Telotristat ethyl compound for use in the treatment of malaria, combination and kit

Abstract

The present invention is related to the telotristat ethyl compound for use in the treatment of malaria and / or the prevention and treatment of the complications thereof. The present invention also concerns a combination of the telotristat ethyl compound and of at least one antimalarial drug conventionally used for treatment of the subject with malaria, and a kit comprising the telotristat ethyl compound inside a container, and one or more antimalarial drugs in one or more containers. Furthermore, the present invention is related to a process for the preparation of the telotristat ethyl.

Description

[0001] TELOTRISTAT ETHYL COMPOUND FOR USE IN THE TREATMENT OF MALARIA, COMBINATION AND KIT

[0002] FIELD OF THE INVENTION

[0003] The present invention is related to the telotristat ethyl compound for use in the treatment of malaria and / or the prevention and treatment of the complications thereof.

[0004] The present invention refers furthermore to a combination of the telotristat ethyl compound and of at least one antimalarial drug conventionally used for treatment of the subject with malaria, and a kit comprising the telotristat ethyl compound inside a container, and one or more antimalarial drugs in one or more containers.

[0005] Furthermore, the present invention is related to a process for the preparation of the telotristat ethyl.

[0006] STATE OF THE ART

[0007] Malaria is an infectious disease caused by a protozoan parasite of the genus Plasmodium transmitted by mosquitoes belonging to the species Anopheles. Malaria represents nowadays the most important parasitosis and the second most important infectious disease in the world in terms of morbidity and mortality after tuberculosis, with 247 million new cases per year (96 percent of which are in tropical Africa). Over the past two decades, progresses toward the control of malaria through the coordinated efforts of international agencies, local governments and private foundations have led to a reduction of more than 40 percent in the global incidence of malaria. However, these improvements have stalled in recent years, and malaria still kills more than 600,000 people each year, two-thirds of whom are children under the age of 5 (World Malaria Report, 2021 ). Children in sub-Saharan Africa are particularly vulnerable to cerebral malaria, the most serious neurological complication of the disease caused by the infection with Plasmodium falciparum. Mortality caused by cerebral malaria ranges from 15 to 20 % and leaves up to 25 % of survivors with long-term neurological sequelae.

[0008] Cerebral malaria is characterized by several mechanisms, such as: sequestration of parasitized red blood cells in brain capillaries, cytokine production, accumulation of immune cells / platelets and release of microparticles, resulting in endothelial injuries of the blood-brain barrier, which contribute to various brain damages such as edema, ischemia and haemorrhages (Dorovini-Zis, K. et al. Am. J. Pathol. 178, 2146-2158 (2011 )).

[0009] In addition to brain injuries induced by P. falciparum, cerebral malaria is also associated with nonparasitic events including oxidative stress and the inflammatory cascade responsible for mortality despite antimalarial treatment during the acute step. Cerebral malaria is associated with elevated pro-inflammatory cytokines in serum and brain tissues and with recruitment of leukocytes into the cerebral microvasculature following sequestration of parasitized red blood cells. The immune response to cerebral malaria is mainly mediated by Th1 lymphocytes and causes a high production of interferon-y (IFN-y) and TNF-o. It has been proven that malaria infection causes an inflammatory reaction by causing release of various chemical mediators such as histamine, glutamate, dopamine, serotonin (5-HT), bradykinins and prostaglandins. These inflammatory processes promote pathological changes in the brain of people with malaria, resulting in damage, neuronal dysfunction and persistent cognitive deficits.

[0010] Current therapy uses antimalarial drugs capable of controlling the growth of the parasite but not the inflammatory cascade. Although the available antimalarial drugs are effective in eliminating parasites from the blood, they have no specific effects against cerebral malaria. Therefore, it is urgent to identify new therapies with the aim of providing a new therapeutic diagram for treatment of cerebral malaria.

[0011] The first intervention toward the control of the oxidative stress and of the inflammatory cascade resulting from infection caused by protozoan parasites of the genus Plasmodium is represented by the use of anti-TNF antibodies, tested in a mouse model of malaria in 1987. Since then about 48 additional interventions have been assessed, i.e. , to be used in association with available antimalarial drugs, which have shown an improvement in the prevention of cerebral malaria in the mouse model, but have yielded poor results in humans. In fact, as of today, clinical studies on the therapy for cerebral malaria have shown no favorable results.

[0012] Therefore, it is evident that it is necessary to have a drug capable of treating the inflammatory processes resulting from the infection caused by protozoan parasites of the genus Plasmodium, to be used preferably in combination with one or more antimalarial drugs.

[0013] Indeed, in addition to effectively killing the parasite, it is necessary to improve the survival rate and overcome cognitive deficits in patients with cerebral malaria and thus reduce the probability of permanent neurological disability or death.

[0014] Therefore, a therapy for treatment of malaria that allows to save patients from the neurological disorders of malaria from death and / or is a primary need.

[0015] SUMMARY OF THE INVENTION

[0016] One of the objects of the present invention is to provide a new drug capable of controlling the oxidative stress and the inflammatory cascade resulting from infection caused by protozoan parasites of the genus Plasmodium, in particular caused by the Plasmodium falciparum.

[0017] The Applicant has surprisingly found that the telotristat ethyl compound is capable of activating a protective and long-lasting immune and / or anti-inflammatory response of the host, counteracting the most serious neurological complication: cerebral malaria caused by P.falciparum. In 2017 the drug Xermelo® comprising telotristat ethyl has been approved by the FDA in combination with somatostatin analogue therapy (SSA) for treatment of adults with diarrhea associated with carcinoid syndrome. The telotristat ethyl acts with a different mechanism of action with respect to measures currently in use to counteract malaria since it blocks the action of enzymes called L-tryptophan hydroxylase (TPH), necessary for serotonin production.

[0018] The Applicant has advantageously demonstrated that pharmacological inhibition of serotonin increases the survival rate of animals in a mouse model of cerebral malaria and the combination of the telotristat ethyl with an antimalarial drug leads to the complete regression of the disease. Therefore, a first aspect of the present invention concerns the telotristat ethyl compound for use in the treatment of malaria and / or the prevention and treatment of the complications thereof. In particular, said complications are the neurological disorders of malaria. Advantageously, as shown in the experimental section, the use of telotristat ethyl improves the clinical picture of the infection, preventing the occurrence of complications associated with cerebral malaria and allowing a longer therapeutic time frame.

[0019] The Applicant's second goal was to provide a new treatment diagram for treatment of malaria.

[0020] As shown in the experimental section, the use of telotristat ethyl in combination with chloroquine, a conventionally used antimalarial drug, increases the survival rate of animals in a mouse model of cerebral malaria and leads to complete regression of the disease.

[0021] Therefore, a second aspect of the present invention refers to a combination of telotristat ethyl and of at least one antimalarial drug conventionally used for treatment of the subject with malaria.

[0022] Currently, the artemisinin-based combination therapy (ACT) is recommended for treatment of malaria by P. falciparum to provide an adequate rate of healing and delay the development of resistance. Fast-acting artemisinin-based compounds are combined with a drug from a different class. The associated drugs comprise lumefantrine, mefloquine, amodiaquine, sulfadoxine / pyrimethamine, piperaquine and chlorproguanil / dapsone. Artemisinin derivatives include dihydroartemisinin, artesunate and artemether. Chloroquine is still the first-line treatment for P.vivax and P. ovale, while primaquine can be used to treat parasites at the liver stage of P.vivax, in areas with low transmission of malaria.

[0023] In particular, the combination according to the invention comprises telotristat ethyl and at least one antimalarial drug selected from chloroquine, primaquine or the artemisininbased compounds or derivatives thereof in association with at least one additional drug selected from lumefantrine, mefloquine, amodiaquine, sulfadoxine / pyrimethamine, piperaquine and chlorproguanil / dapsone. The artemisinin derivatives which can be associated with lumefantrine and / or mefloquine and / or amodiaquine and / or sulfadoxine / pyrimethamine and / or piperaquine and / or chlorproguanil / dapsone include dihydroartemisinin, artesunate and artemether.

[0024] In a particularly preferred embodiment, the combination according to the invention comprises telotristat ethyl and one or more artemisinin-based compounds or derivatives thereof in association with at least one additional drug selected from lumefantrine and / or mefloquine and / or amodiaquine and / or sulfadoxine / pyrimethamine and / or piperaquine and / or chlorproguanil / dapsone.

[0025] A third aspect of the present invention refers to a kit comprising the telotristat ethyl inside a container, and one or more antimalarial drugs in one or more containers.

[0026] A fourth aspect of the present invention is related to a process for the preparation of telotristat ethyl having the following formula (I) characterized in that said telotristat ethyl of formula (I) is obtained by the following two- step process

[0027] Step A

[0028] Step B

[0029] where said step A is conducted in dioxane in the presence of CS2CO3 and said step B is conducted in anhydrous ethanol in the presence of SOCI2.

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] The Applicant has advantageously observed that the telotristat ethyl compound is capable of activating a protective and long-lasting immune and / or anti-inflammatory response, counteracting the most serious neurological complication caused by infection with Plasmodium falciparum-, the cerebral malaria.

[0032] Therefore, the first aspect of the present invention refers to the the telotristat ethyl compound for use in the treatment of malaria and / or the prevention and treatment of the complications thereof.

[0033] In particular, the complications of malaria to which the present invention refers are the neurological disorders resulting from infection by the protozoan P. falciparum.

[0034] In the cerebral malaria, P.falciparum-infected erythrocytes are sequestered in the cerebral microcirculation, adhere to healthy erythrocytes and can obstruct the lumen of cerebral capillaries and venules. As a result, there is cerebral congestion and, in advanced stages, intraparenchymal hemorrhage. From the clinical point of view, the subject with cerebral malaria may present with high fever, headache, drowsiness, delirium, confusional state, convulsions, go into coma and, in severe cases, death. Advantageously, is has been observed on mice model of malaria that the use of telotristat ethyl allows to prevent the occurrence of complications associated with cerebral malaria (see Example 2 and Fig. 2).

[0035] The telotristat ethyl is a compound that acts as a propharmaceutical of telotristat and has the formula (I) indicated below:

[0036] Both the prodrug (telotristat ethyl) and its active metabolite (telotristat) are inhibitors of L-tryptophan hydroxylase TPH1 , one of two isoforms of the enzyme L-tryptophan hydroxylase involved in serotonin production.

[0037] In detail, L-tryptophan hydroxylase intervenes in the hydroxylation at position 5 of tryptophan, leading to the formation of 5-hydroxytryptophan, the intermediate from which serotonin originates.

[0038] In particular, TPH1 synthesizes serotonin in the enterocromaffin cells of the intestinal epithelium, from where it is released and exerts local actions in the intestine, but it is also absorbed by platelets via SERT (Serotonin Transporter). Platelets pack it into their granules and release it upon activation. Smaller amounts of TPH1 have been found in other tissues such as lung, pancreas, fat and vascular wall. In the central nervous system TPH1 is not expressed, but TPH2 is, mainly present in the Raphe nuclei of the brainstem.

[0039] TPH1 enzyme inhibitors have been developed to specifically block the peripheral TPH enzyme and avoid blocking TPH2 which is the equivalent centrally expressed protein. To this end, such inhibitors have been designed so as not to cross the blood-brain barrier and selectively act in the blood and gastrointestinal tract.

[0040] The compound for use according to the invention selectively inhibits the TPH1 enzyme but not the centrally expressed TPH2 enzyme.

[0041] An advantage of telotristat ethyl is that it does not pass through the blood-brain barrier mainly acting in the intestinal tract. The compound for use according to the invention proves advantageous in terms of reducing the immune response and proinflammatory responses (is capable of reducing oxidative stress and the inflammatory cascade resulting from Plasmodium infection ) by not acting on centrally expressed TPH2.

[0042] As previously mentioned, the telotristat ethyl compound present in the drug Xermelo® is useful in the treatment of adults with diarrhea associated with carcinoid syndrome in combination with somatostatin analogue (SSA) therapy.

[0043] The present invention, on the other hand, provides the use of telotristat ethyl for treatment of an infectious disease with different etiopathology with respect to intestinal disorders resulting from carcinoid syndrome.

[0044] From the experiments conducted on an experimental model of malaria and presented in the experimental section is possible to observe that the use of telotristat ethyl compound improves the clinical picture of the infection, preventing the occurrence of complications associated with cerebral malaria and allowing a longer therapeutic time frame (see example 5 and fig. 4A and 4B).

[0045] The synergy of action of telotristat ethyl compound with the antimalarial drugs currently in use implements treatment of malaria and allows to prevent or treat the neurological disorders associated with P. falciparum infection.

[0046] In light of this, an additional aspect ofthe present invention is a combination of the telotristat ethyl compound and of at least one antimalarial drug conventionally used for treatment of the subject with malaria.

[0047] The combination according to the invention comprises telotristat ethyl and at least one antimalarial drug selected from chloroquine, primaquine or artemisinin-based compounds or derivatives thereof in association with at least one additional drug selected from lumefantrine, mefloquine, amodiaquine, sulfadoxine / pyrimethamine, piperaquine and chlorproguanil / dapsone. Artemisinin derivatives which can be associated with lumefantrine and / or mefloquine and / or amodiaquine and / or sulfadoxine / pyrimethamine and / or piperaquine and / or chlorproguanil / dapsone are dihydroartemisinin, artesunate and artemether.

[0048] In a particularly preferred embodiment, the combination according to the invention comprises telotristat ethyl, one or more artemisinin-based compounds or derivatives thereof in association with at least one additional drug selected from lumefantrine and / or mefloquine and / or amodiaquine and / or sulfadoxine / pyrimethamine and / or piperaquine and / or chlorproguanil / dapsone.

[0049] The combination of the telotristat ethyl compound and chloroquine allowed the extension of the survival time of the experimental model of cerebral malaria, exceeding the typical time window of cerebral malaria (see example 5).

[0050] In fact, using the combination of telotristat ethyl and of at least one conventional antimalarial drug is possible to control malaria by acting on the various pathogenetic mechanisms that characterize the evolution of infection: increased inflammatory response, decreased immune response resulting in multi-organ dysfunction, progressive intravascular hemolysis and breakdown of the blood-brain barrier.

[0051] In an embodiment, the telotristat ethyl and one or more antimalarial drugs indicated above, possibly in association with one or more of the additional drugs (lumefantrine, mefloquine, amodiaquine, sulfadoxine / pyrimethamine, piperaquine and chlorproguanil / dapsone) are administered together in a single composition.

[0052] From the experimental evidences presented below, it results that the combined therapy of malaria with the telotristat ethyl compound and a conventional antimalarial drug (chloroquine), administered separately, is effective in the prevention of cerebral malaria in an experimental animal model of cerebral malaria, similar to what is observed in animal models of the disease lacking the enzyme TPH1 .

[0053] Therefore, more preferably, the telotristat ethyl and at least one antimalarial drug conventionally used for treatment of the subject with malaria are administered separately in two or more different compositions or different dosage forms.

[0054] In this preferred embodiment, the administration of telotristat ethyl compound and at least one antimalarial drug conventionally used for treatment of the subject with malaria can occur in any order. For example, the telotristat ethyl compound can be administered concomitantly, before or after the antimalarial drug.

[0055] A further aspect of the present invention is a kit comprising a container for the telotristat ethyl compound, and one or more containers for antimalarial drugs to be combined with treatment with the telotristat ethyl. DESCRIPTION OF FIGURES

[0056] Figure 1. Survival and parasitemia levels in WT and TPH1 KO mice infected with Pb ANKA.

[0057] A) The survival time of the mice (WT, n=26; TPH1 KO, n=29) was monitored daily until the end of the experiments. Statistical analysis was performed by test Log-rank (Mantel- Cox): ****p<0,0001.

[0058] B) The parasitemia levels (WT, n= 39; TPH1 KO, n= 45) were monitored daily until the end of the experiments by microscopic assessement of thin blood smears and Giemsa staining. The data represent the mean ± DS of four experiments.

[0059] Figure 2. TPH1 KO mice do not show cerebral malaria signs.

[0060] The graph represents the scores (“MC Score”) assigned to each clinical sign attributable to the development of cerebral malaria for each group (WT, n=5; TPH1 KO, n=6). Results represent mean ± SD and experiments were repeated twice. Mann-Whitney test **p = 0,022.

[0061] Figure 3. Assessment of blood-brain barrier integrity in WT and TPH1 KO mice after infection with Plasmodium berghei ANKA.

[0062] A) Representative brain images of WT and TPH1 KO mice following staining with Evans blue solution.

[0063] B) Quantification of Evans blue concentration in WT (n = 7) and THP1 KO (n = 8) mice. Mann Whitney test ***p = 0,0003.

[0064] Figure 4. Survival and parasitemia levels in WT mice and treated with telotristat ethyl.

[0065] A) The survival time of mice (WT, n=9; TPH1 inhibitor, n=7) was monitored daily until the end of the experiment. Statistical analysis has been performed test Log-rank (Mantel-Cox) ***p=0,0002.

[0066] B) Parasitemia levels in mice (WT, n = 9; TPH1 inhibitor, n = 7) were monitored all days until the end of the experiment by microscopic assessment of thin blood smears and Giemsa staining. The data represent the mean two experiments ± DS. Figure 5. 1H NMR spectrum of telotristat ethyl compound

[0067] A) 1 H NMR spectrum of telotristat ethyl compound synthesized according to the process of synthesis described in example 4.

[0068] (B) 1 H NMR spectrum of commercial telotristat ethyl compound purchased from the company Asta Tech, Inc.

[0069] Figure 6. 13C NMR spectrum of telotristat ethyl compound

[0070] A) 13C NMR spectrum of telotristat ethyl compound synthesized according to the process of synthesis described in example 4.

[0071] B) 13C NMR spectrum of commercial telotristat ethyl compound purchased from the company Asta Tech, Inc.

[0072] EXAMPLES

[0073] Example 1 : Murine model of cerebral malaria and assessment of survival time and parasitemia in TPH1 KO mice

[0074] The effect of the absence of peripheral serotonin was tested in vivo on an experimental model of cerebral malaria obtained by infecting TPH1 knock-out mice (TPH1 KO) and control mice (wild type, WT) with Plasmodium berghei ANKA (PbA).

[0075] Plasmodium berghei is a single-celled parasite that causes malaria in rodents. It belongs to the subgenus Plasmodium Vinckeia. In particular, Plasmodium berghei is one of four Plasmodium species described in African murine rodents (the others are P. chabaudi, P. vinckei and P. yoelii) and, because of its ability to infect rodents and the relative ease of genetic engineering, P. berghei is the model organism of choice for the study of human malaria. Like all mammalian parasites of malaria, also P. berghei is transmitted by Anopheles mosquitoes and infects the liver after being injected into the bloodstream by the bite of an infected female mosquito. After a short period of development and multiplication, these parasites leave the liver and invade erythrocytes (red blood cells). Multiplication of the parasite in the blood causes diseases such as anemia and damage to essential host organs such as lungs, liver and spleen. P. berghei infections can also affect the brain and be the cause of cerebral complications (murine cerebral malaria) in laboratory mice. The symptoms of PbA-infected mice are comparable to those of cerebral malaria in patients infected with the parasite of human malaria Plasmodium falciparum.

[0076] In detail, for the present experiments were used C57BL / 6N mice provided by the company Charles River (Milano, Italy).

[0077] The mice were infected intravenously with 10,000 PbA sporozoites and, subsequently, survival time and parasitemia levels were assessed daily.

[0078] In particular, the study regarding survival rate was conducted on 26 WT mice and 29 TPH1 KO mice, infected with PbA.

[0079] The Applicant has observed that, after infection, the TPH1 KO mice showed a survival rate of 86% with respect to WT mice, all of which die between the seventh and ninth post-infection day (p.i.) (time window of cerebral malaria) (Fig.lA). The significant increase in survival time in TPH1 KO mice with respect to control mice provides evidence that these mice exceed the temporal window of cerebral malaria that usually occurs between days 6 and 9 p.i.

[0080] The study regarding the percentage of parasitemia was conducted on 39 WT mice and 45 TPH1 KO mice, infected with PbA.

[0081] No significant change was observed on parasitemia levels during the first 9 days of disease between WT and TPH1 KO mice. Subsequently, in TPH1 KO mice, which survive cerebral malaria, parasitemia increases over time reaching about 60% at day 15 p.i. (Fig. 1 B).

[0082] These results demonstrate that the absence of TPH1 enzyme, and consequently of peripheral serotonin, has an effect on the pathogenesis of cerebral malaria induced by PbA.

[0083] Example 2: Assessment of clinical and behavioral signs typical of cerebral malaria in TPH1 KO mice

[0084] To assess the development of cerebral malaria, mice (WT, n=5; TPH1 KO, n=6) were monitored daily and the following physical and behavioral aspects were assessed: matted fur, shaky gait, partial paralysis, seizures and coma. Each of these parameters was assigned a score using an arbitrary scale from 0 (normal) to 4 (dead). Table 1 shows the scores assigned to each of the parameters assessed in TPH1 KO mice and WT mice infected with PbA.

[0085] Table 1 : Clinical signs analyzed and score given for the assessment of development of cerebral malaria

[0086] The Applicant has observed that the TPH1 KO mice showed no visible signs of disease throughout the time window considered, in contrast to the control mice that instead show a progressive worsening from day 6 to day 9 p.i. (see Table 2 and Fig. 2). Table 2 indicated below shows an average of the scores taken considering both “appearance” and “natural behavior” parameters indicated in Table 1.

[0087] Table 2: Average of scores detected in TPH1 KO mice and WT mice over time These observations confirm that, in the absence of the TPH1 enzyme, mice do not show the typical clinical picture of the experimental animal model of cerebral malaria.

[0088] Example 3: Assessment of the integrity of the blood-brain barrier

[0089] The integrity of the blood-brain barrier represents another important indicator for assessing the development of cerebral malaria. In fact, characteristic sign of cerebral malaria are the endothelial lesions of the blood-brain barrier.

[0090] For the present experiment, 7 WT mice and 8 TPH1 KO mice (infected with PbA) were inoculated with Evans Blue solution, which, by binding to plasma albumin that effectively crosses the blood-brain barrier, is capable of staining the brain of mice in the presence of a defect in blood-brain barrier permeability.

[0091] The results showed that the brains of WT mice that die from cerebral malaria turn out to be stained blue while the brains of THP1 KO mice show the absence of staining, indicating an intact blood-brain barrier (Fig. 3A).

[0092] Quantitative analysis of the penetrated dye also confirmed what is evident from the images shown in Fig. 3A: the values of the TPH1 KO group are significantly lower with respect to the values observed for the WT mice group (Fig. 3B).

[0093] It is inferred from the results that TPH1 KO mice have lower blood-brain barrier permeability with respect to WT mice following PbA infection. This suggests that in the absence of the TPH1 enzyme, the mice do not undergo cerebral malaria.

[0094] Example 4: Synthesis of telotristat ethyl compound

[0095] The telotristat ethyl compound was tested on WT mice infected with PbA sporozoites.

[0096] The telotristat ethyl compound was synthesized following a 6-step process of synthesis summarized in diagram I and described in detail below.

[0097] Diagram I: diagram of synthesis of telotristat ethyl compound Step 1 : Synthesis of 1-(2-bromo-4- chlorophenyl)-2,2,2-trifluoroetan-1-one

[0098] In a 100 mL round-bottom flask equipped with a magnetic stirring rod under an atmosphere of anhydrous Argon MeOH (28 mL) was added and SOCI2 (37 mmol, 2.7 mL, 3.7 eq) was slowly dripped at 0 °C. The water and ice bath was removed and 2- bromo-4-chlorobenzoic acid (2) (10 mmol, 2.34 g) was added. The mixture was heated and allowed to reflux. After 12 h the reaction was stopped, the crude concentrate under reduced pressure and dissolved in DCM (15 mL). The organic step was washed with water (10 mL), NaHCCh saturated solution (10 mL) and Brine (15 mL). The organic step was anhydrified with sodium sulfate and concentrated to give the 2-bromo-4- chlorobenzoic acid methyl ester (9.2 mmol, 2.3 g, 92 % yield) which was directly used for the next step without further purification. 2-Bromo-4-chlorobenzoic acid methyl ester (9.2 mmol, 2.3 g) in anhydrous Toluene (36 mL) was chilled to - 70 °C in Argon atmosphere and Trifluoromethyltrimethyl silane (12.9 mmol, 1.83 g, 1.99 mL) was added. TBAF 1 M in THF (0.464 mL, 5 mol %) was added drop by drop and the reaction mixture was allowed to warm up to room temperature for 4 h and then was left under stirring at room temperature 10 h. The reaction was stopped and the reaction mixture concentrated under reduced pressure. The crude was dissolved in MeOH (17 mL) and a 6N HCI solution (17 mL) was dripped at 0 °C. The reaction mixture was left under stirring at 50 °C. After 12 h the reaction was stopped, the MeOH was evaporated and the crude extracted with DCM (37 mL). The pooled organic phases were washed with water (15 mL), NaHCOs (15 mL), Brine (15 mL), dried over sodium sulfate and concentrated under reduced pressure. The crude was purified by PE / EtOAc chromatographic column (EtOAc from 1 % to 5 %) to obtain the pure product (4) 1-(2- bromo-4-chlorophenyl)-2,2,2-trifluoromethane-1 -one (6.27 mmol, 1.8 g, 63 % yield).

[0099] Step 2: Synthesis of (R)-1-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoroethanol

[0100] 4 5

[0101] In a 50 mL round-bottomed flask equipped with a magnetic stirring rod Catecholborane 1 M in THF (11.9 mmol, 11.9 mL) (2 eq) and 2S-methyl-CBS oxazaborolidine (1.19 mmol, 329 mg) (0.2 eq) were added consecutively and the resulting mixture was left under stirring at room temperature. After 20 min, the reaction was cooled to - 78 °C and (4) 1 -(2-bromo-4-chlorophenyl)-2,2,2-trifluoromethane-1 -one (5.95 mmol, 1.7 g) in anhydrous THF (17 mL) was added in 2 h. The reaction mixture was left under stirring until it reached - 30 °C and stirred at this temperature for 48 h. A 3N NaOH solution (10 mL) was added under stirring at - 30 °C, the mixture was left under stirring at 0 °C and a 30 % hydrogen peroxide solution (10 mL) was added drop by drop in 15 min. The reaction mixture was left under stirring at room temperature. After 4 h the reaction was stopped and the aqueous step extracted with DEE (4 x 15 mL). The organic step was washed with NaOH 1 N (4 x 15 mL) and Brine (2 x 15 mL), anhydrified with sodium sulfate, filtered and concentrated under reduced pressure. The crude was purified by chromatographic column (PE / ETOAc (EtOAc from 2 % to 10 %) to obtain the desired product 1 R-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoroethanol (5) (4.42 mmol, 1.28 g, 74 % yield).

[0102] Step 3: Synthesis of (R)-1-(4-chloro-2-(3-methyl-1 H-Dyrazole-1-yl)ohenyl)-2,2,2- trifluoroethanol-1 -ol

[0103] In a pressure tube equipped with a magnetic anchor, 1 R-(2-bromo-4-chloro-phenyl)- 2,2,2-trifluoroethanol (5) (3.45 mmol, 1 g), 3-methylpyrazole (6) (4.14 mmol, 339.39 mg, 0.32 mL), (1.2 eq), Cui (0.69 mmol, 131.41 mg) (0.2 eq), potassium carbonate (7.2 mmol, 995 mg) (2.1 , eq), (1 R, 2R)-N,N'-dimethylcyclohexane-1 ,2-diamine (0.69 mmol, 98.14 mg) (0.2 eq) and Toluene (5.07 mL) were consecutively added. The mixture was left under stirring at 130 °C. After 12 h, the reaction was stopped and cooled to room temperature. EtOAc (30 mL) was added. The organic step was washed with water (4 x 15 mL) and Brine (15 mL), anhydrified with sodium sulfate, filtered and concentrated. The crude was purified by PE / EtOAc chromatographic column (EtOAc from 0 % to 20 %) as eluent to obtain the desired product (7) (R)-1-(4-chloro-2-(3-methyl-1 H-pyrazol- 1 -yl)phenyl)-2,2,2-trifluoroethan-1 -ol (2.36 mmol, 686 mg, 69 % yield).

[0104] Step 4: Synthesis of compound (8) ethyl (S)-3-(4-(2-amino-6-chloropyrimidin-4- yl)phenyl)-2-((ter-butoxycarbonyl)amino)propanoate

[0105] In a vial equipped with a magnetic anchor EtOH (1 .57 mL), SOCL (5.04 mmol, 600 mg, 0.366 mL) (3.5 eq) and (S)-2-amino-3-(4-phenylboronic) acid (10a) (1.44 mmol, 300 mg) were added consecutively at 0 °C, the vial was sealed with a screw cap and the mixture was left under stirring at 25 °C. After 4 h water (2.52 mL) was added slowly at 0 °C to basify the mixture (until it had pH 7), EtOH (4.32 mL), potassium carbonate (398 mg, 2.88 mmol) (2 eq) and (Boc)2O (0.369 mL, 1.73 mmol) (1.2 eq) were added consecutively and the mixture was left under stirring at room temperature. After 2 h, 2- amino-4,6-dichloropyrimidine (2.16 mmol, 354 mg) (1.5 eq) and Pd(PPh3)Cl2 (0.0144 mmol, 10.08 mg) (1 mol %) were consecutively added and the mixture was left under stirring at 70 °C. After 3 h, the reaction was stopped, the solvent was evaporated and water (30 mL) and EtOAc (30 mL) were added (a few drops of 1 N HCI were added until there was pH 7 in the aqueous step). The aqueous step was extracted with (3 x 20 mL) EtOAc and the organic step washed with Brine (20 mL), anhydrified with sodium sulfate, filtered and concentrated under reduced pressure. The crude was purified by chromatographic column (PE / EtOAc from 0 % to 30 %) to obtain the desired product (8) ethyl (S)-3-(4-(2-amino-6-chloropyrimidin- 4-yl)phenyl)-2-((ter-butoxycarbonyl)amino)propanoate (0.42 mmol, 177 mg, 29 % yield).

[0106] Step 5: Synthesis of (S)-3-(4-(2-amino-6-((R)-1 -(4-chloro-2-(3-methyl-1 H-pyrazol-1- yl)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)-2-((ter butoxycarbonyl)amino)propanoic acid

[0107] In a vial equipped with a magnetic anchor the compound (7) (R)-1-(4-chloro-2-(3- methyl-1 H-pyrazol-1 -yl)phenyl)-2,2,2-trifluoroethan-1-ol (0,854 mmol, 2447mg) (1.2 eq), compound (8) ethyl(S)-3-(4-(2-amino-6-chloropyrimidin-4-yl)phenyl)-2-((ter- butoxycarbonyl)amino)propanoate (0.71 mmol, 300 mg), dioxane (3.48 mL) and CS2CO3 (3.45 mmol, 1.11 g) (4.8 eq) were added consecutively, the vial was sealed with screw cap and the mixture was left under stirring at 100 °C. The reaction was stopped after 24 h. EtOAc (20 mL) and water (20 mL) were added to the reaction mixture and the mixture was transferred to a separatory funnel. The water was washed with EtOAc (3 x 20 mL) and with DEE (20 mL), Chloroform (5 mL), THF (5 mL) and DCM (5 mL) to completely extract the product from the aqueous step. The aqueous step was anhydrified with sodium sulfate, filtered and concentrated to obtain the crude. The crude was purified by DCM / EtOH chromatographic column (EtOH from 5 % to 30 %)) to obtain the desired product (9) (S)-3-(4-(2-amino-6-((R)-1 -(4-chloro-2-(3-methyl-1 H-pyrazol-1- yl)phenyl)-2,2,2-trifluoromethoxy)pyrimidin-4-yl)phenyl)-2-((fer-butoxycarbonyl)amino) propanoic acid (208 mg, 0.32 mmol, 45 % yield).

[0108] Step 6: Synthesis of ethyl (S)-2-amino-3-(4-(2-amino-6-((R)-1-(4-chloro-2-(3-methyl-

[0109] 1 H-pyrazol-1 -yl)phenyl)-2,2,2- trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoate y

[0110] In a vial equipped with a magnetic anchor EtOH anhydrous (3 mL) was added. SOCI2 (2.17 mmol, 261 mg, 160 pL) (7 eq) was added. After 10 min, compound (9) (S)-3-(4- (2-amino-6-((R)-1 -(4-chloro-2-(3-methyl-1 H-pyrazol-1 -yl)phenyl)-2,2,2-trifluoroethoxy) oyrimidin-4-yl)ohenyl)-2-((ter butoxycarbonyl)amino)Drooanoic acid (0.31 mmol, 200 mg) was added. The mixture was left under stirring at 45 °C. After 14 h the reaction was stopped and a saturated sodium carbonate solution was added at 0 °C under stirring to raise the pH of the reaction mixture greater than 10. The aqueous step was extracted with EtOAc (5 x 5 mL). The organic step was anhydrified with sodium sulfate, filtered and concentrated under reduced pressure. The crude was purified by chromatographic column (DCM / EtOH, EtOH from 2 % to 4 %) to obtain the final product telotristat ethyl (0.19 mmol, 110 mg, 61 % yield).

[0111] The 1 H NMR spectrum of telotristat ethyl synthesized according to the process of synthesis above described is shown in Fig. 5A and compared with the spectrum of commercial telotristat ethyl purchased from company Asta Tech, Inc (Fig. 5B).

[0112] The 13C NMR spectrum of telotristat ethyl synthesized according to the process of synthesis above described is shown in Fig. 6A and compared with the spectrum of commercial telotristat ethyl purchased from company Asta Tech, Inc (Fig. 6B). Example 5: Survival and parasitemia in mice infected with Pb ANKA and treated with the telotristat ethyl compound

[0113] The telotristat ethyl compound, synthesized according to the process of synthesis described in example 4, was tested on 7 mice infected intravenously with 10,000 PbA sporozoites and subsequently treated intraperitoneally (i.p.) on day 3, 4 5 and 6 p.i. (post infection) with 50 mg / kg of telotristat ethyl dissolved PBS / DMSO (vehicle).

[0114] Such a group of mice is indicated in Fig. 4A and 4B as “TPH1 inhibitor.”

[0115] As a control, a second group of 9 mice infected intravenously with 10,000 PbA sporozoites is treated with the vehicle used for dissolving the drug (2% DMSO in 200pl PBS).

[0116] Such a group of mice is indicated in Fig. 4A and 4B as “WT.”

[0117] Mice treated with telotristat ethyl show a significant 86% survival rate with respect to control mice, all of which die between days 7 and 8 post-infection (time window of cerebral malaria) (Fig. 4A).

[0118] Furthermore, it was observed that treatment with telotristat ethyl does not alter parasitemia levels with respect to control mice (Fig. 4B), as previously observed in THP1 KO mice.

[0119] Because the telotristat ethyl has no direct effect on the parasite, treated animals that are not dead of cerebral malaria then demonstrated an increase in parasitemia levels reaching about 50% around day 15 p.i.

[0120] At this point, mice that survived the infection, and previously treated with the telotristat ethyl, was administered daily, for four days, chloroquine (25mg / kg in distilled water), a drug with direct effect on the parasite conventionally used in the treatment of malaria.

[0121] The results obtained confirmed the trend, in terms of survival and parasitemia levels, previously observed following PbA infection of TPH1 KO mice and described in Example 1.

[0122] In fact, as it is possible to observe from Fig. 4B, mice treated with telotristat ethyl survive beyond day 8 and show 0% parasitemia when, from day 15 p.i. onward, are treated with chloroquine.

[0123] From the present experiment it is possible to conclude that pharmacological inhibition of serotonin increases the survival rate of animals in a mouse model of cerebral malaria and the combination of the telotristat ethyl with an antimalarial drug leads to complete regression of the disease.

Claims

CLAIMS1. The telotristat ethyl compound for use in the treatment of malaria and / or the prevention and treatment of the complications thereof.

2. The telotristat ethyl compound for use according to claim 1 , wherein said complications are the neurological disorders of malaria.

3. The telotristat ethyl compound for use according to claim 1 or 2, wherein said malaria is caused by the protozoan P.vivax, P. ovale or P. falciparum, preferably by P.falciparum.

4. A combination of telotristat ethyl and at least one antimalarial drug conventionally used for the treatment of the subject with malaria, preferably selected from chloroquine, primaquine or artemisinin-based compounds or derivatives thereof in association with at least one additional drug selected from lumefantrine, mefloquine, amodiaquine, sulfadoxine / pyrimethamine, piperaquine and chlorproguanil / dapsone.

5. The combination according to claim 4, wherein said at least one antimalarial drug is an artemisinin-based compound or derivatives thereof in association with at least one additional drug selected from lumefantrine, mefloquine, amodiaquine, sulfadoxine / pyrimethamine, piperaquine, and chlorproguanil / dapsone.

6. The combination according to claim 4 or 5, wherein said artemisinin derivatives include dihydroartemisinin, artesunate and artemether.

7. The combination according to any one of claims 4 to 6, wherein said telotristat ethyl and said at least one antimalarial drug, possibly in association with said additional drug, are administered together in a single composition or separately in two or more different compositions or different dosage forms.

8. The combination according to claim 7, wherein said telotristat ethyl and said at least one antimalarial drug, possibly in association with said additional drug, are administered separately in two or more different compositions or different dosage forms.

9. The combination according to any one of claims 4 to 8, for use in the treatment of malaria and / or the prevention and treatment of the complications thereof, in particular the neurological disorders of malaria.

10. A kit comprising telotristat ethyl compound inside a container, and one or more antimalarial drugs in one or more containers.11 . A process for the preparation of telostristat ethyl having the following formula (I)characterized in that said telostristat ethyl of formula (I) is obtained by the following two- step process Step AStep Bwhere said step A is conducted in dioxane in the presence of CS2CO3 and said step B is conducted in anhydrous ethanol in the presence of SOCI2.

12. The process for the preparation of telostristat ethyl according to claim 11 , where said compound of formula (8) is obtained by the following three-step process:Step AStep Cwhere said step A is conducted in ethanol in the presence of SOCI2, said step B is conducted in a water / ethanol mixture in the presence of K2CO3 and (Boc)2O, and said step C is conducted in a water / ethanol mixture in the presence of 2-amino-4,6- dichloropyrimidine and Pd(PPh3)Cl2 as catalyst.

Images