A Simple Synthesis of Kaurenoic Esters and other Derivatives and Evaluation of their Antifungal Activity

Este trabalho descreve a preparação de ésteres e amidas derivados do ácido caurenóico e a avaliação da atividade antifúngica frente a cepas padronizadas de fungos patogênicos. Ésteres alquílicos e benzílicos foram obtidos com bons rendimentos, sob condições brandas, a partir da esterificação do ácido caurenóico com haletos de alquila em KOH-acetona. Todos os compostos sintetizados foram submetidos aos testes de inibição contra leveduras, hifomicetos e dermatófitos, mas somente o ácido caurenóico e os derivados contendo um grupo carboxilato livre apresentaram atividade moderada frente a dermatófitos.


Introduction
Wedelia paludosa is a herbaceous medicinal plant employed in folk medicine for the treatment of several ailments, including dolorous processes. 1The hypoglycemic, 2 antifungal 3 and antinociceptive 4 action of organic extracts obtained from different parts of W. paludosa was recently described.This behavior was partially associated with the presence of ent-kaur-16-en-19-oic acid (1), a kaurane diterpene 5 containing a rigid tetracyclic skeleton known as kaurenoic acid (Scheme 1).Kauranes are intermediates in the biosynthesis of a number of plant and fungal metabolites, including the gibberellins and other phytohormones involved in the regulation of growth and development of higher plants. 6][10][11][12][13][14][15][16] The relatively high natural abundance of kaurenoic acid (1) and the inexistence of a general method for the synthesis of alkyl kaurenoates by esterification of 1 provides further motivation to investigate its chemical modification 17 in order to obtain derivatives and test their pharmacological activity. 18his paper describes a general synthesis of kaurenoic esters and derivatives as well as their antifungal activity, evaluated against a panel of human opportunistic pathogenic fungi including yeasts, hialohyphomycetes and dermatophytes.

Results and Discussion
A search in literature revealed that preparation of alkyl kaurenoates by esterification of kaurenoic acid is limited to a few examples.Methyl kaurenoate 2 has been prepared by diazotization of 1 in good yields by many research groups, 9,[19][20][21] but the harmful reaction conditions and the restrictions associated with the preparation of other diazoalkanes precluded its general use.On the other hand, synthesis of 3-hydroxypropyl kaurenoate 3 was achieved 22 by treatment of kaurenoic acid 1 with triphenylphosphine in carbon tetrachloride followed by addition of 1,3propanediol to the unstable acyl chloride intermediate (Scheme 1).However, in our hands the utilization of environmentally prohibitive CCl 4 as the chlorinating agent to promote esterification of kaurenoic acid 1 with methanol was unsuccessful.The unavailability of general and simple methods to access alkyl kauranoates for biological screening motivated a more detailed study dealing with esterification of kaurenoic 1.
Attempts to prepare methyl kaurenoate 2 under classical conditions such as sulfuric acid catalyzed esterification of 1 with methanol furnished the 16α-methoxy acid 4 instead of the expected ester 2 (Scheme 2). 23Compound 4 obviously arises from a typical Markovnikov addition of methanol to the tertiary carbocation (C-16) generated by protonation of terminal olefinic carbon (C-17).Observation of only one isolable stereoisomer indicated a high face differentiation by the nucleophile, which could be explained in terms of steric effects, the α-face (Re) being much less crowded than the β-face (Si).An initial effort to extend this reaction to other 16-alkoxy kaurenoic derivatives by treating kaurenoic acid 1 with higher alcohols such as ethanol, n-butanol or ethyleneglycol under the same acidic conditions failed to give the expected addition products.
Another tentative approach to obtain alkyl kaurenoates from 1 involved a one-pot generation of kaurenoyl chloride (from 1 and thionyl chloride) followed by addition of methanol, resulting in esterification of the carboxylic group along with alcohol addition to the double bond to form 16α-methoxy ester 5 (Scheme 2).Structural characterization of product 5 was achieved by 1 H NMR singlets at δ 3.12 and 3.64 which were assigned, respectively, to methoxyl and methoxycarbonyl groups, by IR band at 1720 cm -1 , characteristic of ester carbonyl, and by comparison with previously reported spectral data (synthesized by esterification of 16-methoxy acid 4 with diazomethane). 24s expected, when pyridine was added to the above reaction conditions to prevent the acid-catalyzed methanol addition to the double bond, no detectable formation of adduct 5 was revealed in the NMR of the crude product and methyl ester 2 could be obtained in moderate yield (60-70%).However, the required excess of alcohol and utilization of unpleasant pyridine as an additive are obvious limitations that make this two-step process synthetically unattractive.Moreover, attempts to prepare amides by treating kaurenoyl chloride with an excess of aniline (to act as reagent and acid scavenger) leaded to the formation of the kaurenamide 6, albeit in low yield and purity due to extensive formation of by-products (Scheme 3).Similarly, reactions of kaurenoyl chloride with aliphatic amines also gave rise to complex mixtures of products and attempts to isolate and completely characterize secondary alkyl amides 7 and 8 were unsuccessful.
A much more reliable strategy for esterification of kaurenoic acid 1 was carried out, based on the direct alkylation of carboxylate ions.KOH in polar aprotic solvents has been used for the alkylation of carboxylic acids, amides and phenols with reactive alkyl halides under mild conditions, providing the corresponding esters, Nalkyl amides and aryl ethers in good yields. 25Accordingly, alkylation of kaurenoic acid 1 with a combination of methyl iodide and KOH in acetone resulted in a clean transformation to methyl kaurenoate 2, isolated in 89% yield (not optimized).The potential generality of this reaction was attested by extending the conditions to alkyl bromides, leading to the respective kaurenoates 9-14 in moderate to high yields (Scheme 3).Spectroscopic data are in well agreement with the proposed structures (Table 1).Replacement of benzyl bromide by benzyl chloride decreases the reaction rate and ester 10 was obtained in lower yield.This S N 2 process also shows a remarkable dependence on the solvent being used.Acetone was unique among those tested, usually affording the expected esters in a few hours.However, reaction of 1 with benzyl bromide in KOH-DMF was slow, giving benzyl kaurenoate 10 in low yield and purity.With THF or dichloromethane the corresponding ester was not observed even after days at room temperature, probably because of the poor solubility of KOH in these solvents.
This simple methodology for esterification was extended to 16-oxo-17-norkauranoic acid 15, which is an intermediate for the synthesis of kauranes of biological importance. 18Oxidative cleavage 19,20 of the olefin moiety in 1 gave the keto acid 15 which was methylated with CH 3 I in KOH-acetone to afford methyl norkauranoate 16 quantitatively (Scheme 3).To carry out the antifungal evaluation, concentrations of kaurenoic acid and its derivatives up to 250 μg mL -1 were incorporated to growth media according to NCCLS guidelines. 26None of the compounds screened under the microbroth dilution method presented significant activity against the yeasts Candida albicans, C. tropicalis, Saccharomyces cerevisiae and Cryptococcus neoformans, the filamentous fungi Aspergillus niger, A. flavus and A. fumigatus or the dermatophytes Microsporum canis and M. gypseum.In contrast, some selected compounds of the series showed moderate activity against the dermatophytes Trichophyton rubrum, T. mentagrophytes and Epidermophyton floccosum (Table 2).
Data in Table 2 revealed kaurenoic acid (1) as the most active kaurane derivative, although 16-oxonorkauranoic acid (15) displays comparable action against three dermatophytes studied, being Epidermophyton floccosum the most sensitive species.Since dermatophytes are a group of fungi which characteristically infect the keratinized areas of the body and cause dermatomycoses that are very difficult to eradicate, it is very interesting to note that kaurane derivatives showed selective activity against dermatophytes and not against any other type of fungi.
To evaluate the structure-activity relationships, the effects of three structural changes in the molecule of kaurenoic acid were considered: (a) conversion of the carboxylic acid group into esters (compounds 2, 5, 9-14, 16); (b) methanol addition to the double bond (compounds 4, 5); (c) replacement of the methylene 17 by a 16-oxo group (compounds 15, 16).It is clear that when the carboxylic group is alkylated (change a) the derived esters do not show any activity up to 250 μg mL -1 (compare activity of 1 with that of compounds 2, 9, 11-14).When a methoxyl is added to the double bond (1→4, change b) a decrease in the spectrum of action and an enhancement of MIC values are observed.In addition, introduction of a methyl ester group as in adduct 5 also results in loss of activity.Concerning the products from oxidative cleavage of the olefin moiety (change c) ketone 15 possess almost the same potency as kaurenoic acid 1.The fact that methyl ester 16 displays a lower activity than acid 15 again suggests that the 4-carboxylic Table 1. 13    group (and not the 16-oxo) should be a structural requirement for the antifungal activity in the kaurane series.

Conclusions
A simple method to prepare kaurenoic esters was developed based on alkylation of kaurenoic acids with alkyl halides in KOH-acetone.This mild protocol avoids the use of anhydrous conditions and potential contaminants such as high-boiling solvents and organic additives, leading to reproducible reaction conditions and easy work-up and purification steps.The solvent exerts a dramatic influence on the reaction rates and yields, acetone being the preferred by favoring cleaner reactions in shorter periods.According to the results obtained in microbroth dilution assays, only kaurenoic acid (1) and analogs containing the acidic COOH group displayed a moderate activity against three dermatophytes, indicating that the presence of hydrophilic groups contributes to the observed antifungal activity. 11This synthetic method can be potentially useful for esterification of other biologically important carboxylic acids, and studies dealing with their reactivity and other chemical properties are currently being investigated.

General considerations
All chemicals were of reagent grade and were used as received.Melting points were determined using a Microquímica MG-APF 301 apparatus and are uncorrected.Infrared spectra were acquired with a Perkin-Elmer FT-IR 1600 spectrometer using KBr for solids and film for liquid samples (range 4000-400 cm -1 ). 1 H NMR (200 MHz) and 13   C NMR (50 MHz, fully decoupled) spectra were recorded with a Bruker AC-200F spectrometer.Samples were prepared in CDCl 3 solution containing 1-2% tetramethylsilane (TMS) as internal standard.Chemical shifts are reported in parts per million (δ) relative to TMS.Coupling constants (J) are measured in Hertz (Hz); coupling patterns are designated as s (singlet); d (doublet); t (triplet); q (quartet); m (multiplet); brs (broad singlet).Elemental analyses were performed in a CHN Perkin Elmer 2400 by UFSC-Central Analítica, Departamento de Química, Florianópolis-SC, Brazil.The purity of the synthesized substances was monitored by thin-layer chromatography (TLC) using silica (Macherey-Nagel GmbH, Inc-USA) pre-coated in aluminum plates (Alugram ® SIL G/UV254 layer 0.2 mm) with several solvent systems of different polarity.Compounds were visualized with specific spray reagents anisaldehyde-sulfuric acid and iodine vapor, and were purified by column chromatography over silica gel (Aldrich, 100-200 mesh particle size) eluted with hexane-ethyl acetate gradient.

Extraction of plant material
Different parts of Wedelia paludosa DC. were collected in Florianópolis, State of Santa Catarina, Brazil, in November 2001, and identified by Dr. Ademir Reis (Department of Botany, UFSC, Florianópolis).A voucher specimen was deposited at the Barbosa Rodrigues

General procedure for preparation of alkyl kaurenoates 2, 9-14
Powdered KOH (0.032 g; 0.66 mmol) was added to anhydrous acetone (2.0 mL) and the mixture was stirred for 5 minutes at 25 o C. Kaurenoic acid 1 (0.05 g; 0.165 mmol) was then added to the reaction, followed by the alkyl halide (0.17 mmol).Stirring was continued for the stated time at 25 o C, after which the reaction was poured into water (20 mL) and extracted with ethyl acetate (3 x 20 mL).The combined organic extracts were washed with water (3 x 10 mL), dried over MgSO 4 , filtered and concentrated in a rotary evaporator.The residue was purified by chromatography column with a hexane-ethyl acetate gradient to give the following esters: Methyl kaur-16-en-19-oate (2)

16-oxo-17-norkauran-19-oic acid (15)
To a solution containing kaurenoic acid 1 (0.05 g; 0.165 mmol) in THF-H 2 O (1:1; 5.2 mL) was added NaIO 4 (0.17 g; 0.82 mmol) and one crystal of OsO 4 .After stirring for 12 h at 25 o C, the reaction mixture was poured into water (20 mL), treated with saturated sodium bisulphite and sodium thiosulfate, and extracted with ethyl acetate (3 x 20 mL).The combined organic extracts were washed with water (5 x 10 mL), dried over MgSO 4 , filtered and concentrated in a rotary evaporator.(16)   Powdered KOH (0.032 g; 0.66 mmol) was added to anhydrous acetone (2.0 mL) and the mixture was stirred for 5 minutes at 25 o C. Compound 15 (0.051 g; 0.167 mmol) was then added to the reaction, followed by iodomethane (0.12 g; 0.8 mmol).After stirring for 24 hours at 25 o C, the reaction was poured into water (20 mL) and extracted with ethyl acetate (3 x 20 mL).The combined organic extracts were washed with water (3 x 10 mL), dried over MgSO 4 , filtered and concentrated in a rotary evaporator.The residue was purified by chromatography column to give 0.053 g of 16 as a white solid (100% yield); mp 108-110 o C (lit.: 127-128 o C); 20  C. The strains were maintained on slopes of Sabouraud-dextrose agar (SDA, Oxoid) and subcultured every 15 days to prevent pleomorphic transformations.Spore suspensions were obtained according to reported procedures. 28

Antifungal susceptibility testing
The Minimal Inhibitory Concentration (MIC) of each extract was determined by using broth microdilution techniques following the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS). 26IC values were determined in RPMI 1640 (Sigma, St Louis, Mo, USA) buffered to a pH 7.0 with MOPS.The starting inocula were 1x10 3 to 5x10 3 CFU mL -1 .Microtiter trays were incubated at 35 o C for yeasts and hialohyphomycetes and at 28-30 o C for dermatophyte strains in a moist, dark chamber, and MICs were recorded at 48 h for yeasts, and at a time according to the control fungus growth, for the rest of fungi.The susceptibilities of the standard drugs Ketoconazol, Terbinafine and Amphotericin B were defined as the lowest concentration of drug which resulted in total inhibition of fungal growth.For the assay, extract stock solutions were two-fold diluted with RPMI from 250-0.98 μg mL -1 (final volume = 100 μL) and a final DMSO concentration ≤ 1%.A volume of 100 mL of inoculum suspension was added to each well with the exception of the sterility control where sterile water was added to the well instead.The MIC was defined as the minimum inhibitory concentration of the extract which resulted in total inhibition of the fungal growth.