SYNTHESIS, CHARACTERIZATION, AND IN VITRO ANTIMALARIAL ACTIVITY OF DIHYDROXYLATION DERIVATIVES OF TRICLOSAN

Objective: The emergence of malaria as a global health problem over the past few decades, accompanied by the rise of chemoresistant strains ofPlasmodium falciparum, has emphasized the need for the discovery of new therapeutic drugs against this disease. In this study, enantiomericallyenriched (enantioenriched) analogs of triclosan were synthesized and evaluated for antimalarial activity against P. falciparum cultures.Methods: Enantioselective dihydroxylation of the olefin in amide seven was performed efficiently using chiral quinine ligand (DHQ)2PHAL to yieldenantioenriched dihydroxy propionamide derivative (+)-1 in moderate yields. In a similar way, the chiral quinidine ligand (DHQD)2PHAL was used asstereoselectivity agent yielded the desired enantioenriched (−)-1. The enantioenriched products were used for further in vitro assay, and accordingly thepercent enantiomeric excess (% ee) was not determined. The structures of compounds were proven by spectral data (1H NMR, 13C NMR, and mass spectra).Results: The phenol moiety at the C1 position of triclosan was chemically substituted with a methoxy group, in conjunction with an introducedstereocenter in a 2,3-dihydroxy-propionamide group at C2’ position. Unmodified triclosan inhibited the P. falciparum cultures with an IC50 value of27.2 μM. By contrast, the triclosan analogs, compounds (+)-1 and (−)-1, inhibited the P. falciparum cultures with IC50 values of 0.034 and 0.028 μM,respectively.Conclusion: Collectively, our preliminary in vitro results suggest that these triclosan analogs have potent antimalarial activity and represent apromising new treatment strategy on further development.


INTRODUCTION
To date, malaria remains one of the most devastating diseases of tropical and subtropical countries and is caused by the protozoan parasites of the Plasmodium genus. Worldwide, malaria causes over 500 million new cases each year, with approximately 3 billion people living under the threat of malaria. The disease results in as many as 2.7 million deaths annually, with children mostly affected. In addition, malaria has a striking correlation with social and economic disruption on a grand scale [1][2][3][4][5].
Chemoresistance is of increasing concern, primarily for Plasmodium falciparum, the parasite responsible for cerebral malaria, which is the most serious type of malaria infection. Indeed, this chemoresistance is believed to be a major factor in the worldwide upsurge of malaria [6]; therefore, there is an urgent need for new and potent antimalarial treatments [7].
The trichlorinated biphenyl ether triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol ether] (Fig. 1) is an antimicrobial component sometimes added to consumer products such as toothpastes, mouthwashes, deodorant soaps, lotions, and children toys [8]. Nevertheless, FDA nowadays has a restriction on triclosan as the emerging resistance issues. Triclosan's effectiveness as an antimicrobial agent is believed to be due to its ability to inhibit the enzyme enoylacyl carrier protein reductase, which is involved in bacterial lipid biosynthesis [9][10][11].
In vitro studies confirm that triclosan is effective at killing P. falciparum and curing mice infected with the rodent malaria species Plasmodium berghei, as well as acute bacterial infection [12,13]. Appropriation as their potential efficacy against malaria parasites needs to be developed steadily to get the structure that is pharmacologically suitable for use in humans.

General methods
All starting materials were obtained from Sigma-Aldrich, Wako Pure, or Tokyo Chemical Industries and used as supplied. Solvents for chemical synthesis were acquired from commercial sources and used without further purification unless otherwise stated. Flash column chromatography was performed using Merck silica gel 60, whereas reactions and chromatography fractions were performed using Merck thin-layer chromatography (TLC) plates 60 F 254 . Compounds were visualized by an ultraviolet lamp (254 and 360 nm). Melting points (°C) were determined with a Yanaco micro melting point apparatus and remained uncorrected. Specific rotation, [α] D , was measured on a Jasco Digital Polarimeter. Mass analysis was performed with an electrospray mass JEOL JMS-AX 700 spectrometer, and gas chromatography was coupled with a mass spectrometer of high-resolution. 1 H and 13 C NMR spectral analyses were performed on a JEOL JNM-ECP500 (500 MHz), with tetramethylsilane as the internal standard. Chemical shifts are reported in units of (δ) ppm. The following abbreviations were used to explain the multiplicities: s, singlet; d, doublet; t, triplet; dd, double doublet; m, multiplet, and br, broad.

4-Chloro-1-fluoro-2-nitro-benzene (3)
A three-neck round-bottomed flask was charged with m-CPBA (3.16 g, 14.5 mmol) dissolved in 1,2-dichloroethane (13 mL). The mixture was refluxed until it reached the boiling point of the solvent (84°C). To this mixture, a solution of 5-chloro-2-fluoro-phenylamine 2 (0.4 mL, 3.5 mmol) in 1,2-dichloroethane (3.5 mL) was added dropwise using an addition funnel. After 3 h, the reaction mixture was cooled to room temperature (RT), diluted with CHCl 3 and washed with a 5% NaOH solution, water, and diluted HCl to remove excess and unreacted starting materials. The organic layer was then washed in a brine solution and dried over anhydrous

5-Chloro-2'-(4'-chloro-2-methoxy-phenoxy)-phenylamine (6)
Tin metal (0.18 g) and conc. HCl (0.5 mL) were added to the solution of compound 5 (0.10 g, 0.33 mmol) in EtOH (2 mL) in a 100 mL roundbottomed flask. The mixture solution was refluxed at 100°C for 3 h and then cooled to RT, to which a solution of 30% NaOH was added until the white precipitate was dissolved. The solution was then extracted with ether, washed with water and brine solution, and dried over anhydrous MgSO 4 . The crude product was purified on a silica gel column using 40-70% toluene/hexane to yield 62% (58 mg) of compound 6.

Determination of the antimalarial activity
The antimalarial activity of the synthesized molecules was evaluated against P. falciparum strain 3D7 (Eijkman Institute for Molecular Biology, Indonesia) using sensitive chloroquine and the procedure described by Budimulya [21]. P. falciparum 3D7 in human red blood cells (RBCs) (3% initial parasite density, and 4% hematocrit) was cultured with the test compound (added as 150 µL dimethyl sulfoxide solution) in 1350 µL of the medium (RPMI-1640, 25 µg/mL gentamycin, 50 µg/mL hypoxanthine, 25 mM Hepes buffer, 25 mM NaHCO 3 , and 10% human serum) using a 96-well microtiter plate at 37°C. Tests were carried out simultaneously for three molecules in duplicate. A sealed incubation chamber continuously gassed with a mixture of 2% O 2 , 8% CO 2 , and 90% N 2 was used. Increases in the proportion of infected RBCs were assessed at the end of the 48 h incubation period in control samples and at various concentrations of each drug using Giemsa stained slides. Control samples contained P. falciparum 3D7 without any test compounds. The growth of the parasite was monitored by performing a blood smear fixed with MeOH and stained with a Giemsa stain. The antimalarial activity of each compound was expressed as an IC 50 value, defined as the concentration of the compound causing 50% inhibition of parasite growth relative to the untreated control.

RESULTS AND DISCUSSION
Compound 6 was prepared by three chemical reactions in accordance with a previously reported procedure [18] (Fig. 2). Primary amine 2 in a commercially available form was oxidized with m-CPBA to form the corresponding nitrobenzene 3. Diphenyl ether derivative 5 was obtained by the coupling of the methoxy phenol 4 and nitrobenzene 3 in the presence of K 2 CO 3 and 18-crown-6 in DMF. The nitro group in 5 was easily reduced with Sn/HCl by reflux in ethanol to afford the known compound 6.
In dry THF, the amidation of 6 with acryloyl chloride afforded the desired amide 7. The amidation of 6 with acrylic acids and carbodiimide reagents was also attempted but was unsuccessful to give the desired amide 7. Diamagnetic anisotropy, which is commonly associated with asymmetric monosubstituted alkenes, appeared on the terminal olefin group of compound 7. Each of the three hydrogen atoms contributed inequivalently to form an exceptional splitting pattern (Fig. 3). The resulting peak of each hydrogen atom was expressed in terms of a chemical shift 6.44 ppm (t, J=8.2 Hz), 6.30 ppm (dd, J=16.8 Hz and 10.1 Hz), and 5.78 ppm (dd, J=10.4 Hz and 1.2 Hz).
Enantioselective dihydroxylation of the olefin in amide 7 was performed efficiently using the Sharpless et al. asymmetric dihydroxylation reaction [22] and the chiral quinine ligand (DHQ) 2 PHAL, to yield enantioenriched dihydroxy propionamide derivative (+)-1 in moderate yields, attributed to a specific rotation, [α] (+) 4.0 (c=0.40, in CH 3 OH). Similarly, when the catalyst was changed to the quinidine derivative (DHQD) 2 PHAL, the opposite selectivity was observed, and it yielded the desired enantioenriched (−)-1 with a specific rotation, [α] (−) 3.2 (c=0.37, in CH 3 OH). As reported by Sharpless et al. and associates in 2001, these chiral ligands have proven to be superior to others for the dihydroxylation of olefins with aliphatic substituents [22]. The enantioenriched products were used for further in vitro assays, with the percent of enantiomeric excess (% ee) not determined. The structures of compounds were proven by spectral data ( 1 H NMR, 13 C NMR, and mass spectra).
All inhibitors were tested for their inhibition of growth of the blood stages of the parasite P. falciparum 3D7 culture for 48 h in human blood. The activities of the test compounds compared with those of reference drugs are presented in Table 1. From the biological test results generated, analog (−)-1 appears to possess activity nearly as active as analog (+)-1 at lower micromolar concentrations. Qualitatively, this is understood by the fact that the specific configurations (R, rectus and S, sinister) of the enantiomers of these analogs are both active. This is likely because each of these cinchona ligands produced high enantiomer excess of a certain configuration [23].
Our results indicate that both of the enantioenriched analogs (+)-1 and (-)-1 of the 2'-chloro-and phenol-modified series exhibited better antimalarial activity profiles than the unsubstituted triclosan. Hence, triclosan inhibited P. falciparum cultures with an IC 50 of 27.2 µM, whereas the analogs (+)-1 and (-)-1 inhibited the cultures with IC 50 values of 0.034 and 0.028 µM, respectively. In comparison, the IC 50 value for chloroquine, a well-established drug for the treatment of malaria, is 0.0003 µM. Therefore, given our activity results, it will be necessary to add more chemical attributes to these molecules to make them active at the nanomolar level.

CONCLUSION
We have synthesized novel triclosan analogs that appear to have potent antiplasmodial activity. The design of these new analogs was initiated with the introduction of chirality, and the analogs were synthesized in five steps to obtain a moderate yield. These favorable preliminary in vitro results demonstrating the potency of the synthesized compounds, (+)-1 and (-)-1, indicate that these compounds, particularly with additional modification, may prove to be promising antimalarial leads based on the concept of drug enantioselectivity. Future efforts will seek to define the mode of action of these potent antimalarials while further optimizing their activity with a wider range of substituent groups that may offer improved antimalarial activity.