New Terpenoids from Croton limae (Euphorbiaceae)

An asymmetrical dimer of kaurane diterpene and monoterpene and four novel diterpenes were isolated from the roots of Croton limae, along with kaempferol-3-O-glucoside, ombuin3-O-rutinoside and acetyl aleuritolic acid. The cytotoxic activity of the kaurane diterpene was evaluated against colorectal adenocarcinoma (HCT-116), ovarian carcinoma (OVCAR-8) and glioma (SF-295) cell lines, exhibiting IC50 values of 7.14, 8.19 and > 10 μg mL−1, respectively.


Introduction
Croton is the second largest genus of the Euphorbiaceae family with around 1250 species dividided in 40 sections.The Croton section Argyroglossum comprises about 15 species characterized as shrubs and trees in the neotropics.In South America, there are ten reported species, six of which occurring in the Northeast of Brazil.Croton limae is endemic of this region and was often confused with the most related species C. argyrophyllus and C. tricolor, however, it was recently described by Gomes et al. 1 as a new species belonging to this section.

Experimental
General experimental procedures Infrared (IR) spectra were recorded on a Perkin-Elmer FTIR 1000 spectrometer (Waltham, USA), using NaCl disc.The nuclear magnetic resonance (NMR) spectra were performed on Bruker Avance DRX 500 or DPX 300 instruments, equipped with an inverse detection probe head and z-gradient accessory.All pulse sequences were standard in the Bruker XWIN-NMR software, and all experiments were conducted at room temperature.The 1 H and 13 C chemical shifts are expressed in the d scale and were referenced to tetramethylsilane (TMS) through the residual solvent.High resolution mass spectra were recorded on an UltrOTOF-Q mass spectrometer (LC-IP-TOF model 225-07100-34, Shimadzu) either by positive or negative ionization modes of the ESI source.Optical rotations were obtained on a Perkin-Elmer Q-2000 polarimeter, at 589 nm and 25 °C.Column chromatography was performed over silica gel 60 (Vetec, 70-230 and 40-63 mesh), Sephadex LH-20 (Pharmacia) and cartridge SPE C18 (Phenomenex).Thin layer chromatography (TLC) was performed on precoated silica gel aluminum sheets (Merck) and the compounds were visualized by UV detection and by spraying with vanillin/perchloric acid/EtOH solution, followed by heating.The tested compounds were analysed using the 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) purchased by Sigma Aldrich Co. and high-throughput screening (HTS), Biomek 3000-Beckman Coulter (Inc.Fullerton, California, USA).Absorbance was measured on a DTX 880 Multimode Detector, Beckman Coulter, Inc. Fullerton, CA, USA.

Plant material
Croton limae A. P. Gomes, M. F. Sales & P. E. Berry was collected at Andaraí county, Bahia State, Northeast of Brazil, in December 2009.The specimen was authenticated by Prof Maria Lenise S. Guedes, from the Instituto de Biologia, Departamento de Botânica, Universidade Federal da Bahia (UFBA), Bahia, Brazil, and a voucher specimen (92958) was deposited at the Herbário Alexandre Leal Costa (ALCB), Universidade Federal da Bahia, Bahia, Brazil.

Extraction and isolation
Roots (1.7 kg) of C. limae were pulverized and extracted with hexane at room temperature.The solvent was removed under reduced pressure to yield a residue (40.2 g), which was then extracted with EtOH to yield the correspondent extract (32.1 g).

Results and Discussion
Compound 1, a white solid, has a molecular formula of C 30 H 44 O 3 as established by the ion at m/z 451.3250 [M − H] + in the high-resolution electrospray ionisation mass spectrometry (HRESIMS).The IR spectrum showed bands at 1728 and 1697 cm −1 relative to carbonyl groups and at 3500 cm −1 relative to hydroxyl.The 1 H NMR spectrum of 1 displayed four distinct methyl singlets at d H 1.26 (CH 3 -8'), 1.15 (CH 3 -19), 1.10 (CH 3 -20) and 0.82 (CH 3 -9'), and an olefinic proton at d H 5.21 (br s, H-3'), besides several signals attributable to hydrogens attached to sp 3 carbons (Table 1).The 13 1 and 2).The HMBC spectrum provided evidences for the ABC rings of a kauranetype moiety by the long-range correlations between the hydrogens of the methyl at d From the above data, the two carbonyl groups were undoubtedly located at C-18 and C-15, respectively.On the basis of spectroscopic comparison of the diterpene moiety with those related for the ent-kaur-16-en-15-oxo-18-oic acid a great similarity has been observed between both compounds, and confirmed the partial structure. 9dditionally, were also observed in the HMBC spectrum the correlations of the methine at d ).On the basis of the foregoing evidence and comparison with the literature data, the monoterpene moiety was characterized as α-pinene. 10Thus, the structure of 1 was fully established as an unprecedented asymmetrical dimer of a kaurane diterpene bearing a monoterpene unit at C-16.The relative stereochemistry was elucidated  1).Signals relative to a subsystem constituted by five hydrogens belonging two diastereotopic methylenes at d H 2.56 (dt, J 14.2, 4.9 Hz, H-2β), 2.39 (ddd, J 14.2, 4.9, 1.9 Hz, H-2α), 2.22 (m, H-1β), and 1.68 (m, H-1α), and a methine at d H 2.51 (dd, J 12.4, 2.9 Hz, H-10), were observed by analysis of the 1 H-1 H COSY experiment.The deshielded diastereotopic protons of a methylene at d H 2.56 and 2.39 (2H-2) were attributed to the methylene "α" to a ketone group, due to the constant of 14.2 Hz.Furthermore, the coupling between both methylenes at d H 1.64 (m, H-6 eq ) and 1.50 (m, H-6 ax ), 1.42 (H-7 eq ) and 1.44 (and H-7 ax ), the latter showing correlation with the methine at d H 1.70 (m, H-8), revealed another subsystem of five hydrogens, which were placed between two quaternary sp 3 carbon atoms.In addition, the remaining methylene at d H 1.95 (dd, J 15.7, 9.0 Hz, H-11 eq ) and 1.59 (m, H-11 ax ) showed correlations with the oxymethine at d H 4.92 (dd, J 9.0, 1.5 Hz, H-12).The proposed assignment for the clerodane skeleton contained a β-monosubstituted furan moiety 9,13 was supported by analyzing the HMBC spectrum, through the long-range correlations of the methyl groups at d   New Terpenoids from Croton limae (Euphorbiaceae) J. Braz.Chem.Soc.1570 NOESY experiment (Figure 3).In this spectrum were also observed important correlations between the hydrogen at d H 2.51 (H-10) with the hydrogens at d H 1.37 (H-18) and 1.67 (H-12), as well as correlations between the hydrogens at d H 0.74 (H-20) with the hydrogens at d H 0.81 (H-19) and 1.68 (H-1a) (Figure 3) charactering a trans-decalin system.These data suggested that the lateral chain at C-19 was β-oriented (equatorial), confirming the final structure of compound 2 as the new 3-oxo-15,16-epoxy-4α,12dihydroxy-ent-neo-clerodan-13( 16),14,diene. 14mpound 3 was obtained as a colorless solid.The molecular formula C 20 H 32 O 4 was established by HRESIMS from the ion at m/z 359.2168 [M + Na] + .The IR spectrum showed the presence of a hydroxyl band at 3287 cm −1 .Analysis of the 13 C NMR spectrum of 3 revealed that the chemical shifts were similar to those observed for compound 2 (Table 2), except for the absence of the signals relative to the carbonyl and the presence of an extra oxymethine at d C 77.9 (C-3) (Table 2).The 1 H NMR spectrum corroborated these observations, through the presence of an additional signal for an oxymethine at d H 3.48 (t, J 2.5 Hz, H-3).The suggestion that the carbonyl group at C-3 for compound 2 has been reduced in 3, was confirmed by the diagnostic long-range correlations in the HMBC spectrum observed between the oxymethine proton at d H 3.48 (H-3) and the carbons at d C 18.1(C-1), 43.6 (C-5) and 23.4 (C-18).The relative stereochemistry of the hydroxyl group at C-3 was defined as α-oriented (equatorial) by the small value of the coupling constant (J 2.5 Hz) of the triplet observed for H-3.As for compound 2, the NOESY experiment showed the same correlations between the hydrogens at d H 1.38 (H-10) with the hydrogens at d H 1.06 (H-18) and 4.74 (H-12), as well as correlations between the hydrogens at d H 0.76 (CH 3 -20) with the hydrogens at d H 1.13 (CH 3 -19) and 1.54 (H-1a), and suggested that the lateral chain at C-19 was also β-oriented (equatorial).Thus, the final structure of compound 3 was elucidated as the new 15,16-epoxy-3α,4α,12-trihydroxy-ent-neo-clerodan-13(16)14-diene.
Compound 4 was obtained as a white solid.The molecular formula C 20 H 36 O 4 was established by HRESIMS by the ion at m/z 363.2524 [M + Na] + .The IR spectrum showed hydroxyl and C=C stretching bands at 3349 and 1653 cm −1 , respectively.The 1 H NMR spectrum signals indicated a close relationship with those observed for compound 3, mainly relative to the decalin system (Table 1).The differences found were related to the presence of just one olefinic proton at d H 5.47 (t, J 6.8 Hz, H-14), and two oxymethylenes at d H 4.14 (d, J 6.8 Hz, H-15) and 4.10 (s, 2H-16), besides the disappearance of the signals relative to the furan moiety and the oxymethine at C-12.These data suggested that the furan moiety of compound 3 was opened in 4.This was confirmed by comparison of their 13 2).The NOESY experiment showed correlations between the hydrogen at d H 1.87 (H-10) with the hydrogens at d H 1.50 (H-8), 2.03 (H-2a), 1.67 (H-6b) and 1.19 (CH 3 -18).In addition, it was observed the correlations between the hydrogens at d H 0.75 (CH 3 -20) with hydrogens at d H 1.12 (CH 3 -19), 1.40 (H-11b), 1.49 (H-7a) and 1.58 (H-1a), and the correlation of the hydrogen at d H 3.48 (H-3) with hydrogens at d H 1.19 (CH 3 -18).From the above data we can assume that the hydroxyls groups at C-3 and C-4, and the lateral chain at C-19 have the same configuration as in compound 3, so compound 4 was thus characterized as the new 3α,4α,15,16-tetrahydroxy-ent-neo-(E)-cleroda-13-ene.
Compound 5 was obtained as a white solid with a molecular formula C 26 H 38 O 10 as determined by the peak at m/z 533.2409 [M + Na] + .The IR spectrum implied the presence of hydroxyl and carbonyl groups at 3414 and 1699 cm −1 , respectively.From the 1 H NMR spectrum, a monossubstituted furan system was defined by the typical signals at d H 8.02 (br s, H-16), 7.59 (br s, H-15) and 6.78 (br s, H-14).The magnitude of geminal coupling constants observed for the deshielded methylene groups at d H 2.45 (td, J 14.2, 6.8 Hz, H-2 eq ), 2.33 (dq, J 14.2, 2.0 Hz, H-2 ax ), 2.92 (d, J 16.1 Hz, H-11 eq ), and 2.86 (d, J 16.1 Hz, H-11 ax ) were characteristic of "α" methylene ketone groups (Table 1).In addition, an β-anomeric proton at d H 4.45 (d, J 7.7 Hz, H-1'), and the signals in the region of d H 4.45-3.13,suggested the presence of a sugar unit, that was determined to be the glucose based on the chemical shifts of the 1 H and 13 C NMR spectrum in comparison with literature. 15The β-anomeric configuration was judged by the larger value of the coupling constant (J 7.7 Hz) of the doublet related to the anomeric hydrogen.The location of the hydroxyl group at C-4 was established by the concomitant long-range correlations of hydrogens at d H 0.97 (CH 3 -19), 2.64 (H-10) and 3.95 (H-6) with the carbon at d C 83.9 (C-4), in the HMBC spectrum.Moreover, the correlations between the hydrogen at d H 1.45 (CH 3 -18) with the carbons at d C 51.0 (C-5) and 214.1 (C-3), besides the correlation of the hydrogens at d H 0.97 (CH 3 -19) and the anomeric proton at d H 4.45 (H-1') with the carbon at d C 85.4 (C-6) confirmed the locations of carbonyl and glucosyl at C-3 and C-6, respectively.On the other hand, the correlations observed between the hydrogens at d H 0.87 (CH 3 -20) and 2.64 (H-10) with the carbon at d C 47.8 (C-11), determined the location of the other carbonyl group at C-12.The equatorial orientations of the glucosyl group and hydroxyl groups at C-6 and C-3, respectively, were determined based on the value of the coupling constant observed for the oxymethine H-6 (J 11.6, 3.9 Hz), and by nOe correlations observed between the hydrogens at d H 2.64 (H-10) with those at d H 1.45 (CH 3 -18), 2.05 (H-8) and 3.95 (H-6).Thus, compound 5 was characterized as the new 3,12-dioxo-15,16-epoxy-4α-hydroxy-6-(βglucopyranosyl)-ent-neo-clerodan-13( 16),14-diene.
7][18][19] The occurrence of asymmetrical dimers are more restricted, and generally involve the junction of two diterpene monomers. 20,21n Croton genus, this feature is limited to the species C. tonkinensis and C. micans, as symmetrical structures of ent-kaurene. 22,23The structure of an asymmetrical dimer formed by the junction between diterpene and monoterpene moities, as it is the case for compound 1, is a feature never reported in the literature before.

Conclusions
Chemical investigation of C. limae yielded an unusual asymmetrical dimer of a kaurane derivative with the monoterpene α-pinene (1), besides four new clerodanes (2-5).Based on the data collected from three independent experiments, the results showed that compound 1 exhibited a moderate cytotoxic effect against HCT-116 and OVOCAR-8 cancer cell lines with IC 50 values of 7.14, 8.19 and > 10 µg mL −1 , respectively.These facts are in agreement with the knowledge that the Croton genus is an abundant source of several classes of structurally complex and bioactive diterpenes, which justify the efforts in pursuing the phytochemical study of other Croton species from the northeastern Brazil.
H 2.10 (H-16) with carbons at d C 24.8 (C-12), 53.2 (C-8) and 35.5 (C-10'), besides the correlations of the olefinic methine at d H 5.21 (H-3') with carbons at d C 35.5 (C-10'), 45.8 (C-1') and 41.0 (C-5'), and those of remaining methyl groups at d H 0.82 (H-9') and 1.26 (H-8') with carbons d C 45.8 (C-1') and 41.0 (C-5' H 0.74 (H-20) with carbons at d C 45.9 (C-11), 42.2 (C-10) and 37.3 (C-8), and those of the methyl at d H 0.79 (H-17) with carbons at d C 39.9 (C-9) and 26.8 (C-7).The allocation of the hydroxyl group at C-12 was deduced on the basis of the correlations of the oxymethine at d H 4.92 (H-12) with the carbon at d C 39.9 (C-9), and with the carbons of the furan moiety at d C 108.4 (C-14) and 138.4 (C-16).On the other hand, the correlations of the methyl at d H 1.37 (CH 3 -18) and hydrogens of the methylene at d H 1.68 and 2.22 (2H-1) with the carbonyl at d C 216.0 (C-3), besides the concomitant correlations of the hydrogens at d H 0.81 (CH 3 -19), 2.51 (H-10), 2.39 and 2.56 (2H-2) with the non-hydrogenated oxygenated carbon at d C 81.8 (C-4), are consistent with the presence of a carbonyl at C-3 and a hydroxyl group at C-4.The configuration of the hydroxyl at C-4 α-oriented was definitively determined on the basis of diagnostic nOe correlations observed between the methyl at d H 1.37 (H-18) and the methines at d H 2.51 (H-10) and 4.92 (H-12) in the

Figure 2 .
Figure 2. Important dipolar couplings observed through the NOESY for compound 1.
C NMR data, since the typical furan values of compound 2 were here replaced by two oxymethylene carbons at d C 59.0 (C-15) and 60.5 (C-16), and a double-bond at d C 144.1 (C-13) and 127.3 (C-14) on 3 (Table

Figure 3 .
Figure 3. Dipolar coupling observed through the NOESY experiment for compound 2.

Table 1 .
1 H NMR chemical shifts of compounds 1-5 (d in ppm, J in Hz) Its molecular formula C 20 H 30 O 4 was determined by HRESIMS by the ion at m/z 357.2038 [M + Na] + .The IR spectrum displayed diagnostic absorption bands referring to hydroxyl and carbonyl groups at 3447 and 1707 cm −1 , respectively.The 13 C NMR spectrum displayed twenty signals associated with four methyl groups (d C 15.3, 16.0, 17.9 and 21.9), five methylenes and six methines (one of which oxygenated at d C 63.4 and three olefinic at d C 138.4, 131.5 and 108.4).In addition, five non-hydrogenated carbons including a carbonyl (d C 216.0) were observed (Table 2).The 1 H NMR spectrum revealed signals consistent with the presence of four methyls, three angular at d H 0.74 (s, CH 3 -20), 0.81 (s, CH 3 -19) and 1.37 (s, CH 3 -18), and a tertiary at d H 0.79 (d, J 6.7 Hz, CH 3 -17), an oxymethine at d H 4.92 (dd, J 5.0, 1.5 Hz, H-12), and three olefinic methines, one at d H 6.38 (br s, H-14) and two overlapped at d H 7.38 (m, H-15 and H-16) (Table 11,12NOESY data and comparison with kaurane-type diterpenoids.In this spectrum were observed correlation between the both methyls groups at d H 1.15 (CH 3 -19) and 1.10 (CH 3 -20), as well as the correlation between the hydrogens at d H 1.77 (H-5) and 1.20 (H-9).The orientation of the monoterpene group at C-16 was assigned as β due the strong nOe correlations observed between the hydrogens at d H 2.10 (H-16) and 1.30 (H-14); and d H 2.43 (H-14) and 1.10 (CH 3 -20) (Figure2).This finding was confirmed by the chemical shift observed for C-12 at d C 24.8 that was shielded in comparison to the ent-kaur-16-en-15-oxo-18oic acid at d C 32.4.This effect can be explained by the γ-gauche interaction of the C-12 with the methylene group at C-17, as observed for other reported kaurane dimers with β orientation.11,12Thus,compound 1 was determined to be the new 15-oxo-17(10'-α-pinenyl)-kauran-18-oic acid.Compound 2 was obtained as a yellow solid.