Imidazopyridine-fused [1,3]-diazepinones: Synthesis and antiproliferative activity

Article abstract of DOI:10.1016/j.ejmech.2014.01.044

A series of 15 pyrido-imidazo-1,3-diazepin-5-ones and pyrido-1,3-diazepine- 2,5-diones were synthesized and their anticancer activities were evaluated. Among tested compounds on a cell lines panel, compound 6a presents the best growth inhibition activity on 21 cell lines with a cytotoxic effect on MDA-MB-435 melanoma cells. This compound led to deep cell morphological changes and revealed to be an inhibitor of the Hepatocyte progenitor kinase-like kinase (HGK), which is known to be implicated in the migration, adhesion and invasion of various tumor cells.

Full text of DOI:10.1016/j.ejmech.2014.01.044

European Journal of Medicinal Chemistry 75 (2014) 382e390
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Imidazopyridine-fused [1,3]-diazepinones: Synthesis and
antiproliferative activity
,
,
Audrey Gallud a 1, Ophélie Vaillant a 1, Ludovic T. Maillard a, Dominique P. Arama a,
Joëlle Dubois b, Marie Maynadier a, Vincent Lisowski a, Marcel Garcia a, Jean Martinez a,
Nicolas Masurier a
,
*
a Institut des Biomolécules Max Mousseron, UMR 5247, CNRS, Universités Montpellier I et II, UFR des Sciences Pharmaceutiques et Biologiques, 15 Avenue
Charles Flahault, 34093 Montpellier Cedex 5, France
b Institut de Chimie des Substances Naturelles, UPR 2301 CNRS, Centre de Recherche de Gif, Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 15 pyrido-imidazo-1,3-diazepin-5-ones and pyrido-1,3-diazepine-2,5-diones were synthe-
sized and their anticancer activities were evaluated. Among tested compounds on a cell lines panel,
compound 6a presents the best growth inhibition activity on 21 cell lines with a cytotoxic effect on MDA-
MB-435 melanoma cells. This compound led to deep cell morphological changes and revealed to be an
inhibitor of the Hepatocyte progenitor kinase-like kinase (HGK), which is known to be implicated in the
migration, adhesion and invasion of various tumor cells.
Received 20 December 2013
Received in revised form
17 January 2014
Accepted 18 January 2014
Available online 30 January 2014
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Imidazo[1,2-a]pyridine
Polyfused heterocycles
Diazepinones
Antitumor activity
HGK inhibitors
1. Introduction
activities. In particular, polyfused azepine derivatives led to the
discovery of compounds with anticancer potency. Pyrrolo[1,2-c]
Cancer is a notably complex, widespread and lethal disease ac-
counting for 7.6 million deaths (around 13% of all deaths) in 2008,
that is projected to continue rising, with an estimated 13.1 million
deaths in 2030 [1]. Several lines of evidence support the view that
chemotherapy has become one of the most significant treatment
modalities in cancer management. Classical chemotherapy using
small molecules or bioactive natural products is still the mainstay
of chemotherapy, whereby the major cellular targets are DNA,
tubulin [2,3], along with various kinases [4]. However, the absence
of selectivity and acute toxicity of many antitumor agents beside
the development of cellular drug resistance have been the major
drawback in their usage, prompting the search for new more se-
lective, efficient, and safe antitumor agents [5].
[1,3]benzodiazepines (Fig. 1) revealed to be cytotoxic for Jurkat and
neuronal cells, with induction of DNA cleavage [6]. Hymenialdisine,
a marine natural product, which also has a pyrrolo-azepine core, is
a potent inhibitor of cyclin dependent kinases (CDK) [7]. Paullone
derivatives, which are based on the indolo[3,2-d][1]benzazepinone
system, are CDK inhibitors with very potent antitumor activity
[8,9]. Kenpaullone, alsterpaullone and their analogs were also re-
ported to be inhibitors of glycogen synthase kinase-3 (GSK3) [10e
17] and of Hepatocyte progenitor kinase-like kinase (HGK) [18].
Moreover, some structural isomers of paullones, based on the
indolo[2,3-d][2]benzazepinone framework, were reported to
inhibit tubulin polymerization [19].
Recently, we reported an efficient approach to access polyfused
diazepinone derivatives, based on the imidazo[1,2-a]pyridine (IP)
nucleus, an aza analog of indole [20,21]. In continuation to this
study and since some polyfused IP derivatives have been described
to possess antitumor activities [22,23], we decided to evaluate the
pharmacological potency of IP-fused diazepinones. We reported
herein the synthesis of some pyrido-imidazo [1,3]diazepin-5-ones
Azepine core is considered as a privileged structure to access to
active compounds displaying a wide range of pharmacological
* Corresponding author. Tel.: þ33 4 11 75 96 42; fax: þ33 4 11 75 96 41.
E-mail address: nicolas.masurier@univ-montp1.fr (N. Masurier).
1
These authors contributed equally to the work.
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.044

A. Gallud et al. / European Journal of Medicinal Chemistry 75 (2014) 382e390
383
Scheme 1. Synthesis of pyrido-imidazodiazepinones 4e10 and pyrido-
imidazodiazepinediones 11. Abbreviations used: BocAAOH ¼ Boc-Ala-OH, Boc-DAla-
OH, Boc-Phe-OH or Boc-Pro-OH; EDCI: N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide hydrochloride; HOBt: hydroxybenzotriazole; TEA: triethylamine; CDI:
1,10-carbonyldiimidazole; ACN: acetonitrile. R1, R2 and R3 substituents are referenced in
Table 1.
Fig. 1. Representative examples of azepine derivatives with antitumor potency.
2.2. Antiproliferative activities on cancer cell lines panel
2.2.1. Sulforhodamine B (SRB) assay
and pyrido-imidazo [1,3]diazepine-2,5-diones derivatives and their
evaluation for growth inhibitory activities on cancer cell lines.
Diazepine derivatives 4e11 were evaluated by the NCI towards a
panel of sixty cancer cell lines corresponding to nine different
cancer types, i.e., leukemia, melanoma, as well as lung, colon, central
nervous system, ovarian, renal, prostate and breast cancers. The
2. Results and discussion
compounds were tested at a single concentration (10
mM) and
incubated for 48 h on the cell cultures. Cellular densities were
determined by Sulforhodamine B (SRB) assay, which is based on the
cellular protein content. The SRB test is a direct index of cell density
since the amount of dye incorporated by the cells varies concomi-
tantly with the increase (or the decrease) of the total protein
biomass [28]. The cell growth was evaluated spectrophotometri-
cally after 48 h exposure and reported as the percent of untreated
control cells (Table 1 and SI). Pyrido-imidazodiazepinediones (11a,
11c and 11d), as well as pyrido-imidazodiazepinones (4d, 5c, 5d, 8c
and 10c) appeared inactive whatever the cell line considered. 5a
showed a moderate activity on 4 cell lines (growth < 50%). The best
results were obtained with the bromo analog 6a which inhibited cell
growth (growth < 50%) in 21 cell lines. 6a was particularly active on
MDA-MB-435 and MDA-MB-468 cells with a cytotoxic effect at
2.1. Chemistry
Pyrido-imidazodiazepine derivatives 4e11 were synthesized
according to our previous published methodologies [20,21].
Briefly, 2-amino-imidazo[1,2-a]pyridine 1 [24,25] was selectively
acylated at C-3 position, using four different N-Boc protected
amino acids: Boc-Ala-OH, Boc-D-Ala-OH, Boc-Phe-OH or Boc-Pro-
OH. IP is known to be an electron-rich aromatic ring that could
lead to electrophilic aromatic substitutions at C-3 [26,27].
Therefore, a simple activation at room temperature of a N-Boc
protected amino-acid by EDCI/HOBt in dichloromethane (DCM)
led to the C-3 acylated IP derivatives 2aed in 62e88% isolated
yields (Scheme 1). In accordance with previous results, N-addi-
tion side-products were only detected as traces by LCeMS [20,21].
After Boc removal by HCl treatment, the resulting hydrochloride
salts were neutralized with aqueous ammonia and 2-amino-3-
acyl-imidazo[1,2-a]pyridines 3aed were extracted with chloro-
form and used in the next step without further purification. Di-
10 mM (0% growth). Interestingly, its enantiomer 6b was inactive on
all cell lines suggesting a very specific mechanism of action.
Compound 6a was selected for an advanced assay on the NCI-60
cancer cell lines panel and was tested in a five-dose testing mode, to
determine IC50 values. IC50 for 40 cell lines were lower than 10
(Fig. 2). The best activities were obtained on glioblastoma SNB-75
cells (IC50 ¼ 0.7 M), non-small-cell lung cancer HOP-92 cells
(IC50 ¼ 1.1 M) and melanoma MDA-MB-435 cells (IC50 ¼ 1.3 M).
mM
amines
benzaldehydes in chloroform at reflux overnight. The aminal in-
termediates were then oxidized without isolation, using
3 were then successively reacted with a set of
m
a
m
m
mixture of lead tetraacetate and iodine, to lead to pyrido-
imidazodiazepinones 4e10 in 23e71% isolated yields (Scheme 1,
Table 1). On the other hand, intramolecular carbonylation of di-
2.2.2. MTT assay
amines
3
using carbonyldiimidazole (CDI) led to pyrido-
To confirm these first results, doseeresponse and time-course
experiments were carried out on the most active compound 6a
imidazodiazepinediones 11 in 78e99% isolated yields.

384
A. Gallud et al. / European Journal of Medicinal Chemistry 75 (2014) 382e390
Table 1
Evaluation of cell growth at a single-dose (10
mM) according to the NCI-60 cancer cell lines screening (sulforhodamine B test).
Cell growth (%)a
Leukemia
NSCLCb Colon
cancer
CNS
cancer
Melanoma
Ovarian
cancer
Renal
cancer cancer
Prostate Breast cancer
R1
R2 R3
K562 SR
HOP-92 HCT-116 HT29 SNB-75 MDA-MB-435 SK-MEL-5 OVCAR-3 UO-31 PC-3
MCF-7 MDA-MB-438
4a
5a
6a
(S)-Me
(S)-Me
(S)-Me
H
H
H
H
4-Me
4-Br
108.1 105.7 75.5
98.4
91.1
29.2
112.6 82.8
91.6
25.7
ꢀ13.0
(ꢀ15 ꢁ 3)d
94.4
99.2
81.7
31.9
102.8
90.4
17.3
82.5
45.9
82.5
82.5
72.9
63.2
67.2
71.2
20.9
99.9
91.1
ꢀ0.5
47.1
24.3
42.2
30.0
66.3
59.5
82.8
17.5
68.0
50.1
7a
(S)-Me
(S)-Me
(S)-Me
(R)-Me
(S)-Bn
(S)-Bn
(S)-Bn
(S)
H
H
H
H
H
H
H
4-NMe2 82.2
86.6
80.4
NT
33.0
72.5
NT
100.8
95.9
NT
90.2
94.3
87.9
86.9
97.8
100.9 70.0
95.4
100.2
NT
94.2
86.5
103.7
102.8
99.7
101.3
98.3
NT
95.9
99.3
110.3
107.7
105.3
53.7
55.6
NT
51.5
77.0
83.5
80.6
88.6
83.0
87.3
NT
83.8
80.7
91.8
89.4
92.6
66.0
72.0
NT
65.1
76.1
90.1
84.9
93.0
72.3
76.5
NT
101.9
84.5
98.5
103.6
100.8
8a
4-OMe
4-NO2
4-Br
4-Me
4-OMe
2-Me
H
86.7
98.5
NT
81.9
NT
90.2
9ad
6b
5c
NTc
99 ꢁ 4
84.8
87.9
86.0
77.7
89.7
104.8 95.6
66.6
88.3
104.3 89.3
113.6 80.1
95.4
46.8
51.4
98.8
100.6
8c
10c
4d
117
77.4
113.4 122.4 90.4
123.7 118.4 82.7
115.4 70.8
114.1 88.3
[(CH2)3]c
(S)
5d
4-Me
104.1 88.5
NT NT
105.5 107.4 86.5
123.7 126.0 86.2
78.3
NT
94.0
101.3 90.0
91.1
92.5
96.7
80.3
81.9
89.0
94.85
[(CH2)3]c
11ad (S)-Me
H
H
e
e
e
NT
92.3
89.0
NT
NT
86 ꢁ 3
96.4
94.9
NT
97.2
99.5
NT
100.7
108.0
NT
90.2
95.1
NT
95.2
93.8
NT
95.7
99.9
NT
98.4
93.5
11c
11d (S)
[(CH2)3]c
(S)-Bn
106.0 79.1
105.7 96.5
a
Values for representative cell lines of each cancer type represent % growth compared to the no-drug control, and relative to the time zero number of cells (for all results on
NCI-60 human cancer cell lines panel, see SI).
b
NSCLC: non-small-cell lung cancer.
NT: not tested.
Growth percentages using the MTT assay.
c
d
Fig. 3. Effects of compounds 4a and 6a on melanoma cancer cell lines. (A, B) Cells were
treated with a concentration range of compounds 4a and 6a for 48 h. Cell growth was
determined by MTT assay. (C) Determination of IC50 values on MDA-MB-435 mela-
noma cells of compound 6a by MTT (IC50 ¼ 1.7
Time course with compound 6a on MDA-MB-435 cells. Values are mean
percentage ꢁ standard deviation of three independent experiments.
m
M) or SRB assay (IC50 ¼ 1.3
mM). (D)
Fig. 2. IC50 values of compound 6a on the 40 most sensitive NCI-60 cancer cell lines
(for all data, see Table S2, SI).
5 mM

A. Gallud et al. / European Journal of Medicinal Chemistry 75 (2014) 382e390
385
and its analog without bromine 4a (used as control) using a
different method, the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-
biphenyl tetrazolium bromide) assay. The reduction of MTT by
the mitochondrial succinic dehydrogenase is considered as repre-
sentative of the cell viability [29].
acquired death. These data report that compound 6a possess both
antiproliferative and cytotoxic activities on MDA-MD-435 cells.
2.3. Morphological changes of cells
Considering NCI screening results, the activities of the two
selected compounds were evaluated on three melanoma cell lines;
MDA-MB-435, CAL-1 and SK-MEL-2. Compound 4a induced a 20e
Reorganization and alterations in actin dynamics is a well-
known consequence of signaling. A range of studies has revealed
that actin also plays a key role in programmed cell death regulation
[30]. In the doseeresponse experiment, modification in cell
appearance was highlighted. Dispersed and rounded MDA-MB-435
cells were observed 20 h post-treatment with compound 6a
(Fig. 5A). To evaluate morphological changes, fixed and actin-
immunostained MDA-MB-435 cells were imaged by confocal mi-
croscopy (Fig. 5B). Control cells as well as 4a treated cells (20 h,
30% decrease of all cell growth from 1 to 10
observed with the SRB assay, compound 6a led to a total growth
inhibition of MDA-MB-435 cells at 10 M (Table 1 and Fig. 3B).
Interesting activities were also observed on CAL-1 and SK-MEL-2
cell lines, with only 12% and 32% growth, respectively at 10
concentration of 6a (Fig. 3B). These results confirm a significant
antiproliferative activity of compound 6a. doseeresponse
experiment, indicated an IC50 of 1.7 M (Fig. 3C) on MDA-MB-435
cells for compound 6a, quite similar to that reported using the
SRB assay (IC50 1.3 M).
6a activity was also evaluated on MDA-MB-435 at 5
mM (Fig. 3A). As
m
mM
A
5
m
M concentration) were spread and the tensile forces of actin
stress fibers stretched the cell-adhesive phenotype. In contrast,
cells treated with compound 6a (20 h, 5 M concentration) showed
m
m
m
the most recognizable actin-morphological features. Arrows point
to cell cytoplasm showing alteration induced by actin fibers ag-
gregation. Arrowheads show the nuclei fragmentation and chro-
matin condensation. These results suggest that compound 6a was
involved in diffuse distribution of non-polymerized actin, which
leads to a disorganization of the actin cytoskeleton and altered
malignant melanoma cell morphology.
mM fixed
concentration after 8, 16, 24 or 48 h incubation (Fig. 3D). Decrease
of the percentage of living cells is time-dependent, with 35% after
8 h incubation and 10% after 24 h.
2.2.3. DNA cell cycle analysis
2.4. Mechanism of action study
Cell cycle pattern changes of MDA-MB-435 cells were investi-
gated using fluorescence-activity cell sorting (FACS) (Fig. 4 and
Fig. S1, SI). In this experiment MDA-MB-435 cells were treated with
Although specific inhibitory response of a single cell line may be
relatively uninformative, the pattern of response of a large panel of
different cancer cell lines can be used to classify a compound ac-
cording to the probability of sharing common mechanisms [31,32].
The NCI’s COMPARE algorithm was used to identify a list of com-
pounds having a similar pattern of cellular sensitivity and resis-
tance than compound 6a. However, the results of COMPARE
analysis at the IC50 level for compound 6a showed a poor correla-
tion (r ꢂ 0.5) with various anticancer agents (data not shown),
suggesting that the mode of action of compound 6a might differ
significantly from that of most of the 175 known anticancer agents
in the NCI database.
or without 10 mM compounds 4a and 6a for 16 h or 48 h. The cell
cycle phase distribution was quantified from three independent
sets of measurements. Control and compound 4a treated cells
showed a similar pattern of distribution. After 48 h, compound 6a
induced cell cycle arrest in S phase, (30.8% compared to 22.8% in
control) and increased apoptosis in subG1 phase (8.6% compared to
0.1% in control). Such a synthetic agent that alters the cell cycle is of
particular interest, since cell cycle regulation is the basic mecha-
nism underlying cell fate, i.e., proliferation, differentiation or
As pyrido-imidazodiazepinones show some structural similar-
ities with paullone derivatives (kinases inhibitors) and indolo[2,3-
d][2]benzazepinones (tubulin polymerization inhibitors), we
turned our attention on these two possible mechanisms of action
for compound 6a. We first considered tubulin as a potential target.
Eight pyrido-imidazodiazepinone derivatives, including compound
6a, were evaluated for tubulin assembly inhibition capability at
high concentration (100 mM). However, for all tested compounds,
no notable activity was found (Table 2). Only compound 7a showed
a weak inhibitory effect (55%).
Since the anti-proliferative activity of compound 6a was not
associated with tubulin polymerization, we investigated whether
this compound displays kinase inhibitory activity. Compound 6a
activity was evaluated in duplicate at the single dose of 10 mM on a
panel of 49 representative kinases. The panel covered the different
known kinase families including paullone targeted kinases (CDK,
GSK3 and HGK), tyrosine kinase receptors, serineethreonine kin-
ases. Results are summarized in Fig. 6. Among the 49 tested ki-
nases, compound 6a did not inhibit CDK and GSK3 as paullone
derivatives. However, 6a inhibited the Hepatocyte progenitor
kinase-like kinase (HGK): 50% at 10 mM (Fig. 6). HGK, also called
MAP4K4, is a serineethreonine kinase, broadly expressed in human
tumor cells [33e35], which activates the c-Jun N-terminal kinase
(JNK) pathway playing a crucial role in stress responses, cell pro-
liferation, apoptosis and oncogenesis [36]. It has been demon-
strated recently that HGK is implicated in the migration, adhesion
Fig. 4. Cell cycle analysis. MDA-MB-435 cells were treated with 5 mM 6a, 4a or DMSO
(control) for 16 or 48 h. Panels represent distribution of cells (%) in subG1-, G0/G1-, S-,
G2/M-phases of the cell cycle.

386
A. Gallud et al. / European Journal of Medicinal Chemistry 75 (2014) 382e390
Fig. 5. Morphological changes. (A) Phase contrast microscopy of MDA-MB-435 cells. Cells were imaged 20 h post-treatment with or without 5
mM of 4a or 6a (scale bar 100 mm). (B)
Confocal microscopy of actin localization in fixed MDA-MB-435 cells (scale bar 30 m). Cells were incubated 20 h with or without 5 M of 6a or 4a for 20 h. Cells were co-stained
m
m
with a nuclear marker (Hoechst). AlexaFluor568 actin-marker and nuclei were excited at 543 and 405 nm, and visualized at 620 (red) and 450 nm (blue) respectively. Arrows
pointed to actin aggregation and arrowheads showed nuclei alteration.
and invasion of multiple carcinoma cell lines and that the effect on
migration is mediated through the JNK pathway [37,38]. In partic-
ular, HGK expression studies on the NCI-60 cancer cell lines panel
showed that HGK mRNA was expressed at high levels in 40 of the
60 tumor cell lines, including lung adenocarcinoma HOP-92 and
glioblastoma SNB-75 [38,39]. Several groups also reported that
down-regulation of HGK by small interfering RNAs led to significant
growth reduction of cancer cells [37,40,41]. Moreover, it is known
that ERM proteins (ezrin, radixin and moesin) are HGK substrates
[42]. These proteins regulate cell morphology by cross-linking actin
filaments to the plasma membrane [43]. Therefore, the growth
inhibition induced by 6a and morphological modifications
observed when cells are treated with this compound (Fig. 5), could
be mediated, at least in part, via HGK inhibition. However, the ex-
istence of another target for 6a can’t be excluded at this time and
further experiments have to be carried out to confirm this
hypothesis.
3. Conclusion
A series of 15 polyfused diazepines was synthesized based on a
key C-acylation FriedeleCraft reaction and were evaluated for their
anticancer activity. Compounds were tested at a single dose of
Table 2
Percentage inhibition of tubulin polymerization by 100
derivatives.
mM 4-methyl diazepinone
10
mM at the NCI on a 60 cell lines panel, using SRB assay. Com-
pound 6a was active on 40 different cell lines (IC50 < 10
mM) and
Compound
4a
5a
6a
6b
7a
8a
9a
11a
displayed cytotoxic effects on MDA-MB-435 (cell cycle arrest in S
phase). This result was confirmed using an MTT assay, with an IC50
% of inhibition
7
16
8
26
55
20
30
8

A. Gallud et al. / European Journal of Medicinal Chemistry 75 (2014) 382e390
387
per million relative to TMS or relative to the solvent [1H:
d
(CDCl3) ¼ 7.26 ppm; 13C:
d
(CDCl3) ¼ 77.16 ppm]. The following
abbreviations are used to designate the signal multiplicities: s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br
(broad). Analytical thin-layer chromatography (TLC) was per-
formed using aluminum-backed silica gel plates coated with a
0.2 mm thickness of aluminum oxide 60 F254, neutral. LCeMS
spectra (ESI) were recorded on an HPLC using an analytical Chro-
molith Speed Rod RP-C18 185 Pm column (50 ꢃ 4.6 mm, 5 mm);
solvent A, H2O/HCOOH 0.1%; solvent B, CH3CN/HCOOH 0.1%;
gradient, 0% solvent B to 100% solvent B in solvent A in 3 min; flow
rate, 3.0 mL/min. High-resolution mass spectrometric analyses
were performed with a time-of-flight mass spectrometer fitted
with an electrospray ionization source. All measurements were
performed in the positive ion mode. Melting points (mp) are un-
corrected and were recorded on
apparatus.
a capillary melting point
Compounds 4a, 6a, 9a, 5c, 8c, 10c, 4d, 5d and 11 were synthe-
sized according to our previous reported procedures and the
spectral characteristics were in agreement with the published data
[20,21].
4.2. Synthesis of (2R)-2-Boc-amino-1-(2-aminoimidazo[1,2-a]
pyridin-3-yl)-propan-1-one (2b)
To a suspension of 0.5 g of trifluoro-N-imidazo[1,2-a]pyridin-2-
ylacetamide [20] (2.18 mmol) in 9 mL of aqueous 5 N sodium hy-
droxide solution was added 0.5 mL of THF. The solution was stirred
at 40 ꢄC for 2 h. The solution was extracted with dichloromethane
(3 ꢃ 20 mL). The organic layer was dried over Na2SO4, filtered, and
the solvent was removed in vacuo. The residue was dissolved in
20 mL of dichloromethane and 2.4 mmol of Boc-(
(1.1 equiv), 325 mg (2.4 mmol, 1.1 equiv) of HOBt, 460 mg
(2.4 mmol, 1.1 equiv) of EDCI and 334 L of triethylamine (243 mg,
D)-Ala-OH
m
2.4 mmol, 1.1 equiv) were added at 0 ꢄC. The solution was stirred at
rt for 4 h. The solution was then washed with saturated NaHCO3
solution (2 ꢃ 50 mL). The organic layer was dried over Na2SO4,
filtered, and the solvent was concentrated in vacuo. The residue
was purified by chromatography (Al2O3, DCM/EtOH 99/1 v/v) to
Fig. 6. Percentages of inhibition on a set of 49 representative kinases by 10
compound 6a.
mM of
of 1.7
mM for MDA-MB-435 cells, similar to the IC50 of 1.3 mM,
offer 2b.
measured in the SRB assay. Further evaluation showed that com-
pound 6a led to deep morphological changes on MDA-MB-435
cells, induced cell cycle arrest in S phase and increased apoptosis
in subG1. Evaluation of compound 6a activity on a 49 kinases panel,
covering the different known kinase families, seems to indicate that
6a acts as an HGK inhibitor. As HGK is a kinase implicated in cell
migration, adhesion and invasion of various tumor cells, its in-
hibitors can be considered as potential therapeutic targets to
overcome cancer progression [44]. Further modulations of pyrido-
imidazodiazepinone derivatives are in progress to access to more
potent HGK inhibitors.
20
White solid, m ¼ 451 mg, yield ¼ 68%, [
a
]
¼ þ70.6ꢄ (c 1.0,
D
CHCl3); other data were in agreement with those published for its S
enantiomer [20].
4.3. General procedure for synthesis of diazepinones
A solution of 100 mg of compound 2a or 2b (0.33 mmol) in 3 mL
of 12 N hydrochloric acid was stirred a room temperature for 1 h.
The solution was treated with 28% aqueous ammonia solution and
then extracted with chloroform (3 ꢃ 20 mL). The organic layer was
dried over Na2SO4, filtered, and the solvent was concentrated in
vacuo to lead compounds 3a or 3b, which were used in the next
step without further purification. To a solution of 3a or 3b in 2 mL
chloroform was added 1 equiv of the appropriate aldehyde. The
solution was stirred at 60 ꢄC for 6 h. After cooling, the mixture was
added to a solution of iodine (91 mg, 0.36 mmol, 1.1 equiv) in 4 mL
of chloroform. Finally, a solution of lead tetraacetate (161 mg,
0.36 mmol,1.1 equiv) in 6 mL of chloroform was added. The solution
was stirred at room temperature for 1e6 h (monitoring by TLC). The
solution was washed with 10% m/v sodium thiosulfate aqueous
solution (3 ꢃ 30 mL) and then with aqueous saturated sodium
hydrogen carbonate solution (2 ꢃ 30 mL). The organic layer was
dried over Na2SO4, filtered, and the solvent was evaporated in
vacuo. The residue was purified by chromatography on alumina,
eluted by DCM/EtOH 99/1 v/v.
4. Experimental
4.1. General
Commercially available reagents and solvents were used
without further purification. Reactions were monitored by HPLC
using an analytical Chromolith Speed Rod RP-C18 185 Pm column
(50 ꢃ 4.6 mm, 5 mm) using a flow rate of 3.0 mL/min, and gradients
of 100/0 to 0/100 eluents A/B over 5 min, in which eluents A ¼ H2O/
0.1% TFA and B ¼ CH3CN/0.1% TFA. Detection was at 214 nm using a
Photodiode Array detector. Retention times are reported as follows:
Tr (min). 1H and 13C NMR spectra were recorded at room temper-
ature in deuterated solvents. Chemical shifts (d) are given in parts

388
A. Gallud et al. / European Journal of Medicinal Chemistry 75 (2014) 382e390
4.3.1. (4S)-4-Methyl-2-(4-methylphenyl)-3,4-dihydro-5H-pyrido
4.4.2. MTT assay
[10,20:1,2]imidazo[4,5-d][1,3]diazepin-5-one (5a)
4.4.2.1. Cell culture conditions. Human melanoma (MDA-MB-435)
cells were cultured in a 1:1 mixture of Ham’s F12 and Dulbecco’s
Modified Eagle Medium (DMEM) supplemented with 10% foetal
Pale yellow solid, m ¼ 52 mg, yield ¼ 52%, mp: 137e139 ꢄC.
20
[a
]
¼ ꢀ5.7ꢄ (c 0.2, MeOH); 1H NMR (CDCl3, 300 MHz):
d ppm
D
1.61 (d, 1H, J ¼ 6.8 Hz), 2.31 (s, 3H), 4.10 (q, 1H, J ¼ 6.8 Hz), 6.94 (m,
calf serum (FCS), 50
100
g mLꢀ1 streptomycin. Melanoma cells (SK-MEL-2) were
maintained in RPMI-1640 Medium supplemented with 10% FCS,
100 IU penicillin and 100
g mLꢀ1 streptomycin. Human malignant
melanoma cells (CAL1) were cultured in DMEM supplemented with
2 mM -glutamine, 10% FCS, 200 IU penicillin and 200
g mLꢀ1
m
g mLꢀ1 gentamicin, 100 IU penicillin and
2H), 7.17 (d, 2H, J ¼ 8.4 Hz), 7.32 (dd, 1H, J ¼ 8.4, 7.5 Hz), 7.87 (d, 2H,
m
J ¼ 8.4 Hz), 9.47 (d, 1H, J ¼ 6.8 Hz); 13C NMR (CDCl3, 75 MHz):
d ppm
16.5, 21.5, 60.8, 112.5, 113.6, 115.5, 128.4, 129.4 (4C), 130.0, 132.2,
142.3, 145.9, 155.0, 156.3, 182.9; FT-IR: gmax (cmꢀ1): 739, 764, 825,
1253, 1335, 1421, 1465, 1523, 1624, 2926; HPLC, Tr ¼ 0.94 min; MS
(ESIþ): m/z 305.1 [M þ H]þ; HRMS calcd for C18H17N4O 305.1402,
found 305.1400.
m
L
m
streptomycin. All cell lines were allowed to grow at 37 ꢄC in an
atmosphere containing humidified air with 5% CO2. MDA-MB-435
and SK-MEL-2 cell lines were purchased from ATCC (American
Type Culture Collection, Manassas, VA). The CAL1 cell line was
generously gifted by Pr. P. Cuq.
4.3.2. (4S)-4-Methyl-2-(4-N,N-dimethylamino)-3,4-dihydro-5H-
pyrido[10,20:1,2]imidazo-[4,5-d][1,3]diazepin-5-one (7a)
Brown solid, m ¼ 53 mg, yield ¼ 48.5%, mp: 181 ꢄC dec.
20
[a
]
¼ ꢀ6.8ꢄ (c 0.25, MeOH); 1H NMR (CDCl3, 300 MHz):
d
ppm
4.4.2.2. Cytotoxicity evaluation. Cells were seeded into 96-well
D
1.57 (d, 3H, J ¼ 6.9 Hz), 2.96 (s, 6H), 4.08 (q, 1H, J ¼ 6.9 Hz), 6.62 (d,
2H, J ¼ 9.0 Hz), 6.96 (dd, 1H, J ¼ 7.7, 6.8 Hz), 7.28 (d, 1H, J ¼ 7.7 Hz),
7.40 (dd, 1H, J ¼ 7.7, 7.4 Hz), 7.89 (d, 2H, J ¼ 9.0 Hz), 9.51 (d, 1H,
plates at 104 cells per well in 100
allowed to grow for 24 h. Compounds 4a, 6a, 9a or 11a were freshly
dissolved in media at a concentration of 100 M. Then cells were
incubated for 48 h with compounds in a range of concentrations
from 100 to 0.01 M. Then, an MTT assay was performed to evaluate
the toxicity [46]. Briefly, cells were incubated for 4 h with
0.5 mg mLꢀ1 of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) in media. The MTT media solution was then
removed and the precipitated crystals were dissolved in EtOH/
DMSO (1/1 v/v). The solution absorbance was read at 540 nm.
mL culture medium and
m
J ¼ 6.8 Hz); 13C NMR (CDCl3, 75 MHz):
d ppm 16.2, 40.2, 59.3, 111.4,
112.8, 113.4, 116.0, 121.1, 128.5, 130.1, 130.9, 146.5, 153.1, 156.2, 157.5,
183.5; FT-IR: gmax (cmꢀ1): 761, 824, 1174, 1332, 1483, 1523, 1591,
2919; HPLC, Tr ¼ 1.24 min; MS (ESIþ): m/z 334.1 [M þ H]þ; HRMS
calcd for C19H20N5O 334.1668, found 334.1673.
m
4.3.3. (4S)-4-Methyl-2-(4-methoxy)-3,4-dihydro-5H-pyrido
[10,20:1,2]imidazo[4,5-d][1,3]-diazepin-5-one (8a)
Pale yellow solid, m ¼ 24 mg, yield ¼ 23%, mp: 86 ꢄC dec.
4.4.3. Cell-cycle analysis
Flow cytometric analysis was performed on 350,000 cells
seeded in culture dishes (60 mm diameter) and allowed to grow for
20
[a
]
¼ ꢀ25.3ꢄ (c 0.2, CHCl3); 1H NMR (CDCl3, 300 MHz):
d
ppm
D
1.60 (d, 3H, J ¼ 6.9 Hz), 3.78 (s, 3H), 4.10 (q, 1H, J ¼ 6.9 Hz), 6.88 (d,
2H, J ¼ 8.8 Hz), 6.97 (dd, 1H, J ¼ 7.8, 6.7 Hz), 7.15 (d, 1H, J ¼ 8.1 Hz),
7.39 (dd, 1H, J ¼ 8.1, 7.8 Hz), 7.95 (d, 2H, J ¼ 8.8 Hz), 9.50 (d, 1H,
48 h. Cells were then treated with compound 6a at 5 mM for 16 or
48 h. After treatment cells were harvested and fixed with 70%
ethanol overday. The fixed cells were then incubated with
10 mg mLꢀ1 RNase A and 1 mg mLꢀ1 propidium iodide, in the dark,
for 24 h at 4 ꢄC. Finally, DNA content of the cells was analyzed using
BD FACSCalibur flow cytometer with FlowJo software [47].
J ¼ 6.7 Hz); 13C NMR (CDCl3, 75 MHz):
d ppm 16.4, 55.6, 60.2, 112.6,
113.7, 114.1, 115.8, 127.1, 128.6, 130.2, 131.2, 146.1, 155.2, 156.4, 162.9,
183.0; FT-IR: gmax (cmꢀ1): 742, 840, 1025, 1170, 1251, 1305, 1337,
1428, 1464, 1604, 2930; HPLC, Tr ¼ 0.94 min; MS (ESIþ): m/z 321.1
[M þ H]þ; HRMS calcd for C18H17N4O2 321.1352, found 321.1350.
4.4.4. Immunofluorescent staining for actin localization
4.3.4. (4R)-4-Methyl-2-(4-bromomethyl)-3,4-dihydro-5H-pyrido
To evaluate actin localization, MDA-MB-435 cells allowed to
grow for 24 h where treated with or without compound 4a or 6a at
5 mM for 20 h. Then cells were fixed using Antigenfix for 20 min and
permeabilized using 0.2% Triton X-100 for 4 min at room temper-
ature. Actin was stained using a primary human anti-actin antibody
(made from mouse) and an Alexa Fluor 568-conjugated anti-mouse
antibody. Both of them were incubated 1 h at room temperature.
Negative control was performed on cells stained with Alexa Fluor
568-conjugated antibody alone. Nuclei were counter-stained using
Hoechst 33342. Representative images were obtained under a Zeiss
[10,20:1,2]imidazo[4,5-d]-[1,3]diazepin-5-one (6b)
Orange solid, m ¼ 86 mg, yield ¼ 71%, mp: 141e143 ꢄC.
20
[a
]
¼ þ11.7ꢄ (c 0.25, CHCl3); NMR, IR and MS data were in
D
agreement with those of its enantiomer [20]; HRMS calcd for
C
17H14N4OBr 369.0351, found 369.0349.
4.4. Biological evaluation
4.4.1. SRB assay
Primary anticancer assay was performed on a panel of approx-
imately sixty human tumor cell lines derived from nine neoplastic
diseases, in accordance with the protocol of the Drug Evaluation
Branch, National Cancer Institute, Bethesda, Maryland, USA [45].
Tested compounds were added to the culture at a single concen-
Axioobserver confocal with
objective.
a Plan-Apochromat 63x/1.40 Oil
4.4.5. Evaluation of tubulin assembly
Sheep brain microtubular proteins were purified according to
Shelanski procedure [48] by two cycles of assembly/disassembly at
37 ꢄC/0 ꢄC in MES buffer: 100 mM MES (2-[N-morpholino]-etha-
tration (10 mM) and the cultures were incubated for 48 h. End point
determinations were made with a protein binding dye, sulforhod-
amine B (SRB). Results for each tested compound were reported as
the percent of growth of the treated cells when compared to the
untreated control cells. The percentage growth was evaluated
spectrophotometrically versus controls not treated with test agents.
The cytotoxic and/or growth inhibitory effects of the most active
selected compound 6a were tested in vitro on the full panel of
human tumor cell lines at concentrations ranging from 100 to
nesulfonic acid, pH 6.6), 1 mM EGTA (ethyleneglycol-bis[b-amino-
ethyl ether]-N,N,N0,N0-tetraacetic acid), 0.5 mM MgCl2. Tubulin
assembly was monitored by fluorescence according to reported
procedure [49] using DAPI as fluorescent probe. All samples were
dissolved in DMSO. The evaluated compound (1
microtubular solution (100 L of 10 M tubulin in MES buffer
containing 10 M DAPI), incubated at room temperature for 40 min
mL) was added to
m
m
m
0.01
m
M. 48 h continuous drug exposure protocol was followed and
before addition of 1 mM GTP. Assays were realized on 96-well
plates prepared with Biomek NKMC and Biomek 3000 from
an SRB protein assay was used to estimate cell viability or growth.

A. Gallud et al. / European Journal of Medicinal Chemistry 75 (2014) 382e390
389
Beckman Coulter and read at 37 ꢄC on Wallac Victor fluorimeter
from Perkin Elmer.
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Kinase selectivity was evaluated on a panel of 19 protein tyro-
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Acknowledgments
This work was supported by Institut des Biomolécules Max
Mousseron (IBMM, Montpellier, France) and Région Languedoc
Roussillon (chercheur d’avenir 2011 grant to V.L.). We would like to
thank the National Cancer Institute (NCI), Bethesda, Maryland, USA,
for performing the anticancer testing of diazepinone compounds.
We thank also Pr Pierre Cuq (IBMM) for the CAL1 cell line gift.
[22] M. Andaloussi, E. Moreau, N. Masurier, J. Lacroix, R.C. Gaudreault, J.-
M. Chezal, A. El Laghdach, D. Canitrot, E. Debiton, J.-C. Teulade, O. Chavignon,
Novel imidazo[1,2-a]naphthyridinic systems (part 1): synthesis, anti-
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.044.
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