Synthesis and evaluation of dual antiplatelet activity of bispidine derivatives of N-substituted pyroglutamic acids

Article abstract of DOI:10.1016/j.ejmech.2016.01.019

N-aralkylpyroglutamides of substituted bispidine were prepared and evaluated for their ability to inhibit collagen induced platelet aggregation, both in vivo and in vitro. Some compounds showed high anti-platelet efficacy (in vitro) of which six inhibited

Full text of DOI:10.1016/j.ejmech.2016.01.019

European Journal of Medicinal Chemistry 110 (2016) 1e12
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and evaluation of dual antiplatelet activity of bispidine
derivatives of N-substituted pyroglutamic acids
,
,
Ankita Misra b 1, K.S. Anil Kumar a 1, Manish Jain b, Kirti Bajaj c, Shyamali Shandilya c,
Smriti Srivastava b, Pankaj Shukla b, Manoj K. Barthwal b, Madhu Dikshit b,
Dinesh K. Dikshit a
,
*
a Medicinal and Process Chemistry Division, CSIR- Central Drug Research Institute, Sec-10, Janakipuram, Lucknow 226 031, India
b Pharmacology Division, CSIR-Central Drug Research Institute, Sec-10, Janakipuram, Lucknow 226 031, India
c National Institute of Pharmaceutical Education and Research, Raebareli 229 010, India
a r t i c l e i n f o
a b s t r a c t
Article history:
N-aralkylpyroglutamides of substituted bispidine were prepared and evaluated for their ability to inhibit
collagen induced platelet aggregation, both in vivo and in vitro. Some compounds showed high anti-
platelet efficacy (in vitro) of which six inhibited both collagen as well as U46619 induced platelet ag-
gregation with concentration dependent anti-platelet efficacy through dual mechanism. In particular, the
compound 4j offered significant protection against collagen epinephrine induced pulmonary thrombo-
embolism as well as ferric chloride induced arterial thrombosis, without affecting bleeding tendency in
mice. Therefore, the present study suggests that the compound 4j displays a remarkable antithrombotic
efficacy much better than aspirin and clopidogrel.
Received 29 April 2015
Received in revised form
12 January 2016
Accepted 13 January 2016
Available online 18 January 2016
Keywords:
Pyroglutamic acid
Bispidine
© 2016 Elsevier Masson SAS. All rights reserved.
Anti-platelet
Collagen
U46619
1. Introduction
prove to be useful in terms of improving the outcome of high in-
tensity antithrombotic therapy.
Thrombotic disorders are an important cause of morbidity and
mortality not only in the developed world but also in developing
countries [1]. Platelets play a crucial role in haemostasis and
thrombosis [2e6]. Selective inhibition of the pathways most rele-
vant to the pathological aspects of atherothrombosis can be a
logical approach for therapeutic interventions [2e6]. The prevalent
anti-platelet therapy regimens that include aspirin and clopidogrel
are fraught with increased bleeding risk implying thus the need for
drugs with improved safety margins. A great deal of insight has
been gained into the contribution of collagen, thromboxane A2
(TxA2) and their respective receptors & signaling mechanisms in
promoting platelet adhesion, activation and subsequent thrombus
growth and stability. Hence, targeting against the synergy between
collagen and TxA2 mediated platelet activation pathway could
Building upon the previous findings of nipecotamide derivatives
as inhibitors of platelet aggregation induced by ADP [7], collagen
[8], thrombin [9], epinephrine [10] and stable TxA2 mimetics [11],
we have recently reported tert-butyl ((S)-1-((S)-1-(4-methylbe-
nzyl)-5-oxopyrrolidine-2-carbonyl) piperidin-3-yl)methylcarbam-
ate 1 as a potent inhibitor of collagen induced platelet aggregation
with very potent protection against in vivo pulmonary thrombo-
embolism [12]. Moreover, the N-substituted oxazolidine and thia-
zolidine carboxamides exhibited inhibition of platelet aggregation
by dual mechanism, collagen and thromboxane, but they signifi-
cantly enhanced bleeding tendency by 50e70% [12]. Watson et al.
have recently reported N-substituted bispidine (2) as a potent se-
lective factor Xa inhibitor with a good anticoagulant activity [14].
Further, substituted bispidines [13] (7-diazabicyclo[3.3.1]nonane)
(3) were reported to be useful in the treatment of cardiac ar-
rhythmias [15]. In the present communication, we describe our
work to introduce conformational rigidity on to the 3-
aminomethylpiperidine substructure of 1 by its replacement with
substituted bispidine, leading to synthesis of N-pyroglutamoylbis-
pidines (4) wherein apart from collagen, we observe an additional
* Corresponding author.
E-mail addresses: dk_dikshit@cdri.res.in, coordinating.cell.dfs@gmail.com
(D.K. Dikshit).
1
Equally contributed.
http://dx.doi.org/10.1016/j.ejmech.2016.01.019
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

2
A. Misra et al. / European Journal of Medicinal Chemistry 110 (2016) 1e12
inhibition of thromboxane A2 induced platelet aggregation and
thrombus formation, but without any evident impact on normal
hemostasis or bleeding Fig. 1.
the same model (Table 1).
3.2. Platelet aggregation assay
2. Chemistry
Compounds were assessed for their inhibitory potential
against platelet aggregation in order to identify their probable
mechanism of antithrombotic action. Seven compounds were
found to inhibit collagen induced platelet aggregation in vitro. In
particular compounds 4j (86 3%), 4l (68 6%), 4m (52 8%), 4o
As reported previously N-alkylation of methyl (2S)-pyrogluta-
mate (5) with various arylalkylhalides using LiHMDS [16] and
subsequent hydrolysis of the ester group gave the corresponding N-
substituted pyroglutamic acids 6(a-j) [12].
(57
exhibited significant anti-platelet efficacy at a concentration of
30 M (Table 1).
11%), 4u (67
10%), 4v (61
8%) and 4w (67
8%)
3-(t-butyloxycarbonyl)-7-benzyl-3,7-diazabicyclo [3.3.1] non-
ane (8) was prepared using reported procedure [17]. The Boc group
on 8 was removed using TFA in DCM to give 9, while 10 was pre-
pared by the N-debenzylation of 8 by catalytic hydrogenation over
20% Pd(OH)2 e charcoal and the resulting amines were condensed
with various N-substituted pyroglutamic acids 6(a-j) using one-pot
DCC-HOBt methodology to yield the corresponding pyrogluta-
moylbispidines 4(a-p). Boc protection in pyroglutamoylbispidines
4b and 4c was removed and the resulting amine was converted to
corresponding (N)-acyl, sulfonyl and substituted benzyl derivatives
4(q-w) as shown in Scheme 1.
m
3.3. Dual activity of compounds
Further platelet aggregation studies showed that 6 of the com-
pounds (4j, 4l, 4m, 4u, 4v and 4w) inhibited both collagen as well as
U46619 (thromboxane receptor agonist) induced platelet aggre-
gation, thereby exhibiting dual mechanism of action (Table 1). The
compound 4j was observed to be the most potent among the entire
series and exhibited an IC50 of 1.7
against U46619 induced platelet aggregation (Fig. 2ab). The com-
pound 4j, even up to 300 M, did not exhibit any significant inhi-
mM against collagen and 7.7 mM
3. Biological results
m
bition against other platelet agonists like ADP, thrombin mimetic
SFLLRN (TRAP), GPVI specific collagen related peptide (CRP-XL) and
GP 1b-IX-V agonist Ristocetin induced platelet aggregation (Fig. 2c).
It did not exhibit any inhibition of COX pathway via arachidonic
acid induced platelet aggregation, but at higher concentration
All the synthesized compounds were primarily evaluated in a
murine model of collagen-epinephrine induced pulmonary
thromboembolism (in vivo) as well as collagen induced platelet
aggregation (in vitro) to assess their antithrombotic efficacy. The
compound 4j was found to be most efficacious in inhibiting
collagen and U46619 induced platelet aggregation, and was further
studied in depth.
(300 mM and 500 mM) the compound 4j attenuated platelet ag-
gregation up to 50%. These findings indicate that the compound 4j
might exhibit its anti-platelet efficacy through dual mechanism,
and hence warrants further confirmation regarding its mechanism
of action.
3.1. Collagen epinephrine induced pulmonary thromboembolism
After 1 h of oral administration in mice, it was observed that 13
out of 23 compounds exhibited ~40e60% protection against
collagen epinephrine induced thrombotic challenge at a dose of
3.4. Effect of 4j on pulmonary thromboembolism and bleeding time
in mice
30 mmol/kg (Table 1). Compounds with N-protected tert-butylox-
Interestingly the antithrombotic efficacy of 4j was found to in-
ycarbonyl (4b), benzyl (4j), benzoyl (4q) and tosyl (4s) substitution
at the bicyclic end and with a bromine atom at the ortho position of
phenyl ring were found to be very potent molecules, in vivo, with
50%, 45%, 40%, 60% and 55% protection respectively, while com-
pound 4d and 4v exhibited 55% and 50% protection respectively in
crease from 40% after 1 h to 60% after 4 h of oral administration
(Fig. 3a). In comparison, aspirin (170 mmol/kg) showed only 40%
protection which was effective up to 5 h. Clopidogrel, after 1 h of
dosing, exhibited 70% protection which declined to 50% after 4 h
and became almost similar to that of 4j. Moreover, both aspirin and
clopidogrel exhibited significant increase in bleeding time in mice
(Fig. 3b). However the bleeding time was not significantly altered
from 1 h (3.4 0.2 min) to 4 h (4.3 0.1 min) in 4j treated mice, and
remained comparable to that of control mice (3.5 0.3 min) but
significantly less than aspirin (6.6
0.2 min) and clopidogrel
(9.0 0.5 min) (Fig. 3b).
3.5. Effect of 4j on U46619 induced vasoconstriction
Results from the present study indicated that the maximal
endothelial contraction evoked by U46619 was significantly
reduced in the presence of 4j at 30
m
M concentration (14.2 3.6%)
3.8%) as compared to vehicle treated control
8.5%) (Fig. 4a). Likewise pretreatment of TP receptor
2.9%) significantly inhibited U46619
induced contraction. At lower concentrations (10 M), U46619
shifted the concentration response curve towards right indicating
competitive inhibition against U46619. At 5 M and 3 M con-
and 15
(89.8
mM (21.8
antagonist SQ29548 (8.4
m
m
m
centration, 4j did not show any significant effect. Moreover at the
effective concentrations tested, 4j did not exhibit any significant
effect on KCl response (Fig. 4b).
Fig. 1. Aminomethylpiperidine (1) and bispidine derivatives reported as anti-
coagulants (2) and for the treatment of cardiac arrhythmias (3); Structure of pro-
posed prototype (4).

A. Misra et al. / European Journal of Medicinal Chemistry 110 (2016) 1e12
3
Scheme 1. Reagents and conditions: (a) TFA, dry DCM, 4h; (b) Pd(OH)2eC, H2, methanol, 120 psi, 10h; (c) HOBt, DCC, dry DCM, 00C-RT, 3h; (d) TFA, dry DCM, 5h; (e) R1-Cl, TEA, dry
DCM, 2h; (f) R1-Br, an. K2CO3, dry acetone, reflux, 4 h.
3.6. Effect of 4j on coagulation parameters
vehicle control, it significantly prolonged the thrombin time
(>120sec vs 20.03 0.5 s), prothrombin time (18.7 0.6 s vs
14.3 0.03sec) and activated partial thromboplastin time
To assess the effect of 4j on coagulability of blood, thrombin
time (TT), prothrombin time (PT) and activated partial thrombo-
plastin ime (aPTT) were assessed in the human plasma (in vitro). No
change was observed in TT, PT and aPTT when compared to their
isovolumetric vehicle control (Fig. 4c). Hirudin was used as the
standard anticoagulant for reference and when compared to
(84.3 0.4 s vs 29.6 2.2 s) (Fig. 4c).
3.7. Platelet adhesion over collagen
4j (0e300 mM) exhibited a concentration-dependent inhibition
Table 1
Effect of bispidine derivatives of N-substituted pyroglutamic acid, 4 (a-w), aspirin and clopidogrel on collagen-epinephrine induced pulmonary thromboembolism (mice,
in vivo) and platelet aggregation induced by collagen and U46619 (human, in vitro). Results are expressed as % Protection (in vivo) and % Inhibition (in vitro).
No.
Compound
R/R1
R0
Protection (%)a
Inhibition (%)b,d
Inhibition (%)c,d
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4 m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzoyl
Benzoyl
Tosyl
Benzyl
40
50
40
55
30
30
45
30
30
40
25
40
30
30
40
30
60
40
55
40
30
50
35
06.00 14.00
02.00 12.00
25.00 9.00
11.00 3.00
06.00 11.00
03.00 3.00
10.00 3.00
11.00 4.00
44.00 13.00
86.00 3.41
07.00 7.00
68.00 6.00
52.00 8.00
29.00 1.00
57.00 11.00
10.00 4.00
25.60 3.55
20.50 7.50
16.80 5.66
37.50 10.50
67.00 10.00
61.00 8.00
67.00 8.00
2-Bromobenzyl
4-Methylbenzyl
4-Cyanobenzyl
4-Chlorobenzyl
2,6-dichlorobenzyl
4-Methoxybenzyl
1-Naphthyl
9
Benzyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
2-Bromobenzyl
4-Methylbenzyl
2,6-Dichlorobenzyl
4-Chlorobenzyl
SO2Ph
4-Bromobenzyl
4-Methoxybenzyl
2-Bromobenzyl
4-Methylbenzyl
2-Bromobenzyl
4-Methylbenzyl
4-Methylbenzyl
4-Methylbenzyl
4-Methylbenzyl
79 09
85 03
80 03
Tosyl
2-Bromobenzyl
4-Bromobenzyl
2-Chlorobenzyl
70 08
52 03
81 05
Aspirin
Clopidogrel
DMSO
40 (170
70 (70
m
mol/kg)
m
mol/kg)
25.31 2.59
a
Collagen-epinephrine induced pulmonary thromboembolism in mice (in vivo).
Inhibition of collagen induced platelet aggregation in human platelets (in vitro).
Inhibition of U46619 induced platelet aggregation in human platelets (in vitro).
b
c
d
Compound dose/concentration used: in vivo ¼ 30
m
mol/kg; in vitro: 30
mM; n ¼ 3.

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A. Misra et al. / European Journal of Medicinal Chemistry 110 (2016) 1e12
Fig. 2. Platelet aggregation assay (human, in vitro). Effect of compound 4j against (a) collagen; (b) U46619; (c) ADP, TRAP, Ristocetin, CRP-XL and arachidonic acid induced platelet
aggregation in human platelet rich plasma. Results are expressed as Mean SEM (n ¼ 3) ***P < 0.001, **P < 0.01, *P < 0.05 vs control.
Fig. 3. Time dependent effect of 4j and standard antithrombotic drugs (aspirin and clopidogrel) after single dose administration on (a) collagen-epinephrine induced pulmonary
thromboembolism; (b) bleeding time in mice. Results are expressed as Mean SEM (n ¼ 5, 10 animals/group/experiment).
of platelet adhesion over collagen (Fig. 4d). Threshold inhibitory
effects were evident at 30 M (27 8%), while maximal and almost
complete abrogation of platelet adhesion occurred at 300
~55e60 kDa. These findings suggest that 4j probably obstructs both
collagen and U46619 induced platelet activation thereby disrupting
tyrosine kinase regulated signaling pathway.
m
mM
(Fig. 4d). The compound 4j significantly inhibited both integrin
dependent as well as integrin independent platelet adhesion over
collagen in a concentration dependent manner.
3.9. Effect of 4j on ferric chloride induced arterial thrombosis
The efficacy of 4j to combat pathologic and occlusive thrombus
in vivo was further assessed in another robust model of ferric
chloride induced arterial thrombosis in mice (Fig. 6). The injured
carotid artery upon exposure to 10% FeCl3 was occluded within
3.8. Effect of compound 4j on collagen induced tyrosine
phosphorylation
The activation of platelets by collagen or thromboxane receptor
(TP) agonist U46619 contributes to the assembly and stabilization
of various signaling complexes. Platelets pre-incubated with com-
pound 4j when stimulated with either collagen (Fig. 5a) or U46619
(Fig. 5b) showed reduced phosphorylation level of a number of
proteins compared to the vehicle control. Further the effect of
compound on phosphorylation of different proteins at tyrosine
residues was found to be specific, since it exhibited a concentration
dependent effect at various concentrations of compound ranging
9.5
significantly prolonged the time to occlusion (TTO) upto
13.3 0.5 min and 20 0.8 min at doses of 20 and 30 mol/kg
respectively (P < 0.01 vs control) (Fig. 6). The standard anti-platelet
drug aspirin remained ineffective in this model even at 170 mol/
kg, while clopidogrel increased the TTO up to 23 0.9 min at
70 mol/kg (Fig. 6). The efficacy elicited in this model further
0.4 min. The compound 4j, 4 h after oral administration,
m
m
m
substantiates the anti-thrombotic potential of this compound as
the 4j treated groups exhibited significant prolongation of artery
from 3 to 300
m
M (Fig. 5a,b). The inhibitory effect was particularly
occlusion time by 1.4 and 2.1 fold at 20 and 30
respectively.
mmol/kg,
evident on proteins of molecular weight around 120, 100, 70 and

A. Misra et al. / European Journal of Medicinal Chemistry 110 (2016) 1e12
5
Fig. 4. Effect of 4j or TP receptor antagonist SQ29548 (100 nm) on (a) U46619 induced vasoconstriction (b) Acetylcholine induced vasorelaxation in rat aortic rings in vitro. Data
shown as Mean þ SEM (n ¼ 5) *P < 0.05 [control vs 4j (15 M, 30 M and SQ 100 nM)]; #P < 0.001 [control vs 4j (10 M)]; $ P < 0.01 [(control vs 4j (5 M)]. (c) Effect of compound 4j
or Hirudin on (c) Thrombin time (TT), Prothrombin Time (PT) and activated partial thromboplastin time (aPTT) in human platelet poor plasma. Hirudin (1 g/ml) was used as
m
m
m
m
m
standard anticoagulant and 0.4% DMSO was used as vehicle. (d) Effect of compound 4j on human platelet adhesion on collagen coated surface (in vitro) in presence or absence of
Mgþ2 ions. Data shown as Mean þ SEM. ***p < 0.001, **p < 0.01, *p < 0.05 vs control. vs control.
Fig. 5. Effect of compound 4j at different concentrations on tyrosine phosphorylation of platelet proteins following stimulation with (a) collagen, (b) U46619. Washed platelets pre-
incubated at 37 ꢀC with vehicle (0.4% DMSO) or various concentrations of compound 4j (3, 10, 30, 100
mM) for 5 min, and stimulated with either U46619 (0.25 mg/ml) or collagen
(1 mg/ml). Blots are representative of three separate experiments.
4. Discussion
inhibition was observed at 30 mM with no apparent SAR. The in-
vivo assay in pulmonary thromboembolism assay 12 compounds
displayed 40e60% protection (asprin, 40% protection) with again
no clear correlation hydro which Among active derivatives, 4j
exhibited significant inhibition against collagen induced platelet
Twenty three pyroglutamide derivatives were synthesized and
assessed for their anti-thrombotic activity against collagen-
epinephrine induced pulmonary thromboembolism in mice as
well as collagen induced platelet aggregation in vitro. However, in
the collagen induced platelet aggregation assay only a moderate
aggregation (86.00
3.41%), and ~1.5 fold more antithrombotic
protection than aspirin in vivo. Moreover, when compared to both

6
A. Misra et al. / European Journal of Medicinal Chemistry 110 (2016) 1e12
inhibited both collagen and U46619-induced protein tyrosine
phosphorylation, thereby indicating its participation in switching
off critical signaling mechanisms during platelet activation.
Further investigation pertaining to the evaluation of antith-
rombotic as well as anti-platelet efficacy of 4j was conducted in an
experimental murine model of FeCl3-induced arterial thrombosis. It
is noteworthy that thrombus formation is inhibited by the anti-
platelet drug clopidogrel while aspirin was observed to be devoid
of any significant antithrombotic activity in this model. A similar
lack of activity of aspirin has earlier been described in a rat model
[24]. Thus 4j significantly attenuated arterial thrombosis, with mild
impact on bleeding time.
The present study therefore demonstrates that 4j, a pyrogluta-
mide derivative, possesses the ability to inhibit both thromboxane
A2 and collagen induced platelet activation and signaling. It does
not affect coagulation cascade, and also exhibits in vivo protection
against thrombus formation with little effect on hemostasis, sug-
gesting that it might be a potential candidate for development as an
antithrombotic agent.
Fig. 6. Effect of compound 4j, aspirin and clopidogrel on total time to occlusion (TTO)
in ferric chloride induced arterial thrombosis in mice (n
Mean þ SEM. ***p < 0.001, **p < 0.01, *p < 0.05 vs control.
¼
6). Data shown as
aspirin and clopidogrel, no significant effect of 4j on bleeding time
in mice implicated that 4j might offer a new concept to develop
novel anti-thrombotic, which may not only inhibit arterial throm-
bosis but may also escape the adverse effects of bleeding, a
potentially disturbing issue that limits the clinical application of
presently available antithrombotic drugs.
5. Conclusion
During arterial thrombosis, the cross-talk between endothelial
dysfunction, platelet aggregation, and activation of the coagulation
system plays a major role. Platelet adhesion on subendothelial
collagen followed by aggregation at sites of vascular injury is
recognized as critical aspect for hemostatic plug formation and
thrombosis. Therefore, the effect of compound 4j was assessed on
all the three parameters to delineate its mechanism of action. The
pyroglutamide derivative 4j inhibited platelet aggregation induced
by collagen and U46619, a thromboxane mimetic but exhibited no
effect against ADP-, TRAP, arachidonic acid, CRP-XL- or Ristocetin-
induced platelet aggregation. In fact the compound 4j preferen-
tially inhibited collagen-induced platelet aggregation as compared
Bispidine derivatives of N-arylalkyl pyroglutamides reported
herein showed promising inhibitory effects on platelet activation,
both in vivo and in vitro. However, from the data it is apparent that
the platelet aggregation inhibition data for the compounds is not
amenable to any SAR. This might be due to poor solubility of the
compounds in the test medium. In an in-vivo collagen-epinephrine
induced pulmonary thromboembolism assay, compounds 4a-4w
(Table 1) showed 25 to 60% protection. Among the tested com-
pounds, six compounds exhibited significant anti-platelet effect, via
dual action against both collagen as well as U46619 induced
platelet activation, in vitro. The compound 4j was found to exhibit
significant antithrombotic efficacy and also escapes the unfavorable
events of bleeding risk in mice model when compared to existing
anti-platelet drugs aspirin and clopidogrel. The action of compound
4j is platelet specific, since its presence did not alter the coagula-
bility of blood as assessed by TT, PT and aPTT in human plasma.
Moreover, the efficacy elicited in FeCl3 induced thrombosis further
substantiates the anti-thrombotic potential of this compound.
These results indicate that substitution of N6 of 3,6-diaza-bicyclo
[3.1.1]heptane-3-carboxyl moiety with groups (R/R1) which can
lower the basicity/nucleophilicity are desirable for improved pro-
tection. All compounds, except 4q, followed this trend. Also, an R0
to that induced by U46619 (IC50, 1.7 vs. 7.7 mM, respectively). Since
aspirin is already a clinically proven anti-platelet drug, well-
recognized for its inhibitory effect on COX-1 and the subsequent
production of thromboxane A2, hence these compounds having a
relatively potent efficacy to inhibit both collagen as well as
thromboxane A2 mediated platelet activation could serve as very
useful antithrombotic agent with a distinct mechanism of action.
At the site of vascular injury, platelets adhere over sub-
endothelial collagen and trigger thrombus formation. Thus block-
ing this initial step to prevent arterial thrombosis may be of critical
therapeutic significance [23]. The present study shows that 4j
attenuated platelet adhesion over collagen surface in both integrin
dependent and independent manner as distinguished by the
presence of Mgþ2 ions. However, the anti-platelet effect of 4j
against U46619 induced platelet aggregation further led us to
speculate that the protection conferred by 4j might be due to the
integrated blockage of collagen and thromboxane A2 signaling
machinery. 4j exhibited selective vasorelaxation and restoration of
U46619 induced endothelial contraction. This further substantiates
that 4j might be producing its effect by inhibiting platelet collagen
receptor and TP receptor. The antithrombotic effect of 4j is platelet
specific, since its presence did not alter the coagulability of blood as
assessed by thrombin time, prothrombin time and activated partial
thromboplastin time in human plasma.
substitution which can enhance electronegative character of the
lactam's nitrogen is advantageous for the activity.
g-
6. Experimental section
6.1. Chemistry
Reagents and other chemicals were used as purchased without
further purification. All reactions with moisture/air sensitive re-
actants and solvents were carried out under nitrogen atmosphere
using flame dried apparatus and all the solvents were distilled prior
to use. THF was distilled under N2 over potassium benzopheno-
neketyl radical prior to use. Dichloromethane was dried over P2O5
and methanol using magnesium cake. All the compounds were
vacuum dried in Abderhalden. Reactions were monitored by thin
layer chromatography over pre-coated silica gel plates (60F254, E.
Merck, Germany), using UV, iodine vapour, acidic and basic KMnO4
or Dragendorff's reagent spray as the developing agents. Chro-
matographic separations were performed on flash column using
silica gel (60e120 and 230e400 mesh). All melting points were
taken in open tubes and are uncorrected. IR spectra were recorded
Collagen activates platelets through a tyrosine kinase based
signaling pathway and sequential activation of PLCg2, leading to
intracellular Ca2þ mobilization, as well as the release of throm-
boxane A2, which through autocrine and paracrine manner further
amplifies the platelet activation response [23]. Hence, a well-
established consequence of collagen and thromboxane induced
platelet activation is assembly of numerous downstream kinases,
including tyrosine and MAP kinases. The compound 4j substantially

A. Misra et al. / European Journal of Medicinal Chemistry 110 (2016) 1e12
7
0
on a Perkin-Elmer 881 and FTIR 8201 PC Shimadzu spectropho-
tometers and values are expressed in cmꢁ1. 1H NMR and 13C NMR
spectra were recorded on Bruker WM-200, WM-300 or WM-400
J ¼ 12 Hz, 1H, NC2 HA); 4.09e4.06 (d, J ¼ 15 Hz, 1H, PhCHB; m, 2H,
0
NC2H); 3.81e3.76 (d, J ¼ 15 Hz, 1H, NC8 HA); 3.51e3.47 (d, J ¼ 12 Hz,
0
0
0
0
0
1H, NC2 HB); 3.08e2.86 (m, 4H, PhCHB , NC8 HB, NC4 HA, C6 H2);
0
Spectrometer. The chemical shifts are expressed in
d
using TMS as
2.51e2.40 (m, 2H, C4HA, C4 HB); 2.26e2.16 (m, 1H, C4HB); 2.16e1.89
0
0
0
internal standard. Mass spectra were recorded on a JEOL JMS-D-
3000 spectrometer with an ionization potential of ~70 eV
and FAB on SX 102 instrument.
(m, 4H, C3H2, C3 H, Ca7 H); 1.70 (s, 2H, C9 H); 1.41 (s, 9H, CMe3); 13C
NMR (50 MHz, CDCl3, ppm): d 176.16, 174.85, 135.40, 128.84, 128.53,
127.95, 49.48, 49.00, 45.36, 29.99, 34.49, 28.35, 27.70, 27.32, 22.88;
MS (ESI): m/z ¼ 427.9 (Mþ).
6.1.1. General synthesis of (2S)eN-arylalkyl-pyroglutamic acids
[6(a-j)]
Compound 4(b-p) were obtained following the procedure
described for 4a.
The compounds were prepared via following the same proce-
dure using various substituted benzyl bromides [Ref. [12]].
6.1.5. 7-[1-(2-Bromo-benzyl)-5-oxo-pyrrolidine-2-carbonyl]-3,7-
diaza-bicyclo[3.3.1]nonane-3-caꢀrboxylic acid tert-butyl ester (4b)
Yield: 89%; MP: 138 ꢀC; ½aꢃD27 C: 3.7608 (Methanol, c ¼ 0.2180); IR
(KBr): 3458.8, 2927.9, 1679.6, 1434.4, 1241.5,1172.5, 1134.3 cmꢁ1; 1H
6.1.2. 3-Benzyl-3,7-Diaza-bicyclo [3,3,1]nonane (9)
TFA (2.25 ml, 5eq, 30 mmol) was added to the stirring suspen-
sion of 8 (2.0 g, 1eq, 60 mmol) in DCM at 0 ꢀC and allowed to stir at
room temperature. Then reaction mixture was made alkaline by
adding 20% aq. solution of Na2CO3 and resulting mixture was
extracted with DCM (3 ꢂ 50 ml) and organics were washed with
brine. The combined organics were dried with an. Na2SO4 and
concentrated to obtain yellow oily liquid (1.641 g). Yield: 90%;
NMR (300 MHz, CDCl3, ppm):
d
7.5e7.1 (m, 4H, Ph-H); 5.1e5.0 (d,
0
J ¼ 15 Hz, 1H, PhCHA); 4.5 (d, 1H, NC2 HA); 4.1e4.0 (d, J ¼ 15 Hz, 1H,
0
PhCHB; m, 1H, NC2H); 3.5 (d, J ¼ 15 Hz, 1H, NC8 HB); 3.1e2.8 (m, 4H,
0
0
0
0
0
NC2 HB, NC8 HB, NC4 H, NC6 HA); 2.4e2.3 (m, 3H, C4HA, NC4 H,
0
NC6 HB); 2.2e2.0 (m, 2H, C4HB, C3HA); 1.9 (m, 1H, C3HB); 1.8 (m, 2H,
C3 H, C7 H); 1.7 (s, 2H, C9 H); 1.4 (s, 9H, CMe3); 13C NMR (50 MHz,
0
0
0
IR(Neat): 3451.4, 2924.6, 1610.0, 1450.4 cmꢁ1
;
1H NMR (300 MHz,
CDCl3, ppm): d 135.84, 132.75, 131.39, 129.38, 127.76, 124.13, 79.73,
CDCl3, ppm): 7.37e7.27 (m, 5H, Ph-H); 3.49 (s, 2H, CH2Ph),
d
56.85, 49.59, 46.51, 45.30, 34.64, 28.32, 28.13, 27.70, 27.29, 22.82;
3.36e3.41 (d, 2H, J ¼ 12 Hz, 2 ꢂ NCH); 3.31e3.27 (d, J ¼ 12 Hz, 2H,
2 ꢂ NCH); 3.19e3.11 (d, J ¼ 12 Hz, 2H, 2 ꢂ NCH); 2.49e2.45 (d,
J ¼ 12 Hz, 2H, 2 ꢂ NCH); 2.12e2.07 (m, 2H, 2 ꢂ CH); 1.93e1.89 (d,
J ¼ 12 Hz, 1H, bridge CH); 1.79e1.75 (d, J ¼ 12 Hz, 1H, bridge CH);
MS (ESI): m/z ¼ 217 (Mþ1)þ
MS(ESI): m/z: 528.0 (MþNa)þ.
6.1.6. 7-[1-(4-Methylbenzyl)-5-oxo-pyrrolidine-2-carbonyl]-3,7-
diaza-bicyclo[3.3.1]nonane-3-carboxylic acid tert butyl ester (4c)
ꢀ
Yield: 80%; ½aꢃ2D7 C: ꢁ16.7970 (Methanol, c ¼ 0.0980); IR (Neat):
3412.3, 2925.2, 1667.9, 1423.3, 1364.3, 1245.4, 1172.5, 1135.0 cmꢁ1
;
6.1.3. 3,7-Diaza-bicyclo[3.3.1]nonane-3-carboxylic acid tert-butyl
ester (10)
1H NMR (300 MHz, CDCl3, ppm):
d
7.14e7.11 (d, J ¼ 7.8 Hz, 2H, Ph-
H), 7.07e7.04 (d, J ¼ 7.8 Hz, 2H, Ph-H); 5.13e5.08 (d, J ¼ 15 Hz,
0
Palladium hydroxide (0.5 g) was added portion wise to a sus-
pension of 8 (1.102 g, 36.7 mmol) in methanol (25 ml) in steel parr
under nitrogen atmosphere. The reaction mixture was hydroge-
nated at 55 ꢀC and 150 psi for about 10 h. Then it was allowed to
cool and filtered over sintered funnel with the aid of vacuum and
concentrated to get pale yellow solid. Yield ¼ 93.33%; MP: 75 ꢀC; IR
(KBr): 2979.2, 2919.7, 2858.5, 1679.6, 1402.4, 1240.4,
PhCHA); 4.61e4.56 (m, 1H, NC2 HA); 4.15e4.07 (m, 1H, NC2H);
3.76e3.71 (d, J ¼ 15 Hz, 1H, PhCHB); 3.54e3.50 (d, J ¼ 12 Hz, 1H,
0
0
0
0
0
NC8 HA); 3.04e2.94 (m, 4H, NC2 HB, NC8 HB, NC4 HA, NC6 HA); 2.34
0
0
(s, 1H, CH3); 2.22e2.00 (m, 6H, C4HA, C6 HB, C4 HB, C4HB, C3H2);
0
0
0
1.91e1.90 (m, 2H, C3 H, C7 H); 1.80 (s, 2H, C9 H); 1.42 (s, 9H, CMe3);
13C NMR (50 MHz, CDCl3, ppm):
d
175.68, 168.33, 137.29, 129.28,
128.58, 49.52, 45.03, 34.64, 30.04, 28.35, 27.74, 27.35, 22.81, 21.08;
1172.31131.9 cmꢁ1; 1H NMR (300 MHz, CDCl3, ppm):
d
4.13e4.09 (d,
MS (ESI): m/z ¼ 441.9 (Mþ).
J ¼ 12 Hz, 2H, 2 ꢂ CONCH); 3.14e3.10 (m, 3H, 2 ꢂ CONCH, NCH);
3.01e2.96 (d, J ¼ 15 Hz,1H, NCH); 2.25 (s, 2H, 2 ꢂ NH); 1.92e1.88 (d,
J ¼ 12 Hz, 1H, CH); 1.80e1.76 (d, J ¼ 12 Hz, 1H, CH); 1.67 (s, 2H,
bridge CH2); 1.48 (s, 9H, CMe3); 13C NMR (50 MHz, CDCl3, ppm):
6.1.7. tert-Butyl-7-((S)-1-(4-cyanobenzyl)-5-oxopyrrolidine-2-
carbonyl)-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate (4d)
Yield ¼ 53%; MP: 160e165 ꢀC; 1H NMR (300 MHz, CDCl3, ppm)
d
155.49, 79.78, 51.50, 48.94, 31.39, 28.52, 28.15; MS (ESI):
d
7.64e7.61 (d, J ¼ 7.8 Hz, 2H, Ph-H), 7.28 (d, J ¼ 7.8 Hz, 2H, Ph-H)
227.1481(MþH)þ.
5.17e5.12 (d, J ¼ 15 Hz, 1H, PhCHA), 4.59e4.55 (d, 1H, C(O)
0
NC2 HA), 4.12e3.92 (d,1H, PhCHB), 3.87 (d, J ¼ 15 Hz,1H, NC2H), 3.12
0 0
(m, 1H, PhCHA), 3.10e3.01 (m, 2H, PhCHB , C(O)NC4 HA), 2.60e2.51
6.1.4. 7-(1-Benzyl-5-oxo-pyrrolidine-2-carbonyl)-3,7-diaza-bicyclo
[3.3.1]nonane-3-carboxylic acid tert-butyl ester (4a)
0
0
0
(m, 1H, C(O)NC2 HB), 2.40e2.50 (m, 2H, C(O)NC8 HB, NC6 HA), 2.12
0
0
0
DCC (0.308 g, 1.2 eq, 1.495 mmol) dissolved in dry DCM (5 ml)
was added to the stirring reaction mixture containing N-benzyl
pyroglutamic acid (0.273 g, 1 eq, 1.25 mmol) and HOBt (0.252 g,
1.5 eq, 1.86 mmol) dissolved in dry DCM (10 ml) at 0 ꢀC and
continued to stir for 15 min at same temperature. Then N-Boc
bispidine (0.281 g, 1 eq, 1.25 mmol) dissolved in dry DCM (5 ml)
was added drop wise to the stirring reaction mixture and continued
to stir for a period of 2e3 h. The reaction mixture was then brought
to room temperature and concentrated. The concentrated mass was
then dissolved in diethyl ether and washed successively with dilute
citric acid (1 ꢂ 20 ml), dilute NaHCO3 (1 ꢂ 20 ml), brine and then
extracted with ethyl acetate (3 ꢂ 20 ml). The combined organics
(m, 1H, C4HA), 2.10e1.90 (m, 2H, C4 HB, NC6 HB,0NC8 HA), 1.90e1.82
0
0
(m, 2H, C4HB, C3HA), 1.99e1.97 (bs, 4H, C3HB, C3 H, C7 H, C9 H), 1.43
(s, 9H, (CH3)3); 13C NMR (50 MHz,CDCl3, ppm):
d 175.87, 169.33,
154.89, 142.20, 132.46, 128.87, 127.58, 111.52, 79.93, 56.99, 49.56,
46.54, 45.20, 30.27, 29.69, 29.52, 28.56, 28.38, 27.74, 27.32, 23.09; IR
(KBr): 3896, 3744, 3700, 3576, 3456, 2924, 2859, 2361, 2228, 1679,
1418 cmꢁ1; MS (ESI): m/z ¼ 452.5 (Mþ).
6.1.8. tert-butyl 7-((S)-1-(4-chlorobenzyl)-5-oxopyrrolidine-2-
carbonyl)-3,7-diazabicyclo [3.3.1] nonane-3-carboxylate (4e)
Yield ¼ 77%; 1H NMR (300 MHz, CDCl3, ppm):
d 7.30e7.28 (d,
J ¼ 7.8 Hz, 2H, Ph-H), 7.27e7.12 (d, J ¼ 7.8 Hz, 2H, Ph-H) 5.11e5.04
0
were dried with anhydrous Na2SO4 and concentrated to obtain
(d, J ¼ 15 Hz, 1H, PhCHA), 4.59e4.54 (d, 1H, C(O)NC2 HA), 4.20e4.00
ꢀ
sticky oily product (0.534 g). Yield ¼ 79%; ½aꢃ2D7 C ¼ ꢁ15.1890
(Methanol, c ¼ 0.3160); IR (Neat): 3017.0, 2366.7, 2337.8, 1678.9,
(d, J ¼ 15 Hz, 1H, PhCHB, NC8 HA), 3.79 (d, 12 Hz, 1H, NC2H), 3.56 (d,
0
0
0
0
J ¼ 15 Hz, 1H, PhCHA ), 3.09e3.04 (m, 2H, PhCHB , C(O)NC4 HA),
1432.0, 1217.9 cmꢁ1
;
1H NMR (300 MHz, CDCl3, ppm):
d
7.36e7.14
0 0
2.99e2.96 (m, 1H, C(O)NC2 HB), 2.91e2.86 (m, 2H, C(O)NC8 HB,
0 0 0
NC6 HA), 2.50e2.48 (m, 1H, C4HA), 2.44e2.43 (m, 2H, C4 HB, NC6 HB),
(m, 5H, Ph-H); 5.15e5.10 (d, J ¼ 15 Hz, 1H, PhCHA); 4.59e4.54 (d,

8
A. Misra et al. / European Journal of Medicinal Chemistry 110 (2016) 1e12
0
2.41e2.40 (m, 2H, C4HB, C3HA), 1.94 (bs, 2H, C3HB, C3 H) 1.81(bs, 2H,
6.1.13. 5-(7-Benzyl-3,7-diaza-bicyclo[3.3.1]nonane-3-carbonyl)-1-
C7 H, C9 H), 1.41 (s, 9H, (CH3)3); 13C NMR (50 MHz,CDCl3, ppm);
(2-bromo-benzyl)-pyrrolidin-2-one (4j)
0
0
ꢀ
d
175.57, 169.53, 154.93, 134.94, 133.46, 129.87, 128.78, 79.58, 56.5,
Yield
¼
74%; MP: 131 ꢀC; ½aꢃD27 C
:
þ25.5130 (Methanol,
56.49, 56.47, 49.54, 44.70, 29.81, 28.35, 27.74, 22.92; IR(KBr): 3869,
3759, 3496, 3010, 2926, 2860, 1679, 1423 cmꢁ1; MS(ESI): m/
z ¼ 461.9 (Mþ).
c ¼ 0.2040); IR (KBr): 3464.8, 3354.4, 2911.5, 2802.9, 1690.2, 1638.9,
1442.4, 1342.4, 1285.8, 1254.6 cmꢁ1; 1H NMR (300 MHz, CDCl3,
ppm):
d
7.29e7.22 (m, 9H, 2 ꢂ Ph); 5.14e5.09 (d, J ¼ 15 Hz, 1H,
PhCHA); 4.57e4.53 (d, J ¼ 12 Hz, 1H, NC20HA); 4.21e4.29 (t, 1H,
6.1.9. tert-butyl 7-((S)-1-(2,6-dichlorobenzyl)-5-oxopyrrolidine-2-
carbonyl)-3,7-diazabicyclo [3.3.1] nonane-3-carboxylate (4f)
NC2H) 4.14e4.09 (d, J ¼ 15 Hz, 1H, PhCHB); 3.50e3.43 (m, 2H,
0
0
0
0
PhCHA , NC8 HA); 3.25e3.16 (m, 2H, PhCHB , NC2 HB); 3.0e2.84 (m,
3H, NC80HB, NC40HA, C60HA); 2.61e2.49 (m, 1H, C4HA); 2.35e2.27
(m, 2H, NC40HB, C60HB); 2.05e1.93 (m, 1H, C3HB); 1.89 (m, 2H, C30H,
Yield ¼ 63%; 1H NMR (300 MHz, CDCl3, ppm):
d 7.64e7.61 (m,
2H, Ph), 7.28 (m, 2H, Ph) 5.17e5.12 (d, J ¼ 15 Hz, 1H, PhCHA),
0
0
C7 H); 1.68 (s, 2H, C9 H); 13C NMR (50 MHz, CDCl3, ppm): d 175.72,
0
4.59e4.55 (d, 12 Hz, 1H, C(O)NC2 HA), 4.12e3.92 (d, J ¼ 15 Hz, 1H,
0
PhCHB), 3.87 (d, 12 Hz, 1H, NC2H), 3.12 (m, 1H, PhCHA ), 3.10e3.01
168.34,137.90,136.149,132.84, 128.61, 128.33,127.77,127.01,124.43,
115.35, 63.83, 59.73, 58.56, 56.97, 49.29, 45.67, 34.64, 31.33, 29.20,
21.96; MS (ESI): m/z ¼ 496.2 (MþH)þ.
0
0
0
(m, 2H, PhCHB , C(O)NC4 HA), 2.60e2.51 (m, 1H, C(O)NC2 HB),
2.40e2.50 (m, 2H, C(O)NC8 HB, NC6 HA), 2.12 (m, 1H, C4HA),
0
0
0
0
0
2.10e1.90 (m, 2H, C4 HB, NC6 HB, 0NC8 HA), 1.90e1.82 (m, 2H, C4HB,
0
0
C3HA), 1.99e1.97 (bs, 4H, C3HB, C3 H, C7 H, C9 H), 1.43 (s, 9H, (CH3)3);
IR (KBr): 3896, 3744, 3700, 3576, 3456, 2924, 2859, 2361, 2228,
1679, 1418 cmꢁ1; MS (ESI): m/z ¼ 498.2 (MþH)þ.
6.1.14. 5-(7-Benzyl-3,7-diaza-bicyclo[3.3.1]nonane-3-carbonyl)-1-
(4-methylbenzyl)-pyrrolidin -2- one (4k)
ꢀ
Yield
¼
79%; MP: 133 ꢀC; ½aꢃD27 C
:
þ0.9200 (Methanol,
c ¼ 0.1260); IR (KBr): 3445.7, 2362.3,1637.4,1466.5,1219.1 cmꢁ1; 1H
6.1.10. tert-butyl 7-((S)-1-(4-methoxybenzyl)-5-oxopyrrolidine-2-
carbonyl)-3,7-diazabicyclo[3.3.1] nonane-3-carboxylate (4g)
NMR (300 MHz,CDCl3, ppm):
d
7.30e7.07 (m, 9H, 2 ꢂ Ph); 5.18e5.14
0
(d, J ¼ 15 Hz, 1H, PhCHA); 4.59e4.45 (m, 1H, NC2 HA); 4.17e4.15 (t,
1H, NC2H); 3.79e3.74 (d, J ¼ 15 Hz, 1H, PhCHB); 3.51e3.47 (m, 2H,
Yield ¼ 54%; 1H NMR (300 MHz, CDCl3, ppm):
d 7.28e7.23 (m,
0
0
0
2H, Ph), 6.83e6.72 (m, 2H, Ph), 5.14e5.10 (d, J ¼ 15 Hz, 1H, PhCHA),
PhCHA , NC8 HA); 3.27e3.10 (d, J ¼ 15 Hz,1H, PhCHB ); 3.02e2.86 (m,
0
0 0 0 0
4H, NC2 HB, NC8 HB, NC4 HA, NC6 HA), 2.55 (m, 2H, C4HA), 2.33 (s, 3H,
4.61e4.56 (d, 12 Hz, 1H, C(O)NC2 HA), 4.20e4.04 (m, 2H, PhCHB,
0
0
0
NC2H), 3.85e3.75 (s, 3H, OCH3), 3.74e3.71 (m, 1H, PhCHA ),
CH3); 2.33 (m, 2H, NC4 HB, NC6 HB); 2.09e2.06 (m, 2H, C4HB, C3HA);
0
0
0
0
0
3.60e3.48 (m, 1H, PhCHB ), 3.04e3.00 (m, 2H, C(O)NC2 HB, C(O)
1.97 (m, 1H, C3HB); 1.89 (m, 2H, C3 H, C7 H); 1.70 (s, 2H, C9 H); MS
NC4 HA), 2.94e2.88 (m, 2H, C(O)NC8 HB, NC8 HA), 2.49e2.55 (m, 2H,
(ESI): m/z ¼ 432.2(Mþ1)þ.
0
0
0
0
0
0
C4HA, NC6 HA), 2.31e2.25 (m, 2H, C4 HB, NC6 HB), 2.20 (m, 2H, C4HB,
C3HA), 1.80 (m, 4H, C3HB, C3 H, C7 H, C9 H), 1.42 (s, 9H, (CH3)3); 13C
NMR (50 MHz,CDCl3, ppm): 172.17, 169.73, 159.90, 139.16, 137.77,
129.58, 120.78, 114.11, 114.03, 113.96, 113.22, 113.05, 79.74, 55.21,
55.18, 49.49, 45.31, 33.76, 29.63, 28.32, 22.81; IR (KBr): 3900, 3565,
3366, 3013, 2926, 2856, 2196, 1679, 1434, 1363, 1219 cmꢁ1; MS
(ESI): m/z ¼ 457.5 (Mþ).
6.1.15. (5S)-5-(7-benzyl-3,7-diazabicyclo[3.3.1]nonane-3-
0
0
0
carbonyl)-1-(2,6-dichlorobenzyl)pyrrolidin-2-one (4l)
27ꢀC
Yield ¼ 76%; MP: 50e55 ꢀC; ½aꢃD
¼ þ51.6304 (Chloroform,
c ¼ 0.10); 1H NMR (300 MHz, CDCl3, ppm);
d 7.35e7.24 (m, 8H,
2 ꢂ Ph), 5.31e5.26 (d, J ¼ 15 Hz, 1H, PhCHA), 4.60e4.56 (d, 1H, C(O)
0
NC2 HA), 4.46e4.41 (d, J ¼ 15 Hz, 1H, PhCHB), 4.05e4.02 (dd, 12 Hz,
0
0
1H, NC2H), 3.50e3.46 (m, 2H, PhCHA , NC8 HA), 3.28e3.24 (m, 2H,
0
0
0
6.1.11. tert-butyl 7-((S)-1-(naphthalen-1-ylmethyl)-5-
oxopyrrolidine-2-carbonyl)-3,7-diazabicyclo [3.3.1] nonane-3-
carboxylate (4h)
PhCHB , C(O)NC4 HA), 3.05e3.01 (m, 1H, C(O)NC2 HB), 2.90e2.87 (m,
0
0
0
2H, C(O)NC8 HB, NC6 HA), 2.56 (m, 1H, C4HA), 2.36 (m, 2H, C4 HB,
0
NC6 HB), 2.11e2.01 (m, 2H, C4HB, C3HA), 1.99e1.97 (m, 1H, C3HB),
Yield ¼ 47%; 1H NMR (300 MHz, CDCl3, ppm):
d
8.05e7.37 (m,
1.93 (m, 2H, C3 H, C7 H), 1.73 (s, 2H, C9 H); 13C NMR (50 MHz,CDCl3,
0
0
0
7H, Naphthyl), 5.62e5.67 (d, J ¼ 15 Hz, 1H, PhCHA),5.04e4.97 (d,
ppm): d 175.10, 168.25,151.52, 137.89, 129.57, 128.58, 128.45, 128.33,
0
J ¼ 15 Hz, 1H, PhCHB), 4.62e4.52 (d, 12 Hz, 1H, C(O)NC2 HA),
127.26, 127.01, 63.54, 59.53, 58.47, 56.20, 46.45, 31.08, 29.62, 29.30,
28.55, 21.81; IR (KBr): 3639.2, 3400, 2955, 2800, 1690, 1645, 1439,
1362, 1228, 1154, 1119 cmꢁ1; MS (ESI): m/z ¼ 486.3 (Mþ).
0
0
4.11e4.07 (m, 1H, NC2H, NC8 HA), 3.77e3.76 (m, 1H, PhCHA ),
0
0
3.24e3.20 (m, 1H, PhCHB ), 3.01e2.91 (m, 3H, C(O)NC4 HA, C(O)
0
0
0
0
NC8 HB, C(O)NC2 HB), 2.66e2.42 (m, 3H, NC6 HA1H, C4HA, C4 HB),
0
0
2.39 (m, 1H, NC6 HB), 2.07e2.03 (m, 5H, C4HB, C3HA, C3HB, C3 H,
6.1.16. (5S)-5-(7-benzyl-3,7-diazabicyclo[3.3.1]nonane-3-
0
0
C7 H), 1.70 (bs, 2H, C9 H), 1.42 (s, 9H, (CH3)3); IR (KBr): 3947, 3675,
3484, 3421, 3287, 2923, 2853, 2361, 1674, 1452, 1365 cmꢁ1; MS
(ESI): m/z ¼ 447.5 (Mþ).
carbonyl)-1-(4-chlorobenzyl)pyrrolidin-2-one (4m)
ꢀ
Yield
¼
70%; MP: 131 ꢀC; ½aꢃD27 C
:
þ25.5130 (Methanol,
c ¼ 0.2040); IR (KBr): 3464.8, 3354.4, 2911.5, 2802.9, 1690.2, 1638.9,
1442.4, 1342.4, 1285.8, 1254.6 cmꢁ1
ppm):
;
1H NMR (300 MHz, CDCl3,
6.1.12. 1-Benzyl-5-(7-benzyl-3,7-diaza-bicyclo[3.3.1]nonane-3-
carbonyl)-pyrrolidin-ꢀ2-one (4i)
d
7.29e7.22 (m, 9H, 2 ꢂ Ph); 5.14e5.09 (d, J ¼ 15 Hz, 1H,
0
PhCHA); 4.57e4.53 (d, J ¼ 12 Hz, 1H, NC2 HA); 4.21e4.09 (d,
Yield ¼ 70%; ½aꢃ2D7 C ¼ þ3.44 (Methanol, c ¼ 0.2120); MP: 144 ꢀC;
IR (KBr): 3424.2, 3010.2, 2924.5, 1680.5, 1642.6, 1449.8,
J ¼ 15 Hz, 1H, PhCHB; m, 1H, NC2H); 3.50e3.43 (m, 2H, PhCHA ,
0
0
0
0
NC8 HA); 3.25e3.16 (m, 2H, PhCHB , NC2 HB); 3.0e2.84 (m, 3H,
1218.9 cmꢁ1; 1H NMR (300 MHz, CDCl3, ppm):
d
7.29e7.22 (m, 10H,
NC8 HB, NC4 HA,C6 HA); 2.61e2.49 (m, 1H, C4HA); 2.35e2.27 (m, 2H,
0
0
0
0
0
0
0
2 ꢂ Ph); 5.23e5.18 (d, J ¼ 15 Hz, 1H, PhCHA); 4.60e4.45 (d,
NC4 HB, C6 HB); 2.05e1.93 (m, 1H, C3HB); 1.89 (m, 2H, C3 H, C7 H);
0
0
J ¼ 12 Hz, 1H, NC2 HA); 4.17e4.15 (m, 1H, NC2H); 3.85e3.79 (d,
1.68 (s, 2H, C9 H); 13C NMR (50 MHz, CDCl3, ppm):
d
175.72, 168.34,
0
0
J ¼ 15 Hz, 1H, PhCHB); 3.50e3.45 (m, 2H, PhCHA , NC8 HA);
137.90, 136.149, 132.84, 128.61, 128.33, 127.77, 127.01, 124.43, 115.35,
63.83, 59.73, 58.56, 56.97, 49.29, 45.67, 34.64, 31.33, 29.20, 21.96;
MS (ESI): m/z ¼ 452.2 (MþH)þ.
0
0
3.26e3.22 (d, J ¼ 15 Hz, 1H, PhCHB ); 3.08e2.99 (m, 3H, NC2 HB,
0
0
0
NC8 HB, NC4 HA); 2.85 (m, 1H, NC6 HA); 2.57 (m, 1H, C4HA);
0
0
2.38e2.33 (m, 1H, C4 HB, NC6 HB); 2.10e2.06 (m, 2H, C4HB, C3HA);
1.97 (m, 1H, C3HB); 1.88 (m, 2H, C3 H, C7 H); 1.70 (s, 2H, C9 H); 13C
6.1.17. (7-Benzyl-3,7-diazabicyclo[3.3.1]nonan-3-yl)((S)-1-
0
0
0
NMR (50 MHz,CDCl3, ppm): 175.67, 168.41, 128.64, 128.54, 128.35,
d
(phenylsulfonyl)pyrrolidin-2-yl) methanone (4n)
128.07, 127.58, 127.05, 63.52, 59.35, 58.38, 56.54, 46.44, 45.40,
Yield ¼ 83%; 1H NMR (300 MHz, CDCl3, ppm):
d 7.81e7.79 (d,
31.04, 29.98, 29.19, 28.54, 21.56; MS (ESI): m/z ¼ 418.2 (Mþ1)þ.
J ¼ 7.8 Hz, 2H, SO2Ph) 7.31e7.21 (m, 7H, SO2Ph, Ph), 4.84e4.81 (d,

A. Misra et al. / European Journal of Medicinal Chemistry 110 (2016) 1e12
9
0
0
0
0
12 Hz, 1H, C(O)NC2 HA), 3.86e3.82 (d, J ¼ 15 Hz, 1H, PhCHA ),
NC8 HA); 3.71e3.66 (d, J ¼ 12 Hz, 1H, NC2 HB); 3.24e3.12 (m, 3H,
0
0
0
0
0
0
0
12 Hz, NC6 HB);
3.56e3.32 (m, 4H, PhCHB , C(O)NC4 HA, NC8 HA, NC2H), 3.01e2.89
(m, 3H, C(O)NC2 HB, C(O)NC8 HB, NC6 HA), 2.43 (s, 3H, CH3),
NC8 HB, NC4 HA,,NC6 HA); 2.90e2.86 (d,
J
¼
0
0
0
0
2.52e2.49 (m, 1H, C4HA); 2.44e2.40 (m, 1H, C4 HB); 2.34e2.24 (m,
0
0
0
2.34e2.31 (d, 12 Hz, 1H, C3HA), 2.17e1.69 (m, 10H, C4 HB, NC6 HB,
1H, C4HB); 2.22e2.07 (m, 1H, C3HA); 1.95e1.91 (m, 3H, C3HB, C3 H,
C3 H, C7 H, C3HB, C4H, C5H, C9 H); 13C NMR (200 MHz,CDCl3, ppm):
C7 H); 1.83 (m, 2H, C9 H); 13C NMR (75 MHz, CDCl3, ppm):
d 175.83,
0
0
0
0
0
d
175.61, 168.19, 163.33, 139.25, 129.83, 128.77, 128.62, 128.33,
171.35, 170.21, 135.87, 132.80, 131.37, 129.43, 128.74, 127.84, 126.70,
124.14, 56.95, 52.47, 49.53, 46.56, 46.08, 45.30, 30.85, 29.87, 27.66,
23.39; MS (ESI): m/z ¼ 512 (Mþ3)þ.
127.05, 63.53, 59.34, 58.36, 56.57, 49.22, 46.46, 31.91, 29.79, 29.14,
22.67, 21.58; IR (KBr): 3783, 3448, 3374, 2923, 2361, 2135, 1817,
1640, 1446, 1337, 1224, 1098 cmꢁ1; MS (ESI): m/z ¼ 468.3 (Mþ).
6.1.22. 5-(7-Benzoyl-3,7-diaza-bicyclo[3.3.1]nonane-3-carbonyl)-
6.1.18. (5S)-5-(7-benzyl-3,7-diazabicyclo[3.3.1]nonane-3-
carbonyl)-1-(4-bromobenzyl)pyrrolidin-2-one (4o)
1-(4-methylbenzyl)-pyrrolidin-2-one (4r)
ꢀ
Yield ¼ 89%; ½aꢃ2D7 C: þ2.1583 (Methanol, c ¼ 0.1000); IR (Neat):
Yield ¼ 65%; 1H NMR (300 MHz, CDCl3, ppm):
d 7.45e7.04 (m,
3420.3, 2958.5, 1678.4, 1632.11, 1438.3, 1220.2 cmꢁ1 1H NMR
;
8H, Ph-H) 5.20e5.15 (d, J ¼ 15 Hz, 1H, PhCHA), 4.61e4.56 (d,
(300 MHz,CDCl3, ppm):
d
7.42e7.00 (m, 9H, 2 ꢂ Ph); 5.15e5.04 (d,
0
J ¼ 15 Hz, 1H, C(O)NC2 HA), 4.20e4.12 (dd, 12 Hz, 1H, NC2H),
0
J ¼ 15 Hz, 1H, PhCHA); 4.81e4.55 (d, J ¼ 12 Hz, 1H, NC2 HA); 4.16 (t,
0
3.83e3.71 (d, J ¼ 15 Hz, 1H, PhCHB), 3.52e3.41 (m, 2H, PhCHA ,
0
1H, NC2H); 3.89e3.77 (d, J ¼ 12 Hz, 1H, NC8 HA); 3.67e33.64 (d,
0
0
0
NC8 HA), 3.19e3.15 (m, 2H, PhCHB , C(O)NC4 HA), 2.95 (m, 2H, C(O)
0
0
J ¼ 15 Hz, 1H, PhCHB); 3.33e3.19 (m, 2H, NC2 HB, NC8 HB);
0
0
0
0
NC2 HB, NC8 HB), 2.65 (m, 1H, NC6 HA), 2.15e1.88 (m, 4H, C4 HB,
0
0
3.04e3.02 (m, 1H, NC4 HA); 2.93e2.89 (m, 7H, C4HA, NC6 HB, C4HB,
0
0
0
NC6 HB C4HB, C3HA), 1.83e1.79 (m, 1H, C3HB), 1.74 (m, 2H, C3 H, C7 H,
0
0
0
C4 HB, CH3); 2.22e2.18 (m, 2H, C3H2); 1.95e1.81 (m, 2H, C3 H, C7 H);
bs, 2H, C9 H); 13C NMR (50 MHz,CDCl3, ppm); 175.33, 168.44,137.22,
0
1.27e1.26 (m, 2H, C9 H); 13C NMR (75 MHz, CDCl3, ppm):
d 175.83,
0
136.79, 133.38, 131.46, 130.16, 129.28, 128.54, 127.89, 120.79, 62.72,
59.36, 58.16, 49.20, 46.33, 45.04, 31.05, 29.96, 29.68, 29.40, 29.19,
28.48, 21.10.; IR (KBr): 3870,3777, 3588, 3526, 2924, 2276, 1680,
1451, 1220 cmꢁ1; MS (ESI): m/z ¼ 495.2 (Mþ).
171.28, 170.20, 137.4, 136.08, 136.00, 129.40, 129.22, 128.87, 128.72,
128.65, 128.56, 128.20, 127.22, 126.76, 57.49, 49.47, 46.49, 46.16,
45.92, 45.13, 34.27, 30.40, 27.61, 23.40, 21.29, 21.17; MS (ESI): m/
z ¼ 446.1 (MþH)þ.
6.1.19. (5S)-5-(7-benzyl-3,7-diazabicyclo[3.3.1]nonane-3-
6.1.23. 1-(2-Bromo-benzyl)-5-[7-(toluene-4-sulphonyl)-3,7-diaza-
bicyclo[3.3.1]nonane-3-carbonyl]-pyrrolidin-2-one (4s)
carbonyl)-1-(4-methoxybenzyl)pyrrolidin-2-one, (4p)
Yield ¼ 61%; 1H NMR (300 MHz,CDCl3, ppm):
d 7.29e7.22 (m,
p-Toluene sulphonyl chloride (0.140 g, 1.2 eq, 0.739 mmol) was
added drop wise to the stirring solution of deprotected product
(0.250 g,1eq, 0.616) and triethyl amine (0.197 ml, 2.3 eq, 1.41 mmol)
in dry DCM at 0 ꢀC and allowed to stir for half h. The reaction
mixture was washed with 1 N HCl (1 ꢂ 25 ml), 20% NaHCO3
(1 ꢂ 25 ml). The combined organics were washed with anhydrous
sodium sulphate and concentrated to obtain yellow oily liquid
(0.488 g). The crude product was purified by column chromatog-
9H, 2 ꢂ Ph); 5.14e5.09 (m, 1H, PhCHA); 4.57e4.53 (d, J ¼ 12 Hz, 1H,
0
NC2 HA); 4.21e4.09 (m, 2H, NC2H, PhCHB); 3.79 (s, 3H, OCH3)
0
0
0
3.50e3.43 (m, 2H, PhCHA , NC8 HA); 3.25e3.16 (m, 2H, PhCHB ,
0
0
0
0
NC2 HB); 3.0e2.84 (m, 3H, NC8 HB, NC4 HA,C6 HA); 2.61e2.49 (m, 1H,
0
0
C4HA); 2.35e2.27 (m, 2H, NC4 HB, C6 HB); 2.05e1.93 (m, 1H, C3HB);
1.89 (m, 2H, C3 H, C7 H); 1.68 (s, 2H, C9 H); 13C NMR (50 MHz, CDCl3,
0
0
0
ppm): 175.72, 168.34, 137.90, 136.149, 132.84, 128.61, 128.33,
d
127.77, 127.01, 124.43, 115.35, 63.83, 59.73, 58.56, 56.97, 49.29,
45.67, 34.64, 31.33, 29.20, 21.96; IR (KBr): 3872, 3778, 3578, 3526,
2928, 2276, 1680, 1449, 1220 cmꢁ1; MS (ESI): m/z ¼ 448.2 (MþH)þ.
raphy on silica (Chloroform/Methanol ¼ 7.5/2.5) to obtain the pure
ꢀ
product (0.266 m). Yield ¼ 85%; ½aꢃ2D7 C: ꢁ2.8263 (Methanol,
c ¼ 0.1000); MP: 203e205 ꢀC; IR (KBr): 3451.8, 1638.4 cmꢁ1
;
1H
NMR (300 MHz, CDCl3, ppm):
d
7.58e7.15 (m, 8H, 2 ꢂ Ph);
6.1.20. (Boc deprotection)
5.13e5.08 (d, J ¼ 15 Hz, 1H, PhCHA); 4.66e4.62 (d, J ¼ 12 Hz, 1H,
To a stirred solution 4b or 4c (1.0 eq.) in dry DCM (10 ml), TFA
(5.0 eq.) was added slowly at a temperature of 0 ꢀC and allowed to
stir for 1 h. Reaction mixture was made alkaline by 20% Na2CO3
solution and the resulting mixture was extracted with DCM. It was
washed with water and brine. Organic layer was collected and the
combined fractions were dried over anhydrous sodium sulphate
and concentrated to get yellow oily liquid.
0
NC2 HA); 4.27e4.23 (m, 1H, NC2H); 4.15e4.10 (d, J ¼ 15 Hz, 1H,
0
0
PhCHB); 3.79e3.76 (d, J ¼ 9 Hz, 2H, NC8 HA, NC2 HB); 3.66e3.62 (d,
0
0
J ¼ 12 z, 1H, NC4 HA); 3.20e3.15 (m, 1H, NC8 HB); 2.96e2.91 (m, 1H,
0
0
NC6 HA); 2.71e2.62 (m, 1H, NC4HA); 2.47e2.43 (m, 6H, NC4 HB,
0
0
NC6 HB, C4HB, CH3); 2.34e2.31 (m, 2H, C3H2); 2.28e2.05 (C3 H,
C7 H); 1.98 ppm (m, 2H, C9 H); 13C NMR (75 MHz,CDCl3, ppm):
0
0
d
176.05, 169.32, 143.74, 135.97, 132.80, 131.50, 131.12, 129.67,
129.33, 127.79, 127.75, 124.06, 56.80, 50.59, 48.65, 46.13, 45.38,
6.1.21. 5-(7-Benzoyl-3,7-diaza-bicyclo[3.3.1]nonane-3-carbonyl)-
1-(2-bromobenzyl)-pyrrolid in-2-one (4q)
29.74, 27.73, 27.28, 22.57, 21.51 ppm; MS (ESI): m/z: 562.0 (Mþ1)þ.
Benzoyl chloride (0.104 ml, 1.2 eq, 0.741 mmol) was added drop
wise to the stirring solution of deprotected product (0.25 g, 1eq,
0.617 mmol) and triethylamine (0.198 ml, 2.3 eq, 1.42 mmol) in dry
dichloromethane at 0 ꢀC and allowed to stir for half h. The reaction
mixture was washed with 1 N HCl (1 ꢂ 25 ml), 20% NaHCO3
(1 ꢂ 25 ml). The combined organics were washed with anhydrous
sodium sulphate and concentrated to obtain yellow oily liquid and
the crude product was purified by column chromatography on
6.1.24. 1-(4-Methylbenzyl)-5-[7-(toluene-4-sulphonyl)-3,7-diaza-
bicyclo[3.3.1]nonane-3ꢀ-carbonyl]-pyrrolidin-2-one (4t)
Yield ¼ 91.5%; ½aꢃ2D7 C: ꢁ0.9313 (Methanol, c ¼ 0.1000); IR (Neat):
3449.8, 2953.7, 1641.5, 1443.2, 1220.9 cmꢁ1 1H NMR (300 MHz,
;
CDCl3, ppm):
d
7.60e7.00 (m, 8H, 2 ꢂ Ph), 5.20e5.03 (d, J ¼ 15 Hz,
0
1H, PhCHA), 4.68e4.65 (d, J ¼ 12 Hz, 1H, NC2 HA), 4.24e4.23 (t, 1H,
0
0
NC2H), 3.94e3.64 (m, 3H, PhCHB, NC2 HB, NC8 HA), 3.13e2.97 (m,
silica to obtain the pure product. Yield: 88%; MP: 85 ꢀC;
2H, NC8 HB, NC4 HA), 2.77e2.76 (m, 1H, NC6 HB), 2.72e2.66 (m, 3H,
0
0
0
ꢀ
½aꢃ2D7 C: þ13.73 (Methanol, c ¼ 0.1000); IR (KBr): 3404.0, 2929.5,
C4HA, NC6 HB, C4 HB), 2,29 (s, 6H, 2 ꢂ CH3), 2.45e2.07 (m, 2H, C4HA,
0
0
2365.0, 1629.8, 1429.4, 1351.7, 1246.7, 1085.7 cmꢁ1
;
1H NMR
C3H), 1.92 (m, 2H, C3 H), 1.66 (m, 1H, C7 H), 1.28e1.25 (m, 2H, C9 H);
0
0
0
(300 MHz,CDCl3, ppm):
d
7.57e7.54 (d, J ¼ 7.8 Hz, 1H, Ph-H),
13C NMR (50 MHz, CDCl3, ppm):
d
151.53, 135.80, 129.68, 129.22,
7.42e7.17 (m, 8H, Ph-H); 5.15e5.10 (d, J ¼ 15 Hz, 1H, PhCHA);
128.51, 128.25, 128.65, 128.56, 128.20, 127.22, 126.76, 46.10, 45.01,
34.20, 34.23, 30.34, 27.80, 27.36, 22.64, 21.53, 21.20; MS (ESI): m/z:
496.0 (MþH)þ.
0
4.80e4.75 (d, J ¼ 15 Hz, 1H, NC2 HA); 4.62e4.57 (d, J ¼ 15 Hz, 1H,
PhCHB); 4.22e4.11 (t, 1H, NC2H); 3.88e3.83 (d, J ¼ 12 Hz, 1H,

10
A. Misra et al. / European Journal of Medicinal Chemistry 110 (2016) 1e12
6.1.25. (5S)-5-(2-bromobenzyl-3,7-diazabicyclo[3.3.1]nonane-3-
carbonyl)-1-(4-methylbenzyl)pyrrolidin-2-one (4u)
drugs (asprin and clopidogrel). Before experimental procedure,
vehicle, drugs or test compounds were administered to animals
orally for either 1 h or as indicated.
Yield ¼ 64%; MP: 85e90 ꢀC; 1H NMR (300 MHz, CDCl3, ppm)
d
7.29 (m, 9H, 2 ꢂ Ph), 5.18e5.14 (d, J ¼ 15 Hz,1H, PhCHA), 4.94e4.90
0
(m, 1H, C(O)NC2 HA), 4.61e4.56 (d, J ¼ 15 Hz, 1H, PhCHB), 4.14 (t, 1H,
6.2.2. Collagen-epinephrine induced pulmonary thromboembolism
Pulmonary thromboembolism in mice, following intravenous
administration of collagen epinephrine suspension, was assessed as
described previously [12]. Briefly, the compounds to be tested,
standard drugs (aspirin and clopidogrel) or the vehicle were
administered by oral route at different time points (1, 2, 4, 6, 12,
24 h) prior to the thrombotic challenge. Ten mice were used for
evaluating the effect of test compound, aspirin or clopidogrel, while
a group of 5 mice was used to evaluate the effect of vehicle. A
0
0
NC2H), 3.85e3.80 (m, 2H, PhCHA , NC8 HA), 3.51e3.47 (m, 2H,
0
0
0
0
PhCHB , C(O)NC4 HA), 3.29e3.15 (m, 2H, C(O)NC2 HB, C(O)NC8 HB),
0
0
3.04e2.89 (m, 1H, NC6 HA), 2.57 (m, 1H, C4HA), 2.37 (m, 2H, C4 HB,
0
NC6 HB), 2.33 (s, 3H, CH3), 2.09e2.08 (m, 2H, C4HB, C3HA), 1.98 (m,
1H, C3HB), 1.93 (m, 2H, C3 H, C7 H), 1.72 (m, 2H, C9 H); 13C NMR
0
0
0
(50 MHz,CDCl3, ppm):
d 181.36, 180.12, 169.59, 143.12, 129.42,
128.74, 128.17, 127.64, 114.05, 63.58, 59.23, 58.80, 49.77, 48.27,
46.64, 30.88, 29.83, 29.68, 29.33, 21.52; IR (KBr): 3444, 2925,2857,
2372, 2338, 2141, 1638, 1447, 1355, 1225, 1093 cmꢁ1; MS (ESI): m/
z ¼ 452.3 (Mþ).
mixture of collagen (150
mg/ml) and adrenaline (50 mg/ml) was
injected into the tail vein to induce hind limb paralysis or death.
The number of dead or paralyzed mice was counted for 15 min and
the results are reported as % protection [12].
6.1.26. (5S)-5-(7-(4-bromobenzyl)-3,7-diazabicyclo[3.3.1]nonane-
3-carbonyl)-1-(4-methylbenzyl)pyrrolidin-2-one (4v)
Yield ¼ 67%; 1H NMR (300 MHz, CDCl3, ppm):
d
7.45e7.04 (m,
6.2.3. Bleeding time in mice
8H, Ph-H) 5.20e5.15 (d, J ¼ 15 Hz, 1H, PhCHA), 4.61e4.56 (d,
Mice tail tip (approximately 2 mm) was incised and the blood
oozed was soaked on a filter paper, which was monitored at an
interval of 15 s till the bleeding stops. The time elapsed from the tail
incision to the stoppage of bleeding was determined as the
bleeding time as described previously [12].
0
J ¼ 15 Hz, 1H, C(O)NC2 HA), 4.20e4.12 (t, 1H, NC2H), 3.83e3.71 (d,
0
0
J ¼ 15 Hz, 1H, PhCHB), 3.52e3.41 (m, 2H, PhCHA , NC8 HA), 3.19e3.15
0
0
0
0
(m, 2H, PhCHB , C(O)NC4 HA), 2.95 (m, 2H, C(O)NC2 HB, NC8 HB), 2.65
0
0
0
(m, 1H, NC6 HA), 2.34 (s, 3H, CH3), 2.15e1.88 (m, 4H, C4 HB, NC6 HB
0
0
C4HB, C3HA), 1.83e1.79 (m, 1H, C3HB), 1.74 (m, 2H, C3 H, C7 H, bs, 2H,
C9 H); 13C NMR (50 MHz,CDCl3, ppm):
d
180.33, 173.40, 142.20,
6.2.4. Vasoreactivity studies
0
138.34, 137.69, 134.78, 134.30, 133.53, 132.90, 67.65, 64.34, 63.14,
61.36, 59.96, 54.18, 51.32, 50.03, 36.06, 34.68, 34.17, 26.47; IR (KBr):
3891, 3806, 3708, 3625, 3585, 3446, 2924, 2411, 1676, 1452, 1363,
1221, 1091 cmꢁ1; MS (ESI): m/z ¼ 510.4 (Mþ).
Thoracic aortic rings from male Sprague Dawley rats
(160e180 g) were prepared and equilibrated in KCl Kreb's buffer
followed by assessment of their functions as described previously
[18]. Rings were subsequently incubated with or without vehicle
(0.2%) or test compounds and concentration response curves of
acetylcholine (Ach), U46619 (1 pMe100 nM) or phenylephrine (PE)
6.1.27. (5S)-5-(7-(4-chlorobenzyl)-3,7-diazabicyclo[3.3.1]nonane-
3-carbonyl)-1-(4-methylbenzyl)pyrrolidin-2-one (4w)
(3nMe300mM) was generated as described previously [18].
(5S)-5-(3,7-diazabicyclo[3.3.1]nonane-3-carbonyl)-1-(4-
methylbenzyl)pyrrolidin-2-one (0.250 g, 1.0 eq., 0.76 mmol) was
weighed and taken in round bottom flask, dissolved in dry acetone
(2 ml). 2 g of anhydrous potassium carbonate (K2CO3) was added. 2-
chlorobenzyl chloride (0.182 g, 1.5 eq., 1.14 mmol) was added to the
reaction mixture and refluxed in an oil bath at 60 ꢀC for 2 h with
stirring. The reaction was monitored for completion by TLC. After
the completion of reaction, reaction mixture was filtered to remove
K2CO3 and concentrated in vacuum. The desired product was iso-
lated from the crude reaction mixture by column chromatography.
6.2.5. Ferric chloride induced arterial thrombosis in mice
Swiss albino mice were pretreated with test compound, clopi-
dogrel or vehicle for 60 min and were anesthetized by urethane
(1.25 g/kg, i.p.). The carotid artery was carefully dissected and a
pulsed Doppler flow probe (DBF-120A-CPx) was placed around it to
record the blood flow using Biopac Data Acquisition System (CBI-
8000; Crystal Biotech, USA). Thrombosis was monitored as the time
taken for the cessation of carotid artery blood flow, time to occlu-
sion (TTO) after the application of 10% ferric chloride-soaked filter
paper as described earlier [19,24].
Yield ¼ 62%; 1H NMR (300 MHz, CDCl3, ppm)
d: 7.52e7.49 (m, 1H,
Ph-H) 7.35e7.28 (m, 2H, Ph-H), 7.11e7.00 (m, 4H, Ph-H) 6.85e6.82
(d, 1H, J ¼ 12 Hz, Ph-H) 5.18e5.13 (d, J ¼ 15 Hz, 1H, PhCHA),
6.2.6. Platelet isolation
0
4.64e4.59 (d,12 Hz,1H, C(O)NC2 HA), 4.16e4.12 (t,12 Hz,1H, NC2H),
Blood from healthy volunteers was centrifuged at 1000 rpm for
10 min and platelet-rich plasma (PRP) was obtained. The remaining
blood was further centrifuged at 2500 rpm for 10 min to obtain
platelet poor plasma (PPP). For washed platelets, ACD, apyrase type
0
3.79e3.74 (d, J ¼ 15 Hz, 1H, PhCHB), 3.60e3.41 (m, 2H, PhCHA ,
0
0
0
NC8 HA), 3.16e3.00 (m, 2H, PhCHB , C(O)NC4 HA), 2.91e2.88 (m, 1H,
0
0
C(O)NC2 HB), 2.33 (s, 3H, CH3), 1.98e1.97 (m, 2H, C(O)NC8 HB,
0
0
0
NC6 HA), 1.95e1.93 (m, 2H, C4 HB, NC6 HB), 1.85 (m, 2H, C4HB, C3HA),
VII (0.5 U/mL) and PGI2 (0.5 mM) were added to platelet rich plasma
1.44e1.43 (m, 1H, C3HB), 1.28 (m, 2H, C3 H, C7 H, bs, 2H, C9 H); 13C
NMR (50 MHz,CDCl3, ppm): 175.50, 168.55, 137.20, 136.98, 133.44,
132.65, 129.27, 128.54, 62.45, 59.62, 58.58, 56.41, 54.93, 49.15,
46.36, 45.04, 29.90, 29.90, 29.26, 28.51, 21.10; IR (KBr): 3869, 3441,
3013, 2365, 1679, 1515, 1450, 1218 cmꢁ1; MS (ESI): m/z ¼ 466.01
(Mþ).
and centrifuged at 2200 rpm for 10 min. Platelets were washed and
finally resuspended in Tyrode's HEPES buffer (134 mM NaCl,
2.9 mM KCl, 1.0 mM MgCl2, 10.0 mM HEPES, 5.0 mM glucose,
12.0 mM NaHCO3,0.34 mM Na2HPO4, 5 mM glucose, pH 7.4) as
described previously [20].
0
0
0
d
6.2.7. Platelet aggregation assay
6.2. Biology (materials and method)
A turbidimetric method was applied to measure platelet ag-
gregation, using a four channel-Aggregometer (Chronolog-corp,
Havertown, USA). Platelet rich plasma (1 ꢂ 108 platelets/ml,
0.45 ml) was pre-treated with vehicle (DMSO, 0.4%) or test com-
pounds for 5 min, followed by stimulation with platelet agonists
(collagen, collagen related peptide [CRP-XL] ADP, thrombin recep-
tor activating peptide [TRAP], arachidonic acid [AA], ristocetin and
6.2.1. Animal studies
Male Swiss albino mice (18e22 g) and Sprague Dawley rats
(160e180 g) were obtained from the National Laboratory Animal
Centre of the Institute. The animals were grouped into vehicle (0.5%
carboxymethyl cellulose in water), test compounds or standard

A. Misra et al. / European Journal of Medicinal Chemistry 110 (2016) 1e12
11
U46619) at 37 ꢀC. The reactions were allowed to proceed for at least
Acknowledgements
5e10 min. The percentage of aggregation was calculated by using
Aggrolink Software [20].
We thank Director, CDRI, Lucknow for his kind support. The
study was supported by a financial grant to Madhu Dikshit from the
CSIR project THUNDER (THU0001) and Department of
Biotechnology-INDIGO project (GAP0073), India with CDRI
communication Number 9165.. The award of research fellowship to
Ankita Misra from the Council of Scientific and Industrial Research
(CSIR), New Delhi, India is acknowledged. We thank SAIF, CDRI for
the spectral data and Dr. Surendra Singh, Pharmacology division for
assisting antiplatelet studies.
6.2.8. Coagulation parameters
Coagulation parameters namely thrombin time (TT), pro-
thrombin time (PT), activated partial thromboplastin time (aPTT)
were assayed in human plasma samples using commercial kits
(Stago, France) as per manufacturer's instructions and measured by
using a Coagulometer (Start4 Semi automated, Young Instruments,
Stago, France) [19].
Appendix A. Supplementary data
6.2.9. Platelet adhesion assay
96-well micro titer plates were coated with fibrillar type I
collagen (Chrono-log Corp., USA) overnight, and blocked with BSA
(0.005 g/ml) for 1 h. Washed platelets (1 ꢂ 108 cells/mL) were
preincubated with test compounds or vehicle (DMSO) for
15 min at 37 ꢀC and added to the wells (107/well) for 1 h at room
temperature. Divalent cation-free adhesion buffer was made by
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.01.019.
Conflicts of interest
The authors declare that there is no conflict of interests.
replacing Mg2þ (1 mM) in the Tyrode-HEPES buffer with 50
mM
EDTA. After three washes in Tyrode's buffer, the number of
adherent platelets was evaluated colorimetrically as described by
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