Synthesis, Crystal Structure and Biological Activity of Ethyl 5-(4-Fluorophenyl)-7-(trifluoromethyl)- pyrazolo[1,5-a]pyrimidine-3-carboxylate①

SONG Juan ZHAO Juan DUAN Bo-Feng WU Rui Roy Soumendra 

(Shaanxi Key Laboratory of Catalysis, College of Chemical & Environment Science, Shaanxi University of Technology, Hanzhong 723001, China)

ABSTRACT The title compound, C16H11F4N3O2, was synthesized and structurally characterized by elemental analysis, IR, MS, 1H-NMR and single-crystal X-ray diffraction. This compound has a pyrazolo[1,5-a]pyrimidine skeleton, and it crystallizes in monoclinic system, space group P21/c with a = 20.8547(12), b = 20.5558(10), c = 7.1575(4) Å, β = 96.610(5)°, V = 3047.9(3) Å3 , Z = 4, Dc = 1.540 g·cm–3, F(000) = 1440, μ(MoKα) = 0.14 mm–1, R = 0.0546 and wR = 0.1276 for 5370 reflections with I > 2σ(I). In addition, biological activity determination results indicated that the title compound exhibited poor inhibitory activity on MKN45 and H460 cancer cell lines.

Keywords: pyrazolo[1,5-a], synthesis, X-ray diffraction, biological activity;

1 INTRODUCTION

With the increase of morbidity and mortality worldwide, cancer is a life-threatening disease and remains a major health problem around the globe[1]. Therefore, the search for highly efficient and safe antitumor agents has become more urgent than ever before[2,3]. In recent years, heterocyclic structure has attracted great attention of many scientists because of their important utility in medicine[4,5]. Among them, pyrazolo- [1,5-a]pyrimidines are considered privileged scaffolds in drug discovery with a wide array of biological activities[6]. In the literature, pyrazolo[1,5-a]pyrimidine derivatives have been described as antitumor[7,8], antifungal[9], antibacterial[10], analgesics[11]and anti-inflammatory[12]. Insomnia agent indiplon[13], anticancer agent dinaciclib[14]and type 2 diabetes mellitus agent anagliptin[15]are all approved drugs containing a pyrazolo[1,5-a]pyrimidine moiety.

In the course of our search for new anticancer agents, we have recently reported the synthesis and biological activities of a series of novel fused heterocycles. Several compounds showed excellent broad spectrum anticancer activities[16-18]. The title compound (Scheme 1) was synthesized by a published method in our laboratory[16]. In this paper, we pre- sent the crystal structural analysis of ethyl 5-(4-fluoro- phenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate and evaluated for its anticancer properties against two human cancer cell lines (MKN45 and H460) using MTT assay.

2 EXPERIMENTAL

2. 1 Materials and general method

All melting points were taken on a Beijing Taike X-4 microscopy melting point apparatus and were uncorrected.1H NMR spectra were recorded on a Bruker Biospin 600 MHz instrument using TMS as the internal standard. All chemical shifts were reported in ppm. IR spectra were recorded as KBr pellets on a PerkinElmer Spectrum one FTIR spectrometer. Mass spectra were obtained on a 6460 QQQ mass spec- trometer (Agilent, USA) analysis system (ESI, direct injec- tion). Elemental analysis was carried out on a Carlo Erba 1108 analyser and are found within the range of theoretical value. All reagents used in the synthesis were obtained commercially and used without further purification unless otherwise specified.

Scheme 1. Chemical diagram of ethyl 5-(4-fluorophenyl)-7- (trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

2. 2 Synthesis of the title compound

A solution of ethyl 5-amino-1H-pyrazole-4-carboxylate (1.00 g, 6.45 mmol) and 4,4,4-trifluoro-1-(4-fluorophenyl)- butane-1,3-dione (1.51 g, 6.45 mmol）in acetic acid (50 mL) was heated at reflux for 6 h. After cooling to room temperature, the formed precipitate was filtered off, washed with water, and dried to give 1.97 g of the title compound in 86.5% yield. m.p.: 167～169 °C; IR (KBr) ν: 2965 (Et, m), 1697 (C=O, s), 1634, 1594 (Ar, s), 1570 (Ar, s), 1466 (Ar, m), 1397, 1327 (CF3, s), 1198, 1171, 1027, 848, 778;1HNMR (600MHz, DMSO-d6): δ 8.75 (s, 1H, Ar–H), 8.48 (m, 2H, Ar–H), 8.37(s, 1H, Ar–H), 7.45 (m, 2H, Ar–H), 4.34 (q, J = 7.2 Hz, 2H, CH2), 1.36 (t, J = 7.2 Hz, 3H, CH3); MS (ESI) m/z (%): 354.3 [M+H]+. Anal. Calcd. for C16H11F4N3O2(%): C, 54.40; H, 3.14; N, 11.89. Found (%): C, 54.46; H, 3.13; N, 11.92.

2. 3 Single-crystal X-ray crystallography

The powder of the title compound was dissolved in ethyl acetate/tetrahydrofuran mixed solvents = 3:2 (V/V). After slowly evaporating the solvents for several days, some colourless single crystals suitable for X-ray analysis were obtained. A suitable single crystal of the title compound was mounted on glass fibers for data collection performed on a Bruker APEX-II CCD automatic diffractometer equipped with a graphite-monochromatic MoKα radiation (λ = 0.71073 Å) at 293 K. The data collected were integrated using the SAINT program[19]. Multi-scan absorption corrections were applied using the SADABS program[20]. The structures were solved by direct methods and refined against F2by full-matrix least-squares methods using the SHELXTL package[21]. Location of H atoms was found by geometric calculations, and their positions and thermal parameters were fixed during the structure refinement.

2. 4 In vitro antiancer activity test of the title compound on MKN45 and H460 cell lines

The title compound was evaluated for its in vitro cytotoxic activity against two cancer cell lines (MKN-45 and H460) by the MTT-based assay method under standard conditions using sorafenib tosylate as a positive control (Table 1).

Table 1. In Vitro Anticancer Activity Testa of the Title Compound on MKN45 and H460 Cell Lines

3 RESULTS AND DISCUSSION

The synthesis route of the title compound by one step with good yield was depicted in Scheme 1. This compound was obtained as stable solid and easily purified by filtration, and its structure was elucidated by IR,1H NMR, MS and elemental analysis. IR shows the peak at about 1697 cm−1resulting from the carbonyl group stretching vibration of ester. The1H NMR spectrum for the title compound exhibits a triple peak at 1.36 ppm and a quadruple peak at 4.34 ppm, corresponding to the ethenyl proton of ester. In the mass spectrum, the peak appeared at m/z 354.3 ([M+H]+, 100%), which is in accordance with its molecular formula. IR,1H NMR, MS, elemental analyses and single-crystal X-ray of the target compounds confirmed their structural integrity.

The single crystal of the title compound was cultured using acetate/tetrahydrofuran mixed solvents = 3:2 (V/V) at room temperature and determined by X-ray diffraction method to confirm its structure. Selected bond lengths and bond angles are listed in Table 2.

Table 2. Selected Bond Lengths (Å) and Bond Angles (°) for 1

The title compound crystallizes in monoclinic system with one molecule per asymmetric unit cell. Its crystal structure (Fig.1) shows that the molecule has a pyrazolo[1,5-a]py- rimidine skeleton, in which all bond lengths and bond angles fall in normal ranges. The dihedral angles between pyra- zolo[1,5-a]pyrimidine and phenyl rings is 2.74°, which indicate that the two planes are almost coplanar. Furthermore, there exists a mass of intermolecular and intramolecular hydrogen bonds (Table 3), which plays a major role in stabilizing the molecule. It is worth noting that the crystal packing is further stabilized by weak π-π stacking interactions between the Cg(1)...Cg(3) and Cg(2)...Cg(2) rings, as shown in Fig.2 and Table 4. These interactions together with intermolecular hydrogen bond result in the formation of a three-dimensional framework. In addition, the bioassay results showed the title compound exhibited weak inhibitory activity against MKN45 and H460 cell lines with IC50values of 57.12 and 88.75 μM, which was fairly less potent than sorafenib.

Table 3. Hydrogen Bond Lengths (Å) and Bond Angles (°)

Table 4. π-π Stacking Geometry for the Title Compound

Fig.2. A packing diagram of the title compound

4 CONCLUSION

The title compound 5-(4-fluorophenyl)-7-(trifluorome- thyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate was synthesized and characterized by IR,1H-NMR, MS, elemental analysis and single-crystal X-ray diffraction. The antitumor activities of the title compound were evaluated for cytotoxicity against two human cancer cell lines (MKN-45 and H460), using MTT assay. Cytotoxicity assay results revealed that the title compound showed poor cytotoxicity against MKN-45 and H460 cells.