Unusual 6'-fatty acid esters of (24S)-24-ethylcholesta-5,25-dien-3β-yl β-D-glucopyranoside from Teucrium fruticans

Article abstract of DOI:10.1016/S0031-9422(98)00511-1

An unusual (24S)-24-ethylcholesta-5,25-dien-3β-yl β-D-glucopyranoside possessing a mixture of fatty acid esters at the C-6' position of the sugar moiety has been isolated from Teucrium fruticans. Spectroscopic studies and chemical transformations allowed the characterization of the sterol and sugar parts and palmitic, stearic, oleic, linoleic and linolenic acids as the main constituents of the 6'-O-acyl moiety.

Full text of DOI:10.1016/S0031-9422(98)00511-1

PERGAMON
Phytochemistry 50 (1999) 283±285
Unusual 60-fatty acid esters of (24S)-24-ethylcholesta-5,25-dien-3b-
yl b-D-glucopyranoside from Teucrium fruticans
Gianfranco Fontanaa, Giuseppe Savonaa, *, Benjamõn Rodrõguezb, 1
,
Marõa C. De La Torreb
a
Á
Dipartimento di Chimica Organica dell'Universita, Via Archira® 20, I-90123 Palermo, Italy
b
 Â
Instituto de Quõmica Organica, CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain
Revised 3 July 1998
Abstract
An unusual (24S)-24-ethylcholesta-5,25-dien-3b-yl b-D-glucopyranoside possessing a mixture of fatty acid esters at the C-60
position of the sugar moiety has been isolated from Teucrium fruticans. Spectroscopic studies and chemical transformations
allowed the characterization of the sterol and sugar parts and palmitic, stearic, oleic, linoleic and linolenic acids as the main
constituents of the 60-O-acyl moiety. # 1998 Elsevier Science Ltd. All rights reserved.
Keywords: Teucrium fruticans; Labiatae; Sterol glycoside; 60-Fatty acid esters; (24S)-24-Ethylcholesta-5,25-dien-3b-yl 60-O-acyl-b-D-glucopyranoside
1. Introduction
iments, revealed the presence of a 2,3,4,6-tetra-O-acyl-
b-glucopyranosyl moiety in a 4C1 conformation [dH
4.58 d, J=7.9 Hz (H-10), 4.95 dd, J=7.9, 9.5 Hz (H-
20), 5.20 t, J =9.5 Hz (H-30), 5.04 td, J= 9.7, 2.3 Hz
(H-40, long-range coupled with the HB-60 proton), 3.67
ddd, J= 9.7, 5.3, 2.5 Hz (H-50), 4.12 dd, J=12.1, 2.5
Hz (HA-60), 4.22 ddd, J =12.1, 5.3, 2.3 (HB-60); dC
99.6 d (C-10), 71.5 d (C-20), 72.9 d (C-30), 68.7 d (C-
40), 71.7 d (C-50), 62.0 t (C-60)] attached to the C-3b
position (dH-3a 3.47 m, dC-3 80.1 d) of 24-ethylcholesta-
5,25-dien-3b-yl part [dH 5.35, 1H, br d, J=5.4 Hz
In previous studies, we reported the isolation of sev-
eral neo-clerodane diterpenoids from Teucrium fruti-
cans (Savona et al., 1978a,b; Bruno, de la Torre,
Savona, Piozzi, & Rodrõguez, 1990; Bruno et al.,
1992). An investigation of the more polar chromato-
graphic fractions of an acetone extract of the aerial
parts of this plant has now allowed the isolation of a
steryl glucoside esteri®ed at the C-60 position of the
sugar part with a mixture of fatty acids. We report
here the structural elucidation of this metabolite.
2. Results and discussion
The 1H NMR spectrum of the natural compound
(1, see Section 3) suggested that it was a steryl glyco-
side possessing fatty acid ester groups and not acetoxyl
functions (absence of singlet signals between d 2.3 and
1.8). Treatment of 1 with acetic anhydride±pyridine
yielded a triacetyl derivative (2, singlets at d 2.04, 2.01
1
and 2.00, 3H each) whose H and 13C NMR spectra,
as well as COSY, TOCSY, HMQC and HMBC exper-
* Corresponding author.
1 Corresponding author.
0031-9422/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00511-1

284
G. Fontana et al. / Phytochemistry 50 (1999) 283±285
(H-6), 0.66, 3H, s (Me-18), 0.98, 3H, s (Me-19), 0.89,
3H, d, J =6.6 Hz (Me-21), 4.64, 1H, dq, J26A,26B = 2.5
Hz, J26A,27 = 0.7 Hz (HA-26), 4.72, 1H, sext,
J26B,26A =2.5 Hz, J26B,27 =J26B,24 = 1.3 Hz (HB-26),
1.56, 3H, dd, J =2.5, 1.3 Hz (Me-27), 0.80, 3H, t,
J= 7.5 Hz (Me-29); (for 13C NMR data see Section 3)
(Rubinstein & Goad, 1974; Gaspar, Brito Palma, de la
Torre, & Rodrõguez, 1996; Ahmad, Aliya, Perveen, &
Shameel, 1992; Tori, Yoshimura, Arita, & Tomita,
Section 3 and above), and this peculiarity was not
observed in the tetra-O-acetyl derivative 4 (see Section
3). This di€erent behavior can be explained consider-
ing that in the case of 1 and 2 the presence of long-
chain esters at the C-60 position causes a restriction of
the rotation around the C-50, C-60 bond, thus favoring
a spatial arrangement in which such protons are long-
range coupled.
Finally, acid hydrolysis of 1 yielded the aglycone
which, after treatment with acetic anhydride±pyridine,
was rigorously characterized as (24S)-24-ethylcholesta-
5,25-dien-3b-yl acetate (5) (Rubinstein & Goad, 1974;
Gaspar et al., 1996; Sucrow, Slopianka, & Kircher,
1976) by its melting point, [a]D, 1H NMR and mass
spectra and by comparison (mixed mp, TLC on silver
nitrate impregnated silica gel plates) with an authentic
sample (Gaspar et al., 1996).
1
1977). In addition, the H and 13C NMR spectra of 2
showed typical signals for a mixture of fatty acid esters
[dH 5.35 m (ole®nic), 2.80 t, J= 6.1 Hz (allylic), 2.32 t,
J= 7.4 Hz (a-methylene protons), 1.60 m (b-methylene
protons), 1.25 br s [(CH2)n], 0.86 t, J= 6.8 Hz (o-Me);
dC 173.4 s (C-1), 34.1 t, 34.0 t (C-2), 14.2 q, 14.1 q (o-
Me), 131.9±127.1 (10 signals, d, ole®nic), 25.5 t, 25.3 t
(allylic) and other signals at d 22.7 t, 29.1±29.7 t, 30.1
t, 31.8 t] (Teixeira et al., 1997; Razdan, Kachroo,
Qurishi, Kalla, & Waight, 1996).
The HMBC spectrum of 2 showed connectivities
between the carboxyl carbon of the fatty acid esters (d
173.4 s) and both the C-6 methylene protons of the
glucopyranoside part (d 4.12 and 4.22), whereas the
carboxyl carbons of the three acetates (d 170.3 s,
169.33 s and 169.27 s) were correlated with the sugar
protons at d 5.20 (H-30), 5.04 (H-40) and 4.95 (H-20),
respectively, thus establishing that the fatty acid esters
were attached to the C-6 position of the glucopyrano-
side. The HMBC spectrum of 2 also displayed three
bonds coupling between the C-10 carbon of the glucose
(d 99.6 d) and the H-3a proton (d 3.47 m) of the
sterol, and between the axial H-10 proton of the sugar
(d 4.58 d, J =7.9 Hz) and the C-3 steryl carbon (d
80.1 d). Moreover, the C-2, C-4 and C-5 carbons of
the steryl moiety were up®eld shifted (Dd 2.1, 3.2
and 0.5 ppm, respectively) with respect to those
reported in the literature (Wright et al., 1978) for D5-
sterols, thus con®rming (Tori et al., 1977; Kasai,
Suzuo, Asakawa, & Tanaka, 1977; Faghih et al., 1985)
that 2 was a D5-ster-3b-yl 2,3,4,6-tetra-O-acyl-b-D-glu-
copyranoside.
From all the above data it was evident that the
natural compound and its peracetyl derivative pos-
sessed the structures depicted in 1 and 2, respectively.
Alkaline hydrolysis of 1 gave 3 (which without
characterization was transformed into its tetraacetyl
derivative 4) and an alkali soluble fraction which, after
methylation and GC-MS analysis, allowed the identi®-
cation of the methyl ester of palmitic (34.35%), stearic
(5.38%), oleic (7.98%), linoleic (11.12%) and linolenic
(37.50%) acids together with minor quantities of the n-
alkanoic acid C14 (0.40%), C15 (0.46%), C17 (1.12%),
C20 (0.79%) and C22 (0.64%) methyl esters.
3. Experimental
3.1. General
Mps: uncorr. Plant materials were collected in June
1996 near Palermo, Sicily, and voucher specimens were
deposited in the Herbarium of the Botanic Garden of
Palermo.
3.2. Extraction and isolation of 1
Dried and powdered Teucrium fruticans L. aerial
parts (2 kg) were extracted (3Â) with Me2CO (10 l) at
room temp. for 5 days. The extract (60 g) was sub-
jected to CC (Si gel, deactivated with 15% H2O, w/v,
600 g) eluting with a petrol±EtOAc gradient from 10±
100% EtOAc, yielding the neo-clerodanes previously
reported as constituents of this species (Savona et al.,
1978a,b; Bruno et al., 1992). Final elution with
CH2Cl2±MeOH (49:1) gave 1 (500 mg): soft substance;
[a]1D8 51.58 (CHCl3; c 0.163); IR n
NaCl
max
cm 1: 3400 br
(OH), 3080, 1645, 890 (terminal methylene), 1740
(esters), 2940, 2860, 1470, 1380, 1170, 1080, 1060,
1025, 840, 800, 720; 1H NMR (400 MHz, CDCl3): d
5.35, 1H, br d, J =5.1 Hz (H-6), 4.71, 1H, sext,
J =2.5, 1.3 Hz (HB-26), 4.63, 1H, dq, J=2.5, 0.6 Hz
(HA-26), 4.46, 1H, ddd, J= 12.2, 4.6, 3.5 Hz (HB-60),
4.37, 1H, d, J =7.8 Hz (H-10), 4.25, 1H, dd, J = 12.2,
1.8 Hz (HA-60), 3.56, 2H, m (H-30, H-40), 3.45, 1H, m
(H-3a), 3.43, 1H, ddd, J =9.6, 4.6, 1.8 Hz (H-50),
3.36, 1H, dd, J= 9.0, 7.8 Hz (H-20), 1.56, 3H, dd,
J =2.5, 0.6 Hz (Me-27), 0.99, 3H, s (Me-19), 0.89, 3H,
d, J= 6.6 Hz (Me-21), 0.79, 3H, t, J= 7.5 Hz (Me-
29), 0.66, 3H, s (Me-18); fatty acid esters: d 5.36 m
(ole®nic), 2.79 t, J= 6.0 Hz (allylic), 2.34 t, J =7.2 Hz
(a-methylenes), 1.62 m (b-methylenes), 1.25 br s
[(CH2)n], 0.87 t, J =6.9 Hz (o-Me).
It is of interest to indicate that the H-40 and HB-60
glucopyranosyl protons of 1 and 2 showed a long-
range coupling (4J=3.5 and 2.3 Hz, respectively, see

G. Fontana et al. / Phytochemistry 50 (1999) 283±285
285
3.3. Triacetate 2
3.5. Acid hydrolysis of 1
Treatment of 1 (100 mg) with Ac2O±pyridine (1:1,
10 ml) at room temp. for 48 h yielded 2: mp 118±
Compound 1 (30 mg) was re¯uxed with 2 N HCl in
aqueous MeOH (50%, 30 ml). After 4 h the MeOH
was evaporated under reduced pressure, diluted with
H2O (15 ml) and the hydrolysate was then extracted
with EtOAc (3 Â 20 ml). The extract (12 mg) was trea-
ted with Ac2O±pyridine for 24 h at room temp. The
acetylated compound (5, 10 mg after crystallization
from MeOH) showed mp (127±1288C), [a]2D0 [ 48.98
1208C (from EtOH); [a]1D8 20.88 (CHCl3; c 0.721); IR
KBr
max
n
cm 1: 3080, 1645, 890 (terminal methylene), 1750
br (esters), 1240 (OAc), 2940, 2860, 1470, 1380, 1370,
1170, 1080, 1060, 1040, 910, 840, 800, 720, 690; 1H
NMR (400 MHz, CDCl3): see text; 13C NMR (100
MHz, CDCl3): for the sugar and fatty acid esters moi-
eties see text, aglycone: d 37.2 t (C-1), 29.5 t (C-2),
80.1 d (C-3), 39.0 t (C-4), 140.3 s (C-5), 122.1 d (C-6),
31.8 t (C-7), 31.9 d (C-8), 50.1 d (C-9), 36.7 s (C-10),
21.0 t (C-11), 39.7 t (C-12), 42.3 s (C-13), 56.7 d (C-
14), 24.3 t (C-15), 28.1 t (C-16), 56.0 d (C-17), 12.0 q
(C-18), 19.3 q (C-19), 36.7 d (C-20), 18.6 q (C-21),
35.5 t (C-22), 29.2 t (C-23), 49.5 d (C-24), 147.5 s (C-
25), 111.3 t (C-26), 17.8 q (C-27), 26.5 t (C-28), 11.8 q
(C-29).
1
(CHCl3; c 0.318)], H NMR and MS identical to those
reported previously (Rubinstein & Goad, 1974; Gaspar
et al., 1996; Sucrow et al., 1976) for the acetate of
(24S)-24-ethylcholesta-5,25-dien-3b-ol.
Comparison
[mp, 126±1288C, TLC on AgNO3 impregnated silica
gel plates, petrol±EtOAc (49:1) as eluent] with an auth-
entic sample con®rmed the identity.
Acknowledgements
3.4. Alkaline hydrolysis of 1
The authors thank Ms M.I. Jimenez, CSIC, Madrid,
for GC-MS analyses. This work was supported by
grants from the MURST (`Research Funds 60%',
Italy) and Direccion General de Ensenanza Superior
(PB94-0104 and PB96-0830, Spain).
To a solution of 1 (40 mg) in EtOH (2 ml) was
added a solution of KOH in EtOH (10%, w/v, 10 ml)
and the reaction mixture was left at room temp. for 3
days. After usual work-up, the acids and 3 were separ-
ately recovered. The acid fraction (15 mg) was dis-
solved in Et2O (5 ml) and treated with an excess of an
ethereal solution of CH2N2 for 2 h at room temp. The
solvent was evaporated and the residue subjected to
GC-MS analysis under standard conditions, by using a
Hewlett Packard 5890 gas chromatograph coupled to a
HP 5971A mass detector. The result of this analysis is
reported in Section 2.
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