In vitro and in silico assessment of new beta amino ketones with antiplasmodial activity

ABSTRACT Background: Based on the current need for new drugs against malaria, our study evaluated eight beta amino ketones in silico and in vitro for potential antimalarial activity. Methods: Using the Brazilian Malaria Molecular Targets (BraMMT) and OCTOPUS® software programs, the pattern of interactions of beta-amino ketones was described against different proteins of P. falciparum and screened to evaluate their physicochemical properties. The in vitro antiplasmodial activities of the compounds were evaluated using a SYBR Green-based assay. In parallel, in vitro cytotoxic data were obtained using the MTT assay. Results: Among the eight compounds, compound 1 was the most active and selective against P. falciparum (IC50 = 0.98 µM; SI > 60). Six targets were identified in BraMMT that interact with compounds exhibiting a stronger binding energy than the crystallographic ligand: P. falciparum triophosphate phosphoglycolate complex (1LYX), P. falciparum reductase (2OK8), PfPK7 (2PML), P. falciparum glutaredoxin (4N0Z), PfATP6, and PfHT. Conclusions: The physicochemical properties of compound 1 were compatible with the set of criteria established by the Lipinski rule and demonstrated its potential as a drug prototype for antiplasmodial activity.


INTRODUCTION
Malaria continues to be one of the most important public health problems, with an estimated > 400,000 deaths each year 1,2 . In humans, the disease is caused by the protozoan species of the Plasmodium genus 3 .
Although several substances are used in antimalarial chemotherapy, many of them are no longer used for treatment because of their side effects or the development of parasitic resistance [4][5][6][7][8][9][10][11] . Therefore, new strategies should be used, such as the addition of a third drug with independent antiparasitic activity 10 .
Computational tools have been employed to understand complex interactions in biological models 12 .
The main objective of the computational model is to replicate the patterns of biological systems 13 with a high accuracy. In silico Cellular viability was determined at 540 nm to measure the signal and background (Spectra Max340PC, Molecular Devices, Sunnyvale, California, USA). The minimum lethal dose for 50% of the cells (LD 50 ) was determined as previously described 41 .

Selectivity index (SI)
Selectivity index (SI) is the ratio between the values of LD 50 and IC 50 cytotoxicity of each compound tested. Values greater than 10 were considered low cytotoxic, whereas values below 10 were considered cytotoxic 42 .

Statistical analysis
IC 50 and LD 50 were determined using the equation of the curve obtained by plotting parasitemia reduction (%) or cellular death (%) vs. the concentration of the compound (log scale) using the GraphPad Prism software (version 5.0 for Windows, San Diego, California, USA). Due to the non-normality of the data distribution, the comparison of IC 50 and LD 50 between morpholine and piperidine was analyzed using the nonparametric Mann-Whitney U test. Statistical significance was defined at 5% (p<0.05).

Evaluation of virtual screening and ADMET properties
Description is available in the supplementary material.

Antiplasmodial activity
The antiplasmodial activities of these eight compounds are listed in Table 1. IC 50 values in the in vitro antimalarial tests (W2 strain) ranged from 0.98 μM to 47.95 μM. Compounds 1 and 4 had IC 50 values close to those of chloroquine, a standard antimalarial drug. There was no statistically significant difference in IC 50 or LD 50 between parasites treated with morpholine and piperidine (p = 0.225 and p = 0.593, respectively).

Cytotoxic activity
The compounds did not show cytotoxic activity, since the LD 50 values ranged from 33.97 μM to >100 μM compared to the control drug (chloroquine) ( Table 1). Compounds 2, 3, and 8 were not selective, with SI values below 10 (9.46, 3.07, and 0.81, respectively). Compounds 1, 4, 5, 6, and 7 showed the highest selectivity (SI>10), with values ranging from 20.44 to 128.21 ( Table 1). Therefore, the docking assay and physicochemical properties of this compound were analyzed. Table 2 presents the molecular targets, locations, and enzymatic classes of the 35 proteins listed in the Brazilian Malaria Molecular Targets (BraMMT). Virtual screening of the compounds was performed against all 35 BraMMT. Table 3, six of the 35 targets that make up BraMMT interacted with compounds 1-8, presenting binding energies superior to the crystallographic data. The six targets that were linked to the compounds were: P. falciparum triosphosphate phosphoglycolate complex (1LYX), ferredoxin-NADP + P. falciparum reductase (2OK8), crystal structure of PfPK7 (2PML), oxireduction protein of P. falciparum glutaredoxin (4N0Z), P. falciparum ATPase orthologous calcium pump (PfATP6), and P. falciparum hexose transporter (PfHT). Compound 4 stood out on presenting the most satisfactory result, establishing a connection with the six targets, and presenting a higher binding value than the crystallographic data.
Furthermore, compound 1 stood out in the antimalarial and cytotoxicity tests and was therefore chosen for intermolecular and physicochemical analyses. Figure 2 shows a two-dimensional map of linker-receptor interactions with PfHT and the chemical bonds between compound 1 and the target. Pharmacophoric groups and possible structural improvements in permeability, absorption, and oral bioavailability have been indicated. By analyzing the molecular interactions of 1 with PfHT, it was possible to observe the interaction of 1 with GLN169 and THR145 residues (Figure 2). Molecular anchoring with D-glucose was performed to recognize the interactions at the PfHT binding site.

Tests of physicochemical properties
Supplementary Table 1 shows SwissADME profiles of compound 1 and chloroquine. Compound 1 had a CLogP <5 (1.67) and a molecular weight less than 500 g/mol (219.28 g/mol) (Supplementary Table 1). There were three hydrogen acceptor groups that performed the interactions and no hydrogen donor groups.
Compounds with CLogP values less than 3 present a low risk of side effects and toxicity, which indicates a low risk of retention and storage of the compound. Patterns of mutagenicity, tumorigenicity, and irritability were not associated with the molecular structure of compound 1, corroborating the literature, since this compound had CLogP <3 (Supplementary Table 1).
In addition, compound 1 had a superior synthetic facility compared to chloroquine, standing out for its solubility in water.

DISCUSSION
The committee coordinated by the Global Health Innovative Technology (GHIT) foundation, guided by the specific requirements for the disease and considering the target product and candidate profiles, defined a set of criteria in an attempt to validate new antimalarial candidates. These criteria were divided into validation   and activity/mechanism of action, LD 50 values for cell lines, IC 50 of the parasite, and selectivity index. The compounds must be tested according to the study of the life cycle of Plasmodium to evaluate their mechanism of action, correlating the manifestations of malaria. The main tests must be performed according to the target to ensure the characterization of the potential target candidate 49 .
The in vitro cultivation of P. falciparum strains is the main test model for screening new compounds and monitoring resistance to antimalarial drugs, since it is possible to observe the activity, dosage, and resistance of natural and synthetic compounds through these tests 50 .
IC 50 values must be <1 μM to validate compounds or effective compounds for Plasmodium spp. The selectivity index must be greater than 10 (LD 50 for the cell line relative to the IC 50 of the parasite) for Plasmodium spp 49 . Thus, compound 1 meets two parameters with IC 50 < 0.98 µM and IS=61. Compounds 4, 5, 6, and 7 presented selectivity indices of 128, 20.44, 24, and 40.16, respectively, which are satisfactory; however, the IC 50 was higher than 1 μM.
In a study by Ohashi 52 , it was demonstrated that the potential activity of a given compound varies between the protozoan species, obtaining different SI values according to the species analyzed. These results demonstrate that it is essential to understand the mechanisms of action of the compounds and cell lines used in the cell viability tests, in addition to the activity of the analyzed substances. Compound 1 stood out for its IC 50 of 0.98, as well as an IS of 61. Thus, the physicochemical properties of this compound were analyzed.
Virtual screening of substances derived from the Mannich reaction was performed using 35 molecular targets obtained from BRAMMT. As shown in Table 3, 1LYX, 2OK8, 2PML, 4N0Z, PfATP6, and PfHT targets presented the best binding energy values for the compounds.
Using the BraMMT tool, the identification of a potential target is crucial in the discovery and rational development of new drugs to show their biological importance concurrently with the validation of the methodology to be used 53 . Each P. falciparum target present in the bank was used to perform virtual screening and was identified according to its registration in the Protein Data Bank.
Ferredoxin-NADP + reductase from P. falciparum (PDB code: 2OK8) is associated with apicoplast metabolic pathways, a key organelle for parasite survival. Thus, P. falciparum exhibits a ferredoxin-NADP + reductase, giving rise to reduced ferredoxin for the essential biosynthetic pathways of the apicoplast 54 . All tested substances (1-8) obtained binding energies stronger than the crystallographic ligand with the 2OK8 target.
P. falciparum glutaredoxin 1 (PDB 4N0Z) is an oxireduction protein present in the cytoplasm of the parasite that uses glutathione as a cofactor (glutaredoxin). This protein is exclusively found in Plasmodium species and plays a role in the central performance of maintaining parasite homeostasis 55 . Compounds 1, 2, 3, 4, 6, and 8q exhibited stronger binding energies with the 4N0Z target than with the crystallographic ligand.
P. falciparum ATPase orthologous calcium pump (PDB PfATP6) is present in the sarcoplasmic reticulum of P. falciparum. The cytoplasmic calcium concentration increases with inhibition and therefore compromises the parasite's signaling pathways 56 . Compound 4 obtained a higher binding energy value with the target than that of the crystallographic ligand, presenting a binding energy of -73 kcal.mol -1 compared to the value of -72 kcal.mol -1 of the crystallographic ligand, demonstrating an interaction with PfATP6.
The hexose transporter of P. falciparum (PDB PfHT) is a transmembrane protein responsible for glucose transport. Plasmodium requires glucose as its primary energy source during the erythrocytic cycle. Inhibition of glucose transport to infected cells impairs parasite viability. Therefore, substances that inhibit PfHT can be considered promising templates for developing new antimalarial compounds 57,58,59 .
The P. falciparum Triosphosphate Isomerase Phosphoglycolate complex (PDB 1LYX) was also identified as a P. falciparum Triosephosphate isomerase (PfTIM). This consists of a catalytically active enzyme, which is the subject of many studies because of its particularity at position 96. There is a serine residue in region 96 of triosphosphate isomerase in both humans and other organisms. However, the residue in PfTIM was replaced by a phenylalanine residue, with this enzyme being the only one to have serine in that position; however, so far, no specific role has been attributed to this particularity 60,61 .
Differences were found when comparing the PfTIM structure with other TIM structures, especially that of humans, which can assist in modeling and developing therapeutic targets. One of these differences and particularities of PfTIM is in position 183, which has a completely exposed leucine, different from TIMs of other organisms, which have glutamate. It is believed that this exposed leucine residue, together with the positively charged surrounding adhesive, may be responsible for TIM binding to the erythrocyte membrane, which is important for the energy production of Plasmodium 62 .
The crystalline structure of PfPK7 in complex with an ATP analog (PDB 2PML) is a protein kinase that plays an important role in regulating the development of the parasite, by being involved in the signaling pathway of melatonin and modulating the life cycle of P. falciparum 63,64 . PfPK7 is expressed in both the asexual and sexual stages of the parasite.
Dorin-Semblat et al. 65 demonstrated that interruption of the PfPK7 gene resulted in a reduction in the number of merozoites produced by each schizont, and the ability to produce oocysts in the mosquito was also impaired. These data reinforce the importance of PfPK7 in the parasite's life cycle 63 . Several molecular targets, from subcellular organelles to metabolic pathways, have been described in the literature in an attempt to fight infection 66,67 . Despite the numerous potential targets of pharmacological action, the most used compounds still date back many years: quinine, isolated from the Cinchona sp. in 1820 and artemisinin, purified from Artemisia annua in 1972, both extracted from natural sources 68 .
P. falciparum hexose transporter (PfHT) has recently been characterized as promising for the development of new drugs 59,60,69 . The reason for this is that the parasite is found inside erythrocytes surrounded by the parasitophore vacuole, and glucose molecules must pass through its membrane before being transported to the parasite 70,71 . Therefore, PfHT was characterized among BraMMT with similar or superior binding energy to the crystallographic ligand as a carrier target, which can contribute to the performance of the prototype compounds.
The hexose transporter for P. falciparum (PfHT) was recently described by molecular anchoring, QM/MM, and simulations of molecular dynamics by our research group 59,60 . Figure 2 shows a two-dimensional map of ligand-receptor interactions with PfHT. The chemical bonds between the compound and the target are indicated. The two-dimensional structure of these compounds is also shown. Molecular anchoring with D-Glucose was performed to recognize the interactions at the PfHT binding site.
Identifying and characterizing pharmacologically active molecules is a necessity that has led to efforts in diverse areas of scientific research worldwide. The applicability of such molecules can be quite variable; however, the main objective of the drug development process is to obtain molecules with efficiency and specificity in relation to the therapeutic target, low cost, and absence or reduction of risks to potential users 72 .
The increase in resistance to current antimalarial drugs drives research that aims to understand the mechanism of action of compounds for parasitic diseases with social and economic impacts. This knowledge will assist in screening bioactive compounds with determined pharmacodynamic and/or pharmacokinetic characteristics 73 . The methodologies used in this study have the main objective of characterizing potential targets, and these data and discussions allow us to infer the importance of the present study.
PfHT has characteristics similar to those of the human glucose transporter (GLUT1). However, there are some differences in their interactions with the substrates. PfHT can carry both D-glucose and D-fructose, whereas GLUT1 is selective for D-glucose and GLUT5 for D-fructose. Thus, PfHT can function as a pharmacological target in the development of antimalarials because of its selective affinity for different substrates 74,75 .
In addition, fructose can be used as an energy source in in vitro cultures of P. falciparum. Concentrations at this stage are lower than those of glucose 76 .
The blood phases of the parasite depend on glycolysis for energy production 77,78 . Therefore, PfHT is a potential therapeutic target in the exoerythrocytic phase 69 .
The Lipinski rule is one of the main descriptors used to analyze new prototype compounds for drugs. Thus, the physicochemical profiles of orally administered drugs are mainly based on the Lipinski rule, and it is possible to evaluate the absorption, distribution, metabolism, excretion, and toxicity of the compound, also known as the ADMET property 79,80 . The physicochemical characteristics of a compound can be decisive in the success or failure of its biological activity 81 . New antimalarial candidates should exhibit good oral bioavailability and membrane permeability 82 . The SwissADME tool allows determination of the main physicochemical and pharmacokinetic in silico properties of compounds 47 . Supplementary Table 1 shows SwissADME profiles of compound 1 and chloroquine.
The partition coefficient (CLogP) is recommended for estimating the toxicological factors 83 . It is known that compounds with high molecular weights and an excessive number of hydrogen acceptors and donor groups have higher difficulty crossing the lipid bilayer of cell membranes 84 .
According to Lipinski's rule, compound 1 had a CLogP <5 (1.67) and a molecular weight less than 500 g/mol (219.28 g/mol) (Supplementary Table 1). There were three hydrogen acceptor groups that interacted, and no hydrogen donor groups. These data demonstrated that the ADMET properties of compound 1 were acceptable under Lipinski's rule.
A study by Gleeson 83 demonstrated that compounds with LogP less than 4 and molecular weight less than 400 g/mol had a promising ADMET profile. Thus, compound 1 also fits the presented criteria and therefore presents the appropriate ADMET properties according to Lipinski and Gleeson 80,84 . Compounds with CLogP values less than 3 present a low risk of side effects and toxicity, indicating a low risk of the substance being trapped and stored. When analyzing the toxicological characteristics of compound 1, factors such as mutagenicity, tumogenicity, or irritability were not detected (Supplementary Table 1). In addition, compound 1 has a superior synthetic facility compared to chloroquine, standing out for its solubility in water.
In conclusion, compound 1 has potential as a drug prototype owing to the presentation of appropriate characteristics such as absorption, toxicity, good solubility, low molecular weight, and permeability. These data corroborate the growing interest in synthetic morpholines, as well as the usage diversity of these compounds, as described by Kourounakis 20 , since compound 1 stands out for its antiplasmodial and cytotoxic activity.

SYNTHESIS, PURIFICATION, AND STRUCTURAL ELUCIDATION
The compounds were synthesized according to the classical Mannich reaction using anhydrous ethanol as the solvent and morpholine and piperidine as secondary bases and respective ketones 35 .

3-(morpholin-4-yl)-1-phenylpropan-1-one:
First, 100 mL of anhydrous ethanol, 1.69 g of morpholine hydrochloride (13,66 mmol), 0.41 g paraformaldehyde (13.7 mmol), and 1.64 g acetophenone (13.66 mmol) were stirred under reflux in a sealed vial. The system was turned off after four hours and partitioned three times with water/ethyl acetate. The aqueous phase was collected and dried under vacuum. A small amount of solid residue was crystallized on acetone/ethanol to produce 2.1 g of compound 1 (9.6 mmol of hydrochloride salt) with a yield of 70%.

Compound 2 1-phenyl-3-(piperidin-1-yl)propan-1-one:
Second, 100 mL of anhydrous ethanol, 1.68 g of piperidine hydrochloride (13.82 mmol), 0.414 g paraformaldehyde (13.8 mmol), and 5.0 g of acetophenone (41.7 mmol) were stirred under reflux in a sealed vial. The system was turned off after three hours and partitioned three times with water/ethyl acetate. The aqueous phase was collected and dried under vacuum. The small solid residue was solubilized in acetone and crystalized overnight at a low temperature (-4 ºC). The crystals were filtered and recrystallized to obtain 3.0 g of compound 2 (11.8 mmol, hydrochloride salt) with a yield of 86%.

Compound 3 2-methyl-3-(morpholin-4-yl)-1-phenylpropan-1-one:
Third, 100 mL of anhydrous ethanol, 5.5 g of morpholine hydrochloride (44.5 mmol), 1.35 g paraformaldehyde (45 mmol), and 1.5 g propylphenone (11.2 mmol) were stirred under reflux in a sealed vial. The system was turned off after four hours and partitioned three times with water/ethyl acetate. The aqueous phase was collected and dried under vacuum. The small amount of solid residue was crystallized in ethanol at 0 °C to produce 2.0 g of compound 3 (8.6 mmol of hydrochloride salt) with a yield of 76%.

Compound 4 2-methyl-1-phenyl-3-(piperidein-1-yl)propan-1-one:
Fourth, 100 mL of anhydrous ethanol, 1.37 g of piperidine hydrochloride (11.3 mmol), 0.336 g paraformaldehyde (11.3 mmol), and 4.5 g of propylphenone (33.58 mmol) were stirred under reflux in a sealed vial. The system was turned off after three hours and partitioned three times with water/ethyl acetate. The aqueous phase was collected and dried under vacuum. The small solid residue was solubilized in acetone and crystallized overnight at a low temperature (0 ºC). The crystals were filtered and recrystallized to obtain 2.0 g of compound 4 (7.5 mmol, hydrochloride salt) with a yield of 66%.

Compounds 5 and 6 1-(morpholin-4-yl)decan-3-one and 3-[(morpholin-4-yl) methyl]nonan-2-one:
For compounds 5 and 6, 100 mL of anhydrous ethanol 5.5 g of morpholine hydrochloride (44.5 mmol), 1.35 g paraformaldehyde (45 mmol), and 2.46 g 2-nonanone (17 mmol) were stirred under reflux in a sealed vial. The system was turned off after four hours and partitioned three times with water/hexane. The aqueous phase was collected and dried under vacuum. The small brown solid was solubilized in acetone/methanol and crystallized at low temperature (0 ºC). The crystals were filtered and recrystallized to obtain a mixture of the two isomeric forms (kinetic and thermodynamic) of compounds 5 and 6 in proportions of 48-52 w/w. As a result, a total yield of 3.85 g (14.0 mmol mixture of hydrochloride salt) was obtained with 82% ketone yield.

PURIFICATION
The compounds were purified by preparative reverse phase high performance liquid chromatography (RP-HPLC) using a preparative Shim-pack C18 column and Shimadzu HPLC System and CH 3 CN-H 2 O/MeOH-H 2 O as solvents, normal phase (silica gel 60) using hexane/ethyl acetate as solvents, or selective crystallization by partial solubilization and decreasing solvent temperature. TLC using HF254 silica plates (Merck) and MeOH/ dichloromethane (DCM) as eluents and spots were visualized under visible light or in a UV chamber to monitor the reactions.

STRUCTURAL ELUCIDATION
The compounds were elucidated using high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) of 1H and 13C. HRMS was performed using a maXis ETD highresolution ESI-QTOF mass spectrometer (Bruker) controlled by the Compass1.5 software package (Bruker). Data-dependent fragment