Field of the Invention

The present invention relates to an improved process for the preparation of Luliconazole. The present invention also provides a novel intermediate 1- cyanomethylimidazole methanesulphonate of Formula (V), process for its preparation and its use for the preparation of Luliconazole.

Background of the Invention

Luliconazole is chemically known as (2E)-2-|(4/?)-4-(2.4-dichlorophcn l)- l .3- dithiolan-2-ylidene]-2-imidazol-l-ylacetonitrile, represented by Formula I.

Formula I

Luliconazole is an antifungal azole indicated for the topical treatment of interdigital tinea pedis, tinea cruris, and tinea corporis caused by the organisms Trichophyton rubrum and Epidermophyton floccosum, in patients 18 years of age and older.

Luliconazole was first disclosed in U.S. Patent No. 5,900,488 by Nihon Nohyaku Co., Ltd. U.S. ‘488 discloses a process for the preparation of Luliconazole of Formula (I) by reacting (>S')-2-chloro-l-(2,4-dichlorophenyl)ethyl methanesulfonate of Formula (II) with 1 -cyanomethylimidazole of Formula (III). The process is depicted in the scheme given below:

(.S')-2-Chloro- l -(2.4-dichlorophcnyl)cthyl methanesulfonate of Formula (II) and 1- cyanomethylimidazole of Formula (III) are the key raw materials for the preparation of Luliconazole.

U.S. Publication No. 2015/65522; PCT Publication No. 2016/092478; Bioorganic

& Medicinal Chemistry 2006, 3185-3198 disclose processes for the preparation of 1- cyanomethylimidazole of Formula (III). The inventors of the present invention have reproduced these processes and observed that 1-cyanomethylimidazole obtained is highly hygroscopic, deliquescent and decompose at room temperature and thus becomes unstable for further use.

PCT Publication No. 2017/108972 discloses a use of 1-cyanomethylimidazole hydrochloride in the preparation of Luliconazole. However, preparation of 1- cyanomethylimidazole hydrochloride involves use of hydrogen chloride gas which is costly and highly corrosive in nature. Further, 1-cyanomethylimidazole hydrochloride is highly hygroscopic due to which it is susceptible to convert to 2-( I H-imidazol- 1 -yl (acetamide of Formula (IV) as an impurity. Thus, 1-cyanomethylimidazole hydrochloride is not suitable for commercial purposes.

Formula IV

Therefore, there is a need in the art to develop an improved process for the preparation of Luliconazole.

Summary of the Invention

The present invention relates to an improved process for the preparation of Luliconazole. The present invention also provides a novel intermediate 1- cyanomethylimidazole methanesulphonate of Formula (V), process for its preparation and its use for the preparation of Luliconazole.

The present invention provides cost-effective, environmental friendly and industrially advantageous process for the preparation of Luliconazole in high yield using 1- cyanomethylimidazole methanesulphonate of Formula (V) as a novel intermediate.

Brief Description of the Drawings

Figure 1 : High Performance Liquid Chromatography (HPLC) chromatogram of purity of 1-cyanomethylimidazole hydrochloride, obtained as per Reference Example.

Figure 2: HPLC chromatogram of purity of 1-cyanomethylimidazole methane sulphonate of compound of Formula (V), obtained as per Example 2 of present invention.

Detailed Description of the Invention

The term “about,” as used herein, refers to any value within the range defined by a number up to ±10% of the value. The term “room temperature,” as used herein, refers to a temperature in the range of 25°C to 35°C.

The term “acid addition salt,” as used herein, refers to inorganic salt or organic salt. Examples of inorganic salt include hydrochloric, hydrobromic, hydroiodic, nitric, phosphoric, sulfuric, and trifluoroacetic. Examples of organic salt include acetic, formic, citric, tartaric, benzoic, lactic, malic, fumaric, succinic, methanesulfonic, p- toluenesuphonic, and benzene sulfonic.

A first aspect of the present invention provides a process for the preparation of Luliconazole of Formula (I),

Formula I comprising a) reacting imidazole with chloroacetonitrile to obtain 1 -cyanomethylimidazole of Formula (III);

Formula III b) treating 1 -cyanomethylimidazole of Formula (III) with methanesulfonic acid to obtain 1 -cyanomethylimidazole methane sulphonate of Formula (V);

Formula V c) reacting 1 -cyanomethylimidazole methanesulphonate of Formula (V) with carbon disulfide in the presence of a base and a solvent to form an intermediate of Formula (VI); Formula VI d) reacting the compound of Formula (VI) in-situ with 2,4-dichloro-l-[(l>S')-l,2- dichloroethyl]benzene of Formula (VII) to obtain Luliconazole of Formula (I);

Formula VII e) treating Luliconazole of Formula (I) with suitable acid to obtain acid addition salt of Luliconazole; and f) converting the acid addition salt of Luliconazole to Luliconazole of Formula (I)

A second aspect of the present invention provides a process for the preparation of 1- cyanomethylimidazole methanesulphonate of Formula (V),

Formula V comprising a) reacting imidazole with chloroacetonitrile to obtain 1 -cyanomethylimidazole of

Formula (III); and

Formula III b) treating 1 -cyanomethylimidazole of Formula (III) with methanesulfonic acid to obtain 1 -cyanomethylimidazole methanesulphonate of Formula (V).

A third aspect of the present invention provides 1 -cyanomethylimidazole methane sulphonate of Formula (V).

Formula V

The compound of Formula (VII) may be prepared by any method known in the art, for example, the method as described in U.S. Patent No. 5,900,488.

The reaction of imidazole with chloroacetonitrile to obtain 1 -cyanomethylimidazole of Formula (III) is carried out in the presence of a base in a solvent. The base is selected from the group consisting of inorganic base or an organic base. Examples of inorganic base include sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, sodium hydrogen carbonate, and potassium hydrogen carbonate. Examples of organic base include triethylamine, N, N’ -diisopropylethyl amine, and l,8-diazabicyclo[5.4.0]undec-7- ene.

The solvent is selected from the group consisiting of esters, ethers, alcohols, nitriles, halogenated hydrocarbons, aromatic hydrocarbons, ketones, water, and mixtures thereof. Examples of esters include ethyl acetate, methyl acetate, propyl acetate, isopropyl acetate, and butyl acetate. Examples of ethers include tetrahydrofuran, ethyl methyl ether, diethyl ether, diisopropyl ether, and methyl tert-butyl ether. Examples of alcohols include methanol, ethanol, propanol, iso-propanol, n-butanol, and iso-butanol. Example of nitrile is acetonitrile. Examples of halogenated hydrocarbons include dichloromethane, dichloroethane, chloroform, and carbon tetrachloride. Examples of aromatic hydrocarbons include toluene and xylene. Examples of ketones include acetone, methyl ethyl ketone, and methyl isobutyl ketone.

The reaction of imidazole with chloroacetonitrile is carried out for about 20 hours to about 30 hours. In an embodiment, the reaction of imidazole with chloroacetonitrile is carried out for about 22 hours to about 28 hours. In another embodiment, the reaction of imidazole with chloroacetonitrile is carried out for about 22 hours to about 24 hours.

The reaction of imidazole with chloroacetonitrile is carried out at room temperature to the boiling point of the solvent.

The reaction mixture containing 1 -cyanomethylimidazole of Formula (III) may be used as such or isolated.

1 -Cyanomethylimidazole of Formula (III) may optionally be isolated using conventional techniques, for example, fdtration, decantation, extraction, distillation, evaporation, chromatography, precipitation, concentration, crystallization, centrifugation, and recrystallization. 1 -Cyanomethylimidazole of Formula (III) may be dried using conventional techniques, for example, drying, drying under vacuum, spray drying, suck drying, air drying, or agitated thin fdm drying.

1 -Cyanomethylimidazole of Formula (III) is reacted with methane sulphonic acid in a solvent to obtain 1 -cyanomethylimidazole methane sulphonate of Formula (V).

The solvent is selected from the group consisiting of esters, ethers, alcohols, nitriles, halogenated hydrocarbons, aromatic hydrocarbons, ketones, water, and mixtures thereof. Examples of esters include ethyl acetate, methyl acetate, propyl acetate, isopropyl acetate, and butyl acetate. Examples of ethers include tetrahydrofuran, ethyl methyl ether, diethyl ether, diisopropyl ether, and methyl tert-butyl ether. Examples of alcohols include methanol, ethanol, propanol, iso-propanol, n-butanol, and iso-butanol. Example of nitrile is acetonitrile. Examples of halogenated hydrocarbons include dichloromethane, dichloroethane, chloroform, and carbon tetrachloride. Examples of aromatic hydrocarbons include toluene and xylene. Examples of ketones include acetone, methyl ethyl ketone, and methyl isobutyl ketone.

The reaction of 1 -cyanomethylimidazole of Formula (III) with methanesulphonic acid is carried out at about 0°C to about 15°C. In an embodiment, the reaction of 1- cyanomethylimidazole of Formula (III) with methanesulphonic acid is carried out at about 2°C to about 12°C. In another embodiment, the reaction of 1 -cyanomethylimidazole of Formula (III) with methanesulphonic acid is carried out at about 5°C to about I0°C.

The reaction of 1 -cyanomethylimidazole of Formula (III) with methanesulphonic acid is carried out for about one hour to about 5 hours. In an embodiment, the reaction of 1 -cyanomethylimidazole of Formula (III) with methanesulphonic acid is carried out for about one hour to about 4 hours. In another embodiment, the reaction of 1- cyanomethylimidazole of Formula (III) with methanesulphonic acid is carried out for about 2 hours to about 3 hours.

1 -Cyanomethylimidazole methane sulphonate of Formula (V) may optionally be isolated using conventional techniques, for example, filtration, decantation, extraction, distillation, evaporation, chromatography, precipitation, concentration, crystallization, centrifugation, and recrystallization. 1 -Cyanomethylimidazole methanesulphonate of Formula V may be dried using conventional techniques, for example, drying, drying under vacuum, spray drying, suck drying, air drying, or agitated thin film drying.

The reaction of 1 -cyanomethylimidazole methane sulphonate of Formula (V) with carbon disulphide to obtain the compound of Formula (VI) is carried out in the presence of a base in a solvent.

The base is selected from the group consisting of inorganic base or an organic base. Examples of inorganic base include sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, sodium hydrogen carbonate, and potassium hydrogen carbonate. Examples of organic base include triethylamine, N, N’ -diisopropylethyl amine, and I,8-diazabicyclo[5.4.0]undec-7- ene. The base may be added to the reaction mixture either in solid form or in solution form.

The solvent is selected from the group consisiting of polar aprotic solvent. Examples of polar aprotic solvent include dimethyl sulfoxide, dimethylformamide, dimethylacetamide, acetonitrile, or mixture thereof.

The reaction of 1 -cyanomethylimidazole methane sulphonate of Formula (V) with carbon disulphide is carried out at about -10°C to about 10°C. In an embodiment, the reaction of 1 -cyanomethylimidazole methane sulphonate of Formula (V) with carbon disulphide is carried out at about -7°C to about -3°C. In another embodiment, the reaction of 1 -cyanomethylimidazole methanesulphonate of Formula (V) with carbon disulphide is carried out at about 2°C to about 7°C.

The reaction of 1 -cyanomethylimidazole methane sulphonate of Formula (V) with carbon disulphide is carried out for about 2 hours to about 10 hours. In an embodiment, the reaction of 1 -cyanomethylimidazole methane sulphonate of Formula (V) with carbon disulphide is carried out for about 3 hours to 8 hours. In another embodiment, the reaction of 1 -cyanomethylimidazole methanesulphonate acid of Formula (V) with carbon disulphide is carried out for about 4 hours to about 6 hours.

The reaction mixture containing the compound of Formula (VI) is used as such or without isolation.

The compound of Formula (VI) is reacted with 2,4-dichloro-l-[(l>S')-l,2- dichloroethyl]benzene of Formula (VII) to obtain Luliconazole of Formula (I).

The reaction of the compound of Formula (VI) with 2,4-dichloro-l-[(l>S')-l,2- dichloroethyl] benzene of Formula (VII) is carried out at about -10°C to about room temperature.

The reaction of the compound of Formula (VI) with 2,4-dichloro-l-[(l>S')-l,2- dichloroethyl] benzene of Formula (VII) is carried out for about 2 hours to about 10 hours. In an embodiment, the reaction ofthe compound of Formula (VI) with 2,4-dichloro-l-[(l>S')- l,2-dichloroethyl]benzene of Formula (VII) is carried out for about 4 hours to about 8 hours. In another embodiment, the reaction of the compound of Formula (VI) with 2,4-dichloro-l- |( I.S')- l .2-dichlorocthyl |bcnzcnc of Formula (VII) is carried out for about 5 hours to about 7 hours.

The reaction mass containing Luliconazole of Formula (I) is quenched with water followed by extraction with a suitable organic solvent. The organic solvent is selected from the group consisting of halogenated hydrocarbons, esters, hydrocarbons, and mixtures thereof. Examples of halogenated hydrocarbons include dichloromethane, dichloroethane, chloroform, and carbon tetrachloride. Examples of esters include ethyl acetate, methyl acetate, propyl acetate, isopropyl acetate, and butyl acetate. Examples of hydrocarbons include toluene, xylene, and cyclohexane.

The organic layer is acidified with the addition of suitable acid to obtain an acid addition salt of Luliconazole.

The acid is selected from the group consisting of inorganic acid or organic acid. Examples of inorganic acid include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, sulfuric acid, and trifluoroacetic acid. Examples of organic acid include acetic acid, formic acid, citric acid, tartaric acid, benzoic acid, lactic acid, malic acid, fumaric acid, succinic acid, methanesulfonic acid, and benzenesulfonic acid.

The acid addition salt of Luliconazole may optionally be isolated by filtration, decantation, extraction, distillation, evaporation, chromatography, precipitation, concentration, crystallization, centrifugation, and recrystallization. The acid addition salt of Luliconazole may be dried using conventional techniques, for example, drying, drying under vacuum, spray drying, suck drying, air drying, or agitated thin film drying.

The conversion of the acid addition salt of Luliconazole to Luliconazole of Formula (I) is carried out using a base in a solvent.

The base is selected from the group consisting of organic base or inorganic base. Examples of organic base include aqueous ammonia solution, ammonia gas, triethylamine, and ammonium hydroxide. Examples of inorganic base include sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, and potassium hydrogen carbonate.

The solvent is selected from the group consisiting of esters, ethers, alcohols, nitriles, halogenated hydrocarbons, aromatic hydrocarbons, ketones, water, and mixtures thereof. Examples of esters include ethyl acetate, methyl acetate, propyl acetate, isopropyl acetate, and butyl acetate. Examples of ethers include tetrahydrofuran, ethyl methyl ether, diethyl ether, diisopropyl ether, and methyl tert-butyl ether. Examples of alcohols include methanol, ethanol, propanol, iso-propanol, n-butanol, and iso-butanol. Example of nitrile is acetonitrile. Examples of halogenated hydrocarbons include dichloromethane, dichloroethane, chloroform, and carbon tetrachloride. Examples of aromatic hydrocarbons include toluene and xylene. Examples of ketones include acetone, methyl ethyl ketone, and methyl isobutyl ketone. The pH of the reaction mixture is maintained in the range of about 8.0 to about 12.0. In an embodiment, the pH is maintained in the range of about 9.0 to about 11.0. In another embodiment, the pH is maintained in the range of about 9.5 to about 10.5.

Luliconazole of Formula (I) may optionally be isolated by fdtration, decantation, extraction, distillation, evaporation, chromatography, precipitation, concentration, crystallization, centrifugation, and recrystallization. Luliconazole of Formula (I) may be dried using conventional techniques, for example, drying, drying under vacuum, spray drying, suck drying, air drying, or agitated thin film drying.

The present invention provides a novel intermediate 1 -cyanomethylimidazole methane sulphonate of Formula (V).

Formula V

1 -Cyanomethylimidazole methanesulphonate of Formula (V) is characterized by H- NMR, ¹³ C-NMR, IR, and Mass (m/z).

The inventors of the present invention have observed that 1 -cyanomethylimidazole methane sulphonate of Formula (V) is less hygroscopic, easy to handle, can be stored at room temperature, and less susceptible to conversion to 2-( l/7-imidazol- l -yl)acctamidc of Formula (IV) as an impurity.

HPLC chromatogram (Figure 1) of 1 -cyanomethylimidazole hydrochloride shows the presence of about 12.9% of 2-(lf/-imidazol-l-yl)acetamide of Formula (IV) as an impurity. On the other side, HPLC chromatogram (Figure 2) of 1 -cyanomethylimidazole methane sulphonate of Formula (V) shows the presence of 0.04% of 2-( l/7-imidazol- l - yl)acetamide of Formula (IV) as an impurity.

Further, hygroscopicity studies of 1 -cyanomethylimidazole hydrochloride and 1- cyanomethylimidazole methanesulphonate at 25°C/60%RH shows that 1- cyanomethylimidazole hydrochloride absorbs about 5.8% of water within 6 hours and finally melts after 24 hours.

Hygroscopicity studies are summarized below.

While the present invention has been described in terms of its specific aspects and embodiments, certain modifications and equivalents will be apparent to those skilled in the art, and are intended to be included within the scope of the present invention.

The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Methods

Chromatographic Purity was determined using WATERs e2695 (HPLC-16). The column used was an Inertsil ODS 3V (250 X4.6 mm) 5pm.

^J H NMR spectrum was recorded using a Bruker AVANCE III (300 MHz) NMR spectrometer.

IR was recorded using a Agilent Technologies Spectrum (Model Cary 630 FTIR)

Mass was recored using Pexciex API 2000

EXAMPLES

Reference Example: Preparation of 1-cyanomethylimidazole hydrochloride

Imidazole (100 g) and ethyl acetate (450 mL) were added to a round bottom flask (RBF) at room temperature to obtain a reaction mixture. The reaction mixture was stirred for 10 minutes to 15 minutes and then sodium carbonate (155.5 g) and chloroacetonitrile (160 g) were added. The reaction mixture was heated at 75°C to 80°C for 20 hours to 22 hours, then cooled to 30°C to 35°C and filtered to obtain a filtrate. Activated Carbon (10 g) was added to the filtrate and then stirred for 30 minutes to 35 minutes at room temperature. The reaction mixture was filtered through Hyflo bed and washed with ethyl acetate to obtain a filtrate . The filtrate was distilled under vacuum at 40°C to 45°C (680-720 mmHg) to obtain a residue. Acetone (500 mL) was added to the residue and cooled to 0°C to 5 °C. Hydrogen chloride gas was slowly purged into the mixture until the pH was 1.0. The reaction mixture was stirred for 30 minutes at 0°C to 5°C. Excess hydrogen chloride and acetone were recovered at 40°C to 45°C. Acetone (300 mL) was again added to the reaction mixture and then cooled to 0°C to 5°C and stirred for 60 minutes to obtain a solid. The solid was filtered and washed with chilled acetone (200 mL) at 0°C to 5°C. The solid was dried to obtain 1- cyanomethylimidazole hydrochloride.

Yield: 52%

Example 1: Preparation of 1-cyanomethylimidazole (Formula III)

Imidazole (100 g) and ethyl acetate (350 mL) were added to a round bottom flask (RBF) at room temperature to obtain a reaction mixture. The reaction mixture was stirred for 10 minutes to 15 minutes and then sodium carbonate (155.5 g) and chloro acetonitrile (160 g) were added. The reaction mixture was heated at 75°C to 80°C for 20 hours to 22 hours. The reaction mixture was cooled to 30°C to 35°C and then fdtered to obtain a filtrate. The filtrate was distilled under vacuum at 40°C to 45°C (680-720 mmHg) to obtain a residue. Example 2: Preparation of 1-cyanomethylimidazole methanesulphonate (Formula V)

Acetone (750 mL) was added to the residue (as obtained in Example 1) under nitrogen atmosphere and then cooled to 5°C to 10°C. Methanesulphonic acid (100 g) was added drop wise over a period of 45 minutes to 60 minutes under nitrogen atmosphere and then stirred for 2 hours to 3 hours to obtain a solid. The solid was filtered and washed with chilled acetone (200 mL) under nitrogen atmosphere to obtain a residue. The residue was dried under nitrogen atmosphere and then under vacuum at 40°C to 45°C till moisture content is not more than 2%.

Yield: 62%

Chromatographic purity: 99.5%

¹ HNMR (DMSO-d6, 300MHz): 5 ppm: 9.24 (s, 1H), 7.91-7.92 (d, 1H), 7.76-7.77 (d, 1H), 5.6 (s, 2H), and 2.37 (s, 3H)

¹³ CNMR (CDCh, 300MHz): 36.48, 39.77, 114.97, 120.91, 122.27, and 136.92

IR (KBr): 3434.7, 3144-2980, 1634.4, and 1422-1328 cm’ ¹ ;

Mass (m/z): 202.1 [M-l],

Example 3: Preparation of Luliconazole hydrobromide salt

Dimethylsulfoxide (DMSO; 200 mL) and dimethylformamide (50 mL) were added to a RBF at room temperature to obtain a reaction mixture. 1 -Cyanomethylimidazole methanesulphonate (as obtained in Example 2; 52.5 g; Formula V) was added to the reaction mixture and stirred for 10 minutes. Carbon disulfide (39 g) was then added and flushed with DMSO (14 mL). The reaction mixture was cooled to 2°C to 7°C and an aqueous solution of potassium hydroxide (60 g of potassium hydroxide and 60 mL of DM water) was slowly added over a period of 90 minutes to 120 minutes. The reaction mixture was stirred for 2 hours at room temperature and then cooled to -7°C to -3°C to obtain a thick reaction mass. A solution of 2,4-dichloro-l-[(l>S')-l,2-dichloroethyl]benzene (Formula VII; 100 g) in DMSO (100 mL) was added to the reaction mass over a period of 90 minutes to 120 minutes at -7°C to -3°C. The reaction mass was stirred for 5 hours to 6 hours at 0°C to 5°C. Dichloromethane (750 mL) and DM water (800 mL) precooled to 5°C to 10°C were added to the reaction mixture and stirred at room temperature for 30 minutes. The layers were separated and the aqueous layer was extracted with dichloromethane (200 mL). The combined organic layers were washed with DM water (200 mL) and concentrated under vacuum to obtain a residue. Ethyl acetate (800 mL) was added to the residue and heated at 45°C to 50°C for 30 minutes. Carbon (10 g) was added to the reaction mixture and stirred for 30 minutes. The reaction mixture was fdtered through Hyflo bed and washed with hot ethyl acetate (200 mL). The fdtrate was heated at 45°C to 50°C and 33% acetic acid in hydrobromic acid solution (125 g) was added slowly over a period of 30 minutes to 60 minutes. After the completion of addition, the temperature was raised to 60°C to 65°C and stirred for 3 hours. The reaction mixture was cooled to room temperature and again stirred for 30 minutes to obtain a solid. The solid obtained was filtered and washed with ethyl acetate (200 mL) to obtain wet Luliconazole hydrobromide salt.

Yield: 200-210 g wet material

Example 4: Preparation of Luliconazole (Formula I)

Dichloromethane (500 mL) and DM water (300 mL) were added to Luliconazole hydrobromide salt (as obtained in Example 3) at room temperature to obtain a reaction mixture, which was stirred for 30 minutes at room temperature. 25% Aqueous ammonia solution was added to the reaction mixture until the pH was 9.0 to 9.5 and stirred for 30 minutes. The layers were separated and the aqueous layer was extracted with dichloromethane (200 mL). The combined organic layers were washed with DM water (200 mL) and concentrated under vacuum to obtain Luliconazole.

Yield: 105-108 g wet material

Example 5: Purification of Luliconazole (Formula I)

Ethanol (400 mL) was added to Luliconazole (as obtained in Example 4) and heated at 70°C to 75°C for 30 minutes to obtain a clear solution. Carbon (10 g) was added to the solution and stirred for 30 minutes. The hot solution was filtered through Hyflo bed and washed with hot ethanol (200 mL). The filtrate was heated to 50°C and then slowly cooled to room temperature over a period of 60 minutes to 90 minutes. The solution was stirred for 120 minutes at room temperature and then further cooled to 0°C to 5°C over a period of 90 minutes to 120 minutes. The solution was stirred for 60 minutes and then fdtered and washed with chilled ethanol (100 mL) to obtain a solid. The solid was dried under vacuum at 45°C to 50°C to obtain Luliconazole.

Yield: 65%