Synthesis and characterizations of novel quinoline derivatives having mixed ligand activities at the κ and μ receptors: Potential therapeutic efficacy against morphine dependence

Article abstract of DOI:10.1016/j.bmc.2009.07.024

Based on an established 3D pharmacophore, a series of quinoline derivatives were synthesized. The opioidergic properties of these compounds were determined by a competitive binding assay using ¹²⁵I-Dynorphine, ³H-DAMGO and ¹²⁵

Full text of DOI:10.1016/j.bmc.2009.07.024

Bioorganic & Medicinal Chemistry 17 (2009) 5782–5790
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterizations of novel quinoline derivatives having
mixed ligand activities at the
j and l receptors: Potential therapeutic efficacy
against morphine dependence
Ishani Deb a, Priyankar Paira b, Abhijit Hazra b, Sukdeb Banerjee b, Pradip Kumar Dutta a,
Nirup Bikash Mondal b, Sumantra Das a,
*
a Neurobiology Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, India
b Chemistry Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on an established 3D pharmacophore, a series of quinoline derivatives were synthesized. The opio-
idergic properties of these compounds were determined by a competitive binding assay using 125I-Dynor-
Received 23 May 2009
Revised 11 July 2009
Accepted 14 July 2009
Available online 18 July 2009
phine, 3H-DAMGO and 125I-DADLE for
of activities of the compounds to and
j,
l
, and d receptors, respectively. Results showed varying degree
j
l opioid receptors with negligible interactions at the d receptor.
The compound, S4 was the most successful in inhibiting the two most prominent quantitative features of
naloxone precipitated withdrawal symptoms - stereotyped jumping and body weight loss. Determination
Keywords:
Quinoline
Morphine
Withdrawal
Opioid
of IC50 of S4 revealed a greater affinity towards
atives of S4 like structure offer potential tool for treatment of narcotic addictions.
Ó 2009 Elsevier Ltd. All rights reserved.
l compared to j receptor. In conclusion, quinoline deriv-
Receptor
1. Introduction
Since unlike mu-opioid receptors,
sidered to mediate tolerance and dependence several classes of
-opioid ligands have been explored, such as non peptide -agonist
j-opioid receptors is not con-
Narcotic addiction is perhaps the most common example of
drug abuse worldwide. Unlike treatment of other simple or com-
plex diseases, treatment of addiction has a unique disadvantage
of high incidence of relapse. Therefore, the approach for the treat-
ment of narcotic addiction, over the years, has been aimed at con-
trolling the withdrawal symptoms that occur after prolonged
exposure to the drug of abuse. The first meaningful therapeutic ap-
proach towards treatment of opioid addiction was ushered about
fifty years ago, by the development of methadone,1 which is itself
a behaviorally selective analgesic. However methadone was beset
with the problem of high incidence of relapse when the drug is dis-
continued, although it was effective while the treatment is main-
tained. Subsequently, many other compounds like the methadone
congener LAAM (levo-alpha-acetyl-methadol), clonidine,2 L-type
calcium channel blockers,3 NMDA antagonists,4 Buprenorphine,5
and naltrexone6 have been developed and tried for the treatment
of narcotic addiction with limited success. Additionally, nitric
oxide synthase inhibitors,7 NMDA blockers4,8 or recently isoquino-
line derivatives9 have also been investigated for anti-addictive
properties.
j
j
benzomorphans (cyclazocine, bremazocine and 8-CAC), arylaceta-
mide (U50488, U69593 and R-84760), epoxymorphinans (nalfura-
fine), ibogaine (a naturally occurring indole alkaloid). These drugs
or kappa ligands effectively modulate/attenuate some of the as-
pects of addictive substances, such as cocaine, morphine, metham-
phetamine, etc.10 Unfortunately, these compounds exhibited
behavioral side effects such as sedation, emesis, vomiting11, raising
questions of their uses. Analysis of the effectiveness of
for treatment of addiction versus its side effects revealed that, in
compounds, which have additional agonist/antagonist activity,
j-agonist
l
the side effect is significantly reduced and improvement occurs
in their therapeutic efficacy.12
There is a growing hypothesis suggesting that a ligand having
significant affinity to all three opioid receptors, but with different
combinations of agonistic and antagonistic properties at these
receptors, may prove to be a promising, behaviorally selective
analgesic with diminished side effects.13 Structural analysis of a
series of compounds showing non-specific activity to all three opi-
oid receptors suggested a common 3D pharmacophoric element for
the recognition of the three opioid receptors. This common 3D
recognition pharmacophore at l, j and d opioid receptors consists
of four components—a protonated amine, two hydrophobic groups
and the centroid of an aromatic ring- in a geometric arrangement,
* Corresponding author. Tel.: +91 33 24735197/36793/24049; fax: +91 33
24735197/30284.
E-mail addresses: sdas@iicb.res.in, sumantra00@yahoo.com (S. Das).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.024

I. Deb et al. / Bioorg. Med. Chem. 17 (2009) 5782–5790
5783
common to all binders at the three opioid receptors. A somewhat
similar hypothesis based on Beckett and Casy’s 3-point model sug-
gested a structure containing an amine that is thought to be pro-
tonated at physiological pH, a phenolic ring and a hydrophobic
site in order for the opioid ligand to bind non-selectively to all
the three receptor types.14
Based onthe established3D pharmacophores for opioid ligands, a
series of compounds were synthesized having quinoline motifs. The
present study provided the evidence that quinoline compounds are
effective to attenuate few features of opiate dependence. Few of
these compounds, S4 [C18H17N3O; 2-(2-methylquinolin-4-ylami-
no)-N-phenylacetamide], NS1 [2-(2-methylquinolin-4-ylamino)-
N-p-tolylacetamide], etc., were assessed for their activity on
antagonizing drug dependence which was manifested by their abil-
ity to control abstinence syndrome following naloxone precipitation
in chronically morphine treated mice.
erties by substituting functional groups of the opioid antagonists,
which are used for the treatment of drug abuse, broadens their
therapeutic scope by facilitating their route of administration.16
The enrichment of structure activity relationship study is also ob-
served in the report of synthesis of a series of homo- and hetero-di-
meric ligands containing
pharmacophore joined by a linker chain of varying length.17 Eval-
uation of the binding affinity of these compounds at and d
j-agonist and l-agonist/antagonist
l, j
receptors suggest that the stereochemistry of the pharmacophore,
viz. N-substituents, ester linkages and the spacer length were cru-
cial factors for optimum interactions of such ligands at opioid
receptor binding sites. Analysis of the binding properties of a series
of synthetic dimeric morphinan ligands to three opioid receptors18
showed a few dimeric structures possessing significant
and partial agonist like its monomeric counterpart while another
demonstrated partial agonistic property to both and receptor.
j agonist
l
j
l
The quinoline derivative S4 [C18H17N3O; 2-(2-methylquinolin-
4-ylamino)-N-phenyl acetamide], reported in the present study,
affords a structure, which has similar components of the 3D phar-
macophore proposed by others,14 having a side-arm structure, pos-
sessing a centroid aromatic ring at the free terminal, a hydrophobic
group and a protonated amine. Receptor binding also demon-
2. Results and discussion
2.1. Chemistry
Various chloro-N-phenylacetamides (S4, PP-2, PP-3, PP-5, NS-1)
or chloro-propionamides (PP-7, PP-10) were synthesized by react-
ing chloroacetyl chloride or chloro propionyl chloride with substi-
tuted anilines or cyclohexyl amines (PP-6) or dichloroacetyl
chloride with 4-aminoquinaldine. The dichloro ethanone deriva-
tive (PP-4) was synthesized by Friedel–Crafts acylation of toluene
with dichloroacetyl chloride. These chloro-substituted acetamides
or propionamides or ethanones were then condensed with 4-
aminoquinaldine (PP-11) in the presence of sodium hydride in
dry DMSO under nitrogen atmosphere giving rise to the products
(Scheme 1, Table 1). However, for the preparation of PP-4, where
the condensation was effected between 4-amino quinaldine and
2,2-dichloro-1-p-tolylethanone, an iodoform like reaction took
place giving rise to the benzamide derivatives by the elimination
of dichloromethane due to the presence of two electron withdraw-
ing chlorine atoms in 2,2-dichloro-1-p-tolylethanone (Scheme 2).
For the preparation of PP-6 using N-cyclohexylchloroacetamide,
two units of the acetamide derivative were condensed with 4-
aminoquinaldine or PP-11 (Scheme 3). All the compounds were
separated by column chromatography over silica gel (60–120
mesh) eluting with different ratio of chloroform–methanol mixture
and were characterized by their spectroscopic analysis (IR, 1H, 13C,
and MS).
strated that S4 interacts at the
l- as well as j-opioid receptor
but had no effect at the d receptor (Table 1). We have also looked
at the opioid receptor binding characteristics of similar type of
quinolines where the side arm has been modified (Table 1). In
addition to the side arm, since all the compounds contained a quin-
oline moiety, we also looked at the binding characteristics of PP10,
having only the side chain structure, and at PP11 and PP12, having
only the quinoline core, essentially to establish the contributions of
the two basic components in the structure in inducing opioidergic
activity.
Displacement of specific [3H] DAMGO binding to evaluate the
interactions at the
l-opioid receptor showed that all compounds,
excepting PP12 and PP11, were able to displace the specific [3H]
DAMGO binding. The absence of MOR interaction in both PP11
and PP12 suggested that the quinoline moiety might not contrib-
ute towards l-opioidergic activity. The order of potency of the test
compounds in interacting with MOR was as follows: PP7 > PP3 >
S4 ꢀ NS1 > PP2 > PP6 > PP4 ꢁ PP10 > PP5; PP11 and PP12 were
inactive.
Displacement of specific [125I] Dynorphin binding were carried
out with membranes prepared from C6 glioma cells stably trans-
fected with hKOR (human kappa opioid receptor) primarily due
to its low tissue binding in rodent brain and also rule out any inter-
action of dynorphin with nociceptin receptors.19 While the KOR
2.2. Biology
specific ligand, U50488H, at 10 l
M, inhibited specific [125I] Dynor-
phin binding by approximately 80%, the test compounds, with the
exception of PP6, exhibited different degree of interaction at KOR
binding site at similar concentrations. Among all test compounds,
S4 demonstrated the highest displacement of specific binding of
There have been serious attempts, in the past, to develop com-
pounds having considerable activity at the opiate receptors based
on existing knowledge of activities of similar structures. Enhance-
ment of a particular activity (agonist/antagonist) has been exhib-
ited by introducing the functional group 14-alkoxy in N-methyl-
6-ketomorphinans, which not only enhances its affinity to opioid
receptor, but also increases antinociceptive potency of that com-
pound.15 It is also found that enhancement of the lipophilic prop-
[
125I] Dynorphin binding followed by PP3 and then PP2. Unlike lack
of binding at MOR, PP11 and PP12 showed significant interactions
at KOR suggesting contributions of the quinoline structure for
binding to KOR. On the other hand, the compound PP10, consisting
R
O
(CH2)n
CH3
NH
HN
N
NH2
R
NaH/DMSO
O
NH
(CH2)nCl
N2, 700 C-800 C
N
CH3
PP-11
PP-analogues
Scheme 1. General reaction procedure for preparation of PP-analogues.

5784
I. Deb et al. / Bioorg. Med. Chem. 17 (2009) 5782–5790
Table 1
of only the side arm, displayed binding to both KOR and MOR.
Moreover, displacement of [125I] Dynorphin binding by PP10 was
much greater compared to that by PP11 or PP12, suggesting the
Yield and structure of the quinoline derivatives synthesized in the laboratory
Products
Yield (%)
Structure
importance of the side chain for imparting
j-activity in the com-
O
pounds. PP6, which showed interaction at MOR binding site, had
no effect at KOR site. Overall, the order of potency of the test com-
pounds in interacting with KOR was as follows: S4 > PP3 >
PP2 > PP7 ꢁ PP4 ꢁ PP10 > PP11 > PP5 ꢁ PP12 > NSI; PP6 was inac-
tive (Table 2).
HN
C
N
H
H2
S4
90
N
CH3
O
On the contrary, studies on the interaction of the test com-
pounds at d-opioid receptor showed that none of the compounds,
with the exception of PP7, had any appreciable effect in displacing
specific [125I] DADLE binding. Surprisingly, PP7 increased specific
HN
C
H2
N
H
Cl
PP-2
PP-3
PP-4
PP-5
92
90
90
91
[
125I] DADLE binding by about 70%. Affinity studies for the interac-
N
CH3
tion at the
culating the IC50 values. It was observed that S4 had high affinity
for both - and -opioid receptors, which was comparable to that
l- and j-opioid receptors by S4 were evaluated by cal-
O
l
j
HN
C
H2
N
H
OCH3
of selective ligands like DAMGO and U50488H for the respective
receptors (Fig. 1).
Overall, results from the binding studies suggest a vital influ-
ence of the side-arm structure as well as its linkage with the nitro-
gen-containing ring of quinoloine in imparting opioidergic activity
in the compounds. Of the three essential components of the side-
arm structure, it appears that the presence of phenyl ring (without
any substitution) at the free end of the chain and also free rotation
of this phenyl ring is vital for KOR binding activity since S4 has a
greater activity than other compounds such as PP3, PP2, etc. The
importance of the terminal phenyl ring structure in S4 can be ex-
plained briefly by comparing the same in other compounds, such
as PP2, PP3. The compounds PP2 and PP3 have almost similar
structures, differing only in the substitution in the phenyl ring with
–Cl at ortho position for PP2 and –OMe at para position for PP3. The
N
CH3
O
HN
CH3
N
CH3
Cl
O
HN
N
C
H2
N
H
Cl
comparatively lower activity of PP2 at the
j receptor may be due to
CH3
the presence of –Cl at ortho position causing steric hindrance here-
by the terminal phenyl ring is unable to remain in the same plane
with the rest of the side chain. Considering the structure of PP3
compound, due to the +I effect of –OMe group on the phenyl ring,
that is, the electron donating property, raised the possibility of cre-
ating a partial double bond between the phenyl ring and nitrogen
atom adjacent to the phenyl ring resulting in restricted rotation of
terminal phenyl ring. Interestingly, the importance of free rotation
of terminal phenyl ring has been observed for a number of known
nitrogenous KOR ligand including U69593, and U50488H.
H
N
H
N
N
N
O
O
PP-6
PP-7
88
90
CH3
O
HN
N
C
C
H2 H2
N
H
CH3
For binding to
l-opioid receptors, the phenyl ring of the side-
CH3
O
arm structure also appeared to be important for the tested com-
pounds. It was also supported from the data of PP12, which has a
side chain where the Ph ring is absent, was unable to bind to
PP-10
PP-11
96
—
MOR. A number of
l-specific compounds like morphine, codeine
NH
CH2CH2Cl
and heroin also possess benzene ring with substitutions by –OH,
–OCH3 and –OCOCH3, respectively. In such cases the benzene ring
is restricted in the space, due to its bonding to other components in
the structure. Thus unlike, KOR ligands, free rotation does not ap-
pear to be essential for MOR binding. Even, higher degree of inter-
action of PP3 and PP7 to MOR, compared to S4 could be due to the
presence of restricted rotation of terminal phenyl ring.
NH2
N
O
CH3
Cl
HN
Both
j- and l-opioid receptor agonists and not antagonist’s
PP-12
94
92
causes transient activation on pERK20 in cultured cells within a
few minutes of application. Receptor specific antagonists also
inhibited such stimulatory effect on pERK by the opioid agonists.
The above observations were applied in primary cultures of astro-
cytes with a view to determine the opioid agonistic or antagonistic
property of the test compounds. Astrocyte cultures were treated
with the test compounds for 10 min and pERK levels were esti-
Cl
N
CH3
O
HN
C
H2
N
H
CH3
NS1
mated by western blotting analysis. Like the
U50488H and the -specific ligand DAMGO (data not shown), all
the tested compounds were able to stimulate ERK phophorylation
j-opioid ligand,
N
CH3
l

I. Deb et al. / Bioorg. Med. Chem. 17 (2009) 5782–5790
5785
O
CH3
NH2
N
HN
Cl
NaH/DMSO
Cl
H3C
CH3
N2, 700 C-800 C
O
N
CH3
PP-11
PP-4
Scheme 2. Preparation of PP-4.
H
H
N
N
N
NH2
N
O
O
NaH/DMSO
NH
N2, 700 C-800 C
Cl
N
PP-6
CH3
CH3
O
PP-11
Scheme 3. Preparation of PP-6.
loss that is considered to be quantitatively related to abstinence.21
The selection of the tested compounds for withdrawal testing was
done to some extent on the basis of their interaction to KOR and
MOR. Since both PP4 and PP2 had almost equal interaction at
two receptors, results could provide information on the contribu-
tion of equal interactions on morphine dependence. Since MOR
specific ligands are better in antagonizing physical dependence
during opioid withdrawal, we selected PP7 and NS1. PP7 had great-
er binding to MOR than KOR, where as NS1 had binding mainly to
MOR. PP10 was selected to find the contribution of the side chain
of S4 compound in withdrawal.
The graphical representation of stereotype jumping during nal-
oxone precipitated withdrawal in absence and presence of the test
compounds are presented in Figure 3. It was observed that chronic
morphine treated mice when challenged with naloxone (10 mg/kg)
caused an average of six jumps within the 20 min period of obser-
vation. Daily injection of a single dose (20 mg/kg) of S4, PP2, PP4 or
NS1, throughout the six days of chronic morphine treatment could
completely inhibit jumping response whereas similar treatment
with PP7, PP10 decreased the incidence of stereotype jumping to
a small extent. However, a single injection of S4, just before nalox-
one challenge, had no effect on stereotype jumping.
Physical dependence was also verified by the loss in body
weight following precipitated withdrawal. Graphical representa-
tion of body weight loss in absence and presence of the test com-
pounds are represented in Figure 4. Naloxone precipitated
abstinence in mice caused a significant loss in body weight. S4
(20 mg/kg), at both chronic and acute doses, inhibited this loss in
body weight. Likewise, daily injection of a single dose (20 mg/kg)
of PP4 or PP10 for 6 days could also attenuate naloxone precipi-
tated body weight loss significantly.
Table 2
Effect of quinoline compounds on the specific bindings of
l
,
j
and d opioid receptors
Specific binding (fmol/mg protein)
Compound
l
j
d
—
6.40 0.25 (100)
4.86 0.19 (76)
57.46 1.11 (100)
10.91 0.45 (100)
DAMGO
U50488H
Naloxone
S-4
PP-2
PP-3
PP-4
PP-5
PP-6
PP-7
10.63 0.20 (19)
2.50 0.10 (23)
10.72 0.44 (98)
7.90 0.33 (72)
7.96 0.33 (73)
9.16 0.38 (85)
10.47 0.43 (96)
10.80 0.45 (99)
17.01 0.71 (156)
10.80 0.45 (99)
10.47 0.43 (96)
10.25 0.42 (94)
10.47 0.43 (97)
1.92 0.07 (30)
3.30 0.13 (52)
1.7 0.06 (27)
3.95 0.15 (62)
4.20 0.16 (66)
3.42 0.13 (53)
1.45 0.05 (23)
3.91 0.15 (61)
6.08 0.23 (95)
6.78 0.26 (106)
3.01 0.11 (47)
21.83 0.42 (38)
29.88 0.57 (52)
25.28 0.48 (44)
33.33 0.64 (58)
42.52 0.82 (74)
65.51 1.26 (114)
32.18 0.62 (56)
33.9 0.65 (59)
40.8 0.78 (71)
43.67 0.84 (77)
51.71 0.99 (90)
PP-10
PP-11
PP-12
NS-1
Binding at
l , j
and d opioid receptors were carried out using 3H-DAMGO, 125I-
Dynorphin and 125I- DADLE, respectively, in absence and presence of the test
compounds. For displacement of 3H-DAMGO, concentrations of test compounds
were 5 nM while for 125I- Dynorphin and 125I- DADLE, 5
l
M of test compound was
used. binding at each type of receptor was determined from the difference in
binding in absence and presence of 0.05 nM of unlabeled DAMGO ( ) or 50 M of
U50488H ( ) or 100- M naloxone (d). Specific displacement of test compounds at
l
l
j
l
l, j and d opioid receptors were compared with that by equal concentrations of
unlabeled DAMGO, U50488H and naloxone, respectively. 3H-DAMGO and 125I-
DADLE binding was carried out in mouse brain membrane while 125I- Dynorphin
binding was undertaken in membranes prepared from of C6 cell line stably trans-
fected with
j opioid receptor. Details of methodology are described in Materials
and Methods. Data represent mean SEM from three experiments. Values within
parenthesis represent percentage binding compared to total binding in absence of
any test compound, which is taken as 100.
Interestingly, our results on the effect of the test compounds on
precipitated withdrawal failed to provide any correlation with the
structure of the compounds or the selective contributions of MOR
and KOR, based on our binding studies. It is likely that these two
prominent withdrawal features of stereotype jumping and weight
loss may have other independent controlling systems. A review of
the literature in support of our inference is evident from the report
that NalB, a naloxone derivative and a putative antagonist of noci-
ceptive receptor, reduced the frequency of jumping in nociceptin
receptor knockout mice compared to wild type, although the other
withdrawal behaviour such as forepaw tremor, rearning were
identical in both mice.22 Intrathecal administration (i.t) of
U50488H attenuated three (wet shakes, escaped attempts, teeth
chattering) out of four withdrawal signs, in naloxone-precipitated
(Fig. 2a, b and c). Stimulation of pERK1/2 level by the test com-
pound was also sensitive to the opioid receptor antagonist nalox-
one (10 lM), which could effectively block the stimulation but
by itself had no effect (Fig. 2a).
Having observed that all of the test compounds exhibited opioid
agonistic properties with varying degree of interaction at the
l- and j-opioid receptors, we evaluated by the ability of the com-
pounds to check physical dependence during naloxone precipi-
tated abstinence in mice chronically treated with morphine.
While animals were scored for all the vital signs of abstinence,
such as writhing, wet dog shake, grooming, diarrhea, etc., these
were not quantified for this study. We have reported the effect of
the test compounds on stereotype jumping as well as body weight

5786
I. Deb et al. / Bioorg. Med. Chem. 17 (2009) 5782–5790
Figure 1. Competitive inhibition of specific (a) [3H]DAMGO and (b) [125I]Dynorphin binding to brain membrane by S4. Corresponding displacement curves by selective
agonists, DAMGO (a) and U50488H (b) were also carried out for comparison. Each point represents mean of three individual determinations. Non-linear regression lines were
determined by Graph prism 5 and IC50 concentrations calculated.
Figure 2. Effect of test compounds on induction of pERK activity in primary cultures of astrocytes. Ten-day-old cultures were serum starved for 48 h. The stimulatory effect of
opioid agonist such as
j-opioid receptor agonist, U50488H (1
lM) and the test compounds (40
lM) were evaluated by treating cells for 10 min with the drugs and
quantitating pERK1/2 by western blot analysis. a) Shows stimulation of pERK by S4, which was antagonized by co-treatment with naloxone (10
l
M). The effect of other test
compounds on pERK activation includes PP2, PP12, PP7 in (b) and PP3, PP4, PP5, PP6, PP10, PP11, NS1 in (c). Same blots were probed with ERK2 which served as a loading
control. The relative intensities of the pERK bands, indicated in the graph, were obtained by densitometric scanning and pERK levels were quantitated after normalizing
*
against ERK2. Results are mean SEM of at least 3 blots. P <0.05 versus untreated control.
withdrawal23 but was unable to attenuate weight loss. The
j-
3. Conclusion
selective antagonist nor-BNI, unlike U50488H, potentiated four
withdrawal signs differentially in this study. Nor-BNI significantly
aggravated body weight loss, teeth chattering, wet shakes, but es-
caped attempts were not markedly affected compare to control. In
the present study, none of our test compounds potentiated with-
drawal signs possibly due to their agonistic property at KOR as evi-
dent from our observations of their stimulatory effect on pERK.
Based on present concepts of drug designing, the present study
is an attempt towards developing potential drugs having mixed
opioidergic property and capable of antagonizing physical depen-
dence. A number of quinoline like compounds having typical phar-
macophore for opioidergic activity have been tested and our
observations suggest a potential therapeutic use of quinoline like

I. Deb et al. / Bioorg. Med. Chem. 17 (2009) 5782–5790
5787
trometer at 300 and 74.99 MHz, respectively, with tetramethylsil-
ane as internal standard and the chemical shifts are reported in d
units. 4-Aminoquinalidine and sodium hydride were purchased
from Aldrich Chemical Ltd (USA). Organic solvents used for the
chemical synthesis and for chromatography acquired from E.
Merck (India) were of analytical grade. All chromatographic purifi-
cation was performed with silica gel (60–120 mesh) and was ob-
tained from SRL (India). Thin layer chromatography was
performed on pre-coated silica gel 60 F254 aluminum sheets (E.
Merck, Germany) using the solvent system 1–6% MeOH in CHCl3
and spots were developed using Liebermann–Burchard solution.
4.1.2. General method of preparation of PP analogues
The preparation of each PP analogue was carried out in a three-
necked round bottom flask. The whole system was placed on a
magnetic stirrer with a magnetic fly inside the flask. Sodium hy-
dride was taken in the flask and was washed with dry non-polar
solvent petroleum ether to make it free from suspended oil. Dry di-
methyl sulphoxide was added and the reaction mixture was stirred
(about 20 min) and heated until it reaches at around 40 °C. The
substrate (4-aminoquinalidine) was added to the reaction mixture
and stirring continued for another 20 min. Finally, the reactant
(mono chloro-anilides) was added to the reaction mixture and stir-
ring continued for another 8 h at 70–80 °C. The whole operation
was carried out in an inert atmosphere, viz. nitrogen, argon free
from oxygen and in anhydrous condition. Termination of the reac-
tion was accomplished by adding ice-cold water (taking care that
the temperature of the reactants did not go beyond 4–10 °C). The
whole of the reaction mixture was extracted with ethyl acetate.
The organic layer was washed with water to make it free from al-
kali (neutral to litmus). The organic solvent was removed under re-
duced pressure to furnish PP analogues after crystallization from
methanol.
Figure 3. Demonstration of physical dependence by naloxone-precipitated stereo-
type jumping. Mice (seven per group) in each experiment were treated with chronic
morphine regime. Treatment protocol for test compounds induction of precipitated
withdrawal by naloxone is described in Methods. S4 (S) denotes S4 injected singly
after the last dose of morphine. Otherwise, test drugs were given once daily for
6 days. Stereotype jumping in each mouse was scored within a 20-min period after
each injection. Data are expressed as mean SE of stereotyped jumping in counts
per 20 min of three individual experiments à indicates significantly different (p
<0.001) from control.
4.1.3. 2-(2-Methylquinoline-4-ylamino)-N-phenylacetamide
See Ref. 24.
4.1.4. NS-1: 2-(2-Methylquinoline-4-ylamino)-N-p-
tolylacetamide
Obtained as white solid, mp 228–230 °C; IR (KBr,
mmax) 3410,
1682, 1529, 1324 cmÀ1 1H NMR (DMSO-d6) d 2.25 (s, 3H, –CH3),
;
2.44 (s, 3H, –CH3), 4.13 (d, 2H, J = 5.2 Hz, –CH2), 6.29 (s, 1H,
–NHCH2) 7.12 (d, 2H, J = 7.8 Hz), 7.37–7.51 (m, 4H), 7.59 (m, 1H),
7.73 (d, 1H, J = 8.4 Hz), 8.16 (d, 1H, J = 8.1 Hz), 10.08 (s, 1H,
–CONH).
Figure 4. Effect of naloxone precipitated withdrawal on body weight of mice. Mice
(seven per group) in each experiment were treated with chronic morphine regime.
Treatment protocol for test compounds and induction of precipitated withdrawal
by naloxone are described in Methods. S4 (S) denotes S4 injected singly after the
last dose of morphine. Otherwise, test drugs were given once daily for 6 days. Data
are expressed as mean SE of Body weight loss before naloxone injection and 4 h
after of three individual experiments à indicates significantly different (p <0.05)
from control.
13C NMR (DMSO-d6) d 21.3 (–CH3), 26.1 (–CH3), 47.1 (–CH2),
99.4 (–CH), 118.4 (–C), 120.3 (2 Â –CH), 122.3 (–CH), 124.2
(–CH), 129.2 (–CH), 129.7 (–CH), 130.0 (2 Â –CH), 133.2 (–C),
137.1 (–C), 148.8 (–C), 151.0 (–C), 159.4 (–C), 168.7 (–C@O); MS
(ESI-MS, positive ion) m/z 306 [M+H]+; HRMS m/z [M+H]+
306.1562 [calcd 306.1606].
4.1.5. PP-2: N-(2-Chlorophenyl)-2-(2-methylquinoline-4-
ylamino)acetamide
compounds in the management of the opiate tolerance and with-
drawal syndrome.
Obtained as white solid, mp 177–179 °C; IR (KBr,
mmax) 3250,
1674, 1530, 1436 cmÀ1 1H NMR (DMSO-d6) d 2.50 (s, 3H, –CH3),
;
4. Experimental
4.1. Chemistry
4.20 (d, 2H, J = 5.1 Hz, –CH2), 6.36 (s, 1 H, –NHCH2), 7.20 (t, 1H,
J = 7.8 Hz), 7.34 (t, 1H, J = 7.8 Hz), 7.41 (t, 1H, J = 7.8 Hz), 7.49 (d,
1H, J = 7.8 Hz), 7.57–7.62 (m, 2H), 7.76 (t, 2H, J = 8.4 Hz), 8.18 (d,
1H, J = 8.4 Hz), 9.71 (s, 1H, –CONH).
4.1.1. General methods
13C NMR (DMSO-d6) d 25.2 (–CH3), 46.2 (–CH2), 98.9 (–CH),
117.6 (–C), 121.4 (–CH), 123.4 (–CH), 125.7 (–CH), 126.4 (–C),
126.5 (–CH), 127.6 (–CH), 128.3 (–CH), 128.9 (–CH), 129.5 (–CH),
134.5 (–C), 148.0 (–C), 150.0 (–C), 158.6 (–C), 168.7 (–C@O); MS
(ESI-MS, positive ion) m/z 326[M+H]+; HRMS m/z [M+H]+
326.1085 [calcd 326.1060].
Melting points were determined with a capillary melting point
apparatus and are uncorrected. IR spectra were recorded on a JAS-
CO FTIR (model 410) in KBr pellets. ESI-MS and HRMS (positive)
was conducted using LC-ESI-Q-TOF micro Mass spectrometer. 1H
and 13C NMR spectra were taken on a Bruker 300 MHz DPX spec-

5788
I. Deb et al. / Bioorg. Med. Chem. 17 (2009) 5782–5790
4.1.6. PP-3: N-(4-Methoxyphenyl)-2-(2-methylquinoline-4-
ylamino)acetamide
13C NMR (DMSO-d6) d 21.3 (–CH3), 26.0 (–CH3), 36.3 (–CH2),
39.5(–CH2), 99.1 (–CH), 118.4 (–C), 120.1 (2 Â –CH), 122.2 (–CH),
124.0 (–CH), 129.1 (–CH), 129.6 (–CH), 129.9 (2 Â –CH), 132.9
(–C), 137.5 (–C), 148.8 (–C), 150.7 (–C), 159.6 (–C), 170.3 (–C@O);
MS (ESI-MS, positive ion) m/z 320 [M+H]+; HRMS m/z [M+H]+
320.1729 [calcd 320.1763].
Obtained as white solid, mp 216–218 °C; IR (KBr,
mmax) 3407,
1675, 1536, 1244 cmÀ1 1H NMR (DMSO-d6) d 2.44 (s, 3H, –CH3),
;
3.71 (s, 3H, –OCH3), 4.11 (d, 2H, J = 5.7 Hz, –CH2), 6.29 (s, 1H,
–NHCH2), 6.89 (d, 2H, J = 8.7 Hz), 7.37–7.53 (m, 4H), 7.59 (t, 1H,
J = 7.8 Hz), 7.73 (d, 1H, J = 8.1 Hz), 8.16 (d, 1H, J = 8.1 Hz), 10.04
(s, 1H, –CONH).
4.1.11. PP-10: 3-Chloro-N-phenylpropionamide
Obtained as white solid, mp 102–104 °C.
13C NMR (DMSO-d6) d 26.1 (–CH3), 47.0 (–CH2), 56.0 (–OMe),
99.5 (–CH), 114.8 (2 Â –CH), 118.4 (–C), 121.9 (2 Â –CH), 122.3
(–CH), 124.2 (–CH), 129.1 (–CH), 129.7 (–CH), 132.7 (–C), 148.8
(–C), 151.1 (–C), 156.3 (–C), 159.4 (–C), 168.5 (–C@O); MS (ESI-
MS, positive ion) m/z 322 [M+H]+; HRMS m/z [M+H]+ 322.1540
[calcd 322.1556].
4.1.12. PP-11: 2-Methylquinoline-4-ylamine
Purchased from Aldrich Chemical Ltd (USA).
4.1.13. PP-12: 2,2-Dichloro-N-(2-methylquinoline-4-
yl)acetamide
Obtained as white solid, mp 80–82 °C.
4.1.7. PP-4: 4-Methyl-N-(2-methylquinoline-4-yl)benzamide
Obtained as white solid, mp 137–139 °C; IR (KBr,
mmax) 3297,
1659, 1522, 1282 cmÀ1 1H NMR (DMSO-d6) d 2.42 (s, 3H, –CH3),
;
4.2. Biological studies
2.67 (s, 3H, –CH3), 7.39 (d, 2H, J = 7.8 Hz), 7.55 (m, 1H), 7.73 (m,
1H), 7.83 (s, 1H), 7.97 (m, 3H), 8.17 (d, 1H, J = 8.1 Hz), 10.52 (s,
1H, –CONH).
4.2.1. Materials
DMEM, F12 and FBS were obtained from Gibco-BRL, Life Tech-
nologies. Trypsin, soybean trypsin inhibitor, poly-L-lysine hydro-
13C NMR (DMSO-d6) d 21.9 (–CH3), 26.0 (–C–CH3), 116.4 (–CH),
121.9 (–C), 123.9 (–CH), 125.9 (–CH), 129.0 (2 Â –CH), 129.4 (–CH),
129.9 (2 Â –CH), 130.3 (–CH), 132.3 (–C), 142.9 (–C), 143.1 (–C),
149.3 (–C), 159.7 (–C), 167.2 (–C@O); MS (ESI-MS, positive ion)
m/z 277 [M+H]+; HRMS m/z [M+H]+ 277.1317 [calcd 277.1341].
bromide (molecular weight >300,000), U50488H, Naloxone
hydrochloride, Dynorphin, anti-mouse IgG-HRP conjugated 2nd
antibody were from Sigma Chemical Co. (USA). Primary antibody
of pERK1/2 from Santacruz (USA), Lipofectamine 2000, G418S from
Invitrogen (USA) were purchased. hKOR-pcDNA was from UMR
cDNA Resource Centre, University of Missouri-Rolla and C6 cell line
from National Centre for Cell Sciences, Pune. [3H] DAMGO, NaI125
were from PerkinElmer (Boston, MA). Morphine sulphate was pro-
cured from Gluconate India with the permission of the Excise
Directorate, Government of West Bengal. All other reagents were
of analytical grade and obtained locally.
4.1.8. PP-5: N- (2,5-Dichlorophenyl)-2-(2-methylquinoline-4-
ylamino)acetamide
Obtained as white solid, mp 215–217 °C; IR (KBr,
mmax) 3197,
1676, 1529, 1255 cmÀ1 1H NMR (DMSO-d6) d 2.46 (s, 3H, –C–
;
CH3), 4.24 (d, 2H, J = 5.4 Hz, –CH2), 6.36 (s, 1H, –NHCH2), 7.26
(dd, 1H, J = 2.4 Hz, 8.7 Hz), 7.41 (t, 1H, J = 7.8 Hz), 7.53 (d, 1H,
J = 8.7 Hz), 7.60 (m, 2H), 7.74 (d, 1H, J = 8.4 Hz), 7.94 (d, 1H,
J = 2.4 Hz), 8.17 (d, 1H, J = 8.1 Hz), 9.81 (s, 1H, –CONH).
4.2.2. Animals
The experimental protocols using animals have been approved
by the Institutional Animal Ethics Committee and meet the guide-
lines of the Government of India. For binding experiments as well
as for studies on morphine withdrawal, adult albino Balb/c mice,
20–30 g were used. Animals were housed seven per cage at room
temperature and allowed to adapt to laboratory conditions for at
least 2 days before the initiation of any experiment. The animals
were housed under a standard light dark cycle with free access
to food and water, except during testing.
13C NMR (DMSO-d6) d 25.2 (–CH3), 46.2 (–CH2), 98.9 (–CH),
117.6 (–C), 121.4 (–CH), 123.5 (–CH), 124.3(–C), 124.4 (–CH),
125.9 (–CH), 128.3 (–CH), 128.9 (–CH), 130.8 (–CH), 131.7 (–C),
135.8 (–C), 147.9 (–C), 149.9 (–C), 158.6 (–C), 169.0 (–C@O); MS
(ESI-MS, positive ion) m/z 360 [M+H]+, 362[M+2+H]+; HRMS m/z
[M+H]+ 360.0688 [calcd 360.0670].
4.1.9. PP-6: N-Cyclohexyl-2-[cyclohexylcarbamoylmethyl-(2-
methylquinoline-4-yl)-amino]acetamide
Obtained as white solid, mp 230–232 °C; IR (KBr,
mmax) 3273,
4.2.3. Cell culture and stable transfection
2930, 1653, 1431, 1245 cmÀ1 1H NMR (CDCl3) d 1.05–1.17 (m,
;
C6 cells were transfected with human
j-opioid receptor in
pcDNA using Lipofectamine. Stably transfected G418-resistant
cells were grown in F12 medium containing 10% fetal calf serum,
6H), 1.26–1.38 (m, 4H), 1.54–1.65 (m, 6H), 1.79–1.83 (m, 4H),
2.63 (s, 3H, –CH3), 3.74–3.84 (m, 2H), 3.96 (s, 4H), 6.82 (s, 1H),
6.94 (d, 2H, J = 8.1 Hz), 7.41(t, 1H, J = 7.5 Hz), 7.62 (t, 1H,
J = 7.8 Hz), 7.98 (t, 2H, J = 8.1 Hz, –CONH).
50 lg/ml gentamicin, pen-strep and 50 lg/ml G418 in 5% CO2 at
37 °C.
13C NMR (CDCl3) d 25.0 (4 Â –CH2), 25.7 (2 Â –CH2), 25.9
(–CH3), 33.2 (4 Â –CH2), 48.7 (2 Â –CH), 58.3 (2 Â –CH2), 109.9
(–CH), 121.7 (–C), 123.1 (–CH), 125.4 (–CH), 129.77 (–CH), 129.80
(–CH), 150.1 (–C), 153.7 (–C), 159.4 (–C), 168.4 (2 Â –C@O); MS
(ESI-MS, positive ion) m/z 437 [M+H]+; HRMS m/z [M+H]+
437.2887 [calcd 437.2917]; [M+Na]+ 459.2701 [calcd 459.2736].
4.2.4. Membrane preparation
For opioid receptor binding studies, membranes were prepared
from both the stably transfected cell line (for -opioid receptor
binding) and from mouse brain ( -opioid receptor binding). Stably
j
l
transfected C6 glioma, as described above, were harvested in ice-
cold phosphate-buffered saline and homogenized in 50 mM Tris–
HCl, pH 7.4. The total membrane (TM) fraction was collected upon
centrifugation at 30,000g for 20 min.
In case of membrane from mouse brain, mice was sacrificed by
decapitation, brain dissected out and homogenized in ice-cold
50 mM Tris–HCl buffer (pH 7.4) followed by centrifugation at
20,000 rpm for 30 min. The pellet was resuspended in the same buf-
fer and incubated for 20 min at 37 °C followed by centrifugation as
above. The pellet was resuspended in ice-cold buffer and used for
4.1.10. PP-7: 3-(2-Methylquinoline-4-ylamino)-N-p-tolylpropi-
onamide
Obtained as white solid, mp 241–243 °C; IR (KBr,
mmax) 3365,
1668, 1542, 1351 cmÀ1 1H NMR (DMSO-d6) d 2.24 (s, 3H, –CH3),
;
2.47 (s, 3H, –CH3), 2.73 (t, 2H, J = 6.6 Hz, –CH2), 3.60 (t, 2H,
J = 6.0 Hz, –CH2), 6.44 (s, 1H, –NHCH2), 7.09 (d, 2H, J = 8.1 Hz),
7.16 (m, 1H), 7.33 (t, 1H, J = 7.5 Hz), 7.48–7.58 (m, 3H), 7.70 (d,
1H, J = 8.1 Hz), 8.12 (d, 1H, J = 8.4 Hz), 9.96 (s, 1H, –CONH).

I. Deb et al. / Bioorg. Med. Chem. 17 (2009) 5782–5790
5789
binding assay. Protein concentration was determined by the meth-
od described by25 with bovine serum albumin used as standard.
ated with opioid withdrawal (including ‘biting’, ‘wet dog shakes’,
‘digging’, ‘face washing’, ‘grooming’, ‘rearing’, ‘scratching’, ‘diar-
rhea’, and ‘ptosis’).
4.2.5. Iodination of Dynorphin
For
was labeled with 125I-sodium iodide by chloramines T method.26
Briefly, the reaction was initiated when 10 l of chloramines T
(2 mg/ml) dissolved in a 0.2 M phosphate buffer (pH 7.4) was
added to a vial containing 10 l of the compound (1 mg/ml) dis-
solved in 0.2 M phosphate buffer, 10 l 0.2 M phosphate buffer
and 1 mCi of Na125I. After 40 sec of reaction, 40
l of sodium bisul-
fite (2 mg/ml) was added. The reaction mixture was diluted to 8 ml
with 0.1% aqueous trifluoroacetic acid (TFA) and absorbed in Sep-
Pak. After rinsing with 20 ml of 0.1% aqueous TFA, the mixture of
labeled and unlabeled compound were eluted out in a 1 ml solu-
tion containing 99.9% acetonitrile with 0.1% TFA. Finally, the la-
beled compound was separated from the unlabeled compound by
reverse phase high performance liquid chromatography on a C18
column with elution with a mobile phase of 20:0.2:79.8 acetoni-
trile/TFA/water. Fractions corresponding to monoiodinated com-
pound were pooled and used for subsequent studies.
j
-opioid receptor binding experiments, dynorphin (DPDYN)
4.2.8. Primary cultures of astrocytes from rat brain
Primary cultures of glial cells were prepared from the neocortex
of newborn rat pups after cervical dislocation (<24 h old) as re-
ported earlier.29 Briefly, single-cell suspensions were rinsed with
Dulbecco’s modified Eagle’s medium and then suspended in the
l
l
l
same buffer supplemented with 50
streptomycin, sodium bicarbonate and 10% fetal bovine serum,
pH 7.2.30 Cells were initially plated in 90 mm poly
-lysine hydro-
lg/ml gentamycin, 50 lg/ml
l
L
bromide (MW >300,000; Sigma)-coated tissue culture plates and
kept for 5 min, for preferential attachment of neurons. Two such
attachments yielded a highly purified (>95%) preparation of astro-
cytes. Unattached cells were seeded onto poly L-lysine-coated
plates at 6 Â 106 cells per plate. Cultures were maintained in a For-
ma CO2 incubator (5% CO2, 95% air) at 37 °C for 10 days. For ERK as-
say cells were maintained additionally 48 h in serum free medium.
4.2.9. ERK assay
ERK phosphorylation was measured by immunoblotting as de-
scribed.31 Following starvation for 48 h, cells were treated as indi-
cated. Antagonists were added to the medium 1 h before
stimulation with agonist and compounds. After the indicated stim-
ulation period, medium was removed, and plates were washed
with ice cold PBS. Cell lysates were collected in lysing buffer
(20 mM HEPES, 10 mM EGTA, 40 mM b-glycerophosphate,
2.5 mM MgCl2, 2 mM sodium vanadate, 1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 20 mg/ml aprotinin, and 20 mg/
ml leupeptin, pH 7.5). Cell lysates were centrifuged at 14,000g
for 20 min at 4 °C, and protein concentration of the supernatants
4.2.6. Radioligand binding
As described earlier, KOR binding27 was assayed in the mem-
brane fraction of C6 cells using [125I] Dynorphin while MOR and
DOR binding28 assay was carried out in mouse membrane using
[3H] DAMGO and [125I] DADLE, respectively. For KOR binding,
membranes (100–200
125I] Dynorphin for 30 min at 37 °C in 1 ml of 50 mM Tris–HCl buf-
fer, pH 7.4. Nonspecific binding was determined in the presence of
10- M concentration of unlabeled U-50, 488H. For MOR binding,
membrane homogenates (300 g protein) were incubated for 2 h
lg of protein) were incubated with 2 nM
[
l
l
at 25 °C in 1 ml of 50 mM Tris–HCl (pH 7.4) containing 2 nM [3H]
DAMGO in presence and absence of cold naloxone to determine
specific binding. Following incubation, bound radioligand was col-
lected by filtering under vacuum in a Millipore filtration manifold
using glass-fiber filters (GF/B; Whatman, Clifton, NJ), pretreated
with 0.5% PEI. The filters were washed thrice with ice-cold buffer
and the radioactivities retained on filters were counted in a liquid
scintillation counter (Wallac, model 1409–411, Perkin Elmer, USA).
was determined. Samples (40 lg of protein/lane) were separated
by 10% SDS–PAGE. Proteins were blotted on PVDF membranes
(Millipore Corp., Bedford, MA). Ponceau staining of blots was car-
ried out, prior to immunoblotting, to confirm loading of equal
amount of proteins in the lanes. Non-specific binding to mem-
branes was blocked with 5% non-fat dry milk in Tris-buffered sal-
ine (20 mM Tris, 0.9% NaCl, pH 7.5) for 1 h at 37 °C. The membranes
were then probed with monoclonal antibody against p-ERK
(1:1000 dilution) for 2–3 h, followed by staining with a peroxidase
conjugated secondary antibody.
4.2.7. Evaluation of morphine withdrawal
Development of morphine dependence was assessed from nal-
oxone-precipitated withdrawal. Mice were made tolerant to and
dependent on morphine by receiving morphine sulfate subcutane-
ously thrice daily for six consecutive days with an increment of
5 mg/kg/day with an initial starting dose of 10 mg/kg. Morphinized
animals were divided into groups. Test compounds were dissolved
in DMSO and each group received a particular test compounds
which was injected subcutaneously 30 min before morphine injec-
tion, once per day except that one group received a single acute
dose S4. Control groups received vehicle instead of the test com-
pound. Dosages of the drugs were selected on the basis of previous
result obtained from chronic S4 treatment, that is, 20 mg/kg. On
day 6, a single dose of morphine was given and 6 h later naloxone
(10 mg/kg) was injected ip, whereas tested compounds were given
almost 2 h before naloxone injection. Animals were weighed prior
to and 4 h after giving naloxone. Immediately after giving naloxone
few abstinence syndromes were recorded over a 20-min period.
Physical dependence was measured by quantitation of nalox-
one-precipitated jumping of individual animals using a PlexiglasR
withdrawal chamber over a period of 20 min after naloxone treat-
ment and then the average number of jumps per group was deter-
mined. Physical dependence was also evaluated by measuring
mouse body weight loss after 4 h of administration of naloxone.
Animals were also observed for other signs and symptoms associ-
4.3. Statistical analysis
All results are expressed as means S.E.M. The data were ana-
lyzed by one-way analysis of variance ANOVA followed by Dun-
can’s new multiple range test. Differences with P <0.05 between
experimental groups at each point were considered statistically
significant.
Acknowledgments
Authors would like to thank Department of Science and Tech-
nology (DST), Govt. of India for financial support to carry out the
study and also to The University Grants Commission (UGC) and
Council of Science and Industrial Research (CSIR), Govt. of India,
for providing fellowships to I.D., P.P. and A.H., respectively.
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