Effect of anchoring 4-anilidopiperidines to opioid peptides

Article abstract of DOI:10.1016/j.bmcl.2013.03.065

We report here the design, synthesis, and in vitro characterization of new opioid peptides featuring a 4-anilidopiperidine moiety. Despite the fact that the chemical structures of fentanyl surrogates have been found suboptimal per se for the opioid activity, the corresponding conjugates with opioid peptides displayed potent opioid activity. These studies shed an instructive light on the strategies and potential therapeutic values of anchoring the 4-anilidopiperidine scaffold to different classes of opioid peptides.

Full text of DOI:10.1016/j.bmcl.2013.03.065

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3434–3437
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Effect of anchoring 4-anilidopiperidines to opioid peptides
Ravil R. Petrov a, Yeon Sun Lee a, Ruben S. Vardanyan a, Lu Liu a, Shou-wu Ma b, Peg Davis b, Josephine Lai b,
Frank Porreca b, Todd W. Vanderah b, Victor J. Hruby a,
⇑
a Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA
b Department of Pharmacology, University of Arizona, Tucson, AZ 85721, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here the design, synthesis, and in vitro characterization of new opioid peptides featuring a 4-
anilidopiperidine moiety. Despite the fact that the chemical structures of fentanyl surrogates have been
found suboptimal per se for the opioid activity, the corresponding conjugates with opioid peptides dis-
played potent opioid activity. These studies shed an instructive light on the strategies and potential ther-
apeutic values of anchoring the 4-anilidopiperidine scaffold to different classes of opioid peptides.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 8 January 2013
Revised 11 March 2013
Accepted 20 March 2013
Available online 3 April 2013
Keywords:
Opioid peptide
Dynorphine analog
Bivalent ligand
Fentanyl
Analgesic
The increasing need for effective pain management prompts the
invention of new strategies and pharmacological tools. Among the
resulted in analogs with exceptional properties. One representative
example is biphalin.8 Despite numerous advantages such as low
toxicity, high activity and specificity, opioid peptides are still not
being used as pain-relieving agents in general anesthetic practice
because of main drawbacks such as poor bioavailability after sys-
temic (subcutaneous or oral) administration, limited ability to
cross the blood–brain barrier, and rapid degradation in vivo by
peptidases. Although the fact that fentanyl9–12 is a bioavailable
drug does not directly suggest that its addition to a peptide struc-
ture will yield a bioavailable hybrid, we hypothesized that its
incorporation into opioid peptides may positively impact overall
bioavailability of the latter.
A careful analysis of the literature revealed that in the series of
fentanyl analogs there was no general consensus regarding the ef-
fect of substitution in the propionyl part of the molecule. We thus
set out to prepare the desired compound by using Fmoc- and Pht-
protected amino acid chlorides (Scheme 1). This allowed us to syn-
thesize 3-aminofentanyl and analogs 1–6. The synthesis, NMR
three opioid receptor types (
considered to be essential for efficient pain suppression. However,
-opioids do not provide adequate treatment of chronic pain since
their long-term use results in multiple side effects (e.g.,1). Close
examination of the opioid system conferred that a –d opioid
l, d, and j, the l-opioid receptor is
l
l
receptor heterodimer represents the fundamental signaling unit
responsible for opioid tolerance and dependence.2,3 During the last
two decades, our laboratory has carried out extensive research on
the synthesis of novel opioid analogs. Within the frame of our
works on bivalent opioid ligands there seemed a gap between
small-molecule and peptide-based opioids. We have sought to
bridge this gap by incorporating the 4-ANDP4 scaffolds into opioid
peptides, and also to optimize their physicochemical properties.5–7
Previously, research efforts to produce bivalent opioid peptides
Abbreviations: Aib, aminoisobutyric acid; 4-ANDP, 4-anilidopiperidine; FMPB-AM, 4-
(4-formyl-3-methoxyphenoxy)butyrylaminomethyl; clogP, calculated octanol–water par-
characterization and binding affinity at the
l and d opioid recep-
D
-Ala2,MePhe4,Gly-ol5]enkephalin; DCM, dichloromethane;
tors of compounds 7–12 have been reported previously by us,5
but no results of GPI and MVD assays were provided for this series.
In this report, we provide additional biological data for compounds
7–12. The effects of having the amide substituents in the propionyl
moiety are summarized in Table 1. It is evident from the results
presented in Table 1 that all of these analogs displayed low affinity
tition coefficient; DAMGO, [
DIC, 1,3-diisopropylcarbodiimide; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethyl-
formamide; Dmt, 20,60-dimethyl-(S)-tyrosine; DPDPE, [ -Pen2, -Pen5] enkephalin; EM-1,
D
D
endomorphin-1; F5, pentafluoro; Fmoc, 9-(fluorenylmethoxy)carbonyl; GPI, guinea-
pigileum; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate; HOBt, 1-hydroxybenzotriazole; MBHA, 4-methylbenzhydrylamine; MVD, mouse
vas deferens; NaBH(OAc)3, sodium triacetoxyborohydride; Pht, phthaloyl; PSA, polar surface
area; TEA, triethylamine; TFA, trifluoroacetic acid; TFMSA, trifluoromethane sulfonic acid.
at the
l and d opioid receptors and low activity in MVD and GPI
⇑
Corresponding author. Tel.: +1 520 621 6332; fax: +1 520 621 8407.
E-mail address: hruby@email.arizona.edu (V.J. Hruby).
assays. For the major part, the chemical nature of these fentanyl
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.065

R. R. Petrov et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3434–3437
3435
O
series of analogs (Scheme 2, block A). Although the desired linear
isomers could be obtained in a convenient fashion by the standard
step-by-step solution phase peptide synthesis, it was expected to
be more expedient and efficient to prepare such analogs by apply-
ing solid-phase chemistry. Our attempts to employ FMPB-AM resin
for the synthesis of novel analogs using the reductive amination
technique14 (Scheme 2, block B) resulted in limited success—
although the desired peptide 18 was obtained in the purity exceed-
ing 96%, the yield of the target peptide was rather poor (10%). Our
next alternative methods for making novel peptide conjugates by
the solid phase methods included the use of N- and C-side chain
substitutions. N-side chain modification of a Lys residue was
performed on the resin after assembly of the first amino acid by
removal of the Fmoc or Alloc group followed by treatment with
p-nitrochloroformate and then with 3-aminofentanyl (Scheme 2,
block C). This series also included analogs in which
3-aminofentanyl is linked to a dynorphin A structure.15–17 Further-
more, the Tyr and Phe residues in positions 1 and 4 were replaced
by a Dmt and Phe(4-Cl), respectively, in 21 to facilitate opioid
receptor recognition and enhance metabolic stability. C-side chain
modification of the Glu residue was performed using a combina-
FmocHN
ANDP
Ph
R
HN
N
i
1
: R=H
2: R=Bzl
ii
PhtN
O
H2N
iii
Ph
n
n
ANDP
O
ANDP
4-ANDP
3
: n=1
5
: n=1
4: n=5
6: n=5
O
O
iv
3-Aminofentanyl
R
ANDP
N
H
7-12
Scheme 1. Attachment of Fmoc and phthaloyl amino acids to 4-anilino-1-
phenethyl-piperidine. Reagents and conditions: (i) Fmoc-AA-Cl (AA: Gly, Phe),
DCM/10% NaHCO3 in water, 0 °C (83% and 49%, respectively); (ii) Pht-Gly-Cl, TEA or
DIPEA, DCM, 0 °C; (iii) N2H4ꢀnH2O, ethanol, reflux; (iv) see Ref. 5, R: Me (7), CF3 (8),
Et (9), Ph (10), –NHEt (11), –CH(Bzl)(NHCOCH3) (12).
a
a
tion of N -Fmoc and N -Boc strategies (Scheme 2, block D).
The target compounds were tested for their affinity and potency
in vitro (Table 2). Concerning the functionality of these ligands, no
opioid antagonist activity was observed in the MVD or GPI assays.
Compared to biphalin, analogs 13–18 represent peptide ligands
with higher clogP values (about two- to fourfold) due to the hydro-
phobic character of the fentanyl moiety. As shown in Table 2, the
Table 1
Bioactivities of the 4-anilidopiperidine analogs
d
No.
hDORa
rMORa
IC50
(
l
M)
[3H]DPDPEb
[3H]DAMGOc
Kie
(
lM)
Kie
(
l
M)
MVD (d) GPI (l)
1f
2f
3
1.0
1.0
3.1
2.1
3.2
1.0
0.15
0.45
7.6
n.d.
n.d.
n.d.
n.d.
11%
binding assays reveal that these compounds possess
l receptor
No response
selectivity with binding affinities ranging from 0.09 nM for 14 to
260 nM for 16. In the functional assays, the selectivity of 17 was
13
36
26.4%
14.2%
7.5%
4.1%
4.1%
2.9%
26.1%
6.2%
0.0094
3f
7
5.9 0.89g
5.2 0.21h
3.0 0.89h
1.4 0.48h
3.9 0.22
1.3 0.3
enhanced almost threefold for the d receptor over the
as compared to biphalin and the previously synthesized 13. How-
ever, in this series, despite a slight increase in the receptor selec-
tivity and binding affinity, and the overall lipophilicity, there was a
moderate loss of bioactivity in the GPI functional assays. In con-
l receptor
8.4
12
0.45
4.6
6.6
7.6
8
9
10
11
12
l
0.030
2.7
0.0033
5.6 0.56h
0.0034
l
Fentanyl 0.25
trast, ‘branched’ analogs 23 and 24, in which the 4-ANDP fragment
is attached to the side chain of the Glu residue, showed potency
comparable to that observed with biphalin. These observations
suggested that the branched peptide ligands could be better
accommodated in the opioid binding domain as opposed to the
case of the linear core analogs.
In the endomorphin series, peptides 26 and 27 were very differ-
ent in selectivity and potency from the parent opioid peptide,
EM-1.18 Although 26 and 27 gained moderate binding affinity of
64 and 130 nM, respectively, at the d opioid receptor, both analogs
n.d. not determined.
a
Competition analyses were carried out using membrane preparations from
transfected HN9.10 cells that constitutively expressed the respective receptor
types.
b
Kd = 0.50 0.1 nM.
Kd = 0.85 0.2 nM.
c
d
Concentration at 50% inhibition of muscle concentration at electrically stimu-
lated isolated tissues; these values represent the mean of four tissues within 95%
confidence limit.
e
Competition against radiolabeled ligand, data collected from at least 2 inde-
pendent experiments.
showed a 14- to 28-fold reduction of the affinity for the
l receptor
f
HCl salt.
g
and concomitant reduction in /d potency. Interestingly, the pipe-
l
Naloxone sensitive.
Naloxone insensitive.
h
ridinyl analog 26, which has a longer spacer between the tetrapep-
tide and the 4-ANDP moiety, was slightly more potent than the
analogous 27. Because in both cases the anchoring strategy had
an adverse affect on affinity and potency, it seems that the 4-ANDP
structure disrupts key interactions within the binding site of
endomorphins.
surrogates may not be optimal for the opioid activity in terms of
ionic and hydrophobic interactions.
Although the opioid activities of the fentanyl surrogates 7–12
have turned out to be suboptimal for our original goal, there were
several reasons to further investigate incorporation of these motifs
into opioid peptides. A major reason for the continued interest in
this direction is the fact that even a subtle structural and confor-
mational change of an opioid ligand can greatly influence its over-
all activity profile, physicochemical properties, and biological
efficacy.13 Previously, we showed the synthesis and in vitro data
for the peptide analog 13, which was based on a part of fentanyl
Introduction of the 4-ANDP scaffold into the deltorphin peptide
sequence19,20 (ligands 28 and 29) resulted in significant changes in
binding affinities at the
l and d opioid receptors. The incorporation
of the 4-ANDP through the side chain of the Glu acid residue at a
distance from the opioid message sequence afforded compound
28 with a subnanomolar binding affinity (Ki = 0.28 nM) for d opioid
receptors. As shown in the functional assays, 28 is potent at both d
and
l receptors with IC50 = 6.7 3.9 and 49 16 nM, respectively.
In sharp contrast, the deltorphin analog 29 with the 4-ANDP clo-
sely adjacent to the opioid message region showed a reversed
selectivity compared to deltorphin II and 28. In the binding assay,
structure and ‘one arm’ (the core opioid sequence, H-Tyr-D-
Ala-Gly-Phe) of biphalin.5 In view of the promising bioactivity data
of 13, we then considered replacements for b-alanine for the next
ligand 29 displayed a 16-fold higher selectivity for l over the d

3436
R. R. Petrov et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3434–3437
O
H
OCH3
A
B
N
Ph
O
H
N
5
H-Tyr-D-AA-Gly-Phe-(Leu)m
N
N
n
BAL
Ph
H-Tyr-D-AA-Gly-Phe-Gly
N
N
Resin
HN
13
: AA=Ala, m=0, n=2
O
14: AA=Gly, m=1, n=1
iii, iv
i, ii
15: AA=Abu, m=1, n=1
16: AA=Pro, m=1, n=1
Ph
5
18
N
Ph
Ph
Ph
17: AA=Ala, m=1, n=1
N
C
O
O
O
D
R1HN
R1HN
R4HN
iv, v
i, ii, iii
H
R3
R3
R3
iii
i
ii
NH2
R1
H
R1
R1
R1
H
H
N
H2N
N
O
R2
MBHA
Resin
O
R2
O
Ph
N
Ph
N
Ph
N
R2NH
HN
HN
HN
N
H
O
O
O
N
O
N
H
O
Ph
N
Ph
N
O
H
HN
N
N
O
O
N
Ph
Ph
Ph
23: n=2, R1=Leu, R2=H-Dmt-D-Ala-Gly-(4-Cl)Phe-D-Leu;
N
Ph
O
N
Ph
O
1
2
24
25
26
: n=5, R1=(F5)Phe, R2=H-Tyr-D-Ala-Gly-(4-Cl)Phe;
: n=5, R1=(F5)Phe, R 2=H-Tyr-D-Ala-Gly-Phe-Leu-Arg;
: n=5, R =(4-F)Phe, R =H-Tyr-Pro-Trp-(4-Cl)Phe;
19: R4=H-Tyr-D-Ala-Gly-Phe-D-Leu-Arg, R3=O, method A;
4
3
20
21
22
27: n=1, no R1, R2=H-Tyr-Pro-Trp-Phe;
: R4=H-Tyr-D-Ala-Gly-Phe-D-Leu-Arg-Trp, R =O, method B;3
: R4=H-Dmt-D-Ala-Gly-(4-Cl)Phe-Leu-Arg-D-Arg-Gln-Phe,3R =O, method A;
: R =H-Tyr-D-Ala-Gly-Phe-Leu-Arg-Arg-D-Phe-Arg-Trp, R =Val-NH, method C.
28: n=1, no R1, R2=H-Tyr-D-Ala-Phe-Aib-Val-Val;
1
29
: n=1, R =Val-Val-Gly,R2=H-Tyr-D-Ala-Phe.
Scheme 2. Our strategies to introduce 4-anilidopiperidine functionality to opioid ligands. Reagents and conditions: (A) stepwise solution phase peptide synthesis, analogs
13–17; (B) reductive alkylation on solid support using (i) 4-(4-formyl-3-methoxy-phenoxy)butyryl AM (BAL) resin, NaBH(OAc)3, DMF/DCM (1/3), (ii) Fmoc-AA-OH, HOBt,
HBTU, DIPEA in DMF, (iii) 50% Piperidine in DMF, and (iv) TFA/i-PrSiH3/H2O (90/5/5); (C) N-side chain modification by method A: Merrifield resin (R3@O, R1 = Boc, R2 = Fmoc),
(i) 50% Piperidine in DMF, (ii) p-nitrochloroformate, DIPEA, DCM, 1 h, (iii) 2–3 equiv of 3-aminofentanyl, microwave in a closed vessel for 1 h at 80 °C, (iv) 50% TFA in DCM, (v)
SPPS followed by TFMSA cleavage; method B: 2-chlorotrityl resin (R3@O, R1 = Fmoc, R2 = Alloc), (i) Pd(PPh3)4, PhSiH3 in DCM, (ii) p-nitrochloroformate, DIPEA, DCM, 1 h, (iii) 2–
3 equiv of 3-aminofentanyl, microwave in a closed vessel for 1 h at 80 °C, (iv) 50% piperidine in DMF, (v) SPPS followed by TFA cleavage; method C: MBHA resin (R3 = Val-NH,
R1 = Fmoc, R2 = Boc), (i) 50% TFA in DCM, (ii) p-nitrochloroformate, DIPEA, DCM, 1 h, (iii) 2–3 equiv of 3-aminofentanyl, microwave in a closed vessel for 1 h at 80 °C, (iv) 50%
piperidine in DMF, (v) SPPS followed by TFMSA cleavage; (D) C-side chain modification using (i) Fmoc-Glu(Boc)-OH, HOBt, DIC, DMF ? 50% TFA in DCM ? 5% DIEA in
DCM ? amine (1, 5, 3-aminofentanyl), HOBt, DIC, microwave in DMF for 5 min at 80 °C ? 25% piperidine in DMF, (ii) solid phase peptide synthesis, and (iii) TFMSA cleavage.
opioid receptors. However, 29 was 18- and 39-fold less potent at
d-receptors and -receptors, respectively, than the related
somewhat lower than that observed for 19 (31-fold selectivity
for the receptor). Analog 22 was almost 13-fold more potent than
l
l
compound 28 in the functional assays. In line with the previous
observations, these results indicate that in addition to endowing
molecules with higher lipophilicity, incorporation of the 4-ANDP
the corresponding 19 in the GPI functional assay, although it also
showed a moderate threefold loss of potency in the MVD assay.
Ligands 20 and 25 were endowed with subnanomolar affinities of
0.26 and 0.63 nM, respectively, and selectivity (to twofold in both
cases) for d opioid receptors. Noticeably, most of the dynorphin A
structure confers
l opioid receptor selectivity. However, in the
case of deltorphins, a synergistic or potentiating antinociceptive
effect of anchoring the 4-ANDP structure is questionable.
ligands displayed subnanomolar affinities at the
l opioid receptor.
In the dynorphin A series, potent opioid agonist activities for
both receptors were observed in several ligands with binding affin-
ities in the nanomolar and subnanomolar ranges. In this category,
the introduction of 4-ANDP structure increased the lipophilicity of
Moreover, compounds from this series also maintained good opi-
oid potency with the 4-ANDP modification. This is in agreement
with the earlier model for the dynorphin A binding pocket,21 and
is also consistent with previous results, which showed that intro-
duction of lipophilic residues into dynorphin A structure resulted
in more potent analogs.22 It might be well to point out that
although the high molecular weights and PSA values of the dynor-
phin A analogs are prohibitive of their CNS exposure, the presence
of multiple positively charged residues in their structures suggests
the possibility of their transport across biological membranes via
the adsorptive-mediated endocytosis by binding to negatively
charged sites on the surface of brain capillary endothelial cells.23
In conclusion, binding assays showed that incorporation of the
4-anilidopiperidine moiety strongly impacts both affinity and
functional activity of the opioid peptides. We demonstrated that
anchoring the 4-ANDP structure to short ekephalin-related or
the parent peptide. The binding affinity of 21 at the
l receptor was
subnanomolar (Ki = 0.15 nM), and nanomolar at the d receptor
(Ki = 1.8 nM), which represents almost a 13-fold improvement in
selectivity for
l-opioid receptors as compared to Dyn A-(1-13).
In the functional assays, a reversed trend was observed: analog
21 was about twofold more potent than the parent peptide in
the MVD (IC50 = 3.2 1.3 nM) assay, but threefold less potent in
the GPI (IC50 = 3.8 0.8 nM) assay. In the [35S]GTP-
c-S assays this
compound had a EC50 value of 0.34 at the
l, and 0.2 nM at the d
opioid receptors. How much of an improvement both in affinity
and bioactivity was introduced by the 4-ANDP attachment alone
in this case, however, is a question that remains to be answered.
In terms of useful opioid design, this result is more likely to be
attributed to the introduction of the Dmt residue, as observed in
many cases. A similar trend in affinity was observed for analogs
19 and 22. Analog 22 presents a case where a correlation in selec-
tivity is maintained between binding affinities and bioactivities at
dynorphin
A peptide structures represents a useful tactical
approach for further enhancement of opioid potency. Attachment
of the 4-ANDP moiety to the short ekephalin-related analogs ap-
pears to be a logical choice, considering the relatively small size,
more lipophilic nature and high potency at both
l and d opioid
the
reasonable ninefold selectivity for the
over the receptor (Ki = 4.6 nM) in binding assays, albeit
l
and d opioid receptors. Similarly to 19, ligand 22 showed a
receptors. In case where the fentanyl moiety was conjugated to
the dynorphin A, a superior opioid activity profile was reached to
dynorphin A itself. The present findings suggest, however, that
l
receptor (Ki = 0.54 nM)
d

R. R. Petrov et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3434–3437
3437
Table 2
Bioactivities of the designed peptide analogs
e
No.
hDORa
rMORa
[
35S]GTP-
c
-S binding
IC50 (nM)
[3H]DPDPEb
[3H]DAMGOc
hDORd
rMORd
EC50 (nM)g
g
h
Ki (nM)f
Ki (nM)f
EC50 (nM)
Emax (%)
Emax (%)h
MVD (d)
GPI/LMMP (l)
13i
1.1
0.99
12
0.90
0.97
0.09
0.43
60
60
37
5.8
140
140
49
60
69
45
35
140
140
43
33
35
180 20
22
6
6
110 50
42 16
950 130
97 18
13j
14
15
2.8
22
77
5
16
630
27
4.0
11
0.26
1.8
4.6
0.35
0.51
0.63
84
590
0.28
24
2.6 0.4
5100 660
1.3
260
13
0.73
0.35
0.50
0.15
0.54
0.17
0.28
1.5
64
130
9000
1.5
1.4 0.2
4.6 0.2
500 40
4.0 0.4
n.r.
5.9
46
n.r.
0.59
9.1
2.8
1.5
0.20
1.4
380
2.2
12
56.
7500 700
9.5 2.9
360 170
8.6 1.7
580 150
86 16
150 40
98 24
120 20
3.8 0.8
7.7 2.1
17
64
120
84
19
10
7
11
17
27
13
50
31
59
32
31
45
68
29
21
18k
19
20
21
22
23
24
25
26
2.2
0.22
0.34
8.3
170
1.1
16
4
3.2 1.3
27 8.1
40
8.8 3.0
12
5
12
2
67 15
130 60
480 910
2100 30
49 16
2.8
2
n.r.
n.r.
1.7
90
2.5 0.5
510 130
1000 200
6.7 3.9
27
28
29
Biphalin
Endomorphin-1
Deltorphin II
Dyn A-(1-13)-OH
n.r.
n.r.l
220
6.0 0.2
59
33
27
21
25
120 20
1900 40
8.8 0.3
4
5
27
26
2
3
11
1
0.37 0.04
6.3 0.9
>3000
1.2 0.1
3.8 0.4
n.r. no response.
a
Competition analyses were carried out using membrane preparations from transfected HN9.10 cells that constitutively expressed the respective receptor types.
Kd = 0.50 0.1 nM.
Kd = 0.85 0.2 nM.
Expressed from CHO cell.
b
c
d
e
Concentration at 50% inhibition of muscle concentration at electrically stimulated isolated tissues; these values represent the mean of four tissues within 95% confidence
limit.
f
Competition against radiolabeled ligand, data collected from at least 2 independent experiments in duplicate.
g
h–g
i–h
j–i
Anti-logarithmic value of the respective EC50
Net total bound/basal binding ꢁ 100 SEM.
Compound 13 is a hydrochloride salt of 13.
Effect is not reversed by naloxone.
Tested in the free base form.
.
k–g
l-k
Tested as a trifluoroacetate salt.
7. Lee, Y. S.; Kulkarani, V.; Cowell, S. M.; Ma, S.-W.; Davis, P.; Hanlon, K. E.;
Vanderah, T. W.; Lai, J.; Porreca, F.; Vardanyan, R.; Hruby, V. J. J. Med. Chem.
2010, 54, 382.
8. Horan, P. J.; Mattia, A.; Bilsky, E. J.; Weber, S.; Davis, T. P.; Yamamura, H. I.;
Malatynska, E.; Appleyard, S. M.; Slaninova, J.; Misicka, A.; Lipkowski, A. W.;
Hruby, V. J.; Porreca, F. J. Pharmacol. Exp. Ther. 1993, 265, 1446.
9. Wade, C. L.; Schuster, D. J.; Domingo, K. M.; Kitto, K. F.; Fairbanks, C. A. Eur. J.
Pharmacol. 2008, 587, 135.
due to very weak opioid activity offered by the 4-ANDP conjugates,
other similar sized, lipophilic adducts might be equally effective and
easier to incorporate for the same purpose. Taken together, the re-
sults of these studies shed an instructive light on the development
of mixed classes of opioid agonists as analgesic drug candidates.
10. Wheeler, M.; Birmingham, P. K.; Lugo, R. A.; Heffner, C. L.; Coté, C. J. Anesth.
Analg. 2004, 99, 1347.
Acknowledgment
11. Tamura, M.; Nakamura, K.; Kitamura, R.; Kitagawa, S.; Mori, N.; Ueda, Y. Eur. J.
Anaesthesiol. 2003, 20, 482.
12. Colpaert, F. C.; Tarayre, J. P.; Alliaga, M.; Bruins Slot, L. A.; Attal, N.; Koek, W.
Pain 2001, 91, 33.
The work was supported by Grants from the USDHS, National
Institute on Drug Abuse (DA-12394 and DA-06284).
13. Liu, W. X.; Wang, R. Med. Res. Rev. 2012, 32, 536.
Supplementary data
14. Yamamoto, T.; Nair, P.; Jacobsen, N. E.; Vagner, J.; Kulkarni, V.; Davis, P.; Ma, S.-
W.; Navratilova, E.; Yamamura, H. I.; Vanderah, T. W.; Porreca, F.; Lai, J.; Hruby,
V. J. J. Med. Chem. 2009, 52, 5164.
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.03.
065.
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