WO2016193919A1

WO2016193919A1 - Polymorphic form of ledipasvir

Info

Publication number: WO2016193919A1
Application number: PCT/IB2016/053215
Authority: WO
Inventors: Ramakoteswara Rao Jetti; Amit Singh; Satish BEERAVELLY; Sreenivasarao PATHURI; Nagadurgarao BANDI
Original assignee: Mylan Laboratories Ltd
Current assignee: Mylan Laboratories Ltd
Priority date: 2015-06-05
Filing date: 2016-06-01
Publication date: 2016-12-08
Anticipated expiration: 2017-12-05

Abstract

Novel stable polymorph of ledipasvir designated as form-M1 is disclosed as well as processes for the preparation thereof.

Description

POLYMORPHIC FORM OF LEDIPASVIR

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the earlier filing date of Indian Provisional Patent Application No. 2181/MUM/2015 filed on June 05, 2015.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present disclosure relates generally to active pharmaceutical ingredients and more specifically to a polymorph of ledipasvir designated as form-Mi. The present disclosure also relates to processes for the preparation of ledipasvir form-Mi.

BACKGROUND OF THE INVENTION

Ledipasvir (sometimes known as GS-5885) is an inhibitor of the hepatitis C virus NS5A protein. A combination drug containing ledipasvir and sofosbuvir is currently marketed as HARVONI^® in the United States by Gilead Sciences. HARVONI^® is indicated for the treatment of hepatitis C virus (HCV) genotype 1, 4, 5, or 6 infection. Ledipasvir is chemically known as methyl[(2S)-l-{(6S)-6-[5-(9,9-difluoro-7-{2-

[(lR,3S,4S)-2-{(2S)-2-[(methoxycarbonyl)amino]-3-methylbutanoyl}-2-azabicyclo

[2.2.1]hept-3-yl]-lH-benzimidazol-6-yl}-9H-fluoren-2-yl)-lH-imidazol-2-yl]- 5-azaspiro[2.4]hept-5-yl}-3-methyl-l-oxobutan-2-yl]carbamate and is represented by the following chemical structure:

PCT publication No. WO2013184698A1 discloses various crystalline forms of ledipasvir as well as an amorphous form of ledipasvir.

SUMMARY OF THE INVENTION

One aspect of the present invention provides crystalline ledipasvir form-Ml. Within the context of the present invention, crystalline ledipasvir form-Ml may be characterized by a PXRD pattern having peaks at 2Θ angle positions of 7.1, 9.6, 13.7, 18.2, 20.5 + 0.2 °.

Within the context of the present invention, the crystalline ledipasvir form-Ml may be further characterized by a PXRD pattern as shown in Figure 1. Within the context of the present invention, the crystalline ledipasvir form-Ml may be further characterized by a 13 C-SSNMR spectrum having chemical shift peaks at 125.6, 113.9, and 63.6 ppm + 0.2 ppm.

Within the context of the present invention, crystalline ledipasvir form-Ml may have a degree of crystallinity below 60%. In some embodiments, the crystalline ledipasvir form- Ml has a degree of crystallinity below 30%.

Another aspect of the present invention provides processes for the preparation of ledipasvir form-Ml.

In one embodiment, ledipasvir form-Ml may be prepared by the following steps: a) dissolving ledipasvir in a solvent or mixtures of solvents to form a solution; b) optionally seeding the solution with ledipasvir form-Ml; and

c) isolating ledipasvir form-M 1.

Within the context of this embodiment, the solvent may be, for example, methanol, ethanol, isopropanol, 1-butanol, isobutyl alcohol, 1-pentanol, water, acetonitrile, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethyl sulfoxide, acetone, N-methylpyrrolidone, or mixtures thereof.

In another embodiment, ledipasvir form-Ml may be prepared by the following steps: a) dissolving ledipasvir in an organic solvent at an elevated temperature to form a solution;

b) optionally seeding the solution with form-Ml;

c) adding an anti-solvent to the solution; and

d) isolating ledipasvir form-M 1.

Within the context of this embodiment, the organic solvent may be, for example, a polar protic solvent, a polar aprotic solvents, or mixtures thereof.

Examples of suitable polar protic solvents include methanol, ethanol, isopropanol, 1- butanol, isobutyl alcohol, 1-pentanol, and mixtures thereof.

Examples of polar aprotic solvents include acetonitrile, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethyl sulfoxide, acetone, N- methylpyrrolidone, and mixtures thereof.

Within the context of this embodiment, the anti-may be, for example, an ether solvent, a hydrocarbon solvent, water, or mixtures thereof.

In some embodiments, the ether solvent is isopropyl ether.

In some embodiments, the hydrocarbon solvent is heptane.

In yet another embodiment, ledipasvir form-Ml may be prepared by the following steps: a) dissolving ledipasvir in an organic solvent at an elevated temperature to get a first solution;

b) suspending seeds of ledipasvir form-Ml in an anti-solvent to get a seeded anti- solvent solution;

c) adding the first solution to the seeded anti-solvent solution; and

d) isolating ledipasvir form-M 1.

Examples of suitable polar protic solvents include methanol, ethanol, isopropanol, 1- butanol, isobutyl alcohol, 1-pentanol, and mixtures thereof. Examples of polar aprotic solvents include acetonitrile, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethyl sulfoxide, acetone, N- methylpyrrolidone, and mixtures thereof.

In some embodiments, the ether solvent is isopropyl ether.

In some embodiments, the hydrocarbon solvent is heptane.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure together with additional features contributing thereto and advantages accruing there from will be apparent from the following description of embodiments of the disclosure which are shown in the accompanying drawing figures wherein:

FIG. 1 is a powder X-ray diffraction (PXRD) pattern of ledipasvir form-Ml. FIG. 2 is a differential scanning calorimetry (DSC) thermogram of ledipasvir form-Ml. FIG. 3 is a ¹³C solid-state NMR (¹³C SSNMR) spectrum of ledipasvir form-Ml.

FIG. 4 shows 13 C SSNMR spectra of ledipasvir form-Ml and of amorphous ledipasvir.

FIG. 5 shows modulated DSC thermograms of ledipasvir form-Ml and of amorphous ledipasvir.

DETAILED DESCRIPTION OF THE INVENTION One aspect of the present invention provides crystalline ledipasvir form-Ml.

Another aspect of the present invention provides a process for the preparation of ledipasvir form-Ml.

In one embodiment, ledipasvir form-Ml may be prepared by the following steps: a) dissolving ledipasvir in a solvent or mixtures of solvents to get a solution; b) optionally seeding the solution with ledipasvir form-Mi; and

c) isolating ledipasvir form-M 1.

According to this embodiment, ledipasvir may be dissolved a solvent or mixtures of solvents to get a solution. Within the context of this embodiment, the solvent may be a polar protic or polar aprotic solvent. Examples of suitable polar protic solvents include methanol, ethanol, isopropanol, 1-butanol, isobutyl alcohol, 1-pentanol, water, and mixtures thereof. Examples of suitable polar aprotic solvents include acetonitrile, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethyl sulfoxide, and mixtures thereof. In some embodiments, a mixture of acetonitrile and water is used as the solvent. One of skill in the art will recognize numerous polar aprotic and polar protic solvents that may be useful within the context of this embodiment.

According to this embodiment, this may be carried out at an elevated temperature. For example, in some embodiments, ledipasvir is dissolved in mixture of solvents at a temperature of about 50 °C to about 70 °C.

Next, the solution may be cooled. For example, the solution may be cooled to ambient temperature. In some embodiments, the solution is slowly cooled to about 35 °C to about 45 °C.

Next, the solution may be optionally seeded with ledipasvir form-M 1. In some embodiments, it is found that stirring the solution after seeding is particularly useful.

Next, ledipasvir form-M 1 may be isolated. This may be carried out according to methods well known in the art, for example, by filtering the solution to obtain a solid. The solution may be optionally further cooled prior to isolation of ledipasvir form-M 1. For example, in some embodiments, the reaction mixture is cooled to about 20 °C to about 25 °C and maintained at that temperature for about 10 hours to about 13 hours.

After being obtained, the solid may be further treated, for example, by drying under vacuum to get ledipasvir form-M 1. One of skill in the art would recognize a variety of methods that can be used to isolate the solid. In another embodiment, ledipasvir form-Mi may be prepared by the following steps: a) dissolving ledipasvir in an organic solvent at an elevated temperature to get a solution;

b) optionally seeding the solution with ledipasvir form-Mi;

c) optionally adding an anti-solvent to the solution; and

d) isolating ledipasvir form-M 1.

According to this embodiment, ledipasvir may be dissolved in an organic solvent at an elevated temperature to get a solution. For example, in some embodiments, ledipasvir is dissolved in an organic solvent at a temperature of about 50 °C to about 75 °C.

Within the context of this embodiment, the organic solvent may be a polar protic or polar aprotic solvent. Examples of suitable polar protic solvents include methanol, ethanol, isopropanol, 1-butanol, isobutyl alcohol, 1-pentanol, and mixtures thereof. Examples of suitable polar aprotic solvents include acetonitrile, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethyl sulfoxide, acetone, N-methylpyrrolidone, isopropyl ether, and mixtures thereof. In some embodiments, acetonitrile, methanol, dimethyl sulfoxide, isopropyl ether, or mixtures thereof are used as an organic solvent. One of skill in the art will recognize numerous polar aprotic and polar protic solvents that may be useful within the context of this embodiment.

In some embodiments, the solution may then be cooled. For example, the solution may be cooled to ambient temperature, or to a temperature of about 20 °C to about 35 °C. In some embodiments, it is found that stirring the solution at this temperature for about 10 hours to about 15 hours is particularly useful.

Next, the solution may be optionally seeded with ledipasvir form-M 1.

Next, an anti- solvent may optionally be added. In some embodiments, it is found that adding the anti-solvent slowly is particularly useful and effective at producing the desired solid product.

Within the context of the present embodiment, the steps of optionally seeding and optionally adding an anti-solvent may occur in any order. For example, in some embodiments, the solution may be seeded followed by addition of an anti- solvent. In other embodiments, anti-solvent may be added followed by the addition of seeds of ledipasvir form-Mi.

Within the context of this embodiment, the anti-solvent may be an ether solvent (for example, isopropyl ether), a hydrocarbon solvent (for example, heptane), water, or mixtures thereof.

Next, ledipasvir form-Mi may be isolated. This may be carried out according to methods well known in the art, for example, by filtering the solution to obtain a solid. One of skill in the art would recognize a variety of methods that can be used to isolate the solid.

After being obtained, the solid may be further treated, for example, by drying under vacuum to get ledipasvir form-M 1.

In another embodiment, ledipasvir form-Mi may be prepared by the following steps: a) dissolving ledipasvir in an organic solvent to get a solution;

b) optionally heating the solution;

c) optionally cooling the solution;

d) optionally adding an anti-solvent;

e) isolating ledipasvir form-M 1.

According to this embodiment, ledipasvir may be dissolved in an organic solvent.

In some embodiments, this may be carried at an elevated temperature to get a solution. For example, in some embodiments, ledipasvir is dissolved in an organic solvent at a temperature of about 50 °C to about 75 °C.

In other embodiments this step may be carried out at ambient temperatures.

In yet other embodiments, the solution may be heated after dissolving the ledipasvir in the organic solvent at ambient temperature. For example, in these embodiments, it has been found to be particularly useful to heat the solution to about 110 °C to about 130 °C. Within the context of this embodiment, the organic solvent may be a polar protic or polar aprotic solvent. Examples of suitable polar protic solvents include methanol, ethanol, isopropanol, 1-butanol, isobutyl alcohol, 1-pentanol, and mixtures thereof. Examples of suitable polar aprotic solvents include acetonitrile, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethyl sulfoxide, acetone, N-methylpyrrolidone, isopropyl ether, and mixtures thereof. In some embodiments, acetonitrile, methanol, dimethyl sulfoxide, isopropyl ether, or mixtures thereof are used as an organic solvent. One of skill in the art will recognize numerous polar aprotic and polar protic solvents that may be useful within the context of this embodiment.

In some embodiments where the solution was held at an elevated temperature or the solution was heated, the solution may then be cooled. For example, the solution may be cooled to ambient temperature, or to a temperature of about 20 °C to about 35 °C. In some embodiments, it is found that stirring the solution at this temperature for about 10 hours to about 15 hours is particularly useful.

In other embodiments, it is found that cooling the solution to about 60 °C to about 100 °C before carrying out the next step of optionally adding the anti-solvent is useful. After the anti-solvent is added, further cooling the solution to ambient temperature, or about 20 °C to about 35 °C, may be carried out.

If the first step of dissolving ledipasvir was carried out at ambient temperature and no further heating was carried out, no cooling of the solution is necessary.

After being obtained, the solid may be further treated, for example, by drying under vacuum to get ledipasvir form-Mi. In yet another embodiment, ledipasvir form-Mi may be prepared by the following steps: a) dissolving ledipasvir in an organic solvent at an elevated temperature to get a solution;

b) optionally seeding the solution with ledipasvir form-Mi;

c) adding an anti- solvent to the solution; and

d) isolating ledipasvir form-M 1.

According to this embodiment, ledipasvir may be dissolved in an organic solvent at an elevated temperature. For example, in some embodiments, ledipasvir is dissolved in an organic solvent at a temperature between about 50 °C to about 75 °C.

Within the context of this embodiment, the organic solvent may be a polar protic or polar aprotic solvent. Examples of suitable polar protic solvents include methanol, ethanol, isopropanol, 1-butanol, isobutyl alcohol, 1-pentanol, and mixtures thereof. Examples of suitable polar aprotic solvents include acetonitrile, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethyl sulfoxide, acetone, N-methylpyrrolidone, and mixtures thereof. In some embodiments, acetonitrile is used as an organic solvent. One of skill in the art will recognize numerous polar aprotic and polar protic solvents that may be useful within the context of this embodiment.

In some embodiments, the solution may then be cooled, for example, to ambient temperature, or to a temperature of about 25 °C to about 30 °C.

The solution may then optionally be seeded with ledipasvir form-M 1.

According to this embodiment, an anti-solvent may then be added to the solution. In some embodiments, it is found that adding the anti- solvent slowly at a temperature of about 25 °C to about 30 °C is particularly useful. In some embodiments, it is further found that stirring the solution is particularly useful. For example, in some embodiments, the solution is stirred at about 20 °C to about 35 °C for about 10 hours to about 15 hours.

Within the context of this embodiment, the anti-solvent may be an ether solvent (for example, isopropyl ether), a hydrocarbon solvent (for example, heptane), water, or mixtures thereof. Next, ledipasvir form-Mi may be isolated. This may be carried out according to methods well known in the art, for example, by filtering the solution to obtain a solid. One of skill in the art would recognize a variety of methods that can be used to isolate the solid.

In yet another embodiment, ledipasvir form-M 1 may be prepared by the following steps: a) dissolving ledipasvir in an organic solvent at an elevated temperature to get a first solution;

b) suspending seeds of ledipasvir form-Mi in an anti-solvent to get a seeded anti- solvent solution;

c) adding the first solution to seeded anti-solvent solution; and

d) isolating ledipasvir form-M 1.

According to this embodiment, ledipasvir may be dissolved in an organic solvent to get a solution. Within the context of this embodiment, the organic solvent may be a polar protic or polar aprotic solvent. Examples of suitable polar protic solvents include methanol, ethanol, isopropanol, 1-butanol, isobutyl alcohol, 1-pentanol, and mixtures thereof. Examples of suitable polar aprotic solvents include acetonitrile, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethyl sulfoxide, acetone, N-methylpyrrolidone, and mixtures thereof. In some embodiments, acetonitrile is used as an organic solvent. One of skill in the art will recognize numerous polar aprotic and polar protic solvents that may be useful within the context of this embodiment.

In some embodiments, the solution may then be cooled, for example, to ambient temperature, or to a temperature of about 25 °C to about 30 °C. In a separate reaction vessel, seeds of ledipasvir form-M 1 may be added to an anti- solvent. Within the context of this embodiment, the anti-solvent may be an ether solvent (for example, isopropyl ether), a hydrocarbon solvent (for example, heptane), water, or mixtures thereof. Next, the first solution may be added to the seeded anti-solvent solution. This step may be carried out at ambient temperature, for example, at about 25 °C to about 30 °C. In some embodiments, it is found that adjusting the temperature to about 20 °C to about 35 °C and stirring for about 10 hours to about 15 hours is particularly useful.

After obtaining a solid, the solid may be further treated, for example, by drying under vacuum to get ledipasvir form-Mi.

In yet a further embodiment, ledipasvir form-Mi may be prepared by the following steps:

a) dissolving ledipasvir in an organic solvent at an elevated temperature to get a solution;

b) adding an anti- solvent to the solution;

c) optionally seeding the solution with ledipasvir form-Mi;

d) isolating ledipasvir form-M 1.

According to this embodiment, ledipasvir may be dissolved in an organic solvent at an elevated temperature. For example, in some embodiments, ledipasvir is dissolved in an organic solvent at a temperature of about 50 °C to about 65 °C.

Within the context of this embodiment, the organic solvent may be a polar protic or polar aprotic solvent. Examples of suitable polar protic solvents include methanol, ethanol, isopropanol, 1-butanol, isobutyl alcohol, 1-pentanol, and mixtures thereof. Examples of suitable polar aprotic solvents include acetonitrile, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethyl sulfoxide, acetone, N-methylpyrrolidone, and mixtures thereof. In some embodiments, methanol is used as an organic solvent. One of skill in the art will recognize numerous polar aprotic and polar protic solvents that may be useful within the context of this embodiment.

An anti-solvent may then be added to the solution. In some embodiments, it is found that adding the anti-solvent slowly and that stirring the solution is particularly useful. For example, in some embodiments, the solution is stirred for about 10 minutes to about 15 minutes.

In some embodiments, the solution may then be cooled. For example, in some embodiments, the solution is cooled to ambient temperature. In other embodiments, cooling the solution to a temperature of about 35 °C to about 45 °C was found to be particularly useful. The solution may then be optionally seeded with ledipasvir form-Ml. In some embodiments, it is found that stirring the solution after seeding, for example, for about 1 hour to about 2 hours at the same temperature is useful.

Next, ledipasvir form-Ml may be isolated. This may be carried out according to methods well known in the art, for example, by filtering the solution to obtain a solid. One of skill in the art would recognize a variety of methods that can be used to isolate the solid. In some embodiments, further cooling the solution before isolating ledipasvir form-Ml is found to be useful. For example, in some embodiments, the mixture is cooled to about 20 °C to about 30 °C and stirred for about 15 hours to about 16 hours.

After obtaining a solid, the solid may be further treated, for example, by drying under vacuum to get ledipasvir form-Ml. For example, in some embodiments, the obtained solid is dried under vacuum at about 50 °C to about 60 °C for about 7 hours to about 9 hours to get ledipasvir form-Ml.

The ledipasvir material that is dissolved in a solvent may be crystalline or amorphous and may be prepared by any prior-art process. For example, the solid ledipasvir may be crystalline Form- III or crystalline Form II, as disclosed in WO2013184698.

The purity of ledipasvir form-Ml disclosed herein may be analyzed by HPLC. HPLC analyses were performed on a Waters Alliance 2695 HPLC system with a 2487 UV detector. Data was collected and analyzed with Empower chromatography software or an equivalent. The below parameters were used to analyze samples: Column: X Select FISS CI 8, 250 x 4.6mm, 3.5 um or its equivalent

Detector: UV at 325 nm

Flow rate: l.O mL/minute

Injection volume: 10 pL

Column oven temp.: 40 °C

Run time: 60 minutes

Mobile phase-A: 0.1% TFA in water.

Mobile phase-B: 0.1% TFA in 55:45 acetonitrile: methanol Samples were diluted in a 0.1% TFA 50:50 solution of acetonitrile and water and put in amber-colored glass ware for analysis.

In some embodiments, ledipasvir form-Mi prepared according to the process disclosed herein present may exhibit a purity of more than 99% when analyzed by HPLC.

The polymorphic forms of ledipasvir disclosed herein may be characterized by their X-ray powder diffraction (PXRD) pattern. Thus, the PXRD patterns of the polymorphs of the disclosure were measured on PANalytical, X'Pert PRO powder diffractometer equipped with goniometer of Θ/Θ configuration and X'Celerator detector. The Cu- anode X-ray tube is operated at 40kV and 30mA. The experiments were conducted over the 2Θ range of 2.0°- 50.0°, 0.030° step size, and 50 seconds step time. Within the context of the present invention, crystalline ledipasvir form-Mi may be characterized by a PXRD pattern having significant peaks at 7.1, 9.6, 13.7, 18.2, 20.5 + 0.2 °2Θ.

Within the context of the present invention, ledipasvir form-Mi may be further characterized by a PXRD pattern as shown in Figure 1. In addition to analysis by PXRD, the polymorphs of ledipasvir disclosed herein may also be characterized by differential scanning calorimetry (DSC) or modulated differential scanning calorimetry (MDSC). The DSC measurements were carried out on TA Q1000 of TA instruments. The experiment were performed from 30 to 250 °C at a heating rate of 10.0 °C/minutes with modulation amplitude of + 0.5 °C, for every 80 seconds and nitrogen purging at a flow rate of 50 mL/minute. MDSC experiments were performed from 30-250 °C at a heating rate of 10.0 °C/minutes with modulation amplitude of + 1 °C for every 80 seconds. Standard aluminum pans covered by lids with five pin holes were used.

According to the present invention, ledipasvir form-Mi may be characterized by differential scanning calorimetry (DSC) thermogram as shown in Figure 2. Ledipasvir form-Mi disclosed herein may also be characterized for its water content.

Water content for ledipasvir form-Mi was determined on a Metrohm Coulometer (Model: 774-831, with oven and auto sampler) using a Karl Fisher coulometric reagent. The moisture content was determined at 150 °C with sample mass of about 100 mg.

Ledipasvir form-Mi disclosed herein also may be characterized by 13 C-solid state NMR (¹³C SSNMR). Therefore, ¹³C SSNMR spectra of ledipasvir form-Mi was measured on a Bruker AVANCE III 500 MHz NMR spectrometer with 3.2 mm triple resonance solid state probe. Approximately 80-100 mg of the fine powdered sample was filled into a 4.0 mm zirconium rotor. The sample was oriented at the magic angle and a spinning rate of 10 KHz was applied to remove broadening. Cross polarization technique was used to enhance the 13 C sensitivity by transferring proton magnetization to carbon. All the spectra were acquired for 2 hours and 24 minutes with a spectral width of 301 ppm. The number of average scans was 864. An external reference of KBr was used as a reference.

Within the context of the present invention, ledipasvir form-Mi may be characterized by a 13 C SSNMR spectrum having chemical shift peaks at about 125.6, 113.9, and 63.6 ppm + 0.2 ppm.

Within the context of the present invention, ledipasvir form-Mi may be further characterized by the 13 C-SSNMR spectrum shown in Figure 3.

Within the context of the present invention, ledipasvir form-Mi may be further characterized by a 13 C SSNMR spectrum having chemical shift peaks at 172.4, 170.7, 156.1, 145.4, 140.7, 137.1, 135.6, 130.2, 125.6, 119.3, 113.9, 110.2, 63.6, 57.9, 51.7, 42.9, 38.9, 35.7, 31.1, 18.2, and 4.6 + 0.2 ppm.

Within the context of the present invention, ledipasvir form-Mi may be characterized by comparing a 13 C-SSNMR spectrum of the crystalline form-Mi to a 13 C-

SSNMR spectrum for amorphous ledipasvir. Therefore, 13 C-SSNMR spectra were collected for both ledipasvir form-Ml and amorphous ledipasvir. These two spectra are shown in Figure 4.

Within the context of the present invention, ledipasvir form-Ml may be characterized by comparing an MDSC thermogram of the crystalline form-Ml to an MDSC thermogram for amorphous ledipasvir. Therefore, an MDSC curve for amorphous ledipasvir was also obtained and compared to the MDSC curve for ledipasvir form-Ml. These curves are shown in Figure 5. The MDSC thermograms in Figure 5 for reversible heat flow show the true melting of form-Ml at 168.7 °C whereas the amorphous form shows a glass transition at 160 °C. Ledipasvir form-Ml disclosed herein may, in some embodiments, exhibit long-term physical and chemical stability. The physical and chemical stability of ledipasvir form-Ml was determined by storing the samples at 40 °C/75% relative humidity (RH) and at 25 °C/60% RH for 6 months. The samples were tested for moisture content, stability of crystalline form by PXRD analysis, and purity by HPLC analysis. As an example, Table 1 below is data collected on ledipasvir form-Ml. The stability data shows that ledipasvir form-Ml has no significant change in PXRD pattern, no significant change in purity, and no significant change in moisture content for up to six months when stored at 25 °C/60% RH and 40 °C/75% RH.

Table 1

15 days 2.26 99.77 form-Ml

1 months - 99.81 form-Ml

2 months 2.69 99.85 form-Ml

3 months 2.17 99.81 form-Ml

6 months 2.04 99.80 form-Ml

It is believed that ledipasvir form-Ml obtained by the processes disclosed herein may be a solvated form of ledipasvir. It is further believed that ledipasvir form-Ml obtained by the processes disclosed herein may be a hydrate. Ledipasvir form-Ml disclosed herein and prepared by the disclosed methods may be used to formulate an oral dosage form, such as a tablet or a capsule. When administered to patients, the ledipasvir of the present invention may be useful treatment of individuals infected with hepatitis C. Ledipasvir may be used singly or in combination with other antiviral drugs, such as sofosbuvir. The ledipasvir form-Ml of the present invention may be formulated into a tablet which may contain inactive ingredients such as colloidal silicon dioxide, polyvinylpyrrolidone (copovidone), croscarmellose sodium, lactose monohydrate, magnesium stearate, and microcrystalline cellulose. The tablet may, in some embodiments, be coated with a film that includes additional excipients, artificial colors, and flavors. For example, a coating may contain polyethylene glycol, polyvinyl alcohol, talc, titanium dioxide, and yellow iron oxide. One of skill in the art will be familiar with a variety of excipients and formulations that may be used to prepare desirable dosage forms with desired release characteristics and pharmacokinetic properties without undue experimentation.

Certain specific aspects and embodiments of the present application will be explained in greater detail with reference to the following examples, which are provided only for purposes of illustration and should not be construed as limiting the scope of the disclosure in any manner. In view of the above description and the examples below, one of ordinary skill in the art will be able to practice the invention as claimed without undue experimentation. The foregoing will be better understood with reference to the following examples that detail certain procedures for the preparation of molecules according to the present invention. All references made to these examples are for the purposes of illustration. The following examples should not be considered exhaustive, but merely illustrative of only a few of the many aspects and embodiments contemplated by the present disclosure. EXAMPLES

Example 1: Crystallinity Index

The polymorphs of ledipasvir disclosed herein be characterized for their crystallinity index (CI). The CI of some embodiments of ledipasvir form-Ml was calculated quantitatively from the X-ray powder diffractogram by comparing the area of the crystalline peaks (Ac) to the area under the halo-shaped amorphous peak (AA). The sum of Ac and AA equals the total scattered intensity. The crystallinity may be represented by the formula: CI = Ac* 100/(Ac + AA). CI is estimated with an error of + 5%, primarily due to fluctuations in the baseline.

According to the present invention, ledipasvir form-Ml, prepared by the methods produced herein, may have a degree of crystallinity below 60%. In some preferred embodiments, ledipasvir form-Ml has a degree of crystallinity below 50% as given in Table 2 below.

Table 2

Example 2: Moisture Content

Moisture content was measured for several samples of ledipasvir form-Ml prepared by methods disclosed herein. Table 2 shows that the moisture content of ledipasvir form- Ml has moisture content in the range of about 2% to about 5%.

Table 3

Example 3: Preparation of ledipasvir form-Ml

Ledipasvir form-Ill (1.0 g) was dissolved in acetonitrile (5 mL) at 60 °C. The clear solution was then cooled to 25-30 °C and maintained under stirring at 25-30 °C for 15 hours. The reaction mixture was filtered to obtain a solid which was then dried under vacuum at 40 °C for 15 hours. The solid obtained was identified as ledipasvir form-Ml.

Yield: 0.85 g

Example 4: Preparation of ledipasvir form-Ml

Ledipasvir form-Ill (1.0 g) was dissolved in acetonitrile (5 mL) at 60 °C. The clear solution was then cooled to 25-30 °C and maintained under stirring at 25-30 °C for 15 hours. The reaction mixture was filtered to obtain a solid which was then washed with n- heptane (10 mL) and dried under vacuum at 40 °C for 15 hours. The solid obtained was identified as ledipasvir form-Ml.

Yield: 0.85 g Example 5: Preparation of ledipasvir form-Ml

Ledipasvir form-Ill (1.0 g) was dissolved in acetonitrile (5 mL) at 60 °C. The clear solution was then cooled to 25-30 °C. In another round bottom flask, seeds of ledipasvir form-Ml were suspended in isopropyl ether (15 mL) under stirring at 25-30 °C. The solution of ledipasvir was then slowly added at 25-30 °C. The reaction mass was maintained under stirring for 15 hours at 25-30 °C. The reaction mixture was filtered to obtain a solid which was then washed with isopropyl ether (5 mL) and dried under vacuum at 40 °C for 15 hours. The solid obtained was identified as ledipasvir form-Ml.

Yield: 0.80 g

Example 6: Preparation of ledipasvir form-Ml

Ledipasvir form-Ill (1.0 g) was dissolved in acetonitrile (5 mL) at 60 °C. The clear solution was then cooled to 25-30 °C. In another round bottom flask, seeds of ledipasvir form-Ml were suspended in water (15 mL) while stirring the mixture at 25-30 °C. The solution of ledipasvir was then slowly added at 25-30 °C. The reaction mass was maintained under stirring for 15 hours at 25-30 °C. The reaction mixture was filtered to obtain a solid which was then washed with water (5 mL) and dried under vacuum at 40 °C for 15 hours. The solid obtained was identified as ledipasvir form-Ml. Yield: 0.80 g

Example 7: Preparation of ledipasvir form-Ml

Ledipasvir form-Ill (2.5 g) was dissolved in acetonitrile (13 mL) at 60 °C. The clear solution of was then cooled to 25-30 °C and seeds of ledipasvir form-Ml were added. Then n-heptane (35 mL) was added to the reaction mass and the solution was stirred for 15 hours at 25-30 °C. The reaction mixture was filtered to obtain a solid which was then washed with n-heptane (5 mL) and dried under vacuum at 40 °C for 15 hours. The solid obtained was identified as ledipasvir form-Ml.

Yield: 1.9 g

Example 8: Preparation of ledipasvir form-Ml Ledipasvir form-Ill (0.5 g) was dissolved in dimethyl sulfoxide (3 mL) at 25-30 °C.

The clear solution of was then heated up to 120 °C, maintained for 1 hour, and cooled to 100 °C. Water (10 mL) was added at 100 °C and the solution was stirred for 12 hours. The solution was then further cooled to 80 °C and water (5 mL) was again added at 60 °C. The reaction mixture was filtered to obtain a solid which was then washed with water (3 mL) and dried under vacuum at 80 °C for 15 hours. The solid obtained was identified as ledipasvir form-Mi. Yield: 0.42 g

Example 9: Preparation of ledipasvir form-Mi

Ledipasvir form-Ill (1.0 g) was dissolved in dimethyl sulfoxide (3 mL) at 25-30 °C. Water (9 mL) was then added to the clear solution at 25-30 °C and stirred for 1 hour. The reaction mixture was filtered to obtain a solid which was then washed with water (1 mL) and dried under vacuum at 40 °C for 15 hours. The solid obtained was identified as ledipasvir form-Mi.

Yield: 0.80 g

Example 10: Preparation of crystalline ledipasvir form-Mi

Ledipasvir form- III (1.0 g) was dissolved in methanol (3 mL) at 60 °C. The clear solution of ledipasvir was cooled to 25-30 °C. In another round bottom flask, seeds of ledipasvir form-Mi were suspended in isopropyl ether (10 mL) and the solution of ledipasvir was added slowly at 25-30 °C. The reaction mass was further maintained under stirring for 15 hours at 25-30 °C. The reaction mixture was filtered to obtain a solid which was then washed with isopropyl ether (6 mL) and dried under vacuum at 40 °C for 15 hours. The solid obtained was identified as ledipasvir form-Mi.

Yield: 0.84 g

Example 11: Preparation of ledipasvir form-Mi

Ledipasvir form II (10 g) was dissolved in acetonitrile (40 mL) at 60-70 °C. The clear solution was then cooled to 20-25 °C and seeds of ledipasvir form-Mi (100 mg) were added. Stirring was continued for 20 hours at 20-25 °C. Water (50 mL) was added over 30 minutes and the solution was stirred for 6 hours. The reaction mixture was filtered to obtain a solid which was then washed with water (10 mL) and dried under vacuum at 50 °C for 15 hours. The solid obtained was identified as ledipasvir form-Mi.

Yield: 8 g

Example 12: Preparation of ledipasvir form-Mi Ledipasvir form II (4 g) was dissolved in a mixture of acetonitrile (16 mL) and water

(8 mL) at 50-55 °C. The clear solution was stirred for 10 minutes and then cooled to 45 °C. Seeds of ledipasvir form-Mi (80 mg) were added and the solution was stirred for 30 minutes at 45 °C before cooling to 40 °C where it was maintained for 2 hours. The reaction mixture was then cooled to 20-25 °C and maintained overnight with agitation. The reaction mixture was filtered to obtain a solid, which was then washed with water (8 mL) and dried under vacuum at 50 °C for 8 hours. The solid obtained was identified as ledipasvir form-Mi.

Yield: 3.0 g

Example 13: Preparation of ledipasvir form-Mi

Ledipasvir form II (4 g) was dissolved in methanol (28 mL) at 55-60 °C. Water (11 mL) was added to this solution at the rate of 1 niL/minute. After addition of water, the reaction mass was stirred for 10-15 minutes and then cooled to 45 °C at the rate of 0.5 °C per minute. Seeds of ledipasvir form-Mi (80 mg) were then added into the reaction mass and the reaction mass was stirred for 1 hour at 45 °C. The reaction mass was then cooled to 25 °C over the course of 30-35 minutes and stirred for 15-16 hours at 25 + 2 °C. The reaction mixture was filtered to obtain a solid which was then washed with water (8 mL) and dried under vacuum at 50 °C for 8 hours. The solid obtained was identified as ledipasvir form-Mi.

Yield: 3.2 g

Example 14: Preparation of ledipasvir form-Mi Ledipasvir form II (0.5 g) was dissolved in acetonitrile (2.5 mL) at 60 °C. The clear solution of ledipasvir was cooled to 22-25 °C. In another round bottom flask, seeds of ledipasvir form-Mi were suspended in water (7.5 mL) and the solution was stirred for 10-15 minutes. The clear solution of ledipasvir was then slowly added and resulting reaction mixture was maintained at 27 + 2 °C for 15 hours. The reaction mixture was then filtered to obtain a solid which was then washed with water and dried under vacuum at 40 °C for 15 hours. The solid obtained was identified as ledipasvir form-Ml. Example 15: Preparation of ledipasvir form-Ml

Amorphous form of ledipasvir (0.5 g) was dissolved in acetonitrile (2.5 mL) at 60 °C. The clear solution of ledipasvir was cooled to 22-25 °C. In another round bottom flask, seed of ledipasvir form-Ml (1% w/w) were suspended in water (7.5 mL) and the solution was stirred for 10-15 minutes. The clear solution of ledipasvir was then added and solution was maintained at 27 + 2 °C for 15 hours. The reaction mixture was then filtered to obtain a solid which was washed with water and dried under vacuum at 40 °C for 15 hours. The solid obtained was identified as ledipasvir form-Ml.

Example 16: Preparation of ledipasvir form-Ml

Amorphous ledipasvir (30 g) was dissolved in acetonitrile (120 mL) at 70 °C and then cooled to 22-25 °C. The solution was then filtered to remove any undissolved particulate matter. The clear filtrate was taken in another round bottom flask and seeds of ledipasvir form-Ml (1% w/w) were added at 22-25 °C. The solution was stirred for 15 hours at 22-25 °C. A thick reaction mass was observed. Water (150 mL) was added to the reaction mixture and stirred for 1-2 hours at 22-25 °C. The reaction mixture was filtered to obtain a solid, which was washed with water (40 mL) and dried under vacuum at 60 °C for 15 hours. The solid obtained was identified as ledipasvir form-Ml.

Yield: 29.5 g

Claims

WE CLAIM:

1. Crystalline ledipasvir form-M 1.

2. The crystalline ledipasvir form-Ml of claim 1, characterized by a PXRD pattern having peaks at 2Θ angle positions of 7.1, 9.6, 13.7, 18.2, 20.5 + 0.2 °.

3. The crystalline ledipasvir form-Ml of claim 1, characterized by a PXRD pattern as shown in Figure 1.

4. The crystalline ledipasvir form-Ml of claim 1, characterized by a 13C-SSNMR spectrum having chemical shift peaks at 125.6, 113.9 and 63.6 ppm + 0.2 ppm.

5. Crystalline ledipasvir form-Ml with a degree of crystallinity below 60%.

6. The crystalline ledipasvir form-Ml of claim 5 with a degree of crystallinity below 50%.

7. A process for the preparation of ledipasvir form-Ml, comprising the steps of:

a. dissolving ledipasvir in a solvent or mixtures of solvents to form a solution; and

b. isolating ledipasvir form-Ml.

8. The process according to claim 7, wherein the solvent is selected from the group consisting of methanol, ethanol, isopropanol, 1-butanol, isobutyl alcohol, 1-pentanol, water, acetonitrile, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethyl sulfoxide, acetone, N-methylpyrrolidone, and mixtures thereof.

9. The process according to claim 7, further comprising the step of adding an anti- solvent to the solution after the dissolving step and before the isolating step.

10. The process according to claim 7, further comprising a step of seeding the solution with ledipasvir form-Mi after the dissolving step and before the isolating step.

11. The process according to claim 7, further comprising a step of cooling the solution, before the isolating step.

12. A process for the preparation of ledipasvir form-Mi, comprising the steps of:

a. dissolving ledipasvir in a solvent to get a first solution;

b. suspending seeds of ledipasvir form-Mi in an anti-solvent to get a seeded anti- solvent solution;

c. adding the first solution to the seeded anti-solvent solution; and

d. isolating ledipasvir form-Mi.

13. The process according to claim 7 or 12, wherein the step of dissolving step is carried out at an elevated temperature.

14. The process according to claim 12, further comprising a step wherein the solution is cooled, before the isolating step.

15. The process according to claim 7, wherein the solvent is selected from the group consisting of a polar protic solvent, a polar aprotic solvent, and mixtures thereof.

16. The process according to claim 15, wherein the polar protic solvent is selected from the group consisting of methanol, ethanol, isopropanol, 1-butanol, isobutyl alcohol, 1-pentanol, and mixtures thereof.

17. The process according to claim 15, wherein the polar aprotic solvent is selected from the group consisting of acetonitrile, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethyl sulfoxide, acetone, N-methylpyrrolidone, and mixtures thereof.

18. The process according to claim 9 or 12, wherein the anti-solvent is selected from the group consisting of an ether solvent, a hydrocarbon solvent, water, and mixtures thereof.

19. The process of claim 18, wherein the ether solvent is isopropyl ether.

20. The process of claim 18, wherein the hydrocarbon solvent is heptane.