New 4-( N -cinnamoylbutyl)aminoacridines as potential multi-stage antiplasmodial leads

A novel family of 4-aminoacridine derivatives was obtained by linking this heteroaromatic core to different trans - cinnamic acids. The 4-( N -cinnamoylbutyl)aminoacridines obtained exhibited in vitro activity in the low-or sub-micromolar range against (i) hepatic stages of Plasmodium berghei , (ii) erythrocytic forms of Plasmodium falciparum , and (iii) early and mature gametocytes of Plasmodium falciparum . The most active compound, having a meta -fluorocinnamoyl group linked to the acridine core, was 20-and 120-fold more potent, respectively, against the hepatic and gametocyte stages of Plasmodium infection than the reference drug, primaquine. Moreover, no cytotoxicity towards mammalian and red blood cells at the concentrations tested was observed for any of the compounds under investigation. These novel conjugates represent promising leads for the development of new multi-target antiplasmodials.


Introduction
Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium that remains a major concern in low-income tropical and sub-tropical countries.Despite the steady decrease in the malaria burden during the first decade of the 21st century, progress has stalled in the past five years.Moreover, global warming may favor the migration of the Anopheles mosquitoes, the disease's main vector, to currently malaria-free regions [1].Among the reasons for malaria's ongoing burden are (i) the complex life cycle of the Plasmodium parasite, which hampers the swift and successful development of effective drugs and vaccines, and (ii) the fast selection and spread of drug-resistant parasite strains to all the drugs developed so far [2].In fact, the world is now facing a concerning decline in the effectiveness of the frontline artemisinin-based combination therapies (ACT), as signs of resistance to this therapy has transposed the borders of Southeast Asia and has spread even to Africa [3][4][5][6][7].This highlights the pressing need to discover new potent and safe antimalarial drugs, ideally, cost-effective since malaria mostly affects low-income populations, and actives against multiple stages of the life cycle of different Plasmodium species to decrease the probability of resistance appearance [8].
One cost-effective strategy towards affordable medicines is based on rescuing old antimalarial pharmacophores, through chemical modifications, aiming at their re-introduction in the antimalarial chemotherapy arsenal [9,10].In this regard, our research group have been working on the rescuing of classical antimalarial drugs like quinacrine (QN), chloroquine (CQ), and primaquine (PQ), which their use has been limited due to toxicity and resistance issues [11][12][13][14][15][16][17][18].QN, an acridine-based compound, was the first synthetic antimalarial drug and its potent blood-schizonticidal activity turned it into a promising substitute for quinine, the first effective therapy used against malaria [10,19].However, the serious side effects of QN led to its rapid replacement by CQ, which displayed higher efficiency, oral bioavailability and safety.Still, the use of CQ to treat malaria has been gradually abandoned due to the emergence and spread of CQ-resistant strains of P. falciparum, the most lethal agent of human malaria [19,20].While most antimalarial agents available, such as the case of QN and CQ, target blood-stage parasites, PQ remained, for many years, the only drug in clinical use that (i) acts against liver forms of all Plasmodium species, including the latent hypnozoites of P. vivax and P. ovale that can cause malaria relapses, and (ii) efficiently eliminates gametocytes, the mosquito vector-infective forms of malaria parasites [21].Nevertheless, PQ also presents significant downsides that limit its clinical application, including its high hemotoxicity and low oral bioavailability [22,23].Recently, in 2018, the Food and Drug Administration approved tafenoquine (TQ), which has higher efficacy due to its superior pharmacokinetic properties, but is also hemotoxic and, like PQ, cannot be used in G6PD-unknown or -deficient patients [24].Thus, our latest efforts focused on the development of novel QN analogues merging both the CQ and the PQ cores in a single structure (1a-c, Fig. 1), as potential multi-stage antiplasmodials [17,18].Multi-target drugs have raised considerable interest in the last decade owing to their advantages in the treatment of complex diseases such as malaria and drug resistance issues [25,26].4-N-butylaminoacridine (1a), the best derivative from this family, displayed promising results since the compounds retained the activity of the parent drugs, CQ and PQ, showing significant in vitro activity against both liver and blood stages malaria parasites.Based on this and inspired by the "covalent bitherapy" concept first advanced by Meunier [27,28], which advocates the combination of two pharmacophores in one molecule, our working hypothesis was that conjugation of 4-N-butylaminoacridine (1a) to bioactive trans-cinnamic acids would afford new constructs (2a-l) with improved efficacy and safety in multiple stages of malaria parasite life cycle while being less likely to elicit resistance.This "covalent bitherapy" approach has already been successfully employed in the preparation of compounds with pronounced antimalarial activity [29,30].Cinnamic acid derivatives, due to their α,β-unsaturated carbonyl moiety, can act as Michael acceptors and inhibit cysteine proteases, especially falcipain-2 and falcipain-3, involved in hemoglobin degradation and thus vital for the intraerythrocytic stage of the parasite [31][32][33][34][35][36].In this sense, we choose trans-cinnamic acids not only to introduce the α,β-vinylcarbonyl moiety (Michael acceptor), but also because of their antimalarial potential that was advanced by Kanaani and Ginsburg in 1992, based on cinnamic acids' ability to inhibit lactate transport, the major product of parasite energy metabolism and, thus, vital for the survival and growth of intraerythrocytic parasites.These authors also confirmed that cinnamic acid derivatives hampered the production of ATP by the parasite and inhibited the transport of glucose, glycine, and sorbitol by Plasmodium-infected erythrocytes [37,38].Since then, conjugation of cinnamic acids with classical antimalarials such as CQ, QN and PQ has been studied by different groups [39,40], including ours [15,16,41,42], and has delivering promising in vitro antiplasmodial results [39,43].In view of this, we have coupled a series of twelve different trans-cinnamoyl moieties to the aliphatic amino group of 1a, and evaluated their in vitro activity against the liver, blood and gametocyte stages of Plasmodium parasites, aiming at the establishment of relevant structure-activity relationship (SAR).The data herein obtained demonstrated that the new conjugates preserved and, in some cases, improved multi-stage activity in vitro and that this activity is very dependent of the trans-cinnamoyl moiety coupled to the aliphatic amino group of 1a.

Chemistry
The chemical synthesis of target compounds 2a-l was carried out via the synthetic route presented in Scheme 1. Detailed procedures and relevant spectroscopic data of the final compounds are provided in Section 4 and in Supplementary Information (SI).
The QN analogue 4-(4-aminobutyl)amino-6-chloro-2-methoxy-acridine (1a) was first obtained by a ten-step synthetic route starting from the commercial compound 4-chlorosalicylic acid, as previously reported [17,18].Compound 1a was then coupled to a series of trans-cinnamic acids by a standard in situ amide coupling method, using O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIEA) as a coupling agent and base, respectively, to afford the target compounds 2a-l.To this end, the carboxylic acid group in the trans-cinnamic acid was first activated for 1 h with TBTU and DIEA, in dichloromethane (DCM); after the activation period, 1a was added to the mixture and the reaction allowed to proceed at room temperature (r.t.).The crude products were purified by liquid chromatography on silica-gel columns, to obtain the target compounds in 95-99% purity, as confirmed by high-performance liquid chromatography (HPLC).Structural analyses by electrospray ionization-ion trap mass spectrometry (ESI-IT MS), and by proton ( 1 H) and carbon-13 ( 13 C) nuclear magnetic resonance (NMR) confirmed the expected structures for all target compounds 2a-l, which were obtained in moderate to excellent overall yields (33-95%).

Biological assays 2.2.1. Hepatic stage activity
The in vitro activity previously observed for QN analogue 1a against hepatic forms of P. berghei parasites revealed the importance of including the aminoalkyl side chain in position 4 of the acridine ring, since, according to the literature, QN has poor activity against liver stage parasites and a low selectivity index [16]; moreover, such modification did not abolish the blood stage activity typical of QN and related compounds [18].Advancing drug candidates that eliminate liver stage parasites is highly relevant, as they are not only scarce [22], but may also hamper the development of thousands of erythrocyte-infective merozoites responsible for disease symptoms [44,45].Therefore, based on our previous studies showing that N-cinnamoylation of the classical antimalarial drugs CQ, PQ and QN led to an improvement of liver stage activity, we hypothesized that a similar benefit might result from coupling 1a to trans-cinnamic acids to produce conjugates 2a-l.
Hence, in vitro activity of 1 and 10 μM 2a-l against the hepatic stage of P. berghei infection was evaluated using a previously described bioluminescence-based method to quantify the parasite load of human hepatoma (Huh-7) cells [18].The toxicity of the compounds to this human cell line was also assessed through the fluorescence measurement of cell confluency (Fig. 2), as described in detail in Section 4. Our  The QN analogue 1a was first obtained in ten steps as previously described [17,18], and subsequently coupled to different trans-cinnamic acids (highlighted in blue) under the following conditions: respective trans-cinnamic acid (1.1 equiv), TBTU (1.1 equiv), DIEA (2 equiv) and DCM at r.t.results show that all the compounds 2a-l were more active than the reference drug PQ against hepatic forms of P. berghei (Fig. 2).IC 50 values were then determined for the most active and least cytotoxic (above ~75% confluency relative to control) compounds at the highest concentration tested, 10 μM (2e-f, i-l).All IC 50 values obtained were in the low-or sub-micromolar range (0.496 < IC 50 < 1.189 μM) (Table 1).All N-cinnamoyl conjugates 2a-l were more active than their parent compound 1a (IC 50 = 2.22 μM), which is in support of our initial hypothesis.The IC 50 values were dependent on the substituent of the aryl ring in the cinnamoyl building block (R), and the following observations can be made in this regard.Overall, conjugates 2i and 2l stood out for their higher activity against hepatic forms of P. berghei, being approximately 25-fold more potent than the reference drug PQ, while not showing any significant toxicity to Huh-7 cells up to 10 μM.

Gametocytocidal activity
Like antiplasmodials targeting liver stage Plasmodium parasites, those that are able to kill gametocytes are also highly important, but scarce.Since gametocytes are the only forms of Plasmodium that are infective to the mosquito vector, their elimination hampers the host-tovector transmission, therefore blocking the dissemination of the disease.As such, the gametocytocidal activity of compounds 2a-l was also assessed in vitro against early gametocytes of P. falciparum (3D7 strain) using the test compounds at 5 μM, which is the IC 10 value for the reference drug PQ.Results depicted in Fig. 3 are expressed as % of inhibition of gametocyte growth and show that the inhibitory activity of conjugates 2a-l is comparable to (2a, 2c, 2d, 2f, 2h and 2j, 15-30% Table 1 In vitro activity of compounds 2a-l against liver forms of P. berghei, as well as early gametocytes and ring stages of P. falciparum 3D7.  a Liver-stage antiplasmodial activity was determined against P. berghei, using PQ as reference drug.
b Gametocytocidal activity was determined in P. falciparum NF54-gexp02-Tom strain, using PQ as reference drug.c Blood-stage antiplasmodial activity was determined against the CQ-sensitive P. falciparum 3D7 strain, using CQ as reference drug.d Value taken from Ref. [18].e Not determined due to its high toxicity to Huh-7 cells.M. Fonte et al.

Blood stage activity
Motivated by the encouraging results obtained for the activity of the compounds against both liver and gametocyte stages of Plasmodium parasites, we further tested the blood stage activity of 2e, 2g, 2i, 2k and 2l, i.e., the top-five gametocytocidals, which includes the top-two compounds against liver forms of the parasite.The P. falciparum 3D7 strain was used to assess the effect of the compounds in vitro against blood stage parasites by means of flow cytometry, employing the SYTO 11 green-fluorescent nucleic acid stain.The hemolytic activity of the compounds at 20 μM was assessed in parallel, and none was found significantly hemolytic at this concentration.As for blood antiplasmodial activity (Table 1), except for the unsubstituted (R--H) derivative 2l (IC 50 > 70 μM), all other compounds displayed IC 50 values in the low micromolar range.Still, none of them could match the potent activity of the reference drug CQ against the same P. falciparum 3D7 strain (IC 50 = 0.02).As described for the gametocytocidal activity, no clear SAR could be drawn in regard to blood stage activity because the set of compounds tested was not large enough to allow withdrawing any meaningful conclusions.Nevertheless, it appears that electronwithdrawing R groups, such as p-NO 2 in 2e (IC 50 = 3.82 μM), p-F in 2f (IC 50 = 5.827 μM) and m-F in 2i (IC 50 = 4.34 μM), lead to higher activity than electro-donating ones, as in 2g (R = p-CH 3 ; IC 50 = 10.82 μM) and 2k (R = p-OCH 3 ; IC 50 = 10.3 μM).
The data compiled in Table 1 proves the validity of our working hypothesis, since the second-generation compounds (2a-l) herein reported display the expected multi-stage activity in vitro of with no increase in cytotoxicity, as compared to the parent compound 1a.Actually, the new compounds are more active against liver and gametocyte stages of parasite development than the reference drug, PQ, and more active than the parent drug 1a against the erythrocytic stage.Therefore, the moiety newly introduced clearly has a positive impact on overall antiplasmodial activity.Ongoing studies in vitro (undisclosed data) will allow us to check whether falcipain inhibition may play a role in the slight increase in blood-stage activity of the 2nd generation compounds reported.
The exact mechanism of QN's antimalarial action remains unclear, but it has been demonstrated that QN can interact with β-hematin, consequently inhibiting hemozoin formation and the development of intraerythrocytic parasites.However, other mechanisms of action have been suggested to elucidate the antimalarial activity of QN derivatives, such as (i) inhibition of: the parasite's NF-κB pathway, the mitochondrial bc1 complex, and/or the parasitic DNA topoisomerases II; (ii) activation of the p53 pathway; and/or (iii) binding to DNA, either by intercalation or by groove binding [46,47].On the other hand, as mentioned above, cinnamic acids are reported as inhibitors of lactate, glucose, glycine, and sorbitol transport by Plasmodium-infected erythrocytes and as being able to hinder the production of ATP by the parasite [37].Therefore, both QN and cinnamic acid derivatives are reported to affect crucial pathways in the growth and survival of Plasmodium-infected erythrocytes.Yet, the new compounds herein reported are new molecular entities that may owe their multi-stage antiplasmodial action by effects other than those described to their separate building blocks, or by a combination of (a few) of them.Previous studies in our group focused on similar chloroquine-cinnamic acid conjugates demonstrated their ability to strongly interact with DNA [48], and preliminary studies (undisclosed data) on the compounds now reported point to a similar direction.Further studies will be conducted to deliver conclusive data on mode of action and possible off-target effect.Still, the lack of significant toxicity of the new compounds to either hepatocytes or erythrocytes are suggestive of a selective action.

Concluding remarks
Herein, we report the synthesis and in vitro evaluation of a new family of compounds 2a-l, as promising multi-stage antiplasmodial leads obtained through the coupling of a series of trans-cinnamic acids to the aliphatic amine of the 4-aminoacridine 1a, which we previously found to present dual-stage antiplasmodial activity in vitro [18].Our working hypothesis was that the antimalarial activity of 1a might be improved via such N-cinnamoylation procedure, in light of our earlier findings showing that a similar modification was applied to the classical antimalarial drugs PQ, CQ and QN [15,16,41,42].This hypothesis was confirmed by the data obtained, as they demonstrate that the new conjugates preserve multi-stage activity in vitro, being more active against liver and gametocyte stages of parasite development than the reference drug, PQ.In fact, one compound (2i) showed a 20-and 120-fold higher activity than PQ against liver and gametocyte stages, respectively, alongside low micromolar activity against blood stage P. falciparum parasites.Blood-stage activity of the new compounds is not spectacular; still, some of them represent an improvement over the first-generation compound 1a, while preserving relevant activity against other stages of parasite development.This provides confirmation of our working hypothesis and, together with results from ongoing mechanistic studies, offers an important advance towards the fine tuning of this type of molecular scaffolds envisaging their improvement as multi-stage antiplasmodial leads.These findings are in line with previous reports by other authors on the beneficial role not only of conjugation to trans-cinnamic acids, but also of fluorination in antimalarial drug discovery [49].Moreover, none of the new compounds reported herein showed significant toxicity to either hepatic Huh-7 cells or erythrocytes.
The fact that no compound at 20 μM was found to be significantly hemolytic is very important since the main problem associated to drugs active in liver stages, as PQ, is the hemolytic toxicity in G6PD-unknown or -deficient patients.
Altogether, these are encouraging results highlight scaffolds like 2i as novel potential multi-stage antiplasmodial leads.Considering the importance of halting progression of infection at its very onset in the host, i.e., during the liver stage of the parasite's life cycle, and of avoiding its vector-mediated dissemination from infected to healthy people, these findings are a promising starting point that may have a relevant impact in the future development of antimalarial candidates.

Chemicals and instrumentation
The chemicals and solvents used in this study were purchased from Sigma-Aldrich (trans-4-(trifluoromethyl)cinnamic acid, trans-4-nitrocinnamic acid, trans-3-fluorocinnamic acid and trans-4-(dimethylamino) cinnamic acid), Bachem (TBTU), Alfa Aesar (DIEA, trans-4-isopropylcinnamic acid), Acros organics (trans-cinnamic acid, trans-4chlorocinnamic acid, trans-4-methoxycinnamic acid and trans-4-methylcinnamic acid), PanReac AppliChem (anhydrous Na 2 SO 4 ), Biochem Chemopharma (NaHCO 3 ), VWR international (HPLC gradient grade, LC-MS grade and p.a. grade solvents, i.e., acetonitrile and methanol), LabChem (synthesis grade solvents, i.e., DMF, DCM, hexane and ethyl acetate), and CortecNet (deuterated solvents for NMR analysis).Aluminium foils coated with silica-gel 60 F254 from Merck were used for monitoring reaction progress, purification steps, and determination of compound retention factors (R f ) by thin-layer chromatography (TLC).TLC chromatograms were revealed with ultra-violet light (254 nm), using a Viber Lourmat lamp, model CN-6.The purifications of compounds were performed by normal phase liquid chromatography using silica gel 60 from Sigma-Aldrich as solid-phase and a mixture of hexane and ethyl acetate 1:1 (v/v) as mobile-phase.ESI-IT MS analyses (positive mode) were achieved with solutions of the pure products in methanol, using a Finnigan Surveyor LCQ DECA XP MAX spectrometer, at the Department of Chemistry and Biochemistry of the Faculty of Sciences of the University of Porto. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance III spectrometer at frequencies of 400 mHz and 100 mHz, respectively, and samples were prepared in hexadeuterated dimethylsulfoxide (DMSO-d 6 ) and deuterated chloroform (CDCl 3 ) with tetramethylsilane (TMS) as an internal reference.The determination of the retention times (RT) and purity degrees of compounds n was carried out by analytical HPLC, using a Hitachi-Merck Elite LaChrom system equipped with an L-2130 quaternary pump, an L-2200 thermostatted (Peltier effect) automated sampler, and an L-2455 diode-array detector (DAD).The compounds were dissolved in acetonitrile and injected in a reverse phase column (C18) Purospher star RP-C18 of 125 × 4.0 mm and 5 μm pore size.A gradient elution from 1 to 100% of acetonitrile in 0.05% aqueous (aq.) trifluoroacetic acid (TFA) was carried out for 30 min, at a flow rate of 1 mL min − 1 , and detection was set at 220 nm.

In vitro assays 4.2.1. Hepatic stage activity
The in vitro inhibition of hepatic infection by the test compounds was determined by measuring the luminescence intensity in lysates of Huh7 cells infected with a firefly luciferase-expressing P. berghei line, PbGFP-Luccon, as previously described [50].Huh7 cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI) supplemented with 10% v/v fetal calf serum, 1% v/v penicillin/streptomycin, 1% v/v nonessential amino acids, 1% v/v glutamine, and 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), pH 7, and maintained at 37 • C with 5% CO 2 .For infection assays, Huh7 cells were seeded in 96-well plates the day before drug treatment and infection.Approximately 1 h prior to infection with sporozoites freshly obtained through disruption of salivary glands of infected female Anopheles stephensi mosquitoes, the culture medium in the wells was replaced by infection medium (culture medium supplemented with 50 μg/mL gentamicin and 0.8 μg/mL amphotericin B) containing the appropriate concentration of each compound.In control cells, culture medium was replaced by medium containing the same percentage of DMSO used in compound solutions (0.01%), which is not cytotoxic.After 48 h of infection, inhibition of infection was measured and the effect of the compounds on the viability of Huh7 cells was assessed by the AlamarBlue assay (Invitrogen, UK) using the manufacturer's protocol.Nonlinear regression analysis was employed to fit the normalized results of the dose-response curves, and IC 50 values were determined using GraphPad Prism 8.0 (GraphPad software, La Jolla, CA, USA).Cytotoxicity was extrapolated from the cell confluency data.All data was then normalized to the DMSO control.Therefore, a decrease in cell confluency, as compared to the DMSO control, was indicative of cytotoxicity.

Gametocytocidal activity
P. falciparum parasites of the high-gametocyte productive NF54-gexp02-Tom line [51] (kindly provided by Prof. Alfred Cortés), which has a genome-integrated tandem Tomato (tdTomato) fluorescent marker gene controlled by an early gametocyte promoter, were used.Parasites were grown in group B human erythrocytes at 3% hematocrit in RPMI-based standard medium supplemented with 10% human plasma and 2 mM choline chloride, and incubated at 37 • C under hypoxia (5% O 2 , 5% CO 2 , and 90% N 2 at 37 • C).To induce sexual conversion, choline was removed from the medium, in combination with synchronization by sorbitol lysis.Induced cultures were then maintained without choline until the end of the experiment.The effect on gametocyte development was assessed in two independent replicas by adding growing concentrations (0.0048-100 μM) of each compound to the culture.The appearance of stage V gametocytes was daily monitored by light microscopy.Fifteen days after choline removal, ~80% of gametocytes reached stages IV and V (mature forms).After this time, Giemsa-stained blood smears were prepared and at least 10,000 cells were counted for each condition.A negative control culture without drugs and a positive control treated with the IC 90 of chloroquine and primaquine, where no gametocytes were observed at the end of the experiment, were also included.Growth inhibition data was transformed through sigmoidal fitting and used to determine the compounds' concentrations required for the reduction of P. falciparum viability by 50% (IC 50 ), using the GrapPhad Prism 9 software.

Blood stage activity
Asexual stages of the P. falciparum 3D7 strain were grown in group B human erythrocytes at 3% hematocrit using RPMI supplemented with 0.5% (w/v) Albumax II (Life Technologies, New Zealand) and 2 mM Lglutamine.Throughout the whole experiment, parasites were maintained in standard culturing conditions (37 • C, 5% O 2 , 5% CO 2 , and 90% N 2 ).Before starting the blood stage activity assays, parasites were synchronized in the ring stage with a 5% sorbitol lysis as described in Ref. [52].Afterwards, growing concentrations of the different 4-(N-cinnamoylbutyl)aminoacridines (0.0000256-10 μM) were incubated with the parasite culture during 48 h.Finally, cultures were diluted to ca. 1 to 10 × 10 6 cells/mL and incubated with Syto11 to stain the nuclei.Parasitemia was analyzed by flow cytometry using a LSRFortessa flow cytometer (BD Biosciences, USA) set up with the 4 lasers, 20 parameters standard configuration.The single-cell population was selected on a forward-side scattergram.Syto 11 fluorescence signal was detected by exciting samples at 488 nm and collecting the emission with a 530/30-nm bandpass filter.Growth inhibition was calculated taking as reference values the growth rates of an untreated culture (0% inhibition) and of a chloroquine-treated culture (100% inhibition).Growth inhibition data was transformed through sigmoidal fitting and used to determine the compounds' concentrations required for the reduction of P. falciparum viability by 50% (IC 50 ) using the GraphPad Prism 9 software.

Hemolytic activity
Human blood samples were first centrifuged (Centurion scientific Ltd centrifuge with a BRK1001 rotor) at 2500 rpm for 5 min at 4 • C to remove the plasma.The red blood cells (RBC) thus collected were then washed three times with 0.01 M phosphate-buffered saline, pH 7.4 (PBS, Sigma-Aldrich) and diluted to a final 6% hematocrit in PBS.In a 96-well plate, 100 μL of RBCs at 6% hematocrit were plated in the presence of 100 μL of a solution of each compound at 40 μM in 1% DMSO in PBS, and next incubated at 37 • C for 1 h.1% Triton X-100 in PBS and 1% DMSO in PBS were used as positive and negative controls, respectively.After the 1-h incubation period, the plates were centrifuged (Sigma® 3-30K centrifuge from Sigma Centrifuges GmbH with a swing-out 11222 rotor) at 860×g (2500 rpm), for 10 min at 25 • C. Finally, 80 μL of the supernatants were transferred to a new 96-well plate and absorbance (A) at 450 nm was measured in a microplate reader (Multiskan GO, Thermo Scientific).The percentage of hemolysis was calculated for each test compound using the following equation:

Scheme 1 .
Scheme 1. Synthetic route to target compounds 2a-l a a The QN analogue 1a was first obtained in ten steps as previously described[17,18], and subsequently coupled to different trans-cinnamic acids (highlighted in blue)

Fig. 2 .
Fig. 2. In vitro activity of compounds 2a-l and PQ, at 1 and 10 μM, against liver forms of P. berghei.Cytotoxicity to Huh7 cells (cell confluency scale, blue circles) are also shown.

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.Fonte et al.

Fig. 3 .
Fig. 3.In vitro growth inhibition of early P. falciparum 3D7 gametocytes upon incubation with test compounds 2a-l at 5 μM; PQ was included as the reference gametocytocidal drug.