Synthesis, Crystal Structure and Antiproliferative Activity of Ethyl 1-(Phenylcarbamoyl)-2-3,4,5-trimethoxyphenyl)cyclopropanecarboxylae

ZHANG Jing-Yi ZHONG Hn-Yu SHI Guo-Bing①

1 INTRODUCTION

The microtubule system of eukaryotic cells is an important target for the development of antiproliferative agents[1,2]. Antimitotic agents cause mitotic arrest in eukaryotic cells by interfering with the normal microtubule polymerization/depolymerization process[3]. A water-soluble phosphate prodrug of combretastatin-A4 (CA-4P), the most depolymerization promising compound, is currently in phase III clinical trials for the treatment of solid tumours[4].CA-4, isolated by Pettit from the bark of African willow tree Combretum caffrum in 1982, is a natural cis-stilbene product that strongly inhibits tubulin polymerization by binding to the colchicine binding site[5]. Interference with microtubule dynamics affects the cell signaling pathways involved in regulating and maintaining the cellular cytoskeleton of endothelial cells in tumour vasculature[6]. Extensive studies have been conducted to examine the structure-activity relationship of variously modified CA-4 analogues and many CA-4 analogues show high-level antiproliferative activities both in vitro and in vivo[7,8]. In this study, we continue to synthesize the analogues of CA-4 as novel antiproliferative compounds, and the crystal structure of the title compound is reported here.

2 EXPERIMENTAL

2. 1 Instruments and reagents

All the chemicals and reagents were commercially available without pretreatment and all solvent reagents were analytically pure. Silica gel thin-layer chromatography was performed on precoated plates GF-254 (Qingdao Haiyang Chemical, China).Melting point was determined with an uncorrected RY-2 digital melting point apparatus.1H NMR and13C NMR spectra were recorded on a Bruker Avance 300 spectrometer (Bruker Company, Germany)using TMS as an internal standard and DMSO-d6 as solvents. Chemical shifts were given in ppm (parts per million). For data reporting, the following was used: s = singlet, d = doublet, t = triplet, m = multiplet. High-resolution mass spectra were recorded on a QSTAR XL hybrid MS/MS mass spectrometer(Agilent Technolohies). Crystal structure was determined on a Bruker SMART CCD (Bruker Company, Germany).

2. 2 Synthesis of the title compound

As shown in Scheme 1, commercially available 3,4,5-trimethoxybenzaldehyde (compound 1)reacted with diethyl malonate to give diethyl 2-(3,4,5-trimethoxybenzylidene)malonate (compound 2)through Knoevenagel condensation[9,10]. Then, compound 2 continued to react with trimethylsulfoxonium iodide(TMSOI)to get intermediate 3[11,12]. After that,compound 3 was hydrolyzed by KOH to generate the racemes compound 4. Optical compounds trans-5 and cis-5 were obtained by chromatography on silica gel (eluent:ethyl acetate/petroleum ether)to give the title compound as white solid product (total yield: 65%). Ethyl 1-(phenylcarbamoyl)-2-(3,4,5- trimethoxyphenyl)cyclopropanecarboxylate(trans-5):white solid, m.p: 120.0～120.5 ℃.1H NMR(DMSO-d6, 300 MHZ): δ 10.18 (s, 1H), 7.65～7.62(d, J = 7.8 HZ, 2H), 7.34～7.29 (t, J = 7.8 HZ, 2H),7.09～7.04 (d, J = 7.2 HZ, 1H), 6.54 (s, 2H), 3.85～3.82 (m, 2H), 3.74 (s, 6H), 3.60 (s, 3H), 3.24～3.19(t, J = 7.8 HZ, 1H), 2.22～2.17 (dd, J1= 4.8 HZ, J2=5.1 HZ, 1H), 1.75～1.70 (dd, J1= 5.1 HZ, J2= 4.8 HZ,1H), 0.81～0.76 (t, J = 7.8 HZ, 3H).13C NMR(DMSO-d6, 300 MHZ): δ 168.13, 165.88, 152.45,138.82, 136.76, 130.89, 128.66, 123.56, 119.72,106.54, 60.71, 59.95, 55.87, 37.90, 32.95, 18.59,13.44; HRMS (EI+)calcd. C22H25NO6(M+)3 9 9.1 6 8 2. F o u n d: 3 9 9.1 6 9 2.

Scheme 1. Preparation of 5a

2. 3 Crystal data and structure determination

A single crystal of the title compound suitable for X-ray diffraction study was grown from the mixsolution of ethyl acetate and petroleum ether (V:V =3:1)by slow evaporation at room temperature. X-ray diffraction intensity data were collected on a Bruker SMART APEX CCD diffractometer equipped with a graphite-monochromatic MoKα radiation (λ =1.54178 ?)by using a φ-ω (1.60≤θ≤25.40o)scan mode at 296(2)K. A total of 6348 reflections with 3319 independent ones (Rint= 0.032)were collected,in which 3029 were observed (I ＞ 2σ(I)). The unit cell dimensions were obtained with the full-matrix least-squares refinements. The absorption corrections were carried out by SADABS program and the structure was solved by direct methods using SHELXS-97 program. The final refinement was performed by full-matrix least-squares techniques with anisotropic thermal parameters for the non-hydrogen atoms on F2. The hydrogen atoms were positioned by geometry and refined with riding modal. The final R = 0.0546 and wR = 0.1889 for 3319 observed reflections.

3 RESULTS AND DISCUSSION

3. 1 Synthesis

The title compound was synthesized from original 1. In a 250 mL round-bottomed flask, 3.36 g(21.0 mmol)of diethyl malonate and HOAc (1.5 mL,2.5 mmol)were added into the toluene, in which 3.92 g (2.0 mmol)of compound 1 was dissolved,and the mixture was heated to reflux for 12 h to give compound 2 with the yield of 95%. Then, 6.42 g(19.0 mmol)of 2, 2.30 g (10.5 mmol)of TMSOI and 1.34 g (60% in mineral oil, 20.1 mmol)of NaH were added in a flask with dry DMSO (50.0 m L). The flask was placed in microwave reaction equipment at 60 ℃ for 10 min with the power working to be 400 watt, giving compound 3 in 92% yield. In the next step, 3.56 g (10.0 mmol)of compound 3 reacted with KOH (10.5 mmol)in the solvents of ethanol and water at 40 ℃ to get compound 4 with a yield of 90%. The dry 4 (2.90 g, 9 mmol)and oxalyl chloride(0.86 mL, 9.09 mmol)in dry CH2Cl2were stirred at room temperature. Then, the solvent was removed under reduced pressure and washed with dry CH2Cl2.The obtained acyl chloride was added dropwise to a solution of dry pyridine (2 eq, 10.9 mL, 18.2 mmol)and aniline (1.2 eq, 10.9 mmol)in dry CH2Cl2(4.0 eq, 150 mL). The reaction was periodically monitored by TLC over 6～12 h. After reaction, the organic solvent was removed under reduced pressure and the residue was purified by flash column chromatography to obtain cis and trans compounds 5. The structure of the title compound (5a)was analyzed by1H-NMR,13C-NMR and H RMS and finally confirmed by X-ray diffraction analysis.

3. 2 Crystal structure

The crystal of the title compound suitable for X-ray diffraction, which was grown from ethyl acetate and petroleum ether (V:V = 3:1), is stable in air at room temperature. It is soluble in ethyl acetate,and poorly soluble in petroleum ether. The crystal was determined as a racemate, and selective bond lengths and bond angles are listed in Table 1. The ORTEP drawing of 5a is shown in Fig. 1 and the packing diagram in Fig. 2. As shown in Fig. 1,compound 5a was composed of three molecular moieties: a 3,4,5-trimethoxyphenyl, a phenylcarbamoyl and a cyclopropane. C(14)–C(15)–C(16)–C(17)–C(18)–C(19)and C(8)–C(12)–C(13)form respectively the 3,4,5-trimethoxyphenyl and cyclopropane rings which are linked by C(13)–C(14)bridge. The C(13)–C(14)bond is 1.499(4)(?),indicating that it is a single bond, and the torsion angle of C(19)–C(14)–C(13)–C(12)is 172.4(3)°.The dihedral angle between the cyclopropane and phenylcarbamoyl was linked by C(7)–C(8)in 1.512(3)?. The C(7)–C(8)–C(12)torsion angle is 113.80(14)° and C(7)–C(8)–C(13)113.59(14)°.Meanwhile, Table 2 gives the hydrogen-bonding geometry in 5a. In the crystal, the hydrogen bonds of N(1)–H(1)··O(2)interconnects each molecule in a plane.

Table 1. Selected Bond Distances (?)and Bond Angles (°)for the Title Compound

Table 2. Hydrogen Bonds for the Title Compound

Fig. 1. ORTEP drawings of 5a

Fig. 2. Packing diagram of the title compound in a unit cell

3. 3 Antiproliferative activity

The cytotoxicity of the title compound was evaluated to A549 (non-small-cell lung cancer line),SW480 (human colon cancer), HeLa (human epithelial cervical cancer), K562 (leukemia)and HepG2 cell lines using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay in vitro.5-Fluorouracil (5-Fu)and CA-4 were selected as the positive control substances. IC50(concentration of the drug required to reduce the cell viability by 50%)was used to evaluate the antiproliferative activity.The title compound revealed better biological activity against HepG2 cell lines (IC50= 18.5 μM)than the positive control 5-Fu (IC50= 74.3 μM).

4 CONCLUSION

In summary, we have found a novel route to synthesize the new class antiproliferative compound from the title compound 1. The structure of 1 was identified by NMR and H RMS and its single crystal has been characterized by X-ray crystal diffraction.The results of cytotoxicity to five cancer cell lines in vitro showed that the title compound possessed potential antiproliferative activity.

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