Synthesis of new 1,3,4-benzotriazepin-5-one derivatives and their biological evaluation as antitumor agents

Article abstract of DOI:10.1007/s12272-013-0081-y

New derivatives of 1,3,4-benzotriazepin-5-one were designed and synthesized as structural analogues to the antitumor agents devazepide and asperlicin. An efficient and novel approach to the synthesis of 2-amino-1,3,4-benzotriazepin-5- one 2 was developed and its structure was confirmed. The newly synthesized derivatives were evaluated for their in vitro antitumor activity on 60 different cell lines. Compounds 8 and 9 displayed the most potent antitumor activity against several cell lines specifically ovarian cancer, renal cancer and prostate cancer, while compounds 5, 10 and 12 showed significant activities against UO-31 renal cancer cell line.

Full text of DOI:10.1007/s12272-013-0081-y

Arch. Pharm. Res. (2013) 36:684–693
DOI 10.1007/s12272-013-0081-y
RESEARCH ARTICLE
Synthesis of new 1,3,4-benzotriazepin-5-one derivatives
and their biological evaluation as antitumor agents
•
Azza T. Taher Lamia W. Mohammed
Received: 13 January 2012 / Accepted: 29 March 2012 / Published online: 18 March 2013
Ó The Pharmaceutical Society of Korea 2013
Abstract New derivatives of 1,3,4-benzotriazepin-5-one
were designed and synthesized as structural analogues to
the antitumor agents devazepide and asperlicin. An effi-
cient and novel approach to the synthesis of 2-amino-1,3,4-
benzotriazepin-5-one 2 was developed and its structure was
confirmed. The newly synthesized derivatives were eval-
uated for their in vitro antitumor activity on 60 different
cell lines. Compounds 8 and 9 displayed the most potent
antitumor activity against several cell lines specifically
ovarian cancer, renal cancer and prostate cancer, while
compounds 5, 10 and 12 showed significant activities
against UO-31 renal cancer cell line.
for cancer has been proven elusive since there are more than
100 different types of cancer present. Unfortunately, the
majority of drugs currently in the market are not specific,
which leads to the many common side effects associated
with cancer chemotherapy (Escherich et al. 2001).
There is an increasing interest for oncological applica-
tions of drugs which target specific receptors like cholecys-
tokinin (CCK) receptors, due to their over expression in
cancer cells (De-Luca et al. 2007). CCK antagonists were
studied as growth inhibitors in certain forms of cancer
(Lattmann et al. 2006). Amongst the many non-peptide
ligands that have been devised for CCK receptors, those
based on benzodiazepine ring systems are by far the most
prominent (Sinha et al. 1999). 3-Amino-1,4-benzodiaze-
pines as well as chemically related diverse amines were
screened for the cholecystokinin receptor inhibition in a
radiolabel binding assay (Offel et al. 2006). Devazepide
I (formerly MK-329) (Fig. 1) is considered the most potent
and highly specific CCKA receptor antagonist, with almost
160 times greater affinity for CCKA receptors than for CCKB
receptors (Tashiro et al. 1999). In addition, asperlicin II
(Fig. 1) proved to be a potent nonpeptidal CCK antagonist
where the 1,4-benzodiazepine ring system has served as a
useful tool for delineating the pharmacological actions of
CCK (Saemian et al. 2006). Focusing on the 1,4-benzodi-
azepine ring system that comprises part of the structure of
asperlicin and devazepide and combining the elements of
diazepam and tryptophan which mimic asperlicin (McDon-
ald et al. 2006; Herranz 2003). The synthesis of novel
derivatives of 1,3,4-benzotriazepinone analogous to asper-
licin and devazepide were described in order to achieve new
lead compounds with enhanced the potency of antitumor
agents. In Scheme 1 was introduced new derivatives an
analogues to asperlicin comprising the cyclization of five
and/or six membered rings with 1,3,4-benzotriazepin-5-one
Keywords 1,3,4-benzotriazepin-5-one derivatives ꢀ
Synthesis ꢀ Antitumor activity
Introduction
Benzotriazepinones and benzodiazepinones are commonly
known therapeutic agents and have long been used in
medicine for their anxiolytic, sedative, anticonvulsant,
antidepressant and hypnotic and anticancer activity (Osman
et al. 2002; Araujo et al. 2008; Ophardt 2003). Although
there are many anticancer drugs in the market, a consider-
able amount of research focuses on benzotriazepinones and
benzodiazepinones due to their specificity. They have dis-
tinct mechanism of action which may vary in their effects on
different types of normal and cancer cells. A single ‘‘cure’’
A. T. Taher (&) ꢀ L. W. Mohammed
Department of Organic Chemistry, Faculty of Pharmacy, Cairo
University, P.O. Box 11562, Cairo, Egypt
e-mail: azzataher2005@yahoo.com
123

Synthesis of new 1,3,4-benzotriazepin-5-one derivatives
685
to study the effect of ring size and different substituent’s on
antitumor activity. Schemes 2 and 3 outline the synthesis of
new derivatives analogues to devazepide as different spacer
were engaged 1,3,4-benzotriazepin-5-one as a bioisoster of
1,4-benzodiazepine and different indole derivatives (And-
reani et al. 2010; Joa˜o et al. 2008) to study their biological
effect on several tumor cell lines.
Materials and methods
Chemistry
All melting points were determined in open glass capillaries
on a Gallenkamp melting point apparatus and are uncor-
rected. The IR spectra were recorded on a Perkin-Elmer
Fig. 1 Structures of devazepide
I and asperlicin II
H
H3C
O
O
O
N
N
N
HO
N
NH
N
N
NH
HN
O
O
I
II
Scheme 1 Synthesis of target
compounds 3–7
O
O
N
H
O
1
NH
NH2
H2N
N
H
O
O
O
NH2
NH2
NH
N
NH
N
O
CH2(CN)2
NH
NH
ClCO2COCl
N
N
N
O
N
N
H
NH2
3
7
2
ClCH2COCl
Cl(CH2)2COCl
O
O
ClCHCOCl
CH3
NH
N
NH
N
O
N
N
O
HN
N
NH
H
CH3
O
O
N
4
6
N
N
H
5
123

686
A. T. Taher, L. W. Mohammed
2
a
d
O
NH
NH
b
O
c
N
N
H
NH
O
O
O
NH
NH
NH
NH
NHNH
N
N
N
H
NH
O
N
O
NH
8
N
O
N
O
O
N
H
9
11
10
Scheme 2 Synthesis of target compounds 8–11. a 1H-Indole-2-carbonyl chloride. b 2-Chloro-1-(1H-indol-1-yl)ethanone. c 2-(1H-Indol-3-
yl)acetyl chloride. d 1-(2-Chloroacetyl)indoline-2,3-dione
of the reaction was monitored on ready-made Silica–gel
plates fluorescent (Merck) using CHCl3/CH3OH (9.5:0.5) as
solvent using, UV lamp.
2
e
f
Indole-2-carbonyl chloride (a) (Shakila et al. 2010),
indole-1-acetyl chloride (b) (Mutschler and Winkler 1978),
indole-3-acetyl chloride (c) (Rogers and Stern 1992), ethyl
2-(1H-indol-1-yl)acetate (d) (Huntress and Bornstein
1949), ethyl 2-(2,3-dioxoindolin-1-yl)acetate (e) (Rekhter
2005) and1-(2-chloroacetyl)indoline-2,3-dione (f) (Abd-
Elrahman et al. 2008) were prepared according to reported
procedures.
O
NH
NH
O
NH
NH
N
NH
O
O
N
NH
N
O
O
N
2-Amino-3,4-dihydro-1,3,4-benzotriazepin-5-one (2)
To a suspension of isatoic anhydride 1 (0.01 mol, 1.63 g)
in acetic acid (25 mL), aminoguanidine bicarbonate
(0.01 mol, 0.77 g) was added and the mixture was heated
under reflux for 4 h.The reaction mixture was cooled and
poured into ice-cold water (50 mL), the solution was then
neutralized with sodium bicarbonate. Aqueous ammonia
(10 mL) was added and the solution was left overnight.
The precipitated product was then filtered, washed with
ice-cold water (20 mL) and crystallized from dimethyl-
formamide/water (1:1).
m.p. 240–241 °C, yield 82 %, IR(KBr, cm-1): 3425,
3248, 3221(NH and NH2), 3150 (CH aromatic), 1647
(C=O); 1H NMR 300 MHz (DMSO-d6): d 2.02 (s, 2H, NH2
exchanged by D2O), 6.48–6.76 (m., 1H, C7 ArH),
12
13
Scheme 3 Synthesis of target compounds 12 and 13. e Ethyl 2-(2,3-
dioxoindolin-1-yl)acetate and f ethyl 2-(1H-indol-1-yl)acetate
spectrophotometer using potassium bromide discs. The
H-NMR spectra were recorded on a Varian Gemini
300 MHz using DMSO-d6 as solvent. The chemical shifts
were reported as parts permillion d ppm, tetramethylsilane
(TMS) as an internal standard. Mass spectra were obtained
on a Jeol-SX-102 instrument. Elemental analysis was per-
formed on a Perkin-Elmer 2400 C, H, N analyzer and values
were within 0.4 % of theoretical percentages. The progress
123

Synthesis of new 1,3,4-benzotriazepin-5-one derivatives
687
7.02–7.29 (m., 1H, C8 ArH), 7.89 (s., 1H, C6 ArH), 8.57 (s.,
1H, C9 ArH), 15.95, 16.21 (2 s., 2H, NH exchanged by
D2O); EIMS: M? (m/z) 176 (9.44 %). Anal Calcd. for
C8H8N4O (176.18): C, 54.54; H, 4.58; N, 31.80; Found C,
54.44; H, 4.31; N, 31.50.
57.39; H, 4.38; N, 24.34; Found C, 57.57; H, 4.58; N,
24.74.
3,4-Dihydropyrimido[2,1-b]1,3,4-benzotriazepine-2,7-
(1H,6H)-dione (6)
General synthetic procedure for 3, 4, 5 and 6
m.p.[300 °C, yield 75 %, IR(KBr, cm-1): 3414, 3224(NH),
3107 (CH aromatic),1695, 1647 (2C=O); 1H NMR 300 MHz
(DMSO-d6): d 2.26 (t., 2H, NHCH2), 3.59 (t., 2H, CO–CH2),
6.98–7.16 (s., 1H, C9 ArH), 7.45 (m., 1H, C10 ArH), 7.68 (s.,
1H, C8 ArH), 8.58 (s., 1H, C11 ArH), 10.44, 11.64 (2 s., 2H,
NH exchanged by D2O); EIMS: M-1 (m/z) 229 (2.10 %),
M? m/z 230 (1.50 %). Anal Calcd. for C11H10N4O2
(230.22): C, 57.39; H, 4.38; N, 24.34; Found C, 57.66; H,
4.18; N, 24.11.
A mixture of 2 (1.76 g, 0.01 mol) and each of oxalyl
chloride, chloroacetyl chloride, 2-chloropropionyl chloride
and 3-chloropropionyl chloride (0.015 mol) in dry benzene
(25 mL) and anhydrous potassium carbonate (0.2 mol) was
heated under reflux for 4, 5, 5, 7 h respectively. The sol-
vent was evaporated under reduced pressure to dryness.
The residue was dissolved in water (10 mL), filtered, dried
and crystallized from methanol.
2,4-Diaminopyrimido[2,1-b]1,3,4-benzotriazepine-
7-(1H)-one (7)
1H-Imidazo[2,1-b]1,3,4-benzotriazepine-2,3,6-
(5H)-trione (3)
A mixture of 2 (1.76 g, 0.01 mol), malononitrile (6.60 g,
0.01 mol) and triethylamine (3 mL) in absolute ethyl alcohol
(30 mL) was heated under reflux for 12 h. The solvent was
evaporated under reduced pressure to dryness and cooled.
The precipitate formed was filtered, washed with water
(2 9 10 mL), dried and crystallized from methanol.
m.p.[300 °C, yield 77 %, IR(KBr, cm-1): 3406, 3221(NH),
3087 (CH aromatic), 1710, 1690, 1651(3C=O); H-NMR
1
300 MHz (DMSO-d6): d 6.47–6.78 (m., 1H, C8 ArH),
7.03–7.23 (m., 1H, C9 ArH), 7.55 (s., 1H, C7 ArH), 8.57 (s.,
1H, C10 ArH), 11.43, 12.29 (2s., 2H, NH exchanged by D2O);
EIMS: M-1 m/z 229 (4.72 %), M? (m/z) 230 (1.50 %). Anal
Calcd. for C10H6 N4O3 (230.18): C, 52.18; H, 2.63; N, 24.34;
Found C, 52.39; H, 2.74; N, 24.82.
m.p. 270–271 °C, yield 54 %, IR(KBr, cm-1): 3480,
3252, 3224 (NH and NH2), 3077 (CH aromatic), 1652
1
(C=O); H NMR 300 MHz (DMSO-d6): d 1.99 (br.s, 4H,
2NH2 exchanged by D2O), 6.49-6.66 (m., 1H, C9 ArH),
7.03-7.48 (m., 1H, C10 ArH), 7.81 (s., 1H, C8 ArH), 8.40
(s., 1H, C11 ArH), 8.55 (s., 1H, C3 ArH), 15.22 (s., 1H, NH
exchanged by D2O); EIMS: M ? (m/z) 242 (33.43 %);
Anal Calcd. for C11H10N6O (242.24): C, 54.54; H, 4.16; N,
34.69; Found C, 54.83; H, 4.32; N, 34.95.
1H-Imidazo[2,1-b]1,3,4-benzotriazepine-2,6-(3H,5H)-
dione (4)
m.p. 232–233 °C, yield 75 %, IR(KBr, cm-1): 3441, 3221
(NH), 3147 (CH aromatic), 2978, 2939 (CH aliphatic),1708,
1
1651 (2C=O); H NMR 300 MHz (DMSO-d6): d 3.06 (s.,
2H, N–CH2–CO), d 7.07–7.25 (m., 1H, C8 ArH), 7.43–7.62
(m., 1H, C9 ArH), 7.80 (s., 1H, C9 ArH), 8.35 (s., 1H, C10
ArH), 10.59, 11.78 (2 s., 2H, NH exchanged by D2O); EIMS:
M? (m/z) 216 (22.75 %), M? 1 m/z 217 (12.60 %). Anal
Calcd. for C10H8N4O2 (216.20): C, 55.55; H, 3.73; N, 25.91;
Found C, 55.18; H, 3.53; N, 26.11.
General synthetic procedure for 8, 9, 10 and 11
A mixture of 2 (1.76 g, 0.01 mol) and freshly prepared a, b, c
and d (0.01 mol) of each in dry benzene (25 mL)/triethyl-
amine (0.5 mL) was heated under reflux for 2,3,2,4 h
respectively. The solvent was evaporated under reduced
pressure to dryness. Water (5 mL) was added and the formed
precipitate was filtered, dried and crystallized from ethanol.
3-Methyl-1H-imidazo[2,1-b]1,3,4-benzotriazepine-2,6-
(5H)-dione (5)
N-(5-Oxo-4,5-dihydro-3H-1,3,4-benzotriazepin-2yl)-1H-
indole-2-carboxamide (8)
m.p. 238–240 °C, yield 80 %, IR(KBr, cm-1): 3422, 3221
(NH), 3128 (CH aromatic), 2982 (CH aliphatic),1711,
1
1651(2C=O); H-NMR 300 MHz (DMSO-d6): d 1.19 (d.,
m.p. 250–252 °C, yield 50 %, IR (KBr, cm-1): 3380,
3221(NH), 3103 (CH aromatic), 1690, 1651 (2C=O); H-
1
3H, CH3), 3.07 (q., 1H, CH), 6.71–6.82 (m., 1H, C8 ArH),
7.36–7.61 (m., 1H, C9 ArH), 7.67 (s., 1H, C6 ArH), 8.39 (s.,
1H, C10 ArH), 10.22, 11.61 (2 s., 2H, NH exchanged by
D2O); EIMS: M? 1 (m/z) 231 (23.26 %), M? m/z 230
(22.09 %). Anal Calcd. for C11H10N4O2 (230.22): C,
0
0
0
NMR 300 MHz (DMSO-d6): d 6.42–6.79 (m., 4H, C4 ,5 , 6 ,
0
C7 ArH), 7.11 (s, 1H, C3 ArH), 7.25–7.60 (m., 1H, C8
0
ArH), 7.79 (s., 1H, C6 ArH), 8.23 (br s., 1H, C7 ArH), 8.48
(s., 1H, C9 ArH), 10.40, 11.41, 11.62, 12.24 (4 s., 4H, NH
123

688
A. T. Taher, L. W. Mohammed
exchanged by D2O); EIMS: M? (m/z) 242 (33.43 %); Anal
Calcd. for C17H13N5O2 (319.32): C, 63.94; H, 4.10; N,
21.93; Found C, 64.17; H, 4.08; N, 21.53.
cooled and the formed precipitate was filtered, dried and
crystallized from ethanol.
N-(4,5-dihydro-5-oxo-3H-1,3,4-benzotriazepin-2-yl)-2-
(2,3-dioxoindolin-1-yl)acetamide (12)
2-(2-(1H-indol-1-yl)-2-oxoethylamino)-3,4-dihydro-
1,3,4-triazepin-5-one (9)
m.p. 120–122 °C, yield 42 %, IR (KBr, cm-1): 3418, 3395
(NH), 3039 (CH aromatic), 2985, 2947 (CH aliphatic),
1751, 1740, 1675, 1647 (4C=O); 1H NMR 300 MHz
(DMSO-d6): d 1.23 (s, 1H, NH exchanged by D2O); 4.13,
4.60 (2s, 2H, CO–CH2), 7.15–7.19 (m., 4H, ArH),
7.59–8.01 (m., 4H, ArH); EIMS: M - 2 m/z 361
(75.48 %), M - 1 362 (22.50 %), M ? 363 (6.10 %).
Anal Calcd. for C18H13N5O4 (363.33): C, 59.50; H, 3.61;
N, 19.28; Found C, 59.72; H, 3.92; N, 19.02.
m.p. 155–156 °C, yield 49 %, IR(KBr, cm-1): 3400–3221
(NH), 3101 (CH aromatic), 2986 (CH aliphatic), 1692, 1651
(2C=O); 1H NMR 300 MHz (DMSO-d6): d 3.07 (s, 2H, CO–
0
CH2NH), 6.48–7.43 (m., 4H, C4 ,5 , 6 , C7 ArH), 7.46 (s, 1H,
0
0
0
C8 ArH), 7.59–7.68 (m., 1H, C3 ArH), 7.85 (s., 1H, C6 ArH),
0
8.15 (br s., 1H, C7 ArH), 8.24 (s., 1H, C9 ArH), 8.44 (s., 1H,
0
C2 ArH),10.23, 11.33, 12.00 (3s., 3H, NH exchanged by
D2O); EIMS: M - 1 (m/z) 332 (1.59 %), M? (m/z) 333
(1.81 %), M ? 1 (m/z) 334 (8.29). Anal Calcd. for
C18H15N5O2 (333.34): C, 64.86; H, 4.54; N, 21.01; Found C,
64.88; H, 4.32; N, 20.97.
N-(4,5-dihydro-5-oxo-3H-1,3,4-benzotriazepin-2-yl)-2-
(1H-indol-1yl)acetamide (13)
N-(5-Oxo-4,5-dihydro-3H-1,3,4-benzotriazepin-2yl)-
1H-indole-3-acetamide (10)
m.p. 203–204 °C, yield 46 %, IR(KBr, cm-1): 3421, 3385
(NH), 3066 (CH aromatic), 1671, 1647 (2C=O); H NMR
1
300 MHz (DMSO-d6): d 4.23, 4.77 (2s, 2H, CO–CH2),
m.p. 195–197 °C, yield 52 %, IR(KBr, cm-1): 3406, 3224,
3213 (NH), 3127 (CH aromatic), 2974, 2939 (CH ali-
1
phatic), 1691,1643 (2C=O); H NMR 300 MHz (DMSO-
6.59–7.19 (m., 4H, C4 ,5
6 , C7 ArH), 7.34–7.39 (m., 1H,
0
0
0
,
0
C8 ArH), 7.55 (br s., 1H, C6 ArH), 7.88 (s., 1H, C7 ArH),
0
8.21 (br s., 1H, C9 ArH), 8.46 (d., 1H, C3 ArH), 8.59 (d.,
0
0
0
0
1H, C2 ArH), 10.63, 11.83, 12.41 (3s., 3H, NH exchanged
d6): d 3.09 (s, 2H, CO–CH2), 6.57–7.43 (m., 4H, C4 ,5
C7 ArH), 7.46–7.53 (m., 1H, C8 ArH), 7.68 (s., 1H, C6
,
,
6
by D2O); EIMS: m/z M ? 333 (19.48 %). Anal Calcd. for
C18H15N5O2 (333.34): C, 64.86; H, 4.54; N, 21.01; Found
C, 64.72; H, 4.36; N, 21.02.
0
ArH), 8.15 (br s., 1H, C7 ArH), 8.24 (s., 1H, C9 ArH), 8.49
0
(s., 1H, C2 ArH), 10.41, 11.60, 11.95, 12.42 (4s., 4H, NH
exchanged by D2O). Anal Calcd. for C18H15N5O2 (333.34):
C, 64.86; H, 4.54; N, 21.01; Found C, 65.09; H, 4.32; N,
21.32.
Antitumor screening
Under a sterile condition, cell lines were grown in RPMI
1640 media (Gibco, NY, USA) supplemented with 10 %
fetal bovine serum (Biocell, CA, USA), 5 9 105 cell/ml
was used to test the growth inhibition activity of the syn-
thesized compounds. The concentrations of the compounds
ranging from 0.01 to 100 lM were prepared in phosphate
buffer saline. Each compound was initially solubilised in
dimethyl sulfoxide (DMSO), however, each final dilution
contained less than 1 % DMSO. Solutions of different
concentrations (0.2 mL) were pipetted into separate well of
a microtiter tray in duplicate. Cell culture (1.8 mL) con-
taining a cell population of 6 9 104 cells/ml was pipetted
into each well. Controls, containing only phosphate buffer
saline and DMSO at identical dilutions, were also prepared
in the same manner. These cultures were incubated in a
humidified incubator at 37 °C. The incubator was supplied
with 5 % CO2 atmosphere. After 48 h, cells in each well
were diluted 10 times with saline and counted by using a
coulter counter. The counts were corrected for the dilution.
The data reported as mean graph of the percent growth
inhibition of the treated cells, and presented as percentage
1-[2-(4,5-Dihydro-5-oxo-3H-1,3,4-benzotriazepin-2yl-
amino) acetyl] indoline-2,3-dione (11)
m.p. 220–222 °C, yield 47 %, IR(KBr, cm-1): 3500–3350
(NH), 3123 (CH aromatic), 2978, 2931, 2893 (CH ali-
phatic), br. 1700–1635 (4C=O); 1H NMR 300 MHz
(DMSO-d6): d 3.91 (s, 2H, CO–CH2NH), 6.91–6.96 (m.,
0
0
1H, C5 ArH), 7.23–7.28 (m., 1H, C6 ArH), 7.33–7.48 (m.,
1H, C7 ArH), 7.62–7.66 (m., 1H, C8 ArH), 7.75 (s., 1H, C6
0
ArH), 7.82 (d., 1H, C4 ArH), 8.54 (br s., 1H, C9 ArH), 8.70
0
(d., 1H, C7 ArH), 11.56, 11.94, 12.19 (3s., 3H, NH
exchanged by D2O); EIMS: M ? 4 (m/z) 367 (0.94 %).
Anal Calcd. for C18H13N5O4 (363.33): C, 59.50; H, 3.61;
N, 19.28; Found C, 59.18; H, 4.02; N, 19.42.
General synthetic procedure for 12 and 13
A mixture of 2 (1.76 g, 0.01 mol) and freshly prepared
e and f (0.01 mol) of each in absolute ethanol (25 mL) was
heated under reflux for 7 h. The reaction mixture was
123

Synthesis of new 1,3,4-benzotriazepin-5-one derivatives
689
growth inhibition (GI %). The obtained results of the tested
triazepinone analogues 2–13; showed distinctive potential
pattern of selectivity, as well as broad-spectrum antitumor
activity. (Grever et al. 1992; Monks et al. 1991; Boyd and
Paull 1995; Skehan et al. 1990) (Table 1).
Fetouh 1988). Consequently, the reaction of 2 with mal-
ononitrile in absolute ethanol and calculated amount of
triethylamine afforded 7 with IR spectrum showing forked
bands at 3330–3250 cm-1 corresponding to 2 NH2 groups
and mass spectrum showing molecular ion peak in 33.43 %
abundance. In addition to 1H NMR spectrum which showed
distinct two singlet signals exchangeable with D2O at
1.9 ppm equivalent to the 2 NH2 groups. In Schemes 2 and
3, target compounds 8–13 were prepared by reaction of 2
with freshly prepared reported indole acid chlorides and/or
esters. Firstly, the reagents a and c were prepared by
reaction of either 1H-indole 2-carboxylic acid or indole
3-acetic acid reacted with thionyl chloride. Secondly,
indole and isatin (indole 2,3-dione) reacted with chloro-
acetyl chloride producing intermediates b and d. Finally,
e and f intermediates were obtained upon the reaction of
indole and isatin with ethyl chloroacetate in boiling ethanol
[c.f experimental part]. The new final compounds 8–13
were synthesized via the reaction of compound 2 with each
of the previously obtained intermediates a–f. The structure
of the new derivatives was consistent with the proposed
structures and was proved via IR spectra by the disap-
pearance of the forked peak at 3248, 3221 equivalent to
NH2 group and via 1H-NMR spectra showed a peak of CH2
of acetyl linkage around 3.06–4.77 ppm, in addition to the
molecular ion peak obtained in each case.
Results and discussion
Synthetic routes for novel compounds are depicted in
Schemes 1, 2 and 3. The structure and synthesis of
2-amino-1,3,4-benzotriazepin-5-one (2) was unavailable
through previously described methods. Literature survey
revealed that, the reaction of isatoic anhydride with glycine
afforded 3H-1,4-benzodiazepin-2,5-(1H,4H)-dione (III)
(Fig. 2) (Mohiuddin et al. 1985). Moreover, derivatives of
4-methyl benzotriazepinone (IV) (Fig. 2) were reported by
two steps reaction (Leiby and Heindel 1977).
From the previous findings the unsubstituted 2-amino-
1,3,4-benzotriazepin-5-one was synthesized using one pot
reaction of isatoic anhydride with 1-aminoguanidine
bicarbonate affording 82 % yield of the target compound.
The possible mechanism of the regioselective formation of
2 is shown in (Fig. 3).
In order to prove the structure of 2-amino-1,3,4-ben-
1
zotriazepin-5-one IR, mass, H NMR spectra and micro-
analysis were performed. In addition, the reaction of isatoic
anhydride with 1-aminoguanidine bicarbonate in refluxing
ethanol (a previously reported procedure for the synthesis
of V (Fig. 4) has been used as an evidence for the cycli-
zation to eliminate the possible formation of the open
structure (Bozena and Stanislaw 1962).
Preliminary in vitro antitumor screening
The antitumor screening of all novel synthesized com-
pounds 2–13 was carried out in the National Cancer
Institute (NCI), Bethesda, MD, USA. They were subjected
to the NCI’s disease-oriented human cell lines screening
assay to be evaluated for their in vitro antitumor activity. A
single dose (10 lM) of the test compounds were used in
the full NCI 60 cell lines panel assay which includes nine
tumor subpanels namely; leukaemia, non-small cell lung,
colon, CNS, melanoma, ovarian, renal, prostate, and breast
cancer cells (Grever et al. 1992; Monks et al. 1991; Boyd
and Paull 1995; Skehan et al. 1990).
According to the fact that several diamino compounds
upon reaction with chloroacid chlorides leads to the for-
mation of cyclised derivatives,(Taher and Raafat 2005) the
reaction of compound 2 with oxalyl chloride, chloroacetyl
chloride, 2-chloropropionyl chloride and 3-chloropropionyl
chloride in dry benzene in presence of potassium carbonate
resulted in the formation of the cyclised target compounds
3, 4, 5 and 6 in 75–85 % yield.The IR spectra of the
products revealed the disappearance of absorption bands of
NH2 group and the appearance of one absorption band at
3,450–3,220 cm-1 due to NH group. Beside the appear-
ance of an absorption band at 1690–1670 cm-1 due to C=O
The results revealed that compound 2 showed moderate
activity against non cell small lung cancer HOP-92 and
renal cancer UO-31 with percentage growth inhibition
(GI %) 16 %. Compound 5 showed moderate activity
against ovarian cancer and breast cancer MCF7, GI %
21–26 %, respectively and displayed promising activity
against renal cancer UO-31, GI % 36 %. Compound 8
possessed moderate activity against lung cancer HOP-92,
H-322M and H-522, GI % 15, 19 and 17 % respectively. In
addition, it recorded significant activity against leukaemia
CCRF-CEM, CNS cancer U-251, colon cancer HCT-116
and ovarian LGROVI, GI % 26, 28, 23 and 22 %,
respectively. Moreover, compound 8 showed an excellent
1
groups. H NMR was also used as an evidence for the
1
formed derivatives structure, H NMR of 4 showed a sin-
glet peak at d 2.99 ppm corresponding to CH2 group, while
compound 5 showed doublet peak at 1.19, quartet signal at
3.07 ppm due to CH3 and CH groups. Compound 6 showed
two signals at 2.26–3.59 ppm due to 2 CH2 groups.
Moreover, the cyclization of several compounds by the use
of malononitrile in the presence of triethylamine was pre-
viously reported (El-Enany et al. 2011; Ried and Aboul-
123

690
A. T. Taher, L. W. Mohammed
Table 1 Inhibition percent of the tested compounds 2–13 (10 lMolar) on different 60 cell lines
Subpanel tumor cell lines
% Growth inhibition (GI %)a
2
3
4
5
6
7
8
9
10
11
12
13
Leukemia
CCRF-CEM
HL-60(TB)
K-562
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
26
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
15
17
–
–
MOLT-4
–
–
–
–
RPMI-8226
SR EKVX
Non-small cell lung cancer
Hop-62
10
9
–
15
–
–
–
–
31
–
–
–
13
–
–
–
–
–
–
–
–
Hop-92
ATTC
–
14
16
–
–
–
–
–
–
–
–
12
–
–
–
–
–
–
–
–
–
14
15
–
10
41
10
10
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Hop-92
–
10
–
–
NCI-H460
NCI-H226
NCI-H23
NCI-322M
NCI-H522
COLO-205
Colon cancer
HCT-116
HCT-15
–
–
–
–
–
–
–
19
–
–
10
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
17
–
10
–
–
–
–
–
–
10
–
–
–
–
–
–
–
–
–
23
–
10
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
19
–
HT29
–
–
–
–
KM12
–
–
–
–
–
HCC-2988
SW-620
CNS cancer
SF-268
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
SF-295
–
–
–
–
–
–
SF-539
–
–
11
–
–
–
–
18
–
SNB-19
–
–
12
–
–
–
SNB-75
14
–
11
–
–
–
22
–
–
U251
–
28
13
–
Melanoma
LOX IMVI
MALME-3 M
M14
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10
–
–
–
–
–
–
–
–
–
–
–
18
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGORV-1
OVCAR-3
OVCAR-4
OVCAR-5
–
–
10
–
–
–
–
–
–
–
–
–
10
–
–
–
–
21
–
–
40
31
13
11
–
–
123

Synthesis of new 1,3,4-benzotriazepin-5-one derivatives
691
Table 1 continued
Subpanel tumor cell lines
% Growth inhibition (GI %)a
2
3
4
5
6
7
8
9
10
11
12
13
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
22
15
–
16
16
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
A498
12
–
–
13
–
–
–
–
–
–
–
ACHN
–
–
–
–
13
10
–
–
–
CAKI-1
–
–
10
–
16
–
15
–
11
–
19
–
–
SN12C
–
–
–
TK-10
–
–
–
–
–
–
–
–
–
UO-31
16
36
13
31
30
35
18
41
14
Prostate cancer
PC-3
–
–
–
–
–
–
32
29
21
–
–
–
DU-145
Breast cancer
MCF7
–
–
–
–
–
–
26
10
–
–
–
–
33
19
18
13
–
–
–
–
–
–
–
–
MDA-MB-231/ATCC
MDA-MB-231/ATCC
HS-578T
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
BT-549
–
–
–
–
T-47D
10
–
–
11
–
13
–
MDA-MB-468
19
– dashes means; growth inhibition GI % \ 10
O
O
CH3
O
NH
R
N
O
..
H2N
+
NH2
N
N
NH
H
N
H
O
N
NH2
N
H
O
R'
-CO2
CH3COOH/NH3
IV
III
O
O
R= H, Cl, Br, NO2
R'= H,CH3
NH
NH
NH
-NH3
NH
Fig. 2 Structures of previously prepared some benzoazepine
derivatives
NH
..
2
NH2
NH
N
NH2
activity against ovarian cancer, renal cancer UO-31, pros-
tate cancer PC-3 and breast cancer MCF-7, GI % 40, 31,
31, 33 %, respectively. Compound 9 showed highly
activity against non small cell lung HOP-92, ovarian can-
cer, renal cancer UO-31 and prostate cancer PC-3 of GI %
41, 31, 30 and 29 %, respectively. Compound 10 displayed
high activity against renal cancer UO-31 in GI % 35 %
Fig. 3 Proposed mechanism of synthesis of 2-amino-1,3,4-ben-
zotriazepin-5-one
while it registered moderate activity against prostate cancer
PC-3 in ratio 21 %. Compound 11 showed moderate
activity against leukaemia K-562 and MOLT-4 in ratios 15
and 17 %, respectively. In addition, it possessed good
123

692
A. T. Taher, L. W. Mohammed
O
Research Highlights
H
N
NH2
Synthesis of novel 1,3,4-benzotriazepines derivatives.
In vitro antitumor evaluation of all the synthesized
compounds was on 60 different cell lines.
Compounds 8 and 9 displayed the most potent antitumor
activity.
N
H
NH
NH2
Elemental analysis and spectroscopic characterization of
newly synthesized compounds.
V
Fig. 4 Structure of N-(o-aminobenzoyl)-hydrazinecarboximidamide
Acknowledgments Thanks are due to the NCI, Bethesda, MD, and
Chemotherapeutic Agents Repository. Attn: Thelma Dizon, USA for
performing the antitumor testing of the synthesized compounds.
activity toward CNS CNB-75, melanoma MAL-3M an d
renal cancer UO-31, GI % 22, 18 and 18 %, respectively.
Compound 12 showed high activity against renal cancer
UO-31 in GI 41 % inhibition, while displayed moderate
activity toward renal cancer CAKI-1 in 18 % inhibition.
Compound 13 showed promising activity against leu-
kaemia RPMI-88226 in GI % 31 % while showed mod-
erate inhibition activity toward CNS-SF539 and colon
cancer HCT-116 in GI % 18 and 19 %, respectively.
From all the data presented by NCI, it appears that
amongst the tested compounds, compounds 8 and 9
displayed the higher inhibition against most of the tested
cell lines as leukaemia, ovarian, renal UO-31, prostate
and breast cancer. While higher activity against leukae-
mia CCRF-EM and SR is displayed by compounds 8 and
13. Best inhibition activity towards ovarian and prostate
PC-3 is presented by compounds 8 and 9. While the
most promising inhibition activity against renal cancer
UO-31 is demonstrated by compounds 5, 8–12. Com-
pounds 5 and 8 showed the most inhibition against breast
cancer MCF-7.
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