Cytokine production inhibitors

Details Cytokine production inhibitors Cytokine production inhibitors

2001-06-07

2004-08-31

Lankford, Jr., Leon B. (Department: 1651)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

C435S171000, C435S134000, C435S071100, C424S274100

Reexamination Certificate

active

06784203

ABSTRACT:

The present invention relates to 5,6-dihydro-&agr;-pyrones useful as cytokine production inhibitors, to the preparation of these compounds and to pharmaceutical and veterinary compositions containing them.
We have now discovered that fermentation of a strain of the fungus Phomopsis sp. in a nutrient medium produces two 5,6-dihydro-&agr;-pyrones esterified in the 5-position with an unsaturated C
14
fatty acid and also the free C
14
fatty acid. We have also discovered that fermentation of a strain of the fungus Paecilomyces sp. in a nutrient medium produces a useful phomalactone.
The present invention therefore provides a 5,6-dihydro-&agr;-pyrone of formula (I):
wherein R is CO
2
H or CH
3
and each of R
1
and R
2
is H; or R is CO
2
H, one of R
1
and R
2
is H and the other is OH; or, when R is CO
2
H, a pharmaceutically or veterinarily acceptable salt thereof.
Preferred compounds of the invention are:
3-((5S,6S)-5,6-dihydro-5-((6S)-4,6-dimethyldodeca-2E,4E-dienoyl)-2H-pyran-2-on-6-yl)-prop-2E-enoic acid; and
3-((5S,6S)-5,6-dihydro-5-((6S)-4,6-dimethyldodeca-2E,4E-dienoyl)-2H-pyran-2-on-6-yl)-prop-2E-ene.
The present invention provides a process for the preparation of a 5,6-dihydro-&agr;-pyrone of formula (I) or a pharmaceutically or veterinarily acceptable salt thereof, which process comprises:
(i) fermenting, in a source of carbon, nitrogen and inorganic salts, fungal strain Phomopsis sp. 22502 (CBS 313.96) or a mutant thereof which produces a said 5,6-dihydro-&agr;-pyrone;
(ii) isolating a said 5,6-dihydro-&agr;-pyrone from the fermentation broth; and
(iii) if desired, when the isolated 5,6-dihydro-&agr;-pyrone is the compound of formula (I) wherein R is CO
2
H, converting the said 5,6-dihydro-&agr;-pyrone into a pharmaceutically or veterinarily acceptable salt thereof.
The compounds of formula (I) have been isolated from a microorganism which we have designated X22502 and which has been identified as a strain of the genus Phomopsis (Saccardo) Bubák on the basis of the following morphological data with reference to the description given by SUTTON, B. C., 1980 (The Coelomycetes. Farnham Royal: Commonwealth Agricultural Bureaux):
The fungal strain Phomopsis sp. (X22502) (CBS 313.96) is a coelomycete isolated from tropical freshwater foam which produces a dense, dark grey-olivaceous (Flora of British Fungi Colour Identification Chart, 1969, Edinburgh: HMSO) mycelium with a white lobate margin at 24° C. on 2% malt extract agar with glucose and peptone (MEA: composition per liter of distilled water: Difco malt extract, 20 g; Bacto-peptone, 1 g; agar, 20 g). After 7 days the mycelium attains a diameter of 2.5-3.5 cm.
Conidiomatal development is stimulated by exposure to near-UV light. Conidiomata are solitary, carbonaceous, unilocular, ostiolate and measure 1.5-2.0 mm wide and 1.25-2.0 mm high. Conidiogenous cells are borne on branched conidiophores which line the conidiogenous cavity. These cells are hyaline, obclavate to cylindrical, integrated, phialidic and measure 16-20 &mgr;m×1.5-2.0 &mgr;m. Conidia are hyaline, aseptate and generally of three types: A-conidia (5.5-7.5 &mgr;m×1.5-3.0 &mgr;m) are ellipsoid to fusiform, usually with acute apices and a guttule at each end; B-conidia (20-32 &mgr;m×<1 &mgr;m) are hamate and filiform; C-conidia (9.5-11.5 &mgr;m×1.5-3.0 &mgr;m) are obclavate with acute apices and usually at least three guttules. All three conidial types can be found within a single conidioma. The fluvial origin and observed microscopical characters did not allow further classification to species.
The 5,6-dihydro-&agr;-pyrones of formula (I) are associated primarily with the mycelium on termination of the fermentation. They may be recovered and purified from the medium. The separation and purification of the compounds from the fermentation broth and their recovery can be achieved using solvent extraction followed by chromatographic fractionation. The 5,6-dihydro-&agr;-pyrone of formula (I) in which R is CO
2
H may be converted into pharmaceutically or veterinarily acceptable salts by conventional methods. Suitable salts include salts with alkali metals such as sodium or potassium and ammonium salts.
The 5,6-dihydro-&agr;-pyrone of formula (I) wherein R is CH
3
can, alternatively, be produced by the esterification of the phomalactone of formula (II):
with (6S)-4,6-dimethyldodeca-2E,4E-dienoic acid which is a fatty acid of formula (IIIa):
Preferably the reaction is carried out in the presence of a dehydrating agent such as DCC (dicyclohexylcarbodiimide) or EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and dimethylaminopyridine. The reaction is typically carried out in an inert solvent such as dichloromethane or tetrahydrofuran.
The reagents are generally mixed with stirring for example at a low temperature such as −78° C. The reaction is then allowed to warm to room temperature (20-25° C.) and stirred until complete. The reaction may be monitored chromatographically by thin layer chromatography or reversed phase high performance liquid chromatography and is typically complete within sixteen hours. Other dehydrating agents such as an alkylchloroformate and triethylamine, phenyldichlorophosphate, 2-chloro-1,3,5-trinitrobenzene and pyridine, and chlorosulphonyl isocyanate can also be used under similar conditions.
Alternatively an excess of the phomalactone of formula (II) can be reacted with the acid of formula (IIIa). The water formed can be removed by azeotropic distillation. Suitable solvents include toluene and 1,4-dioxane. The reaction is typically catalysed by acids such as sulphuric acid and ptoluenesulphonic acid.
It is advantageous to produce the 5,6-dihydro-&agr;-pyrone of formula (I) wherein R is CH
3
by this route as both the phomalactone and the fatty acid can be produced by fermentation in larger quantities than the 5,6-dihydro-&agr;-pyrone of formula (I) wherein R is CH
3
.
The fatty acid of formula (IIIa) is one of a group of fatty acids of the following formula (III):
wherein one of R
1
and R
2
is H and the other is H or OH. These fatty acids can be obtained by fermentation of the fungal strain Phomopsis sp 22502 (CBS 313.96) or a mutant thereof. In accordance with the present invention, therefore, the fatty acid of formula (III) can be produced by a process which comprises:
(i) fermenting, in a source of carbon, nitrogen and inorganic salts, strain Phomopsis sp. 22502 (CBS 313.96) or a mutant thereof which produces the said fatty acid; and
(ii) isolating the said fatty acid of formula (III) from the fermentation broth.
The fatty acid of formula (III) may, of course, be isolated from the same fermentation broth as the 5,6-dihydro-&agr;-pyrones of formula (I). The fatty acid, like the 5,6-dihydro-&agr;-pyrones, is primarily associated with the mycelium on termination of fermentation.
Some of the fatty acids of formula (III) are novel. The present invention therefore further provides a fatty acid of formula (IIIb):
wherein one of R
1
and R
2
is H and the other is OH
The phomalactone of formula (II) can be synthesised by methods known in the prior art, for example those disclosed in Krivobok, S. et al, Pharmazie, (1994): 49, H8, 605-607; Guirand, P. et al, Pharmazie, (1994): 49, H8, 279-281; Krasnoff, S. B. et al, J. Chem. Ecol., (1994): 20, 293-302 and Murayama, T. et al, Agric. Biol. Chem., (1987): 51, 2055-2060.
The present invention does however provide a new process for the preparation of the phomalactone of formula (II), which process comprises:
(i) fermenting, in a source of carbon, nitrogen and inorganic salts, fungal strain Paecilomyces sp. 3527 (CBS 314.96) or a mutant thereof which produces the said phomalactone; and
(ii) isolating the said phomalactone from the fermentation broth.
The phomalactone is found primarily in the culture liquor on termination of the fermentation and may be recovered and purified. The separation and purification of the compound from the fermentation broth and its recovery can be achieved using solvent extraction followed by application of conventional

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