Croton sublyratus, belonging to the Euphorbiaceae family, is called “Plau-Noi” in Thai. An acyclic diterpene alcohol, plaunotol, was isolated from this plant [1] , which is already on the market as an anti-ulcerative agent called Kelnac [2] . Diterpenelactones, plaunols A and B [3] [4] , and ent-labdane and ent-kaurane [5] have also been isolated from C. sublyratus. From this plant, isolation of a furanoid diterpene was also reported [6] [7] . Our reinvestigation of constituents of C. sublyratus resulted in the isolation of two cembrane-type diterpenoids, named sublylactones A (1) and B (2), and a phenolic compound (3) together with a known cembrane-type diterpenoid, laevigatlactone E (4), which has also been isolated from Croton laevigatus [8].

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]_D²³ +3.90, was isolated as an amorphous powder and its elemental composition was determined to be C₂₀H₃₀O₄ by the observation of a quasi-molecular ion (C₂₀H₃₀O₄Na) 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 ¹H-NMR spectrum, signals assignable to two singlet methyls, two doublet methyls and five olefinic protons were observed (Table 1). The ¹³C-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. ¹H-¹H correlation spectroscopy (COSY) established five partial structures, A: H-2 - H-3, B: H-5 - H-7, C: H-9 - H-11, D: H₂-13 - H₂-14 and E: H₃-16 - H-15 - H₃-17, 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: H₃-18 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: H₃-19 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, H₂-10 to C-12, and H₂-14 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 H₂-14 to C-1. The dimethyl group (partial structure E) was placed on C-1 by the HMBC correlations from H₃-16 and H₃-17 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 ¹³C-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-lae- vigatlactone 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 H₂-13 (δ_H 2.48) in the phase-sensitive NOESY spectrum (Figure 3). The relative structures of the methyl groups at the 4- and 8-positions were estimated to be in a β orientation from the NOE correlation peaks between H₃-18 and H-7, H₃-19 and H-7, and H₃-19 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]_D²⁵ ‒0.47, was isolated as an amorphous powder and its elemental composition was determined to be C₁₉H₂₂O₆ 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 ¹H NMR together with the ¹³C 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

Data in brackets are for CD₃OD. In parentheses, number of hydrogen are specified, when they are not 1H. Letters and figures are multiplicities and J in Hz).

Data in parentheses are for CD₃OD.

(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 ¹H-¹H COSY spectrum, two proton chains were observed, these are oxymethylene (H₂-9)-methylene (H₂-8)-oxymethine (H-7) and H-2' through H-4'. In the HMBC spectrum, oxymethylene protons (δ_H 4.36 and 4.41) showed significant correlation cross peaks with the carbonyl carbon (δ_C 166.5), to which the aromatic protons of H-2' and 6' [δ_H 8.02 (2H, dd, J = 8, 1 Hz)] were also correlated. The oxymethylene protons further correlated with C-7 (δ_C 81.2) and methylene protons (δ_H 2.08 and 2.25) with C-1 (δ_C 132.9). The positions of the methoxy groups were assigned as on C-3 and 5, and C-7 by the HMBC correlation shown in Figure 4. Therefore, the structure of 3 was elucidated to be 7-methoxydihydrosynapyl alcohol 9-O-benzoyl ester, as shown in Figure 1. Due to the very small optical rotation value, it is uncertain whether 3 is a racemic, partially racemic or chiral compound. A related compound, (7S)-7-methoxydihydro- synapyl alcohol (5) was isolated from Acer truncatum [9]. The optical rotation value of 5 was reported to be ‒6.8 in CHCl₃ ([M]_D ‒16.4, where molecular rotation is calculated as [M]_D=[α]_D × MW./100). Thus, 3 ([M]_D ‒5.0) was estimated to be nearly a racemic compound in which an S-form is slightly dominant.

The authors are grateful for access to the superconducting NMR instrument (JEOL JNM α-400) and an Applied Biosystem QSTAR XL system ESI (Nano Spray)-MS at the Analysis Center of Life Science of the Graduate School of Biomedical Sciences, Hiroshima University. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japan Society for the Promotion of Science. Thanks are also due to the Research Foundation for Pharmaceutical Sciences and the Takeda Science Foundation for the financial support.