The invention relates to compounds, in particular derivatives of the diterpenoids sempervirol and 7-keto-sempervirol, and their use in therapy, including in pharmaceutical compositions and combinations, especially in the treatment and/or prophylaxis of the tropical diseases schistosomiasis and/or fascioliasis, e.g. in humans or in non-human land mammals such as sheep, cattle, other ruminants, or goats. Background of the Invention

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Schistosomiasis (also known as bilharzia, snail fever or Katayama fever) and fascioliasis (also known as fasciolosis, fasciolasis, distomatosis or liver rot) are Neglected Tropical Diseases (NTDs) caused by related parasitic blood fluke trematodes (Schistosoma species such as Schistosoma mansoni, Schistosoma intercalatum, Schistosoma haematobium or Schistosoma japonicum) and parasitic liver fluke trematodes (Fasciola species such as Fasciola hepatica or Fasciola gigantica) found within the phylum Platyhelminthes. These NTDs are responsible for insidious conditions of biomedical as well as veterinary significance and collectively affect a considerable proportion of the world's human and animal populations. Globally, approximately 200 million people are currently afflicted by schistosomiasis, with this chronic disease being most prominent in tropical and subtropical rural areas. While fascioliasis is one of the most important parasitic diseases of ruminant livestock animals, approximately 2.4 million humans are also affected. This particular NTD is rapidly becoming recognised as an emerging public health problem due to its role in driving chronic liver pathologies in infected humans. Schistosomiasis is generally spread by contact with water that contains the Schistosoma parasites; these parasites are typically released from freshwater snails that have been infected. Similarly, the life cycle of Fasciola parasites generally includes freshwater snails as an intermediate host of the parasite.

The life cycles of flukes, such as Schistosoma sp., involve a number of distinct developmental stages. Significantly, the sensitivity of flukes in their various developmental stages to chemotherapeutic treatment can vary. Chemotherapeutic agents that are active against one developmental stage of the fluke often prove ineffective against another. As a result, populations that are treated for parasitic infection with a drug that is ineffective against all fluke developmental stages can remain infected. Infected populations may therefore require repeated treatment to eradicate the parasitic fluke infection and this can lead to increased cost and compliance issues with treatment. The lifecycle of the liver fluke Fasciola sp. in animals is classified as having three district stages: mature, immature and early immature. Early immature fluke are defined as those fluke that are 4-week or younger. These fluke have hatched from the infective stage, which encysts on grass (metacercariae). After hatching in the gut the early immature flukes burrow through the gut wall and penetrate the liver wherein they begin to eat their way through the liver to migrate to the bile ducts. Immature fluke are defined as those fluke that are older than 4 weeks but not yet adult. These immature fluke are at the later stage of migration and from 7 weeks of age are entering the bile ducts. The mature fluke stage is generally attained after 10 to 12 weeks of development and these fluke generally reside in the bile ducts of the liver where they actively lay eggs. As well as being a significant human and animal health problem, parasitic fluke infections have a significant economic impact in the livestock industry. For example, the global impact of liver fluke infestation across the livestock sector as a whole is estimated at over US$3.2 billion a year. In Australia, lost production has been estimated to cost US$50-80 million a year (1999 estimate) and the rate of infection in NW Europe is estimated at greater than 30%. In cattle, losses result from declines in the condition and the performance of affected animals, including poor feed conversion and reduced weight gain, lower rates of fertility in breeding cows, liver condemnation in slaughtered beef animals and reduced milk yield in dairy cows. For example, a low grade infection of just 100 flukes has been shown to reduce milk yield by 400 litres per cow per lactation. Liver flukes are estimated to cost UK livestock industry alone over £300 million per year. Fluke occurrence in the UK is on the rise with increases of over 300% seen in both sheep and cattle (over five years), and it is likely to become even more prevalent as temperatures rise and the host snails proliferate. The annual cost to the UK cattle industry is estimated at £23 million; the cost of lost milk production is estimated at £5.8 million.

Control of schistosomiasis and fascioliasis remains largely centred on the use of large-scale chemotherapy administered to infected individuals in high prevalence areas. Praziquantel (PZQ) ( ) is presently the gold standard drug for schistosomiasis control due to its safety, low cost, and activity towards the adult life stage of the three major, human- infective species (S. mansoni, Schistosoma haematobium and Schistosoma japonicum). For fascioliasis

chemotherapy, triclabendazole (TBZ) remains the drug of choice and is the only available compound effective against both adult and juvenile liver fluke lifecycle stages. In both cases, the over reliance of a single drug class for maintaining the future of blood and liver fluke control has generated significant concerns that PZQ or TBZ-resistant parasites could develop.

M. Yakoot, Arq. Gastroenterol., 2010, vol. 47, no.4, October/December 2010, pp. 393-394 gives a short review of the anthelmintic role of Mirazid™, which is a preparation derived from the oleo-resin of myrrh, which is a plant that has been used in folk medicine since the era of ancient Egyptians. Phase III human clinical trials suggest Myrazid is safe and efficacious in the treatment of schistosomiasis and/or fascioliasis, such that Myrazid was registered by the ministry of health in Egypt. As a result of the factors detailed above there is a need for new drugs for the treatment of schistosomiasis and/or fascioliasis in humans and animals. New drugs would ideally display activity against Schistoma sp or Fasciola sp across more than one of its/their various developmental stages. This is advantageous as it offers the potential to overcome or substantially reduce problems associated with rapid reinfection of treated patient groups. Advantageously, new anthelmintic drugs would display activity against both Schistoma sp and Fasciola sp, as such compounds could be used for the treatment of both schistosomiasis and fascioliasis. Agents that exhibit activity against both Schistoma sp and Fasciola sp in more than one of their developmental stages would be particularly advantageous. US 4,282,253 (Steck, assignee USA Army) discloses a method for preventing schistosomiasis in a mammal exposed to schistosome-infected water comprising applying to the skin of the mammal prior to exposure a composition comprising dehydroabietylamine or a salt or adduct thereof.

(+)-Dehydroabietylamine is available from Aldrich and has the following structure:

673-675 discloses the isolation of

the brief synthesis of

derivatives (in which R =

C(0)CH3). No medicinal uses are disclosed in Mangoni and Caputo for sempervirol, 7-ketosempervirol or the acetate derivatives thereof.

It is desirable to discover compounds that exhibit activity against either schistosomiasis, or fascioliasis, or schistosomiasis and fascioliasis, e.g. in a mammal (in particular a human or a non-human land mammal), especially where in the drug displays activity against one or more of the various developmental forms of parasitic fluke (in particular Schistosoma or Fasciola species) which are capable of causing schistosomiasis and/or fascioliasis. Summary of the Invention

In a first aspect, the invention provides a compound of the formula (I) for use as a therapeutically active substance, preferably for use as a therapeutically active substance in a mammal (in particular a human or a non-human land mammal):

wherein: i and 2 are, independently, C1-C6 alkyl optionally substituted with 1, 2 or 3 substituents independently being: hydroxy, Ci-C4alkoxy, halogen (in particular fluorine), NH2, N(H)-Ci-C4 alkyl, N(Ci- C ₄ alkyl) ₂ , carbonyl (=0), -C0 ₂ H, or -C(0)OCi-C ₄ alkyl;

R ₃ is selected from Ci-C ₆ alkyl optionally substituted with one substituent being: hydroxy, =0, - C0 ₂ H, -C(0)OCi-C ₄ alkyl or C C ₄ alkoxy; R ₄ is selected from C1-C6 alkyl, C1-C6 alkoxy, N(H)-Ci-6 alkyl, and N(Ci-C ₄ alkyl) ₂ ;

X is selected from hydrogen, hydroxy, C1-C6 alkoxy, Ci-C3alkoxy-CH ₂ CH ₂ 0-, -OC(0)Ci-C ₄ alkyl, carbonyl (=0), NH2, N(H)-Ci-6 alkyl, N(Ci _-6 alkyl) ₂ , =NOH, alkyl) and -NC(0)(Ci-C ₆ alkyl); and

Y is selected from hydrogen, hydroxy, C1-C6 alkoxy, -C(0)Ci-C6 alkyl, -OC(0)Ci-C6 alkyl, -OC(0)OCi-C ₄ alkyl, -SC(0)Ci-C ₄ alkyl, halogen, cyano, NH ₂ , N(H)-Ci _-6 alkyl, N(Ci _-6 alkyl) ₂ , -NC(0)(Ci-C ₆ alkyl) and nitro.

The invention also encompasses salts, preferably pharmaceutically acceptable salts, of compounds of formula (I). Therefore, throughout this specification, and generally in the present invention, the compound of formula (I) (or the compound of (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li) or (Ij) as defined below) is optionally in the form of a salt, preferably a pharmaceutically acceptable salt, of the compound; and compounds according to or used in the present invention, such as a compound of formula (I) or (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li) or (Ij), should be interpreted as including salts e.g. pharmaceutically acceptable salts thereof except where the context clearly indicates otherwise. The present invention provides a compound of formula (I) (or a compound of (la), (lb), (Ic), (Id), (le), (If) _/ (lg) _/ Oh) _/ (li) or (Ij) as defined below) for use in medicine and/or for use as a therapeutically active substance in a mammal (in particular a human or a non-human land mammal). The invention provides a compound of formula (I) (or a compound of (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li) or (Ij) as defined below) for use in non-human mammals that are members of the Bovidae, Equidae, Suidae, Camelini and Cervidae families.

Preferably, the invention provides a compound of formula (I) (or a compound of (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li) or (Ij) as defined below) for use in the treatment or prophylaxis of schistosomiasis, or fascioliasis, or schistosomiasis and fascioliasis, preferably in a mammal (in particular a human or a non- human land mammal). Preferably, the schistosomiasis and/or fascioliasis is caused by one or more of the various developmental forms (e.g. mature, immature or early immature) of a Schistosoma and/or Fasciola species capable of causing schistosomiasis and/or fascioliasis.

"Therapeutically active substance" includes a substance which is active when given as therapy by treatment, or as therapy by prophylaxis, or as therapy by treatment and prophylaxis.

The invention provides, in another aspect, the use of a compound (I) (or a compound of (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li) or (Ij) as defined below) for the manufacture of a medicament. The invention also provides the use of a compound of formula (I) (or a compound of (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li) or (Ij) as defined below) for the manufacture of a medicament for the treatment or prophylaxis of schistosomiasis, or fascioliasis, or schistosomiasis and fascioliasis in a mammal (in particular a human or a non-human land mammal). The invention also provides the use of a compound of formula (I) (or a compound of (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li) or (Ij) as defined below) or the manufacture of a medicament for the treatment or prophylaxis of schistosomiasis, or fascioliasis, or schistosomiasis and fascioliasis in a mammal of the Bovidae, Equidae, Suidae, Camelini and Cervidae families. The invention provides, in another aspect, a method of treatment of schistosomiasis and/or fascioliasis in human or non-human land mammals in need thereof comprising administering to the patient a pharmacologically effective dose of a compound of formula (I) (or a compound of (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li) or (Ij) as defined below) for the manufacture of a medicament. The invention also provides a method of treatment or prophylaxis of schistosomiasis, or fascioliasis, or schistosomiasis and fascioliasis in a mammal of the Bovidae, Equidae, Suidae, Camelini and Cervidae families involving administering to the patient a pharmacologically effective dose of a compound of formula (I) (or a compound of (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih), (li) or (Ij) as defined below). The invention also provides, in another aspect, a pharmaceutical composition (in particular a tablet or capsule) comprising a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih), (li) or (Ij), and preferably a pharmaceutically acceptable carrier, excipient or diluent (more preferably a solid carrier, excipient or diluent). Such compositions may be administered (e.g. to a human or non-human mammal) alone, or in conjunction with other therapeutic agents which optionally may be included in the same pharmaceutical composition, or which optionally be administered separately.

The invention also provides for, in another aspect, a pharmaceutical composition (in particular a tablet or capsule), for use in the treatment and/or prophylaxis of schistosomiasis and/or fascioliasis, preferably in a mammal (in particular a human or a non-human land mammal), comprising a comprising a compound of formula (I) or (la), and preferably a pharmaceutically acceptable carrier, excipient or diluent (more preferably a solid carrier, excipient or diluent). The compounds (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih), (li) or (Ij) for use in treatment or for use in manufacturing a medicine or for use in methods of treatment of treatments described herein can also be advantageously combined with an anthelmintic agent, for example praziquantel or triclabendazole, to provide an enhanced anti- parasitic activity.

In a further as ect, the invention provides a compound of formula (la):

i and 2 are, independently, C1-C6 alkyl optionally substituted with 1, 2 or 3 substituents independently being: hydroxy, Ci-C ₄ alkoxy, halogen (in particular fluorine), N H2, N(H)-Ci-C4 alkyl,

N(Ci-C ₄ alkyl) ₂ , carbonyl (=0), -C0 ₂ H, or -C(0)OCi-C ₄ alkyl; 3 is selected from C1-C6 alkyl optionally substituted with one substituent being: hydroxy, =0, - CO2H, -C(0)OCi-C ₄ alkyl or C1-C4 alkoxy; 4 is selected from C1-C6 alkyl, C1-C6 alkoxy, N(H)-Ci-6 alkyl, or N(Ci-C4 alkyl)2 ;

X is selected from hydrogen, hydroxy, C1-C6 alkoxy, Ci-Csalkoxy-ChbCI-bO-, -OC(0)Ci-C4 alkyl, carbonyl (=0), NH2, N(H)-Ci-6 alkyl, N(Ci _-6 alkyl) ₂ , =NOH, alkyl) and -NC(0)(Ci-C ₆ alkyl); and Y is selected from hydrogen, hydroxy, Ci-C ₆ alkoxy, -C(0)C C ₆ alkyl, -OC(0)d-C ₆ alkyl, -OC(0)OCi-C ₄ alkyl, -SC(0)Ci-C ₄ alkyl, halogen (in particular fluorine), cyano, NH ₂ , N(H)-Ci_ ₆ alkyl, N(Ci_ ₆ alkyl) _2, -NC(0)(C C ₆ alkyl) and nitro; provided that when 4 is isopropyl, then Y is not OH, OCH3 or 0(CO)CH3 ; and wherein the compound of formula (la) is optionally in the form of a salt, preferably a pharmaceutically acceptable salt, of the compound.

Brief Description of the Drawings

In order that the invention can be fully understood, the invention and embodiments thereof are described with reference to the following Figures, which are not intended to limit the scope of the invention.

Figure 1 shows the structural formula of 7-keto-sempervirol. Its structure is also shown here:

Figure 2 shows the results obtained from a Helminth Fluorescent Bioassay evaluation of the activity of 7-keto-sempervirol (100μΜ-1.575 μΜ) on schistosomula viability during a 24 hour in vitro co-culture. Figure 3 shows results obtained in a phenotypic assessment of the activity of 7-keto-sempervirol against the adult blood fluke (S. mansoni). This assay uses a phenotypic assessment of movement indices and phenotypic discrepancies. Figure 4 illustrates the effect that 7-keto-sempervirol has on the outer tegument of the male parasite at 100 μΜ prior to 72 hr cultivation in the presence of the compound. SEM microscopy of the adult male worms co-cultured in the presence of the diterpenoid compound (100 μΜ) for 72 hrs reveals holes appearing on the outer tegument of the parasite (Figure 4b). Figure 5 presents in utero confocal images of the internal structure of the female adult worms in control and 7-keto-sempervirol treated fluke. This image reveal irregular egg architecture after a 24 hr cultivation period with 7-keto-sempervirol at a final concentration of 100 μΜ (Figure 5b).

Figure 6 shows the effect of 7-keto-sempervirol treatment on egg output and morphology for S. mansoni.

Figure 7 shows the effect of 7-keto-sempervirol on F. hepatica NEJs.

Figure 8 illustrates the phenotypic alterations upon the adult F. hepatica life stage following 7-keto- sempervirol treatment.

Detailed Description of the Invention

The general structure of the compounds of Formula (I) according to or as used in the present invention is presented below. The numberin of the ring-carbon atoms within formula (I) is also shown.

Formula (I)

In the compound of formula (I) shown above, the substituent Y may be attached to any of the ring- carbons numbered 11, 13 or 14 in Formula (I). Preferably, the substituent Y is attached to ring-carbon numbered 13 in the formula shown above. In the compound of formula (I) shown above, the substituent X may be attached to any the ring- carbons numbered 6 or 7 in Formula (I). Preferably, the substituent X is attached to ring-carbon numbered 7 in the formula shown above. The chemical definitions used herein will be familiar to those skilled in the art. For clarity, some general (e.g. preferred or particular) definitions are provided hereinbelow and/or hereinabove.

The preferred, particular, suitable, advantageous, exemplary and/or optional values of the substituents in or other features of a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih), (li) or (Ij) as described above and/or below, in particular i, R ₂ , R3, R4, R5, X and/or Y, and any substituents or other features within Ri, R ₂ , R3, R4, R5, X and/or Y, are set out hereinbelow (and/or hereinabove) and can be taken either alone or taken together with one or more of any other preferred, particular, suitable, advantageous, exemplary and/or optional features in any combination(s) thereof. In this paragraph and the preceding paragraph, "preferred" is intended to encompass more preferred, even or still or yet more preferred, particularly or highly preferred, most preferred and all similar terms.

Alkyl, such as C1-C6 alkyl, as used herein refers to straight chain or branched chain alkyl (e.g. C1-C6 alkyl), such as, without limitation, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert- butyl, n-pentyl, iso-pentyl, neo-pentyl, n-hexyl, iso-hexyl or neo-hexyl. In some embodiments, alkyl refers to straight chain alkyl. In other embodiments, branched alkyl groups are preferred. Examples of preferred branched alkyl groups include iso-propyl, iso-butyl, iso-pentyl or neo-pentyl. In some cases the alkyl groups have from one to six, or from one to four, carbon atoms. In some preferred instances the alkyl groups have from one to three carbon atoms. Preferably, Ci-C ₆ alkyl is C1-C4 alkyl, more preferably C1-C3 alkyl or Ci-C ₂ alkyl.

Alkoxy as used herein refers to straight or branched chain alkoxy, for example methoxy, ethoxy, propoxy, iso-propoxy or butoxy. In general, the alkoxy groups of the compounds of the invention have from one to six carbon atoms. Preferably, C1-C6 alkoxy is C1-C4 alkoxy, more preferably C1-C3 alkoxy or C1-C2 alkoxy. In one embodiment alkoxy relates to straight chain alkoxy. In another embodiment alkoxy relates to branched chain alkoxy.

Alkylamino and dialkylamino as used herein refers to amino groups substituted with (respectively) one or two straight or branched C1-C6 alkyl groups (preferably one or two straight or branched C1-C4 alkyl or C1-C3 alkyl or C1-C2 alkyl groups). Halo or halogen includes fluoro, chloro, bromo or iodo, in particular fluoro, chloro or bromo, preferably fluoro or chloro, most preferably fluoro (fluorine). AlkyI substituted by halo (haloalkyi) as employed herein refers to alkyl groups substituted by 1 to 6 independent halogen atoms or more preferably substituted by 1, 2, 3, 4 or 5 (in particular 1, 2 or 3) independent halogens. The independent halogens can e.g. independently be fluorine or chlorine, but preferably are fluorine. Haloalkyi can e.g. be perhaloalkyl, in particular perfluoroalkyl. Preferably, haloalkyi is C1-C4 or C1-C3 haloalkyi (more preferably C1-C2 haloalkyi) substituted by 1, 2, 3, 4 or 5 (more preferably 1, 2 or 3) independent halogens (in particular fluorine), more preferably Ci haloalkyi substituted by 1, 2 or 3 independent halogens in particular -CF ₂ CF ₃ , -CH ₂ CF ₃ , CH ₂ F, CHF ₂ or CF ₃ .

Particularly preferably, haloalkyi is fluoroalkyl, i.e. is an alkyl group substituted by 1 to 6 fluorine atoms, preferably by 1, 2, 3, 4 or 5 fluorine atoms, or more preferably by 1, 2 or 3 fluorine atoms. Preferably, fluoroalkyl is C1-C4 or C1-C3 fluoroalkyl, more preferably Ci-C ₂ or Ci fluoroalkyl, still more preferably -CF ₂ CF3 , -CH ₂ CF3 , CH ₂ F, CHF ₂ or CF3, or most preferably CH ₂ F, CHF ₂ or CF3.

Haloalkoxy (in particular fluoroalkoxy) means an alkoxy group substituted by 1 to 6 independent halogen (in particular fluorine) atoms, preferably by 1, 2, 3, 4 or 5 independent halogen (in particular fluorine) atoms, or more preferably by 1, 2 or 3 independent halogen (in particular fluorine) atoms. Preferably, haloalkoxy or fluoroalkoxy is C1-C4 or C1-C3 haloalkoxy or fluoroalkoxy, more preferably Ci-C ₂ or Ci haloalkoxy or fluoroalkoxy, still more preferably -OCF ₂ CF3 , -OCH ₂ CF3 , OCH ₂ F, OCHF ₂ or OCF3, or most preferably OCH ₂ F, OCHF ₂ or OCF ₃ . Alkyl substituted by hydroxy (hydroxyalkyi) as employed herein refers to alkyl substituted by 1, 2 or 3 hydroxy groups, in particular by 1 or 2 hydroxy groups; and preferably is C1-C3 or Ci-C ₂ hydroxyalkyi, more preferably -CH ₂ CH ₂ OH, -C(CH ₃ )CH ₂ OH, -C(CH ₃ ) ₂ CH ₂ OH or similar.

Unless otherwise specified, alkylene as employed herein is a straight chain or branched chain bivalent carbon linking group, for example comprising methylenes, between two other moieties. For the avoidance of doubt, the term "n-alkylene", when used herein, refers to straight chain alkylene.

C1-C6 alkyl or haloalkyi or fluoroalkyl can be Ci, C ₂ , C3, C4, C5 or C6. C1-C4 alkyl or haloalkyi or fluoroalkyl can be Ci, C ₂ , C3 or C4. C1-C3 alkyl or haloalkyi or fluoroalkyl can be Ci, C ₂ or C3. C1-C6 alkoxy, haloalkoxy or fluoroalkoxy can be Ci, C2, C3, C4, C5 or C6. C1-C4 alkoxy, haloalkoxy or fluoroalkoxy can be Ci, C2, C3 or C4. C1-C3 alkoxy, haloalkoxy or fluoroalkoxy can be Ci, C2 or C3.

Preferably, there is provided a compound (or compound for use) of formula (la)

wherein Ri, R2, R3, R4, X and Y are as defined as above for the compound of formula (la), wherein R ₄ is any Ci-C ₆ alkyl excluding isopropyl (i.e. is methyl, ethyl, n-propyl or C ₄ -C ₆ alkyl), or is C C ₆ alkoxy, N(H)-Ci_ ₆ alkyl, or N(d _-6 alkyl) ₂ ; and/or wherein Y is selected from hydrogen, C ₂ -C ₆ alkoxy, -C(0)C ₂ -C ₆ alkyl, -OC(0)Ci-C ₆ alkyl, -OC(0)OCi-C ₄ alkyl, -SC(0)Ci-C ₄ alkyl, halogen (e.g. fluorine), cyano, NH ₂ , N(H)-Ci- ₆ alkyl, N(Ci _-6 alkyl) ₂ , NCO(Ci-C ₆ alkyl) and nitro.

Preferably, there is provided a compound (or compound for use) of formula (lb)

(lb)

wherein R3, R4, X and Y are as defined herein eg as above for compounds (I) or (la). Preferably, there is provided a compound (or compound for use) of formula (lc)

(lc)

wherein 4, X and Y are as defined herein eg as above for compounds (I) or (la).

More preferabl there is provided a com ound (or compound for use of formula (Id), (le) or (If)

(Id) (le) (If) wherein R4, X and Y are as defined herein eg as above for compounds (I) or (la).

Even more preferably, there is provided ompound for use) of the formula (Ig)

(ig)

wherein X and Y are as defined herein eg as above for compounds (I) or (la).

Still more preferably, there is provided a compound (or compound for use) of the formula (Ih)

(Ih)

wherein X is as defined herein eg as above for compounds (I) or (la), and 5 is selected from H, C1-C6 alkyi and C(0)Ci-C6 alkyi (preferably 5 is selected from H, C1-C4 alkyi and C(0)Ci-C ₄ alkyi).

Yet more preferably, there is provided a compound (or compound for use) of the formula (li)

(«)

wherein 5 is selected from H, C1-C6 alkyi or C(0)Ci-C6 alkyi (preferably R5 is selected from H, C1-C4 alkyi and C(0)d-C ₄ alkyi).

In one most preferred embodiment, in the compound of formula (Ih) or (li), R5 is H. In another most preferred embodiment, in the compound of formula (Ih) or (li), R5 is C1-C3 alkyi or C(0)Ci-C3 alkyi.

In the compounds of the formula (I) and (la), preferably Ri, R ₂ and R ₃ here are C1-C4 alkyi in particular methyl, ethyl, or propyl. In the compound of formula (I), (la), (lb), (Ic), (Id), (le) or (If), preferably, R4 is C1-C4 alkyi, in particular methyl, ethyl, isopropyl, n-propyl, sec-butyl, isobutyl or t-butyl. In one preferred embodiment, R4 is methyl, ethyl, n-propyl, sec-butyl, isobutyl or t-butyl; in particular ethyl, n-propyl, sec-butyl, isobutyl or t-butyl. In another preferred embodiment, R4 is isopropyl. Preferably in all cases, when R4 is isopropyl, then Y is not OH, OCH ₃ or 0(CO)CH ₃ .

In the compound of formula (I), (la), (lb), (Ic), (Id), (le), (If) or (Ig) or (Ih), preferably, X is selected from hydrogen, hydroxy, Ci-C ₃ alkoxy, -OC(0)Ci alkyi, carbonyl (=0), NH ₂ , N(H)-Ci alkyi, N(Ci alkyl) ₂ , =NOH, alkyi) and -NC(0)(Ci alkyi). More preferably, X is selected from hydrogen, hydroxy, C1-C3 alkoxy, carbonyl (=0), =NOH and alkyi); most preferably carbonyl (=0).

In the compound of formula (I), (la), (lb), (Ic), (Id), (le), (If) or (Ig), preferably, Y is selected from hydrogen, hydroxy, C1-C3 alkoxy, -C(0)Ci-C3 alkyi, -OC(0)Ci-C3 alkyi, halogen (in particular chlorine or fluorine), cyano, NH ₂ , N(H)-Ci alkyi, N(Ci alkyl) ₂ and -NC(0)(Ci-C ₆ alkyi). More preferably, Y is selected from hydrogen, hydroxy, Ci-C ₃ alkoxy and -OC(0)Ci-C3 alkyl; most preferably OH. Preferably in all cases, when 4 is isopropyl, then Y is not OH, OCH3 or 0(CO)CH3 .

In one particular embodiment the invention relates a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (lg), (lh) or (li), optionally in the form of a pharmaceutically acceptable salt thereof, for use in medicine and/ or as a therapeutically active substance. In one particular embodiment the invention relates a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (lg), (lh) or (li), optionally in the form of a pharmaceutically acceptable salt thereof, for use in medicine and/ or as a therapeutically active substance for the Bovidae, Equidae, Suidae, Camelini and Cervidae families. In one embodiment the invention relates to a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (lg), (lh) or (li), or a pharmaceutically acceptable salt of these compounds, for use in the treatment and/or prophylaxis of schistosomiasis. In one embodiment the invention relates to a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (lg), (lh) or (li) for use in the treatment of fascioliasis. In a further embodiment the invention relates to a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (lg), (lh) or (li) for use in the treatment and/or prophylaxis schistosomiasis and fascioliasis. Advantageously the compounds of the present invention display activity against Schistosoma sp or Fasciola sp in more than one of their various developmental stages. In one advantageous embodiment the compounds of formula (I), (la), (lb), (lc), (Id), (le), (If), (lg), (lh) or (li) display activity against the juvenile life stages of the S. mansoni and/or F. hepatica parasites.

Advantageously the compounds of the present invention display activity against both Schistosoma sp and Fasciola sp in more than one of their various developmental stages. In one advantageous embodiment, compounds according to the present invention can be used to decrease motility of adult male parasite. In a further advantageous embodiment compounds according to the present invention can be used to prevent adult parasite male/female pairs from remaining in copula thus inhibiting parasite reproduction thereby rendering the compounds of the present invention useful for treating schistosomiasis and/or fascioliasis. In another advantageous aspect the compounds detailed above can be used to inhibit or substantially reduce egg output of S. mansoni and/or F. hepatica parasites thereby rendering the compounds of the present invention useful for treating schistosomiasis and/or fascioliasis. In another advantageous aspect the compounds detailed above can be used to increase abnormal egg output of S. mansoni and/or F. hepatica parasites thereby rendering the compounds of the present invention useful for treating schistosomiasis and/or fascioliasis. Advantageously the effects of compounds of the present invention on egg output of S. mansoni and/or F. hepatica parasites can be used to reduce disease transmission in treated subject populations. The advantageous properties of the compounds of the present invention render them suitable for use with agents that have activity against other common parasitic infections including agents that are used for treatment of schistosomiasis and fascioliasis. The pharmaceutically acceptable salts of the compounds of the invention include acid addition salts of compounds of formulas (I), (la), (lb), (lc), (Id), (le), (If), (Ig) that contain a basic group such as an amine and are meant to comprise the therapeutically active non-toxic acid addition salts that the compounds of formula (I) are able to form. These pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the free base form with such appropriate acids in a suitable solvent or mixture of solvents. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulphuric, nitric, phosphoric acids and the like; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p- toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic acid and the like. Conversely said salt forms can be converted by treatment with an appropriate base into the free base form. Other pharmaceutical salts include those incorporating a phenolate or carboxylate anion in conjunction with a non-toxic cation species.

The invention provided herein extends to prodrugs of the compound of formula (I), that is to say compounds which break down and/or are metabolised in vivo to provide an active compound of formula (I). General examples of prodrugs include simple esters, and other esters such as mixed carbonate esters, carbamates, glycosides, ethers, acetals and ketals.

In a further aspect of the invention there is provided one or more metabolites of the compound of formula (I), in particular a metabolite that retains one or more of the therapeutic activities of the compound of formula (I). A metabolite, as employed herein, is a compound that is produced in vivo from the metabolism of the compound of formula (I), such as, without limitation, oxidative metabolites and/or metabolites generated, for example, from O-dealkylation. The compounds of the disclosure include those where the atom specified is a naturally occurring or non-naturally occurring isotope. In one embodiment the isotope is a stable isotope. Thus the compounds of the disclosure include, for example deuterium containing compounds and the like.

The disclosure also extends to all polymorphic forms of the compounds herein defined. Processes for preparing compounds of formula (I) or (la)

According to another aspect of the invention there is provided a process for preparing a compound of formula (I) or (la), as defined herein, comprising any one of the final-stage (or final- and penultimate- stage, or or final- and penultimate- and antepenultimate-stage) preparative processes to the compound of formula (I) or (la) which are disclosed hereinbelow.

The ring numbering for the compound of formula (I) or (la) is shown again, for reference with respect

Sempervirol ) is a natural product that can be obtained from Cypressus sempervirens as described in the literature (see L. Mangoni and R. Caputo, Tetrahedron Letters, 1967, no. 8, pp. 673-675)

7-Ketosempervirol ) can be obtained (i) by extraction from Lycium chinense, e.g. as described herein, or (ii) by synthesis e.g. by conversion of sempervirol to the acetate derivative (

, wherein R is C(0)CH3), chromic acid oxidation of this acetate to give the keto- acetate derivative, and hydrolysis of the keto-acetate to give 7-ketosempervirol (as described in L. Mangoni and R. Caputo, Tetrahedron Letters, 1967, no. 8, pp. 673-675). Two total syntheses of sempervirol have been published and are reviewed in "The Total Synthesis of Natural Products", Volume 8, edited by John ApSimon, 1992, John Wiley & Sons Inc., New York. In the first synthesis (see schemes below), in which sempervirol and its positional isomer ferruginol were synthesized, Matsumoto and Usui prepared the cyclization substrate (compound 255, shown below) by a Wittig sequence starting from a-cyclocitral, compound 256. Treatment of compound 255 with aluminium chloride affords a 1:1 mixture of tricyclic products, 257, from one of which ferruginol, which is compound 206, may be prepared. In a similar fashion, the cyclisation substrate 258 can be and/or was prepared, and then can be and/or was converted into sempervirol, which is compound 252 shown below, by cyclisation with AICI3, and then conversion of the methoxy group to a (phenolic) hydroxy group using BBr3, BzCI, separation of isomers, and then LAH (lithium aluminium hydride, UAIH4).

ferruginol

252

sempervirol

In the second synthesis of sempervirol, by R. Caputo and L. Mangoni (shown in the scheme below), sempervirol was prepared from arylbutanoic acid 259 by standard annelation methods for the generation of an A/B-irons-fused abietane derivative. Cyclisation of 259 followed by methylation and ketone transposition yields compound 260. A sequence of Robinson annelation, methylation, reduction and ether cleavage yields sempervirol, which is compound 252 shown below.

sempervirol The above-mentioned syntheses of sempervirol and 7-keto-sempervirol can also be used as part of the synthesis of derivatives of sempervirol or 7-keto-sempervirol, which are within formulae (I) or (la) of the invention, in which 4 is isopropyl. Alternatively, the above-mentioned syntheses of sempervirol and 7-keto-sempervirol can be modified to synthesize compounds of formula (I) or (la) in which 4 is any Ci-C6alkyl (in particular Ci-C6alkyl t non-cyclised starting material (such as

, compound 259) in which the

aromatic isopropyl group is replaced with a Ci-C ₆ alkyl group other than isopropyl.

Therefore, in general terms a compound of formula (I) or (la) can be prepared as follows:

Additionally, or alternatively, a compound of formula (I) or (la) can be prepared as follows:

Chemical reaction of the two available starting materials (sempervirol and 7-ketosempervirol) can be used to generate various compounds of the invention (e.g. the compound of formula (I) or (la)). For example, the hydroxyl group of sempervirol and related compounds can be reacted with an alkyl halide in the presence of base to generate a Y substituent that is an alkoxy group. Reactions of this type are known as the Williams ether synthesis. In another example, the hydroxyl group of sempervirol and related compounds can be reacted with a carboxylic acid in the presence of a catalyst, for example an acid catalyst, or a coupling reagent, to generate an ester group. Alternative reactions for forming an ester are known in the art, for example an acid anhydride such as acetic anhydride may be reacted with an alcohol, optionally in the presence of a base such as pyridine and/or dimethylaminopyridine, to deliver an acetate ester. Acid chlorides can be used in similar ester forming reactions.

The hydroxyl group of sempervirol can be converted to other useful intermediates to give access to a range of other substituents. For example the hydroxyl group can be reacted with triflic anhydride to afford a triflate that can in turn be reacted with various amines under e.g. palladium catalysis to provide access to various amine structures. Once an amine is incorporated into the structure amides can be formed under standard conditions for example by reacting with a carboxylic acid in the presence of a coupling reagent such as EDCI. The triflate intermediate can also be converted to various aromatic halides under palladium catalysis to give access to the claimed aromatic halides. The aromatic halides, such as the bromine, can be hydrogenated under a hydrogen atmosphere to provide the compounds wherein Y is hydrogen, these compounds in turn can undergo electrophilic substitution to afford compounds featuring the claimed substituent either directly, or by standard functional group interconversion to provide access to the compounds of invention wherein the Y substituent is located at carbon-11 or carbon-14. For example a nitro group could be introduced by reaction with concentrated nitric acid, and this could then be reduced to the corresponding amine. The resultant amine could then be activated to nucleophilic substitution by diazotization to provide access to e.g. halide via the Sandmeyer or related reactions. Alternatively the amine could be converted to the amide under the conditions described above or could be alkylated by e.g. reductive amination. Further oxygen functions can be introduced by reaction of the compounds wherein Y is hydrogen with benzoyl peroxide and then performing a functional group interconversion under standard conditions. Alternatively an acetate can be installed (under e.g. Friedel Crafts conditions) then converted to the phenol with the Baeyer Villiger oxidation.

The carbonyl group of 7-ketosempervirol and related compounds is a useful handle for further synthetic modification. Reduction of the keto group can provide access to the hydroxyl group, reagents that are useful for this transformation include metal hydrides such as sodium borohydride. Selective reduction of the carbonyl group of 7-keto-sempervirol and related compounds can be achieved by using chiral reducing agents such as selectride or oxazaborolizidines, other chiral reducing agents can equally be applied. The alcohol products of the above reactions can be converted to alkoxy groups using the Williams ether synthesis. If an amine group is required as the X substituent the carbonyl group can be subjected to a reductive amination reaction, for example with a primary or secondary amine in a solvent such as dioxane in the presence of a reducing agent such as sodium cyanoborohydride. Alternatively, the carbonyl group can be reacted with an hydroxylamine, for example an O-alkyl hydroxylamine to form an oxime. The oxime group can be reduced to an amine with for example lithium aluminium hydride. The resultant amine could then be converted to an amide using the standard amide coupling reactions, for example using EDCI as described above.

The carbonyl group of 7-ketosempervirol and related compounds can be used to functionalise in the C-6 position. For example, oxidation, halogenation and alkylation are all possible under standard conditions. Once the functionality at C-6 has been installed the carbonyl group can be reduced to a methylene group as appropriate by using a reaction such as the Huang Minion reaction. The C-6 substituent can then be subjected to functional group interconversion to afford access to the desired substituent, for example the hydroxyl group could be alkylated or oxidised to give the alkoxy and keto- derivative respectively. The 6-keto derivative can then be subjected to the series of transformations described above for the 7-keto compound list.

Compositions, uses, dosage regimens, et al.

Furthermore, the present invention provides a pharmaceutical composition comprising a compound according to the disclosure optionally in combination with one or more pharmaceutically acceptable diluents or carriers.

Diluents and carriers may include those suitable for parenteral, oral, topical, mucosal and rectal administration.

The present invention also provides a process for preparing such a pharmaceutical composition (for example a pharmaceutical composition for parenteral, oral, topical, mucosal or rectal administration), said process comprising mixing the ingredients. As mentioned above, such compositions may be prepared e.g. for parenteral, subcutaneous, intramuscular, intravenous, intra-articular or peri-articular administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; for topical e.g. pulmonary or intranasal administration, particularly in the form of powders, nasal drops or aerosols and transdermal administration; for mucosal administration e.g. to buccal, sublingual or vaginal mucosa, and for rectal administration e.g. in the form of a suppository.

The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA., (1985). Formulations for parenteral administration may contain as excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Formulations for nasal administration may be solid and may contain excipients, for example, lactose or dextran, or may be aqueous or oily solutions for use in the form of nasal drops or metered sprays. For buccal administration typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like.

Compositions of compounds of formula (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih) or (li) that are suitable for oral administration may comprise one or more physiologically compatible carriers and/or excipients and may be in solid or liquid form. Tablets and capsules may be prepared with binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, microcrystalline cellulose or poly- vinylpyrollidone; fillers, such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine; lubricants, such as magnesium stearate, talc, polyethylene glycol, or silica; and surfactants, such as sodium lauryl sulfate and/or a tablet dispersant or disintingrant. Liquid compositions may contain conventional additives such as suspending agents, for example sorbitol syrup, methyl cellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or edible fats; emulsifying agents such as lecithin, or acacia; vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil; preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Liquid compositions may be encapsulated in, for example, gelatin to provide a unit dosage form.

Solid oral dosage forms include tablets, two-piece hard shell capsules and soft elastic gelatin (SEG) capsules. An alternative strategy is to design treatment paradigms in which the drug is dosed directly to the inflamed organ, that is, to exploit topical administration.

In topical therapy, one way in which efficacy can be achieved is by the use of a drug that has a sustained duration of action and is retained in the relevant organ, thereby minimizing the risk of systemic toxicity. Alternatively, in some cases, a formulation can be developed that generates a "reservoir" of the active drug which is available to sustain its desired effects. In one aspect of the disclosure the compounds of formula (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih) or (li) are particularly suitable for topical delivery.

A dry shell formulation typically comprises of about 40% to 60% w/w concentration of gelatin, about a 20% to 30% concentration of plasticizer (such as glycerin, sorbitol or propylene glycol) and about a 30% to 40% concentration of water. Other materials such as preservatives, dyes, opacifiers and flavours also may be present. The liquid fill material comprises a solid drug that has been dissolved, solubilized or dispersed (with suspending agents such as beeswax, hydrogenated castor oil or polyethylene glycol 4000) or a liquid drug in vehicles or combinations of vehicles such as mineral oil, vegetable oils, triglycerides, glycols, polyols and surface-active agents.

The compositions administered according to the present invention may also include various other ingredients, including, but not limited to, tonicity agents, buffers, surfactants, stabilizing polymer, preservatives, co-solvents and viscosity building agents. Preferred pharmaceutical compositions of the present invention include the inhibitor with a tonicity agent and a buffer. The pharmaceutical compositions of the present invention may further optionally include a surfactant and/or a palliative agent and/or a stabilizing polymer.

Surfactants may optionally be employed to deliver higher concentrations of the compounds for use. The surfactants function to solubilise the inhibitor and stabilise colloid dispersion, such as micellar solution, microemulsion, emulsion and suspension. Examples of surfactants which may optionally be used include polysorbate, poloxamer, polyosyl 40 stearate, polyoxyl castor oil, tyloxapol, triton, and sorbitan monolaurate. The medical practitioner, or other skilled person, will be able to determine a suitable dosage for the compounds of the invention, and hence the amount of the compound of the invention that should be included in any particular pharmaceutical formulation (whether in unit dosage form or otherwise). Certain compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih) or (li) described above are likely to have therapeutic activity. In a further aspect, the present invention provides a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih) or (li) described above for use as a medicament. Thus, in a further aspect, the present invention provides a compound as described herein for use in the treatment of one or more of the above mentioned conditions. In a further aspect, the present invention provides use of a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih) or (li) described above for the manufacture of a medicament for the treatment of one or more of the above mentioned conditions.

In a further aspect, the present invention provides a method of treatment of one or more of the above mentioned conditions which comprises administering to a subject an effective amount of a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih) or (li) described above or a pharmaceutical composition comprising the one of these compounds.

The word "treatment" is intended to embrace prophylaxis as well as therapeutic treatment. Treatment of conditions or disorders also embraces treatment of exacerbations thereof.

A compound of the disclosure may also be administered in combination with one or more other active ingredients e.g. active ingredients suitable for treating the above mentioned conditions or indeed other conditions.

In one aspect of the present invention a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih) or (li) may be used in combination with praziquantel (PZQ) and/or mefloquine, for treatment of schistosomiasis. This is of particular interest because compounds of formula (I), (la), (lb), (lc), (Id), (le), (If) _/ (Ig) _/ (Ih) ^or (li) h ^ave b ^een found to target juvenile schistosomula and adult fluke whereas PZQ displays activity towards the adult life stage. The combination of the compounds of the invention and the compounds for use of the invention with other anthelmintic agents can therefore provide a synergistic anthelmintic effect.

Hence another aspect of the invention provides a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih) or (li) in combination with one or more further active ingredients, for example one or more active ingredients described above. In one embodiment a suitable unit dose of (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (Ih) or (li) (e.g. oral unit dose) of 0.02 to 1000 mg or 0.05 to 1000 mg, for example 0.1 to 200 mg such as 1.0 to 200 mg, and/or for example 0.02 to 200 mg or 0.05 to 200 mg such as 0.05 to 45 mg or 0.1 to 45 mg, of the compound or the pharmaceutically acceptable salt of the invention (measured as the "free base" compound), may be used, for example in a pharmaceutical composition (e.g. in an oral pharmaceutical composition, and/or e.g. in a unit dose form) of the invention. In one embodiment, such a unit dose is for administration once a day, e.g. orally and/or to a mammal such as a human; alternatively such a unit dose may be for administration more than once a day, for example two or three times a day, e.g. orally and/or to a mammal such as a human. Such therapy may extend for a number of weeks, months or years.

Similarly, another aspect of the invention provides a combination product comprising:

(A) a compound of the present invention (i.e. a compound of formula (I), (la), (lb), (lc), (Id), (le), (If) _/ (lg) _/ (lh) ^or (li) _/ ^as defined above, or a pharmaceutically acceptable salt thereof); and (B) another therapeutic agent, wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically- acceptable adjuvant, diluent or carrier. In this aspect of the invention, the combination product may be either a single (combination) pharmaceutical formulation or a kit-of-parts.

Thus, this aspect of the invention encompasses a pharmaceutical formulation including a compound of the present invention and another therapeutic agent, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier (which formulation is hereinafter referred to as a "combined preparation").

It also encompasses a kit of parts comprising components:

(i) a pharmaceutical formulation including a compound of formula (I), (la), (lb), (lc), (Id), (le), (If), (Ig), (lh) or (li) in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier; and

(ii) a pharmaceutical formulation including another therapeutic agent, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, which components (i) and (ii) are each provided in a form that is suitable for administration in conjunction with the other. Component (i) of the kit of parts is thus component (A) above in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. Similarly, component (ii) is component (B) above in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. The aspects of the invention described herein (e.g. the above-mentioned compound, combinations, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, be longer acting than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, have a better pharmacokinetic and/or pharmacodynamic profile than, have more suitable solid state morphology than, have better stability than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise. In some aspects the compounds of the invention display activity against Schistosoma sp and/or Fasciola sp that are resistant to the prior art agents.

EXAMPLES

The present invention is illustrated by the following examples, which are not intended to limit the scope of the invention. Exemplary methods and biological activity

Isolation of 7-keto-sempervirol

The diterpenoid 7-keto-sempervirol was isolated from the temperate plant Lycium chinense as a secondary metabolite. Nine fractions were obtained from CH ₂ CI ₂ extracted plant material that had been subjected to Biotage™ 75 flash chromatography (silica gel, eluted with a step gradient of increasing polarity: n-hexane - EtOAc - MeOH). 7-keto-sempervirol was isolated from these fractions via reverse-phase preparative HPLC (Cis preparative column, eluted with a gradient- water: acetonitrile: 0.1% TFA in acetonitrile, with UV detection). The compound structure was then determined by UV ¹ H NMR, ¹³ C NMR and LC-MS analyses and also through direct comparison with the literature.

Schistosoma mansoni schistosomula culture

Schistosoma mansoni (NRMI, Puerto Rican strain) cercariae were shed from infected Biomphalaria glabrata snails (NMRI strain) by exposure to 2 hours of light in an artificially heated room (24°C). Collected cercariae were mechanically transformed into schistosomula as previously described (Colley DG, Wikel SK (1974) Schistosoma mansoni: simplified method for the production of schistosomules. Experimental Parasitology 35: 44-51) and resuspended in culture media containing DMEM (Dulbecco's Modified Eagle Medium, Sigma-Aldrich, UK) lacking phenol red, but containing 4500 mg/l glucose, 2 mM L-glutamine, 200 U/ml penicillin and 200 μg/ml streptomycin (all Sigma-Aldrich, UK). Schistosomula were then transferred to a black sided, flat-bottom (optically clear) 96-well microtiter plate (Star Lab, UK) at a density of 1000 parasites per 100 μΙ well. The plate was then incubated at 37°C and 5% CO ₂ for 24 hr to allow parasite equilibration. Adult Schistosoma mansoni culture

S. mansoni adult parasites were recovered by hepatic portal perfusion from TO (Tuck Ordinary) mice (Harlan Laboratories, UK) experimentally infected seven weeks earlier with 250 cercariae. Washed adult worms were cultured in DMEM containing phenol red, 4500 mg/l glucose, supplemented with 10% foetal calf serum, 2 mM L-glutamine, 200 U/ml penicillin, 200 μg/ml streptomycin (all Sigma- Aldrich, UK). Schistosome cultures were maintained at 37°C in a humidified atmosphere containing 5% C0 ₂ for 24 hr prior to further manipulations. For egg laying experiments, five adult worm pairs per ml of culture medium (48-well tissue culture plates) were cultivated as above for a total of 72hr (in the presence/absence of 7-keto-sempervirol). Eggs were enumerated after 72 hr and classified as normal (oval and containing a fully-formed lateral spine) or abnormal (lacking an oval shape and fully formed lateral spine).

Fasciola hepatica newly excysted juvenile (NEJ) culture

Fasciola hepatic metacercariae (Baldwin Aquatic, Inc., OR, USA) were transformed into newly excysted juveniles (NEJs) and equilibrated for 4 hr in Fasciola saline according to established methodologies (McGonigle et al. (2008) The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration. International Journal for Parasitology 38: 149-155). After equilibration, NEJs were distributed into black sided, flat-bottom (optically clear) 96- well microtiter plates (Star Lab) at a density of 100 parasites per ΙΟΟμί well and cultured at 37°C in a humidified atmosphere containing 5% C0 ₂ subject to further treatments.

Adult Fasciola hepatica culture

F. hepatica adult flukes were recovered from sheep livers (Ridgeway Research, UK), washed and cultured in media containing DMEM (lacking phenol red,) containing 4500 mg/l glucose (Sigma, UK). This was supplemented with 2.2 mM Ca(C ₂ H ₃ 02)2, 2.7 mM MgS0 ₄ , 61 mM glucose, 15 mM Hepes, 1 μΜ serotonin and 5 μg/ml gentamycin (all Sigma, UK). Adult fluke were placed in 12-well plates, 1 parasite per well in 1ml of culture media and cultivated for 48 hr at 37°C in a humidified atmosphere containing 5% CO2.

HepG2 cell culture and CellTitre-Glo assay

The human HepG2 cell line purchased from the European collection of cell cultures (ECACC 85011430) was grown to confluency in culture media containing EMEM (Eagle's Minimum Essential Medium, Sigma Aldrich, UK) supplemented with 1% bovine calf serum, 1% 200 mM L-glutamine, 1% nonessential amino acid solution and 1% penstrep (10000 U of penicillin and 10000 μg of streptomycin/ml, Invitrogen). Cells were distributed into black sided, flat-bottom (optically clear) 96-well microtiter plates (Star Lab, UK) at 50 μΙ/well (lxlO ⁵ cells/ well). The plate was equilibrated in a humidified atmosphere containing 5% C0 ₂ and 37°C for 2 hrs and then test compounds were added to relevant wells to create a total well volume of 100 μΙ/well. Negative and positive controls were included within each plate to compensate for gain set by the luminescent plate reader (BMG Labtech Polarstar Omega Plate Reader). Positive controls composed of cells incubated in the same concentration (1% v/v) of dimethyl sulfoxide (DMSO) that was used within the experimental (compound treated) wells. Cells incubated with 1% v/v Triton X-100 (TX-100) (Sigma-Aldrich) were utilised as the negative control for this assay (Dayeh VR et al (2004) Evaluating the toxicity of Triton X-100 to protozoan, fish, and mammalian cells using fluorescent dyes as indicators of cell viability. Ecotoxicology and Environmental Safety 57: 375-382). Tissue culture plates were returned to a humidified atmosphere containing 5% C0 ₂ and 37°C for a further 24 hrs.

The CellTitre-Glo™ reagents were prepared according to the manufacturer's instructions (Promega, UK). Tissue culture plates were equilibrated to RT for 30 minutes before CellTitre-Glo™ reagents were distributed into each plate well at 100 μΙ/well (total volume 200 μΙ/well) and mixed on an orbital shaker for 2 minutes. Cells were then stabilised for 10 minutes at RT and bubbles that may have formed were removed using a sterilised needle. After this time luminescent signal was read utilising the BMG Labtech Polarstar Omega Plate Reader and exported into Microsoft Excel™ for further manipulation and conversion into percentage viability. Helminth Fluorescent Bioassay (HFB)

A HFB was utilised to objectively determine the viability of schistosomula and NEJ parasites co- cultured in the presence of 7-keto-sempervirol, auranofin (70 μΜ), DMSO (1% v/v/) or media only. . A slight modification of the HFB was applied to NEJs and included assaying only 100 parasites/well (as opposed to 1000 schistosomula/well). A total of 100 μΙ of test substance (varying concentrations) was added to each well containing 100 μΙ of suspended parasites and cultured for 24 hr in a humidified atmosphere at 37°C before the HFB was performed.

Phenotypic Measurements

The in vitro activity of 7-keto-sempervirol on S. mansoni adult worms and F. hepatica NEJs was assessed by measuring motility disturbances and morphological variations in comparison to an appropriate untreated control (DMSO, 1% v/v). The scoring matrix used to assess S. mansoni adult worms viability was based on the standard operating procedure for compound screening at the Special Programme for Research and Training in Tropical Diseases, World Health Organization, WHO-TDR. Motility was numerically scored from 0-4 with 0 being total absence of all motility, 1 indicating absence of motility other than gut peristalsis, 2 representing minimal activity such as occasional head and tail movement, 3 demonstrating slow activity and 4 signifying normal activity. Morphological descriptors of the parasite were also recorded during phenotypic assessment and included 'knots' developing along the normally cylindrical body line of the adult worms and the sloughing, blebbing or turbercle swelling of the tegument. Scoring matrix assessment of the worms was performed at 24, 48 and 72 hr post treatment.

NEJ phenotyping (a supportive metric of the HFB) was performed using values derived from both movement indices and morphologic formats. The movement score ranges from 1-5 with 1 representing good/normal movement and 5 representing a complete absence of movement. The morphologic score ranges from 1-6 with 1 representing a good/normal phenotype and 6 signifying a severely dissolved/granulated parasite. This scoring matrix assessment (values derived from summation of both movement and morphology metrics) was conducted at 24 hr post treatment. Scanning electron, laser confocal scanning and high content imaging microscopy

Prior to scanning electron microscopy (SEM) analysis, adult schistosomes and liver flukes were first fixed in 2.5% (w/v) gluteraldehyde in PBS for 24 hr at 22-24°C. After fixation, worms were washed 3 times in 1 X phosphate buffered saline (PBS, pH 7.5) and stored in the same buffer at 4°C until use. Fixed parasite material was subsequently washed 2 times in double distilled water and dehydrated in ascending acetone concentrations (30, 50, 70, 80, 90, 95, and 100%) for 15 minutes each. Dehydrated worms were then critically dried (Polaron Critical Point Dryer E3000) for 1 hr in 100% acetone (critical point of CO2 is 7.38 mPa/ or 72.9 atm at a critical temperature of 31°C). Critically dried worms were mounted on aluminium stubs, sputter coated with platinum/palladium and observed under a Hitachi S-4700 field emission scanning electron microscope. For laser confocal scanning microscopy (LCSM), adult schistosomes were first fixed in a solution containing 2% (v/v) acetic acid, 25% (v/v) formalin, 48% (v/v) ethanol and 25% (v/v) H2O at room temperature for 24 hr. After fixation, worms were stained with Langeron's Carmine. Worms were then mounted on glass microscope slides in DPX (distyrene, plasticiser, xylene) and observed using a Leica TCS SP5II laser scanning confocal microscope, equipped with a 40X oil immersion objective and a 488 nm Argon laser and a 561nm laser.

S. mansoni eggs laid by adult females after 72 hr in vitro cultures (+/- 6-keto-sempervirol) were fixed in a 10% formaldehyde solution to prepare the biological tissue for microscopy. Fixed eggs were then phenotypically assessed by an ImageXpress™ micro XL High Content Imager. Fluorescent images were obtained using a FITC filter (40x magnification). Schistosomula subjected to the HFB were visualised on the lmageXpres™s micro XL High Content Imager using FITC (to identify fluorescein diacetate positive parasites) and T ITC (to identify propidium iodide positive parasites) filters (10X magnification).

Statistical analysis

A One Way ANOVA was utilised to identify any significant differences between treatment groups (>n=2) followed by post hoc testing with the Tukey's test to identify significantly different means (means that do not share a letter are significantly different). The Student's t-test was utilised to determine significant differences between treatment groups (n=2).

Results Anti-schistosomal 7-keto-sempervirol properties

The potential anthelmintic activity of 7-keto-sempervirol, a diterpenoid purified from Lycium chinense (Fig. 1) was assessed. Employing the HFB described above, a titration series of 7-keto-sempervirol (100μΜ-1.575 μΜ) was first used to assess the ability of this diterpenoid to affect schistosomula viability during in vitro co-culture (Fig. 2). At high 7-keto-sempervirol concentrations (100 - 25 μΜ), almost all of the schistosomula were killed or severely affected as indicated by low percent viability values and fluorescent microscopic images of individual parasites (propidium iodide positive parasites > fluorescein acetate positive parasites) (Fig. 2). Lower 7-keto-sempervirol concentrations (between 6.25 μΜ - 1.575 μΜ) had very little effect on schistosomula viability and phenotype when compared to the DMSO (untreated) control parasites (Fig. 2). Based on these titration experiments, an LD50 of 19.1 μΜ was calculated for 7-keto-sempervirol on the schistosomula lifecycle stage. A parallel set of titration experiments was additionally performed with the human HepG2 cell line (data not shown) and demonstrated that 7-keto-sempervirol was minimally toxic after 24 hr co-cultivation. 7-Keto-sempervirol's ability to affect adult schistosome motility, surface-tegument morphology and egg development was measured using both WHO-adopted indices and microscopic measures. Here, 7-keto-semperivol displayed a significant effect on both male and female worm motility at the highest concentration used in this study (100 μΜ) at 24, and 48 hr post treatment compared to the untreated control group (Fig. 3). For both genders, there was no significant difference between 48 hr and 72 hr treatments at this concentration (Fig. 3). Interestingly, female worms (unlike males) displayed a 7- keto-semperivol-induced hyperactivity at 24 hr post-treatment (Fig.3). There were motility and phenotypic discrepancies observed for some individuals cultured in the presence of 10 μΜ 7-keto- sempervirol, but these differences were not significantly different compared to the untreated control group (data not shown). Scanning electron microscopy (SEM) of adult male worms co-cultured in the presence of 7-keto-sempervirol (100 μΜ for 72 hr) further revealed tubercle swelling, spine loss, spine shortening and surface holes throughout the tegument (Fig. 4).

Laser scanning confocal microscopy (LSCM) of adult females cultured in the presence of 7-keto- sempervirol (100 μΜ for 24 hr) indicated the presence of irregularly shaped in utero eggs (Fig. 5). When compared to control eggs (parasites treated with 1% v/v DMSO, Fig. 5A), these abnormal eggs lacked regular autofluorescence as well as fully formed eggshells and were missing the characteristic lateral spines indicative of the species (Fig. 5B). Due to these phenotypic deficiencies in egg development, the effect that 7-keto-sempervirol had on in vitro schistosome oviposition was also assessed (Fig. 6). Here, 7-keto-sempervirol induced a dose-dependent (ΙΟΟμΜ > 10μΜ) ability to inhibit the deposition of phenotypically normal schistosome eggs with a complete lack of oviposition observed in wells containing the highest level of diterpenoid (100 μΜ) (Fig. 6A). When compared to control wells (schistosomes co-cultured with 1% v/v DMSO), eggs deposited in wells containing 7-keto- sempervirol (10 μΜ) displayed a range of abnormal phenotypes (Fig. 6B) similar to those observed in utero (Fig. 5B) These phenotypes included non-oval shapes, lack of lateral spines and patchy autofluorescence.

Anti-Fasciola 7-keto-sempervirol activities

To assess 7-keto-sempervirol's activity on F. hepatica NEJs, two complementary methodologies were employed (Fig. 7). The first methodology, using motility and phenotypic metrics indicated that 7-keto- sempervirol induced a negative dose-dependent effect on NEJ movement and viability (Fig. 7A). This finding was supported by fluorescent microscopy of NEJs co-stained with the discriminatory viability dyes FDA and PI (Fig. 7A). The second methodology, using the HFB method for determining NEJ viability, confirmed this dose-dependent effect and established an LD50 of 17.7 μΜ for 7-keto- sempervirol against F. hepatica NEJs (Fig. 7B). When compared to the mammalian HepG2 cell line (data not shown), 7-keto-sempervirol displayed a selective anti-NEJ effect. To identify whether 7-keto- sempervirol also affected the surface integrity of F. hepatica adults, similar to S. mansoni (Fig. 4), SEM analyses were performed on adult liver flukes co-cultured with this diterpenoid (Fig. 8). Here, in comparison to control flukes incubated with 1% (v/v) DMSO, prolonged (48 hr) exposure to 7-keto- sempervirol (50 μΜ) induced substantial spine shortening and spine loss that was most apparent on the ventral side of the organism.