European Journal of Medicinal Chemistry 79 (2014) 260e265
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel building blocks of benzosuberone bearing coumarin moieties and their evaluation as potential anticancer agents Bandi Yadagiri a, Uma Devi Holagunda a, Rajashaker Bantu a, Lingaiah Nagarapu a, *, C. Ganesh Kumar b, Sujitha Pombala b, B. Sridhar c a b c
Organic Chemistry Division II, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India Centre for Medicinal Chemistry & Pharmacology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India Centre for X-ray Crystallography, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 December 2013 Received in revised form 19 March 2014 Accepted 4 April 2014 Available online 5 April 2014
A series of novel benzosuberone bearing coumarin moieties 5aec have been synthesized and their structures were determined by analytical and spectral (FT-IR, 1H NMR, 13C NMR, HRMS) studies. The newly synthesized compounds were evaluated for their cytotoxicity against four human cancer cell lines, A549 (Human alveolar adenocarcinoma cell line), HeLa (Human cervical cancer cell line), MDA-MB-231 (Human breast adenocarcinoma cell line), MCF-7 (Human breast adenocarcinoma cell line) and normal cell line HEK293 (Human embryonic kidney cell line). Compound 5a exhibited promising cytotoxicity with IC50 values ranging from 3.35 to 16.79 mM against all the cancer cell lines like A549, HeLa, MCF-7 and MDA-MB-231, while compound 5c showed significant cytotoxicity against HeLa and MDA-MB-231 with IC50 values of 6.72 and 4.87, respectively. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Benzosuberone Vilsmeier HaackeArnold reaction Cytotoxicity Cell lines
1. Introduction Colchicine, a major natural alkaloid derived from the plant Colchicum autumnal, is a benzosuberone derivative exhibiting antitumour activity, due to the binding of aromatic ring of colchicine with hydrophobic domain of tubulin. Colchicine was the first drug reported to function through a tubulin binding mechanism in the late 1930s [1]. However, colchicine was eventually withdrawn from clinical use for cancer treatment due to significant in vivo toxicity [2]. Nonetheless, colchicine has been used in the treatment of gout at very low doses since the late 1950s [3]. Recent investigations on benzosuberone-containing moiety showed that chemokine receptor CCR5, is expressed in prostate cancer specimens [4]. Benzosuberone derivatives also showed a wide range of biological activities like antimicrobial [5], anti-inflammatory [6], anti-estrogenic [7], anti-malarial [8] and anti-platelet aggregation [9] but their anticancer effects have been reported to be more potent against a majority of human cancer cell lines. Coumarins are an elite class of oxygen heterocycles, which occur naturally in many food sources including citrus fruits, herbs and vegetables [10]. Some important coumarin members have been
* Corresponding author. E-mail address: [email protected] (L. Nagarapu). http://dx.doi.org/10.1016/j.ejmech.2014.04.015 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.
isolated from microbial sources, e.g. novobiocin and coumermycin from Streptomyces, and aflatoxins from Aspergillus species and (þ)-Calanolide A, a natural product isolated from several tropical plants of the genus Calophyllum as shown in Fig. 1. Pharmacologically, they belong to neoflavonoid class of compounds, which have been found to exhibit a wide variety of biological activities, usually associated with low toxicity and have raised considerable interest because of their potential beneficial effects on human health. In the recent years, they have attracted immense interest due to their diverse pharmacological properties like anti-HIV [11] anticoagulant [12], anti-bacterial [13], and anti-malarial [14]. In addition, few coumarin derivatives have the special ability to show potent anti-tumour activity against several human tumour cell lines. The coumarins are extremely variable in structure, due to various types of substitutions in their basic structure, which in turn can influence their biological activity. In continuation to our ongoing research program [15e21] to discover and develop tumour growth inhibitors and apoptotic inducers as potential new anti-cancer agents, we have designed and synthesized novel benzosuberone-bearing coumarin moiety analogues (using VilsmeiereHaack and Suzuki cross-coupling reactions) and evaluated their in vitro cytotoxicity against different human cancer cell lines like A549, HeLa, MDA-MB-231 and MCF-7. Significantly, the compound 5a showed promising cytotoxicity against the HeLa and MCF-7 cancer cell lines with IC50 values of
B. Yadagiri et al. / European Journal of Medicinal Chemistry 79 (2014) 260e265
O
O
O
O
O
O O
O O
O
O
OH
HN
O
O
OH OH
O
261
O
O
N H OH
NH 2
O Colchicine
(+)- Calanolide A
Novabiocine
Fig. 1. Structures of some previously isolated important natural alkaloids.
3.35 and 9.63 mM, respectively, and compound 5c displayed the most potent selectivity against HeLa and MDA-MB-231 cell lines with IC50 values of 6.72 and 9.63 mM, respectively. 2. Results and discussion 2.1. Chemistry The detailed reaction conditions for the synthesis of crosscondensed coumarin moieties are illustrated in Scheme 1. The synthesis of compounds 2aec was easily carried out from the corresponding ketones by VilsmeiereHaackeArnold reaction [22]. The vinyl chlorides obtained above, were activated by the presence of an electron-withdrawing group, which in turn allowed their easy coupling with o-anicyl boronic acids in the presence of 2 mol % of palladium (II) acetate, tetrabutylammonium bromide and potassium carbonate, with water as the reaction medium at 45 C for 3 h afforded compound 3aec in 79.1e90.7% yields. Oxidation of aldehydes 3aec with sodium chlorite in the presence of 30% H2O2 in acetonitrile at room temperature allowed the formation of acids 4aec in 70.2e74.9% yields. Finally, the acids were converted to the corresponding acyl chlorides, followed by cyclization with aluminium chloride in dichloromethane at room temperature to furnish the desired tetra cyclic systems 5aec in 81.5e88.7% yields. All the new synthesized compounds were characterized using NMR, IR and mass spectrometry (see supporting information). Spectral data of all synthesized compounds were in good agreement with the proposed structures. IR spectrum revealed the presence of C]O bond in lactone (1712e1716 cm1) and CH (2928e2934 cm1) functional groups in the synthesized compounds. This was further confirmed by 1H NMR spectrum (300 MHz) of 5aec recorded in CDCl3 displayed the disappearance of eOCH3 substituent at d 3.30e3.40 ppm in the starting compound indicating that the cyclization occurred in the final step. Two additional triplets around d 2.43e3.41 ppm and multiplet at d 1.81e2.30 ppm in seven membered ring were observed. The compound 5b was obtained as colourless crystal after recrystallisation in ethanol and the structure was confirmed by X-ray crystallography. The X-ray diffraction analysis of the single crystal of 5b (Fig. 1) proved the presence of chlorine substitution at ring D; whereas the compounds 5a and 5c lack it; herein we studied the amount of catalyst in different equivalents. The molecular formula of all the synthesized compounds was confirmed by HRMS data analysis (Fig. 2). 2.2. X-ray crystallography and spectral studies 2.2.1. X-ray crystal data for compound AI41 (5b) Data were collected at room temperature using a Bruker Smart Apex CCD diffractometer with graphite monochromated MoKa
radiation (l ¼ 0.71073 A) with u-scan method [23]. Preliminary lattice parameters and orientation matrices were obtained from four sets of frames. Unit cell dimensions were determined using 5417 reflections in the range of 2.25 < q < 27.95 . Integration and scaling of intensity data were accomplished using SAINT [24] program. The structure was solved by Direct Methods using SHELXS97 [24] and refinement was carried out by full-matrix least-squares technique using SHELXL97 [24]. All hydrogen atoms were positioned geometrically and were treated as riding on their parent carbon atoms, with CeH distance of 0.93e0.98 A, with Uiso(H) ¼ 1.2Ueq (C) or 1.5Ueq(C). Crystal data for AI41: C20H17ClO2, M ¼ 324.79, colourless plate, 0.14 0 0.12 0 0.06 mm3, triclinic, space group P-1 (No. 2), a ¼ 9.8232 (11), b ¼ 9.9810 (11), c ¼ 18.362 (2) A, a ¼ 84.591 (2), b ¼ 81.371 (2), g ¼ 64.395 (2) , V ¼ 1604.2 (3) A3, Z ¼ 4, Dc ¼ 1.345 g/cm3, F000 ¼ 680, CCD Area Detector, MoKa radiation, l ¼ 0.71073 A, T ¼ 294 (2)K, 2qmax ¼ 50.0 , 15,411 reflections collected, 5635 unique (Rint ¼ 0.0156). Final GooF ¼ 1.030, R1 ¼ 0.0370, wR2 ¼ 0.1010, R indices based on 5037 reflections with I > 2s (I) (refinement on F2), 418 parameters, 0 restraints, m ¼ 0.245 mm1. Crystallographic data has been deposited for compound AI41 with the Cambridge Crystallographic Data Centre [CCDC No. 933434]. Copies of the data can be obtained free of charge at www.ccdc.cam. ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: þ44 (0) 1223 336 033; email: [email protected]]. 2.3. Effects of the compounds on the viability of human cancer cells The results of the assay are summarized in Table 1. To examine the effects of the compounds on the viability of different human cancer cell lines, the in vitro antitumour activity was evaluated against A549, HeLa, MDA-MB-231 and MCF-7 by the standard MTT assay method. The inhibitory activity was expressed in micromolar concentrations of the compound, which caused 50% inhibition (IC50 value) under the assay conditions and Doxorubicin was used as a reference drug. Data in Table 1 showed that the majority of the synthesized compounds displayed antitumour activities against the tested four human cancer cell lines, which could support the rationale of the drug design. An attempt was made to draw some considerations concerning the structureeactivity relationship (SAR). Compound 5a with no substitution on the benzene ring A (Scheme 1) exhibited promising antitumour effect against A549 and MCF-7 cell lines with IC50 values of 3.35 and 9.63 mM, respectively. Introduction of electron donating groups (eCH3, eOCH3) on benzene ring A was associated with a noticeable increase in the cytotoxic effect against all the four tested cell lines. On the contrary, compound 5b with an electron-donating group (eCl) at benzene ring D effected moderate anti-tumour activity against all the tested
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Scheme 1. Synthesis of compounds 5aec.
significantly more active than compounds 5a and 5b. Further, these compounds did not show any cytotoxicity against the normal cancer cell line, HEK293. Compounds 5a, 5b, and 5c differed structurally only at the ring A and ring D. This could suggest the importance of the substituted benzene ring A of this molecular modification for exhibiting the bioactivity. Finally, we conclude that the substitutions on ring A played an important role in the exhibiting cytotoxicity due its conjugation effect on the coumarin moiety (ring C) and were more favourable than having substitutions on ring D. 3. Pharmacology 3.1. In vitro cytotoxicity assay
Fig. 2. The molecular structure of 5b, with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. The asymmetric unit contains two crystallographically independent molecules (labelled with suffix A and B) and for clarity molecule B has been omitted.
cell lines as compared to the compounds 5a and 5c. Although, compound 5c with a stronger electron-donating group (-OCH3) at benzene ring A displayed promising anti-tumour activity as compared to doxorubicin with IC50 values of 6.72 and 4.87 mM against HeLa and MDA-MB-231 cell lines, respectively, it was
Cytotoxicity of all the synthesized compounds was determined on the basis of measurement of in vitro growth inhibition of tumour cell lines in 96 well plates by cell-mediated reduction of tetrazolium salt to water insoluble formazan crystals using doxorubicin as a standard. The cytotoxicity as assessed against a panel of four different human tumour cell lines: A549 derived from human alveolar adenocarcinoma epithelial cells (ATCC No. CCL-185), HeLa derived from human cervical cancer cells (ATCC No. CCL-2), MDAMB-231 derived from human breast adenocarcinoma cells (ATCC No. HTB26), MCF7 derived from human breast adenocarcinoma cells (ATCC No. HTB22) and HEK293 (normal human embryonic kidney cells) (ATCC No. CRL-1573) using the MTT assay [25]. The IC50 values (50% inhibitory concentration) were calculated from the plotted absorbance data for the doseeresponse curves. IC50 values (in mM) are indicated as means SD of three independent experiments.
B. Yadagiri et al. / European Journal of Medicinal Chemistry 79 (2014) 260e265 Table 1 IC50 of the tested compounds against human cancer and normal cell lines. Compounds
a b
red liquid. The product was purified by column chromatography using 60e120 mesh silica gel (hexane/EtOAc).
IC50 ina mM A549
5a 5b 5c Doxorubicinb
263
He La
MDA-MB-231 MCF-7
3.35 0.05 14.07 ± 0.07 16.79 ± 0.08 10.77 ± 0.04 12.69 ± 0.06 13.28 ± 0.07 >200 6.72 ± 0.05 4.87 ± 0.06 0.46 ± 0.02 0.51 ± 0.02 0.91 ± 0.02
9.63 ± 0.06 15.76 0.06 >200 1.07 ± 0.03
HEK293 >200 >200 >200 >200
Data presented is the mean value of three independent experiments. Positive control.
4. Conclusion In conclusion, we have reported a simple synthesis of novel benzosuberone-bearing coumarin compounds using Vilsmeiere Haack and Suzuki cross-coupling followed by cyclization and evaluated there in vitro cytotoxicity against HeLa, A549, MCF-7, MDA-MB-231 and normal HEK293 cell lines. All the synthesised compounds indicated significant growth inhibitory effects. In addition, the compounds 5a, 5b and 5c exhibited potent activity against the four tested human cancer cell lines. Moreover, the compound 5a was more potent against A549 and MCF-7 cell lines and compound 5c was selectively potent against HeLa and MDAMB-231 cell lines. Introduction of substitution on ring A was associated with a distinct tendency to increase the in vitro cytotoxic potential. This study provides valuable information for further drug design and in developing more promising anticancer agents. 5. Experimental protocols 5.1. General information All of the solvents and reagents were purchased from Sigmae Aldrich. Melting points were determined in capillaries and are uncorrected. Nuclear Magnetic Resonance (1H and 13C NMR) spectra were recorded on Varian 300 MHz or Bruker WH 75 MHz spectrometers, using tetramethylsilane (TMS) as the internal standard. Chemical shifts were expressed in (ppm) down field from TMS. IR spectra were recorded on PerkineElmer model 683 or 1310 spectrometers with sodium chloride optics or KBr pellets with neat. ESI-MS was recorded on Thermo Finnigan LCQ ion trap mass spectrometer equipped with electron spray ionization. High Resolution Mass spectra were recorded on QSTAR mass spectrometer (Applied BioSystems, USA) at 5 or 7 K resolution using polyethylene glycol as an internal reference compound. Visualization of the spots on TLC plates was achieved either by exposure to UV (254 nm) light, iodine vapour and by dipping the plates in phosphomolybdic acidceric (IV) sulphate-sulphuric acid solution (PMA solution) and heating the plates at 120 C.Finally, we used 60e120 mesh silica gel in column chromatography. 5.2. General procedures for the synthesis of compounds (2aec) Phosphorus oxychloride (0.9 g, 8.1 mmol) was added to a flask containing N, N-dimethylformamide (5 mL) in an ice bath and stirred for 10 min. The ice bath was replaced with an ambient temperature water bath and stirred for an additional 10e15 min. The substituted benzosuberone (1.0 g, 6.2 mmol) was added and stirred for 15 min. The reaction mixture was heated to 80 C and stirred for an additional 3 h. The orange solution was diluted with ice water and neutralised with 20% sodium acetate solution and with diethyl ether (3 10e20 mL). The combined organic extracts were washed with saturated aqueous sodium bicarbonate solution (50 mL), brine 50 mL, and water (3 50 mL). The organic layer was dried with sodium sulphate, and concentrated in vacuum to yield a
5.2.1. 5-Chloro-8,9-dihydro-7H-benzocycloheptene-6carbaldehyde(2a) Brown liquid. Yield: 84.4% (5.4 g), IR (NEAT, v cm1): 2936, 2860, 1671, 1580, 1448, 1262, 919, 750, 1H NMR (300 MHz, CDCl3): d 2.11e 2.25 (m, 4H, 2CH2), 2.62 (t, J ¼ 6.4, 6.9 Hz, 2H, CH2), 2.60 (t, J ¼ 6.4, 6.6 Hz, 2H, CH2), 7.19e7.27 (m, 1H, AreH), 7.28e7.37 (m, 2H, AreH), 5.90e7.68 (m, 1H, AreH), 10.36 (s, 1H, CHO), 13C NMR (75 MHz, CDCl3): d 22.5, 32.0, 33.8, 96.2, 126.5, 126.7, 128.4, 128.8, 129.0, 130.5, 140.7, 189.3, ESI-MS: m/z ¼ 207 [M þ H]þ, 229 [M þ Na]þ. 5.2.2. 5-Chloro-2,3-dimethyl-8,9-dihydro-7H-benzocycloheptene6-carbaldehyde (2b) Brown liquid. Yield: 67.6% (4.7 g): IR (KBr, v cm1): 2932, 1670, 1582, 1447, 1266, 749, 1H NMR (300 MHz, CDCl3): d 2.07e2.12 (m, 2H, CH2), 2.21 (t, J ¼ 6.4, 3.5 Hz, 2H, CH2), 2.29 (s, 3H, CH3), 2.30 (s, 3H, CH3), 2.50 (t, J ¼ 6.6, 7.1 Hz, 2H, CH2), 6.69 (s, 1H, AreH), 7.38 (s, 1H, AreH), 10.33 (s, 1H, CHO), 13C NMR (75 MHz, CDCl3): d 19.3, 19.6, 22.6, 31.3, 33.7, 129.3, 130.2, 134.9, 135.0, 135.8, 138.5, 139.7, 147.8, 190.2, ESI-MS: m/z ¼ 235 [M þ Na]þ. 5.2.3. 5-Chloro-2,3-dimethoxy-8,9-dihydro-7Hbenzocycloheptene-6-carbaldehyde (2c) Brown liquid. Yield:79.9% (3.5 g): IR (KBr, v cm1): 2929, 2854, 1658, 1512, 1457, 1267, 1214, 1121, 859, 768, 1H NMR (300 MHz, CDCl3): d 2.11e2.18 (m, 2H, CH2), 2.23 (t, J ¼ 6.7 Hz, 2H, CH2), 2.55 (t, J ¼ 7.2, 6.7 Hz, 2H, CH2), 3.91 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 6.69 (s, 1H, AreH), 7.11 (s, 1H, AreH), 10.32 (s, 1H, CHO), 13C NMR (75 MHz, CDCl3): d 22.8, 31.8, 55.9, 56.0, 111.2, 111.6, 115.0, 129.4, 135.0, 135.8, 147.5,147.6,150.6,190.2, ESI-MS: m/z ¼ 267 [M þ H]þ, 289 [M þ Na]þ. 5.3. General procedures for the synthesis of compounds (3aec) The compounds 2aee (0.6 g, 2.9 mmol), boronicacid (0.4 g, 3.2 mmol), tetrabutylammonium bromide (0.9 g, 2.2 mmol), palladium acetate (2 mol%) and potassium carbonate (1.0 g, 7.2 mmol) were added to the round-bottom flask. Deionised H2O (5 mL) was added, and the reaction mixture was stirred vigorously for 3 h at 45 C. The reaction mixture turned dark and nonhomogenous. The mixture was diluted with 20 mL H2O, and the product was extracted with EtOAc (3 20 mL). The organic products were stirred over charcoal for 30 min followed by drying over sodium sulphate. Then the organic products were filtered and concentrated. The product was purified by column chromatography using 60e120 mesh silica gel (hexane/EtOAc). 5.3.1. 5-(2-Methoxy-phenyl)-8,9-dihydro-7H-benzocycloheptene6-carbaldehyde (3a) Pale Yellow semi-solid. Yield: 86.2% (2.24 g): IR (KBr, v cm1): 2934, 2856, 1664, 1596, 1457, 1244, 753. 1H NMR (300 MHz, CDCl3): d 2.58e2.26 (m, 2H, CH2), 2.49e2.6 (m, 1H, CH), 2.60e2.72 (m, 1H, CH), 2.79 (t, J ¼ 8.0 Hz, 1H, CH), 3.60 (s, 3H, OCH3), 6.79e6.91 (m, 2H, AreH), 6.94e7.02 (m, 1H, AreH), 7.06e7.23 (m, 4H, AreH), 7.31e7.42 (m,1H, AreH), 9.55 (s,1H, CHO), 13C NMR (75 MHz, CDCl3): d 21.4, 31.8, 34.5, 55.5, 109.8, 111.2, 120.0, 121.1, 125.8, 128.2, 128.6, 130.1, 132.4, 136.7, 138.6, 141.5, 156.3, 157.3, 192.7, ESI-MS: m/z ¼ 279 [M þ H]þ, HRMS calcd for C19H19O2[M þ H]þ: calcd 279.1379, Found 279.1373. 5.3.2. 2,3-Di methyl-5-(2-methoxy-phenyl)-8,9-dihydro-7Hbenzocycloheptene-carbaldehyde (3b) Pale yellow semi-solid. Yield: 90.7% (1.5 g) IR (KBr, v cm1): 3420, 2929, 2858, 1657, 1453, 1241, 1021, 758, 1H NMR (300 MHz, CDCl3): d 1.99e2.10 (m, 2H, CH2), 2.11 (s, 3H, CH3), 2.24 (s, 3H, CH3),
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2.45e2.64 (m, 2H, CH2), 2.65e2.77 (m, 2H, CH2), 3.63 (s, 3H, OCH3), 6.53 (s, 1H, AreH), 6.88 (d, J ¼ 8.3 Hz, 1H, AreH), 6.92e7.01 (m, 3H, AreH), 7.09 (dd, J ¼ 1.5 Hz, 1H, AreH), 7.30e7.39 (m, 1H, AreH), 13C NMR (75 MHz, CDCl3): d 13.6, 19.1, 19.7, 21.6, 24.1, 31.3, 34.5, 55.5, 111.1, 120.2, 129.4, 130.0, 129.9, 132.3, 133.8, 137.3, 138.3, 139.0, 156.6, 157.3, 192.7, ESI-MS: m/z ¼ 307[M þ H]þ, 329[M þ Na]þ, HRMS calcd for C21H23O2[M þ H]þ: calcd 307.1692, Found 307.1686. 5.3.3. 2,3-Dimethoxy-5-(2-methoxy-phenyl)-8,9-dihydro-7Hbenzocycloheptene-6-carbaldehyde (3c) Pale yellow semi solid. Yield: 79.1% (1.55 g): IR (KBr, v cm1): 2926, 2837, 1667, 1256, 1211, 1110, 1029, 870, 758, 1H NMR (300 MHz, CDCl3): d 2.11e2.25 (m, 1H, CH2), 2.24e2.62 (m, 2H, CH2), 2.65e2.78 (m, 2H, CH2), 3.62 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 6.29 (s, 1H, AreH), 6.74 (s, 1H, AreH), 6.89 (d, J ¼ 8.3 Hz, 1H, AreH), 6.97 (t, J ¼ 7.3 Hz, 1H, AreH), 7.11 (d, J ¼ 7.3 Hz, 1H, AreH), 7.34 (t, J ¼ 8.3, 9.6 Hz, 1H, AreH), 13C NMR (75 MHz, CDCl3): d 21.7, 31.7, 34.9, 55.4, 55.6, 55.7, 111.0, 111.8, 111.8, 120.0, 126.8, 130.0, 132.2, 132.7, 135.2, 138.2, 148.9, 156.3, 157.2, 192.4, ESIMS: m/z ¼ 339[M þ H]þ, 361[M þ Na]þ. HRMS calcd for C21H23O4 [M þ H]þ: calcd 339.1590, Found 339.1586. 5.4. General procedures for the synthesis of the compounds (4aec) Reactions were carried out by addition of aqueous NaClO2 (0.7 g, 7.92 mmol) to a solution of aldehyde 3aee (1.5 g, 4.5 mmol) and (0.16 g, 4.6 mmol) equiv of 34% H2O2 in aqueous acetonitrile, at 0 C, buffered with KH2PO4 at pH 4.3. The reaction mixture was stirred overnight. After complete reaction, a small amount of the Na2SO3 (w0.5 g) was added to destroy the unreacted HOCl and H2O2. Acidification with 10% HCl, the mixture was diluted with H2O (10e 20 mL). The compound was extracted with Et2O (10 mL). The organic layer was separated and extracted with water and brine and then dried over sodium sulphate and concentrated. The product was purified by column chromatography using 60e120 mesh silica gel (hexane/EtOAc). 5.4.1. 5-(2-Methoxy-phenyl)-8,9-dihydro-7H-benzocycloheptene6-carboxylicacid (4a) Yellow solid. Yield: 71.3% (1.2 g): mp 179 C: IR (KBr, v cm1): 2927, 2860, 1657, 1422, 1021, 752, 1H NMR (300 MHz, CDCl3): d 2.18e 2.26 (m, 2H, CH2), 2.32 (d, J ¼ 6.3, 2H, CH2), 2.76 (t, J ¼ 6.3 Hz, 2H, CH2), 3.60 (s, 3H, OCH3), 6.72 (d, J ¼ 8.4 Hz, 1H, AreH), 6.78e6.84 (m, 5H, AreH), 6.87 (t, J ¼ 7.3 Hz, 1H, AreH), 6.96 (t, J ¼ 7.3 Hz, 1H, Are H), 13C NMR (75 MHz, CDCl3): d 27.1, 31.4, 34.8, 55.4, 111.0, 112.1, 120.2, 125.9, 128.0, 128.2, 128.4, 128.7, 128.8, 128.9, 130.1, 130.3, 141.4, 147.8, 174.0. ESI-MS: m/z ¼ 295 [M þ H]þ, 317[M þ Na]þ, HRMS calcd for C19H19O3 [M þ H]þ: calcd 295.1328, Found 295.1324. 5.4.2. 2,3-Di methyl-5-(2-methoxy-phenyl)-8,9-dihydro-7Hbenzocycloheptene-6-carboxylic acid (4b) Yellow solid. Yield: 74.9% (1.7 g): mp 209 C: IR (KBr, v cm1): 2931, 2856, 1669, 1245, 751, 1H NMR (300 MHz, CDCl3): d 2.10 (s, 3H, CH3), 2.10e2.20 (m, 2H, CH2), 2.20 (s, 3H, CH3), 2.23 (t, J ¼ 6.6, 6.4 Hz, 2H, CH2), 2.65 (t, J ¼ 6.2, 6.6 Hz, 2H, CH2), 3.63 (s, 3H, OCH3), 6.52 (s, 1H, AreH), 6.70e6.96 (m, 4H, AreH), 7.15e7.24 (m, 1H, AreH), 13C NMR (75 MHz, CDCl3): d 19.2, 19.5, 27.1, 27.2, 30.9, 34.8, 55.4, 55.8, 111.0, 112.1, 120.2, 128.7, 129.6, 129.9, 130.3, 130.9, 133.7, 136.5, 138.9, 156.6, 174.1, ESI-MS: m/z ¼ 323[M þ H]þ, 345[M þ Na]þ, HRMS calcd for C21H22O3Na [M þ Na]þ: calcd 345.1461, Found 345.1454. 5.4.3. 2,3-Dimethoxy-5-(2-methoxy-phenyl)-8,9-dihydro-7Hbenzocycloheptene-6-carboxylic acid (4c) Yellow solid. Yield of 70.2%, mp 173 C: IR (KBr, v cm1): 2937, 2857, 1658, 1437, 1258, 1210, 1021, 755, 1H NMR (300 MHz, CDCl3):
d 1.97e2.09 (m, 1H, CH), 2.20 (t, J ¼ 6.6 Hz, 2H, CH2), 2.32 (d, J ¼ 6.4 Hz, 1H, CH), 2.68 (t, J ¼ 6.4, 6.2 Hz, 2H, CH2), 3.60 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.28 (s, 1H, AreH), 6.78e6.99 (m, 3H, AreH), 7.21 (d, J ¼ 6.6 Hz, 1H, AreH), 13C NMR (75 MHz, CDCl3): d 19.1, 27.4, 31.4, 35.4, 55.4, 55.7, 55.8, 111.3, 112.3, 120.2, 128.7, 128.9, 129.7, 130.3, 130.0, 130.8, 133.2, 135.0, 147.8, 148.5, 156.6, ESI-MS: m/z ¼ 355[M þ H]þ,377[M þ Na]þ, HRMS calcd for C21H22O5Na [M þ Na]þ: calcd 377.1354, Found 377.1350. 5.5. General procedures for the synthesis of the compounds (5aec) To a solution of acid as a starting material (0.1 g, 0.34 mmol) in DCM (5 mL), SOCl2 (1 mL) was added and the mixture was stirred at reflux conditions for 3 h. After formation of the corresponding acid chloride reaction as shown by the TLC, the excess SOCl2 was distilled out, then DCM (5 mL) and AlCl3 (0.09 g, 0.68 mmol) were added and the mixture was stirred at room temperature conditions for half an hour. The reaction mixture was then diluted with H2O (5 mL) and extracted with DCM (3 10 mL). The combined DCM extracts were washed with H2O (2 10 mL), the organic phase dried over Na2SO4 and filtered. The solvent was then evaporated under reduced pressure and the residue purified by column chromatography using 60e120 mesh silica gel (hexane/EtOAc). 5.5.1. 8,9-Dihydro-7H-5-oxa-benzo [6,7] cyclohepta [1,2-a] naphthalen-6-one (5a) Pale yellow solid. Yield: 83.4% (1.4 g): mp 130 C: IR (KBr, v cm1): 3447, 2929, 2858, 1716, 1603, 1159, 757, 1H NMR (300 MHz, CDCl3): d 1.83e1.96 (m, 1H, CH), 2.16e2.36 (m, 2H, CH2), 2.48e2.71 (m, 2H, CH2), 3.09 (dd, J ¼ 4.5, 5.2 Hz, 1H, CH), 7.13e7.55 (m, 8H, Are H), 13C NMR (75 MHz, CDCl3): d 23.5, 23.7, 31.0, 32.8, 116.9, 118.4, 123.0, 125.8, 126.2, 126.5, 128.6, 128.8, 129.2, 129.4, 129.7, 130.5, 141.1, ESI-MS: m/z ¼ 263[M þ H]þ, 285[M þ Na]þ, HRMS calcd for C18H14O2Na [M þ Na]þ: calcd 285.0900. Found 285.0910. 5.5.2. 2-Chloro-11, 12-di methyl-8,9-dihydro-7H-5-oxa-benzo [6,7] cyclohepta [1,2-a] naphthalen-6-one (5b) Colourless solid. Yield: 88.7% (78 g): mp 151 C: IR (KBr, v cm1): 2928, 2858, 1712, 1446, 1069, 755, 1H NMR (300 MHz, CDCl3): d 1.82e1.96 (m, 1H, CH), 2.1e2.29 (m, 2H, CH2), 2.24e2.26 (m, 2H, CH2), 3.06 (dd, J ¼ 5.2, 5.4 Hz, 1H, CH), 2.34 (s, 6H, 2CH3), 7.07e7.21 (m, 2H, AreH), 7.28e7.57 (m, 3H, AreH), 13C NMR (75 MHz, CDCl3): d 19.4, 19.6, 23.8, 23.9, 29.6, 30.6, 32.7, 116.9, 118.3, 123.8, 126.0, 129.6, 130.0, 130.4, 130.5, 130.7, 134.0, 138.1, 138.6, 153.4, ESI-MS: m/ z ¼ 325[M þ H]þ, 347[M þ Na]þ, HRMS calcd for C20H18O2Cl [M þ H]þ: calcd 325.0981, Found 325.0995. 5.5.3. 11,12-Dimethoxy-8,9-dihydro-7H-5-oxa-benzo [6,7] cyclohepta [1,2-a] naphthalen-6-one (5c) Pale yellow solid. Yield:81.5% (1.7 g): mp 200 C: IR (KBr, v cm1): 3394, 2934, 1708, 1510, 1215, 773, 1H NMR (300 MHz, CDCl3): d 1.86e1.98 (m, 1H, CH), 2.1e2.23 (m, 2H, CH2), 2.45e2.60 (m, 2H,eCH2), 2.19e3.15 (dd, J ¼ 3.7, 3.9 Hz, 1H, CH), 3.86 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.82 (s, 1H, AreH), 6.91 (s, 1H, AreH), 7.18 (t, J ¼ 6.9, 1H, AreH), 7.38 (d, J ¼ 7.3 Hz, 1H, AreH), 7.48 (t, J ¼ 6.9, 1H, AreH), 7.57 (d, J ¼ 7.9 Hz, 1H, AreH), 13C NMR (75 MHz, CDCl3): d 23.8, 30.8, 33.3, 55.1, 56.1, 112.1, 112.2, 117.0, 119.1, 123.8, 124.7, 125.3, 126.3, 130.5, 134.6, 146.8, 149.1, 149.5, 153.3, 161.7, ESI-MS: m/ z ¼ 323[M þ H], 345[M þ Na]þ, HRMS calcd for C20H19O4 [M þ H]þ: calcd 323.1270, Found 323.1283. Acknowledgements The authors are gratefully acknowledge DST-SERB/EMEQ-078/ 2013 for the financial support and the Council of Scientific and
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Industrial Research (CSIR), New Delhi for the award of fellowship to BY. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.04.015. References [1] E. Boyland, M.E. Boyland, Biochemical Journal 31 (1937) 457. [2] D.J. Chaplin, S.A. Hills, International Journal of Radiation Oncology Biology Physics 54 (2002) 1491. [3] G.F. Falasca, Clinics in Dermatology 24 (2006) 498. [4] J.E. Koenig, T. Senge, E.P. Allhoff, W. Koenig, Analysis of the inflammatory network in benign prostate hyperplasia and prostate cancer, Prostate 58 (2004) 121. [5] J. Crutze, K. Thomas, Jerchl, Dietrich, South African Patent 68 (1967) 173. [6] S. Chungwong, S. Sasso, H. Jones, J.J. Kaminski, Journal of Medicinal Chemistry 27 (1984) 20. [7] B. Rolf, K. Jorg, Ger. Offen, D. E, vol. 19, 2000, p. 833. [8] A. Rosowsky, K.K.N. Chen, M. Lin, M.E. Nadael, R. St. Amand, S.A. Yeager, Journal of the Chemical Society, Perkin Transactions 1 (1971) 789. [9] B. Alberto, L. Mario, M.M. Emilia, Farmaco 46 (1961) 461. [10] R. Murray, J. Mendez, S. Brown, The Natural Coumarins: Occurrence, Chemistry and Biochemistry, John Wiley and Sons, New York, 1982, p. 97.
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[11] T. Ma, L. Liu, H. Xue, L. Li, C. Han, L. Wang, Z. Chen, G. Liu, Journal of Medicinal Chemistry 51 (2008) 1432. [12] A.G. Kidane, H.A. Salacinski, K.R. Tiwari, A.M. Bruckdorfer, Biomolecules 5 (2004) 798. [13] G. Appendino, E. Mercalli, N. Fuzzati, L. Arnoldi, M. Stavri, S. Gibbons, M. Ballero, A. Maxia, Journal of Natural Products 67 (2004) 2108. [14] Y.Z. Yang, A. Ranz, H.Z. Pan, Z.N. Zhang, X.B. Lin, S.R. Meshnick, American Journal of Tropical Medicine and Hygiene 46 (1992) 15. [15] L. Nagarapu, H.K. Gaikwad, K. Sirikonda, J. Mateti, R. Bantu, P.S. Raghu, K.M. Mahduri, S.V. Kalivendi, European Journal of Medicinal Chemistry 46 (2010) 4720. [16] L. Nagarapu, J. Mateti, H.K. Gaikwad, R. Bantu, M. Shibarani, N.J.P. Shubhasini, Bioorganic & Medicinal Chemistry Letters 21 (2011) 4138. [17] L. Nagarapu, H.K. Gaikwad, R. Bantu, S.R. Manikonda, European Journal of Medicinal Chemistry 46 (2011) 2152. [18] L. Nagarapu, V. Paparaju, A. Satyenar, Bioorganic & Medicinal Chemistry Letters 18 (2008) 2351. [19] L. Nagarapu, A. Satyendar, B. Rajashaker, K. Srinivas, P. Ruparani, K. Rahika, G. Shubhashini, Bioorganic & Medicinal Chemistry Letters 18 (2008) 1167. [20] L. Nagarapu, S. Vanaparthi, R. Bantu, C.G. Kumar, European Journal of Medicinal Chemistry 69 (2013) 817. [21] L. Nagarapu, B. Yadagiri, R. Bantu, C.G. Kumar, S. Pombala, European Journal of Medicinal Chemistry 71 (2014) 91. [22] Z. Arnold, J. Zemlicka, Collection of Czechoslovak Chemical Communications 24 (1959) 2385. [23] Bruker, SAINT (Version 6.28a) & SMART (Version 5.625), Bruker AXS Inc, Madison, Wisconsin, USA, 2001. [24] G.M. Sheldrick, Acta Crystallographica A64 (2008) 112e122. [25] T. Mossman, Journal of Immunological Methods 65 (1983) 55.
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel building blocks of benzosuberone bearing coumarin moieties and their evaluation as potential anticancer agents Bandi Yadagiri a, Uma Devi Holagunda a, Rajashaker Bantu a, Lingaiah Nagarapu a, *, C. Ganesh Kumar b, Sujitha Pombala b, B. Sridhar c a b c
Organic Chemistry Division II, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India Centre for Medicinal Chemistry & Pharmacology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India Centre for X-ray Crystallography, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 December 2013 Received in revised form 19 March 2014 Accepted 4 April 2014 Available online 5 April 2014
A series of novel benzosuberone bearing coumarin moieties 5aec have been synthesized and their structures were determined by analytical and spectral (FT-IR, 1H NMR, 13C NMR, HRMS) studies. The newly synthesized compounds were evaluated for their cytotoxicity against four human cancer cell lines, A549 (Human alveolar adenocarcinoma cell line), HeLa (Human cervical cancer cell line), MDA-MB-231 (Human breast adenocarcinoma cell line), MCF-7 (Human breast adenocarcinoma cell line) and normal cell line HEK293 (Human embryonic kidney cell line). Compound 5a exhibited promising cytotoxicity with IC50 values ranging from 3.35 to 16.79 mM against all the cancer cell lines like A549, HeLa, MCF-7 and MDA-MB-231, while compound 5c showed significant cytotoxicity against HeLa and MDA-MB-231 with IC50 values of 6.72 and 4.87, respectively. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Benzosuberone Vilsmeier HaackeArnold reaction Cytotoxicity Cell lines
1. Introduction Colchicine, a major natural alkaloid derived from the plant Colchicum autumnal, is a benzosuberone derivative exhibiting antitumour activity, due to the binding of aromatic ring of colchicine with hydrophobic domain of tubulin. Colchicine was the first drug reported to function through a tubulin binding mechanism in the late 1930s [1]. However, colchicine was eventually withdrawn from clinical use for cancer treatment due to significant in vivo toxicity [2]. Nonetheless, colchicine has been used in the treatment of gout at very low doses since the late 1950s [3]. Recent investigations on benzosuberone-containing moiety showed that chemokine receptor CCR5, is expressed in prostate cancer specimens [4]. Benzosuberone derivatives also showed a wide range of biological activities like antimicrobial [5], anti-inflammatory [6], anti-estrogenic [7], anti-malarial [8] and anti-platelet aggregation [9] but their anticancer effects have been reported to be more potent against a majority of human cancer cell lines. Coumarins are an elite class of oxygen heterocycles, which occur naturally in many food sources including citrus fruits, herbs and vegetables [10]. Some important coumarin members have been
* Corresponding author. E-mail address: [email protected] (L. Nagarapu). http://dx.doi.org/10.1016/j.ejmech.2014.04.015 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.
isolated from microbial sources, e.g. novobiocin and coumermycin from Streptomyces, and aflatoxins from Aspergillus species and (þ)-Calanolide A, a natural product isolated from several tropical plants of the genus Calophyllum as shown in Fig. 1. Pharmacologically, they belong to neoflavonoid class of compounds, which have been found to exhibit a wide variety of biological activities, usually associated with low toxicity and have raised considerable interest because of their potential beneficial effects on human health. In the recent years, they have attracted immense interest due to their diverse pharmacological properties like anti-HIV [11] anticoagulant [12], anti-bacterial [13], and anti-malarial [14]. In addition, few coumarin derivatives have the special ability to show potent anti-tumour activity against several human tumour cell lines. The coumarins are extremely variable in structure, due to various types of substitutions in their basic structure, which in turn can influence their biological activity. In continuation to our ongoing research program [15e21] to discover and develop tumour growth inhibitors and apoptotic inducers as potential new anti-cancer agents, we have designed and synthesized novel benzosuberone-bearing coumarin moiety analogues (using VilsmeiereHaack and Suzuki cross-coupling reactions) and evaluated their in vitro cytotoxicity against different human cancer cell lines like A549, HeLa, MDA-MB-231 and MCF-7. Significantly, the compound 5a showed promising cytotoxicity against the HeLa and MCF-7 cancer cell lines with IC50 values of
B. Yadagiri et al. / European Journal of Medicinal Chemistry 79 (2014) 260e265
O
O
O
O
O
O O
O O
O
O
OH
HN
O
O
OH OH
O
261
O
O
N H OH
NH 2
O Colchicine
(+)- Calanolide A
Novabiocine
Fig. 1. Structures of some previously isolated important natural alkaloids.
3.35 and 9.63 mM, respectively, and compound 5c displayed the most potent selectivity against HeLa and MDA-MB-231 cell lines with IC50 values of 6.72 and 9.63 mM, respectively. 2. Results and discussion 2.1. Chemistry The detailed reaction conditions for the synthesis of crosscondensed coumarin moieties are illustrated in Scheme 1. The synthesis of compounds 2aec was easily carried out from the corresponding ketones by VilsmeiereHaackeArnold reaction [22]. The vinyl chlorides obtained above, were activated by the presence of an electron-withdrawing group, which in turn allowed their easy coupling with o-anicyl boronic acids in the presence of 2 mol % of palladium (II) acetate, tetrabutylammonium bromide and potassium carbonate, with water as the reaction medium at 45 C for 3 h afforded compound 3aec in 79.1e90.7% yields. Oxidation of aldehydes 3aec with sodium chlorite in the presence of 30% H2O2 in acetonitrile at room temperature allowed the formation of acids 4aec in 70.2e74.9% yields. Finally, the acids were converted to the corresponding acyl chlorides, followed by cyclization with aluminium chloride in dichloromethane at room temperature to furnish the desired tetra cyclic systems 5aec in 81.5e88.7% yields. All the new synthesized compounds were characterized using NMR, IR and mass spectrometry (see supporting information). Spectral data of all synthesized compounds were in good agreement with the proposed structures. IR spectrum revealed the presence of C]O bond in lactone (1712e1716 cm1) and CH (2928e2934 cm1) functional groups in the synthesized compounds. This was further confirmed by 1H NMR spectrum (300 MHz) of 5aec recorded in CDCl3 displayed the disappearance of eOCH3 substituent at d 3.30e3.40 ppm in the starting compound indicating that the cyclization occurred in the final step. Two additional triplets around d 2.43e3.41 ppm and multiplet at d 1.81e2.30 ppm in seven membered ring were observed. The compound 5b was obtained as colourless crystal after recrystallisation in ethanol and the structure was confirmed by X-ray crystallography. The X-ray diffraction analysis of the single crystal of 5b (Fig. 1) proved the presence of chlorine substitution at ring D; whereas the compounds 5a and 5c lack it; herein we studied the amount of catalyst in different equivalents. The molecular formula of all the synthesized compounds was confirmed by HRMS data analysis (Fig. 2). 2.2. X-ray crystallography and spectral studies 2.2.1. X-ray crystal data for compound AI41 (5b) Data were collected at room temperature using a Bruker Smart Apex CCD diffractometer with graphite monochromated MoKa
radiation (l ¼ 0.71073 A) with u-scan method [23]. Preliminary lattice parameters and orientation matrices were obtained from four sets of frames. Unit cell dimensions were determined using 5417 reflections in the range of 2.25 < q < 27.95 . Integration and scaling of intensity data were accomplished using SAINT [24] program. The structure was solved by Direct Methods using SHELXS97 [24] and refinement was carried out by full-matrix least-squares technique using SHELXL97 [24]. All hydrogen atoms were positioned geometrically and were treated as riding on their parent carbon atoms, with CeH distance of 0.93e0.98 A, with Uiso(H) ¼ 1.2Ueq (C) or 1.5Ueq(C). Crystal data for AI41: C20H17ClO2, M ¼ 324.79, colourless plate, 0.14 0 0.12 0 0.06 mm3, triclinic, space group P-1 (No. 2), a ¼ 9.8232 (11), b ¼ 9.9810 (11), c ¼ 18.362 (2) A, a ¼ 84.591 (2), b ¼ 81.371 (2), g ¼ 64.395 (2) , V ¼ 1604.2 (3) A3, Z ¼ 4, Dc ¼ 1.345 g/cm3, F000 ¼ 680, CCD Area Detector, MoKa radiation, l ¼ 0.71073 A, T ¼ 294 (2)K, 2qmax ¼ 50.0 , 15,411 reflections collected, 5635 unique (Rint ¼ 0.0156). Final GooF ¼ 1.030, R1 ¼ 0.0370, wR2 ¼ 0.1010, R indices based on 5037 reflections with I > 2s (I) (refinement on F2), 418 parameters, 0 restraints, m ¼ 0.245 mm1. Crystallographic data has been deposited for compound AI41 with the Cambridge Crystallographic Data Centre [CCDC No. 933434]. Copies of the data can be obtained free of charge at www.ccdc.cam. ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: þ44 (0) 1223 336 033; email: [email protected]]. 2.3. Effects of the compounds on the viability of human cancer cells The results of the assay are summarized in Table 1. To examine the effects of the compounds on the viability of different human cancer cell lines, the in vitro antitumour activity was evaluated against A549, HeLa, MDA-MB-231 and MCF-7 by the standard MTT assay method. The inhibitory activity was expressed in micromolar concentrations of the compound, which caused 50% inhibition (IC50 value) under the assay conditions and Doxorubicin was used as a reference drug. Data in Table 1 showed that the majority of the synthesized compounds displayed antitumour activities against the tested four human cancer cell lines, which could support the rationale of the drug design. An attempt was made to draw some considerations concerning the structureeactivity relationship (SAR). Compound 5a with no substitution on the benzene ring A (Scheme 1) exhibited promising antitumour effect against A549 and MCF-7 cell lines with IC50 values of 3.35 and 9.63 mM, respectively. Introduction of electron donating groups (eCH3, eOCH3) on benzene ring A was associated with a noticeable increase in the cytotoxic effect against all the four tested cell lines. On the contrary, compound 5b with an electron-donating group (eCl) at benzene ring D effected moderate anti-tumour activity against all the tested
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Scheme 1. Synthesis of compounds 5aec.
significantly more active than compounds 5a and 5b. Further, these compounds did not show any cytotoxicity against the normal cancer cell line, HEK293. Compounds 5a, 5b, and 5c differed structurally only at the ring A and ring D. This could suggest the importance of the substituted benzene ring A of this molecular modification for exhibiting the bioactivity. Finally, we conclude that the substitutions on ring A played an important role in the exhibiting cytotoxicity due its conjugation effect on the coumarin moiety (ring C) and were more favourable than having substitutions on ring D. 3. Pharmacology 3.1. In vitro cytotoxicity assay
Fig. 2. The molecular structure of 5b, with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. The asymmetric unit contains two crystallographically independent molecules (labelled with suffix A and B) and for clarity molecule B has been omitted.
cell lines as compared to the compounds 5a and 5c. Although, compound 5c with a stronger electron-donating group (-OCH3) at benzene ring A displayed promising anti-tumour activity as compared to doxorubicin with IC50 values of 6.72 and 4.87 mM against HeLa and MDA-MB-231 cell lines, respectively, it was
Cytotoxicity of all the synthesized compounds was determined on the basis of measurement of in vitro growth inhibition of tumour cell lines in 96 well plates by cell-mediated reduction of tetrazolium salt to water insoluble formazan crystals using doxorubicin as a standard. The cytotoxicity as assessed against a panel of four different human tumour cell lines: A549 derived from human alveolar adenocarcinoma epithelial cells (ATCC No. CCL-185), HeLa derived from human cervical cancer cells (ATCC No. CCL-2), MDAMB-231 derived from human breast adenocarcinoma cells (ATCC No. HTB26), MCF7 derived from human breast adenocarcinoma cells (ATCC No. HTB22) and HEK293 (normal human embryonic kidney cells) (ATCC No. CRL-1573) using the MTT assay [25]. The IC50 values (50% inhibitory concentration) were calculated from the plotted absorbance data for the doseeresponse curves. IC50 values (in mM) are indicated as means SD of three independent experiments.
B. Yadagiri et al. / European Journal of Medicinal Chemistry 79 (2014) 260e265 Table 1 IC50 of the tested compounds against human cancer and normal cell lines. Compounds
a b
red liquid. The product was purified by column chromatography using 60e120 mesh silica gel (hexane/EtOAc).
IC50 ina mM A549
5a 5b 5c Doxorubicinb
263
He La
MDA-MB-231 MCF-7
3.35 0.05 14.07 ± 0.07 16.79 ± 0.08 10.77 ± 0.04 12.69 ± 0.06 13.28 ± 0.07 >200 6.72 ± 0.05 4.87 ± 0.06 0.46 ± 0.02 0.51 ± 0.02 0.91 ± 0.02
9.63 ± 0.06 15.76 0.06 >200 1.07 ± 0.03
HEK293 >200 >200 >200 >200
Data presented is the mean value of three independent experiments. Positive control.
4. Conclusion In conclusion, we have reported a simple synthesis of novel benzosuberone-bearing coumarin compounds using Vilsmeiere Haack and Suzuki cross-coupling followed by cyclization and evaluated there in vitro cytotoxicity against HeLa, A549, MCF-7, MDA-MB-231 and normal HEK293 cell lines. All the synthesised compounds indicated significant growth inhibitory effects. In addition, the compounds 5a, 5b and 5c exhibited potent activity against the four tested human cancer cell lines. Moreover, the compound 5a was more potent against A549 and MCF-7 cell lines and compound 5c was selectively potent against HeLa and MDAMB-231 cell lines. Introduction of substitution on ring A was associated with a distinct tendency to increase the in vitro cytotoxic potential. This study provides valuable information for further drug design and in developing more promising anticancer agents. 5. Experimental protocols 5.1. General information All of the solvents and reagents were purchased from Sigmae Aldrich. Melting points were determined in capillaries and are uncorrected. Nuclear Magnetic Resonance (1H and 13C NMR) spectra were recorded on Varian 300 MHz or Bruker WH 75 MHz spectrometers, using tetramethylsilane (TMS) as the internal standard. Chemical shifts were expressed in (ppm) down field from TMS. IR spectra were recorded on PerkineElmer model 683 or 1310 spectrometers with sodium chloride optics or KBr pellets with neat. ESI-MS was recorded on Thermo Finnigan LCQ ion trap mass spectrometer equipped with electron spray ionization. High Resolution Mass spectra were recorded on QSTAR mass spectrometer (Applied BioSystems, USA) at 5 or 7 K resolution using polyethylene glycol as an internal reference compound. Visualization of the spots on TLC plates was achieved either by exposure to UV (254 nm) light, iodine vapour and by dipping the plates in phosphomolybdic acidceric (IV) sulphate-sulphuric acid solution (PMA solution) and heating the plates at 120 C.Finally, we used 60e120 mesh silica gel in column chromatography. 5.2. General procedures for the synthesis of compounds (2aec) Phosphorus oxychloride (0.9 g, 8.1 mmol) was added to a flask containing N, N-dimethylformamide (5 mL) in an ice bath and stirred for 10 min. The ice bath was replaced with an ambient temperature water bath and stirred for an additional 10e15 min. The substituted benzosuberone (1.0 g, 6.2 mmol) was added and stirred for 15 min. The reaction mixture was heated to 80 C and stirred for an additional 3 h. The orange solution was diluted with ice water and neutralised with 20% sodium acetate solution and with diethyl ether (3 10e20 mL). The combined organic extracts were washed with saturated aqueous sodium bicarbonate solution (50 mL), brine 50 mL, and water (3 50 mL). The organic layer was dried with sodium sulphate, and concentrated in vacuum to yield a
5.2.1. 5-Chloro-8,9-dihydro-7H-benzocycloheptene-6carbaldehyde(2a) Brown liquid. Yield: 84.4% (5.4 g), IR (NEAT, v cm1): 2936, 2860, 1671, 1580, 1448, 1262, 919, 750, 1H NMR (300 MHz, CDCl3): d 2.11e 2.25 (m, 4H, 2CH2), 2.62 (t, J ¼ 6.4, 6.9 Hz, 2H, CH2), 2.60 (t, J ¼ 6.4, 6.6 Hz, 2H, CH2), 7.19e7.27 (m, 1H, AreH), 7.28e7.37 (m, 2H, AreH), 5.90e7.68 (m, 1H, AreH), 10.36 (s, 1H, CHO), 13C NMR (75 MHz, CDCl3): d 22.5, 32.0, 33.8, 96.2, 126.5, 126.7, 128.4, 128.8, 129.0, 130.5, 140.7, 189.3, ESI-MS: m/z ¼ 207 [M þ H]þ, 229 [M þ Na]þ. 5.2.2. 5-Chloro-2,3-dimethyl-8,9-dihydro-7H-benzocycloheptene6-carbaldehyde (2b) Brown liquid. Yield: 67.6% (4.7 g): IR (KBr, v cm1): 2932, 1670, 1582, 1447, 1266, 749, 1H NMR (300 MHz, CDCl3): d 2.07e2.12 (m, 2H, CH2), 2.21 (t, J ¼ 6.4, 3.5 Hz, 2H, CH2), 2.29 (s, 3H, CH3), 2.30 (s, 3H, CH3), 2.50 (t, J ¼ 6.6, 7.1 Hz, 2H, CH2), 6.69 (s, 1H, AreH), 7.38 (s, 1H, AreH), 10.33 (s, 1H, CHO), 13C NMR (75 MHz, CDCl3): d 19.3, 19.6, 22.6, 31.3, 33.7, 129.3, 130.2, 134.9, 135.0, 135.8, 138.5, 139.7, 147.8, 190.2, ESI-MS: m/z ¼ 235 [M þ Na]þ. 5.2.3. 5-Chloro-2,3-dimethoxy-8,9-dihydro-7Hbenzocycloheptene-6-carbaldehyde (2c) Brown liquid. Yield:79.9% (3.5 g): IR (KBr, v cm1): 2929, 2854, 1658, 1512, 1457, 1267, 1214, 1121, 859, 768, 1H NMR (300 MHz, CDCl3): d 2.11e2.18 (m, 2H, CH2), 2.23 (t, J ¼ 6.7 Hz, 2H, CH2), 2.55 (t, J ¼ 7.2, 6.7 Hz, 2H, CH2), 3.91 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 6.69 (s, 1H, AreH), 7.11 (s, 1H, AreH), 10.32 (s, 1H, CHO), 13C NMR (75 MHz, CDCl3): d 22.8, 31.8, 55.9, 56.0, 111.2, 111.6, 115.0, 129.4, 135.0, 135.8, 147.5,147.6,150.6,190.2, ESI-MS: m/z ¼ 267 [M þ H]þ, 289 [M þ Na]þ. 5.3. General procedures for the synthesis of compounds (3aec) The compounds 2aee (0.6 g, 2.9 mmol), boronicacid (0.4 g, 3.2 mmol), tetrabutylammonium bromide (0.9 g, 2.2 mmol), palladium acetate (2 mol%) and potassium carbonate (1.0 g, 7.2 mmol) were added to the round-bottom flask. Deionised H2O (5 mL) was added, and the reaction mixture was stirred vigorously for 3 h at 45 C. The reaction mixture turned dark and nonhomogenous. The mixture was diluted with 20 mL H2O, and the product was extracted with EtOAc (3 20 mL). The organic products were stirred over charcoal for 30 min followed by drying over sodium sulphate. Then the organic products were filtered and concentrated. The product was purified by column chromatography using 60e120 mesh silica gel (hexane/EtOAc). 5.3.1. 5-(2-Methoxy-phenyl)-8,9-dihydro-7H-benzocycloheptene6-carbaldehyde (3a) Pale Yellow semi-solid. Yield: 86.2% (2.24 g): IR (KBr, v cm1): 2934, 2856, 1664, 1596, 1457, 1244, 753. 1H NMR (300 MHz, CDCl3): d 2.58e2.26 (m, 2H, CH2), 2.49e2.6 (m, 1H, CH), 2.60e2.72 (m, 1H, CH), 2.79 (t, J ¼ 8.0 Hz, 1H, CH), 3.60 (s, 3H, OCH3), 6.79e6.91 (m, 2H, AreH), 6.94e7.02 (m, 1H, AreH), 7.06e7.23 (m, 4H, AreH), 7.31e7.42 (m,1H, AreH), 9.55 (s,1H, CHO), 13C NMR (75 MHz, CDCl3): d 21.4, 31.8, 34.5, 55.5, 109.8, 111.2, 120.0, 121.1, 125.8, 128.2, 128.6, 130.1, 132.4, 136.7, 138.6, 141.5, 156.3, 157.3, 192.7, ESI-MS: m/z ¼ 279 [M þ H]þ, HRMS calcd for C19H19O2[M þ H]þ: calcd 279.1379, Found 279.1373. 5.3.2. 2,3-Di methyl-5-(2-methoxy-phenyl)-8,9-dihydro-7Hbenzocycloheptene-carbaldehyde (3b) Pale yellow semi-solid. Yield: 90.7% (1.5 g) IR (KBr, v cm1): 3420, 2929, 2858, 1657, 1453, 1241, 1021, 758, 1H NMR (300 MHz, CDCl3): d 1.99e2.10 (m, 2H, CH2), 2.11 (s, 3H, CH3), 2.24 (s, 3H, CH3),
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2.45e2.64 (m, 2H, CH2), 2.65e2.77 (m, 2H, CH2), 3.63 (s, 3H, OCH3), 6.53 (s, 1H, AreH), 6.88 (d, J ¼ 8.3 Hz, 1H, AreH), 6.92e7.01 (m, 3H, AreH), 7.09 (dd, J ¼ 1.5 Hz, 1H, AreH), 7.30e7.39 (m, 1H, AreH), 13C NMR (75 MHz, CDCl3): d 13.6, 19.1, 19.7, 21.6, 24.1, 31.3, 34.5, 55.5, 111.1, 120.2, 129.4, 130.0, 129.9, 132.3, 133.8, 137.3, 138.3, 139.0, 156.6, 157.3, 192.7, ESI-MS: m/z ¼ 307[M þ H]þ, 329[M þ Na]þ, HRMS calcd for C21H23O2[M þ H]þ: calcd 307.1692, Found 307.1686. 5.3.3. 2,3-Dimethoxy-5-(2-methoxy-phenyl)-8,9-dihydro-7Hbenzocycloheptene-6-carbaldehyde (3c) Pale yellow semi solid. Yield: 79.1% (1.55 g): IR (KBr, v cm1): 2926, 2837, 1667, 1256, 1211, 1110, 1029, 870, 758, 1H NMR (300 MHz, CDCl3): d 2.11e2.25 (m, 1H, CH2), 2.24e2.62 (m, 2H, CH2), 2.65e2.78 (m, 2H, CH2), 3.62 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 6.29 (s, 1H, AreH), 6.74 (s, 1H, AreH), 6.89 (d, J ¼ 8.3 Hz, 1H, AreH), 6.97 (t, J ¼ 7.3 Hz, 1H, AreH), 7.11 (d, J ¼ 7.3 Hz, 1H, AreH), 7.34 (t, J ¼ 8.3, 9.6 Hz, 1H, AreH), 13C NMR (75 MHz, CDCl3): d 21.7, 31.7, 34.9, 55.4, 55.6, 55.7, 111.0, 111.8, 111.8, 120.0, 126.8, 130.0, 132.2, 132.7, 135.2, 138.2, 148.9, 156.3, 157.2, 192.4, ESIMS: m/z ¼ 339[M þ H]þ, 361[M þ Na]þ. HRMS calcd for C21H23O4 [M þ H]þ: calcd 339.1590, Found 339.1586. 5.4. General procedures for the synthesis of the compounds (4aec) Reactions were carried out by addition of aqueous NaClO2 (0.7 g, 7.92 mmol) to a solution of aldehyde 3aee (1.5 g, 4.5 mmol) and (0.16 g, 4.6 mmol) equiv of 34% H2O2 in aqueous acetonitrile, at 0 C, buffered with KH2PO4 at pH 4.3. The reaction mixture was stirred overnight. After complete reaction, a small amount of the Na2SO3 (w0.5 g) was added to destroy the unreacted HOCl and H2O2. Acidification with 10% HCl, the mixture was diluted with H2O (10e 20 mL). The compound was extracted with Et2O (10 mL). The organic layer was separated and extracted with water and brine and then dried over sodium sulphate and concentrated. The product was purified by column chromatography using 60e120 mesh silica gel (hexane/EtOAc). 5.4.1. 5-(2-Methoxy-phenyl)-8,9-dihydro-7H-benzocycloheptene6-carboxylicacid (4a) Yellow solid. Yield: 71.3% (1.2 g): mp 179 C: IR (KBr, v cm1): 2927, 2860, 1657, 1422, 1021, 752, 1H NMR (300 MHz, CDCl3): d 2.18e 2.26 (m, 2H, CH2), 2.32 (d, J ¼ 6.3, 2H, CH2), 2.76 (t, J ¼ 6.3 Hz, 2H, CH2), 3.60 (s, 3H, OCH3), 6.72 (d, J ¼ 8.4 Hz, 1H, AreH), 6.78e6.84 (m, 5H, AreH), 6.87 (t, J ¼ 7.3 Hz, 1H, AreH), 6.96 (t, J ¼ 7.3 Hz, 1H, Are H), 13C NMR (75 MHz, CDCl3): d 27.1, 31.4, 34.8, 55.4, 111.0, 112.1, 120.2, 125.9, 128.0, 128.2, 128.4, 128.7, 128.8, 128.9, 130.1, 130.3, 141.4, 147.8, 174.0. ESI-MS: m/z ¼ 295 [M þ H]þ, 317[M þ Na]þ, HRMS calcd for C19H19O3 [M þ H]þ: calcd 295.1328, Found 295.1324. 5.4.2. 2,3-Di methyl-5-(2-methoxy-phenyl)-8,9-dihydro-7Hbenzocycloheptene-6-carboxylic acid (4b) Yellow solid. Yield: 74.9% (1.7 g): mp 209 C: IR (KBr, v cm1): 2931, 2856, 1669, 1245, 751, 1H NMR (300 MHz, CDCl3): d 2.10 (s, 3H, CH3), 2.10e2.20 (m, 2H, CH2), 2.20 (s, 3H, CH3), 2.23 (t, J ¼ 6.6, 6.4 Hz, 2H, CH2), 2.65 (t, J ¼ 6.2, 6.6 Hz, 2H, CH2), 3.63 (s, 3H, OCH3), 6.52 (s, 1H, AreH), 6.70e6.96 (m, 4H, AreH), 7.15e7.24 (m, 1H, AreH), 13C NMR (75 MHz, CDCl3): d 19.2, 19.5, 27.1, 27.2, 30.9, 34.8, 55.4, 55.8, 111.0, 112.1, 120.2, 128.7, 129.6, 129.9, 130.3, 130.9, 133.7, 136.5, 138.9, 156.6, 174.1, ESI-MS: m/z ¼ 323[M þ H]þ, 345[M þ Na]þ, HRMS calcd for C21H22O3Na [M þ Na]þ: calcd 345.1461, Found 345.1454. 5.4.3. 2,3-Dimethoxy-5-(2-methoxy-phenyl)-8,9-dihydro-7Hbenzocycloheptene-6-carboxylic acid (4c) Yellow solid. Yield of 70.2%, mp 173 C: IR (KBr, v cm1): 2937, 2857, 1658, 1437, 1258, 1210, 1021, 755, 1H NMR (300 MHz, CDCl3):
d 1.97e2.09 (m, 1H, CH), 2.20 (t, J ¼ 6.6 Hz, 2H, CH2), 2.32 (d, J ¼ 6.4 Hz, 1H, CH), 2.68 (t, J ¼ 6.4, 6.2 Hz, 2H, CH2), 3.60 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.28 (s, 1H, AreH), 6.78e6.99 (m, 3H, AreH), 7.21 (d, J ¼ 6.6 Hz, 1H, AreH), 13C NMR (75 MHz, CDCl3): d 19.1, 27.4, 31.4, 35.4, 55.4, 55.7, 55.8, 111.3, 112.3, 120.2, 128.7, 128.9, 129.7, 130.3, 130.0, 130.8, 133.2, 135.0, 147.8, 148.5, 156.6, ESI-MS: m/z ¼ 355[M þ H]þ,377[M þ Na]þ, HRMS calcd for C21H22O5Na [M þ Na]þ: calcd 377.1354, Found 377.1350. 5.5. General procedures for the synthesis of the compounds (5aec) To a solution of acid as a starting material (0.1 g, 0.34 mmol) in DCM (5 mL), SOCl2 (1 mL) was added and the mixture was stirred at reflux conditions for 3 h. After formation of the corresponding acid chloride reaction as shown by the TLC, the excess SOCl2 was distilled out, then DCM (5 mL) and AlCl3 (0.09 g, 0.68 mmol) were added and the mixture was stirred at room temperature conditions for half an hour. The reaction mixture was then diluted with H2O (5 mL) and extracted with DCM (3 10 mL). The combined DCM extracts were washed with H2O (2 10 mL), the organic phase dried over Na2SO4 and filtered. The solvent was then evaporated under reduced pressure and the residue purified by column chromatography using 60e120 mesh silica gel (hexane/EtOAc). 5.5.1. 8,9-Dihydro-7H-5-oxa-benzo [6,7] cyclohepta [1,2-a] naphthalen-6-one (5a) Pale yellow solid. Yield: 83.4% (1.4 g): mp 130 C: IR (KBr, v cm1): 3447, 2929, 2858, 1716, 1603, 1159, 757, 1H NMR (300 MHz, CDCl3): d 1.83e1.96 (m, 1H, CH), 2.16e2.36 (m, 2H, CH2), 2.48e2.71 (m, 2H, CH2), 3.09 (dd, J ¼ 4.5, 5.2 Hz, 1H, CH), 7.13e7.55 (m, 8H, Are H), 13C NMR (75 MHz, CDCl3): d 23.5, 23.7, 31.0, 32.8, 116.9, 118.4, 123.0, 125.8, 126.2, 126.5, 128.6, 128.8, 129.2, 129.4, 129.7, 130.5, 141.1, ESI-MS: m/z ¼ 263[M þ H]þ, 285[M þ Na]þ, HRMS calcd for C18H14O2Na [M þ Na]þ: calcd 285.0900. Found 285.0910. 5.5.2. 2-Chloro-11, 12-di methyl-8,9-dihydro-7H-5-oxa-benzo [6,7] cyclohepta [1,2-a] naphthalen-6-one (5b) Colourless solid. Yield: 88.7% (78 g): mp 151 C: IR (KBr, v cm1): 2928, 2858, 1712, 1446, 1069, 755, 1H NMR (300 MHz, CDCl3): d 1.82e1.96 (m, 1H, CH), 2.1e2.29 (m, 2H, CH2), 2.24e2.26 (m, 2H, CH2), 3.06 (dd, J ¼ 5.2, 5.4 Hz, 1H, CH), 2.34 (s, 6H, 2CH3), 7.07e7.21 (m, 2H, AreH), 7.28e7.57 (m, 3H, AreH), 13C NMR (75 MHz, CDCl3): d 19.4, 19.6, 23.8, 23.9, 29.6, 30.6, 32.7, 116.9, 118.3, 123.8, 126.0, 129.6, 130.0, 130.4, 130.5, 130.7, 134.0, 138.1, 138.6, 153.4, ESI-MS: m/ z ¼ 325[M þ H]þ, 347[M þ Na]þ, HRMS calcd for C20H18O2Cl [M þ H]þ: calcd 325.0981, Found 325.0995. 5.5.3. 11,12-Dimethoxy-8,9-dihydro-7H-5-oxa-benzo [6,7] cyclohepta [1,2-a] naphthalen-6-one (5c) Pale yellow solid. Yield:81.5% (1.7 g): mp 200 C: IR (KBr, v cm1): 3394, 2934, 1708, 1510, 1215, 773, 1H NMR (300 MHz, CDCl3): d 1.86e1.98 (m, 1H, CH), 2.1e2.23 (m, 2H, CH2), 2.45e2.60 (m, 2H,eCH2), 2.19e3.15 (dd, J ¼ 3.7, 3.9 Hz, 1H, CH), 3.86 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.82 (s, 1H, AreH), 6.91 (s, 1H, AreH), 7.18 (t, J ¼ 6.9, 1H, AreH), 7.38 (d, J ¼ 7.3 Hz, 1H, AreH), 7.48 (t, J ¼ 6.9, 1H, AreH), 7.57 (d, J ¼ 7.9 Hz, 1H, AreH), 13C NMR (75 MHz, CDCl3): d 23.8, 30.8, 33.3, 55.1, 56.1, 112.1, 112.2, 117.0, 119.1, 123.8, 124.7, 125.3, 126.3, 130.5, 134.6, 146.8, 149.1, 149.5, 153.3, 161.7, ESI-MS: m/ z ¼ 323[M þ H], 345[M þ Na]þ, HRMS calcd for C20H19O4 [M þ H]þ: calcd 323.1270, Found 323.1283. Acknowledgements The authors are gratefully acknowledge DST-SERB/EMEQ-078/ 2013 for the financial support and the Council of Scientific and
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Industrial Research (CSIR), New Delhi for the award of fellowship to BY. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.04.015. References [1] E. Boyland, M.E. Boyland, Biochemical Journal 31 (1937) 457. [2] D.J. Chaplin, S.A. Hills, International Journal of Radiation Oncology Biology Physics 54 (2002) 1491. [3] G.F. Falasca, Clinics in Dermatology 24 (2006) 498. [4] J.E. Koenig, T. Senge, E.P. Allhoff, W. Koenig, Analysis of the inflammatory network in benign prostate hyperplasia and prostate cancer, Prostate 58 (2004) 121. [5] J. Crutze, K. Thomas, Jerchl, Dietrich, South African Patent 68 (1967) 173. [6] S. Chungwong, S. Sasso, H. Jones, J.J. Kaminski, Journal of Medicinal Chemistry 27 (1984) 20. [7] B. Rolf, K. Jorg, Ger. Offen, D. E, vol. 19, 2000, p. 833. [8] A. Rosowsky, K.K.N. Chen, M. Lin, M.E. Nadael, R. St. Amand, S.A. Yeager, Journal of the Chemical Society, Perkin Transactions 1 (1971) 789. [9] B. Alberto, L. Mario, M.M. Emilia, Farmaco 46 (1961) 461. [10] R. Murray, J. Mendez, S. Brown, The Natural Coumarins: Occurrence, Chemistry and Biochemistry, John Wiley and Sons, New York, 1982, p. 97.
265
[11] T. Ma, L. Liu, H. Xue, L. Li, C. Han, L. Wang, Z. Chen, G. Liu, Journal of Medicinal Chemistry 51 (2008) 1432. [12] A.G. Kidane, H.A. Salacinski, K.R. Tiwari, A.M. Bruckdorfer, Biomolecules 5 (2004) 798. [13] G. Appendino, E. Mercalli, N. Fuzzati, L. Arnoldi, M. Stavri, S. Gibbons, M. Ballero, A. Maxia, Journal of Natural Products 67 (2004) 2108. [14] Y.Z. Yang, A. Ranz, H.Z. Pan, Z.N. Zhang, X.B. Lin, S.R. Meshnick, American Journal of Tropical Medicine and Hygiene 46 (1992) 15. [15] L. Nagarapu, H.K. Gaikwad, K. Sirikonda, J. Mateti, R. Bantu, P.S. Raghu, K.M. Mahduri, S.V. Kalivendi, European Journal of Medicinal Chemistry 46 (2010) 4720. [16] L. Nagarapu, J. Mateti, H.K. Gaikwad, R. Bantu, M. Shibarani, N.J.P. Shubhasini, Bioorganic & Medicinal Chemistry Letters 21 (2011) 4138. [17] L. Nagarapu, H.K. Gaikwad, R. Bantu, S.R. Manikonda, European Journal of Medicinal Chemistry 46 (2011) 2152. [18] L. Nagarapu, V. Paparaju, A. Satyenar, Bioorganic & Medicinal Chemistry Letters 18 (2008) 2351. [19] L. Nagarapu, A. Satyendar, B. Rajashaker, K. Srinivas, P. Ruparani, K. Rahika, G. Shubhashini, Bioorganic & Medicinal Chemistry Letters 18 (2008) 1167. [20] L. Nagarapu, S. Vanaparthi, R. Bantu, C.G. Kumar, European Journal of Medicinal Chemistry 69 (2013) 817. [21] L. Nagarapu, B. Yadagiri, R. Bantu, C.G. Kumar, S. Pombala, European Journal of Medicinal Chemistry 71 (2014) 91. [22] Z. Arnold, J. Zemlicka, Collection of Czechoslovak Chemical Communications 24 (1959) 2385. [23] Bruker, SAINT (Version 6.28a) & SMART (Version 5.625), Bruker AXS Inc, Madison, Wisconsin, USA, 2001. [24] G.M. Sheldrick, Acta Crystallographica A64 (2008) 112e122. [25] T. Mossman, Journal of Immunological Methods 65 (1983) 55.
