Synthesis and biological evaluation of C-12 triazole and oxadiazole analogs of salvinorin A

Article abstract of DOI:10.1016/j.bmcl.2009.01.078

Salvinorin A (1), the main active ingredient of Salvia divinorum, is a potent and selective κ-opioid receptor (KOPR) agonist. A series of C-12 triazole analogs and the oxadiazole (4) analog of 1 are synthesized and screened for binding affinity at κ, μ (M

Full text of DOI:10.1016/j.bmcl.2009.01.078

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1301–1304
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of C-12 triazole and oxadiazole analogs
of salvinorin A
Lu Yang a, Wei Xu b, Feng Chen a, Lee-Yuan Liu-Chen b, Zhongze Ma a, David Y. W. Lee a,
*
a Bio-Organic and Natural Products Laboratory, McLean Hospital, Harvard Medical School, 115 Mill Street, Belmont, MA 02478, USA
b Department of Pharmacology and Center for Substance Abuse Research, School of Medicine, Temple University, 3420 N. Broad Street, Philadelphia, PA 19140, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Salvinorin A (1), the main active ingredient of Salvia divinorum, is a potent and selective j-opioid receptor
(KOPR) agonist. A series of C-12 triazole analogs and the oxadiazole (4) analog of 1 are synthesized and
Received 17 December 2008
Revised 21 January 2009
Accepted 22 January 2009
Available online 9 February 2009
screened for binding affinity at (MOPR), or d (DOPR). Surprisingly, all triazole analogs have shown neg-
j, l
ligible binding affinity at opioid receptors and the oxadiazole 4, a reported MOPR and KOPR antagonist,
exhibits very low affinities to opioid receptors and no antagonism in our binding assays. These results sug-
gest that electronic factors that may affect either the electron density of hydrogen bond acceptor at C-12 or
hydrophobic interactions between C-12 moiety and KOPR are critical to C-12 analog’s affinity for KOPR.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Salvinorin A
Triazole
Antagonist
Salvinorin
A
(1),
a
naturally occurring hallucinogen, has
drugs are withdrawn, the upregulated KOPR system becomes un-
masked and the dysphoric states are manifested, which drives
the patient’s craving for the drug to normalize the mood.6 There-
fore, it is reasonable to conclude that selective KOPR antagonists
can be useful relapse prevention agents for drug abuse patients.
They are also potential anxiolytics and antidepressants. Moreover,
small-molecule selective KOPR antagonists are invaluable tools in
the understanding of pharmacological functions of KOPR.7
attracted much attention due to its potent KOPR agonist activity.
It represents the first non-alkaloid opioid subtype-selective drug.1
The unique structural and biological features of 1 make it an attrac-
tive probe for exploring opioid pharmacology. It can also function
as a potential template for development of novel psychotherapeu-
tic drugs.
O
O
N
O
12
N
O
O
8
AcO
O
2
O
O
O
O
H
H
RO
AcO
O
O
Salvinorin A (1)
O
OMe
2 R=
3 R=
O
O
4
O
OMe
O
OMe
Prior structure–activity relationship studies of 1 by our group
and others have largely focused on its A ring.2 These studies have
led to the synthesis of methoxymethyl ether (2)3 and ethoxy-
methyl ether (3).4 Currently, 2 and 3 are the only salvinorin deriv-
atives with a higher binding affinity and potency than 1 at KOPR.
Compound 2 also exhibits longer duration of action than 1 in vivo.5
Interestingly, prolonged use of drugs such as cocaine and amphet-
amine has been found to upregulate the KOPR system. When the
O
O
OH
O
O
H
AcO
O
5
O
OMe
* Corresponding author. Tel.: +1 617 855 2038; fax: +1 617 855 2040.
E-mail address: DLee@mclean.harvard.edu (D.Y.W. Lee).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.078

1302
L. Yang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1301–1304
Recent studies by Prisinzano et al.8 have shown that the furan
opposite is observed for oxygen content.12 Here, we report the
synthesis and biological evaluation of a series of C-12 triazole ana-
logs of 1. To provide a direct in vitro comparison of the triazoles
with the known KOPR antagonist, 4 is synthesized by following
the reported procedure and isolated with negligible amount of C-
8 epimer.8b
ring modified analogs of 1, oxadiazole (4) and salvidivin A (5), ex-
hibit antagonist activities at MOPR and KOPR. The reduced furan
ring stereochemistry of 5 may also affect its efficacy.8 According
to Roth’s binding model of 1 at KOPR, the furan oxygen may act
as a hydrogen bond acceptor to tyrosine moieties of the KOPR.9 It
is then conceivable that either a substitution of the furan oxygen
with other hetero-atoms or a modification of the stereochemistry
of the oxidized furan ring may cause a change in the binding
behavior of 1 at the KOPR. This could then lead to changes in 1’s
efficacy at the receptors leading to alterations in its subsequent
coupling to G proteins. Therefore, we feel that optimization of
the hydrogen bond acceptor properties of the five-membered ring
in 1, especially those heterocycles that may interact with residues
in the binding pocket of KOPR, might be a viable way to find novel
selective KOPR antagonists. To this end, a series of C-12 modified
analogs of 1 ought to be synthesized in particular the five-mem-
bered heterocycles that resemble the furan ring in 1 would have
priority.
As evidenced, 1,2,3-triazole pharmacophores have displayed a
broad spectrum of biological activities associated with antibacte-
rial, antifungal and herbicidal, antiallergic, and anti-HIV.10 We
were also encouraged by the advances in click chemistry, particu-
larly the copper (I)-catalyzed 1,3-dipolar cycloaddition of organic
azides and alkynes which allows efficient construction of the tria-
zole framework under mild conditions.11 Furthermore, replacing of
the furan ring of 1 with a triazole ring would increase its drug-like
property if considering that the average nitrogen content per mol-
ecule in natural products is lower than in drug molecules, and the
Salvinorin A is isolated from Salvia divinorum as previously de-
scribed.13 It is degraded by ruthenium-catalyzed oxidation to acid
(6),14 which provides an ideal template for further modification at
C-12 position (Scheme 1). Based on our previous work in chemose-
lective reduction of the C-4 acid to alcohol,15 6 is reduced with bor-
ane to primary alcohol (7) at 55 °C. Notably, forcing conditions, for
example, excess borane or higher temperature, have no effect in
improving the yield of 7 or leading to C-8 epimerization that nor-
mally observed under basic conditions. Attempts to oxidize 7 with
Swern or PCC conditions form a complex mixture of products.
Alternatively, it is treated with Dess–Martin periodinane at 0 °C
to provide the desired aldehyde (8) in good yield. We find quench-
ing the reaction with sodium bicarbonate significantly lowers the
yield of 8, presumably due to the hydrolysis of C-2 acetate. To elab-
orate the synthesis of the five-membered triazole analogs, 8 is con-
verted to terminal alkyne (9) by Ohira–Bestmann alkynylation,16
which sets the stage for 1,3-dipolar cycloaddition click reaction.
Interestingly, basic Ohira–Bestmann condition is well tolerated in
the homologation and no C-8 epi alkyne or C-2 deacetylation prod-
uct is isolated. Subsequent coupling of 9 with various azides under
the copper (I)-catalyzed ‘click reaction’ condition affords 1,4-
disubstituted-1,2,3-triazoles (10), (11), and (12) in high yields.
N-unsubstituted 1,2,3-triazole heterocycles are found to have
O
O
OH
O
CH2OH
CHO
O
O
O
O
O
H
O
O
H
H
H
AcO
AcO
AcO
AcO
a
O
b
c
O
O
O
8
1
7
6
O
OMe
O
OMe
O
OMe
N
O
OMe
R
N
N
O
O
O
O
H
H
d
AcO
e
AcO
O
O
R= Me (10)
Et (11)
9
O
OMe
f
O
OMe
Bn (12)
O
N
N
N
NH
N
N
N
N
O
O
NH
O
O
O
O
O
O
H
H
H
g
AcO
AcO
AcO
O
O
13
14
15
O
OMe
O
OMe
O
OMe
Scheme 1. Reagents and conditions: (a) Ref. 14, 65%; (b) BH3, THF, 55 °C, 3 h, 82%; (c) DMP, CH2Cl2, 0 °C, 2 h, 43%; (d) Ohira–Bestmann reagent, 61%; (e) RN3,CuSO4 (cat.), Na
asorbate, t-BuOH/H2O (1:1), 88%; (f) azidomethyl pivalate, CuSO4 (cat.), Na asorbate, t-BuOH/H2O (1:1), 88%; (g) i—NaOH(2.2 equiv) MeOH/H2O (1:1); ii—1 M HCl (2.2 equiv);
iii—Ac2O, Pyr, 54% for 3 steps 14:15 (4:6).

L. Yang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1301–1304
1303
significant nevertheless different biological properties from their
100
75
50
25
0
substituted analogs.17 This prompts us to synthesize the N-unsub-
stituted triazole analog of 1. Compound 9 undergoes facile cycload-
dition with azidomethyl pivalate to give the pivalyl triazole (13).
Treatment of 13 with 1 N NaOH at room temperature removes
both N-pivalyl group and the C-2 acetyl group18 and also leads to
C-8 epimerization. Acetylation of the epimeric mixture followed
by preparative TLC separation furnishes two epimeric N-unsubsti-
tuted triazoles (14) and (15) with a ratio of 4:6.
Compound 4
U50,488H
Compounds 4 and 10–15 are evaluated for their affinities for
MOPR, DOPR, and KOPR by competitive inhibition of [3H]diprenor-
phine (ꢀ0.3 nM) binding to membranes prepared from Chinese
hamster ovary (CHO) cell lines stably transfected with rat MOPR,
FLAG-mouse DOPR, and human KOPR. The non-selective opioid
receptor agonist, etorphine, is used as the reference compound in
the binding assays. The binding data for compounds 10–15 is listed
in Table 1. Surprisingly, none of the C-12 triazole analogs of 1
-25
10-7
10-6
10-5
10-4
Concentration (M)
Figure 1. Effect of the compound 4 on [3H]diprenorphine binding to the mem-
branes prepared from Chinese hamster ovary cells (CHO) stably transfected with
the human KOPR (hKOPR).
shows significant binding affinities for l, d, and j-opioid receptors
(Ki > 1000 nM). Roth’s binding model of salvinorin A-KOPR three
point interactions suggests that KOPR interacts with tyrosines
320 or 119 via hydrogen bonding with the C-12 furanyl substituent
of 1 and the hydrogen bond between furan oxygen and tyrosines is
weaker than a normal hydrogen bond due to the delocalization of
the lone-pair electrons of the oxygen atom in the furan ring.9 De-
spite the triazole moiety of analogs 10–15 offers two nitrogen
sp2 lone-pairs for hydrogen bonding, inductive effects of the three
vincinal nitrogens decrease the electron density of the lone-pairs
and make triazole moiety a much weaker hydrogen bond acceptor
than furan moiety, which might explain the loss of binding affinity
at KOPR for compounds 10–15. A more recent model by Kane et
al.19 suggests the tyrosines from TM II and TM VII stabilize salvino-
rin A through hydrophobic effects, either by pi-stacking or other
electronic effects. In this case, substitution of the furan with tria-
zole would not significantly affect pi-stacking, however, the intro-
duction of the triazole group changes the electronics of the
environment from neutral to basic, which does not favor hydro-
phobic interaction in the KOPR binding pocket that this group
seems to occupy. For N-unsubstituted triazoles 14 and 15, rapid
NH tautomerization could be an additional unfavorable electronic
factor for binding. Interestingly, the oxadiazole analog 4, reported
to have antagonist activities at MOPR and KOPR, shows negligible
affinities for MOPR, DOPR, and KOPR in our binding assays
properties of the 4, we have used [3H]diprenorphine and 50 mM
Tris–HCl buffer containing 1 mM EGTA (pH 7.4) for our binding
assay.
To examine the possibility that EGTA in our buffer may induce
protonation of the oxadiazole group of 4 by its acidic hydrogens,
we have evaluated inhibition of [3H]diprenorphine binding to
KOPR by 4 in Tris buffer (50 mM, pH 7.4) in the presence or ab-
sence of EGTA. Compound 4 at 3 lM induces the same extent of
inhibition (about 50%, Ki > 1000 nM) with and without EGTA, indi-
cating that EGTA does not affect affinity of 4 for the KOPR.
As [3H]diprenorphine is an antagonist, we have also evaluated
inhibition of [3H]DAMGO (2 nM), an agonist, binding to MOPR by
4 in Tris buffer (50 mM, pH 7.4). Compound 4 at 3
lM induces only
8% inhibition of [3H]DAMGO binding, compared with 99% inhibi-
tion by the reference compound etorphine (1 lM), indicating that
4 does not bind to MOPR (Ki > 1000 nM).
Although Simpson et al.8a reported 4 to be a weak KOPR antag-
onist in antagonizing agonist-induced increase of [35S]GTP
ing as (Ke = 360 nM), they did not examine the effect of 4 alone.
Here we find that compound increases KOPR-mediated
35S]GTP
S binding in a dose-dependent manner with an EC50 va-
lue of >1 M (Fig. 2). Its maximal response is similar to that of
cS bind-
4
[
c
l
U50,488H, a prototypical KOPR full agonist. These results indicate
that compound 4 is a full agonist with very weak potency, but
not an antagonist, of KOPR. It is not surprising 4 has low affinities
at KOPR given that oxadiazole group is weakly basic and the elec-
tron density of the three sp2 lone-pairs are delocalized due to
inductive effect, which is in keeping with that found in triazole
analogs.
(Ki > 1000 nM for all three). Compound 4 at 3 lM only induces
about 50% inhibition on [3H]diprenorphine binding to the hKOPR
(Fig. 1).
The discrepancy may result from differences in radioligand and
binding buffer used. While Simpson et al.8a used [125I] IOXY in
50 mM Tris–HCl buffer, pH 7.4, containing a protease inhibitor
cocktail [bacitracin (100
lg/mL), bestatin (10
lg/mL), leupeptin
(4 lg/mL) and chymostatin (2
lg/mL) in characterizing binding
400
U50,488H
Compound 4
Table 1
300
Ki values (in nM) of compounds 4 and 10–15 binding to the rMOPR, mDOPR, and
hKOPR stably expressed in CHO cellsa
200
100
Compound
rMOPR
mDOPR
hKOPR
4
10
11
12
13
14
15
Etorphine
>1000
>1000
>1000
>1000
>1000
>1000
>1000
0.14 0.02
>1000
>1000
>1000
>1000
>1000
>1000
>1000
2.9 0.6
>1000
>1000
>1000
>1000
>1000
>1000
>1000
0.5 0.05
0
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4
Compound (M)
a
Competitive inhibition by 4 and 10–15 of [3H]diprenorphine (ꢀ0.3 nM) binding
to MOPR, DOPR, and KOPR is conducted and their Ki values are determined by the
program Prism. Etorphine is used as the reference compound.
Figure 2. Agonist-induced increase in [35S]GTP
cS binding. Reference compound
U50,488 is a full agonist of KOPR. Compound 4 showed to possess KOPR agonist
properties with much weaker potency and similar efficacy as U50,488H.

1304
L. Yang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1301–1304
4. Munro, T. A.; Duncan, K. K.; Xu, W.; Wang, Y.; Liu-Chen, L. Y.; Carlezon, W. A.;
In conclusion, C-12 triazole analogs 10–15 have been synthe-
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pharmacophore to the salvinorin A template by ‘click chemistry’.
Binding assay results indicate that replacement of furan with the
triazoles result in loss of affinity at KOPR. Similar binding affinity
result has been obtained on oxadiazole analog 4. These results sug-
gest that either electron density of the hetero-atom acting as
hydrogen bond acceptor on C-12 or hydrophobic interactions be-
tween C-12 moiety and KOPR is critical to salvinorin A templated
analog’s binding affinity at KOPR. Efforts in optimizing the hydro-
gen bond accepting capability and hydrophobic properties of C-12
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Acknowledgments
We thank Dr. Yongxuan Su of University of California, San Diego
for HR-MS measurements of all samples and National Institutes of
Health for financial support (RO1DA019688).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.01.078.
13. Munro, T. A.; Rizzacasa, M. A. J. Nat. Prod. 2003, 66, 703.
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