CN108623568B

CN108623568B - Salt form of 9,10 dihydrophenanthrene hepatitis C virus inhibitor and preparation thereof

Info

Publication number: CN108623568B
Application number: CN201810227152.3A
Authority: CN
Inventors: 赵立文; 储刚; 罗放; 王志强; 于振磊
Original assignee: Nanjing Huicheng Pharmaceutical Co ltd
Current assignee: Nanjing Huicheng Pharmaceutical Co Ltd
Priority date: 2017-03-21
Filing date: 2018-03-20
Publication date: 2022-04-19
Anticipated expiration: 2038-03-20
Also published as: CN108623568A

Abstract

The invention relates to a salt form of a hepatitis C virus inhibitor having a 9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthrene structure and a preparation method thereof, in particular to N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptane-6-yl) -1H-imidazole-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthrene-2-yl) -1H-benzo [ d ] imidazole-2-yl) pyrrolidine-1-yl) -3-methyl-1-oxobutane-2- Base) salt forms of methyl carbamate, and methods of preparation and use.

Description

Salt form of 9,10 dihydrophenanthrene hepatitis C virus inhibitor and preparation thereof

Technical Field

Background

Viral Hepatitis C (Viral Hepatitis C) is an infectious disease of acute and chronic inflammation of the liver caused by Hepatitis C Virus (HCV), and chronic liver diseases such as chronic Hepatitis, liver cirrhosis, liver cancer and the like are very easy to develop after HCV infection, which seriously affects the health of people.

HCV belongs to the flaviviridae family, and can be currently divided into 6 genotypes and different subtypes, and according to the international popular method, the HCV genotypes are represented by arabic numerals, and the gene subtypes are represented by lowercase english letters, wherein the genotype 1 shows global distribution, accounting for more than 70% of all HCV infections, and the main infection type of the chinese population is HCV 1b subtype. It was found that both the 5 'and 3' ends of the positive strand RNA of HCV contain noncoding regions (UTRs) between which is a large polyprotein Open Reading Frame (ORF). The ORF encodes a polyprotein precursor of about 3000 amino acids in length, which is cleaved into the various HCV mature proteins by the combined action of host-encoded signal peptidases and HCV-encoded proteases. The HCV mature proteins include 4 structural proteins and 6 non-structural proteins, of which 6 are designated NS2, NS3, NS4A, NS4B, NS5A, and NS5B, respectively. Research shows that 6 nonstructural proteins play a very important role in HCV replication, such as NS3, regulating the activity of NS3 serine protease, NS5A is a phosphorylated protein containing interferon sensitivity determining regions, and plays an important role in interferon therapeutic effect prediction, virus replication, antiviral resistance, hepatocellular carcinoma change and the like, and has become the focus of HCV nonstructural protein research.

CN104744444A discloses an NS5A inhibitor having a 9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthrene structure, the compound structure of which is shown in formula I (hereinafter referred to as "compound of formula I"), and the name of the compound is N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptane-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-yl -oxobutan-2-yl) carbamic acid methyl ester. The compound of the formula I has good inhibitory activity to hepatitis C virus, low toxicity to host cells, high effectiveness and good safety, and is very promising to be a medicament for treating and/or preventing diseases related to HCV infection. The entire contents of which are incorporated herein by reference,

however, the different existing forms of the drug have large differences in solubility and physicochemical stability, therefore, intensive research is required to find methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate which is suitable for pharmaceutical use.

Disclosure of Invention

The object of the present invention is to provide an amorphous form of the NS5A inhibitor methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate, represented by formula I below,

the inventor of the invention carries out crystal form screening experiments on the compound of the formula I under the condition of not less than hundreds of conditions, wherein the screening modes comprise a volatilization experiment, a crystal slurry experiment, a solvent resistance experiment, a cooling crystallization experiment, a melting cooling experiment, a diffusion experiment, a high molecular template experiment or a water vapor stress experiment and the like, and the experimental result shows that the amorphous substance form of the obtained compound of the formula I is the most stable form.

Without limitation, the X-ray powder diffraction pattern of the compound of formula I in amorphous form is shown in FIG. 1. In a particular embodiment, the present invention provides an amorphous form of the compound of formula I having an X-ray powder diffraction pattern as shown in figure 1.

In some embodiments, amorphous forms of the compounds of formula I of the present invention are prepared using a volatilization assay in which a sample is dissolved in a solvent and the clear solution is then allowed to evaporate to dryness at various temperatures. The solvent is preferably selected from alcohols, ketones, nitriles, ethers, esters and the like with less than 6 carbon atoms; the solvent is more preferably methanol, ethanol, trifluoroethanol, isopropanol, n-propanol, sec-butanol, n-butanol, nitromethane, acetone, butanone, diethyl ether, ethyl acetate, methyl tert-butyl ether, isopropyl acetate, tetrahydrofuran, 1, 4-dioxane, acetonitrile, dichloromethane, chloroform, toluene, dimethyl sulfoxide, or the like. The volatilization condition can be volatilization at any temperature condition, and in some specific embodiments, room temperature volatilization is selected; in other embodiments, volatilization at elevated temperatures of 40-60 ℃ may also be selected.

In some embodiments, amorphous forms of the compounds of formula I of the present invention are prepared using a slurry experiment in which a supersaturated solution of a sample (with undissolved solids present) is stirred in various solvents for a period of time. The solvent is selected from one or more of ethanol, sec-butyl alcohol, water, acetone, butanone, diethyl ether, ethyl acetate, methyl tert-butyl ether, tetrahydrofuran, dichloromethane, chloroform, 1, 4-dioxane, toluene, n-heptane and methylcyclohexane, the reaction temperature can be carried out at any temperature, preferably at room temperature or high temperature (40-60 ℃) or low temperature (4 ℃), and the reaction time is preferably 2 days to 4 days.

In some embodiments, the amorphous form of the compound of formula I of the present invention is prepared using an anti-solvent test in which a sample is dissolved in a good solvent, an anti-solvent is added, a solid is precipitated, and the solid is filtered off immediately after stirring. The good solvent is preferably an alcohol, an ester, a ketone, tetrahydrofuran, 1, 4-dioxane, a nitrile, chlorohydrocarbon or an aromatic ring solvent; the antisolvent is selected from water, alkane or ether solvents. The good solvent is more preferably methanol, ethanol, trifluoroethanol, isopropanol, acetone, tetrahydrofuran, 1, 4-dioxane, acetonitrile, ethyl acetate, methyl tert-butyl ether, chloroform, toluene, etc., and the anti-solvent is more preferably water, n-heptane or isopropyl ether. The mixing ratio of the solvents is preferably 1:1 to 6 (good solvent: antisolvent).

In some embodiments, the amorphous form of the compound of formula I of the present invention is prepared by a cooling crystallization experiment, which comprises two ways, the first way is to dissolve a certain amount of sample into a corresponding solvent at a high temperature, and then directly stir at room temperature or low temperature for crystallization; the second method is to dissolve the sample in a proper solvent under high temperature, filter, put the filtrate in a glass vial, and let the solution stand at low temperature for crystallization. The solvent is preferably a mixed solvent of an alcohol, an ester, a ketone, tetrahydrofuran, 1, 4-dioxane, a nitrile, chlorohydrocarbon or aromatic ring solvent and water or an alkane or ether solvent. Non-limiting examples of the solvent are a mixed solution of methanol or dimethyl sulfoxide or tetrahydrofuran or 1, 4-dioxane or acetonitrile with water, a mixed solvent of 1, 4-dioxane or acetonitrile or n-butanol with n-heptane, a mixed solvent of ethyl acetate with methylcyclohexane, and the like.

In some embodiments, the amorphous form of the compound of formula I of the present invention is prepared using a diffusion crystallization experiment in which a defined amount of an amorphous sample is placed in a small container (e.g., a centrifuge tube) and placed in a large container containing various solvents for diffusion by standing. The solvent is preferably water or ethers; non-limiting examples of the solvent are water, diethyl ether or methyl tert-butyl ether.

In some embodiments, the amorphous form of the compound of formula I of the present invention is prepared using a polymer template assay, where the sample is dissolved in a suitable solvent in a polymer template laboratory, 5% to 10% of the polymer template material is added, left open to allow natural evaporation at room temperature. The solvent is preferably ethanol; the polymer template material is preferably selected from povidone K30, polyethylene glycol 6000, polymethyl methacrylate, polyallylamine hydrochloride or polyvinyl alcohol 124.

In some embodiments, the amorphous form of the compound of formula I of the present invention is prepared using a moisture stress test in which a sample is placed in an environment at room temperature and humidity. An amount of a sample (e.g., 20mg) is placed in an environment at room temperature and a relative humidity of 33% RH, 52% RH, 75% RH, 85% RH, or 97% RH, 40 ℃ to 75% RH, or 60 ℃ to dry, and X-ray powder diffraction tests are performed on the solid at different times, indicating that the solid obtained is amorphous.

In some embodiments, amorphous forms of the compounds of formula I of the present invention are prepared using a melt cooling experiment. In a specific embodiment, the melting and cooling experiment comprises the steps of placing the sample in a DSC pan, raising the temperature until the sample melts, and then bringing the temperature to room temperature at a certain rate. In another specific embodiment, the melting and cooling experiment includes a step of dropping an organic solvent containing a polymer into the melted sample after melting the sample at a high temperature. In yet another embodiment, the melt cooling experiment comprises the steps of mixing the sample and the polymer, heating to melt, and cooling to room temperature.

In the process for preparing amorphous form of the compound of formula I according to the present invention, there is no limitation on the form in which the compound of formula I is present, and any crystalline form or amorphous solid may be used.

It is another object of the present invention to provide a pharmaceutically acceptable salt of the NS5A inhibitor methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutanoyl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate, represented by formula I below,

in the salt formation studies of the compounds of formula I, a variety of inorganic and organic acids are selected for salt sieve experiments, preferably selected from benzenesulfonic acid, citric acid, D-gluconic acid, glycolic acid, lactic acid, L-malic acid, malonic acid, phosphoric acid, succinic acid, sulfuric acid, L-tartaric acid, p-toluenesulfonic acid, acetic acid, oxalic acid, fumaric acid, alpha-ketoglutaric acid, hippuric acid, hydrochloric acid, maleic acid, methanesulfonic acid, D-tartaric acid, and the like. A plurality of salt type screening experiments are carried out by adopting different solvent systems and different molar ratios to feed materials.

In more than 100 salt sieve experiments, the inventor of the invention surprised to obtain medicinal salts with better specific properties, compared with free alkali, the solubility, hygroscopicity, stability and the like of the salt forms of the compound shown in the formula I are improved, the medicinal salts are preferably sulfate and D-tartrate, the melting point is improved after salt formation, the melting range is shortened, the thermal stability is improved after salt formation, and the impurity removal effect is more obvious.

In some embodiments, the present invention provides a compound of formula I having a salt formation ratio of free base to sulfuric acid in the sulfate salt of 1: x and X are preferably 1-2.

Because the compound of the formula I has two salt forming sites, after the compound is subjected to salt formation with one sulfuric acid, the sulfuric acid can be dissociated into one proton to form one sulfate, and can also be dissociated into two protons to form the sulfate after the compound is subjected to salt formation. So the salt formation ratio after salt formation in equal proportions is 1:1, which is described herein as the monosulfate salt. The salt formation ratio after salt formation with two sulfuric acids is 1:2, which is described herein as hydrogen disulfate.

The salt forming ratio of the sulfate provided by the invention is 1: x; x is preferably 1-2.

The sulfate salt of the compound of formula I provided by the present invention is preferably a monosulfate salt of the compound of formula I or a bisbisulfate salt of the compound of formula I or a mixture of the monosulfate and the bisbisulfate salt of the compound of formula I.

The salt formation ratio of free alkali to D-tartaric acid in the D-tartrate provided by the invention is preferably 1:1.

It is another object of the present invention to provide a crystalline form of a pharmaceutically acceptable salt of the NS5A inhibitor methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate (structure shown in formula I), the crystalline forms of the pharmaceutically acceptable salts are preferably amorphous forms of the sulfate salt, preferably the monosulfate or bisulfate or mixtures of the monosulfate and bisulfate, and the D-tartrate salt.

In some embodiments, different crystal sieve experiments are adopted, the crystallization experiment method comprises a volatilization experiment, a crystal slurry experiment, an anti-solvent experiment, a cooling crystallization experiment, a melting cooling experiment, a diffusion experiment, a polymer template experiment, a water vapor stress experiment and the like, the crystallization solvent is preferably alcohols, ketones, chloroform and the like, and the obtained salt has amorphous crystal forms.

Without limitation, the X-ray powder diffraction patterns of the sulfate and the D-tartrate provided by the invention are shown in figures 2, 5 and 8.

Another object of the present invention is to provide a process for preparing a pharmaceutically acceptable salt of N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamic acid methyl ester (structure shown in formula I) which is an inhibitor of NS5A, comprising the step of reacting a compound of formula I in free base form with the corresponding acid to form a salt. The pharmaceutically acceptable salts of the compounds of formula I can be prepared according to art-conventional salt-forming procedures.

In some embodiments, the process for preparing a pharmaceutically acceptable salt of a compound of formula I according to the present invention, wherein the salt-forming reaction is carried out in a reaction solvent, preferably a lower organic solvent, preferably an alcohol having less than 6 carbon atoms or a ketone having less than 6 carbon atoms, preferably methanol, ethanol, propanol, butanol, sec-butanol, isopropanol or acetone. In some embodiments, the molar ratio of the compound of formula I to the corresponding acid in the salt-forming reaction is about 1:0.5 to 5; in some preferred embodiments, the molar ratio of the compound of formula I to the corresponding acid is preferably about 1: 1-3.

According to the invention, a process for the preparation of sulfates is provided, wherein the sulfates obtained are preferably monosulfate or bisulfate or a mixture of monosulfate and bisulfate. In a specific embodiment, a solution of concentrated sulfuric acid in isopropanol is added dropwise to a solution of the compound of formula I in isopropanol with stirring, stirred, centrifuged and dried under vacuum at room temperature. In some embodiments, the molar ratio of the compound of formula I in free base form to concentrated sulfuric acid is preferably about 1: 1-4, more preferably about 1: 1.1-2.2.

In a specific embodiment, according to the sulfate preparation method of the present invention, an isopropanol solution of concentrated sulfuric acid is added dropwise to an isopropanol solution of the compound of formula I under stirring, stirred, centrifuged, and vacuum-dried at room temperature. Wherein the molar ratio of free basic I compound to concentrated sulfuric acid is preferably from about 1:1.1, the reaction time is preferably from 10min to 30 min.

In a specific embodiment, according to the sulfate preparation method of the present invention, an isopropanol solution of concentrated sulfuric acid is added dropwise to an isopropanol solution of the compound of formula I under stirring, stirred, centrifuged, and vacuum-dried at room temperature. Wherein the molar ratio of free basic I compound to concentrated sulfuric acid is preferably from about 1: 2.2, the reaction time is preferably 1 to 2 h.

In a specific embodiment, according to the preparation method of the D-tartaric acid salt of the present invention, an isopropanol solution of D-tartaric acid is added dropwise to an isopropanol solution of the compound of formula I under stirring, stirred, centrifuged, and vacuum-dried at room temperature. Wherein the molar ratio of free basic I compound to D-tartaric acid is preferably from about 1:1.1, the reaction temperature is preferably 0-10 ℃ at low temperature, and the reaction time is preferably 12-36 h.

In the preparation method of the medicinal salt provided by the invention, the existence form of the compound in the formula I is not limited at all, and any crystal form or amorphous solid can be used.

Another object of the present invention is to provide a pharmaceutical composition comprising a pharmaceutically acceptable salt of the NS5A inhibitor methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate (structure shown in formula I) and a pharmaceutically acceptable carrier And (3) a body. The pharmaceutically acceptable salt is preferably a sulfate or D-tartrate salt of the compound of formula I provided by the invention. The sulfate or D-tartrate salt of the compound of formula I is mixed with a pharmaceutically acceptable carrier to prepare a pharmaceutical formulation suitable for oral or parenteral administration. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The formulations may be administered by any route, for example by oral administration, by infusion or bolus injection, by a route of absorption through epithelial or cutaneous mucosa (e.g. oral mucosa or rectum, etc.). Administration may be systemic or local. Examples of the formulation for oral administration include solid or liquid dosage forms, specifically, tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions and the like. The formulations may be prepared by methods known in the art and include carriers conventionally used in the art of pharmaceutical formulation.

Another object of the present invention is to provide a method for treating and/or preventing liver diseases caused by hepatitis c virus by using the pharmaceutically acceptable salts of the compound of formula I or the pharmaceutical composition containing the pharmaceutically acceptable salts of the present invention and the use thereof in preparing a medicament for preventing and/or treating liver diseases caused by hepatitis c virus, which comprises administering the pharmaceutically acceptable salts of the present invention or the pharmaceutical composition containing the pharmaceutically acceptable salts to patients with liver diseases caused by hepatitis c virus, so as to effectively inhibit HCV and prevent the progression of the disease. In some embodiments, the present invention provides a method for the treatment and/or prevention of an infection caused by hepatitis c virus, said method comprising administering to a subject in need thereof a therapeutically and/or prophylactically effective amount of a pharmaceutically acceptable salt of the present invention, or a pharmaceutical composition comprising a pharmaceutically acceptable salt. The pharmaceutically acceptable salts of the present invention, preferably the sulfate or D-tartrate salts provided herein, or a pharmaceutical composition comprising the pharmaceutically acceptable salts, can be administered to a mammal in need thereof to inhibit HCV and prevent progression of the disease process.

In other embodiments, the method or use for treating and/or preventing an infection caused by a hepatitis c virus further comprises administering to the subject a pharmaceutically acceptable salt or a pharmaceutical composition comprising a pharmaceutically acceptable salt of the present invention, preferably the sulfate or D-tartrate salt provided herein, and at least one other compound having anti-HCV activity before, after or simultaneously with the pharmaceutically acceptable salt or the pharmaceutical composition comprising a pharmaceutically acceptable salt of the present invention. In some embodiments, at least one of the additional compounds is an interferon or a ribavirin. In some specific embodiments, the interferon is selected from interferon alpha 2B, PEG, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau. In other embodiments, at least one of the additional compounds is selected from interleukin 2, interleukin 6, interleukin 12, interfering RNA, antisense RNA, imiquimod, ribavirin, an inosine 5' -monophospate dehydrogenase inhibitor, amantadine, and rimantadine. In other embodiments, at least one of the additional compounds is effective to inhibit the function of a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV NS5B protein, HCV entry, HCV assembly, HCV egress, HCV NS3/4A protein, and IMPDH for the treatment of an HCV infection.

Description of terms:

the room temperature conditions of the present invention are generally room temperature (10-30 ℃); the solvent ratio means a volume ratio.

As used herein, "monosulfate" refers to the salt of a compound of formula I with sulfuric acid by dissociation of two protons, i.e., the salt of a compound of formula I with a sulfuric acid salt, i.e., an equivalent ratio of salts, at a salt ratio of 1:1.

As used herein, "bisulphate" refers to the salt of the compound of formula I with the sulphate by dissociation of one proton from the sulphate, i.e. the salt of the compound of formula I with two sulphuric acids in a ratio of 1: 2.

Description of the drawings:

FIG. 1: an X-ray powder diffraction pattern of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate.

FIG. 2: x-ray powder diffraction pattern of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate sulfate (salt formation ratio near 1: 1).

FIG. 3: TGA spectrum of N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamic acid methyl ester sulfate (salt formation ratio near 1: 1).

FIG. 4: DSC spectra of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate sulfate (salt formation ratio near 1: 1).

FIG. 5: x-ray powder diffraction pattern of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate sulfate (salt formation ratio near 1: 2).

FIG. 6: TGA spectrum of N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamic acid methyl ester sulfate (salt formation ratio close to 1: 2).

FIG. 7: DSC spectra of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate sulfate (salt formation ratio close to 1: 2).

FIG. 8: an X-ray powder diffraction pattern of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ D ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate D-tartrate.

FIG. 9: a TGA profile of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ D ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate D-tartrate.

FIG. 10: DSC profile of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ D ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate D-tartrate.

Detailed Description

The present invention is explained in more detail below with reference to examples, which are provided only for illustrating the technical solutions of the present invention and are not intended to limit the scope of the present invention.

The reagents used in the following examples are all commercially available.

Test instrument for experiments

X-ray powder diffraction Spectroscopy (XRPD)

The instrument model is as follows: bruker D8Advance X-ray Diffractometer

The technical indexes are as follows: k alpha radiation (40kV, 40mA) with copper target wavelength of 1.54nm, theta-2 theta goniometer, Mo monochromator, Lynxeye

Scanning range: 3-40 degrees 2 theta

Step length: 0.02 degree 2 theta

2. Hot platform Polarized Light Microscope (PLM)

The instrument model is as follows: XP-500E

The manufacturer: shanghai rectangular optical instruments Ltd

3. Differential thermal analysis scanner (DSC)

The instrument comprises the following steps: TA Instruments Q200DSC

And (3) control software: thermal Advantage

Analysis software: universal Analysis

Sample pan: aluminum crucible (with cover and hole)

Protective gas: nitrogen gas

Gas flow rate: 40ml/min

The detection method comprises the following steps: equilibrate at 20 ℃; ramp 10 deg.C/min to 300 deg.C

4. Thermogravimetric analyzer (TGA)

The instrument comprises the following steps: TA Instruments Q500TGA

And (3) control software: thermal Advantage

Analysis software: universal Analysis

Sample pan: platinum crucible

Protective gas: nitrogen gas

Gas flow rate: 40ml/min

The detection method comprises the following steps: Hi-Res sensitivity 3.0; ramp 10 ℃/min, res 5.0to 150 ℃; ramp 10 deg.C/min to 350 deg.C.

5. Ion Chromatograph (IC)

The instrument comprises the following steps: dionex ICS-900

A workstation: chromeleon

6. Dynamic moisture adsorption instrument

The instrument comprises the following steps: TA Instruments Q5000TGA

And (3) control software: thermal Advantage

Analysis software: universal Analysis

Sample pan: platinum crucible

Detecting the amount of a sample: 1-10mg

Protective gas: nitrogen gas

Gas flow rate: 10mL/min

A detection method; equilibrate at 25 ℃;

Humidity 0％；

Isothermal for 300min；

Abort next iso if weight(％)<0.0100for 15.00min；

Step humidity 10％every 120min to 80％；

Abort next iso if weight(％)<0.0100for 15.00min

Step humidity 10％every 120min to 0％；

example 1: purification of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate

Step 1: synthesis of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate

Methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate was prepared according to the procedure of CN104744444A specification example 12.

Step 2: purification of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate

A100L reactor was charged with methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate obtained in step 1 (5.2kg,5.696mol) and IPOAc (36L), stirred at room temperature and dissolved. The temperature is raised to 75 ℃, active carbon (1080g) is added into the system, and the mixture is stirred for 2 hours. The mixture was filtered while hot, the filter cake was washed with IPOAc (7.2L), and the filtrate was slowly added dropwise to n-heptane (129.6L) with stirring at room temperature, and after dropwise addition, the mixture was stirred for 2 hours. Centrifuging and taking solid. Drying under reduced pressure in vacuum gave a white solid (5.13kg, yield 98.7%). After jet milling, drying for 48h to obtain white powder (4135.0g, yield 79.5%), purity 98.6%, total impurities 1.1%, specific rotation-132.7 deg.

The X-ray powder diffraction pattern of the compound of the formula I is shown in figure 1, and the compound of the formula I obtained in the way is an amorphous substance.

Example 2: sulfate salt of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate (salt formation ratio approaching 1:1)

The method comprises the following steps: dissolve the compound of formula I (364.75mg,0.40mmol) in 3.0mL of isopropanol at room temperature; concentrated sulfuric acid (43.95mg,0.44mmol) was dissolved in 3.0mL of isopropanol; dropwise adding the isopropanol solution of sulfuric acid into the isopropanol solution of the compound shown in the formula I under the stirring condition, wherein turbidity appears in the dropwise adding process, supplementing 2.0mL of isopropanol after stirring for 5min, wherein a large amount of turbidity appears, continuously stirring for 10min, centrifuging, and vacuum-drying the solid at room temperature for 24h to obtain 260mg of amorphous sulfate.

The method 2 comprises the following steps: dissolve the compound of formula I (150.19mg,0.16mmol) in 1.2mL of isopropanol at room temperature; concentrated sulfuric acid (19mg,0.18mmol) was dissolved in 3.0mL of isopropanol; dropwise adding the isopropanol solution of sulfuric acid into the isopropanol solution of the compound of the formula I under the stirring condition, wherein turbidity appears in the dropwise adding process, the system is too viscous, 0.8mL of isopropanol is supplemented, stirring is continued for 24h, centrifuging is carried out, and the solid is dried in vacuum at room temperature for 24h to obtain 156mg of amorphous sulfate.

The ion chromatography and HPLC results show that the salt formation ratio of the obtained sulfate is close to 1:1.

The PLM test showed that the sulphate obtained according to method 1 or method 2 of example 1 was fine particulate. The X-ray powder diffraction test of the sulfate salt (salt formation ratio is close to 1:1) of the compound of the formula I is shown in figure 2, namely the obtained sulfate salt is amorphous. TGA testing of the sulfate salt (salt formation ratio close to 1:1) of the compound of formula 1 referring to fig. 3, weight loss was 5.1% before 150 ℃. With reference to FIG. 4, no melting peak was observed.

Example 3: sulfate salt of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate (salt formation ratio approaching 1:2)

The compound of formula I ((200mg,0.22mmol)) was dissolved in 1.6mL of isopropanol at room temperature. Concentrated sulfuric acid (48.20mg, 0.48mmol) was dissolved in 1.6mL of isopropanol. Dropwise adding the isopropanol solution of the compound shown in the formula I into the isopropanol solution of sulfuric acid, enabling the isopropanol solution to be turbid and stick to the wall in the dropwise adding process, continuously stirring after dropwise adding 1.0mL of isopropanol, stirring for 1.5h, enabling the isopropanol solution to be completely adhered to the wall of a bottle, standing, removing liquid in a suction mode, adding 1.0mL of isopropyl ether into the solid, stirring and slurrying into a flowing solid, centrifuging, washing the solid with isopropyl ether, and then carrying out vacuum drying for 24h at room temperature to obtain 172.67mg of sulfate. Ion chromatography and HPLC results show that the free base in the sulfate salt is approximately 1:2 salt with sulfuric acid.

The PLM test showed the sulfate to be a fine particulate. The X-ray powder diffraction test of the sulfate salt (salt formation ratio is close to 1:2) of the compound of the formula I is shown in figure 5, namely the obtained sulfate salt is amorphous. TGA testing of the sulfate salt (salt formation ratio close to 1:2) of the compound of formula I referring to fig. 6, weight loss was 4.65% before 150 ℃. With reference to FIG. 7, no melting peak was observed.

Example 4: d-tartrate salt of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ D ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate

The method comprises the following steps: dissolving a compound of formula I ((100mg,0.11mmol) in 0.4mL of isopropanol at room temperature, dissolving D-tartaric acid ((18.27mg,0.12mmol) in 0.4mL of isopropanol, dropwise adding the isopropanol solution of D-tartaric acid into the isopropanol solution of the compound of formula I under stirring, wherein turbidity appears and sticky walls appear quickly in the dropwise adding process, supplementing 0.4mL of isopropanol, dissolving clear solution, placing at 4 ℃, stirring for 24h, precipitating a large amount of solids, dropwise adding 3.0mL of isopropyl ether, continuously stirring for 6h at 4 ℃, centrifuging to separate the solids, and vacuum-drying at room temperature for 24h to obtain 49.21mg of D-tartrate.

The method 2 comprises the following steps: dissolving a compound of formula I ((108.19mg,0.12mmol) in 0.4mL of isopropanol at room temperature, dissolving D-tartaric acid ((19.56mg,0.13mmol) in 0.4mL of isopropanol, dropwise adding the isopropanol solution of D-tartaric acid into the isopropanol solution of the compound of formula I under stirring, allowing the solution to be turbid during dropwise addition, stirring at 4 ℃ for 24h to precipitate a large amount of solid, dropwise adding 3.0mL of isopropyl ether, continuously stirring at 4 ℃ for 1h, centrifuging to separate the solid, and vacuum-drying at room temperature for 24h to obtain 85.98mg of D-tartrate.

¹H-NMR spectrum shows that the salt formation ratio of free alkali to D-tartaric acid in the D-tartrate obtained by the two methods is 1:1.

The D-tartrate salt obtained above was subjected to X-ray powder diffraction test, and XRPD pattern showed that the D-tartrate salt was amorphous (see FIG. 8). TGA testing (see fig. 9) showed that D-tartaric acid lost 7.8% of weight before 160 ℃. DSC testing of the D-tartrate salt (see figure 10) showed no melting absorption peak.

Example 5: crystalline morphology study of pharmaceutically acceptable salts of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate

The magma experiment was performed by stirring a supersaturated solution of the sample (with undissolved solids present in the solution) in a different solvent system for a period of time.

The sulfate and D-tartrate obtained in examples 2 to 4 were respectively put in different solvent systems for crystal slurry experiments, filtered and vacuum-dried at room temperature. X-ray powder diffraction tests are carried out on the obtained salts, and the results show that the solids obtained by carrying out crystal slurry experiments under different solvent conditions are all amorphous substances, and the amorphous substances are shown in Table 1.

Table 1: salt form of the compound of formula I

Example 6: solubility and hygroscopicity studies of pharmaceutically acceptable salts of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate

Solubility test method: weighing a known amount of sample at 25 ℃, adding a solvent into the sample in portions, stirring or assisting the dissolution by ultrasound until the sample is visually dissolved, recording the consumed solvent amount, and if the sample is not dissolved yet at a specific concentration, indicating the solubility of the sample by a specific concentration of < ">.

Moisture absorption test method: about 10mg of sample was taken and its hygroscopicity was measured by a dynamic moisture adsorption apparatus.

Water solubility: very soluble (greater than 1 g/mL); easily soluble (greater than 100mg/mL, but less than or equal to 1 g/mL); dissolution (greater than 33.3mg/mL, but less than or equal to 100 mg/mL); sparingly soluble (greater than 10mg/mL, but less than or equal to 33.3 mg/mL); slightly soluble (greater than 1mg/mL, but less than or equal to 10 mg/mL); very slightly dissolved (greater than 0.1mg/mL, but less than or equal to 1 mg/mL); almost insoluble or insoluble (less than 0.1 mg/mL).

Description of hygroscopicity characteristics and definition of hygroscopicity increase (directive of hygroscopicity test for drugs, appendix XIX J, 2010 edition, Chinese pharmacopoeia, No.: 25 ℃. + -. 1 ℃, 80% relative humidity).

The water solubility and hygroscopicity of the compounds of formula I (as free base), the sulfate salts, the D-tartrate salts, and other salts of the compounds of formula I were determined using the solubility and hygroscopicity test methods described above, and the water solubility and hygroscopicity results for the compounds of formula I and the sulfate and D-tartrate salts of the compounds of formula I are given in Table 2.

Table 2: water solubility and hygroscopicity of pharmaceutically acceptable salts of compounds of formula I

Sample (I)	Water solubility	Hygroscopicity (within the range of 0% RH-80% RH)
			A compound of formula I	5.36μg/mL	40.8％
Sulfate (salt ratio close to 1:1)	55.32μg/mL	8.0％
			Sulfate (salt ratio close to 1:2)	30.58μg/mL	9.1％
D-tartrate salt	10.23μg/mL	9.1％

The results show that the water solubility and hygroscopicity of the sulfate and D-tartrate salts of the compound of formula I are greatly improved compared to the free base and other salt forms.

Example 7: stability test of a pharmaceutically acceptable salt of methyl N- ((2S) -1- ((S) -2- (6- (7- ((S) -2- (5- ((S) -2- ((methoxycarbonyl) amino) -3-methylbutyryl) -5-azaspiro [2.4] heptan-6-yl) -1H-imidazol-5-yl) -9,9,10, 10-tetrafluoro-9, 10-dihydrophenanthren-2-yl) -1H-benzo [ d ] imidazol-2-yl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) carbamate

Taking a proper amount of a compound (free alkali form) in the formula I, sulfate (salt forming ratio is close to 1:1), sulfate (salt forming ratio is close to 1:2) and a D-tartrate sample, placing the sample in a watch glass, paving the sample into a thin layer with the thickness of about 3-5 mm, processing the thin layer according to the following requirements, sampling the sample in 5 days and 10 days respectively under the conditions of high temperature, high humidity, illumination, oxidation and 5 accelerated influence factors, detecting and inspecting the change conditions of main peaks and impurities by HPLC, and detecting the purity of the sample shown in Table 3.

The placing conditions are respectively as follows:

1) high temperature experiment: and (5) blowing a drying box at 60 ℃, and sealing and protecting from light.

2) High humidity experiment: 25 ℃, 90% +/-5% RH hygrometer, open to the sun.

3) And (3) illumination experiment: 25 ℃, 5000Lux, open mouth.

4) Oxidation experiment: and (4) at 40 ℃, sealing a drier for the urea hydrogen oxide, and placing the drier open.

5) Accelerated testing: 40-75% RH, constant temperature and humidity box, and open to the sun.

Table 3 stability testing of pharmaceutically acceptable salts

Test results show that the purity of the compound shown in the formula I, the sulfate and the D-tartrate of the compound is not obviously changed after 10 days under the conditions of high temperature, high humidity, illumination, oxidation and acceleration, and the stability of the sulfate and the D-tartrate of the compound shown in the formula I is better than that of the compound shown in the formula I and other salts thereof under the conditions of oxidation and illumination.

Claims

1. A pharmaceutically acceptable salt of a compound of formula I, wherein the pharmaceutically acceptable salt is a sulfate or D-tartrate salt,

wherein the sulfate is monosulfate of the compound in the formula I or bisulfate of the compound in the formula I or a mixture of the monosulfate and the bisulfate of the compound in the formula I, and the salt formation ratio of the D-tartrate is 1:1.

2. A pharmaceutically acceptable salt as in claim 1, wherein said pharmaceutically acceptable salt is an amorphous form.

3. A process for the preparation of a pharmaceutically acceptable salt as claimed in claim 1 or 2, which process comprises the step of reacting a compound of formula I with the corresponding acid to form the salt.

4. The process according to claim 3, wherein the reaction is carried out in an alcoholic solvent having less than 6 carbon atoms or a ketone solvent having less than 6 carbon atoms.

5. The process of claim 4, wherein the solvent is selected from one or more of methanol, ethanol, propanol, n-butanol, sec-butanol, isopropanol, and acetone.

6. The process according to any one of claims 3 to 5, wherein the molar ratio of the compound of formula I to the corresponding acid is from 1:0.5 to 5.

7. The process of claim 6, wherein the molar ratio of the compound of formula I to the corresponding acid is 1: 1-3.

8. A pharmaceutical composition comprising a pharmaceutically acceptable salt as claimed in claim 1 or 2 and a pharmaceutically acceptable carrier.

9. Use of a pharmaceutically acceptable salt as defined in claim 1 or 2 or a composition as defined in claim 8 for the manufacture of a medicament for the treatment and/or prevention of a disease caused by hepatitis c virus.