Substituted quinazolinones as kinase inhibitors endowed with anti-fibrotic properties

Article abstract of DOI:10.1016/j.ejmech.2016.03.053

Some new 3-substituted quinazolinones were synthesized and evaluated as inhibitors of kinases involved in fibrogenic process. The compounds were tested against a panel of both tyrosine and serine-threonine kinases. The profile of selectivity of some representative compounds was investigated through molecular docking studies. The most interesting compounds were also evaluated in vitro as potential agents for the treatment of fibrotic diseases. Quinazolinone derivatives reduced proliferation and expression of genes involved in the fibrogenic process in hepatic stellate cells (HSCs) and intestinal subepithelial myofibroblasts (ISEMFs). Furthermore some compounds downregulated phosphorylation of p38MAPK. Our findings provide evidences that 3-substituted quinazolinones target multiple essential pathways of the fibrogenic process.

Full text of DOI:10.1016/j.ejmech.2016.03.053

European Journal of Medicinal Chemistry 115 (2016) 416e425
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Substituted quinazolinones as kinase inhibitors endowed with anti-
fibrotic properties
,
Giovanni Marzaro a *, Ignazio Castagliuolo b, Giulia Schirato b, Giorgio Palu' b,
Martina Dalla Via a, Adriana Chilin a, Paola Brun b
a Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, 35131 Padova, Italy
b Department of Molecular Medicine, University of Padova, Via Gabelli 63, 35121 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Some new 3-substituted quinazolinones were synthesized and evaluated as inhibitors of kinases
involved in fibrogenic process. The compounds were tested against a panel of both tyrosine and serine
ethreonine kinases. The profile of selectivity of some representative compounds was investigated
through molecular docking studies. The most interesting compounds were also evaluated in vitro as
potential agents for the treatment of fibrotic diseases. Quinazolinone derivatives reduced proliferation
and expression of genes involved in the fibrogenic process in hepatic stellate cells (HSCs) and intestinal
subepithelial myofibroblasts (ISEMFs). Furthermore some compounds downregulated phosphorylation
of p38MAPK. Our findings provide evidences that 3-substituted quinazolinones target multiple essential
pathways of the fibrogenic process.
Received 11 January 2016
Received in revised form
17 March 2016
Accepted 18 March 2016
Available online 19 March 2016
Keywords:
Quinazolinones
Tyrosine kinases
Multi kinase inhibitors
Fibrosis
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
receptor (FGFR1), and the cytoplasmic enzymes Abl1 and Src play
crucial roles in cell proliferation as well as in cancer onset and
Protein kinases are ubiquitous enzymes devoted to the regula-
tion of almost all cellular events. The kinases catalyze the transfer of
a phosphate group from the ATP to specific substrate eventually
leading to transduction and propagation of cellular signal [1].
Deregulated activity, mutation or over-expression of these enzymes
have been correlated to cancer [2], chronic inflammatory disorders
[3], diabetes [4], cardiovascular diseases [5] and hypertension [6].
According to the targeted amino acids, kinases are commonly
grouped in two major families: the tyrosine kinases (TKs) and the
serineethreonine kinases (STKs). Among the TKs, the epidermal
growth factor receptor (EGFR), the type 2 vascular endothelial re-
ceptor (VEGFR2 or KDR), the type 1 fibroblast growth factor
progression [7]. On the other hand, the phosphatidylinositol-3 ki-
nase (PI3K) is mainly a lipid kinase that, along with the mammalian
target of rapamycin (mTOR), is included in the STK family. Indeed,
by phosphorylation of serine or threonine containing proteins the
mTOR/AKT/PI3K pathway controls several cellular functions
including inflammatory responses and cancer development [8].
Remarkably, TKs and STKs -induced intracellular signals are
important modulators of fibrogenic process in lung, liver, pancreas,
heart, and gut. Fibrosis can occur during tissue repair or inflam-
mation as a result of persistent activation of fibrogenic cells, which
leads to aberrant extracellular matrix (ECM) deposition and pro-
gressive substitution of the normal parenchyma by scar tissue [9].
For instance, persistent liver injury and unrestrained inflammatory
cascade lead to EGFR-mediated proliferation and migration of he-
patic stellate cells (HSCs), the cellular population involved in the
deposition of ECM in the liver [10]. In addition, release of specific
growth factors triggers the synergistic activation of a number of
protein kinase pathways that serve different biological roles related
to fibrogenesis [11]. Inhibition of KDR by neutralizing monoclonal
antibody ameliorated carbon tetrachloride induced hepatic fibrosis
in mice not only by suppressing the neovascularisation but also by
Abbreviations: CAN, cerium ammonium nitrate; CD, Crohn's Desease; COL1A1,
prepro-alpha 1 collagen; DMF, dimethylformamide; ECM, extracellular matrix;
EGFR, epidermal growth factor receptor; FGFR1, type 1 fibroblast growth factor
receptor; FN1, fibronectin 1; HSC, hepatic stellate cells; ISEMF, intestinal sub-
epithelial myofibroblasts; KDR, type
2 vascular endothelial receptor; mTOR,
mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor;
PI3K, phosphatidylinositol-3 kinase; STK, serineethreonine kinases; TFA, tri-
fluoroacetic acid; TIMP1, tissue inhibitor of metalloproteinase 1; TK, tyrosine ki-
nases; VEGFR2, type 2 vascular endothelial receptor.
* Corresponding author.
E-mail address: giovanni.marzaro@unipd.it (G. Marzaro).
reducing the
a1(I)-procollagen mRNA expression in HSCs [12].
Likewise, preliminary randomized double-blind clinical trials
http://dx.doi.org/10.1016/j.ejmech.2016.03.053
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

G. Marzaro et al. / European Journal of Medicinal Chemistry 115 (2016) 416e425
417
indicate that sirolimus and everolimus, two mTOR inhibitors,
induce remission in refractory Crohn's Disease (CD) by decreasing
the number of intestinal subepithelial myofibroblasts (ISEMFs) and
the expression of pro-fibrotic cytokines [13e15]. Currently, the
pharmacological care in patients with tissue fibrosis relies on cor-
ticosteroids and immunosuppressant drugs but fibrosis-related
consequences remain a major causes of morbidity and mortality
[16].
was substituted with several lipophilic moieties (2-bromopyridine,
biphenyl, halophenyl) linked to the quinazolinone nitrogen with a
bridge of variable sizes and chemical properties (methylene, 2-
hydroxyethylene, 2-oxoethylene). According to the type of substi-
tution at the positions 6 and 7, the newly synthesized compounds
were grouped in three classes (Table 1). All the compounds have
been synthesized starting from the appropriate aniline derivatives
through quinazoline intermediates, taking advantage of an already
reported synthetic strategy [36] (Scheme 1).
The pivotal role of kinases as targets for novel drugs is clearly
demonstrated by the huge amount of ATP-mimic kinase inhibitors,
mainly TK inhibitors, developed in the last two decades [17]. The
ATP-mimic kinase inhibitors belong to different chemical classes of
compounds. However they share a common pharmacophore [17]
that is generally composed by: i) a scaffold (mainly a nitrogen
containing heterocycle) able to interact through an H-bond with
the kinase hinge region; ii) a lipophilic moiety (mainly an aromatic
or an heteroaromatic system) that occupy a pocket opened by the
so called gatekeeper residue; iii) a spacer between the heterocycle
and the hydrophobic moiety; iv) solvent exposed residues. As part
of our novel bioactive compounds discovery projects [18e20] and
due to our experience in quinazoline compounds [18,19,21], we
decided to investigate whether it was possible to develop novel
quinazolinone-based kinase inhibitors able to restrain the activa-
tion of fibrogenic cells. Quinazolinone compounds are endowed
with a number of biological activities comprising antiviral [22],
antitubercular [23], antimicrobial [24], antitubulin [25], antifolate
[26], anticonvulsivant [27], anti-inflammatory [28], antifibrotic [29]
and anticancer [30e32] properties. Their kinase inhibitory activity
however has not been extensively explored.
Briefly, anilines 16a-c were protected as carbamates, submitted
to condensation with hexametylenetetramine in TFA under mi-
crowave irradiation and then to aromatization with potassium
ferricyanide in hydroalcoholic KOH at reflux. The obtained quina-
zoline 18a-c were oxidized with CAN in acetic acid to quinazoli-
nones 19a-c [18], which were finally condensed with the suitable
halobenzyl derivatives, aryloxyrane or haloacetophenones and NaH
in DMF under microwave irradiation give the final products 1e15.
2.2. Kinase screening
To outline the profile of activity/selectivity, all the synthesized
compounds were preliminarily screened for their ability to coun-
teract the kinase activity of a selected panel of kinases (both TKs
and STKs) involved in fibrosis [37e43]. Thus, in this study the
synthesized compounds have been tested at 1 mM against a panel of
six tyrosine kinases and two serineethreonine kinases. Vatalanib
(PTK787/ZK-22258, a poly-tyrosine kinase inhibitor endowed with
anti-fibrotic and anti-neoplastic activities) [44,45] has been used as
positive control. The results of the screening are summarized in
Table 2.
Many compounds inhibited the activity of KDR or EGFR, though
to a lower extent than vatalanib. The majority of these compounds
(6, 10, 11, and 14) were dual KDR and EGFR inhibitors, whereas
Herein we report the synthesis and the preliminary evaluation
of several 3-substituted quinazolinones against a panel of kinases
mainly involved in the fibrogenic process. The binding mode of
several compounds with the target kinases was investigated by
means of molecular docking studies. The anti-fibrotic activity of the
compounds was assessed in vitro.
compound 1 was a dual EGFR/PDGFRb (Platelet-Derived Growth
Factor Receptor -isoform) inhibitor. Moreover compounds 7 and
b
11 were also active against one of the tested STKs. All the com-
pounds were inactive against the cytoplasmic kinases Abl1 and Src
and the receptor kinase FGFR1.
2. Results and discussion
2.1. Chemistry
2.3. Molecular docking
The general structure of the novel compounds is reported in
Fig. 1A.
To rationalize the activity profile of some representative com-
pounds, molecular modelling studies were performed. In particular
we focused on compounds 1 (active against EGFR; inactive against
KDR), 11 (active against KDR, EGFR and PI3K; inactive against
mTOR) and 7 (active against mTOR; moderately active against
PI3K).
As depicted in Fig. 2A, compound 1 was expected to interact
through the N1-quinazoline nitrogen and the pyridine nitrogen
with the hinge region residue M793 and with the gatekeeper T790
The 3-substituted quinazoline compounds are structurally
related to another well known class of kinases inhibitors, the 4-
anilinoquinazolines [34,35] (Fig. 1B), as demonstrated by the su-
perimposition with erlotinib in its binding conformation with EGFR
(Fig. 1C and D). According to our previous studies [19], the 6 and 7
positions of the quinazolinone scaffold were functionalized with
dimethoxy functions or with fused dialkoxy rings, namely a
dioxane and a dioxolane ring. The 3 position of the quinazolinone
Fig. 1. (A) General structure of quinazolinone derivatives. (B) General structure of 4-anilinoquinazoline kinase inhibitors. (C) Superimposition of the 3-benzylquinazolin-4-one
scaffold (green carbon sticks) and erlotinib (cyan carbon sticks). (D) Superimposition of the 3-(benzoylmethyl)quinazolin-4-one scaffold (yellow carbon sticks) and erlotinib
(cyan carbon sticks). The tridimensional structure of erlotinib was extracted by the crystallographic complex with EGFR (PDB ID: 1M17) [33]. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

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G. Marzaro et al. / European Journal of Medicinal Chemistry 115 (2016) 416e425
Table 1
of EGFR respectively. No consistent binding mode for compound 1
Structures of the synthesized compounds.
in KDR was obtained. Probably, the presence of the V916 as gate-
keeper in KDR was detrimental for the activity: when the pose
obtained for compound 1 in EGFR was inserted in KDR a clash
between the valine and the lipophilic moiety was observed
(Fig. 2B).
The presence of a longer bridge with H-bond acceptor feature in
compound 11 was probably the key to achieve dual EGFR/KDR in-
hibition (Fig. 3). Again, the N1-quinazoline nitrogen was expected to
form an H-bond with the hinge residues (M790 in EGFR, Fig. 3A;
C918 in KDR, Fig. 3B). In the case of EGFR, an additional H-bond
between the alcoholic function of T790 and the carbonyl of 11 was
suggested by molecular docking, whereas in KDR the bromophenyl
function probably established lipophilic interaction with the gate-
keeper (V916).
Notably, a similar behaviour was found also for compound 5 and
10, bearing the same CH2C]O bridge (data not shown).
Besides being a dual EGFR/KDR inhibitor, compound 11 was also
able to inhibit PI3K while it was inactive against the other tested
STK, mTOR. On the contrary, the shorter bridge in compounds 7 and
8 led to compounds endowed with moderate dual mTOR/PI3K
inhibitory activity. As depicted in Fig. 4, 7 (Fig. 4A) and 11 (Fig. 4B)
showed comparable binding modes in PI3K. Docking studies sug-
gested that the dioxane oxygens could establish H-bonds with the
hinge residue V848, the N1-quinazoline nitrogen atoms could
interact with the K799 and the lipophilic moieties may be involved
in T-shaped areneearene interactions with the W781 aromatic side
chains.
Conversely, in mTOR kinase, compound 7 was expected to
interact with the hinge residue V2240 through an H-bond, with the
W2239 through a T-shaped areneearene interaction and with the
DFG-motif (Fig. 5A). A similar pose was not obtained for compound
11. Indeed, the alignment of compound 11 on the pose obtained for
compound 7 in mTOR revealed two clashes between the carbonyl
in the bridge and the W2239 and between the bromine atom and
the I2163 side chain (Fig. 5B).
Finally, we used molecular docking studies also to investigate
the differences in potencies of compounds 1, 3 and 4 against EGFR.
Indeed, very small variation in structures (compounds mainly
differed for the relative position of the bromine atom and for the
presence of a pyrimidine in place of a phenyl function at position 3)
caused quite high differences. As shown in Fig. 6A, there were
probably no differences in binding modes for compounds 1 and 3.
Hence, the lower activity of 3 could be ascribed to the lack of
interaction with the T790 (due to the absence of the nitrogen atom
in the benzyl portion) rather than to the bromine atom position. On
Compd
Series
A
X
Ar
1
-CH2-
2
A
-CH2-
3
4
A
A
-CH2-
-CH2-
5
6
7
A
A
B
-CH2CO-
-CH2CH(OH)-
-CH2-
8
9
B
B
-CH2-
-CH2-
10
B
-CH2CO-
11
12
13
B
B
C
-CH2CO-
-CH2CH(OH)-
-CH2-
14
15
C
C
-CH2-
-CH2-
aReagents and conditions: (i) ClCOOEt, TEA, THF, rt, 30 min; (ii) 1. HMTA, TFA, MW, 110
°C, 10 min. 2. K3Fe(CN)6, KOH 10%, EtOH/H2O 1/1, reflux, 4 h; (iii) CAN, AcOH, H2O, rt, 5
min; (iv) 2-bromo-3-bromethylpyridine (1) or 3-phenyl-benzylbromide (2, 14) or 3-
bromobenzylbromide (3, 8) or 4-bromobenzylbromide (4, 9, 13) or 4-phenylbenzylchloride (7,
15) or 2,4’-dibromoaceto-phenone (5, 10) or 2,3’-dibromo-acetophenone (11), or 2-(4-
bromophenyl)oxirane (6) or 2-(3-chlorophenyl)oxirane (12), NaH, anhydrous DMF, MW, 120
°C, 5 min.
Scheme 1. General strategy for the synthesis of quinazolinone derivatives as kinase inhibitorsa.

G. Marzaro et al. / European Journal of Medicinal Chemistry 115 (2016) 416e425
419
Table 2
Kinase inhibition profile.
the contrary, docking studies suggested a worse complementarity
with the ATP pocket for compound 4 (Fig. 6B), probably caused by
the hindrance of the para-bromine atom.
2.4. In vitro anti-fibrotic activity
As many substituted quinazolinones are endowed with good/
strong inhibition activity towards kinases involved in the fibrogenic
process (Table 2), we decided to assess the biological effect of the
active compounds in modulating the activation of fibrogenic cells,
namely HSCs and ISEMFs. Primary cells were isolated from human
liver or gut specimens and cultured in plastic support to induce
in vitro fibrogenic activation. As reported in Fig. 7, at 1 mM com-
pounds 6, 7, 10, 11, and 14 were the most effective in reducing the
mRNA levels specific for COL1A1 in HSCs and ISEMFs. The mRNA
transcript levels specific for FN1 and TIMP1 were decreased also by
compounds 1 and 5. Noteworthy, compounds 6, 7, 10, 11, and 14
showed a unique profile of activity in the series since they were
able to inhibit KDR with strong/good potency and at least another
kinase (Table 2). Compounds 1 and 5 effectively reduced the mRNA
expression levels of TIMP1 and FN1 in pathological samples (i.e.
ISEMFs cultured from patients suffering from Crohn's disease) with
poor effect on myofibroblasts obtained from healthy subjects in
which the fibrogenic activation is probably less important. For all
the tested genes, the reduction in the mRNA expression was com-
parable to one reported for vatalanib. Finally, compounds endowed
Fig. 2. (A) Binding mode proposed for 1 in EGFR. (B) Clash (black arrow) between 1 and V916 in KDR (V916 is represented as mesh surface). In both cases, the kinase hinge region is
on the left side of the pictures, while the kinase solvent accessible region is on the right side.
Fig. 3. (A) Binding mode proposed for 11 in EGFR. (B)Binding mode proposed for 11 in KDR (V916 is represented as mesh surface). In both cases, the kinase hinge region is on the left
side of the pictures, while the kinase solvent accessible region is on the right side.

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G. Marzaro et al. / European Journal of Medicinal Chemistry 115 (2016) 416e425
Fig. 4. (A) Binding modes proposed for 7 in PI3K. (B) Binding mode proposed for 11 in PI3K. In both cases, the kinase hinge region is on the left side of the pictures, while the kinase
solvent accessible region is on the right side.
Fig. 5. (A) Binding proposed for 7 in mTOR. (B) Clashes (black arrows) between 11 and W2239 and I2163 in mTOR (11, W2239 and I2163 are depicted as mesh surfaces). In both
cases, the kinase hinge region is on the left side of the pictures, while the kinase solvent accessible region is on the right side.
Fig. 6. (A) Comparison between the binding modes suggested by docking studies for 1 (yellow carbon sticks) and 3 (cyan carbon sticks) in EGFR. (B) Comparison between binding
modes of 1 (yellow carbon sticks) and 4 (pink carbon sticks) in EGFR. In both cases, the kinase hinge region is on the left side of the pictures, while the kinase solvent accessible
region is on the right side. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
with weak inhibitor activity (3, 12, 13) did not significantly reduce
the expression of genes involved in the fibrogenic process.
Compounds 1, 6, and 13 also inhibited proliferation in HSCs and
ISEMFs. Consistent with previous reports, VEGF induces prolifera-
tion in HSCs [46]. As reported in Fig. 8, treatment of cells with 1 mM
for 72 h significantly decreased VEGF-induced proliferation. The
effect was more evident in cells treated with compounds 1 and 13,
the only quinazolinone derivatives endowed with good inhibition
of PDGFR.
Most important, the treatment for 72 h with compounds at 1 mM
did not compromise cell viability (Fig. 9). On the contrary, under the
same conditions vatalanib reduced cell viability, as previously re-
ported [47].
The anti-fibrogenic and anti-proliferative effects of compounds

G. Marzaro et al. / European Journal of Medicinal Chemistry 115 (2016) 416e425
421
Fig. 7. Expression levels of mRNA transcripts specific for prepro-alpha 1 collagen (COL1A1), fibronectin (FN1), and tissue inhibitor of metalloproteinase 1 (TIMP1) assessed by
quantitative RT-PCR on hepatic stellate cells (HSCs) and intestinal subepithelial myofibroblasts (ISEMFs) cultured from specimens obtained by healthy subjects and patients
suffering from Crohn's disease (CD). Data are reported as mean SE. * denotes P < 0.05 vs untreated, control cells (CTR). Vat: vatalanib.
1, 6, and 11 were associated with reduced phosphorylation of p38
MAPK. Thus, phosphorylation of p38MAPK is augmented in acti-
vated HSCs mediating the upregulation of COL1A1 mRNA levels
[48]. Indeed, phospho-p38MAPK levels mainly decreased in HSCs
quinazolinone derivatives. Our data revealed that the synergistic
inhibition of EGFR and KDR efficaciously reduced the levels of
mRNA transcripts involved in the fibrogenic activation of both HSCs
and ISEMFs, the cell populations mainly involved in the fibrosis of
liver and gut, respectively. Indeed, compounds 6, 10, 11 and 14,
showing the dual EGFR/KDR inhibitory activity, significantly
reduced COL1A1 mRNA expression as compare to non-treated
in vitro activated cells to an extent comparable to or even greater
than vatalanib. Previous studies reported the involvement of EGFR
and KDR in the regulation of HSCs [10,12] but to our knowledge this
is the first time that dual inhibitors of TKs efficaciously control
expression of fibrogenic genes. The simultaneous inhibition of KDR
and EGFR however is not mandatory for the anti-fibrotic effect.
Indeed, compounds 1, 5, and 7 revealed different gene-related and
cellular specific effects. In particular, compound 7 endowed with
dual KDR/mTOR inhibitory activity, significantly downregulated the
expression of pro-fibrogenic genes. Thus, several soluble factors
and signalling pathways are central to the fibrotic process. Besides
EGFR and KDR, PDGFR has been reported as the most potent pro-
liferative factor toward HSCs and myofibroblasts [49]. Because of
their combined role in fibrosis, inhibition of both proliferation and
fibrogenesis is an attractive target for antifibrotic therapy. Among
treated for 4 h with compounds 1, 6 and 11 at 1 mM as compare to
untreated cells (Fig. 10). Similar results were obtained in ISEMFs
cultured from patients suffering from Crohn's disease (data not
shown).
3. Conclusions
The evaluation of kinase inhibitors bearing the quinazolinone
core has not yet been extensively studied, although this scaffold is
present in a number of biologically active compounds. In this work
the synthesis and the preliminary biological evaluation of quina-
zolinone derivatives as kinase inhibitors have been described. The
synthesized compounds have been tested towards a panel of six
tyrosine kinases and two serineethreonine kinases. The profile of
selectivity of some representative compounds has been rational-
ized through molecular docking studies, furnishing useful sugges-
tions for further development of this class of compounds.
The kinase inhibition profile evaluated by in vitro assay (Table 2)
prompted us to investigate the anti-fibrotic properties of
our synthetized quinazolinone derivatives only compound
1

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G. Marzaro et al. / European Journal of Medicinal Chemistry 115 (2016) 416e425
Fig. 9. Percentage of vital HSCs and ISEMFs following treatment with compounds for
72 h. Data are reported as mean SE. * denotes P < 0.05 vs untreated, control cells
(CTR). Vat: vatalanib.
Fig. 8. Cell proliferation assessed as carboxyfluorescein diacetate succinimidyl ester
(CFSE) positive cells (percentage). HSCs and ISEMFs were treated with 1 ng/ml VEGF
and compounds for 72 h. Fluorescence was then evaluated by FACS analysis. Data are
ꢀ
reported as mean SE. denotes P < 0.05 vs untreated, control cells (CTR). * denotes
P < 0.05 vs VEGF-treated cells. Vat: vatalanib.
inhibits both PDGFR and EGFR (Table 2). Consistently, compound 1
reported good anti-fibrotic activity, reducing FN1 and TIMP1 mRNA
transcript levels and also inhibited VEGF-induced cell proliferation.
However, even if at lower extent compound 6 reported anti-
proliferative activity, too, proving that quinazolinone derivatives
inhibit activated fibrogenic cells by complex mechanisms. Indeed,
compounds 1, 6, and 11 exert their anti-fibrotic and anti-
proliferative effects on p38MAPK pathway although we can not
exclude other intracellular signaling cascades.
Fig. 10. Inhibition of p38MAPK phosphorylation assessed by western blot analysis in
HSCs untreated (CTR) or treated for 4 h with compounds 1 mM.
properties of quinazolinone derivatives. Fully exhaustive relation-
ship between kinases inhibition profile and biological activity is
however not derivable mainly because i) the expression of the
On the basis of the acquired data and of the docking studies,
novel compounds with improved inhibitory activity against STKs
will be synthesized, with the aim to strengthen the anti-fibrotic

G. Marzaro et al. / European Journal of Medicinal Chemistry 115 (2016) 416e425
423
different ECM proteins is driven by several intracellular pathways
and ii) different kinases are specifically elicited in distinct cell
populations (i.e. HSCs and ISEMFs) as well as at different step of the
fibrogenic process (compare effects on ISEMFs from healthy sub-
jects and patients suffering for Crohn's disease). Indeed, a complex
network of extracellular factors (e.g. endocrine system, metabolism,
growth factor and metalloproteinase secretion) characterizes
fibroblast activation and ECM deposition in every tissue-related
fibrogenic process [50,51]. The acquired data indicate compounds
1, 6, and 11 as novel promising anti-fibrotic compounds endowed
with multiple kinases inhibitory properties. The synthesis of novel
compounds as well as a more exhaustive biological investigation on
this novel class of multi-kinase inhibitors is in progress. Overall, our
data suggest that the quinazolinone is an attractive scaffold for the
development of novel inhibitors of kinases involved in the fibro-
genic process.
provided by Gibco, Milan, Italy). Purity of cultured HSCs was
assessed by immunocytochemistry using anti-
(Sigma).
aSMA antibody
Human intestinal subepithelial myofibroblasts (ISEMFs) were
isolated from non-pathological colonic biopsies collected during
colonoscopy for cancer screening and from patients with Crohn's
disease collected during routine follow-up endoscopy program.
Tissue samples were processed separately. Biopsies were diced and
digested for 30 min at 37 ꢀC in collagenase (0.25 mg/ml, Sigma).
Recovered cells were suspended in DMEM with 20% vol/vol FBS,
2 mM
L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential
amino acids, 100 U/ml penicillin, 100
mg/ml streptomycin and 2 ng/
mL fungizone (Gibco). Purity of cultured ISEMFs was ascertained by
fluorescence-activated cell sorting (FACS) analysis using anti-CD90
antibody (ImmunoTools, Germany). HSCs and ISEMFs were
cultured at 37 ꢀC in a 5% CO2 humidified incubator. At confluence,
cells were detached using 0.05% Trypsin-EDTA (Gibco). The study
protocol followed the principles expressed in the Declaration of
Helsinki and was approved by the Ethical Committee of the Uni-
versity Hospital of Padova. Each patient was provided with detailed
information about the study aims and protocols, and gave their
written, informed consent.
4. Experimental
4.1. Chemistry
See Supplementary Data for general synthetic methods, for the
synthesis of 2-bromo-3-bromethylpyridine and for the analytical
details (mp, NMR, HRMS, elemental analyses) of all compounds.
Purity of all tested compounds was determined by elemental ana-
lyses and was found equal or more than 95%. Compounds 19a [21]
and 19b-c [18] were synthesized as previously reported.
General procedure for quinazolinones 1e15. A mixture of quina-
zolinone 19a [21] or 19b-c [18] (1.0 mmol) and NaH in DMF was
maintained under N2 for 5 min. The appropriate haloderivative or
aryloxirane was added (1.0 mmol) and the mixture was microwave
irradiated at 120 ꢀC (power 250 W; hold time 5 min). After cooling,
the mixture was poured in sat. NH4Cl solution (30 mL) and the
obtained precipitate were collected by filtration. The solid was
purified by crystallization to give the title compounds (yields
10e52%).
4.3.3. RNA isolation and quantitative polymerase chain reaction
HSCs and ISEMFs were cultured in 6 well plates and treated for
24 h with 1 mM of test compounds or vatalanib. Total RNA was
extracted using the SV total RNA isolation system (Promega, Italy)
according to the manufacturer's instructions. Contaminating DNA
was removed by DNase I treatment (Promega). cDNA was synthe-
sized using 2 mg RNA as template, random hexamer primers and
MuLV Reverse Transcriptase (Applied Biosystems, Milan, Italy).
Gene expression was evaluated by quantitative polymerase chain
reactions (qPCR) using ABI Prism 7700 Sequence Detection System
(Applied Biosystems), TaqMan qPCR Master Mix (Applied Bio-
systems), and specific oligonucleotides and probes (Universal Probe
Library system, UPL, Roche Applied Science, Monza, Italy) for
prepro-alpha 1 collagen (COL1A1), fibronectin 1 (FN1), and tissue
inhibitor of metalloproteinase 1 (TIMP1). The expression of the
target gene was normalized to the level of the housekeeping gene
18S ribosomal RNA (18SrRNA). Experiments were performed in
triplicate for each isolated cell batch. The relative changes in gene
expression were analyzed using the DDCT method. Oligonucleo-
tides and probes are reported in Supplementary Data.
4.2. Computational methodologies
The computational experiments were performed on a 4 CPUs
(Intel Core2 Quad CPU Q9550 @ 2.83 GHz) ACPI Â64 Linux work-
station (operating system: Ubuntu ver. 12.04). Protein structures
were handled with Chimera 1.5.3 software [52]. The structures of
the quinazolinone compounds were prepared using MarvinSketch
5.5.0.1 software [53] and OpenBabel 2.2.3 softwares [54]. The
docking studies were conducted with AutoDock 4.2 software [55]
as previously reported [19]. Further details are given in
Supplementary Data.
4.3.4. Proliferation assay
HSCs and ISEMFs were incubated at 37 ꢀC for 10 min in pre-
warmed PBS containing 0.1% vol/vol BSA (Sigma) and 25 mM car-
boxyfluorescein diacetate succinimidyl ester (CFSE, Molecular
Probe, Invitrogen). Staining was quenched by adding 5 volumes of
ice-cold culture media. Sixteen hrs later the cells were washed and
4.3. Biology
treated with 1 mM compounds or vatalanib for 72 h. Cell prolifer-
4.3.1. In vitro kinase assays
ation was evaluated by the partitioning of fluorescent dye between
daughter cells using BD FACS-Calibur flow cytometer. Experiments
were performed in duplicate for each isolated cell batch. Results
were analyzed using the WinMDI 2.9 (Windows Multiple Docu-
ment Interface for Flow Cytometry) program.
Synthesized compounds were tested in vitro for inhibition of a
panel of TKs and STKs, as previously reported [19]. See
Supplementary Data for a detailed description.
4.3.2. Cell isolation and culture
Human hepatic stellate cells (HSCs) were freshly isolated from
non-pathological fragments of liver tissue collected during surgical
procedures. Samples were processed separately and HSCs were
cultured as previously described [56]. Briefly, following digestion
with collagenase and pronase, HSCs were isolated by centrifugation
over a gradient of Percoll (Amersham Biosciences, Sweden) and
cultured in DMEM containing 10% vol/vol fetal bovine serum, 2 mM
4.3.5. Cell viability assay
To evaluate cell viability, HSCs and ISEMFs were seeded into 96-
well plates at 2 Â 103 cells/well. Cells were then exposed for 72 h to
1 mM tested compounds or vatalanib and finally added with 3-(4,5-
dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT,
0.50 mg/ml, Sigma) for 4 h. Formazan crystals were dissolved in 10%
w/vol SDS containing 0.01 M HCl. Optical densities were measured
at 450 nm using a microplate reader (Sunrise, Tecan; Switzerland).
L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin, (all

424
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HSCs and ISEMFS isolated from gut specimens of patients
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pounds at 1
mM. For Western blot analysis total proteins from
cultured cells were extracted in RIPA buffer (150 mM NaCl, 50 mM
TriseHCl, 0.25% wt/vol sodium deoxycholate, 0.1% Nonidet P-40,
100
10
m
M NaVO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride,
m
g/ml aprotinin, 10 g/ml leupeptin). Particulate material was
m
removed by centrifugation. Proteins were separated with 10% SDS
PAGE and then transferred to nitrocellulose membranes (BioRad,
Italy). Membranes were probed with phospho-p38 MAP Kinase
antibody (Cell Signaling) and then incubated with anti-rabbit HRP-
conjugated secondary antibodies. Immune complexes were visu-
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were then re-probed with p-38 MAP Kinase antibody as loading
control. Experiments were performed in duplicate. Images were
captured using Hyper Film MP (GE Healthcare).
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4.3.7. Statistical analysis
Statistical analysis was performed using GraphPad Prism 3.03
software (San Diego, California, USA). One-way analysis of variance
followed by the Bonferroni post-hoc test was used to compare
multiple experimental groups. P values < 0.05 were considered
statistically significant.
Acknowledgment
The present work has been carried out with the financial sup-
port of the University of Padova “Progetto di Ricerca di Ateneo
2008” CPDA084954/08 to A.C. and “Progetto Giovani Studiosi 2012”
to G.M.. M.D.V. thanks the Fondazione Cariparo for a PhD student
grant.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.03.053.
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Author contributions
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of the
manuscript.
discovery
of
N-cyclopropyl-4-methyl-3-[6-(4-methylpiperazin-1-yl)-4-
oxoquinazolin-3(4H)-yl]benzamide (AZD6703), a clinical p38alpha MAP ki-
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