WO2017072523A1 - Treatment of parasitic disease - Google Patents

Abstract

The present invention provides compounds such as delamanid for use in the treatment of a parasitic disease caused by or associated with parasites selected from -the Order Kinetoplastida consisting of species belonging to the genera Leishmania and Trypanosoma.

Description

TREATMENT OF PARASITIC DISEASE

The present invention relates to products, compositions, methods and uses which are useful in the prevention, alleviation or treatment of diseases caused by parasites selected from the Order Kinetoplastida consisting of species belonging to the genera ^{Leishmanra and} Trypanosomes. In particular the products, compositions and uses of the present invention are useful in the treatment of visceral leishmaniasis, mucocutaneous leishmaniasis, cutaneous leishmaniasis, African trypanosomiasis and Chagas' disease. The products and compositions include one or more 2,3-dihydro-6-nitroimidazo[2,1-b]oxazole compounds. BACKGROUND TO THE INVENTION

The present teachings relate to a drug therapy for the treatment of a patient, such as a human or a dog, with a nitroaromatic compound. There is an urgent requirement for safe, cost effective drugs for the treatment of kinetoplastid diseases such as visceral leishmaniasis, cutaneous leishmaniasis and Chagas' disease. Visceral leishmaniasis results from infection with the protozoan parasites Leishmania donovani or L. infantum. There are approximately 200,000-400.000 cases of visceral leishmaniasis per year, with the vast majority of cases in South America, East Africa and the Indian subcontinent (Bangladesh, Brazil, Ethiopia, India, South Sudan and Sudan). Cutaneous leishmaniasis is caused by many species of leishmania, including L. major, L. tropica, L. aethiopica and L. mexicana. The majority of cutaneous Leishmaniasis cases occur in Afghanistan, Algeria, Brazil, Colombia, the Islamic Republic of Iran, Pakistan, Peru, Saudi Arabia and the Syrian Arab Republic. Cutaneous leishmaniasis is characterized by disfiguring skin lesions that are usually self-healing within months or years, but can become chronic. It is estimated that there are 700,000-1,300,000 new cases each year. Chagas' disease is caused by Trypanosoma cruzi and is endemic to 21 countries in Latin America. Approximately 8 million people carry the parasite, which is characterised by acute, indeterminate and chronic phases of the infection. Of those that survive the acute phase, about 30% will develop to the chronic phase, characterised by heart disease or enlargement and dysfunction of the oesophagus and colon. Nitroaromatic drugs such as delamanid are known anti-mycobacterial agents. Delamanid has recently been approved for the treatment of multidrug resistant TB in Europe. STATEMENT OF INVENTION

According to a first aspect of the present invention, there is provided a method of treating a parasitic disease comprising the administration in a therapeutically effective amount of one or more compounds of Formula I. In various embodiments, the parasitic disease is caused by or associated with parasites selected from the Order Kinetoplastida consisting of species b_{elonging to the genera Leishmania} ^{and Trypanosoma. Particular mention may be made of} Leishmania donovani, L. infantum, L. niajor, L. tropica, L. aethiopica, L. mexicana ^and Trypanosoma cruzi. The methods can include treating the parasitic disease, or alleviating one or more symptoms thereof in a patient. According to a further aspect of the present invention, there is provided a method of treating a disease selected from the groups consisting of. visceral leishmaniasis, mucocutaneous leishmaniasis, cutaneous leishmaniasis. African trypanosomiasis and Chagas' disease, or alleviating one or more of the symptoms associated therewith comprising the administration in a therapeutically effective amount of one or more compounds of Formula I. According to a further aspect of the present invention, there is provided one or more compounds of Formula I for use in the treatment of a parasitic disease caused by or associated with parasites selected from the Order Kinetoplastida consisting of specieselonging to the genera Leishmania and Trypanosoma. The uses can include treating the parasitic disease, or alleviating one or more symptoms thereof in a patient. According to a further aspect of the present invention, there is provided one or more compounds of Formula I for use in the treatment of a disease selected from the groups consisting of visceral leishmaniasis, mucocutaneous leishmaniasis. African trypanosomiasis, cutaneous leishmaniasis and Chagas' disease, or alleviating one or more of the symptoms associated therewith. In yet another aspect, the present teachings include a kit, where the kit includes one or more of the compounds of Formula I, and instructions for use thereof The foregoing as well as other features and advantages of the present teachings will be more fully understood from the following description, Examples and Claims. DETAILED DESCRIPTION

The present teachings can enable the treatment of parasitic diseases such as visceral leishmaniasis, cutaneous leishmaniasis, mucocutaneous leishmaniasis, African

trypanosomiasis and Chagas' disease through the administration of compounds of Formula I, in particular 2-Methyl-6-nitro-2-[(4-{414-(trifluoromethoxy)phenoxy]-1- piperidinyllphenoxy)methyl]-2,3-dihydroimidazo[2,1-b][1,3]oxazole, (generic name, delamanid, also known during development as OPC-67683), sold under the trade name D_{eltyba. Particular mention may be made of the R} ^{enantiomer of delamanid, although the use} of the S enantiomer is also envisaged. Throughout the Application, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present teachings also consist essentially of, or consist of, the recited components, and that the processes of the present teachings also consist essentially of, or consist of, the recited process steps. In the Application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. Further, it should be understood that elements and/or features of a composition, an apparatus, or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present teachings, whether explicit or implicit herein. The use of the terms "include," "includes", "including," "have," "has," or "having" should be generally understood as open-ended and non-limiting unless specifically stated otherwise. The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise. In addition, where the use of the term "about" is before a quantitative value, the present teachings also include the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present teachings remain operable. Moreover, two or more steps or actions may be conducted simultaneously. As used herein, a "compound" (including a specifically named compound e.g., delamanid) refers to the compound itself and its pharmaceutically acceptable salts and hydrates unless otherwise understood from the context of the description or expressly limited to one particular form of the compound, i.e., the compound itself, or a pharmaceutically acceptable salt or hydrate thereof Compounds for administration in accordance with the methods and uses of the present invention have the formula:

w_{here R.1 represents a hydrogen atom or a CI to} ^{C6 alkyl group, generally a CI to C3 alkyl} group, typically methyl or ethyl, suitably methyl. Generally R^{I represents a C: to C6 alkyl} group and the S- or the R- enantiomer is used, or a racemic mixture is used. Suitably the R- enantiomer is of particular interest. N represents an integer of 0 to 6, generally 0 to 3, suitably 1 or 2, more suitably 1. R2 represents any group of the formulae (A) to (G) as described below.

wherein R3 represents any groups of the following (1) to (6): ₁₎ a phenoxy group (wherein the phenyl ring may, optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen- substituted CI-6 alkyl group and an optionally halogen-substituted ^C1-6 alkoxy group); 2) a phenyl Ch6 alkoxy group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted C1-6 alkyl group and an optionally halogen-substituted CI-6 alkoxy group);

3) NR4R5 group, wherein R4 represents a hydrogen atom or C1.6 alkyl group and R⁵ represents a phenyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting a halogen atom, an optionally h_{alogen-substituted} C1-6 alkyl group and an optionally halogen-substituted CI-6 alkoxy group);

4_{) a phenyl} C1-6 alkyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted C1-6 alkyl group and an optionally halogen-substituted C1-6 alkoxy group);

5_{) a phenoxy} Ci_6 alkyl group (wherein the phenyl rim may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted CI-6 alkyl group and an optionally halogen-substituted C1-6 alkoxy group) ; and

6₎ a benzofuryl C1_6 alkyl group (wherein the benzofuran ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted C1_6 alkyl group and an optionally halogen-substituted Ci_6 alkoxy group); The group represented by the general formula (B) is:

w_{herein R6} represents a phenyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted CI.6 alkyl group and an optionally halogen-substituted C1-6 alkoxy group). The group represented by the general formula (C) is:

wherein R7 represents a phenyl C2-M alkenyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted C1.6 alkyl group and an optionally halogen-substituted CI-6 alkoxy group) or a biphenyl C1.6 alkyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted CI-6 alkyl group and an optionally halogen-substituted C1.6 alkoxy group). The group represented by the general formula (D) is:

w_{herein R8 represents a phenyl C} ^{1.6 alkyl group (wherein the phenyl ring may optionally be} substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted C1_6 alkyl group and an optionally halogen-substituted C1_6 alkoxy group). The group represented by the general formula (E) is:

wherein le is as described above. The group represented by the general formula (F) is:

wherein R6 is as described above. The group represented by the general formula (G) is:

wherein 128 is as described above. Generally R2 represents a group according to formula (A). S_{uitably, R}3 represents group (1) or group (2). Suitably R3 represents a phenoxy aroup, generally a substituted phenoxy group. Typically the phenoxy group is substituted at one or two positions, generally one position. Suitably the phenoxy group is substituted with an optionally halogen-substituted C1.6 alkoxy group, generally an optionally halogen-substituted C1.3 alkoxy group, typically optionally halogen- substituted C1-2 alkoxy group. The C1-6 alkoxy group is generally substituted with one to three halogen groups, typically two or three halogen groups, suitably three halogen groups. The, or each halogen group may be independently selected from the group consisting of chlorine and fluorine. The C1_6 alkoxy group may suitably be substituted with one or more fluorine groups, generally three fluorine groups. Alternatively, R3 may represent a phenyl C1-6 alkoxy group generally a substituted phenyl group. Typically the phenyl group is substituted at one or two positions, generally one position. Suitably the phenyl group is substituted with an optionally halogen-substituted C1_6 alkoxy group, generally an optionally halogen-substituted C1-3 ^{alkoxy group, typically} optionally halogen-substituted C1.2 alkoxy group. The C1.6 alkoxy group is generally substituted with one to three halogen groups, typically two or three halogen groups, suitably three halogen groups. The, or each halogen group may be independently selected from the group consisting of chlorine and fluorine. The Ci.6 alkoxy group may suitably be substituted with one or more fluorine groups, generally three fluorine groups. According to one embodiment, R3 represents a phenoxy group substituted with a halogen- substituted CI,6 alkoxy group, generally substituted with—0CF3. According to one embodiment, the compound of Formula I has the structure of delamanid. As used herein, "delamanid" refers to a compound having the formula:

and pharmaceutically acceptable salts and hydrates thereof Delamanid can be referred to by other names, for example (2R)-2-Methyl-6-nitro-2-[(4-{4-[4- (trifluoromethoxy)phenoxy]-1 -piperidinyl } phenoxy)methy1]-2,3 -dihydroimidazo [2,1 - b][1,3]oxazole, or its trade name in Europe, Deltyba. Delamanid is also known in some literature as OPC-67683. The methods and uses of the present invention generally include the administration of the free base of delamanid. The methods and uses of the present invention may comprise the administration of delamanid in either enantiomeric form, or as a racemate. According to one embodiment, the methods and uses of the present invention comprise the administration of the R-enantiomer of delamanid. However, administration of the S- enantiomer is also of interest. It has been found that the R-enantiomer has an efficacy of at least double (generally up to ten times greater) than that of the S-enantiomer in treating parasitic disease caused by or associated with parasites selected from the Order Kinetoplastida consisting of species belonging to the genera Leishmania and Trypanosoma The compounds of Formula I have the potential to be repurposed as a short course oral therapy for the treatment of kinetoplastid diseases, in particular visceral leishmaniasis, cutaneous leishmaniasis and Chagas' disease. As used herein, "therapeutic combination" refers to a combination of one or more active drug substances, i.e., compounds having a therapeutic utility. Typically, each such compound in the therapeutic combinations of the present teachings can be present in a pharmaceutical composition comprising that compound and a pharmaceutically acceptable carrier. The compounds in a therapeutic combination of the present teachings can be administered simultaneously, together or separately, or separately at different times, as part of a regimen. The present teachings also provide pharmaceutical compositions that include at least one compound of Formula 1 as described herein, in particular delamanid, or a therapeutic combination, and one or more pharmaceutically acceptable carriers, excipients, or diluents. Examples of such carriers are well known to those skilled in the art and can be prepared in accordance with acceptable pharmaceutical procedures, such as, for example, those described _in Remington: The Science and Practice of Pharmacy, 20th edition, ed. Alfonso R. Gennaro, Lippincott Williams & Wilkins, Baltimore, MD (2000), the entire disclosure of which is incorporated by reference herein for all purposes. As used herein, "pharmaceutically acceptable" refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological perspective and does not adversely interact with the active ingredient. Accordingly, pharmaceutically acceptable carriers are those that are compatible with the other ingredients in the formulation and are biologically acceptable. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions. The pharmaceutical formulations of the present teachings can include other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, and osmo-regulators. Because the present teachings provide pharmaceutical formulations and their intended use is with patients such as humans, each of the ingredients or compounds of a pharmaceutical formulation described herein can be a pharmaceutically acceptable ingredient or compound. As used herein. "therapeutically effective" refers to a substance or an amount that elicits a desirable biological activity or effect. Method of Treatment

According to a first aspect of the present invention, there is provided a method of treating a parasitic disease comprising the administration in a therapeutically effective amount of one or more compounds of Formula I wherein the parasitic disease is caused by or associated with parasites selected from the Order Kinetoplastida consisting of species belonging to the genera _{Leishmania and Trypanosoma, in particular} ^{Leishmania donovani, L. infantum, L. major and} _{Trypanosoma cruzi.} The methods can include treating the parasitic disease, or alleviating one or more symptoms thereof in a patient. Generally the method includes the administration in a therapeutically effective amount of delamanid. According to a further aspect of the present invention, there is provided a method of treating a kinetoplasmid disease, in particular a disease selected from the groups consisting of visceral leishmaniasis, cutaneous leishmaniasis and Chagas' disease, or alleviating one or more of the symptoms associated therewith comprising the administration in a therapeutically effective amount of one or more compounds of Formula I. Generally the method includes the administration in a therapeutically effective amount of delamanid. _{The parasitic disease is suitably caused by} ^{Leishmania species, and is typically visceral} leishmaniasis, mucocutaneous leishmaniasis or cutaneous leishmaniasis, generally visceral lei shmaniasi s. There is an urgent requirement for safe, oral and cost-effective drugs for the treatment of visceral leishmaniasis (VL). We report that delamanid (OPC-67683), an approved drug for multi-drug resistant tuberculosis, is a potent inhibitor of Leishmania donovani both in vitro and in vivo. Twice-daily oral dosing of delamanid at 1 mg kg-I for 10 days resulted in sterile cures in a mouse model of VL, with 86% suppression of parasite liver burden after 5-days. Blood delamanid levels were consistent with those achieved in tuberculosis patients. Treatment with higher doses revealed a U-shaped (hormetic) dose-response curve with >90% suppression with 30 mg kg-1 (5 days) or 10 mg kg-1 (10 days). Alternatively, the parasitic disease may be caused by Trypanosoma species and is typically Chagas' disease. Compounds and therapeutic combinations of the present teachings can be useful for treating a pathological condition or disorder in a patient, for example, a human. As used herein, "treating" refers to partially or completely alleviating and/or ameliorating the condition and/or symptoms thereof The present teachings accordingly include a method of providing to a patient a pharmaceutical composition that includes a compound or therapeutic combination of the present teachings in combination or association with a pharmaceutically acceptable carrier. Compounds and therapeutic combinations of the present teachings can be administered alone or in combination with other therapeutically effective compounds or therapies for the treatment of a pathological condition or disorder. As used herein, "patient" refers to a mammal, such as a human although mention may also be made of dogs, cats, horses and camels. The one or more compound of Formula I may be administered in the form of a pharmaceutical composition. Generally the method includes the administration of a pharmaceutical composition comprising delamanid. When administered for the treatment or inhibition of a particular disease state or disorder, it is understood that an effective dosage can vary depending upon many factors such as the particular compound or therapeutic combination utilized, the mode of administration, and severity of the condition being treated, as well as the various physical factors related to the individual being treated. In therapeutic applications, a compound or therapeutic combination of the present teachings can be provided to a patient already suffering from a disease, for example, bacterial infection, in an amount sufficient to cure or at least paftially ameliorate the: symptoms of the disease and its complications. The dosage to be used in the treatment of a specific individual typically must be subjectively determined by the attending physician. The variables involved include the specific condition and its state as well as the size, age and response pattern of the patient. The duration of the course of treatment depends on the condition to be treated and its severity. The method of the present invention may be undertaken whenever required. The compounds having therapeutic activity may be administered at least once daily, generally at least three times daily, and typically up to five times daily. The compound of Formula I is generally administered at a once to five times daily dose of from about 0.5 to about 50 mg/kg, generally a twice daily dose of about 0.5 to about 50 mg/kg. Generally the course of treatment is 3 to 30 days. The compounds of Formula I tend to act within a course of treatment of five or ten days in greatly reducing the parasite burden on the patient. A significant suppression of the parasites is generally observed after 5 days where an efficacious dose has been administered. However, the method of treatment is generally continued for 20 to 30 days to ensure that the parasite population is not reestablished. Surprisingly it has been found that lower doses of compounds of Formula I, in particular delamanid were more efficacious in suppressing the parasite burden than higher doses. In particular twice daily doses of 0.5 to 2 mg/kg were associated with a mean suppression of parasites of over 80% after 5 days and even higher after 10 days. The methods and uses of the present invention generally involve the compound(s) of Formula I being administered at a twice daily dose of 0.5 to 2 mg/kg for at least 5 days. In contrast twice daily doses of 3 to 8 mg/kg were associated with a mean suppression of parasites of 15 to 50% after 5 days, and this remained approximately equivalent after 10 days. The efficacy of the compounds of Formula I in suppressing the parasite burden was observed to increase to over 80% once again where the dose was further increased to twice daily doses of over 10 mg/kg for 10 days, or twice daily doses of 20 mg/kg to 50 mg/kg for 5 days. The methods and uses of the present invention generally involve the compound(s) of Formula I being administered at a twice daily dose of 10 to 15 mg/kg for at least 10 days. The methods and uses of the present invention generally involve the compound(s) of Formula I being administered at a twice daily dose of 20 to 50 mg/kg for at least 5 days. The compounds of Formula I, in particular delamanid is associated with a U shaped (hormetic) dose response curve, with over 90% parasite suppression with twice daily doses of 30 mg/kg after 5 days, and with twice daily doses of 10 mg/kg after 10 days. This hormetic dose-response may be related to the finding that compounds of Formula I (in particular delamanid) is rapidly metabolized by parasites via an enzyme. Suitably the compounds of Formula I are in a form suitable for oral administration. The composition generally comprises from at least 0.1% to no more than 20 wt. % compound of Formula I. According to one embodiment, a unit dose of the composition of the present invention comprises 50 to 500 mg, generally 100 to 200 mg compound of Formula I. According to one embodiment, a unit dose of the composition of the present invention comprises 50 to 500 mg, generally 100 to 200 mg delamanid. The form and physical properties of the composition depend on the method of administration. When the compounds of Formula I are prepared for oral administration, they are generally combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. For oral administration, the compounds of Formula I may be in the form of a tablet, capsule or liquid. The compounds of Formula I may be present as a powder, a granular formation, a solution, a suspension, an emulsion or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum. The compounds of Formula I may also be presented as a bolus, electuary or paste. Orally administered compounds of Formula I can also be formulated for sustained release, e.g., the compounds of Formula I can be coated, micro-encapsulated, or otherwise placed within a sustained delivery device. The total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation. Pharmaceutical formulations comprising the compounds of Formula I can be prepared by procedures known in the art using well-known and readily available ingredients. For example, the compounds of Formula I can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives. Binding agents can also be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates. gelatine, and polyvinylpyrrolidone. Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium carbonate and sodium bicarbonate. Agents for retarding dissolution can also be included such as paraffin. Resorption accelerators such as quaternary ammonium compounds can also be included. Surface active agents such as cetyl alcohol and glycerol monostearate can be included. Adsorptive carriers such as kaolin and bentonite can be added. Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols can also be included. Preservatives may also be added. The compositions of the invention can also comprise thickening agents such as cellulose - and/or cellulose derivatives. They may also include gums such as xanthan, guar or carbo gum or gum arable, or alternatively polyethylene glycols, bentones and montmorillonites, and the like. For example, tablets or caplets comprising the compounds of Formula I can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Suitable buffering agents may also include acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt. Caplets and tablets can also include inactive ingredients such as cellulose, pregelatinized starch, silicon dioxide, hydroxyl propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate, and the like. Hard or soft gelatine capsules comprising one or more compounds of Formula I may comprise inactive ingredients such as gelatine, microcrystalline cellulose, sodium lauryl sulphate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric-coated caplets or tablets comprising one or more compounds of Formula I are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum. The compounds of Formula I can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous, intraperitoneal or intravenous routes. The pharmaceutical formulations of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension or salve. Liquid carriers can be used in preparing solutions, suspensions, and emulsions. A compound described herein can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, or a mixture of both, or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, and osmo-regulators. Examples of liquid carriers for parenteral administration include water, alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the carrier can be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are used in sterile liquid form compositions for parenteral administration. Thus, the compounds of Formula I may be formulated for parenteral administration (e.g. by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers. As noted above, preservatives can be added to help maintain the shelve life of the dosage form. The compounds of Formula I and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may comprise formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the compounds of Formula I and other ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water before use. These formulations can include pharmaceutically acceptable carriers, vehicles and adjuvants that are well-known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, acetic acid, ethanol, isopropyl alcohol, dimethyl sulphoxide, glycol ethers such as the products sold under the name "Dowanol", polyglycols and polyethylene glycols, Cl - C4 alkyl esters of short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol", isopropyl mytrisate, animal, mineral and vegetable oils and polysiloxanes. According to one embodiment, the pharmaceutical formulations of the invention can also take the form of a solvent or diluent comprising the compounds of Formula I. Solvents or diluents may include acid solutions, dimethylsulphone, N-(2- mercaptopropionyl) glycine, 2- n-nony1-1,3-dioxolane and ethyl alcohol. Suitably the solvent/diluent is an acidic solvent, for example, acetic acid, citric acid, boric acid, lactic acid, propionic acid, phosphoric acid, benzoic acid, butyric acid, malic acid, malonic acid, oxalic acid, succinic acid or tartaric acid. According to one embodiment, the method may include the administration of a therapeutic combination. Generally the therapeutic combination includes delamanid and one or more additional compounds of therapeutic utility. Each compound of therapeutic utility of the therapeutic combination may be administered simultaneously, separately or sequentially. According to a further embodiment, the pharmaceutical formulations of the invention are in the form of a composition suitable for topical administration, in particular a liquid, cream, ointment or gel. In particular such topical formulations may be applied directly to a skin lesion. The therapeutic combination may include more than one compound of Formula I. Generally the therapeutic combination includes delamanid and a second or third compound of Formula I.

Alternatively the therapeutic combination may include a compound of Formula I and a second or third compound having therapeutic utility falling outside the scope of Formula I. The ratio of compound of Formula I to other compound having therapeutic activity may be 1:4 to 4:1 The abovementioned compounds having therapeutic utility may be administered as free or fixed combinations. Free combinations may be provided as combination packages containing all the active agents in free combinations. The active agents of the present invention may be administered by any suitable route known to those skilled in the art, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment or prevention intended. According to one embodiment, the compounds having therapeutic activity may be administered via different routes. For instance, the compound of Formula I may be administered orally and the other compound(s) having therapeutic activity may be administered via intravenous injection. Kit of Parts

According to a further aspect of the present invention, there is provided a kit of parts for use in the treatment or alleviation of a parasitic disease, said kit of parts comprising one or more compounds of Formula I (generally in the form of a composition) and instructions for use. The kit of parts may comprise the active agents in dosage units containing a particular amount of the active agent. The dosage units may comprise one or more active agents. Generally the kit includes instructions for use, for example the nature of administration. Examples

The present invention will now be described by way of Example only with reference to the accompanying Figures. Figure 1 provides a graph showing the effects of delamanid drug treatment on the parasite burden of mice infected with L. donovani at different dosages over 5 and 10 days. Figure 2 provides a graph detailing ED50 for delamanid in a mouse model of VL. Figure 3 provides four graphs showing the phannacokinetic behaviour of delamanid in infected mice after different lengths of treatment.

Figure 4 provides four graphs showing the effects of delamanid on L. donovani promastigotes.

Figure 5 provides two graphs showing the different modes of action between delamanid and nifurtimox.

Figure 6 provides four graphs showing delamanid metabolism in L. donovani promastigotes.

Figure 7 provides two graphs showing the mean suppression of parasite burden as a _{function of}

for the penultimate dose and extrapolated AUC(0-24h) for the last day of the 5- and 10-day treatment regimens, respectively.

Figure 8A provides a Schematic representation of the generation of a (R)-PA-824 _{resistant cell line in} Leishman ct donovani. Each passage of cells in culture (black circles) is indicated. Figure 8B Dose-response curves for WT (closed circles) and RES III resistant cells (open circles). The curves are the non-linear regression fits using a two-parameter EC50 equation, yielding EC50 values of 262 ± 14 nM and > 100 MM for (R)-PA-824 against WT and RES III cells, respectively. Data are the mean ± standard deviation of triplicate cultures in a single experiment.

Figure 9 provides the chemical structures of the bicyclic and monocyclic compounds used.

F_{igure 10 illustrates the role of} ^{NTR2 in resistance to (R)-PA-824 in Leishmania.} Figure 11 illustrates the metabolism of nitroheterocyclics in cultures of WT and NTR2-overexpressing promastigotes.

Figure 12 provides a characterisation of ^LdNTR2. Figure 13 Investigates cellular localisation of NTR2.

F_{igure 14 investigates the modulation of} ^{NTR2 levels and its effect on (R)-PA-824} potency, compound metabolism and infectivity.

Figure 15 demonstrates the susceptibility of RES III and WT parasites to (R)-PA-824 in the intra-macrophage amastigote stage. WT (open circles) and RES III (closed circles) metacyclic promastigotes were used to infect starch-elicited, mouse peritoneal macrophages. Dose response curves are the non-linear regression fits using a four- _{parameter ECso equation, yielding EC5} ^{0 values of 2.1 ± 0.151iM and > 50 RIVI for (R)-} PA-824 against WT and RES III cells, respectively.

Figure 16 shows levels of NTR2 in WT, RESIII and NTR2 overexpressing

promastigotes. (A) Immunoblots of whole cell extracts (equivalent of 5x10^{6 parasites} in each lane) from L. donovani mid-log promastigotes (WT) and mid-log

p_{romastigotes overexpressina NTR2} ^{(OE) were probed with LdNTR2-specific} polyclonal antiserum. (B) Immunoblots of whole cell extracts from L. donovani mid- log promastigotes (WT) and resistant promastigotes (clone RESIII) were probed with LdNTR2-specific polyclonal antiserum. NTR-specific bands are indicated with double-headed black arrows and non-specific bands are indicated with chevrons. Figure 17 provides the sequence listing of the protein identified (Lin.I.12.0730).

Figure 18 represents the nucleic acid provides the nucleic acid sequence of a nucleic acid molecule comprising Linn 2.0730.

Figure 19 demonstrates the growth inhibitiory effect of the S-enantiomer of delamanid on epimastigotes.

Figure 20 shows delamanid induced parasite death over time. Cell lines and culture conditions

T_{he clonal Leishmania donovani} ^{cell line LdBOB (derived from MHOM/S.D./62/1S-CL2D)} was grown as promastigotes at 24°C in 10% FCS, as previously described (Goyard et al., 2003), except when investigating the effect of serum concentration on drug efficacy, in which case 5, 10, or 20% FCS was used. L. major promastigotes (Friedlin strain; WHO designation: MHOWIL /81 /Friedlin) were grown in M199 medium (Caisson Laboratories) with supplements, as previously described (Oza et al., 2005). Transgenic LdBOB promastigotes expressing the L. major nitroreductase (LmjF.05.0660) enzyme (Wyllie et al., 2012) were cultured under identical conditions. L. donovani (LV9 strain; WHO designation: MHOM/ET/67/HU3) ex vivo amastigotes were used in both in vitro and in vivo drug sensitivity assays. Amastigotes were derived from hamster spleens, as previously described (Wyllie and Fairlamb, 2006). Chemical synthesis of delamanid and analogues

Delamanid was prepared as shown in the synthetic route below.

Synthetic route towards delamanid (7) and (S)-delamanid (13). Reagents and conditions; a) 2-bromo-4-nitro-1H-imidazole, DIPEA, Et0Ac, 65°C, 20 h; b) K2CO3, Me0H, room temp., 16 h; c) MsCI, pyridine, CH2C12, 0°C—*room temp., 16 h; d) DBU, Et0Ac, room temp., 16 h; e) 6, NaH, DMF, 0—>50°C, 1.5-4 h. Modification of the delamanid synthetic route afforded (S)-delamanid and des-nitro- delamanid. The relevant synthetic route is provided below.

Synthetic route towards des-nitro-delamanid (18). Reagents and conditions; a) DIPEA, 2- nitroimidazole, Et0Ac/MeCN, 77°C, 44 h, 72 %; K2CO3, MeOH, room temp., 20 h, 83%; MsCl, pyridine, CH2C12, 0°C--*room temp., 16 h; DBU, Et0Ac, room temp., 16 h, 15% over 2 steps; NaH, DMF, 0—>50°C, 5 h, 30%. Compound purity was determined by liquid chromatography-mass spectrometry, with all compounds found to be >95% pure. For in vivo experiments, delamanid was further analysed by ultra high-performance liquid chromatography-mass spectrometry (UPLC-MS), with all batches found to be of >98% purity. The optical rotation of delamanid was in close agreement to the published value (Sasaki et al., 2006), confirming the optical purity of the material used in this study. In vitro drug sensitivity assays against promastigotes

To examine the effects of test compounds on growth, triplicate cultures were seeded with 1 x 105 parasites m1-1. Parasites were grown in the presence of drug for 72 h, after which 50 uM resazurin was added to each well and fluorescence (excitation of 528 nm and emission of 590 nm) measured after a further 2 h incubation (Jones et al., 2010). Data were processed using GRAFIT (version 5.0.13; Erithacus software) and fitted to a 2-parameter equation, where the data are corrected for background fluorescence, to obtain the effective concentration inhibiting growth by 50% (EC5o):

In this equation [I] represents inhibitor concentration and in is the slope factor. Experiments were repeated at least two times and the data is presented as the mean plus standard deviation (Young, 1962). When investigating the speed of drug-mediated cell killing, parasites were grown in the presence of drug for 24, 48, or 72 h in an otherwise identical assay. The same assay was used to investigate the effect of seeding density upon drug efficacy, except that the number of parasites used to seed the assays was varied to be either 103, 104 or 106 parasites m1-1. Cytocidal effects of delamanid on L. donovani promastigotes

Delamanid was added to early-log cultures of LdBOB promastigotes (-1 x 106 m1P-1) at concentrations equivalent to 10 times its ECBsou value. At intervals, the cell density was determined, samples of culture (500 ul) removed, washed and resuspended in fresh culture medium in the absence of drug. The viability of drug-treated parasites was monitored for up to 24 h and the point of irreversible drug toxicity determined by microscopic examination of subcultures after 5 days. In vitro drug sensitivity assays in mouse macrophages and toxicity to HepG2 cells In macrophage drug sensitivity assays were carried out using starch-elicited mouse peritoneal macrophages and hamster-derived ex vivo amastigotes, as previously described (Wyllie et al., 2012). Assays to determine the sensitivity of HepG2 cells to test compounds were carried out precisely as previously described (Patterson et al., 2013). In vitro pharmacokinetic and biophysical properties

The PPB of delamanid was determined by the equilibrium dialysis method (Jones et al., 2010). The aqueous solubility of delamanid was measured using a laser nephelometry-based method (Patterson et al., 2013). In vivo drug sensitivity

Groups of female BALB/c mice (5 per group) were inoculated intravenously (tail vein) with approximately 2 x 107 L. donovani LV9 amastigotes harvested from the spleen of an infected hamster (Wyllie and Fairlamb, 2006). From day 7 post-infection, groups of mice were treated with either drug vehicle only (orally), with miltefosine (30 mg kg' orally), or with delamanid (1, 3, 10, 30 or 50 mg kg' orally). Miltefosine was administered once daily for 5, or 10 days, with vehicle and delamanid administered twice daily over the same period. Drug dosing solutions were freshly prepared each day, and the vehicle for delamanid was 0.5% hydroxypropylmethylcellulose, 0.4% Tween 80, 0.5% Benzyl alcohol, and 98.6% deionized water. On day 14 (for 5 day dosing experiments), or day 19 post-infection (for 10 day dosing experiments), all animals were humanely euthanized and parasite burdens were determined by counting the number of amastigotes/500 liver cells (Wyllie et al., 2012). Parasite burden is expressed in Leishman Donovan Units (LDU): mean number of amastigotes per 500 liver cells x mg weight of liver (Bradley and Kirkley, 1977). The LDU of drug-treated samples are compared to that of untreated samples and the % inhibition calculated. ED50 values were determined using GRAFIT (version 5.0.13; Erithacus software) by fitting data to a 2- parameter equation, as described above. Determination of delamanid exposure in infected mice after oral dosing

Blood samples (10 RI) from 3 of 5 infected mice (see in vivo drug sensitivity above) in each dosing group were collected from the tail vein and placed into Micronic tubes (Micronic BV) containing deionized water (20 R1). Samples were taken following the first dose on the first (day 7 post-infection) and last day of dosing (day 11, or 16 post-infection) at 0.5, 1, 2, 4 and 8 h post-dose. Diluted blood samples were freeze-thawed three times prior to bioanalysis. The concentration of delamanid in mouse blood was determined by UPLC-MS/MS on a Xevo TQ-S (Waters, UK) by modification of that described previously for the analysis of fexinidazole (Sokolova et al., 2010). Rate of delamanid metabolism in L. donovani promastigotes

Rate of metabolism studies were carried out at 15, 45 and 150 nM delamanid (equivalent to I-, 3- and 10-times EC50) in culture medium alone and in the presence of wild type L. donovani promastigotes (1 x 107 parasites m1-1). At 0, 0.5, I, 2, 4, 6, 8 and 24 h aliquots were removed, precipitated by addition of a 3-fold volume of acetonitrile and centrifuged (1,665 x g, 10 min, room temperature). The supernatant was diluted with water to maintain a final solvent concentration of 50% and stored at -20°C prior to UPLC-MS/MS analysis, as described below. UPLC-MS/MS was performed On a Waters Acquity UPLC interfaced to a Xevo TQ-S MS. Chromatographic resolution was achieved on a 2.1 x 50 mm Acquity BEH C18, 1.7 pm column which was maintained at 40°C with an injection volume of 8 µ1. The mobile phase consisted of A: deionized water plus 0.01% (v/v) formic acid and B: acetonitrile plus 0.01% (v/v) formic acid at a flow rate of 0.6 ml mm-i. The initial gradient was 5% B held for 0.5 min before increasing to 95% B from 0.5-2 min, where it was held from 2-2.6 min before decreasing back to 5% B from 2.6-3 min. Mass spectra were obtained using electrospray ionization (EST), in positive ion mode with the following conditions: capillary 3.5 kV; desolvation temperature 600°C; source temperature 150°C; desolvation gas flow (nitrogen) 1000 I h-I and collision gas (argon) gas of 0.15 ml min-1. Multiple reaction monitoring (MRM) was performed for delamanid using the transition 535.02> 351.80 at a cone voltage of 16 V and collision energy of 33 V. Data was processed using the TargetLynx feature of Mass Lynx v4.1. In vitro sensitivity of L. donovani to (S)- and (R)-delamanid

The anti-tubercular drug delamanid and its corresponding S-enantiomer were synthesized as described above and assessed for anti-leishmanial activity. The potency of both compounds was determined in vitro against L. donovani (LdBOB) promastigotes and against intracellular amastigotes (LV9) in mouse peritoneal macrophages. The (5)-enantiomer of delamanid showed promising anti-leishmanial activity against both developmental stages of the-parasite (EC50 values of 147 ± 4 and 1332 ± 106 nM against promastigotes and amastigotes, respectively). However, delamanid (the R-enantiomer) proved to be an order of magnitude more potent against promastigotes, axenic amastigotes and intracellular amastigotes with ECso values of 15.5, 5.4 and 86.5 nM, respectively (Table 1). Both compounds were found to be inactive (EC51) >50 !AM) in a counter screen against the mammalian cell line 1-lepG2 (Table 1). Table 1 Activity of delamanid against laboratory and clinical isolates of ^{L. donovani in} v_{itro. EC90} values are calculated from the 8C50, Hill slopes and the molecular weight of delamanid.

Physicochemical Properties of delamanid

The plasma protein binding.of delamanid was measured and found to be high (Fu=0.0045), in agreement with that reported previously (Committee for Medicinal Products for Human Use, 2013). A kinetic solubility assay demonstrated that delamanid possesses sufficient aqueous solubility (>250 pM in 2.5% DMSO) for use in in vitro assays. Efficacy of delamanid in a murine model of visceral leishmaniasis

The efficacy of delamanid was assessed in a murine model of VL. Groups of infected B_{ALB/c mice (seven days post infection with ex vivo L.} ^{donovani LV9 amastigotes) were} dosed twice-daily, for five consecutive days with an oral formulation of delamanid (1, 3, 10, 30, or 50 mg kg-I ). On day 14 post-infection, the parasite burdens in the livers of infected mice were determined and compared with those of control animals. The only current oral a_{nti-leishmanial therapy miltefosine (30 mg kg} ^{-I , once-daily, 5 days) was included as a} positive control. Both delamanid and miltefosine were well tolerated at these doses, with no mice displaying any overt signs of toxicity. An initial experiment showed that treatment with _{delamanid at 50 mg kg -I} effectively cured the study mice, with no detectable parasites in the liver smears, whereas control mice dosed with vehicle alone showed a high level of infection (Figure 1). In Figure 1, Groups of mice (five per group) infected with L. donovani (strain LV9) were dosed with drug vehicle (orally), miltefosine (orally) or delamanid (twice daily, orally) on day 7 post-infection and for a total of 5 or 10 days. Two days after the final dose, animals were humanely euthanized and parasite burdens were determined microscopically by examining Giemsa-stained liver smears. A second in vivo study with mice dosed twice-daily at 30, 10, or 3 mg kg ^{-J suppressed} infection in the murine model by 99.5%, 63.5% and 16.0%, respectively, establishing a dose- dependent anti-leishmanial effect within the range of 3-50 mg kg-1. These results give an estimated ED50 and ED90 of 7.3 and 21.5 mg kg-I, respectively (Figure 2). In Figure 2, results are based on dosing in the range 3 to 50 mg kg-1 b.i.d. oral for 5 days. At 30 and 50 mg kg-1 delamanid compares favourably with miltefosine (98.8-99.8% suppression at 30 mg kg-1), which exemplifies the therapeutic potential of delamanid. A third in vivo study with a further reduced delamanid dose of 1 mg kg-1 resulted in a suppression of parasitaemia of 86.3% compared with control mice, proving unexpectedly superior to dosing at 3, or 10 mg kg-1 (Figure 1). The same hormetic effect was observed in a fourth experiment when delamanid was instead dosed twice-daily for 10 days at 10, 3, or I mg kg-1, with the suppression of infection being 92.3%, 24.3% and >99.9%, respectively. In Figure 7, (A and B) Mean suppression of parasite burden as a function of Cmax for the penultimate dose and extrapolated AUC(0-24h) for the last day of the 5- and 10-day treatment regimens, respectively. Open and closed symbols represent data combined from the 5- and 10-day studies, respectively. The dashed and dotted line in (A) is the EC90 value obtained for infected macrophages after 72-h exposure. The dotted line in (A) and (B) represents the mean delamanid Crnax (175 ng m1-1) and mean AUC(0-24h), ( 2,500 h*ng ml- 1) obtained in TB patients after 14 days treatment with 100 mg, oral, once daily (Diacon et al., 2011). Blood levels of orally dosed delamanid in a mouse model

In order to understand the pharmacokinetics of delamanid in L. donovani-infected mice, the blood levels of the drug were measured at intervals (up to 8 h post dose) during the in vivo efficacy studies. Figure 3A and B show the blood levels of delamanid following the first oral dose on day 1 (3A) and the penultimate oral dose on day 5 (3B) for 3, 10 and 30 mg kg1 b.i.d. (complete line, dotted line and dashed and dotted line respectively). Error bars are SEM (n=3). Figure 3C and D show the relationship between dose with Cm. or AUC(0-8h), respectively, after the first oral dose on day 1 (complete line), or the penultimate dose on day 5 (dotted line) or day 10 (dotted and dashed line). Error bars in (C) are SEM (n=3). Lines in (C) and (D) are best fits by linear regression. Specimen data for the first and ninth dose in a 5-day twice daily treatment experiment (Figure 3A, B) show a dose dependent response with accumulation over time. More detailed analysis of the combined PK data from four experiments (including a 10-day treatment study) shows a linear relationship between dose and peak blood concentration (Cmax) or area under the curve (AUC(0-t)) with accumulation from day 1 through to day 10 (Figure 3C, D). The 10 and 30 ^{mg kg-1} doses should provide adequate coverage over the EC90 (120 ne m1-1) as measured ^{for the L. donovani} isolate LV9 in macrophages over a 3 day exposure (Table 1). However, due to high protein binding, the free fraction (Fu = 0.0045) cannot account for biological activity in vivo at any dose. Delamanid-mediated cell killing

To determine whether delamanid was cytostatic or cytotoxic, mid-log promastigotes were incubated with drug concentrations equivalent to 10 times the EC50 value (Figure 4A). Growth of drug-treated cultures ceased almost immediately with cell numbers declining after 8 h and no live parasites visible at 24 h. To determine the actual point where treated cells lost viability, at defined intervals parasites were washed and sub-cultured without drug. No viable parasites could be recovered after 12 h in the presence of drug, confirming that delamanid is rapidly leishmanicidal. In support of this apparent rapid mechanism of cell killing, EC50 values determined after 24, 48 and 72 h were essentially identical (Figure 4B). In addition, the potency (EC50 value) of delamanid was found to be dependent on the initial cell density (Figure 4C) and on the assay serum concentration (Figure 4D). Figure 4A shows delamanid causes rapid cell killing. Promastigotes were exposed to delamanid (10 times ECHO and samples removed, at intervals to determine cell density and cell viability. Complete line: no inhibitor; dotted line: plus drug; >° the point of irreversible drug toxicity. Figure 4B shows drug sensitivity is independent of exposure beyond 24 h. Complete line, dotted line and dashed and dotted line are EC50 curves determined after-24, 48 and 72 If, respectively. Figure 4C shows drug sensitivity is cell-density dependent. Complete line, dotted line and dashed and dotted line are EC50 curves determined after 72 h, with initial seeding densities of 103, 104 and 105 cells m1-1, respectively. Figure 4D shows drug sensitivity is serum dependent. Complete line, dotted line and dashed and dotted line are EC50 curves determined after 72 h in the presence of 5, 10 and 20% FCS, respectively. Delamanid— mode of action studies

Many nitroheterocyclics require bio-activation of their nitro groups to become biologically active. In Mycobacterium tuberculosis, delamanid is assumed to be reductively activated by the same unusual deazaflavin (F420)-dependent nitroreductase (Ddn) known to activate the closely related nitroimidazo-oxazine drug PA-824 (Manjunatha et al., 2006; Singh et al., 2008; Manjunatha et al., 2009). In the absence of a Ddn homologue in Leishmania, we assessed whether the reduction of delamanid is catalysed by the NADH-dependent bacterial- like nitroreductase (NTR) already shown to activate the nitroimidazoles fexinidazole and nifurtimox in these parasites (Wyllie et al., 2012). The potency of delamanid was determined against parasites overexpressing NTR. Increased concentrations of NTR in these transgenic parasites were confirmed by a 13-fold increase in their sensitivity to nifurtimox (EC50 of 8.0 ± 0.2 and 0.61 ± 0.006 uM for WT and transgenic parasites, respectively Figure 5A), known to undergo two-electron reduction by NTR (Hall et al., 2011). However, overexpression of NTR in promastigotes did not significantly alter their sensitivity to delamanid (EC50 of 4.5 ± 0.004 and 4.1 ± 0.003 nM for WT and transgenic parasites, respectively) (Figure 5B). In addition, in studies to be published elsewhere, LV9 promastigotes which had been selected for resistance to fexinidazole retained sensitivity to delamanid (3.24 ± 0.04 and 3.09 ± 0.03 nM for WT and resistant lines, respectively). These findings show that NTR does not play a role in the activation of delamanid in L. donovani and that the mechanism of action of this nitroheterocyclic drug is different from that of fexinidazole. In Figure 5A and B Susceptibility to nifurtimox (A) is increased in NTR-overexpressing parasites (dotted line), but not to delamanid (B) compared to WT cells (complete line). Data are the mean of triplicate cultures from a single experiment. Proteomic analysis of (R)-PA-824 resistant promastigotes

Bio-activation of nifurtirnox and fexinidazole sulfone is catalysed by an oxygen-insensitive _{nitroreductase (NTR) in both} T. brucei [10] and Leishmania [7] and loss of, or mutations in, this enzyme has been shown to play a key role in drug resistance mechanisms in the trypanosomatids [16]. Therefore, we hypothesised that should an alternative nitroreductase be involved in the bio-activation of (R)-PA-824 in Leishmania, changes in this enzyme may be evident in parasites resistant to (R)-PA-824. Thus, a comparative proteomic analysis of drug- resistant and WT promastigotes was conducted using stable isotope labelling by amino acids in cell culture (S1LAC). WT parasites were grown in modified SDM-79 medium in the presence of normal L-arginine and L-lysine (ROKO) and mixed 1:1 with RES III cells grown for at least 6 cell divisions in the presence of stable isotopes of L-arginine and L-lysine (R6K4) to achieve uniform labelling. Additionally, a label-swap experiment in which the 'heavy' and 'light' culture media were reversed was also carried out. Cell cultures of WT and RES III were harvested by centrifugation and cell pellets extracted with detergent. Extracted lysates were then pooled, fractionated by SDS-PAGE, digested with trypsin and analyzed by LC-MS/MS. Individual peptides with a heavy to light ratio of 1.0 indicate an equal abundance in both populations. In the combined proteomic dataset, 2119 proteins were identified by at least one uniquely mapped peptide, prior to filtering the datasets and combining the label-swap experiments. This resulted in the identification of 1472 proteins with a quantifiable expression change between the parental and the RES III cell line. Statistical significance was assessed using significance B, leading to the identification of 38 proteins with significantly altered expression levels compared to the wild type (Si table). The most striking change was of a hypothetical NADI-I:FMN-dependent oxidoreductase (Uniprot: E9AGH7; GeneDB: LinJ.12.0730) identified to be—16-fold less abundant in RES III parasites (Fig 3A). Figure 10 illustrates the role of NTR2 in resistance to (R)-PA-824 in Leishmania. (A) Plot of the proteomics data from (R)-PA-824 resistant clone RES III. Each protein identification is represented by a point plotted as the Log2 of their heavy-to-light isotope ratio (x axis) versus the Logi° value of the intensities of the peptides belonging to each protein (y axis). Proteins plotted in open circles were determined to have significantly different expression levels compared to those in WT parasites, with the most significantly changed protein shown labelled as LinJ.12.0730 (LinJ.12.0730; NADH:flavin oxidoreductase/NADH oxidase, putative). The sequence of the protein analysed is shown in Figures 17 and 18. The identity of the proteins found to be consistently over- or under-expressedwere identified. (B) Dose- response curve of WT promastigotes (closed circles) and promastigotes overexpressing NTR2 (open circles) to (R)-PA-824. EC50 values of 140 ± 4.6 and 3.5 ± 0.2 nM were determined for WT and NTR2-overexpressing parasites, respectively. Overexpression of NTR2 was confirmed by western blotting. (C) The susceptibility of RES III parasites (open circles) and RES III parasites overexpressing NTR2 to (R)-PA-824 (closed circles). RES III parasites were insensitive to (R)-PA-824 at concentrations up to and including 100 ftM while these promastigotes overexpressing NTR2 returned an EC50 value of 1 + 0.02 nM. Data are the mean LI SD of triplicate cultures. (D) Representation of the frame shift and premature termination of NTR2 translation that results from deletion of a cytosine at genomic position 483544 in (R)-PA-824-resistant clones. The frame shift results in a shortened open reading frame and corresponding amino acid sequence (highlighted in bold), with the greyed out portion indicating the frame-shifted part of this sequence. Genomic analysis of NTR2 in (R)-PA-824-resistant parasites

In an attempt to understand the mechanisms involved in the depletion of ^{NTR2 from our drug} resistant cell lines and also to identify additional factors that may be involved in resistance, the complete genomes of each independently derived resistant clone (RES I, II and III) were sequenced. ^ Surprisingly, first-pass analysis revealed only 12 single nucleotide polyrnorphisms (SNPs) resulting in nonsynonymous changes in 3 ORFs in the drug-resistant clones (S2 Table). At this point in our studies, SILAC analysis focused our attention on the role of NTR2 in nitroheterocyclic drug activation and resistance. Complementing this finding, _{closer examination of} NTR2 and its flanking sequences identified the deletion of a single cytosine (genomic position 483544 on chromosome 12 in 1.,dBPK; C457 in the open reading frame) within NTR2 that results in a frame shift and premature termination of NTR2 translation (Fig 3D). Despite the fact that each clone appeared to be genetically distinct (S2 Table), this deletion was identified in both allelic copies of ^{NTR2 in all 3 independently} generated resistant clones. The reason for this unusual finding is not clear. Nonetheless, these _{data, alongside our failure to detect full length} ^{NTR2 in RES III parasites (S2 Fig.), confirm} _{that each (R)-PA-824-resistant clone is effectively NTR2} ^{null and further strengthen our} hypothesis that NTR2 is principally responsible for (R)-PA-824 bio-activation. A comprehensive analysis of the sequencing data from our (R)-PA-824-resistant parasites will be reported in a subsequent publication. Can NTR2 activate other nitroheterocyclic drugs?

Having established its role in the bio-activation of (R)-PA-284, we assessed the possible role of NTR2 in the activation of other Leishmania-active nitroheterocyclic compounds (Table 1). The potencies of these compounds were determined against WT promastigotes and WT transgenic parasites overexpressing NTR2, where hypersensitivity in transgenic parasites is indicative of a compound activated via NTR2. As expected, the (8)-enantiomer of PA-824, an anti-tubercular clinical candidate, was 27-fold more potent against parasites with elevated levels of NTR2 than WT. Structurally-related compounds including delamanid, CGT-17341 and DNDI-VL-2098 are also activated by NTR2. NTR2-overexpressing parasites showed a <2-fold increase in susceptibility to fexinidazole sulfone suggesting that this nitroimidazole, previously shown to be activated by NTRI, is unlikely to be an efficient substrate for NTR2. However, another known substrate of NTRI, nifurtimox, was 15-fold more potent against NTR2 overexpressing parasites suggesting that this compound may be a substrate of both enzymes in L. donovani. Metabolism of (R)-PA-824 and DNDI-VL-2098 in L. donovani

Levels of (R)-PA-824 metabolism in WT and NTR2-overexpressing parasites were monitored by UPLC-MS/MS in cultures of promastigotes treated with 160 TIM of drug over a 24-h period. (R)-PA-824 was essentially stable in culture medium alone with a t112 of >24 h (Fig 11A). The addition of L. donovani promastigotes to culture medium resulted in a marked increase in the rate of disappearance of the drug (biz= 14 h) associated with the appearance of several drug metabolites. Metabolism was further increased in cultures of parasites overexpressing NTR2 (tu2 = 0.5 h), such that drug levels had dropped below the limit of quantification (0.31 nM) by 4 h. Similar rates of drug metabolism were also observed in cultures incubated with both 15 nM delamanid (EC50 value, Fig I 1B) and 20 nM DNDI-VL- 2098 (10 x ECso value Fig 11C). However, the instability of these compounds in medium alone was higher than those seen with (R)-PA-824. This instability can be explained by the fact that delamanid is known to be primarily metabolised in plasma by albumin. Likewise, DNDI-VL-2098 is reportedly unstable in plasma presumably for the same reason. Fig 11. Metabolism of nitroheterocyclics in cultures of WT and NTR2-overexpressing promastigotes. Metabolism of 160 nM (R)-PA-824 (A), 15 nM delamanid (B) and 20 nM DNDI-VL-2098 (C) in media alone (closed circles), wild type L. donovani promastigotes (squares) and NTR2-overexpressing L. donovani promastigotes (open circles). The half-life of (R)-PA-824 in media alone, in cultures of WT promastigotes and cultures of promastigotes overexpressing NTR2 were >24 h, 14 h and 0.5 h, respectively. DNDI-VL-2098 incubated in media alone had a half-life of 3.1 hand half-lives of 0.83 h and 0.13 bin cultures of WT and NTR2°E parasites, respectively. The half-life of delamanid was 12 h in media alone and 1.6 h in WT promastigotes. As the disappearance of delamanid in NTR2°E parasites was plotted to a double exponential decay, two half-lives were calculated as 0.096 h for

and 0.64 h for k2. .Enzymatic analysis of recombinant NTR2

T_{o further study the substrate specificity of NTR2,} ^{the recombinant enzyme was expressed} and purified to homogeneity in three chromatographic steps to obtain a yield of 15 mg 1-1 of aellow product (Fig 12A), indicative of a flavoprotein. ^ Using an established spectrophotometric method, FMN was confirmed as the bound co-factor in NTR2. Analysis of the recombinant protein by size-exclusion chromatography revealed that NTR2 elutes primarily as a monomer at—40 kDa (Fig 12B), close to the predicted molecular mass of 39.6 kDa (Fig 12B). This was confirmed by MS to be 39.4 kDa for the recombinant protein by MALDI—TOF analysis. Fig 12. Characterisation of LdNTR2. (A) Purification of recombinant LdNTR2 from E. colt BL21(DE3)pLysS [pET15b-LdNTR2]. Lane 1, insoluble fraction; lane 2, soluble fraction; lane 3, pooled fractions from Ni2taffinity chromatography; lane 4, soluble protein following removal of histidine tag; and lane 5, pooled fractions from size-exclusion chromatography. MALDI analysis confirmed that the minor bands represent NTR2 degradation products. (B) Gel filtration profile of the LdNTR2. The inset shows a plot of elution volume against the log molecular mass (Mw) of a standard protein mixture (closed circles). The open circle represents the elution volume of NTR2. (C) Metabolism of nitroheterocyclic compounds by recombinant NTR2. Initial rates of metabolism were measured in assays containing 100 µM nitro-compound, 100 )1M NADPH and 500 nM NTR2. Rates of metabolism with NADPH and NADH alone were 0.0124 ± 0.001 and 0.0084 ± _{0.0006 p.mol} ^mind mg-1, respectively. Rates represent the mean ± SD of triplicate measurements. (D) Immunoblots of whole cell extracts (equivalent of 5 x10^{6 parasites in each} _{lane) from L. donovani} mid-log promastigotes (L), metacyclic promastigotes (M) and axenic amastigotes (A) were probed with LdNTR2-specific polyclonal antiserum. Known amounts of purified recombinant LdNTR2 were loaded as standards for the quantification of the cellular levels of NTR2. Our preliminary studies indicate that this enzyme was able to utilise NADH or NADPH as a reductant. The ability of NTR2 to reduce a variety of nitroheterocyclic compounds was then assessed in the presence of 100 pM NADPH (Fig 12C). The highest rates of activity were observed with the bicyclic nitro-compounds structurally related to (R)-PA-824. Further, the highest rates of NTR2 metabolism broadly correlate with the most potent anti-leishmanial compounds, DNDI-VL-2098 (15.7 ± 0.4 pmol min' mg") and delamanid (7.2 ± 0.7 pmol min' mg"). In keeping with our overexpression studies, recombinant NTR2 was able to reduce nifurtimox, albeit at a comparatively low rate (0.30 + 0.02 pmol min' mg") but showed little activity with fexinidazole sulfone (0.004 ± 0.0002 pmol min" mg"). Quantitation of cellular NTR2 levels

Using a NTR2-specific polyclonal antiserum generated against our purified recombinant protein, we were able to confirm that NTR2 is expressed in all developmental stages of the _Leishmania parasite by probing an immunoblot of whole cell lysates (Fig 12D). Single bands of approximately 40 kDa, close to the predicted molecular mass of NTR2 (39.6 kDa), were detected in cell lysates of log phase promastigotes (the dividing insect stage), metacyclic promastigotes (the insect stage infective to mammals) and axenic amastigotes (the intracellular mammalian stage). The cellular concentration of NTR2 in each of these parasite stages was determined by densitometry. NTR2 levels in each developmental stage were found to be remarkably similar with concentrations of 1.70 pM, 1.73 pM and 1.75 µM in promastigotes, metacyclics and amastigotes, respectively. Cellular localisation of NTR2

I_{mmunolluorescence studies confirm that} ^{L. donovani NTR2 localises to the cytosol of mid-} log promastigotes (Fig.13). Staining of promastigotes with an anti-NTR2 polyclonal antibody showed extensive and even staining throughout the cells, except for the nucleus and kinetoplast, demonstrating the cytosolic location of this enzyme (Fig.13C and D). Fig.13. Cellular localisation of NTR2. Phase contrast images of ^{L. donovani promastigotes} (A), promastigotes stained with DAPI (4,6-diamidino-2-phenylindole), immunofluorescence staining of promastigotes with NTR2 anti-serum (FITC) (C) and a merged FITC and DAPI image (D). The kinetoplastid (K) and nucleus (N) are indicated in the DAPI stained image. Assessing the essentiality of ^NTR2

The studies described above show that loss of NTR2 is strongly associated with resistance to (R)-PA-824. However, this does not exclude the possibility that additional genes may also be involved. To address this issue, we investigated the impact of NTR2 ^{loss by gene deletion in a} _{WT genetic background. Thus,} ^{NTR2 null parasites were generated by classical gene} replacement. Both copies of the NTR2 gene were sequentially replaced with hygromycin and puromycin drug resistance genes. Southern blot analysis of genomic DNA from putative _{double knockout (DKO) cells confirmed that they were} ^{NTR2 null (Fig 14A). Loss of both} _{copies of NTR2} had no obvious effects on the viability of these parasites with DKO promastigotes growing at the same rate in culture as WT and achieving similar cell densities. DKO parasites were found to be completely refractory to (R)-PA-824 at concentrations up to and including 1001.1M (Fig 11B), as found in our resistant lines obtained by drug selection. Similar results were found for (S)-PA-824, delamanid and DNDI-VL-2098 (Table 21). Susceptibility to fexinidazole sulfone remained unchanged and susceptibility to nifurtimox increased marginally in good agreement with RES III. Adding back an exogenous copy of NTR2 to DKO null parasites entirely recovered sensitivity to (R)-PA-824 is necessary and sufficient for activation of toxicity with compounds such as (R)-PA-824, delamanid or DNDI-VL-2098. Fig 14 Modulation of NTR2 levels and its effect on (R)-PA-824 potency, compound metabolism and infectivity. (A) (i) Schematic representation of the stepwise generation of the NTR2 DKO cell line in L. donovani. One allele of NTR2 was replaced with the puromycin resistance gene (PAC) by homologous recombination; the remaining allele was replaced with a hygromycin resistance gene (HYG) by homologous recombination resulting in a NTR2 ^null cell line. (ii) Southern-blot analysis of XhoI-digested genomic DNA (-5 jig) from wild-type L. donovani (LdBOB) cells (lane 1), NTR2-double knockout clone 1 (DK01) cells (lane 2) and NTR2-double knockout clone 2 (DK02) cells (lane 3). The ^{NTR2 ORF probe shows} allelic LdNTR2 at 10 kb. (B) EC50 values were determined for (R)-PA-824 against WT (closed circle), DKO1 (open circle), DKO2 parasites (square) and DKO1 parasites with an NTR2 add-back (triangle): An EC5r) value of 115 ± 3 nM was determined for (R)-PA-824 against WT parasites, DKO1 and DKO2 were unaffected by the drug at concentrations up to and including 10 µM, while an ECso value of 5.5 ± 0.03 nM was determined for DKO1 parasites expressing an NTR2 add-back. (C) Metabolism of 160 nM (R)-PA-824 in media _{alone (closed circles), wild type} ^{L. donovani promastigotes (square), DKO parasites (open} _{circles) and DKO parasites plus} ^{NTR2 add-back (triangle). The half-life of (R)-PA-824} metabolism in cultures of WT parasites is 12.5 It, while the half-life of DKO parasites with an NTR2 add-back is 0.5 h. (D) Mean numbers of WT, DKO (clone 1) and RES (clone RES III) amastigotes infecting mouse peritoneal macrophages. White bars, invasion after 24 h; black bars, replication after 72h. Differences in the replication of WT versus RES and DKO amastigotes in macrophages were confirmed as highly significant (P values equal to 0.0057 and 0.0087, respectively) using an un-paired student t-test (**). The 'impact of NTR2 deletion on drug metabolism was determined by measuring the concentration of (R)-PA-824 in cultures of WT and DKO over a 24-h period. Samples of culture were removed at defined intervals and the supernatants analysed by UPLC-MS/MS, as previously described (Fig 14C). WT parasites metabolised (R)-PA-824 at a similar rate to that seen in our earlier study (tu2 = 12.5 h). In contrast, rates of metabolism in medium alone and in cultures of DKO promastigotes were negligible over the same 24-h period. The addition of an NTR2 add-back to DKO parasites recovered the ability of these cells to metabolise (R)-PA-824 (tv2 = 0.5 h). These data confirm that NTR2 ^{alone is necessary and} sufficient for metabolic conversion of bicyclic nitro-drugs. Loss of functional NTR2 did not have a material effect on the ability of DKO or RES metacyclic prornastigotes to infect peritoneal macrophages, as determined by comparing the mean numbers of amastigotes per infected macrophage to that seen in WT-infected macrophage cultures 24h following infection (Fig 14D). However, there did appear to be a moderate but statistically significant effect on the ability of NTR2-deficient parasites to replicate within peritoneal macrophages with mean numbers of amastigotes per infected macrophage considerably lower in DKO and RES cultures at 72h. The reduced ability of NTR2 null amastigotes to replicate within macrophages was entirely alleviated by the addition of an NTR2-addback. Collectively, these data suggest that, while ^{NTR2 is not} essential for L. donovani survival, null parasites do appear to suffer a moderate but statistically significant loss of "fitness" in macrophage infections that may have implications for the propagation of NTR2-related drug resistance in the field. Metabolism of delamanid in L. donovani

Given that NTR does not activate delamanid in L. donovant promastigotes and the requirement of the nitro group for biological activity, it was important to determine if the drug is metabolised in culture. To address this issue, the concentration of delamanid was determined by UPLC-MS/MS in cultures of promastigotes over a 24 h period. Delamanid is known to be primarily metabolised in plasma by albumin and to a lesser extent by CYP3A4, CYP1A1, CYP2D6 and CYP2E1 (Committee for Medicinal Products for Human Use, 2013). Thus, the concentration of delamanid in culture medium without parasites was measured over the same time period as a control. In the presence of medium alone, delamanid decreased linearly in a concentration-dependent manner (Figure 6A). However, in the presence of L. donovani promastigotes the rate of disappearance of delamanid was markedly increased, such that the drug had essentially disappeared by 6 h (Figure 6B). The net amount of delamanid metabolised by parasites as a function of time is also linear and dependent on the initial concentration in the medium (Figure 6C). Linear regression of these data revealed that the rate of cell metabolism is not saturated up to the top concentration tested (Figure 6D). In Figure 6, (A) Medium plus delamanid alone and (B) cells incubated in medium plus delamanid. Delamanid concentrations added are 15, 45 and 150 nM (complete line, dotted line and dotted and dashed line respectively). The lines represent best fits by linear regression for all data points in (A) and 0 to 5 h in (B). The dotted line in (B) is the best fit by non-linear regression to a single exponential decay. (C) Net metabolism of delamanid by cells was obtained by subtraction of (A) from (B). Data fitted by linear regression gave correlation coefficients of 0.996, 0.991 and 0.951 for delamanid concentrations of 15, 45 and 150 nM, respectively. (D) Rates of delamanid metabolism obtained from (C) are linear up to 150 nM (correlation coefficient 0.996, explicit errors used in fit). Growth inhibition of epimastigotes

The growth inhibition of epimastigotes was investigated. Epimastigotes seeded at 1 x 105 nil- ' in RTH/FCS were incubated with drug over 120 11. Resazurin was added after 96 h incubation. The S-enantiomer of delamanid displays bi-phasic dose-response. Delamanid metabolism product P3 shows some activity but is only an n=2. Some of the results are shown in the table below.

72 h Trypomastigote cidal assay

Trypomastigotes taken from an infected monolayer were seeded at a density of 5 x 106 ^ml with 3 pM delamanid or nifurtimox(10p,M) for 72 h. Parasites were harvested and washed in PBS prior to incubation with a dead cell stain. The percentage of live and dead cells were determined by flow cytometry. The results were compared with nifurtimox (dead control), and DMSO (live control). T-test between DMSO and delamanid dead populations is significant p = <0.002

Time course measuring delamanid induced parasite death

Trypomastigotes seeded at 1 x 106 m1-1 were incubated with delamanid (3µM) or nifurtimox (10 uM) over 116 hours in DMEM/FCS. Parasites in DMEM/FCS gradually transformed into epimastigotes over the time course. However, it shows that delamanid is cidal over a much longer time than nifurtimox. Delamanid and DMSO samples were carried out in triplicate whilst nifurtmox is reported as single values. The results are provided in Figure 20. Delamanid Metabolism

The metabolism route of delamanid is provided below.

Delamanid has an ECso of 417 nM against T. cruzi (X10/7 Al), cultured as amastigotes in Vero cells.

Future anti-leishmanial therapies will be required to demonstrate a broad spectrum of activity against different Leishmania strains and against drug resistant parasites (Patterson and _{Wyllie, 2014). With this in mind, L. donovani} ^{clinical isolates DD8 (WHO reference strain),} BHU1 (Indian antimony resistant isolate) and SUKA 001 (recent Sudanese isolate) were assessed for their sensitivity to delamanid (Table 1). These clinical isolates were marginally less sensitive to delamanid than our laboratory strain LV9, but at the ECso varied by only 3- fold (Table 1). The corresponding des-nitro analogue was also synthesized as provided above and assayed _{against L. donovani} promastigotes. Des-nitro-delamanid was found to be inactive (EC50 >50 p.M), which is consistent with the nitro group being involved in the mechanism of action, or having a role in the binding of delamanid to its molecular target(s) in L. donovani. Throughout the description and Claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All documents referred to herein are incorporated by reference. Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following Claims.

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Claims

CLAIMS 1. One or more compounds according to Formula I for use in the treatment of a parasitic disease caused by or associated with parasites selected from the Order Kinetoplastida consisting of species belonging to the genera Leishmania and Trypanosoma.

Formula I

where R1 represents a hydrogen atom or a C I to C6 alkyl group, generally a C1 to C3 alkyl group, typically methyl or ethyl, suitably methyl.

N represents an integer of 0 to 6, generally 0 to 3, suitably 1 or 2, more suitably 1. R2 represents any group of the formulae (A) to (G) as described below.

A

wherein 12.5 represents any groups of the following (1) to (6):

1) a phenoxy group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted C1_6 alkyl group and an optionally halogen-substituted C1.6 alkoxy group);

2₎ a phenyl CI-6 alkoxy group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted C1-6 alkyl group and an optionally halogen- substituted Ci_6 alkoxy group);

3₎ ^{NR4R5 group, wherein R4} _{represents a hydrogen atom or CI} ^{-6 alkyl group and} R5 represents a phenyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting a halogen atom, an optionally halogen-substituted CI^-6 alkyl group and an optionally halogen-substituted C1-6 alkoxy group); ₄₎ a phenyl C1.6 alkyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted CI-6 alkyl group and an optionally halogen- substituted C1.6 alkoxy group);

5) a phenoxy C1-6 alkyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted Ci_6 alkyl group and an optionally halogen- substituted C1.6 alkoxy group) ; and

6₎ a benzofuryl C1.6 alkyl group (wherein the benzofuran ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted CI-6 alkyl group and an optionally halogen- substituted CI.6 alkoxy group); the group represented by the general formula (B) is:

wherein R6 represents a phenyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted C1.6 alkyl group and an optionally halogen- substituted C1.6 alkoxy group); the group represented by the general formula (C) is:

wherein R7 represents a phenyl C2.10 alkenyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted C1.6 alkyl group and an optionally halogen-substituted C1.6 alkoxy group) or a biphenyl C1-6 alkyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen- substituted CI-6 alkyl group and an optionally halogen-substituted C16 alkoxy group); the group represented by the general formula (D) is:

w_{herein R8} represents a phenyl CI-6 alkyl group (wherein the phenyl ring may optionally be substituted with one or more groups selected from the group consisting of a halogen atom, an optionally halogen-substituted CI-6 alkyl group and an optionally halogen-substituted C16 alkoxy group); the group represented by the general formula (E) is:

wherein R8 is as described above; the group represented by the general formula (F) is:

wherein R6 is as described above; the group represented by the general formula (G) is:

wherein R8 is as described above.

_2. A compound for use as claimed in Claim 1 wherein R2 represents a group according to formula (A). _{3. A compound for use as claimed in Claim 2 wherein R} ^{3 represents group (1) or group} (2). 4. A compound for use as claimed in either one of Claims 2 and 3 wherein R3 represents a phenoxy group substituted with one or more halogen-substituted C1_6 alkoxy groups. 5. A compound for use as claimed in either one of Claims 2 and 3 wherein R3 represents a phenyl Ci_6 alkoxy group, the phnyl groups being substituted with one or more halogen-substituted C1-6 alkoxy groups. 6. A compound for use as claimed in any preceding Claim having the structure:

7. A compound for use as claimed in any preceding Claim wherein the R-enantiomer is used. 8 A compound for use as claimed in any preceding Claim wherein the disease is selected from the group consisting of visceral leishmaniasis, mucocutaneous leishmaniasis, cutaneous leishmaniasis, African trypanosomiasis and Chagas' disease. 9. A compound for use as claimed in any preceding Claim wherein the disease is visceral leishmaniasis.

10. A compound as claimed in any one of Claims 1 to 9 wherein the parasite is selected from the group consisting of Leishmania donovani, L. infaraum, L. major, L. tropica, L. aeihiopica, L. mexicana and Trypanosoma cruzi _11. A compound for use as claimed in any preceding Claim wherein the one or more compounds of Formula I are in a form suitable for oral administration. 12. A compound for use as claimed in any preceding Claim wherein the compound is administered to a mammal, typically a human, dog, cat, horse or camel. 13. A compound for use as claimed in any preceding Claim administered at a twice daily dose of 0.5 to 2 mg/kg for at least 5 days. 14. A compound for use as claimed in any one of Claims 1 to 13 administered at a twice daily dose of 10 to 15 mg/kg for at least 10 days. 15. A compound for use as claimed in any one of Claims 1 to 13 administered at a twice daily dose of 20 to 50 mg/kg for at least 5 days. 16. A method of treating a parasitic disease comprising the administration in a therapeutically effective amount of one or more compounds of Formula I wherein the parasitic disease is caused by or associated with parasites selected from the Order Kinetoplastida consisting of species belonging to the genera Leishmania and Trypanosoma. _17. A kit of parts for use in the treatment or alleviation of a parasitic disease caused by or associated with parasites selected from the Order Kinetoplastida consisting of species belonging to the genera Leishmania and Trypanosoma, said kit of parts comprising one or more compounds of Formula I (generally in the form of a composition) and instructions for use.