A. L. Pelotte et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2811–2814
2813
OH
and carbamates alike generally displayed a bias for the MOR. All
compounds also bound to the KOR with nanomolar affinity ranging
from approximately 1–28 nM. Conversely, tosylate 13 was the only
compound of those synthesized that demonstrated low nanomolar
binding (Ki = 5.16 nM) to the DOR.
N
OR
O
H
(13), R= Ts
(14), R= Ms
OH
The receptor binding data for 13 are particularly interesting.
Due to 13’s sub- to low-nanomolar affinity for all opioid receptors
Figure 4. Proposed stable conformation of 6b-OTs and -OMs.
investigated, including
a
slight preference for the MOR
this stability to Portoguese’s previous insight9 that the preferred
chair conformation of Ring C places the 6b-sulfonate ester in an
unfavorable orientation to undergo an SN2 or E2 reaction (Fig. 4).11
Once obtained, the resulting sulfonate ester (11 and 12) and
carbamate (7 and 8) intermediates were debenzylated using stan-
dard hydrogenolysis conditions (H2, Pd/C, 3:1:CH2Cl2:CH3OH) to
yield final compounds 9 and 10 and 13 and 14 in good yields
(Fig. 3). All intermediate and final compounds were purified using
silica gel chromatography and spectroscopically characterized
using NMR (1H, 13C, and 2D NMR spectroscopy), high resolution
mass spectrometry, and IR.12 The isolated yields reported in this
work are unoptimized.
The four compounds synthesized were next screened in in vitro
opioid receptor binding assays according to procedures outlined by
Fontana et al.13 Data is provided in Table 1. All four compounds
synthesized exhibited affinity for the MOR better than the stan-
dard, 6b-naltrexol HCl. Our most potent compounds for binding
at the MOR, diphenylcarbamate 9 (Ki = 0.56 nM) and tosylate 13
(Ki = 0.79 nM), both contained one or more phenyl rings. Based
on Ki data, the order of MOR affinity for the sulfonates and carba-
mates is as follows: 9 > 13 > 14 > 10 > 6b-naltrexol HCl. These data
suggests that bulky, hydrophobic substituents protruding from the
oxygen atom off of C6 may create favorable interactions that could
enhance the affinity of compounds for the MOR.
Even though dimethylcarbamate 10 did not possess the greatest
affinity for the MOR over other compounds studied, 10 was the most
selective compound for binding to the MOR subtype. Compound 10
was 180 times more selective for the MOR than the DOR, and 15
times more selective for the MOR versus the KOR. Since the MOR is
known to be involved in the therapeutically relevant pathways lead-
ing to the manifestation of pain and addiction, we are encouraged by
the subtype specificity and affinity trends observed with 10 and
other compounds in the sulfonate and carbamate series. Our data
suggest that the absence of a hydrogen-bond donor group on the
oxygenatom connectedtoC6 does notappeartonegativelyinfluence
in vitro affinity of naltrexol derivatives for the MOR.
(Ki = 0.79 nM), 13 may make a non-selective in vitro probe for gen-
eral opioid receptor function (Table 1). Furthermore, other investi-
gators are interested in opioid ligands with mixed receptor binding
and functional profiles for varied applications in pain and addiction
therapies. For example, opioid ligands possessing
l-agonist/d-
antagonist profiles are known to produce analgesia with less toler-
ance and dependence.14 Clearly, present insights from this research
into the relationship between structure and receptor binding for
the sulfonate ester and carbamate derivatives will be further in-
formed by functional binding experiments using the [35S]GTP-
c-S
assay.15 Functional binding assay data and in vivo efficacy data
on selected compounds from the sulfonate esters and carbamates
will be disclosed in due course.
Acknowledgments
A.M.D. gratefully acknowledges the support of the University of
New England (UNE) for University and College of Arts & Sciences
(CAS) Mini-Grants and the Chemistry & Physics Department for
monies to support research & conference travel. A.M.D. also thanks
the American Society for Experimental Therapeutics (ASPET) for a
Young Investigator Award. R.M.S. thanks ASPET and UNE’s College
Of Osteopathic Medicine for fellowship support. A.L.P. thanks the
CAS Dean’s Office for summer research grant support. The authors
thank Mallinckrodt, Inc. for the generous donation of naltrexol free
base and naltrexone. Portions of this work were supported by the
Intramural Research Program, National Institute on Drug Abuse,
NIH, DHHS.
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
Although we were seeking l-selective compounds as potential
1. (a) Contet, C.; Kieffer, B. L.; Befort, K. Curr. Opin. Neurobiol. 2004, 14, 370; (b)
Wang, Z.; Bilsky, E. J.; Porreca, F.; Sadée, W. Life Sci. 1994, 54(20), 339; (c)
Waldhoer, M.; Bartlett, S. E.; Whistler, J. L. Ann. Rev. Biochem. 2004, 73, 953; (d)
Dar, D. E.; Zangen, A. Central Nervous Syst. Agents Med. Chem. 2006, 6, 1; (e)
Narita, M.; Funada, M.; Suzuki, T. Pharmacol. Ther. 2001, 89, 1.
2. (a) Andersen, G.; Christrup, L.; Sjøgren, P. J. Pain Symptom. Manage. 2003, 25(1),
74; (b) Donnelly, S.; Davis, M. P.; Walsh, D.; Naughton, M. Supportive Care
Cancer 2002, 10, 13.
treatments for addiction in this research, we are also interested
in the receptor subtype selectivity profiles for the sulfonate ester
and carbamate derivatives synthesized. Overall, we found the
selectivity rank for MOR over DOR to be: 10 > 9 > 6b-naltrexol
HCl > 14 > 13, while the preference for MOR versus KOR binding
follows: 9 > 10 > 14 > 6b-naltrexol HCl > 13. Both sulfonate esters
Table 1
Data from radioligand displacement assays with
l, d, or j-opioid receptors and subtype selectivity ratios for compounds 9, 10, 13, and 14.
Compound
l
opioid receptora (MOR)
d opioid receptora (DOR)
[3H]-DADLE
Ki (nM) SD
j
opioid receptora (KOR)
Opioid Receptor Subtype Selectivity
d/
[3H]-DAMGO
[3H]-U69,593
Ki (nM) SD
Ki (nM) SD
l
j/l
9
10
13
14
0.56 0.09
1.85 0.24
0.79 0.06
1.11 0.13
2.12 0.29
74.6 3.2
334 22
5.16 0.38
93.1 5.5
213 18
14.7 1.0
28.2 1.5
1.66 0.15
6.75 0.73
7.42 0.45
133
180
6.5
83.8
100
26.3
15.2
2.1
6.1
3.5
6b-naltrexol HCl
a
Radioligand-based binding assays were performed using Chinese hamster ovary (CHO) cells that were stably transfected with either human
l, d, or
j
DNA and expressed
the human
l, d, or j
-opioid receptor, respectively.10 All results are standard deviation (SD) with n = 3. Ki values were determined by fitting the pooled data of three curves
(30 data points) to the two parameter logistic equation for the best-fit estimates of the IC50. The Ki value was calculated from the IC50 using standard equations. Assays were
conducted as described in Fontana et al.13