A new 3,4-seco-lupane derivative from Lasianthus gardneri

Article abstract of DOI:10.1021/np030374g

A new seco-ring A lupane triterpene derivative (1), along with lupenone, lupeol, β-sitosterol, ursolic acid, and stigmasterol 3-O-β -D-glucoside, were isolated from a methanol extract of mature stems of Lasianthus gardneri, a shrub from the family Rubiaceae growing in Sri Lanka. The structure and stereochemistry of the new compound were determined using a combination of ¹³C and ¹H homo- and heteronuclear 2D NMR experiments and from mass spectral data. The structure of 1 was confirmed by partial synthesis from lupeol.

Full text of DOI:10.1021/np030374g

J . Nat. Prod. 2004, 67, 911-913
911
A New 3,4-seco-Lu p a n e Der iva tive fr om La sia n th u s ga r d n er i
Sabrina Dallavalle,† Lalith J ayasinghe,*,‡ B. M. M. Kumarihamy,‡ Lucio Merlini,*,† Loana Musso,† and
Leonardo Scaglioni†
Dipartimento di Scienze Molecolari Agroalimentari, Universita` di Milano, Via Celoria 2, 20133 Milano, Italy, and
Institute of Fundamental Studies, Hantana Road, Kandy, Sri Lanka
Received August 14, 2003
A new seco-ring A lupane triterpene derivative (1), along with lupenone, lupeol, â-sitosterol, ursolic acid,
and stigmasterol 3-O-â-D-glucoside, were isolated from a methanol extract of mature stems of Lasianthus
gardneri, a shrub from the family Rubiaceae growing in Sri Lanka. The structure and stereochemistry
1
of the new compound were determined using a combination of 13C and H homo- and heteronuclear 2D
NMR experiments and from mass spectral data. The structure of 1 was confirmed by partial synthesis
from lupeol.
Ta ble 1. 13C and 1H NMR Data (CDCl3) for Compound 1
As a part of our studies on Rubiaceae plants endemic to
Sri Lanka1 we have investigated the methanol extract of
position
δC
δH, J (Hz)
HMBC (H f C)
9, 11
1, 10
1, 2
Lasianthus gardneri (Thw.) Hook., a shrub growing in
mountain forests. There are about 80 species of the genus
Lasianthus2 around the world, and nine of them found in
Sri Lanka are considered to be endemic.1 To the best of
our knowledge no phytochemical or bioactivity work has
been previously reported on L. gardneri.
In this paper we describe the isolation and characteriza-
tion of a new triterpene derivative (1) with a seco-lupane
skeleton, together with lupenone, lupeol, â-sitosterol, ur-
solic acid, and stigmasterol 3-O-â-D-glucoside. The struc-
ture of 1 was elucidated mainly by 1D and 2D NMR
experiments and by comparison with an authentic sample
prepared starting from lupeol.
1
2
3
35.2 t 1.25-1.14
25.2 t 1.59
63.7 t 3.55
4
148.3 s
5, 24
5
6
7
8
50.4 d 2.02 dd (13.0, 2.8)
24.7 t 1.76 m (12.4, 2.8) 1.32
32.8 t 1.41-1.32
40.6 s
40.7 d 1.60 dd (12.6, 3.1)
39.1 s
21.5 t 1.33-1.22
29.7 t 1.24
38.2 d 1.66
43.2 s
6, 25, 23, 10, 24, 4
5, 7
5, 26
26, 27
9
26, 12, 25a
25, 9, 5
10
11
12
13
14
15
16
15, 27, 8
27,26
16,27
27.5 t 1.65-1.02
35.5 t 1.48 ddd (12.9, 2.5, 4.6) 15, 28, 17
1.35
Dried ground mature stems of L. gardneri were extracted
with methanol. Purification of the extract by passage on a
series of silica gel columns and by preparative thin-layer
chromatography afforded six compounds. Five of them were
identified as lupenone, lupeol, â-sitosterol, ursolic acid, and
stigmasterol 3-O-â-D-glucoside, respectively, from mass
17
18
19
20
21
22
23
43.0 s
48.2 d 1.37
18, 28, 22
28, 16, 17, 13, 19
48.0 d 2.37 ddd (11.0, 11.0, 5.8) 18, 22, 13, 30, 29, 20
151.0 s
29.8 t 1.91-1.29
40.0 t 1.37-1.18
112.8 t 4.80 dq (2.7, 1.5)-4.62
d (2.7)
19, 30, 18
22, 30
21, 19
1
spectral and H and 13C NMR data and by direct compari-
son with authentic samples. Moreover, the identification
of lupenone was confirmed by the oxidation of lupeol to
lupenone with pyridinium chlorochromate (PCC), whereas
stigmasterol glucoside was subjected to acid hydrolysis to
confirm the presence of stigmasterol as the aglycon and
glucose as the sugar.
The molecular formula of compound 1 was determined
as C30H50O, from high-resolution mass spectrometry (m/z
426.3834) as well as from its DEPT NMR spectrum. In the
1H NMR spectrum the presence of two methyl groups (δH
1.70 and 1.68) located at sp2 carbons and four protons (δH
4.80, 4.68, 4.62, and 4.56), whose chemical shifts and small
coupling constants were typical of terminal double bonds,
confirmed the presence of two isopropenyl moieties. Using
combined 1H and 13C NMR data and DEPT experiments,
six singlet methyl groups, 13 methylenes, five methines,
and six quaternary carbons, for a total of 30 carbons, were
identified (Table 1). No carbonyl or carboxylic carbons were
detected. Considering the molecular formula C30H50O, two
of the implied six degrees of unsaturation were explained
5, 24
24
25
26
27
28
29
23.1 q 1.70 d (1.5)
20.5 q 0.79 s
16.0 q 1.06 s
14.5 q 0.95 s
18.0 q 0.78 s
5, 23
5, 1, 10, 9
9, 7, 8,14
13, 15, 14, 8
22, 16, 17, 18
19, 30
109.4 t 4.68 d (2.5)-4.56 dq
(2.5, 1.3)
30
19.3 q 1.68 d (1.3)
19, 29
a
Only observed in the spectrum measured in pyridine-d5.
by the two isopropenyl groups. The absence of carbon-
oxygen double bonds led us to hypothesize that 1 is a
tetracyclic triterpene. Furthermore, closer examination of
HMQC, HMBC, TOCSY, and NOESY experiments revealed
that the compound was a lupeol-type triterpene3-5 (Table
1).
Prominent differences in the 1H and 13C NMR data
between 1 and lupeol were observed only in the A ring
region; these were the presence of a CH2 instead of a CH
group linked to an alcoholic oxygen atom (hydrogen ex-
changeable with D2O), a further isopropenyl moiety, and
the absence of a geminal dimethyl group at C-4. From these
considerations we deduced that these differences arose
from the cleavage of C-3,C-4 bond (seco-lupane A ring). The
* To whom correspondence should be addressed. (L.J .) Tel: +94-8-
2232002. Fax: +94-8-2232131. E-mail: lalith@ifs.ac.lk. (L.M.) Tel: +39
0250316812. Fax: +39 0250316801. E-mail: lucio.merlini@unimi.it.
† Universita` di Milano.
‡ Institute of Fundamental Studies, Kandy, Sri Lanka.
10.1021/np030374g CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/27/2004

912 J ournal of Natural Products, 2004, Vol. 67, No. 5
Notes
hyde intermediate 2 afforded a product that showed
physical and spectroscopic data completely identical to
those of compound 1, thus confirming the assignment of
its structure. To the best of our knowledge compound 1 has
never been described before.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. All reagents and
solvents were reagent grade or were purified by standard
methods before use. Melting points were determined in open
capillaries on
a Bu¨chi melting point apparatus and are
F igu r e 1. Mass spectral fragment ions of 1 at m/z 189 (C14H21) and
uncorrected. NMR spectra were recorded in CDCl3 (when not
otherwise stated) with a Bruker AMX-600 spectrometer, at
600.13 MHz for 1H and 150.92 MHz for 13C. Chemical shifts
(δ values) and coupling constants (J values) are given in ppm
and Hz, respectively. EIMS and HREIMS were recorded at
an ionizing voltage of 70 eV on a Finnigan TQ70 spectrometer.
The relative intensities of mass spectrum peaks are listed in
parentheses. Column chromatography (when not otherwise
stated) was carried out on flash silica gel (Merck 230-400
mesh). TLC analysis was conducted on silica gel plates (Merck
60F254). All reactions requiring anhydrous conditions were
performed under a positive nitrogen flow, and all glassware
was oven-dried and/or flame-dried. Authentic lupeol was
purchased from Extrasynthe`se, Genay, France.
203 (C15H23).
F igu r e 2. Selected NOESY correlations for 1.
P la n t Ma ter ia l. Aerial parts of Lasianthus gardneri were
collected from Horton Plains, Central Province of Sri Lanka,
in April 1997. A voucher specimen (No. IFS/97/LG1) has been
deposited at Institute of Fundamental Studies, Hantana Road,
Kandy, Sri Lanka.
remaining part of the skeleton of lupeol remained intact,
as shown by the two diagnostic fragmentations6,7 at m/z
189 [M - C16H29O]+ and 203 [M - C15H27O]+ in the mass
spectrum corresponding to species A and B (Figure 1).
From these data compound 1 was characterized as 3,4-seco-
lupa-4(23),20(29)-dien-3-ol. Among natural compounds with
a seco-lupane structure, canaric acid (3)8 shows spectro-
scopic properties9 very similar to compound 1, apart from
the presence in 1 of a hydroxymethyl group in position C-3
instead of a carboxylic function. The relative configuration
of 1 was assigned on the basis of NOESY data. The trans
B/C/D ring junctions were consistent with the strong NOE
correlations between the H-9R/Me-27; Me-27/H-18R and
between Me-26/H-13; H-13/Me-28. The absence of a NOE
between H-5R and Me-25 together with a strong NOE
between Me-25/Me-26 and H-5/H-9R was consistent with
a seco-ring A in a diequatorial conformation. The NOE
correlation between H-13 and H-19 allowed the assignment
of the configuration at C-19 (Figure 2).
Extr a ction a n d Isola tion . Ground mature stems (1.3 kg)
were extracted with methanol (5 L) to give a brown solid (180
g), which was partitioned between n-butanol (1.2 L) and water.
Evaporation of n-butanol gave a crude solid (11.2 g). Chroma-
tography of this residue on Merck 60 silica gel (0.040-0.063
mm) with hexane, then hexane with increasing (from 10% to
100%) percentage of dichloromethane, then dichloromethane-
methanol, 95:5 (total amount of eluent 1.2 L), followed by
preparative thick-layer chromatography afforded six com-
pounds. Five of them were identified as lupenone, lupeol,
â-sitosterol, ursolic acid, and stigmasterol 3-O-â-D-glucoside,
from mass spectral and 1H and 13C NMR data, and by direct
comparison with authentic samples. Compound 1 (65 mg) was
eluted with 60%-100% dichloromethane from the column and
purified by preparative TLC with dichloromethane.
Oxid a tion of Lu p eol.12 Lupeol (10 mg, 0.02 mmol) was
dissolved in 10 mL of dichloromethane. After addition of 10
mg of pyridinium chlorochromate, the solution was stirred 2
h at room temperature. The reaction mixture was then filtered
through a Celite pad. Evaporation of the solvent afforded a
light yellow solid, which appeared identical to lupenone by
comparison with an authentic sample.13
To confirm unambiguously the structure and stereo-
chemistry of compound 1, we undertook a synthesis of 3,4-
seco-lupa-4(23),20(29)-dien-3-ol starting from lupeol. Ac-
cording to a fragmentation reaction initiated by an alkoxy
radical,10,11 treatment of lupeol with lead tetraacetate
followed by immediate reduction with LiAlH4 of the alde-
Acid Hyd r olysis of Stigm a ster ol Glu cosid e.14 Stigmas-
terol glucoside (5 mg) was refluxed in 2 mL of 4 N HCl for 2

Notes
J ournal of Natural Products, 2004, Vol. 67, No. 5 913
h. Extraction with EtOAc (5 mL × 2) led to the identification
in the organic layer of stigmasterol14 as the aglycon by TLC
analysis. The aqueous phase was then adjusted to pH 6 with
NaHCO3 and evaporated to dryness. The residue was redis-
solved in pyridine. TLC analysis showed the presence of
glucose (eluent: CHCl3-MeOH, 2:1).
(29)-dien-3-ol, which showed physical and spectroscopic char-
acteristics identical to those of compound 1.
Ack n ow led gm en t. We are indebted to the Italian Minis-
try for University and Research (MIUR) for a grant to support
scientific cooperation between Italy and Sri Lanka.
Com p ou n d 1: sticky oil, [R]25 +42.6° (c 0.5, CHCl3); 13C
Refer en ces a n d Notes
D
and 1H NMR data, see Table 1; EIMS m/z 426 [M]+ (54) 367
[M - CH2CH2CH2OH] (29), 345 (100), 315 (13), 245 (11), 231
(15), 217 (14), 203 (15), 191 (11), 189 (17), 181 (17), 163 (14),
149 (18), 135 (13), 129 (13), 109 (20), 85 (42); HREIMS m/z
426.383 (calcd for C30H50O, 426.3862).
(1) Bandaranayake, W. M.; Sultanbawa, M. U. S. A List of Endemic
Plants of Sri Lanka; Forestry Information Service, Forest Depart-
ment: Colombo, Sri Lanka, 1993.
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London, 1895; p 364
(3) Wenkert, E.; Baddeley, G. V.; Burfitt, I. R.; Moreno, L. M. Org. Magn.
Reson. 1978, 11, 337-343.
Syn th esis of 1 fr om Lu p eol. Lupeol (50 mg, 0.12 mmol),
212 mg (0.48 mmol) of lead tetraacetate, and CaCO3 (2 mg) in
8 mL of anhydrous toluene were heated under reflux for 2.5
h. The mixture was filtered, and the filtrate was washed with
a 5% aqueous solution of KI, a 10% solution of Na2S2O3, and
water. Then the toluene solution was evaporated to dryness.
The crude product (56 mg) was purified by flash chromatog-
raphy (hexane-Et2O, 97:3; 500 mL) to obtain 20 mg of the
aldehyde 2, as an oil: 1H NMR (CDCl3, 300 MHz) δ 0.70 (3H,
s, Me-25), 0.77 (3H, s, Me-28), 0.87 (3H, s, Me-27), 0.99 (3H,
s, Me-26), 1.59 (3H, dd, J ) 0.8, 1.5 Hz, Me-24), 1.61 (3H, dd,
J ) 0.7, 1.35 Hz, Me-30), 2.3 (2H, m, H-2), 4.48-4.60 (2H, m,
H-29), 4.52-4.73 (2H, m, H-23), 9.62 (1H, t, J ) 2.05 Hz,
-CHO) that was immediately dissolved in 1 mL of anhydrous
THF and treated with 4.8 mL of a solution of 1 M LiAlH4 in
THF. The solution was kept 1.5 h at room temperature, then
cooled at 0 °C. After addition of 4 mL of water, the mixture
was filtered and the filtrate was evaporated to dryness.
Purification by flash chromatography (hexane-Et2O, 85:15;
300 mL) afforded 19 mg (yield 89%) of 3,4-seco-lupa-4(23),20-
(4) Patra, A.; Mukhopadhyay, A. K.; Mitra, A. K. Org. Magn. Reson. 1981,
17, 166-168.
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NP030374G