GU Wen MIAO Ting-Ting WANG Shi-Fa HAO Yun ZHANG Kang-Ping JIN Xiao-Yan

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Synthesis, Crystal Structure and Antitumor Activity of a New 3-Phenanthro-[2,1-]imidazole Derivative of Dehydroabietic Acid①

GU Wen②MIAO Ting-Ting WANG Shi-Fa HAO Yun ZHANG Kang-Ping JIN Xiao-Yan

(210037)

The title compound (C19H24BrN3O2, 3) was synthesized from dehydroabietic acid and its crystal structure was determined by single-crystal X-ray diffraction. The compound is of monoclinic system, space group2 with= 24.830(5),= 7.1410(14),= 13.981(3) ?,= 107.68(3)°,= 4,= 2361.9(8) ?3,M= 482.42,D= 1.357 Mg/m3,= 1.003 ,= 1.772 mm-1,(000) = 1008, the final= 0.0660 and= 0.1402 for 2244 observed reflections (> 2()). The crystal structure is stabilized by four hydrogen bonds (O(W)–H(WA)···O(3), O(W)–H(WB)···O(1), N(2)–H(2A)···O(W) and N(3)–H(3A)···N(1)) formed among the title compound, the crystal water and acetone molecules. The preliminary antitumor assay showed the title compound exhibited considerable inhibitory activity against HepG2 and SMMC-7721 cell lines with the IC50values of 17.1 and 10.2 μM, respectively.

dehydroabietic acid, 3-phenanthro[2,1-]imidazole, crystal structure, antitumor activity;

1 INTRODUCTION

As an important class of nitrogen-containing heterocycles, imidazole constitutes numerous natural and synthetic molecules with diverse structures[1]. The structural feature of imidazole ring with desirable electron-rich property is beneficial for imidazole derivatives to bind with a variety of enzymes and receptors in biological systems, thereby exhibiting broad bioactivities[2]. Imidazole derivatives were found to possess a wide range of biological and pharmacological properties including anticancer[3], antimicrobial[4], antiviral[5], anti- inflammatory[6], antihypertensive[7], anticonvulsant[8], antihistaminic[9], antiparasitic[10]and antiobesity[11]activities. The research on imidazole-based medi- cinal chemistry has become a rapidly developing and increasingly highlighted topic in recent years[2].

Dehydroabietic acid (DAA, 1), a major com- ponent of natural diterpene resin acids, can be readily obtained fromrosin or commercial disproportionated rosin. In recent years, its natural or synthetic derivatives have attracted great interest for their broad-spectrum biological activities, such as antimicrobial, antitumor, antiulcer, antioxidant and BK-channel opening activities[12-16], which indicate its potential as a starting material for the discovery of new pharmacologically important products. In continuation of our studies on novel bioactive heterocyclic dehydroabietate derivatives[17, 18], we are focusing on the introduction of imidazole moiety into the molecule. In this study, we report the synthesis, characterization and crystal structure of a new imidazole derivative of DAA. In addition, theantitumor activity of the title compound is also presented.

2 EXPERIMENTAL

2. 1 Reagents and measurements

The melting point was determined by means of an XT-4 apparatus (Taike Corp., Beijing, China) without correction. IR spectrum was recorded on a Nexus 870 FT-IR spectrometer. The ESI-MS spec- trum was measured on a Mariner System 5304 mass spectrometer. NMR spectra were accomplished in CDCl3on a Bruker AV-500 spectrometer using TMS as the internal standard. Elemental analyses were performed on a CHN-O-Rapid instrument within ±0.4% of the theoretical values. Reactions were monitored by TLC which was carried out on TLC Silica gel 60 F254sheets from EMD Millipore Co., USA and visualized in UV light (254 and 365 nm). Silica gel (300～400 mesh) for column chroma- tography was purchased from Qingdao Marine Chemical Factory, China. The reagents and chemi- cals of AR grade were purchased from commercial suppliers and used without further purification. Disproportionated rosin was provided by Zhongbang Chemicals Co., Ltd. (Zhaoqing, China), from which dehydroabietic acid (97%) was isolated according to the published method[19].

2. 2 Synthesis of the title compound (3)

The starting material (1) was synthesized from dehydroabietic acid according to the procedure previously reported[20], which was further treated as follows to afford compounds 2 and 3 (Scheme 1). To a solution of compound 1 (0.44 g, 1.0 mmol) in 20 mL of EtOH was added reduced iron powder (0.6 g, 10.6 mmol), H2O (2mL) and 10 drops of con- centrated HCl. The mixture was stirred under reflux for 4 h. After cooling, the mixture was filtered to remove the iron powder. The solution was neutra- lized with aqueous NaOH (2 mol/L) and then concentrated. The residue was purified by column chromatography on silica gel, eluting with CH2Cl2-MeOH (20:1, v/v) to give compound 2 as a yellow resin (0.20 g, yield 52%). The spectral data of the product were in accordance with those of the previous literature[20].

To a solution of compound 2 (0.18 g, 0.5 mmol) in EtOH (10 mL) was added BrCN (0.26 g, 2.5 mmol) and H2O (5 mL). The mixture was stirred under reflux for 5 h. Thereafter, the solution was concentrated, and the residue was extracted with CH2Cl2(3 × 50 mL). The organic layer was combined, washed with water and brine, dried over anhydrous Na2SO4and concentrated. The residue was purified by column chromatography on silica gel, and eluted with petroleum ether-acetone (3:1, v/v) to afford compound 5 as a light brown powder (0.12 g, 0.3 mmol). The solid was recrystallized in acetone to yield yellowish prisms suitable for X-ray analysis. Yield 60%; m.p.: 218～220 ℃. Anal. Calcd. (%) for C19H24BrN3O2: C, 56.28; H, 5.97; N, 10.37. Found (%): C, 56.32; H, 5.99; N, 10.34. IR (KBr):3360, 3150, 3051, 2931, 2863, 1724, 1642, 1558, 1456, 1255, 1129, 736 cm-1.1H-NMR (CDCl3, 500 MHz):1.22 (s, 3H), 1.28 (s, 3H), 1.49 (m, 2H), 1.60～1.95 (m, 4H), 2.17～2.31 (m, 3H), 2.84 (m, 2H), 3.67 (s, 3H, COOCH3), 5.02 (brs, 3H), 7.11 (s, 1H, H-7); ESI-MS/406.1, 408.1 [M+H]+.

2. 3 X-ray structure determination

A yellowish single crystal of the title compound with dimensions of 0.20mm ×0.10mm×0.10mm was mounted on the top of a glass fiber. X-ray diffraction data were collected using an Enraf-Nonius CAD-4 diffractometer equipped with graphite-monochromated Moradiation (= 0.71073 ?) by using an/2scan mode in the range of 1.53≤≤25.38°(0≤≤29,-8≤≤8,-16≤≤16) at 293(2) K. A total of 4450 reflections were collected, of which 4344 were independent (int= 0.0948) and 2244 were observed with> 2(). The structure was solved by direct methods using SHELXS-97[21]and refined by full-matrix least- squares procedure on2with SHELXL-97[22]. All non-hydrogen atoms were refined anisotropically, and hydrogen atoms were positioned geometrically. The final refinement gave= 0.0660,= 0.1402 (= 1/[2(2) + (0.0570)2], where= (F2+ 2F2)/3),= 1.003, (Δ/)max= 0.000, (Δ)max= 0.278 and (Δ)min=-0.327 e·?-3. The selected bond lengths and bond angles are given in Table 1.

Table 1. Selected Bond Lengths (?) and Bond Angles (°) of Compound 3

2. 4 Cytotoxic activity

Compound 3 was further tested for its antitumor activity using the typical MTT assay according to the literature[23]. Doxorubicin was co-assayed as positive control.

3 RESULTS AND DISCUSSION

The structure of the title compound was charac- terized on the basis of elemental analysis, IR, MS and NMR techniques. Its molecular formula was determined to be C19H24BrN3O2through the ESI-MS spectrum (/406.1 [M+H]+) and elemental analysis. Two isotopic peaks at/406.1 and 408.1 indicate the presence of a bromine atom in the molecule. The IR spectrum of 3 exhibits strong absorption at 3360 cm-1corresponding to the N–H stretch of amino group and strong to medium absorptions around 3000～2860 cm-1for the C–H stretch of3carbon atoms. The strong absorption band at 1724 cm-1is due to the C=O stretch vibration of the methyl ester moiety. In its1H-NMR spectrum, three singlets at1.22, 1.28 and 3.67 ppm can be observed corres- ponding to methyl protons at C(16), C(17) and the ester group, respectively. The broad singlet at5.02 ppm containing three protons can be attributed to the signals of the amino group and N–H on the imida- zole ring. In addition, the singlet at7.11 ppm can be attributed to the only aromatic hydrogen at C(7). All the1H-NMR data are in good agreement with the structure of the title compound.

The compound crystallizes in the monoclinic space group2 with= 24.830(5),= 7.1410(14),= 13.981(3) ?,= 107.68(3)°,= 4,= 2361.9(8) ?3,M= 482.42,D= 1.357 Mg/m3,= 1.003,= 1.772 mm-1,(000) = 1008, the final= 0.0660 and= 0.1402 for 2244 observed reflections (> 2()). The perspective view of the title compound with atomic numbering scheme is given in Fig. 1, and the selected bond lengths and bond angles are listed in Table 1. It can be seen from Fig. 1 that each molecule of compound 3 is accompanied with one crystal water and one acetone molecules. The molecule of compound 3 contains two-fused cyclohexane rings, a phenyl ring and an imidazole ring. One cyclohexane ring (C(1)～C(5), C(15)) exhibits a classicalconformation with two methyl groups (C(16) and C(17)) in the axial positions, while the other (C(5), C(6), C(12)～C(15)) adopts a half-chair conformation due to the fusion with the phenyl ring. The phenyl and imidazole rings are approximately coplanar (dihedral angle 1.96(16)°) because of the conjugated structure. The amino group (N(3)) is also coplanar with the imidazole ring. The N(3)-C(10) bond is 1.363(8) ?, which is shorter than the isolated N-C single bond (1.471 ?) but longer than the double bond (1.273 ?), indicating p-conjugation effect between the amino group and the aromatic ring system. Due to the presence of heavy atom bromine in the molecule, the final refinement resulted in a small Flack parameter-0.015(17), permitting the assignments of the absolute configuration as (1, 5, 15)[24].

Fig. 1. Molecular diagram of compound 3 showing atom labeling scheme. Displacement ellipsoids were drawn at the 30% probability level

The molecular packing diagram of 3 is displayed in Fig. 2, in which four kinds of hydrogen bonds present in the crystal structure. Each title compound is connected with solvent water and acetone mole- cules by two hydrogen bonds (O(W)-H(WA)···O(3) and O(W)-H(WB)···O(1)) (See Table 2). The O(W) atom of water also acts as a hydrogen bond receptor to the N(2) atoms of an adjacent compound 3 (N(2)-H(2A)···O(W)). These hydrogen bonds make the molecules stack along theaxis. In addition, two molecules of 3 along theaxis are also connected with each other by two intermolecular hydrogen bonds (N(3)-H(3A)···N(1)). All the intermolecular contacts link the molecules into a three-dimensional network.

Table 2. Hydrogen Bond Lengths (?) and Bond Angles (°) of Compound 3

Symmetry codes: (a) –+1/2,+1/2, –+2; (b) –1,, –+2

Scheme 1. Synthetic route of the title compound

Fig. 2. Perspective view of the molecular packing of compound 3

Compounds 1～3 were assayed for theirantitumor activities via the MTT colorimetric me- thod against two human hepatocarcinoma cells (HepG2 and SMMC-7721). As a result, compounds 1 and 2 showed moderate cytotoxic activities against two cell lines (See Table 3). Compound 3 exhibited stronger cytotoxicity against two cell lines than those of 1 and 2, with IC50values to be 17.1 and 0.2 μM, respectively. The IC50values of the positive control doxorubicin were 2.7 and 4.2 μM, respec- tively. These results indicated that the introduction of imidazole moiety into the molecule of dehydro- abietic acid could be beneficial to the antitumor activity and the title compound could be a promising lead compound for the discovery of novel antitumor agents.

Table 3. IC50 Values of Compounds 1～3 against Two Hepatocarcinoma Cells

(1) Jallapally, A.; Addla, D.; Yogeeswari, P.; Sriram, D.; Kantevari, S. 2-Butyl-4-chloroimidazole based substituted piperazine-thiosemicarbazone hybrids as potent inhibitors of.2014, 24, 5520-5524.

(2) Zhang, L.; Peng, X. M.; Damu, G. L. V.; Geng, R. X.; Zhou, C. H. Comprehensive review in current developments of imidazole-based medicinal chemistry.2014, 34, 340–437.

(3) Dao, P.; Smith, N.; Tornkiewicz-Raulet, C.; Yen-Pon, E.; Carnacho-Artacho, M.; Lietha, D.; Herbeuval, J. P.; Commoul, X.; Garbay, C.; Chen, H. X. Design, synthesis, and evaluation of novel imidazo[1,2-a][1,3,5]triazines and their derivatives as focal adhesion kinase inhibitors with antitumor activity.2015, 58, 237-251.

(4) Ribeiro, A. I.; Gabriel, C.; Cerqueira, F.; Maia, M.; Pinto, E.; Sousa, J. C.; Medeiros, R.; Proenca, M. F.; Dias, A. M. Synthesis and antimicrobial activity of novel 5-aminoimidazole-4-carboxamidrazones.2014, 24, 4699-4702.

(5) Saudi, M.; Zmurko, J.; Kaptein, S.; Rozenski, J.; Neyts, J.; Van Aerschot, A. Synthesis and evaluation of imidazole-4,5- and pyrazine-2,3-dicarboxamides targeting dengue and yellow fever virus.2014, 87, 529-539.

(6) Wang, X. Y.; Bynum, J. A.; Stavchansky, S.; Bowman, P. D. Cytoprotection of human endothelial cells against oxidative stress by 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im): application of systems biology to understand the mechanism of action.2014, 734, 122-131.

(7) García, G.; Serrano, I.; Sánchez-Alonso, P.; Rodríguez-Puyol, M.; Alajarín, R.; Griera, M.; Vaquero, J. J.; Rodríguez-Puyol, D.; álvarez-Builla, J.; Díez-Marqués, M. L. New losartan-hydrocaffeic acid hybrids as antihypertensive-antioxidant dual drugs: Ester, amide and amine linkers.2012, 50, 90-101.

(8) Ahangar, N.; Ayati, A.; Alipour, E.; Pashapour, A.; Foroumadi, A.; Emami, S. 1-[(2-Arylthiazol-4-yl)methyl]azoles as a new class of anticonvulsants: design, synthesis, in vivo screening, and in silico drug-like properties.2011, 78, 844-852.

(9) Ishikawa, M.; Shinei, R.; Yokoyama, F.; Yamauchi, M.; Oyama, M.; Okuma, K.; Nagayama, T.; Kato, K.; Kakui, N.; Sato, Y. Role of hydrophobic substituents on the terminal nitrogen of histamine in receptor binding and agonist activity: development of an orally active histamine type 3 receptor agonist and evaluation of its antistress activity in mice.2010, 53, 3840-3844.

(10) Liu, Z. Y.; Wenzler, T.; Brun, R.; Zhu, X. H.; Boykin, D. W. Synthesis and antiparasitic activity of new bis-arylimidamides: DB766 analogs modified in the terminal groups.2014, 83, 167-173.

(11) Kim, J. Y.; Seo, H. J.; Lee, S. H.; Jung, M. E.; Ahn, K.; Kim, J.; Lee, J. Diarylimidazolyl oxadiazole and thiadiazole derivatives as cannabinoid CB1 receptor antagonists.2009, 19, 142-145.

(12) Savluchinske-Feio, S.; Curto, M. J. M.; Gigante, B.; Roseiro, J. C. Antimicrobial activity of resin acid derivatives.2006, 72, 430-436.

(13) Huang, X. C.; Wang, M.; Pan, Y. M.; Tian, X. Y.; Wang, H. S.; Zhang, Y. Synthesis and antitumor activities of novel-aminophosphonates dehydroabietic acid derivatives.2013, 23, 5283-5289.

(14) Sepúlveda, B.; Astudillo, L.; Rodríguez, J. A.; Yá?ez, T.; Theoduloz, C.; Schmeda-Hirschmann, G. Gastroprotective and cytotoxic effect of dehydroabietic acid derivatives.2005, 52, 429-437.

(15) Esteves, M. A.; Narender, N.; Marcelo-Curto, M. J.; Gigante, B. Synthetic derivatives of abietic acid with radical scavenging activity.2001, 64, 761-766.

(16) Lv, X. S.; Cui, Y. M.; Wang, H. Y.; Lin, H. X.; Ni, W. Y.; Ohwada, T.; Ido, K.; Sawada, K. Synthesis and BK channel-opening activity of novel-acylhydrazone derivatives from dehydroabietic acid.2013, 24, 1023-1026.

(17) Gu, W.; Qiao, C.; Wang, S. F.; Hao, Y.; Miao, T. T. Synthesis and biological evaluation of novel-substituted 1-dibenzo[a,c]carbazole derivatives of dehydroabietic acid as potential antimicrobial agents.2014, 24, 328-331.

(18) Gu, W.; Hao, Y.; Chen, H. T.; Wang, S. F. Synthesis, crystal structure and cytotoxic activity of a new-vinyl-1-dibenzo[a,c]carbazole derivative of the dehydroabietic acid.2013, 32, 1904-1910.

(19) Halbrook, N. J.; Lawrence, R. V. The isolation of dehydroabietic acid from disproportionated rosin.1966, 31, 4246-4247.

(20) Fonseca, T.; Gigante, B.; Marques, M. M.; Gilchrist, T. L.; De Clercq, E. Synthesis and antiviral evaluation of benzimidazoles, quinoxalines and indoles from dehydroabietic acid.2004, 12, 103-112.

(21) Sheldrick, G. M.. University of G?ttingen, Germany 1997.

(22) Sheldrick, G. M.. University of G?ttingen, Germany 1997.

(23) Wang, C. J.; Delcros, J. G.; Biggerstaff, J.; Phanstiel, O. Synthesis and biological evaluation of1-(anthracen-9-ylmethyl)triamines as molecular recognition elements for the polyamine transporter.2003, 46, 2663-2671.

(24) Flack, H. D. On enantiomorph-polarity estimation.1983, A39, 876-881.

① Project supported by the National Natural Science Foundation of China (31000273), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The authors would thank the Modern Analysis and Testing Center, Nanjing Forestry University for NMR test

② Corresponding author. Gu Wen, born in 1979, Ph. D., associate professor, majoring in synthetic medicinal chemistry. E-mail: njguwen@163.com

10.14102/j.cnki.0254-5861.2011-0766

4 April 2015; accepted15 July 2015 (CCDC 1040587)