WO2017093980A1

WO2017093980A1 - Process for preparation of testosterone

Info

Publication number: WO2017093980A1
Application number: PCT/IB2016/057335
Authority: WO
Inventors: Vishal Sukhadeo MAWALE; Naresh Daga PATIL; Dabeer Rauf Karnalkar; Pravin Raghunath Mahajan; Gurvinder Pal Singh; Purna Chandra Ray; Girij Pal Singh
Original assignee: Lupin Limited
Priority date: 2015-12-05
Filing date: 2016-12-03
Publication date: 2017-06-08

Abstract

The present invention relates to a process for purification of 4-androsten-3,17-dione (II) and its conversion to testosterone (I) having purity > 99 % as measured by HPLC.

Description

PROCESS FOR PREPARATION OF TESTOSTERONE

FIELD OF THE INVENTION

The present invention relates to a process for preparation of testosterone (I) having HPLC purity > 99 %.

BACKGROUND OF THE INVENTION

Testosterone (I) is a steroid hormone that belongs to androgen group and is found in all vertebrates. It plays a key role in development of reproductive tissues and secondary sexual characteristics as well as prevention of osteoporosis. Testosterone is chemically known as (8R,9S,10R,13S,14S,17S)-17-hydroxy-10,13-dimethyl-

1,2,6,7,8,9,11,12,14,15,16, 17-dodeca hydrocyclopenta[a]phenanthren-3-one and represented by formula as shown below.

(I) (ID

The synthesis of testosterone is described in several publications, some of these are:

R E Marker et al., in Journal of American chemical society (1941), 63, 777; Norymberski, J. K. and Woods, Gilbert F. in Journal of the Chemical Society (1955), 3426-30; K Morita, in Bulletin of the Chemical Society of Japan (1958), 31, 450-3. The patents relevant to synthesis of testosterone are US 2294433 and 2679502; Indian patent application IN 1483/DEL/2003; PCT applications WO 2007118644 Al and WO 2011000693 Al. Although several documents exist in literature but none of them describe process to obtain highly pure testosterone (I).

It is always desirable to prepare pharmaceutical product of a high purity having a minimum amount of impurities, in order to reduce adverse side effects and to improve the shelf life of active ingredient, as well as its formulation. In some cases it has been found that high purity also facilitates in formulation process.

Therefore, the present invention is directed to provide an improved synthetic process for the preparation of testosterone, having minimum amount of impurities. SUMMARY OF THE INVENTION

The objective of the present invention is to provide a process for purification of 4- androsten-3,17-dione (II).

Another objective of the present invention relates to a process for preparing testosterone (I) having purity > 99 % as measured by HPLC, comprising purification of 4-androsten- 3,17-dione (II) from mixture of tetrahydrofuran and cyclohexane; and its conversion to testosterone (I).

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides testosterone (I) having purity > 99 % as measured by HPLC. The testosterone (I) obtained in the present invention has total impurity content less than 1 %, preferably less than 0.5 %, more preferably less than 0.25%, measured as area percentage by HPLC.

The present invention further provides a process for purification of 4-androsten-3,17- dione (II) comprising the steps of:

i) contacting 4-androsten-3,17-dione (II) with mixture of tetrahydrofuran and cyclohexane,

heating reaction mixture of step i) to 70-80°C,

iii) cooling of step ii) to 25-35 °C,

iv) isolating pure 4-androsten-3,17-dione (II). Purification of 4-androsten-3,17-dione (II) is carried out by contacting it with mixture of tetrahydrofuran and cyclohexane to form the slurry. The slurry is heated to 70-80°C for 1- 2 hrs, then cooled to 30-35°C followed by filtration to obtain 4-androsten-3,17-dione (II) having purity > 98 % as measured by HPLC (phytosterol content < 0.1 %).

The present invention further provides a process for preparation of testosterone (I) having purity > 99 % when measured by HPLC comprising the steps of:

ii) heating reaction mixture of step i) to 70-80°C,

iii) cooling of step ii) to 25-35 °C,

iv) isolating pure 4-androsten-3,17-dione (II), and

v) converting pure 4-androsten-3, 17-dione (II) to testosterone (I)

Pure 4-androsten-3,17-dione (II) of the present invention can be converted to testosterone (I) by the methods known in the literature.

ii) heating reaction mixture of step i) to 70-80°C,

iii) cooling of step ii) to 25-35 °C,

iv) isolating pure 4-androsten-3,17-dione (II),

v) reacting pure 4-androsten-3,17-dione (II) with triethyl orthoformate in alcohol using pyridine hydrochloride to get 17-Keto-3-enol ether (III),

Etcr

(III)

reducing 17-Keto-3-enol ether (III) with reducing agent in alcohol to obtain 3 enol ether (IV),

(IV)

vii) dealkylating 3-enol ether (IV) using hydrochloric acid to testosterone (I) viii) optionally carrying out the purification of testosterone (I).

Reaction of pure 4-androsten-3,17-dione (II) with triethyl orthoformate in alcohol using pyridine hydrochloride to obtain 17-Keto-3-enol ether (III) is carried out at 0-15°C, more preferably 5-10°C for 4-6 hrs.

Reaction of 17-Keto-3-enol ether (III) with reducing agent in alcohol is carried out in presence of base triethyl amine at 0-15°C, more preferably at 5-10°C for 4-6 hrs to obtain 3-enol ether (IV). In situ dealkylation of 3-enol ether (IV) is carried out with inorganic acid such as hydrochloric acid or sulfuric acid at 0-5°C to obtain crude testosterone (I).

The alcohol used in step (v) and step (vi) is selected from group comprising of one or more methanol, ethanol, isopropyl alcohol, n-propyl alcohol, n-butanol, isobutanol and mixture thereof. More preferably methanol or ethanol is used.

The reducing agent used in step (vi) is selected from group comprising of one or more sodium borohydride, potassium borohydride, lithium borohydride, lithium aluminium hydride, dimethyl aluminium hydride, sodium metal, dimethyl aluminium, semicarbazide and mixture thereof. More preferably sodium borohydride or lithium borohydride is used.

The testosterone (I) obtained according to the present invention is optionally purified by contacting testosterone (I) with mixture of methanol and triethylamine, adding water to reaction mixture, heating the reaction mixture at 65-80°C, more preferably 70-75°C, cooling the reaction mixture to 20-30°C, followed by filtration to obtain pure testosterone (I) wherein the phytosterol content is less than 0.1% and the total impurity content is less than 1 %, preferably less than 0.5 %, more preferably less than 0.25%, measured as area percentage by HPLC. The isolated testosterone may be dried using different techniques of drying like tray drying and rotatory drying techniques with or without application of vacuum and/or under inert condition.

The presence of impurities in testosterone may pose a problem for formulation in that impurities often affect the safety and shelf life of a formulation. Therefore, the testosterone prepared by the process of the present invention might be ideal for pharmaceutical formulation, since it is substantially pure.

Testosterone (I) obtained by the process of the present invention may be formulated in conventional manner using one or more pharmaceutically acceptable carriers, excipients, or diluents.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention and specific examples provided herein, without deviating from the scope of the invention. Therefore, it is intended that the scope of the present invention covers the modifications and/or variations that are equivalents.

Examples:

HPLC (High Performance Liquid Chromatography) Chromatographic conditions: Instrument : HPLC equipped with Pump, Injector, UV detector and

Recorder

Column : Zorbax SB C-8 (4.6x150 mm) 3.5 μπι

Wavelength : UV at 200 nm

Flow rate : 2.0 mL/min

Injection volume : 20 μΙ,

Column oven temp : 25 °C

Auto sampler temp : 25 °C

Buffer : Not applicable.

Mobile phase : Water: Acetonitrile (10:90) v/v. Example 1: Purification of 4-androsten-3,17-dione (II)

To a slurry of 200 gm of 4-androsten-3,17-dione (HPLC purity < 97 %) in 150 ml of tetrahydrofuran was added 1000 ml of cyclohexane. The mixture was then stirred at 70- 80°C for 1.5 to 2hrs. The mixture was the cooled to 30-35°C. The solid was filtered , washed with 200 ml of cyclohexane and dried to give 182.6 gm of purified 4-androsten- 3,17-dione (HPLC purity = 99.36%, Retention time = 19.1 min).

Example 2: Purification of 4-androsten-3,17-dione (II)

To a slurry of 200 gm of 4-androsten-3,17-dione (HPLC purity < 97 %) in 150 ml of tetrahydrofuran was added 1000 ml of cyclohexane. The mixture was then stirred at 70- 80°C for 1.0 to 1.5 hrs. The mixture was the cooled to 30-35°C and maintained for 1-2 hrs. The solid was filtered , washed with 200 ml of cyclohexane and dried to give 185.6 gm of purified 4-androsten-3,17-dione (HPLC purity = 99.35 %, Retention time = 19.1 min).

Example 3: Preparation of 17-Keto-3-enol ether (III)

A mixture of 87 ml (78 gm) of triethyl orthoformate and 13 gm was prepared in 350 ml of absolute ethanol and cooled to 5-10°C. To this mixture lOOgm of purified 4-androsten- 3,17-dione was added and stirred at 5-10°C for 6 hrs. 90 ml of triethylamine was the added and mixture is stirred 5-15°C for 20 min. 300 ml of water was then added and the mixture is stirred 25-30°C for 1 hr. The solid obtained was filtered, washed with water and dried to give 98.4 gm of 17-Keto-3-enol ether (III) (HPLC purity = 98.7 %). Example 3: Preparation of testosterone (I)

90 gm of 17-Keto-3-enol ether (III) was added to a mixture of 540 ml of ethanol and 4.5 ml of triethylamine at 0-5 °C and then stirred for 10 min. 10.8 gm of sodium borohydride was then added to this mixture at 0-5°C and then the reaction was maintained at 5-10°C for 6 hrs. To this 450 ml of IN hydrochloric acid was added dropwise at 0-5 °C and after addition the mixture is stirred at 25-30°C for 4-5 hrs. 180 ml of water was added and stirred the slurry at 25-30°C for 1 hr. the solid obtained was filtered, washed with water and dried to give 73.8 gm of testosterone (off white crystalline powder) (HPLC purity = 99.92 %).

Example 4: Purification of testosterone (I)

Testosterone (70 gm) was added to mixture of 70 ml of triethylamine and 500 ml of methanol at 25-30°C, stirred for 20-30 min and filtered. To the clear filtrate 420 ml of water was added with stirring and maintained the slurry formed at 70-75 °C for 20-30 mins, cooled to 25-30°C and stirred for 1 hr. Solid obtained was filtered, washed with water and dried to give 57 gm of pure testosterone (off white crystalline solid) (HPLC purity = 99.95 %, Retention time = 16 min). Melting range = 153.6 - 154.4°C.

Claims

process for purification of 4-androsten-3,17-dione (II) comprising the steps of:

(ID

ii) heating reaction mixture of step i) to 70-80°C,

iii) cooling of step ii) to 25-35°C,

iv) isolating pure 4-androsten-3 , 17-dione (II) .

A process for preparation of testosterone (I) having purity > 99 % when measured by HPLC, comprising the step

(I)

i) contacting 4-androsten-3,l 7-dione (II) with mixture of tetrahydrofuran and cyclohexane,

ii) heating reaction mixture of step i) to 70-80°C,

iii) cooling of step ii) to 25-35°C,

iv) isolating pure 4-androsten-3,l 7-dione (II),

v) converting pure 4-androsten-3,17-dione (II) to testosterone (I).

A process for preparation of testosterone (I) having purity > 99 % when measured by HPLC, comprising the steps of:

ii) heating reaction mixture of step i) to 70-80°C, cooling of step ii) to 25-35°C,

isolating pure 4-androsten-3,17-dione (II),

reacting pure 4-androsten-3,17-dione (II) with triethyl orthoformate alcohol using pyridine to-3-enol ether (III),

(Hi)

reducing 17-Keto-3-enol ether (III) with reducing agent in alcohol to obtain 3-enol ether (IV),

(IV)

vii) dealkylating 3-enol ether (IV) using hydrochloric acid to testosterone (I), viii) optionally carrying out the purification of testosterone (I).

The process according to claim 3, wherein the alcohol used in step (v) and step (vi) is selected from one or more methanol, ethanol, isopropyl alcohol, n-propyl alcohol, n-butanol, isobutanol and mixture thereof.

The process according to claim 4, wherein the alcohol is methanol or ethanol.

The process according to claim 3, wherein the reducing agent used in step (vi) is selected from one or more sodium borohydride, potassium borohydride, lithium borohydride, lithium aluminium hydride, dimethyl aluminium hydride, sodium metal, dimethyl aluminium, semicarbazide and mixture thereof.

The process according to claim 6, wherein the reducing agent is sodium borohydride.

The process according to claim 3, wherein the purification of testosterone (I) in step (viii) comprising the steps of: i) Contacting testosterone (I) with mixture of trimethylamine and methanol, ii) adding water to reaction mixture of step i),

iii) heating the reaction mixture of step ii),

iv) cooling of step iii),

v) isolating testosterone (I).

The process according to claim 8, wherein the heating the reaction mixture of step ii) is carried out at 65-80°C and cooling of step iii) is carried out at 20-30°C.

Testosterone (I) obtained by the process of claim 2, claim 3 and claim 8 having total impurities less than 0.25% when measured by HPLC.