A facile method for the preparation of deuterium labeled salvinorin A: Synthesis of [2,2,2-2H3]-salvinorin a

Article abstract of DOI:10.1016/j.bmcl.2004.07.081

Salvinorin A is a novel hallucinogen isolated from the widely available leaves of Salvia divinorum. Based on its mechanism of action, salvinorin A has shown potential as a stimulant abuse therapeutic. However, there are no methods for the detection of salvinorin A or its metabolites in biological fluids. In order to begin developing salvinorin A as a potential therapeutic, an understanding of its metabolism is needed. Here, a straightforward synthesis of a deuterium labeled analog of salvinorin A and its utility as an internal standard for the detection of salvinorin A and its metabolites in biological fluids by LC-MS is described.

Full text of DOI:10.1016/j.bmcl.2004.07.081

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5099–5102
A facile method for the preparation of deuterium labeled
salvinorin A: synthesis of [2,2,2-2H3]-salvinorin A
Kevin Tidgewell,a Wayne W. Harding,a Mark Schmidt,b Kenneth G. Holden,c
Daryl J. Murryb and Thomas E. Prisinzanoa,*
aDivision of Medicinal and Natural Products Chemistry, College of Pharmacy, The University of Iowa, Iowa City, IA 52242, USA
bDivision of Clinical and Administrative Pharmacy, College of Pharmacy, The University of Iowa, Iowa City, IA 52242, USA
cHolden Laboratories, Carmel, CA 93923, USA
Received 13 July 2004; revised 28 July 2004; accepted 28 July 2004
Available online 21 August 2004
Abstract—Salvinorin A is a novel hallucinogen isolated from the widely available leaves of Salvia divinorum. Based on its mechanism
of action, salvinorin A has shown potential as a stimulant abuse therapeutic. However, there are no methods for the detection of
salvinorin A or its metabolites in biological fluids. In order to begin developing salvinorin A as a potential therapeutic, an under-
standing of its metabolism is needed. Here, a straightforward synthesis of a deuterium labeled analog of salvinorin A and its utility
as an internal standard for the detection of salvinorin A and its metabolites in biological fluids by LC–MS is described.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Salvinorin A (1a) (Fig. 1) is a neoclerodane diterpene
originally isolated from the leaves of Salvia divinorum
O
O
1
´
by Ortega et al. in 1982 and soon thereafter, Valdes
CH3
H
III et al.2 S. divinorum is a plant from the Sage family
that has been used in traditional spiritual practices by
the Mazatec Indians of Oaxaca, Mexico to produce
ÔmysticalÕ or hallucinogenic experiences.3 A smoked dose
of 200–500lg of salvinorin A, the presumed active
ingredient, produces profound hallucinations lasting
up to 1h.4,5 Thus, it has a potency that is similar to
the highly active synthetic hallucinogen LSD (2).
N
N
O
O
H
H
O
R
O
CO2Me
N
H
1a: R = Ac
1b: R = H
2
N
OH
Recreational use of the Mexican mint, S. divinorum, has
recently increased rapidly.6 Young adults and adoles-
cents have begun to smoke the leaves and leaf extracts
of the plants to induce powerful hallucinations. Recipes
for leaf extracts, elixirs, and tinctures may be found
posted on the Internet.7 Currently, Mexican youths have
begun to use the plant as a substitute for marijuana.8 In
addition, a large number of S. divinorum plants were
seized at a large-scale plantation in Switzerland, indicat-
ing this plantÕs increasing use as a recreational drug in
Europe.8 Due to the recent increase in the popularity
of the plant among both European and American teens,
O
O
N
O
N
CH3
O
HO
N
H3C
O
3
4
Figure 1. Structures of salvinorin A (1a), salvinorin B (1b), LSD (2),
U69,593 (3), and TRK-820 (4).
the DEA has recently placed it on the list of drugs of
concern.6
Keywords: Salvinorin A; Deuterium; Salvia divinorum; Hallucinogen.
*
Currently, S. divinorum is unregulated in most countries
and available throughout the world by purchasing it
Corresponding author. Tel.: +31 93356920; fax: +31 93358766;
e-mail: thomas-prisinzano@uiowa.edu
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.081

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K. Tidgewell et al. / Bioorg. Med. Chem. Lett. 14(2004) 5099–5102
over the Internet but is listed as a controlled substance
in Denmark and Australia. Obtaining S. divinorum is
easy in countries where it is unregulated and it is a
cheap, easy solution for many youths who wish to exper-
iment with drugs and perception altering substances. At
present, US laws for controlled substances do not ban
the use of S. divinorum or its active components. This
has resulted in various on-line botanical companies
advertising and selling S. divinorum as a legal alternative
to other regulated plant hallucinogens. As mentioned
earlier, these botanical companies also sell extracts, sal-
vinorin A enhanced extracts, and S. divinorum tinctures
and elixirs.7 It is predictable that its misuse will increase
rapidly.
O
O
O
O
H
H
O
O
D
O
H
H
D
R
O
b
O
O
D
O
CO2Me
1a: R = Ac
CO2Me
5
a
1b: R = H
Scheme 1. Reagents and conditions: (a) Na2CO3, MeOH, 77%; (b)
(Cd3CO2)2O, DMAP, CH2Cl2, 80%.
Curiously, salvinorin A does not act at the presumed
molecular target responsible for the actions of classical
hallucinogens, the serotonin 5-HT2A receptor.9–12 Rather,
studies have shown that salvinorin A is a potent and selec-
tive j opioid receptor agonist. Interestingly, salvinorin A
(1a) shares little structural similarity with other j agonists
such as U69,593 (3) and TRK-820 (4).
rin A compared to previously described methodology.
Efforts were then shifted to the preparation of salvinorin
B. Basic hydrolysis of the C-2 acetate using sodium car-
bonate in MeOH afforded salvinorin B in 77% yield.19
Curiously, these conditions do not result in the cleavage
of the C-18 methyl ester as noted by the presence of a
methyl singlet at 3.7ppm in the 1H NMR spectrum. This
is likely due to the C-18 position being more sterically
hindered than the C-2 position. More vigorous condi-
tions, such as heat and NaOH, are required for the
cleavage of this group. However, this leads to the open-
ing of the lactone ring, as well as epimerization of the 8-
position. Reacetylation of salvinorin B using d6-acetic
anhydride in the presence of a catalytic amount of
DMAP afforded [2,2,2-2H3]-salvinorin A (5) in 80%
yield.20
Selective j opioid receptor agonists have been explored
as analgesics with potential for reduced tolerance and
dependence. However, psychotomimesis, dysphoria,
and diuresis are associated with their therapeutic use.
Interestingly, a large body of evidence indicates that j
receptors may be involved in the modulation of some
abuse related effects of CNS stimulants.13–16 These find-
ings indicate that j opioid receptors may be involved in
the antagonism of some abuse related effects of cocaine,
offering a novel pharmacological approach to treat co-
caine abuse.
Analysis of isotopic purity of 5 was performed by tabu-
lating mass intensities from the peak top and subtracting
mass intensities from a nearby blank region of the chro-
matogram.21 Approximately 86% of the total ion inten-
sity is due to the d3 species, most of the remainder
corresponds to the d2 species (Fig. 2).
As part of our program to develop novel stimulant
abuse therapeutics, we sought to investigate the psycho-
pharmacology of salvinorin A. Presently, the identity of
the metabolites of salvinorin A are unknown. It has
been suggested that salvinorin B (1b) (Fig. 1) is a meta-
bolite of salvinorin A.5 However, this has not been
shown definitively either in vitro or in vivo. In order
to better study the metabolism of salvinorin A in vivo,
the development of a validated liquid chromato-
graphy–mass spectrum (LC–MS) method for the deter-
mination of salvinorin A and its potential metabolites,
such as salvinorin B, in biological fluids was needed.
As is generally agreed, stable isotopically labeled ana-
logs with the same molecular structure of a compound
are the best internal standards for the LC–MS assay.
To this end, we describe the synthesis of an analog that
is specifically labeled with deuterium starting from sal-
vinorin A (Scheme 1).
A calibration curve for salvinorin A was constructed by
spiking eight 0.25mL aliquots of human plasma with
100ng of 5 and 1, 5, 10, 25, 50, 100, 250, or 500ng sal-
vinorin A, respectively. Controls at 40, 200, and 400ng,
as well as a blank and a blank 0 were prepared in the
same manner. All samples were extracted using Waters
21
Oasis SPEcartridges.
Salvinorins were eluted with
75% CH2Cl2/CH3CN. After evaporation, samples were
reconstituted in 0.1mL of 75% CH3CN/H2O. Analysis
was done with a Shimadzu LCMS-2010A with an APCI
100000
80000
60000
40000
Rather than begin a lengthy total synthesis of salvinorin
A, we initially focused on extracting salvinorin A from
the dried leaves of S. divinorum. Commercially available
leaves were extracted as previously described1,2,17 and
afforded salvinorin A. However, we then set out to fur-
ther improve the bioyield of salvinorin A. Modification
of extraction procedure18 resulted in the isolation of
7.5g of salvinorin A from 1.5kg of dried leaves. This
process has resulted in an improved bioyield of salvino-
20000
0
431.3
432.3
433.3
434.3
M/Z Ratio
Figure 2. Analysis of isotopic purity of 5.

K. Tidgewell et al. / Bioorg. Med. Chem. Lett. 14(2004) 5099–5102
7. Valdes L. J., III. Personal communication, 2003.
5101
8
7
6
5
4
3
2
1
0
8. Giroud, C.; Felber, F.; Augsburger, M.; Horisberger, B.;
Rivier, L.; Mangin, P. Forensic Sci. Int. 2000, 112, 143–
150.
y = 0.0133x - 0.0107
r2 = 0.999
9. Glennon, R. A.; Titeler, M.; McKenney, J. D. Life Sci.
1984, 35, 2505–2511.
10. Titeler, M.; Lyon, R. A.; Glennon, R. A. Psychopharma-
cology 1988, 94, 213–216.
11. Egan, C. T.; Herrick-Davis, K.; Miller, K.; Glennon,
R. A.; Teitler, M. Psychopharmacology 1998, 136, 409–
414.
12. Nichols, D. E. Pharmacol. Ther. 2004, 101, 131–
181.
13. Mello, N. K.; Negus, S. S. Ann. N Y Acad. Sci. 2000, 909,
104–132.
0
100
200
300
400
500
600
14. Shippenberg, T. S.; Chefer, V. I.; Zapata, A.; Heidbreder,
C. A. Ann. N Y Acad. Sci. 2001, 937, 50–73.
15. Schenk, S.; Partridge, B.; Shippenberg, T. S. Psychophar-
macology 1999, 144, 339–346.
ng of Salvinorin A added
Figure 3. Calibration curve for 1a in human plasma.
16. Schenk, S.; Partridge, B.; Shippenberg, T. S. Psychophar-
macology 2000, 151, 85–90.
17. Munro, T. A.; Rizzacasa, M. A. J. Nat. Prod. 2003, 66,
703–705.
probe operating in negative ion mode. [MÀ1] ions were
obtained and used for quantitation. Separation was
done using a Phenomenex Synergi Polar-RP column
(150mm · 2mm, 4lm), eluted isocratically with 50%
CH3CN/4mM NH4OAc adjusted to pH7.2. The cali-
bration curve was linear with a coefficient (r2) value of
0.999 (Fig. 3).
18. Dried Salvia divinorum leaves (1.5kg), obtained commer-
cially from Ethnogens.com, were ground to a fine powder
and percolated with acetone (5 · 4L). The acetone extract
was concentrated under reduced pressure to afford a crude
green gum (93g), which was subjected to column chro-
matography on silica gel with elution in n-hexanes
containing increasing amounts EtOAc. Fractions eluting
in 20% n-hexanes/EtOAc contained salvinorin A (TLC)
and other minor diterpenes and some pigmented material.
These fractions were pooled and concentrated in vacuo to
give a green gum (24g). A mixture of the crude green gum,
acetic anhydride (50mL, 530mmol) and DMAP (0.2g) in
CH2Cl2 (250mL) was stirred at rt overnight. The CH2Cl2
solution was washed sequentially with 1N HCl (2 ·
500mL), 2N NaOH (100mL), and H2O (2 · 100mL).
The CH2Cl2 solution was dried (Na2SO4) and the solvent
was removed under reduced pressure to afford a yellow-
green gum (23g). The resulting gum was subjected to
column chromatography on silica gel. Elution was per-
formed in 1000mL aliquots of a mixture of n-hexanes/
EtOAc in increments of 10% EtOAc with the final elution
in neat EtOAc. Fractions eluting in 30% n-hexanes/EtOAc
and subsequent fractions were pooled and the solvent
was removed under reduced pressure affording salvinorin
A (7.5g, 0.5%) as a green powder, mp 235–238ꢁC
(lit.1,2 240–242ꢁC).
19. A mixture of 1a (3.5g, 8.0mmol) and Na2CO3 (3.4g,
32.2mmol) in absolute MeOH (150mL) was stirred at
room temperature for 4h. The solvent was removed under
reduced pressure and CH2Cl2 (500mL) was added to the
crude residue. The organic extract was washed successively
with 2N HCl (50mL) and saturated NaCl (50mL) and
dried (Na2SO4). The solvent was removed under reduced
pressure and MeOH (100mL) was added to the residue.
The resulting solid was collected by filtration and dried to
afford 2.4g (77%) of 1b as a white solid, mp 211–214ꢁC
(lit.2 213–216ꢁC).
In conclusion, an improved extraction method for the
isolation of salvinorin A from the leaves of S. divinorum
is described. Synthesis of [2,2,2-2H3]-salvinorin A (5)
was achieved in two steps from salvinorin A. An LC–
MS method employing deuterium-labeled 5 was also
developed and found to be suitable for the detection
of salvinorin A and its metabolites in biological fluids.
This method allows us and others to better monitor
the concentration of salvinorin A in biological fluids.
Additional studies characterizing the structure–activity
relationships and metabolism of salvinorin A are cur-
rently under investigation and will be presented in due
course.
Acknowledgements
The authors thank the College of Pharmacy and the Bio-
logical Sciences Funding Program of the University of
Iowa for financial support.
References and notes
1. Ortega, A.; Blount, J. F.; Manchand, P. S. J. Chem. Soc.,
Perkin Trans. 1 1982, 2505–2508.
2. Valdes, L. J., III; Butler, W. M.; Hatfield, G. M.; Paul,
A. G.; Koreeda, M. J. Org. Chem. 1984, 49, 4716–
4720.
20. A solution of 1b (0.1g, 0.3mmol), d6-acetic anhydride
(0.1g, 1.3mmol) and a catalytic amount of DMAP in CH2
Cl2 (20mL) was stirred at room temperature for 2h.
Absolute MeOH (15mL) was added and the solvent was
removed under reduced pressure. CH2Cl2 (25mL) was
added to the residue and the solution was washed with
10% HCl (3 · 20mL) and saturated NaCl (3 · 20mL) and
dried (Na2SO4). Removal of the solvent under reduced
pressure afforded 0.09g (80%) of 5 as a white solid, mp
´
3. Valdes, L. J., III; Diaz, J. L.; Paul, A. G. J. Ethnophar-
macol. 1983, 7, 287–312.
4. Siebert, D. J. J. Ethnopharmacol. 1994, 43, 53–56.
5. Valdes, L. J., III; Chang, H. M.; Visger, D. C.; Koreeda,
M. Org. Lett. 2001, 3, 3935–3937.
6. National Drug Intelligence Center. Salvia divinorum. In
Information Bulletin; US Department of Justice: Johns-
town PA, 2003.

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1
237–240ꢁC: H NMR (300MHz, CDCl3) d 1.12 (s, 3H),
1.46 (s, 3H), 1.80 (m, 1H), 2.07 (dd, J = 3.0, 11.6Hz, 1H),
2.31 (m, 2H), 2.51 (dd, J = 5.4, 13.2Hz, 1H), 2.75 (dd,
J = 6.3, 10.2Hz, 1H), 3.73 (s, 3H), 5.15 (dd, J = 9.9,
9.9Hz, 1H), 5.53 (dd, J = 4.8, 12.0Hz, 1H), 6.37 (dd,
J = 0.9, 1.5Hz, 1H), 7.39 (dd, J = 1.5, 1.8Hz, 1H), 7.41
(dd, J = 0.9, 1.5Hz, 1H).
21. Schmidt, M. S.; Tidgewell, K; Harding, W. W.; Butelman,
E. R.; Prisinzano, T.; Murry, D. J. J. Chromatogr. B.,
submitted for publication.