Dinor Casearin X , a New Cytotoxic Clerodane Diterpene from Casearia sylvestris

A new clerodane-type diterpene, named dinor casearin X, and four known casearins (A, B, G and J) were isolated from leaves of Casearia sylvestris (Salicaceae). These compounds were evaluated for cytotoxic activity against five human cancer cell lines (A2058, HL-60, HCT, MCF-7 and HeLa) as well as against a murine melanoma cell line (B16F10-Nex2). Among these compounds, dinor casearin X exhibited the highest cytotoxic activity against HL-60 cells with IC50 of 0.51 ± 0.11 μg mL, whereas casearin A exhibited the highest cytotoxic activity against HCT cells (IC50 1.84 ± 0.14 μg mL).


Introduction
Casearia sylvestris Swartz (Salicaceae), popularly known as "guaçatonga", is geographically distributed throughout Latin America, 1 where it has been used by native communities to treat several diseases. 2The use of this plant in traditional medicine and subsequent scientific investigations have highlighted the importance of C. sylvestris extracts due to their antiulcer, antiinflammatory, antiophidian and antitumor properties. 3Chemically, C. sylvestris extracts are rich in clerodane-type diterpenes, known as casearins and casearvestrins. 3,4Casearins A-F have been described as antitumor compounds, with casearin C exhibiting the highest cytotoxicity against V-79 cells in vitro. 5Moreover, casearin B exhibited chemoprotective effect against DNA damage. 6Other studies reported the occurrence of antitumoral casearins G-R, 7 DNA-damaging casearins S and T, 8 and cytotoxic casearins U and V. 9 Cytotoxic effects of casearin X were demonstrated in leukemia cells where it triggered apoptosis. 10,11Furthermore, cytotoxic casearvestrins A-C have also been isolated in C. sylvestris. 4 As a part of our continuous study aiming at the discovery of novel bioactive compounds from C. sylvestris, 12,13 this work reports the isolation and characterization of casearins A, B, G and J, as well as a new derivative named dinor casearin X, which displayed cytotoxic activity against the human leukemia cell line HL-60.

Experimental
General experimental procedures 1 H and 13 C nuclear magnetic resonance (NMR) spectra of compounds 1-4 were recorded at 300 and 75 MHz, respectively, in a Bruker Ultrashield 300 Avance III spectrometer. 1 H and 13 C NMR spectra of compound 5 were recorded in a Bruker AIII-500 (500 MHz for 1 H) spectrometer and in a Bruker Avance III Ultrashield Plus spectrometer (150 MHz for 13 C) with cryo-probe of 5 mm.CD 3 OD (Aldrich) was the solvent and the residual resonance peaks at d H 3.3 ( 1 H ) and d C 49.0 ( 13 C) were used as internal standard.Optical rotation was recorded in a Schmidt+Haensch polartronic H100 (automatic high resolution circular).Fourier transform infrared (FTIR) spectrum was recorded on a Shimadzu Prestige-21 FTIR spectrometer.High resolution electrospray ionization mass spectra (HRESIMS) were obtained in a Bruker Daltonics ultrOTOFq (ESI/time-of-flight (TOF), positive mode).High performance liquid chromatography (HPLC) analysis was performed in a Dionex Ultimate 3000 chromatograph, using a Luna Phenomenex RP-18 column (3 μm, 150 × 5 mm) and an UV-diode array detector (DAD).Silica gel (Merck, 230-400 mesh) and Sephadex LH-20 (Sigma-Aldrich) were used for the column chromatographic (CC) separation, while silica gel 60 PF 254 (Merck) was used for analytical and preparative thin-layer chromatography (TLC).

Cytotoxicity assays
The murine melanoma cell line B16F10 was originally obtained from the Ludwig Institute for Cancer Research (São Paulo, Brazil).The melanotic B16F10-Nex2 subline, which is characterized by low immunogenicity and moderate virulence, was identified at the Experimental Oncology Unit (UNIFESP).Human melanoma (A2058), leukemia (HL-60), colon cancer (HCT) and breast cancer (MCF-7) cell lines were obtained from the Ludwig Institute for Cancer Research.Human cervical carcinoma (HeLa) was acquired from PhD Hugo Pequeno Monteiro (UNIFESP).

Results and Discussion
Bioactivity guided chromatographic fractionation of the hexane phase obtained from the MeOH extract of C. sylvestris leaves led to the isolation of known casearins A (1), 5 B (2), 5,15 G (3) 7,8 and J (4) 7 as well as one new derivative named dinor casearin X (5) (Figure 1).Identification was carried out by analysis of their spectral data and comparison with those reported in the literature. 5,7,8,15inor casearin X (5) was isolated as an amorphous white solid, with a molecular formula C 28 H 40 O 9 , as determined from the HRESIMS (positive mode) adduct ion [M + Na] + at m/z 543.2564 (calcd.543.2570).The IR spectrum showed the presence of one carbonyl ketone conjugated to a double bound at 1617 cm -1 and one hydroxyl broad band at 3445 cm -1 .The 13 C NMR spectrum exhibited 28 signals (Table 1), several of which were similar to casearins 1-4, suggesting a substructure containing the decalinic system (rings A and B) and diacetalic ring C. Analysis of the heteronuclear multiple-bond correlation (HMBC) spectrum indicated the partial structure formed by the side chain containing one a,β-unsaturated system (double bond at  2).Trans configuration of the double bond at C-11 was confirmed by the coupling constant of doublets assigned to H-11 and H-12 (J 16.5 Hz) (Table 1). 16Additionally, the 13 C NMR spectrum displayed signals at d C 169.9, 173.0 and 173.3, which were assigned to one acetate and two butanoate groups, respectively. 10The acetate group was attached to C-17 of the diacetalic ring C, as deduced from the HMBC correlations between the signal at d H 6.31 (H-17) and that at d C 169.9 (C-1''').Furthermore, the signal at d H 6.31 (H-17) exhibited correlations with those at d C 145.7 (C-4), 53.4 (C-5), 71.5 (C-6) and 95.0 (C-16) (Figure 2).The assignment of the structure was straightforward with exception of the butyrate groups, since HMBC correlations between H-2, or H-16, and the carbonyl groups were not observed, in accordance with previously reported data. 17,18owever, the downfield chemical shift of C-2 (d C 66.3) and C-16 (d C 95.9) indicated that the butanoate groups were attached at C-2 and C-16 of the A ring. 10,17   23.5 (C-14), which confirmed the partial structure of the side chain.An additional cross peak between the signal at d H 1.17 (H-7) with that at d C 36.2 (C-7) suggested that there was no substituent in this carbon, a similar pattern previously observed for casearin X, 10 caseanigrescen D, 17 and argutin A. 19 Thus, considering that the structure of 5 differs from that of casearin X with regards to its C-9 side chain, which is shortened by two carbons, the name dinor casearin X was proposed for this substance.
1][22] The cis configuration of the A/B ring junction was deduced from chemical shift of Me-18 (d C 25.8). 10,21Cis clerodane diterpenes that are not substituted at C-7 show chemical shifts around d C 15 and 26 for Me-17 and Me-20, respectively, when these groups are in a trans relationship, 5   around d C 16 and 18 for Me-17 and Me-20, respectively, characterize a cis relationship. 7,10Considering that in the structure of dinor casearin X there is no substituent at C-7, the carbon resonances of Me-15, and Me-18 indicated their trans relationship.The carbon chemical shift for C-2 was determined to be d C 66.3, which is consistent with a C-2 substituent having an a-orientation.When the substituent has β-orientation the chemical shift is around d C 70. 17,19,20,23,24 Moreover, the J values of 7.5 and 14.0 Hz for the coupling H-10/H-1 revealed that H-10 has an axial orientation. 21he crude MeOH extract from the leaves of C. sylvestris and its respective hexane partition phase displayed cytotoxic effects on B16F10-Nex2, A2058, HL-60, HCT, MCF-7, and HeLa tumor cell lines.Bioguided fractionation afforded compounds 1-5 as being responsible for the activity (Table 2).Based on the IC 50 values casearins 1, 2 and 4 showed cytotoxic activities for all tested cell lines, while casearin G (3) exhibited cytotoxic activity only for HL-60, HeLa and HCT.Dinor casearin X (5) was the less active compound against the tested cell lines in comparison with casearins 1-4, except for the HL-60 cell line, thus indicating a probable selective activity of this compound.Therefore, studies upon the mechanism of action of this new compound on HL-60 cells must be addressed in order to eventually propose dinor casearin X as a lead antileukemic agent.

Conclusions
One novel clerodane diterpene, named dinor casearin X, was isolated from leaves of Casearia sylvestris.Dinor casearin X reduced cell viability of the HL-60 tumor cell line, indicating a selective cytotoxic activity of this compound when compared to that displayed against other cells lines (A2058, HCT, MCF-7 and HeLa).These findings suggest that dinor casearin X could be considered as a lead anti-leukemic agent.

C- 11
and carbonyl group at C-13) attached at C-9.The proposed structure was confirmed by correlations of the hydrogen signal at d H 7.05 (H-11) with carbon resonances at d C 41.5 (C-8), 41.0 (C-9), 25.8 (C-18), 35.7 (C-10) and 200.1 (C-13).Additional cross peaks between the signal at d H 6.15 (H-12) with those at d C 41.0 (C-9) and d C 200.1 (C-13), and between the signal at d H 2.20 (H-14) with the resonance of the carbonyl group at d C 200.1 (C-13), confirmed the connectivity of the carbon side chain at C-9 (Figure

Table 2 .
Cytotoxic activity of compounds 1-5 against human tumor cell lines Dox: Doxorubicin; nd: not determined.