Antimalarial agents against both sexual and asexual parasites stages: structure-activity relationships and biological studies of the Malaria Box compound 1-[5-(4-bromo-2-chlorophenyl)furan-2-yl]-N-[(piperidin-4-yl)methyl]methanamine (MMV019918) and analogues
Therapies addressing multiple stages of Plasmodium falciparum life cycle are highly desirable for implementing malaria elimination strategies. MMV019918 (1, 1-[5-(4-bromo-2-chlorophenyl)furan-2-yl]-N-[(piperidin-4-yl)methyl]methanamine) was selected from the MMV Malaria Box for its dual activity against both asexual stages and gametocytes. In-depth structure-activity relationship studies and cytotoxicity evaluation led to the selection of 25 for further biological investigation. The potential transmission blocking activity of 25 versus P. falciparum was confirmed through the standard membrane-feeding assay. Both 1 and 25 significantly prolonged atrioventricular conduction time in Langendorff-isolated rat hearts, and showed inhibitory activity of Ba2+ current through Cav1.2 channels. An in silico target-fishing study suggested the enzyme phosphoethanolamine methyltransferase (PfPMT) as a potential target. However, compound activity against PfPMT did not track with the antiplasmodial activity, suggesting the latter activity relies on a different molecular target. Nevertheless, 25 showed interesting activity against PfPMT, which could be an important starting point for the identification of more potent inhibitors active against both sexual and asexual stages of the parasite.
1. Introduction<!>2.1. Chemistry<!>2.2. Antiplasmodial and gametocytocidal activities and structure-activity relationships<!>2.2.1. Hydrophobic head<!>2.2.2. Heterocyclic linker<!>2.2.3. Pendant basic chain<!>2.3. In vitro cytotoxicity<!>2.4. Standard membrane-feeding assay<!>2.5. Evaluation of activity of compound 25 against P. berghei liver stages<!>2.6. Evaluation of cardiac toxicity and calcium antagonist activity of 1 and 25<!>2.7. In silico approach for target identification of compounds 1 and 25<!>2.7.1. Evaluation of 1, 25 and analogues as inhibitors of PfPMT<!>3. Conclusions<!>4.1. Chemistry<!>4.1.1. 5-(4-Bromo-2-chlorophenyl)furan-2-carbaldehyde (5a)<!>4.1.2. 5-Phenylfuran-2-carbaldehyde (5b)<!>4.1.3. 5-(4-Bromophenyl)furan-2-carbaldehyde (5c)<!>4.1.4. 5-(4-Fluorophenyl)furan-2-carbaldehyde (5d)<!>4.1.5. 5-(2-Methoxyphenyl)furan-2-carbaldehyde (5e)<!>5-(2-Benzyloxyphenyl)furan-2-carbaldehyde (5f)<!>5-(2-Hydroxyphenyl)furan-2-carbaldehyde (6)<!>4.1.6. 1-(5-(4-Bromo-2-chlorophenyl)furan-2-yl)-N-(piperidin-4-ylmethyl)methanamine (1)<!>4.1.7. 1-(5-Phenylfuran-2-yl)-N-piperidin-4-ylmethyl-methanamine (8)<!>4.1.8. 1-(5-(4-Bromophenyl)furan-2-yl)-N-(piperidin-4-ylmethyl)methanamine (9)<!>4.1.9. 1-(5-(4-Fluorophenyl)furan-2-yl)-N-(piperidin-4-ylmethyl)methanamine (10)<!>4.1.10. 1-(5-(2-Methoxyphenyl)furan-2-yl)-N-(piperidin-4-ylmethyl)methanamine (11)<!>4.1.11. 1-(5-(2-Hydroxyphenyl)furan-2-yl)-N-(piperidin-4-ylmethyl)methanamine (12)<!>4.1.12. tert-Butyl 4-formylpiperidine-1-carboxylate (14)<!>4.1.13. tert-Butyl 4-(azidomethyl)piperidine-1-carboxylate (15)<!>4.1.14. tert-Butyl 4-(aminomethyl)piperidine-1-carboxylate (7)<!>4.1.15. 5-(4-Chloro-2-bromophenyl)furan-2-carbaldehyde (18b)<!>4.1.16. 5-(2-Chlorophenyl)furan-2-carbaldehyde (18c)<!>4.1.17. 5-(4-(Trifluoromethyl)phenyl)furan-2-carbaldehyde (18d)<!>4.1.18. 4-(5-Formylfuran-2-yl)benzonitrile (18e)<!>4.1.19. 5-(4-Phenyl-2-chlorophenyl)furan-2-carbaldehyde (19a)<!>4.1.20. 5-(2-Phenyl-4-chlorophenyl)furan-2-carbaldehyde (19b)<!>4.1.21. 1-(5-(2-Bromo-4-chlorophenyl)furan-2-yl)-N-(piperidin-4-ylmethyl)methanamine (21)<!>4.1.22. 1-(5-(2-Chlorophenyl)furan-2-yl)-N-(piperidin-4-ylmethyl)methanamine (22)<!>4.1.23. 1-(5-(4-Trifluoromethylphenyl)furan-2-yl)-N-(piperidin-4-ylmethyl)methanamine (23)<!>4.1.24. 1-(5-(4-Cyanophenyl)furan-2-yl)-N-(piperidin-4-3ylmethyl)methanamine (24)<!>4.1.25. 1-(5-(2-Phenyl-4-chlorophenyl)furan-2-yl)-N-(piperidin-4-ylmethyl)methanamine (25)<!>4.1.26. 1-(5-(4-Phenyl-2-chlorophenyl)furan-2-yl)-N-(piperidin-4-ylmethyl)methanamine (26)<!>4.1.27. 5-(4-Bromo-2-chlorophenyl)thiophene-2-carbaldehyde (28a)<!>4.1.28. 5-(2-Bromo-4-chlorophenyl)thiophene-2-carbaldehyde (28b)<!>4.1.29. 1-(5-(4-Bromo-2-chlorophenyl)thiophen-2-yl)-N-(piperidin-4-ylmethyl)methanamine (29)<!>4.1.30. 1-(5-(2-Bromo-4-chlorophenyl)thiophen-2-yl)-N-(piperidin-4-ylmethyl)methanamine (30)<!>4.1.31. 1-(5-Bromobenzothiophen-2-yl)-N-(piperidin-4-ylmethyl)methanamine (32)<!>4.1.32. 4-Benzyl-3-(4-chlorophenyl)-4H-1,2,4-triazole (34)<!>4.1.33. (4-Benzyl-5-(4-chlorophenyl)-4H-1,2,4-triazol-3-yl)methanol (35)<!>4.1.34. 4-Benzyl-3-(chloromethyl)-5-(4-chlorophenyl)-4H-1,2,4-triazole (36a)<!>4.1.35. 2-(2-Bromo-4-chlorophenyl)-5-(chloromethyl)-1,3,4-oxadiazole (36b)<!>4.1.36. 1-(5-(4-Chlorophenyl)-4H-1,2,4-triazol-3-yl)-N-(piperidin-4-ylmethyl)methanamine (37)<!>4.1.37. 1-(5-(2-Bromo-4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-N-(piperidin-4-ylmethyl)methan amine (38)<!>4.1.38. 2-Methyl-6-(4-(trifluoromethyl)phenyl)pyridine (41)<!>4.1.39. 2-(Bromomethyl)-6-(4-trifluoromethyl)phenylpyridine (42)<!>4.1.40. 1-(Piperidin-4-yl)-N-((6-(4-(trifluoromethyl)phenyl)pyridin-2-yl)methyl)methanamine (43)<!>4.1.41. N\xe2\x80\xb2-[(5-(4-Bromo-2-chlorophenyl)furan-2-yl)methyl]-N\xe2\x80\xb2,N\xe2\x80\xb2-dimethylpropane-1,3-diamine (45)<!>4.1.42. tert-Butyl [(5-(4-chloro-2-bromophenyl)furan-2-yl-methyl]-3-dimethylaminopropyl carbamate (46)<!>4.1.43. 3-[(5-(4-Chloro-2-bromophenyl)furan-2-yl-methyl)amino]-N,N,N-trimethylpropan-1-ammonium chloride (47)<!>4.1.44. N-Benzyl-3-[(5-(4-chloro-2-bromophenyl)furan-2-yl-methyl)amino]-N,N-dimethylpropan-1-ammonium chloride (48)<!>4.2. Pan-assay interference compounds<!>4.3.1. Parasite growth of D10 and W2 strains<!>4.3.2. Antiplasmodial activity against D10 and W2 strains<!>4.3.3. Gametocytocidal activity against P. falciparum 3D7 strain 3D7elo1-pfs16-CBG99<!>4.3.4. NF54<!>4.3.5. NF54 Gametocytes<!>4.4. Liver-stage analysis<!>4.5.1. Animals<!>4.5.2. Isolated rat heart preparation and perfusion<!>4.6.1. Cell isolation procedure and whole-cell patch-clamp recording<!>4.7. Enzyme Assays and IC50 Analysis<!>4.8. Computational details<!>4.8.1. Protein and ligands preparation<!>4.8.2. Molecular Docking<!>4.8.3. Evaluation of ligand binding energy<!>4.8.4. Picture preparation
<p>Despite recent advances in the field of antimalarial treatment, malaria is still responsible for a high number of infections and related deaths in tropical and subtropical regions.[1] The antimalarial agenda has currently shifted from malaria control to eradication/elimination strategies. To reach this ambitious goal and to complement appropriate vector-control measures, such as larvicidal compounds and insecticide-treated bed nets, prioritization of new approaches, targeting the transmission stages of Plasmodium falciparum life cycle (i.e. the gametocytes) is necessary.[2] Development of dual acting drugs active against asexual intraerythrocytic parasites, responsible for the clinical symptoms of malaria, and gametocytes, the sexual forms of the parasite responsible for malaria transmission, is a challenging goal but necessary for the implementation of the above-mentioned strategies.</p><p>High-throughput screening (HTS) assays have been successfully applied to P. falciparum asexual stages for the discovery of novel hits endowed with innovative modes of action.[3–14] Moreover, such approaches have been adapted for the discovery of compounds active as gametocytocidal agents.[15–24] Screening campaigns using these assays allowed a better analysis and characterization of the anti-gametocyte properties of known antimalarials and for the discovery of potential hit compounds useful for the development of transmission-blocking therapies.[25] The implementation of novel methodologies of phenotypic screening for gametocytocidal compounds was recently boosted by the non-profit foundation Medicine for Malaria Venture (MMV), which produced and delivered a collection of 400 commercial compounds divided into 200 drug-like and 200 probe-like structures, i.e., the "Open Access Malaria Box".[26] The structures present in the Malarial Box were selected using results from phenotypic screening of large compound collections against P. falciparum.[3, 27, 28] These phenotypic screens identified nearly 20,000 compounds that were active against P. falciparum 3D7 and endowed with reasonable toxicity. Selection criteria for compounds included in the Malaria Box were commercial availability, maximization of the structural diversity, and potency.</p><p>A cell-based assay with P. falciparum gametocytes expressing a potent luciferase and a lactate dehydrogenase (LDH)-based gametocyte assay were used to screen known antimalarial compounds and those of the MMV Malaria Box.[23, 25, 26] From this initial study, compound MMV019918 (1, Chart 1)[26] demonstrated dual activity against P. falciparum asexual and sexual stages with IC50 < 1000 nM; an in vitro IC50 < 1000 nM against late stages Plasmodium gametocytes consistently found in different published assays (and also in the structurally related compound N-[(5-(3-chloro-4-fluorophenyl)furan-2-yl)methyl]-N′,N′-dimethylpropane-1,3-diamine (MMV020505, 2));[25] activity on both male and female NF54 gametes; and properties favorable for drug development, including a low molecular weight and a scaffold suitable for optimization of efficacy. Starting from compound 1 and combining synthetic and biological efforts, we investigated the structure-activity relationships (SAR) of this class of compounds with the aim of understanding the general structural features important for the observed activity. Our goal was also to investigate and possibly improve the toxicity profile of the prepared analogues since the selectivity index (SI) of 1 was rather low based on the available information, and a certain degree of hERG activity (83% of inhibition at 50 μM) was also known for this compound.[25]</p><p>The SAR investigation of 1 was based on the possibility of disconnecting its scaffold into three modules: a hydrophobic head, a heterocyclic linker, and a pendant basic chain (Chart 1). We introduced different substituents, at the hydrophobic head to explore the role of the two halogen substituents, replaced the central linker with different heterocyclic scaffolds, and replaced the cyclic piperidine moiety with linear lateral chains. Compound 25, showing comparable potency with respect to 1, characterized by the presence of and extra aromatic ring at the hydrophobic head and endowed with reasonable TC50, was then selected for further biological studies.</p><!><p>We identified a synthetic strategy for the preparation of the hit compound 1 that was also suitable for the preparation of a broad series of analogues bearing different substitution patterns at the phenyl ring.</p><p>As displayed in Scheme 1, the first step to the synthesis of 1 and analogues 8–12 was a Suzuki-Miyaura coupling between aryliodides 3a–f and the organoboronic acid 4,[29] to afford aldehydes 5a–f. In the case of derivatives 5a,c,d, this palladium-catalyzed cross coupling reaction turned out to be exquisitely regioselective for the formation of reaction products arising from attack at the most reactive iodine with respect to other halides. In the subsequent steps of the synthesis, aldehydes 5a–f and 6 (in turn obtained from 5f by deprotection) were reacted with the primary amine 7 and the resulting imine intermediates were reduced using sodium cyanoborohydride. Final removal of the piperidine protecting group upon exposure to acidic conditions led to the final compounds 1 and 8–12.</p><p>Amine 7 was prepared starting from N-Boc-piperidone 13, which was subject to a Wittig reaction using (methoxymethyl)triphenylphosphonium chloride and sodium bis(trimethylsilyl)amide[30] resulting in an intermediate vinylmethyl ether that was hydrolyzed using cerium chloride and sodium iodide[31] to afford aldehyde 14. This latter compound was reduced to the corresponding alcohol, which was converted into methanesulfonate and reacted with sodium azide to furnish 15. The final catalytic hydrogenation, using palladium on carbon, led to the formation of the desired piperidinyl-methanamine 7 in satisfactory yield.</p><p>Because of the unavailability of the starting aryliodides, a second set of compounds, modified at the hydrophobic head, was synthesized using a different chemical route (Scheme 2). For the synthesis of aldehydes 18a–e, the Meerwein arylation protocol,[32] consisting in a copper-catalyzed arylation of furan derivatives with arenediazonium salts, was conveniently used. According to this procedure, anilines 16a–e were converted into the corresponding diazonium salts, and these latter intermediates were coupled in situ with furan-carboxaldehyde 17. Intermediats 18a,b were also submitted to a Suzuki coupling to afford the biphenyl-derivatives 19a,b, respectively. Aldehydes 18a–e, and 19a,b were finally converted into final compounds 21–26 using the protocol previously described for the introduction of the pendant basic chain.</p><p>Starting from 16a,b, the Meerwein arylation protocol was also applied to the thiophen-carboxaldehyde 27 to obtain intermediates 28a,b. These latter compounds were then converted to the final compounds 29,30, representing a first set of analogues of the hit compound 1 bearing modification of the heterocyclic linker.</p><p>For the synthesis of the benzothiophene derivative 32 (Scheme 3), aldehyde 31[33, 34] was submitted to the reductive amination reaction, which upon acidic Boc deprotection, led to the preparation of the desired final compound 32. Five-membered heterocycles 37 and 38 were prepared starting from arylhydrazide 33. Cyclization of 33 was performed through a Pellizzari-like reaction, consisting in the condensation between acyl hydrazine 33 with benzyl amine.[35] Only the dehalogenated product 34 was obtained from this reaction. The alcoholic function of 35, introduced by a Prince electrophilic addition using formaldehyde, was activated to the corresponding chloride 36a which was alkylated with amine 7. Oxidative N-debenzylation[36] followed by acid-mediated deprotection of the Boc-group furnished the triazole 37. For the synthesis of oxadiazole 38, the chloromethyl intermediate 36b was obtained by cyclization of 33 in the presence of chloroacetic acid and POCl3.[37]</p><p>Another substitution of the central linker is represented by the pyridine derivative 43 (Scheme 4). A C2 selective Suzuki-Miyaura arylation was applied starting from the commercially available 2-bromo-6-methylpyridine 39 and 4-trifluoromethylphenylboronic acid 40. The methyl group at C6 of the pyridine ring was then subjected to a bromination reaction using N-bromosuccinimide (NBS) and azobisisobutyronitrile (AIBN). The brominated intermediate 42 was used to alkylate amine 7. The final Boc deprotection gave in high yield the final derivative 43.</p><p>The open-chain analogue 45 was synthesized as described in Scheme 5 starting from intermediate 5a and the commercially available N,N′-dimethyl-1,3-propanediamine 44. Starting from 18b, reductive amination with 44 followed by protection of the secondary amine as Boc-derivative led to intermediate 46. The tertiary amine was subsequently converted to a quaternary ammonium salt using benzyl bromide and iodomethane, respectively. The final Boc-deprotection using acetyl chloride and methanol gave the two final derivatives 47 and 48 in satisfactory yields.</p><!><p>The antiplasmodial activity of the newly synthesized compounds was tested against two laboratory P. falciparum strains, the chloroquine-sensitive (CQ-S) D10 and the chloroquine-resistant (CQ-R) W2, according to the described procedures.[38, 39] Activity against gametocytes (GCT) was assessed using a luciferase-based assay, as previously described.[23]</p><p>Selected compounds were also tested against P. falciparum strain NF54 asexual blood stage parasites using a Sybrgreen DNA replication assay. NF54 gametocyte assays were performed using reporter strain NF54-HGL.[40] The results of the biological assays are reported in Tables 1–3.</p><!><p>In order to understand if the antiplasmodial and antigametocytocidal activities depended upon the substitution pattern of the aromatic head, we removed one or both alogens of 1 (compounds 9, 22 and 8, respectively). Moreover, we inverted the position of the Cl or Br substituent obtaining the regioisomeric derivative 21, and introduced different electron-withdrawing groups at the p-position such as –F (10), -CN (24), and –CF3 (23). We also placed electrondonating and H-bond donor/acceptor groups such as -OMe and –OH (11 and 12, respectively). A second aromatic ring, giving rise to regioisomers 25 and 26 was placed along the halogen present in the original structure in order to explore the tolerance of the system to bulky substituents. From this study we derived interesting SARs: as shown in Table 1, the substituents at the hydrophobic head seems to play a key role for in vitro activity against both asexual and sexual parasite stages. In particular, the presence of both halogens is important for activity since the unsubstituted analogue 8, as well as the mono-halogenated derivatives 9, 10 and 22 are less potent antiplasmodial and gametocytocidal agents than the hit compound 1. Inversion of chlorine and bromine seems not to have a significant effect on potency as demonstrated by the regioisomeric derivative 21. Among the other electron-withdrawing substituents investigated, the trifluoromethyl derivative 23 maintained a potency similar to 1, while with a cyano group at para position (24) a drop of potency was observed, especially against the gametocytes. Also the introduction of electron-donating substituents such as methoxy- (11) or hydroxyl- (12) groups resulted in a dramatic drop of potency against gametocytes. Interestingly, introduction of an aromatic ring at either C2 or C4, as in compounds 25 and 26, respectively, was tolerated and antiplasmodial and gametocytocidal activity comparable to the hit compound 1 were observed.</p><!><p>The role of the heterocyclic tether on activity was evaluated through the synthesis of several 5-membered heterocyclic systems as isosteric derivatives of the furan ring such as thiophene- (29, 30), triazole- (37), and oxadiazole- (38) derivatives (Table 2). The pyridine-derivative 43 was conceived in order to explore the effect on activity of a 6-membered ring bearing an heteroatom able to function as H-bond acceptor analogously to the furan oxygen, while the fusion of the pendant phenyl ring with the thiophene of 29 resulted in the benzothiophene-derivative 32. The sulfur-based heterocycles were tolerated and in some cases led to a slight improvement of activity. Also the six-membered pyridine-derivative (43) showed reasonable potency, especially against the CQ-S strain, while the other heterocycles investigated led to a dramatic drop of potency.</p><!><p>Derivative 45, bearing the same basic chain as 2, showed a potency comparable to 1 (Table 3). The ammonium quaternary salts 47 and 48 were synthesized in an attempt to increase potency since quaternary ammonium salt derivatives were previously displayed as interesting antiplasmodial agents.[41] Both compounds displayed a drop of potency against gametocytes. Interestingly, benzyl-derivative 48 maintained its potency against the asexual forms of the parasite, but not against the gametocytes.</p><p>For all compounds synthesized, molecular properties calculated in silico by QikProp (version 4.3; Schrödinger, LLC: New York, 2015) such as lipophilicity, polar and solvent accessible surface areas, solubility and membrane permeability were all in the recommended range values for known drugs (Table S1 in the Supplementary Information file).</p><!><p>After investigation of SAR, we selected a representative set of analogues characterized by various degrees of antiplasmodial and gametocytocidal potency for cytotoxicity assays. Cell viability was evaluated in vitro against NIH3T3 after 24 h of contact, by Neutral Red Uptake (NRU) test. The cell line has been chosen because is a standard fibroblast cell line.</p><p>Results of cytotoxicity tests are reported in Table 4. Compounds 11, 23 and 25 resulted the least toxic of the series. The reasonable toxicity of compound 25, combined to the structure of the inhibitor presenting an extra aromatic ring that could be further decorated, prompted us to select this compound for further biological investigation as detailed in the next paragraphs.</p><!><p>The evaluation of the ability of compounds to inhibit the development of oocysts in the mosquito and thus reduce the mosquito's ability to transmit malaria parasites can be confidently assessed using the standard membrane-feeding assay (SMFA) which remains the assay of choice for this kind of investigation. The assay consists in feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the presence of the test compound and measuring subsequent mosquito infection. The hit compound 1 was recently shown to have an EC50 in the SMFA assay of 0.07 μM.[21] Evaluation of compound 25 in the SMFA assay resulted in an IC50 of 0.21 μM on infection intensity, and 0.83 μM on infection prevalence at a baseline infection intensity of 30 oocysts/midgut (Figure 1). These data confirm that the gametocytocidal activity of 25 as observed in the NF54 gametocyte luciferase assay translates into an inability to infect the mosquito vector. The IC50 on infection intensity is slightly higher than expected on basis of the NF54 gametocyte luciferase assay. This may be explained by the difference in incubation time (24 h in the SMFA vs 72 h in the gametocyte assay) and a relatively slow mode of action of the compound.</p><!><p>We also investigated if compound 25 could have activity against the liver stages of P. berghei parasites. Primary mouse hepatocytes have been infected with mCherry expressing P. berghei sporozoites. 2 h post infection (hpi) the cells were exposed to different concentrations of 25 for 48 hpi with medium change at 24 hpi. Neither parasite numbers (Figure 2A) nor parasite sizes (Figure 2B) were significantly influenced at the concentration tested. Compound 25 was also used to analyze detached cell formation, which is an in vitro assay for final development (equivalent to merosome formation in vivo). Living parasites were counted at 48 h and from the same wells detached cells at 65 hpi evaluated. Neither concentration tested significantly inhibited detached cell formation, as indicated by the similar rates of detached cells compared to DMSO-treated control cells at 48 h (Figure 2C).</p><!><p>Considering the potential hERG liability previously highlighted for compound 1, we decided to evaluate the cardiotoxic potential of both compounds 1 and 25. Accordingly, their effect on cardiac mechanical function and electrocardiogram (ECG) in Langendorff-isolated rat hearts was assessed, as previously described.[39, 42]</p><p>Compounds 1 and 25 reduced left ventricular pressure (LVP) in a concentration-dependent manner (Figure 3), leaving unaltered coronary perfusion pressure (CPP, data not shown). They significantly prolonged atrioventricular conduction time (PQ) and intraventricular conduction time (QRS) in ECG at the maximum concentration tested (10 μM; Table 5). However, QTc values did not vary over the drug range tested (Table 5). Compound 1 significantly decreased heart rate (HR), increased cycle length (RR) interval (Table 5) and caused a sinus arrhythmia in 3 out of 4 hearts.</p><p>Since channel blockers, by inhibiting the Cav1.2 channel currents, may prolong conduction and refractoriness in the atrioventricular node,[43] the increased PQ interval obtained in presence of both compounds prompted us to investigate the effects of 1 and 25 on Cav1.2 channel currents. Compound 1 inhibited IBa1.2 (Ba2+ current through Cav1.2 channels), measured at 0 mV from a holding potential (Vh) of −50 mV, in a concentration-dependent manner (Figure 4). At 10 μM concentration, inhibition amounted to 57%. Compound 25 blocked the current at 30 μM concentration and showed an IC50 value of 3.3 ± 0.5 μM (n = 5).</p><!><p>Despite recent advances in the field, target identification remains a challenging task that could be approached through several methodologies such as direct biochemical methods, genetic interactions, computational approaches, or a combination of methods. We performed a preliminary computational investigation aimed at identifying a potential target of compounds 1 and 25 from structures in the Protein Data Bank (PDB). As a part of a broad investigation aimed at repurposing FDA-approved drugs against malaria, we prepared all the P. falciparum X-ray crystal structures present in PDB at May 2016, to be used in a high-throughput docking (HTD) procedure for identifying potential antimalarial drug-targets targeted by known drugs. Employing a reverse-docking procedure (also known as reverse virtual screening or target fishing),[44] we retrieved from the PDB database (and prepared by Protein Preparation Wizard as previously reported[42, 45]) all enzymes and proteins relevant for the plasmodium biology, some of which are established drug targets. Compounds 1 and 25 were then used to "fish" for potential targets by using Glide software.[46–48] The results of our in silico study for these compounds indicated a very limited number of potential targets with low docking scores among the examined docking complexes. Surprisingly, for one protein (P. falciparum phosphoethanolamine methyltransferase; PfPMT) we observed for both compounds significant docking scores and calculated ligand-binding energies, as found by Glide and Prime (see Experimental part for further details).[46, 49–51] In fact, for 1 and 25 we found computational scores (1, Glide XP score = −10.52 kcal/mol, ΔGbind = −57.38 kcal/mol; 25, Glide XP score = −11.28 kcal/mol, ΔGbind = −67.54 kcal/mol) comparable to those found for Sinefungin (Glide XP score = −13.16 kcal/mol, ΔGbind = −84.77 kcal/mol) and S-adenosylmethionine (SAM) (Glide XP score = −10.57 kcal/mol, ΔGbind = −82.42 kcal/mol), especially for the docking scores. The most relevant differences were related to the ΔGbind, for which we observed less negative values of 1 and 25 with respect to the reference ligands. So, given the satisfactory in silico scores, we decided to test them against PfPMT along with two related derivatives 24 and 48.</p><!><p>Compounds 1, 24, 25 and 48 were tested as potential inhibitors of PfPMT using a radiochemical assay performed with purified recombinant protein, which was expressed and purified as previously reported (Figure 5A).[52, 53] Compounds 1, 24, and 48 showed weak activity against PfPMT, PfPMT, while compound 25 displayed an inhibitory effect against PfPMT with an IC50 = 44.3 ± 5.4 μM (Figure 5B). The experimental data is in good agreement with the computational output. This latter indicates compound 25 as the best performing compound among the tested molecules, although with slight reduced activity with respect to the reference compounds [53]. Considering that at the moment only few PfPMT inhibitors have been described, mainly related to the natural substrate SAM, compound 25 could be used as a starting point to develop PfPMT inhibitors as potential antimalarials. Figure 6 depicts the docking output found by Glide software of 25 into the PfPMT active site. In particular, the amino-groups of the compound may form hydrogen bonds with D85 and R127 with the biaryl system fitting into in a hydrophobic sub-pocket and the furan group stacking with H132. Notably, these interactions are part of the main contacts found for the above-mentioned reference compounds [53].</p><p>Despite the interesting findings, the inhibition of PfPMT experimentally observed was not correlated to the antiplasmodial and gametocytocidal activity of the compound and from these data we can rule out PfPMT as the target (or at least the main target) of this series of compounds. Our results are in line with a study recently published[25] in which PfPMT has been excluded as the target of 1 in an indirect yeast-mediated assay.</p><!><p>Here we described a detailed SAR analysis of hit compound 1 and its analogue 25 for their activity against asexual and sexual life cycle stages of P. falciparum. From our SAR studies, the electron withdrawing substituents at the hydrophobic head were identified as important for activity. Moreover, the presence of nitrogen-based heterocyclic linkers resulted detrimental for activity being the thiophene and the furan rings the best performing linkers. A small set of side-chain analogues was explored highlighting that the open-chain analogue retained antiplasmodial potency, while it was interesting to note that among the two quaternary ammonium salts prepared, the benzyl-derivate was active against the asexual life cycle stage. Among the set of compounds synthesized, analogue 25 was selected for a more in depth biological investigation, due to its antiplasmodial potency in the low micromolar range, reasonable cytotoxicity and the presence of an extra aromatic ring that could offer a handle for further scaffold decoration. In particular, compound 25 was active in the SMFA assay with potency comparable to 1. On the other hand, 25 did not show activity against P. berghei hepatocytic life-cycle stage at the concentration tested, even though this could be a species specific effect. Unfortunately, cardiac toxicity was observed for both 1 and 25. Even though the biological activity of 1 and 25 against gametocytes and asexuals P. falciparum stages remains interesting and worth of further investigation, exploitation of this scaffold will only be possible by identifying its target at the molecular level. This is a necessary pre-requisite for investigating if dissociation of in vitro toxicity from the specific gametocytocidal and schizontocidal activities is a feasible task. Accordingly, we reported herein our preliminary in silico study devoted at identifying the target for this series of compounds. Driven from this study we subsequently found that analogue 25 has an interesting in vitro inhibitory activity against PfPMT, a key enzyme for phospholipid biosynthesis in P. falciparum.[54] Compound 25 could serve as a starting molecule, for the structure-based design of novel inhibitors as potential antiplasmodial and transmission-blocking agents.</p><!><p>Starting materials and solvents were purchased from commercial suppliers and used without further purification. Reaction progress was monitored by TLC using silica gel 60 F254 (0.040 – 0.063 mm) with detection by UV. Silica gel 60 (0.040 – 0.063 mm) was used for column chromatography 1H NMR and 13C NMR spectra were recorded on a Varian 300 MHz, or a Bruker 400 MHz spectrometer using the residual signal of the deuterated solvent as internal standard. Splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), and broad (br); the value of chemical shifts (δ) are given in ppm and coupling constants (J) in Hertz (Hz). Mass spectra were recorded utilizing electron spray ionization (ESI) Agilent 1100 Series LC/MSD spectrometer. Melting points were determined using a Büchi Melting point B-540. Yields refer to purified products and are not optimized. All moisture-sensitive reactions were performed under argon atmosphere using oven-dried glassware and anhydrous solvents. All compounds that were tested in the biological assays were analyzed by combustion analysis (CHN) to confirm the purity > 95%.</p><!><p>Method A: To a solution of 3a (150 mg, 0.47 mmol) in a mixture of dry DME (3 mL) and dry EtOH (3 mL), bis(triphenylphosphine)palladium(II) dichloride (17 mg, 0.02 mmol) was added. After 30 min, a solution of sodium carbonate (299 mg, 2.82 mmol) in H2O (2 mL) and a solution of 5-formyl-2-furanylboronic acid 4 (93 mg, 0.66 mmol) in dry EtOH (1.5 mL) were added in this order. The reaction was heated under reflux for 12 h. After this time, H2O (4 mL) was added at 25 °C and the organic phase was extracted with EtOAc (3 × 5 mL), dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (2% EtOAc in petroleum ether) to give 5a as orange solid (79 mg, 60%). Method B: To a suspension of 16a (1.0 g, 4.8 mmol) in H2O (20 mL), HCl (37%, 2 mL) was added. The resulting solution was cooled at 0 °C and a solution of sodium nitrite (397 mg, 5.76 mmol) in H2O (2 mL) was added dropwise. After 1 h, a solution of 17 (400 μL, 4.8 mmol) in acetone (2 mL) and solid copper(II) chloride (128 mg, 0.96 mmol) were added. The mixture kept at 25 °C for 12 h. EtOAc was added (3 × 10 mL) and the organic phase was separated, dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (2% EtOAc in petroleum ether) to give 5a as orange solid (1.23 g, 90%). mp (EtOAc/n-hexane) 141–144 °C; 1H NMR (300 MHz, CDCl3) δ 9.69 (s, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.65 (s, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.33 (d, J = 3.8 Hz, 1H), 7.30 (d, J = 3.8 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 177.6, 154.6, 151.8, 133.7, 132.4, 130.7, 130.2, 126.8, 123.6, 123.0, 113.6. MS (ESI) m/z 308 [M + Na]+.</p><!><p>Starting from 3b (100 mg, 0.49 mmol) and 4 (96 mg, 0.68 mmol), the title compound was prepared following the procedure described for the synthesis of compound 5a (Method A). The crude product was purified by flash chromatography on silica gel (5% EtOAc in petroleum ether) to give 5b as a yellow oil (70 mg, 83%).1H NMR (400 MHz, CDCl3) δ 9.61 (s, 1H), 7.78 (d, J = 7.6 Hz, 2H), 7.38 (ddd, J = 14.4, 7.9, 4.3 Hz, 3H), 7.28 (d, J = 2.4 Hz, 1H), 6.80 (d, J = 2.4 Hz, 1H); MS (ESI) m/z 173 [M + H]+, 195 [M + Na]+.</p><!><p>Starting from 3c (100 mg, 0.35 mmol) and 4 (65 mg, 0.46 mmol), the title compound was prepared following the procedure described for the synthesis of compound 5a (Method A). The crude product was purified by flash chromatography on silica gel (5% EtOAc in petroleum ether) to give 5c as a yellow solid (60 mg, 68%). mp (EtOAc/n-hexane) 152–155 °C; 1H NMR (400 MHz, CDCl3) δ 9.62 (s, 1H), 7.63 (d, J = 6.8 Hz, 2H), 7.53 (d, J = 4.8 Hz, 2H), 7.27 (d, J = 3.6 Hz, 1H), 6.80 (d, J = 3.6 Hz, 1H); MS (ESI) m/z 252 [M + H]+.</p><!><p>Starting from 3d (100 mg, 0.45 mmol) and 4 (88 mg, 0.63 mmol), the title compound was prepared following the procedure described for the synthesis of compound 5a (Method A). The crude product was purified by flash chromatography on silica gel (5% EtOAc in petroleum ether) to give 5d as a yellow oil (70 mg, 82%).1H NMR (400 MHz, CDCl3) δ 9.59 (s, 1H), 7.78 – 7.72 (m, 2H), 7.27 (d, J = 3.5 Hz, 1H), 7.14 – 7.04 (m, 2H), 6.74 (d, J = 3.4 Hz, 1H); MS (ESI) m/z 191 [M + H]+, 213 [M + Na]+.</p><!><p>Starting from 3e (160 mg, 0.75 mmol) and 4 (147 mg, 1.05 mmol), the title compound was prepared following the procedure described for the synthesis of compound 5a (Method A). The crude product was purified by flash chromatography on silica gel (5% EtOAc in petroleum ether) to give 5e as a yellow oil (50 mg, 33%). 1H NMR (300 MHz, CDCl3) δ 9.63 (s, 1H), 8.03 (d, J = 5.2 Hz, 1H), 7.40 – 7.34 (m, 1H), 7.32 (dd, J = 3.8, 1.2 Hz, 1H), 7.12 (dd, J = 3.7, 1.1 Hz, 1H), 7.08 – 6.96 (m, 2H), 3.93 (s, 3H); MS (ESI) m/z 225 [M + Na]+.</p><!><p>Starting from 3f (250 mg, 0.80 mmol) and 4 (158 mg, 1.12 mmol), the title compound was prepared following the procedure described for the synthesis of compound 5a (Method A). The crude product was purified by flash chromatography on silica gel (5% EtOAc in petroleum ether) to give 5f as a yellow oil (122 mg, 55%). 1H NMR (300 MHz, CDCl3) δ 9.58 (s, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.47 – 7.35 (m, 5H), 7.35 – 7.26 (m, 1H), 7.26 (d, J = 1.1 Hz, 1H), 7.08 – 7.01 (m, 3H), 5.14 (s, 2H); MS (ESI) m/z 301 [M + Na]+.</p><!><p>To a solution of 5f (122 mg, 0.43 mmol), in dry dichloromethane (DCM) (7 mL), boron trichloride (395 μL, 4.38 mmol) was added at −78 °C. After 3 h, the mixture warmed to 25 °C and the solvent was removed in vacuo. The crude product was purified by flash chromatography on silica gel (2% MeOH in DCM) to give 6 as yellow oil (97 mg, 55%). 1H NMR (75 MHz, CDCl3) δ 9.63 (s, 1H), 7.60 (d, J = 6.5 Hz, 1H), 7.26 (d, J = 1.1 Hz, 1H), 7.13 – 7.08 (m, 1H), 6.92 – 6.84 (m, 2H), 6.76 (d, J = 3.3 Hz, 1H), 6.28 (d, J = 2.0 Hz, 1H); MS (ESI) m/z 189 [M + H]+.</p><!><p>A solution of 5a (90 mg, 0.31 mmol) and 7 (67 mg, 0.31 mmol) in MeOH (10 mL), was heated under reflux for 12 h. After this time, the reaction was cooled to 0 °C and sodium borohydride (12 mg, 0.31 mmol) was added. After 1 h, the solvent was removed in vacuo, EtOAc was added (10 mL) and the organic phase was washed with a saturated solution of ammonium chloride (1 × 3 mL), dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (2% MeOH in DCM) to give the N-Boc protected intermediate as yellow oil (90 mg, 60%). 1H NMR (300 MHz, CDCl3) δ 7.65 (d, J = 8.6 Hz, 1H), 7.53 (d, J = 1.9 Hz, 1H), 7.37 (dd, J = 8.5, 1.9 Hz, 1H), 7.02 (d, J = 3.3 Hz, 1H), 6.27 (d, J = 3.3 Hz, 1H), 4.06 (d, J = 9.6 Hz, 2H), 3.79 (s, 2H), 2.66 (dd, J = 22.1, 9.9 Hz, 2H), 2.50 (d, J = 6.6 Hz, 2H), 1.67 (d, J = 13.0 Hz, 2H), 1.61 – 1.49 (m, 1H), 1.47 – 1.36 (m, 9H), 1.08 (d, J = 16.2, Hz, 2H); 13C NMR (75 MHz, CDCl3) δ 155.1, 152.9, 148.9, 133.4, 130.6, 130.3, 128.6, 128.4, 120.6, 112.3, 111.0, 79.5, 59.6, 57.6, 50.8, 34.7, 30.6, 28.7; MS (ESI) m/z 506 [M + Na]+. A solution 1 N HCl was prepared using acetyl chloride (355 μL, 4.97 mmol) in MeOH (4.64 mL). This solution (540 μL, 0.54 mmol) was added to the above described intermediate (90 mg, 0.18 mmol) at 0 °C and the mixture kept at 25 °C for 4 h. The solvent was removed in vacuo, a saturated solution of sodium bicarbonate was added and the organic phase was extracted with EtOAc (3 × 4 mL), dried over sodium sulfate, filtered and evaporated in vacuo, giving the product 1 as yellow oil without further purification (68 mg, 100%). 1H NMR (300 MHz, CD3OD) δ 7.88 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 1.8 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H), 7.19 (d, J = 3.3 Hz, 1H), 6.85 (d, J = 3.3 Hz, 1H), 4.42 (s, 2H), 3.44 (d, J = 12.7 Hz, 2H), 3.05 (dd, J = 17.7, 9.1 Hz, 2H), 2.17 (brs, 1H), 2.06 (d, J = 14.3 Hz, 2H), 1.55 (dd, J = 23.5, 11.6 Hz, 2H); 13C NMR (75 MHz, CD3OD) δ 150.6, 147.1, 133.1, 130.8, 130.4, 129.2, 127.8, 121.4, 113.7, 112.37, 51.9, 43.9, 43.4, 31.9, 26.4. MS (ESI) m/z 383 [M + H]+. Anal. (C17H20BrClN2O) C, H, N.</p><!><p>Starting from 5b (30 mg, 0.17 mmol) and 7 (36 mg, 0.17 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1 to afford 8 as yellow oil (29 mg, 64% over two steps). 1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 7.6 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.22 (dd, J = 12.1, 4.9 Hz, 1H), 6.55 (d, J = 3.1 Hz, 1H), 6.23 (d, J = 3.1 Hz, 1H), 3.80 (s, 2H), 3.33 (d, J = 12.5 Hz, 2H), 2.78 (t, J = 11.6 Hz, 2H), 2.56 (d, J = 6.5 Hz, 2H), 1.90 (d, J = 13.5 Hz, 2H), 1.64 (d, J = 3.0 Hz, 1H), 1.54 – 1.40 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 154.0, 153.3, 131.1, 128.8, 127.3, 123.8, 109.2, 105.8, 55.7, 46.8, 36.8; MS (ESI) m/z 271 [M + H]+. Anal. (C17H22N2O) C, H, N.</p><!><p>Starting from 5c (64 mg, 0.25 mmol) and 7 (53 mg, 0.17 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 9 as yellow oil (38 mg, 64% over two steps). 1H NMR (400 MHz, CD3OD) δ 7.59 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 3.3 Hz, 1H), 6.68 (d, J = 3.2 Hz, 1H), 4.34 (s, 2H), 3.38 (d, J = 12.5 Hz, 2H), 3.02 (d, J = 6.8 Hz, 2H), 2.95 (d, J = 11.4 Hz, 2H), 2.11 (brs, 1H), 2.01 (d, J = 13.6 Hz, 2H), 1.51 (dd, J = 23.4, 11.3 Hz, 2H); 13C NMR (75 MHz, CD3OD) δ 154.8, 144.8, 131.8, 129.3, 125.6, 121.6, 114.9, 106.8, 51.3, 43.7, 43.2, 31.3, 26.2; MS (ESI) m/z 349 [M + H]+. Anal. (C17H21BrN2O) C, H, N.</p><!><p>Starting from 5f (32 mg, 0.17 mmol) and 7 (36 mg, 0.17 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 10 as yellow oil (37 mg, 75% over two steps). 1H NMR (400 MHz, CD3OD) δ 7.76 (d, J = 5.6 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 6.79 (d, J = 3.2 Hz, 1H), 6.74 (d, J = 3.2 Hz, 1H), 4.40 (s, 2H), 3.44 (d, J = 12.1 Hz, 2H), 3.07 (d, J = 6.6 Hz, 2H), 3.00 (d, J = 13.2 Hz, 2H), 2.15 (brs, 1H), 2.04 (d, J = 12.3 Hz, 2H), 1.54 (dd, J = 23.4, 11.3 Hz, 2H); 13C NMR (75 MHz, CD3OD) δ 164.4, 161.1, 155.0, 144.5, 144.5, 126.8, 126.7, 126.0, 125.9, 115.7, 115.4, 114.9, 105.9, 105.9, 51.3, 43.7, 43.2, 31.3, 26.2; MS (ESI) m/z 289 [M + H]+. Anal. (C17H21FN2O) C, H, N.</p><!><p>Starting from 5e (50 mg, 0.25 mmol) and 7 (53 mg, 0.25 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 11 as yellow oil (49 mg, 65% over two steps). 1H NMR (300 MHz, CD3OD) δ 7.81 (d, J = 7.8 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.07 – 6.94 (m, 2H), 6.84 (d, J = 3.2 Hz, 1H), 6.33 (d, J = 2.6 Hz, 1H), 3.92 (s, 3H), 3.80 (s, 2H), 3.05 (d, J = 12.4 Hz, 2H), 2.61 (t, J = 12.5 Hz, 2H), 2.50 (d, J = 6.6 Hz, 2H), 1.77 (d, J = 13.0 Hz, 2H), 1.65 (m, 1H), 1.21 – 1.08 (m, 2H); MS (ESI) m/z 301 [M + H]+, 323 [M + Na]+. Anal. (C18H24N2O2) C, H, N.</p><!><p>Starting from 6 (80 mg, 0.42 mmol) and 7 (90 mg, 0.42 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 16 as yellow oil (72 mg, 60% over two steps). 1H NMR (300 MHz, CD3OD) δ 7.75 – 7.71 (m, 1H), 7.09 – 7.02 (m, 1H), 6.91 – 6.81 (m, 3H), 6.32 (d, J = 2.7 Hz, 1H), 3.78 (s, 2H), 3.00 (d, J = 12.4 Hz, 2H), 2.57 (d, J = 12.2 Hz, 2H), 2.48 (d, J = 6.7 Hz, 2H), 1.72 (d, J = 13.2 Hz, 2H), 1.66 – 1.55 (m, 1H), 1.21 – 1.02 (m, 2H); MS (ESI) m/z 287 [M + H]+. Anal. (C17H22N2O2) C, H, N.</p><!><p>To a solution of (methoxymethyl)triphenylphosphonium chloride (6.3 g, 18.3 mmol) in dry tetrahydrofuran (THF, 20 mL), sodium bis(trimethylsilyl)amide (1 M in dry THF, 45 mmol) was added dropwise at 0 °C. After 1 h, a solution of 13 (2.8 g, 14.1 mmol) in dry THF (20 mL) was slowly added at 0 °C and the mixture kept at 25 °C for 12 h. The solvent was removed in vacuo, H2O and EtOAc were added and the organic phase was separated, washed with an aqueous solution of HCl 1 N (1 × 5 mL) and with a saturated solution of sodium bicarbonate (1 × 5 mL), dried over sodium sulfate, filtered and evaporated in vacuo. The crude reaction mixture was purified by flash chromatography on silica gel (2% EtOAc in petroleum ether) to give the enolether intermediate tert-butyl 4-(methoxymethylene)piperidine-1-carboxylate as yellow oil (1.28 g, 40%). 1H NMR (300 MHz, CDCl3) δ 5.81 (s, 1H), 3.51 (s, 3H), 3.33 (t, J = 6.3 Hz, 4H), 2.20 (t, J = 6.0 Hz, 2H), 1.96 (t, J = 6.0 Hz, 2H), 1.42 (s, 9H); MS (ESI) m/z 250 [M + Na]+. To a solution of the above-described compound (807 mg, 3.6 mmol) in CH3CN (20 mL), cerium chloride (530 mg, 1.4 mmol) and sodium iodide (160 mg, 1.1 mmol) were added. The reaction mixture was heated under reflux for 12 h. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 14 as yellow oil (667 mg, 87%). 1H NMR (300 MHz, CDCl3) δ 9.57 (s, 1H), 3.88 (d, J = 13.3 Hz, 2H), 2.84 (t, J = 10.9 Hz, 2H), 2.39 – 2.28 (m, 1H), 1.80 (d, J = 13.5, 2H), 1.49 – 1.41 (m, 2H), 1.36 (s, 9H). MS (ESI) m/z 268 [M + MeOH + K]+.</p><!><p>To a solution of 14 (660 mg, 3.1 mmol) in MeOH (10 mL), sodium borohydride (117 mg, 3.1 mmol) was added at 0 °C and the mixture kept at 25 °C for 1 h. The solvent was removed in vacuo, EtOAc was added (15 mL) and the organic phase was washed with a saturated solution of ammonium chloride (1 × 5 mL), dried over sodium sulfate, filtered and evaporated in vacuo. The alcohol tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate was obtained as yellow oil without further purification (666 mg, 100%). 1H NMR (300 MHz, CDCl3) δ 4.03 – 3.90 (d, J = 12.6 Hz, 2H), 3.31 (d, J = 6.2 Hz, 2H), 2.56 (t, J = 12.8 Hz, 2H), 1.67 – 1.54 (m, 2H), 1.54 – 1.42 (m, 1H), 1.32 (s, 9H), 1.07 – 0.90 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 154.9, 79.3, 67.6, 67.0, 43.7, 38.8, 38.7, 37.7, 28.4; MS (ESI) m/z 238 [M + Na]+. To a solution of the above-described alcohol (666 mg, 3.1 mmol) in dry DCM (30 mL), triethylamine (2.6 mL, 18.6 mmol) and methanesulfonyl chloride (959 μL, 12.4 mmol) were added dropwise at 0 °C. Reaction kept at 25 °C for 12 h. A saturated solution of sodium bicarbonate (5 mL) was added and the organic phase was extracted with DCM (3 × 10 mL), dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (2% MeOH in DCM) to give mesylate intermediate as yellow oil (900 mg, 99%). 1H NMR (300 MHz, CDCl3) δ 3.98-3.73 (m, 2H), 2.97 – 2.84 (m, 2H), 2.79 (s, 3H), 2.48 (t, J = 13.1 Hz, 2H), 1.67 (br s, 1H), 1.50 (d, J = 13.2 Hz, 2H), 1.21 (s, 9H), 1.05 – 0.92 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 154.8, 79.6, 73.7, 43.3, 37.3, 36.0, 31.8, 28.5, 28.3; MS (ESI) m/z 294 [M + H]+. To a solution of the above compound (900 mg, 3.1 mmol) in dry N,N-dimethylformamide (DMF) (10 mL), sodium azide (400 mg, 6.2 mmol) was added at 0 °C. The reaction was heated under reflux at 90 °C for 12 h. The solvent was removed in vacuo and the product 15 was obtained as yellow oil without further purification (0.52 g, 70%). 1H NMR (300 MHz, CDCl3) δ 4.01 (d, J = 12.1 Hz, 2H), 3.08 (d, J = 6.3 Hz, 2H), 2.57 (t, J = 12.8 Hz, 2H), 1.68 – 1.49 (m, 3H), 1.34 (s, 9H), 1.15-0.96 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 111.3, 79.7, 57.2, 43.4, 36.7, 29.8, 28.6; MS (ESI) m/z 263 [M + Na]+.</p><!><p>To a solution of 15 (520 mg, 2.1 mmol) in MeOH (15 mL), a catalytic amount of palladium on carbon 10 wt.% was added under argon atmosphere. The reaction environment was saturated with hydrogen gas. The mixture was stirred at 25 °C for 3 h. The suspension was filtered on paper and concentrated in vacuo giving 7 as colorless oil without further purification (427 mg, 95%). 1H NMR (300 MHz, CDCl3) δ 3.88 (d, J = 13.1 Hz, 2H), 2.50 (d, J = 17.4 Hz, 4H), 2.45-2.33 (m, 2H), 1.50 (d, J = 13.0 Hz, 2H), 1.38-1.27 (m, 1H), 1.27 (s, 9H), 0.90-0.45 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 155.0, 79.3, 48.1, 43.4, 39.7, 29.9, 28.6; MS (ESI) m/z 215 [M + H]+.</p><!><p>Starting from 16b (2.0 g, 9.7 mmol) and 40 (802 μL, 9.7 mmol), the title compound was prepared following the procedure described for the synthesis of compound 5a (Method B). The crude product was purified by flash chromatography on silica gel (5% EtOAc in petroleum ether) to give 18b as an orange solid (2.5 g, 90%). mp (EtOAc/n-hexane) 141–144 °C; 1H NMR (300 MHz, CDCl3) δ 9.69 (s, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 7.40 (d, J = 8.6, Hz, 1H), 7.35 (q, J = 3.8 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ 177.6, 154.6, 151.8, 133.7, 132.4, 130.7, 130.2, 126.8, 123.6, 123.0, 113.6. MS (ESI) m/z 308 [M + Na]+.</p><!><p>Starting from 16c (100 mg, 0.78 mmol) and 17 (65 μL, 0.78 mmol), the title compound was prepared following the procedure described for the synthesis of compound 5a (Method B). The crude product was purified by flash chromatography on silica gel (10 % EtOAc in petroleum ether) to give 18c as a yellow solid (112 mg, 70%). mp (EtOAc/n-hexane) 68–72 °C; 1H NMR (400 MHz, CDCl3) δ 9.66 (s, 1H), 7.97 (d, J = 5.9 Hz, 1H), 7.44 (d, J = 6.5 Hz, 1H), 7.36 – 7.26 (m, 4H); MS (ESI) m/z 207 [M + H]+, 228 [M + Na]+.</p><!><p>Starting from 16d (300 mg, 1.86 mmol) and furaldehyde 17 (154 μL, 1.86 mmol), the title compound was prepared following the procedure described for the synthesis of compound 5a (Method B). The crude product was purified by flash chromatography on silica gel (5 % EtOAc in petroleum ether) to give 18d as yellow solid (312 mg, 70%). mp (EtOAc/n-hexane) 64–66 °C; 1H NMR (300 MHz, CDCl3) δ 9.65 (s, 1H), 7.85 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 3.8 Hz, 1H), 6.91 (d, J = 3.8 Hz, 1H); MS (ESI) m/z 263 [M + Na]+.</p><!><p>Starting from 16e (600 mg, 5.1 mmol) and furaldehyde 17 (421 μL, 1.86 mmol), the title compound was prepared following the procedure described for the synthesis of compound 5a (Method B). The crude product was purified by flash chromatography on silica gel (15 % EtOAc in petroleum ether) to give 18e as brown solid (753 mg, 75%). mp (EtOAc/n-hexane) 145–150 °C; 1H NMR (300 MHz, CDCl3) δ 9.71 (s, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.76 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 3.4 Hz, 1H), 6.97 (d, J = 3.4 Hz, 1H); MS (ESI) m/z 220 [M + Na]+.</p><!><p>Triphenylphosphine (220 mg, 0.84 mmol) and palladium (II) acetate (12 mg, 0.05 mmol) were dissolved in dry toluene (3 mL) under argon atmosphere. After 30 min, a solution of 5a (300 mg, 1.05 mmol) in dry toluene (5 mL) was added. After further 30 min, a solution of sodium carbonate (701 mg, 6.6 mmol) in H2O (3 mL) and powered phenyl boronic acid (128 mg, 1.05 mmol) were added in this order. Reaction was heated under reflux for 12 h. The solvent was removed under vacuo. H2O (2 mL) and EtOAc (8 mL) were added, the organic phase was separated, dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (5 % EtOAc in petroleum ether) to give 19a as yellow oil (254 mg, 85%). 1H NMR (300 MHz, CDCl3) δ 9.69 (s, 1H), 8.08 (d, J = 1.5 Hz, 1H), 7.70 (d, J = 1.6 Hz,1H), 7.62 – 7.56 (m, 3H), 7.50 – 7.36 (m, 3H), 7.35 (d, J = 0.8 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ 177.6, 157.7, 151.5, 142.6, 140.0, 135.4, 131.1, 129.8, 129.4, 129.0 (2C), 127.5, 127.3, 126.4 (2C), 115.6, 112.3; MS (ESI) m/z 305 [M + Na]+.</p><!><p>Starting from 18b (80 mg, 0.28 mmol) and phenyl boronic acid (35 mg, 0.28 mmol), the title compound was prepared following the procedure reported for compound 19a. The crude product was purified by flash chromatography on silica gel (5 % EtOAc in petroleum ether) to give 19b as yellow oil (50 mg, 63%). 1H NMR (300 MHz, CDCl3) δ 9.55 (s, 1H), 7.93 (d, J = 5.3 Hz, 1H), 7.44 – 7.40 (m, 5H), 7.33 (d, J = 2.7 Hz, 2H), 7.02 (d, J = 3.1 Hz, 1H), 5.62 (d, J = 3.7 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 177.6, 157.7, 151.5, 142.6, 140.0, 135.4, 131.1, 129.8, 129.4, 129.0 (2C), 127.5, 127.4, 126.4, 120.4, 115.6, 112.3; MS (ESI) m/z 305 [M + Na]+.</p><!><p>Starting from 18b (50 mg, 0.18 mmol) and 7 (38 mg, 0.18 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 21 as yellow oil (48 mg, 70% over two steps). 1H NMR (300 MHz, CDCl3) δ 7.65 (d, J = 1.2 Hz, 1H), 7.61 (s, 1H), 7.28 (d, J = 2.2 Hz, 1H), 7.05 (s, 1H), 6.27 (s, 1H), 3.78 (s, 2H), 3.30 (d, J = 11.1 Hz, 2H), 2.74 (t, J = 11.8 Hz, 2H), 2.53 (d, J = 5.2 Hz, 2H), 1.86 (d, J = 12.9 Hz, 2H), 1.61 (s, 1H), 1.48 – 1.30 (m, 2H); 13C NMR (75 MHz, CD3OD) δ 153.7, 149.5, 133.1, 132.9, 129.8, 129.1, 127.5, 118.9, 111.5, 109.2, 54.3, 45.3, 44.7, 34.9, 29.2; MS (ESI) m/z 383 [M + H]+. Anal. (C17H20BrClN2O) C, H, N.</p><!><p>Starting from 18c (45 mg, 0.22 mmol) and 7 (47 mg, 0.22 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 22 as yellow oil (48 mg, 68% over two steps). 1H NMR (300 MHz, CD3OD) δ 7.91 (dd, J = 7.8, 1.7 Hz, 1H), 7.51 – 7.47 (m, 1H), 7.41 – 7.26 (m, 2H), 7.12 (d, J = 3.5 Hz, 1H), 6.75 (d, J = 3.5 Hz, 1H), 4.32 (s, 2H), 3.40 (d, J = 2.4 Hz, 2H), 3.30 (dt, J = 2.3, 1.6 Hz, 2H), 3.04 – 2.94 (m, 2H), 2.15 – 2.07 (m, 1H), 2.02 (d, J = 14.3 Hz, 2H), 1.52 (td, J = 14.7, 4.0 Hz, 2H); 13C NMR (75 MHz, CD3OD) δ 151.7, 146.3, 130.7, 130.2,.6, 128.2, 127.1, 113.8, 111.8, 51.7, 43.9, 43.3, 31.7, 26.4; MS (ESI) m/z 305 [M + H]+. Anal. (C17H21ClN2O) C, H, N.</p><!><p>Starting from 18d (100 mg, 0.41 mmol) and 7 (89 mg, 0.41 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 23 as yellow oil (90 mg, 65% over two steps). 1H NMR (300 MHz, CD3OD) δ 7.86 (d, J = 8.1 Hz, 2H), 7.66 (d, J = 7.6 Hz, 2H), 6.89 (d, J = 3.3 Hz, 1H), 6.42 (d, J = 3.3 Hz, 1H), 3.83 (s, 2H), 3.16 (d, J = 11.7 Hz, 2H), 2.72 (t, J = 12.4 Hz, 2H), 2.53 (d, J = 6.3 Hz, 2H), 1.85 (d, J = 14.0 Hz, 2H), 1.70 (brs, 1H), 1.28 – 1.15 (m, 2H); 13C NMR (75 MHz, CD3OD) δ 154.3, 151.6, 134.2, 128.5, 128.1, 125.4, 125.3, 125.3, 125.2, 123.3, 109.7, 107.8, 54.6, 45.3, 45.1, 35.4, 30.0; MS (ESI) m/z 338 [M + H]+. Anal. (C18H21F3N2O) C, H, N.</p><!><p>Starting from 18e (51 mg, 0.26 mmol) and 7 (55 mg, 0.41 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 24 as yellow oil (51 mg, 67% over two steps). 1H NMR (300 MHz, CD3OD) δ 7.88 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H), 7.03 (d, J = 3.5 Hz, 1H), 6.77 (d, J = 3.5 Hz, 1H), 4.41 (s, 2H), 3.47 – 3.37 (m, 2H), 3.08 (t, J = 7.7 Hz, 2H), 3.02 – 2.93 (m, 2H), 2.14 (brs, 1H), 2.08 – 2.00 (m, 2H), 1.51 (dd, J = 18.7, 8.6 Hz, 2H); 13C NMR (75 MHz, CD3OD) δ 153.7, 146.3, 134.2, 132.6, 131.9, 124.4, 118.5, 115.15, 110.6, 109.4, 51.4, 43.6, 43.2, 31.3, 26.2; MS (ESI) m/z 296 [M + H]+. Anal. (C18H21N3O) C, H, N.</p><!><p>Starting from 19b (50 mg, 0.17 mmol) and 7 (38 mg, 0.17 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 25 as yellow oil (52 mg, 80% over two steps). 1H NMR (300 MHz, CD3OD) δ 7.76 (d, J = 8.5 Hz, 1H), 7.43 – 7.35 (m, 4H), 7.27 – 7.21 (m, 3H), 6.10 (d, J = 3.1 Hz, 1H), 5.60 (d, J = 3.3 Hz, 1H), 3.63 (s, 2H), 3.05 (d, J = 12.4 Hz, 2H), 2.60 (t, J = 11.7 Hz, 2H), 2.39 (d, J = 6.7 Hz, 2H), 1.72 (d, J = 13.0 Hz, 2H), 1.64 – 1.53 (m, 1H), 1.20 – 1.04 (m, 2H); 13C NMR (75 MHz, CD3OD) δ 171.8, 152.7, 151.4, 141.1, 141.1, 132.7, 130.3, 128.7, 128.4, 128.2, 127.6, 127.5, 110.0, 109.2, 53.7, 45.3, 44.0, 33.9, 27.4; MS (ESI) m/z 381 [M + H]+. Anal. for (C23H25ClN2O) C, H, N.</p><!><p>Starting from 19a (50 mg, 0.17 mmol) and 7 (38 mg, 0.17 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on alumina gel (2% MeOH, 0.5% NH4OH, in DCM) to give 26 as yellow oil (33 mg, 51% over two steps). 1H NMR (300 MHz, CD3OD) δ 7.97 (d, J = 8.3 Hz, 1H), 7.72 (d, J = 1.9 Hz, 1H), 7.68 – 7.60 (m, 3H), 7.54 – 7.34 (m, 3H), 7.11 (d, J = 3.3 Hz, 1H), 6.44 (d, J = 3.3 Hz, 1H), 3.84 (s, 2H), 3.06 (d, J = 12.2 Hz, 2H), 2.61 (t, J = 11.6 Hz, 2H), 2.52 (d, J = 6.9 Hz, 2H), 1.78 (d, J = 13.6 Hz, 2H), 1.66 (brs, 1H), 1.25 – 1.08 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 154.3, 149.4, 140.9, 139.4, 133.4, 130.4, 130.3, 129.3, 129.1, 128.8, 128.2, 128.1, 127.1, 125.6, 112.0, 109.4, 55.7, 46.8, 46.7, 36.8, 31.6; MS (ESI) m/z 381 [M + H]+. Anal. for (C23H25ClN2O) C, H, N.</p><!><p>Starting from 16a (300 mg, 1.5 mmol) and 27 (135 μL, 1.5 mmol), the title compound was prepared following the procedure described for the synthesis of compound 5a (Method B). The crude product was purified by flash chromatography on silica gel (10 % EtOAc in petroleum ether) to give 28a as yellow oil (360 mg, 80%). 1H NMR (300 MHz, CDCl3) δ 9.93 (s, 1H), 7.75 (d, J = 3.9 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.41 (s, 1H), 7.38 – 7.35 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 183.1, 148.7, 144.2, 136.3, 133.5, 133.5, 132.4, 131.1, 130.7, 129.1, 123.5; MS (ESI) m/z 302 [M + H]+.</p><!><p>Starting from 16b (200 mg, 0.97 mmol) and 27 (87 μL, 0.97 mmol), the title compound was prepared following the procedure described for the synthesis of compound 5a (Method B). The crude product was purified by flash chromatography on silica gel (10 % EtOAc in petroleum ether) to give 28b as yellow oil (227 mg, 78%). 1H NMR (300 MHz, CDCl3) δ 9.93 (s, 1H), 7.75 (d, J = 3.9 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.41 (s, 1H), 7.38 – 7.35 (m, 2H). 13C NMR: (75 MHz, CDCl3) δ 183.1, 148.7, 144.2, 136.3, 133.5, 133.5, 132.4, 131.1, 130.7, 129.1, 123.5. MS (ESI) m/z 302 [M + H]+.</p><!><p>Starting from 28a (88 mg, 0.4 mmol) and 7 (85 mg, 0.4 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 29 as yellow oil (115 mg, 72% over two steps). 1H NMR (300 MHz, CDCl3) δ 7.62 (s, 1H), 7.38 (d, J = 1.6 Hz, 1H), 7.26 (d, J = 0.9 Hz, 1H), 7.20 (d, J = 2.4 Hz, 1H), 6.90 (d, J = 2.7 Hz, 1H), 3.98 (s, 2H), 3.08 (d, J = 12.2 Hz, 2H), 2.57 (d, J = 6.5 Hz, 2H), 1.74 (m, 4H), 1.61 (brs, 2H), 1.29 – 1.07 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 207.2, 146.4, 137.9, 133.2, 132.6, 132.3, 130.3, 127.8, 125.0, 121.4, 55.6, 49.0, 46.3, 36.6, 31.1; MS (ESI) m/z 400 [M + H]+. Anal. (C17H20BrClN2S) C, H, N.</p><!><p>Starting from 28b (48 mg, 0.2 mmol) and 7 (43 mg, 0.2 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 30 as yellow oil (50 mg, 63% over two steps). 1H NMR (300 MHz, CD3OD) δ 7.73 (s, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.40 – 7.37 (m, 1H), 7.14 (d, J = 1.5 Hz, 1H), 6.98 (d, J = 2.7 Hz, 1H), 3.95 (s, 2H), 3.06 (d, J = 12.8 Hz, 2H), 2.62 (t, J = 13.3, Hz, 2H), 2.51 (d, J = 6.6 Hz, 2H), 1.78 (d, J = 13.2 Hz, 2H), 1.65 (m, 1H), 1.28 – 1.08 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 207.2, 146.4, 137.9, 133.2, 132.6, 132.29, 130.3, 127.8, 125.0, 121.4, 55.6, 49.0, 46.3, 36.6, 31.1; MS (ESI) m/z 400 [M + H]+. Anal. (C17H20BrClN2S) C, H, N.</p><!><p>Starting from 31 (40 mg, 0.21 mmol), the N-Boc-protected intermediate was prepared and deprotected as described for the synthesis of 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 32 as yellow oil (41 mg, 60% over two steps). 1H NMR (300 MHz, CD3OD) δ 7.97 (s, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.41 (d, J = 4.2, Hz, 1H), 7.20 (s, 1H), 4.01 (s, 2H), 3.03 (d, J = 12.4 Hz, 2H), 2.58 (t, J = 12.4, Hz, 2H), 2.49 (d, J = 6.7 Hz, 2H), 1.75 (d, J = 13.2 Hz, 2H), 1.71 – 1.56 (m, 1H), 1.13 (m, 2H); 13C NMR (75 MHz, CD3OD) δ 145.9, 141.6, 138.9, 127.4, 124.5, 124.3, 121.5, 117.3, 54.8, 45.4, 35.9, 30.4; MS (ESI) m/z 362 [M + Na]+. Anal. (C15H19BrN2S) C, H, N.</p><!><p>To a solution of 33 (239.0 mg, 0.95 mmol) in MeCN (10 mL), DMF-DMA (128 μL, 0.95 mmol) was added. The reaction was heated to 50 °C for 1 h. After this time, benzyl amine (101 μL, 0.85 mmol) and acetic acid (2 mL) were added and reaction was heated at 120 °C for 12 h. The solvent was removed under vacuo. The crude product was purified by flash chromatography on silica gel (2% MeOH in EtOAc) to give 34 (198 mg, 60%) as yellow oil. 1H NMR (300 MHz, CDCl3) δ 8.19 (s, 1H), 7.46 (dd, J = 8.5, 2.1 Hz, 2H), 7.41 – 7.33 (m,2H), 7.30 – 7.23 (m, 3H), 7.06 – 6.96 (m, 1H), 5.17 (s, 2H); MS (ESI) m/z 350 [M + H]+.</p><!><p>A solution of 34 (100 mg, 0.29) in formaldehyde (aq. sol. 37%, 2.8 mL) was heated under reflux for 12 h. The solvent was removed under vacuo. The crude product was purified by flash chromatography on silica gel (2% MeOH in EtOAc) to give 35 as yellow oil. 1H NMR (300 MHz, CD3OD) δ 7.55 – 7.44 (m, 4H), 7.32 – 7.26 (m, 2H), 7.03 – 6.97 (m, 2H), 5.45 (s, 2H), 4.81 (d, J = 1.2 Hz, 2H). (43 mg, 50%). MS (ESI) m/z 300 [M + H]+.</p><!><p>To a solution of 35 (40 mg, 0.13 mmol) in dry DCM (4 mL), pyridine (12 μL, 0.14 mmol) and thionyl chloride (13 μL, 0.16 mmol) were added at 0 °C. After 3 h an aqueous solution of sodium bicarbonate was added and the organic phase was extracted with DCM, dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (2% MeOH in DCM) to give 36a as yellow oil (20 mg, 50%). 1H NMR (300 MHz, CD3OD) δ 7.55 – 7.44 (m, 4H), 7.32 – 7.26 (m, 2H), 7.03 – 6.97 (m, 2H), 5.43 (s, 2H), 4.92 (s, 2H); MS (ESI) m/z 319 [M + H]+.</p><!><p>To a solution of 33 (200 mg, 0.8 mmol) in phosphoryl chloride (2 mL), chloroacetic aid (76 mg, 0.8 mmol) was added. The reaction mixture was heated under reflux for 12 h. After this time the mixture was cooled at 0 °C, and an aqueous solution of sodium bicarbonate (2 mL) was slowly added. The organic phase was extracted with EtOAc (3 × 5 mL), dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (15% EtOAc in petroleum ether) to give 36b as yellow oil (49 mg, 20%). 1H NMR (300 MHz, CD3OD) δ 8.04 (d, J = 8.7 Hz, 1H), 7.94 – 7.89 (m, 1H), 7.60 (d, J = 8.0 Hz, 1H), 4.96 (s, 2H); 13C NMR (75 MHz, CD3OD) δ 139.13, 134.11, 132.61, 131.86, 130.09, 129.56, 128.36, 122.05, 32.49. MS (ESI) m/z 308 [M + H]+, 330 [M + Na]+.</p><!><p>To a solution of 36a (19 mg, 0.060 mmol) in dry MeCN (1 mL), a solution of 7 (20 mg, 0.065 mmol) in dry MeCN (2 mL), potassium carbonate (134 mg, 0.97 mmol) and a catalytic amount of potassium iodide were slowly added. The reaction mixture was heated under reflux for 1 h. After this time, the solvent was removed in vacuo. H2O (1 mL) and EtOAc (5 mL) were added and the organic phase was separated, dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (2% MeOH in DCM) to give 4-(4-benzyl-5-(4-chlorophenyl)-4H-1,2,4-triazol-3-yl)methylamino-N-tert-butoxy carbonylpiperidine as colorless oil (21 mg, 72%). 1H NMR (300 MHz, CD3OD) δ 7.55 – 7.44 (m, 5H), 7.34 – 7.26 (m, 2H), 6.98 (d, J = 8.0 Hz, 2H), 5.47 (s, 2H), 4.02 (d, J = 13.4 Hz, 2H), 3.91 (s, 2H), 2.69 (s, 2H), 2.49 (s, 2H), 1.65 (d, J = 13.5 Hz, 2H), 1.61 – 1.50 (m, 1H), 1.44 (s, 9H), 1.11 – 0.94 (m, 2H); MS (ESI) m/z 497 [M + H]+, 535 [M + K] +. To a solution of the above product (20 mg, 0.04 mmol) in DMSO (30 μL), a solution of potassium tert-butoxide (34 mg, 0.30 mmol) in dry THF (1 mL) was added. The reaction environment was saturated with oxygen. After 2 h an aqueous solution of ammonium chloride (0.5 mL) was added and the organic phase was extracted with EtOAc (3 × 3 mL), dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (2% MeOH in DCM) to give [5-(4-chlorophenyl)-4H-1,2,4-triazol-3-yl]methylamino-methyl-N-tert-butoxy-carbonyl piperidine as yellow oil (14 mg, 87%). 1H NMR (300 MHz, CD3OD) δ 7.97 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 7.9 Hz, 2H), 4.07 (d, J = 13.2, 2H), 3.96 (s,2H), 2.77 (d, J = 12.0 Hz, 2H), 2.58 (d, J = 6.0 Hz, 2H), 1.76 (d, J = 12.4 Hz, 2H), 1.44 (s, 9H), 1.31 – 1.21 (m, 2H), 1.15 – 1.04 (m, 1H). MS (ESI) m/z 407 [M + H]+. Starting from the above compound (15 mg, 0.03 mmol) and HCl 1 N (120 μL, 0.12 mmol), the title compound was deprotected as described for the synthesis of 1. The crude product 37 was obtained as yellow oil without further purification (11 mg, 84%). 1H NMR (300 MHz, CD3OD) δ 8.03 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 4.62 (s, 2H), 3.46 (d, J = 12.1 Hz, 2H), 3.11 (t, J = 11.8 Hz, 2H), 2.83 (brs, 2H), 2.32 (brs, 1H), 2.14 (d, J = 11.9 Hz, 2H), 1.68 (d, J = 11.1 Hz, 2H); MS (ESI) m/z 397 [M + H]+. Anal. (C15H20ClN5) C, H, N.</p><!><p>To a solution of 36b (12 mg, 0.06 mmol) in dry MeCN (1 mL), a solution of 84 (20 mg, 0.065 mmol) in dry MeCN (2 mL), potassium carbonate (134 mg, 0.97 mmol) and a catalytic amount of potassium iodide were slowly added. The reaction mixture was heated under reflux for 1 h. After this time, the solvent was removed in vacuo. H2O (1 mL) and EtOAc (5 mL) were added and the organic phase was separated, dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (2% MeOH in DCM) to give (5-(2-bromo-4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methylaminomethyl-N-tert-butoxy carbonylpiperidine as yellow oil (25 mg, 81%). 1H NMR (300 MHz, CD3OD) δ 8.02 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 5.0 Hz, 1H), 7.59 (dd, J = 8.5, 1.9 Hz, 1H), 4.08 (d, J = 5.8 Hz, 2H), 4.03 (s, 2H), 2.74 (s, 2H), 2.57 (s, 2H), 1.74 (d, J = 13.9 Hz, 2H), 1.69 – 1.61 (m, 1H), 1,44 (s, 9H) 1.17 – 0.99 (m, 2H); MS (ESI) m/z 508 [M + H]+. Starting from the above compound (20 mg, 0.04 mmol) and HCl 1 N (160 μL, 0.16 mmol), the title compound was prepared following the procedure reported for compound 37. The solvent was removed in vacuo. A saturated solution of sodium bicarbonate (1 mL) was added and the organic phase was extracted with EtOAc (3 × 3 mL), dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 38 as yellow oil (13 mg, 82%). 1H NMR (300 MHz, CD3OD) δ 8.03 (d, J = 8.3 Hz, 1H), 7.91 (d, J = 4.9, 1H), 7.59 (d, J = 8.5 Hz, 1H), 5.48 (s, 2H), 3.39 (d, J = 12.5 Hz, 2H), 3.32 – 3.27 (m, 2H), 2.97 (t, J = 12.4 Hz, 2H), 2.07 – 1.98 (m, 2H), 1.90 – 1.82 (m, 1H), 1.50 – 1.24 (m, 2H); MS (ESI) m/z 386 [M + H]+. Anal. for (C15H18BrClN4O) C, H, N.</p><!><p>Triphenylphosphine (366 mg, 1.4 mmol) and palladium (II) acetate (20 mg, 0.1 mmol) were dissolved in a mixture of dry DMF (7 mL) and H2O (3 mL) under argon atmosphere. After 30 min, 39 (198 μL, 1.7 mmol) was added. After further 30 min, potassium carbonate (721 mg, 5.2 mmol) and powered 4-trifluoromethyl-phenyl boronic acid 40 (331 mg, 1.7 mmol) were added in this order. The reaction mixture was heated at reflux for 12 h. The solvent was removed under vacuo. H2O (2 mL) and EtOAc (8 mL) were added, the organic phase was separated, dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (2 % EtOAc in petroleum ether) to give 41 as white solid (336 mg, 81%). 1H NMR (300 MHz, CDCl3) δ 8.13 – 8.01 (d, J = 7,83 Hz, 2H), 7.67 (d, J = 8.6 Hz, 2H), 7.56 (t, J = 7.73 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.07 (d, J = 7.8 Hz, 1H), 2.61 (s, 3H). MS (ESI) m/z 238 [M + H]+.</p><!><p>To a solution of 41 (336 mg, 1.4 mmol) in CCl4 (14 mL), NBS (904 mg, 5.1 mmol) and AIBN (98 mg, 0.6 mmol) were added portion wise. The reaction was heated under reflux for 12 h. The mixture was filtered on paper and the filtrate was concentrated under vacuo. The crude product was purified by flash chromatography on silica gel (20 % DCM in petroleum ether) to give 42 as white solid (132 mg, 30%). 1H NMR (300 MHz, CDCl3) δ 8.13 (d, J = 8.00 Hz, 2H), 7.81 (t, J = 7.8 Hz, 1H), 7.73 (d, J = 8.12 Hz, 2H), 7.68 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 4.63 (s, 2H); MS (ESI) m/z 317 [M + H]+.</p><!><p>To a solution of 7 (66 mg, 0.3 mmol) in dry MeCN (1 mL), was slowly added a solution of 42 (49 mg, 0.2 mmol) in dry MeCN (3 mL) and powered potassium carbonate (171 mg, 1.2 mmol). After 12 h, the solvent was removed in vacuo. H2O (1 mL) and EtOAc (5 mL) were added and the organic phase was washed with an aqueous solution NaOH (1 N, 2 mL) dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (2 % MeOH in DCM) to give the alkylation product 6-[4-(trifluoromethyl)phenyl-pyridin-2-yl]methylamino-methyl-N-tert-butoxycarbonyl piperidine as colorless oil. (18 mg, 20%). 1H NMR: (300 MHz, CD3OD) δ 8.25 (d, J = 8.1 Hz, 2H), 7.87-7.82 (m, 2H), 7.78 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 7.0 Hz, 1H), 4.06 (d, J = 13.5 Hz, 2H), 3.99 (s, 2H), 2.76 (t, J = 12.7 Hz, 2H), 2.60 (d, J = 6.2 Hz, 2H), 1.78 (d, J = 12.0 Hz, 2H), 1.44 (s, 9H), 1.28 (s, 1H), 1.17 – 1.04 (m, 2H); MS (ESI) m/z 450 [M + H]+, 472 [M + Na]+. Starting from the above alkylation product (30 mg, 0.06 mmol) and HCl 1 N (240 μL, 0.24 mmol), the title compound was prepared following the deprotection procedure reported for compound 1. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 43 as yellow oil (20 mg, 98%). 1H NMR (300 MHz, CD3OD) δ 8.25 (d, J = 8.2 Hz, 2H), 7.87 (d, J = 7.3 Hz, 2H), 7.78 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 7.1 Hz, 1H), 3.96 (s, 2H), 3.21 (d, J = 12.4 Hz, 2H), 2.78 (t, J = 11.4 Hz, 2H), 2.58 (d, J = 6.6 Hz, 2H), 1.91 (d, J = 13.4 Hz, 2H), 1.80 (brs, 1H), 1.27 (m, 2H); 13C NMR (75 MHz, CD3OD) δ 162.1, 159.1, 155.3, 149.1, 143.0, 138.0, 130.8, 130.4, 127.4, 126.3, 125.5, 125.4, 119.5, 54.7, 54.1, 44.7, 35.0, 29.5, 29.0, 21.8, 17.6, 13.2, 7.1, −9.3. MS (ESI) m/z 350 [M + H]+. Anal. f(C19H22F3N3) C, H, N.</p><!><p>To a solution of N1,N1-dimethylpropane-1,3-diamine 44 (22 μL, 0.17 mmol) in dry DCM (2 mL), was added a solution of aldehyde 5a (45 mg 0.16 mmol) in dry DCM (5 mL). After 1 h sodium triacetoxyborohydride (51 mg, 0.24 mmol) was added at 0 °C and the mixture kept at 25 °C for 12 h. After this time, sodium cyanoborohydride (20 mg, 0.32 mmol) was added and the solution was maintained at the same temperature for further 1 h. A saturated solution of sodium bicarbonate (3 mL) was added and the organic phase was extracted with DCM (3 × 5 mL), dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give 45 as yellow oil (50 mg, 85%). 1H NMR (300 MHz, CD3OD) δ 7.82 (d, J = 6.1 Hz, 1H), 7.66 (s, 1H), 7.51 (d, J = 6.5 Hz, 1H), 7.09 (d, J = 3.2 Hz, 1H), 6.43 (d, J = 3.1 Hz, 1H), 3.83 (d, J = 2.1 Hz, 2H), 2.66 (t, J = 6.1 Hz, 2H), 2.43 – 2.36 (m, 2H), 2.26 (s, 6H), 1.74 – 1.70 (m, 2H); 13C NMR (75 MHz, CD3OD) δ 153.7, 148.7, 132.9, 130.3, 130.3, 128.8, 128.4, 120.4, 112.2, 109.8, 57.4, 46.9, 45.2, 44.1, 25.6; MS (ESI) m/z 394 [M + Na]+. Anal. (C16H20BrClN2O) C, H, N.</p><!><p>Starting from 44 (65 μL, 0.5 mmol) and 18b (130 mg, 0.5 mmol), the title compound was prepared following the procedure reported for compound 45. The crude product was purified by flash chromatography on silica gel (10% MeOH, 1% NH4OH, in DCM) to give N1-[(5-(4-chloro-2-bromophenyl)furan-2-yl-methyl]-N3,N3-dimethylpropane-1,3-diamine as yellow oil (92 mg, 50%). 1H NMR (300 MHz, CD3OD) δ 7.80 (d, J = 8.6 Hz, 1H), 7.68 (s, 1H), 7.43 (d, J = 9.0 Hz, 1H), 7.12 (d, J = 3.4 Hz, 1H), 6.41 (d, J = 3.4 Hz, 1H), 3.87 (s, 2H), 2.64 (t, J = 7.3 Hz, 2H), 2.41 – 2.26 (m, 2H), 2.22 (s, 6H), 1.80 (t J = 6.0 Hz, 2H); 13C NMR (75 MHz, CD3OD) δ 153.7, 148.7, 132.9, 130.3, 130.3, 128.8, 128.4, 120.4, 112.2, 109.8, 57.4, 46.86, 45.2, 44.1, 25.6; MS (ESI) m/z 394 [M + Na]+. To a solution of the above secondary amine (57 mg, 0.2 mmol) in dry MeOH (4 mL), triethylamine (63 μL, 0.5 mmol) and di-tert-butyl dicarbonate (49 mg, 0.2 mmol) were added. The reaction mixture was kept at 25 °C for 4 h. After this time, the solvent was removed in vacuo. H2O (2 mL) was added and the organic phase was extracted with EtOAc (3 × 3 mL), dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (5% MeOH in DCM) to give 46 as yellow oil (82 mg, 87%). 1H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 8.5 Hz, 1H), 7.60 (s, 1H), 7.27 (d, J = 8.5,1H), 7.06 (s, 1H), 6,28 (br s, 1H), 4.42 (br s, 2H), 3.29 (br s, 2H), 2.19 (s, 6H), 1.67 (brs, 4H), 1.44 (s, 9H); MS (ESI) m/z 394 [M + H]+.</p><!><p>To a solution of 46 (30 mg, 0.1 mmol) in dry acetone (2 mL), iodomethane (16 μL, 0.3 mmol) was added. Reaction refluxed for 72 h. The solvent was removed under vacuo. The crude product was purified by flash chromatography on alumina gel (33% MeCN in DCM) to give (5-(2-bromo-4-chlorophenyl)furan-2-yl)methyl)(tert-butoxycarbonyl)amino)-N,N,N-trimethylpropan-1-ammonium iodide as yellow oil (29 mg, 60%). 1H NMR (300 MHz, CD3OD) δ 7.78 – 7.72 (m, 2H), 7.44 (d, J = 8.6 Hz, 1H), 7.12 (d, J = 3.4 Hz, 1H), 6.49 (d, J = 3.4 Hz, 1H), 4.54 (s, 2H), 3.43 (t, J = 6.9 Hz, 2H), 3.30 (s, 2H), 3.10 (s, 9H), 2.03 (br s, 2H), 1.50 (s, 9H). MS (ESI) m/z 485 [M + H]+. To a solution of the above compound (10 mg, 0.02 mmol) in MeOH (2 mL), a solution of HCl 1 N (110 μL), prepared using acetyl chloride (355 μL, 4.97 mmol) in MeOH (4.64 mL) was added at 0 °C. The reaction mixture was kept at 25 °C for 4 h. The solvent was removed under vacuo to give the 47 as colorless oil without further purification (7 mg, 95%). 1H NMR (300 MHz, CD3OD) δ 7.89 (d, J = 7,01 1H), 7.48 (s,1H), 7.21 (d, J = 3.4 Hz, 1H), 6.85 (d, J = 3.4 Hz, 1H), 4.46 (s, 2H), 3.53 (br s, 4H), 3.20 (s, 9H), 2.28 (br s, 2H). (ESI) m/z 385 [M + H]+. Anal. for (C17H23BrCl2N2O) C, H, N.</p><!><p>To a solution of 46 (30 mg, 0.1 mmol) in dry acetone (2 mL), benzyl bromide (38 μL, 0.3 mmol) was added. The reaction mixture was heated under reflux for 72 h. The solvent was removed under vacuo. The crude product was purified by flash chromatography on silica gel (5% MeOH in DCM) to give the benzyldimethylammonium bromide salt as yellow oil (32 mg, 58%). 1H NMR (300 MHz, CD3OD) δ 7.80 – 7.62 (m, 2H), 7.57 – 7.45 (m, 5H), 7.41 (d, J = 8.5 Hz, 1H), 7.09 (d, J = 3.5 Hz, 1H), 6.47 (d, J = 3.3 Hz, 1H), 4.54 (s, 2H), 4.49 (s, 2H), 3.46 (t, J = 6.7 Hz, 1H), 3.38 – 3.21 (m, 2H), 3.00 (s, 6H), 2.14 (t, J = 9.0 Hz, 2H), 1.49 (s, 9H); MS (ESI) m/z 562 [M + H]+. To a solution of the above compound (21 mg, 0.04 mmol) in MeOH (2 mL), a solution of HCl 1 N (222 μL), prepared using acetyl chloride (355 μL, 4.97 mmol) in MeOH (4.64 mL) was added at 0 °C. The reaction mixture was kept at 25 °C for 4 h. The solvent was removed under vacuo to give 48 as yellow oil without further purification (17 mg, 94%). 1H NMR (300 MHz, CD3OD) δ 7.90 (d, J = 7.7 Hz, 1H), 7.73 (s, 1H), 7.65-7.42 (m, 6H), 7,20 (s, 1H), 6.85 (s, 1H), 4.60 (s, 2H), 4.45 (s, 2H), 3.51 (br s, 2H), 3.22 (br s, 2H), 3.09 (s, 6H), 2.39 (br s, 2H); MS (ESI) m/z 462 [M + H]+. Anal. (C23H27BrCl2N2O) C, H, N.</p><!><p>Reference (1) and synthesized compounds were investigated for their potential capability to behave as pan-assay interference compounds (PAINS) by means of FAFDrugs4.0.[55, 56] Remarkably, none of compounds contain sub-structural features that would label them as "frequent hitters" in high throughput screens.</p><!><p>The CQ sensitive (D10) and the CQ resistant (W2) strains of P. falciparum were maintained in vitro at 5% hematocrit (human type A-positive red blood cells) in RPMI 1640 (EuroClone, Celbio) medium with the addition of 1% AlbuMax (Invitrogen, Milan, Italy), 0.01% hypoxanthine, 20 mM HEPES, and 2 mM glutamine, at 37 °C in a standard gas mixture consisting of 1% O2, 5%CO2, and 94% N2.[39]</p><!><p>Compounds were dissolved in DMSO and then diluted with medium to achieve the required concentrations (final DMSO concentration <1%, which is nontoxic to the parasite). Drugs were placed in 96 well flat-bottom microplates (COSTAR) and serial dilutions made. Asynchronous cultures with parasitemia of 1–1.5% and 1% final hematocrit were aliquoted into the plates and incubated for 72 h at 37 °C. Parasite growth was determined spectrophotometrically (OD650) by measuring the activity of the parasite lactate dehydrogenase (pLDH), according to a modified version of Makler's method in control and drug-treated cultures.[39] Antiplasmodial activity is expressed as the 50%inhibitory concentrations (IC50). Each IC50 value is the mean ± standard deviation of at least three separate experiments performed in duplicate.</p><!><p>Gametocytes were obtained from the transgenic P. falciparum 3D7 strain 3D7elo1-pfs16-CBG99 expressing the Pyrophorus plagiophthalamus CBG99 luciferase under the gametocyte specific promoter pfs16. Late-stage gametocytes (stage IV–V, evaluated by Giemsa staining) were exposed to compounds at day 8–10 after N-acetylglucosamine (NAG) addition. For drug susceptibility assay, compounds were prepared by serial dilution, in 96-well plate, in complete medium. Epoxomicin or methylene blue were used as reference drugs. After 72 h incubation, drug-treated gametocyte samples at 2% haematocrit were transferred to 96-well black microplates and D-luciferin (1 mM in citrate buffer 0.1 M, pH 5.5) was added at a 1:1 volume ratio. After 10 min incubation, gametocytes viability was measured as luciferase activity using a Sinergy 4 (Biotek) microplate reader (500 ms integration time). The IC50 was extrapolated from the non-linear regression analysis of the concentration–response curve.[23]</p><!><p>Asexual blood stage parasites were seeded at a density of 0.83% in 1.5% haematocrit in RPMI1640 medium with 10% human serum and combined with compounds serially diluted in DMSO and RPMI1640 medium to reach a final DMSO concentration of 0.1% in a volume of 60 μL. Following a 72 h incubation at 37 °C, 3% O2, 4% CO2, 30 μL of diluted Sybrgeen reagent was added according to the instructions of the manufacturer (Life Technologies) and fluorescence intensity was quantified using a Biotek Synergy 2 plate reader.</p><!><p>Gametocytes were obtained from a culture flask inoculated with 1% asexual blood stage parasites in 5% haematocrit in RPMI1640 medium with 10% human serum. From day 4 to day 9 post-inoculation, cultures were treated with 50 mM NAG to eliminate asexual blood stage parasites. At day 11 post inoculation, gametocytes (predominantly stage IV) were isolated by Percoll density gradient centrifugation as described previously (PMID 25667405). Gametocytes were seeded at a density of 5000 cells/well in a 384 well plate and combined with compound diluted in DMSO and subsequently in RPMI1640 medium to reach a final DMSO concentration of 0.1% in a volume of 60 μL RPMI1640 medium with 10% human serum. Following a 72 h incubation at 37 °C, 3% O2, 4% CO2, 30 μL of ONE-Glo reagent (Promega) was added and luminescence was quantified using a Biotek Synergy 2 reader.</p><!><p>20'000 mCherry expressing P. berghei (ANKA strain) sporozoites (PbmCherryhsp70,[57]) were used to infect mouse primary hepatocytes grown on 96-well plate (isolated as published in Prado et al.[58]). 2 hpi the cells were exposed to different concentrations of 25, as control DMSO (equal to the highest concentration of 25) was used. Medium/Drug was renewed at 24 hpi. At 48 hpi parasite number and size of unfixed cells were determined by automated microscopy (INcell Analyzer 2000, Gelifesciences). At 65 hpi detached cells (DC) were transferred to new wells and counted by fluorescent microscopy (Leica DMI 6000B). The detached cell assay is presented as detached cell formation rate (DC number in % of 48 h number). Statistical analysis was done by performing One-way ANOVA with Dunnet's Multiple Comparisons (Prism, GraphPad) (* p < 0.05, ** p < 0.01, *** p < 0.001, ns/not significant > 0.05).</p><!><p>All animal care and experimental protocols conformed to the European Union Guidelines for the Care and the Use of Laboratory Animals (European Union Directive 2010/63/EU) and were approved by the Italian Department of Health (666/2015-PR). Male Wistar rats (300–350 g, Charles River Italia, Calco, Italy) were anaesthetized (i.p.) with a mixture of Zoletil® 100 (7.5 mg/kg tiletamine HCl + 7.5 mg/kg zolazepam HCl; Virbac srl, Milano) e Rompun® (4 mg/kg xylazine HCl; Bio 98, San Lazzaro, Bologna), containing heparin (5000 U/kg), decapitated and exsanguinated.</p><!><p>The hearts, spontaneously beating, were rapidly explanted and mounted on a Langendorff apparatus for retrograde perfusion via the aorta at a constant flow rate of 10 mL/min with a Krebs-Henseleit solution of the following composition (mM): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11.5, Na pyruvate 2, and EDTA 0.5, bubbled with a 95% O2-5% CO2 gas mixture (pH 7.4), and kept at 37 °C, as described elsewhere.[59] The hearts were allowed to equilibrate for at least 20 min before drug exposure. Heart contractility was measured as LVP by means of latex balloon, inserted into the left ventricle via the mitral valve and connected to a pressure transducer (BLPR, WPI, Berlin, Germany). The balloon was inflated with deionized water from a microsyringe until a left ventricular end diastolic pressure of 10 mmHg was obtained. Alteration in CPP, arising from changes in coronary vascular resistance, were recorded by pressure transducer (BLPR, WPI, Berlin, Germany) placed in the inflow line. A surface ECG was recorded at a sampling rate of 1 kHz by means of two steel electrodes, one placed on the apex and the other on the left atrium of the heart. The ECG analysis included the following measurements: RR (cycle length), HR (frequency), PQ (atrioventricular conduction time), QRS (intraventricular conduction time), and QT (overall action potential duration).[60] LVP, CPP, and ECG were recorded with a digital PowerLab data acquisition system (PowerLab 8/30; ADInstruments, Castle Hill, Australia) and analyzed by using Chart Pro for Windows software (PowerLab; ADInstruments, Castle Hill, Australia). LVP was calculated by subtracting the left ventricular diastolic pressure from the left ventricular systolic pressure. As the QT interval is affected by heart rate changes (e.g., it shortens when heart rate increases), Bazett's formula normalized to average rat RR (corrected QT, QTc=QT/(RR/f)1/2)[61] was routinely used to correct it, in order to avoid confounding effects. In our experiments, "f", the normalization factor according to the basal RR duration was 242.23 ms for compound 1 and 217.15 ms for compound 25, as it was the average cardiac cycle length. Analysis of data was accomplished using GraphPad Prism version 5.04 (GraphPad Software, San Diego, USA). Statistical analyses and significance as measured by repeated measures ANOVA (followed by Dunnett's post test) were obtained using GraphPad InStat version 3.06 (GraphPad Software, San Diego, USA). In all comparisons, P < 0.05 was considered significant. Compounds 1 and 25 were dissolved in DMSO. Solvent failed to alter the response of the preparations (data not shown).</p><!><p>Rat tail was cut immediately, cleaned of skin and placed in physiological solution (namely external solution). The tail main artery was dissected free of its connective tissue and cells or rings prepared as detailed below. Smooth muscle cells were freshly isolated from the tail main artery under the following conditions: a 5-mm long piece of artery was incubated at 37 °C for 40–45 min in 2 mL of 0.1 mM Ca2+ external solution (in mM: 130 NaCl, 5.6 KCl, 10 Hepes, 20 glucose, 1.2 MgCl2, and 5 Na-pyruvate; pH 7.4) containing 20 mM taurine (prepared by replacing NaCl with equimolar taurine), 1.35 mg/mL collagenase (type XI), 1 mg/mL soybean trypsin inhibitor, and 1 mg/mL bovine serum albumin (Sigma Chimica, Milan, Italy), which was gently bubbled with a 95% O2 - 5% CO2 gas mixture to gently stir the enzyme solution, as previously described.[62] Cells, stored in 0.05 mM Ca2+ external solution containing 20 mM taurine and 0.5 mg/mL bovine serum albumin at 4 °C under normal atmosphere, were used for experiments within two days after isolation.[63] Cells were continuously superfused with external solution containing 0.1 mM Ca2+ and 30 mM tetraethylammonium (TEA, Sigma Chimica, Milan, Italy) using a peristaltic pump (LKB 2132, Bromma, Sweden), at a flow rate of 400 μL/min. The conventional whole-cell patch-clamp method was employed to voltage-clamp smooth muscle cells. Recording electrodes were pulled from borosilicate glass capillaries (WPI, Berlin, Germany) and fire-polished to obtain a pipette resistance of 2–5 MΩ when filled with internal solution. The internal solution (pCa 8.4) consisted of (in mM): 100 CsCl, 10 HEPES, 11 EGTA, 2 MgCl2, 1 CaCl2, 5 Na-pyruvate, 5 succinic acid, 5 oxaloacetic acid, 3 Na2-ATP and 5 phosphocreatine; pH was adjusted to 7.4 with CsOH. An Axopatch 200B patch-clamp amplifier (Molecular Devices Corporation, Sunnyvale, USA) was used to generate and apply voltage pulses to the clamped cells and record the corresponding membrane currents. At the beginning of each experiment, the junction potential between the pipette and bath solution was electronically adjusted to zero. Current signals, after compensation for whole-cell capacitance and series resistance (between 70% and 75%), were low-pass filtered at 1 kHz and digitized at 3 kHz prior to being stored on the computer hard disk. Electrophysiological responses were tested at room temperature (20–22 °C). The current through Cav1.2 channels was recorded in external solution containing 30 mM TEA and 5 mM Ba2+. Current was elicited with 250-ms clamp pulses (0.067 Hz) to 0 mV from a Vh of −50 mV. Data were collected once the current amplitude had been stabilized (usually 7–10 min after the whole-cell configuration had been obtained). Under these conditions, the current did not run down during the following 40 min.[64] K+ currents were blocked with 30 mM TEA in the external solution and Cs+ in the internal solution. Current values were corrected for leakage and residual outward currents using 10 μM nifedipine (Sigma Chimica, Milan, Italy), which completely blocked ICa1.2. The osmolarity of the 30 mM TEA- and 5 mM Ba2+-containing external solution (320 mosmol) and that of the internal solution (290 mosmol) were measured with an osmometer (Osmostat OM 6020, Menarini Diagnostics, Florence, Italy). Acquisition and analysis of data were accomplished using pClamp 9.2.1.9 software (Molecular Devices Corporation, Sunnyvale, USA) and GraphPad Prism version 5.04 (GraphPad Software, San Diego, USA). Compounds 1 and 25 were dissolved in DMSO (below 0.01). Solvent failed to alter the response of the preparations (data not shown).</p><!><p>For biochemical analysis of compounds, PfPMT was expressed in and purified from E. coli, as previously described.[53] Methyltransferase activity was monitored using a radiochemical assay.[52] Standard reaction conditions were 0.1 M Hepes KOH (pH 8.0), 2 mM Na2EDTA, 10% (v/v) glycerol, 5% (v/v) DMSO, 30 μM SAM (100 nCi of [methyl-14C] SAM), and 55 μM phosphoethanolamine in 100 μL with 2 μg of purified protein. Initial screening of compounds 1, 24, 25 and 48 as potential PfPMT inhibitors was performed at 100 μM. For determination of the IC50 for compound 25 versus PfPMT, assays were performed with varied inhibitor concentration (0–250 μM; final 5% (v/v) DMSO) with 100% activity defined as the control reaction (conditions as above)). Data were plotted as percentage of activity versus inhibitor concentration and were fit to y = 100/(1 + ([I]/IC50)) using Kaleidagraph (Synergy Software), where IC50 is the inhibitor concentration at 50% activity.</p><!><p>All calculations in this work were performed on a system comprising 72 Intel Xeon E5-2695 v4@2.10 GHz processors and two NVIDIA GeForce 1070 GTX GPU with Ubuntu 16.04 LTS (long-term support) operating system, running Schrödinger Molecular Modelling Environment Release 2015. Among the software in the molecular modelling suite we used Maestro, version 10.1; MacroModel, version 10.7; LigPrep, version 3.3; SiteMap version 3.4; Glide, version 6.6; Prime version 4.1 and PyMOL v1.8.4.0.</p><!><p>We retrieved from the PDB database all proteins relevant for the Plasmodium biology for a total of 108 crystal structures. All the crystal structures were analyzed, removing the water molecules, ions not involved in the enzymatic reactions and molecules used for the crystallization process. For the proteins with the co-crystallized ligands (inhibitors, substrates etc) we extracted them for a re-docking procedure to assess the reliability of the docking protocol (see next paragraph for further details). The selected crystal structures were prepared by means of Protein Preparation Wizard (PPW) protocol implemented in Maestro, for acquiring appropriate starting complexes for the subsequent computational analyses. In particular, the protocol includes three steps to: (1) add hydrogens, (2) optimize the orientation of hydroxyl groups, Asn, and Gln, and the protonation state of His, and (3) perform a constrained refinement by employing impref software (max RMSD = 0.30), consisting of a cycles of energy minimization based on the impact molecular mechanics engine employing OPLS_2005 as force field. Regarding PfPMT, the crystal structure with the code 3UJ8 from PDB containing the inhbitor sinefungin (adenosyl-ornithine) was selected. For the redocking procedure we consider also the natural substrate SAM extracted from the PfPMT crystal structure 3UJ6. Compounds 1 and 25 as well as all the ligands in the Plasmodium proteins (Sinefungin and SAM in the case of PfPMT) were treated by means of MacroModel for retrieving the lower energy conformers to use as input in molecular docking experiments. The calculation was performed using OPLS-2005 as force field. The Generalized-Born/Surface-Area (GB/SA) model for simulating the solvent effects was used. No cutoff for non-bonded interactions was used. PRCG method was employed with 1000 maximum iterations and 0.001 gradient convergence threshold for performing the molecular energy minimizations. MCMM (Monte Carlo Multiple Minimum) was employed as torsional sampling method for the conformational searches, performing automatic setup with 21 kJ/mol (5.02 Kcal/mol) in the energy window for saving structure and 0.5 Å was used as cutoff distance for redundant conformers. The lower conformers were treated by LigPrep application, generating the most plausible ionization state at cellular pH value (7.4 ± 0.2).</p><!><p>Molecular Docking was carried out by Glide using the ligands and the protein prepared as above-mentioned, applying Glide extra precision (XP) method. Energy grid was prepared using default value of protein atom scaling factor (1.0 Å) within a cubic box centered on the crystallized ligands were present, while for the Plasmodium proteins without any ligand we selected the binding site by analysing the literature data and/or performing a SiteMap calculation to find potential binding sites. Considering PfPMT, the cubic box centered on the sinefungin. After grid generation, the ligands and the crystallized inhibitor were docked into the enzymes with default parameters (no constraints were added). The number of poses entered to post-docking minimization was set to 50. Glide XP score was evaluated. The interactions of compounds with proteins were assessed using ligand-interaction diagram and a script for displaying hydrophobic interactions (display_hydrophobic_interactions.py) downloaded from Schrödinger website and implemented in Maestro. The RMSD between the docked poses and the co-crystallized compounds was calculated by using the script rmsd.py available in Maestro. We chose the Glide XP protocol since it demonstrated in this virtual screening procedure better accuracy in retrieving the binding mode of the crystallized ligands, showing lower RMSD over the Glide standard precision (SP) protocol as highlighted also for PfPMT. In fact, although the docking protocols were able to correctly accommodate the considered ligands (sinefungin and SAM), the Glide XP protocol showed low RMSD between the docked poses and crystallized ligands (sinefungin Glide XP RMSD = 0.21; Glide SP RMSD = 0.66. SAM Glide XP RMSD = 0.48; Glide SP RMSD = 1.05).</p><!><p>The Prime/MM-GBSA method (Prime software) computes the difference between both the states, free and complex, of the ligand and the protein after energy minimization as reported [50]. The calculated absolute values are not necessarily in agreement with observed binding affinities. Nonetheless, the ranking of the ligands based on the binding energies calculations (MM/GBSA ΔGbind) can agree reasonably well with ranking based on observed binding affinity, particularly in the case of congeneric series. As the MM/GBSA binding energies are approximate free energies of binding, a more negative value indicates stronger binding. The technique was used on the docked complexes (ligand-protein). For each ligand the software was employed to assess its ligand binding energy (ΔGbind) exploiting the following equation: 
ΔGbind=ΔEMM+ΔGsolv+ΔGSA</p><p>ΔEMM is the difference in the minimized energies calculated for the complex and the sum of the energies of the unbounded protein and ligand. ΔGsolv represents the difference in the GBSA solvation energy of the complex and the sum of these energies for the unbounded protein and ligand. ΔGSA is the difference in the surface area (SA) energies for the complex and the sum of the SA energies for the unbounded protein and ligand.</p><!><p>Figure 6 was prepared by means of PyMOL (The PyMOL Molecular Graphics System, v1.8.4.0, Schrödinger LLC, New York, 2015).</p>