alexa 3and#8242;,5and#8242;-Dibromo-2and#8242;,4and#8242;-dihydroxy Substituted Chal
ISSN: 2161-0401
Organic Chemistry: Current Research
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3,5-Dibromo-2,4-dihydroxy Substituted Chalcones: Synthesis and in vitro Trypanocidal Evaluation

K. L. Ameta1*, Nitu S. Rathore1, Biresh Kumar1, Edith S. Maalaga M2, Manuela Veraastegui2 and Robert H. Gilman3
1Department of Chemistry, FASC, Mody Institute of Technology & Science, Lakshmangarh-332311, Rajasthan, India
2Department of Microbiology, Faculty of Science and Philosophy, Universidad Peruana Cayetano Heredia, Lima, Peru
3Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland-21205, USA
Corresponding Author : K. L. Ameta
Department of Chemistry
Faculty of Arts Science and Commerce (FASC)
MITS University, Lakshmangarh
Sikar, Rajasthan-332311 India
Tel: +91-9414682501
Fax: +91-1573225044
E-mail: [email protected], [email protected]
Received July 11, 2012; Accepted August 16, 2012; Published August 20, 2012
Citation: Ameta KL, Rathore NS, Kumar B, Maalaga MES, Veraastegui M, et al. (2012) 3′,5′-Dibromo-2′,4′-dihydroxy Substituted Chalcones: Synthesis and in vitro Trypanocidal Evaluation. Organic Chem Curr Res 1:107. doi:10.4172/2161-0401.1000107
Copyright: © 2012 Ameta KL, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

A new series of 3′, 5′-dibromo-2′, 4′-dihydroxy substituted chalcones was synthesized by the classical Claisen- Schmidt condensation of 3, 5-dibromo-2, 4-dihydroxyacetophenone and variously substituted aromatic aldehydes via conventional as well as non-conventional inorganic solid supported microwave irradiation methods. The non- conventional methodology, has the advantages of being rapid, eco-friendly, easy to handle, requiring shorter reaction time, and quite general. All newly synthesized compounds were evaluated for their inhibitory effect against Trypanosoma cruzi (Chagas disease). Compounds 3c, 3g and 3m showed 85.53, 85.03 and 83.34 in-vitro percentage growth of inhibition respectively; while compounds 3b, 3e, 3i and 3l showed 71.23, 71.95, 67.53 and 68.88 percentage growth of inhibition respectively with nifurtimox and benznidazole as reference drugs. 3l was the compound with a good anti- trypanocidal activity, the lower cytotoxicity, higher therapeutic index 14.5, and was the best candidate in comparison with the others. The structures of the newly synthesized compounds (3a-t) were determined by elemental analysis, FTIR, 1H-NMR, 13C-NMR and mass spectroscopic studies.

Keywords
Chalcones; Trypanosoma cruzi; Chagas disease; Inhibition
Introduction
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-diaryl- 2-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.
The most widely used classical method for synthesizing chalcones is the Claisen-Schmidt condensation or through microwave irradiation [15-17] and ultrasonic irradiation because of their rapidity and improvement in yields [18,19]. In a continuation of our earlier endeavour [20-22] to design and synthesize novel bioactive heterocycles, and to consider the biological and medicinal importance of chalcones, herein, we reported a new series of chalcones via conventional as well as nonconventional microwave irradiation method and their trypanocidal evaluation.
Results and Discussion
Chemistry
3′,5′-dibromo-2′,4′-dihydroxyacetophenone (1) was treated with substituted aromatic aldehydes (2) (Note: From the structure, they are not aromatic aldehydes) to give substituted chalcones (3a-t) as shown in Scheme 1, with 74-84% yields. The structures of the newly synthesized compounds (3a-t) were determined on the basis of analytical and spectroscopic data. Thus, FTIR spectrum showed bands at 1688-1722 (C=O), 1642-1654 (-CH=CH), 1H NMR spectrum revealed the presence of a doublet at δ 7.48-7.98 corresponding to α Hydrogen and doublet at δ 8.11-8.42 corresponding to β Hydrogen and 13C NMR spectrum revealed the presence of Cα group (122.96-131.94), Cβ group (133.36- 155.64), C=O group (187.87-192.40) ppm. All these newly synthesized compounds were evaluated for their in vitro trypanocidal evaluation using nifurtimox and benznidazole as reference drugs.
Biological evaluation
The results of percentage growth of inhibition are summarized in (Table 1). The compounds 3a, 3d, 3f, 3h, 3j, 3k, 3n, 3o, 3r, 3s, 3t don’t have trypanocidal activity but there are nine active compounds that do have trypanocidal activity, which were evaluated for their IC50 and their cytotoxicity. Table 2 shows the compounds’ IC50, cytotoxicity, therapeutic index, and the values of the compounds with more anti trypanocidal activity. When the range of therapeutic index is short, testing in vivo is not recommended; we found that compounds 3b, 3c, 3e, 3g, 3i, 3l, 3m, 3p, and 3q have a good IC50 and their cytotoxicity is low, which means that these compounds could be used 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.
Experimental section
General: All melting points (m.p.) were determined in open capillaries on Veego (VMP – PM) melting point apparatus and are uncorrected. The purity of the compounds was routinely checked by thin layer chromatography (TLC) with Silica Gel-G (Merck).The instruments used for spectroscopic data are the IR spectrophotometer Brucker Alpha-Zn-Se, 1HNMR and 13CNMR (CDCl3) on 500 MHz FT-NMR spectrometer Bruker AV III, GC-MS (EI-MS fragment) performed on JEOL GC Mate spectrometer and elemental analysis was carried out on a Carlo Erba 1108 analyzer within ± 0.5% of the theoretical values. Column chromatography was performed on silica gel (Merck, 60-120 mesh). Microwave assisted reaction was carried out on a commercially modified MW synthesis system model CATA-R, operating 700W, generating 2450 MHz frequency.
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.
Spectroscopic data of the synthesized compounds are shown below:
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-phenylprop- 2-en-1-one
(3a). Yellow solid; Yield (?) m.p. 128-129°C. IR (KBr, cm-1): 3350 (Ar-OH), 3071, 3009 (Ar-H), 1688 (-C=O), 1642 (-CH=CH), 862 (CBr). 1HNMR (500 MHz, CDCl3) δ: 7.20-7.40 (m, 5H, Ar-H), 7.51 (d, αH, J=15.4Hz), 8.32 (d, βH, J=15.4Hz), 8.10 (s, 1H, Ar-H), 11.10 (s, 2H, 2 x Ar-OH). 13CNMR (125 MHz, CDCl3) δ: 79.55, 99.01, 103.62, 116.20, 123.96 (Cα), 126.14, 129.07, 130.48, 132.34, 133.37, 139.84 (Cβ), 160.37, 161.70, 190.95 (C=O). MS: m/z 397.80 (M+). Calcd. for C15H10Br2O3: C, 45.25, H, 2.51%. Found: C, 45.30, H, 2.46%.
(2E)-3-(2-chlorophenyl)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl) prop-2-en-1-one
(3b). Orange solid; Yield (?) m.p. 120-121°C. IR (KBr, cm-1): 3393 (Ar-OH), 3061, 3006 (Ar-H), 1693 (-C=O), 1645 (-CH=CH), 862 (CBr), 753 (C-Cl). 1HNMR (500 MHz, CDCl3) δ: 7.28-7.61 (m, 4H, Ar- H), 7.78 (d, αH, J=15.6Hz), 8.13 (s, 1H, Ar-H), 8.37 (d, βH, J=15.6Hz), 11.05 (s, 2H, 2 x Ar-OH). 13CNMR (125MHz, CDCl3) δ: 79.55, 97.91, 105.92, 116.20, 124.89 (Cα), 127.04, 130.30, 131.03, 132.72, 133.38, 140.06 (Cβ), 160.97, 188.15 (C=O). MS: m/z 432.30 (M+). Calcd. for C15H9Br2O3Cl: C, 41.64, H, 2.08%. Found: C, 41.69, H, 2.13%.
(2E)-3-(3-chlorophenyl)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl) prop-2-en-1-one
(3c). Yellow solid; Yield (?) m.p. 141-142°C. IR (KBr, cm-1): 3368 (Ar-OH), 3071, 3009 (Ar-H), 1695 (-C=O), 1642 (-CH=CH), 862 (CBr), 758 (C-Cl). 1HNMR (500 MHz, CDCl3) δ: 7.22-7.42 (m, 4H, Ar-H), 7.48 (d, αH, J=15.6Hz), 10.72 (s, 2H, 2 x Ar-OH), 8.06 (s, 1H, Ar-H), 8.34 (d, βH, J=15.6Hz). 13C-NMR (125 MHz, CDCl3): = 81.55, 97.97, 106.12, 116.43, 124.81 (Cα), 128.54, 130.30, 133.79, 134.03, 138.07 (Cβ), 148.22, 161.93, 187.87 (C=O). MS: m/z 432.30 (M+). Calcd. for C15H9Br2O3Cl: C, 41.64, H, 2.08%. Found: C, 41.59, H, 2.12%.
(2E)-3-(4-chlorophenyl)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl) prop-2-en-1-one
(3d). Brown solid; Yield (?) m.p. 129-130°C. IR (KBr, cm-1): 3365 (Ar-OH), 3054, 3011 (Ar-H), 1701 (-C=O), 1648 (-CH=CH), 862 (CBr), 753 (C-Cl). 1H-NMR (500 MHz, CDCl3) δ: 7.26-7.64 (m, 4H, Ar- H), 7.89 (d, αH, J=15.6Hz), 10.89 (s, 2H, 2 x Ar-OH), 8.08 (s, 1H, Ar- H), 8.40 (d, βH, J=15.6Hz). 13C NMR (125 MHz, CDCl3) δ: 88.37, 94.61, 104.43, 115.19, 124.89 (Cα), 128.54, 130.71, 133.38, 136.19, 140.06 (Cβ), 149.21, 163.93, 188.15 (C=O). MS: m/z 432.30 (M+). Calcd. for C15H9Br2O3Cl: C, 41.64, H, 2.08%. Found: C, 41.59, H, 2.12%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(2, 4-dichlorophenyl) prop-2-en-1-one
(3e). Orange solid; Yield (?). m.p. 124-125°C. IR (KBr, cm-1): 3394 (Ar-OH), 3081, 3005 (Ar-H), 1699 (-C=O), 1652 (-CH=CH), 862 (C-Br), 783,754 (C-Cl). 1HNMR (500 MHz, CDCl3) δ: 7.36-7.69 (m, 3H, Ar-H), 7.75 (d, αH, J=15.6Hz), 8.08 (s, 1H, Ar-H), 8.37 (d, βH, J=15.6Hz), 11.05 (s, 2H, 2 x Ar-OH). 13CNMR (125 MHz, CDCl3) δ: 87.19, 96.27, 108.76, 115.19, 125.40 (Cα), 128.04, 131.93, 133.08, 135.40, 143.72 (Cβ), 149.21, 163.93, 188.22 (C=O). MS: m/z 466.80 (M+). Calcd. for C15H8Br2O3Cl2: C, 38.56, H, 1.71%. Found: C, 38.62, H, 1.76%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(2-fluorophenyl) prop-2-en-1-one
(3f). Brown solid; Yield (?), m.p. 102-103°C. IR (KBr, cm-1): 3410 (Ar-OH), 3092, 3009 (Ar-H), 1710 (-C=O), 1651 (-CH=CH), 862 (C-Br), 1256 (C-F). 1HNMR (500 MHz, CDCl3) δ: 7.28-7.52 (m, 4H, Ar-H), 7.82 (d, αH, J=15.6Hz), 10.95 (s, 2H, 2 x Ar-OH), 8.06 (s, 1H, Ar-H), 8.25 (d, βH, J=15.6Hz). 13CNMR (125 MHz, CDCl3) δ: 79.73, 98.98, 103.44, 115.23, 127.82 (Cα), 129.86, 130.92, 133.67, 145.26 (Cβ), 149.29, 155.66, 163.88, 188.65 (C=O). MS: m/z 415.80 (M+). Calcd. for C15H9Br2O3 F: C, 43.29, H, 2.16%. Found: C, 43.34, H, 2.11%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(4-fluorophenyl) prop-2-en-1-one
(3g). Brown solid; Yield (?), m.p. 158-159°C. IR (KBr, cm-1): 3372 (Ar-OH), 3089, 3005 (Ar-H), 1705 (-C=O), 1652 (-CH=CH), 862 (C-Br), 1261 (C-F). 1HNMR (500 MHz, CDCl3) δ: 7.26-7.61 (m, 4H, Ar-H), 7.89 (d, αH, J=15.6Hz), 10.90 (s, 2H, 2 x Ar-OH), 8.09 (s, 1H, Ar-H), 8.34 (d, βH, J=15.6Hz). 13CNMR (125 MHz, CDCl3) δ: 81.23, 99.09, 103.49, 116.29, 127.83 (Cα), 130.86, 132.29, 133.37, 145.96 (Cβ), 155.66, 158.35, 163.57, 190.95 (C=O). MS: m/z 415.80 (M+). Calcd. for C15H9Br2O3F: C, 43.29, H, 2.16%. Found: C, 43.24, H, 2.22%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(2-methylphenyl) prop-2-en-1-one
(3h). Yellow solid; Yield (?), m.p. 119-120°C. IR (KBr, cm-1): 3395 (Ar-OH), 3289 (CH3), 3093, 3007 (Ar-H), 1710 (-C=O), 1649 (-CH=CH), 862 (C-Br). 1HNMR (500 MHz, CDCl3) δ: 2.63 (3H, s, -CH3), 7.27-7.74 (m, 4H, Ar-H), 7.89 (d, αH, J=15.6Hz), 8.11 (s, 1H, Ar-H), 8.42 (d, βH, J=15.6Hz), 11.08 (s, 2H, 2 x Ar-OH). 13CNMR (125 MHz, CDCl3) δ: 21.21 (CH3), 80.33, 99.47, 103.24, 117.86, 125.98 (Cα), 130.15, 132.28, 134.84, 146.70 (Cβ), 155.61, 158.57, 161.63, 189.11 (C=O). MS: m/z 411.80 (M+). Calcd. for C16H12Br2O3: C, 46.62, H, 2.91%. Found: C, 46.55, H, 2.96%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(4-methylphenyl) prop-2-en-1-one
(3i). Yellow solid; Yield (?), m.p. 117-118°C. IR (KBr, cm-1): 3391 (Ar-OH), 3283 (CH3), 3091, 3005 (Ar-H), 1699 (-C=O), 1645 (-CH=CH), 862 (C-Br). 1HNMR (500 MHz, CDCl3) δ: 2.67 (3H, s, -CH3), 7.29-7.79 (m, 4H, Ar-H), 7.91 (d, αH, J=15.6Hz), 10.92 (s, 2H, 2 x Ar-OH), 8.07 (s, 1H, Ar-H), 8.39 (d, βH, J=15.6Hz). 13CNMR (125 MHz, CDCl3) δ: 21.65 (CH3), 81.41, 99.66, 105.41, 117.81, 127.36 (Cα), 131.56, 133.36, 138.84, 147.43 (Cβ), 155.82, 160.37, 161.65, 191.16 (C=O) ppm. MS: m/z 411.80 (M+). Calcd. for C16H12Br2O3: C, 46.62, H, 2.91%. Found: C, 46.67, H, 2.87%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(3-nitrophenyl) prop-2-en-1-one
(3j). Orange solid; Yield (?), m.p. 159-160°C. IR (KBr, cm-1): 3389 (Ar-OH), 3091, 3011 (Ar-H), 1715 (-C=O), 1654 (-CH=CH), 1531 (Asy Ar-NO2), 1350 (Sym Ar-NO2), 862 (C-Br). 1HNMR (500 MHz, CDCl3) δ: 7.25-7.78 (m, 4H, Ar-H), 7.97 (d, αH, J=15.6Hz), 10.90 (s, 2H, 2 x Ar-OH), 8.01 (s, 1H, Ar-H), 8.11 (d, βH, J=15.6Hz). 13CNMR (125 MHz, CDCl3) δ: 83.47, 99.61, 103.98, 116.87, 124.46 (Cα), 130.14, 133.36, 138.84, 144.25 (Cβ), 148.22, 152.11, 161.22, 186.79 (C=O). MS: m/z 442.80 (M+). Calcd. for C15H9Br2O5N: C, 40.65, H, 2.03, N, 3.16%. Found: C, 40.59, H, 2.08, N, 3.12%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(4-hydroxyphenyl) prop-2-en-1-one
(3k). Brown solid;Yiled (?), m.p. 101-102°C. IR (KBr, cm-1): 3395 (Ar-OH), 3087, 3007 (Ar-H), 1710 (-C=O), 1648 (-CH=CH), 862 (CBr). 1HNMR (500 MHz, CDCl3) δ: 7.32-7.83 (m, 4H, Ar-H), 7.98 (d, αH, J=15.6Hz), 8.12 (s, 1H, Ar-H), 8.19 (d, βH, J=15.6Hz), 11.02 (s, 2H, 2 x Ar-OH), 11.50 (s, 1H, Ar-OH). 13CNMR (125 MHz, CDCl3) δ: 80.20, 99.41, 115.25, 116.13, 123.88 (Cα), 131.09, 133.38, 155.64 (Cβ), 160.35, 162.81, 190.92 (C=O). MS: m/z 413.80 (M+). Calcd. for C15H10Br2O4: C, 43.50, H, 2.42%. Found: C, 43.55, H, 2.37%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(4-methoxyphenyl) prop-2-en-1-one
(3l). Red solid; Yiled (?). m.p. 159-160°C. IR (KBr, cm-1): 3402 (Ar- OH), 3089, 3007 (Ar-H), 2830 (OCH3), 1715 (-C=O), 1651 (-CH=CH), 862 (C-Br). 1HNMR (500 MHz, CDCl3) δ: 3.86 (s, 3H, OCH3), 7.00- 7.48 (m, 4H, Ar-H), 7.84 (d, αH, J=16.0Hz), 10.82 (s, 2H, 2 x Ar-OH), 8.08 (s, 1H, Ar-H), 8.17 (d, βH, J=16.0Hz). 13CNMR (125 MHz, CDCl3) δ: 56.29 (OCH3), 80.25, 99.84, 115.05, 116.04, 123.87 (Cα), 129.31, 131.71, 133.40, 138.26 (Cβ), 156.20, 160.35, 161.77, 190.77 (C=O). MS: m/z 427.80 (M+). Calcd. for C16H12Br2O4: C, 44.88, H, 2.80%. Found: C, 44.83, H, 2.74%.
(2E)-3-(4-bromophenyl)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl) prop-2-en-1-one
(3m). Orange solid; Yield (?). m.p. 130-131°C. IR (KBr, cm-1), 3418 (Ar-OH), 3087, 3009 (Ar-H), 1718 (-C=O), 1652 (-CH=CH), 862 (CBr). 1HNMR (500 MHz, CDCl3) δ: 7.04-7.58 (m, 4H, Ar-H), 7.88 (d, αH, J=15.6Hz), 10.93 (s, 2H, 2 x Ar-OH), 8.06 (s, 1H, Ar-H), 8.14 (d, βH, J=15.6Hz). 13CNMR (125 MHz, CDCl3) δ: 77.28, 99.57, 115.51, 118.13, 124.14 (Cα), 129.75, 132.19, 133.04, 136.16 (Cβ), 153.66, 160.35, 161.97, 188.79 (C=O). MS: m/z 476.70 (M+). Calcd. for C15H9Br3O3: C, 37.76, H, 1.89%. Found: C, 37.70, H, 1.93%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(3-hydroxy-4- methoxyphenyl) prop-2-en-1-one
(3n). Orange solid; Yield (?). m.p. 152-153°C. IR (KBr, cm-1) :3398 (Ar-OH), 3087, 3011 (Ar-H), 2830 (OCH3), 1720 (-C=O), 1649 (-CH=CH), 862 (C-Br). 1HNMR (500 MHz, CDCl3) δ: 3.87 (s, 3H, OCH3), 7.11-7.61 (m, 4H, Ar-H), 7.91 (d, αH, J=15.6Hz), 8.00 (s, 1H, Ar-H), 8.11 (d, βH, J=15.6Hz), 11.13 (s, 2H, 2 x Ar-OH). 13CNMR (125 MHz, CDCl3) δ: 56.39 (OCH3), 80.25, 99.84, 116.04, 119.56, 122.96 (Cα), 129.87, 132.12, 133.16, 145.08 (Cβ), 153.66, 160.36, 161.68, 192.40 (C=O). MS: m/z 443.80 (M+). Calcd. for C16H12Br2O5: C, 43.26, H, 2.70%. Found: C, 43.32, H, 2.65%.
(2E)-3-(5-bromo-2-hydroxyphenyl)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl) prop-2-en-1-one
(3o). Brown solid; Yield (?). m.p. 121-122°C. IR (KBr, cm-1): 3432 (Ar-OH), 3089, 3009 (Ar-H), 1721 (-C=O), 1652 (-CH=CH), 862 (CBr). 1HNMR (500 MHz, CDCl3) δ: 7.03-7.68 (m, 4H, Ar-H), 7.94 (d, αH, J=15.6Hz), 10.78 (s, 2H, 2 x Ar-OH), 8.01 (s, 1H, Ar-H), 8.16 (d, βH, J=15.6Hz). 13CNMR (125 MHz, CDCl3) δ: 77.27, 99.03, 116.20, 119.64, 125.75 (Cα), 129.81, 132.29, 133.37, 135.85 (Cβ), 155.73, 158.25, 160.36, 190.71 (C=O). MS: m/z 492.70 (M+). Calcd. for C15H9Br3O4: C, 36.53, H, 1.83%. Found: C, 36.58, H, 1.76%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(3, 4-dimethoxyphenyl) prop-2-en-1-one
(3p). Brown solid; Yield (?). m.p. 151-152°C. IR (KBr, cm-1): 3436 (Ar-OH), 3091, 3009 (Ar-H), 2832 (OCH3), 1699 (-C=O), 1649 (-CH=CH), 862 (C-Br). 1HNMR (500 MHz, CDCl3) δ: 3.93 (s, 6H, 2 x OCH3), 6.99-7.48 (m, 3H, Ar-H), 7.98 (d, αH, J=15.6Hz), 10.97 (s, 2H, 2 x Ar-OH), 8.02 (s, 1H, Ar-H), 8.14 (d, βH, J=15.6Hz). 13CNMR (125 MHz, CDCl3) δ: 56.77 (OCH3), 60.81, 99.89, 105.78, 109.64, 115.05, 116.15, 126.51 (Cα), 129.31, 132.41, 132.97, 135.68 (Cβ), 153.13, 160.35, 162.17, 189.21 (C=O). MS: m/z 457.80 (M+). Calcd. for C17H14Br2O5: C, 44.56, H, 3.06%. Found: C, 44.51, H, 3.11%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(3,4,5-trimethoxyphenyl) prop-2-en-1-one
(3q).Orange solid; Yield (?) m.p. 128-129°C. IR (KBr, cm-1): 3438 (Ar-OH), 3091, 3009 (Ar-H), 2832 (OCH3), 1699 (-C=O), 1649 (-CH=CH), 862 (C-Br). 1HNMR (500 MHz, CDCl3) δ: 3.87 (s, 9H, 3 x OCH3), 6.97-7.53 (m, 2H, Ar-H), 7.98 (d, αH, J=15.6Hz), 8.05 (s, 1H, Ar-H), 8.16 (d, βH, J=15.6Hz), 11.05 (s, 2H, 2 x Ar-OH). 13CNMR (125 MHz, CDCl3) δ: 56.17 (OCH3), 56.45, 60.89, 99.81, 105.16, 110.64, 113.38, 120.56, 126.57 (Cα), 128.09, 131.31, 134.63 (Cβ), 155.13, 160.35, 162.17, 191.12 (C=O). MS: m/z 487.80 (M+). Calcd. for C18H16Br2O6: C, 44.28, H, 3.28% Found: C, 44.32, H, 3.23%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(4-hydroxy-3- methoxyphenyl) prop-2-en-1-one
(3r).Yellow solid; Yiled (?) m.p. 110-111°C. IR (KBr, cm-1): 3438 (Ar-OH), 3089, 3009 (Ar-H), 2830 (OCH3), 1718 (-C=O), 1651 (-CH=CH), 862 (C-Br). 1HNMR (500 MHz, CDCl3) δ: 3.89 (s, 3H, OCH3), 7.09-7.58 (m, 4H, Ar-H), 7.96 (d, αH, J=15.6Hz), 10.88 (s, 2H, 2 x Ar-OH), 8.02 (s, 1H, Ar-H), 8.13 (d, βH, J=15.6Hz). 13CNMR (125 MHz, CDCl3) δ: 56.17 (OCH3), 99.40, 108.76, 110.42, 114.38, 119.31, 124.24 (Cα), 127.54, 129.61, 133.38 (Cβ), 155.36, 160.34, 161.58, 190.91 (C=O). MS: m/z 443.80 (M+). Calcd. for C16H12Br2O5: C, 43.26, H, 2.70%. Found: C, 43.21, H, 2.75%.
(2E)-3-(3-bromo-4-hydroxy-5-methoxyphenyl)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl) prop-2-en-1-one
(3s). Brown solid; Yield (?) m.p. 99-100°C. IR (KBr, cm-1): 3435 (Ar-OH), 3092, 3005 (Ar-H), 2833 (OCH3), 1720 (-C=O), 1652 (-CH=CH), 862 (C-Br). 1HNMR (500 MHz, CDCl3) δ: 3.94 (s, 3H, OCH3), 7.03-7.63 (m, 4H, Ar-H), 7.89 (d, αH, J=16.0Hz), 8.03 (s, 1H, Ar-H), 8.13 (d, βH, J=16.0Hz), 10.95 (s, 1H, Ar-OH), 11.27 (s, 2H, 2 x Ar-OH). 13CNMR (125MHz, CDCl3) δ: 56.53 (OCH3), 77.28, 99.11, 108.21, 110.42, 115.14, 119.31, 122.56, 129.85 (Cα), 130.09, 130.47, 133.37 (Cβ), 155.87, 160.37, 162.61, 189.67 (C=O). MS: m/z 526.70 (M+). Calcd. for C16H11Br3O5: C, 37.21, H, 2.09%. Found: C, 37.26, H, 2.04%.
(2E)-1-(3′, 5′-dibromo-2′, 4′-dihydroxyphenyl)-3-(furan-2-yl) prop-2-en-1-one
(3t). Yellow solid; Yield (?) m.p. 132-133°C. IR (KBr, cm-1):3435 (Ar-OH), 3087, 3009 (Ar-H), 1722 (-C=O), 1649 (-CH=CH), 862 (CBr). 1HNMR (500 MHz, CDCl3) δ: 7.09-7.59 (m, 3H, Ar-H), 7.91 (d, αH, J=16.0Hz), 8.00 (s, 1H, Ar-H), 8.16 (d, βH, J=16.0Hz), 11.09 (s, 2H, 2 x Ar-OH). 13CNMR (125 MHz, CDCl3) δ: 99.42, 113.10, 117.96, 131.94 (Cα), 132.29, 133.36 (Cβ), 151.31, 155.63, 160.36, 162.64, 190.74 (C=O). MS: m/z 387.80 (M+). Calcd. for C13H8Br2O4: C, 40.22, H, 2.06%. Found: C, 40.16, H, 2.11%.
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% CO2, 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 5×104 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 (IC50). 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% CO2.
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 IC50 of the compound was calculated by logarithmic regression of the values of OD obtained, compared with the untreated control. Those samples showing IC50 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 x104 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 GI50 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 IC50 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.
Acknowledgements
The authors are thankful to Prof. B. L. Verma, M.L.S. University and Dr. Sunil Jhakoria, Dean, FASC, MITS University for their constant encouragement during this work. Authors are also thankful to the Head, Sophisticated Analytical Instrument Facility, Indian Institute of Technology, Madras for spectral analysis.
References


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