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Title:
TRIAMINOPYRIMIDINE DERIVATIVES WITH ANTIPARASITIC ACTIVITY
Document Type and Number:
WIPO Patent Application WO/2020/188437
Kind Code:
A1
Abstract:
The present invention relates to compounds with a 5-(2-nitroethyI)-2,4,6- triaminopyrimidine structure, derivatised at the 1 position on the ethylene chain with heteroaromatic or phenyl-benzylether rings. The invention also relates to the use of said compounds as antiparasitic agents against infections caused by Trypanosoma brucei and Trypanosoma cruzi.

Inventors:
COSTI MARIA PAOLA (IT)
COSTANTINO LUCA (IT)
FERRARI STEFANIA (IT)
LINCIANO PASQUALE (IT)
CORDEIRO DA SILVA ANABELA (PT)
Application Number:
PCT/IB2020/052304
Publication Date:
September 24, 2020
Filing Date:
March 13, 2020
Export Citation:
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Assignee:
UNIV DEGLI STUDI DI MODENA E REGGIO EMILIA (IT)
UNIV DO PORTO (PT)
IBMC INST DE BIOLOGIA MOLECULAR E CELULAR (PT)
International Classes:
C07D239/50; A61K31/506; A61P33/02; C07D403/06; C07D407/06; C07D409/06
Other References:
ABEDAWN I KHALAF ET AL: "Structure-based design and synthesis of antiparasitic pyrrolopyrimidines targeting pteridine reductase 1", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 57, 9 July 2014 (2014-07-09), pages 6479 - 6494, XP002784100, ISSN: 0022-2623, DOI: 10.1021/JM500483B
HARDY L W ET AL: "Biochemical and genetic tests for inhibitors of Leishmania pteridine pathways", EXPERIMENTAL PARASITOLOGY, NEW YORK, NY, US, vol. 87, no. 3, 1 November 1997 (1997-11-01), pages 157 - 169, XP002234573, ISSN: 0014-4894, DOI: 10.1006/EXPR.1997.4207
Attorney, Agent or Firm:
BERTUCCIO, Silvia (IT)
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Claims:
CLAIMS

1. Compound of general formula (I):

wherein

Het is selected from:

a heteroaryl of formula (A1) bonded at the 2, 3 or 4-position:

wherein

X is an atom of S, O or N;

n is an integer from 0 to 1 ;

R is selected from -H, -OH, -OR1, -CH3, Cl, Br, F, -NO2, -NH -CN and -COOR2, wherein R1 and R2 are independently straight or branched C 1-4 alkyl groups; or

- a bicyclic heterocycle of formula (A2) bonded at the 2, 3 or 4-position:

wherein

X is an atom of C, S, O or N;

n and m are independently an integer from 0 to 1;

Y 1 and Y2 are independently or simultaneously a -CH- or an atom of O;

R is selected from -H, -OH, -OR1, -CH3, Cl, Br, F, -NO2, -NH2, -CN and -COOR2, where R1 and R2 are independently straight or branched C 1-4 alkyl groups; or

- a substituent of formula (B) bonded at the 6-position:

wherein

X is an atom of S, O or N bonded at the 1', 2’ or 3’ -position of the aromatic ring; R1, R2, R3, R4 and R5 are independently selected from -H, -OH, -OR1, -CH3, Cl, Br, F, -NO2, -NH2, -CN and -COOR2, wherein R1 and R2 are independently straight or branched C 1-4 alkyl groups

and the pharmaceutically acceptable salts thereof,

2, Compound according to claim 1, wherein Het is a substituent of formula (A2) or

(B)·

3. Compound according to claim 1 , wherein the substituent of formula (A1) is selected from;

4. Compound according to claim 1 or 2, wherein the substituent of formula (A2) is selected from:

5. Compound according to claim 1 or 2, wherein in the substituent of formula (B):

X is O bonded at the 1’ or 3’ -position;

R1 is H or NO2;

R2 is H or Cl;

- R3 is H or Cl;

R4 is H;

- R5 is H.

6. Compound according to claim 1, selected from the group consisting of:

5-(1-(benzo[b]thiophen-3-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine (F252)

5 -(1 -(2, 3 -dihydrobenz[b] [1,4] dioxin-6-yl)-2-nitroethyl)pyrimidine-2,4,6-tn amine (F253)

5-(1-(benzo[d] [1 ,3]dioxol-4-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine (F254)

5-(1-(1H-indol-3-yl)-2-nitroethypl)yrimidine-2,4,6-triamine (F255)

5-(1-(benzo[d][1,3]dioxol-5-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine (F262)

5-(1-(5-methylthiophen-2-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine (F271) 5-(2-nitro-1-(thiophen-2-yl)ethyl)pyrimidine-2,4,6-triamine (F272)

5-(1-(2,3-dihydrobenzofuran-5-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine (F273)

5-(1-(furan-2-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine (F277)

5-(2-nitro-1-(pyridin-3-yl)ethyl)pyrimidine-2, 4, 6-triamine

5-(2-nitro-1-(pyridin-2-yl)ethyl)pyrimidine-2,4,6-triamine

5-(2-nitro-1-(pyridin-4-yl)ethyl)pyrimidine-2,4,6-triamine

5-(1-(2,4-dichloro-pyridin-3-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine

5-(1-(2-chloro-4-methylpyridin-3-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine 5 -( 1 -(6-methylpyridin-3 -yl)-2-nitroethyl)pyrimidine-2,4,6-triamine

5-(1-(benzo[b]thiophen-2-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine 5-(1-(naphthalen-2-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine

5-(1-(naphthalen-l-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine

5-(1-(benzofuran-2-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine

5 -(1 -(benzofuran-3 -yl)-2-nitroethyl)pyrimidine-2,4,6-triamine

5-(1-(5-methylbenzo[b]thiophen-3-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine

5-(1-(5-chlorobenzo[b]thiophen-3-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine 5-(1-(6-chloro-1H-indol-3-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine.

7. Compound according to claim 1 , selected from the group consisting of:

5-(1-(2-((3,4-dichlorobenzyl)oxty)phenyl)-2-nitroethyl)pyrimidine-2,4,6-triamine

(F220)

5-(1-(2-(benzyloxy)phenyl)-2-nitroethyl)pyrimidine-2,4,6-triamine (F242) 5-(2-nitro-1-(2-((2-nitrobenzyl)oxy)phenyl)ethyl)pyrimidine-2,4,6-triamine (F244)

5-(1-(2-((3-chlorobenzyI)oxy)phenyl)-2-nitroethyl)pyrimidine-2,4,6-triamine (F248)

5-(1-(4-((3,4-dichlorobenzyl)oxy)phenyl)-2-nitroethyl)pyrimidine-2,4,6-triamine

(F250)

methyl 4-((2-(2-nitro-1-(2,4,6-triaminopyrimidin-5-yl)ethyl)phenoxy) methyl)benzoate

4-((2-(2-nitro-1-(2,4,6-triaminopyrimidin-5-yl)ethyl)phenoxy)methyl)benzoic acid

5-(1-(2-((4-fluorobenzyl)oxy)phenyl)-2-nitroethyl)pyrimidine-2,4,6-triamine 5-(1-(2-(benzylthio)phenyl)-2-nitroethyl)pyrimidine-2,4,6-triamine

5-(1-(2-((3,4-dichlorobenzyl)thio)phenyl)-2-nitroethyl)pyrimidine-2,4,6-triamine

5-(1-(2-((3,4-dichlorobenzyl)amino)phenyl)-2-nitroethyl)pyrimidine-2,4,6- triamine

5-(1-(3-((3,4-dichlorobenzyl)oxy)phenyl)-2-nitroethyl)pyrimidine-2,4,6-triamine

5-(1-(3-((3,4-dichlorobenzyl)thio)phenyl)-2-nitroethyl)pyrimidine-2,4,6-triamine

5-(1-(4-((3,4-dichlorobenzyl)thio)phenyl)-2-nitroethyl)pyrimidine-2,4,6-triamine methyl 4-(((4-(2-nitro-1-(2,4,6-triaminopyrimidin-5-yl)ethyl)phenyl)thio)- methyl)benzoate

4-(((4-(2-nitro-1-(2,4,6-triaminopyrimidin-5-yl)ethyl)phenyl)thio)methyl)-benzoic acid,

8, Compound according to claim 1, selected from:

9. Pharmaceutical composition comprising at least one compound of general formula (I) according to claims 1-8 and at least one pharmaceutically acceptable excipient and/or carner,

10, Compound according to claims 1-8 for use as a medicament

11. Compound according to claims 1-8 for use as inhibitor of parasitic enzyme PTR1.

12. Compound according to claims 1-8 for use as an antiparasitic agent.

13, Compound for use according to claim 12, against Trypanosoma brucei and

Trypanosoma cruzi parasites.

14. Compound according to claims 1-8 for use in the treatment of Human African Trypanosomiasis (sleeping sickness) or Chagas disease.

Description:
TRIAMINOPYRIMIDINE DERIVATIVES WITH ANTIPARASITIC ACTIVITY

Technical field of invention

The present invention relates to compounds with a 5-(2-nitroethyl)-2,4,6- triaminopyrimidine structure, derivatised at the 1 position on the ethylene chain with heteroaromatic or phenyl-benzylether rings, which are useful as antiparasitic agents.

State of the art

“Neglected” tropical diseases (NTDs) are a heterogeneous group of disorders mainly occurring in the tropical and sub-tropical areas of the world, associated with conditions of extreme poverty that affect the populations of poor and developing countries. The WHO currently includes 17 disorders caused by micro- or macroparasites in the list of neglected tropical diseases. Diseases caused by microparasites include those with a trypanosomatid protozoan etiology such as Chagas disease, human African trypanosomiasis and leishmaniasis.

Trypanosomatids (class Kinetoplastea, order Trypanosomatida) are unicellular flagellated organisms belonging to the Leishmania and Trypanosoma genera. These primitive eukaryotic parasites represent the etiological agent of diseases such as leishmaniasis (caused by various species of Leishmania), human African trypanosomiasis (caused by Trypanosoma brucei gambiense and rodesiense), and Chagas disease (caused by Trypanosoma cruzi).

The acronym HAT (Human African Trypanosomiasis, or sleeping sickness) refers to the disease caused by the protozoan Trypanosoma brucei (T b ). Only two of the various sub-species are pathogenic for humans: Tb. rhodesiense and T.b. gambiense , The two parasites are morphologically indistinguishable, making a classification based on morphological characteristics impossible. A third sub-species, T.b. brucei, is widely used in research due to the fact that it is non-pathogenic to humans, but maintains all the other characteristics of the two pathogenic species. The two species of Trypanosoma pathogenic for humans, which are also morphologically identical, occur in various parts of sub-Saharan Africa, and give rise to the development of clinical symptoms that differ in terms of clinical course and severity. T brucei gambiense is associated with a chronic form of the disease, with a slower clinical course and delayed appearance of symptoms after infection. It is mainly present in central and western Africa, and involves a level of parasitaemia (circulating parasite levels) lower than 100/ml of blood, which makes microscopic identification of the parasitic cells in the blood difficult. Conversely, T. brucei rhodesiense is associated with an acute form of the disease with a more rapid course, and tends to be fatal within 2 weeks of infection. It is present in eastern and southern Africa, and causes higher levels of parasitaemia. In humans, the first symptoms of HAT appear after at least 5 days, at the point of inoculation, From this time, the infection evolves in two distinct clinical stages, The first stage involves systemic parasitic infection with symptoms similar to those of the common cold, with fever, headache and joint pain, The second stage begins several weeks (sspp rhodesiense ) or months (sspp gambiense) after the infection, when the parasite crosses the blood-brain barrier by a still unidentified mechanism, and infects the central nervous system. This stage leads to chronic encephalopathy with brain damage giving rise to neuropsychiatric disorders, poor concentration, disruption of the sleep-wake cycle and prolonged states of drowsiness, whence the common name of the disease. The infection eventually leads to coma and the patient’s death. The medicaments mainly used during the first stage of the infection are pentamidine and suramin. The medicaments mainly used to treat infections at the second stage are melarsoprol, eflomithine and nifurtimox,

Finally, Chagas disease is caused by Trypanosoma cruzi , and is often known as American trypanosomiasis. The parasite mainly tends to colonise organs such as the liver, spleen and heart tissue, causing serious systemic dysfunctions which can lead to death if the infection is not treated rapidly. Chagas disease currently represents a serious medical and socioeconomic problem in the Latin American countries in which it is endemic,

Studies conducted on said parasites demonstrate that these protozoa belonging to the Leishmania and Trypanosoma genera are auxotrophic for folate and other compounds with a pterin structure required for the biosynthesis of nucleic acids and proteins. During their development, these parasites have developed a sophisticated biochemistry of procurement and reuse of said compounds from the infected host, Consequently, in the past, antifolates were believed to be useful medicaments to treat leishmaniasis and trypanosomiasis, and the failure of these treatments caused surprise and disappointment in the medical profession [Nare, B., Lua, J., Hardy, L.W., and Beverley, S,, (1997) New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity. Parasitology 114: S101-S110.

Hardy, L.W., Matthews, W., Nare, B., and Beverley, S.M., (1997) Biochemical and genetic tests for inhibitors of Leishmania pteridine pathways. Exp Parasitol 87: 157-169,

Vickers, T.J., Beverley, S.M., Folate Metabolic Pathways in Leishmania. Essays Biochem. 2011, 51, 63-80.

Dawson, A,, Gibellini, F., Siehkiewicz, N., Tulloch, L.B., Fyfe, P.K., McLuskey, K., Fairlamb, A.H., Hunter, W.N., Structure and Reactivity of Trypanosoma Brucei Pteridine Reductase: Inhibition by the Archetypal Antifolate Methotrexate. Mol. Microbiol. 2006, 61, 1457-1468],

However, the medicaments currently on the market are characterised by high toxicity and limited efficacy, and require long treatment periods. Moreover, many of said medicaments have been used therapeutically for over 50 years, leading to the onset of resistance.

There is still a pressing need to identify new compounds active against the parasites Trypanosoma brucei and Trypanosoma cruzi which cause human African trypanosomiasis (sleeping sickness) and Chagas disease.

Brief description of the figure

Figure 1 shows the ratio between the plasma concentration of compound F252 normalised against the corresponding EC 50 towards T. brucei after administration.

Summary of the invention

The object of the invention is a compound of general formula (I):

wherein Het is a heteroaryl of formula (Al) or a bicyclic heterocycle of formula (A2) or a substituent of formula (B) as defined in the detailed description below, and the pharmaceutically acceptable salts thereof.

A further object of the invention is compositions containing at least one compound of general formula (I) as defined above, and at least one pharmaceutically acceptable excipient and/or carrier.

The invention also relates to a compound of general formula (I) as defined above for use as a medicament, in particular as an inhibitor of the parasitic enzyme PTR1.

The invention further relates to a compound of general formula (I) as defined above for use as an antiparasitic agent.

Detailed description of the invention

It has surprisingly been found that compounds of general formula (I) with a 5-(2-nitroethyl)-2,4,6-triaminopyrimidine structure derivatised at the 1 position on the ethylene chain with heteroaromatic or phenyl-benzylether rings have antiparasitic activity against Trypanosoma brucei (T. brucei ) and Trypanosoma cruzi (T. cruzi).

The object of the present invention is a compound of general formula (I):

wherein Het is a heteroaryl of formula (Al) or a bicyclic heterocycle of formula (A2) or a substituent of formula (B):

- Het can be a heteroaryl of formula (Al) bonded at the 2, 3 or 4 position:

wherein

X is an S, O or N atom;

n is an integer between 0 and 1 ;

R is selected from -H, -OH, -OR 1, -CH 3 , Cl, Br, F, -NO 2 , -NH 2 , -CN and -COOR 2 , wherein R 1 and R 2 are independently straight or branched C 1- 4 alkyls; or

- Het can be a bicyclic heterocycle of formula (A2) bonded at the 2, 3 or 4 position:

wherein

X is a C , S, O or N atom;

n and m are independently an integer between 0 and 1;

Y 1 and Y 2 are independently or simultaneously a -CH- or an O atom;

R is selected from -H, -OH, -OR 1 , -CH 3 , Cl, Br, F, -NO 2, -NH 2, -CN and -COOR 2 , wherein R 1 and R 2 are independently straight or branched C 1- 4 alkyls; or

- Het can be a substituent of formula (B) bonded at the 6 position:

wherein

X is an S, O or N atom bonded at the 1', 2’ or 3’ position on the aromatic ring;

R 1 , R 2 R 3 , R 4 and R 5 are selected independently from -H, -OH, -OR 1 , -CH 3 , Cl, Br, F, -NO 2 , -NH 2 , -CN and -COOR 2 , wherein R 1 and R 2 are independently straight or branched C 1-4 alkyls,

and the pharmaceutically acceptable salts thereof.

Het is preferably a substituent of formula (A2) or (B),

According to a preferred aspect, a substituent of formula (A1) is selected from

According to another preferred aspect, a substituent of formula (A2) is selected from

According to a further preferred aspect, in a substituent of formula (B):

- X is O bonded at the 1' or 3’ position;

- R 1 is H or NO 2 ;

- R 2 is H or Cl;

- R 3 is H or Cl;

- R 4 is H;

- R 5 is H. The compounds of general formula (I) wherein Het is a substituent of formula (Al) or (A2) are preferably, for example:

5-(1-(benzo[b]thiophen-3-yl)-2-nitroethyl)pyrimidine-2,4,6-t riamine (F252)

5-(1-(2,3-dihydrobenzo[b][1,4]dioxm-6-yl)-2-nitroethyI)py rimidine-2,4,6-triamine (F253)

5-(1-(benzo[d][1,3]dioxol-4-yl)-2-nitroethyl)pyrimidine-2,4, 6-triamine (F254)

5-(1-(1H-indol-3-yl)-2-nitroethyl)pyrimidine-2,4,6-triami ne (F255)

5-(1-(benzo[d][1,3]dioxol-5-yl)-2-nitroethyl)pyrimidine-2,4, 6-triamine (F262)

5-(1-(5-metylthiophen-2-yl)-2-nitroethyl)pyrimidine-2,4,6 -triamine (F271 ) 5-(2-nitro-1-(thiophen-2-yl)ethyl)pyrimidine-2,4,6-triamine (F272)

5-(1-(2,3-dihydrobenzofuran-5-yl)-2-nitroethyl)pyrimidine-2, 4,6-triamine (F273)

5-(1-(furan-2-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine (F277)

5-(2-nitro-1-(pyTidin-3-yl)ethyl)pyrimidine-2,4,6-triamme

5-(2-nitro-1-(pyridin-2-yl)ethyl)pyrimidine-2,4,6-triamine

5-(2-nitro-1-(pyridin--4-yl)ethyl)pyrimidine-2,4,6-triamine

5-(1-(2,4-dichloropyridin-3-yl)-2-iiitroethyl)pyrimidine-2,4 ,6-triamme

5-(1-(2-chloro-4-methylpyridin-3-y])-2-nitroethyl)pyrimidine -2,4,6-triamme 5 -(1-(6-methylpyridin-3-yl)-2-nitroethyl)pyrimidine-2,4,6-tri amine

5-(1-(benzo[b]thiophen-2-yl)-2-nitroethyl)pyrimidine-2,4,6-t riamine

5-(1-(naphthalen-2-yl)-2-nitroethyl)pyrimidine-2,4,6-triamin e

5-(1-(naphthalen-1-yl)-2-nitroethyl)pyrimidine-2,4,6-triamin e

5-(1-(benzofuran-2-yl)-2-nitroethyl)pyrimidine-2,4,6-triamin e

5-(1-(benzofuran-3-yl)-2-nitroethyl)pyrimidine-2,4,6-triamin e

5-(1-(5-methylbenzo[b]thiophen-3-yl)-2-nitroethyl)pyrimidine -2,4,6-triamine

5-(1-(5-chlorobenzo[b]thiophen-3-yl)-2-nitroethyl)pyrimid ine-2,4,6-triamine

5-(1-(6-chloro-1H-indol-3-yl)-2-nitroethyl)pyrimidine-2,4 ,6-triamine

According to a preferred aspect, the compounds of general formula (I) wherein Hetstituent of formula (B) are, for example:

5-(1-(2-((3,4-dichlorobenzyl)oxy)phenyl)-2-nitroethyl)pyrimi dine-2,4,6-triamine (F220)

5-(1-(2-(benzylox:y)phenyl)-2-nitroethyl)pyrimidine-2,4,6-tr iamine (F242) 5-(2-nitro-1-(2-((2-nitrobenzyl)oxy)phenyl)ethyl)pyrimidine- 2,4,6-triamine (F244) 5-(1-(2-((3 -chlorobenzyl)oxy)phenyl)-2-nitroethyl)pyrimidine-2,4,6-tria mine (F248) 5-(1-(4-((3,4-dichlorobenzyl)oxy)phenyl)-2-nitroethyl)pyrimi dine-2,4,6-triamine (F250)

methyl 4-((2-(2-nitro-l-(2,4,6-triaminopyrimidin-5-yl)ethyl) phenoxy)methyl)benzoate

4-((2-(2-nitro-1-(2,4,6-triaminopyrimidin-5-yl)ethyl)phen oxy)methyl)benzoic acid 5-(1-(2-((4-fluorobenzyl)oxy)phenyl)-2-nitroethyl)pyrimidine -2,4,6-triamine

5-(1-(2-(benzylthio)phenyl)-2-nitroethyl)pyrimidine-2,4,6 -triamine

5-(1-(2-((3,4-dichlorobenzy l)thio)phenyl)-2-nitroeth.yl)pyrimidine-2,4,6-triamine

5-(1-(2-((3,4-dichlorobenzyl)amino)phenyl)-2-nitroethyl)p yiimidine-2,4,6- triamine

5-(1-(3-((3,4-dichlorobenzyl)oxy)phenyl)-2-nitroethyl)pyrimi dine-2,4,6-triamine

5-(1-(3-((3,4-dichlorobenzyl)thio)phenyl)-2-nitroethyl)py rimidine-2,4,6-triamine

5-(1-(4-((3,4-dichlorobenzyl)thio)phenyl)-2-nitroethyl)py rimidine-2,4,6-triamine methyl 4-(((4-(2-nitro-l-(2,4,6-triaminopyrimidm-5-yl)ethyl)phenyl) thio)- methyl)benzoate

4-(((4-(2-nitro-1-(2,4,6-triaminopyrimidin-5-yl)ethyl)phenyl )thio)methyl)benzoic acid

The compounds of formula:

are particularly preferred.

A further object of the invention is compositions containing at least one compound of general formula (I) as defined above, and at least one pharmaceutically acceptable excipient and/or carrier.

A further object of the present invention is a compound of general formula (I) as defined above for use as a medicament, in particular as an inhibitor of the parasitic enzyme

PTR1.

The invention also relates to a compound of general formula (I) as defined above for use as an antiparasitic agent, in particular against the parasites Trypanosoma brucei and Trypanosoma cruzi.

According to a further embodiment, the invention relates to a compound of general formula (I) as defined above for use in the treatment of a disorder selected from human African trypanosomiasis (sleeping sickness) and Chagas disease.

The invention also relates to a method of treatment of a patient (human or animal) infected with Trypanosoma brucei or Trypanosoma cruzi which comprises administration to the patient of an effective dose of a compound of general formula (I) as defined above, In particular, the patient may suffer from human African trypanosomiasis (sleeping sickness) or Chagas disease.

The following examples further illustrate the invention. EXAMPLES

SYNTHESIS PART

Materials and methods

All the commercially available reagents and solvents were used without prior purification, unless otherwise specified. Reactions were monitored by thin-layer chromatography (60F-254, E. Merck) and displayed with a UV lamp, iodine vapours, cerium ammonium sulphate solution or alkaline potassium permanganate solution. The following solvents and reagents were abbreviated as shown in brackets: tetrahydrofuran (THF), diethyl ether (Et 2 O), dimethyl sulphoxide (DMSO), ethyl acetate (EtOAc), dichloromethane (DCM), dimethyl formamide (DMF) and methanol (MeOH) , All reactions were conducted by traditional techniques or irradiation with microwaves. The NMR spectra were acquired on a Bruker 400 spectrometer. The 1H spectra were acquired at 400.134 MHz, and the 13C spectra at 100.62 MHz. The chemical shift of the proton refers to TMS used as internal standard. The chemical shifts are expressed in parts per million (ppm, d). Coupling constants J are expressed in Hertz (Hz). Splitting is described as s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublet), m (multiplet) and b (broad signal). The mass spectra were acquired on 6520 Accurate-Mass Q-TOF LC/MS and 6310A Ion-Trap LC-M$(n) mass spectrometers. The melting points were determined with a Stuart melting-point block, SMP3 (Barloworld Scientific Limited, Stone, Staffordshire, UK), and are not corrected.

Example 1 - General procedure for synthesis of 5-(2-nitro-1-aryl-ethyl)-2,4,6- triaminopirimidines (1A-9A, 1B-5B) according to the invention

Scheme 1.

a) benzyl-chloride (1 eq.), K 2 CO 3 (1 eq.), DMF 80°C, yield: 95%;

b) NH 4 AcO (1.5 eq.), nitromethane, reflux, yield 75-90%;

c) 2,4,6-triaminopyrimidine (1 eq.), H 2 O:AcOEt 1:1, 60°C, yield 45-70%.

2,4,6-triaminopyrimidine (1 eq.), solubilised in an equal volume of water, is added to a solution of nitrostyrene (1 eq,) in ethyl acetate. The mixture is left to react at 60°C under strong stirring for 18 hours, whereafter it is decanted and the organic phase is recovered and washed with water and a saturated solution of sodium chloride. The organic phase is dried with anhydrous Na 2 SO 4 and concentrated. The residue crystallises from ethyl ether to provide the desired product.

Example 2 - Characterisation of the compound 5-(l-(benzo[b]thiophen-3-yl)- 2-nitrocthyl)pyrimidine-2,4,6-triamine (F252, 1A)

1 N HMR (400 MHz, DMSO-d 6 ) d 5.12 - 5.27 (m, 2H), 5.34 (s, 2H), 5.47 - 5.54 (m, 5H), 7,35 (pd, J = 1,5, 7.1 Hz, 2H), 7.75 (ddd, . , 3.1, 4.6 Hz, 2H), 7,96 - 8.00 (m, 1H). 13 C NMR (101 MHz, DMSO) d 33.80, 75.03, 82.61, 121.54, 122.60, 122.78, 124.01, 124.68, 134.42, 138.09, 139.53, 161.08, 162.22, HRMS m/z [M+H]+ calculated for C14H15N6O2S; 331.0972, found: 331.0972. MP [dec. 153°C].

Example 3 - Characterisation of the compound 5-(1-(2,3- dihydrobenzo[b][1,4]dioxm-6-yl)-2-nitroethyI)pyrimidme-2,4,6 -triamine (F253, 2A) 1H NMR (400 MHz, DMSO-d 6 ) d 4.21 (s, 4H), 4.92 (t, J = 7.9 Hz, 1H), 5.11 (dd, J = 8.9, 13.0 Hz, 1H), 5.39 (dd, J = 6.9, 13.0 Hz, 1H), 5.51 (d, J = 18,4 Hz, 5H), 6.60 - 6.88 (m, 3H), 13 C NMR (101 MHz, DMSO) d 36.77, 63.99, 64.07, 75.63, 84.30, 115.60, 116.88, 119.53, 131.95, 142.02, 143.17, 161.17, 162.28. HRMS m/z [M+H]+ calculated for C14H17N6O4: 333.1306, found: 333.1305. MP [dec, 139°C].

Example 4 - Characterisation of the compound 5-(1-(benzo[d][l,3]dioxol-4-yl)- 2-nitroethyl)pyrimidine-2,4,6-triamine (F254, 3A)

1 H NMR (400 MHz, DMSO-d 6 ) d 4.94 - 5.18 (m, 2H), 5.36 (dd, J = 6.9, 13.3 Hz, 1H), 5.41 (s, 2H), 5.48 (s, 4H), 6.00 (d, J = 2.0 Hz, 2H), 6.71 (dd, J = 1.6, 7,6 Hz, 1H), 6.77 - 6,90 (m, 2H). 13 C NMR (101 MHz, DMSO) d 34,41, 74.25, 82.62, 100.58, 107.26, 120.38, 120.88, 121.73, 144.62, 147,06, 161.27, 162.27. HRMS m/z [M+H]+ calculated for C13H15N6O4: 319.1149, found: 319.1150. MP [dec. 150°C]

Example 5 - Characterisation of the compound 5-(l-(lH-indol-3-yI)-2- nitro ethyl)pyrimidine-2,4,6-triamme (F255, 4A)

1 N NMR (400 MHz, DMSO-d 6 ) d 5.08 - 5.25 (m, 2H), 5.30 - 5.44 (m, 3H), 5.45 - 5.60 (m, 4H), 6.84 - 6.96 (m, 1H), 7.08 (ddd, J = U, 7.0, 8.3 Hz, 1H), 7.31 - 7.41 (m, 4H),

I I.00 (d, J = 2.4 Hz, 1H). 13 C NMR (101 MHz, DMSO) d 31.32, 64.87, 75.55, 83.83,

I I I.36, 112,57, 118,37, 119.39, 121.32, 121.55, 126.20, 136 57, 160.65, 162.04. HRMS m/z [M+H]+ calculated for C14H16N7O2: 314,1360, found: 314.1362. MP [dec. 163 °C].

Example 6 - Characterisation of the compound 5-(l-(benzo[d][l,3]dioxol-5-yl)- 2-nitroethyl)pyrimidine-2,4,6-triamine (F262, 5A)

1 N NMR (400 MHz, DMSO-d 6 ) d 4.94 (dd, J = 7,0, 8,9 Hz, 1H), 5.11 (dd, J = 8.9, 13.1 Hz, 1H), 5.38 (m, 7H), 5.95 - 6.02 (m, 2H), 6,73 (dd, J = 1.8, 8.1 Hz, 1H), 6.80 (d, J = 1.7 Hz, 1H), 6.86 (d, 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO) d 37.15, 75.60,

100.93, 107.67, 107.95, 119.48, 132.99, 145.82, 147.42, 161.22, 162.26. HRMS m/z [M+H]+ calculated for C13H15N6O4: 319,1149, found: 319.1150. MP [dec. 147°C].

Example 7 - Characterisation of the compound 5-(1-(5-methylthiophen-2-yl)- 2-nitroethyl)pyrimidine-2,4,6-triamine (F271, 6 A)

1 N NMR (400 MHz, DMSO-d 6 ) d 2,09 (s, 3H), 5.11 (d, . Hz, 2H), 5.40 (d,

J = 46.0 Hz, 7H), 6.07 (s, 1H), 6.63 (d, J = 3.4 Hz, 1H), 6.76 (d, J = 3,5 Hz, 1H). HRMS m/z [M+H]+ calculated for C11H15N6O2S: 295.0972, found: 295.0975. MP [dec. 142 º C], Example 8 - Characterisation of the compound 5-(2-nitro-1-(thiophen-2- yl)ethyl)pyrimidine-2,4,6-triamine (F272, 7A)

1H NMR (400 MHz, DMSO-d 6 ) d 5.08 - 5.27 (m, 2H), 5.36 - 5.45 (m, 1H), 5.52 (s, 6H), 6.94 - 7.04 (m, 2H), 7.43 (dd, J = 1.3, 5.0 Hz, 1H). 13C NMR (101 MHz, DMSO) d 34.31, 75.81, 84.31, 123.91, 125.20, 126.66, 143.72, 160.86, 161.92. HRMS m/z [M+H]+ calculated for C10H13N6O2S: 281.0815, found: 281.0815. MP [dec. 149°C]. Example 9 - Characterisation of the compound 5-(1-(2,3-dihydrobenzofuran- 5-yl)-2-nitroethyl)pyrimidine-2,4,6-triamine (F273, 8A)

1H NMR (400 MHz, DMSO-d6) d 3.14 (t, J = 8.7 Hz, 2H), 4.50 (t, J = 8.7 Hz, 2H), 4.95 (t, J = 7.9 Hz, 1H), 5.10 (dd, J = 9.0, 12.9 Hz, 1H), 5,37 (m, 7H), 6,71 (d, J = 8.3 Hz, 1H), 6.98 (dd, J = 2.1, 8.3 Hz, 1H), 7.10 (d, J = 1 ,9 Hz, 1H). HRMS m/z [M+H]+ calculated for C14H17N6O3: 317.1357, found: 317.1360. MP [dec. 140°C].

Example 10 - Characterisation of the compound 5-(1-(furan-2-yl)-2- nitroethyl)pyrimidine-2, 4 ,6-triamine (F277, 9A)

1 H NMR (400 MHz, DMSO-d 6 ) d 5.04 (q, J = 4.1, 5.8 Hz, 2H), 5.27 (d, J = 4,8 Hz, 1H), 5.43 (s, 2H), 5.51 (s, 4H), 6.28 (d, J = 3.3 Hz, 1H), 6.37 - 6,49 (m, 1H), 7.60 (s, 1H). 13 C NMR (101 MHz, DMSO) d 33.23, 74.54, 81.74, 106.42, 110.35, 142.31, 152.08, 161.19, 162.26. HRMS m/z [M+H]+ calculated for C10H13N6O3: 265.1044, found: 265.1045. MP [dec. 149°C]

Example 11 - Characterisation of the compound 5-(1-(2-((3,4- dichlorobenzyl)oxy)phenyl)-2-nitroethyl)pyrimidine-2,4,6-tri amine (F220, IB)

1 H NMR (400 MHz, DMSO-d 6 ) d 4.98 - 5.10 (m, 2H), 5.16 (s, 2H), 5.28 - 5.43 (m, 7H), 6.94 (td, J = 1.1, 7.5 Hz, 1H), 7.03 (dd, 1.1, 8,4 Hz, ΪH), 7.23 (ddd, J = 1.6, 7.4, 8,8 Hz, 1H), 7.31 (dd, J = 1.6, 7.7 Hz, 1H), 7,38 (dd, J = 2.0, 8.3 Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.72 (d, J = 2,0 Hz, 1H). 13 C NMR (101 MHz, DMSO) d 67,71, 74.83, 83.70, 112.42, 120.64, 127.03, 127.56, 127.60, 128.00, 128.07, 129.28, 130.15, 130.42, 131 ,00, 133.91, 138.11, 155.51, 161.12, 162,23. C19H19[35C1]2N6O3: 449.0890, found: 449,0890; calculated for C19H19[35C1] [37C1]N6O3: 451.0861, found: 451.0862. MP [dec. 130°C]

Example 12 - Characterisation of the compound 5-(1-(2-(benzyloxy)phenyl)-2- nitroethyl)pyrimidine-2,4,6-triamine (F242, 2B)

1 H NMR (400 MHz, DMSO-d 6 ) d 4.98 - 5, 10 (m, 2H), 5.17 (s, 2H), 5,33 (d, J = 5.1 Hz, 7H), 6.92 (td, J = 1.1, 7.5 Hz, 1H), 7.06 (dd, J = 1.1, 8.3 Hz, 1H), 7.22 (ddd, 1.6, 7.4, 8.9 Hz, 1H), 7.27 - 7.34 (m, 2H), 7.34 - 7.40 (m, 2H), 7.40 - 7.50 (m, 2H). 13 C NMR (101 MHz, DMSO) d 34.42, 69.36, 74.89, 83.89, 112.55, 120-48, 126.87, 127.41, 127.66, 127.83, 128.05, 128.32, 136.81, 155.80, 161.15, 162.28, 164.44. HRMS m/z [M+H]+ calculated for C19H21N6O3: 381.1670, found: 381.1665, MP [dec, 127°C].

Example 13 - Characterisation of the compound 5-(2-nitro-1-(2-((2- nitrobenzyl)oxy)phenyl)ethyl)pyrimidine-2,4,6-triamine (F244, 3B)

1 N NMR (400 MHz, DMSO-d 6 ) d 5.08 (d, J = 9.0 Hz, 2H), 5.31 - 5.50 (m, 7H), 5.53 (s, 2H), 6.97 (dd, J = 6.9, 8.0 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H), 7.23 (td, J = 1.6, 7.4, 7.9 Hz, 1H), 7.34 (dd, J = 1.6, 7.8 Hz, 1H), 7.60 (ddd, J = 1.9, 7.0, 8.7 Hz, 1H), 7.69 (dq, J = 7.1, 8.6 Hz, 2H), 8.17 (dd, J = 1.3, 8.1 Hz, 1H). HRMS m/z [M+H]+ calculated for C19H20N7O5: 426.1520, found: 426.1515. MP [dec. 154°C],

Example 14 - Characterisation of the compound 5-(1-(2-((3- chlorobenzyl)oxy)phenyl)-2-nitroethyl)pyrimidme-2,4,6-triami ne (F248, 4B)

1 H NMR (400 MHz, DMSO-d 6 ) d 5.02 - 5.13 (m, 2H), 5.18 (d, J = 1.9 Hz, 2H), 5.28 - 5.47 (m, 7H), 6.94 (t, 7.4 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H), 7.23 (td, J = 1.6, 7.8

Hz, 1H), 7.30 (dd, J = 1.6, 7.7 Hz, 1H), 7.37 (t, 1.8 Hz, 3H), 7.54 (d, 2.1 Hz, 1H).

13 C NMR (101 MHz, DMSO) d 34.36, 68.44, 74.92, 83.70, 112.48, 120.61, 125.87, 126.93, 127.05, 127.59, 127.76, 128,09, 130.19, 133.06, 139.47, 155.65, 161.18, 162.30. HRMS mix [M+H]+ calculated for C19H20C1N6O3: 415.1280, found: 415.1281. MP [dec. 161°C].

Example 15 - Characterisation of the compound 5-(1-(4-((3,4- dichlorobenzyl)oxy)phenyl)-2-nitroethyl)pyrimidine-2,4,6-tri amine (F250, 5B)

1 H NMR (400 MHz, DMSO-d 6 ) d 4.96 - 4.99 (m, 1H), 5,09 - 5.13 (m, 3H), 5.36 - 5.42 (m, 7H), 6.97 (d, J = 7.4 Hz, 2H), 7.18 (d , J = 7.4 Hz, 2H), 7.45 (d, J = 6.8 Hz, 1H), 7.66 (d, J = 6.8 Hz, 1H), 7.72 (s, 1H), 13 C NMR (101 MHz, DMSO) d 36.68, 67.57, 75.59,

84.56, 114.72, 115.55, 127.74, 127.97, 129.38, 130.26, 130.64, 131.04, 131.49, 138.37,

156.56, 161.16, 162.25. HRMS m/z [M+H]+ calculated for C19H19[35C1]2N6O3: 449.0890, found: 449.0887; calculated for C19H19[35C1][37C1]N6O3: 451.0861, found: 451.0860. MP [dec, 89°C]. BIOLOGICAL EVALUATION

Example 16 - Inhibition of PTR1 parasitic enzyme

Materials and methods

The in vitro assay used in this study to evaluate inhibition of the TbPTRl parasitic enzymes is reported in the literature (Shank et al.). The assay involves spectrophotometrically monitoring, at the wavelength of 550 nm, the reduction of cytochrome c Fe3+ to Fe2+ following oxidation of tetrahydrobiopterin H4B, the product of the enzymatic reaction catalysed by PTR1, to quinoid dihydrobiopterin qH2B. In the presence of an inhibitor, the reduced catalytic activity of PTR1 gives rise to the formation of a smaller amount of H4B, which can become the substrate of the oxidation reaction catalysed by cytochrome c Fe3+. This will involve the consequent formation of a smaller amount of cytochrome c Fe2+, with a consequent reduction of the signal in the photometric reading, Inhibition of the PTR1 enzyme is evaluated in a 20 mM citrate buffer at pH 6.0. The final reaction mixture contains the test compound at the desired concentration, enzyme TbPTR1 /LmPTR1 (at 6,0 nM and 12 nM respectively), H2B (0,3 mM/3 mM), cytochrome c (100 mM/100 mM) and NADPH (500 mM/500 mM), making a total volume of 50 mL. The assay is conducted in 384-well plates; the test compound (in 100% of DMSO) is added to each well, followed by addition of 45 mL of the reaction mixture (enzyme, H2B, and cytochrome c in 20 mM citrate buffer). A pre-reading of the plate is taken at 550 nm using an EnVision® Multilabel Reader 2103 (PerkinElmer Inc, US), followed by incubation of the plate at 30ºC for 10 minutes. The enzymatic reaction is triggered by adding 5 mL of NADPH (from a 5 mm mother solution in ultrapure water), and monitoring of the enzymatic reaction begins immediately at 550 nm. Five readings are taken after 10, 20, 30, 40 and 50 minutes, and the slope of the reaction curve is determined, The data are analysed with Activity Base (IDBS), and the 3 -sigma method is used to eliminate outliers in the control wells. On the basis of the slope, the data are normalised against the values obtained for methotrexate (positive control) and against a 1% solution of DMSO (negative control), so that the percentage inhibition of each sample can be calculated. The measurement at time zero is used to evaluate optical interference in the sample. Each sample is tested in triplicate, and the pIC 50 , standard deviation, slope, minimum signal and maximum signal values for each dose-response curve are obtained by using a 4-parameter logistic calculation in the XE module of ActivityBase (IDBS),

Assay

The compounds listed in Tables A and B were tested in vitro for their ability to inhibit parasitic enzyme PTR1 of T. brucei. The compounds were initially tested at the single concentration of 50 pM. The compounds exhibited inhibitory enzymatic activity greater than 80% at the concentration tested, and the dose-response curve, for the calculation of the IC 50 , was determined experimentally. The enzymatic inhibition against the PTR1 of T. brucei is shown in Table 1.

The compounds proved to be powerful inhibitors at the concentration of 50 mM against the PTR1 of T. brucei, with experimental IC 50 values in the low-medium nanomolar range (33-600 nM).

Example 17 - Antiparasitic activity and Selectivity Index

Materials and methods

- In-vitro evaluation of activity against the parasite T. brucei

The efficacy of the compounds against the circulating parasite T. brucei was evaluated by modifying the resazurin assay (see Bowling, T, et. al. Application of a resazurin-based high-throughput screening assay for the identification and progression of new treatments for human African trypanosomiasis, Int J Parasitol Drugs Resist. 2012, 2, 262-270).

The forms circulating in the blood were added to an equal volume of serial dilutions of the compound in an HMI-9 medium until a cell density of 5x10 3 /mL was obtained. After 72 hours’ incubation at 37°C, 5% CO 2 , 20 mL of an 0.5 mM solution of resazurin was added, and the plates were incubated for a further 4 hours under the same conditions. The fluorescence was measured at 540 nm and 620 nm (excitation and emission wavelengths respectively) using a Synergy 2 Multi-Mode Reader (Biotek), The anti-trypanosoma effect was evaluated by determining the IC 50 value (the concentration required to inhibit the growth of the parasite by 50%) and calculated by non-linear regression analysis using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com, The IC 50 reported corresponds to the average of the results obtained in at least two independent experiments.

- In-vitro evaluation of activity against the parasite T. cruzi

The assay is based on determination of antiparasitic activity on osteosarcoma U20S cell lines infected with T. cnizi. The U2OS cells were cultured in 6-well plates (3 x 106 HG39 cells/well) for 24 hours and infected with circulating trypomastigotes of T. cruzi Y, in the ratio of 1 : 1. After four hours, the cells were washed three times with PB S and a fresh medium was added (RPMI supplemented with Glut/Pen/Strep and 20% heat-inactivated FCS, Sigma-Aldrich). The infected cells were incubated at 37°C and 5% CO 2 . 24 hours after infection, the cells were treated with the compound and incubated for 72 hours. The plates were analysed by high-content imaging, and the activity of the compound was normalised against the control represented by infected and hoh-infected cells (see C.B. Moraes et al., Sci. Rep. no. 4 (2014) 4703).

- Evaluation of cytotoxicity towards THP-1 macrophages

The effect of the compounds on macrophages derived from THP-1 was evaluated with the MTT colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide). Briefly, 1×10 6 THP-1 cells were differentiated into macrophages with the addition of 20 ng/mL of phorbol-myristate 13 -acetate (PMA, Sigma Aldrich) for 18 hours, followed by substitution with a fresh medium for 24 hours. The cells were incubated with the compound (concentration between 100 and 3 mM) after dilution in an RPMI medium containing a maximum of 1% DMSO. After incubation for 72 hours at 37°C and 5% CO 2 , the medium was removed and an 0.5 mg/mL MTT solution was added. The plates were incubated for a further 4 hours to enable the viable cells to convert MTT to purple formazan. The formazan crystals were solubilised by adding 2-propanol, and the absorbance was read at 570 nm using a Synergy 2 Multi-Mode reader (Biotek). Cytotoxicity was evaluated by determining the CC 50 value (concentration of medicament that reduces the percentage of viable cells by 50%) and calculated by non-linear regression analysis using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, California, USA, www. graphpad.com.

Assay

The compounds were assayed for antiparasitic activity against parasitic lines of T. brucei and the amastigote cell phase of T cruzi. The compounds were initially tested at 10 mM against T. brucei and at 50 mM against amastigote forms of T. crusi. As the latter is an intracellular species, and in view of the difficulty at international scientific level of identifying compounds active against this parasitic species, it was preferred to test the compounds at a higher concentration.

The compounds that exhibited antiparasitic activity exceeding 80% at the concentration selected were further tested to determine the EC 50 (concentration able to induce the death of 50% of the parasites).

In addition, to evaluate toxicity towards human cells, the compounds were tested against THP-1 human macrophages. The NOAEL (No- Adverse-Effect-Level, i.e. the maximum concentration of compound that does not produce toxic effects) was taken as the measurement of the toxicity of the compounds towards human cells and used to calculate the selectivity index (SI), defined as the ratio between NOAEL and the parasitic EC 50 .

The complete profiles of antiparasitic activity against T. brucei and the corresponding toxicity are set out in Table 2, and antiparasitic activity against T. cruzi in

Table 3.

Compounds F220, F242, F248, F250, F252, F262, F266 and F270 proved to be powerful antiparasitic agents against T, brucei , with EC 50 values ranging between 0.09 and 0.65 mM and a high selectivity index, ranging between 31 and 167, against human macrophage ΊΉR-1, and are therefore usable for treating human African trypanosomiasis.

The compounds tested proved active against T. cruzi, with special reference to compound F252, which exhibits a parasitic EC 50 of 14 mM, They can therefore also be used to treat Chagas disease.

Compounds F252 and F220 are particularly preferred:

Compounds F252 and F220 have a broad action spectrum and are active against T. brucei (F252 EC 50 = 0.09 mM, F220 EC 50 = 0.30 mM) and the amastigote form of T. cruzi (F252 EC 50 = 14, 1 mM), The two compounds have an activity comparable to that of pentamidine (T. brucei EC 50 =1.5nM) and benznidazole (T. cruzi EC 50 =2,4 mM), two of the medicaments currently most widely used to treat HAT and Chagas disease respectively. They also exhibit low toxicity against human THP1 cells (NOAEL > 25 mM), with a consequently high selectivity index towards human cells (F252 SI = 138, F220 SI = 83).

Compounds F252 and F220 are therefore suitable for use as a medicament, in particular in the treatment of human African trypanosomiasis (sleeping sickness) and Chagas disease. Compounds F252 and F220 are therefore an antiparasitic agent against the parasites T. brucei and T. cruzi.

Example 18 - Toxicity evaluation (ADME-Tox)

Materials and methods

- Test for cardiotoxieity towards hERG

The Invitrogen Predictor™ hERG Fluorescence Polarisation assay was used in a

384-well format (Greiner bio-one, 784076) to test the compounds. 100 nL of the compound (or control) was added to each well of the plate, followed by 5 ml of a homogenised membrane solution (undiluted), followed by a further addition of 5 ml of tracer (1 nM: final concentration in the assay). The plates were incubated for 2 hours at 25 °C in an incubator with controlled humidity, and the polarisation fluorescence was measured with an EnVision® Multilabel Reader 2103 (PerkinElmer Inc, US), The data were analysed with ActivityBase (IDBS), and the 3-sigma method was applied to eliminate outliers in the control plate. The negative control (0% inhibition) and the positive control E-4031, an hERG potassium channel blocker (100% inhibition), were used to normalise the raw data. Each compound was tested in triplicate, and pIC 50 , standard deviation, slope of curve, minimum signal and maximum signal for each dose-response curve were obtained with an ActivityBase XE module (IDBS) All the IC 50 values were associated with a standard deviation <10%,

- Cytochrome P450 1A2, 2C9, 209, 2D6 and 3A4 inhibition assay

The assay, based on P450-Glo™ luminescence (Promega), was used in a 384-well format (Greiner bio-one, 784076) to test the compounds. The selected panel of P450 cytochromes includes the microsomal preparation of cytochromes P450 1 A2, 2C9, 2C19, 2D6 and 3A4 (Coming) from insect cells infected with baculovirus (BTI-TN-5B1-4), which expresses P450 (CYP) cytochromes and cytochrome c reductase (and cytochrome b5 for 3A4). The compounds (or controls) were added to an empty 384-well plate (100 nL/ v/v 1% DMSO) using Echo® Liquid Handler (Labcytelnc); 5 mL/well of CYP/Luciferin- substrate was then added, and incubated for 30 minutes at 37°C. The reaction was triggered by adding 5 mL/well of mixture containing NADPH. After 30 minutes at 37°C the reaction with CYP was stopped, and the luciferase reaction was started by adding 10 ml/well of Luciferin Detection Reagent. After 30 minutes at 37°C, the luminescence reading was performed with the Infinite® M1000 PRO microplate reader (Tecan). Outliers were eliminated with the 3 -sigma method.

The negative controls (0% inhibition) only include the carrier (v/v 1% DMSO), a specific standard CYP inhibitor; CYP 1A2 inhibitor - a-naphtho-flavone (Sigma- Aldrich, 15 nM), CYP 2C9 inhibitor - sulfaphenazole (Sigma- Aldrich, 67 nM), CYP 2C19 inhibitor - troglitazone (Sigma- Aldrich, 3.2 mM), CYP 2D6 inhibitor - quinidine (Sigma Aldrich, 2 nM) and CYP 3 A4 inhibitor - ketoconazole (Sigma Aldrich, 54 nM) were used as positive controls (100% inhibition). Each compound was tested in triplicate, and pIC 50 , standard deviation, slope of curve, minimum signal and maximum signal for each dose-response curve were obtained with an ActivityBase XE module (IDBS), All the IC 50 values were associated with a standard deviation <10%.

- Assay of cytotoxicity towards A549 cells

This assay was conducted with the Promega Corp CellTiter-Glo assay. The A549 cells were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). The cells were incubated at 37°C in the presence of 5% CO 2 and collected at 80-90% confluence. Each compound tested (200 nl of 10 mM in 100% v/v DMSO) was added to 384-well cell culture plates (cat. no. 781073, 384 CellStar, Greiner Bio One, AT) using Echo 550® liquid handler (Labcyte Inc., US). 1.5 ml of trypsin/EDTA (cat. no. LI 1-004, 0.5 mg/mL and 0.22 mg/mL respectively, PAA Laboratories GmbH, AT) were added for each T175 vessel to collect the cells, and incubated at 37°C in the presence of 5% CO 2 for 2 minutes. The isolated cells were then resuspended in a pre-heated medium until a density of 0.2x106 cells/mL was reached. 20 ml per well was added to said cell suspension in a 384-well plate until a final concentration of 100 mM and 0.1% v/v DMSO was obtained, After 48 hours’ incubation at 37°C in the presence of 5% CO 2 , 20 ml of CellTiter-Glo® reagent (cat. no. #G7571, Promega Inc., US) per well was added, and the plate was placed on a linear stirrer for one minute at room temperature and further incubated at room temperature without mechanical stirring for 10 minutes. The luminescence was read with an EnVision® Multilabel Reader 2103 (PerkinEImer Inc, US) with 0.5 seconds’ reading time per well. Each plate also contained 16 wells for the positive control (cells prepared by treating with cisplatin: 200 nl of a 300 mM stock solution in 100% v/v DMSO to obtain a final concentration of 3 mM of cisplatin). The 16 wells per plate with the negative control were prepared by treating the cells with DMSO only (200 nL). Each compound was tested in triplicate, and pIC 50 , standard deviation, slope of curve, minimum signal and maximum signal for each dose-response curve were obtained with an ActivityBase XE module (IDBS).

Assay

Inhibitory activity was evaluated at 10mM of 1A and IB against hERG and against five isoforms of cytochrome P450 (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3 A4). Cytotoxicity towards A549 cells and mitochondrial toxicity were also evaluated. The toxicity profile for the two compounds and the selectivity index (SI) calculated for each parameter in relation to the corresponding antiparasitic activity is summarised in Table

Both compounds exhibited a highly satisfactory toxicity profile, with selectivity indexes normalised against the corresponding antiparasitic activities exceeding 10, and are therefore suitable for subsequent pharmacokinetics studies.

Example 19 - Pharmacokinetics evaluation with SNAP-PK

Formulation

Compound F252 was complexed with hydroxypropyl-p-cyclodextrins (50% weight/volume) (Cavasol® W7 HP Pharma, Ashland) to increase its solubility and bioavailability for subsequent pharmacokinetics studies.

The pharmacokinetics of compound F252 were determined with the SNAP-PK technique. Plasma samples originating from 6 B ALB/c mice, taken 5, 15, 30 and 45 minutes and 1, 3, 24, 48 and 72 hours after administration of the test compound, were analysed with LC-QQQMS to quantitate the compound in the blood. All the compounds, complexed with cyclodextrin, were administered per os at the dose of 20 mg/Kg. The pharmacokinetic profile is shown in Table 5.

Compound F252 exhibits a good pharmacokinetic profile. If the plasma concentration of the compound is compared with the corresponding EC 50 against T. brucei (Figure 1), it will be seen that in the first hour after administration, compound F252 exceeds the parasitic EC 50 30-fold,

F252, in particular, maintains a plasma concentration 10-30 times greater than the EC 50 against T. brucei (90 nM) for the first 5 hours after administration, and will be used in subsequent in vivo studies of antiparasitic activity on models of mice infected with T. brucei.