J. Nat. Prod. 2003, 66, 703-705 703
Salvinorins D-F, New Neoclerodane Diterpenoids from Salvia divinorum, and
an Improved Method for the Isolation of Salvinorin A
Thomas A. Munro and Mark A. Rizzacasa*
School of Chemistry, The University of Melbourne, Victoria, 3010, Australia
Received December 11, 2002
Three new neoclerodane diterpenoids, salvinorins D-F(4-6), have been isolated from the leaves of Salvia
divinorum. The structures were elucidated by chemical and spectroscopic methods, particularly 1D and
2D NMR. A simplified isolation method using chromatography on activated carbon also gave improved
yields of the controlled substance salvinorin A (1) and of salvinorin C (3).
Salvia divinorum Epling & Játiva (Lamiaceae) is a sage in the crude extract proved troublesome; the green-black
native to Oaxaca, Mexico. An infusion prepared from the color made it almost impossible to distinguish the phases
leaves is used by the Mazatec Indians for the treatment of during the solvent partitioning procedure employed by
various illnesses including headache and diarrhea. At high Valdes and co-workers.3 Some coloration persisted even
doses, the infusion induces hallucinations.1 Salvinorins A after chromatography and recrystallization, as noted previ-
(1),2 B(2),3 and C(3)4 have been isolated from the leaves; ously,12 and the numerous pigment spots greatly compli-
in mice, 1 produces sedation, while 2 is inactive.3 The cated TLC analysis. We therefore decided to decolorize the
status of 3 remains unclear; a mixture of 1 and 3 was crude extract by chromatography on activated carbon.13
significantly more potent than 1 alone, but the pure This gave an amber terpenoid mixture from which pure 1
compound was not tested, and the results of this investiga- was isolated by recrystallization from methanol (giving a
tion appear to have been confounded, possibly by toxicity.4 total yield of 3.4 g/kg). Repeated flash chromatography of
Salvinorin A (1) binds potently and selectively to the kappa the mother liquor on silica gel gave 2-4 and a mixture of
opioid receptor5 and produces effects typical of º-opioids,6 5 and 6, which were separated by normal-phase HPLC.
including confusion, depersonalization, and hallucina- Compounds 3, 5, and 6 were isolated as clear resins.
tions.7,8 A recent case report suggests S. divinorum may Attempted recrystallizations from methanol and hexane/
have antidepressant activity.9 This is in accord with EtOAc were unsuccessful and caused substantial decom-
another recent study which found the º-opioid U-50,488H position. Compound 4 was triturated in diethyl ether, and
effective against an animal model of depression.10 Simi- it was subsequently found that this is also effective for 1,
larly, the relief of abdominal pain (particularly gut disten- which could greatly reduce the decomposition accompany-
sion) by º-opioids11 suggests a plausible basis for Mazatec ing recrystallization from alcohols.
use of S. divinorum against abdominal swelling.1
The structures of salvinorins D, E, and F (4-6) were
During the development of an improved procedure for
assigned by comparison of NMR data with the published
the isolation of 1 and 3 from S. divinorum, three new
values for 3 and analogues,4 along with decoupling, HMQC,
compounds were isolated: salvinorins D, E, and F (4-6).
HMBC, and DEPT experiments. HRESIMS established the
Using several solvents, Gruber demonstrated12 that extrac- 1
molecular formula of 4 and 5 as C23H28O8. The H NMR
spectra were also extremely similar. Compared to 3, the
spectra of 4 and 5 showed only one acetyl peak and gained
one new peak (´ 2.01 in 4; ´ 1.94 in 5) which exchanged
with D2O. The presence of a hydroxyl group was confirmed
by IR spectroscopy (3475 cm-1 in 4; 3510 cm-1 in 5). These
data suggested that the compounds were the two possible
monoacetates of 3.
1
The two compounds were readily differentiated by H
NMR. Relative to 3, the H-2 signal of 4 was shifted upfield
from ´ 5.55 to ´ 4.44 and showed a strong coupling to the
hydroxylic proton (J ) 6.7 Hz) as well as the expected
couplings to H-1 (5.6 Hz) and H-3 (2.4 Hz). The HMBC
spectrum showed correlations between H-2 and C-4, as well
as H-1 and C-5. The proposed structure of 4 was verified
by acetylation, which proceeded smoothly to give 3, identi-
cal in all respects with the isolated material (work with
cis-diol derivatives of 1 showed that while the 2-position
tion at room temperature gives a higher recovery than the
is readily esterified, the 1-position is unreactive).4,14 The
refluxing solvent used in the original procedures.2,3 His
1
implied structure of 5 was confirmed by H NMR. Relative
results suggest that 1 decomposes rapidly in hot solution.
to 3, the H-1 signal of 5 was shifted from ´ 5.76 to ´ 4.46.
We found that extraction at room temperature in acetone
Irradiation sharpened the H-10 singlet. The HMBC spec-
gave an excellent recovery. However, the pigments present
trum again showed a correlation between H-2 and C-4.
The molecular formula of compound 6, C21H26O6, was
* To whom correspondence should be addressed. Tel: +61-3-8344-6488.
1
Fax: +61-3-9347-5180. E-mail: masr@unimelb.edu.au. established by HRESIMS. In contrast to 4 and 5, H NMR
10.1021/np0205699 CCC: $25.00 © 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/18/2003
704 Journal of Natural Products, 2003, Vol. 66, No. 5 Notes
1
Table 1. H NMR Data (400 MHz, CDCl3) of Compounds 4-6 [´ (ppm), m, J (Hz)]
proton 45 6
1 5.70 br d (5.6) 4.46 ddd (4.7,1.6,1.3) 4.52 ddd (5.5,4.6,2.4)
2R 2.35 br dd (20.1,4.7)
2 4.44 ddd (6.7,5.6,2.4) 5.40 dd (4.7,2.4) 2.60 ddd (20.1,5.5,3.0)
3 6.54 dd(2.4,1.2) 6.43 dd (2.4,1.3) 6.67 ddd (4.7,3.0,2.4)
6R 2.56 dt (13.3,3.4) 2.52 ddd (12.5,3.0,2.6) 2.53 dt (13.3,3.2)
6 1.19 m 1.16-1.25 m 1.18 td (13.3,3.4)
7R 1.78 m 1.84 m 1.82 m
7 2.09-2.17 m 2.07-2.18 m 2.08-2.17 m
8 2.13 dd (13.5,3.5) 2.07-2.18 m 2.08-2.17 m
10 1.42 br s 1.30 br s 1.25 br s
11R 2.54 dd (13.1,5.6) 2.46 dd (13.1,6.0) 2.50 dd (13.2,5.8)
11 1.64 dd (13.1,11.2) 1.62 dd (13.1,11.2) 1.62 dd (13.2,11.2)
12 5.53 dd (11.2,5.6) 5.60 dd (11.2,6.0) 5.60 dd (11.2,5.8)
14 6.40 br s 6.41 br s 6.41 d (1.6)
15 7.42 t (1.6) 7.42 m 7.42 t (1.6)
16 7.44 br s 7.44 br s 7.44 br s
19 1.69 s 1.72 s 1.71 s
20 1.22 s 1.47 s 1.48 s
CO2CH3 3.74 s 3.73 s 3.72 s
OCOCH3 2.15 s 2.17 s
OH 2.01 d (6.7) 1.94 br s 1.29 d (4.6)
13
Table 2. C NMR (100 MHz) of Compounds 4-6 [´ (ppm)]
showed no acetyl peak and only one oxygenated methine
carbon 456
signal, with two new diastereotopic protons at ´ 2.35 and
2.60 coupling to H-1 and H-3. This implied the 2-deoxy
1 66.5 64.3 63.9
structure shown. Protons H-2R and H-2 were distin- 2 68.7 72.3 38.0
3 135.7 131.5 133.4
guished by their coupling constants: molecular modeling15
4 141.2 143.4 140.6
predicted J1 ,2 ) 5.4 Hz (observed: 5.5 Hz) and J1 ,2R )
5 37.6 37.8 36.6
1.5 Hz (irradiation of H-1 sharpened the H-2R peaks by
6 37.0 37.0 37.3
0.4 Hz).
7 18.3 18.4 18.6
A number of neoclerodane diterpenoids, very similar in
8 51.8 51.7 52.2
9 37.0 37.5 37.7
structure to the salvinorins, display a broad range of
10 52.5 54.0 54.8
activities against insects.16 Salvinorins C-F (3-6) fit an
11 43.9 44.4 44.4
experimentally determined pharmacophore for antifeedant
12 71.6 71.7 71.7
activity against Tenebrio molitor,17 while 1 and 2 contain
13 125.4 125.8 125.9
the key features for antifeedant activity against Spodoptera
14 108.4 108.4 108.4
littoralis.18 Research into S. divinorum has thus yielded
15 143.8 143.9 143.8
16 139.4 139.3 139.3
promising leads in a remarkably wide range of areas of
17 171.5 169.8 172.1
therapeutic and agrochemical interest. It should be noted,
18 166.2 166.0 166.9
however, that S. divinorum and salvinorin A (1) are now
19 21.6 21.9 21.6
controlled substances in Australia and research involving
20 15.6 16.2 16.4
them requires a State Health Department permit.19
CO2CH3 51.7 51.8 51.5
OCOCH3 21.2 21.0
Experimental Section
OCOCH3 171.5 171.8
General Experimental Procedures. Uncorrected melting
tography (FCC) on an equal mixture of activated carbon and
points were determined using a Reichert hot-stage apparatus.
filter aid, eluting with a gradient from acetone to petrol, to
Optical rotations were measured on a JASCO DIP-1000 digital
give an amber semicrystalline mass (5.73 g). Several recrys-
polarimeter. IR spectra were recorded using a Bio-Rad FTS
1 13 tallizations from methanol and ethanol gave 1 (2.64 g). The
165 FT-IR spectrophotometer. H NMR (400 MHz) and C
mother liquor was purified by FCC on silica gel (5-50%
NMR (100 MHz) spectra were recorded on Varian Inova 400
acetone/CH2Cl2 gradient). This was divided by TLC into three
and Unity Plus 400 instruments. HRESIMS were run on a
series: A (656 mg), B (150 mg), and C (359 mg).
Bruker 4.7T BiOAPEX FTMS. TLC was conducted on Merck
Series A. In a typical case, 71 mg was subjected to FCC on
silica gel 60 F254 plates visualized with phosphomolybdic acid
silica gel (14 g), eluting with a gradient from 50 to 80% Et2O/
in ethanol. HPLC was performed on a Spherex 5 µm silica
petrol, to give 3 (total yield 219 mg, 0.25 g/kg) and additional
column (250 × 10 mm) at a flow rate of 2 mL min-1. Column
1, which was recrystallized from ethanol (total yield 2.9 g, 3.4
chromatography was performed on Merck silica gel 60 or
g/kg).
Merck activated carbon 2183, at an adsorbent:solute mass
Series B. FCC on silica gel (35 g) in 70-90% Et2O/petrol
ratio of 30:1 unless otherwise indicated. Petrol refers to the
and recrystallization from methanol gave 2 (13 mg). 1 and 2
1
fraction boiling at 40-60 °C.
were identified by H NMR and mp.3
Plant Material. Dried S. divinorum leaves, cultivated in
Series C. Trituration in Et2O gave 4 (75 mg). The mother
Oaxaca, Mexico, were purchased in April 2002 from Salvia
liquor was subjected to repeated FCC on silica gel (Et2O/
Space Ethnobotanicals (Berkeley, CA). Voucher specimens
petrol). Final purification by HPLC (60% EtOAc/petrol; tR 9.8
were deposited at the National Herbarium of Victoria (acces- min for 5 and 10.7 min for 6) and drying under high vacuum
sion number MEL 2101361) and the University of Melbourne
(96 h at 45 °C) gave 5 (2.8 mg) and 6 (1.1 mg).
Herbarium (MELU sn).
Salvinorin D (4): fine colorless crystals, mp 185-187 °C;
Extraction and Isolation. Dried S. divinorum leaves (860 [R]17D +66.6° (c 1.0, CH2Cl2); IR (thin film) ½max 3475, 3146,
g) were powdered and steeped for 1 h in acetone (3 × 1 L). 2952, 2861, 1723, 1505, 1435, 1371, 1228, 1142, 1027, 875, 788
1 13
Filtration and evaporation under reduced pressure gave a dark cm-1; H and C NMR, see Tables 1 and 2; HRESIMS m/z
green tar (30.5 g). This was purified by flash column chroma- 455.1672 (calcd for C23H28O8Na, 455.1676); TLC, see Table S1.
Notes Journal of Natural Products, 2003, Vol. 66, No. 5 705
(3) Valdés, L. J., III; Butler, W. M.; Hatfield, G. M.; Paul, A. G.; Koreeda,
Salvinorin E (5): clear resin; [R]17D +46.4° (c 0.14, CHCl3);
M. J. Org. Chem. 1984, 49, 4716-4720.
IR (thin film) ½max 3510, 3144, 2952, 2858, 1722, 1505, 1436,
(4) Valdés, L. J., III; Chang, H. M.; Visger, D. C.; Koreeda, M. Org. Lett.
1 13
1374, 1228, 1142, 1070, 1029, 875, 805 cm-1; H and C NMR,
2001, 3, 3935-3937.
see Tables 1 and 2; HRESIMS m/z 455.1687 (calcd for
(5) Roth, B. L.; Baner, K.; Westkaemper, R.; Siebert, D.; Rice, K. C.;
C23H28O8Na, 455.1676); TLC, see Table S1.
Steinberg, S.; Ernsberger, P.; Rothman, R. B. Proc. Natl. Acad. Sci.
Salvinorin F (6): clear resin; [R]16D -20° (c 0.05, CHCl3); U.S.A. 2002, 99, 11934-11939.
(6) Walsh, S. L.; Strain, E. C.; Abreu, M. E.; Bigelow, G. E. Psychophar-
IR (thin film) ½max 3514, 3147, 2951, 2857, 1712, 1505, 1436,
1 13 macology 2001, 157, 151-162.
1372, 1318, 1232, 1144, 1070, 1028, 875, 797 cm-1; H and C
(7) Siebert, D. J. J. Ethnopharmacol. 1994, 43, 53-56.
NMR, see Tables 1 and 2; HRESIMS m/z 397.1610 (calcd for
(8) Ott, J. Curare 1995, 18, 103-129.
C21H26O6Na, 397.1622); TLC, see Table S1.
(9) Hanes, K. R. J. Clin. Psychopharmacol. 2001, 21, 634-635.
Acetylation of 4. Ac2O (250 µL, 2.6 mmol) was added to a (10) Ukai, M.; Suzuki, M.; Mamiya, T. J. Neural Transm. 2002, 109, 1221-
1225.
solution of 4 (11 mg, 25.4 µmol) in dry pyridine (2.5 mL) and
(11) Delvaux, M. Expert Opin. Invest. Drugs 2001, 10, 97-110.
stirred under argon. After 3.5 h, TLC (10% acetone/CH2Cl2)
(12) Gruber, J. W. Quantification of Salvinorin A from Tissues of Salvia
indicated completion. The reaction mixture was diluted with
divinorum (Epling & Játiva-M.). M.S. Thesis, Philadelphia College
ice water and extracted with Et2O (×3). The organic phase
of Pharmacy and Science, Philadelphia, 1997; pp 37, 46-53.
was washed with saturated NaHCO3, saturated CuSO4, water,
(13) Gordon, A. J.; Ford, R. A. The Chemist s Companion: a Handbook of
and brine and dried over MgSO4. Filtration and evaporation Practical Data, Techniques, and References; Wiley: New York, 1972;
pp 371-376.
in vacuo gave a cloudy resin (12 mg). FCC on silica gel (55%
(14) Koreeda, M.; Brown, L.; Valdés, L. J., III Chem. Lett. 1990, 2015-
Et2O/petrol) gave 3 as a clear resin (8.4 mg, 70%), identical
2018.
1 13
with the natural material by TLC, H and C NMR, IR, and
(15) PC Model v5.04; Serena Software: Bloomington, IN, 1994.
optical rotation: [R]16D +69.4° (c 0.40, CHCl3); natural mate-
(16) Esquivel, B.; Sánchez, A. A.; Aranda, E. In Phytochemicals and
rial: [R]16D +70.5° (c 0.55, CHCl3); lit.:4,20 [R]22D +49.3° (c 0.61, Phytopharmaceuticals; Shahidi, F., Ho, C. T., Eds.; AOCS Press:
Champaign, IL, 2000; pp 371-385.
CHCl3).
(17) Enriz, R. D.; Baldoni, H. A.; Zamora, M. A.; Jáuregui, E. A.; Sosa, M.
E.; Tonn, C. E.; Luco, J. M.; Gordaliza, M. J. Agric. Food Chem. 2000,
Acknowledgment. We thank the Melbourne University
48, 1384-1392.
Research Grants Scheme for financial support, and the Com-
(18) Simmonds, M. S. J.; Blaney, W. M.; Esquivel, B.; Rodriguez-Hahn,
monwealth Department of Education, Science and Training
L. Pestic. Sci. 1996, 47, 17-23.
for an Australian Postgraduate Award (T.M.) (19) Standard for the Uniform Scheduling of Drugs and Poisons; Com-
monwealth Department of Health and Ageing: Canberra, 2002; Vol.
17; pp 230, 232. See under the (incorrect) systematic name: 8-
Supporting Information Available: Table of TLC data for
13 METHOXYCARBONYL-4A,8A-DIMETHYL-6-ACETOXY-5-KETO-
compounds 1-6 and NMR spectra (1H, C, and DEPT) for compounds
3,4,4B,7,9,10,10A-SEPTAHYDRO-3-(4-FURANYL)-2,1-NAPHTHO-
3-6. This material is available free of charge via the Internet at http://
[4,3-E]PYRONE . Among many other errors, note the use of septa"
pubs.acs.org.
for hepta and the incorrect numbering, apparently based on phenan-
threne.
References and Notes
1
(20) Our spectroscopic data were otherwise in accord, except for the H
NMR assignments of H-19 and H-20, which should be reversed
(1) Valdés, L. J., III; Hatfield, G. M.; Koreeda, M.; Paul, A. G. Econ. Bot.
1987, 41, 283-291. (HMQC and HMBC data).
(2) Ortega, A.; Blount, J. F.; Manchand, P. S. J. Chem. Soc., Perkin Trans.
1 1982, 2505-2508. NP0205699
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