ANTIPARASITIC AGENTS
This invention relates to antiparasitic agents, related to the milbe ycins and to processes for their preparation and compositions thereof.
The avermectins and milbemycins form an important group of broad spectrum antiparasitic agents possessing anthelmintic, ectoparasiticidal, insecticidal, antibacterial and antifungal activity with application in the areas of animal and human health, agriculture and horticulture. The avermectins are a group of macrolide compounds (previously referred to as C-076) isolated from the fermentation broth of an aver ectin producing strain of Streptomyces avermitilis such as those deposited strains ATCC 31267, ATCC 31271, ATCC 31272 as described in US patents 4310519 and 4429042. European patents 214731 and 276103 disclose a related series of compounds differing from the C-076 compounds in the nature of the substituent at C-25. The milbemycins form another group of related acrolides which are distinguished from the avermectins in lacking a sugar residue attached at C-13. Examples of such compounds are the B-41 series as described in UK patent 1390336, the LL-F28249 series described in European patent EP 170006, the E225 compounds described in European patent 254583 and the N787-182 compounds described in European patent application 334484. In addition to these fermentation products a large number of publications describe compounds derived semi-synthetically from these fermentation products many of which possess useful antiparasitic activities. Some of this chemistry is reviewed by Davies, E . G . 'and Green, R.H. in Natural Product Reports (1986) , 2, 87-121.
Our European Patent Application 410615 describes a compound, herein designated UK-86956 of formula:
It may be made by fermentation of a micro-organism deposited at the American Type Culture Collection under accession number ATCC 53928.
It has been found that certain semi-synthetic derivatives of the above-mentioned compound UK-86956, also have antiparasitic activity. Thus one aspect of this invention provides compounds of formula (I) :
where R1 and R2 are independently selected from H, Cj-C8 alkyl, alkenyl, alkynyl, C2-C8 alkanoyl, alkenoyl and alkynoyl, arylalkanoyl, arylcarbonyl and Cj-C8 alkylsulphonyl and arylsulphonyl groups, wherein all these groups other than H may optionally be substituted with at least one hydroxy, Cj-Cg alkoxy, carboxy or halo group with the proviso that at least one of R1 and R2 are other than hydrogen. The alkane, alkene and alkyne
groups may be straight or branched-chain. Halo means F, Cl, Br or I.
Compounds within this aspect of the invention include those in which R1 is methyl and R2 is H or methyl.
Another aspect of this invention provides compounds of formula (II) :
where R3 is oxo, optionally O-substituted oximino or methylene optionally substituted with cyano or a C,-C8 alkyl, cycloalkyl, aryl, aralkyl or C,-C8 alkoxycarbonyl group which groups may optionally be substituted by a halo, carboxy and/or other groups; R4 is 0-R5 where R5 is as defined for R1 in formula (I) and the oxygen is attached at C-5 by a single bond or R4 is oxo, optionally substituted imino or optionally O-substituted oximino, the broken line representing an double bond.
The oximino groups may be O-substituted by a wide variety of substituents including linear or branched Cj-Cg alkyl, alkenyl, alkynyl, trialkylsilyl and aralkyl groups which may themselves be substituted by halo, carboxy and/or other groups.
Compounds within this aspect of the invention include those in which R3 is methylene and R4 is OH or oximino and those in which R3 is oxo and R4 is hydroxy, methoxy or oximino.
A further aspect of the invention provides compounds of formula (III) :
where R1 is as defined for formula (I) and R4 is oxo or oximino optionally substituted as for compounds of formula (II) .
Compounds within this aspect of the invention include that in which R1 is -OH and R4 is oximino.
The compounds of the invention have several asymmetric centres, the number of such centres depending on the nature of substituents R1-R4, and accordingly may exist as several pairs of stereoisomers. Compound UK 86956, when prepared by fermentation of micro-organism ATCC 53928 is believed to have the following relative stereochemistry.
The compounds of the invention may be prepared by replacement of one or more of the hydroxy groups at the 5 and 22 positions of compound UK 86956 by the appropriate
substituents. These substitutions may be carried out by means of reactions which are known in the art. However, in preparing these compounds it is necessary to react selectively at one of the 5 and 22 hydroxy groups. In some instances this may be achieved by choice of appropriate reagent, for example manganese dioxide which selectively converts the 5-hydroxy group to a 5-oxo group. Alternatively selective reaction may be achieved using suitable protecting groups such as acyl or silyl derivatives. A particularly preferred protecting group is the tert-butyldi ethylsilyl group (TBDMS) which is readily removed when reguired using either an acid in a protic solvent such as methanol or with a fluoride ion source such as tetrabutylammonium fluoride dissolved in a suitable inert solvent such as tetrahydrofuran.
Alkylation of compound UK-86,956 (or a derivative thereof retaining a hydroxy group at position 5 or 22) may be achieved using conventional methodology such as treatment with an alkyl halide, preferably an alkyl iodide, in the presence of a silver salt or silver oxide. Alternatively other reactive alkylating agents known to those skilled in the art such as alkyl triflates or diazoalkanes may be employed and may show differing tendencies to react at the free hydroxy groups at the 5 and 22 positions. Similarly acylation or sulphonylation of UK-86,956 or its derivatives can be achieved using standard procedures for example by using an acyl or sulphonyl halide or anhydride in the presence of a base such as triethylamine, pyridine or diiεopropylamine.
Oxidation of the hydroxy group at C-22 may be carried out using a suitable oxidising agent such as dimethyls-ulphoxide/oxalyl chloride. Using these conditions the C-5 hydroxy group will also be oxidised if
not protected for example using the TBDMS group. The oxo group at C-5 or C-22 may be reduced to a hydroxy group although the stereochemical outcome of this reaction will depend on the choice of reducing agent. For example sodium borohydride will reduce either a C-5 or C-22 oxo group to a hydroxy group possessing substantially the same stereochemistry as that found in the UK-86,956 starting compound. However, other reducing agents such as lithium tri-sec-butylborohydride (L-selectride, Aldrich Chemical Company) will reduce the C-22 oxo group to give a hydroxy group with the opposite stereochemistry to that found in the starting compound.
The oxo group at either C-5 or C-22 is a useful functional group for performing further semi-synthetic modification to the compounds. For example reaction between a phosphorane of the general formula XYZP=CHR6, where X, Y and Z are substituents such as phenyl, phenoxy, alkoxy or oxo and a 22-oxo derivative of UK- 86,956 in an inert solvent yields a 22-methylene or when R6 is other than H, a mono-substituted methylene derivative. R6 is H or a substituent such as an alkyl, cyano, phenyl, alkanoyl or alkoxycarbonyl group. Alternatively the oxo group at C-5 and/or C-22 may be converted to an oximino group or O-substituted oximino group by reaction with an optionally O-substituted hydroxyla ine or salt thereof in a suitable solvent such as methanol, dioxane or water or combination thereof. 0- substituted oximino derivatives may alternatively be prepared by reaction of the oximino derivative with a suitable alkylating or acylating agent. Oximino and substituted methylene derivatives may exist as E or Z- isomers and both stereoisomers are within the scope of this invention. In addition those derivatives of UK-86,956 which are singly bonded at C-22 may be prepared with two possible geometries and both are within the scope of this invention (including the epi-hydroxy compound) .
The compounds of the invention are highly active antiparasitic agents having particular utility as anthelmintics, ectoparasiticides, insecticides and acaricides.
Thus the compounds are effective in treating a variety of conditions caused by endoparasites including, in particular, helminthiasis which is most freguently 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 and various parasites of dogs and cats which can infect humans including gastro-intestinal parasites such as Ancylostoma. Necator, Ascaris, Stronαyloides, Trichinella. 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 Strongyloides 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 swine, sheep, cattle and horses as well as affecting domestic animals and poultry.
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, aphidε, caterpillars and against migratory orthopterans such as locusts.
The compounds of the invention possess a number of beneficial properties compared to similar compounds such as the natural product UK-86,956 in terms of their efficacy, phar acokinetics and toleration. The benefits ..that arise from this unexpected combination of properties include efficacy against the important parasitic worms or arthropods afflicting livestock, domesticated animals or humans at lower doses than are currently employed for related compounds and, in addition, the ability to treat animals previously regarded as sensitive to this class of macrolide with a greater margin of safety.
The compounds of formula (I) , (II) or (III) are 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 compounds are preferably administered by injection, either subcutaneously or intramuscularly, alternatively they may be administered orally in the form of a capsule, bolus, tablet, chewable tablet or liquid drench, or they may be administered as a pour-on formulation or as an implant. Such formulations are prepared in a conventional manner in accordance with standard veterinary practice. Thus 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. 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 0.001 to 10 mg 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 an insecticide and for treating agricultural pests the compounds are applied as sprays, dusts, pour-on formulations, emulsions and the like in accordance with standard agricultural practice.
For human use the compounds may be administered as a pharmaceutically acceptable formulation of conventional type.
The preparation of compounds according to the invention and certain intermediate compounds is illustrated by the following Examples. "Silica" refers to Kieselgel 60, 230-400 mesh, Merck, Art. 9385. The reaction steps described in the Examples are illustrated by accompanying Figure 1.
EXAMPLE 1 5-Keto-UK-86.956 UK-86,956 (500mg) was dissolved in diethyl ether (anhydrous, 50ml) and manganese dioxide (activated, Aldrich Chemical Co. Ltd., 2.0g) was added. The suspension was stirred at room temperature for 18h and a further aliquot of manganese dioxide (l.Og) was added. After a further 8h the mixture was filtered, the solid washed with ethyl acetate and the combined filtrate evaporated under vacuum to give an off-white powder which was used directly in the following Example 2.
EXAMPLE 2 5-Oximino-UK-86 -956 5-Keto-UK-86,956 (300mg) was dissolved in methanol (7.5ml) -at room temperature and then was added, in sequence, dioxan (7.5ml), hydroxylamine hydrochloride (300mg) and water (7.5ml). After stirring for 18h the mixture was added to diethyl ether and water, the upper layer was separated and washed twice with water. The lower layer was extracted with diethyl ether and the combined organic layers were dried over anhydrous sodium sulphate and evaporated under vacuum to give a colourless glass (460mg) . Final purification was achieved by column chromatography on silica (Kieselgel 60, 230-400 mesh, Merck) (50g) eluting with methylene chloride-methanol 50:1 to give the pure title compound. Mass and NMR spectra were fully consistent with the structure shown in Figure 1.
EXAMPLE 3 5-MethQXV-UK-86,956 To a solution of UK-86,956 (500mg) in anhydrous diethyl ether (125ml) was added iodomethane (10ml) and freshly prepared silver (I) oxide (2.5g). The mixture was stirred for 60h at a room temperature, filtered and evaporated under vacuum to give the crude product (600mg) . This was purified by high pressure liquid chro atography on a Dynamax column (41.4 x 300mm, 8μm ODS-silica, Rainin) eluting with methanol-water 87:13 at 70ml per minute to give .the title compound (198mg) and 5,22-dimethoxy-UK-86,956 (200mg) from later fractions. Mass and NMR spectra were fully consistent with the proposed structures.
EXAMPLE 4 5-Tertbutyldimethylsilyoxy-UK-86.956 To a solution of UK-86,956 (4.9g) in anhydrous methylene chloride (100ml) was added tertbutyl- dimethylεilyl chloride (3.62g) and imidazole (3.2g) and the mixture was heated under reflux for 2h. Water (50ml) was then added and the organic layer separated. The aqueous layer was extracted once with methylene chloride (20ml) and the .combined organic solution was washed with dilute hydrochloric acid, water and then dried over anhydrous sodium sulphate before evaporating to dryness under vacuum to give the crude product (7.58g) as a yellow foam. This material was purified by column chromatography twice on silica (Kieselgel 60, 230-400 mesh, Merck) (50g) eluting with methylene chloride to give the title compound. 3.77g as a white foam. The NMR spectrum.of this material was fully consistent with the proposed structure.
EXAMPLE 5 5-Tertbutyldimethylsilyloxy-22-keto-UK-86.956 To a solution of oxalyl chloride (0.5ml) in anhydrous methylene chloride (5ml) under nitrogen at -78°C was added a solution of dimethylsulphoxide (0.4ml) in anhydrous methylene chloride (10ml) at such a rate that the temperature did not rise above -60°C. After 10 minutes gas evolution had ceased and a solution of 5-tertbutyldimethylsilyloxy-UK-86,956 (150mg) obtained from Example 4 in anhydrous methylene chloride (5ml) was added quickly. After stirring for lh at -78°C a solution of diisopropylethylamine (2.0ml) in anhydrous methylene chloride (5ml)- was added. The yellow solution was then allowed to warm to room temperature. After 30 minutes diethyl ether (50ml) was added and the solution was washed quickly with ice cold dilute hydrochloric acid (50ml) . The aqueous layer was extracted with diethyl ether (20ml) and the combined organic solutions were washed with water (4 x 20ml) , dried over anhydrous sodium sulphate and evaporated under vacuum to give the crude product (292mg) as a yellow oil. This material was purified by column chromatography on silica (Kieselgel 60, 230-400 mesh, Merck) (30g) eluting with methylene chloride to give the pure title compound (125mg) . The NMR spectrum was fully consistent with the proposed structure.
EXAMPLE 6 22-Keto-UK-86.956 5-Tertbutyldimethylsilyloxy-22-keto-UK-86,956 (116mg) from Example 5 was dissolved in methanol and p- toluenesulphonic acid (58mg) was added. The mixture was stirred at room temperature for lh then a mixture of saturated aqueous potassium hydrogen carbonate solution and diethyl ether
was added. The organic layer was separated and the aqueous layer was extracted with diethyl ether. The combined organic solution was washed successively with water and saturated sodium chloride solution, dried over anhydrous sodium sulphate solution and evaporated under vacuum to give the crude product as a white solid (120mg) . Final purification was achieved by column chromatography on silica (12g) eluting initially with methylene chloride followed by methylene chloride: methanol 100:1 to give the pure title compound (lOOmg) . Mass and NMR spectra were fully consistent with the proposed structure.
EXAMPLE 7 5-Methoxy-22-keto-UK,86.956 To a solution of 22-keto-UK-86,956 from Example 6 (lOOmg) in anhydrous ether (25ml) was added iodomethane (2.0ml) and silver (I) oxide (0.5g) . The mixture was stirred at room temperature for 18h then filtered and evaporated to dryness under vacuum. The crude product was purified by column chromatography on silica (12g) eluting initially with methylene chloride followed by methylene chloride: methanol 100:1 to give the desired product (77.9mg) . Final purification was achieved by reverse phase high pressure liquid chromatography on a Zorbax column (21.2 x 250mm, 8μm ODS-silica, Dupont) eluting with acetonitrile:water 85:15 to give the pure title compound (30mg) . Mass and NMR spectra were fully consistent with the proposed structure.
EXAMPLE 8 22-Methylene-UK-86.956 n-Butyllithium (1.6M in hexaneε, 0.375ml) was added dropwise to a stirred suspension of triphenylmethyl- phoεphonium bromide in anhydrous tetrahydrofuran (10ml)
under nitrogen at 0°C. After 15 minutes a solution of 5- tertbutyldimethylsilyloxy-22-keto-UK-86,956 (Example 5) in anhydrous tetrahydrofuran (5ml) was added. The mixture was allowed to warm to room temperature and ..stirred for lh before adding saturated ammonium chloride solution (50ml) and methylene chloride (20ml) . The organic layer was separated and the aqueous layer extracted with methylene chloride. The combined organic solution was dried over anhydrous sodium sulphate and evaporated to dryness under vacuum to give crude 5- tertbutyldimethylsilyloxy-22-methylene-UK-86,956 (216mg) which was purified by column chromatography on silica (20g) eluting with hexane: diethyl ether 2:1. This material (60mg) was dissolved in methanol (1.5ml) containing p-toluenesulphonic acid (30mg) and stirred at 40°C for 30 minutes. Potassium hydrogen carbonate solution was then added followed by diethyl ether and' he organic layer was separated, washed with water and saturated sodium chloride solution and dried over anhydrous sodium sulphate. The crude product was purified by column chromatography on silica (lOg) eluting with methylene chloride:methanol 50:1 to give the pure title compound (47.5mg). The mass and NMR spectra were fully consistent with the proposed structure.
The product of Example 8 may be converted to the corresponding 5-oxo and 5-oximino compounds by the methods described in Examples 1 and 2.
Figure 1
Reagents
1. Manganese dioxide, diethyl ether.
2. Hydroxylamine hydrochloride, methanol, dioxane, water.
3. Methyl iodide, silver (I) oxide, diethyl ether.
4. tert-Butyldimethylsilyl chloride, imidazole, methylene chloride.
5. Oxalyl chloride, dimethyl sulphoxide, diisopropylethylamine, methylene chloride.
6. p-Toluenesulphonic acid, methanol.
7. Triphenylphosphonium bromide, n-butyl lithium, tetrahydrofuran.