This invention relates to a novel class of compounds, related to the plant growth regulator, G, contained in the leaves of Eucalyptus grandis , which have been found to have an elmintic and/or nematocidal activity. The invention also relates to methods for the use of these compounds as an ehnintics and/or nematocides, and to compositions comprising these compounds as active ingredients.

Collaborative studies in natural products chemistry (Crow et al. 1971) and plant physiology (Paton et al. 1970) have led to identification of some plant growth regulators (PGRs) that occur in eucalypt leaves. One such regulator is G (Fig. 1) which is found in large amounts in the leaves of adult E. grandis (from which it gets its name). Extraction and purification methods for examining G activity in various bioassays have been described (Dhawan et al. 1979), and high yields have been obtained in synthetic studies (Bolte et al. 1982a, 1982b, 1982c).

Much of the physiological work refers to total G content embracing three main components, G _lt G ₂ and G ₃ (Fig. 1) with UV absorption at 242 nm. Recent HPLC work (Bolte et al. 1987) indicates the existence of G ₄ (Fig. 1), a natural precursor of G ₃ : the existence of analogous precursors of G _j and G ₂ is also envisaged.

Adult leaves of E. grandis contain up to lOmg/g fr.wt. of the plant growth regulator, G. The maximum leaf content occurs during winter when pre-dawn levels increase in inverse proportion to the minimum temperature of the previous night. Leaf content decreases during the following day: for the main G-regulator, G ₃ , this diurnal fluctuation involves reciprocal changes in G ₄ , a less active compound than G ₃ and a precursor for G ₃ in synthetic studies. G ₃ is known to react with amines to form Mannich bases that are capable of oxidising thiols to disulphides, forming a reduction product that is closely related to G ₄ .

Comparable reaction between G ₃ and proteins in vivo is believed to be involved in the low temperature hardening of E. grandis , when the high content of active G during the cold winter nights is available to stabilise protein structure by preserving disulphide bonds, thus avoiding damage during freezing and the associated dehydration. During the cyclic regeneration of G ₃ associated with this phenomenon, the molecule presumably passes through a stage where a Mannich base is formed.

In work leading to the present invention during studies involving the reaction of the plant growth regulator, G, with amines in vitro and with proteins in vivo to form Mannich bases, it has been found that such G-Mannich bases in particular have anmelmintic and/or nematocidal activity.

Since Mannich bases are involved in the reaction of G (or its precursors) with almost any suitable amine, including protein such as nematode protein, the observed inactivation of free living larval nematodes by G presumably involves interference with redox reactions similar to that postulated to occur in frost resistance. The reversibility of the effects observed in frost resistance are perhaps not feasible in the nematode system.

Throughout this specification, the term "G" is used to include the naturally occurring G,, G^ G ₃ and G ₄ , as well as natural and synthetic structural analogues, precursors and derivatives thereof.

According to one aspect of the present invention, there are provided compounds having anthelmintic and/or nematocidal activity, having the general formula I or II:

wherein; X and Y, which may be the same or different, each represents a heteroatom, preferably O;

R,, R ₃ and R ₄ , which may be the same or different, each represents H or alkyl (preferably C,-C ₆ alkyl which may be substituted) or aryl (preferably phenyl which may be substituted);

R ₂ represents H or alkyl (preferably Cι-C ₆ alkyl which may be substituted) or aryl (preferably phenyl which may be substituted), or W;

the group A ... B completes a carbocyclic or heterocyclic ring, which may be substituted; and

W represents a protein residue linked through an amine group, or a group of the formula OR ₅ , -SR _j or -NR _j R^, wherein R _j and R^, which may be the same or different, each represents H or alkyl (preferably C,-C ₆ alkyl which may be substituted) or aryl (preferably phenyl which may be substituted), or R _j and Rg together with the nitrogen atom to which they are attached form a saturated 5- or 6- membered heterocyclic ring which may be substituted and may contain one or more other hetero atoms.

As described above, the group A...3 completes a carbocyclic or heterocyclic ring, particularly a 5- or 6- membered ring. Preferably, the group A...JB is a group of the formula

-CH ₂ -CR ₇ R ₈ -CH ₂ -;

-NR ₇ -C-NR ₈ - ; or

O

-CR ₇ Rg-C-CR ₇ Rg-

O

wherein R ₇ and R ₈ , which may be the same or different, each represents H or alkyl (preferably C,-C ₆ alkyl which may be substituted) or aryl (preferably phenyl which may be substituted).

Where the alkyl or aryl groups, or the carbocyclic or heterocyclic rings of compounds of the general formulae I or II are substituted, typically they are substituted by one or more - alkyl or alkenyl, C _j -Cg alkoxy or alkenyloxy, hydroxy, cyano, amino, -Cg ai lamino or di-C _j - aUcylamino groups.

In the compounds of general formula II above where R ₄ is hydrogen, enolisation is structurally permitted as exemplified in general formula in below. Accordingly, the compounds having the general formula II are to be taken to include the enolic forms as exemplified by formula IE:

III

wherein X, Y, R _1? R ₃ and W are as defined above.

Preferably, in the compounds of general formula II above, W is a dialkylamino group (more preferably, dimethylamino or diethylamino) or a pyrrolidinyl group. Preferably also, in these compounds, R ₃ and R ₄ are H, X and Y are O, A ... B is a -CH ₂ -CR ₇ R ₈ -CH ₂ - group (more preferably, a -CH ₂ -CMe ₂ - CH ₂ - group) and R _j represents a C C ₆ alkyl or phenyl group which is optionally substituted.

Preferably also, in the compounds of general formula I above, A ... B is a -

CR ₇ R ₈ -CO-CR ₇ R ₈ - group (more preferably a -CMe^CO-CMe^r group), X and Y are O, R _t is a -Cg alkyl group and R ₂ represents a dialkylamino group (more preferably, dimethylamino or dieώylamino) or a pyrrolidinyl group.

Compounds of the formulae I and II above may be prepared in accordance with the general synthetic approaches described by Bolte et.al. (1982a, 1982b, 1982c), followed where appropriate by formation of the Mannich base by reaction with a protein or other group represented by W in general formula II.

The compounds of the present invention as described herein have been found to have anthelmintic and/or nematocidal activity. Accordingly, the present invention extends to a method of anthelmintic and/or nematocidal treatment of an animal, which comprises administration to the animal of an effective amount of a compound of the general formula I or II as described above.

In another aspect, the present invention also provides a composition for use in the anthelmintic and/or nematocidal treatment of an animal, comprising a effective amount of a compound of the general formula I or II as described above, together with an acceptable pharmaceutical or veterinary carrier or diluent.

Animals which may be treated in accordance with the present invention include both wild and domestic animals, particularly domestic animals such as horses, cats and dogs as well as sheep, cattle and other ruminant animals.

The compounds of the present invention may be administered by any suitable route, for example, orally or parenteraUy by intraperitoneal, subcutaneous or intravenous injection. Suitable effective dosages may range for example from 0.1 to 100 mg/kg (more particularly from 1 to 50 mg/kg) body weight of the animal to be treated, and will be readily determined by simple trial protocols.

The following Examples illustrate the preparation and in vitro and in vivo activity of compounds of the present invention.

EXAMPLE 1

In an in vitro larval assay, third stage infective larvae of a laboratory strain of the sheep nematode Haemonchus contortus which has not been exposed to anthelmintic treatment for 15 years are used as the test larvae. These larvae are incubated in the presence of compound and efficacy is determined from the percent reduction in the number of active larvae compared with untreated controls after 3 days incubation. The compounds are tested in concentrations ranging from 0.1-100 mM, in triplicate. Log concentration/probit curves are drawn up and the LD^ calculated. The compounds tested and results are set out in Table 1.

EXAMPLE 2

In a secondary in vitro screening test, adult parasites were isolated from their hosts and sensitivity to selected compounds (selected on the basis of high activity in the in vitro larval assay) in in vitro culture determined. Selected structures with low LD^ against larvae were also chosen and their effect against adult parasites compared. Three parasite types (trematode, cestode and nematode) were tested: Fasciola hepatica (sheep trematode), Hymenolepis diminuta (rat tapeworm), Nematospiroides dubius (mouse nematode), Haemonchus contortus and Trichostrongylus colύbr ormis (sheep nematodes). Structural activity was ranked according to the time taken to immobilise the adult worms. Concentrations ranging from 0.1 mM to 10 mM were tested in triplicate. The results are shown in Tables 2A and 2B.

Table 2A shows the time in minutes or hours for 10 mM of the compound to immobilise the adult worms. No anthehnintic compounds were administered i the control experiment. Table 2B shows the activity score for the parasites after being treated with 1 mM of each compound (again no compound was administered in the control experiment).

TABLE 2A - in vitro susceptibility of adult parasites.

- time to immobility (min or hours if specified) in vitro

TABLE 2B - in vitro susceptibility of adult parasites, activity score at 180 minutes in vitro

5 - activity of parasites in control medium; 0 - no movement detectable in parasites.

EXAMPLE 3

Compound No.252 was chosen for testing in in vivo laboratory models based on efficacy in (a) the in vitro larval assay and (b) the in vitro susceptibility of adult parasites over a broad spectrum - trematodes, cestodes and nematodes. For the in vivo tests the compound was administered: an oral aqueous suspension, an intraperitoneal injection of aqueous suspension, in a subcutaneous suspension in oil, single or repeated dose, iv an intravenous injection in serum or albumin.

Oral dosing or intraperitoneal injection of an aqueous suspension had no effect on parasites in vivo . Similarly, subcutaneous aclministration in oil had no effect on parasite burdens. When compound No. 252, suspended in rat albumin was administered by intravenous injection, 72% fewer worms were recovered from

treated rats compared with untreated control rats (Table 3). Table 4 shows a repeated experiment in rats with Compound No. 252. The compound administered in this experiment had been prepared as a large batch and increased particle size was noted. It is considered that the lower efficacy of Compound No.252 against N ras iensis was most likely due to particle size. Polyvinyl- pyrrolidine (PVP) was used for modification of Compound No. 252, with the compound absorbed in PVP at the rate of 10:1. 0.2g of this 252/PVP was suspended in 1 ml of water and the suspension administered orally to mice infected with the mouse nematode Nematospiroides d bius . The number of worms recovered from mice treated with 252/PVP and PVP alone are shown in Table 5. A 27.9% reduction in worm burden is noted (sig. at 95%).

TABLE 3 Nippostrongylus brasiliensis/ rat.

one way analysis of variance, significant at 95%

TABLE 4 Nippostrongylus brasiliensisl rat.

TABLE 5 Nematospiroides dubiusl mouse.

one way analysis of variance, significant at 95%

EXAMPLE 4

(a) Compounds of the present invention which are Mannich bases may be prepared by the following general procedure for Mannich bases derived from pyrrolidine and related amines:

To a solution of the aldehyde (7.2 mmol) and amine (7.2 mmol) in dichloromethane (10 mL) is added the diketone (7.2 mmol). The mixture is stirred for 30 min and the resultant solution evaporated to dryness. The solid residue is washed with ether to provide the Mannich base (75 - 100%).

By way of example, Compound No. 204 was prepared by this general procedure, as follows:

To a solution of p -tolualdehyde (0.84 mL, 7.2 mmol) and pyrrolidine (0.60mL, 7.2 mmol) in dichloromethane (10 mL) was added dimedone (1 g, 7.2 mmol). After 30 min the solvent was evaporated and the residue washed with ether to provide the Mannich base as a pale yellow solid (2.0 g, 90%).

(b) Alternatively, the compounds may be prepared by the following general procedure for Mannich bases derived from dimethylamine:

Dimethylamine is bubbled into an ice cold suspension of the diketone (7.0 mmol) in dichloromethane (40 mL) under nitrogen. After all the solid material has dissolved, a solution of the aldehyde (7.0 mmol) in dichloromethane (20 mL) is added. The ice bath is removed and the reaction mixture stirred for 30 min. The solution is evaporated to dryness and the solid residue washed with ether to give the dimethylamine Mannich base (70 - 90%).

(c) Compounds of the present invention which are enediones may be prepared by the following general procedure for making enediones:

A mixture of the Mannich base (1.6 mmol) and p -toluenesulfonic acid (3.2 mmol) in dichloromethane (15 mL) is stirred under nitrogen for 10 min.

The reaction mixture is evaporated to dryness, then dissolved in ethyl acetate (30 mL) and washed with water until the pH of the aqueous layer is - 5. The organic extract is dried (MgS0 ₄ ) and the solvent removed to provide the enedione (80 - 95%).

The enedione may then be treated with a variety of nitrogen and sulfur nucleophiles affording the appropriate Mannich bases or their sulphur analogues, as exemplified below.

(i) Procedure for chloroform /dichloromethane soluble nucleophiles:

Compound No. 269 was prepared by the following procedure.

To a stirred solution of the enedione obtained from Compound No.

204 (240 mg, 1.0 mmol) in dichloromethane (10 mL) under nitrogen, was added ethanethiol (0.07 mL). After 30 min the pale yellow solution was evaporated to dryness and the residue washed with ether. This yielded the desired product (210 mg, 70%).

(ii) Procedure for chloroform/dichloromethane insoluble nucleophiles: Compound No. 275 was prepared by the following procedure. To a stirred solution of the enedione obtained from Compound No.

251 (340 mg, 1.0 mmol) in dry methanol (10 mL) under nitrogen, was added thiosalicylic acid (154 mg, 1 mmol). After 2h the pale yellow solution was evaporated to dryness and the residue washed with ether to provide the desired compound (400 mg, 81%) as a cream solid.

EXAMPLE 5

Compound No. 252 was dissolved in sterile sheep serum and administered intravenously to one sheep infected with Haemonchus contortus (nematode) and Monezia expansa (cestode).

Dose rate 26.5 mg/kg bodyweight Total dose 500 mg.

Results

epg = eggs per gram faeces

** increase in cestode egg counts indicates a parasite under stress.

REFERENCE&

1. Bolte, M.L., Crow, WJD., and Yoshida, S. (1982a). Plant growth regulators in E.grandis TV: synthetic approaches to G-regulators analogues. AustJ.Chem 35, 1411-19.

2. Bolte, M.L., Crow, W.D., and Yoshida, S. (1982b). Plant growth regulators in E.grandisV: synthesis of G-regulators. AustJ.Chem. 35, 1421-1429.

3. Bolte, M.L., Crow, W.D., and Yoshida, S. (1982c). Plant growth regulators in E.grandis VI: reaction of G-regulators with nucleophiles. AustJ.Chem 35, 1431-1437.

4. Bolte, M.L., Crow, W.D., and Paton, D.M. (1987). Frost hardiness in Eucalyptus grandis a possible molecular mechanism. In Plant Cold Hardiness, ed.Paul, H. Li; Alan R.Liss, NY, pp.129-40.

5. Crow, W.D., Nicholls, W., Sterns, M. (1971). Root inhibitors in Eucalyptus grandis. Tetrahedron Letters, 1353-6.

6. Dhawan, A.K., Paton, D.M., and Willing, R.R. (1979). Occurrence and bioassay responses of G: a plant growth regulator in Eucalyptus and other Myrtaceae. Planta 146: 419-422.

7. Paton, D.M., Willing, R.R., Nicholls, W., Pryor, L.D. (1970). Rooting of stem cuttings of Eucalyptus: a rooting inhibitor in adult tissue. AustJ ot 18: 175-183.