STRUCTURE OF A NEW TRITERPENE GLYCOSIDE FROM CENTROSEMA BRACTEOSUM*

ABSTRACT

A new triterpene 3-O-[α-L-Rhamnopyranosyl (1 ->2)-(β-D-xy-lopyranosyl 16β,23 (R): 16α,30α-diepoxy-20(S)-hydraxydammar-24-ene (1) was isolated from roots of Centrosema bracteosum. The structure of this triterpenoid glycoside was elucidated by spectroscopic analysis of the natural product (1) and its peracetyl derivative (1a), including 2D NMR experiments.

Keywords:
Centrosema bracteosum; Leguminosae-Faboideae; triterpene glycoside; spectral data.

RESUMO

Um novo Triperpeno 3-0-[α -L-Rhamnopiranosil (1 ->2)-(β-D-xylopiranosil 16 β, 23(R):16α, 30α - diepoxi-20(2)-hidroxidammar-24-eno(1) foi isolado a partir das raizes de centrosema bracteosum. A estrutura deste glicosídeo triterpenoidico foi elucidada por análise espectroscópica do produto natural (1) e seu derivado peracetil (1a), incluindo experimentos de 2D RMN.

Palavras chave:
Centrosema bracteosum, Leguminosae-Faboideae; Glicosídeo Triterpenoidico; Dados espectrais.

INTRODUCTION

Centrosema bracteosum is a medicinal plant used as stomatic in popular medicine and was submitted to biological studies. These additional information contributed to the chemical investigation of a specimen of this species, as part of our continuing research with brazilian plants. We have isolated from the ethanolic roots extract a new pentacyclic triterpenoid saponin 1. The structure of this triterpene glycoside was deter mined by spectrometric data analysis of 1 and its peracetyl derivative (1a).

RESULTS AND DISCUSSION

The IR spectrum of triterpene glycoside 1 revealed the absence of absorption due to ester or ketone carbonyl group and showed a strong absorption band (3400cm^-1) for hydroxyl functions. The molecular formula C60H88O23 for the peracetyl derivative 1a was determined on the basis of its molecular ton at m/z 1176, deduced by fast atom bombardment mass spectrum [FABMS: (SCH₂CH₂OH)₂ matrix reference, negative ion mode] in combination with the ¹H and ¹³C NMR(PND = Proton-Noise Decoupled and DEPT = Distortionless Enhancement by Polarization Transfer) spectra (Tables 1 and 2). The ¹H and ¹³C NMR spectra showed the presence of three anomeric carbons [δ_Η (CDCI₃) 4.46 (d, J=5.9 Hz, H-1'), 4.63 (d, J-6.6 Hz, H-1") and 4.89 (br s, H-1"'); δ_C 103.47 (d, C-1'), 100.94 (d, C-1") and 97.94 (d, C-1"')], signals for five tertiary methyl protons attached to sp3 carbons [dH (CDCI₃ 1.14 (s), 1.06 (s), 0.94 (s), 0.80 (s) and 0.77 (s)] and two to sp² [δ_Η 1.67 (br s) and 1.63 (br s)], along with one bound to a secondary sp3 carbon (δ_Η 1.15 (d, J=5.8 Hz) corresponding to the methyl group of the rhamnose, and seven acetyl functions [δ_Η (CDCI₃) 2.08 (s), 2.06 (s), 2.05 (s), 2.01 (s), 1.99 (s), 1.97 (s) and 1.94 (s)] (Tables 1 and 2). The mass spectrum obtained for electron impact (EIMS) of the peracetyl derivative 1a did not show the peak corresponding to molecular ion (M* 1176) but revealed peaks at m/z 489 (6%), 437 (6%), 273 (99%), 213 (24%) and 153 (100%) which were attributed to the fragment ions 2-6, respectively. The fast atom bombardment mass spectrum [FABMS: SCH₂CH₂OH)₂ matrix reference, negative ion mode] of the natural product 1 exhibited a molecular ion peak at m/z 882 base peak), consistent with a molecular formula and peaks at m/z 736 (15%), 266 (22%) and 185 (41 %), which were correlated with fragment ions 7, 7a and 8, corresponding to the loss of terminal 6-dehydroxylhexose (7) and the production of the ions pentose-pentose disaccharide (7a) and hemiacetal (8). These sugars were characterized as L-rhamnose and D-xylose (two molecules) with the aid of ¹H and ¹³C NMR spectra (Table 1) including 2D-shift-correlated ¹Hx¹H-COSY (homonuclear) and ¹Hx¹³C-COSY-¹J_CH(heteronuclear, modulated to ¹J_CH)². In fact upon hydrolysis, 1 furnished L-rhamnose and D-xylose (1:2) which were identified through thin layer chromatography (TLC) by comparison with authentic samples. Thus, the presence of the seven acetyl groups observed during the ¹H NMR spectrum analysis of 1a represents the esterification of the seven hydroxyl groups of the sugar moiety and, consequently, the aglycone skeleton does not sustain hydroxy group for acetylation in the conditions utilized. From the above arguments the sugar moiety was classified as a trisaccharide containing one L-rhamnose molecule and two D xylose molecules. The ¹H and ¹³C NMR sugar signals (Table 1) of peracetyl derivative 1a were in agreement with a terminal L-rhamnose unit linked at C-2 of an inner D-xylose unit and the inner D-xylose linked at C-2 of another inner D-xylose unit. As shown in Table 1, the chemical shifts of the H-2' [δ (CDCI₃) 3.8 (dd, J=5.9 and 8.4 Hz)], H-2" [δ(CDCI3) 3.57 (dd, J=6.6 and 8.6 Hz)], along with C-2' [δ 74.51 (d)] and C-2" [δ 75.85 (d)], indicated that the acetylation did not affect these signals. Acetylation deshields all the CH α to acetates, appearing in the 4.5 - 5.5 ppm region, and leaves unaltered all the α protons to branching points (α to ether functions). This sequencing of the sugar chain on the peracetylated 1a was confirmed by ¹H x ¹H-COSY spectrum, which clearly revealed the interactions of the H-1' [δ(CDCI3) 4.46 (δ, J=5.9 Hz)] with H-2' [δ (CDCI3) 3.80] and H-1" [d (CDCI3) 4.63 (δ, J=6.6 Hz)] with H-2", [d (CDCI3) 3.57] and the NOEs observed for the H-1" (6.4%) and H-I" (11%) upon irradiation of the H-2' and H-2", respectively. All ¹H and ¹³C NMR signals of the sugar moiety were as signed as listed in Table 1 by the DEPT experiment, ¹Hx¹H-COSY and ¹Hx¹³C-COSY spectra. The chemical shifts of the four methylenic protons (δ 3.3-42) and two carbon atoms C-5' and C-5" (δ 62.01 and 61.48) were used to established the presence to two molecules of pentose D-xylose as D-xylopyranosyl units. Additional confirmation of terminal system α-L-rhamnopyranosyl (1 ->2)-xylopyranosyl was obtained by comparison of the ¹³C NMR chemical shifts of 1 with the values described for the model compound 13³. Based on the evidences mentioned above, the sugar moiety was defined as -Ο-α-L-rhamnopyranosyl (1->2)-b-D-xylopyranosyl (1->2)-β-D-xylopyranoside peracetylated, corresponding to the partial molecular formula C₃₀H₄₁O₂₀.

The remaining ¹³C NMR signal represents the aglycone moiety (Table 2). The molecular formula C₃₀H₄₇O₃ for this aglycone was also deduced by difference between the molecular formula of 1a (C₆₀Η₈₈Ο₂₃) and the sugar moiety which analysed in combination with the ¹H and ¹³C NMR and mass data allowed clearly to classify the aglycone moiety as triterpenoid. The presence of two methyl singlet (broad) signals attached to sp2 carbon atom (δ 1.67 and 1.63) in the ¹H NMR spectrum and only one double bond (trisubstituted) revealed by ¹³C NMR spectrum [δ125.04 (d) and 135.28 (s)] pointed to the localization of this unsaturation between carbon atoms C-24 and C-25 and, consequently, allowed to classify the triterpene as tetracarbocyclic with side-chain carbons at C-17. The difference between the unsaturation number (seven) obtained of the partial molecular formula (C₃₀H₄₇O₃) and those corresponding to tetracarbocyclic system (four) and to double bond (one) indicated the existence of two additional rings. The ¹³C NMR spectra showed also the presence of signals attributed to carbon atoms sustaining oxygen atoms at δ 109.48 (s), 89.69 (d), 69.23 (s), 68.60 (d), and 65.72 (t). The linkage between the trisaccharide and the aglycone was shown to be at C-3 by the ¹³C absorption at δ 89.69 in comparison with the absorption at d 88.10 in the model compound 94, containing the same aglicone (vide infra), demonstrating by the observed downfield field (∆δ=10.79) that this carbon sustains an ether glycosidation (eg. 10 and 11, Table 2). This deduction was confirmed by an upfield shift of C-2 [δ 25 85 (t)] when compared with the signal at δ 27.40 of the model compound 10⁵, the chemical shift of the H-3 (δ 3.1-2.9) in the ¹H NMR spectrum and by biogenetic arguments because of the frequent presence of oxi group at C-3 of triterpenoids⁶. The location of a tertiary hydroxyl group at C-20 was derived from the signal at δ 69.23 (s, quaternary carbon) in the ¹³C NMR and a methyl singlet signal at δ 1.14 in the ¹H NMR spectrum which was 2D-shift-correlated2 by the heteronuclear ¹H x ¹³C-COSY-¹J_CH spectrum with the carbon methyl signal at δ 29.84 (Table 2). The remaining two oxygen atoms were used to justify the presence of a ketal, an oxymethylene and oxymethine functions and two additional rings in the aglycone on the basis of the chemical shifts at δ 109.48 (s), 65.72 (f), and 68.60 (d), respectively, in the ¹³C NMR and unsaturation number. The homonuclear coupling of the oxymethine H-23 (δ 4.60) with H-24 (δ 5.15) and with H-22 (d 1.6-1.4) were revealed by 2D-shift-correlated (¹H x ¹H-COSY) spectra. These data are consistent with an ether function 16,23-epoxy and ketal group was established with another ether group 16,30-epoxy, containing the oxymethylene as show in 1 and 1a. Thus, the aglycone was defined as a triterpene with the basic skeleton of the dammarane type.

The data from ¹H and ¹³C NMR uni (1D) and two-dimensionals (2D) spectra of the natural product 1, recorded in pyridine-d₅ (Tables 1 and 2), together with comparison of the sugar moiety with the model saponin 13³ containing an analogous terminal system α-L-rhamnopyranosyl (1->2)-β-D-xylopyranosyl (Table 1) revealed themselves consistent with the deduction described above.

The assignment of an equatorial-position for the O-trisaccharide moiety at C-3 was deduced from the chemical shifts of carbons 1 to 5, 28 and 29 of 1a (Table 2) when compared with the model compounds 9⁴,10,12⁷ and 11⁵.

The chemical shifts [δ_C 29.84 (la); 30.27 (1)] of the methyl group at C-20 in the ¹³C NMR spectra of 1 and 1a suggested an equatorial configuration (and axial hydroxy, 1b). The signal of a methyl group at an axial-position in a hexacyclic system appears with a smaller chemical shift than δ_c20 because of g-effects (e.g. in the Table 2). The assignment of an equatorial-orientation for the CH=CMe₂ group at C-23 was deduced from the coupling constant J=8.9 Hz observed in the signal of H-23 [δ_Η 5.27 (dd, J=8.9, 6.0 Hz)] in the ¹H NMR spectrum of 1. This value of J=8.9 Hz is only consistent with H-23 at an axial-position (lb).

Based upon the above data, the structure of new triterpene saponin 1 was established as 3b-O-[α-L-rhamnopyranosyl (1 ->2)-β-D-xylopyranosyl (1->2)-β-D-xylopyranosyl]-16β,23(R): 16α,30α-diepoxy-20(S)-hydraxydammar-24-ene (1) and its peracetyl derivative as 3β-O-[α-L-2'",3'",4'"-tri-O-acetyirharrincpyranosyl (1->2)-b-D-3",4"-di-O-acetylxylopyranosyl (1->2)-b-D-3",4"-di-O-acetylxylopyranosyl-16β,23(R): 16α,30α-diepoxy-20(S)-hydroxyldammar-24-ene (1a). All ¹³C NMR signals were reasonably assigned as listed in Tables 1 and 2 through the chemical shifts, multiplicity deduced of DEPT, comparison with models (e.g. compounds 9-11), application of the usual shift parameters and 2D NMR spectra (¹H x ¹H-COSY,¹H x ¹³C-COSY). Furthermore, the stereochemistry of the aglycone (1 b) of 1 and 1a is in agreement with the prediction on the basis of a biogenetic route of the secondary metabolism for the biaproduction of dammarane skeleton6. The NOE difference experiments were used in the case of the sequencing of the sugar chain, which also indicated the preferred conformation to establish the adequate spatial proximity of H-2' to H-1" and of H-2" to H-1as shown in 1 and la.

The biosynthesis of this new triterpene has not yet been investigated but it might involve a sequence of reactions enzymatically controlled as described in the literature6. Suggestion about the secondary biocyclization to produce the two heterocycle rings (16β,23-epoxy and 16α,30α-epoxy) include oxidation at C-16 (carbonyl or hydroxy groups), C-23 (hydroxyl or carbonyl groups) and C-30 (hydroxyl group), enzimatically induced, to formation of a precursor 16-oxo-23,30-dihydroxy or 16,23-dioxo-30-hydroxy. These precursors present the appropriate functional groups for the biocyclization and production of the two heterocycle rings involving two ethers and one ketal functions (Chart 1). The stereochemistries at chiral carbon atoms C-5, C-8, C-9, C-13, C-14, and C-17 are those antecipated by the biogenetic route of dammarane skeletons. Since the configuration of the hydroxymethylene at C-14 is a, the attack of its hydroxy group on C-16 can only be carried out by the same side, consequently leading to the formation 16_'",30_'"-epoxy function. The glycosidation is a common process of secondary metabolism⁶.

After having submitted this work for publication (January 5,1994) which returned to us with some corrections and suggestions from two referees, we received J. Nat. Prod. (¹⁹⁹⁴), 57 (2) with a paper which reported the structure elucidation of three triterpenoid glycosides containing the same aglycone (jujubogenin) of 1⁸. In that paper, the configuration at C-17 of the aglycone had the hydrogen atom with b orientation (and D/E-frans ring fusion) on the basis of a comparison with the literature: "the structure of a derivative has been confirmed by chemical evidence as well as by X-ray crystallography⁹,¹⁰. The comparison of the NMR spectral data, mainly the ¹³C NMR data, indicated that the aglycone of the three glycosides⁶ is identical to the one presente in 1, including H-23 and hydroxyl group at C-20 at axial position. After this comparison and additional evaluation of the papers⁹,¹⁰ (cited in reference 10 as 6 and 7) we remain convinced of the D/E-cis ring fusion (1b), for which the structure defined by X-ray crystallographyl0 is more suitable. This configuration is also the one suggested by the biogenetic route of a dammarane skeleton⁶ and by the smaller chemical shift of C-13 methine carbon [δ_c 36.85 (d)] of 1 (g-effect of hydraxyi groups at C-20) when compared with the model 10 [δ_c 42.3 (C-13)]⁷ (Table2).

EXPERIMENTAL

General experimental procedures. Melting points were determined with a Kofler hot-stage apparatus and are uncorrected. The ¹H (200 MHz) and ¹³C (50.3 MHz) NMR spectra were obtained in C₅D₅N, CDCI₃ and C₆D₆ with TMS as an internal standard, employing a Broker AC-200 spectrometer; standard Bruker pulse sequences were used to perform ¹H x ¹H-COSY and ¹H x ¹³C-COSY; proton-noise decoupled and DEPT - ¹³C NMR were utilized to recognize the number of attached protons for each carbon atom. IR spectra were recorded as KBr discs on a Perkin-Elmer spectrometer. Low resolution mass spectra were obtained on a Hewlett Packard - 5890/5988A GC/MS instrument operating at 70 eV and FABMS on a Kratos MS-50 in the negative ion mode. Column chromatography was run with silica gel 60 (70-230 mesh, E. Merck, Darmstadt, Germany). Thin layer chromatography was performed on silica gel 60 F 254 (Merck).

Plant material. A specimen of Centrosema bracteosum Bentham, Leguminosae-Faboideae, was collected in Reserva Ecológica do Instituto Brasileiro de Geografia e Estatística, Brasília, DF, and identified by Drs. Ezequias Paula Heringer and Geraldo Ismael Rocha. A voucher specimen is deposited at the herbarium of the Instituto Brasileiro de Geografia e Estatística.

Extraction and isolation. The air-dried powdered roots (3.0 kg) were extracted exhaustively with EtOH in a Soxhlet apparatus. The EtOH solution was concentrated in vacuo to yield a residue (320 g). A portion of this residue (21.4 g) was dissolved in hot MeOH and precipitated after addition of EtOAc. The precipitate was filtered and washed with hot EtOAc to yield 5.0 g of material which was chromatographed on a silica gel (200 g) column using CHCI3-MeOH (7:3) as eluent. Twenty two fractions of 50 ml each were collected. Fractions 8 to 11 afforded 1 (2.5 g) as an amorphous solid colourless, after drying and trituration.

3-O-[α-L-Rhamnopyranosyl ( 1 -> 2 )-b-D-xylopyranosyl (1 >2)β-D-xylopyranosyl]- 16β, 23(R): 16α, 30 α-diepoxy-20(S)-hydroxy-dammar-24-ene (1). Mp 188-200 °C, colourless amorphous solid. IR v^KBr_maxcm^-1: 3400 (OH), 1680, 840 (CH=C), 1150, 1080, 1040 (C-O). FABMS [(S CH₂CH₂OH)₂ matrix, negative ion mode] m/z (rel. int.) 882 (100, M⁺), 736 (15, 7), 266 (22, 7a), 185(41, 8).

Acetylation of compound 1. The peracetyl derivative 1a was prepared by treatment of [1] (110 mg) with Ac2O (2.5 ml) and pyridine (1 ml). The usual work-up, after the solution had been allowed to stand for 12 hr at room temperature, followed by chromatography on a silica gel column gave peracetyl derivative 1a (106 mg), mp 142-144 °C. IR v^KBr_maxcm^-1: 1750 (C=O ester), 1240, 1220, 1090, 1050 (C-O) ¹H NMR: Table I. ¹³C NMR: Table 2 EIMS m/z (rel. int.): 489 (6, 2), 437 (6, 3), 274 (13), 273 (99, 4), 259 (13), 213 (24,5), 201 (7), 199 (6), 187 (7), 173(5), 171 (27), 169 (6), 161 (14), 159 (6), 157 (27), 154 (9), 153 (100, 6), 149 (5), 147 (7), 145 (8), 143 (8), 142 (5), 140 (6), 139 (21), 137 (10), 135 (8), 133 (7), 129 (8), 127(9), 125(18), 123(6), 121 (7), 119 (8), 115 (14), 113 (7), 112 (6), 111 (62), 109 (16), 107 (10); FABMS [SCH₂CH₂OH)₂ matrix, negative ion mode] m/z (rel. int.): 1328 [34, M+ (SCH₂CH₂OH)₂-2H], 1208 [11, M+ (SCH₂CH₂OH-2H-2AcOH], 1153[19, M+ (SCH₂CH₂OH)-2H-2AcOH-CH=CMe₂], 405 (28tri-O-acetyl-α-L-rharnnopyranosyl(1->2)-[β-D-xylopyranosyl-OH), 266 (53, 7a), 185(100, 8).

Acid hydrolysis of 1. Compound 1 (250 mg) was dissolved in 20 ml of MeOH-H₂O (1:1) with concentrated HCl (10 ml) and the solution was refluxed for 2 hr. The mixture was extracted with CHCl₃. The CHCl₃ layer was washed with H₂O, dried over anydrous Na₂SO₄ and concentrated under reduced pressure to yield an impure aglycone. The H₂O solution was concentrated, under reduced pressure, to dryness after addition of acetone and the sugar were identified as rhamnose and xylose (1:2) by comparison with authentic samples by thin layer chromatography (TLC) using silica gel G (Merck) impregnated with 5% of NaOAc¹¹ as adsorbent and EtOAc-isoPrOH-H2O (3.5:3.9:2.6) as eluent. Spots were visualized by spraying with a freshly pre pared solution of diphenylamine (4% in EtOH), aniline (4% in EtOH), and concentrated phosphoric acid (5:4:1), after heating for 10 min¹².

Table 1
¹H and ¹³C chemicals shift values (δ)" of lhe sugar moiety of peracetyl 1a and 1 compared with the model 13³.

1		13c
C	CDCI3 δHb	C6D6	δC CDCI3	δC C6D5N	δHb C6D5N	δC C6D5N	δH C6D5N
1'	4.46(d,J=5.9)	4.70(d,J=5.7)	103.47	106.62	4.86(d,J=6.7)
2'	3.80(dd, J=5.9,8.4)	4.2-3.9	74.51	80.93	4.2
3'	5.2-5.0	5.43(t,J=8)	69.14	79.29	4.2
4'	5.0-4.8	5.2-5.0	72.76	72.72	4.6
5'	3.37(t,J=8.6)	3.18(dd,J=8.6,11.6)	62.01	66.91	4.3
1"	4.2-3.9	4.2-3.9	-	-
2"	4.63(d,J=6.6)	4.79(d,J=6.2)	100.94	103.19	5.72(d,J=6.6)	102.60
	5.70(d,J=7.6)
3"	3.57(dd,J=6.6,8.6)	3.82(dd,J=6.2,8.3)	75.85	80.93	4.3	79.50
4"	5.2-4.9	5.41(t,J=8.3)	69.36	78.52	4.7	78.24
5"	5.0-4.8	5.2-5.0	72.76	71.68	4.1	70.90
1"'	3.31 (t,J=8.6)	3.31(dd,J=7.2,11.3)	61.48	66.91	4.3	66.90
2"'	4.2-3.9	4.2-3.9	-	-
3"'	4.89(br s)	5.24(br s)	97.94	102.31	6.35(s)	102.40	6.37(br
s)
4"'	5.07(br s)	5.50(d,J=2.9)	69.78	72.52	4.7	72.40
5"'	5.2-4.9	5.67(dd.J=2.9.9.6)	67.87	72.72	4.6	72.40
6"'	5.2-4.9	5.49(t, J=9.6)	70.65	74.43	4.3	74.36
OAc	4.1-3.9	4.47(dq,J=9.6,6.1)	66.84	69.72	4.9	69.51
	1.15(d,J=5.8)	1.53(d, J=6.1)	17.09	19.09	1.79(d, J=5.9)	18.91
	2.08(s)	2.01(s)	170.24	-	-
	2.06(s)	1.97(s)	170.11	-	-
	2.05(s)	1.79(s)	169.88	-	-
	2.01 (s)	1.77(s)	169.60	-	-
	1.99(s)	1.77(s)	20.72	-	-
	1.97(s)	1.72(s)	20.67	-	-
	1.94(s)	1.72(s)	20.60	-	-

a Chemical shifts in d (ppm) and TMS as internal standard. Coupling constants (J) in Hz. The assignments were made with the aid of the DEPT and 2D-shift-correlated (1H x 1H-COSY and 1H x 13C-COSY) spectra (except for acetyl groups).b 1H chemical shifts described with only one decimal correspond to approximate values of 1 and 1a obtained of the 1H x 13C-COSY spectra.c We described only useful values for comparison.NOTAS
* In attention to an invitation formulatet by the Managing Editor-Concil of Scientific Research Integration (Trivandrum, India) through a letter of September 12, 1991, this paper was found suitable for publication in Life Science Advances - Phytochemistry sice October 13, 1992, and was returned (December, 1993) because we had not funds to cover the cost of 100 reprints.

Table 2
¹H and ¹³C NMR chemical shifts values (d)^a of the aglycone of peracetyl 1a and 1 compared with the models 9⁴ 10, 12⁷ and 11⁵

1a(CDCI3)	1(C5D5N)	9b	CDCI3	C5D5N
C	δC δH	δC δH	δC	10 11c 12c
1	38.49		39.90		39.00	39.0	39.5
	33.7
2	25.85		27.03		26.70	27.4	26.8
	25.5
3	89.69	3.1-2.9	89.54	3.25(m)	88.10	78.9	89.5
	75.9
4	39.37	-	40.11	-	39.70	39.0	39.8
	37.6
5	55.89	0.7	56.48	0.7	56.30	55.9	56.7
	49.5
6	17.88		18.64		18.30	18.3	18.6
7	35.52		37.33		36.00	35.3	35.3
8	37.13	-	37.49		37.30	40.4	40.3
9	52.51	0.8	53.29	0.8	53.00	50.7	50.4
10	36.98	-	37.49	-	37.30	37.1	37.2
11	21.30		22.03		21.80	21.6
12	27.91		28.77		28.60	25.4
13	36.85		38.96	2.80(m)	35.70	42.3
14	53.29	-	53.96		53.70	50.3
15	35.39	1.6-1.4	37.77	2.48(d, J=11.7)	37.10	31.2
16	109.48	-	110.87	-	110.60	27.6
17	52.62	1.1	54.15	1.4	53.90	49.9
18	18.51	1.06(s)	19.15	1.04(s)	18.30	16.2*	16.2*
19	15.88	0.77(s)	16.58	0.77(s)	16.40	15.5*	16.2*
20	69.23	-	68.72	-	68.50	75.4
21	29.84	1.14(s)	30.27	1.29(s)	30.00	24.9
22	44.36	1.6-1.4	45.60	1.7	45.30	40.5
23	68.60	4.60	68.93	5.27(dd, J=8.9,6.0)	68.50	22.6
24	125.04	5.15	127.21	5.51(d,J=6.0)	127.00	124.8
25	135.28	-	134.56	-	134.2	131.5
26	18.26	1.63(br s)	19.09	1.66(s)	25.50	17.7
27	25.51	1.67(br s)	25.91	1.64(s)	18.90	25.7
28	27.60	0.94(s)	28.77	1.22(s)	28.00	28.0	28.2
	28.5
29	16.02	0.80(s)	16.71	1.04(s)	16.80	15.4*	16.6*
	22.1
30	65.72		66.12		65.8	16.5

a Chemical shifts in d (ppm), coupling constants (J) in Hz and TMS as internal standard. Assignments were made with the aid of the DEPT and 2D-shift-correlated [1H x 13C-COSY, optimized for one-bond couplings (1JCH)] spectra. 1H Chemical shifts described with only one decimal correspond to approximate values of 1a and 1 and may correspond to only one proton in the case of the methylene groups.b The assignments of carbon atoms C-7, C-8, C-10, C-13, and C-15 need to be re-examined.c We described only useful values for comparison.
• ^*
  From the authors of the paper: "values in any vertical column may be reversed although those given here are preference".

Chart 1
Suggested biosynthetic pathway for the formation of the two heterocyclic rings in 1.

ACKNOWLEDGMENTS

This work was supported by CNPq research follcwships (M.G. de C. and R. B. F.) and by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). The authors are also grateful to professor Massayoshi Yoshida, Instituto de Química - Universidade de São Paulo, São Paulo, for acquisition of the electron impact mass spectrum (EIMS) and Dr. Michael S. Tempesta, Shaman Pharmaceuticals, Analytical Service Center, IOCD, Chemistry & Discovery, San Carlos, California, for recording the fast atom bombardment massa spectra (FABMS).

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Publication Dates

• Publication in this collection
  26 Jan 2024
• Date of issue
  1997