Synthesis and identification of chiral aminomethylpiperidine carboxamides as inhibitor of collagen induced platelet activation

Article abstract of DOI:10.1016/j.ejmech.2014.05.017

A series of chiral lactam carboxamides of aminomethylpiperidine were synthesized and investigated for the collagen induced in vitro anti-platelet efficacy and collagen plus epinephrine induced in vivo pulmonary thromboembolism. The compound 31a (30 μM/kg) displayed a remarkable antithrombotic efficacy (60% protection) which was sustained for more than 24 h and points to its excellent bioavailability. The compounds 31a (IC₅₀ = 6.6 μM) and 32a (IC₅₀ = 37 μM), as well as their racemic mixture 28i (IC₅₀ = 16 μM) significantly inhibited collagen-induced human platelet aggregation in vitro. Compound 34c displayed dual mechanism of action against both collagen (IC₅₀ = 3.3 μM) and U46619 (IC₅₀ = 2.7 μM) induced platelet aggregation. The pharmacokinetic study of 31a indicated very faster absorption, prolonged and constant systemic exposure and thereby exhibiting better therapeutic response.

Full text of DOI:10.1016/j.ejmech.2014.05.017

Accepted Manuscript
Synthesis and Identification of Chiral Aminomethylpiperidine Carboxamides as
Inhibitor of Collagen Induced Platelet Activation
K.S. Anil Kumar , Ankita Misra , Tanveer Irshad Siddiqi , Stuti Srivastava , Manish
Jain , Rabi Sankar Bhatta , Madhu Dikshit , Dinesh K. Dikshit
PII:
S0223-5234(14)00431-0
10.1016/j.ejmech.2014.05.017
EJMECH 6973
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 September 2013
Revised Date: 1 May 2014
Accepted Date: 4 May 2014
Please cite this article as: K. Anil Kumar , A. Misra, T.I. Siddiqi, S. Srivastava, M. Jain, R.S. Bhatta, M.
Dikshit, D.K Dikshit, Synthesis and Identification of Chiral Aminomethylpiperidine Carboxamides as
Inhibitor of Collagen Induced Platelet Activation, European Journal of Medicinal Chemistry (2014), doi:
10.1016/j.ejmech.2014.05.017.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Synthesis and Identification of Chiral Aminomethylpiperidine
Carboxamides as Inhibitor of Collagen Induced Platelet Activation
a#
b#
a
a
b
Anil Kumar K S , Ankita Misra ,Tanveer Irshad Siddiqi , Stuti Srivastava , Manish Jain , Rabi
c
b
a,*
Sankar Bhatta , Madhu Dikshit , Dinesh K Dikshit
a
Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Sec-10,
Janakipuram Ext., Sitapur Road, Lucknow-226 031, India
b
Pharmacology Division, CSIR-Central Drug Research Institute, Sec-10, Janakipuram Ext.,
Sitapur Road, Lucknow-226 031, India
c
Pharmacokinetics & Metabolism Division, CSIR-Central Drug Research Institute, Sec-10,
Janakipuram Ext., Sitapur Road, Lucknow-226 031, India
∗
Corresponding author. Tel.: +91-522-262-0713; fax: +91-522-262-3405; e-mail:
dk_dikshit@cdri.res.in (D.K. Dikshit)
#
equally contributed

ACCEPTED MANUSCRIPT
ABSTRACT
A series of chiral lactamcarboxamides of aminomethylpiperidine were synthesized and
investigated for the collagen induced in vitro anti-platelet efficacy and collagen plus epinephrine
induced in vivo pulmonary thromboembolism. The compound 31a (30 ꢀM/Kg) displayed a
remarkable antithrombotic efficacy (60% protection) which was sustained for more than 24
hours and points to its excellent bioavailability. The compounds 31a (IC = 6.6µM) and 32a
5
0
(
IC50=37µM), as well as their racemic mixture 28i (IC50=16µM) significantly inhibited collagen-
induced human platelet aggregation in vitro. Compound 34c displayed dual mechanism of action
against both collagen (IC =3.3µM) and U46619 (IC =2.7µM) induced platelet aggregation.
5
0
50
The pharmacokinetic study of 31a indicated very faster absorption, prolonged and constant
systemic exposure and thereby exhibiting better therapeutic response.
Keywords
Thrombosis, Antiplatelet, Pyroglutamic acid, Aminomethylpiperidine, Collagen, Epinephrine

ACCEPTED MANUSCRIPT
1
. Introduction
Arterial thrombosis is the acute complication that develops on the chronic lesions of
atherosclerosis and causes heart attack and stroke, today the most common causes of mortality in
the world [1]. Search of more selective and specific antithrombotic agents for the prevention and
treatment of acute occlusive arterial thrombosis is, therefore, a valid approach. Platelet
interaction [2-4] is the first step in the haemostatic process, where, extracellular matrix (ECM) is
exposed at sites of injury. Among the macromolecular constituents of the ECM, collagen is
considered to play a major role in this process, as in vitro it not only supports platelet adhesion
through direct and indirect pathways but it also directly activates the cells in initiating
aggregation and coagulant activity. The primary targets of existing anti-platelet therapy are
molecules involved in platelet activation and aggregation. At present, there are no drugs in
clinical use that block the initial tethering and adhesion of platelets to collagen and von
Willebrand factor and hence their arrest on the blood vessel wall. The inhibition of this early step
in thrombus formation is more likely to reduce or prevent the incidence of arterial thrombosis in
patients of cardiovascular diseases [5]. Though several reports of peptide and proteins inhibitors
of platelet aggregation by inhibiting platelet activation by collagen and other endothelial derived
activating molecules [6-10], only a few small molecules inhibitors of collagen mediated platelet
aggregation have been reported [11-13]
The present study originates in the serendipitous observation of collagen induced platelet
aggregation inhibition by pyroglutamylpiperazines 1 and 2 in a random screening of CDRI
library of compounds. Observation of antithrombotic potential in an in vivo model and their
effect on bleeding time prompted an old fashioned systemic search for more efficacious
compounds. We also took note of the published anti-adhesive property of nipecotamide
derivatives, 3 [14-25] and α β -integrin (VLA-4) inhibitors possessing a pyroglutamyl moiety 4
4
1
[26] and antiplatelet activity of various five and six membered heterocycles [27, 28] to arrive at
possible modifications (Fig. 1) and also to make use of the combined the therapeutic benefit [29].

ACCEPTED MANUSCRIPT
R
N
O
R''
H
N
N
N
O
O
N
R'
N
R2
R1
O
N
O
R
X
3
1
; R = 2-Cl Ph; 40 % protection in vivo
4 % inhibition in vitro
; R = 2-F Ph; 50% protection in vivo
7 % inhibition in vitro
4
7
2
5
Fig. 1. Structure of pyroglutamylpiperazines (1 & 2), nipecotamides (3) possessing anti-platelet
activity and N-substituted pyroglutamic acid derivatives (4) as α β -integrin inhibitors.
4
1
The planned exploratory sequential modification of three sub-structural components of the
molecule was planned as shown in Fig.2.
Modification of A
X
X
O
N
O
N
X = CH , O, S
2
X = CH , O, S
2
B
R
A
N
Modification of B
N
N
N
N R
O
N
NHR
O
NHR
HN
R = Boc, Cbz, tosyl
R = Boc, Cbz, pivaloyl,
R = Boc, Cbz
COCH NHCbz, tosyl
2
C
Modification of C
Y = CH , CO, CH CH=CH
2
2
Z = 4-methyl, H, 3-methyl, 4- bromo,
-bromo, 4-chloro, 2,6-dichloro,
3,4-dichloro, 3-cyano, 4-cyano,
Y
2
Z
4
-methoxyl, 3-methoxy,
2
-methoxyl, 2-chloro
Fig. 2. Proposed structural modifications
2
. Results and discussion
2
.1. Chemistry
Various lactamcarboxylate esters 10-14 were prepared by reported methodologies [30-33]
starting from corresponding of L-amino acids 5-9. LiHMDS mediated N-deprotonation of these

ACCEPTED MANUSCRIPT
esters and subsequent alkylation with different arylalkylhalides gave the corresponding N-
substituted lactam esters which were hydrolysed to yield the corresponding lactam carboxylic
acids 16-20. (Scheme 1). N-benzylserine 15a and N-benzylthreonine 15b were cyclized with
chloroacetyl chloride in aqueous alkali solution [34] to the corresponding 5-oxomorpholine-3-
carboxylic acids 21a and 22a respectively (Scheme 1).
COOH
NH2
COOH
COOH
COOH
NH2
HOOC
COOH
R'
NH2
HOOC
NH2
NH2
OH
9a; R = H; L-serine
b; R = Me; L-threonine
XH
SH
5
, L-glutamic acid
6a; x = o; L-serine
7
, L-cysteine
8, L-aspartic acid
9
6
b; x = s; L-cysteine
ref. 30
ref. 31
ref. 32
ref. 34
ref. 33
R'
OH
NH
X
S
HOOC
O
CO CH3
O
CO CH3
CO CH
2 3
N
H
2
N
H
2
N
H
O
N
H
CO CH
2
3
1
0
11a; X = O
13
14
R
1
2a; X = S
1
1
5a; R = H
5b; R = Me
(
i) a, b
(i) a, b
(ii) c
(
(
i) a, b
ii) c
(i) a, b
(ii) c
(ii) c
d
O
N
R'
X
S
O
COOH
COOH
O
COOH
N
N
O
N
COOH
N
Y
O
COOH
O
R
R
R
R
R
2
2
1a; R = H
2a; R = Me
1
1
7a; X = O
8a; X = S
1
6(a-n)
19
20
Scheme 1. Synthesis of N-arylalkyl acids. Reagents and conditions: (a) LiHMDS, Dry THF, -15
0
0
C, 1h; (b) R-Br, dry THF, 4h, 0 C to room temperature; (c) 20 % aq. Na CO solution, 4 hrs,
2
3
room temperature; (d) 2.5 N NaOH, chloroacetyl chloride.
N-substituted oxothiomorpholine carboxylic acid, 26 was prepared in a multistep reaction
sequence (Scheme 2). A solution of L-cysteine 23 in dry DMF was treated with t-butyl-2-
bromoacetate in the presence of DIPEA to provide (S)-t-butoxycarbonylmethyl cysteine ethyl
ester 24. Reaction of 24 with p-methyl benzaldehyde under reductive amination condition gave
2
5 which was converted to corresponding carboxylic acid, cyclised intramolecularly using DIC-
HOBt and the resultant cyclized ester was hydrolysed to give thiomorpholinone carboxylic acid
6.
2

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O
S
O
S
OH
SH
S
OH
OtBu
O
N
a
b, c
EtO2C
NH
d, e
O
EtO2C
NH2. HCl
EtO2C
NH2
2
3
24
25
26
Scheme 2. Synthesis of thiomorpholinone carboxylic acid. Reagents and conditions: (a) DIPEA,
0
dry DMF, BrCH COOtBu, -10 C; (b) NaOAc, p-tolualdehyde, NaCNBH , dry methanol; (c)
2
3
4
0% TFA, dry DCM; (d) DIC, HOBt, Et N, dry DCM; (e) 15 % aq.K CO , 4h
3 2 3
Initially only a few differentially N-substituted pyroglutamic acids were condensed with various
- or 4-aminomethylpiperidines to give corresponding pyroglutamides which were evaluated for
3
in vitro (inducer, collagen), in vivo (inducer, collagen plus epinephrine) antithrombotic properties
and their effect on bleeding time in mice (Scheme 3, Table-1).
NHR'
N
NHR'
O
COOH
O
N
Y
a
N
Y
+
O
N
H
X
X
1
6
27
2
8 (a-j) R' = 3-Boc, 4-Boc, Pivaloyl, tosyl
-Cbz, 4-Cbz & COCH NHCbz
3
2
Scheme 3. Synthesis of diamine coupled recemic pyroglutamides. Reagents and conditions: (a)
0
DCC, HOBt, dry DCM, 0 C, 3h
It became evident that the carboxamides having substituted 3-aminomethylpiperidine had far
superior bioactivity profile taking account both ex vivo platelet aggregation and in vivo
protection against thromboembolism (Table-1). Therefore our attention was directed to the most
promising compound 28i which despite being a diastereomeric mixture was not resolvable
chromatographically even by HPLC. This prompted us to synthesize both the diastereomers in
pure form using chiral 3-N-Boc-aminomethylpiperidine (29, 30) which were prepared using
literature procedures [35]. Condensation of several substituted N-benzylpyroglutamic acids
1
6(a-n) individually with (3R)-N-Boc-3-aminomethylpiperidine, 29 and (3S)-N-Boc-3-
aminomethylpiperidine, 30 gave 31(a-n) and 32(a-n) respectively (Scheme 4, Table-2). Further
modification of 3-aminomethyl substituent was carried out as shown in Fig. 6 to give 31(o-s)
having substitutions other than t-butyloxycarbonyl (Boc) group.

ACCEPTED MANUSCRIPT
a
O
N
b, c
N
O
N
N
O
O
NHBoc
(
R)
HN
R'HN
N
H
29
O
R
O
O
COOH
N
3
1(a-n)
3
1(o-s)
R
31o; R' = COC(NHZ)CH(CH3)2
N
3
1p; R' = COC(NHZ)CH CH(CH )
3 2
2
O
a
N
31q; R' = COC(NHZ)CH Ph
2
1
6(a-n)
O
31r; R' = COPh-4-CH3
NHBoc
(
S)
HN
31s; R' = Tosyl
O
N
H
R
30
O
3
2(a-n)
Scheme 4. Synthesis of chiral pyroglutamides. Reagents and conditions: (a) Coupling agent; (b)
TFA, dry DCM, room temperature, 5h.; (c). R’-Cl, TEA, dry DCM, 2h
Employing similar methodology, various lactam carboxylic acids 17-22 and 26 were also
condensed with 29 and 30 to provide another set of carboxamides 33-36 (Fig. 3, Table 2 & 3).
X
X
Y
Y
N
N
N
Z
N
Z
O
O
BocHN
BocHN
3
3(a-d)
34(a-d)
X
Y
Z
X
Y
Z
3
3a
O
C=O CH2
34a O C=O CH
34b S C=O CH
2
2
2
3
3
3
3b S C=O CH2
3c S CH2 CH2
3d S CH2 C=O
34c S CH
34d S CH
CH
C=O
2
2
X
R
X
R
N
NHBoc
N
NHBoc
O
N
O
N
O
O
R1
R1
3
5(a-d)
36(a-d)
R
R1
X
R
R1
X
3
3
3
3
5a H
5b H
5c CH3 CH3
5d CH3
CH3 CH2
36a H
36b H
36c CH3 CH3
36d CH3
CH3 CH2
CH3
S
CH3
S
O
O
O
O
H
H
Fig. 3. N-Substituted oxazolidine and thiazolidine carboxamides and higher homologs.

ACCEPTED MANUSCRIPT
1
All the compounds were isolated in good yields and were characterized on the basis of their H
1
3
and C NMR, IR and mass spectra. The purity and diastereomeric composition were determined
by Merck HPLC using Chiralpak 1A column.
2
.2 Biology
The in vitro anti-platelet efficacy was investigated on the human platelet aggregation in platelet-
rich plasma induced by collagen (Born’s method) and in vivo by collagen-epinephrine induced
pulmonary thromboembolism in mice (Table 1). Compounds 28a, 28b, 28c, 28d, 28e, 28f, 28g,
2
8i and 28j exhibited considerably high potency in inhibiting collagen induced platelet
aggregation than aspirin, in vivo. Furthermore, compounds 28b, 28c and 28i exhibited excellent
inhibitory activity against collagen induced platelet aggregation, in vitro. Among them,
compounds with 3-substituion in the piperidine ring were the most potential leads in this series
with a range of 67-70 % protection in vivo at 30µΜ concentration and above 60 % inhibition of
platelet aggregation, in vitro at 30µΜ concentration. Compound 28i was found to be the
extremely promising among this series both in vivo (60%) and in vitro (77.3 + 10%). Moreover,
this molecule offered moderately less impact on bleeding tendency (50-60%) (Table 1) and
therefore it merits comprehensive study.
Table 1
In vivo (inducer, collagen+epinephrine) in vitro (inducer, collagen) antiplatelet and
antithrombotic properties of compounds 28(a-j).
%
increase in
a
b
No Compound
X
Y
R’
% protection
% inhibition
bleeding time
1
2
3
4
5
6
7
8
9
1
28a
28b
28c
28d
28e
28f
2-Cl
2,6-Cl
4-Br
4-CN
H
CH2
CH2
CH2
CH2
3-Boc
70
70
67
40
30
50
40
25
60
15 + 8
80 + 9
66.4 + 5
34 + 8
42 + 3
2.4 + 2.4
12 + 5
72 + 9
77.3 + 10
11+9
60-70
50
4-Boc
3-Boc
60-70
0
4-Boc
CH
2
2
-CH=CH
3-Boc
0
H
CH
3-Pivaloyl
41
28g
28h
28i
H
CH2
CH2
CH2
CH2
3-COCH
2
NHCbz
13
H
3-Tosyl
3-Boc
4-Cbz
0
4-CH3
4-Br
50-60
25
0
28j
70
a
Collagen-epinephrine induced pulmonary thromboembolism (in vivo) in mice; Compound conc. = 30 µΜ/kg
Inhibition of collagen induced platelet aggregation (in vitro) in human platelets; Compound conc. = 30 µM; n (number of experiment) = 3
b

ACCEPTED MANUSCRIPT
Carboxamides were prepared in chirally pure form and compound with a para-substituted methyl
group at the phenyl ring showed 60 % and 50% protection in diastereomers 31a and 32a
respectively, induced by collagen plus epinephrine at 30µΜ concentration in vivo. Compound
3
2e with meta-substituted methyl group also showed significant antithrombotic activity by 70%
protection in vivo. Moreover, compounds with 4-cyano (31n, 60%), 4-chloro (31h, 50%), 2-
bromo (31i and 32i, 50%) and 4-bromo (32j, 50%) substituted molecules also exhibited crucial
anti-platelet activity in vivo. Modification of the piperidine 3-substituent of the most promising
analog, 31a was executed to give 31r (N-acyl), 31s (N-sulfonyl) and 31(o-q) (N-substituted
hydrophobic amino acids) and observed a decline in the in vivo activity at the same
concentrations and therefore further modification of 3-piperidine substituent of ring-B was
established insignificant.
All the compounds were evaluated for their ability to inhibit human platelet aggregation in vitro
using Born’s method. Compound 31a showed significant activity with a percent inhibition of
platelet aggregation value 49±4 % even at 10µΜ concentration and highly promising compared
to its diastereomer, 32a (30±09 %). Meanwhile, 32e showed very low inhibition of percent
aggregation in vitro. The series 31(a-n) were found to be more dynamic than the series 32(a-n)
to the extent that in vitro collagen induced percentage inhibition was concerned (Table 2).
Therefore diastereomers of 28i, that is, 31a and 32a were taken as the lead molecule and
preceded for further studies.
Table 2
In vivo (inducer, collagen + epinephrine) in vivo (inducer, collagen) anti-platelet and anti-
thrombotic properties of (S,R) diastereomers 31(a-s) and (S,S) diastereomers 32(a-n) of N-Boc-
3
-aminomethylpiperidine derivatives of N-substituted pyroglutamic acid and higher homologs.
(S,S) diastereomers, 31(a-s)
(S,R) diastereomers, 32(a-n)
a
b
a
b
No compound
X
R
% protection
% inhibition
49±04
% protection
% inhibition
30±09
1
a
b
c
d
e
f
4-CH3
4-OCH3
COOC(CH3)3
COOC(CH3)3
60
20
ns
50
2
3
4
5
6
7
07±05
20
25±05
3-OCH
3
COOC(CH
COOC(CH3)3
COOC(CH
COOC(CH3)3
3
)
3
34±08
22.3
50
06±04
2-OCH3
40
30
20
30
32±11
22±05
3-CH
3
3
)
3
28±08
70
13±04
H
26±08
50
17±08
g
Naphthyl COOC(CH
3
)
3
29±09
22.3
22±09

ACCEPTED MANUSCRIPT
8
9
1
1
1
1
1
1
1
1
1
1
h
i
4-Cl
COOC(CH3)3
COOC(CH
COOC(CH3)3
COOC(CH
50
50
40
20
20
30
60
10
30
30
30
40
35±07
27±11
20±06
27±12
24±14
06±05
08±04
nd
15
50
50
30
10
20
10
nd
nd
nd
nd
nd
21±04
06±05
04±02
24±07
22±05
19±09
25±05
nd
2-Br
3 3
)
0
1
2
3
4
5
6
7
8
9
j
4-Br
k
l
3-4-Cl
2,6-Cl
3-CN
4-CN
4-CH3
3 3
)
COOC(CH3)3
m
n
o
p
q
r
COOC(CH3)3
COOC(CH3)3
COC(NHZ)CH(CH3)2
4-CH
4-CH3
4-CH
4-CH3
3
COC(NHZ)CH
2
CH(CH
3
)
2
nd
nd
COC(NHZ)CH Ph
nd
nd
2
3
COPh-p-CH
3
nd
nd
s
SO Ph-p-CH3
nd
nd
2
a
Collagen-epinephrine induced pulmonary thromboembolism (in vivo) in mice; Compound conc. = 30 µΜ/kg; nd, not done
Inhibition of collagen induced platelet aggregation (in vitro) in human platelets; Compound conc. = 10 µΜ; n = 3
b
Among all the eight N-substituted oxazolidine and thiazolidine carboxamides, 33c and exhibited
maximum antithrombotic protection (50% and 60%, respectively). Other compounds including
3
3b and 34b also exhibited significant protection (40% and 50%, respectively) while the
remaining compounds either remained inactive or offered weaker antithrombotic activity
compared to aspirin. All the four active compounds 33b, 34b, 33c and 34c at 30µM/kg had
relatively less impact on bleeding tendency (43%, 50%, 50% and 72% respectively) than aspirin.
The results indicate that these compounds satisfactorily preserve the haemostasis with minor
alterations in the pre-clinical mice model and hence, they might promise some improvisation
with respect to the critical disadvantage of bleeding risk endured by the current anti-platelet
strategies. Therefore, all the four compounds were further pursued for their probable mode of
action in platelet aggregation assay (in vitro).
The active compounds were evaluated for their ability to inhibit human platelet aggregation in
vitro induced by collagen. As can be seen from Table 3, all the four active compounds 33b, 34b,
3
3c and 34c exhibited significantly high potency in inhibiting collagen induced platelet
aggregation than aspirin (34c>34b>33b>33c), without affecting ADP, TRAP (thrombin receptor
activated peptide) or ristocetin induced platelet aggregation. Moreover, the order of their in vitro
potency was identical to the observations in mice model of pulmonary thromboembolism (Table
3
and 4). Among all, 34c displayed the best anti-platelet potential against collagen with an IC50
of 3.3µM. Nevertheless, the anti-platelet activity found for 34c, when the platelets were induced
either by arachidonic acid (AA) or U46619, indicated that 34c might be operating by a dual

ACCEPTED MANUSCRIPT
mechanism of action and showed a better anti-platelet potential than aspirin against both
collagen and U46619 induced platelet aggregation. Moreover, it’s IC regarding arachidonic
5
0
acid induced platelet aggregation was almost comparable to that of aspirin (6.2 µM vs 5µM).
Thus 34c might offer better protection against thrombotic complications than aspirin and hence
should be further pursued for its mechanism of action and binding site.
Table 3
In vivo antithrombotic properties and bleeding tendency of N-substituted oxazolidine and
thiazolidine carboxamides 33(a-d) and 34(a-d).
Bleeding Time
a
No
Compound
X
Y
Z
% protection
b
%
increase
1
2
3
4
5
6
7
8
33a
33b
33c
33d
34a
34b
34c
34d
O
S
S
S
O
S
S
S
C=O
C=O
CH2
CH2
30
40
50
10
20
50
60
40
54
CH
2
43
50
8
CH2
C=O
CH2
CH2
CH2
C=O
CH
2
C=O
C=O
CH2
CH2
10
50
72
50
a
collagen-epinephrine induced pulmonary thromboembolism in mice (in vivo); compound dose = 30 µM/kg
bleeding time by tail excision in mice; compound dose. = 30 µM/kg; n = 3
b
Table 4
Effect of active N-substituted oxazolidine and thiazolidine carboxamides on in vitro aggregation
of human platelet rich plasma induced by Collagen (1µg/ml), Arachidonic acid (0.5mM) and
U46619 (2.5 µg/ml).
Collagen
Arachidonic Acid
% Inhibition
15±6
U46619
% Inhibition
75±3
Compound
%
Inhibition
IC50
6.4
8.0
6.0
3.3
IC50
>100
>100
>100
6
IC50
6.4
3
3
3
3
3b
3c
4b
4c
81±5
74±4
83±2
87±5
25±4
96±8
4.7
8±5
64±9
10.2
2.7
93±8
100±7
Although the other three compounds 33b, 34b and 33c exhibited almost negligible inhibitory
effect on AA induced platelet aggregation, but all three selectively attenuated collagen and
U46619 induced platelet activation in a concentration dependent manner, thereby indicating
towards the structurally defined anti-platelet activity. The present study therefore might provide

ACCEPTED MANUSCRIPT
a basis for the development of new candidates with potent anti-platelet and antithrombotic
activities.
Compounds 35a and 36a may be considered as six membered analogs of the pyroglutamate
based amide derivatives 31a and 32a, respectively. Results revealed the fact that both
diastereomers could be a comparable antithrombotic agent, although with a slightly decreased
activity in terms of percent protection in vivo (40 and 50 %, respectively). However there is
much considerable reduction in the in vitro activity in comparison to their lower homologs.
Similarly, activity of thiomoprpholinone 35(b-d) and oxomorpholinone carboxamides 36(b-d)
were also displayed a considerable drop. Thus, expansion of ring size of the lactam scaffold
seems to have least effect as far as in vitro and in vivo anti-platelet activity results were
concerned (Table 5).
Table 5
In vivo and in vitro antiplatelet and antithrombotic properties of six-membered analogs 35(a-d)
and 36(a-d)
a
b
No Compound
R
H
H
R1
X
% protection
% inhibition
18±9
15±0
nd
1
2
3
4
5
6
7
8
35a
35b
35c
35d
36a
36b
36c
36d
CH
3
CH
2
40
30
20
20
CH3
S
CH3 CH3
O
CH
H
3
H
O
nd
CH3
CH3
CH2 50
22±10
18±0
nd
H
S
30
30
20
CH
3
CH
3
O
O
CH3
H
nd
a
collagen-epinephrine induced pulmonary thromboembolism in mice (in vivo); compound conc. = 30 µm
inhibition of collagen induced platelet aggregation (in vitro) in human platelets; compound conc. = 10 µm; n = 3; nd = not done
b
2
.2.1. Antithrombotic efficacy of 31a in collagen-epinephrine induced thrombosis in mice (in
vivo)
3
1a and 32a are the diastereomers while 28i is the racemic mixture of the two. After one hour of
administration by oral route at a dose of 30 ꢀM/Kg, the three compounds viz. 28i, 31a and 32a
offered significant protection in mice against thrombotic challenge.. All the three compounds
exhibited better antithrombotic efficacy than standard drug Aspirin (40% protection at 170 µm),
therefore, all of them were further tested in order to identify the best among them. The

ACCEPTED MANUSCRIPT
compound 31a (30 ꢀM/Kg) displayed a remarkable antithrombotic efficacy (60% protection)
which was sustained for more than 24 hours and thus highlights its excellent bioavailability. The
compound 28i exhibited almost similar efficacy like compound 31a but only upto 18 hours after
which it became ineffective, while compound 32a displayed relatively weaker antithrombotic
potential throughout and remained effective only upto 12 hours, when compared against
compound 31a. However, the standard anti-platelet drugs Aspirin (170µM/kg, 40% protection)
and Clopidogrel (70 µM/kg, 60% protection) were effective only upto 5 hours after which their
effect was perished and that too at a very high dose sufficient enough to cause bleeding
complications (Fig. 4).
Fig. 4. Time dependent effect of compounds (28i, 31a and 32a) and standard antithrombotic
drugs (Aspirin and Clopidogrel) after single dose administration (30µM/kg, p.o.) on collagen-
epinephrine induced pulmonary thromboembolism in mice. Results are expressed as Mean±SEM
(n=5, 10 animals/group/ experiment).
2
.2.2. Effect on Bleeding Time in mice
Tail bleeding time was significantly prolonged in standard anti-platelet drugs, Aspirin (2.2
fold) and Clopidogrel (2.3 fold) treated mice as compared to control. The diastereomers 31a
and 32a as well as their mixture 28i (30ꢀM/kg) although exhibited a mild prolongation in
bleeding time (~1.5 fold) but it was considerably less in comparison to both the standard anti-
platelet drugs, Aspirin and Clopidogrel (Fig. 5).

ACCEPTED MANUSCRIPT
Fig. 5. Time dependent effect of CDRI candidate compounds (28i, 31a, 32a) and standard
antithrombotic drugs (Aspirin and Clopidogrel) after single dose administration (30µM/kg, p.o.)
on tail bleeding time in mice. Results are expressed as Mean±SEM (n=5, 10
animals/group/experiment).
2
.2.3. Aggregation in human platelet rich plasma (in vitro)
Various platelet agonists were used in order to identify their anti-platelet action and also the
selectivity of their pharmacological target over platelet surface. The compounds 31a (IC = 6.6µM)
5
0
and 32a (IC50=37µM) as well as their racemic mixture 28i (IC50=16µM) significantly inhibited
collagen induced human platelet aggregation but had no effect on ADP, TRAP and Arachidonic
acid induced platelet aggregation. The involvement of platelet GP 1b-IX-V was also ruled out as the
compound 31a did not show any inhibitory effect on Ristocetin induced platelet aggregation. The
aggregation observed with type 1 fibrillar collagen is primarily mediated by the functional GPVI
receptor on the surface of platelets. Therefore, specific agonists, Convulxin (snake venom protein)
and CRP-XL (cross linked collagen related peptide) were used to examine the interaction between
compound and GPVI, which activate platelets selectively via GPVI. The compound 31a had no
effect on Convulxin induced platelet aggregation but at higher concentrations, it attenuated the
CRP-XL induced platelet aggregation at concentrations up to 500µM. This suggests that the
compound 31a inhibits collagen induced platelet aggregation with a better affinity over CRP-XL
and convulxin (Fig. 6).

ACCEPTED MANUSCRIPT
Fig. 6. (a) Effect of Aspirin, compound 28i, 31a and 32a on aggregation of human platelet rich
plasma (in vitro) induced by Collagen, (b) effect of compound 31a on aggregation of human
platelet rich plasma (in vitro) induced by ADP, TRAP and Arachidonic acid, (c) effect of
compound 31a on aggregation of human platelet rich plasma (in vitro) induced by ristocetin,
collagen, Convulxin and CRP-XL (n=3) Data shown as Mean + SEM. ***p<0.001,**p<0.01 vs
control.

ACCEPTED MANUSCRIPT
2
.3 Oral pharmacokinetic study in NZ rabbits
The oral pharmacokinetic study of 31a was carried out in healthy male New Zealand (NZ)
rabbits (~3kg) at 20 mg/kg dose. The animals were obtained from Laboratory Animal Services
Division of CDRI, and housed in cages in standard laboratory conditions with a regular 12 h day-
night cycle. The prescribed diet was given and water ad libitum. The animals were acclimatized
for two days before carrying out the pharmacokinetic study. The 31a suspension formulation was
prepared using 0.5 % w/v carboxymethyl cellulose in a mortar and pestle. Blood samples from
marginal ear vein were collected at 0.25 to 48 hr post oral dosing. Plasma was separated and
stored at –80°C until analysis by HPLC-UV method. The pharmacokinetic parameters Cmax , tmax
clearance (CL/F), volume of distribution (V /F), MRT and area under curve (AUC) for all the
d
analytes were determined using non-compartmental analysis (Fig. 7, Table 6).
The pharmacokinetic data of the compound 28i indicate very fast absorption leading to Cmax of
9
47.02 ± 237.4 ng/ml which appears to be a very high concentration therefore on repeated dosing
for prolonged durations may lead to plasma and peripheral accumulation and toxicity. On the
other hand compound 31a, owing to its higher solubility (416.41 ± 62.35 µg/ml) than compound
3
2a (71.75 ± 13.45 µg/ml) exhibits comparatively faster absorption (Cmax 149.49 ± 53.12 at tmax
0
.75 ± 0.144 hrs) and prolonged and constant systemic exposure and more likely maintaining the
desired therapeutic levels, for more than 24 hours than compound 32a (tmax 8.67 ± 0.66 hrs)
thereby exhibiting better therapeutic response.
Fig. 7. Plasma concentration time profiles of compounds 28i, 31a and 32a

ACCEPTED MANUSCRIPT
Table 6: Estimates of pharmacokinetic parameters of compounds 28i, 31a and 32a in male NZ
rabbits at a dose of 20 mg/kg body weight.
PK Parameters Compound 28i
Compound 31a
149.49 ± 53.12
0.75 ± 0.144
Compound 32a
112.40 ± 10.25
8.67 ± 0.66
Cmax (ng/ml)
Tmax (h)
947.02 ± 237.4
0.68 ± 0.29
AUC (ng.h/ml) 6524.15 ± 1502.7
MRT (h) 16.20 ± 1.46
874.8 ± 232.01
5.25 ± 1.75
1112.81 ± 169.77
16.20 ± 1.46
3
. Conclusion
In the present study, a novel class of various amino acids derived, constrained lactam
carboxamides having chirally pure cyclic diamines were synthesized. The entire series of
compounds were subjected to a preliminary screening for their antithrombotic activity in mice
model of collagen epinephrine induced pulmonary thromboembolism. This is a simple
preclinical test model based upon the synergistic effect of collagen and epinephrine to induce
thrombosis, and is of great value for studying antithrombotic agents which work primarily by
inhibiting pulmonary thromboembolism through their effect on platelet aggregation. The
compound 28i and its diastereomers 31a and 32a exhibited remarkable antithrombotic efficacy in
mice model of collagen epinephrine induced pulmonary thromboembolism, which was better
than standard drug Aspirin and almost comparable to Clopidogrel. After one hour of oral
administration, 28i and one of its diastereomer 31a exhibited almost identical efficacy, while 32a
showed relatively less antithrombotic potential. However, the compound 31a offered significant
protection for a longer duration, i.e., upto 24 hours, while the activity of 28i gradually declined
after 18 hours. This observation was also in complete agreement with the pharmacokinetic
properties of these compounds, which indicates that unlike 28i and 32a, the compound 31a not
only exhibits faster absorption but also maintains a prolonged and constant systemic exposure
thereby maintaining the desired therapeutic levels for more than 24 hours. Moreover, its anti-
platelet action of these compounds was specific as it did not inhibit other platelet activation
pathways as evidenced by its ineffectiveness to alter ADP, arachidonic acid or thombin mimetic
TRAP induced platelet aggregation. Further, compound 34c displayed promising anti-platelet
potential via dual mechanism of action against both collagen (IC =3.3µM) and U46619
5
0
(
IC50=2.7µM) induced platelet aggregation.

ACCEPTED MANUSCRIPT
The existing anti-thrombotic therapy suffers from a major drawback of bleeding risk that further
leads to haemorrhage. Thus the objective of the ideal anti-thrombotic therapy is to prevent
thrombosis but to leave haemostasis sufficiently intact to prevent bleeding complications. The
three active compounds were therefore compared to the standard drugs aspirin and clopidogrel
regarding bleeding complications in mice. Both aspirin and clopidogrel at their respective
efficacy dose, showed a >2 fold prolongation in bleeding time in mice, while all the three test
compounds exhibited a relatively milder increase (1.3 fold) in bleeding. Therefore, these
compounds satisfactorily preserve the haemostasis with minor alterations in the pre-clinical mice
model and hence they might promise some improvisation with respect to the critical
disadvantage of bleeding risk endured by the current anti-platelet strategies.
4
4
4
. Experimental
.1. Chemistry
.1.1. General
Reagents and other chemicals were used as purchased without further purification. All reactions
with moisture/air sensitive reactants 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 N over potassium benzophenoneketyl radical prior to use. Dichloromethane was dried
2
over P
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 KMnO or
2 5
O and methanol using magnesium cake. All the compounds were vacuum dried in
4
Dragendorff's reagent spray as the developing agents. Chromatographic separations were
performed on flash column using silica gel (60-120 and 230-400 mesh). All melting points were
taken in open tubes and are uncorrected. IR spectra were recorded on a Perkin-Elmer 881 and
-1 1
FTIR 8201 PC Shimadzu spectrophotometers and values are expressed in cm . HNMR and
1
3
CNMR spectra were recorded on Bruker WM-200, WM-300 or WM-400 Spectrometer. The
chemical shifts are expressed in δ using TMS as internal standard. Mass spectra were recorded
on a JEOL JMS-D-3000 spectrometer with an ionization potential of ~70 eV and and FAB on
SX 102 instrument. Purity and diastereomeric ratios were determined by HPLC using Chiralpak-
1
A (Diacel) column with gradient elution (acetonitrile/2-propanol).

ACCEPTED MANUSCRIPT
4
4
.1.2. Synthesis of (2S)-N-arylalkyl acids.
.1.2.1. (2S)-N-(4-methylbenzyl) pyroglutamic acid, (16a)
A solution of methyl pyroglutamate, 10 (2.0 gm, 1 eq, 13.9 mmol) and THF (100 ml) was taken
0
in a three necked RBF fitted with rubber septa, N
2
inlet and cooled to -20 C. LiHMDS (14 ml,
1
.2 eq, 16.7 mmol) was added through a syringe to that solution and allowed to stir for 1h.
o
Substituted benzylbromide (1.1 eq) was added and stirring was continued for 4h. from 0 C to
room temperature. The reaction was quenched by addition of 1N HCl (10 ml) and extracted with
ethyl acetate (3 x 25 ml). The organic layer was washed with brine (2 x 25 ml), dried over
Na
2
SO
4
and concentrated under reduced pressure to give an oily ester. The ester was then re-
0
dissolved in methanol (10 ml) and cooled to 0 C. 20% sodium carbonate solution was then added
to the reaction mixture portion wise. The reaction mixture was then stirred at room temperature
for 5 hours. Methanol was then distilled off and the reduced reaction mixture was then extracted
with ether (1 x 25 ml). The mixture was acidified with conc. HCl and extracted with ethyl acetate
2
7 0
C
3 x 30 ml). The organic layer was dried and concentrated. Yield: 90%; [α] : - 3.97 (c = 0.24 ;
D
(
-1 1
Methanol); IR (KBr): 3408.3, 2366.5, 1647.2, 1456.2, 1220.3 cm ; H NMR (CDCl , 200 MHz):
3
δ 2.05-2.18 (m, 1H, 3-H ); 2.20-2.27 (m, 1H, 3-H ); 2.50- 2.60 (m, 2H, 4-H); 3.93-3.98 (d, 1H, -
a
b
1
3
NCHPh); 4.02-4.04 (m, 1H, 2-H); 5.09-5.17 (d, 1H, -NCHPh); 7.12 (s, 5H, Ph-H); C NMR (50
MHz, CDCl ): 21.52, 23.26, 30.10, 45.83, 59.01, 61.00, 128.95, 129.92, 132.62, 138.11, 174.74,
3
+
1
2
76.90; FAB MS (m/z): 234 (M+H) ; HRMS: (m/z) calc. for C H NO : 234.1052. Found:
1
3
15
3
34.1085
.1.2.2 (S)-3-(4-methylbenzyl)-2-oxooxazolidine-4-carboxylic acid, [17a]: Prepared from 11a.
4
Yield: 58 %; IR (KBr): 3469, 3019, 2926, 2601, 1912, 1759, 1645, 1515, 1420, 1226, 1086, 1049
-1 1
3 3
cm ; HNMR (CDCl , 300 MHz): δ 2.35 (s, 3H, CH ); 4.09-4.12 (t, 1H, 5H); 4.18-4.23 (d, 1H,
NCHPh); 4.39-4.42 (m, 2H, 5H); 4.92-4.97 (d, 1H, NCHPh); 7.17 (s, 4H, Ph-H); FABMS: m/z :
+
2
36 [M+1] ; HRMS: (m/z) calc. for C12
H13NO
4
: 236.0845. Found: 235.0851
4
.1.2.3. (R)-3-(4-methylbenzyl)-2-oxothiazolidine-4-carboxylic acid, [18a]: Prepared from 12a.
Yield: 70 %; IR (KBr): 3015, 2930, 2504, 2504, 1925, 1731, 1666, 1513, 1440, 1387, 1297,
-1 1
1
231, 1160, 1018 cm ; HNMR (CDCl
3 3
, 300 MHz): δ 2.34 (S, 3H, -CH ); 3.43-3.50 (m, 2H, 5-
H); 4.00-4.05 (d, 1H, NCHPh); 4.13-4.17(dd, 1H, 4H); 5.15-5.20 (d, 1H, NCHPh); 7.15 (s, 4H,
1
3
Ph-H); CNMR (CDCl , 75 MHz): 21.09, 29.14, 31.80, 36.92, 47.43, 59.11, 128.40, 129.51,
3

ACCEPTED MANUSCRIPT
+
1
32.74, 137.71, 163.44, 171.94; FABMS: m/z : 252 [M+1] ; HRMS: (m/z) calc. for
C H NO S: 252.3015. Found: 252.3045
12
13
3
4
.1.2.4. (R)-3-(4-methylbenzoyl)thiazolidine-4-carboxylic acid, (19): Step-1: (R)-methyl
thiazolidine-4-carboxylate, 13 (0.50 g, 1 eq, 3.40 mmol) was dissolved in dry DCM (10 ml) and
0
cooled to 0 C in an ice bath. TEA (1.1 ml, 2.3 eq, 7.82 mmol) was added drop wise to the
reaction mixture and further stirred for 15 min. 4-methylbenzoyl chloride (0.60 g, 1.1 eq, 3.74
mmol) dissolved in dry DCM (10 ml) was added to the reaction mixture maintaining the
0
temperature at 0 C. The stirring was continued for 3 hrs at room temperature. The reaction
mixture was then washed with dilute citric acid 1 x 25 ml), saturated aq.NaHCO
3
solution (1 x 25
ml) and then with brine. The organic layer was separated, dried over Na SO , filtered and
2
4
concentrated. Step-2: The sticky oil obtained from the above step was dissolved in minimum
0
quantity of methanol (5 ml) and cooled to 0 C. 20 % aq.Na
2
CO
3
solution (25 ml) was then added
portion wise and the resulting solution was stirred for 4hrs at room temperature. Methanol
present in the reaction mixture was evaporated and aqueous solution was then acidified with
con.HCl. The turbid solution thus obtained was extracted with ethyl acetate (3 x 25 ml). The
organic layer was separated, dried over Na SO , filtered and concentrated. 2.1.2.4. (R)-3-(4-
2
4
methylbenzyl)thiazolidine-4-carboxylic acid, (19a): Prepared from 13: Yield: 82 %; IR (KBr):
-
1 1
3
013, 2928, 2854, 1908, 1720, 1621, 1514, 1448, 1379, 1218, 1137, 1049, 949 cm ; HNMR
CDCl , 300 MHz): δ 2.37 (s, 3H, CH ); 3.26-3.29 (m, H, 5-H ); 3.46-3.50 (m, H, 5-H ); 3.73-
.77 (d, 1H, 2-H ); 3.84-3.88 (d, 1H, NCH Ph); 4.02-4.03 (t, 1H, 4H); 4.05 (d, 1H, 2-H ); 4.11-
(
3
3
a
b
3
4
2
a
a
b
.14 (d, 1H, NCH Ph); 7.21-7.28 (m, 4H, Ph-H); ; HRMS: (m/z) calc. for C H NO S:
b
12 13
3
52.3015. Found: 252.3032
4
.1.2.5. (S)-1-(4-methylbenzyl)-6-oxopiperidine-2-carboxylic acid, [20]: Prepared from 14.
2
7 0
C
0
[α]
D
Yield: 90.3 %; MP: 140-142 C;
925, 2534, 1952, 1710, 1594, 1499, 1445, 1411, 1333, 1267, 1181, 1004 cm ; H NMR
CDCl ; 300MHz): δ 1.85 (m, 2H, 4-H); 1.90 (m, 1H, 3-H ); 2.12 (m, 1H, 3-H ); 2.34 (s, 3H, Ph-
: +40.80 (c = 0.10; Chloroform); IR (KBr): 3449, 2964,
-1 1
2
(
3
a
b
CH
3
); 2.51-2.69 (m, 2H, 3-H); 3.63-3.68 (d, 1H, Ph-CH); 4.06 (t, 1H, 3-H); 5.59-5.64 (d, 1H, Ph-
+
CH); 7.13 (s, 4H, Ph-H); FAB-MS: (m/z): 248, [M+H] ; HRMS: (m/z) calc. for C H NO :
1
4
17
3
2
47.1208. Found: 234.1025

ACCEPTED MANUSCRIPT
4
.1.2.6. (3S)-4-benzyl-2-methyl-5-oxomorpholine-3-carboxylic acid, [21a]
N-benzyl serine, 15 (5g) was stirred with 35 ml aq. NaOH (2.5M) at -10°C for 10-15 min. Chloro
o
acetyl chloride (4.5 ml) was added drop wise, after 10-15 min stirring at 0 C the pH was adjusted
o
to 8.5 using NaOH (7.5M) aq. solution. The reaction was then carried out at 30 C for 2-3 hrs and
o
then cooled to 0 C and added HCl (conc.) for precipitation. The precipitate was filtered and
washed with hexane and ether and dried in Abdel-Halden under vacuum. (same procedure was
o
followed for the preparation of 21b, starting from N-benzyl threonine) Yield: 60%; MP: 127 C;
-1 1
IR (KBr): 3427, 3020, 2360, 1719, 1638, 1515, 1439, 1216, 1049, 928, 761, 670 cm ; HNMR
(
CDCl
3
, 300 MHz): δ 1.24 (d, 3H, CH
3
), 3.78-3.90 (m, 2H, H-2), 4.20-4.26 (m, 2H, H-6), 4.80
+
(
m, 1H, H-3), 5.61-5.65 (d, 2H, NCH Ar), 7.27-7.35 (m, 5H, Ph); FABMS m/z : 250 [M+1] ;
2
HRMS: (m/z) calc. for C13
4
H15NO : 250.1001. Found: 234.1038
4
.1.2.7. (S)-tert butoxy carbonyl methyl cysteine ethyl ester, [24]: L-cysteine ethyl ester
o
hydrochloride (11 gm, 0.059 mol) was taken in dry DMF (30 ml) and at -10 C DIPEA (26 ml,
0
.148 mol) was added; when half of the DIPEA added the reaction mixture turned into yellow
o
slurry and after the complete addition BrCH COOtBu (10 ml, 0.065 mol) was added at 0 C
2
slowly through a dropping funnel. Reaction mixture turned gradually within 3-4 hr as white
slurry. Reaction mixture was poured into a large excess of (250 ml) of chilled ice water with
stirring and the aqueous layer then extracted with ethyl acetate (3 x 150 mL). All the organic
layers were separately washed with brine water and then combined and dried over activated
Na SO . Concentrated at reduced pressure gave a crude oil which was purified using flash
2
4
chromatography with 28% ethyl acetate in hexane as an eluant. Yield: 70 %; IR (neat): 3432,
-1 1
3
3
330, 1976, 1735, 1623, 1498, 1408, 1289, 1206, 1153, 909 cm ; HNMR (CDCl , 300 MHz): δ
1
.25-1.32 (t, 3H, CH CH ), 1.45 (s, 9H, t-Butyl-H), 2.91-2.94 (d, 2H, α-CHCH S), 3.17 (s, 2H,
2
3
2
SCH
2
COOtBu), 3.6 (m, 1H, α-CHCH
2
S), 4.2 (q, 2H, COOCH
2
CH
3
), 2.1 (br, 2H, NH); FABMS
+
+
(
m/z): 264 [M+1] , 208 [(M+1)-57]
2
.1.2.8. N-p-Methyl benzyl (S)-tert butoxy carbonyl methyl cysteine ethyl ester, [25]: Compound
(
3 gm, 0.011 mol) was taken in dry methanol and under N environment added baked NaOAc (2
2
o
gm, 0.025 mol) followed by tolualdehyde (1.4 ml, 0.013 mol) and the slurry was stirred at 0 C
for 10 min after which NaCNBH (1.6 gm, 0.025 mol) was gradually added in portions and the
3
reaction mixture was allowed to stir over night. Reaction mixture was quenched with dil. citric

ACCEPTED MANUSCRIPT
acid and the reaction contents were concentrated prior to washing in a biphasic system of water-
ethyl acetate. Organic layer was again washed with brine and dried over activated sodium sulfate
and flash chromatographed using 12% ethyl acetate in hexane as an eluent gave the pure oily
-
product. Yield: 40%; IR (neat): 3432, 3330, 1976, 1735, 1623, 1498, 1408, 1289, 1206, 1180 cm
1
1
;
HNMR (CDCl , 300 MHz): δ 1.25-1.32 (t, 3H, COOCH CH ), 1.48 (s, 9H, COOtBu), 2.34
3
2
3
(
s, 3H, CH
3
), 2.91-2.94 (d, 2H, α-CHCH
2
S), 3.19 (s, 2H, SCH
2
COOtBu), 3.43 (m, 2H,
NHCH Ar), 3.82 (m, 1H, α-CHCH S), 4.22 (q, 2H, COOCH CH ), 7.11-7.24 (dd, 4H, Ar), 2.00
2
2
2
3
+
(br, 1H, NH).; FABMS m/z: 368 [M+1]
4
.1.2.9. N-p-methyl benzyl thiomorpholinone carboxylic acid, (26): Compound (0.4 gm, 1.1
o
mmol) was dissolved in dry DCM (12 ml), and treated with 40% TFA in DCM (5 ml) at 40 C
with stirring for about 3-4 h until the TLC indicated the complete removal of t-butyl ester group.
Reaction mixture was then concentrated at ambient temperature under vacuum to remove the
solvent and traces of unreacted acid. The crude (without purification) was again dissolved in dry
DCM and added Et N (0.27 ml, 1.94 mmol), followed by HOBt (0.17 gm, 1.34 mmol)) and then
3
through syringe DIC (0.24 ml, 1.52 mmol) was added under nitrogen atmosphere. Reaction was
stirred over night and quenched with saturated ammonium chloride solution. The compound was
o
taken in a THF: Methanol mixture and cooled to 0 C. A methanolic solution of K
2
CO
3
(15%) and
few pellets of KOH at 0°C was added to the above solution and made it to stir at the same
temperature for about 5-6 hrs. The reaction mixture was then concentrated and washed twice
with ether. The aqueous layer was made acidic and then extracted with ethyl acetate. The
combined organic layers were washed with brine and dried over sodium sulfate and concentrated
0
under reduced pressure. Yield: 50%; MP: 128-130 C; IR (KBr): 3432, 3330, 1976, 1750, 1623,
-1 1
1
498, 1408, 1289, 1190, 1180 cm ; HNMR (CDCl ): δ 2.35 (s, 3H, CH ), 3.10-3.20 (m, 2H, α-
3 3
CHCH S), 3.53-3.61 (m, 1H, α-CHCH S), 3.42 (d, 2H, SCH CO), 4.33 (s, 2H, NCH Ar), 7.11-
2
2
2
2
7
4
4
.24 (dd, 4H, Ar); HRMS: (m/z) calc. for C H NO S: 266.0773. Found: 234.0745
13 15 3
.1.3. Synthesis of chiral carboxamide derivatives
.1.3.1. tert-butyl ((S)-1-((S)-1-(4-methylbenzyl)-5-oxopyrrolidine-2-carbonyl) piperidin-3-
yl)methylcarbamate (31a): Step-1: (2S)-N-(4-methylbenzyl)-pyroglutamic acid (3.50 g, 1 eq, 15
0
mmol) was dissolved in dry DCM (50 ml) and cooled to 0 C. Oxalyl chloride (1.3 ml, 1.2 eq, 18
0
mmol) was then added drop wise at 0 C and the mixture was allowed to warm up to room

ACCEPTED MANUSCRIPT
temperature and stirred for 12 hours. The reaction mixture was then concentrated over reduced
pressure. Step-2: (3R)-N-Boc-3-aminomethyl-piperidine (3.53 g, 1.1 eq, 16 mmol) was dissolved
0
in dry DCM (50 ml) and cooled to 0 C. TEA (4.82 ml, 2.3 eq, 35 mmol) was then added to the
reaction mixture and stirred for 10 min. The acid chloride (from step-1) dissolved in dry DCM
0
(
25 ml) was then added drop wise at 0 C and stirred at room temperature for 3 hrs. The reaction
mixture was then washed with saturated sodium bicarbonate (1 x 50 ml), ice cold 1N HCl (1 x 50
ml) and then with brine. The organic layer was separated, dried and concentrated and flash
27 C
0
[α]
0
chromatographed. Yield: 75 %;
D
: + 35.00 (c = 0.098; Chloroform); MP: 138-140 C; IR
(
KBr): 3274, 2977, 2934, 2860, 1714, 1674, 1630, 1541, 1449, 1365, 1272, 12489, 1176, 1144,
-1 1
1
3
039, 997, 958 cm ; H NMR (CDCl ; 600MHz): δ 1.119-1.390 (m, 2H, 4’-H); 1.448 (s, 9H, -O-
C(CH ) ); 1.704 (m, 1H, 3’-H); 1.725-1.887 (m, 2H, 5’-H); 2.138-2.175 (m, 1H, 3-H ); 2.305 (s,
3
3
a
3
H, Ph-CH3), 2.361-2.398 (m, 1H, 3-H
b
2
); 2.496-2.555 (m, 2H, 4-H); 2.646-2.757 (m, 2H, -CH -
NH-Boc); 2.833-3.090 (m, 2H, 6’-H); 3.324-3.428 (m, 1H, 2’-H ) 3.731-3.769 (m, 1H, 2’-H );
a
b
4
7
2
5
1
.110-4.200 (m, 2H, -CH -Ph & 2-H); 4.871 (s, 1H. NH); 5.133-5.138 (d, 1H, -CH -Ph); 7.060-
2
2
1
3
.090 (dd, 4H, Ph-H); C NMR (CDCl
3
; 100 MHz): 21.063, 21.086, 22.651, 24.614, 27.898,
8.287, 28.639, 29.748, 35.955, 38.330, 42.728, 43.118, 43.332, 45.065, 45.646, 49.273, 55.673,
5.841, 79.331, 128.427, 128, 733, 129.351, 133.078, 137.339, 156.028, 168.810, 169.253,
+
+
75.164; FAB MS (m/z): 430 (M+1) , 330, 374, 452 (M+Na) ; HRMS: (m/z calcd. for
C H N O , 430.2628; Found, 430.2661.
24
35
3
4
4
.1.3.2. tert-butyl ((R)-1-((S)-1-(4-methylbenzyl)-5-oxopyrrolidine-2-carbonyl) piperidin-3-
27 C
0
[α]
yl)methylcarbamate (32a): Same as 31a. Yield: 85 %;
D
: +5.69 (c = 0.99; Chloroform); MP:
0
1
1
9
2
2
1
80-182 C; IR (KBr): 3330, 2974, 2935, 2857, 1710, 1677, 1642, 1528, 1444, 1363, 1274, 1173,
-
1 1
146, 1080, 997, 958 cm ; H NMR (CDCl ; 600MHz): δ 1.177-1.270 (m, 2H, 4’-H); 1.424 (s,
3
H, -O-C(CH ) ); 1.668 (m, 1H, 3’-H); 1.768-1.856 (m, 2H, 5’-H); 2.138-2.175 (m, 1H, 3-H );
3
3
a
.305 (s, 3H, Ph-CH ), 2.361-2.398 (m, 1H, 3-H ); 2.496-2.555 (m, 2H, 4-H); 2.646-2.757 (m,
3
b
H, -CH -NH-Boc); 2.833-2.984 (m, 2H, 6’-H); 3.324-3.428 (dd, 1H, 2’-H ); 3.731-3.769 (m,
2
a
H, 2’-H
b
); 4.160-4.180 (m, 2H, -CH
2
2
-Ph & 2-H); 4.871 (s, 1H. NH); 5.133-5.138 (m, 1H, -CH -
1
3
Ph); 7.060-7.090 (dd, 4H, Ph-H); C NMR (CDCl ; 100 MHz): 21.086, 22.728, 23.866, 24.614,
3
2
5
8.096, 28.303, 28.700, 29.784, 36.405, 37.322, 42.828, 43.011, 43.263, 45.042, 45.852, 48.441,
5.986, 56.047, 79.316, 128.389, 128.710, 129.336, 133.048, 137.339, 156.080, 168.810,

ACCEPTED MANUSCRIPT
+
+
1
69.001, 175.072; FABMS (m/z): 429[M ],330, 452 (M+Na) ; HRMS: (m/z) calcd. for
C H N O ) 430.2628; Found, 430.2684.
24
35
3
4
4
.1.3.3. tert-butyl ((S)-1-((S)-1-(4-methoxybenzyl)-5-oxopyrrolidine-2-carbonyl) piperidin-3-
27 C
0
[α]
yl)methylcarbamate [31b]: Same as 31a. Yield: 84 %;
D
: + 32.50 (c = 0.24; Chloroform); IR
-
1 1
(
(
KBr): 3349, 3008, 2934, 2361, 1684, 1513, 1455, 1365, 1250, 1174, 1035, 963 cm ; H NMR
CDCl
3
; 300MHz): δ 1.16-1.29 (m, 2H, 4’-H); 1.43 (s, 9H, -O-C(CH
3
)
3
); 1.63 (m, 1H, 3’-H);
1
.76-1.87 (m, 2H, 5’-H); 2.15-2.18 (m, 1H, 3-H ); 2.36-2.39 (m, 1H, 3-H ); 2.52-2.57 (m, 2H, 4-
a
b
H); 2.61-2.65 (m, 2H, -CH
2
-NH-Boc); 2.88-3.00 (m, 2H, 6’-H); 3.34-3.43 (m, 1H, 2’-H
a
); 3.73-
3
5
7
1
.86 (m, 1H, 2’-H ); 3.78 (s, 3H, -OCH3); 4.15-4.22 (m, 2H, 2-H & -CH -Ph); 4.87 (s, 1H. NH);
b
2
1
3
.10-5.13 (d, 1H, -CH -Ph); 6.84-6.89 (d, 2H, Ph-H); 7.06-7.09 (d, 2H, Ph-H); C NMR (CDCl ;
2
3
5 MHz): 21.06, 22.65, 24.61, 27.89, 28.28, 29.74, 43.33, 55.67, 55.84, 79.33, 112.81, 126.94,
+
26.53, 128.86, 155.02, 168.78, 175.14; FAB MS (m/z): 446 (M+H) ; HRMS: (m/z) calc. for
C
13
H
15NO
4
: 446.1252. Found: 446.1285
4
.1.3.4. tert-butyl (R-1-((S)-1-(4-methoxybenzyl)-5-oxopyrrolidine-2 carbonyl) piperidine-3-
27 C
0
[α]
yl)methylcarbamate [32b]: Same as 31a. Yield: 75 %;
D
: - 15.00 (c = 0.10; Chloroform); IR
-1 1
(
KBr): 3328, 2975, 2937, 2362, 1685, 1652, 1516, 1451, 1366, 1251, 1175, 1026, 961 cm ; H
NMR (CDCl ; 300MHz): δ 1.25 (m, 2H, 4’-H); 1.46 (s, 9H, -O-C(CH ) ); 1.60 (m, 1H, 3’-H);
3
3 3
1
.82-1.92 (m, 2H, 5’-H); 2.19-2.23 (m, 1H, 3-H
a
); 2.40-2.47 (m, 1H, 3-H
b
); 2.52-2.58 (m, 2H, 4-
H); 2.64-2.79 (m, 2H, -CH -NH-Boc); 2.91-3.01 (m, 2H, 6’-H); 3.36-3.45 (m, 1H, 2’-H ); 3.74-
2
a
3
5
2
1
.83 (m, 1H, 2’-H
b
); 3.81 (s, 3H, -OCH
3
); 4.15-4.27 (m, 2H, 2-H & -CH
2
-Ph); 4.83 (s, 1H. NH);
1
3
.12-5.17 (m, 1H, -CH -Ph); 6.84-7.15 (m, 4H, Ph-H); C NMR (CDCl ; 75 MHz): 22.78, 23,98,
2
3
4.61, 28.13, 29.73, 42.87, 45.31, 48.58, 55.27, 56.11, 113.07, 114.42, 120.61, 129.72, 137.68,
+
56.10, 159.92, 169.01, 175.24; ESI MS (m/z): 446 (M+H) , 345 (M-Boc), 390 (M-t_butyl), 486
+
(
M+Na) ; HRMS: (m/z) calc. for C H NO : 446.1252. Found: 446.1258
1
3
15
4
4
.1.3.5. tert-butyl ((S)-1-((S)-1-(3-methoxybenzyl)-5-oxopyrrolidine-2-carbonyl) piperidin-3-
27 C
0
[α]
yl)methylcarbamate [31c]: Same as 31a. Yield: 82 %;
D
: + 47.37 (c = 0.19; Chloroform); IR
-1 1
(
KBr): 3451, 3010, 2937, 2361, 1684, 1647, 1512, 1456, 1365, 1256, 1218, 1169, 1044 cm ; H
NMR (CDCl ; 300MHz): δ 1.16-1.38 (m, 2H, 4’-H); 1.45 (s, 9H, -O-C(CH ); 1.62 (m, 1H, 3’-
H); 1.76-1.89 (m, 2H, 5’-H); 2.17-2.24 (m, 1H, 3-H ); 2.37-2.47 (m, 1H, 3-H ); 2.52-2.66 (m, 2H,
3
3 3
)
a
b
4
-H); 2.80-3.02 (m, 2H, -CH -NH-Boc); 2.88-3.04 (m, 2H, 6’-H); 3.34-3.49 (m, 1H, 2’-H ); 3.78-
2 a

ACCEPTED MANUSCRIPT
3
5
.81 (m, 1H, 2’-H
b
2
); 3.80 (S, 3H, -OCH3); 4.08-4.25 (m, 2H, 2-H & -CH -Ph); 4.89 (s, 1H. NH);
1
3
.13-5.18 (m, 1H, -CH -Ph); 6.74-6.84 (m, 3H, Ph-H); 7.20-7.28(m, 1H, Ph-H); C NMR
2
(
CDCl ; 75 MHz): 21.42, 21,81, 27.01, 27.11, 44.10, 44.40, 53.98, 54.36, 112.69, 113.42, 119.42,
3
+
1
28.43, 128.54, 136.52, 158.68, 168.00, 173.95; ESI MS (m/z): 446 (M+H) , 345 (M-Boc), 390
+
(
M-t_butyl), 486 (M+Na) ; HRMS: (m/z) calc. for C H NO : 446.1252. Found: 446.1261
1
3
15
4
4
.1.3.6. tert-butyl((R)-1-((S)-1-(3-methoxybenzyl)-5-oxopyrrolidine-2-carbonyl) piperidin-3-
27 C
0
[α]
yl)methylcarbamate [32c]: Same as 31a. Yield: 78 %;
D
: -12.00 (c = 0.15; Chloroform); IR
(
KBr): 3330, 2974, 2935, 2857, 1710, 1677, 1642, 1528, 1444, 1363, 1274, 1173, 1146, 1080,
-1 1
9
97, 958 cm ; H NMR (CDCl ; 300MHz): δ 1.24 (m, 2H, 4’-H); 1.43 (s, 9H, -O-C(CH ) ); 1.67
3 3 3
(m, 1H, 3’-H); 1.77-1.86 (m, 2H, 5’-H); 2.15-2.19 (m, 1H, 3-H ); 2.40 (m, 1H, 3-H ); 2.49-2.57
a b
2
(m, 2H, 4-H); 2.60-2.79 (m, 2H, -CH -NH-Boc); 2.82-3.09 (m, 2H, 6’-H); 3.34-3.48 (dd, 1H, 2’-
H ); 3.73 (m, 1H, 2’-H ); 3.76(s, 3H, -OCH ) 4.14-4.23 (m, 2H, 2-H & -CH -Ph); 4.90 (s, 1H.
a
b
3
2
1
3
NH); 5.10-5.15 (m, 1H, -CH
2
-Ph); 6.72-6.81 (m, 3H, Ph-H); 7.18-7.24 (m, 1H, Ph-H); C NMR
(
CDCl ; 75 MHz): 22.75, 24.61, 28.38, 29.64, 36.60, 43.08, 45.39, 55.23, 56.11, 79.37, 113.06,
3
+
1
14.41, 120.58, 129.72, 137.60, 159.89, 168.98, 175.29; ESI MS (m/z): 446 (M+H) , 345 (M-
+
Boc), 390 (M-t_butyl), 486 (M+Na) ; HRMS: (m/z) calc. for C13
H15NO
4
: 446.1252. Found:
4
46.1221
4
.1.3.7. tert-butyl ((S)-1-((S)-1-(2-methoxybenzyl)-5-oxopyrrolidine-2-carbonyl) piperidin-3-
27 C
0
[α]
yl)methylcarbamate [31d]: Same as 31a. Yield: 80 %;
D
: +8.15 (c = 0.26; Chloroform); IR
-1 1
(
KBr): 3454, 3342, 3008, 1235, 2361, 1681, 1498, 1461, 1249, 1171, 1030 cm ; H NMR
CDCl ; 300 MHz): δ 1.26-1.33 (m, 2H, 4’-H); 1.43 (s, 9H, -O-C(CH ); 1.60-1.66 (m, 1H, 3’-
(
3
3 3
)
H); 1.71-1.91 (m, 2H, 5’-H); 2.16-2.175 (m, 1H, 3-H ); 2.40-2.42 (m, 1H, 3-H ); 2.50-2.60 (m,
a
b
2
3
H, 4-H); 2.84-2.90 (m, 2H, -CH -NH-Boc); 2.95-3.00 (m, 2H, 6’-H); 3.32-3.58 (m, 1H, 2’-H );
2
a
.74-3.79 (m, 1H, 2’-H
b
); 3.80 (s, 3H, -OCH
3
); 4.02-4.07 (d, 1H, -CH
2
-Ph); 4.24-4.31 (m, 1H, 2-
H); 4.91 (s, 1H. NH); 4.96-5.04 (d, 1H, -CH -Ph); 6.85-6.94 (m, 3H, Ph-H); 7.19-7.28 (m, 1H,
2
1
3
Ph-H); C NMR (CDCl
3
; 75 MHz): 21.42, 21.81, 27.01, 27.11, 44.10, 44.40, 53.98, 54.36,
1
09.14, 113.42, 119.46, 127.78, 129.36, 136.52, 158.68, 168.00, 173.95; FAB MS (m/z): ESI MS
+
+
(
m/z): 446 (M+H) , 345 (M-Boc), 390 (M-t_butyl), 486 (M+Na) ; HRMS: (m/z) calc. for
13 4
C H15NO
: 446.1252. Found: 446.1233

ACCEPTED MANUSCRIPT
4
.1.3.8. tert-butyl((R)-1-((S)-1-(2-methoxybenzyl)-5-oxopyrrolidine-2-carbonyl) piperidin-3-
27 C
0
[α]
yl)methylcarbamate [32d]: Same as 31a. Yield: 85 %;
D
: - 6.25 (c = 0.24; Chloroform); IR
-
1 1
(
(
KBr) 3337, 3005, 2930, 2361, 1684, 1496, 1460, 1365, 1249, 1172, 1119, 1029 cm ; H NMR
CDCl ; 300MHz): δ 1.17-1.26 (m, 2H, 4’-H); 1.45 (s, 9H, -O-C(CH ) ); 1.66-1.73 (m, 1H, 3’-
3
3 3
H); 1.81-1.88 (m, 2H, 5’-H); 2.15-2.18 (m, 1H, 3-H
a
b
); 2.32-2.42 (m, 1H, 3-H ); 2.47-2.56 (m, 2H,
4
-H); 2.60-2.67 (m, 2H, -CH -NH-Boc); 2.85-3.01 (m, 2H, 6’-H); 3.39 (dd, 1H, 2’-H ); 3.45-3.80
2 a
(m, 1H, 2’-H ); 3.78 (s, 3H, -OCH ); 4.02-4.06 (d, 1H, -CH -Ph); 4.23-4.29 (m, 1H, 2-H); 4.90
b 3 2
1
3
(
s, 1H. NH); 4.95-5.03 (d, 1H, -CH -Ph); 6.84-6.93 (m, 2H, Ph-H); 7.20-7.28 (m, 2H, Ph-H); C
2
NMR (CDCl ; 75 MHz): 22.78, 23.98, 28.13, 29.73, 42.87, 45.41, 45.93, 48.58, 55.27, 56.11,
3
7
4
9.31, 113.07, 113.99, 121.00, 129.72, 137.68, 156.10, 159.92, 169.01, 175.24; FAB MS (m/z):
+
46 (M+H) ; HRMS: (m/z) calc. for C H NO : 446.1252. Found: 446.1227
1
3
15
4
4
.1.3.9. tert-butyl ((S)-1-((S)-1-(3-methylbenzyl)-5-oxopyrrolidine-2-carbonyl) piperidin-3-
27 C
0
[α]
yl)methylcarbamate [31e]: Same as 31a. Yield: 80 %;
D
: +23.00 (c = 0.20; Chloroform); IR
-1 1
(
KBr): 3451, 3012, 2933, 2360, 1688, 1512, 1455, 1251, 1219, 1170 cm ; H NMR (CDCl ; 300
3
MHz): δ 1.17-1.25 (m, 2H, 4’-H); 1.42 (s, 9H, -O-C(CH ) ); 1.66 (m, 1H, 3’-H); 1.76-1.86 (m,
3
3
2
H, 5’-H); 2.13-2.17 (m, 1H, 3-H ); 2.30 (s, 3H, Ph-CH3), 2.36-2.39 (m, 1H, 3-H ); 2.49-2.55
a
b
(m, 2H, 4-H); 2.64-2.75 (m, 2H, -CH
2
-NH-Boc); 2.83-3.00 (m, 2H, 6’-H); 3.32-3.42 (m, 1H, 2’-
H ); 3.73-3.76 (m, 1H, 2’-H ); 4.14-4.16 (m, 2H, 2-H &-CH -Ph); 4.85 (s, 1H. NH); 5.13-5.20
a
b
2
1
3
(
m, 1H, -CH -Ph); 6.89-7.26 (m, 4H, Ph-H); C NMR (CDCl ; 75 MHz): 21.10, 22.67, 23.07,
2
3
2
1
4.71, 27.91, 28.40, 29.77, 35.98, 38.32, 43.12, 45.08, 45.66, 49.29, 55.70, 79.32, 128.44,
+
28.74, 129.37, 133.10, 137.38, 156.11, 168.83, 175.32; FAB MS (m/z): 429 (M ), 330, 374, 452
+
(
M+Na) ; HRMS: (m/z) calcd. for C24
H
35
N
3
O
4
) 430.2628; Found, 430.2672
4
.1.3.10. tert-butyl ((R)-1-((S)-1-(3-methylbenzyl)-5-oxopyrrolidine-2-carbonyl) piperidin-3-
27 C
0
[α]
yl)methylcarbamate [32e]: Same as 31a. Yield: 85 %;
D
: - 7.14 (c = 0.34; Chloroform); IR
(
KBr): 3453, 3349, 3010, 2932, 2862, 2362, 1695, 1658, 1511, 1450, 1394, 1366, 1253, 1170,
-1 1
1
042, 964 cm ; H NMR (CDCl ; 300MHz): δ 1.13-1.25 (m, 2H, 4’-H); 1.42 (s, 9H, -O-
3
3 3 a
C(CH ) ); 1.68 (m, 1H, 3’-H); 1.76-1.85 (m, 2H, 5’-H); 2.14-2.19 (m, 1H, 3-H ); 2.35 (s, 3H, Ph-
CH3), 2.33-2.39 (m, 1H, 3-H ); 2.47-2.59 (m, 2H, 4-H); 2.62-2.77 (m, 2H, -CH -NH-Boc); 2.80-
b
2
2
.98 (m, 2H, 6’-H); 3.31-3.40 (m, 1H, 2’-H
a
); 3.72-3.77 (m, 1H, 2’-H
b
); 4.12-4.20 (m, 2H, 2-H
1
3
&
-CH -Ph); 4.93 (s, 1H. NH); 5.09-5.14 (m, 1H, -CH -Ph); 6.93-7.28 (dd, 4H, Ph-H); C NMR
2
2

ACCEPTED MANUSCRIPT
(
CDCl
3
; 75 MHz): 21.10, 22.75, 23.89, 24.64, 28.32, 29.80, 36.43, 37.36, 42.85, 45.88, 48.47,
+
5
3
6.01, 79.33, 128.72, 129.37, 133.05, 137.44, 156.10, 169.01, 175.11; FAB MS (m/z): 429 (M ),
+
30, 374, 452 (M+Na) ; HRMS: (m/z) calcd. for C H N O ) 430.2628; Found, 430.2684.
24
35
3
4
4
.1.3.11. tert-butyl ((S)-1-((S)-1-benzyl-5-oxopyrrolidine-2-carbonyl)piperidin-3-yl)methyl
carbamate [31f]: (2S)-N-(phenylmethyl)-pyroglutamic acid (0.1 g, 1 eq, 0.46 mmol) and HOBt
(0.13 g, 1.5 eq, 0.68 mmol) were dissolved in dry DCM (25 ml) in a three necked round bottom
0
flask fitted with N
2
inlet. The reaction mixture was cooled to 0 C in an ice-salt bath. DCC (0.13
g, 1.2 eq, 0.55 mmol) dissolved in dry DCM (10 ml) was added to it and stirred while being at
0
0
C for 15 min. (3R)-N-Boc-3-aminomethylpiperidine (0.098 g, 1 eq, 0.46 mmol) dissolved in
dry DCM (10 ml) was added drop wise to the reaction mixture and stirring was continued for 3
0
hrs at 0 C. The reaction mixture was then brought to room temperature and concentrated under
reduced pressure. The residue was dissolved in ethyl acetate, filtered and the filtrate was washed
successively with dil. citric acid (3 x 25 ml), dil. Na CO solution (3 x 25 ml) and brine (1 x 25
2
3
ml). The organic layer was separated, dried and concentrated and the crude material was flash
27 C
0
[α]
chromatographed to get pure compound. Yield: 80 %;
KBr): 3353, 2932, 2360, 1687, 1517, 1454, 1364, 1255, 1171 cm ; H NMR (CDCl
δ 1.17-1.38 (m, 2H, 4’-H); 1.44 (s, 9H, -O-C(CH ) ); 1.65 (m, 1H, 3’-H); 1.72-1.88 (m, 2H, 5’-
D
: + 27.85 (c = 0.28; Chloroform); IR
-1 1
(
3
; 300MHz):
3
3
H); 2.12-2.21 (m, 1H, 3-H
a
); 2.40-2.47 (m, 1H, 3-H
b
); 2.51-2.63 (m, 2H, 4-H); 2.70-2.84 (m, 2H,
-
CH -NH-Boc); 2.88-2.93 (m, 2H, 6’-H); 3.29-3.45 (m, 1H, 2’-H ); 3.72-3.93 (m, 1H, 2’-H );
2
a
b
4
5
3
1
.10-4.21 (m, 2H, 2-H & -CH -Ph); 4.92 (s, 1H. NH); 5.13-5.27 (d, 1H, -CH -Ph); 7.14-7.32 (m,
2 2
1
3
3
H, Ph-H); C NMR (CDCl ; 75 MHz): 21.06, 21.06, 22.65, 24.61, 27.88, 28.28, 28.69, 29.74,
5.95, 38.33, 42.72, 43.11, 43.33, 45.06, 45.64, 49.27, 55.67, 55.84, 79.31, 128.47, 128.73,
+
29.35, 133.07, 137.33, 156.02, 168.81, 169.25, 175.16; ESI MS (m/z): 416 (M+H) , 316 (M-
+
Boc), 439 (M+Na) ; HRMS: (m/z) calc. for C H N O : 416.2471. Found: 416.2485
2
3
33
3
4
4
.1.3.12. tert-butyl ((R)-1-((S)-1-benzyl-5-oxopyrrolidine-2-carbonyl)piperidin-3-yl)methyl
27 C
0
[α]
carbamate [32f]: Same as 31f. Yield: 80 %;
D
: - 9.35 (c = 0.32; Chloroform); IR (KBr): 3353,
-1 1
2
932, 2360, 1687, 1517, 1454, 1364, 1255, 1171 cm ; H NMR (CDCl ; 300MHz): δ 1.17-1.38
3
(m, 2H, 4’-H); 1.44 (s, 9H, -O-C(CH
3
)
3
); 1.65 (m, 1H, 3’-H); 1.72-1.88 (m, 2H, 5’-H); 2.12-2.21
(
m, 1H, 3-H ); 2.40-2.47 (m, 1H, 3-H ); 2.51-2.63 (m, 2H, 4-H); 2.70-2.84 (m, 2H, -CH -NH-
a
b
2
Boc); 2.88-3.01 (m, 2H, 6’-H); 3.29-3.45 (m, 1H, 2’-H
a
); 3.72-3.93 (m, 1H, 2’-H
b
); (m, 1H, -

ACCEPTED MANUSCRIPT
CH
2
2
-Ph); 4.10-4.21 (m, 1H, 2-H); 4.92 (s, 1H. NH); 5.13-5.27 (m, 1H, -CH -Ph); 7.14-7.32 (m,
1
3
5
3
1
H, Ph-H); C NMR (CDCl ; 75 MHz): 21.06, 21.06, 22.65, 24.61, 27.88, 28.28, 28.69, 29.74,
3
5.95, 38.33, 42.72, 43.11, 43.33, 45.06, 45.64, 49.27, 55.67, 55.84, 79.31, 128.47, 128.73,
+
29.35, 133.07, 137.33, 156.02, 168.81, 169.25, 175.16; ESI MS (m/z): 416 (M+H) , 316 (M-
+
Boc), 439 (M+Na) ; HRMS: (m/z) calc. for C H N O : 416.2471. Found: 416.2485
2
3
33
3
4
4
3
.1.3.13. tert-butyl ((S)-1-((S)-1-(naphthalen-2-ylmethyl)-5-oxopyrrolidine-2-carbonyl) piperidin-
27 C
0
[α]
-yl)methylcarbamate [31g]: Same as 31f. Yield: 90 %;
D
: + 8.95 (c = 0.19; Chloroform); IR
(
KBr): 3344, 3088, 2979, 2931, 2859, 2361, 1682, 1512, 1459, 1365, 1253, 1170, 1082, 1047,
-1 1
1
016, 962 cm ; H NMR (CDCl ; 300MHz): δ 1.12-1.25 (m, 2H, 4’-H); 1.45 (s, 9H, -O-
3
C(CH ) ); 1.66 (m, 1H, 3’-H); 1.73-1.79 (m, 2H, 5’-H); 1.99-2.10 (m, 1H, 3-H ); 2.37-2.41 (m,
3
3
a
1
3
1
H, 3-H
b
); 2.49-2.55 (m, 2H, 4-H); 2.62-2.77 (m, 2H, -CH
2
-NH-Boc); 2.86-2.90 (m, 2H, 6’-H);
.08-3.14 (m, 1H, 2’-H ); 3.79-3.93 (m, 1H, 2’-H ); 4.05-4.15 (dd, 1H, -CH -Ph); 4.20-4.27 (m,
a
b
2
1
3
2
H, 2-H); 4.83 (s, 1H. NH); 5.62-5.72 (dd, 1H, -CH -Ph); 7.26-8.01 (m, 7H, Naph-H); C NMR
(
CDCl ; 75 MHz): 23.05, 28.28, 28,63, 28.84, 30.10, 30.25, 45.01, 56.26, 56.32, 124.25, 126.60,
3
1
4
26.84, 127.29, 127.56, 128.95, 129.08, 129.34, 123.28, 155.17, 167.56, 173.89; ESI MS (m/z):
+
+
+
+
66 (M+H) , 366 (M-Boc) , 410 (M-t_butyl) , 488 (M+Na) ; HRMS: (m/z) calc. for
C H N O : 466.2628. Found: 466.2620
27
35
3
4
4
.1.3.14. tert-butyl ((R)-1-((S)-1-(naphthalen-2-ylmethyl)-5-oxopyrrolidine-2-carbonyl)
27 C
0
[α]
piperidin-3-yl)methylcarbamate [32g]: Same as 31f. Yield: 86 %;
D
: + 26.32 (c = 0.19;
Chloroform); IR (KBr): 3343, 3049, 2930, 2859, 2364, 1685, 1655, 1514, 1481, 1259, 1169,
-
1 1
1
1
2
1
023, 962 cm ; H NMR (CDCl ; 300MHz): δ 1.23 (m, 2H, 4’-H); 1.45 (s, 9H, -O-C(CH ) );
3 3 3
.668 (m, 1H, 3’-H); 1.71-1.77 (m, 2H, 5’-H); 1.90-2.10 (m, 1H, 3-H ); 2.38 (m, 1H, 3-H ); 2.43-
a
b
.53 (m, 2H, 4-H); 2.64-2.75 (m, 2H, -CH
2
-NH-Boc); 2.83-2.98 (m, 2H, 6’-H); 3.32-3.48 (dd,
H, 2’-H ); 3.90 (m, 1H, 2’-H ); 4.22-4.29 (m, 1H, 2-H); 4.22-4.29 (d, 1H, -CH -Naphthyl); 4.90
a
b
2
1
3
(s, 1H. NH); 5.64-5.71 (d, 1H, -CH
2
-Naphthyl); 7.26-7.87 (m, 7H, Naphthyl-H); C NMR
(
CDCl ; 75 MHz): 21.57, 22.96, 23.50, 27.42, 28.82, 35.50, 36.45, 42.30, 44.97, 47.40, 55.12,
3
7
8.31, 122.73, 124.11, 125.90, 126.73, 127.30, 127.95, 130.79, 132.76, 155.17, 167.87, 173.92;
+
+
ESI MS (m/z): 488 (M+Na) , 466 (M+H) , 366 (M-Boc) ; HRMS: (m/z) calc. for C27
66.2628. Found: 466.2605
35 3 4
H N O :
4