New Oxidized ent-Kaurane and ent-Norkaurane Derivatives from Kaurenoic Acid

Kauranes are an important class of diterpenes containing a rigid tetracyclic skeleton and exhibiting a wide variety of biological activities such as antitumor, anti-HIV, trypanocidal and antimicrobial, among others. For this reason, the development of new strategies for the synthesis of novel kaurane derivatives may be considered as one of the interesting challenges in Chemistry of Natural Products. Indeed, many naturally occurring bioactive kauranes have been transformed using chemical and microbial methods in order to improve their bioactivity. Kaurenoic acid (ent-kaur-16-en-19-oic acid, 1) is an intermediate in the biosynthesis of numerous plants and fungal secondary metabolites, including gibberellins, the phytohormones involved in the regulation of growth and development of higher plants, found abundantly in some Brazilian species as Wedelia paludosa D.C. (Asteraceae), Xylopia frutescens and Annona glabra (Annonaceae). We have reported, in a previous phytochemical study of Wedelia paludosa D.C., the isolation of kaurenoic acid (1) as the main ent-kaurane diterpene in this species, besides other related diterpenes and triterpenes as minor constituents. Among more recently biological activities reported for 1, we can stand out the antimicrobial, antiplatelet aggregation, analgesic, antifungal, smooth muscle relaxant, hypoglycemic, cytotoxic and embryotoxic effects. Considering these biological effects, along with our special interest on novel kaurane derivatives, we carried on the synthesis of ent-kaurane aldehydes methyl ent17-oxokauran-19-oate (4) and methyl ent-17-oxo-16βkauran-19-oate (5), important as semisynthetic coupling intermediates, starting from kaurenoic acid (1). In addition, we describe here, for the first time, the synthesis of methyl ent-17-oxokauran-19-oate (4), ent-19methoxy-19-oxokauran-17-oic acid (7), methyl ent-16βhydroxy-17-norkauran-19-oate (8) and methyl ent-16oxo-17-norkauran-19-oate (9), from the usual PDC oxidation of methyl ent-17-hydroxykauran-19-oate (6). The nomenclature and numbering of ent-kaurane derivatives obtained in this work follow the IUPAC recomendations.


Introduction
Kauranes are an important class of diterpenes containing a rigid tetracyclic skeleton and exhibiting a wide variety of biological activities such as antitumor, anti-HIV, trypanocidal and antimicrobial, among others. 1 For this reason, the development of new strategies for the synthesis of novel kaurane derivatives may be considered as one of the interesting challenges in Chemistry of Natural Products. Indeed, many naturally occurring bioactive kauranes have been transformed using chemical and microbial methods in order to improve their bioactivity. 2,3 Kaurenoic acid (ent-kaur-16-en-19-oic acid, 1) is an intermediate in the biosynthesis of numerous plants and fungal secondary metabolites, including gibberellins, the phytohormones involved in the regulation of growth and development of higher plants, 1 found abundantly in some Brazilian species as Wedelia paludosa D.C. (Asteraceae), Xylopia frutescens and Annona glabra (Annonaceae). 4 We have reported, in a previous phytochemical study of Wedelia paludosa D.C., the isolation of kaurenoic acid (1) as the main ent-kaurane diterpene in this species, besides other related diterpenes and triterpenes as minor constituents. 4,5 Among more recently biological activities reported for 1, we can stand out the antimicrobial, 6 antiplatelet aggregation, 7 analgesic, 8 antifungal, 3,9 smooth muscle relaxant, 10 hypoglycemic, 11 cytotoxic and embryotoxic 12 effects.

Results and Discussion
Kaurenoic acid (1), isolated from aerial parts of Wedelia paludosa D.C., 4,5 was esterified with diazomethane to the corresponding methyl ester 2, which was subjected to two different pathways of chemical transformation (Scheme 1). 623 Batista et al. Vol. 18, No. 3, 2007 The first one was the epoxidation of 2 with MCPBA taking place stereoselectively at the more accesible side of the double bond, to yield exclusively the ent-16β,17epoxide 3, what was confirmed by X-ray crystallography. 14 Further rearrangement of 3 employing Lewis acids such as InCl 3 or BF 3 afforded a 1:1 mixture of epimer aldehydes 4 and 5, in moderate to quantitative yields (43% and 100%, respectively), that could not be separated by column chromatography. A mixture of products is usually obtained from rearrangement of epoxides to carbonyl compounds, due to lack of regioselectivity in the ring opening step. 15 Moreover, methyl ester 2 was subjected to hydroboration reaction with NaBH 4 and BF 3 •Et 2 O, followed by NaOH and H 2 O 2 oxidation, giving exclusively the hydroxymethyl group at the ent-α side of the derivative 6. These results, affording stereoselectively the alcohol 6 with an ent-16α configuration, are in agreement with literature data 16 and are justified by the regio-and stereoselectivity of the hydroboration-oxidation reaction, with a syn-addition taking place at the less hindered face of the double bond. Next, the PDC oxidation of 6 yielded the aldehyde 4 as the major product, together with the ent-kaurane 7 and ent-norkauranes 8 and 9 as minor products. The isomerization of 4 into its more stable epimer 5 was satisfactoriously performed by hydrochloric and acetic acids condition.
At the best of our knowledge, this is the first report of the kaurane and norkaurane derivatives 4, 7, 8 and 9 by the oxidation of methyl ent-17-hydroxykauran-19-oate (6) under PDC conditions. These products may be considered as subsequent oxidized compounds from alcohol 6. The initial oxidation of 6 afforded the expected aldehyde 4, which in the presence of the chromate underwent further oxidation to the acid 7. This acid can be considered the precursor of the norkauranes 8 and 9 according to the mechanism proposed in Scheme 2. As seen in this scheme, the key step of this mechanism is pointed to be the nucleofilic addition between HCrO 3 -(1 mol) and the acid 7, followed by intra-SN 2 rearrangement of this intermediate and finally a decarboxylation-oxidation step, respectively. So, it is possible to explain the synthesis of the norkaurane alcohol 8, bearing an ent-β configuration at C-16, in opposite to the other kaurane derivatives 4, 6 and 7, that stand an ent-16α configuration.
All products were characterized by mass, NMR and IR spectroscopies. Known compounds 1, 2, 3, 6, 7 and 9 were identified by comparison of their spectral properties (MS, 1 H NMR and, except for 3, 13 C NMR) with those reported in literature. [16][17][18][19][20] Compounds 4, 5 and 8, along with 13 C NMR data for compound 3, are reported here for the first time as far as the authors know.
Structures of compounds 4 and 5 were established on the basis of their IR, NMR ( 1 H NMR, 13 C NMR) and mass spectral data. FAB-HRMS data indicated molecular masses for 4 (333.2417) and for 5 (333.2428), both in agreement with the molecular formula C 21 H 32 O 3 (calculated = 333.2430). Aldehyde functions of 4 and 5 were evident in their IR spectra, with a C-H stretching band of -CHO group at 2701 cm -1 and in their 1 H NMR spectra, by characteristic signals at δ 9.89 and δ 9.65 (1H each), respectively ( Table  1). The ent-α and ent-β orientations of aldehydes 4 and 5, respectively, were deduced from the chemical shifts and multiplicity of H-17, which was deshielded as a singlet at δ 9.89 (4) or shielded as a 2 J 1.8 doublet at δ 9.65 (5) by the carbonyl group, according to literature data. 21 The shielded chemical shift of C-12 observed for 4 (δ 27.0), in comparison to that for 5 (δ 30.9), indeed ensure this assignement ( Table 2).
The norkaurane pattern of methyl esters 8 and 9 was confirmed by both 13 C NMR (20 signals each compound) and FAB-HRMS (C 20 H 32 O 3 and C 20 H 30 O 3 , respectively) methods. There is a close similarity between 1 H NMR and 13 C NMR (Tables 1 and 2) data of these compounds, the differences being those related to the alcohol (8) and ketone (9) functions at C-16. The location of the -OH group at C-16 in structure 8 was confirmed by the correlations in the COSY spectrum between H-16 (δ 4.14, d, J 6.0), H-15α (δ 1.90, m) and H-15β (δ 1.20, m), in addition to the helpfull HMQC spectrum data. The ent-β configuration of the -OH group at C-16 position was assigned in terms of gauche interactions, by comparison of its C-12 (δ 28.7) and C-14 (δ 36.1) chemical shifts with those (δ 29.0 and δ 38.5, respectively) from the epoxide 3 (Table 2). This assignement is also corroborated by the multiplicity observed for the H-16 signal (Table 1), since the doublet format is understandable if there is a

Conclusions
This work reports the synthesis of new oxidized entkaurane (4 and 5) and ent-norkaurane (8) derivatives starting from kaurenoic acid (1). In addition, we describe here, for the first time, the synthesis of compounds 4, 7, 8 and 9 by the oxidation of methyl ent-17-hydroxykauran-19-oate (6) under PDC conditions.

General experimental procedures
Melting points were taken with a Microquímica apparatus APF-301 and were uncorrected. Optical rotations were measured with a Perkin-Elmer 241 digital polarimeter. IR spectra were obtained on a Shimadzu IR-400 and Nicolet Impact 410 spectrophotometer. NMR spectra were recorded at 200 MHz for 1 H and 50 MHz for 13 C in deuterochloroform, added of TMS as internal reference, on a Bruker AC 200. The assignments of carbon signals were made by comparison with literature data and by means of 2D NMR 1 H and 13 C single bond correlation studies, on a Bruker Advance DRX400 (400 MHz for 1 H and 100 MHz for 13 C in deuterochloroform). Chemical shift values are expressed in ppm and coupling constants (J) in Hz. Column chromatography (CC) and flash column chromatography (FCC) were performed on silica gel Merck 60 (0.063-0.200 and 0.040-0.063 mm, respectively). HRMS were run in a VG TS-250 spectrometer working at 70 eV. TLC were carried out on silica gel Merck 60 F 254 (0.25 mm thick). Solvents and reagents were purified by standard procedures as necessary.

Procedures for preparation of compounds 4, 5, 7, 8 and 9
Step c, Scheme 1 A solution of epoxide 3 (40 mg, 0.12 mmol) in THF (3 mL) was added to a stirred suspension of InCl 3 (16 mg, 0.07 mmol) in THF (2 mL) at room temperature and stirring was continued for 1 h for reaction completion (TLC). The mixture was concentrated under reduced pressure. The recovered product was purified by FCC eluting with n-hexane-EtOAc (93:7) to afford a 1:1 mixture of aldehydes 4 and 5 (17 mg, 43%).
Step d, Scheme 1 The BF 3 ·Et 2 O complex (10 μL, 0.08 mmol) was added to a solution of epoxide 3 (41 mg, 0.12 mmol) in benzene (5 mL), and the system was stirred at room temperature under nitrogen by 30 min. The mixture was concentrated under reduced pressure, affording a 1:1 mixture of aldehydes 4 and 5 (42 mg, 100%).