Synthesis, antiplatelet aggregation activity, and molecular modeling study of novel substituted-piperazine analogues

Article abstract of DOI:10.1007/s00044-010-9411-5

New carbamoylpyridine and carbamoylpiperidine analogues containing nipecotic acid scaffold were designed, synthesized, and evaluated for their platelet aggregation inhibitory activity. Molecular modeling investigation was performed and the impact of lipophilicity on activity was also discussed. Structure activity relationship among this series was obtained. N ¹-[1-(4-bromobenzyl)-3-piperidinocarbonyl]- N⁴-(2- chlorophenyl)-piperazine hydrobromide (20), and 1,4-bis-[3-[N ⁴-(2-chlorophenyl)-N¹-(piperazinocarbonyl)]- piperidin-1-yl-methyl]-benzene dibromide (30) are the most active antiplatelet aggregating compounds in this study, both at concentration of 0.06 lM. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9411-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:898–911
DOI 10.1007/s00044-010-9411-5
ORIGINAL RESEARCH
Synthesis, antiplatelet aggregation activity, and molecular
modeling study of novel substituted-piperazine analogues
•
Khairia M. Youssef Mohamed A. Al-Omar Hussein I. El-Subbagh
•
•
•
Laila A. Abou-zeid Abdel-Galil M. Abdel-Gader
•
•
Nadia G. Haress Ali S. Al-Tuwaijri
Received: 21 December 2009 / Accepted: 16 August 2010 / Published online: 2 September 2010
Ó Springer Science+Business Media, LLC 2010
Abstract New carbamoylpyridine and carbamoylpiperi-
dine analogues containing nipecotic acid scaffold were
designed, synthesized, and evaluated for their platelet aggre-
gation inhibitory activity. Molecular modeling investigation
was performed and the impact of lipophilicity on activity was
also discussed. Structure activity relationship among this
series was obtained. N1-[1-(4-bromobenzyl)-3-piperidino-
carbonyl]-N4-(2-chlorophenyl)-piperazine hydrobromide
(20), and 1,4-bis-[3-[N4-(2-chlorophenyl)-N1-(piperazino-
carbonyl)]-piperidin-1-yl-methyl]-benzene dibromide (30)
are the most active antiplatelet aggregating compounds in
this study, both at concentration of 0.06 lM.
Introduction
Platelets are involved in the pathogenesis of many car-
diovascular and thromboembolic diseases (Chatelain and
Massingham, 1997; Fuster et al., 1992). Their hyperactivity
increases the risk of various vaso-occlusive diseases, such
as unstable angina, acute myocardial infarction, transient
ischemic attacks, and complications following percutane-
ous coronary intervention (Fuster et al., 1992). Platelets
can be activated by a number of agonists, such as adeno-
sine 5(-diphosphate (ADP), thrombin, platelet-activating
factor (PAF), serotonin, thromboxane A2 (TxA2), collagen,
and catecholamines (Majerus and Tollesfsen, 2001). ADP
plays a relevant role in platelet function. It can trigger
platelet activation, which is mediated by at least three
purinergic receptors (P2Y1, P2Y12, and P2X) showing dis-
tinct specificity; ADP is also responsible for the secondary
wave of platelet aggregation, followed by ADP release
from dense granules which potentiates the aggregation
response induced by other agents (Weksler, 2000; Mills,
1996).
Keywords Synthesis ꢀ Piperazine analogues ꢀ
Antiplatelet aggregation ꢀ Molecular modeling studies
Antiplatelet aggregation agents, including aspirin and
thromboxane modulators, e.g., ridogrel; ADP antagonists,
like the thienopyridine derivatives ticlopidine and clopi-
dogrel; phosphodiesterase inhibitors, e.g., dipyridamole and
cilostazol; and platelet glycoprotein IIb/IIIa antagonists,
such as tirofiban and sibrafiban; are useful in the prophy-
laxis and treatment of thromboembolic diseases (Kunapuli,
1998; Gachet, 2001; Van De Graaff and Steinhubl, 2000;
K. M. Youssef (&)
Department of Pharmaceutical Chemistry, Faculty
of Pharmaceutical Sciences & Pharmaceutical Industries,
Future University, 12311 Cairo, Egypt
e-mail: khairia_m@yahoo.com
M. A. Al-Omar ꢀ H. I. El-Subbagh ꢀ N. G. Haress
Department of Pharmaceutical Chemistry, College of Pharmacy,
King Saud University, Riyadh 11451, Saudi Arabia
´
Patrono et al., 2001; Dogne et al., 2001). Nevertheless,
L. A. Abou-zeid
Department of Pharmaceutical Organic Chemistry, Faculty
of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
currently available, orally administered antiplatelet drugs
have limitations, especially with regard to side effects and
broad clinical utility, as well as interference with physio-
logical platelet function in hemostasis (Antithrombotic
Trialists’ Collaboration, 2002). Despite their efficacy, the
A.-G. M. Abdel-Gader ꢀ A. S. Al-Tuwaijri
Department of Physiology, College of Medicine, King Saud
University, Riyadh 11451, Saudi Arabia
123

Med Chem Res (2011) 20:898–911
899
administration of thienopyridines is associated with some
undesired side effects, such as neutropenia, thrombocyto-
penia, aplastic anemia, and thrombotic thrombocytopenic
purpura (Storey, 2001). Moreover, most probably due to
selective inhibition of some pathways of platelet activation
and recruitment by aspirin and thienopyridines, a number of
patients have been shown to be resistant to these drugs
(Berger, 1999; McKee et al., 2002). These limitations are
among the reasons stimulating the search for new anti-
platelet drugs.
In this study, a series of new modified carbamoyl-pyri-
dine and carbamoyl-piperidine analogues were designed
and synthesized which related to the C-terminal c-chain of
fibrinogen, containing a nipecotic acid scaffold. The
molecular modeling features of the designed compounds
and their recognition profiles with the active site of
thrombin receptor were investigated.
Chemistry
The interest in antiplatelet agents stemmed from the
finding that some bis-3-carbamoyl-piperidine derivatives,
namely a,a0-bis[3-(N,N-diethyl-carbamoyl)-piperidino]-p-
xylenes (1, Chart 1), inhibit in vitro human platelet
aggregation, and are effective in thrombosis models in
vivo (Tucker et al., 1997; Youssef and Al-Shafie, 1998;
De Marco et al., 2004; De Candia et al., 2005; Papado-
poulou et al., 2005; Leoncini et al., 1997, 2004; Cody
et al., 1999; Binisti et al., 2001; Heath et al., 2004). Due
to their lipophilicity and surface activity, bis-nipecota-
mides can penetrate the platelet membranes and interact
with anionic phospholipids (mainly phosphatidylinositol,
PI, and phosphatidylserine, PS), thus rendering them
resistant to hydrolysis catalyzed by phospholipase-C to
the second messengers inositol 1,4,5-triphosphate (IP3)
and s,n-1,2-diacylglycerol (DAG). They also inhibit
phosphoinositide turnover. As a consequence, the levels
of IP3 and of cytosolic Ca2? concentrations, necessary for
myosin phosphorylation and subsequent platelet activa-
tion, are reduced (Youssef and Al-Shafie, 1998; Heath
et al., 2004; Feng et al., 1992; Dillingham et al., 1989).
Phospholipases A2 (PLA2s) catalyze the hydrolysis of
glycerol-phospholipids at the sn-2 position and generate
free fatty acids and lysophospholipids (Slotboom et al.,
1982). This can provide, in some cases, substrates for the
biosynthesis of prostaglandins, thromboxanes, leukotri-
enes, and other oxygenated metabolites of arachidonic
acid (AA), i.e., eicosanoids, as well as PAF (Binisti et al.,
2001; Sallem et al., 2006) well-known mediators of
inflammatory processes and tissue injury. It was reported
that the piperazine derivative PMS 832 (2, Chart 1)
showed remarkable inhibitory potency of PLA2 activity
with IC50 values in the micromolar range.
The synthesis of the target compounds is depicted in
Scheme 1. Nicotinoyl chloride (3) was reacted with differ-
ent 1-aryl and 1-aroyl-piperazines to furnish the car-
bamoyl-pyridines N1-(3-pyridino-carbonyl)-N4-aryl-pipera-
zine hydrochlorides (4, 5). Quaternization of 4 and 5 was
carried out in acetone solutions of different alkyl or aroyl-
halides in the presence of K2CO3 to afford the corresponding
quaternary ammonium salts N1-(1-aralkyl- or 1-aroyl-pyr-
idinium-3-yl-carbonyl)-N4-aryl-piperazine halides (6–16).
Catalytic hydrogenation of the obtained pyridinium quater-
naryammoniumsalts,usingplatinumoxideaffordedthetarget
compounds N1-(1-aralkyl or 1-aroyl-3-piperidino-carbonyl)-
N4-aryl-piperazine hydrohalides (17–27) (Table 1). Two
molar equivalents of N1-(3-pyridino-carbonyl)-N4-aryl-
piperazine hydro-chlorides (4, 5) were allowed to react with
one mole equivalent of a,a0-dibromo-p-xylene to give the
quaternary ammonium salts 1,4-bis-[3-(N4-aryl-N1-piperazi-
no-carbonyl)-pyridinium-1-yl-methyl]-benzene dibromides
(28, 29), which were then subjected to catalytic hydrogenation
using platinum oxide to afford the target compounds 1,4-bis-
[3-(N4-aryl-N1-piperazino-carbonyl)-piperidin-1-yl-methyl]-
benzene dibromides (30, 31) (Table 2). Two molar equiva-
lents of nicotinoyl chloride (3) were reacted with anhy-
drous piperazine to furnish the corresponding N1,N4-bis-[3-
pyridino-carbonyl]-piperazine dihydrochloride (32). The
acetone solution of the starting amide 32 was reacted with
different alkyl or aroyl-halides in the presence of K2CO3 to
afford the corresponding quaternary ammonium salts N1,N4-
bis-(1-aralkyl- or 1-aroyl-pyridinium-3-yl-carbonyl)-piper-
azine dihalides (33–39), which were then subjected to
catalytic hydrogenation using platinum oxide to afford the
target compounds N1,N4-bis-(1-aralkyl- or 1-aroyl-piper
idino-3-yl-carbonyl)-piperazine dihydrohalides (40–46)
(Table 3).
OCH3
O
N
O
N
R
N
N
R
HN
N
Results and discussion
R
R
O
2HBr
n-C13H27
Platelet aggregation involves the cohesion of platelets to each
other and this process is basic to form the hemostatic platelet
plug. In vitro it can be induced by a number of agonists
PMS 832 (2)
1
Chart 1 Literature cited antiplatelet aggregation agents
123

900
Med Chem Res (2011) 20:898–911
O
O
H2/PtO2
HX
N
N
+
N
N
N
-
X
N
R
Ar
Ar
R
6-16
17-27
RX
O
O
-
Br
Br
Ar
2 Br
+
NH
COCl Ar
N
N
N
N
N
N
N
N
N
N
O
N
Ar
HCl
+
Ar
4,5
28,29
3
H2/PtO2
HN NH
O
N
Ar
N
2 HX
N
N
O
2 HCl
N
N
N
N
N
O
O
N
Ar
30, 31
32
Ar = 2-Cl-Ph, 2-Pyrimidinyl
R = Bn, 2-F-Bn, 4-F-Bn, 4-Br-Bn, 4-NO2-Bn, 3,4,5-(CH3O)3-Bz, 4-Br-Bz.
Bn = Benzyl, Bz = Benzoyl
RX
R
N +
R
N
H2/PtO2
O
O
-
2 X
2 HX
N
N
N
N
O
O
+ N
R
N
R
33-39
40-46
Scheme 1 Synthesis of the target compounds 4–46
including adenosine-50-diphosphate (ADP), adrenaline, col-
lagen, thrombin, and other non-physiological compounds
such as ristocetin. In this study, platelet aggregation was
measured in response to ADP (20.0, 2.0, and 1.0 lmol/l),
adrenaline (100 lmol/l), collagen (0.19 g/l), arachidonic
acid (AA, 1.64 mmol/l), and ristocetin (1.5, 1.2, and 1.0 g/l)
as reported. All these concentrations of agonists represent
final concentrations obtained by adding 20 ll of the aggre-
gating agent to 180 ll of platelet-rich plasma (PRP).
and N1-(1-aralkyl- or 1-aroyl-pyridinium-3-yl-carbonyl)-
N4-aryl-piperazine halides (6–9, 11, and 13) showed low
antiaggregating effects. Compound 4 showed antiaggre-
gating effect for ADP, AA, adrenaline, and collagen at
concentration of 0.4 lM. Compound 5 inhibited the
aggregating effects of all agonists at concentration of
3.0 lM. Compound 6 showed antiaggregating effect for
ristocetin only at concentration of 2.5 lM. Compounds 7,
8, 9, 11, and 13 inhibited the aggregating effect of ADP,
AA, adrenaline, and collagen at concentration of 2.0, 10.0,
1.0, 0.5, and 10.0 lM, respectively. Compounds 7, 11,
The antiaggregating effect of compounds N1-(3-pyri-
dino-carbonyl)-N4-aryl-piperazine hydrochlorides (4, 5)
123

Med Chem Res (2011) 20:898–911
901
Table 1 Physicochemical properties of the new synthesized compounds 4–27
O
O
O
N
N
N
+
N
N
N
N
Ar
N
Ar
N
R
Ar
-
HCl
X
HX
R
4, 5
6-16
17-27
Compd
Ar
R
Yield (%)
MP (°C)
Molecular formulae
log P
4
2-Cl-Ph
–
–
62
–
225–227
–
C16H17Cl2N3O
2.46
0.48
–
5
2-Pyrimidinyl
2-Cl-Ph
Youssef and Al-Shafie (1998)
6
Bn
55
57
60
65
58
41
61
78
81
51
59
53
56
59
64
57
42
60
77
80
60
58
[300
C
C
C
C
23H23Cl2N3O
23H22Cl2FN3O
23H22Cl2FN3O
23H22Br2ClN3O
7
2-Cl-Ph
2-F-Bn
102–104
246–248
195–197
172–174
204–206
170–172
218–220
[300
–
8
2-Cl-Ph
4-F-Bn
–
9
2-Cl-Ph
4-Br-Bn
4-NO2-Bn
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
2-Cl-Ph
C23H22BrClN4O3
C26H27Cl2N3O5
–
2-Cl-Ph
3,4,5-(CH3O)3-Bz
4-Br-Bz
–
2-Cl-Ph
C
C
23H20BrCl2N3O2
21H21Br2N5O
–
2-Pyrimidinyl
2-Pyrimidinyl
2-pyrimidinyl
2-Pyrimidinyl
2-Cl-Ph
4-Br-Bn
–
4-NO2-Bn
3,4,5-(CH3O)3-Bz
4-Br-Bz
C21H21BrN6O3
C24H26ClN5O5
–
220–222
240–242
189–191
160–162
195–197
191–193
135–137
170–172
176–178
118–120
197–199
102–104
196–198
–
C
C
C
C
C
21H19BrClN5O2
23H29Cl2N3O
–
Bn
4.22
4.38
4.38
5.05
3.22
3.28
4.48
3.07
0.84
1.29
2.50
2-Cl-Ph
2-F-Bn
23H28Cl2FN3O
23H28Cl2FN3O
23H28Br2ClN3O
2-Cl-Ph
4-F-Bn
2-Cl-Ph
4-Br-Bn
2-Cl-Ph
4-NO2-Bn
3,4,5-(CH3O)3-Bz
4-Br-Bz
C23H28BrClN4O3
C26H33Cl2N3O5
2-Cl-Ph
2-Cl-Ph
C
C
23H26BrCl2N3O2
21H27Br2N5O
2-Pyrimidinyl
2-Pyrimidinyl
2-Pyrimidinyl
2-Pyrimidinyl
4-Br-Bn
4-NO2-Bn
3,4,5-(CH3O)3-Bz
4-Br-Bz
C21H27BrN6O3
C24H32ClN5O5
C
21H25BrClN5O2
and 13 inhibited the aggregating effect of ristocetin at
concentration of 5.0, 1.0, and 20.0 lM, respectively
(Table 4).
0.125 lM. Ristocetin-induced aggregation response was
inhibited at 53% at this concentration. At a concentration
of 0.06 lM compound 20 showed 20% disaggregation
against ADP, and total inhibition of the aggregating
activity of AA, adrenaline, and collagen, but ristocetin-
induced activity was inhibited by only 62%. When the
concentration of compound 20 was decreased to 0.03 lM, it
elicited 55 and 56% inhibition of aggregation for ADP and
AA, respectively, a total aggregation inhibition for adrena-
line, collagen and 67% inhibition for ristocetin. Compounds
21, 23, 24, and 26 inhibited the aggregating effect of ADP,
AA, adrenaline, and collagen at concentration of 0.5, 0.5,
1.0, and 0.8 lM, respectively. Compound 23 completely
inhibited ristocetin-induced aggregation at concentration of
2.5 lM (Table 5).
Compounds N1-(1-aralkyl- or 1-aroyl-3-piperidino-
carbonyl)-N4-aryl-piperazine hydrohalides showed high
antiaggregating effect. Compound 17 showed aggregation
inhibition for all agonists at concentration of 5.0 lM. It
inhibited the aggregating effect of ADP, AA, adrenaline,
and collagen at concentration of 0.25 lM. Similarly,
compounds 18 and 19 showed the same potency at con-
centration of 0.25 lM. Ristocetin-induced aggregation
response was inhibited by compounds 18 and 19 at 18 and
68%, respectively at the same concentration. Compound 20
elicited maximum inhibition of aggregation against
ADP, AA, adrenaline, and collagen at a concentration of
123

902
Med Chem Res (2011) 20:898–911
Table 2 Physicochemical properties of the new synthesized compounds 28–31
O
Ar
+
O
N
Ar
N
N
N
N
N
N
N
N
O
N
N
N
O
+
-
2 HX
2 Br
Ar
Ar
30,31
28,29
Compd
Ar
2-Cl-Ph
Yield (%)
MP (°C)
Molecular formulae
C40H40Br2Cl2N6O2
log P
28
29
30
31
72
78
70
75
238–240
240–242
185–187
212–214
–
2-Pyrimidinyl
2-Cl-Ph
C
36H38Br2N10O2
40H52Br2Cl2N6O2
36H50 Cl2N10O2
–
C
C
6.15
2.44
2-Pyrimidinyl
Table 3 Physicochemical properties of the new synthesized compounds 32–46
N
R N
R
N +
O
O
N
O
N
N
N
N
N
O
O
O
N
R
2 X-
N
+ N
R
2 HX
2 HCl
32
40-46
33-39
Compd
R
Yield (%)
MP (°C)
Molecular formulae
C16H18Cl2N4O2
log P
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
–
60
84
81
78
79
72
61
69
59
58
61
64
57
54
68
195–197
220–222
230–232
[300
–
Bn
C
C
C
C
30H30Cl2N4O2
–
2-F-Bn
4-F-Bn
4-Br-Bn
30H28 Cl2F2N4O2
30H28 Cl2F2N4O2
30H28Br4N4O2
–
–
240–242
188–190
150–152
297–299
173–175
135–137
136–138
270–272
173–175
182–184
178–180
–
4-NO2-Bn
C30H28Cl2N6O6
C36H38Cl2N4O10
–
3,4,5-(CH3O)3-Bz
4-Br-Bz
–
C
C
C
C
C
30H24 Br2Cl2 N4O4
30H42Cl2N4O2
–
Bn
3.17
3.49
3.49
4.83
1.95
1.29
3.70
2-F-Bn
30H40Cl2F2N4O2
30H40 Cl2F2N4O2
30H40Br4N4O2
4-F-Bn
4-Br-Bn
4-NO2-Bn
3,4,5-(CH3O)3-Bz
4-Br-Bz
C30H40Cl2N6O6
C36H50Cl2N4O10
C30H36 Br2Cl2N4O4
The antiaggregating effect of 1,4-bis-[3-(N4-aryl-N1-
piperazino-carbonyl)-pyridinium-1-yl-methyl]-benzene
dibromides (28 and 29) showed the lowest antiaggregating
activity in this study. Compounds 28 and 29 inhibited the
aggregating effect of ADP, AA, adrenaline, and collagen at
concentration of 5.0 and 50 lM, respectively. They
inhibited the aggregating effect of ristocetin at 51 and 73%,
respectively, at the same concentrations. While compounds
1,4-bis-[3-(N4-aryl-N1-piperazino-carbonyl)-piperidin-1-yl-
methyl]-benzene dibromides (30 and 31) inhibited the
aggregating effect of ADP, AA, adrenaline, and collagen
at concentration of 0.125 and 1.0 lM, respectively.
They inhibited the aggregating effect of ristocetin at 31
and 58%, respectively, at the same concentrations. The
123

Med Chem Res (2011) 20:898–911
903
Table 4 The antiaggregating activity of the new synthesized com-
pounds 4–9, 11, and 13
Table 5 The antiaggregating activity of the new synthesized com-
pounds 17–21, 23, 24, and 26
Compd Conc.(lM) ADP AA Adrenaline Collagen Ristocetin
Compd Conc.(lM) ADP AA Adrenaline Collagen Ristocetin
4
N
70
#
69 65
81
#
84
23*
71
77
99
#
17
N
75
#
72 67
79
#
76
#
1.0
0.5
0.4
N
#
#
#
#
#
#
5.0
1.0
0.5
0.25
0.1
N
#
#
#
#
#
#
#
#
#
#
#
22
26
30
90
87
50
73
18*
50
96
40
67
68
100
94
39
53
62
67
68
23
50*
58
66
83
#
#
#
#
#
#
5
88
#
90 81
98
#
#
#
#
3.0
1.0
0.6
N
#
#
#
#
#
86
81
#
23
100
68
#
13
72
58
74
82
80
86
#
74
76
82
#
18
19
20
21
77 71
40 20*
1.0
0.5
0.25
0.1
N
#
#
#
#
#
#
6
7
83
83
83
73
#
81 71
53 12
67 75
86 74
#
#
2.5
1.0
N
38*
74
#
#
89
88
#
59
#
89 100 100
89
#
5.0
2.0
1.0
0.5
N
#
#
#
#
#
#
1.0
0.5
0.25
0.125
N
#
#
#
#
#
#
#
#
#
#
#
#
66
85
89
79
24
87
88
85
69
78
84
84
85
#
#
55*
71
71
#
80
71
#
#
#
90
75
#
80*
79
#
72
66
#
8
9
77 70
70 70
10.0
5.0
4.0
N
#
#
#
#
#
#
1.0
0.125
0.06
0.03
N
#
#
#
#
#
#
40*
60*
74
#
#
#
#
#
20*
55
59
#
#
#
77 78
79
#
56
#
1.0
0.5
0.25
0.125
N
#
#
#
#
#
#
55 63
65
#
52*
74
84
71
#
#
1.0
0.5
0.25
0.1
N
#
#
#
#
#
#
#
#
#
77
67
75
#
#
#
68
28*
57
79
#
#
11
13
77 78
58
85
#
1.0
0.5
0.25
N
#
#
#
#
#
#
23
24
69 72
#
#
20
79
65
#
2.5
0.5
0.25
N
#
#
#
#
#
#
#
#
#
#
#
#
#
#
75
81
77
24
63
81
91
69
#
58
#
57 56
50
#
47*
67
#
#
#
20.0
10.0
5.0
1.0
#
#
#
#
#
#
71
#
72
#
#
#
27
43
60
1.0
0.5
0.25
0.1
N
64
60
#
#
18 28*
65 22*
#
52
64
64
67
70
#
76
64
#
72
Results expressed as maximum aggregation %
N neat, ADP adenosine 50-diphosphate, AA arachidonic acid
26
74 68
2.0
1.0
0.8
0.5
0.1
#
#
#
#
#
#
#
# Total aggregation inhibition; * disaggregation
#
#
75
55
68
77
#
#
#
50
72
54
76
78
67
antiaggregating effects of N1,N4-bis-(1-aralkyl- or 1-aroyl-
pyridinium-3-yl-carbonyl)-piperazine dihalides, showed
low antiaggregating activity. Compound 33 inhibited the
aggregating effect of ADP, AA, adrenaline, collagen, and
ristocetin at concentration of 2.0 lM. In the same manner,
compounds N1,N4-bis-(1-aralkyl- or 1-aroyl-piperidino-3-
yl-carbonyl)-piperazine dihydrohalides (40 and 41) showed
complete antiaggregating effect against ADP, AA, adren-
aline, and collagen at a concentration of 2.0 and 1.0 lM,
Results expressed as maximum aggregation %
N neat, ADP adenosine 50-diphosphate, AA arachidonic acid
# Total aggregation inhibition, * disaggregation
respectively, while ristocetin-induced aggregation respon-
ses were inhibited at 9 and 64%, respectively, at the same
concentrations (Table 6).
123

904
Med Chem Res (2011) 20:898–911
aryl-piperazine hydrohalides (17–21, 23, 24, and 26) with
increased antiaggregating activity. N1-(1-benzyl-3-piperidino-
carbonyl)-N4-(2-chlorophenyl)-piperazine hydrohalides (17)
proved to be active at a concentration of 0.25 lM. Replace-
ment of the 2-chlorophenyl group of 17 by 2-pyrimidinyl
moiety as in 24 resulted in a decrease in the antiaggregating
effect (1.0 lM). N1-(1-(1-(4-fluorobenzyl)-3-piperidino-car-
bonyl)-N4-(2-chloro-phenyl)-piperazine (19) proved to be
more active than N1-(1-(1-(4-fluorobenzyl)-3-piperidino-
carbonyl)-N4-(2-pyrimidinyl)-piperazine (26) at concen-
trations of 0.25 and 0.8 lM, respectively. With respect to
substitutions at the 1-aralkyl or the 1-aroyl moieties, it was
found that 1-(4-fluorobenzyl)- (19) suites activity rather
than 1-(2-fluorobenzyl)- (18) (0.25 vs. 0.5 lM, respec-
tively). Replacement of the 1-benzyl group by a 1-benzoyl
group resulted in an increase in the magnitude of the
antiaggregating activity. Compound N1-(1-(1-(4-bromo-
benzyl)-3-piperidino-carbonyl)-N4-(2-chlorophenyl)-piper-
azine (20) showed a remarkable activity at 0.06 lM; while,
its counterpart N1-(1-(1-(4-bromobenzoyl)- (23) showed
activity at 0.5 lM. The antiaggregating effect of 1,4-bis-[3-
(N4-aryl-N1-piperazino-carbonyl)-pyridinium-1-yl-methyl]
-benzene dibromides showed an obvious decrease in
activity. Compounds 28 and 29 showed their potency at
concentrations of 5.0 and 50.0 lM, respectively. Reduction
of 28 and 29 afforded 1,4-bis-[3-(N4-aryl-N1-piperazino-
carbonyl)-piperidin-1-yl-methyl]-benzene dibromides (30
and 31) with a remarkable activity. Compound 30, with its
N4-(2-chlorophenyl)- and 31 with its N4-(2-pyrimidinyl)-
showed antiaggregating activity at concentrations of 0.06,
0.5 lM, respectively, which empathized the contribution
of the 2-chlorophenyl moiety to activity rather than the
2-pyrimidinyl analogues. The carbamoylpyridines, qua-
ternary ammonium salt series N1-(1-aralkyl- or 1-aroyl-
pyridinium-3-yl-carbonyl)-N4-aryl-piperazine halides (6–16),
proved to be the least active antiaggregating agents. This low
activity may be attributed to their diminished lipophylic
character. On the contrary, the carbamoyl-piperidines N1-(1-
aralkyl- or 1-aroyl-3-piperidino-carbonyl)-N4-aryl-piperazine
hydro-halides (17–27) and 1,4-bis-[3-(N4-aryl-N1-piperazi-
Table 6 The antiaggregating activity of the new synthesized com-
pounds 28–31, 33, 40, and 41
Compd Conc.(lM) ADP AA Adrenaline Collagen Ristocetin
28
29
30
N
77
68 73
97
#
89
51
65
78
82
73
82
78
71
30
35
31
61
78
82
58
92
88
#
5.0
2.0
1.0
N
13*
#
#
79 16* 21
26
84
79
#
91
72
#
40 22
76 70
50.0
10.0
1.0
N
#
#
71
70
75
#
71 28
70 73
66 66
82
73
76
#
1.0
0.5
0.125
0.06
0.03
N
#
#
#
#
#
#
#
#
#
#
#
#
27*
84
69
#
#
#
58
76
72
#
31
33
74 70
1.0
0.5
N
#
#
#
#
#
23
82
#
54
#
75 69
2.0
1.0
0.25
N
#
#
#
#
25*
28
69
#
#
73
80
89
9
71 66
80 70
79
77
#
40
41
2.0
N
#
#
63
#
62 63
59
#
72
#
2.5
1.0
#
#
#
#
#
#
64
Results expressed as maximum aggregation %
N neat, ADP adenosine 50-diphosphate, AA arachidonic acid
# Total aggregation inhibition; * disaggregation
Structure activity correlation
The obtained results revealed that N1-(3-pyridino-car-
bonyl)-N4-aryl-piperazine hydrochlorides (4 and 5) showed
moderate activities as antiaggregating agents. Alkylation of
the pyridine ring to give the corresponding quaternary
ammonium salts namely, N1-(1-aralkyl- or 1-aroyl-pyr-
idinium-3-yl-carbonyl)-N4-aryl-piperazine halides (6–9, 11,
and 13), showed a decrease in the antiaggregating activity.
Comparing the activities within this series revealed that
N1-[1-(4-bromo-benzyl)-pyridinium-3-yl-carbonyl)-N4-(2-
chlorophenyl)-piperazine (9) is more active than N1-[1-(4-
bromo-benzyl)-pyridinium-3-yl-carbonyl)-N4-(2-pyrimidinyl)
-piperazine (13), at a concentrations of 1.0 and 5.0 lM,
respectively. Reduction of those quaternary ammonium salts
afforded N1-(1-aralkyl- or 1-aroyl-3-piperidino-carbonyl)-N4-
no-carbonyl)-piperidin-1-yl-methyl]-benzene
dibromides
(30, 31) proved to be the most active members in this study.
This activity could be attributed to their lipophilic charac-
ters. The antiaggregating effect of N1,N4-bis-(1-aralkyl-
or 1-aroyl-pyridinium-3-yl-carbonyl)-piperazine dihalides
showed a decrease in activity as represented by 33
(2.0 lM). Their reduction products, N1,N4-bis-(1-benzyl-
piperidino-3-yl-carbonyl)-piperazine dihydrohalides (40)
and N1,N4-bis-[1-(2-fluoro-benzyl)-piperidino-3-yl-carbo-
nyl]-piperazine dihydrohalides (41), showed a more or less
the same potency at of 2.0 and 1.0 lM, respectively.
123

Med Chem Res (2011) 20:898–911
Molecular modeling study
905
Compounds bearing benzoyl group substitution behaved
uniquely by rotation around the carbonyl group to generate
the distinctive conformation that allowed the oxygen atom
of the benzoyl group to be hooked between two bifurcated
H-bonds with N2-His57 and Ser214. Also, allowed the
benzene ring geometrically oriented parallel to the phenyl
fused ring of Trp215 performing favorable lipophilic
interaction. The introduction of trimethoxybenzyl sub-
stituent was the reason behind the decreased antiaggre-
gating activity due to their bulkiness which shifts the
molecule out from binding to the key amino acids in the
receptor site.
Computational studies have been performed to the
designed compounds (4–46) to examine their degree of
recognition at the binding active site with the conserved
amino acids of the thrombin receptor. Studying the con-
formational recognition of the test compounds indicated
that, in general, the oxygen atom of the carbonyl group
performed bifurcated H-bonds with two crucial amino
acids; His57 and Ser195. The N1 atom of the piperazine
ring favorably oriented and exhibited bifurcated H-bonds
with Ser195 and Ser214. Replacing the N4-2-chlorophenyl
moiety with 2-pyrimidinyl group improved the recognition
where the nitrogen atom of the pyrimidine ring extended
two H-bonds with two glycine amino acids, namely:
Gly216 and Gly219.
To gain a better insight into the molecular structures of
the most active compounds 20 and 30 and the most inactive
compound 29 the binding performance was studied
(Fig. 1). Compound 20 indicated that the presence of the
piperidino-carbonyl-piperazine function is responsible for
the proper recognition of the compound with the conserved
amino acid residues Ser195 and Gly219 at the active site
that are essential for expressing high thrombin inhibition
activity. In addition, the existence of the same group in bis-
form, as in case of compounds 29 and 30 changes the
conformational form making a turn-over-like shape, to be
Compounds possessing benzyl substituent showed free
rotation around its methylene group, this conformational
twist allowed the phenyl group to lie at the proper angle in
the active site without causing any improper deviation to
the neighboring residues. The introduction of 4-nitro group
at those benzyl functions improved the binding interaction
by forming an extra H-bond with the residue Gly216.
Fig. 1 Binding mode for
compounds 20, 29, and 30
docked and minimized in the
thrombin receptor binding
pocket, showing residues
involved in its recognition
123

906
Med Chem Res (2011) 20:898–911
accommodated within the enzyme cavity. In case of 29, the
two carbonyl function groups were recognized with
Ser195, Gly216, and Gly219, the three conserved amino
acids; however, the bis-pyridine and the bis-pyrimidine
parts represent source of high hydrophobic interaction
that reduce the stability and divert the proper interaction
with the pocket amino acids (Fig. 1). In compound 30, the
significance of the oxygen of the carbonyl groups in the
recognition with Ser195 and Gly219, where the 2-chloro-
phenyl moiety improves the ligand receptor interaction at
the active site by interaction with both Gly216 and Gly219,
the two conserved residues at the binding active site. In
addition, most of the conserved amino acids at the binding
site expressed proper recognition for the bis-piperidine
groups including Trp60 and Asp189. In other words,
comparing the conformational behavior of 29 and 30
indicated that the aromatic areas of pyridine and pyrimi-
dine rings (29) severely decline the binding interaction and
magnify the clash interaction. The carbocyclic areas of
piperidine and piperazine rings (30) provide a hydrophobic
interaction without conflict with the overall electrostatic
interaction (Fig. 1). The data of molecular docking of the
tested compounds indicated that the piperidino-carbonyl-
piperazine functional group was recognized by the key
amino acid residues Ser195 and Gly219 and are essential
for expressing high thrombin inhibition activity.
Experimental
Synthesis
Melting points (°C) were determined on Mettler FP 80
melting point apparatus and are uncorrected. Microanaly-
ses were performed on a Perkin Elmer 240 elemental
analyzer at the Central Research Laboratory, College of
Pharmacy, King Saud University. All the new compounds
were analyzed for C, H, and N and agreed with the pro-
posed structures within 0.4% of the theoretical values. 1H
and 13C-NMR spectra were recorded on a Brucker XL
500 MHz FT spectrometer in DMSO-d6; chemical shifts
are expressed in d ppm in reference to TMS. Mass spectral
(MS) data were obtained on a Shimadzu GC/MS QP 5000
apparatus. Thin layer chromatography was performed on
precoated (0.25 nm) silica gel GF254 plates (E. Merck,
Germany), compounds were detected with 254 nm UV
lamp. Silica gel (60–230 mesh) was employed for routine
column chromatography separations. Compounds were
reduced using Shaker Type Hydrogenation Apparatus
Series 3911/3916. ADP, adrenaline, collagen, arachidonic
acid, and ristocetin are purchased from Bio/Data, Corp.
USA, and used for the biological evaluation. Compound 5
was previously reported (Youssef and Al-Shafie, 1998).
Molecular modeling study of the newly synthesized com-
pounds was conducted using Hyperchem: Molecular
Modeling System, Hypercube, Inc., Release 6, Florida,
USA, 1999. log P was calculated for each compound using
ChemDraw Ultra 10.0.
Conclusion
Carbamoylpyridine and carbamoylpiperidine analogues con-
taining nipecotic acid scaffold were designed, synthesized, and
evaluated for their platelet aggregation inhibitory activity.
Molecular modeling investigation was performed and the
impact of lipophilicity on activity was also discussed. Structure
activity relationship among this series was obtained. Figure 2
shows compounds N1-[1-(4-bromobenzyl)-3-piperidino-car-
bonyl]-N4-(2-chlorophenyl)-piperazine hydrobromide (20)and
1,4-bis-[3-[N4-(2-chlorophenyl)-N1-(piperazino-carbonyl)]-
piperidin-1-yl-methyl]-benzene dibromide (30), which proved
to be the most active antiplatelet aggregating compounds in
this study, both at concentration of 0.06 lM. Those new car-
bamoylpiperidine analogues could be used as template for
future development to get more active derivatives.
N1-(3-Pyridino-carbonyl)-N4-aryl-piperazine
hydrochlorides (4, 5)
Thionyl chloride (35.7 g, 0.3 mol) was added dropwise to a
cold stirred mixture of 36.9 g (0.3 mol) nicotinic acid in
pyridine (50 ml) and toluene (15 ml). The reaction mixture
was gradually heated to and maintained at 90°C for 1 h.
The appropriate 1-substituted piperazine hydrochloride
(0.3 mol) in toluene (50 ml) was added gradually into the
reaction mixture. The stirred mixture was maintained at
60°C for 3 h and at 90°C for 1 h, after which the toluene
layer containing the product was separated and washed
with 1-N HCl (4 9 250 ml). The pH of the combined
Fig. 2 Structures of the most
active antiplatelet aggregating
agents, compounds 20 and 30
O
O
N
Cl
2 HBr
N
N
N
N
Cl
N
HBr
N
N
N
O
Cl
Br
20, 0.06 µM
30, 0.06 µM
123

Med Chem Res (2011) 20:898–911
907
aqueous acidic solution was adjusted to pH 9.0 with 30%
aqueous Na2CO3, and the product was extracted with tol-
uene (4 9 250 ml). The extract was dried (MgSO4), fil-
tered, and concentrated to give the free base as an oil. A
solution of the free base 4 and 5 in 100 ml of anhydrous
ether was acidified to pH 5.0 with a saturated solution of
dry HCl gas in diethyl ether at 0°C. The formed precipitate
was recrystallized from ethanol to give 4, 5 as hydro-
chlorides (Table 1). 4: 1H-NMR; d 3.21 (s, 4H, piperazine-
H), 3.51 (s, 4H, piperazine-H), 7.06–8.00 (m, 4H, Ar),
8.65–9.77 (m, 5H, pyridine-H & exchangeable-H). 13C-
NMR; d 43.5, 48.1, 121.5, 116.7, 124.9, 126.3, 128.1,
145.1, 134.5, 142.0, 145.1, 148.4, 149.1, 165.1. MS; m/z
119.7, 124.6, 127.2, 129.5, 131.2, 134.3, 141.5, 144.2,
147.0, 148.5, 151.7, 164.71. MS; m/z 518 (0.5%). 11:
1H-NMR; d 3.16 (s, 4H, piperazine-H), 3.34 (s, 4H,
piperazine-H), 3.76 (s, 3H, OCH3), 3.81 (s, 6H, OCH3),
6.84–7.22 (m, 4H, ArH), 7.24 (s, 2H, ArH), 8.61–9.58 (m,
4H, pyridine-H). 13C-NMR; d 42.8, 45.4, 56.5, 106.8,
114.8, 115.9, 119.7, 125.9, 128.8, 131.2, 134.5, 151.8,
142.0, 147.0, 149.6, 153.2, 157.8, 165.4, 184.4. MS; m/z
532 (0.8%). 12: 1H-NMR; d 2.50–3.79 (m, 8H, piperazine-
H), 6.86–7.25 (m, 4H, ArH), 7.97–7.99 (m, 4H, ArH),
8.72–9.61 (m, 4H, pyridine-H). 13C-NMR; d 42.8, 45.4,
114.8, 115.9, 119.7, 126.8, 129.5, 131.2, 131.9, 132.3,
134.5, 131.9, 143.3, 145.9, 148.4, 151.8, 164.9, 167.1. MS;
m/z 520 (18%) M - 1. 13: 1H-NMR; d 3.13 (s, 4H,
piperazine-H), 3.98 (s, 4H, piperazine-H), 6.53 (s, 2H,
CH2Ph), 6.78–8.48 (m, 7H, pyrimidine-H and ArH),
8.92–9.57 (m, 4H, pyridine-H). 13C-NMR; d 40.8, 42.8,
61.6, 111.7, 126.9, 132.0, 134.4, 129.8, 142.1, 145.5,
158.5, 158.6, 160.6, 160.9, 165.3. MS; m/z 520 (1%)
M ? 1. 14: 1H-NMR; d 3.13 (s, 4H, piperazine-H), 4.02 (s,
4H, piperazine-H), 6.25 (s, 2H, CH2Ph), 6.80–8.47 (m, 7H,
pyrimidine-H and ArH), 8.82–9.91 (m, 4H, pyridine-H).
13C-NMR; d 41.0, 42.5, 60.0, 111.4, 127.3, 128.8, 130.0,
144.4, 144.6, 147.0, 158.3, 159.7, 164.4. MS; m/z 485
(30%). 15: 1H-NMR; d 3.12 (s, 4H, piperazine-H), 3.49 (s,
4H, piperazine-H), 3.81 (s, 9H, OCH3), 6.73–8.48 (m, 3H,
pyrimidine-H), 7.22 (s, 2H, ArH), 8.89–9.65 (m, 4H, pyr-
idine-H). 13C-NMR; d 40.7, 42.8, 56.5, 106.9, 111.7, 125.6,
128.3, 140.4, 148.3, 151.1, 158.6, 161.1, 165.9. MS; m/z
500 (5%). 16: 1H-NMR; d 3.13 (s, 4H, piperazine-H), 3.43
(s, 4H, piperazine-H), 6.77–8.03 (m, 7H, pyrimidine-H and
ArH), 8.44–9.73 (m, 4H, pyridine-H). 13C-NMR; d 40.3,
42.3, 111.6, 127.0, 127.4, 129.6, 129.9, 132.0, 143.7, 145.6,
148.0, 158.3, 160.0, 164.8, 168.8. MS; m/z 488 (9%).
1
301 (1%). 5: H-NMR; d 3.13 (s, 4H, piperazine-H), 3.97
(s, 4H, piperazine-H), 6.72–9.07 (m, 7H, ArH), 10.61 (s,
1H, exchangeable-H). 13C-NMR; d 40.6, 42.7, 111.6,
124.2, 127.3, 137.4, 150.7, 153.6, 158.5, 161.3, 166.7.
N1-(1-Aralkyl- or 1-aroyl-pyridinium-3-yl-carbonyl)-N4-
aryl-piperazine halides (6–16)
To a stirred solution of 4 or 5 (0.02 mol) in absolute eth-
anol (50 ml), 0.02 mol of the appropriate aralkyl- or aroyl
halide in acetone (50 ml) was added. The reaction mixture
was heated under reflux for 9 h, the separated solids were
filtered, dried, and recrystallized from ethanol to afford
1
6–16 (Table 1). 6: H-NMR; d 3.20 (s, 4H, piperazine-H),
3.50 (s, 4H, piperazine-H), 6.07 (s, 2H, CH2Ph), 7.08–8.33
(m, 9H, ArH), 8.98–9.81 (m, 4H, pyridine-H). 13C-NMR; d
43.6, 48.1, 63.6, 121.7, 125.2, 126.9, 128.2, 128.6, 128.7,
129.2, 129.4, 129.6, 129.9, 130.8, 134.4, 136.2, 145.6,
146.2, 148.1, 148.8, 164.9. MS; m/z 430 (0.5%) M ? 2. 7:
1H-NMR; d 3.06 (s, 4H, piperazine-H), 3.79 (s, 4H,
piperazine-H), 6.18 (s, 2H, CH2Ph), 7.04–8.05 (m, 8H,
ArH), 8.30–9.55 (m, 4H, pyridine-H). 13C-NMR; d 43.4,
48.1, 58.6, 116.4, 116.5, 124.9, 126.9, 128.2, 128.7, 129.3,
129.7, 130.9, 132.2, 132.6, 132.7, 136.2, 144.9, 146.5,
N1-(1-Aralkyl- or 1-aroyl-3-piperidino-carbonyl)-N4-aryl-
piperazine hydrohalides (17–27)
1
148.4, 148.8, 162.1. MS; m/z 447 (0.6%) M ? 1. 8: H-
NMR; d 3.21 (s, 4H, piperazine-H), 3.54 (s, 4H, pipera-
zine-H), 6.07 (s, 2H, CH2Ph), 7.06–8.32 (m, 8H, ArH),
8.71–9.64 (m, 4H, pyridine-H). 13C-NMR; d 43.6, 48.1,
62.8, 116.6, 121.7, 124.9, 125.2, 126.6, 128.2, 128.6,
128.7, 130.7, 130.8, 132.2, 132.4, 129.2, 142.8, 144.6,
N1-(1-Aralkyl- or 1-aroyl-pyridinium-3-yl-carbonyl)-N4-
aryl-piperazine halides (6–16, 0.05 mol) were dissolved in
absolute ethanol (20 ml), and hydrogenated using 0.2 g of
PtO2 at a maximum pressure of 43 p.s.i. When hydrogen
absorption was ceased, the catalyst was filtered, and the
solvent was removed under reduced pressure. The obtained
products were recrystallized from absolute ethanol to give
17–27 (Table 1). 17: 1H-NMR; d 1.72–3.58 (m, 17H,
piperazine-H and piperidine-H), 4.33–4.37 (m, 2H,
CH2Ph), 6.95–7.03 (m, 5H, ArH), 7.23–7.26 (m, 4H, ArH),
9.14 (brs, 1H, exchangeable-H). MS; m/z 398 (2%). 18: 1H-
NMR; d 1.18–3.59 (m, 17H, piperazine-H and piperidine-
H), 5.3 (s, 2H, CH2Ph), 7.20–7.31 (m, 4H, ArH), 7.43–7.45
(m, 4H, ArH), 9.57 (brs, 1H, exchangeable-H). MS; m/z
1
148.0, 148.9, 163.4. MS; m/z 447 (0.9%) M ? 1. 9: H-
NMR; d 3.21 (s, 4H, piperazine-H), 3.43 (s, 4H, pipera-
zine-H), 6.03 (s, 2H, CH2Ph), 7.21–8.31 (m, 8H, ArH),
8.98–9.57 (m, 4H, pyridine-H). 13C-NMR; d 43.8, 48.3,
63.2, 116.5, 116.8, 121.6, 125.4, 128.8, 128.9, 131.0,
132.2, 132.3, 129.4, 131.0, 146.1, 146.5, 148.4, 162.14.
1
MS; m/z 551 (0.6%). 10: H-NMR; d 3.18 (s, 4H, pipera-
zine-H), 3.43 (s, 4H, piperazine-H), 6.25 (s, 2H, CH2Ph),
6.91–8.28 (m, 8H, ArH), 8.78–9.31 (m, 4H, pyridine-H).
13C-NMR; d 42.9, 45.4, 63.3, 114.8, 115.9, 116.7, 117.5,
1
416 (6%). 19: H-NMR; d 1.71–2.51 (m, 9H, piperidine-
123

908
Med Chem Res (2011) 20:898–911
H), 2.89 (s, 4H, piperazine-H), 3.89 (s, 4H, piperazine-H),
3.65 (s, 2H, CH2Ph), 7.07–7.45 (m, 8H, ArH), 9.17 (s, 1H,
exchangeable-H). MS; m/z 416 (12%). 20: 1H-NMR; d
1.14–1.89 (m, 9H, piperidine-H), 3.35 (s, 4H, piperazine-
H), 3.64 (s, 4H, piperazine-H), 5.17 (s, 2H, CH2Ph),
6.71–7.48 (m, 8H, ArH), 9.48 (s, 1H, exchangeable-H).
13C-NMR; d 18.5, 21.0, 22.0, 23.9, 26.0, 43.4, 46.3, 58.2,
115.0, 116.9, 121.2, 130.5, 131.8, 132.5, 133.0, 135.1,
151.1, 170.8. MS; m/z 477 (5%). 21: 1H-NMR; d 1.50–1.94
(m, 9H, piperidine-H), 3.17 (s, 4H, piperazine-H), 3.40 (s,
4H, piperazine-H), 4.50 (s, 2H, CH2Ph), 7.12–8.18 (m, 8H,
ArH), 9.48 (s, 1H, exchangeable-H). MS; m/z 443 (10%).
(s, 8H, piperazine-H), 5.41 (s, 4H, CH2Ph), 6.06–7.92 (m,
12H, ArH), 8.64–9.23 (m, 8H, pyridine-H). 13C-NMR; d
42.7, 45.3, 63.1, 114.7, 115.8, 117.5, 119.6, 126.0, 128.8,
130.2, 131.2, 134.4, 141.5, 147.2, 149.8, 151.7, 165.3. 29:
1H-NMR; d 3.13 (s, 8H, piperazine-H), 3.99 (s, 8H,
piperazine-H), 5.52 (s, 4H, CH2Ph), 6.77–8.46 (m, 10H,
ArH), 9.03–9.69 (m, 8H, pyridine-H). 13C-NMR; d 40.9,
42.7, 63.3, 111.6, 127.1, 129.8, 158.5, 160.3, 129.8, 144.0,
145.3, 147.7, 148.8, 164.7.
1,4-bis-[3-(N4-aryl-N1-piperazino-carbonyl)-piperidin-1-
yl-methyl]-benzene dibromides (30, 31)
1
22: H-NMR; d 1.58–2.50 (m, 9H, piperidine-H), 2.93–
3.65 (m, 8H, piperazine-H), 3.71 (s, 3H, OCH3), 3.79 (s,
6H, OCH3), 7.07–7.45 (m, 6H, ArH), 9.17 (s, 1H,
exchangeable-H). 13C-NMR; d 21.5, 24.4, 28.1, 38.3, 41.8,
44.9, 48.2, 56.5, 60.5, 105.1, 114.5, 115.5, 119.1, 125.7,
129.3, 132.1, 134.4, 139.0, 153.3, 169.4, 171.4. MS; m/z
Compounds 1,4-bis-[3-(N4-aryl-N1-piperazino-carbonyl)-
pyridinium-1-yl-meth-yl]-benzene dibromides (28 or 29,
0.05 mol) were dissolved in ethanol (20 ml), and hydro-
genated using 0.2 g of PtO2 at a maximum pressure of 43
p.s.i. When hydrogen absorption ceased, the catalyst was
filtered, and the solvent was removed under reduced pres-
sure. The obtained products were recrystallized from eth-
anol to afford 30 or 31 (Table 2). 30: 1H-NMR; d
1.72–3.58 (m, 34H, piperazine-H, and piperidine-H), 4.30
(s, 2H, CH2Ph), 4.32 (s, 2H, CH2Ph), 6.95–7.03 (m, 4H,
ArH), 7.23–8.26 (m, 8H, ArH), 9.14 (s, 1H, exchangeable-
H), 9.25 (s, 1H, exchangeable-H). 31: 1H-NMR; d
1.74–2.29 (m, 18H, piperidine-H), 3.14 (s, 8H, piperazine-
H), 3.97 (s, 8H, piperazine-H), 4.32 (s, 4H, CH2Ph),
6.74–9.17 (m, 10H, ArH), 9.45 (s, 2H, exchangeable-H).
1
502 (6%). 23: H-NMR; d 1.72–1.95 (m, 9H, piperidine-
H), 2.88 (s, 4H, piperazine-H), 3.20 (s, 4H, piperazine-H),
7.09–7.46 (m, 8H, ArH), 10.17 (s, 1H, exchangeable-H).
13C-NMR; d 21.6, 25.5, 35.2, 42.8, 43.3, 45.4, 48.9, 114.9,
115.8, 115.9, 119.5, 119.7, 131.1, 134.4, 152.1, 170.8,
173.8. MS; m/z 490 (2%) M - 1. 24: 1H-NMR; d
1.74–2.76 (m, 9H, piperidine-H), 3.13 (m, 8H, piperazine-
H), 4.33 (s, 2H, CH2Ph), 6.72–8.44 (m, 7H, ArH), 9.43 (s,
1H, exchangeable-H). 13C-NMR; d 38.2, 39.2, 39.5, 39.8,
40.1, 40.3, 42.7, 56.4, 111.6, 125.8, 128.8, 129.5, 158.6,
160.7, 173.8. MS; m/z 444 (1%). 25: 1H-NMR; d 2.49–3.97
(m, 17H, piperazine-H and piperidine-H), 6.00 (s, 2H,
CH2Ph), 6.76–6.78 (m, 3H, pyrimidine-H), 8.44–8.46 (m,
4H, ArH), 9.52 (s, 1H, exchangeable-H). MS; m/z 410
N1,N4-bis-[3-Pyridino-carbonyl]-piperazine
dihydrochloride (32)
1
(5%). 26: H-NMR; d 1.02–3.78 (m, 17H, piperazine-H
and piperidine-H), 3.82 (s, 9H, OCH3), 6.54–6.67 (m, 3H,
pyrimidine-H), 7.42–7.45 (m, 2H, ArH), 9.68 (s, 1H,
exchangeable-H). 13C-NMR; d 40.7, 42.8, 56.5, 106.9,
111.7, 125.9, 128.3, 140.4, 148.3, 151.1, 158.6, 161.2,
Thionyl chloride (35.7 g, 0.3 mol) was added dropwise to a
cold stirred mixture of 36.9 g (0.3 mol) nicotinic acid, in
pyridine (50 ml) and toluene (15 ml). The reaction mixture
was gradually heated to and maintained at 90°C for 1 h.
piperazine (0.15 mol) in toluene (50 ml) was added grad-
ually into the reaction mixture. The stirred mixture was
maintained at 60°C for 3 h and at 90°C for 1 h, after which
the toluene layer containing the product was separated and
washed with 1-N HCl (4 3 250 ml). The pH of the com-
bined aqueous acidic solution was adjusted to pH 9.0 with
30% aqueous Na2CO3, and the product was extracted with
toluene (4 3 250 ml). The extract was dried (MgSO4),
filtered, and concentrated to give the free base as an oil. A
solution of the free base in 500 ml of anhydrous ether was
acidified to pH 5.0 with a saturated solution of dry HCl gas
in diethyl ether at 0°C, and the precipitate was recrystal-
lized from absolute ethanol to give 32 (Table 3). 1H-NMR;
d 3.87 (s, 8H, piperazine-H), 7.54–7.56 (m, 2H, pyridine-
H), 8.43–8.45 (m, 2H, pyridine-H), 8.79–8.80 (m, 2H,
1
165.9. MS; m/z 469 (7%). 27: H-NMR; d 2.49–2.81 (m,
9H, piperidine-H), 2.82–3.98 (m, 8H, piperazine-H)
6.75–8.46 (m, 7H, ArH),), 9.57 (s, 1H, exchangeable-H).
MS; m/z 458 (14%).
1,4-bis-[3-(N4-Aryl-N1-piperazino-carbonyl)-pyridinium-1-
yl-methyl]-benzene dibromides (28, 29)
To a stirred solution of N1-(3-pyridino-carbonyl)-N4-aryl-
piperazine hydro-chlorides (4 or 5, 0.02 mol) in absolute
ethanol (50 ml), a,a0-dibromo-p-xylene (2.6 g, 0.01 mol)
was added. The reaction mixture was heated under reflux
for 9 h. The separated product was filtered, dried, and
recrystallized from ethanol to give 28 or 29 as dibromides
(Table 2). 28: 1H-NMR; d 3.17 (s, 8H, piperazine-H), 3.45
123

Med Chem Res (2011) 20:898–911
909
pyridine-H), 9.07 (s, 2H, pyridine-H), 9.35 (s, 2H,
exchangeable-H). 13C-NMR; d 39.5, 40.0, 40.3, 45.7,
124.1, 124.3, 127.0, 131.9, 135.4, 137.4, 148.7, 156.7,
151.0, 153.8, 166.7, 167.6.
piperazine-H), 3.68 (s, 4H, CH2Ph), 7.16–7.23 (m, 8H,
ArH), 9.80 (brs, 2H, exchangeable-H). 42: 1H-NMR; d
1.74–2.94 (m, 18H, piperidine-H), 2.94–3.32 (m, 8H,
piperazine-H) 3.45 (s, 4H, CH2Ph), 7.18–7.64 (m, 8H,
ArH), 9.36 (brs, 1H, exchangeable-H), 9.40 (brs, 1H,
exchangeable-H). 43: 1H-NMR; d 1.14–2.87 (s, 18H,
piperidine-H), 2.91 (m, 4H, piperazine-H), 3.31 (m, 4H,
piperazine-H), 3.45 (s, 4H, CH2Ph), 7.18–7.64 (m, 8H,
ArH), 9.20 (brs, 1H, exchangeable-H), 9.42 (brs, 1H,
exchangeable-H). 44: 1H-NMR; d 1.74-2.80 (m, 18H,
piperidine-H), 2.90 (s, 4H, piperazine-H), 3.13 (s, 4H,
piperazine-H), 3.17 (s, 4H, CH2Ph), 7.15–8.22 (m, 8H,
ArH), 9.38 (s, 2H, exchangeable-H). 13C-NMR; d 21.6,
25.5, 38.5, 39.2, 40.9, 42.0, 43.4, 60.4, 124.0, 125.9, 128.8,
N1,N4-bis-(1-Aralkyl- or 1-aroyl-pyridinium-3-yl-
carbonyl)-piperazine dihalides (33–39)
To a stirred solution of N1,N4-bis-[3-pyridinocarbonyl]-
piperazine dihydro-chloride (32, 0.02 mol) in absolute eth-
anol (50 ml), 0.02 mol of the appropriate aralkyl- or aroyl
halide in acetone (50 ml) was added. The reaction mixture
was heated under reflux for 9 h, the separated solids were
filtered, dried, and recrystallized from ethanol to afford 33–
1
1
39 (Table 3). 33: H-NMR; d 3.21 (s, 4H, piperazine-H),
3.78 (s, 4H, piperazine-H), 5.51 (s, 4H, CH2Ph), 7.05–7.42
129.4, 173.9. 45: H-NMR; d 2.29–3.44 (m, 26H, pipera-
zine-H, and piperidine-H), 3.89 (s, 18H, OCH3), 7.96–7.99
(m, 4H, ArH), 9.14 (s, 1H, exchangeable-H), 9.99 (s, 1H,
exchangeable-H). 46: 1H-NMR; d 1.47–2.74 (m, 18H,
piperidine-H), 3.68 (s, 8H, piperazine-H), 7.42–7.80 (m,
8H, ArH), 9.25 (s, 1H, exchangeable-H), 9.46 (s, 1H,
exchangeable-H). 13C-NMR; d 21.5, 27.9, 29.1, 39.4, 40.5,
44.8, 48.2, 129.5, 130.5, 132.0, 140.5, 168.9, 176.9.
1
(m, 10H, ArH), 7.66–9.17 (m, 8H, pyridine-H). 34: H-
NMR; d 5.48 (s, 4H, CH2Ph), 3.13 (s, 4H, piperazine-H),
3.99 (s, 4H, piperazine-H), 6.77–8.46 (m, 8H, ArH),
9.03–9.69 (m, 8H, pyridine-H). 13C-NMR; d 40.9, 42.7,
63.3, 111.6, 127.1, 129.8, 158.5, 160.3, 129.8, 145.3, 147.7,
1
148.8, 164.7. 35: H-NMR; d 3.48 (s, 4H, piperazine-H),
3.78 (s, 4H, piperazine-H), 5.52 (s, 4H, CH2Ph), 7.05–7.42
1
(m, 8H, ArH), 7.66–9.17 (m, 8H, pyridine-H). 36: d H-
The antiaggregating effect of the new synthesized
compounds
NMR; 5.56 (s, 4H, CH2Ph), 3.72 (s, 8H, piperazine-H),
6.74–7.77 (m, 8H, ArH), 8.42–9.71 (m, 8H, pyridine-H). 37:
1H-NMR; d 3.50 (s, 8H, piperazine-H), 5.70 (s, 4H, CH2Ph),
6.20–8.33 (m, 8H, ArH), 8.81–9.67 (m, 8H, pyridine-H).
13C-NMR; d 48.0, 62.6, 124.1, 126.7, 130.8, 133.5, 135.7,
Reagents, methodology, and the turbidimetric procedure
employed in the aggregometric determinations were repor-
ted previously (Gader et al., 1988). Certain laboratory con-
ditions were carefully standardized and these include the
number of platelets in PRP (200–400 9 109/l); temperature
(37°C) and rate of stirring (1000 rpm), time interval between
venepuncture and the measurement of platelet aggregation
(\2 h). Aggregation was recorded in an Aggregation Pro-
filer (PAP4, Bio/Data, USA), which registers the slopes
(S) of the aggregation curves. The minimal concentration of
adenosine diphosphate (ADP) and arachidonic acid (AA)
eliciting full biphasic aggregation (8.16 0.19 lM for 64
plasma samples) was determined using platelet-rich plasma
(PRP) from each donor immediately prior to the determi-
nation of aggregation inhibition induced by the test com-
pounds. The platelet count of PRP was adjusted to
250,000–300,000 platelet/mm3 using autologous PPP. Test
compounds were reconstituted to appropriate concentrations
in redistilled 95% ethanol. Solubility permitting, 95% eth-
anol–water (1:1, 1:2, or 1:4) will be used in the aggregation
system. Total ethanol concentration up to 0.190% was
reported to be without effect on platelet aggregation. Control
cuvettes receive equal volumes of the appropriate vehicle
without test compound. Test compounds (50 lg) was added
to PRP after 15 s of stirring (1100 rpm) in a Payton Asso-
ciate Dual Chanel Aggregometer equipped with a Fisher
1
141.2, 144.4, 146.5, 148.7, 163.5. 38: H-NMR; d 3.42 (s,
8H, piperazine-H), 3.79 (s, 18H, OCH3), 6.75 (s, 4H, ArH),
7.21–9.44 (m, 8H, pyridine-H). 13C-NMR; d 49.8, 60.5,
107.1, 124.1, 128.3, 131.4, 135.5, 131.1, 135.5, 146.7,
1
148.2, 149.9, 153.3, 167.60. 39: H-NMR; d 3.21 (s, 4H,
piperazine-H), 3.78 (s, 4H, piperazine-H), 7.05–7.42 (m,
8H, ArH), 7.66–9.17 (m, 8H, pyridine-H).
N1,N4-bis-(1-Aralkyl- or 1-aroyl-piperidino-3-yl-
carbonyl)-piperazine dihydro-halides (40–46)
N1,N4-bis-(1-aralkyl- or 1-aroyl-pyridinium-3-yl-carbo-
nyl)-piperazine dihalides (33–39, 0.05 mol) were dissolved
in ethanol (20 ml), and hydrogenated using 0.2 g of PtO2 at
a maximum pressure of 43 p.s.i. When hydrogen absorp-
tion ceased, the catalyst was filtered, and the solvent was
removed under reduced pressure. The obtained products
were recrystallized from ethanol to afford 40–46 (Table 3).
40: 1H-NMR; d 2.30 3.45(m, 26H, piperazine-H, and
piperidine-H), 3.94–4.33 (m, 4H, 4 CH2Ph), 7.16–8.21 (m,
10H, ArH), 9.26 (brs, 1H, exchangeable-H), 9.50 (brs, 1H,
exchangeable-H). 41: 1H-NMR; d 1.14–2.49 (m, 18H,
piperidine-H), 3.17 (s, 4H, piperazine-H), 3.22 (s, 4H,
123

910
Med Chem Res (2011) 20:898–911
References
Dual Chanel OmniScribe recorder and transferred back to
the Masters constant temperature block 15 s after the addi-
tion of compound. After 1 min and 45 s, the cuvette was
returned to the aggregometer and the agonist (ADP and AA,
50 ll) was added after 4 min incubation with compound.
Changes in light transmission associated with platelet
change and aggregation were recorded for an additional
5.5 min. The (control) cuvette containing the vehicle-treated
PRP will be initiated 1 min after the (treated) cuvette con-
taining plasma treated with the appropriate test compound
and follow the same sequence of events. Inhibition of
aggregation (%) was expressed as the difference in maxi-
mum responses of the paired treated and control cuvettes as a
percent of the control response.
Antithrombotic Trialists’ Collaboration (2002) Collaborative meta-
analysis of randomized trials of antiplatelet therapy for preven-
tion of death, myocardial infarction, and stroke in high risk
patients. Br Med J 324:71–86
Banner DW, Hadvary P (1991) Crystallographic analysis at 3.0-A
resolution of the binding to human thrombin of four active site-
directed inhibitors. J Biol Chem v266:20085–20093
Berger PB (1999) The thienopyridines in coronary artery disease.
Curr Cardiol Rep 1:192–198
Binisti C, Assogba L, Touboul E, Mounier C, Huet J, Ombetta J-E,
Dong CZ, Redeuilh C, Heymans F, Godfroid J-J (2001)
Structure–activity relationships in platelet-activating factor
(PAF). 11-From PAF-antagonism to phospholipase A2 inhibi-
tion: syntheses and structure–activity relationships in 1-arylsul-
famido-2-alkylpiperazines. Eur J Med Chem 36:809–828
Budin N, Majeux N, Caflisch A (2001) Fragment-based flexible
ligand docking by evolutionary optimization. Biol Chem
382:1365–1372
Molecular modeling calculations
Chatelain P, Massingham R (1997) Cardiac ischaemia: possibilities
for future drug therapy. Eur J Med Chem 32:687–707
Cody WL, Cai C, Doherty AM, Edmunds JJ, He JX, Narasimhan LS,
Plummer JS, Rapundalo ST, Rubin JR, Van Huis CA, St-Denis
Y, Winocour PD, Siddiqui MA (1999) The design of potent and
selective inhibitors of thrombin utilizing a piperazinedione
template: part 1. Bioorg Med Chem Lett 9:2497–2502
De Candia M, Fossa P, Cellamare S, Mosti L, Carotti A, Altomare C
(2005) Insights into structure-activity relationships from lipo-
philicity profiles of pyridine-2(1H)-one analogs of the cardio-
tonic agent milrinone. Eur J Pharm Sci 26:78–86
Molecular modeling studies of the new analogues in com-
plex with 1DWD have been done to delineate features that
differentiate their mode of interaction. Starting coordinate of
the X-ray crystal structure of the thrombin 1DWD pdb
receptor in complex is obtained from the RCSB Protein Data
Bank of Brookhaven National Laboratory (Banner and
Hadvary 1991). All the hydrogen were added and thrombin
structure was subjected to a refinement protocol in which the
constraints on the 1DWD were gradually removed and
De Marco A, de Candia M, Carroti A, Cellamare S, De Candia E,
Altomare C (2004) Lipophilicity-related inhibition of blood
platelet aggregation by nipecotic acid anilides. Eur J Pharm Sci
22:153–164
˚
minimized until the RMS gradient was 0.01 kcal/mol A. The
energy minimization was carried out using the molecular
mechanics force field ‘‘AMBER.’’ The energy-minimized
structure was used for molecular dynamics studies. The new
analogues were constructed from fragment libraries in the
Hyperchem Program (1999). The partial atomic charges for
each analog were assigned with the semiempirical mechan-
ical calculation method ‘‘AM1’’ implemented in Hyperchem
6.0. Conformational search was performed around all the
rotatable bonds with an increment of 10° using conforma-
tional search module as implemented in HyperChem 6.0. All
the conformers were minimized until the RMS deviation was
Dillingham EO, Lasslo A, Carter-Burks G, Bond SE, Gollamudi R
(1989) Relationships between chemical structure and inhibition
of ADP-stimulated human thrombocyte release of serotonin and
platelet factor 4. Biochim Biophys Acta 990:128–132
´
Dogne J-M, de Leval X, Benoit P, Delarge J, Masereel B, David J-L
(2001) Recent advances in antiplatelet agents. Curr Med Chem
9:577–589
Feng Z, Gollamudi R, Dillingham EO, Bond SE, Lyman BA, Purcell
WP, Hill RJ, Korfmacher WA (1992) Molecular determinants of
the platelet aggregation inhibitory activity of carbamoylpiperi-
dines. J Med Chem 35:2952–2958
Fuster V, Badimon L, Badimon JJ, Chesebro JH (1992) The
pathogenesis of coronary artery disease and the acute coronary
syndromes. N Engl J Med 326:242–250
Gachet C (2001) ADP receptors of platelets and their inhibition.
Thromb Haemost 86:222–232
Gader AMA, Bahakim HM, Jabbar FA, Lambourne AL, Gaafar TH,
Edrees YB (1988) Dose-response aggregometry in maternal/
neonatal platelets. Thromb Haemostat 60:314–318
Heath JA, Mehrotra MM, Chi S, Yu J-C, Hutchaleelaha A,
Hollenbach SJ, Giese NA, Scarborough RM, Pandey A (2004)
Corrigendum to ‘‘Identification of 4-piperazin-1-yl-quinazoline
template based aryl and benzyl thioureas as potent, selective, and
orally bioavailable inhibitors of platelet-derived growth factor
(PDGF) receptor. Bioorg Med Chem Lett 14:6133–6142
Hyperchem (1999) Molecular Modeling System, Hypercube, Inc.,
Release 6, Florida, USA
˚
0.01 kcal/mol A. For each of the chosen analogs, energy
minimizations (EM) were performed using 1,000 steps of
steepest descent, followed by conjugate gradient minimiza-
˚
tion to a RMS energy gradient of 0.01 kcal/mol A. The
docking was carried out on using a flexible fitting module in
the program. Distance between the hydrogen bond donor and
the acceptor atoms within the pocket surrounding the ligand
and the ligand itself were defined and restrained. Each
inhibitor was geometrically optimized in the enzyme-bind-
ing pocket (Budin et al., 2001).
Acknowledgment The financial support of King Abdulaziz City for
Science and Technology, Project No. AR-25-50 is greatly appreci-
ated.
Kunapuli SP (1998) Multiple P2 receptor subtypes on platelets: a new
interpretation of their function. Trends Pharmacol Sci 19:
391–394
123

Med Chem Res (2011) 20:898–911
911
Leoncini G, Signorello MG, Roma G, Di Braccio M (1997) Effect of
2(1-piperazinyl)-4H-pyrido[1, 2-a]pyrimidin-4-one (AP155) on
human platelets in vitro. Biochem Pharmacol 53:1667–1672
Leoncini G, Signorello MG, Bruzzese D, Di Braccio M, Grossi GC,
Roma G (2004) Mechanisms involved in the antiplatelet activity
of 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H–1-benzo-
pyran-2-one (RC414). Biochem Pharmacol 67:911–918
Majerus W, Tollesfsen DM (2001) Anticoagulant, thrombolytic, and
antiplatelet drugs. In: Goodman LS, Gilman AG, Limbird LE
(eds) The Goodman Gilman’s pharmacological basis of thera-
peutics. McGraw-Hill, New York, pp 1519–1537
McKee SA, Sane DC, Deliargyris EN (2002) Aspirin resistance in
cardiovascular disease: a review of prevalence, mechanisms, and
clinical significance. Thromb Haemost 88:711–715
Mills DC (1996) ADP receptors on platelets. Thromb Haemost
76:835–856
activating factor. Part 14: synthesis and biological evaluation of
piperazine derivatives with dual anti-PAF and anti-HIV-1
activity. Bioorg Med Chem Lett 14:7999–8013
Slotboom AJ, Verheij HM, De Haas GH (1982) Specific transforma-
tions at the N-terminal region of phospholipase A2. In: Ansell
GB (ed) Phospholipids. Elsevier, Amsterdam, pp 359–434
Storey F (2001) The P2Y12 receptor as a therapeutic target in
cardiovascular disease. Platelets 12:197–209
Tucker TJ, Lumma WC, Mulichak AM, Naylor-Olsen AM, Lewis
SD, Lucas R, Freidinger RM, Kuo LC (1997) Design of highly
potent noncovalent thrombin inhibitors that utilize a novel
lipophilic binding pocket in the thrombin active Site. J Med
Chem 40:830–832
Van De Graaff E, Steinhubl SR (2000) Antiplatelet medications and
their indications in preventing and treating coronary thrombosis.
Ann Med 32:561–571
Papadopoulou C, Geronikaki A, Hadjipavlou-Litina D (2005) Syn-
thesis and biological evaluation of new thiazolyl/benzothiazolyl-
amides, derivatives of 4-phenylpiperazine. Farmaco 60:969–973
Patrono C, Coller B, Dalen JE, FitzGerald GA, Fuster V, Gent M,
Hirsh J, Roth G (2001) Platelet-active drugs: the relationships
among dose, effectiveness, and side effects. Chest 119:39S–63S
Sallem W, Serradji N, Dereuddre-Bosquet N, Dive G, Clayette P,
Heymans F (2006) Structure–activity relationships in platelet-
Weksler BB (2000) Antiplatelet agents in stroke prevention. Com-
bination therapy: present and future. Cerebrovasc Dis 10:41–48
Youssef KM, Al-Shafie FS (1998) ADP-and arachidonic acid (AA)-
induced platelet aggregation inhibitory activity of carbamoylpyri-
dines and carbamoylpiperidines. Al-Azhar Bull Sci 9:569–582
123