Synthesis and anticancer activity of (+)-nopinone-based 2-amino-3-cyanopyridines

Twelve (+)-nopinone-based 2-amino-3-cyanopyridines 4a–l were synthesized from (–)-β-pinene. The structures of these compounds were characterized by FT-IR, H NMR, and ESI-MS. All the compounds were tested for their anticancer activity against lung cancer cell line A549, gastric cancer cell line MKN45 and breast cancer cell line MCF7 by MTT method, respectively. The results showed that compounds 4f, 4j and 4k had promising anticancer activity against these cancer cell lines, in particular, compound 4f exhibited broad-spectrum and highly efficient anticancer activity against cell lines A549, MKN45 and MCF7 with IC50 of 23.78, 67.61 and 53.87 μmol$L, respectively. The preliminary analysis of the structure activity relationship implied that the Br or Cl substituted group of the benzene ring in these derivatives significantly contributed to the anticancer activity.


Introduction
Natural products are important source for drug development in all major disease areas due to their structure diversity and low side effects [1] . β-Pinene is a naturally occurring monoterpene and commonly occurs in many natural essential oils, including turpentine, tarragon and tea tree oils [2][3][4] . Research has shown that β-pinene and its derivatives displayed broad-spectrum biological activity, such as anticancer, antibacterial, antifungal, antidiabetic and antiallergic activity [5][6][7][8][9][10] . Hence, exploiting β-pinene and its derivatives for their biological activities has significant potential.
2-amino-3-cyanopyridines are known to have a range of biological activity [11][12][13][14] . The anticancer activity of these compounds has especially attracted great interest because of their different modes of action [15] . With both β-pinene and 2-amino-3-cyanopyridine having potential anticancer activity, there is a possibility that synthesis of the β-pinene derivatives with a 2-amino-3-cyanopyridine core may lead to more active anticancer agents.

Material and methods
(-)-β-Pinene was purchased from the domestic spice company Jiangxi Jishui Hongda Natural Perfume Co., Limited, China, and other compounds were of reagent grade. Melting points were determined in a WRS-2 melting point apparatus (Shanghai Precision and Scientific Instrument Co. Ltd., China) and were uncorrected. IR spectra were recorded on a Nicolet IS10 FT-IR spectrometer (Nicolet, Madison, USA). 1 H NMR spectra were recorded on a Bruker DKX500 NMR spectrometer (Bruker, Karlsruhe, Germany) using CDCl 3 or DMSO-d6 as solvent, and TMS as internal standard. Mass spectra were recorded on an Agilent-5973 spectrometer (ESI source). Purity of compounds was detected by Agilent 1260 high performance liquid chromatography (Agilent, Santa Clara, USA) and Fuli GC-9750 gas chromatography (Zhejiang Fuli analysis instrument Co. Ltd., China). All reactions were followed by thin layer chromatography (TLC). Cancer cell lines (human lung cancer cell A549, human gastric cancer cell MKN45, human breast cancer cell MCF7) were supplied by the Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Science, Chinese Academy of Sciences, China. Neurobasal medium (RPMI 1640) and fetal calf serum were supplied by Gibco TM , USA and (4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by AMRESCO, USA.

Anticancer activity evaluation
The cells were thawed and grown in RPMI 1640 medium with 10% fetal bovine serum and 0.25% trypsin at 37°C in a humidified (95%) incubator with CO 2 (5%). Then, the cells in logarithmic phase was collected, trypsinized with 0.25% trypsin/0.02% EDTA and diluted into cell suspensions. The cell suspensions were aliquoted (90 µL per well) into 96-well plates, incubated for 24 h, and then treated with 10 µL testing compounds at different concentrations (3.13, 6.25, 12.5, 25, 50 and 100 µmol$L -1 ). The treated 96-well plates were incubated for 48 h at 37°C in a humidified 5% CO 2 /95% air mixture. Ten microliter of 5 mg$mL -1 MTT was added to the cell cultures and incubated for 4 h. Formed formazan crystals were dissolved in 150 µL dimethyl sulfoxide, and the plates were analyzed using a microplate reader at 570 nm wavelength. The experiment was repeated three times. Cell inhibition rate was calculated by: Half maximal inhibitory concentration (IC 50 ) was calculated according to the improved Karber method [16] .

Synthesis and characterization
According to the literature, the optimum reaction conditions to synthesize compound 2 were to add 1 equivalent (-)-β-pinene, 3 equivalent KMnO 4 , 0.162 equivalent H 2 SO 4 in acetone, then stirring at 15-25°C for 5 h [17] . In the present research, the reaction time was extended to 8 h in order to deplete the (-)-β-pinene, and the obtained (+)nopinone was directly used in the following reaction without further purification. The structure of (+)-nopinone was confirmed by FT-IR, 1 H NMR and ESI-MS, and values were in good agreement with those reported in the literature [18] .
one-pot multicomponent reaction. Furthermore, a diversity of substitutions including methyl-, methoxy-, fluoro-, chloro-and bromo-groups was introduced into the derivatives for high throughput biological screening. The characterization data of these synthesized compounds are given in Table 1.

Anticancer activity evaluation
Anticancer activity evaluation were carried out for all compounds. The inhibition rates at different concentrations and the IC 50 values of the compounds against cell lines A549, MKN45 and MCF7 are given in Table 2 Table 1 Characterization data of (+)-nopinone and (+)-nopinone-based 2-amino-3-cyanopyridines MCF7. Its IC 50 was 53.87 µmol$L -1 , which was better than that of dasatinib (with IC 50 of 55.07 µmol$L -1 ). Generally, among these (+)-nopinone-based 2-amino-3-cyanopyridine derivatives, compounds 4f, 4j and 4k showed promising anticancer activity against the selected cancer cells. Compound 4f, in particular, was effective against all of the selected cancer cells. The concentrations of test compounds had great impact on their inhibition rates. Figure 2 shows the inhibition rates of compounds 4f, 4j, 4k and dasatinib against cell lines A549 and MKN45 at different test concentrations. The inhibition rates of these compounds against cell lines A549 and MKN45 were observed to increase with the increasing test concentration. For cell line A549 at a test concentration of 100 µmol$L -1 , the inhibition rates of compounds 4f and 4k were comparable to that of dasatinib.

Synthesis
For the four component reaction between an aldehyde, a ketone, malononitrile and ammonium acetate, earlier  studies have found that para-substituted and metasubstituted aromatic aldehydes tolerated the reaction and yielded considerable products, but ortho-substituted aromatic aldehydes only gave trace products [19] . However, in this study, we successfully obtained relevant products using ortho-substituted aromatic aldehydes by the one-pot four-component reactions (compounds 4b and 4c). One possible reason for this result was that the steric hindrance effect of the pinane ring was smaller than that of two neighboring substituents in the pyridine ring.

Structure activity relationship
A preliminary analysis of the structure activity relationship of the (+)-nopinone-based 2-amino-3-cyanopyridine derivatives showed that the substituted groups in the benzene ring influenced the anticancer activity. A derivative with a Br or Cl substituted group at the meta-position or paraposition of the benzene ring normally had anticancer activity, such as compounds 4f, 4j and 4k. Furthermore, compound 4f with Br substituted group at the metaposition of the benzene ring showed better anticancer activity than the derivatives with other substituted groups at the same position (such as compounds 4d and 4e).

Conclusions
A series of (+)-nopinone-based 2-amino-3-cyanopyridine derivatives were synthesized and evaluated for their anticancer activity against A549, MKN45 and MCF7. The result showed that compounds 4f, 4j and 4k showed promising anticancer activity against the selected cancer cells. Compound 4f exhibited broad spectrum and highly efficient anticancer activity against cancer cell lines A549, MKN45 and MCF7 with IC 50 of 23.78, 67.61 and 53.87 µmol$L -1 , respectively. Compound 4j showed moderate anticancer activity against cell lines A549 and MKN45 with IC 50 of 91.29 and 79.61 µmol$L -1 , respectively. Compound 4k exhibited moderate anticancer activity against cell lines A549 and MKN45 with IC 50 of 37.35 and 91.66 µmol$L -1 , respectively. Compared with dasatinib, compound 4f obtained comparable anticancer activity against A549 and better anticancer activity against MCF7. The preliminary analysis of the structure activity relationship implied that the substituted groups in the benzene ring exert an influence on the anticancer activity. The findings of this study could aid in the further design and development of new anticancer analogs of monoterpene derivatives with enhanced bioactivity.