1,5-Benzodiazepine derivatives as potential antimicrobial agents: Design, synthesis, biological evaluation, and structure-activity relationships

Article abstract of DOI:10.1039/c5ob00655d

36 Novel 1,5-benzodiazepine derivatives were rationally designed and synthesized according to the principle of superposition of bioactive substructures by the combination of 1,5-benzodiazepines, thiophene or thiazole and ester group. The structures of the target compounds have been characterized by IR, ¹H NMR, ¹³C NMR, MS and elemental analysis. The structure of 1v was further determined using X-ray single crystal diffraction. All synthesized 1,5-benzodiazepine derivatives were evaluated for their in vitro antimicrobial activity against C. neoformans, C. neoformans clinical isolates, C. albicans, E. coli and S. aureus. The bioactive assay results revealed that most of the 1,5-benzodiazepine derivatives exhibited considerable potency against all of the tested strains. In particular, compounds 1v and 1w (MIC: 2-6 μg mL^-1, MFC: 10-14 μg mL^-1) exhibited excellent antifungal activity and were found to be 32-64 and 9-12.8 times more potent than the reference drugs against C. neoformans, respectively. Moreover, compound 1v (MIC: 40 μg mL^-1) displayed equipotent antibacterial activity against E. coli and S. aureus compared to the reference drugs. The most potent of the synthesized compounds 1v and 1w were further studied by evaluating their cytotoxicities, and the results showed that they had relatively low level cytotoxicity for BV2 cell. A preliminary study of the structure-activity relationship revealed that substituents in the phenyl ring and the thiophene ring had a great effect on the antimicrobial activity of these compounds. In addition, the thiazole ring at C₂ may be a pharmacophore of these compounds and COOC₂H₅ group at C₃ is the best substituent for the maintenance of antimicrobial activities at low concentrations (1.5625 μg per disc).

Full text of DOI:10.1039/c5ob00655d

Organic &
Biomolecular Chemistry
View Article Online
View Journal
PAPER
1,5-Benzodiazepine derivatives as potential
antimicrobial agents: design, synthesis, biological
Cite this: DOI: 10.1039/c5ob00655d
evaluation, and structure–activity relationships†
Lan-Zhi Wang,* Xiao-Qing Li and Ying-Shuang An
36 Novel 1,5-benzodiazepine derivatives were rationally designed and synthesized according to the prin-
ciple of superposition of bioactive substructures by the combination of 1,5-benzodiazepines, thiophene
or thiazole and ester group. The structures of the target compounds have been characterized by IR,
1H NMR, 13C NMR, MS and elemental analysis. The structure of 1v was further determined using X-ray
single crystal diffraction. All synthesized 1,5-benzodiazepine derivatives were evaluated for their in vitro
antimicrobial activity against C. neoformans, C. neoformans clinical isolates, C. albicans, E. coli and
S. aureus. The bioactive assay results revealed that most of the 1,5-benzodiazepine derivatives exhibited
considerable potency against all of the tested strains. In particular, compounds 1v and 1w (MIC: 2–6 µg
mL−1, MFC: 10–14 µg mL−1) exhibited excellent antifungal activity and were found to be 32–64 and 9–12.8
times more potent than the reference drugs against C. neoformans, respectively. Moreover, compound 1v
(MIC: 40 µg mL−1) displayed equipotent antibacterial activity against E. coli and S. aureus compared to the
reference drugs. The most potent of the synthesized compounds 1v and 1w were further studied by evalu-
ating their cytotoxicities, and the results showed that they had relatively low level cytotoxicity for BV2 cell.
A preliminary study of the structure–activity relationship revealed that substituents in the phenyl ring and
the thiophene ring had a great effect on the antimicrobial activity of these compounds. In addition, the thi-
azole ring at C2 may be a pharmacophore of these compounds and COOC2H5 group at C3 is the best sub-
stituent for the maintenance of antimicrobial activities at low concentrations (1.5625 μg per disc).
Received 2nd April 2015,
Accepted 7th April 2015
DOI: 10.1039/c5ob00655d
www.rsc.org/obc
the pharmacological properties of the compounds, and this
Introduction
effect is attributed to their high hydrophobicity.5
1,5-Benzodiazepines is a class of outstanding organic mole-
Moreover five-membered heterocycle compounds, such as
cules with a wide array of biological activities and therapeutic thiophene or thiazole were reported to be significant structural
functions. They are commonly used as anxiolytic and anti- units of potent antimicrobial agents, and they were also vital
convulsive drugs.1 The use of 1,5-benzodiazepines for thera- pharmacodynamic heterocyclic nuclei.6,7 1,5-Benzodiazepines
peutic purposes is not confined to the treatment of anxiety composed of five-membered heterocycle compounds show
and stress conditions because minor changes in their struc- superior biological activity.8
tures can produce various biological activities, and novel appli-
In view of the above-mentioned findings, and as a continu-
cations of these compounds are continuously emerging.2 ation of our effort to identify new candidates that may be
Recently, 1,5-benzodiazepines and their derivatives have been valuable in designing new, potent, selective, and less toxic
also reported to exhibit excellent antibacterial and antifungal antimicrobial agents, we have reported herein the synthesis of
properties.3,4 In addition, previous studies on 1,5-benzo- 36 novel 1,5-benzodiazepine derivatives by incorporating a five-
diazepines have indicated that the free ester group present at membered S-heterocyclic group and an ester group. All the syn-
different positions in the nuclei of the molecules can enhance thesized compounds were screened for their in vitro antimicro-
bial activities against fungi (C. neoformans ATCC 32264 and
C. neoformans clinical isolates, C. albicans ATCC 10231) and a
College of Chemistry & Material Science, Hebei Normal University, Shijiazhuang
representative Gram-negative bacterium (E. coli ATCC 44752)
and Gram-positive bacteria (S. aureus ATCC 25923). This com-
bination was suggested to investigate the influence of such
hybridization and structure variation on the anticipated bio-
050024, People’s Republic of China. E-mail: wanglanzhi@126.com;
Fax: +86 311 80787431
†Electronic supplementary information (ESI) available. CCDC 969639. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
logical activities, hoping to add some synergistic biological sig-
c5ob00655d
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 1 Structures of compounds 1–4.
nificance to target compounds. The structures of the com- aldehyde or thiazol aldehyde in ethanol at 0 °C in the presence
pounds 1–4 are shown in Fig. 1.
of a catalytic amount of phosphomolybdic acid with a yield of
80–90%. Compounds 2, 3 and 4 were obtained via a similar syn-
thetic route in which ethyl acetoacetate was replaced by the
corresponding ester (methyl acetoacetate, acetyl propyl acetate
and acetyl isopropyl acetate). All of the compounds were charac-
Results and discussion
Synthesis of 1,5-benzodiazepine derivatives 1–4
1
terized by IR, H NMR, 13C NMR, MS and elemental analysis.
To correctly define the structure of 1v, X-ray analysis was per-
formed. Fig. 2 shows the ORTEP of 1v.
The novel 1,5-benzodiazepine derivatives 1–4 described in the
present study were synthesized as shown in Scheme 1. The
nucleophilic addition of substituted o-PDA with ethyl aceto-
acetate under solvent-free, catalyst-free and room-temperature
Biological activity
conditions yielded approximately 95% N-o-aminoaryl-β-enamino The 36 target compounds 1–4 were evaluated for their in vitro
esters 5. Compound 5 was smoothly converted to the 1,5-benzo- antibacterial activities using the disk-diffusion method at a
diazepine derivatives 1 via reaction with substituted thiophene concentration of 200 μg per disc.9 The diameter of the inhi-
Scheme 1 Synthesis of benzodiazepines 1–4.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
In order to further explore the influence of substituent Y at
the C3-position on the antimicrobial activity of this series of
1,5-benzothiazepines, compounds 2–4 were synthesized by
modifying the ester group that caused compounds 1 to exhibit
high antimicrobial activities. Compounds 2, 3 and 4 have
Y groups of COOCH3, COOCH2CH2CH3 and COOCH(CH3)2,
respectively. The results of the preliminary antibacterial testing
of compounds 2–4 are also shown in Table 1.
The data shown in Table 1 demonstrate that 8 compounds
2, 3 and 4 exhibited good inhibition activity. Compound 2b
exhibited larger inhibition zones against bacteria than against
fungi and displayed the highest antibacterial activity against
S. aureus but no antifungal activity against the C. neoformans
clinical strain. Compounds 2c and 2d exhibited slight anti-
microbial activity against S. aureus. Compounds 2e and 2f
showed high to moderate antimicrobial activities against
C. neoformans, E. coli and S. aureus, but 2g (R = Br) did not
exhibit any activity against any of the tested microorganisms.
These results illustrate that a Br atom may reduce the anti-
microbial activity of this series of compounds. Overall, the
inhibition zones of compounds 2 against bacteria were larger
than their zones against fungi. Therefore, compounds 2 exhibi-
ted better sensitivity against bacteria than against fungi. Fur-
thermore, compounds 2 showed better activity against Gram-
positive bacteria than against Gram-negative bacteria. Com-
pounds 3a and 3b inhibited all of the tested microorganisms
and showed an efficient broad-spectrum inhibitory effect.
These compounds exhibited excellent activity against the
Gram-positive bacteria S. aureus and C. neoformans clinical
strain. Compound 4a was also an efficient broad-spectrum
antibiotic and exhibited excellent inhibition activities against
the C. neoformans clinical strain and S. aureus. In contrast,
compound 4b had no activity against any of the tested
microorganisms.
Fig. 2 Crystal structure of 1v.
bition zone was measured as an indicator of each compound’s
activity. For ease of visualisation, the zone values obtained
from these assays indicate the average diameter (from three
trials) of the growth inhibition zones. The margin of error of
these measurements was 1 mm. The antibacterial activity was
classified as highly active (>14 mm), moderately active
(10 mm–14 mm), slightly active (6 mm–10 mm) and inactive
(<6 mm). The zones of the 36 compounds are reported in
Table 1.
Table 1 reveals 19 compounds with good antimicrobial
activities. Among compounds 1a–1c with 3-thienyl group at
the C2-position, compounds 1a and 1b showed moderate to
high activity against all tested fungi and slight broad-spectrum
activity against all tested bacteria, whereas compound 1c was
found inactive against the tested microorganisms. In general,
1a and 1b exhibited better antimicrobial activity against fungi
than against bacteria. Of the compounds 1d–1f with a
2-thienyl group at the C2-position, 1d exhibited high activity
against the C. neoformans clinical strain, slight activity against
S. aureus, and no activity against E. coli. Compounds 1h–1l pre-
sented a methyl substituent at positions 3 or 5 on the thio-
phene ring. These compounds showed obvious inhibition
potency and specificity to C. albicans alone except 1j.
Concentration gradient
Compounds 1v–1y, which were composed of 2-thiazolyl at
the C2-position, showed good to excellent antimicrobial activity
against all of the tested microorganisms. Compounds 1v–1y
presented the largest zone activity against C. neoformans clini-
cal isolates compared with their activity against the remaining
four microorganisms. Compound 1v in particular exhibited a
zone of inhibition of 28.63 mm against C. neoformans clinical
isolates. In general, the activities of 1v–1y against bacteria or
fungi were in the order 1v > 1w > 1y > 1x. Compounds 1v–1y
exhibited better antimicrobial activities against fungi com-
pared with their corresponding activities against bacteria and
better activities against Gram-positive bacteria than Gram-
negative bacteria.
Compounds 1a–1c and 1v–1y exhibited reduced inhibition
activities, indicating that their activity decreased when R = Br,
F. These data suggest that the introduction of Br or F is detri-
mental to the antimicrobial activity of these compounds. Com-
pounds 1h–1i and 1k–1l (X = 3-methyl-2-thienyl or 5-methyl-2-
thienyl) show unique antifungal activity against C. albicans
among the five tested microorganisms.
Compounds 1v, 1w, 2e, 3a, 3b and 4a, which exhibited remark-
able antimicrobial activity, were screened for their in vitro anti-
microbial activities using different concentration gradients
against the various strains through the standard two-fold
serial dilution method using agar media.10 The changes in the
inhibition zone size with the concentration of the compounds
are shown in Table 2.
From Table 2, it can be observed that the antimicrobial
activity of the compounds was reduced with decreasing com-
pound concentration. Compounds 1v and 1w exhibited slight
activity at doses <1.5625 μg per disc, but 2e, 3a and 3b did not
exhibit any activity at doses <25 μg per disc, which indicates
that the ethoxycarbonyl (–COOCH2CH3) is more conducive to
the maintenance of biological activity at low concentrations.
The antimicrobial capacity of 1v against the tested micro-
organisms was in the following order: C. neoformans clinical
strain > C. neoformans > S. aureus > E. coli. The antimicrobial
capacity of 1w for the tested microorganisms was in the same
order as 1v. The same order of antimicrobial capacity was
observed for most of the compounds in our present study.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Table 1 Antimicrobial activity results (zone of inhibition) of the newly synthesized compounds (1–4)
Zone of inhibitiona (mm), dose (200 μg per disc)
Compd
R
X
Y
C. n.b
C. n. C.b
C. a.b
E. c.b
S. a.b
1a
H
COOCH2CH3
9.90 0.20
13.03 0.15
12.10 0.22
6.00 0.00
8.80 0.10
1b
1c
CH3
Br
COOCH2CH3
COOCH2CH3
10.37 0.15
6.00 0.00
12.73 0.25
6.00 0.00
10.73 0.10
6.00 0.00
8.50 0.10
6.00 0.00
9.23 0.15
6.00 0.00
1d
1e
1f
H
COOCH2CH3
COOCH2CH3
COOCH2CH3
COOCH2CH3
7.97 0.05
6.00 0.00
6.00 0.00
6.00 0.00
10.40 0.06
6.00 0.00
6.00 0.00
6.00 0.00
9.03 0.10
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
8.47 0.05
6.00 0.00
6.00 0.00
6.00 0.00
CH3
Br
H
1g
1h
1i
CH3
Br
COOCH2CH3
COOCH2CH3
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
18.73 0.32
17.03 0.25
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
1j
H
COOCH2CH3
COOCH2CH3
COOCH2CH3
COOCH2CH3
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
18.67 0.30
17.47 0.42
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
1k
1l
CH3
Br
H
1m
1n
1o
1p
1q
1r
CH3
Br
COOCH2CH3
COOCH2CH3
COOCH2CH3
COOCH2CH3
COOCH2CH3
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
H
CH3
Br
1s
1t
H
COOCH2CH3
COOCH2CH3
COOCH2CH3
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
CH3
Br
1u
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
Table 1 (Contd.)
Zone of inhibitiona (mm), dose (200 μg per disc)
Compd
R
X
Y
C. n.b
C. n. C.b
C. a.b
E. c.b
S. a.b
1v
H
COOCH2CH3
24.27 0.20
28.63 0.30
15.00 0.10
19.70 0.25
21.17 0.20
1w
1x
1y
2a
CH3
Br
F
COOCH2CH3
COOCH2CH3
COOCH2CH3
COOCH3
21.00 0.21
12.07 0.12
15.83 0.32
6.00 0.00
24.07 0.23
15.70 0.25
15.90 0.25
6.00 0.00
14.37 0.15
11.70 0.20
13.33 0.15
6.00 0.00
15.03 0.19
12.03 0.10
12.67 0.20
6.00 0.00
17.10 0.30
13.30 0.15
19.00 0.32
6.00 0.00
H
2b
CH3
COOCH3
9.03 0.35
6.00 0.00
7.30 0.20
10.27 0.35
13.53 0.42
2c
2d
2e
H
COOCH3
COOCH3
COOCH3
6.00 0.00
6.00 0.00
13.57 0.26
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
6.00 0.00
17.37 0.32
8.83 0.15
7.83 0.05
22.47 0.43
CH3
H
2f
CH3
Br
COOCH3
11.20 0.10
6.00 0.00
11.73 0.26
16.93 0.15
11.87 0.23
6.00 0.00
6.00 0.00
6.00 0.00
27.27 0.32
29.87 0.35
23.87 0.30
6.00 0.00
6.00 0.00
6.00 0.00
17.10 0.24
20.53 0.30
10.47 0.12
6.00 0.00
14.23 0.06
6.00 0.00
16.30 0.25
17.73 0.30
13.73 0.15
6.00 0.00
19.70 0.23
6.00 0.00
28.77 0.42
26.60 0.35
26.67 0.32
6.00 0.00
2g
3a
3b
4a
4b
COOCH3
H
COO(CH2)2CH3
COO(CH2)2CH3
COOCH(CH3)2
COOCH(CH3)2
CH3
H
CH3
a The values indicate the average diameters in mm (of three trials) for the zone of growth inhibition observed after 24 h of incubation at 30 °C.
b C. 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.
Table 2 Inhibition zones at different dose levels for compounds 1v, 1w, 2e, 3a, 3b and 4a
(Zone of inhibition/mm)
1v
1w
3a
3b
4a
2e
Dose (μg per disc) C. n.
C. n. C. E. c.
S. a.
C. n.
C. n. C. S. a.
C. n. C. S. a.
C. n. C. C. a.
S. a.
C. n. C. S. a.
200
100
50
25
12.5
6.25
3.125
1.5625
25.20 28.97
20.67 21.93 21.63 24.23
20.33 21.47 20.00 23.97
19.23 20.90 18.67 23.23
18.90 17.70 17.90 22.93
14.73 14.73 17.03 20.37
22.47 27.27
19.63 21.23
16.93 18.13
28.77 29.87
12.27 21.20
11.60 20.03
10.00 13.23
20.53 26.60 23.87
15.40 14.27 14.03
12.97 12.47 12.13
26.67
12.77
10.77
6.00
6.00
6.00
24.73 27.50
22.27 27.30
19.63 21.83
16.67 17.60
15.70 16.87
13.20 13.53
6.50
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
7.97 10.37
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
9.03
6.00
6.00
6.00
7.93
6.00
6.00
6.93 14.80 17.03
6.00 13.20 13.63
6.00
6.00
8.37
8.77
6.00
6.00
8.20
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Table 3 MIC, MIC80 and MFC values for 1v, 1w and fluconazole (μg mL−1
)
1v
1w
Fluconazole
Fungal straina
MIC
MIC80
MFC
MIC
MIC80
MFC
MIC
MIC80
MFC
C.n
C. n. C.
2.0
6.0
1.0
5.0
10.0
14.0
4.0
3.0
3.0
1.5
14.0
12.0
>128.0
>128.0
2.0
2.0
>128.0
>128.0
a C.n.: C. neoformans ATCC 32264; C. n. C.: C. neoformans clinical strain.
The MIC and MFC determination
The MIC is defined as the minimum concentration of a com-
pound required to exhibit complete inhibitions of bacterial
and fungal growth. MIC80 was recorded as the concentration
that produced 80% growth reduction compared to wells with
no compound present. The MFC is the concentration at which
fungi failed to grow in the liquid nutrient medium. Flucon-
azole and ciprofloxacin were used as the standard drugs
against fungi and bacteria, respectively.11 Compounds 1v and
1w maintained biological activities even at low concentration
(1.5625 μg per disc). Compounds 1v and 1w, which had promi-
nent antimicrobial activities, were subjected to further
pharmacological evaluation, including determining MIC,
MIC80, and MFC using standard techniques.10 The MIC, MIC80
and MFC values of the compounds tested are listed in Table 3.
To our surprise, compounds 1v demonstrated excellent
inhibitory activity against C. neoformans and emerged as the
most effective antifungal agents with 64 times lower MIC (2 µg
mL−1) and 12.8 times lower MFC (10 µg mL−1) than the refer-
ence drug fluconazole (MIC > 128.0 μg mL−1, MFC > 128.0 μg
mL−1). It should be emphasized that compounds 1w also
exhibited especially interesting inhibitory activity against C.
neoformans with 32 times lower MIC (4 µg mL−1) and 9.1 times
lower MFC (14 µg mL−1) than the reference drug fluconazole
(MIC > 128.0 μg mL−1, MFC > 128.0 μg mL−1). Furthermore,
compounds 1v and 1w showed especially higher antifungal
potential than the reference drug fluconazole against C. neofor-
mans clinical strain. These results indicated that compounds
1v and 1w may become lead molecules of antifungal drug
against C. neoformans.
Fig. 3 Effects of compounds 1v, 1w and fluconazole on the survival of
cells.
DMSO was used as a control. The results (Fig. 3) showed that
compounds 1v and 1w had no cytotoxic activity at concen-
trations ≤12.5 μg mL−1 and low cytotoxicities within the range
of 12.5–25 μg mL−1, which was not significantly different from
fluconazole. This indicated that compounds 1v and 1w did not
affect the cell viability of the BV2 cell line at their MIC values
(2 and 4 μg mL−1) against the C. neoformans and MIC values
(6 and 3 μg mL−1) against the C. neoformans clinical strain,
respectively. The compounds 1v and 1w also exhibited less
effect on the survival of the BV2 cell line at their MIC values
against the S. aureus and E. coli. But the relative survival rate of
the cells significantly decreased with compounds 1v and 1w at
concentrations ≥25.0 μg mL−1 (p < 0.01) which indicated that
compounds 1v and 1w were cytotoxic at high concentrations.
The disparity between the cytotoxicities and antibacterial
activities of compounds 1v and 1w suggested that these com-
pounds exhibited high in vitro antimicrobial activities at non-
cytotoxic concentrations.
In addition, 1v had a MIC value of 40 μg mL−1 against both
S. aureus and E. coli and demonstrated equipotent antibacter-
ial activity with reference drug ciprofloxacin (MIC = 28 μg
mL−1 for S. aureus and MIC = 36 μg mL−1 for E. coli). In
general, compounds 1v and 1w demonstrated better antifungal
activity than antibacterial activity. The antimicrobial activity of
1v is better than that of 1w.
Cytotoxicities of compounds 1v and 1w
SAR
To investigate whether the antimicrobial activities of com-
pounds 1v and 1w related specifically to their selective toxicity To analyze structure–activity relationships, three structural
toward the tested strains, we evaluated their cytotoxicities components were considered: the nature of the five-membered
using a BV2 cell line and a 3-(4,5-dimethylthiazol-2-yl)-2,5- heterocycle nucleus (X) at the C2 position, the length and size
diphenyltetrazolium bromide (MTT) assay. The cytotoxicity of of the ester group chain (Y) at the C3 position and the elec-
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
tronic properties of substitution (R) at the C8 position.
bacterial screening results led to the assumption that the
activity of the compounds was in the order R = –H > R = –CH3
> R = –F > R = –Br. In addition, it seems that sizes of substitu-
ents are important. The compounds containing larger substi-
tuents (Br 1c, 1f) at the C8 position are the least active. The
electron-withdrawing group –Br at the C8 position, which is
larger than –CH3, –H, increased the volume of the compound
and lowered the electron density of the seven-membered ring,
which resulted in reduced biological activity. Similar results
The presence of a thienyl group at the C2 position resulted have been reported previously.14
in high selectivity and specificity to C. albicans. The introduc-
The observed antimicrobial activity of compounds could be
tion of the thiazole nucleus at the C2 position also led to a sig- attributed to the synergistic effect of the five-membered
nificant increase of activity. The influence of X at the C2 heterocycle nucleus, 1,5-benzodiazepine moieties and ester
position was easily observed as compounds with the thiazole group as antimicrobial moieties.
moiety (1v and 1w) showed much superior activities over the
thienyl group (1a and 1b). The lowest MIC value (2.0 μg mL−1
)
was obtained with 1v which appeared as the most active com-
pound against the C. neoformans. This value is 64 times lower
Conclusions
than the control (fluconazole), indicating the powerful and very In conclusion, 36 novel 1,5-benzodiazepine derivatives were
interesting antifungal potential of this structure on C. neoformans. synthesized by incorporating a five-membered S-heterocyclic
The thiazole moiety in this type of 1,5-benzodiazepine plays a key group and an ester group and investigated for their anti-
role in enhancing the efficacy of the antimicrobial activity. Similar microbial properties and cytotoxicities with an anticipation of
results have been reported in the literature.12 Therefore, 2-thi- generating new structural leads serving as potent antimicrobial
azolyl may be a pharmacophore group in these compounds.
agents. Among the 36 synthesized compounds screened, com-
The types of thiophene ring (X) at the C2 position and sub- pounds 1v, 1w, 2e, 3a, 3b and 4a exhibit remarkable antimicro-
stituents also affected the activity of the compounds. Overall, bial activities at different concentrations. The results also
when X = 3-thienyl, the compounds exhibited better activities show that most of the target compounds 1–4 with a thiazole
compared with the activity observed when X = 2-thienyl. The ring exhibited considerable potency against all of the tested
introduction of an electron-donating group (CH3) at the thio- strains at microgram concentrations, especially compounds 1v
phene improved the antimicrobial activity of compounds and 1w. The most active compound 1v (MIC: 2–6 µg mL−1 and
against C. albicans slightly, but no improvement in the anti- MFC: 10–14 µg mL−1) was found to be 21–64 times and 9–12.8
bacterial activity of the compounds against the other tested times more potent than fluconazole against C. neoformans and
microorganisms was observed. The role of the electron-donat- the C. neoformans clinical strain, respectively. Compound 1w
ing group in improving antimicrobial activities has been (MIC: 3–4 µg mL−1 and MFC: 10–14 µg mL−1) was found to be
reported previously.13
9–42.7 times more potent than fluconazole against C. neofor-
The ester group chain length and size at the C3 position, on mans and the C. neoformans clinical strain. Moreover, the MIC
the other hand, appeared to be much more important for bio- value (40 μg mL−1) of 1v against both S. aureus and E. coli indi-
logical activity.
cates that it exhibits equipotent antibacterial activity compared
An increase in the ester group chain length [–COOCH3, to ciprofloxacin. Simultaneously, compounds 1v and 1w were
–COOC2H5, –COOCH2CH2CH3] can increase the antibacterial accompanied with relatively low levels of cytotoxicity. There-
activity and broad spectrum of the compounds. However, fore, 1v and 1w are very attractive antimicrobial leads and
replacement of the small size n-propyl ester group should be excellent scaffolds for further study.
(–COOCH2CH2CH3) at the C3 position with a bigger isoproply
ester group (COOCH(CH3)2) resulted in a decrease in the anti- that compounds with a thiazole moiety at the C2 position
microbial activity. showed much superior activities over the thienyl group. The
The structure–activity relationship (SAR) results indicated
The substituent (R = CH3, H, F and Br) at the C8 position introduction of an electron-withdrawing group instead of an
plays a key role in varying the efficacy of antimicrobial activity. electron-donating group at the thiophene ring at the C2 posi-
Compounds without a substituent (R = H) at the C8 position tion was apparently detrimental to their activity. The variations
provided the best antimicrobial action, which demonstrates of R at the C8 position of the 1,5-benzodiazepine also have a
that C8, in the class of compounds studied, should be un- great effect on the antimicrobial activity of these compounds.
substituted. The influence of the electronic properties of sub- The activity based on different R groups against all of the
stitution (R) at the C8 position was also important. It was microorganisms tested was in the order hydrogen (1v) >
noticed that compounds possessing an electron-donating methyl (1w) > fluoro (1y) > bromine (1x). The results of the
group (CH3) at the C8 position are more active against all tested concentration gradients showed that COOC2H5 is the
tested bacteria and fungi than compounds having electron- best substituent for the maintenance of biological activities at
withdrawing substituents (F and Br). On the whole, the anti- low concentrations (1.5625 μg per disc). Furthermore,
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
lengthening the carbon chain at the end of the ester group can cipitate that formed was filtered, washed with ethanol and
increase the antibacterial activity and broaden the spectrum of recrystallised from dry ethanol to yield 1a–1x.
the compounds. However, increasing the number of branched
chains at the end may decrease or eliminate the activity of diazepine-3-carboxylate (1a). Yield: 82%, mp 52–54 °C. 1H
the compounds. NMR (CDCl3, 500 MHz) δ = 6.49–7.09 (7H, m, –C6H4, –C4H3S),
Ethyl
4-methyl-2-(thiophen-3-yl)-2,5-dihydro-1,5-benzo-
Findings from the SAR and toxicity studies have encouraged 5.96 (1H, s, –NH), 5.89 (1H, s, –CH), 4.41 (1H, s, –NH), 4.12
us to make some modifications to the basic structure of the (2H, q, J = 7.5 Hz, –COOCH2), 2.52 (3H, s, –CH3), 1.21 (3H, t,
obtained compounds 1v and 1w to achieve selective, more J = 7.5 Hz, –CH2CH3) ppm. 13C NMR (CDCl3, 125 MHz) δ =
active and non-toxic derivatives in ongoing studies. In addition, 14.49, 25.32, 59.89, 60.46, 102.12, 110.45, 119.73, 121.31,
the further investigations on these findings can be useful for 121.54, 122.42, 124.79, 129.23, 130.86, 136.03, 150.19, 151.31,
medicinal chemists to synthesize similar compounds.
167.86 ppm. IR (KBr, cm−1) ν = 3340 (N–H), 1674 (CvO), 1632
(CvC). MS (m/z): 315 [M + H]+; C17H18N2O2S: found: C, 64.90;
H, 5.79; N, 8.89; Calcd: C, 64.94; H, 5.77; N, 8.91.
Ethyl 4,8-dimethyl-2-(thiophen-3-yl)-2,5-dihydro-1,5-benzo-
diazepine-3-carboxylate (1b). Yield: 83%, mp 68–70 °C. 1H
NMR (CDCl3, 500 MHz) δ = 6.31–7.09 (6H, m, –C6H3, –C4H3S),
5.93 (1H, s, –NH), 5.87 (1H, s, –CH), 4.35 (1H, s, –NH), 4.12
(2H, q, J = 7.0 Hz, –COOCH2), 2.50 (3H, s, –CH3), 2.14 (3H, s,
–CH3), 1.22 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. 13C NMR
(CDCl3, 125 MHz) δ = 14.64, 19.68, 25.29, 58.65, 59.62, 116.23,
119.19, 121.92, 122.31, 125.89, 126.47, 128.51, 131.15, 138.05,
150.69, 151.62, 161.91, 170.66 ppm. IR (KBr, cm−1) ν = 3337
(N–H), 1672 (CvO), 1625 (CvC). MS (m/z): 329 [M + H]+.
C18H20N2O2S: found: C, 65.80; H, 6.15; N, 8.54; Calcd: C, 65.83;
H, 6.14; N, 8.53.
Experimental
General
The melting points (°C) were determined with a PXT-4 digital
melting point apparatus. The 1H NMR spectra were recorded
on a Bruker AVANCE-500 MHz spectrometer; the chemical
shifts (δ) are reported in ppm using SiMe4 as the internal stan-
dard when measured in CDCl3. The signal multiplicities are
represented by s (singlet), d (doublet), t (triplet), m (multiplet),
and q (quartet). Low-resolution mass spectra were recorded on
a Thermo DSQ II mass spectrometer. The IR spectra (in KBr
pellets) were recorded on a BIO-RAD PE-M-1730 IR spectrophoto-
meter. The elemental analysis was performed with a Vario
EL-III-CHN-0 Elemental Analyser. The structure of 1v was
further determined by single-crystal X-ray diffraction on a
Bruker Smart-1000 diffractometer. Thiazol aldehyde, substi-
tuted thiophene aldehyde and o-PDA were purchased from
Aladdin, and the other chemicals used were commercial pro-
ducts of analytical grade. The solvents were dried and purified
according to the literature as necessary. The reactions were
monitored by thin-layer chromatography (TLC) on pre-coated
silica gel GF254 plates.
Ethyl
8-bromo-4-methyl-2-(thiophen-3-yl)-2,5-dihydro-1,5-
benzodiazepine-3-carboxylate (1c). Yield: 84%, mp 88–90 °C.
1H NMR (CDCl3, 500 MHz) δ = 6.45–7.14 (6H, m, –C6H3,
–C4H3S), 5.89 (1H, s, –NH), 5.86 (1H, s, –CH), 4.46 (1H, s,
–NH), 4.13 (2H, q, J = 7.0 Hz, –COOCH2), 2.50 (3H, s, –CH3),
1.23 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. 13C NMR (CDCl3,
125 MHz) δ = 14.45, 24.87, 58.87, 59.88, 103.93, 114.81, 119.12,
120.58, 121.47, 122.11, 124.60, 129.47, 130.67, 137.06, 150.08,
151.51, 168.07 ppm. IR (KBr, cm−1) ν = 3297 (N–H), 1630
(CvO), 1583 (CvC). MS (m/z): 393 [M + H]+. C17H17BrN2O2S:
found: C, 51.90; H, 4.37; N, 7.14; Calcd: C, 51.92; H, 4.36;
N, 7.12.
Synthesis
Ethyl
4-methyl-2-(thiophen-2-yl)-2,5-dihydro-1,5-benzo-
Synthesis of N-o-aryl amino-β-enamino esters 5a–5j. A
mixture of ethyl acetoacetate (methyl acetoacetate, acetyl
propyl acetate or acetyl isopropyl acetate) (30 mmol) and
various o-PDAs (o-PDA, 4-methyl-o-PDA or 4-bromo-o-PDA)
(25 mmol) was stirred at room temperature for approximately
50 min. The solids were collected by filtration under reduced
pressure to give the corresponding crude N-o-aryl amino-
β-enamino esters 5a–5j (∼98%), which were used directly
without further purification.
diazepine-3-carboxylate (1d). Yield: 81.8%, mp 62–64 °C.
1H NMR (CDCl3, 500 MHz) δ = 6.52–7.03 (7H, m, –C6H4,
–C4H3S), 6.09 (1H, s, –CH), 6.04 (1H, s, –NH), 4.39 (1H, s,
–NH), 4.13 (2H, q, J = 7.5 Hz, –COOCH2), 2.53 (3H, s, –CH3),
1.23 (3H, t, J = 7.5 Hz, –CH2CH3) ppm. 13C NMR (CDCl3,
125 MHz) δ = 14.45, 25.22, 56.41, 59.77, 103.29, 119.54, 121.46,
123.10, 124.12, 124.57, 126.24, 127.67, 132.29, 136.63, 148.46,
150.52, 167.81 ppm. IR (KBr, cm−1) ν = 3341 (N–H), 1673 (CvO),
1629 (CvC). MS (m/z): 315 [M + H]+. C17H18N2O2S: found: C,
64.98; H, 5.75; N, 8.89; Calcd: C, 64.94; H, 5.77; N, 8.91.
General procedure for the preparation of ethyl 4-methyl-
2-(thiophen-2-yl, thiophen-3-yl or thiazol-2-yl)-2,5-dihydro-
1,5-benzodiazepine-3-carboxylate (1a–1x)
Ethyl 4,8-dimethyl-2-(thiophen-2-yl)-2,5-dihydro-1,5-benzo-
diazepine-3-carboxylate (1e). Yield: 82.6%, mp 74–76 °C.
1H NMR (CDCl3, 500 MHz) δ = 6.34–7.03 (6H, m, –C6H3,
Ethyl acetoacetate and o-PDA, 4-methyl-o-PDA or 4-bromo-o- –C4H3S), 6.07 (1H, s, –CH), 6.00 (1H, s, –NH), 4.33 (1H, s,
PDA yield 5a–5c. A mixture of compound 5a (5b or 5c) –NH), 4.13 (2H, q, J = 7.5 Hz, –COOCH2), 2.51 (3H, s, –CH3),
(2 mmol) and a substituted thiophene aldehyde or thiazol 2.15 (3H, s, –CH3), 1.23 (3H, t, J = 7.5 Hz, –CH2CH3) ppm.
aldehyde (2 mmol) in dry ethanol (15 ml) was stirred at 0 °C 13C NMR (CDCl3, 125 MHz) δ = 14.46, 20.49, 25.33, 58.62,
for approximately 8 h. After the reaction was complete, the pre- 59.69, 102.77, 119.07, 119.45, 121.65, 122.14, 124.08, 124.49,
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
128.97, 132.79, 136.38, 148.58, 152.61, 167.87 ppm. IR (KBr, –NH), 4.09 (2H, q, J = 7.0 Hz, –COOCH2), 2.54 (3H, s, –CH3),
cm−1) ν = 3336 (N–H), 1670 (CvO), 1625 (CvC). MS (m/z): 329 1.20 (3H, t, J = 7. 0 Hz, –CH2CH3) ppm. 13C NMR (CDCl3,
[M + H]+. C18H20N2O2S: found: C, 65.80; H, 6.12; N, 8.55; 125 MHz) δ = 14.10, 15.97, 19.69, 50.17, 61.39, 85.17, 115.36,
Calcd: C, 65.83; H, 6.14; N, 8.53.
Ethyl
117.02, 118.35, 123.35, 126.19, 127.39, 132.09, 142.27, 145.45,
8-bromo-4-methyl-2-(thiophen-2-yl)-2,5-dihydro-1,5- 150.26, 161.51, 167.12 ppm. IR (KBr, cm−1) ν = 3335 (N–H),
benzodiazepine-3-carboxylate (1f). Yield: 83.4%, mp 82–84 °C. 1673 (CvO), 1638 (CvC). MS (m/z): 329 [M
+
H]+.
1H NMR (CDCl3, 500 MHz) δ = 6.49–7.06 (6H, m, –C6H3, C18H20N2O2S: found: C, 65.85; H, 6.15; N, 8.55; Calcd: C, 65.83;
–C4H3S), 6.05 (1H, d, J = 6.0 Hz, –CH), 5.95 (1H, s, –NH), 4.42 H, 6.14; N, 8.53.
(1H, d, J = 6.0 Hz, –NH), 4.14 (2H, q, J = 7.0 Hz, –COOCH2),
Ethyl 4,8-dimethyl-2-(5-methylthiophen-2-yl)-2,5-dihydro-1,5-
2.51 (3H, s, –CH3), 1.23 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. benzodiazepine-3-carboxylate (1k). Yield: 80.2%, mp 66–68 °C.
13C NMR (CDCl3, 125 MHz) δ = 14.32, 25.01, 56.03, 59.87, 1H NMR (CDCl3, 500 MHz) δ = 6.43–6.89 (5H, m, –C6H3,
103.80, 114.94, 123.30, 124.00, 124.35, 124.59, 125.56, 126.32, –C4H2S), 6.40 (1H, s, –NH), 5.96 (1H, s, –CH), 4.30 (1H, s,
130.57, 137.88, 147.73, 150.15, 167.58 ppm. IR (KBr, cm−1) ν = –NH), 4.14 (2H, q, J = 7.0 Hz, –COOCH2), 3.48 (3H, s, –CH3),
3296 (N–H), 1629 (CvO), 1580 (CvC). MS (m/z): 393 [M + H]+. 2.49 (3H, s, –CH3), 2.33 (3H, s, –CH3), 1.23 (3H, t, J = 7.0 Hz,
C17H17BrN2O2S: found: C, 51.95; H, 4.35; N, 7.11; Calcd: C, –CH2CH3) ppm. 13C NMR (CDCl3, 125 MHz) δ = 14.66, 15.99,
51.92; H, 4.36; N, 7.12.
Ethyl
20.76, 20.93, 50.78, 58.58, 86.48, 119.12, 119.16, 121.99,
4-methyl-2-(3-methylthiophen-2-yl)-2,5-dihydro-1,5- 123.77, 124.29, 125.24, 128.49, 132.78, 134.73, 149.18, 152.68,
benzodiazepine-3-carboxylate (1g). Yield: 78.8%, mp 95–97 °C. 169.89 ppm. IR (KBr, cm−1) ν = 3346 (N–H), 1674 (CvO), 1646
1H NMR (CDCl3, 500 MHz) δ = 6.67–6.84 (6H, m, –C6H4, (CvC). MS (m/z): 343 [M + H]+. C19H22N2O2S: found: C, 66.66;
–C4H2S), 6.41 (1H, d, J = 6.5 Hz, –CH), 6.20 (1H, s, –NH), 4.31 H, 6.51; N, 8.16; Calcd: C, 66.64; H, 6.48; N, 8.18.
(1H, d, J = 7.0 Hz, –NH), 4.08 (2H, q, J = 7.0 Hz, –COOCH2),
Ethyl
8-bromo-4-methyl-2-(5-methylthiophen-2-yl)-2,5-
2.53 (3H, s, –CH3), 2.30 (3H, s, –CH3), 1.17 (3H, t, J = 7.0 Hz, dihydro-1,5-benzodiazepine-3-carboxylate (1l). Yield: 81.8%,
–CH2CH3) ppm. 13C NMR (CDCl3, 125 MHz) δ = 14.40, 20.87, mp 60–62 °C. 1H NMR (CDCl3, 500 MHz) δ = 6.37–6.72 (5H, m,
20.91, 55.18, 59.72, 103.47, 117.98, 121.01, 121.81, 123.23, –C6H3, –C4H2S), 5.97 (1H, s, –NH), 5.96 (1H, s, –CH), 4.28 (1H,
124.67, 129.70, 132.66, 136.71, 142.18, 149.98, 151.97, s, –NH), 4.15 (2H, q, J = 7.0 Hz, –COOCH2), 2.52 (3H, s, –CH3),
167.83 ppm. IR (KBr, cm−1) ν = 3330 (N–H), 1626 (CvO), 1611 2.21 (3H, s, –CH3), 1.25 (3H, t, J = 7.0 Hz, –CH2CH3) ppm.
(CvC). MS (m/z): 329 [M + H]+. C18H20N2O2S: found: C, 65.86; 13C NMR (CDCl3, 125 MHz) δ = 14.44, 15.36, 20.83, 56.22,
H, 6.16; N, 8.50; Calcd: C, 65.83; H, 6.14; N, 8.53.
59.91, 103.85, 117.74, 120.03, 121.25, 123.28, 124.43, 128.33,
Ethyl 4,8-dimethyl-2-(3-methylthiophen-2-yl)-2,5-dihydro-1,5- 133.77, 138.09, 145.22, 150.09, 153.46, 167.72 ppm. IR (KBr,
benzodiazepine-3-carboxylate
(1h). Yield:
79.4%,
mp cm−1) ν = 3296 (N–H), 1670 (CvO), 1647 (CvC). MS (m/z): 407
86–88 °C. 1H NMR (CDCl3, 500 MHz) δ = 6.53–6.86 (5H, m, [M + H]+. C18H19BrN2O2S: found: C, 53.12; H, 4.68; N, 6.90;
–C6H3, –C4H2S), 6.31 (1H, s, –CH), 6.23 (1H, s, –NH), 6.04 (1H, Calcd: C, 53.08; H, 4.70; N, 6.88.
s, –NH), 4.09 (2H, q, J = 7.5 Hz, –COOCH2), 2.53 (3H, s, –CH3),
2.31 (3H, s, –CH3), 2.18 (3H, s, –CH3), 1.18 (3H, t, J = 7.5 Hz, benzodiazepine-3-carboxylate
Ethyl
2-(3-bromothiophen-2-yl)-4-methyl-2,5-dihydro-1,5-
(1m). Yield: 81.6%, mp
–CH2CH3) ppm. 13C NMR (CDCl3, 125 MHz) δ = 14.10, 14.64, 132–134 °C. H NMR (CDCl3, 500 MHz) δ = 6.55–6.90 (6H, m,
19.68, 21.27, 50.16, 61.39, 84.82, 116.72, 117.67, 119.13, –C6H4, –C4H2S), 6.32 (1H, s, –NH), 6.03 (1H, d, J = 6.0 Hz,
121.72, 121.94, 123.14, 128.82, 130.98, 134.68, 148.07, 148.98, –CH), 4.88 (1H, d, J = 6.0 Hz, –NH), 4.07 (2H, q, J = 7.0 Hz,
167.12 ppm. IR (KBr, cm−1) ν = 3353 (N–H), 1673 (CvO), 1620 –COOCH2), 2.56 (3H, s, –CH3), 1.16 (3H, t, J = 7.0 Hz,
(CvC). MS (m/z): 343 [M + H]+. C19H22N2O2S: found: C, 66.66; –CH2CH3) ppm. 13C NMR (CDCl3, 125 MHz) δ = 14.37, 25.15,
1
H, 6.45; N, 8.15; Calcd: C, 66.64; H, 6.48; N, 8.18.
Ethyl
56.36, 59.80, 101.68, 107.97, 119.47, 121.28, 122.08, 123.53,
8-bromo-4-methyl-2-(3-methylthiophen-2-yl)-2,5- 123.73, 129.89, 132.49, 136.62, 143.22, 151.24, 167.58 ppm.
dihydro-1,5-benzodiazepine-3-carboxylate (1i). Yield: 80.4%, IR (KBr, cm−1) ν = 3311 (N–H), 1667 (CvO), 1627 (CvC). MS
mp 58–60 °C. 1H NMR (CDCl3, 500 MHz) δ = 6.79–6.94 (5H, m, (m/z): 393 [M + H]+. C17H17BrN2O2S: found: C, 51.90; H, 4.34;
–C6H3, –C4H2S), 6.71 (1H, d, J = 6.5 Hz, –NH), 6.56 (1H, d, J = N, 7.11; Calcd: C, 51.92; H, 4.36; N, 7.12.
7.0 Hz, –CH), 6.04 (1H, s, –NH), 4.10 (2H, q, J = 7.0 Hz,
–COOCH2), 2.53 (3H, s, –CH3), 2.30 (3H, s, –CH3), 1.18 (3H, t, benzodiazepine-3-carboxylate
Ethyl 2-(3-bromothiophen-2-yl)-4,8-dimethyl-2,5-dihydro-1,5-
(1n). Yield: 83.4%, mp
J = 7.0 Hz, –CH2CH3) ppm. 13C NMR (CDCl3, 125 MHz) δ = 130–132 °C. H NMR (CDCl3, 500 MHz) δ = 6.36–6.93 (5H, m,
14.31, 18.15, 20.96, 54.88, 59.87, 88.39, 117.73, 120.00, 120.56, –C6H3, –C4H2S), 6.06 (1H, s, –NH), 6.02 (1H, d, J = 5.0 Hz,
122.27, 123.14, 124.45, 129.40, 130.58, 139.15, 143.57, 152.58, –CH), 4.80 (1H, d, J = 5.0 Hz, –NH), 4.08 (2H, q, J = 7.0 Hz,
166.17 ppm. IR (KBr, cm−1) ν = 3311 (N–H), 1680 (CvO), 1633 –COOCH2), 2.57 (3H, s, –CH3), 2.13 (3H, s, –CH3), 1.18 (3H, t,
(CvC). MS (m/z): 407 [M + H]+. C18H19BrN2O2S: found: C, J = 7.0 Hz, –CH2CH3) ppm. 13C NMR (CDCl3, 125 MHz) δ =
1
53.05; H, 4.68; N, 6.91; Calcd: C, 53.08; H, 4.70; N, 6.88.
Ethyl
14.39, 20.52, 25.31, 56.21, 59.69, 101.30, 107.96, 119.29, 121.61,
4-methyl-2-(5-methylthiophen-2-yl)-2,5-dihydro-1,5- 122.72, 123.69, 124.34, 129.81, 133.27, 136.42, 143.30, 151.07,
benzodiazepine-3-carboxylate (1j). Yield: 80.0%, mp 90–92 °C. 167.55 ppm. IR (KBr, cm−1) ν = 3296 (N–H), 1670 (CvO), 1647
1H NMR (CDCl3, 500 MHz) δ = 6.42–6.85 (6H, m, –C6H4, (CvC). MS (m/z): 407 [M + H]+. C18H19BrN2O2S: found: C, 53.11;
–C4H2S), 6.12 (1H, s, –NH), 6.07 (1H, s, –CH), 4.30 (1H, s, H, 4.68; N, 6.90; Calcd: C, 53.08; H, 4.70; N, 6.88.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Ethyl 8-bromo-2-(3-bromothiophen-2-yl)-4-methyl-2,5-dihydro- 150.89, 167.62 ppm. IR (KBr, cm−1) ν = 3320 (N–H), 1630
1,5-benzodiazepine-3-carboxylate (1o). Yield: 84.8%, mp (CvO), 1611 (CvC). MS (m/z): 395 [M + H]+. C17H17BrN2O2S:
72–74 °C. 1H NMR (CDCl3, 500 MHz) δ = 6.84–6.95 (5H, m, found: C, 51.90; H, 4.36; N, 7.11; Calcd: C, 51.92; H, 4.36; N,
–C6H3, –C4H2S), 6.70 (1H, d, J = 7.0 Hz, –NH), 6.54 (1H, d, J = 7.12.
7.0 Hz, –CH), 6.01 (1H, s, –NH), 4.10 (2H, q, J = 7.0 Hz,
Ethyl 2-(5-bromothiophen-2-yl)-4,8-dimethyl-2,5-dihydro-1,5-
–COOCH2), 2.56 (3H, s, –CH3), 1.17 (3H, t, J = 7.5 Hz, benzodiazepine-3-carboxylate (1t). Yield: 84.0%, mp 88–90 °C.
–CH2CH3) ppm. 13C NMR (CDCl3, 125 MHz) δ = 14.34, 25.08, 1H NMR (CDCl3, 500 MHz) δ = 6.36–6.70 (5H, m, –C6H3,
56.04, 59.94, 102.40, 108.27, 118.31, 120.53, 123.47, 123.89, –C4H2S), 5.98 (1H, s, –NH), 5.96 (1H, s, –CH), 4.31 (1H, s,
124.75, 129.02, 131.78, 137.93, 142.39, 150.57, 167.27 ppm. –NH), 4.14 (2H, q, J = 7.0 Hz, –COOCH2), 2.50 (3H, s, –CH3),
IR (KBr, cm−1) ν = 3311 (N–H), 1670 (CvO), 1629 (CvC). MS 2.18 (3H, s, –CH3), 1.23 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. 13C
(m/z): 473 [M + H]+. C17H16Br2N2O2S: found: C, 43.22; H, 3.43; NMR (CDCl3, 125 MHz) δ = 14.48, 20.55, 25.34, 56.29, 59.83,
N, 5.94; Calcd: C, 43.24; H, 3.42; N, 5.93.
102.18, 119.68, 120.24, 121.53, 122.38, 124.78, 128.64, 129.21,
Ethyl 2-(4-bromothiophen-2-yl)-4-methyl-2,5-dihydro-1,5-benzo- 133.11, 136.03, 150.22, 151.14, 167.74 ppm. IR (KBr, cm−1) ν =
diazepine-3-carboxylate (1p). Yield: 82.4%, mp 95–97 °C. 1H 3317 (N–H), 1645 (CvO), 1596 (CvC). MS (m/z): 409 [M + H]+.
NMR (CDCl3, 500 MHz) δ = 6.74–6.93 (6H, m, –C6H4, –C4H2S), C18H19BrN2O2S: found: C, 53.07; H, 4.72; N, 6.90; Calcd: C,
6.54 (1H, d, J = 7.0 Hz, –NH), 6.06 (1H, s, –CH), 6.02 (1H, s, 53.08; H, 4.70; N, 6.88.
–NH), 4.13 (2H, q, J = 7.0 Hz, –COOCH2), 2.53 (3H, s, –CH3),
Ethyl 8-bromo-2-(5-bromothiophen-2-yl)-4-methyl-2,5-dihydro-
1.22 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. 13C NMR (CDCl3, 1,5-benzodiazepine-3-carboxylate (1u). Yield: 85.6%, mp
125 MHz) δ = 14.45, 25.33, 56.27, 59.91, 102.48, 108.59, 119.73, 66–68 °C. 1H NMR (CDCl3, 500 MHz) δ = 6.42–6.88 (5H, m,
121.34, 121.63, 121.76, 123.39, 127.24, 131.26, 136.13, 149.77, –C6H3, –C4H2S), 5.97 (1H, s, –NH), 5.94 (1H, s, –CH), 4.39 (1H,
150.99, 167.61 ppm. IR (KBr, cm−1) ν = 3311 (N–H), 1673 s, –NH), 4.13 (2H, q, J = 7.0 Hz, –COOCH2), 2.49 (3H, s, –CH3),
(CvO), 1627 (CvC). MS (m/z): 393 [M + H]+. C17H17BrN2O2S: 1.23 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. 13C NMR (CDCl3,
found: C, 51.90; H, 4.34; N, 7.11; Calcd: C, 51.92; H, 4.36; 125 MHz) δ = 14.35, 24.93, 55.99, 59.99, 103.38, 110.65, 115.20,
N, 7.12.
120.84, 123.09, 124.13, 124.87, 125.80, 129.32, 137.57, 149.44,
Ethyl 2-(4-bromothiophen-2-yl)-4,8-dimethyl-2,5-dihydro-1,5- 150.76, 167.46 ppm. IR (KBr cm−1) ν = 3319 (N–H), 1674
benzodiazepine-3-carboxylate (1q). Yield: 83.7%, mp 70–72 °C. (CvO), 1648 (CvC). MS (m/z): 473 [M + H]+. C17H16Br2N2O2S:
1H NMR (CDCl3, 500 MHz) δ = 6.35–6.93 (5H, m, –C6H3, found: C, 43.25; H, 3.43; N, 5.92; Calcd: C, 43.24; H, 3.42;
–C4H2S), 6.02 (1H, s, –CH), 6.00 (1H, s, –NH), 4.33 (1H, s, N, 5.93.
–NH), 4.13 (2H, q, J = 7.0 Hz, –COOCH2), 2.51 (3H, s, –CH3),
Ethyl 4-methyl-2-(thiazol-2-yl)-2,5-dihydro-1,5-benzodiazepine-
2.17 (3H, s, –CH3), 1.23 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. 13C 3-carboxylate (1v). Yield: 83.8%, mp 166–168 °C. 1H NMR
NMR (CDCl3, 125 MHz) δ = 14.38, 20.52, 25.31, 56.21, 59.69, (CDCl3, 500 MHz) δ = 6.58–7.64 (5H, m, –C6H3, –C3H2NS), 6.11
101.30, 107.96, 119.30, 119.90, 121.61, 122.72, 123.69, 129.86, (1H, s, –NH), 6.15 (1H, s, –CH), 4.86 (1H, s, –NH), 4.13 (2H, q,
133.27, 136.42, 143.30, 151.07, 167.55 ppm. IR (KBr, cm−1) ν = J = 7.0 Hz, –COOCH2), 2.60 (3H, s, –CH3), 1.18 (3H, t, J = 7.0
3313 (N–H), 1668 (CvO), 1621 (CvC). MS (m/z): 407 [M + H]+. Hz, –CH2CH3) ppm. 13C NMR (CDCl3, 125 MHz) δ = 14.41,
C18H19BrN2O2S: found: C, 53.08; H, 4.70; N, 6.88; Calcd: C, 25.38, 58.88, 59.85, 100.83, 118.59, 119.63, 121.64, 121.73,
53.10; H, 4.72; N, 6.90.
Ethyl 8-bromo-2-(4-bromothiophen-2-yl)-4-methyl-2,5-dihydro- IR (KBr cm−1) ν = 3321 (N–H), 1680 (CvO), 1632 (CvC). MS
1,5-benzodiazepine-3-carboxylate (1r). Yield 84.6%,
mp (m/z): 316 [M + H]+. C16H17N3O2S: found: C, 60.95; H, 5.44; N,
80–82 °C. 1H NMR (CDCl3, 500 MHz) δ = 6.52–6.97 (5H, m, 13.34; Calcd: C, 60.93; H, 5.43; N, 13.32.
–C6H3, –C4H2S), 5.99 (1H, s, –NH), 5.98 (1H, s, –CH), 4.44 (1H, Ethyl 4,8-dimethyl-2-(thiazol-2-yl)-2,5-dihydro-1,5-benzo-
123.53, 131.37, 136.71, 142.97, 152.17, 167.59, 175.08 ppm.
s, –NH), 4.14 (2H, q, J = 7.0 Hz, –COOCH2), 2.51 (3H, s, –CH3), diazepine-3-carboxylate (1w). Yield: 91.8%, mp 162–164 °C. 1H
1.24 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. 13C NMR (CDCl3, NMR (CDCl3, 500 MHz) δ = 6.40–7.65 (5H, m, –C6H3,
125 MHz) δ = 14.44, 25.11, 55.92, 60.12, 102.98, 108.73, 115.31, –C3H2NS), 6.16 (1H, s, –NH), 6.12 (1H, s, –CH), 4.81 (1H, s,
120.94, 121.98, 123.35, 124.35, 127.35, 130.36, 137.52, 149.18, –NH), 4.12 (2H, q, J = 7.0 Hz, –COOCH2), 2.57 (3H, s, –CH3),
150.95, 167.57 ppm. IR (KBr, cm−1) ν = 3310 (N–H), 1672 2.12 (3H, s, –CH3), 1.17 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. 13C
(CvO), 1632 (CvC). MS (m/z): 475 [M + H]+. C17H16Br2N2O2S: NMR (CDCl3, 125 MHz) δ = 14.44, 20.44, 25.21, 58.68, 59.73,
found: C, 43.26; H, 3.42; N, 5.92; Calcd: C, 43.24; H, 3.42; 100.16, 118.65, 119.65, 121.86, 122.36, 128.84, 133.20, 136.56,
N, 5.93.
Ethyl
142.89, 152.63, 167.71, 175.34 ppm. IR (KBr, cm−1) ν = 3317
2-(5-bromothiophen-2-yl)-4-methyl-2,5-dihydro-1,5- (N–H), 1674 (CvO), 1621 (CvC). MS (m/z): 330 [M + H]+.
benzodiazepine-3-carboxylate (1s). Yield: 82.8%, mp 63–65 °C. C17H19N3O2S: found: C, 61.96; H, 5.83; N, 12.78; Calcd: C,
1H NMR (CDCl3, 500 MHz) δ = 6.44–6.81 (6H, m, –C6H4, 61.98; H, 5.81; N, 12.76.
–C4H2S), 6.10 (1H, s, –CH), 5.99 (1H, s, –NH), 4.42 (1H, s,
Ethyl 8-bromo-4-methyl-2-(thiazol-2-yl)-2,5-dihydro-1,5-benzo-
1
–NH), 4.15 (2H, q, J = 7.0 Hz, –COOCH2), 2.53 (3H, s, –CH3), diazepine-3-carboxylate (1x). Yield: 89.4%, mp 168–170 °C. H
1.25 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. 13C NMR (CDCl3, NMR (CDCl3, 500 MHz) δ = 6.48–7.67 (5H, m, –C6H3,
125 MHz) δ = 14.46, 25.32, 56.47, 59.91, 102.75, 110.51, 119.71, –C4H2NS), 6.12 (1H, s, –CH), 6.11 (1H, s, –NH), 4.91 (1H, s,
121.28, 121.70, 123.41, 124.84, 129.20, 131.22, 136.23, 150.00, –NH), 4.13 (2H, q, J = 7.0 Hz, –COOCH2), 2.57 (3H, s, –CH3),
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
1.18 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. 13C NMR (CDCl3, 122.67, 124.59, 124.69, 126.87, 131.65, 138.41, 150.19, 153.13,
125 MHz) δ = 14.30, 24.90, 58.70, 59.96, 102.27, 113.34, 118.64, 168.14 ppm. IR (KBr, cm−1) ν = 3262 (N–H), 1680 (CvO), 1632
121.99, 122.81, 126.00, 132.65, 135.95, 143.04, 151.44, 167.34, (CvC). MS (m/z): 301 [M + H]+. C16H16N2O2S: found: C, 64.01;
174.44 ppm. IR (KBr, cm−1) ν = 3340 (N–H), 1670 (CvO), 1625 H, 5.35; N, 9.36; Calcd: C, 63.98; H, 5.37; N, 9.33.
(CvC). MS (m/z): 394 [M + H]+. C16H16BrN3O2S: found: C,
48.76; H, 4.10; N, 10.68; Calcd: C, 48.74; H, 4.09; N, 10.66.
Methyl 4,8-dimethyl-2-(thiophen-2-yl)-2,5-dihydro-1,5-benzo-
diazepine-3-carboxylate (2d). Yield: 81.4%, mp 94–96 °C. 1H
Ethyl 8-fluoro-4-methyl-2-(thiazol-2-yl)-2,5-dihydro-1,5-benzo- NMR (CDCl3, 500 MHz) δ = 6.34–7.03 (6H, m, –C6H3, –C4H3S),
diazepine-3-carboxylate (1y). Yield: 89.7%, mp 168–169 °C. 1H 6.06 (1H, s, –CH), 6.02 (1H, s, –NH), 4.34 (1H, s, –NH), 3.66
NMR (CDCl3, 500 MHz) δ = 6.33–7.66 (5H, m, –C6H3, (3H, s, –CH3), 2.52 (3H, s, –CH3), 2.15 (3H, s, –CH3) ppm. 13C
–C4H2NS), 6.16 (1H, s, –CH), 6.12 (1H, d, J = 6.5 Hz, –NH), 5.01 NMR (DMSO, 125 MHz) δ = 20.69, 23.87, 50.99, 54.82, 101.89,
(1H, d, J = 6.0 Hz, –NH), 4.12 (2H, q, J = 5.0 Hz, –COOCH2), 120.23, 120.67, 120.75, 124.58, 124.63, 126.88, 129.13, 131.43,
2.57 (3H, s, –CH3), 1.17 (3H, t, J = 7.0 Hz, –CH2CH3) ppm. 13C 138.22, 150.33, 153.18, 168.13 ppm. IR (KBr, cm−1) ν = 3316
NMR (CDCl3, 125 MHz) δ = 14.39, 25.31, 58.64, 59.90, 100.52, (N–H), 1674 (CvO), 1617 (CvC). MS (m/z): 315 [M + H]+.
118.69, 120.52, 127.65, 138.25, 138.33, 143.13, 152.08, 157.93, C17H18N2O2S: found: C, 64.94; H, 5.76; N, 8.94; Calcd: C, 64.94;
159.86, 167.47, 174.65 ppm. IR (KBr, cm−1) ν 3356 (N–H), 1670 H, 5.77; N, 8.91.
(CvO), 1616 (CvC). MS (m/z): 334 [M + H]+. found: C, 57.67;
H, 4.86; N, 12.62; Calcd: C, 57.64; H, 4.84; N, 12.60.
Methyl
4-methyl-2-(thiazol-2-yl)-2,5-dihydro-1,5-benzo-
1
diazepine-3-carboxylate (2e). Yield: 81.6%, mp 172–174 °C. H
NMR (CDCl3, 500 MHz) δ = 6.57–7.65 (6H, m, –C6H4,
–C4H2NS), 6.27 (1H, s, –NH), 6.12 (1H, d, –CH), 4.92 (1H, d,
–NH), 3.64 (3H, s, –CH3), 2.59 (3H, s, –CH3) ppm. 13C NMR
(CDCl3, 125 MHz) δ = 25.40, 51.27, 58.76, 100.60, 118.67,
General procedure for the preparation of methyl 4-methyl-
2-(thiophen-2-yl, thiophen-3-yl or thiazol-2-yl)-2,5-dihydro-
1,5-benzodiazepine-3-carboxylate (2a–2g)
Methyl acetoacetate and o-PDA, 4-methyl-o-PDA or 4-bromo-o- 119.67, 121.64, 121.72, 123.59, 131.22, 136.70, 143.01, 152.52,
PDA yield 5d–5f. A mixture of compound 5d (5e or 5f) 168.03, 174.97 ppm. IR (KBr, cm−1) ν = 3306 (N–H), 1693
(2 mmol) and thiophene aldehyde or thiazol aldehyde (CvO), 1638 (CvC). MS (m/z): 302 [M + H]+. C15H15N3O2S:
(2 mmol) in dry ethanol (15 ml) was stirred in an ice-salt bath found: C, 59.81; H, 5.01; N, 13.96; Calcd: C, 59.78; H, 5.02;
for approximately 5 h. After the reaction was complete, the pre- N, 13.94.
cipitate that formed was filtered, washed with cool ethanol
and recrystallised from dry ethanol to yield 2a–2g.
Methyl 4,8-dimethyl-2-(thiazol-2-yl)-2,5-dihydro-1,5-benzo-
diazepine-3-carboxylate (2f). Yield: 85.6%, mp 170–172 °C. H
1
Methyl
4-methyl-2-(thiophen-3-yl)-2,5-dihydro-1,5-benzo- NMR (CDCl3, 500 MHz) δ = 6.40–7.66 (5H, m, –C6H3,
diazepine-3-carboxylate (2a). Yield: 80.9%, mp 78–80 °C. 1H –C4H2NS), 6.12 (1H, s, –CH), 6.11 (1H, s, –NH), 4.81 (1H, d,
NMR (CDCl3, 500 MHz) δ = 6.49–7.10 (7H, m, –C6H4, –C4H3S), –NH), 3.65 (3H, s, –CH3), 2.59 (3H, s, –CH3), 2.12 (3H, s, –CH3)
6.00 (1H, s, –NH), 5.88 (1H, s, –CH), 4.49 (1H, s, –NH), 3.66 ppm. 13C NMR (CDCl3, 125 MHz) δ = 20.45, 25.40, 51.20,
(3H, s, –CH3), 2.53 (3H, s, –CH3) ppm. 13C NMR (CDCl3, 58.56, 100.04, 118.70, 119.59, 121.90, 122.39, 128.62, 133.33,
125 MHz) δ = 25.24, 51.15, 56.56, 103.06, 118.49, 119.37, 136.49, 142.97, 152.73, 168.08, 175.14 ppm. IR (KBr, cm−1) ν =
121.22, 121.48, 123.05, 131.43, 136.74, 143.55, 145.02, 150.55, 3318 (N–H), 1701 (CvO), 1625 (CvC). MS (m/z): 316 [M + H]+.
151.87, 168.53 ppm. IR (KBr, cm−1) ν = 3343 (N–H), 1684 C16H17N3O2S: found: C, 60.96; H, 5.40; N, 13.34; Calcd: C,
(CvO), 1638 (CvC). MS (m/z): 301 [M + H]+. C16H16N2O2S: 60.93; H, 5.43; N, 13.32.
found: C, 63.96; H, 5.38; N, 9.36; Calcd: C, 63.98; H, 5.37;
N, 9.33.
Methyl
8-bromo-4-methyl-2-(thiazol-2-yl)-2,5-dihydro-
1,5-benzodiazepine-3-carboxylate (2g). Yield: 84.8%, mp
Methyl 4,8-dimethyl-2-(thiophen-3-yl)-2,5-dihydro-1,5-benzo- 180–182 °C. 1H NMR (CDCl3, 500 MHz) δ = 6.10–6.50,
1
diazepine-3-carboxylate (2b). Yield: 81.6%, mp 107–109 °C. H 7.07–7.68 (5H, m, –C6H3, –C4H2NS), 6.81 (1H, d, –CH), 6.76
NMR (CDCl3, 500 MHz) δ = 6.31–7.10 (6H, m, –C6H3, –C4H3S), (1H, s, –NH), 4.92 (1H, d, –NH), 3.66 (3H, s, –CH3), 2.58 (3H, s,
5.94 (1H, s, –CH), 5.86 (1H, s, –NH), 4.37 (1H, s, –NH), 3.65 –CH3) ppm. 13C NMR (CDCl3, 125 MHz) δ = 25.35, 51.38,
(3H, s, –CH3), 2.51 (3H, s, –CH3), 2.14 (3H, s, –CH3) ppm. 13C 58.49, 101.75, 118.83, 120.75, 122.00, 122.90, 123.86, 124.41,
NMR (DMSO, 125 MHz) δ = 19.03, 20.69, 23.84, 55.20, 56.50, 138.00, 143.30, 152.04, 167.78, 174.31 ppm. IR (KBr, cm−1) ν =
101.39, 120.00, 120.36, 121.28, 125.83, 128.19, 129.17, 131.14, 3255 (N–H), 1675 (CvO), 1638 (CvC). MS (m/z): 382 [M + H]+.
138.69, 146.73, 152.72, 168.45 ppm. IR (KBr, cm−1) ν = 3315 C15H14BrN3O2S: found: C, 47.40; H, 3.70; N, 11.07; Calcd: C,
(N–H), 1673 (CvO), 1631 (CvC). MS (m/z): 315 [M + H]+. 47.38; H, 3.71; N, 11.05.
C17H18N2O2S: found: C, 64.97; H, 5.75; N, 8.93; Calcd: C, 64.94;
H, 5.77; N, 8.91.
General procedure for the preparation of propyl 4-methyl-
2-(thiazol-2-yl)-2,5-dihydro-1,5-benzodiazepine-3-carboxylate
(3a–3b)
Methyl
4-methyl-2-(thiophen-2-yl)-2,5-dihydro-1,5-benzo-
1
diazepine-3-carboxylate (2c). Yield: 80.8%, mp 174–176 °C. H
NMR (CDCl3, 500 MHz) δ = 6.53–7.03 (7H, m, –C6H4, –C4H3S), Propyl acetoacetate and o-PDA, 4-methyl-o-PDA or 4-bromo-
6.07 (1H, s, –CH), 6.05 (1H, s, –NH), 4.40 (1H, s, –NH), 3.67 o-PDA yield 5g–5h. A mixture of compound 5g or 5h (2 mmol)
(3H, s, –CH3), 2.54 (3H, s, –CH3) ppm. 13C NMR (DMSO, and thiazol aldehyde (2 mmol) in ethanol (15 ml) was stirred
125 MHz) δ = 19.04, 23.81, 55.07, 56.50, 102.34, 120.20, 120.61, in an ice-salt bath for approximately 7 h. After the reaction was
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
complete, the precipitate that formed was filtered, washed 0.85 (3H, d, J = 6.5 Hz, –CH(CH3)2) ppm. 13C NMR (CDCl3,
with ethanol and recrystallised from ethanol to yield products 125 MHz) δ = 10.49, 20.44, 22.15, 25.33, 58.79, 65.51, 100.10,
3a and 3b.
Propyl
118.55, 119.58, 121.91, 122.44, 128.91, 133.28, 136.52, 142.88,
4-methyl-2-(thiazol-2-yl)-2,5-dihydro-1,5-benzo- 152.19, 152.40, 167.74, 175.39 ppm. IR (KBr, cm−1) ν = 3273
diazepine-3-carboxylate (3a). Yield: 82.8%, mp 154–156 °C. H (N–H), 1667 (CvO), 1635 (CvC). MS (m/z): 344 [M + H]+.
NMR (CDCl3, 500 MHz) δ = 6.56–7.64 (6H, m, –C6H4, C18H21N3O2S: found: C, 62.97; H, 6.18; N, 12.23; Calcd: C,
–C3H2NS), 6.22 (1H, s, –NH), 6.14 (1H, d, J = 6.0 Hz, –CH), 4.90 62.95; H, 6.16; N, 12.23.
1
(1H, d, J = 6.0 Hz, –NH), 4.01 (2H, t, J = 6.5 Hz, –CH2CH2CH3),
Biological evaluations
2.59 (3H, s, –CH3), 1.52–1.60 (2H, m, –CH2CH2CH3), 0.81 (3H,
t, J = 7.5 Hz, –CH2CH2CH3) ppm. 13C NMR (CDCl3, 125 MHz)
δ = 10.39, 22.04, 25.33, 58.88, 65.50, 100.66, 118.41, 119.50,
121.58, 121.70, 123.48, 131.37, 136.59, 132.87, 151.92, 167.51,
175.02 ppm. IR (KBr, cm−1) ν = 3304 (N–H), 1734 (CvO), 1668
Cytotoxicity assays was performed using an Epoch microplate
spectrophotometer (BioTek, USA). Membrane permeability was
measured using a DDS-307 conductivity meter.
(CvC). MS (m/z): 330 [M + H]+. C17H19N3O2S: found: C, 61.94; Evaluation of cytotoxicity
H, 5.82; N, 12.78; Calcd: C, 61.98; H, 5.81; N, 12.76.
The BV2 cells were cultured at 37 °C in 96-well plates at a
density of 4 × 104 cells per well with 5% CO2 in Dulbecco’s
Modified Eagle Medium (Invitrogen, GIBCO) supplemented
with 10% fetal bovine serum (Invitrogen) for 72 h. Compounds
1v, 1w and fluconazole were added to each well at final concen-
trations of 10.0, 20.0, 50.0 and 100.0 μg mL−1. An equivalent
volume of DMSO without the compound was added as a
control. The cells were incubated for 48 h. Subsequently, 10 μL
of aqueous MTT solution (5.0 mg mL−1) and the mixture were
incubated at 37 °C for 4 h. The MTT solution was carefully de-
canted off, and 100 μL of DMSO was added to each well. The
color was measured with an Epoch microplate spectropho-
tometer at 490 nm with the reference filter set to 620 nm. All
MTT assays were repeated three times. Each measurement con-
tained six parallel treatments (wells).
Propyl
4,8-dimethyl-2-(thiazol-2-yl)-2,5-dihydro-1,5-benzo-
1
diazepine-3-carboxylate (3b). Yield: 83.6%, mp 120–122 °C. H
NMR (CDCl3, 500 MHz) δ = 6.38–7.64 (5H, m, –C6H3, –C4H2S),
6.17 (1H, s, –NH), 6.12 (1H, s, –CH), 4.82 (1H, s, –NH), 4.00
(2H, t, J = 6.5 Hz, –CH2CH2CH3), 2.56 (3H, s, –CH3), 2.11 (3H,
s, –CH3), 1.54–1.58 (2H, m, –CH2CH2CH3), 0.81 (3H, t, J = 7.0
Hz, –CH2CH2CH3) ppm. 13C NMR (CDCl3, 125 MHz) δ = 10.40,
22.05, 25.17, 58.69, 65.40, 99.96, 118.47, 119.52, 121.80,
122.33, 128.84, 133.16, 136.44, 142.79, 152.39, 167.64,
175.29 ppm. IR (KBr, cm−1) ν = 3343 (N–H), 1671 (CvO), 1656
(CvC). MS (m/z): 344 [M + H]+. C18H21N3O2S: found: C, 62.98;
H, 6.18; N, 12.24; Calcd: C, 62.95; H, 6.16; N, 12.23.
General procedure for the preparation of isopropyl 4-methyl-
2-(thiazol-2-yl)-2,5-dihydro-1,5-benzodiazepine-3-carboxylate
(4a–4b)
Relative survival rate = (treatment A490/negative control
A620) × 100%.
Isopropyl acetoacetate and o-PDA, 4-methyl-o-PDA or 4-bromo-
o-PDA yield 5i–5j. A mixture of compound 5i or 5j (2 mmol)
and thiazol aldehyde (2 mmol) in ethanol (15 ml) was stirred
in an ice-salt bath for approximately 8 h. After the reaction was
complete, the precipitate that formed was filtered, washed
with ethanol and recrystallised from ethanol to yield products
4a and 4b.
Acknowledgements
We are grateful for the financial support provided by the
National Natural Science Foundation of China (21276064), the
Natural Science Foundation of Hebei Education Department
(2008320), and the Science Foundation of Hebei Normal Uni-
versity (2011Y04).
Isopropyl
4-methyl-2-(thiazol-2-yl)-2,5-dihydro-1,5-benzo-
1
diazepine-3-carboxylate (4a). Yield: 81.8%, mp 168–170 °C. H
NMR (CDCl3, 500 MHz) δ = 6.58–7.63 (6H, m, –C6H4,
–C3H2NS), 6.19 (1H, s, –CH), 6.13 (1H, s, –NH), 4.99 (1H, t, J =
5.0 Hz, –CH(CH3)2), 4.90 (1H, s, –NH), 2.58 (3H, s, –CH3), 1.21
(3H, d, J = 5.0 Hz, –CH(CH3)2), 1.06 (3H, d, J = 5.0 Hz, –CH
(CH3)2) ppm. 13C NMR (CDCl3, 125 MHz) δ = 25.12, 58.98,
66.81, 100.80, 118.43, 119.57, 121.54, 123.37, 126.47, 128.29,
131.54, 136.68, 142.78, 151.90, 167.03, 175.26 ppm. IR (KBr,
cm−1) ν = 3326 (N–H), 1730 (CvO), 1669 (CvC). MS (m/z): 330
[M + H]+. C17H19N3O2S: found: C, 62.02; H, 5.83; N, 12.78;
Calcd: C, 61.98; H, 5.81; N, 12.76.
Notes and references
1 (a) M. D. Braccio, G. Roma, G. C. Ghia and E. Mereto,
Eur. J. Med. Chem., 1990, 25, 681–687; (b) G. de Sarro,
E. D. di Paola, U. Aguglia and A. De Sarro, Pharmacol.,
Biochem. Behav., 1996, 55, 39–48; (c) W. M. Aly,
Y. A. Mohamed, K. A. M. El-Bayouki, W. M. Basyouni and
S. Y. Abbas, Eur. J. Med. Chem., 2010, 45, 3265–3273;
(d) G. D. Sarro, A. Chimirri, A. D. Sarro, R. Gitto, S. Grasso
and M. Zappala, Eur. J. Med. Chem., 1995, 30, 925–929;
(e) X. Zhou, M. Y. Zhang, S. T. Gao, J. J. Ma, C. Wang and
C. Liu, Chin. Chem. Lett., 2009, 20, 905–908.
Isopropyl 4,8-dimethyl-2-(thiazol-2-yl)-2,5-dihydro-1,5-benzo-
1
diazepine-3-carboxylate (4b). Yield: 82.4%, mp 150–152 °C. H
NMR (CDCl3, 500 MHz) δ = 6.37–7.73 (5H, m, –C6H3,
–C3H2NS), 6.12 (1H, s, –NH), 5.59 (1H, d, –CH), 4.76 (1H, t, J =
6.0 Hz, –CH(CH3)2), 4.52 (1H, d, J = 4.0 Hz, –NH), 1.96 (3H, s,
–CH3), 2.21 (3H, s, –CH3), 0.97 (3H, d, J = 6.5 Hz, –CH(CH3)2),
2 (a) W. Zhang, J. P. Williams, Y. Lu, T. Nagashima and
Q. Chu, Tetrahedron Lett., 2007, 48, 563–565; (b) K. D. Surya
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
and A. G. Richard, Tetrahedron Lett., 2005, 46, 1811–1813; 10 MICs were determined as described by the NCCLS (see:
(c) C. W. Kuo, S. V. More and C. F. Yao, Tetrahedron Lett.,
2006, 47, 8523–8528; (d) D. V. Jarikote, S. A. Siddiqui,
R. Rajagopal, T. Daniel, R. J. Lahoti and K. V. Srinivasan,
Tetrahedron Lett., 2003, 44, 1835–1838; (e) T. W. Ku,
F. E. Ali, W. F. Bondinell, K. F. Erhard, W. F. Huffman,
J. W. Venslavsky and C. K. Y. Catherine, Tetrahedron Lett.,
1997, 18, 3131–3134.
National Committee for Clinical Laboratory Standards, Per-
formance Standards for Antimicrobial Susceptibility
Testing: 11th Informational Supplement, vol. 21, National
Committee for Clinical Laboratory Standards, Wayne, PA,
2001, M100-S11). The MIC was defined as the lowest con-
centration of each compound resulting in inhibition of
visible growth of bacteria after incubation at 35 °C for 18 h.
3 S. G. Konda, B. M. Shaikh, S. A. Chavan and B. S. Dawane, 11 (a) N. Zanatta, D. M. Borchhardt, S. H. Alves, H. S. Coelho,
Chin. Chem. Lett., 2011, 22, 65–68.
4 M. Babu, K. Pitchumani and P. Ramesh, Med. Chem. Res.,
2014, 23, 2070–2079.
5 M. M. Heravi, F. Derikvand, L. Ranjbar and F. F.
Bamoharram, J. Mol. Catal. A: Chem., 2007, 261, 156–159.
A. M. C. Squizani, T. M. Marchi, H. G. Bonacorso and
M. A. P. Martins, Bioorg. Med. Chem., 2006, 14, 3174–3184;
(b) E. Rajanarendar, M. N. Reddy, S. R. Krishna,
K. R. Murthy, Y. N. Reddy and M. V. Rajam, Eur. J. Med.
Chem., 2012, 55, 273–283.
6 S. Bondock, W. Fadaly and M. A. Metwally, Eur. J. Med. 12 X. Li, W. L. Zhao, J. D. Jiang, K. H. Ren, N. N. Du, Y. B. Li,
Chem., 2010, 45, 3692–3701.
7 H. M. Aly, N. M. Saleh and H. A. Elhady, Eur. J. Med. Chem.,
2011, 46, 4566–4572.
Y. X. Wang, C. W. Bi, R. G. Shao and D. Q. Song, Bioorg.
Med. Chem. Lett., 2011, 21, 5251–5254.
13 R. Ramachandran, M. Rani, S. Senthan, Y. T. Jeong and
S. Kabilan, Eur. J. Med. Chem., 2011, 46, 1926–1934.
8 J. G. Ghogare, S. V. Bhandari, K. G. Bothara,
A. R. Madgulkar, G. A. Parashar, B. G. Sonawane and 14 (a) S. R. Tong, W. G. Wang, C. P. Ma, M. F. Ge and
P. R. Inamdar, Eur. J. Med. Chem., 2010, 45, 857–863.
9 E. Yamuna, R. A. Kumar, M. Zeller and K. J. R. Prasad,
Eur. J. Med. Chem., 2012, 47, 228.
D. X. Wang, Sci. China, Ser. B, 2009, 11, 1932–1937;
(b) B. Samir, N. Tamer and A. A. Yousry, Eur. J. Med. Chem.,
2013, 62, 270–279.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.