GB2552919A - Stable amorphous form of daclatasvir - Google Patents

Abstract

A process for the preparation daclatasvir or a pharmaceutically acceptable salt thereof, wherein the process includes the synthetic step of reacting a compound of Formula (I) with a compound of Formula (II): Wherein LG is a leaving group and Z is a protecting group. The protecting group Z may be selected from Boc, Cbz, Tosyl, Mesyl, Benzyl, Fmoc, substituted acetyl, Benzoyl and Tolyl. A process for the production of amorphous daclatasvir-2 HCl is also provided, comprising the steps of contacting daclatasvir-2 HCl with a pharmaceutically acceptable solvent; filtering off the solution or dispersion of the first step; and the immediate removal of the solvent from the filtered solution or dispersion of the second step, characterised in that the solvent removal is performed within a timescale of 10 seconds to 5 hours after the filtering step.

Description

(71) Applicant(s):

AZAD Pharmaceutical Ingredients AG Durachweg 15, Schaffhausen 8200, Switzerland (72) Inventor(s):

Sven Haferkamp Roman Gerber Aeschbacher Miriam Oberholzer Armine Aramayisyan David Hambardzumyan Arsen Sahakyan Viktorya Rstakyan (56) Documents Cited:

WO 2011/060000 A1 WO 2008/021928 A2

WO 2009/102568 A1 WO 2008/021927 A2 (58) Field of Search:

INT CLA61K, C07D

Other: WPI, EPODOC, CAS Online (74) Agent and/or Address for Service:

Michalski Huttermann & Partner Patentanwalte, Hafenspitze, Speditionstr.21, 40221 DUsseldorf NRW, Germany (54) Title of the Invention: Stable amorphous form of daclatasvir

Abstract Title: Process for the preparation of Daclatasvir and pharmaceutically acceptable salts thereof (57) A process for the preparation daclatasvir or a pharmaceutically acceptable salt thereof, wherein the process includes the synthetic step of reacting a compound of Formula (I) with a compound of Formula (II):

Wherein LG is a leaving group and Z is a protecting group. The protecting group Z may be selected from Boc, Cbz, Tosyl, Mesyl, Benzyl, Fmoc, substituted acetyl, Benzoyl and Tolyl. A process for the production of amorphous daclatasvir-2 HCI is also provided, comprising the steps of contacting daclatasvir-2 HCI with a pharmaceutically acceptable solvent; filtering off the solution or dispersion of the first step; and the immediate removal of the solvent from the filtered solution or dispersion of the second step, characterised in that the solvent removal is performed within a timescale of 10 seconds to 5 hours after the filtering step.

At least one drawing originally filed was informal and the print reproduced here is taken from a later filed formal copy.

/6

Figure 1

ΥΊ

·· ·· • · · • ·

o.

•O

2/6

Figure 2

3/6

Figure 4

Figure 5 ·· ·· • * · • · • ·

I ' I ' I ' I

15,0

13,0

10,5 9,5 fi(_ppm) ⁷'°

5,0

2,5

0,5

4/6

Figure 6

TG /% DSC /(mW/mg)i

Temperatur /°C • Figure 7

Temperatur/°C

5/6

Figure 8

» · » ♦ » ··· ···«·· <w · ^1-1 ’“I ’ ’ '-·-1-1--*.....«-’“Ί-1-·-1-1-1· ».....«—1—I-Γ“’-’-’-«-1-1' » '¹-1-1-1-1-1-1-1—

10 20 30 40

Theta [·] ······ • * • · ·· #· • * · » · «·«·«*

Figure 9

Theta [·]

6/6

»· • * • ···

•	• Λ
	•
•	♦
	•
······
•	•
··	• 4
•	»* ·
•	•
	•

STABLE AMORPHOUS FORM OF DACLATASVIR

FIELD OF THE INVENTION

The present invention relates to a route of synthesis of pharmaceutically acceptable daclatasvir salts and a process for the production of amorphous daclatasvir-2 HCI. Furthermore, the present invention concerns the use of the amorphous daclatasvir-2 HCI in pharmaceutical compositions and the use of the amorphous drug in the treatment of HCV (hepatitis C virus)-infections.

BACKGROUND

Chronic hepatitis C virus (HCV) infection affects approximately 180 million individuals worldwide and is a common cause of chronic liver disease and hepatocellular carcinoma (HCC) in Japan, the United States, and many European countries. Currently, the standard treatment approach for HCV includes 24—48 weeks of treatment with pegylated interferon a (pegIFNa) in combination with ribavirin. This treatment has nonspecific and largely unknown mechanisms of action and common drawbacks are side-effects (e.g. flu-like symptoms, fatigue, neuropsychiatric effects, autoimmune phenomena and hemolytic anemia), nonresponsiveness (e.g. genotype 3-infected patients) and the risk of developing resistance-associated variants. In the recent past new target structures in the virus life cycle have been identified, which can selectively be addressed by so-called direct acting agents (DAA). Four different DAA classes for the HCV have been established differing by their mode of action and specific therapeutic target, i.e. nonstructural proteins 3/4A (NS3/4A) protease inhibitors (Pis), NS5B nucleoside polymerase inhibitors (NPIs), NS5B non-nucleoside polymerase inhibitors (NNPIs), and NS5A inhibitors. In certain cases it has been proven in clinical trials that these compound classes are able to avoid some of the drawbacks of the established treatment regime.

One new therapeutic group of compounds following the DAA pathway is for instance disclosed in WO 2008/021927, wherein substituted biphenyl-imidazoles are utilized as direct antiviral agents (DAA) for anti-HCV virus treatments. Within this document also the special drug daclatasvir

amino] -3 -methylbutanoyl} -2-pyrrolidinyl] -1 H-imidazol-4-yl} -4biphenylyl)-1Himidazol-2-yl]-l-pyrrolidinyl}-3-methyl-l-oxo-2 butanyl]carbamate) is disclosed in the form of a pharmaceutically acceptable salt, i.e. especially in the form of the dihydrochloride salt. The dihydrochloride salt form has been established since the pure organic compound is difficult to crystallize and hence formation of the pure product (without the hydrochloride) has not been reproducible. Therefore, it is necessary to include a re-crystallization step in order to achieve the crystalline drug form.

Such finding is also explicitly addressed by a further patent document of the same applicant, US 2009/0041716 Al, disclosing a particular polymorph N-2 of daclatasvir-2 HC1 which can repeatedly be crystallized into that particular polymorph, offering high aqueous solubility and an excellent purification capacity.

In contrast to the proposed use of crystalline forms of daclatasvir-2 HC1, the German utility model DE 21 2012 000 197 U1 explicitly teaches the use of amorphous solid dispersion (ASD) of a plethora of DAAs, wherein the presence of a polymeric carrier is mandatory to yield an amorphous configuration of the drug compound in the form of a polymer/drug adduct. The dispersion is either obtainable by solvent evaporation via spray drying of the polymer/drug mixture or by amorphous crystallization from the melt. The main disadvantage of this method is that the presence of an additional excipient is required, which complicates the overall production process and might influence the bioavailability and/or the storage behavior of the formulation.

Nevertheless, besides the existing ways of processing there is still the need for new synthesis routes and new pharmaceutically acceptable forms of daclatasvir salts, comprising improved shelf-life, dissolution rate, hygroscopicity, tableting properties, compactibility, density, hardness, powder flow ability, triboelectrical properties (electrostatic charging), chemical stability, optical stability, taste, odor, and regulatory unambiguousness. It is therefore an object of the present invention to provide such reliable route of synthesis and such a process for the production of essentially pure amorphous daclatasvir-2 HC1. It is further an object of the invention to provide a pharmaceutical composition utilizing the amorphous form and to provide the use of such amorphous composition for the use in the treatment of HCVinfections.

BRIEF DESCRIPTION OF THE INVENTION

It has been found that the above mentioned task is inventively fulfilled by a process for the preparation of a compound according to the following formula (13):

the following synthesis steps:

a) reacting a compound (2) comprising the leaving groups LG

O

and a compound (7) ^NH2

wherein X is a halide ion and Z is a nitrogen protecting group, in order to obtain compound (8)

b) subjecting compound (8) to a deprotecting agent and

c) reacting the product of step b) and compound (11)

O

in order to obtain compound (13). Such route of synthesis is able to provide compound (13) with high yields and only small amounts of unwanted by-products. Especially it is possible to follow that route at low temperature conditions reducing the risk of epimerization of chiral compounds and side reactions. This in consequence leads to a better control of impurities and higher overall yields. In particular, the intermediates are crystalline and can easily be recrystallized, allowing a better control of critical impurities and easier transfer to a large scale manufacturing process. In addition, the synthesis of compound (11) avoids methane chloroformate (acyl chloride), which is very reactive and reacts exothermic. Furthermore, compound (13) is easily convertible into pharmaceutically acceptable salts of compound (13) by addition of inorganic or organic acids, for instance by addition of mineral, carboxylic- or sulphonic acids. Mineral acids can for instance be hydrochloric-, hydrobromic-, hydroiodic- or hydrofluoric-acids. Acceptable nonminer alic acids for the transformation of compound (13) into the respective salt form can be sulphuric-, phosphoric-, methanesulphonic-, ethanesulphonic-, ptoluenesulphonic-, benzenesulphonic-, naphthaline-disulphonic-, acetic-, propionic-, lactic-, tartaric-, citric-, fumaric-, maleic- or benzoic acid. The anion to cation stoichiometry in the salt form may vary with respect to the chosen acid and can be in between 1 and 4, preferably larger or equal 1 and smaller or equal 2. It is also possible to use the proposed route of synthesis with minor modifications to obtain tautomeric forms of compound (13), and the invention likewise encompasses these forms.

In step a) compound (2) and compound (7) can be reacted in a pharmaceutically acceptable solvent in order to obtain compound (8). Suitable solvents for this reaction may be protic or aprotic solvents and can be chosen from the group consisting water, ethanol, methanol, isopropanol, 1-propanol, dioxane, THF, DMF, DMSO, DMAc, acetonitrile or mixtures thereof. In a preferred embodiment the reaction can be performed under reflux-conditions in a 70:10 dioxane:water mixture. Such mixture is preferred, because the dioxane/water boiling points are very similar and this mixture of highly polar aprotic and protic solvents seems to facilitate product formation.

The deprotection of compound (8) in step b) can be performed according to methods known to the skilled artisan. Suitable reaction mechanisms for deprotection might include hydrogenolysis, acid or base catalyzed deprotection or the use of reducing agents. Preferably the deprotection is achieved in the presence of a mineral acid in a temperature range between 25°C and 70°C, preferably between 40°C and 60°C. Within a preferred embodiment of the invention the mineral acid can be hydrochloric acid.

In a first embodiment of the invention the nitrogen protecting group Z can be selected from the group consisting of Boc, Cbz, Tosyl, Mesyl, Benzyl, Fmoc, substituted or unsubstituted Acetyl, Benzoyl, Tolyl, -C(O)CF₃. Such protecting groups are able to effectively protect the nitrogen function of the compounds (4) (7) and (8) and can be removed under gentle conditions, not interfering with the following transformation steps. Preferred groups Z may further be selected from the group of Boc, Tosyl, Mesyl.

In another aspect of the process the anion X of compound (7) can be selected from the group consisting of F, Cl , Br . Especially the halide anions have been proven useful to achieve a fast reaction kinetics and a low concentration of side-products for the reaction of compound (7) to compound (8).

In a further characteristic of the process the leaving group LG can be selected from the group consisting of -F, -Cl, -Br, -I, -OH, -NH₂, mesylate, triflate, tosylate, diazonium salts, alkyl- or aryl sulfonates, phosphates, phosphonic acids or phosphonic esters.

Within a preferred embodiment of the invention the compound (7) can be prepared via the following route of synthesis:

a) reacting L-Proline and a nitrogen protecting agent in order to obtain the nitrogen protected compound (4)

b) reacting compound (4) and an amination reagent in order to obtain compound (5)

c) reacting compound (5) and an acid in a pharmaceutically acceptable solvent in order to obtain compound (6)

\

Z

d) reacting compound (6) and a C1-C6 alcoholate in order to obtain compound (7a)

wherein R¹ is a C1-C6 alkyl,

e) reacting compound (7a) and an amination reagent in order to obtain compound (7)

Within the route of synthesis for the preparation of compound (7) a nitrogen protected L-Proline is generated in step a). Suitable protection groups may be selected from the same group as given above and the protection reaction may be performed in a protic solvent at room temperature. In a preferred embodiment the protic solvent can be a 50:50 vol.% water/ethanol mixture. The resulting compound (4) can be used in the further synthesis steps either after a purification step or as the crude product. Especially the route without an in-between purification is preferred, because the amidine can easily be purified at the end via re-crystallization, resulting in an overall higher yield.

In step b) a carbonic acid amide is formed. The reaction can be performed in a pharmaceutically acceptable solvent, wherein polar solvents are preferred. Suitable solvents may for instance be selected from the group consisting of protic or aprotic polar solvents like pyridine, tetrahydrofuran (THF), ethyl acetate (EtOAc), acetone, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), formic acid, n-butanol, isopropanol (IPA), nitromethane, ethanol (EtOH), methanol (MeOH), acetic acid (AcOH), water or mixtures thereof, dioxane/pyridine mixtures being further preferred. Suitable amination reagents for forming the carbonic acid amide in step b) can be ammonium-salts, because these salts are available at low costs and the handling is much easier compared to ammonia-gas. This reaction can further be catalyzed by addition of a base. Suitable bases can be chosen from organic or inorganic compounds, such as pyridine and functionalized pyridines (such as 4dimethylaminopyridine, lutidines, aminopyridines), mono-, di- and trisubstituted amines or mixtures thereof.

In step c) the carbonic acid amide is dehydrated to a nitrile. Suitable reagents for achieving this reaction may be anhydrides, such as TFAA. The dehydration reaction can be performed at room temperature in suitable pharmaceutically acceptable solvents. Preferred solvents can be selected from the group consisting of DMF, DMAc, dichloromethane, chloroform, DMSO, THF, dioxane, acetonitril, wherein THF is preferred.

In step d) the nitrile is transformed by addition of an alcohol or alcoholate to an amidate. Such reaction may in general be performed in acid or alkaline medium, the alkaline medium being preferred. Especially this reaction can be performed using the same alcohol as solvent and in the form of the alcoholate. Suitable sources of the deprotonated alcohol may be for instance the alkali-alcoholates, sodium alcoholates being preferred.

In step e) the amidate is transformed to the salt form of compound (7). Suitable anions X are given above. Suitable amination reagents for forming compound (7) from compound (7a) can be selected from the group consisting of ammonium-salts,

NH₄C1, NH₄Br, NH₄HCO₃, (NH₄)₂CO₃, ammonium acetate, (NH₄)PF₆ and NH₄NO₃ or mixtures thereof. This reaction can be performed acid or base catalyzed, the acid catalyzation being preferred. Suitable catalysts can for instance be AcOH, HC1, TFA, NH₄C1, acetic acid being preferred.

In another aspect of the process the amination in step e) can be achieved within a two-step procedure, wherein in a first step compound (7a) is subjected to a pharmaceutically acceptable acid and an ammonium-halide salt and, in a second step, the resulting solution is neutralized by a base. Suitable pharmaceutically acceptable acids for the first step can be selected from the group consisting of acetic acid, HC1, TFA. The ammonium-halide salt can be chosen from the group consisting of ammonium chloride, -bromide, fluoride and iodide, wherein the chloride salt is preferred.

Furthermore, in a preferred embodiment of the process the reaction of step d) and e) can be performed in a one-pot synthesis without isolation of the intermediate compound (7a). Such route of reaction decreases the loss of product compared to a two-pot reaction, wherein an in-between purification is performed.

Within a further embodiment of the process the compound (13) can be treated with aqueous hydrochloric acid solution to achieve daclatasvir-2 HC1.

It has additionally been found that daclatasvir-2 HC1 prepared according to the invention can be transformed into a purely amorphous form. This is achieved by a process at least comprising the steps of a) contacting daclatasvir or daclatasvir-2 HC1 and a pharmaceutically acceptable solvent; b) filtering of the solution or dispersion of step a); c) immediate removal of the solvent from the filtered solution or dispersion of step b), wherein the solvent removal is performed within a timescale of > 10 seconds and < 5 h after the filtering step b). Surprisingly it has been found that in comparison to crystalline forms the amorphous form of daclatasvir-2 HC1 shows better production characteristics. Among such characteristics are hygroscopicity, dissolution behavior, compatibility with other excipients, chemical stability with respect to temperature and pressure, bioavailability and resulting from that an overall better storage stability. Furthermore, the amorphous form can easily and reliably be produced at low costs and no further excipients or auxiliary substances are needed in order to achieve the amorphous form. This is especially helpful, because the complexity of the system is reduced due to the fact that it is unnecessary to include other substances like polymers which are known in the state of the art to stabilize the amorphous form of daclatasvir-2 HC1. Such auxiliary substances may influence other physical or chemical characteristics of the formulation or may even influence the bioavailability of the drug. Therefore, the addition of such auxiliary substances to induce the formation of amorphous phases is not within the scope of the present invention. Furthermore, it is surprising that the amorphous form precipitates immediately from the filtered solution although the N-2 form is thermodynamically preferred. Without being bound by the theory it is believed that the removal of potential crystallization centers in the filtration step and the immediate and fast precipitation result in the formation of the amorphous phase instead of any of the favored crystalline daclatasvir-2 HC1 phases. Such process is different to the crystallization steps performed in the state of the art, which are directed to long crystallizations including the presence of seed crystals. Usually also a long and sophisticated temperature program is used, which is directed to the formation of the thermodynamically more stable crystalline forms. Inasmuch as, the crystalline form is thermodynamically favored it is surprising that it is possible to achieve the pure amorphous form of daclatasvir-2 HC1 without such complicated processes or any auxiliary substances. Furthermore, it is believed that the better dissolution of the amorphous form and the higher pressure stability are direct results of the less organized solid state, i.e. amorphous structures allowing better hydration due to the fact that no crystal packing forces have to be destroyed during dissolution and amorphous forms are less affected by pressure tensors. Furthermore, the present process results in a special size distribution of the amorphous aggregates, which are highly compactable, exhibit low hygroscopicity and remain free flowing even after long storage times. In the case that daclatasvir is used as starting substance the solvent in step a) has to comprise a sufficient amount of hydrochloric acid in order to yield the dihydrochloride salt of daclatasvir.

Amorphous daclatasvir-2 HC1 is the dihydrochloride salt of above given structure and IUPAC name which do not show any kind of symmetric short or long range structure between the single molecules, i.e. in the dry solid form the single molecules are randomly oriented and/or aggregated. The presence of such amorphous structure can especially be assumed if a powder X-ray diffraction pattern exhibits no isolated peaks, but instead only a broad, unstructured halo in the whole 2-theta range between 2 and 50°. In the case that the skilled artisan is in doubt whether a pattern exhibits a peak or not it is in the meaning of the present application that a diffraction pattern is absent of any peak if the ratio between the integral intensity assessed over a 2-theta interval of 0.5° (of the presumed peak) and both adjacent 2 theta-ranges of the same size is smaller than 0.1 (excluding border and purely statistical effects, which may be caused for instance by a short measurement time; especially baseline uncorrected diffraction pattern are considered). The 2-theta interval is defined starting from the 2-theta position of the maximum of the (presumed) peak and extending 0.25° to the left and the right. It is especially within the scope of the invention to provide an amorphous daclatasvir-2 HCI which exhibits a powder x-ray diffraction pattern at a temperature between 20°C and 25 °C especially absent of peaks at any of the 2-theta values (±0.1°) of 10.3, 12.4, 12.8, 13.3, 13.6, 15.5, 20.3, 21.2, 22.4, 22.7 and 23.7°.

Within the inventive process daclatasvir-2 HCI and a pharmaceutically acceptable solvent are contacted. Such contacting is achieved by addition of a solvent to daclatasvir or daclatasvir-2 HCI, wherein in the first case (starting from the free base) also hydrochloric acid may be present in the solvent. The resulting suspension or dispersion may further be mechanically treated, e.g. by stirring, shaking or ultrasonic treatment in order to assist the dissolution of the daclatasvir or daclatasvir2 HCI, if necessary. Furthermore, the suspension or dispersion can be heated to temperatures up to 60°C, preferably 50°C and even more preferred to 40°C. Pharmaceutically acceptable solvents which are suitable for the process can for instance be found in the Q3C guidance for the industry document of the ICH (February 2012) and can be selected from the group of solvents in Class 2 or 3 (solvents which should be limited in pharmaceutical products). Preferably, the solvent may be selected from, but not limited to, the group consisting of water, (halogenated-) alkanes like chloroform, 1,2-dichloroethane, dichloromethane, 1,1,2trichloroethane, cyclohexane, methylcyclohexane, 1,2-dimethoxyethane, pentane, hexane, heptane, toluene; ethers like diethylether, MTBE, THF, 1,4-dioxane; organic acids and esters like acetic acid, formic acid, butylacetate, ethylaceteate, ethylformate, isobutyl acetate, isopropyl acetate, methyl acetate, propyl acetate; alcohols like methanol, ethanol, iso-butanol, 1-pentanol, 1-propanol, 2-propanol, 2methoxyethanol, 1-butanol, 2-butanol; ketones like acetone, MEK, MIBK, nitriles like acetonitrile; pyridine, nitromethane or mixtures thereof.

In a preferred embodiment of the invention the amorphous daclatasvir-2 HCI is obtained by precipitation of daclatasvir-2 HCI synthesized according to inventive route of synthesis.

Furthermore, in a preferred embodiment the solvent can be selected from the group consisting of halogenated and non-halogenated C(l-8) alkanes like chloroform, 1,211 dichloroethane, dichloromethane, 1,1,2-trichloroethane, cyclohexane, methylcyclohexane, 1,2-dimethoxyethane, pentane, hexane, heptane, toluene; or (Cl-6) ethers like diethylether, MTBE, THF, 1,4-dioxane or mixtures thereof.

In addition, in another aspect of the invention the solvent can be selected from the group consisting of (C1-C6) organic acids and esters like acetic acid, formic acid, butylacetate, ethylaceteate, ethylformate, isobutyl acetate, isopropyl acetate, methyl acetate, propyl acetate or mixtures thereof.

In another aspect of the invention the solvent can be selected from the group consisting of heptane, MTBE, acetic acid, formic acid, butylacetate, ethylaceteate, ethylformate, isobutyl acetate, isopropyl acetate, methyl acetate, propyl acetate; alcohols like methanol, ethanol, iso-butanol, MEK, MIBK. These solvents exhibit a good solubility of the API and a preferred toxicology.

In a further aspect the solvent can be selected from the group consisting of heptane MTBE, acetic acid, formic acid, butylacetate, ethylacetate and MEK. Besides a sufficient solubility, low costs, excellent toxicology profile these solvents do exhibit a favorable vapor pressure, favoring the formation of the essentially amorphous form of daclatasvir-2 HCI. The vapor pressure of the solvent may be in the range of larger or equal 5 hPa and smaller or equal 500 hPa, preferably larger or equal 10 hPa and smaller or equal 300 hPa. Solvents comprising a smaller vapor pressure might yield an insufficient evaporation rate, resulting in the formation of a higher proportion of crystalline daclatasvir-2 HCI polymorphs.

It is further a preferred aspect of the invention that the solvent is selected from the group consisting of (Cl-6) alcohols like methanol, ethanol, iso-butanol, 1-pentanol, 1-propanol, 2-propanol, 2-methoxyethanol, 1-butanol, 2-butanol or mixtures thereof.

In another embodiment the solvent can be selected from the group consisting of (ClC6) ketones like acetone, MEK, MIBK, nitriles like acetonitrile; pyridine, nitromethane or mixtures thereof.

Within the filtering step the undissolved, i.e. still in solution aggregated daclatasvir is removed from the solvent in order to achieve a daclatasvir solution without any significant undissolved amounts, which may for instance act as seed crystals. Preferably the used filter is a size exclusion filter, being able to exclude sizes (largest dimension of the particle) from passing the filter unit of larger than 1 pm, larger than 0.75 pm, larger than 0.5 pm, larger than 0.4 pm, larger than 0.3 pm or larger than 0.2 pm. This exclusion size range has been proven to provide filtered solution without significant amounts of larger aggregates, enabling the formation of amorphous daclatasvir-2 HC1 in the following drying step. Larger exclusion sizes are not preferred because larger particles increase the risk of precipitating daclatasvir-2 HC1 crystals of defined symmetry. Suitable materials for the filter may be selected from any pharmaceutical acceptable filter material like active carbon, nylon, cellulose, diatomaceous earth, silica, provided that a defined size exclusion is achievable.

After the filtration step the removal of the solvent is started immediately. In the sense of this application this means that after the last solvent of a batch has passed the filter unit the solvent removal is started without any purposeful added waiting period, i.e. a period wherein no means are performed in order to remove the solvent. Especially it is not intended that the solvent is for instance left standing or stirred or tempered or otherwise conditioned without any additional means for solvent removal. This procedure is in contrast to standard re-crystallization steps, wherein always a certain time period is included, wherein the crystals are subjected to an Oswald ripening. Immediate in the sense of the invention especially means that the removal of the solvent is initiated on a timescale of > 10 seconds and < 5 h, i.e. the first solvent molecules are irreversibly removed from the filtered solution in the above given timescale. In addition, it is feasible to further define that at least 1 % of the solvent mass has to be removed within 30 minutes after passing the filter unit. The removal of the solvent may for instance be achieved at ambient or elevated temperatures, vacuum assisted or at ambient pressures. Surprisingly, it has been found that the immediate removal of the solvent within this process yields essentially pure amorphous daclatasvir-2 HC1 without any significant crystalline amounts. Means for the removal of the solvent are for instance evaporation of the solvent from a vessel comprising a large surface area or via a rotary evaporator. In addition to the timescale of > 10 seconds and < 5 h, the solvent may be removed within > 30 seconds and < 2.5 h, preferably > 1 minute and < 1.5 h and even more preferred > 5 minutes and < 1 h.

In another aspect of the invention the filtered solution is essentially free of undissolved daclatasvir-2 HC1. In order to avoid any seed crystals or pre-build nucleation centers in the filtered solution it has proven useful, that the filtered solution does not comprise any larger aggregates which may act in that sense. The filtered solution can be considered essentially free of any undissolved daclatasvir-2 HC1 particles if the solution is optically clear, i.e. not turbid. The solution is not turbid in the sense of the invention if the solution exhibits a turbidity of > 0,001 und < 50 NTU, preferably > 0,01 und < 25 NTU and even more preferred >0,1 und <10 NTU measured at 20°C according to DIN EN ISO 7027. This turbidity range has been found to result in the formation of essentially amorphous daclatasvir-2 HC1.

Furthermore, in a preferred embodiment of the invention the pharmaceutically acceptable solvent can be selected from the group consisting of water, acetonitrile, methanol, ethanol, 1-propanol, acetone, MEK, ethylacetate, 1,4-Dioxan, THF, MIBK or mixtures thereof. Especially the above mentioned solvents have been found to be pharmaceutically acceptable and are able to dissolve a sufficient amount of daclatasvir or daclatasvir-2 HC1 on a very short timescale. Furthermore, these solvents are able to be evaporated at low temperatures, resulting in essentially dry amorphous products. In a preferred embodiment the solvent can be selected from the group consisting of water and ethanol.

Within a further inventive characteristic the filtering step b) can be performed by passing the solution or dispersion through a filter comprising a pore size of < 0.4 pm. Preferably the removal of possible nucleation centers is achieved by a size exclusion filtering step, wherein the filter is able to remove aggregates exhibiting dimensions (largest distance from point to point within the aggregate) larger than 0.4 pm. Such aggregates may induce the formation of crystalline instead of amorphous daclatasvir-2 HC1 and hence may result in the formation of larger crystalline daclatasvir-2 HC1 proportions. The minimum exclusion size may for practical reasons be set to 100 nm at least allowing the passing of single daclatasvir molecules.

In a further characteristic of the process the solution or dispersion of step a) is heated to a temperature of > 20 °C and < 65 °C. It has been found that a slight warming of daclatasvir-2 HC1 solutions before the filtering step is able to increase the resulting yield of amorphous daclatasvir-2 HC1. This finding may be related to a reduction of the content of larger daclatasvir-2 HC1 aggregates in the course of the heating. Higher temperatures are less preferred, because degradation processes of the daclatasvir-2 HC1 might be induced. Furthermore, the temperature can be > 25 °C and < 60 °C, preferably > 30 °C and < 55 °C and even more preferred > 35 °C and < 50 °C.

In a preferred embodiment the solvent removal in step c) can be performed at the same temperature as the contacting in step a). Surprisingly, it has been found that an isothermal process of contacting, filtering and solvent removal is able to deliver essentially amorphous daclatasvir-2 HC1 of a preferred particle size, without the need of special stabilizers, comprising an advantageous dissolution behavior and a low hygroscopicity. Without being bound by the theory this is achievable in above defined temperature range, because this range enables a formation kinetics which is fast enough to result in the formation of amorphous daclatasvir-2 HC1 and slow enough to achieve a sufficient particle size.

A preferred embodiment of the inventive process includes the solvent removal at room temperature. For the given filtered daclatasvir-2 HC1- solvent-system it has been found suitable to keep the solution during the evaporation step at room temperature. Without being bound by the theory the amount of crystalline daclatasvir-2 HC1 and the particle size of the resulting amorphous daclatasvir-2 HC1 is in a preferred range, achieving a fast production process and essentially amorphous daclatasvir-2 HC1 is achieved. Furthermore, the obtainable amorphous daclatasvir-2 HC1 exhibits a fast dissolution profile and a suitable particle size. Especially the obtainable particle size distribution results in excellent tableting properties and a very low pressure sensitivity.

According to another aspect of the invention the contacting in step a) can be performed under stirring for a time interval of > 10 minutes and < 6 days. In order to achieve efficient dissolution of the daclatasvir or daclatasvir-2 HC1 in the contacting step it has been proven useful to combine the substance and the solvent for a longer time period und gentle mechanical agitation. Such treatment might increase the amount of dissolved drug and/or reduce the amount of larger aggregates, resulting in a more efficient production process due to the fact that less product might be lost in the filtering step. Shorter time periods may be unsuitable because most of the substance is lost in the filter, resulting in an insufficient yield. In addition, the time interval can be set to > 10 minutes and < 6 days, preferably > 30 minutes and < 3 days or > 1 h and < 1 days.

It is also within the scope of the invention to provide amorphous daclatasvir-2 HC1 prepared according to the inventive process, wherein the daclatasvir-2 HC1 is essentially amorphous and exhibits no characteristic peaks in a powder X-Ray diffraction pattern. In comparison to the crystalline forms of daclatasvir-2 HC1 the amorphous daclatasvir-2 HC1 exhibits improved processing characteristics and is especially less prone to changes in a tableting process, thus yielding more defined and stable tableting results. This can be attributed to the feature “amorphous” of the daclatasvir-2 HC1. Furthermore, without being bound by the theory this improved tableting behavior may also be based on the fact that the amorphous daclatasvir-2 HC1 according to the presented process additionally comprise a special particle size distribution of the amorphous particles. This particle size distribution may further contribute to the disclosed processing characteristics. The term “essentially amorphous” is understood as defined above. Specifically, the absence of peaks in an powder x-ray diffraction pattern at a temperature between 20°C and 25 °C at any of the 2-theta values of 10.3+0.1, 12.4+0.1, 12.8+0.1, 13.3+0.1, 13.6+0.1, 15.5+0.1, 20.3+0.1, 21.2+0.1, 22.4+0.1, 22.7+0.1 and 23.7°+0.1° has to be understood as prove for the essentially pure amorphous form.

In another embodiment of the invention the amorphous daclatasvir-2 HC1 can be essentially free of solvents. After the evaporation process in step c) preferably an amorphous daclatasvir-2 HC1 is achieved comprising a very low solvent content. Such low solvent content might improve the stability of the amorphous phase and hence contribute to the storage stability and the shelf life of the drug. The solvent content of the amorphous daclatasvir-2 HC1 after the evaporation step can be in the range of 0,0001 up to 5 weight%, 0,0001 up to 3 weight%, preferably between > 0,001 und < 2 weight% and even more preferred between > 0,01 und < 1 weight%. This solvent content might be achievable within a decent processing time scale and is sufficient to assure an acceptable storage stability without any change from the amorphous to the crystalline state of daclatasvir-2 HC1. The solvent content in the dried product can be assessed according to methods known to the skilled artisan. If the solvent is water for instance Karl-Fischer can be used for determination.

Furthermore, it is within the scope of the invention to provide a pharmaceutical composition comprising essentially amorphous daclatasvir-2 HC1. This pharmaceutical composition may optionally comprise further pharmaceutical acceptable excipients. The inventively achievable amorphous daclatasvir-2 HC1 is especially suitable for use in a pharmaceutical composition because the amorphous daclatasvir-2 HC1 can be processed in a more reproducible way compared to the crystalline forms. Hence, pharmaceutical composition are accessible comprising an improved shelf life and more homogeneous characteristics.

In a preferred embodiment the pharmaceutical composition further may comprise another direct antiviral agent selected from the group consisting of boceprevir, telaprevir, simeprevir, faldaprevir, asunaprevir, danoprevir, sofosbuvir, mericitabin, deleobuvir, setrobuvir, ledipasvir, meravirsen or mixtures thereof. Especially the inventively prepared amorphous daclatasvir-2 HC1 is suitable to be administered with additional DAAs which might be classified as a) NS3/NS4A-PIs, like boceprevir, telaprevir, simeprevir, faldaprevir, asunaprevir, danoprevir, b) NS5BPolymerase-inhibitors, like sofosbuvir, mericitabin, deleobuvir, setrobuvir, c) NS5Ainhibitors, like ledipasvir or d) Anti-miR122, like meravirsen. Such combination might increase the anti-HCV activity and even reduce the SVR.

Within a further aspect the pharmaceutical composition can be an oral dosage form. Due to the solubility and compactibility of the amorphous form of daclatasvir-2 HC1 it is especially possible to establish a production process wherein oral dosage forms can reliably and reproducibly be produced, resulting in homogenous, well defined and storage stable oral dosage forms like pills, tablets, powders, films or capsules.

It is further within the scope of the invention to disclose the use of the inventive pharmaceutical composition for the treatment of HCV infections.

With respect to additional advantages and features of the previously described pharmaceutical composition and amorphous daclatasvir-2 HC1 it is explicitly referred to the disclosure of the inventive process. In addition, also aspects and features of the inventive amorphous daclatasvir-2 HC1 shall be deemed applicable and disclosed to the inventive pharmaceutical composition and the inventive use. Furthermore, all combinations of at least two features disclosed in the claims and/or in the description are within the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures 1 to 10 exhibit:

1. a schematic representation of the inventive route of synthesis;

2. a PXRD of the essentially amorphous daclatasvir-2 HC1 prepared according to the invention;

3. a PXRD of the essentially amorphous daclatasvir-2 HC1 after 4 weeks storage time;

4. a NMR-spectrum of the essentially amorphous daclatasvir-2 HC1 freshly prepared;

5. a NMR-spectrum of the essentially amorphous daclatasvir-2 HC1 after 4 weeks storage time;

6. a DSC/TGA measurement of the essentially amorphous daclatasvir-2 HC1 freshly prepared;

Ί. a DSC measurement of the essentially amorphous daclatasvir-2 HC1 after 4 weeks storage time;

8. a PXRD of daclatasvir-2 HC1 processed not according to the invention after 4 weeks storage time;

9. a PXRD of the essentially amorphous daclatasvir-2 HC1 prepared according to the invention in a tableting experiment;

10. a PXRD of the N2-form of daclatasvir-2 HC1 in a tableting experiment.

Figure 1 displays a schematic representation of the inventive route of synthesis including the formation of a pharmaceutically acceptable salt form of compound (13). This salt form is just chosen as one example out of the possible pharmaceutically acceptable salt forms of compound (13). Furthermore, this scheme does not display the reaction of compound (12) to the essentially amorphous form of daclatasvir-2 HC1.

In Figure 2 the PXRD pattern of the essentially amorphous daclatasvir-2 HC1 prepared according to the invention is displayed in the 2-theta range from 2° up to 50°. The sample was prepared according to example 1 and placed in a standard glass capillary (0 - 0.7 mm). This measurement and also the PXRD-measurements discussed below were recorded at room temperature with a D8 Bruker Advance o

Diffractometer (Cu-Καΐ = 1.54059 A, Johansson primary beam monochromator, position sensitive detector) in transmission mode with rotation of the sample. The measurement time is 2 h. The PXRD pattern in Figure 1 exhibits no isolated peaks but instead a broad halo in the range from 20° to 30°, indicating amorphous material without any regular arrangements.

In Figure 3 the PXRD patterns of the essentially amorphous daclatasvir-2 HC1 freshly prepared (lower curve) and after 4 weeks storage time at 40°C and 75%rh (upper curve) are displayed. It can be deduced from the similarity of the X-ray patterns, that the amorphous form is stable and does not change over time. Hence, a storage stable amorphous form is achieved by the inventive process.

Figures 4 and 5 depict NMR-spectra of the essentially amorphous daclatasvir-2 HC1 freshly prepared (figure 3) and after 4 weeks storage time at 40°C and 75%rh (figure 4). The NMR spectra show a small amount of MeOH, because the amorphous form was precipitated from MeOH. The NMR-spectra remain essentially unaltered, indicating that the purity and the chemical structure of the essentially amorphous daclatasvir-2 HC1 are not affected upon storage.

Figure 6 show a DSC and a TGA spectrum of essentially amorphous daclatasvir-2 HC1 freshly prepared. The DSC-measurements were performed using a Netzsch DSC 204 Fl Phoenix instrument, heating rate 10 K/min, Al-pan in a nitrogen atmosphere. The TGA was measured using a Perkin Elmer Pyris 6 TGA, heating rate 10 K/min, open ceramic-pan in a nitrogen atmosphere. As it can be deduced from the TGA there are two mass changes visible. A first change in the sample weight from beginning of the experiment up to approx. 150°C (first plateau) in the range of approximately 1.96 wt.-%, followed by a second weight change of approx. 15% from 150 up to 290 °C.

Figure 7 displays a DSC thermogram of essentially amorphous daclatasvir-2 HC1 after 4 weeks storage time at 40°C and 75%rh. The sample was heated from -50°C at a rate of lOK/min. No glass transition or melting process is visible and the sample starts to decompose at 170°C. Therefore, this DSC-thermogram is an indicator that the amorphous from is storage stable and does not change into a defined crystalline polymorph.

Figure 8 exhibits a PXRD pattern of daclatasvir-2 HC1 prepared not according to the invention. 100 mg of daclatasvir-2 HC1 was dissolved at 50°C in 2-propanol and seed-crystals were added (approximately 10 wt.-% of the overall sample amount) to the resulting solution. The solvent was evaporated until dryness and the resulting powder was subjected to a PXRD-measurement. As it can be deduced from the diffraction pattern the sample starts to crystallize, resulting in the first appearance of distinct peaks in the pattern (at 2-theta 10-15° and 32-53°).

Figure 9 depicts the PXRD-pattern of essentially amorphous daclatasvir-2 HC1 prepared according to the invention (experimental example see below) before and after a tableting step at various pressures and time intervals. The lowest curve represents the initial essentially amorphous daclatasvir-2 HC1 prepared according to the invention. The curve above (the second) depicts a sample after a tableting step at 5 t for 30 seconds, the third after a tableting step at 10 t for 30 seconds and the uppermost curve after a tableting step at 10 t for 10 minutes. As it can be deduced from a comparison of the patterns there is no significant difference in the structure of amorphous daclatasvir-2 HC1 as a function of a tableting step. This is a clear indicator that the amorphous daclatasvir-2 HC1 structure is not prone to changes in the structure and the arrangement as a function of the tableting processing conditions, assuming a reproducible and stable processing behavior of the essentially amorphous daclatasvir-2 HC1 prepared according to the invention.

Figure 10 exhibits the PXRD patterns of the crystalline N2-form of daclatasvir-2 HC1 before and after a tableting step at various pressures and time intervals. The diffraction pattern with the lowest intensity is recorded from the N2-form as is and the other diffraction patterns (upper curves in ascending intensity) are recorded after subjecting the daclatasvir-2 HC1 polymorph to a pressure step of 5 t for 30 seconds, 10 t for 30 seconds and 10 t for 10 minutes. A comparison of the patterns clearly reveals that the peaks become broader and less defined after the pressure step indicating an uncontrolled loss of crystallinity. An analysis of the full width of half maximum (FWHM) of the pressurized vs. the non-pressurized daclatasvir-2 HC1 reveals for the peak at 10° an increase from 0.194° (initial) up to 0.261°, 0.300° and 0.242° FWMH; for the peak at approx. 15.5° an increase from 0.222° (initial) up to 0.313°, 0.323°, 0.303° FWHM; and for the peak at 24° an increase from 0.251° (initial) up to 0.349°, 0.353° and 0.309° FWMH. Such increase in FWHM is a clear indicator that the crystalline forms of daclatasvir-2 HC1 are pressure sensitive and that the crystal structure may be subject to change as a function of the processing conditions during tableting.

Experimental Examples

Synthesis

I. Preparation of compound (2)

2-Bromo-l-[4-[4-(2-bromoacetyl)phenyl]phenyl] ethanone

0.5 g (2.09 mmol. 1 eq) of l-[4-(4-acetylphenyl)phenyl]ethanone (1) was suspended in 6 ml of glacial acetic acid and heated to 50°C. A solution consisting of 0.66 g (4.18 mmol 2 eq) bromine in 4 ml glacial acetic acid was then added dropwise at 50°C during 3 h. After completion of the bromine addition the reaction mixture was cooled to room temperature and a yellowish precipitate was filtered off and washed with 3 ml glacial acetic acid. Recrystallization in 15 ml THF yielded 0.44 g (56%, 1.12 mmol) off white crystals of (2). 'H NMR (300 MHz, DMSO/CC1₄ -1/3, ppm) δ

4.70 (4H, s, COCH₂), 7.81-7.86 (4H, m, C₆H₄), 8.09-8.14 (4H, m, C₆H₄). ¹³C NMR (75 MHz, DMSO/CC1₄ -1/3, ppm) δ 31.8 (CH₂), 127.0 (CH), 129.2 (CH), 133.2, 143.6, 190.0 (CO). Melting point 215°C.

II. Preparation of compound (7)

Tert-butyl (2S)-2-(l -aminovinyl)pyrrolidine-1 -carboxylate

Compound (7) is prepared starting from L-Proline (3) via a 5-step synthesis [(3)->(4)—>(5)—>(6)—>(7a)—>(7)]. Within this example a Boc-group is chosen as protecting group Z.

Ila. Preparation of compound (4) (2S)-1 -tert-butoxycarbonylpyrrolidine-2-carboxylic acid

0.5 g (4.3 mmol, 1 eq.) of L-Proline (3) was dissolved in a mixture of 7.5 ml of water and 7.5 ml of ethanol. Then, 2.9 g (34.5 mmol, 8 eq.) of sodium bicarbonate and 1.14 g of di-tert-butyldicarbonate (5.2 mmol, 1.2 eq.) were added and the solution was stirred at room temperature for 15 h. After completeness of the reaction the mixture was filtered and the filter cake was washed with additional 4 ml of ethanol. The filtrate was neutralized with 20% citric acid to neutral pH and evaporated to dryness. The resulting colourless crystals were dried in a drying oven at 60°C and 180 mbar for 20 h. The crude compound (4) was transferred without further purification to the next step. Yield of crude compound (4): 1.1 g (5.1 mmol, 118 %, colorless crystals). 'H NMR (300 MHz, DMSO/CC1₄ -1/3, ppm) δ 1.36 (3H, s, CH₃), 1.41 (6H, s, 2CH₃), 1.65-1.75 (1H, m, CH₂), 1.78-1.99 (3H, m, CH₂), 3.23.43 (2H, m, NCH₂), 3.38-3.91 (1H, m, NCH). ¹³C NMR (75 MHz, DMSO/CC1₄ 21

1/3, ppm) δ 23.6 (CH₂), 27.9 (CH₃), 30.11 (CH₂), 45.842 (NCH₂), 58.267 (NCH), 77.988 (O-C=), 173.79 and 173.33 (C=O).Melting point 114°C.

lib. Preparation of compound (5)

7eri-butyl (2S)-2-carbamoylpyrrolidine-1 -carboxylate

10.24 g (47.6 mmol, 1 eq.) of compound (4), 5.73 g (72 mmol, 1.5 eq.) of NH₄HCO₃ and 15.5 g (72 mmol, 1.5 eq.) of di-tert-butyldicarbonate were dissolved in 15 ml of dioxane. While stirring at room temperature a solution of 10.24 ml (129 mmol, 2.7 eq.) of pyridine in 190 ml of dioxane was added dropwise during 1 h to the reaction mixture. After complete addition of pyridine, the mixture was stirred at room temperature for 15 h and filtered. The filter cake was washed with 15 ml of dichloromethane and the filtrate was extracted with 3x30 ml of dichloromethane, and the combined organic phases were washed once with 20% a citric acid solution, once with a 1 M sodium bicarbonate solution and once with a saturated NaCI solution. Drying of the organic phase over MgSO₄ and evaporation resulted in yellowish oil which was further dried in a drying oven for 15 h. The crude compound (5) was transferred to the next step, without further purification. Yield of crude compound (5): 9.77 g (46 mmol, 97 %, oil). 'H NMR (300 MHz, DMSO/CC1₄ -1/3, ppm) δ 1.40 (9H, s, 3CH₃), 1.74-2.19 (4H, m, 2CH₂), 3.25-3.35 (1H, m, NCH₂), 3.38-3.47 (1H, m, NCH₂), 3.93-4.10 (1H, m, NCH), 6.60 (1H, br. NH₂), 6.97 (1H, br. NH₂) lie. Preparation of compound (6)

Teri-butyl (2S)-2-cyanopyrrolidine-1 -carboxylate y—CN \

Z

4.1 g (19 mmol, 1 eq.) of compound (5) were dissolved in 50 ml of THF and 3.86 ml (48 mmol, 2.5 eq.) of pyridine was added to the solution. After having stirred the mixture for 5 min, 6.87 ml (48 mmol, 2.5 eq.) of TFAA was added dropwise to the reaction mixture during 1 h, maintaining a temperature of 25 °C. After the complete addition of TFAA, the reaction mixture was stirred for additional 15 h at room temperature. The reaction mixture was then neutralized with saturated solution of NaHCO₃ to adjust a pH of approximately 8 and extracted with 2x35 ml of EtOAc. The combined organic layers were dried over MgSO₄ and evaporated to dryness. The crude compound (6) 4.1 g (110%, yellowish oil) was transferred to the next step without further purification. 'H NMR (300 MHz, DMSO/CC14 -1/3, ppm) δ 1.48 (9H, s, CH₃), 1.96-2.10 (2H, m, CH₂), 2.14-2.31 (2H, m, CH₂), 3.31 (1H, dt, J₁₌10.3, J₂=7.7, NCH₂), 3.44 (1H, ddd, J₁₌10.3, J₂=6.6, J₃=5.1, NCH₂), 4.44-4.59 (1H, m, NCH). ¹³C NMR (75 MHz, DMSO/CC1₄ -1/3, ppm) δ 24.1 (CH₂), 27.7 (CH₃), 31.0 (CH₂), 45.5 (NCH₂), 46.4 (NCH), 79.4 (O-C=), 118.5 (CN), 152.5 (C=O).

lid. Preparation of compound (7) via compound (7a)

Teri-butyl (2S)-2-(l-aminovinyl)pyrrolidine-l-carboxylate (7) via Methyl (2S)-l-(3,3-dimethylbutanoyl)pyrrolidine-2-carboximidate (7a)

4.1 g of crude compound (6) from the previous step was dissolved in 130 ml MeOH and 3.4 g (57 mmol) NaOMe was added. After having stirred the mixture at room temperature for 5 h, 3.3 ml of AcOH and lg (19 mmol) of NH₄C1 was added and stirring at room temperature was continued for additional 15 h. The reaction volume was concentrated to 2/3 of the initial amount and 30 ml of a saturated NaHCO₃ solution was added to adjust the acidic solution to pH = 7. The reaction mixture was then extracted with 2x50 ml of EtOAc, dried over MgSO₄ and evaporated to dryness. The resulting yellowish mass was further dried in a drying oven, yielding 0.96 g of dry crude compound (7) which was recrystallized from MeOH. Yield 0.9 g (21.5 %, white crystals). 'H NMR (300 MHz, DMSO/CC1₄ -1/3, ppm) δ 1.43 (9H, s, 3CH₃), 1.83-1.99 (3H, m, CH₂), 2.26-2.40 (1H, m, CH₂), 3.28-3.43 (1H, m, NCH₂), 3.513.63 (1H, m, NCH₂), 4.30-4.39 (1H, m, NCH), 9.49 (4H, br. NH, NH₂, HC1). ¹³C NMR (75 MHz, DMSO/CC1₄ -1/3, ppm) δ 23.24 (CH₂), 24.66 (CH-CH₂), 27.8 (3CH₃), 46.85 (N-CH₂), 57.67 (N-CH), 79.5 (C-(CH₃)₃), 171.67 (C=NH), 172.24 (C=O). Melting point 194-195°C.

III. Preparation of compound (12)

Methyl [(2S)-1 -{(2S)-2-[4-(4’-{2-[(2S)-l-{(2S)-2-[(methoxycarbonyl)amino]-3methylbutanoyl} -2-pyrrolidinyl] -1 H-imidazol-4-yl} -4-biphenylyl)-1 H-imidazol2-yl] -1 -pyrrolidinyl} -3 -methyl-1 -oxo-2-butanyl]carbamate

Ilia. Preparation of compound (8) by reacting (7) and (2) ieri-Butyl (2S)-2- [4-(4'- {2- [(2S)-1 -tert-butoxycarbonylperhydro-1 H-pyrrol-2-yl] 3H-imidazol-4-yl} -4-biphenylyl)-3H-imidazol-2-yl]perhydro-1 H-pyrrole-1 carboxylate dihydrochloride

0.21 g (0.756 mmol, 2.5 eq.) of (7) and 0.8 g (8 mmol, 10 eq.) of potassium bicarbonate were dissolved in a mixture consisting of 3 ml of dioxane and 2 ml of water. After having heated the solution to reflux, a solution of 0.12 g (0.3 mmol, 1 eq.) (2) in 15 ml dioxane was added dropwise over 1 h into the reaction mixture and refluxing was continued for additional 6 h until all of the starting materials were consumed. The mixture was cooled to room temperature and evaporated to dryness. The product was then extracted from the solid with 10 ml of chloroform, separated from residual solids by decantation and evaporated to dryness. The solid was then dissolved again in MeOH and upon slow evaporation under ambient conditions, yellow crystals were formed. 'Η NMR (300 MHz, DMSO/CC1₄ -1/3, ppm) δ 1.24 (9H, br. S, 3-CH₃); 1.45 (9H, br. s,3- CH₃); 1.82-2.40 (8H, m, β, γ-Cfh, Pro.); 3.36-3.46 (2H, m) and 3.51-3.68 (2H, m, 5-CH₂, Pro.); 4.80-4.91 (2H, m, α-CH, Pro); 7.24 (2H, s, =CHNH); 7.55-7.61 (4H, m) and 7.71-7.77 (4H, m, C₆H₄); 11.59 (2H, br. s, NH). ¹³C

NMR (75 MHz, DMSO/CC1₄ -1/3, ppm) δ 23.0 and 23.9 (y-CH₂, Pro.), 27.8 and 28.0 (CH₃), 30.6 and 33.1 (β-(ΖΗ₂, Pro.), 46.1 and 46.2 (5-CH₂, Pro), 54.0 and 55.1 (α-CH, Pro.); 77.9 and 77.2 (OC); -114 (br.); 124.4 and 125.9 (CH, C₆H₄); 132.6 (br.); -137 (br.); 137.6 (CHNH); 149.1 and 150.2; 153.0 and 153.7.

Illb. Preparation of (9)

2-|(2.S')-Pcihydi'o- l//-pyrrol-2-yl] -5-(4'-{2-[(2>Sj-perhydro-1//-pyrrol-2-yl] -3//imidazol-4-yl} -4-biphenylyl) -1 //-imidazole

Boc deprotection of (8) was carried out in-situ by adding 1 ml of 1 M HCI and heating the solution for an additional hour at 50°C. During this process off-white crystals are obtained which were filtered off. Yield 0.1 g (100%, crude crystals). 'H NMR (300 MHz, DMSO/CC1₄-1/3, ppm) δ 1.97-2.11 (2H, m, CH₂); 2.12-2.29 (2H, m, CH₂); 2.32-2.56 (4H, m,CH₂); 3.22-3.49 (4H, m, CH₂), 3.51 (2H, br,NH); 4.96 (2H, t, J=8.1, CH); 7.83-7.89 (4H, m) and 7.95-8.00 (4H, m, C₆H₄) =CHNH); 8.03 (2H, s, =CH), 9.58 (2H, br., HCI), 10.20 (2H, br., s„ NH).

IIIc. Preparation of (12) by reacting (9) and (11)

IIIcl Pre-step: Preparation of (11) via compound (10) (2S)-2-(methoxycarbonylamino)-2,3-dimethyl-butanoic acid

O O

2.5 g (21 mmol, 1 eq.) of L-Valine (10) was dissolved in 60 ml of absolute MeOH. While stirring at room temperature, a solution consisting of 3.25 g (30 mmol, 1.4 eq.) dimethyldicarbonate dissolved in 15 ml of absolute MeOH was added dropwise into the reaction mixture and stirring was continued for 35 h. After completion of the reaction the solvent was evaporated to dryness. Yield 3.93 g of crude (11) (140 %, crude product, colourless amorphous solid). Drying in drying oven yielded 3.81 g of crude (11) (135.5%). Recrystallization of crude (11) from hexane:EtOAc:2.5:l mixture provided pure (11) as colourless crystals. First crop yield 2.18 g (57%). Second crop yielded 0.36 g (9.5%) with m.p. 103.5°C. 'H NMR (300 MHz, DMSO/CCU -1/3, ppm) δ 0.90 (3H, d, j=6.9 CHCHfr, 0.94 (3H, d, j=6.9, CHCHfr, 2.0-2.17 (1H, M, CHCHA 3.59 (3H, s, OCH3), 3.91 (1H, dd, j₁₌8.6, j₂=5.5, NHCH), 6.56 (1H, j=8.6, NH), 12.28 (1H, m, OH). ¹³C NMR (75 MHz, DMSO/CCU 1/3, ppm) δ 17.56 (CH₃), 18.86 (CH₃), 29.79 (CH(CH₃)₂), 50.98 (O-CH3), 58.82 (CH-NH), 156.24 (COOMe), 172.84 (COOH). Melting point 106 °C.

IIIc2 Preparation of (12) g (6.57 mmol, 2.5 eq) hydroxybenzotriazole hydrate, 1.11 g (6.32 mmol, 2.4 eq) N-(methoxycarbonyl)-L-valine (11) and 1.21g (6.32 mmol, 2.4 eq) 1-(3dimethyaminopropyl)-3-ethylcarbodiimide hydrochloride were dissolved in 14.7 ml of acetonitrile. The resulting solution was agitated at 20 °C for 1 hour and charged with 1.5 g (2.63 mmol, 1 eq) of 9. The slurry was cooled to about 0 °C and 1.36 g (10.5mmol, 4 equiv) diisopropylethylamine were added over 30 minutes while maintaining a temperature below 10 °C. The solution was slowly heated to 15 °C over 3 hours and held at 15 °C for 12 hours. The resulting solution was charged with 8.82 ml 13 wt % aqueous NaCI and heated to 50 °C for 1 hour. After cooling to 20 °C, 7.35 ml of isopropyl acetate was added. The biphasic solution was filtered through a 0.45 pm filter and the aqueous and organic phases were separated. The rich organic phase was washed with 2x17.65 ml of a 0.5N NaOH solution containing 13 wt % NaCI followed by 8.82 ml 13 wt % aqueous NaCI. The mixture was then solvent exchanged into isopropyl acetate by vacuum distillation with a target volume of 29.5 ml. The resulting turbid solution was cooled to 20 °C and filtered through a 0.45 pm filter. The clear solution was then solvent exchanged into ethanol by vacuum distillation with a target volume of 10.3 ml. While maintaining a temperature of 50° C, 4.88 ml (6.05 mmol, 2.3 eq) of a 1.24 M HCI solution in ethanol was added.

Precipitation of the amorphous form

100 mg daclatasvir-2 HC1 prepared according to the invention was dissolved for approximately 5 minutes under gentle stirring in 4 ml methanol. The resulting optically clear solution was filtered through a filter of 0.2 pm pore-size into a crystallizing dish and after the filtration step the optically clear solution was immediately (no waiting period) subjected to drying at room temperature. The resulting dry solid product (solvent content <0.1 %) was brittle, easy to crush and a yield of 81% was achieved. The amorphous product is 99% pure.

mg daclatasvir-2 HC1 prepared according to the invention was dissolved for approximately 5 minutes under gentle stirring in 3 ml MEK. The resulting optically clear solution was filtered through a filter of 0.2 pm pore-size into a crystallizing dish and after the filtration step the optically clear solution was immediately (no waiting period) subjected to drying at room temperature. The resulting dry solid product was brittle, easy to crush and a yield of 80% was achieved. The amorphous product is 99% pure.

mg daclatasvir-2 HC1 prepared according to the invention was dissolved at 50°C for approximately 5 minutes under gentle stirring in 3 ml THF. The resulting optically clear solution was filtered through a filter of 0.2 pm pore-size into a crystallizing dish and after the filtration step the optically clear solution was immediately subjected to drying at 50°C. The resulting dry solid product was brittle, easy to crush and a yield of 85% was achieved. The amorphous product is 99% pure.

mg daclatasvir-2 HC1 prepared according to the invention was dissolved at 50°C for approximately 5 minutes under gentle stirring in 3 ml water. The resulting optically clear solution was filtered through a filter if 0.2 pm pore-size into a crystallizing dish and after the filtration step the optically clear solution was immediately subjected to drying at 50°C. The resulting dry solid product was brittle, easy to crush and a yield of 88% was achieved. The amorphous product is 99% pure.

Claims (25)

What is claimed:

1) Process for the preparation of a compound according to the following formula (13):

a) reacting a compound (2) comprising the leaving groups LG O and a compound (7) wherein X is a halide ion and Z is a nitrogen protecting group, in order to obtain compound (8)

b) subjecting compound (8) to a deprotecting agent and

c) reacting the product of step b) and compound (11)

Ο in order to obtain compound (13).

2) Process according to claim 1, wherein the nitrogen protecting group Z is selected from the group consisting of Boc, Cbz, Tosyl, Mesyl, Benzyl, Fmoc, substituted or unsubstituted Acetyl, Benzoyl, Tolyl.

3) Process according to any of the preceding claims, wherein the anion X of compound (7) is selected from the group consisting of F , Cl, Br .

4) Process according to any of the preceding claims, wherein the leaving group LG is selected from the group consisting of -F, -Cl, -Br, -I, -OH, -NH2, mesylate, triflate, tosylate, diazonium salts, alkyl- or aryl sulfonates, phosphates, phosphonic acids or phosphonic esters.

5) Process according to any of the preceding claims, wherein compound (7) is prepared via the following route of synthesis:

a) reacting L-Proline and a nitrogen protecting agent in order to obtain the nitrogen protected compound (4)

b) reacting compound (4) and an amination reagent in order to obtain compound (5)

c) reacting compound (5) and an acid in a pharmaceutically acceptable solvent in order to obtain compound (6)

d) reacting compound (6) and a C1-C6 alcoholate in order to obtain compound (7a) wherein R1 is a C1-C6 alkyl.

e) reacting compound (7a) and an amination reagent in order to obtain compound (7)

6) Process according to claim 5, wherein in step e) the amination is achieved within a two-step procedure, wherein in a first step compound (7a) is subjected to a pharmaceutically acceptable acid and an ammonium-halide salt and, in a second step, the resulting solution is neutralized by a base.

7) Process according to claim 5 or 6, wherein the reaction of step d) and e) is performed in a one-pot synthesis without isolation of the intermediate compound (7a).

8) Process according to any of the preceding claims, wherein compound (13) is treated with aqueous hydrochloric acid solution to achieve daclatasvir-2 HCI.

9) Process for the production of amorphous daclatasvir-2 HCI, at least comprising the steps of

a) Contacting daclatasvir or daclatasvir-2 HCI and a pharmaceutically acceptable solvent,

b) filtering off the solution or dispersion of step a),

c) immediate removal of the solvent from the filtered solution or dispersion of step b), characterized in that the solvent removal is performed within a timescale of > 10 seconds and < 5 h after the filtering step b).

10) Process according to claim 9, wherein the daclatasvir-2 HCI is prepared according to a process of any of the claims 1-8.

11) Process according to any of the claims 9-10, wherein the filtered solution is essentially free of undissolved daclatasvir-2 HC1.

12) Process according to any of the claims 9-11, wherein the pharmaceutically acceptable solvent is selected from the group consisting of water, acetonitrile, methanol, ethanol, 1-propanol, acetone, MEK, ethylacetate, 1,4-Dioxan, THF, MIBK or mixtures thereof.

13) Process according to any of the claims 9-12, wherein the filtering step b) is performed by passing the solution or dispersion through a filter comprising a pore size of < 0.4 pm.

14) Process according to any of the claims 9-13, wherein the solution or dispersion of step a) is heated to a temperature of > 20 °C and < 65 °C.

15) Process according to any of the claims 9-14, wherein the solvent removal is performed at room temperature.

16) Process according to any of the claims 9-15, wherein the contacting in step a) is performed under stirring for a time interval of > 10 minutes and < 6 days.

17) Process according to any of the claims 9 - 16, wherein the solvent removal in step c) is performed at the same temperature as the contacting of step a).

18) Amorphous daclatasvir-2 HC1 prepared according to a process according to any of the claims 9 17, characterized in that the daclatasvir-2 HC1 is essentially amorphous and exhibits no characteristic peaks in a powder X-Ray diffraction pattern.

19) Amorphous daclatasvir-2 HC1, characterized in that the daclatasvir-2 HC1 is essentially amorphous and exhibits no characteristic peaks in a powder X-Ray diffraction pattern.

20) Amorphous daclatasvir-2 HC1 prepared according to a process according to any of the claims 9 17, characterized in that the daclatasvir-2 HC1 is essentially amorphous and exhibits no characteristic peaks in a powder X-Ray diffraction pattern at 2-theta values of 10.3+0.1, 12.4+0.1, 12.8+0.1, 13.3+0.1, 13.6+0.1, 15.5+0.1, 20.3+0.1, 21.2+0.1, 22.4+0.1, 22.7+0.1 and 23.7°+0.1°.

21) Amorphous daclatasvir-2 HC1 according to any of the claims 18 - 20, wherein the daclatasvir-2 HC1 is essentially free of solvents.

22) A pharmaceutical composition, comprising essentially amorphous daclatasvir-2 HC1 according to any of the claims 18-21.

23) Pharmaceutical composition according to claim 22, wherein the composition further comprises another direct antiviral agent selected from the group consisting of boceprevir, telaprevir, simeprevir, faldaprevir, asunaprevir, danoprevir, sofosbuvir, mericitabin, deleobuvir, setrobuvir, ledipasvir, meravirsen or mixtures thereof.

24) Pharmaceutical composition according to any of the claims 22-23, wherein the composition is an oral dosage form.

25) Use of a pharmaceutical composition according to any of the claims 22 - 24 for the treatment of HCV infections.

Intellectual

Property

Office

Application No: GB1520346.6 Examiner: Mr Aaron Butt