Synthesis and biological evaluation of new salvinorin A analogues incorporating natural amino acids

Article abstract of DOI:10.1016/j.bmcl.2010.11.046

The synthesis and in vitro evaluation of a new series of salvinorin A analogues substituted at the C(2) position with natural amino acids is reported. Compound 12, containing Val, displayed high affinity and full agonist activity at the kappa-opioid recep

Full text of DOI:10.1016/j.bmcl.2010.11.046

Bioorganic & Medicinal Chemistry Letters 21 (2011) 160–163
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of new salvinorin A analogues
incorporating natural amino acids
Jakub Fichna a,b, Kevin Lewellyn a, Feng Yan c, Bryan L. Roth c, Jordan K. Zjawiony a,
⇑
a Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS, USA
b Department of Biomolecular Chemistry, Medical University of Lodz, Lodz, Poland
c Department of Pharmacology, University of North Carolina Medical School, Chapel Hill, NC, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and in vitro evaluation of a new series of salvinorin A analogues substituted at the C(2)
position with natural amino acids is reported. Compound 12, containing Val, displayed high affinity
and full agonist activity at the kappa-opioid receptor. Analogues with bulky and/or aromatic residues
were inactive, showing the importance of size and electronegativity of C(2)-substituents for binding
affinity of salvinorin A derivatives.
Received 2 October 2010
Revised 5 November 2010
Accepted 8 November 2010
Available online 11 November 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Salvia divinorum
Salvinorin B
Neoclerodane diterpenoid
Natural amino acids
Opioid receptor ligands
A neoclerodane diterpenoid salvinorin A (SA) (1), the active
ingredient of the Mexican plant Salvia divinorum, is a selective kap-
pa-opioid receptor (KOR) agonist with potent in vivo activity
(Fig. 1).1 Recent reports point out a potential role in the treatment
of functional gastrointestinal (GI) disorders, which is in accord with
the traditional use of S. divinorum extracts. SA potently inhibits
smooth muscle contractility and motility in vivo in the upper and
lower GI, as well as influences the epithelial ion transport in a
KOR-dependent manner under both physiological and pathophysio-
logical conditions.2 Pharmacokinetic studies in animals and in hu-
mans indicate that SA is rapidly degraded and that its in vivo
effects are short-lasting, regardless of route of administration.3
Therefore numerous attempts have been made to synthesise SA
derivatives with potent affinity at KOR and prolonged in vivo
actions.2
The structure–activity relationship (SAR) studies of SA have
demonstrated that the size and electronegativity of the substituent
at the C(2) position is critical for activity at opioid receptors. It was
shown that SA derivatives with spatially restrained (3–4 carbon
atoms) lipophilic groups at C(2) display highest binding affinity
and potency at KOR.4–9 However, incorporation of non-lipophilic
substituents, such as amines and amino acids also resulted in po-
tent and selective KOR ligands.6,8 Interestingly, bulkier substitu-
ents tend to reduce KOR affinity and in some cases increase mu-
opioid receptor (MOR) affinity.2
In an attempt to further investigate the role of C(2) in SA bind-
ing at KOR we synthesized a series of derivatives substituted in this
position with natural non-aromatic and aromatic amino acids and
evaluated their in vitro affinities and potencies. Previous studies
showed that the introduction of an aromatic moiety in position 2
increased affinity at MOR.10 Therefore, N-(9-fluorenylmethoxycar-
bonyl) (Fmoc)-protected derivatives were also synthesized and
their affinities compared with those of respective non-protected
analogues.
SA (1) was isolated from the leaves of S. divinorum and purified as
previously described.11 Compound 1 was converted to salvinorin B
(2), which served as the starting material for the preparation of the
C(2)-modified analogues 3–14 (Table 1). Compounds 3–8 were syn-
thesized from N-Fmoc-protected amino acids using N-methylmor-
pholine as a catalyst and TBTU as a coupling reagent (Scheme 1).12
Analogues 9–14 were obtained from respective N-Fmoc-protected
derivatives by deprotection with piperidine.13,14
Spectral data (1H NMR, 13C NMR, high-resolution mass) consis-
tent with the proposed structures were obtained for all the com-
pounds prepared in this study.
The KOR radioligand binding assays were conducted according
to previously described procedures and the results are presented
in Tables 2 and 3.15
⇑
Corresponding author. Address: Department of Pharmacognosy and Research
Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi,
PO Box 1848, University, MS 38677-1848, USA. Tel.: +1 662 915 7290; fax: +1 662
915 6975.
[
35S]GTP
cS binding assays at MOR, DOR and KOR were per-
formed for analogue 12 and SA, which was used as a reference
E-mail address: jordan@olemiss.edu (J.K. Zjawiony).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.046

J. Fichna et al. / Bioorg. Med. Chem. Lett. 21 (2011) 160–163
161
compound. The in vitro pharmacological data for SA and 12 are
listed in Table 4.
O
R:
9
Gly ( )
H2N
H2N
O
Preliminary in vitro binding data in opioid receptor-expressing
cells showed that the introduction of N-Fmoc-protected amino
acids at the C(2) position of SA resulted in loss of affinity at KOR,
depending on the amino acid incorporated and the radiolabelled li-
gand used (Table 2). Only in the case of the Val-substituted ana-
logue (12) did the removal of the Fmoc protecting group
significantly increase its ability to displace selective ligands at
KOR binding sites, making it almost equipotent with the parent
compound, SA. Interestingly, the Ala-derivative (10) displayed
good affinity at KOR against [3H]U69,593, but did not displace
KOR antagonist binding, as assessed with [3H]diprenorphine. Con-
centration–response curves showed that analogue 12 had high
affinity and selectivity at KOR (Ki values of 42.0 2.05 and
0.75 0.62 nM for 12 and SA, respectively; Table 3), while the
affinity of 10 was in a micromolar range (Table 3). To determine
O
O
Ala (10)
15
O
O
O
14
16
13
11
)
GABA (
H2N
11
9
12
O
1
O
17
H
H
R
O
2
10
8
20
5
O
7
3
4
Val (12)
H2N
6
18C
19
O
O
O
O
O
the agonist activity of 12 at KOR, [35S]GTP
cS binding assays were
performed. The potency of Val-substituted derivative (12) was
lower than that of the parent compound (see Table 4); however,
the novel analogue was a full agonist at KOR (Table 4).
Amino acid analogues of SA substituted at the C(2) position
were reported earlier, but only N-Boc-Gly-derivative displayed
good affinity at KOR (90 10 nM).8 The introduction of N-Ac-Gly,
N,N-diMeGly and N,N-diMeGly gave inactive compounds, while
the Gly and Sar analogues were unstable.6 In our study the modi-
fication with Val resulted in a potent and selective KOR agonist,
with affinity in the nanomolar range. These data suggest that the
C(2) position in the structure of SA is important for its interaction
R
1
2
O
O
AcO
OH
Thr(tBu) (13)
H2N
H2N
14
Phe (
)
O
Figure 1. Structure of salvinorin
A (1), salvinorin B (2) and new amino acid
analogues.
Table 1
Physicochemical data for amino acid analogues of salvinorin A
Analogue no.
Amino acid residue
TLC
HPLC trc [min]
FAB-MS
MW
Formula
[M+H]+
3
4
5
6
7
Fmoc-Gly
Fmoc-Ala
Fmoc-GABA
Fmoc-Val
Fmoc-Thr(tBu)
Fmoc-Phe
Gly
Ala
GABA
Val
Thr(tBu)
Phe
0.54a
0.75a
0.39a
0.84a
0.62a
0.56a
0.56b
0.65b
0.77b
0.62b
0.67b
0.61b
17.6
19.0
17.7
17.8
19.2
19.3
12.6
12.8
13.1
14.1
18.7
17.6
C38H39NO10
C39H41NO10
C40H43NO10
C41H45NO10
C44H51NO11
C45H45NO10
C23H29NO8
C24H31NO8
C25H33NO8
C26H35NO8
C29H41NO9
C30H35NO8
669.72
683.74
697.77
711.79
769.87
759.83
447.48
461.50
475.53
489.56
547.64
537.60
670.25
684.37
698.30
712.31
770.51
760.32
448.06
462.07
476.19
490.08
548.30
538.21
8
9
10
11
12
13
14
a
b
c
Rf values for TLC in hexane/EtOAc (1:1).
Rf values for TLC in CH2Cl2/MeOH (49:1).
HPLC elution on a Waters RP C18 column (7.8 ꢀ 100 mm, 5
lm) using the solvent system of water (A)/acetonitrile (B) and a linear gradient of 30–100% solvent B over
25 min at flow rate of 1 mL/min.
O
O
O
O
C
O
O
C
O
O
C
O
a
b
H
O
H
H
H
H
H
HO
Fmoc-RO
RO
O
O
O
O
O
O
O
O
2
3-8
9-14
Scheme 1. Synthesis of amino acid analogues of salvinorin A. Reagents and conditions: (a) salvinorin B (2), appropriate N-Fmoc-amino acids, TBTU, N-methylmorpholine,
CH2Cl2; (b) deprotection with piperidine in CH2Cl2.

162
J. Fichna et al. / Bioorg. Med. Chem. Lett. 21 (2011) 160–163
Table 2
Opioid receptor binding data for amino acid analogues of salvinorin A
Analogue no.
Amino acid residue
MORa [%]
DORb [%]
KORc [%]
KORd [%]
3
4
5
6
7
8
9
10
11
12
13
14
Fmoc-Gly
Fmoc-Ala
Fmoc-GABA
Fmoc-Val
Fmoc-Thr(tBu)
Fmoc-Phe
Gly
Ala
GABA
Val
Thr(tBu)
Phe
20
24
27
15
18
0.5
ꢁ5
ꢁ1.6
1
5
2
ꢁ6
100
—
16
15
24
22
8
4
21
10
39
28
0
41
82
48
103
6
10
0
0
0
12
34
0
0
0
91
0
0
—
—
100
3
17
18
28
48
11
13
—
19
—
—
DAMGO
Natrindole
SA
100
—
—
100
Data are mean of three experiments performed in duplicate. All compounds were tested at a concentration of 10
l
l
M.
M).
M).
M).
Displacement of [3H]diprenorphine in KOR-expressing cells, presented as % effect of respective control (SA, 10
a
Displacement of [3H]DAMGO in MOR-expressing cells, presented as % effect of respective control (DAMGO, 10
Displacement of [3H]DADLE in DOR-expressing cells, presented as % effect of respective control (DADLE, 10
l
b
c
Displacement of [3H]U69,593 in KOR-expressing cells, presented as % effect of respective control (SA, 10
l
d
l
M).
Table 3
Opioid receptor binding affinity and selectivity for selected amino acid analogues of salvinorin A
Analogue no.
Amino acid residue
Kia [MOR, nM]
Kib [DOR, nM]
Kic [KOR, nM]
MOR/KOR
DOR/KOR
10
12
SA
Ala
Val
—
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
3784 1284
42.0 2.05
0.75 0.62d
>2.64
>238
>10,000
>2.64
>238
>10,000
Data are mean of three experiments performed in duplicate.
a
Determined against [3H]DAMGO.
b
Determined against [3H]DADLE.
c
Determined against [3H]U69,593.
Data from Ref. 15.
d
relative ease of our approach and the availability of reagents the
synthetic strategy used in this study may result in a vast library
of analogues and could become the method of choice in prepara-
tion of novel derivatives based on SA structure.
Table 4
Agonist activity of analogue 12 in [35S]GTP
cS binding assay
Analogue no. Amino acid residue EC50 [nM]
Emax [%]
213 15.0 828 275 117.74
0.2 0.1 703 179 100.00
Relative Emax
12
SA
Val
—
Data are mean SEM of four experiments.
Acknowledgements
This work was supported by the NIH Grant R01 DA017204 and
the NIMH Psychoactive Drug Screening Program. The authors wish
to thank Lukasz Kutrzeba for his help in extraction of salvinorin A
from plant material.
with the opioid receptors and that only small, non-aromatic amino
acids fit into the binding pocket of KOR.
This is in good agreement with a recently published Letter by
McGovern et al. on molecular modelling of C(2)-analogues of
SA.16 A region of bulk tolerance, falling within a receptor hydro-
phobic pocket, was observed around the C(2) position, extending
approximately three carbon atoms in length from the carbonyl car-
bon. It has also been shown that the methyl and carbonyl groups,
as well as a hydrophobic moiety at a terminal position of the
C(2) substituent are important for optimal ligand affinity. Interest-
ingly, the structure of Val-derivative, which seems to be well-toler-
ated by the putative binding pocket, accommodates all above
mentioned criteria.
References and notes
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Chen, Y.; Beguin, C.; Carlezon, W. A., Jr.; Cohen, B. Bioorg. Med. Chem. Lett. 2005,
15, 3744.
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Jr.; Cohen, B. M.; Beguin, C. Bioorg. Med. Chem. 2008, 16, 1279.
6. Beguin, C.; Richards, M. R.; Wang, Y.; Chen, Y.; Liu-Chen, L. Y.; Ma, Z.; Lee, D. Y.;
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Dersch, C. M.; Parrish, D.; Deschamps, J. R.; Rothman, R. B.; Prisinzano, T. E. J.
Nat. Prod. 2006, 69, 914.
Unexpectedly, in the study of Beguin et al.6 and in our assays,
the Ala-derivative did not displace [3H]diprenorphine from its
binding at KOR. However, here we observed that the Ala-substi-
tuted
analogue
displayed
micromolar
affinity
against
[3H]U69,593, clearly demonstrating that the choice of the radiola-
beled compound can influence the reported binding affinity data.
In conclusion, we were able to obtain a series of novel amino
acid analogues of SA, of which the Val-derivative displayed highest
affinity at KOR, comparable to that of the parent compound. Due to
9. Stewart, D. J.; Fahmy, H.; Roth, B. L.; Yan, F.; Zjawiony, J. K.
Arzneimittelforschung 2006, 56, 269.

J. Fichna et al. / Bioorg. Med. Chem. Lett. 21 (2011) 160–163
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10. Harding, W. W.; Tidgewell, K.; Byrd, N.; Cobb, H.; Dersch, C. M.; Butelman, E. R.;
Rothman, R. B.; Prisinzano, T. E. J. Med. Chem. 2005, 48, 4765.
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Pharm. Biol. 2009, 47, 1078.
14. Synthesis of 2-(20-(S)-amino-3-methylbutanoate) salvinorin B (12): yield 76.9%
starting from 2, a white solid, Rf 0.62 (1:1, hexane/EtOAc); 1H NMR (400 MHz,
CDCl3) d 1.02–1.04 (d, J = 6.8 Hz, 3H), 1.07–1.10 (d, J = 6.8 Hz, 3 Hz), 1.14 (s,
3H), 1.47 (s, 3H), 1.57–1.75 (m, 4H), 1.77–1.84 (m, 1H), 2.07–2.11 (dd, J = 10.8,
1.6 Hz, 1H), 2.16–2.21 (m, 2H), 2.30–2.36 (m, 2H), 2.48–2.55 (dd, J = 13.4,
5.2 Hz, 1H), 2.75–2.80 (dd, J = 13.0, 3.5 Hz, 1H), 3.44–3.46 (d, J = 4.4 Hz, 1H),
3.75 (s, 3H), 5.17–5.22 (dd, J = 12.6, 7.5 Hz, 1H), 5.53–5.57 (dd, J = 11.6, 5.1 Hz,
1H), 6.41 (s, 1H), 7.41 (t, J = 2.7 Hz, 1H), 7.45 (s, 1H); 13C NMR (100 MHz, CDCl3)
15.2, 16.4, 16.9, 17.0, 18.1, 19.4, 30.9, 34.4, 35.5, 38.2, 42.1, 43.4, 51.4, 52.0,
53.6, 64.0, 71.2, 75.2, 108.5, 125.5, 139.5, 143.7, 171.1, 171.6, 172.1,
202.0 ppm; HRESIMS m/z [M+H]+ 490.08 (calcd for C26H35NO8 489.56).
15. Bikbulatov, R. V.; Yan, F.; Roth, B. L.; Zjawiony, J. K. Bioorg. Med. Chem. Lett.
2007, 17, 2229.
12. General procedure for synthesis of N-Fmoc-amino acid analogues of salvinorin A
(3–8): To a stirred solution of 2 (20 mg, 0.05 mmol, 1 equiv) in CH2Cl2 (2 mL),
the N-Fmoc-amino acid (3 equiv), and N-methylmorpholine (3 equiv) was
added TBTU (3 equiv). The solution was stirred at 50 °C (24 h), concentrated
under reduced pressure and purified by column chromatography (SiO2; 2:1,
hexane/EtOAc) to obtain the desired product.
13. General procedure for deprotection of N-Fmoc-amino acid analogues of salvinorin
A (9–14): Respective N-Fmoc-amino acid derivative of salvinorin A (1 equiv)
was dissolved in CH2Cl2 and piperidine (3 equiv) was added. The solution was
stirred at rt (1.5 h), concentrated under reduced pressure and purified by
column chromatography (SiO2; 2:1, hexane/EtOAc) to obtain the desired
product.
16. McGovern, D. L.; Mosier, P. D.; Roth, B. L.; Westkaemper, R. B. J. Mol. Graphics
Modell. 2010, 28, 612.