PROCESS FOR PREPARATION OF ATOVAQUONE AND THE CONVERSION OF CIS-ISOMER TO TRANS- ISOMER

The present invention relates a process for the preparation of Atovaquone of formula (I).

More particularly, the present invention relates to the process for the preparation of Atovaquone substantially free from its cis isomer. The present invention also provides the process for the preparation of purification of Atovaquone. BACKGROUND OF THE INVENTION

Pneumocystis carinii is a parasite, which has a natural habitat in lung tissue, in a host with normal immune system. Without treatment Pneumocystis carinii pneumonia is almost always fatal in immune compromised host. U.S. Pat. No. 4,981,874 discloses the process of preparation and the activity of the Atovaquone.

U.S. Pat. No. 5053432 disclosed route of synthesis of atovaquone. In condensation of 2-chloro-l, 4-naphthoquinone and 4-(4-chlorophenyl) cyclohexane-1- carboxylic acid in presence of silver nitrate and ammonium persulphate then extracted with chloroform also treated with acetonitrile gives a poor yield 35%. Here cis-isomer more generated, another disadvantage is last steps treated with KOH give 57% yield. Thus obtained Atovaquone so in this patent several disadvantages.

The process for the preparation of Atovaquone was also reported in Tetrahedron Letters, Vol. 39, 7629-7632 (1998), article as shown in below scheme:

As above disclosed process suffers with several drawbacks, as it requires to purification by recrystallization from acetonitrile. The solubility of Atovaquone in acetonitrile is very less and the high amount acetonitrile required. Moreover, it provides crystalline floppy material, which is difficult to handle at formulation development end.

WO2006008752 discloses three polymorphic forms of Atovaquone designated as Form-I, Form-II and Form-Ill. The patent application discloses that Form-I is prior art form.

OBJECTS OF THE INVENTION

It is an important object of the present invention to provide a process for the preparation of Atovaquone of formula (I). Another object of the present invention is to provide a process for the preparation of Atovaquone substantially free of its c/s-ismer.

Yet another object of the present invention is to provide a process for the purification of Atovaquone of formula (I).

Another embodiment of invention cis-isomer converted to trans-isomer. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. I XRPD of cis-isomer of 2-Chloro-3-[4-(4-chloro-phenyl)-cyclohexyI]-

[ 1 ,4]naphthoquinone.

FIG. π DSC of cis-isomer of 2-Chloro-3-[4-(4-chloro-phenyl)-cyclohexyl]-

[ 1 ,4]naphthoquinone. FIG. πi XRPD of trans-isomer of 2-Chloro-3-[4-(4-chloro-phenyl)-cyclohexyl]-

[ 1 ,4]naphthoquinone.

FIG. IV DSC of trans-isomer of 2-Chloro-3-[4-(4-chloro-phenyl)-cyclohexyl]-

[ 1 ,4]naphthoquinone.

FIG. V XRPD of Cis-isomer of Atovaquone.

FIG. VI DSC of Cis-isomer of Atovaquone. FIG. VII XRPD of trans-isomer of Atovaquone. FIG. Viπ DSC of trans-isomer of Atovaquone.

FIG. IX particle size of trans-isomer of 2-Chloro-3-[4-(4-chloro-phenyl)-cyclohexyl]- [l,4]naphthoquinone.

FIG. X Particle size of Cis-isomer of Atovaquone. FIG. XI Particle size of trans-isomer of Atovaquone. FIG. XII HPLC graph of free from cis-isomer. DETAILED DESCRIPTION OF THE INVENTION As used herein the term "substantially pure atovaquone" means it contains less than about 0.1% of individual impurity.

As used here in the term "atovaquone substantially Pure" means it atovaquone containing less than about 0.1% of cis-atovaquone.

According to the present invention, there is provided a process for the preparation of Atovaquone of formula (I), which comprises the steps of

(i) condensing 2-chloro-l,4-naphthoquinone of formula (II) with 4-(4- chlorophenyl)cyclohexane-l-carboxylic acid of formula (III) in presence of silver nitrate and ammonium persulfate to provide 2-[4-(4- chlorophenyl)cyclohexyl]-3-chloro- 1 ,4-naphthoquinone of formula (IV)

(II) (III)

(ii) treating 2-[4-(4-chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone of formula (IV) with alkali base in mixture of alcoholic solvent and halogenated solvent followed by treatment with mineral acid to provide

Atovaquone of formula (I).

(IV)

The step of preparing the compound of formula (IV) by reacting 2-chloro-l,4- naphthoquinone of formula (II) with 4-(4-chlorophenyl)cyclohexane-l-carboxylic acid of formula (III) is carried out at a temperature in the range of 70-100 ⁰ C, preferably, temperature range of 75-85°C. It is preferable to avoid solvent like sulpholane. This reaction condition gives high yield and purity.

According to an embodiment of the present invention, 2-chloro-l,4-naphthoquinone of formula (II) is reacted with 4-(4-chlorophenyl)cyclohexane-l-carboxylic acid of formula (III) in the presence of silver nitrate and ammonium persulfate to provide 2-[4- (4-chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone of formula (IV). The reaction is preferably carried out in polar solvent such as acetonitrile. Thus obtained 2- [4-(4-chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone of formula (IV) may contain a mixture of cis and trans isomer. The ratio of trans: cis isomer may be in the range of 99:1 to about 1:99, preferably it may be in the range of 70:30 to 60:40

In another embodiment of present invention is formula (IV), any cis-isomer produced is converted to trans-isomer by dissolving it in organic solvent and heating it to reflux temperature.

The organic solventa are selected from group comprising of ketones like acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl isopropyl ketone and methyl tert-butyl ketone; alcohols like methanol, ethanol, isopropyl alcohol and tert- butyl alcohol etc., esters like methyl acetate, ethyl acetate, n-butyl acetate, tert-butyl

acetate, halogenated alkanes like dichloromethane, aromatic hydrocarbons like toluene, xylene etc., preferably xylene.

The alkali metal base is selected from sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, potassium tert-butoxide. The preferred alkali metal base is potassium hydroxide.

According to an embodiment of the present invention, the solvent system of step (i) comprises of a mixture of alcoholic solvent and halogenated solvent. The preferred alcoholic solvent is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol and halogenated solvent selected from methylene dichloride, ethylene dichloride, chloroform, carbon tetrachloride.

An another embodiment of the invention is treating 2-[4-(4-chlorophenyl) cyclohexyl]-3-chloro-l,4-naphthoquinone of formula (IV) with alkali base in mixture of alcoholic solvent and halogenated solvent followed by treatment with mineral acid to provide Atovaquone of formula (I).

In another embodiment of present invention is formula (IV), any cis-isomer produced is converted to trans-isomer by dissolving it in organic solvent and heating it to reflux temperature.

m-Atovaquone

/rαns-Atovaquone

The organic solvents are selected from group comprising of ketones like acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl isopropyl ketone and methyl tert-butyl ketone; alcohols like methanol, ethanol, isopropyl alcohol and tert- butyl alcohol etc., esters like methyl acetate, ethyl acetate, n-butyl acetate, tert-butyl acetate, halogenated alkanes like dichloromethane, aromatic hydrocarbons like toluene, xylene etc., preferably xylene.

The alkali metal base is selected from sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, potassium ter/-butoxide. The preferred alkali metal base is potassium hydroxide.

According to embodiment of the present invention, the solvent system of step (ii) comprised of mixture of alcoholic solvent and halogenated solvent. The preferred alcoholic solvent is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol and halogenated solvent selected from methylene dichloride, ethylene dichloride, chloroform, carbon tetrachloride.

The present invention further provides a process for the purification of atovaquone, which comprises treating atovaquone with alkali base in suitable solvent to form solution of alkali salt of atovaquone and treating it with acid to provide "substantially pure atovaquone". According to the preferred embodiment of the present invention, atovaquone is treated with alkali base selected from sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, potassium tert- butoxide to form solution of alkali salt of atovaquone.

The reaction is preferably carried out in suitable solvent. The solvent system is preferably selected so as to facilitate the salt reaction and to provide solution of alkali salt of atovaquone or subsequent separation of the alkali salt of atovaquone. Advantageously, both atovaquone and the alkali base are dissolvable, at least partly, in the solvent system, at least at elevated temperatures. In the process, a mixture, slurry, or solution of atovaquone and a solvent may be contacted with alkali base, or conversely, a mixture, slurry, or solution of alkali base and a solvent may be contacted with atovaquone. In another embodiment, both atovaquone and alkali base combined with a solvent system prior to being contacted together, whereby the solvent system used for alkali base may be identical with or different from the solvent system used for atovaquone. The solvent system can be comprised of a single solvent or a mixture of solvents. Suitable solvents include water, a lower alcohol (Cl- C6) such as methanol, ethanol, isopropanol, n-propanol, n-butanol, iso-butanol, /er/-butanol; ester such as ethyl acetate, isopropyl acetate, butyl acetate, iso-butyl acetate; ketone such as acetone, methyl ethyl ketone, methyl /ert-butyl ketone; ether such as tetrahydrofuran, di ethyl ether, diisoproipyl ether, dioxane and the like.

The temperature of contact of atovaquone and alkali base in the solvent system is from ambient to the boiling point of the solvent system, with elevated temperatures, but generally less than the boiling point, being preferred. It is not required that a

complete solution is formed in this step, i.e. a slurry is also possible, though a single solution is generally preferred.

The atovaquone alkali salt can be isolated or recovered from the salt forming reaction by any convenient means. For example, the atovaquone alkali metal salt can be precipitated out of a solution or reaction mixture. The precipitation may be spontaneous depending upon the solvent system used and the conditions. Alternatively, the precipitation can be induced by reducing the temperature of the solvent, especially if the initial temperature at contact is elevated.

Thus obtained alkali salt of atovaquone is treated with mineral acid or organic acid such as HCl, H ₂ SO ₄ , HNO ₃ , and acetic acid to provide substantially pure atovaquone. Thus obtained atovaquone is substantially pure from and having individual impurities less than 0.1%. Figure-XII typically shows the HPLC chromatogram of the substantially pure Atovaquone.

According to the another embodiment of the present invention, there is provided a process for the preparation of cis- isomer of 2-[4-(4-chlorophenyl) cyclohexyl]-3- chloro-l,4-naphthoquinone of formula (IV). 2-[4-(4-chlorophenyl) cyclohexyl]-3- chloro-l,4-naphthoquinone treated with organic solvent and then treated with alcoholic solvent.

The preferred alcoholic solvent is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol and organic solvent is a halogenated solvent selected from methylene dichloride, ethylene dichloride, chloroform, carbon tetrachloride, preferably methylene dichloride and methanol.

According to the another embodiment of the present invention, there is provided a process for the preparation of trans- isomer of 2-[4-(4-chlorophenyl) cyclohexyl]-3- chloro-l,4-naphthoquinone of formula (IV). 2-[4-(4-chlorophenyl) cyclohexyl]-3- chloro-l,4-naphthoquinone treated with organic solvent.

The preferred organic solvent is selected from group comprising of ketones like acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl isopropyl ketone and methyl tert-butyl ketone; alcohols like methanol, ethanol, isopropyl alcohol and tert- butyl alcohol etc., esters like methyl acetate, ethyl acetate, n-butyl acetate, tert-butyl acetate, halogenated alkanes like dichloromethane, ether such as tetrahydrofuran, diethyl ether, disopropyl ether, dioxane and the like, nitriles such as acetonitrile preferably tetrahydro furan and acetonitrile.

According to the another embodiment of the present invention, there is provided a process for the preparation of cis isomer of Atovaquone formula (I). Cis/trans isomer of Atovaquone treated with organic solvent .

The organic solvent is selected from halogenated alkanes like dichloromethane and nitriles such as acetonitrile preferably dichloromethane and acetonitrile.

According to another embodiment of the present invention, there is provided control synthesis of atovaquone drug substance to insure that level of cw-atovaquone is at low levels. According to the present invention, a level of not greater than about 0.1% of cw-atovaquone. The present invention pertains to atovaquone compositions comprising levels of c/s-atovaquone of up to but not greater than about 0.1% and to methods for controlling the levels of cw-atovaquone to up to but not greater than about 0.1% in a atovaquone composition.

The preset invention further relates to atovaquone composition comprising low levels of cw-atovaquone, preferably not greater than about 0.1% c/s-atovaquone, more preferably not greater than about 0.05% c/s-atovaquone, and even more preferably not greater than about cis- Atovaquone 0.01% c/s-atovaquone.

In a more specific embodiment, this invention provides a method of synthesizing atovaquone composition that comprises an amount of c/s-atovaquone of not greater than about 0.1%, which method comprises: a) obtaining one or more samples of one or more 2-[4-(4-chlorophenyl)cyclohexyl]-3- chloro- 1 ,4-naphthoquinone batches; b) measuring the level of c/s-2-[4-(4-chlorophenyl)cyc!ohexyl]-3-chloro-l,4- naphthoquinone impurity in each of the samples of (a); c) selecting a 2-[4-(4-chlorophenyI)cyclohexyl]-3-chloro-l,4-naphthoquinone batch that comprises a level of cis isomeric impurity of not greater than about 0.4% based on the measurement or measurements conducted in (b); and d) using the batch selected in (c) to synthesize said atovaquone composition.

In one embodiment, step (c) comprises selecting a 2-[4-(4- chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone batch that comprises a level of c/s-isomeric impurity of not greater than about 0.15%. In a preferred embodiment, step (c) comprises selecting a 2-[4-(4-chlorophenyl)cyclohexyl]-3-chloro-l,4- naphthoquinone batch that comprises a level of c/s-isomeric impurity of not greater than about 0.05%.

The level of cw-isomeric impurity in a sample of a batch of 2-[4-(4- chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone can be determined using

Standard analytical techniques known to those of ordinary skill in the art. For example, the level of cw-isomeric impurity may be determined by normal phase HPLC, reverse phase HPLC, or gas chromatography methods.

This invention also provides a method of synthesizing atovaquone composition that comprises an amount of cw-atovaquone of not greater than about 0.1%, which method comprises: a) condensing 2-chloro-l,4-naphthoquinone of formula (II) or its salts with 4-(4- chlorophenyl)cyclohexane-l-carboxylic acid of formula (III) or its salts in presence of silver nitrate and ammonium persulfate to provide 2-[4-(4-chlorophenyl)cyclohexyl]-3- chloro-l,4-naphthoquinone of formula (IV); b) isolating a sample of the composition resulting from (a); c) measuring the quantity of cis- 2-[4-(4-chlorophenyl)cyclohexyl]-3-chloro-l,4- naphthoquinone impurity in the isolated sample from (b); d) determining whether or not the quantity in (c) is not greater than about 0.40%; and e) purifying by treating the composition resulting from (a) with organic solvent, if the quantity measured in (c) is greater than about 0.40% until the quantity of cis- 2-[4-(4- chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone impurity is not greater than about 0.40%, and synthesizing a atovaquone composition from the composition so purified; or, f) if the quantity in (c) is not greater than about 0.40%, synthesizing a atovaquone composition from the composition of (a).

In one embodiment, the purification of a composition comprising 2-[4-(4- chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone in step (e) is carried out by slurrying or suspending or dissolving in organic solvent at about 0° C to elevated temperature for sufficient time.

According to the present invention, there is provided a process for the preparation of atovaquone substantially free from cw-atovaquone impurity, which comprises treating crude atovaquone with organic solvent selected from ketones, Cl-

C5 alcohols, esters, halogenated solvent, aromatic hydrocarbons and isolating atovaquone substantially free from cw-isomer.

According to the preferred embodiment of the present invention, crude atovaquone is slurried or suspended or dissolved in organic solvent selected from ketones, C1-C5 alcohols, esters, halogenated solvent, aromatic hydrocarbons at about 0° C to elevated temperature for sufficient time to form atovaquone substantially free from c/s-isomer, which is isolated by conventional process such as evaporation of solvent, cooling followed by filtration, by addition of anti solvent or freeze drying.

The present invention further relates to the substantially pure cw-isomer of 2-[4- (4-chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone, which can be used as reference marker for the determination of purity of 2-[4-(4-chlorophenyl)cyclohexyl]-3- chloro-l,4-naphthoquinone. The substantially pure c/s-isomer of 2-[4-(4- chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone is characterized by its powder X-ray diffraction pattern having peaks expressed as 2θ at about 7.3, 15.0, 17.9, 20.0, 25.4±0.2 degrees. The substantially pure e/s-isomer of 2-[4-(4- chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone is further characterized by its powder X-ray diffraction pattern as shown in Figure- 1. FIG. 2 shows DSC analysis of typical substantially pure c/s-isomer of 2-[4-(4-chlorophenyl)cyclohexyl]-3-chloro-l,4- naphthoquinone.

The present invention further relates to the substantially pure trans-isomer of 2- [4-(4-chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone, which can be used for the formation of substantially pure atovaquone. The substantially pure trans-isomer 2- [4-(4-chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone is characterized by its powder X-ray diffraction pattern having peaks expressed as 2θ at about 7.2, 21.6, 25.2, 28.9±0.2 degrees. The substantially pure trans-isomer of 2-[4-(4- chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone is further characterized by its powder X-ray diffraction pattern as shown in Figure-Ill. FIG. IV shows DSC analysis of typical substantially pure trans-isomer of 2-[4-(4-chlorophenyl)cyclohexyl]-3- chloro-l,4-naphthoquinone. The particle size of trans-isomer of 2-[4-(4- chlorophenyl)cyclohexyl]-3-chloro-l,4-naphthoquinone is analyzed and found to be D(0.50):25.33μm, D(0.90):91.83μm as shown in Fig.IX. The present invention further relates to the substantially pure cw-atovaquone, which can be used as reference marker for the determination of purity. The substantially pure cw-atovaquone is characterized by its powder X-ray diffraction pattern having peaks expressed as 2θ at about 1 1.1, 17.9, 23.1, 24.3, 27.8±0.2 degrees.

The substantially pure cw-atovaquone is further characterized by its powder X-ray diffraction pattern as shown in Figure-V. FIG. VI shows DSC analysis of typical substantially pure c/s-atovaquone.

The particle size of cis isomer of Atovaquone is analyzed and found to be D(0.50):81.03μm, D(0.90):183.04μm as shown in Fig.X.

The present invention further relates to the substantially pure Atovaquone. The substantially pure Atovaquone is characterized by its powder X-ray diffraction pattern having peaks expressed as 2θ at about 10.7, 19.3, 19.9, 21.1, 24.6±0.2 degrees. The substantially pure atovaquone is further characterized by its powder X-ray diffraction pattern as shown in Figure- VII. FIG. VIII shows DSC analysis of typical substantially pure atovaquone. The particle size of atovaquone is analyzed and found to be

D(0.50):81.03μm, D(0.90):183.04μm as shown in Fig.X.

The HPLC analysis performed as follows:

Column & Packing : Kromosil C 18 (250 x 4.6 mm, 5μ) Mobile Phase : Acetonitrile :Methanol : water : ortho phosphoric acid

(500:175:325:5)

Flow rate : 30 mL/min

Wavelength : 220 nm

Injection Volume : 2 1 μL Run time : 60 min

Diluent : Acetonitrile: water (80:20)

The present invention is demonstrated in examples illustrated below. These examples are provided as illustration only and therefore should not be construed as limitation of the scope of invention. Working Examples:

Example-1 (a): Process for preparation of 2-Chloro-3-[4-(4-chIoro-phenyl)- cyclohexyl]-[l,4]naphthoquinone (cis/trans isomer).

2-Chloro-[l,4]naphthoquinone(600g 1.0 mole), trans-4-(p-chlorophenyl)- cyclohexane carboxylic acid (75Og 1.0 mole) ,water(3L) and acetonitrile(6L) were added to the reaction vessel and stirred at 25-35 ⁰ C. Silver nitrate (160.5g 0.3mole) was added and heated 75-85°C after then aq. Ammonium persulphate(1837.5g 2.6mole)was added at reflux temperature 75-85 ⁰ C for 5 hours. Then stirred for lhrours, after reaction

was completed the acetonitrile was distilled, then cooled at, 10-20 ⁰ C and washed with water (500ml). Product was dried at 50-55 ⁰ C for 12 hours and 579g of dry product was obtained. (47% Yield)

Example-1 (b): Purification of 2-ChIoro-3-[4-(4-chloro-phenyl)-cyclohexyl]- [l,4]naphthoquiπone (trans isomer).

579g of cured product of of 2-Chloro-3-[4-(4-chloro-phenyl)-cyclohexyl]-

[l,4]naphthoquinone dissolved in dichloromethane (2.8 liter) at 25-35 ⁰ C and stirred for

1 hour. Filter the product, filtrate use for next step.

Example-2(a): Preparation of Atovaquone (cis/trans isomer). As per example 1 collect the MDC then added methanol (3.8 lit) at 25-35°C.

Prepare the KOH solution (KOH (374 g 7.0v/w) added water (1.5Ht)), then this solution added for lor 2 hours time at 25-35 ⁰ C. Then stirred for 18 hours at 35-4O ⁰ C, after the complies of reaction then added con. HCl (450ml) adjust P ^H =2 then stirred for 30 min.

Filter and wash with water (500ml). Dry the product at 50-55 ⁰ C for 12 hours. 600mg Yield = 67%.

Example-2 (b): Purification of Atovaquone (trans isomer).

As per example 2 dissolved in THF (3 Lit) stirred for 30 min, added charcoal (15g) stirred for 1 hrs, filter and distilled THF at temperature 65-7O ⁰ C. added acetonitrile (6 lit.) and stirred for 1 hours cooled then filter it wash with acetonitrile (300 ml) for two times. Dry the product at 55-65 ⁰ C for 12 hours, 126g product obtains. Yield 22%. Example 3: Preparation of Atovaquone (Form I).

To the compound of disclosed in example 2(b) (332g dissolved in methanol (2Lit)) was added a solution of methanolic KOH (102g in 1 Lit, 10% solution) slowly for 1 hour at temperature of 20-30 ⁰ C followed by filtration. The filtrate was neutralized with the help of cone. HCl (P ^H =2) then water was added (675 ml) and stirred for 1 hours till solid was obtained. It was filtered and washed with water (200ml) and acetone (100 ml). The product was dried at 50-55 ⁰ C for 12 hours. Yield 98%. Example 4: Preparation of conversion of Cis 2-Chloro-3-[4-(4-chloro-phenyl)- cyclohexyl]-[l,4]naphthoquinone to trans 2-Chloro-3-[4-(4-chloro-phenyl)- cyclohexyl]- [ 1,4] naphthoquinone.

2.0g of2-Chloro-3-[4-(4-chloro-phenyl)-cyclohexyl]-[l,4]naphthoqu inone(cis) was added to xylene(10 ml) at temperature 20-30 ⁰ C, then refluxed at 120-136 ⁰ C for 18 hours. Cooled at 20-30 ⁰ C and the product was filtered. Yield: 1.8-g. 90%, Purity 96.91%. Example 5: Preparation of conversion of Cis Atovaquone to trans Atovaquone. To 5.0g of Atovaquone (cis) xylene (30 ml) was added at temperature 20-30 ⁰ C, then refluxed at 120-136 ⁰ C for 24 hours. Cooled at 20-30 ⁰ C and filtered the product 3.6-g. Yield 72% Purity 83.20%.

Example 6: Preparation of trans 2-Chloro-3-[4-(4-chloro-phenyl)-cyclohexyl]- [1,4] naphthoquinone.

32.2 g 2-Chloro-3-[4-(4-chloro-phenyl)-cyclohexyl]-[l,4]naphthoquin one was taken and to it THF (162 ml) added at temperature 20-30 ⁰ C, then refluxed at 63-66°C for 1 hour. Added acetonitrile (324 ml) at temperature 63-65 ⁰ C. Cooled at 20-30 ⁰ C and again cooled to 0-10 ⁰ C. The product was filtered. 16.4-g. Yield 50.6%.[99.61 % trans isomer/ 0.06 cis isomer]

Example 7: Preparation of cis 2-Chloro-3-[4-(4-chloro-phenyl)-cyclohexyl]- [l,4]naphthoquinone.

14.8 g 2-Chloro-3-[4-(4-chloro-phenyl)-cyclohexyl]-[l,4]naphthoquin one was taken and to it, MDC (70 ml) was added at temperature 20-30 ⁰ C, then refluxed at 30- 42°C for 1 hour. It was cooled at 0-10 ⁰ C again. Filtered the product 3.6-g wash with methanol. Yield 24.3%.[75.7 % cis isomer/ 6.04 trans isomer] Example 8: Preparation of cis-isomer of Atovaquone a) 40 g cis/trans isomer mixture was taken and to it MDC (70 ml) was added. The reaction mass was stirred for 30 min. Further, charcoal 92.0 g) was added and the reaction mixture was filtered and solvent was distilled. To the reaction mixture acetonitrile (40 mL) was added and stirred. Finally, reaction mixture was filtered to obtain cis-isomer of Atovaquone (86% cis and 12% trans). b) 27 g of crude cis isomer of Atovaquone as obtained in a) was taken and treated with acetonitrile and refluxed at 75-81 ⁰ C. The reaction mass was filtered to obtain the cis-isomer of Atovaquone. (93.4% cis and 6.3% trans).