Synthesis and Antiproliferative Activity of Some Novel Triazole Derivatives from Dehydroabietic Acid

Dehydroabietic acid (DHA) is a naturally occurring diterpene with different and relevant biological activities. Previous studies have shown that some DHA derivatives display antiproliferative activity. However, the reported compounds did not include triazole derivatives. Starting from DHA (8,11,13-abietatrien-18-oic acid), and its alcohol dehydroabietinol (8,11,13-abietatrien-18-ol), four alkyl esters were prepared. The alkyl terpenes were treated with different aromatic azides to synthesize hybrid compounds using click chemistry. Some 16 new DHA hybrids were thus synthesized and their structures were confirmed by spectroscopic and spectrometric means. The antiproliferative activity of the new compounds was assessed towards human cell lines, namely normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), lung cancer (SK-MES-1) and bladder carcinoma (J82) cells. Better antiproliferative effect was found for compound 5, with an IC50 of 6.1 μM and selectivity on SK-MES-1 cells. Under the same experimental conditions, the IC50 of etoposide, was 1.83 µM.


Introduction
Synthesis or modification of known anticancer drugs is an important aspect of research. However, to date a vast amount of synthetic work has contributed to relatively small improvements over the OPEN ACCESS

Results and Discussion
Starting from DHA and its alcohol, four alkyl esters were prepared and then treated with different aromatic azides using click chemistry to produce 16 hybrid abietane-triazole compounds (Schemes 1 and 2). The new compounds were obtained in moderate to good yields. The method proved to be suitable to obtain series of derivatives from diterpenes and can be applied to other natural products to increase structural diversity. Compounds 1-16 are described for the first time. The structures of all the compounds were confirmed by spectroscopic and spectrometric means. The compounds were then assessed for antiproliferative activity towards the following human cell lines: normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), lung cancer (SK-MES-1) and bladder carcinoma (J82) cells. IC 50 values > 100 µM were considered inactive (Table 1). Among the triazoles prepared with the diterpene acid, the derivatives 1 and 4 presented antiproliferative activity towards all of the selected cell lines. The effect, however, is low. Compounds 2 and 3, differing in the number of CH 2 groups of the linker or in the aromatic moiety were inactive against MRC-5 and SK-MES1cells. When comparing the pairs 1-2 and 3-4 bearing one or two CH 2 units as linkers, the effect of 1 was higher than that of 2, suggesting that a longer chain decreases activity when R is a phenyl group. However, an opposite effect was found for the methyl phenyl sulfide derivatives. The best compound of this group was the derivative 4, with a (CH 2 ) 2 linker and a methyl phenyl sulfide substituent in the triazole ring. More derivatives with longer linker chains need to be prepared to have a clearer picture of the effect of this structural feature on the antiproliferative effect.
The compounds 5-16, were prepared with the diterpene alcohol. Among them, the compounds 11-14 were inactive. All of them contain electron donors such as a methoxy group or a chlorine atom in the aromatic ring. Compounds 7 and 8 showed similar activity against AGS, SK-MES-1 and J82 cells and were inactive against fibroblasts, showing a good selectivity. This fact suggests that the length of the CH 2 linker from 1 to 3 units do not have a relevant influence in the antiproliferative activity. The same trend was observed for the products 9 and 10, differing in the linker. For the pair 15 and 16, the best effect was observed for compound 16 with a longer linker. Overall the activity for the compounds should be considered as moderate.
The hybrid compound 5 with a phenyl ring was the most active product designed in this study, presenting IC 50 values of 17.1 and 6.1 µM against MRC-5 and SK-MES-1 cells. The compound differs from 1 in the identity of the diterpene moiety and in the nature of the linker. In the slightly active compound 1, the diterpene is a carboxylic acid, while in 5, the terpene moiety is an alcohol. The effect of compound 5 was associated with some selectivity against the SK-MES-1 cells. The SK-MES-1 are lung cancer cells (HTB-58) derived from metastatic site. The selectivity of compound 5 towards SK-MES-1 cells suggest potential of the compound against lung cancer cells and encourages further studies using in vivo models. The difference in effect of compound 5 and 7 against SK-MES-1 cells can be related to the differences in the phenyl vs. benzyl ring in the triazole moiety. However, the effect is inverse for J82 bladder carcinoma cells. In summary, the new compounds present different degrees of antiproliferative activity, with derivative 5 displaying relevant effect against lung cancer cell line SK-MES-1, with an IC 50 value of 6.1 µM. Under the same experimental conditions, the IC 50 value of etoposide was 1.83 µM.

Obtention of Dehydroabietic Acid Derivatives
Dehydroabietic acid (DHA) was obtained from commercial rosin as described previously [22]. Some 300 g of rosin with 300 mg of 5% Pd/C were heating at 280 °C under a constant flow of N 2 for 2 h. The generated crude product was purified by CC on silica gel with petroleum ether-ethyl acetate (8:2) to obtain dehydroabietic acid as colorless crystals after recrystallization in MeOH/H 2 O (164 g, 55% w/w yield). DHA was methylated with a solution of CH 2 N 2 in ethyl ether to afford the corresponding dehydroabietic acid methyl ester (94%). Reduction of the methyl ester with LiAlH 4 in dry tetrahydrofuran at reflux gave dehydroabietinol (78% yield).

Preparation of Alkynyl Esters
Esterification of DHA and dehydroabietinol was performed using DCC/DMAP and appropriate alcohol (propargyl alcohol or 3-butyn-1-ol) or acid (4-pentynoic acid or 6-heptynoic acid) according to reference [18]. Briefly, DHA or alkynyl acid (1 eq) was dissolved in dry CH 2 Cl 2 at room temperature under constant stirring. Then, DCC (1 eq) was added, followed by a catalytic amount of DMAP and alkynyl alcohol or dehydroabietinol (1 eq) dissolved in dry CH 2 Cl 2 . The reaction was stopped by adding H 2 O, extracted with CH 2 Cl 2 , dried over Na 2 SO 4 , concentrated and purified (60%-81% yield).