As a part of our program on the discovery of new anticancer agents from plants, an ethyl acetate-soluble extract has been investigated of the stem bark of D. gelonioides (Roxb.) Engl., collected in 1994 on Palawan Island in the Philippines. Only one phytochemical investigation has been reported previously on this species, resulting in the isolation of the triterpenoid, friedelin.¹¹ Activity-guided fractionation using the LNCaP (hormone-dependent human prostate cancer) cell line led to the isolation of two new dichapetalin-type triterpenoids [dichapetalins I (2), and J (3)], and the parent compound, dichapetalin A (1), along with two dolabrane norditerpenoids (6,7), and zeylanol (8), 28-hydroxyzeylanol (9), and betulinic acid. In spite of the above-mentioned strong cytotoxicity of dichapetalin A for L1210 cells, highly functionalized triterpenoids of this class have not been subjected to evaluation against a human cancer cell panel previously. Thus, it was found in our work that dichapetalins A, I, and J all exhibited promising selective activity in a human tumor panel against the SW626 (human ovarian adenocarcinoma) cell line. Therefore, these compounds were prioritized for a more detailed biological evaluation of their antitumor potential.

Since all supplies of dichapetalins A, I, and J (1–3), along with the plant material of origin, were exhausted in the initial phytochemical and biological work, it was decided to obtain a recollection of the stem bark of D. gelonioides in order to perform follow-up biological testing on one or more of these compounds. However, subsequent to the initial collection of the plant in January, 1994, bioprospecting in the Philippines has been restricted under Executive Order 247 (EO247), effective from May 18, 1995.^12,13 In order to comply with these new regulations, a four-party Memorandum of Agreement for this specific plant recollection had to be signed (see Experimental Section). Further to this agreement, 12 kg of the stem bark of D. gelonioides were collected in Palawan Island in August, 2003, and an ethyl acetate extract was prepared at the University of the Philippines, Manila, and shipped to the United States for compound purification work. Altogether, 47 mg of dichapetalin A (1) were isolated from the recollected plant material. However, dichapetalins I (2) and J (3) were absent from the recollection, although two additional analogs were purified, namely, dichapetalins K (4) and L (5). In the present contribution, we describe the isolation, characterization, and biological testing of the constituents of D. gelonioides stem bark in a human tumor panel, as well as the evaluation of the parent phenylpyranotriterpenoid, dichapetalin A (1), in an in vivo hollow fiber test.¹⁴

Compound 1 was identified as dichapetalin A, a substance of previously established structure and absolute configuration, by comparing its spectroscopic data to values reported in the literature.⁸

The molecular formula of 2 was confirmed by HREIMS ([M]⁺ at m/z 602.3607) as C₃₈H₅₀O₆. Prominent IR absorption bands at 3454 and 1753 cm⁻¹ indicated the presence of one or more hydroxy function(s) and a five-membered lactone ring. The ¹H NMR spectroscopic data of 2 demonstrated an unsubstituted phenyl ring (δ_H 7.27–7.38, m, 5H), two double bonds (δ_H 5.51, 5.38), four methyl groups (δ_H 1.06, 1.18, 1.31, 1.74), and a cyclopropyl methylene (δ_H 0.43 and 0.76). Also, there were observed four oxymethine signals (δ_H 5.14, 4.26, 4.31, 3.84) and two oxymethylene groups [δ_H 4.05 (2H), 3.57, 3.77]. The ¹³C NMR spectrum of 2 showed 35 signals, which supported the presence of a phenyl ring, a cyclopropyl ring, two double bonds, and all the other structural elements mentioned above. The assignment of the two overlapping symmetric carbons of the phenyl ring at δ_C 125.8 and 128.3, and two methylene signals at δ_C 25.7 were clarified from their HMQC NMR spectrum. These data and other 2D NMR experiments conducted (COSY, HMBC) revealed a phenylpyranotriterpenoid skeleton with a γ-lactone side chain. Compared to 1, 2 was found to lack a Δ^11,12-double bond, but could be assigned one more secondary hydroxyl group. The position of this hydroxy group was determined at C-12 because of the long-range heteronuclear coupling of H-12 (J = 5 Hz) in a selective INEPT NMR experiment. The configuration of the hydroxy group at C-12 was defined as β based on a ROESY NMR experiment, which demonstrated a cross peak between H-12 and H-18. Therefore, the structure of 2 was assigned as (4α,6′α,7α,12β,17α,20S,23R,24E)-2′,3′,5′,6′-tetrahydro-7,12,23,26-tetrahydroxy-6′-phenyl-13,30-cyclo-29-nordammara-2,24-dieno[4,3-c]pyran-21-oic acid γ-lactone, and has been named dichapetalin I, according to a previous convention.⁸

The protonated molecular ion ([M+1]⁺) of a second new dichapetalin derivative (3) was indicated by CIMS at m/z 633, and confirmed by both positive- and negative-FABMS, consistent with an elemental formula of C₃₉H₅₂O₇. Attempts to obtain high-resolution mass spectral data for this compound were unsuccessful. The IR spectrum displayed hydroxy (3391 cm⁻¹) and a γ-lactone (1746 cm⁻¹) absorption bands. The ¹H NMR spectrum of 3 exhibited a very similar pattern to that of 2, except in the aromatic region as a result of the presence of a methoxy singlet at δ_H 3.79. The phenyl protons appeared as a pair of doublets (δ_H 6.87, d, J = 8.6 Hz; 7.29, d, J = 8.6 Hz) integrating for four protons, a typical pattern for a para-substituted aromatic ring. The ¹³C NMR spectrum of 3 exhibited one more resonance at δ_C 55.3 compared to 2. Several 2D NMR experiments (COSY, HMQC, HMBC) were also performed on 3 to permit the unambiguous interpretation of its ¹H and ¹³C NMR spectra. Compound 3 (dichapetalin J) was assigned the structure (4α,6′α,7α,12β,17α,20S,23R,24E)-2′,3′,5′,6′-tetrahydro-7,12,23,26-tetrahydroxy–6′-p-methoxyphenyl-13,30-cyclo-29-nordammara-2,24-dieno[4,3-c]pyran-21-oic acid γ-lactone.

Compound 4 exhibited a molecular formula of C₃₉H₅₀O₆ from its HREIMS. The ¹H NMR spectrum of 4 was very similar to that of 1, except for the presence of signals consistent with a methoxy group at δ_H 3.80 (3H, s) at C-4″. In the ¹³C NMR spectrum of 4, when compared with that of 1, a methoxyl carbon at δ_C 55.3 was observed, in place of the signal at C-4″. Also, a HMBC correlation between the methoxy methyl proton signal and C-4″ was apparent. Other COSY and HMBC correlations were consistent with the structure proposed. Thus, compound 4 was determined as (4α,6′α,7α,17α,20S,23R,24E)-2′,3′,5′,6′-tetrahydro-7,23,26-trihydroxy–6′-p-methoxyphenyl-13,30-cyclo-29-nordammara-2,12,24-trieno[4,3-c]pyran-21-oic acid γ-lactone (dichapetalin K).

The spectroscopic data of compound 5 were similar to those of 2. Compound 5 exhibited a molecular formula of C₃₈H₅₀O₅ from its HRESIMS, suggesting the loss of a hydroxy group. In the ¹³C NMR spectrum of 5, a methylene carbon (δ_C 25.0) was observed instead of an oxygen-bearing methine carbon (δ_C 66.3) at C-12, indicating a loss of the hydroxy group at C-12. Also upfield shifts were observed at C-11 and C-13 (11.6 and 7.4 ppm, respectively). The HMBC spectrum showed cross-peaks between H-11 and C-9 and C-12, between H-12 and C-11 and C-13, between H-18 and C-13, and between H-30 and C-7, C-9, and C-14. Other correlations in the HMBC and COSY spectra supported the structure proposed and the NMR spectroscopic assignments made. Therefore, compound 5 was characterized as (4α,6′α,7α,17α,20S,23R,24E)-2′,3′,5′,6′-tetrahydro-7,23,26-trihydroxy-6′-phenyl-13,30-cyclo-29-nordammara-2,24-dieno[4,3-c]pyran-21-oic acid γ-lactone (dichapetalin L).

Thus, it was readily apparent that compounds 3 and 4 represent methoxylated variants of dichapetalins I (2) and A (1), respectively. While it is possible that these are extraction artifacts due to the initial use of methanol as a solvent, samples of compounds 1 and 2 were not available to attempt chemical interconversion experiments by storage of these substances in methanol, since their supplies were consumed in the biological testing aspects of this study.

Compound 6 was isolated and identified as a known dolabrane norditerpenoid,^15–17 possessing a diosphenol system in the A ring, which has been found in both diterpenes^15,17,18 and triterpenes.¹⁹ This isolate from D. gelonioides exhibited the same ¹H-, ¹³C NMR, and MS data as “ent-16-nor-3-oxodolabr-1,4(18)-diene-2-ol-15-oic acid”, isolated from Endospermum diadenum Airy Shaw (Euphorbiaceae).¹⁵ However, no optical rotation was published for the E. diadenum isolate, making it unclear whether 6 and this known compound are the same or are enantiomeric.²⁰ The “dolabrane” (8,15-freido-pimarane) diterpenoid nomenclature was first proposed by Ansell et al. in 1993,²¹ and is based on dolabradiene, a diterpenoid isolated from Thujopsis dolabrata Sieb. et Zucc.²² In earlier literature, the same type of skeleton was named “dolabradane”.²³ Dolabradiene and a related compound, erythroxydiol Y, have the same carbon skeleton and the same configuration at C-13, but are enantiomeric at C-5, C-8, C-9, and C-10.²⁴ They have opposite specific rotations, with dolatradiene being negative (−70°) and erythoxydiol Y positive (+87°).^22,25 Fagonone, an analogue whose structure was proved by X-ray crystallography, has the same skelton as erythroxydiol Y, but with an A/B cis junction. The structure of fagonone was proposed by comparison of its CD spectrum to 7-oxosteroids with the same absolute configuration at C-5 and C-10, and it was shown to exhibit a positive optical rotation (+26°).²⁶ Therefore, because of its observed positive optical rotation ([α]_D +158°; c 0.46, MeOH), it was concluded that 6 is the same compound as “ent-16-nor-3-oxodolabr-1,4(18)-diene-2-ol-15-oic acids” from E. diadenum.¹⁵ On methylation using diazomethane, 6 afforded a monomethyl ester (6a). The structure and relative stereochemistry of 6 were confirmed in the present investigation by single-crystal X ray diffraction. The two carbon–carbon double bonds are 1.340 Å (Δ^1,2) and 1.318 Å (Δ^4,18), and rings B and C adopt a normal chair conformation while the A-ring is a half boat (Figure 1). It appears that many of the semi-systematic names of dolabrane diterpenoids are inconsistent in the literature, since they do not specify the configuration at the C-13 position. Therefore, 6 was identified as ent-16-nor-5α,14α(methyl)-3-oxodolabra-1,4(18)-dien-2-ol-15-oic acid.

Figure 1 ORTEP drawing of 6 with thermal ellipsoids at 50% probability for non-H atoms and open circles for H-atoms.

Since it also exhibited a positive optical rotation ([α]_D +47.4°; c 0.29, CHCl₃), compound 7 was identified as another known constituent of E. diadenum, namely “ent-16-nor-5a-2-oxodolabr-3-ene-3-ol-15-oic acid”,¹⁵ which was also published without any optical rotation. However, the semi-systematic name of this compound should be changed to ent-16-nor-5α,13α(methyl)-2-oxodolabra-3-en-3-ol-15-oic acid.

Zeylanol (8) was identified by comparing its spectroscopic data to values reported in the literature,^27,28 but its ¹³C NMR data were assigned for the first time in this investigation.

The molecular formula of compound 9 was indicated as C₃₀H₅₀O₃ by CIMS, with a [M+1]⁺ peak occurring at m/z 459. This was supported by HREIMS based on the [M-CH₂OH]⁺ peak at m/z 427 (C₂₉H₄₇O₂). The IR spectrum suggested the presence of hydroxyl group(s) (br 3453 cm⁻¹) and a cyclic ketone group (1715 cm⁻¹) similar to those of 8. The presence of primary and secondary hydroxyl groups was suggested from the ¹H-, ¹³C-, and DEPT NMR spectra of 9. Although the ¹H NMR signals of the H-6 and the CH₂-28 overlapped at δ_H 3.90, they could be correlated to the two hydroxyl proton signals (δ_H 5.64, d, J = 5.3 Hz; 5.99, br s) in the COSY NMR spectrum. The secondary hydroxyl group was assigned as C-6β, because the ¹H NMR data were in good agreement with the analogous data of 8. Confirmation was obtained from a ROESY NMR experiment, which showed spatial interaction between OH-6 and Me-24, OH-6 and Me-23, and H-4 and H-6. The primary hydroxyl group was assigned to C-28 because of the downfield shift of the C-17 resonance (+5.8 ppm), and the upfield shifts of the C-16 (−6.2 ppm), C-18 (−3.3 ppm), C-22 (−7.0 ppm) resonances compared to those of 8. The presence of a C-28 hydroxymethyl group was confirmed by comparing the data to those published for model compounds²⁹ and by a HMBC NMR experiment, indicating long-range correlations between C-28 and H-15, and C-28 and H-16. Compound 9 was therefore assigned as 6β,28-dihydroxyfriedelan-3-one.

Betulinic acid was identified by direct comparison to the standard compound,³⁰ and by comparing its spectroscopic data (MS, ¹H NMR, ¹³C NMR, UV, IR) to published values.^31–33

All compounds isolated (1–9 and betulinic acid) and the methylated derivative of 6 (6a) were screened for cytotoxicity against a panel of human cancer cell lines according to established protocols.^34,35 The dolabrane diterpenoid 6 and its methyl ester 6a were broadly cytotoxic against all cell lines tested. The cytotoxic potency of 6 was increased by methylation, with the methyl ester 6a being two- to eleven-fold more active than the parent compound. In contrast, the second dolabrane diterpenoid isolated (7), with a modified A ring when compared to 6, was not significantly active in any of the cell lines tested. Dolabrane diterpenoids have been found to inhibit mouse ear edema induced by 12-O-tetradecanoylphorbol acetate (TPA), and to inhibit mouse paw edema induced by carrageenan.³⁶ They also inhibited myeloperoxidase activity, an index of leukocyte migration, suggesting a mechanism of blocking the recruitment of neutrophils into inflammatory lesions.³⁶ Cytotoxicity against cancer cell lines with this type of compound is reported here for the first time. The triterpenoids, dichapetalins A (1), I (2), and J (3) showed selective and significant cytotoxicity (IC₅₀: 0.2–0.5 μg/mL) against the SW626 human ovarian cancer cell line, while dichapetalins K (4) and L (5) showed more broad cytotoxicity against the cell lines tested. Compounds 8 and 9 did not show any cytotoxicity for any of the cancer cell lines tested. Betulinic acid was reported to be a melanoma-specific cytotoxic agent in cell culture against the MEL-1 (ED₅₀: 1.1 μg/mL) and MEL-1 (ED₅₀: 2.0 μg/mL) cell lines.^30,37 In a series of studies performed with athymic mice injected subcutaneously with MEL-1 and MEL-2 cells, betulinic acid completely inhibited tumor development and growth in vivo.³⁰ In the current study, this isolate did not exhibit any cytotoxicity against the cancer cell lines tested, owing to the omission of a melanoma cell line from the panel.

Dichapetalin A (1) was evaluated in the in vivo hollow fiber model (i.p. and s.c.),¹⁴ at doses of 1, 2, 4, and 6 mg/kg. However, no significant growth inhibition was observed for any of the LNCaP (hormone-dependent prostate), Lu1 (lung), MCF-7 (breast adenocarcinoma), or SW626 (ovarian adenocarcinoma) human cell types used (data not shown). As a result of these negative data, dichapetalin A (1) and its phenylpyranotriterpenoid analogs do not seem to be very promising for their potential anticancer activity. Accordingly, dichapetalin A (1) will not be subjected to any more advanced biological testing through our plant anticancer drug discovery program.

The recollected dried stem bark of D. gelonioides (12 kg) was extracted four times with MeOH at room temperature. The resultant extracts were combined, concentrated under reduced pressure, and diluted with H₂O to afford a 75% aqueous MeOH extract, and washed with hexane. The lower layer was concentrated to dryness under reduced pressure and partitioned between 5% MeOH/H₂O and EtOAc. The EtOAc-soluble extract [42 g, ED₅₀ 3.4 μg/mL against the Lu1 cell line (human lung cancer cell line)] was subjected to silica gel CC and eluted with a gradient mixture of CHCl₃-MeOH to give 12 pooled fractions. Additional chromatographic separation of fraction 6 over silica gel using gradient mixtures of CHCl₃-acetone as eluents yielded seven subfractions (fractions 6A–6G). Further purification of subfraction 6C, using HPLC with an ODS-AQ column (250 × 20 mm; YMC, Inc., Willington, NC) using 83% CH₃CN-H₂O as eluant at a flow rate of 6 mL/min, afforded compound 1 (47 mg, t_R 29.1 min) and 5 (19 mg, t_R 42.4 min). Compound 4 (21 mg) was obtained from subfraction 6E using HPLC with 77% CH₃CN-H₂O (t_R 29.9 min).

Dichapetalin A, (4α,6′α,7α,17α,20S,23R,24E)-2′,3′,5′,6′-Tetrahydro-7,23,26-trihydroxy–6′-phenyl-13,30-cyclo-29-nordammara-2,12,24-trieno[4,3-c]pyran-21-oic acid γ-lactone (1): crystals; mp 213–214 °C; [α]_D²⁰ +31.5° (c 1.0, CHCl₃); lit.⁸ colorless crystals, mp 211–214 °C, [α]_D²⁰ +35° (c 1.5, CHCl₃); exhibited comparable spectroscopic (UV, IR, 1H NMR, 13C NMR) data to published values.⁸ CIMS and 2D-NMR experiments (COSY, HETCOR, NOESY) were performed to confirm the structural identity of 1.

Dichapetalin I, (4α,6′α,7α,12β,17α,20S,23R,24E)-2′,3′,5′,6′-Tetrahydro-7,12,23,26-tetrahydroxy–6′-phenyl-13,30-cyclo-29-nordammara-2,24-dieno[4,3-c]pyran-21-oic acid γ-lactone (2): crystals; mp 192–193 °C; [α]_D²⁰ +13.1° (c 0.11, CHCl₃); UV (CHCl₃) λ_max (log ε) 239 (2.95), 282 (2.90) nm; IR (dry film) ν_max 3454, 3017, 2926, 2854, 1753, 1585, 1216, 758 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.38 (2H, m, H-2″, H-6″), 7.34 (2H, m, H-3″, H-5″), 7.27 (2H, m, H-4″), 5.51 (1H, dd, J = 8.5, 1.4 Hz, H-24), 5.38 (1H, d, J = 6.9 Hz, H-2), 5.14 (1H, m, H-23), 4.31 (1H, m, H-12), 4.26 (1H, dd, J = 11.6, 2.5 Hz, H-6′), 4.05 (2H, s, H-26), 3.84 (1H, br s, H-7), 3.77 (1H, d, J = 10.7 Hz, H-2′), 3.57 (1H, d, J = 10.7 Hz, H-2′), 2.85–2.86 (2H, overlapped, H-17, H-20), 2.39 (1H, m, H-22), 2.18 (2H, dd, J = 13.4, 2.5 Hz, H-5′), 1.95–2.00 (4H, overlapped, H-1, H-5, H-11, H-15), 1.78–1.83 (2H, overlapped, H-6, H-22), 1.74 (3H, d, J = 0.8 Hz, H-27), 1.58–1.64 (3H, overlapped, H-1, 6, 15), 1.41 (2H overlapped, H-9, H-11), 1.31 (3H, s, H-28), 1.18 (3H, s, H-30), 1.06 (3H, s, H-19), 0.98 (2H, m, H-16), 0.76 (1H, d, J = 5.4 Hz, H-18), 0.43 (1H, d, J = 5.4 Hz, H-18); ¹³C NMR (90 MHz, CDCl₃), see Table 1; EIMS m/z [M]+ 602 (22), 447 (61), 260 (50), 131 (63), 105 (100); HREIMS m/z [M]⁺ 602.3607 (calcd for C₃₈H₅₀O₆, 602.3600).

Table 1 13C NMR Spectroscopic Data of Dichapetalins I (2) – L (5) (CHCl3, 90 MHz).

Dichapetalin J, (4α,6′α,7α,12β,17α,20S,23R,24E)-2′,3′,5′,6′-Tetrahydro-7,12,23,26-tetrahydroxy-6′-p-methoxyphenyl-13,30-cyclo-29-nordammara-2,24-dieno[4,3-c]pyran-21-oic acid γ-lactone (3): crystals; mp 210–212 °C; [α]_D²⁰ +14.0° (c 0.10, CHCl₃); UV(CHCl₃) λ_max (log ε) 240 (3.49), 275 (3.29), 281 (sh, 3.24) nm; IR (dry film) ν_max 3391, 2921, 2847, 1746, 1613, 1514, 1035 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 7.29 (2H, d, J = 8.6 Hz, H-2″, H-6″), 6.87 (2H, m, H-3″, H-5″), 5.51 (1H, dd, J = 7.9, 1.4 Hz, H-24), 5.37 (1H, d, J = 6.8 Hz, H-2), 5.13 (1H, m, H-23), 4.31 (1H, m, H-12), 4.20 (1H, dd, J = 11.6, 2.5 Hz, H-6′), 4.06 (2H, s, H-26), 3.81 (1H, br s, H-7), 3.79 (3H, s, OMe-4″), 3.76 (1H, d, J = 10.7 Hz, H-2′), 3.55 (1H, d, J = 10.7 Hz, H-2′), 2.84 (2H, overlapped, H-17, H-20), 2.40 (1H, m, H-22), 2.15 (2H, dd, J = 13.4, 2.5 Hz, H-5′), 1.96–2.00 (4H, overlapped, H-1, H-5, H-11, H-15), 1.78–1.83 (2H, overlapped, H-6, H-22), 1.74 (3H, s, H-27), 1.55–1.64 (3H, overlapped, H-1, H-6, H-15), 1.41 (2H overlapped, H-9, H-11), 1.31 (3H, s, H-28), 1.18 (3H, s, H-30), 1.05 (3H, s, H-19), 0.98 (2H, m, H-16), 0.76 (1H, d, J = 5.3 Hz, H-18), 0.43 (1H, d, J = 5.3 Hz, H-18); ¹³C NMR (90 MHz, CDCl₃), see Table 1; EIMS [M] 448 (13), 330 (37), 315 (85), 285 (79), 117 (100); CIMS m/z [M+1]⁺ 633 (1); FABMS m/z [M−1]⁺ 631 (51), [M+1]⁺ 633 (1).

Dichapetalin K, (4α,6′α,7α,17α,20S,23R,24E)-2′,3′,5′,6′-Tetrahydro-7,23,26-trihydroxy–6′-p-methoxyphenyl-13,30-cyclo-29-nordammara-2,12,24-trieno[4,3-c]pyran-21-oic acid γ-lactone (4): crystals; mp 116–118 °C; [α]_D²⁰ +34.1° (c 0.5, CHCl₃); UV (CHCl₃) λ_max (log ε) 241 (3.65), 265 (3.50), 318 (2.94) nm; IR (dry film) ν_max 3400, 2918, 2850, 1734, 1457, 1214 cm⁻¹; ¹H NMR (360 MHz, CDCl₃) δ 7.30 (2H, d, J = 8.7 Hz, H-2″, H-6″), 6.88 (2H, d, J = 8.7 Hz, H-3″, H-5″), 6.14 (1H, dd, J = 10.0, 2.9 Hz, H-12), 5.53 (1H, dq, J = 8.4, 1.3 Hz, H-24), 5.46 (1H, dd, J = 10.0, 2.1 Hz, H-11), 5.39 (1H, br d, J = 7.0, H-2), 5.14 (1H, ddd, J = 14.0, 7.2, 5.5 Hz, H-23), 4.22 (1H, dd, J = 11.7, 2.4 Hz, H-6′), 4.05 (2H, br s, H-26), 3.94 (1H, dd, J = 2.3, 2.3 Hz, H-7), 3.80 (3H, s, OMe), 3.76 (1H, d, J = 10.7 Hz, H-2′), 3.58 (1H, d, J = 10.7 Hz, H-2′), 3.10 (1H, ddd, J = 13.0, 8.2, 4.9 Hz, H-20), 2.59–2.66 (2H, overlapped, H-17, H-5′), 2.39 (1H, ddd, J = 13.0, 8.2, 5.5 Hz, H-22), 2.17 (1H, dd, J = 13.4, 2.5 Hz, H-5′), 2.10 (1H, dd, J = 16.4, 7.1 Hz, H-1), 2.01–2.04 (2H, overlapped, H-5, 15), 1.97 (1H, m, H-9), 1.76–1.86 (3H, overlapped, H-6, H-16, H-22), 1.74 (3H, d, J = 0.8 Hz, H-27), 1.64–1.73 (3H, overlapped, H-1, H-6, H-15), 1.32 (3H, s, H-28), 1.19 (1H, d, J = 5.2 Hz, H-18), 1.14 (1H, m, H-16), 1.08 (3H, s, H-19), 0.91 (3H, s, H-30), 0.78 (1H, d, J = 5.2 Hz, H-18); ¹³C NMR (90 MHz, CDCl₃), see Table 1; EIMS m/z [M]⁺ 614 (29), 612 (38), 596 (100), 584 (57); HREIMS m/z [M]⁺ 614.3607 (calcd for C₃₉H₅₀O₆, 614.3607).

Dichapetalin L, (4α,6′α,7α,17α,20S,23R,24E)-2′,3′,5′,6′-Trihydro-7,23,26-tetrahydroxy-6′-phenyl-13,30-cyclo-29-nordammara-2,24-dieno[4,3-c]pyran-21-oic acid γ-lactone (5): crystals; mp 110–112 °C; [α]_D²⁰ +16.8° (c 0.2, CHCl₃); UV(CHCl₃) λ_max (log ε) 240 (2.77), 267 (2.61), 318 (2.31) nm; IR (dry film) ν_max 3466, 2916, 2848, 1772, 1457, 1215, 1029 cm⁻¹; ¹H NMR (360 MHz, CDCl₃) δ 7.38 (2H, m, H-2″, H-6″), 7.34 (2H, m, H-3″, H-5″), 7.27 (2H, m, H-4″), 5.51 (1H, dd, J = 8.4, 1.1 Hz, H-24), 5.37 (1H, d, J = 6.8 Hz, H-2), 5.12 (1H, ddd, J = 14.1, 8.7, 5.6 Hz, H-23), 4.26 (1H, dd, J = 11.7, 2.4 Hz, H-6′), 4.05 (2H, br s, H-26), 3.83 (1H, dd, J = 2.3, 2.3 Hz, H-7), 3.76 (1H, d, J = 10.7 Hz, H-2′), 3.59 (1H, d, J = 10.7 Hz, H-2′), 2.98 (1H, ddd, J = 12.7, 8.4, 4.3 Hz, H-20), 2.62 (1H, m, H-5′), 2.55 (1H, m, H-17), 2.31 (1H, ddd, J = 13.1, 8.3, 4.8 Hz, H-22), 2.18 (1H, dd, J = 13.4, 2.5 Hz, H-5′), 1.93–2.00 (4H, overlapped, H-1, H-5, H-12, H-15), 1.83 (1H, m, H-22), 1.74 (3H, s, H-27), 1.57–1.65 (4H, overlapped, H-1, H-6, H-15, H-16), 1.50 (1H, dd, J = 13.5, 1.6 Hz, H-6), 1.36–1.44 (2H overlapped, H-11, H-12), 1.32 (3H, s, H-28), 1.25–1.30 (2H, overlapped, H-9, H-11), 1.07 (3H, s, H-30), 1.03 (3H, s, H-19), 0.96 (2H, m, H-16), 0.67 (1H, d, J = 4.6 Hz, H-18), 0.47 (1H, d, J = 4.6 Hz, H-18); ¹³C NMR (90Hz, CDCl₃), see Table 1; EIMS m/z [M]⁺ 586 (31), 584 (35), 574 (17), 566 (100), 562 (19); HREIMS m/z [M]⁺ 586.3673 (calcd for C₃₈H₅₀O₅, 586.3658).

ent-16-Nor-5α,13α(methyl)-3-oxodolabra-1,4(18)-dien-2-ol-15-oic acid (6): pale yellow crystals; mp 87–89 °C; [α]_D²⁰ +157.8° (c 0.45, MeOH); UV (MeOH) λ_max (log ε) 305 (3.75) nm; IR (dry film) ν_max 3416 (br), 2932, 2863, 1698, 1661, 1414, 1223 cm⁻¹; ¹H and ¹³C NMR data, consistent with literature values;¹⁵ EIMS m/z [M]⁺ 318 (44), 300 (6), 257 (4), 181 (19), 151 (79), 150 (54), 137 (80), 135 (100), 107 (28); HREIMS m/z [M]⁺ 318.1806 (calcd for C₁₉H₂₆O₄, 318.1831). This compound was obtained previously as a gum, with no [α]_D, UV, IR, or HRMS data obtained.¹⁵

ent-16-Nor-5α,13α(methyl)-2-oxodolabra-3-en-3-ol-15-oic acid (7): crystals; mp 73–74 °C; [α]_D²⁰ +47.4° (c 0.29, CHCl₃); UV(MeOH) λ_max (log ε) 283 (3.29) nm; IR (dry film) ν_max 3423, 3190, 2930, 2865, 1698, 1665, 1638, 1395, 756 cm⁻¹; ¹H and ¹³C NMR data, consistent with literature values;¹⁵ EIMS m/z [M]⁺ 320 (33), 305 (58), 151 (18), 137 (100), 121 (23); HREIMS m/z [M]⁺ 320.1979 (calcd for C₁₉H₂₈O₄, 320.1986). This compound was obtained previously as a semi-solid, with no [α]_D, UV, IR, or HRMS data obtained.¹⁵

Zeylanol [6β-Hydroxyfriedelan-3-one] (8): crystals; mp 254 °C [lit.²¹ 274–276 °C]; [α]_D²⁰ +9.5° (c 0.16, pyridine) [lit.21 [α]_D²⁰ +17° (CHCl₃)]; IR and ¹H NMR data consistent with literature values;^{21 13}C NMR (75 MHz, pyridine-d₅) δ 212.0 (s, C-3), 78.7 (d, C-6), 58.5 (d, C-4), 58.4 (d, C-10), 49.7 (d, C-8), 47.9 (s, C-5), 42.8 (d, C-18), 41.5 (t, C-2), 39.7 (s, C-13), 39.2 (t, C-22), 38.0 (s, C-14), 37.3 (s, C-9), 36.0 (t, C-16), 35.5 (t, C-11), 35.3 (t, C-19), 34.8 (q, C-30), 32.8 (t, C-21), 32.3 (t, C-15), 32.0 (q, C-28), 31.7 (q, C-29), 30.5 (t, C-12), 29.9 (s, C-17), 29.8 (t, C-1), 28.1 (s, C-20), 22.0 (t, C-7), 20.1 (q, C-27), 18.6 (q, C-26), 17.5 (q, C-25), 11.1 (q, C-23), 9.7 (q, C-24); EIMS m/z [M]⁺ 442 (95), 370 (48), 318 (79), 273 (100), 205 (88), 123 (93).

28-Hydroxyzelanol [6β,28-Dihydroxyfriedelan-3-one] (9): crystals; mp 282 °C; [α]_D²⁰ +2.8° (c 0.84, pyridine); UV (MeOH) λ_max (log ε) 251 (3.40), 257 (3.43), 263 (3.27) nm; IR (dry film) ν_max 3453, 2942, 2872, 1715, 1443, 1389, 1026, 710 cm⁻¹; ¹H (300 MHz, pyridine-d₅), δ 5.99 (1H, br s, OH-28), 5.64 (1H, d, J = 5.3 Hz, OH-16), 3.90 (2H, overlapped, H-28), 3.87 (1H, overlapped, H-6), 1.60 (3H, d, J = 6.7 Hz, H-23), 1.21 (3H, s, H-27), 1.08 (3H, s, H-29), 1.08 (3H, s, H-30), 1.03 (3H, s, H-24), 0.99 (3H, s, H-26), 0.86 (3H, s, H-25); ¹³C NMR (90 MHz, pyridine-d₅) δ 212.0 (s, C-3), 78.8 (d, C-6), 67.2 (t, C-28), 58.5 (d, C-4), 58.5 (d, C-10), 49.2 (d, C-8), 47.9 (s, C-5), 41.5 (t, C-2), 39.5 (s, C-13), 39.5 (d, C-18), 38.0 (s, C-14), 37.5 (s, C-9), 35.7 (s, C-17), 35.5 (t, C-11), 34.8 (t, C-19), 34.3 (q, C-30), 33.6 (t, C-21), 32.9 (q, C-29), 32.2 (t, C-22), 31.6 (t, C-15), 30.2 (t, C-12), 29.8 (t, C-1), 29.7 (t, C-16), 28.2 (s, C-20), 22.0 (t, C-7), 19.4 (q, C-27), 19.0 (q, C-26), 17.7 (q, C-25), 11.1 (q, C-23), 9.7 (q, C-24); EIMS m/z [M]⁺ 472 (41, M⁺-CH2OH), 409 (32), 289 (29), 137 (100); CIMS m/z [M+1]⁺ 459; HREIMS m/z 427(M⁺-CH₂OH, calcd for C₂₉H₄₇O₂, 427.3576).

Betulinic acid: crystals; mp 281–283 °C; [α]_D²⁰ +5.7° (c 0.84, pyridine) [lit.31 mp 275–278 °C; [α]_D²⁰ +7.9° (c 0.057, pyridine)]; UV, IR, ¹H, ¹³C NMR, and EIMS data, consistent with literature values.^31–33

This investigation was supported by grant U19 CA-52956, funded by the National Cancer Institute (NCI), NIH, Bethesda, Maryland. We acknowledge the NCI for the transfer of plant material and Palawan Council for Sustainable Development, Puerto Princesa, Palawan, Philippines for the recollection of the plant sample. We thank Ms. C. A. Crot, Research Resources Center, University of Illinois at Chicago and Mr. R. B. Dvorak, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago for the mass spectral data. We are grateful to the Nuclear Magnetic Resonance Laboratory of the Research Resources Center, University of Illinois at Chicago, for the provision of certain NMR spectroscopic facilities used in this investigation.