Synthesis, Antiproliferative and Antifungal Activities of 1,2,3-Triazole-Substituted Carnosic Acid and Carnosol Derivatives

Abietane diterpenes exhibit an array of interesting biological activities, which have generated significant interest among the pharmacological community. Starting from the abietane diterpenes carnosic acid and carnosol, twenty four new triazole derivatives were synthesized using click chemistry. The compounds differ in the length of the linker and the substituent on the triazole moiety. The compounds were assessed as antiproliferative and antifungal agents. The antiproliferative activity was determined on normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), lung cancer (SK-MES-1) and bladder carcinoma (J82) cells while the antifungal activity was assessed against Candida albicans ATCC 10231 and Cryptococcus neoformans ATCC 32264. The carnosic acid γ-lactone derivatives 1–3 were the most active antiproliferative compounds of the series, with IC50 values in the range of 43.4–46.9 μM and 39.2–48.9 μM for MRC-5 and AGS cells, respectively. Regarding antifungal activity, C. neoformans was the most sensitive fungus, with nine compounds inhibiting more than 50% of its fungal growth at concentrations ≤250 µg∙mL−1. Compound 22, possessing a p-Br-benzyl substituent on the triazole ring, showed the best activity (91% growth inhibition) at 250 µg∙mL−1 In turn, six compounds inhibited 50% C. albicans growth at concentrations lower than 250 µg∙mL−1.


Introduction
Metabolites isolated from natural sources, mainly from plants, remain a major source of compounds with pharmacological properties that can be modified to generate new drugs with better effects and lower toxicity [1]. Among the terpenes investigated for pharmacological properties, the abietane diterpenes are a promising group due to their abundance in Nature and occurrence in medicinal plants and industrial wastes [2]. A review of the biological activities of natural and synthetic abietane diterpenes has been published recently [3]. It has been reported that some abietane terpenes are cytotoxic and antiproliferative, leading to new studies with the aim to identify the mechanisms of action of these molecules. A recent review that focused on molecular targets of these terpenes in cancer cells, pointed out the potential of abietanes from Salvia as pro-apoptotic agents [4]. An important source of abietane diterpenes is Rosmarinus officinalis L. (rosemary), being carnosic acid and carnosol the main phenolic diterpenes from the leaves of this plant [5]. These compounds demonstrated antioxidant [6], antibacterial [7], antifungal [8,9], and cytotoxic activities [10]. A recent review of carnosol as an anticancer and antiinflammatory agent has been published [11].
In previous work, we investigated the gastroprotective activity and cytotoxicity of carnosic acid γ-lactone derivatives [12] as well as carnosic acid derivatives [13] and their gastroprotective mechanisms of action in human cells [14]. In the present report we used click chemistry reactions to prepare new carnosic acid and carnosol derivatives. Recent reports show that click chemistry is a very useful tool for drug discovery and gene therapy [15] that simplifies the synthesis of compounds through the use of simple and selective chemical transformations. Click chemistry reactions can be used for the generation of dimers, chimeras and multivalent drugs. The triazole in this case could be seen as an inactive linker or spacer, although it cannot be excluded that, at times, it may act as a biological entity on its own. Different compounds containing 1,2,3-triazoles with interesting antiproliferative activity have been reported [16][17][18][19][20]. This has recently led us to investigate the synthesis and antiproliferative activity of different terpenes coupled to triazole rings by the click chemistry technique [21][22][23]. The antifungal activity of triazoles is well known, being fluconazole, itraconazole, voriconazole and posaconazole the most used agents in the clinic [24]. However, their continued use has generated resistance from fungi making it necessary to find alternative antifungal compounds. Recent research has used click chemistry in the search for novel antifungal compounds [25][26][27][28]. Herein, we report an efficient method for the synthesis of novel carnosic acid and carnosol derivatives using click chemistry. The new compounds were assessed as antiproliferative and antifungal agents using human cell lines and reference microorganisms.

Results and Discussion
A series of new abietane derivatives was synthesized by click chemistry. The diterpene carnosic acid (CA) was methylated using diazomethane in diethyl ether to obtain carnosic acid methyl ester (CAM). Previously we reported that treating CA with DCC/DMAP generated the corresponding carnosic acid γ-lactone (CAL) by an intramolecular esterification [12]. In this work six alkyl esters were prepared starting from CA, its methyl ester and carnosol (C), and then treated with different aromatic azides using click chemistry to produce 24 new compounds (Scheme 1). Compounds 1-24 are described for the first time. All the products were characterized by spectroscopic means.

Antiproliferative Assay
The antiproliferative activity towards the following human cell lines was determined: normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), lung cancer (SK-MES-1) and bladder carcinoma (J82) cells. IC50 values > 100 µM were considered inactive. The hybrid compounds of carnosic acid γ-lactone (compound 1-8) showed variable antiproliferative activity (Table 1). Compounds 1 and 2, differing in the number of CH2 groups of the linker and presenting a methyl phenyl sulfide in the aromatic moiety showed about the same antiproliferative activity against MRC-5 (IC50 values 45.1 and 46.9 μg·mL −1 ) and AGS cells (IC50 values 39.2 and 41.0 μg·mL −1 ). Both compounds were also active against lung cancer cells SK-MES-1, with IC50 values of 81.7 and 76.0 μg·mL −1 , respectively. When comparing the pairs 3-4, 5-6 and 7-8 differing in one CH2 unit in the linker, the activity decreased with linker length. The benzyl derivative 3 with two CH2 units in the linker was active towards MRC-5 and AGS cells, while the compound 4 presenting three CH2 units in the linker was inactive.
For the carnosic acid methyl ester (compounds 9-16) and carnosol (compounds 17-24) derivatives, only compounds 11 and 23 showed weak antiproliferative activity against AGS cells (IC50 value: 89.4 μM and 99.4 µM respectively). All other compounds should be regarded as inactive on all cell lines tested. Overall, selectivity against MRC-5 and AGS cells was observed for some of the new compounds.

Antifungal Assays
The antifungal properties of compounds 1-24 against two clinical important fungal species, C. albicans ATCC 10231 and C. neoformans ATCC 32264 were investigated. Results were expressed as the percentages of inhibition of each fungus in the range 250-3.9 μg·mL −1 by using the standardized microbroth dilution method M-27A3 of Clinical and Laboratory Standards Institute [29] which assures reliable and reproducible results. Results are shown in Tables 2 and 3.
The minimum inhibitory concentration of compound 1-24 necessary to completely inhibit (MIC100) the growth of the selected opportunistic pathogenic fungi was >250 μg·mL −1 . However, when considering less stringent end-points such as the minimum concentration required to inhibit 50% microbial growth (MIC50), there were interesting effects towards C. albicans ATCC 10231 and C. neoformans ATCC 32264.
On the other hand, compounds 2, 4, 9-12, 14, 22 and 23 inhibited >50% fungal growth (53.4%-91.3%) at 250 μg·mL −1 against C. neoformans (Table 3)  From the results of Table 2, some structure/activity relationships can be inferred. Compounds 2, 4, 12 and 18, that showed the best activities against C. albicans, possess the following common features (i) the linker to the diterpene moiety contained three CH2 units while the corresponding derivatives with two CH2 units were devoid of activity (compound 1 and 3) or showed weak effect (compounds 11 and 17) at the assayed concentrations; (ii) in the triazole rings, R1 was either a benzyl (compounds 4 and 12) or a methyl phenyl sulfide (compounds 2 and 18); (iii) the activity was almost the same for the four compounds, regardless of the presence of a lactone (carnosic acid γ-lactone derivatives 2 and 4 and carnosol derivative 18) and one (carnosic acid γ-lactone derivatives 2 and 4) or two triazole rings (carnosic acid methyl ester derivative 12 and carnosol derivative 18); (iv) when R1 joined to the triazole ring was p-bromobenzyl or p-nitrobenzyl, the corresponding derivatives were inactive.
The structure/activity trends observed for the compounds on C. neoformans indicate that the nature of the substituent on the triazole ring is relevant for the effect and different than for C. albicans. For compound 2 (74.8% inhibition growth at 250 μg·mL −1 ) and 4 (53.4%) the γ-lactone appears to be important for activity. In the carnosic acid methyl ester derivatives group, compounds 9-12 and 14 were active in the range 57.2%-67.4% inhibition at 250 μg·mL −1 . For the pairs 9-10 (R1: methyl phenyl sulfide) and 11-12 (R1: benzyl) bearing two or three CH2 units as linkers, the effect was similar.
When comparing 11-12 with 13-14 (R1: p-bromobenzyl), the occurrence of a bromine in the aromatic ring did not change the activity when the length of the linker is three CH2 units, but it diminishes when the linker contains two CH2 units. When comparing the activity of 11-12 with 15-16, presenting a nitro group in the aromatic ring (R1: p-nitrobenzyl), the activity of the nitro compounds is lower.  The most active carnosol derivative was the p-bromobenzyl derivative 22, which reduced the growth of C. neoformans by about 91% at 250 μg·mL −1 while compound 23, with a p-nitrobenzyl unit decreased fungal growth by about 71% at the same concentration. The results indicate some selectivity for the different fungi and that the placement of the lactone (either C-20, C-11 or C-20, C-7) is important for the effect. Further studies including additional biological models are advisable to find novel activities for the new synthetic compounds.

General Procedure for the Synthesis of Compounds 1-24
Carnosol and carnosic acid (CA) were isolated from the aerial parts of Rosmarinus officinalis as described previously [12]. Methylation of CA was performed using diazomethane in diethyl ether (Et2O). The compounds 1-24 were prepared treating carnosol, carnosic acid and carnosic acid methyl ester with the appropriate alkyne acid/DCC/DMAP to obtain the esters. Treatment with the appropriate azide yielded the corresponding triazole.

Preparation of Alkynyl Esters
Esterification of carnosol, carnosic acid and carnosic acid methyl ester was performed using DCC/DMAP and appropriate acid (4-pentynoic acid or 5-hexynoic acid) according to references [22,23]. Briefly, alkynyl acid (1 eq) was dissolved in dry CH2Cl2 at room temperature under constant stirring. Then, DCC (1 eq) was added, followed by a catalytic amount of DMAP and the corresponding terpene (0.5 eq) dissolved in dry CH2Cl2. The reaction was stopped by adding H2O, extracted with CH2Cl2, dried over Na2SO4, concentrated and purified (58%-76% yield).