Cembrane-Type Diterpenoids and a Phenolic Compound from the Leaves of a Thai Medicinal Plant , Croton sublyratus Kurz

From the leaves of a Thai medicinal plant, Croton sublyratus, collected in Thailand, two new cembrane-type diterpenoids, named sublylactones A and B, and a phenolic compound were isolated from the EtOAc-soluble fraction of a MeOH extract. Their structures were elucidated on the basis of spectroscopic evidence.


Results and Discussion
From the EtOAc-soluble fraction of a MeOH extract, two new cembrane-type diterpenes (1 and 2) and a phenolic compound (3) (Figure 1) were isolated by a combination of various types of chromatography.Their structures were elucidated from spectroscopic evidence.
Sublylactone A (1), [α] D 23 +3.90, was isolated as an amorphous powder and its elemental composition was determined to be C 20 H 30 O 4 by the observation of a quasi-molecular ion (C 20 H 30 O 4 Na) on high-resolution (HR)electrospray ionization (ESI)-mass spectroscopy (MS).The IR spectrum exhibited absorption bands for hydroxyl groups (3423 cm -1 ), C-H (2968, 2929 and 2881 cm -1 ), a lactone (1697 cm -1 ), and double bonds (1631 cm -1 ), and the UV spectrum indicated the presence of a conjugated system (234 nm).In the 1 H-NMR spectrum, signals assignable to two singlet methyls, two doublet methyls and five olefinic protons were observed (Table 1).The 13 C-NMR spectrum showed twenty signals that were assignable to four methyls, five methylenes, one methine, three oxygenated tertiary carbons, three double bonds and a carbonyl carbon (Table 2).Of the three double bonds, two were disubstituted ones and their geometry was determined to be E from the coupling constants of olefinic protons on them. 1 H-1 H correlation spectroscopy (COSY) established five partial structures, A: H-2 -H-3, B: H-5 -H-7, C: H-9 -H-11, D: H 2 -13 -H 2 -14 and E: H 3 -16 -H-15 -H 3 -17, and these partial structures were connected by heteronuclear multiple-bond correlation spectroscopy (HMBC) (Figure 2).Partial structures A and B were connected through an oxygenated tertiary carbon at δ C 72.5 (C-4) by the following HMBC correlations: H 3 -18 to C-3, C-4 and C-5, partial structures B and C through an oxygenated tertiary carbon at δ C 72.9 (C-8) by the correlations: H 3 -19 to C-7, C-8 and C-9, and partial structures C and D through C-12 including the position of the carbonyl group by the following correlations: H-11 to C-20, H-13 (δ H 2.40) to C-20, H 2 -10 to C-12, and H 2 -14 to C-12.Finally, partial structures A and D were connected through the remaining oxygenated tertiary carbon at δ C 87.4 by the following correlations: H-2 to C-1 and H 2 -14 to C-1.The dimethyl group (partial structure E) was placed on C-1 by the HMBC correlations from H 3 -16 and H 3 -17 to C-1.Thus, a cyclic structure was proposed for sublylactone A (1), and the remaining one degree of unsaturation was expected to be compensated for by the formation of a lactone ring between the C-20 carboxylic acid and the hydroxy group at the C-1 position.Structurally related lactone cembranoids were isolated from Croton laevigatus as laevigatlac tones A-E and the relative structure of laevigatlactone A was determined by X-ray crystallographic analysis [8].From the above evidence, the structure of sublylactone A (1) was expected to be that of a stereomeric isomer of laevigatlactone E (4), which was simultaneously isolated from this plant.On comparison of 13 C-NMR spectral data for sublylactone A (1) and laevigatlactone E (4) (Table 2), C-2 to C-7 showed some differences, C-2 by 2.1 ppm, C-4 by 1.6 ppm, C-6 by 0.7 and C-7 by 0.7 ppm.The most prominent difference was observed between the C-18 methyls, 6.2 ppm (Table 2).Therefore, the structure of sublylactone A (1) was elucidated to be 4-epi-laevigatlactone E, as shown in Figure 1.
Sublylactone B (2), [M] D -1.45, was isolated as an amorphous powder and its elemental composition was the same as that of 1, NMR spectroscopic data also indicating that 2 possessed the same functionality as that of 1.The geometry of two disubstituted double bonds (C-2=C-3 and C-6=C-7) was assigned as E from the coupling constants of their olefinic protons.The remaining double bond (C-11=C-12) was assigned to Z geometry from the significant evidence of the NOE correlation between the olefinic proton at H-11 (M H 5.91) and H 2 -13 (δ H 2.48) in the phase-sensitive NOESY spectrum (Figure 3).The relative structures of the methyl groups at the 4and 8-positions were estimated to be in a β orientation from the NOE correlation peaks between H 3 -18 and H-7, H 3 -19 and H-7, and H 3 -19 and H-10 (δ H 3.47) (Figure 3).Therefore, the relative structure was elucidated to be as shown in Figure 1.
Phenolic compound (3), [α] D 25 -0.47, was isolated as an amorphous powder and its elemental composition was determined to be C 19 H 22 O 6 by positive-ion HR-ESI-MS.The IR spectrum exhibited distinct absorptions assignable to hydroxy groups (3445 cm -1 ) and an ester functional group (1701 cm -1 ).The 1 H NMR together with the 13 C spectral data suggested the presence of monosubstituted and symmetrically tetrasubstituted aromatic rings as well as one methoxy signal (δ H 3.87) for six protons, and one methoxy signal (δ H 3.23) for three protons   (Table 3).From their chemical shifts, the former were expected to be on the aromatic carbons and the latter on the aliphatic carbon.The remaining signals comprised those of one methylene, one oxygenated methylene, one oxygenated methine and a carbonyl carbon.In the

Plant Material
Leaves of C. sublyratus were collected in the Botanical Garden of the Faculty of Pharmacy, Chiang Mai University, Thailand in July 2008.A voucher specimen was deposited in the Herbarium of the Faculty of Pharmacy, Chiang Mai University(CS-CMU-July-2008).

General Experimental Proceddures
Optical rotations were measured on a JASCO P-1030 digital polarimeter.IR and UV spectra were measured on Horiba FT-710 and JASCO V-520 UV/Vis spectrophotometers, respectively. 1H-and 13 C-NMR spectra were taken on a JEOL ECA-600 at 600 MHz and 150 MHz with tetramethylsilane as an internal standard.Positiveion HR-ESI-MS was performed with an Applied Biosystems QSTAR XL NanoSprayTM System.Silica gel CC was performed on silica gel 60 (E.Merck, Darmstadt, Germany).

Extraction and Isolation
Powdered and air-dried leaves of C. sublyratus (450 g) were extracted with MeOH (2 L × 3) and the total extracts were concentrated to 1 L. The concentrated MeOH extract was washed with n-hexane (1 L, 7.35 g) and then the remaining MeOH layer was concentrated to a viscous gum.The viscous gum was suspended in H 2 O (1 L), and then partitioned successively with EtOAc (1 L) and 1-BuOH (1 L) to give EtOAc-soluble (24.0 g) and 1-BuOH-soluble (7.88 g) fractions, respectively.The H 2 O layer was evaporated to leave 16.3 g of a residue.