Synthesis and in vitro pharmacological evaluation of salvinorin A analogues modified at C(2)

Article abstract of DOI:10.1016/j.bmcl.2005.03.113

Salvinorin A is the only known non-nitrogenous and specific κ-opioid agonist. A series of salvinorin A derivatives were prepared and tested for in vitro activity at the κ-opioid receptor. Unsubstituted carbamate 9 was a potent κ-agonist (EC₅₀ = 6.2 nM) and should be more stable than salvinorin A toward metabolic transformations. Compound 10, containing an N-methyl carbamate at C(2), showed partial agonist activity with 81% efficacy when compared with the full agonist U50,488H. No antagonist ligands were observed.

Full text of DOI:10.1016/j.bmcl.2005.03.113

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2761–2765
Synthesis and in vitro pharmacological evaluation of salvinorin A
analogues modified at C(2)
a
C e´ cile B e´ guin, Michele R. Richards, Yulin Wang, Yong Chen, Lee-Yuan Liu-Chen,
a
b
b
b
c
Zhongze Ma, David Y. W. Lee, William A. Carlezon, Jr. and Bruce M. Cohen
c
d
a,
*
a
Molecular Pharmacology Laboratory, McLean Hospital, 115 Mill St., Belmont, MA 02478, USA
Department of Pharmacology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140, USA
b
c
Bio-Organic and Natural Products Research Laboratory, McLean Hospital, 115 Mill St., Belmont, MA 02478, USA
d
Behavioral Genetics Laboratory, McLean Hospital, 115 Mill St., Belmont, MA 02478, USA
Received 4 January 2005; revised 29 March 2005; accepted 30 March 2005
Abstract—Salvinorin A is the only known non-nitrogenous and specific j-opioid agonist. A series of salvinorin A derivatives were
prepared and tested for in vitro activity at the j-opioid receptor. Unsubstituted carbamate 9 was a potent j-agonist (EC50 = 6.2 nM)
and should be more stable than salvinorin A toward metabolic transformations. Compound 10, containing an N-methyl carbamate
at C(2), showed partial agonist activity with 81% efficacy when compared with the full agonist U50,488H. No antagonist ligands
were observed.
Ó 2005 Elsevier Ltd. All rights reserved.
6
j-Opioid receptor agonists appear to affect mood in hu-
tive and potent agonist at the j-receptor. Salvinorin A
makes an attractive lead compound for several reasons.
1
,2
man subjects, and recent studies indicate that mole-
cules that selectively bind to j-opioid receptors have
behavioral effects in tests that may reflect mood states
It is an active j-agonist (K = 1.3 nM) that seems to
i
7
cross the blood–brain barrier. A recent study suggests
8,9
3
,4
in rodents. In particular, Carlezon and co-workers
demonstrated that the selective j-agonist U69,593 pro-
duces depressive-like effects in behavioral models often
used in rats to study depression. In contrast, the selec-
tive j-antagonists norBNI and ANTI produce antide-
that salvinorin A has low toxicity in rodents; simi-
larly, both salvinorin A and its parent plant, Salvia
1
0
divinorum, seem to be non-toxic and to induce mood
9
effects in humans.
,11
3
,4
pressant-like effects in these models.
We are
interested in designing and testing novel agents that
selectively bind to j-opioid receptors in order to explore
whether selective j-ligands may be useful to treat symp-
toms of depression and bipolar disorder.
Classes of compounds known to bind to the j-receptor
include morphinans, benzomorphans, 4-phenyl-piper-
idines, and arylacetamides. The structure–activity rela-
tionships (SARs) for these compounds have been well
5
characterized. Roth and co-workers recently found that
the neoclerodane diterpene, salvinorin A (1), was a selec-
Little is known regarding the SAR of salvinorin A toward
the j-receptor. In HallÕs open field tests conducted when
salvinorin A was first isolated and characterized, the
C(2) acetate appeared to be important for activity:
salvinorin A is behaviorally active, while the deacetylated
Keywords: Salvinorin; Kappa-opioid receptor; Structure–activity rela-
tionship; Agonist; Partial agonist.
*
Corresponding author. Tel.: +1 617 855 3227; fax: +1 617 855
670; e-mail: cohenb@mcleanpo.Mclean.org
3
0
960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.113

2762
C. B e´ guin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2761–2765
1
2
salvinorin B is not. Another recent report indicates that
the inactive salvinorin B is the major metabolite of salvi-
epimer, it could be separated from 2 by chromatography
or crystallization.
1
3
norin A, which might explain salvinorin AÕs short dura-
tion of action. Chavkin et al. made a series of esters and
The C(2) esters (3–8) were synthesized using standard
acylation procedures (Scheme 1). Salvinorin B (2) was
treated with propionyl or butyryl chloride in the pres-
ence of pyridine to give 3 and 4. In order to test the roles
charge and hydrogen bond donors may play in binding,
several small amino acids were added at C(2) as well. Es-
ters 5 and 6 were formed from 2 and N-acetylglycine or
N,N-dimethylglycine, respectively, using dicyclo-
hexylcarbodiimide (DCC) as the coupling agent, and 4-
(dimethylamino)pyridine (DMAP) as a catalyst. Ester
6 was then precipitated from CH Cl as the hydrochlo-
1
4
carbonates at the C(2) site. The binding data suggest
that the size of the C(2) substituent has a significant effect
on binding to the j-receptor, with smaller substituents
increasing binding affinity. We further explored the role
the C(2) position plays in binding to j-receptors. We pre-
pared a series of compounds to determine the effect of var-
ious functional groups in vitro with two goals: first, to
identify candidate compounds with increased stability
over salvinorin A in vivo, and second, to investigate if it
is possible to change the biological profile of a salvinorin
A-based ligand from full agonist at j-receptors to partial
agonist or antagonist. We introduced various ester (3–8,
Fig. 1), carbamate (9–11), amine (12–14), and ether (15–
2
2
ride salt 7. Ester 8 was prepared in two steps: 2 was
0
treated with 9 -fluorenylmethoxycarbonyl-N-(L)-alanine
in the presence of DMAP and 1-(3-dimethylaminoprop-
yl)-3-ethylcarbodiimide hydrochloride (EDCI), then was
deprotected with piperidine. The glycine and sarcosine
analogues were synthesized using a similar sequence,
but the free amines were unstable.
2
0) substituents at this site and evaluated their in vitro
j-receptor activities.
Salvinorin A (1) was isolated from dry S. divinorum
leaves and purified using a modification of procedures
described previously.
1
2,15
Compound 1 was then con-
Carbamates 10 and 11 were prepared by reacting 2 with
methyl isocyanate and ethyl isocyanate, respectively
(Scheme 2). For the preparation of 9, we used the car-
verted to salvinorin B (2), which served as the starting
material for the preparation of the C(2)-modified
salvinorins 3–20. Methanolysis of 1 to 2 was best
accomplished using a modification of the published
procedure. During the course of the reaction, forma-
tion of a minor side product was consistently observed.
Careful analysis of the 1D and 2D NMR data indicated
that epimerization occurred at C(8), which is consistent
1
8
bamoylation procedure reported by Kocovsky: 2 was
first treated with trichloroacetyl isocyanate followed by
in situ hydrolysis of the trichloroacetyl moiety to give
9 as a stable white solid in 91% yield from 2.
1
6
Scheme 3 describes the two-step synthesis of secondary
and tertiary amines 12–14 from 2. Accordingly, treat-
ment of 2 with trifluoromethanesulfonic anhydride
provided an activated triflate intermediate, which was
displaced by methylamine, ethylamine, or dimethyl-
amine to form 12, 13, and 14, respectively. The structure
1
7
with earlier observations reported by Koreeda et al.
While we did not eliminate the formation of the C(8)
Scheme 1. Syntheses of C(2) esters. Reagents and conditions: (a)
appropriate acyl chlorides, pyridine, 36–61%; (b) appropriate amino
acids, DCC or EDCI, DMAP, CH
2
Cl
piperidine for the preparation of 8), 51–91%; (c) HCl, Et
2
(followed by deprotection with
O, 58%.
Figure 1. Salvinorin derivatives selected for study.
2

C. B e´ guin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2761–2765
2763
pounds 15–20 in good yields. Our attempts to prepare
branched alkyl ethers failed under these reaction
conditions.
1
13
Spectral data ( H NMR, C NMR, high-resolution
mass) consistent with the proposed structures were
obtained for all the compounds prepared in this study.
The binding affinities of 1–20 were determined by com-
petitive inhibition of [ H]-diprenorphine binding to
membranes prepared from Chinese hamster ovary cells
3
(
CHO-hKOR) stably transfected with the human j-opi-
19
oid receptor (hKOR). The potencies and efficacies of
–20 on hKOR were determined by their abilities to
1
3
5
regulate [ S]GTPcS binding to membranes of CHO-
2
0
hKOR cells. The selective full j-agonist, U50,488H,
was used as a reference compound. The in vitro pharma-
cological data for 1–20 are listed in Table 1.
When the seven C(2) esters (1, 3–8) were evaluated in in
vitro studies, we observed a decrease in affinity as we in-
creased the size of the alkyl ester substituent. These data
are consistent with the earlier observations of Chavkin
Scheme 2. Syntheses of C(2) carbamates. Reagents and conditions: (a)
CCl C(O)NCO, CH Cl ; (b) Al , 91%, over two steps; (c) appro-
priate isocyanates, DMAP, pyridine, 53–68%.
3
2
2
2 3
O
1
4
et al. Accordingly, C(2) n-propyl (3) and C(2) n-butyl
4) esters showed similar affinity to the j-receptor in
(
binding (K values (nM): 3, 7.2 ± 0.5; 4, 4.9 ± 0.6) and
i
efficacy (EC50 values (nM): 3, 20.4 ± 3.4; 4, 9.9 ± 0.6),
with an average 4-fold lower affinity than salvinorin A
(K = 1.3 ± 0.5 nM; EC = 4.5 ± 1.2 nM). Introduction
i 50
of various small amino acids at C(2) (5–8) led to a com-
plete loss of binding activity (K >10 lM), with the nat-
i
ure of the amino acid having no effect on the in vitro
binding properties at j-receptors.
In the C(2) carbamate substituted compounds (9–11),
we observed an important effect on in vitro pharmaco-
logical properties as we varied the C(2) moiety. Unsub-
stituted carbamate 9 was a potent full agonist
Scheme 3. Syntheses of C(2) amines. Reagents and conditions: (a)
1
2
(
CF
3 2 2 2 2
SO ) O, pyridine, CH Cl ; (b) R R NH, THF, 26–29%, over two
steps.
(
Its K and EC were comparable with salvinorin A
K = 3.2 ± 0.2 nM; EC = 6.2 ± 1.4 nM, 99% efficacy).
i 50
of 12 was determined by 1D NOESY experiments,
which confirmed that the nucleophilic substitution pro-
ceeded with inversion of configuration at C(2).
i
50
and U50,488H (K = 1.4 ± 0.3 nM; EC = 4.5 ± 1.2 nM,
i
50
100% efficacy). Introduction of an N-methylcarbamate
substituent at C(2) led to decreased affinity for the
j-receptor: thus carbamate 10 had lower affinity than
Incorporation of ethers at C(2) was accomplished accord-
ing to Scheme 4. Treatment of 2 with the appropriate
alkyl halides in the presence of Ag O provided com-
2
9
and 1 in the j-receptor binding assay (26- and 64-fold,
respectively). Interestingly, 10 displayed partial agonist
3
5
properties in the [ S]GTPcS binding assay (81% efficacy
when compared with U50,488H). Increasing the size of
the C(2) carbamate substituent by one additional carbon
led to a significant loss of in vitro activity. Thus, ethyl
carbamate 11 did not show any appreciable potency
at j-receptors.
The size of the alkyl substituents in the amine series (12–
14) was also critical for activity. Introduction of a
methylamine at C(2) gave 12, which was devoid of bind-
ing activity (K >10 lM). However, increasing the size
i
of the C(2) secondary amine (13) or converting it to a
tertiary amine (14) significantly increased the affinity
for j-receptors (>100-fold).
Scheme 4. Syntheses of C(2) ethers. Reagents and conditions: (a)
appropriate alkyl iodide (or bromide for 20), Ag O, CH CN, 36–77%.
Similar SAR patterns were observed in the ether series
(15–20). Methyl ether 15 and salvinorin B (2) showed
2
3

2764
C. B e´ guin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2761–2765
Table 1. Affinities (K
i
), potencies (EC50), and efficacies of C(2)-substituted salvinorins at the j-opioid receptor
3
5
very low affinities and potencies at the j-receptor.
Increasing the size of the C(2) ether substituent by one
carbon led to a pronounced increase in affinities and
potencies at j-receptors. Ethyl ether 16 was the most po-
the [ S]GTPcS binding assay. We will determine the
selectivities of these j-ligands toward l- and d-opioid
receptors before they can be considered as candidates
for evaluation in behavioral models used to study mood.
Such compounds might stabilize the activity of neural
systems that regulate mood, and thus be useful clinically
if they are tolerated by humans.
tent j-agonist in the ether series (K = 7.9 ± 0.3 nM;
i
EC50 = 18.6 ± 2.6 nM, 103% efficacy). Further homolo-
gation of the carbon side chain led to compounds with
weaker activities (17, 18). Introduction of unsaturation
at C(2) had little effect on j-agonist activity: allyl ether
1
values. The bulkier benzyl ether 20 displayed modest
9 and its isostere, 17, had comparable K and EC50
Acknowledgements
i
in vitro activity and potency.
This work was supported by the Stanley Medical Re-
search Institute and by NIH Grants DA 04745, DA
17302, and P30 DA 13429.
These studies suggest that the size of the substituent at
the C(2) position is critical for activity at the j-receptor,
with three-atom branched chains giving the highest
affinities and potencies (i.e., compounds 1 and 9). Larger
substituents gradually lost affinity, which is consistent
Supplementary data
1
4
with the findings of Chavkin et al. Smaller substituents
2, 12, and 15) were inactive. The addition of a hydrogen
bond acceptor improved binding 2–7-fold (1 vs 16, 3 vs
7, and 4 vs 18), while the addition of a hydrogen bond
Supplementary data associated with this article can be
found, in the online version at doi:10.1016/j.bmcl.
2005.03.113.
(
1
donor decreased activity (e.g., 9 vs 1 and 12 vs 15).
Finally, configuration at C(2) may not be a critical
factor for binding, though more compounds are needed
to confirm this. In summary, unsubstituted carbamate 9
was almost as potent as salvinorin A and should be
more stable toward metabolic deacetylation. This deriv-
ative suggests potential modifications to salvinorin A
that may increase biological stability and maintain high
activity. Also of particular interest was the observation
that methyl carbamate 10 was a partial j-agonist in
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