iso-Kaurenoic acid from Wedelia paludosa D.C

A recent reinvestigation of aerial parts of Wedelia paludosa D.C. is described and reports, for the first time, the isolation of iso-kaurenoic acid from this species.


INTRODUCTION
The genus Wedelia (Asteraceae, tribe Heliantheae, subtribe Ecliptinae) consists of about 60 species spread in tropical and warm temperate regions, including Brazil, India, Burma, Ceylon, China and Japan.Many plants of this genus, which are used as traditional herbal medicines throughout the world, have been reported to possess hepatoprotective, antipyretic-analgesic, bactericidal and molluscicidal activities (Li et al. 2007, García et al. 2007).
The present paper describes a recent reinvest of aerial parts of W. paludosa D.C. and reports, first time, the occurrence in this species of ent-ka en-19-oic acid (iso-kaurenoic acid) (3).The isola the methyl ester of 3, from a mixture of 1+2+3, reported.

PREPARATION OF SILICA GEL IMPREGNATED WITH SILVER NITRATE (20%)
Column chromatography silica gel (64 g) was added to a solution of silver nitrate (16 g) in deionized water (100 mL).The aqueous mixture was concentrated in a rotary evaporator and dryed in an oven at 150 • C for 12 hr.The resulting grey powder was stored in vacuo in the dark for further use.

RESULTS AND DISCUSSION
ISOLATION AND IDENTIFICATION OF iso-KAURENOIC ACID After extraction with a hexane-dichloromethane mixture (1:1), the aerial parts of W. paludosa were extracted with ethanol to give the ethanol extract (EE) that, after successive CC over silica gel, has yielded a mixture of diterpenes 1, 2 and 3, as described in the experimental  to esterification with diazomethane to the corresponding mixture of methyl esters 4, 5 and 6, in the same proportion (14:17:8) of the starting material, according to the analysis of integral values observed on its 1 H NMR spectrum (Fig. 1).The composition of this mixture was evident by the presence of characteristic signals at δ 4.79 and 4.74 (s, H-17 α,β ) for compound 4; δ 5.24 (bs, H-11), 4.91 and 4.79 (s, H-17 α,β ) for compound 5; and, finally, δ 5.06 (s, H-15) for methyl iso-kaurenoate 6 (Wada et al. 1981, Batista et al. 2005, 2007).The unsuccessful attempts to isolate each constituent from the mixture 4+5+6 by usual CC on silica gel is in agreement with literature data, since a mixture of 4 and 6 was previously considered as an inseparable one (Wada et al. 1981).Thus, CC of this mixture on silica gel impregnated with iso-kaurenoate 6, the minor constituent of the mixture of acids 1+2+3, was successfully isolated with a 50% yield from the mixture of the methyl esters 4+5+6.
1 H and 13 C NMR data of compound 6 were found to be in agreement with those available for methyl isokaurenoate (Wada et al. 1981, Yamasaki et al. 1976).
Besides the presence of the characteristic singlet at δ 5.06 (1H, H-15) on the 1 H NMR spectrum of methyl isokaurenoate (6), in opposite to two singlets at δ 4.74 and 4.79 (1H each, H-17 α,β ) observed for methyl kaurenoate (4), compound 6 can also be distinguished from 4 by comparison of their C-8, C-9, C-12, C-13, C-15, C-16 and C-17 chemical shifts (Table I), whose differences are due to the presence of the double bond at C-15/C-16 (6) or C-16/C-17 (4) positions.Recent data on isokaurenoic acid (3) and methyl iso-kaurenoate (6) have not been found in the literature.Miles et al. (1990) stated that physical and chemical properties of iso-kaurenoic acid (3) had been previously reported by Bohlmann et al. (1981) and Hayman et al. (1986), but such data were also not found in these references.
IPP is transformed via an isomerase-catalyzed reaction into dimethylallyl-PP.In head-to-tail condensations, three molecules of IPP are sequentially added to this compound to form geranyl diphosphate (GPP, C 10 ), farnesyl diphosphate (FPP, C 15 ), and, finally, the C 20 compound (Scheme 2) occurs in a very similar way to the c tion that produces cyclic triterpenes.However, t no previous epoxidation step as occurs in triterpe clization, but the double bond protonation on the isopropylidene unit of the GGPP chain leads to tw hydronaphtalene bicyclical intermediates [Scheme palyl diphosphate (CPP, I) and ent-copalyl dipho (ent-CPP, II)], resulting in the two enantiomeric that differ from each other in their inverted con tions of the carbons C-5, C-9 and C-10.Name "normal" series are the structures whose fusion b A and B rings occurs in the same way as in st "main" -2010/11/4 -11:13 -page 828 -#6
Kaurene synthase (KS) catalyzes the cyclization of ent-copalyl diphosphate (ent-CPP, II) to ent-kaur-16-ene (ent-kaurene, 11) through a multiple-step reaction mechanism that is depicted in Scheme 3.According to this scheme, diphosphate ionization-initiated cyclization of Scheme 3 -Cyclization mechanism for pimaradienes, kaurenes and atiserenes (Xu et al. 2007).KS is found in all higher plants because kaurene is an intermediate in the route to the diterpenoid gibberellin phytohormones required for normal growth and development.Hence, this enzyme participates in primary metabolism (Xu et al. 2007).
Kaurenoic acid (1), an ent-kaurane diterpene, discloses a wide spectrum of bioactivities such as antiin-quantification in these plant species in order to ena of them to be used as natural sources of this dit (García et al. 2007).It is one of the intermediat pounds in the biosynthesis of diverse kaurane dite including gibberellins, a group of growth phytohor (Rademacher 2000).Therefore, it is not surprisi many naturally occurring kauranes act as growth tors in plants (García et al. 2007).

CONCLUSION
W. paludosa is an abundant source of ent-kaurenoic acid (1), which is a bioactive diterpene showing a wide spectrum of biological effects and of interest as a starting compound for the production of bioactive derivatives.The presence in this species of iso-kaurenoic acid (3), which is a diterpene of very restricted occurrence, along with its effect as total feeding inhibitor of boll weevils and very potent antifungal compound against soil-borne fungi, brings naturally occurring kauranes to an important position as starting materials for the synthesis of new derivatives of promising agrochemical application as pesticides.