Opioid Receptor Modulators with a Cinnamyl Group

Article abstract of DOI:10.1021/acs.jmedchem.7b00643

To obtain selective and potent opioid receptor ligands, we synthesized dehydro derivatives of alvimopan and found compound (28f), a selective but modest affinity MOR antagonist weaker than alvimopan (1). We replaced the arylpiperidine unit by an arylpiperazine to obtain the 1-(α-carboxycinnamyl)-4-arylpiperazines like 13h, which to our surprise had no MOR or DOR activity but was a KOR agonist with moderate affinity. In contrast, literature examples of arylpiperazines 4 and 5 were reported to be pan opioid receptor antagonists, while 6 was a MOR agonist. Two compounds (13l and 11b) showed analgesic response in tail flick test which was blocked by pretreatment with norbinaltorphimine (norBNI). Among 10 1-(α-carboxycinnamyl)-4-arylpiperidines, compound 28g and five others were specific MOR antagonists. Interestingly, compound 26b of this series was found to be more potent than naloxone but weaker than 1. Docking studies have explained differential activities of the above piperazines and piperidines.

Full text of DOI:10.1021/acs.jmedchem.7b00643

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Article
Opioid receptor modulators with a cinnamyl group
Lokesh Ravilla, N. Venkata Subba Naidu, Shalini Dogra, Deepmala Umrao, Prem N.
Yadav, Ansuman Biswas, Daliah Michael, Kanagaraj Sekar, and Kuppuswamy Nagarajan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00643 • Publication Date (Web): 20 Jul 2017
Downloaded from http://pubs.acs.org on July 20, 2017
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Opioid receptor modulators with a cinnamyl group
a,b
b
c
c
Lokesh Ravilla , N. Venkata subba Naidu , Shalini Dogra , Deepmala Umrao , Prem N.
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c,*
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a,*
Yadav , Ansuman Biswas , Daliah Michael , Kanagaraj Sekar , Kuppuswamy Nagarajan
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Alkem laboratories Ltd. R&D Centre, Peenya Ind. Area, 3rd stage, Bangaloreꢀ560 058, India.
Department of Chemistry, S.V.University, Tirupatiꢀ517 502, India.
b
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Pharmacology Division, CSIRꢀCentral Drug Research Institute, Lucknowꢀ226 031, India.
Department of Physics, Indian Institute of Science, Bangaloreꢀ560 012, India.
e
Department of Computational and Data Sciences
12, India.
, Indian Institute of Science, Bangaloreꢀ560
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Abstract
To obtain selective and potent opioid receptor ligands, we synthesized dehydro derivatives of
alvimopan and found compound (28f), a selective but modest affinity MOR antagonist, weaker
than alvimopan (1). We replaced the aryl piperidine unit by an aryl piperazine to obtain the 1ꢀ(αꢀ
carboxycinnamyl)ꢀ4ꢀarylpiperazines like (13h), which to our surprise had no MOR or DOR
activity but was a KOR agonist with moderate affinity. In contrast, literature examples of aryl
piperazines (4) and (5) were reported to be pan opioid receptor antagonists, while (6) was a
MOR agonist. Two compounds (13l) and (11b) showed analgesic response in tail flick test which
was blocked by pretreatment with norbinaltorphimine (norBNI). Among ten 1ꢀ(αꢀ
carboxycinnamyl)ꢀ4ꢀarylpiperidines, compound (28g) and five others were specific MOR
antagonists. Interestingly, Compound (26b) of this series was found to be more potent than
naloxone, but weaker than (1). Docking studies have explained differential activities of the above
piperazines and piperidines.
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Introduction
Opioid receptors are member of Gꢀprotein coupled receptors (GPCRs) that couple to inhibitory
G protein (Gi/o) isoforms and therefore cause overall inhibitory response upon agonist binding.
These receptors are broadly classified into three subtypes: mu (MOR), kappa (KOR) and delta
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1
ꢀ3
(DOR). Being involved in several biological functions, selective opioid receptor modulators
(
both agonists and antagonists) are proposed to have beneficial therapeutic effects in certain
disease conditions like pain and depression. Hence a large number of opioid compounds have
4
been synthesized and studied. Among these a subclass of molecules with a core of 4ꢀ
arylpiperazine or 4ꢀarylpiperidine has been found to have interesting opioid properties relevant to
this study and are shown below (Figure 1).
Figure 1. Piperidines and piperazines with opioid receptor activities
5
, 6
Nꢀ(3ꢀHydroxyꢀ4ꢀphenyl) piperidines: 1 (Alvimopan), a selective MOR antagonist
was
7
, 8
approved in 2008 by USFDA for the treatment of post operative ileus. 2 (LY255582), a MOR,
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KOR and DOR antagonist with potent anorectic properties was discontinued in phaseꢀIII clinical
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10ꢀ12
trials. 3 (JDTic), a selective KOR antagonist with potential antidepressant properties
was
discarded during phaseꢀI in cocaine abuse clinical trials due to unfavorable ADME properties.
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Nꢀ(3ꢀHydroxyphenyl) piperazines: Compound (4) with a phenylpropyl side chain and similar
1
3
compounds were found to be pure antagonists of MOR, KOR and DOR. Compound (5)
inspired by (3) was the most potent in a class of compounds which inhibited agonist stimulated
[
35S] GTPγS binding in cloned human MOR, KOR and DOR. Compound (5) had a high
1
4, 15
selectivity for MOR over KOR and DOR.
Much earlier to the discovery of (4) and (5) T. Komoto et al had published MOR agonist activity
1
6
for a series of aryl piperazines replacing the aryl piperidines in loperamide. Interestingly in this
series, the 2ꢀmethoxyphenyl compound (6) was more active than the 2ꢀ, 3ꢀ and 4ꢀ hydroxy
phenyl compounds.
Like other opioid receptor (MOR and DOR) agonists, KOR agonists are known to produce
analgesia without having dependence and abuse liability, but are not approved for clinical use as
most of them induce sedation, hallucinations and dysphoria in human as well as in preclinical
models. Therefore, various groups worldwide are still trying to obtain KOR agonists based on
distinct templates in a hope to get new molecules with analgesic properties but devoid of adverse
effects. Multiple lines of evidence have also revealed that local application of synthetic opioid
receptor agonists could produce analgesic effect by activating peripheral opioid receptors
17
particularly in inflamed tissues. The peripheral opioid agonists bind to opioid receptors
expressed on sensory nerve terminals and produce analgesia by modulating the excitability of
sensory nerves and the release of proꢀinflammatory neuropeptides (e.g. substance P, calcitonin
gene related peptide). Since such analgesic activity occurs by engaging the opioid receptor
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locally in peripheral tissues, adverse effects such as respiratory depression and addiction are
absent. Thus, such observations have opened new frontiers of research on developing
1
8
peripherally restricted opioid agonists that lack CNS effects. Our studies were aimed at using
these concepts to find a useful analgesic.
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Chemistry
During the course of our engagement in the chemistry of (1), we observed that in all the above
publications, a cinnamyl group had not been studied in place of a phenyl propyl or a benzamide
group. On the other hand, we had developed a high yielding synthesis of Nꢀaryl piperazines from
19
anilines. Most of these piperazines are significant components of important drugs and were
available to us. Therefore, we decided to fill the vacant chemical space by constructing
molecules with the template (7), where in X was OR, OH, NHC(R)COOR. The aryl piperidine
was chosen from the structure of (1) or loperamide and the aryl piperazines were chosen from
medicinal chemistry literature. e.g. 9b (2, 3ꢀdichlorophenyl piperazine) present in antipsychotic
2
0
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aripiprazole, 9e (4ꢀflurophenyl piperazine) present in sedative niaprazine, 9h (2ꢀ
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hydroxyphenyl piperazine) present in antipsychotic bifeprunox, 9j (3ꢀchlorophenyl piperazine)
2
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present in antidepressant trazodone and 14 (benisothiazolyl piperazine) present in antipsychotic
2
4
ziprasidone. We were aware of a class of antiallergic drugs like cinnarizine and flunarizine
which are 1ꢀcinnamyl piperazines with a benzyhydryl group at position 4. Some examples of 7
(figure 2) were also hydrogenated to give new phenylpropyl compounds.
Figure 2. General structure of synthesized molecules
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Their synthesis and assay for MOR, KOR and DOR modulatory activities are discussed below.
The study yielded new molecules with moderate KOR and MOR activity in vitro. Interestingly, a
couple of piperazinesꢀ (13l) and (11b) were found to have KOR agonist activities apparently
unknown earlier and exhibited potent analgesic activity. It was also observed that a cinnamyl
piperidine (26b) was equipotent with naloxone as a MOR antagonist.
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Scheme 1. Synthesis of phenyl piperazine molecules
R3
R2
R1
O
N
O
N
O
O
OH
LiOH, THF, Methanol,H2O
25-30 0C, 2-4 h
O
K2CO3, NaI, Acetonitrile
5-30 0C, 10-12 h
N
N
2
N
N
Br
H.HCl
R1
R3
R1
R3
11a-11l
R2
R2
R
8
9a-9l
10a-
10l
1
2
3
9a-11a R , R , R = H
HATU
DIPEA
DMF
9b-11b R , R = Cl, R3 = H
1
2
O
c-11c R , R = H, R3 = Cl
1
2
H2N
HCl
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5-30 0
2
C
d-11d R , R = H, R3 = OH
1
2
O
10-12 h
e-11e R , R = H, R3 = F
1
2
f-11f R1 = OCH3, R , R = H
2
3
1
2
3
g-11g R , R = H, R = OCH3
h-11h R1 = H, R2 = OH, R3 = H
O
N
R
O
N
R
O
OH
N
i-11i R1 = Cl, R , R = H
9j-11j R1 = H, R = Cl, R = H
9k-11k R1 = H, R2 = OCH3, R =H
2
3
N
H
LiOH, THF, Methanol,H2O
5-30 0C, 2-4 h
H
O
O
2
3
2
N
3
N
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3
R1
R3
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l-11l R1 = F, R , R = H
R1
R3
R2
R2
13a-13af
12a-12af
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1
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2a-13a R, R , R , R = H
12b-13b R = CH(CH3)2, R , R , R = H
12c-13c R = CH2-CH(CH3)2, R , R , R = H
1
2
3
1
2
3
12f-13f R = H, R , R = Cl, R3 = H
1
2
2d-13d R = CH(CH3)CH2CH3, R , R , R = H
12e-13e R = (CH2)2CH3, R , R , R = H
1
2g-13g R = CH(CH3)2, R , R = Cl, R3 = H
1
2
12h-13h R = CH2-CH(CH3)2, R , R2 = Cl, R3 = H
12i-13f i = CH(CH3)CH2CH3, R , R2 = Cl, R3 = H
1
12j-13j R = (CH2)2CH3, R , R = Cl, R3 = H
1
2
12k-13k R, R , R = H, R3 = Cl
1
2
12l-13l R = CH(CH3)2, R , R = H, R3 = Cl
1
2
2m-13m R = CH2-CH(CH3)2, R , R = H, R3 = Cl
1
2
12n-13n R = CH(CH3)CH2CH3, R , R = H, R3 = Cl
1
2
1
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1
1
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1
12o-13o R = (CH2)2CH3, R , R ,= H, R = Cl
2p-13p R, R , R = H, R3 = OH
1
2
12q-13q R = CH(CH3)2, R1, R2 = H, R3 = OH
12t-13t R= (CH2)2CH3, R , R2 = H, R3 = OH
12r-13r R= CH2-CH(CH3)2, R , R = H, R3 = OH
1
2
2s-13s R= CH(CH3)CH2CH3, R , R = H, R3 = OH
1
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1
12u-13u R, R , R = H, R3 = F
1
2
2v-13v R = CH(CH3)2, R , R = H, R3 = F
1
2
12w-13w R = CH2-CH(CH3)2, R , R = H, R3 = F
1
2
12x-13x R = CH(CH3)CH2CH3, R , R = H, R3 = F
1
2
2y-13y R = (CH2)2CH3, R , R = H, R3 = F
1
2
12z-13z R = H, R = OCH3, R , R = H
1
2
3
12aa-13aa R, R ,R = H, R3 = OCH3
1
2
12ab-13ab R, R1 = H, R2 = OH, R3 = H
12ac-13ac R = H, R = Cl, R , R = H
1
2
3
12ad-13ad R, R1 = H, R2 = Cl, R3 = H
2ae-13ae R, R1 = H, R2 = OCH3 , R3 = H
1
2
3
11b R , R = Cl, R3 = H
1
2
1
1
12af-13af R = H, R = F, R , R = H
1c R , R = H, R3 = Cl
1
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Ethyl 2ꢀbromomethylcinnamate (8) was condensed with aryl/hetaryl piperazines (schemes 1, 2)
o
and piperidines (scheme 4) at 25ꢀ30 C to give (10), (15) and (25). The retention of Eꢀgeometry
during displacement of bromine was demonstrated as follows in the case of (10b). When the
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o
reaction was conducted at 70 C two products were formed; one was (10b) and the other was the
Zꢀisomer as shown by NMR studies (see supplementary information).
Compounds (10), (15) and (25) were hydrolyzed to carboxylic acids (11), (16) and (26)
respectively which were coupled to ethyl/methyl esters of αꢀaminoacids (Lꢀglycine, Lꢀvaline, Lꢀ
leucine, Lꢀisoleucine, Lꢀnorvaline) to offer (12), (17) and (27). A second hydrolysis offered the
target compounds (13), (18) and (28). (28f) is the dehydro analogue of alvimopan and on
catalytic hydrogenation yielded a diasterioisomeric mixture from which (1) was isolated in
modest yield. (28f) has been recently synthesized by another route but its activity has not been
25
reported. Palladiumꢀcatalyzed hydrogenation of (10a), (10fꢀ10h) led to the reduced products,
(
20a), (20fꢀ20h) with minimal concurrent hydrogenolysis of the cinnamyl group (which
happened when the reaction was done in an agitated hydrogenator). Hydrolysis to (21) followed
by coupling with ethyl glycinate using HATU provided amides (22a), (22fꢀ22h) (Scheme 3)
which were transformed to target molecules (23a), (23fꢀ23h) which are piperazine analogues of
(
1). Apart from them, compounds (19a) and (19b) were also synthesized for SAR studies. (19a)
was obtained by alkylation of (14) with benzyl chloride and (19b) by alkylation with ethylꢀ3ꢀ
chloro propionate and hydrolysis to the acid followed by coupling with Lꢀvaline.
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Scheme 2. Synthesis of benisothiazolyl piperazine derivatives
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Scheme 3: Synthesis of reduced phenyl piperazine analogues
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Scheme 4. Synthesis of phenyl piperidine molecules
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Results and discussion
A total of 44 piperazines listed in Scheme 1ꢀ3 and 11 piperidines listed in Scheme 4 were
evaluated on a panel of 11 GPCRS (Opioid receptors: KOR, DOR, MOR; Dopamine receptors:
D R, D R and D R; Serotonin receptor 5ꢀHT6; Histamine Receptors: H R, H R and H R;
1
2
5
1
2
3
Orphan Receptor GPR40) for agonist and antagonist activity. In the first stage, all the
compounds were evaluated at only 10 µM concentration in triplicate for agonist or antagonist
activity at these receptors individually, and if any compound exhibited minimum 50% of
inhibition or stimulation in comparison to control then that particular compound was further
evaluated in a concentration dependent (10 µM to 10 pM) response to determine the EC /IC
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values. The following eleven compounds (Table 1 and 2) were active by the above definition in
these tests.
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Table 1. IC50 values of cinnamyl piperazine derivatives at KOR. These compounds did not
exhibit any activity at MOR or DOR.
Compounds Codes
logIC50 ( IC50: µM)
ꢀ6.09 ± 0.27 (0.8)
ꢀ5.83 ±0.16 (1.4)
ꢀ5.73 ±0.15 (1.8)
ꢀ6.041± 0.17 (0.91)
ꢀ5.72±0.18 (1.8)
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U50488
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Compounds with a cinnamyl piperazine template 11b, 13g, 13h, 13l and 18b (table 1) showed
agonist activity at KOR without any activity at MOR or DOR in contrast to piperidines. To
determine the IC of these cinnamyl piperazine template compounds, we measured inhibition of
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forskolin (10 µM, FSK) induced cAMP formation using GloSensor assay in HEK293T cells
26
transiently transfected with hKOR. 29 (U50488) was used as standard KOR agonist for cAMP
assay. Results presented in table 1 show that five analogues of 28f (dehydroalvimopan) with an
aryl piperazine instead of aryl piperidine were modest KOR agonists with about 3 orders of
magnitude less potency than the standard (29). (IC : 0.3 nM, Figure 3). The aryl piperazines
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involved have 2,3ꢀdichlorophenyl, 4ꢀchlorophenyl or 3ꢀbenisothiazolyl groups present in the
antipsychotics aripiprazole, haloperidol and ziprasidone, respectively. It is interesting to note that
our piperazines with an αꢀcarboxycinnamyl substituent did not exhibit Dopamine D2 receptor
(known to have antipsychotic activity) and histamine H1 receptor activities like 1ꢀbenzhydrylꢀ4ꢀ
cinnamyl piperazine (cinnarizine). Compound (11b) that showed KOR agonist activity
equivalent to the earlier three is quite interesting as it lacks the peptide bond which seems to
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have no special contribution. Surprisingly though, the 3ꢀhydroxy phenyl piperazine derivative
(
13ab) was found inactive unlike the literature compound (5). Further, none of the other phenyl
piperazine derivatives containing an oxygen function in the phenyl residue such as 3ꢀmethoxy
13ae), 4ꢀhydroxy (13pꢀ13t), 2ꢀmethoxy (13z) and 4ꢀmethoxy (13aa) had any activity in KOR
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(
and MOR assays in contrast to the MOR agonist activity exhibited when they were present in
structures like (6). Four dihydro derivatives (23a), (23fꢀ23h) were also devoid of activity on
KOR and MOR.
Similar to MOR and DOR agonists, KOR agonists are also widely known to have analgesic
properties and are devoid of gastrointestinal associated adverse effects. Therefore, we sought to
determine the analgesic effects of two of our best KOR agonists that is (13l) and (11b) in tail
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flick assay, a standard test to measure analgesic response. Although compounds (13l) and (11b)
had modest affinity at KOR (IC ≤ 1 µM), we observed significant and time dependent analgesic
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response at 10 mg/kg dose, in comparison to the standard KOR agonist (29) at 5 mg/kg dose.
This observation clearly indicates that both compounds readily cross the blood brain barrier.
While (13l) exhibited modest analgesic response, (11b) showed a strong analgesic response
equivalent to (29) (Figure 4). This activity was blocked by the KOR selective antagonist
norBNI. Further work is warranted to fully characterize the biological properties of (13l) and
(11b) and to improve their activity.
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Figure 3. Agonist activity of 11b, 13g, 13h, 13l and 18b at KOR. HEK293T cells transiently
transfected with KOR were stimulated with varying concentrations of (29) and test compounds
(10 µMꢀ10pM) followed by forskolin (FSK, 10 µM) to determine the inhibition of FSK induced
cAMP formation in live cells via GloSensor assay. The relative luminescence units for cAMP
with test compounds or (29) were normalized to % control (FSK alone) and IC values were
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generated by nonꢀlinear regression analysis using Graphpad Prism 5.0. For presentation purpose,
normalized values of two independent experiments (performed in triplicates) were pooled and
presented as % mean ± standard error of mean (SEM).
Figure 4: Analgesic effect of (29) and two novel KOR agonists: 13l and 11b. (A) Time
dependent analgesic effect of (29) (5 mg/kg), 13l and 11b (10 mg/kg, i.p.). Tail withdrawal
latency was determined and presented as % baseline (tail flick measured at 15 min prior to
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vehicle or drug administration). (B) Bar graph showing area under the curve of tail flick test
graph.*p < .01 by unpaired student’s tꢀtest (n=5ꢀ6 mice/group). (C) KOR selective antagonist
norBNI (10 mg/kg, i.p.) blocked the analgesic effect of (29) as well as of two novel compounds
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3l and 11b. A separate set of animals (5ꢀ6/group) were administered norBNI (10 mg/kg, i.p.) 30
min prior to (29) or 13l or 11b administration, and tail withdrawal latency was determined at 30
min time point.
Table 2. IC50 values of cinnamyl piperidine derivatives at MOR. These compounds did not
exhibit any activity at KOR or DOR.
Compounds Codes
logIC50 ( IC50: µM)
ꢀ7.73 ± 0.27 (0.018)
ꢀ5.89 ± 0.07 (1.26)
ꢀ6.18 ±0.07 (0.65)
ꢀ6.10 ±0.09 (0.78)
ꢀ5.79 ± 0.07 (1.60)
ꢀ5.63 ± 0.07 (2.32)
ꢀ8.50 ± 0.22 (0.003)
ꢀ7.34 ± 0.26 (0.045)
26b
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8f
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28j
Alvimopan (1)
Naloxone
Among the piperidines screened, besides (1), the reference compound, (26b) and (28fꢀ28j)
showed antagonist activity to MOR (table 2), but lack any activity at KOR and DOR in
GloSensor assay (figure 5). Interestingly, all these have the framework of (1), characterized by
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ꢀmꢀhydroxyphenylꢀ3,4ꢀdimethyl piperidine residue. The other set of analogues having the 4ꢀ(4ꢀ
chlorophenyl)ꢀ4ꢀhydroxy piperidine (28aꢀ28e) moiety present in loperamide were inactive.
Hence, the former group was taken up for IC determination. The activity was evaluated in
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HEK293T cells transiently expressing MOR for suppression of DAMGO (1 µM) induced
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inhibition of cAMP production using GloSensor assay. Results presented in table 2 show that
28f) and (28gꢀ28j) wherein the glycine residue in (28f) has been replaced by higher homologues
(
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Lꢀvaline, Lꢀleucine, Lꢀisoleucine, Lꢀnorvaline were less active than (1) by a factor of 2.5 to 3 log
units. Interestingly, compound (26b) which is dehydroalvimopan truncated by the removal of
glycine is more potent than the others (IC ≤ 20 nM) which is about one sixth of the activity of
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1) and slightly surpasses the potency of naloxone (IC ≤ 50 nM). These observations suggest
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that manipulation of structure (26b) could be a fruitful strategy for newer MOR antagonists.
Figure 5. Antagonist activity of 28f, 28g, 28h, 28i, 28j and 26b at MOR. HEK293T cells
transiently transfected with MOR were stimulated with Naloxone and test compounds (10 µMꢀ
10 pM) followed by DAMGO (1 µM) and forskolin to determine the inhibition of DAMGO
response on cAMP formation in live cells via GloSensor assay. The relative luminescence units
for cAMP with test compounds or Naloxone were normalized to % control (DAMGO +
forskolin) and IC50 values were generated by nonꢀlinear regression analysis using Graphpad
Prism 5.0. For presentation purpose, normalized values of two independent experiments
(performed in triplicates) were pooled and presented % mean ± standard error of mean (SEM).
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Methods of Pharmacological studies:
Animals: The in vivo analgesia experiments with mice in this study were approved by the
institutional animal ethics committee (IAEC) of CSIRꢀ Central Drug Research Institute,
Lucknow, India. Male C57BL/6J mice (6ꢀ8 weeks old) weighing 22ꢀ25 g were used in this study.
Animals were housed on a 12ꢀh light/dark cycle (lights on at 8.00 am). Food and water were
provided ad libitum.
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GloSensor Assay for cAMP measurement: HEK293T cells were cultured in Dulbecco's
Modified Eagle Medium (DMEM) with 25 mM Glucose and 20 mM HEPES supplemented with
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0% FBS at 37 C and 5% CO in a humidified incubator. The cultured HEK293T cells were
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transfected with KOR or DOR or MOR, and pGloSensor™ꢀ22F plasmid (Promega corp.) using
calcium phosphate method of transfection. Briefly, 5 µg of receptor cDNA plasmid and 5µg of
pGloSensor™ꢀ22F plasmid DNA were added to 61.3 µl of 2 M CaCl and water to final volume
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of 500 µl. This mixture was added to 500 µl of 2X HBS slowly and was incubated at room
temperature for ten minutes. After incubation, the mix was added drop wise to the cells in dish.
After overnight incubation, the cells were dislodged from the dish by multiple pipetting and
resuspended in drug buffer (1X HBSS with 20 mM HEPES, pH7.4) comprising sodiumꢀluciferin
solution (10 µg/µl) and were plated in 96 well plate for 90 minutes. Thereafter, cells were treated
with various concentrations (10000nM to 0.01nM) of reference compound ((29) for KOR,
DAMGO for MOR and DADLE for DOR) and test compounds in triplicates and were incubated
in a humidified tissue culture incubator at 37°C with 5% CO for 15ꢀ20 minutes to get a steady
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state condition. After 15 minutes, cells were again treated either with final 10 µM of forskolin
(
FSK) (to test compounds for agonistic activity) or agonist (1µM) followed by addition of FSK
10 µM; to test compounds for antagonistic activity) and luminescence per well was measured
(
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using luminescence plate reader (BMG Labtech). The relative luminescence units (RLU) were
plotted as percent inhibition of FSK response using non linear regression fit analysis equation of
GraphPad prismꢀ5.0.
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Tail flick test for analgesia: For assessment of thermal analgesia, tail flick test was done as
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described previously. Briefly, for each testing session, mice were individually acclimatized into
the restrainers for one minute without tail immersion. After acclimatization, each mouse was
gently introduced into the restrainers and the distal oneꢀthird of the tail was dipped into the hot
water bath (temp 52° ± 0.5°C) and tail withdrawal latencies were recorded thrice at a time point
by an observer unknown to the drug treatment in the mice.
In silico analysis:
The crystal structures of MOR and KOR are available in the ligandꢀbound forms with morphinan
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antagonist (PDB ID 4DKL) and with JDTic antagonist (PDB ID 4DJH), respectively. These
two protein structures were selected for docking simulations. In an attempt to understand the
moderate MOR antagonistic activity of (28f) and the KOR agonist activity of (18b), docking
simulations were performed, after removing the existing ligands (βꢀFNA and (3)) of the proteins.
Further, in silico analysis of the binding site and the interacting residues revealed the following
findings.
βꢀFNA is the ligand that binds with the MOR (PDB ID 4DKL) and among the interacting
residues, Asp147 and Tyr326 are known to be conserved in MORs. Asp147 is involved in a
chargeꢀcharge interaction with the amine group of the ligand and also forms hydrogen bond with
Tyr326. His297 is found to interact with the aromatic ring of βꢀFNA (not hydrogen bonds). After
docking of (28f) with MOR, the conformation of the docked protein molecule was selected based
on low Binding Energy (BE) and conserved interactions. The residues that contribute to the
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different interactions of the ligand are listed in Table 3 (see supplementary information) and can
be viewed in Figure 6. It is seen in Table 3, that most of the interacting residues are conserved
even in the docked structure of the MOR with (28f). Conserved His297 forms hydrogen bonds
with (28f) and conserved Asp147 along with Lys233 forms salt bridges in the docked structure.
Such conserved and important interactions are the contributing factors for the high Binding
Energy and favorable binding of the docked structure. It is also interesting to note that when
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(28f) binds to MOR, two antiparallel βꢀstrands get extended to a region which was earlier a loop.
A similar docking of (1) was done with MOR and the interactions are presented in Table 3 (see
supplementary information) and represented in Figure 8.The binding energies of (28f) and (1)
are ꢀ8.87 kcal/mol and ꢀ9.38 kcal/mol, respectively. This agrees with the observed IC50 only
qualitatively, thus exposing a need for further tweaking the docking model. It is noteworthy, that
(28f) fits exactly in the existing pocket and in a similar orientation to that of βꢀFNA. Thus,
considering all factors, (28f) is clearly a suitable choice for MOR.
In the available structure of KOR (PDB ID 4DJH), the residue Asp138 is crucial in binding of
the ligand (3) in the active site of protein. It is also suggested that the characteristic Vꢀshape bend
of (3) is due to the interaction of the two aromatic rings with conserved residue Asp138. Four
other residues (Val108, Val118, Ile294 and Tyr312) interacting with (3) are thought to be
specific to KOR binding with ligands. The desirable conformation of the docked protein
molecule, (18b) with KOR, was selected based on the optimal BE. Table 3 (see supplementary
information) lists the interacting residues and Figure 7 represents the graphical view. Apart from
the interacting residues being conserved in the docked structure, it is interesting and important to
note that the ligand (18b) adopts a similar Vꢀshape like (3). This bend in the ligand is probably
due to the charged interaction of conserved Asp138. A study involving siteꢀdirected mutagenesis
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of Asp138 in KOR (D138A and D138N) resulted in reduced binding affinities to the respective
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agonists . This undeniably manifests the significance of residue Asp138, which forms a salt
bridge with 18b in the docked structure. In addition, one of the hydrogen bonds formed by
Tyr312 is conserved in the binding site of the protein bound to (3), as well as to (18b). It is due
to the interactions of these conserved residues that the Binding Energy of the docked structure is
high, explaining the observed KOR activity of (18b). It is worth mentioning that (3) and (18b),
both, interact strongly with Cys210 (βꢀstrand). We were unable to efficiently dock 18b in MOR
and 28f in KOR.
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Thus, the in silico studies provide favorable binding energies for the synthesized small molecules
in the respective sites and support the results from the biological studies. Favorable BE indicates
proper binding of the ligands in the pocket of the protein molecule and the interaction of the
residues in the active site.
Figure 6. Image of all the interactions of the MOR ꢀ (28f) docked protein structure, obtained
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from PLIP. The blue lines represent the hydrogen bonds, the grey dotted lines represent
hydrophobic interactions and the yellow dotted lines represent salt bridges.
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Figure 7. Image of all the interactions of the KOR ꢀ (18b) docked protein structure, obtained
from PLIP. The blue lines represent the hydrogen bonds, the grey dotted lines represent
hydrophobic interactions the yellow dotted lines represent salt bridges and black dotted lines
represent piꢀinteractions.
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Figure 8. Image of all the interactions of the MOR ꢀ (1) docked protein structure (obtained from
PLIP). The blue lines represent the hydrogen bonds, the grey dotted lines represent hydrophobic
interactions and the yellow dotted lines represent salt bridges.
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Conclusion:
Our studies have shown that replacement of the piperidine moiety by a piperazine in structures
like (28f) results in a dramatic change in the opioid properties of the molecules from MOR
antagonism to KOR agonism (28f vs. 13l). This also reveal that replacing a phenyl propyl group
in substituted piperazines like (4) by a αꢀcarboxy cinnamyl group (13g) changes from KOR
antagonist property to agonist. Further the antipsychotic properties of aryl piperazines
incorporated in drugs disappear when the side chain is replaced by αꢀcarboxy cinnamyl group
leading to KOR agonist properties. Among the piperazines studied (13l) and (11b) had moderate
in vitro but potent in vivo analgesic activity comparable to (29). Among the piperidines with
MOR antagonist activity (28f) was less potent than (1). Compound (26b) lacking the peptide
chain was more active than (28f) and was slightly more potent than naloxone as MOR
antagonist. Thus, observations made in this study provides valuable insight for designing and
synthesis of novel and selective opioid receptor ligands that can further optimized as lead
candidates for the treatment of pain disorders. In silico docking studies offered explanations for
the KOR activity of piperazine (18b) as against the MOR activity of (1)
Experimental Section
All chemicals and solvents were commercially purchased and used as such without further
purification. The progress of all reactions was monitored by thin layer chromatography using
Merck TLC silica gel 60 F254 plates with ethyl acetate/nꢀhexane solvent systems as mobile phase.
Column purification was carried on Globe chem silica gel (100ꢀ200 mesh); spots were identified
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with UV light of wavelength 254 nm. Purity assessment and mass spectra (MS) data were
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obtained using Shimadzu lcms2020 using electrospray ionization (ESI) for detection. H and C
NMR spectra were measured on a Bruker 300 and 400 MHz instrument. Chemical shifts are
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reported in δ ppm relative to TMS. ( H NMR: TMS δ
= 0.00 ppm, CDCl δ = 7.26 ppm, DMSOꢀ
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d δ = 2.50 ppm; C NMR (APT): TMS δ = 0.00 ppm, CDCl δ = 77.16 ppm, DMSOꢀd δ =
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9.52 ppm). Coupling constants (J) are given in hertz (Hz). The following abbreviations were
used to express the multiplicities: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet;
dd = doublet of doublets; dt = doublet of triplet; td = triplet of doublet; bs = broad singlet.
General procedure for synthesis of 10aꢀ10l, 15 and 25aꢀ25b (General procedure A)
To a solution of ethyl 2ꢀ(bromomethyl)ꢀ3ꢀphenylacrylate 8 (3.7 mmol) in 15 mL of acetonitrile
were added potassium carbonate (7.4 mmol), sodium iodide (1.8 mmol) and Nꢀaryl piperazine
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aꢀ9l, 14 (4.1 mmol) or aryl piperidine 24aꢀ24b (4.1 mmol) and the reaction mass was stirred at
o
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5ꢀ30 C for 10 ꢀ 12 h. Completion of reaction was monitored by TLC. The reaction mass was
filtered through celite bed and the bed was washed with 10 mL of acetonitrile. The solution was
removed of its solvent by rotary evaporation. The remaining residue was partitioned between
water (40 mL) and DCM (30 mL). The aqueous layer was extracted twice with DCM (20 mL).
The pooled DCM layer was dried over anhydrous sodium sulphate and concentrated to get crude
product, which was purified by silica gel column chromatography, elution being done with 10%
ethyl acetate in nꢀhexane.
Ethyl (E)ꢀ3ꢀphenylꢀ2ꢀ((4ꢀphenylpiperazinꢀ1ꢀyl)methyl)acrylate (10a)
Following the above general procedure A, reaction of 8 and 9a resulted in 10a as a syrup (Yield:
1
9
0%); H NMR (400 MHz, CDCl ) δ ppm : 1.34 ꢀ 1.37 (t, J = 7.12 Hz, 3 H), 2.62 (s, 4 H), 3.14
3
(
s, 4 H), 3.46 (s, 2 H), 4.27 ꢀ 4.32 (q, J = 7.08 Hz, 2 H), 6.73 ꢀ 6.83 (m, 1 H), 6.87 ꢀ 6.98 (m, 2
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+
(
ESI): found: [M + H] , 351.25.
Ethyl (E)ꢀ2ꢀ((4ꢀ(2,3ꢀdichlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylate (10b)
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Following the above general procedure A, reaction of 8 and 9b resulted in 10b as a gummy
1
material (Yield: 89%); H NMR (400 MHz, CDCl ) δ ppm : 1.35 ꢀ 1.38 (t, J = 7.12 Hz, 3 H),
3
2.67 (s, 4 H), 3.04 (s, 4 H), 3.44 (s, 2 H), 4.28 ꢀ 4.33 (q, J = 7.12 Hz, 2 H), 6.92 ꢀ 6.95 (dd, J =
6.76, 2.76 Hz, 1 H), 7.10 ꢀ 7.15 (dd, J = 6.92, 2.52 Hz, 2 H), 7.33 ꢀ 7.42 (m, J = 7.28 Hz, 3 H),
+
7.66 ꢀ 7.68 (d, J = 7.12 Hz, 2 H), 7.86 (s, 1 H); MS (ESI): found: [M + H] , 420.25.
Ethyl (E)ꢀ2ꢀ((4ꢀ(4ꢀchlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylate (10c)
Following the above general procedure A, reaction of 8 and 9c resulted in 10c a viscous oil
1
(Yield: 92%); H NMR (400 MHz, CDCl ) δ ppm : 1.34 ꢀ 1.37 (t, J = 7.12 Hz, 3 H), 2.61 ꢀ 2.64
3
(t, J = 7.12 Hz, 4 H), 3.12 ꢀ 3.14 (t, J = 7.12 Hz, 4 H), 3.42 (s, 2 H), 4.27 ꢀ 4.32 (q, J = 7.12 Hz, 2
H), 6.80 ꢀ 6.83 (dd, J = 7.12 Hz, 2 H), 7.17 ꢀ 7.19 (dd, J = 7.12 Hz, 2 H), 7.35 ꢀ 7.41 (m, J = 7.12
+
Hz, 3 H), 7.66 ꢀ 7.68 (d, J = 7.12 Hz, 2 H), 7.87 (s, 1 H); MS (ESI): found: [M + H] , 385.20.
Ethyl (E)ꢀ2ꢀ((4ꢀ(4ꢀhydroxyphenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylate (10d)
Following the above general procedure A, reaction of 8 and 9d resulted in 10d a syrup (Yield:
1
9
6%); H NMR (400 MHz, CDCl ) δ ppm : 1.34 ꢀ 1.37 (t, J = 3.63 Hz, 3 H), 2.65 (s, 4 H), 3.05
3
(s, 4 H), 3.49 (s, 2 H), 4.27 ꢀ 4.32 (q, J = 7.08 Hz, 2 H), 4.92 (s, 1 H), 6.73 ꢀ 6.75 (d, J = 8.84 Hz,
2 H), 6.81 ꢀ 6.83 (d, J = 8.76 Hz, 2 H), 7.33 ꢀ 7.41 (m, J = 7.04 Hz, 3 H), 7.64 ꢀ 7.66 (d, J = 7.24
+
Hz, 2 H), 7.87(s, 1 H); MS (ESI): found: [M + H] , 367.30.
Ethyl (E)ꢀ2ꢀ((4ꢀ(4ꢀfluorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylate (10e)
Following the above general procedure A, reaction of 8 and 9e resulted in 10e a viscous oil
1
(
Yield: 92%); H NMR (300 MHz, CDCl ) δ ppm : 1.26 ꢀ 1.31 (t, J = 7.12 Hz, 3 H), 2.65 (s, 4
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H), 3.15 ꢀ 3.18 (t, J = 6.0 Hz, 4 H), 3.53 (s, 2 H), 4.15 ꢀ 4.22 (q, J = 7.08 Hz, 2 H), 6.83 ꢀ 6.88
(m, 2 H), 6.92 ꢀ 6.98 (t, J = 8.76 Hz, 2 H), 7.26 ꢀ 7.32 (t, J = 9.04 Hz, 2 H), 7.34 ꢀ 7.41 (m, 3 H),
+
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.99 (s, 1 H), 10.18 (s, 1 H); MS (ESI): found: [M + H] , 369.15.
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Ethyl (E)ꢀ2ꢀ((4ꢀ(3ꢀhydroxyphenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylate (10h)
Following the above general procedure A, reaction of 8 and 9h resulted in 10h as a gummy mass
1
(
Yield: 92%); H NMR (400 MHz, CDCl ) δ ppm : 1.34 ꢀ 1.37 (t, J = 7.12 Hz, 3 H), 2.61 ꢀ 2.63
3
(t, J = 4.92 Hz, 4 H), 3.14 ꢀ 3.17 (t, J = 4.84 Hz, 4 H), 3.42 (s, 2 H), 4.27 ꢀ 4.30 (q, J = 7.12 Hz, 2
H), 4.75 (s, 1 H), 6.28 ꢀ 6.30 (dd, J = 7.92, 2.08 Hz, 1 H), 6.37 ꢀ 6.38 (t, J = 2.20 Hz, 1 H), 6.47 ꢀ
6.50 (dd, J = 7.72, 2.18 Hz, 1 H), 7.06 ꢀ 7.12 (t, J = 8.08 Hz, 1 H), 7.34 ꢀ 7.41 (m, J = 9.04 Hz, 4
+
H), 7.66 ꢀ 7.68 (d, J = 6.84 Hz, 2 H), 7.87 (s, 1 H); MS (ESI): found: [M + H] , 367.2.
Ethyl (E)ꢀ2ꢀ((4ꢀ(3ꢀchlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylate (10j)
Following the above general procedure A, reaction of 8 and 9j resulted in 10j a syrup (Yield:
1
9
2%); H NMR (400 MHz, CDCl ) δ ppm : 1.36 ꢀ 1.39 (t, J = 7.12 Hz, 3 H), 2.66 (s, 4 H), 2.87
3
(s, 4 H), 3.45 (s, 2 H), 3.70 (s, 1 H), 4.28 ꢀ 4.34 (q, J = 7.08 Hz, 2 H), 6.82 ꢀ 6.86 (m, 1 H), 6.92 ꢀ
6.94 (d, J = 8.00 Hz, 1 H), 7.04 ꢀ 7.08 (t, J = 9.04 Hz, 1 H), 7.14 ꢀ 7.16 (d, J = 7.60 Hz, 1 H),
+
7.35 ꢀ 7.44 (m, 3 H), 7.66 ꢀ 7.68 (d, J = 6.88 Hz, 2 H), 7.88 (s, 1 H); MS (ESI): found: [M + H] ,
385.2.
Ethyl (E)ꢀ2ꢀ((4ꢀ(2ꢀfluorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylate (10l)
Following the above general procedure A, reaction of 8 and 9l resulted in 10l a viscous oil
1
(
Yield: 87%); H NMR (400 MHz, CDCl ) δ ppm : 1.34 ꢀ 1.39 (t, J = 7.11 Hz, 3H), 2.66 ꢀ 2.69
3
(t, J = 6.00 Hz, 4 H), 3.07 ꢀ 3.10 (t, J = 6.00 Hz, 4 H), 3.44 (s, 2 H), 4.27 ꢀ 4.34 (q, J = 7.09 Hz, 2
H), 6.89 ꢀ 7.07 (m, 4 H), 7.32 ꢀ 7.45 (m, 3 H), 7.64 ꢀ 7.68 (m, 2 H), 7.70 (s, 1 H); MS (ESI):
+
found: [M + H] , 369.15.
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General procedure for synthesis of 11aꢀ11l, 16, 21a, 21fꢀ21h and 26aꢀ26b (General
procedure B)
To a solution of 10aꢀ10l, 15, 20a, 20fꢀ20h or 25aꢀ25b (3.2 mmol) in 14 mL (14 v) 1:1 mixture
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of methanol and THF was added pre dissolved LiOH.H O (9.7 mmol) in minimum quantity of
2
o
water. The reaction mass was stirred at 25ꢀ30 C for 2 ꢀ 4 h. Completion of reaction was
monitored by TLC. THF was distilled under reduced pressure, 20v water was added and the pH
was adjusted up to 7 using 1N HCl. The product was extracted twice with ethyl acetate. The total
ethyl acetate layer was dried with anhydrous sodium sulphate and concentrated to get an acid as
white color solid which was used as such without purification for next step.
(E)ꢀ2ꢀ((4ꢀ(2,3ꢀDichlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylic acid (11b)
11b was synthesized from 10b by following the general procedure B as a white solid (Yield:
1
8
7
7
9%); H NMR (400 MHz, DMSOꢀd ) δ ppm : 2.61 (s, 4 H), 2.97 (s, 4 H), 3.42 (s, 2 H), 7.11 ꢀ
6
.14 (dd, J = 5.48, 3.96 Hz, 1 H), 7.27 ꢀ 7.30 (dd, J = 10.08, 2.00 Hz, 2 H), 7.33 ꢀ 7.36 (q, J =
.2 Hz, 1 H), 7.39 ꢀ 7.43 (q, J = 7.56 Hz, 2 H), 7.63 ꢀ 7.65 (d, J = 7.40 Hz, 2 H), 7.72 (s, 1 H);
+
MS (ESI): found: [M + H] , 391.05.
(E)ꢀ2ꢀ((4ꢀ(4ꢀChlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylic acid (11c)
11c was synthesized from 10c by following the general procedure B as a white solid (Yield:
1
9
2%); H NMR (400 MHz, CDCl ) δ ppm : 2.69 ꢀ 2.74 (t, 4 H), 3.15 ꢀ 3.19 (t, 4 H), 3.66 (s, 2 H),
3
13
6.78 ꢀ 6.85 (t, 2 H), 7.18 ꢀ 7.19 (d, 2 H), 7.26 (s, 1 H), 7.38 (m, 4 H), 8.04 (s, 1 H); C NMR
(
DMSOꢀd )   ppm : 167.78, 150.12, 141.90, 135.48, 130.71, 129.36, 129.07, 128.96, 122.90,
6
+
1
17.41, 53.71, 52.33, 48.49; MS (ESI): found: [M + H] , 357.10.
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(
E)ꢀ2ꢀ((4ꢀ(4ꢀHydroxyphenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylic acid (11d)
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9
6
1d was synthesized from 10d by following the general procedure B as a white solid (Yield:
1
3%); H NMR (400 MHz, DMSOꢀd ) δ ppm : 2.96 (s, 4 H), 3.23 (s, 4 H), 3.99 (s, 2 H), 6.68 ꢀ
6
10
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.70 (d, J = 8.6 Hz, 2 H), 6.81 (s, 2 H), 7.30 ꢀ 7.39 (m, J = 7.96 Hz, 5 H), 8.11 (s, 1 H), 8.31 (s, 1
+
H); MS (ESI): found: [M + H] , 339.20.
(
E)ꢀ2ꢀ((4ꢀ(2ꢀMethoxyphenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylic acid (11f)
1
1f was synthesized from 10f by following the general procedure B as a white solid (Yield:
1
72%); H NMR (300 MHz, CDCl ) δ ppm : 2.97 (s, 8 H), 3.75 ꢀ 3.76 (d, J = 1.38 Hz, 2 H), 3.85
3
(
(
(
s, 3 H), 6.87 ꢀ 6.95 (m, 3 H), 7.00 ꢀ 7.13 (m, 1 H), 7.29 (dd, J = 5.41, 2.11 Hz, 2 H), 7.36 ꢀ 7.50
+
m, 3 H), 8.07 (s, 1 H); MS (ESI): found: [M + H] , 353.20.
E)ꢀ2ꢀ((4ꢀ(4ꢀMethoxyphenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylic acid (11g)
1
1g was synthesized from 10g by following the general procedure B as a white solid (Yield:
1
8
0%); H NMR (300 MHz, CDCl ) δ ppm : 2.83 (s, 4 H), 3.17 (s, 4 H), 3.72 ꢀ 3.73 (d, J = 1.38
3
Hz, 2 H), 3.78 (s, 3 H), 6.80 ꢀ 6.92 (m, 4 H), 7.25 ꢀ7.33 (m, 2 H), 7.35 ꢀ 7.49 (m, 3 H), 8.07 (s, 1
+
H); MS (ESI): found: [M + H] , 353.20.
(E)ꢀ2ꢀ((4ꢀ(3ꢀHydroxyphenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylic acid (11h)
1
1h was synthesized from 10h by following the general procedure B as a white solid (Yield:
1
8
6%); H NMR (400 MHz, CDCl ) δ ppm : 2.81 (s, 4 H), 3.25 (s, 4 H), 3.82 (s, 2 H), 6.28 ꢀ 6.35
3
(m, J = 8.6 Hz, 3 H), 6.96 ꢀ 7.00 (t, J = 7.92 Hz, 1 H), 7.27 (s, 1 H), 7.30 ꢀ 7.39 (m, J = 6.72 Hz ,
+
3
H), 8.06 (s, 1 H); MS (ESI): found: [M + H] , 339.20.
(
E)ꢀ2ꢀ((4ꢀ(2ꢀChlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylic acid (11i)
1
1i was synthesized from 10i by following the general procedure B as a white solid (Yield:
1
7
2%); H NMR (300 MHz, CDCl ) δ ppm : 2.62 (s, 4 H), 3.73 (s, 4 H), 3.50 (s, 2 H), 6.73 ꢀ 6.81
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(
(
(
d, 1 H), 6.84 ꢀ 6.94 (t, 2 H), 7.12 – 7.24 (t, 1 H), 7.36 ꢀ 7.50 (m, 3 H), 7.63 ꢀ 7.70 (d, 2 H), 7.80
+
s, 1 H), 13.50 (s, 1 H); MS (ESI): found: [M + H] , 357.10.
E)ꢀ2ꢀ((4ꢀ(3ꢀChlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacrylic acid (11j)
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1j was synthesized from 10j by following the general procedure B as a white solid (Yield:
1
86%); H NMR (400 MHz, DMSOꢀd ) δ ppm : 2.61 ꢀ 2.63 (s, 4 H), 2.90 ꢀ 3.10 (s, 4 H), 3.5 (s, 2
6
H), 6.90 ꢀ 7.10 (t, 1 H), 7.10 ꢀ 7.20 (t, 1 H), 7.20 ꢀ 7.30 (t, 1 H), 7.40 ꢀ 7.50 (m, 4 H), 7.66 ꢀ 7.70
+
(d, 2 H), 7.87 (s, 1H); MS (ESI): found: [M + H] , 357.10.
General procedure for synthesis of 12aꢀ12af, 17aꢀ17e, 22a, 22fꢀ22h and 27aꢀ27j (General
procedure C)
To 11aꢀ11l, 16, 21a, 21fꢀ21h or 26a, 26b (0.6 mmol) in 3 mL (3 v) of DMF were added
ethyl/methyl ester of αꢀaminoacid hydrochloride (0.9 mmol), HATU (0.9 mmol) and DIPEA
o
(1.86 mmol), the reaction mass was stirred at 25ꢀ30 C for 10ꢀ12 h. Reaction completion was
checked by TLC. DMF was concentrated under reduces pressure. To the residue ice water was
added and the mixture was extracted three times with DCM. The combined DCM layers were
washed with ice water and brine, dried with anhydrous sodium sulphate and concentrated to get
crude product. This was then subjected to silica gel column chromatography, elution being done
with ethyl acetate in nꢀhexane. The fraction containing desired product was evaporated to
dryness to get an oily mass.
Methyl (E)ꢀ(3ꢀphenylꢀ2ꢀ((4ꢀphenylpiperazinꢀ1ꢀyl)methyl)acryloyl)glycinate (12a)
Following above general procedure C, reaction of 11a and methyl glycinate hydrochloride
1
resulted in 12a as a syrup (Yield: 87%); H NMR (300 MHz, DMSOꢀd ) δ ppm : 1.20 (s, 3 H),
6
2
.52 ꢀ 2.60 (m, 4 H), 3.17 (s, 4 H), 3.46 (s, 2 H), 4.02 (d, J = 5.59 Hz, 2 H), 4.07 ꢀ 4.17 (m, 2 H),
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.71 ꢀ 6.82 (m, 1 H), 6.88 ꢀ 7.00 (m, 2 H), 7.16 ꢀ 7.25 (m, 2 H), 7.31 ꢀ 7.39 (m, 1 H), 7.45 (d, J =
4
.31 Hz, 4 H), 7.63 ꢀ 7.69 (m, 1 H), 9.61 ꢀ 9.71 (m, 1 H).
Methyl (E)ꢀ(3ꢀphenylꢀ2ꢀ((4ꢀphenylpiperazinꢀ1ꢀyl)methyl)acryloyl)valinate (12b)
10
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Following above general procedure C, reaction of 11a and methyl valinate hydrochloride
1
resulted in 12b as a viscous oil (Yield: 70%); H NMR (300 MHz, DMSOꢀd ) δ ppm : 0.93 (t, J
6
=
6.92 Hz, 6 H), 2.10 ꢀ 2.22 (m, 1 H), 2.53 ꢀ 2.63 (m, 4 H), 3.12 ꢀ 3.25 (m, 4 H), 3.50 ꢀ 3.55 (m, 2
H), 3.66 (s, 3 H), 4.37 ꢀ 4.47 (m, 1 H), 6.73 ꢀ 6.83 (m, 1 H), 6.92 (d, J = 8.07 Hz, 2 H), 7.20 (s, 2
H), 7.32 ꢀ 7.52 (m, 5 H), 7.71 (s, 1 H), 9.95 ꢀ 10.05 (m, 1 H).
Methyl (E)ꢀ(3ꢀphenylꢀ2ꢀ((4ꢀphenylpiperazinꢀ1ꢀyl)methyl)acryloyl)leucinate (12c)
Following above general procedure C, reaction of 11a and methyl leucinate hydrochloride
1
resulted in 12c as a syrup (Yield: 70%); H NMR (300 MHz, DMSOꢀd ) δ ppm : 0.90 (dd, J =
6
6.37, 2.43 Hz, 6 H), 1.48 ꢀ 1.66 (m, 2 H), 1.67 ꢀ 1.83 (m, 1 H), 2.52 ꢀ 2.60 (m, 4 H), 3.15 (s, 4 H),
3.48 (s, 2 H), 3.65 (s, 3 H), 4.40 ꢀ 4.53 (m, 1 H), 6.78 (s, 1 H), 6.93 (d, J = 8.07 Hz, 2 H), 7.21 (s,
2 H), 7.33 ꢀ 7.40 (m, 1 H), 7.43 (s, 4 H), 7.61 (s, 1 H), 9.69 ꢀ 9.77 (m, 1 H).
Methyl
(E)ꢀ3ꢀmethylꢀ2ꢀ(3ꢀphenylꢀ2ꢀ((4ꢀphenylpiperazinꢀ1ꢀyl)methyl)acrylamido)
pentanoate (12d)
Following above general procedure C, reaction of 11a and methyl 2ꢀaminoꢀ3ꢀmethylpentanoate
1
hydrochloride resulted in 12d as a viscous oil (Yield: 75%); H NMR (300 MHz, DMSOꢀd ) δ
6
ppm : 0.79 ꢀ 0.99 (m, 1 H), 1.24 (s, 1 H), 1.12 ꢀ 1.30 (m, 1 H), 1.32 ꢀ 1.51 (m, 1 H), 1.81 ꢀ 1.94
(m, 1 H), 2.55 (d, J = 4.49 Hz, 1 H), 2.52 ꢀ 2.64 (m, 1 H), 3.17 (s, 1 H), 3.49 ꢀ 3.56 (m, 1 H),
3
.65 (s, 1 H), 4.39 ꢀ 4.53 (m, 1 H), 6.78 (s, 1 H), 6.92 (d, J = 8.07 Hz, 1 H), 7.14 ꢀ 7.28 (m, 1 H),
.32 ꢀ 7.52 (m, 1 H), 7.70 (s, 1 H), 10.00 (d, J = 8.25 Hz, 1 H).
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Ethyl (E)ꢀ2ꢀ(3ꢀphenylꢀ2ꢀ((4ꢀphenylpiperazinꢀ1ꢀyl)methyl)acrylamido)pentanoate (12e)
Following above general procedure C, reaction of 11a and ethyl 2ꢀaminopentanoate
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hydrochloride resulted in 12e as a syrup (Yield: 78%); H NMR (300 MHz, CDCl ) δ ppm : 0.88
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(
s, 3 H), 1.19 (t, J = 7.11 Hz, 3 H), 1.32 ꢀ 1.42 (m, 2 H), 1.64 ꢀ 1.79 (m, 2 H), 2.53 ꢀ 2.63 (m, 4
H), 3.11 ꢀ 3.24 (m, 4 H), 3.49 (s, 2 H), 4.07 ꢀ 4.19 (m, 2 H), 4.36 ꢀ 4.48 (m, 1 H), 6.69 ꢀ 6.82 (m,
H), 6.88 ꢀ 7.01 (m, 2 H), 7.13 ꢀ 7.27 (m, 2 H), 7.42 (s, 5 H), 7.62ꢀ 7.68 (m, 1 H), 9.80 ꢀ 9.92
m, 1 H).
Ethyl
12f)
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(
(E)ꢀ(2ꢀ((4ꢀ(2,3ꢀdichlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacryloyl)glycinate
(
Following above general procedure C, reaction of 11b and ethyl glycinate hydrochloride
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resulted in 12f as a viscous oil (Yield: 61%, purity by LCꢀMS: >99%); H NMR (300 MHz,
CDCl ) δ ppm : 1.18 ꢀ 1.33 (t, J = 7.15 Hz, 3 H), 2.68 (s, 4 H), 3.11 (s, 4 H), 3.56 (s, 2 H), 4.19
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(d, J = 4.95 Hz, 2 H), 4.21 ꢀ 4.29 (q, J = 5.95 Hz ,2 H), 6.96 ꢀ 6.99 (dd, J = 6.33, 3.30 Hz, 1 H),
7.10 ꢀ 7.18 (m, 2 H), 7.27 ꢀ 7.29 (d, J = 1.19 Hz, 2 H), 7.31 ꢀ 7.45 (m, 3 H), 7.99 (s, 1 H), 10.28 ꢀ
+
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0.30 (t, J = 3.05 Hz,1 H); MS (ESI): found: [M + H] , 476.20.
Methyl
12g)
(E)ꢀ(2ꢀ((4ꢀ(2,3ꢀdichlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacryloyl)valinate
(
Following above general procedure C, reaction of 11b and methyl valinate hydrochloride
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resulted in 12g as a thick liquid (Yield: 73%, purity by LCꢀMS: >99%); H NMR (300 MHz,
CDCl ) δ ppm : 0.98 ꢀ 1.04 (dd, J = 11.28, 6.88 Hz, 6 H), 2.25 ꢀ 2.31 (m, J = 6.85, 4.72 Hz, 1 H),
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.67 ꢀ 2.74 (m, 4 H), 3.09 ꢀ 3.14 (d, J = 13.94 Hz, 4 H), 3.49 ꢀ 3.63 (m, 2 H), 3.75 (s, 3 H), 4.69 ꢀ
.73 (dd, J = 8.76, 4.63 Hz, 1 H), 6.94 ꢀ 6.98 (dd, J = 6.46, 3.16 Hz, 1 H), 7.14 ꢀ 7.18 (m, 2 H),
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Journal of Medicinal Chemistry
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.26 ꢀ 7.30 (m, 3 H), 7.31 ꢀ 7.44 (m, 3 H), 7.98 (s, 1 H), 10.22 ꢀ 10.25 (d, J = 8.80 Hz, 1 H); MS
+
(ESI): found: [M + H] , 504.25.
Ethyl (E)ꢀ(2ꢀ((4ꢀ(4ꢀchlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacryloyl)glycinate (12k)
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Following above general procedure C, reaction of 11c and ethyl glycinate hydrochloride resulted
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in 12k as a thick liquid (Yield: 68%, purity by LCꢀMS: >99%); H NMR (300 MHz, CDCl ) δ
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ppm : 1.19 ꢀ 1.23 (t, J = 6.00 Hz, 3 H), 2.54ꢀ2.57 (t, J = 4.54 Hz, 4 H), 3.12 ꢀ 3.16 (t, J = 6.24
Hz, 4 H), 3.45 (s, 2 H), 4.08 ꢀ 4.17 (m, 4 H), 6.73 ꢀ 6.76 (m, 2 H), 7.09 ꢀ 7.13 (m, 2 H), 7.18 ꢀ
7.21 (m, 2 H), 7.25 ꢀ 7.34 (m, 3 H), 7.93 (s, 1 H), 10.09 ꢀ 10.12 (t, J = 4.36 Hz, 3 H); found: [M
+
+
H] , 442.20.
Methyl (E)ꢀ(2ꢀ((4ꢀ(4ꢀchlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacryloyl)valinate (12l)
Following above general procedure C, reaction of 11c and methyl valinate hydrochloride
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resulted in 12l as a thick liquid (Yield: 72%, purity by LCꢀMS: >99%); H NMR (300 MHz,
CDCl ) δ ppm : 0.93 ꢀ 0.96 (d, J = 6.88 Hz, 3 H), 0.99 ꢀ 1.01 (d, J = 6.88 Hz, 3 H), 2.22 ꢀ 2.28
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(
m, J = 6.83, 4.68 Hz, 1 H), 2.60 ꢀ 2.62 (d, J = 4.95 Hz, 2 H), 2.66 ꢀ 2.72 (m, 2 H), 3.18 ꢀ 3.24
m, 4 H), 3.47 ꢀ 3.60 (q, J = 10.50, 8.00 Hz, 2 H), 3.73 (s, 1 H), 4.66 ꢀ 4.71 (q, J = 8.71, 4.58 Hz,
(
1 H), 6.78 ꢀ 6.86 (m, 2 H), 7.16 ꢀ 7.23 (m, 2 H), 7.25 ꢀ 7.29 (m, 2 H), 7.30 ꢀ 7.43 (m, 3 H), 7.98
+
(
s, 1 H), 10.09 ꢀ 10.12 (d, J = 7.89 Hz, 1 H); found: [M + H] , 470.25.
Ethyl (E)ꢀ(2ꢀ((4ꢀ(4ꢀchlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylacryloyl)leucinate (12m)
Following above general procedure C, reaction of 11c and ethyl leucinate hydrochloride resulted
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in 12m as a thick liquid (Yield: 57%, purity by LCꢀMS: >99%); H NMR (300 MHz, CDCl ) δ
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ppm : 0.97 ꢀ 1.01 (t, J = 6.14 Hz, 6 H), 1.28 ꢀ 1.32 (t, J = 7.15 Hz, 4 H), 1.59 ꢀ 1.80 (m, 3 H),
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.60 ꢀ 2.65 (m, 2 H), 2.67 ꢀ 2.72 (m, 2 H), 3.18 ꢀ 3.28 (m, 4 H), 3.49 ꢀ 3.60 (q, J = 6.00 Hz, 2 H),
.17 ꢀ 4.24 (q, J = 7.15 Hz, 2 H), 4.68 ꢀ 4.75 (m, J = 8.23, 5.00 Hz, 1 H), 6.81 ꢀ 6.86 (m, 2 H),
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