Field of the invention

The present invention relates to an improved synthesis of 5-hydroxyproline. More specifically, such an improved process of the present invention is to provide a commercially viable intermediate for the synthesis of 5-hydroxytryptophan.

Back ground of the invention

3-Hydroxyproline and 4-hydroxyproline are the basic components of collagen, the most abundant protein in vertebrates (Krane, S. M., Amino Acids, 2008, vol. 35, 703). Both of these proline derivatives are non-proteinogenic amino acids and play an important role in the stabilization of the collagen triple helix (Friess,W., Ed., Adv. Drug Delivery Rev. 2003, vol. 55, pp 1529-1530 ; Kotch, F.W.; J. Am. Chem. Soc. 2008, vol. 130, 2952). On the other hand, 5-hydroxyproline accumulates in injured tissues and its concentration is a parameter associated with their wound healing capacity (Agalar, F., Hamaloglu,E., Daphan, C, Tarim, A., Onur, R., Renda, N., Sayek, I., Aust. N. Z. J. Surg., 2000, vol. 70, 739 ; Eroglu, E., Agalar, F., Altuntas, I., Eroglu, F. T., J. Exp. Med. 2004, vol. 204, 11). In addition, 5-hydroxyproline has been used as a building block for the preparation of other proline derivatives (Chung, J. Y. L., Wasicak, J. T., Arnold, W. A., May, C. S., Nadzan, A. M., Holladay, M. W., J. Org. Chem. 1990, vol. 55, 270; Sacchi, A., de Caprariis, P., Mayol, L., De Martino, G., J. Heterocycl. Chem. 1995, vol.32, 1067 ) and a versatile precursor for the synthesis of conformationally restricted peptidomimetics (Manzoni, L., Colombo, M., May, E., Scolastico, C, Tetrahedron 2001, vol. 57, 249 ; Belvisi, L., Colombo, L., Colombo, M., Di Giacomo, M., Manzoni, L., Vodopivec, B., Scolastico, C, Tetrahedron 2001, vol. 57, 6463; Belvisi, L., Colombo, L., Manzoni, L., Potenza, D., Scolastico, C, SynLett, 2004, 1449; Manzoni, L., Arosio, D., Belvisi, L., Bracci, A., Colombo, M., Invernizzi, D., Scolastico, C, J. Org. Chem. 2005, vol. 70, 4124).

5-Hydroxyproline is a key intermediate for the synthesis of 5-hydroxytryptophan (Kunihiko, I., Akihiko I., Tohru N., Tokuro O-I., Chem. Pharm. Bull., 1984, vol. 32, 2126-2139). 5-Hydroxytryptophan is a naturally occurring amino acid, commercially obtained from the seeds of the Griffonia simplicifolia, an African medicinal plant. 5-hydroxytryptophan (5-HTP) has been used in Alternative Medicine as an aid to relieve the symptoms of anxiety, depression, fibromyalgia, insomnia, chronic headaches, premenstrual syndrome, binge-eating related to obesity, attention deficit disorder and chronic headaches. 5-HTP has also been used in treating certain seizures and symptoms of Parkinson's disease. 5-HTP is often sold as a dietary supplement (http://www.drugs.com/mtm/5-hydroxytryptophan.html)

Different processes for obtaining 5-hydroxyproline, by synthesis, are known in the art. For instance Chiesa, M. V., Manzoni, L., Scolastico, C, SynLett, 1996, 441 discloses the reduction of the gamma-lactamic carbonyl group of a protected pyroglutamic acid, using DIBAL-H or LiEt3BH, the synthesis as shown in the Scheme 1. However, the use of these expensive reagents (DIB AL-H or LiEt3BH) and the difficulty in handling the air and moisture sensitive reagents make the process unsuitable for large scale manufacturing.

Scheme-1

The second method reported in the prior art starts from L- glutamic acid, but, suffers from same disadvantage because of using expensive and air sensitive DIBAL-H or LiEt3BH (Scheme 2).

Scheme-2

Third method reported in literature starts from substituted cyclobutane alcohol (Scheme -3), which are difficult to obtain in a commercial scale. The synthesis of 5-hydroxyproline derivatives using cyclobutane serine analogs as starting materials was reported in the literature (Marta P-F., Alberto A., Jesus H. B., Jesus M. P., Fernando R, ARKIVOC 2010 (iii) 191-202).

The methods available in the prior art utilize either expensive and air and moisture sensitive reagents like DIBAL-H or LiEt3BH, which are non-suitable for the scaled up synthesis of 5-hydroxylproline derivatives. Even the approach of using cyclobutane derivatives is not practical for manufacturing. Since 5-hydroxyproline is a key intermediate for a commercially marketed 5- hydroxytryptophan , there is a need to develop a cost effective and scalable process for making 5-hydroxyproline.

OBJECTIVE OF THE INVENTION

The present invention consists of the use of commercially available and inexpensive reagent, sodium borohydride, for the reduction of lactamic carbonyl group in a suitably protected pyroglutamic acid ester. The innovation is based on the understanding that N-acyl protected lactams behave more like ketones and such groups can be reduced with reagents like sodium borohydride which is safe to handle and inexpensive. Sodium borohydride a commercially available reagent. The present invention relates to novel reduction process and the process for making 5-hydroxyproline and its derivatives. Other reagents that are suitable for the reduction of a keto group can also be used for this process.

Synthetic Scheme:

(Where in R is alkyl, aryl, aralkyl and Rx is alkyl, aryl, alkoxy, arylalkoxy etc)

DETAILED DESCRIPTION

The main aspect of the of the present invention is to use N-acyl / N-alkoxycarbonyl protection to L-pyroglutamic acid ester, which renders resistant lactamic carbonyl into a susceptible keto group thus facilitating the reduction by the inexpensive and commercially available sodium borohydride or other keto group reducing agents. The method is general and could be used for the reduction of any lactamic carbonyl groups.
The present invention relates to an improved synthesis of 5-hydroxyproline. More specifically, such an improved process of the present invention is to provide a commercially viable intermediate for the synthesis of 5-hydroxytryptophan.
5-hydroxytryptophan can be synthesized by following two process:

Process I : Process for preparing protected 5-hydroxyproline which comprises the following steps.

a) N-alkoxycarbonyl /acyl protected L-pyroglutamic acid ester was dissolved in methanol and cooled to less than 0°C ;

b) adding sodium borohydride in 0.5 to 5.0 mole ratio, preferably 1.0 to 2.0 mole ratio at a temperature of-30°C to 0°C ;

c) treating the reaction mixture with aqueous NaHC03 solution (15g of NaHC03 in 115 ml of water);

d) isolation of the product, protected 5-hydroxyproline, by extraction with organic solvent such as dichloromethane and removal of the solvent to yield a desired product as a pale yellow viscous oil. The product is a mixture of isomers.

Process II : Process for preparing protected 5-hydroxyproline which comprises the following steps.

a) N-alkoxycarbonyl /acyl protected L-pyroglutamic acid ester was dissolved in methanol and cooled to 0°C.

b) adding sodium borohydride 0.5 to 5.0 moles at 0-5°C and stirring for 1 h.

c) the reaction mixture was quenched with acetic acid and solvent was removed.

d) The residue obtained was dissolved in ethyl acetate and washed the ethyl acetate layer with water and brine solution. The organic layer was dried over sodium sulphate and concentrated under vacuum . The crude reaction product was purified further by column chromatography.

The N-alkoxycarbonyl groups as described in the above process are COOR, where R is alkyl, preferably methyl or t-butyl. The N-acyl groups as described in the above process are COR, where R is alkyl, preferably methyl.

The N-alkoxycarbonyl /acyl protected L-pyroglutamic acid esters are alkyl, preferably, methyl, ethyl, t-butyl or benzyl. The reduction process is carried out between -30°C to 20°C, preferably at - 10°C.

Best method of performing the invention is described in example 4. The following examples are illustrative uses of the invention but not limitative to the scope there of.

Example 1: Synthesis of N-BOC- L-pyroglutamic acid methyl ester

In a 500 mL two- necked RB flask, L-Pyro glutamic acid methyl ester (8.9 g, 0.0622 mol) was dissolved in dichloromethane (100 mL)at room temperature. Triethylamine (17.4 mL, 0.124 mol) of and catalytic amount of dimethylaminopyridine (200 mg) were added to the reaction mixture and cooled the reaction contents to 0 - 5°C. Di-tert- butyl dicarbonate (16.2 g, 0.743 mol) was added to the reaction mixture at the same temperature, slowly. The reaction mixture was allowed to raise to room temperature and stirred for overnight. After completion of the reaction (monitored by thin layer chromatography), the reaction mixture was diluted with dichloromethane (500 mL) and washed successively, with water and brine solution. The organic layer was separated, dried over sodium sulphate and concentrated under vacuum. The crude product obtained was purified by column chromatography using methanol and chloroform mixtures, as eluents. The pure product was obtained in the range of 40-80% yield.

Characterization data:

HNMR (CDC13): 8 1.50 (s, 9H); 3.80 (s, 3H); 4.60-4.65 (d, 1H); 2.10-2.15 (t, 1H); 2.30-2.31 (t, 1H); 2.45-2.50 (t, 1H); 2.60-2.70 (t, 1H)

Example 2: Synthesis of N-(t-butyloxycarbonyl)- L-pyroglutamic acid methyl ester
In a 5 L four-necked RB Flask, L-Pyro glutamic acid methyl ester (350 g, 2.447 mol) of was dissolved in dichloromethane (1750 mL) at room temperature. Triethylamine (482 g , 4.772 mol) and dimethylaminopyridine (10 g) were added to the reaction mixture and cooled the contents of the reaction to 0-5°C. Di-tert.-butyl dicarbonate (640 g, 2.935 mol) was added to the reaction mixture at the same temperature, slowly and the reaction mixture was allowed to raise to room temperature and stirred for further 26 h. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water (500mL) and the organic layer was separated and the organic layer was washed successively with aq. HC1 (1%, 500 mL) and of brine ( 500 mL) solution. The organic layer was concentrated under vacuum. The concentrate was triturated with Hexane (750 mL) at 10 to 15°C and the product formed was filtered, dried at room temperature to obtain the desired product in 50 - 90 % Yield. Recrystallization from mixtures of dichloromethane and hexane afforded pure colorless crystalline solid in 40 - 80% yield.
Example 3 : Synthesis of 1-tert-butyl- 2-methyl 5-hydroxypyrrolidine -1,2-dicarboxylate
In a 250 mL two-necked RB flask, N-(t-butyloxycarbonyl)-L-pyroglutamic acid methyl ester (6.3 g, 0.0259 mol) was taken and dissolved in of methanol (100 mL). The reaction contents were cooled to 0°C and at this temperature, was added sodiumborohydride (37.5 g, 0.02592 mol), slowly over a period of 15 min. The reaction mixture was stirred at 0-5°C for further 1 h. After completion of the reaction (monitored by TLC ), the reaction mixture was quenched with acetic acid and methanol was removed under vacuum. The residue obtained was dissolved in ethyl acetate and washed the ethyl acetate layer with water and brine solution. The organic layer was dried over sodium sulphate and concentrated under vacuum. The crude reaction product was purified further by column chromatography using methanol and chloroform mixtures, as eluents. The pure product (1-tert-butyl- 2-methyl 5-hydroxypyrrolidine -1,2-dicarboxylate) was obtained in 60-85% yield.

Characterization data:

'HNMR (CDC13): 8 1.50 (s, 9H); 3.80 (s, 3H); 5.50-5.60 (d, 1H); 4.20-4.40 (m, 1H); 2.00-2.30 (m, 4H)

Example 4: Synthesis of 1-tert-butyl- 2-methyl 5-hydroxypyrrolidine -1,2-dicarboxylate
In a 1000 mL four-necked RB flask, N-(t-butyloxycarbonyl)-L-Pyroglutamic acid methyl ester (50g, 0.2055 mol) was dissolved in methanol (500 mL) under stirring at room temperature. The methanol solution was cooled to - 10 deg C with ice-salt cooling bath followed by the lot wise addition of solid NaBH4 (9.6g, 0.256 moles, in six lots) during 1.0 h at -10 deg C. After the complete addition of NaBH4, the clear solution of reaction mass was maintained under stirring for further period of 2 h at -10 deg C. After this period, the reaction mixture was treated slowly with aq. NaHC03 solution (15g of NaHC03 in 115 ml of water) for 10 min and stirring was continued for another 15 min and the salts formed were filtered, washed with methanol. The filtrate was diluted with water (110 mL) and extracted with dichloromethane (150 mL x 3 times). The combined dichloromethane layer was washed with water ( llOmL) and separated the organic layer . T he organic layer was distilled off under reduced pressure to give 40-48 g ( 80-95 % yield) of the desired product as a pale yellow viscous oil. The product is a mixture of isomers.

Example 5: Preparation of L-Pyro glutamic acid benzyl ester :

In a 250 mL three necked RB flask, to a mixture of L-Pyro glutamic acid (30 g, 0.2325 mol), dimethyl formamide (23.5 mL), potassium carbonate (40g, 0.2829 mol) was added Benzyl bromide (39.7 g, 0.2321 mol) dropwise at room temperature and stirred for further 2 h at same temperature. The reaction mixture was heated to 80°C and maintained at the same temperature for further 4 h. After completion of reaction (monitored by TLC), the reaction mixture was cooled to room temperature, diluted with water (100 mL) and extracted with dichloromethane (2X 200 mL; 1X100 mL; 1X50 mL). The combined organic layer was washed with water (100 mL) and dried over sodium sulfate. The solvent was distilled off under vacuum to obtain the desired product as pale yellow liquid in 60-85% yield.

Example 6: Preparation of N-(t-butyIoxycarbonyI)-L-Pyro glutamic acid benzyl ester:
In a 250 mL three necked round bottomed flask, L-Pyro glutamic acid benzyl ester (30g, 0.1369 mol) was dissolved in dichloromethane (lOOmL), cooled to 10°C and added triethylamine (29 g, 0.2871 mol), catalytic amount of dimethylaminopyridine (1 g) and di-tert.-butyldicarbonate (35.15 g, 0.1612 mol) slowly at 10 to 15°C. Reaction mass allowed to raise to room temperature, stirred for further 24 h at the same temperature, diluted with water (150 mL). The organic layer was separated , washed successively with aq. HC1 (1%, 150 mL), brine (150mL) and concentrated under vacuum to obtain the desired product as pale yellow liquid in 70-90% yield. The oily product slowly converted into a solid on storage for a week at room temperature.

WE CLAIM:

1. Process for preparing protected 5-hydroxyproline which comprises the following
steps.

a) dissolving N-alkoxycarbonyl /acyl protected L-pyroglutamic acid ester in methanol and cooling to -30°C to 0°C ;

b) adding 0.5-5.0 mole ratio of sodium borohydride at a temperature of-30°C to 0°C;

c) treating the reaction mixture with aqueous NaHC03 solution ;

d) isolating protected 5-hydroxyproline, by extracting with organic solvent and removing the solvent.

2. The process as claimed claim 1, wherein the amount of Sodium borohydride is added preferably in 1.0 to 2.0 mole ratio.

3. The process as claimed claim 1, wherein the N-alkoxycarbonyl groups are COOR, where R is alkyl, preferably methyl or t-butyl.

4. The process as claimed claim 1, wherein the N-acyl groups are COR, where R is alkyl, preferably methyl.

5. The process as claimed claim 1, wherein the esters are alkyl, preferably, methyl, ethyl, t-butyl or benzyl.

6. The process as claimed claim 1,wherein the temperature of reduction is -30°C to 0°C, preferably-10°C.