Method for bioconversion of synthesis of trans-hydroxysulfonic (options)

 

(57) Abstract:

New way (two options microbial bioconversion) is suitable for synthesis of TRANS-hydroxysulfonic, which is the predecessor of inhibitors carbonatehydroxide local action. The method of bioconversion carried out in an aqueous carbohydrate medium in the presence of the microorganism Rhodotorula rubra was ATSS 74283 or Rhodotorula piliminae 32762 and substrate connection. The process of cultivation are in aerobic conditions at 20-50oC and pH 4.5 to 8.0, followed by separation of the target product. The method provides diastereomeric surplus of more than 95%, which greatly simplifies and reduces the production of pharmaceuticals for the treatment of ocular hypertension. 2 s and 5 C.p. f-crystals.

Glaucoma is an eye disorder associated with elevated intraocular pressure that is too high for the normal functioning and can lead to irreversible vision loss. If untreated, glaucoma may lead to blindness. Ocular hypertension, i.e., the condition of elevated intraocular pressure without damage to the optic nerve or characteristic of glaucoma field defects, as it is now believed by many modern oualie diastereoisomers, individual enantiomers or mixtures thereof, or ophthalmologist acceptable salt it, where:

A is carbon or nitrogen,

Z - other or-OR,

R - C1-6alkyl linear or branched chain,

R1and C1-5alkyl linear or branched chain, especially n-propyl or isobutyl,

b) C3-5alkenyl, especially allyl,

in) C3-5quinil, especially propargyl,

g) hydrogen or

d) C1-4alkoxy-C1-4alkyl, and

X - -SO2or-C(O)-;

known from U.S. patent N 4797413 and 5157129. Compounds known as inhibitors of carbonatehydroxide local action (TCAI's), useful in the treatment of ocular hypertension. The synthesis of compounds includes the restoration of sulfolane to predecessor TRANS-hydroxysulfonic for the above mentioned compounds. However, the methods of synthesis described for their production, give diastereomeric or racemic products which must be separated and allocated with concomitant loss of at least 50% of the product to get the most active enantiomer.

Now with the present invention provided a new microbiological method of bioconversion intermediate compounds sulfolane in the intermediate compound TRANS-hydrox is sulfone, having the structural formula

< / BR>
in which A and R1- above.

TRANS-hydroxysulfonic is a precursor of the final product, inhibitor carbonatehydroxide the above formula I. the Final product has a local effect in the treatment of ocular hypertension and glaucoma. The method includes the fermentation substrate sulfolane in the presence of the microorganism Rhodotorula rubra, (ATCC 74283) or Rhodotorula piliminae (ATCC 32762), preferably Rhodotorula rubra. The bioconversion is carried out in aerobic conditions immersion in an aqueous carbohydrate medium containing a nutrient nitrogen, at a pH of about 4.5 to 8.0, preferably of 6.0, in a period of time sufficient to obtain a compound of structural formula II.

Received similar TRANS-hydroxysulfonic detects diastereomeric surplus of more than 95%. A key stage in this new way (i.e. control diastereomeric excess of hydroxysulfonic) consists in regulation of the residual concentration of sulfolane in the reaction medium of bioconversion.

Thus, the object of the present invention is a microbiological method for the synthesis of intermediate compounds TRANS-hydroxysulfonic.

The invention concerns a synthesis of C5 alkyl linear or branched chain, especially n-propyl or isobutyl,

b) C3-5alkenyl, especially allyl,

in) C3-C5quinil, especially propargyl,

g) hydrogen or

d) C1-4alkoxy-C1-4alkyl,

which is the precursor compounds of the formula

< / BR>
the individual diastereomers, the individual enantiomers or mixtures thereof, or ophthalmologist acceptable salts, where:

A is carbon or nitrogen,

Z - other or-OR,

R - C1-6alkyl linear or branched chain,

R1and C1-5alkyl linear or branched chain, especially n-propyl or isobutyl,

b) C3-C5alkenyl, especially allyl,

in) C3-C5quinil, especially propargyl,

g) hydrogen or

d) C1-4alkoxy-C1-4alkyl, and

X - -SO2or-C(O)-.

The new method of this invention involves the fermentation of the microorganism Rhodotorula rubra or Rhodotorula piliminae, preferably Rhodotorula rubra in the presence of the substrate of compound III, as shown

< / BR>
in which R2< / BR>
a) C1-5alkyl linear or branched chain, especially n-propyl or isobutyl,

b) C3-5alkenyl, especially allyl,

in) C3-5AI environment under aerobic conditions before the formation of substantial amounts of compound II and isolation of the compounds obtained in the usual way. Compounds of structural formula I are suitable for the treatment of glaucoma.

The preferred embodiment of this invention, in which the compound represented by formula II

< / BR>
in which A is carbon or nitrogen and R represents:

a) C1-5alkyl linear or branched chain, especially n-propyl or isobutyl,

b) C3-5alkenyl, especially allyl,

in) C3-5quinil, especially propargyl,

g) hydrogen or

d) C1-4alkoxy-C1-4alkyl,

which is a precursor compounds represented by formula I

< / BR>
the individual diastereomers, the individual enantiomers or mixtures thereof, or ophthalmologist acceptable salt, where A is carbon or nitrogen,

Z - other or-OR,

R - C1-6alkyl linear or branched chain,

R1and C1-5alkyl linear or branched chain, especially n-propyl or isobutyl,

b) C3-5alkenyl, especially allyl,

in) C3-5quinil, especially propargyl,

g) hydrogen or

d) C1-4alkoxy-C1-4alkyl, and

X - -SO2or-C(O)-;

includes the stage of culturing a microorganism Rhodotorula rubra, (ATCC 74283) in a nutrient medium containing assimilated sources nitrogen is tlenol chain especially n-propyl or isobutyl,

b) C3-5alkenyl, especially allyl,

in) C3-5quinil, especially propargyl,

g) hydrogen or

d) C1-4alkoxy-C1-4alkyl,

in aerobic conditions until, until it forms a significant amount of compound II, and the selection of the obtained compound, and the substrate is dissolved in approximately 1-15%.about. ethanol, methanol or DMSO, the number of the loaded substrate is about 1-3, l, a temperature of 20-50 supportoC and a pH of about 4.5 to 8.0.

Substrate compound III can be synthesized as not limiting the invention method:

Methyl-3(R)-hydroxyhexanoate.

Methyl-3-clohexane (44,7 g, 310 mmol) was diluted with methanol (75 ml) and 0.2 n HCl (2 ml). Add Et2NH2+ Ru2Cl5-(BINAP)2(200 mg) and the mixture is heated at 60oC under hydrogen pressure 7,0307 kg/cm2(100 lb/DM2within 2 hours Add toluene (100 ml) and the mixture concentrated to education oil weighing 75 g

Methyl-3(R)-tolylsulfochloride.

The crude solution of methyl-3(R)-hydroxyhexanoate (310 mmol) dissolved in pyridine (100 ml) and add n-tolylsulfochloride (59 g, 3100 mmol). The mixture paramesh bytony reagent. The mixture is then poured into 20% toluene/hexane (600 ml) and washed with water (3 x 250 ml). The organic layer is concentrated to obtain 83 g of the product in the form of oil 95% purity.

Methyl-3(S)-(2-tiofoto)hexanoate.

n-Utility (1,64 M, which is 97.6 ml, 160 mmol) is added to the thiophene (15,1 g, 180 mmol) in THF (100 ml) at -20oC. After stirring for 30 min add sulfur (5,23 g) in parts. Then after 1 h, add deoxygenating formamide (100 ml) followed by addition of methyl-3(R)-tolylsulfochloride (40 g, 33.3 mmol). The mixture is stirred at room temperature for 24 h and then diluted with ethyl acetate (100 ml) and water (50 ml). The layers are separated and the hold back extraction of the aqueous portion with ethyl acetate (100 ml). The combined organic substances concentrated to obtain the product as a yellow oil weighing 23,9, the Total yield of methyl-3-ketohexose is 74%.

5,6-Dihydro-6(S)-(propyl)-4H-thieno[2,3 b]thiopyran-4-one.

Methyl-3(S)-(2-tiofoto)hexanoate (125 g, 513 mmol) is heated at 100oC with acetic acid (150 ml) and concentrated HCl (150 ml) for 96 hours, the Mixture is extracted with toluene (2 x 300 ml). The combined organic layers are washed and concentrated to obtain a dark brown masotti (126 g, 600 mmol). After 45 min the mixture was washed with water (2 x 200 ml) and concentrate to obtain 151 g of oil, which is transferred to the next stage without purification.

5,6-Dihydro-6(S)-(propyl)-4H-thieno[2, 3b]thiopyran-4-one-7,7-dioxide

5,6-Dihydro-6(S)-(propyl)-4H-thieno[2, 3b] thiopyran-4-one (4,25 g, 20 mmol) is dissolved in ethyl acetate (80 ml). Add the sodium tungstate (660 mg, 2 mmol), 30% hydrogen peroxide (8.2 ml, 80 mmol) and 10 drops of sulfuric acid. After 24 h the reaction mixture was diluted with ethyl acetate (100 ml) and washed with 10% solution of Na2SO3and a saturated solution of NaHCO3. The organic layer is concentrated and triturated with ethanol to obtain 4.5 g of product (95%).

The microorganisms.

Biologically pure sample of Rhodotorula piliminae separated from the larvae Drosophyla piliminae, Hawaii, and he is publicly available on the basis of the Budapest agreement in the permanent collection of cultures American type culture collection, 12301 Parklawn Drive in Rockville, Maryland, in which it is registered under the number ATCC 32762. Biologically pure sample of Rhodotorula rubra is publicly available in the permanent collection of cultures American type culture collection, 12301 Parklawn Drive in Rockville, Maryland, in which it is registered under the number ATCC 74283. Any limitation Rhodotorula rubra ATCC 74283 isolated from infected cultured cream.

Analytical methods, which are mainly used in the present invention, but not limit it, are the following:

Analytic methods.

Measurements of biomass.

Biomass was measured by optical density and dry weight of cells. Optical density measurement performed using a Hewlett Packard 8451A kit spectrophotometer with diode unit at 660 nm. The mass of dry cells determined using milliprobe filter type, pore size of 0.45 nm.

The glucose.

Glucose monitor liquid chromatograph, equipped with a classic Macintosh computer for data logging and data accumulation, the detector of the absolute refractive index, autoinjection A1-2 Dynamax, analytical HP pump, pressure module and a Biorad Aminex HPX-87H ion-exclusion column (300 x 78 mm), heated to 60oC. the Eluent consists of 0.005 M sulfuric acid at 0.7 ml/min

Extraction of the broth.

Whole broth is extracted by adding an equal volume of ethyl acetate. The mixture is placed in a shaker for 5 min, then centrifuged at 2000 Rev/min for 10 min using a centrifuge Beckman TJ-6. The obtained supernatant is dried and re-suspended in methanol DL is layer chromatography using F254 plates of silica gel 60 with a preliminary coating Russ. name. The mobile phase consists of 94% methylene chloride, 5% methanol and 1% ammonium hydroxide. Samples applied to the plate and dried. One edge of the plate is immersed in the mobile phase and allow the solvent to rise over the plate until, until it reaches approximately 25.4 mm from the top of the plate.

Liquid chromatography high resolution.

GHUR performed with a Rainin system, equipped with classic Macintosh computer for data logging and data accumulation, Dynamax detector optical density UV-M, autoinjection A1-2 Dynamax, two analytical HP pumps, pressure module with Bond Aminex RX-C8 column (a 4.6 x 250 nm), which is supported at room temperature. The method uses two eluent, methanol and water (with 0.1% vol./about. H3PO4) at a total flow rate of 1.5 ml/min and UV detection at 254 nm. The method includes the gradient of from 30/70 methanol/acidified water (vol./about.) to 70/30 (vol./about.) within 15 minutes the Way parts of CIS-hydroxysulfonic, TRANS-hydroxysulfonic and sulfonate 9.5, 10 and 12.8 min, respectively.

Example 1.

Methods of cultivation.

Production of TRANS-hydroxysulfonic occurs, for example, during the cultivation with uzivanjem or in shake flask, to orogenic glycerine cell suspensions (1 ml) in an Erlenmeyer flask with a capacity of 250 ml, containing 50 ml of Sabouraud's dextrose broth. Sabouraud's dextrose broth commercially available and contains 10 g/l Difco of neoperene and 20 g/l Bacto dextrose. Flasks incubated for 24 h at 28oC under stirring with a speed of 220 Rev/min in order to obtain sufficient biomass for use as inoculum. The inoculum is transferred (5%/25 ml) in 2-liter Erlenmeyer flask containing 500 ml of Sabouraud's dextrose broth. Then the culture is incubated at 28oC with stirring at 180 Rev/min for 42 hours, the Cells centrifuged, washed with MES buffer at pH 6.0 and re-suspended in MES buffer at pH of 6.0 before adding sulfolane.

Bioreactor cultivation is carried out in a fermenter with a capacity of 23 litres, which inoculant 2-liter flask Sabouraud's dextrose broth containing Rhodotorula rubra ATCC 74283, cultured for 42 h, as described above. The parameters of the fermenter following: temperature 28oC, stirring at 200 Rev/min (minimum starting point), aeration (10 l/min and back pressure 0,0422 kg/cm2. Pressure of dissolved oxygen support mixing at the level of at least 40%. When the rate of oxygen uptake (OUR) falls below 5 mmol/l/h, the cells are harvested under sterile conditions.

Bioconversion and isolation of TRANS-hydroxysulfonic II (5,6-dihydro-4 (S)-hydroxy-6(S)-propyl-4H-thieno-[2, 3b]thiopyran-7,7-dioxide).

Aliquot samples of the broth (10 or 50 ml) containing cells of Rhodotorula rubra, centrifuged at 4000 Rev/min for 10 min using a centrifuge Beckman TJ-6 and decanted supernatant. The pellet re-suspended in 0.5 M 2-[N-morpholino] acanalonia acid buffer (MES) at pH 6.0 and centrifuged again. The washed cells are re-suspended in 50 ml of MES buffer. If necessary, the cells are diluted to facilitate analysis of the degree of reaction in ethanol (3% vol./vol.), add to the flask containing the washed cells. The flask is incubated at 32,5oC in a water bath, which continuously shaken. The broth is collected by extraction with chloroform (1: 1, vol/about.) or ethyl acetate (1:1, vol/vol.), dried and re-suspended in methanol. Then conduct analyses of extracts of thin-layer and liquid chromatography high resolution (IHVR). The retention time GHUR for TRANS-hydroxysulfonic, CIS-hydroxysulfonic and sulfolane to 9.5, and 10.0 and 12.8, respectively.1H NMR results for TRANS-hydroxysulfonic (250 MHz, CDCl3) is shown below: Delta 7,58(d, J=5,1 Hz, 1H), was 7.08 (d, J=5,1 Hz, 1H), 4,94 (t, J=3,6 Hz, 1H), 3,66 (m, 1H), 2,6-2,1 (m, 3H), 1,7-1,5 (m, 3H), 1,01 (t, J=7,01, 3H).

Cells after bioreactor cultivation harvested when OUR falls below 5 mmol/l/h, centrifuged and washed with 0.5 M MES buffer at pH of 6.0. Cells re-suspended in 0.5 M MES buffer at pH 6 and returned to the fermenter. The parameters of the fermenter set for the bioconversion, the following: temperature of 32.5oC, stirring at 400 Rev/rpm and aeration of 6 l/min and back pressure 0,0422 kg/cm2. Sulfonate (1.5 g/l) dissolved in DMSO and added to the fermenter (3% vol. /about. ). Samples are taken periodically to monitor the activity of bioconversion by GHWR. Speed bierny excess, reach for collecting cells. The broth is extracted with ethyl acetate (0,5:1, vol/vol.), and phase solvent concentrated using a rotary evaporator.

Example 2.

Bioconversion of TRANS-hydroxysulfonic (5,6-dihydro-4(3)-hydroxy-6(S) -methyl-4H-thieno-[2, 3b] thiopyran-7, 7-dioxide).

Cell culture ATCC 74283 stored on Sabouraud's dextrose beveled agars at 4oC, is used to inoculate Erlenmeyer flask with a capacity of 250 ml containing 50 ml of Sabouraud's dextrose broth. After incubation for 20 h at 28oC with shaking the ketosulfone, dissolved in ethanol (33.3 mg/ml), added to the flask (1 ml per flask). Culture returned in the same conditions of incubation for an additional 24 hours Residual ketosulfone and produced hydroxysulfonic extracted from the broth one volume of chloroform. The TLC analyses and GHUR show the complete transformation of sulfolane in TRANS-hydroxysulfonic. Yarm analyses confirm the structure of TRANS-hydroxysulfonic: Delta of 7.6 (d, 1H, C2-H), and 7.1 (d, 1-H, C3(H) a 4.9 (m, 1H, C4-H), and 3.8 (m, 1H, C6-H), 3,5-3,0 (user. s, 1H, HE), and 2.6 (m, 1H, C5(H) to 2.4 (m, 1H, C5(H) a 1.5 (d, 3H, C6-CH3).

Example 3.

In this example, the method of obtaining compounds fusim way.

Stage 1. Procedure:

TRANS-hydroxysulfonic (7,32 g, 29.7 mmol) suspended in acetonitrile (38 ml) in a round bottom flask with a capacity of 100 ml, equipped with a magnetic stirrer, thermocouple and nitrogen inlet. The solution is cooled to 0oC and add sulfuric acid (5 ml, 88.7 mmol) in parts. The reaction mixture was then stirred at room temperature. The mixture is cooled to 0-5oC. the Flask 500 ml load water (34 ml) and acetonitrile (43 ml), the mixture is then cooled to 0-5oC and mix well. Then gently add the reaction mixture so that the temperature remained below 5oC. Saturated potassium carbonate (30 ml) were then added to establish a pH of the water layer between 7-8 or as long as you do not stop the emissions of carbon dioxide. The organic layer was concentrated in crude oily material (14.1 g), pilot analysis of 42.5 wt.%. The output is of 5.99 g (73%).

Stage 2. Procedure:

A round flask with a capacity of 100 ml, equipped with a magnetic stirrer and a thermocouple download chlorosulfonic acid (13,14 ml) and the acid is cooled to 0-5oC. Acetaminoohen (6,57 g) added in portions over 30 min so that the internal temperature was below 15oC. the Dark reaction mixture is then heated honowai acid and sulphonylchloride) acetaminoohen, the mixture is cooled to room temperature. Then added dropwise thionyl chloride (13,14 ml). After adding the dark mixture is heated to 45oC. After 16 h remains < 0.5% square sulfonic acids. The mixture is cooled to 0-5oC. Liter flask is charged with water (325 ml) and cooled to 0oC. the Mixture to chlorination then added dropwise within 30 min to mix well cooled solution, so that the internal temperature remains below the 5oC. the Mixture is stirred for 45 min and then filtered. The wet cake is washed with cold water (10 ml) and dried in a stream of nitrogen. Concentrated aqueous ammonia (24 ml) and THF (43 ml) are loaded into a flask of 250 ml, equipped with a magnetic stirrer and a thermocouple. The mixture is cooled to about -10oC. the Crude wet solid sulphonylchloride add portions over 1 h, maintaining the internal temperature below 0oC. After 2 h remains less than 1.8% of sulphonylchloride.

The excess ammonia is neutralized aqueous hydrochloric acid (about 50 ml). The aqueous layer was washed with THF twice. Layers of THF are combined and concentrated. The material is suspended again in THF and carefully add water. Formed brown crystals and remains yellow Vodnye 3. Procedure:

Acetamidomalonate (4,21 g, 11.5 mmol) is dried by distillation with THF (2 x 100 ml portions) in the flask 250 ml Flask supplied with a magnetic stirrer, thermocouple and nitrogen inlet. Suspension of acetamidomalonate 22.5 ml of THF then cooled to 0-5oC. borane-THF (51 ml, 51 mmol) is added dropwise over 45 min, keeping the internal temperature below 5oC. After the evolution of hydrogen (20 min) the solution is heated to 30-35oC. After completion of the reaction (3 h) the mixture is cooled to room temperature. A round bottom flask with a capacity of 250 ml, equipped with a magnetic stirrer, thermocouple and nitrogen inlet, loaded with sulfuric acid (60 ml) and cooled to 0-5oC. the Reaction mixture is then carefully dispense in a well-mixed acid solution, maintaining the internal temperature below 20oC. After the addition the mixture is stirred at room temperature until completion of hydrogen evolution. The flask is then set for distillation (1 ATM) and the mixture is concentrated until the internal temperature reaches > 97oC. After distillation, the mixture is cooled to 20oC. the Mixture was then neutralized aqueous potassium bicarbonate and extracted with ethyl acetate (100 ml). The organic layer is concentrated with paragraph 1. The method of obtaining the compound represented by formula II

< / BR>
in which A is carbon or nitrogen and R1is: a) C1-5alkyl linear or branched chain, especially n-propyl or isobutyl; b) C3-5alkenyl, especially allyl;) C3-5quinil, especially propargyl; g) hydrogen or d) C1-4alkoxy-C1-4alkyl,

characterized in that it comprises the stage of culturing a microorganism Rhodotorula rubra was ATSS 74283 or Rhodotorula piliminae ADS 32762 in a nutrient medium containing assimilated sources of nitrogen and carbon and the substrate compound of formula III

< / BR>
in which (a) C1-5alkyl linear or branched chain, especially n-propyl or isobutyl; b) C3-5alkenyl, especially allyl;) C3-5quinil, especially propargyl; g) hydrogen or d) C1-4alkoxy-C1-4alkyl,

in aerobic conditions until, until it forms a significant amount of compound II, and the allocation of the compounds obtained.

2. The method according to p. 1, characterized in that the microorganism is Rhodotorula rubra was ATSS 74283, the substrate is dissolved in 1 to 15% vol./about. ethanol, methanol or DMSO when the number of the loaded substrate 1 to 3 g/L.

3. The method according to p. 2, characterized in that the substrate rustwater, the process is conducted at a temperature of 20 - 50oC and pH 4.5 to 8.0.

5. The method according to p. 1, wherein the process is conducted at a temperature of 30 - 35oC and pH from about 5.5 to 6.5.

6. The method of obtaining the compound represented by formula

< / BR>
in which A is carbon or nitrogen and R1is: a) C1-5alkyl linear or branched chain, especially n-propyl or isobutyl; b) C3-5alkenyl, especially allyl;) C3-5quinil, especially propargyl; g) hydrogen or d) C1-4alkoxy-C1-4alkyl,

characterized in that it comprises the stage of culturing a microorganism Rhodotorula rubra was ATSS 74283 in a nutrient medium containing assimilated sources of nitrogen and carbon substrate and the compound III

< / BR>
where (a) C1-5alkyl linear or branched chain, especially n-propyl or isobutyl; b) C3-5alkenyl, especially allyl;) C3-5quinil, especially propargyl; g) hydrogen or d) C1-4alkoxy-C1-4alkyl,

in aerobic conditions until, until it forms a significant amount of compound II, and the selection of the obtained compound, and the substrate is dissolved in 1 to 15% vol./about. ethanol, methanol or DMSO when the number of the loaded substrate 1 to 3 g/l, the process Eaut 1 - 3% vol. /about. DMSO when the number of the loaded substrate 1 to 3 g/l and the process is conducted at a temperature of 30 - 35oC and pH 5.5 to 6.5.

 

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