and III.4 were not substrates for such efflux pumps.

The growth of MDA-MB-231 cancer cells was not particularly affected by the P.

madagascariensis abietane diterpenes. This cell line is a highly metastatic triple negative

breast cancer cell line, not displaying estrogenic receptors (ER), progesterone receptors (PR) or human epidermal growth factor receptor 2 (HER2), and thus, being clinically difficult to target313_{. It was known that the ER negative cells have a higher expression of}

PKC classic isoforms when compared to ER positive cell lines314,315_{. The upregulation of}

the classic isoform PKCα promotes the invasiveness and metastasis formation315–317

along with increased drug resistance318 _{in breast cancers. On the other hand, the PKCδ}

activation supports both prosurvival319 _{and proapoptotic functions in breast cancer}

cells320,321_{. Also, some abietane diterpenes (coleon U}156 _{and a benzyloxy derivative of}

7α-acetoxy,6β-hydroxyroyleanone322_{) have demonstrated to exert proapoptotic effects}

by the specific activation of the PKCδ. In this cell line, where its verified an overexpression of classic PKC isoforms, in detriment to new PKC isoforms, the preferential mechanism of apoptosis induction by coleon U (and eventually of other abietanes) by activation of PKCδ should be less effective, which justifies a lower growth inhibition of this cell line by such compounds.

There have been demonstrated some relationship between the PKC overexpression and the drug resistance by MDR1. Following the treatment with the PKC activator TPA, a cellular increase of the MDR1 expression was verified, which was suppressed by the use of a PKC inhibitor (staurosporine)323_{. Also, the use of a PKC inhibitor, bryostatin 1,}

potentiate the cytotoxic effects of anticancer drugs transported by efflux pumps as vincristine, by the reduction of MDR1 expression324_{. Those findings could be especially}

relevant in the case of abietane diterpenes not transported by MDR1, in which the PKC activation and the secondary MDR1 overexpression shouldn’t increase the cell resistance to those compounds.

87

Table 3.4. Growth inhibitory effects of the P. madagascariensis major compounds in different cell lines.

Cell line / GI50 (µM)

MDA-MB-231 MCF-7 HCT116 NCI-H460 NCI-H460/R MCR-5 SI#

III.1 ˃ 100 nt nt ˃ 100 ˃ 100 ˃ 100 -

III.2 ˃ 100 26.0 ± 0.6 ≥ 50 25 ± 2 25 ± 2 91 ± 13 4.3

III.3 ˃ 100 7.9 ± 0.8 7.9 ± 1.2 14.9 ± 2.9 nt nt -

III.4 ˃ 100 6.4 ± 0.4* - 2.7 ± 0.4 3.1 ± 0.4 8.6 ± 0.4 3.2

III.5 46.9 5.5 ± 0.8* - 3.0 ± 0.2* nt nt -

#_{Selectivity index. SI=GI}

50 (MCR-5)/GI50 (NCI-H460); nt – not tested; *Previously published results from our group39.

3.4. Structure activity relationships

The conjugation of the new data on abietane diterpene cytotoxicity (Table 3.4) with other studies on the cytotoxicity of this compound classes (Table 3.5) allowed the prospection of some structure-activity relationships (SAR). The compounds III.2-III.4 present as main difference the polarity of the group 7α (Figure 3.3). In this SAR, a clear tendency could be verified for the increasing cytotoxicity with the higher lipophilicity of the 7α substituent. The same tendency was verified between horminone (Figure 3.6, R1=H, R2=OH and R3=OH) and its more cytotoxic 7α-acetoxy derivate (Figure 3.6, R1=H,

R2=OAc and R3=OH)325,326.

The bibliographic exhaustive surge of reported cytotoxicity of royleanone-type compounds, bearing a p-benzoquinone moiety in the C-ring (Figure 3.6), additional tendencies could be explored (Table 3.5). Burmistrova et al. (2013) studied the cytotoxic effects from a series of derivatives of the 7α-acetoxy-6β-hydroxyroyleanone (III.4)40_{. The}

overall compounds affect the cell proliferation with an intensity apparently cell-type dependent. When such compounds were displayed by their log P value, a strong tendency for higher cytotoxic effects was verified for log P values between 2 and 5.5. As the “Lipinski rule of 5”, establish that the log P for an oral bioavailable compound should be under 5, the useful compounds must be considered in the 2-5 range of log P327_{. The}

only exception for this trend was royleanone (Figure 3.6, R1=H, R2=H and R3=OH) that

showed only slight cytotoxic effects in some cell lines40,325,328_{. This could indicate that}

the presence of a lipophilic substituent was needed at position 6 and/or 7 for the cytotoxic effects to take place. However, the royleanone 6,7dehidro derivative has showed some potent cell-type-specific cytotoxic effects329–331_{. This way, the presence of}

Table 3.5. Cytotoxicity of several royleanone derivatives, expressed as GI50 (µM) in different cell lines.

Compound features / Cell line (GI50 in µM) [reference] Breast CNS Colon Gastric Leukaemia/Lymphoma Melanoma Papil. Pancreas Renal Lung Normal

R1 R2 R3 logP MCF-7 _[39,40] SF-268 HCT116 AGS _[328] HL-60 _[329,330] P-388 _[331] U937 _[40] Molt-3 _[40] MEL-1 _[40] UACC-62 _[39] MV-3 _{[325] [331]}KB PaCa-2 _[325] TK-10 _[39] H460 MCR-5 _[328]

OH OH OH 0.85 + - - - +

OH OCHO OH 0.97 ++ ++ ++

OH OCOCH3 OH 1.08 ++ + + + + +++ ++ +++ ++

OCOCH3 OCOCH3 OH 1.31 + + + ++ +

OCOCH3 OCOCH3 OCOCH3 1.54 ++ ++ ++ +++ ++

H =O OH 1.57 - - H βOH OH 1.74 - + H OH OH 1.74 + +++ + + + + OCOCH2CH3 OCOCH3 OH 1.96 + + ++ ++ ++ H OCOCH3 OH 1.97 ++ +++ OH OCH3 OH 2.10 +++ + OH OCOCH3 OCOBzNO2(p) 2.30 + ++ +++ ++ ++ OCO(CH2)2CH3 OCOCH3 OH 2.38 ++ ++ +++ +++ +++ H6,7 _H6,7 _{OH 2.51 - +++/+ +++ ++ +/-} H H OH 2.83 - - - + - - - +

OCOCH2CH3 OCOCH3 OCOCH2CH3 2.85 + +++ +++ + +

OH OCOCH3 OCOBzCH3(p) 3.69 +++ +++ +++ +++ ++

OH OCOCH3 OCOBzCl(p) 3.77 +++ +++ +++ +++ +++

OCOBzNO2(p) OCOCH3 OH 4.31 ++ +++ +++ +++ ++

OCOBz OCOCH3 OCOBz 5.33 + +++ +++ ++ +++

OCOBzCl(p) OCOCH3 OCOBzCl(p) 6.45 - ++ ++ - -

OH O-FA OH >7 - - - - -

Otherwise stated all R2 conformation was β; logP values estimated using ChemBioDraw; –: not active (GI50>30); +: low cytotoxic (10<GI50≤30); ++: cytotoxic (5<GI50≤10); +++: highly cytotoxic (GI50≤

Papil. – papilloma; Bz – benzene; FA – fatty acid.

89

Figure 3.6. Structure and proposed SAR of 6,7,12-substituted royleanone-type abietane diterpenes based on data

compilated at Table 3.5.

3.7. Chapter conclusions

This chapter study presented the preparation of several P. madagascariensis

extracts, which have been screened for their cytotoxic activity. The main compounds in these extracts were identified by HPLC-DAD and the compound III.3 was isolated and spectroscopically characterized. The acetonic maceration extract (E5) showed high yields of abietane diterpenes, presenting also the highest cytotoxic effect in MDA-MB- 231 breast cancer cells (IC50 of 63.9 µg/mL). The observed cytotoxic activity may be

justified by the presence of known cytotoxic diterpenes, namely 7α,6β- dihydroxyroyleanone (III.2, 3.19 mg/g), 7α-formyloxy-6β-hydroxyroyleanone (III.3, 6.74 mg/g), 7α-acetoxy-6β-hydroxyroyleanone (III.4, 1.21 mg/g) and coleon U (III.5, 5.77 mg/g). To the best of our knowledge this is the first report of the presence of compounds