New Cytotoxic Diterpenes from Cassipourea madagascariensis from the Madagascar Rainforest

Bioassay-directed fractionation of ethanol extracts of the roots and leaves of the plant Cassipourea madagascariensis resulted in the isolation of the two new terpenoids cassipourol (1) and cassipouryl acetate (2) in addition to the three known compounds, 3β,30-dihydroxylup-20(29)-ene (3), 30-hydroxylup-20(29)-ene-3-one (4), and combretol (5). The structures of the two new compounds were established on the basis of 1D and 2D NMR spectroscopic data and chemical conversion. All the isolated compounds were tested against the A2780 human ovarian cancer cell line; the two diterpenes (1 and 2) showed moderate cytotoxic activity while the three known compounds (3–5) were weakly active.

In our continuing research on the isolation of bioactive compounds from the Suriname and Madagascar rainforests as a part of the mission of the International Cooperative Biodiversity Group (ICBG),² we obtained a sample of Cassipourea madagascariensis DC. (Rhizophoraceae) from our collection work in Madagascar. C. madagascariensis is a shrub of 3 m height, with orange tri-lobed fruit, called “tavolopika” by the local population. The genus Cassipourea is a rich source of a variety of sulfur-containing amides,³ but C. madagascariensis has not previously been investigated. Ethanol extracts of the roots and leaves of C. madagascariensis, designated MG1871 and 1873, respectively, were selected for bioassay-guided fractionation on the basis of their cytotoxicity, with an IC₅₀ of 18.2 µg/mL against the A2780 ovarian cancer cell line for the leaf extract, and also because of the absence of any phytochemistry on the species.

Initial liquid-liquid partition of MG1871 and MG1873 indicated that the activity was concentrated in the hexane and CHCl₃-soluble fractions of the hexane/MeOH and CHCl₃/aqueous MeOH partitions. The combined hexane and CHCl₃ soluble fractions were subjected to chromatography over MCI gel followed by reversed-phase preparative TLC and HPLC, and furnished the two new bioactive diterpenoids, cassipourol A (1) and cassipourin A (2), in addition to the three known compounds, 3β,30-dihydroxylup-20(29)-ene (3),⁴ 30-hydroxylup-20(29)-ene-3-one (4),⁵ and combretol (5).⁶

Compound 1 was isolated as a colorless optically active viscous liquid whose molecular formula was established as C₂₀H₃₈O from its HRFABMS, ¹³C NMR and APT (attached proton test) spectroscopic data. These data indicated that the compound had two degrees of unsaturation, and an IR absorption band at 3380 cm⁻¹ indicated the presence of a hydroxyl functional group. The ¹H NMR spectrum showed the presence of an olefinic proton as a quartet of triplets at δ 5.40 (J = 1.4, 7.0 Hz), an oxymethylene group as a doublet at δ 4.14 (J = 7.0 Hz); three methyl singlets at δ 1.66, 0.866, and 0.853; two methyl doublets at δ 0.846 (J = 7.1 Hz), and 0.838 (J = 6.6 Hz); and eight methylenes and three methines between δ 1.05 and 1.99. The ¹³C NMR signals were assigned for all twenty carbons on the basis of APT, HMQC and HMBC spectroscopic data, and these assignments are shown in Table 1. Since the ¹³C NMR spectrum indicated the presence of only one double bond, 1 must be a monocyclic diterpene. A search of the literature indicated that the ¹H and ¹³C NMR spectroscopic data of 1 were similar to those of the monocyclic diterpenoids viridiol A (6) and viridiol B (7) isolated from the red alga, Laurencia viridis.⁷

Table 1.

¹H and ¹³C NMR Data for Compounds 1 and 2 (CDCl₃)^a

position	1		2
	1H	13C	1H	13C
1		36.74		36.72
2	1.28 m, 1.05 m	39.45	1.28 m, 1.04 m	39.45
3	1.26 m	24.87	1.26 m	24.88
4	1.24 m	37.44	1.24 m	37.43
5	1.50 m	28.06	1.44 m	28.06
6	1.38 m	32.88	1.36 m	32.88
7	1.28 m	24.55	1.26 m	24.54
8	1.34 m	37.44	1.33 m	37.43
9	1.40 m	32.78	1.38 m	32.75
10	1.38 m, 1.28 m	37.51	1.36 m, 1.26 m	37.51
11	1.53 m	25.21	1.52 m	25.11
12	1.99 m, 1.13 m	39.95	2.01 m, 1.08 m	39.94
13		140.42		142.89
14	5.40 (qt, 1.4, 7.0)	123.16	5.32 (qt, 1.2, 7.0)	118.05
15	4.14 (d, 7.0)	59.52	4.57 (d, 7.0)	61.52
16	0.866 s	22.80b	0.866 s	22.80d
17	0.853 s	22.70b	0.853 s	22.70d
18	0.846 (d, 7.1)	19.83c	0.845 (d, 6.9)	19.83e
19	0.838 (d, 6.6)	19.80c	0.838 (d, 6.4)	19.80e
20	1.66 br s	16.26	1.68 br s	16.45
OCOCH3				171.23
OCOCH3			2.04 s	21.16

^aAssignments made on the basis of HMQC, and HMBC spectroscopic data and in comparison with the literature values.⁷

^b–eValues having the same superscript in the respective columns may be interchanged.

A comparison of the ¹H and ¹³C NMR data of 1 with those of 6 and 7 indicated that they had very similar diterpenoid skeletons, differing only at carbons C-12 to C-15 and C-20. The primary hydroxyl group and the trisubstituted double bond in 1 were assigned to C-15 and C-13/C-14, respectively, based on COSY (H-3/H-2, H-4; H-5/H-4, H-6, H-18; H-7/H-6, H-8; H-9/H-8, H-10, H-19; H-11/H-10, H-12; H-14/H-15) and HMBC (H-2/C-1, C-3, C-16, C-17; H-5/C-1, C-3, C-4, C-6, C-7, C-18; H-7/C-1, C-5, C-6, C-8, C-9; H-9/C-8, C-10, C-11, C-19; H-14/C-12, C-13, C-15, C-20) correlations; selected HMBC correlations are shown in Fig. 1. The configurations of the three methyl groups at C-16, C-17 and C-18, and of the chiral centers at C-5 and C-6 were assigned to be the same as those of 6 and 7 based on their almost identical coupling constants and ¹³C NMR values. This was supported by the ROESY spectrum of 1, in which the methyl group at C-16 showed a correlation to the methine proton at C-6, and the methyl groups at C-17 and C-18 showed correlation to the methylene group at C-7. A comparison of the ¹³C NMR values of C-12 – C-15 and C-20 in 1 with the values of the corresponding carbons of crotonadiol isolated from Croton zambesicus⁸ suggested the E stereochemistry of the double bond between C-13 and C-14. This was supported by the ROESY spectrum of 1, in which the methyl group at C-20 did not show any correlation to the olefinic proton at C-14 but did show a correlation to the oxymethylene group at C-15. The stereochemistry of the secondary methyl group at C-9 position has not been established. On the basis of the above spectroscopic data, the structure of cassipourol was assigned as 1.

Figure 1.

Selected HMBC correlations of compound 1

The molecular formula of 2 was established as C₂₂H₄₀O₂ from HRFABMS (m/z 337.3120 [M+H]⁺, calcd for C₂₂H₄₁O₂, 337.3107) and ¹³C NMR spectroscopic data. The IR spectrum of 2 showed the absence of the absorption band corresponding to that of hydroxyl group and the appearance of an absorption band corresponding to an ester carbonyl at 1742 cm⁻¹. A comparison of the ¹H and ¹³C NMR spectra of 2 with those of 1 (Table 1) showed that they were identical except for the presence of signals for an acetate group [δ_H: 2.04 (s, 3H) and δ_C: 171.23 and 21.16] in the spectra of 2. The acetate group was thus assigned to C-15, and this was supported by from the key HMBC correlations: H-14/C-13, C-15, OCOCH₃; H-15/C-13, C-14, OCOCH₃; and OCOCH₃/C-15, OCOCH₃. Finally, acetylation of 1 with Ac₂O/pyr furnished a product that was identified as 2 on the basis of co-TLC and ¹H NMR, confirming the structure of 2 as cassipouryl acetate.

All the isolated compounds were tested against A2780 human ovarian cancer cell line. The two new diterpenoids 1 and 2 were modestly active with IC₅₀ values of 2.4 and 2.8 µg/mL, respectively, while the three known compounds 3–5 had weaker activity with IC₅₀ values of 12.2, 16.4, and 8.7 µg/mL, respectively.

Experimental Section

General Experimental Procedures

Optical rotations were recorded on a Perkin-Elmer 241 polarimeter. IR and UV spectra were measured on MIDAC M-series FTIR and Shimadzu UV-1201 spectrophotometers, respectively. NMR spectra were obtained on a JEOL Eclipse 500 spectrometer. The chemical shifts are given in δ (ppm) with TMS (tetramethylsilane) as internal reference and coupling constants are reported in Hz. MCI gel (CHP20P) was used for column chromatography. Mass spectra were obtained on a JEOL HX-110 instrument. Reversed-phase preparative TLC was performed on Baker Si-C₁₈F plates. HPLC was performed on a Shimadzu LC-10AT instrument with an ODS A323 column (250 × 10 mm).

Cytotoxicity Bioassays

The A2780 ovarian cancer cell line assay was performed at Virginia Polytechnic Institute and State University as previously reported.^1,9 The concentration of each compound that inhibited 50% of the growth of the A2780 mammalian cell line was determined; actinomycin D (IC₅₀ 1–3 ng/mL) was used as the positive control.

Plant Material

Samples of Cassipourea madagascariensis DC. (Rhizophoraceae) were collected in May 2003 by Fidy Ratovoson et al. in a forest adjacent to the national park of Zahamena, 3 km west of Ambatoharanana (17° 33′ 34″ S and 48° 53′ 42″ E, elevation between 750 – 900 m), in the province of Toamasina, on 03 May 2003. Duplicate voucher specimens (Ratovoson.F 694, TROPICOS specimen id 01902919¹⁰) were deposited at Centre National d'Application des Recherches Pharmaceutiques (CNARP) and Direction des Recherches Forestières et Piscicoles Herbarium in Antananarivo, Madagascar (TEF), Missouri Botanical Garden, St. Louis, Missouri (MO), and Museum National d'Histoire Naturelle in Paris, France (P).

Extraction and Isolation

Dried roots of C. madagascariensis (470 g) were extracted with ethanol to give extract MG1871 (15.6 g), and dried leaves (295 g) were extracted with ethanol to give extract MG1873 (11.2 g). Extract MG1871 (0.48 g) was suspended in aqueous MeOH (9:1 MeOH-H₂O, 100 mL) and extracted with three 100 mL portions of hexane. The aqueous layer was then diluted to 70% MeOH (v/v) with H₂O and extracted with three 100 mL portions of CHCl₃. The aqueous layer was concentrated and the residue obtained was suspended in H₂O (25 mL) and extracted with two 25 mL portions of n-BuOH. The hexane- and CHCl₃-soluble fractions were found to be active. The leaf extract MG1873 (0.90 g) was treated similarly to give active hexane- and CHCl₃-soluble portions. Both the hexane and the CHCl₃ fractions of the roots and leaves were similarly cytotoxic and had similar TLC and ¹H NMR patterns, so all four fractions were combined. The combined residue (0.50 g) was fractionated over MCI gel using MeOH/H₂O (1:1 to 100:0) and MeOH/CHCl₃ (100:0 to 70:30) to furnish twenty fractions (A–T), of which fraction O was found to be the most active. Fraction O upon further chromatography over MCI gel using MeOH/H₂O (1:1 to 100:0) and MeOH/CHCl₃ (100:0 to 80:20) furnished nine sub-fractions (O-1 to O-9), of which fractions O-4 to O-7 were the most cytotoxic. Fractions O-4 and O-5 were combined and separated by preparative reversed-phase TLC (MeOH-H₂O, 80:20) to yield the known flavanoid combretol (5, 1.4 mg) and the known triterpene 30-hydroxylup-20(29)-ene-3-one (4, 2.6 mg). Similarly, fractions O-6 and O-7 were combined and subjected to reversed-phase HPLC with the mobile phase MeOH:H₂O (75:25), thereby furnishing the two new diterpenes 1 (2.1 mg) and 2 (1.6 mg) in addition to the known triterpene, 3β,30-dihydroxylup-20(29)-ene (3, 1.8 mg). The structures of the known compounds (3–5) were identified by comparison of their spectroscopic data with literature values.^4–6

Cassipourol (1)

colorless liquid; [α]_D +10.9° (c 0.042, CHCl₃); UV (MeOH) λ_max 208.2 nm (ε 11 400); IR ν_max 3380, 2926, 1461, 1377, 1366, 1215, 1004, 757 cm⁻¹; ¹H and ¹³C NMR, see Table 1; HRFABMS m/z 295.2983 [M+H]⁺ (calcd for C₂₀H₃₉O, 295.3001).

Acetylation of 1

Compound 1 (0.8 mg) was dissolved in pyridine (0.2 mL) and acetic anhydride (0.2 mL) and the mixture was stirred for 16 h at room temperature. The product was dried under vacuum and the residue obtained was purified over reversed-phase preparative HPLC with the mobile phase MeOH-H₂O (80:20) furnished a product (0.7 mg), which was identified as 2 by the co-TLC, and ¹H NMR spectroscopic data.

Cassipourylacetate (2)

colorless liquid; [α]_D +10.6° (c 0.034, CHCl₃); UV (MeOH) λ_max 204.6 nm (ε 13 300); IR ν_max 2926, 1742, 1462, 1453, 1378, 1365, 1229, 1215, 1021, 747 cm⁻¹; ¹H and ¹³C NMR, see Table 1; HRFABMS m/z 337.3120 [M+H]⁺ (calcd for C₂₂H₄₁O₂, 337.3107).