Synthesis, Molecular Docking and Antiplasmodial Activities of New Tetrahydro-β-Carbolines

Malaria is still one of the most dangerous infectious diseases and the emergence of drug resistant parasites only worsens the situation. A series of new tetrahydro-β-carbolines were designed, synthesized by the Pictet–Spengler reaction, and characterized. Further, the compounds were screened for their in vitro antiplasmodial activity against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Moreover, molecular modeling studies were performed to assess the potential action of the designed molecules and toxicity assays were conducted on the human microvascular endothelial (HMEC-1) cell line and human red blood cells. Our studies identified N-(3,3-dimethylbutyl)-1-octyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b] indole-3-carboxamide (7) (a mixture of diastereomers) as the most promising compound endowed with the highest antiplasmodial activity, highest selectivity, and lack of cytotoxicity. In silico simulations carried out for (1S,3R)-7 provided useful insights into its possible interactions with enzymes essential for parasite metabolism. Further studies are underway to develop the optimal nanosized lipid-based delivery system for this compound and to determine its precise mechanism of action.


Introduction
Malaria is an infectious disease caused by the protozoan parasite of the genus Plasmodium, with Plasmodium falciparum and Plasmodium vivax being predominantly responsible for mortality and morbidity. In 2019, the global tally of malaria cases was 229 million, an annual estimate that has remained virtually unchanged over the last 4 years [1]. Despite the COVID-19 pandemic, an analysis of malaria prevention showed that the 2020 campaigns to control and eradicate malaria were realized as planned. Lastly, in October of 2021, the WHO has recommended Mosquirix, the RTS,S/AS01 malaria vaccine, for the prevention of P. falciparum malaria in children living in regions with moderate to high transmission [2]. However, disruptions to continued access to effective antimalarial treatment could lead to considerable loss of life [3].
First-line malaria treatments include several artemisinin-based combination therapies (ACTs), such as artemether-lumefantrine (AL), artesunate-amodiaquine (AS-AQ), artesunate-sulfadoxine-pyrimethamine (AS+SP), artesunate-mefloquine (AS-MQ), and dihydroartemisinin-piperaquine (DHA-PPQ). The malaria problem is evolving, dynamic, tesunate-sulfadoxine-pyrimethamine (AS+SP), artesunate-mefloquine (AS-MQ), and dihydroartemisinin-piperaquine (DHA-PPQ). The malaria problem is evolving, dynamic, and diverse, mainly due to the intrinsic ability of P. falciparum to acquire resistance against drugs. P. falciparum has developed resistance to nearly all currently available antimalarial drugs, such as sulfadoxine/pyrimethamine, mefloquine, halofantrine, and quinine [4]. The observed decrease in the effectiveness of artemisinin is correlated with mutation in the P. falciparum Kelch13 gene [5]. In addition, there is resistance against artemisinin partner drugs, such as mefloquine and piperaquine [6,7]. Thus, there is an urgent need to find a replacement for artemisinin or novel artemisinin partner-drugs active against known-resistant strains. Currently, all new molecules are tested against a wide variety of resistant laboratory strains of P. falciparum, and activity in these assays is a key requirement for further evaluation.
Considering these promising antimalarial activities of various THβC derivatives, we designed and synthesized a new series of compounds with aromatic and/or aliphatic side chains in the 1-position and an amide bond in the 3-position of THβC ( Figure 2). The known THβC derivatives active against the drug-resistant P. falciparum contain mostly bulky aromatic or heteroaromatic substituents (such as piridyl, piperonyl, thiophene, or di-chlorophenyl moieties) at the C1 position and small methyl esters at the C3 position. Consequently, we decided to change the physicochemical properties of the substituents at the C1 and C3 positions, namely, from bulky to small at the C1 position and from small to bulky at the C3 position. The target compounds in the further biological investigations Similarly, compound MMV008138 ( Figure 1) exhibits potent antimalarial activity and inhibits the growth of the P. falciparum Dd2 strain with an IC 50 of 250 nM [10]. Gorki et al. explored the antimalarial activity of a β-carboline derivative, compound 9a (Figure 1), against P. falciparum. Compound 9a inhibited both the 3D7 and RKL-9 strains of P. falciparum with an IC 50 < 2.86 µM, respectively, was nontoxic to normal dermal fibroblasts, and its selectivity index was >10 against both strains [11]. Further, Gellis et al. reported on a series of 1-phenyl-substituted-β-carboline derivatives with significant antimalarial activity (0.7 < IC 50 < 1.7 mM) against the W2 multidrug-resistant strain of P. falciparum [12].
Considering these promising antimalarial activities of various THβC derivatives, we designed and synthesized a new series of compounds with aromatic and/or aliphatic side chains in the 1-position and an amide bond in the 3-position of THβC ( Figure 2). The known THβC derivatives active against the drug-resistant P. falciparum contain mostly bulky aromatic or heteroaromatic substituents (such as piridyl, piperonyl, thiophene, or di-chlorophenyl moieties) at the C1 position and small methyl esters at the C3 position. Consequently, we decided to change the physicochemical properties of the substituents at the C1 and C3 positions, namely, from bulky to small at the C1 position and from small to bulky at the C3 position. The target compounds in the further biological investigations that were conducted, were used as undetermined mixtures of enantiomers and as pure diastereomers or as a mixture of diastereomers. that were conducted, were used as undetermined mixtures of enantiomers and as pure diastereomers or as a mixture of diastereomers. The synthesized compounds were evaluated for in vitro activity against D10 (CQsensitive) and W2 (CQ-resistant) strains of P. falciparum. Subsequently, the cytotoxic activity on human microvascular endothelial cells and hemolytic effects on human erythrocytes were also investigated, to obtain more information concerning their safety against mammalian cells. Moreover, compounds 2-14 were also characterized by their physicochemical parameters (Log D). Next, we focused on the probable mechanism of action of the most active compound. Thus, molecular modeling studies were performed to assess the potential binding modes of one enantiomer of compound 7 (that is (1S,3R)-7) with a series of different enzymes essential for parasite metabolism. Phosphoethanolamine methyltransferase (PMT) plays a critical function in parasite development and differentiation but is absent in mammals. PMT catalyzes the synthesis of phosphatidylcholine-the major phospholipid constituent of the membranes of parasites during the sexual and asexual stages of Plasmodium [13]. Lactate dehydrogenase (LDH) is synthesized by parasites in the blood-stage of malaria as the terminal enzyme in the glycolytic pathway of Plasmodium [14]. Cytosolic malate dehydrogenase (MDH) converts malate to oxaloacetate and, as a result, generates NADH or NADPH, two reducing equivalents to the respiratory chain of Plasmodium [15]. Falcipain-2 (FP2) and falcipain-3 (FP3) are critical hemoglobinases of P. falciparum, which catalyze the degradation of hemoglobin into hemozoin [16].
For this, five different enzymes of P. falciparum were selected for docking studies with the view of investigating a possible multitargeting mode of action. Such a strategy would constitute a promising solution to P. falciparum drug resistance. To our knowledge, this is the first study describing the syntheses of this type of tetrahydro-β-carboline series as well as their antiplasmodial activities and the in silico approach used to study their probable mechanisms of action on this parasite.

Chemistry
The synthesis of the designed THβC derivatives (2-14) commenced with the Pictet-Spengler reaction of racemic tryptophan and an appropriate aldehyde in the presence of sulfuric acid (H2SO4) in water ( Figure 3) [17]. The resultant 1-substituted-tetrahydro-βcarboline-3-carboxylic acids (1a-e) were further reacted with diverse amines in the pres- The synthesized compounds were evaluated for in vitro activity against D10 (CQsensitive) and W2 (CQ-resistant) strains of P. falciparum. Subsequently, the cytotoxic activity on human microvascular endothelial cells and hemolytic effects on human erythrocytes were also investigated, to obtain more information concerning their safety against mammalian cells. Moreover, compounds 2-14 were also characterized by their physicochemical parameters (Log D). Next, we focused on the probable mechanism of action of the most active compound. Thus, molecular modeling studies were performed to assess the potential binding modes of one enantiomer of compound 7 (that is (1S,3R)-7) with a series of different enzymes essential for parasite metabolism. Phosphoethanolamine methyltransferase (PMT) plays a critical function in parasite development and differentiation but is absent in mammals. PMT catalyzes the synthesis of phosphatidylcholine-the major phospholipid constituent of the membranes of parasites during the sexual and asexual stages of Plasmodium [13]. Lactate dehydrogenase (LDH) is synthesized by parasites in the blood-stage of malaria as the terminal enzyme in the glycolytic pathway of Plasmodium [14]. Cytosolic malate dehydrogenase (MDH) converts malate to oxaloacetate and, as a result, generates NADH or NADPH, two reducing equivalents to the respiratory chain of Plasmodium [15]. Falcipain-2 (FP2) and falcipain-3 (FP3) are critical hemoglobinases of P. falciparum, which catalyze the degradation of hemoglobin into hemozoin [16].
For this, five different enzymes of P. falciparum were selected for docking studies with the view of investigating a possible multitargeting mode of action. Such a strategy would constitute a promising solution to P. falciparum drug resistance. To our knowledge, this is the first study describing the syntheses of this type of tetrahydro-β-carboline series as well as their antiplasmodial activities and the in silico approach used to study their probable mechanisms of action on this parasite.

Log D Calculation
Compounds 2-14 are partially ionized; thus, the distribution coefficients D (Log D) were calculated at fixed pH. The chosen pH (7.4, 7.2, and 5.5) represent the physiological conditions found in the blood, human erythrocyte cytoplasm, and P. falciparum food vacuoles. The data in Table 1 show that, for Log D at pH 7.4, the compounds fall in the range 1.73-5.65, and in the range of 1.59-5.53 at pH 7.2, whereas at pH 5.5, the compounds were in the range of 0.05-3.93. Only compound 3 displayed a gradient of Log D similar to CQ, however, the values of Log D for compound 3 were almost twice as large as those for CQ itself.  The applied synthetic methods facilitated the synthesis of intermediates (1a-e), and compounds (2-14), with yields of 15-74% and the purity of the final compounds above 95%.

Log D Calculation
Compounds 2-14 are partially ionized; thus, the distribution coefficients D (Log D) were calculated at fixed pH. The chosen pH (7.4, 7.2, and 5.5) represent the physiological conditions found in the blood, human erythrocyte cytoplasm, and P. falciparum food vacuoles. The data in Table 1 show that, for Log D at pH 7.4, the compounds fall in the range 1.73-5.65, and in the range of 1.59-5.53 at pH 7.2, whereas at pH 5.5, the compounds were in the range of 0.05-3.93. Only compound 3 displayed a gradient of Log D similar to CQ, however, the values of Log D for compound 3 were almost twice as large as those for CQ itself. ence of 4-dimethylaminopyridine (DMAP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 1-hydroxybenzotriazole (HOBt), and pyridine. The final compounds were purified by crystallization from hexane or by column chromatography. The structures and purity of the newly synthesized compounds were characterized by 1 H NMR, 13 C NMR spectroscopy, and UPLC/MS spectrometry. Compounds were isolated as an undetermined mixture of enantiomers (3RS-2, 1RS,3SR for 8-11 and 14), pure diastereomers (1RS,3RS-13, 1RS,3SR-13) or as a mixture of diastereomers (1, 3-7, 12) with various diastereoselectivity (ds) (Section Materials and Methods, Section 3.1). The applied synthetic methods facilitated the synthesis of intermediates (1a-e), and compounds (2-14), with yields of 15-74% and the purity of the final compounds above 95%.

Log D Calculation
Compounds 2-14 are partially ionized; thus, the distribution coefficients D (Log D) were calculated at fixed pH. The chosen pH (7.4, 7.2, and 5.5) represent the physiological conditions found in the blood, human erythrocyte cytoplasm, and P. falciparum food vacuoles. The data in Table 1 show that, for Log D at pH 7.4, the compounds fall in the range 1.73-5.65, and in the range of 1.59-5.53 at pH 7.2, whereas at pH 5.5, the compounds were in the range of 0.05-3.93. Only compound 3 displayed a gradient of Log D similar to CQ, however, the values of Log D for compound 3 were almost twice as large as those for CQ itself. ence of 4-dimethylaminopyridine (DMAP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 1-hydroxybenzotriazole (HOBt), and pyridine. The final compounds were purified by crystallization from hexane or by column chromatography. The structures and purity of the newly synthesized compounds were characterized by 1 H NMR, 13 C NMR spectroscopy, and UPLC/MS spectrometry. Compounds were isolated as an undetermined mixture of enantiomers (3RS-2, 1RS,3SR for 8-11 and 14), pure diastereomers (1RS,3RS-13, 1RS,3SR-13) or as a mixture of diastereomers (1, 3-7, 12) with various diastereoselectivity (ds) (Section Materials and Methods, Section 3.1). The applied synthetic methods facilitated the synthesis of intermediates (1a-e), and compounds (2-14), with yields of 15-74% and the purity of the final compounds above 95%.

Log D Calculation
Compounds 2-14 are partially ionized; thus, the distribution coefficients D (Log D) were calculated at fixed pH. The chosen pH (7.4, 7.2, and 5.5) represent the physiological conditions found in the blood, human erythrocyte cytoplasm, and P. falciparum food vacuoles. The data in Table 1 show that, for Log D at pH 7.4, the compounds fall in the range 1.73-5.65, and in the range of 1.59-5.53 at pH 7.2, whereas at pH 5.5, the compounds were in the range of 0.05-3.93. Only compound 3 displayed a gradient of Log D similar to CQ, however, the values of Log D for compound 3 were almost twice as large as those for CQ itself.  The applied synthetic methods facilitated the synthesis of intermediates (1a-e), and compounds (2-14), with yields of 15-74% and the purity of the final compounds above 95%.

Log D Calculation
Compounds 2-14 are partially ionized; thus, the distribution coefficients D (Log D) were calculated at fixed pH. The chosen pH (7.4, 7.2, and 5.5) represent the physiological conditions found in the blood, human erythrocyte cytoplasm, and P. falciparum food vacuoles. The data in Table 1 show that, for Log D at pH 7.4, the compounds fall in the range 1.73-5.65, and in the range of 1.59-5.53 at pH 7.2, whereas at pH 5.5, the compounds were in the range of 0.05-3.93. Only compound 3 displayed a gradient of Log D similar to CQ, however, the values of Log D for compound 3 were almost twice as large as those for CQ itself.  The applied synthetic methods facilitated the synthesis of intermediates (1a-e), and compounds (2-14), with yields of 15-74% and the purity of the final compounds above 95%.

Log D Calculation
Compounds 2-14 are partially ionized; thus, the distribution coefficients D (Log D) were calculated at fixed pH. The chosen pH (7.4, 7.2, and 5.5) represent the physiological conditions found in the blood, human erythrocyte cytoplasm, and P. falciparum food vacuoles. The data in Table 1 show that, for Log D at pH 7.4, the compounds fall in the range 1.73-5.65, and in the range of 1.59-5.53 at pH 7.2, whereas at pH 5.5, the compounds were in the range of 0.05-3.93. Only compound 3 displayed a gradient of Log D similar to CQ, however, the values of Log D for compound 3 were almost twice as large as those for CQ itself.

Antiplasmodial Activities
Next, the screening of antiplasmodial activities in in vitro parasite cultures was performed using standard procedures. For this purpose, we tested the susceptibility of two strains of P. falciparum differing in resistance to CQ, namely D10 (chloroquine-sensitive) and W2 (chloroquine-resistant). The inhibitory effect on the growth of these strains, expressed as IC50 values, is presented in Table 2. The synthesized derivatives have IC50 values in the range of 4.00 ± 0.53-35.36 ± 4.86 µ M. The highest antiplasmodial activity among the tested series was shown by compound 7, which is also characterized by one of the highest Log D values in the series of compounds. Moreover, many compounds have a similar magnitude of inhibitory activity against the two P. falciparum strains, as reported for other tetrahydro-β-carboline derivatives by Eagon et al. [18]. The important fact is also that, except for compound 6, the determined values of IC50 are lower for the CQ-resistant

Antiplasmodial Activities
Next, the screening of antiplasmodial activities in in vitro parasite cultures was performed using standard procedures. For this purpose, we tested the susceptibility of two strains of P. falciparum differing in resistance to CQ, namely D10 (chloroquine-sensitive) and W2 (chloroquine-resistant). The inhibitory effect on the growth of these strains, expressed as IC50 values, is presented in Table 2. The synthesized derivatives have IC50 values in the range of 4.00 ± 0.53-35.36 ± 4.86 µ M. The highest antiplasmodial activity among the tested series was shown by compound 7, which is also characterized by one of the highest Log D values in the series of compounds. Moreover, many compounds have a similar magnitude of inhibitory activity against the two P. falciparum strains, as reported for other tetrahydro-β-carboline derivatives by Eagon et al. [18]. The important fact is also that, except for compound 6, the determined values of IC50 are lower for the CQ-resistant 4

Antiplasmodial Activities
Next, the screening of antiplasmodial activities in in vitro parasite cultures was performed using standard procedures. For this purpose, we tested the susceptibility of two strains of P. falciparum differing in resistance to CQ, namely D10 (chloroquine-sensitive) and W2 (chloroquine-resistant). The inhibitory effect on the growth of these strains, expressed as IC50 values, is presented in Table 2. The synthesized derivatives have IC50 values in the range of 4.00 ± 0.53-35.36 ± 4.86 µ M. The highest antiplasmodial activity among the tested series was shown by compound 7, which is also characterized by one of the highest Log D values in the series of compounds. Moreover, many compounds have a similar magnitude of inhibitory activity against the two P. falciparum strains, as reported for other tetrahydro-β-carboline derivatives by Eagon et al. [18]. The important fact is also that, except for compound 6, the determined values of IC50 are lower for the CQ-resistant 3

Antiplasmodial Activities
Next, the screening of antiplasmodial activities in in vitro parasite cultures was performed using standard procedures. For this purpose, we tested the susceptibility of two strains of P. falciparum differing in resistance to CQ, namely D10 (chloroquine-sensitive) and W2 (chloroquine-resistant). The inhibitory effect on the growth of these strains, expressed as IC50 values, is presented in Table 2. The synthesized derivatives have IC50 values in the range of 4.00 ± 0.53-35.36 ± 4.86 µ M. The highest antiplasmodial activity among the tested series was shown by compound 7, which is also characterized by one of the highest Log D values in the series of compounds. Moreover, many compounds have a similar magnitude of inhibitory activity against the two P. falciparum strains, as reported for other tetrahydro-β-carboline derivatives by Eagon et al. [18]. The important fact is also that, except for compound 6, the determined values of IC50 are lower for the CQ-resistant 4

Antiplasmodial Activities
Next, the screening of antiplasmodial activities in in vitro parasite cultures was performed using standard procedures. For this purpose, we tested the susceptibility of two strains of P. falciparum differing in resistance to CQ, namely D10 (chloroquine-sensitive) and W2 (chloroquine-resistant). The inhibitory effect on the growth of these strains, expressed as IC50 values, is presented in Table 2. The synthesized derivatives have IC50 values in the range of 4.00 ± 0.53-35.36 ± 4.86 µ M. The highest antiplasmodial activity among the tested series was shown by compound 7, which is also characterized by one of the highest Log D values in the series of compounds. Moreover, many compounds have a similar magnitude of inhibitory activity against the two P. falciparum strains, as reported for other tetrahydro-β-carboline derivatives by Eagon et al. [18]. The important fact is also that, except for compound 6, the determined values of IC50 are lower for the CQ-resistant 5

Antiplasmodial Activities
Next, the screening of antiplasmodial activities in in vitro parasite cultures was performed using standard procedures. For this purpose, we tested the susceptibility of two strains of P. falciparum differing in resistance to CQ, namely D10 (chloroquine-sensitive) and W2 (chloroquine-resistant). The inhibitory effect on the growth of these strains, expressed as IC50 values, is presented in Table 2. The synthesized derivatives have IC50 values in the range of 4.00 ± 0.53-35.36 ± 4.86 µ M. The highest antiplasmodial activity among the tested series was shown by compound 7, which is also characterized by one of the highest Log D values in the series of compounds. Moreover, many compounds have a similar magnitude of inhibitory activity against the two P. falciparum strains, as reported for other tetrahydro-β-carboline derivatives by Eagon et al. [18]. The important fact is also that, except for compound 6, the determined values of IC50 are lower for the CQ-resistant 5

Antiplasmodial Activities
Next, the screening of antiplasmodial activities in in vitro parasite cultures was performed using standard procedures. For this purpose, we tested the susceptibility of two strains of P. falciparum differing in resistance to CQ, namely D10 (chloroquine-sensitive) and W2 (chloroquine-resistant). The inhibitory effect on the growth of these strains, expressed as IC50 values, is presented in Table 2. The synthesized derivatives have IC50 values in the range of 4.00 ± 0.53-35.36 ± 4.86 µ M. The highest antiplasmodial activity among the tested series was shown by compound 7, which is also characterized by one of the highest Log D values in the series of compounds. Moreover, many compounds have a similar magnitude of inhibitory activity against the two P. falciparum strains, as reported for other tetrahydro-β-carboline derivatives by Eagon et al. [18]. The important fact is also that, except for compound 6, the determined values of IC50 are lower for the CQ-resistant 4

Antiplasmodial Activities
Next, the screening of antiplasmodial activities in in vitro parasite cultures was performed using standard procedures. For this purpose, we tested the susceptibility of two strains of P. falciparum differing in resistance to CQ, namely D10 (chloroquine-sensitive) and W2 (chloroquine-resistant). The inhibitory effect on the growth of these strains, expressed as IC50 values, is presented in Table 2. The synthesized derivatives have IC50 values in the range of 4.00 ± 0.53-35.36 ± 4.86 µ M. The highest antiplasmodial activity among the tested series was shown by compound 7, which is also characterized by one of the highest Log D values in the series of compounds. Moreover, many compounds have a similar magnitude of inhibitory activity against the two P. falciparum strains, as reported for other tetrahydro-β-carboline derivatives by Eagon et al. [18]. The important fact is also that, except for compound 6, the determined values of IC50 are lower for the CQ-resistant 0

Antiplasmodial Activities
Next, the screening of antiplasmodial activities in in vitro parasite cultures was performed using standard procedures. For this purpose, we tested the susceptibility of two strains of P. falciparum differing in resistance to CQ, namely D10 (chloroquine-sensitive) and W2 (chloroquine-resistant). The inhibitory effect on the growth of these strains, expressed as IC50 values, is presented in Table 2. The synthesized derivatives have IC50 values in the range of 4.00 ± 0.53-35.36 ± 4.86 µ M. The highest antiplasmodial activity among the tested series was shown by compound 7, which is also characterized by one of the highest Log D values in the series of compounds. Moreover, many compounds have a similar magnitude of inhibitory activity against the two P. falciparum strains, as reported for other tetrahydro-β-carboline derivatives by Eagon et al. [18]. The important fact is also that, except for compound 6, the determined values of IC50 are lower for the CQ-resistant 5.58 5.53 4.31

Antiplasmodial Activities
Next, the screening of antiplasmodial activities in in vitro parasite cultures was performed using standard procedures. For this purpose, we tested the susceptibility of two strains of P. falciparum differing in resistance to CQ, namely D10 (chloroquine-sensitive) and W2 (chloroquine-resistant). The inhibitory effect on the growth of these strains, expressed as IC 50 values, is presented in Table 2. The synthesized derivatives have IC 50 values in the range of 4.00 ± 0.53-35.36 ± 4.86 µM. The highest antiplasmodial activity among the tested series was shown by compound 7, which is also characterized by one of the highest Log D values in the series of compounds. Moreover, many compounds have a similar magnitude of inhibitory activity against the two P. falciparum strains, as reported for other tetrahydro-β-carboline derivatives by Eagon et al. [18]. The important fact is also that, except for compound 6, the determined values of IC 50 are lower for the CQ-resistant strain in comparison to the CQ-sensitive strain, reflected in their RI (resistance index) values. This could suggest superior sensitivity of the designed compounds towards the CQ-resistant parasite strain. The obtained results are very encouraging, especially since malaria control is threatened by the emergence of drug resistance to the artemisinin derivatives [19]. The data also indicate a different relationship between chemical structure and antiplasmodial activity that is not directly related to Log D values and the types of chemical substituents.

Biocompatibility Studies
Results from the preliminary antiplasmodial activity screening highlighted the necessity for the evaluation of the safety of these agents to human cells. Hence, we chose two model cell systems: the human microvascular endothelial (HMEC-1) cell line and human erythrocytes, to obtain greater insights into their cell toxicity potential. In these studies, we focused only on derivatives that had exhibited the highest antiplasmodial activities, namely 3, 6, 7, 8, 9, 12, 1RS,3RS-13, and 1RS,3SR-13. The results concerning their cytotoxic effects on HMEC-1 cells as well as their calculated selectivity index (SI), namely the ratio between the IC 50 values on the endothelial cells and that on the Plasmodium strains, are summarized in Table 3. Based on the analysis of the obtained results, it can be concluded that the determined IC 50 values fall within a fairly wide range from 17.95 ± 9.46 to 157.18 ± 42.50 µM, even though they all have their IC 50 in the range of low micromolar concentrations against both strains of Plasmodium. A similar phenomenon has already been described for methoxy-thiazinoquinones by Imperatore et al. [20]. Interestingly, the most active compound against the parasite (7) is also safe to HMEC-1 cells and is highly selective, also exhibiting the highest SI value of the whole compound series. Table 3. Cytotoxicity of compounds 3, 6, 7, 8, 9, 12, 1RS,3RS-13, 1RS,3SR-13, and CQ on the HMEC-1 cell line and relevant selectivity indexes (SI). We also sought to investigate the hemolytic potential of the synthetized compounds. For this experiment, we selected three compounds, namely 3, 7, and 12, which exhibited the highest selectivity. We incubated these compounds at a concentration of 10 µM, with human erythrocytes, and then, by measuring the released hemoglobin, we assessed their potential harmful effects on these cells. The measured level of hemolysis was less than 5% for all agents, which proves that the compounds are not hemolytic. It is worth emphasizing at this point that this type of test is extremely important to confirm that the observed inhibitory activity against Plasmodium is a result of a compound acting directly upon it and not as a result of red blood cell lysis. Taking into account the fact that the tested concentration of 7 in this test was, respectively, 2.2 and 2.5 times higher than the IC 50 determined for the Plasmodium strains, we can conclude that the observed effect on the parasite is not due to activity on erythrocytes. In conclusion, compound 7 is, therefore, biocompatible, since no hemolytic effects were detected in the mammalian cells tested.
In the complex of compound (1S,3R)-7 and PMT that was obtained by IFD simulations ( Figure 4A), a π-cation interaction between Tyr27 of PMT and the protonated amine group of tetrahydropyridine, as well as hydrogen-bonding between the side chain of Lys247 of PMT and the oxygen in the amide moiety of compound (1S,3R)-7 were observed. Moreover, both mentioned amino acid residues participated in forming interactions with phosphocholine in the PMT crystal. In addition, a π-π interaction was observed between Tyr19 of PMT and the aromatic benzene ring from the β-carboline moiety. This additional interaction was not observed in the PMT crystal structure.
The obtained complex of compound (1S,3R)-7 and falcipain-2 ( Figure 4B) formed H-bonds between the main chain of Gly83 in PMT and the protonated amine group of tetrahydropyridine and the oxygen in the amide moiety of compound (1S,3R)-7. A hydrogen-bond was also observed between the main chain of Asn173 of PMT and the hydrogen on the amide grouping in compound (1S,3R)-7. All the amino acid residues discussed previously formed interactions with epoxysuccinate in the FP2 crystal. However, the H-bond between the side chain of Asp234 and the amine group of the center ring of β-carboline was not observed in the FP3 crystal structure.
The complex of FP3 ( Figure 4C) and compound (1S,3R)-7 formed H-bonds between the main chain of Asn182 and the protonated amine group of tetrahydropyridine, as well as with hydrogen from the amide of compound (1S,3R)-7. In addition, a hydrogen bond between the main chain of Gly92 of FP3 and the amide's oxygen of compound (1S,3R)-7 was observed. It is noteworthy that all the mentioned amino acid residues participated in interactions with leupeptin in the FP3 crystal. The obtained complex of compound (1S,3R)-7 and falcipain-2 ( Figure 4B) formed Hbonds between the main chain of Gly83 in PMT and the protonated amine group of tetrahydropyridine and the oxygen in the amide moiety of compound (1S,3R)-7. A hydrogenbond was also observed between the main chain of Asn173 of PMT and the hydrogen on the amide grouping in compound (1S,3R)-7. All the amino acid residues discussed previously formed interactions with epoxysuccinate in the FP2 crystal. However, the H-bond between the side chain of Asp234 and the amine group of the center ring of β-carboline was not observed in the FP3 crystal structure.
The complex of FP3 ( Figure 4C) and compound (1S,3R)-7 formed H-bonds between the main chain of Asn182 and the protonated amine group of tetrahydropyridine, as well as with hydrogen from the amide of compound (1S,3R)-7. In addition, a hydrogen bond between the main chain of Gly92 of FP3 and the amide's oxygen of compound (1S,3R)-7 was observed. It is noteworthy that all the mentioned amino acid residues participated in interactions with leupeptin in the FP3 crystal.
An ionic bond (salt bridge) was observed between the side chain of Asp53 in LDH and the protonated amine group of tetrahydropyridine of compound (1S,3R)-7, as well as a hydrogen bond between the side chain of Asp53 and the hydrogen of the amide group of compounds (1S,3R)-7, and an additional H-bond identified between the main chain of Gly99 and the oxygen of the amide group of compounds (1S,3R)-7 ( Figure 4D). All the mentioned amino acid residues participated in forming interactions with NADH in the LDH crystal.
Compound (1S,3R)-7 ( Figure 4E) formed a hydrogen bond between the main chain of MDH's Gly78 and the protonated amine group of tetrahydropyridine, along with an H-bond between the main chain of Thr76 and the protonated amine group of tetrahydropyridine and the hydrogen of the amide moiety of compound (1S,3R)-7. A further H-bond exists between the main chain of Gln11 and the oxygen on the amide moiety of compound An ionic bond (salt bridge) was observed between the side chain of Asp53 in LDH and the protonated amine group of tetrahydropyridine of compound (1S,3R)-7, as well as a hydrogen bond between the side chain of Asp53 and the hydrogen of the amide group of compounds (1S,3R)-7, and an additional H-bond identified between the main chain of Gly99 and the oxygen of the amide group of compounds (1S,3R)-7 ( Figure 4D). All the mentioned amino acid residues participated in forming interactions with NADH in the LDH crystal.
Compound (1S,3R)-7 ( Figure 4E) formed a hydrogen bond between the main chain of MDH's Gly78 and the protonated amine group of tetrahydropyridine, along with an H-bond between the main chain of Thr76 and the protonated amine group of tetrahydropyridine and the hydrogen of the amide moiety of compound (1S,3R)-7. A further H-bond exists between the main chain of Gln11 and the oxygen on the amide moiety of compound (1S,3R)-7, and an H-bond between the side chain of Asp32 and the amine group of the center ring of β-carboline of compound (1S,3R)-7. Among the mentioned amino-acid interactions, only Thr76 was not involved in forming interactions with NAD in the 6R8G crystal.
Results from IFD revealed that each H-bond donor/acceptor fragment present in compound (1S,3R)-7, such as the amine group of the center ring of β-carboline, the protonated amine group on tetrahydropyridine, and the oxygen, nitrogen, and hydrogen atoms of the amide moiety of compound (1S,3R)-7, were involved in forming an interaction with at least one amino-acid target in the group of enzymes investigated. Moreover, the aliphatic chains of compound (1S,3R)-7 additionally stabilized the position of the entire molecule within the enzymes. Observations from in silico simulations highlight that compound (1S,3R)-7 has structural features enabling it to interact with five described molecular targets within sites occupied originally by other ligands (products/inhibitors/cofactors). The results are a good starting point for further in vitro evaluation of compound (1S,3R)-7.
Medium and supplements for P. falciparum culture and cell culture (RPMI 1640 Medium, Glutamine, Hepes Buffer, Fetal calf serum) were from EuroClone (Milan, Italy). AlbuMax and MCDD 131 medium were from Invitrogen (Milan, Italy). Unless stated otherwise, all reagents were from Sigma Italia (Milan, Italy).

Synthetic Procedures
3.2.1. General Method for Synthesis of the 1-Substituted-tetrahydro-β-carboline-3-carboxylic Acids (1a-e) According to the literature, a mixture of 0.5 N H 2 SO 4 (2.5 mL) and H 2 O (200 mL), D,L-tryptophan (2.04 g, 10 mmol), and appropriate aldehydes (30 mmol) was stirred at room temperature overnight and the synthesis products detected by TLC. The precipitate was filtered and washed well with H 2 O and dried in a vacuum. The material was used without further purification for the following steps.