Discovery of biarylaminoquinazolines as novel tubulin polymerization inhibitors

Article abstract of DOI:10.1021/jm500034j

Cell cycle experiments with our previously reported 4- biphenylaminoquinazoline (1-3) multityrosine kinase inhibitors revealed an activity profile resembling that of known tubulin polymerization inhibitors. Novel 4-biarylaminoquinazoline analogues of comp

Full text of DOI:10.1021/jm500034j

Article
pubs.acs.org/jmc
Discovery of Biarylaminoquinazolines as Novel Tubulin
Polymerization Inhibitors
Giovanni Marzaro,†,# Antonio Coluccia,‡,# Alessandro Ferrarese,† Paola Brun,§ Ignazio Castagliuolo,§
Maria Teresa Conconi,† Giuseppe La Regina,‡ Ruoli Bai,∥ Romano Silvestri,‡ Ernest Hamel,∥
and Adriana Chilin*,†
†Dipartimento di Scienze del Farmaco, Universita
̀
degli Studi di Padova, via Marzolo 5, 35131 Padova, Italy
‡Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur−Fondazione Cenci Bolognetti, Sapienza Universita
̀
di Roma,
Piazzale Aldo Moro 5, I-00185 Roma, Italy
§Department of Molecular Medicine, University of Padova, via Gabelli 63, 35121 Padova, Italy
∥Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick
National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702,
United States
S
* Supporting Information
ABSTRACT: Cell cycle experiments with our previously
reported 4-biphenylaminoquinazoline (1−3) multityrosine kinase
inhibitors revealed an activity profile resembling that of known
tubulin polymerization inhibitors. Novel 4-biarylaminoquinazoline
analogues of compound 2 were synthesized and evaluated as
inhibitors of several tyrosine kinases and of tubulin. Although
compounds 1−3 acted as dual inhibitors, the heterobiaryl
analogues possessed only anti-tubulin properties and targeted
the colchicine site. Furthermore, molecular modeling studies
allowed the rationalization of the pharmacodynamic properties of
the compounds.
INTRODUCTION
■
The discovery of the anticancer drugs erlotinib1 and gefitinib2
in the early 2000s prompted intensive research on 4-
anilinoquinazoline compounds, leading to the development of
new attractive compounds such as lapatinib,3 vandetanib,4 and
afatinib.5 Several published patents and articles showed the
Figure 1. Previously reported quinazoline-based multi-TK inhibitors.
feasibility of the anilinoquinazoline scaffold for the develop-
ment of tyrosine kinase (TK) inhibitors (TKIs).6,7 The main
targets than the TKs. In particular, the effects of compounds 1−
biomolecular target of this class of compounds remains
epidermal growth factor receptor (EGFR), although some
compounds do not show high selectivity for it. For example,
lapatinib is a dual EGFR/Her-2 inhibitor, whereas vandetanib
inhibits the kinase activities of both EGFR and VEGFR-2. In
this regard, we have recently reported that the functionalization
of the quinazoline scaffold with both a fused dioxygenated ring
3 on cell cycle progression (vide post) were quite similar to
known tubulin inhibitors, suggesting an interaction with
cytoskeletal components.
Interestingly, in 2008, Sirisoma et al. reported some N4-
methyl-N4-phenyl-4-aminoquinazolines (e.g., N-(4-methoxy-
phenyl)-N,2-dimethylquinazolin-4-amine; see Figure S1, Sup-
porting Information) endowed with potent cytotoxic effects,
at the 6 and 7 positions and a 3-biphenylamino function at the
4 position leads to multi-TKIs.8 In particular, compound 2
which were discovered through a cell-based anticancer
screening apoptosis platform.10 The authors proposed and
(Figure 1) was found to inhibit the kinase activities of EGFR,
FGFR-1, PDGFRβ, Abl1, and Src at submicromolar concen-
validated tubulin as the main target of these compounds. In
fact, although the structural similarity to other substituted
trations and also showed EC50’s against several cancer cell lines
in the low nanomolar range.9 For example, in A431 cells, 2 was
anilinoquinazolines, such as erlotinib or gefitinib, might suggest
about 10 times more cytotoxic than analogues 1 and 3. Further
investigations on the mechanism of action of the biphenylami-
noquinazolines indicated that they could have other molecular
Received: January 8, 2014
Published: May 6, 2014
© 2014 American Chemical Society
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EGFR as being a plausible target, the N4-methyl and the
presence of a substituent at the 2 position of N-(4-
methoxyphenyl)-N,2-dimethylquinazolin-4-amine impaired the
ability of the compounds to act also as kinase inhibitors.11
Combining the results of cell cycle analysis with Sirisoma’s
finding, we were even more convinced that, besides the anti-TK
activity, the previously described 4-anilinoquinazolines probably
inhibited tubulin polymerization. Here, we demonstrate that
biphenylaminoquinazolines possess anti-tubulin effects as well
as TKI properties. Thus, they are promising examples of dual
inhibitors. Moreover, in order to establish the molecular
determinants required to achieve dual TK/tubulin inhibition or
to be selective anti-tubulin compounds, we synthesized and
evaluated several heterobiaryl analogues of compound 2 in
which one of the two benzene rings of the biphenylamino
moiety was replaced by a 5- or 6-atom heterocycle. Finally, all
of the synthesized compounds were evaluated by a molecular
modeling approach, with the aim of rationalizing the structure−
activity relationship.
Chloroquinazoline intermediate 11 was condensed with the
appropriate heterobiaryl amines through two different micro-
wave-assisted protocols according to the type of the nucleus
bearing the amine function. Final products 4−6, in which the
nucleus bearing the amine was a benzene, were obtained as
hydrochlorides using i-PrOH as solvent, whereas compounds 7
and 8, in which the nucleus bearing the amino group was a
heterocycle, were obtained as free bases using NaH in THF. In
fact, because of the low nucleophilicity of the amine function,
when 2-amino-4-phenylpyrimidine or 2-amino-4-phenylthiazole
was mixed with chloroquinazoline in i-PrOH and irradiated, no
products were formed. Consequently, for these two heteroaryl-
amines, a stronger alkaline medium was required.
For the synthesis of compound 9, the 3-(2-pyrrolyl)aniline
synthon was prepared starting from 3-bromoaniline (12),
protected as ethyl carbamate 13, which was condensed with N-
boc-2-pyrroleboronic acid via Suzuki coupling.
Intermediate 14 was deprotected at both amine functions in
alkaline medium to give the desired derivative 15, which was
finally condensed with chloroquinazoline 11 in i-PrOH to
afford compound 9 as the hydrochloride (Scheme 2).
RESULTS AND DISCUSSION
■
To investigate the structural determinants required for the
inhibition of kinases and tubulin polymerization, we planned
the synthesis of some novel analogues of compound 2, which
was the most cytotoxic (among the three biphenylaminoqui-
nazolines previously discovered) against several cell lines
(Figure 2).8
a
Scheme 2. Synthesis of Compound 9
a
Reagents and conditions: (a) ClCOOEt, TEA, THF, reflux, 1 h; (b)
N-boc-2-pyrroleboronic acid, Na2CO3(aq), ethylene glycol dimethyl
ether, Pd(PPh3)4, reflux, 4 h; (c) KOH(aq), reflux, 4 h; (d) compound
11, i-PrOH, MW, 80 °C, 15 min.
All of the newly synthesized compounds, together with 1−3,
were evaluated against a panel of isolated tyrosine kinases and
against tubulin (Table 1). Compounds 1−3 inhibited both
tubulin polymerization and the tested TKs. Moreover, the
biphenylaminoquinazolines were found to bind at the
colchicine site, as demonstrated by their ability to inhibit the
binding of [3H]colchicine to tubulin. Conversely, heterobiaryl
analogues 4−9 were almost inactive at the tested concen-
trations (IC50’s > 10 μM) against the TKs, whereas they
maintained varying anti-tubulin activity. Compound 10
(bearing the biphenylamino moiety but not bearing the fused
dioxygenated ring on the quinazoline core) was inactive against
all of the tested targets. Interestingly, the inhibitory activity
against tubulin polymerization of compounds 1−3, 5, 6, and 9,
especially that of compounds 1 and 5, was comparable with that
of combretastatin A-4 (C A-4).
Figure 2. Newly synthesized quinazoline compounds.
Compounds 1−3 and 10 were prepared as previously
described.8 Compounds 4−8 were synthesized starting from 4-
chloro-7,8-dihydro[1,4]-dioxino[2,3-g]quinazoline (11)
(Scheme 1).
In view of the data acquired on isolated targets, compounds
1−3, 5, 6, and 9 were also tested on human ovarian carcinoma
cell line OVCAR-8 and its cognate P-glycoprotein (P-gp)-
overexpressing cell line, NCI/ADR-RES (Table 2).
a
Scheme 1. Synthesis of Compounds 4−8
All compounds, except the paclitaxel control, had essentially
identical antiproliferative activities in both the parental and P-
gp-overexpressing multidrug-resistant cells. Moreover, the
activity of the compounds against the OVCAR-8 cell line
may indicate a hormone-independent mechanism of action for
the tested compounds.12 In the cell-based assays, only
compounds 1 and 5 exerted effects similar to that of C A-4.
Contrary to what was previously reported for other cell lines,
compound 1 was more active than compound 2 in inhibiting
a
Reagents and conditions: (a) method A: 3-(2-furyl)aniline, 3-(2-
thienyl)aniline, or 3-(2-pyridinyl)aniline, i-PrOH, MW, 80 °C, 15 min;
method B: 2-amino-4-phenylpyrimidine or 2-amino-4-phenylthiazole,
NaH, THF, MW, 80 °C, 15 min. See Figure 2 for Ar specification.
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Table 1. In Vitro Evaluation of Quinazoline Compounds
a
IC50 (μM)
b
c
compd
EGFR
FGFR1
Abl1
Src
tubulin
colchicine binding inhibition (%)
72 0.4
1
2
3
4
5
6
7
8
9
0.002 0.001
0.042 0.005
0.027 0.012
0.063 0.015
0.178 0.030
0.063 0.025
0.051 0.010
0.024 0.010
0.056 0.016
0.032 0.008
0.642 0.105
0.645 0.085
1.1 0.03
4.0 0.2
2.3 0.02
12 0.4
1.2 0.06
3.2 0.1
>20
41
34
nd
7
1
e
e
e
e
d
>10
>10
>10
>10
e
e
e
e
>10
>10
>10
>10
75 0.8
e
e
e
e
>10
>10
>10
>10
25
nd
nd
33
nd
nd
4
7
e
e
e
e
d
d
>10
>10
>10
>10
e
e
e
e
>10
>10
>10
>10
>20
e
e
e
e
>10
>10
>10
>10
3.0 0.2
>20
e
e
e
e
d
d
10
>10
>10
>10
>10
vandetanib
C A-4
0.190 0.085
>10
>10
>10
0.151 0.024
0.044 0.016
>10
>20
>10
>10
>10
>10
1.1 0.1
99 0.7
f
f
paclitaxel
>10
nd
nd
a
b
Values are reported as the mean SD of at least three independent experiments. Inhibition of tubulin polymerization with tubulin at 10 μM.
c
d
Percentage of inhibition of [3H]colchicine binding. Tubulin was 1 μM. Both [3H]colchicine and the inhibitor were 5 μM. nd, not determined
e
because tubulin polymerization’s IC50 was greater than 10 μM. The percentage of inhibition at 10 μM is reported in the Supporting Information.
f
Paclitaxel stimulates tubulin assembly.
confirming the previous findings. In particular, 1 and 5, the
two most active compounds against tubulin and as cytotoxic
agents, significantly increased the proportion of cells arrested at
G2/M as compared to that of control cells (P < 0.05).
The 4-anilinoquinazoline moiety represents a widely studied
scaffold in the field of TKIs,7 and a large number of examples
are available for both the type I and II TKIs.14 However, in the
field of tubulin polymerization inhibitors, the 4-anilinoquinazo-
line core is not commonly used, and N-(4-methoxyphenyl)-
N,2-dimethylquinazolin-4-amine represents, to our knowledge,
the most active published derivative.10 Because this chemotype
is quite unexplored as a class of tubulin inhibitors, we initiated a
molecular modeling study with the goal of rationalizing the
pivotal pharmacophore features required to achieve dual
inhibition of tubulin polymerization and TKs. Some attempts
to draw a common SAR were previously published,10,11 but
these were aimed mainly at determining differences between
the requirements of the two targets.
Thus, the binding mode of 1−9 was evaluated by docking
studies. As a consequence of the structural similarity with N-(4-
methoxyphenyl)-N,2-dimethylquinazolin-4-amine and on the
basis of their inhibition of colchicine binding, we modeled them
into the colchicine site of tubulin. From among the available
crystal structures with colchicine site inhibitors,15 we selected
1SA0 (tubulin with DAMA-colchicine)16 and 3N2G (tubulin
with ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-
b]-pyrazin-7-yl-carbamate).17 3N2G showed a larger binding
site than 1SA0 because the Leu255β residue moves its side
chain far from the binding site, thereby opening a small
subpocket deeply buried within the tubulin β-subunit (Figure
4). We docked compounds 1−9 into both structures, and we
observed two very similar binding modes for each compound
(data not shown). The poses obtained with the 3N2G structure
had the best docking score.
Table 2. Cytotoxic Activity of Quinazoline Compounds
a
EC50 (nM) SD
b
compd
OVCAR-8
NCI/ADR-RES
15
c
1
30
0
0
7
2
500
350 80
3
550 70
10
300 100
5
0
4.0
1
6
150 70
4000 1000
5.5 0.7
250 70
9
3600 200
paclitaxel
C A-4
5000
13
0
4
18
4
a
b
Data were obtained in at least two independent experiments. A cell
c
line isogenic with OVCAR-8 except that it overexpresses P-gp.
standard deviation of 0 means that the same value was obtained in all
experiments.
A
the proliferation of both the OVCAR-8 and NCI/ADR-RES
cell lines.
Because of the above results, compounds 1−3, 5, 6, and 9
were also subjected to analysis for their effects on cell cycle
distribution. All compounds were added to the cells at a
concentration of 1.0 μM. In the OVCAR-8 cell line, these
experiments revealed a profile of activity typically reported for
inhibitors of tubulin polymerization (Figure 3),13 further
The cell cycle results strongly indicated that the cytotoxic
effects of the reported derivatives result from interactions of the
compounds with tubulin. In further support of our hypothesis,
we superimposed the selected binding poses of the 4-
biphenylamino-quinazolines with the Nguyen seven-point
pharmacophore model (Figure 4).18 Not all of the model
requirements for polar interactions were met, but the
Figure 3. Effect of quinazoline compounds on cell cycle progression.
Data are presented as the mean
SEM of three independent
experiments.
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established nonpolar interactions with Leu248β, Asn258β,
Met259β, Lys352β, and Val181α. Molecular dynamics carried
out for 2 indicated that the described interactions were stable
(data not shown).
For the essentially inactive derivatives 7 and 8, AutoDock
provided some binding poses similar to those found for the
active compounds. As a consequence, we suggest that a stable/
lasting binding was also related to the logP of the bridge cycle.
The mainly hydrophobic features of the colchicine site were
matched better by phenyl than by pyrimidine (7) or thiazole
(8). To confirm this hypothesis, we are preparing additional
compounds. In contrast, with derivative 10 (characterized by
two methoxy functions in the quinazoline moiety instead of a
fused dioxygenated ring) we did not observe any binding pose
consistent with either reference compound.
Similar modeling experiments were also performed for the
studied kinases (EGFR, FGFR1, Abl1, and Src; Table 1). We
previously performed docking analyses with compounds 1−3
and 10,8 from which it was concluded that more reliable
binding conformations were obtained using kinase crystal
structures with the DFG-out conformation. The in/out
movement of the DFG triplet (aspartic−phenylalanine−
glycine) largely affects the morphology of the ATP binding
site. The kinases in the DFG-out form have, as an important
feature, a closed conformation of the activation loop, and this
prevents ATP binding and exposes an additional hydrophobic
site.19 Using derivative 2 as reference, we noted three major
contacts (Figure 6): (i) the quinazoline nitrogen N1 forms an
H-bond with the hinge region; (ii) the terminal phenyl moiety
forms an “edge-to-face” phenyl−phenyl interaction with the
phenylalanine of the DFG triplet; and (iii) the aniline ring lies
in a lipophilic cleft formed by hydrophobic residues. (EGFR:
Val716, Ala743, Leu844, and Thr854; FGFR: Val492, Lys514,
and Phe489; Abl1: Ala269, Lys271, and Ala380; Src: Lys295,
Val281, and Thr338).
Figure 4. Tubulin structures are shown as ribbons: green/brown for
the β-subunits and yellow/red for the α-subunits of 1SA0 and 3N2G,
respectively. The binding mode of 2 (cyan) is shown. Global view of
the Nguyen pharmacophore model: the red, pink, and yellow balls are
H-bond acceptors; the magenta ball is an H-bond donor; the blue and
skyblue balls are hydrophobic points; and the green ball is a planar
group. DAMA−colchicine (purple) is also shown. Residue Leu255 is
shown as a stick (green and brown for 1SA0 and 3N2G, respectively).
hydrophobic requirements were fully satisfied. Hence, our
modeling data strongly support the view that derivatives 1−9
are tubulin polymerization inhibitors, in agreement with the
biological results presented above.
The specific amino acid residue interactions of derivative 2
(tubulin assembly IC50 = 4.0 μM) are shown in Figure 5.
Among the AutoDock proposed binding conformations of
4−9, we observed a cluster of poses similar to the one selected
for active compounds, but this cluster never scored as one of
the best or the most populated. In spite of this, we observed a
very poor correlation between docking score and experimental
biological activity. In an attempt to readily rationalize their
inactivity, we hypothesize that for compound 7 the loss of
kinase inhibition was due to the polarization induced by the
pyrimidine ring. In contrast, in the case of compounds 4−6 and
9, we hypothesize that the heterocycles impair the T-shaped
interaction, probably because of the different partial charge
distributions. Indeed, it was reported that heterocycles tend to
form a “face-to-face” arene−arene interaction in contrast to the
“edge-to-face” interaction that is preferred by benzene rings.20
Summarizing, the 4-anilinoquinoline scaffold can be used for
the development of both TKIs and tubulin polymerization
inhibitors, with the opportunity to modulate these biological
activities through the selection of appropriate substituents.
Merging the results of the computational analysis with the
biological data, we can define the requirements for dual activity
(Figure 7):
Figure 5. Binding mode of compound 2 in the colchicine site.
Residues forming the hydrophobic interactions are shown using a
color code: in green are residues interacting with the phenyl moiety; in
magenta are those interacting with the aniline ring; and in white are
those interacting with the dioxoquinazoline moiety.
The binding mode analysis was carried out by splitting the
structure of 2 into three different portions: the terminal phenyl
ring, the aniline ring, and the quinazoline moiety. The terminal
phenyl ring bound in the subpocket opened by the shift of the
Leu255β side chain in 3N2G. In this cleft, the phenyl ring was
strongly stabilized by hydrophobic contacts with Tyr202β,
Asn167β, Val238β, Leu242β, and Leu252β. The aniline ring
was well-stabilized by a series of hydrophobic contacts with
Leu255β, Ala316β, and Ile387β. Lastly, the quinazoline moiety
(1) The carbon atom linker between the oxygen atoms may
be varied from one carbon to three, but the dioxygenated
function must be included in a cycle;
(2) The substitution of the aniline benzene (ring A) with any
other heteroaryl is not allowed;
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Figure 6. Binding mode of derivative 2 in the ATP binding site of: EGFR (green), FGFR (cyan), Src (magenta), and Abl (yellow). Residues of the
hinge region are shown. The Phe residue of the DFG triplet is also shown. In all cases, the biphenyl moiety is stabilized in a lipophilic cleft (EGFR:
Val716, Ala743, Leu844, and Thr854; FGFR: Val492, Lys514, and Phe489; Abl1: Ala269, Lys271 and Ala380; Src: Lys295, Val281, and Thr338).
modeling studies allowed us to rationalize our structure−
activity relationship findings.
Specifically, we found that (1) the 3-phenylaniline moiety
was essential for the dual TK/tubulin polymerization inhibition,
but substituents could further enhance hydrophobic inter-
actions; (2) generally, the introduction of an heteroatom in the
biphenyl moiety was detrimental for anti-TK activity; (3) the
substitution of the ring A with an heteroaryl (compounds 7 and
8) caused the abrogation of both anti-TK and anti-tubulin
activities; (4) the introduction of a heteroatom in ring B did
Figure 7. Structure−activity relationship for biarylaminoquinazolines.
not affect anti-tubulin activity; and (5) the fused dioxygenated
ring was essential for anti-tubulin activity (compare compounds
1−3 with 10), as previously reported for inhibition of multiple
tyrosine kinases.8
(3) The terminal cycle (ring B) represents the switching
force between the two activities: with a heterocycle, we
observed selective anti-tubulin activity, whereas with a
phenyl ring, we obtained dual anti-tubulin/anti-TK
activity.
The functionalization of the biphenyl moiety with lipophilic
substituents (halogen atoms or methyl or methoxy function) is
in progress, with the aim to investigate more thoroughly the
pharmacophore features of dual TK/tubulin inhibitors. We are
also planning the synthesis of heteroaryl analogues of
compound 1, because it showed higher inhibitory activity
against the OVCAR-8 and NCI/ADR-RES cell lines than
reference compound 2. Moreover, because the novel
compounds did not show anti-TK activities, we will also
synthesize the N4-methyl analogues of the compounds
described here.
CONCLUSIONS
■
The previously reported biphenylaminoquinazoline derivatives
(compounds 1−3), initially developed as multi-TKIs, were also
found to inhibit tubulin polymerization. By examining the
inhibition of the binding of [3H]colchicine to tubulin, we
demonstrated that the quinazolines bound in the colchicine site
of tubulin. In order to establish a structure−activity relation-
ship, several heteroaryl analogues were synthesized and
evaluated as TKIs and as tubulin polymerization inhibitors.
The newly synthesized compounds were all found to be almost
inactive against the isolated kinases, whereas some derivatives
maintained inhibitory activity on tubulin polymerization.
Consequently, we found that it is possible to obtain both
dual TK/tubulin polymerization inhibitors and selective tubulin
inhibitors bearing the same quinazoline scaffold. Molecular
EXPERIMENTAL SECTION
■
Chemistry. All commercial chemicals and solvents used were
analytical grade and were used without further purification. Analytical
thin-layer chromatography (TLC) was performed on precoated silica
gel plates (Merck 60-F-254, 0.25 mm). Preparative column
chromatography was performed using silica gel 60 (0.063−0.100
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mm; Merck), eluting with CHCl3. Melting points were determined on
a Gallenkamp MFB-595-010 M melting point apparatus and are
uncorrected. The H NMR spectra were recorded on a Bruker 300-
134.17, 129.16, 125.53, 124.54, 123.29, 122.93, 120.96, 110.87, 107.97,
105.03, 65.01, 64.08. Anal. Calcd for C21H17ClN4O2: C, 64.21; H,
4.36; Cl, 9.02; N, 14.26. Found: C, 64.21; H, 4.30; Cl, 9.07; N, 14.27.
HRMS (ESI-TOF) for C21H17N4O2 [M + H]+: calcd, 357.1346;
found, 357.1318
(7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)-(4′-phenyl-
pyrimidin-2″yl)amine (7). From 2-amino-4-phenylpyrimidine with
method B: Yield 5%. mp > 300 °C. 1H NMR (300 MHz, DMSO-d6):
δ 8.95 (d, J = 5.4 Hz, 1H, 6′-H), 8.87 (s, 1H, 2-H), 8.46 (s, 1H, 5-H or
10-H), 8.32−8.26 (m, 2H, 2″-H and 6″-H), 8.04 (d, J = 5.4 Hz, 1H,
5′-H), 7.67−7.58 (m, 3H, 3″-H, 4″-H and 5″-H), 7.44 (s, 1H, 5-H or
10-H), 4.57−4.45 (m, 4H, OCH2CH2O). 13C NMR (75 MHz,
DMSO-d6): δ 164.06, 158.96, 158.92, 149.69, 143.89, 136.19, 131.09,
128.90, 127.09, 112.33, 111.26, 109.75, 64.52, 64.07. Anal. Calcd for
C20H16ClN5O2: C, 60.99; H, 4.09; Cl, 9.00; N, 17.78. Found: C, 70.01;
H, 4.07; Cl, 9.03; N, 17.76. HRMS (ESI-TOF) for C20H16N5O2 [M +
H]+: calcd, 358.1299; found, 358.1204
1
AMX spectrometer with TMS as an internal standard. Coupling
constants are given in hertz (Hz), and the relative area peaks were in
agreement with all assignments. Elemental analyses were characterized
on a PerkinElmer 2400 analyzer. Mass spectra were recorded on an
Applied Biosystem Mariner System 5220 with direct injection of the
sample. Microwave-assisted reactions were performed on a CEM
Discover monomode reactor with the temperature monitored by a
built-in infrared sensor and automatic control of power; all reactions
were performed in closed devices with pressure control. Compounds
1−3 and 10 were prepared as previously described.8 Starting product
11 was synthesized as previously reported.21 All of the amine reagents
were commercial products except for 3-(2-pyrrolyl)aniline, which was
synthesized as described below. The purity for all of the tested
compounds was determined by elemental analyses and was found to
be equal to or greater than 95%.
General Procedure for Aminoquinazolines 4−9. Method A. A
mixture of 11 (1.0 mmol) and 3-(2-heteroaryl)aniline (1.0 mmol) in i-
PrOH (3 mL) was microwave-irradiated at 80 °C (power set point, 60
W; ramp time, 1 min; hold time, 15 min). After cooling, the resulting
precipitate was collected by filtration to give 4−6 as hydrochlorides.
Method B. To a solution of 2-amino-4-phenylheteroaryl derivatives
(1.0 mmol) in anhydrous THF (6 mL) was added NaH (4.5 mmol),
and the mixture was stirred at room temperature for 30 min.
Compound 11 (1.0 mmol) was then added, and the mixture was
microwave-irradiated at 80 °C (power set point, 60 W; ramp time, 1
min; hold time, 15 min). After cooling, the mixture was diluted with
water (100 mL) and extracted with EtOAc (3 × 100 mL). The organic
phase was evaporated under reduced pressure, and the residue was
crystallized from i-PrOH to give 7 or 8.
(7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)-(4′-phenyl-
thiazol-2″yl)amine (8). From 2-amino-4-phenylthiazole with
1
method B: yield 10%. mp 278 °C. H NMR (300 MHz, DMSO-d6):
δ 12.14 (broad s, 1H, NH). 8.68 (s, 1H, 2-H), 8.37 (s, 1H, 5-H or 10-
H), 7.99 (d, J = 7.7 Hz, 2H, 2″-H and 6″-H), 7.69 (s, 1H, 5-H or 10-
H), 7.46 (t, J = 7.7 Hz, 2H, 3″-H and 5″-H), 7.35 (t, J = 7.7 Hz, 1H,
4″-H), 7.28 (s, 1H, 5′-H), 4.47−4.38 (m, 4H, OCH2CH2O). 13C
NMR (75 MHz, DMSO-d6): δ 151.32, 151.16, 149.54, 143.96, 128.60,
127.60, 125.68, 125.56, 122.15, 112.58, 112.55, 109.74, 109.38, 108.91,
108.69, 64.42, 63.99. Anal. Calcd for C19H15ClN4O2S: C, 57.21; H,
3.79; Cl, 8.89; N, 14.05; S, 8.04. Found: C, 57.24; H, 3.81; Cl, 8.90; N,
14.04; S, 8.06. HRMS (ESI-TOF) for C19H15N4O2S [M + H]+: calcd,
363.0910; found, 363.0861
(7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)-[3′-(pyrrol-
2″-yl)phenyl]amine Hydrochloride (9). From 3-(2-pyrrolyl)aniline
1
(15) with method A: yield 67%. mp 270 °C. H NMR (300 MHz,
(7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)-[3′-(furan-
DMSO-d6): δ 11.38 (broad s, 1H, NH). 11.10 (broad s, 1H, NH);
8.81 (s, 1H, 2-H), 8.34 (s, 1H, 5-H or 10-H), 7.89 (broad s, 1H, 2′-H),
7.58 (dt, J = 7.7, 2.0 Hz, 1H, 4′-H or 6′-H), 7.46 (t, J = 7.7 Hz, 1H, 5′-
H), 7.46−7.41 (m, 1H, 4′-H or 6′-H), 7.33 (s, 1H, 5-H or 10-H), 6.88
(dd, J = 4.1, 2.7 Hz, 1H, 5″-H), 6.54 (dd, J = 5.1, 4.1 Hz, 1H, 4″-H),
6.14 (dd, J = 5.1, 2.7 Hz, 1H, 3″-H), 4.55−4.43 (m, 4H, OCH2CH2O).
13C NMR (75 MHz, DMSO-d6): δ 158.49, 151.25, 149.41, 144.97,
137.15, 134.68, 133.61, 130.38, 129.07, 121.77, 121.24, 119.83, 119.71,
110.51, 109.15, 107.92, 106.02, 105.48, 65.02, 64.13. Anal. Calcd for
C20H17ClN4O2: C, 63.08; H, 4.50; Cl, 9.31; N, 14.71. Found: C, 63.06;
H, 4.52; Cl, 9.29; N, 14.71. HRMS (ESI-TOF) for C20H17N4O2 [M +
H]+: calcd, 345.1346; found, 345.1364
2″-yl)phenyl]amine Hydrochloride (4). From 3-(2-furyl)aniline
1
with method A: yield 52%. mp 75 °C. H NMR (300 MHz, DMSO-
d6): δ 11.02 (broad s, 1H, NH), 8.83 (s, 1H, 2-H), 8.33 (s, 1H, 5-H or
10-H), 8.05 (d, J = 1.7 Hz, 1H, 5″-H), 7.81 (s, 1H, 2′-H), 7.71−7.62
(m, 2H, 4′-H and 6′-H), 7.53 (t, J = 7.7 Hz, 1H, 5′-H), 7.32 (s, 1H, 5-
H or 10-H), 7.00 (d, J = 3.4 Hz, 1H, 3″-H), 6.64 (dd, J = 3.4, 1.7 Hz,
1H, 4″-H), 4.55−4.43 (m, 4H, OCH2CH2O). 13C NMR (75 MHz,
DMSO-d6): δ 158.36, 152.26, 151.22, 149.41, 144.93, 143.16, 137.41,
134.76, 130.74, 129.27, 123.33, 121.23, 119.22, 112.10, 110.53, 108.01,
106.40, 105.44, 64.97, 64.08. Anal. Calcd for C20H15N3O3·HCl: C,
62.91; H, 4.22; Cl, 9.29; N, 11.01. Found: C, 62.94; H, 4.26; Cl, 9.20;
N, 11.04. HRMS (ESI-TOF) for C20H16N3O3 [M + H]+: calcd,
346.1186; found, 346.1135.
(7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)-[3′-(thien-
2″-yl)phenyl]amine Hydrochloride (5). From 3-(2-thienyl)aniline
with method A: yield 70%. mp > 300 °C. 1H NMR (300 MHz,
DMSO-d6): δ 10.96 (broad s, 1H, NH), 8.82 (s, 1H, 2-H), 8.33 (s, 1H,
5-H or 10-H), 8.01 (d, J = 1.7 Hz, 1H, 2′-H), 7.71 (dd, J = 8.0, 1.7 Hz,
1H, 4′-H or 6′-H), 7.63 (m, 1H, 4′-H or 6′-H), 7.61 (dd, J = 5.1, 1.2
Hz, 1H, 5″-H), 7.55 (dd, J = 3.6, 1.2 Hz, 1H, 3″-H), 7.51 (t, J = 8.0
Hz, 1H, 5′-H), 7.32 (s, 1 H, 5-H or 10-H), 7.18 (dd, J = 5.1, 3.6 Hz,
1H, 4″-H), 4.55−4.44 (m, 4H, OCH2CH2O). 13C NMR (75 MHz,
DMSO-d6): δ 158.37, 151.21, 149.34, 144.92, 142.52, 137.56, 134.78,
134.19, 129.41, 128.49, 126.06, 124.05, 123.44, 123.07, 121.23, 110.63,
108.07, 105.43, 64.98, 64.08. Anal. Calcd for C20H16ClN3O2S: C,
60.37; H, 4.05; Cl, 8.91; N, 10.56; S, 8.06. Found: C, 60.35; H, 4.06;
Cl, 8.96; N, 10.55; S, 8.09. HRMS (ESI-TOF) for C20H16N3O2S [M +
H]+: calcd, 362.0958; found, 362.0896.
Ethyl 3-Bromophenylcarbamate (13). To a solution of 3-
bromoaniline (12) (0.4 g, 2.0 mmol) in THF (20 mL) and TEA (1.0
mL, 8.0 mmol) was added ethyl chloroformate (0.8 mL, 8.0 mmol)
dropwise. The mixture was heated at reflux for 1 h. After cooling, the
precipitate was removed by filtration, and the solution was evaporated
under reduced pressure. The solid was suspended in H2O (100 mL)
and extracted with EtOAc (3 × 50 mL). The organic phase was
evaporated under reduced pressure to give 13 (0.5 g, yield 84%) as an
1
oil. H NMR (300 MHz, DMSO-d6): δ 9.82 (broad s, 1H, NH), 7.74
(t, J = 1.3 Hz, 1H, 2-H), 7.41 (dt, J = 8.1, 1.3 Hz, 1H, 4-H or 6-H),
7.23 (t, J = 8.1 Hz, 1H, 5-H), 7.16 (dt, J = 8.1, 1.3 Hz, 1H, 4-H or 6-
H), 4.13 (q, J = 7.0 Hz, 2H, CH2CH3), 1.24 (t, J = 7.0 Hz, 3H,
CH2CH3). Anal. Calcd for C9H10BrNO2: C, 44.29; H, 4.13; Br, 32.74;
N, 5.74. Found: C, 44.31; H, 4.15; Br, 32.70; N, 5.78.
Ethyl [3-(1′-tert-Butyloxycarbonylpyrrol-2′-yl)phenyl]-
carbamate (14). To a mixture of ethyl 3-bromophenylcarbamate
(13) (0.4 g, 1.6 mmol) and N-Boc-2-pyrroleboronic acid (0.4 g, 2.0
mmol) in ethylene glycol dimethyl ether (25 mL) were added a
solution of Na2CO3 (0.8 g, 7.2 mmol) in H2O (2.0 mL) and
Pd(PPh3)4 (100 mg). The mixture was heated at reflux for 4 h. After
cooling, the mixture was poured into H2O (100 mL) and extracted
with CH2Cl2 (3 × 100 mL). The organic phase was evaporated under
reduced pressure and purified by column chromatography to give 14
(7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)-[3′-(pyridin-
yl-2″-yl)phenyl]amine Hydrochloride (6). From 3-(2-pyridinyl)-
aniline with method A: yield 56%. mp > 300 °C. 1H NMR (300 MHz,
DMSO-d6): δ 11.01 (broad s, 1 H, NH), 8.82 (s, 1 H, 2-H), 8.70 (d, J
= 3.2, 1H, 6″-H), 8.45 (s, 1H, 2′-H), 8.34 (s, 1H, 5-H or 10-H), 8.05−
7.82 (m, 4H, 4′-H, 5′-H, 6′-H and 3″-H), 7.60 (t, J = 7.6 Hz, 1H, 4″-
H), 7.41 (dd, J = 7.6, 3.2 Hz, 1H, 5″-H), 7.30 (s, 1H, 5-H or 10-H),
4.55−4.42 (m, 4H, OCH2CH2O). 13C NMR (75 MHz, DMSO-d6): δ
158.49, 154.50, 151.32, 149.11, 148.56, 145.00, 138.55, 138.11, 137.35,
1
(0.35 g, yield 64%). mp 126 °C. H NMR (300 MHz, DMSO-d6): δ
9.66 (broad s, 1H, NH), 7.44−7.40 (m, 2H, 2-H and 4-H or 6-H),
4603
dx.doi.org/10.1021/jm500034j | J. Med. Chem. 2014, 57, 4598−4605

Journal of Medicinal Chemistry
Article
7.33 (dd, J = 3.6, 1.7 Hz, 1H, 3′-H or 5′-H), 7.26 (t, J = 7.6 Hz, 1H, 5-
H), 6.93 (d, J = 7.6 Hz, 1H, 4-H or 6-H), 4.12 (q, J = 7.3 Hz, 2H,
CH2CH3), 1.29 (s, 9H, C(CH3)3), 1.24 (t, J = 7.3 Hz, 3H, CH2CH3).
Anal. Calcd for C18H22N2O4: C, 65.44; H, 6.71; N, 8.48. Found: C,
65.40; H, 6.74; N, 8.48.
different compounds (1 μM). Cells were harvested, washed twice with
PBS, and fixed in 70% cold ethanol (30 min at −20 °C). Cells were
then washed once in citrate phosphate buffer (0.2 N Na2HPO4 and 0.1
M citric acid, 24:1) followed by PBS and were lastly incubated in an
RNase solution (100 μg/mL in PBS). After 30 min at 37 °C, the cells
were incubated in a propidium iodide solution (100 μg/mL in PBS,
Sigma) at room temperature for a further 30 min. Samples were
analyzed on a BD FACS Calibur flow cytometer, collecting 10 000
events. Results of cell cycle analysis were examined using WinMDI 2.9.
Statistical Analysis. Biological results are reported as means
standard error. Statistical analysis was performed by using one-way
analysis of variance. A P value of less than 0.05 was considered to be
statistically significant.
Molecular Modeling. All molecular modeling studies were
performed on a MacPro dual 2.66 GHz Xeon running Ubuntu 12.
The tubulin structure was downloaded from the Protein Data Bank
(http://www.rcsb.org/; PDB IDs: 1SA016 and 3G2N17). Hydrogen
atoms were added to the protein using Molecular Operating
Environment (MOE) 2007.09,26 structures were minimized, and all
heavy atoms were kept fixed until an rmsd gradient of 0.05 kcal mol−1
Å−1 was reached. Ligand structures were built with MOE and
minimized using the MMFF94x force field until an rmsd gradient of
0.05 kcal mol−1 Å−1 was reached. The docking simulations were
performed using AutoDock27 using a 80:80:80 grid box and a spacing
of 0.375 Å, with the grid center obtained by the average of
cocrystallized inhibitor coordinates. Molecular dynamics was per-
formed with the AMBER 9 package28 using the settings previously
reported.29
3-(2-Pyrrolyl)aniline (15). A solution of ethyl [3-(1′-tert-
butyloxycarbonylpyrrol-2′-yl)phenyl]carbamate (14) (0.33 g, 1.0
mmol) in 5% KOH(aq) (100 mL) was heated at reflux for 4 h.
After cooling, the mixture was poured into H2O (100 mL) and
extracted with CH2Cl2 (3 × 50 mL). The organic phase was
evaporated under reduced pressure to give 14 (0.15 g, yield 96%). mp
1
89 °C. H NMR (300 MHz, DMSO-d6): δ 11.08 (broad s, 1H, NH),
6.98 (t, J = 7.7 Hz, 1H, 5-H), 6.82−6.74 (m, 3H, 2-H and 4-H or 6-H
and 3′-H or 5′-H), 6.38 (dt, J = 7.7, 1.5 Hz, 1H, 4-H or 6-H), 6.33−
6.29 (m, 1H, 3′-H or 5′-H), 6.06 (dd, J = 5.0, 2,6 Hz, 1H, 4′-H), 4.98
(broad s, 2H, NH2). Anal. Calcd for C10H10N2: C, 75.92; H, 6.37; N,
17.71. Found: C, 75.95; H, 6.35; N, 17.74.
Biology. Cell Lines and Culture Conditions. Human cell lines,
ovarian carcinoma cells OVCAR-8 and NCI/ADR-RES, used in the
studies were generously provided by the National Cancer Institute
Drug Screen, Frederick, MD. Cells were grown in RPMI 1640 medium
supplemented with 5% fetal bovine serum (FBS) at 37 °C in a 5%
CO2 atmosphere for 96 h. Cell protein was measured using
sulforhodamine B.
In Vitro Kinase Assays. Novel compounds were tested for in vitro
kinase activity as previously described8 using purified EGFR
(Invitrogen), FGFR-1, Src, and Abl1 (the latter three from Sigma).
Kinase reactions were performed in precooled microcentrifuge tubes
in a 25 μL final volume of kinase buffer containing 200 ng/mL active
kinase, 0.5 mg/mL myelin basic protein (Sigma), 50 μM unlabeled
ATP (Sigma), and 1 μCi/mL [γ-32P]ATP (PerkinElmer Italia).
Compounds were added at concentrations ranging from 10 μM to 10
nM. Negative controls were prepared replacing the substrate solution
with water, whereas positive controls were setup by replacing the
tested compound with water. The reactions were carried out at 30 °C
for 30 min and stopped by addition of loading buffer containing 0.25
mM β-mercaptoethanol. Reactions were then subjected to electro-
phoresis on a 12% SDS-PAGE gel. Gels were dried, and
phosphorylated myelin basic protein was identified by autoradiog-
raphy.
Tubulin Studies. Electrophoretically homogeneous bovine brain
tubulin was purified as described previously in detail.22 The tubulin
assembly assay was performed with 10 μM (1.0 mg/mL) tubulin and
0.4 mM GTP in 0.8 M monosodium glutamate (pH of 2 M stock
solution adjusted to 6.6 with HCl). Reaction mixtures contained
varying compound concentrations in 4% (v/v) dimethyl sulfoxide, and
control reaction mixtures contained only the solvent. Reaction
mixtures were preincubated without GTP at 30 °C for 15 min and
chilled on ice. GTP was added, and the reaction mixtures were placed
at 0 °C in cuvettes in Beckman DU7400 and DU7500 recording
spectrophotometers equipped with electronic temperature controllers.
After baselines at 350 nm were established, the reaction temperature
was rapidly increased to 30 °C. The IC50 was defined as the compound
concentration that inhibited the extent of assembly, measured
turbidimetrically, by 50% after 20 min at 30 °C. Reaction mixture
volume was 0.25 mL.23
The kinase structures were downloaded from the Protein Data Bank
(http://www.rcsb.org/; PDB IDs: EGFR, 1XKK;3 FGFR1, 3C4F;30
Abl1, 2QOH;31 and SRC, 3ENF32). Docking studies were carried out
by AutoDock using already reported settings.8 The images in the
article were created with PyMol.33
ASSOCIATED CONTENT
* Supporting Information
■
S
Structure of N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-
amine; kinase inhibition data; and 1H NMR, 13C NMR, and MS
spectra of the tested compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 049 8275349; Fax: +39 049 8275366; E-mail:
adriana.chilin@unipd.it.
■
Author Contributions
#G. Marzaro and A. Coluccia contributed equally to this work
and should be considered cofirst authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The present work was carried out with financial support from
the University of Padova, “Progetto di Ricerca di Ateneo 2008”
CPDA084954/08 to A. Chilin. A. Coluccia thanks Dr. A.
Brancale for his support and helpful suggestions.
Inhibition of [3H]colchicine binding to tubulin was performed as
described previously24 using reaction conditions that strongly stabilize
the colchicine binding activity of tubulin.25 Reaction mixtures (0.1
mL) contained 1.0 μM (0.1 mg/mL) tubulin, 5.0 μM [3H]colchicine,
5.0 μM potential inhibitor, 5% (v/v) dimethyl sulfoxide, and the
stabilizing components described previously.25 Incubation was for 10
min at 37 °C, a time selected so that the control reaction without
inhibitor was 40−60% complete.
Fluorescence-Activated Cell Sorting Analysis. OVCAR-8 cells
were grown at 37 °C in a humidified incubator in the presence of 5%
CO2. Cells were grown in Dulbecco’s modified Eagle’s medium
supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin G
and 100 μg/mL streptomycin (all provided by Gibco). Cells were
cultured for 24 h in a drug-free medium or supplemented with
ABBREVIATIONS USED
■
C A-4, combretastatin A-4; compds, compounds; DFG, Asp−
Phe−Gly; MW, microwave; i-PrOH, isopropanol; TEA,
triethylamine; THF, tetrahydrofuran; TK, tyrosine kinase
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