3′,5′-Dibromo-2′,4′-dihydroxy Substituted Chalcones: Synthesis and in vitro Trypanocidal Evaluation

Currently, an infectious disease crisis of global proportion is threatening hard-won gains in health and life expectancy. Infectious diseases are the world’s largest killer of children and young adults. Chagas disease is one of them which are caused by the protozoan parasite T. cruzi. It is a major cause of illness, morbidity, long-term disability, and death in Latin America. This disease is the third largest parasitic disease burden in the world and an estimated 10 million people are infected with this disease worldwide, mostly in Latin America [1]. In Latin America, infection with T. cruzi is responsible for Chagas disease, which is the leading cause of heart disease [2]. Despite the alarming health, economic and social consequences of this parasite infection and the limited existing drug therapy (nifurtimox and benznidazole) suffer from a combination of drawbacks including poor efficacy and serious side effects. Therefore, there is an urgent need for new chemotherapeutic agents with novel mechanisms of action [3-6]. Chalcones are a diverse group of compounds that can be synthesized or obtained from natural sources. This type compounds is 1, 3-diaryl2-propen-1-ones and belong to the flavonoid family. These compounds are small molecules that exert various biological activities [7-10]. Moreover, they provide an opportunity for chemist to synthesize a wide variety of bioactive heterocycles [11-14] due to the presence of α, β-unsaturated carbonyl functionality. However, the search for an efficient synthesis for chalcones remains a challenging task.

*Each value is the mean of three experiments * a IC 50 : concentration that produces 50% inhibitory effect, b cytotoxicity TD: dose required to produce of 50% cell LLCMK2, c TI: therapeutic index: IC 50, LLC-MK2 /IC 50, T. cruzi .

Synthetic procedures for (3a-t):
• Conventional solution phase method A mixture of 3′,5′-dibromo-2′,4′-dihydroxyacetophenone 1 (0.01 mol) and substituted aromatic aldehydes 2 (Note: From the structure, they are not aromatic aldehydes ) (0.01 mol) was stirred in 30 mL ethanol and then 15mL 40% KOH solution was added to it. The mixture was kept overnight at room temperature and then it was poured into crushed ice and acidified with HCl. The solid was obtained by filtering and then it was crystallized from ethanol and offered the analytical samples of (3a-t).

• Non-conventional solid phase method
To a solution of 3′,5′-dibromo-2′,4′-dihydroxyacetophenone 1 (0.01 mol) and substituted aromatic aldehyde 2 (Note: From the structure, they are not aromatic aldehydes ) (0.01 mol) in l mL DMF placed in 100 mL borosil flask, was added 4 g basic alumina. The mixture was uniformly mixed with glass rod and dried to remove the solvent under air. Adsorbed material was irradiated inside a microwave oven for 4-6 min. at medium power level (700 W). After completion of the reaction (monitored by TLC), the reaction mixture was cooled at room temperature and the product was extracted with dichloromethane (2×20 mL). Removal of the solvent and subsequent recrystallization from ethanol afforded analytical samples of (3a-t). The synthesis of title compounds is shown in Scheme 1 and the comparison of reaction times and yields of compounds (3a-t) under microwave and classical method are showed in Table 3.        13

Biological evaluation: materials and method
The parasite: T. cruzi (Tulahuen C4) transfected with β-galactosidase (Lac Z) gene was obtained from Institute of Scientific Research and Advanced High Technology services -Panama (AIP). The strain was maintained in monolayer Vero cells (African Green Monkey cells line (ATCC/CCL-81)) in complete RPMI 1640 medium without phenol red (Sigma company, St. Louis MO modified -R8755), supplemented with 10% heat inactivated fetal bovine serum. All cultures and assays were conducted at 37 ºC under an atmosphere of 5% CO 2, 95% air mixture.

Invitro trypanocidal evaluation:
The anti-trypanocidal activity was evaluated by the colorimetric method based on reducing of the substrate chlorophenolred-β-d-galactopyranoside (CPRG) for β-galactosidase resulting from the expression of the gene for T. cruzi (Tulahuen C4) [22]. The assay was realised in 96 wells plates containing monolayer VERO cells which were infected with 5×10 4 trypomastigotes (Tulahuen C4). We grew the parasite using VERO cells that were infected with T. cruzi trypomastigotes. The parasite is in the trypomastigote stage before it infects the cells. Once it infects the Vero cells, it enters an amastigotes stage and begins to reproduce as amastigotes. When it is released from the cells, it returns to the original trypomastigote stage to infect new cells. All the active compounds showed anti-trypanocidal activity, passed through a second test for determining the inhibitory concentration of 50% growth of the parasites (IC 50 ). These compounds were evaluated at 10, 2, 0.4, 0.8 and 0.16 ug/mL and incubated for 5 days at 37°C, relative humidity 95% and 5% CO 2 .
The intensity of colour resulting from the cleavage of CPRG by T. cruzi (Tulahuen C4) β-galactosidase was measured at 570 nm using a reader boards VersaMax Micro™ microplate reader. The IC 50 of the compound was calculated by logarithmic regression of the values of OD obtained, compared with the untreated control. Those samples showing IC 50 values <50ug/mL, have been further tested for cytotoxicity evaluation. Nifurtimox (Bayer) was used as a control at concentrations of 0.1, 1 and 10ug/mL. Negative Control is comprised of 50uL of a solution containing DMSO, equivalent to the DMSO contained in samples (working dilution).
Cytotoxicity assay: Active Compounds were screened for cytotoxicity against VERO cell line, at a maximum concentration of 50μg / mL. Briefly, Vero cell line were seeded into 96-well plate at a total concentration of 12 x10 4 cells/well in 100 uL of RPMI-1640 media without phenol red with 10% FBS. Cells were allowed to attach for 24 hrs. The wells were incubated with five decreasing concentrations, diluted in RPMI 1640 modified media or RPMI 1640 modified media alone, used as control. After 72 hrs, a colorimetric MTT assay was performed. Wells were incubated for 4 hrs with 5mg/mL of the tetrazolium salt MTT (3 -[4.5 dimethylthiazol-2-y1] -2, 5-diphenyl tetrazolium bromide/ Aldrich company, St. Louis MO). The sobrenadant were removed and cells lysed with 100% isopropanol. The absorbance was measured using an ELISA microplate reader (VersaMax Micro™ microplate reader) at 570nm. Tetrazolium salts are cleaved to formazan by mitochondrial enzymes in viable cells. Therefore, an increase in the OD reading, as a result of production of formazan, indirectly measures cell viability. The GI 50 value was defined as the concentration of test sample resulting in a 50% reduction of absorbance as compared with untreated controls that received a serial dilution of the solvent in which the test samples were dissolved, and was determined by linear regression analysis.

Conclusions
Two noteworthy features are apparent from our study project on the synthesis of small molecules of medicinal interest. Firstly, a novel series of substituted chalcones has been synthesized and it is concluded from Table 2 that classical procedure is tedious, time consuming, low yield and requiring an appreciable amount of solvent as compare to the environmentally benign synthetic procedure utilizing microwave irradiation (MWI) under solvent free conditions, over inorganic solid support. Secondly, it was observed from the results obtained by the trypanocidal evaluation that compounds: 3b, 3c, 3e, 3g, 3i, 3l, 3m, 3p, and 3q have a good IC 50 and their cytotoxicity is low, this means that these compounds could be used in future in vivo studies. 3l was the compound with a good anti-trypanocidal activity, the lower cytotoxicity, higher therapeutic index 14.5, and is the best candidate in comparison with the others. This compound has a therapeutic index lower than that of benznidazole and nifurtimox. Our results demonstrate the potential of these compounds as a new class of small molecule inhibitors of T. cruzi. The further biologically assay of the tested compounds gives an idea about the possible development for a new encouraging framework in this field that may lead to the discovery of potent trypanocidal drug.