Guaianolides from Salvia nubicola (Lamiaceae)

Article abstract of DOI:10.1248/cpb.54.1235

A new sesquiterpene-lactone (nubenolide) belonging to the guaianolide class along with its acetate (nubenolide acetate) and a dimer (bisnubenolide) have been isolated from Salvia nubicola collected from Quetta, Pakistan. Structures of all three new metabolites were elucidated with the aid of spectroscopic techniques including 2D-NMR. However, the structure of nubenolide was finally confirmed via single-crystal X-ray diffraction method.

Full text of DOI:10.1248/cpb.54.1235

September 2006
Chem. Pharm. Bull. 54(9) 1235—1238 (2006)
1235
Guaianolides from Salvia nubicola (Lamiaceae)
Muhammad Shaiq ALI, ,a Waqar AHMED,a Andrea Florence ARMSTRONG,b Syed Amir IBRAHIM,a
*
b
Shakeel AHMED,a and Masood PARVEZ
a H. E. J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi; Karachi-
75270, Pakistan: and b Department of Chemistry, University of Calgary; 2500 University Drive NW, Calgary, Alberta,
Canada T2N IN4. Received December 12, 2005; accepted April 30, 2006
A new sesquiterpene-lactone (nubenolide) belonging to the guaianolide class along with its acetate (nubeno-
lide acetate) and a dimer (bisnubenolide) have been isolated from Salvia nubicola collected from Quetta, Pak-
istan. Structures of all three new metabolites were elucidated with the aid of spectroscopic techniques including
2D-NMR. However, the structure of nubenolide was finally confirmed via single-crystal X-ray diffraction method.
Key words Salvia nubicola; Lamiaceae; guaianolide; 2D-NMR; X-ray
Salvia is the largest genus of the family Lamiaceae having via high resolution (HR)-EI-MS. The other fragments are
about 800 species throughout the world.1) Most of the plants mentioned in the experimental section.
1
of this genus are well known for their biologically active con-
Most of the signals in H-NMR spectrum showed confus-
stituents, specifically those with anti-tumor activity.2) Several ing multiplicities with minor coupling constants inferring the
species of Salvia are used in folk medicines, for instance, existence of long-range couplings in the molecule. Therefore
Salvia cavaleriei is used for the treatment of dysentery, the spectrum was simplified by recording at low temperature
haemoptysis, boils, and fall injuries,3) Salvia desoleria for (10 °C). A singlet at d 6.30 was assigned to H-3. A pair of
the treatment of menstrual, digestive, and central nervous doublets having the coupling constants 10.1 Hz appeared at d
system diseases,4) and Salvia bucharica as a traditional medi- 3.45 and 4.55 due to H-5 and H-6. An ABX system was
cine for the treatment of liver disorders. The plants of this found in the molecule due to H-8, H-9a, and 9b at d 4.76
genus are rich in essential oils and among their constituents, (dd , 11.8, 3.4), 2.50 (dd, 12.9, 11.8), and 2.68 (dd, 12.9, 3.4),
1,8-cineol and guaiane-type mono- and sesquiterpenes are respectively. The three quaternary methyls appeared at d
very common.5,6)
In continuation of our research on the chemical con- ture of the proton NMR spectrum is given in Table 1.
stituents of Salvia,7—14) herein wish to report the isolation The 13C-NMR spectrum showed 15 carbon signals alto-
2.37 (H-13), 2.45 (H-14), and 2.44 (H-15). A complete pic-
and spectroscopic characterization of three new guaianolides: gether which were resolved into three methyls, a methylene,
nubenolide (1), bisnubenolide (2), and nubenolide acetate (3) four methines, and seven other quaternary carbons with the
from Salvia nubicola collected from Quetta, Pakistan.
aid of Distortionless Enhancement by Polarization Transfer
(DEPT) experiments. The signals due to the ketone and lac-
tone carbonyls were located in the spectrum at d 195.8 and
174.2, respectively. The carbinylic carbon appeared at d
75.1. The olefinic methine was found at d 135.3. The
olefinic-quaternary carbons, which are also the part of ring
junctions, resonated at d 135.4 (C-1) and 164.6 (C-7). The
signals of the three remaining olefinic-quaternary carbons to
which methyls are attached appeared at d 173.1(C-4), 141.7
(C-10), and 122.7 (C-11). Their corresponding methyls ap-
peared at d 20.1, 21.0 and 10.5, respectively. An oxy-me-
thine and the only methylene in the molecule resonated at d
77.6 (C-8) and 41.7 (C-9). Detailed 13C-NMR data are given
in Table 2.
Results and Discussion
The obtained information through NMR spectra were fur-
1
ther correlated via H–1H correlation spectroscopy (COSY)
The ethyl acetate-soluble part of the methanolic extract of and heteronuclear multiple-bond correlations (HMBC) (Fig.
S. nubicola collected from Quetta (Pakistan) yielded a gua- 1) experiments. The HMBC connectivity of H-6 (d 4.55)
ianolide (1, nubenolide), its dimer (2, bisnubenolide), and with C-7 (d 164.6) clearly confirmed the position of hy-
aceteate (3, nubenolide acetate).
droxyl function at C-6. The structure of 1 was elucidated
Nubenolide (1) Compound 1 was obtained as colorless when the information gathered from COSY and HMBC ex-
needles. This was melted at 110—112 °C. The IR spectrum periments was coupled with obtained NMR spectral data. Fi-
exhibited presence of hydroxyl (3440 cmꢀ1), a,b-unsaturated nally, the relative stereochemistry of chiral centers (C-5, C-8)
ketone and/or lactone (1750 cmꢀ1) and olefinic (1608 cmꢀ1) and complete structure of 1 were cross-checked by means of
functionalities in the molecule. The molecular ion peak was single-crystal X-ray crystallography (Fig. 2).
observed at m/z 260 in field desorption mass spectrum (FD-
However, the (S) absolute configuration at C-6 was estab-
MS) and the formula of this peak was depicted as C15H16O4 lished by modified Mosher’s method.15) Esterification of 1
∗ To whom correspondence should be addressed. e-mail: shaiq303@hotmail.com
© 2006 Pharmaceutical Society of Japan

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Vol. 54, No. 9
Table 1. 1H-NMR Spectral Data of Nubenolide (1), (R) MTPA-Ester (1a), (S) MTPA-Ester (1b), Bisnubenolide (2) and Nubenolide Acetate (3)
Nubenolide
(J in Hz)
(R) MTPA-ester
(J in Hz)
(S) MTPA-ester
(J in Hz)
Bisnubenolide
(J in Hz)
Nubenolide acetate
H. No.
(J in Hz)
3
5
6
6.30 (s)
6.29 (s)
6.29 (s)
5.99 (s)
3.19 (d, 10.8)
4.10 (d, 10.8)
4.45 (d, 11.8)
2.54 (d, 11.8)
—
1.95 (s)
2.24 (s)
2.20 (s)
—
6.21 (s)
3.45 (d, 10.1)
4.55 (d, 10.1)
4.76 (dd, 11.8, 3.4)
2.50 (dd, 12.9, 11.8)
2.68 (dd, 12.9, 3.4)
2.37 (s)
2.45 (s)
2.44 (s)
—
—
3.39 (d, 10.1)
5.23 (d, 10.1)
4.76 (dd, 11.8, 3.4)
2.52 (dd, 12.9, 11.8)
2.68 (dd, 12.9, 3.4)
2.37 (s)
2.44 (s)
2.42 (s)
—
7.22 (m)
3.45 (d, 10.1)
5.26 (d, 10.1)
4.76 (dd, 11.8, 3.4)
2.50 (dd, 12.9, 11.8)
2.69 (dd, 12.9, 3.4)
2.34 (s)
2.44 (s)
2.44 (s)
—
7.23 (m)
3.47 (d, 10.6)
5.35 (d, 10.6)
4.69 (dd, 12.1, 3.3)
2.43 (dd, 13.3, 12.1)
2.78 (dd, 13.3, 3.3)
1.97 (s)
2.45 (s)
2.23 (s)
2.20 (s)
—
8
9a
9b
13
14
15
OAc
Ph
OMe
—
—
—
3.58 (s)
3.56 (s)
—
At 300 MHz in CDCl3.
Table 2. 13C-NMR Spectral Data of Nubenolide (1), Bisnubenolide (2)
and Nubenolide Acetate (3)
Nubenolide
(125 MHz)
Nubenolide acetate
(75 MHz)
Bisnubenolide
(100 MHz)
C. No.
1
2
3
4
5
6
7
8
135.4
195.8
135.3
173.1
53.3
75.1
164.6
77.6
41.7
141.7
122.7
174.2
10.5
21
133.9
194.8
137
167.5
50.5
75.4
158.5
77
134.6
196.6
134.7
173.7
52.7
74.5
163.8
77.3
41.3
142.5
122.8
174.6
9.4
9
10
11
12
13
14
15
CH3CO
CH3CO
42
143.3
122.5
172.9
9.6
20.7
19.9
168.7
20.9
20.4
20.1
—
with R-(ꢀ)MTPA-Cl and S-(ꢁ)MTPA-Cl afforded the corre-
sponding esters 1a and b, respectively. The positive value of
D(dSꢀdR) for H-5 indicated (S)-configuration at C-6 in 1.
This new guaianolide has not been reported so far from
any natural source and is named nubenolide. Guaianolides
are very common natural sesquiterpene-lactones found in
various terrestrial plants.16—18)
20.1
—
—
—
In CDCl3.
Bisnubenolide (2) Compound 2 was obtained as a white
powder. Spectral analysis of 2 suggested the structure as a
dimer of 1. Brief mass and IR spectral data are given in the
experimental section.
Compound 2 was melted at 131 °C. The molecular mass
and corresponding formula were confirmed by FD and HR-
EI mass spectra at m/z 518 as C30H30O8. As compared with
the NMR spectra of 1, only disappearance of methylene and
appearance of an additional methine were observed in the
NMR spectra of 2 (see Tables 1, 2). Instead of ABX system,
Fig. 1. Important HMBC Interactions in 1
1
the H-NMR spectrum showed a mutually coupled pair of
doublets at d 4.45 (H-8) and 2.45 (H-9) with the coupling
constant 11.8 Hz. Similarly, 13C-NMR spectrum displayed an
extra methine at d 41.3 with the loss of methylene signal.
Thus the structure of 2 was concluded as a dimer of 1 with
the carbon-carbon bond across C-9 of both monomeric units.
With the aid of X-ray data of nubenolide (1) (CCDC
1768121), the dihedral angles of 9-Ha and 9-Hb with H-8b
could be calculated and were found to be 184.3° and 76.5°,
respectively. Since 9-Hb is missing in bisnubenolide (2) de-
picted by the disappearance of 3.4 Hz coupling in the H-8
Fig. 2. X-Ray Structure of 1

September 2006
1237
NMR signal (see Table 1), so the linkage of the other AcOH]ꢁ and 227 [MꢀAcOHꢁCH3]ꢁ. FD-MS m/z: 302. HR-EI-MS m/z:
302.1159 (Calcd for C17H18O5: 302.1154). 1H-NMR: see Table 1. 13C-NMR:
see Table 2.
Fractions eluted with hexane–ethyl acetate, 17 : 3 yielded colorless nee-
dles, which on washing with methanol yielded 1 (9.13 mg).
monomeric unit in 2 should be from b-face. The molecular
model of 2 also suggested the b-orientation of attachment to
avoid the steric factor from the a-face. This new dimer is
named bisnubenolide.
Nubenolide (1): mp 110—112 °C. [a]D28 ꢁ16.1° (cꢃ0.613, CHCl3). IR
(KBr) cmꢀ1: 3440 (OH), 1750 (broad, a,b-unsaturated ketone and ester
CꢃO) and 1608 (CꢃC). EI-MS m/z: 260 [M]ꢁ, 245 [MꢀCH3], 242
[MꢀH2O], and 227 [MꢀH2OꢁCH3]. FD-MS m/z: 260. HR-EI-MS m/z:
260.1044 (Calcd for C15H16O4: 260.1048), 245.0808 (Calcd for C14H13O4:
245.0813), 242.0938 (Calcd for C15H14O3: 242.0942), and 227.0704 (Calcd
for C14H11O3: 227.0708). 1H-NMR: see Table 1. 13C-NMR: see Table 2.
HMBC: see Fig. 1. X-ray: a colorless crystal with dimensions 0.16ꢂ0.15ꢂ
0.13 mm was selected for the crystallographic measurements. C15H16O4:
Mr 260.28; monoclinic, aꢃ6.468(3), bꢃ16.952(9), cꢃ12.070(7) Å, bꢃ
101.42(3)°, Vꢃ1297.2 (12) Å3, space groupꢃP21, Zꢃ4, Dxꢃ1.333 mg/m3,
F(000)ꢃ552, MoKaꢃ0.71073 Å. Unit cell dimensions were determined by
least-square fit of 352 reflections measured at 293(2) K using MoKa radia-
tions on Nanius Kappa CCD diffractometer. The intensity data within (q)
range of 4.0 to 27.5° were collected at 293(2) K. A total of 5057 reflections
were collected, of which 3052 reflections were monitored on the basis of
Iꢄ2s(I). The structure was solved by the direct method and expanded using
Fourier transformation technique. The structure was refined by full-matrix
Nubenolide Acetate (3) Compound 3 was obtained as
an off-white powder melted at 128 °C. Most of the spectral
data were matched with the data of 1 except for a few sig-
nals/peaks described here. The FD-MS of 3 displayed the
molecular weight 302 atomic mass unit and the formula was
observed as C17H18O5 in the HR-EI-MS. The status of an ad-
ditional two carbons, an oxygen, and two hydrogen atoms in
the molecule was inferred by the presence of an acetate moi-
ety that was cross-checked via NMR spectra. The carbon
spectrum of 3 showed two extra signals at d 20.9 and 168.7
due to the methyl and carbonyl carbons of acetoxyl function.
The methyl corresponds to the acetoxyl function appearing in
the proton NMR spectrum at d 2.20. Detailed NMR spectral
data of 3 are given in Tables 1 and 2. The slight shift of
carbinylic signal towards downfield in the 1H-NMR spectrum least-square calculation on F2 with the aid of program SHELXL 97. The
final R and Rw factors were measured as 0.040 and 0.079, respectively. The
figure was plotted with the aid of ORTEP program. See Fig. 2. [The X-ray
data are deposited with the X-ray Crystallographic Centre, Cambridge, U.K.
(CCDC 176812).]
suggested the position of the acetate moiety at C-6. In the
13C-NMR spectrum, the upfield shift of both signals adjacent
to C-6 carbons at d 50.5 (C-5) and 158.5 (C-7) additionally
supports the position of the acetate moiety at C-6. Further-
more, the position of this additional moiety was reconfirmed
with the aid of HMBC and Heteronuclear Multiple-Quantum
Coherence (HMQC) experiments. This acetate-guaianolide
named nubenolide acetate is another new addition in the con-
stituents of S. nubicola.
The NMR spectra of chemically acetylated product of 1
exactly matched with 3, confirming the S-configuration at C-
6 in 3.
Preparation and Purification of Mosher’s Esters R-(ꢀ)MTPA-Cl/S-
(ꢁ)MTPA-Cl (10 ml) was treated with a stirred solution of 1 (0.01 mmol) in
pyridine (200 ml) for 8 h. After removal of the solvent, the products (1a, b)
were purified by preparative TLC (CHCl3 : hexane 9.5 : 0.5) as gums. See
1H-NMR data in Table 1.
Acetylation of 1 Compound 1 (0.01 mmol) was dissolved in pyridine
(0.2 ml) and treated with acetic anhydride (0.2 ml) overnight. Then the reac-
tion mixture was quenched with ice-cold water and the unreacted pyridine
removed by adding saturated CuSO4 solution. The organic mass was recov-
ered in ethyl acetate and the organic layer was washed with water, condensed
under reduced pressure, and chromatographed.
Fractions eluted with hexane–ethyl acetate, 4 : 1 yielded a white powder,
which on washing with methanol yielded 2 (8.43 mg).
Experimental
Bisnubenolide (2): mp 131 °C. [a]D28 ca. 0° (cꢃ0.666, CHCl3). IR (KBr)
cmꢀ1: 3435 (OH), 1735 (broad, a,b-unsaturated ketone and ester CꢃO) and
1615 (CꢃC). FD-MS m/z: 518. HR-EI-MS m/z: 518.1945 (Calcd for
C30H30O8: 518.1940). 1H-NMR: Table 1 and 13C-NMR: Table 2.
General Procedures IR spectra were recorded by Jasco-320-A spec-
1
trometer. H-NMR spectra were recorded in CDCl3 by a Bruker AM-300
spectrometer while the 13C-NMR spectra were recorded by Bruker AM-300,
AM-400 and AM-500 spectrophotometers at 75, 100, and 125 MHz in the
same solvent. Chemical shifts are given relative to TMS (d: 0.00) as internal
standard (1H) and d: 77.0 (ppm) from CDCl3 as standard (13C). Optical rota-
tions were measured by a JASCO DIP-360 digital polarimeter in CHCl3
using a 10 cm cell tube. Mass spectra (EI, FD, and HR-EI-MS) were meas-
ured in electron impact mode by Finnigan MAT 12 or MAT 312 spectrome-
ters and ions are given in m/z (%). TLC was performed with precoated silica
gel G-25-UV254 plates and detection was done by spraying with ceric sul-
phate in 10% H2SO4. Silica gel (E. Merck, 230—400 mesh) was used for
column chromatography. Melting points were determined by Gallenkemp
apparatus and are uncorrected.
Acknowledgments We thank Dr. R. B. Tareen, Department of Botany,
Baluchistan University, Quetta, for the collection and identification of the
plant material. S.A.I. acknowledges the enabling role of the Higher Educa-
tion Commission Islamabad, Pakistan and appreciates its financial support
through “Merit Scholarship Scheme for Ph.D. Studies in Science & Technol-
ogy (200 Scholarship).”
References
1) Chadha Y. R., “The Wealth of India,” Publication and Information Di-
rectorate, CSIR, New Delhi, 1972, p. 327.
2) Fujita E., Node M., Prog. Chem. Org. Nat. Prod., 46, 77—157 (1984).
3) Zhang H. J., Li L. N., Planta Med., 60, 70—72 (1994).
4) Peana A., Satta M., Moretti M. D., Orecchioni M., Planta Med., 60,
478—479 (1994).
Plant Material S. nubicola was collected from Urhat-Juniper forest,
near Zearat (Quetta), Pakistan, in the month of July (1999) and identified by
Dr. Rasool Baksh Tareen, Department Botany, Baluchistan University,
Quetta, where the voucher specimen (No. 613) of the plant is deposited in
the herbarium.
Extraction and Isolation The fresh plant material (all parts, 13 kg) was
dried under shade (6.5 kg) for a period of 2 weeks then soaked in hexane
(12 lꢂ2) and methanol (12 lꢂ2) for 10 d in each solvent. Solvents were
evaporated through vacuum distillation. The condensed and crude methano-
lic extract (217 g) was then partitioned between water and ethyl acetate. The
ethyl acetate layer was again condensed (167 g) and subjected to column
chromatography using hexane; hexane–ethyl acetate; and ethyl acetate and
ethyl acetate–methanol as mobile phase.
5) Rustaiyan A., Komeilizadeh H., Masoudi M., Jassbi A. R., J. Essent.
Oil, 9, 713—714 (1997).
6) Rustaiyan A., Masoudi S., Jassbi A. R., J. Essent. Oil, 9, 599—600
(1997).
7) Ahmad V. U., Zahid M., Ali M. S., Iqbal M. Z., Z. Naturforsch., 54b,
415—418 (1999).
8) Ahmad V. U., Zahid M., Ali M. S., Choudhary M. I., Akhtar F., Ali Z.,
Iqbal M. Z., Tetrahedron Lett., 40, 7561—7564 (1999).
9) Ahmad V. U., Zahid M., Ali M. S., Ali Z., Jassbi A. R., Abbas M.,
Clardy J., Lobkovsky E., Tareen R. B., Iqbal M. Z., J. Org. Chem., 64,
8465—8467 (1999).
Fractions eluted with hexane–ethyl acetate, 19 : 1 yielded off-white pow-
der 3 (8.30 mg).
Nubenolide Acetate (3): mp 128 °C. [a]D28 ꢁ21.7 ° (cꢃ0.733, CHCl3). IR
(KBr) cmꢀ1: 1756 (broad, ester, a,b-unsaturated ketone and ester CꢃO),
and 1618 (CꢃC). EI-MS m/z: 302 [M]ꢁ, 260 [Mꢀketene]ꢁ, 242 [Mꢀ
10) Ahmad V. U., Zahid M., Ali M. S., Ahmad S., Ali Z., Iqbal M. Z.,
Tareen R. B., Sci. Pharm., 68, 429—433 (2000).

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Vol. 54, No. 9
11) Ahmad V. U., Zahid M., Ali M. S., Jassbi A. R., Ali Z., Ahmad S.,
Iqbal M. Z., Nat. Prod. Sci., 6, 66—69 (2000).
15) Seco J. M., Quinoa E., Riguera R., Tetrahedron: Asymmetry, 12,
2915—2925 (2001).
12) Chou N. H.-H., Pervez M., Ali M. S., Ahmad S., Ahmed W., Acta
Cryst., E58, 0285—0287 (2002).
16) Bohlmann F., Zdero C., Ahmad M., Phytochemistry, 21, 1679—1691
(1982).
13) Ali M. S., Dardass A. K. Y., Ahmad S., Saleem M., Firdous S., Ahmad
V. U., Fitoterapia, 71, 347—352 (2000).
17) Romo J., Joseph-Nathan P., Vivar A. R. D., Alvarez C., Tetrahedron,
23, 529—534 (1967).
14) Ahmad V. U., Zahid M., Ali M. S., Jassbi A. R., Abbas M., Ali Z.,
Iqbal M. Z., Phytochemistry, 52, 1319—1322 (1999).
18) Zdero C., Bohlmann F., Phytochemistry, 28, 3105—3120 (1989).