DE2348471C3 - Process for the preparation of 4 (R) -hydroxycyclopentane-1,3-diones substituted in the 2-position - Google Patents

Description

in which R is a hydrogen atom or a group the general formula

- (CH ₂ ) ^ - COOR '

means, where χ is an integer from 2 to 8, or a compound of the general formula exercise venous, reproductive, kidney and gastrointestinal systems of animals and humans.

The availability of natural prostaglandins is currently quite limited, and methods of using them Manufacture are long and tedious and only result in extremely small amounts of the compounds. A Another disadvantage of the known processes for the production of prostaglandins and their analogs are the common separation processes that must be carried out ίο in order to obtain the desired optically active Get connections.

It is the object of the present invention to provide a process for the preparation of important intermediate products for the synthesis of prostaglandins and their analogues to develop the desired have optical activity.

The invention relates to a process for the preparation of 4-hydroxy-cyclopentane-1,3-diones substituted in the 2-position, in which one compounds of the general formula

OCH ₃

in which R is a hydrogen atom or a group of the general formula

CH ₂ -CH = CH- (CH ₂ ) ,. - COOR '

is, in which y is an integer from 3 to 5 and R 'denotes the radical of an alkanol with 1 to 6, preferably 1 to 2 carbon atoms, in the presence of a microorganism of the groups Endomycetales, Mucorales, Moliliales and Eurotiales from the class of Ascomycetes are fermented under otherwise customary conditions and the desired reaction product is separated off in the customary manner, such as by column chromatography.

The invention relates to a process for the preparation of 4-hydroxy-cyclopentane-1,3-diones substituted in the 2-position the important intermediates for the production of natural prostaglandins and whose analogs are.

The invention relates to a process for the preparation of 4-hydroxy-cyclopentane-1,3-diones substituted in the 2-position, which is characterized by it. that one substituted in the 2-position cyclopentane-1,3,4-triones or 3-alkoxy-2-cyclic-1, 4-diones substituted in the 2-position exposes certain microorganisms to the enzymatic action of fermentation, whereby the desired optically active compounds are obtained.

The prostaglandins, which are cyclic, oxidized C ₂₀ fatty acids on the basis of the prostate acid chain, and their analogues have achieved great importance because of the wide range of physiological effects they have on the cardiovascular system. Respiratory, endocrine, hijmostalischc. Ner-

in which R is a hydrogen atom or a group of the general formula (CH ₂ ) ^ - COOR 'in which χ is an integer from 2 to 8, or a compound of the general formula

in which R is a hydrogen atom or a group of the general formula

CH ₂ - CH = CH - (CH ₂ ) ,. - COOR '

means in which y is an integer from 3 to 5, and R 'is the radical of an alkanol having 1 to 6, advantageously 1 to 2 carbon atoms in the presence of a microorganism from the groups Endomycetales, Mucorales, Moliliales and Eurotiales from the Ascomycetes class fermented under otherwise customary conditions and the desired reaction product is separated off in the customary manner, such as by column chromatography.

Even if the free acids themselves can be used, it has been shown that with Use of esters gives higher yields. The preferred starting compounds are those in which ν = 6 and R 'is an alkyl radical with 1 to 6 carbon atoms means.

Some microorganisms have estcrase activity.

which cause the partial hydrolysis of the ester group in the radical R. can. However, this activity can be increased by adding a small amount of allyl alcohol too the fermentation medium are completely inhibited.

It is known that the Kctofunklion in the 4-position of the above compounds by hydrogenation in the presence of a palladium-carbon catalyst can be reduced (see R. Pappo.

ρ Collins and C. Jung, Annals of New York Academy of Sciences, Vol. 180, 64 [1971]). However, the hydroxy compound obtained is a racemate. In contrast, the hydrogenation of the oxo group in the 4-position can be carried out stereospecifically by utilizing the fermentative action of a microorganism according to the method according to the invention, the 4 (R) configuration (according to the Cahn-Ingold-Prelog name) being obtained This configuration is necessary for the conversion of the manufactured compounds into the natural prostaglandins. An example of such a conversion, in which the final product obtained is prostaglandin PGE ₁ , is shown schematically below:

(D

ι ο

according to the invention

CO ₂ CH ₃

Q ₁ H ₅ COCIzN (C ₂ H ₅ ).,

O - CO - QH ₅

CO ₂ CH ₃

(Uli

Na [AlH ₂ (OCH ₂ CH ₂ OCH ₃ I ₂ ]

CO ₂ CH ₃

(iv:

CO ₂ CH.,

(V)

HO

The conversion of IV to V can easily be carried out according to the procedure described by Charles J. S ih et al. In J. Amer. Chem. Soc, 94, 3643 (1972). From the stereospecific 2- (6'-carbomethoxyhexyl) -4 (R) -hydroxy-cyclopentane-1,3-dione produced according to the invention, the product corresponding to _{the natural prostaglandin E 1 can therefore be produced without difficulty while maintaining the particular slereospecific configuration.}

In the following examples, the stereoisomerism of the 4-hydroxyl group was determined by circular dichroism analysis. The R epimer shows a negative Cotton effect ([Θ] · 10 " ³ -85 °) at / · 281πΐμ and a positive Cotton effect ([θ · 10 ~ ³ + 87 °) at / · 262πΐμ, which is characteristic is for such a system.

The suitability of a particular organism for the reduction according to the invention can easily be determined can be determined by the following examination procedure.

General investigation procedure

to determine the reduction effect

of a special organism

Sabouraud's agar slants or other agar-based media suitable for growth were inoculated with the microorganism. The inoculated agar slant was placed in an incubator kept at 25 C and left there for 1 week. It was then removed and 15 cm ^{3 of} sterile water was added. The spores and plant growth were removed from the agar with a sterile needle. The suspension was placed in a flask ^{containing 50 cm 3 of} the soy dextrose medium described below. The flask was then placed on a rotary shaker in an incubator maintained at 25 ° C and shaken at 210 rpm for 24 hours. After this initial period (first inoculum) 5 cm ^{3 of} the submerged culture were placed in each double flask with 3 different media, namely soy dextrose, cerelose edamin, and dxtrin corn water, the composition of which is given below. The flasks were placed in a shaker and the microorganisms were allowed to grow at 25 ° C for 24 to 48 hours. 25 mg of cyclopentane-1,3,4-triene, substituted in the 2-position, in 25 cm ^{3 of} dimethylformamide were added to each of a pair of flasks as substrate. To the other flask of each pair was added 0.25 cm ^{3 of} dimethylformamide for comparison. All flasks were shaken under the same conditions for an additional 24 hours and then removed from the shaker. The growth properties and pH were noted, the entire broth in each flask was acidified to pH 2.0 with dn-HCl and extracted once with a volume of ethyl acetate equal to the volume of the fermentation broth. The solvent was removed from each drawer by heating it in a water bath to approximately 60 ° C. The residues were each dissolved in 1 cm ^{3 of} acetone and analyzed using thin-layer chromatography.

The samples examined were then all placed on plates with silica gel G using an appropriate Systems chromatography, with a preferred solvent system ethyl acetate-acetic acid

re-isooctane-Η, Ο (110: 20: 50: 100) was. After During development, the plates were viewed under ultraviolet light. The following Rf values were obtained for the compounds of interest.

CO ₂ CH ₂ CH,

Rf = 0.57

CO ₂ CH ₃

O O Rf = 0.48

CO ₂ H

O O Rf = 0.32

CO ₂ CH ₇ CH

' ₂ <~ JI ₂ ^ n,

HO O Rf = 0.18

HO O Rf = r 0.09

Dextrin corn water

Dextrin I ⁽ⁱ ü

Corn Wasaer SO g

KH ₇ PO ₄ 1 g

NaCl 5 g

Water 11

pH 7.0

Autoclave 30 minutes at 1.1 kg cm ³ .

IO The general test method given above, the nutrient media and the fermentation method in the following examples are only exemplary and can vary in different ways!

v / earth. So can other nitrogen and carbon sources in the nutrient media (e.g. corn flour, oat flour, meat extract) or other protein hydrolysates or sucrose, glucose. Maltose. Strength. Molasses can be used instead of dextrin.

Other modifications customary in fermentations can also be carried out - the addition time of the substrate after the addition of the medium can be varied, the initial pH value for the addition and the conversion of the substrate can be varied from about 5.0 to about 7.5, the amount of the substrate and the stirring speed can be changed.

The products obtained in the following examples were determined using the ultraviolet, infrared and nuclear magnetic Resonance spectra and characterized by thin-layer chromalography.

example 1

2 - (6 '- carbomethoxyhexyl) - 4 (R) - hydroxy - cyclopentane-1,3-dione (II) was made as follows:

CO ₂ CH,

CO ₇ H

40

The composition of typical nutrient media, the door to the above-mentioned examination method and The batch fermentation in the following examples are suitable are the following:

Soy dexirose

Soybean meal 5 g

Dextrose 20 ti

NaCl 5 g

K ₂ HPO ₄ 5g

Yeast 5 g

Water 11

pH 7.0

Autoclave at 1.1 kg / cm ^{1 for} 15 minutes.

S5

CO ₂ CH.,

Ccielosc-iidamine

Cerelose 'raw dextrose) 50 g

Edamin *)

Corn water

water

pH value 7.0

*) Enzymal hydrolyzate of milk prolein.

A. Fermentation

The surface growth of 1 week old Dipodascus uninucleatus agar slant was suspended in 5 cm ^{3 of} saline (0.85%). 2 cm ³ portions of this suspension were used to ^{inoculate 50 cm 3 of} the above soybean dextrose medium in 250 cm ³ ErIcnmeyer flasks (FI stage). The flasks were incubated for 24 hours at 25 ° C. on a rotary shaker (250 revolutions minute, radius 5.1 cm) and then 10 percent by volume of this with each of four 2-1 Frlency flasks (F-2 stage). containing 500 cm ^{3 of} the soybean dextrose medium. After incubation for 24 hours on a rotary shaker,

250 mg of 2 - (6 '- carbomethoxyhexyl) - cyclopentane-1,3,4-trione (I) (J. Katsube and M. Matsui, J. Agr. Biol. Chem., 33, 1078 [1969]) in 2 cm ^{3 of} dimethylformamide was added to each flask. The F-2 flasks were then incubated for an additional 24 hours under the conditions indicated for the incubation of the FI stage.

B. Isolation

24 hours after the addition of the 2-carbomethoxyhexyl-cyclopentane-1,3,4-trione (I), the cells were centrifuged off. The supernatant liquid was brought to a pH of 2.0 with 6N HCl and extracted three times with 1.51 ethyl acetate. The ethyl acetate was _{dried over Na 2} SO ₄ and evaporated to an oil. This residue was dissolved in 5 cm ^{3 of} benzene-ethyl acetate (1: 1) and applied to a column (32 × 2.5 cm) which had been charged with silica-diatomaceous earth (85:15). The column was eluted with a gradient system consisting of 500 cm ^{3 of} 50% ethyl acetate-benzene in the mixing chamber and 50 cm ^{3 of} pure ethyl acetate in the storage container and the eluate was ^{collected in 7 cm 3} fractions. Fractions 25 to 104, which contained the desired product, were combined and concentrated to dryness, 1.2 g of a crystalline residue being obtained. Recrystallization from ethyl acetate-petroleum ether gave 750 mg of 2-carbomethoxyhexyl-4 (R) -hydroxy-cyclopentane-1,3-dione (II), melting point 90 to 92 ° C; [<0 "= +19.93" (C ₁ = 1.02CHCl ₃ ); /.[ “■
[0] x 10 ^-3 = -85 ° at
+ 87 ° at /. 262 πΐμ).

B e i s ρ i c 1 c 2 to 67

The procedure of Example 1 was repeated using that given in the following table Microorganism was applied. The desired product 2-carbomethoxyhexyl-4 (R) -hydroxy-cyclopentane-1,3-dione was obtained in each case in the yield given in the table.

The abbreviations Door the depository given in the table mean:

WB = Professor Kenneth B.R a per, Dept. of

Bacterology, University of Wisconsin, ^4S Madison, Wis. 53 706 USA. ATCC = American Type Culture Collection, 12 301

Parklawn Dr., Rockville, Md. USA. CBS = Centraalbureau voor Schimmelcultures, Baarn, Holland.

NRRL = Nothein Regional Research Labs, U.S. D.A., Peoria, III. USA.

Example

Introductory no.

organism

Order - - Endomycetales

yield

WB 1125 Byssoclamys fulva 57%

WB 1629 Dipodascus 70%

uninucleatus

ATCC 14625 Dipodascus aggrcgatus 17%

ATCC 12934 Dipodascus albidus 11%

WB Y 22 Zygosaccharomyces 50%

priorianus

WBYl 598 Zygosaccharomyces 19% ashbya

example

16 17

18th

²⁷² ™ µ (23,500); CD, /. 281 π \ μ and [fi>] · 10 " ³ =

19 20 21

25th

55

32 33 34 35 36 37

60 Laying-down organism
No.

Order hndomveciales

38 39 WB 1304
WB X196
WB 1839
WB 1489

WB 1593
WB 1554

CBS 317

CBS 320
ATCC 4798
WBY87
WBY301
WB 1828

WB 1752

WB1118
WB 1695
WB 10045
WB 1982
WB 1085
WB 5057

WB 5058
WB 4896

yield

WBY24 Saccharomyccs

cerevisiae
ATCC 10607 Saccharomyces

cerevisiae var. odessa WBY610 Saccharomyces ■ -

cerevisiae fragilis
WBYlOIl Saccharomyces -

cerevisiae acidifaciens WB Y1140 Saccharomyces -

cerevisiae lactis

WB Y1974 Saccharomyces

cerevisiae dobzanskii

WB Y 25 Endomycopsis fibuliger 44% WB Y1483 Endomycopsis 38%

javaanesis

WB Y 37 Hansenula anomala 38% CSS 352 Schizosaccharomyces 31% pombe

WBY854 Schizosaccharomyces 27% octosporum

With- deposit organism Ausspiel Nr. Booty

Order Mucorales

Absidia blakesleeana Absidia regnieri
Mucor rammannianus Zygorhynchus
heterogamus (±)
Phascolomyces
articulosus
Phycomyces
blakesleeanus

Order Molilialcs

Rhodotorula
aurantiaca
Rhodotorula pallida Geotrichum candidum Torulopsis pulcherrima Candida krusei
Gliocladium
fimbriatum
Gliocladium
vermoeseni
Paecilomyces varioti Stachybotrys lobulata Trichoderma viride Memnoniella echinata Gliocladium roseum Fusarium
decemcellulare
Alternaria tenuis
Gliocladium
catenulatum

609 616/300

Io

at Backing organism Out game No. prey Order burotialcs 40 WB717 Pcnicillium striatum 37% 41 WB 2031 P. claviforme 21 ",, 42 WB 5680 P. pseudostromaticuin 19 ': ,, 43 WB 4717 P. roqueforti 22% 44 WB 874 P. cascicolum 3% 45 WB 973 P. expansum 17% 46 WB 1061 P. purpurogenum 47 WB1115 P. varioti

20 "/ ,,

15%

16% 38% 26 "/" 15% 25 "/ ,, 7% 14%

I I

40 ",, oxy-2-cyclopcntcn-1,4-dione was used as a substrate for the conversion into the desired product.

Example 69

2 - (6 '- Carboethoxyhexyl) - 4 (R) - hydroxy - cyclopentane-1,3-dione (IV) was made as follows:

IS

48 NRRL 2033 P. frequenlans

49 WB 2020 P. duclauxi

50 WB 2059 P. multicolor

51 WB 2074 P. sclerotiorum

52 WB 2036 P. granulatum

53 WB 2099 P. vermiculatum

54 NRRL 2067 P. terlikowskii

55 WB1900 P. italicum

56 WB 1974 Aspergillus ustus

57 WB 154 A. restriclus

58 WB 216 A. ungins

59 WB 265 A. terreus

60 WB 356 A. luchensis

61 WB 2254 A. clavatus

62 WB 2256 A. ornaHis

63 WB 2276 A. miyakoensis

64 WB 2292 A. citrisporus

65 WB 1787 A. janua

66 WBA952 Dactylomyces

thermophilus

67 WB 1697 Thiclavia

sependonium

Example 68

2 - (6 '- Carbomethoxyhxyl) - 4 (R) - hydroxy - cyclopenlan-1,3-dione was prepared according to the procedure given in Example 1, with the exception that 2- (6'-Carbomethoxyhcxyl) -3-meth-

40

CO ₂ C ₂ H ₅

(III)

CO ₂ C ₂ H ₅

(IV)

A. Fermentation

The surface growth of one week old agar dishes of Sehizosaccharomyces pombe (CBS 352) was ^{suspended in 5 cm 3 of} 0.85% saline. 2 cm ³ portions of this suspension were used ^{to inoculate 50 cm 3 of} the above soybean dextrose medium in 250 cm ³ Erlenmeyer flasks (FI). The flasks were shaken for 24 hours at 25 ° C. in a rotary shaker (250 revolutions / minute, radius 5.1 cm) and then 10 percent by volume in a 2-1 Erlenmeyer flask (F-2) containing 500 cm ^{3 of} the soybean dextrose -Medium contained. After 24 hours of incubation on a rolatory shaker, 250 mg of 2 - (6 '- carboethoxyhexyl) - cyclopenian -1.3. 4-trione (111) (J. K at su be and M. M atsu, J. Anr. Biol. Chem. [Japan] 33, 1078, [1969]) in 2 cm ^{3 of} dimethylformamide was added to the flask. The F-2 flask was then incubated for an additional 24 hours under the conditions specified for incubation of the F-1 flask.

B. Isolieruni;

24 hours after the addition of the Trion substrate (III), the yeast cells were centrifuged off. The supernatant liquid was brought to a pH value of 2 with 6N HCl and extracted three times ^{with 500 cm 3 of ethyl acetal.} The ethyl acetate layer was _{dried over Na 2} SO ₄ and evaporated to give an oily residue. The residue was dissolved in 2 cm ^{3 of} benzene-ethyl acetate (1: 1) and placed on a column of 2.5 × 25 cm which was charged with silica-dialomene crude (85:15). The column was eluted with a gradient system.

consisting of 300 cm ^{3 of} ethyl acetate-benzene (1: 1) in the mixing chamber and 300 cm ^{3 of} ethyl acetate in the storage container, and 7 cm ³ fraction were collected. Fractions 19 to 31 were brought together and unified. 39 ml IV: mp. ILV to 85 C: [«] '? +14 (C ₁ = 0.47. CHCl ₃ J>. ^ ^OH 272 times. X 20,000).

In the process according to the invention, the optically active 4-H> substituted in the 2-position is obtained Droxy-cyclopentane-1,3-diones in high yields, where a 70-80% conversion is possible

Claims (1)

Claim: Process for the preparation of 4 (R) - hydroxy - cyclopentani, 3-dione, substituted in the 2-position, characterized in that cyclopentane-1,3,4-triones which are substituted in the 2-position the general formula