Quinazoline-based multi-tyrosine kinase inhibitors: Synthesis, modeling, antitumor and antiangiogenic properties

Article abstract of DOI:10.1016/j.ejmech.2013.06.057

In this work the synthesis and the biological evaluation of some novel anilinoquinazoline derivatives carrying modifications in the quinazoline scaffold and in the aniline moiety were reported. Preliminary cytotoxicity studies identified three derivatives

Full text of DOI:10.1016/j.ejmech.2013.06.057

European Journal of Medicinal Chemistry 67 (2013) 373e383
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Quinazoline-based multi-tyrosine kinase inhibitors: Synthesis,
modeling, antitumor and antiangiogenic properties
,
,
Maria Teresa Conconi a 1, Giovanni Marzaro a 1, Luca Urbani a, Ilenia Zanusso a,
Rosa Di Liddo a, Ignazio Castagliuolo b, Paola Brun b, Francesca Tonus a,
Alessandro Ferrarese a, Adriano Guiotto a, Adriana Chilin a
,
*
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:
In this work the synthesis and the biological evaluation of some novel anilinoquinazoline derivatives
carrying modifications in the quinazoline scaffold and in the aniline moiety were reported. Preliminary
cytotoxicity studies identified three derivatives, carrying dioxygenated rings fused on the quinazoline
portion and the biphenylamino substituent as aniline portion, as the most effective compounds. Further
investigations revealed that these compounds exhibited antiproliferative activity on a wide panel of
human tumor cell lines through the inhibition of both receptor and nonreceptor TKs. Furthermore, the
compound bearing the dioxolane nucleus was also able to inhibit in vivo tumor growth. Molecular
modeling of these compounds into kinase domain suggested that the phenyl group allows favorable
interaction energies with the target proteins: this feature is favored by fused dioxygenated ring at the 6,7
positions, whereas free rotating functions do not allow the correct placement of the molecule, thus
impairing the inhibitory potency. Finally, the biphenylamino derivatives, at noncytotoxic concentrations,
acted as antiangiogenic agents both in in vitro and in vivo assays.
Received 16 May 2013
Received in revised form
27 June 2013
Accepted 29 June 2013
Available online 6 July 2013
Keywords:
Cancer therapy
Multi-kinase inhibitors
Angiogenesis
Anilinoquinazoline
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Alterations in cell cycle regulation may cause cancer onset,
progression and metastasis [1]. Among the proteins involved in
Abbreviations: ADT, AutoDock Tools; BSA, bovine serum albumin; CAN, cerium
ammonium nitrate; DAPI, 40,6-diamidino-2-phenylindole; DMEM, Dulbecco’s
modified eagle medium; DMF, dimethylformamide; DTT, dithiothreitol; EC, endo-
thelial cell; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor;
EGFR, epidermal growth factor receptor; EGTA, ethylene glycol tetraacetic acid;
ELISA, enzyme-linked immunoassorbent assay; FBS, fetal bovine serum; FCS, fetal
calf serum; FGF, fibroblast growth factor; FGFR-1, fibroblast growth factor receptor
1; GFR, growth factor reduced; HMTA, hexamethylenetetramine; HUVEC, human
umbilical vein endothelial cell; IGF, insulin-like growth factor; MOPS, 3-(N-mor-
signal recognition, transduction and amplification, tyrosine kinases
(TKs) play a key role [2,3]. These protein enzymes catalyze the
transfer of a phosphate group from an ATP molecule to a tyrosine
residue of the target protein, thus leading to signal transduction.
Both receptor TKs (RTKs, such as EGFR, FGFR-1, VEGFR-1 and 2 and
PDGFRb) and cytoplasmic (or soluble) TKs (such as Src, Abl and Lck)
have been to date discovered and characterized [4]. The RTKs are
cell-surface receptors that are activated through the binding to
specific growth factors, such as epidermal growth factor (EGF),
insulin-like growth factor (IGF) and vascular endothelial growth
factor (VEGF). In particular, the RTK activation causes receptor
dimerization, phosphorylation of the proper substrate and then
activation of the signal transduction cascade. Thus, the final effect of
RTKs activation results in stimulation of cell growth and division [5].
In normal cells, TKs activity is tightly regulated to preserve the
proper balance between proliferation and apoptosis. In almost all
tumor tissues an overexpression or a gain-of-function mutations of
several TKs have been detected [6,7]. Over the past two decades, the
pholino)propanesulfonic
dimethyltetrazolium bromide; PDB, Protein Data Bank; PDGF, platelet-derived
growth factor; PDGFR , platelet-derived growth factor receptor ; RTK, receptor
acid;
MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-
b
b
tyrosine kinase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electro-
phoresis; TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TK,
tyrosine kinase; TKI, tyrosine kinase inhibitor; VEGF, vascular endothelial growth
factor; VEGFR-1 and 2, vascular endothelial growth factor receptor 1 and 2.
* Corresponding author. Department of Pharmaceutical Sciences, University of
Padova, Via Marzolo 5, 35131 Padova, Italy. Tel.: þ39 0498275349; fax: þ39
0498275366.
E-mail address: adriana.chilin@unipd.it (A. Chilin).
First two authors contributed equally to this work and should be considered co-
1
first authors.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.057

374
M.T. Conconi et al. / European Journal of Medicinal Chemistry 67 (2013) 373e383
strong efforts to develop useful strategies to inhibit TKs have
allowed to identify inhibitors, such as small organic compounds
(also known as TKs inhibitors, TKIs) and monoclonal antibodies
(mAbs), that have been approved for therapy or have entered clin-
ical trials [8]. The TKIs act as ATP-mimic inhibitors or growth factor
binders, thus avoiding ATP binding. On the contrary, the mAbs act as
growth factor-mimic inhibitors, so preventing the RTK activation.
Among the TKIs, 4-anilinoquinazolines have been intensively
studied, leading to a number of active molecules already approved
by FDA (e.g. erlotinib [9], gefitinib [10], lapatinib [11], vandetanib
[12]) or under advanced clinical experimentation (e.g. afatinib [13],
canertinib [14]). However, the development of novel analogs con-
stitutes a field of great interest, as demonstrated by the number of
patents filed and of the paper published in the last years [15].
Although the main target of 4-anilinoquinazoline is EGFR, some
derivatives possess a poor selectivity profile targeting several ki-
nases. For example, vandetanib is a dual EGFR/VEGFR-2 inhibitor,
whereas afatinib and lapatinib target EGFR and Her2.
Continuing our studies in the field of quinazoline derivatives [16],
herein we present the synthesis and the biological evaluation of
some novel derivatives carrying modifications in the quinazoline
scaffold and in the aniline moiety. These compounds have been
initially screened for their cytotoxic activities against two cell lines,
A431 (overexpressing EGFR) and NIH3T3 (not expressing EGFR). The
most active compounds have been then submitted to in vitro assay
on a panel of isolated TKs and molecular modeling studies have been
carried out to figure out their interaction with TKs. Their effective-
ness to inhibit in vivo tumor growth in a xenograft mouse model as
well as their anti-angiogenic properties have been also evaluated.
on the quinazoline portion. All the compounds showed predicted
binding poses comparable to the binding mode of lapatinib (see
Fig. S2 in Supplementary data).
With respect to the aniline substituent, we considered both well
known functionalization (as m-bromoaniline, m-toluidine and m-
trifluoridine; see compounds 10e18) [19] as well as some previ-
ously unreported lipophilic functions (aminopicolines, m-meth-
anesulphonamidoaniline, m-aminobiphenyl; see compounds 1e9).
In fact it is well known that TKs present a deep lipophilic pocket
accommodating the substituted aniline moiety [20].
All the compounds were prepared according to a common
synthetic strategy (Scheme 1), that consisted in: 1) synthesis of the
quinazoline nucleus, starting from the appropriate aniline de-
rivatives, by the HMTA/TFA/K3Fe(CN)6 method [21]; 2) oxidation of
the quinazoline nucleus to quinazolin-4(3H)-one by cerium
ammonium nitrate (CAN) in aqueous acetic acid [22]; 3) chlorina-
tion at the 4 position with phosphorous oxychloride in the presence
of triethylamine; 4) condensation of the 4-chloroquinazoline in-
termediates with the suitable substituted anilines. Compounds 1e4
were obtained as free bases, from chloroquinazolines and amino-
picolines in the presence of sodium hydride in tetrahydrofuran,
while compounds 5e18 were obtained directly as hydrochlorides
irradiating the chloroquinazolines and the appropriate anilines in
isopropanol, thus taking advantage of the microwave assisted
organic chemistry technique.
The compounds of the A series were obtained from 8-chloro-
[1,3]dioxolo[4,5-g]quinazoline (23a), 4-chloro-7,8-dihydro[1,4]dio
xino[2,3-g]quinazoline (23b), and 4-chloro-8,9-dihydro-7H-[1,4]
dioxepino[2,3-g]quinazoline (23c), previously described [16],
which were condensed with 2-amino-4-picoline, 2-amino-6-
picoline, 3-methansulfonamidoaniline or m-biphenylamine afford
ing compounds 1e9.
2. Result and discussion
For the compounds of the B series, the starting 3-allyloxy-4-
methoxyaniline (19d) was prepared from 3-hydroxy-4-metho
xynitrobenzene (24), which was condensed with allyl bromide in
alkaline dimethylformamide to give the O-allylether 25, finally
reduced with stannous chloride in ethanol to the desired 19d
(Scheme 2).
2.1. Chemistry
We planned to explore the effect of the different parts of the
anilinoquinazoline nucleus on inhibition of cell proliferation,
introducing some structural modifications in the quinazoline
portion and in the aniline portion (Fig. 1).
The aniline intermediate 19e for the synthesis of compounds of
the C series was synthesized starting from o-phenylendiamine (26),
whose amino groups were protected as carbamates with ethyl
chloroformate in tetrahydrofuran in the presence of triethylamine
(Scheme 3). The N,N0-diprotected aniline 27 was nitrated with 65%
nitric acid in acetic acid at reflux and the nitroderivative 28 was
treated with methyl iodide in dimethylformamide in the presence
of sodium hydride. Besides the desired methylation of the carbamic
function, also the cyclization of the imidazolinone ring occurred.
This unpredicted reaction probably proceeded through an initial N-
monodeprotection with the formation of methylamine function,
able to carry out a nucleophilic attack on the second carbamate
group. The consequent elimination of ethanol furnished the desired
compound. The nitroimidazolinone derivative 29 was finally sub-
mitted to reduction with stannous chloride in ethanol to achieve
compound 19e.
The newly synthesized compounds were grouped in four series,
according to the type of substituent in the quinazoline portion
(Table 1) that were chosen on the basis of our previously reported
work and on the analysis of the ATP binding pocket features in
EGFR. In fact, the 6,7-fused dioxygenated rings (present in the A
series) were found to improve the biological properties of the
quinazoline derivatives [17]. On the other hand, the co-crystalized
structure of EGFR with lapatinib (PDB ID: 1XKK) showed that the
6, 7 and 8 positions of quinazoline scaffold are surrounded by
lipophilic residues (Leu718, from Leu792 to Cys797, Met1002; see
Fig. S1 in Supplementary data). In particular Met793-Gly796 form a
lipophilic cleft that could accommodate planar and lipophilic sub-
stituent, such as the allyloxy (see B series) or the phenyl (see D
series) moieties. Moreover, Shen et al. have recently reported some
monoalkylated imidazolinonequinazolines as EGFR inhibitors [18].
However, since no N,N-dialkylated imidazolinonequinazoline
appeared in literature, we also considered the synthesis of the
compounds, belonging to the C series. Preliminary molecular
modeling studies were performed on compounds 10, 13 and 16 in
order to evaluate in silico the effects of the different substitutions
For the synthesis of compounds of the D series, the benzo[h]
quinazoline 21f was prepared starting from 6-bromobenzo[h]qui-
nazoline (30) previously reported (Scheme 4) [23]. Debromination
was accomplished by MW irradiation of compound 30 in the
presence of sodium ethylate in ethanol.
2.2. Biology
2.2.1. In vitro screening of synthesized compounds
The compounds listed in Table 1 were screened for their anti-
proliferative effects on two cell lines: A431 cells and NIH3T3 cells,
Fig. 1. Points of modifications in the anilinoquinazoline nucleus.

M.T. Conconi et al. / European Journal of Medicinal Chemistry 67 (2013) 373e383
375
Table 1
Structures of title compounds.
Compound
Series
n
R
Compound
Series
n
R
1
2
3
4
5
6
7
A
A
A
A
A
A
A
1
2
1
2
1
2
1
10
11
12
13
14
15
16
B
B
B
C
C
C
D
e
e
e
e
e
e
e
8
9
A
A
2
3
17
18
D
D
e
e
that over-express or lack EGFR, respectively. PD153035, one of the
most potent EGFR inhibitor (IC50 ¼ 25pM) [24] sharing with the
novel compounds the aminoquinazoline moiety, was used as
reference. The inhibitory effects on cell proliferation were deter-
mined by MTT assay (Table 2). The compounds belonging to the C
and D series were almost ineffective on both cell lines, while the 7-
allyloxy derivatives 10, 11, and 12, bearing methoxy and allyloxy
groups in the 6 and 7 positions respectively of the quinazoline
nucleus, showed IC50 values against A431 cells comparable to
PD153035 and to our previously reported compounds carrying a
dioxolane or dioxepine rings condensed on the quinazoline portion
[16]. In the A series, compound 2 showed an inhibitory activity
comparable to that of the reference compound, whereas com-
pounds 1, 3, 4, 5 and 6 seemed to be ineffective. On the contrary,
derivatives 7e9, carrying dioxygenated rings fused on the quina-
zoline portion and the biphenylamino substituent as aniline
portion, exhibited a very strong antiproliferative activity, their IC50
values being about 5e50 times lower than that determined for
PD153035. Furthermore, the dioxane derivative was more effective
than the corresponding dioxolane and dioxepine derivatives, in
agreement with our previous findings [16,17].
Interestingly, the biphenyl derivatives exhibited cytotoxic ef-
fects also in NIH3T3 cells lacking EGFR. IC50 values for compounds 7
and 9 were quite similar to those determined in A431 cells.
Although antiproliferative activity of compound 8 seemed to be
lower in NIH3T3 cells respect to A431 ones, the corresponding IC50
value was comparable to those detected for compounds 7 and 9.
The maintenance of cytotoxic activity on NIH3T3 cells suggested
that these compounds could exert their effects by interacting with
additional cell target, other than EGFR. Since all eukaryotic tyrosine
kinases share a conserved catalytic domain able to transfer the
phosphate group of ATP to aminoacid residues on protein sub-
strates [20], we have hypothesized that the above mentioned
compounds could act as multi-TKIs.
Scheme 1. General strategy for the synthesis of quinazoline derivatives. Reagent 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)
POCl3, TEA, reflux, 3 h; (v) RNH2, NaH, THF, 1 h (for cpds 1e4) or RNH2, i-PrOH, MW,
80 ꢀC, 15e30 min (for cpds 5e18). Letter specifications: a ¼ dioxolane derivatives;
b
¼
dioxano derivatives;
c
¼
dioxepino derivatives;
d
¼
allyloxy derivatives;
To verify this hypothesis, compounds 7e9 were tested against a
panel of both receptor (EGFR, FGFR-1, VEGFR and PDGFRb) and
e ¼ imidazolinone derivatives; f ¼ benzoquinazoline derivatives. See Table 1 for R
specification.

376
M.T. Conconi et al. / European Journal of Medicinal Chemistry 67 (2013) 373e383
Table 2
IC50 values for 72 h exposure of cells to the compounds.
Compound
IC50 (m
M)a against cell lines
A431
NH3T3
Scheme 2. Synthesis of the aniline intermediate 19d. Reagent and conditions: (i) allyl
bromide, K2CO3, DMF, reflux, 16 h; (ii) SnCl2$2H2O, EtOH, reflux, 6 h.
1
2
3
4
5
6
>10b
>10b
5.17 ꢁ 0.84
6.60 ꢁ 1.07
>10b
>10b
>10b
>10b
>10b
>10b
>10b
>10b
7
8
9
10
11
12
13
14
15
16
17
18
0.81 ꢁ 0.06
0.08 ꢁ 0.01
0.66 ꢁ 0.08
4.49 ꢁ 0.62
8.45 ꢁ 0.55
5.95 ꢁ 0.01
>10b
0.60 ꢁ 0.02
0.60 ꢁ 0.20
0.92 ꢁ 0.06
>10b
6.60 ꢁ 0.70
>10b
>10b
>10b
>10b
>10b
>10b
>10b
>10b
Scheme 3. Synthesis of the aniline intermediate 19e. Reagent and conditions: (i)
ClCOOEt, TEA, THF, rt, 1 h; (ii) 65% HNO3, AcOH, reflux, 1 h; (iii) MeI, NaH, DMF, rt,
10 min; (iv) SnCl2$2H2O, EtOH, reflux, 6 h.
>10b
>10b
>10b
>10b
PD153035
4.40 ꢁ 1.30
>10b
a
The values are the mean ꢁ SD of at least three independent experiments.
b
IC50 not determined because less than 50% inhibition was observed at the
soluble (Abl1 and Src) TKs. All the derivatives were found to possess
a broad inhibitory activity (Table 3) although they hindered RTKs
and soluble TKs at different extents. In particular, the dioxolane
derivative 7 seemed to be the most effective, showing IC50 values at
sub-micromolar concentrations on all tested TKs.
highest tested concentration (10 mM). Higher concentrations were not used to avoid
precipitation of the compounds in the culture medium.
Fibroblast Growth Factor (bFGF), Platelet Derived Growth Factor
(PDGF), VEGF and EGF, are released thus inducing the angiogenesis
process that in turn creates the basic conditions for cancer growth
and metastasis [26,27]. The binding between these factors and the
TKs leads the cells to enter in G2/M phases and also inhibits
apoptosis processes. Thus, the endothelial cell (EC) growth rate is
dramatically accelerated, happening as rapidly as every few days,
whereas under physiological conditions ECs divide every years [28].
In the last years great attention has been paid to the angiogenesis for
the development of novel anticancer targeted therapies.
Our data demonstrated that compounds 7e9 are able to inhibit
the phosphorylation of some TKs involved in the formation of new
blood vessels. VEGF-A and VEGFR-2 are currently the main targets
for anti-angiogenic efforts [29]. Together with FGF and its receptor,
they are overexpressed in different types of tumors and their
signaling promotes the proliferation, migration, and differentiation
2.2.2. Effects of compounds 7e9 on cancer cell lines and in vivo
tumor growth
Since the three biphenyl derivatives appeared the most prom-
ising compounds, further investigations were performed on a panel
of human tumor cell lines derived from breast (MCF-7) and colon
(HT-29) adenocarcinomas, cervical cancer (HeLa), and on the mu-
rine hepatocellular carcinoma cell line BNL (BNL 1 ME A.7R.1).
Overall, in MCF-7, HT-29, and HeLa cells, compounds 7e9 presented
IC50 values about 6e10 times lower than those determined for
PD153035, except for compound 9 showing an antiproliferative
activity in Hela cells comparable to reference compound (Table 4).
Furthermore, also in the BNL cells all compounds showed a com-
parable activity and inhibited cell proliferation to the same degree
of PD153035.
of vascular ECs [30]. PDGFRb and its ligands induce vessel matu-
We next determined whether the compounds inhibited tumor
growth in a typical transplanted model of syngenic hepatocellular
carcinoma in Balb/c mice. As reported in Fig. 2, daily intraperitoneal
administration of 50 mg/kg of compound 7 significantly reduced
tumor growth as compared to not treated animals whereas com-
pound 8 did not achieve a statistical difference in tumor growth.
Administration of compound 9 caused severe body weight loss in
animals therefore they were suppressed after 3 days of treatment.
ration and the recruitment of pericytes [31]. On the other hand,
EGFR pathway acts as an indirect regulator of angiogenesis because
its activation stimulates the production of pro-angiogenic factors,
such as VEGF.34 Nonreceptor TKs, namely Src and Abl1, can be
activated also by growth factors through receptor TKs, like EGFR,
PDGFRb, FGFR and VEGFR, leading to an increased expression of
proangiogenic cytokines [32e36].
Starting from these considerations, it seemed noteworthy to
verify whether the most active compounds, that is the biphenyl
derivatives 7e9, could affect the angiogenic process. The effects of
these compounds on the proliferation, migration and tube forma-
tion of human umbilical vein cord cells (HUVECs) have been in vitro
verified, thus mimicking the various steps of new blood vessels
formation [17]. The biphenyl derivatives presented IC50 values
lower than that determined for PD153035, as shown in Table 4, and
in particular compound 8 was much more cytotoxic than com-
pounds 7 and 9.
2.2.3. Antiangiogenic properties of compounds 7e9
Tumor development and maintenance require a permissive
microenvironment. In the absence of new blood vessel formation
the cancer can’t grow over a volume of 1e2 mm3 [25]. As a conse-
quence of cell hypoxia, several pro-angiogenic factors, such as basic
The antiproliferative activity seemed to be due to a pro-
apoptotic effect. Indeed, after a 24 h incubation period, all com-
pounds (10 mM), except for compound 7, induced significant de-
creases in cell proliferation (Fig. 3a) and increases in the apoptotic
rate (Fig. 3b) when compared to not treated cultured.
Scheme 4. Synthesis of benzo[h]quinazoline 21f. Reagent and conditions: (i) EtONa,
EtOH, MW, 105 ꢀC, 15 min.

M.T. Conconi et al. / European Journal of Medicinal Chemistry 67 (2013) 373e383
377
Table 3
Kinase inhibition profile of the compounds.a
Compounds
IC50
(m
M) or % inhibition ([I] ¼ 1
m
M) against isolated kinases
VEGFR-2
EGFR
FGFR-1
PDGFR
b
Src
Abl
7
8
9
0.002 ꢁ 0.001
20%
0.027 ꢁ 0.012
0.063 ꢁ 0.015
0.178 ꢁ 0.030
0.063 ꢁ 0.025
0.848 ꢁ 0.110
0.243 ꢁ 0.056
0.100 ꢁ 0.045
40%
0.032 ꢁ 0.008
0.642 ꢁ 0.105
0.645 ꢁ 0.085
0.051 ꢁ 0.010
0.024 ꢁ 0.010
0.056 ꢁ 0.016
50%
30%
a
The values represent the mean ꢁ SD of at least three independent experiments.
To avoid direct cytotoxic effects, migration and morphogenesis
assays were carried out treating cultures with the highest not cyto-
toxic concentrations of the tested compounds, as determined from
MTT assay (data not shown). Cell migration was significantly
The compounds 34, 35, and 36 exerted poor antiproliferative
activity on A431 and NIH3T3 cells: 10 M compounds induced
about 70% reduction in cell number respect to that determined in
untreated cultures. Similarly, the three novel compounds were
unable to counteract the kinase activity of EGFR, FGFR-1, VEGFR-2,
m
decreasedby1mMPD153035andbiphenylderivatives(Fig. 4a). After
18 h from seeding on Matrigel, HUVECs gave rise to tubule-like
structures (Fig. 4b) that was disrupted by incubation with com-
pound 8 (Fig. 4c). Image analysis confirmed these observation,
showing that compound 8 significantly decreased both dimensional
and topological parameters (Fig. 4d). On the other hand, PD153035
and other compounds affected only the number of meshes. The
inhibitory effect on in vitro angiogenesis was achieved using com-
pound concentration not affecting cell proliferation and viability. To
date, the main side effects of TKIs target the cardiovascular system,
and are inpart related to direct effects on normal ECs. Although most
normal blood vessels remain quiescent, however growth factor
signaling maintains EC survival and vascular integrity. Thus, cardio-
vascular toxicity may be a consequence of an impaired EC renewal
capacity [37]. The maintenance of HUVEC viability in the presence of
compound concentration able to induce anti-angiogenic effects may
suggest a reduced toxicity of biphenyl derivatives.
PDGFRb, Abl1 and Src (IC50 higher than 1 mM in all the cases).
Thus, we concluded that the remarkable biological properties of
compounds 7e9 derived from the presence of both the fused
dioxygenated ring and the biphenylamino moiety. To rationalize
this behavior, molecular modeling studies were performed. Com-
pounds 7 and 34 were docked in the DFG-out crystallographic
structure of EGFR, FGFR-1, VEGFR-2, Src and Abl1 (PDB IDs: 1XKK,
3C4F, 3EWH, 3EN6, 2QOH respectively). The visual inspection of the
binding poses revealed that the terminal phenyl ring of compound
7 was able to establish “edge-to-face” areneearene interactions
with the phenylalanine of the DFG-motif of EGFR, FGFR-1, Abl1 and
Src (Fig. 5aed). On the contrary, the DFG-motif of VEGFR-2 was not
involved in the interaction (Fig. 5e). This observation is consistent
with the data reported in Table 3: the IC50 against VEGFR-2 was
bout 15e400 times higher than the IC50 against the other TKs
considered in the docking studies.
To verify in vivo the anti-angiogenic activity of compounds,
Matrigel plugs containing FGF-2 (200 ng/plug) alone or with the
compounds (1 mM) were implanted subcutaneously into the flank
of C57/BL6 mice. The mean hemoglobin concentration in
compound-treated Matrigel plugs was significantly lower than
FGF-2-treated Matrigel plugs, but higher than that determined in
control. The reference compound was unable to inhibit the FGF-2-
induced neovascularization (Fig. 4e).
In the case of compound 34 the molecular docking studies
revealed that the steric hindrance generated by the bulkier free-
rotating methoxy functions caused a rotation of the quinazoline
scaffold of 34 in order to maintain the correct placement of the
biphenyl function. This feature determines a larger distance be-
tween Met793 and the quinazoline nitrogen of compound 34 in
comparison with that of compound 7 (Fig. 5feh). This was detri-
mental for the formation of the H-bond between the quinazoline
nucleus and the protein backbone (that is known to be fundamental
[20]), thus causing a dramatic decrease in the ability to counteract
the kinase activity.
2.3. Synthesis of analogs of compounds 7e9 and molecular
modeling
On the basis of the above described results and to deeply
investigate the role of the biphenylamino function in the anti-TKs
activity, we synthesized some “hybrid” compounds bearing this
substituent on the quinazoline scaffold of known tyrosine kinase
inhibitors, such as PD153035, erlotinib and gefitinib. The hybrid
compounds 34e36 were synthesized according to the same
synthetic route described in Scheme 1, starting from the opportune
4-chloroquinazoline intermediates 31e33 already reported
(Scheme 5) [22].
3. Conclusions
In this work the synthesis and biological evaluation of four
classes of aminoquinazoline derivatives have been reported. Taken
together, our results indicate that the functionalization of the ani-
line moiety with a further benzene ring together with dioxy-
genated rings fused to quinazoline core lead to compounds (7, 8,
and 9) characterized by high antiproliferative activity through the
inhibition of a wide panel of both receptor and nonreceptor TKs.
Table 4
IC50 values for 72 h exposure of tumor cell lines and HUVECs to the compounds.
Compounds
IC50
(
m
M)a against cell lines
A431
NIH3T3
MCF-7
b
HT-29
HeLa
BNL
HUVEC
7
8
9
0.81 ꢁ 0.06
0.08 ꢁ 0.01
0.66 ꢁ 0.08
4.42 ꢁ 1.29
0.61 ꢁ 0.02
0.60 ꢁ 0.20
0.92 ꢁ 0.06
>10b
0.77 ꢁ 0.02
0.74 ꢁ 0.06
0.70 ꢁ 0.11
8.21 ꢁ 0.71
0.95 ꢁ 0.06
0.68 ꢁ 0.36
0.71 ꢁ 0.01
>10b
0.77 ꢁ 0.02
0.62 ꢁ 0.01
5.49 ꢁ 0.62
>10b
2.80 ꢁ 0.65
2.70 ꢁ 0.35
3.00 ꢁ 0.80
9.50 ꢁ 0.40
0.75 ꢁ 0.01
0.08 ꢁ 0.01
0.91 ꢁ 0.06
4.06 ꢁ 0.19
PD153035
a
The values are the mean ꢁ SD of at least three independent experiments.
b
IC50 not determined because less than 50% inhibition was observed at the highest tested concentration (10
of the compounds in the culture medium.
mM). Higher concentrations were not used to avoid precipitation

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M.T. Conconi et al. / European Journal of Medicinal Chemistry 67 (2013) 373e383
4.1.1. General procedure for carbamates 20dee
A mixture of aniline (19dee) (13.0 mmol), ethyl chloroformate
(2.5 mL, 26.0 mmol) and Et3N (3.6 mL, 26.0 mmol) in anhydrous
THF (150 mL) was stirred at room temperature for 30 min. The solid
was filtered off and the solvent was evaporated under reduced
pressure. The residue was dissolved in CH2Cl2 (100 mL) and the
organic layer was washed with water (3 ꢂ 50 mL). The organic
phase was evaporated under reduced pressure to give 20dee in
quantitative yield (98%).
4.1.2. General procedure for quinazolines 21dee
A mixture of ethyl carbamate (20dee) (10.0 mmol) and hexa-
methylenetetramine (1.4 g, 10.0 mmol) in TFA (30 mL) was micro-
wave irradiated at 110 ꢀC (power set point 80 W; ramp time 1 min;
hold time 10 min). After cooling the mixture was diluted with
aqueous ethanolic (water/EtOH: 1/1) 10% KOH (300 mL), added
with K3Fe(CN)6 (26.3 g, 80.0 mmol) and stirred to reflux for 4 h.
After cooling the mixture was diluted with water (300 mL) and
extracted with CH2Cl2 (3 ꢂ 200 mL). The organic phase was evap-
orated under reduced pressure to give the quinazoline 21dee
(yields 40e91%).
Fig. 2. Inhibition of tumor growth in vivo by compounds 7 and 8. Male mice were
injected subcutaneously with a syngenic hepatocellular carcinoma cell line, BNL 1ME
A.7R.1 cells. Tumor-bearing mice were injected daily i.p. with vehicle as a control or
50 mg/kg of compound 7 or 8. The figure shows the average measured tumor volumes
after 7 days of treatment. Data are presented as the mean ꢁ SEM of tumor volume for
five animals per group. *p < 0.05 vs control.
Molecular modeling of these compounds into kinase domain has
suggested that the phenyl group allows favorable interaction en-
ergies with the target proteins and that this feature is favored by
the reduced steric hindrance of the fused dioxygenated ring at the
6,7 positions with respect to different alkoxy functions. Among the
biphenyl derivatives, compound 7, bearing the dioxolane nucleus,
has been shown to significantly reduce in vivo tumor growth.
Interestingly, compounds 7e9, at noncytotoxic concentrations,
exerted also anti-angiogenic effects both in vitro and in vivo assays.
Thus, we can suppose that they may affect neo-vessel formation
without impairing the viability of ECs, leading then to reduced
potential cardiovascular side effects. This hypothesis will be veri-
fied by further in vivo investigations. In conclusion, our findings
indicate that the biphenyl derivatives, acting as multi-kinase in-
hibitor, may possess therapeutic potential for cancer treatment.
4.1.3. General procedure for quinazolin-4(3H)-ones 22def
To a solution of quinazoline (21def) (5.0 mmol) in AcOH (2.0 mL)
a solution of CAN (10.8 g, 20.0 mmol) in water (10 mL) was added
dropwise. A white precipitate was formed, which was collected by
filtration affording compounds 22def (yields 43e98%).
4.1.4. General procedure for 4-chloroquinazolines 23def
A suspension of quinazolinone (22def) (2.0 mmol) in POCl3
(4.0 mL) and TEA (1.0 mL) was heated to reflux for 3 h. After cooling
the mixture was concentrated under reduced pressure and
the solid residue was dissolved in EtOAc (50 mL) and washed with
sat. NaHCO3 solution (2 ꢂ 20 mL). The organic phase was evapo-
rated under reduced pressure affording compounds 23def (yields
63e93%).
4. Experimental section
4.1. Chemistry
4.1.5. General procedure for 4-pyridylaminoquinazolines 1e4
To a solution of 4-chloroquinazoline 23a [16] or 23b [16]
(0.5 mmol) in THF (4 mL), 2-amino-4-picoline or 2-amino-6-
picoline (60 mg, 0.6 mmol) and NaH (60 mg, 2.5 mmol) were
added. The mixture was stirred at room temperature for 5 min and
then refluxed for 1.5 h. After cooling the mixture was poured in
water (5 mL) and the precipitate was collected by filtration to give
the title compounds (yields 15e75%).
See Supplementary data for general synthetic methods, for the
synthesis of 19dee, 21f, 25, 27, 28, 29 and for the analytical details
(mp, NMR, HRMS, elemental analyses) of all compounds. Com-
pounds 19aec [16], 20aec [16], 21aec [16], 22aec [16], 23aec [16],
30 [23], 31e33 [22] were synthesized as previously reported.
Compounds 24, 26 and all the anilines used for the final conden-
sation were commercially available.
9
9
*
*
8
*
8
7
*
7
*
*
PD153035
PD153035
*
*
-80
-70
-60
-50
-40
-30
-20
-10
0
10
20
-50
0
50
100
150
200
250
300
% changes from control
% changes from control
a)
b)
Fig. 3. Pro-apoptotic effects of compounds on HUVECs. After a 24 h incubation period with compounds (10 mM), both cell viability (a) and apoptotic rate (b) were determined by
MTT assay and assessment of mono- and oligo-nucleosomes formation, respectively. Results, expressed as percent change from control cultures (taken as 0), are the mean ꢁ SD of at
least three independent experiments. * ¼p < 0.05 vs control cultures, Student t test.

M.T. Conconi et al. / European Journal of Medicinal Chemistry 67 (2013) 373e383
379
9
*
b)
8
*
7
*
PD153035
*
c)
-60
-40
-20
0
20
a)
% changes from control
*
*
meshes
*
*
*
*
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
branching
points
*
*
*
lenght
*
% area
FGF-2
-120
-100
-80
-60
-40
-20
0
20
40
control
7 + FGF2
8 + FGF2
9 + FGF2
% changes from control
e)
d)
PD153035 + FGF2
Fig. 4. Effects on in vitro and in vivo angiogenesis. For the in vitro experiments, cells were treated with compounds at the highest concentration not inducing decrease in cell viability
(1 and 0.1
M for migration and morphogenesis assays, respectively). Bars are means ꢁ S.D. of three separate experiments. * ¼p < 0.05 vs control cultures, Student t test. a): HUVEC
migration through a 5.0 m pore after 4 h from seeding. Results are expressed as percent change from control not treated cultures (taken as 0). Phase contrast micrographs of
HUVEC cultures on Matrigel nontreated (b) or incubated with 0.1 M compound 8 (c). d): Quantitative analysis of the effects of compounds on the dimensional (percent area
m
m
m
covered by HUVECs and total length per field), and topological parameters (number of branching points per field, number of mesh per field and percent area inside meshes) of the
HUVEC meshwork. Bars: black, PD153035; dark gray, compound 7; light gray, compound 8; white, compound 9. e): Quantitative hemoglobin (Hb) content in the Matrigel plug was
determined using the Drabkin’s Reagent kit.
4.1.6. General procedure for 4-anilinoquinazolines 5e18, 34e36
A mixture of 4-chloroquinazoline (23aec [16], 23def, 31e33
[22]) (0.5 mmol) and the appropriate aniline (0.5 mmol) in i-PrOH
(1 mL) was microwave irradiated at 80 ꢀC (power set point 60 W;
ramp time 1 min; hold time 15 min). After cooling, the obtained
precipitate was collected by filtration to give compounds 5e18 and
33e35 as hydrochlorides (yields 52e93%).
ADT, thus preparing the appropriate PDBQT files for the molecular
docking studies.
Each crystallographic complex of the TKs in complex with in-
hibitors (1XKK for EGFR, 3EWH for VEGFR-2, 3C4F for FGFR-1, 3EN6
for Src and 2QOH for Abl1) has been initially independently loaded
in Chimera software. PDGFRß was not considered since the crys-
tallographic structure of the kinase domain has not yet been
resolved. The water molecules, the co-factors and the ligand were
4.2. Computational methodology
All the computational studies were carried out on a 4 CPUs
(Inteol Core2 Quad CPU Q9550 @ 2.83 GHz) ACPI x64 Linux work-
station with Ubuntu 12.04 operating system. All X-ray structure
data of EGFR, VEGFR-2, FGFR-1, Src and Abl1 have been downloaded
from Protein Data Bank (PDB) and have been analyzed and handled
with Chimera 1.5.3 software [38]. The structure of compounds 7, 10,
13, 16 and 34 has been prepared using MarvinSketch 5.5.0.1 soft-
ware [39]. The lowest energy conformations were determined at
pH 7.4 with OpenBabel 2.2.3 software [40], using the MMFF94s
force field. All docking studies have been performed with AutoDock
4.2 software, employing AutoDock Tools (ADT) 1.5.4 graphical
interface [41]. For the ligands, the Gasteiger charges were
computed and the nonpolar hydrogen atoms were merged with
Scheme 5. Synthesis of quinazoline derivatives 34 (PD153035-like), 35 (erlotinib-like)
and 36 (gefitinib-like). Reagent and conditions: (i) 3-aminobiphenyl, i-PrOH, MW, 80 ꢀC,
15 min.

380
M.T. Conconi et al. / European Journal of Medicinal Chemistry 67 (2013) 373e383
Fig. 5. Binding modes predicted for compound 7 in EGFR (a), FGFR1 (b), Abl1 (c), Src (d), VEGFR-2 (e); details of the distance between Met793 and the quinazoline nitrogen for
compound 7 (f) and for compound 34 (g); superimposition of the binding modes for the two ligands (h). Note the difference in the quinazoline scaffold placement due to steric
hindrance generated by the two methoxylic functions.
ꢀ
removed. The handled protein structure was then saved as PDB file
and loaded in ADT software. The hydrogen atoms were added and
the Gasteiger charges were computed. After the addition of the
AutoDock atom types the nonpolar hydrogen atoms were merged
and the protein was saved in PDBQT format required for AutoDock
to work. AutoDock makes use of two packages, AutoGrid (that
computes several atom-specific grids) and AutoDock (that per-
forms the conformational analysis based on the pre-calculated
grids). In all cases, the grids have been calculated using following
parameter settings: atom types ¼ A, C, N, NA, OA; center of the grids
on the center of the co-crystallized ligand; number of point on x, y
and z-dimensions ¼ 60; spacing ¼ 0.375 A (the resulting grids were
ꢀ
thus computed in a cube with sides of 22.5 A). The conformational
searches have been performed setting the default parameters for
Lamarckian Genetic Algorithm search method.
4.3. Biology
4.3.1. In vitro kinase assays
Novel compounds were tested in vitro for inhibition of selected
TKs. Recombinant EGFR (Invitrogen, Milan, Italy), FGFR-1 and
VEGFR-2 (purchased from Sigma, Milan, Italy) were diluted in kinase

M.T. Conconi et al. / European Journal of Medicinal Chemistry 67 (2013) 373e383
381
dilution buffer (5 mM MOPS pH 7.2, 2.5 mM glycerol 2-phosphate,
5 mM MgCl2, 0.4 mM EGTA, 0.4 mM EDTA, 0.05 mM DTT, 0.5 mM
BSA). Active kinases (final concentration 200 ng/mL) were added to
To verify the reversibility of the antiproliferative activity,
HUVECs (1.5 ꢂ 104 cell/cm2) were seeded into a 96-well plate. After
a 24 h incubation period, cultures were treated with 10 mM of the
compounds at concentrations ranging from 1
final reaction mixture (25 L), prepared in pre-cooled micro-
centrifuge tubes, contained 0.2 mg/ml substrate solution (Myelin
Basic Protein, MBP, Sigma), 0.05 mM ATP and 0.25 Ci of [
32P]ATP
(PerkinElmer, Monza, MI). Negative controls were prepared replac-
ing the substrate solution with water whereas positive controls were
set up replacing inhibitors tested with water. Reactions were carried
out at 30 ꢀC for 20 min and stopped by addition of loading buffer
m
M to 0.01
m
M. The
tested compounds for 4 h. Media were removed and, after washing
with phosphate buffer (PBS; Gibco-Invitrogen Corporation, Milano,
Italy), cells were allowed to recover for 68 h in compound-free
medium. To determine cell viability the (MTT)-tetrazolium dye
assay was performed as described above. Results were expressed as
percent change from control non-treated cultures.
m
m
g-
The linearity of absorbance of formazan over a range of 5 ꢂ 103e
5 ꢂ 105 cells was established by determining the linear coefficient
(0.9799).
containing 0.25 mM b-mercaptoethanol. Samples were then sub-
jected to electrophoresis on 10% w/v SDS-PAGE gel. Gels were dried,
and phosphorylated myelin basic protein was identified by autora-
diography. The VersaDoc Quantity One software (BioRad) was used
4.3.4. Evaluation of pro-apoptotic activity
HUVECs (2.5 ꢂ 104 cell/cm2) were seeded into a 96-well plate
for densitometric analysis. Results were expressed as IC50 (mM) or %
and incubated at 37 ꢀC for 24 h. Then, cultures were washed and
inhibition ([I] ¼ 1
m
M) against purified kinases.
treated with compounds (10 mM) for 24 h. Apoptosis was detected
by an enzyme-linked immunoassorbent assay (ELISA) using the Cell
Death Detection ELISAPLUS Kit (Roche Diagnostics S.p.A., Monza,
Italy) according to the manufacturer’s instructions. Briefly, at the
end of incubation period, cells were lysated and then centrifuged.
Aliquots of supernatant were transferred to a streptavidin-coated
well of a microtiter plate. Apoptosis detection was based on a
quantitative sandwich-enzyme immunoassay using peroxidase-
labeled monoclonal antibodies directed against DNA and histones
of mono- and oligo-nucleosomes in supernatants of cell lysates.
Peroxidase retained in immunocomplexes was measured photo-
metrically at 405 nm wavelength using diammonium 2,20-azino-
bis(3-ethylbenzothiazoline-6-sulfonate) as substrate. Results were
expressed as percent change from control non-treated cultures.
4.3.2. Cell culture
A431 human vulvar squamous carcinoma cells overexpressing
HER-1 [42] and NIH3T3 murine embryonic fibroblast-like cells
lacking EGF receptors [42], human colonic adenocarcinoma HT-29
and murine hepatocarcinoma BNL 1ME A.7R.1 were purchased by
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-
Romagna (Brescia, Italy), whereas human MCF-7 and HeLa were
obtained from American Tissue Culture Collection (ATCC, Manassas,
Virginia). A431, HT-29, HeLa, MCF-7, and BNL cell lines were grown
in monolayer culture with DMEM High Glucose medium (Euro-
clone S.p.A., Pavia, Italy) supplemented with 10% FBS (Euroclone
S.p.A., Pavia, Italy), 100 U/ml penicillin and 100 mg/mL streptomycin
(Sigma Aldrich S.r.l., Milano, Italy). NIH3T3 cells were maintained in
DMEM High Glucose enriched with 10% FCS (Promocell, Heidelberg,
4.3.5. Cell migration
Germany), 100 U/ml penicillin and 100
m
g/mL streptomycin. Cul-
Cell migration was evaluated using a modified Boyden chamber.
tures were incubated at 37 ꢀC in a humidified atmosphere.
Primary cultures of human umbilical vein endothelial cell
(HUVEC) were obtained by enzymatic digestion of umbilical vein
endothelial layer with a 0.1% collagenase IV solution (Sigma
Aldrich, Milan, Italy). The cells were seeded on Petri dishes (BD,
HUVECs (3 ꢂ 104 cell/cm2) were seeded on the upper side of 5.0
mm
pore Transwell insert (Corning Inc.) in basal medium supplemented
with 1% FCS with or without noncytotoxic concentration of the
tested compounds (1 mM). Inserts were placed in a 24-well plate
containing medium supplemented with growth factors (lower
chamber). After 4 h, cultures were fixed in 10% formaldehyde
(Sigma) and the upper membrane of the insert was swabbed to
remove non-migrated cells. The membrane was cut from the insert
and mounted with DAPI. HUVEC migration was quantified by
counting the number of nuclei in the lower side of the membrane in
five random fields per insert (magnification ꢂ100) by fluorescence
microscopy. Experiments were repeated five times. Results were
expressed as percent change from control cultures incubated with
basal medium supplemented only with growth factors.
Franklin Lakes, NJ USA) previously coated with fibronectin (1 mg/
mL; Sigma) and cultured with Endothelial Cell growth Medium
MV2 (basal medium, Promocell, Hidelberg, Germany) supple-
mented with 5% FCS, ascorbic acid (1
hEGF (5 ng/mL), hydrocortisone (0.2
m
g/mL), hFGF-2 (10 ng/mL),
m
g/mL), R3-IGF-1 (20 ng/mL),
VEGF (0.5 ng/mL) (endothelial MV2 medium kit, Promocell), and 1%
antibiotic solution (Sigma), containing streptomycin sulfate (10 ng/
mL), amphotericin-B (250 ng/mL) and penicillin G (100 U/mL).
Cultures were incubated at 37 ꢀC in a humidified atmosphere. The
endothelial cells were used up to the fifth passage and harvested at
80% confluence.
4.3.6. Morphogenesis assay
Morphogenesis analysis was carried out seeding HUVECs on
Matrigel (BD Biosciences). Matrigel was thawed on ice overnight,
4.3.3. Evaluation of anti-proliferative activity
Cells were seeded into a 96-well plate atthe followingcelldensity:
1.5 ꢂ 104 cell/cm2 (A431), 6 ꢂ 103 cell/cm2 (NIH3T3), 2.5 ꢂ 104 cell/
cm2 (MCF-7, HT-29, HeLa, BNL), and 1 ꢂ104 cell/cm2 (HUVECs). Aftera
24 h incubation period, media were replaced with ones containing or
spread evenly over each well (50 mL) of a 24-well plate, and allowed
to polymerized for 30 min at 37 ꢀC. HUVECs (2.5 ꢂ 104 cells/cm2)
were seeded on Matrigel and cultured in basal medium supple-
mented with 1% FCS, with or without not cytotoxic concentrations of
not various concentrations (ranging from 0.01 to 10
m
M) of the tested
the tested compounds (0.1 mM). Other wells were evenly coated with
compounds. After 20 h or 68 h incubation, cells were treated with 3-
(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT)
(0.50 mg/ml, Sigma) for 4 h. Formazan precipitates were dissolved in
2-propanol acid (0.04 M HCl in 2-propanol; Sigma) and optical den-
sity was measured at 570 nm, using a Microplate autoreader EL 13
(BIO-TEK instruments Inc., Winooski, Vermont USA). Results were
expressed as IC50, i.e. the concentrations of the agents (mM) which
induced a 50% reduction of viability in comparison with non-treated
cultures.
Matrigel GFR (Growth Factor Reduced). HUVECs (2.5 ꢂ 104 cells/cm2)
were seeded on Matrigel and cultured in basal medium supple-
mented with 1% FCS with or without not cytotoxic concentrations of
the tested compounds (0.1 m
M). After 18 h of incubation at 37 ꢀC,
cultures were fixed in 2% glutaraldehyde in cacodylate buffer, pH 7.2
and then photographed (5 fields for each well: the four quadrants
and the center) at a magnification ꢂ50. Phase contrast images were
recorded using a digital camera and image analysis was carried out
using the ImageJ image analysis software. The following dimensional

382
M.T. Conconi et al. / European Journal of Medicinal Chemistry 67 (2013) 373e383
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meshes, and branching points per fields) were estimated. Values
were expressed as percent change from control non-treated cultures.
4.3.7. In vivo antitumor activity
The in vivo antitumor activity of compounds 7e9 was evaluated
in Balb/c mice using a syngenic murine hepatocellular carcinoma
cell line (BNL 1ME A.7R.1) [43]. Briefly, male mice, 10 weeks old,
were purchased from Harlan (S. Pietro al Natisone Udine, Italy), and
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mL of sterile PBS were injected
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divided into four groups, and starting on the seventh day, they were
daily dosed intraperitoneally (ip) with 10 50 mg/kg body weight in
vehicle (0.9% NaCl containing 5% polyethylene glycol 400 and 0.5%
Tween 80), of either compound 7, 8 or 9 or just vehicle. Tumor sizes
were measured daily for 7 days using a pair of callipers and the
tumor volume (V) was calculated by the rotational ellipsoid for-
mula: V ¼ AB2/2, where A is the longer diameter (axial) and B is the
shorter diameter (rotational). All experimental procedures fol-
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4.3.8. In vivo Matrigel plug assay
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which warrants care of experimental animals in Italy. The research
project was approved by the Italian Health Department according
to the art. 7 of above mentioned D.L. Three-month-old C57/BL6
mice were anesthetized with isoflurane (2e3%) via nose cone and
100% oxygen used as carrier gas; the flank was shaved and dis-
infected with ethyl alcohol. A total of 0.5 mL of Matrigel GFR, mixed
with 12 U.I. heparin (Vister) with or without 200 ng/plug FGF-2 and
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mM tested compounds, was injected subcutaneously into the
flank. After injection, the Matrigel polymerized forming a plug.
After 7 days, the animals were sacrificed and the plugs were care-
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fully removed and steeped in 300 mL/plug Brij-35 0.1% solution in
PBS overnight at 4 ꢀC. Hemoglobin concentration was analyzed
using Drabkin’s Reagent kit (Sigma). The optical density was read at
540 nm using a Microplate autoreader EL 13. The results were
expressed as mg/m haemoglobin.
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4.3.9. Statistical analysis
Unless otherwise indicated, the results are presented as the
mean ꢁ SEM. The differences between different treatments were
analyzed using the two-sided Student’s t test. P values of less than
0.05 were considered significant.
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Acknowledgments
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.. G.M. thanks financial support from
the University of Padova for a post-doc senior grant.
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