Opioids and efflux transporters. Part 1: P-Glycoprotein substrate activity of N-substituted analogs of meperidine

Article abstract of DOI:10.1016/j.bmcl.2006.12.042

P-Glycoprotein (P-gp) is an efflux transporter which is up-regulated at the blood-brain barrier in both morphine- and oxycodone-tolerant rats. Numerous studies have shown that many clinically employed opioid analgesics are substrates for 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 compounds, and show that a meperidine analog of greater potency, N-phenylbutyl-N-normeperidine, has low activity as a P-gp substrate and has the potential to be utilized as a tool to study the contribution of P-gp to the development of central tolerance to opioids.

Full text of DOI:10.1016/j.bmcl.2006.12.042

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1160–1162
Opioids and efflux transporters. Part 1: P-Glycoprotein substrate
activity of N-substituted analogs of meperidine
Susan L. Mercer, Hazem E. Hassan, Christopher W. Cunningham,
Natalie D. Eddington and Andrew Coop*
Department of Pharmaceutical Sciences, University of Maryland, School of Pharmacy, 20 Penn Street, Room 637,
Baltimore, MD 21201, USA
Received 14 November 2006; revised 9 December 2006; accepted 11 December 2006
Available online 21 December 2006
Abstract—P-Glycoprotein (P-gp) is an efflux transporter which is up-regulated at the blood–brain barrier in both morphine- and
oxycodone-tolerant rats. Numerous studies have shown that many clinically employed opioid analgesics are substrates for 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 compounds, and show that a meperidine analog of
greater potency, N-phenylbutyl-N-normeperidine, has low activity as a P-gp substrate and has the potential to be utilized as a tool to
study the contribution of P-gp to the development of central tolerance to opioids.
Ó 2006 Elsevier Ltd. All rights reserved.
The development of improved agents for the treatment
of chronic pain remains an important goal in public
health.1 The vast majority of currently employed agents
for the treatment of severe chronic pain are opioid anal-
gesics, which act as agonists at mu opioid receptors in
the CNS.2 Unfortunately, clinically employed opioid
analgesics suffer from the development of tolerance,
necessitating escalating doses to maintain the patient
in a pain-free state,3 thereby leading to escalated side-ef-
fects such as constipation.4,5 Numerous mechanisms at
the receptor and cellular level have been indicated in
the development of tolerance to mu opioid receptor ago-
nists,6 but recent reports have suggested that efflux
transporters at the blood–brain barrier (BBB) may also
contribute toward the development of central toler-
ance.7,8 P-glycoprotein (P-gp) is an efflux transporter
which is located in numerous tissues,9 and its function
at the BBB is to actively remove xenobiotics from the
CNS.9 Two commonly employed opioid analgesics,
morphine (1) and oxycodone (2) (Fig. 1), are known
substrates for this transporter and rats tolerant to both
morphine8 and oxycodone7 show up-regulation in P-gp
level at the BBB. Thus, on chronic administration, the
up-regulated P-gp would be expected to result in lower
brain concentrations of opioid thereby exacerbating tol-
erance to the central analgesic effects. P-gp knockout
animals are available and offer a useful model to study
the effects of P-gp on opioids,10 but an alternative ap-
proach in wild-type animals is the development of mu
opioid receptor agonists which are not substrates for
P-gp. These compounds would allow a full investigation
of the contribution of up-regulated P-gp to opioid toler-
ance as full cross-tolerance between morphine and the
opioid with no P-gp substrate activity would not be
anticipated to occur, and also potentially be developed
into opioid analgesics with lower degrees of tolerance.
The mu opioid analgesic meperidine (3) has been shown
to possess low activity as a P-gp substrate,11 but only
moderate antinociceptive activity in vivo,12,13 thus initial
investigations described herein are focused on delineat-
ing the structure–activity relationships (SAR) of the
NMe
H
NMe
OH
O
O
HO
OH
MeO
O
Keywords: Opioid; Tolerance.
*
2
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 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.042

S. L. Mercer et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1160–1162
1161
Table 1. Compounds prepared, salt form, yield, and melting points
R
N
H
N
R
Ester
Salt, yield (%), mp (°C)
a, b
CH3
314
516
617
717
814
918
1019
11
Oxalate, 7, 190–192
Oxalate, 33, 205–206
Oxalate, 14, 225
O
(CH2)2(C6H5)
(CH2)3(C6H5)
(CH2)4(C6H5)
CH2(C6H5)
N
O
Oxalate, 25, 170
4
3, 5-14
Oxalate, 46, 204–205
Oxalate, 40, 213–214
Oxalate, 57, 215–216
Oxalate, 67, 173–177
Oxalate, 24, 190–192
Oxalate, 35, 180–181
Oxalate, 55, 165–167
CH2CH@CH2
(CH2)2CH3
Scheme 1. Reagents and condition: (a) RX, K2CO3, DMF; (b) H2SO4,
EtOH, reflux.
CH2CH@CHCH3
(CH2)3CH3
1219
13
CH2C(CH3)@CH2
CH2CH(CH3)2
14
N-substituent in this class for low P-gp substrate activi-
ty, while increasing mu opioid potency based on known
SAR for potency in this series.13
Citations reference of previously known compounds.
lower than the control (NT) are P-gp substrates,
whereas test compounds significantly higher than the
NT are P-gp inhibitors. Compounds which are not sig-
nificantly different from the NT are neither P-gp sub-
strates nor inhibitors.
A range of previously reported and novel N-substituted
analogs of meperidine were prepared from nitrile 4
(obtained from Sigma–Aldrich, Inc.), via alkylation with
alkyl halides in DMF in the presence of K2CO3, followed
by hydrolysis of the nitrile to the ethyl esters (5–14)
through treatment with H2SO4 and EtOH14 (Scheme 1).
The alkyl substituents were chosen based on known active
meperidine analogs and also following a series as previ-
ously described for other classes of opioids,15 and include
arylalky, alkyl, and branched alkyl groups (Table 1).
The P-gp substrate activity of the esters showed differ-
ences depending on the nature of the N-substituent.
Most analogs were substrates for P-gp like verapamil,
with the exception of meperidine itself and N-phenylbu-
tyl-N-normeperidine (7). These results show a distinct
SAR for P-gp substrate activity in this series as N-phen-
ylalkyl analogs of shorter length (phenethyl (5), phenyl-
propyl (6)) were substrates.
All esters were converted to oxalate salts (Scheme 1).
Drug stimulated P-gp ATPase activity was estimated
by Pgp-Glo assay system20 (Promega, Madison, WI)
and the results are shown in Figure 2. 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 orthovana-
date was used as a P-gp ATPase inhibitor, whereas
verapamil was used as a positive control. Test com-
pounds (all tested at 200 lM) which are significantly
Previous studies have shown that 7 has twice the antin-
ociceptive activity as meperidine.13 Thus, 7 has the pro-
file of low P-gp substrate activity and greater potency
than meperidine required for use as a tool to study the
contribution of P-gp upregulation to the development
of opioid tolerance and cross-tolerance between opioids
with P-gp substrate activity and opioids that are not
P-gp substrates. Studies on this compound are currently
underway and will be reported in due course.
2500.00
*
*
= P value <0.05
2000.00
*
*
*
*
*
*
*
1500.00
*
*
*
1000.00
500.00
0.00
Figure 2. Results of compounds and standards in the Pgp-Glo assay system; all compounds assayed at 200 lM. Data are represented as
means SEM (n = 4). Indicates significant difference from the control at p < 0.05 as indicated by t-test.
*

1162
S. L. Mercer et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1160–1162
6. Kieffer, B. L.; Evans, C. J. Cell 2002, 108, 587.
Acknowledgments
7. Hassan, H. E.; Myers, A. L.; Lee, I. J.; Coop, A.;
Eddington, N. D. J. Pharm. Sci. (accepted for
publication).
8. Ambudkar, S. V.; Kimchi-Sarfaty, C.; Sauna, Z. E.;
Gottesman, M. M. Oncogene 2003, 22, 7468.
9. Aquilante, C. L.; Letrent, S. P.; Pollack, G. M.; Brouwer,
K. L. Life Sci. 2000, 66, 47.
10. Xie, R.; Hammarlund-Udenaes, M.; de Boer, A. G.; de
Lange, E. C. Br. J. Pharmacol. 1999, 128, 563.
11. Dagenais, C.; Graff, C. L.; Pollack, G. M. Biochem.
Pharmacol. 2004, 67, 269.
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.
AC is supported by an Independent Scientist Award
(K02 DA019634).
Supplementary data
12. Janssen, P. A. J.; Eddy, N. B. J. Med. Pharm. Chem. 1960,
2, 31.
13. Casy, A. F.; Parfitt, R. T. Opioid Analgesics: Chemistry
and Receptors; Plenum Press: New York, 1986, Chapter 6.
14. Eisleb, O. Chem. Ber. 1941, 74, 1433.
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.
2006.12.042.
15. McLamore, S.; Ullrich, T.; Rothman, R. B.; Xu, H.;
Dersch, C.; Coop, A.; Davis, P.; Porreca, F.; Jacob-
son, A. E.; Rice, K. C. J. Med. Chem. 2001, 44,
1471.
16. Perrine, T. D.; Eddy, N. B. J. Org. Chem. 1956, 21,
125.
References and notes
1. Tabakian, H. M. Mol. Med. 2005, 102, 456.
2. Pasternak, G. W. Neuropharmacology 2004, 47, 312.
3. Zieglgansberger, W.; Tolle, T.; Zimprich, A.; Hollt, V.;
Spanagel, R. Pain Brain 1995, 22, 439.
17. Elpern, B.; Gardner, L. N.; Grumbach, L. J. Am. Chem.
Soc. 1957, 79, 1951.
4. McNicol, E.; Horowicz-Mehler, N.; Fisk, R. A.; Bennett,
K.; Gialeli-Goudas, M.; Chew, P. W.; Lau, J.; Carr, D.
J. Pain 2003, 4, 231.
5. De Luca, A.; Coupar, I. M. Pharmacol. Ther. 1996, 69,
103.
18. Thorp, R. H.; Walton, E. J. Chem. Soc. 1947, 559.
19. Walton, E.; Green, M. B. J. Chem. Soc. 1945, 315.
20. Ambudkar, S. V.; Dey, S.; Hrycyna, C. A.; Ramachandra,
M.; Pastan, I.; Gottesman, M. M. Annu. Rev. Pharmacol.
Toxicol. 1999, 39, 361.