Dammarane triterpenes from Gardenia aubryi vieill.

Article abstract of DOI:10.1002/hlca.201000286

Three new dammarane triterpenoids, gardaubryones A-C (1-3), were isolated from the gum collected on the aerial parts of Gardenia aubryi Vieill., together with the known compounds hydroxydammarenone II (4), ocotillone (5), cabraleone, and hollongdione. The structures of the novel compounds were established on the basis of mass spectrometry, NMR experiments, and chemical-correlation reactions. Copyright

Full text of DOI:10.1002/hlca.201000286

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Helvetica Chimica Acta – Vol. 94 (2011)
Dammarane Triterpenes from Gardenia aubryi Vieill.
by Raphae¨l Grougneta)b), Prokopios Magiatisa), Sofia Mitakua), Alexios-Leandros Skaltsounis*a),
Pierre Cabalionc), FranÅois Tillequinb), and Sylvie Michelb)
a) Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of
Athens, Panepistimiopolis-Zografou, Athens GR-15771
(phone: þ 30-210-7274598; fax: þ 30-210-7274594; e-mail: skaltsounis@pharm.uoa.gr)
b
´
) Laboratoire de Pharmacognosie de lꢀUniversite Paris Descartes, UMR/CNRS 8638,
´
Faculte de Pharmacie, 4 Avenue de lꢀObservatoire, F-75006 Paris
c
´
) Laboratoire des Substances Naturelles Terrestres et Savoirs Traditionnels, Center IRD de Noumea,
´
98800 Noumea, New Caledonia
Three new dammarane triterpenoids, gardaubryones A – C (1 – 3), were isolated from the gum
collected on the aerial parts of Gardenia aubryi Vieill., together with the known compounds
hydroxydammarenone II (4), ocotillone (5), cabraleone, and hollongdione. The structures of the novel
compounds were established on the basis of mass spectrometry, NMR experiments, and chemical-
correlation reactions.
Introduction. – The genus Gardenia (Rubiaceae) includes some two hundred
species of trees and shrubs widely distributed in the warm and tropical regions of the
Old World. Six endemic species occur in New Caledonia [1][2]. The leaves and flower
buds of two of these, Gardenia aubryi Vieill. and Gardenia urvillei Montrouz., are
covered with an exudate locally used for chewing, especially by children. In a
continuation of our chemical studies on secondary metabolites of New Caledonian
Gardenia species [3], we report here the structure elucidation of three new dammarane
triterpenoids, gardaubryone A – C (1 – 3), isolated from Gardenia aubryi gum, along
with the known hydroxydammarenone II (4), ocotillone (5), cabraleone, and
hollongdione.
Results and Discussion. – Repeated column chromatography on silica gel of G.
aubryi exudate resulted in the isolation of the three novel dammarane triterpenoids,
gardaubryones A – C (1 – 3), together with four known dammaranes, which were
identified as hydroxydammarenone II (¼20-hydroxy-dammar-24-en-3-one; 4 (20S))
[4][5], ocotillone (¼(24R)-20,24-epoxy-25-hydroxydammaran-3-one; 5 (20S)) [6 – 8],
cabraleone [6 – 8], and hollongdione [9][10] by comparison with literature data.
Gardaubryone A (1) was obtained as a colorless amorphous solid, whose empirical
formula was determined as C30H50O2 by HR-EI-MS. Inspection of the 13C-NMR
spectrum revealed the presence of a C¼O s and 29 sp3 C-atoms (six quaternary C, four
1
CH, eleven CH2, and eight Me). In the H-NMR spectrum, the eight Me signals
appeared as ss. A typical set of five Me resonances, correlated in the HMQC plot, at
d(C)/d(H) 15.1/0.98, 16.1/0.91, 16.5/0.84, 21.0/1.01, and 26.7/1.13, was associated with
ꢁ 2011 Verlag Helvetica Chimica Acta AG, Zꢂrich

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657
the Me(18), Me(19), Me(30), Me(29), and Me(28) groups, respectively, of a
dammarane skeleton bearing a C¼O group at C(3) (Tables 1 and 2) [5][11][12].
Indeed, these data were closely related to those previously reported for the dammarane
core present in ocotillone (5) [7] and in cabraleone [7], (24S)-20,24-epoxydammar-
25(26)-en-3-one [7], 20-hydroxydammaran-3-one [5], and hydroxydammarenone II (4)
[5]. The 13C-NMR resonances of the side chain at C(17), associated with a 2,2,6-
trimethyltetrahydro-2H-pyran moiety, were observed at d(C) 76.5 (C(20)), 26.0
(C(21)), 31.2 (C(22)), 16.4 (C(23)), 37.0 (C(24)), 70.9 (C(25)), 28.2 (C(26)), and 32.9
(C(27)). They were similar to the corresponding signals present in the spectrum of
(20S)-panaxadiol (¼(3b,12b)-20,25-epoxydammarane-3,12-diol (20S)) [13] and
strongly differed from those of (20R)-panaxadiol (¼(3b,12b)-20,25-epoxydammar-
ane-3,12-diol) [13 – 15]. These data permitted to depict the structure of gardaubryone
A as 20,25-epoxydammaran-3-one (20S) (1).
Gardaubryone B (2) was found to possess a molecular formula C30H50O3 by HR-EI-
MS. The 13C-NMR spectrum indicated the presence of a C¼O, two sp2 C-atoms (one
quaternary C, one CH) corresponding to the C¼C bond, and 27 sp3 C-atoms (five
quaternary C, four CH, ten CH2, and eight Me). The signals observed were typical for a
dammaran-3-one derivative and closely related to those of 5, except for the resonances
associated with the side chain at C(17) (Table 2). In the 1H-NMR spectrum, the signal
of the Me(21) group at the side chain was strongly deshielded (d(H) 1.56), whereas
those of the two geminal ones, Me(26) and Me(27), were only slightly deshielded.
Typical 1H,13C-HMBC cross-peaks, associated with a 6-substituted 2-methylhept-5-ene-
2,3-diol moiety were noticed between Me(21) and the quaternary and tertiary sp2 C(20)
and C(22) (d(C) 141.8 and 120.5, resp.) on the one hand, and between Me(26) and
Me(27) and the quaternary and tertiary oxygenated C(25) and C(24) (d(C) 72.6 and
78.2, resp.) on the other hand. The side-chain structure was confirmed by the
observation of a t at d(H) 5.21 and a dd at d(H) 3.38, attributable to HꢀC(22) and

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Helvetica Chimica Acta – Vol. 94 (2011)
Table 1. 1H-NMR Data (400 MHz, CDCl3) of Compounds 1 – 3. d in ppm, J in Hz. In the case of
overlapped signals or H-atoms observed as m, only the d(H) is presented. The d(H) of overlapped H-
atom signals is derived from HSQC-DEPT data.
1
2
3
CH2(1)
CH2(2)
1.93, 1.46
2.47, 2.42
1.35
1.52, 1.43
1.52, 1.26
1.40
1.45, 1.26
1.93, 1.26
1.69
1.34, 0.98
1.64, 1.42
1.67
0.98 (s)
0.92 (s)
1.11 (s)
1.42, 1.32
1.73, 1.59
1.51, 1.34
1.14 (s)
1.18 (s)
1.01 (s)
1.05 (s)
0.84 (s)
1.91, 1.44
2.50, 2.41
1.36
1.53, 1.44
1.56, 1.30
1.38
1.49, 1.18
1.84, 1.28
1.59
1.47, 1.05
1.66, 1.44
2.20
0.99 (s)
0.92 (s)
1.56 (s)
5.21 (t, J ¼ 7)
2.20
3.38 (dd, J ¼ 8, 5.5)
1.17 (s)
1.22 (s)
1.02 (s)
1.06 (s)
0.86 (s)
1.90, 1.44
2.48, 2.43
1.36
1.53, 1.45
1.54, 1.29
1.42
1.48, 1.26
1.80, 1.27
1.61
1.44, 1.10
1.74, 1.47
1.79
0.97 (s)
0.92 (s)
1.15 (s)
1.73, 1.60
1.61, 1.41
3.37 (br. d, J ¼ 8.5)
1.22 (s)
1.17 (s)
1.02 (s)
1.07 (s)
0.87 (s)
HꢀC(5)
CH2(6)
CH2(7)
HꢀC(9)
CH2(11)
CH2(12
HꢀC(13)
CH2(15)
CH2(16)
HꢀC(17)
Me(18)
Me(19)
Me(21)
CH2(22) or HꢀC(22)
CH2(23)
CH2(24) or HꢀC(24)
Me(26)
Me(27)
Me(28)
Me(29)
Me(30)
Table 2. 13C-NMR Data (50 MHz, CDCl3) of Compounds 1 – 3. d in ppm.
1
2
3
1
2
3
C(1)
39.9
34.1
39.9
34.1
39.8
34.1
C(16)
C(17)
C(18)
C(19)
C(20)
C(21)
C(22)
C(23)
C(24)
C(25)
C(26)
C(27)
C(28)
C(29)
C(30)
25.2
51.4
15.1
16.1
76.5
26.0
31.2
16.4
37.0
70.9
28.2
32.9
26.7
21.0
16.5
27.3
50.6
15.2
15.8
25.6
49.9
15.1
16.0
75.6
25.2
36.9
24.9
78.8
73.2
23.2
26.6
26.7
21.0
16.3
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
C(9)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
218.2
47.4
55.3
19.7
34.6
40.3
50.2
36.9
22.2
27.6
42.0
50.0
31.2
218.2
47.4
55.3
19.6
34.7
40.4
50.2
36.9
21.9
25.8
44.5
49.2
31.6
218.2
47.4
55.3
19.6
34.5
40.2
50.0
36.8
22.0
27.4
42.6
50.2
31.1
140.3
13.3
120.5
30.4
78.5
72.6
23.7
26.6
26.7
21.0
16.1
HꢀC(24), respectively. These data permitted to depict the structure of gardaubryone B
as 24,25-dihydroxydammar-20(22)-en-3-one (2), in good agreement with the 13C-NMR

Helvetica Chimica Acta – Vol. 94 (2011)
659
data reported for the analogous dammar-20(22)-ene-24,25-diol moiety present in the
triterpene cordialin B (¼(3b,11a,20E,24S)-3,19-epoxydammar-20(22)-ene-3,11,24,25-
tetrol) [16]. The absolute configuration at C(24) of gardaubryone B could not be
unambiguously established despite several chemical-correlation trials due to the low
amount of isolated material.
It should be noted that a structure similar to that established here for gardaubryone
B has been recently postulated for the triterpene ailexcelone isolated from Ailanthus
excelsa Roxb. [17]. However, the spectral data reported for this latter compound are
not consistent with the proposed structure. Specifically, the d(C) values of the signals of
the 13C-NMR spectrum of ailexcelone unambiguously exclude a dammarane basic core
but are in full agreement with a tirucall-7-ene (¼(13a,14b,17a,20S)-lanost-7-ene)
structure. Consequently, ailexcelone should be regarded as one of the epimers at C(24)
of 24,25-dihydroxytirucall-7-en-3-one [18]. Similarly, the structure of ailexcelol,
presented as the corresponding secondary alcohol at C(3), should be revised to one
of the C(24) epimers of (3S)-tirucall-7-ene-3,24,25-triol. It should also be emphasized
that (3S,24S)-tirucall-7-ene-3,24,25-triol had been previously isolated from Ailanthus
excelsa Roxb. [19].
The empirical formula of gardaubryone C (3) was deduced as C30H52O4 from the
HR-EI-MS. The 13C-NMR spectrum indicated the presence of a C¼O and 29 sp3 C-
atoms (six quaternary C, five CH, ten CH2, and eight Me). It exhibited, as those of 1
and 2, the 22 signals of a dammaran-3-one tetracyclic skeleton. A set of additional
resonances associated with a 6-substituted 2-methylheptan-2,3,6-triol moiety was
noticed at d(C) 75.6 (C(20)), 25.2 (C(21)), 36.9 (C(22)), 24.9 (C(23)), 78.8 (C(24)),
73.2 (C(25)), 23.2 (C(26)), and 26.6 (C(27)), similar to analogous signals in the
spectrum of various glycosides of dammarane-3,12,20,24,25-pentol [20] and cyclo-
lanostane-3,16,20,24,25-pentol [21 – 23]. Thus, gardaubryone C could be described as a
20,24,25-trihydroxydammaran-3-one. Catalytic OsO4 oxidation of hydroxydammare-
none II (4) gave a 20,24,25-trihydroxydammaran-3-one mixture of two diastereoisom-
ers epimeric at C(24) (Scheme). One of these triols was identical to gardaubryone C
(3), permitting to establish its (20S) configuration. Upon acid treatment, gardaubryone
C was converted to a mixture of the two corresponding 20,24-epoxy derivatives
epimeric at C(20) [20]. One of these compounds was identical to ocotillone (5), giving
evidence for the (24R) configuration of gardaubryone C, the structure of which was
therefore established as (24R)-20,24,25-trihydroxydammaran-3-one (20S).
Experimental Part
General. Liquid column chromatography (CC): silica gel 60 (SiO2, 40 – 63 mm, Merck). Optical
rotations: Perkin-Elmer-341 polarimeter. IR Spectra: Perkin-Elmer-Paragon-500 instrument; ˜n in cmꢀ1
.
NMR Spectra: Bruker-DRX-400 and Bruker-AC-200 spectrometers; at 400 (1H) and 50 MHz (13C); d in
ppm rel. to Me4Si as internal standard, J in Hz; 2D-NMR experiments were performed with standard
Bruker microprograms. EI-MS: HP-6890 spectrometer; in m/z. HR-EI-MS: AEI-MS-902 mass
spectrometer; in m/z.
Plant Material. Aerial parts of Gardenia aubryi were collected on July 9, 2003, in New Caledonia. A
´
voucher sample (JWHJ-75) is kept with the Herbarium of the Center IRD of Noumea, New Caledonia.
Extraction and Isolation. Gum (2.35 g), collected manually from fresh Gardenia aubryi aerial parts
(1.4 kg), was dissolved in CH2Cl2 (35 ml) and filtered. The solvent was evaporated, and an aliquot of the

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Helvetica Chimica Acta – Vol. 94 (2011)
Scheme
solid residue (2.10 g) was subjected to liquid vacuum chromatography (SiO2, stepwise gradient AcOEt/
cyclohexane, 10-ml fractions). Fractions of similar composition (TLC) were pooled and further purified
by CC (SiO2 (20 – 45 mm), cyclohexane/CH2Cl2/MeOH (of increasing polarity): gardaubryone A (1;
28 mg), hollongdione (8 mg), ocotillone (5; 17 mg), cabraleone (24 mg), hydroxydammarenone II (4;
157 mg), gardaubryone B (2; 4 mg), and gardaubryone C (3; 12 mg), in this order.
Gardaubryone A (¼20,25-Epoxydammaran-3-one; 1 ((20S) configuration)): Amorphous solid.
[a]2D0 ¼ þ81 (c ¼ 0.2, MeOH). EI-MS: 442 (Mþ). HR-EI-MS: 442.3817 (Mþ, C30H50Oþ2 ; calc. 442.3811).
Gardaubryone B (¼24,25-Dihydroxydammar-20(22)-en-3-one; 2): Amorphous solid. [a]2D0 ¼ þ84
(c ¼ 0.2, MeOH). EI-MS: 458 (Mþ). HR-EI-MS: 458.3758 (Mþ, C30H50Oþ3 ; calc. 442.3760).
Gardaubryone C (¼(24R)-20,24,25-Trihydroxydammaran-3-one; 3 ((20S) configuration)): Amor-
phous solid. [a]2D0 ¼ þ57 (c ¼ 0.2, MeOH). EI-MS: 476 (Mþ). HR-EI-MS: 442.3869 (Mþ, C30H52Oþ4 ; calc.
442.3866).
OsO4 Oxidation of Hydroxydammarenone II (4). To a soln. of 4 (25 mg) in t-BuOH/THF/H2O
10 :3 :1 (5 ml) was added a 2.5% (w/v) soln. of OsO4 in t-BuOH (0.15 ml) and 4-methylmorpholine 4-
oxide (30 mg). The mixture was stirred for 48 h at 258. A sat. aq. NaHSO3 soln. (20 ml) was added, and
the mixture was stirred for 1 h. Then, the mixture was extracted with CH2Cl2 (20 ml) and H2O (20 ml),
the org. layer dried (Na2SO4) and concentrated, and the residue submitted to flash chromatography
(SiO2, CH2Cl2/MeOH 99 :1) affording 3 (8 mg), identical to the natural product isolated from Gardenia
aubryi, and a mixture of 3 with its (24S) epimer which could not be separated.
Cyclization of Gardaubryone C (3). To a soln. of 3 (8 mg) in THF (5 ml) was added H2SO4
(0.05 ml). The mixture was stirred for 24 h at 258. Then, the solvent was evaporated and the residue
purified by prep. TLC: ocotillone (5; 5 mg).
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Received May 7, 2010