Synthesis, antileukemic and antiplatelet activities of 2,3-diaryl-6,7-dihydro-5H-1,4-diazepines

Article abstract of DOI:10.1016/j.ejmech.2007.11.023

The synthesis, antileukemic and antiplatelet activity evaluation of 2,3-diaryl-6,7-dihydro-5H-1,4-diazepines are described. In general, it was found that compound 17o showed moderate antileukemic activity against MOLT3 human leukemic cancer cell lines. An arachidonic acid induced platelet aggregation effect on washed rat platelets was studied. Compound 17i was found to be the most potent. The antiplatelet properties may be mediated by interference with the arachidonic acid pathway.

Full text of DOI:10.1016/j.ejmech.2007.11.023

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2004e2010
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, antileukemic and antiplatelet activities of
2,3-diaryl-6,7-dihydro-5H-1,4-diazepines
a
a
b
R. Ramajayam a, Rajani Giridhar a, , M.R. Yadav , R. Balaraman , Hakim Djaballah ,
*
David Shum b, Constantin Radu b
a Pharmacy Department, Faculty of Technology and Engineering, The M.S University of Baroda, Kalabhavan, Vadodara 390 001, India
b HTS Core Facility, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 487, New York, NY 10583, USA
Received 14 August 2007; received in revised form 26 November 2007; accepted 27 November 2007
Available online 11 January 2008
Abstract
The synthesis, antileukemic and antiplatelet activity evaluation of 2,3-diaryl-6,7-dihydro-5H-1,4-diazepines are described. In general, it was
found that compound 17o showed moderate antileukemic activity against MOLT3 human leukemic cancer cell lines. An arachidonic acid in-
duced platelet aggregation effect on washed rat platelets was studied. Compound 17i was found to be the most potent. The antiplatelet properties
may be mediated by interference with the arachidonic acid pathway.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Diaryldiazepines; Antileukemic; Antiplatelet activity
1. Introduction
imidazole structure 5 shows remarkable selectivity for the allo-
steric modulation of GABAA receptor [5], whereas 1,2-diphe-
nylimidazolyl piperazine derivatives 6 as antipsychotic agents
are endowed with substantial affinities for both D2 dopamine
receptors as well as 5-HT1A and 5-HT2A serotonin receptors
[6]. The 4,5-bis(4-hydroxyphenyl)-2-imidazoline scaffold 7
displays selective estrogen receptor modulating (SERM) by
either agonistic or antagonistic effects [7], while compound 8
acts as a selective p38a inhibitor in inflammatory disease [8],
diarylfurazans 9 (combretastatin analogs) act as antimitotic
agents [9] and compound 10 acts as non-nucleoside reverse-
transcriptase inhibitor [10,11]. Diaryl five membered heterocy-
clic compounds 11 and pyrazino thiadiazine derivatives 12 are
reported for antiplatelet activity [12e15]. Fig. 1 shows the vic-
inal diaryl system with various heterocyclic cores for diverse
therapeutic categories.
Syntheses of diverse pools of small drug-like molecules
possessing vicinal diaryl motifs are of recent interest in the
field of medicinal chemistry. The vicinal diaryl template 1
has recently received wide attention in the pharmaceutical
industry as a privileged substructure. Synthesis of COX-II in-
hibitors containing various five, six membered and fused het-
erocyclic systems [1,2] with a vicinal diaryl motif 2 have
been described over the past few years. Until now, two com-
pounds, celecoxib and rofecoxib, have been launched for the
treatment of inflammatory processes. The cannabinoid receptor
antagonists 3 were reported with the above structural require-
ments with slight modifications [3]. Molecules incorporating
the unique structure of this scaffold have demonstrated activity
across many therapeutic classes, viz., 2,3-diphenyl-1,2,4-
thiadiazole 4 which has shown to act as a potential adenosine
A1 receptor antagonist [4], while substituted 1,2-diphenyl
Recently, our research on the vicinal diaryl systems 13 and
14 gave a large number of compounds (Fig. 1) endowed with
interesting pharmacological activity such as COX II inhibition
[16,17]. 1,4-Diazepines are reported for various types of phar-
macological activity [18]. However, 1,4-diazepines containing
the vicinal diaryl system is very scantily reported [19]. In this
* Corresponding author. Tel.: þ91 0265 2434187; fax: þ91 0265 2423898.
E-mail address: rajanimsu@rediffmail.com (R. Giridhar).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.11.023

R. Ramajayam et al. / European Journal of Medicinal Chemistry 43 (2008) 2004e2010
2005
H3CO2S/H2NO2S
HN
O
R
X
X
X
X = carbocyclic,
5/6 membered/fused ring
3
1
2
N
S
N
N
R
OC2H5
N
N
N
N
F
N
N
O
H3CO
4
5
6
R
OCH3
HO
R2
H3CO
H
H
S
CH3
O
N
N
R = halogens
X=CH/N
S
N
H3CO
HO
N
X
R3
N
H
O
N
O
N
N
H
N
8
R1
9
7
10
OCH3
NH2
X
X
N
N
N
X
O
N
R
O
S
N
N
N
N
N
R
X = S/N
O
O
12
11
Y
14
13
Y
Fig. 1. Structures of vicinal diaryl heterocycles.
paper we report 2,3-diaryl-6,7-dihydro-5H-1,4-diazepines for
anticancer and antiplatelet activities.
exhibit in the IR spectrum a peak at 1650e1670 cmꢀ1. The
ethanedione derivatives (16aer) and 1,3-propanediamine in
equimolar amounts in the presence of glacial acetic acid in
ethanol under reflux condition afforded the cyclised product.
It is clearly stated in literature that synthesis of these analogs
is difficult and occasionally unsuccessful [21]. The yield of the
cyclised compound is low to moderate due to formation of the
undesired uncyclised product as described in the literature [22]
and partial recovery of starting material. The IR spectrum of
the uncyclised product obtained shows an intense peak at
1670 cmꢀ1 which may be due to multiple condensation of
ethanedione with 1,3-propanediamine even when equimolar
quantities of ethanedione and 1,3-propanediamine are reacted
[22]. The cyclised products are confirmed by the appearance
of C]N stretching in the IR spectrum at 1590e1610 cmꢀ1
and absence of carbonyl stretching. The proton NMR shows
a broad hump for the methylene protons around d 3.5, adjacent
2. Chemistry
Phenylacetic acid derivatives were synthesized by Wilger-
odt method and hydrolysis of benzylcyanide derivatives. Frie-
deleCrafts acylation reaction of substituted/phenylacetic acid
with substituted aromatic compounds produced ethanone
derivatives (15aer) in good yield. The IR spectrum showed
an intense peak at 1685e1690 cmꢀ1 for carbonyl stretching.
Unlike conventional methods, microwave assisted selenium
dioxide oxidation of ethanone derivatives using DMSO as
solvent simplifies the workup procedure and reduction of the
reaction time [20]. Some ethanedione derivatives obtained as
either oily or sticky compound were used in the next step with-
out purification. The a-dicarbonyl (ethanedione) compounds

2006
R. Ramajayam et al. / European Journal of Medicinal Chemistry 43 (2008) 2004e2010
to the imine (]NeCH2eCH2, 4H). This may be due to tem-
perature coalescence or ring flip. The methylene protons adja-
cent to the imine of the compounds (17n and 17o) appear
clearly as a multiplet (triplet). This clearly indicates that the
hump is due to the ring flip [23]. The presence of the 2-chloro
substituent does not allow the ring flip. This was confirmed by
the NMR of the cyclised compound (17q) formed from change
of position of substituent in the phenyl acetic acid, i.e. 3-chlor-
ophenylacetic acid. A broad hump was observed in the NMR
spectra for the methylene protons adjacent to the imine of the
compound 17q. The other methylene protons (]NeCH2e
CH2, 2H) appear as a multiplet at d 2.32e2.39. The mass spec-
trum shows the M þ 1 peak for all compounds. An M þ 2 peak
is observed for all halo compounds in the mass spectrum. The
synthesis is depicted in the scheme below:
3.2. Antiplatelet activity
The 2,3-diaryl-6,7-dihydro-5H-1,4-diazepines 17aer were
evaluated to inhibit platelet aggregation of female Spraguee
Dawley rat platelet-rich plasma induced by arachidonic acid
[26]. Test compounds (500 mM) 17aer were used in the
preliminary screening (Table 2). Aspirin was used as standard
drug and showed maximum inhibition (97%) at 10 mM. From
the preliminary screening, compounds 17c, 17d, 17h, 17i, 17k,
17m and 17o were selected for further screening at low con-
centrations (100 mM). The results of the screening are dis-
played in Table 3. From these studies, 17i was the most
active one with 36.4% inhibition. Compound 17o showed
28.3% inhibition with twofold greater activity as compared
to aspirin (14%). The compounds 17h, 17k and 17m show
moderately good activity.
COOH
X
X
X
N
N
O
O
SOCl2
SeO2, M.W
NH2(CH2)3NH2
AcOH, EtOH
X
+
Y
AlCl3
DMSO
O
Y
Y
Y
15a-r
16a-r
17a-r
3. Results and discussion
4. Conclusion
3.1. Antileukemic activity
In summary, substituted 2,3-diaryl-1,4-diazepines were
synthesized and evaluated for antileukemic and antiplatelet ac-
tivities. Among the compounds, 17h, 17i, 17j and 17o showed
moderate antileukemic activity against Molt3 leukemic cancer
cell lines. The above compounds screened for antiplatelet
activity resulted in the identification of new and potent antipla-
telet agents. Further studies are in progress in our laboratories
Initial evaluation of 17aer was carried out at Memorial
Sloan Kettering Cancer Center (MSKCC), New York (USA).
The cytotoxic effects of 2,3-diaryl-1,4-diazepine derivatives
were tested using Alamar blue assay [24,25]. Alamar blue
(Resazurin) is commonly employed as an indicator of cell
number and viability, since it is reduced to a pink fluorescent
dye (Resorufin) in the medium by cell activity (possibly by ox-
ygen consumption through metabolism). Alamar Blue is non-
toxic to cells and does not necessitate killing of cells to obtain
measurements, as is the case with 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-tetrazolium bromide (MTT).
Table 1
Antileukemic activity IC50 (mM) values of 2,3-diaryl-6,7-dihydro-5H-1,4-
diazepines (17aer)
Compound
Jurkat
HL60
Molt-3
NCEB-1
K562
17a
17b
17c
17d
17e
17f
>100
48.60
40.59
71.87
NT
>100
45.0
41.0
>100
31.25
24.37
82.0
NT
>100
70.0
54.0
>100
64.0
49.0
O
N
N
59.0
75.0
68.0
>100
>100
39.0
>100
>100
>100
54.0
>100
>100
27.22
39.19
43.83
36.59
44.0
O
O
OH
O
O
OH
NT
NT
17g
17h
17i
30.1
51.78
33.72
26.67
29.0
48.26
39.73
>100
44.0
NT
22.6
27.6
22.47
21.4
22.64
28.75
25.13
>100
21
Resorufin
(pink)
Resazurin
(blue)
46.0
47.0
57.0
40.0
The various human leukemic cancer cell lines Jurkat,
17j
30.0
40.0
HL60, Molt3, NCEB-1 and K562 were incubated with various
concentrations of the 2,3-diaryl-1,4-diazepines for 72 h. The
results of IC50 values of the compound are summarized in
Table 1. Compound 17o showed moderate cytotoxic activity
with IC50 value of 21.0 mM against Molt3 cell lines. For the
same cell line, the other compounds 17g, 17i and 17j showed
antileukemic activity with IC50 values of 22.6, 22.47 and
21.4 mM, respectively.
17k
17l
56.0
>100
65.0
67.0
32.0
51.54
49.18
>100
37.79
>100
>100
>100
17m
17n
17o
17p
17q
17r
>100
38.0
>100
45.0
>100
>100
>100
NT
NT
82.88
>100
>100
NT
NT
NT
NT ¼ Not tested.

R. Ramajayam et al. / European Journal of Medicinal Chemistry 43 (2008) 2004e2010
2007
Table 2
Inhibitory data (500 mM) for 2,3-diaryl-1,4-diazepines against arachidonic
acid induced platelet aggregation
exposure to iodine vapors. Solvents were purified using stan-
dard methods.
Compound
% Inhibition
5.1. General procedure for synthesis of ethanone
derivatives (15aer)
17a
17b
17c
17d
17e
17f
18.5
35.7
42.5
60.7
35.7
34.0
34.0
44.3
77.8
36.4
44.6
43.0
52.6
22.0
58.2
NT
2-Chloro/3-chloro/4-chloro/4-nitro/4-methyl phenylacetic
acid (1 mol) was dissolved in excess quantity of thionyl
chloride (2 mol) and allowed to reflux on steam bath for 3 h.
The excess of thionyl chloride was recovered under vacuum.
The resulting acid chloride solution was cooled and added
dropwise into the cooled mixture of AlCl3 (1.5 mol) and
substituted aromatic compounds. The reaction mixture was
stirred for 45 min at room temperature. The reaction mixture
is quenched with cold-HCl, extracted with chloroform (3 ꢁ
20 ml). The combined organic extracts were washed with
sodium bicarbonate solution, water and dried over anhydrous
sodium sulfate. Recrystallization from methanol after solvent
removal gave the ethanone derivatives. The results are summa-
rized in Table 4.
17g
17h
17i
17j
17k
17l
17m
17n
17o
17p
17q
17r
22.1
0.0
NT ¼ Not tested.
to exploit the preliminary results of antiplatelet activity for the
synthesis of a larger library, incorporating substitution or/and
bioisosteric replacement of the phenyl group in the diazepine
system.
5.2. General procedure for synthesis of ethanedione
derivatives (16aer)
Selenium dioxide (0.15 mol) was added into the solution of
ethanone derivatives (0.1 mol) in DMSO (15 ml) and irradi-
ated in the microwave oven (domestic household oven
1000 W) for the specified time as given in Table 4. The hot
mixture was filtered to remove the selenium metal and filtrate
was poured over crushed ice. The resulting precipitate was
filtered, dried and recrystallized from methanol to get the etha-
nedione derivatives. The results are summarized in Table 4.
5. Experimental
Melting points were determined in capillaries using Tosh-
niwal melting point apparatus and are uncorrected. IR (in
cmꢀ1) spectra in KBr pellets on a Shimadzu 8300 instrument;
1
and H NMR spectra were recorded in CDCl on a Bruker
¨
3
spectrometer (300 or 400 MHz), using tetramethylsilane as
an internal standard. Chemical shift data are reported in parts
per million (d in ppm) where s, br, t and m designate singlet,
broad, triplet and multiplet, respectively. Elemental analyses
were recorded on a PerkineElmer PE 2400 CHNS analyzer.
Mass spectra were recorded on APISciEX mass spectrometer
equipped with an electrospray ionisation (ESI) interface.
Column chromatography was carried out using silica gel
(100e200 mesh). Thin-layer chromatography (TLC) was per-
formed on precoated Silica gel Merck plates. Compounds
were visualized by illuminating with UV light (254 nm) or
Table 4
General structure of 1,2-diarylethanone and ethanedione derivatives
Cpd
X
Y
IR (cmꢀ1
)
Reaction
time (s)
O
Ar
Ar
Ar
Ar
O
(%Yield)
O
(%Yield)
15a, 16a
15b, 16b
15c, 16c
15d, 16d
15e, 16e
15f, 16f
15g, 16g
15h, 16h
15i, 16i
H
H
1676 (70)
1680 (59)
1686 (60)
1685 (62)
1674 (45)
1681 (60)
1676 (77)
1690 (59)
1683 (80)
1672 (53)
1684 (73)
1689 (62)
1685 (78)
1680 (80)
1691 (77)
1670 (53)
1685 (59)
1685 (66)
1654 (84)
1652 (77)
1668 (57)
1664 (75)
1668 (85)
1666 (81)
1666 (81)
1658 (57)
1663 (73)
1652 (74)
1667 (87)
1664 (68)
1662 (76)
1661 (74)
1674 (81)
1659 (52)
1660 (94)
1677 (63)
30
30
H
4-CH3
4-Br
4-F
H
95
80
H
H
4-OCH3
4-SCH3
4-CH3
4-Cl
35
35
H
Table 3
Inhibitory data (100 mM) for 2,3-diaryl-1,4-diazepines against arachidonic
acid induced platelet aggregation
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-NO2
4-NO2
2-Cl
2-Cl
4-CH3
3-Cl
50
100
80
4-F
Compound
% Inhibition
15j, 16j
15k, 16k
15l, 16l
4-SCH3
H
4-Br
115
55
17c
17d
18.5
18.8
24.0
36.4
24.0
24.2
28.3
14.0
155
20
15m, 16m
15n, 16n
15o, 16o
15p, 16p
15q, 16q
15r, 16r
H
17h
17i
4-CH3
4-Cl
35
40
17k
4-SCH3
4-CH3
4-CH3
40
40
17m
17o
55
Aspirin

2008
R. Ramajayam et al. / European Journal of Medicinal Chemistry 43 (2008) 2004e2010
5.3. General procedure for synthesis of diaryl
diazepines (17aer)
1247, 1174, 1029, 945, 813, 786 cmꢀ1 1H (CDCl3,
;
400 MHz): 3.77 (s, 3H, OCH3), 2.31e2.38 (m, 2H, NeCH2e
CH2e), 3.2 (br, 4H, NeCH2eCH2e), 6.8e7.3 (m, 9H, ArH).
MS m/z; 279 (MHþ). Anal. Calcd for C18H18N2O: C, 77.67;
H, 6.52; N, 10.06. Found: C, 77.84; H, 6.38; N, 10.0.
The equimolar mixture of ethanedione derivatives, 1,3-pro-
panediamine and glacial acetic acid (1:1:1) were dissolved in
ethanol (30 ml). The mixture was allowed to reflux for 24e
30 h. The reaction was monitored throughout by TLC and
the solvent was evaporated under vacuum after completion.
The resulting sticky compound was stripped with silica gel
and chromatography with benzene gives a liquid product,
with partial recovery of starting material, which on trituration
with petroleum ether afforded a solid product. This was re-
crystallized from a suitable solvent to afford the desired com-
pounds (17aer).
5.3.6. 2-Phenyl-3-(4-methylsulfanylphenyl)-6,7-dihydro-
5H-1,4-diazepine (17f)
Yield 29%, Mp 115e117 ꢂC (brownish white crystals from
pet ether), IR (KBr) nmax: 1600, 1593 (C]N), 1550, 1247,
1
1180, 1097, 960, 823, 785 cmꢀ1; H (CDCl3, 400 MHz): 2.4
(s, 3H, SCH3), 2.34e2.37 (m, 2H, NeCH2eCH2e), 3.5 (br,
4H, NeCH2eCH2e), 7.1e7.5 (m, 9H, ArH). MS m/z; 295
(MHþ). Anal. Calcd for C18H18N2S: C, 73.43; H, 6.16; N,
9.51. Found: C, 73.14; H, 6.08; N, 9.42.
5.3.1. 2,3-Diphenyl-6,7-dihydro-5H-1,4-diazepine (17a)
Yield 10%, Mp 115e118 (114e116 ꢂC) [22] (colorless crys-
tals from MeOH), IR (KBr) nmax: 1605 (C]N), 1510, 1332,
5.3.7. 2-(4-Chlorophenyl)-3-(40-methylphenyl)-
6,7-dihydro-5H-1,4-diazepine (17g)
1
1272, 825, 780 cmꢀ1; H (CDCl3, 400 MHz): 2.31e2.40 (m,
Yield 34%, Mp 102e104 ꢂC (colorless crystals from
MeOH), IR (KBr) nmax: 1610 (C]N), 1600, 1593, 1400,
2H, NeCH2eCH2e), 3.47 (br, 4H, NeCH2eCH2e), 7.2e7.6
(m, 10H, ArH). MS m/z; 249 (MHþ). Anal. Calcd for
C17H16N2: C, 82.22; H, 6.49; N, 11.28. Found: C, 82.12; H,
6.74; N, 11.40.
840, 821, 761 cmꢀ1 1H (CDCl3, 300 MHz): 2.3 (s, 3H,
;
CH3), 2.32e2.39 (m, 2H, NeCH2eCH2e), 3.4 (br, 4H, Ne
CH2eCH2e), 7.1e7.5 (m, 8H, ArH). MS m/z; 297 (MHþ).
Anal. Calcd for C18H17ClN2: C, 72.84; H, 5.77; N, 9.44.
Found: C, 72.83; H, 5.62; N, 9.49.
5.3.2. 2-Phenyl-3-(4-methylphenyl)-6,7-dihydro-
5H-1,4-diazepine (17b)
Yield 23%, Mp 82e84 ꢂC (brown crystals from MeOH), IR
(KBr) nmax: 1608, 1592 (C]N), 1510, 1332, 1272, 825,
5.3.8. 2,3-(4,40-Dichlorophenyl)-6,7-dihydro-
5H-1,4-diazepine (17h)
1
780 cmꢀ1; H (CDCl3, 400 MHz): 2.32 (s, 3H, CH3), 2.34e
Yield 17%, Mp 111e113 ꢂC (colorless crystals from
MeOH), IR (KBr) nmax: 1614 (C]N), 1591, 1467, 1398,
2.41(m, 2H, NeCH2eCH2e), 3.5 (br, 4H, NeCH2eCH2e),
7.1e7.6 (m, 9H, ArH). MS m/z; 263 (MHþ). Anal. Calcd
for C18H18N2: C, 82.41; H, 6.92; N, 10.68. Found: C, 82.28;
H, 7.02; N, 10.84.
1
1080, 840, 769 cmꢀ1; H (CDCl3, 300 MHz): 2.31e2.40 (m,
2H, NeCH2eCH2e), 3.5 (br, 4H, NeCH2eCH2e), 7.2e7.5
(m, 8H, ArH). MS m/z; 318 (MHþ). Anal. Calcd for
C17H14Cl2N2: C, 64.37; H, 4.45; N, 8.83. Found: C, 64.24;
H, 4.35; N, 8.98.
5.3.3. 2-Phenyl-3-(4-bromphenyl)-6,7-dihydro-
5H-1,4-diazepine (17c)
Yield 6%, Mp 93e95 ꢂC (colorless crystals from pet ether),
IR (KBr) nmax: 1598 (C]N), 1585, 1392, 1272, 1068, 945,
5.3.9. 2-(4-Chlorophenyl)-3-(40-fluorophenyl)-
6,7-dihydro-5H-1,4-diazepine (17i)
825 cmꢀ1 1H (CDCl3, 300 MHz): 2.33e2.41 (m, 2H, Ne
;
CH2eCH2e), 3.5 (br, 4H, NeCH2eCH2e), 7.1e7.6 (m, 9H,
ArH). MS m/z; 329 (M þ 2). Anal. Calcd for C17H15BrN2: C,
62.4; H, 4.62; N, 8.56. Found: C, 62.56; H, 4.60; N, 8.24.
Yield 41%, Mp 104e106 ꢂC (colorless crystals from
MeOH), IR (KBr) nmax: 1614 (C]N), 1587, 1272, 1090,
1
945, 850, 763 cmꢀ1; H (CDCl3, 400 MHz): 2.32e2.39 (m,
2H, NeCH2eCH2e), 3.5 (br, 4H, NeCH2eCH2e), 6.98e
7.31 (m, 8H, ArH). MS m/z; 301 (MHþ). Anal. Calcd for
C17H14ClFN2: C, 67.89; H, 4.69; N, 9.31. Found: C, 68.12;
H, 4.66; N, 9.54.
5.3.4. 2-Phenyl-3-(4-fluorophenyl)-6,7-dihydro-
5H-1,4-diazepine (17d)
Yield 13%, Mp 77e78 ꢂC (colorless crystals from pet
ether), IR (KBr) nmax: 1598 (C]N), 1587, 1506, 1288,
1159, 1080, 943, 850, 783 cmꢀ1 1H (CDCl3, 300 MHz):
;
2.31e2.4 (m, 2H, NeCH2eCH2e), 3.5 (br, 4H, NeCH2e
CH2e), 6.9e7.6 (m, 9H, ArH). MS m/z; 267 (MHþ). Anal.
Calcd for C17H15FN2: C, 76.67; H, 5.68; N, 10.52. Found:
C, 76.74; H, 5.72; N, 10.56.
5.3.10. 2-(4-Chlorophenyl)-3-(40-methylsulfanylphenyl)-
6,7-dihydro-5H-1,4-diazepine (17j)
Yield 13%, Mp 120e122 ꢂC (colorless crystals from pet
ether), IR (KBr) nmax: 1610 (C]N), 1589, 1396, 1274,
1
1095, 941, 835, 815 cmꢀ1; H (CDCl3, 400 MHz): 2.45 (s,
5.3.5. 2-Phenyl-3-(4-methoxyphenyl)-6,7-dihydro-
5H-1,4-diazepine (17e)
Yield 13%, Mp 106e108 ꢂC (colorless crystals from
MeOHepet ether), IR (KBr) nmax: 1600 (C]N), 1570, 1508,
3H, SCH3), 2.28e2.43 (m, 2H, NeCH2eCH2e), 3.51 (br,
4H, NeCH2eCH2e), 7.1e7.7 (m, 8H, ArH). MS m/z; 329
(MHþ). Anal. Calcd for C18H17ClN2S: C, 65.74; H, 5.21; N,
8.52. Found: C, 65.91; H, 5.15; N, 8.82.

R. Ramajayam et al. / European Journal of Medicinal Chemistry 43 (2008) 2004e2010
2009
5.3.11. 2-(4-Chlorophenyl)-3-phenyl-6,7-dihydro-
5H-1,4-diazepine (17k)
NeCH2eCH2e), 3.60e3.63 (t, 2H, J ¼ 12 Hz, NeCH2e
CH2e), 3.70e3.73 (t, 2H, J ¼ 12 Hz, NeCH2eCH2e), 7.1e
7.3 (m, 8H, ArH). MS m/z; 329 (MHþ). Anal. Calcd for
C18H17ClN2S: C, 65.74; H, 5.21; N, 8.52. Found: C, 65.94;
H, 5.16; N, 8.48.
Yield 38%, Mp 72e74 ꢂC (colorless crystals from MeOH),
IR (KBr) nmax: 1611, 1592 (C]N), 1456, 1338, 1272, 1092,
1013, 944, 835, 786 cmꢀ1 1H (CDCl3, 300 MHz): 2.32e
;
2.41(m, 2H, NeCH2eCH2e), 3.5 (br, 4H, NeCH2eCH2e),
7.2e7.6 (m, 9H, ArH). MS m/z; 283 (MHþ). Anal. Calcd
for C17H15ClN2: C, 72.21; H, 5.35; N, 9.91. Found: C,
72.04; H, 5.62; N, 9.72.
5.3.17. 2,3-(4,40-Dimethylphenyl)- 6,7-dihydro-
5H-1,4-diazepine (17q)
Yield 16%, Mp 94e95 ꢂC (colorless crystals from pet
ether), IR (KBr) nmax: 1608 (C]N), 1593, 1566, 1319, 1278,
5.3.12. 2-(4-Chlorophenyl)-3-(40-bromophenyl)-
6,7-dihydro-5H-1,4-diazepine (17l)
1176, 1083, 945, 852, 800 cmꢀ1 1H (CDCl3, 400 MHz):
;
2.32 (s, 6H, (CH3)2), 2.34e2.37 (m, 2H, NeCH2eCH2e),
3.5 (br, 4H, NeCH2eCH2e), 7.11e7.52 (m, 8H, ArH). MS
m/z; 277 (MHþ). Anal. Calcd for C19H20N2: C, 82.57; H,
7.29; N, 10.14. Found: C, 82.72; H, 7.12; N, 10.32.
Yield 16%, Mp 140e142 ꢂC (pale yellow flakes from
MeOH), IR (KBr) nmax: 1612 (C]N), 1585, 1490, 1310,
1174, 1068, 943, 827, 769 cmꢀ1 1H (CDCl3, 300 MHz):
;
2.3e2.4 (m, 2H, NeCH2eCH2e), 3.5 (br, 4H, NeCH2e
CH2e), 7.2e7.5 (m, 8H, ArH). MS m/z; 363 (M þ 2). Anal.
Calcd for C17H14BrClN2: C, 56.46; H, 3.9; N, 7.75. Found:
C, 56.50; H, 3.83; N, 7.81.
5.3.18. 2-(3-Chlorophenyl)-3-(40-methylphenyl)-
6,7-dihydro-5H-1,4-diazepine (17r)
Yield 38%, Mp 119e121 ꢂC (colorless crystals from pet
ether), IR (KBr) nmax: 1608 (C]N), 1595, 1564, 1471, 1255,
1
5.3.13. 2-(4-Nitrophenyl)-3-phenyl-6,7-dihydro-
5H-1,4-diazepine (17m)
1091, 943, 829, 802, 761 cmꢀ1; H (CDCl3, 400 MHz): 2.33
(s, 3H, CH3), 2.35e2.40 (m, 2H, NeCH2-CH2e), 3.53 (br,
4H, NeCH2eCH2e), 7.1e7.7 (m, 8H, ArH). MS m/z; 297
(MHþ). Anal. Calcd for C18H17ClN2: C, 72.84; H, 5.77; N,
9.44. Found: C, 72.62; H, 5.72; N, 9.52.
Yield 11%, Mp 99e101 ꢂC (brown crystals from MeOH),
IR (KBr) nmax: 1608, 1600 (C]N), 1574, 1520, 1347, 854,
794 cmꢀ1 1H (CDCl3, 300 MHz): 2.3e2.4 (m, 2H, Ne
;
CH2eCH2e), 3.5 (br, 4H, NeCH2eCH2e), 7.2e7.5 (m,
9H, ArH). MS m/z; 293 (Mþ). Anal. Calcd for C17H15N3O2:
C, 69.61; H, 5.15; N, 14.33. Found: C, 69.94; H, 5.03; N,
14.10.
5.4. Antiproliferative activity
The results of the cytotoxic potencies of the synthesized
compounds tested in vitro against five leukemic cell lines
Jurkat, HL60-DS, Molt-3, NCEB-1 and K562 are summarized
in Table 1. The synthesized compounds were dissolved in
DMSO (5 ml of each compound at 10 mM final concentration)
and dispensed into an assay plate using accustom built low
volume 384-well head tool. The assay plates are then loaded
with 45 ml of cells and allowed to incubate for 48 h at
37 ꢂC. Then, 5 ml of alamar blue reagent was added to the as-
say plate and incubated for 24 h at 37 ꢂC. Alamar blue reduc-
tion was measured on CCD-based optical imaging reader.
For initial screening, 1% DMSO was utilized as the high
control to represent maximum reduction of alamar blue from
cellular metabolism. The cytotoxic agent staurosporine is uti-
lized at 50 mM to represent minimal reduction of alamar blue
as a result of total cellular killing. These controls are used to
calculate Z0 as a test of the functionality of the assay and to
determine its range, robustness and reliability. For these cell
lines, the controls gave a Z0 factor of 0.598 or better and
a good signal to noise ratio indicating a broad dynamic range
making the study reliable in our initial screening.
5.3.14. 2-(4-Nitrophenyl)-3-(40-methylphenyl)-
6,7-dihydro-5H-1,4-diazepine (17n)
Yield 17%, Mp 161e163 ꢂC (yellow crystals from MeOH),
IR (KBr) nmax: 1595 (C]N), 1514, 1344, 1259, 1160, 1090,
1
973, 850 cmꢀ1; H (CDCl3, 300 MHz): 2.33 (s, 3H, CH3),
2.34e2.4 (m, 2H, NeCH2eCH2e), 3.58 (br, 4H, NeCH2e
CH2e), 7.1e8.1 (m, 8H, ArH). MS m/z; 307 (Mþ). Anal.
Calcd for C18H17N3O2: C, 70.34; H, 5.58; N, 13.67. Found:
C, 70.2; H, 5.72; N, 13.88.
5.3.15. 2-(2-Chlorophenyl)-3-(40-chlorophenyl)-
6,7-dihydro-5H-1,4-diazepine (17o)
Yield 8%, Mp 115e116 ꢂC (colorless crystals from
MeOH), IR (KBr) nmax: 1608 (C]N), 1589, 1562, 1490,
1320, 1100, 1060, 945, 850, 775 cmꢀ1
;
1H (CDCl3,
300 MHz): 2.43e2.52 (m, 2H, NeCH2eCH2e), 3.58e3.62
(t, 2H, J ¼ 12 Hz, NeCH2eCH2e), 3.70e3.74 (t, 2H,
J ¼ 12 Hz, NeCH2eCH2e), 7.2e8.6 (m, 8H, ArH). MS m/
z; 318 (MHþ). Anal. Calcd for C17H14Cl2N2: C, 64.37; H,
4.45; N, 8.83. Found: C, 64.55; H, 4.32; N, 9.12.
5.5. Antiplatelet activity
5.3.16. 2-(2-Chlorophenyl)-3-(40-methylsulfanylphenyl)-
6,7-dihydro-5H-1,4-diazepine (17p)
5.5.1. Preparation of platelet rich plasma (PRP) and
platelet poor plasma (PPP)
The blood was collected from healthy female Sprague-
Dawley rats in tubes containing 4% trisodium citrate solution.
Samples were centrifuged at 250g for 15 min to obtain PRP.
Yield 11%, Mp 119e121 ꢂC (colorless crystals from
MeOHepet ether), IR (KBr) nmax: 1610 (C]N), 1589, 1465,
1490, 1309, 1274, 1184, 1097, 943, 856, 750 cmꢀ1 1H
;
(CDCl3, 400 MHz): 2.43 (s, 3H, SCH3), 2.46e2.49 (m, 2H,

2010
R. Ramajayam et al. / European Journal of Medicinal Chemistry 43 (2008) 2004e2010
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5.5.2. Platelet aggregation study
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continuous stirring at 900 rpm and then stimulated with arach-
idonic acid solution (5 ml in ethanol, 200 mM). The standard
drug aspirin and diazepine derivatives 17aer and the vehicle
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before addition of aggregating agent. The change in optical
density was monitored continuously every 30 s for 5 min at
560 nm using microplate reader. The whole study was com-
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aggregation curve (AUC) over 5 min was calculated using
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%inhibition ¼ AUCðcontrolÞ
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´
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Acknowledgements
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We thank Indian Council of Medical Research (ICMR),
New Delhi, India, for the award of Senior Research Fellowship
to RR, and All India Council for Technical Education
(AICTE), New Delhi, India, for financial support in the form
of research promotion scheme [File No. 8023/RID/RPS.46/
2004e05] to RG.
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