taken, EDTA-K2 is anticoagulant, immediately places blood sample in ice, 4 DEG C are transferred in 30min, centrifugal separation plasma under the conditions of 4000rpm, 10min.It is to be measured in -20 DEG C of preservations immediately after the whole blood plasma of collection.

Draw 40 μ L test plasma samples and standard sample, the acetonitrile solution of 200 μ L containing the internal standards is added, oscillation mixes 5min, 13000rpm is centrifuged 10min, take 80 μ L of supernatant to sample introduction plate, 80 μ L, 50% acetonitrile solution is added, mixes, draws 1 μ L for LC-MS/MS and measures (liquid-phase condition: chromatographic column: ACQUITY UPLC BEH C18 (1.7 μm of 2.1 × 50mm)；Water phase: 0.1% aqueous formic acid, organic phase: methanol solution；Flow velocity: 0.3ml/min, gradient condition: the ratio 0-0.5min:90% of water phase；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: it is detected using LC-MS/MS (Waters TQ-S), ion source: ESI+, orifice potential: 85V, capillary voltage: 3.2kV, ion source temperature: 150 DEG C, desolvation temperature: 400 DEG C, desolvation gas flow velocity: 800L/hr.The selective reaction of Elbasvir detects ion [M+H] m/z 882.5 → 656.42, and the selective reaction of compound 7 detects ion [M+H] m/z 883.43 → 657.37.Internal standard diazepam selective reaction detects m/z285.06 → 154.04 ion [M+H]), record chromatogram.

Pass through the oral administration biaavailability of Rats pharmacokinetics experimental evaluation the compounds of this invention.

The medicine of 4 test compound of table is for parameter

Claims (12)

1. One kind such as formula Ι compound represented or its pharmaceutically acceptable salt:

   Wherein, R₁Or R₂Separately it is selected from CH₃、CH₂D、CHD₂Or CD₃, R₃、R₄、R₅、R₆、R7、R₈Or R₉It is independently selected from H or D, condition is R₁~R₉In contain at least one D-atom.
2. The compound of claim 1, wherein R₁、R₂It is simultaneously CH₃Or CD₃。
3. The compound of claim 1, wherein R₅、R₆、R₇、R₈、R₉It is simultaneously H or D.
4. The compound of claim 1, wherein such as formula Ι compound represented are as follows:

   Compound R₁ R₂ R₃ R₄ R₅ R₆ R₇ R₈ R₉ 1 CD₃ CD₃ H H H H H H H 2 CD₃ CD₃ D H H H H H H 3 CD₃ CD₃ H D H H H H H 4 CD₃ CD₃ D H D D D D D 5 CD₃ CD₃ D D H H H H H 6 CD₃ CD₃ D D D D D D D 7 CH₃ CH₃ D H H H H H H 8 CH₃ CH₃ D D H H H H H 9 CH₃ CH₃ D H D D D D D 10 CH₃ CH₃ D D D D D D D 11 CH₃ CH₃ H D H H H H H
5. The compound of claim 1, wherein such as formula Ι compound represented are as follows:
6. A kind of pharmaceutical composition, it includes compound of any of claims 1-5 or its pharmaceutically acceptable salts and pharmaceutically acceptable excipient.
7. The purposes of compound of any of claims 1-5 or its pharmaceutically acceptable salt, pharmaceutical composition as claimed in claim 6 in the drug of preparation prevention or treatment hepatitis c virus infection.
8. The purposes of claim 7, wherein the hepatitis C virus is selected from following genotype: 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a or 6a.
9. For preventing or treating the pharmaceutical composition containing compound of any of claims 1-5 or its pharmaceutically acceptable salt of hepatitis c virus infection.
10. The pharmaceutical composition of claim 9, wherein the hepatitis C virus is selected from following genotype: 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a or 6a.
11. A method of prevention or treatment hepatitis c virus infection, wherein, the method includes compound of any of claims 1-5 or its pharmaceutically acceptable salt, the pharmaceutical composition containing compound shown in formula I or its pharmaceutically acceptable salt are given to the patient of needs.
12. The method of claim 11, wherein the hepatitis C virus is selected from following genotype: 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a or 6a.