WO1992000300A1

WO1992000300A1 - Antiparasitic agents related to paraherquamide

Info

Publication number: WO1992000300A1
Application number: PCT/EP1991/001170
Authority: WO
Inventors: David Austen Perry
Original assignee: Pfizer Limited; Pfizer Inc.
Priority date: 1990-06-26
Filing date: 1991-06-21
Publication date: 1992-01-09
Also published as: PT98074A; GB9014194D0; IE912199A1

Abstract

Antiparasitic compounds of formula (IV) wherein R is H, -OH, -OR?3, -OCOR4¿ or -OCONHR?4, R1 R2 R3 and R4¿ may be variety of organic substituents and X is O or a direct link, or wherein R is -OH, R1 is O, R2 is H and X is O, are made by fermentation of Penicillium paraherquei CM1 68220 followed if necessary by synthetic steps.

Description

ANTIPARASITIC AGEOTS RELATED TO PARAHERQUOTDE

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

Paraherquamide is a cσrrpound produced under certain conditions by the fungal organism Penicillium paraherquei. Details of the structural and spectroscopic characteristics of paraherquamide are described in Yamazaki et. al. Tetrahedron Letters, 22, 135-136 (1981) . Subsequently it has been discovered that paraherquamide together with some related compounds are produced by the fungal organism Penicillium charlesii and fiirthermore these compounds possess potent activity against a range of endo- and ecto-parasites as described in EP-A-0301742 an EP-A-0322937.

We have now discovered a family of novel compounds produced by fermentation under controlled conditions of the organism Penicillium paraherquei which is a known microorganism deposited at the C.A.B. International Mycological Institute, Ferry Lane, Kew, London, under the deposit number CMI 68220. These novel ∞rφounds possess potent activity against a broad range of endo- and ecto-parasites afflicting animals, man and plants. Thus in one aspect the present invention provides compounds of the formula:-

and their non-toxic salts, in which (i) X is O and is CH,

(ii) X is 0 and R is H, or (ϋi) X is a direct link and R is H.

The compounds (i) , (ii) and (iii) are identified, respectively, by the Pfizer references UK-110123, UK-109692, and UK-105166.

In a further aspect the present invention provides a compound of the formula (II) identified by the Pfizer reference UK-112847.

and its non-toxic salts. The compounds (I) are distinguished from previously known paraherquamides in that they lack a methyl substituent at N-30, extending the numbering system described in Blizzard et. al. , Journal of Organic Chemistry, 54, 2657-2663 (1989) . The stereochemistry of the compounds (I) and (II) has not been determined absolutely but is inferred by comparison with the related compounds described in the literature. However this invention relates to those cxmpounds produced by the fermentation as subsequently described and as defined by their specific spectroscopic and civrαmatographic properties and is not intended to be limited to compounds having the particular stereochemical structures shown in formulae (IA) and (IIA) below although these are believed to be correct:-

(IIΛ).

It is disclosed in EP-A-0301742 and EP-A-354615 that certain synthetic derivatives of the natural product paraherquamide also possess antiparasitic activity. 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-<iihyά oparaherquamide, is an active antiparasitic agent. We have also found that the analogous 24,25-dihydro derivatives correspondingly obtained from the compounds of formula (I) have antiparasitic activities. Thus in a further aspect the invention provides conpounds of the formula (III) :-

and their non-toxic salts, wherein (i) X is O and R is OH,

(ii) X is O and R is H, or (iϋ) X is a direct link and R is H. In a yet further aspect of this invention we have found that the hydrogen atom attached to N-30 in the conpounds of the formulae (I) and (III) can be replaced by certain alkyl, alkanoyl or arylsulphonyl substituents to obtain antiparasitic derivatives of paraherquamide not previously known. This can be achieved by treating a solution of a c sipound of the formula (I) or (III) with a strong base such as lithium diisσpropylamide, 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 or sulphonating 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 may be protected conventionally, for example by prior reaction with diazomethane to give the 2 )-methyliιιιidate 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 tefcrahy<drofuran. In the case of compound UK-110123, an additional competing reaction may occur at the 14-hydroxy group. If this is not desired, the hydroxy group can be protected conventionally, for example by silylation using trimethylchlorosilane or trimethylsilyl triflate in the presence of a base such as triethylamine or with bis(triιrøthylsilyl)ac^tamide in a suitable solvent. Such a protecting group can subsequently be removed conventionally at the end of the reaction sequence by treatment with a dilute mineral acid (including hydrofluoric acid) in a water-miscible solvent such as tetrahydrofuran, or by treatment with a solution of tefcrabutyl ammonium fluoride in 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 can be an optionally substituted benzenesulphonyl chloride or anhydride. Thus in this aspect the invention provides conpounds of the general formula (IV) , which also possess significant antiparasitic and anthelmintic activity, of the formula:-

and their non-toxic salts; where R is H, -OH, -OR³ , -OCOR⁴ or -OQONHR⁴ ;

3 . .

R is (1) C,-C_R alkyl optionally substituted by halo, hydroxy, C.-C alkoxy or phenyl, (ii) alkenylalkyl having 3 to 8 carbon atoms in total, (iii) alkynylalkyl having 3 to 8 carbon atoms in total, or (iv) benzyl;

4 .

R is C, -C„ alkyl optionally substituted by halo, C,-C. alkoxy or phenyl;

R is (i) H, (ii) C„-C₀ alkyl optionally substituted by halo, hydroxy, C,-C alkoxy or phenyl, (iii) alkenylalkyl having 3 to 8 carbon atoms in total, (iv) alkynylalkyl having 3 to 8 carbon atoms in total, (v)

5 5 benzyl (vi) a group of the formula -COR or -CONHR

5 . where R is either (a) C,-C alkyl optionally substituted by halo, C,-C alkoxy or phenyl, or (b) phenyl or (vii) -SO .phenyl;

2 . R is (l) C,-C_ alkyl optionally substituted by halo, i. o hydroxy, C.-C alkoxy or phenyl (ii) C -C alkenyl (iii) C_-C„ alkynyl, (iv) benzyl, (v) a group of the formula -COR⁶ or -CONHR⁶ where R⁶ is either (a) C,-C_ alkyl

1 o optionally substituted by halo, C.-C. alkoxy or phenyl or (b) phenyl, or (vi) -SO .phenyl;

X is O or a direct link with the provisos that (a) when

R is other than H, X is O and also (b) when R is H or

1 2 . OH, at least one of R and R is other than H; 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 fom C,-C. alkyl, C,-C. alkoxy, hydroxy and halo.

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

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

The non-toxic salts of the compounds of the above-mentioned compounds include pharmaceutically-acceptable acid addition salts such as the hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or hydrogen phosphate, acetate, besylate, citrate, fumarate, gluconate, lactate, maleate, mesylate, pamoate, succinate and tartrate salts. For a more comprehensive list of 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 conpounds (III) and (IV) are also believed to have the following stereochemistry : -

and

The compounds of the formula (I) can be prepared by the aerobic fermentation of Penicillium paraherquei CMI 68220 in stirred aqueous or static solid nutrient media. Such conditions are similar to those conventionally employed to produce antibiotics 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 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 cσπponents, aeration conditions and pH. The progress of the fermentation can be monitored by an appropriate analytical technique such as high pressure liquid c±iromatαgraphy (hplc) coupled with a suitable detector such as a U.V. spectroiiotometer. Significant amounts of the desired conpounds 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 conpounds UK-110123, UK-109692, UK-105166 and UK-112847 in a range of organic solvents than in water provided that the pH is adjusted to be greater than 4. Thus in one recovery procedure the whole fermentation broth is extracted, preferably with a water-iiπmiscible 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 chrcmatograjάry.

In an alternative recovery procedure the whole fermentation broth may be filtered and the mycelium and filtrate extracted separately. In this case the mycelium may be extracted by a range of water-miscible solvents such as methanol, ethanol, 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 c±rcmatography. The filtrate derived from the whole broth may be recovered in an exactly similar mariner to the whole 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 Coπpany) 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 the conpounds. The crude mixture containing the conpounds UK-110123, UK-109692, ϋK-105166 and UK-112847 as obtained by any of the above procedures is further enriched using column chrcaxiatography, thin layer chrαnatography or similar technique using for example silica gel as the chromatographic 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 conpounds may be achieved by repeated column chrαmatography, hplc or preparative thin layer chromatograph . Alternatively, the cultivation of Penicillium paraherquei CMI 68220 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 provides a process for producing the conpounds UK-110123, UK-109692, UK-105166 and UK-112847 as previously defined, which comprises cultivating the microorganism Penicillium paraherquei CMI 68220, or a mutant, genetically transformed or recoπbinant form thereof having the ability to produce the compounds OK-110123, OK-109692, UK-105166 and UK-112847 in stirred aqueous or static solid culture media contai-ning an assimilable source of carbon, nitrogen and inorganic salts, under aerobic fermentation conditions until a recoverable amount of said (.xinpound is 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-metJyl-N-nitroso-urethane, nitrosoguanidine and ethane methane sulphate, etc. Genetically transformed and reccmbinant forms include mutants and genetic variants produced by genetic engineering techniques, including for example recombination, transformation, transduction, and protoplast fusion, etc. As previously mentioned the conpounds of the formulae (I) , (II) , (III) and (IV) are highly active antiparasitic agents having particular utility as anthel intics, ectoparasiticides, insecticides and acaricides.

Thus the ccnpounds (I) , (II) , (III) and (TV) are useful in treating a variety of conditions caused by endoparasites including, in particular, heliriinthiasis 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 conpounds 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 Ancylostσma_f Negator, Ascaris, Strongyloides, Trichinella, Capillaria, Tridhuris, Enterobius and parasites which are found in the blood or other tissues and organs such as filiarial worms and the extra intestinal stages of Stronαyloides and Trichinella.

The compounds are also of value in treating ectoparasite infections including in particular arthropod ectoparasites of animals and birds such as ticks, mites, lice, fleas, blowfly, biting insects and migrating dipterous larvae which can affect cattle and horses. The conpounds 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 and animal being treated and the parasite or insect involved. For use as an anthe_l__mintic the conpounds 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 int-THmuscularly. 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 compound 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 administration a dose of from about 1 to 100 irg per kg of animal body weight given as a single dose or in divided doses for a period of from 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 conpounds can be applied as sprays, dusts, pour-on formulations, emulsions and the like in accordance with standard agricultural practice.

For human use the conpounds 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 . conpounds UK-110123, UK-109692, UK-105166 and UK-112847:-

"Ardamine" was supplied by Champlain of Clifton, New Jersey, U.S.A. Baσto-Casitone was supplied by the Difco Corporation of Detroit, Michigan.

Ultraviolet spectra were recorded using a Hewlett Packard HP1090A diode array spectrophotometer.

Mass spectroscopy was performed using a VG Trio 3 mass spectrometer. Samples were introduced using a thermospray interface in a matrix consisting of water, methanol and ammonium acetate. Nuclear magnetic resonance spectral data were obtained using a General Electric GN500 spectrometer, a Nicolet GE 300 spectrcaneter or a Bruker AC300 spectrometer.

EXAMPLE The culture Penicillium paraherquei CMI 68220, supplied by the C.A.B. International Mycological Institute, Ferry Lane, Kew, London, England, was maintained on agar slants prepared from a medium comprising glucose (10 g) , starch (20 g) , "Bacto-Casitone" (Trade Mark) (5 g) , yeast extract (5 g) , calcium carbonate (1 g) and agar (20 g) made up to 1 litre with tap water and adjusted to pH 7.3 before autoclaving. An inoculum was prepared by washing a slant culture with 10 ml of sterile water into a 3 litre Fernbach Erlenmeyer flask containing a medium comprising glycerol (50 g) , peptonised milk (5 g) , lactic casein (10 g) and "Ardamine" (1 g) (Trade Mark) made up to 1 litre with tap water and adjusted to pH 7.0. The inoculum was incubated at 28°C on a rotary shaker with a 2.5 cm throw at 170 r.p.m. for 144 hours. The total contents were then transferred to a 100 litre mechanically agitated vessel containing 70 litres of the same medium and incubated at 28°C until the first signs of pellet formation. This second stage seed culture (10 litres) was used to inoculate a 2000 litre mechanically agitated fermenter containing 1200 litres of the same medium. The fermentation was maintained at 28°C with aeration at 1200 litres per minute and agitation at 250 r.p.m. and the addition of dilute sulphuric acid if necessary at such a rate to prevent the pH rising above 6.4. After 6 days the broth was filtered using a plate and frame press and the mycelium was discarded. The filtrate (1600 litres) was pumped into ethyl acetate (800 litres) and briefly stirred. After settling the aqueous layer was discarded and the organic extract was washed with saturated brine (60 litres) before evaporating to dryness. The residue (125 g) was suspended in ethyl acetate (125 ml) and filtered. The filtrate was added slowly to petroleum ether (b.p. 40-60°C, 2 litres) with stirring and the precipitate was recovered by filtration. The solid precipitate was then chroπatographed on a column of silica gel (600 g, Merck "Kieselgel 60" [Trade Mark], 230-400 mesh) eluting with dichloromethane containing an increasing proportion of ethyl acetate. Fractions rich in the conpounds UK-110123, UK-109692 and UK-105166 co-eluted when the solvent coπposition was approximately 1:1 and UK-112847 eluted when the solvent was changed to c±iloroform-inethanol 4:1 and were recognised by silica thin layer chrαmatography as U.V.-absorbing spots with RFS as tabulated below and giving a characteristic red-purple colouration on spraying with vanillin and sulphuric acid in ethanol and heating. Fractions containing the desired conpounds were αsmbined 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 mixtures as known to those skilled in the art. The composition of the fractions collected was monitored by analytical h.p.l.c. using a Beckman Ultrasphere-ODS (Trade Mark) (5μm) h.p.l.c. column (4.6 x 250 mm) eluting with a water-methanol linear gradient from 1:1 to 100% methanol over 30 minutes at a flow rate of 0.85 ml per minute and monitoring by ultraviolet absorbance at 230 nm. Using these conditions the individual components eluted at the following times:- UK-112847 at 11.7 minutes UK-110123 at 14.3 minutes, UK-109692 at 20.6 minutes, and UK-105166 at 21.6 minutes.

Thin layer cihrαmatography (Merck (T.M.) Kieselgel 60)

The novel components were distinguished and characterised by their spectroscopic properties as follows:-

Claims

Claims 1. A compound of formula (IV) or a non-toxic salt thereof:(a) wherein R is H, -OH, -OR3, -OCOR4 or -OCONHR4;3 R is (l) C,-C8 alkyl cptionally substituted by halo, hydroxy, C,-C. alkoxy or phenyl, (ii) alkenylalkyl having 3 to 8 carbon atoms in total, (iii) alkynylalkyl having 3 to 8 carbon atoms in total, or (iv) benzyl;ΕC is C--C alkyl optionally substituted by halo, C,-C alkoxy or phenyl;R is (i) H, (ii) -C alkyl optionally substituted by halo, hydroxy, C,-C. alkoxy or phenyl, (iii) alkenylalkyl having 3 to 8 carbon atoms in total, (iv) alkynylalkyl having 3 to 8 carbon atoms in total, (v)5 5 benzyl (vi) a group of the formula -COR or -CONHR5 . . where R is either (a) C,~CR alkyl optionally substituted by halo, C--C alkoxy or phenyl, or (b) phenyl or (vii) -SO .phenyl; 2R is (i) C,-C alkyl optionally substituted by halo, hydroxy, C,-C alkoxy or phenyl (ii) C -C alkenyl (iii) C_,-C0 alkynyl, (iv) benzyl, (v) a group of the formula -COR6 or -O0NHR6 where R6 is either (a) C,-C0 alkyl1 o optionally substituted by halo, C,-C alkoxy or phenyl or (b) phenyl, or (vi) -SO .phenyl;X is O or a direct link with the proviso that when R is other than H, X is O; and the dotted line represents an optional bond; or

1 . 2 . (b) wherein R is -CH, R is 0, R is H, X is 0 and the dotted line represents a bond, said phenyl groups, and the phenyl portion of said benzyl groups, all being optionally substituted by up to three substituents each independently selected f om

C -C alkyl, C -C alkoxy, hydroxy and halo.

1 2

2. A compound a x>rding to claim 1, wherein R and R are H, the dotted line represents a bond and either (i) X is 0 and R is OH, or (ii) X is 0 and R is H, or (iii) X is a direct link and R is H.

3. A compound according to claim 1, in which said alkyl, alkoxy, alkenyl and/or alkynyl groups have up to 4 carbon atoms.

4. A compound preparable by fermentation of Penicillium paraherquei CMI 68220 and having one of the following spectroscopic characteristics(i) to (iv) :-

5. An antiparasitic compound or coπposition, substantially as hereinbefore described with reference to the Examples.

6. An antiparasitic composition, coπprising a compound according to any one of the preceding claims.

7. An antiparasitic coπposition, coπprising a compound according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient or carrier.

8. A cσπpound according to any one of claims 1 to 5 for use in human or veterinary medicine.

9. Use of a cαπpαund according to any one of claims 1 to 5 for making a medicament for treatment or prevention of parasitic infestations.

10. A process for making a compound according to claim 2, 4 or 5, which comprises fermenting in a medium containing assimilable sources of carbon, nitrogen and trace elements micro-organism CMI 68220 or a mutant or recσmbinant form thereof having the ability to produce said compound, and isolating said compound from the medium.

11. A process according to claim 10, in which said fermentation is carried out under aerobic conditions in a stirred aqueous or static solid nutrient medium.

12. A method of preventing or treating parasitic infestations, comprising application of a coπpound according to any one of claims 1 to 5.