Design, synthesis and inhibition activity of novel cyclic peptides against protein tyrosine phosphatase A from Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2010.09.052

Mycobacterium tuberculosis, the causative agent for tuberculosis has employed several signalling molecules to sense the host cellular environment and act accordingly. For example, protein tyrosine phosphatase A (MPtpA) of M. tuberculosis, a signalling pro

Full text of DOI:10.1016/j.bmc.2010.09.052

Bioorganic & Medicinal Chemistry 18 (2010) 8365–8373
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and inhibition activity of novel cyclic peptides against
protein tyrosine phosphatase A from Mycobacterium tuberculosis
⇑
⇑
Koushik Chandra, Debajyoti Dutta, Amit K. Das , Amit Basak
Department of Chemistry, Department of Biotechnology, Indian Institute of Technology, Kharagpur 721 302, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2010
Revised 21 September 2010
Accepted 22 September 2010
Available online 7 October 2010
Mycobacterium tuberculosis, the causative agent for tuberculosis has employed several signalling
molecules to sense the host cellular environment and act accordingly. For example, protein tyrosine
phosphatase A (MPtpA) of M. tuberculosis, a signalling protein belonging to the tyrosine phosphatase
superfamily, is involved in phagocytosis and is active in virulent mycobacterial form. Starting from a
b-lactam framework a new class of structure based cyclic peptide (CP) inhibitors was designed. The syn-
thesis involves a crucial intramolecular transamidation via a ring opening reaction. All the compounds
show moderate to good inhibitory activities against MPtpA in micromolar concentrations. The results
of inhibition kinetics suggest mixed mode of inhibition. The binding constant determined from circular
dichroism (CD) and fluorescence quenching studies shows strong binding of the hydrophilic side chain
of CPs with the enzyme active site residues. All these are well supported by docking studies.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Peptide
Mycobacterium
Inhibition
Phosphatase
Tuberculosis
1. Introduction
active site (Fig. 1). Its mechanism of action1,6 as shown in Scheme 1
involves the nucleophilic attack by cysteine thiol followed by
Protein phosphorylation and dephosphorylation are two impor-
tant processes that are pivotal for the sustenance of the biological
systems.1 They have been identified as the key mediators of cell
signalling and are required for cell division, control of metabolic
activity, environmental response and pathogenicity. Understand-
ing the role of these in enzymes and controlling their activities
have been the focus of a rapidly growing research in the area of
medicinal chemistry. Several pathogenic bacteria produce phos-
phatases which have been implicated to their virulence. One such
example2 is Mycobacterium tuberculosis which is the causative
agent for tuberculosis (TB) and is a major cause of mortality
throughout the world. Analysis of M. tuberculosis genome revealed
the presence of MPtpA and MPtpB genes encoding protein tyrosine
phosphatases MPtpA and MPtpB.3 The bacillus releases MPtpA dur-
ing its infection to macrophages and turns down the phagocytosis
process.4 Though the exact mechanism of its action is still un-
known, the MPtps are now identified as potential targets for devel-
opment of anti-tuberculosis agents and development of inhibitors
of these enzymes is believed to be one of the ways to tackle the
menace of TB. In this paper, we describe the synthesis of a series
of novel cyclic peptides (CP). Their inhibitory activity against MPt-
pA have been evaluated and studied by different techniques and
the results are also included.
hydrolysis of the resulting thioacyl intermediate. Based upon the
all these aspects and also the reported7 inhibitory activity of
cyclodepsipeptides, we have designed a new class of b-amino
based cyclic peptides (CPs) as depicted in Figure 2. The rationale
for our design is the possibility of H-bond and hydrophobic inter-
actions that may occur with the CPs. The synthesized CPs were
classified into three types (A–C) depending upon the number of
a-amino acids connecting the b-amino acid to form the cyclic net-
The crystal structure of MPtpA5 shows a PTP loop as a signature
motif and the presence of Cys11, Arg17 and Asp126 as a part of the
⇑
Corresponding authors. Tel.: +91 3222 283300; fax: +91 3222 282252.
E-mail address: absk@chem.iitkgp.ernet.in (A. Basak).
Figure 1. Cartoon structure of MPtpA showing the PTP loop (red) along with the
active site residues Cys11, Arg17 and Asp126.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.052

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K. Chandra et al. / Bioorg. Med. Chem. 18 (2010) 8365–8373
Asp
Asp
Asp
OH
OH
HO-H
O
H
H
O
OH
O
Arg
O
Arg
O
Arg
O
O
O
S
O
P
O
S
P
P
O
O
O
NH
O
NH
NH
O
O
H2N
H2N
Cys
H2N
S
NH2
NH2
NH2
Cys
Cys
Scheme 1. The mechanism of the dephosphorylation catalysed by MptpA.
pled with N-Boc protected peptide to provide the b-lactam based
acyclic peptides (24–30). These were deprotected with TFA and
the salt was converted to the free amine by washing with sodium
carbonate solution (31–37). Final transamidation step10 involving
intramolecular ring opening of b-lactam was carried out under dif-
ferent reaction conditions shown in Table 1. The table clearly
shows that the cyclization becomes more facile in terms of yield
and reaction time when acetic acid and triethyl amine were used
as a buffer in EtOH. For higher member ring, the cyclization was
even more facile possibly due to proper conformational alignment
during cyclization.
Ph
Ph
H
NHPh
O
NHPh
O
H
H
H
Type 1: n = 1
Type 2: n = 2
Type 3: n = 3
NH
NH
(AA)n
(AA)n
Figure 2. Prepared as a inseparable mixture of diastereomers.
work. These are shown in Figure 3 (for compounds 8–11, please re-
All the compounds gave characteristic NMR and mass spectral
data. The purity of the peptides was checked by hplc; however,
in spite of all our attempts, the two diastereomers could not be
separated, even by hplc. These were characterized as a mixture
of two diastereomers (besides the ones reported here in the man-
uscript, four more CPs were synthesized and studied. The results
are included in Supplementary data).
fer to Supplementary data).
2. Results and discussion
2.1. Synthesis
The various steps leading towards the synthesis of CPs are
shown in Scheme 2. The key step is the intramolecular nucleophilic
ring opening of a b-lactam with the free amine of an amino acid
appendage. As a consequence, the b-amino acids were derived
from the b-lactam skeleton itself. Thus in one step, we have been
able to prepare the cyclic network along with the incorporation
of the b-amino acid framework. The key intermediate, namely
the cis 3-aminomethyl b-lactams (16–17)8 (synthesized from the
known hydroxyl-analogue via the Kinugasa reaction9) were cou-
2.2. VT NMR study
Since H-bond interactions between the peptides and the en-
zyme should significantly contribute to their mutual binding, it is
important to know the presence of any strong intramolecular H-
bond, if any, in the CPs. Existence of any such interactions within
the small molecules may rule out participation of the amide groups
NHPh
Ph
H
H
Ph
H
O
Ph
NHPh
H
NHPh
H
O
H
CH3
O
H
HN
O
NH
O
O
H3C
H
NH
HN
NH
H
HN
NH
N
O
H
O
H
H3C
COOH
1
7
2
Type A
Type C
Ph
NHPh
H
Ph
H
Ph
H
NHPh
NHPh
NHPh
H
O
H
H
H
H
OH
O
O
O
H
O
H
NH
NH
NH
HN
NH
HN
HN
O
HN
O
CH3
O
O
O
O
N
O
N
H
N
H
O H3C
N
H
H
H
6
3
4
5
Type B
Figure 3. Synthesized cyclic peptides with different amino acid residues.

K. Chandra et al. / Bioorg. Med. Chem. 18 (2010) 8365–8373
8367
H
N
H
H
H
H
Z
Z
H
H
H
Z
H
Z
(i)
O
(iii)
(iv)
23
N3
HO
H2N
(ii)
N
N
R1
NH
NH
N
Ph
O
Ph
O
Ph
O
Ph
O
Z= Ph, 12
Z= furan, 13
O
Z= Ph, 14
Z= furan, 15
Z= Ph, 16
Z= furan, 17
R2
HN
R3
(iv)
20, 21, 22
18,19 (iv)
O
H
N
NHBoc
H
H
H
Z
O
N
H
H
30
Z
O
(v)
R1
(CH2)n
NH
N
H
N
Ph
O
O
N
O
H
H
N
Ph
Z
O
NHBoc
R
R2
24, 25
26, 27,28, 29
BocHN
R1
NH
(v)
Ph
O
(v)
O
H
N
H
R2
N
H
H
Z
H
H
Z
O
O
HN
R3
O
(CH2)n
NH2
R1
NH
N
N
O
Ph
O
Ph
NH2
O
R
37
33, 34,35, 36
31, 32
R2
(vi)
H2N
(vi)
(vi)
H
Z
NHPh
H
Z
NHPh
O
H
H
Z
NHPh
O
H
H
O
NH
HN
R3
O
O
NH
HN
NH
HN
NH
R1
R2
H
NH
R
O
R1
N
O
(CH2)n
1, 38
O
O
R2
2
3, 4, 5, 6
7
(vii)
For clarity, only one diastereomer is shown here
For 24, 31, 1: Z= Ph, R= H, n = 0
For 25, 32, 38 : Z= Ph, R=COOBn, n = 2
For 2 : Z= Ph, R= COOH, n = 2
For 26, 33, 3 : Z= Ph, R 1= H, R2= H
For 27, 34, 4 : Z= 2-furyl, R1= H, R2= H
For 28, 35, 5 : Z= Ph, R 1= CH3, R2= CH3
BocNHCH2COOH, 18
BocNHCH(COOBn)CH2(CH2)(COOH), 19
BocNHCH2CONHCH2COOH, 20
BocNHCH(CH3)CONHCH(CH3)COOH, 21
BocNHCH2Ph(p-OH)CONHCH2COOH, 22
BocNHCH(CH3)CONHCH(CH3)CONHCH(CH3)COOH, 23
For 29, 36, 6 : Z= Ph, R 1= H, R2= CH2Ph(p-OH)
For 30, 37, 7 : Z= Ph, R 1= CH3, R2= CH3, R3= CH3
Reagents and conditions: (i) MsCl, Et3N, DCM, 0 oC, 10 mins, 93-96% (ii) NaN3, dry DMF, rt, 3 h, 93-95% (iii)
PPh3, moist THF, rt, overnight, 81-83% (iv) EDC.HCl, HOBT, DIPEA, dry DCM/DMF, rt, 6 h, 75-78% (v)TFA,
dry DCM, 0 oC, then aqueous Na2CO3, 30 min, 69-72% (vi) see Table 1, 65-70% (vii) H2, 10% Pd-charcoal, dry
EtOH, rt, 85%
Scheme 2. Synthesis of Types 1, 2 and 3 CPs as diastereomeric mixture from cis b-lactam. Reagents and conditions: (i) MsCl, Et3N, DCM, 0 °C, 10 min, 93–96%; (ii) NaN3, dry
DMF, rt, 3 h, 93–95%; (iii) PPh3, moist THF, rt, overnight, 81–83%; (iv) EDCꢀHCl, HOBT, DIPEA, dry DCM/DMF, rt, 6 h, 75–78%; (v)TFA, dry DCM, 0 °C, then aqueous Na2CO3,
30 min, 69–72%; (vi) see Table 1, 65–70%; (vii) H2, 10% Pd–charcoal, dry EtOH, rt, 85%.
Table 1
Reaction parameters for the intramolecular transamidation reaction for the synthesis of CPs
Entry
CPs (from amines)
Additives
Concn (M)
Temp (°C)
Reaction time (h)
1
2
3
4
5
6
7
8
9
1–6
7
1–6
7
1
2–7
7
None
None
0.5(M)Et3N
0.5(M)Et3N
0.5(M)Et3N/0.05(M)AcOH
0.5(M)Et3N/0.05(M)AcOH
0.5(M)Et3N/0.05(M)AcOH
1.0(M)Et3N/0.5(M)AcOH
1.1 mmol K2CO3
2 ꢁ 10ꢂ2
2 ꢁ 10ꢂ2
2 ꢁ 10ꢂ2
3 ꢁ 10ꢂ3
2 ꢁ 10ꢂ2
2.5 ꢁ 10ꢂ3
1.5 ꢁ 10ꢂ3
4 ꢁ 10ꢂ2
4 ꢁ 10ꢂ2
55
50
50
50
45
45
45
60
60
150
95
105
55
38
25
18
88–110
72–120
1–7
1–7

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K. Chandra et al. / Bioorg. Med. Chem. 18 (2010) 8365–8373
in H-bonding interactions with the enzyme. In order to check that
possibility, VT NMR was carried out with some of the peptides. The
2000000
1500000
1000000
500000
0
temperature coefficients (Dd/D
T)11 of the various NHs were deter-
I=160μM
I=120μM
I=0μM
mined in CDCl3 and are shown in Table 2.
Except for one NH of peptide 7, which has a temperature coef-
ficient of 3.1, there was no evidence of any strong intramolecular
H-bonding meaning that the NHs and consequently the amide car-
bonyls are available for H-bonding with the enzyme residues (for
plots of chemical shift vs temperature, see Supplementary data).
-500000
-0.1
0
0.1
0.2
2.3. Inhibition study
1/[S] mM-1
Recombinant wild type MPtpA was purified as described previ-
ously.12 Enzyme purity was assessed by SDS PAGE and protein con-
centration was determined from the absorbance at 280 nm. The
phosphatase activity was measured at 25 °C using p-nitrophenol
phosphate (pNPP) as substrate keeping a fixed concentration of
Figure 4. Lineweaver–Burk plot for CP 6.
Table 3
Inhibitory constants (Ki) of CPs
protein (1 lM) in a reaction mixture of Tris buffer containing
0.1 M Tris, 0.15 M NaCl and 1 mM EDTA of pH 7.5. Synthetic CPs
were dissolved in methanol and the methanol concentration was
adjusted so as not to exceed 3% (v/v) in the final reaction mixture.
This ensured the phosphatase activity of the enzyme to remain
unaffected. The release of p-nitrophenol at 405 nm was quantified
both in case of control and in presence of the inhibitors. The effec-
tive inhibitor and substrate concentrations were usually varied
Compound
Ki (lM)
1
2
3
4
5
6
7
140.2 10
36.3 8.5
85.5 7.3
215.4 12
220.2 11
8.0 .20
199.1 10
from 10–50 lm to 100–300 lm, respectively. Each assay was re-
peated in triplicate.
2.4. Data analysis
of 50 nm/min with a response period of 4 s. The spectra for MPt-
pA–CP 6 complex in far UV were obtained by subtracting the cor-
responding buffer blank containing CP 6 from the respective
samples. CD spectra for each MPtpA–ligand complex were re-
corded by varying the concentration ratio up to 1:6. Finally the
secondary structure was determined using CONTINLL of DICHRO-
WEB,13 an online server for protein secondary structure analysis
from CD spectroscopic data.
Using pNPP as a substrate, we have screened the CPs for possi-
ble inhibition of MPtpA activity. Values of the kinetic parameters
and inhibitor constants were obtained from the Lineweaver–Burk
plot (Fig. 4). For different concentration of same inhibitor, double
reciprocal plots were taken into consideration to conclude the
mode of inhibition. Inhibitory constant (Ki) was determined from
ꢀ
ꢁ
Km
Vmax
the plot of
vs [I] where [I] is the inhibitor concentration,
Km is the Michaelis–Menten constant and Vmax is the maximum
reaction velocity. All the compounds were found to behave as
mixed inhibitors with Ki values in micromolar range (Table 3). Thus
reversible binding interaction enabled the conformation change in
PTP loop of MPtpA which in turn reflects in the rate of inhibition.
However, such conformational irregularity became more pro-
nounced in case of polar CPs bearing tyrosine or glutamic acid sub-
unit compared to that observed for hydrophobic analogues. The
tyrosine-based CP exerted maxima perturbation on MPtpA. How-
ever, the trans analogues of CPs were found to be comparatively
less effective in inhibiting MPtpA.
2.6. Determination of binding constant from CD data
Changes in structural properties of MPtpA caused by the bind-
ing of above synthesized CPs are essential in understanding the
mechanism of action and regulation of biological activity. For this
it is important to observe any change in the secondary structural
motif like
a-helix of MPtpA which appears as negative bands in
the far UV-region with the minima at 208 nm and 222 nm. The
CPs alone in Tris buffer did not exhibit any significant peaks in
these particular regions. Then we performed the titration based
kinetics studies between CPs and MPtpA in such a way that the
concentration ratio was varied upto 6:1 (one representative CD-
titration curve for compound 6 is shown in Figure 5; for others,
please refer to Supplementary data).
2.5. CD measurement
CD measurement was accomplished on a Jasco-810 automatic
recording spectropolarimeter using a quartz cell of path length
10 mm with the sample temperature maintained at 25 °C. The
spectra were recorded in the range of 195–240 nm with a scan rate
It was found that the regions characteristic of the a-helix were
affected due to gradual increase in concentration of all cyclic pep-
tides which substantiates the unfolding of the protein. Such con-
formational changes were even more pronounced in case of
those CP, which contain polar components like carboxylic acid or
phenolic-OH group etc. These findings clearly suggest that these
Table 2
Amide proton NMR chemical shift temperature dependence in CDCl3 (1 mM)
CP have the tendency to interact strongly with the a-helix associ-
ated with PTP loop in MPtpA by virtue of complementary binding
sites.14 The red shift of the band in CD spectra also indicates the
formation of stable transition state complex. A similar phenome-
non was observed when we performed the time dependent CD
spectroscopy between MPtpA and compound 6 (Table 4). As the
Peptides Ring
size
Amide-NH (1)
dNH T)
(ppb/K)
Amide-NH (2)
dNH T)
(ppb/K)
Amide-NH (3)
dNH T)
(ppb/K)
(ꢂ
D
/
D
(ꢂ
D
/
D
(ꢂ
D
/D
1
4
6
7
7
10
10
13
6.4
9.2
10.5
3.1
6.7
6.1
5.6
5.5
—
—
—
5.8
time proceeds, the disruption of
a random coil conformation.
a-helix takes place ending up in

K. Chandra et al. / Bioorg. Med. Chem. 18 (2010) 8365–8373
8369
2.7. Fluorescence studies
30
20
Intrinsic tryptophan fluorescence of native and chemically
denatured samples were monitored by Hitachi spectrofluorometer
using a quartz cuvette of 10 mm path length at a fixed temperature
of 20 °C, equipped with a constant temperature water circulator
bath (NesLab). Baseline correction was made by subtracting the
spectra of CP from respective measurements. The protein was ex-
cited at 295 nm and the emission spectra were recorded starting
from 305 nm to 550 nm with emission maxima at 340 nm. Multi-
ple scans were taken in each case. The spectrum of MPtpA shows
maxima at 340 nm indicating the presence of tryptophan residue.
There was a quenching of fluorescence in presence of the inhibitor
and the interactions were explained in terms of Stern–Volmer
equation15 and Scatchered plot.16
10
0
-10
-20
-30
-40
-50
200
210
220
230
240
wavelength (nm)
2.8. Steady state fluorescence quenching
Figure 5. Overlayed deconvoluted CD spectra recorded during complexation
between MptpA and different concentration of CP-6. Upward arrow shows the
gradual increase in concentration ratio (MPtpA/CP-6) (i) only MPtpA; (ii) 2:1; (iii)
1:1; (iv) 1:2; (v) 1:3; (vi) 1:4; (vii) 1:5; (viii) 1:6.
The origin of intrinsic fluorescence of MPtpA is guided by the
presence of aromatic amino acid tryptophan where the indole moi-
ety is responsible for UV absorbance and the fluorescence of the
protein. Henceforth it provides a fundamental tool to extract infor-
mation about the mode of interaction of MPtpA. Based on this ap-
proach, fluorometric titration of MPtpA and CP were performed by
Table 4
Changes in secondary structure of MPtpA. Upon interaction with CP-6 at different
time interval
varying the concentration from 2.5 lM to 20 lM. The intensity
maximum at 340 nm was found to diminish gradually with
increasing concentration of CP. One representative fluorescence-
titration curve for compound 6 is shown in Figure 6 (for others, re-
fer to Supplementary data). The quenching phenomenon was well
observed up to the concentration ratio 1:10. This offers a valuable
explanation that CP interacts with the PTP loop in such a way that
two tryptophan residues simultaneously get buried leading to fluo-
rescence quenching. In addition, the polar subunits like carboxylic
acid or phenolic-OH interact even strongly resulting in a greater
extent of quenching behaviour. The induction of polar residues to
CP not only brought about the conformational change of MPtpA
but it also throws light to the probable route of the interaction pro-
cess. The time dependent interaction studies between CP and MPt-
pA also reflected the similar behaviour. Finally, the binding
constants have been calculated for each complex by direct titration
method.
Time (min)
Helix (%)
Strands (%)
Turns (%)
Unordered (%)
0
5
42
8.9
12
16.8
20.9
15.2
17.9
19.1
21.4
13.9
18.3
17.7
38.7
32.2
25.5
33.5
25.3
30.9
23.0
36.5
56.5
46.8
43.7
41.5
36.1
28.9
22.7
16.3
12.3
2.1
10
15
20
30
45
60
90
180
15.2
27.7
27.3
39.2
37.3
25.0
31.5
16.9
0.9
0
We have also calculated the binding constants (Table 5) of the
CPs upon complexation with the protein by direct titration meth-
od. For such interaction it is assumed that there is only 1:1 com-
plex formation in which the MPtpA. CP complex is in equilibrium
with unreacted free MPtpA and CP, that is, ([MPtpA] + [CP] = [MPt-
pA. CP]. The equation used for the calculation is
1=½hꢃ ¼ 1=½hꢃmax þ 1=½hꢃmaxKbCP in which CP represents the initial
concentration of CP, [h] is the difference in absorption in presence
and in absence of inhibitor and Kb is the binding constant between
MPtpA and CPs. The double reciprocal plot of 1/[h] versus 1/CP is
linear and the binding constant (Kb) is obtained by taking the ratio
of intercept to the slope. The higher the binding constant, the
greater is the perturbation. From the binding constant values (Ta-
ble 5) it is again the tyrosine and glutamic acid-based inhibitors
The quenching of fluorescence was analyzed (Fig. 7) following
the classical Stern–Volmer equation. F0/F = 1 + Ksv[Q] where the
F0 and F are the fluorescence intensity in absence and in presence
of a quencher, [Q] is the molar concentration of quencher and Ksv is
the collision quenching constant [Mꢂ1]. The MPtpA concentration
was kept 2 lM throughout the experiment. Binding constant was
700
600
500
400
300
200
100
0
that exerted relatively greater extent of perturbation on
as compared to other non-polar hydrophobic analogues.
a-helix
Table 5
Binding constant of CPs from CD
CPs
Kb (Mꢂ1) at 222 nm
1
2
3
4
5
6
7
3.6 ꢁ 104
8.5 ꢁ 104
4.7 ꢁ 104
2.95 ꢁ 104
1.1 ꢁ 104
1.2 ꢁ 105
3.3 ꢁ 104
350
400
450
500
550
Wavelength (nm)
Figure 6. Overlayed deconvulated fluorescence spectra of the complexation
between MptpA and different concentration of CP-6. Downward arrow shows the
quenching.

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K. Chandra et al. / Bioorg. Med. Chem. 18 (2010) 8365–8373
of availability of the maximum number of hydrogen bonding as well
assuitablehydrophobicand -stackinginteractionshowninTable7.
The probable interactions of CP 6 with different residues of MPtpA
are shown in Figure 8. The cyclic peptide backbone is stabilized by
the hydrogen binding interaction with Arg17 and Thr12 located at
the two ends of the PTP loop. Perturbation of the PTP loop associated
2
1.5
1
6
2
3
1
4
7
p
5
a-helix is already confirmed by CD experiments. The stacking inter-
action of N-Phenyl group in CP 6 with Tyr128 and the van der Wall
interaction with Trp48 are also notable. Interaction between Trp48
and CP 6 is also confirmed by the fluorescence quenching experi-
ments. In addition, two aryl rings of CP 6 fit into the hydrophobic
groove of MPtpA causing restricted movement of PTP loop.
Thus comparative docking analysis was able to throw light on
the nature of interaction of the CPs with the protein and was also
in agreement with other physical measurements.
0.5
0.00E+00
5.00E-06
1.00E-05
[Q](M)
1.50E-05
2.00E-05
Figure 7. Overlayed deconvulated Stern–Volmer plots.
determined from the equation, log (F0 ꢂ F)/F = log Kb + n log [Q],
where Kb is the binding constant and n is the number of binding
site. The binding constants as shown in Table 6, are high for tyro-
sine and glutamic acid-based CP which substantiates the fact that
2.11. Conclusion
A new class of CPs has been identified as a potential candidate for
MPtpA inhibitor. Three types depending on the number of amino
acids of CPs were synthesized from b-lactam intermediates via novel
intramolecular transamidation reaction. These CPs constitute a core
where a common residue b-alanine is attached to various polar and
non-polar amino acids. The functional groups in polar and non-polar
residuesimpart a significant contributiontowards the complexation
with target MPtpA. VT NMR reveals the absence of strong intramo-
lecular H-bond except for compound 11 which showed strong intra-
molecular H-bond for one of the NHs. Therefore with the presence of
sufficient number of hydrogen bonding locations available for the
interaction with enzyme, all CPs interacted strongly with MPtpA
as reflected in their Ki values. Although the exact mode of interaction
by possible X-ray structure of co-crystal is currently under investi-
gation, CD studies and fluorescence quenching of tryptophan resi-
dues predicted significant perturbation of helix structure. Docking
experiments suggest that all the CPs bind strongly to the PTP loops
of the signature motif which influences the associated helix and
hydrophobic tryptophan residue to a greater extent. However, small
modification to the chemical structure generates profound altera-
tionsin the mode of interaction to the target enzyme. While thetyro-
sine-based CP showed much impact on the phosphorylation state of
the target enzyme, their trans analogues have little influence on the
enzyme as evident from kinetics of inhibition, CD and fluorescence
study. Further studies are on the way to bring down the inhibition
to nanomolar range by suitable changes in the structure of CPs.
hydrophilic together with p-stacking interactions play a dominant
role upon complexation with MPtpA. All the interactions support
1:1 binding indicating that the inhibitor binds to a specific site of
MPtpA.
2.9. Docking materials
Coordinates of MPtpA were retrieved from Protein Data Bank
(PDB ID 1U2P). Docking was performed using Dock617 in association
with Chimera18 visualization interface. Accessible surface was cal-
culated using PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/
pistart.html). Protein model illustration with bound inhibitor was
carried out using Pymol (http://www.pymol.sourceforge.net). The
goal of the docking studies was to substantiate the facts related to
the conformational aspects of preferred binding with MPtpA and
the molecular mechanism of its inhibitory effect on MPtpA.
2.10. Docking study
In MPtpA, the PTP loop functions as the key element for its flex-
ible accessibility of the substrate to the active site and also direct-
ing the catalytic mechanism pivotal for various types of interaction
with different substances. The loop undergoes a large conforma-
tional change housing two active site residues Cys11 and Arg17.
Therefore, the ligand induced flexibility of the PTP loop is impor-
tant to exert catalysis via an optimized interaction with polar res-
idues. The important substitution in PTP loop of M. tuberculosis is
Leu12 instead of Thr present in mammalian PTP loop.
3. Experimental
As inferred from the docking studies that although most of the
CPs are effective in recognizing the active site and its surroundings,
their effectiveness depends on their ring substitutions. The mode of
the binding depends on the relative perturbation of the interacting
residues. Apart from the active site residues, Glu125, Trp48, Thr12
are also able to strengthen the binding of the CP. Among all, tyro-
sine-based CP 6 which exhibited the highest inhibition, was found
to have greater impact on secondary structure of MPtpA in terms
All 1H NMR and 13C NMR spectra were recorded in CDCl3 at
400 MHz unless otherwise mentioned. IR was recorded using Ther-
mo Nicolet FT-IR. Mass spectra were analyzed by Waters LCT mass
spectrometer. Enzyme purity was assessed by SDS–PAGE and pro-
tein concentration was determined from the absorbance at
280 nm. 0.1 M Tris–HCl pH 7.5 having ionic strength 0.15 M of
NaCl and 1 mM EDTA was used for the kinetic study with all the
synthesized compounds. CD and fluorescence measurement were
recorded on Jasco-810 automatic recording spectropolarimeter
and Hitachi spectrofluorometer respectively using a quartz cell of
path length 10 mm.
Table 6
Binding constant of CPs from fluorescence
Compound
Kb (Mꢂ1) at 295 nm
1
2
3
4
5
6
7
3.41 ꢁ 104
8.89 ꢁ 104
4.87 ꢁ 104
2.99 ꢁ 104
1.0 ꢁ 104
3.1. General procedure for the synthesis of b-lactam linear
peptides conjugates
To a solution of N-Boc protected free acid of linear peptide
(1 equiv) in 2:1 DCM/DMF mixture (10 mL/5 mL), 1-[3-dimethyl
aminopropyl]-3 ethyl carbodiimide hydrochloride (EDCꢀHCl)
1.5 ꢁ 105
3.27 ꢁ 104

K. Chandra et al. / Bioorg. Med. Chem. 18 (2010) 8365–8373
8371
Table 7
Docking interaction analysis during complexation between MPtpA and CP 6
Interacting protein residues (atom)
Interacting counterpart of CP 6
Interaction distance (Å)
Remarks
Thr12 (OG1)
Arg17 (NH2)
Tyr128
Glu125 (OE1)
Trp48
Amide oxygen
Amide oxygen
NHPh group
Tyrosine oxygen
Two outer Ph-rings
Amide nitrogen
3.2
2.9
4.2
3.9
4.7
3.9
Present in PTP loop (H-bonding)
Located at C-terminal part of the PTP loop (H-bonding)
Aromatic -stacking interaction
p
Polar interaction
Hydrophobic interaction
Polar interaction
Asp126 (OD2)
Figure 8. CP 6 docked on MPtpA. (A) CP 6 sits in the cleft near the active site. Interacting residues are shown in salmon colour. Thr12 and Arg17 are part of the PTP–loop. (B)
Different interactions between the MPtpA residues with CP 6 are shown.
(1.2 equiv) followed by 1-hydroxy benztriazole (HOBT) (1.2 equiv)
were stirred at 0 °C for 15 min. The b-lactam containing free amine
(1 equiv) were added dropwise at 0 °C followed by diisopropyl ethyl
amine (DIPEA) (3 equiv). The whole mixture was left stirring for
overnight at room temperature. After partitioning between DCM
and water, the organic layer was washed first with aq NaHCO3, dil
HCl, brine and finally dried over sodium sulfate and evaporated.
The oily crude mixture was purified by column chromatography
(60–120 silica gel) using 2:1 hexane/EtOAC mixture.
3.1.3. [({[(2-Oxo-1,4-diphenyl-azetidin-3-ylmethyl)-
carbamoyl]-methyl}-carbamoyl)-methyl]-carbamic acid tert-
butyl ester (26)
Yield 78%; off-white solid product; c
max (KBr/cmꢂ1) 1642, 1750,
2373, 3435. dH 1.45 (9H, s), 3.06–3.12 (1H, m), 3.22–3.29 (1H, m),
3.80–3.94 (5H, m), 5.24 (1H, d, J = 5.6 Hz), 5.32 (1H, br s), 6.45 (1H,
br s), 6.93 (1H, br s), 7.04–7.09 (1H, m), 7.22–7.39 (9H, m); dC 28.2,
36.1, 42.8, 44.4, 52.4, 57.0, 80.5, 117.1, 124.1, 125.8, 126.6, 128.7,
129.0, 133.8, 137.1, 165.7, 168.4, 169.9; HRMS calcd for
C
25H30N4O5 + H+ 467.2294 found 467.2298.
3.1.1. {[(2-Oxo-1,4-diphenyl-azetidin-3-ylmethyl)-carbamoyl]-
methyl}-carbamic acid tert-butyl ester (24)
3.1.4. [({[(2-Furan-2-yl-4-oxo-1-phenyl-azetidin-3-ylmethyl)-
carbamoyl]-methyl}-carbamoyl)-methyl] carbamic acid
tert-butyl ester (27)
Yield 78%; off-white solid; cmax (KBr/cmꢂ1) 1645, 1743, 2372,
3436; dH 1.45 (9H, s), 3.16–3.27 (2H, m), 3.70–3.73 (2H, m), 3.83
(1H, q, J = 7.2 Hz), 5.25 (1H, d, J = 6.0 Hz), 5.32 (1H, br s), 6.41
(1H, br s), 7.04–7.08 (1H, m), 7.22–7.39 (9H, m); dC 28.3, 31.6,
36.0, 52.8, 57.0, 80.4, 117.1, 124.2, 126.7, 128.7, 129.1, 134.0,
Yield 74%; off-white solid; cmax (KBr/cmꢂ1) 1643, 1749, 2370,
3427. dH 1.35 (9H, s), 3.40–3.55 (2H, m), 3.80–3.95 (5H, m), 5.22
(1H, d, J = 5.6 Hz), 5.31 (1H, br s), 6.39 (2H, m), 6.63 (1H, br s),
6.99 (1H, br s), 7.05–7.09 (1H, m), 7.24–7.27 (4H, m), 7.42 (1H, br
s); dC 28.2, 31.5, 36.0, 42.9, 51.1, 53.0, 81.2, 110.4, 110.7, 116.8,
124.2, 124.9, 129.0, 131.1, 137.1, 143.5, 147.9, 165.6, 168.8,
170.1; HRMS calcd for C23H28N4O6 + H+ 457.2087 found 457.2092.
137.2, 156.5, 165.8, 169.3; HRMS calcd for
410.2080 found 410.2084.
C
23H27N3O4 + H+
3.1.2. 2-tert-Butoxycarbonylamino-4-[(2-oxo-1, 4-diphenyl-
azetidin-3-ylmethyl)-carbamoyl]-butyric acid (25)
Yield 77%; off-white solid; isolated as inseparable mixture of
diastereomers; cmax (KBr/cmꢂ1) 1645, 1745, 2365, 3431; dH 1.44
(9H, s), 1.84–2.48 (6H, m), 3.26 (1H, m), 3.69–3.82 (1H, m), 4.03
(1H, br s), 4.29 (1H, br s), 5.10–5.25 (3H, m), 7.05–7.08 (1H, m),
7.26–7.45 (9H, m); dC [Major isomer] 27.9, 28.2, 35.9, 37.1, 51.7,
52.8, 57.0, 67.1, 80.2, 110.2, 116.7, 117.0, 124.0, 124.1, 125.8,
126.6, 128.4, 128.5, 128.6, 128.7, 128.9, 129.0, 129.1, 130.8,
133.9, 137.2, 143.5, 165.5, 165.7, 171.2, 171.2; [Minor isomer]
27.8, 28.8, 35.9, 38.6, 51.1, 52.9, 58.7, 68.0, 80.2, 110.8, 116.7,
117.0, 124.0, 124.1, 125.8, 126.6, 128.3, 128.5, 128.6, 128.7,
128.9, 129.0, 129.1, 130.8, 133.9, 137.2, 143.5, 165.5, 165.7,
171.2, 171.2; HRMS calcd for C26H31N3O6 + H+ 482.2291 found
482.2293.
3.1.5. (1-{1-[(2-Oxo-1,4-diphenyl-azetidin-3-ylmethyl)-
carbamoyl]-ethylcarbamoyl}-ethyl)-carbamic acid
tert-butyl ester (28)
Yield 76%; yellowish solid; isolated as inseparable mixture of
diastereomers; cmax (KBr/cmꢂ1
) 1643, 1746, 2366, 3432; dH
1.25–1.31 (6H, m), 1.45 (9H, s, Boc), 3.06–3.29 (2H, m), 3.78–3.86
(1H, m), 4.14 (1H, m), 4.28–4.40 (1H, m), 5.26 (1H, d, J = 6.0 Hz),
6.25 (1H, br s), 6.35 (1H, br s), 6.70 (1H, br s), 7.04–7.08 (1H, m),
7.23–7.38 (9H, m); dC [Major isomer] 18.3, 18.3, 28.3, 31.5, 36.0,
48.8, 52.6, 57.0, 80.2, 117.1, 117.1, 124.0, 124.1, 126.7, 126.7,
126.8, 128.6, 129.0, 133.9, 134.0, 137.2, 165.7, 172.0, 172.3,
172.7; [Minor isomer] 18.3, 18.3, 28.3, 31.5, 36.2, 48.7, 52.8, 57.0,
80.2, 117.1, 117.1, 124.0, 124.1, 126.7, 126.7, 126.8, 128.6, 129.1,

8372
K. Chandra et al. / Bioorg. Med. Chem. 18 (2010) 8365–8373
133.9, 134.0, 137.2, 165.7, 172.1, 172.3, 172.7; HRMS calcd for
27H34N4O5 + H+ 495.2607 found 495.2612.
3.3.1. 6-(Phenyl-phenylamino-methyl)-[1,4]diazepane-2,5-
dione (1)
C
Yield 73%; yellowish semi-solid; cmax (KBr/cmꢂ1) 1654, 1741,
2345, 3431; dH 3.14–3.28 (2H, m), 3.75–3.89 (3H, m), 5.25 (1H, d,
J = 5.6 Hz), 6.12 (1H, br s), 6.28 (1H, br s), 7.05–7.09 (1H, m),
7.26–7.36 (9H, m); dC 36.0, 43.0, 52.6, 57.0, 117.1, 124.0, 124.2,
126.7, 127.0, 128.7, 128.8, 129.1, 129.1, 133.9, 134.3, 137.1,
168.9, 170.7; HRMS: calcd for C18H19N3O2 + H+ 310.1556 found
310.1559.
3.1.6. [2-(4-Hydroxyphenyl)-1-({[(2-oxo-1,4-diphenyl-azetidin-
3-ylmethyl)-carbamoyl]-methylcarbamoyl)-ethyl]-carbamic
acid tert-butyl ester (29)
Yield 75%; white semi-solid; isolated as inseparable mixture of
diastereomers; cmax (KBr/cmꢂ1) 1628, 1749, 2375, 3630; dH 1.42
(9H, s), 2.87–3.15 (4H, m), 3.65–3.81 (3H, m), 4.50 (1H, m), 5.14
(1H, m,), 6.34 (1H, br s), 6.70–7.34 (14H, m); dC [Major isomer]
28.2, 35.9, 44.1, 52.6, 53.2, 54.2, 57.0, 80.2, 110.7, 115.6, 116.8,
117.1, 121.5, 121.7, 124.1, 126.6, 127.4, 128.6, 128.9, 129.0,
130.2, 130.3, 133.7, 133.8, 137.1, 143.5, 155.5, 166.0, 169.7,
171.0; [Minor isomer] 28.2, 37.2, 44.1, 53.0, 54.0, 54.3, 56.9, 80.2,
110.5, 115.6, 116.8, 117.1, 121.5, 121.7, 124.1, 126.6, 127.4,
128.6, 128.9, 129.0, 130.2, 130.3, 133.7, 137.0, 137.1, 143.5,
3.3.2. 4,9-Dioxo-3-(phenyl-phenylamino-methyl)-
[1,5]diazonane-6-carboxylic acid (2)
Yield 68%; yellowish powder; was obtained as an inseparable
mixture of diastereomer; cmax (KBr/cmꢂ1) 1631, 1755, 2336,
3670; dH 2.08–2.45 (4H, m), 2.95–3.29 (1H, m), 3.85 (1H, m),
4.03–4.11 (1H, m), 5.25 (1H, d, J = 4.8 Hz), 6.61 (1H, br s), 6.82
(1H, br s), 7.07 (1H, m), 7.23–7.44 (9H, m); dC: [Major isomer]
25.7, 29.1, 36.2, 52.3, 56.8, 57.0, 116.8, 117.1, 124.3, 125.8, 126.6,
128.7, 128.8, 129.1, 129.2, 133.8, 137.1, 143.7, 155.9, 167.6,
172.0; [Minor isomer] 25.7, 29.2, 36.2, 52.2, 56.8, 57.0, 116.8,
117.1, 124.3, 125.8, 126.6, 128.7, 128.8, 129.1, 129.2, 133.8,
137.1, 143.8, 154.0, 159.0, 166.3; HRMS calcd for C21H24N3O4 + H+
382.1767 found 382.1770.
155.2, 165.9, 170.5, 170.9; HRMS calcd for
573.2713 found 573.2715.
C
32H36N4O6 + H+
3.1.7. [1-(1-{1-[(2-Oxo-1,4-diphenyl-azetidin-3-ylmethyl)-
carbamoyl]-ethylcarbamoyl} ethyl carbamoyl) ethyl]-carbamic
acid tert-butyl ester (30)
Yield 75%; yellowish semi-solid; isolated as inseparable mixture
of diastereomer; cmax (KBr/cmꢂ1) 1645, 1748, 2375, 3442; dH
1.25–1.47 (18H, m), 3.07–3.27 (2H, m), 3.94 (1H, m), 4.13–4.41
(3H, m), 5.08 (1H, br s), 5.26 (1H, dd, J = 5.6 Hz, J = 6.0 Hz), 6.51
(1H, br s), 6.94 (1H, br s), 7.03–7.08 (1H, m), 7.25–7.38 (9H, m);
dC [Major isomer] 17.9, 18.1, 18.4, 28.3, 36.0, 48.9, 49.1, 51.4,
52.9, 57.4, 80.5, 117.1, 124.0, 124.1, 126.7, 126.9, 128.5, 128.6,
128.9, 129.1, 133.9, 134.0, 137.4, 156.1, 165.9, 172.1, 172.2,
173.8; [Minor isomer] 17.9, 18.1, 18.4, 28.3, 36.0, 49.1, 49.7, 51.2,
52.5, 57.2, 80.5, 117.1, 124.0, 124.1, 126.7, 126.9, 128.5, 128.6,
128.9, 129.1, 133.9, 134.0, 137.2, 156.1, 166.0, 172.0, 172.3,
173.1; HRMS calcd for C25H31N5O4 + H+ 466.2454 found 466.2455.
3.3.3. 9-(Phenyl-phenylamino-methyl)-[1,4,7]triazecane-2,5,8-
trione (3)
Yield 73%; off-white semi-solid; cmax (KBr/cmꢂ1) 1654, 1741,
2369, 3432; dH 3.03 (1H, m), 3.27 (1H, m), 3.81–3.97 (5H, m),
5.25 (1H, d, J = 5.6 Hz), 6.51 (1H, br s), 6.95 (1H, br s), 7.08 (1H,
m), 7.26–7.37 (9H, m); dC 36.2, 42.8, 43.4, 52.2, 57.0, 117.1,
124.2, 126.5, 128.7, 129.0, 129.1, 133.7, 137.0, 166.2, 168.5,
169.4; HRMS calcd for C20H22N4O3 + H+ 367.1770 found 367.1776.
3.3.4. 9-(Furan-2-yl-phenylamino-methyl)-[1,4,7]triazecane-
2,5,8-trione (4)
Yield 72%; off-white semi-solid; cmax (KBr/cmꢂ1) 1645, 1751,
2368, 3410; dH 3.31–3.69 (7H, m), 4.65 (1H, br s), 5.22 (1H, d,
J = 5.6 Hz), 6.40 (2H, m), 6.75 (1H, br s), 7.08 (1H, m), 7.26 (4H,
m), 7.43 (1H, br s); dC 36.0, 44.4, 51.2, 52.9, 62.1, 110.4, 110.7,
116.8, 116.8, 124.4, 129.1, 137.1, 143.6, 143.6, 147.8, 167.7,
167.9, 169.8; HRMS calcd for C18H20N4O4 + H+ 357.1563 found
357.1567.
3.2. General procedure for the deprotection of N-Boc b-lactam
peptides conjugates
The N-Boc-protected b-lactam-peptides conjugates (1 equiv)
dissolved in 10 ml dry DCM was treated dropwise with TFA
(20 equiv) at 0 °C for 30 min. The mixture was evaporated in vacuo,
washed twice with benzene and finally diluted with Na2CO3
solution followed by EtOAc. The solution was stirred for another
30 min. The organic layer was separated, washed with brine and
dried over anhydrous sodium sulfate solution. Finally organic layer
was concentrated in vacuo. The crude product obtained can be
treated as pure solid starting material (Rf = 0.3, 5:1 DCM/MeOH)
without being further purification by column chromatography.
3.3.5. 3,6-Dimethyl-9-(phenyl-phenylamino-methyl)-[1,4,7]-
triazecane-2,5,8-trione (5)
Yield 70%; off-white semi-solid product which was isolated as
inseparable mixture of diastereomer; cmax (KBr/cmꢂ1
) 1646,
1742, 2368, 3435; dH 1.25–1.46 (6H, m), 3.12–3.27 (2H, m),
3.45–3.50 (1H, m), 3.69–3.82 (2H, m), 5.24 (1H, m), 6.74 (1H, br
s), 7.06 (1H, m), 7.23–7.39 (9H, m); dC [Major isomer] 17.8, 17.9,
36.2, 48.7, 52.5, 52.5, 56.9, 117.1, 124.1, 126.6, 128.6, 128.6,
129.0, 129.1, 131.9, 131.9, 132.8, 133.9, 137.1, 166.2, 170.9,
179.2. [Minor isomer] 17.8, 17.9, 36.1, 48.9, 52.3, 52.5, 56.9,
117.0, 124.1, 126.6, 128.3, 128.5, 129.0, 129.0, 131.8, 131.9,
132.9, 133.9, 137.1, 166.1, 170.9, 172.3; calcd for C22H26N4O3 + H+
395.2083 found 295.2085.
3.3. General procedure for the synthesis of cyclic peptides
The b-lactam containing peptidyl free amine (1 equiv) was dis-
solved under high dilution condition in distiled EtOH (20 ml) in
presence of a buffer mixture of Et3N and acetic acid. The overall
mixture was stirred at 40–60 °C for several hours as indicated in
Table 1. After checking the TLC the reaction was quenched by
adding saturated NaHCO3 solution. It was washed with 1 N HCl
followed by brine and finally extracted with EtOAc before drying
over anhydrous sodium sulfate solution. The organic layer was
concentrated in rotavapor and the crude mixture was then purified
in flash chromatography (230–400 silica gel) using DCM/MeOH
mixture (50:1–10:1) solvent (Rf = 0.3) yielding 65–73% non separa-
ble mixture of diastereomeric products. All the diastereomers has
been well characterized by NMR and Mass spectrometry after fur-
ther purified by HPLC.
3.3.6. 6-(4-Hydroxy-benzyl)-9-(phenyl-phenylamino-methyl)-
[1,4,7]triazecane-2,5,8-trione (6)
Yield 70%; off-white semi-solid; was isolated as inseparable
mixture of diastereomer; cmax (KBr/cmꢂ1) 1646, 1742, 2368,
3435. dH 2.97–3.17 (4H, m), 3.73–3.82 (3H, m), 5.16 (1H, d,
J = 5.2 Hz), 5.22–5.27 (1H, m), 6.40 (1H, br s), 6.51 (1H, br s),
6.78–7.36 (14H, m). dC [Major isomer] 29.6, 36.0, 36.9, 52.5, 54.5,
57.0, 110.7, 115.6, 117.1, 124.2, 125.9, 126.6, 127.7, 128.5, 128.6,
128.8, 129.0, 130.3, 132.0, 133.7, 137.0, 143.5, 155.3, 166.3,

K. Chandra et al. / Bioorg. Med. Chem. 18 (2010) 8365–8373
8373
2. (a) Koul, A.; Choidas, A.; Treder, M.; Tyagi, A. K.; Drlica, K.; Singh, Y.; Ullrich, A. J.
Bacteriol. 2000, 182, 5425; (b) Cowley, S. C.; Babakaiff, R.; Av-gay, Y. Res.
Microbiol. 2002, 153, 233.
3. Cole, S. T.; Brosch, R.; Parkhill, J.; Garnier, T.; Churcher, C.; Harris, D.; Gordon, S.
V.; Eiglmeier, K.; Gas, S.; Barry, C. E.; Tekaia, F.; Badcock, K.; Basham, D.; Brown,
D.; Chillingworth, T.; Connor, R.; Davies, R.; Devlin, K.; Feltwell, T.; Gentles, S.;
Hamlin, N.; Holroyd, S.; Hornsby, T.; Jagels, K.; Krogh, A.; McLean, J.; Moule, S.;
Murphy, I.; Oliver, K.; Osborne, J.; Quail, M. A.; Rajandream, M. A.; Roger, J.;
Rutter, S.; Seeger, K.; Skelton, J.; Squares, R.; Squares, S.; Sulston, J. E.; Taylor, K.;
Whitehead, S.; Barell, B. G. Nature 1998, 393, 537.
171.1; [Minor isomer] 29.2, 36.2, 37.2, 52.5, 54.5, 57.0, 110.7,
115.6, 117.1, 124.2, 125.9, 126.6, 127.7, 128.5, 128.6, 128.8,
129.0, 130.3, 132.0, 133.7, 137.0, 143.5, 155.3, 166.3, 171.1; HRMS
calcd for C27H28N4O4 + H+ 473.2190 found 473.2195.
3.3.7. 3,6,9-Trimethyl-12-(phenyl-phenylamino-methyl)-
1,4,7,10 tetraaza-cyclotridecane-2,5,8,11-tetraone (7)
Yield 75%; off-white semi-solid; isolated as inseparable mixture
of diastereomer; cmax (KBr/cmꢂ1) 1644, 1748, 2345, 3428; dH
1.24–1.30 (9H, m), 2.95–3.27 (3H, m), 3.82 (1H, m), 4.34–4.37
(2H, m), 5.22 (1H, dd, J1 = J2 = 5.6 Hz), 6.63 (1H, br s), 6.83 (1H,
br s), 7.04 (1H, m), 7.24–7.33 (9H, m), 7.80 (1H, br s). dC [Major iso-
mer] 17.6, 17.7, 17.7, 36.0, 48.8, 49.0, 50.6, 52. 5, 57.0, 117.1, 124.1,
126.6, 126.6, 128.4, 128.5, 128.9, 129.0, 129.0, 133.8, 133.8, 137.1,
166.1, 172.1, 172.6, 176.4; [Minor isomer] 17.6, 17.7, 17.7, 36.2,
48.8, 48.9, 49.0, 52.3, 57.0, 117.1, 124.1, 126.6, 126.6, 128.4,
128.5, 128.9, 129.0, 129.1, 133.8, 133.8, 137.1, 166.2, 166.3,
172.3, 172.6; HRMS calcd for C25H31N5O4 + H+ 466.2454 found
466.2458.
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K.C. and D.D. thank Council of Scientific and Industrial Research
(CSIR) and Department of Biotechnology (DBT), Government of
India, respectively for fellowship. Central Research Facility and
Department of Chemistry, IIT Kharagpur are thanked for providing
all the instrumental facilities. A.B. and A.K.D. gratefully acknowl-
edge the support of DBT for providing the necessary research grant.
Supplementary data
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Supplementary data associated with this article can be found, in
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