Efficient Synthesis and Biological Evaluation of a Novel Series of 1,5-Benzodiazepine Derivatives as Potential Antimicrobial Agents

Article abstract of DOI:10.1111/cbdd.12739

A series of novel 1,5-benzodiazepine derivatives were rationally designed and synthesized following the principle of the superposition of bioactive substructures by the combination of 1,5-benzodiazepine, pyridine (phenyl), and an ester group. The structur

Full text of DOI:10.1111/cbdd.12739

Chem Biol Drug Des 2016; 88: 110–121
Research Article
Efficient Synthesis and Biological Evaluation of
a Novel Series of 1,5-Benzodiazepine Derivatives
as Potential Antimicrobial Agents
Received 20 September 2015, revised 22 January 2016 and
accepted for publication 26 January 2016
Ying-shuang An, Zhen-fang Hao, Xiu-jun Zhang
and Lan-zhi Wang*
College of Chemistry & Material Science, Hebei Normal
University, Shijiazhuang 050024, China
*Corresponding author: Lan-zhi Wang,
wanglanzhi@126.com
The effect of bacterial and fungal resistance to antimicro-
bial agents on human health is reaching alarming levels
(1). Infectious disease caused by microorganisms is a
primary cause of death worldwide. The search for new
antibacterial and antifungal drugs is ongoing because of
the increasing resistance of microbial pathogens. It is
desirable to discover new drugs with superior potency and
wide activity spectrum. Among various heterocyclic
systems, 1,5-benzodiazepines have gained importance
because of their pharmacological and biological activities.
Over recent years, an increasing number of studies on the
antimicrobial activity of 1,5-benzodiazepines have been
reported. To date, a significant number of 1,5-benzodiaze-
pines analogs with antimicrobial activity has been synthe-
sized (2–5). For example, series of 1,5-benzodiazepines
were described by Pandaram et al. as potent antimicrobial
agents (6). Therefore, the research on the synthesis and
antimicrobial activity of 1,5-benzodiazepines has become
a popular topic.
A series of novel 1,5-benzodiazepine derivatives were
rationally designed and synthesized following the
principle of the superposition of bioactive substruc-
tures by the combination of 1,5-benzodiazepine, pyri-
dine (phenyl), and an ester group. The structures of
the target compounds were determined by 1H NMR,
13C NMR, MS, IR, and elemental analysis. All the
synthesized compounds were evaluated for their
antimicrobial activities in vitro against the fungi
C. neoformans, C. neoformans clinical isolates (ATCC
32264), C. albicans (ATCC 10231), Gram-negative
bacterium E. coli (ATCC 44752), and Gram-positive
bacterium S. aureus (ATCC 25923). The results of the
bioactive assay demonstrated that most of the tested
compounds exhibited variable inhibitory effects on
the growth of the tested microorganisms. All the
active compounds showed better antifungal activity
than antibacterial activity. Notably, compound 2b dis-
played the highest activity (MIC = 30 lg/mL) against
C. neoformans and (MIC = 31 lg/mL) against C. neo-
formans clinical isolates. In addition, compound 2a
also showed excellent activity against C. neoformans
and C. neoformans clinical isolates with minimum
inhibitory concentration of 35 and 36 lg/mL, respec-
tively. Compounds 2a and 2b were further studied
by evaluating their cytotoxicities, and the results
showed that they have relatively low level cytotoxic-
ity for BV2 and 293T cell. Preliminary structure-
activity relationship study on three diverse sets (C-2,
C-3, and C-8 positions) of 1,5-benzodiazepines was
performed. The results revealed that the presence of
On one hand, previous studies indicated that the free ester
group at different positions of the nuclei of the molecules
could enhance their pharmacological properties due to its
high hydrophobicity and fat-solubility (7). On the other
hand, the pyridine group is also known as an important
scaffold in various biologically active molecules, possess-
ing antimicrobial, antiviral, anti-HIV, anti fungal, and
antimycobacterial activities (8–13).
Encouraged by these observations, we synthesized a series
of novel 1,5-benzodiazepine derivatives based on the prin-
ciple of superposition (14). The structures of these newly
synthesized 1,5-benzodiazepine derivatives are shown in
Figure 1. In our design, we emphasized the strategy of
combining three molecule fragments, 1,5-benzodiazepine,
pyridine and an ester group, into one frame. To the best of
our knowledge, the synthesis and the antimicrobial activity
of compounds 2–5 is reported first. This library compounds
represent a new starting point in the synthesis of many
other useful 1,5-benzodiazepine derivatives. The new com-
pounds we synthesized were screened for their in vitro
antimicrobial activity against Cryptococcus neoformans,
a
-CH3 group at the C-8 position had a positive
effect on the inhibitory activity of these compounds.
Additionally, the 2-pyridyl group at the C-2 position
may be
a pharmacophore and -COOC2H5 at C-3
position is the best substituent for the maintenance
of antimicrobial activities.
Key words: 1,5-benzodiazepine, antimicrobial activity, struc-
ture-activity relationship, synthesis
110
ª 2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12739

Novel Series of 1,5-Benzodiazepine Derivatives
Figure 1: Structures of compounds 2–5.
Cryptococcus neoformans clinical isolates, Candida albi-
cans, Escherichia coli, and Staphylococcus aureus. The
aim of the present research was to determine the exact
moiety of the molecules that affects the activity and to
determine whether the change of R1, R2, and X causes any
significant changes in antimicrobial activity. The results of
the bioactive assay showed that most of the synthesized
compounds displayed variable inhibitory effects on the
growth of all of the tested strains. Notably, compound 2b
showed the highest activity against C. neoformans, and
C. neoformans clinical isolates.
(30 mmol) and various o-PDAs (25 mmol) was stirred for
approximately 50 min at room temperature. The progress
and situation of the reaction conditions were monitored by
TLC. The solids were collected by filtration under reduced
pressure to give the corresponding crude N-o-aryl amino-
b-enamino esters 1 (~95%) after recrystallization with EtOH
as a solvent for purification.
Synthesis of 1,5-benzodiazepines (2–5)
Ethyl acetoacetate and various o-PDAs were used to yield
1. A mixture of compound 1 (20 mmol), a substituted pyri-
dine aldehyde or benzonic aldehyde (20 mmol) and PMA
(5 mol%) in dry ethanol (50 mL) was stirred at 0 °C for
15–60 min. The progress and situation of the reaction
conditions were monitored by TLC. After the reaction was
complete, the formed precipitate was filtered, washed with
ethanol, and recrystallized from dry ethanol to yield com-
pounds 2–5.
Experimental Section
Starting materials were purchased from commercial
sources and were used without further purification. Sol-
vents were dried according to standard procedures. The
reaction progress was monitored by thin layer chromatog-
raphy. The melting of these new compounds was deter-
mined by an open capillary using a Veego Precision Digital
Melting points apparatus (MP-D) and the results were
uncorrected. The IR spectra (in KBr pellets) were recorded
Ethyl-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-benzodiaze-
pine-3-carboxylate (2a, C18H19N3O2): White solid: yield
(95%); mp: 129–131 °C; Rf = 0.16 (Petroleum ether: ethy-
lacetate = 3:1); IR (KBr cmꢀ1): m = 3327 (N–H), 1672
(C=O), 1630 (C=C); 1H NMR (DMSO-d6, 500 MHz):
d = 1.07 (t, J = 7.0 Hz, 3H, -CH3), 2.48 (s, 3H, -CH3),
3.93 (q, J = 7.0 Hz, 2H, -COOCH2-), 5.65 (d, J = 6.0 Hz,
1H, -CH), 6.11 (d, J = 6.0 Hz, 1H, -NH), 6.47–8.36 (m,
8H, -C6H4, -C5H4N), 8.37 (s, 1H, -NH) p.p.m.; 13C NMR
(DMSO-d6, 125 MHz): d = 14.39, 23.51, 58.62, 61.64,
98.61, 119.55, 119.57, 120.21, 120.78, 121.17, 121.96,
131.90, 135.89, 138.00, 148.69, 152.10, 163.02,
167.68 p.p.m.; MS (ESI-TOF) m/z: [M + H+] Calcd for
1
on a BIO-RAD PE-M-1730 IR spectrophotometer. H NMR
spectra were recorded on a Bruker DRX-500 MHz spec-
trometer using Tetramethylsilane (TMS) as the internal
standard with DMSO-d6 as the solvent. Chemical shift val-
ues are expressed as parts per million downfield from
TMS, and J values are in Hertz. Splitting patterns are indi-
cated as s, singlet; d, doublet; t, triplet; q, quartet, and m,
multiplet. Mass spectra were recorded on a Thermo DSQ
II mass spectrometer. The results of the elemental analysis
were obtained on a Vario EL-III-CHN-0 elemental analyzer.
C
18H19N3O2 310, found 310; Anal. Calcd for C18H19N3O2:
C, 69.88; H, 6.19; N, 13.58; Found: C, 69.92; H, 6.14; N,
13.67.
General method for the synthesis of 1,5-
benzodiazepines (2–5)
Ethyl-4,8-dimethyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-benzo-
diazepine-3-carboxylate (2b, C19H21N3O2): White solid:
yield (95%); mp: 142–143 °C; Rf = 0.09 (Petroleum ether:
ethylacetate = 3:1); IR (KBr, cmꢀ1) m = 3317 (N–H), 1670
Synthesis of N-o-aryl amino-b-enamino esters 1
A mixture of ethyl acetoacetate (methyl acetoacetate,
acetyl propyl acetate, or acetyl isopropyl acetate)
Chem Biol Drug Des 2016; 88: 110–121
111

An et al.
(C=O), 1618 (C=C). 1H NMR (DMSO-d6, 500 MHz):
d = 1.07 (t, J = 7.0 Hz, 3H, -CH3), 1.98 (s, 3H, -CH3),
2.46 (s, 3H, -CH3), 3.96 (q, J = 7.0 Hz, 2H, -CH2), 5.64
(d, J = 7.0 Hz, 1H, -CH), 6.03 (d, J = 7.0 Hz, 1H, -NH),
6.29–8.32 (m, 7H, -C6H3, -C5H4N), 8.37 (s, 1H, -NH)
p.p.m.; 13C NMR (DMSO-d6, 125 MHz): d = 14.41, 20.13,
23.56, 58.52, 61.40, 98.37, 119.56, 120.24, 120.35,
120.75, 121.16, 122.74, 130.71, 135.86, 137.78, 148.69,
152.17, 163.09, 167.68 p.p.m.; MS (ESI-TOF) m/z:
[M + H+] Calcd for C19H21N3O2 324, found 324; Anal.
Calcd for C19H21N3O2: C, 70.57; H, 6.55; N, 12.99;
Found: C, 70.62; H, 6.59; N, 12.59.
found 309; Anal. Calcd for C19H20N2O2: C, 74.00; H,
6.54; N, 9.08; Found: C, 74.02; H, 6.52; N, 9.11.
Ethyl4,8-dimethyl-2-phenyl-2,5-dihydro-1,5-benzodiaze-
pine-3-carboxylate (2f, C20H22N2O2): White solid: yield
(95%); mp: 132–134 °C; Rf = 0.30 (Petroleum ether: ethy-
lacetate = 3:1); IR (KBr, cmꢀ1) m = 3306 (N–H), 1673
(C=O), 1630 (C=C). 1H NMR (DMSO-d6, 500 MHz):
d = 1.29 (t, J = 7.0 Hz, 3H, -CH3), 2.46 (s, 3H, -CH3),
4.23 (q, J = 7.0 Hz, 2H, -CH2), 5.60 (d, J = 6.5 Hz, 1H, -
CH), 6.13 (d, J = 6.5 Hz, 1H, -NH), 6.30–7.13 (m, 7H, -
C6H3, -C5H4N), 8.23 (s, 1H, -NH) p.p.m.; 13C NMR
(DMSO-d6, 125 MHz): d = 14.92, 20.64, 23.89, 49.08,
59.02, 59.25, 100.46, 119.96, 120.44, 120.55, 126.24,
127.83, 128.21, 129.58, 131.13, 131.96, 138.45, 145.21,
152.49, 168.18 p.p.m.; MS (ESI-TOF) m/z: [M + H+] Calcd
for C20H22N2O2 323, found 323; Anal. Calcd for
Ethyl-8-fluoro-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-
benzodiazepine-3-carboxylate (2c, C18H18FN3O2): White
solid: yield (93%); mp: 135–137 °C; Rf = 0.17 (Petroleum
ether: ethylacetate = 3:1); IR (KBr, cmꢀ1) m = 3359 (N–H),
1672 (C=O), 1623 (C=C). 1H NMR (DMSO-d6, 500 MHz):
d = 1.08 (t, J = 7.0 Hz, 3H, -CH3), 2.46 (s, 3H, -CH3),
3.94 (q, J = 6.5 Hz, 2H, -CH2), 5.64 (d, J = 6.5 Hz, 1H,
-CH), 6.31 (d, J = 6.5 Hz, 1H, -NH), 6.34–8.39 (m, 7H, -
C6H3, -C5H4N), 8.45 (s, 1H, -NH), p.p.m.; 13C NMR
(DMSO-d6, 125 MHz): d = 14.86, 23.85, 59.09, 61.64,
99.10, 106.24, 120.86, 120.94, 121.27, 121.79, 128.85,
136.53, 140.20, 149.23, 152.48, 163.10, 168.02 p.p.m.;
MS (ESI-TOF) m/z: [M + H+] Calcd for C18H18FN3O2 328,
found 328.
C
20H22N2O2: C, 74.51; H, 6.88; N, 8.69; Found: C, 74.48;
H, 6.87; N, 8.72.
Ethyl-8-bromo-4-methyl-2-phenyl-2,5-dihydro-1,5-benzo-
diazepine-3-carboxylate (2g, C19H19BrN2O2): Brown solid:
yield (93%) mp: 134–135 °C; Rf = 0.36 (Petroleum ether:
ethylacetate = 3:1); IR (KBr, cmꢀ1) m = 3303 (N–H), 1682
(C=O), 1627 (C=C). 1H NMR (DMSO-d6, 500 MHz):
d = 1.11 (t, J = 7.0 Hz, 3H, -CH3), 2.45 (s, 3H, -CH3),
3.96 (q, J = 7.0 Hz, 2H, -CH2), 5.59 (d, J = 6.5 Hz, 1H,
-CH), 6.48 (d, J = 6.5 Hz, 1H, -NH), 6.64–7.17 (m, 7H, -
C6H3, -C5H4N), 8.39 (s, 1H, -NH) p.p.m.; 13C NMR
(DMSO-d6, 125 MHz): d = 14.68, 23.65, 56.54, 59.28,
99.88, 113.87, 121.59, 122.24, 122.96, 123.99, 128.45,
132.23, 139.87, 152.52, 159.76, 161.71, 167.58 p.p.m.;
MS (ESI-TOF) m/z: [M + H+] Calcd for C19H19BrN2O2 388,
found 388; Anal. Calcd for C19H19BrN2O2: C, 58.93; H,
4.95; N, 7.23; Found: C, 58.89; H, 4.90; N, 7.28.
Ethyl-8-bromo-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-
1,5-benzodiazepine-3-carboxylate (2d,
Brown solid: yield (93%); mp: 134–135 °C; Rf = 0.16
(Petroleum ether: ethylacetate = 3:1); IR (KBr, cmꢀ1
C18H18BrN3O2):
)
m = 3341 (N–H), 1671 (C=O), 1618 (C=C). 1H NMR
(DMSO-d6, 500 MHz): d = 1.09 (t, J = 7.0 Hz, 3H, -CH3),
2.46 (s, 3H, -CH3), 3.93 (q, J = 7.0 Hz, 2H, -CH2), 5.63
(d, J = 6.5 Hz, 1H, -CH), 6.44 (d, J = 6.5 Hz, 1H, -NH),
6.65–8.40 (m, 7H, -C6H3, -C5H4N), 8.45 (s, 1H, -NH)
p.p.m.; 13C NMR (DMSO-d6, 125 MHz): d = 14.87, 23.94,
59.05, 62.06, 99.35, 120.00, 120.64, 121.24, 121.63,
122.40, 132.34, 136.35, 138.45, 149.14, 152.52, 163.47,
168.12 p.p.m.; MS (ESI-TOF) m/z: [M + H+] Calcd for
Methyl-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-benzodia-
zepine-3-carboxylate (3a, C17H17N3O2): White solid: yield
(95%); mp:135–137 °C; Rf = 0.08 (Petroleum ether: ethy-
lacetate = 3:1); IR(KBr cmꢀ1): m = 3327 (N–H), 1674 (C=O),
1637 (C=C); 1H NMR (DMSO-d6, 500 MHz): d = 2.49 (s,
3H, -CH3), 3.48 (s, 3H, -COOCH3), 5.65 (d, J = 6.5 Hz, 1H,
-CH), 6.13 (d, J = 6.5 Hz, 1H, -NH), 6.48–8.38 (m, 8H, -
C6H4, -C5H4N), 8.41 (s, 1H, -NH) p.p.m.; 13C NMR (DMSO-
d6, 125 MHz): d = 23.48, 50.50, 61.47, 98.61, 119.55,
119.59, 120.20, 120.80, 121.26, 122.01, 131.77, 135.97,
138.00 148.74, 152.74, 162.84, 168.05 p.p.m.; MS (ESI-
TOF) m/z: [M + H+] Calcd for C17H17N3O2 296, found 296;
Anal. Calcd for C17H17N3O2: C, 69.14; H, 5.80; N, 14.23;
Found: C, 69.03; H, 5.92; N, 14.11.
C
18H18BrN3O2 390, found 390; Anal. Calcd for
C18H18BrN3O2: C, 55.68; H, 4.67; N, 10.82; Found: C,
55.74; H, 4.63; N, 10.79.
Ethyl4-methyl-2-phenyl-2,5-dihydro-1,5-benzodiazepine-3-
carboxylate (2e, C19H20N2O2): White solid: yield (95%);
mp: 130–132 °C; Rf = 0.30 (Petroleum ether: ethylac-
etate = 3:1); IR (KBr, cmꢀ1) m = 3345 (N–H), 1667 (C=O),
1630 (C=C). 1H NMR (DMSO-d6, 500 MHz): d = 1.12 (t,
J = 7.0 Hz, 3H, -CH3), 2.45 (s, 3H, -CH3), 4.03 (q,
J = 7.0 Hz, 2H, -CH2), 5.62 (d, J = 6.5 Hz, 1H, -CH),
6.19 (d, J = 6.5 Hz, 1H, -NH), 6.47–7.12 (m, 8H, -C6H3,
-C5H5), 8.28 (s, 1H, -NH) p.p.m.; 13C NMR (DMSO-d6,
125 MHz): d = 14.50, 23.45, 58.96, 61.28, 100.38,
110.44, 121.10, 121.46, 121.51, 121.81, 124.18, 133.63,
136.27, 137.63, 148.96, 151.70, 162.69, 167.69 p.p.m.;
MS (ESI-TOF) m/z: [M + H+] Calcd for C19H20N2O2 309,
Methyl-4,8-dimethyl-2-(pyridin-2-yl)-2,5-dihydro-1,5diaze-
pine-3-carboxylate (3b, C18H19N3O2): White solid: yield
(95%); mp:129–130 °C; Rf = 0.10 (Petroleum ether: ethy-
lacetate = 3:1); IR (KBr, cmꢀ1) m = 3318 (N–H), 1672
(C=O), 1617 (C=C). 1H NMR (DMSO-d6, 500 MHz):
d = 1.98 (s, 3H, -CH3), 2.47 (s, 3H, -CH3), 3.47 (s, 3H, -
CH3), 5.63 (d, J = 6.5 Hz, 1H, -CH), 6.03 (d, J = 6.5 Hz,
112
Chem Biol Drug Des 2016; 88: 110–121

Novel Series of 1,5-Benzodiazepine Derivatives
1H, -NH), 6.30–8.35 (m, 7H, -C6H3, -C5H4N), 8.38 (s, 1H,
-NH) p.p.m.; 13C NMR (DMSO-d6, 125 MHz): d = 20.56,
23.98, 49.09, 50.86, 61.75, 98.57, 120.02, 120.82,
121.22, 121.70, 129.68, 131.25, 136.42, 138.24, 149.19,
153.01, 163.38, 168.53 p.p.m.; MS (ESI-TOF) m/z:
[M + H+] Calcd for C18H19N3O2 310, found 310; Anal.
Calcd for C18H19N3O2: C, 69.88; H, 6.19; N, 13.58;
Found: C, 69.80; H, 6.27; N, 13.67.
ethylacetate = 3:1); IR (KBr, cmꢀ1) m = 3319 (N–H), 1671
(C=O), 1617 (C=C). 1H NMR (DMSO-d6, 500 MHz):
d = 0.73 (t, J = 7.0 Hz, 3H, -CH3), 1.42–1.47 (m,
2H, -CH2), 1.98 (s, 3H, -CH3), 2.47 (s, 3H, -CH3), 3.84 (t,
J = 6.5 Hz, 2H, -COOCH2), 5.65 (d, J = 6.0 Hz, 1H, -CH),
6,02 (d, J = 6.0 Hz, 1H, -NH), 6.28–8.32 (m, 7H, -C6H3,
-C5H4N), 8.37 (s, 1H, -NH) p.p.m.; 13C NMR (DMSO-d6,
125 MHz): d = 10.90, 20.60, 22.28, 23.96, 62.04, 64.67,
98.63, 120.82, 121.11, 121.61, 123.22, 129.89, 131.19,
136.32, 138.20, 149.10, 152.57, 163.66, 168.11 p.p.m.;
MS (ESI-TOF) m/z: [M + H+] Calcd for C20H23N3O2 338,
found 338; Anal. Calcd for C20H23N3O2: C, 71.19; H,
6.87; N, 12.45; Found: C, 71.24; H, 6.94; N, 12.36.
Methyl-8-fluoro-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-
benzodiazepine-3-carboxylate (3c, C17H16FN3O2): White
solid: yield (93%); mp:155–156 °C; Rf = 0.09 (Petroleum
ether: ethylacetate = 3:1); IR (KBr, cmꢀ1) m = 3326 (N–H),
1670 (C=O), 1637 (C=C). 1H NMR (DMSO-d6, 500 MHz):
d = 2.47 (s, 3H, -CH3), 3.49 (s, 3H, -CH3), 5.63 (d,
J = 6.5 Hz, 1H, -CH), 6.32 (d, J = 6.5 Hz, 1H, -NH),
6.34–8.40 (m, 7H, -C6H3, -C5H4N), 8.43 (s, 1H, -NH)
p.p.m.; 13C NMR (DMSO-d6, 125 MHz): d = 23.86, 50.97,
61.58, 98.83, 106.30, 120.90, 120.98, 121.28, 121.85,
128.77, 136.57, 140.24, 149.29, 152.87, 162.96,
168.41 p.p.m.; MS (ESI-TOF) m/z: [M + H+] Calcd for
C17H16FN3O2 314, found 314.
Propyl-8-fluoro-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-
benzodiazepine-3-carboxylate (4c, C19H20FN3O2): White
solid: yield (90%); mp: 139–141 °C; Rf = 0.18 (Petroleum
ether: ethylacetate = 3:1); IR (KBr cmꢀ1): m = 3358 (N–H),
1675 (C=O), 1635 (C=C); 1H NMR (DMSO-d6, 500 MHz):
d = 0.74 (t, J = 7.0 Hz, 3H, -CH3), 1.42–1.48 (m, 2H, -CH2),
2.47 (s, 3H, -CH3), 3.86 (t, J = 6.0 Hz, 2H, -COOCH2), 5.65
(d, J = 6.5 Hz, 1H, -CH), 6.11 (d, J = 6.5 Hz, 1H, -NH),
6.30–8.42 (m, 7H, -C6H3, -C5H4N), 8.47 (s, 1H, -NH) p.p.m.;
13C NMR (DMSO-d6, 125 MHz): d = 10.42, 21.79, 23.35,
61.38, 64.30, 98.38, 105.84, 120.52, 120.71, 120.83,
121.31, 128.51, 136.02, 139.79, 148.73, 152.06, 156.60,
162.74, 167.55 p.p.m.; MS (ESI-TOF) m/z: [M + H+] Calcd
for C19H20FN3O2 342, found 342.
Methyl-8-bromo-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-
benzodiazepine-3-carboxylate (3d, C17H16BrN3O2): Brown
solid: yield (93%); mp:154–155 °C; Rf = 0.12 (Petroleum
ether: ethylacetate = 3:1); IR (KBr, cmꢀ1) m = 3336 (N–H),
1673 (C=O), 1638 (C=C). 1H NMR (DMSO-d6, 500 MHz):
d = 2.46 (s, 3H, -CH3), 3.50 (s, 3H, -CH3), 5.62 (d,
J = 6.0 Hz, 1H, -CH), 6.45 (d, J = 6.5 Hz, 1H, -NH), 6.65–
8.41 (m, 7H, -C6H3, -C5H4N), 8.48 (s, 1H, -NH) p.p.m.; 13C
NMR (DMSO-d6, 125 MHz): d = 23.77, 51.08, 61.45,
99.92, 113.67, 121.36, 121.55, 121.95, 122.19, 131.64,
136.68, 140.29, 149.37, 152.67, 162.75, 168.40 p.p.m.;
MS (ESI-TOF) m/z: [M + H+] Calcd for C17H16BrN3O2 374,
found 374; Anal. Calcd for C17H16BrN3O2: C, 54.56; H,
4.31; N, 11.23; Found: C, 54.47; H, 4.38; N, 11.29.
Propyl-8-bromo-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-
benzodiazepine-3-carboxylate (4d, C19H20BrN3O2): Brown
solid: yield (90%); mp:132–133 °C; Rf = 0.22 (Petroleum
ether: ethylacetate = 3:1); IR (KBr, cmꢀ1) m = 3338 (N–H),
1670 (C=O), 1618 (C=C). 1H NMR (DMSO-d6, 500 MHz):
d = 0.74 (t, J = 7.5 Hz, 3H, -CH3), 1.43–1.50 (m,
2H, -CH2), 2.47 (s, 3H, -CH3), 3.87 (t, J = 7.5 Hz, 2H, -
COOCH2), 5.64 (d, J = 6.5 Hz, 1H, -CH), 6.45 (d,
J = 6.5 Hz, 1H, -NH), 6.66–8.40 (m, 7H, -C6H3, -C5H4N),
8.47 (s, 1H, -NH) p.p.m.; 13C NMR (DMSO-d6, 125 MHz):
d = 10.90, 22.25, 23.75, 61.74, 64.90, 99.98, 113.67,
121.26, 121.55, 121.86, 122.23, 131.85, 136.58, 140.23,
149.28, 152.27, 163.00, 168.00 p.p.m.; MS (ESI-TOF) m/
z: [M + H+] Calcd for C19H20BrN3O2 404, found 404; Anal.
Calcd for C19H20BrN3O2: C, 56.73; H, 5.01; N, 10.45;
Found: C, 56.79; H, 5.08; N, 10.50.
Propyl-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-benzodia-
zepine-3-carboxylate (4a, C19H21N3O2): White solid: yield
(90%); mp:132–134 °C; Rf = 0.31 (Petroleum ether: ethy-
lacetate = 3:1); IR(KBr cmꢀ1): m = 3325 (N–H), 1671
(C=O), 1622 (C=C); 1H NMR (DMSO-d6, 500 MHz):
d = 0.73 (t, J = 7.0 Hz, 3H, -CH3), 1.44–1.46 (m, 2H,
-CH2), 2.49 (s, 3H, -CH3), 3.84 (t, J = 6.0 Hz, 2H, -
COOCH2), 5.66 (d, J = 6.5 Hz, 1H, -CH), 6.10 (d,
J = 6.5 Hz, 1H, -NH), 6.45–8.36 (m, 8H, -C6H4, -C5H4N),
8.38 (s, 1H, -NH) p.p.m.; 13C NMR (DMSO-d6, 125 MHz):
d = 10.43, 21.80, 23.44, 61.74, 64.27, 98.64, 119.53,
119.60, 120.20, 120.67, 121.17, 121.97, 131.99, 135.86,
137.95, 148.64, 152.06, 163.09, 167.62 p.p.m.; MS (ESI-
TOF) m/z: [M + H+] Calcd for C19H21N3O2 324, found
324; Anal. Calcd for C19H21N3O2: C, 70.57; H, 6.55; N,
12.99; Found: C, 70.65; H, 6.48; N, 12.89.
Isopropyl-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-benzo-
diazepine-3-carboxylate (5a, C19H21N3O2): White solid:
yield (88%); mp:133–134 °C; Rf = 0.16 (Petroleum ether:
ethylacetate = 3:1); IR (KBr cmꢀ1): m = 3330 (N–H), 1672
(C=O), 1621 (C=C); 1H NMR (DMSO-d6, 500 MHz):
d = 0.96 (d, J = 6.0 Hz, 3H, -CH3), 1.14 (d, J = 6.0 Hz,
3H, -CH3), 2.47 (s, 3H, -CH3), 4.77–4.82 (m, 1H, -CH),
5.64 (d, J = 6.5 Hz, 1H, -CH), 6.07 (d, J = 6.5 Hz,
1H, -NH), 6.45–8.33 (m, 8H, -C6H4, -C5H4N,) 8.37 (s, 1H,
-NH) p.p.m.; 13C NMR (DMSO-d6, 125 MHz): d = 21.78,
22.02, 23.46, 61.82, 65.39, 99.11, 119.52, 119.56,
120.20, 120.70, 121.09, 121.89, 132.03, 135.80, 137.95,
Propyl-4,8-dimethyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-benzo-
diazepine-3-carboxylate (4b, C20H23N3O2): White solid:
yield (90%); mp:130–131 °C; Rf = 0.31 (Petroleum ether:
Chem Biol Drug Des 2016; 88: 110–121
113

An et al.
148.61, 151.63, 163.17, 167.25 p.p.m.; MS (ESI-TOF) m/
z: [M + H+] Calcd for C19H21N3O2 324, found 324; Anal.
Calcd for C19H21N3O2: C, 70.57; H, 6.55; N, 12.99;
Found: C, 70.65; H, 6.48; N, 12.90.
(antibacterial and antifungal) activities of 19 1,5-benzodia-
zepine derivatives we synthesized. Five microorganisms
have been used including C. neoformans (ATCC 32264),
C. neoformans clinical isolates, C. albicans (ATCC 10231),
E. coli (ATCC 44752), and S. aureus (ATCC 25923).
Isopropyl-4,8dimethyl-2-(pyridin-2-yl)-2,5-dihydro-1,5-ben-
zodiazepine-3-carboxylate (5b, C20H23N3O2): White solid:
yield (88%); mp:135–136 °C; Rf = 0.20 (Petroleum ether:
ethylacetate = 3:1); IR (KBr, cmꢀ1) m = 3337 (N–H), 1671
(C=O), 1620 (C=C). 1H NMR (DMSO-d6, 500 MHz):
d = 0.97 (d, J = 6.0 Hz, 3H, -CH3), 1.13 (d, J = 6.5 Hz,
3H, -CH3), 1.98 (s, 3H, -CH3), 2.45 (s, 3H, -CH3), 4.76–
4.81 (m, 1H, -CH), 5.62 (d, J = 6.0 Hz, 1H, -CH), 5.99 (d,
J = 6.5 Hz, 1H, -NH), 6.28–8.27 (m, 7H, -C6H3, -C5H4N),
8.37 (s, 1H, -NH) p.p.m.; 13C NMR (DMSO-d6, 125 MHz):
d = 20.61, 22.28, 22.51, 23.99, 62.05, 65.74, 99.10,
120.80, 121.16, 121.56, 129.91, 131.09, 136.29, 138.20,
149.08, 152.16, 163.73, 167.72 p.p.m.; MS (ESI-TOF) m/
z: [M + H+] Calcd for C20H23N3O2 338, found 338; Anal.
Calcd for C20H23N3O2: C, 71.19; H, 6.87; N, 12.45;
Found: C, 71.25; H, 6.95; N, 12.49.
In the disc diffusion method, sterile paper discs (Φ 6 mm)
impregnated with compounds dissolved in DMSO at con-
centrations of 200 mg/disc were used. Discs containing
DMSO were used as controls. Paper discs impregnated
with a solution of the compound tested were placed on
the surface of the media inoculated with the microorgan-
isms. The plates were incubated at 37 °C for 24 h for cul-
ture of microorganisms. After incubation, the growth
inhibition zones around the discs were observed, which
indicated that the examined compound inhibited the
growth of microorganisms. Each assay in this experiment
was repeated three times.
MIC assays were performed in sterile 96-well plates using
the method described by Sarmiento (15). The MIC is
defined as the minimum concentration of a compound
required to exhibit complete inhibition of bacterial and fun-
gal growth. MIC80 was recorded as the concentration that
produced 80% growth reduction compared with wells with
no compound present. The MFC is the concentration at
which fungi failed to grow in the liquid nutrient medium. It
was determined that the solvent had no antifungal activi-
ties against any of the tested microorganisms. MFC
assays were also conducted in sterile 96-well plates with
different compound concentrations inoculated with 200 lL
of fungi suspension (2 9 103 CFU/mL). The plates were
incubated for 36 h at 30 °C, and growth was visually
observed. Approximately 200 lL of fungi suspension from
the wells that did not show growth was plated on nutrient
agar. The MFC is the concentration at which fungi failed to
grow in the liquid nutrient medium and nutrient agar inocu-
lated with 200 lL of suspension. The MIC and MFC
assays were repeated three times.
Isopropyl-8-fluoro-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-
1,5-benzodiazepine-3-carboxylate (5c, C19H20FN3O2):
White solid: yield (85%); mp:159–160 °C; Rf = 0.19 (Petro-
leum ether: ethylacetate = 3:1); IR (KBr, cmꢀ1) m = 3329
(N–H), 1667 (C=O), 1615 (C=C). 1H NMR (DMSO-d6,
500 MHz): d = 0.98 (d, J = 6.0 Hz, 3H, -CH3), 1.14 (d,
J = 6.0 Hz, 3H, -CH3), 2.45 (s, 3H, -CH3), 4.79–4.82 (m,
1H, -CH), 5.62 (d, J = 6.5 Hz, 1H, -CH), 6.30 (d,
J = 6.5 Hz, 1H, -NH), 6.32–8.35 (m, 7H, -C6H3, -C5H4N),
8.39 (s, 1H, -NH) p.p.m.; 13C NMR (DMSO-d6, 125 MHz):
d = 22.27, 22.48, 23.84, 61.82, 65.92, 99.38, 106.25,
106.45, 120.85, 120.92, 121.24, 121.73, 128.98, 136.47,
140.17, 149.17, 152.07, 163.26, 167.63 p.p.m.; MS (ESI-
TOF) m/z: [M + H+] Calcd for C19H20FN3O2 342, found 342.
Isopropyl-8-bromo-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-
1,5-benzodiazepine-3-carboxylate (5d,
C19H20BrN3O2):
Brown solid: yield (85%); mp:141–142 °C; Rf = 0.25 (Pet-
roleum ether: ethylacetate = 3:1); IR (KBr, cmꢀ1) m = 3329
(N–H), 1671 (C=O), 1617 (C=C). 1H NMR (DMSO-d6,
500 MHz): d = 0.99 (d, J = 6.0 Hz, 3H, -CH3), 1.15 (d,
J = 6.0 Hz, 3H, -CH3), 2.45 (s, 3H, -CH3), 4.79–4.84 (m,
1H, -CH), 5.62 (d, J = 6.0 Hz, 1H, -CH), 6.42 (d,
J = 6.0 Hz, 1H, -NH), 6.66–8.40 (m, 7H, -C6H3, -C5H4N),
8.42 (s, 1H, -NH) p.p.m. 13C NMR (DMSO-d6, 125 MHz):
d = 21.69, 21.88, 23.19, 61.11, 65.52, 99.88, 112.97,
120.76, 120.94, 121.24, 121.58, 121.64, 131.27, 135.99,
139.65, 148.68, 151.27, 162.49, 167.02 p.p.m.; MS (ESI-
TOF) m/z: [M + H+] Calcd for C19H20BrN3O2 404, found
404; Anal. Calcd for C19H20BrN3O2: C, 56.73; H, 5.01; N,
10.45; Found: C, 56.65; H, 5.09; N, 10.50.
The BV2 cells were cultured at 37 °C in 96-well plates at a
density of 4 9 104 cells per well with 5% CO2 in Dulbecco’s
Modified Eagle Medium (Invitrogen, GIBCO, Grand Island,
NY, USA) supplemented with 10% fetal bovine serum (Invit-
rogen) for 72 h. The 293T cells were incubated in the same
conditions with BV2 cells in RPMI 1640 supplemented with
10% fetal bovine. Compounds 2a, 2b, and fluconazole were
added to each well at final concentrations of 10.0, 20.0,
35.0, 50.0, 100.0, and 200.0 lg/mL. An equivalent volume
of DMSO without the compound was added as a control.
The cells were incubated for 48 h. Subsequently, 10 lL of
aqueous MTT solution (5.0 mg/mL) and the mixture were
incubated at 37 °C for 4 h. The MTT solution was carefully
decanted off, and 100 lL of DMSO was added to each
well. The color was measured with an Epoch microplate
spectrophotometer at 490 nm with the reference filter set to
620 nm. All MTT assays were repeated three times. Each
measurement contained six parallel treatments (wells).
Biological evaluation
Assay of antimicrobial activity in vitro. The standardized
disc diffusion methods were used to test the antimicrobial
114
Chem Biol Drug Des 2016; 88: 110–121

Novel Series of 1,5-Benzodiazepine Derivatives
Scheme 1: Synthesis of this novel series of
1,5-benzodiazepines 2–5.
time could be shortened by refluxing or increasing the
dosage of the catalyst, the application of milder conditions
(0 °C, PMA as a catalyst) proved to be more favorable
which can avoid the formation of the by-product benzimi-
dazole (Figure 2). In conclusion, higher temperature, high
acidity, and excess catalyst would reduce the yields of
1,5-benzodiazepines. Therefore, the reaction was con-
ducted using PMA as a catalyst in the appropriate amount
under optimal conditions (0 °C and EtOH as a solvent).
The products 2 were obtained after stirring of the mixtures
for 15–60 min at 0 °C in yields of 93–95%. Compounds 3,
4 and 5 were obtained via a similar synthetic route in
which ethyl acetoacetate was replaced by the correspond-
ing ester (methyl acetoacetate, acetyl propyl acetate, and
acetyl isopropyl acetate) in yields of 85–95%. Simple oper-
ation, short reaction time, high yields, and environment
friendliness are the advantages of this method. These
features make this method an attractive choice for the
synthesis of 1,5-benzodiazepines. The structures of the
synthesized analogs 2–5 were confirmed by 1H NMR, 13C
NMR, IR, MS, and elemental analysis.
Relative survival rate
¼ ðtreatment A490/negative control A620Þ ꢁ 100%
Results and Discussion
Chemistry
The novel 1,5-benzodiazepine derivatives 2–5 were syn-
thesized in two steps as shown in Scheme 1. First, the
condensation of substituted 1,2-phenylenediamines and
ethyl acetoacetate was performed under solvent-free, cat-
alyst-free, and room temperature conditions to yield
approximately 95% N-o-aminoaryl-b-enamino esters 1.
The addition of the solvents leads to a decrease of the
yield of compound 1. It is worth mentioning that adding
catalyst or increasing the temperature promotes the for-
mation of the by-product benzimidazole (Figure 2). There-
fore, the reaction was conducted under solvent-free,
catalyst-free, and room-temperature conditions.
Then, the target compounds 1,5-benzodiazepine
derivatives 2 were obtained via the reaction between 2-
pyridinecarboxaldehyde or benzaldehyde and compound 1
(N-o-aminoaryl-b-enamino esters). This step occurred
under various reaction conditions (e.g., catalyst, solvent,
and reaction temperature), which were investigated in
preliminary experiments. At the beginning of this study, the
reaction of 2-pyridinecarboxaldehyde and N-o-aminoaryl-
b-enamino esters was selected as a model to establish
optimum reaction conditions. The results are displayed in
Table 1. In the absence of the catalyst, only a trace
amount of the desired product was observed even after
1 h (Table 1, entry 1), which demonstrated that catalyst
plays an important role in this reaction. Then, we tried to
optimize the reaction conditions with different catalysts
and found that the reaction gave satisfying results in the
presence of phosphomolybdic acid (PMA). Other catalysts,
such as p-TsOH, acetic acid (HOAc), silicotungstic acid
(STA, H4SiW12O40), CeCl3ꢂ7H2O, NiCl2ꢂ6H2O and I2
afforded the target compounds in low yields. Several sol-
vents including acetonitrile, dichloromethane, chloroform,
methanol, ethanol, benzene, and toluene were tested for
the reaction. Compared with other solvents, EtOH was
found to be superior in terms of reaction time, environ-
mental protection, and significant yields. Although the rate
of the transformation could be enhanced and the reaction
Overall, we have proposed an efficient, clean, simple, and
economical procedure for the synthesis of novel 1,5-ben-
zodiazepine. Mild reaction conditions, short reaction time,
simple work-up, high selectivity, and excellent yields of the
products make this methodology highly significant. This
procedure represents a powerful green technology using
an environmentally solvent and avoids unwanted waste
production for the synthesis of new 1,5-benzodiazepine
derivatives. Surprisingly, the products were isolated with-
out column chromatography, which is beneficial for
industrial production.
Based on the above results and previous studies, a plausi-
ble mechanism for this reaction is presented in Scheme 2.
The first step involves the nucleophilic attack of the para-
amino group in substituted 1,2-phenylenediamines on the
carbonyl carbon of the b-keto ester to generate com-
pounds 1 by the loss of a water molecule. Compounds 1
Figure 2: Structure of by-product benzimidazole.
Chem Biol Drug Des 2016; 88: 110–121
115

An et al.
Table 1: Optimization of the reaction conditions for the synthesis of 2aa
N
CHO
H
N
NH2
HC COOCH2CH3
N
+
COOCH2CH3
CH3
N
H
N
H
CH3
Entry
Catalyst
Amount of catalyst (mol%)
Solvent
Temperature (°C)
Time (min)
Yieldb (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
None
HOAc
STA
CeCl3ꢂ7H2O
p-TsOH
NiCl2
I2
PMA
PMA
PMA
PMA
–
5
5
5
5
5
5
5
5
5
5
5
5
5
5
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
CH3CN
CH2Cl2
MeOH
Toluene
Benzene
EtOH
0 °C
0 °C
0 °C
0 °C
0 °C
0 °C
0 °C
0 °C
0 °C
0 °C
0 °C
0 °C
0 °C
r. t
60
25
30
32
30
38
50
15
40
35
20
50
45
10
10
–
72
88
76
85
70
75
95
73
75
90
55
50
75
70
PMA
PMA
PMA
PMA
EtOH
Reflux
PMA, phosphomolybdic acid.
aReaction conditions: N-o-aminoaryl-b-enamino esters (1 mmol), 2-pyridinecarboxaldehyde (1 mmol), Solvent (5 mL).
bIsolated yield.
were confirmed by IR, 1H NMR, MS, and elemental
analysis. Then, the other amino group of substituted 1,2-
phenylenediamines attacks the carbonyl carbon of 2-pyri-
dinecarboxaldehyde or benzaldehyde to give the interme-
diate I. The latter may undergo a cyclization reaction by
hydrogen transfer to yield the target product 2–5 (16,17).
trails) of the growth inhibition. The margin of error of these
measurements is ꢃ1 mm. The antimicrobial activity was
classified as highly active (>14 mm), moderately active
(10–14 mm), slightly active (6–10 mm), and inactive
(≤6 mm). The zone data of the 19 1,5-benzodiazepines
are reported in Table 2.
From Table 2, it could be observed that most of the syn-
thesized 1,5-benzodiazepines showed different inhibitory
effects on the growth of all tested microbial strains. The
synthesized compounds with antimicrobial activity exhib-
ited better antifungal activities than antibacterial activities.
The results suggest that these new series of 1,5-benzodia-
zepines are biologically active and have obvious specificity
to the tested fungi, especially C. neoformans and C. neo-
formans clinical isolates.
Antimicrobial activity
In this study, all the synthesized 1,5-benzodiazepine
derivatives were tested against five microbial strains using
disc diffusion methods, as follows: C. neoformans (ATCC
32264), C. neoformans clinical isolates, C. albicans (ATCC
10231), E. coli (ATCC 44752), and S. aureus (ATCC
25923). To facilitate visualization, the zone scope from
these assays indicates the average diameter (from three
Scheme 2: Plausible mechanism for the
synthesis of products 1–5.
116
Chem Biol Drug Des 2016; 88: 110–121

Novel Series of 1,5-Benzodiazepine Derivatives
Table 2: Antimicrobial activity of compounds 2–5
X
H
N
1
9
R1
2
4
8
7
COOR2
3
5
N
H
6
CH3
Zone of inhibitiona (mm) Dose (200 lg/disc)
Compd.
R1
X
R2
C. n.b
C. n.C.b
C. a.b
E. c.b
S. a.b
2a
2b
2c
2d
2e
2f
H
CH3
F
Br
H
CH3
Br
H
CH3
F
Br
H
CH3
F
Br
H
N
N
N
N
C
C
C
N
N
N
N
N
N
N
N
N
N
N
N
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH3
23.53 ꢃ 0.05
25.13 ꢃ 0.03
21.63 ꢃ 0.12
20.67 ꢃ 0.07
6.00 ꢃ 0.00
6.00 ꢃ 0.00
6.00 ꢃ 0.00
21.50 ꢃ 0.09
23.50 ꢃ 0.05
19.60 ꢃ 0.09
6.00 ꢃ 0.00
15.57 ꢃ 0.11
21.67 ꢃ 0.09
11.07 ꢃ 0.12
6.00 ꢃ 0.00
6.00 ꢃ 0.00
13.03 ꢃ 0.09
6.00 ꢃ 0.00
6.00 ꢃ 0.00
24.80 ꢃ 0.08
26.97 ꢃ 0.05
21.49 ꢃ 0.09
20.13 ꢃ 0.05
14.30 ꢃ 0.05
15.60 ꢃ 0.10
15.60 ꢃ 0.12
21.09 ꢃ 0.05
23.23 ꢃ 0.12
19.23 ꢃ 0.12
6.00 ꢃ 0.00
15.43 ꢃ 0.05
22.13 ꢃ 0.05
11.90 ꢃ 0.11
6.00 ꢃ 0.00
6.00 ꢃ 0.00
12.93 ꢃ 0.05
6.00 ꢃ 0.00
6.00 ꢃ 0.00
14.33 ꢃ 0.12
17.77 ꢃ 0.11
13.90 ꢃ 0.10
12.78 ꢃ 0.10
7.20 ꢃ 0.10
16.60 ꢃ 0.05
11.33 ꢃ 0.10
12.93 ꢃ 0.12
13.50 ꢃ 0.10
12.47 ꢃ 0.05
6.00 ꢃ 0.00
11.13 ꢃ 0.09
12.33 ꢃ 0.10
11.10 ꢃ 0.05
6.00 ꢃ 0.00
6.00 ꢃ 0.00
8.47 ꢃ 0.12
6.00 ꢃ 0.00
6.00 ꢃ 0.00
12.20 ꢃ 0.15
13.10 ꢃ 0.09
11.67 ꢃ 0.15
10.00 ꢃ 0.13
6.00 ꢃ 0.00
6.00 ꢃ 0.00
10.10 ꢃ 0.11
9.81 ꢃ 0.12
11.71 ꢃ 0.12
6.00 ꢃ 0.00
6.00 ꢃ 0.00
9.63 ꢃ 0.10
11.57 ꢃ 0.09
6.00 ꢃ 0.00
6.00 ꢃ 0.00
6.00 ꢃ 0.00
8.10 ꢃ 0.10
6.00 ꢃ 0.00
6.00 ꢃ 0.00
13.73 ꢃ 0.06
13.30 ꢃ 0.10
13.40 ꢃ 0.10
10.67 ꢃ 0.15
10.02 ꢃ 0.11
11.17 ꢃ 0.12
10.30 ꢃ 0.12
12.00 ꢃ 0.05
12.80 ꢃ 0.11
6.00 ꢃ 0.00
6.00 ꢃ 0.00
6.00 ꢃ 0.00
11.03 ꢃ 0.12
6.00 ꢃ 0.00
6.00 ꢃ 0.00
6.00 ꢃ 0.00
9.54 ꢃ 0.11
6.00 ꢃ 0.00
6.00 ꢃ 0.00
2g
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
CH3
CH3
CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH3
F
Br
aThe values indicate the average diameters in mm (of three trials) for the zone of growth inhibition observed after 24 h of incubation at
37 °C.
bC. n.: C. neoformans ATCC 32264; C. n. C.: C. neoformans clinical strain; C. a: C. albicans ATCC 10231; E. c.: E. coli ATCC 44752;
S. a.: S. aureus ATCC 25923.
Among compounds 2a–2d containing a 2-pyridyl group at
the C-2 position, 2b was the most active derivative with
inhibition zone data of 25.13 and 26.97 mm against C. ne-
oformans and C. neoformans clinical isolates, respectively.
The derivatives 2a, 2c, 2d also exhibited high activities
against C. neoformans and C. neoformans clinical isolates.
Compounds 2e–2g containing a phenyl group at the C-2
position, displayed only weak or moderate activity against
C. neoformans and C. neoformans clinical isolates. The
results demonstrated that the pyridine ring was essential
for the antifungal (C. neoformans and C. neoformans clini-
cal isolates) activity of this family of compounds. All the
compounds 2 showed moderate to low antimicrobial activ-
ity against C. albicans, E. coli and S. aureus. Particularly,
compounds 2a–2b inhibited all the tested microorganisms
and showed a broad-spectrum inhibitory effect.
benzothiazepines. Compounds 3, 4, and 5 contain R2
groups of -CH3, -CH2CH2CH3, and -CH(CH3)2, respec-
tively. Compounds 3a–3c also showed good activity
against C. neoformans and C. neoformans clinical isolates
and moderate activity against C. albicans, E. coli, and
S. aureus, but compound 3d (R1 = -Br) did not exhibit any
activity against any of the tested microorganism. Com-
pound 4b (R1 = -CH3) is also an efficient broad-spectrum
antibiotic and exhibits excellent inhibition activities against
the C. neoformans and C. neoformans clinical isolates. In
contrast, compounds 4a, 4c, and 4d showed weak or
moderate activity against the tested microorganisms.
Compounds 5 failed to show any significant inhibition with
the exception of compound 5b (R1 = -CH3), which
showed low to moderate antimicrobial activity against all
the tested microorganisms. In conclusion, compounds 2a–
2d with an ethyl ester group at the C-3 position of the
seven-membered ring display better antimicrobial activity
than compounds 3–5. This indicated that the ethyl ester
group in the region is favorable.
Compounds 3–5 with a 2-pyridyl unit were synthesized by
modifying the length and size of R2 to further explore its
influence on the antimicrobial activity of this series of 1,5-
Chem Biol Drug Des 2016; 88: 110–121
117

An et al.
Table 3: Inhibition zones at different dose levels for compounds 2a, 2b, 2c, 2d, 3a, and 3b
(Zone of inhibition/mm)
2a
2b
2c
2d
3a
3b
Dose (lg/disc) C. n.
C. n. C. C. n.
C. n. C. C. n.
C. n. C. C. n.
C. n. C. C. n.
C. n. C. C. n.
C. n. C.
200
100
50
23.53 24.80
18.73 18.50
12.27 12.30
25.13 26.97
19.33 19.47
13.23 13.90
21.63 21.49
17.00 17.97
11.67 11.23
20.67 20.13
16.63 16.23
11.93 11.13
21.50 21.09
16.27 16.20
11.60 11.03
23.50 23.23
18.40 18.27
12.97 12.47
35
30
25
7.63
6.90
6.00
7.83
6.10
6.00
8.90
7.00
6.00
8.70
6.86
6.00
6.90
6.00
6.00
6.93
6.00
6.00
6.50
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.23
6.00
6.00
6.97
6.00
6.00
6.37
6.00
6.00
C. n.: C. neoformans ATCC 32264; C. n. C.: C. neoformans clinical strain.
To explore the antimicrobial activity of 1,5-benzodiazepine
and to identify promising lead compounds. Compounds
2a, 2b, 2c, 2d, 3a, and 3b, which exhibited remarkable
antifungal activity in this study, were selected to investigate
the effect of dose levels on antifungal activity against C. ne-
oformans, C. neoformans clinical isolates. The inhibition
zones (Table 3) showed that the concentrations of the
compounds were ranged from 25 lg to 200 lg. It was also
found that the antimicrobial activity of the compounds was
reduced with decreasing in concentration. Compounds 2a
and 2b exhibited slight activity at 30 lg/disc, but 2c, 2d,
3a, and 3b did not exhibit any activity at 30 lg/disc. Com-
pounds 2a and 2b exhibited inactivity at 25 lg/disc.
64 lg/disc in the case of C. neoformans and C. neofor-
mans clinical isolates. These results indicated that com-
pounds 2a and 2b may become lead molecules of
antifungal drug against C. neoformans.
Cytotoxicities of compounds 2a and 2b
The synthesized compounds 2a and 2b were screened for
their cytotoxicity against two cell lines (BV2 and 293T)
using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide) assay. The cytotoxicity of DMSO was
used as a control. The results are given in Figures 3 and
4. A similar tendency was observed for the cytotoxic activ-
ity of compounds 2a and 2b against the two cell lines. It
was found that compounds 2a and 2b showed no cyto-
toxic activity at concentrations ≤35 lg/mL, which was not
significantly different from fluconazole. This result indicated
that compounds 2a and 2b did not affect the cell viability
of the tested cell lines (BV2 and 293T) at their MIC values
(35 and 30 lg/mL) against C. neoformans and MIC values
(36 and 31 lg/mL) against the C. neoformans clinical
strain, respectively. The relative survival rate of BV2 signifi-
cantly decreased with compounds 2a and 2b at concen-
trations ≥50.0 lg/mL (p < 0.01). Compounds 2a and 2b
showed significant cytotoxic effects against 293T at con-
centrations ≥100.0 lg/mL (p < 0.01). The results indicated
that compounds 2a and 2b showed slightly lower cyto-
toxic activity against 293T than BV2. The disparity
between the cytotoxicities and antimicrobial activities of
compounds 2a and 2b suggested that these compounds
exhibited their high in vitro antibacterial activities at non-
cytotoxic concentrations.
In addition, the MIC, MIC80, and MFC values were deter-
mined to explore the antifungal activity of compounds 2a
and 2b, which show the highest activity. Flucanazole was
used as the standard drug against fungi. The results are
summarized in Table 4. Surprisingly, the MIC values of
compound 2b were 30 lg/disc and 31 lg/disc against
C. neoformans and C. neoformans clinical isolates,
respectively. Compound 2a displayed similar fungicidal
effect with MIC values of 35 lg/disc and 36 lg/disc
against C. neoformans and C. neoformans clinical isolates,
respectively. By comparison, the MIC values of com-
pounds 2a and 2b were 3–4 fold more potent than the
reference drug fluconazole (MIC >128.0 lg/mL). The MFC
involves an additional set of steps performed once the
MIC is determined. The antimicrobials are typically
regarded as bactericidal/fungicidal if the MFC is not >4
times the MIC (18). The MFC values of compounds 2a
and 2b were approximately 29 MIC over the range of 52–
Table 4: MIC, MIC80 and MFC values for compounds 2a, 2b, and fluconazole (lg/mL)
2a
2b
Fluconazole
MIC
Fungal straina
MIC
MIC80
MFC
MIC
MIC80
MFC
MIC80
MFC
C. n
C. n. C.
35.0
36.0
33.0
35.0
60.0
64.0
30.0
31.0
25.5
28.0
52.0
54.0
>128.0
>128.0
2.0
2.0
>128.0
>128.0
aC. n.: C. neoformans ATCC 32264; C. n. C.: C. neoformans clinical strain.
118
Chem Biol Drug Des 2016; 88: 110–121

Novel Series of 1,5-Benzodiazepine Derivatives
pyridyl unit plays an important role in the antifungal activity
against C. neoformans and C. neoformans clinical isolates.
The subsequent SAR analysis focused on the ester group
at the C-3 positions of the seven-membered ring of 1,5-
benzodiazepine. Compounds 3, 4, and 5 have R2 groups
of -CH3, -CH2CH2CH3 and -CH(CH3)2 at the C-3 posi-
tions, respectively. Overall, compounds 3–5 displayed
lower antimicrobial activity than compounds 2a–2d
containing the -CH2CH3 group. A decrease or increase in
the length of R2 can result in low or no activity. Both in the
-CH2CH2CH3 series (compounds 4) and in the -CH(CH3)2
series (compounds 5), the activity generally decreases as
the size of the side carbon chain increases. The results
indicate that the substituent size appears to be an impor-
tant factor for biological activity. This is presumably
because the bulky groups at these positions hindered the
crossing of the molecular over the bacterial cell wall or hin-
dered its interaction with intracellular bacterial molecular
(19).
Figure 3: Effects of compounds 2a, 2b, and fluconazole on the
survival of BV2. *Significantly different between compounds 2a,
2b and standard drug fluconazole.
In another variation, an SAR study was conducted to
explore R1 at the C-8 position of the 1,5-benzodiazepine.
Hence, we planned to introduce electron withdrawing
and donating groups at the C-8 position to study their
influence on activity. As shown in Table 2, compounds
2–5 carrying R1 with different electronic properties (-CH3,
-H, -F and -Br) exhibited different inhibitory activities. This
indicated that the electronic properties of R1 exerted
important influence on their antimicrobial activity. The
antibacterial results lead to the assumption that the activ-
ity of the compounds was in the order of R1 = -
CH3 > R1 = -H > R1 = -F > R1 = -Br. It should point out
that the compounds (2b, 3b, 4b, and 5b) with a -CH3
group at the R1 position also display a broad-spectrum
inhibitory effect against all the tested microorganisms.
The electron-donating substituent (R1 = -CH3) exhibited
high inhibitory activity, indicating that -CH3 group exert an
enhanced positive influence on the antimicrobial activity.
However, compounds with electron-withdrawing groups,
such as fluorine and bromine (3d, 4c, 4d, 5c, and 5d)
exhibited low to no activity. The results revealed that the
electron withdrawing groups attached at the C-8 position
reduce the activity. This loss in activity may be because
the high electronegativity impedes the intracellular trans-
port (20).
Figure 4: Effects of compounds 2a, 2b, and fluconazole on the
survival of 293T. *Significantly different between compounds 2a,
2b and standard drug fluconazole.
Structure-activity relationship study
The structure-activity relationship (SAR) was studied, con-
sidering the following: the nature of the substitution at the
C-2 position, the length and size of the side chain at the
C-3 position, and the electronic properties of the substitu-
tion of R1 at the C-8 position.
Thus, the various substituent groups exhibited diverse
effects on the biological activity of the synthesized com-
pounds. However, the effects on biological activity were
caused by the joint action of these substituent groups.
First, the influences of the substituent at the C-2 position
were explored. Generally, the compounds 2a–2d with a 2-
pyridyl group at the C-2 position exhibit higher antimicrobial
activity than the compounds 2e–2g with a phenyl group at
the C-2 position. In this study, the presence of a phenyl
moiety at the C-2 position induces only a slightly improve-
ment of antimicrobial activity. However, the 2-pyridyl group
at the C-2 position significantly increased the antimicrobial
effectiveness, especially against C. neoformans and C. ne-
oformans clinical isolates. The results suggest that the 2-
Conclusion
In this study, 19 novel 1,5-benzodiazepine derivatives
were synthesized and their biological activities were pre-
liminarily evaluated in vitro. The synthesis scheme is sim-
Chem Biol Drug Des 2016; 88: 110–121
119

An et al.
ple and has only a limited number of steps, making this
economical process. The results of the primary in vitro
antimicrobial bioactivity assay and the preliminary SAR
analysis revealed that the 1,5-benzodiazepine derivatives
containing pyridine ring had good antimicrobial activity.
The results also showed that the -COOC2H5 group at the
C-3 position is the best substituent for the maintenance
of biological activities. In addition, the electronic properties
of the R1 at the C-8 position exert an important influence
on the activity of this series of 1,5-benzodiazepines.
Among all the synthesized compounds, 2b showed the
highest antimicrobial activity, and compounds 2a, 2c, 2d,
3a, 3b, 3c, and 4b showed excellent antimicrobial
activity.
1,5-benzodiazepine derivatives.
Soc;73:937–943.
J
Serb Chem
5. Al-Hiari Y.M., Abu-Dahab R., El-Abadelah M.M. (2008)
Heterocycles [h]-fused onto 4-oxoquinoline-3-car-
boxylic acid, Part VIII [1]. Convenient synthesis and
antimicrobial properties of substituted hexahydro[1,4]di-
azepino[2,3-h]quinoline-9-carboxylic acid and its
tetrahydroquino[7,8-b]benzodiazepine
Molecules;13:2880–2893.
analog.
6. Palanisamy P., Jenniefer S.J., Muthiah P.T., Kumare-
san S. (2013) Synthesis, characterization, antimicrobial,
anticancer, and antituberculosis activity of some new
pyrazole, isoxazole, pyrimidine and benzodiazepine
derivatives thiochromeno and benzothiepino moieties.
RSC Adv;3:19300–19310.
Because the ester-4-methyl-2-(pyridin-2-yl)-2,5-dihydro-
1,5-benzodiazepine -3-carboxylate represents a new type
of 1,5-benzodiazepine with antimicrobial activity, further
structural modifications and optimizations are required to
obtain more information about the SARs. The results of
this study will aid the design and development of new
antimicrobial agents. Further evaluation of the in vitro
antimicrobial activity properties of the compounds, partic-
ularly the mechanisms underlying their enhanced antimi-
crobial activity, should be performed in future
investigations.
7. Arya K., Dandia A. (2008) The expedient synthesis of
1,5-benzothiazepines as a family of cytotoxic drugs.
Bioorg Med Chem Lett;18:114–119.
8. Patel N.B., Agravat S.N., Shaik F.M. (2011) Synthesis
and antimicrobial activity of new pyridine derivatives-I.
Med Chem Res;20:1033–1041.
9. Patel N.B., Agravat S.N. (2009) Synthesis and antimi-
crobial studies of new pyridine derivatives. Chem
Heterocycl Compd;45:1343–1353.
10. El-Emary T.I., El-Mohsen S.A.A. (2012) Multi-compo-
nent one-pot synthesis and antimicrobial activities of
3-methyl-1,4-diphenyl-7-thioxo-4,6,8,9-tetrahydropyr-
azolo[5,4-b]pyrimidino[5,4-e]pyridine-5-one and related
derivatives. Molecules;17:14464–14483.
11. Bernardino A.M.R., De Azevedo A.R., Pinheiro L.C.D.,
Borges J.C., Carvalho V.L., Miranda M.D., De Mene-
ses M.D.F., Nascimento M., Ferreira D., Rebello M.A.
(2007) Synthesis and antiviral activity of new 4-(pheny-
lamino)/4-[(methylpyridin-2-yl)amino]-1-phenyl-1Hpyra-
zolo[3,4-b]pyridine-4-carboxylic acids derivatives. Med
Chem Res;16:352–369.
Acknowledgments
We are grateful for financial support from the National
Natural Science Foundation of China (21276064), the Nat-
ural Science Foundation of Hebei Education Department
(2008320), and the Science Foundation of Hebei Normal
University (2011Y04).
12. Tucker T.J., Sisko J.T., Tynebor R.M., Williams T.M.,
Felock P.J., Flynn J.A., Lai M., Liang Y., McGaughey
G., Liu M. (2008) Discovery of 3-{5-[(6-Amino-1H-pyr-
azolo[3,4-b]pyridine-3-yl)methoxy]-2-chlorophenoxy}-5-
References
1. Ilic D., Damljanovic I., Stevanovic D., Vukicevic M.,
Radulovic N., Kahlenberg V., Laus G., Vukicevic R.D.
(2010) Synthesis, spectral characterization, electro-
chemical properties and antimicrobial screening of
sulfur containing acylferrocenes. Polyhedron;29:1863–
1869.
2. Thakrar S., Bavishi A., Radadiya A., Parekh S., Bhavsar
D., Vala H., Pandya N., Shah A. (2013) Microwave-
assisted rapid synthesis of novel 1,5-benzodiazepine
derivatives as potent antimicrobial agent. J Heterocycl
Chem;50:E73–E79.
chlorobenzonitrile (MK-4965):
a
potent, orally
bioavailable HIV-1 non-nucleoside reverse transcrip-
tase inhibitor with improved potency against key
mutant viruses. J Med Chem;51:6503–6511.
13. Mamolo M.G., Zampieri D., Falagiani V., Vio L., Fer-
meglia M., Ferrone M., Pricl S., Banfi E., Scialino G.
(2004) Antifungal and antimycobacterial activity of new
N1-[1-aryl-2-(1Himidazol-1-yl and 1H-1,2,4-triazol-1-
yl)-ethylidene]-pyridine-2-carboxamidrazone derivatives:
a
combined experimental and computational
approach. Arkivoc;5:231–250.
3. Konda S.G., Shaikh B.M., Chavan S.A., Dawane B.S.
(2010) Polyethylene glycol (PEG-400): an efficient and
recyclable reaction medium for the synthesis of novel
1,5-benzodiazepines and their antimicrobial activity.
Chin Chem Lett;22:65–68.
14. Ji D., Lu J.R., Lu B.W., Xin C.W., Mu J.B., Li J.F.,
Peng C.Y., Bao X.R. (2013) Efficient synthesis and
antimicrobial activity of some novel S-b-D-glucosides of
5-aryl-1,2,4-triazole-3-thiones derivatives. Bioorg Med
Chem Lett;23:1997–2000.
4. Kumar R., Joshi Y.C. (2008) Synthesis and antimicro-
bial, antifungal and anthelmintic activities of 3H-
15. Sarmiento G.P., Vitale R.G., Afeltra J., Moltrasio G.Y.,
Moglioni A.G. (2011) Synthesis and antifungal activity
120
Chem Biol Drug Des 2016; 88: 110–121

Novel Series of 1,5-Benzodiazepine Derivatives
of some substituted phenothiazines and related com-
pounds. Eur. J. Med Chem;46:101–105.
16. Li X.Q., Wang L.Z. (2014) Highly efficient one-pot,
three-component synthesis of 1,5-benzodiazepine
derivatives. Chin Chem Lett;25:327–332.
17. Pasquale A.G., Ruiz M.D., Jios L., Autino J.C.J.,
Romanelli P.G. (2013) Preyssler catalyst-promoted
rapid, clean, and efficient condensation reactions for
3H-1,5-benzodiazepine synthesis in solvent-free condi-
tions. Tetrahedron Lett;54:6574–6579.
nowski R., Andes R.D., Kaczmarek L. (2014) Search-
ing for new derivatives of neocryptolepine: synthesis,
antiproliferative, antimicrobial and antifungal activities.
Euro J Med Chem;78:304–313.
20. Jadhav G.R., Shaikh M.U., Kale R.P., Shiradkarc
M.R., Gill C.H. (2009) SAR study of clubbed [1,2,4]-
triazolyl with fluorobenzimidazoles as antimicrobial
and antituberculosis agents. Euro
J Med Chem;
44:2930–2935.
18. Seenaiah D., Reddy P.R., Reddy G.M., Padmaja A.,
Padmavathi V., Krishna N.S. (2014) Synthesis, antimi-
crobial and cytotoxic activities of pyrimidinyl benzoxa-
zole, benzothiazole and benzimidazole. Euro J Med
Chem;77:1–7.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
19. Sidoryk K., Jaromin A., Edward J.A., Switalska M.,
Stefanska J., Cmoch P., Zagrodzka J., Szczepek W.,
Peczynska-Czoch W., Wietrzyk J., Kozubek A., Zar-
Appendix S1. Spectrum data of target compounds.
Chem Biol Drug Des 2016; 88: 110–121
121