Imidazopyridine-fused [1,3]diazepinones: modulations of positions 2 to 4 and their impacts on the anti-melanoma activity

Article abstract of DOI:10.1080/14756366.2020.1748024

A series of 19 novel pyrido-imidazodiazepinones, with modulations of positions 2, 3 and 4 of the diazepine ring were synthesised and screened for their in vitro cytotoxic activities against two melanoma cell lines (A375 and MDA-MB-435) and for their poten

Full text of DOI:10.1080/14756366.2020.1748024

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Imidazopyridine-fused [1,3]diazepinones:
modulations of positions 2 to 4 and their impacts
on the anti-melanoma activity
Paul Le Baccon-Sollier, Yohan Malki, Morgane Maye, Lamiaa M. A. Ali, Laure
Lichon, Pierre Cuq, Laure-Anaïs Vincent & Nicolas Masurier
To cite this article: Paul Le Baccon-Sollier, Yohan Malki, Morgane Maye, Lamiaa M. A. Ali,
Laure Lichon, Pierre Cuq, Laure-Anaïs Vincent & Nicolas Masurier (2020) Imidazopyridine-
fused [1,3]diazepinones: modulations of positions 2 to 4 and their impacts on the anti-
melanoma activity, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 935-949, DOI:
10.1080/14756366.2020.1748024
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 935–949
https://doi.org/10.1080/14756366.2020.1748024
RESEARCH PAPER
Imidazopyridine-fused [1,3]diazepinones: modulations of positions 2 to 4 and their
impacts on the anti-melanoma activity
a
a
a
a,b
, Laure Lichona, Pierre Cuqa
,
ꢀ
ꢀ
Paul Le Baccon-Sollier , Yohan Malki , Morgane Maye , Lamiaa M. A. Ali
Laure-Anaïs Vincenta
and Nicolas Masuriera
a
ꢀ
ꢀ
Institut des Biomolecules Max Mousseron, UMR 5247, CNRS, Universites Montpellier, UFR des Sciences Pharmaceutiques et Biologiques,
b
Montpellier, France; Department of Biochemistry, Medical Research Institute, University of Alexandria, Alexandria, Egypt
ABSTRACT
ARTICLE HISTORY
Received 23 December 2019
Revised 4 March 2020
Accepted 22 March 2020
A series of 19 novel pyrido-imidazodiazepinones, with modulations of positions 2, 3 and 4 of the diaze-
pine ring were synthesised and screened for their in vitro cytotoxic activities against two melanoma cell
lines (A375 and MDA-MB-435) and for their potential toxicity against NIH-3T3 non-cancerous cells.
Selected compounds were also evaluated on the NCI-60 cell line panel. The SAR study revealed that the
molecular volume and the cLogP of compounds modified at position 2 were significantly correlated with
the activity of these compounds on melanoma cell lines. Moreover, introduction of a heterocyclic group
at position 2 or an azido-alkyl chain at position 4 led to compounds displaying a significantly different
activity profile on the NCI-60 cell line panel, compared to phenyl-substituted compounds at position 2 of
the diazepinone. This study provides us crucial information for the development of new derivatives active
against melanoma.
KEYWORDS
Antitumor activity;
diazepinones; imidazo
[1,2-a]pyridine; melanoma;
polyfused heterocycles
Introduction
parallel, immunotherapies (inhibitors of CTLA4, PD1 and PDL1)
have shown very interesting results, since some treated patients
have not presented any relapse to date7. Unfortunately, no test
can identify potentially responders and these therapies are not
free of toxicity8. Finally, association between targeted therapies
and immunotherapies confers a longer progression-free survival
and duration of response, but led to a higher rate of serious
toxicity9. Therefore, the development of new drugs that could
be used in combination with mentioned therapies is still in
demand to overcome the extreme chemoresistance of meta-
static melanoma.
Recently, we reported the discovery of JMV5038, an original
pyrido-imidazodiazepinone derivative, using a screening on the
NCI-60 cancer cell line panel10. This compound showed a promis-
ing activity on several cancer cell lines, with a growth inhibition of
50% (GI50) in the low micromolar range on melanoma cells and
with no significant cytotoxic activity on NIH-3T3 fibroblasts, even
at 100 mM. Further investigations revealed that JMV5038 increased
apoptosis in subG1 phase and microscopic observations of treated
Melanoma is the most aggressive skin cancer and remains a thera-
peutic challenge. Its global incidence is increasing continuously,
with 287,723 new cases in 2018, growing up to 301,694 in 2020
and 466,914 in 20401,2. The prognosis of melanoma depends on
how early the diagnosis is. While surgical resection of local forms
is usually curative, metastatic melanoma (MM) requires systemic
treatment and is associated with very poor prognosis. Indeed, the
extreme chemoresistance of MM makes it less sensitive to conven-
tional chemotherapy. Since 2011, new therapeutic strategies have
emerged and offer promising results3. Small molecules, so-called
“targeted” therapies, have been developed to specifically inhibit
the V600E mutated BRAF kinase found in 50% of MM
(Vemurafenib, Dabrafenib, Encorafenib)4. However, the rapid emer-
gence of drug resistance to initially responsive cancers has led to
only marginal patient benefit. To limit these resistances, mainly
due to the reactivation of the MAPK pathway, MEK inhibitors
(Trametinib, Cobimetinib, Binimetinib) are used in combination5.
Nevertheless therapeutic escape are inevitably encountered6. In cells showed morphological changes, associated with an increase
ꢀ ꢀ
Institut des Biomolecules Max Mousseron, UMR 5247, CNRS, Universites Montpellier, UFR des
CONTACT Nicolas Masurier
nicolas.masurier@umontpellier.fr
Sciences Pharmaceutiques et Biologiques, 15 Avenue Charles Flahault, Montpellier Cedex 5, 34093, France
ꢀ
These authors contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

936
P. L. BACCON-SOLLIER ET AL.
Figure 1. Structure of JMV5038, SAR studies from references10 and11 and new studied modulations.
1
of the membrane permeability and disruption of the actin net- 13 C), CD3OD ¼ 3.31 ppm for H (49.00 ppm for 13 C) or DMSO d6
work. We then started a structure-activity relationship (SAR) study ¼ 2.50 ppm for 1H (39.52 ppm for 13 C). The following abbrevia-
on these series11. Whereas positions 3 and 5 of the diazepine ring
tions were used: s (singlet), d (doublet), t (triplet), q (quartet), m
could not be modified without loss of the cytotoxic activity, posi-
tions 2 and 4 of the diazepine cycle tolerated several modifica-
tions (Figure 1). Concerning position 2, we showed that large
hydrophobic groups, such as a 1- or a 2-naphthyl ring, as well as
halogenophenyl groups, did not modify the biological activity of
JMV5038. For position 4, several modifications were tolerated with
preservation of JMV5038 cytotoxic activity, like short elongation of
the alkyl chain or introduction of bulky groups such as a tert-buty-
loxymethyl substituent (Figure 1). To further explore the SAR on
these series, we decided to pursue the modulations of position 2
by introducing different hydrophobic groups and, of the position
4, with the introduction of different alkyl chains. Among others,
we decided to study the introduction of azido or alkyne groups
on these two positions, such groups could be of interest to inves-
tigate, in the future, further modulations using click chemistry. For
example, such approach could permit to introduce a biotin resi-
due to identify the pharmacological target of our compounds,
using “clickable” cell-permeable activity-based probe12.
In this paper, we report the synthesis of new analogues of
JMV5038. Their cytotoxic activity on melanoma cell lines and their
potential toxicity against NIH-3T3 non-cancerous cells were eval-
uated. Subsequently, a statistical study was carried out to high-
light the physicochemical parameters influencing the activity
profile of our molecules. Selected compounds were then eval-
uated by the NCI on the sixty cancer cell lines panel. Further
in vitro functional evaluations were finally performed on the most
relevant compounds.
(multiplet), br (broad). Analytical thin-layer chromatography (TLC)
was performed using 60 F254 aluminium-backed silica gel plates
coated with a 0.2 mm thickness of neutral aluminium oxide.
Column chromatography was performed on silica gel 60 (70–230
mesh) or on alumina gel (activated, neutral, Brockmann I). RP-pre-
parative HPLC purification was performed on a Waters HPLC 4000
instrument, equipped with a UV detector 486 and Waters Delta-
Pack 40ꢀ100 mm, 15 Å, 100 lm, reversed-phase column. Standard
conditions were eluent system A (H2O/0.1% TFA), system B
(CH3CN/0.1% TFA). A flow rate of 50 ml/min and a gradient of
(0–40)% B over 30 min were used, detection was performed at
214 nm. The LC/MS system consisted of a Waters Alliance 2695
HPLC, coupled to a Micromass (Manchester, UK) ZQ spectrometer
(positive electrospray ionisation mode, ESIþ). All the analyses
were carried out using a Merck Chromolith Speed rod C18,
25 ꢁ 4.6 mm reversed-phase column. A flow rate of 3 ml/min and
a gradient of (0–100)% B over 3 min (or over 15 min) were used.
Eluent A: water/0.1% HCO2H; eluent B: acetonitrile/0.1% HCO2H.
Retention times (RT) are given in minutes. Nitrogen was used for
both the nebulising and drying gas. The data were obtained in a
scan mode ranging from 100 to 1000 m/z in 0.1 s intervals; 10
scans were summed up to get the final spectrum. High-resolution
mass spectrometry analyses were performed with a Waters Synapt
G2-S time-of-flight mass spectrometer fitted with an electrospray
ionisation source. All measurements were performed in the posi-
tive ion mode. Melting points (Mp) are uncorrected and were
recorded on a Stuart capillary melting point apparatus SMP3.
Compounds 2a13, 2c–d14, 2f–g14, 3i14, JMV503813, Boc-azido-
norleucine 2915 and compound 2311 were synthesised according
to previous reported procedures and their physical characteristics
were in agreement with the published data.
Materials and methods
Instruments and reagents
Commercially available reagents and solvents were used without
further purification. Reactions were monitored by an analytical
Waters Alliance 2690 HPLC instrument, equipped with a photo-
diode array and an analytical Chromolith Speed Rod RP-C18
185 Pm column (50 ꢁ 4.6 mm, 5 mm), at a flow rate of 3.0 ml/min,
and gradients of 100/0–0/100 eluents A/B for 5 min (eluent
A ¼ H2O/0.1% TFA and B ¼ CH3CN/0.1% TFA). Detection was per-
formed at 214 nm. Retention times (Tr) are reported in min. 1H
and 13 C NMR spectra were recorded on Bruker spectrometers
(300 or 400 MHz) at room temperature in deuterated solvents.
Chemistry
Synthesis of 4-benzoyl-benzaldehyde 25
To a solution of 4-benzoylbenzoic acid (1 g, 4.42 mmol) in 30 ml of
ethanol and 30 ml of cyclohexane, 2 ml of sulphuric acid were
added. The solution was refluxed for one night. After cooling to
room temperature, the solution was washed with a saturated
NaHCO3 aqueous solution (2 ꢁ 30 ml). The organic layer was dried
over Na2SO4, filtered, and the solvent was evaporated in vacuo to
Chemical shifts (d) are expressed in parts per million (ppm), rela- offer ethyl 4-benzoylbenzoate as a white powder (1.09 g, 97%
tive to the resonance of CDCl3 ¼ 7.26 ppm for 1H (77.16 ppm for yield), with spectral data in agreement with the literature16.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
937
Ethyl 4-benzoylbenzoate (1 g, 3.94 mmol) was dissolved in DCM/EtOH 99/1 v/v for compound 2b and 2h or SiO2, DCM/AcOEt
10 ml of dry THF and cooled to 0 ꢂC. LiAlH4 (388 mg, 10.2 mmol,
9/1 v/v for compound 2e).
2.6 equiv) was added by fractions and the mixture was stirred at
0 ꢂC for 3 h. 50 ml of water were added and the solution was
(2S)-2-Boc-amino-1-(2-aminoimidazo[1,2-a]pyridin-3-yl)-hexan-1-
extracted with ethyl acetate (3 ꢁ 50 ml). The organic layer was
one (2 b)
dried over Na2SO4, filtered, and the solvent was evaporated in
vacuo. The residue was then dissolved in 10 ml of chloroform and
20
Beige solid (536 mg, yield 71%); mp: 174–175 ꢂC; [a]D ¼ ꢃ7.9ꢂ (c
4 equiv. of manganese(II) oxide were added. The mixture was 1.1, CHCl3); 1H NMR (CDCl3, 400 MHz): d ppm 0.83 (t, 3H,
stirred at reflux for 4 h. After cooling to room temperature, the
mixture was filtered through a pad of celite and washed with
J ¼ 6.7 Hz), 1.28 (m, 4H), 1.40 (s, 9H), 1.67 (m, 1H), 1.85 (m, 1H),
4.92 (q, 1H, J ¼ 7.2 Hz), 5.50 (d, 1H, J ¼ 9.0 Hz), 6.01 (br, 2H), 6.85
chloroform. The filtrate was then evaporated in vacuo. The residue
(td, 1H, J ¼ 6.8 Hz, 1.3 Hz), 7.33 (d, 1H, J ¼ 8.6 Hz), 7.39 (dd, 1H,
was purified by chromatography on silica gel, eluted by DCM/
J ¼ 8.6 Hz, 6.8 Hz), 9.63 (br, 1H); 13 C NMR (CDCl3, 75 MHz): d ppm
EtOH 99/1 v/v to offer 4-benzoyl-benzaldehyde 25 as a white solid
(145 mg, 18% yield), with spectral data in agreement with the lit-
erature17; HPLC, RT¼ 1.67 min; MS (ESIþ) calcd for C14H11O2: 211.1,
found m/z: 211.1 [M þ H]þ
14.1, 22.7, 28.0, 28.5, 54.5, 80.5, 108.3, 113.2, 114.4, 129.5, 130.7,
147.9, 156.9, 158.9, 186.1; HPLC, Tr¼ 1.62 min; MS (ESIþ) calcd for
C18H27N4O3 347.2, found m/z 347.2 [M þ H]þ.
(2S)-2-Boc-amino-1-(2-aminoimidazo[1,2-a]pyridin-3-yl)pent-4-yn-
1-one (2e)
Synthesis of quinoline-6-carbaldehyde 26
20
White solid (257 mg, yield 36%); mp: 147–148 ꢂC; [a]D ¼ ꢃ59.1ꢂ
To a solution of 6-quinolinecarboxylic acid (1 g, 5.71 mmol) in
40 ml of ethanol, 2 ml of sulphuric acid were added. The solution
was refluxed for one night. After cooling to room temperature,
100 ml of water were added and the solution was basified using
K2CO3. The solution was then extracted with dichloromethane
(3 ꢁ 30 ml). The organic layer was dried over Na2SO4, filtered, and
the solvent was evaporated in vacuo to offer ethyl 6-quinolinecar-
boxylate 25 (1.1 g, 95% yield) as a white powder. Spectral data
were in agreement with the literature18.
Ester 25 (1.1 g, 5.47 mmol) was then dissolved in 10 ml of dry
THF and cooled to 0 ꢂC. LiAlH4 (375 mg, 10.9 mmol, 2 equiv) was
added by fractions and the mixture was stirred at 0 ꢂC for 2 h.
50 ml of water were added and the solution was extracted with
ethyl acetate (2 ꢁ 50 ml). The organic layer was dried over Na2SO4,
filtered, and the solvent was evaporated in vacuo. The residue was
(c 0.1, CHCl3); 1H NMR (CDCl3, 300 MHz): d ppm 1.41 (s, 9H), 1.96
(t, 1H, J ¼ 2.6 Hz), 2.70 (ddd, 2H, J ¼ 6.8 Hz, 6.8 Hz, 2.6 Hz), 5.14 (m,
1H), 5.60 (d, 1H, J ¼ 9.7 Hz), 6.86 (td, 1H, J ¼ 6.8 Hz, 1.3 Hz), 7.33 (d,
1H, J ¼ 8.6 Hz), 7.41 (dd, 1H, J ¼ 8.6 Hz, 6.8 Hz), 9.59 (br, 1H); 13 C
NMR (CDCl3, 75 MHz): d ppm 22.6, 28.4, 55.6, 71.0, 79.8, 81.0, 98.5,
113.4, 114.4, 129.7, 131.2, 148.0, 156.6, 158.9, 182.9; HPLC, RT¼
1.33 min; MS (ESIþ) calcd for C17H21N4O3 329.1, found m/z
329.0 [M þ H]þ.
(2S)-2-Boc-amino-1-(2-aminoimidazo[1,2-a]pyridin-3-yl)-6-azido-
hexan-1-one (2 h)
20
White solid (340 mg, yield 70%); mp: 74–76 ꢂC; [a]D ¼ þ1.3ꢂ (c
1.0, CHCl3); 1H NMR (CDCl3, 300 MHz): d ppm 1.12–1.21 (m, 1H),
1.34 (m, 10H), 1.50 (m, 2H), 1.70 (m, 1H), 1.84 (m, 1H), 3.13 (t, 1H,
J ¼ 6.7 Hz), 4.92 (q, 1H, J ¼ 7.2 Hz), 6.37 (br, 3H), 6.80 (m, 1H), 7.33
(m, 2H), 9.60 (d, 1H, J ¼ 7.8 Hz); 13 C NMR (CDCl3, 75 MHz): d ppm
22.9, 28.3, 28.6, 32.0, 51.1, 54.1, 80.3, 108.0, 113.1, 114.1, 129.4,
131.0, 147.8, 157.0, 159.0, 185.4; HPLC, RT¼ 1.38 min; MS (ESIþ)
calcd for C18H26N7O 388.2, found m/z 387.8 [M þ H]þ.
then dissolved in 10 ml of chloroform and
4 equiv. of
manganese(II) oxide were added. The mixture was stirred at reflux
for 4 h. After cooling to room temperature, the mixture was fil-
tered through a pad of celite and washed with chloroform. The fil-
trate was then evaporated in vacuo. The residue was purified by
chromatography on silica gel, eluted by DCM/nHexane 9/1 v/v to
offer 6-quinolinecarboxaldehyde 26 as a white solid (343 mg, yield
30%); mp: 73–75 ꢂC (litt.19 75–76 ꢂC); 1H NMR (CDCl3, 300 MHz): d
ppm 7.50 (dd, 1H, J ¼ 8.3 Hz, 8.3 Hz), 8.18 (m, 2H), 8.29–8.33 (m,
2H), 9.03 (dd, 1H, J ¼ 4.2 Hz, 1.7 Hz), 10.18 (s, 1H); 13 C NMR
(CDCl3, 75 MHz): d ppm 122.4, 127.0, 127.9, 130.8, 131.0, 133.8,
134.5, 137.7, 153.3, 191.7; HPLC, Tr¼ 0.84 min; MS (ESIþ): calcd for
C10H8NO 158.1, found m/z 157.8 [M þ H]þ.
General procedure for the synthesis of diazepinones 4–20
Method A (for compounds 5–13 and 15–20). A solution of com-
pound 2a–i (0.33 mmol) in a 50/50 v/v mixture of trifluoroacetic
acid and dichloromethane (5 ml) was stirred at room temperature
for 1 h. After completion of the reaction (HPLC analysis), the solu-
tion was concentrated to dryness and co-evaporated several times
with acetonitrile to completely remove TFA. Then, were added
THF (8 ml), K2CO3 (182 mg, 4 equiv.), iodine (251 mg, 3 equiv.) and
the appropriate aldehyde (1 equiv.). The mixture was stirred under
heating at 70 ꢂC for one night. THF was then removed under
reduce pressure and 30 ml of dichloromethane were added. The
organic phase was successively washed with a 10% m/v sodium
thiosulphate aqueous solution (2 ꢁ 30 ml) and then with a satu-
rated NaHCO3 aqueous solution (2 ꢁ 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, DCM/EtOH 99/1 v/v and finally DCM/
EtOH 98/2 v/v.
Synthesis of compounds 2
To a suspension of trifluoro-N-imidazo[1,2-a]pyridin-2-acetamide13
(0.5 g, 2.18 mmol) in 5 N aqueous sodium hydroxide (9 ml), was
added THF (0.5 ml). The solution was stirred at 40 ꢂC for 2 h. The
solution was extracted with dichloromethane (3 ꢁ 20 ml). The
organic layer was dried over Na2SO4, filtered, and the solvent was
removed in vacuo. The residue was dissolved in dichloromethane
(20 ml) and either Boc-azidonorleucine 29 or Boc-Pra-OH or Boc-
Nle-OH (2.4 mmol, 1.1 equiv), HOBt (325 mg, 2.4 mmol, 1.1 equiv),
EDCI (460 mg, 2.4 mmol, 1.1 equiv), or triethylamine (334 ml,
2.4 mmol, 1.1 equiv), were added at 0 ꢂC. The solution was stirred
at room temperature for one night. The solution was then washed
with a saturated NaHCO3 solution (2 ꢁ 50 ml). The organic layer
was dried over Na2SO4, filtered, and the solvent was concentrated
Method B (for compounds 4 and 14). A solution of compound 2a
in vacuo. The residue was purified by chromatography (Al2O3, or compound 2e (0.33 mmol) in
a 50/50 v/v mixture of

938
P. L. BACCON-SOLLIER ET AL.
trifluoroacetic acid and dichloromethane (5 ml) was stirred at 400 MHz): d ppm 1.58 (d, 3H, J ¼ 6.8 Hz), 3.46 (s, 1H), 4.17 (d, 1H,
room temperature. After completion of the reaction (HPLC ana- J ¼ 6.8 Hz), 7.00 (dd, 1H, J ¼ 6.8 Hz, 6.0 Hz), 7.26 (dd, 1H, J ¼ 6.6 Hz,
lysis), the solution was concentrated to dryness and co-evaporated 7.7 Hz), 7.36 (t, 1H, J ¼ 7.2 Hz), 7.49 (m, 2H), 7.63 (d, 2H, J ¼ 7.6 Hz),
several times with acetonitrile to completely remove TFA. Then, 7.73 (s, 1H), 9.52 (d, 1H, J ¼ 6.8 Hz); 13 C NMR (CDCl3, 100 MHz): d
5 ml of water were added and the solution was basified by K2CO3 ppm 16.6, 58.7, 111.1, 111.8, 114.1, 116.5, 121.2, 122.9, 124.1,
and then extracted by chloroform (3 ꢁ 10 ml). The organic layer 124.4, 127.0, 128.4, 128.7, 130.6, 146.7, 150.0, 155.4, 182.7; HPLC,
was dried over Na2SO4, filtered and the solvent was evaporated in RT¼ 1.17 min; MS (ESIþ): m/z 331.0 [M þ H]þ; HRMS calcd for
vacuo. The crude was then dissolved in 20 ml of chloroform and 1 C19H15N4O2 331.1195, found 331.1199.
equiv of the appropriate aldehyde was added. The solution was
stirred at 80 ꢂC for 6 h. After cooling, DDQ (0.44 mmol, 2 equiv)
was added to the reaction mixture. The solution was stirred at
(4S)-4-methyl-2-benzothiophen-3-yl-3,4-dihydro-5H-pyrido[1’,2’:
1,2]imidazo[4,5-d][1,3]diazepin-5-one (8). Brown solid (175 mg,
20
1
room temperature for 6 h (monitoring by TLC). The solution was
washed with aqueous saturated sodium hydrogen carbonate solu-
tion (2 ꢁ 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 and then DCM/
EtOH 99/1 v/v for compound 4 or by preparative HPLC for com-
pound 14 (gradient of 100% H2O/0.1% TFA to 40% CH3CN/
0.1% TFA).
yield 31%); mp: 135–137 ꢂC; [a]D ¼ þ6.5ꢂ (c 1.0, CHCl3); H NMR
(DMSO-d6, 500 MHz): d ppm 1.39 (br, 3H), 4.07 (q, 1H, J ¼ 6.1 Hz),
7.19 (td, 1H, J ¼ 1.4 Hz, J ¼ 6.8 Hz), 7.47 (t, 1H, J ¼ 7.7 Hz), 7.53 (t,
1H, J ¼ 7.3 Hz), 7.67 (td, 1H, J ¼ 1.3 Hz, J ¼ 6.8 Hz), 7.71 (d, 1H,
J ¼ 8.5 Hz), 8.07 (d, 1H, J ¼ 7.9 Hz), 8.52 (br, 1H), 8.81 (br, 1H), 9.50
(br, 1H), 11.21 (br, 1H); 13 C NMR (DMSO-d6, 500 MHz): d ppm
15.0, 56.6, 113.3, 115.6, 122.5, 124.6, 124.7, 125.0, 127.5, 130.0,
133.4, 137.0, 139.7, 146.1, 146.5, 148.0, 153.6, 157.6, 183.3; HPLC,
RT¼ 1.98 min; MS (ESIþ): m/z 347 [M þ H]þ; HRMS calcd for
C19H15N4O2 347.0975, found 347.0961.
(4S)-4-methyl-2-(4-trimethylsilylethynylphenyl)-3,4-dihydro-5H-pyri-
do[1’,2’:1,2]imidazo[4,5-d][1,3]diazepin-5-one (4). Brown solid
20
(23 mg, yield 18%); mp: 115–116 ꢂC; [a]D ¼ þ43.8ꢂ (c 0.4, CHCl3);
(4S)-4-methyl-2-(1-boc-indol-3-yl)-3,4-dihydro-5H-pyrido[1’,2’:1,2]i-
midazo[4,5-d][1,3]diazepin-5-one (9). Yellow solid (70 mg, yield
1H NMR (CDCl3, 500 MHz): 2 conformers d ppm 0.23 (s, 4.5H), 0.24
(s, 4.5H), 1.70 (d, 3H, J ¼ 6.6 Hz), 4.10 (m, 1H), 7.0 (dd, 1H,
J ¼ 6.7 Hz, 6.6 Hz), 7.41 (m, 1H), 7.49 (d, 1H, J ¼ 8.2 Hz), 7.53 (d, 1H,
J ¼ 8.2 Hz), 7.66 (d, 1H, J ¼ 8.6 Hz), 7.87 (d, 1H, J ¼ 8.2 Hz), 7.99 (d,
1H, J ¼ 8.2 Hz), 9.47 (d, 0.5H, J ¼ 6.6 Hz), 9.55 (d, 0.5H, J ¼ 6.6 Hz),
9.97 (br, 1H); 13 C NMR (CDCl3, 125 MHz): 2 conformers d ppm
15.5, 57.4, 97.4, 97.9, 104.2, 113.8, 115.2, 117.0, 126.5, 126.8, 127.1,
128.6, 128.8, 130.2, 130.5, 132.3, 132.5, 134.6, 135.4, 145.5, 147.3,
152.6, 152.9, 157.6, 158.3, 182.5, 183.2; HPLC, RT¼ 1.32 min; MS
(ESIþ) calcd for C22H23N4OSi 387.1, found m/z 387.0 [M þ H]þ.
16%); mp: 155–157 ꢂC; [a]D ¼ ꢃ16.4ꢂ (c 0.8, CHCl3); 1H NMR
20
(DMSO-d6, 400 MHz): d ppm 1.38 (d, 3H, J ¼ 6.5 Hz), 1.68 (s, 9H),
4.00 (q, 1H, J ¼ 6.5 Hz), 7.18 (td, 1H, J ¼ 1.4 Hz, J ¼ 6.8 Hz),
7.36–7.44 (m, 2H), 7.66 (td, 1H, J ¼ 1.2 Hz, J ¼ 6.7 Hz), 7.71 (d, 1H,
J ¼ 8.7 Hz), 8.13 (d, 1H, J ¼ 7.5 Hz), 8.49–8.69 (m, 3H), 9,47 (br, 1H);
13 C NMR (DMSO-d6, 400 MHz): d ppm 15.3, 27.6, 56.8, 84.9, 113.6,
114.6, 115.7, 122.9, 123.4, 125.1, 127.7, 128.1, 129.5, 130.4, 133.6,
134.1, 135.2, 136.9, 146.2, 148.7, 157.8, 195.5; HPLC, RT¼ 2.60 min;
MS (ESIþ): m/z 430.1 [M þ H]þ; HRMS calcd for C24H24N5O3
430.1879, found 430.1886.
(4S)-4-methyl-2-(4-azidophenyl)-3,4-dihydro-5H-pyrido[1’,2’:1,2]imi-
dazo[4,5-d][1,3]diazepin-5-one (5). Brown solid (28 mg, yield 26%);
(4S)-4-methyl-2-quinolin-6-yl-3,4-dihydro-5H-pyrido[1’,2’:1,2]imi-
dazo[4,5-d][1,3]diazepin-5-one (10). Brown solid (9 mg, yield 8%);
20
mp: 235–236 ꢂC (dec.); [a]D ¼ þ113ꢂ (c 0.1, CHCl3); 1H NMR
20
mp: 165–166 ꢂC; [a]D ¼ þ52.4ꢂ (c 0.25, CHCl3); 1H NMR (CDCl3,
(CDCl3, 500 MHz): d ppm 1.69 (br, 3H), 4.09 (q, 1H, J ¼ 6.6 Hz), 6.08
(br, 1H), 7.00 (t, 1H, J ¼ 7.0 Hz), 7.05 (d, 1H, J ¼ 7.9 Hz), 7.45 (br,
2H), 7.65 (br, 1H), 7.95 (m, 2H), 9.51 (d, 1H, J ¼ 7.0 Hz); 13C NMR
(CDCl3, 125 MHz): d ppm 35.2, 62.7, 113.9, 116.9, 119.4, 123.1,
123.4, 125.5, 126.4, 128.7, 130.3, 130.7, 152.8, 155.5, 181.7; HPLC,
RT¼ 1.05 min; MS (ESIþ): m/z 332.1 [M þ H]þ; HRMS calcd for
C17H14N7O 332.1260, found 332.1262.
500 MHz): 2 conformers d ppm 1.54 (d, 1.5H, J ¼ 6.1 Hz), 1.74 (d,
1.5H, J ¼ 6.1 Hz), 4.19 (q, 1H, J ¼ 6.1 Hz), 6.95 (m, 2H), 7.37 (m, 1H),
7.49 (dd, 0.5H, J ¼ 6.7 Hz, 7.9 Hz), 7.65 (d, 0.5H, J ¼ 7.7 Hz),
8.06–8.16 (m, 2H), 8.27 (d, 1H, J ¼ 8.6 Hz), 8.35 (br, 0.5 H), 8.57 (br,
0.5H), 8.89 (d, 1H, J ¼ 2.9 Hz), 9.43 (d, 0.5H, J ¼ 5.4 Hz), 9.52 (d,
0.5H, J ¼ 5.4 Hz), 10.52 (br, 1H); 13 C NMR (CDCl3, 100 MHz): 2 con-
formers d ppm 16.3, 17.0, 64.0, 113.9, 115.0, 116.8, 122.1, 127.8,
128.6, 129.0, 129.7, 129.9, 130.0, 130.2, 130.3, 130.5, 133.0, 133.7,
(4S)-4-methyl-2-(4-benzoylphenyl)-3,4-dihydro-5H-pyrido[1’,2’:1,2]i-
midazo[4,5-d][1,3]diazepin-5-one (6). Yellow solid (45 mg, yield 136.9, 137.3, 145.4, 147.2, 149.4, 149.6, 152.1, 152.2, 152.7, 153.0,
20
35%); mp: 132–133 ꢂC; [a]D ¼ þ18.7ꢂ (c 1.0, MeOH); 1H NMR 157.8, 158.2, 182.4, 183.3; HPLC, RT¼ 0.92 min; MS (ESIþ): m/z
(DMSO d6, 500 MHz): d ppm 1.35 (br, 1.8H), 1.54 (br, 1.2H), 4.05 (q, 342.2 [M þ H]þ; HRMS calcd for C20H16N5O 342.1355,
1H, J ¼ 6.7 Hz), 7.19 (dd, 1H, J ¼ 6.6 Hz, 7.9 Hz), 7.60 (t, 2H, found 342.1356.
J ¼ 7.6 Hz), 7.63–7.73 (m, 3H), 7.78 (d, 2H, J ¼ 7.0 Hz), 7.87 (m, 2H),
8.20 (d, 1H, J ¼ 6.4 Hz), 8.77 (s, 1H), 9.39 (br, 0.4H), 9.45 (br, 0.6H);
(4S)-4-methyl-2-quinolin-2-yl-3,4-dihydro-5H-pyrido[1’,2’:1,2]imi-
13 C NMR (DMSO d6, 125 MHz): d ppm 15.5, 56.6, 113.8, 116.0,
dazo[4,5-d][1,3]diazepin-5-one (11). Yellow solid (92 mg, yield
20
82%); mp: 170–171 ꢂC; [a]D ¼ þ22.5ꢂ (c 0.6, CHCl3); 1H NMR
127.8, 128.6, 128.7, 129.3, 129.5, 129.7, 130.4, 133.0, 136.7, 139.3,
146.3, 149.1, 156.9, 157.6, 183.2, 195.4; HPLC, RT¼ 1.25 min; MS
(ESIþ): m/z 395.1 [M þ H]þ; HRMS calcd for C24H19N4O2 395.1508,
found 395.1507.
(CDCl3, 400 MHz): (2 conformers) d ppm 1.56 (d, 2H, J ¼ 6.6 Hz),
1.68 (d, 1H, J ¼ 4.9 Hz), 4.26 (m, 0.7H), 4.36 (m, 0.3H), 7.00 (dd, 1H,
J ¼ 7.0 Hz, 6.7 Hz),7.49 (ddd, 1H, J ¼ 8.8 Hz, 8.3 Hz, 1.2 Hz), 7.58 (dd,
1H, J ¼ 7.2 Hz, 7.6 Hz), 7.67 (d, 1H, J ¼ 8.3 Hz), 7.74 (ddd, 1H,
J ¼ 7.0 Hz, 7.0 Hz, 1.2 Hz), 7.86 (d, 1H, J ¼ 7.3 Hz), 8.21–8.30 (m, 2H),
(4S)-4-methyl-2-benzofuran-3-yl-3,4-dihydro-5H-pyrido[1’,2’:1,2]imi-
dazo[4,5-d][1,3]diazepin-5-one (7). Yellow solid (21 mg, yield 19%); 8.66 (br, 1H), 8.88 (s, 1H, J ¼ 8.3 Hz), 9.52 (d, 0.3H, J ¼ 6.2 Hz), 9.60
20
mp: 164–165 ꢂC; [a]D ¼ þ23.2ꢂ (c 1.0, CHCl3); 1H NMR (CDCl3, (d, 0.7H, J ¼ 6.2 Hz); 13 C NMR (CDCl3, 100 MHz): d ppm 17.1, 57.2,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
939
113.8, 116.6, 119.7, 127.9, 128.0, 128.8, 129.4, 129.7, 129.9, 130.2, 147.2, 153.7, 182.9; HPLC, RT¼ 1.37 min; MS (ESIþ): m/z 452.0
130.3, 132.4, 137.4, 146.5, 150.9, 152.2, 163.0, 183.0; HPLC, RT¼ [M þ H]þ, 454.0 [M þ 2 þ H]þ; HRMS calcd for C20H19N7O79Br
1.36 min; MS (ESIþ): m/z 342.2 [M þ H]þ; HRMS calcd for 452.0834, found 452.0835.
C20H16N5O 342.1355, found 342.1356.
(4R)-4-(4-azidobutyl)-2-(4-bromophenyl)-3,4-dihydro-5H-pyrido
(4S)-4-methyl-2-coumarin-6-yl-3,4-dihydro-5H-pyrido[1’,2’:1,2]
[1’,2’:1,2]imidazo[4,5-d][1,3]diazepin-5-one (17). Yellow solid
20
1
imidazo[4,5-d][1,3]diazepin-5-one (12). Yellow solid (46 mg, yield (45 mg, yield 30%); [a]D ¼ ꢃ39.1ꢂ (c 0.3, CHCl3); H NMR (CDCl3,
20
39%); mp: 132.5–133.5 ꢂC; [a]D ¼ ꢃ8.4ꢂ (c 0.6, DMSO); 1H NMR 400 MHz): d ppm 1.57–1.70 (m, 6H), 3.31 (m, 2H), 3.93 (m, 1H),
(DMSO d6, 500 MHz): d ppm 1.42 (d, 3H, J ¼ 6.7 Hz), 4.08 (q, 1H, 7.00 (dd, 1H, J ¼ 6.2 Hz, 6.8 Hz), 7.45 (m, 2H), 7.54 (d, 2H,
J ¼ 6.7 Hz), 6.60 (d, 1H, J ¼ 9.7 Hz), 7.23 (td, 1H, J ¼ 6.5 Hz, 2.0 Hz), J ¼ 8.4 Hz), 7.85 (br d, 2H), 9.48 (d, 1H, J ¼ 6.2 Hz), 10.41 (br, 1H);
7.56 (d, 1H, J ¼ 8.7 Hz), 7.71 (m, 2H), 8.20 (m, 2H), 8.36 (s, 1H), 9.47 13 C NMR (CDCl3, 100 MHz): d ppm 23.7, 29.0, 29.9, 30.3, 51.5,
(d, 1H, J ¼ 6.5 Hz); 13 C NMR (DMSO d6, 125 MHz): d ppm 15.4, 114.0, 115.4, 116.2, 122.0, 128.7, 130.0, 130.6, 132.2, 134.1, 139.8,
58.0, 112.3, 113.0, 114.3, 115.7, 116.5, 117.1, 118.5, 127.9, 129.6, 147.2, 153.7, 182.9; HPLC, RT¼ 1.37 min; MS (ESIþ): m/z 452.0
130.1, 130.6, 131.0, 132.9, 144.1, 145.5, 155.8, 159.6, 182.3; HPLC, [M þ H]þ, 454.0 [M þ 2 þ H]þ; HRMS calcd for C20H19N7O79Br
RT¼ 0.99 min; MS (ESIþ): m/z 359.1 [M þ H]þ; HRMS calcd for 452.0834, found 452.0835.
C20H15N4O3 359.1144, found 359.1147.
(4S)-4-(4-azidobutyl)-2-phenyl-3,4-dihydro-5H-pyrido[1’,2’:1,2]imi-
(4S)-4-butyl-(4-bromophenyl)-3,4-dihydro-5H-pyrido[1’,2’:1,2]imi-
dazo[4,5-d][1,3]diazepin-5-one (18). Yellow solid (45 mg, yield
dazo[4,5-d][1,3]diazepin-5-one (13). Brown gum (72 mg, yield 30%); mp: 116–117 ꢂC; [a]D ¼ þ20.0ꢂ (c 0.5, CHCl3); 1H NMR
20
53%); [a]D ¼ þ89.1ꢂ (c 0.6, MeOH); 1H NMR (CDCl3, 400 MHz): d (CDCl3, 400 MHz): d ppm 1.57–1.71 (m, 4H), 2.05 (m, 2H), 3.32 (t,
20
ppm 0.80 (t, 3H, J ¼ 7.2 Hz), 1.21–1.37 (m, 4H), 1.65 (m, 2H), 4.08 (t, 2H, J ¼ 6.4 Hz), 3.93 (m, 1H), 6.97 (dd, 1H, J ¼ 7.5 Hz, 6.6 Hz), 7.38
1H, J ¼ 7.2 Hz), 7.34 (t, 1H, J ¼ 6.8 Hz), 7.37 (d, 2H, J ¼ 8.6 Hz), 7.82 (m, 1H), 7.43 (dd, 1H, J ¼ 7.8 Hz, 6.6 Hz), 7.49 (d, 2H, J ¼ 7.2 Hz),
(m, 3H), 7.91 (m, 2H), 9.57 (d, 1H, J ¼ 6.8 Hz); 13 C NMR (CDCl3, 7.98 (d, 1H, J ¼ 6.7 Hz), 9.48 (d, 1H, J ¼ 6.6 Hz), 10.60 (br, 1H); 13 C
100 MHz): d ppm 13.9, 22.2, 27.9, 30.0, 63.4, 111.4, 113.7, 117.3, NMR (CDCl3, 100 MHz): d ppm 23.6, 28.9, 29.7, 30.1, 51.4, 113.6,
129.2, 129.3, 130.8, 131.4, 132.2, 134.6, 141.2, 149.0, 160.6, 181.8; 115.5, 128.4, 128.8, 129.0, 129.7, 130.2, 131.7, 135.1, 139.7, 145.7,
HPLC, RT¼ 1.35 min; MS (ESIþ): m/z 411.1 [M þ H]þ, 413.1 147.0, 182.0; HPLC, RT¼ 0.97 min; MS (ESIþ): m/z 373.9 [M þ H]þ;
[M þ 2 þ H]þ; HRMS calcd for C20H20N4O79Br 411.0820, HRMS calcd for C20H20N7O 374.1729, found 374.1731.
found 411.0821.
(4S)- benzyl [2-(4-bromophenyl)-5-oxo-4,5-dihydro-3H-pyrido[1’,2’:
(4S)-4-propargyl-2-(4-bromophenyl)-3,4-dihydro-5H-pyrido[1’,2’:1,
1,2]imidazo[4,5-d][1,3]diazepin-4-yl]acetate (19). Orange solid
20
2]imidazo[4,5-d][1,3]diazepin-5-one (as trifluoroacetate salt) (14). (22 mg, yield 13%); mp: 72.5–73.5 ꢂC; [a]D ¼ þ10ꢂ (c 0.1, CHCl3);
20
Pale yellow solid (8 mg, yield 5%); [a]D ¼ þ17.0ꢂ (c 0.1, CHCl3); 1H NMR (CDCl3, 400 MHz): d ppm 3.17 (dd, 1H, J ¼ 16.3 Hz, 7.6 Hz),
1H NMR (CD3OD, 500 MHz): d ppm 2.60 (t, 1H, J ¼ 2.3 Hz), 2.69 (m, 3.44 (dd, 1H, J ¼ 16.3 Hz, 6.2 Hz), 4.50 (dd, 1H, J ¼ 7.6 Hz, 6.2 Hz),
1H), 2.80 (m, 1H), 4.16 (dd, 1H, J ¼ 4.5 Hz, 4.4 Hz), 7.24 (td, 1H, 5.24(m, 2H), 6.49 (d, 1H, J ¼ 8.9 Hz), 6.98 (t, 1H, J ¼ 6.6 Hz),
J ¼ 6.8 Hz, 1.0 Hz), 7.70–7.76 (m, 4H), 8.04 (d, 2H, J ¼ 8.3 Hz), 9.57 7.26–7.34 (m, 6H), 7.39 (d, 2H, J ¼ 6.6 Hz), 7.47 (d, 2H, J ¼ 8.4 Hz),
(d, 1H, J ¼ 6.8 Hz); 13 C NMR (CD3OD, 125 MHz): d ppm 24.9, 67.6, 7.70 (d, 2H, J ¼ 8.4 Hz), 9.42 (d, 1H, J ¼ 6.7 Hz); 13 C NMR (CDCl3,
72.4, 83.3, 121.8, 122.5, 124.8 (d, J ¼ 284 Hz, TFA), 125.2, 126.6, 100 MHz): d ppm 36.7, 64.9, 66.5, 111.7, 114.0, 114.9, 126.4, 127.1,
128.0, 137.4, 139.6, 140.1, 140.5, 142.4, 153.6, 155.0, 165.0 (TFA), 128.3, 128.4, 128.7, 130.7, 131.1, 132.1, 133.7, 136.4, 145.4, 152.9,
191.9; HPLC, RT¼ 1.23 min; MS (ESIþ): m/z 393.0 [M þ H]þ, 395.0 153.7, 172.4, 180.0; HPLC, RT¼ 1.60 min; MS (ESIþ): m/z 503.1
[M þ 2 þ H]þ; HRMS calcd for C19H14N4O79Br 393.0351, [M þ H]þ, 505.1 [M þ 2 þ H]þ; HRMS calcd for C25H20N4O379Br
found 393.0352.
503.0719, found 503.0720.
(4S)-4-isobutyl-2-(4-bromophenyl)-3,4-dihydro-5H-pyrido[1’,2’:1,2]i-
7–(4-bromophenyl)-9,10,11,11a-tetrahydro-12H-pyrido[2’,1’:2,3]imi-
midazo[4,5-d][1,3]diazepin-5-one (15). Yellow solid (133 mg, yield dazo[4,5-f]pyrrolo[1,2-c][1,3]diazepin-12-one (20). Brown gum
20
1
20
98%); mp: 72–73 ꢂC; [a]D ¼ þ33.0ꢂ (c 0.4, CHCl3); H NMR (CDCl3, (128 mg, yield 98%); [a]D ¼ ꢃ75.5ꢂ (c 1.0, MeOH); 1H NMR
400 MHz): d ppm 0.94 (d, 6H, J ¼ 6.4 Hz), 1.65–2.07 (m, 3H), 4.07 (CDCl3, 400 MHz): d ppm 1.77–1.87 (m, 1H), 1.98–2.14 (m, 2H), 3.15
(m, 1H), 6.98 (td, 1H, J ¼ 6.7 Hz, 1.0 Hz), 7.42 (m, 1H), 7.52 (d, 2H, (m, 1H), 3.30 (td, 1H, J ¼ 7.3 Hz, 1.2 Hz), 3.85 (ddd, 1H, J ¼ 6.4 Hz,
J ¼ 8.4 Hz), 7.63 (m, 1H), 7.86 (br, 2H), 9.50 (d, 1H, J ¼ 6.7 Hz), 10.63 6.4 Hz, 6.4 Hz), 4.08 (dd, 1H, J ¼ 6.4 Hz, 1.5 Hz), 6.96 (td, 1H,
(br, 1H); 13 C NMR (CDCl3, 100 MHz): d ppm 21.9, 25.0, 39.3, 60.8, J ¼ 6.8 Hz, 1.3 Hz), 7.46 (ddd, 1H, J ¼ 8.9 Hz, 6.8 Hz, 1.3 Hz), 7.55 (s,
113.9, 115.0, 116.9, 127.0, 128.6, 130.3, 130.6, 132.1, 132.4, 134.9 4H), 7.63 (d, 1H, J ¼ 8.9 Hz), 9.50 (d, 1H, J ¼ 6.8 Hz); 13 C NMR
(4 C), 156.8, 158.0, 186.1; HPLC, RT¼ 1.34 min; MS (ESIþ): m/z 411.1 (CDCl3, 100 MHz): d ppm 24.7, 25.6, 53.4, 64.9, 113.6, 114.7, 116.9,
[M þ H]þ, 413.1 [M þ 2 þ H]þ; HRMS calcd for C20H20N4O79Br 125.4, 128.4, 130.1, 131.6 (2 C), 131.8 (2 C), 135.3, 146.9, 158.8,
411.0820, found 411.0821.
159.9, 179.8; HPLC, RT¼ 1.19 min; MS (ESIþ): m/z 395.0 [M þ H]þ,
397.0 [M þ 2 þ H]þ; HRMS calcd for C19H16N4O79Br 395.0507,
found 395.0510.
(4S)-4-(4-azidobutyl)-2-(4-bromophenyl)-3,4-dihydro-5H-pyrido[1’,
2’:1,2]imidazo[4,5-d][1,3]diazepin-5-one (16). Yellow solid (45 mg,
yield 30%); mp: 127–128 ꢂC; [a]D ¼ þ39.7ꢂ (c 0.4, CHCl3); 1H
20
Synthesis of (4S)-4-methyl-2–(4-ethynylphenyl)-3,4-dihydro-5H-pyr-
NMR (CDCl3, 400 MHz): d ppm 1.57–1.70 (m, 6H), 3.31 (m, 2H), 3.93 ido[1’,2’:1,2]imidazo[4,5-d][1,3]diazepin-5-one (4’). To a solution of
(m, 1H), 7.00 (dd, 1H, J ¼ 6.2 Hz, 6.8 Hz), 7.45 (m, 2H), 7.54 (d, 2H, 70 mg of compound 4 (0.18 mmol) in 2 ml of THF, 181 mL of a
J ¼ 8.4 Hz), 7.85 (br d, 2H), 9.48 (d, 1H, J ¼ 6.2 Hz), 10.41 (br, 1H); solution of 2 M tetrabutylammonium fluoride in THF were added.
13 C NMR (CDCl3, 100 MHz): d ppm 23.7, 29.0, 29.9, 30.3, 51.5, The solution was stirred at room temperature for 2 h until comple-
114.0, 115.4, 116.2, 122.0, 128.7, 130.0, 130.6, 132.2, 134.1, 139.8, tion of the deprotection (HPLC analysis). The solution was then

940
P. L. BACCON-SOLLIER ET AL.
evaporated to dryness in vacuo. The residue was dissolved in ethyl monohydrate (101 mg, 0.66 mmol, 1.1 equiv.), EDCI (126 mg,
acetate and washed with an aqueous saturated solution of ammo- 0.66 mmol, 1.1 equiv.), 4-pentynoic acid (59 mg, 0.6 mmol, 1 equiv.)
and triethylamine (184 mL, 1.3 mmol, 2.2 equiv.) were added at
0 ꢂC. The solution was stirred at 0 ꢂC for 30 min and then at room
temperature for one night. The solution was washed with an
aqueous saturated NaHCO3 solution and then with brine. 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 to offer compound 22
as a brown gum (280 mg, yield 94%); two conformers detected by
nium chloride. The organic layer was dried over Na2SO4, filtered
and the solvent was evaporated in vacuo to offer compound 40 as
20
a yellow solid (56 mg, yield qt). Mp: 128–129 ꢂC; [a]D ¼ þ20.4ꢂ (c
1
0.5, CHCl3); H NMR (CDCl3, 300 MHz): d ppm 1.63 (br, 3H), 3.17 (s,
1H), 4.10 (q, 1H, J ¼ 6.7 Hz), 6.57 (br, 1H), 6.97 (dd, 1H, J ¼ 7.6 Hz,
6.6 Hz), 7.37 (m, 1H), 7.50 (d, 2H, J ¼ 8.3 Hz), 7.94 (d, 2H, J ¼ 8.3 Hz),
9.49 (d, 1H, J ¼ 6.6 Hz); 13 C NMR (CDCl3, 75 MHz): d ppm 16.7,
66.0, 79.9, 82.9, 113.7, 113.9, 125.6, 128.5 (2 C), 128.6, 129.2, 130.4,
132.4, 132.5 (2 C), 135.4, 153.3, 152.3, 180.5; HPLC, RT¼ 1.07 min;
MS (ESIþ): m/z 315.1 [M þ H]þ; HRMS calcd for C19H15N4O
315.1246, found 315.1248.
1
NMR in CDCl3; H NMR (CDCl3, 500 MHz): d ppm 1.44–1.77 (m, 6H),
2.30–2.51 (m, 4H), 3.21 (m, 1H), 3.35 (m, 1H), 3.85–4.06 (m, 1H),
6.05 (t, 1H, J ¼ 5.1 Hz), 6.60 (d, 1H, J ¼ 8.7 Hz), 6.98 (t, 1H,
J ¼ 6.9 Hz), 7.29–7.37 (m, 1H), 7.52 (d, 2H, J ¼ 8.4 Hz), 7.78 (d, 1H,
J ¼ 7.8 Hz), 7.96 (d, 1H, J ¼ 7.5 Hz), 9.45 (m, 1H), 10.91 (br, 1H); 13 C
NMR (CDCl3, 125 MHz): d ppm 15.1, 23.1, 29.6, 30.3, 35.6, 39.5,
67.9, 69.6, 83.2, 114.0, 115.0, 116.7, 126.9, 128.7, 130.5, 130.8,
132.1, 134.7, 147.3, 153.3, 158.2, 171.6, 183.1; HPLC, RT¼ 1.21 min;
MS (ESIþ): m/z 506.1 [M þ H]þ, 508.2 [M þ 2 þ H]þ; HRMS calcd for
C25H25N5O279Br 506.1192, found 506.1193.
Synthesis of compound (4S)-4-methyl-2-indol-3-yl-3,4-dihydro-5H-
pyrido[1’,2’:1,2]imidazo[4,5-d][1,3]diazepin-5-one (9’). A solution of
50 mg of compound 9 (0.12 mmol) in a 50/50 v/v mixture of tri-
fluoroacetic acid and dichloromethane (5 ml) was stirred at room
temperature. After completion of the reaction (HPLC analysis), the
solution was concentrated to dryness and co-evaporated several
times with acetonitrile to completely remove TFA. Then, 5 ml of
water were added and the solution was basified by K2CO3 and
then extracted by DCM (3 ꢁ 10 ml). The organic layer was dried
over Na2SO4, filtered, and the solvent was evaporated in vacuo to
offer compound 90 as a white solid (33 mg, yield 83%); mp:
General procedure for diazo transfer. To a solution of compound
23 as a bromhydrate salt (0.24 mmol) in 6 ml of methanol, were
added K2CO3 (110 mg, 0.8 mmol, 3.3 equiv.), copper II sulfate pen-
tahydrate (0.5 mg, 0.002 mmol, 0.01 equiv) and imidazole-1-sul-
fonyl azide hydrochloride (0.26 mmol, 1.1 equiv.). The solution was
stirred at room temperature for 24 h. The solution was then
evaporated in vacuo. 5 ml of water were added and the solution
was extracted with ethyl acetate (3 ꢁ 15 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 nHex/AcOEt 60/40 v/v and then 50/50 v/v to offer azido
compound 16 as a pale yellow powder (35 mg, yield 32%).
Spectral characteristics are in agreement with those
described below.
20
164.5–165.5 ꢂC; [a]D ¼ þ12.1ꢂ (c 1.0, MeOH); 1H NMR (DMSO-d6,
500 MHz): d ppm 1.31 (d, 3H, J ¼ 7.0 Hz), 3.98 (q, 1H, J ¼ 5.6 Hz),
7.11 (t, 1H, J ¼ 6.5 Hz), 7.16–7.20 (m, 2H), 7.47 (d, 1H, J ¼ 6.8 Hz),
7.58–7.65 (m, 2H), 8.25 (d, 1H, J ¼ 4.6 Hz), 8.30 (s, 1H), 8.62 (d, 1H,
J ¼ 6.5 Hz), 9.51 (d, 1H, J ¼ 6.7 Hz), 11.96 (s, 1H); 13 C NMR (DMSO-
d6, 500 MHz): d ppm 15.0, 56.4, 111.7, 112.2, 112.7, 115.4, 120.2,
121.9, 122.1, 126.1, 127.5, 128.6, 129.7, 130.2, 136.7, 146.4, 155.9,
158.7, 184.3; HPLC, RT¼ 1.81 min; MS (ESIþ): m/z 330 [M þ H]þ;
HRMS calcd for C19H16N5O 330.1363, found 330.1349.
Synthesis of compound (4S)-[2-(4-bromophenyl)-5-oxo-4,5-dihydro-
3H-pyrido[1’,2’:1,2]imidazo[4,5-d][1,3]diazepin-4-yl]acetic acid (21).
To a solution of compound 19 (20 mg, 0.04 mmol) in 5 ml of DCM
was added at 0 ꢂC a mixture of HBr 33% in acetic acid (5 ml). The
solution was then stirred at room temperature. After completion
of the reaction (HPLC analysis), the solution was concentrated to
dryness and the crude product was dissolved in 2 ml of DMSO
and purified by preparative HPLC (gradient of 100% H2O/0.1% TFA
to 40% CH3CN/0.1% TFA). The fractions containing the product
were collected. The ACN was evaporated in vacuo. The aqueous
residue was basified by NaHCO3 and extracted with DCM
(3 ꢁ 25 ml). The organic layer was dried over Na2SO4, filtered, and
the solvent was evaporated in vacuo to offer compound 21 as a
Biological evaluation
Cell lines and culture
Melanoma human cancer cell line (A375 and MDA-MB-435) were
obtained from ATCC. The NIH-3T3 cell line was generously gifted
by the ANSM (French National Agency for Medicines and Health
Products Safety, Vendargues, France). Cells were cultured in
Dulbecco’s Modified Eagle Medium (DMEM, Sigma Aldrich) supple-
mented with 10% heat-inactivated foetal calf serum (FCS, Gibco),
2 mM glutamax (Gibco), 100 IU/mL penicillin G sodium (Sigma
Aldrich), 100 lU/mL streptomycin sulphate (Sigma Aldrich). The
cells were allowed to grow at 37 ꢂC in a fully humidified atmos-
phere containing 5% CO2.
white solid (5 mg, yield 12%); [a]D ¼ ꢃ57,2ꢂ (c 0.25, DMSO); 1H
20
NMR (DMSO-d6, 500 MHz):
d
ppm 2.90 (dd, 1H, J ¼ 8.2 Hz,
J ¼ 16.9 Hz), 3.05 (dd, 1H, J ¼ 5.6 Hz, J ¼ 16.7 Hz), 4.38 (t, 1H,
J ¼ 5.5 Hz), 7.37 (t, 1H, J ¼ 6.0 Hz), 7.83–7.89 (m, 6H), 9.45 (d, 1H,
J ¼ 6.7 Hz), 12.71 (s, 1H); 13 C NMR (DMSO-d6, 500 MHz): d ppm
33.5, 59.8, 112.0, 115.1, 115.5, 117.9, 127.2, 128.2, 132.3, 132.5,
144.0, 152.2, 157.8, 158.1, 171.9, 179.3; HPLC, RT¼ 1.85 min; MS
(ESIþ): m/z 413 [M þ H]þ; HRMS calcd for C18H14N4O379Br 413.0244,
found 413.0257.
Neutral red uptake (NRU) viability assay
A viability assay was used to evaluate the cytotoxic activity on
A375 and MDA-MB-435 melanoma cells as previously described20.
According to OCDE/ICCVAM guidance nꢂ129, NRU assay was also
used to evaluate the toxicity on the non-cancerous NIH-3T3
cells21. Cells were seeded at a final concentration of 2000 cells/
well (A375), 3000 cells/well (NIH-3T3) or 7500 cells/well (MDA-MB-
435) in 96-well microtiter plates for 24 h. Then, cells were exposed
to two negative controls (1% DMSO in culture medium (vehicle)
and culture medium alone), or to different compounds freshly dis-
Synthesis of (4S)-N-f4-[2-(4-bromophenyl)-5-oxo-4,5-dihydro-3H-
pyrido[1’,2’:1,2]imidazo[4,5-d][1,3]diazepin-4-yl]butylgpent-4-yna-
mide (22). To a solution of compound 23 as a bromhydrate salt11 solved in DMSO, in a range of concentration from 0.01 to 40 mM
(300 mg, 0.6 mmol) in 15 ml of DCM, hydroxybenzotriazole (six wells per concentration). After 72 h of incubation, cells were

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
941
Table 1. Structure and IC50 values of compounds on the A375, MDA-MB-435 and NIH-3T3 cell lines.
IC50 A375
(mM)
IC50 MDA-MB-435
IC50 NIH-3T3
Entry
Compound
R2
4-Br-C6H4
4-HCC-C6H4
4-N3-C6H4
4-Bz-C6H4
3-benzofuranyl
3-benzothiophenyl
3-indolyl
6-quinolinyl
2-quinolinyl
6-coumarinyl
4-Br-C6H4
4-Br-C6H4
4-Br-C6H4
4-Br-C6H4
4-Br-C6H4
Ph
4-Br-C6H4
R3
R4
(mM)
(mM)
1
2
JMV5038
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
(S)-Me
(S)-Me
(S)-Me
(S)-Me
(S)-Me
(S)-Me
(S)-Me
(S)-Me
(S)-Me
(S)-Me
(S)-n-Bu
(S)-CH2CCH
2.10 0.42
10.23 0.55
7.13 0.51
>40
8.47 0.21
7.97 0.65
>40
9.76 0.95
15.57 0.7
>40
15.29 1.29
>40
1.30 0.53
5.14 0.08
4.75 0.21
>40
25.92 0.17
10.12 0.08
>40
35.49 0.21
30.71 0.19
>40
30.83 0.09
>30
>100
>40
>40
NTa
>40
>40
NT
>40
>40
NT
>40
NT
40
3
5
4
6
5
6
7
7
8
90
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
10
11
12
13
14
15
16
17
18
19
20
(S)-CH2CH(CH3)2
(S)-(CH2)4N3
(R)-(CH2)4N3
(S)-(CH2)4N3
>40
>30
NT
3.68 0.63
8.54 0.22
>40
4.12 0.22
30.90 0.11
>40
9.90 0.51
13.71 0.34
NT
(S)-CH2COOBn
9.78 0.83
19.44 0.64
>40
4-Br-C6H4
(S)-[(CH2)3]c
>40
>40
NT
4-Br-C6H4
4-Br-C6H4
H
H
(S)-CH2COOH
(S)-(CH2)4NHCO(CH2)3CCH
>40
>40
>40
>40
NT
NT
aNT: not tested.
Bold values correspond to compounds number.
stained with new medium containing 50 mg/mL of neutral red (3- 70% ethanol. The fixed cells were then incubated with 10 mg/mL
amino-7-dimethylamino-2-methylphenazine hydrochloride, Sigma RNase A (Sigma Aldrich) and 1 mg/mL propidium iodide (Sigma
Aldrich) at 37 ꢂC for 2 h. The neutral red medium solution was Aldrich), in the dark, for 24 h at 4 ꢂC. Finally, DNA content of the
then removed and the wells were washed with PBS. Finally, cells was analysed using Gallios Beckman Coulter flow cytometer
100 mL of solution containing ethanol, acetic acid and water (50/1/ with FlowJo software v1.0.
49 v/v/v respectively) were added into the wells and the absorb-
ance was measured at 540 nm using the microplate reader 352
Multiscan MS (Labsystems). Cell survival rates were normalised to
the absorbance values of untreated control cells. IC50 was
and stress actin fibres, an immunofluorescence imaging of cells
obtained using a logistic regression model on Graphpad Prism 5.0
software. Results are given as the mean standard deviation (SD)
seeded on cover slips in petri dishes and allowed to grow for
of three independent experiments. Statistical analysis was per-
formed using the Student’s t-test.
Immunofluorescent staining for actin localisation
To evaluate the effect of compound treatment on cell morphology
was performed as previously described10. MDA-MB-435 cells were
24 h. After cell growth, cells were treated with the vehicule (con-
trol cells) or with JMV5038, compound 5 or 8 at 5 mM for 20 h.
Then cells were fixed using Antigenfix for 20 min and permeabi-
Sulforhodamine B (SRB) assay
lized using 0.2% Triton X-100 for 4 min at room temperature.
Primary anticancer assay was performed on a panel of 60 human Actin was stained using a primary human anti-actin antibody
cancer cell lines derived from nine neoplastic diseases, in accord- (made from mouse) and a secondary Alexa Fluor 568-conjugated
ance with the protocol of the Drug Evaluation Branch, National goat anti-mouse antibody. Both of them were incubated 1 h at
Cancer Institute, Bethesda, Maryland, USA22. Cells were incubated room temperature. Nuclei were counter-stained using Hoechst
with tested compounds at 10 mM for 48 h. The growth percentage 33342. Representative images were obtained using a Zeiss LSM
was evaluated spectrophotometrically using a protein binding 780 inverted confocal microscopy. Confocal fluorescence micros-
dye, SRB. Results for each tested compound were reported as the copy was performed on fixed cells under a 543 nm wavelength
percent of growth of treated cells when compared to untreated excitation for AlexaFluor568, and 405 nm for nuclei, and visualised
control cells.
at 620 nm (red) and 450 nm (blue) respectively.
Cell cycle analysis
Statistical analysis
The cell cycle analysis was performed as previously described to Data set of physicochemical properties (cLogP, TPSA and molecu-
compare data with our previous results10. Flow cytometric analysis lar volume, Table 2) were calculated using the online available
was performed on 350,000 MDA-MB-435 cells seeded in culture molsoft cheminformatic tool23. IC50 values of the compounds
dishes (60 mm diameter) and allowed to grow for 48 h. Cells were included in the test are presented in Table 1 for the correlations
then treated with compound 5 or 8 at 5 mM concentration for between IC50 and physicochemical properties of compounds
16 h and 48 h. After treatment cells were harvested and fixed with modified in position 2 (Figure 2), and in Table S1 (SI) for the

942
P. L. BACCON-SOLLIER ET AL.
trifluoroacetate salts, and used without further purification.
Diamines 3a-i were then successively reacted with a set of alde-
hydes, using two strategies. For compounds bearing an alkyne
group (compounds 4 and 14), a 3-step procedure was used.
Firstly, the trifloroacetate salts of the diamines were neutralised
and converted into their corresponding free bases. Secondly,
these latter were then refluxed successively in chloroform with
the adequate aldehyde to lead to the corresponding gem-diamine
intermediate, which was then oxidised using dichloro-dicyano-1,4-
benzoquinone (DDQ). Finally, pyrido-imidazodiazepinones
4
(Scheme 3) and 14 (Table 1) were isolated in low yields (5–18%),
after purification by chromatography on alumina gel or by pre-
parative chromatography. Indeed, the presence of the alkyne
group led to several unidentified by products, which complicated
the purification step.
For other derivatives, diamines 3a-i, as trifluoroacetate salts,
were directly reacted successively with a set of aldehydes with
potassium carbonate and iodine at 70 ꢂC overnight. Initially, tert-
butanol was used as the solvent. However, we found that tetra-
hydrofurane (THF) gave the same results and as it is easier to
remove, this solvent was preferred for the cyclisation step.
Pyrido-imidazodiazepinones 5–13 and 15–20 were finally iso-
lated in 8–98% yield, after purification by chromatography on
alumina gel. The introduction of a chiral center alpha to the car-
bonyl group might lead to partial racemization. However, using
chiral HPLC analysis, we previously demonstrated that no epi-
merization occured during the synthesis of diazepine
derivatives13
.
For several compounds, the necessary aldehydes used in the
cyclisation step were synthesised (Scheme 2). For compounds 6
and 10, where R2 is a 4-benzophenyl or a 6-quinolinyl substituent
respectively, the corresponding aldehydes 24 and 26 were pre-
pared, using a 3-step procedure, starting from their respective car-
boxylic
acids.
Briefly,
4-benzoyl-benzoic
acid
or
6-
quinolinecarboxylic acid were esterified using ethanol in acidic
medium. The ethyl esters 23 or 25 were then reduced into their
corresponding alcohols, using lithium aluminium hydride. These
latter were then oxidised using manganese (II) oxide to generate
4-benzoyl-benzaldehyde 25 and quinoline-6-carbaldehyde 26. For
compound 40 bearing an alkyne group, the TMS protected 4-for-
mylphenylacetylene 27 was prepared from 4-bromobenzaldehyde,
using a Sonogahira reaction, according to the procedure described
by Cummings et al. (Scheme 3)24. Boc protecting-3-indolecarboxal-
dehyde 28 was prepared from 3-indolecarboxaldehyde and di-tert-
butyl dicarbonate, using the procedure described by Pelcman
et al. (Scheme 3)25. After cyclisation and oxidation, compounds 40
and 90 were then unprotected, using respectively a solution of tet-
rabutylammonium fluoride in THF or a mixture of TFA/DCM,
according to Scheme 3.
Figure 2. Correlations between IC50, determined in two melanoma cell lines, and
molecular volume of compounds modified in position R2 (A and B) or between
IC50 and cLogP (C and D). ᭿: A375 cell line; ᭜: MDA-MB-435 cell line; r:
Pearson’s correlation coefficient, p values: level of significance, n: sample size; the
dotted line separates the most active compounds (IC50 < 15 mM) from the less
active ones (IC50 > 15 mM), except for compound 9’ (see the text).
correlations between in vitro anti-cancer activity of JMV5038, com-
pound 5 and compound 7 on the NCI cell line panel (Figure 4).
Pearson’s correlation coefficient r and level of significant p values
were calculated using Origin Pro 2019.
Results and discussion
For 4-azidobutyl derivatives 16–18, introduction of the azido
group was studied using two strategies. Initially, the azido group
was introduced in the final step of the sequence by diazo transfer
on the free amino derivative 23, using imidazo-1-sulfonyl azide in
presence of potassium carbonate and copper sulfate (Scheme 4).
The imidazolyl reactant was preferred to the classical sodium
azide, because this reagent is more stable and easier to handle26.
The diazo transfer reaction proceeded smoothly and led to several
by-products, which complicated the purification step. Compounds
16–18 were finally isolated in 10% yields from compound 1 (5-
step procedure). In a second approach, Boc-6-azido-Nle-OH 29
was prepared from Boc-Lys-OH, according to the procedure
Chemistry
Pyrido-imidazodiazepinones 4–20 were synthesised starting from
2-amino-imidazo[1,2-a]pyridine 1, by selective C-3 acylation of the
imidazo[1,2-a]pyridine nucleus (Scheme 1), using 9 different N-Boc
amino-acids (Boc-Ala-OH, Boc-Nle-OH, Boc-Leu-OH, Boc-Asp(Bn)-
OH, Boc-Pra-OH, S or R Boc-Lys(Cbz)-OH, Boc-6-azido-Nle-OH or
Boc-Pro-OH), according to our previously reported method-
ology10,11,13,14. C-3 acylated compounds 2a–i were isolated in
36–97% yield after chromatography on alumina gel. Only traces of
the corresponding N-acylated derivatives were detected in the
crude mixture by LC-MS analysis. After Boc removal using a mix-
ture of trifluoroacetic acid/dichloromethane (50/50 v/v), the result- described by Shoonen et al.15 Acylation of 1 with compound 29
ing 2-amino-3-acyl-imidazo[1,2-a] pyridines 3a-i were isolated as led to compound 3 h in 71% yield. The Boc group was then

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
943
Scheme 1. Synthesis of pyrido-imidazodiazepinones 4–20. Conditions and reagents: Boc-AA-OH: Boc-Ala-OH, Boc-Nle-OH, Boc-Leu-OH, Boc-Asp(Bn)-OH, Boc-Pra-OH,
Boc-Lys(Cbz)-OH, R-Boc-Lys(Cbz)-OH, Boc-azidonorleucine 29 or Boc-Pro-OH; i: 1. NH4OH, H2O; 2. R2CHO, CHCl3, 60 ꢂC; 3. DDQ, rt; ii: R2CHO, K2CO3, I2, THF, 65 ꢂC. For
compounds 4–20, R2, R3 and R4 substituents are referenced in Table 1.
Scheme 2. Synthesis of aldehydes 24 and 26.
Scheme 3. Synthesis of compounds 4’ and 9’.
Structure-activity relationships
deprotected, using a mixture of trifluoroacetic acid/dichlorome-
thane (50/50 v/v) and the resulting diamine was cyclized with 4-
bromobenzaldehyde to offer compound 16 in 30% yield.
Compared with the initial 5-step sequence, the 4-step procedure
led to a better global yield from compound 1 (21% vs 10%) and
compounds were easier to purify. Finally, alkyne derivative 22 was
prepared in 94% yields by acylation of the amine 23 with penty-
noic acid (Scheme 4).
The in vitro cytotoxic activity of diazepine derivatives was meas-
ured towards A375 and MDA-MB-435 melanoma cell lines, using
neutral red uptake viability assay. The half maximal inhibitory con-
centrations (IC50) values are reported in Table 1.
We started our investigation by studying the position 2 of the
diazepine ring. From our previous studies, we observed that 4-
bromophenyl, 4-iodophenyl, 2,4-dichlorophenyl and 1- or 2-

944
P. L. BACCON-SOLLIER ET AL.
Scheme 4. Synthesis of compounds 16 and 22.
naphthyl substituents were the most preferred groups in this pos- compounds 10, 11 and 12 possess a greater polar surface
ition11, which could suggest that such substituents occupy a large (Topological Polar Surface Area, TPSA) and a weaker cLogP (Table
hydrophobic pocket in the target. To further study this position, 2). Whereas an increase of the polar surface was well tolerated in
we investigated the substitution of this position by several other the case of the azido compound 5 (TPSA increase of 185% for
hydrophobic groups.
compound 5 compared to JMV5038) suggesting that this param-
eter is not crucial for the activity of these series of compounds,
the lipophilicity seems to be more important. Indeed, the cLogP
of compounds 10 to 12 (2.43, 2.53 and 1.84 respectively) were
weaker or slightly weaker than those calculated for JMV5038 and
its 1- or 2-naphthyl analogues (2.83 and 3.31/3.43, Table 2).
Concerning the [6 þ 5] derivatives, best activities on A375 cell line
Firstly, an alkyne, an azido group or a phenylketone were intro-
duced in para position of the phenyl substituent, because we pre-
viously demonstrated that the para position was the most
favourable position for the phenyl substitution11. The results are
reported in Table 1 (entries 2–4). The activities were comparable
for the two tested melanoma cell lines, even if the MDA-MB-435
cell line was more sensitive to compounds 40 and 5, as also
observed with JMV5038 (Table 1, entry 1). Whereas the substitu-
tion of the bromine atom of JMV5038 by an azido group (com-
pound 5) or, in a lesser extent, by an alkyne group (compound 40)
were obtained with the benzothiophene derivative 8 (IC50
¼
¼
7.97 mM), followed by the benzofuran derivative
7 (IC50
8.47 mM). Indolyl derivative 90 was totally inactive on the two mel-
anoma cell lines. Among the three [6 þ 5] analogues tested, which
maintained the cytotoxic activity in the two melanoma cell lines are known to be bioisosters of the naphthyl group27, indole
derivative 90 is slightly more hydrophilic (cLogP ¼ 2.20) than the
tested, the benzophenone derivative 6 was totally inactive, prob-
ably due to the steric hindrance brought by the supplementary
benzofuran and the benzothiophene derivatives 7 and 8 (cLogP ¼
phenyl ring. Indeed, the molecular volume of compound 6, pre- 2.47 and 2.93 respectively, Table 2), confirming that the lipophilic-
dicted using the molsoft cheminformatic tool, is about 20%
ity is a crucial parameter for the choice of substituents to intro-
greater than that of JMV5038 (397 vs 317 Å3, Table 2), whereas for duce in position 2.
compounds 40 and 5, the molecular volume increase is only 2%
To further evaluate the influence of some physicochemical
and 4% respectively (324 and 330 Å3), suggesting that the parameters on the cytotoxic activity of compounds modified at
molecular volume of the substituent occupying the position 2 of position 2 (i.e. compounds 40212), their IC50 values were graphed
the diazepine ring is a crucial parameter for the biological activity. as a function of three commonly used molecular descriptors
Introduction of [6 þ 6] or [6 þ 5]-heterocycles was then investi- (Figure 2), namely cLogP, molecular volume and TPSA. Whereas
gated at position 2. Surprisingly, whereas the MDA-MB-435 cell no correlation were observed between TPSA and IC50 (correlation
line was more sensitive to JMV5038 and to compounds 40 and 5 coefficient of 0.22 and 0.13 for A375 and MDA-MB-435 cell lines
than the A375 cell line (see entries 1–3, Table 1), these com- respectively, p values > 0.1, data not shown), a statistically signifi-
pounds were around 3 times less active on MDA-MB-435 than on cant correlation was obtained when the IC50 values were com-
A375 cells (see for example, compounds 7, 10 or 11, entries 5, 8 pared to the molecular volume of compounds 40212 and
and 9, Table 1). This result could suggest that the molecular JMV5038, with a Pearson correlation coefficient of 0.75 (p values
mechanism involved in the cytotoxic activity of compounds 7, 10 ¼ 0.01) for A375 cell line and 0.65 (p values ¼ 0.04) for MDA-MB-
and 11 is not totally similar to the one involved in the activity of 435 cell line. Thus, to obtain some activity on the two melanoma
JMV5038. For [6 þ 6] derivatives, the coumarinyl group totally cell lines, the molecular volume of the diazepinone derivative
abolished the activity (compound 12), whereas the 2- or 6-quino- need to be less than 350 Å3 (except for compound 90, see below)
linyl groups were tolerated, even if the biological activities were and with a cut off of around 330 Å3 to obtain an IC50 less than
weaker than those obtained with their previously described naph- 10 mM (Figure 2(A,B)). Thus, a large substituent like a benzophe-
thyl analogues (IC50 ¼ 35.49 and 30.71 mM for compounds 10 and none (compound 6) or a coumarinyl group (compound 12) led
11 respectively, compared to IC50 of 1.4 mM for 1- or 2-naphthyl probably to a steric clash with the target and to the loss of the
derivatives on MDA-MB-435 cell line11). Indeed, compound 12 activity. Then, the influence of the cLogP was investigated. After
possesses a large molecular volume (370.59 Å3), which could be removing compounds 6 and 12 for a too high molecular volume,
detrimental for the activity, as observed with compound 6. a statistically significant correlation was obtained when the IC50
Moreover, in comparison with their naphthyl analogues, were compared to the cLogP, with a correlation coefficient of ꢃ

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
945
Table 2. Some calculated physicochemical parameters of compounds modified
on position 2.
Molecular
Entry
Compound
cLogPa
TPSA (Å2)a
volume (Å3)a
1
2
3
4
5
6
7
8
JMV5038
2.83
3.31
3.43
2.53
2.74
3.24
2.47
2.93
2.20
2.43
2.53
1.84
43.63
43.36
43.36
43.63
80.94
57.10
53.19
44.38
53.50
52.39
51.96
64.04
316.84
344.46
344.32
330.95
324.19
397.26
329.84
339.6
333.93
339.34
339.34
370.59
1-naphthyl derivativeb
2-naphthyl derivativeb
40
5
6
7
8
9
90
10
11
12
10
11
12
aCalculated using the molsoft chemoinformatic tool; bNaphthyl derivatives were
11
described in ref.
.
Bold values correspond to compounds number.
Figure 3. Correlation 3 D-plot between calculated molecular volume, cLogP and
cytotoxic activity of compounds modified in position R2 (mean of IC50 determined
on MDA-MB-435 and A375 melanoma cell lines).
Finally, a 3D diagram was built to visualise the influence of
these two physicochemical parameters on the cytotoxic activity of
compounds modified at position 2 (mean of the IC50 obtained on
the two melanoma cell lines). Figure 3 shows clearly that a cLogP
between ꢅ2.5 and 2.8, associated with a molecular volume
between 310 and 350 Å3 are necessary to obtained active com-
pounds in these series of compounds.
To resume the RSA study of the position 2, compound 5
showed the best activity of all derivatives modified in this pos-
ition, with cytotoxic activities against the two tested melanoma
cell lines closed to those obtained with JMV5038. Azido group is
known to exhibit some properties in common with halogens and
in particular this group resembles a bromo substituent in terms of
approximate size, polarity and electronic properties28, which could
explain that compound 5 presented a similar profile of activity
to JMV5038.
Then, we turned our attention to the SAR study of the position
4 of the diazepine ring, keeping constant the 4-bromophenyl
group at R2 (i.e. compounds 13–17 and 19–22). We previously
demonstrated that position 4 could tolerate short linear alkyl
chains (methyl, ethyl or hydroxymethyl), but also bulky groups
like tert-butyl, but not too close to the diazepine ring11. To further
study this position, several other chains were introduced into pos-
ition 4. n-Butyl and n-azidobutyl chains are tolerated (compounds
13 and 16 respectively), with a good activity obtained for com-
pound 16, close to the activity obtained with JMV5038 (mean of
IC50 values in MDA-MB-435 cells are 4.12 mM for compound 16
compared to 1.30 mM for JMV5038, entries 1 and 14, Table 1).
Surprisingly, we observed that the stereochemistry is not so cru-
cial for the activity when an n-azidobutyl chain was introduced at
position 4 of the diazepine ring. Whereas a R-methyl group at
position 4 led to a totally inactive compound10, a R-azidobutyl
chain led only to a slight decrease of the activity on A375 cell
line, and to a more significant decrease for the MDA-MB-435 cell
line (see entries 14 and 15, Table 1). This result could suggest that
the n-azidobutyl derivatives were not positioned exactly in the
same way as the methyl analogues in the target or that the mech-
anisms of action involved in the cytotoxicity are not totally similar.
However, as also observed with the 4-methyl derivatives, the pres-
ence of a bromophenyl substituent at R2 is still necessary, because
compound 18 is totally inactive (entry 16, Table 1). Introduction of
a ramified isobutyl chain (compound 15) led also to a loss of the
activity (entry 13, Table 1). This result is not surprising, because
Figure 4. Evaluation of in vitro anti-cancer activity of JMV5038, compounds 5, 8
and 16 on the NCI-60 cell line panel, after 48 h of exposure at a single dose of
10 mM of each tested compound. Presented results are cell growth level deter-
mined for the 4 compounds on the 27 most JMV5038 sensitive cell lines (i.e.
mean growth % – growth % > 0). For all data (60-cell lines), see Table S1, SI.
The difference between the growth level of a specific cell line and the mean
growth is represented by a bar. Cell lines that were more sensitive are displayed
as bars that project to the right of the mean. Cell lines that were less sensitive
are displayed with bars projected to the left.
0.76 (p values ¼ 0.03) for A375 and ꢃ 0.81 (p values ¼ 0.01) for
MDA-MB-435 (Figure 2(C,D)). Thus, a cLogP greater than 2.4 was
required to obtain a cytotoxic activity (IC50 ꢄ 10 mM) within these
series of compounds. This result could explain that the indole
derivative 90, even if it possesses a molecular volume less than
350 Å3, presents a cLogP slightly too low to be active (namely we already reported that an isopropyl chain in this position is
2.20, Table 2).
unfavourable for the activity11, suggesting that substituents borne

946
P. L. BACCON-SOLLIER ET AL.
possible modification of the mechanism of action was hypothes-
ised for these two derivatives (see above), this phenomenon could
probably be also correlated with the cytotoxicity observed on the
NIH-3T3 cell line.
In vitro antiproliferative activities on the NCI-60 cancer cell
line panel
From the SAR study of the position 2 of the diazepine ring con-
ducted on A375 and MDA-MB-435 cell lines, it appeared that the
sensitivity of the two melanoma cell lines tested towards several
compounds was quite different.
Whereas a substituted phenyl group in position 2 of the diaze-
pine (compounds 40 and 5) led to a similar response than
JMV5038 (i.e. more active on MDA-MB-435 than on A375 cell lines,
Table 1, entries 2–3), introduction of [6 þ 6] or [6 þ 5]-heterocycles
in this position led to compounds more active on A375 than on
MDA-MB-435 cell lines (compounds 7–8, 10–11, Table 1, entries
5–6 and 8–9). Even if the mechanism of action and the target of
JMV5038 are not elucidated at this moment, the specificity of the
activity profiles on different cell lines could suggest that these
new derivatives did not share exactly the same mechanism
involved in the cytotoxic activity. To better compare the activity
profile of these two series of compounds, compounds 5 and 8
were selected and then evaluated towards a panel of sixty cancer
cell lines corresponding to nine different cancer types, i.e. leukae-
mia, melanoma, as well as lung, colon, central nervous system,
ovarian, renal, prostate and breast cancers (NCI evaluation).
Indeed, it is now well established that the growth inhibitory pat-
terns of anticancer drugs against the NCI-60 cell line panel corre-
lated well with their modes of action29. For example, compounds
that bind to tubulin have similar growth inhibition patterns
regardless of which site on tubulin they bind30. Compounds 5 and
8 were chosen because they are the most active compounds in
the two series of 2-modified derivatives (Table 1, entries 3 and 6).
Compounds were tested using the sulforhodamine B (SRB) assay
that provides a direct cell density index. The amount of dye incor-
porated by the cells increases with the increase of the total pro-
tein biomass. Cells were incubated for 48 h with compounds at
10 mM concentration, then cell growth was evaluated spectro-
photometrically and presented as the percent of untreated control
cells. Values of the most sensitive cell lines were compared to
those obtained with JMV5038 (Figure 4, for all data see SI).
As suggested by our results obtained on A375 and MDA-MB-
435 cell lines, the activity profile on the NCI-60 cell line panel
revealed a great difference of sensitivity between the two tested
compounds. Compound 5 presented a profile similar to that
obtained with JMV5038 (statistically significant correlation,
r ¼ 0.72, p values < 0.01), with the best activity obtained against
MDA-MB-435 melanoma cell line (growth inhibition percentage of
87%), as already observed with JMV5038 and several other ana-
logues11. On the other hand, the activity profile obtained with
compound 8 was very different from that of JMV5038 and no cor-
relation was obtained (r ¼ 0.07, p values ¼ 0.63), confirming that
the mechanism of action involved in the cytotoxic activity is prob-
ably not totally comparable to that of JMV5038.
Figure 5. Flow cytometric analyses showing the cell cycle distribution of MDA-
MB-435 cells treated with DMSO or 5 mM concentration of compound 5 or 8 for
16 h or 48 h. The panels represent distribution of cells (%) in Sub G1-, G1-, S-,
G2/M- phase of the cell cycle.
by position 4 of the diazepine ring occupied a relatively narrow
pocket in the target. In the same way, a less flexible chain bearing
an alkyne group (compound 14) was not tolerated, as well as a
long chain (compound 22) (Table 1, entries 12 and 20 respect-
ively). Surprisingly, some activity was recovered with the bulky
benzyl ester derivative (compound 19, entry 17, Table 1), whereas
compounds with chains bearing a phenyl ring (namely a benzyl or
a -(CH2)4NHCbz substituent) were previously determined to be
inactive10,11. This result could suggest that compound 19 could
be a pro-drug. Indeed, compound 19 was partially converted into
its corresponding acid 21, with a hydrolysis rate of 65.5%, deter-
mined by HPLC-MS, after 72 h in the presence of melanoma cells
and culture medium. The acid derivative 21 was prepared and
tested, but this compound revealed to be inactive (Table 1, entry
19), which seems to invalidate the pro-drug hypothesis. At this
time we could not exclude that the main problem with com-
pound 21 is not its activity towards the target, but its increase of
polarity (cLogP of 2.1 for compound 21, compared to 3.97 for the
ester 19); this parameter revealing to be important for the bio-
logical activity. However, contrary to what was observed with
modulations of the position 2 of the diazepine ring, no significant
correlation could be highlighted between the activity of the 4-
modified compounds and their cLogP or their molecular volume
(data not shown).
Finally, the introduction of a fused tetrahydropyrrolo cycle
between positions 3 and 4 (compound 20) led to the loss of the
activity. This result confirms that the position 3 of the diazepine
ring needs to be unsubstituted, as already observed with the N3-
methyl derivative of JMV503811.
Concerning the compounds modified in position 4 (compounds
13–22), the response towards the two tested melanoma cell lines
appeared also different (Table 1, entries 11–20). Compounds 13,
Then, all active compounds on the two melanoma cell lines
(IC50 < 40 mM) were assessed on the NIH-3T3 mouse fibroblast cell
line, in order to check the toxicity of our compounds on non-can-
cerous cells. Globally, the diazepine compounds were not toxic on 16, 17 and 19 were more active on A375 cell line than on MDA-
this cell line (IC50 > 40 mM), except for compounds 16 and 17 MB-435 (inverse of the JMV5038 profile). Moreover, compounds
bearing an n-azidobutyl chain, where a not negligible toxicity was 16 and 17 led also to some toxicity on NIH-3T3 cell line, indicat-
noted (IC50 around 10 mM, Table 1, entries 14 and 15). As a ing a probably modified mechanism of action, compared to

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
947
Figure 6. Confocal microscopy of actin localised in fixed MDA-MB-435 cells. Cells were incubated with 5 mM concentration of JMV5038, compound 5, compound 8 or
DMSO (control) for 20 h. Nuclei were stained with Hoechst 33342. AlexaFluor568 and nuclei were excited at 543 and 405 nm, and visualised at 620 (red) and 450 nm
(blue) respectively.
JMV5038. This result was confirmed by analysing the activity pro- were treated with or without 5 mM concentration of compound 5
file of compound 16 on the NCI-60 cancer cell line panel (Figure or 8 for 16 h and 48 h.
After 16 h, compound 5 increased the proportion of cells in
4), where no correlation was obtained with JMV5038 (r ¼ 0.06, p
G2/M phase (33.1% compared to 14.3% in control). At 48 h, this
increase was still present, but less marked (18.4% vs 14.3%). A
values ¼ 0.66).
slightly increase of apoptosis in subG1 phase was also observed
(2.4% compared to 1.2% in control). These induced changes in the
DNA cell cycle analysis
cell cycle were quite comparable with those already observed in
cells treated with JMV503810. For compound 8, a slightly increase
Compounds 5 and 8 were then selected for further characterisa-
tion, because these two compounds showed different activity pro-
files (similar to that of JMV5038 for compound 5 and different for
compound 8). Thus, compound 5 could probably shared a com-
mon mechanism of action with JMV5038, contrary to compound
8. DNA cell cycle analysis was performed using MDA-MB-435 cells
treated with either compound 5 or compound 8, and compared
to that obtained with cells treated with vehicle (DMSO) as a nega-
tive control10. Cell cycle pattern changes of MDA-MB-435 cells
of the proportion of cells in G2/M phase was also observed
(20.8% at 16 h and 15.4% at 48 h vs 14.3% in control). However,
compound 8 increased the proportion of cells in phase G1 at 16 h
(53.0% compared to 47.1% in control). This profile was opposite
from that observed with compound 5, which led to a decrease of
cells in G1 phase at 16 h. The activity profile of compounds 5 and
8 on the NCI-60 cancer cell lines was quite different and this dis-
similarity was also observable through the analysis of the cell
were investigated using fluorescence-activated cell sorting (FACS) cycle, with a slightly shifted distribution of DNA, particularly in the
(Figure 5 and Figure S1, SI). In this experiment MDA-MB-435 cells G1 phase.

948
P. L. BACCON-SOLLIER ET AL.
Morphological changes of cells
facility MRI, member of the national infrastructure France-
BioImaging infrastructure supported by the French National
Research Agency (ANR-10-INBS-04, “Investissements d’avenir”). The
authors wish to especially thank Christophe Duperray for his tech-
nical assistance within the platform. NM thanks Eva Beltrando and
Sabrina Fellous for technical assistance.
In our previous study, we reported that JMV5038 led to changes
in the cell morphology, concomitant with a disruption of the actin
network10. Morphological changes in MDA-MB-435 cells induced
by compounds 5 or 8 were investigated and compared to those
induced by JMV5038. For this purpose, fixed and actin immunos-
tained MDA-MB-435 cells were imaged by confocal microscopy
(Figure 6). Control cells treated with DMSO (20 h, 5 mM concentra-
tion) were spread and the tensile forces of actin stress fibres
stretched the cell-adhesive phenotype. In contrast, cells treated
with compounds 5 or 8 (20 h, 5 mM concentration) showed the
most recognisable actin-morphological features, comparable with
those obtained with cells treated with JMV5038. Even if com-
pounds 5 and 8 showed a different profile of activity on the two
tested melanoma cell lines and on the NCI-60 cell line panel, the
morphological changes induced by the three tested compounds
were very similar. A diffuse distribution of non-polymerized actin,
associated with a disorganisation of the actin cytoskeleton and
altered malignant melanoma cell morphology were observed.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was publicly funded through ANR (the French National
Research Agency) under the “Investissements d’avenir” pro-
gramme with the reference ANR-16-IDEX-0006 and by the “Ligue
ꢀ
ꢀ
Nationale contre le Cancer (Comite de l’Herault)”.
ORCID
Lamiaa M. A. Ali
Pierre Cuq http://orcid.org/0000-0002-5440-9165
http://orcid.org/0000-0003-1176-5335
Conclusion
Laure-Anaïs Vincent
Nicolas Masurier
http://orcid.org/0000-0003-3613-859X
http://orcid.org/0000-0001-9169-8149
Nineteen new pyrido-imidazodiazepinone derivatives were syn-
thesised, bearing various substituents at positions 2, 3 and 4 of
the diazepine ring. Their cytotoxic activities against A375 and
MDA-MB-435 melanoma cell lines were evaluated, as well as their
potential toxicity against NIH-3T3 fibroblast cells. Introduction of a
4-ethynyl-, a 4-azidophenyl substituent was tolerated onto the
position 2 of the diazepine ring and led to compounds with a
similar in vitro activity on melanoma cells and with no apparent
toxicity on NIH-3T3 cells. On the other hand, introduction of a
bicyclic and heteroaromatic group on this position led to com-
pounds possessing a significant difference of activity towards the
two tested melanoma cell lines. These results were confirmed by
comparing the activity profile of compounds 5 and 8 on the NCI-
60 cell line panel, indicating that the mechanisms of action of
these two compounds are not exactly similar. This dissimilarity
was also observed through a slightly shifted distribution of the
cell cycle phases. However, both compounds led to comparable
modifications on cells morphology. SAR studies revealed also that
the molecular volume of the substituent introduced onto the pos-
ition 2 of the diazepine ring, as well as the cLogP of the molecule,
were two crucial parameters to maintain the activity. Thus, a
molecular volume less than 350 Å3 and a cLogP greater than
ꢅ2.5 were necessary to maintain the activity. These results will
guide the choice of the substituents to be introduced at this pos-
ition in next studies. Then, modulations of the position 4 of the
diazepinone revealed that a butyl chain, an azidobutyl chain or an
ester group were tolerated, even if the azido-alkyl group led to
some toxicity on the NIH-3T3 cells. However, such azido com-
pounds could be particularly interesting to introduce further mod-
ulations using click-chemistry. In particular, introduction of a
biotin or a fluorescent moiety on these compounds could be help-
ful to determine their cellular localisation as well as their pharma-
cological target using a proteomic approach.
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