Synthesis of 12,12a-dihydro-7,12a-diazacyclohepta[cd]-benz[g]azulen-12-one (cyclohepta[mn]pyrrolo[2,1-c][1,4]-benzodiazepin-12-one) and evaluation of cytotoxic activity against HeLa S3 cells

Article abstract of DOI:10.3987/COM-11-12207

12,12a-dihydro-7,12a-diazacyclohepta[cd]benz[g]azulen-12-one (cyclohepta[mn]pyrrolo[2,1-c][1,4]benzodiazepin-12-one) derivative (7) was synthesized by the intramolecular cyclization of methyl 2-(3-phenyl-1-azaazulen- 8-yl)aminobenzoate. Compound (7) showe

Full text of DOI:10.3987/COM-11-12207

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HETEROCYCLES, Vol. 83, No. 6, 2011, pp. 1409 - 1415. © The Japan Institute of Heterocyclic Chemistry
Received, 11th March, 2011, Accepted, 5th April, 2011, Published online, 20th April, 2011
DOI: 10.3987/COM-11-12207
SYNTHESIS OF 12,12a-DIHYDRO-7,12a-DIAZACYCLOHEPTA[cd]-
BENZ[g]AZULEN-12-ONE (CYCLOHEPTA[mn]PYRROLO[2,1-c][1,4]-
BENZODIAZEPIN-12-ONE) AND EVALUATION OF CYTOTOXIC
ACTIVITY AGAINST HeLa S3 CELLS
Eiko Yoshioka,a Hiroyuki Fujii,b Toshihiro Murafuji,a Reiko Ikeda,c Takeo
Konakahara,c Takahiro Gunji,c and Noritaka Abec*
aGraduate School of Medicine, Yamaguchi University, Yoshida, Yamaguchi
b
753-8512, Japan; Science Research Center, Yamaguchi University, Yoshida,
c
Yamaguchi 753-8512, Japan; Department of Pure and Applied Chemistry,
Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba
278-8510, Japan: E-mail noriabe@rs.noda.tus.ac.jp
Abstract
(cyclohepta[mn]pyrrolo[2,1-c][1,4]benzodiazepin-12-one) derivative (7) was
synthesized by the intramolecular cyclization of methyl
–
12,12a-dihydro-7,12a-diazacyclohepta[cd]benz[g]azulen-12-one
2-(3-phenyl-1-azaazulen-8-yl)aminobenzoate. Compound (7) showed extremely
strong cytotoxic activity against HeLa S3 cells (IC50=0.543±0.063 M).
The 3a,9-diazabenz[f]azulene (pyrrolo[2,1-c][1,4]benzodiazepine: PBD) ring system (1), a skeleton of a
group of potent naturally occurring antitumor antibiotics from Streptomyces species, are of considerable
interest for their potentiality as antitumor agents, gene regulators, and DNA probs. Many synthetically
and pharmacological investigations related to PBD and its analogs were reported.1,2
N
N
1
O
1-Azaazulenes3 are attracted attention for their pharmaceutical and biological interests4,5 as well as their
characteristic physical and chemical properties. We also reported that the cytotoxic activities against

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HETEROCYCLES, Vol. 83, No. 6, 2011
HeLa S3 cells of 1-zaazulene derivatives.6-8 In the line of our interests for the synthesis of antitumoral
1-azaazulene derivatives, we proceeded to synthesize of fused 1-azaazulenes containing PBD skeleton.
It is considered that 12,12a-dihydro-7,12a-diazacyclohepta[cd]benz[g]azulene (cyclohepta[mn]pyrrolo-
[2,1-c][1,4]benzodiazepine) system would be derived from 2-(1-azaazulen-8-yl)aminobenzoic acid
derivative by intramolecular cyclization. It is known that 8-(arylamino)-1-azaazulene derivatives were
easily obtained by nucleophilic substitution of 8-halo-1-azaazulenes with arylamines.9,10 Therefore, we
synthesized methyl 2-(3-phenyl-1-azaazulen-8-yl)aminobenzoate (2) by the treatment of
8-chloro-3-phenyl-1-azaazulene (3) with methyl 2-aminobenzoate (4) in dioxane for 30min at rt in 89%
yield. In the IR spectrum of 2, an ester carbonyl peak was seen at 1725 cm-1 and an NH peak was seen at
3433 cm-1. In its 1H NMR spectrum, an ester methyl singlet was seen at  3.93 (s) and an NH signal was
seen at  8.24 (br s). In the aromatic protons resonated region, protons owing to 1-azaazulene ring were
seen at  7.03 (like t, J = 9.2, H-5), 7.46 (like t, J = 9.5, H-6), 7.56 (s, H-2), 7.56 (d, J = 9.6, H-7), and
8.21 (d, J = 9.6, H-4) together with phenyl protons (9H). From these results as well as HRMS (m/z
355.1418 [M+H]+), we assigned the structure.
7
Ph
4
3'
NH2
HN
+
N
N
Ph
CO2Me
CO2Me
6'
Cl
4
2
3
Scheme 1
The reaction can expand to other substituted benzoic acid, and the reaction of 3 with methyl
2-mercaptobenzoate (5) gave 6 in 66% yield (Scheme 2).
Ph
SH
S
+
N
N
Ph
CO2Me
CO2Me
Cl
5
3
6
Scheme 2
Previously, we reported the successful cyclization reaction of ethyl heteroarylamino-1-azaazulene-3-
carboxylate with POCl3-polyphosphoric acid (PPA).6 Therefore, we adequate the condition to the

HETEROCYCLES, Vol. 83, No. 6, 2011
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annulation of 2. Thus, we treated 2 with POCl3-PPA for 1 h at 150 ºC under argon atmosphere and
obtained 2-phenyl-12,12a-dihydro-7,12a-diazacyclohepta[cd]benz[f]azulen-
12-one (7) in 43% yield as red needles (Scheme 3). In the IR spectrum of 7, a carbonyl peak was seen at
1631 cm-1, which is equitable to an amide carbonyl. In its 1H NMR spectrum, signals owing to azaazulene
ring moiety at  7.56 (ddd, J = 10.0, 9.6, and 1.6, H-4), 7.59 (ddd, 10.8, 10.0, and 0.8, H-5), 8.34 (s, H-1),
8.70 (dd, J = 9.6 and 0.8, H-3), and 9.30 (d, J = 10.8, H-6) and signals owing to benzene moiety 7.40 (ddd,
J = 8.0, 7.2, and 1.2, H-9), 7.57 (dd, J = 8.0 and 1.6, H-8), 7.78 (ddd, J = 8.0, 7.2, and 1.6, H-10), and
8.47 (dd, J = 8.0 and 1.2, H-11) were seen together with phenyl protons (7.43 (tt, J = 7.6 and 1.6, H-p-Ph),
7.54 (dd, J = 8.4 and 7.6, H-m-Ph), and 7.61(dd, J = 8.4 and 1.6, H-o-Ph)). From these results as well as
HRMS (m/z 345.0971 [M+Na]+), we assigned the structure.
Similar treatment of 6 with POCl3-PPA resulted in complex results and did not give distinct products.
5
6
4
7
N
HN
8
3
POCl3 - PPA
9
2
N
12
Ph
N
Ph
CO2Me
10
1
11
O
2
7
Scheme 3
The cytotoxic activity against HeLa S3 cells of obtained compounds (2, 6, and 7) were evaluated by MTT
assay. Compounds (2 and 6) showed medium cytotoxic activity and 7 showed extremely strong cytotoxic
activity as expected.
Table 1. Cytotoxic evaluation of compounds (2a, 6, and 7a) expressed in M
2
6
7
31.1 ± 2.3a
12.9 ± 2.3a
0.543 ± 0.063
IC50
a The precipitates were appeared at the concentration of 1% DMSO of the culture fluid.
In summary, we presented a facile synthetic method for new annulated pyrrolo[2,1-c][1,4]benzodiazepine
system having a strong cytotoxic activity against HeLa S3 cells, and the system is expected as a leading
compound for new anticancer drugs.

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EXPERIMENTAL
Melting points were determined with a Yanagimoto micro-melting point MP JP-3 apparatus and were
1
uncorrected. H NMR spectra were recorded on a Bruker AVANCE 400S (400 MHz) using CDCl3 as a
solvent with tetramethylsilane as an internal standard; J values are recorded in Hz. IR spectra were
recorded for KBr pellets on a Nicolet FT-IR AVTAR 370DTGS. Mass specra (ESI-MS) were taken with
JEOL JMS-T100CS. Merck Kieselgel 60 was used for column chromatography.
Reaction of 8-chloro-3-phenyl-1-azaazulene (3) with methyl 2-aminobenzoate (4)
A mixture of 3 (0.106 g, 0.44 mmol) and 4 (0.117 g, 0.77 mmol) in dry dioxane (5 mL) was stirred for 30
min at rt. To the mixture hexane (30 mL) was added. The precipitates were corrected by filtration and
HCl salt of 2 was obtained. The salt was dissolved in water, then the solution was neutralized with
NaHCO3 and extracted with CHCl3. Evaporation of the extract to give 2 (0.140 g, 89%).
2: Orange scales (from hexane-CHCl3), mp 147.0-147.5 ºC; 1H NMR  3.93 (3H, s, OMe), 7.03 (1H, like
t, J = 9.2, H-5), 7.20 (1H, dd, J = 8.0 and 7.8, H-5’), 7.37 (1H, t, J = 7.3, H-p-Ph), 7.46 (1H, like t, J = 9.5,
H-6), 7.50 (2H, t, J = 7.3, H-m-Ph), 7.55 (1H, dd, J = 8.0 and 7.8, H-4’), 7.56 (1H, d, J = 9.6, H-7), 7.56
(1H, s, H-2), 7.56 (1H, d, J = 7.8, H-3’), 7.57 (2H, d, J = 7.3, H-o-Ph), 8.10 (1H, d, J = 7.8, H-6’), 8.21
(1H, d, J = 9.6, H-4), and 8.24 (1H, br s, NH); max/cm-1 3214 (OH), 1633 (C=O). MS m/z 355 ([M+H]+,
100%). HRMS: Calcd. for C23H19N2O2: 355.1447. Found: m/z 355.1418. HCl salt of 2: Yellow needles
(from hexane-CH2Cl2), mp 170 ºC (decomp); 1H NMR  3.78 (3H, s, OMe), 7.24 (1H, d, J = 11.6, H-7),
7.28 (1H, dd, J = 10.4 and 9.2, H-5), 7.46 (1H, td, J = 7.6 and 1.6, H-p-Ph), 7.48 (2H, dd, J = 7.2 and 1.6,
H-o-Ph), 7.53 (1H, d, J = 8.0, H-3’), 7.54 (1H, ddd, J = 8.0, 7.3 and 1.4, H-4’), 7.55 (2H, dd, J = 7.6 and
7.2, H-m-Ph), 7.66 (1H, dd, J = 11.6 and 9.2, H-6), 7.72 (1H, dd, J = 8.0 and 7.3, H-5’), 8.02 (1H, d, J =
2.8, H-2), 8.17 (1H, dd, J = 8.0 and 1.4, H-6’), 8.28 (1H, d, J = 10.4, H-4), 12.56 (1H, s, NH), and 15.25
(1H, br s, NH).
Reaction of 8-chloro-3-phenyl-1-azaazulene (3) with methyl 2-mercaptobenzoate (5)
A mixture of 3 (0.100 g, 0.42 mmol) and 5 (0.122 g, 0.73 mmol) in dry dioxane (5 mL) was stirred for 30
min at rt. To the mixture hexane (30 mL) was added. The precipitates were corrected by filtration and
HCl salt of 6 was obtained as yellow powders. The salt was dissolved in water, then the solution was
neutralized with NaHCO3 and extracted with CHCl3. Evaporation of the extract to give 6 (0.099 g, 66%)
was obtained as orange needles.
6 : Red powders (from hexane-CHCl3), mp 52.0-53.9 ºC; 1H NMR  3.78 (3H, s, OMe), 7.23 (1H, d, J =
8.8, H-7), 7.40 (1H, dd, J = 8.0 and 6.8, H-5’), 7.43 (1H, d, J = 9.0, H-4), 7.52 (2H, like t, J = 6.4,
H-m-Ph), 7.56 (1H, like t, J = 8.0, H-p-Ph), 7.59-7.62 (4H, m, H-3’, 4’, and H-o-Ph), 7.75-7.79 (1H, m,

HETEROCYCLES, Vol. 83, No. 6, 2011
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H-5), 7.95-7.99 (1H, m, H-6), 8.61 (1H, d, J = 8.0, H-6’), and 8.73 (1H, s, H-2); max/cm-1 1719 (C=O).
MS m/z 372 (([M+H]+, 100%), HRMS: Calcd. for C23H18N2O2S: 372.1058. Found: m/z 372.1044. HCl
1
salt of 6: Orange-yellow prisms (from hexane-CH2Cl2), mp 154 ºC (decomp); H NMR  3.82 (3H, s,
OMe), 7.48-7.61 (5H, m, Ph), 7.57 (1H, d, J = 9.6, H-7), 7.72 (1H, ddd, J = 7.6, 7.2, and 2.4, H-5’) , 7.75
(1H, ddd, J = 7.6, 6.4, and 2.4, H-4’), 7.81 (1H, like t, J = 9.6, H-5), 7.86 (1H, dd, J = 6.4 and 2.4, H-3’),
7.89 (1H, dd, J = 10.0 and 9.6, H-6), 8.09 (1H, dd, J = 7.2 and 2.4, H-6’), 8.78 (1H, s, H-2), 8.81 (1H, dd,
J = 10.0 and 1.2, H-4), and 14.50 (1H, br s, NH).
Cyclization of methyl 2-(3-phenyl-1-azaazulen-8-yl)aminobenzoate (2)
Under argon atmosphere, a mixture of 2 (0.034 g, 0.10 mmol), PPA (5 mL), and POCl3 (5 mL) was
heated for 1 h at 150 ºC, then ice-water (20 mL) was added. The mixture was neutralized with aq Na2CO3,
and extracted with CHCl3. The extract was dried over Na2SO4 and evaporated. Chromatography of the
residue with hexane-AcOEt (1:1) and recrystallization from hexane-CH2Cl2 gave 7 (0.014 g, 43%).
1
7: Red needles (from hexane-CH2Cl2), mp 230 ºC (decomp); H NMR  7.40 (1H, ddd, J = 8.0, 7.2, and
1.2, H-9), 7.43 (2H, tt, J = 7.6 and 1.6, H-p-Ph), 7.54 (2H, dd, J = 8.4 and 7.6, H-m-Ph), 7.56 (1H, ddd, J
= 10.0, 9.6, and 1.6, H-4), 7.57 (1H, dd, J = 8.0 and 1.6, H-8), 7.61(1H, dd, J = 8.4 and 1.6, H-o-Ph), 7.59
(1H, ddd, J = 10.8, 10.0, and 0.8, H-5), 7.78 (1H, ddd, J = 8.0, 7.2, and 1.6, H-10), 8.34 (1H, s, H-1), 8.47
(1H, dd, J = 8.0 and 1.2, H-11), 8.70 (1H, dd, J = 9.6 and 0.8, H-3), and 9.30 (1H, d, J = 10.8, H-6);

max/cm-1 1631 (C=O). HRMS: Calcd. for C22H14N2NaO: 345.1004. Found: m/z 345.0971 ([M+Na]+).
Biological assay
HeLa S3 cells were obtained from AIST and used after cultivation. The cultivated HeLa S3 cells were
cell counted and the culture fluid was prepared to the cell consistency of 2 × 104 cell/mL. The compounds
were added to the medium in DMSO solutions. To the aliquot of the culture fluid, which was incubated
for 3 h at 37 ºC, the test sample was added and then the culture fluid was incubated for 72 h. To the
culture fluid, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) solution was added,
and incubated for 4 h. Then the sample was centrifuged at 3000 rpm for 10 min at 4 ºC, and the solvent
was evaporated. Then DMSO was added to obtained mixture. The MTT-formazan was dissolved by
plate-mixing and OD540 was measured. The rate of outlive determined to refer with un-dosed control.
Dose-response curve was drawn up and IC50 was pursued. Every experiment in the cytotoxic assay was
replicated four times in order to define the IC values.
ACKNOWLEDGEMENTS
This work was partially supported by Research Fund of Tokyo University of Science.

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