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.
Croton sublyratus, belonging to the Euphorbiaceae family, is called “Plau-Noi” in Thai. An acyclic diterpene alcohol, plaunotol, was isolated from this plant [
From the EtOAc-soluble fraction of a MeOH extract, two new cembrane-type diterpenes (1 and 2) and a phenolic compound (3) (
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 (
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 (MH 5.91) and H2-13 (δH 2.48) in the phase-sensitive NOESY spectrum (
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 (δH 3.87) for six protons, and one methoxy signal (δH 3.23) for three protons
H | 1 | 2 | 3 | |
---|---|---|---|---|
2 | 5.41 (d, 16) | [5.48] (d, 16) | 5.60 (d, 16) | [5.54] (d, 16) |
3 | 5.45 (d, 16) | [5.36] (d, 16) | 5.48 (d, 16) | [5.23] (d, 16) |
5 | 2.34 (2H, d, 7) | [2.31] (2H, m) | 2.09 (dd, 13, 12) | [2.18] (m) |
2.45 (ddd, 13,2,2) | [2.32] (ddd, 15, 3, 3) | |||
6 | 5.45 (d, 16) | [5.38] (br d,16) | 5.63 (ddd, 15, 12, 3) | [5.64] (ddd, 16, 11, 3) |
7 | 5.49 (d, 16) | [5.41] (dd,16, 2) | 5.35 (dd, 15, 2) | [5.34] (dd, 16,2) |
9 | 1.87 (m) | [1.84] (2H, m) | 1.66 (dd, 14, 14) | [1.85 (2H, m) |
1.90 (m) | 1.89 (m) | |||
10 | 2.16 (2H, m) | [2.18] (2H, m) | 2.20 (m) | [2.15 (dd, 12, 3) |
3.47 (m) | [2.24] (m) | |||
11 | 6.95 (m) | [6.89] (ddd, 11, 2, 2) | 5.91 (ddd, 11, 2, 2) | [6.83] (ddd, 11, 2, 2) |
13 | 2.35 (2H, dd, 14, 6) | [2.22] (m) | 2.48 (2H, m) | [2.24] (m) |
[2.40] (ddd, 17, 2, 2) | [2.44] (ddd, 17, 6, 5, 2) | |||
14 | 1.79 (ddd, 14, 14, 6) | [1.76] (ddd, 14, 14, 6) | 1.79 (m) | [1.75] (ddd, 14, 14, 6) |
1.95 (dd, 14, 6) | [2.04] (dd, 14, 6) | 1.88 (m) | [2.01] (dd, 14, 6) | |
15 | 1.88 (m) | [1.86] (m) | 1.84 (m) | [1.84] (m) |
16 | 0.98 (3H, d, 7) | [0.98] (3H, d, 7) | 0.95 (3H, d, 7) | [0.97] (3H, d, 7) |
17 | 0.96 (3H, d, 7) | [0.99] (3H, d, 7) | 0.96 (3H, d, 7) | [0.97] (3H, d, 7) |
18 | 1.40 (3H, s) | [1.39] (3H, s) | 1.39 (3H, s) | [1.28] (3H, s) |
19 | 1.29 (3H, s) | [1.28] (3H, s) | 1.21 (3H, s) | [1.31] (3H, s) |
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).
C | 1 | 2 | 4 | ||
---|---|---|---|---|---|
1 | 85.5 | (87.4) | 86.7 | 85.7 | (87.7) |
2 | 127.2 | (128.4) | 124.6 | 125.2 | (126.3) |
3 | 138.5 | (140.0) | 139.1 | 138.8 | (140.3) |
4 | 72.5 | (73.0) | 73.6 | 73.7 | (74.1) |
5 | 46.6 | (48.4) | 49.1 | 46.2 | (47.6) |
6 | 123.9 | (125.0) | 122.8 | 124.3 | (125.7) |
7 | 138.0 | (139.1) | 140.4 | 137.7 | (138.4) |
8 | 72.4 | (72.9) | 72.0 | 72.4 | (73.0) |
9 | 41.0 | (42.4) | 42.2 | 41.4 | (42.5) |
10 | 25.6 | (25.9) | 25.1 | 25.0 | (25.9) |
11 | 145.9 | (148.5) | 150.4 | 146.1 | (148.4) |
12 | 124.3 | (125.5) | 123.2 | 124.4 | (125.6) |
13 | 21.0 | (21.9) | 24.9 | 21.1 | (22.0) |
14 | 26.8 | (28.2) | 28.2 | 27.8 | (28.9) |
15 | 37.1 | (38.3) | 36.9 | 37.0 | (38.1) |
16 | 17.2 | (17.7) | 17.3 | 17.4 | (17.7) |
17 | 16.8 | (16.9) | 16.6 | 16.5 | (16.8) |
18 | 25.1 | (24.7) | 28.3 | 31.1 | (30.9) |
19 | 26.5 | (25.8) | 31.4 | 24.7 | (24.7) |
20 | 167.7 | (170.6) | 167.4 | 169.0 | (170.7) |
Data in parentheses are for CD3OD.
(
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).
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 13C-NMR spectra were taken on a JEOL ECA-600 at 600 MHz and 150 MHz with tetramethylsilane as an internal standard. Positive- ion 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).
C | H | |
---|---|---|
1 | 132.9 | - |
2, 6 | 103.3 | 6.55 (2H, s) |
3, 5 | 147.3 | - |
4 | 134.3 | - |
7 | 81.2 | 4.22 (1H, dd, 8, 6) |
8 | 37.4 | 2.08 (1H, m) |
2.25 (1H, m) | ||
9 | 62.4 | |
4.41 (1H, m) | ||
1’ | 130.4 | - |
2’, 6’ | 129.5 | 8.02 (2H, dd, 8, 1) |
3’, 5’ | 128.4 | 7.44 (2H, dd, 8, 8) |
4’ | 132.9 | 7.56 (1H, tt, 8, 1) |
7’ | 166.5 | - |
4’-OH | 5.49 (1H, s) | |
3, 5-OMe | 56.4 | 3.87 (6H, s) |
7Me | 56.6 | 3.23 (3H, s) |
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 H2O (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 H2O layer was evaporated to leave 16.3 g of a residue.
The residue (24.0 g) of the EtOAc-soluble fraction was subjected to silica gel (500 g) CC with a solvent system consisting of n-hexane (3 L), n-hexane-EtOAc [9:1 (3 L), 4:1 (3 L), 7:3 (3 L), 3:2 (3 L), 1:1 (3 L), and 3:7 (3 L)], EtOAc (3 L), and MeOH (3 L), 1 L fractions being collected. The residue (3.51 g) in fractions 6 - 8 was fractionated by ODS CC (Cosmosil 75 C18OPN) (Φ = 40 mm, L = 250 mm), by elution with H2O-MeOH [(3:7, 800 mL), (1:3, 800 mL), (1:4, 800 mL), (3:17, 800 mL), (1:9, 800 mL), and (1:19, 800 mL)], MeOH (800 mL), (CH3)2CO (800 mL), and EtOAc (800 mL), 800 mL fractions being collected. The residue (745 mg) in fraction 1 was further separated by ODS CC (Cosmosil 75 C18OPN) (Φ = 40 mm, L = 250 mm) using H2O-MeOH [(7:3, 800 mL), (3:2, 800 mL), (1:1, 800 mL), (2:3, 800 mL), (7:13, 800 mL), and (3:7. 800 mL)], and MeOH (800 mL), 800 mL-fractions being collected. The residue (108 mg) in fraction 4 was again purified by silica gel CC (Φ = 10 mm, L = 30 cm) with a linear gradient solvent system from n-hexane (250 mL) to n-hexane-EtOAc (1:1, 250 mL), 4-gram fractions being collected. From fractions 109‒120, 5.6 mg of 2 was obtained.
The residue (869 mg) in fractions 11 - 12 obtained on the first silica gel CC was separated by ODS CC with H2O-MeOH [(7:3, 800 mL), (3:2, 800 mL), (1:1, 800 mL), (2:3, 800 mL), (7:13, 800 mL), (3:7. 800 mL), (1:3, 800 mL), (1:4, 800 mL), (3:17, 800 mL), (1:9, 800 mL), and (1:19, 800 mL)], MeOH (800 mL), (CH3)2CO (800 mL), and EtOAc (800 mL), 800 mL fractions being collected. The residue (203 mg) in fraction 3 was purified by silica gel CC (Φ = 10 mm, L = 40 cm) with a linear gradient solvent system from n-hexane (250 mL) to n-hexane-EtOAc (1:1, 250 mL), 4-gram fractions being collected. Further amounts of n-hexane-EtOAc (1:1, 750 mL) and MeOH (250 mL) were eluted and 250 mL-fractions were collected. From the third n-hexane-EtOAc (1:1, 250 mL) fraction, 11.0 mg of 4 was obtained. The residue (276 mg) in fraction 4 was separated by silica gel CC (Φ = 10 mm, L = 40 cm) with a linear gradient solvent system from n-hexane (250 mL) to n-hexane- EtOAc (1:1, 250 mL), 4-gram fractions being collected. From fractions 67 - 74, 20.0 mg of 3 was obtained.
The residue (1.17 g) in fractions 13 - 14 obtained on the first silica gel CC was separated by ODS CC with H2O-MeOH [(7:3, 800 mL), (3:2, 800 mL), (1:1, 800 mL), (2:3, 800 mL), (7:13, 800 mL), (3:7. 800 mL), (1:3, 800 mL), (1:4, 800 mL), (3:17, 800 mL), (1:9, 800 mL), and (1:19, 800 mL)], MeOH (800 mL), (CH3)2CO (800 mL), and EtOAc (800 mL), 800 mL fractions being collected. The residue (121 mg) in fraction 3 was purified by silica gel CC (Φ = 10 mm, L = 34 cm) with a linear gradient solvent system from n-hexane (250 mL) to n-hexane-EtOAc (1:1, 250 mL), and n-hexane-EtOAc (1:1, 700 mL). Further amounts of n-hexane-EtOAc (1:1, 250 mL) and MeOH (250 mL) were eluted. From the final 250 mL n-hexane-EtOAc fraction, 8.2 mg of 1 was obtained.
The residue (779 mg) in fractions 17 - 18 obtained on the first silica gel CC was separated by ODS CC with H2O-MeOH [(7:3, 800 mL), (3:2, 800 mL), (1:1, 800 mL), (2:3, 800 mL), (7:13, 800 mL), (3:7. 800 mL), (1:3, 800 mL), (1:4, 800 mL), (3:17, 800 mL), (1:9, 800 mL), and (1:19, 800 mL)], MeOH (800 mL), (CH3)2CO (800 mL), and EtOAc (800 mL), 800 mL fractions being collected. The residue (74.2 mg) in fraction 3 was purified by silica gel CC (Φ = 10 mm, L = 28 cm) with a linear gradient solvent system from n-hexane (250 mL) to n-hexane-EtOAc (1:1, 250 mL), 4-gram fractions being collected. From fractions 101 - 124, a further amount of 2 (17.2 mg) was obtained.
Amorphous powder, [α]D23 +3.90 (c 0.28, CHCl3); IR νmax (film) cm‒1: 3423, 2968, 2929, 2881, 1697, 1631, 1469, 1371, 1320, 1267, 1094, 977; UV λmax (MeOH) nm (log ε): 234 (3.56); 1H-NMR (CDCl3 and CD3OD, 600 MHz):
Amorphous powder, [α]D25 ‒1.45 (c 0.46, CD3OD); IR νmax (film) cm‒1: 3445, 2967, 2929, 2881, 1693, 1630, 1455, 1381, 1320, 1244, 1118, 978; UV λmax (MeOH) nm (log ε): 233 (3.76); 1H-NMR (CDCl3, 600 MHz):
Amorphous powder, [α]D25 ‒0.47 (c 0.54, CHCl3); IR νmax (film) cm‒1: 3445, 2962, 2934, 2841, 1701, 1274; UV λmax (MeOH) nm (log ε): 212 (4.20), 227 (4.14); 1H-NMR (CDCl3, 600 MHz):
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.