Pyrido-imidazodiazepinones as a new class of reversible inhibitors of human kallikrein 7

Article abstract of DOI:10.1016/j.ejmech.2015.02.008

The human tissue kallikrein-7 (KLK7) is a chymotryptic serine protease member of tissue kallikrein family. KLK7 is involved in skin homeostasis and inflammation. Excess of KLK7 activity is also associated with tumor metastasis processes, especially in ovarian carcinomas, prostatic and pancreatic cancers. Development of Kallikrein 7 inhibitors is thus of great interest in oncology but also for treating skin diseases. Most of the developed synthetic inhibitors present several drawbacks such as poor selectivity and unsuitable physico-chemical properties for in vivo use. Recently, we described a practical sequence for the synthesis of imidazopyridine-fused [1,3]-diazepines. Here, we report the identification of pyrido-imidazodiazepinone core as a new potential scaffold to develop selective and competitive inhibitors of kallikrein-related peptidase 7. Structure-activity relationships (SAR), inhibition mechanisms and selectivity as well as cytotoxicity against selected cancer cell lines were investigated.

Full text of DOI:10.1016/j.ejmech.2015.02.008

European Journal of Medicinal Chemistry 93 (2015) 202e213
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Pyrido-imidazodiazepinones as a new class of reversible inhibitors of
human kallikrein 7
,
,
Dominique P. Arama a 1, Feryel Soualmia b 1, Vincent Lisowski a, Jean-François Longevial a,
Elodie Bosc b, Ludovic T. Maillard a, Jean Martinez a, Nicolas Masurier a
,
,
*
,
*
Chahrazade El Amri b
a
ꢀ
ꢀ
Institut des Biomolecules Max Mousseron, UMR 5247, CNRS, Universite de Montpellier, UFR des Sciences Pharmaceutiques et Biologiques,
15 Avenue Charles Flahault, 34093 Montpellier Cedex 5, France
b
ꢀ
Sorbonne Universites, UPMC Univ Paris 06, UMR 8256, B2A, Biological Adaptation and Ageing, Integrated Cellular Ageing and Inflammation,
Molecular & Functional Enzymology, 7 Quai St Bernard, F-75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The human tissue kallikrein-7 (KLK7) is a chymotryptic serine protease member of tissue kallikrein
family. KLK7 is involved in skin homeostasis and inflammation. Excess of KLK7 activity is also associated
with tumor metastasis processes, especially in ovarian carcinomas, prostatic and pancreatic cancers.
Development of Kallikrein 7 inhibitors is thus of great interest in oncology but also for treating skin
diseases. Most of the developed synthetic inhibitors present several drawbacks such as poor selectivity
and unsuitable physico-chemical properties for in vivo use. Recently, we described a practical sequence
for the synthesis of imidazopyridine-fused [1,3]-diazepines. Here, we report the identification of pyrido-
imidazodiazepinone core as a new potential scaffold to develop selective and competitive inhibitors of
kallikreinerelated peptidase 7. Structure-activity relationships (SAR), inhibition mechanisms and selec-
tivity as well as cytotoxicity against selected cancer cell lines were investigated.
Received 9 October 2014
Received in revised form
3 February 2015
Accepted 6 February 2015
Available online 7 February 2015
Keywords:
Imidazo[1,2-a]pyridine
Polyfused heterocycles
Diazepinones
© 2015 Elsevier Masson SAS. All rights reserved.
Serine protease
Human tissue kallikrein 7
Non-covalent inhibitors
1. Introduction
extracellular proteases, which are able to cleave one or more of
these cellular adhesion proteins. Among these various families of
Tumor invasion and dissemination are complex and multi-step
processes involving several key cellular events. Among the many
events leading to metastasis, loss of intercellular adhesion is one of
the earliest phenomenon, which could lead to cells dissemination
[1]. One of the most well characterized mechanisms by which tu-
mor cells can lose adhesion is by up-regulating the expression of
proteases, the human tissue kallikrein serine proteases (KLKs) are
known to be over-expressed in various cancers [2,3].
In this context, kallikrein 7 (KLK7) a chymotryptic enzyme [4,5],
was reported to be implicated in several tumor metastasis pro-
cesses, especially in ovarian carcinomas, pancreatic cancers, by the
hydrolysis of several cadherin subtypes [6,7]. KLK7 facilitates
metastasis via degradation of extracellular matrix components
such as fibronectin, desmosomal proteins particularly desmogleins
1 and 2, thus reducing cellecell adhesion. In prostatic cancers, KLK7
induces epithelial to mesenchymal transition-like changes in
prostate carcinoma cells, implying a role in prostate cancer invasion
and progression [8]. Furthermore, KLK7 like other kallikreins such
as KLK5, KLK8, KLK14 and matriptase plays an important role in
normal skin desquamation [9e11]. Such enzymes can hydrolyze
extracellular proteins that are part of intercellular adhesion struc-
tures such as corneodesmosomes in the stratum corneum [12]. As a
consequence, mutation in the Spink5 gene of LEKTI, the physio-
logical inhibitor of skin kallikreins, is responsible of the Netherton
Abbreviations: AMC, 7-amino-4-methylcoumarin; DCM, dichloromethane;
DMEM, Dulbecco's Modified Eagle Medium; DMSO, dimethyl sulfoxide; EDCI, 1-
Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; HOBt, hydrox-
ybenzotriazole; IP, imidazo[1,2-a]pyridine; KLK5, kallikrein-related peptidase 5;
KLK7, kallikrein-related peptidase 7; KLK8, kallikrein-related peptidase 8; KLK14,
kallikrein-related peptidase 14; LEKTI, lymphoepithelial Kazal-typeerelated in-
hibitor; PDB, Protein Data Bank; TFA, trifluoroacetic acid; THF, tetrahydrofuran; XTT,
2,3-Bis-(2-Methoxy-4-Nitro-5-sulfophenyl)-2H-Tetrazolium-5-Carboxanilide.
* Corresponding authors.
E-mail addresses: nicolas.masurier@univ-montp1.fr (N. Masurier), chahrazade.
el_amri@upmc.fr (C. El Amri).
1
These authors contributed equally to the work.
http://dx.doi.org/10.1016/j.ejmech.2015.02.008
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

D.P. Arama et al. / European Journal of Medicinal Chemistry 93 (2015) 202e213
203
syndrome, a lethal recessive autosomal skin disorder [13]. Recently,
in an organotypic human skin culture used as a model system of
Netherton Syndrome, it has been shown that genetic inhibition of
KLK5 and KLK7 ameliorates epidermal architecture demonstrating
the relevance of these two enzymes as targets for treating this
disease [14]. On the other hand, KLK7 transgenic mice exhibit a
phenotype comparable to psoriasis with cutaneous hyper-
proliferation and immune cell infiltration [15]. Obese subjects often
suffer from psoriatic lesions associated with high levels of adipo-
kines such as chemerine. Beck-Sickinger and coll. have recently
shown that KLK7 mediated pro-chemerine processing contributing
thus to the development of these psoriatic lesions [16].
Consequently, the development of KLK7 inhibitors to control
unopposed proteolytic cascades could represent a potential thera-
peutic approach to avoid or decrease metastasis associated with
over-expression of KLK7 as well as for treating some skin disorders.
Among the reported inhibitors of KLK7 [17], a few consist in peptide
derivatives such as chloromethylketones [5], LEKTI-derived pep-
tides [18] and sunflower trypsin inhibitor (SFTI-1) peptide de-
rivatives [19]. More recently some heterocyclic compounds such as
natural isocoumarins (as 8,80-paepalantine) [20] isomannide de-
rivatives [21,22] or a mercaptobenzimidazolyl-quinazoline deriva-
tive [23] have also shown significant activities against KLK7 (Fig. 1).
All of them are reversible KLK7 inhibitors with moderate activity
potential inhibiting activity against KLK7. We report herein the
identification of the pyrido-imidazodiazepinone structure as a new
potential scaffold to develop selective and competitive KLK7 in-
hibitors. Structure-activity relationship (SAR), inhibition mecha-
nisms and selectivity, as well as biological evaluations against
selected cancer cells were investigated.
1.1. Screening of the diazepinone library and rationale for the
synthesis of analogs
Due to the heterogeneity of the heterocyclic scaffolds used to
obtain KLK7 inhibition, 65 heterocyclic derivatives from our inter-
nal library were chosen for an initial screening (data not shown),
including imidazo[1,2-a]pyridine (IP) and 1,3-thiazole derivatives,
due to our particular interest in these heterocyclic scaffolds
[25e31].
Compounds were all tested at the single dose of 50
them, pyrido-imidazodiazepin-5-one showed an inhibition
capability higher than 30% (compound 1a, Table 1)
(IC50 ¼ 57 1.0 M). The reversibility of the inhibition was evalu-
mM. Among
a
m
ated by dilution of the reaction mixture after 15 and 60 min incu-
bation of 1a with KLK7 at pH8 and 37 ꢀC. The enzymatic activity
was immediately restored in agreement with no formation of a
covalent bond between the protease and the inhibitor. Line-
weavereBurk double reciprocal plot was used to determine the
mechanism of action of compound 1a. Plots are consistent with a
competitive inhibitor behavior with an inhibition constant Ki of
(10 < IC50 < 200
mM). Additionally, some acyl-1,2,4-triazoles were
reported to be more potent inhibitors (0.04 < IC50 < 3.5
mM), with
the ability to form transient covalent complexes (acyl-enzymes)
with both KLK7 and KLK5 [24]. Since, most of the identified in-
hibitors suffer from low selectivity, moderate activity and unsuit-
able physico-chemical properties for in vivo use, the identification
of new chemical scaffold for potent and reversible inhibition of
KLK7 is thus needed.
A survey of the literature shows that some compounds bearing
various heterocyclic pharmacophores can be found as KLK7 in-
hibitors. However, very few structure-activity relationships were
carried out in these series and no co-crystallization of small mol-
ecules in the active site of KLK7 has been reported yet. So, crucial
determinants for KLK7 specific targeting and inhibition still need to
be addressed. All these points prompted us to screen some het-
erocyclic derivatives from our internal library to study their
50.6 0.8
mM (see Fig. S1, SI). It is worth noting that no noticeable
inhibition was observed at 50
mM on other screened serine pro-
teases (KLK5, KLK8 and KLK14), meaning that compound 1a had a
selective action on KLK7.
A SAR study was then initiated, starting from compound 1a. A
set of 30 new pyrido-imidazodiazepinones as well as 9 acyl-IP
derivatives, which can be seen as “opened pyrido-imidazodiaze-
pinones”, were synthesized and their potency was evaluated
against KLK7 and other serine proteases to check selectivity.
1.2. Rationale for the synthesis of optimized diazepinone analogs
Modifications at positions 2, 3, 4 and 5 of the diazepinone core
were envisioned (Fig. 2). Synthesis of “opened-diazepinones” that
possessed a higher flexibility was also investigated.
Pyrido-imidazodiazepinones 1a-ad were synthesized starting
from 2-amino-imidazo-[1,2-a]pyridine 5 by selective C-3 acylation
of the IP nucleus (Scheme 1), using 7 different N-Boc amino-acids
(Boc-Phe-OH, Boc-D-Phe-OH, Boc-Ala-OH, Boc-Ser(Bn)-OH, Boc-
1-Nal-OH, Boc-Tyr(Bn)-OH, Boc-N-Me-Phe-OH) according to our
previously reported methodology [25,32]. C-3 acylated compounds
4a-g were isolated in 27%e88% yield after chromatography on
alumina gel. Only traces of the corresponding N-acylated de-
rivatives were detected by LC-MS analysis in the crude. After Boc
removal in acidic media (by using concentrated hydrochloric acid
or a mixture of trifluoroacetic acid/dichloromethane (50/50 v/v)),
the resulting 2-amino-3-acyl-imidazo-[1,2-a]pyridines 3a-g were
isolated in basic medium, without further purification. Diamines
3a-g were then successively reacted with a set of aldehydes in
chloroform at reflux for one night. Intermediate aminals were then
oxidized without isolation, using a mixture of lead tetraacetate and
iodine, to lead to pyrido-imidazodiazepinones 1a-1w that were
purified by chromatography on alumina gel.
The pyrido-imidazodiazepine-2,5-dione 1aa was synthesized by
intracarbonylation of diamine 2a, using 1,10-carbonyldiimidazole in
acetonitrile at 60 ꢀC and was isolated in 78% yield [32]. “Opened
derivatives” 2a-i were synthesized by a coupling reaction between
the diamine 3a and a set of commercially available carboxylic acids.
Fig. 1. Heterocyclic compounds reported as KLK7 inhibitors.

204
D.P. Arama et al. / European Journal of Medicinal Chemistry 93 (2015) 202e213
Table 1
Compounds 2a-i were isolated after purification on an alumina gel
column, in 20e89% yield. As the diamine 3a possesses two free
amines, the position of the N-acylation was investigated. The
structure of the resulting coupling product was ascertained by
1He13C HMBC analysis of compound 2a (R1 ¼ Ph), which showed a
correlation between the CHa of the phenylalanine residue
(5.85 ppm) and the carbonyl signal of the amide (168.7 ppm),
which unambiguously proved the N-acylation position (see Fig. S2,
SI). This result confirmed that the aromatic amine in position 2 of
the IP nucleus was poorly reactive, as previously observed [32].
Benzyl protected derivatives 1u and 1v were treated by a solu-
tion of 33% bromhydric acid in acetic acid to offer the corre-
sponding hydroxy derivatives 1ab and 1ac in 93 and 99% yields
respectively (Scheme 2). The 5-hydroxy derivative 1ad was ob-
tained as a mixture of diastereoisomers after reduction of com-
pound 1o, using sodium borohydride in methanol (Scheme 2).
Finally, phenol derivatives 1x-z were obtained by demethylation of
the methoxy derivatives 1l-n in 45e81% yields, using a 1 M boron
tribromide dichloromethane solution (Scheme 1).
Structure and IC50 against KLK7 of compounds 1 and 2 after 15 min of incubation at
pH 8.0 and 37 ꢀC.1: ni ¼ no inhibition (<30% inhibition at 50
mM).
Compounds
IC50 (mM)
R1
R2
R3
R4
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
2-Me-C6H4
3-Me-C6H4
4-Me-C6H4
4-OMe-C6H4
2-Me-C6H4
C6H5
4-BrC6H4
4-NO2C6H4
3-pyridinyl
CH2CH(CH3)2
2,4-diMe-C6H3
2-OMe-C6H4
3-OMe-C6H4
2,5-diOMe-C6H3
3,4-diOMe-C6H3
3,4,5-triOMe-C6H2
2-Me-C6H4
2-Me-C6H4
3,4-diMeO-C6H3
2-Me-C6H4
3,4-diMeO-C6H3
3,4-diMeO-C6H3
3,4-diMeO-C6H3
2-HO-C6H4
3-HO-C6H4
2,5-diHO-C6H3
]O
3,4-diMeO-C6H3
3,4-diMeO-C6H3
3,4-diMeO-C6H3
Ph
2-pyridinyl
4-pyridinyl
2-pyrrolyl
2-Me-C6H4
3,4-diOMe-C6H3
CH2-Ph
CH]CHePh
CH2eCH2ePh
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
e
e
e
e
e
e
e
e
e
(S)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(R)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(S)-(1-naphtyl)CH2
(S)-Me
(S)-Me
(S)-BnOCH2
(S)-BnOCH2
(S)-4-BnOC6H4CH2
(S)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(S)-Bn
(S)-HOCH2
(S)-4-HOC6H4CH2
(S)-Bn
e
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
]O
OH
e
57.0 1.0
ni1
68.0 3.1
72.0 1.9
95.9 1.7
ni
ni
ni
ni
ni
83.9 1.7
ni
ni
80.9 2.5
33.5 1.5
55.7 2.3
ni
ni
ni
ni
ni
ni
ni
ni
1.3. Structure-activity relationships
As our initial hit (compound 1a) possesses a chiral center, the
significance of the stereochemistry was first investigated.
Compared to compound 1a, which showed a moderate activity
(IC50 ¼ 57
1
m
M), its (R)-enantiomer (compound 1e) led to a
decrease of the inhibitory potency (IC50 ¼ 95.9
1.7 M), sug-
m
gesting that the (S) configuration is important to maintain inhibi-
tion in these series (Table 1). Modulation of position 2 of the
diazepinone ring (R1) was then studied. Replacement of the aryl
group by an aliphatic substituent (compound 1j) or by a carbonyl
group (urea derivative 1aa) completely abolished the inhibition.
Introduction of a phenyl (compound 1f) or a 3-pyridinyl group
(compound 1i) also led to a loss of KLK7 inhibition. Introduction of
an electro-withdrawing group on the para position of the phenyl
group was also unfavorable (compounds 1g and 1h). The inhibiting
capacity was preserved when a para-methyl- or a para-methox-
yphenyl group was present (compounds 1c or 1d), although the
1s
1t
1u
1v
1w
1x
1y
1z
1aa
1ab
1ac
1ad
2a
2b
2c
2d
2e
2f
2g
2h
2i
124.8 1.4
ni
ni
ni
ni
ni
ni
ni
activity was lower than that of compound 1a (IC50 ~70 mM).
e
e
The inhibitory potency observed with compound 1a, 1c and 1d
prompted us to further investigate compounds bearing methyl- or
methoxyphenyl substituents at R1. In the methyl series, the meta
derivative (compound 1b) was inactive, whereas the 2,4-dimethyl
derivative (compound 1k) showed a weaker activity compared to
the two corresponding monomethyl derivatives (1a and 1c). In the
methoxy series, the ortho-methoxy and the meta-methoxyphenyl
derivatives (1l and 1m) were inactive. Some inhibition was recov-
ered in the case of 2,5-dimethoxy- and 3,4,5-trimethoxyphenyl
e
e
ni
ni
e
e
e
e
135.4 9.2
ni
149.2 19.2
ni
ni
e
e
e
e
e
e
e
e
Fig. 2. Study of the compound 1a pharmacomodulation.

D.P. Arama et al. / European Journal of Medicinal Chemistry 93 (2015) 202e213
205
Scheme 1. Synthesis of compounds 1a-w, 1aa and 2a-i (R1, R2 and R3 substituents are referenced in Table 1). Reagents and conditions: Boc-AA-OH ] Boc-Phe-OH, Boc-D-Phe-OH,
Boc-Ala-OH, Boc-Ser(Bn)-OH, Boc-1-Nal-OH, Boc-Tyr(Bn)-OH or Boc-N-Me-Ala-OH.
derivatives (compounds 1n and 1p). The best result was obtained
with the 3,4-dimethoxyphenyl derivative 1o, which showed the
Finally, methylation of the diazepine N-3 or reduction of the
carbonyl in position 5 of the most active compound 1o led to a loss
of activity (compounds 1w and 1ad respectively). These results
suggest that the NH and the carbonyl of the diazepine ring could be
implicated in important interactions with the KLK7 binding site.
Finally, “opened diazepinone” series was investigated (com-
pounds 2a-i). Only two derivatives bearing a benzyl or a 2-
methylphenyl substituent at R1 (compounds 2e and 2g, Scheme 1
higher potency (IC50 ¼ 33.5
mM). Whereas some methoxy de-
rivatives exhibited different inhibition potencies, their corre-
sponding phenol derivatives (compounds 1x to 1z) were totally
inactive. These results suggest that one or several methoxy groups
can be involved in hydrogen bonding within the KLK7 active site.
Study of the position 4 of the diazepine ring was then carried
out, keeping constant the 2-methylphenyl or the 3,4-
dimethoxyphenyl groups at R1 (Fig. 2). Replacement of the benzyl
group at R3 by a methyl group (compounds 1r and 1s) or a
hydroxymethyl group (compound 1ab) led to a loss of activity,
suggesting that an aromatic group at this position is important for
the interaction with the target. Introduction of bulkier groups like
1-naphtylmethyl or 4-benzyloxybenzyle (compounds 1q and 1v) or
short elongation of the chain (compounds 1t and 1u) also led to
inactive compounds. A 4-hydroxybenzyl substituent in R3 was also
unfavorable for KLK7 inhibition (compound 1ac). These results
suggest that no modification is tolerated at R3 and a benzyl group is
crucial to obtain KLK7 inhibition.
and Table 1) showed
a
weak KLK7 inhibition activity
(IC50 > 100 M). Although the flexibility of compounds 2 was higher
m
than that of the diazepinone derivatives, which could eventually
lead to a better molecular recognition, the increase of conforma-
tional entropy in such molecules was unfavorable to the activity.
This result suggests that the presence of the diazepine ring is
crucial for the inhibition potency.
Starting from compound 1a, which showed moderate inhibition
potency against KLK7 (IC50 ¼ 57.0
mM), modifications of positions 2,
3, 4 and 5 of the diazepinone ring were investigated. The SAR study
indicated that position 3, 4 and 5 could not be modified to preserve
potency. Replacement of the 2-methylphenyl substituent by a 3,4-
dimethoxyphenyl group led to a slight increase of the inhibition
potency i.e. compound 1o (IC50 ¼ 33.5
mM) (Fig. 3A), which was
selected for further studies.
1.4. Mechanism of inhibition by compound 1o
As already observed with compound 1a, when using compound
1o, the inhibition was reversed by dilution, in agreement with no
formation of a stable covalent adduct between KLK7 and compound
1o (Fig. 3BeC). LineweavereBurk double reciprocal plots
showed that compound 1o is a competitive inhibitor indicating that
1o exclusively binds to the free enzyme (Fig. 3C). The Ki value for
KLK7 was determined to be 27
1.8 mM corresponding to an
acceptable good affinity. The selectivity of compound 1o was then
evaluated against three other human tissue kallikreins (KLK5, KLK8
and KLK14). No significant inhibition was observed at a 50
mM
concentration, indicating that compound 1o was selective for
Scheme 2. Synthesis of compounds 1ab-1ad.
kallikrein 7.

206
D.P. Arama et al. / European Journal of Medicinal Chemistry 93 (2015) 202e213
Fig. 3. Inhibition of KLK7 by compound 1o. A- IC50 curve after 15 min incubation of KLK7 (7.6 nM) at pH 8.0 and 37 ꢀC in 50 mM TRIS-HCl pH 8.0, Tween 20 0.01% (v/v) and 1 M NaCl.
The enzyme activity was monitored at 40 M final concentration of substrat (Suc-Leu-Leu-Leu-Val-Tyr-AMC). B- Remaining activity of hK7 in absence of inhibitor (black), after
15 min incubation with 50 M of compound 1o (grey), after dilution (1/40) of the formed complex KLK7-1o (striped). C-LineweavereBurk plot at different concentrations of 1o:
0 (:); 6.25 M ( ); 12.5 M (-); 25 M (C); 50 M ( ).
m
m
m
m
m
m
A
;
1.5. Structural basis for the inhibition
including compound 1o were evaluated on three human cancer cell
lines, i.e. the HeLa tumor cell line, the human prostatic PC-3 cell line
[33] and the human colorectal adenocarcinoma SW-620 [34]. In
particular, a recent study pointed out that aberrant expression of
KLK7 in colon cancer cells and tissues is involved in cell prolifera-
tion resulting in human colon tumorogenesis [35].
Models of 1o/KLK7 complexes were built to corroborate the
mechanistic data and to propose some structural basis for the in-
hibition. The lowest energy model of docking of compound 1o into
KLK7 showed several intermolecular interactions that are repre-
sented in Fig. 4. The docking results suggest that the IP nucleus of
compound 1o occupies the S1’ pocket of the protease active site, the
diazepinone ring being located in the S1 pocket while the benzyl
group is directed toward pocket S2. The catalytic residue Ser195 of
KLK7 might also be implicated in two hydrogen bonds with the two
nitrogens of the IP ring. Finally, a key hydrogen bond between Asn
189 and one of the two methoxy groups could explain the higher
affinity obtained with compound 1o, compared to 1m that bears
only one meta methoxy group. This polar contact will be taken into
account in the design of further optimized derivatives.
Compounds were first screened at a 100 mM concentration by
measuring cellular death using the colorimetric XTT assay. Only
derivatives exhibiting cytotoxic effects over 50% of cell death were
retained. Several derivatives were found cytotoxic on cancer cell
lines, including compounds 1k, 1n, 1o and 1p (Table 2) and their
EC50 determined.
Overall, the HeLa cell line was the most sensitive toward these
molecules, followed by PC-3 and SW-620. Interestingly compound
1o reported as the best KLK7 inhibitor displayed a noticeable
cytotoxic effect on both HeLa and PC-3 cells lines. Moreover, one
can also notice that 1o is the only compound that displays an effect,
although moderate, on PC-3 cells (EC50 ¼ 64.8
mM), while com-
1.6. Cytotoxicity potency towards cancer cell lines
pound 1k is specific of SW-620 cell line. This selective behavior of
1o on PC-3 is particularly of great interest since KLK7 is highly
expressed in prostate tissue and is believed to participate to cancer
progression by its role in the induction of the
Since KLK7 is involved in tumor progression through its pro-
teolytic activity, we investigated the anticancer potential of the
most potent compounds. The cytotoxic effects of 23 derivatives

D.P. Arama et al. / European Journal of Medicinal Chemistry 93 (2015) 202e213
207
0
Fig. 4. Putative binding mode of compound 1o within KLK7 active site. A- Positioning of 1o (CPK colors) in the S1 , S1 and S2 pockets of KLK7. B-Residues (sticks, carbon in gray)
nearby of 1o (sticks, carbon in green). Hydrogen bonds (black dotted lines) between residues Asn189, Gly193, Ser195 and 1o. C- Binding topology of compound 1o within the KLK7
0
active site. The protease surface is colored in light brown and the ligands are shown in stick representation. Subsites S1, S1 and S2 are indicated. The chymotrypsinogen numbering is
used; the residues Ser195, His57 and Asp102 form the catalytic triad. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)
epithelialemesenchymal transition on in vitro cellular models [8].
diazepine, a carbonyl group in position 5, a NH in position 3 gave
the best results. The phenyl ring at position 2 tolerates some
modulations, i.e. substitution by one or two methyl or methoxy
groups led to the most potent compounds. This study led to the
discovery of compound 1o, which is approximately twice more
2. Conclusion
Starting from an initial screening of 65 in-house heterocyclic
derivatives, a pyrido-imidazodiazepinone 1a was identified to be a
active than our initial hit 1a (Ki ¼ 27
mM compared to 50 mM for
compound 1a). Moreover some analogs, including compound 1o,
were shown to exhibit a cytotoxic effect, although moderate,
against selected colon and prostate cancer cell lines. A detailed
study of the cellular events involving KLK7 with optimized com-
pounds could be of great interest to address the correlation be-
tween KLK7 inhibition and potential anticancer activity.
moderate inhibitor of KLK7 (IC50 ¼ 57
mM and Ki ¼ 50
mM). Enzy-
matic studies showed that this derivative is a reversible and
competitive KLK7 inhibitor. Compound 1a was shown to have se-
lective action toward KLK7, when compared to others tested serine
proteases, suggesting its potential as a good starting point to design
potent selective, competitive and reversible KLK7 inhibitors. A SAR
program was then initiated to identify the structural requirement
for KLK7 inhibition. Modulation of positions 2, 3, 4 and 5 of the
diazepinone ring and “opened diazepine” derivatives were inves-
tigated. First, the diazepinone ring has shown to be crucial for KLK7
inhibition. In addition, a (S)-benzyl substituent in position 4 of the
3. Experimental
3.1. 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 ꢁ 4.6 mm, 5 mm) using a flow rate of 3.0 mL/min, and a
gradient of 100/0 to 0/100 eluents A/B over 5 min, in which eluents
A ¼ H2O/0.1% TFA and B¼CH3CN/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
Table 2
Evaluation of cytotoxic effect of compounds 1k, 1n-p on human cancer cell lines
HeLa, PC-3 and SW-620. Cytotoxicity is expressed as EC50, which refers to the
concentration that induces 50% of cell death determined by XTT assay.
Compounds
Cell lines
HeLa
PC-3
SW-620
1k
1n
1o
1p
Nca
40.0 1.5
24.3 1.6
53.8 3.5
nc
nc
67.8 3.4
temperature in deuterated solvents. Chemical shifts (d) are given in
Nc
Nc
nc
parts per million relative to the solvent [1H:
(DMSO d6) 2.50 ppm; (CD3OD)
(CDCl3)
d
(CDCl3) ¼ 7.26 ppm,
64.8 2.3
nc
¼
¼
3.31 ppm; 13C:
a
nc: non cytotoxic.
d
¼
77.2 ppm, (DMSO d6)
¼
39.5 ppm;

208
D.P. Arama et al. / European Journal of Medicinal Chemistry 93 (2015) 202e213
(CD3OD) ¼ 49.0 ppm]. The following abbreviations are used to
designate the signal multiplicities: s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), bs (broad singlet). Analytical
thin-layer chromatography (TLC) was performed using aluminium-
backed silica gel plates coated with a 0.2 mm thickness of silica gel
or with aluminium oxide 60 F254, neutral. LC-MS spectra (ESI) were
recorded on a HPLC using an analytical Chromolith Speed Rod RP-
C18 185 Pm column (50 ꢁ 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-1.
High-resolution mass spectrometric analyses were performed with
a 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 capillary melting point apparatus.
3.2.3. (2S)-2-Boc-amino-1-(2-aminoimidazo[1,2-a]pyridin-3-yl)-
3-(4-benzyloxy-phenyl)propan-1-one (4f)
Light yellow solid (m ¼ 0.561 g, 53%); mp: 108.1e120.0 ꢀC;
20
[a
]
¼ þ64.08ꢀ (c 0.76, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm
D
1.36 (s, 9H), 2.95 (dd, 1H, J ¼ 15.0, 9.0 Hz), 3.11 (dd, 1H, J ¼ 15.0,
9.0 Hz), 4.95 (s, 2H), 5.20 (bs, 1H), 5.50 (d, 1H, J ¼ 6.0 Hz), 5.89 (bs,
1H), 6.83 (m, 3H), 7.12 (d, 2H, J ¼ 9.0 Hz), 7.27e7.42 (m, 8H), 9.60
(bs, 1H); 13C NMR (CDCl3, 100 MHz):
d ppm 28.5 (3C), 38.0, 55.7,
70.2, 80.7, 108.5, 113.2, 114.3, 115.0 (2C), 127.6 (2C), 128.1, 128.7 (2C),
129.3, 129.4, 130.4 (2C), 130.8, 137.3, 147.7, 156.7, 157.8, 158.7, 185.1;
FT-IR (cmꢂ1): 3330, 3207, 2976, 1681, 1581, 1510, 1496, 1450, 1366,
1343, 1242, 1161, 1074, 1045, 1013, 857, 762, 735, 695; HPLC,
Tr ¼ 3.38 min; MS (ESIþ): m/z 487.3 [MþH]þ; HRMS calcd for
C28H31N4O4 487.2346, found 487.2345.
Compounds 5, 3a-c, 4a-c, 1a-d, 1f-j and 1aa were synthesized
according to our previous reported procedures and the spectral
characteristics were in agreement with the published data [25,32].
3.2.4. (2S)-2-Boc-N-methylamino-1-(2-aminoimidazo[1,2-a]
pyridin-3-yl)-3-(4-phenyl)propan-1-one (4g)
Yellow solid (m
¼
0.578 g, 67%), mp: 58.5e61.4 ꢀC;
20
[
a
]
¼ ꢂ164.9ꢀ (c 1.05, CHCl3); 1H NMR (CDCl3, 400 MHz):
d ppm
D
1.31 (s, 9H), 2.83 (s, 3H), 3.12 (m, 2H), 5.63 (t, 1H, J ¼ 8.0 Hz), 6.27 (s,
2H), 6.82 (t,1H, J ¼ 8.0 Hz), 7.14 (t,1H, J ¼ 8.0 Hz), 7.22e7.29 (m, 5H),
7.37 (t, 1H, J ¼ 8.0 Hz), 9.75 (bs, 1H); 13C NMR (CDCl3, 100 MHz):
3.2. Synthesis of compounds 4d-g
To a suspension of 0.5 g of trifluoro-N-imidazo[1,2-a]pyridin-2-
ylacetamide (2.18 mmol) in 9 mL of an aqueous 5 N sodium hy-
droxide solution was added 0.5 mL of THF. The solution was stirred
at 40 ꢀC 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
20 mL of DCM and Boc-1-Nal-OH (2.4 mmol, 1.1 equiv.) [or Boc-
Tyr(Bn)-OH or Boc-Ser(Bn)-OH or Boc-N-Me-Phe-OH], HOBt
(325 mg, 2.4 mmol, 1.1 equiv.), EDCI (460 mg, 2.4 mmol, 1.1 equiv.)
d
ppm 28.2 (3C), 29.7, 34.5, 59.2, 81.0,108.5,112.9,113.9,126.5,128.4
(2C), 129.4, 129.8 (2C), 130.8, 137.5, 147.8, 157.7, 158.1, 182.3; FT-IR
(cmꢂ1): 3331, 3216, 2974, 2928, 1666, 1627, 1591, 1524, 1496,
1444, 1387, 1366, 1341, 1264, 1140, 1060, 1012, 951, 849, 760, 697;
HPLC, Tr ¼ 3.36 min; MS (ESIþ): m/z 395.0 [MþH]þ; HRMS calcd for
C22H27N4O3 395.2083, found 395.2081.
3.3. General procedure for the synthesis of diazepinones
and triethylamine (334 m
L, 2.4 mmol, 1.1 equiv.) were added at 0 ꢀC.
A solution of compound 4a-f (0.33 mmol) in 12 N aqueous hy-
drochloric acid (3 mL) was stirred a room temperature for 1 h. The
solution was treated with 28% aqueous ammonia solution until pH
12 and then extracted with chloroform (3 ꢁ 20 mL). The organic
layer was dried over Na2SO4, filtered, and the solvent was
concentrated in vacuo to lead compounds 3a-f, which were used in
the next step without further purification. To a solution of 3a-f in
chloroform (2 mL) was added 1 equiv. of the appropriate aldehyde.
The solution was stirred at 60 ꢀC for 6 h. After cooling, the mixture
was pooled to a solution of iodine (91 mg, 0.36 mmol, 1.1 equiv) in
chloroform (4 mL). Finally, a solution of lead tetraacetate (161 mg,
0.36 mmol, 1.1 equiv) in chloroform (6 mL) 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 ꢁ 30 mL) and then with saturated 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/EtOH 99/1
v/v.
The solution was stirred at rt for 4 h. The solution was then washed
with saturated NaHCO3 aqueous solution (2 ꢁ 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 offer compounds 4d-g.
3.2.1. (2S)-2-Boc-amino-1-(2-aminoimidazo[1,2-a]pyridin-3-yl)-3-
(1-naphtyl)propan- 1-one (4d)
Yellow solid (m ¼ 0.297 g, 63.5%); mp: 208.0e210.2 ꢀC;
20
[
a]
¼ þ3.6ꢀ (c 0.5, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 1.33
D
(s, 9H), 3.50e3.70 (m, 2H), 5.46 (t, 1H, J ¼ 8.4 Hz), 5.57 (d, 1H,
J ¼ 9.3 Hz), 5.86 (bs, 2H), 6.85 (bs, 1H), 7.29e7.49 (m, 6H), 7.66 (d,
1H, J ¼ 7.8 Hz), 7.78 (d, 1H, J ¼ 7.8 Hz), 8.12 (d, 1H, J ¼ 8.7 Hz), 9.60
(bs, 1H); 13C NMR (CDCl3, 75 MHz):
d ppm 28.1 (3C), 35.6, 54.7, 80.3,
113.0, 113.9, 115.8, 123.3, 125.1, 125.5, 126.2, 127.3, 127.5, 128.7, 129.1,
130.7, 132.0, 132.7, 133.7, 147.2, 156.4, 158.1, 184.8; FT-IR (cmꢂ1):
3329, 3189, 2971, 1679, 1588, 1497, 1449, 1366, 1342, 1253, 1162,
1055, 762; HPLC, Tr ¼ 1.81 min; MS (ESIþ): m/z 431.3 [MþH]þ;
HRMS calcd for C25H27N4O3 431.2083, found 431.2085.
3.3.1. (4R)-4-benzyl-2-(2-methylphenyl)-3,4-dihydro-5H-pyrido
3.2.2. (2S)-2-Boc-amino-1-(2-aminoimidazo[1,2-a]pyridin-3-yl)-
3-benzyloxy-propan- 1-one (4e)
[10,20:1,2]imidazo[4,5-d][1,3]diazepin-5-one (1e)
Yellow solid (m
¼
0.117 g, 37.5%); mp: 135.5e136.5 ꢀC;
20
Yellow solid (m
¼
0.488 g, 54.5%); mp: 123e124 ꢀC;
[a
]
¼ þ9.1ꢀ (c 1, CHCl3); other data were in agreement with those
D
20
[
a]
¼ þ3.66ꢀ (c 1.5, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 1.46
published for its S enantiomer [25].
D
(s, 9H), 3.77 (t, 1H, J ¼ 9.0 Hz), 3.89 (dd, 1H, J ¼ 6.0, 9.0 Hz), 4.54 (s,
2H), 5.30 (dd, 1H, J ¼ 6.0, 12.0 Hz), 5.79 (d, 1H, J ¼ 9.0 Hz), 5.89 (bs,
2H), 6.90 (t, 1H, J ¼ 9.0 Hz), 7.21e7.33 (m, 5H), 7.37e7.47 (m, 2H),
3.3.2. (4S)-4-benzyl-2-(2,4-dimethylphenyl)-3,4-dihydro-5H-
pyrido[10,20:1,2]imidazo [4,5-d][1,3]diazepin-5-one (1k)
9.62 (d, 1H, J ¼ 6.0 Hz); 13C NMR (CDCl3, 75 MHz):
d
ppm 28.4 (3C),
Yellow solid (m
¼
0.080 g, 38%); mp: 107.0e108.0 ꢀC;
20
54.1, 71.7, 73.6, 80.2, 109.1, 113.1, 114.3, 127.7 (2C), 127.9, 128.4 (2C),
129.4, 130.7, 137.2, 147.8, 155.8, 159.1, 184.1; FT-IR (cmꢂ1): 3356,
3204, 2970, 2849, 1678, 1631, 1578, 1456, 1297, 1163, 1057, 985, 763,
699; HPLC, Tr ¼ 1.71 min; MS (ESIþ): m/z 411.3 [MþH]þ; HRMS
calcd for C22H27N4O4 411.2032, found 411.2035.
[a
]
¼ ꢂ20.2ꢀ (c 1, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 2.12
D
(s, 3H), 2.21 (s, 3H), 3.25 (dd, 1H, J ¼ 9.0, 12.0 Hz), 3.51 (dd, 1H,
J ¼ 6.0, 12.0 Hz), 4.20 (bs, 1H), 5.93 (bs, 1H), 6.89 (m, 3H), 7.19 (m,
8H), 9.40 (d, 1H, J ¼ 6.0 Hz); 13C NMR (CDCl3, 75 MHz):
d ppm 20.4,
21.4, 36.9, 60.5, 111.9, 113.9, 115.2, 126.5, 126.8 (2C), 128.4, 128.5

D.P. Arama et al. / European Journal of Medicinal Chemistry 93 (2015) 202e213
209
(2C), 129.9, 130.1, 131.6, 132.0, 138.5, 141.5, 145.4, 152.6, 152.7, 156.8,
3.3.7. (4S)-4-benzyl-2-(3,4,5-trimethoxyphenyl)-3,4-dihydro-5H-
159.2, 181.6; FT-IR (cmꢂ1): 3027, 2956, 2923, 1732, 1645, 1633, 1556,
1523, 1480, 1421, 1335, 1299, 1247, 1122, 1094, 1073, 1044, 949, 898,
820, 761; HPLC, Tr ¼ 1.60 min; MS (ESIþ): m/z 395.1 [MþH]þ; HRMS
calcd for C25H23N4O 395.1872, found 395.1858.
pyrido[10,20:1,2] imidazo[4,5-d][1,3]diazepin-5-one (1p)
Yellow solid (m
¼
0.192 g, 59%); mp: 119.0e120.0 ꢀC;
20
[a
]
¼ þ22.98ꢀ (c 0.87, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm
D
3.35 (bs, 1H), 3.70 (s, 6H), 3.82 (s, 4H),4.31 (bs, 1H), 6.82 (bs, 2H),
7.03 (td, 1H, J ¼ 14.5, 6.9 Hz), 7.33 (m, 8H), 9.58 (bs, 1H); 13C NMR
3.3.3. (4S)-4-benzyl-2-(2-methoxyphenyl)-3,4-dihydro-5H-pyrido
[10,20:1,2]imidazo[4,5-d][1,3]diazepin-5-one (1l)
(CDCl3, 100 MHz): d ppm 29.9, 36.5, 56.4 (2C), 61.0, 105.1, 106.8 (2C),
112.1, 114.2, 115.9, 127.1, 128.8 (2C), 128.9, 129.9 (2C), 130.9, 141.7,
146.2, 153.1 (2C), 159.7, 166.5, 182.3; FT-IR (cmꢂ1): 2937, 2836, 1719,
1625, 1559, 1462, 1409, 1333, 1232, 1122, 1000, 763, 700;
HPLC,Tr ¼ 1.50 min; MS (ESIþ): m/z 457.1 [MþH]þ; HRMS calcd for
Yellow solid (m
¼
0.255 g, 72%); mp: 104.0e105.0 ꢀC;
20
[
a]
¼ ꢂ24.86ꢀ (c 1.5, CHCl3); 1HNMR (CDCl3, 300 MHz):
d ppm
D
3.09 (m, 1H), 3.32 (m, 1H), 3.60 (s, 3H), 4.24 (dd, 1H, J ¼ 9.0, 6.0 Hz),
6.80 (d, 1H, J ¼ 9.0 Hz), 6.99 (m, 2H), 7.20e7.24 (m, 6H), 7.35e7.46
(m, 3H), 8.22 (bs, 1H), 9.50 (d, 1H, J ¼ 6.0 Hz); 13C NMR (CDCl3,
C25H25N4O4 457.1876, found 457.1874.
75 MHz):
d
ppm 36.5, 56.0, 66.4, 111.6, 112.6, 113.8, 116.0, 121.5,
3.3.8. (4S)-4-(1-naphthyl)methyl-2-(2-methylphenyl)-3,4-dihydro-
5H-pyrido[10,20:1,2] imidazo[4,5-d][1,3]diazepin-5-one (1q)
122.3, 126.7 (2C), 128.6, 128.7 (2C), 129.8, 129.9, 130.6, 133.1, 133.3,
138.1, 146.5, 156.3, 158.1, 181.9; FT-IR (cmꢂ1): 660, 680, 694, 722,
741, 842, 904, 921, 978, 1024, 1042, 1073, 1106, 1126, 1163, 1182,
1240, 1269, 1297, 1335, 1453, 1481, 1524, 1562, 1601, 1622, 1645,
2971; HPLC, Tr ¼ 1.34 min; MS (ESIþ): m/z 397.1 [MþH]þ; HRMS
calcd for C24H21N4O2 397.1665, found 397.1655.
Yellow solid (m
¼
0.105 g, 45%); mp: 134.4e135.4 ꢀC;
20
[a
]
¼ þ29.4ꢀ (c 0.5, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 2.20
D
(s, 3H), 3.71 (bs, 1H), 4.18 (bs, 2H), 6.9 (t, 1H, J ¼ 9.0 Hz), 7.12e7.19
(m, 4H), 7.30e7.48 (m, 5H), 7.61 (bs,1H), 7.76 (d, 1H, J ¼ 9.0 Hz), 7.85
(dd, 2H, J ¼ 9.0, 3.0 Hz), 8.05 (dd, 1H, J ¼ 9.0, 3.0 Hz), 9.48 (d, 1H,
J ¼ 9.0 Hz); 13C NMR (CDCl3, 75 MHz):
d ppm 20.4, 33.9, 53.5 (from
3.3.4. (4S)-4-benzyl-2-(3-methoxyphenyl)-3,4-dihydro-5H-pyrido
[10,20:1,2]imidazo[4,5-d][1,3]diazepin-5-one (1m)
1He13C HSQC correlation), 113.7, 115.3, 120.2, 121.8, 124.0, 125.6,
126.0 (2C), 127.3, 128.4, 128.6, 128.9, 130.3, 130.7, 131.2, 132.3 (2C),
134.1, 135.6, 138.4, 145.6, 156.1, 157.4, 159.5, 181.2; FT-IR (cmꢂ1):
2927, 1691, 1632, 1556, 1523, 1480, 1421, 1335, 1301, 1247, 1162, 1124,
760; HPLC, Tr ¼ 1.69 min; MS (ESIþ): m/z 431.2 [MþH]þ; HRMS
calcd for C28H23N4O 431.1872, found 431.1873.
Brown dough (m
¼
0.130 g, 94%); mp: 66.0e67.0 ꢀC;
20
[
d
a
]
¼ þ3.2ꢀ (c 0.375, DMSO); 1H NMR (DMSO-d6, 400 MHz):
D
ppm 2.96 (dd, 1H, J ¼ 12.0, 8.0 Hz), 3.26 (dd, 1H, J ¼ 12.0, 4.0 Hz),
3.75 (s, 3H), 4.56 (dd, 1H, J ¼ 12.0, 4.0 Hz), 6.97e7.03 (m, 1H),
7.13e7.50 (m, 10H), 7.90e7.97 (m, 2H), 9.54 (d, 1H, J ¼ 8.0 Hz); 13C
NMR (DMSO-d6, 100 MHz):
d
ppm 34.7, 55.4, 63.7, 111.7, 114.8, 115.1,
3.3.9. (4S)-4-methyl-2-(2-methylphenyl)-3,4-dihydro-5H-pyrido
[10,20:1,2]-imidazo[4,5-d][1,3]diazepin-5-one (1r)
116.4, 120.2, 122.7, 127.0 (2C), 128.5, 128.6 (2C), 129.4, 129.8, 133.4,
136.1, 143.4, 150.1, 158.0, 158.3, 159.0, 180.3; FT-IR (cmꢂ1): 2963,
1648, 1596, 1580, 1519, 1488, 1466, 1454, 1417, 1332, 1292, 1259,
1177, 1129, 1029, 762; HPLC, Tr ¼ 1.42 min; MS (ESIþ): m/z 397.1
[MþH]þ; HRMS calcd for C24H21N4O2 397.1665, found 397.1654.
White solid (m
¼
0.023 g, 8%); mp: 124.0ꢀCe125.0 ꢀC;
20
[a
]
¼ ꢂ41.04ꢀ (c 0.57, CHCl3); 1H NMR (CDCl3, 400 MHz):
d ppm
D
1.68 (bs, 3H), 2.42 (s, 3H), 4.09e4.16 (m, 1H), 6.93 (t, 1H, J ¼ 8.0 Hz),
7.17e7.35 (m, 6H), 7.61 (d,1H, J ¼ 8.0 Hz), 9.48 (d,1H, J ¼ 8.0 Hz); 13
C
NMR (CDCl3, 100 MHz):
d ppm 16.6, 20.3, 63.4, 111.7, 113.4, 114.8,
3.3.5. (4S)-4-benzyl-2-(2,5-dimethoxyphenyl)-3,4-dihydro-5H-
126.0, 128.2, 130.0 (2C), 130.5, 131.0, 131.1, 135.6, 137.9, 145.4, 154.6,
182.5; FT-IR (cmꢂ1): 2929, 1645, 1627, 1558, 1523, 1482, 1419, 1337,
1300,1248, 757; HPLC, Tr ¼ 1.14 min; MS (ESIþ): m/z 305.3 [MþH]þ;
HRMS calcd for C18H17N4O 305.1402, found 305.1404.
pyrido[10,20:1,2]imidazo [4,5-d][1,3]diazepin-5-one (1n)
Yellow solid (m
¼
0.089 g, 39%); mp: 155.0e156.0 ꢀC;
20
[
a]
¼ ꢂ12.0ꢀ (c 1, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 3.13
D
(m, 1H), 3.31 (m, 1H), 3.59 (s, 3H), 3.79 (s, 3H), 4.25 (m, 1H), 6.76 (d,
1H, J ¼ 9.0 Hz), 6.92e7.03 (m, 2H), 7.16e7.24 (m, 6H), 7.47 (t, 2H,
J ¼ 6.0 Hz), 7.80 (bs, 1H), 9.54 (d, 1H, J ¼ 6.0 Hz); 13C NMR (CDCl3,
3.3.10. (4S)-4-methyl-2-(3,4-dimethoxyphenyl)-3,4-dihydro-5H-
pyrido[10,20:1,2] imidazo[4,5-d][1,3]diazepin-5-one (1s)
75 MHz):
d
ppm 36.5, 54.2 (from 1He13C HSQC correlation), 56.2,
Brown solid (m
¼
0.006 g, 6%); mp: 102.0e103.0 ꢀC;
20
56.7, 112.6, 113.4, 113.9, 116.0, 120.3, 126.5, 126.7, 128.6, 128.7, 129.8,
129.9, 130.7, 134.2, 138.0, 143.2, 146.4, 152.5, 154.1, 182.0; FT-IR
(cmꢂ1): 3019, 2928, 2832, 1647, 1635, 1565, 1526, 1480, 1460,
1448, 1412, 1335, 1318, 1268, 1240, 1220, 1178, 1099, 1074, 1042,
1024, 961, 941, 902, 870, 813, 755, 740, 697, 676,; HPLC,
Tr ¼ 1.66 min; MS (ESIþ): m/z 427.1 [MþH]þ; HRMS calcd for
[
a
]
¼ ꢂ40.8ꢀ (c 0.25, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm
D
1.61 (bs, 3H), 3.90 (s, 3H), 3.94 (s, 3H), 4.66 (bs, 1H), 6.84 (d, 1H,
J ¼ 9.0 Hz), 6.97 (t, 1H, J ¼ 9.0 Hz), 7.45e7.48 (m, 3H), 7.67 (bs, 1H),
8.07 (bs, 1H), 9.53 (d, 1H, J ¼ 6.0 Hz); 13C NMR (CDCl3, 75 MHz):
d
ppm 16.4, 56.1, 56.2, 56.3, 109.7, 110.6, 112.3, 113.7, 115.9, 116.1,
117.6, 121.5, 128.0, 128.6, 129.9, 130.2, 149.3, 152.5, 183.1; HPLC,
C
25H23N4O3 427.1770, found 427.1778.
Tr ¼ 1.10 min; MS (ESIþ): m/z 351.2 [MþH]þ; HRMS calcd for
C19H19N4O3 351.1457, found 351.1458.
3.3.6. (4S)-4-benzyl-2-(3,4-dimethoxyphenyl)-3,4-dihydro-5H-
pyrido[10,20:1,2]imidazo [4,5-d][1,3]diazepin-5-one (1o)
3.3.11. (4S)-4-benzyloxymethyl-2-(2-methylphenyl)-3,4-dihydro-
Yellow solid (m
¼
0.115 g, 77%); mp: 136.0e137.0 ꢀC;
5H-pyrido[10,20:1,2] imidazo[4,5-d][1,3]diazepin-5-one (1t)
20
[
a]
¼ þ36.8ꢀ (c 1, DMSO); 1H NMR (DMSO-d6, 400 MHz):
d
ppm
Yellow solid (m
¼
0.036 g,31%); mp: 102.0e103.0 ꢀC;
D
¼ ꢂ8.25ꢀ (c 0.4, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 2.41
20
2.90e3.17 (m, 3H), 3.70 (s, 3H), 3.81 (s, 3H), 4.15 (bs,1H), 6.97 (d,1H,
J ¼ 8.0 Hz), 7.14 (bs, 1H), 7.17 (td, 1H, J ¼ 8.0, 4.0 Hz), 7.24e7.32 (m,
6H), 7.64e7.70 (m, 2H), 9.47 (d, 1H, J ¼ 8.0 Hz); 13C NMR (DMSO-d6,
[a
]
D
(bs, 3H), 4.10 (bs,1H), 4.25 (dd,1H, J ¼ 9.0, 6.0 Hz), 4.54 (m,1H), 4.71
(bs, 2H), 6.93 (t, 1H, J ¼ 6.0 Hz), 7.17 (dd, 3H, J ¼ 9.0, 6.0 Hz),
7.29e7.39 (m, 8H), 7.63 (d, 1H, J ¼ 6.0 Hz), 9.47 (d, 1H, J ¼ 6.0 Hz);
100 MHz):
d ppm 35.1, 54.9, 55.4, 55.6, 108.4, 110.8, 112.3, 113.6,
115.7, 122.6, 125.3, 126.5, 127.3 (2C), 127.8, 128.3 (2C), 129.5, 129.7,
130.5, 136.7, 146.1, 147.9, 151.8, 180.1; FT-IR (cmꢂ1): 2925, 2853, 699,
739, 764, 814, 840, 937, 973, 1021, 1080, 1140, 1173, 1231, 1261, 1335,
1435, 1462, 1496, 1510, 1557, 1599, 1623; HPLC, Tr ¼ 1.33 min; MS
(ESIþ): m/z 427.0 [MþH]þ; HRMS calcd for C25H23N4O3 427.1770,
found 427.1774.
13C NMR (CDCl3, 100 MHz):
d ppm 29.8, 69.9, 73.7, 73.8, 113.8, 115.3,
126.0, 126.1, 126.2, 127.8 (2C), 128.0, 128.5 (2C), 128.6, 130.4, 130.8,
130.9, 131.2, 131.3, 135.6, 138.3, 138.4, 145.7, 180.6; FT-IR (cmꢂ1):
3055, 3023, 2923, 2853, 2246, 1634, 1558, 1523, 1421, 1336, 1250,
1097, 907, 762, 729, 696; HPLC, Tr ¼ 1.55 min; MS (ESIþ): m/z 411.3
[MþH]þ; HRMS calcd for C25H23N4O2 411.1821, found 411.1825.

210
D.P. Arama et al. / European Journal of Medicinal Chemistry 93 (2015) 202e213
3.3.12. (4S)-4-benzyloxymethyl-2-(3,4-dimethoxyphenyl)-3,4-
dihydro-5H-pyrido [10,20:1,2]imidazo[4,5-d][1,3]diazepin-5-one
(1u)
118.2, 126.8, 128.0, 128.4 (2C), 129.4 (2C), 131.6, 134.7, 136.5, 145.8,
150.1, 152.8, 159.1, 160.9, 181.2; FT-IR (cmꢂ1): 3034, 2924, 1724,
1636, 1611, 1583, 1515, 1488, 1454, 1424, 1340, 1310, 1257, 1152, 1117,
1080, 1031, 749; HPLC, Tr ¼ 1.56 min; MS (ESIþ): m/z 383.0 [MþH]þ;
HRMS calcd for C23H19N4O2 383.1502, found 383.1511.
Yellow solid (m
¼
0.069 g, 47%); mp: 80.5e81.5 ꢀC;
20
[
a]
¼ ꢂ27.0ꢀ (c 0.8, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 3.82
D
(s, 3H), 3.89 (bs, 5H), 4.26 (bs, 1H), 4.60 (bs, 2H), 6.79 (d, 1H,
J ¼ 6.0 Hz), 6.98 (t, 1H, J ¼ 6.0 Hz), 7.30e7.52 (m, 8H), 7.66 (bs, 2H),
3.4.2. (4S)-4-benzyl-2-(3-hydroxyphenyl)-3,4-dihydro-5H-pyrido
9.53 (bs,1H); 13C NMR (CDCl3, 100 MHz):
d
ppm 55.8 (2C), 60.6, 67.1,
[10,20:1,2]imidazo-[4,5-d][1,3]diazepin-5-one (1y)
73.5, 110.2, 111.7, 113.5, 116.5, 121.9, 122.9, 127.9 (2C), 128.4 (2C),
130.1, 137.0, 142.5, 147.1, 148.9, 150.8, 152.3, 158.5, 159.0, 160.7, 181.1;
FT-IR (cmꢂ1): 3216, 2931, 2853, 1622, 1559, 1508, 1462, 1427, 1334,
1233, 1132, 1094, 1020, 764, 738, 697; HPLC, Tr ¼ 1.54 min; MS
(ESIþ): m/z 457.3 [MþH]þ; HRMS calcd for C26H25N4O4 457.1876,
found 457.1873.
Yellow solid (m
¼
0.030 g, 45%); mp: 145.0e146.0 ꢀC;
20
[a
]
¼ ꢂ5.0ꢀ (c 1.0, MeOH); 1H NMR (CD3OD, 300 MHz):
d ppm
D
3.03 (dd, 1H, J ¼ 12.0, 9.0 Hz), 3.37 (dd, 1H, J ¼ 12.0, 6.0 Hz), 4.68
(dd, 1H, J ¼ 12.0, 6.0 Hz), 6.83 (d, 1H, J ¼ 6.0 Hz); 6.98 (bs, 1H), 7.15
(dd, 2H, J ¼ 9.0, 6.0 Hz), 7.29e7.38 (m, 7H), 7.43 (t, 1H, J ¼ 6.0 Hz),
7.85e7.96 (m, 2H), 9.66 (d, 1H, J ¼ 6.0 Hz); 13C NMR (CD3OD,
75 MHz): d ppm 36.9, 66.2,116.4,117.7,118.2,122.5,123.1,128.8 (2C),
3.3.13. (4S)-4-(4-benzyloxy)benzyl-2-(3,4-dimethoxyphenyl)-3,4-
129.6 (2C), 129.9, 130.2, 130.3, 130.7, 131.4, 131.8, 132.1, 134.8, 136.7,
159.5, 163.7, 181.4; FT-IR (cmꢂ1): 2930, 1779, 1650, 1594, 1530, 1450,
1420, 1331, 1296, 1264, 1244, 1197, 1126, 1080, 1033, 795; HPLC,
Tr ¼ 1.32 min; MS (ESIþ): m/z 383.1 [MþH]þ; HRMS calcd for
dihydro-5H-pyrido [10,20:1,2]imidazo[4,5-d][1,3]diazepin-5-one (1v)
Orange solid (m
¼
0.134 g, 65%); mp: 129.0e130.0 ꢀC;
20
[
a]
¼ þ3.93ꢀ (c 1.5, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm
D
3.71e3.78 (bs, 4H), 3.81e3.88 (bs, 4H), 4.18 (bs, 1H), 5.02 (s, 2H),
6.64 (d, 1H, J ¼ 6.0 Hz), 6.90 (d, 2H, J ¼ 9.0 Hz), 6.98 (t, 1H,
J ¼ 6.0 Hz), 7.19e7.45 (m, 12H), 9.53 (d, 1H, J ¼ 6.0 Hz); 13C NMR
C23H19N4O2 383.1508, found 383.1495.
3.4.3. (4S)-4-benzyl-2-(2,5-dihydroxyphenyl)-3,4-dihydro-5H-
(CDCl3, 100 MHz):
d
ppm 30.2, 31.4, 35.8, 56.5, 70.6, 110.8, 112.8,
pyrido[10,20:1,2]imidazo-[4,5-d][1,3]diazepin-5-one (1z)
113.1, 113.2, 114.4, 115.4, 115.6 (2C), 116.5, 123.0, 127.4, 127.9 (2C),
128.5, 129.1 (2C), 131.0, 131.2 (2C), 132.5, 137.4, 146.7, 149.4, 153.1,
153.8, 158.4, 183.0; FT-IR (cmꢂ1): 3031, 2932, 2837, 1730, 1624, 1557,
1510, 1461, 1425, 1335, 1260, 1231, 1140, 1020, 811, 764, 736, 695;
HPLC, Tr ¼ 3.08 min; MS (ESIþ): m/z 533.2 [MþH]þ; HRMS calcd for
Yellow solid (m
¼
0.037 g, 79%); mp: 260.5e261.5 ꢀC;
20
[
d
a
]
¼ þ81,48ꢀ (c 0.405, DMSO); 1H NMR (DMSO-d6, 300 MHz):
D
ppm 2.82e3.01 (m, 3H), 3.61 (bs, 1H), 4.25 (m, 1H), 6.76 (m, 1H),
6.91 (bs, 1H), 7.14e7.25 (m, 7H), 7.76e7.78 (m, 2H), 8.64 (d, 1H,
J ¼ 3.4 Hz), 9.46 (d, 1H, J ¼ 3.4 Hz); 13C NMR (DMSO-d6, 100 MHz):
C
32H29N4O4 533.2194, found 533.2189.
d ppm 35.1, 63.3, 112.6, 113.6, 114.2, 114.9, 116.2, 118.2, 122.2, 126.7,
128.3, 128.4 (2C), 129.0, 129.3 (2C), 131.0, 136.6, 146.4, 148.7, 154.7,
158.9, 182.2; FT-IR (cmꢂ1): 3234, 2396, 1724, 1622, 1544, 1480, 1417,
1340, 1249, 1219, 1130, 823; HPLC, Tr ¼ 1.358 min; MS (ESIþ): m/z
399.2 [MþH]þ; HRMS calcd for C23H19N4O3 399.1457, found
399.1459.
3.3.14. (S)-4-benzyl-2-(3,4-dimethoxyphenyl)-3-methyl-3H-pyrido
[10,20:1,2]imidazo [4,5-d][1,3]diazepin-5(4H)-one (1w)
Yellow solid (m
¼
0.120 g, 54%); mp: 217.0e218.0 ꢀC;
20
[
a]
¼ þ174.1ꢀ (c 0.39, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm
D
2.69 (bs,1H), 2.73 (bs,1H), 2.98 (s, 3H), 3.71 (s, 3H), 3.86 (s, 3H), 4.32
(bs, 1H), 6.67 (d, 1H, J ¼ 8.4 Hz), 6.85 (s, 2H), 6.98 (td, 1H, J ¼ 5.7,
6.9 Hz), 7.28 (bs, 5H), 7.50 (td, 1H, J ¼ 7.1, 14.6 Hz), 7.69 (d, 1H,
J ¼ 8.9 Hz), 9.55 (d, 1H J ¼ 6.9 Hz); 13C NMR (CDCl3, 100 MHz):
3.5. General procedure for synthesis of deprotected derivatives 1ab-
ac
d
ppm 29.9, 34.3, 45.6, 56.2, 72.4, 110.3, 112.8, 113.7, 113.9, 116.9,
A solution of compound 1u-v (95 mg) in DCM was cooled at 0 ꢀC.
A mixture of HBr 33% in acetic acid (5 mL) was added and the
resulting solution was stirred for 3 h (monitoring by TLC) at room
temperature. The solvent was then removed under reduced pres-
sure to afford compound 1ab-ac.
125.4, 127.4, 128.4 (2C), 128.6, 129.0 (2C), 129.6, 130.4, 136.7, 147.4,
148.5, 151.95, 158.5, 160.7, 183.0; FT-IR (cmꢂ1): 3055, 3000, 2928,
2837, 1624, 1543, 1515, 1478, 1455, 1436, 1359, 1340, 1262, 1126,
1019, 879, 817, 757, 706; HPLC, Tr ¼ 2.45 min; MS (ESIþ): m/z 441.2
[MþH]þ; HRMS calcd for C26H25N4O3 441.1931, found 429.1927.
3.5.1. (4S)-4-hydroxymethyl-2-(3,4-dimethoxyphenyl)-3,4-
dihydro-5H-pyrido[10,20:1,2] imi-dazo[4,5-d][1,3]diazepin-5-one
3.4. General procedure for synthesis of phenol derivatives 1x-z
(1ab)
20
To a solution of compound 1l-n (100 mg) in DCM (10 mL) at 0 ꢀC,
was added a solution of boron tribromide 1 M in DCM (6 equiv.).
The resulting mixture was stirred at rt for 3 h (monitoring by TLC).
The reaction was quenched by adding distilled water (30 mL). The
solution was extracted with ethyl acetate; the organic layer was
dried over Na2SO4, filtered and the solvent was removed under
reduced pressure. The residue was dissolved in ethyl acetate and
precipitated by addition of diethyl ether. The precipitate was then
filtered over Buchner funnel and washed with diethyl ether to
afford phenol derivatives 1x-z.
Brown gum (m ¼ 0.030 g, 93%); [
a]
¼ þ16.0ꢀ (c 0.175, DMSO);
D
1H NMR (DMSO-d6, 400 MHz):
d
ppm 3.81e3.86 (m, 4H), 3.91 (s,
6H), 4.44 (m, 1H), 7.28 (d, 1H, J ¼ 8.0 Hz), 7.42e7.49 (m, 1H), 7.56 (d,
1H, J ¼ 4.0 Hz), 7.63 (dd, 1H, J ¼ 12.0 Hz, J ¼ 4.0 Hz), 7.92 (d, 2H,
J ¼ 4.0 Hz), 9.52 (d, 1H, J ¼ 8.0 Hz); 13C NMR (DMSO-d6, 100 MHz):
d
ppm 55.9, 56.2, 60.0, 65.3, 111.5, 112.1, 113.9, 115.8, 116.4, 120.1,
125.6, 128.6, 133.2, 144.6, 148.3, 148.8, 154.6, 160.9, 179.4; FT-IR
(cmꢂ1): 3380, 3206, 3027, 2928, 1708, 1647, 1589, 1512, 1489,
1319, 1272, 1222, 1170, 1013, 810, 747; HPLC, Tr ¼ 1.7 min; MS
(ESIþ): m/z 367.2 [MþH]þ; HRMS calcd for C19H19N4O4 367.1406,
found 367.1405.
3.4.1. (4S)-4-benzyl-2-(2-hydroxyphenyl)-3,4-dihydro-5H-pyrido
[10,20:1,2]imidazo-[4,5-d][1,3]diazepin-5-one (1x)
3.5.2. (4S)-4-(4-hydroxyphenyl)-2-(3,4-dimethoxyphenyl)-3,4-
dihydro-5H-pyrido [10,20:1,2]imidazo[4,5-d][1,3]diazepin-5-one
(1ac)
Brown solid (m
¼
0.117 g, 81%); mp: 187.0e188.0 ꢀC;
20
[
d
a]
¼ þ35.2ꢀ (c 0.25, DMSO); 1H NMR (DMSO-d6, 300 MHz):
D
ppm 2.90 (m, 1H), 3.15 (m, 1H), 4.41 (m, 1H), 6.70 (m, 1H), 6.86 (m,
Yellow solid (m
¼
0.080 g, 99%); mp: 167.0e168.0 ꢀC;
20
1H), 7.17 (m, 9H), 7.35 (m, 1H), 7.80 (bs, 2H), 9.47 (m, 1H); 13C NMR
(DMSO-d6, 75 MHz): ppm 35.0, 64.0, 112.1, 114.8, 114.9, 116.1, 117.6,
[a
]
¼ þ36.4ꢀ (c 0.75, DMSO); 1H NMR (DMSO-d6, 300 MHz):
D
d
d ppm 2.72 (m, 1H), 2.97 (m, 1H), 3.72 (s, 3H), 3.83 (s, 3H), 4.25 (d,

D.P. Arama et al. / European Journal of Medicinal Chemistry 93 (2015) 202e213
211
20
1H, J ¼ 8.1 Hz), 6.68 (d, 1H, J ¼ 6.0 Hz), 7.04e7.34 (m, 9H), 7.74 (bs,
1H), 9.29 (s, 1H), 9.48 (d, 1H, J ¼ 6.0 Hz); 13C NMR (DMSO-d6,
[a
]
¼ þ62.8ꢀ (c 0.53, CHCl3); 1H NMR (CDCl3, 300 MHz):
d
ppm
D
3.23 (dd, 1H, J ¼ 13.6, 7.4 Hz), 3.40 (dd, 1H, J ¼ 13.7, 7.0 Hz), 5.78 (dd,
1H, J ¼ 16.9, 7.3 Hz), 6.86 (td, 1H, J ¼ 6.8, 1.4 Hz), 7.11e7.44 (m, 10H),
7.8 (td, 1H, J ¼ 7.7, 1.8 Hz), 8.10 (d, 1H, J ¼ 7.9 Hz), 8.57 (d, 1H,
J ¼ 4.0 Hz), 8.78 (d, 1H, J ¼ 9.5 Hz), 9.64 (bs, 1H); 13C NMR (CDCl3,
100 MHz):
d ppm 34.2, 55.4, 55.8, 64.0, 111.0, 112.0, 112.7, 114.5,
115.2 (2C), 115.7, 123.6, 123.9, 126.7, 128.0, 130.4 (2C), 131.3, 135.2,
145.7, 148.0, 152.6, 156.2, 158.0, 182.1; FT-IR (cmꢂ1): 3230, 3087,
3031, 2916, 1722, 1613, 1591, 1557, 1510, 1462, 1337, 1236, 1175, 1141,
1111, 1021, 843, 811, 762, 694; HPLC, Tr ¼ 2.44 min; MS (ESIþ): m/z
443.2 [MþH]þ; HRMS calcd for C25H23N4O4 443.1722, found
443.1719.
75 MHz):
d ppm 38.6, 54.5, 108.7, 113.2, 114.4, 122.5, 126.7, 126.8,
127.4, 128.6 (2C), 129.3 (2C), 130.9, 136.9, 137.5, 147.9, 148.6, 149.1,
159.1, 165.2, 183.7; FT-IR (cmꢂ1): 3312, 3187, 3026, 1659, 1577, 1523,
1495, 1448, 1339, 1265, 1223, 1146, 1063, 753, 698; HPLC,
Tr ¼ 1.59 min; MS (ESIþ): m/z 386.2 [MþH]þ; HRMS calcd for
3.6. Synthesis of (4S)-4-benzyl-2-(3,4-dimethoxyphenyl)-4,5-
dihydro-3H-pyrido [10,20:1,2]imidazo[4,5-d][1,3]diazepin-5-ol (1ad)
C22H20N5O2 386.1617, found 386.1618.
3.7.3. (S)-N-(1-(2-amino-imidazo[1,2-a]pyridin-3-yl)-1-oxo-3-
A solution of compound 1o (200 mg, 0.047 mmol) in MeOH
(2 mL) was cooled at 0 ꢀC. Then NaBH4 (3 equiv., 53.5 mg, 1.4 mmol)
was added and the mixture was stirred at 0 ꢀC for 3 h (monitoring
by TLC). After reaction completion, 1 mL of saturated NaHCO3
aqueous solution was added to quench the reaction. The solvent
was then removed and the resulting residue was dissolved in ethyl
acetate. The solution was washed with saturated NaHCO3 aqueous
solution. The organic layer was dried over Na2SO4, filtered and the
solvent was removed under reduced pressure to afford compound
phenylpropan-2-yl)isonicotinamide (2c)
Yellow solid (m
¼
0.086 g, 80%); mp: 205.0e206.0 ꢀC;
20
[a
]
¼ þ69.5ꢀ (c 0.4, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 3.30
D
(dd, 1H, J ¼ 13.8, 7.4 Hz), 3.4 (dd, 1H, J ¼ 13.8, 6.7 Hz), 5.76 (dd, 1H,
J ¼ 15.5, 6.9 Hz), 5.98 (s, 2H), 6.9 (td, 1H, J ¼ 6.9, 1.3 Hz), 7.14e7.23
(m, 4H), 7.30 (d, 1H, J ¼ 8.7 Hz), 7.44 (td, 1H, J ¼ 7.0, 1.1 Hz), 7.53 (d,
4H, J ¼ 6.0 Hz), 8.67 (dd, 2H, J ¼ 4.6, 1.4 Hz), 9.55 (bs, 1H); 13C NMR
(CDCl3, 75 MHz):
d ppm 38.6, 55.1, 108.5, 113.5, 114.5, 121.0 (2C),
127.2, 128.5, 128.7 (2C), 129.3 (2C), 129.8, 131.4, 136.3, 140.8, 148.1,
150.7 (2C), 159.2, 166.4; FT-IR (cmꢂ1): 3311, 3188, 1650, 1579, 1495,
1455, 1339, 1310, 1265, 1063, 846, 752, 696, 667; HPLC,
Tr ¼ 1.19 min; MS (ESIþ): m/z 386.2 [MþH]þ; HRMS calcd for
1ad as a light yellow solid (m ¼ 30 mg, 15%). Mp: 174.0ꢀCe175.0 ꢀC;
20
[
d
a
]
¼ ꢂ45.31ꢀ (c 0.49, DMSO); 1H NMR (DMSO-d6, 400 MHz):
D
ppm 1.23 (s,1H), 3.72 (s, 4H), 3.81 (s, 5H), 5.40 (s,1H), 5.87 (bs,1H),
6.99 (bs, 2H), 7.00 (bs, 1H), 7.11e7.35 (m, 5H), 7.49 (bs, 3H), 8.36 (bs,
C22H20N5O2 386.1617, found 386.1619.
1H); 13C NMR (DMSO-d6, 100 MHz):
d
ppm 36.1, 55.5, 55.6, 60.0,
66.5, 110.9, 112.1, 112.3, 112.9, 115.5, 118.9, 122.8, 124.4, 125.0, 126.5,
128.5 (2C), 129.4 (2C), 138.9, 141.2, 144.9, 148.0, 152.0, 158.0; FT-
IR(cmꢂ1): 3379, 2921, 2869, 2853,1702, 1597, 1570,1501, 1455, 1414,
1350, 1267, 1100, 1027, 948, 746, 707; HPLC, Tr ¼ 2.27 min; MS
(ESIþ): m/z 429.2 [MþH]þ; HRMS calcd for C25H25N4O3 429.1924,
found 429.1927.
3.7.4. (S)-N-(1-(2-amino-imidazo[1,2-a]pyridin-3-yl)-1-oxo-3-
phenylpropan-2-yl)-1H-pyrrole-2-carboxamide (2d)
Yellow solid (m
¼
0.117 g, 88%); mp: 197.5e198.5 ꢀC;
20
[
d
a
]
¼ þ15.8ꢀ (c 0.54, DMSO); 1H NMR (DMSO-d6, 300 MHz):
D
ppm 3.15 (m, 2H), 5.46 (dd, 1H, J ¼ 13.0, 7.0 Hz), 6.1 (m, 1H), 6.87
(m,1H), 6.95 (t,1H, J ¼ 8.1 Hz), 7.02 (m,1H), 7.1e7.25 (m, 7H), 7.33 (t,
1H, J ¼ 7.2 Hz), 7.48 (t, 1H, J ¼ 7.4 Hz), 8.8 (d, 1H, J ¼ 8.7 Hz), 9.55 (d,
3.7. General procedure for synthesis of opened derivatives 2a-i
1H, J ¼ 6.6 Hz), 11.48 (s, 1H); 13C NMR (DMSO-d6, 75 MHz):
d ppm
37.0, 54.3, 107.3, 108.8, 111.6, 112.6, 113.6, 122.1, 124.8, 126.2, 128.0
(2C), 128.3, 129.0 (2C), 130.7, 138.0, 147.2, 158.8, 161.2, 184.0; FT-IR
(cmꢂ1): 3305, 3194, 1577, 1556, 1495, 1442, 1338, 1264, 1193, 1129,
1041, 844, 733, 696; HPLC, Tr ¼ 1.54 min; MS (ESIþ): m/z 374.2
[MþH]þ; HRMS calcd for C21H20N5O2 374.1617, found 374.1616.
To a solution of diamine derivative 3a (100 mg) in DCM (10 mL)
at 0 ꢀC, were added HOBt (1.1 equiv.), EDCI (1.1 equiv.) and Et3N
(1.1 equiv.) and the resulting mixture was stirred at 0 ꢀC for 30 min.
The solution was then stirred at rt for 6 h (monitoring by TLC). The
solution was washed with saturated NaHCO3 aqueous solution
(3 ꢁ 30 mL). The organic layers were combined, dried over Na2SO4,
filtered and the solvent was removed under reduced pressure. The
residue was dissolved in dichloromethane and filtered through
alumina gel using sintered glass Buchner funnel. The solvent was
removed in vacuo to afford pure opened derivatives 2a-i.
3.7.5. (S)-N-(1-(2-amino-imidazo[1,2-a]pyridin-3-yl)-1-oxo-3-
phenylpropan-2-yl)-2-methylbenzamide (2e)
20
White solid (m ¼ 0.111 g, 76%); mp: 87.0e88.0 ꢀC; [
a
]
¼ þ31.1ꢀ
D
(c 1.0, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 1.86 (bs, 1H), 2.21
(s, 3H), 3.26 (dd, 1H, J ¼ 13.9, 8.3 Hz), 3.33 (dd, 1H, J ¼ 13.9, 6.3 Hz),
5.80 (dd, 1H, J ¼ 13.9, 8.3 Hz), 6.31 (s, 2H), 6.84 (td, 1H, J ¼ 13.8,
3.7.1. (S)-N-(1-(2-amino-imidazo[1,2-a]pyridin-3-yl)-1-oxo-3-
phenylpropan-2-yl)benzamide (2a)
6.9 Hz), 7.22 (m,10H), 7.38 (td,1H, J ¼ 15.8, 6.9 Hz), 9.55 (bs,1H); 13
C
NMR (CDCl3, 75 MHz): d ppm 19.7, 38.4, 54.8, 113.2, 114.3, 125.7,
White solid (m
¼
0.038 g, 68%); mp: 214.0e215.0 ꢀC;
126.9, 127.0, 127.3, 128.6 (2C), 128.7, 129.4 (2C), 130.2, 131.0, 131.1,
135.3, 136.3, 136.8, 148.1, 159.3, 170.9, 184.0; FT-IR (cmꢂ1): 3331,
3179, 2828, 1731, 1633, 1578, 1526, 1496, 1452, 1338, 1318, 1203,
1158, 1056, 735, 698; HPLC, Tr ¼ 1.70 min; MS (ESIþ): m/z 399.1
[MþH]þ; HRMS calcd for C24H23N4O2 399.1821, found 399.1820.
20
[
a]
¼ þ96.59ꢀ (c 0.44, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm
D
3.24 (dd, 1H, J ¼ 9.0, 6.0 Hz), 3.35 (dd, 1H, J ¼ 9.0, 6.0 Hz), 5.78 (dd,
1H, J ¼ 12.0, 3.0 Hz), 6.22 (bs, 2H), 6.65 (td, 1H, J ¼ 6.0, 3.0 Hz), 7.13
(t, 1H, J ¼ 6.0 Hz), 7.20 (t, 2H, J ¼ 6.0 Hz), 7.26e7.30 (m, 4H), 7.33 (t,
2H, J ¼ 6.0 Hz), 7.39e7.48 (m, 2H), 7.68 (d, 2H, J ¼ 6.0 Hz), 9.60 (d,
1H, J ¼ 6.0 Hz); 13C NMR (CDCl3, 75 MHz):
d
ppm 38.6, 54.8, 108.5,
3.7.6. (S)-N-(1-(2-amino-imidazo[1,2-a]pyridin-3-yl)-1-oxo-3-
113.2, 114.3, 127.0, 127.2 (2C), 128.7 (4C), 129.3 (2C), 129.8, 131.1,
132.1, 133.6, 136.7, 148.0, 159.3, 168.3, 184.2; FT-IR (cmꢂ1): 3311,
3184, 1633,1574, 1526, 1495, 1455, 1339, 1263, 1054, 760, 695; HPLC,
Tr ¼ 1.62 min; MS (ESIþ): m/z 385.2 [MþH]þ; HRMS calcd for
phenylpropan-2-yl)-3,4-dimethoxybenzamide (2f)
20
White solid (m ¼ 0.141 g, 89%), mp: 85.0e86.0 ꢀC; [
a
]
¼ þ47.2ꢀ
D
(c 1.0, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 1.99 (bs, 1H), 3.25
(dd, 1H, J ¼ 12.0, 6.0 Hz), 3.34 (dd, 1H, J ¼ 15.0, 6.0 Hz), 3.83 (s, 3H)
3.86 (s, 3H), 5.77 (dd, 1H, J ¼ 15.0, 6.0 Hz), 6.27 (bs, 2H), 6.72 (d, 1H,
J ¼ 9.0 Hz), 6.82 (td, 1H, J ¼ 6.0, 3.0 Hz), 7.11e7.24 (m, 4H), 7.29e7.42
C
23H21N4O2 385.1665, found 385.1665.
3.7.2. (S)-N-(1-(2-amino-imidazo[1,2-a]pyridin-3-yl)-1-oxo-3-
(m, 5H) 9.59 (bs, 1H); 13C NMR (CDCl3, 75 MHz):
d ppm 38.6, 54.8,
phenylpropan-2-yl)picolinamide (2b)
56.1 (2C), 110.4, 110.7, 113.2, 114.4, 120.0, 126.0, 126.9, 128.6 (2C),
128.9, 129.3 (2C), 130.3, 131.1, 136.7, 148.0, 149.1, 152.3, 159.3, 167.9,
Yellow solid (m
¼
0.045 g, 53%); mp: 74.0e75.0 ꢀC;

212
D.P. Arama et al. / European Journal of Medicinal Chemistry 93 (2015) 202e213
184.5; FT-IR (cmꢂ1): 3331, 2925, 2852, 1724, 1577, 1542, 1494, 1440,
1339, 1262, 1229, 1055, 1019, 757, 698; HPLC, Tr ¼ 1.35 min; MS
(ESIþ): m/z 445.1 [MþH]þ; HRMS calcd for C25H25N4O4 445.1876,
found 445.1877.
0.2 nM (KLK14), 10 mM (Boc-Val-Pro-Arg-AMC). Compounds were
tested in triplicate for different concentrations to detect their
inhibitory potency. The enzyme and the inhibitors were incubated
for 15 min before the determination of the enzyme activity. Initial
rates determined in control experiments (V0) were considered to be
100% of the proteinase activity; initial rates (Vi) that were below
100% in the presence of a tested compound were considered to be
inhibitions. The inhibitory activity of compounds was expressed as
IC50 (inhibitor concentrations giving 50% inhibition). The values of
IC50 were calculated by fitting the experimental data either to
3.7.7. (S)-N-(1-(2-amino-imidazo[1,2-a]pyridin-3-yl)-1-oxo-3-
phenylpropan-2-yl)-2-phenylacetamide (2g)
Yellow solid (m
¼
0.120 g, 84%); mp: 161.0e162.0 ꢀC;
20
[
a]
¼ þ23.8ꢀ (c 0.5, DMSO); 1H NMR (DMSO d6, 300 MHz):
d ppm
D
3.06 (dd, 1H, J ¼ 13.6, 8.0 Hz), 3.15 (dd, 1H, J ¼ 13.6, 6.0 Hz), 3.35 (bs,
1H), 3.48 (d, 1H, J ¼ 14.0 Hz), 3.55 (d, 1H, J ¼ 14.0 Hz), 5.33 (dd, 1H,
J ¼ 13.0, 8.0 Hz), 6.96 (s, 2H), 7.02 (t, 1H, J ¼ 6.8 Hz), 7.21 (m, 10H),
7.36 (d,1H, J ¼ 8.7 Hz), 7.54 (t,1H, J ¼ 8.0 Hz), 9.15 (d,1H, J ¼ 8.0 Hz);
equation (1): % Inhibition ¼ 100 ꢁ (1-Vi/V0) ¼ 100 [I]0/(IC50 þ [I]0),
nH
or equation (2), % Inhibition ¼ 100 [I]n0H/(IC þ [I]0nH) with nH ¼ Hill
50
number.
13C NMR (DMSO d6, 75 MHz):
d ppm 37.1, 41.5, 54.4, 107.1, 112.7,
113.6, 126.2, 126.3, 128.0 (2C), 128.1 (2C), 129.0 (2C), 129.1 (2C),
130.8, 135.8, 136.9, 137.6, 147.2, 158.8, 171.4, 183.7; FT-IR (cmꢂ1):
694, 716, 759, 1065, 1264, 1317, 1337, 1454, 1494, 1574, 1522, 1646,
1630, 2922, 3003, 3195, 3326; HPLC, Tr ¼ 1.6 min; MS(ESIþ): m/z
399.1 [MþH]þ; HRMS calcd for C24H23N4O2 399.1821, found
399.1818.
3.9. Mechanisms of inhibition
Reversibility was analyzed by diluting the reaction mixtures
(dilution factor of 40) after 15 and 60 min preincubation of the
enzyme with inhibitor. Aliquots of reaction mixtures (2.5
mL) were
added to 97.5 L of buffer containing the fluorogenic substrate
m
(experimental conditions identical to the routine protocol used for
a given enzyme). The mechanism of inhibition was determined by
varying substrate and inhibitor concentrations and using classical
LineweavereBurk plots.
3.7.8. (S)-N-(1-(2-amino-imidazo[1,2-a]pyridin-3-yl)-1-oxo-3-
phenylpropan-2-yl)cinnamamide (2h)
Yellow solid (m
¼
0.017 g, 20%); mp: 85.0e86.0 ꢀC;
20
[
a]
¼ þ16.5ꢀ (c 0.6, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 3.08
D
(dd, 1H, J ¼ 13.8, 7.5 Hz), 3.21 (dd, 1H, J ¼ 13.9, 6.6 Hz), 5.64 (dd, 1H,
J ¼ 15.9, 7.4 Hz), 6.15 (s, 2H), 6.28 (d, 1H, J ¼ 15.6 Hz), 6.76 (t, 1H,
J ¼ 6.8 Hz), 6.98e7.30 (m, 13H), 7.44 (d, 1H, J ¼ 15.6 Hz), 9.50 (bs,
3.10. Structure-based molecular docking
The docking study of compound 1o was carried out using the
docking engine Molegro virtual docker version 5.5.0. The enzyme
structures used for the docking were obtained from 2QXH PDB file
corresponding to KLK7 complexed with succinyl-Ala-Ala-Pro-Phe
chloromethylketone inhibitor. Heteroatoms and ligands were
removed from the crystal structures and hydrogen atoms were
added. The scoring function Moldock Score and the Moldock SE
search algorithm were used; the resolution of the grid for estima-
tion of the computed binding affinity was 0.3 Å with a radius of 15 Å
around the catalytic triad residues. Fifteen runs were launched and
up to 50 (maximum) poses subsequently analyzed. Figures were
prepared with Pymol (DeLano Scientific LLC, San Carlos, CA, USA).
1H); 13C NMR (CDCl3, 75 MHz):
d ppm 38.6, 54.6, 108.6, 113.3, 114.4,
119.7, 127.0, 128.0 (2C), 128.3, 128.6 (2C), 128.9 (2C), 129.3 (2C),
130.1, 131.1, 134.6, 136.7, 142.4, 148.2, 159.6, 166.8, 184.6; FT-IR
(cmꢂ1): 3285, 3185, 2974, 1720, 1574, 1494, 1455, 1337, 1265, 1221,
979, 753, 731, 683; HPLC, Tr ¼ 1.5 min; MS (ESIþ): m/z 411.1
[MþH]þ; HRMS calcd for C25H23N4O2 411.1821, found 411.1821.
3.7.9. (S)-N-(1-(2-amino-imidazo[1,2-a]pyridin-3-yl)-1-oxo-3-
phenylpropan-2-yl)-3-phenylpropanamide (2i)
White solid (m
¼
0.085 g, 59%); mp: 69.0ꢀCe70.0 ꢀC;
20
[
a]
¼ þ69.0ꢀ (c 1.0, CHCl3); 1H NMR (CDCl3, 300 MHz):
d ppm 2.43
D
(t, 2H, J ¼ 9.0 Hz), 2.83 (t, 2H, J ¼ 9.0 Hz), 3.04 (dd, 1H, J ¼ 12.0,
6.0 Hz), 3.18 (dd, 1H, J ¼ 15.0, 6.0 Hz), 5.58 (dd, 1H, J ¼ 15.0, 6.0 Hz),
6.29 (bs, 2H), 6.81 (td, 1H, J ¼ 9.0, 3.0 Hz), 6.94e7.18 (m, 11H), 7.28
(bs, 1H), 7.37 (td, 1H, J ¼ 9.0, 3.0 Hz), 9.58 (bs, 1H); 13C NMR (CDCl3,
3.11. Cell culture and cytotoxicity assay
PC3 and SW-60 cells were kindly supplied by Dr Dalila Darmoul
ꢀ
ꢀ
75 MHz):
d
ppm 31.6, 38.0, 38.3, 54.2, 108.4, 113.2, 114.3, 126.3,
(INSERM U976, Universite Paris Diderot, and Sorbonne Paris Cite).
Cells were spread at a rate of 5000 cells per well of 96-well plates in
126.9, 128.3 (4C), 128.4, 128.6 (2C), 129.2 (2C), 131.1, 136.8, 140.5,
148.0, 159.4, 173.2, 184.3; FT-IR (cmꢂ1): 3325, 3178, 3028, 1734,
1644, 1571, 1495, 1452, 1339, 1264, 1065, 762; HPLC, Tr ¼ 1.6 min;
MS (ESIþ): m/z 413.3 [MþH]þ; HRMS calcd for C25H25N4O2
413.1978, found 413.1981.
100
plemented with fetal bovine serum (10%), penicillin (100
and streptomycin (100
g/mL), at 37 ꢀC and 5% of CO2. 20 h after
seeding the cells, 1 L of different concentrations of each inhibitor
mL of medium/well consisting of DMEM medium com-
mg/mL)
m
m
previously dissolved in DMSO was added to the wells. An equiva-
lent volume of DMSO was used in controls for a final DMSO con-
centration of 1% (v/v). For each condition, at least three wells were
measured. 48e72 h after incubation, culture medium was removed
3.8. Enzyme and inhibition assays
The enzyme activities of KLK5, KLK7, KLK8 and KLK14 (R&D
Systems, France) were determined using 96-well plates monitored
using a BMG Fluostar apparatus. The hydrolysis of the appropriate
fluorogenic substrate was measured (lexc ¼ 360 nm, lem ¼ 460 nm)
for 15 min at 37 ꢀC in the presence of untreated enzyme (control)
and treated enzyme incubated with a test compound. Substrates
(Bachem, Weil am Rhein, Germany) were previously dissolved in
DMSO, with the final solvent concentration kept constant at 2% (v/
v). The composition of the activity buffers was (pH 8.0): 50 mM
Tris-HCl, Tween 20 0.01% (v/v) and 150 mM NaCl for KLK5, 1 M NaCl
for KLK7 and KLK14. The final concentrations were 0.6 nM (KLK5),
and replaced by 100
mL of new medium/well (without phenol red)
supplemented with XTT (0.3 mg/mL) and PMS (8.3
mM). After 3 h
incubation the absorbance at 485 nm was read. The XTT assay is
based on the mitochondrial deshydrogenase activity. The relative
cell viability was expressed as a percentage to the viability of cells
treated with DMSO only.
Author contributions
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of the
manuscript.
100
mM (Boc-Val-Pro-Arg-AMC); 7.6 nM (KLK7), 40
mM (Suc-Leu-
M (Boc-Val-Pro-Arg-AMC);
Leu-Val-Tyr-AMC); 1 nM (KLK8), 100
m

D.P. Arama et al. / European Journal of Medicinal Chemistry 93 (2015) 202e213
213
overexpression of stratum corneum chymotryptic enzyme in mice: a model
for chronic itchy dermatitis, J. Invest. Dermatol. 118 (2002) 444e449.
[16] S. Schultz, A. Saalbach, J.T. Heiker, R. Meier, T. Zellmann, J.C. Simon, A.G. Beck-
Sickinger, Proteolytic activation of prochemerin by kallikrein 7 breaks an ionic
linkage and results in C-terminal rearrangement, Biochem. J. 452 (2013)
271e280.
[17] P. Goettig, V. Magdolen, H. Brandstetter, Natural and synthetic inhibitors of
kallikrein-related peptidases (KLKs), Biochimie 92 (2010) 1546e1567.
[18] C. Deraison, C. Bonnart, F. Lopez, C. Besson, R. Robinson, A. Jayakumar,
F. Wagberg, M. Brattsand, J.P. Hachem, G. Leonardsson, A. Hovnanian, LEKTI
fragments specifically inhibit KLK5, KLK7, and KLK14 and control desqua-
Notes
The authors declare no competing financial interest.
Acknowledgments
This work was supported by Universite of Montpellier, Institut
ꢀ
ꢀ
ꢀ
National pour la Recherche Medicale (INSERM), Universite Pierre et
Marie Curie (UPMC), Centre National de la Recherche Scientifique
mation through
a pH-dependent interaction, Mol. Biol. Cell. 18 (2007)
ꢀ
(CNRS), and Region Languedoc-Roussillon (Chercheur d'avenir 2011
3607e3619.
[19] S.J. de Veer, S.S. Ukolova, C.A. Munro, J.E. Swedberg, A.M. Buckle, J.M. Harris,
Mechanism-based selection of a potent kallikrein-related peptidase 7 inhib-
itor from a versatile library based on the sunflower trypsin inhibitor SFTI-1,
Biopolymers (2013).
grant to V.L.). The authors are grateful to the French Ministry of
Research and Education for F. S. and the Mali Government for D.A
ꢁ
PhD fellowships. CE and NM thanks Prof Michele Reboud-Ravaux
[20] T.S. Teixeira, R.F. Freitas, O. Abrahao Jr., K.F. Devienne, L.R. de Souza, S.I. Blaber,
M. Blaber, M.Y. Kondo, M.A. Juliano, L. Juliano, L. Puzer, Biological evaluation
and docking studies of natural isocoumarins as inhibitors for human kallikrein
5 and 7, Bioorg. Med. Chem. Lett. 21 (2011) 6112e6115.
and Dr Alain Chavanieu for fruitful discussions and are grateful to
Christina Lymperopoulou and Isabelle Bequignon for technical
assistance.
[21] R.F. Freitas, T.S. Teixeira, T.G. Barros, J.A. Santos, M.Y. Kondo, M.A. Juliano,
L. Juliano, M. Blaber, O.A. Antunes, O. Abrahao Jr., S. Pinheiro, E.M. Muri,
L. Puzer, Isomannide derivatives as new class of inhibitors for human kalli-
krein 7, Bioorg. Med. Chem. Lett. 22 (2012) 6072e6075.
Appendix A. Supplementary data
[22] J.P. Oliveira, R.F. Freitas, L.S. Melo, T.G. Barros, J.A. Santos, M.A. Juliano,
S. Pinheiro, M. Blaber, L. Juliano, E.M. Muri, L. Puzer, Isomannide-based pep-
tidomimetics as inhibitors for human tissue kallikreins 5 and 7, ACS Med.
Chem. Lett. 5 (2014) 128e132.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.02.008.
[23] X. Tan, C. Bertonati, L. Qin, L. Furio, C. El Amri, A. Hovnanian, M. Reboud-
Ravaux, B.O. Villoutreix, Identification by in silico and in vitro screenings of
small organic molecules acting as reversible inhibitors of kallikreins, Eur. J.
Med. Chem. 70 (2013) 661e668.
[24] X. Tan, L. Furio, M. Reboud-Ravaux, B.O. Villoutreix, A. Hovnanian, C. El Amri,
1,2,4-Triazole derivatives as transient inactivators of kallikreins involved in
skin diseases, Bioorg. Med. Chem. Lett. 23 (2013) 4547e4551.
[25] N. Masurier, R. Aruta, V. Gaumet, S. Denoyelle, E. Moreau, V. Lisowski,
J. Martinez, L.T. Maillard, Selective C-acylation of 2-Aminoimidazo[1,2-a]
pyridine: application to the synthesis of imidazopyridine-fused [1,3]Dia-
zepinones, J. Org. Chem. 77 (2012) 3679e3685.
[26] N. Masurier, E. Debiton, A. Jacquemet, A. Bussiere, J.M. Chezal, A. Ollivier,
D. Tetegan, M. Andaloussi, M.J. Galmier, J. Lacroix, D. Canitrot, J.C. Teulade,
R.C. Gaudreault, O. Chavignon, E. Moreau, Imidazonaphthyridine systems
(part 2): functionalization of the phenyl ring linked to the pyridine pharma-
cophore and its replacement by a pyridinone ring produces intriguing dif-
ferences in cytocidal activity, Eur. J. Med. Chem. 52 (2012) 137e150.
[27] N. Masurier, E. Moreau, C. Lartigue, V. Gaumet, J.-M. Chezal, A. Heitz, J.-
C. Teulade, O. Chavignon, New opportunities with the Duff reaction, J. Org.
Chem. 73 (2008) 5989e5992.
[28] 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, antiproliferative
and DNA-intercalating activities, Eur. J. Med. Chem. 43 (2008) 2505e2517.
[29] G. Chaubet, L.T. Maillard, J. Martinez, N. Masurier, A tandem aza-Friedel-Crafts
reaction/Hantzsch cyclization: a simple procedure to access polysubstituted
2-amino-1,3-thiazoles, Tetrahedron 67 (2011) 4897e4904.
[30] L.T. Maillard, S. Bertout, O. Quinonero, G. Akalin, G. Turan-Zitouni, P. Fulcrand,
F. Demirci, J. Martinez, N. Masurier, Synthesis and anti-Candida activity of
novel 2-hydrazino-1,3-thiazole derivatives, Bioorg. Med. Chem. Lett. 23
(2013) 1803e1807.
[31] A. Gallud, O. Vaillant, L.T. Maillard, D.P. Arama, J. Dubois, M. Maynadier,
V. Lisowski, M. Garcia, J. Martinez, N. Masurier, Imidazopyridine-fused [1,3]-
diazepinones: synthesis and antiproliferative activity, Eur. J. Med. Chem. 75
(2014) 382e390.
[32] D.P. Arama, V. Lisowski, E. Scarlata, P. Fulcrand, L.T. Maillard, J. Martinez,
N. Masurier, An efficient synthesis of pyrido-imidazodiazepinediones, Tetra-
hedron Lett. 54 (2013) 1364e1367.
References
ꢀ
[1] G.P. Gupta, J. Massague, Cancer metastasis: building a framework, Cell 127
(2006) 679e695.
[2] C.A. Borgono, E.P. Diamandis, The emerging roles of human tissue kallikreins
in cancer, Nat. Rev. Cancer 4 (2004) 876e890.
[3] C.A. Borgono, I.P. Michael, E.P. Diamandis, Human tissue kallikreins: physio-
logic roles and applications in cancer, Mol. Cancer Res. 2 (2004) 257e280.
[4] M. Debela, V. Magdolen, N. Schechter, M. Valachova, F. Lottspeich, C.S. Craik,
Y. Choe, W. Bode, P. Goettig, Specificity profiling of seven human tissue kal-
likreins reveals individual subsite preferences, J. Biol. Chem. 281 (2006)
25678e25688.
[5] M. Debela, P. Hess, V. Magdolen, N.M. Schechter, T. Steiner, R. Huber, W. Bode,
P. Goettig, Chymotryptic specificity determinants in the 1.0 A structure of the
zinc-inhibited human tissue kallikrein 7, Proc. Natl. Acad. Sci. U. S. A. 104
(2007) 16086e16091.
[6] V.C. Ramani, R.S. Haun, Expression of kallikrein 7 diminishes pancreatic cancer
cell adhesion to vitronectin and enhances urokinase-type plasminogen acti-
vator receptor shedding, Pancreas 37 (2008) 399e404.
[7] M. Devetzi, T. Trangas, A. Scorilas, D. Xynopoulos, M. Talieri, Parallel over-
expression and clinical significance of kallikrein-related peptidases 7 and 14
(KLK7KLK14) in colon cancer, Thromb. Haemost. 109 (2013) 716e725.
[8] L. Mo, J. Zhang, J. Shi, Q. Xuan, X. Yang, M. Qin, C. Lee, H. Klocker, Q.Q. Li, Z. Mo,
Human kallikrein 7 induces epithelial-mesenchymal transition-like changes
in prostate carcinoma cells: a role in prostate cancer invasion and progression,
Anticancer Res. 30 (2010) 3413e3420.
[9] C.A. Borgono, I.P. Michael, N. Komatsu, A. Jayakumar, R. Kapadia, G.L. Clayman,
G. Sotiropoulou, E.P. Diamandis, A potential role for multiple tissue kallikrein
serine proteases in epidermal desquamation, J. Biol. Chem. 282 (2007)
3640e3652.
[10] T. Egelrud, M. Brattsand, P. Kreutzmann, M. Walden, K. Vitzithum, U.C. Marx,
W.G. Forssmann, H.J. Magert, hK5 and hK7, two serine proteinases abundant
in human skin, are inhibited by LEKTI domain 6, Br. J. Dermatol 153 (2005)
1200e1203.
[11] M. Brattsand, K. Stefansson, C. Lundh, Y. Haasum, T. Egelrud, A proteolytic
cascade of kallikreins in the stratum corneum, J. Invest. Dermatol. 124 (2005)
198e203.
[12] C. Caubet, N. Jonca, M. Brattsand, M. Guerrin, D. Bernard, R. Schmidt,
T. Egelrud, M. Simon, G. Serre, Degradation of corneodesmosome proteins by
two serine proteases of the kallikrein family, SCTE/KLK5/hK5 and SCCE/KLK7/
hK7, J. Invest. Dermatol. 122 (2004) 1235e1244.
[33] M.E. Kaighn, K.S. Narayan, Y. Ohnuki, J.F. Lechner, L.W. Jones, Establishment
and characterization of a human prostatic carcinoma cell line (PC-3), Invest.
Urol. 17 (1979) 16e23.
[34] A. Leibovitz, J.C. Stinson, W.B. McCombs III, C.E. McCoy, K.C. Mazur,
N.D. Mabry, Classification of human colorectal adenocarcinoma cell lines.
Cancer Res. 36 (1976) 4562e4569.
[13] A. Hovnanian, Netherton syndrome: skin inflammation and allergy by loss of
protease inhibition, Cell. Tissue Res. 351 (2013) 289e300.
[14] S. Wang, S. Olt, N. Schoefmann, A. Stuetz, A. Winiski, B. Wolff-Winiski, SPINK5
knockdown in organotypic human skin culture as a model system for Neth-
erton syndrome: effect of genetic inhibition of serine proteases kallikrein 5
and kallikrein 7, Exp. Dermatol (2014).
[35] F. Walker, P. Nicole, A. Jallan, A. Soosaipillai, V. Mosbach, K. Oikonomopoulou,
E.P. Diamandis, V. Magdolen, D. Darmoul, Kallikrein-related peptidase
7
(KLK7) is a proliferative factor that is aberrantly expressed in human colon
cancer, Biol. Chem. 395 (2014) 1075e1086.
[15] L. Hansson, A. Backman, A. Ny, M. Edlund, E. Ekholm, B. Ekstrand Hammar-
strom, J. Tornell, P. Wallbrandt, H. Wennbo, T. Egelrud, Epidermal