Amino acid-based enantiomerically pure 3-substituted 1,4-benzodiazepin-2-ones: A new class of anti-ischemic agents

Article abstract of DOI:10.1016/j.bmcl.2006.12.001

A series of 3-substituted 1, 4-benzodiazepin-2-ones derived from S and R amino acids were evaluated for their anti-ischemic activity in vitro. Treatment with compounds 7h, 16, 9d, and 17 decreased the apoptotic neuronal number, however increased the neuronal viability. The compounds decreasing apoptosis could protect neurons from the ischemic injury. The difference in the activities of 1,4-benzodiazepin-2-ones derived from S- and R-amino acids is discussed and explained on the basis of molecular modeling studies.

Full text of DOI:10.1016/j.bmcl.2006.12.001

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1326–1331
Amino acid-based enantiomerically pure 3-substituted 1,4-
benzodiazepin-2-ones: A new class of anti-ischemic agentsq
Jitendra Kumar Mishra,a Puja Garg,b Preeti Dohare,b Ashutosh Kumar,c
Mohammad Imran Siddiqi,c Madhur Rayb and Gautam Pandaa,*
aMedicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow 226001, UP, India
bPharmacology Division, Central Drug Research Institute, Lucknow 226001, UP, India
cMolecular and Structural Biology Division, Central Drug Research Institute, Lucknow 226001, UP, India
Received 1 September 2006; revised 28 November 2006; accepted 1 December 2006
Available online 3 December 2006
Abstract—A series of 3-substituted 1, 4-benzodiazepin-2-ones derived from S and R amino acids were evaluated for their anti-ische-
mic activity in vitro. Treatment with compounds 7h, 16, 9d, and 17 decreased the apoptotic neuronal number, however increased the
neuronal viability. The compounds decreasing apoptosis could protect neurons from the ischemic injury. The difference in the activ-
ities of 1,4-benzodiazepin-2-ones derived from S- and R-amino acids is discussed and explained on the basis of molecular modeling
studies.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Brain injury by transient or permanent ischemia afflicts
H C
3
3
OR
H
N
O
OCH
H
3
N
4
a large number of patients with death or permanent dis-
ability.1 With the onset of ischemia, critical balance be-
tween the demand and supply of oxygen and substrates
fails, leading to the damage of reversible or irreversible
cellular interdependent pathways and it can be managed
by timely protection offered by the drugs.
R
1
2
R
R
Cl
N
N
CON
O
2, Anthramycin (R4=CH3, R3= R2=R1=H)
3, Porothramycin B (R4=H, R3= R2=R1=CH3)
1
Ro5-4864
Figure 1. Biologically active 1, 4-benzodiazepine derivatives.
Benzodiazepine derivatives are of considerable interest
because of their wide range of biological activities such
as anticonvulsant,2 cholecystokinin receptor A and
receptor B antagonists,3 platelet-activating factor antag-
onists,4 GPIIb/IIIa inhibitors,5 and Ras farnesyltrans-
ferase inhibitors.6 Another well-known member of
benzodiazepine family is pyrrolo[2,1-c][1,4]benzodiaze-
pines (PBDs), known for their potential as antitumor
agents, gene regulators, and DNA probes.7 Although di-
verse biological activities of 1,4-benzodiazepines are
known, to the best of our knowledge there is no report
on anti-ischemic efficacy of 3-substituted 1,4-ben-
zodiazepin-2-ones (Fig. 1).
Toward the objective of finding new anti-ischemic
agents, we have synthesized a series of S and R-amino
acid-based enantiomerically pure 3-substituted 1,4-ben-
zodiazepin-2-ones8 (Schemes 1 and 2) and screened for
anti-ischemic activity in vitro following the literature
methods.9–12 Compounds 7a–h, 14, 15, and 16 derived
from S-amino acids were evaluated for apoptotic/ne-
crotic quantification of neuronal population in vitro
(Fig. 2 and Table 1). Control neurons (LL) showed
no FITC/PI-positive staining (0.05%/0.6%). The num-
ber of FITC-positive neurons (LR) was increased to
30.22% in ischemic neurons. Compounds 7h and 16
derived from S-methionine and 4-hydroxy-S-proline
decreased the number of FITC-positive neurons to
9.08% and 3.79%, respectively. The number of viable
neurons was also increased by the treatment of
compounds. In the ischemic group without treatment,
Keywords: Anti-ischemic agents; 1,4-Benzodiazepin-2-ones; Amino
acids.
q
CDRI communication No. 6935.
Corresponding author. Tel.: +915222364635; fax: +915222623938;
e-mail: gautam.panda@gmail.com
*
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.001

J. K. Mishra et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1326–1331
7a, R= CH
1327
3
R
7b, R= C H N
7c, R= CH SCH C H
2 6 5
7d, R= CH COOCH
7e, R= CH C H (p-OCH C H )
7f, R= CH CH CONH
2 2 2
7g, R= CH C H
2 6 5
7h, R= CH CH SCH
3
9
8
ClH.H2N
COOCH3
H
N
2
O
(S-amino acids methyl
ester hydrochloride)
NO
2
2
3
COOCH
R
3
c
R
2
6
4
2 6 5
b
NH
O
N
H
NO
NO
Br
2
2
a
7a-h
6a-h
NO
2
2
H
N
CH
3
2
COOCH
R
3
b
9a, R= CH
3
9b, R= C H N
9 8
c
4
5
R
R
N
H
NH
9c, R= CH C H
6 5
9d, R= CH CH SCH
3
2
ClH.H2N
COOCH3
8a-d
9a-d
(R-amino acids methyl
ester hydrochloride)
2
2
Scheme 1. Syntheses of 3-substituted 1,4-benzodiazepin-2-ones derived from S- and R-amino acids. Reagents and conditions: (a) NBS,
(C6H5CO)2O2, CCl4, 80 ꢁC, 69%; (b) DIPEA, DMF, rt, 2 h, 62–78%; (c) Fe, AcOH, 110 ꢁC, 30 min, 52–85%.
H
N
O
NO2
S
COOCH3
N
H
b
H
a,
N S
N
78%
68%
10
11
14
COOCH3
H
N
O
S
NO2
O
H3COOC
O
N
H
b
H
NS
N
90%
a,
84%
15
NO
2
H3COOC
NO2
O
HO
Br
H
O
S
COOCH3
N
N
H
b
H
5
OH
S
N
89%
a,
87%
N
12
OH
16
H3COOC
NO2
H
N
R
O
COOCH3
N
H
b
H
N R
N
a,
64%
62%
13
COOCH3
17
Scheme 2. Reagents and conditions: (a) DIPEA, DMF, rt, 2 h; (b) Fe, AcOH, reflux, 30 min.
Figure 2. Representative dot plots of Flow cytometric profile observed in 7h, 16 and 14. The staining of cells simultaneously with FITC-Annexin V
(green fluorescence) and the nonvital dye propidium iodide (red fluorescence), that is, bivariate analysis allows the discrimination of intact cells. The
lower left quadrant (LL) represents viable cells (FITCꢀPIꢀ) whereas lower right quadrant (LR) is for early apoptotic (FITC+PIꢀ). On the other
hand, the upper right quadrant (UR) is for late apoptotic (FITC+PI+) and the upper left quadrant (UL) is for necrotic cells (FITCꢀPI+). The dot
plots represent two measurement parameters on the x- and y-axes. Cell count height and the particle counts are shown by density gradient and dot
density, respectively. Dot plot displays FL1-FITC on the x-axis and FL2-PI on the y-axis. Logarithmic amplification is used to measure fluorescence
in cells as this expands the scale for weak signals and compresses the scale for strong or specific fluorescence signals.

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J. K. Mishra et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1326–1331
Table 1. In vitro assay of 1,4-benzodiazepin-2-ones derived from S-amino acids for apoptotic/necrotic quantification of neuronal population
UL (necrosis)
0.88 0.4
UR (necrosis + delayed apoptosis)
2.34 0.8
LL (normal viable)
91.31 3.8
LR (apoptosis)
6.47 0.9
Control
Ischemia
1.57 0.2
5.35 0.6
7.82 1.3
4.8 0.9
60.39 2.9
60.38 3.1
30.22 1.5
29.47 2.1
H
N
O
1
2
CH3
3
4NH
7a
H
O
N
1
2
3
4.33 1.2
5.05 0.8
4.73 0.5
3.55 1.1
4.58 1.3
4.28 0.8
61.26 4.3
64.36 5.2
65.24 5.6
30.86 0.9
26.01 1.7
25.75 1.8
4NH
N
H
7b
H
N
O
CH2SCH2C6H5
CH2COOCH3
NH
O
7c
H
N
NH
O
7d
H
N
4.99 0.5
4.83 1.2
66.55 5.7
23.63 1.4
NH
OCH2C6H5
7e
7f
H
N
O
CH2CH2CONH2
4.75 0.3
2.07 0.3
4.73 0.7
5.56 0.6
5.51 0.4
5.68 0.8
3.12 0.7
1.21 1.3
3.5 0.8
67.2 3.7
70.14 4.6
82.69 6.3
66.07 4.8
66.18 5.1
87.45 6.7
24.93 2.1
25.58 1.1
9.08 0.4
24.62 1.1
23.27 1.2
3.79 0.3
NH
H
O
N
1
2
3
4 NH
7g
H
O
N
1
2
3
CH2CH2SCH3
4NH
7h
15
H
N
O
H
3.75 0.8
5.04 1.4
3.08 1.0
N
O
O
H
N
H
H
1
2
3
4
N
14
H
N
O
N
OH
16
UL, upper left quadrant; UR, upper right quadrant; LL, lower left quadrant; LR, lower right quadrant.
neuronal viability was 61.11% (cells falling in lower
left quadrant). Among all the compounds tested 7h
and 16 showed increase in neuronal viability to
82.69 and 87.45%, respectively. The 1,4-benzodiaze-
pin-2-ones derived from S-alanine, S-aspartic acid,
S-glutamine, and S-methionine showed 60.38%,
65.24%, 67.20% and 82.69% neuronal viability, respec-
tively. On the other hand, 1,4-benzodiazepin-2-ones
derived from S-amino acids containing aromatic side
chains such as tryptophan, cysteine (S-Bn), tyrosine
(–OBn), and phenylalanine showed 61.26%, 64.36%,
66.55%, and 70.14% neuronal viability, respectively.
To test the difference in the activities of enantiomers, we
synthesized 1,4-benzodiazepin-2-ones derived from R-
amino acids following the method reported by our
group (Schemes 1 and 2).8 In this context 9a, 9b, 9c,
9d and 17 were evaluated for apoptotic/necrotic quanti-
fication of neuronal population in vitro (Fig. 3 and
Table 2). Careful analysis of Tables 1 and 2 suggests that

J. K. Mishra et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1326–1331
1329
Figure 3. Representative dot plots of flow cytometric profile observed in 9a, 9b, 9c, 9d, and 17.
Table 2. In vitro assay of 1,4-benzodiazepin-2-ones 9a, 9b, 9c, 9d, and 17 derived from R-amino acids for apoptotic/necrotic quantification of
neuronal population
20
Yield, ½aꢁD
UL (necrosis) UR (necrosis + delayed apoptosis) LL (normal viable) LR (apoptosis)
Control
1.21 0.7
0.745 0.3
0.98 0.14
2.8 1.3
6.6 0.3
3.2 0.4
93.34 2.3
65.89 2.79
68.96 2.42
2.6 0.2
26.76 2.1
26.85 1.88
Ischemia
H
N
O
1
2
52%, ꢀ15ꢁ (c 1, CH2Cl2)
72%, ꢀ42ꢁ (c 1, CH2Cl2)
85%, +23ꢁ (c 1, CH2Cl2)
63%, ꢀ12ꢁ (c 1, CH2Cl2)
64%, ꢀ21ꢁ (c 1, CH2Cl2)
CH3
3
4NH
9a
H
O
N
1
2
3
0.5 0.2
0.68 0.41
0.52 0.29
0.64 0.36
3.47 0.33
0.68 0.41
3.98 0.39
3.10 0.34
74.97 2.04
79.01 0.58
77.85 2.29
84.53 0.80
21.06 2.1
17.26 0.13
16.96 0.94
11.72 0.79
4 NH
N
H
9b
H
O
N
1
2
3
4NH
9c
H
O
N
SCH3
1
2
3
4NH
9d
H
O
N
1
2
3
4
N
17
S-methionine-derived 1,4-benzodiazepine-2-one 7h is
more active than its R-isomer 9d, whereas in the case
of proline R-isomer 17 is more active as compared to
its S-isomer 14. To gain insight into the difference in
activities of R- and S-isomers, docking studies were car-
ried out with ligand binding core of AMPA receptor

1330
J. K. Mishra et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1326–1331
with the help of modern docking engine LigandFit avail-
able with Cerius 2 4.10.13
docking studies were taken from Protein DataBank
(PDB). Charges were assigned to the protein molecule
and the docked ligands using the Cff1.02 force field.16
A number of benzodiazepines act as negative modulators
of AMPA receptor14 and inhibit AMPA-induced current
in a noncompetitive fashion.15 The negative allosteric
modulators regulate the frequency and amplitude of the
excitatory neurotransmitters independent of the concen-
tration of excitatory amino acids or polarization state of
the synaptic membrane. The negative modulators of
AMPA receptor bind at the same site as the positive mod-
ulators of AMPA, so we have taken the crystal structure
of ligand binding core of AMPA receptor in complex with
noncompetitive positive modulator, aniracetum (PDB
The binding site of modulators is located in a solvent-
filled crevice, at the dimer interface and it is proximal
to the transdomain-strands that undergo conformation-
al changes during agonist binding and domain closure.17
Strikingly, a key element of the modulator-binding site in-
volves a U-shaped crevice that is formed by main-chain
residues Pro 494 through Ser 497. Pro 494 forms the apex
of the crevice, whereas Ser 497 and 729 define its base.
˚
entry 2AL5, resolution: 1.65 A), as the crystal structure
in complex with noncompetitive negative modulator
was not available. Reference protein coordinates for
A qualitative analysis of the binding mode found in the
docked benzodiazepin-2-ones-AMPA receptor complex
revealed that both the enantiomers bind in different con-
Figure 4. Docked conformations of 1,4-benzodiazepine-2-ones (green) 7h, 9d, 14, and 17 with ligand binding core of AMPA receptor noncompetitive
antagonist binding site.

J. K. Mishra et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1326–1331
Table 3. Docking scores using different scoring functions
1331
2. Sternbach, L. H. J. Med. Chem. 1979, 22, 1.
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Int. Patent Appl., WO 93/00095.
6. James, G. L.; Goldstein, J. L.; Brown, M. S.; Rawson, T.
E.; Somers, T. C.; McDowell, R. S.; Crowley, C. W.;
Lucas, B. K.; Levinson, A. D.; Marsters, J. C. Science
1993, 260, 1937.
Scores
Compounds
7h
43.883
9d
41.213
14
17
51.118
Dock_Score
LigScore1
LigScore2
ꢀPLP1
13.173
2.290
2.760
59.270
60.410
3.200
ꢀ61.480
461
1.990
1.770
74.660
74.520
1.690
ꢀ22.870
385
3.420
4.830
83.690
79.480
1.610
ꢀ30.960
428
3.920
4.710
72.760
72.180
1.280
ꢀ33.250
491
ꢀPLP2
JAIN
PMF
LUDI
Cons_Score
11
12
6
12
7. Advances in the Study of Pyrrolo[2,1-c][1,4]benzodiaze-
pine (PBD) Antitumour Antibiotics. In Molecular
Aspects of Anticancer Drug–DNA Interactions; Neidle,
S., Waring, M. J., Eds.; The Macmilan: London, 1993;
Vol. 1, p 54.
8. Mishra, J. K.; Panda, G. Synthesis 2005, 1881.
9. Oyama, Y.; Chikahisa, L.; Ueha, T.; Kanemaru, K.;
Noda, K. Brain Res. 1996, 712, 349.
10. Sureda, F. X.; Camins, A.; Trullas, R.; Camarasa, J.;
Escubedo, E. Brain Res. 1996, 723, 110.
11. Boldyrev, A. A.; Carpenter, D. O.; Huentelman, M. J.;
Peters, C. M.; Johnson, P. Biochem. Biophys. Res. Com-
mun. 1999, 256, 320.
12. Experimental procedure: The forebrain neurons were
isolated. Three groups were assigned to the isolated
neuron at 1 · 106 neuronal cells/100 ll: (1) no treatment,
(2) hydrogen peroxide treated, and (3) Pretreated with test
compound followed by H2O2 treatment. For H2O2 treat-
ment neurons were treated with equal volume of H2O2
(concentration of 300 lM) for one hour at 37 ꢁC. This
treatment resulted in significant number of death of the
neurons either by apoptosis or necrosis mode of death.
For pretreated group neurons were treated with each
compound (100 lM) separately for 20 min at 37 ꢁC
followed by H2O2 treatment. For no treatment group
neurons were maintained for 1 h and 20 min at 37 ꢁC. The
concentration of no treatment, H2O2, and/or compound
treated neurons is 1 · 106 cells/100 ll. Neurons were
washed twice with cold PBS and treated with binding
buffer, FITC-labeled Annexin V and Propidium iodide
(PI) essentially following the manufacturer’s protocol
(Oncogene, Apoptosis Detection kit). After incubation at
room temperature for 15 min in the dark, fluorescence of
10,000 neuronal cells was acquired, as it is possible to
identify and quantitate apoptotic cells on a single-cell basis
by flow cytometry. Neuronal fluorescence was deter-
mined16 at an excitation wavelengths of 488 nm and
emission wavelength of 530 and 670 nm with Flow
Cytometer (Becton–Dickinson, UK) and analyzed in a
Cell Quest program. Those compounds showing >80%
viable population of neurons, falling in the left lower
quadrant, are considered active. Acquisition and analysis
were performed with the Cell Quest software package
(Becton–Dickinson, UK, Ltd).
formation and it may be responsible for the difference in
the activities of 1,4-benzodiazepin-2-ones. The nitrogen
atom N4 of 17 interacts with backbone nitrogen of
Gly 731 forming a direct hydrogen bond and it also
forms water-mediated hydrogen bonds with oxygen of
Ser 729, oxygen of Lys 730, and nitrogen of Gly 731
while 14 binds in a different orientation (Fig. 4, 17 and
14). The difference in binding affinity is also revealed
by comparing the docking scores. The compound 17 is
having LigandFit Dock_Score of 51.118 as compared
to 14 with 13.173 (Table 3). S- and R-isomers of methi-
onine-based 1,4-benzodiazepin-2-ones 7h and 9d exhibit
similar LigandFit Dock score (Table 3, 7h, 43.883; 9d,
41.213) and bind with the receptor in almost similar
fashion. Both isomers form direct hydrogen bonds with
backbone oxygen of Lys 730 and backbone nitrogen of
Gly 731 (Fig. 4, 7h and 9d).
In summary, a series of 3-substituted 1,4-benzodiazepin-
2-ones derived from S- and R-amino acids were evaluat-
ed for anti-ischemic activity in vitro. Among the com-
pounds tested 7h, 16, 9d, and 17 showed promising
activity. The molecular modeling studies demonstrate
that the stereochemistry of 3-substituted benzodiaze-
pin-2-ones may affect the anti-ischemic efficacy.
Acknowledgments
Jitendra, Puja, Preeti, and Ashutosh thank CSIR for
providing fellowships. We thank Mr. A. L.Vishwakar-
ma for flow cytometric analysis. The DST, New Delhi,
India, supported this project.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.12.001.
13. Cerius2 Version 4.10, Accelrys Inc.: 9685 Scranton Rd.,
San Diego, CA 92121, U.S.A., http://www.accelrys.com.
14. Tarnawa, I.; Molnar, P.; Gaal, L.; Andrasi, F. Acta
Physiol. Hung. 1992, 79, 163.
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