Synthesis and biological evaluation of benzo[b]furo[3,4-e][1,4]diazepin-1-one derivatives as anti-cancer agents

Article abstract of DOI:10.1016/j.bioorg.2018.07.023

A new series of novel Podophyllotoxin-like benzo[b]furo[3,4-e][1,4]diazepin-1-ones possessing structural elements of 4-aza-2,3-didehydropodophyllotoxins with central diazepine ring was designed and synthesized as anti-cancer agents. In initial assessment,

Full text of DOI:10.1016/j.bioorg.2018.07.023

Accepted Manuscript
Synthesis and biological evaluation of benzo[b]furo[3,4-e][1,4]diazepin-1-one
derivatives as anti-cancer agents
Sina Omid Malayeri, Zahra Tayarani-Najaran, Fatemeh Shaebani Behbahani,
Roghayeh Rashidi, Saeedeh Delpazir, Razieh Ghodsi
PII:
S0045-2068(18)30472-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.07.023
YBIOO 2442
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
14 May 2018
12 June 2018
18 July 2018
Please cite this article as: S.O. Malayeri, Z. Tayarani-Najaran, F.S. Behbahani, R. Rashidi, S. Delpazir, R. Ghodsi,
Synthesis and biological evaluation of benzo[b]furo[3,4-e][1,4]diazepin-1-one derivatives as anti-cancer agents,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.07.023
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Synthesis and biological evaluation of benzo[b]furo[3,4-e][1,4]diazepin-1-one derivatives as
anti-cancer agents
Sina Omid Malayeriab, Zahra Tayarani-Najaranc, Fatemeh Shaebani Behbahanic,
Roghayeh Rashidic, Saeedeh Delpazirb and Razieh Ghodsi*ab
aBiotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of
Medical Sciences, Mashhad, Iran
bDepartment of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical
Sciences, Mashhad, Iran
cDepartment of Toxicology, School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran
*Corresponding author: Tel: +98 51 38823255; Fax: +98 51 38823251; E-mail:
ghodsir@mums.ac.ir
Abstract
A new series of novel Podophyllotoxin-like benzo[b]furo[3,4-e][1,4]diazepin-1-ones possessing
structural elements of 4-aza-2,3-didehydropodophyllotoxins with central diazepine ring was
designed and synthesized as anti-cancer agents. In initial assessment, the cytotoxic activity of the
synthesized compounds was evaluated against three cancer cell lines including MCF-7, PC3 and
B16-F10 employing the MTT assay. Some of compounds (12h, 13a, 13c and 14b) showed
significant cytotoxic activity. So, we investigated the cytotoxicity of compounds 12h, 13a, 13c
and 14b, along with podophyllotoxin as the reference drug in different cancer cell lines including
A549, A2780, DU145, HeLa, and normal Huvec cell line .Among these four compounds, 13c
showed promising antiproliferative activity against all cancer cells stronger than the other
compounds and comparable to reference drug podophyllotoxin in some cancer cells. All these
four compounds did not show significant cytotoxicity on normal Huvec cell line. The flow
cytometry analysis of the MCF-7, PC3 and A2780 human cancer cell lines treated with 13c
1

showed that 13c, induced apoptosis in the MCF-7, PC3 and A2780 human cancer cell lines,
which is in good agreement to its cytotoxic activity as well. Compound 13c did not show
significant influence on tubulin assembly and exert its cytotoxic effects via induction of
apoptosis and has potent and selective cytotoxic effects in cancer cells.
Key words; Podophyllotoxin, benzo[b]furodiazepin-1-ones, anti-cancer, cytotoxic, synthesis,
apoptosis, tubulin inhibitor
1. Introduction,
Cancer is a major public health problem in the world and it is now the second leading cause of
death in the United States, and is predicted to surpass heart diseases as the leading cause of death
in the futures [1] Consequently; increasing interest has been devoted to the design and discovery
of more effective anticancer agents in current medicinal chemistry [2-9]. Podophyllotoxin (1) is
the main component of podophyllum resin, a naturally occurring antimitotic cyclolignan which
displays strong anticancer activity against numerous cancer cell lines[10]. Although
podophyllotoxin possesses notable in vitro antitumor effects, it is not used as an anticancer drug
because of its various side effects such as nausea, vomiting and damage of normal tissues.
Though it’s unfavorable pharmacological profiles, podophyllotoxin can still be used as a lead
compound for the discovering of possible anticancer drugs. Etoposide (2), teniposide (3), and
etoposide phosphate (4) are semisynthetic derivatives of 4-epipodophyllotoxin. They have
numerous limitations, such as poor water solubility, metabolic inactivation, development of drug
resistance and toxic effects. To overcome such problems, extensive synthetic efforts have been
carried out by a number of researchers. This has led to the development of TOP-53 (5) and
F14512 (6). Recently novel podophyllotoxin derivatives have been reported by researchers [11-
17]. 4-aza-2,3-didehydropodophyllotoxins (7) have been also reported as anticancer agents [18-
21], which displayed cytotoxicity comparable to that of podophyllotoxin. In the present study
we report the synthesis of novel podophyllotoxin-like benzo[b]furo[3,4-e][1,4]diazepin-1-ones
possessing structural elements of 4-aza-2,3-didehydropodophyllotoxins with central
dihydrodiazepine ring (Fig.1). The synthesized compounds were initially evaluated for their
cytotoxic activity towards three different cancer cell cancer cell lines including MCF-7, PC3 and
B16-F10 cancer cells employing the MTT assay. Then we investigated the cytotoxicity of
compounds showed the most antiproliferative activity in initial assessment, in different human
2

cancer cell lines including A549, A2780, DU145, HeLa, and normal Huvec cell line. The effect
of the most cytotoxic compounds to induce apoptosis was evaluated by apoptosis assay and
compounds 13c was evaluated for tubulin polymerization inhibitory effect.
Fig. 1. Structures of podophyllotoxin, its semisynthetic analogs and our designed compounds
2. Results and discussion
2.1. Synthesis
As illustrated in Scheme 1, benzene-1,2-diamine 8 and tetronic acid 9 were stirred in ethanol in
the presence of catalytic amount of acetic acid to afford 4-((2-aminophenyl)amino)furan-2(5H)-
one 10, then substituted benzaldehydes 11 and compound 10 were dissolved in chloroform and
combined in the presence of catalytic amount of acetic acid under microwave irradiation [22] to
obtain the target benzo[b]furo[3,4-e][1,4]diazepin-1-ones 12, then chloroacetyl derivatives 13
were prepared using compounds 12 and chloroacetyl chloride in THF [23]. Reaction of 13 with
morpholine or N-methylpiperazine in THF afforded the target compounds 14. The compounds
3

13
were characterized by nuclear magnetic resonance (1H-NMR and C-NMR), infrared and mass
spectrometry.
Scheme1. Reagents and conditions; a, EtOH , AcOH, room temperature, 10 h. b, CHCl3 , AcOH,
Microwave, 30 min, c. Chloroacetyl chloride, THF, reflux. d. Morpholine (N-Methyl
piperazine), THF, reflux.
2.2. Biological evaluation
2.2.1. In vitro anticancer activity
The cytotoxic activity of the synthesized compounds was evaluated against MCF-7 (human
breast cancer cells), PC3 (human prostate cancer cells), and B16-F10 (Melanoma cells) cancer
cells employing the MTT assay. As depicted in Table 1, concentrations of 5 and 50 μM of
compounds were used for evaluation of cytotoxicity of these compounds. Initial evaluation
showed that most of the compounds were not potent cytotoxic agents at concentrations below 50
4

μM, among the compounds, 13c, 2-chloroacetyl analog of 12j possessing p-methyl phenyl
showed significant cytotoxic activity in all three cancer cells. Compound 12h possessing p-
Hydroxy phenyl, 13a, 2-chloroacetyl analog of 12a possessing phenyl and 14b, 2-(4-
methylpiperazin-1-yl)acetyl analog of 12a, also showed cytotoxic activity in cancer cells. As 2-
chloroacetyl analogs showed more cytotoxicity in cancer cells compared to 2-morpholinoacetyl
and 2-(4-methylpiperazin-1-yl)acetyl analogs, it can be concluded that the higher cytotoxic
activity of 2-chloroacetyl analogs could be attributed to their higher lipophilicity. Among the
compounds 12a-12k, Compound 12h possessing p-Hydroxy phenyl was the most cytotoxic agent
in MCF-7 and B16-F10 cancer cells. Among the chloroacetyl derivatives 13a-13c, 13c,
possessing p-tolyl, was the most cytotoxic agents. Surprisingly, compound 13b possessing 3,4,5-
trimethoxyphenyl similar to podophyllotoxin, showed the least cytotoxic activity among the
series, it might be because of more steric hindrance occurred by three methoxy groups. So it can
be concluded that in 2-chloroacetyl analogs, small lipophilic group in para position of phenyl
ring can increase the cytotoxic activity in cancer cells. Since researchers reported 4-aza-2,3-
didehydropodophyllotoxins (7) as anticancer agents [18-21], which displayed cytotoxicity
comparable to that of podophyllotoxin and more than those of our compounds 12a-12k, we can
conclude that expansion of the central ring of 4-aza-2,3-didehydropodophyllotoxins from six-
membered ring (dihydroquinoline) to seven-membered ring (dihydrodiazepine) can decrease the
cytotoxic activity of these compounds. Among the 2-morpholinoacetyl and 2-(4-
methylpiperazin-1-yl)acetyl derivatives 14a-14e, 14b, 2-(4-methylpiperazin-1-yl)acetyl analog
of 12a, showed the highest cytotoxicity. So we investigated the cytotoxicity of compounds 12h,
13a, 13c and 14b and podophyllotoxin as the reference drug in six cancer cell lines including
A549 (adenocarcinoma human alveolar basal epithelial cells), A2780 (human ovarian cancer
cells), DU145 (human prostate cancer cells), HeLa (cervical cancer cells), MCF-7 (human breast
cancer cells), PC3 (human prostate cancer cells), and normal Huvec cell line (Human Umbilical
Vein Endothelial Cells) (Table 2) .Among these four compounds, chloroacetyl derivatives 13a
showed significant cytotoxic activity in all cancer cells. Compound 13c showed promising
antiproliferative activity against all cancer cells stronger than the other compounds and
comparable to reference drug podophyllotoxin in some cancer cells. All these four compounds
did not show significant cytotoxicity on normal Huvec cell line. Compounds 13a and 13c
showed potent and selective cytotoxic effects in cancer cells.
5

Table 1. In-vitro cytotoxicity of compounds (Percent survival of cancer cells (Mean ± SD)
following exposure to 5 μM and 50 μM concentrations of compounds)
PC3
MCF-7
MCF-7
B16F10
B16F10
Compound PC3 (5μM)
(50μM)
104.001±5.
43
(5μM)
(50μM)
(5μM)
(50μM)
103.83±3.51
99.10±7.16
87.01±3.04 65.45±9.07
99.28±6.61
54.94±0.27
60.80±6.43
59.62±3.69
94.84±4.79
12a
12b
12c
12d
97.31±15.9
79.71±2.07
5
104.97±13.3
5
99.82±7.41
73.42±16.3
5
107.24±15.16
101.07±3.60
64.61±2.31
97.45±4.46
99.05±8.39
46.12±7.06
74.28±1.67
68.58±4.95
102.15±7.9 91.36±12.7
3
2
99.48±14.8
5
96.88±1.26
103.47±7.21
97.78±7.80
102.87±9.03
96.82±4.39
80.43±5.56
101.18±5.04 98.163±5.16
12e
12f
12g
12h
12i
95.33±6.89 86.69±4.86 89.89±6.161 99.46±5.88
58.08±0.93
102.03±6.5
104.9±8.25 95.20±11.44 96.68±4.90
8
77.91±5.91
90.65±3.62 62.14±8.28
96.221±5.9 103.32±1.2
97.21±0.87
54.85±6.58
18.65±9.26
100.11±20.6
0
97.63±8.37
59.11±2.18
1
7
63.432±4.5
5
90.90±14.36
88.03±7.29 86.30±4.13
103.61±5.64 63.52±17.22
12j
100.95±15.80
61.91±4.28
94.37±1.97 89.89±1.81 90.33±4.42
98.93±2.98
100.41±5.19
12k
13a
86.18±5.49 10.28±2.29
89.63±10.40
6.28±4.69
40.55±7.22
78.82±16.6
86.75±4.22
5
92.69±4.76
71.27±8.89
90.34±11.82 70.21±6.87
13b
54.44±13.0
6
13c
14a
58.88±2.27
11.31±3.68
1.60±0.38
27.53±6.60
6.92±3.67
6
101.19±5.29
88.98±14.7 87.46±5.96
105.62±5.44 99.23±11.17
47.02±4.10

5
105.62±11.2
9
56.13±14.5
5
86.65±8.01
93.77±3.37
97.06±4.03
99.64±6.37
86.88±7.77
105.38±7.14
14b
14c
14d
14e
44.01±13.54
102.03±8.7 105.50±1.1
77.28±11.31 87.68±10.62 99.58±7.65
6
7
97.31±15.6
7
80.67±8.11
83.74±6.51
82.46±6.05
98.34±5.16
98.22±6.46
100.172±2. 105.18±11.
97.75±7.19
ND
95.14±3.08
ND
99
14
podophyllotoxin 19.45±3.64
ND
15.62±2.84 ND
ND: not determined
7

Table 2. The in vitro anti-proliferative activities (IC50 (µM)) of compounds, and
podophyllotoxin against human cancer cell lines and normal Huvec cell line
Compound
A549
A2780
DU145
PC3
HeLa
MCF-7
Huvec
>50
>50
>50
20.27±2.04 >50
>100
12h
48.34±4.1
2
20.38±1.02
10.46±1.34
48.53±3.82
1.64±0.16
5.1±1.01
3.9±0.42
7.13±2.17 10.43±2.02 12.64±1.03
13a
13c
50.17±3.0
4
2.72±0.33 3.89±0.45 6.19±2.65 3.51±1.21
9.37±2.07
44.73±2.4
4
>50
>50
>50
46.23±5.24 >100
14b
45.67±3.8
7
podophyllotoxin
0.86±0.25 0.97±0.13 1.26±0.17 1.21±0.14
1.02±0.13 1.82±0.17
IC50: compound concentration required to inhibit tumor cell proliferation by 50%. Data are
presented as the mean ± SD from the dose−response curves of three independent experiments
2.2.2. Apoptosis assay
2.2.2.1. PI staining
To investigate whether compound 13c and 14b induce apoptosis of cells, the effect of 13c and
14b to induce apoptosis was evaluated by apoptosis assay with flow cytometry using PI staining
test [24]. Cells were treated with 5μM and 50μM concentrations of 13c and 14b for 48 h. the
percentage of sub-G1 apoptotic cells in 13c and 14b treatments was determined by flow
cytometry. As shown in Fig. 2, the results proved that 13c, in addition to its anti-proliferative
activity, also induced apoptosis in MCF-7 and PC3 cancer cell lines, which is in good agreement
to its cytotoxic activity as well. The percentage of apoptotic cells induced by 13a in MCF-7 cells
increased from 10.5% (control) to 33% (5 μM) and 83.9% (50 μM) respectively and the
percentage of apoptotic cells induced by 14b increased from 10.5% (control) to 14.6% (5 μM)
and 18.5% (50 μM), respectively. Cell cycle analysis showed that the ratio of apoptosis in
8

PC3cells for compound 13c at 5 and 50 μM was 71% and 83.73% respectively, and for
compound 14b at 5 and 50 μM it was found to be 26.1% and 23.9% respectively.
A
B
Fig. 2. PI staining and flow cytometry analysis of compound 13c and 14b (5 and 50μM) induced
apoptosis (A) in MCF-7 cells and (B) in PC3 cells.
2.2.2.2. Apoptosis assay (Annexin V binding staining)
As compounds 13a and 13c showed the most cytotoxic effect in A2780 cancer cells, to quantify
the percentage of cells undergoing apoptosis by compound 13a and 13c, Annexin V-
9

FITC/Propidium iodide staining assay was performed in A2780 cancer cells according to
reported procedures [25]. This assay enables the detection of live cells (Q4), early apoptotic cells
(Q3), late apoptotic cells (Q2) and necrotic cells (Q1). A2780 cells were treated with 10 μM, and
50 μM concentrations of compounds and stained with Annexin V-FITC/Propidium iodide.
Results from Fig. 3 showed that the percentage of total apoptotic cells (early and late apoptotic
cells) induced by 13a increased from 0.224% (control) to 21.14% (10 μM) and 76.16% (50 μM)
respectively and the percentage of total apoptotic cells induced by 13c increased from 0.224%
(control) to 76.84% (10 μM) and 78.73% (50 μM) respectively. Therefore, the percentage
increase in early and late apoptotic cells induced by 13a and 13c indicates that these compounds
induced apoptosis in A2780 cancer cells.
13a (10μM)
13a (50μM)
Control
13c (10μM)
13c (50μM)
10

Fig. 3. Effects of 13a and 13c on the apoptosis of A2780 cancer cells using Annexin V/PI double
staining test by flow cytometry.
2.2.3. Tubulin polymerization assay
To investigate whether the cytotoxic activity of compound 13c was related to the binding to
tubulin, compounds 13c and reference compounds podophyllotoxin (polymerization suppressor)
and a polymerization promoter (paclitaxel) were evaluated for tubulin polymerization inhibitory
effect. Compound 13c which was most cytotoxic compound did not show significant influence
on tubulin assembly in comparison to the reference compounds podophyllotoxin (Fig. 4). This
may be explained by other mechanisms involved in cytotoxicity other than tubulin inhibition.
25000
PODO-10μM
20000
PODO-5μM
15000
10000
5000
0
neg control
13c-20 μM
13c-50μM
paclitaxel-3μM
1
11
21
31
41
51
61
Time (min)
Fig. 4. Effect of compounds (13c, podophyllotoxin and Paclitaxel) on in vitro tubulin
polymerization
3. Conclusions
A new series of novel Podophyllotoxin-like like benzo[b]furo[3,4-e][1,4]diazepin-1-one
analogues was synthesized and evaluated for their cytotoxic activity against different cancer cell
lines. Some of compounds (12h, 13a, 13c and 14b) showed significant cytotoxic activity.
Among these four compounds, 13c showed promising antiproliferative activity against all six
11

cancer cell lines including A549, A2780, DU145, HeLa, MCF-7 and PC3, stronger than the other
compounds (with IC50 in the range of 2.72-10.46 μM) and comparable to reference drug
podophyllotoxin in some cancer cells. All these four compounds (12h, 13a, 13c and 14b) did not
show significant cytotoxicity on normal Huvec cell line. The flow cytometry analysis of the
MCF-7 and PC3 and A2780 human cancer cell lines treated with 13c showed that 13c, induced
apoptosis in the MCF-7, PC3 and A2780 human cancer cell lines, which is in good agreement to
its cytotoxic activity as well. We conclude that compound 13c exert its cytotoxic effects via
induction of apoptosis and has potent and selective cytotoxic effects in cancer cells. The data
resulted from compound13c seems promising and have prompted further evaluation of this
compound.
4. Experimental section
4.1. Chemistry
All chemicals, reagents and solvents used in this study were purchased from Merck AG and
Aldrich Chemical. Melting points were determined with a Thomas–Hoover capillary apparatus.
Infrared spectra were acquired using a Perkin Elmer Model 1420 spectrometer. Bruker FT-400
and 300 MHz instruments (Brucker Biosciences, USA) were used to acquire 1HNMR spectra and
13
A Bruker FT-300 MHz instrument was used to acquire CNMR spectra with TMS as internal
standard. Chloroform-D and DMSO-D6 were used as solvents. Coupling constant (J) values are
assessed in hertz (Hz) and spin multiples are given as s (singlet), d (double), t (triplet), q
(quartet), m (multiplet). The mass spectral measurements were performed on a 3200 QTRAP
LCMS triple quadrupole mass spectrometer (LCMS) with an electrospray ionization (ESI)
interface.
4.2. preparation of 4-((2-aminophenyl)amino)furan-2(5H)-one[23]
A solution of 1 g of benzene-1,2-diamine 8 (10mmol) and 1.084 g tetronic acid 9 were stirred in
10 ml ethanol and acetic acid (0.25 ml) for 10 hours, then the produced precipitate was filtered
and washed with ethanol and recrystallized in ethanol to obtain 1g of 10.
12

1
Yield: 53%; mp= 225-227 °C; IR (KBr): v(cm-1)3403-3231(NH), 1676-1613(C=O); H
NMR(400 MHz, DMSO-d6): δ(ppm) 8.77 (s, 1H, NH), 7.05-6.56 (m, 4H, ArH), 5.04-5.02 (d,
1H, NH2, J=7.2 Hz), 4.82 (s, 2H, CH2), 4.55 (s, 1H, CH).
4.3. General procedure for preparation of 10- substituted phenyl-3,4,9,10-tetrahydro-1H-
benzo[b]furo[3,4-e][1,4]diazepin-1-one (12a-12j)
4-((2-aminophenyl)amino)furan-2(5H)-one 10 (2.1 mmol), appropriate benzaldehyde 11 (2.1 mmol),
acetic acid (0.3 ml) and chloroform (5 ml) were mixed. The mixture was subjected to microwave
irradiation at 200 W. Upon completion, monitored by TLC, the reaction mixture was cooled to room
temperature then produced precipitate was filtered and washed with chloroform and hexane to obtain
the pure compounds.
4.3.1. 10-phenyl-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-1-one (12a)[22]
Yield: 65%; mp= 200-202 °C; IR (KBr): v(cm-1)3352-3201(NH), 1717-1669(C=O); 1H
NMR(400 MHz, DMSO-d6): δ(ppm) 9.84 (s, 1H, NH), 7.20-7.08 (m, 5H, ArH), 6.91-6.89
(d,1H, ArH, J=7.6 Hz), 6.75-6.66 (m, 2H, ArH), 6.60-6.58 (d, 1H, ArH, J=7.2 Hz), 6.08-6.07 (d,
1H, NH, J=4.2 Hz), 5.07-5.05 (d, 1H, CH, J=4.4 Hz), 4.90 (s, 2H, CH2).
4.3.2.
10-(3,4,5-trimethoxyphenyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-
e][1,4]diazepin-1-one (12b)[22]
1
Yield: 52%; mp= 259-260 °C; IR (KBr): v(cm-1)3429(NH), 1717-1618(C=O); H NMR(500
MHz, DMSO-d6): δ(ppm) 9.779 (s, 1H, NH), 6.87-6.85 (m, 1H, ArH), 6.73-6.67 (m, 2H, ArH),
6.62-6.60 (dd, 1H, ArH, J=9.21, J=1.76 Hz), 6.37 (s, 2H, ArH), 5.96-5.95 (d, 1H, NH, J=4 Hz),
4.94-4.93 (d, 1H, CH, J=4.0 Hz), 4.87-4.80 (d, 2H, CH2, J=15.1 Hz), 3.55 (s, 6H, OCH3), 3.51 (s,
3H, OCH3).
4.3.3. 10-(3,4-dimethoxyphenyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-1-
one (12c)[23]
1
Yield: 60%; mp= 230-232 °C; IR (KBr): v(cm-1)3419(NH), 1720-1621(C=O); H NMR (500
MHz, DMSO-d6): δ(ppm) 9.72 (s, 1H, NH), 6.85-6.82 (m, 2H, ArH), 6.69-6.63 (m, 3H, ArH),
13

6.58-6.56 (m, 1H, ArH), 6.45-6.43 (m, 1H, ArH), 5.96 (s, 1H, NH), 4.95 (s, 1H, CH), 4.86-4.80
(m, 2H, CH2), 3.59 (s, 3H, OCH3) 3.57 (s, 3H, OCH3).
10-(3-hydroxy-4-methoxyphenyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-
1-one (12d)
1
Yield: 66%; mp= 263-265 °C; IR (KBr): v(cm-1)3320(NH), 1711-1616(C=O); H NMR (300
MHz, DMSO-d6): δ(ppm)9.84 (s, 1H, NH), ), 8.78 (s, 1H, OH), 6.94-6.91 (m, 1H, ArH),6.80-
6.71 (m, 3H, ArH), 6.66-6.61 (m, 2H, ArH), 6.58-6.55 (m, 1H, ArH), 5.96-5.95 (d, 1H, NH,
J=4.4 Hz), 4.95 (s, 1H, CH), 4.92 (s, 2H, CH2), 3.72 (s, 3H, OCH3); 13C NMR (75 MHz, DMSO-
d6): δ(ppm) 55.98, 57.27, 66.44, 97.61, 112.07, 115.27, 118.34, 119.89, 120.99, 123.29, 131.89,
136.95, 137.96, 146.27, 146.72, 159.00, 173.30. LC-MS (ESI): 325.1 (M+1).
10-(4-hydroxy-3-methoxyphenyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-
1-one (12e)
1
Yield: 67%; mp= 252-254 °C; IR (KBr): v(cm-1)3241(NH), 1725-1622(C=O); H NMR (400
MHz, DMSO-d6): δ(ppm)9.77 (s, 1H, NH), ), 8.75 (s, 1H, OH), ), 6.84-6.61 (m, 7H, ArH), 5.97
13
(s, 1H, NH), 4.96 (s, 1H, CH), 4.87 (s, 2H, CH2), 3.62 (s, 3H, OCH3). C NMR (75 MHz,
DMSO-d6): δ(ppm) 55.89, 57.54, 66.49, 97.56, 112.26, 115.07, 119.53, 119.86, 121.15, 123.31,
123.47, 132.18, 135.26, 138.13, 145.60, 147.71, 159.27, 173.41. LC-MS (ESI): 325.1 (M+1).
10-(3-hydroxyphenyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-1-one (12f)
1
Yield: 71%; mp= 280-282 °C; IR (KBr): v(cm-1)3636(OH), 3319(NH), 1714-1647(C=O); H
NMR(500 MHz, DMSO-d6): δ(ppm)9.74 (s, 1H, NH), 9.11(s,1H,OH), 6.91 (t, 1H, ArH), 6.84-
6.82 (m, 1H, ArH), 6.68-6.64 (m, 2H, ArH), 6.56-6.52 (m, 2H, ArH), 6.46-6.44 (m, 2H, ArH),
5.96-5.95 (d, 1H, NH, J=4.5 Hz), 4.899-4.890 (d, 1H, CH, J=4.5 Hz), 4.85-4.79 (d, 2H, CH2,
J=15.5 Hz); 13C NMR (75 MHz, DMSO-d6): δ(ppm) 57.68, 66.48, 97.26, 113.90, 114.68,
118.41, 119.94, 121.01, 123.24, 123.32, 129.33, 131.75, 137.82, 145.70, 157.43, 159.06, 173.30.
LC-MS (ESI): 295.0 (M+1).
4.3.4. 10-(4-methoxyphenyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-1-one
(12g)
14

1
Yield: 63%; mp= 222-224 °C; IR (KBr): v(cm-1)3563-3417(NH), 1718-1667(C=O); H NMR
(400 MHz, DMSO-d6): δ(ppm) 9.80 (s, 1H, NH), 7.06-7.03 (m, 2H, ArH), 6.89-6.87 (m, 1H,
ArH), 6.75-6.68 (m, 4H, ArH), 6.608-6.604 (m, 1H, ArH), 5.96-5.95 (d, 1H, NH, J=4.4 Hz),
5.02-5.01 (d, 1H, CH, J=3.2 Hz), 4.88 (m, 2H, CH2), 3.65 (s, 3H, OCH3). 13C NMR (75 MHz,
DMSO-d6): δ(ppm) 55.35, 57.26, 66.56, 97.52, 113.79, 119.98, 121.20, 123.37, 123.40, 128.68,
132.10, 136.29, 137.86, 158.35, 159.23, 173.36. LC-MS (ESI): 309.0 (M+1).
4.3.5. 10-(4-hydroxyphenyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-1-one
(12h)
1
Yield: 73%; mp= 263-265 °C; IR (KBr): v(cm-1)3377(OH), 3235(NH), 1729-1617(C=O); H
NMR(400 MHz, DMSO-d6): δ(ppm) 9.75 (s, 1H, NH), 9.15(s,1H,OH), 6.92-6.90(d,2 H,
ArH,J=8.4 Hz), 6.87-6.85 (d, 1H, ArH, J=1.6 Hz), 6.76-6.65 (m, 2H, ArH), 6.58-6.56, (d, 1H,
ArH, J=1.6 Hz), 6.55-6.53 (d, 2H, ArH, J=8.4Hz), 5.93-5.92 (d, 1H, NH, J=4.4 Hz), 4.96-4.95(d,
13
1H, CH,J=4.4 Hz), 4.86(s, 2H, CH2) C NMR (75 MHz, DMSO-d6): δ(ppm) 57.31, 66.51,
97.67, 115.188, 119.92, 121.13, 123.31, 123.42, 128.64, 132.11, 134.64, 137.97, 156.400,
159.18, 173.38. LC-MS (ESI): 295.0 (M+1).
4.3.6. 10-(4-acetamidophenyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-1-
one (12i)
Yield: 65%; mp > 300 °C; IR (KBr): v(cm-1)3316, 3281(NH), 1705-1690(C=O); 1H NMR (400
MHz, DMSO-d6): δ(ppm)9.67 (s, 2H, NH), 7.17-6.51 (m, 8H, ArH), 5.96-5.95 (d, 1H, NH,
J=4.4 Hz), 4.75 (s, 2H, CH2), 4.25 (s, 1H, CH), 1.85 (s, 3H, OCH3). 13C NMR (75 MHz, DMSO-
d6): δ(ppm) 19.036, 56.54, 66.54, 97.20, 119.12, 119.94, 121.20, 123.34, 123.39, 127.80,
132.12, 137.81, 138.21, 138.84, 159.35, 168.58, 173.32. LC-MS (ESI): 336.0 (M+1).
4.3.7. 10-(p-tolyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-1-one (12j) [22]
Yield: 67%; mp: 290-292 °C.
4.3.8. 10-(2,4-dihydroxyphenyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-1-
one (12k)
15

1
Yield: 81%; mp = 291-292 °C; IR (KBr): v(cm-1)3295-3115(NH), 1719-1655(C=O); H NMR
(400 MHz, DMSO-d6): δ(ppm)9.74-9.71 (m, 2H, OH),9.01(s, 1H, NH), 6.86-6.84 (m, 1H, ArH),
6.76-6.59 (m, 2H, ArH), 6.50-6.48 (m, 1H, ArH), 6.32-6.28(m, 2H, ArH), 5.89-5.86(m, 1H,
ArH), 5.96-5.95 (d, 1H, NH, J=4.4 Hz ), 5.03 (s, 1H, CH), 4.90-4.88 (d, 2H, CH2, J=5.2 Hz). 13C
NMR (75 MHz, DMSO-d6): δ(ppm) 53.46, 66.57, 96.82, 102.91, 105.71, 116.84, 120.41,
121.47, 123.27, 123.41, 127.65, 132.18, 137.91, 156.48, 157.70, 160.28, 173.30. LC-MS (ESI):
311.0 (M+1).
4.4. General procedure for preparation of 9-(2-chloroacetyl)-10-substituted phenyl-
3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-1-one (13a-13c)
Chloroacetyl chloride (2.2 mmol) was added to a suspension of compound 12 (2 mmol) in dry
THF (30 mL). The mixture was refluxed with stirring for 3 h. On cooling, the precipitate that
formed was filtered off and washed with hexane and ether to give compound 13.
4.4.1.
9-(2-chloroacetyl)-10-phenyl-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-
e][1,4]diazepin-1- one (13a)
1
Yield:79%; mp = 165-167 °C; H NMR (300 MHz, DMSO-d6): δ(ppm) 3.76-3.81 (d, 1H,
CH2Cl, J=13.5 Hz), 4.20-4.24 (d, 1H, CH2Cl, J=13.5 Hz), 5.01 (m, 2H, CH2), 6.82-6.86 (m, 2H,
CH & ArH), 6.92-6.95 (dd, 1H, ArH, J=8.1, J= 1.2 Hz), 7.05-7.26 (m, 7H, ArH), 10.22(s, 1H,
NH); 13C NMR (75 MHz, DMSO-d6): δ(ppm) 56.10, 67.49, 96.63, 120.77, 120. 85, 123.67,
127.83, 128.08, 128.47, 128.73, 129.87, 132.39, 138.83, 138.89, 138.93, 159.76, 165.59, 172.89.
LC-MS (ESI): 355.1 (M+1).
4.4.2.
9-(2-chloroacetyl)-10-(3,4,5-trimethoxyphenyl)-3,4,9,10-tetrahydro-1H-
benzo[b]furo[3,4-e][1,4]diazepin-1-one (13b)
1
Yield:75%; mp = 256-258 °C H NMR (300 MHz, DMSO-d6): δ(ppm) 3.56 (s, 3H, OCH3),
3.62(s, 6H, OCH3), 3.75-3.80 (d, 1H, CH2Cl, J=13.8 Hz), 4.21-4.25 (d, 1H, CH2Cl, J=13.8 Hz),
4.94-5.06 (dd, 2H, CH2, J=15.3 Hz), 6.27 (s, 2H, ArH), 6.75 (s, 1H, CH), 6.89-6.94 (dt, 1H, ArH,
J= 8.1, J=1.2 Hz), 7.00-7.03 (dd, 1H, ArH, J=7.8, J= 1.2 Hz), 7.11-7.14 (dd, 1H, ArH, J=7.8, J=
13
1.2 Hz), 7.25-7.31 (dt, 1H, ArH, J= 8.1, J=1.2 Hz), 10.21(s, 1H, NH); C NMR (75 MHz,
DMSO-d6): δ(ppm) 43.48, 56.40, 60.44, 66.95, 96.82, 106.14, 120.70, 120.78, 123.78, 127.97,
16

128, 129.89, 132.44, 134.63, 137.36, 138.89, 138.99, 153.07, 159.65, 159.78, 165.36, 172.88.
LC-MS (ESI): 445.1 (M+1).
4.4.3.
9-(2-chloroacetyl)-10-(p-tolyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-
e][1,4]diazepin-1-one (13c)
Yield:68 %; mp = 262-264°C 1H NMR (300 MHz, DMSO-d6): δ(ppm) 2.19 (s, 3H, CH3), 3.75-
3.79 (d, 1H, CH2Cl, J=13.8 Hz), 4.18-4.22 (d, 1H, CH2Cl, J=13.8 Hz), 4.94-5.06 (dd, 2H, CH2,
J=15.3 Hz), 6.81 (s, 1H, CH), 6.82-6.88 (t, 1H, ArH, J= 7.2 Hz), 6.92-6.94 (m, 3H, ArH), 7.00-
7.03 (d, 2H, ArH, J=7.8Hz), 7.084-7.11 (d, 1H, ArH, J=8.1Hz), 7.25-7.31 (t, 1H, ArH, J=
8.1Hz), 10.22(s, 1H, NH). LC-MS (ESI): 369.1 (M+1).
4.5. General procedure for preparation of 9-(2-morpholino or methylpiperazinyl acetyl)-
10-(sustitutedphenyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-e][1,4]diazepin-1-one (14a-
14e)
Morpholin or N-methylpiperazin (5 mmol) was added to a suspension of compound 13 (1 mmol)
in dry THF (10 mL). The mixture was refluxed with stirring overnight. The solvent was removed
under reduced pressure. It was diluted with water and extracted with ethyl acetate. The combined
organic phases was washed with saturated solution of sodium bicarbonate and water, dried over
Na2SO4, and evaporated under reduced pressure. Then the product recrystallized in ethanol.
4.5.1.
9-(2-morpholinoacetyl)-10-phenyl-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-
e][1,4]diazepin-1-one (14a)
Yield: 56%; mp = 145-147°C 1H NMR (300 MHz, DMSO-d6): δ(ppm) 1.99-2.05(m, 2H, CH2-
morpholine), 2.19-2.23 (m, 2H, CH2-morpholine), 2.80-2.94 (dd, , 2H, COCH2, J= 14.4 Hz),
3.05-3.09 (t, 2H, CH2-morpholine, J=5.1), 3.77-3.80 (t, 2H, CH2-morpholine, J=5.1),4.89-5.05
(dd, 2H, CH2, J=15.3 Hz), 6.79-7.22 (m, 10H, CH & ArH), 10.45(s, 1H, NH); 13C NMR (75
MHz, DMSO-d6): δ(ppm) 43.21, 53.19, 60.73, 63.79, 66.42, 96.75, 120.31, 123.23, 127.80,
128.48, 128.65, 128.92, 129.46, 132.04, 139.26, 139.50, 159.92, 168.95, 173.03. LC-MS (ESI):
406.2 (M+1).
4.5.2.
9-(2-(4-methylpiperazin-1-yl)acetyl)-10-phenyl-3,4,9,10-tetrahydro-1H-
benzo[b]furo[3,4-e][1,4]diazepin-1-one (14b)
17

1
Yield: 48%; mp = 155-157°C H NMR (300 MHz, DMSO-d6): δ(ppm) 2.05-2.19 (m, 11H, N-
methyl piperazine), 2.79-2.93 (dd, 2H, CH2N, J=14.4 Hz), 4.89-5.04 (dd, 2H, CH2, J=15.3 Hz),
6.77-7.22 (m, 10H, CH & ArH), 10.15(s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): δ(ppm)
46.15, 52.71, 54.89, 5524, 60.48, 66.94, 96.85, 120.29, 123.21, 127.79, 128.47, 128.64, 128.90,
129.49, 132.09, 139.14, 139.54, 159.87, 169.13, 173.03. LC-MS (ESI): 419.1 (M+1).
4.5.3.
9-(2-morpholinoacetyl)-10-(3,4,5-trimethoxyphenyl)-3,4,9,10-tetrahydro-1H-
benzo[b]furo[3,4-e][1,4]diazepin-1-one (14c)
1
Yield: 61%; mp = 205-207°C H NMR (300 MHz, DMSO-d6): δ(ppm) 1.9-2.23 (m, 4H, CH2-
morpholine), 2.78-2.92 (m, 2H, CH2N), 3.35-3.37 (m, 4H, CH2-morpholine), 3.56 (s, 3H,
OCH3), 3.62(s, 6H, OCH3), 4.88-5.04 (dd, 2H, CH2, J=15.0 Hz), 6.25 (s, 2H, ArH), 6.76(s, 1H,
CH), 6.84-6.94 (m, 2H, ArH), 7.08-7.11 (d, 1H, ArH, J=7.8 Hz), 7.19-7.24 (t, 1H, ArH, J= 7.8
13
Hz), 10.24(s, 1H, NH). C NMR (75 MHz, DMSO-d6): δ(ppm) 53.19, 55.59, 56.36, 60.42,
60.86, 66.81, 96.99, 106.12, 120.12, 123.38, 128.98, 132.02, 135.23, 137.19, 139.14, 139.24,
153.01, 159.77, 168.70, 173. LC-MS (ESI): 496.2 (M+1).
4.5.4. 9-(2-(4-methylpiperazin-1-yl)acetyl)-10-(3,4,5-trimethoxyphenyl)-3,4,9,10-tetrahydro-
1H-benzo[b]furo[3,4-e][1,4]diazepin-1-one (14d)
1
Yield:54 %; mp = 195-197°C H NMR (300 MHz, DMSO-d6): δ(ppm) 2.05-2.21 (m, 11H, N-
methyl piperazine), 2.78-2.92 (dd, 2H, CH2N, J=14.4 Hz), 3.56 (s, 3H, OCH3), 3.62(s, 6H,
OCH3), 4.87-5.04 (dd, 2H, CH2, J=15.3 Hz), 6.25 (s, 2H, ArH), 6.77 (s, 1H, CH), 6.84-6.93 (m,
2H, ArH), 7.07-7.09 (d, 1H, ArH, J=6.9 Hz), 7.19-7.24 (t, 1H, ArH, J= 6.9 Hz), 10.19(s, 1H,
13
NH); C NMR (75 MHz, DMSO-d6): δ(ppm) 46, 52.60, 54.81, 56.49, 60.42, 66.82, 96.97,
106.11, 120.13, 123.32, 128.93, 129.59, 132.07, 135.30, 137.18, 139.06, 139.16, 153.01, 159.63,
168.89, 173.00. LC-MS (ESI): 509.3 (M+1).
4.5.5.
9-(2-morpholinoacetyl)-10-(p-tolyl)-3,4,9,10-tetrahydro-1H-benzo[b]furo[3,4-
e][1,4]diazepin-1-one (14e)
Yiled:67 %; mp = 233-235°C 1H NMR (300 MHz, DMSO-d6): δ(ppm) 1.99-2.03(m, 2H, CH2-
morpholine), 2.18 (m, 5H, CH2-morpholine & CH3), 2.80-2.94 (dd, , 2H, COCH2, J= 14.4 Hz),
3.38-3.40 (m, 4H, CH2-morpholine), 4.88-5.02 (dd, 2H, CH2, J=15.3 Hz), 6.78-7.07 (m, 8H, CH
18

& ArH), 7.15-7.19 (t, 1H, ArH, J= 6.9 Hz), 10.24(s, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
δ(ppm) 43.23, 53.20, 55.34, 60.73, 63.79, 66.43, 66.99, 99.77, 123.24, 127.80, 128.49, 128.66,
128.92, 129.46, 132.05, 139.28, 139.51, 159.93, 168.97, 173.05; LC-MS (ESI): 420.2 (M+1),
839.4 (2M+1).
4.6. Cytotoxicity assay
4.6.1. General procedure
The MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) based assay[26] was
carried out by seeding 5000 cancer cells per 180 µL RPMI complete culture medium in each
well of 96-well culture plates. The day after seeding, culture medium was replaced with medium
containing standard anti-tumor agent as well as different concentrations of newly synthesized
quinolines and RPMI control (no drug). Cells were then incubated at 37 ºC in 5% Co2 incubator
for 48h. Then 25 µL of MTT solution (4mg Ml-1) were added to each well and further incubated
at 37 ºC for 3h. At the end of incubation, formazan crystals were dissolved in 100 µL of DMSO
and plates were read in a plate reader (Synergy H4, USA) at 540 nm. This experiment was
performed in triplicate determination each time.
4.7. PI staining
MCF-7 and PC3 cells were seeded in each well of a 24-well plate and treated with 13c and 14b
at concentration of 5 µM and 50 µM and Doxorubicin at concentration of 5 µM or vehicle alone
(0.05% DMSO) for 48 h. Floating and adherent cells were then harvested and incubated at 4 ◦C
overnight in the dark with 750 μL of a hypotonic buffer (50 μg/mL PI in 0.1% sodium citrate
plus 0.1% Triton X-100) before flow cytometric analysis using a FAC Scan flow cytometer
(Becton Dickinson).
4.8. Annexin V binding assay
A2780 cancer cells were seeded in six-well plates for 24h. The medium was then replaced with
complete medium containing compound 13a and 13b. After 24 h of treatment, cells from the
supernatant and adherent monolayer cells were harvested by trypsinization, washed with PBS at
3000 rpm. Then the cells were processed with Annexin V-assay kit (FITC Annexin V Apoptosis
Detection Kit, (Cat. No. ab14085, Abcam, UK)) according to the manufacturer's instruction.
Further, flow cytometric analysis was performed using a flow cytometer (Becton Dickinson).
4.9. Tubulin polymerization assay
19

Tubulin polymerization assay were performed by employing a commercial kit (Tubulin
Polymerization Assay Kit (Porcine tubulin and Fluorescence based Kit (Cat. No. BK011P,
Cytoskeleton, USA)), according to the manufacturer's protocol [15]. Briefly, tubulin protein was
suspended in tubulin buffer (80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP, 60%
(v/v) glycerol, PH 6.9) then added to wells on a 96-well plate containing the cytotoxic
compounds or vehicle and mixed well. The effect of compound 13c at 50 and 100 μM
concentrations on tubulin polymerization was evaluated. Tubulin polymerization was followed
by monitoring the fluorescence enhancement due to the integration of a fluorescence reporter
into microtubules as polymerization happens. Polymerization was measured by excitation at 360
nm and emission at 420 nm for 1 h at 1 min intervals in a plate reader (Synergy H4, USA).
Paclitaxel at 3 μM concentration and podophyllotoxin at 5 and 10 μM concentrations were used
as positive stabilizing and destabilizing controls, respectively.
Acknowledgments
We are grateful to Research deputy of Mashhad University of Medical Sciences for financial
support of this research as part of thesis of Sina Omid Malayeri.
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21

Synthesis and biological evaluation of benzo[b]furo[3,4-e][1,4]diazepin-1-one derivatives as
anti-cancer agents
Sina Omid Malayeriab, Zahra Tayarani-Najaranc, Fatemeh Shaebani Behbahanic,
Roghayeh Rashidic, Saeedeh Delpazirb, and Razieh Ghodsi*ab
aBiotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
bDepartment of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical
Sciences, Mashhad, Iran
cDepartment of Toxicology, School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran
The percentage of total apoptotic cells induced by 13c increased
from 0.224% (control) to 76.84% (10 μM) in A2780 cancer cells.
Compound
A549
A2780
DU145
PC3
HeLa
MCF-7
Huvec
10.46±1.34
1.64±0.16
2.72±0.33
0.86±0.25
3.89±0.45
0.97±0.13
6.19±2.65
1.26±0.17
3.51±1.21 9.37±2.07 44.73±2.44
1.21±0.14 1.02±0.13 1.82±0.17
13c
podophyllotoxin
The in vitro anti-proliferative activities (IC50 (µM)) of 13c, and podophyllotoxin against human cancer
cell lines and normal Huvec cell line

Highlights:
. Novel Podophyllotoxin-like furobenzodiazepines were designed and synthesized as anti-
cancer agents
. Some of compounds (12h, 13a, 13c and 14b) showed significant cytotoxic activity on
cancer cells
. 13c showed promising antiproliferative activity against all cancer cells stronger than the
other compounds.
.
The percentage of total apoptotic cells induced by 13c was 76.84% at concentration of 10
μM
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