Synthesis and biological evaluation of 2-alkyl-2-methoxymethyl-salvinorin ethers as selective κ-opioid receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2015.06.092

The synthesis of a new series of C-2-alkyl-2-methoxymethyl-salvinorin ethers and their binding affinities at κ-, μ-, and δ-opioid receptors are presented. We have developed a synthesis that enables installation of alkyl-substituents at C-2 while maintaining the integrity of the C-2 methoxymethyl ether and retaining κ-opioid receptor binding activity. Among these new compounds, 2-methyl-2-methoxymethyl-salvinorin ether (9a) is a potent full agonist at the κ receptor and shows comparable potency in K_i and EC₅₀ with salvinorin A and U50488H. These C2-alkylated analogs have been identified as full κ agonists.

Full text of DOI:10.1016/j.bmcl.2015.06.092

Accepted Manuscript
Synthesis and Biological Evaluation of 2-Alkyl-2-Methoxymethyl-Salvinorin
Ethers as Selective κ-Opioid Receptor Agonists
David Y.W. Lee, Gang Deng, Zhongze Ma, Wei Xu, Lu Yang, Jing Liu,
Ronghua Dai, Lee-Yuan Liu-Chen
PII:
S0960-894X(15)00697-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.092
BMCL 22882
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 February 2015
23 June 2015
25 June 2015
Please cite this article as: Lee, D.Y.W., Deng, G., Ma, Z., Xu, W., Yang, L., Liu, J., Dai, R., Liu-Chen, L-Y.,
Synthesis and Biological Evaluation of 2-Alkyl-2-Methoxymethyl-Salvinorin Ethers as Selective κ-Opioid Receptor
Agonists, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.06.092
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Synthesis and Biological Evaluation of 2-Alkyl-2-Methoxymethyl-Salvinorin
Ethers as Selective κ-Opioid Receptor Agonists
David Y.W. Leea,*, Gang Denga, Zhongze Maa, Wei Xub, Lu Yanga, Jing Liua,
Ronghua Daia, and Lee-Yuan Liu-Chenb
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
Abstract: The synthesis of a new series of C-2-alkyl-2-methoxymethyl-salvinorin ethers and their
binding affinities at κ-, µ-, and δ-opioid receptors are presented. We have developed a synthesis
that enables installation of alkyl-substituents at C-2 while maintaining the integrity of the C-2
methoxymethyl ether and retaining κ-opioid receptor binding activity. Among these new
compounds, 2-methyl-2-methoxymethyl-salvinorin ether (9a) is a potent full agonist at the κ
receptor and shows comparable potency in Ki and EC50 with salvinorin A and U50488H. These
C2-alkylated analogs have been identified as full κ agonists.
Keywords: κ-opioid, receptor, affinity, agonist
Introduction
The κ-opioid receptor (KOR) is one of the three types of opioid receptors which belong to the
seven-transmembrane receptor family; the other two are δ (DOR) and µ (MOR).1 Activation of
endogenous KOR results in analgesia, dysphoria, anti-pruritis, and water diuresis, among other
effects.2 Many selective KOR agonists have been synthesized, including HZ-2,3 U50,488H,4
U69,593,5 U62,066,2 and enadoline,6 as shown in Figure 1. Structurally, each of these compounds
contains at least one nitrogen atom. Salvinorin A (1), a neoclerodane diterpene isolated from
Salvia divinorum, is the first known natural product acting on KOR selectively.7 Notably, it does
not contain a basic nitrogen atom in the structure. Salvinorin A is a potent and selective KOR
agonist. It binds to KOR with a higher affinity (Ki=1.3 nM) than U50,488H (Ki=2.7 nM), a
prototypic highly selective KOR agonist, but does not show significant affinities to DOR, MOR,
and nociceptin/orphanin FQ receptors.8 It has also been reported that salvinorin A possesses higher
selectivity for KOR than U50,488H and U69,593.9 However, the duration of action of salvinorin A
in vivo is short, which is likely due to the hydrolysis of the acetate by esterase to produce
Salvinorin B (2), a compound with a comparatively much lower affinity to KOR. We have
previously reported that 2-methoxymethyl salvinorin
B
(3)10 and corresponding
2-ethoxymethyl-salvinorin B11 both have greater potency (x2) than salvinorin A and much longer
durations of action in vivo.
1

O
O
O
N
O
O
N
O
O
O
O
O
O
O
H
H
H
H
H
H
O
C
N
O
AcO
HO
MOMO
O
O
O
N
O
OMe
O
OMe
O
OMe
3
4
HZ-2
1
Salvinorin A
2
Salvinorin B
O
Cl
Cl
Cl
Cl
O
N
O
O
N
N
N
O
N
N
N
N
.CH3SO3H
O
O
O
8
enadoline
5
U50,488H
7
U62,066
6
U69,593
Figure 1. Structures of KOR agonists
To date, all previous modifications at the C-2 position of salvinorin A have been limited to
either replacing the C-2 acetate with various ester or ether derivatives, or with halogens.11
Alkylation at C-2 has not been reported because of the difficulties encountered in introducing an
alkyl group at this position, which is adjacent to a carbonyl group and ester. In our attempt to fully
explore the effect of 3D-alignment at C-2 position to structure-activity relationship (SAR) of
salvinorin A, a series of 2-alkyl-2-methoxymethyl-salvinorin ethers were synthesized and
evaluated for affinity, potency, and efficacy as KOR ligands. As shown in Table 1,
2-methyl-2-methoxymethyl-salvinorin ether (9a) was found to be a potent full agonist at the KOR,
and shows a comparable potency in Ki and EC50 to salvinorin A and U50488-H. In addition, 9a,
which lacks a labile ester moiety, is much more stable than salvinorin A. This report opens up a
synthetic strategy for introducing a series of stable C2-β-alkylated derivative of salvinorin A for
pharmacological evaluation.
Table 1
Affinities (Ki) at the κ-opioid receptors
Ki SEM (nM) a,b
Compound
R
κ
δ
µ
-CH3
4.7 0.3
20.5 6.5
301 24
210 22
>1,000
>1,000
48 6
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
9a
-CH3
9b
-CH2CH3
10a
10b
11a
11b
12a
12b
13a
-CH2CH3
-CH2CH2CH3
-CH2CH2CH3
-CH2CH=CH2
-CH2CH=CH2
-CH2(CH2)3CH2I
250 51
>1,000
2

-CH2(CH2)3CH2I
>1,000
108 19
316 70
0.5 0.05
1.3 0.5
0.73 0.06
>1,000
>1,000
>1,000
2.9 0.6
>1,000
>1,000
>1,000
>1,000
13b
-CH2CH2OCH2CH2I
-CH2CH2OCH2CH2I
14a
14b
>1,000
Etorphine
1 (Salvinorin A)
3
0.14 0.02
>1,000
>1,000
a Inhibition of [3H]diprenorphine binding hKOR in membranes of CHO-hKOR cells;
b Mean s.e.m. of at least three independent experiments performed in duplicate.
Chemistry
Salvinorin A (1) was isolated from dry Salvia divinorum leaves by following the
chromatographic procedures worked out in our laboratory.8 As shown in Scheme 1, deacetylation
of 1 using a reported procedure provided the Salvinorin B (2) and the C8-epimer almost in 1:1
ratio.12 It would be ideal to be able to control the epimerization and shift the ratio to the desirable
natural isomer.13 In order to improve the ratio of C8-epimerization and increase the overall yield of
the intermediate compound 2, an efficient and mild deacetylation method (H2O2/NaHCO3) for
compounds with an adjacent α carbonyl group14 similar to the structure of 1 was employed.
Unfortunately, a significant amount of the starting material (~40%) was recovered even under
various concentrations and ratios of reagents or prolonged reaction time. However, this problem
was overcome by simply adding the sodium-selective complexing agent, 15-crown-5, and
prolonged vigorous stirring (36 h) to afford a nearly quantitative yield of salvinorin B. Notably, no
C8-epimer product was detected following this deacetylation procedure, affording salvinorin B
exclusively in good yield.
3

Scheme 1. Reagents and conditions: a) NaHCO3, H2O2, 15-Crown-5, THF:MeCN=1:1, rt; b)
MOMCl, DIPEA, DMF, rt; c) NaH, Alkyl halide , THF, -50 oC; d) K2CO3, MeOH, rt.
With a sufficient amount of intermediate 2 in hand, derivatization of the C2-hydroxyl as the
methoxymethyl ether proceeded smoothly employing previously reported methods.15 The next
step involved alkylation at the C-2 position with various groups. With a bulky group introduced at
C-2, we were concerned about the reactivity of C-2 under standard alkylation conditions.
Therefore, we chose iodomethane as the model substrate to react with the enolate anion generated
by various bases including LDA, triphenylmethyllithium, sodium hydride, lithium
bis(trimethylsilyl)amide, lithium 2,2,6,6-tetramethylpiperidide and lithium hexamethyldisilazide.
Unsurprisingly, employment of most of these bases resulted in either the C8-epimerization of
2-methoxymethyl-salvinorin B, or the decomposition of the starting material even at -78oC.
Interestingly, only NaH gave the desired 2-methyl-2-methoxymethyl-salvinorin ether product
without C8-epimerization (Scheme 1, Table 1). The configuration of the methyl group at C-2
position was confirmed to be oriented in the β position by 2D NOESY. This may due to the fact
that axial methyl groups at C-5 and C-9 along with the C-5 carboxyl methyl ester group block the
attack of iodomethane from the α face. Encouraged by this result, we successfully employed a
series of alkylating agents with various carbon chain lengths, affording yields from 30% to 70%.
However, the C8-epimer of the 2-alkyl-2-methoxymethyl-salvinorin ether always accompanied
the product, and even comprised the sole product in one case (10b). Nevertheless, epimerization
of the undesired C8-epimer to its natural isomer was easily achieved through use of K2CO3 in
methanol at room temperature. All spectral data (1H-NMR, 13C-NMR and high-resolution mass)
4

obtained were consistent with the structures proposed.
Results and discussion
All these compounds were evaluated for binding affinities to the KOR, DOR, and MOR by
competitive inhibition of [3H] diprenorphine binding to membranes prepared from Chinese
hamster ovary cells (CHO) stably transfected with either rat MOR, mouse DOR, or human KOR
(hKOR). Inhibition of [3H] diprenorphine binding was first performed with 3 µM of each
compound, and those exhibiting >50% inhibition were further evaluated for determination of Ki
values.16 The potencies and efficacies on hKOR were determined by their abilities to enhance
[35S]GTPγS binding to the membranes of CHO-hKOR cells. The selective full κ agonist
U50,488H was used as the reference compound with a relative efficacy of 100. Data are shown in
Table 1 and Table 2.
Table 2
Affinities (Ki), potencies (EC50) and efficacies at the κ-opioid receptor
d
Ki (nM)a,b
Compound
R
EC50 (nM)b,c
Emax
-
1.3 0.5
0.73 0.06
4.7 0.3
20.5 6.5
301 24
210 22
>1,000
>1,000
48 6
4.5 1.2
0.6 0.2
19.2 2.0
80.9 8.7
NDe
NDe
NTf
NTf
NDe
NDe
NTf
110 7
115 15
123 16
103 16
83 at 1 µM
81 at 1 µM
NTf
1
H
3
-CH3
9a
-CH3
9b
-CH2CH3
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
U50,488H
-CH2CH3
-CH2CH2CH3
-CH2CH2CH3
-CH2CH=CH2
-CH2CH=CH2
-CH2(CH2)3CH2I
-CH2(CH2)3CH2I
-CH2CH2OCH2CH2I
-CH2CH2OCH2CH2I
-
NTf
99 at 1 µM
84 at 1 µM
NTe
250 51
>1,000
>1,000
108 19
316 70
2.7 0.2
NTf
NDe
NDe
NTe
98 at 1 µM
85 at 1 µM
100
10.6 1.8
a Inhibition of [3H]diprenorphine binding hKOR in membranes of CHO-hKOR cells;
b Mean s.e.m. of at least three independent experiments performed in duplicate;
c Enhancement of [35S]GTPγS binding to membranes of CHO-hKOR cells;
d Emax values were determined as the percentage of maximal response to U50,488H;
e ND: not determined because the response did not reach a plateau or it was tested at only 1 µM
f NT: not tested due to low affinity.
Most of the newly synthesized C-2 alkyl analogs have submicromolar affinity and potency.
The compounds with Ki values < 1 µM exhibited agonist activity, but no antagonist activity was
detected. When the aliphatic 2-alkyl derivatives (9a-11a, 9b-11b) were evaluated, we found that
by increasing the size of alkyl groups, affinities to the KOR were decreased accordingly. Similar
results at C-2 alpha position were reported by Chavkin et al.17 and Béguin et al.18 For instance, the
5

affinity of 9a was about 6-fold lower (Ki value = 4.7 0.3 nM) than lead compound 3 (Ki value =
0.73 0.06 nM), which lacks an alkyl substituent. Subsequently, compound, 9a was found to be a
potent full agonist at the KOR (123% efficacy relative to the full agonist U50,488H) and showed a
2- to 5-fold higher Ki and EC50 than 1 (Ki values (nM): 9a, 4.7 0.3; 1, 1.3 0.5; EC50 values (nM):
9a, 19.2 2.0; 1, 4.5 1.2) and than U50,488H (Ki values (nM): 2.7 0.2; EC50 values (nM):
10.6 1.8). This suggests that 9a, if labeled with 14C-methyl, may serve as a molecular probe for
mapping the KOR in the brain. Introduction of an ethyl group (10a and 10b) at the C-2 position
led to decreased affinity (about 70-fold lower than 9a). Extending the aliphatic alkyl group to
n-propyl at C-2(11a and 11b) resulted in the loss of binding activity (Ki >1.0 µM). When the
unsaturated allyl group was introduced at C-2(12a, 12b), a pronounced increase in affinity (Ki
values (nM): 48 6 and 250 51, respectively) to the KOR was observed compared to its
corresponding saturated analog (11a, 11b, Ki values (nM): >1,000 for both), and both of them
showed agonist activities (Emax values of 99% and 84%, respectively, compared to U50,488H).
These data are different from the C-2 allyl ether reported by Béguin et al,17 which showed a 2-fold
decrease in affinity and potency compared to its isostere. When a 5-iodopentyl group was
introduced, the affinity to KOR was abolished (Ki values (nM) >1,000). Interestingly, affinity to
KOR was somewhat recovered when the linear 2’-iodoethoxyethyl group (14a, 14b, Ki values
(nM): 108 19, 316 70, respectively) was introduced at C-2.
Most of the C2-alkyl analogs with the natural configuration (8β-H) show better affinity and
potency than its corresponding epimer (8α-H), except 10a, 10b, in which the natural configuration
10a (Ki values (nM): 301 24) shows 1.5-fold lower affinity than its unnatural epimer (10b, Ki
values (nM): 210 22). This result was also reported in our previous publication where salvinorin
B showed 3-fold lower affinity than its epimer.
In summary, the present results suggest that the affinity and potency at KOR is closely related
to the size of the alkyl substituent at the C-2 position. Introduction of alkyl groups at C-2 led to
decrease in binding affinity and potency compared to lead compound 3, especially with a bulky
alkyl group. This may due to the fact that the 3D-alignment at C-2 affected interaction with
Tyr313.19 In addition, an ether functionality at C2 (14a, 14b) was found to enhance affinity to the
KOR compared to alkyl substituents lacking an ethereal oxygen (13a, 13b). Since the 2-methyl
substituted derivative 9a shows only 2- to 5-fold lower affinity than salvinorin A and U50,488H,
its 14C-labeled 9a would be an excellent molecular probe for mapping the KOR in the brain, a
highly interesting approach which is being investigated.
Acknowledgements
We thank to Dr. Yong-Xuan Su at University of California, San Diego for MS analysis and
NIH grant (NIH-DA-0019688) for financial support.
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Graphic abstract
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