Opioids and efflux transporters. Part 3: P-glycoprotein substrate activity of 3-hydroxyl addition to meperidine analogs

Article abstract of DOI:10.1016/j.bmcl.2008.04.046

Numerous studies have shown that many clinically employed opioid analgesics are substrates for P-glycoprotein (P-gp), suggesting that up-regulation of P-gp may contribute to the development of central tolerance to opioids. The studies herein focus on the development of SAR for P-gp substrate activity in the meperidine series of opioids. Addition of a 3-OH to meperidine and the ketone analog of meperidine yielding bemidone and ketobemidone, respectively, significantly increased P-gp substrate affinity. The results of this study have implications in the development of novel analgesics to be utilized as tools to study the contribution of P-gp on the development of central tolerance to opioids.

Full text of DOI:10.1016/j.bmcl.2008.04.046

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3638–3640
Opioids and efflux transporters. Part 3: P-glycoprotein
substrate activity of 3-hydroxyl addition to meperidine analogs
Susan L. Mercer, Christopher W. Cunningham, Natalie D. Eddington and Andrew Coop*
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 Penn Street, Room 543,
Baltimore, MD 21201, USA
Received 4 March 2008; revised 18 April 2008; accepted 22 April 2008
Available online 25 April 2008
Abstract—Numerous studies have shown that many clinically employed opioid analgesics are substrates for P-glycoprotein (P-gp),
suggesting that up-regulation of P-gp may contribute to the development of central tolerance to opioids. The studies herein focus on
the development of SAR for P-gp substrate activity in the meperidine series of opioids. Addition of a 3-OH to meperidine and the
ketone analog of meperidine yielding bemidone and ketobemidone, respectively, significantly increased P-gp substrate affinity. The
results of this study have implications in the development of novel analgesics to be utilized as tools to study the contribution of P-gp
on the development of central tolerance to opioids.
Ó 2008 Elsevier Ltd. All rights reserved.
Introduction. There is a growing body of evidence that
suggests efflux transporters, specifically P-glycoprotein
(P-gp), may play a role in the development of opioid re-
lated central tolerance and constipation.1–6 Recent stud-
ies have shown that opioids are substrates for P-gp,
although to differing extents,7 and P-gp is up-regulated
at the blood-brain barrier (BBB) of morphine3 (1) and
oxycodone2 (2) (Fig. 1) tolerant rats. Upon chronic
administration, the up-regulated P-gp would be ex-
pected to result in lower brain concentrations of opioid,
thereby exacerbating tolerance to the central analgesic
effects. P-gp knockout animals8 are available and offer
a useful model to study the effects of P-gp on opioids;
however, an alternative approach in wild-type animals
is the development of mu opioid receptor agonists which
are not P-gp substrates. These compounds would allow
a full investigation of the contribution of up-regulated
P-gp to opioid tolerance, as full cross-tolerance between
morphine and the opioid lacking P-gp substrate activity
would not be anticipated to occur. Additionally, opioids
lacking P-gp substrate activity may potentially be devel-
oped into analgesics with lower degrees of tolerance.
activity.7 Therefore, our investigations are focused on
delineating the structure-activity relationship (SAR)
for the addition of a m-OH, while increasing mu opioid
potency based on known SAR for this series.10
Results and discussion. The compounds synthesized are
readily known in the literature as mu opioid analge-
sics;10 however, the syntheses described here are novel
approaches. Meperidine (3) was prepared from nitrile
4 (obtained from Sigma–Aldrich, Inc.), via alkylation
with MeI in DMF in the presence of K2CO3, followed
by aqueous NH4Cl hydrolysis of the N-methyl nitrile 5
to the ethyl ester through treatment with H2SO4 and
EtOH. Treatment of 5 with EtMgBr, via a Grignard
reaction,11 produced the ketone meperidine analog 6
(Scheme 1).
Bemidone 9 was prepared from the condensation of
mechloroethamine hydrochloride and 3-methoxyphenyl-
N
N
H
OH
Meperidine (3), a moderately potent mu opioid analge-
9,10
sic,
has been reported to possess low P-gp substrate
O
O
2
HO
OH
MeO
O
Keywords: Meperidine; P-glycoprotein; Opioids; Tolerance.
*
1
Corresponding author. Tel.: +1 410 706 2029; fax: +1 410 706
5017; e-mail: acoop@rx.umaryland.edu
Figure 1. Morphine (1) and oxycodone (2).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.046

S. L. Mercer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3638–3640
3639
was estimated using the Pgp-Glo assay system13 (Prome-
ga, Madison, WI) and the results are shown in Table 1.
Briefly, this method relies on the ATP dependence of the
light-generating reaction of firefly luciferase where ATP
consumption is detected as a decrease in luminescence,
the greater the decrease in signal the higher the P-gp
activity. Sodium orthovanadate was used as a P-gp
ATPase inhibitor, whereas verapamil was used as a po-
sitive control. All test compounds were analyzed at
200 lM and fold stimulation values were calculated
using Eq. 1. Fold stimulation values greater than 2.0
indicate a P-gp substrate.14
H
N
N
N
(a)
(b)
O
N
N
O
3
4
5
(c)
N
Fold stimulation by a test compound
Test compound stimulated P-gp activity
O
6
¼
ð1Þ
Basal P-gp activity
Scheme 1. Reagents and conditions: (a) MeI, K2CO3, DMF; (b)
H2SO4, EtOH, reflux; (c) EtMgBr, NH4Cl hydrolysis.
The addition of a m-OH into the phenyl ring signifi-
cantly increases the P-gp fold stimulation of meperidine
analogs. Meperidine itself has a P-gp fold stimulation
value of 1.78 and increases to 2.64 with the m-OH addi-
tion (bemidone, 9). Whereas the ketone analog 6, with a
P-gp fold stimulation value of 1.37, increases to 4.89
with the m-OH addition (ketobemidone, 11). Thus, the
addition of a m-OH increases the P-gp substrate activity
of these meperidine analogs, which are members of the
4-phenylpiperidine class of opioids.
acetonitrile (both reagents obtained from Sigma–Aldrich,
Inc.) with NaH and NaOH to yield 7. O-dealkylation of 7
was performed with BBr3 and NH4OH,12 converting the
methoxy group to a phenol 8, followed by nitrile hydroly-
sis to give the m-OH ethyl ester as previously described.4
Treatment of 7 with an EtMgBr Grignard reagent,11 fol-
lowed by aqueous NH4Cl hydrolysis produced 10, which
then underwent treatment with BBr3 to produce ketob-
emidone (11) (Scheme 2).
The hydroxylated meperidine analogs were initially pur-
sued to investigate the relationship between P-gp and in-
creased opioid potency. Interestingly, these results are
consistent with previous studies in our laboratory which
All compounds were converted to their respective salts
(see Table 1). Drug stimulated P-gp ATPase activity
N
N
N
N
(a)
(c)
(b)
HO
O
Cl
O
Cl
O
HO
N
N
O
8
9
7
N
(d)
N
N
(b)
HO
O
O
O
11
10
Scheme 2. Reagents: (a) NaH, NaOH; (b) BBr3, NH4OH; (c) H2SO4, EtOH; (d) EtMgBr, NH4Cl hydrolysis.
Table 1. Fold stimulation values of test compounds prepared, salt form, yield, and melting point
Compound
Name
Salt
Yield (%)
Mp (°C)
Fold stimulation SEM
Non-treated (control)
Meperidine
1.00
3
6
Oxalate
Citrate
Oxalate
Oxalate
7
56
36
51
190–192
170–171
200–202
233–235
1.78 0.39*
1.37 0.19*
2.64 0.82*
4.89 1.94*
Ketone analog
Bemidone
Ketobemidone
9
11
All compounds assayed at 200 lM. Data are represented as fold stimulation SEM (n = 3). *Significant difference (p < 0.05) from control (non-
treated) as determined from t-test. All compounds gave satisfactory CHN ( 0.4%) and spectral analysis (see Supporting Information).

3640
S. L. Mercer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3638–3640
showed that removal of the 3- and 6-OH from morphine
resulted in decreased P-gp substrate activity,5 as mor-
phine is a P-gp substrate.7 These studies attest that the
m-OH substituent increases P-gp substrate activity
across the phenylpiperidine and morphinan classes of
opioids. Furthermore, the development of opioids lack-
ing P-gp substrate activity should not possess a m-OH
substituent. The interaction between opioids and P-gp
is currently under investigation and these results aid in
further SAR development. The ultimate goal is to devel-
op a potent opioid with low P-gp substrate activity for
use as a tool to study the contribution of P-gp up-regu-
lation to the development of opioid tolerance and cross-
tolerance between opioids with P-gp substrate activity
and those without.
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Acknowledgments
The authors thank the National Institute on Drug
Abuse, National Institutes of Health (NIDA, NIH) for
financial support (DA-13583), and also the University
of Maryland School of Pharmacy for partial support.
SLM is supported by a Fellowship from the University
of Maryland, School of Pharmacy. A.C. is supported
by an Independent Scientist Award (K02 DA019634).
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2008.
04.046.
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