11-Phenyl-[b,e]-dibenzazepine compounds: Novel antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2008.11.001

A series of 11-phenyl-[b,e]-dibenzazepine compounds were synthesized and shown to be inhibitors of tumor cell proliferation with IC₅₀ values ranging from submicromolar to micromolar concentrations. Flow cytometric analyses of several active com

Full text of DOI:10.1016/j.bmcl.2008.11.001

Bioorganic & Medicinal Chemistry Letters 19 (2009) 104–107
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
11-Phenyl-[b,e]-dibenzazepine compounds: Novel antitumor agents
Raed A. Al-Qawasmeh a, Yoon Lee b, Ming-Yu Cao b, Xiaoping Gu b, Stéphane Viau b, Jeff Lightfoot b,
,
*
Jim A. Wright b, Aiping H. Young b
a Department of Chemistry, The University of Jordan, Amman-11942, Jordan
b Lorus Therapeutics Inc., 2 Meridian Road, Toronto, Ont., Canada M9W 4Z7
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 11-phenyl-[b,e]-dibenzazepine compounds were synthesized and shown to be inhibitors of
tumor cell proliferation with IC50 values ranging from submicromolar to micromolar concentrations.
Flow cytometric analyses of several active compounds demonstrated inhibition of cell cycle progression
at the G0–G1 phase transition resulting in G0–G1 arrest.
Received 22 September 2008
Revised 31 October 2008
Accepted 3 November 2008
Available online 6 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Clotrimazole
Dibenzazepines
Heterocycles
Antitumor
Cell cycle
For small organic molecules, nitrogen-containing heterocycles
have received considerable attention in the literature, mainly due
to a broad range of biological properties and a detailed history as
important medicinal pharmacophores.1 Of these heterocycles, the
synthesis, reaction, and biological activity of nitrogen containing
seven-membered ring compounds is an expanding area of research
in medicinal chemistry. There has been an extensive analysis of
benzdiazepine compounds, which exhibit a wide range of biologi-
cal activities including antidepressive, analgesic, antipsychotic,
antihistaminic, and antimuscarinic activities.2a–e Although, various
scaffolds of dibenzazepines 3, 4, and 5 (Fig. 1) show diverse biolog-
ical activities,2f–h these compounds, especially scaffold 4, have not
been as widely investigated.
the Ca2+ stores-regulated Ca2+ channels, and inhibits Ca2+ uptake
into intracellular stores. The sustained depletion of intracellular
Ca2+ activates RNA-dependent protein kinase (PKR), resulting in
phosphorylation of eIF2
tion. Inactivation of eIF2
plex between Met-tRNA, eIF2
a
a
at serine 51 and its concomitant inactiva-
inhibits formation of the ternary com-
, and GTP, the rate-limiting step in
a
translation initiation, and abrogates ribosomal synthesis of growth
promoting proteins such as cyclin A, E, and D1 leading to cell
growth arrest in early G1.5,3b
HN
As an ongoing program aimed at discovery and development of
effective antiproliferative agents, a study was initiated to assess
clotrimazole 1 (Fig. 1) and its analogs for anticancer activity based
on a novel mechanism of action.3
N
N
Cl
Cl
Clotrimazole (CLT) 1, a drug marketed to fight fungal infections,
inhibits cell growth and proliferation by blocking the cell cycle in
G0–G1 through Ca2+ store-mediated inhibition of translation initia-
tion.4 In particular, CLT reduces synthesis and expression of G1 cyc-
lins and thereby inhibits associated cyclin-dependent kinase
activity, which is required for progression into S phase.4 CLT
induces the release of Ca2+ from intracellular stores in the endo-
plasmic reticulum, blocks the influx of extracellular Ca2+ through
1
2
N
H
N
HN
3
[b,f]dibenzazepine
4
[b,e]dibenzazepine
5
[b,d]dibenzazepine
* Corresponding author.
Figure 1. Structure of Clotrimazole1 and different scaffolds of dibenzazepine
E-mail address: jlightfoot@lorusthera.com (J. Lightfoot).
compounds.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.001

R. A. Al-Qawasmeh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 104–107
105
Table 1
Previous studies on CLT concluded that the antiproliferative
Various dibenzazepine compounds, synthesized in this study.
activity depends on the triaryl methane moiety and in particular
the ‘propeller’ type configuration around the methane.3a In a
continued effort aimed at generation of more active compounds
with reduced toxicity, compounds were synthesized based on the
11-aryl-[b,e]-dibenzazepine 2 scaffold, resulting in a series of cyto-
static antiproliferative agents. Herein, we describe the synthesis
and in vitro antiproliferative activity of a series of the 11-aryl-
substituted [b,e]-dibenzazepine compounds and their potential
for use as anticancer agents. Given that the 11-aryl-[b,e]-dib-
enzazepine scaffold 2 and CLT 1 (Fig. 1) have a similar configura-
tion at the central carbon C-11, while lacking the imidazole
moiety associated with the known hepato-toxicity of CLT 1, we
hypothesized that the new compounds would have similar activity
with lower toxicity.
The dibenzazepine scaffold has been constructed as shown in
Scheme 1.6 The synthesis starts with benzaldehyde 6 and aniline,
where reductive amination of aniline and the corresponding alde-
hyde gave the desired amino derivative 7 in a quantitative yield.
The alkylation of secondary aniline was selectively achieved
through the intermediate anilinodichloboranes derivatives.7 The
reaction of this intermediate, which formed in situ, with benzalde-
hyde derivative in the presence of tertiary amine yielded the sec-
ondary aniline derivative 8 in 70% yield.8 Subsequently, the
compounds were cyclized using concentrated sulfuric acid in
dichloromethane at room temperature. The 11-substituted dib-
enzazepine system 2 was generated as a racemic mixture, which
was used for further reactions. The final compounds were prepared
through a standard method for alkylation and acylation of the aze-
pine ring. Table 1 shows the compounds that were prepared for
this study.
Compound
R1
R2
R3
R4a
R5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Cl
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
CH3
CO2Me
CO2Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Bz
H
3-OMe3Bz
3-OMe3Bz
Bz
Bz
Bn
4-NH2Bz
4-NHMe3Bz
4-NMe2Bz
4-NO2Bz
4-OMeBz
CONH(4-NO2Ph)
4-NO2Bz
4-NO2Bz
Cl
Cl
H
Cl
Cl
H
H
H
Cl
Cl
Cl
Cl
OMe
a
Bz is COPh, Bn is CH2Ph.
2 was 1.2 lM compared to 5.1 l
M for CLT 1.3 SRB assays performed
with different cell lines demonstrated a broad range of activity for
these compounds. Table 2 shows the IC50 values in SRB assays of
the synthesized compound tested against up to a total of 9 tumor
cell lines. As shown in Table 2, compounds 9, 22, 23, 25, and 26
have a submicromolar activity against different cell lines, while
the rest of the synthesized compounds show an activity in the
micromolar range.
It has been demonstrated that the antiproliferative activity of
CLT 1 depends, in large part, on the conformation of the triaryl
methane moiety. This led to the hypothesis that the similar triaryl
structure of the dibenzazepine at position 11 would have antipro-
liferative activity but decreased systemic toxicity compared to CLT
due to the absence of the imidazole moiety of 1. A sulforhodamine
B (SRB) assay,9 using human colon adenocarcinoma cells (HT-29),
was performed to test the antiproliferative activity of compound
2, the core of the designed compounds. The IC50 value of compound
Compounds 9, 25, and 26 have been screened in the NCI 60 cell
line assay for antitumor activity.10 The results demonstrate prom-
ising activity with a unique spectrum of activity against the panel
of cell lines. The NCI COMPARE algorithm11 suggests that this class
of compounds has distinguishing activity that may indicate a novel
mechanism of action and subsequently novel anticancer activity.
To further investigate the effect of this class of compounds, the
effect of compound 9 (chosen as a representative of the com-
pounds with submicromolar activity against the panel of cell lines
used) was tested on cell cycle progression. DNA content analysis of
cultured human colon carcinoma cells (HT-29) was determined
O
H
N
HN
a
b, c
OH
Cl
after treatment with or without compound 912 at 25 and 50
lM
6
7
8
(Fig. 2). When cultures were treated with vehicle alone (DMSO)
in complete medium for 16 h, cell cycle progression was evident
with 37.91% and 13.78% at S and G2–M phases, respectively (A).
Under identical conditions, cell cycle progression was dramatically
blocked in G0–G1 when cells were treated with compound 9. In the
NO2
d
presence of 25
phase (B; 63.68%) and consequently fewer cells progressed to S
phase (B; 25.79%). Higher concentrations of compound 9 (50 M)
lM of compound 9, fewer cells exited out of G0–G1
O
HN
N
e
l
resulted in a dose dependent increase in the number of cells
remaining in G0–G1 phase (C; 69.22%). As expected, there was a
concomitant decrease in the number of cells in S phase. There
was no indication that treatment with compound 9 induced apop-
tosis, as there was no increase in the sub-G1 population of cells.
In this study, the dibenzazepine compounds were prepared as a
racemate. To see whether there is a differential effect for the pure
enantiomers and whether it is worth separating the enantiomers
forfurtherdevelopmentofthisclassofcompound, bothenantiomers
Cl
Cl
9
2
Scheme 1. Reagents and conditions: (a) aniline, Et3N, NaBH4, MeOH; (b) BCl3,
toluene, À10 C; (c) 2-chlorobenzaldehyde, Et3N; (d) H2SO4, DCM, rt, 2 h; (e) p-
nitrobenzoic chloride, Et3N, DCM.

106
R. A. Al-Qawasmeh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 104–107
Table 2
IC50 (l
M) values of the synthesized compounds.a
Compound
A549
HT29
MMRU
MCF7
HepG2
U118MG
Hela
HTB 174
MDA-MB-231
2
9
5.4
0.8
11.4
>10
—
>10
>5
3.7
2.5
>5
>5
3.7
2
2.1
—
1.1
>5
1.2
0.3
9.7
>10
5
>10
1.7
1.7
0.6
2.6
>5
1.7
1.1
1.0
0.7
0.4
4.8
0.6
0.2
—
2.7
0.6
1.7
—
4.2
—
4.5
3.4
1.5
4.2
4.3
1.1
1.7
1.7
—
0.5
2.4
1.3
0.7
0.9
0.3
—
—
3.6
—
2.6
1.2
0.8
3.4
4.5
1.5
1.0
1.0
0.9
0.4
4.5
0.9
0.4
1.4
0.5
—
—
—
—
—
2.5
1.3
—
1.1
0.3
—
—
—
—
0.1
—
—
—
—
3.6
—
—
2.6
—
—
—
—
0.7
0.3
4.0
0.5
0.2
1.3
0.5
—
—
—
0.2
4.6
10
—
>10
2.5
—
—
2.7
—
—
—
—
0.5
0.2
3.1
0.5
0.2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
—
—
—
—
1.4
0.9
—
>5
0.5
0.4
0.9
0.6
0.2
—
1.5
1.2
—
>5
1.2
1.3
1.7
—
—
—
0.9
0.4
—
1.6
1.8
2.6
0.9
0.4
—
1.5
0.6
0.8
0.4
0.5
0.2
a
The assay was done using SRB assay method, (—), not tested.
Figure 2. DNA content analysis of HT-29 colon cancer cells by flow cytometry. HT-29 cells were treated with various concentrations (25 and 50
l
M) of compound 9 for 16 h.
Cells were stained with propidium iodide and analyzed by flow cytometry. The percentage of cells at different stages of the cell cycle (G0–G1, S, G2–M) is indicated below.
Table 3
of compound 9 were separated (enantiomer 9a and enantiomer 9b;
the enantiomeric identity was not characterized). Both enantiomers
have been screened in the NCI screening assay, the results did not
show any difference of either enantiomer to that of the racemate
compound 9. The average logGI50 values of 9a and 9b were À5.36
and À5.18, respectively, while that of 9 was À5.71. To further
confirm this finding, the cell cycle effect of 9a and 9b on the H-460
Effect of 9a, 9b, and 9 on cell cycle progression.a
Treatment
DMSO
G0–G1
G2–M
S
40.92
71.06
74.96
73.95
11.55
5.09
5.76
5.24
47.53
23.86
19.29
20.81
9a (50
9b (50
9
l
l
M)
M)
a
cell line was compared to that of 9 at a concentration of 50 lM fol-
DNA content analysis of H-460 non-small cell lung cancer cells by flow
lowing serum-starvation for 72 h. As shown in Table 3, no significant
difference was observed between the two enantiomers and the race-
mate. The extent of the arrest was similar to that of the racemate (%
of G0–G1 population in the range of ꢀ70% for all compounds com-
pared to ꢀ40% for the vehicle control). These results confirmed that
there is no significant difference between the two enantiomers and
their ability to cause cell cycle arrest.
In conclusion, a series of 11-aryl-substituted [b,e]-dibenzazepine
compounds were synthesized and the in vitro antiproliferative
activity investigated. The synthetic dibenzazepine compounds were
effective in growth inhibition at submicromolar to micromolar
cytometry. H-460 cells were serum-starved for 72 h, and then treated with 50 lM
of 9a, 9b, and 9 for 16 h. Cells were stained with propidium iodide and analyzed by
flow cytometry. Percentage of cells under different stages of cell cycles (G0–G1, S,
G2–M) is shown.
concentrations. Compound 9, as a representative compound from
those in the series with submicromolar activity, was shown to spe-
cifically arrest the cell cycle in G0–G1 phase. Pure enantiomers of 9
did not show any advantage over the racemate, in antiproliferative
activity or in the ability to arrest the cell cycle at G0–G1 stage. These
results together with those from an ongoing investigation into the

R. A. Al-Qawasmeh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 104–107
107
of compound 2 and 8. Boron trichloride (1 equiv, 1 M in Heptane) was added
over 10 min via a canulla to stirred solution of 7 (1 mmol) at 0 °C in dry Toluene
under nitrogen atmosphere. When the addition was completed, the ice water
bath was replaced by an oil bath, and the reaction mixture was heated to reflux
for 30 min. The heating bath was replaced by an ice bath to bring the internal
molecular mechanism of action will form the basis for further devel-
opment of this class of compounds as anticancer therapeutics.
Acknowledgment
temperature to 5 °C.
A solution of 2-chlorobenzaldehyde (1.1 equiv) and
triethylamine (2 equiv) in toluene (6 ml) was added over 30 min. The resulting
slurry was stirred at room temperature for 1 h. Deionized water (20 ml)
followed by 10% aqueous sodium hydroxide (100 ml) was added, the aqueous
layer was adjusted to pH 8. The organic layer was isolated and the aqueous
layer was extracted with toluene (2Â 10 ml). The combined organic layers were
washed with water, dried and concentrated to dryness by rotary evaporation.
Column chromatography separation gave 70% yield. NMR (300 MHz, CDCl3)
7.52 (1H, dd) 7.38 (1H, m), 7.1–7.4 (6H, m), 6.8 (2H, dm), 6.65 (2H, m), 6.1 (1H,
bs), 5.1 (1H, bs), 4.6 (2H, bs). (2-chloro)-[2-(benzylamino)-phenyl]-methan-1-
ol 8, (1 mmol) was suspended in 5 ml DCM. To the resulting slurry, sulfuric acid
(95–98%, 5.5 equiv) was added over 5 min the reaction mixture turned from
slurry to a solution. The viscous solution was stirred at room temperature for
2.5 h until TLC showed the consumption of the starting material. DCM (125 ml)
was added and the biphasic reaction mixture was cooled to 10 °C. Ten percent
aqueous sodium hydroxide was added to adjust the aqueous layer to pH 10.
The organic layer was isolated and the aqueous layer was extracted with DCM
(2Â 60). The organic layer was combined and evaporated to dryness; the
resultant material was subjected to chromatographic separation to give pure 2,
NMR (300 MHz, CDCl3) 7.62 (bd, 1H), 7.48 (bd, 1H), 7.32–7.04 (m, 8H), 6.68 (bt,
1H), 6.58 (bd, 1H), 5.36 (bs,1H), 4.64 (d,1H), 4.25 (bs,1H), 3.65 (bd,1H).
9. Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.;
Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589.
10. Based on the results of the 60-cell line assay performed by NCI, these three
compounds have been referred to the biological committee in order to study
their spectrum of activity as compounds with novel activity. This resulted in a
recommendation by the NCI to further evaluate these compounds in preclinical
stages.
We thank Joe Covey at the National Cancer Institute, Maryland,
U.S.A., for his support and constructive discussions throughout the
development of this class of compound.
References and notes
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containing 0.1%DMSO, 10, 25, and 50 lM 9. Cells were harvested 16 h
following treatment, and washed with cold PBS twice. Cells were fixed in
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for 4 min at 4 °C, washed with cold PBS containing 2% FBS twice, treated with
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8. All reactions were performed under dry conditions and the products gave a
satisfactory physico-chemical analysis, herein the procedure for the synthesis
3 mg/ml ribonuclease (Sigma Chemical Co) and 50 lg/ml propidium iodide (PI)
(Sigma Chemical Co) for 30 min at 37 °C. Flow cytometry analysis was
performed on the Becton Dickinson fluorescence-activated sorter FACS Scan
by using the Becton Dickinson Cell Quest program. Data were evaluated using
Modfit software (Verity Software House, Topsham, ME).