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

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

(Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China)

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], antiinflammatory[6], antihypertensive[7], anticonvulsant[8],antihistaminic[9], antiparasitic[10]and antiobesity[11]activities. The research on imidazole-based medicinal chemistry has become a rapidly developing and increasingly highlighted topic in recent years[2].

Dehydroabietic acid (DAA, 1), a major component of natural diterpene resin acids, can be readily obtained from Pinus rosin 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, the in vitro antitumor 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 spectrum 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 chromatography was purchased from Qingdao Marine Chemical Factory, China. The reagents and chemicals 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 concentrated HCl. The mixture was stirred under reflux for 4 h. After cooling, the mixture was filtered to remove the iron powder. The solution was neutralized with aqueous NaOH (2 mol/L) and then concentrated in vacuo. 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 in vacuo, 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 in vacuo. 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): ν 3360, 3150, 3051, 2931,2863, 1724, 1642, 1558, 1456, 1255, 1129, 736 cm-1.1H-NMR (CDCl3, 500 MHz): δ 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 m/z 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 Mo Kα radiation (λ =0.71073 ?) by using an ω/2θ scan mode in the range of 1.53≤θ≤25.38° (0≤h≤29, -8≤k≤8, -16≤l≤16) at 293(2) K. A total of 4450 reflections were collected, of which 4344 were independent (Rint=0.0948) and 2244 were observed with I > 2σ(I). The structure was solved by direct methods using SHELXS-97[21]and refined by full-matrix leastsquares procedure on F2with SHELXL-97[22]. All non-hydrogen atoms were refined anisotropically,and hydrogen atoms were positioned geometrically.The final refinement gave R = 0.0660, wR = 0.1402(w = 1/[σ2(Fo2) + (0.0570P)2], where P = (Fo2+2Fc2)/3), S = 1.003, (Δ/σ)max= 0.000, (Δρ)max=0.278 and (Δρ)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 characterized on the basis of elemental analysis, IR, MS and NMR techniques. Its molecular formula was determined to be C19H24BrN3O2through the ESI-MS spectrum (m/z 406.1 [M+H]+) and elemental analysis.Two isotopic peaks at m/z 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 of sp3carbon 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 at δ 1.22, 1.28 and 3.67 ppm can be observed corresponding to methyl protons at C(16), C(17) and the ester group, respectively. The broad singlet at δ 5.02 ppm containing three protons can be attributed to the signals of the amino group and N–H on the imidazole ring. In addition, the singlet at δ 7.11 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 group C2 with a = 24.830(5), b = 7.1410(14),c = 13.981(3) ?, β = 107.68(3)°, Z = 4, V = 2361.9(8)?3, Mr= 482.42, Dc= 1.357 Mg/m3, S = 1.003, μ =1.772 mm-1, F(000) = 1008, the final R = 0.0660 and wR = 0.1402 for 2244 observed reflections (I >2σ(I)). 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 trans-fused cyclohexane rings, a phenyl ring and an imidazole ring. One cyclohexane ring (C(1)～C(5), C(15))exhibits a classical chair conformation 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-π 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 (1R, 5S, 15R)[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 molecules 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 the b axis. In addition, two molecules of 3 along the a axis 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

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 their in vitro antitumor activities via the MTT colorimetric method 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, respectively. These results indicated that the introduction of imidazole moiety into the molecule of dehydroabietic 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

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