WO2001009365A1

WO2001009365A1 - Process for the preparation of 2-cyano-5-hydroxypyrazine

Info

Publication number: WO2001009365A1
Application number: PCT/EP2000/006862
Authority: WO
Inventors: Andreas Kiener; Toru Magasawa; Eva Maria Urban
Original assignee: Lonza AG
Current assignee: Lonza AG
Priority date: 1999-07-29
Filing date: 2000-07-18
Publication date: 2001-02-08
Anticipated expiration: 2002-01-29
Also published as: AU6275300A

Abstract

Described is a process for the preparation of 2-cyano-5-hydroxypyrazine of formula (I), which process comprises reacting 2-cyanopyrazine of formula (II) with a microorganism selected from the genera Alcaligenes, Comamonas, Delftia, Pseudomonas, Serratia, Acinetobacter, Agrobacterium, Brevibacterium, Micrococcus and Rhodocossus or with relevant enzyme extracts derived from any of the above microorganisms.

Description

Process for the preparation of 2-cyano-5-hy roxypyrazine

Description

The invention relates to a novel process for the microbiological preparation of 2-cyano-5- hydroxypyrazine of the formula

from 2-cyanopyrazine of the formula

2-Cyano-5-hydroxypyrazine is a promising intermediate for the preparation of analogues of pyrazineamide for the treatment of tuberculosis (Cynamon et al , J Med Chem 39, 1996, 3394-3400)

Access to 2-cyano-5-hydroxypyrazine is conceivable via chemical routes, however such a process suffers from being not economically feasible

EP-A 0 558 022 describes the conversion of 3-cyanopyridine to 3-cyano-6-hydroxypyridine by means of microorganisms of the species Micrococcus morrhuae (IAM 171 1), Comamonas acidovorans (NCIMB 9289), Comamonas testosteroni (ATCC 1 1996), Pseudomonas dacunhae (ATCC 13261), Pseudomonas maltophila (ATCC 13637), Pseudomonas chlororaphis (IFO 3904), Pseudomonas hydantoinophilum (FER.MP-4347), Pseudomonas putida (ATCC 21244), Sarcina lutea (ATCC 9341), Serratia liquefaciens (IFO 12979), Serratia marcescens (IFO 3054), Serratia marcescens (IFO 12648) and Xanthobacter flavus (NCIMB 10071) The conversion of 2-cyanopyrazine to 2-cyano-5-hydroxypyrazine is not exemplified The object of the present invention, therefore, was to provide a simple and environmentally friendly method for regioselectively producing 2-cyano-5-hydroxypyrazine from 2-cyanopyrazine in excellent quality

This object is achieved with the process according to claim 1

Microorganisms used in the present invention are selected from the genera Alcaligenes, Comamonas, Delftia, Pseudomonas, Serratia, Acinetobacter, Agrobacterium, Brevibacterium, Micrococcus, Kocuria and Rhodococcus Alternatively, also the relevant enzyme extracts derived from the microorganisms mentioned above can be used Methods for recovering the enzyme extracts from the cells such as ultrasonic- or frenchpress-method are known to those skilled in the art

Possible microorganisms for the biotransformation belonging to the genus Alcaligenes are Alcaligenes faecalis or Alcaligenes xylosoxydans as exemplified by the species Alcaligenes faecalis IAM 1446 (Research Institute of Applied Microbiology, Tokyo, Japan), Alcaligenes faecalis IAM 12369 (Research Institute of Applied Microbiology, Tokyo, Japan), Alcaligenes faecalis IFO 14479 (Institute for Fermentation Osaka, Japan), Alcaligenes xylosoxydans IFO 13495 (Institute for Fermentation Osaka, Japan) and Alcaligenes sp IFO 14130 (Institute for Fermentation Osaka, Japan)

Possible microorganisms for the biotransformation belonging to the genus Comamonas are Comamonas testosteroni as exemplified by the species Comamonas testosteroni IAM 12419 (Research Institute of Applied Microbiology, Tokyo, Japan) and Comamonas acidovorans (Pseudomonas fluorescens)

Possible microorganisms for the biotransformations belonging to the genus Pseudomonas are Pseudomonas fluorescens or Pseudomonas putida as exemplified by the species Pseudomonas fluorescens TN5 (Isolation described in Nagasawa et al , Biosci Biotech Biochem 58(4), 1994, 665-668), this one was also identified belonging to Delftia acidovorans, formerly Comamonas acidovorans, Pseudomonas putida 870, and Pseudomonas putida CRl-1 (Isolation described in Nagasawa et al , J Biol Chem 257 (22), 1982, 13749-13756) Possible microorganisms for the biotransformation belonging to the genus Serratia are Serratia marcescens or Serratia fonticola as exemplified by

Serratia marcescens IAM 1162 (Research Institute of Applied Microbiology, Tokyo, Japan), Serratia marcescens IAM 12143 (Research Institute of Applied Microbiology, Tokyo, Japan), Serratia marcescens IAM 13543 (Research Institute of Applied Microbiology, Tokyo, Japan), Serratia fonticola IAM 13541 (Research Institute of Applied Microbiology, Tokyo, Japan)

Possible microorganisms for the biotransformation belonging to the genus Acinetobacter are Acinetobacter cycloclastes as exemplified by

Acinetobacter cycloclastes IAM 1013 (Research Institute of Applied Microbiology, Tokyo, Japan)

Possible microorganisms belonging to genus Agrobacterium are Agrobacterium tumefaciens as exemplified by

Agrobacterium tumefaciens IAM 12048 (Research Institute of Applied Microbiology, Tokyo, Japan)

Possible microorganisms for the biotransformation belonging to the genus Brevibacterium are Brevibacterium acetylicum or Brevibacterium lines as exemplified by Brevibacterium acetylicum IAM 1790 (Research Institute of Applied Microbiology, Tokyo, Japan) and Brevibacterium lines IAM 12437 (Research Institute of Applied Microbiology, Tokyo, Japan)

Possible microorganisms for the biotransformation belonging to the genus Micrococcus are Micrococcus roseus or Micrococcus varians as exemplified by

Micrococcus roseus IAM 13 15 (Research Institute of Applied Microbiology, Tokyo, Japan), this one was also identified belonging to Kocuria rosea and Micrococcus varians IAM 13594 (Research Institute of Applied Microbiology, Tokyo, Japan)

Possible microorganisms for the biotransformation belonging to the genus Rhodococcus are Rhodococcus luteus or Rhodococcus sp as exemplified by

Rhodococcus luteus NCIB 1 1743 (National Collection of Industrial Bacteria, Aberdeen, UK). Rhodococcus sp NCIB 10554 (National Collection of Industrial Bacteria, Aberdeen, UK) Preferred microorganisms for the biotransformation are microorganisms of the species Serratia fonticola IAM 13541 , deposited at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig) on June 4, 1999 under DSM No 12855, Delftia acidovorans (Comamonas acidovorans, Pseudomonas fluorescens TN 5), deposited at the DSMZ on June 4, 1999 under DSM No 12853, Kocuria rosea (Micrococcus roseus) IAM 1315, deposited at the DSMZ on June 4, 1999 under DSM No 12854, and its functional equivalent variants and mutants

The most preferred microorganisms for the biotransformation are Serratia fonticola IAM 13541 (DSM 12855) and Kocuria rosea (Micrococcus roseus) JAM 13 15 (DSM 12854)

The term "functional equivalent variants and mutants" designates microorganisms having essentially the same properties and functions as the original microorganism Such variants and mutants can be formed incidentally e g by UV radiation

Scientific description of microorganism DSM 12854 identified as Kocuria rosea (Micrococcus roseus)

Properties of the strain

characteristica gram-positive coccs Motility Spores Catalase +

Aerobic Growth + Anaerobic Growth +w

Peptidoglycan-Typ A3α, L-Lys-L-Ala₂. 16S rDNA Sequence Similarity 100 % with Kocuria rosea

+w = weak / retarded reaction Scientific description of microorganism DSM 12855 identified as Serratia fonticola

Properties of the strain

Cell form rods

Width μm 0,7 - 0,8

Length μm 1,2 - 2,0

Motility +

Gram-Reaction

Lysis by 3 % KOH + Aminopeptidase (Cerny) +

Oxidase

Catalase +

Acid from (ASS)

Glucose +

D-Xylose +

Erythritol +

Adonitol + D-Mannose +

Rhamnose +

Inositol + α-Methyl-D-glucosid +

Cellobiose Maltose +

Lactose +

ONPG (O-nitrophenylgalactosidase) + ADH (alcohol dehydrogenase) LDC (lactate decarboxylase) +

ODC (ornithine decarboxylase) +

VP (Voges Proskauer) Indole

Malonate +

Use of Citrate degradation (Simmons) + Phenylalanindesaminase (24 h) Urease

Methyl red test +

Hydrolysis of DNA Tween 80

Scientific description of microorganism DSM 12853 identified as Delftia acidovorans

Properties of the strain

Cell form rods Width μm 0 7 - 0 8

Lenght μm 2 5 - 4 0

Gram-Reaction Lysis by 3 % KOH + Aminopeptidase (Cerny) +

Spores

Oxidase + Catalase +

ADH Urease Hydrolysis by Gelatine

Nitrate reduction +

Use of Substrates

Adipate + Citrate

Glycolate +

Malate +

Phenylacetate +

D-Glucose Maltose

Fructose +

Mannitol + β-Alanine +

L-Histidine + Benzoate

The microorganisms are usually cultivated before the biotransformation takes place However, it is also possible that cultivation and biotransformation is performed during the growth of the

The cultivation is conveniently performed in standard media known to the skilled artisan such as for example low molecular weight phosphate buffers, HEPES buffers, citrate buffers, borate buffers, Tris-HCl or Glycine-NaOH containing conventional nutrients like the conventional carbon-, nitrogen-, energy- and mineral sources, or in complete media

Preferred media are mineral salt media with a defined carbon- and nitrogen source, for example complete media containing, for example, yeast extract as exemplified in example 1 Cultivation is expediently effected under aerobic conditions at a temperature from 15 °C to 50 °C and at a pH from 4 0 to 9 0 over a period of up to 96 h

Suitably, the effective enzymes are induced before the biotransformation takes place Effective inducers are, e g , nicotinic acid, pyrazinecarboxylic acid, 3-cyanopyridine, 2-cyanopyrazine, nicotinamide or pyrazine amide

The biotransformation then takes place with either batchwise or continuous substrate (2-cyanopyrazine) addition

The biotransformation can be performed with growing or with resting cells, the biotransformation with resting cells is preferred

Appropriate concentration of 2-cyanopyrazine is between 0 1 % by weight and 30 % by weight, preferably between 0 5 % by weight and 15 % by weight

The biotransformation is expediently carried out under aerobic conditions at a temperature from 15 °C to 50 °C, preferably from 25 °C to 45 °C, and at pH values in the range of 4 0 to 9 0, preferably 5 0 to 8 0

The biotransformation can be performed in standard media known in the art as mentioned above or in complete media such as "Nutrient Yeast Broth" (NYB)

In general conversion is complete after 1 h to 48 h

Examples:

Example 1: Hydroxylation activity for 2-cyanopyrazine with different microorganisms

To a 500 ml shaking flask 40 ml of a medium containing per 100 ml water 1 0 g nicotinic acid, 1 0 g meat extract, 0 1 g yeast extract, 1 0 g malic acid, 0 1 g

and 1 ml of a metal salt mixture containing per 1000 ml water 400 mg CaCl₂ x 2 H₂O, 300 g H3BO3, 40 mg CuSO₄ x 5 H₂O, 100 mg KJ, 200 mg FeSO₄ x 7H₂O, 400 mg MnSO₄ x 7 H₂O, 200 mg

NaMoO₄ x 2 H₂O and 10 ml HC1 cone The pH of the final medium was adjusted to 7 0 and one of the following microorganism listed in Table 1 was added The microorganisms Pseudomonas fluorescens TN 5 and Pseudomonas putida CR l - 1 were isolated according to the receipt given in Biosci Biotech Biochem 58(4), 1994, 665-668 and J Biol Chem 257(22), 1982, 13749-13756 All other microorganisms were obtained from the depositories indicated The mixture was incubated for 24 hours at 28 °C

The standard reaction mixture (2 ml) was composed of 0 4 ml of an 1 M aqueous solution of 2-cyanopyrazine, 0 6 ml of 0 3 M potassium phosphate buffer (pH 6 5) and 1 ml of the concentrated cell suspension obtained after centrifugation from 10 ml of the culture broth mentioned above The reaction started with the addition of the 2-cyanopyrazine as substrate and was carried out at 35 °C for 30 minutes Addition of 2 ml acetonitrile stopped the reaction Table 1 shows the results of the hydroxylation activity of each microorganism

HPLC analysis conditions

Column water spherisorb S5 OD S2 (4 6 x 150 cm)

Solvent 10 mM KH₂PO₄ x H₃PO₄ (pH 2 5) CH₃CN = 9 1

Flowrate 1 0 ml/min

Detection 230 nm Injection 5 μl

Retention time of 2-cyanopyrazine was measured to be 3 3 mi 11 and 2-cyano-5-hydroxy- pyrazine was measured to be 2 min Table 1

Table 1 (continued)

Example 2:

Hydroxylation of 2-cyanopyrazine with Comamonas acidovorans (Pseudomonas fluorescens) TN 5

10 ml of a standard reaction mixture (see example 1) containing 200 mM 2-cyanopyrazine and 300 mg of the wet cells was shaken at 35 °C at 180 rpm for 24 h

After removing the cells the reaction mixture (30 ml) was subsequently evaporated, whereby remaining 2-cyanopyrazine could be eliminated 100 ml distilled water was then added to the residue to dissolve the remaining product The mixture was then applied to a Dowex 1 x 2 (H⁺ form) column ( 13 x 230 mm) The column was washed with distilled water and then with 0,01 N acetic acid to elute 2-cyano-5-hydroxy- pyrazine The product fractions were combined and evaporated Crystallisation of the product in ethanol led to 45 mg of the title product Fig 1 shows the accumulation of 2-cyano- 5-hydroxypyrazine by Pseudomonas fluorescens TN5

Structure was confirmed by Η-NMR and ^I3C-NMR

Η-NMR (DMSO-d_fi, MHz 400) δ 8 01 (1H, d, J = 1 2 Hz) 8 36 (1H, d, J = 1 2 Hz)

13 0 (1H, br s)

^I C-NMR (DMSO-d₆, MHz 400) δ 155,0 (s)

149 9 (d) 137 6 (s)

116 6 (s) 107 3 (d) Example 3:

Hydroxylation of 2-cyanopyrazine with Serratia fonticola IAM 13541

The effect of various concentrations of 2-cyanopyrazine on the rate of hydroxylation was investigated for Serratia fonticola IAM 13541. For these experiments, the amount of 2-cyanopyrazine was changed with the standard reaction mixture The final volume and optical density of the individual tests were kept constant by using a 2 M solution of 2-cyanopyrazine and defined amount of water to adjust the volume. The incubation was carried out at 35 °C for 30 min The highest conversion rate during this initial period of 30 min at an optical density of 4 7 was determined to be 24 nmol/min/ml (Fig 2) in presence of 250 mM 2-cyanopyrazine After 24 h the molar conversion of 200 mM 2-cyanopyrazine to 2-cyano-5-hydroxypyrazine was determined to be 50 % or 100 mM (Fig 3) 2-Cyano-5-hydro\ypyrazine was isolated from this bioconversion as described in Example 2 Structure was confirmed by H-NMR and ^"C-NMR

Example 4:

Hydroxylation of 2-cyanopyrazine with Serratia marcescens IAM 1162 at different pH values

The effect of pH on the hydroxylation was investigated The potassium phosphate buffer 90 mM (pH 6 5) in the standard reaction mixture (see example 1 ) was replaced by the following buffers at a final concentration of 90 mM

Sodium citrate-citrate (pH 4 0, 5 0, 5 5 and 6 0), KH₂PO₄-K₂HPO₄ (pH 6 0, 6 5, 7 0, 7 5), Tris-HCl (pH 7 5, 8 0, 8 5, 9 0) and Glycine-NaOH (pH 9 0, 9 5, 10 0) The optimum pH for the hydroxylation of 2-cyanopyrazine by Serratia marcescens IAM 1 162 was determined to be 5 5 (Fig 4) Example 5:

Temperature dependence of 2-cyanopyrazine hydroxylation by Serratia marcescens

IAM 1162

The optimum temperature was tested in 2 ml of the standard reaction mixture (see Example 1 ) containing 90 mM sodium citrate-citrate buffer (pH 5 5) and 200 mM 2-cyanopyrazine and 68.2 mg of the respective cells (dry weight) The reaction was performed for 30 min on a shaker (180 rpm's) at various temperatures. As shown in Fig 5, the optimum temperature was determined to be 50 °C

Example 6:

Tests of example 3 (best hydroxylation rate ), 4 (pH optimum) and 5 (temperature optimum) were also performed with Micrococcus roseus IAM 1315 and Serratia fonticola IAM 13541

Claims

Process for the preparation of 2-cyano-5-hydroxypyrazine of the formula

which process comprises reacting 2-cyanopyrazine of the formula V-^CN II

^<*>

N with a microorganism selected from the genera Alcaligenes, Comamonas, Delftia, Pseudomonas, Serratia, Acinetobacter, Agrobacterium, Brevibacterium, Micrococcus, Kocuria and Rhodococcus or with relevant enzyme extracts derived from any of the above microorganisms

Process according to claim 1, characterised in that the l eaction is effected under aerobic conditions at a temperature of 15 °C to 50 °C and at a pH of 4 0 to 9 0

Process according to claim 1 or 2, characterised in that the concentration of 2-cyanopyrazine in the medium is between 0 1 % by weight and 30 % by weight

Process according to any one of claims 1 to 3, characterised in that the microorganisms are selected from the species Serratia fonticola IAM 1 541 (DSM 12855) and Kocuria rosea (Micrococcus roseus) IAM 1315 (DSM 12854) and its functional equivalent variants and mutants