Novel 2-chloro-4-anilino-quinazoline derivatives as EGFR and VEGFR-2 dual inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.10.058

Novel 2-chloro-4-anilino-quinazolines designed as EGFR and VEGFR-2 dual inhibitors were synthesized and evaluated for inhibitory effects. EGFR and VEGFR-2 are validated targets in cancer therapy and combined inhibition might be synergistic for both antitumor activity and resistance prevention. The biological data obtained proved the potential of 2-chloro-4-anilino-quinazoline derivatives as EGFR and VEGFR-2 dual inhibitors, highlighting compound 8o, which was approximately 7-fold more potent on VEGFR-2 and approximately 11-fold more potent on EGFR compared to the prototype 7. SAR and docking studies allowed the identification of pharmacophoric groups for both kinases and demonstrated the importance of a hydrogen bond donor at the para position of the aniline moiety for interaction with conserved Glu and Asp amino acids in EGFR and VEGFR-2 binding sites.

Full text of DOI:10.1016/j.ejmech.2013.10.058

European Journal of Medicinal Chemistry 71 (2014) 1e14
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 2-chloro-4-anilino-quinazoline derivatives as EGFR and
VEGFR-2 dual inhibitors
,
,
Maria Letícia de Castro Barbosa a b, Lídia Moreira Lima a b, Roberta Tesch a,
Carlos Mauricio R. Sant’Anna c, Frank Totzke d, Michael H.G. Kubbutat d,
Christoph Schächtele d, Stefan A. Laufer e, Eliezer J. Barreiro a
,b,
*
a Laboratory of Evaluation and Synthesis of Bioactive Substances (LASSBio), Federal University of Rio de Janeiro, P.O. Box 68024, 21944-971 Rio de Janeiro,
RJ, Brazil1
b Graduate Program of Chemistry (PGQu), Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
c Department of Chemistry, Federal Rural University of Rio de Janeiro (UFRRJ), Seropédica, RJ, Brazil
d ProQinase GmbH, Freiburg, Germany
e Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Eberhard-Karls-University Tübingen, Tübingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 2-chloro-4-anilino-quinazolines designed as EGFR and VEGFR-2 dual inhibitors were synthesized
and evaluated for inhibitory effects. EGFR and VEGFR-2 are validated targets in cancer therapy and
combined inhibition might be synergistic for both antitumor activity and resistance prevention. The
biological data obtained proved the potential of 2-chloro-4-anilino-quinazoline derivatives as EGFR and
VEGFR-2 dual inhibitors, highlighting compound 8o, which was approximately 7-fold more potent on
VEGFR-2 and approximately 11-fold more potent on EGFR compared to the prototype 7. SAR and docking
studies allowed the identification of pharmacophoric groups for both kinases and demonstrated the
importance of a hydrogen bond donor at the para position of the aniline moiety for interaction with
conserved Glu and Asp amino acids in EGFR and VEGFR-2 binding sites.
Received 29 April 2013
Received in revised form
14 October 2013
Accepted 23 October 2013
Available online 31 October 2013
Keywords:
Quinazoline
EGFR
VEGFR-2
Antitumor
Cancer
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
In particular, the epidermal growth factor receptor (EGFR, also
called ErbB1, HER1) is a transmembrane receptor tyrosine kinase
Several cellular signaling pathways are involved in the evolu-
tion, aggressiveness and metastatic potential of malignant tumors.
These diverse mechanisms generate heterogeneity, redundancy
and the possibility for tumors to bypass a signal transduction
blockade resulting in primary or acquired resistance [1]. Therefore,
the multifactorial nature of cancer illustrates the need for multi-
functional therapeutic tools, e.g. single compounds that can
modulate different pathogenic pathways [2].
that belongs to the ErbB family [4], a four member family of tyro-
sine kinase growth factor receptors that include EGFR (HER1),
ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4) [2]. Binding of
a specific set of ligands/growth factors to the extracellular domain
of ErbB receptors promotes dimerization and autophosphorylation,
resulting in activation of the cytoplasmic tyrosine kinase domains
and leading to downstream signaling pathways that regulate cell
differentiation, growth, migration and apoptosis [4,5].
Protein kinases play important roles in the regulation of
numerous cellular processes, including proliferation, differentia-
tion and survival [3].
EGFR mediated signal transduction has been implicated in
several human malignances by promoting growth, local invasion,
angiogenesis and metastasis of tumor cells. EGFR overexpression
and/or mutation are clinical features of numerous solid tumors and
are usually associated with a poor prognosis [1,2,4]. EGFR signaling
pathways also stimulate vascular endothelial growth factor (VEGF),
which is the primary inducer of functional angiogenesis [6].
VEGF has also been described as the most common inducer of
tumor angiogenesis. VEGF promotes endothelial cell activation,
proliferation and migration and increases vascular permeability in
* Corresponding author. Laboratório de Avaliação e Síntese de Substâncias Bio-
ativas, Universidade Federal do Rio de Janeiro, P.O. Box 68024, 21944-971 Rio de
Janeiro, RJ, Brazil. Tel.: þ55 21 25626644; fax: þ55 21 25626478.
E-mail
addresses:
ejbarreiro@ccsdecania.ufrj.br,
ejbarreiro@gmail.com
(E.J. Barreiro).
1
http://www.farmacia.ufrj.br/lassbio/.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.058

2
M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
Fig. 1. Structural design of the target 2-chloro-4-anilino-quinazoline derivatives 8aep.
solid tumors. The key step in this process is mediated through a
specific VEGF receptor, which is the kinase insert domain-
containing receptor, also known as vascular endothelial growth
factor receptor 2 (VEGFR-2) [7].
of PD153035 (4), the most potent known inhibitor of EGFR
(IC50 ¼ 25 pM), have been developed through the insertion of
solubilizing groups in an attempt to improve their aqueous solu-
bility and, consequently, their oral bioavailability [4].
EGFR and VEGFR-2 are closely linked, sharing common down-
stream signal transduction pathways and playing key roles in tu-
mor growth and angiogenesis [1]. Their functional relationship in
cancer therapy is well known, i.e. inhibition of VEGFR-2 signaling
pathway contributes to the antitumoral effect of EGFR inhibitors;
whereas activation of VEGF expression independent of EGFR
signaling is thought to be one of the resistance mechanisms to anti-
EGFR therapy [1,8].
Therefore, tyrosine kinases EGFR and VEGFR-2 are validated
targets in cancer therapy and several inhibitors have been approved
by the FDA for clinical use in EGFR and/or VEGFR-2 overexpressing
solid tumors, including the ATP-mimetic tyrosine kinase inhibitors
(TKIs) (Chart 1) gefitinib (2) and lapatinib (3) for EGFR [2,3,5,6] and
sorafenib (5) [9] for VEGFR-2.
Secondary resistance following the initial benefits of treatment
with approved EGFR inhibitors remains a challenge in cancer
therapy and demonstrates the need for the development of novel
therapeutic alternatives [10]. Therefore, dual inhibition of EGFR and
VEGFR-2 represents a promising approach to cancer treatment
because VEGFR-2 inhibition increases the efficacy of EGFR TKIs by
exerting a synergistic effect [8]. Combined VEGFR-2 and EGFR in-
hibition is currently being evaluated in several preclinical and
clinical trials [8,10e12] and this novel therapeutic approach can be
exemplified by the dual tyrosine kinase inhibitor vandetanib (1)
(Chart 1), targeting EGFR and VEGFR-2, which was recently
approved in April 2011 by FDA for treatment of late-stage meta-
static medullary thyroid cancer [13].
Among EGFR TKIs either already approved by the FDA or
currently in clinical trials, the 4-anilinoquinazoline class of com-
pounds stands out. Many 4-anilinoquinazoline TKIs have been
described in the literature and generally show a potent inhibitory
effect in vitro at nanomolar concentrations. However, many of these
compounds show limited activity in vivo. Therefore, several analogs
Aiming to identify novel inhibitors with different structural
features, Abouzid and Shouman [14] recently described the syn-
thesis of a new class of 2-chloro-4-anilino-quinazoline compounds
designed as EGFR inhibitors and selected by virtual screening. In
particular, compound 7 (Fig. 1) exhibited a potent anti-proliferative
effect on the MCF-7 human breast carcinoma cell line over-
expressing EGFR (IC50 ¼ 0.13 nM). However, no inhibition data for
EGFR was reported. We evaluated compound 7 and found it to be
only a weak inhibitor of EGFR (Table 1).
Compound 7 differs from the first generation of EGFR inhibitors,
including those commercially available (2 and 3), by the presence of
a 2-chloro-quinazoline core containing a para-sulfonamide group
at the aniline moiety with a heterocyclic substituent instead of
ordinary halogenated rings. This provides several additional inter-
action points for binding to the therapeutic target, e.g. hydrogen
bond donor and acceptors.
Hydrogen bonds involving para-substituents in the aniline
moiety of EGFR inhibitors with the amino acids in the ATP binding
site of this tyrosine kinase have not been extensively explored.
However, this type of binding is known as an important feature for
the selectivity and potency of several urea VEGFR-2 TKIs [7,9,15],
such as sorafenib (5) and tivozanib (6) [9] (Chart 1), which form a
hydrogen bond between the urea NH groups and the side chains of
amino acids Glu855 and Asp1046 in the binding site of VEGFR-2.
Therefore, considering the great interest in associating EGFR and
VEGFR-2 inhibition for cancer therapy and noticing the presence of
structural features previously described as favorable for the inter-
action with VEGFR-2 binding site in compound 7 (Fig. 1) [14], this
compound was selected to evaluate this structural pattern as a basis
for the design of novel dual inhibitors of the tyrosine kinases EGFR
and VEGFR-2, which are structurally and clinically related.
Starting from the structure of compound 7, the 2-chloro-4-
anilino-quinazoline scaffold was preserved in the design concept

M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
3
Table 1
overall yield of 65% from the corresponding anthranilic acids 11a
and 11b as described by Hess [17]. By contrast, the 6,7-
methylenedioxy-2,4-dichloro-quinazoline 9c was prepared from
piperonilic acid (12) by regioselective nitration [18] followed by
classical functional group interconversions [19,20]. This resulted in
the anthranilamide 15, which was easily converted to the desired
6,7-methylenedioxy-2,4-quinazolinedione 10c with a yield of 89%
according to the procedure of Tollefson [21].
EGFRwt and VEGFR-2 inhibition by the 2-chloro-4-anilino-quinazoline derivatives 7
and 8aep.
The target 4-anilino-quinazoline compounds (8aep) were syn-
thesized by a key condensation step between the 2,4-dichloro-
quinazoline intermediates (9aec) and the corresponding aniline
derivatives through either a nucleophilic aromatic substitution
[14,22] or a BuchwaldeHartwig amination [23] (Scheme 2).
The mass spectra of the 2-chloro-4-anilino-quinazoline prod-
ucts 8aep revealed the isotopic clusters predicted for mono-
chlorinated molecules. The spectra displayed ions with a 3:1
relative abundance, which is the m/z expected for the 35Cl and 37Cl
isotopic ratio. Moreover, the chemical shift range observed for the
typical signal of the 4-anilino-quinazoline NH group from com-
pounds 8aep in the 1H NMR spectra, which were determined in
DMSO-d6 solution, varied from 9.55 to 10.42 ppm and is consistent
with the value of 10.19 ppm previously described for prototype 7
[14].
All 2-chloro-4-anilino-quinazoline derivatives (8aep) were
characterized by 1H and 13C nuclear magnetic resonance (NMR),
infrared spectroscopy and fast atom bombardment mass spec-
trometry (FAB-MS) and showed spectral data consistent with the
assigned structures.
Finally, whereas most of the anilines could be obtained from
commercial sources, the 4-aminobenzenesulfonamides 16a and
16b could be easily prepared in high yields by the synthetic
pathway depicted in Scheme 3. The 4-(acetylamino)benzene-
sulfonyl chloride 19 was synthesized as previously described [24].
The desired sulfonamides were produced via condensation of 19
with the corresponding amines followed by deprotection of the
amino group through alkaline hydrolysis of the acetamide moiety
[25].
Compound
R1, R2
Y
W
EGFRwta,b
IC50 M)
VEGFR-2a,b
IC50 M)
(m
(m
7c
OCH3, OCH3
OCH2O
SO2
SO2
9.70
7.79
8a
>100
>100
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8nc
8o
8p
OCH3, OCH3
OCH2O
H, H
OCH3, OCH3
OCH2O
OCH3, OCH3
OCH2O
SO2
SO2
SO2
SO2
SO2
SO2
SO2
e
N(CH3)2
N(CH3)2
N(CH3)2
CH3
CH3
NH2
67.2
18.3
>100
61.5
>100
2.37
43.3
23.4
>100
>100
>100
1.02
26.9
39.3
>100
0.85
2.10
>100
>100
1.17
1.99
NH2
34.6
36.0
OCH3, OCH3
OCH2O
N(CH3)2
N(CH3)2
NHCH3
OH
OH
OH
e
>100
1.63
4.30
>100
>100
0.90
37.6
OCH3, OCH3
OCH3, OCH3
OCH3, OCH3
OCH3, OCH3
OCH3, OCH3
OCH3, OCH3
SO2
e
SO2
C]O
C]O
e
NH2
NHCOCH3
a
A radiometric protein kinase assay (33PanQinaseÒ Activity Assay) was used to
measure the kinase activity of the protein kinases EGFRwt and VEGFR-2.
b
The IC50 values were calculated using Quattro Workflow V3.1.0 (Quattro
Research GmbH, Munich, Germany; www.quattroresearch.com) and are in mM.
c
Previously described by Abouzid & Shouman, Bioorganic & Medicinal Chemistry
16 (2008) 7543e7551.
2.2. EGFR and VEGFR-2 inhibition
of the novel series of 8aep derivatives (Fig. 1). Initial structural
modifications were performed in the substitution pattern of the
pharmacophoric heterocyclic system in fragment A, including the
removal of oxygenated substituents and the incorporation of both
6- and 7-methoxy groups into a dioxolane ring, employing the ring
closure strategy, based on positive results described by Chilin and
colleagues, who have studied the role of fused dioxygenated rings
on the quinazoline scaffold of EGFR inhibitors [4] (Fig. 1).
The para-substituent of the aniline moiety (fragment B) was
varied, containing whether or not a sulfone or carbonyl group (Y),
which are hydrogen bond acceptor sites. Furthermore, the W sub-
stituents, including acid groups, hydrogen bond donors or accep-
tors, and alkyl or aryl groups, provided structural diversity in the
search for suitable pharmacophoric points of interaction with the
selected targets (Fig. 1).
Compound 7 was initially synthesized as previously reported
[14]. The inhibitory effect of compound 7 on the tyrosine kinase
activity of the selected targets was compared with the new 2-
chloro-4-anilino-quinazoline derivatives 8aep.
Compound 7 was previously identified as an EGFR inhibitor
candidate by virtual screening and displayed a significant anti-
proliferative effect on the MCF-7 human breast carcinoma cell
line overexpressing EGFR with an IC50 of 0.13 nM [14]. However, 7
exhibited only weak inhibition of both EGFR (IC50 ¼ 9.70
mM) and
VEGFR-2 (IC50 ¼ 7.79 M) (Table 1), indicating that an alternative
m
mechanism of action may be involved in the anti-proliferative ac-
tivity of this compound described by Abouzid and Shouman [14].
By contrast, 7 can modulate the tyrosine kinase activity of the
selected targets with similar potencies (Table 1). This finding sug-
gests that optimization of the inhibitory effect observed for 7 pro-
vides a tool for the development of a new series of EGFR and
VEGFR-2 dual inhibitors.
2. Results and discussion
2.1. Chemistry
As shown in Table 1, a direct correlation between the inhibitory
potencies against EGFR and VEGFR-2 was generally observed
except for the acetamide derivative 8p (LASSBio-1820;
The synthesis of the designed 4-anilino-quinazoline derivatives
8aep employed the corresponding 2,4-dichloro-quinazolines 9aec
as key intermediates (Scheme 2). These intermediates were ob-
tained from the 2,4-quinazolinedione precursors 10aec through
the synthetic methodology depicted in Scheme
Dichloro-quinazolines 9a and 9b were both synthesized with an
IC50 ¼ 37.6
mM for EGFRwt and 1.99 mM for VEGFR-2), which was
almost 20 times more potent for VEGFR-2 inhibition (Table 1).
Moreover, the loss of inhibitory effect on both tyrosine kinases
for compound 8d compared to analogs 8b and c indicated the
relevance of the dioxygenated quinazoline scaffold for interaction
1 [16]. 2,4-

4
M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
Scheme 1. Reagents and conditions: a) (1) AcOH, H2O, 35 ꢀC, 150 (2) KOCN, H2O, 35 ꢀC, 300 (3) NaOH (4) HClaq 37%, r.t., 85e87%; b) conc. HNO3, cat. H2SO4, 0 ꢀC, 2 h, 67%; c) (1) oxalyl
chloride, CH2Cl2, cat. DMF, r.t., 2 h (2) NH3 7 N in methanol, r.t., 200, 100%; d) Fe0, NH4Cl, EtOH, H2O, 78 ꢀC, 12 h, 85%; e) CDI, THF, 66 ꢀC, 12 h, 89%; f) POCl3, 100 ꢀC, 24 h, 71e77%.
with the ATP binding site (Table 1). On the other hand, replacement
of the 6,7-dimethoxy substituent by a dioxolane ring was unfa-
vorable for interaction with the enzymes binding sites in four of five
evaluated cases, resulting in a loss of activity for three compounds,
8a, f and j, compared to the corresponding 6,7-dimethoxy-analogs,
7, 8e and i.
suggests that the acidity or hydrogen bond donating property of the
primary sulfonamide is an important structural feature.
Results for the methyl-sulfonamide 8k (LASSBio-1816;
IC50 ¼ 1.63
mM for EGFRwt and 0.85 mM for VEGFR-2) have shown
equivalent potencies in inhibition compared to the primary sul-
fonamide 8g. Additionally, it is possible to conclude that the ability
to bind the active site of the structurally related tyrosine kinases
EGFR and VEGFR-2 does not depend on the acidity of the para
substituent because the primary amide 8o (LASSBio-1819;
Those derivatives containing a hydrogen bond donor at the para
position of the aniline moiety presented lower IC50 values, high-
lighting compounds 8g (LASSBio-1814; IC50 ¼ 2.37
and 1.02
M for VEGFR-2), 8k (LASSBio-1816; IC50 ¼ 1.63
EGFRwt and 0.85 for VEGFR-2) and 8o (LASSBio-1819;
M for EGFRwt and 1.17 M for VEGFR-2) as dual in-
mM for EGFRwt
m
mM for
IC50 ¼ 0.90
mM for EGFRwt and 1.17 mM for VEGFR-2) displayed a
mM
significant inhibitory effect on both tyrosine kinases, whereas the
corresponding sulfonic (8m) and carboxylic (8n) acids were not
inhibitors.
IC50 ¼ 0.90
m
m
hibitors of both the EGFR and VEGFR-2 tyrosine kinases.
The primary sulfonamide 8g (LASSBio-1814; IC50 ¼ 2.37
m
M for
Therefore, the biological data have demonstrated the relevance
of a hydrogen bond donating substituent at the para position of the
aniline (Table 1) for interaction with the EGFR and VEGFR-2 tyro-
sine kinase domain binding sites.
EGFRwt and 1.02
m
M for VEGFR-2) has increased potency compared
to the corresponding dimethyl-sulfonamide 8b (IC50 ¼ 67.2
EGFRwt and 43.3
(IC50 ¼ 61.5 M for EGFRwt and >100
m
M for
m
M
for VEGFR-2) and methylsulfone 8e
M for VEGFR-2). This result
m
m
2.3. Molecular docking
A docking study of this new class of ligands with the tyrosine
kinase domain of EGFRwt (Fig. 2) was performed to elucidate the
molecular reasons behind the observed inhibition profile, including
which amino acids in the EGFR active site would be involved in the
anticipated hydrogen bonding interactions.
According to the results obtained with the semi-rigid docking
approach, the novel 2-chloro-4-anilino-quinazoline compounds
interact in the EGFR ATP binding site with the amino acid Met793
through a hydrogen bonding, adopting an orientation similar to
that of the co-crystallized drug gefitinib (2) in the reference crys-
tallographic structure (PDB: 2ity), as shown in Fig. 2A for the pri-
mary sulfonamide 8g.
Scheme 2. Reagents and conditions: g) DIPEA, dioxane, 80 ꢀC, 12 h, 60e66%; h) iso-
propyl alcohol, 82 ꢀC, 24 h, 67e72%; i) ethanol, 78 ꢀC, 24 h, 64e73%; j) Pd(oAc)2, XPhos,
t
tBuONa, BuOH, toluene, 90 ꢀC, 1 h, 45e55%.
Furthermore, for all evaluated ligands, hydrophobic interactions
were observed with amino acids in the active site of EGFR,
including Leu718, Val726, Leu792 and Leu844 (Fig. 2BeF).
As shown in Fig. 2, the comparable potencies of the dimethyl-
sulfonamide 8b (IC50 ¼ 67.2
mM; Fig. 2C) and the methylsulfone
8e (IC50 ¼ 61.5 M; Fig. 2D) against EGFR could be explained by
m
similar binding interactions, including those mentioned above and
a hydrogen bond between the sulfone group and amino acid
Lys745.
As previously suggested, the increased potency of compound 8g
of almost 30-fold against EGFR (IC50 ¼ 2.37
mM) compared to 8b
and e could be related to the hydrogen bond donating ability of the
primary sulfonamide, which establishes additional interactions
with the side chains of amino acids Glu762 and Asp855. Moreover,
Scheme 3. Reagents and conditions: k) acetic anhydride, glacial acetic acid, CH3CO2Na
anhydrous, r.t., 300, 90%; l) HSO3Cl, 60 ꢀC, 300, 85%; m) R0NHCH3 (R0 ¼ H or CH3), CH2Cl2,
r.t., 300, 70e95%; n) KOH, H2O, MeOH, 65 ꢀC, 24 h, 73e75%.

M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
5
Fig. 2. (A) Superposition of 8g (yellow) and gefitinib (2, purple) poses inside the EGFR tyrosine kinase ATP binding site. (B) Binding interactions of 8g (yellow) with EGFRwt. (C)
Binding interactions of 8b (magenta) with EGFRwt. (D) Binding interactions of 8e (green) with EGFRwt. (E) Binding interactions of 8k (blue) with EGFRwt. (F) Binding interactions of
8o (light pink) with EGFRwt. Docking studies were performed with the GOLD 5.1 program. Apolar hydrogen atoms were omitted to improve clarity. The images were generated with
PyMol software.
the sulfone group of 8g forms a hydrogen bond with the eNHe
from the Asp855 backbone, and there is a catione interaction
between the aniline aromatic ring and Lys745 side chain (Fig. 2B).
For the secondary sulfonamide derivative 8k (IC50 ¼ 1.63 M),
which had a potency similar to the primary sulfonamide 8g
M), the hydrogen bond interaction with the side chain
of Asp855 was maintained; additionally, the hydrogen bond
formed between the sulfone group of 8g and the Asp855 backbone
was replaced by a stronger bond between the same group and the
Lys745 side chain in 8k (Fig. 2E).
According to results shown in Fig. 3, the novel 2-chloro-4-
anilino-quinazoline derivatives interact in VEGFR-2 ATP binding
site with the amino acid Cys919 eNHe backbone through hydrogen
bonding, adopting an orientation similar to the co-crystallized
tivozanib (6) in the reference crystallographic structure (PDB:
4ase), as shown in Fig. 3A for the primary sulfonamide 8g.
Moreover, all tested ligands have shown interaction modes
analogous to those described above for EGFR binding. These include
hydrophobic interactions with corresponding amino acids in the
VEGFR-2 binding site, i.e. Leu840, Val848, Phe918 and Leu1035,
and several hydrogen bond interactions illustrated in Fig. 3 for
compounds 8g, k and o (Fig. 3BeE).
p
m
(IC50 ¼ 2.37
m
Finally, for the primary amide 8o (IC50 ¼ 0.90
mM), which
showed the greatest inhibitory effect, a catione interaction be-
p
tween the aniline aromatic ring and the amino acid Lys745 was
detected as well as an optimal geometry for the formation of
hydrogen bonds between the eNH2 of the amide group and the
Glu762 and Asp855 side chains (Fig. 2F).
Subsequently, the interaction modes of the identified dual in-
hibitors were also studied by molecular docking with VEGFR-2
(Fig. 3).
The primary sulfonamide 8g (IC50 ¼ 1.02
mM) formed a hydrogen
bond with the side chain of Glu855. Furthermore, the peptidic e
NHe from the Asp1046 backbone formed a hydrogen bond with the
sulfone group of 8g, and the C]O from the Asp1046 backbone
formed a hydrogen bond with the eNH2 of 8g. These findings
support results previously described in the literature [7,9,15] that
indicate the relevance of hydrogen bond interactions with these

6
M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
Fig. 3. (A) Superposition of 8g (yellow) with tivozanib (6, orange) inside the VEGFR-2 tyrosine kinase ATP binding site. (B) Binding interactions of 8g (yellow) with VEGFR-2. (C)
Binding interactions of 8k (blue) with VEGFR-2 showing the best score value (Score: 71.82). (D) Binding interactions of 8k (blue) with VEGFR-2 showing the possibility of a hydrogen
bond with the sulfonamide eNHe as a donor (Score: 69.29). (E) Binding interactions of 8o (light pink) with VEGFR-2. Docking studies were performed with the GOLD 5.1 program.
Apolar hydrogen atoms and the Phe918 residue were omitted to improve clarity. The images were generated with PyMol software.
amino acids for VEGFR-2 potent inhibition. A catione
between the aniline aromatic ring and Lys868 was detected, as well
as an additional edge-to-face -stacking interaction with Phe1047
M), hydrogen
bonds between the eNH2 of the amide and the Glu855 and
Asp1046 side chains were also observed (Fig. 3E).
Notably, for the secondary sulfonamide 8k two hydrogen bond
interactions between the sulfone group as hydrogen acceptor and
the amino acids Lys868 and Asp1046 were favored in the semi-
rigid docking model (Fig. 3C; Score value: 71.82), although other
poses of 8k in the binding site with lower score values have shown
the possibility of hydrogen bonds with the sulfonamide eNHe as a
donor (Fig. 3D; Score value: 69.29).
p
interaction
interaction with the enzymes binding sites in four of five evaluated
cases, resulting in a loss of activity for three compounds, 8a, f and j,
compared to the corresponding 6,7-dimethoxy-analogs, 7, 8e and i.
According to results shown in Fig. 4, the comparison between
the binding modes of the 6,7-dimethoxy-derivative 8g (LASSBio-
p
(Fig. 3B). For the primary amide 8o (IC50 ¼ 1.17
m
1814; IC50 ¼ 2.37
6,7-methylenedioxy-derivative 8h (LASSBio-1815; IC50 ¼ 34.6
for EGFRwt and 26.9 M for VEGFR-2) demonstrates that the
mM for EGFRwt and 1.02 mM for VEGFR-2) and the
mM
m
structural modification performed has led to the loss of important
hydrophobic interaction with Val726, Leu718 and Leu792 amino
acid residues in EGFRwt binding site; and with Val848, Leu840 and
Phe918 amino acid residues in VEGFR-2 binding site.
Finally, the docking studies have indicated the molecular rea-
sons for the observed decrease of the inhibitory potencies as a
consequence for the replacement of the 6,7-dimethoxy sub-
stituents by a dioxolane ring in the quinazoline scaffold. As already
mentioned, this structural modification was unfavorable for
3. Conclusion
This study has described the synthesis and biological testing of a
novel series of 2-chloro-4-anilino-quinazolines as EGFR and
VEGFR-2 dual inhibitors. The associated modulation of these two

M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
7
Fig. 4. (A) Binding interactions of 8g (yellow) with EGFRwt. (B) Binding interactions of 8h (gray) with EGFRwt. (C) Binding interactions of 8g (yellow) with VEGFR-2. (D) Binding
interactions of 8h (gray) with VEGFR-2. Docking studies were performed with the GOLD 5.1 program. Apolar hydrogen atoms were omitted to improve clarity. The images were
generated with PyMol software.
tyrosine kinases represents a promising therapeutic approach to
overcome and prevent resistance in cancer therapy due to a syn-
ergistic effect.
Moreover, this study identified pharmacophoric groups for
binding to the selected therapeutic targets and demonstrated the
importance of a hydrogen bond donor at the para position of the
aniline moiety for interaction with the conserved Glu and Asp
amino acids in the EGFR and VEGFR-2 binding sites, which pro-
motes a significant increase in potency.
The structural modifications performed optimized the initial
inhibitory activities found for the prototype 7 [14] with the analogs
8g, k, l and o, highlighting compound 8o, approximately 7-fold
more potent in inhibition of VEGFR-2 and approximately 11-fold
more potent in inhibition of EGFR compared to this prototype.
by thin-layer chromatography (TLC) in silica gel (F245 Merck
plates) and the products visualized with ultraviolet lamp (254 and
365 nm). 1H and 13C nuclear magnetic resonance (NMR) spectra
were determined in DMSO-d6, D2O or CDCl3 solutions using a
Bruker AC-200 or a Bruker Avance 400 spectrometer. The chemical
shifts are given in parts per million (d) from solvent residual peaks
and the coupling constant values (J) are given in Hz. Signal multi-
plicities are represented by: s (singlet), d (doublet), dd (double
doublet), t (triplet), dt (double triplet), q (quadruplet), qu (quin-
tuplet), m (multiplet) and br (broad signal). Infrared spectra were
obtained using a Thermo Nicolet’s Avatar 330 FT-IR spectrometer
equipped with smart endurance diamond ATR unit for direct
measurements. Mass spectra were obtained from a Hewlette
Packard HP 6890 series GC-system equipped with a HP-5MS
capillary column (0.25
m
M film thickness 30 m ꢁ 0.25 mm) and a
4. Experimental section
HewlettePackard HP 5973 mass selective detector (70 eV). FAB-MS
(fast atom bombardment mass spectrometry) data were obtained
from the Department for Mass Spectrometry, Institute of Organic
Chemistry, Eberhard-Karls-University of Tübingen. Melting points
were determined using a Büchi Melting Point B-545 and are un-
corrected. Flash chromatography was performed using a LaFlash
4.1. Synthesis and characterization of compounds
4.1.1. General
All commercially available reagents and solvents were used
without further purification. Reactions were routinely monitored
system (VWR) with Merck silica gel (25e40 mm). The purity of the

8
M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
Chart 1. Several inhibitors of EGFR and VEGFR tyrosine kinases.
testing compounds was determined by HPLC (Merck Hitachi L-
6200 intelligent pump, Merck Hitachi AS-2000 autosampler, Merck
Hitachi L-4250 UVevis detector) using a ZORBAX Eclipse XDB C8
column (5 mm), employing a gradient of 0.01 M KH2PO4 (pH 2.3)
and methanol as solvent system with a flow rate of 1.0 mL/min and
detection at 230 and 254 nm.
All testing compounds, i.e. 2-chloro-4-anilino-quinazoline de-
rivatives (8aep), were characterized by 1H and 13C nuclear mag-
netic resonance (NMR), infrared spectroscopy and fast atom
bombardment mass spectrometry (FAB-MS), showing spectral data
according to the assigned structures. Compounds 8aep presented
>95% purity determined by HPLC.
4.1.4. 6-Nitrobenzo[d][1,3]dioxole-5-carboxylic acid (13)
In 100 mL flask containing piperonylic acid (2.00 g,
a
12.04 mmol), concentrated nitric acid (15.17 g, 10 mL, 240.78 mmol)
was carefully added under magnetic stirring at 0 ꢀC. Next, 16 drops
concentrated sulfuric acid were slowly added resulting in a gradual
change of color from colorless to yellow. After 2 h, compound
isolation was performed by adding crushed ice to the flask resulting
in the precipitation of a yellow solid, which was filtered and
washed with cold water. The product was purified by stirring in hot
hexane and filtering, giving a yellow solid with a yield of 67% yield,
m.p. 167 ꢀC (dec). The melting point, 1H NMR and 13C NMR data for
compound 13 are consistent with previous reports [29,30].
4.1.2. Quinazoline-2,4(1H,3H)-dione (10a)
4.1.5. 6-Nitrobenzo[d][1,3]dioxole-5-carboxamide (14)
In a 100 mL flask equipped with a reflux condenser, a suspension
of anthranilic acid (0.82 g, 5.98 mmol) in distilled water (36 mL)
and glacial acetic acid (0.7 mL) was stirred and heated at 35 ꢀC for
15 min. Next, potassium cyanate (1.21 g, 14.92 mmol) was dissolved
in water and then slowly added to the reaction medium, which was
constantly stirred at 35 ꢀC for 30 min. Subsequently, sodium hy-
droxide (10.68 g, 267 mmol) was carefully added. The reaction
medium was cooled to room temperature and the pH was adjusted
to 4.0 with concentrated HCl. The white solid precipitate was
filtered and washed with cold water, resulting in the desired
compound with a yield of 87% and m.p. >300 ꢀC. The melting point,
1H NMR and 13C NMR data for compound 10a are consistent with
previous reports [26,27].
In a 250 mL flask, compound 13 (1.00 g, 4.74 mmol) was dis-
solved in dichloromethane (50 mL). Next, oxalyl chloride (0.66 g,
477 ml, 5.21 mmol) and 3 drops of dimethylformamide were added.
The resulting solution was stirred at room temperature for 2 h.
Subsequently, the second step was performed in one pot through
the addition of 2.0 mL 7 N solution of ammonia in methanol. The
reaction medium was stirred at room temperature for 20 min, and
completion of the reaction was detected by TLC. The isolation was
performed by adding hexane to the reaction medium and filtering
the precipitate, which resulted in the desired product with a yield
of 100% at the completion of both reaction steps. The product was a
light yellow solid with m.p. of 192 ꢀC. The melting point for com-
pound 14 is consistent with previous reports [31].
1H NMR (200 MHz, DMSO-d6)
d (ppm): 6.26 (s, 2H, OCH2O); 7.12
4.1.3. 6,7-Dimethoxy-quinazoline-2,4(1H,3H)-dione (10b)
(s, 1H, H6); 7.58 (br, 1H, D2O exchangeable, RCONHaHb); 7.62 (s, 1H,
H3); 7.96 (br, 1H, D2O exchangeable, RCONHaHb).
d (ppm): 103.6 (OCH2O); 104.7
(C6); 107.7 (C3); 129.3 (C5); 141.3 (C4); 148.0 (C2); 151.1 (C1); 166.8
(CONH2).
Compound 10b was prepared according to the procedure
described above for the synthesis of 10a with a yield of 85% and
m.p. >300 ꢀC. The melting point, 1H NMR and 13C NMR data for
compound 10b are consistent with previous reports [28].
13C NMR (50 MHz, DMSO-d6)

M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
9
IR (ATR: cmꢂ1): 3362, 3179, 2921, 1653, 1526, 1347.
13C NMR and DEPT 135 (100 MHz, CDCl3)
d
(ppm): 56.6 (OCH3);
56.9 (OCH3); 102.9 (C5); 106.4 (C8); 118.0 (C4a); 150.7 (C8a); 151.8
(C6); 153.8 (C2); 158.0 (C4); 160.3 (C7).
4.1.6. 6-Aminobenzo[d][1,3]dioxole-5-carboxamide (15)
In a 250 mL flask equipped with a reflux condenser, the reaction
mixture containing 14 (2.00 g, 9.52 mmol), metallic iron dust
(3.72 g, 66.62 mmol), ammonium chloride (1.02 g, 19.03 mmol),
100 mL ethanol and 20 mL distilled water was stirred and heated at
78 ꢀC for 12 h. Completion of the reaction was verified by TLC. The
product was isolated by filtration on CeliteÒ followed by washing
with ethanol. Subsequently, the ethanol was concentrated under
reduced pressure and crushed ice was added to the flask resulting
in the precipitation of a dark yellow solid, which was filtered and
washed with cold water, resulting in the desired compound with a
yield of 85% and m.p. of 172e173 ꢀC. The melting point for com-
pound 15 is consistent with previous reports [31].
IR (ATR: cmꢂ1): 3023, 2978, 2945, 1610, 1552, 1507, 1459, 747.
GCeMS (EI): t.r. ¼ 13.3 min; m/z 258 [M]þ (100%); 260 [M þ 2]þ
(64%); 262 [M þ 4]þ (10%).
4.1.8.3. 2,4-Dichloro-6,7-methylenedioxy-quinazoline
(9c).
Compound 9c was obtained from 10c as a white powder with a
yield of 71% after purification and m.p. of 217e219 ꢀC. The melting
point for compound 9c is consistent with previous reports [17].
1H NMR (400 MHz, CDCl3)
1H, H5); 7.44 (s, 1H, H8).
d
(ppm): 6.24 (s, 2H, OCH2O); 7.24 (s,
13C NMR and DEPT 135 (100 MHz, CDCl3)
d
(ppm): 101.2 (C5);
103.3 (OCH2O); 104.7 (C8); 119.6 (C4a); 150.0 (C8a); 152.5 (C6);
154.1 (C2); 156.0 (C4); 160.9 (C7).
1H NMR (200 MHz, DMSO-d6)
d (ppm): 5.88 (s, 2H, OCH2O); 6.27
(s, 1H, H3); 6.63 (br, 2H, D2O exchangeable, RCONH2); 7.13 (m, 3H,
H6, ArNH2).
IR (ATR: cmꢂ1): 3053, 2921, 1607, 1556, 1468, 1420, 715.
GCeMS (EI): t.r. ¼ 12.6 min; m/z 242 [M]þ (100%); 244 [M þ 2]þ
(64%); 246 [M þ 4]þ (10%).
13C NMR (50 MHz, DMSO-d6)
d (ppm): 96.6 (C3); 100.6 (OCH2O);
104.4 (C6); 107.0 (C5); 137.3 (C1); 148.2 (C4); 150.5 (C2); 170.8
(CONH2).
4.1.9. N-Phenyl-acetamide (18)
IR (ATR: cmꢂ1): 3380, 3284, 3189, 2910, 1644, 1618.
A solution of 2.1 g (25.6 mmol) anhydrous sodium acetate in
8.0 mL (139.9 mmol) glacial acetic acid was prepared. Aniline
(17) (8.0 mL, 87.8 mmol) was slowly added to the solution fol-
lowed by 8.5 mL (90.1 mmol) acetic anhydride. The reaction
mixture was stirred for 30 min at room temperature and was
then poured into a water and ice mixture. The N-phenyl-acet-
amide (18) was then filtered through a Büchner funnel and
washed twice with 200 mL water, resulting in a white shiny
powder with a yield of 90% and m.p. of 116 ꢀC. The melting point,
1H NMR and IR data for compound 18 are consistent with pre-
vious reports [34,35].
4.1.7. 6,7-Methylenedioxy-quinazoline-2,4(1H,3H)-dione (10c)
In a 100 mL flask equipped with a reflux condenser, the reaction
mixture containing 15 (1.00 g, 5.55 mmol),1,10-carbonyldiimidazole
(CDI) (1.08 g, 6.66 mmol) and 20 mL anhydrous THF was stirred and
heated at 66 ꢀC for 12 h under an argon atmosphere. Completion of
the reaction was confirmed by TLC. The product was isolated by the
addition of crushed ice to the flask resulting in the precipitation of a
white solid, which was filtered and washed with cold water,
resulting in the desired compound with a yield of 89% and m.p.
>300 ꢀC. The melting point for compound 10c is consistent with
previous reports [17].
4.1.10. 4-(Acetylamino)-benzenesulfonyl chloride (19)
1H NMR (200 MHz, DMSO-d6)
d
(ppm): 6.11 (s, 2H, OCH2O); 6.64
Chlorosulfonic acid (5.2 mL, 78.27 mmol) was slowly added to
2.0 g (14.80 mmol) N-phenyl-acetamide (18). The resulting mixture
was stirred and heated at 60 ꢀC for 30 min. After cooling, the re-
action medium was poured into a water and ice mixture. The 4-
(acetylamino)-benzenesulfonyl chloride (19) was then filtered
through a Büchner funnel and washed twice with 100 mL water
resulting in a white powder with a yield of 85% and m.p. of 143e
148 ꢀC. The melting point and IR data for compound 19 are
consistent with previous reports [36,37].
(s, 1H, H8); 7.21 (s, 1H, H5); 11.00 (br, 1H, D2O exchangeable, H3);
11.16 (br, 1H, D2O exchangeable, H1).
13C NMR (50 MHz, DMSO-d6)
d (ppm): 95.3 (C8); 102.3 (OCH2O);
104.0 (C5); 107.6 (C4a); 138.2 (C8a); 143.6 (C6); 150.2 (C7); 153.2
(C2); 162.1 (C4).
IR (ATR: cmꢂ1): 3141, 3083, 3009, 2954, 1725, 1673, 1626, 1497,
1453.
4.1.8. General methodology for the synthesis of 2,4-
dichloroquinazolines
4.1.11. General procedure for the synthesis of sulfonamides 20a and
b
In a 100 mL flask equipped with a reflux condenser, the reaction
mixture containing the quinazolinediones (1.00 g) and POCl3
(15 mL; 24.77 g; 161.53 mmol) was stirred and heated at 100 ꢀC for
24 h. Isolation was performed by the slow addition of the reaction
medium over a mixture of ice and water with vigorous stirring. The
resulting precipitate was filtered under a vacuum and purified by
filtration through silica gel using dichloromethane as the eluent.
The corresponding amines (5.0 mmol) were added to a solution
of sulfonylchloride derivative 19 (500 mg, 2.14 mmol) in 50 mL
methylene chloride. The reaction mixture was stirred for 30 min at
room temperature, and completion of the reaction was confirmed
by TLC. The sulfonamides were isolated by the addition of 50 mL
methylene chloride and extraction followed by washing of the
organic layer with an aqueous solution of HCl (pH ¼ 3). The organic
layer was then dried over anhydrous sodium sulfate, filtered and
concentrated at reduced pressure.
4.1.8.1. 2,4-Dichloroquinazoline (9a). Compound 9a was obtained
from the quinazolinedione 10a as a white powder with a yield of
75% after purification and m.p. of 117e120 ꢀC. The melting point, 1H
NMR and 13C NMR data for compound 9a are consistent with
previous reports [27,32].
4.1.11.1. N-[4-(Methylamino-4-ylsulfonyl)phenyl]acetamide
(20a).
Compound 20a was obtained via condensation of 19 with
methylamine (2 M solution in THF) resulting in a white shiny
powder with a 70% yield and m.p. of 185 ꢀC. The melting point for
compound 20a is consistent with previous reports [38].
4.1.8.2. 2,4-Dichloro-6,7-dimethoxyquinazoline (9b).
Compound 9b was obtained from 10b as a light pink powder with a
yield of 77% after purification and m.p. of 172 ꢀC. The melting point
for compound 9b is consistent with previous reports [33].
1H NMR (400 MHz, DMSO-d6)
d (ppm): 2.08 (s, 3H, NHCOCH3);
2.38 (d, 3H, J ¼ 4.9 Hz, RSO2NHCH3); 7.28 (q, 1H, D2O exchangeable,
J ¼ 4.9 Hz, RSO2NHCH3); 7.69 (d, 2H, J ¼ 8.8 Hz, H3 and H5); 7.78 (d,
2H, J ¼ 8.8 Hz, H2 and H6); 10.31 (s, 1H, NHCOCH3).
1H NMR (400 MHz, CDCl3)
d
(ppm): 4.07 (s, 3H, OCH3); 4.08 (s,
3H, OCH3); 7.27 (s, 1H, H5); 7.35 (s, 1H, H8).

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M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
13C NMR and DEPT-135 (100 MHz, DMSO-d6)
d
(ppm): 24.0
were combined, washed with saturated saline solution, and dried
over anhydrous Na2SO4. The solvent was removed under vacuum,
and the residue was purified by recrystallization from a mixture
of dichloromethane and hexane.
(NHCOCH3); 28.6 (RSO2NHCH3); 118.6 (C3 and C5); 127.7 (C2 and
C6); 132.9 (C1); 142.7 (C4); 168.9 (NHCOCH3).
IR (ATR: cmꢂ1): 3317, 3159, 3055, 2980, 2918, 1682, 1586, 1528,
1492, 1456, 1316, 1151, 836.
4.1.13.1. 4-((2-Chloro-6,7-dimethoxyquinazolin-4-yl)amino)-N,N-
dimethyl benzenesulfonamide (8b). Compound 8b was synthesized
via condensation of 9b with 16b resulting in a yellow powder
with a 53% yield after precipitation from a mixture of dichloro-
methane and hexane, and the m.p. was 258e259 ꢀC.
4.1.11.2. N-[4-(Dimethylamino-4-ylsulfonyl)phenyl]acetamide (20b).
Compound 20b was obtained via condensation of 19 with dime-
thylamine (2 M solution in THF) resulting in a beige solid with a 95%
yield and m.p. of 143 ꢀC. The melting point for compound 20b is
consistent with previous reports [39].
1H NMR (400 MHz, DMSO-d6)
d (ppm): 2.64 (s, 6H,
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 2.09 (s, 3H, NHCOCH3);
RSO2N(CH3)2); 3.93 (s, 3H, OCH3); 3.96 (s, 3H, OCH3); 7.20 (s, 1H,
H8); 7.81 (d, 2H, J ¼ 8.5 Hz, H30 and H50); 7.87 (s, 1H, H5); 8.08 (d,
2H, J ¼ 8.5 Hz, H20 and H60); 10.04 (s, 1H, NH).
2.57 (s, 6H, RSO2N(CH3)2); 7.68 (d, 2H, J ¼ 8.6 Hz, H3 and H5); 7.82
(d, 2H, J ¼ 8.6 Hz, H2 and H6); 10.37 (s, 1H, NHCOCH3).
13C NMR and DEPT-135 (100 MHz, DMSO-d6)
d
(ppm): 24.1
13C NMR and DEPT 135 (100 MHz, DMSO-d6)
d (ppm): 37.7
(NHCOCH3); 37.5 (RSO2N(CH3)2); 118.6 (C3 and C5); 128.2 (C1);
128.6 (C2 and C6); 143.3 (C4); 169.0 (NHCOCH3).
IR (ATR: cmꢂ1): 3305, 3256, 3060, 2924, 1668, 1589, 1536, 1494,
1404, 1317, 1158, 833.
(RSO2N(CH3)2); 56.1 (OCH3); 56.3 (OCH3); 102.1 (C8); 106.7 (C5);
107.5 (C4a); 121.5 (C30 and C50); 128.4 (C20 and C60); 129.0 (C10);
143.1 (C40); 148.6 (C8a); 149.3 (C6); 153.7 (C2); 155.3 (C7); 157.4
(C4).
IR (ATR: cmꢂ1): 3398, 2935, 2828, 1597, 1572, 1512, 1420, 1327,
1234, 1149, 838, 723.
4.1.12. General procedure for deprotection of the amino group
A solution of potassium hydroxide (1.62 g, 28.89 mmol) in
5 mL water was slowly added to a solution of the corresponding
acetamide intermediate (4 mmol) in 25 mL methanol. The re-
action mixture was stirred and heated at 65 ꢀC for 24 h.
Completion of the reaction was verified by TLC. The aniline
derivative was isolated by concentration at reduced pressure
followed by filtration of the precipitate through a Büchner
funnel.
MS: posFAB: m/z 423 [M þ 1]þ; 425 [M þ 1 þ 2]þ.
4.1.13.2. 4-((2-Chloro-6,7-methylenedioxy-quinazolin-4-yl)amino)-
N,N-dimethylbenzenesulfonamide (8c). Compound 8c was synthe-
sized via condensation of 9c with 16b resulting in a yellow powder
with a 45% yield after precipitation from a mixture of dichloro-
methane and hexane, and the m.p. was 245e247 ꢀC.
1H NMR (400 MHz, DMSO-d6)
d (ppm): 2.63 (s, 6H,
RSO2N(CH3)2); 6.29 (s, 2H, OCH2O); 7.22 (s, 1H, H8); 7.79 (d, 2H,
J ¼ 8.7 Hz, H30 and H50); 8.04 (s, 1H, H5); 8.10 (d, 2H, J ¼ 8.7 Hz, H20
and H60); 9.96 (s, 1H, NH).
4.1.12.1. 4-(Methylamino-4-ylsulfonyl)aniline (16a). Compound 16a
was obtained from alkaline hydrolysis of the acetamide precursor
20a resulting in a beige solid with a 75% yield and m.p. of 107e
108 ꢀC. The melting point for compound 16a is consistent with
previous reports [40].
13C NMR and DEPT 135 (100 MHz, DMSO-d6)
d (ppm): 37.6
(RSO2N(CH3)2); 99.2 (C8); 102.9 (OCH2O); 104.2 (C5); 109.0 (C4a);
121.2 (C30 and C50); 128.4 (C20 and C60); 129.1 (C10); 143.1 (C40);
147.8 (C8a); 150.2 (C6); 153.6 (C2); 154.0 (C7); 157.8 (C4).
IR (ATR: cmꢂ1): 3323, 2917, 2848, 1595, 1568, 1499, 1458, 1325,
1260, 1158, 837, 731.
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 2.33 (d, 3H, J ¼ 5.2 Hz,
RSO2NHCH3); 5.90 (br, 2H, D2O exchangeable, ArNH2); 6.62 (d, 2H,
J ¼ 8.8 Hz, H3 and H5); 6.90 (q, 1H, D2O exchangeable, J ¼ 5.2 Hz,
RSO2NHCH3); 7.40 (d, 2H, J ¼ 8.8 Hz, H2 and H6).
MS: negFAB: m/z 405 [M ꢂ 1]ꢂ; 407 [M ꢂ 1 þ 2]ꢂ.
13C NMR (100 MHz, DMSO-d6)
d (ppm): 28.6 (RSO2NHCH3);
112.6 (C3 and C5); 124.4 (C1); 128.5 (C2 and C6); 152.4 (C4).
IR (ATR: cmꢂ1): 3460, 3345, 2927, 2904, 2816, 1627, 1592, 1508,
1400, 1289, 1136, 821.
4.1.13.3. 4-((2-Chloroquinazolin-4-yl)amino)-N,N-dimethylbenzene-
sulfonamide (8d). Compound 8d was synthesized via condensation
of 9a with 16b resulting in a yellow powder with a 55% yield after
precipitation from a mixture of dichloromethane and hexane, and
the m.p. was 245e246 ꢀC.
4.1.12.2. 4-(Dimethylamino-4-ylsulfonyl)aniline
(16b).
Compound 16b was obtained from alkaline hydrolysis of the acet-
amide precursor 20b resulting in a white crystalline solid with a
73% yield and m.p. of 169e170 ꢀC. The melting point for compound
16b is consistent with previous reports [41].
1H NMR (400 MHz, DMSO-d6)
d (ppm): 2.64 (s, 6H,
RSO2N(CH3)2); 7.75 (m, 4H, H5, H6, H30 and H50); 7.93 (t, 1H,
J ¼ 7.5 Hz, H7); 8.15 (d, 2H, J ¼ 8.7 Hz, H20 and H60); 8.62 (d, 1H,
J ¼ 7.5 Hz, H8); 10.42 (s, 1H, NH).
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 2.51 (s, 6H, RSO2(CH3)2);
13C NMR and DEPT 135 (100 MHz, DMSO-d6)
d (ppm): 37.6
6.05 (br, 2H, D2O exchangeable, ArNH2); 6.65 (d, 2H, J ¼ 8.0 Hz, H3
(RSO2N(CH3)2); 113.8 (C4a); 121.8 (C30 and C50); 123.5 (C8); 126.8
(C6); 127.0 (C5); 128.3 (C20 and C60); 129.6 (C10); 134.3 (C7); 142.6
(C40); 151.0 (C8a); 155.6 (C2); 159.1 (C4).
and H5); 7.36 (d, 2H, J ¼ 8.0 Hz, H2 and H6).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 37.6 (RSO2(CH3)2); 112.7
(C3 and C5); 119.3 (C1); 129.5 (C2 and C6); 153.0 (C4).
IR (ATR: cmꢂ1): 3460, 3362, 2969, 2905, 2871, 1634, 1593, 1506,
1453, 1305, 1135, 829.
IR (ATR: cmꢂ1): 3357, 2972, 2835, 1597, 1564, 1494, 1408, 1327,
1279, 1157, 838, 727.
MS: posFAB: m/z 363 [M þ 1]þ; 365 [M þ 1 þ 2]þ.
4.1.13. General procedure for BuchwaldeHartwig amination
4.1.13.4. 2-Chloro-6,7-dimethoxy-N-(4-(methylsulfonyl)phenyl)qui-
nazolin-4-amine (8e). Compound 8e was synthesized via conden-
sation of 9b with 4-methyl-sulfonylaniline hydrochloride resulting
in a beige powder with a 50% yield after precipitation from a
mixture of dichloromethane and hexane, and the m.p. was 184e
186 ꢀC.
A mixture of 0.63 mmol aryl chloride, 0.63 mmol aniline,
1.89 mmol t-BuONa, 0.19 mmol 2-(dicyclohexylphosphino)-
20,40,60-triisopropylbiphenyl (X-Phos) and 0.03 mmol Pd(OAc)2 in
6 mL tert-butanol and 10 mL toluene was stirred at 90 ꢀC for 1 h
under an argon atmosphere. When the reaction was complete,
the mixture was cooled to room temperature, diluted with water
and subsequently extracted with dichloromethane. The extracts
1H NMR (400 MHz, DMSO-d6)
3.94 (s, 3H, OCH3); 3.97 (s, 3H, OCH3); 7.22 (s, 1H, H8); 7.89 (s, 1H,
d (ppm): 3.23 (s, 3H, RSO2CH3);

M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
11
H5); 7.97 (d, 2H, J ¼ 8.4 Hz, H30 and H50); 8.05 (d, 2H, J ¼ 8.4 Hz, H20
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 2.90 (s, 6H, RN(CH3)2);
and H60); 10.11 (s, 1H, NH).
6.23 (s, 2H, OCH2O); 6.77 (d, 2H, J ¼ 8.7 Hz, H30 and H50); 7.09 (s, 1H,
H8); 7.46 (d, 2H, J ¼ 8.7 Hz, H20 and H60); 7.92 (s,1H, H5); 9.55 (s,1H,
NH).
13C NMR and DEPT 135 (100 MHz, DMSO-d6)
d (ppm): 43.8
(RSO2CH3); 56.1 (OCH3); 56.3 (OCH3); 102.2 (C8); 106.7 (C5);
107.5 (C4a); 121.8 (C30 and C50); 127.8 (C20 and C60); 135.2 (C10);
143.4 (C40); 148.6 (C8a); 149.3 (C6); 153.7 (C2); 155.3 (C7); 157.5
(C4).
13C NMR and DEPT 135 (100 MHz, DMSO-d6)
d (ppm): 40.3
(RN(CH3)2); 99.2 (C8); 102.5 (OCH2O); 104.0 (C5); 108.4 (C4a);
112.3 (C30 and C50); 124.3 (C20 and C60); 127.5 (C10); 147.1 (C40);
147.9 (C8a); 149.4 (C6); 152.8 (C2); 155.0 (C7); 158.5 (C4).
IR (ATR: cmꢂ1): 3257, 2902, 2853, 1601, 1564, 1519, 1453, 1238,
819, 690.
IR (ATR: cmꢂ1): 3369, 2916, 2835, 1597, 1572, 1505, 1423, 1338,
1283, 1134, 834, 738.
MS: posFAB: m/z 394 [M þ 1]þ; 396 [M þ 1 þ 2]þ.
MS: negFAB: m/z 341 [M ꢂ 1]ꢂ; 343 [M ꢂ 1 þ 2]ꢂ.
4.1.13.5. 2-Chloro-6,7-methylenedioxy-N-(4-(methylsulfonyl)phenyl)
quinazolin-4-amine (8f). Compound 8f was synthesized via
condensation of 9c with 4-methyl-sulfonylaniline hydrochloride
resulting in a yellow solid with a 52% yield after precipitation from
a mixture of dichloromethane and hexane, and the m.p. was
>300 ꢀC.
4.1.14.3. 4-((2-Chloro-6,7-dimethoxyquinazolin-4-yl)amino)phenol
(8l). Compound 8l was synthesized via condensation of 9b with 4-
aminophenol. After extraction with ethyl acetate at neutral pH, the
organic layer was dried with anhydrous sodium sulfate, filtered and
evaporated, resulting in the desired compound as a beige solid with
a 60% yield and m.p. >300 ꢀC.
1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.21 (s, 3H, RSO2CH3);
6.27 (s, 2H, OCH2O); 7.17 (s, 1H, H8); 7.91 (d, 2H, J ¼ 8.7 Hz, H30 and
1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.91 (s, 3H, OCH3); 3.92
H50); 8.03 (m, 3H, H5, H20 and H60); 10.04 (s, 1H, NH).
(s, 3H, OCH3); 6.82 (d, 2H, J ¼ 8.8 Hz, H30 and H50); 7.12 (s, 1H, H8);
7.41 (d, 2H, J ¼ 8.8 Hz, H20 and H60); 7.82 (s, 1H, H5); 9.39 (s, 1H,
OH); 9.68 (s, 1H, NH).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 43.8 (RSO2CH3); 99.5
(C8); 102.8 (OCH2O); 104.0 (C5); 109.5 (C4a); 121.6 (C30 and C50);
127.7 (C20 and C60); 134.5 (C10); 144.6 (C40); 147.5 (C8a); 150.0 (C6);
153.4 (C2); 154.3 (C7); 158.0 (C4).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 55.8 (OCH3); 56.2
(OCH3); 102.3 (C8); 106.6 (C5); 107.0 (C4a); 115.1 (C30 and C50);
125.1 (C20 and C60); 129.6 (C40); 147.8 (C8a); 148.8 (C6); 154.5 (C2);
154.6 (C10); 154.7 (C7); 158.3 (C4).
IR (ATR: cmꢂ1): 3361, 2935, 2905, 1590, 1557, 1509, 1449, 1286,
1234, 1138, 871, 764.
MS: negFAB: m/z 376 [M ꢂ 1]ꢂ; 378 [M ꢂ 1 þ 2]ꢂ.
IR (ATR: cmꢂ1): 3490, 3328, 2968, 2925, 1625, 1582, 1511, 1463,
1234, 820, 732.
MS: posFAB: m/z 378 [M þ 1]þ; 380 [M þ 1 þ 2]þ.
MS: posFAB: m/z 332 [M þ 1]þ; 334 [M þ 1 þ 2]þ.
4.1.14. General procedure for base-catalyzed SNAr
A mixture of 0.60 mmol aryl chloride, 0.60 mmol aniline
and 2.18 mmol N,N-diisopropylethylamine (DIPEA) in 6 mL
dioxane was stirred at 80 ꢀC for 12 h under an argon atmo-
sphere. When the reaction was complete, the mixture was
cooled to room temperature. The mixture was diluted with
water and subsequently extracted with ethyl acetate. The ex-
tracts were combined, washed with saturated saline solution,
and dried over anhydrous Na2SO4. The solvent was removed
under vacuum, and the residue was purified as described
below.
4.1.15. General procedure for SNAr
A mixture of 0.80 mmol aryl chloride and 0.80 mmol aniline in
20 mL ethanol or isopropyl alcohol was stirred at reflux for 24 h.
The resulting precipitate was filtered while hot and purified as
described below.
4.1.15.1. 4-((2-Chloro-6,7-dimethoxyquinazolin-4-yl)amino)-N-
(thiazol-2-yl)benzenesulfonamide hydrochloride (7). Compound 7
was synthesized via condensation of 9b with sulfathiazole in iso-
propyl alcohol solution resulting in a white solid with a 72% yield
after recrystallization from methanol, and the m.p. was >300 ꢀC.
The 1H NMR, 13C NMR and IR data for compound 7 are consistent
with previous reports [14].
4.1.14.1. N1-(2-Chloro-6,7-dimethoxyquinazolin-4-yl)-N4,N4-
dimethyl benzene-1,4-diamine (8i). Compound 8i was synthesized
via condensation of 9b with 4-(N,N-dimethylamino)-aniline
resulting in a yellow shiny powder with a 65% yield after purifi-
cation by flash chromatography (silica gel; dichlor-
omethane:methanol, 99:1), and the m.p. was 171e173 ꢀC.
MS: negFAB: m/z 476 [M ꢂ 1]ꢂ; 478 [M ꢂ 1þ2]ꢂ.
MS: posFAB: m/z 478 [M þ 1]þ; 480 [M þ 1 þ 2]þ.
4.1.15.2. 4-((2-Chloro-6,7-methylenedioxy-quinazolin-4-yl)amino)-
N-(thiazol-2-yl)benzenesulfonamide
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 2.91 (s, 6H, RN(CH3)2);
hydrochloride
(8a).
3.91 (s, 3H, OCH3); 3.92 (s, 3H, OCH3); 6.78 (d, 2H, J ¼ 8.3 Hz, H30
and H50); 7.11 (s, 1H, H8); 7.44 (d, 2H, J ¼ 8.3 Hz, H20 and H60); 7.83
(s, 1H, H5); 9.69 (s, 1H, NH).
Compound 8a was synthesized via condensation of 9c with sulfa-
thiazole in isopropyl alcohol solution resulting in a yellow solid
with a 69% yield after filtration from isopropyl alcohol and washing
with methanol, and the m.p. was >300 ꢀC.
13C NMR and DEPT 135 (100 MHz, DMSO-d6)
d (ppm): 40.3
(RN(CH3)2); 55.9 (OCH3); 56.2 (OCH3); 102.3 (C8); 106.6 (C5); 107.1
(C4a); 112.3 (C30 and C50); 124.6 (C20 and C60); 127.4 (C10); 147.8
(C40); 147.9 (C8a); 148.7 (C6); 154.6 (C2); 154.8 (C7); 158.3 (C4).
IR (ATR: cmꢂ1): 3317, 2950, 2850, 1608, 1579, 1509, 1453, 1242,
808, 767.
1H NMR (400 MHz, DMSO-d6)
d (ppm): 5.80 (br, 2H, D2O
exchangeable, RSO2NHAr and H from hydrochloride); 6.27 (s, 2H,
OCH2O); 6.83 (d, 1H, J ¼ 4.6 Hz, H500); 7.18 (s, 1H, H8); 7.25 (d, 1H,
J ¼ 4.6 Hz, H400); 7.81 (d, 2H, J ¼ 8.6 Hz, H30 and H50); 7.97 (d, 2H,
J ¼ 8.6 Hz, H20 and H60); 8.11 (s, 1H, H5); 10.08 (s, 1H, NH).
MS: posFAB: m/z 359 [M þ 1]þ; 361 [M þ 1 þ 2]þ.
13C NMR (100 MHz, DMSO-d6)
d (ppm): 37.6 (RSO2N(CH3)2);
99.5 (C8); 102.9 (OCH2O); 104.0 (C5); 108.1 (C500); 108.9 (C4a); 121.6
(C30 and C50); 124.4 (C10); 126.5 (C20 and C60); 137.0 (C400); 142.0
(C40); 147.7 (C8a); 150.0 (C6); 153.5 (C2); 154.1 (C7); 158.0 (C4);
168.7 (C200).
4.1.14.2. N1-(2-Chloro-6,7-methylenedioxy-quinazolin-4-yl)-N4,N4-
dimethyl benzene-1,4-diamine (8j). Compound 8j was synthesized
via condensation of 9c with 4-(N,N-dimethylamino)-aniline
resulting in a shiny yellow powder with a 66% yield after pu-
rification by flash chromatography (silica gel; dichlor-
omethane:methanol, 99:1), and the m.p. was 254e256 ꢀC.
IR (ATR: cmꢂ1): 3143, 2913, 2846, 1642, 1597, 1553, 1501, 1468,
1331, 1279, 1145, 856, 697.
MS: posFAB: m/z 462 [M þ 1]þ; 464 [M þ 1 þ 2]þ.

12
M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
4.1.15.3. 4-((2-Chloro-6,7-dimethoxyquinazolin-4-yl)amino)benze-
nesulfonamide hydrochloride (8g). Compound 8g was synthesized
via condensation of 9b with sulfanilamide in isopropyl alcohol
solution resulting in a white solid with a 68% yield after recrys-
tallization from methanol, and the m.p. was >300 ꢀC.
1H NMR (400 MHz, D2O)
d (ppm): 3.26 (s, 3H, OCH3); 3.43 (s, 3H,
OCH3); 5.89 (s, 1H, H8); 6.35 (s, 1H, H5); 7.43 (d, 2H, J ¼ 8.4 Hz, H30
and H50); 7.62 (d, 2H, J ¼ 8.4 Hz, H20 and H60).
13C NMR (100 MHz, D2O)
d (ppm): 55.6 (OCH3); 55.9 (OCH3);
99.7 (C8); 104.6 (C5); 105.9 (C4a); 121.3 (C30 and C50); 126.2 (C20
and C60); 138.0 (C10); 140.3 (C40); 146.1 (C8a); 148.2 (C6); 153.3
(C2); 154.0 (C7); 155.9 (C4).
1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.80 (br, 1H, D2O
exchangeable, H from hydrochloride); 3.93 (s, 3H, OCH3); 3.97 (s,
3H, OCH3); 7.19 (s, 1H, H8); 7.32 (br, 2H, D2O exchangeable,
RSO2NH2); 7.86 (d, 2H, J ¼ 8.7 Hz, H30 and H50); 7.95 (m, 3H, H5, H20
and H60); 10.13 (s, 1H, NH).
IR (ATR: cmꢂ1): 3065, 2978, 2946, 1635, 1552, 1520, 1468, 1346,
1251, 1147, 830, 700.
MS: negFAB: m/z 394 [M ꢂ 1]ꢂ; 396 [M ꢂ 1þ2]ꢂ.
13C NMR (100 MHz, DMSO-d6)
d (ppm): 56.0 (OCH3); 56.4
(OCH3); 102.3 (C8); 106.6 (C5); 107.4 (C4a); 122.0 (C30 and C50);
126.3 (C20 and C60); 139.0 (C10); 141.7 (C40); 148.4 (C8a); 149.2 (C6);
153.9 (C2); 155.2 (C7); 157.7 (C4).
4.1.15.7. 4-((2-Chloro-6,7-dimethoxyquinazolin-4-yl)amino)-benzoic
acid hydrochloride (8n). Compound 8n was synthesized via
condensation of 9b with 4-aminobenzoic acid in ethanol solution
resulting in a beige solid with a 73% yield after filtration from
ethanol and washing with methanol, and the m.p. was >300 ꢀC. The
melting point, 1H NMR and IR data for compound 8n are consistent
with previous reports [14].
IR (ATR: cmꢂ1): 3391, 3331, 2979, 2939, 1594, 1568, 1509, 1449,
1323, 1234, 1149, 834, 701.
MS: posFAB: m/z 395 [M þ 1]þ; 397 [M þ 1 þ 2]þ.
4.1.15.4. 4-((2-Chloro-6,7-methylenedioxy-quinazolin-4-yl)amino)
benzene sulfonamide hydrochloride (8h). Compound 8h was syn-
thesized via condensation of 9c with sulfanilamide in isopropyl
alcohol solution resulting in a white solid with a 67% yield after
recrystallization from methanol, and the m.p. was >300 ꢀC.
13C NMR (100 MHz, DMSO-d6)
d (ppm): 56.0 (OCH3); 56.5
(OCH3); 102.8 (C8); 106.5 (C5); 107.6 (C4a); 121.4 (C30 and C50);
125.5 (C10); 129.8 (C20 and C60); 143.0 (C40); 148.4 (C8a); 149.1 (C6);
153.8 (C2); 155.1 (C7); 157.6 (C4); 166.8 (C]O).
MS: posFAB: m/z 360 [M þ 1]þ; 362 [M þ 1 þ 2]þ.
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 3.43 (br, 1H, D2O
MS: negFAB: m/z 358 [M ꢂ 1]ꢂ; 360 [M ꢂ 1 þ 2]ꢂ.
exchangeable, H from hydrochloride); 6.27 (s, 2H, OCH2O); 7.18 (s,
1H, H8); 7.31 (br, 2H, D2O exchangeable, RSO2NH2); 7.84 (d, 2H,
J ¼ 8.4 Hz, H30 and H50); 7.94 (d, 2H, J ¼ 8.4 Hz, H20 and H60); 7.99 (s,
1H, H5); 9.93 (s, 1H, NH).
4.1.15.8. 4-((2-Chloro-6,7-dimethoxyquinazolin-4-yl)amino)benza-
mide hydrochloride (8o). Compound 8o was synthesized via
condensation of 9b with 4-aminobenzamide in ethanol solution
resulting in a yellow solid with a 69% yield after recrystallization
from a mixture of methanol and ethanol, and the m.p. was 285 ꢀC.
13C NMR and DEPT 135 (100 MHz, DMSO-d6)
d (ppm): 99.2 (C8);
102.8 (OCH2O); 104.1 (C5); 108.8 (C4a); 121.7 (C30 and C50); 126.3
(C20 and C60); 138.9 (C10); 141.7 (C40); 147.6 (C8a); 150.0 (C6); 153.5
(C2); 154.2 (C7); 158.0 (C4).
1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.92 (s, 3H, OCH3); 3.97
(s, 3H, OCH3); 4.37 (br, 1H, H from hydrochloride); 7.18 (s, 1H, H8);
7.32 (br, 1H, D2O exchangeable, RCONHaHb); 7.91 (m, 5H, RCON-
HaHb, H20, H30, H50 and H60); 8.03 (s, 1H, H5); 10.26 (s, 1H, NH).
IR (ATR: cmꢂ1): 3369, 3343, 2998, 1601, 1571, 1523, 1446, 1327,
1231, 1145, 838, 620.
MS: posFAB: m/z 379 [M þ 1]þ; 381 [M þ 1 þ 2]þ.
13C NMR (100 MHz, DMSO-d6)
d (ppm): 56.0 (OCH3); 56.5
(OCH3); 102.7 (C8); 106.2 (C5); 107.4 (C4a); 121.6 (C30 and C50);
128.0 (C20 and C60); 129.6 (C10); 141.3 (C40); 147.8 (C8a); 149.2 (C6);
153.7 (C2); 155.2 (C7); 157.8 (C4); 167.4 (C]O).
IR (ATR: cmꢂ1): 3323, 3156, 2978, 2927, 1674, 1591, 1516, 1456,
1159, 854, 700.
4.1.15.5. 4-((2-Chloro-6,7-dimethoxyquinazolin-4-yl)amino)-N-
methylbenzene sulfonamide hydrochloride (8k). Compound 8k was
synthesized via condensation of 9b with 16a in ethanol solution
resulting in a white solid with a 68% yield after recrystallization
from methanol, and the m.p. was >300 ꢀC.
MS: posFAB: m/z 359 [M þ 1]þ; 361 [M þ 1 þ 2]þ.
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 2.45 (d, 3H, J ¼ 5.2 Hz,
RSO2NHCH3); 3.73 (br, 1H, D2O exchangeable, H from hydrochlo-
ride); 3.93 (s, 3H, OCH3); 3.97 (s, 3H, OCH3); 7.20 (s, 1H, H8); 7.38 (q,
1H, D2O exchangeable, J ¼ 5.2 Hz, RSO2NHCH3); 7.82 (d, 2H,
J ¼ 8.8 Hz, H30 and H50); 7.90 (s, 1H, H5); 8.00 (d, 2H, J ¼ 8.8 Hz, H20
and H60); 10.08 (s, 1H, ArNHAr).
4.1.15.9. N-(4-((2-Chloro-6,7-dimethoxyquinazolin-4-yl)amino)
phenyl) acetamide (8p). Compound 8p was synthesized via conden-
sation of 9b with 4-aminoacetanilide in ethanol solution resulting in a
pale yellow solid after filtration from the warm reaction medium. The
purification was performed through a flash chromatographic column
with 0.1% NH3 and 5% MeOH in AcOEt as the eluent resulting in the
desired compound with a 64% yield and m.p. of 246e247 ꢀC.
13C NMR (100 MHz, DMSO-d6)
d (ppm): 28.7 (RSO2NHCH3); 56.0
(OCH3); 56.3 (OCH3); 102.3 (C8); 106.6 (C5); 107.5 (C4a); 121.8 (C30
and C50); 127.4 (C20 and C60); 133.8 (C10); 142.3 (C40); 148.5 (C8a);
149.2 (C6); 153.8 (C2); 155.3 (C7); 157.5 (C4).
1H NMR (400 MHz, DMSO-d6)
d (ppm): 2.06 (s, 3H, NHCOCH3);
3.91 (s, 3H, OCH3); 3.94 (s, 3H, OCH3); 7.14 (s, 1H, H5); 7.61 (m, 4H,
H20, H30, H50 and H60); 7.89 (s, 1H, H8); 9.88 (s, 3H, NHCOCH3);
10.02 (s, 1H, ArNHAr).
IR (ATR: cmꢂ1): 3398, 3008, 2974, 2932, 1599, 1565, 1508, 1463,
1315, 1285, 1145, 842, 694.
MS: posFAB: m/z 409 [M þ 1]þ; 411 [M þ 1 þ 2]þ.
13C NMR (100 MHz, DMSO-d6)
d (ppm): 23.9 (NHCOCH3); 55.9
(OCH3); 56.2 (OCH3); 102.4 (C5); 106.6 (C8); 107.2 (C4a); 119.1 (C30
and C50); 123.4 (C20 and C60); 133.4 (C10); 135.9 (C40); 148.0 (C8a);
148.9 (C6); 154.4 (C2); 154.8 (C7); 158.1 (C4); 168.1 (C]O).
IR (ATR: cmꢂ1): 3349, 3280, 2938, 1665, 1634, 1573, 1504, 1458,
1147, 833, 729.
4.1.15.6. 4-((2-Chloro-6,7-dimethoxyquinazolin-4-yl)amino)benze-
nesulfonic acid hydrochloride (8m). Compound 8m was synthesized
via condensation of 9b with sulfanilic acid in ethanol solution
resulting in a beige solid with a 66% yield after recrystallization
from a mixture of dimethylsulfoxide and methanol, and the m.p.
was >300 ꢀC.
MS: posFAB: m/z 373 [M þ 1]þ; 375 [M þ 1 þ 2]þ.
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 3.33 (br, 2H, H from
4.2. Biochemical protein kinase assays
hydrochloride and RSO3H); 3.93 (s, 3H, OCH3); 3.96 (s, 3H, OCH3);
7.18 (s, 1H, H8); 7.65 (m, 4H, H20, H30, H50 and H60); 7.90 (s, 1H, H5);
9.92 (s, 1H, NH).
A radiometric protein kinase assay (33PanQinaseÒ Activity
Assay) was used to measure the kinase activity of the protein

M.L.deC. Barbosa et al. / European Journal of Medicinal Chemistry 71 (2014) 1e14
13
kinases. All kinase assays were performed in 96-well FlashPlatesTM
(Perkin Elmer, Boston, MA, USA) in a 50 l reaction volume. The
assay buffer for all enzymes contained 70 mM HEPES-NaOH, pH 7.5,
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The EGFRwt and VEGFR-2 crystallographic structures available
ꢀ
in the Protein Data Bank with codes 2ITY (resolution: 3.42 A) [5]
ꢀ
and 4ASE (resolution: 1.83 A) [9], respectively, were used for
docking runs with the GOLD 5.1 program (CCDC; License Key: G/
414/2006). There are four fitness functions available in the pro-
gram, ASP [42], ChemPLP [43], ChemScore [44,45] and GoldScore
[46,47], and all were evaluated for re-docking of the co-crystallized
ligands to identify the most adequate fitness function for the
docking studies into VEGFR-2 and EGFR. Crystallographic waters
were maintained fixed during the docking runs, and the set of
amino acid residues selected as the binding site was determined
ꢀ
within a 15 A distance from the amino acid residues Gln781 for
2ITY and Asp1046 for 4ASE. After docking, the RMSD between the
best result for each fitness function and the crystallographic
structure was calculated.
The fitness function with the best performance in the re-docking
test, which was the consensus docking scoring GoldScore-
ChemPLP, was used for the docking study of the compounds
described in this study.
Poses presenting the best score for each compound were
analyzed to identify potential interactions with amino acids in the
active site of each enzyme. The amino acids with more favorable
interactions were then selected and a semi-rigid docking was
performed, which allowed for flexibility of the side chains of
Lys716, Lys728, Lys745, Glu762, Thr854 and Asp855 for EGFRwt
and of Asp1046, Glu850, Lys868, Glu885 and Phe918 for VEGFR-2.
The final poses selected were those with the best scores after the
semi-rigid docking runs for each enzyme.
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Acknowledgments
The authors would like to thank CNPq (BR), DAAD (DE), FAPERJ
(BR) and INCT-INOFAR (BR, Grant CNPq 573.564/2008-6 and FAPERJ
E-26/170.020/2008) for fellowship and financial support.
Appendix A. Supplementary data
1H-pyrazolo[4,3-d]pyrimidines as potent phosphodiesterase
hibitors, Bioorg. Bioorg. Med. Chem. Lett. 20 (2010) 3125e3128.
5 (PDE5) in-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.058.

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