Synthesis and Antiproliferative Evaluation of Novel Hybrids of Dehydroabietic Acid Bearing 1,2,3-Triazole Moiety

To discover novel potent cytotoxic diterpenoids, a series of hybrids of dehydroabietic acid containing 1,2,3-triazole moiety were designed and synthesized. The target compounds were characterized by means of FT-IR, 1H NMR, 13C NMR, ESI-MS and elemental analysis techniques. The in vitro cytotoxicity of these compounds was evaluated by standard MTT (methyl thiazolytetrazolium) assay against CNE-2 (nasopharynx), HepG2 (liver), HeLa (epithelial cervical), BEL-7402 (liver) human carcinoma cell lines and human normal liver cell (HL-7702). The screening results revealed that most of the hybrids showed significantly improved cytotoxicity over parent compound DHAA. Among them, [1-(3-fluorobenzyl)-1H-1,2,3-triazole-4-yl]dehydroabietic acid methyl ester (3c), and [1-(2-nitrobenzyl)-1H-1,2,3-triazole-4-yl]dehydroabietic acid methyl ester (3k) displayed better antiproliferative activity with IC50 (50% inhibitory concentration) values of 5.90 ± 0.41 and 6.25 ± 0.37 µM toward HepG2 cells compared to cisplatin, while they exhibited lower cytotoxicity against HL-7702. Therefore, the 1,2,3-triazole-hybrids could be a promising strategy for the synthesis of antitumor diterpenoids and it also proved the essential role of 1,2,3-triazole moiety of DHAA in the biological activity.


Introduction
Nowadays, cancer has a serious impact on human health with a high mortality rate [1]. The rapid development of drug resistance and the acute side effects of clinical used anticancer drugs are still the major obstacles to effective chemotherapy [2,3]. Therefore, the discovery and development of new drugs and therapies with high efficacy and low side effects is always the basic mission for medicinal chemists. Natural products have played a dominant role in drug discovery, according to their chemically structural diversity, good biological activities and biocompatibility. There has been increased interest in the search of antitumor drugs from natural sources, and many natural or natural based antitumor drugs such as vinblastine, etoposide and paclitaxel were found and clinically used in recent years [4].
On the other hand, 1,2,3-triazole, a privileged building block in the discovery of new anticancer agents, plays a key role in enhancing the cytotoxicity towards the cancer cells because of its improved solubility, cell permeability and pharmacokinetic parameters at the binding site [23,24]. It has been reported that hybridization of 1,2,3-triazole framework other anticancer pharmacophores has the potential to provide novel anticancer candidates [25][26][27][28]. Moreover, some of 1,2,3-triazole containing compounds such as Cefatrizine and Carboxyamidotriazole have already been applied in clinics or under clinical trials for fighting against cancers. Click chemistry is considered to be a nearly perfect synthesizing strategy for reaction between azides and alkynes to afford 1,2,3-triazoles under mild conditions. Therefore, it has been widely applied in many aspects of drug discovery, ranging from the design of lead compounds to tagging of biological systems [29,30].
Considering the above benefits and in continuation of our interest in searching for the pharmacological effects of terpenoid derivatives [31][32][33][34]. We envisioned that the combination of the DHAA framework with 1,2,3-triazole unit may afford the desired high-performance anticancer agents. In this study, a series of novel DHAA-1,2,3-triazole hybrids were synthesized and their cytotoxic activities were assessed in vitro against CNE-2 (Nasopharynx), HepG2 (liver), HeLa (epithelial cervical), and BEL-7402 (liver) human cancer cell lines and HL-7702 normal human liver cell line.

Synthesis and Characterization
DHAA derivatives containing 1,2,3-triazole moiety were synthesized as presented in Scheme 1. All 16 compounds have been confirmed by FT-IR, 1 H NMR, 13  The FT-IR spectra of the target compounds 3a-p exhibited characteristic moderate absorption bands at about 1716 cm −1 attributed to the stretching vibrations of the C=O. The bands at 1600-1450 cm −1 were assigned to the vibration of the skeleton in benzene rings. The bands at 2950-2850 cm −1 were assigned to the stretching vibrations of the C-H in methyl or methylene. The 1 H NMR spectra of the target compounds 3a-p showed characteristic signals at about 7.5 ppm assigned to the triazole protons and signals at 8.30-6.80 ppm assigned to the benzene protons. The 13 C NMR spectra of the target compounds 3a-p showed peaks for C=O at about 178 ppm, and for aromatic rings at 160-113 ppm.

In Vitro Assay of Antiproliferative Activity
The antiproliferative activities of the 16 library members were tested against four human cancer cell lines including human nasopharyngeal carcinoma cell (CNE-2), human liver cancer cell (HepG2), human hepatocellular carcinoma cell (BEL-7402), human cervical cancer cells (HeLa), and human normal liver cell (HL-7702) using MTT assay according the reported literature method [35]. The tested results were shown in Table 1. It was found from Table 1 that some of the newly synthesized compounds had significant antineoplastic activity against four tested cancer cell lines, indicating that the introduction of 1,2,3-triazole moiety on the DHAA skeleton increased anti-tumor activity. In particular, compounds 3c and 3k exhibited good antitumor activity against HepG2 with IC 50 values of 5.90 ± 0.41 and 6.25 ± 0.37 µM, better than those of positive control cisplatin. Moreover, compounds 3c (R = m-F), 3d (R = p-F) and 3l (R = m-NO 2 ) against CNE-2 with IC 50 values of 10.92 ± 0.21, 12.20 ± 0.33 and 11.45 ± 0.18 µM, respectively. However, compound 3d and 3j showed moderate activity active against BEL-7402 with IC 50 values of 14.84 ± 075 and 14.53 ± 0.62 µM, respectively. These results declared that introduction of an electron withdrawing group was superior to an electron donating group on the benzene ring against CNE-2 (nasopharynx) cell line. The benzene ring that possessed an electron withdrawing group in meta-positions was better than in orthoand parapositions. Compound 3b exhibited the best antitumor activity against Hela (epithelial cervical) with values of 17.76 ± 0.31 µM. Most compounds showed more potent anticancer activities against the HepG2 tumor cells compared to the parent compound. It was important to note that compounds 3a-p showed a certain selectivity against four tumor cell lines and low cytotoxicity on the human normal liver cell (HL-7702).

General Information
All commercially available reagents including substituted benzyl chloride/bromide were purchased from Energy Chemicals and used without further purification. Optical rotations were measured on a WZZ-3 polarimeter in CH 2 Cl 2 at 20 • C (Shanghai Shenguang Instrument, Co., Ltd., Shanghai, China). FT-IR spectra was recorded on a Prestige-21 spectrometer, and samples were prepared as KBr plates (Shimadzu, Co., Ltd., Tokyo, Japan). 1 H NMR and 13 C NMR spectra were obtained using a Bruker Avance 600/400 MHz spectrometer in CDCl 3 with tetramethylsilane (TMS)as an internal standard (Brucker Co., Ltd., Zurich, Switzerland). Elemental analyses were carried out on a PE 2400II elemental analyzer (Perkin Elmer Instruments Co., Ltd., Waltham, MA, USA). Melting points were determined on WRX-4 digital melting point apparatus (uncorrected) (Shanghai YiCe Apparatus & Equipment, Co., Ltd., Shanghai, China). Mass spectra were carried out with a Shimazu liquid chromatograph mass spectrometer (Shimadzu, Co., Ltd., Tokyo, Japan). NMR and IR spectra of compounds 3 can be found in the Supplementary Materials.

Synthesis of Dehydroabietic Acid Propynyl Ester 2
In a round bottom flask, anhydrous potassium carbonate (9.4 g) and propargyl bromide (2.8 mL) were added to a solution of compound 1 (9.3 g) in dry DMF (40 mL), and the reaction mixture was stirred at room temperature for 4 h. Reaction was monitored by TLC and the crude product was poured into ice water (200 mL), then the mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic layer was washed with saturated salt water three times, dried over anhydrous sodium sulfate and purified through silica gel chromatography (petroleum ether-EtOAc

Conclusions
Taken together, a series of dehydroabietic acid coupled 1,2,3-triazole derivatives (3a-p) were synthesized by a convenient one-pot Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction from propynyl ester 2 and a variety of readily available benzyl chloride/bromide without isolation of potentially unstable organic azide. The synthesized compounds were screened for cytotoxic activity against a panel of four human cancer cell lines and the human HL-7702 normal cell line using an MTT assay. Some compounds exhibited better anticancer activity against the tested cancer cell lines compared to positive controls cisplatin and low cytotoxicity on human normal liver cell HL-7702. Among these compounds, compounds 3c (IC 50 = 5.90 ± 0.41 µM) and 3k (IC 50 = 6.25 ± 0.37 µM) were the most promising derivatives. The antitumor activity in vivo and the mechanism in antitumor activity of dehydroabietic acid-1,2,3-triazole hybrids are under investigation.