HK1092782B - Process for preparing racimatic of optically active 4-(hydroxymethyl)-2-cyclopentene derivative - Google Patents

Description

Process for preparing racemic or optically active 4- (hydroxymethyl) -2-cyclopentene derivatives

This application is filed under the application number 03123433.x, filed on divisional filing date 28/2003 and filed under the name "(1R, 4S) -or (1S, 4R) -1-amino-4- (hydroxymethyl) -2-cyclopentene derivative enantiomer preparation method", which was filed under the application number 98108865.1, filed on filed under the application number 1998 5/13/1998 and filed under the name "(1S, 4R) -or (1R, 4S) -4- (2-amino-6-chloro-9H-purin-9-yl) -2-cyclopentene-1-methanol or salt thereof".

Technical Field

The present invention relates to a novel process for the preparation of (1S, 4R) -or (1R, 4S) -4- (2-amino-6-chloro-9H-purin-9-yl) -2-cyclopentene-1-methanol or its salts represented by formula I or II,

and a novel process for producing an optically active compound represented by the general formula XVI or XVII.

Background

(1S, 4R) -4- (2-amino-6-chloro-9H-purin-9-yl) -2-cyclopentene-1-methanol is an important intermediate for the preparation of 2-aminopurine nucleosides, for example for the preparation of (1S, 4R) -4- (2-amino-6-cyclopropylamino-9H-purin-9-yl) -2-cyclopentene-1-methanol (W0/95/21161) or 1592U89(J.Org.Chem., 1996, 61, 4192. -.

WO/95/21161 describes a process for the preparation of (1S, 4R) -4- (2-amino-6-chloro-9H-purin-9-yl) -2-cyclopentene-1-methanol from (1S, 4R) -4-amino-2-cyclopentene-1-methanol. The disadvantage of this process is that the precursor (1S, 4R) -4-amino-2-cyclopentene-1-methanol can only be prepared from (. + -.) -2-aza-bicyclo [2.2.1.] hept-5-en-3-one substituted with a noble BOC protecting group (tert-butoxycarbonyl protecting group) (J.org.chem., 1995, 60, 4602-one 4616).

Disclosure of Invention

The object of the present invention is to provide a simple, low-cost and more economical process for the preparation of (1S, 4R) -or (1R, 4S) -4- (2-amino-6-chloro-9H-purin-9-yl) -2-cyclopentene-1-methanol or its salts.

The object of the invention has been achieved by the novel process as claimed in claim 1.

The first step of the novel process is the acylation of (±) -2-aza-bicyclo [2.2.1.] hept-5-en-3-one represented by formula III,

producing a (±) -2-aza-bicyclo [2.2.1.] hept-5-ene 3-one derivative represented by formula IV,

in the formula R¹Is represented by C_1-4Alkyl radical, C_1-4Alkoxy, aryl or aryloxy.

C_1-4Alkyl groups may be substituted or unsubstituted. Hereinafter, substituted C_1-4Alkyl means C substituted by halogen atoms_1-4An alkyl group. The halogen atom can be F, Cl, Br or I. C_1-4Examples of alkyl are methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, isopropyl, chloromethyl, bromomethyl, dichloromethyl, dibromomethyl. As C_1-4Preferred alkyl groups are methyl, ethyl, propyl, butyl, isobutyl or chloromethyl.

Can be used as C_1-4Examples of alkoxy are methoxy, ethoxy, propoxy, butoxy, tert-butoxy or isobutoxy. Preferably used C_1-4Alkoxy is tert-butoxy.

As the aryl group, there may be used, for example, a phenyl group or a benzyl group, preferably a phenyl group. Benzyloxy or phenoxy can be used, for example, as aryloxy.

The precursor (. + -.) -2-aza-bicyclo [2.2.1.] hept-5-en-3-one can be prepared as disclosed in EP-A0508352.

Acylation may be with a carbonyl halide (carbonyl halide) of formula XI

Or by using a carboxylic anhydride of the formula XII,

in the formula R¹Has the above meaning, and X represents a halogen atom. Fluorine, chlorine, bromine or iodine may be used as the halogen atom. Preferably, chlorine or fluorine is used.

Examples of carbonyl halides are: acetyl chloride, chloroacetyl chloride, butyryl chloride, isobutyryl chloride, phenylacetyl chloride, benzyl chloroformate (Cbz-Cl), propionyl chloride, benzoyl chloride, allyl chloroformate, or tert-butoxycarbonyl fluoride. Examples of carboxylic acid anhydrides are: di-tert-butyl peroxydicarbonate (di-tert-butyl dicarbonate), butyric anhydride, acetic anhydride or propionic anhydride.

The acylation may be carried out without a solvent or with the addition of an aprotic solvent.

The acylation is preferably carried out in an aprotic solvent. Examples of suitable aprotic solvents are diisopropyl ether, pyridine, acetonitrile, dimethylformamide, triethylamine, tetrahydrofuran, toluene, dichloromethane, N-methylpyrrolidone or mixtures thereof.

The acylation is preferably carried out at a temperature of-80 to 50 deg.C, preferably 0 to 25 deg.C.

In the second step of the novel process, the (. + -.) -2-aza-bicyclo [2.2.1.] hept-5-ene 3-one derivative represented by formula IV is reduced to produce a cyclopentene derivative represented by formula V,

in the formula R¹Have the meaning indicated above.

The reduction is preferably carried out with alkali metal or alkaline earth metal borohydrides, alkali metal or alkaline earth metal alanates or Vitride (sodium bis (2-methoxyethoxy) dihydroaluminate). Sodium or potassium aluminum hydride may be used as the alkali metal alanate. Sodium or potassium borohydride may be used as the alkali metal borohydride. Calcium borohydride can be used as an alkaline earth metal borohydride. Aluminum nitride, for example, can be used as the nitride.

The reduction is preferably carried out in a protic solvent. Protic solvents which may be used are lower aliphatic alcohols, such as methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, tert-butanol, isobutanol or water, or mixtures of these alcohols with water.

The reduction is preferably carried out at a temperature of-40 to 40 ℃ and preferably 0 to 20 ℃.

The third step of the novel process is the conversion of a cyclopentene derivative represented by the general formula V into (1R, 4S) -or (1S, 4R) -1-amino-4- (hydroxymethyl) -2-cyclopentene represented by the formula VI or VII by a microorganism, an enzyme having N-acylaminoalcohol hydrolase activity or penicillin G acylase,

. This biotransformation converts the acylated (1S, 4R) or (1R, 4S) -amino alcohol derivative into (1R, 4S) -or (1S, 4R) -1-amino-4- (hydroxymethyl) -2-cyclopentene (general formulae VI, VII).

All microorganisms which are capable of using the cyclopentene derivative represented by the formula V as the only nitrogen source, the only carbon source or the only carbon-nitrogen source are suitable for the bioconversion. These microorganisms can be isolated from soil samples, sludges or waste waters using conventional microbiological techniques. By incorporating cyclopentene derivatives represented by the formula V in a conventional manner

In the formula R¹Has the meaning as described above and has,

● as the sole carbon and nitrogen sources

● as the sole nitrogen source and a suitable carbon source or

● as sole carbon source and suitable nitrogen source in a nutrient medium.

Detailed Description

Examples of suitable cyclopentene derivatives represented by the formula V are: n-acetyl-, N-propionyl-, N-isobutyryl-, N-tert-butoxycarbonyl- (N-BOC), N-butyryl-or N-phenylacetyl-1-4 amino-4-hydroxymethyl-2-cyclopentene.

The microorganisms can use, for example, ammonium, nitrate, amino acid or urea propagation substrates as suitable nitrogen sources. The microorganism may be, for example, sugar alcohol, C₂-C₄-carboxylic acid or amino acid propagation substrates are used as suitable carbon sources. Hexoses or pentoses such as glucose can be used as the sugar. Glycerol is used, for example, as a sugar alcohol. Acetic acid or propionic acid may be used, for example, as C₂-C₄-a carboxylic acid. Leucine, alanine, asparagine can be used, for example, as amino acids.

The selection medium and the culture medium which can be used are the medium commonly used by those skilled in the art (such as the medium described in Table 1) or a complete medium (a medium containing a yeast extract) (such as nutrient yeast soup (NYB), preferably the yeast soup described in Table 1).

During the cultivation and selection process, the active enzymes of the microorganism are conveniently induced. Cyclopentene derivatives represented by the formula V can be used as inducers of enzymes.

The culturing and selection are generally carried out at a temperature of 20 to 40 ℃ and preferably 30 to 38 ℃ and at a pH of 5.5 to 8 and preferably 6.8 to 7.8.

The bioconversion is preferably carried out using a microorganism in which the (1R, 4S) isomer of the cyclopentene derivative is used as the sole nitrogen source, the sole carbon source or the sole carbon and nitrogen source.

The biotransformation is preferably carried out with microorganisms of the genera Alcaligenes/Bordetella, Rhodococcus, Arthrobacter, Alcaligenes, Agrobacterium/Rhizobium, Bacillus, Pseudomonas or Gordonia, in particular Alcaligenes/Bordetella FB 188(DSM 11172), Rhodococcus erythropolis CB101(DSM 10686), Arthrobacter HSZ5(DSM 10328), Rhodococcus FB 387(DSM11291), xylose-oxidizing Alcaligenes denitrifying subspecies HSZ17(DSM 10329), Agrobacterium/Rhizobium HSZ30, Bacillus simplex K2, Pseudomonas putida K32 or Gordonia CB 100(DSM 10687) and functionally equivalent variants and mutants thereof. According to the Budapest treaty, microorganisms DSM 10686 and 10687 were deposited at 20.05.1996 at the German Collection of microorganisms and cell cultures company (Maschererweg 1b, D38124Braunschweig), microorganisms DSM 10328 and 10329 were deposited at 06.11.1995, microorganisms DSM11291 at 08.10.1996 and microorganisms DSM 11172 at 20.09.1996 at this company.

"functionally equivalent variants and mutants" refer to microorganisms having essentially the same properties and functions as microorganisms.

Alcaligenes/Bordetella FB 188(DSM 11172) Specification

Cellular shape bacillus

Width, micron 0.5-0.6

Length, micron 1.0-2.5

Dynamic property

With peripheral hair protruding from flagellum

Gram reaction-

Is dissolved by 3 percent KOH

Aminopeptidase (Cerny) +

Spore-

Oxidase +

Catalase +

ADH (alcohol dehydrogenase) -

Derived from NO₃NO of₂ -

Denitrification-

Urease-

Hydrolysis of gelatin

Acids generated from the following compounds (OF test)

Glucose-

Fructose-

Arabinose-

Adipate (salt) +

Sebacate (salt) +

Citrate ester (salt) +

Malate (++)

Mannitol-

Classification of Rhodococcus erythropolis CB101(DSM 10686)

1. Morphology and color of colonies: short branching hyphae, which divide into bacilli (rods) and cocci upon aging, with flashing and partial fusion of colonies, with a pale pink primary color, RAL 1001;

2. amino acids detected from peptidoglycan: meso-diaminopimelic acid;

3. and (3) mycolic acid: rhodococcus fungal acid; chain length of mycolic acid was determined (C32-C44) and the data was compared to data entries in DSM mycolic acid database and found to have great similarity (similarity: 0.588) to the Rhodococcus erythropolis strain pattern (pattern).

4. Fatty acid profile: straight, saturated and unsaturated fatty acids and tuberculostearic acid.

5. When the 16S rDNA part of the strain is sequenced, the sequence is highly consistent with the sequence of a specific region of the Rhodococcus erythropolis (100%).

This identification is unambiguous because three independent methods (mycolic acid, fatty acid, 16SrDNA) have established that the strain is Rhodococcus erythropolis.

Classification of Gordonia sp.CB 100(DSM 10687)

1. Morphology and color of colonies: short branching hyphae, which divide into bacilli and cocci when aged, the colonies being light orange yellow, (RAL 2008);

2. amino acids detected from peptidoglycan: meso-diaminopimelic acid;

3. menaquinone map: MK-9 (H)₂)100％；

4. And (3) mycolic acid: gordonia fungal acid; determination of the chain Length of mycolic acid by high temperature gas chromatography (C)₅₀-C₆₀) The profile corresponds to the profile found in a gordonia sample.

5. Fatty acid profile: straight, saturated and unsaturated fatty acids and tuberculostearic acid.

6. When the 16S rDNA portion of the strain was sequenced, only a low identity (98.8%) with the sequence of the specific region of Gordonia rubrum was found.

Based on these available results (menaquinone profile, mycolic acid, fatty acid, 16S rDNA), although this isolate could be unambiguously identified as gordonia, it could not be determined from these results that it is a known gordonia. Thus, strain DSM10687 was assumed to be a new, undescribed species of gordonia.

Classification of the xylose oxidizing Alcaligenes denitrifying subspecies HSZ17(DSM 10329)

Properties of the Strain

Cellular shape bacillus

Width, micron 0.5-0.6

Length, micron 1.5-3.0

Dynamic property

With peripheral hair protruding from flagellum

Gram reaction-

Is dissolved by 3 percent KOH

Aminopeptidase (Cerny) +

Spore-

Oxidase +

Catalase +

Anaerobic growth-

ADH (alcohol dehydrogenase) +

Derived from NO₃NO of₂ +

Denitrification effect +

Urease-

Hydrolysis of gelatin

Hydrolysis of Tween 80

Acids generated from the following compounds (OF test)

Glucose oxygen demand-

Xylose 80-

Substrate utilization

Glucose-

Fructose-

Arabinose-

Citrate ester (salt) +

Malate (++)

Mannitol-

Classification of Arthrobacter HSZ5(DSM 10328)

And (3) characterization and identification: gram with significant bacilli-cocci growth cycle

(ii) a positive irregular bacillus; strictly aerobic; in grape

No acid or gas is formed in the glucose.

Motility-

Spore-

Catalase +

Meso-diaminopimelic acid in cell walls: yuan

Peptidoglycan type: a3 alpha, L-Lys-L-Ser-L-Thr-L-Ala

16S rDNA sequence similarity: by nourishing Arthrobacter, Mycobacterium and Othrobacter

When bacilli sequence the most variable regions

The maximum value found was 98.2%

Classification of Agrobacterium/Rhizobium HSZ30

Cell shape polytype bacillus

Width (micrometer) 0.6-1.0

Length (micrometer) 1.5-3.0

Gram reaction-

Is dissolved by 3 percent KOH

Aminopeptidase C

Spore-

Oxidase +

Catalase +

Dynamic property

Anaerobic growth-

Nitrite production from nitrate-

Denitrification-

Urease +

Hydrolysis of gelatin

Generating an acid from:

l-arabinose +

Galactose-

Melezitose-

Fucose of

Arabitol-

Mannitol-

Erythritol-

Alkalization of litmus juice

Ketolactose-

Partial sequencing of 16S rDNA revealed greater similarity to samples of Agrobacterium and Rhizobium, approximately 96%. It is not possible to unambiguously identify them as species within these genera.

Classification of Bacillus simplex K2

Cellular shape bacillus

Width (micrometer) 0.8-1.0

Length (micrometer) 3.0-5.0

Spore-

Ellipsoid-

Round body-

Sporangium-

Catalase +

Anaerobic growth-

VP reacts n.g.

Maximum temperature

Temperature at normal growth of 40 DEG C

Negative growth temperature, ° c 45

Growth in pH 5.7 Medium

NaCl2％ +

5％ -

7％ -

10％ -

Lysozyme culture medium +

Production of Acid (ASS) from

D-glucose +

L-arabinose +

D-xylose-

D-mannitol +

D-fructose +

Gas generated from fructose-

Lecithinase-

Hydrolysis of the following

Starch + C

Gelatin

Casein-

Tween 80 +

Esculin-

Using the following substances

Citrate + C

Propionate-

Nitrite generated from nitrate

Indole-

Phenylalanine deaminase-

Arginine dehydroxylase-

Analysis of cellular fatty acids determined it to be of the genus bacillus.

Partial sequencing of 16S rDNA showed 100% similarity to B.simplex.

Classification of Pseudomonas putida K32

Cellular shape bacillus

Width (micrometer) 0.8-0.9

Length (micrometer) 1.5-4.0

Dynamic property

Flagellum protrusion polarity > 1

Gram reaction-

Is dissolved by 3 percent KOH

Aminopeptidase (Cerny) +

Spore-

Oxidase +

Catalase +

Anaerobic growth-

Pigment

Fluorescent lamp

Pustulosu-

ADH +

Nitrite from nitrate-

Denitrification-

Urease-

Hydrolysis of gelatin

Substrate utilization

Adipate (or salt)

Citrate ester (or salt) +

Malic acid ester (or salt) +

D-mandelate ester (or salt) +

Phenyl acetate (or salt) +

D-tartrate (or salt)

D-glucose +

Trehalase-

Mannitol-

Benzoyl formate (or salt) -

Propylene glycol+

Bufonis um

Benzylamine

Tryptamine-

Acetamide +

Hippurate (or ester) +

The cellular fatty acid profile is characteristic of Pseudomonas putida.

Partial sequencing of 16S rDNA showed approximately 98% similarity to Pseudomonas mendocina and Pseudomonas alcaligenes, 97.4% similarity to Pseudomonas putida.

Classification of Rhodococcus sp.FB 387(DSM11291)

1. Morphology and color of colonies: short branching hyphae, which divide into bacilli (rods) and cocci when aged, with dull colonies, pale pink-orange RAL 2008;

2. amino acids detected from peptidoglycan: meso-diaminopimelic acid;

3. and (3) mycolic acid: rhodococcus fungal acid;

the chain length of mycolic acid was determined (C32-C44) and comparison of this data with data entries in the DSMZ mycolic acid database showed only minor similarity to the Rhodococcus erythropolis spectrum (similarity: 0.019). This correlation coefficient is too small to be used for species identification.

4. Fatty acid profile: straight, saturated and unsaturated fatty acids and tuberculostearic acid.

This fatty acid profile is valuable for the judgment of all Rhodococcus samples and their close relatives (e.g., Mycobacterium, Nocardia and Gordonia). in order to differentiate to species level, attempts were made to account for qualitative and quantitative differences in fatty acid profiles. Since there is only a small similarity (0.063) to the above-mentioned Rhodococcus species, Rhodococcus sp.FB 387 could not be confirmed by this method.

5. When the 16S rDNA portion of the strain was sequenced, 96-818 was identified as Rhodococcus cloudiness due to a correlation of 97.9% this sequence identity was much lower than 99.5% required for unequivocal identification of the species at the time of classification.

From the results obtained, it can be assumed that the strain Rhodococcus sp.FB 387 is a novel, undescribed Rhodococcus species.

After a conventional initial culture of these microorganisms, the biotransformation can be carried out using resting cells (non-growing cells which no longer require a carbon or energy source) or growing cells.

The enzymes having N-acylaminoalcohol hydrolase activity suitable for such biotransformation can be isolated, for example, from the above-mentioned microbial cells by disruption methods customary in the art, for which, for example, the ultrasonic method or the French press method can be used. Preferably, these enzymes are isolated from the microorganism Rhodococcus erythropolis CB101(DSM 10686).

Suitable penicillin G acylases are obtainable from a number of microorganisms, such as bacteria and actinomycetes; in particular, it can be obtained from the following microorganisms: escherichia coli ATCC 9637, Bacillus megaterium, Streptomyces lavendulae ATCC 13664, Nocardia sp.ATCC 13635, providencia rettgeri ATCC 9918, Arthrobacter viscosus ATCC 15294, Rhodococcus luteus ATCC 12975, Streptomyces unbutton or divest one's upper garment producer ATCC21289, Achromobacter ATCC 23584, and Micrococcus roseus ATCC 416. In particular, commercially available penicillin G acylases are used, such as penicillin G acylase EC 3.5.1.11 from E.coli (Boehringer Mannheim) or Bacillus megaterium.

In a preferred embodiment, an immobilized penicillin G acylase is used.

The biotransformation can be carried out in media commonly used in the art, such as low molarity phosphate, citrate or Hepes buffers, water, nutrient yeast juices and the like or complete media as indicated in the tables. The biotransformation is preferably carried out in the medium indicated in Table 1 or in a low molar phosphate buffer.

The biotransformation is preferably carried out under conditions such that the concentration of the cyclopentene derivative (formula V) is not more than 10% by weight, preferably not more than 2% by weight, added in one portion or continuously.

The pH at the time of biotransformation may be from 5 to 9, preferably from 6 to 8. The temperature for the biotransformation is preferably 20 to 40 ℃ and more preferably 25 to 30 ℃.

In a fourth step, N- (2-amino-4, 6-dichloro-5-pyrimidinyl) carboxamide of formula VIII

Converting the cyclopentene derivative (formula VI or VII) into (1S, 4R) -or (1R, 4S) -4- [ (2-amino-6-chloro-5-formylamino-4-pyrimidinyl) -amino ] -2-cyclopentene 1-methanol represented by the formula IX or X.

N- (2-amino-4, 6-dichloro-5-pyrimidinyl) carboxamide may be prepared as disclosed in WO 95/21161.

The reaction of the fourth step is preferably carried out in the presence of a base, an organic base or an inorganic base may be used, and trialkylamine may be used as the organic base. Examples of inorganic bases which can be used are alkali metal carbonates or alkaline earth metal carbonates, or alkali metal hydrogencarbonates or alkaline earth metal hydrogencarbonates, such as potassium carbonate and sodium hydrogencarbonate.

The reaction in the fourth step is preferably carried out in a protic solvent, a lower alkyl alcohol may be used as the protic solvent, such as methanol, ethanol, propanol, isopropanol, butanol or isobutanol.

The temperature at which the fourth reaction step is carried out is preferably 0 to 150 ℃ and more preferably 20 to 100 ℃.

In a fifth step, (1R, 4S) -or (1S, 4R) -4- [ (2-amino-6-chloro-5-carboxamido-4-pyrimidinyl) -amino ] -2-cyclopentene 1-methanol (formula IX, X) is cyclized according to a known method disclosed in WO95/21161 to give a final product represented by formula I or II.

The cyclisation is generally carried out by dissolution in a trialkyl orthoformate in the presence of a concentrated aqueous acid (aquous acid). Trialkyl orthoformates which can be used are trimethyl orthoformate or triethyl orthoformate. For example hydrochloric acid, sulfuric acid or methanesulfonic acid can be used as aqueous acid.

The present invention also relates to a process for producing an optically active compound represented by the general formula XVI or XVII,

in the formula R¹Have the meaning indicated above. These compounds can be prepared by reacting cyclopentene derivatives represented by the general formula V with the above-mentioned microorganisms, N-acylaminohydrolase or penicillin G acylase

In the formula R¹Having the meaning indicated above, to (1R, 4S) -or (1S, 4R) -1-amino-4- (hydroxymethyl) -2-cyclopentene represented by the formula VI or VII,

the latter is acylated to a compound represented by formula XVI or XVII.

Both the microbial transformation and the acylation are carried out under the same conditions as described above.

The temperature for acylation is preferably 20 to 100 ℃ and more preferably 0 to 80 ℃.

Examples of optically active compounds prepared by the process are: (1R, 4S) -N-tert-butoxycarbonyl-1-amino-4-hydroxymethyl-2-cyclopentene with an enantiomeric excess of 98%, (1R, 4S) -N-acetyl-1-amino-4-hydroxymethyl-2-cyclopentene with an enantiomeric excess of 98%, the enantiomeric excess was 98% of (1R, 4S) -N-butyryl-1-amino-4-hydroxymethyl-2-cyclopentene and optically active (1S, 4R) -N-acetyl-1-amino-4-hydroxymethyl-2-cyclopentene and optically active (1S, 4R) -N-butyryl-1-amino-4-hydroxymethyl-2-cyclopentene.

Among these compounds, optically active (1R, 4S) -N-butyryl-1-amino-4-hydroxymethyl-2-cyclopentene is a compound which has not been reported in the literature. The invention therefore also relates to an enantiomeric excess of more than 0%, preferably at least 80%, 90% or 95%, in particular at least 98%, of optically active N-butyryl-1-amino-4-hydroxymethyl-2-cyclopentene. These optically active compounds can be racemized by methods known to those of ordinary skill in the art to produce rac-N-butyryl-1-amino-4-hydroxymethyl-2-cyclopentene, which has not been reported in the literature.

The racemic or optically active 4- (hydroxymethyl) -2-cyclopentene derivative represented by the formula XIII can be prepared as follows

In the formula R¹Having the meaning described above, in a first step with a carbonyl halide of the formula XI

In the formula R¹And X has the meaning described above, a racemic cyclopentene-4-carboxylic acid represented by the formula XIV or one of its optically active isomers

Acylation to form racemic or optically active cyclopentene-4-carboxylic acid derivatives represented by the general formula XV,

the latter is reduced in a second step to give the desired product represented by formula XIII.

The optically active 4- (hydroxymethyl) -2-cyclopentene derivative (formula XIII) and the optically active cyclopentene-4-carboxylic acid derivative (formula XV) refer to the corresponding (1R, 4S) or (1S, 4R) isomers.

The first step (acylation) of the process is carried out with a carbonyl halide of formula XI the carbonyl halides which may be used are the same as described above. Preferably, tert-butyloxycarbonyl fluoride is used.

The first reaction is preferably carried out at a pH of from 8 to 14, preferably from 12 to 14, and at a temperature of from 0 to 50, preferably from 15 to 25 ℃.

Suitable solvents are mixtures of water and ethers which may be used are dioxane, tetrahydrofuran, diethyl ether, ethylene glycol dimethyl ether or ethylene glycol diethyl ether.

The second step (reduction) of the process may be carried out using alkali metal alanates, boranes/diC_1-4Lithium, sodium or potassium aluminum hydride may be used as the alkali metal alanate. Lithium aluminum hydride is preferably used. Borane/dimethylsulfide, borane/diethylsulfide, borane/dipropylthiosulfide or borane/dibutylthiosulfide adducts as borane/di-C_1-4The borane/dimethylsulfide adduct is preferably used.

In the second step, one of the above-mentioned anhydrous ethers is preferably used as a solvent.

The temperature of the second reaction may be-50 to 5 deg.C, preferably-25 to-10 deg.C.

Examples

Example 1

Preparation of (+ -) -2-acetyl-2-azabicyclo [2.2.1] hept-5-en-3-one

100 g (. + -.) -2-azabicyclo [2.2.1] hept-5-en-3-one were dissolved in acetonitrile (800 ml) and pyridine (161.26 ml) under nitrogen. 104.5 g of acetyl chloride were added dropwise at 12 ℃ over a period of 2 hours. The mixture was then stirred at room temperature for 4.5 hours. To this mixture, 800 ml of water were added and the acetonitrile was distilled off in vacuo. The aqueous phase is extracted three times with 400 ml of ethyl acetate each time. The combined organic phases were washed with 1N HCl (400 ml), water (400 ml), saturated sodium chloride (400 ml), dried over magnesium sulfate and evaporated to dryness. The residue was dissolved in dichloromethane and filtered over silica gel the filtrate was concentrated and the product was purified by distillation. 107.76 g of a clear liquid product are obtained, yield 71%.

Boiling point (0.07 torr): 51 deg.C

¹H-NMR(CDCl₃)：δ[ppm]400HHz 2.25(AB syst.，2H)

2.8(s，3H)

3.42(m，1H)

5.30(m，1H)

6.89(m，1H)

6.92(m，1H)

Example 2

Preparation of (+ -) -2-butyryl-2-azabicyclo [2.2.1] hept-5-en-3-one

100.3 g (. + -.) -2-azabicyclo [2.2.1] hept-5-en-3-one were dissolved in acetonitrile (720 ml) and pyridine (142 ml) under nitrogen. At 12 ℃, 141.8 g of butyryl chloride are added dropwise over 1 hour, the mixture is then stirred at room temperature for 3 hours, 720 ml of water are added to the mixture and the phases are separated, the acetonitrile is distilled off under vacuum, and the aqueous phase is extracted three times with 300 ml of ethyl acetate. The combined organic phases were washed with 1N HCl (350 ml), saturated sodium chloride (500 ml) and water (400 ml), dried over magnesium sulphate, evaporated completely and the product was purified by distillation. 107.76 g of a clear liquid product are obtained. The yield was 85%.

Boiling point (0.05 torr): 70 deg.C

¹H-NMR(CDCl₃)：δ[ppm]400HHz 0.98(t，J＝8.5Hz，3H)

1.58-1.65(2H)

2.23(AB syst.，2H)

2.82-2.90(23H)

3.42(m，1H)

5.30(m，1H)

6.62(m，1H)

6.90(m，1H)

Example 3

Preparation of (+ -) -2-phenylacetyl-2-azabicyclo [2.2.1] hept-5-en-3-one

33.4 g (. + -.) -2-azabicyclo [2.2.1] hept-5-en-3-one were dissolved in acetonitrile (24Q ml) and pyridine (48.3 ml) under nitrogen. At 12 deg.C, 68.6 g of phenylacetyl chloride are added dropwise over a period of 30 minutes, the mixture is then stirred at room temperature for 3.5 hours, 240 ml of water are added to the mixture, and the acetonitrile is distilled off in vacuo. The aqueous phase is extracted three times with 150 ml of ethyl acetate each time. The combined organic phases were washed with 1N HCl (150 ml), saturated sodium chloride (150 ml) and water (150 ml), dried over magnesium sulphate and evaporated completely the crude product was filtered through silica gel (hexane: ethyl acetate 1: 1) to yield 78.34 g of crude product as a yellow oil.

Example 4

Preparation of (+ -) -2-propionyl-2-azabicyclo [2.2.1] hept-5-en-3-one

47 g (. + -.) -2-azabicyclo [2.2.1] hept-5-en-3-one were dissolved in acetonitrile (325 ml) and pyridine (41 ml) at 12 ℃ 43.9 g propionyl chloride were added dropwise over 1h, then the mixture was stirred at room temperature for 5 h, 145 ml water was added to the mixture, acetonitrile was evaporated off under vacuum, the aqueous phase was extracted three times with ethyl acetate, 115 ml each time, the combined organic phases were washed with 1N HCl (140 ml), saturated sodium bicarbonate (40 ml) and sodium chloride solution (40 ml), dried over sodium sulfate, evaporated completely, the purified residue was distilled off to give 55.8 g of the title product, solidified on standing, yield 81.6%.

Boiling point (2.8 mbar): 75-80 deg.C

Melting point: 54-56 deg.C

¹H-NMR(DMSO-d₆)：δ[ppm]400Hz 0.95(t，3H)；

2.10(quart.1H)；

2.28(quart.1H)；

2.64(m，2H)；

3.42(s，1H)；

5.16(s，1H)；

6.78(m，1H)；

6.96(m，1H).

Example 5

Preparation of (+ -) -2-isobutyryl-2-azabicyclo [2.2.1] hept-5-en-3-one

45.1 g (. + -.) -2-azabicyclo [2.2.1] hept-5-en-3-one were dissolved in acetonitrile (310 ml) and pyridine (39 ml) under nitrogen. 54.1 g of isobutyryl chloride were added dropwise at 10 ℃ over a period of 1 hour. The mixture was then stirred at room temperature for 5 hours. To this mixture 140 ml of water were added and the phases were separated, the acetonitrile was distilled off under vacuum, the aqueous phase was extracted four times with 120 ml each time, the combined organic phases were washed with 1N HCl (50 ml), saturated sodium bicarbonate solution (50 ml) and sodium chloride (50 ml), dried over magnesium sulphate and evaporated completely. The residue was boiled under reflux in n-hexane (240 ml) to which activated carbon had been added, the activated carbon was filtered off, the filtrate was cooled to 0 ℃ and the title compound was filtered to give 54.5 g of product in 76% yield.

Melting point: 41-42 deg.C

¹H-NMR(DMSO-d₆)：δ[ppm]400HHz 0.92(d，3H)

1.06(d，3H)

2.10(m，1H)

2.28(m，1H)

3.40(m，2H)

5.16(s，1H)

6.78(m，1H)

7.92(m，1H)

Example 6

Preparation of (+ -) -2-chloroacetyl-2-azabicyclo [2.2.1] hept-5-en-3-one

10.1 g (. + -.) -2-azabicyclo [2.2.1] hept-5-en-3-one are dissolved under nitrogen at 10 ℃ in a mixture of dichloromethane (10 ml), pyridine (8.4 ml) and 0.22 g 4-N, N-dimethylaminopyridine 13.5 g chloroacetyl chloride are added dropwise over 1h, the temperature is raised to 44 ℃ and the mixture is stirred for a further 2 h at room temperature, 100 ml water is added to this solution, the aqueous phase is extracted with 100 ml dichloromethane after separation of the phases. The combined organic phases were dried over sodium sulfate and evaporated completely. The residue is boiled under reflux in 100 ml of isopropyl ether in the presence of 1 g of activated carbon for 10 minutes. After hot filtration, the filtrate was cooled to room temperature, and the solid was filtered off and dried. 10.35 g of the title product are obtained, yield 60%.

The melting point is 86-88 DEG C

¹H-NMR(CDCl₃)：δ[ppm]400HHz 2.28(d，1H)；

2.40(d，1H)；

3.48(s，1H)；

4.56(d，2H)；

5.30(s，1H)；

6.70(d，1H)；

6.94(m，1H).

Example 7

Preparation of (+/-) -1-acetamido-4-hydroxymethyl-2-cyclopentene

79.56 g (. + -.) -2-acetyl-2-azabicyclo [2.2.1] hept-5-en-3-one were dissolved in ethanol (450 ml) under nitrogen, cooled to-10 ℃ and 19.8 g sodium borohydride were added in portions over 45 minutes.

The reaction was stirred at 0 ℃ for 3 hours and then the pH was adjusted to 1.8 with concentrated sulfuric acid, to this mixture ethyl acetate (200 ml) was added, the solid was filtered off, then evaporated completely, the residue was taken up in water, washed with dichloromethane and evaporated, the crude product was purified by filtration over silica gel. 51.83 g of product were obtained as a white solid. The yield was 64% based on (. + -.) -2-acetyl-2-azabicyclo [2.2.1] hept-5-en-3-one used.

¹H-NMR(DMSO-d₆)：δ[ppm]400MHz 1.18(m，1H)；

1.78(s，3H)；

2.29(m，1H)；

2.66(m，1H)；

3.25(s，2H)；

4.58(s，1H)；

4.72(m，1H)；

5.61(d，1H)；

5.85(d，1H)；

7.83(d，1H).

Example 8

Preparation of (+/-) -1-butyrylamino-4-hydroxymethyl-2-cyclopentene

73.87 g (. + -.) -2-butyryl-2-azabicyclo [2.2.1] hept-5-en-3-one were dissolved in ethanol (400 ml) under nitrogen and cooled to-10 ℃. 15.68 g of sodium borohydride were added in portions over 45 minutes. The reaction was stirred at 0 ℃ for 3 hours and then the pH was adjusted to 1.5 with concentrated sulfuric acid. To the mixture was added ethyl acetate (200 ml), and the solid was filtered off. The residue was taken up in water, washed with dichloromethane and evaporated to dryness in vacuo. 60.55 g of product were obtained, 80% based on (. + -.) -2-butyryl-2-azabicyclo [2.2.1] hept-5-en-3-one used. Melting point: 71-72 deg.C

¹H-NMR(CDCl₃)：δ[ppm]400MHz 0.98(t，J＝8.5Hz，3H)；

1.40-1.50(1H)；

1.58-1.68(2H)；

2.10-2.18(2H)；

2.42-2.55(1H)；

2.85(m，1H)；

3.62(AB syst.，2H)；

4.98(m，1H)；

5.78-5.82(2H)；

6.38(m，1H).

Example 9

Preparation of (+/-) -1-phenylacetylamino-4-hydroxymethyl-2-cyclopentene

77 g of crude (. + -.) -2-phenylacetyl-2-azabicyclo [2.2.1] hept-5-en-3-one are dissolved in ethanol (450 ml) under nitrogen, cooled to-10 ℃ and 13.2 g of sodium borohydride are added in portions over 1h, the reaction is stirred at room temperature for 3.5 h, the pH is then adjusted to 1.8 with concentrated sulfuric acid, the mixture is purified by silica gel filtration (hexane: ethyl acetate 2: 8) and, after recrystallization from ethyl acetate, 15.89 g of a white solid are obtained. The yield was 80% based on (. + -.) -2-phenylacetyl-2-azabicyclo [2.2.1] hept-5-en-3-one used.

¹H-NMR(CDCl₃)：δ[ppm]400MHz 1.28-1.35(1H)；

1.40(m，1H)；

2.38-2.45(1H)；

2.79(m，1H)；

3.50(AB syst.，2H)；

3.52(s，3H)；

4.98(m，1H)；

5.75(m，2H)；

5.98(m，1H)；

7.20-7.38(5H).

Example 10

Preparation of (+ -) -1-BOC-amino-4-hydroxymethyl-2-cyclopentene (BOC ═ t-butyloxycarbonyl)

At room temperature, 15 g of crude (. + -.) -1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride (prepared as described in J.org.chem.1981, 46, 3268) were dissolved in a mixture of 150 ml water and 150 ml dioxane under nitrogen atmosphere. The pH of the solution was adjusted to 14 with 1N Na OH, then a solution of tert-butyloxycarbonyl fluoride (BOC-F, 20% excess) in ether was added and the mixture was stirred at room temperature for 3 hours (BOC-F was prepared as described in Synthesis 1975, 599). The pH was adjusted to 2 with concentrated hydrochloric acid, 50 ml of water were added to the residue after distilling off the organic solvent, and the mixture was extracted 3 times with 100 ml of ethyl acetate, the combined organic phases were evaporated completely. The residue was crystallized from a mixture of 100 ml of diisopropyl ether and 80 ml of n-hexane to give 11.95 g of the title product. Yield 56%. melting point: 68-70 ℃.

¹H-NMR(DMSO-d₆)：δ[ppm]400MHz 1.18(m，1H)；

1.38(s，9H)；

2.26(m，1H)；

2.65(m，1H)；

3.33(t，2H)；

4.45(m，1H)；

4.55(t，1H)；

5.62(m，1H)；

5.79(m，1H)；

6.73(d，1H).

Example 11

Preparation of (+/-) -1-propionylamino-4-hydroxymethyl-2-cyclopentene

16.6 g (. + -.) -2-propionyl-2-azabicyclo [2.2.1] hept-5-en-3-one were dissolved in water (140 ml) and 2-butanol (66 ml) under nitrogen, cooled to-5 ℃ 3 g sodium borohydride were added in portions over 2 h, the mixture was stirred at 10 ℃ for 2.5 h, then the pH was adjusted to 2.2 with a mixture of concentrated hydrochloric acid and water (1: 1), the solution was evaporated to 40 g, the pH was adjusted to 6.2 with 2N NaOH, the mixture was extracted 5 times with dichloromethane, 50 ml each time, the combined organic phases were evaporated completely and the residue was recrystallized in toluene (150 ml). 11.1 g of the title product are obtained. The yield was 65%.

Melting point: 67-68 ℃.

¹H-NMR(DMSO-d₆)：δ[ppm]400MHz 0.96(t，3H)；

1.16(quint.，1H)；

2.04(quart.，2H)；

2.26(m，1H)；

2.66(m，1H)；

3.34(m，2H)；

4.58(t，1H)；

4.72(m，1H)；

5.61(m，1H)；

5.84(m，1H)；

7.72(d，1H).

Example 12

Preparation of (+/-) -1-isobutyrylamino-4-hydroxymethyl-2-cyclopentene

9 g of (+ -) -2-isobutyryl-2-azabicyclo [2.2.1] hept-5-en-3-one are dissolved in water (32 ml) and 2-butanol (84 ml) under nitrogen, cooled to 0 ℃ and 1.37 g of sodium borohydride are added in portions over 3.5 h, the mixture is stirred at 20 ℃ for a further 3h, the pH is then adjusted to 2.5 with a mixture of concentrated hydrochloric acid and water (1: 1) and then neutralized with 2N NaOH, the solution is evaporated to 40 g, the residue is extracted 3 times with dichloromethane, 80 ml each time, the combined organic phases are evaporated completely and the resulting solid is crystallized in toluene (25 ml) to give 6.8 g of the title product in 73.6% yield.

Melting point: 80-81 ℃.

¹H-NMR(DMSO-d₆)：δ[ppm]400MHz 0.98(d，3H)；

1.16(quint.，1H)；

2.30(m，2H)；

2.68(m，1H)；

3.32(t，2H)；

4.58(t，1H)；

4.70(m，1H)；

5.61(m，1H)；

5.82(m，1H)；

7.68(d，1H).

Example 13

Preparation of (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene with penicillin G acylase

Penicillin G acylase EC 3.5.1.11(Boehringer Mannheim)165U (units)/G derived from E.coli or penicillin G acylase 3.5.1.11 derived from Bacillus megaterium were used for the biotransformation.

For this purpose, 1% by weight of non-racemic 1-phenylacetamido-4-hydroxymethyl-2-cyclopentene and 400 mg of the appropriate penicillin G acylase were incubated at 37 ℃ in 50mM sodium phosphate buffer solution (pH 5-9, 4 ml).

Samples were taken after certain time intervals and analyzed by thin-layer chromatography (silica gel 60, butanol: water: glacial acetic acid 3: 1; ninhydrin detection), gas chromatography (capillary column, HP-5, 5% phenylmethylsiloxane) or High Pressure Liquid Chromatography (HPLC), which removes the phenylacetyl group with high activity, releasing up to 40% of the corresponding amino alcohol. The free amino alcohol is obtained in an enantiomeric excess of 80%.

Example 14

Preparation of (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene by microorganism

Sludge from ASA water treatment plants from Visp was incubated at 14.137 ℃ with shaking in a + N medium containing 0.5% by weight of 1-acetyl, 1-propionyl, 1-isobutyryl or 1-butyrylamino-4-hydroxymethyl-2-cyclopentene (see table 1). The formation of (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene was followed by thin layer chromatography.

The microorganisms Alcaligenes/Bordetella FB 188(DSM 1172), Rhodococcus erythropolis CB101(DSM 10686), Gordonia sp.CB 100(DSM 10687) and Rhodococcus sp.FB 387(DSM11291) were isolated in this way.

14.2 the microorganisms isolated in this way were cultivated in a medium containing 0.5% by weight of 1-acetyl, 1-propionyl, 1-isobutyryl or 1-butyrylamino-4-hydroxymethyl-2-cyclopentene (see Table 1). The cells obtained in this way were harvested in the late exponential growth phase and washed with 10mM phosphate buffer solution, since they had grown to an Optical Density (OD) of 2 to 3 within 24 to 36 hours.

The subsequent biotransformation is carried out in 50mM phosphate buffer solution (pH4.5-9) containing 1% by weight of 1-acetyl, 1-isobutyryl or 1-butyrylamino-4-hydroxymethyl-2-cyclopentene. 50% of the substrate was hydrolyzed to (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene as determined by thin layer chromatography. HPLC analysis revealed an enantiomeric excess of 80-93%.

When 1-butyrylamino-4-hydroxymethyl-2-cyclopentene was used as a substrate and when grown on A + N medium, the bioconversion rate of the strain DSM 10686 was 0.14(g/l/h/OD), and when grown on NYB (Yeast Nutrition juice) medium containing 1-butyrylamino-4-hydroxymethyl-2-cyclopentene, the bioconversion rate was 0.03 (g/l/h/OD).

When the same transformation was carried out with the strain DSM10687 at a substrate (1-butyrylamino-4-hydroxymethyl-2-cyclopentene) concentration of 200mM, the biotransformation rate was 0.161(g/l/h/OD).

TABLE 1A + N Medium

MgCl₂0.4 g/l

CaCl₂0.014 g/l

FeCl₃0.8 mg/l

Na₂SO₄0.1 g/l

KH₂PO₄1 g/l

Na₂HPO₄2.5 g/l

NaCl 3 g/l

Vitamin solution 1 ml/l

Trace element solution 1 ml/l

pH7.5

At 14.330 deg.C, Rhodococcus erythropolis DSM 10686 was cultured to OD on minimal medium (see Table 2) using ammonium acetate (3 g/l) as carbon and nitrogen source in a 6 l fermenter₆₅₀(iii) a cell density of > 25. during cell growth, 50% acetic acid was continuously added as an additional carbon source. then to induce enzyme activity, 60 g of (+/-) -1-acetamido-4-hydroxymethyl-2-cyclopentene was added, and the culture was continued for several hours.finally, 40 g of (+/-) -1-acetamido-4-hydroxymethyl-2-cyclopentene was added, and then the culture was continued for 10 hours. The biotransformation process was followed by on-line HPLC and when the analytical yield was 40% calculated from the racemic substrate used and the enantiomeric excess reached 85%, the fermentation was stopped by addition of acid.

TABLE 2

Composition of culture medium

Concentration of the component

Yeast extract 0.5 g/L

Peptone M660.5 g/l

KH₂PO₄4.0 g/l

Na₂HPO₄2H₂O0.5 g/l

K₂SO₄2.0 g/l

Ammonium acetate 3.0 g/l

CaCl₂0.2 g/l

MgCl₂·6H₂O1.0 g/l

Trace element solution 1.5 ml/L

(see below)

PPG (Polypropylene glycol) 0.1 g/L

Solution of trace elements

KOH 15.1 g/l

EDTA·Na₂·2H₂O100.0 g/l

ZnSO₄·7H₂O9.0 g/l

MnCl₂·4H₂O4.0 g/l

H₃BO₃2.7 g/l

CoCl₂·6H₂O1.8 g/l

CuCl₂·2H₂O1.5 g/l

NiCl₂·6H₂O0.18 g/l

Na₂MoO₄·2H₂O0.27 g/l

14.4 the microorganisms Arthrobacter sp.HSZ 5(DSM 10328), Rhodococcus sp.FB 387(DSM11291), Alcaligenes xylosoxidans denitrifying subspecies HSZ17(DSM 10329), Agrobacterium/Rhizobium HSZ30, Bacillus simplex K2 and Pseudomonas putida K32 were cultured in a similar manner to example 14.3 on sodium acetate in a medium (Table 1) with or without 1-acetyl, 1-propionyl, 1-isobutyryl or 1-butyrylamino-4-hydroxymethyl-2-cyclopentene (hereinafter abbreviated to amino alcohol).

The following results (HPLC analysis) were obtained with exponential cells cultured in the absence of amino alcohol:

bacterial strains	Speed [ mmol/OD.h]	ee/conversion (%)
			HSZ 5(DSM10328)	0.05	88.7/16
HSZ 17(DSM10329)	0.005	95/23
			K32	0.05	54/1
CB101(DSM 10686)	0.1	84/39

Strains K2 and K17 were grown and harvested and subjected to biotransformation for 60 hours.

Bacterial strains	Speed [ mmol/OD.h]	ee/transformation
			K2	-	92/10
HSZ 30	-	93/3.5

Each batch of exponential cells (exponential cells) and static cells (static cells) was collected and used as the quiescent cells for biotransformation. There was no significant difference in the initial velocity of the cells induced or not with amino alcohol according to thin layer chromatography.

Example 15

Purification of N-acetamido-alcohol hydrolase from Rhodococcus erythropolis CB101(DSM 10686)

The enzyme was purified as described below until SDS-PAGE (Pharmacia Phast gel, 10-15% gradient) had only one protein band with a molecular weight of 50kD.

Rhodococcus erythropolis CB101(DSM 10686) cells were washed with 50mM Tris buffer (pH 6.2) and then concentrated to optical density OD_650nm190. after adding phenylmethanesulfonyl fluoride (PMSF) to a final concentration of 1mM and DNase, the cells were treated with a French press to obtain a crude extract. Centrifugation yielded a protein concentration of 4.8 milligram ml^-1The cell-free extract of (1).

960 mg of the cell-free extract was loaded onto a HiLoad 26/10Q-Sepharose ion exchange column (Pharmacia), which had been equilibrated with 50mM Tris buffer (pH8.0) containing 1mM Dithiothreitol (DTT).

After the column was washed with the same buffer, the protein was eluted with a linear buffer gradient (1500 ml; gradient: 50mM Tris buffer (pH8.0) containing 1mM DTT-50 mM Tris buffer (pH 7.0) containing 1mM DDT and 1M NaCl the enzyme was eluted from the column between 370 and 430mM NaCl at pH 7.6. the active fractions were collected and concentrated to 9 ml. the protein content was 41 mg.

For further purification, the protein solution was loaded on a HiLoad 26/60Superdex 75 gel filtration chromatography column (Pharmacia) equilibrated with 50mM Tris buffer containing 50mM NaCl and 1mM DTT. The active fractions were combined and the total protein content was 10.9 mg.

The protein solution was loaded on a Mono Q HR5/5 column (Pharmacia). The column was equilibrated with 50mM Tris buffer (pH8.5) containing 1mM DTT. The protein was eluted with a linear gradient (40 ml) of 50mM Tris buffer (pH8.5) containing 1mM DTT, 50mM Tris buffer (pH8.5) containing 1mM DDT and 1M NaCl. The enzyme eluted between 390-440mM NaCl. The active fraction contained 1.4 mg of protein.

In the final purification step, the same column equilibrated with the same buffer was used. The elution gradient used was the same buffer containing 0-500mM NaCl and pH 7.0-8.5. 430. mu.g of pure enzyme can be isolated in this way.

The N-terminal order of the enzymes was determined directly from Western blotting. The following sequence of 20 amino acids was obtained: Thr-Glu-Gln-Asn-Leu-His-Trp-Leu-Ser-Ala-Thr-Glu-Met-Ala-Ala-Ser-Val-Ala-Ser-Asn.

This sequence has no homology to known proteins.

Example 16

Characterization of the enzymes

Enzyme characterization was performed using purified enzyme and cell-free extract that had been desalted using Sephadex G-25 column (PD-10, Pharmacia).

The protein concentration in the cell-free extract was 7.3 mg ml^-1The concentration of protein in the purified enzyme was 135. mu.g ml^-1. No PMSF was added to the cell-free extract.

16.1K_mMeasurement of

Performing K in cell-free extract_mMeasurement reaction K of substrate 1-acetamido-4-hydroxymethyl-2-cyclopentene at pH7.0 and temperature 30 deg.C_mIt was 22.5mM.

16.2 optimum pH value

The optimum pH for hydrolysis of 1-acetamido-4-hydroxymethyl-2-cyclopentene (25mM) was determined in the following buffer solution with purified enzyme and cell-free extract at pH range of 6.2-9.0.

Tris buffer 100mM pH 9.0; 8.5; 8.0; 7.5; 7.0

Citrate/phosphate buffer 100mM pH7.0; 6.55; 6.2

The activity measurement was carried out for 24 hours.

The optimum pH for the reaction to prepare the 1R, 4S and 1S, 4R enantiomers is from pH7.0 to 7.5.

The optimum pH value for the activity of the cell-free extract is pH 7.0. However, selectivity is better between pH 6.0 and 7.0.

FIG. 1 shows the activity of N-acetyl amino alcohol hydrolase (cell-free extract) extracted from Rhodococcus erythropolis CB101(DSM 10686) as a function of pH the optimum temperature for the reaction shown in example 16.2 is 25-30 ℃.

FIG. 2 shows the activity of N-acetyl amino alcohol hydrolase (cell-free extract) extracted from Rhodococcus erythropolis CB101(DSM 10686) as a function of temperature.

16.4 molecular weight 50kD as determined by SDS-PAGE.

16.5 hydrolysis of the following substrates: 1-acetamido-4-hydroxymethyl-2-cyclopentene, 1-butyrylamino-4-hydroxymethyl-2-cyclopentene, 1-propionylamino-4-hydroxymethyl-2-cyclopentene, 1-isobutyrylamino-4-hydroxymethyl-2-cyclopentene.

Example 17

Preparation of (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride

374.1 g of (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene solution (prepared in a similar manner to example 14) was evaporated to 123.7 g. A solution containing 60.2 mmol of the above-mentioned compound (HPLC) is adjusted from pH2 to pH11.7 with 30% strength NaOH. It was then extracted three times with 70 ml of isobutanol each time. The combined isobutanol extracts were adjusted to pH1 with hydrogen oxide gas, concentrated to 65 g and filtered (to remove solid impurities). 60 ml of acetone are added dropwise to the vigorously stirred filtrate at 20 ℃. To the cloudy mixture was added seed crystals of the title compound and stirred at 5 ℃ for 1 hour. After filtration and drying, 5.2 g of product are obtained, yield 58%.

Melting point: 125 ℃ C. and 127 ℃ C

Enantiomeric excess of 98% (calibrated chiral high pressure liquid chromatography column)

¹H-NMR(DMSO-d₆)：δ[ppm]400Mhz 1.44(m，1H)

2.35(m，1H)；

2.83(m，1H)p

3.42(m，2H)；

4.10(s，1H)；

4.80(d，1H)；

5.80(d，1H)；

6.06(d，1H)；

8.13(s，3H).

Example 18

Preparation of (1R, 4S) -1-BOC-amino-4-hydroxymethyl-2-cyclopentene

A solution of 75 g of (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene (prepared analogously to example 14; 44.6 mmol of the compound) was adjusted to pH8 with 30% strength NaOH and 6 g of sodium bicarbonate were added. The mixture was heated to 52 ℃ with vigorous stirring, 60 ml of diisopropyl ether were added and then a solution of 11.12 g of tert-butoxycarbonyl acid anhydride in 18.2 ml of diisopropyl ether was added dropwise over 2 hours, the mixture was filtered over Celite (Celite), the phases were separated and the aqueous phase was extracted with 65 ml of diisopropyl ether. The combined organic phases were washed with 45 ml of water and evaporated to 37.5 g, heated to 50 ℃. To the solution was added dropwise 31 ml of n-hexane. Then slowly cooled to 0 deg.C (2 hours), the title product was filtered off, washed with 12 ml of n-hexane/diisopropyl ether (1/1), and dried. 6.75 g of product are obtained. The yield was 71%.

Melting point: 70-71 deg.C

Enantiomeric excess of 98% (calibrated chiral high pressure liquid chromatography column)

¹H-NMR(DMSO-d₆)：δ[ppm]400Mhz 1.18(m，1H)；

1.27(s，9H)；

2.28(m，1H)；

2.63(m，1H)；

3.33(q，2H)；

4.43(m，1H)；

4.56(t，1H)；

5.62(m，1H)；

5.78(m，1H)；

6.72(d，3H).

Example 19

Preparation of (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride

87.8 g of (1R, 4S) -1-BOC-amino-4-hydroxymethyl-2-cyclopentene were dissolved in 270 ml of 2N hydrochloric acid and 1340 ml of methanol, the mixture was heated under reflux for 4.5 hours, after distilling off the methanol, the residue was dissolved in 800 ml of water, the aqueous solution was extracted twice with ethyl acetate, 340 ml each time, the aqueous phase was evaporated completely (50 ℃/60 mbar), the solid was dried under vacuum at 50 ℃ and suspended in 150 ml of diethyl ether, filtered and washed twice with 50 ml of diethyl ether, yielding the title product after drying in 95% yield (58.4 g).

The physical and spectral data of the product were the same as in example 17.

Example 20

Preparation of (1R, 4S) -1-acetamido-4-hydroxymethyl-2-cyclopentene

25 g of (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride were dissolved in 182 ml of acetic anhydride, and at 0 ℃ a solution of 18.25 g of triethylamine in 60 ml of acetic anhydride was added, and at 80 ℃ the mixture was stirred for 3 hours and then cooled to room temperature. The triethylamine hydrochloride was filtered off and washed with 120 ml of n-hexane. The filtrate was evaporated, and the residue was mixed with 300 ml of toluene, and after adding 5.2 g of activated carbon and 13 g of celite, it was stirred at room temperature for 20 minutes. After filtration and washing of the filter cake (3X 40 ml) toluene, the solvent was evaporated off completely and the residue was mixed with 180 ml methanol and 15.5 g potassium carbonate and stirred at room temperature for 10 hours. The residue is suspended in 750 ml of isopropyl acetate, 0.5 g of activated carbon is added and the mixture is heated under reflux for 1.5 hours. After filtering off the activated carbon (70-80 ℃), the filtrate was cooled at 0 ℃ overnight. The title compound was filtered off, washed with 80 ml of cold isopropyl acetate and dried in vacuo. 17.2 g of product are obtained. The yield was 66%.

Melting point: 77-80 deg.C

Enantiomeric excess of 98% (calibrated chiral high pressure liquid chromatography column)

¹H-NMR(DMSO-d₆)：δ[ppm]400MHz 1.15(m，1H)

1.78(s，3H)；

2.25(m，1H)；

2.66(m，1H)；

3.35(m，2H)；

4.58(t，1H)；

4.70(m，1H)；

5.62(m，1H)；

5.85(m，1H)；

7.80(d，3H).

Example 21

Preparation of (1S, 4R) -1-acetamido-4-hydroxymethyl-2-cyclopentene

The title enantiomer was prepared in 68% yield by the method of example 18 starting with 25 g of (1S, 4R) -1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride. The spectral and physical data of the product were the same as in example 20.

Example 22

Preparation of (1R, 4S) -1-butyrylamino-4-hydroxymethyl-2-cyclopentene

34.7 g of (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride and 2 g of 4-N, N-dimethylaminopyridine were dissolved in 600 ml of dichloromethane, and the solution was cooled to 0 ℃. Then 52 g triethylamine (5 min) were added dropwise. The mixture was stirred for a further 30 minutes. To the mixture was added dropwise over 1 hour at 0 ℃ a solution of 35.2 g of butyryl chloride dissolved in 60 ml of dichloromethane. The mixture was stirred at 0-20 ℃ for a further 1.5 h, then 600 ml of water were added. After phase separation, the aqueous phase is extracted with 600 ml of dichloromethane and the combined organic phases are washed three times with 500 ml of 10% strength sodium hydroxide. It was then evaporated completely. The dried solid was dissolved in 120 ml of methanol, the solution was mixed with 5 g of potassium carbonate and stirred at room temperature for a further 2 hours. The inorganic salts were filtered off and washed with 20 ml of methanol. The filtrate was neutralized with 2N hydrochloric acid. The suspension is filtered and the filter cake is washed with 20 ml of methanol. The filtrate was evaporated completely. The solid residue was dried and crystallized from 150 ml of toluene, 28.5 g of the title compound were obtained. The yield was 67%.

Melting point: 71-72 deg.C

Enantiomeric excess of 98% (calibrated chiral high pressure liquid chromatography column)

¹H-NMR(DMSO-d₆)：δ[ppm]400MHz 0.85(t，3H)；

1.15(m，1H)；

1.50(q，2H)；

2.03(d，2H)；

2.28(m，1H)；

2.67(m，1H)；

3.35(d，2H)；

4.62(s，1H)；

4.76(m，1H)；

5.63(m，1H)；

5.85(m，1H)；

7.77(d，1H).

Example 23

Preparation of (1S, 4R) -1-butyrylamino-4-hydroxymethyl-2-cyclopentene

The title enantiomer (yield 63%) was prepared in the same manner as in example 20 using 34.7 g of (1S, 4R) -1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride as starting material the spectral data and physical data of the product were the same as in example 22.

Example 24

Preparation of (1R, 4S) -1- [ (2-amino-6-chloro-5-formylamino-4-pyrimidinyl) amino ] -4-hydroxymethyl-2-cyclopentene

2.07 g of N- (2-amino-4, 6-dichloro-5-pyrimidinyl) formamide are heated to 80 ℃ in 40 ml of isobutanol (white suspension). To this mixture was added a solution of 1.97 g of (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride, 3.8 g of triethylamine and 15 ml of isobutanol, and the mixture was stirred at 80 ℃ for another 13 hours. To this clear solution was added 10 ml of 1N sodium hydroxide at 20 ℃ and then evaporated to dryness, and the residue was subjected to flash chromatography (column on silica gel 60, length 8 cm, diameter 6.5 cm, eluent ethyl acetate/methanol 95/5). After evaporation of the eluent and drying of the residue, 2.1 g of the title product are obtained. The yield was 74%.

Melting point: 174 deg.C 176 deg.C

Enantiomeric excess of 98% (calibrated chiral high pressure liquid chromatography column)

¹H-NMR(DMSO-d₆)：δ[ppm]400MHz 1.37(m，1H)；

2.35(m，1H)；

2.73(m，1H)；

3.38(t，2H)；

4.68(m，1H)；

5.08(m，1H)；

5.70(d，1H)；

5.85(d，1H)；

6.40; 6.55 and 6.65(s, dd, 3H total);

7.78 and 8.10(d and s, total 1H);

8.55 and 8.95(d and s, total 1H).

Example 25

Preparation of (1R, 4S) -1- [ (2-amino-6-chloro-5-formylamino-4-pyrimidinyl) amino ] -4-hydroxymethyl-2-cyclopentene

A solution of 145.2 ml of (1R, 4S) -1-amino-4-hydroxymethyl-2-cyclopentene (prepared in a similar manner to example 14) was concentrated to 25.5 ml, and filtered through celite. The filter cake is washed with 7.5 ml of water, the pH of the filtrate containing 17.7 mmol of the above-mentioned compound (HPLC) is adjusted from 6.6 to 1 with concentrated hydrochloric acid and then extracted three times with isobutanol, 20 ml each time, and the organic phase is discarded. The pH of the aqueous phase is adjusted to 12 with 30% strength sodium hydroxide, extracted 3 times with isobutanol, 10 ml each time, the combined organic phases are evaporated to 15 ml, 2.53 g of triethylamine are added, a solution of 2.07 g of N- (2-amino-4, 6-dichloro-5-pyrimidinyl) formamide in 40 ml of ethanol is added to the mixture as described in example 24, and the mixture is stirred at 80 ℃ for 16 hours. Working-up was carried out as in example 22 to give 2.4 g of the title product in 85% yield.

The physical and spectral data of the product are the same as in example 24.

Example 26

Preparation of (+ -) -1-BOC-amino-2-cyclopentene-4-carboxylic acid

16.4 g of crude (. + -.) -1-amino-2-cyclopentene-4-carboxylic acid hydrochloride (prepared as described in J.org.chem.1981, 46, 3268) were dissolved in a mixture of 80 ml water and 80 ml dioxane at room temperature under nitrogen atmosphere. The pH of this mixture was adjusted to 14 with 1N sodium hydroxide, followed by addition of a solution of tert-butyloxycarbonyl fluoride (BOC-F, 20% excess) in diethyl ether (BOC-F was prepared as described in Synthesis 1975, 599). The mixture was stirred at room temperature for a further 5 hours and the pH was adjusted to 2 with concentrated HCl. After distilling off the organic solvent, 50 ml of water were added to the residue, the mixture was extracted three times with 100 ml of ethyl acetate, the organic phase was evaporated to 50 ml and diluted with 25 ml of toluene. After cooling (0-10 ℃ C.), the title product was filtered off and dried, giving 14.3 g of product in 63% yield.

Melting point: 126 ℃ and 127 DEG C

¹H-NMR(DMSO-d₆)：δ[ppm]400MHz 1.14(s，9H)；

1.70(m，1H)；

2.40(m，1H)；

3.40(m，1H)；

4.47(m，1H)；

5.70(t，1H)；

4.87(t，1H)；

6.88(d，1H)；

12.30(d，1H).

Example 27

Preparation of (+ -) -1-BOC-amino-4-hydroxymethyl-2-cyclopentene from (+ -) -1-BOC-amino-2-cyclopentene-4-carboxylic acid

250 ml of tetrahydrofuran and 10.02 g (0.164 mol) of LiAlH₄Into a 500 ml stirring apparatus, at-10 ℃ a solution of 30.0 g (0.132 mol) (. + -.) -1-BOC-amino-2-cyclopentene-4-carboxylic acid in 75 ml tetrahydrofuran is added dropwise over 1 hour, then 10 g water, 10 g 15% strength sodium hydroxide solution and 20 g water are added and filtration is carried out again, the residue is washed twice with 100 ml each of tert-butyl methyl ether, the combined organic phases are evaporated to dryness with 80 ml hexane and seed crystals of the compound of example 10 and the crystalline solid of the title compound is isolated. Yield 15.84 grams (56%). physical and spectral data were identical to example 10.

Example 28

Preparation of (1R, 4S) -1-BOC-amino-4-hydroxymethyl-2-cyclopentene from (1R, 4S) -1-BOC-amino-2-cyclopentene-4-carboxylic acid

80 ml of tetrahydrofuran and 11.38 g (50.07 mmol) of (1R, 4S) -1-BOC-amino-2-cyclopentene-4-carboxylic acid (prepared as described in Tetrahedron: Asymmetry 1993, 4, 1117) are introduced into a 500 ml stirring apparatus, 5 ml of borane/dimethyl sulfide adduct are added dropwise at-15 ℃ over a period of 1 hour, and the mixture is then stirred at this temperature for 3 hours. A solution of 4 g of sodium hydroxide dissolved in 60 ml of water is added, warmed to room temperature, extracted with toluene, filtered over silica gel and then crystallized in ethyl acetate/n-hexane 1: 1 to give 5.4 g of the title product as a white crystalline solid, corresponding to a yield of 57%. The physical and spectral data were the same as in example 10. enantiomeric excess was 99% (same chiral high pressure liquid chromatography column).

Claims (1)

1. A process for the preparation of racemic or optically active 4- (hydroxymethyl) -2-cyclopentene derivatives represented by the formula XIII,

in the formula R¹Is represented by C_1-4Alkyl radical, C_1-4Alkoxy, aryl or aryloxy, characterized in that in a first step a carbonyl halide of the formula XI is used

In the formula R¹Is represented by C_1-4Alkyl radical, C_1-4Alkoxy, aryl or aryloxy group, and X represents a halogen atom, and a racemic cyclopentene-4-carboxylic acid represented by the formula XIV or one of its optically active isomers

Acylation to form racemic or optically active cyclopentene-4-carboxylic acid derivatives represented by the general formula XV,

the latter is reduced in a second step to give the desired product represented by formula XIII.