"Antiparasitic Compounds"

Field of the Invention

The present invention relates to anti-parasitic compounds and their use to treat parasite infections or diseases and more particularly to dinitrobiphenyl compounds and their use as parasiticides.

Background Art

Diseases caused by parasite infections far outnumber diseases caused by other infectious agents. It is estimated that one billion people are infected with the helminthic parasite roundworm alone and that eight hundred million people are infected with hookworm. This does not include the billions of animals infected with parasites.

Parasitic infection vary from severe to very mild infections; and may sometimes be so mild that no clinical or subclinical infection can be detected by measuring weight loss, food consumption, blood parameters, or histopathology. The infectious parasites affect the host in many ways depending on the tissue tropism (site organ and tissue preference) of the specific parasite; the number of parasitic oocysts ingested by the subject in the initial infection; and the pathogenicity of the parasitic species. Often, minimal to no clinical evidence of infection is observed; but the loss to the subject is primarily as a depressed growth and impaired feed conversion. In animals destined for consumption by humans, the infection often results in a downgrading of quality at processing of the carcass, such that the animal no longer qualifies as food fit for human consumption.

Parasite infections in subjects, including humans, are typically treated by chemical drugs. However, the approach of repeated administration of drugs, both prophylactically and therapeutically, has resulted in the selection and survival of drug-resistant parasitic strains that no longer respond to treatment. Furthermore, many of the previously used and currently employed chemical drugs interfere with the host metabolism and are harmful to the subject being treated, often resulting in toxicity or decreased weight gains and feed efficiency when used at high doses. As larger doses become required due to the build up of resistance, the side effects become even greater. Additionally, the life cycle of most parasites includes a variety of life forms, each of which presents different targets, and challenges, for chemical therapy.

Parasitic infections are of great economic significance both directly to humans and to the animals we breed for food, companionship, and other economic benefits Parasitic protozoal diseases include leishmaniasis, including visceral leishmaniasis, mucocutaneous leishmaniasis, and cutaneous leishmaniasis; Chagas disease; human African trypanosomiasis, also known as African sleeping sickness; animal trypanosomiasis; helminthic parasites, Giardia, Cryptosporidium, malarial species etc.

Leishmaniasis currently threatens 350 million men, women and children in 88 countries around the world. The leishmaniases are parasitic diseases with a wide range of clinical symptoms: cutaneous, mucocutaneous and visceral. Visceral leishmaniasis-also known as kala azar is characterized by irregular bouts of fever, substantial weight loss, swelling of the spleen and liver, and anemia

(occasionally serious). If left untreated, the fatality rate can be as high as 100%. In mucocutaneous forms of leishmaniasis, lesions can lead to partial or total destruction of the mucose membranes of the nose, mouth and throat cavities and surrounding tissues. These disabling and degrading forms of leishmaniasis can result in victims being humiliated and cast out from society. Cutaneous forms of the disease normally produce skin ulcers on the exposed parts of the body such as the face, arms and legs. The disease can produce a large number of lesions- sometimes up to 200-causing serious disability and invariably leaving the patient permanently scarred, a stigma which can cause serious social prejudice.

The leishmaniases are caused by different species belonging to the genus

Leishmania a protozoa transmitted by the bite of a tiny 2 to 3 millimeter-long insect vector, the phlebotomine sandfly. Of 500 known phlebotomine species, only some 30 of them have been positively identified as vectors of the disease. Only the female sandfly transmits the protozoan, infecting itself with the Leishmania parasites contained in the blood it sucks from its human or mammalian host in order to obtain the protein necessary to develop its eggs. During a period of 4 to 25 days, the parasite continues its development inside the sandfly where it undergoes major transformation. When the now infectious female sandfly feeds on a fresh source of blood, its painful sting inoculates its new victim with the parasite, and the transmission cycle is completed.

Human African trypanosomiasis, known as sleeping sickness, is a vector-borne parasitic disease. Trypanosoma, the parasites concerned, are protozoa transmitted to humans by tsetse flies (glossina). Tsetse flies live in Africa, and they are found in vegetation by rivers and lakes, gallery-forests and vast stretches of wooded savannah. Sleeping sickness occurs only in sub-Saharan Africa, in regions where tsetse flies are endemic. For reasons as yet unknown, there are many regions where tsetse flies are found, but sleeping sickness is not. The rural populations that live in such environments and depend on them for agriculture, fishing, animal husbandry or hunting are the most exposed-along with their livestock~to the bite of the tsetse fly.

Sleeping sickness affects remote and rural areas where health systems are least effective, or non-existent. It spreads with socio-economic problems such as political instability, displacement of populations, war and poverty. It develops in foci whose size can range from a village to an entire region. Within a given focus, the intensity of the disease can vary considerably from one village to the next.

Human African trypanosomiasis takes two forms, depending on the parasite involved; Trypanosoma brucei gambiense (T.b. gambiense) is found in central and West Africa. It causes chronic infection, which does not mean benign. A person can be infected for months or even years without obvious symptoms of the disease emerging. When symptoms do emerge, the disease is already at an advanced stage. Trypanosoma brucei rhodesiense (T.b. rhodesiense) is found in southern and east Africa. It causes acute infection that emerges after a few weeks. It is more virulent than the other strain and develops more rapidly, which means that it is more quickly detected clinically.

Other sub-species of the parasite cause animal trypanosomiasis, which are pathogenic to animals and are often different from those that cause the disease in humans. Animals can carry parasites, especially T.b. rhodesiense; domestic and wild animals are a major reservoir. They can also be infected with T.b. gambiense, though the precise role of this reservoir is not well known. The two human and animal forms of the disease remain a major obstacle to the development of rural regions of sub-Saharan Africa: human loss, decimation of cattle and abandonment of fertile land where the disease is rife.

Cryptosporidiosis infection varies with host immune competence from mild, self- limiting diarrhoea to life-threatening enteritis complicated by extraintestinal disease. There is no reliable therapy for cryptosporidiosis. The problems of developing in vitro and in vivo methods of screening drugs, such as limited availability and poor reproducibility, have contributed to this lack of effective treatment. However, the major hindrance has been a lack of understanding of the parasite, its virulence and its interactions with the host's immune system.

Insofar as is presently known, therefore, both conventionally known major approaches to treating parasitic infections continue to impose major drawbacks and difficulties regarding their use, efficacy, and concomitant undesirable consequences. For these reasons, new methods for prophylactically and/or therapeutically treating parasitic infections in subjects such as humans and animals intended for human consumption have long been sought without apparent success. Accordingly, the introduction of efficacious anti-parasitic approaches and methods which are relatively simple, rapid, and easy to employ with large numbers of animals would be recognized by practitioners skilled in this art as a major advance and improvement in this field.

Other objects, features, and advantages of the instant invention may be determined from the following description and examples. Summary of the Invention

The present invention provides a compound of Formula A:

wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl, wherein the heteroaryl or substituted heteroaryl group is connected to the 2,4-dinito-6- (trifluoromethyl)phenyl ring via a carbon-carbon bond.

The present invention provides a compound of Formula A:

wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl, wherein the heteroaryl or substituted heteroaryl group is connected to the 2,4-dinito-6- (trifluoromethyl)phenyl ring via a carbon-carbon bond and wherein C6 aryl is substituted.

Z may be substituted with one or more substituent Y, where Y may be independently selected from R ₁ R2, R3, R4 or R5 where:

R is H, C1-C8 alkyl, C3-C8 branched alkyl, C3-C8 cycloalkyl, C2-C8 alkenyl, C3-C8 branched alkenyl, C5-C8 cycloalkenyl, C2-C8 alkynyl, C1-

C8 haloalkyl, C3-C8 branched haloalkyl, C3-C8 cyclohaloalkyl, C2-C8 haloalkenyl, C3-C8 branched haloalkenyl, phenyl, substituted phenyl, benzyl, or substituted benzyl.

R2 is F, Cl, Br, I ₁ CN, NO ₂ or B(OR) ₂ .

R3 is OR5, OCOR5, O-NR5 ₂ , NR5 ₂ , NR5COR5, NR5CONR5 ₂ , NR5CO ₂ R5, NR5COCO ₂ R5, NR5S(O) _X R5, NR5-OR5, NR5C(=NR5)-NR5 ₂ ,

NR5C(=NR5)-R5, N(OR5)COR5, S(O) _X R5, S(O) _X NR5 ₂₎ COR5, CO ₂ R5, CONR5 ₂₎ CR5=N-R5, CR5=N-OR5, C(=NR5)NR5 _2> CON(OR5)R5 or CONR5NR5 ₂ , where x = 0, 1 , or 2.

R4 is (CR ₂ ) _n -R3, CR=CR-(CR ₂ ) _m -R3, CR=CR-CO ₂ R5, CR=CR-CONR5 ₂ or CR=CR-COHET2, where n = 1 , 2, 3, or 4 and m = 1 or 2.

R5 is R, HET2, (CR ₂ ) _n -HET2, (CR ₂ ) _n -OR, (CR ₂ ) _n -OCOR, (CR ₂ ) _n -O-NR ₂ , (CR ₂ )n-NR ₂₎ (CR ₂ ) _n -NRCOR, (CR ₂ ) _n -NRSO ₂ R, (CR ₂ ) _n -NRCONR ₂ , (CR ₂ ) _n - NRCO ₂ R, (CR ₂ )n-NRC(=NR)-NR ₂ , (CR ₂ ) _n -NRC(=NR)-R, (CR ₂ ) _n -CO ₂ R, (CR ₂ )n-CONR ₂ , (CR ₂ ) _n -CONRNR ₂ or (CR ₂ ) _n -CO-HET2, where n = 1 , 2, 3, or 4.

The heteroaryl or substituted heteroaryl may be a 5 or 6 membered monocyclic heteroaryl ring, a 5,6-fused bicyclic heteroaryl ring or a 6,6-fused bicyclic heteroaryl ring, wherein the heteroaryl or substituted heteroaryl contains at least one carbon atom and is connected to the 2,4-dinitro-6-(trifluoromethyl)phenyl ring via a carbon-carbon bond. Examples include, but are not limited to, furan, thiophene, pyrrole, oxazole, thiazole, pyrazole, purine, imidazole, triazole, pyridine, pyridazine, pyrimidine, pyrazine, triazine, benzofuran, benzothiophene, indole, benzoxazole, benzothiazole, benzimidazole, benzofurazan, quinoline, isoquinoline, quinoxaline and quinoxaline.

Where Z is substituted with two substituents, Y which are ortho to each other may, where chemistry allows, be connected by a bond to form a fused ring. Examples include, but are not limited to, indane, tetralin, methylenedioxyphenyl and benzoxazine.

HET2 may be 5 or 6 membered heterocyclic ring, either unsubstituted or substitued. HET2 may be bonded either via a carbon atom or, if appropriate, a nitrogen atom. Examples include, but are not limited to, furan, thiophene, pyrrolidine, pyrrole, oxazole, thiazole, imidazole, pyrazole, triazole, tetrazole, piperidine, morpholine, thiomorpholine, piperazine, pyridine, pyridazine, pyrimidine, pyrazine, and triazine.

In a preferred embodiment of the invention, the present invention provides the compound of the structure:

The present invention also provides a method for preparing a compound of Formula A, the method comprising the step of reacting 2-chloro-3,5- dinitrobenzotrifluoride with the corresponding aryl, substituted aryl, heteroaryl, or substituted heteroaryl organometallic reagent.

The compounds of the present invention may be formulated into compositions for administration. Thus, the present invention also provides a composition comprising a therapeutically-effective amount of a compound of Formula A and a pharmaceutically acceptable carrier or diluent.

The compounds of the present invention have broad antiparasitic activity, and thus are useful for treating or preventing parasitic infections. Thus, the present invention also provides a method of treating a parasitic infection in a subject comprising the step of administering to the subject an effective amount of a compound of Formula A. The present invention further provides a method for the prophylactic or therapeutic treatment of a parasitic infection or disease in a subject comprising the step of administering to the subject an effective amount of a compound of formula A, wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl and wherein the heteroaryl or substituted heteroaryl connected to a 2,4-dinito-6-(trifluoromethyl)phenyl ring via a carbon-carbon bond.

The present invention further provides for the use of a compound of formula A or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylactic or therapeutic treatment of parasitic infection or disease in a subject in need thereof, where in Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl and wherein the heteroaryl or substituted heteroaryl connected to a 2,4-dinito-6-(trifluoromethyl)phenyl ring via a carbon-carbon bond.

Brief Description of the Drawings

Figure 1 is a table of the in vitro antiparasitic activity of the heteroaryl analogue compounds of the present invention against Cryptosporidium, L. donovani, T. b. rhodesiense, and T. cruzi.;

Figure 2 is a table of the in vitro antiparasitic activity of the simple phenyl analogue compounds of the present invention against Cryptosporidium, L. donovani, T. b. rhodesiense, and T. cruzi.;

Figure 2A is a table of the in vitro antiparasitic activity of the disubstituted and fused aryl analogue compounds of the present invention against Cryptosporidium, L. donovani, T. b. rhodesiense, and T. cruzi.;

Figure 3 is a table of the in vitro antiparasitic activity of the 4- carboxamidophenyl analogue compounds of the present invention against 7. b. rhodesiense, and T. cruzi.; Figure 4 is a table of the in vitro antiparasitic activity of the 4- (aminomethyl)phenyl analogue compounds of the present invention against T. b. rhodesiense, and T. cruzi.;

Figure 5 is a table of the in vitro antiparasitic activity of the 3- carboxamidophenyl analogue compounds of the present invention against T. b. rhodesiense, and T. cruzi.;

Figure 6 is a table of the in vitro antiparasitic activity of the 3- (aminomethyl)phenyl analogue compounds of the present invention against T. b. rhodesiense, and T. cruzi.; and

Figure 7 is a table of the in vivo antiparasitic activity of compound #9 (Figure 2) of the present invention as determined in mice infected with T. b. rhodesiense.

Description of the Invention

Compounds

The present invention provides a compound of Formula A:

-wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl, wherein the heteroaryl or substituted heteroaryl group is connected to the 2,4-dinito-6- (trifluoromethyl)phenyl ring via a carbon-carbon bond.

The present invention provides a compound of Formula A:

(A)

wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl, wherein the heteroaryl or substituted heteroaryl group is connected to the 2,4-dinito-6- (trifluoromethyl)phenyl ring via a carbon-carbon bond and wherein C6 aryl is substituted.

The compound of the present invention is preferably selected from the group comprising the compounds listed in Figure 1.

In a preferred form of the invention, the compound of the present invention is a compound according to Formula A, wherein Z is selected from the group comprising 2-thiophene, 3-thiophene, 3-furan, 4-pyrazole, 2-methyl-4-pyrazole, 2- (2-methylpropyl)-4-pyrazole, 2,6-dimthoxy-3-pyridine, 2,4-dimethoxy-5-pyrimidine, 1-benzyl-1 ,2,3-triazol-4-yl, 1-(4-methoxybenzyl)-1 ,2,3-triazol-4-yl,5-indole, 1- phenyl-1 ,2,3-triazol-4-yl,1 -cyclopropyl-1 ,2,3-triazol-4-yl,1 -(2-(morpholin-4-yl)ethyl)- 1 ,2,3-triazol-4-yl,1-(indan-4-yl)-1 ,2,3-triazol-4-yl,1-methyl-5-indole and 5- benzooxadiazole.

Z may be substituted with one or more substituent Y, where Y is independently selected from R, R2, R3, R4 or R5 where:

R is H, C1-C8 alkyl, C3-C8 branched alkyl, C3-C8 cycloalkyl, C2-C8 alkenyl, C3-C8 branched alkenyl, C5-C8 cycloalkenyl, C2-C8 alkynyl, C1- C8 haloalkyl, C3-C8 branched haloalkyl, C3-C8 cyclohaloalkyl, C2-C8 haloalkenyl, C3-C8 branched haloalkenyl, phenyl, substituted phenyl, benzyl, or substituted benzyl. R2 is F, Cl, Br, I, CN, NO ₂ or B(OR) ₂

R3 is OR5, OCOR5, O-NR5 ₂ , NR5 ₂ , NR5COR5, NR5CONR5 ₂ , NR5CO ₂ R5, NR5COCO ₂ R5, NR5S(O) _X R5, NR5-OR5, NR5C(=NR5)-NR5 ₂ , NR5C(=NR5)-R5, N(OR5)COR5, S(O) _X R5, S(O) _X NR5 ₂ , COR5, CO ₂ R5, CONR5 ₂ , CR5=N-R5, CR5=N-OR5, C(=NR5)NR5 _2> CON(OR5)R5 or

CONR5NR5 ₂ , where x = 0, 1 , or 2.

R4 is (CR ₂ ) _n -R3, CR=CR-(CR ₂ ) _m -R3, CR=CR-CO ₂ R5, CR=CR-CONR5 ₂ or CR=CR-COHET2, where n = 1, 2, 3, or 4 and m = 1 or 2.

R5 is R, HET2, (CR ₂ ) _n -HET2, (CR ₂ ) _n -OR, (CR ₂ ) _n -OCOR, (CR ₂ ) _n -O-NR ₂ , (CR ₂ )n-NR ₂₎ (CR ₂ ) _n -NRCOR, (CR ₂ ) _n -NRSO ₂ R, (CR ₂ ) _n -NRCONR ₂₎ (CR ₂ ) _n -

NRCO ₂ R, (CR ₂ )n-NRC(=NR)-NR ₂ , (CR ₂ ) _n -NRC(=NR)-R, (CR ₂ ) _n -CO ₂ R, (CR ₂ ) _n -CONR ₂ , (CR ₂ ) _n -CONRNR ₂ or (CR ₂ ) _n -CO-HET2, where n = 1 , 2, 3, or 4.

The heteroaryl or substituted heteroaryl may be a 5 or 6 membered monocyclic heteroaryl ring, a 5,6-fused bicyclic heteroaryl ring or a 6,6-fused bicyclic heteroaryl ring, wherein the heteroaryl or substituted heteroaryl contains at least one carbon atom and is connected to the 2,4-dinitro-6-(trifluoromethyl)phenyl ring via a carbon-carbon bond. Examples include, but are not limited to, furan, thiophene, pyrrole, oxazole, thiazole, pyrazole, purine, imidazole, triazole, pyridine, pyridazine, pyrimidine, pyrazine, triazine, benzofuran, benzothiophene, indole, benzoxazole, benzothiazole, benzimidazole, benzofurazan, quinoline, isoquinoline, quinoxaline and quinoxaline.

Where Z is substituted with two substituents, Y which are ortho to each other may, where chemistry allows, be connected by a bond to form a fused ring. Examples include, but are not limited to, indane, tetralin, methylenedioxyphenyl and benzoxazine. HET2 may be 5 or 6 membered heterocyclic ring, either unsubstituted or substitued. HET2 may be bonded either via a carbon atom or, if appropriate, a nitrogen atom. Examples include, but are not limited to, furan, thiophene, pyrrolidine, pyrrole, oxazole, thiazole, imidazole, pyrazole, triazole, tetrazole, piperidine, morpholine, thiomorpholine, piperazine, pyridine, pyridazine, pyrimidine, pyrazine, and triazine.

In a preferred form of the invention, the present invention provides a compound of Formula B:

In a preferred form of the invention, the compound of the present invention according to Formula B is selected from the group comprising the compounds listed in Figure 2, wherein X1 may be a substituent selected from the substituents listed in Figure 2.

In a preferred form of the invention, the compound of the present invention according to Formula B is selected from the group comprising the compounds listed in Figure 2, wherein X1 is a substituent selected from the group comprising 2-Me ₁ 3-Me, 4-/-Pr, 4-f-Bu, 3-F, 4-F, 3-CI, 4-CI, 3-OH, 4-OH, 2-OMe, 3-OMe, 4- OMe, 4-O-/-Pr, 4-OCF ₃ , 3-OCH ₂ CO ₂ Et, 2,4-di-OMe, , 3,4-methylenedioxy, , 3-NH _2, 4-NH ₂ , 4-NMe ₂ , 3-NHAc, 4-NHAc, 4-NHCOCO ₂ Me, 4-NHCOCO ₂ H, 4-NHCO ₂ -J-Bu, 4-NHSO ₂ Me, 4-SO ₂ Me, 4-SO ₂ NH ₂ , 3-CH ₂ OH, 4-CH ₂ OH, 3-CO ₂ H, 4-CO ₂ H, 4- CO ₂ Me, 4-CONHOH, 3-CHO, 4-CHO, 3-CN, 4-CN, 4-C(=NH)NH ₂ HCI, 3- CH=NOH, 4-CH=NOH, 4-CH=N-(I -piperidyl) HCI and 4-(5-tetrazole). In a preferred form of the invention, the present invention provides a compound of the structure:

In another form of the invention, the compound of the present invention is selected from the group comprising the compounds as identified by their chemical formula listed in Figures 1 to 6.

The compound of the present invention may be selected from the group consisting of: 3-[2,4-dinitro-6-(trifluoromethyl)phenyl]thiophene; 2,4-dinitro-6-

(trifluoromethyl)biphenyl; 2',4'-dinitro-6'-(trifluoromethyl)biphenyl-4-carbaldehyde;

2',4 ¹ -dinitro-6'-(trifluoromethyl)biphenyl-4-carboxylic acid; 2',4'-dinitro-6'-

(trifluoromethyl)biphenyl-4-carbonyl chloride; ty/V-bisføropyO^'^'-dinitro-θ'-

(trifluoromethyl)biphenyl-4-carboxamide; and N-{[2' ,4'-d\r\\\ro-Q'- (trifluoromethyl)biphenyl-4-yl]methyl}-Λ/-propylpropanamine HCI.

In a further form of the invention, the present invention provides a compound of Formula C:

wherein X2 and X3 may be substituents selected from the substituents listed in Figure 3. In a preferred form of the invention, the compound of the present invention according to Formula C is selected from the group comprising the compounds listed in Figure 3, wherein Z is p-benzamide, the amide substituted with X2 and X3, wherein X2 is a substituent selected from the group comprising Pr, CH ₂ CH ₂ OH, CH ₂ CH ₂ NEt ₂ , 3-pyridyl, CH ₂ CH ₂ -(Λ/-morpholinyl), /V-piperidyl, CH ₂ CH ₂ CH ₂ CH ₂ , CH ₂ CH ₂ OCH ₂ CH ₂ , CH ₂ CH ₂ SCH ₂ CH ₂ , CH ₂ CH ₂ N(Me)CH ₂ CH ₂ , CH ₂ CH ₂ N(Et)CH ₂ CH ₂ , CH ₂ CH ₂ N(Ac)CH ₂ CH ₂ , CH ₂ CH ₂ N(CO ₂ Et)CH ₂ CH ₂ , CH ₂ CH ₂ N(CH ₂ CH ₂ OH)CH ₂ CH ₂ , CH ₂ CH ₂ N(2-pyrimidyl)CH ₂ CH ₂₎

CH ₂ CH ₂ CH(OH)CH ₂ CH ₂ and CH ₂ CH ₂ CH(Λ/-pyrrolidinyl)CH ₂ CH ₂ and X3 is a substituent selected from the group comprising Pr, CH ₂ CH ₂ OH, Et and H.

In a further form of the invention, the present invention provides a compound of Formula D:

wherein X4 and X5 may be substituents selected from the substituents listed in Figure 4.

In a preferred form of the invention, the compound of the present invention according to Formula D is selected from the group comprising the compounds listed in Figure 4, wherein Z is p-benzyl amine, the amine substituted with X4 and X5, wherein X4 is a substituent selected from the group comprising Pr(HCI salt), CH ₂ CH ₂ OH(HCI salt), CH ₂ CH ₂ NEt ₂ (HCI salt), Me(HCI salt), CH ₂ CO ₂ Et(HCI salt), cyclopropyl(HCI salt), CH ₂ -(3-pyridyl)(HCI salt), CH ₂ -(4-methoxyphenyl)(HCI salt), CH ₂ CH ₂ CH ₂ CH ₂ (HCI salt), CH ₂ CH ₂ OCH ₂ CH ₂ , CH ₂ CH ₂ N(Me)CH ₂ CH ₂ , CH ₂ CH(Me)OCH(Me)CH ₂ (HCI salt), CH ₂ CH ₂ N(Ac)CH ₂ CH ₂ ,

CH ₂ CH ₂ N(CO ₂ Et)CH ₂ CH ₂ , CH ₂ CH ₂ N(CH ₂ CH ₂ OH)CH ₂ CH ₂ (di HCI salt), CH ₂ CH ₂ CH(OH)CH ₂ CH ₂ and CH ₂ CH ₂ CH(CH ₂ OH)CH ₂ CH ₂ (HCI salt) and X5 is a substituent selected from the group comprising Pr, CH ₂ CH ₂ OH, Et, Me and H.

In a further form of the invention, the present invention provides a compound of Formula E:

wherein X6 and X7 may be substituents selected from the substituents listed in Figure 5.

In a preferred form of the invention, the compound of the present invention according to Formula E is selected from the group comprising the compounds listed in Figure 5, wherein Z is m-benzamide, the amide substituted with X6 and X7, wherein X6 is a substituent selected from the group comprising Pr ₁ 3-pyridyl, CH ₂ CH ₂ -(Λ/-morpholinyl), /V-piperidyl, CH ₂ CH ₂ CH ₂ -(I -imidazyl), CH ₂ CH ₂ CH ₂ CH ₂ , CH ₂ CH ₂ OCH ₂ CH ₂ , CH ₂ CH ₂ SCH ₂ CH ₂ , CH ₂ CH ₂ N(Me)CH ₂ CH ₂ ,

CH ₂ CH ₂ N(Et)CH ₂ CH ₂ , CH ₂ CH ₂ N(Ac)CH ₂ CH ₂ , CH ₂ CH ₂ N(CO ₂ Et)CH ₂ CH ₂ , CH ₂ CH ₂ N(2-pyrimidyl)CH ₂ CH ₂ and CH ₂ CH ₂ CH(OH)CH ₂ CH ₂ and X7 is a substituent selected from the group comprising Pr and H.

In a further form of the invention, the present invention provides a compound of Formula F:

wherein X8 and X9 may be substituents selected from the substituents listed in Figure 6.

In a preferred form of the invention, the compound of the present invention according to Formula F is selected from the group comprising the compounds listed in Figure 6, wherein Z is m-benzyl amine, the amine substituted with X8 and X9, wherein X8 and X9 are substituents selected from group comprising CH ₂ CH ₂ OCH ₂ CH ₂ , CH ₂ CH ₂ N(Me)CH ₂ CH ₂ , CH ₂ CH ₂ N(CH ₂ CH ₂ OH)CH ₂ CH ₂ and CH ₂ CH ₂ N(2-pyrirnidyl)CH ₂ CH _2.

In a further embodiment of the invention, the present invention provides a compound of Formula G:

(G)

The compound of the present invention may be provided in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts for the purposes of the present invention include non-toxic acid addition salts formed with pharmaceutically acceptable acids. Examples include, but are not limited to, hydrochloride, hydrobromide, sulphate and phosphate, acetate, borate, nitrate, citrate, fumarate, gluconate, lactate, maleate, succinate and tartrate salts. Other salts include pharmaceutically acceptable metal salts such as non-toxic alkali metal salts, with bases. Examples include, but are not limited to sodium and potassium salts, ammonium and alkylammonium salts including tetralkylammonium salts.

Methods of preparing compounds

The present invention provides a method for preparing a compound of Formula A wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl; the method comprising the step of reacting 2-chloro-3,5-dinitrobenzotrifluoride with the corresponding aryl, substituted aryl, heteroaryl, or substituted heteroaryl moiety in the form of an organoboron or organometallic reagent.

The present invention provides a method for preparing a compound of Formula A; the method comprising the reaction:

(A)

wherein K is 2-chloro-3,5-dinitrobenzotriflouride, in the presence of compounds comprising, for example but not at the exclusion of others, a catalyst, a base and where Z-B(OR)2 is a organoboron compound.

The catalyst may be provided as a palladium or nickel catalyst.

The present method also provides a method for preparing a compound of Formula A comprising use of the Suzuki Coupling method (Suzuki, A. (1999) Journal of Organometallic Chemistry. 576: 147-168).

The organoboron compound may be, but is not limited to, boronic acids or boronate esters, particularly pinacol esters.

Organoboron compounds may be prepared by the method comprising, but not limited to, treatment of an organomagnesium or organolithium reagent with a trialkyl borate followed by hydrolysis of the intermediate borate ester, if required.

Bis(pinacolato)diboron may be used as a reagent for preparation of pinacol esters. In an embodiment of the invention, the organometallic reagent comprises organomagnesium or organozinc, amongst others.

It will be appreciated that use of an organometallic reagent may require a suitable transition metal catalyst.

The present method provides a method for preparing a compound of Formula B wherein the biaryl bond connecting the aryl or heteroaryl ring to the 2,4-dinitro-6- (trifluoromethyl)phenyl ring is formed before X1 has been introduced.

Other related methods for forming the biaryl bond comprise treating 2-chloro-3,5- dinitrobenzotrifluoride with other suitable organometallic reagents including, but not limited to, organomagnesiums or organozincs, in the presence of suitable transition metal catalysts.

The present method provides a further method for preparing a compound of Formula B comprising the reactions:

wherein the aldehyde M is used to prepare the compounds comprising, but not limited to, benzylamines N, benzyl alcohol P, oxime and oxime ethers Q, or carboxylic acid L; or

wherein carboxylic acid L can be further transformed into carboxamides O.

Those skilled in the art will be aware of other functional group transformations which could form part of a compound of Formula B.

In some instances it is convenient to construct the heteroaryl moieties from a suitably substituted 3,5-dinitrobenzotrifluoride. An example of this strategy is illustrated in the following synthesis of triazoles (S) from the intermediate alkyne (R). The initroduction of the ethynyl group into (K) to give (R) is readilly achieved using a Sonogashira coupling, a reaction well known in the art (Chincilla and Najera, Chem. Rev., 2007, 107(3), 874-922). The subsequent formation of the triazole ring to give the desired compounds (S) is readily achieved using a azide- alkyne Huisgen cycloaddition, a reaction also well known in the art (Sharpless et a/., Angew. Chem. Int. Ed. 2001 , 40(11 ), 2004-2021 ).

RN,

Cu(II) + ascorbate

Compositions

The compounds of the present invention may be formulated into compositions for administration. Thus, the present invention also provides a composition comprising a therapeutically-effective amount of a compound of Formula A:

(A)

wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl; and a pharmaceutically acceptable carrier or diluent.

The present invention also provides a composition comprising a therapeutically- effective amount of a compound of Formula A:

wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl and wherein C6 aryl is substituted.; and a pharmaceutically acceptable carrier or diluent.

The precise composition of the present invention will vary according to a wide range of commercial and scientific criteria. Methods for the preparation of pharmaceutical compositions comprising one or more active ingredients are generally known in the art. Such compositions will generally be formulated for the mode of delivery that is to be used and will usually include one or more pharmaceutically acceptable carriers.

Generally, examples of suitable carriers, excipient and diluents include, without limitation, water, saline, ethanol, dextrose, glycerol, lactose, dextrose, sucrose sorbitol, mannitol, starches, gum acacia, calcium phosphates, alginate, tragacanth, gelatine, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate and mineral oil or combinations thereof. The formulations can additionally include lubricating agents, pH buffering agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavouring agents.

(a) Topicals

The pharmaceutical composition may be adapted for topical application. In this regard, various topical delivery systems may be appropriate for administering the compositions of the present invention depending upon the preferred treatment regimen. Topical formulations may be produced by dissolving or combining the compound of the present invention in an aqueous or nonaqueous carrier. In general, any liquid, cream, or gel, or similar substance that does not appreciably react with the compound or any other of the active ingredients that may be introduced into the composition and which is non-irritating is suitable. Appropriate non-sprayable viscous, semi-solid or solid forms can also be employed that include a carrier compatible with topical application and have a dynamic viscosity preferably greater than water.

Suitable formulations are well known to those skilled in the art and include, but are not limited to, solutions, suspensions, emulsions, creams, gels, ointments, powders, liniments, salves, aerosols, transdermal patches, etc, which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, emulsifiers, wetting agents, fragrances, colouring agents, odour controllers, thickeners such as natural gums etc. Particularly preferred topical formulations include ointments, creams or gels.

Ointments generally are prepared using either (1 ) an oleaginous base, i.e., one consisting of fixed oils or hydrocarbons, such as white petroleum or mineral oil, or

(2) an absorbent base, i.e., one consisting of an anhydrous substance or substances which can absorb water, for example anhydrous lanolin. Customarily, following formation of the base, whether oleaginous or absorbent, the active ingredient is added to an amount affording the desired concentration.

Creams are oil/water emulsions. They consist of an oil phase (internal phase), comprising typically fixed oils, hydrocarbons and the like, waxes, petroleum, mineral oil and the like and an aqueous phase (continuous phase), comprising water and any water-soluble substances, such as added salts. The two phases are stabilised by use of an emulsifying agent, for example, a surface active agent, such as sodium lauryl sulfite; hydrophilic colloids, such as acacia colloidal clays, veegum and the like. Upon formation of the emulsion, the compound can be added in an amount to achieve the desired concentration.

Gels comprise a base selected from an oleaginous base, water, or an emulsion- suspension base. To the base is added a gelling agent that forms a matrix in the base, increasing its viscosity. Examples of gelling agents are hydroxypropyl cellulose, acrylic acid polymers and the like. Customarily, the compound is added to the formulation at the desired concentration at a point preceding addition of the gelling agent.

The amount of compound incorporated into a topical formulation is not critical; the concentration should be within a range sufficient to permit ready application of the formulation such that an effective amount of the compound is delivered.

(b) Oral Formulations

The pharmaceutical composition may be adapted for oral delivery. In this regard, the compound can be administered as an oral preparation adapted in such a manner that facilitates delivery of a therapeutically effective concentration of the compound.

The effective dosages of the compound, when administered orally, must take into consideration the diluent, preferably water. The composition preferably contains 0.05% to about 100% by weight active ingredient and more preferably about 10% to about 80% by weight. When the compositions are ingested, desirably they are taken on an empty stomach. Contemplated for use herein are oral solid dosage forms including tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions. Liposomal encapsulation may be used and the liposomes may be derivatised with various polymers. In general, the formulation will include the compound and inert ingredients that allow for protection against the stomach environment and release of the biologically active material in the intestine.

The location of release may be the stomach, the small intestine (the duodenum, the jejunem, or the ileum), or the large intestine. One skilled in the art has available formulations that will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the composition or by release of the compound beyond the stomach environment, such as in the intestine.

To ensure full gastric resistance, a coating impermeable to at least pH 5.0 may be used. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S and Shellac. These coatings may be used as mixed films.

A coating or mixture of coatings that are not intended for protection against the stomach can also be used on tablets. This can include sugar coatings, or coatings that make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatine) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatine shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, moulded tablets or tablet triturates, moist massing techniques can be used.

One may dilute or increase the volume of the composition with an inert material.

These diluents could include carbohydrates, especially mannitol, alpha-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the compound into a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatine, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants is insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders may be used to hold the composition together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatine. Others include methylcellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the compound.

An antifrictional agent may be included in the formulation to prevent sticking during the formulation process. Lubricants may be used as a layer between the compound and the die wall and these can include but are not limited to: stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights and Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the composition during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the compound, a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation either alone or as a mixture in different ratios.

Controlled release formulations may be desirable. The compounds can be incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms i.e., gums. Slowly degenerating matrices may also be incorporated into the formulation. Another form of a controlled release formulation is by a method based on the Oros therapeutic system (Alza Corp.), i.e. the composition is enclosed in a semipermeable membrane which allows water to enter and push the composition out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.

A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidised bed or by compression coating.

The compound can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The compound could be prepared by compression.

(c) Injectable formulations

The compound can also be formulated for parenteral delivery. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water- soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Alternatively, the compounds of the invention may be encapsulated in liposomes and delivered in injectable solutions to assist their transport across cell membrane. The solution may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures thereof and vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatine.

Sterile injectable solutions may be prepared by incorporating the active compounds in the required amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the compound into a sterile vehicle that contains the basic dispersion medium and the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques that yield a powder of the compound plus any additional desired ingredient from previously sterile-filtered solution thereof.

Thus, the present invention also provides an injectable, stable, sterile composition comprising a compound of Formula A, or a salt thereof, in a unit dosage form in a sealed container. The compound or salt may be provided in lyophilised form capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 10 mg to about 10 grams of the compound or salt thereof. When the compound or salt is substantially water- insoluble, a sufficient amount of emulsifying agent which is physiologically acceptable may be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

(d) Aerosols

Pharmaceutical compositions are also provided which are suitable for administration as an aerosol, by inhalation. These compositions comprise a solution or suspension of the desired compound or a salt thereof or a plurality of solid particles of the compound or salt. The desired composition may be placed in a small chamber and nebulized. Nebulization may be accomplished by compressed air or by ultrasonic energy to form a plurality of liquid droplets or solid particles comprising the compounds or salts.

The solid particles can be obtained by processing solid compound or a salt thereof, in any appropriate manner known in the art, such as by micronization. Commercial nebulizers are also available to provide liquid droplets of any desired size.

The liquid droplets or solid particles should have a particle size in the range of about 0.5 to about 5 microns, preferably from about 1 to about 2 microns. Most preferably, the size of the solid particles or droplets will be from about 1 to about 2 microns. Such particles or droplets may be dispensed by commercially available nebulisers or by other means known to the skilled person.

When the pharmaceutical composition suitable for administration as an aerosol is in the form of a liquid, the composition will comprise a water-soluble form of the compound or a salt thereof, in a carrier that comprises water. A surfactant may be present which lowers the surface tension of the composition sufficiently to result in the formation of droplets within the desired size range when subjected to nebulization.

In addition, the pharmaceutical composition may also include other agents. For example, preservatives, co-solvents, surfactants, oils, humectants, emollients, chelating agents, dyestuffs, stabilizers or antioxidants may be employed. Water soluble preservatives that may be employed include, but are not limited to, benzalkonium chloride, chlorobutanol, thimerosal, sodium bisulfate, phenylmercuric acetate, phenylmercuric nitrate, ethyl alcohol, methylparaben, polyvinyl alcohol, benzyl alcohol and phenylethyl alcohol. A surfactant may be Tween 80. Other suitable additives include lubricants and slip agents, such as, for example, magnesium stearate, stearic acid, talc and bentonites, substances which promote disintegration, such as starch or crosslinked polyvinylpyrrolidone, binders, such as, for example, starch, gelatin or linear polyvinylpyrrolidone, and dry binders, such as microcrystalline cellulose.

Other vehicles that may be used include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose, purified water, etc. Tonicity adjustors may be included, for example, sodium chloride, potassium chloride, mannitol, glycerin, etc. Antioxidants include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydraxyanisole, butylated hydroxytoluene, etc. The indications, effective doses, compositions, contraindications, vendors etc, of the compounds in the compositions are available or are known to one skilled in the art. These agents may be present in individual amounts of from about 0.001% to about 5% by weight and preferably about 0.01% to about 2%.

Electrolytes such as, but not limited to, sodium chloride and potassium chloride may also be included in the composition.

Further, the compositions may contain microbial preservatives. Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the composition is placed in a vial designed for multidose use.

Excipients which may be used are all the physiologically acceptable solid inert substances, either inorganic or organic in nature. Inorganic substances are, for example, sodium chloride, carbonates, such as calcium carbonate, bicarbonates, aluminium oxides, silicic acids, aluminas, precipitated or colloidal silicon dioxide and phosphates. Organic substances are, for example, sugars, cellulose, foodstuffs and feedstuffs, such as milk powder, animal flours, cereal flours and shredded cereals and starches.

Finally, it will be appreciated that the compositions of the present invention may comprise a plurality of compounds as described herein. Method of treatment

The compounds of the present invention have broad antiparasitic activity, and thus are useful for treating or preventing parasitic infections. Thus, the present invention also provides a method of treating a parasitic infection in a subject comprising the step of administering to the subject an effective amount of a compound of Formula A:

wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl and wherein the heteroaryl or substituted heteroaryl group is connected to the 2,4- dinito-6-(trifluoromethyl)phenyl ring via a carbon-carbon bond; or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of treating a parasitic infection in a subject comprising the step of administering to the subject an effective amount of a compound of Formula A:

wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl and wherein the heteroaryl or substituted heteroaryl group is connected to the 2,4- dinito-6-(trifluoromethyl)phenyl ring via a carbon-carbon bond and wherein C6 aryl is substituted; or a pharmaceutically acceptable salt thereof. For the purposes of the present invention the term "treating" means ameliorating the deleterious effects of a parasitic infection, inhibiting the onset, growth, or spread of the infection, causing regression of the infection, curing the infection, or otherwise improving the general well-being of an infected subject. This may be achieved by killing or inactivating the parasites or by inhibiting or preventing their reproduction. Preferably, treatment according to the present invention involves the eradication of the parasite and thus cures the infection. For the purposes of the present invention "treating" also encompasses preventing infection or reinfection with a parasite and thus also covers the prophylactic use of the compound.

The parasitic infection may be an infection caused by an endoparasite or an ectoparasite.

Preferably, the parasitic infection is caused by a parasite selected from the group consisting of: trypanosomes; haemoprotozoa and parasites capable of causing malaria; enteric and systemic cestodes including taeniid cestodes; enteric coccidians; enteric flagellate protozoa; filarial nematodes; gastrointestinal and systemic nematodes and hookworms.

More preferably, the trypanosomes are selected from the genera Trypanosoma and Leishmania; the enteric coccidians are selected from the genera Cryptosporidium and Eimeria; the haemoprotazoa are selected from the genera Plasmodium; the taeniid cestodes are selected from the genera Echinococcus; the enteric flagellate protozoa are selected from the genera Giardia; the gastrointestinal and systemic nematodes and hookworms are selected from the genera Amidostomum, Trichostrongylus, Tenorastrongylus, Nippostrongylus, Heligmonina, Boreostrongylus, Ancylostoma; and the filarial nematodes are selected from the genera Wucherieria, Onchocera and Dirofilaria.

Even more preferably, parasitic infection is caused by a parasite selected from the group comprising: Cryptosporidium andersoni, Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium hominis, Cryptosporidium wrairi, Cryptosporidium felis, Cryptosporidium canis, Cryptosporidium baileyi, Cryptosporidium meleagridis, Cryptosporidium galli, Cryptosporidium serpentis, Cryptosporidium saurophilum and Cryptosporidium molnari; Echinococcus granulosus, E. multilocularis, E. vogeli, E. oligarthrus; Trypanosome brucei rhodesiense, T. brucei gambiense, T brucei, T. cruzi, T. evansi, T. equiperdum, T. vivax; G. intestinalis; L brasiliensis, L. donavani, L. ethiopica L. mexicana L. peruviana, L. tropica, L. major, L. infantum, Plasmodium falciparum, P. humain et simian; Caenorhabditis elegans, Caenorhabditis briggsae, Caenorhabditis drosophilae, Caenorhabditis japonica, Caenorhabditis maupasi, Caenorhabditis plicata, Caenorhabditis remanei, Caenorhabditis sonorae, Caenorhabditis sp. CB5161, Caenorhabditis sp. DF5070, Caenorhabditis sp. PS1010, Caenorhabditis sp. SB341 Caenorhabditis vulgaris, Amidostomum fulicae, A. acutum, Trichostrongylus colubriformis Tenorastrongyfus josephi, Nippostrongylus brasiliensis, Nippostrongylus witenbergi, Heligmonina nevoi; Boreostrongylus seurati, Boreostrongylus minutes, Heligmosomoides polygyrus, Wuchereria bancrofti, Onchocerca volvulus, Dirofilaria immitis, Schistosoma mansoni, S. haematobium, S. japonicum, Blastocytis hominis, Pediculus humanis capitis, Onchocera volvulus, Sarcoptes scabei, Trichomonas vaginalis, Toxocaria canis, T. cati and Toxoplasma gondii.

For example, the infection may be caused by a parasite selected from the group consisting of: Trypanosoma brucei rhodesiense, T. brucei, T. cruzi, Leishmania donovani, or Cryptosporidium.

The present invention also provides a method of treating a parasitic disease in a subject comprising the step of administering to the subject an effective amount of a compound as herein described, wherein said disease is selected from the group comprising: trypanosomiasis, cryptosporidiosis, leishmaniasis, malaria, coccidiosis, giardiasis, hookworm infection, Chagas disease, Schistosomiasis (bilharzia), Blastocystosis, filariasis, head, pubic and body lice infection, ascariasis, onchocerciasis (River blindness), scabies, toxocariasis and toxoplasmosis. The subjects treated by the method of the present invention may be human but are typically non-human subjects such as animals and in particular livestock and other economically important animals that are farmed or otherwise managed by humans for commercial gain. Thus, subjects encompass any animal, preferably a vertebrate and more preferably a mammal or a bird. Non-mammalian vertebrates include reptiles and freshwater and salt water fish, such as, for example, trout, carp and eels. Also included are insects, such as, for example, honey bees and silk worms. Mammals include, but are not limited to, humans, sport animals, livestock such as cows, horses, sheep, pigs, goats, camels, water buffalo, donkeys, rabbits, fallow deer and reindeer, fur-bearing animals, such as, for example, mink, chinchillas and raccoons. Also included are pets such as dogs, cats and horses and laboratory and experimental animals such as mice, rats, guinea pigs and hamsters. Birds include, but are not limited to, avian livestock such as chickens, geese, turkeys and ducks and pet birds such as pigeons and song birds.

The compound may be administered via any route as deemed appropriate by a suitably qualified practitioner including orally, by inhalation, topically, intramuscularly or intravenously.

As indicated above the compounds of the present invention may also be used to protect a subject against parasite infection, the method comprising the step of administering a prophylactically effective amount of the compound. Such administration may be desired, for example, if the subject is in contact with other subjects which have the infection, or is going to enter an area where the parasite is known to be present.

The effective amounts and dosage requirements (i.e., the amount of each dose, the concentration of the compound used and the frequency of administration) of a subject undergoing treatment of the invention may vary depending on the nature of the compound, the clinical condition of the subject, the diluent, severity and nature of the infection, the response of the subject, the route of delivery or delivery device selected, the side effects and the stability of the compound in the composition. Thus, the skilled person administering the composition comprising a compound of the invention will employ the appropriate preparation containing the appropriate concentration of the compound and select the amount of composition administered, depending upon clinical experience with the subject in question or with similar subjects.

Administration of the compounds of the invention can be carried out in single or multiple doses. As a general proposition, a dosage from about 0.1 to 50, 0.5 to 40 or 1 to 30mg/kg will have therapeutic efficacy, with still higher dosages potentially being employed for oral and/or aerosol administration. Toxicity concerns at the higher level may restrict intravenous dosages to a lower level, all weights being calculated based upon the weight of the active compound, including the cases where a salt is employed. Typically a dosage from about 0.5 mg/kg to about 5 mg/kg will be employed for intravenous or intramuscular administration. A dosage from about 10 mg/kg to about 100 mg/kg may be employed for oral administration.

The duration of the treatment may be once daily for a period of two to three weeks or until the infection is essentially controlled. Lower doses given less frequently can be used to prevent or reduce the incidence or recurrence of the infection.

The pharmaceutically effective compositions of the present invention may be administered to a subject by any method that leads to delivery of the compound to the site of the infection. The invention is therefore not limited to any one form of delivery in that it includes topical (e.g. application to the skin), systemic (e.g. orally), parenteral (e.g. by intramuscular, intravenous, intraocular or intranasal injection), by inhalation (e.g. using aerosols) or by other means known to the skilled person provided that a sufficient amount of the active compound achieves contact with the site of the endoparasitic infection.

Where the composition comprises two or more active agents (e.g. two or more compounds as described herein, or a compound as described herein and another agent), the active agents may be administered as a mixture, as an admixture, in the same composition, in separate compositions, in extended release compositions, liposomes, microcapsules, or any of the previously described embodiments. For example, the composition may be administered topically, or may be injected, or one active agent may be administered topically and the other agent(s) may be injected.

Administration of the composition may be parenteral means (by chemical delivery system or invasive device) to a subject, although other modes of administration may be effective. Parenteral administration is used in appropriate circumstances apparent to the practitioner. Preferably, the compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts.

Parenteral delivery encompasses injection through a variety of routes, such as subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intravenous, intranasal etc. The compound to be delivered may be in a depot form. Other parenteral routes of administration and injection sites and forms are also contemplated and are within the scope of the invention.

Additionally, it is also possible to administer the compounds of the present invention topically to an affected part such as the skin. Topical application of compositions of the invention for the treatment or prevention of endoparasitic infections may be as an ointment, gel, eye drops, creams, lotions etc. Preferably a penetrating composition comprising the active compound is used.

Such solutions for use on the skin may be dripped on, brushed on, massaged in, sprinkled on or sprayed on. Pour-on formulations are poured or sprinkled onto limited areas of the skin, the active compound penetrating through the skin and having a systemic action.

The topical composition may further be an in situ gellable aqueous composition. Such a composition comprises a gelling agent in a concentration effective to promote gelling upon contact with the skin, mucous membranes etc. Suitable gelling agents include, but are not limited to, thermosetting polymers such as tetra-substituted ethylene diamine block copolymers of ethylene oxide and propylene oxide (e.g., poloxamine); polycarbophil; and polysaccharides such as gellan, carrageenan (e.g., kappa-carrageenan and iota-carrageenan), chitosan and alginate gums.

The phrase "in situ gellable" as used herein embraces not only liquids of low viscosity that form gels upon contact with the skin, mucous membranes etc, but also more viscous liquids such as semi-fluid and thixotropic gels that exhibit _t substantially increased viscosity or gel stiffness upon administration to the skin, mucous membranes etc. Indeed, it can be advantageous to formulate a composition of the invention as a gel, to minimize loss of the composition immediately upon administration. Although it is preferred that such a composition exhibit further increase in viscosity or gel stiffness upon administration, this is not absolutely required if the initial gel is sufficiently resistant to dissipation to provide the effective residence time specified herein.

The composition may be also administered as a slow release composition, with a carrier composition such as microspheres, microcapsules, liposomes, etc., as a topical ointment or solution, an intravenous solution or suspension, or in an intraocular injection, as known to one skilled in the art to treat or prevent an endoparasitic infection.

A time-release drug delivery system may be administered by a method such as parenteral administration, topical delivery, oral delivery etc to result in sustained release of the compound over a period of time. The composition may be in the form of a vehicle, such as a micro- or macro-capsule or matrix of biocompatible polymers such as polycaprolactone, polyglycolic acid, polylactic acid, polyanhyd rides, polylactide-co-glycolides, polyamino acids, polyethylene oxide, acrylic terminated polyethylene oxide, polyamides, polyethylenes, polyacrylonitriles, polyphosphazenes, poly(ortho esters), sucrose acetate isobutyrate (SAIB), and other polymers or lipids that may be formulated as microspheres or liposomes. Delayed or extended release properties may be provided through various compositions of the vehicle (coated or uncoated microsphere, coated or uncoated capsule, lipid or polymer components, unilamellar or multilamellar structure, and combinations of the above, etc.). The composition and loading of microspheres, microcapsules, liposomes, etc. and their delivery are standard techniques known by one skilled in the art.

The invention also provides a pharmaceutical composition comprising: a compound of the present invention or a pharmaceutically acceptable salt thereof, in a biocompatible, biodegradable matrix, for delivery as an implant. When the compound is delivered as an implant, it may be incorporated in any known biocompatible biodegradable matrix as a liquid, or in the form, for example, of a micelle using known chemistry or as microparticles.

Slow or extended-release delivery systems include any of a number of biopolymers (biological-based systems), systems employing liposomes, colloids, resins, and other polymeric delivery systems or compartmentalized reservoirs, can be utilized with the compositions described herein to provide a continuous or long term source of therapeutic compound.

In any slow release device prepared, the active compound is preferably present in an amount of about 10% to 90% by weight of the implant. More preferably, the compound is from about 50% to about 80% by weight of the implant. In a preferred embodiment, the active compound for treatment or prevention of an endoparasitic infection comprises about 50% by weight of the implant. In a particularly preferred embodiment, the active compound comprises about 70% by weight of the implant.

In one form, implants used in the method of the present invention are formulated with particles of the compound entrapped within the bio-erodible polymer matrix. Release of the agent is achieved by erosion of the polymer followed by exposure of previously entrapped agent particles to the vitreous, and subsequent dissolution and release of agent. The release kinetics achieved by this form of drug release are different than that achieved through compositions which release drug through polymer swelling, such as with hydrogels such as methylcellulose. In that case, the drug is not released through polymer erosion, but through polymer swelling, which releases drug as liquid diffuses through the pathways exposed. The parameters which determine the release kinetics include the size of the drug particles, the water solubility of the drug, the ratio of drug to polymer, the method of manufacture, the surface area exposed, and the erosion rate of the polymer.

Exemplary biocompatible, non-biodegradable polymers of particular interest include polycarbamates or polyureas, particularly polyurethanes, polymers which may be cross-linked to produce non-biodegradable polymers such as cross-linked polyvinyl acetate) and the like. Also of particular interest are ethylene-vinyl ester copolymers having an ester content of 4 to 80% such as ethylene-vinyl acetate (EVA) copolymer, ethylene-vinyl hexanoate copolymer, ethylene-vinyl propionate copolymer, ethylene-vinyl butyrate copolymer, ethylene-vinyl pentantoate copolymer, ethylene-vinyl trimethyl acetate copolymer, ethylene-vinyl diethyl acetate copolymer, ethylene-vinyl 3-methyl butanoate copolymer, ethylene-vinyl 3-3-dimethyl butanoate copolymer, and ethylene-vinyl benzoate copolymer.

Additional exemplary naturally occurring or synthetic non-biodegradable polymeric materials include polymethylmethacrylate), poly(butylmethacrylate), plasticized poly(vinylchloride), plasticized poly(amides), plasticized nylon, plasticized soft nylon, plasticized poly(ethylene terephthalate), natural rubber, silicone, poly(isoprene), poly(isobutylene), poly(butadiene), poly(ethylene), poly(tetrafluoroethylene), poly(vinylidene chloride), poly(acrylonitrile, cross-linked poly(vinylpyrrolidone), poly(trifluorochloroethylene), chlorinated poly(ethylene), poly(4,4'-isopropylidene diphenylene carbonate), vinylidene chloride-acrylonitrile copolymer, vinyl chloridediethyl fumarate copolymer, silicone, silicone rubbers

(especially the medical grade), poly(dimethylsiloxanes), ethylene-propylene rubber, silicone-carbonate copolymers, vinylidene chloride-vinyl chloride copolymer, vinyl chloride-acrylonitrile copolymer, vinylidene chloride-acrylonitrile copolymer, poly(olefins), poly(vinyl-olefins), poly(styrene), poly(halo-olefins), poly(vinyls), poly(acrylate), poly(methacrylate), poly(oxides), poly(esters), poly(amides), and poly(carbonates).

Diffusion of the compound from the implant may also be controlled by the structure of the implant. For example, diffusion from the implant may be controlled by means of a membrane affixed to the polymer layer comprising the compound. The membrane layer will be positioned intermediate to the polymer layer comprising the compound and the desired site of therapy. The membrane may be composed of any of the biocompatible materials indicated above and may vary with the compound employed, the presence of agents in addition to the compound present in the polymer, the composition of the polymer comprising the compound, the desired rate of diffusion and the like. For example, the polymer layer will usually comprise a very large amount of compound and will typically be saturated. Such saturated polymers may generally release the compound at a very high rate. In this situation, the release of the compound may be slowed by selecting a membrane which is of a lower compound permeability than the polymer. Due to the lower permeability of the membrane, the compound will remain concentrated in the polymer and the overall rate of diffusion will be determined by the permeability of the membrane. Therefore, the rate of release of the compound from the implant is reduced, providing for a more controlled and extended delivery to the site of therapy.

The compositions of the present invention may alternatively be delivered with the aid of shaped articles containing the active compound, such as, for example, strips, plates, tapes, collars, ear tags, limb bands or marking devices.

The skilled reader will appreciate that the duration over which any of the compositions used in the method of the invention will dwell in the tissues of the subject will depend, inter alia, on such factors as the pharmacological properties of the compounds employed in the composition, the concentration of the compound employed, the bioavailability of the compound, the infection to be treated, the mode of administration and the preferred longevity of the treatment. Where that balance is struck will often depend on the longevity of the effect required and the particular parasite infection being treated.

The frequency of treatment according to the method of the invention is determined according to the infection being treated, the deliverable concentration of the active compound and the method of delivery. Once a therapeutic result is achieved, the compound can be tapered or discontinued. Occasionally, side effects warrant discontinuation of therapy. In general, an effective amount of the compound is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by a suitably qualified practitioner.

Use of compounds

The present invention further provides for the use of a compound of Formula A:

or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylactic or therapeutic treatment of parasitic infection or disease in a subject in need thereof, wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl and wherein the heteroaryl or substituted heteroaryl group is connected to the 2,4-dinito-6-(trifluoromethyl)phenyl ring via a carbon-carbon bond; or a pharmaceutically acceptable salt thereof, to prepare a medicament to treat a parasitic infection in a subject.

The present invention further provides for the use of a compound of Formula A:

(A)

or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylactic or therapeutic treatment of parasitic infection or disease in a subject in need thereof, wherein Z is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl and wherein the heteroaryl or substituted heteroaryl group is connected to the 2,4-dinito-6-(trifluoromethyl)phenyl ring via a carbon-carbon bond and wherein C6 aryl is substituted; or a pharmaceutically acceptable salt thereof, to prepare a medicament to treat a parasitic infection in a subject.

The parasitic infection is preferably caused by an endoparasite or an ectoparasite.

The parasitic infection may be caused by a parasite selected from the group comprising trypanosomes; haemoprotozoa and parasites capable of causing malaria; enteric and systemic cestodes including taeniid cestodes; enteric coccidians; enteric flagellate protozoa; filarial nematodes; gastrointestinal and systemic nematodes and hookworms.

The trypanosomes may be selected from the genera Trypanosoma and Leishmanial the enteric coccidians may be selected from the genera Cryptosporidium and Eimeria; the haemoprotazoa may be selected from the genera Plasmodium; the taeniid cestodes may be selected from the genera Echinococcus; the enteric flagellate protozoa may be selected from the genera Giardia; the gastrointestinal and systemic nematodes and hookworms may be selected from the genera Amidostomum, Trichostrongylus, Tenorastrongylus, Nippostrongylus, Heligmonina, Boreostrongylus, Ancyiostoma; and the filarial nematodes may be selected from the genera Wucherieria, Onchocera and Dirofilaria. The parasite may be selected from the group consisting of: Cryptosporidium andersoni, Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium hominis, Cryptosporidium wrairi, Cryptosporidium felis, Cryptosporidium canis, Cryptosporidium baileyi, Cryptosporidium meleagridis, Cryptosporidium galli, Cryptosporidium serpentis, Cryptosporidium saurophilum and Cryptosporidium molnari; Echinococcus granulosus, E. multilocularis, E. vogeli, E. oligarthrus; Trypanosome brucei rhodesiense, T. brucei gambiense, T brucei, T. cruzi, T. evansi, T. equiperdum, T. vivax; G. intestinalis; L. brasiliensis, L. donavani, L ethiopica L. mexicana L. peruviana, L. tropica, L. major, L. infantum, Plasmodium falciparum, P. humain et simian; Caenorhabditis elegans, Caenorhabditis briggsae, Caenorhabditis drosophilae, Caenorhabditis japonica, Caenorhabditis maupasi, Caenorhabditis plicata, Caenorhabditis remanei, Caenorhabditis sonorae, Caenorhabditis sp. CB5161, Caenorhabditis sp. DF5070, Caenorhabditis sp. PS 1010, Caenorhabditis sp. SB341 Caenorhabditis vulgaris, Amidostomum fulicae, A. acutum, Trichostrongylus colubriformis Tenorastrongylus josephi, Nippostrongylus brasi/iensis, Nippostrongylus witenbergi, Heligmonina nevoi; Boreostrongylus seurati, Boreostrongylus minutes, Heligmosomoides polygyrus, Wuchereria bancrofti, Onchocerca volvulus, Dirofilaria immitis, Schistosoma mansoni, S. haematobium, S. japonicum, Blastocytis hominis, Pediculus humanis capitis, Onchocera volvulus, Sarcoptes scabei, Trichomonas vaginalis, Toxocaria canis, T. cati and Toxoplasma gondii.

For example, the parasitic infection may be caused by a parasite selected from the group consisting of: Trypanosoma brucei rhodesiense, T.brucei, T. cruzi, Leishmania donovani, or Cryptosporidium.

The disease may be selected from the group comprising: trypanosomiasis, cryptosporidiosis, leishmaniasis, malaria, coccidiosis, giardiasis, hookworm infection, Chagas disease, Schistosomiasis (bilharzia), Blastocystosis, filariasis, head, pubic and body lice infection, ascariasis, onchocerciasis (River blindness), scabies, toxocariasis and toxoplasmosis. The disease may be selected from the group comprising: trypanosomiasis, coccidiosis, and leishmaniasis.

General

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.

The invention described herein may include one or more range of values (e.g. size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

For the purposes of the present invention an "endoparasite" is a parasite that has at least one lifecycle stage that lives intracellular^ and/or within the tissues of its host. Such parasites are dependent on at least one gene or its product from that host to complete their own life-cycle. A "pharmaceutically acceptable carrier" is a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the active agent(s) without causing unacceptable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

Other definitions for selected terms used herein may be found within the description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

Examples

Further features of the present invention are more fully described in the following non-limiting Examples. This detailed description is included solely for the purposes of exemplifying the present invention. It should not be understood in any way as a restriction on the broad description of the invention as set out above.

Example 1 : 3-[2.4-dinitro-6-(trifluoromethyl)phenvnthiophene

A solution of 2-chloro-3,5-dinitrobenzotrifluoride (214mg, 0.79mmol) and 3- (4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)thiophene (178mg, 0.85mmol) in toluene (3mL) was treated with 2M aqueous sodium carbonate (0.445mL, 0.89mmol). The mixture was flushed with nitrogen and tetrakis(triphenylphosphine)palladium(0) (44mg, 0.04mmol) was added. The mixture was then refluxed under nitrogen for 67 hours. The reaction was allowed to cool and saturated aqueous sodium carbonate (8ml_) was added. The mixture was extracted with dichloromethane (3 x 3OmL) and the combined organic extract was dried over MgSO ₄ before being evaporated to dryness. The crude product was chromatographed over silica gel. Elution with ethyl acetate/hexanes (1 :9) gave the title compound as a pale yellow solid (96mg, 38%), m.p. 104-5 ⁰ C. ¹ HNMR (300 MHz, CDCI ₃ ): 8.81 , 8.73 (ABq, 2H, J=2.2), 7.46 (dd, 1H, J=5.0, 3.0), 7.35 (br s, 1 H), 7.09 (d, 1 H, J=5.0).

Example 2: 2,4-dinitro-6-(trifluoromethyl)biphenyl

Nitrogen was bubbled through a mixture of 2-chloro-3,5-dinitrobenzotrifluoride (5.Og, 18.5mmol), phenylboronic acid (2.5g, 20.4mmol), etrakis(triphenylphosphine)palladium (0) (1.1g, 0.9mmol) in toluene (5OmL) and water (1OmL). Sodium carbonate (2.16g, 20.4mmol) was added to the mixture which was then refluxed under nitrogen for 20 hours. The reaction mixture was allowed to cool to room temperature and saturated sodium carbonate solution was added. The mixture was extracted with ethyl acetate and the organic layers were combined, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The crude material was then chromatographed over silica gel. Elution with hexanes/ethyl acetate (19:1 ) gave a yellow oil that was dissolved in hot ethyl acetate and triturated with hexanes. The resulting solid was collected by filtration and dried under high vacuum overnight to give 2,4-dinitro-6-(trifluoromethyl)biphenyl (3.19g, 55%) as yellow crystals, m.p.100 - 102 ⁰ C. ¹ HNMR (300 MHz, CDCI ₃ ) 8.82, 8.76 (ABq, 2H, J=2.2), 7.51 - 7.44 (m, 3H), 7.27 - 7.24 (m, 2H). Mass spectrum m/z 312 (70%, M+), 284 (71 %), 226 (58%), 219 (96%), 210 (100%), 201 (49%), 199 (42%), 188 (36%), 170 (34%), 139 (39%).

Example 3: 2',4'-dinitro-6'-(trifluoromethyl)biphenyl-4-carbaldehvde

Nitrogen was bubbled through a mixture of 2-chloro-3,5-dinitrobenzotrifluoride (0.5Og, 1.85mmol), 4-formylphenylboronic acid (0.55g, 2.22mmol) and tetrakis(triphenylphosphine)palladium (0) (0.11g, 0.09mmol) in toluene (7.8ml_). 2M aqueous sodium carbonate (1.1 ml_) was added and the mixture was then refluxed under nitrogen for 20 hours. The reaction mixture was allowed to cool to room temperature and saturated sodium carbonate solution (2OmL) was added. The mixture was extracted with ethyl acetate (3 x 3OmL), and the organic layers were combined, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The crude material was then chromatographed over silica gel. Elution with hexanes/ethyl acetate (9:1 ) gave a yellow oil that was dissolved in hot ethyl acetate and triturated with hexanes. The resulting solid was collected by filtration and dried under high vacuum overnight to give 2',4 ^I -dinitro-6'-(trifluoromethyl)biphenyl-4- carbaldehyde (0.2Og, 46%) as a pale yellow powder, m.p. 110-112 ⁰ C. ¹ HNMR (300 MHz, CDCI ₃ ): 10.11 (s, 1 H), 8.87 (s, 2H), 8.09 - 7.40 (AA ¹ BB ¹ , 4H, J=8.0). Found: C 49.8, H 2.3, N 8.0%. Calculated: C 49.4, H 2.1 , N 8.2%.

Example 4: 2'.4'-dinitro-6'-(trifluoromethyl)biphenyl-4-carboxylic acid

2 ^I ,4'-dinitro-6'-(trifluoromethyl)biphenyl-4-carbaldehyd e (5.5g, 16mmol) was dissolved in acetone (3.6mL) and a solution of potassium permanganate (3.8g, 24.0mmol) in a mixture of water (15.7mL) and acetone (54mL) was added with stirring at room temperature over 2 min. After 1 hour a further aliquot of potassium permanganate (1.3g, 8.2mmol) was added to the reaction. After another hour the reaction mixture was filtered through a celite pad and the filtrate was concentrated under reduced pressure. The resulting residue (yellow solid) was dissolved in water and the pH was adjusted to 2 with 4M HCI solution. The resulting solid was collected by filtration, dissolved in hot ethyl acetate and triturated with hexanes. The resulting solid was collected by filtration and dried under high vacuum overnight to give 2 ^I ,4'-dinitro-6'-(trifluoromethyl)biphenyl-4-carboxylic acid (4.1 g, 71%) as pale yellow powder, m.p. 192-193.5 ⁰ C. ¹ HNMR (300 MHz, CDCI ₃ ): 8.86 (s, 2H), 8.24, 7.41 (AA ¹ BB ¹ , 4H, J=8.1).

Example 5: 2'.4'-dinitro-6'-(trifluoromethyl)biphenyl-4-carbonyl chloride

2 ¹ ,4 ¹ -dinitro-6'-(trifluoromethyl)biphenyl-4-carboxylic acid (4.10g, 11.5mmol) in oxalyl chloride (4.5mL, 52mmol) was treated with Λ/,Λ/-dimethylformamide (0.1 mL). After the initial vigorous evolution of gas subsided, the solution was heated under reflux for three hours. After this time the reaction was concentrated under vacuum and the resulting residue (pale yellow solid) was used directly without purification.

Example 6: N.N-bis(propyl)-2'.4 ¹ -dinitro-6 ¹ -(trifluoromethyl)biphenyl-4-carboxamide

2',4 ¹ -Dinitro-6'-(trifluoromethyl)biphenyl-4-carbonyl chloride (0.61 mmol) in dichloromethane (0.7ml_) was added dropwise to a stirred solution of dipropylamine (0.61 mmol) in dichloromethane (1mL) at room temperature. Triethylamine (0.085mL, 0.61 mmol) was then added to the mixture which was stirred overnight at room temperature. The reaction was poured into saturated sodium bicarbonate solution and worked up by ethyl acetate extraction in the usual way. The crude product was dried under high vacuum overnight to provide Λ/,Λ/-bis(propyl)-2',4 ¹ -dinitro-6 ^l - (trifluoromethyl)biphenyl-4-carboxamide (113mg, 42%) as white crystals. ¹ HNMR (300 MHz, CDCI ₃ ): 8.84, 8.81 (ABq, 2H, J=2.1 ), 7.45, 7.30 (AA ¹ BB ¹ , 4H, J=7.9), 3.62 - 3.35 (m, 2H), 3.28 - 3.00 (m, 2H), 2.83 - 1.40 (m, 4H), 1.12 - 0.89 (m, 3H), 0.89 - 0.60 (m, 3H). m.p. 169 - 171 ⁰ C. Example 7: N-{r2\4'-dinitro-6'-(trifluoromethyl)biphenyl-4-yl1methyl}-N - Dropylpropanamine HCI

2 ^I ,4 ^f -dinitro-6'-(trifluoromethyl)biphenyl-4-carbaldehyde (0.2Og, 0.59mmol) and di- n-propylamine (0.62mmol) in 1 ,2-dichloroethane (0.3mL) were treated portion wise with sodium triacetoxyborohydride (0.19g, 0.88mmol). The mixture was stirred at room temperature for two hours after which time a further aliquot of sodium triacetoxyborohydride (0.06g, 0.28mmol) was added. The reaction was stirred for a further hour before being poured into water and worked up by ethyl acetate extraction in the usual way. The crude material was chromatographed over silica gel. Elution with hexanes/ethyl acetate (9:1 ) gave a yellow oil. Concentrated hydrochloric acid was then added to the oil and the mixture evaporated to dryness in vacuo to give a foam that was triturated in diethyl ether. The resulting solid was collected by filtration and dried under high vacuum overnight to give /V-fβ^'-dinitro-β'-ftrifluoromethylJbiphenyM-y^methylJ-Λ/ - propylpropanamine HCI (0.08g, 31%) as white crystals, m.p. 172-174 ⁰ C. ¹ HNMR (300 MHz, DMSO): 10.11 (bb, 1 H), 9.21 , 8.81 (ABq, 2H, J=2.2), 7.74, 7.48 (AA ¹ BB ¹ , 4H, J=8.0), 4.41 (d, 2H, J=5.4), 2.85 - 3.06 (m, 4H), 1.81 - 1.55 (m, 4H), 0.85 (t, 6H, J=7.4).

Example 8: 2-(trimethylsilylethvnyl)-3,5-dintrobenzotrifluoride

A dry 25 ml_ round bottom flask was charged with trimethylsilylacetylene (1.35g, 13.7 mmol), anhydrous THF (10 ml_), CuI (500mg, 2.6 mmol), triethylamine (1.31g, 12.9 mmol), palladium chloride (100mg, 0.56 mmol) and 2-chloro-3,5- dinitrobenzotrifluoride (2.Og, 7.4 mmol) under nitrogen. The dark brown mixture was stirred and heated to 4O ⁰ C, causing an exotherm and the solvent to reflux briefly. After cooling to room temperature the mixture was analysed by TLC (10% ethyl acetate/90% hexane) showing consumption of the starting material. The reaction mixture was filtered through a plug of silica, followed by rinsing with ethyl acetate (100 ml_). The filtrate was reduced to dryness and the brown residue was purified by chromatography on silica (2% ethyl acetate/98% hexane), affording a brown oil. This oil was further purified by chromatography on silica (8% toluene/92% hexane), affording an orange oil (0.87g, 2.6 mmol, 35%). ¹ H NMR (CDCI ₃ , 300 MHz): δ 8.9 (d, 1 H, J = 2.2 Hz), 8.7 (d, 1 H, J = 2.2 Hz), 0.3 (s, 9H).

Example 9: 4J2,4-dinitro-6-(trifluoromethyl)phenyll-1-phenyl-1H-1,2,3-t riazole

A 5ml_ round bottom flask was charged with aniline (213 mg, 2.3 mmol), A- dimethylaminopyridine (250 mg, 2.05 mmol), imidazole-1-sulfonylazide hydrochloride (430 mg, 2.05 mmol) and methanol (1 ml_). CuSO ₄ (0.1 ml_ of 1 M aqueous solution, 0.1 mmol) was added and the mixture was stirred at room temperature for 30 min, after which time TLC analysis (20/80 ethyl acetate- hexane) showed completion of the reaction. The reaction mixture was added to 4M HCI (10 ml_) and diethyl ether (10 ml_) and the organic layer was washed with saturated NaHCO ₃ solution (2 x 20 ml_) and water (20 mL). Drying over MgSO ₄ and cautious concentration to half the volume afforded an orange solution. This was diluted with methanol (10 mL) and the diethyl ether removed cautiously on the rota-vap. The remaining methanol solution was used for the next step.

The above methanolic phenyl-azide solution (5 mL, ~1 mmol of PhN ₃ ) was combined with 2-trimethylsilylethynyl-3,5-dinitrobenzotrifluoride (160 mg, 0.48 mmol) in methanol (1.5 mL) in a 20 mL microwave reactor vial. Cu(OAc) ₂ (87 mg, 0.48 mmol) was dissolved in H ₂ O (3 mL) and this solution was added to the vial followed by copper powder (30 mg, 0.48 mmol). The mixture was heated in a microwave reactor at 8O ⁰ C for 30 min. The reaction mixture was then added to ethyl acetate (20 mL) followed by drying over MgSO ₄ and filtering. The filtrate was cautiously reduced then co-evaporated with dichloromethane 3 times, eventually leaving 3 mL solution. Addition of hexane (5 mL) gave a chunky brown precipitate. The supernatant was decanted and the solid was washed with more hexane (10 mL). Decanting of the hexane followed by addition of diethyl ether (10 mL) gave a brown powder that was filtered and washed with diethyl ether (10 mL). The powder was dried at room temperature under high vacuum for 1 hr affording the desired triazole (88 mg, 0.23 mmol, 48%). Melting point 167-168 ⁰ C. ¹ H NMR (CDCI ₃ , 300 MHz): δ 8.9 (s, 2H), 8.3 (s, 1 H), 7.8 (d, 2H ₁ J = 7.3 Hz), 7.6 (m, 3H). Example 10: 4-[2.4-dinitro-6-(trifluoromethyl)Dhenyll-1-(4-methoxybenzyl )-1H- 1 ,2,3-triazole

A 5 mL flask was charged with 4-methoxybenzylamine (40mg, 0.29 mmol), CuSO ₄ .5H ₂ O (10mg, 40 μmol), methanol (2 mL) and imidazole-1-sulfonylazide hydrochloride (50mg, 0.24 mmol). With stirring was added NEt ₃ (0.03 mL, 0.22 mmol), and immediate TLC analysis (20% ethyl acetate/80% hexane) showed consumption of the diazo-transfer reagent. To this reaction mixture was added 2- trimethylsilylethynyl-3,5-dinitrobenzotrifluoride (80mg, 0.24 mmol) in methanol (1 mL), followed by water (2 mL) and sodium ascorbate (0.05 mL of 2M aqueous solution, 0.1 mmol). The brown mixture was heated in the microwave for 30 minutes at 8O ⁰ C. The reaction was then worked up by adding ethyl acetate (20 mL), drying over MgSθ ₄ , filtering and concentrating to afford a brown oil. This was purified by chromatography on silica (20% ethyl acetate/80% hexane) to afford the desired product as a white solid (20mg, 47 μmol, 13%). Melting point: 149-151 ⁰ C. ¹ H NMR (CDCI ₃ , 300 MHz): δ 8.85 (d, 1H, J = 2.2 Hz), 8.82 (d, 1H ₁ J = 2.2 Hz), 7.7 (s, 1 H), 7.2 (d, 2H, J = 11.6 Hz), 6.9 (d, 2H, J = 11.6 Hz), 5.6 (s, 2H).

Example 11 : Screening against Trypanosomes

Materials/Methods

A. Cell culture/initial screening

Trypanosome (comprising the species Leishmania donovani, Trypanosoma brucei rhodesiense, and Trypanosoma cruzi) were grown in HMI-9 media with blood stream form-supporting factors (BSF-S factors) and 20% Fresh Horse Serum.

Drugs are screened against Trypanosomes in 96 well plates at 100μM concentrations. The final liquid volume per well is 100μl. IC ₅ O Diminazene controls are included on each plate (10ng/ml). The Trypanosomes are exposed to the drug for 72 hours in total. The alamarBlue is added after 48 hours incubation allowing 24 hours for metabolism and colour change prior to the plate being read at 72 hours.

1. Using a haemocytometer, a 10 ml culture of L. donovani, T. b. rhodesiense, or T. cruzi was counted at optimum growth (i.e. high in numbers without any short and stumpy forms apparent). Calculate the dilution factor needed to achieve a concentration of organisms at about 1X10 ⁵ /ml. The Trypanosomes will be diluted a further 1 :2 during plate set up, so that the final concentration of organisms should be about 5X10 ⁴ /ml.

2. Drugs to be screened are pre-diluted in a 24 or 48 well plate to a concentration of 200μM. A volume sufficient to allow 50μl to be dispensed to each screening and drug blank well is prepared (1 :50 dilution of 1OmM to give 500μl final volume i.e. 10μl drug + 490μl media).

3. Add 50μl of 200μM drug to the appropriate screening wells and drug blank wells on the screening plate.

4. Dilute 1 μg/ml Diminazene stock 1 :50 to give 2OnM (8μl Dimin. + 396μl Media) and dispense 50μl of this dilution to each of the Diminazene control wells.

5. Add 100μl of warm media to the Media only and Media + aB wells.

6. Add 50μl of warm media to the Drug + Media blank, Trypanosomes only and Diminazene Control wells.

7. Dilute the 10 ml culture of Trypanosomes previously counted to achieve 1X10 ⁵ /ml with warm media. Add 50μl of Trypanosomes suspension to all wells except the media only controls. The addition of Trypanosomes suspension completes the dilution of the drug screening and control wells to 100μM and 10ng/ml respectively.

8. Incubate plate for 48 hours. 9. After about 48 hours alamarBlue (aB) should be added to the plate as follows. Add 10μl of aB to all of the drug screening wells, the Trypanosomes only wells, the Diminazene control wells and the Media + aB wells (DO NOT add the aB to the Drug + Media blank wells or the Media only wells).

10. Return to the incubator for a further 72 hrs.

11. Once a total of 72 hours has elapsed, read the plate on a Biorad microplate reader at 570nm and 630nm.

The data is used to calculate the %lnhibition for each drug and the Diminazene control. Those drugs giving over 50% inhibition go on to have a dose response curve assay.

B. Dose response curves against Trypanosomes.

As with screening, dose response curves against Trypanosomes are performed in 96 well plates with 3 curves per plate. Initial curves start at 10μM and decrease to 0.01 μM.

1. Using a haemocytometer count a 10 ml culture of Trypanosomes at optimum growth (i.e. high in numbers without any short and stumpy forms apparent). Calculate the dilution factor needed to achieve a concentration of organisms at about 1X10 ⁵ /ml. The Trypanosomes will be diluted a further 1 :2 during plate set up, so that the final concentration of organisms should be about 5X10 ⁴ AnI.

2. Drugs are pre-diluted in a 48 well plate. As with the screening plates, a volume sufficient to allow 50μl to be dispensed to each test and drug blank well is prepared. Dilute Diminazene stock to give 10ng/ml

3. Add 100μl of warm media to the Media only and Media + aB wells. 4. Add 50μl of warm media to the Drug + Media blank wells, Trypanosome only and Diminazene Control wells.

5. Add 50μl of drug dilution to the appropriate test wells and drug blank wells on the DRC plate.

6. Add 50μl of Trypanosome suspension to all wells excluding Drug + Media blanks. The addition of Trypanosomes completes the dilution of the drugs. Incubate plate at 37 ⁰ C and 5% CO ₂ for 48 hours.

7. After about 48 hours alamarBlue (aB) should be added to the plate as follows. Add 10μl of aB to all of the drug screening wells, the Trypanosome only wells, the Diminazene control wells and the Media + aB wells (DO

NOT add the aB to the Drug + Media blank wells or the Media only wells).

8. Return to the incubator for a further 24 hrs.

9. Once a total of 72 hours has elapsed, read the plate on a Biorad microplate reader at 570nm and 630nm.

Results

The results are set out in Figures 1 to 6.

Example 12: in vivo screening of the compounds against Trypanosomes

6-8 week old ARC (Swiss) mice 28-30 grams were randomised into groups of 5. Each animal was infected with 2 x 104 Trypanosoma brucei rhodesiense on day 0. Animals were dosed once a day at 100mg/kg of compound dissolved in 10% DMSO in corn oil on days 1 to 4. Parasitaemia was checked using tail blood smears examined microscopically on days 2 to ten and then twice a week until day 30. The experiment was terminated at day 30. Parasitaemia and day of death was recorded for all animals. Results

The results of the in vivo screen are set out in Figure 7.