DESC:FIELD OF THE INVENTION
The present invention relates to a process for the preparation of ledipasvir a compound of formula I, which is useful as an antiviral agent.
BACKGROUND
Hepatitis C is recognized as a chronic viral disease of the liver which is characterized by liver disease. Inhibitors of hepatitis C virus (HCV) are useful to limit the establishment and progression of infection by HCV as well as in diagnostic assays for HCV.
Ledipasvir, also known as Methyl [(2S)-1-{(6S)-6-[5-(9,9-difluoro-7{2-[(1R,3S,4S)-2-{(2S)-2-[(methoxycarbonyl)amino]-3-methylbutanoyl}-2azabicyclo[2.2.1]hept-3-yl]-1H-benzimidazol-6-yl}-9H-fluoren-2-yl)-1H-imidazol-2-yl]-5azaspiro[2.4]hept-5-yl}-3-methyl-1-oxobutan-2-yl]carbamate, is represented by compound of formula I.

I
HARVONI™ is a fixed-dose combination tablet containing ledipasvir and sofosbuvir for oral administration. Ledipasvir is an HCV NS5A inhibitor and sofosbuvir is a nucleotide analog inhibitor of HCV NS5B polymerase.
Regulatory authorities worldwide require that drug manufacturers isolate, identify and characterize the impurities in their products. Side products and by-products of the reaction and adjunct reagents used in the reaction will, in most cases, be present in the product mixture. It is required to control the levels of these impurities in the final drug compound. The impurity can be identified by its position in the chromatogram, which is conventionally measured in minutes between injection of the sample on the column and elution of the particular component through the detector, known as the “retention time” (“RT”). This time period varies based upon the condition of the instrumentation and many other factors. To mitigate the effect that such variations have upon accurate identification of an impurity, “relative retention time” (“RRT”) is used to identify impurities. The RRT of an impurity is its retention time divided by the retention time of a reference marker.The present invention provides an efficient process for the preparation of ledipasvir in a purity of at least 99% as determined by HPLC without using chromatographic techniques.
SUMMARY OF THE INVENTION
The present invention provides a process for the preparation of ledipasvir a compound of formula (I) or a pharmaceutically acceptable salt thereof comprising:

I
(a) preparing an acid addition salt of compound of formula II;

II
(b) reacting the acid addition salt of compound of formula II with a base to obtain the
compound of formula II in a purity of at least 90% w/w as determined by HPLC;
(c) reacting the compound of Formula II in a purity of at least 90% w/w, with a
deprotecting agent to obtain a compound of formula III or an acid addition salt
thereof; and

III
(d) reacting the compound of formula III or an acid addition salt thereof, with a
compound of formula IV

IV
to obtain ledipasvir a compound of formula I.
BRIEF DESCRIPTION OF FIGURES
Fig 1: 1H NMR of Ledipasvir phosphate salt.
DETAILED DESCRIPTION OF INVENTION
In one embodiment, in step “a” an acid addition salt of compound of formula II is prepared by a process comprising reacting a compound of formula II with an acid.
In one embodiment, the acid used may be an organic or an inorganic acid.In one embodiment, the organic acid used to provide an acid addition salt may include an acid such as formic acid, acetic acid, oxalic acid, citric acid, tartaric acid, bitartaric acid, benzoic acid, lactic acid, malic acid, fumaric acid, succinic acid, gluconic acid, pamoic acid, methanesulfonic acid, benzenesulfonic acid, p toluene sulfonic acid, cyclamic acid, trifluoroacetic acid, and the like and the inorganic acid used, to provide an acid addition salt, may include an acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, sulfuric acid and the like.The reaction may be carried out in presence of a solvent. The solvent may be selected from the group consisting of alcohols such as methanol, ethanol, n-propanol, 2-propanol; esters such as ethyl acetate, butyl acetate, isopropyl acetate; hydrocarbons such as toluene, cyclohexane, xylene; halogenated hydrocarbons such as ethylene dichloride, methylene dichloride, ethers such as diethyl ether, di-isopropyl ether, tetrahydrofuran; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone; water or mixtures thereof. In one embodiment, the oxalate salt of compound of formula II is prepared by a process comprising reacting a compound of formula II with oxalic acid.In one embodiment, the phosphate salt of compound of formula II is prepared by a process comprising reacting a compound of formula II with phosphoric acid.
In one embodiment, in step “b” the acid addition salt of compound of formula II is reacted with a base to obtain the compound of formula II in a purity of at least 90% w/w as determined by HPLC.The suitable base may be selected from, but not limited to ammonia, hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide; alkoxides such as sodium methoxide, potassium methoxide, sodium tert-butoxide, potassium tert-butoxide; carbonates such as sodium carbonate, potassium carbonate; bicarbonates such as sodium bicarbonate, potassium bicarbonate and the like organic bases such as triethylamine and the like.
In one embodiment, the process of the present invention provides the compound of formula II in a purity of at least 90% w/w as determined by HPLC by dissolving in a solvent like ethyl acetate and adding an antisolvent selected from alkanes like hexane, heptane and the like or cycloalkanes like cyclohexane. In one embodiment, the process of the present invention provides the compound of formula II in a purity of at least 90% w/w and wherein the impurity compound E, at RRT 0.87,

Compound E
is less than 2 area% as determined by HPLC.
In one embodiment, the process of the present invention provides the compound of formula II in a purity of at least 95% w/w and wherein the impurity compound E, at RRT 0.87, is less than 1 area% as determined by HPLC by a process comprising reacting a compound of formula II with a phosphoric acid to form phosphate salt of compound II, which is reacted with base to obtain compound of formula II.
In one embodiment, in step c, the compound of formula II, in a purity of at least 90% w/w is reacted with a deprotecting agent selected from hydrochloric acid, trifluoroacetic acid, sulfuric acid, phosphoric acid, tetrabutylammonium fluoride, and the like to obtain compound of formula III or salt thereof.In one embodiment, in step c, the compound of formula II in a purity of at least 90% w/w is deprotected with hydrochloric acid to obtain hydrochloride salt of compound of formula III. The deprotection reaction may be carried out in a solvent as discussed supra.In one embodiment, in step c, the compound of formula II in a purity of at least 90% w/w is deprotected in acetonitrile with hydrochloric acid to obtain hydrochloride salt of compound of formula III and wherein the level of impurity Compound C and/or compound D and or compound F

Compound C Compound D

Compound F
is less than 0.15% w/w as measured by HPLC.
In one embodiment, the process of the present invention provides crystalline isolated hydrochloride salt of compound of formula III. In one embodiment, the process of the present invention provides crystalline isolated hydrochloride salt of compound of formula III in a purity of at least 95% w/w and wherein the level of impurity compound C, compound D,is less than 0.15- area % as determined by HPLC.
In one embodiment, step d comprises reacting the compound of formula III or an acid addition salt thereof with a compound of formula IV and a condensing agent to obtain a reaction mixture comprising ledipasvir, compound of formula I. In one embodiment, base is not used in step d. In one embodiment, step d comprises reacting the compound of formula III or an acid addition salt thereof with a compound of formula IV in presence of a base and a condensing agent to obtain a reaction mixture comprising ledipasvir, compound of formula I.In one embodiment, step d is carried out in an organic solvent as discussed supra. The organic solvent may also be selected from sulfoxides like dimethyl sulfoxide, amides like dimethyl formamide, N-methylpyrrolidine, halogenated hydrocarbons such as ethylene dichloride, methylene dichloride.In one embodiment, the condensing agent may be selected from the group consisting of N,N"-dicyclohexylcarbodiimide (DCC), N’N’-diisopropylcarbodiimide (DIC), 6-chloro-2,4-dimethoxy-s-triazine (CDMT), O-benzotriazole-N’N’N’N-tetramethyl-uronium-hexafluoro-phosphate (HBTU), 1-Hydroxy-7-azabenzotriazole(HOAt), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide or HCl thereof(EDC) and 2-(7-Aza-1H-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU). In one embodiment the base may be selected as discussed supra. In one embodiment base may be an amine base selected from tertiary amines, triethylamine, diisopropylethylamine, 2,6-lutidine, pyridine, dicyclohexylmethylamme, and N- methylmorpholine (NMM).In one embodiment, step d comprises (i)reacting the compound of formula III or an acid addition salt thereof with a compound of formula IV in an organic solvent, in presence of a base and a condensing agent to obtain a reaction mixture comprising ledipasvir, compound of formula I;(ii) subjecting the reaction mixture comprising ledipasvir, compound of formula I to treatment with a strong base; and(iii) obtaining ledipasvir, the compound of formula I .having a purity of at least 95% as determined by HPLC. In one embodiment treatment with strong base comprises washing the reaction mixture or slurrying the reaction mixture or extracting the reaction mixture comprising ledipasvir with a strong base. The strong base may be an alkali metal hydroxide such a sodium hydroxide, potassium hydroxide and the like.
In one embodiment, of the process of the present invention the reaction mixture containing ledipasvir, compound of formula I is subjected to treatment with a strong base to obtain ledipasvir, the compound of formula I having a purity of at least 95% as determined by HPLC and wherein ledipasvir compound of formula I, does not have detectable level of impurity when measured by HPLC at RRT of 1.61.
In one embodiment, present invention provides a process comprising(a) reacting ledipasvir, the compound of formula I with an acid to obtain ledipasvir acid addition salt; (b) treating the ledipasvir acid addition salt with a base to obtain a reaction mixture containing ledipasvir; (c) subjecting the reaction mixture containing ledipasvir to treatment with an acid, to maintain the pH of the reaction mixture to less than 3; and (d) obtaining ledipasvir with a purity of at least 99% as determined by HPLC.In one embodiment, the acid used in step a for formation of acid addition salt may be selected from the group consisting of formic acid, acetic acid, oxalic acid, citric acid, tartaric acid, bitartaric acid, benzoic acid, lactic acid, malic acid, fumaric acid, succinic acid, gluconic acid, pamoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluene sulfonic acid, cyclamic acid, trifluoroacetic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, sulfuric acid and the like. In one embodiment the acid is p-toluene sulfonic acid.
The base used in step (b) may be as discussed supra.The acid used in step c, for adjusting the pH may be selected from, hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid and the like. In one embodiment, the pH of the reaction mixture in step c is adjusted in the range of 1.5-2.5.In one embodiment, the process of the present invention provides ledipasvir by a process comprising (a)subjecting the ledipasvir to an aqueous acid wash followed by an aqueous base wash;(b) reacting ledipasvir, the compound of formula I with an acid to obtain ledipasvir acid addition salt; and(c) treating the ledipasvir acid addition salt with a base to obtain ledipasvir.
In one embodiment, step a may be optional.In step a, the ledipasvir dissolved in organic solvent is subjected to aqueous acid wash followed by an aqueous base wash. The organic solvent may be selected from halogenated hydrocarbons like methylene dichloride, esters, alcohols and the like. The acid may be selected from hydrochloric acid, sulphuric acid, nitric acid and the like. The base for the aqueous wash may be any base so that pH of the aqueous solution is in the range of 9-9.5. The ledipasvir present in the organic layer of step a may be isolated by removal of organic solvent or by precipitating it out by addition of another organic solvent.In step b, the ledipasvir obtained from step (a) or from any previous reaction mixture is subjected to treatment with acid to obtain ledipasvir acid addition salt.In one embodiment, the acid used in step (b) for formation of acid addition salt may be selected from the group consisting of formic acid, acetic acid, oxalic acid, citric acid, tartaric acid, bitartaric acid, benzoic acid, lactic acid, malic acid, fumaric acid, succinic acid, gluconic acid, pamoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluene sulfonic acid, cyclamic acid, trifluoroacetic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, sulfuric acid and the like. In one embodiment the acid is phosphoric acid.The base used in step (c) may be selected from, but not limited to ammonia, hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide; alkoxides such as sodium methoxide, potassium methoxide, sodium tert-butoxide, potassium tert-butoxide; carbonates such as sodium carbonate, potassium carbonate; bicarbonates such as sodium bicarbonate, potassium bicarbonate and the like organic bases such as triethylamine and the like.
In one embodiment, the process of the present invention provides ledipasvir wherein, the level of compounds A or B

Compound A Compound B
is less than 0.15%w/w and impurity at RRT 0.83 is less than 0.15 area % as determined by HPLC. The compound A corresponds to RRT 1.04 and compound B corresponds to RRT 0.92 as measured by HPLC.
In one embodiment, present invention provides a process comprising
(a) reacting ledipasvir, the compound of formula I with p-toluenesulfonic acid to obtain ledipasvir tosylate salt; (b) treating the ledipasvir tosyalte salt with a base to obtain a reaction mixture containing ledipasvir; (c) subjecting the reaction mixture containing ledipasvir to treatment with an acid, to maintain the pH of the reaction mixture in the range of 1.5-2.5; and (d) obtaining ledipasvir with a purity of at least 99% as determined by HPLC.
In one embodiment, the present invention provides ledipasvir in a purity of at least 99% as determined by HPLC wherein, the level of one or more compounds selected from the group consisting of A, B,G, H, I, J

Compound G Compound H

Compound I Compound J
is less than 0.15%w/w as determined by HPLC.
In one embodiment, the process of the present invention provides ledipasvir in a purity of at least 99% as determined by HPLC wherein, the level of one or more compounds selected from the group consisting of A, G, H, I, J is less than 0.15%w/w as determined by HPLC, by a process comprising(a)subjecting the reaction mixture comprising ledipasvir in an organic solvent to treatment with an aqueous inorganic acid, followed by an aqueous base wash;(b)isolating the ledipasvir from step a by adding ethyl acetate and acetone to obtain solid ledipasvir;(c)reacting the solid ledipasvir, obtained in step b with phosphoric acid to obtain ledipasvir phosphate; and(d) treating the ledipasvir phosphate with a base to obtain ledipasvir.
In one embodiment, the present invention provides a process for isolating the amorphous form of ledipasvir by a process comprising the steps of (a) treating the ledipasvir in a solvent mixture comprising acetonitrile and ethyl acetate or methylene dichloride, to distillation; (b) adding acetonitrile to step (a) to form a solution; (c) adding the solution of step (b) to water; and (d) isolating the amorphous form of ledipasvir.In one embodiment, the present invention provides a method for preparing ledipasvir, or salt thereof, suitable for pharmaceutical use, comprising the steps of: (a) providing a batch of ledipasvir or a salt thereof; (b) assessing the purity of said batch of ledipasvir or salt thereof, by using compound A or compound B as a reference marker to determine the level of compound A or compound B impurity; and (c) selecting the batch of ledipasvir only if the percentage of compound A or compound B is less than 0.15% w/w as determined by HPLC. In one embodiment, the present invention provides a method for assessing the purity of ledipasvir or salt and pharmaceutical compositions containing them comprising the steps of: (a) providing a standard solution of compound A or B; and (b) using the solution as a reference marker to determine the level of compound A or B impurity. In one embodiment, the present invention provides a method for preparing ledipasvir, or salt thereof, suitable for pharmaceutical use, comprising the steps of:(a) providing a batch of ledipasvir or a salt thereof;(b) assessing the purity of said batch of ledipasvir or salt thereof, by using one or more compounds selected from the group consisting of compound A,G, H, I and J as a reference marker to determine the level of compounds A,G,H, I and J impurity; and(c) selecting the batch of ledipasvir only if the percentage of compound A, G, H, I and J is less than 0.15% w/w as determined by HPLC.
In one embodiment, the present invention provides a method for assessing the purity of ledipasvir or salt and pharmaceutical compositions containing them comprising the steps of: (a) providing a standard solution of one or more compounds selected from the group consisting of compound A, G, H, I and J (b) using the solution as a reference marker to determine the level of compound A,G,H,I and J impurity.
In one embodiment, the present invention provides Compound A.
In one embodiment, the present invention provides Compound B characterized by 1H NMR (300MHz), DMSO-d6): 8.007-7.844 (6.7H), 7.573-6.950 (4.49H), 5.203 (1H), 4.607 (1H), 4.55 (1H), 4.167-4.002 (2H), 3.77 (2.2H), 3.36 (6.8H), 2.65 (1H), 2.4-1.221 (12H), 0.98-.576 (14H).
In one embodiment, the present invention provides Compound G characterized by 1H NMR (400MHz), DMSO-d6): 0.58 (4H), 0.85-0.99d (12H), 1.46-1.48d (6H), 1.93- 2.51d (12H), 3.56-4.56d (5H), 5.19-5.57d (3H), 7.22-7.86d (12 H), 12d (2H).
In one embodiment, the present invention provides Compound H characterized by 1H NMR (300MHz), DMSO-d6): 0.57 d(4H), 0.84-0.94d (12 H), 1.21-2.63d (11 H), 3.3 -3.98 d (9H), 4.5-5.18d (6H), 7.19-8.37d (12 H) ,11.63d (2H).In one embodiment, the present invention provides Compound I characterized by 1H NMR (300MHz), DMSO-d6): 0.56-0.71d (4H), 0.91d (12 H), 1.46-2.65d (11 H), 3.3-4.16d (9H), 4.54-5.2d (3H), 6.47-6.65d (2H) ,7.22-8.02d (12 H), 12.15d (2H).In one embodiment, the present invention provides Compound J as hydrochloride characterized by 1H NMR (300MHz), DMSO-d6): 0.61-0.69 d(4H), 0.76-0.98 d (12H), 1.0-2.88 d (11 H), 4.04-5.43 d (6 H),7.89-8.38 (12 H) ,11.63 (2H).In one embodiment, the present invention provides ledipasvir tosylate salt. In one embodiment, the present invention provides ledipasvir ditosylate salt, characterized by 1H NMR 300MHz), DMSO-d6): 8.29-7.89 (9.3H), 7.51-7.37 (5.4H), 7.13-7.10 (3.9), 5.27 (1.2H), 4.86 (1.2H), 4.56 (1.2H), 4.22-3.38 (14.4H), 3.087-2.87 (1.4H), 2.5-1.6 (16H), 1.16-0.64 (14H).In one embodiment, the present invention provides ledipasvir phosphate. In one embodiment, the present invention provides ledipasvir phosphate, characterized by 1H NMR 400MHz), DMSO-d6): 0.52-0.63d (4H), 0.70-0.87d (12H), 1.77-2.4d (8H), 3.55-4.55d(8H), 5.18-5.20d (8H), 7.21-8.07(12 H), 12d (2H).In one embodiment, the present invention provides oxalic acid salt of compound of Formula II. In one embodiment, the present invention provides phosphate salt of compound of Formula II, characterized by 1H NMR (400MHz), DMSO-d6): 0.52-0.61d (4H), 0.85-1.04d (10H), 1.14-1.65d (4H), 2.04-2.5d (4H), 3.51-3.80d(4H) 4.0-4.52d (3H), 5.19-5.21d (1H), 6.50-6.85d(9H), 7.31-8.07d(12H)
In one embodiment, the present invention provides, isolated crystalline HCl salt of compound of Formula III, characterized by 1H NMR (300MHz), DMSO-d6, ??): 15.69 (1H), 15.03 (1H), 10.4 (1H), 9.32 (1.6H), 8.32-7.768 (8.1), 7.4-7.27 (1H), 5.74-5.316 (6.5H), 4.82 (1H), 4.16-4.02 (2.4H), 3.63-3.141 (4.3H), 2.48-2.37 (1H), 2.096-1.963 (3.3H), 1.76-1.71 (3.3H), 0.908-0.305 (7.5H).In one embodiment, the present invention provides Ledipasvir with a purity of 99%, wherein the ledipasvir does not have detectable level of impurity when measured by HPLC at RRT of 1.61.In one embodiment, the process of the present invention provides ledipasvir that does not have detectable level of impurity at RRT of 1.61, when measured by HPLC and wherein the level of compounds A and/or B is less than 0.15% w/w and impurity at RRT 0.83 is less than 0.15 area % as measured by HPLC. In one embodiment, the process of the present invention provides ledipasvir with a purity of 99%, that does not have detectable level of impurity when measured by HPLC at RRT of 1.61 and wherein the level of compounds A and/or B, is less than 0.15% w/w and impurity at RRT 0.83 is less than 0.15 area % as measured by HPLC, without using column chromatography. In one embodiment, the process of the present invention provides the amorphous ledipasvir without the formation of crystalline ledipasvir or without the formation of crystalline ledipasvir solvates. In one embodiment, the process of the present invention provides ledipasvir wherein level of one or more ledipasvir isomer compounds wherein stereochemistry at stereocentres of ledipasvir isomers is as below, is less than 0.15% w/w as determined by HPLC.
1. (1S)(6S)[(1R,3S,4S)-2-[(2R) 2. (1S)(6S)[(1R,3S,4R)-2-[(2S)
3.(1S)(6S)[(1R,3S,4R)-2-[(2R) 4.(1S)(6S)[(1S,3R,4S)-2-[(2S)
5.(1S)(6S)[(1S,3R,4S)-2-[(2R) 6.(1S)(6S)[(1S,3R,4R)-2-[(2S)
7.(1S)(6S)[(1S,3R,4R)-2-[(2R) 8.(1R)(6R)[(1R,3S,4S)-2-[(2S
9.(1R)(6R)[(1R,3S,4S)-2-[(2R) 10.(1R)(6R)[(1R,3S,4R)-2-[(2S)
11. (1R)(6R)[(1R,3S,4R)-2-[(2R) 12.(1R)(6R)[(1S,3R,4S)-2-[(2S)
13.(1R)(6R)[(1S,3R,4S)-2-[(2R) 14.1R)(6R)[(1S,3R,4R)-2-[(2S)
15.(1R)(6R)[(1S,3R,4R)-2-[(2R) 16. 1R)(6S)[(1R,3S,4S)-2-[(2S)
17.1R)(6S)[(1R,3S,4S)-2-[(2R) 18.(1R)(6S)[(1R,3S,4R)-2-[(2S)
19.(1R)(6S)[(1R,3S,4R)-2-[(2R) 20.(1R)(6S)[(1R,3R,4S)-2-[(2S)
21.(1R)(6S)[(1R,3R,4S)-2-[(2R) 22.1R)(6S)[(1S,3R,4R)-2-[(2S)
23.(1R)(6S)[(1S,3R,4R)-2-[(2R) 24.(1S)(6R)[(1R,3S,4S)-2-[(2S)
25.(1S)(6R)[(1R,3S,4S)-2-[(2R) 26.1S)(6R)[(1R,3S,4R)-2-[(2S)
27.(1S)(6R)[(1R,3S,4R)-2-[(2R 28.(1S)(6R)[(1R,3S,4S)-2-[(2S)
29.(1S)(6R)[(1R,3S,4S)-2-[(2R 30.(1S)(6R)[(1R,3S,4R)-2-[(2S)]
31.(1S)(6R)[(1R,3S,4R)-2-[(2R)
In one embodiment, the present invention provides a process for the preparation of ledipasvir a compound of formula (I) or a pharmaceutically acceptable salt thereof comprising: a) preparing an acid addition salt of compound of formula II; b) reacting the acid addition salt of compound of formula II with a base to obtain the compound of formula II in a purity of at least 90% w/w as determined by HPLC; c) reacting the compound of Formula II in a purity of at least 90% w/w, with a deprotecting agent to obtain a compound of formula III or an acid addition salt thereof; d) reacting the compound of formula III or an acid addition salt thereof with a compound of formula IV to obtain a reaction mixture comprising ledipasvir, compound of formula I; e) subjecting the reaction mixture comprising ledipasvir, compound of formula I to treatment with a strong base; and obtaining ledipasvir, the compound of formula I having a purity of at least 95% as determined by HPLC; f) reacting ledipasvir, the compound of formula I of step e with an acid to obtain ledipasvir acid addition salt; g) treating the ledipasvir acid addition salt with a base to obtain a reaction mixture containing ledipasvir; h) isolating the ledipasvir with a purity of at least 99% as determined by HPLC; wherein, the level of compounds A or B, or G is less than 0.15%w/w as determined by HPLC; and i) optionally converting ledipasvir to amorphous ledipasvir. In one embodiment, the present invention provides a process for ledipasvir wherein the compound of formula B is below detection limit or absent, by a process comprising treating the reaction mixture containing the compound III or the reaction mixture containing ledipasvir with a reducing agent or hydrazine hydrate. The reducing agent may be sodium borohydride and the like. In one embodiment, the present invention provides a process for compound III wherein the compound of formula C is below detection limit or absent, by a process comprising treating the reaction mixture containing the compound III with a reducing agent.
In one embodiment, the compound of formula II, is prepared by reacting a compound of formula V with a compound of formula VI in presence of a base and palladium catalyst.

V VI
The palladium catalyst may be selected from Pd(0) and Pd(II) compounds.
The suitable base may be selected from, but not limited to ammonia, hydroxides of alkali or alkaline earth metals; alkali metal alkoxides; alkali or alkaline earth metals carbonates or bicarbonates. In one embodiment, the compound of formula II, is isolated from the reaction mixture before reacting with an acid. In one embodiment, the compound of formula II, prepared by reacting a compound of formula V with a compound of formula VI is not isolated from the reaction mixture before reacting it with an acid.
Related substances by HPLC for Ledipasvir: Reagents and Solvents: Sodium perchlorate monohydrate (AR Grade); Perchloric acid (AR Grade) Acetonitrile (HPLC Grade); Water (Milli Qor equivalent)Chromatographic Conditions: Apparatus: A HPLC equipped with quaternary gradient pumps, variable wavelength UV detector attached with data recorder and integrator software Column: Inertsil ODS 3V, 250 X 4.6mm, 5? ; Column temperature: 30°C.Sample Cooler temperature: 25°C Mobile Phase: Mobile phase A = Buffer; Buffer: 0.01M Sodium perchlorate monohydrate in water. Adjust pH to 3.0 with perchloric acid. Mobile phase B = Acetonitrile. Diluent: Acetonitrile: Water (70: 30, v/v). The retention time of main peak i.e. ledipasvir is about 36.0 minutes under these conditions.
The examples that follow are provided to enable one skilled in the art to practice the invention and are merely illustrative of the invention. The examples should not be read as limiting the scope of the invention as defined in the features and advantage

EXAMPLES
EXAMPLE 1: Preparation of compound of formula II
1,2-dimethoxy ethane (125mL), compound of formula V (25g), compound of formula VI (22.8g) and palladium catalyst Pd-101(3.75g) were added to a RBF. The reaction mixture was stirred and maintained at 20-30°C under nitrogen. In another flask potassium carbonate was dissolved in water and solution of potassium carbonate was added to the above reaction mixture at 20-30°C. The reaction mixture was heated at 80-85°C and maintained for about 8-10h. Further the reaction mixture was quenched with water. The solid obtained was dried to give crude compound II, which was added to acetone and stirred. Activated charcoal was added to above solution and filtered through hyflo bed and acetone layer was transferred to another RBF and anhydrous oxalic acid (15g) was added. The reaction mixture was stirred, filtered and washed with acetone. The solid obtained was dried to yield the oxalate salt of compound of formula II.1H NMR (300MHz), DMSO-d6,): 8.16-7.32 (12.5H), 5.22 (1H), 4.559 (1H), 4.285-3.42 (6.6H), 2.68-2.5 (1.1H), 2.20-2.02 (9H), 1.65-1.349 (9.5H), 1.13-0.57 (13.1H).
HPLC analysis of oxalate salt of compound II:
RRT 0.23 0.33 0.4 0.44 0.53 0.64 0.72 0.73
Analysis of Crude comp II (Area%) 0.01 0.01 0.03 1.77 0.29 0.02 0.01 0.02
Analysis of Oxalate Salt of comp II (Area%) BDL BDL BDL 0.8 0.13 0.05 BDL 0.04

RRT 0.83 0.87 0.89 0.92 0.94 0.97 0.99 1.0
(Compound II)
Analysis of Crude comp II (Area%) 0.25 5.41 0.04 0.14 0.79 0.26 1.85 86.85
Analysis of Oxalate Salt of comp II (Area%) 0.15 1.71 BDL 0.03 0.3 0.25 0.79 94.1

RRT 1.03 1.05 1.11 1.13 1.16 1.18 1.22 1.29
Analysis of Crude comp II (Area%) 0.05 0.18 0.43 0.37 0.05 0.52 0.02 0.41
Analysis of Oxalate Salt of comp II (Area%) 0.05 0.2 0.13 0.36 0.05 0.26 0.03 0.45
BDL-Below detection limit
The oxalate salt of compound of formula II (39g), ethyl acetate (250mL) and water (250mL) were mixed and aqueous ammonia (15mL) was added. The aqueous layer was extracted with ethyl acetate and combined ethyl acetate layer was washed with water followed by washing with 10% sodium chloride solution. The ethyl acetate layer distilled under vacuum below 45°C to obtain residue. Again ethyl acetate was added to a residue and stirred. Cyclohexane was added to a clear ethyl acetate solution with stirring. Filtered and washed with cyclohexane to yield compound of formula II. HPLC Purity - 94.88%.
Preparation of compound of formula II from compound V & VI was carried out under different reaction conditions:
Solvent Base Catalyst
1, 2-dimethoxy ethane Sodium bicarbonate Palladium (Pd-101)
Toluene Potassium carbonate Palladium (Pd-101)
1, 2-dimethoxy ethane Sodium hydroxide Palladium (Pd-101)
1, 2-dimethoxy ethane Sodium carbonate Palladium (Pd-100)

EXAMPLE 2: Preparation of HCl salt of compound of formula III
Acetonitrile (120mL) was added to a compound of formula II (15g) at 20-30°C with stirring and mixture was heated to 60-65°C. The HCl solution (1.5N) was added slowly and the reaction mixture was stirred for about 2 hrs and then cooled to 45°C. Acetonitrile was added and mixture was cooled to 25-30°C and stirred. The solid obtained was filtered; washed with acetonitrile and dried under vacuum at 20-30°C to obtain HCl salt of compound of formula III. HPLC purity 97.6%.
1H NMR (300MHz), DMSO-d6, ??): 15.69 (1H), 15.03 (1H), 10.4 (1H), 9.32 (1.6H), 8.32-7.768 (8.1), 7.4-7.27 (1H), 5.74-5.316 (6.5H), 4.82 (1H), 4.16-4.02 (2.4H), 3.63-3.141 (4.3H), 2.48-2.37 (1H), 2.096-1.963 (3.3H), 1.76-1.71 (3.3H), 0.908-0.305 (7.5H).
RRT 0.78 0.83 0.91 1.0
(compound III HCl salt) 1.17 1.35 1.38 1.58 1.64
Analysis of Reaction Mass (Area %) 0.03 0.41 0.06 88.4 6.26 1.25 0.42 0.18 0.31
Analysis of Isolated HCl salt comp III
(Area %) 0.04 0.14 0.06 99.31 0.01 BDL BDL BDL BDL
Preparation of compound of formula III or acid addition salt thereof by reacting compound II with different deprotecting agents in presence of different solvents
Solvent Deprotecting agent Compound obtained
Dichloromethane Aq. Hydrochloric acid compound of formula III HCl
Toluene Aq. Hydrochloric acid compound of formula III HCl
Dichloromethane Trifluoro acetic acid Compound of formula III trifluoroacetate
Dichloromethane Ethyl acetate/HCl compound of formula III HCl
Isopropyl alcohol Isopropyl alcohol/HCl compound of formula III HCl

EXAMPLE 3: Preparation of compound of formula I (amorphous form)
DMF (45mL) was added to a compound of formula III (9g) and DIPEA (1.9g) and stirred for about 30min in a RBF. In another flask DMF (45mL) was added to N-methoxy carbonyl L-valine (3.9g), HATU (8.4g) and DIPEA (2.9g) and stirred. HATU complex solution was added to a solution of compound of formula III at 20-30°C. Ethyl acetate and water were added to the reaction mixture and stirred. The aqueous layer was extracted with ethyl acetate and combined ethyl acetate layer was washed with water. The ethyl acetate layer was subsequently washed with 10% sodium hydroxide solution and 10 % sodium chloride solution. Sample was analyzed by HPLC.
Impurities removed with 10 % sodium hydroxide solution washing:
RRT 0.27 0.82 1.0 (ledipasvir ) 1.61
Analysis of Reaction Mass
(Area%) 0.28 0.71 92.36 5.16
Analysis of Reaction Mass
After NaOH washing
(Area%) 0.11 BDL 97.92 BDL
Then p-toluene sulfonic acid was added to an ethyl acetate layer and stirred. Filtered the tosylate salt of compound of formula I and washed with ethyl acetate. Sample was analyzed by HPLC.
RRT 0.27 0.83 0.86 1.0
(ledipasvir ) 1.04 1.06 1.08 1.11
Analysis of Reaction Mass Before PTSA Salt (Area%) 0.11 1.03 0.11 97.92 0.15 0.11 0.22 0.05
Analysis of PTSA salt of ledipasvir (Area%) BDL 0.24 BDL 99.11 0.06 BDL BDL BDL

The tosylate salt of compound of formula I was transferred to another RBF containing water and ethyl acetate. The aqueous ammonia was added to the above mixture and stirred. The separated organic layer was washed with water and pH was adjusted to 1.5-3 with HCl. The organic layer separated and distilled out under vacuum below 45°C till residue remains in RBF. Acetonitrile was added and distilled out organic layer under vacuum below 45°C. Again acetonitrile was added. In another RBF containing water, added above acetonitrile reaction mass drop wise with stirring. Filtered the solid and washed with water. The solid was dried in air oven at 45-50°C. Dry Wt. 4.5 gm. HPLC purity 99.23%.
RRT 0.27 0.31 0.36 0.4 0.46 0.74 0.78 0.8 0.82
Before acid wash (area%) BDL BDL BDL BDL BDL BDL BDL BDL 0.04
After acid wash (area%) BDL 0.03 BDL BDL BDL BDL BDL BDL 0.05

RRT 0.83 0.86 0.9 0.92 0.93 0.95 1.0
(Ledipasvir)
Before acid wash (area%) 0.34 0.17 0.02 0.12 BDL 0.01 98.74
After acid wash (area%) 0.05 0.02 0.01 0.08 BDL 0.02 99.24

RRT 1.04 1.06 1.08 1.11 1.13 1.3 1.34 1.37 1.61
Before acid wash (area%) 0.03 0.03 0.05 0.18 0.02 0.01 0.05 0.04 BDL
After acid wash (area%) 0.04 0.07 0.07 0.04 0.02 0.03 0.04 BDL BDL

Example 4: Preparation of ditosylate salt of Ledipasvir
To 10g of Ledipasvir in 50mL of ethyl acetate 4.2g of p-toluene sulfonic acid was added and the reaction mixture was stirred. The generated solid was isolated by filtration and washed with ethyl acetate to obtain 10.5g of ditosylate salt of ledipasvir
1H NMR (300MHz), DMSO-d6,): 8.29-7.89 (9.3H), 7.51-7.37 (5.4H), 7.13-7.10 (3.9), 5.27 (1.2H) 4.86 (1.2H), 4.56 (1.2H), 4.22-3.38 (14.4H), 3.087-2.87 (1.4H), 2.5-1.6 (16H), 1.16-0.64 (14H).
Example 5: Preparation of different acid addition salts of Ledipasvir:
Preparation of different acid addition salts of Ledipasvir was carried out by reacting Ledipasvir in different solvents with respective acids as described in example 4.
Solvent Acid Compound obtained
Ethyl acetate Cyclamic acid Ledipasvir cyclamate
Ethyl acetate Phosphoric acid Ledipasvir phosphate
Ethyl acetate Dioxane/Hydrochloric acid Ledipasvir Hydrochloride
Isopropyl alcohol Camphor sulphonic acid Ledipasvir camphor sulfonate
Isopropyl alcohol Citric acid Ledipasvir citrate
Ethyl acetate Succinic acid Ledipasvir succinate

Example 5: Preparation of compound A
The compound D (1g) was dissolved in DMF (10mL) and DIPEA (1.25mL) was charged and stirred for 15 min. In another flask dissolved N-methoxy carbonyl L-valine (0.55g) and HATU (1.19g) in DMF (10mL) in the presence of base DIPEA (1.25mL). This solution was added to the compound D solution and stirred for 24hrs. The reaction mass was quenched with water (50mL) and stirred for 30min. the solid was filtered and washed with water. The crude material was dried at 50-60°C. Acetone 15mL was charged and the reaction mass was heated to 45-50°C followed by cooling to 20-30° and stirring for 6 hours. The solid obtained was filtered and washed with acetone and dried at 45°C Wt. - 0.8g.
Example 6: Preparation of compound B.
The compound of formula I (1.5g) and Trifluoro acetic acid (15mL) were stirred and heated to 65-70°C and maintained for 48h.The reaction mixture was distilled under vacuum at 50-60°C. Ethyl acetate (20mL) and water (20mL) were charged to the above reaction mixture followed by stirring and basifying with aqueous ammonia (7mL). The ethyl acetate layer was washed with water and brine solution and distilled under vacuum at 45-50°C. wt – 1.2g HPLC purity - 96.83% .
1H NMR (300MHz), DMSO-d6): 8.007-7.844 (6.7H), 7.573-6.950 (4.49H), 5.203 (1H), 4.607 (1H), 4.55 (1H), 4.167-4.002 (2H), 3.77 (2.2H), 3.36 (6.8H), 2.65 (1H), 2.4-1.221 (12H), 0.98-0.576 (14H)
Example 7: Preparation of Phosphate Salt of compound II
Compound V (30.0g) and compound VI (23.1g), toluene (300mL), palladium catalyst Pd-100 (0.45g) and triphenyl phosphine (0.45g) were charged in RBF and stirred. Solution of potassium carbonate was added to the reaction mass at 20-30°C. The reaction mixture was heated at 90-105°C and maintained for about 9-12h. Further the reaction mass was cooled to 55-60°C and water was added to the reaction mass. The reaction mass was stirred and toluene layer was separated. Activated charcoal was added to toluene layer at 55-60°C, filtered through hyflo bed and distilled under vacuum below 50°C. The solid (40.0g) obtained was dried under vacuum at 45-55°C.. HPLC purity–93.97%. Above obtained solid (40.0g), ethyl acetate and water were mixed and the pH of the mixture was adjusted to 2.0-3.0 with dilute hydrochloric acid. The aqueous layer was extracted with ethyl acetate. Aqueous ammonia was added to the ethyl acetate layer. The mixture was stirred and layers were separated. Combined ethyl acetate layer was distilled under vacuum 50°C to obtain residue. Acetone was added to the residue and stirred to get clear solution. A solution of phosphoric acid (14.73g) in acetone was added to the above clear acetone solution, stirred and the precipitated solid was filtered and washed with acetone to yield phosphate salt of compound II. The solid obtained was dried under vacuum at 45-55°C. Dry wt.-35.0g. HPLC purity – 95.68%
In a modified process the reaction was carried out without triphenyl phosphine
Example 8: Preparation of compound of formula II
Phosphate salt of compound II (60.gm) and 1, 2 dimethyl ethane (500mL) were mixed, stirred and aqueous ammonia was added. The solution was added to distilled water, stirred and maintained for 2h. The precipitates obtained were filtered and washed with distilled water and dried under vacuum at 45-55°C. Dry wt. - 51.0g HPLC purity -96.95%
Example 9: Preparation of Ledipasvir Phosphate
EDC.HCL (16.4g), Moc-L-Valine (14.96g) and HOAT (11.6g) were added to a RBF containing 500ml of methylene dichloride at 20-30°C under stirring. The reaction mass was stirred at 20-30°C and maintained for 90 min at 20-30°C. Compound III HCl salt (15.0gm) and diisopropylethylamine (26.5 ml) were added to above reaction mixture, stirred and maintained at 20-30°C. After completion of reaction distilled water was added to it, stirred and the layers were separated. The methylene dichloride layer was washed with sodium hydroxide solution followed by washing with water.
To the methylene dichloride layer water was added and acidified with 3 N HCl solution to pH 2.0-3.0. The layers were separated and methylene dichloride layer was washed with distilled water. Again the methylene dichloride layer was mixed with distilled water and basified with aqueous ammonia solution to pH 9.0-9.5. The layers were separated and methylene dichloride layer was washed with distilled water and distilled under vacuum to obtain solid residue. To the residue obtained was added ethyl acetate and heated to 45-55°C to get a clear solution. Acetone was added to the clear ethyl acetate solution and heated to 45-55°C for 15-20 min. Then the mixture was cooled to 20-25°C and maintained for 12 h. The slurry mass was filtered, washed with acetone and dried under vacuum. Acetonitrile was added to the solid obtained and the reaction mass was stirred to get a clear solution. A solution of phosphoric acid in acetonitrile was added dropwise at 20-25°C to the above clear solution. The reaction mass was stirred for 2h, filtered and washed with acetonitrile and Di-isopropyl ether dried in vacuum oven at 50-55°C for 12h to get Ledipasvir phosphate
HPLC Purity : > 99.0 %
Example 10: Preparation of Ledipasvir : Ledipasvir phosphate (50g) and distilled water were mixed (1000 ml), the reaction mixture was cooled to 10-20°C and basified with aqueous ammonia solution to pH 9.0-9.5. The reaction mixture was stirred and extracted with methylene dichloride at 20-30°C. The separated methylene dichloride layer was washed with distilled water to obtain solid residue. To the solid residue obtained was added ethyl acetate and heated to 40-50°C to get clear solution and cooled to 25-30°C. Acetone was added to the above clear solution and heated to 40-50°C for 15-20 min. Thereafter reaction mass was cooled to 20-25°C and maintained for 12h. The solid precipitates were filtered and washed with acetone and dried under vacuum. The solid wet cake obtained was added to acetonitrile, stirred and reaction mass was heated to 40-50° to get clear solution. This clear acetonitrile solution was added drop wise into distilled water at 20-30°C. The reaction mass was stirred for 3-4 h, filtered and washed with water and dried in vacuum oven at 50-55°C for 12h to get the Ledipasvir Amorphous form. HPLC purity: > 99.5%
Example 11: Preparation of Ledipasvir:Ledipasvir phosphate (1.2g) and distilled water (24mL) were mixed, the reaction mixture was cooled to 10-15°C and basified with aqueous ammonia solution. The reaction mixture was stirred and extracted with methylene dichloride. The methylene dichloride layer obtained was again extracted with distilled water and distilled atmospherically at 40-45°C to obtain solid residue. To the solid residue obtained was added acetonitrile at 40-45°C. In another RBF distilled water was added and solution of acetonitrile was added slowly to it. The reaction mixture was stirred for 2h, filtered and washed with distilled water and dried in vacuum tray dryer. Wt-1.1gm ,CLAIMS:1. A process for the preparation of ledipasvir a compound of formula (I) or a pharmaceutically acceptable salt thereof comprising:

I
(a) preparing an acid addition salt of compound of formula II;

II
(b) reacting the acid addition salt of compound of formula II with a base to obtain the
compound of formula II in a purity of at least 90% w/w as determined by HPLC;
(c) reacting the compound of Formula II in a purity of at least 90% w/w, with a
deprotecting agent to obtain a compound of formula III or an acid addition salt
thereof; and

III
(d) reacting the compound of formula III or an acid addition salt thereof with a
compound of formula IV

IV
to obtain ledipasvir a compound of formula I.
2. The process of claim 1, wherein the compound of formula II is obtained in a
purity of at least 90% w/w and wherein the impurity compound E

Compound E
is less than 2 area %, as determined by HPLC.
3. The process of claim 1, wherein the acid addition salt of compound of formula II is prepared by reacting a compound of formula II with an organic acid or inorganic acid.
4. The process of claim 1, wherein step d comprises
(i) reacting the compound of formula III or an acid addition salt thereof with a compound of formula IV in an organic solvent, in presence of a base and a condensing agent to obtain a reaction mixture comprising ledipasvir, compound of formula I;
(ii) subjecting the reaction mixture comprising ledipasvir, compound of formula I to treatment with a strong base; and
(iii) obtaining ledipasvir, the compound of formula I having a purity of at least 95% as determined by HPLC.

5. The process of claim 1, further comprising a step of
(a) optionally subjecting the resultant ledipasvir to an aqueous acid wash, followed by an aqueous base wash;
(b) reacting the resultant ledipasvir, the compound of formula I with an acid to
obtain ledipasvir acid addition salt; and
(c) treating the ledipasvir acid addition salt with a base to obtain ledipasvir.
6. The process of claim 5, wherein the level of one or more compounds selected from the group consisting of A, G, H, I and J

Compound A

Compound G Compound H

Compound I Compound J

in the ledipasvir obtained is less than 0.15% w/w as determined by HPLC.
7. The process of claim 6, wherein ledipasvir acid addition salt is ledipasvir tosylate or phosphate.
8. The process of claim 7, further comprising isolating amorphous form of ledipasvir by a process comprising the steps of :
(a) treating the resultant ledipasvir in a solvent mixture comprising acetonitrile and ethyl acetate or methylene dichloride to distillation ;
(b) adding acetonitrile to step (a) to form a solution;
(c) adding the solution of step (b) to water; and
(d) isolating the amorphous form of ledipasvir.
9. A method for preparing ledipasvir, or salt thereof, suitable for pharmaceutical use,
comprising the steps of:
(a) providing a batch of ledipasvir or a salt thereof;
(b) assessing the purity of said batch of ledipasvir or salt thereof, by using one or more compounds selected from the group consisting of compound A,G, H, I and J as a reference marker to determine the level of compounds A,G, H, I and J impurity; and(c) selecting the batch of ledipasvir only if the percentage of compound A, G, H, I and J is less than 0.15% w/w as determined by HPLC.
10. A method for assessing the purity of ledipasvir or salt and pharmaceutical compositions containing them comprising the steps of:
(a) providing a standard solution of one or more compounds selected from the
group consisting of compound A,G, H, I and J ; and(b) using the solution as a reference marker to determine the level of compound A, G, H, I and J impurity.
Dated this 16th day of February, 2017
(Signed)____________________
DR. MADHAVI KARNIK
SENIOR GENERAL MANAGER-IPM
GLENMARK PHARMACEUTICALS LIMITED