Furanoditerpenes from Pterodon pubescens Benth with Selective in vitro Anticancer Activity for Prostate Cell Line

O fracionamento biomonitorado do extrato diclorometânico das sementes de Pterodon pubescens Benth forneceu o 6α-acetóxi-7β-hidróxi-vouacapano 1 (inédito), além de quatro diterpenos furânicos (2, 3, 4 e 5). A atividade antiproliferativa dos compostos foi avaliada in vitro contra as linhagens de células tumorais humanas UACC-62 (melanoma), MCF-7 (mama), NCI-H460 (pulmão), OVCAR-03 (ovário), PC-3 (próstata), HT-29 (colon), 786-0 (rim), K562 (leucemia) e NCI-ADR/RES (ovário com fenótipo de resistência a múltiplos fármacos). Os resultados foram expressos em três concentrações efetivas GI 50 (concentração para que ocorra 50% de inibição de crescimento), TGI (concentração que resulta em inibição total de crescimento) e LC 50 (concentração que resulta em 50% de morte celular). A citotoxicidade in vitro foi avaliada também frente a uma linhagem de célula murina normal (3T3). Este é o primeiro relato de atividade anticâncer para os compostos 1, 4 e 5, que apresentaram grande seletividade, dependente da concentração, para PC-3. O composto 1 foi 26 vezes mais potente para inibir 50% do crescimento (GI 50 ) de PC-3, 15 vezes mais citostático (TGI) e 6 vezes menos tóxico (LC 50 ) quando comparado com Doxorrubicina (controle).

Among the many compounds identified for cancer treatment Taxol, isolated from Taxus brevifolia, has proven to be an important chemotherapeutic agent. 1 Medicinal plants still play an important role as source of new targets for drug discovery. The huge structural diversity of natural compounds and their bioactivity potential have meant that several products isolated from plants, marine flora and microorganisms can serve as "lead" compounds for improvement of their therapeutic potential by molecular modification. 2,3 Pterodon genus compromises four species native to Brazil: P. abruptus Benth., P. apparucuri Pedersoli, P. pubescens Benth. (P. emarginatus Vog.) and P. polygalaeflorus Benth. Initially scientific studies of these plants were motivated by evidence that the seed's oil had cercaricidal 4 and anti-microbial activity. 5 Pterodon pubescens Benth. (Leguminosae) known, as Sucupira Branca is widespread throughout Goiás, Minas Gerais and São Paulo states in Brazil. The seeds are commercially available in Brazilian medicinal flora market. Plant's crude alcoholic extracts are used in folk medicine as anti-inflammatory, analgesic and anti-rheumatic preparations. [5][6][7] Phytochemical studies of Pterodon genus have revealed the presence of alkaloids, isoflavones and diterpenes. Furanditerpenes were identified and isolated from Pterodon fruits. [8][9][10][11] Some authors have suggested that furan-diterpenes possessing vouacapan skeleton are involved with antiinflammatory properties of Pterodon pubescens seeds´ oil. 5,12,13 Diterpenes 6α-hydroxyvouacapan-7β-17β-lactone and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester, present in P. emarginatus and P. polygalaeflorus seeds, respectively, were previously found to be associated with anti-inflammatory activity of these species. 12 Another compound, acid 6,7-dihydroxyvouacapan-17β-oic, was suggested to be one of the possible compounds involved with anti-inflammatory activity, since this compound was identified in the active fraction that exhibited antiedematogenic activity when tested in carrageenin-induced paw edema or in Croton oil-induced ear edema assays. 13 Evidence of biogenic amines involved with antinociceptive effect of a vouacapans extracted from P. polygalaeflorus Benth was studied by Duarte et al. 14 suggested that the pharmacological activity was triggered by catecholaminergic system.
Coelho et al. 15 studied Pterodon seed extract's antinociceptive activity suggesting that both peripheral and central inhibitory mechanisms are involved.
In the present study we report the isolation by activityguided fractionation, identification and in vitro anticancer activities of vouacapans from Pterodon pubescens Benth that are herein reported for the first time.

Results and Discussion
C o m p o u n d s 6 α , 7 β -d i a c e t o x y vo u a c a p a n 2 , 7β-diacetoxyvouacapan 3, 6α,7β-dihydroxyvouacapan-17β-oate methyl ester 4, and 6α,7β-dihydroxyvouacapan-17β-methylene-ol 5 (Figure 1) were identified based on comparison of experimental 1 H and 13 C-NMR with reported spectral data. 9,10,16,17 Novel compound 1 was deduced as having an elemental formula C 22  3 Hz indicating a transdiaxial relationship. When compound 1 was acetylated with excess acetic anhydride /pyridine, this compound showed identical 1 H and 13 C-NMR spectral data to compound 6α,7β-diacetoxyvouacapane 2 9 (HREI-MS 402.2630), suggesting that compound 1 has the same relative configuration to 6α,7β-diacetoxyvouacapane 2 with a hydroxy group attached β to C-7 whereas the acetyl group positioned α at C-6 ( Table 1).
The activity-guided fractionation of methylene chloride soluble fraction was monitored by in vitro anticancer activity assay in UACC-62 (melanoma), MCF-7 (breast), NCI-H460 (lung, non-small cells), OVCAR-3 (ovarian), PC-3 (prostate), HT-29 (colon), 786-0 (renal), K562 (leukemia) and NCI-ADR/RES (ovarian expressing phenotype multiple drugs resistance) cancer cell lines. A 48 h SRB (Sulforhodamine B) cell viability assay was performed to determine growth inhibition and cytotoxic properties of fractions and compounds. Cells were treated with at least four different concentrations levels (0.25 to 250 µg mL -1 ) with determination of three endpoints, concentration inhibiting the growth of 50% of the cells (GI50), concentration for total growth inhibition (TGI) and concentration needed to kill 50% of the cells (Table 2). 18 Compounds 2 and 3 were equally not potent based on GI 50 , TGI and LC 50 values.
A mean graph for compounds 1-5 corroborated the selectivity of compounds 1, 4 and 5 for PC-3 human prostate cancer cell lines ( Figure 2). The mean graph was developed by NCI emphasize differential effects of test compounds on various human tumor cell lines. This graph is generated from a set of GI 50 , TGI, or LC 50 values.
Positive values project to the right of the vertical line and represent cellular sensitivities to the test agent that exceed the mean. Negative values project to the left and represent cell line sensitivities to the test agent that are less than the average value. 19 Based on the three graphics, PC-3 cell line was high sensible to compounds 1, 4 and 5 . The interesting thing to notice is that compound 1 was more potent than 4 and 5 to inhibit cellular growth in 50% (GI 50 , Figure 2A), whereas compounds 1 and 5 showed almost same potency in causing cytostatic (TGI, Figure 2B). On the other hand, when cytotoxicity parameter (LC 50 , Figure  2C) was evaluated, compound 5 was more toxic whereas 1 and 4 were similarly toxics to PC-3 cell line. This high selectivity to PC-3 cell line suggests that furanoditerpenes 1, 4 and 5 may share a similar action mechanism, probably evolving androgenic receptors.
Compound 4 was able to reduce in 50% cellular growth of MCF-7 and NCI-H460 cell lines and also presented activity against NCI/ADR-RES cell line measured by all three parameters.
Compound 1 demonstrated selectivity 26 fold more potent than the positive control (doxorubicin) for PC-3 (prostrate) cell line based on GI 50 values, causing cytostatic effect (TGI value) at a concentration fifteen times inferior than positive control (doxorubicin). Moreover comparison Cytotoxicity in normal cell lines of compounds 1, 4, 5 were evaluated against 3T3 cell line (mouse embryonic fibroblasts) assessing mitochondrial functions by MTT reduction with succinate dehydrogenase in order to obtain cell viability. Compound 1 (IC 50 = 34.33 µg mL -1 ) demonstrated to be slightly less cytotoxic than compounds 4 (IC 50 = 22.83 µg mL -1 ) and 5 (IC 50 = 23.55 µg mL -1 ). All these values were higher than almost all GI 50 and TGI obtained for promissory compounds.
Cyproterone, a steroid formed by the mevalonate pathway is a known drug used as anti-androgen for prostrate cancer treatment. 20 Cyproterone is a steroidal antiandrogen agent that inhibits the action of adrenal and testicular androgens on prostate cells, seminal vesicles, testes, and the vas deferens. Additionally causes a centrally mediated reduction in testicular secretion of androgens. This drug is indicated for treatment of prostate cancer, androgen induced disorders of the skin (acne, seborrhoea, hirsutism, alopecia), precocious puberty and sexual disorders in men. 21,22 Male rats treated during two weeks with 100 and 300 mg kg -1 doses of Pterodon pubescens dichloromethane crude extract decreased body weight gain by 57 and 75% respectively. 23 That difference in body weight gain may have a relationship with antiandrogen activity of vouacapan type compounds found in the crude dichloromethane UACC-62 (melanoma), MCF-7 (breast), NCI-H460 (lung non-small cells), OVCAR-3 (ovarian), PC-3 (prostate), HT29 (colon), 786-0 (renal), K562 (leukemia) and NCI-ADR/RES (ovarian expressing phenotype multiple drugs resistance). GI 50 : concentration (µg mL -1 ) inhibiting the growth of 50% of the cells; TGI: concentration (µg mL -1 ) total growth inhibition; LC 50 : concentration (µg mL -1 ) need to kill 50% of the cells. 18 extract. Decrease of mean final body weight was also observed with cyproterone after 15 days treatment. 24 This data corroborates with the hypothesis that vouacapan type compounds interact with testosterone receptors. Further animal studies shall evaluate these findings. Considering that compounds 1, 4 and 5, furanditerpenoid, also originates from the mevalonate biosynthetic pathway, 20

Preparation of plant extract (EB) and fraction purification
Freeze-dried seeds (100g) were grinded prior to use on a Stephen mill (model UM 40 ) and extracted with dichloromethane three times during two hour periods, with 5:1 solvent/plant ratio, at room temperature. The extract was dried over anhydride Na 2 SO 4 filtered and concentrated under vacuum (Buchi RE 120), with 32% yield of crude seed oil extract (EB).
This crude oil (18. The resulting fractions were monitored by thin layer chromatography (TLC), exposed with anysaldehyde reagent (50 mL acetic acid, 0.5 mL sulfuric acid and 0.5 mL anysaldehyde) followed by heating at 110 o C. According to TLC profile the fractions were group and submitted to biological assay. The in vitro anticancer model on nine human cell lines determined the fractions that were further purified. Among the fractions isolated by column chromatography, Fraction 7 presented the best anticancer in vitro activity (data not shown).

Conclusions
Considering the data presented herein the chemotherapeutic potential of compounds 1, 4 and 5 were determined as possible candidates of new agents with high selectivity for prostate cancer. Further in vivo studies and in vitro assays are needed to establish pharmacological mechanism, toxicity and production viability.