Method for production of 4-[4-[4-(hydroxybiphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]- α,α-dimethylphenylacetic acid

FIELD: biotechnology.

SUBSTANCE: 4-[4-[4-(hydroxybiphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylphenylacetic acid is produced by incubation of mixture, containing terphenadine and microorganism capable to produce said acid followed by isolation of target product.

EFFECT: simplified process with increased yield.

20 cl, 2 tbl

 

The technical field

This invention relates to a method for production of piperidine derivatives with the use of microorganisms.

Prior

Terfenadine, 1-(p-tert-butylphenyl)-4-[-4'-(α-hydroxydiphenylmethyl)-1'-piperidinyl]butanol is nnegative antihistaminic agent. Described that it is a specific receptor antagonist N1that is also devoid of anticholinergic, antiserotonergic and antiadrainergicakimi actions both in vitro and in vivo. Cm. D. McTavish, K. L. Goa, M. Ferrill, Drugs, 1990, 39, 552; C. R. Kingsolving, N. L. Monroe, A. A. Carr, Pharmacologist, 1973, 15, 221; J. K. Woodward, N. L. Munro, Arzneim-Forsch, 1982, 32, 1154; K. V. Mann, K. J. Tietze, Clin. Pharm., 1989, 6, 331. Significant efforts have been made to study the relationship of structure - activity of analogues of terfenadine, they are reflected in numerous U.S. patents that describe this connection and related structures, namely:

U.S. patent No. 3687956, Zivkovic

U.S. patent No. 3806526, Carr, et al

U.S. patent No. 3829433, Carr, et al

U.S. patent No. 3862173, Carr, et al

U.S. patent No. 3878217, Carr, et al

U.S. patent No. 3922276, Duncan, et al

U.S. patent No. 3931197, Carr, et al

U.S. patent No. 3941795, Carr, et al

U.S. patent No. 3946022, Carr, et al

U.S. patent No. 3956296, Duncan, et al

U.S. patent No. 3965257, Carr, et al

U.S. patent No. 4742175, Fawcett, et al.

Metabolism studies in animals and humans showed that Ter is anadin undergoes extensive hepatic metabolism of the first cycle and, after the usual dosage he could not be determined in the plasma, if not only apply the tests with very high sensitivity. Specific hepatic cytochrome P-450 turns terfenadine in the main metabolite 4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylphenoxy acid, also known as the carboxylic acid metabolite of terfenadine. This metabolite can be easily determined in the plasma and is considered the active form of the oral input terfenadine.

The side effects described for terfenadine, include heart arrhythmia (ventricular tachyarrhythmia, Torsade de pointes, ventricular fibrillation), sedation, gastrointestinal upset, dry mouth, constipation and/or diarrhea. The most serious among them and potentially life-threatening are cardiac arrhythmias that are associated with the ability of terfenadine to prolong the cardiac QT interval and described only for patients who entered terfenadine suffering from liver disease who are also antifungal agent ketoconazole or antibiotic erythromycin.

So as cardiac side effects of terfenadine described for patients with impaired liver function, as well as for patients who are taking antibiotics, which are known to inhibit the function of p is chanoch enzymes I believe that cardiac side effects occur because of the accumulation of terfenadine, and not due to the accumulation of carboxylic acid metabolite of terfenadine. "Patch-clamp" studies on isolated ventricular myocyte cats confirm the assertion that terfenadine, instead of the carboxylic acid metabolite responsible for cardiac side effects. At a concentration of 1 μm terfenadine causes more than 90% inhibition of the delayed rectifying potassium current. At concentrations up to 5 μm, the carboxylic acid metabolite of terfenadine no significant action on the potassium current in this study (see R. L. Woosley, Y. Chen, Frieman J. P. and R. A. Gillis, JAMA, 1993, 269, 1532). Because the inhibition of ion transport associated with cardiac anomalies, such as arrhythmia, these results indicate that the carboxylic acid of terfenadine in all probability not responsible for heart arrhythmia at levels of dose at which there is a risk of such side effects caused by terfenadine.

Carebastine, 4-[4-[4-(diphenylmethoxy)-1-piperidinyl]-1-oxobutyl]-α,α-dimethylferrocene acid is a carboxylic acid metabolite of ebastine, 1-(p-tert-butylphenyl)-4-[4'-(α-diphenylmethoxy)-1'-piperidinyl]butanol. Both compounds possess potent selective blocking action is at the H 1-receptor histamine and properties of a calcium antagonist, and proved that they are useful in the treatment of various respiratory, allergic and cardiovascular illnesses.

These compounds relax bronchial and cardiac smooth muscle in vitro and in vivo and inhibit the compressive action of norepinephrine, potassium ions, and various other drugs-agonists. The compounds also inhibit the reaction of the intestinal and tracheal preparations histamine, acetylcholine and barium chloride and block bronchostenosis caused by aerosol of histamine in Guinea pigs at doses less than 1 mg/kg animal body weight when administered orally. They also have antianaphylactic properties in rats, inhibit skin lesion variety of anaphylactic mediators (histamine, 5-hydroxytryptamine, bradykinin, LCD4etc) and are antagonists of the reaction Schultz-Dale in sensitive Guinea pigs.

Derivatives of piperidine, related to the carboxylic acid metabolite of terfenadine, is described in the following U.S. patents:

U.S. patent No. 4254129, Carr, et al

U.S. patent No. 4254130, Carr, et al

U.S. patent No. 4285957, Carr, et al

U.S. patent No. 4285958, Carr, et al.

In these patents 4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylbenzoxazole acid and related compounds get al what Yerevanian substituted piperidine derivative of the formula:

α-halogenosilanes phenylketones formula:

where the substituents halogen, R1, R2, n, Z and R6described in column 6 of U.S. patent No. 4254130.

Similarly, in U.S. patent No. 4550116, Soto et al. describes the obtaining of piperidine derivatives related carebastine interaction α-halogenosilanes of phenylketone with substituted derivatives of hydroxypiperidine formula:

In U.S. patent No. 4254130 stated that α-galogenzameshchennye phenylketone, in which Z is hydrogen, produced by interaction of a suitable low1-6Olkiluoto straight or branched chain of ester α,α-dimethylphenylacetic acid with a compound of the following formula:

under normal conditions of acylation Friedel-where halogen and m is described in column 11 of U.S. patent No. 4254129. The reaction is carried out in carbon disulfide as the preferred solvent.

Other synthetic methods to obtain the carboxylic acid metabolite of terfenadine described in U.S. patents№ 5578610, 5581011, 5589487, 5663412, 5750703 and 5994549, as well as in PCT application no WO 95/00492, WO 94/03170 and WO 95/00480.

Another approach to the formation of compounds such as carboxylic acid metabolite of terfenadine in the cancel the transformation of compounds, like terfenadine, using fungi. This technique is described in U.S. patent No. 5204249, Schwartz et al and U.S. patent No. 5990127, Meiwes et al. In the patent Schwartz fungi of the genus Cunninghamella used for the transformation of ebastine in carebastine. In the patent Meiwes used fungi species of the genus Cunninghamella and Absidia for the conversion of terfenadine in its acid metabolite. Although it was found that these methods are useful for producing compounds such as carboxylic acid metabolite of terfenadine, the initial output of these products in this process is quite low and limitations of the hyphomycetes of the above genera lead to undesirable with the limits for a commercially viable way.

This invention relates to an improved process for the preparation of carboxylic acid metabolite of terfenadine and derivatives of carebastine using microbial catalysis.

Brief description of the invention

This invention relates to the production of compounds having formula IA and/or IB:

where

n means 0 or 1;

R1is hydrogen or hydroxy;

R2is hydrogen;

or if n is 0, R1and R2taken together form a second bond between the carbon atoms bearing R1and R2provided that when n is 1, R1and R2each is hydrogen is;

R3is-COOH or-COOR4;

R4is alkyl or aryl;

A, b and D are the substituents on the respective rings, which may be different or the same and are selected from the group comprising hydrogen, halogen, alkyl, hydroxy and alkoxy.

The method involves incubation of the parent compound, having formula IIA and/or IIB:

where R3* is-CH3and R1, R2A , B and D such as defined above,

in the presence of the microorganism under conditions effective to obtain the target compound. The microorganism may be selected from the genus Streptomyces, Stemphylium, Gliocladium, Bacillus, Botrytis, Cyathus, Rhizopus, Pycniodosphora, Pseudomonas, Helicostylum, Aspergillus, Mucor, Gelasinospora, Rhodotorula, Candida, Mycobacterium, or Penicillium.

This invention also relates to the production of the target compounds having a structure according to formula IA and/or IB, incubation of the parent compound, having a structure according to formula IIA and/or IIB in the presence of Cunninghamella bainieri under conditions effective to obtain the target compound.

This invention represents an alternative and/or improved method of producing carboxyterminal of terfenadine. The selectivity and yield of carboxyterminal obtained with the use of strains and methods in accordance with given the first invention, can be higher than those values obtained by applying the known strains. In addition, the identification of many strains, especially strains of bacteria (gram positive and gram negative) for the target transformations, enables a significant improvement of the strain, the advantages in processing and manufacturing in comparison with previously used strains of hyphomycetes.

Importantly and unexpectedly, that Streptomyces, Bacillus and Pseudomonas are gram-positive and gram-negative eubacterial strains, there is an area that is completely different from hyphomycetes, which were previously used for the targeted transformation. Techniques to improve strains and genetic manipulation of strains of bacteria, especially including species of Streptomyces, Bacillus and Pseudomonas, much easier and better designed compared to fungi, such as strains of Cunninghamella. Moreover, the industrial processing senitivity microorganisms, including venicevenice fungi, yeast and eubacteria, allows for many additional and more economical fermentors and cleaning methods compared to those that apply only to hyphomycetes.

Moreover, many of microbial biocatalysts are used for conversion into a variety of structural options. In addition, identification of various strains with genes and enzymes is useful for such transformations, is an important prerequisite for the application of modern techniques of molecular biology for further optimization of micro-organisms as industrial catalysts for production of piperidine derivatives.

Detailed disclosure of the invention

This invention relates to the production of target compounds having formula IA and/or IB:

where

n means 0 or 1;

R1is hydrogen or hydroxy;

R2is hydrogen;or when n is 0, R1and R2taken together form a second bond between the carbon atoms bearing R1and R2provided that when n is 1, R1and R2each is hydrogen;

R3is-COOH or-COOR4;

R4is alkyl or aryl;

A, b and D are the substituents on the respective rings, which may be different or the same and are selected from the group comprising hydrogen, halogen, alkyl, hydroxy and alkoxy.

The method involves incubation of the parent compound, having formula IIA and/or IIB:

where R3* is-CH3and R1, R2A , B and D such as defined above, in the presence of the microorganism under conditions effective to obtain the target connection is in. The microorganism may be selected from the genus Streptomyces, Stemphylium, Gliocladium, Bacillus, Botrytis, Cyathus, Rhizopus, Pycniodosphora, Pseudomonas, Helicostylum, Aspergillus, Mucor, Gelasinospora, Rhodotorula, Candida, Mycobacterium, or Penicillium.

This invention also relates to the production of the target compounds having a structure according to formula IA and/or IB, incubation of the parent compound, having a structure according to formula IIA and/or IIB in the presence of Cunninghamella bainieri under conditions effective to obtain the target compound.

The method in accordance with this invention is carried out in a liquid nutrient medium. The composition of a suitable nutrient medium depends on the specific microorganism and goals and well-known specialists in this field of technology. In General, the nutrient medium contains sources of carbon, such as dextrose, sucrose, citrate and/or starch, and nitrogen sources such as soybean meal, yeast extract, tripton, malt extract and/or ammonium acetate. In addition, the nutrient medium contains inorganic salts such as sodium phosphate, potassium phosphate, sodium chloride, calcium chloride, calcium sulfate, calcium carbonate and/or magnesium sulfate, and trace elements such as salts of iron, zinc, copper, molybdenum, manganese or other metals.

The microorganisms used in accordance with this invention, can be selected from the following genera: Streptomyces, Stemphylium, Gliocladium, Bacillus, Borytis, Cyathus, Rhizopus, Pycniodosphora, Pseudomonas, Helicostylum, Aspergillus, Mucor, Gelasinospora, Rhodotorula, Candida, Mycobacterium, or Penicillium. For the genus Streptomyces suitable species include Streptomyces catenulae, Streptomyces cavourensis, Streptomyces rimosus and Streptomyces griseus. For the genus Stemphylium appropriate form is Stemphylium consortiale. Beneficial species include Aspergillus Aspergillus aliaceus, Aspergillus carbonarium (Bainier) Thom, Aspergillus flavipes, Aspergillus fumigatus, Aspergillus ochraceous and Aspergillus terricola. For the genus Gliocladium especially useful is the view of Gliocladium deliquescens. Of the genus Bacillus for implementing the method in accordance with this invention can be applied to the species Bacillus cereus, Bacillus subtilis and Bacillus fusiformis. A suitable form of Botrytis is Botrytis allii. Of the genus Cyathus can be used Cyathus striatus. Rhizopus oryzae is a member of the genus Rhizopus, which can be used in accordance with this invention. Beneficial species include Pseudomonas Pseudomonas putida. From the kind of Pycniodosphora can be applied Pycniodosphora dispersa. From the kind of Helicostylum for implementing the method in accordance with this invention can be applied Helicostylum piriforme. Of the genus Mucor for implementing the method in accordance with this invention can be applied Mucor circinelloides f. griseo-cyanus, Mucor recurvatus and Mucor mucedo. View Gelasinospora autosteria is a member of the genus Gelasinospora, which is suitable for implementing the method in accordance with this invention. Of the genus Rhodotorula can be applied view of Rhodotorula rubra. Of the genus Penicillium in the practice of the method in accordance with the data of what Britanie can be applied to the species Penicillium notatum and Penicillium chyrsogenum. Of Candida can be applied to the species Candida guilliermondii yeast, Candida lipolytica and Candida parasilosis var. quercus. Suitable species include Mycobacterium Mycobacterium bisrymcum.

For each strain, the invention relates to the use of the whole organism and its components, including but not limited to, extracts of cells, microsome assay, isolated enzymes and genes for chemo - and regioselective oxidation of compounds of formula IIA and/or IIB to the products of formula IA and/or IB.

Moreover, mutants and selectany microbes listed genera and, especially, specific strains listed above, are also suitable for use in the method in accordance with this invention. Mutants can be obtained by classical methods of mutagenesis for improvement of strain, such as non-specific mutagenesis, mediaready chemicals or electromagnetic waves, or the modern methods of genetic manipulation, such as PCR with bronirovannymi discrepancies, mutagenesis of the codon or rearrangement of genes.

Another aspect of this invention relates to the use of species Cunninghamella bainieri for implementing the method in accordance with this invention.

This invention also relates to the detection and use of microorganisms of the genus Streptomyces, Gliocladium and Stemphyllium as agents for the selective oxidation of terfenadine (formula IIA/IIB) to carboxide is enedina (formula IA/IB), superior fungi of the genus Cunninghamella, and Absidia.

In addition, microbial strains of the genera Botrytis, Rhizopus, Cyathus, Bacillus, Pycniodosphora, Pseudomonas, Helicostylum, Aspergillus, Gelasinospora, Rhodotorula, Penicillium and Candida are also identified as oxidizing terfenadine to carboxitherapy with the release of more than 3% without optimization. In previous experiments Meiwes et al. identified only two strains, which give output 3% or more at the time of initial screening.

Moreover, it was found that microorganisms of the genus Ascoidia, Enterococcus, Fusidium, Lentinus, Lophotrichus, Mycobacterium, Polyporus, Spicaria and Trichophyton are biocatalysts that can oxidize terfenadine to carboxytherapy.

All of these microorganisms are easily available from public culture collections. The specific types and sources of microbial cultures described in the examples below.

Microbial culture used in accordance with this invention, can be stored using methods well known to specialists in this field, such as in hard environment, conservation of mineral oil and lyophilization or freeze.

Microbial culture can be stored in a suitable solid medium, such as 30 grams per liter of broth, Sabouraud dextrose and 20 g/liter of agar. Preferably, some strains obtaining inoculum, including cryopreservation at low temperature and thawing (i.e. methodology Cyoready"), is used to improve the approach to transform the source material into the product of piperidine in accordance with this invention that reduces the time to obtain a suitable inoculum and increases the yield of piperidine. After growing the culture in a suitable liquid medium microbial suspension is centrifuged, the spent liquid medium is removed and the concentrated sludge cells in vitro re-suspended in equal volume of sterile 20% glycerol, initial solution and fresh broth with obtaining a cell suspension in 10% glycerol.

From the solid medium the microorganisms are first diluted in one or more stages in a neutral liquid culture medium suitable for growth of specific strains (for example, the methods of the "Multistage"). A typical environment for the original breeding contains 20 g/l glucose, 5 g/l yeast extract, 5 g/l soybean flour, 5 g/l NaCl and 5 g/l K2HPO4. The initial phase of microbial cultures incubated at 29°C and 250 rpm for 48 to 72 hours. Further stage inoculant heavy inoculum (1-20% V/V, especially 10% V/V microbial suspension liquid culture with the previous stage in fresh liquid medium).

For carrying out the reaction stage heavy inoculum (1-20% V/V, particularly, 10% V/V) of microbial suspension or thawed cryopreserved cells inoculant in a fresh environment. Microorganisms Kul is everyt at temperatures from about 20° With up to 80°C, preferably from 25°to 37°and pH from 4 to 9, especially at pH 5 to 8, depending on the particular microorganism used for the conversion. Incubation of microorganisms can be performed during 2-240 hours, preferably from 75 to 170 hours. The reaction is carried out aerobically, first in parallel, in a multiple-cell reaction chambers, in which continuously served the air or enriched oxygen, and mix. Therefore, by this method of fermentation can be carried out on an industrial scale in shake flasks and then in fermentors under stirring and aeration.

Adding original material to microbial culture should be performed within 0-72 hours of inoculation reaction medium obtained by the inoculum, preferably after about 8-48 hours, and particularly preferably after 24 hours of incubation. Adding the source material is most appropriate to carry out from a solution in a suitable organic solvent, but it can also be added in the form of a solid powder or in suspension. From the solution of the original material is most preferably added to dimethylformamide (DMF), but also in ethanol, dimethylsulfoxide (DMSO), dimethylacetamide (DMA), acetonitrile, tetrahydrofuran (THF) and formamide (for example, dibutil-, aminobutiramida - or diethyl-), pyrrolidone (e.g., 1-m is l, 1-ethyl-, 1-cyclohexyl-), 4-formylmorpholine, 1-formylpiperidine, 1-formylpyridine, tetramethyl-, tetraethyl-, terbutylazine, the phosphine oxide (e.g., Trimeperidine or triprolidine-), sulfolane, N-methylcaprolactam or their mixtures. To the reaction medium containing the microorganism can also be added biocompatible organic solubilization, such as cyclodextrins or surfactants (such as Tween 80 or Pluronic F38).

Compounds of formula IA and/or IB can be isolated directly from microbial broth or purified liquid after separation of the cells, for example, by centrifugation or filtration. These products can be isolated by extraction with organic solvents or by adsorption on hydrophobic resins or ion exchangers.

In additional embodiments of the present invention can be applied to the microorganisms and standard methods and conventional methods incubation of microorganisms and reactions performed as described in the public guidelines. For example, the methods described Demain, A. L. and J. E. Davies, in Manual of Industrial Microbiology and Biotechnology, 2ndEd. (1999) and Crueger, W. and A. Crueger, Biotechnology: A Textbook of Industrial Microbiology (1984), are used to obtain cultures and implementation of the method in accordance with this invention.

Of particular importance are compounds of formula IIIA and/or IIIB:

where R1, R2, R3A , B and D such as defined above. Among these compounds, particularly preferred is 4-(4-(4-hydroxydiphenyl)-1-piperidinyl)-1-hydroxybutyl)-α,α-dimethylferrocene acid.

Other preferred compounds are compounds of formula IVA and/or IVB:

where R1, R2, R3A , B and D such as defined above. Among these compounds, particularly preferred is 4-[4-[4-(diphenylmethoxy)-1-piperidinyl]oxobutyl]-α,α-dimethylferrocene acid.

This invention also relates to a method more analogues of formula IA and/or IB, based on the compounds of the formulae IIA and/or IIB with microorganisms in accordance with this invention.

Other illustrative examples of compounds obtained by the method in accordance with this invention, are:

4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylbenzanthracene acid;

4-[4-[4-(diphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylbenzanthracene acid;

4-[4-[4-(diphenylmethylene)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylbenzanthracene acid;

4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-hidroxi the Teal]-α thatα-dimethyl-3-hydroxyanthracene acid;

4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethyl-2-hydroxyanthracene acid;

4-[4-[4-(diphenylmethylene)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethyl-3-hydroxyanthracene acid;

4-[4-[4-(diphenylmethylene)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylbenzanthracene acid;

ethyl 4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylphenylacetate;

n-pentyl 4-[4-[4-(diphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylphenylacetate;

ethyl 4-[4-[4-(diphenylmethylene)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylphenylacetate;

methyl 4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylphenylacetate;

ethyl 4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethyl-(3-hydroxybenzoyl)acetate;

n-propyl 4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethyl-(2-hydroxybenzoyl)acetate;

n-hexyl 4-[4-[4-(diphenylmethylene)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethyl-(3-hydroxybenzoyl)acetate;

ethyl 4-[4-[4-(diphenylmethylene)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylphenylacetate;

4-[4-[4-(diphenylmethoxy)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylbenzanthracene acid;

4-[4-[4-(difen is metoxi)-1-piperidinyl]-1-hydroxybutyl]-α thatα-dimethyl-3-hydroxyanthracene acid;

4-[4-[4-(diphenylmethoxy)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethyl-2-hydroxyanthracene acid;

4-[4-[4-(diphenylmethoxy)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethyl-3-hydroxyanthracene acid;

4-[4-[4-(diphenylmethoxy)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylbenzanthracene acid;

n-pentyl 4-[4-[4-(diphenylmethoxy)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylphenylacetate;

ethyl 4-[4-[4-(diphenylmethoxy)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylphenylacetate;

ethyl 4-[4-[4-(diphenylmethoxy)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethyl-(3-hydroxybenzoyl)acetate;

n-propyl 4-[4-[4-(diphenylmethoxy)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethyl-(2-hydroxybenzoyl)acetate;

n-hexyl 4-[4-[4-(diphenylmethoxy)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethyl-(3-hydroxybenzoyl)acetate and

ethyl 4-[4-[4-(diphenylmethoxy)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylanthracene.

This invention also relates to a method more analogs of compounds of formula IA and/or IB, based on the compounds of the formulae IIA and/or IIB with microorganisms used in accordance with the method of the present invention (or almost equivalent), described in this specification.

Especially predpochtitelnye compounds of the formulas:

and

Optional both diphenylene group of the piperidine can be substituted by alkyl (e.g., stands) in the para-position to the methylene, for example,

or

Compounds obtained by methods in accordance with this invention may be pharmaceutically acceptable salts, in the form of inorganic or organic acidic or basic additive salts of the above compounds. Suitable inorganic acids include, for example, hydrochloric, Hydrobromic, sulfuric and phosphoric acid. Suitable organic acids include carboxylic acids such as acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, fumaric, malic, tartaric, citric, ciclamino, ascorbic, maleic, hydroxymaleimide, dihydroxytoluene, benzoic, phenylacetic, 4-aminobenzoic, Anthranilic, cinnamic, salicylic, 4-aminosalicylic, 2-phenoxybenzoic, 2-acetoxybenzoic and almond acid. Also suitable acids are sulfonic acids, such as methanesulfonate, econsultancy and β-hydroxyethanesulfonic acid. Detoxi the basic salts of the compounds of the above formulas, formed with inorganic and organic bases include, for example, salts of alkali metals such as sodium, potassium and lithium, salts of alkaline earth metals, e.g. calcium and magnesium salts of light metals, such as aluminum, salts of organic amines such as primary, secondary or tertiary amines, for example, cyclohexylamine, ethylamine, pyridine, methylaminoethanol and piperazine. These salts receive by conventional means, for example, processing of piperidine derivatives of the formula IA and/or IB:

where A, B, D, n, R1, R2and R3such as defined above, with a suitable acid or base.

Derivatives of piperidine, obtained by methods in accordance with this invention, can be used as biologically active compounds in pharmaceutical compositions. These compounds are useful as antihistamines, antiallergic agents and bronchodilators. They can be introduced in pure form or with suitable pharmaceutical carriers, and can be in solid or liquid form, for example in the form of tablets, capsules, powders, solutions, suspensions or emulsions.

Compounds obtained by methods in accordance with this invention, can be administered orally, parenterally, for example after inserting the tenderly, intravenously, intramuscularly, intraperitoneally, by using intranasal instillator or by application to mucous membranes, such as the mucous membranes of the nose, throat and bronchial tubes. Application to mucous membranes can be carried out using an aerosol spray containing small particles of compounds in accordance with this invention in the form of drops or as a dry powder.

The number of input connections depends on the patient and the route of administration and can be any effective amount. The number of input connections can vary in a wide range with receiving a single dose of an effective amount of from 0.01 to 20 mg/kg of body weight of the patient per day to achieve the desired effect. For example, desirable antihistamines, antiallergic and bronchodilatators effect can be achieved with unit dosage forms, such as a tablet, containing 1-50 mg of the compounds in accordance with this invention taken 1-4 times a day.

Solid unit dosage forms can be of conventional type. This solid form may be in the form of capsules, such as gelatin capsules containing the compound in accordance with this invention and a carrier, for example, lubricants and inert fillers such as lactose, sucrose, or cornstarch. In another embodiment, the data connect the deposits formed into tablets with the usual bases for tablets such as lactose, sucrose, or cornstarch in combination with binders agents, such as the Arabian gum, corn starch or gelatin, disintegrants, such as corn starch, potato starch or alginic acid, and lubricants such as stearic acid or magnesium stearate.

The compounds obtained in accordance with this invention may also be administered in the form of a dosage form for injection in the form of solutions or suspensions of the compounds in accordance with this invention in a physiologically acceptable diluent with a pharmaceutical carrier. Such carriers include sterile liquids, such as water or oil, with or without addition of a surfactant and other pharmaceutically acceptable adjuvants. Illustrative oils include oils, petrochemicals, animal, vegetable or synthetic origin, such as peanut oil, soybean oil or mineral oil. Basically, water, saline, aqueous dextrose and solutions related sugars and glycols, such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

For use as aerosols compounds in solution or suspension may be packaged in an aerosol container under what t together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The connection data can be entered in the form, not under pressure, such as a nebulizer or atomizer.

The compounds obtained in accordance with this invention, can be used for the treatment of warm-blooded animals, birds, and mammals. Examples of such creatures include humans, cats, dogs, horses, sheep, cows, pigs, lambs, rats, mice and Guinea pigs.

The following examples are illustrative and do not limit the invention.

EXAMPLES

EXAMPLE 1 - the effective Screening of microbial strains for transformation

Microbial culture for reactions inoculant by the above methods, such cultures are described in table 2. The reaction inoculate receive for each microorganism listed in table 2, as follows: 2.5 ml of each inoculum added to 22.5 ml medium in 125 ml DeLong flask and incubated on an orbital mixer for 24 hours at 29°and 225 revolutions per minute (rpm). After a specified time the pH of each culture register and 0.5 ml culture is transferred into individual wells of a 48-hole polypropylene tablet standard format (nominal volume of 5 ml/well), covered with glass wool, gauze, covered with Teflon cloth or other item is chodashim for gas permeable barrier, and initiate the reaction by adding 5 ál of 25 g/l initial solution of the acid metabolite of terfenadine in DMF (final reaction concentration of 250 mg/l). The reaction tablets incubated at 29°C and 225 rpm within the incubation chambers with controlled atmosphere and in camera at 1 cm3/min serves gas containing 95% oxygen and 5% CO2saturated water in the chamber for humidifying spray.

Aliquots of the samples collected from all cultures in reactions between 2 and 168 hours. To 100 μl of the reaction sample is transferred into the appropriate wells of the net advance of the tablet, add 100 ál of acetonitrile and the plate rotate for 1 minute. To each well add 250 ál of ethyl acetate and tablets rotate, then treated with ultrasound for 4 minutes. Tablets centrifuged at 3500 rpm for 5 minutes and 200 μl of the resulting organic phase is transferred into corresponding wells of 96-well plate. Ekstragirovanie with ethyl acetate repeat a second time on the reactivity of the sample and the combined organic phases are dried in a vacuum without heating. The resulting residue re-dissolved in 150 μl of DMF.

Samples analyzed by high-performance liquid chromatography (HPLC) with mass spectrometry with chemical ionization at atmospheric pressure (MS-head, APCI-MS) 5 μm column (Luna C8(2) (500 mm length x 2.0 mm di the meter) manufactured by Phenomenex (table 1).

Table 1
StageSo time (min)Duration (min)Flow (µl/min)GradientThe solvent AndThe solvent In
0-0,100,101000090%10%
10,000,501000090%10%
20,502,501000140%60%
33,002,00100010%100%
45,001,00100010%100%
56,000,501000190%10%
66,500,401000090%10%
76,900,101000090%10%
Solvent: A = water + 0,4% acetic acid

B = acetonitrile + 0.4% UKS is SNA acid

Gradient: 0 = speed gradient; 1 = linear gradient

Qualifiers: UV at 230 nm with MS-MS-head (APCI-MS-MS) (Triple Quadripole Mass Spectrometer, model API 2000 from Perkin-Elmer Sciex)

The output count of the indicators of integration area for each chromatographic peak corresponding to a particular molecular ion in the positive ionization MS-head. Molecular ions for terfenadine (compound 1) and the acid metabolite of terfenadine (compound 2) are shown in table 2. The response factors of the acid metabolite of terfenadine were adopted are identical to those of the terfenadine.

Table 2 shows that the conversion of up to 54% of terfenadine acid metabolite of terfenadine can be achieved with some of the investigated strains.

Table 2

Catalysts for oxidation to terfenadine acid metabolite of terfenadine (KMT)
The biocatalystno culture collectionsA-DType strainGetting biocatalystEthe pH of the cultureGetting KMT after 6 days
Streptomyces rimosusNRRL-2234gram+multistage554%
Stempliylium consortiale UI-4136fungimultistage750%
Gliocladium deliquescensNRRL-1086fungicryoready739%
Cunninghamella bainieriSC-3065fungicryoready727%
Bacillus cereusUI-1477gram+cryoready725%
Cunninghamella bainieriSC-3065fungimultistage718%
Botrytis alliiNRRL-2502fungimultistage518%
Cyathus striatusMR-356fungimultistage511%
Streptomyces rimosusNRRL-2234gram+cryoready511%
Rhizopus sp.MR-224fungimultistage510%
Pycniodosphora dispersaMR-346fungimultistage710%
Absidia spinosa var. biappendiculataMR-7600fungimultistage79%
Rhizopus oryzae MR-ROfungicryoready68%
Cunninghamella echinulataNRRL-1386fungimultistage78%
Cunninghamella echinulataNRRL-3655fungimultistage58%
Gliocladium deliquescensNRRL-1086fungimultistage77%
Pseudomonas spDG-9816gram-multistage76%
Helicostylum piriformeQM-6945fungicryoready75%
Aspergillus flavipesATCC-1030fungimultistage74%
Mucor circmelloides f griseo-cyanusIFO-4563fungimultistage74%
Gelasinospora autosteriaMR-GAfungimultistage63%
Bacillus fusiformisATCC-7055gram+83%
Streptomyces griseusmutant of ATCC-13273gram+multistage53%
Rhootorula rubra ATCC-36994yeastcryoready73%
Cunninghamella echinulata(+)fungicryoready63%
Cunninghamella echinulatafungicryoready73%
Mucor mucedoATCC-7941fungimultistage73%
Pennicillium chrysogenumUI-251fungicryoready73%
Candida parasilosis var quercusATCC-56466yeastmultistage73%
Streptomyces gliseus10137-ATCCgram+cryoready72%
Bacillus cereus14591-NRRL-Bgram+cryoready72%
Streptomyces cavourensis27732-ATCCgram+cryoready72%
Mucor recurvatus36-MRfungicryoready72%
Pennicillium notatum36740-ATCCfungicryoready72%
Aspergilus carbonarium (Bainier) Thorn 6277-ATCCfungicryoready52%
Candida lipolytica8661-UIyeastcryoready42%
AscoidiaMR-Ascfungimultistage72%
Lentinus lepidiusMR-LLfungimultistage72%
Pseudomonas putida (Whiled)9866-NCIMBbacteriacryoready62%
Trichophyton gallinae1210-MRfungicryoready71%
Streptomyces griseus13968-ATCCgram+cryoready71%
Lophotrichus martinii177-MRfungicryoready71%
Penicilliun notatum18233-ATCCfungicryoready71%
Aspergillus ochraceous18500-ATCCfungicryoready61%
Streptomyces catenulae23893-ATCCgram+cryoready71%
Bacillus subtilis 2485-UIgram+cryoready71%
Aspergillus alliaceus315-UIfungicryoready71%
Mycobacterium sp.3683-NRRLfungicryoready71%
Spicaria violacea3702-MRfungicryoready71%
Mycobacterium bisrymcum463-AMfungicryoready71%
Aspergillus fumigatus51-MRfungicryoready71%
Candida lipolytica746-IFOyeastcryoready41%
Polyporus anceps784-F-Sfungicryoready71%
Candida guilliermondii yeast9058-UIyeastcryoready61%
Cunninghamella elegans9245-ATCCfungicryoready71%
Pseudomonas sp (naphthalene wild type)9816-DGgram-cryoready71%
Aspergilus terricola MR-Atfungicryoready71%
Hansends cadaver yeastMR-Hansyeastcryoready71%
Pseudomonas putida (Trevisan), kind of toluene33015-ATCCbacteriacryoready51%
Fusidium coccineum14700-ATCCfungicryoready71%
Enterococcus faecium51558-ATCCbacteriacryoready41%
Streptomyces griseus mutant13273-ASFZbacteriacryoready71%
Streptomyces griseus mutant13273-#11bacteriacryoready61%

AndATS = American collection of type cultures, 10801 University Boulevard, Manassas, VA 20110-2209

InDSM = Deutsche Samlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures), Grisebachstrasse, 8, D-34 Goettingen, Braunschweig, Germany

CUI SC, MR, DG and QM = culture Collection of the University of Iowa, Iowa City IA, 52240

DNRRL = agricultural research Service, USDA, 1815 N. University Ave. Peoria IL, 60604

EIndicate "multistage" and "cryoready" refers to a certain way is, applied in a specific example for obtaining microbial inoculum for the reaction. A detailed description of each method is given in the section "description of the invention" of this specification.

Example 2

25 ml of the medium based on soy flour 125 ml DeLong flask inoculant Streptomyces rimosus (NRRL-2234), obtained from solid culture on the sloped agar, as described in example 1. After incubation at 29°C and 225 rpm for 24 h in 500 μl of solution culture (pH 5.0) is transferred into wells of a 48-hole deep tablet and add to the culture of 125 mg of terfenadine, dissolved in 5 μl of DMF. After further cultivation in the incubation chamber at 29°within 7 days of the obtained microbial broth is extracted with acetonitrile and ethyl acetate. The organic phase is dried over sodium sulfate and then the solvent is removed. The residue re-dissolved in DMF and analyzed by HPLC-MS (HPLC-MS). Integration shows that 76% of the selected material is KMT.

Example 3

As described above, 2.5 ml of the frozen culture of Gliocladium deliquescens cultivated in 25 ml of culture medium at pH 7 for 24 hours. 500 μl of liquid culture is transferred into wells of a 48-hole deep tablet and add to the culture of 125 mg of terfenadine, dissolved in 5 μl of DMF), and incubated at 29°C for 1 week in the incubation chamber. The selection of the product and the analysis shows the Ute, that output is 39% KMT.

Example 4

As described in example 2, 125 μl of terfenadine, dissolved in 50 ml of DMF, are added to 500 μl of solution culture Stemphylium Consortiale (4136-UI) in the reactor with advance tablet. The selection of the product and the analysis show that the output is 50% of the KMT.

Example 5

A two-week culture of Streptomyces rimosus (ATSS-14673) in solid agar inoculant in 25 ml of soy medium in 125 ml DeLong flask for 72 hours at 29°C and 225 rpm 2.5 ml of the liquid culture is transferred into 22.5 ml of medium on the basis of soy flour at pH 5 and cultured at 29°C, 225 rpm for 24 hours. Culture add 12.5 mg of terfenadine, dissolved in 250 μl DMF) and incubated for 1 week. The selection of the product and the analysis show that output in accordance with the procedure of example 2 is 72% KMT.

Although this invention is described for illustration, it is understood that such detailed description is given only for this purpose and specialists in this field of technology can be made variations that fall within the scope and essence of the present invention, which is defined by the claims.

1. The method of obtaining 4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-hydroxybutyl]-α,α-dimethylphenylacetic acid, where the method includes: incubation of 1-(p-tert-butylphenyl)-4-[4'-(α-hydroxydiphenyl-m is Teal)-1'-piperidinyl]butanol in the presence of the microorganism under conditions effective to obtain the target compound, where the microorganism is chosen from the group comprising Stemphylium consortiale, Gliocladium deliquescens, Bacillus cereus, Bacillus subtilis, Bacillus fusiformis, Botrytis allii, Cyathus striatus, Rhizopus oryzae, Pycniodosphora dispersa, Pseudomonas putida, Helicostylum piriforme, Mucor circmelloides f. griseo-cyanus, Mucor recurvatus, Mucor mucedo, Gelasinospora autosteria, Rhodotorula rubra, Mycobacterium bisrymcum, Candida guilliermondii yeast, Candida lipolytica, Candida parasilosis var. quercus, Penicillium notatum and Penicillum chrysogenum.

2. The method according to claim 1, wherein the microorganism is a Stemphylium consortiale.

3. The method according to claim 1, wherein the microorganism is a Gliocladium deliquescens.

4. The method according to claim 1, wherein the microorganism is chosen from the group of Bacillus cereus, Bacillus subtilis. Bacillus fusiformis.

5. The method according to claim 1, wherein the microorganism is a Botrytis allii.

6. The method according to claim 1, wherein the microorganism is a Cyathus striatus.

7. The method according to claim 6, in which the microorganism is a Rhizopus oryzae.

8. The method according to claim 1, wherein the microorganism is a Pycniodosphora dispersa.

9. The method according to claim 1, wherein the microorganism is a Pseudomonas putida.

10. The method according to claim 1, wherein the microorganism is a Helicostylum piriforme.

11. The method according to claim 1, wherein the microorganism is chosen from the group including Mucor circinelloides f. griseo-cyanus, Mucor recurvatus and Mucor mucedo.

12. The method according to claim 1, wherein the microorganism is a Gelasinospora autosteria.

13. The method according to claim 1, wherein the microorganism represents the t a Rhodotorula rubra.

14. The method according to claim 1, wherein the microorganism is a Mycobacterium bisrymcum.

15. The method according to claim 1, wherein the microorganism is chosen from the group including Candida guilliermondii yeast, Candida lipolytica, Candida parasilosis var. quercus.

16. The method according to claim 1, wherein the microorganism is chosen from the group comprising Penicillium notatum and Penicillum chrysogenum.

17. The method according to claim 1, in which the indicated incubation is carried out at a temperature of from 20 to 80°C.

18. The method according to claim 1, in which the indicated incubation is carried out at a pH from 4 to 9.

19. The method according to claim 1, in which the indicated incubation carried out for from 2 to 240 hours

20. The method according to claim 1, wherein before the specified incubation of the microorganism is subjected to cryoconservation or multi-stage induction in liquid culture.



 

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