Kappa-opioid receptor-selective dicarboxylic ester-derived salvinorin A ligands

Article abstract of DOI:10.1016/j.bmcl.2013.03.111

Salvinorin A, the active ingredient of the hallucinogenic plant Salvia divinorum is the most potent known naturally occurring hallucinogen and is a selective κ-opioid receptor agonist. To better understand the ligand-receptor interactions, a series of dicarboxylic ester-type of salvinorin A derivatives were synthesized and evaluated for their binding affinity at κ-, δ- and μ-opioid receptors. Most of the analogues show high affinity to the κ-opioid receptor. Methyl malonyl derivative 4 shows the highest binding affinity (K_i = 2 nM), analogues 5, 7, and 14 exhibit significant affinity for the κ-receptor (K_i = 21, 36 and 39 nM).

Full text of DOI:10.1016/j.bmcl.2013.03.111

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2860–2862
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Kappa-opioid receptor-selective dicarboxylic ester-derived salvinorin
A ligands
Prabhakar R. Polepally a, Kate White b, Eyal Vardy b, Bryan L. Roth b, Daneel Ferreira a,
Jordan K. Zjawiony a,
⇑
a Department of Pharmacognosy, and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677-1848, USA
b Department of Pharmacology, School of Medicine and Division of Chemical Biology and Medicinal Chemistry, School of Pharmacy,
NIMH Psychoactive Drug Screening Program, University of North Carolina, Chapel Hill, NC 27599, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Salvinorin A, the active ingredient of the hallucinogenic plant Salvia divinorum is the most potent known
Received 4 February 2013
Revised 22 March 2013
Accepted 27 March 2013
Available online 4 April 2013
naturally occurring hallucinogen and is a selective
ligand–receptor interactions, a series of dicarboxylic ester-type of salvinorin A derivatives were synthe-
sized and evaluated for their binding affinity at -, d- and -opioid receptors. Most of the analogues show
high affinity to the -opioid receptor. Methyl malonyl derivative 4 shows the highest binding affinity
(Ki = 2 nM), analogues 5, 7, and 14 exhibit significant affinity for the -receptor (Ki = 21, 36 and 39 nM).
j-opioid receptor agonist. To better understand the
j
l
j
j
Keywords:
Salvia divinorum
Ó 2013 Elsevier Ltd. All rights reserved.
Salvinorin A and B
Dicarboxylic ester-type ligands
Kappa-opioid receptor agonists
The neoclerodane diterpenoid salvinorin A (1) isolated from the
leaves of hallucinogenic sage Salvia divinorum, is a potent and
selective
large number of analogues have been prepared by semi-synthesis
to probe the pharmacophore and mode of binding.3 Some of these
analogues present interesting pharmacological profiles from full
Salvinorin A (1) was isolated from dried leaves of Salvia divino-
rum and purified as previously reported.7 It was hydrolyzed to
salvinorin B (3), which served as the starting material for the
preparation of the C(2)-modified dicarboxylic ester analogue
library 4–16 (Schemes 1 and 2). Compounds 4–10, 14, and 15
were prepared8 in yields of 57–69% by reacting 3 with the corre-
sponding acid halides in the presence of Et3N in DCM (Schemes 1
and 2). Analogues 11–13 and 16 were synthesized9 in yields of
40–51% via the reaction of 3 with appropriate anhydrides using
j
-opioid receptor (KOR) agonist.1,2 Since its discovery, a
KOR agonist to partial d-opioid receptor (DOR) or l-opioid receptor
(MOR) agonist and antagonists. The current objective is to utilize
the knowledge about salvinorin A–KOR interactions to rationally
design salvinorin A derivatives with different pharmacological pro-
files and therapeutic potential. In the course of our work on the
molecular mechanism of interaction of salvinorin A with KOR, we
reported irreversible binding of 22-thiocyanatosalvinorin A (2)
DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) as base (Schemes
1
and 2). The physical data (1H, 13C NMR and HR-ESIMS) were
consistent with the proposed structures.
(Fig. 1) with the sulfhydryl group of Cys-315 at the j-opioid recep-
tor.4,5 Our previous work on the KOR model and analysis of the
mode of binding of 2 suggest that Cys-315 may be a good anchor-
ing amino acid at the binding site in KOR.4,6 The presence of ester
substituents at C-22 may enhance electrophilicity of this center
and thereby leads to stronger binding with the thiol group of
Cys-315 of KOR.
O
15
O
16
14
13
12
H 11
H
8
O
O
O
O
H
H
22
9
O
RO
1
R
17O
O
O21
7
10
2
Herein, we report the synthesis of series of new dicarboxylic es-
3
4
ter salvinorin A derivatives and their binding affinity to
-opioid receptors.
j-, d- and
6
l
O
OMe
O
OMe
R = H, Salvinorin A (1)
R = H, Salvinorin B (3)
R = SCN, 22-Thiocyanato salvinorin A (2)
Figure 1. ThestructuresofsalvinorinA, 22-thiocyanatosalvinorinA, andsalvinorin B.
⇑
Corresponding author.
E-mail address: jordan@olemiss.edu (J.K. Zjawiony).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.111

P. R. Polepally et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2860–2862
2861
Table 1
O
O
Binding affinities of C(2)-dicarboxylic ester-derived salvinorin A analogues (4–16) at
MOR, DOR, and KOR
Compound Affinitiesa
MORb
Ki SD (nM)
Selectivity
O
O
O
O
H
H
H
H
DORc
KORd
MOR/KOR DOR/KOR
HO
RO
O
a
O
n
O
1
>10,000
>10,000
0.9 0.3
ND
6.2 2.2
ND
ND
O
O
Naltrindole ND
DAMGO
0.9 0.2
O
OMe
O
2e
3
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
0.6 0.2
>10,000
OMe
(3)
(4-12)
4
5
6
7
4320 680 2.0 0.3
>5000
>476
>67
2160
>476
>67
>277
>23
>33
>17
>2
4 :
n = 1, R = Me
>10,000
>10,000
>10,000
>10,000
21.0 5.6
148 32
5 :
n = 1, R = Et
6 : n = 1, R = t-butyl
7 : n = 2, R = Me
8 : n = 2, R = Et
36
8
>277
>23
16
8
437 129
302 94
9 :
n = 3, R = Me
9
5012 1400 >10,000
10 : n = 4, R = Me
11 : n = 2, R = H
12 : n = 3, R = H
10
11
12
13
14
15
16
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
575 183
4070 1300 >2
263 86
>10,000
39 11
>17
4570 1300
>10,000
711 134
724 137
>10,000
17
1
18
2
>38
1
>256
>23
>4
Scheme 1. Synthesis of dicarboxylic ester-type salvinorin
Reagents and conditions: (a) appropriate acid chloride, Et3N, dry DCM, N2, rt,
2–3 h, or appropriate acid anhydride, DBU, dry DCM, N2, rt, 6–8 h.
A analogues 4–12.
427 134
2291 492
>4
ND—not determined.
The
j-, d- and l-opioid receptor binding affinities of synthe-
a
Ki determined against [3H]U69,553 ligand.
sized compounds 4–16 were evaluated at the NIMH-sponsored
Psychoactive Drug Screening Program (PDSP), University of North
Carolina at Chapel Hill using radioligand binding assays conducted
as previously described,1,5 and the data are collated in Tables 1 and
2. For comparison purposes, opioid binding affinity data for
compounds 1–3 and positive controls for MOR and DOR are in-
cluded in Table 1. Most of the dicarboxylic ester derivatives show
appreciable binding affinity to KOR, and no affinity to MOR and
DOR. The methyl malonyl derivative 4 had higher affinity than 1
at KOR (Ki = 2.0 vs 6.2 nM) (Table 1). The ethyl malonyl and methyl
succinyl ligands 5 and 7, also have significant affinities to KOR,
these being respectively three and sixfold reduced compared to 1
(Ki = 21.0 and 36, respectively, vs 6.2 nM). The KOR affinities of
analogues 6, 8, and 9 were decreased 24-, 70- and 49-fold as
compared to 1 (Ki = 148, 437 and 302, respectively, vs 6.2 nM).
Similarly, compounds 10, 12, and 15 show reduced affinity
(Ki = 575, 263, and 427 nM, respectively) at KOR. Interestingly,
b
c
See Ref. 4, Ki of 2 is 0.6 versus 1.9 nM of 1 for the KOR.
Data are mean of three experiments performed in duplicate.
d
e
The binding affinity constant (Ki) determined against [3H]DAMGO ligand.
Ki determined against [3H]DADLE ligand.
Table 2
Kappa-opioid receptor functional assay: Efficacies of Gai activa-
tion in live HEK293 cells using a cAMP biosensor Glosensor-22F
(Promega)
Compound
EC50 SD (nM)a
1
4
5
7
5
3
137 15
52 23
855 130
70 25
14
a
Data are mean of four experiments.
O
O
R3
O
O
H
H
O
R1O
O
R2
O
O
O
OMe
(13-15)
13 : Z; R1, R2, R3 = H
O
O
H
H
a
14 : E; R1 = Me, R2, R3 = H
15 : E; R1 = Et, R2, R3 = H
HO
O
O
O
OMe
O
O
(3)
O
O
OH
O
H
H
O
O
OMe
16
Scheme 2. Synthetic route for the designed ligands 13–16. Reagents and conditions: (a) appropriate acid chloride, Et3N, dry DCM, N2, rt, 3 h, or appropriate acid anhydride,
DBU, dry DCM, N2, rt, 6–8 h.

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P. R. Polepally et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2860–2862
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Biochemistry 2009, 48, 6898.
the methyl fumaryl ligand 14 had a sixfold reduced but still consid-
erable affinity for the -opioid receptor compared to 1 (Ki = 39 vs
6.2 nM). Analogues 11 and 16 exhibited less affinity for the
receptor (Ki = 4070 and 2291 nM, respectively), and derivative 13
has no affinity for any of the opioid receptors.
To characterize the relative efficacy of these dicarboxylic ester
ligands, compounds 4, 5, 7, and 14 together with salvinorin A (1)
were selected for a functional assay. Table 2 shows the EC50 values
of these ligands in stimulating Gai signaling mediated by the
j
j
-
j-opioid receptor.
In summary, a series of new dicarboxylic ester-type analogues
of salvinorin A were synthesized in an effort to explore the effects
of C-2 substitution towards selectivity at -, d-, and -opioid
receptors. Most of the analogues showed high binding affinity to
the -opioid receptor with little affinity for d- and -opioid
j
l
j
l
receptors. Compound 4 possessing a C-2 methyl malonyl group
exhibited increased selectivity for KOR compared to the parent
salvinorin A (1).
5. Wu, H.; Wacker, D.; Mileni, M.; Katritch, V.; Han, G. W.; Vardy, E.; Liu, W.;
Thompson, A. A.; Huang, X. P.; Carroll, F. I.; Mascarella, S. W.; Westkaemper, R.
B.; Mosier, P. D.; Roth, B. L.; Cherezov, V.; Stevens, R. C. Nature 2012, 485, 327.
6. Yan, F.; Mosier, P. D.; Westkaemper, R. B.; Stewart, J.; Zjawiony, J. K.; Vortherms,
T. A.; Sheffler, D. J.; Roth, B. L. Biochemistry 2005, 44, 8643.
Acknowledgments
7. Kutrzeba, L. M.; Karamayan, V. T.; Speth, R. C.; Williamson, J. S.; Zjawiony, J. K.
Pharm. Biol. 2009, 47, 1078.
This work was supported by the NIH Grant R01 DA017204 and
the NIMH Psychoactive Drug Screening Program (PDSP), University
of North Carolina at Chapel Hill, NC 27599. We thank Mr. Daniel J.
Siebert from the Salvia divinorum Research and Information Center
for supplying the plant material, and Dr. Bin Wang for the assis-
tance with HRESIMS.
8. Procedure for the synthesis of analogues 4–10, 14 and 15: Compound 3 (15 mg,
0.05 mmol) and a catalytic amount of triethylamine were dissolved in DCM
(3 mL). An appropriate acid chloride (0.125 mmol) was added, and the reaction
mixture was stirred for 2–3 h at rt. After TLC indicated completion of the
reaction, the mixture was quenched with water and the organic layer separated.
The organic phase was washed with dilute aqueous HCl (0.01 mol/L, 2 mL)
followed by saturated NaHCO3 (2 mL). The organic layer was dried over
anhydrous Na2SO4, evaporated, and the residue was purified by column
chromatography (SiO2; eluent:n-hexane/EtOAc) to obtain the target product.
In some cases purification was done by preparative HPLC (C18 column, MeCN–
water).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.03.
111.
9. Method for the synthesis of derivatives 11–13 and 16: To a solution of compound 3
(15 mg, 0.05 mmol) in DCM (3 mL),
a catalytic amount of DBU and an
appropriate acid anhydride (0.15 mmol) were added. The mixture was stirred
at room temperature for 6–8 h. After TLC indicated completion of the reaction,
the mixture was concentrated under reduced pressure and the residue was
purified by column chromatography (SiO2; eluent:n-hexane/EtOAc) and HPLC
(C18 column, MeCN–water) to yield the target product.
References and notes
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2. Roth, B. L.; Baner, K.; Westkaemper, R.; Siebert, D.; Rice, K. C.; Steinberg, S.;
Ernsberger, P.; Rothman, R. B. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11934.