Design, Synthesis and Antiparasitic Evaluation of Click Phospholipids

A library of seventeen novel ether phospholipid analogues, containing 5-membered heterocyclic rings (1,2,3-triazolyl, isoxazolyl, 1,3,4-oxadiazolyl and 1,2,4-oxadiazolyl) in the lipid portion were designed and synthesized aiming to identify optimised miltefosine analogues. The compounds were evaluated for their in vitro antiparasitic activity against Leishmania infantum and Leishmania donovani intracellular amastigotes, against Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the substituents of the heterocyclic ring (tail) and the oligomethylene spacer between the head group and the heterocyclic ring was found to affect the activity and toxicity of these compounds leading to a significantly improved understanding of their structure–activity relationships. The early ADMET profile of the new derivatives did not reveal major liabilities for the potent compounds. The 1,2,3-triazole derivative 27 substituted by a decyl tail, an undecyl spacer and a choline head group exhibited broad spectrum antiparasitic activity. It possessed low micromolar activity against the intracellular amastigotes of two L. infantum strains and T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes, while its cytotoxicity concentration (CC50) against THP-1 macrophages ranged between 50 and 100 μM. Altogether, our work paves the way for the development of improved ether phospholipid derivatives to control neglected tropical diseases.


Introduction
Neglected tropical diseases (NTDs) are a group of infections primarily affecting people in developing regions worldwide, mainly in Africa, Asia and Latin America, due to the poor sanitation, lack of potable water and limited or no access to health care [1]. According to the WHO, NTDs such as leishmaniasis (Leishmania sp.), Chagas disease (Trypanosoma cruzi) and human African trypanosomiasis (HAT) or sleeping sickness as it is commonly called (Trypanosoma brucei) affect more than one billion people in 149 countries [2]. The new road map, launched by WHO, aiming to end the suffering caused by NTDs by the end of 2030, creates grounds for optimism regarding the fight against these diseases [3].
Leishmaniasis is a vector-borne disease caused by the protozoan Leishmania parasites, which are transmitted by the bite of infected female phlebotomine sandflies [4,5]. Leishmaniasis is endemic in areas of the tropics, subtropics and Southern Europe, whereas 900,000-1.3 million new cases and 20,000-30,000 deaths are estimated annually. The main forms of the disease are visceral (VL, also known as kala-azar), cutaneous (CL, the most common) and mucocutaneous. VL is the most severe form of Leishmaniasis, caused by Leishmania infantum and Leishmania donovani. Undoubtedly, it represents an emergent threat with high morbidity and mortality rates, since VL is fatal if left untreated in over 95% of cases. Unfortunately, the standard treatment of Leishmaniasis depends on poorly tolerated drugs with high toxicity. In addition, a major problem encountered in clinical practice is the increased chemoresistance of the parasite [6].
Pentavalent antimonials (sodium stibogluconate and meglumine antimonate) remain the first-line treatment for both VL and CL. However, they exhibit poor oral absorption and therefore they are administered intravenously or intramuscularly [7]. An improvement towards the treatment of Leishmaniasis has been the development of liposomal formulations of amphotericin B [8,9], which are more effective and less toxic. However, the high cost constitutes a stumbling block for people in developing countries. Another antileishmanial drug, pentamidine, currently used as a first-line treatment for CL caused by Leishmania guyanensis, is administered intravenously with severe side effects such as hypoglycaemia, myocarditis or hypotension [10,11].
Miltefosine (hexadecylphosphocholine) is an alkylphosphocholine analogue initially developed as an anticancer drug, which was later shown to exhibit antileishmanial activity. Miltefosine is currently the only oral drug available for the treatment of VL and CL. It has a long half-life (100-200 h) in humans and a low therapeutic index. It reaches steady-state concentrations in the plasma only a few days before the end of treatment, when viable parasites may still be present. Its major downside is that it induces teratogenesis in animals and, thus, cannot be administered to pregnant women [12,13]. Furthermore, miltefosine can result in severe gastrointestinal side effects such as vomiting, diarrhoea, nausea or loss of appetite [14].
Trypanosomiasis is a serious parasitic disease transmitted by insects caused by protozoa of the genus Trypanosoma. There are two forms of the disease: Chagas disease caused by T. cruzi and human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense. Chagas disease is encountered mainly in South America with more than 25 million people being at risk and approximately 10,000 deaths annually [15]. Treatment of the disease is challenging since the majority of symptoms are elusive. Often the patient's health deteriorates and death from sudden cardiac arrest is an outcome. There are only two drugs currently available for the treatment of Chagas disease, namely, nifurtimox and benznidazole. These drugs are used for both the acute and chronic stages of the disease, albeit with low cure rates in particular during the chronic phase. In addition, they are associated with serious side effects especially in elderly patients, with dermatitis due to hypersensitivity and digestive intolerance being the most frequent [16][17][18].
HAT is endemic in sub-Saharan Africa where over 70 million people are at risk [19]. The available treatments of HAT are few depending on the stage of the disease, with limitations mainly due to toxicity and route of administration. Pentamidine and suramin are the treatment of choice for the haemolymphatic early stage of HAT. Melarsoprol, despite its high toxicity, is widely used against the second meningo-encephalitic stage of HAT caused by T. b. rhodesiense during which, the parasite invades the central nervous system (CNS) [20,21]. At the same time, eflornithine is the drug of choice for the CNS stage of HAT caused by T. b. gambiense.
Undoubtedly, the existing medications for the treatment of leishmaniasis and trypa nosomiasιs suffer from a variety of problems including serious side-effects, lengthy course of treatment, high cost and development of parasite resistance.
In the context of the present work, a series of 17 novel ether phospholipid derivatives was synthesized and structure-activity relationships were obtained. A systematic chemical investigation was performed considering the following modifications: (a) conformational restriction by incorporating 5-membered heteroaromatic rings in the lipid portion namely, 1,2,3-triazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole and 3,5-disubstituted isoxazole, (b) variable length of the oligomethylene spacer between the heterocyclic ring and the phosphate head group, (c) replacement of choline by homocholine in the phosphate polar head-group and (d) attachment of alkyl, cycloalkyl, alkylcarbamate and carboxyalkyl groups as substituents of the heterocyclic ring ( Figure 1). tients, with dermatitis due to hypersensitivity and digestive intolerance being the most frequent [16][17][18].
HAT is endemic in sub-Saharan Africa where over 70 million people are at risk [19]. The available treatments of HAT are few depending on the stage of the disease, with limitations mainly due to toxicity and route of administration. Pentamidine and suramin are the treatment of choice for the haemolymphatic early stage of HAT. Melarsoprol, despite its high toxicity, is widely used against the second meningo-encephalitic stage of HAT caused by T. b. rhodesiense during which, the parasite invades the central nervous system (CNS) [20,21]. At the same time, eflornithine is the drug of choice for the CNS stage of HAT caused by T. b. gambiense.
Undoubtedly, the existing medications for the treatment of leishmaniasis and trypanosomiasιs suffer from a variety of problems including serious side-effects, lengthy course of treatment, high cost and development of parasite resistance.
In the context of the present work, a series of 17 novel ether phospholipid derivatives was synthesized and structure-activity relationships were obtained. A systematic chemical investigation was performed considering the following modifications: (a) conformational restriction by incorporating 5-membered heteroaromatic rings in the lipid portion namely, 1,2,3-triazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole and 3,5-disubstituted isoxazole, (b) variable length of the oligomethylene spacer between the heterocyclic ring and the phosphate head group, (c) replacement of choline by homocholine in the phosphate polar head-group and (d) attachment of alkyl, cycloalkyl, alkylcarbamate and carboxyalkyl groups as substituents of the heterocyclic ring ( Figure 1).

Chemistry
The general synthetic procedure for the synthesis of the new ether phospholipids 25-36, 53, 54, 65 and 66 involves the phosphorylation of the appropriate alcohols 17-24, 40, 51, 52, 63 and 64, respectively with phosphorus oxychloride in the presence of triethylamine in THF to afford the corresponding phosphoric acid derivatives. These in turn are treated with pyridine to form the pyridinium salts, which are coupled with choline or homocholine tosylate in the presence of 1-(mesitylenesulfonyl)-3-nitro-1,2,4-triazolide (MSNT) or 2,4,6-tri-isopropylbenzenesulfonyl chloride (TPSCl) as a condensing agent to afford the phospholipid derivatives of the present study.
In particular, the synthesis of the 1,2,3-triazole-substituted alcohols 17-24 was affected through a click reaction between azides 7-9 and alkynes 10-16 and is described in The required alkynes were either commercially available (compounds 10-13) or prepared as described in Scheme 2. Alkyne 15 was obtained by esterification of 10-undecynoic acid (14) with benzyl bromide in DMF using Cs2CO3 as a base in 93% yield. Alkyne 16 was synthesized through a Curtius rearrangement reaction from 10-undecynoic acid (14) using diphenylphosphoryl azide followed by the addition of benzyl alcohol in 66% yield [38]. Following the synthesis of the desired alkynes (10-13, 15 and 16) and azides (7-9), alcohols 17-24 incorporating a 1,2,3-triazole ring were prepared through a copper catalysed Huisgen cycloaddition reaction in 51-80% yield (Scheme 3) [39]. The reaction was carried out in the presence of CuSO4•5H2O and sodium ascorbate in t BuOH/H2O (4:1). Finally, the desired ether phospholipids (25)(26)(27)(28)(29)(30)(31)(32)(33) were obtained according to the procedure described above (Scheme 3). fected through a click reaction between azides 7-9 and alkynes 10-16 and is described in Schemes 1-3. Thus, commercially available ω-bromo carboxylic acids 1-3 were treated with BH3•Me2S in THF to afford the corresponding bromoalcohols 4-6 in 95-100% yield [37], which were in turn reacted with NaN3 in DMF to give ω-azidoalcohols 7-9 in 92-97% yield. (Scheme 1). The required alkynes were either commercially available (compounds 10-13) or prepared as described in Scheme 2. Alkyne 15 was obtained by esterification of 10-undecynoic acid (14) with benzyl bromide in DMF using Cs2CO3 as a base in 93% yield. Alkyne 16 was synthesized through a Curtius rearrangement reaction from 10-undecynoic acid (14) using diphenylphosphoryl azide followed by the addition of benzyl alcohol in 66% yield [38]. Following the synthesis of the desired alkynes (10-13, 15 and 16) and azides (7)(8)(9), alcohols 17-24 incorporating a 1,2,3-triazole ring were prepared through a copper catalysed Huisgen cycloaddition reaction in 51-80% yield (Scheme 3) [39]. The reaction was carried out in the presence of CuSO4•5H2O and sodium ascorbate in t BuOH/H2O (4:1). Finally, the desired ether phospholipids (25)(26)(27)(28)(29)(30)(31)(32)(33) were obtained according to the procedure described above (Scheme 3). The library of click phospholipids was further expanded with the synthesis of selected new derivatives in which the 1,2,3-triazole ring was replaced by isoxazole, 1,2,4-or 1,3,4-oxadiazole rings. The synthesis of phospholipid 41 incorporating an isoxazole ring is depicted in Scheme 5. Aldehyde 37, which was obtained after PCC-mediated oxidation of 11-bromoundecanol (6) [40], was converted to the corresponding oxime 38 upon treatment with NH2OH•HCl in t BuOH:H2O (1:1). 38 was then subjected to a 1,3-dipolar nitrile oxide/alkyne cycloaddition reaction with commercially available 1-dodecyne in the presence of NaOCl to afford the isoxazole derivative 39. Subsequently, bromide 39 was converted to the corresponding alcohol 40 in the presence of K2CO3 in H2O/DMSO (2:1), which was phosphorylated using POCl3 and further reacted with choline tosylate in the presence of MSNT as a condensing agent to obtain the desired phospholipid derivative 41 (Scheme 5). The library of click phospholipids was further expanded with the synthesis of selected new derivatives in which the 1,2,3-triazole ring was replaced by isoxazole, 1,2,4-or 1,3,4-oxadiazole rings. The synthesis of phospholipid 41 incorporating an isoxazole ring is depicted in Scheme 5. Aldehyde 37, which was obtained after PCC-mediated oxidation of 11-bromoundecanol (6) [40], was converted to the corresponding oxime 38 upon treatment with NH 2 OH·HCl in t BuOH:H 2 O (1:1). 38 was then subjected to a 1,3-dipolar nitrile oxide/alkyne cycloaddition reaction with commercially available 1-dodecyne in the presence of NaOCl to afford the isoxazole derivative 39. Subsequently, bromide 39 was converted to the corresponding alcohol 40 in the presence of K 2 CO 3 in H 2 O/DMSO (2:1), which was phosphorylated using POCl 3 and further reacted with choline tosylate in the presence of MSNT as a condensing agent to obtain the desired phospholipid derivative 41 (Scheme 5). The preparation of phospholipids 53 and 54 bearing a 1,2,4-oxadiazole ring is described in Scheme 6. Nonanitrile (43) was obtained upon treatment of 1-bromooctane (42) with NaCN in DMF and was subsequently reacted with NH2-OH•HCl in the presence of NaHCO3 to obtain amidoxime 44 in 59% yield. The required active esters 45 and 46 were synthesized from the commercially available ω-bromo acids 2 and 3, respectively, after reaction with NHS and DCC in THF. Subsequently, the formation of the 1,2,4-oxadiazolecontaining bromides 47 and 48 was accomplished in 52% and 64% yield, respectively, through the reaction of 44 with 45 or 46 under microwave irradiation. Thereafter, reaction of bromides 47 and 48 with CH3CO2K and hydrolysis of the obtained acetates 49 and 50 The preparation of phospholipids 53 and 54 bearing a 1,2,4-oxadiazole ring is described in Scheme 6. Nonanitrile (43) was obtained upon treatment of 1-bromooctane (42) with NaCN in DMF and was subsequently reacted with NH2-OH•HCl in the presence of NaHCO3 to obtain amidoxime 44 in 59% yield. The required active esters 45 and 46 were synthesized from the commercially available ω-bromo acids 2 and 3, respectively, after reaction with NHS and DCC in THF. Subsequently, the formation of the 1,2,4-oxadiazolecontaining bromides 47 and 48 was accomplished in 52% and 64% yield, respectively, through the reaction of 44 with 45 or 46 under microwave irradiation. Thereafter, reaction of bromides 47 and 48 with CH3CO2K and hydrolysis of the obtained acetates 49 and 50 with LiOH afforded the required alcohols 51 and 52, which were phosphorylated following the procedure described above to obtain phospholipids 53 and 54, respectively (Scheme 6). The synthesis of phospholipids 65 and 66 bearing a 1,3,4-oxadiazole ring is reported in Scheme 7. Commercially available ethyl octanoate (55) or ethyl dodecanoate (56) were converted to the corresponding hydrazides 57 and 58 under microwave irradiation using NH2NH2•H2O in 33% and 54% yield, respectively, and were subsequently coupled with 11-bromoundecanoic acid (3) in the presence of POCl3 to furnish the 1,3,4-oxadiazole-substituted bromides 59 and 60 in 60% and 77% yield, respectively. These in turn were converted to the corresponding alcohols 63 and 64 following an analogous procedure as described above for alcohol 51. Eventually, phospholipids 65 and 66 were obtained from the The synthesis of phospholipids 65 and 66 bearing a 1,3,4-oxadiazole ring is reported in Scheme 7. Commercially available ethyl octanoate (55) or ethyl dodecanoate (56) were converted to the corresponding hydrazides 57 and 58 under microwave irradiation using NH 2 NH 2 ·H 2 O in 33% and 54% yield, respectively, and were subsequently coupled with 11-bromoundecanoic acid (3) in the presence of POCl 3 to furnish the 1,3,4-oxadiazolesubstituted bromides 59 and 60 in 60% and 77% yield, respectively. These in turn were converted to the corresponding alcohols 63 and 64 following an analogous procedure as described above for alcohol 51. Eventually, phospholipids 65 and 66 were obtained from the phosphorylation of alcohols 63 and 64 and reaction with choline tosylate in the presence of MSNT (Scheme 7).

Biological Evaluation
In vitro evaluation of antileishmanial activity: Initially, the antileishmanial activity of the 1,2,3-triazolyl-substituted phospholipids 25-29 was evaluated against the intracellular amastigotes of L. infantum strain MHOM/TN/80/LEM235 (see Table S1). Interestingly, the oligomethylene spacer between the heterocyclic ring and the phosphocholine head group and the aliphatic substituent on the 1,2,3-triazole ring (tail) influenced the activity. Thus,

Biological Evaluation
In vitro evaluation of antileishmanial activity: Initially, the antileishmanial activity of the 1,2,3-triazolyl-substituted phospholipids 25-29 was evaluated against the intracellular amastigotes of L. infantum strain MHOM/TN/80/LEM235 (see Table S1). Interestingly, the oligomethylene spacer between the heterocyclic ring and the phosphocholine head group and the aliphatic substituent on the 1,2,3-triazole ring (tail) influenced the activity. Thus, compound 25 bearing a pentyl tail and an undecyl spacer was less potent (IC 50 = 9.33 ± 0.99 µM) against L. infantum than 26, substituted by a decyl tail and a hexyl spacer (IC 50 = 5.21 ± 0.95 µM), although the total number of carbons of the tail and spacer of the two compounds were identical. Furthermore, maintaining the decyl tail, the length of the spacer was increased to eleven carbons leading to a further improvement in the potency of compound 27 against L. infantum (IC 50 = 1.54 ± 0.22 µM). Based on the above data, the undecyl spacer was kept and the aliphatic tail was replaced by cycloalkyl rings aiming to reduced compound flexibility. Thus, 28 bearing a cyclopentylmethylene tail possessed similar potency to 27 with IC 50 = 1.5 ± 0.4 µM while increasing the ring size to a cyclohexyl group was beneficial to the antileishmanial activity of 29 (IC 50 = 0.5 ± 0.2 µM) that was 13-fold more potent than miltefosine. Since it was observed that different Leishmania strains were associated with diverse susceptibility to the same compound [41,42], the effect of derivatives 25-29 was assessed against the intracellular amastigotes of a second L. infantum strain, namely MHOM/MA/67/ITMAP-263 (Table 1). However, this strain was not as sensitive to click phospholipids 25-29. In particular, when tested at a single dose of 10 µM 28 was inactive while, 25, 26, 27 and 29 inhibited L. infantum amastigotes by 32%, 44%, 78% and 43%, respectively. Only 27 resulted in >50% inhibition at 10 µM and the IC 50 value was determined to be 4.23 ± 0.94 µM, which was slightly inferior to that against L. infantum LEM235 amastigotes but similar to that of miltefosine. Thus, it was decided to proceed with the evaluation of the remaining new analogues only against L. infantum (MHOM/MA/67/ITMAP-263) amastigotes since it is more resistant to the click phospholipids (Table 1). Prompted by this initial SAR, additional 1,2,3-triazole containing phospholipids were synthesized by introducing polar head groups to the lipophilic tail. Thus, an ester (compound 30), a carbamate (compounds 31 and 32), a carboxy (compound 34) and amino groups (compounds 35 and 36) were introduced. An undecyl oligomethylene spacer was maintained for the ester and acid-substituted derivatives 30 and 34. Two different spacers (the pentyl and undecyl groups) were studied for the carbamate and corresponding amino analogues. Unfortunately, the presence of polar groups as terminal substituents of the lipophilic tail abolished the activity against L. infantum (MHOM/MA/67/ITMAP-263) amastigotes while the toxicity against THP1 macrophages was not affected (see Table 1, compounds 30-36). In addition, replacement of the choline head group by a homocholine in analogue 29 (compound 33) did not improve the antileishmanial activity. This structural modification was based on previous reports that erufosine (ErPC3), which contains a homocholine head group, displays reduced bone marrow toxicity than miltefosine [43,44]. Subsequently, we investigated the effect of other five-membered heterocyclic rings on the antileishmanial activity. Thus, the 1,2,3-triazole moiety was replaced by isoxazole (41), 1,2,4-oxadiazole (53,54) and 1,3,4-oxadiazole (65,66) with concomitant variations of the length of the lipophilic tail and the oligomethylene spacer. Interestingly, the activity of the new compounds against L. infantum (MHOM/MA/67/ITMAP-263) amastigotes ranged between 92 and 100% inhibition at 10 µM. However, the high level of activity was not observed at a 1 µM concentration except for the 1,2,4-oxadiazole derivative 54 substituted by an octyl tail and a decyl oligomethylene spacer, which exhibited 67.1% inhibition at 1 µM and an IC 50 value of 0.8 ± 0.18 µM. Thus, compound 54 was 4-fold more potent than miltefosine. A common characteristic of compounds 41, 53, 54, 65 and 66 was the higher toxicity against THP1 macrophages, which ranged between 12.5 and 25 µM for 41, 53 and 65 and 25-50 µM for 66. Although the cytotoxicity of the potent derivative 54 was similar to that of miltefosine (CC 50 > 10 µM), the corresponding selectivity index was 5.6-fold higher (SI = 13.6 and SI = 4.9, respectively). To acquire a more comprehensive understanding of the range of activity of the new click phospholipids, the activity of the 1,2,3-triazolyl-substituted derivatives 25 and 28-36 were also screened at 5 µM concentration against L. donovani amastigotes in infected bone marrow-derived macrophages using Taqman qPCR of the Leishmania actin gene over the mouse actin gene ( Figure 2). These derivatives exhibited higher activity than against L. infantum (MHOM/MA/67/ITMAP-263) amastigotes. Compound 29 bearing a cyclohexylmethyl tail reduced the parasite load by 41.7% similar to miltefosine (44.3%) and was also found to be active against L. infantum (MHOM/TN/80/LEM235) amastigotes. Replacing choline in 29 with homocholine in 33 did not reduce the percentage (%) of parasite load. Conversely, the length of the oligomethylene spacer in the two 1,2,3-triazolylcarbamate derivatives 31 and 33 played a critical role in the activity against L. donovani amastigotes. 31 substituted by a pentyl spacer resulted in a 59.2% parasite load reduction at 5 µM, while the undecyl spacer abolished the activity (compound 32). Furthermore, deprotection of the carbamate in 31 resulted in the amino derivative 35 with increased potency to a 69.1% parasite load reduction, corresponding to a 1.5-fold higher activity than miltefosine ( Figure 2).
Conversely, the length of the oligomethylene spacer in the two 1,2,3-triazolyl-carbamate derivatives 31 and 33 played a critical role in the activity against L. donovani amastigotes. 31 substituted by a pentyl spacer resulted in a 59.2% parasite load reduction at 5 μΜ, while the undecyl spacer abolished the activity (compound 32). Furthermore, deprotection of the carbamate in 31 resulted in the amino derivative 35 with increased potency to a 69.1% parasite load reduction, corresponding to a 1.5-fold higher activity than miltefosine (Figure 2). Early ADMET profiling: The early ADMET profiling of the new derivatives involved a group of assays widely used in drug development as part of the preclinical in vitro toxicity studies. In particular our panel included hERG inhibition, five cytochromes P450 (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), two cell-lines (A549, epithelial human lung adenocarcinoma, WI38 and foetal lung fibroblasts), Aurora B kinase and mitochondrial toxicity, in addition to the parasite relevant toxicity against THP1 macrophages. The compounds were tested at 10 μΜ, whilst the miltefosine control was tested at 10 μΜ or 1 μM. The toxicity of the new click phospholipids is depicted in Figure 3 in a traffic light format (from red through yellow to green for decreasing ADMET liabilities). The values are included in Table S2. Early ADMET profiling: The early ADMET profiling of the new derivatives involved a group of assays widely used in drug development as part of the preclinical in vitro toxicity studies. In particular our panel included hERG inhibition, five cytochromes P450 (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), two cell-lines (A549, epithelial human lung adenocarcinoma, WI38 and foetal lung fibroblasts), Aurora B kinase and mitochondrial toxicity, in addition to the parasite relevant toxicity against THP1 macrophages. The compounds were tested at 10 µM, whilst the miltefosine control was tested at 10 µM or 1 µM. The toxicity of the new click phospholipids is depicted in Figure 3 in a traffic light format (from red through yellow to green for decreasing ADMET liabilities). The values are included in Table S2.  The compound with the best profile is that with dark green boxes in all assays. The grey colour corresponds to non-tested compounds. * Average % of mitochondrial toxicity 10 μM. ** Cytotoxicity THP-1 macrophages.
Ideally, for the perfect lead compound all results should be green. None of the compounds tested was associated with CYP1A2, or A549 liability. Only 41 was slightly toxic against hERG. Derivatives Figure 3. In vitro evaluation of toxicities (hERG, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and A549 and WI38 cytotoxicities) of compounds at 10 µM and miltefosine (10 µM or 1 µM). The compound with the best profile is that with dark green boxes in all assays. The grey colour corresponds to non-tested compounds. * Average % of mitochondrial toxicity 10 µM. ** Cytotoxicity THP-1 macrophages.
In vitro evaluation of antitrypanosomal activity against T. b. brucei: Most of the synthesized compounds were tested for their antitrypanosomal activity against the T. b. brucei L427 WT bloodstream form in a single dose assay at 10 µM using a modified resazurin-based assay. Pentamidine was used as an internal control for the fitness of the parasite (quality control of the assay). All compounds tested were associated with a minimum or no activity against T. b. brucei (see Table S3).
In vitro evaluation of antitrypanosomal activity against T. cruzi: Selected compounds (25, 28-36, 41, 53, 54, 65 and 66) were tested at 10 µM against T. cruzi Y strain intracellular amastigotes propagated through a HG39 cell-line ( Figure 4). As a general observation among the 1,2,3-triazolyl-substituted phospholipids, the presence of a carbamate, acid or amino terminal group at the lipophilic tail abolished activity (31, 32 and 34-36). Furthermore, replacement of the choline head group by homocholine in the cyclohexylmethyl derivative 29 was detrimental to activity against T. cruzi amastigotes, in particular 29 resulted in 69.7% parasite load reduction similar to miltefosine (71.9% parasite load reduction) while 33 was inactive. The isoxazole-substituted analogue 41 induced 63.3% parasite load reduction, while, for the two 1,2,4-oxadiazole-substituted derivatives 53 and 54, the latter showed a higher antitrypanosomal potency (90.5% vs. 44.5% parasite load reduction). Finally, between the 1,3,4-oxadiazole derivatives 65 and 66 compound 65 bearing a shorter heptyl tail was very potent (92.2% parasite load reduction) in comparison to 66, which was substituted by an undecyl tail. Interestingly, among compounds 41, 53, 54, 65 and 66, 53 with a pentyl spacer was the least potent highlighting that the activity was impacted by the oligomethylene spacer.  Subsequently, a more in-depth evaluation against all the parasite developmental stages of T. cruzi [45] was performed for a small number of 1,2,3-triazole-substituted derivatives (25, 27 and 29) based on their antileishmanial activity and reduced toxicity against THP-1 derived macrophages. In particular, T. cruzi epimastigotes (Y strain) were obtained by axenic cultivation in the LIT medium, supplemented with 10% FBS (Cultilab) and the effect of compounds 25, 27 and 29 was determined after 24 h, 48 h and 72 h treatment ( Table 2). The most potent derivative was 27 substituted by a decyl tail, an undecyl spacer and a choline head group with IC50 values in the submicromolar range following 48 h or 72 h of treatment at 7.8 μΜ. Shortening the tail length to a pentyl group (25)    Subsequently, a more in-depth evaluation against all the parasite developmental stages of T. cruzi [45] was performed for a small number of 1,2,3-triazole-substituted derivatives (25, 27 and 29) based on their antileishmanial activity and reduced toxicity against THP-1 derived macrophages. In particular, T. cruzi epimastigotes (Y strain) were obtained by axenic cultivation in the LIT medium, supplemented with 10% FBS (Cultilab) and the effect of compounds 25, 27 and 29 was determined after 24 h, 48 h and 72 h treatment ( Table 2). The most potent derivative was 27 substituted by a decyl tail, an undecyl spacer and a choline head group with IC 50 Figure 5). Light microscopy observations of treated macrophages containing intracellular amastigotes revealed a gradual reduction in the number of amastigotes but no changes in the morphology of the host cell, which even remained attached to the substrate.  In order to determine if compounds 25, 27 and 29 were able to lyse T. cruzi trypomastigotes, this developmental stage was treated for 24 h with various compound concentrations. Significant lysis of the parasites was observed only with the 27 treatment (LD50 = 1.02 μΜ, Figure 6), highlighting its broad spectrum antiparasitic profile. In order to determine if compounds 25, 27 and 29 were able to lyse T. cruzi trypomastigotes, this developmental stage was treated for 24 h with various compound concentrations. Significant lysis of the parasites was observed only with the 27 treatment (LD 50 = 1.02 µM, Figure 6), highlighting its broad spectrum antiparasitic profile. In order to determine if compounds 25, 27 and 29 were able to lyse T. cruzi trypomastigotes, this developmental stage was treated for 24 h with various compound concentrations. Significant lysis of the parasites was observed only with the 27 treatment (LD50 = 1.02 μΜ, Figure 6), highlighting its broad spectrum antiparasitic profile.

Morphological analysis of epimastigotes and amastigotes treated with click phospholipid 27:
Since compound 27 was the most potent against all the developmental stages of T. cruzi, scanning and transmission electron microscopy were employed to analyse the effects on epimastigotes and intracellular amastigotes. Treatment of T. cruzi epimastigotes with 27 for 72 h (100 nM) resulted in changes in their morphology, gradually losing the typical elongated form and acquiring a rounded shape ( Figure 7B,C), confirming initial observations made by light microscopy (data not shown).  Furthermore, observation of the untreated epimastigotes by transmission electron microscopy (TEM) showed cells with an elongated shape, a smooth cell surface and intact organelles ( Figure 8A). Micrographs of epimastigotes treated with compound 27 (100 nM, for 72 h) revealed drastic alterations in the Golgi complex in particular disorganization of its cisternae, which had adopted dilated and empty appearances ( Figure 8B). Mitochondrial swelling and membrane blebs on the parasite flagellum were also evident ( Figure 8C,D).  Flow cytometry studies in T. cruzi epimastigotes treated with 27: Flow cytometry was subsequently employed to assess T. cruzi epimastigotes' type of death induced by exposure to 27 at 5 μM for 24 h. The labelling of 86% of the population of T. cruzi epimastigotes with annexin V indicated that these parasites probably were involved in an apoptotic process. However, the absence of labelling with propidium iodide was suggestive that their plasma membranes were intact ( Figure 10). Following 48 h of treatment, 13% of epimastigotes were labelled only with propidium iodide, thereby demonstrating a necrotic death of this population. Nevertheless, phosphatidylserine (PS) exposure was still present, indicating that death by apoptosis was also occurring. Flow cytometry studies in T. cruzi epimastigotes treated with 27: Flow cytometry was subsequently employed to assess T. cruzi epimastigotes' type of death induced by exposure to 27 at 5 µM for 24 h. The labelling of 86% of the population of T. cruzi epimastigotes with annexin V indicated that these parasites probably were involved in an apoptotic process. However, the absence of labelling with propidium iodide was suggestive that their plasma membranes were intact ( Figure 10). Following 48 h of treatment, 13% of epimastigotes were labelled only with propidium iodide, thereby demonstrating a necrotic death of this population. Nevertheless, phosphatidylserine (PS) exposure was still present, indicating that death by apoptosis was also occurring. To confirm the occurrence of apoptotic death after 24 h of treatment, epimas were assessed using the Click-iT TUNEL assay for apoptosis, followed by fluor microscopy. The labelling of the epimastigotes with Annexin V/Alexa 488 was ob suggesting that PS exposure and apoptotic death had taken place ( Figure 11).

Programmed cell death by autophagy in intracellular amastigotes treated with compo
Electron microscopy observations of T. cruzi intracellular amastigotes treated (data not shown) revealed that the parasites were surrounded by profiles of the en mic reticulum, indicating the presence of autophagosomes. Therefore, we decided t itor the autophagy process. To this end, infected macrophages were treated with To confirm the occurrence of apoptotic death after 24 h of treatment, epimastigotes were assessed using the Click-iT TUNEL assay for apoptosis, followed by fluorescence microscopy. The labelling of the epimastigotes with Annexin V/Alexa 488 was observed, suggesting that PS exposure and apoptotic death had taken place ( Figure 11). To confirm the occurrence of apoptotic death after 24 h of treatment, epimastig were assessed using the Click-iT TUNEL assay for apoptosis, followed by fluoresc microscopy. The labelling of the epimastigotes with Annexin V/Alexa 488 was obse suggesting that PS exposure and apoptotic death had taken place ( Figure 11). Programmed cell death by autophagy in intracellular amastigotes treated with compoun Electron microscopy observations of T. cruzi intracellular amastigotes treated wi (data not shown) revealed that the parasites were surrounded by profiles of the endo mic reticulum, indicating the presence of autophagosomes. Therefore, we decided to m Electron microscopy observations of T. cruzi intracellular amastigotes treated with 27 (data not shown) revealed that the parasites were surrounded by profiles of the endoplasmic reticulum, indicating the presence of autophagosomes. Therefore, we decided to monitor the autophagy process. To this end, infected macrophages were treated with the IC 50 concentration of 27 and the anti-LC3B antibody was used to identify autophagic structures by fluorescence microscopy (Figure 12). Approximately 30% of amastigotes inside macrophages appeared strongly labelled after the treatment. No labelling was observed in untreated amastigotes, thereby suggesting that the treatment with 27 induces amastigote autophagy.
Molecules 2021, 26, x FOR PEER REVIEW concentration of 27 and the anti-LC3B antibody was used to identify autophagic str by fluorescence microscopy (Figure 12). Approximately 30% of amastigotes inside phages appeared strongly labelled after the treatment. No labelling was observed treated amastigotes, thereby suggesting that the treatment with 27 induces amastig tophagy.

General
Melting points were determined with a Buchi 510 apparatus (Buchi, Flawil, S land) and are uncorrected. NMR spectra were recorded on Varian spectrometers ( Palo Alto, CA, USA). 1 H NMR spectra were recorded at 600 or 300 MHz, 13 C NMR at 150 or 75 MHz and were internally referenced to residual solvent signals. 31

General
Melting points were determined with a Buchi 510 apparatus (Buchi, Flawil, Switzerland) and are uncorrected. NMR spectra were recorded on Varian spectrometers (Varian, Palo Alto, CA, USA). 1 H NMR spectra were recorded at 600 or 300 MHz, 13 C NMR spectra at 150 or 75 MHz and were internally referenced to residual solvent signals. 31 P NMR were recorded at 121.44 MHz. Chemical shifts are reported in δ units, parts per million (ppm). Electrospray ionization (ESI) mass spectra were recorded on a LC-MSn Fleet spectrometer (Thermo Scientific, Waltham, MA, USA) using MeOH as a solvent. HRMS spectra were recorded, in the ESI mode, on a UPLC-MSn Orbitrap Velos-Thermo spectrometer (Thermo Scientific, Waltham, MA, USA). Reactions under microwave irradiation were performed in a CEM Discover Lab Mate reactor (CEM Corporation, Matthews, NC, USA). Flash column chromatography (FCC) was performed on Merck silica gel 60 (230-400 mesh) and TLC on Merck 60 F254 films (0.2 mm) precoated glass plates. Spots were visualized with UV light at 254 nm and a PMA stain. All solvents were dried and/or purified according to standard procedures prior to use. All reagents employed in the present work were purchased from commercial suppliers and used without further purification. Reactions were run in flame-dried glassware under an atmosphere of argon. Active esters 45 [46] and 46 [47] were synthesized according to published procedures.

General Procedure for the Synthesis of Alcohols 4-6
To an ice-cold solution of acid 1-3 (20 mmol) in THF (30 mL), a borane dimethyl sulphide complex (2.7 mL, 28 mmol) was added dropwise. The solution was warmed at room temperature and stirred for 1 h at 45 • C. Then, the reaction was cooled at 0 • C, quenched with MeOH (60 mL) and concentrated under reduced pressure. The residue was dissolved in EtOAc, washed with brine, dried over Na 2 SO 4 and concentrated in vacuo to obtain the projected alcohols 4-6, which were used in the next step without further purification.

5-(5-bromopentyl)-3-octyl-1,2,4-oxadiazole (47)
It was synthesized according to the general procedure described above from active ester 45. Pale-yellow oil; yield: 0.306 g, 52%; 1 H-NMR (300 MHz, CDCl 3 )  A solution of 47 or 48 (1 equiv, 1.0 mmol) and CH 3 CO 2 K (4 equiv) in DMF (0.1 M) was heated at 70-80 • C for 4 h. The solvent was removed under reduced pressure, the residue was taken up in EtOAc and washed with water and brine. The organic layer was dried over Na 2 SO 4 and concentrated in vacuo to afford acetates 49 and 50, which were used to the next step without further purification.
To a solution of 49 or 50 (1 equiv, 1.0 mmol) in THF/MeOH (1:3, 0.05 M), a solution of LiOH (1.3 equiv) in H 2 O (0.2 M) was added dropwise. The mixture was stirred at rt for 2 h. Upon completion, the solvents were removed in vacuo, the residue was taken up in EtOAc and washed with water and brine. The organic layer was dried over Na 2 SO 4 and concentrated in vacuo. Pure alcohols 51 and 52 were obtained after FCC purification. 5-(3-octyl-1,2,4-oxadiazol-5-yl)pentan-1-ol (51) It was synthesized according to the general procedure described above from bromide 47. Pale-yellow oil; yield: 0.236 g, 82%; 1  To a solution of ester 55 or 56 (1 equiv, 1.0 mmol) in absolute ethanol (5.8 M), placed in a tube (10 mL pressure-rated reaction vial), hydrazine monohydrate (4.5 equiv) was added. The resulting mixture was irradiated using a CEM microwave reactor for 17 min at 120 • C. After cooling to rt, the solid was filtered to give compounds 57 and 58. These compounds were used to the next step without further purification.
n-octanoic Acid Hydrazide (57) It was synthesized according to the general procedure described above from ester 55. To a rb-flask containing hydrazides 57 or 58 (1 equiv, 1.0 mmol) and 3 (1.2 equiv), POCl 3 (6.5 equiv) was added and the resulting mixture was refluxed for 2 h. Then, it was cooled to 0 • C, H 2 O was added dropwise and it was extracted with EtOAc. The organic layer washed with water and brine, dried over Na 2 SO 4 and concentrated under reduced pressure. Pure compounds 59 and 60 were obtained after FCC purification.
T. cruzi epimastigotes (Y strain) were obtained by axenic cultivation in the LIT medium, supplemented with 10% FBS (Cultilab, São Paulo, Brazil). Cultures were maintained at 280 • C for 4-5 days until they reached the log growth phase. New cultures were made every 4 days from the initial inoculum of 2 × 106 parasites/mL. T. cruzi trypomastigotes (Y strain) were maintained in culture by the infection of LLC-MK2 (ATTC-CCL7) epithelial cells. This cell line was cultured in 175 cm 3 tissue culture bottles (TPP) in RPMI 1640 medium (Gibco), (Thermo Fischer Scientific, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco) (Thermo Fischer Scientific, Carlsbad, CA, USA). Cultures were maintained at 37 • C under 5% CO 2 atmosphere, and RPMI 1640 medium and SFB were exchanged every 48 h. After confluence, the cultures were treated with 1 mL of a mixture of trypsin and versene (Sigma, São Paulo, Brazil) (0.2 and 0.002%, respectively). After 24 h, a subconfluence culture was infected with the trypomastigotes in the approximate proportion of 10:1 parasites/cell. From five to seven days post infection, the trypomastigote stage was obtained from the culture supernatant. Alternatively (Figure 2), T. cruzi strain Y was maintained as previously described [56].

In-Vitro Evaluation of Activity Against L. donovani Intramacrophage Amastigotes
Activity against L. donovani strain BPK190 was tested in vitro using bone marrowderived macrophages [57] as host cells. Infection was established for 24 h prior to the addition of compounds from 10 mM stocks in DMSO. Parasite load was determined 48 h later by quantifying Leishmania actin DNA relative to mouse actin DNA using real time qPCR as described [58].
3.2.3. In-Vitro Evaluation of Activity Against L. infantum MHOM/TN/80/LEM235 Intramacrophage Amastigotes THP-1 cells were differentiated with 1 mM retinoic acid (Sigma, Saint Louis, MI, USA) for 3 days at 37 • C and 5% CO 2 . Infection of THP-1 cells with promastigotes was achieved by mixing the THP-1 and the parasite cultures at a ratio of 1:4 and incubation at 37 • C and 5% CO 2 . The next day cells were washed by centrifugation at 400 × g for 10 min, the pellet was resuspended in RPMI medium, and the suspension was overlaid with an equal volume of Histopaque 1077 (Invitrogen, Waltham, MA, USA). The free promastigotes were removed by centrifugation at 1000 × g for 20 min. The cell layer was washed with PBS twice and resuspended in RPMI at 4 × 105 cells/mL. Infection rate was assessed microscopically by Giemsa staining of the infected THP-1 suspension. An amount of 200 µL of infected THP-1 was plated in a 24-well tissue culture plate (Cellstar®, Greiner) (Sigma-Aldrich Chemie GMBH, Taufkirchen, Germany) and mixed with an equal volume of the appropriate compound concentration (compounds were dissolved in DMSO/ethanol, 50/50 v/v, to a final concentration of 65 mM and linear 5-fold dilutions ranging from 100 to 0.8 µM were done in the culture medium). Incubation proceeded for 72 h at 37 • C and 5% CO 2 , and the percentage of infected cells was monitored microscopically after Giemsa staining.

In-Vitro Evaluation of Activity Against L. infantum MHOM/MA/67/ITMAP-263 Intramacrophage Amastigotes
The efficacy of compounds against L. infantum intracellular amastigotes was determined according to the literature [59] with slight modifications. Briefly, 1 × 106 THP-1 cell/mL were seeded in a 96 well flat bottom plate (100 µL/well) and differentiated into macrophages by addition of 20 ng/mL of phorbol-myristate 13-acetate (PMA, Sigma, Saint Louis, MI, USA) for 18 h, followed by replacement with fresh medium for 24 h. Then these cells were infected with luciferase-expressing L. infantum axenic amastigotes in a macrophage:amastigotes ratio of 1:10 for 4 h at 37 • C, 5% CO 2 . Non-internalized parasites were washed and compounds were added at different concentrations in a final volume of 100 µL. After 72 h of incubation, the media was substituted by 100 µL of PBS. Then of 25 µL of Glo-lysis buffer from the Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA) was added mixed and incubated for 10 min with agitation at 100 rpm. Finally, 30 µL of the Steady-Glo reagent (Promega, Madison, WI, USA) was then added to the plate and was incubated for 15 min with agitation at 100 rpm and then the content of each well was transferred to white-bottom 96-well plates. Luminescence intensity was read using a Synergy 2 Multi-Mode Reader (Biotek, Winooski, VT, USA). The antileishmanial effect was evaluated by the determination of the IC 50 value (concentration required to inhibit growth in 50%) and calculated by the non-linear regression analysis using GraphPad Prism version 8.1.1 for Windows (GraphPad Software, San Diego CA, USA).

In-Vitro Evaluation of Activity Against T. brucei Bloodstream Forms
The efficacy of compounds against T. brucei bloodstream forms was evaluated using a modified resazurin-based assay previously described [60]. Mid-log bloodstream forms were added to an equal volume of serial dilutions of compounds in supplemented complete HMI-9 medium at a final cell density of 5 × 103/mL. Following incubation for 72 h at 37 • C 5% CO 2 , 20 µL of a 0.5 mM resazurin solution was added and plates were incubated for a further 4 h under the same conditions. Fluorescence was measured at 540 nm and 620 nm excitation and emission wavelength, respectively, using a Synergy 2 Multi-Mode Reader (Biotek, Winooski, VT, USA). For pentamidine, the antitrypanosomatid effect was evaluated by the determination of the IC 50 value (concentration required to inhibit growth in 50%) and calculated by non-linear regression analysis using GraphPad Prism version 8.1.1 for Windows (GraphPad Software, San Diego CA, USA).

In-Vitro Evaluation of Antitrypanosomal Activity
The trypomastigotes were obtained from the supernatant of a previously infected cell line (LLC-MK2). Between 5 and 7 days after infection, protozoa were collected from the supernatants of the infected LLC-MK2 cells and were then incubated with fresh RPMI 1640 medium supplemented with 0.5% FBS, with or without the compounds, for 24 h at 37 • C under a 5% CO 2 atmosphere. The concentration of the compounds at which 50% of the parasites were lysed (LC50) was calculated by counting the cells in a Neubauer chamber when trypomastigotes were treated with 100 nM, 500 nM, 1.0 µM, 1.5 µM and 3.0 µM. The experiment was performed in duplicate for each of the three different experiments. For the antiproliferative assay involving epimastigotes, 106 parasites/mL were cultivated in LIT medium supplemented with 10% FBS. After 24 h of epimastigote growth, different concentrations of 50 nM to 50 µM were added to the culture and incubated for 120 h at 28 • C. Cells were collected every 24 h for counting in a Neubauer chamber. Two controls were used and consisted of liver infusion broth-tryptose (LIT) supplemented with 10% FBS and LIT added with 0.01% DMSO. To investigate the effect of the compounds on intracellular amastigotes, peritoneal macrophages from Swiss mouse (CF1) plated in 96 wells plates (Costar®, Sigma, São Paulo, Brazil) were incubated for 2 h with T. cruzi trypomastigotes at a ratio of 10 parasites to 1 cell. The non-internalized parasites were removed by washing with the RPMI 1640 medium, and the host cells were incubated for 24 h at 37 • C to allow full internalization and differentiation of trypomastigotes to amastigotes. Fresh 10% FBS-RPMI 1640 medium with 1 µg/mL Hoescht 3348 and 1 µg/mL of WGA-FITC (control) or the same solution containing the inhibitors was added to the infected cells, which were then incubated for 96 h at 37 • C. Every 24 h samples were taken and analysed in high content equipment (InCell Analyser 2000, GE Healthcare, Chicago, IL, USA) with 20× air objective (NA 0.4) using respectively filters to excitation and emission to DAPI (405/455 nm), FITC (488/505 nm) and Cy3 (514/605 nm). Six images were collected for each well for reliable statistical analysis. No significant differences were observed in cell or amastigote numbers among images in different locations within wells. The infection index (i.e., the percentage of infected host cells multiplied by the average number of intracellular amastigotes per infected host cell) was determined by counting a total of 500 host cells. Inhibitor activity was calculated using the SigmaPlot (version 10) program (Systat Software, Inc., San Jose, CA, USA). The results are expressed as the mean values from three independent experiments. The IC 50 was calculated for promastigotes and intracellular amastigotes by fitting the values to a non-linear curve analysis. The regression analyses were performed with SigmaPlot 10 (version 10) program (Systat Software, Inc., San Jose, CA, USA).

Cytotoxicity Assessment against THP-1 Macrophages
The effect of compounds selected compounds on THP-1-derived macrophages was assessed by the colorimetric MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Briefly, 1 × 105 THP-1 cells/mL were differentiated into macrophages by addition of 20 ng/mL of phorbol-myristate 13-acetate (PMA, Sigma, Saint Louis, MI, USA) for 18 h, followed by replacement with fresh medium for 24 h. Cells were incubated with 100 µL of compounds ranging from 100 to 1 µM after dilution in the RPMI complete medium containing a maximum amount of 1% DMSO. After incubation for 72 h, the medium was removed and 100 µL of 0.5 mg/mL MTT solution diluted in RPMI was added. Plates were incubated for an additional 4 h to allow viable cells to convert MTT into a purple formazan product. Solubilization of formazan crystals was achieved by the addition of 2-propanol and absorbance was read at 570 nm using a Synergy 2 Multi-Mode Reader (Biotek, Winooski, VT, USA). Cytotoxicity was evaluated by the determination of the CC50 value (drug concentration that reduced the percentage of viable cells in 50%) and calculated by non-linear regression analysis using GraphPad Prism version 8.1.1 for Windows (GraphPad Software, San Diego, CA, USA).

Early ADMET Profiling
Cytotoxicity assay: A549 cells and WI-38 cells were grown on surface-modified T175 cell culture flasks in Dulbecco's modified Eagle medium with 10% foetal calf serum (FCS), streptomycin (100 µg/mL) and 100 U/mL penicillin G. At about 80% confluency, cells were washed, trypsinized, resuspended and counted in RPMI-1640 medium before seeding (in triplicate) into white 384-well microtiter plates (20 µL) at 500 cells/well and incubated at 37 • C in the presence of 5% CO 2 for 24 h. A total of 20 µL/well of CellTiter-Glo (CTG) reagent (Promega Corp., Madison, WI, USA) was added to each well, and plates were read using an EnVision Multilabel 2103 Reader after a 10-min incubation in the dark. Doseresponse assays for compounds made use of 11-point curves. Each test compound (200 nL of 10 mM top concentration in 100% v/v DMSO) was added to cells seeded in polystyrene 384-well cell culture microtiter plates using the Echo 550 Liquid Handler and read after 24 h of incubation at 37 • C in the presence of 5% CO 2 using CTG as described above. The compounds/positive control (paclitaxel with final concentration of 10 µM and 1% v/v DMSO) and no compound (final 1% v/v DMSO) were added into the 384-well plates (200 nL/well; 1% v/v DMSO) using the Echo 550 Liquid Handler. The raw luminescence signal of each sample was converted into the percentage of cell growth.
Aurora B kinase assay: Inhibition of Aurora B kinase was determined using the ADP-Glo Kinase Enzyme System (Promega Corp., Madison, WI, USA). The positive control was SU6656 at a final concentration of 1 µM, with the NC being DMSO at the same concentration (v/v). An enzyme master mix containing 1 × buffer, 50 µM DTT and 17.5 ng/µL (35 µL/well) Aurora B (all reagents provided in the kit) was prepared. A substrate master mix containing 1 × buffer, 36 µM adenosine triphosphate (ATP) and 7.5 ng/µL (15 ng/well) myelin basic protein (MBP) as a substrate (buffer and MBP were provided in the Aurora B Kinase Enzyme System; ultrapure ATP was provided in the ADP-Glo Kinase Assay System) was prepared. Two microliters of the enzyme master mix and 2 µL of the substrate master mix were added to each well of a 384-well low-volume plate. The plate was sealed using Thermowell sealing tape (Corning Inc., Corning, NY, USA) and incubated for 45 min at rt. The enzymatic reaction was stopped by adding 4 µL of ADP-Glo reagent (provided in the ADP-Glo Kinase Assay System) and the plate sealed using Thermowell sealing tape and incubated for 40 min at rt. Following this, 8 µL of detection reagent was added to each well, and the plate was sealed again and incubated for 45 min at rt, with the luminescence measured using the EnVision Multilabel 2103 Reader (PerkinElmer, Waltham, MA. USA). DMSO concentration was tolerated up to 2% v/v final.
hERG cardiotoxicity assay: The Predictor®hERG fluorescence polarization assay (Thermo Fischer Scientific, Waltham, MA, USA) was used to test compounds for potential cardiotoxicity. To each well of the assay plate, 100 nL of the test/control compound was added followed by addition of 5 µL homogenized membrane solution (undiluted) and 5 µL of tracer (1 nM final concentration in the assay). The plates were incubated for 2 h at 25 • C in a humidity controlled incubator, and the fluorescence polarization was measured using an EnVision Multilabel 2103 Reader (PerkinElmer, Waltham, MA. USA). The NCs (0% inhibition) and positive controls with E-4031, a blocker of hERG-type potassium channels (yielding 100% inhibition), were used to normalize the raw data.
Mitochondrial toxicity assay: This assay made use of the MitoTracker Red CMXRos dye (Thermo Fischer Scientific, Waltham, MA, USA), which stains the mitochondria in live cells and its accumulation is dependent on the presence of a membrane potential. The renal carcinoma 786-0 cell line was used for mitochondrial toxicity screening. Cells were harvested from a 75 cm 2 flask at 80% confluency by washing once with 5 mL of rt PBS and incubating with 1 mL of 0.05% v/v trypsin/0.02% v/v EDTA for 3 min. Cells were suspended in 10 mL of prewarmed cell culture media (RPMI-1 640 supplemented with 10% v/v FCS, 100 U/mL penicillin and 100 µg/mL streptomycin) and counted using a Scepter (Merck Millipore, Darmstadt, Germany). The 786-0 cells were diluted to 75,000 cells/mL and 20 µL of this suspension added to each well of a 384-well plate. Cells were incubated for 36 h at 37 • C and 5% CO 2 , and compounds were added using a predilution plate. The positive control was valinomycin at a final concentration of 1 µM, with the negative control being DMSO at the same concentration. Ten microliters of compounds and controls were added to cells and incubated for 6 h at 37 • C and 5% CO 2 in a humidity controlled atmosphere. After incubation, 10 µL of a 200 nM solution of MitoTracker Red CMXRos in prewarmed cell culture media was added to each well, and the 786-0 cells were incubated for an additional 45 min at 37 • C and 5% CO 2 . MitoTracker Red CMXRos uptake was measured using an Opera Imaging System. To facilitate automatic image analysis, the layout containing the compound area and the valinomycin and DMSO control areas were created and stored. A sublayout of five evenly dispersed fields per well were used. These settings also included a measurement height of 1 µm, which was stored in an exposure file format. By using the stored settings and files, an automated run was repeatedly created and executed. The images were transferred to a file server and uploaded into Columbus 2.4.0 (PerkinElmer, Waltham, MA. USA) using the built-in helper function and analysed therein.
Data analysis: The screening data were obtained in triplicate and analysed using ActivityBase (IDBS, Guildford, UK), and outlier elimination in the control wells was performed using the 3-sigma method. Unless stated, dose-response experiments were performed in the 11-point format with the IC 50 value, Hill slope, minimum signal and maximum signal for each dose-response curve obtained using a four-parameter logistic fit in the XE module of ActivityBase (IDBS).

Transmission Electron Microscopy
Epimastigotes and trypomastigotes were cultivated and treated as previously described (treatment with 0.5 µM-72 h and 1 µM-24 h of 27 for epimastigotes and trypomastigotes, respectively). Intracellular amastigotes were allowed to grow inside peritoneal macrophages platted in 60 mm 2 Petri dishes (TPP, Trasadingen, Switzerland) and treated for 72 h with 0.25 µM of 27. After the experimental procedure, the cells were washed and then fixed and post fixed as described above for SEM; then, they were dehydrated in increasing concentrations of acetone and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under a Jeol 1200EX (Jeol Tokyo, Japan) or Zeiss EM900 (Carl Zeiss A.G., Jena, Germany) transmission electron microscope.

Flow Cytometry and Fluorescence Microscopy for Apoptosis Detection
All epimastigotes of T. cruzi (i.e., those treated with compound 27 0.5 µM for 24 or 48 h) were washed, suspended in Annexin V binding buffer and were incubated at room temperature for 15 min with Annexin V-Alexa 488 (Molecular Probes, Eugene, OR, USA) at the concentration indicated by the manufacturer (BD Bioscience, San Jose, CA, USA). At the moment of acquisition, 10 µg/mL of propidium iodide (Molecular Probes, Eugene, OR, USA) was added to the samples. Data were collected by Cellquest Pro ® in a BD FACSCalibur ® and analysed using Summit 4.3 (Dako Colorado Inc., Fort Collins, CO, USA). Ten thousand gated events were harvested from each sample. The control consisted of epimastigotes permeabilized with 0.1% Triton X100 and incubated with 10 µg/mL propidium iodide and/or Annexin V. Two independent experiments and 10,000 events were analysed for each culture condition.

Conclusions
In summary, 5-membered heterocyclic rings were introduced on the miltefosine scaffold to investigate their effect on antiparasitic activity and toxicity. In detail, seventeen novel ether phospholipid analogues, containing 1,2,3-triazolyl, isoxazolyl, 1,3,4-oxadiazolyl and 1,2,4-oxadiazolyl in the lipid portion, were synthesized. The library was evaluated for their in vitro antiparasitic activity against L. infantum and L. donovani intracellular amastigotes, against T. b. brucei and against T. cruzi different developmental stages. The nature of the substituents of the heterocyclic ring (tail) and the oligomethylene spacer between the head group and the heterocyclic ring strongly affected the activity and toxicity of these compounds. The evaluation of the early toxicological profile of the new derivatives pointed out the overall safe profile for the potent compounds.
Our study pinpointed compound 27, a 1,2,3-triazole derivative substituted by a decyl tail, an undecyl spacer and a choline head group, as the most promising derivative of the series. 27 exhibited a broad spectrum antiparasitic activity, showing submicromolar IC 50 against the intracellular amastigotes of two Leishmania infantum strains and T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes. The compound cytotoxicity against THP-1 macrophages ranged between 50 < CC 50 < 100 µM. Thus, the click phospholipid 27 showed a lower toxicity compared to miltefosine and a selectivity index >10. Replacement of the 1,2,3-triazole moiety by isoxazolyl, 1,3,4-oxadiazolyl or 1,2,4-oxadiazolyl rings resulted in some cases to more potent compounds accompanied however, by toxicity against THP-1 macrophages. This extensive SAR study paves the way for the understanding of the chemical features required for the development of an optimized miltefosine analogue.
Supplementary Materials: The following are available online. Table S1: In vitro evaluation of antiparasitic activity against L. Infantum in tricellular amastigotes, Table S2: In vitro evaluation of toxicities of compounds at 10 µM and miltefosine (at 1 or 10 µM), Table S3: In vitro evaluation of antiparasitic activity against the T. brucei L427 WT bloodstream form. In addition, copies of 1 H, 13 C and 31 P NMR of the final compounds are included, Figure S1. 1 H-NMR of compound 25 in CDCl 3 at 600 MHz, Figure S2. 13