GB2247016A - Antiparasitic agents - Google Patents

Abstract

Antiparasitic agents of formulae III: <IMAGE> where X is 0 or a direct link, the dotted line is an optional bond and R<1> and R<2> may be H or specified organic substituents, are made by fermentation of Penicillium roqueforti- B2b followed if necessary by synthetic steps.

Description

ANTIPARASITIC AGENTS This invention relates to new antiparasitic agents and in particular to a family of novel compounds related to the marcfortines and to a process for their preparation, to ccmpositions containing them, and to their use in veterinary or human medicine.

Marcfortines A, B and C are natural products produced under certain conditions by the fungal organism Penicillium roqueforti B26. Details of the structural and spectro:cepic characteristics of the marcfortines are described by Polonsky et. al. J.C.S., Chem. Comm., 601-602 (1980), and Polonsky et. al. Tetrahedron Letters 22, 1977-1980, (1981). Subsequently it has been discovered that marcfortines A, B and C possess potent activity against a range of endo- and ecto-parasites affecting man, animal and plants as described in US-Pat 4,866,060.

We have now discovered a family of novel compounds produced by fermentation under controlled conditions of the organism, Penicillium roaueforti - B26 wfiich is a known microorganism available from the Unite de Biologie et Biochemie, Parasitaires et Fongiques, Inserm, Villeneuve d'Ascq, France. These novel compounds possess potent activity against a broad range of endoand ecto-parasites affecting animals, man and plants.

Thus in one aspect the present invention provides compounds of the fouttiffa:-

and their non-toxic salts in which (i) X is 0 and R is or (ii) X is a direct link and R is H The compounds (i) and (ii) are identified, respectively, by the Pfizer references UK-111866 and UK-108433.

The compounds of formula (I) are distinguished from previously known marcfortines in that the pipecolic acid-derived piperidine ring is replaced by the proline-derived pyrrolidine ring. The stereochemistry of the compounds (I) has not been determined absolutely but is inferred by comparison with the related compcans described in the literature.However this invention relates to those compounds produced by the fermentation as subsequently described and as defined by their specific spectroscopic and chromatographic properties and is not intended to be limited to compounds having the particular stereochemical structure shown in formula (IA) below although this is believed to

It is disclosed in EP-A-0301742 and EP-A-754615 that certain synthetic derivatives of the antiparasitic natural product, paraherquamide, a closely related structure to marcfortine, also possess antiparasitic activities. For example, the compound obtained from paraherquamide by catalytic hydrogenation under the conditions described by Yamazaki et. al., Tetrahedron Letters, 22, 135-136 (1981), i.e. 24,25-dihydroparaherquamide, is an active antiparasitic agent.We have also found that analogous 24,25-dihydro derivatives correspondingly obtained from the compeerds of formula (I) have significant antiparasitic activities. Thus in a further aspect the invention provides compounds of the formula (II):

and their non-toxic salts wherein (i) X is 0 and R is CH3 or (ii) X is a direct link and R is H In a yet further aspect of this invention we have found that the group R in the compounds of formulae (I) and (II) can be replaced by certain alkyl, acyl or sulphonyl substituents to obtain further antiparasitic derivatives.This can be achieved by treating a solution of a compound of the formula (I) or (II) where R is H with a strong base such as lithium diisopropylamide, n-butyl lithium, sodium hydride, potassium hydride, potassium hydroxide or potassium tert-butoxide in a suitable organic solvent such as tetrahydrofuran, toluene, dimethylsulphoxide, dimethylformamide or ether, followed by the addition of an appropriate alkylating or acylating reagent.

Depending on the precise conditions used and the choice of alkylating or acylating reagent additional reaction may also take place at position N-l. If this is not desired, this position should be protected conventionally, for example by prior reaction with diazomethane to give the 2-0-methylimidate using conditions described in the EP-A-0354615. At the end of the reaction sequence, the protecting group can be removed conventionally, e.g.

by treatment with a mineral acid such as hydrochloric acid in an organic solvent such as tetrahydrofuran. The alkylating agent used may be an alkyl iodide, alkyl bromide or an alkyl sulphonate.

The acylating agent may be an acyl chloride, acyl bromide, acyl anhydride, a chloroformate ester or an isocyanate.

The sulphonating agent used may be an aryl sulphonyl halide.

Thus in this aspect the invention provides oampounds of the general formula (III), which also possess significant antiparasitic activity, of the formula: -

and their non-toxic salts where R1 is (i) H (ii) C28 alkyl optionally substituted by halo, hydroxy, 1-C4 alkoxy or phenyl, (iii) alkenylalkyl having 3 to 8 carbon atoms in total, (iv) alkynylalkyl having 3 to 8 carbon atoms in total (v) benzyl (vi) a group of the formula - COR3 or CONHR3 where R3 is either (a) C1-C8 alkyl optionally substituted by halo, C1-C4 alkoxy or phenyl, or (b) phenyl or (vii) - SO2 phenyl;; R2 is (i) C1-C8 alkyl optionally substituted by halo, hydroxy, C1-C4 alkoxy or phenyl (ii) C2-C8 alkonyl (iii) C1-C8 alkynyl, (iv) benzyl, (v) a group of the formula or -CONHR4 where R4 is either (a) C1-C8 alkyl optionally substituted by halo, C1-C4 alkoxy or phenyl or (b) phenyl or (vi) - sO2 - phenyl; and X is 0 or a direct link and the dotted line represents an optional bond; said phenyl groups, and the phenyl portion of said benzyl groups, all being optionally substituted by up to three substituents each independently selected from C1-C4 alkyl, C1-C4 alkoxy, hydroxy and halo.

When appropriate, the alkyl, alkoxy, alkenyl and alkynyl groups can be straight or branched chain. Halo means F, C1, Br or I.

In the compounds (III), the preferred alkyl, alkoxy, alkenyl or alkynyl groups have up to 4 carbon atoms.

The non-toxic salts of the compounds of formula (I), (II) and (III) include pharmaceutically-acceptable acid addition salts such as the hydrochloride, hydrooromide, sulphate or bisulphate, phosphate. For pharmaceutically acceptable salts see, for example, the Journal of Pharmaceutical Sciences, Vo. 66, No. 1, January 1977, pages 1-19. These salts can be prepared conventionally, e.g. by mixing a solution of the free base and the acid in a suitable solvent, e.g. ethanol, and recovering the acid addition salt either as a precipitate, or by evaporation of the solution.

The ccgrpounds (II) and (III) are also believed to have the same stereochemistry as compound (I):

The compounds of the formula (I) can be prepared by the aerobic fermentation of Penicillium rppueforti - B26 in stirred aqueous or static solid nutrient media. Such conditions are similar to those conventionally employed to produce natural products by fermentation.

Cultivation may take place in an aqueous nutrient medium containing suitable sources of carbon, nitrogen and trace elements for a period of several days under aerobic conditions, typically at a temperature in the range of from 24 to 36"C. As with the majority of antibiotic fermentations, the amount of the compound produced will vary with changing fermentation conditions especially with regard to nutrient components, aeration conditions and pH.The progress of the fermentation can be monitored by an appropriate analytical technique such as high pressure liquid chromatography (hplc) coupled with a suitable detector such as a U.V. spectrophotometer. Significant amounts of the desired compounds are generally found in both the mycelial and aqueous phases of the whole broth although the proportion in each phase will depend on the fermentation conditions. Isolation of the products is facilitated by the greater solubility of the compounds (I) in a range of organic solvents than in water provided that the pH is adjusted to be greater than 7.Thus in one recovery procedure the wfiole fermentation broth is extracted, preferably with a water-immiscible solvent such as methylene chloride, chloroform, hexane, petroleum spirit, ethyl acetate, butanol or methyl isobutyl ketone. Maximum recovery is achieved by repeating the extraction several times. The solvent extract is concentrated to dryness and the crude product is further purified as necessary by chromatography.

In an alternative recovery procedure the whole fermentation broth may be filtered and the my mycelium and filtrate extracted separately. In this case the mycelium may be extracted by a range of water-miscible solvents such as methanol, ethanol, a acetone and the like in addition to those water-immiscible solvents listed above. After filtration the solvent extract is concentrated and either freeze-dried or further extracted with a water-immiscible solvent to give a crude material suitable for further purification by chromatography.The filtrate derived from the wfiole broth may be recovered in an exactly similar manner to the wfiole broth as described above or, alternatively, it may be passed through a column containing a suitable polystyrene resin such as "Diaion HP20" (Trade Mark - available from Mitsubishi Chemical Company) which retains the desired products but allows many impurities to pass through. After washing the column with water, the column is eluted with an organic solvent such as ethanol, methanol or acetone.The solvent is then evaporated to give a crude mixture containing ccsnpounds UK-111866 and UK-108433 which is further enriched using column chromatography, thin layer chromatography or similar technique using for example silica gel as the chranatographic medium and eluting with a suitable solvent or combination of solvents as is known to those skilled in the art.

Final purification of the individual compounds may be achieved by repeated column chromatography, hplc or preparative thin layer chromatography.

Alternatively, the cultivation of Penicillium ropueforti B-26 may take place on a static nutrient medium under aerobic conditions at a temperature typically in the range of from 24 to 36"C for several days. The medium together with the mycelial growth is then extracted with an organic solvent such as methanol or ethyl acetate, filtered and concentrated to give a crude product which is further purified by the techniques described above.

Thus the present invention also providers a process for producing the compounds UK-111866 and UK-108433 as previously defined, which comprises cultivating the micro-organism Penicillium ropueforti - B-26, or a mutant, genetically transformed or reccobinant form thereof having the ability to produce the compounds UK-111866 and UK-108433 in stirred aqueous or static solid culture media containing an assiinilable source of carbon, nitrogen and inorganic salts, under aerobic fermentation conditions until a recoverable amount of said compounds are obtained.

The term mutant includes any mutant strain which arises spontaneously or by the application of known techniques, such as exposure to ionising radiation, ultraviolet light, and/or chemical mutagens such as N-methyl-N-nitroso-urethane, nitrosoguanidine and ethane methane sulphate, etc. Genetically transformed and recoobinant forms include mutants and genetic variants produced by genetic engineering techniques, including for example recombination, trans formation, transduction, and protoplast fusion, etc.

As previously mentioned the compounds of the formulae (I), (11) and (III) are highly active antiparasitic agents having particular utility as anthelmintics, ectoparasiticides, insecticides and acaricides.

Thus the compounds (I), (II) and (III) are useful in treating a variety of conditions caused by endoparasites including, in particular, helminthiasis which is most frequently caused by a group of parasitic worms described as nematodes and which can cause severe economic losses in swine, sheep, horses and cattle as well as affecting domestic animals and poultry.The compounds are also effective against other nematodes which affect various species of animals including, for example, Dirofilaria in dogs and various parasites which can infect humans including gastro-intestinal parasites such as Ancylostoma, Necator, Ascaris, Stromyloides, Trichinel la, Capillaria, Trichuris, Enterobius and parasites which are found in the blood or other tissues and organs such as filiarial worms and the extra intestinal stages of Stronqvloides and Trichinella.

The compounds are also of value in treating ectoparasite infections including in particular arthropod ectopasites of animals and birds such as ticks, mites, lice, fleas, blowfly, biting insects and migrating dipterous larvae which can affect cattle and horses.

The compounds are also insecticides active against household pests such as the cockroach, clothes moth, carpet beetle and the housefly as well as being useful against insect pests of stored grain and of agricultural plants such as spider mites, aphids, caterpillars and against migratory orthopterans such as locusts.

The compounds may be administered as a formulation appropriate to the specific use envisaged and to the particular species of host animal being treated and the parasite or insect involved. For use as an anthelmintic the ccuçrunds may be administered orally in the form of a capsule, bolus, tablet or liquid drench, or alternatively, they may be administered as a pour-on or by injection, either subcutaneously or intramuscularly.

Capsules, boluses or tablets may be prepared by mixing the active ingredient with a suitable finely divided diluent or carrier, additionally containing a disintegrating agent and/or binder such as starch, lactose, talc, or magnesium stearate. A drench formulation may be prepared by dispersing the active ingredient in an aqueous solution together with dispersing or wetting agents and injectable formulations may be prepared in the form of a sterile solution or emulsion.

Pour-on and injection formulations are prepared in a conventional manner in accordance with standard veterinary practice. These formulations will vary with regard to the weight of active cooped depending on the species of host animal to be treated, the severity and type of infection and the body weight of the host. Generally for oral adminirtration a dose of fram about 1 to 100 mg per kg of animal body weight given as a single dose or in divided doses for a period of fram 1 to 5 days will be satisfactory but of course there can be instances where higher or lower dosage ranges are indicated and such are within the scope of this invention.

As an alternative the compounds may be administered with the animal feedstuff and for this purpose a concentrated feed additive or premix may be prepared for mixing with the normal animal feed.

For use as insecticides and for treating agricultural pests the compounds can be applied as sprays, dusts, pour-on formulations, emulsions and the like in accordance with standard agricultural practice.

For human use the compounds are administered as a pharmaceutically-acceptable formulation in accordance with standard medical practice.

The invention is illustrated by the following Example which describes the preparation, isolation and identification of the campounds UK-111866 and UK-108433.

Bacto-Cesitone was supplied by the Difco Corporation of Detroit, Michigan.

Ultraviolet spectra were recorded using a Hewlett Fackard HP109OA diode array spectrophotometer.

Mass spectroscopy was performed using a VG Trio 3 mass spectrxaeter. Samples were introduced using a thermospray interface in a matrix consisting of water, methanol and ammonium acetate. Electron impact mass spectra were obtained using a Krato Concept 15 mass spectrometer.

Nuclear magnetic resonance spectral data were obtained using a Nicolet GE 300 spectraneter, or a Bruker AC 300 spectrcmeter.

EXAMPLE The culture Penicillin rocueforti B-26 was maintained on agar slants prepared fran a medium comprising glucose (log), starch (20g), "Bacto-Casitone" (Trade Mark) (5g), yeast extract (5g), potassium monohydrogenphosphate (0.5g), calcium carbonate (41g) and agar (20g) made up to 1 litre with tap water and adjusted to pH 7 before autoclaving.An inoculum was prepared by washing a slant culture with lOml of sterile water into a 250ml Erlenmeyer flask containing a medium comprising glucose (0.25g), corn starch (0.5g), "Bacto-casitone" (Trade Mark) (0.125g), yeast extract (0.125g), wheat embryo (0.125g) and calcium carbonate (O.lg) made up to 50ml tap water and adjusted to PE 6.5. The inoculum was incubated at 28 C on a rotary shaker with a 2.5cm throw at 170 r.p.m. for 48 hours. The resultant vegetative growth was used to inoculate a 3 litre Fernbach Erlenmeyer flask containing 1 litre of the above seed medium and incubated under the same condition for a further two days.The total contents were then transferred to a 150 litre mechanically agitated vessel containing 100 litres of a production medium comprising ammonium succinate (3kg), mannitol (5kg), KH2P04 (lOOg) and MgSO4 (30g). The fermentation was maintained at 28 C with aeration at 40 litres per minute and agitation at 250 r.p.m. After 6 days the broth was filtered using a plate and frame press. The filtrate (140 litres) was pumped into ethyl acetate (100 litres) and briefly stirred. The aqueous layer was discarded and the organic extract was washed with saturated brine (50 litre). The mycelial solids were slurried with ethyl a acetate (50 litres). The mycelial solids were removed by filtration and the organic extract washed with saturated brine (50 litres). The filtrate and mycelial extracts were combined and evaporated to dryness.The oily residue was added slowly to petroleum ether (b.p. 40-60"C, 3 litres) with stirring and the precipitate was recovered by filtration. The solid precipitate was then chrcmatographed in a column of silica gel (200g, Mercy "Kieselgel 60") (Trade Mark) eluting with ethyl acetate containing an increasing proportion of methanol. Compounds B26 factors 1, 2 and 3 eluted from the column when the solvent composition was approximately 1:1. Fractions containing the derived compounds were combined and evaporated to dryness. The individual compounds were separated by preparative h.p.l.c. using a C-18 bonded phase column and eluting with water-methanol Inixtues as known to those skilled in the art. The composition of the fraction collected was monitored by analytical h.p.l.c. using a Beckman Ultrasphere-ODS (Trade Mark) (5 m) h.p.l.c. column (4.6 x 250mm) eluting with water:methanol (65.35) at a flow rate of Imi per minute and monitoring by ultraviolet absorbance at 226mm.Using these conditions the individual components eluted at the following times: UK-111866 12.6 minutes UK-108433 14.0 The novel compounds were distinguished and characterised by their spectroscopic properties as follows:-

UK-111866 UK-108433 463 (n+) 433 (+) Mass Spectrum Theoretical 463 Theoretical 433 U.V. Spectrum 226 246 (n.m.) 260 (sh) 275 (sh) 290 (sh) N.M.R. Spectrum 7.55 (1H, bs) 7.88 (1H, bs) (CD)Cl3, solution)~ 6.85 (1H, d) 6.92 (1H, d) (in part) 6.74 (1H, d) 6.5 (1H, bs) 6.72 (1H, d) 6.46 (1H, d) 6.35 (1H, d) 6.28 (1H, d) 4.92 (1H, d) 5.72 (1H, d) 3.68 (1H, d) 3.7 (1H, d) 3.1 (3H, s) 3.12 (1H, m) 2.75 (1H, d) 2.75 (1H, d) 2.22 (1H, dd) 1.52 (1H, s) 1.49 (3H, s) 1.49 (1H, s) 1.48 (3H, s) 1.14 (1H, s) 1.18 (3H, s) 0.9 (1H, s) 0.9 (3H, s) It will be understood fran the foregoing that the inventions described in this application include, for example, the following: - (1) The compounds of the formulae (I), (II) and (III) and their non-toxic salts, and processes for their preparation; (2) A process for preparing the compounds of the formula (I) which comprises fermenting in a medium containing assimilable sources of carbon, nitrogen and inorganic salts the strain Penicillum roqueforti B-26; and (3) A composition useful for the prevention or treatment of ecto or endo-parasite infections which comprises a carrier and a ccrmpound nd of the formula (I), (II) or (III) or a non-toxic salt thereof.

Claims (1)

1. CIAIMS

   1. A compound of general formula (III).

   and its non-toxic salts where R1 is (i) H (ii) C1-C8 alkyl optionally substituted by halo, hydroxy, C1-C4 alkoxy or phenyl, (iii) alkenylalkyl having 3 to 8 carbon atoms in total, (iv) alkynylalkyl having 3 to 8 carbon atoms in total (v) benzyl (vi) a group of the formula - COR3 or CONHR3 where R3 is either (a) C1-C8 alkyl optionally substituted by halo, C14 alkoxy or phenyl, or (b) phenyl or (vii) - sO2 phenyl; R2 is (i) C18 alkyl optionally substituted by halo, hydroxy, C1-C4 alkoxy or phenyl (ii) C2-C8 alkonyl (iii) C1-C8 alkynyl, (iv) benzyl, (v) a group of the formula or -CONHR4 where R4 is either (a) C1-C8 alkyl optionally substituted by halo, C1-C4 alkoxy or phenyl or (b) phenyl or (vi) - sO2 - phenyl or (vii) H; and X is O or a direct link and the dotted line represents an optional bond; said phenyl groups, and the phenyl portion of said benzyl groups, all being optionally substituted by up to three substituents each independently selected from C1-C4 alkyl, C1-C4 alkoxy, hydroxy and halo.