HER2 Kinase-Targeted Breast Cancer Therapy: Design, Synthesis, and in Vitro and in Vivo Evaluation of Novel Lapatinib Congeners as Selective and Potent HER2 Inhibitors with Favorable Metabolic Stability

Article abstract of DOI:10.1021/acs.jmedchem.0c01647

HER2 kinase as a well-established target for breast cancer (BC) therapy is associated with aggressive clinical outcomes; thus, herein we present structural optimization for HER2-selective targeting. HER2 profiling of the developed derivatives demonstrated

Full text of DOI:10.1021/acs.jmedchem.0c01647

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Article
HER2 Kinase-Targeted Breast Cancer Therapy: Design, Synthesis,
and In Vitro and In Vivo Evaluation of Novel Lapatinib Congeners as
Selective and Potent HER2 Inhibitors with Favorable Metabolic
Stability
Tamer A. Elwaie, Safinaz E. Abbas, Enayat I. Aly, Riham F. George, Hamdy Ali, Nikolai Kraiouchkine,
Khaldoun S. Abdelwahed, Tamer E. Fandy, Khalid A. El Sayed, Zakaria Y. Abd Elmageed,*
and Hamed I. Ali*
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ABSTRACT: HER2 kinase as a well-established target for breast cancer (BC) therapy is associated with aggressive clinical
outcomes; thus, herein we present structural optimization for HER2-selective targeting. HER2 profiling of the developed derivatives
demonstrated potent and selective inhibitions (IC50: 5.4−12 nM) compared to lapatinib (IC50: 95.5 nM). Favorably, 17d exhibited
minimum off-target kinase activation. NCI-5-dose screening revealed broad-spectrum activities (GI50: 1.43−2.09 μM) and 17d had a
remarkable selectivity toward BC. Our compounds revealed significant selective and potent antiproliferative activities (∼20-fold)
against HER2+ (AU565, BT474) compared to HER2(−) cells. At 0.1 IC50, 15i, 17d, and 25b inhibited pERK1/2 and pAkt by
immunoblotting. Furthermore, 17d demonstrated potent in vivo tumor regression against the BT474 xenograft model. Notably, a
metastasis case was observed in the vehicle but not in the test mice groups. CD-1 mice metabolic stability assay revealed high
stability and low intrinsic clearance of 17d (T1/2 > 145 min and CLint(mic) < 9.6 mL/min/kg).
domain.10 Unlike EGFR (ErbB1/HER1), ErbB2 (HER2) does
INTRODUCTION
■
not have a recognized activating ligand (ligandless receptor),11
Cancer is the second main cause of death in the United States
accordingly its overexpression can lead to cell transformation
and worldwide.1−6 The most common cancers among men and
without the need of ligand interaction.12,13 HER2 signalings
women are prostate and breast cancers (BCs), respectively.1
are initiated by homo- or hetero-dimerization and these dimers
BC is the most commonly diagnosed cancer and the main
have a lower dissociation rate with persistent signal trans-
cause of cancer deaths among women globally. Approximately,
duction compared to other dimers. Subsequently, these
two million patients are diagnosed with BC and more than
intracellular cascades activate cell survival, proliferation,
600,000 deaths occur each year.7 In the United States, 276,480
motility, and differentiation.14,15 Dysregulation of the HER2
new cases of female BC have been estimated and 42,170 deaths
gene disrupts cell proliferation and leads to the formation of
were reported in 2020.8 The ErbB (HER) proteins are related
aggressive tumor cells.16 The HER2 RTK is amplified in
to subclass I of the superfamily of receptor tyrosine kinases
(RTKs) which plays a vital role in signal transduction pathways
and the initiation of malignancy of several solid tumors. The
Received: September 21, 2020
ErbB family contains epidermal growth factor receptor (EGFR;
also named ErbB1/HER1), ErbB2/HER2, ErbB3/HER3, and
ErbB4/HER4.9 All ErbBs are closely similar in their structures
and consist of three domains, an extracellular ligand-binding
domain, a transmembrane domain, and an intracellular kinase
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Figure 1. Binding mode of EGFR/HER2 inhibitors into their corresponding kinases and the fragments used for their design.
Figure 2. Chemical structure for the FDA-approved EGFR and dual EGFR/HER2 kinase inhibitors.
approximately 15−30% of BC and is associated with worse
aggressive tumor forms.17 HER2 overexpression has also been
reported with high frequencies in the lung (NSCLC),
gastroesophageal, ovarian, melanoma, and oral cancers.18−20
HER2-positive (HER2+) BC often has a more aggressive
phenotype than HER2-negative (HER2−) BC.21 Worldwide,
approximately 15−20% of BC cases are HER2+.22 Because of
the identification of the HER2 receptor amplification as an
adverse prognostic factor in a special subtype of the metastatic
BC, there has been a substantial improvement in the survival of
these patients because of the development of anti-HER2-
targeted therapies.23 HER2 is a well-established therapeutic
target for the treatment of BC.24 Currently, there is a variety of
FDA-approved HER2-targeted therapies for the treatment of
HER2+ BCs including anti-HER2 monoclonal antibodies, such
as trastuzumab and pertuzumab, antibody−drug conjugate
such as trastuzumab-emtansine (T-DM1), and small-molecule
pan-HER tyrosine kinase inhibitors (TKIs) such as lapatinib
and neratinib.23 The small-molecule TKIs have potential
advantages over monoclonal antibody therapies, including their
ability to inhibit multiple kinases and p95 HER2 (highly active
truncated HER2 lacking most of the extracellular domain).23,25
Likewise, TKIs have lower cardiopulmonary toxicities
commonly associated with monoclonal antibody therapies26−28
and have a higher ability to penetrate the blood−brain barrier
with a greater therapeutic potential against brain metastases.20
B
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Figure 3. Docking mode of lapatinib (ball and sticks) and TAK285 (orange lines) into the HER2 kinase domain (PDB: 3PP0). Hydrophobic
region 1 (L726, A751, L800, G804, L852, and M801); hydrophobic selectivity pocket (M774, L785, L796, T798, D863, and F864); solvent-
accessible region for TAK285 (G727, S728, and V734); solvent-accessible region for lapatinib (C805 and D808); α-C helix (P761-V777), and the
gatekeeper amino acid (T798).
In addition, the majority of BC patients who have an initial
therapeutic response to the HER2-targeted antibody trastuzu-
mab reveal poor prognosis within one year.29 Unlike lapatinib,
neratinib is a potent irreversible pan-HER kinase inhibitor but
its clinical use may result in hepatotoxicity.30 In favor of this
strategy, four of the 25 FDA-approved medications in 2019 are
kinase inhibitors.31 The development of kinase inhibitors is
based on the fundamental understanding of the general
pharmacophoric features of these inhibitors (summarized in
Figure 1) which are composed of (a) core scaffold serving as a
hinge binder; (b) hydrophobic bulky moiety extended into the
back pocket revealing HER2 kinase selectivity; and (c)
hydrophilic solubilizing fragment harbored into the solvent-
accessible region to conduct its pharmacokinetic role through
balancing the logP of the inhibitors. The solvent-accessible
region can tolerate a wide range of solubilizing groups
modulating the cellular activity without affecting the enzymatic
activity. Moreover, introducing particular functionalities into
the solubilizing moiety would optimize the potency and
selectivity profiles.32 The 4-anilinoquinazoline is the most
commonly used hinge binder scaffolds for EGFR kinase
inhibitors, represented by the clinically approved drugs:
gefitinib, erlotinib, and lapatinib (Figure 2).33−35 Lapatinib
was developed by GSK as the first dual EGFR/HER2 inhibitor
and approved by the FDA in 2007 for treatment of BC. Its
bulky 4-benzyloxyaniline substituent is essential for EGFR/
HER2 dual inhibition. However, smaller anilines such as 3-
ethynylaniline in erlotinib and 3-chloro-4-fluoro-aniline in
gefitinib only inhibit EGFR.36 Lapatinib binds to the inactive
EGFR closed conformation displacing the αC-helix to “out”
and gives the 3-fluorobenzyloxy group access to the hydro-
phobic back pocket.32,37 This extra binding in the back pocket
contributes to the dual EGFR/HER2 inhibition and has longer
inhibition time when compared to gefitinib and erlotinib which
do not have access into the back pocket.38 In this regard,
Abouzid et al. reported enhanced EGFR/HER2 activities of
quinazoline derivatives through incorporating bulkier anilino
moieties at position 4 in a manner similar to lapatinib. These
moieties include 4-(4-aryl-2-imino pyridino) anilines, which
could be oriented deep in the back of the ATP-binding site and
mimicking the 3-chloro-4-[(3-fluorobenzyl)oxy]aniline group
of lapatinib.39 The HER2T798I(M) gatekeeper mutation and its
analogous EGFRT790M have been shown to confer the most
common clinical resistance against lapatinib.40 This resistance
emerges from replacing a smaller polar Thr798 with bulkier
Ile798 or Met798 residues, which causes unfavorable steric
clashes between the gatekeeper mutant Ile798 amino acid and
lapatinib.40 Targeting other HER family members, such as
EGFR (HER1), does not functionally suppress HER2 signaling
and may introduce off-target toxicities such as diarrhea and
rash.41 Moreover, in BC cells that overexpress HER2, the
inhibition of HER family members EGFR or HER4 does not
functionally suppress HER2 signaling and consequently does
not prevent tumor cell proliferation or survival.42,43 Note-
worthy, lapatinib, as an EGFR inhibitor, has another serious
drawback by causing severe skin rash and diarrhea. Skin
toxicity is typically manifested as mild to severe papulopustular
rash in the majority of patients (45−100%) receiving EGFR
inhibitors.44−46 These adverse effects reveal serious physical
and psycho-social complications, particularly for female
patients with BC. For all the aforementioned limitations of
current medications, there is an eventual need for developing
novel selective HER2 inhibitors with enhanced efficacy against
mutant isoforms to overcome the reported limitations of
lapatinib. Based on the reported key binding features of
lapatinib as a dual EGFR/HER2 inhibitor, guided by its
binding mode through flexible docking into the HER2 kinase
domain (Figure 3) and inspired by the importance of HER2
overexpression in BC progression, in this study, we present the
design and development of new series of 6-substituted-4-
anilinoquinazolines (10a−c, 15a−l, 17a−d, and, 25a−h) as
promising candidates for HER2 inhibition with improved
potency and selectivity. The developed candidates can
preferentially inhibit HER2 kinase and reduce the potential
of EGFR-associated toxicities. In addition, our strategy is
oriented to developing a variety of lapatinib analogues with
C
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Figure 4. Rational design enhanced using CADD of the newly synthesized compounds.
some structural flexibility, this flexibility of the designed
compounds may diminish the steric clashes within the
gatekeeper amino acids of the HER2T798I mutant BC and
consequently provide better treatment options.
compound (10a; Figure 4) via incorporating a flexible spacer
at position 6 of the quinazoline ring, namely, 6-acetamido, to
mimic neratinib. This flexible design is aimed to avoid the
steric clashes within the gatekeeper T798I(M) mutant kinase.
A hydrophilic ethanolamine tail was introduced to the flexible
spacer at position 6 through nucleophilic substitution. At
position 4 “N-(4-(3-chloro-4-(3-fluorobenzyl)oxy)phenyl)-
amino” moiety of lapatinib was retained. The lead compound
(10a) revealed superior inhibition against HER2 kinase (IC50
= 10.4 nM) compared to lapatinib which exhibited inferior
inhibition (IC50 = 95.5 nM). WST-8 assay for the lead
compound was conducted using AU565 (only HER2+) and
BT474 {triple positive, HER2+, estrogen (ER+), and
progesterone (PR+)} BC cell lines. The lead compound
RESULTS AND DISCUSSION
■
Rational Design. The rational design of our compounds
was based on the fundamental pharmacophoric features for the
kinase inhibitors summarized in Figure 3 and according to the
analysis of the binding pattern of the FDA-approved kinase
inhibitors. Our template kinase inhibitors include the dual
EGFR/HER2 inhibitors (lapatinib and neratinib) and the
investigational selective HER2 inhibitor (CP-724,714).47 Our
efforts were focused on the design and synthesis of new hybrid
molecules with the 6-substituted 4-anilinoquinazoline core to
potentiate the selectivity against the target HER2 kinase by
reversible inhibition and tackling the emergence of lapatinib
acquired resistance. Guided by the reported EGFR/HER2
kinase inhibitors, we initially designed and synthesized the lead
(10a) exhibited promising antiproliferative activities (IC50
=
0.22 and 0.24 μM, respectively). These preliminary results
have prompted us to enhance the efficiency of the lead
compound through lead optimization, as depicted in Figure 4.
To increase the selectivity of HER2 inhibition, the “N-(4-(3-
D
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Scheme 1. Synthesis of 2-Chloro-4-nitro-1-(3-(substituted)phenoxy/benzyloxy)benzene (3a−d) and 3-Chloro-4-(3-
a
(substituted)phenoxy/benzyloxy)aniline (4a−d)
a
Reagents and conditions: (a) DMF, K2CO3, 85 °C, 3 h; (b) Fe, CaCl2, EtOH, H2O, 80 °C, 8 h; (c) acetonitrile, K2CO3, 85 °C, 5 h; (d) SnCl2,
MeOH, reflux, under N2, 2.5 h.
Scheme 2. Synthesis of N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)-2-Substituted Acetamide
a
(10a−c)
a
Reagents and conditions: (a) DMF-DMA, reflux 2 h; (b) glacial HOAc, reflux, 3 h; (c) Fe, CaCl2, EtOH, H2O, 90 °C, 12 h; (d) ClCH2COCl,
NaHCO3, acetone, 0 °C, under N2, 1 h; (e) ethanolamine (5 equiv), MeOH, TEA, reflux, 4 h or appropriate secondary amine (50 equiv), reflux, 2
h.
chloro-4-(3-fluorobenzyl)oxy)phenyl)amino” moiety of lapati-
nib was replaced by phenoxy phenyl analogues that fit well into
the back pocket of HER2. Optimization of HER2 inhibitory
activity was conducted through replacing ethanolamine by
morpholine, N-methyl piperazine, diethylamine, and dimethyl-
amine at position 6 of the quinazoline scaffold to improve the
pharmacokinetic properties. This hydrophilic tail was designed
to fit into the ribose and phosphate region of the kinase ATP-
binding site and improve the druggability. Different flexible
spacers of acetamido, benzamido, and propoxy moieties were
bridged between quinazoline and the hydrophilic tail. The
flexibility of the used spacers is engendered by replacing the
rigid 6-C−C bond in lapatinib by more flexible C−O or C−N
bonds in the newly synthesized compounds, as shown in detail
in Figure 4. The structural optimization was conducted using
computer-aided drug design (CADD). A small library of 189
compounds (data are presented in the Supporting Information,
Table S1) was designed by changing the substituents at
position 4 and 6 of the quinazoline nucleus based on the
literature database. At position 4, the lipophilic moiety was
either substituted benzyloxy or phenoxy anilino moieties with
the variables, including n = 0 or 1, X = H, Cl, or Br and R = o,
m, or p CH3, CF3, CN, and F. At position 6, the flexible spacer
was the acetamido, benzamido, or propoxy moiety and the
hydrophilic terminal was the ethanolamino, morpholino, N-
methyl piperazino, diethylamino, dimethylamino, methylsulfo-
nylethylamino, immidazo, or pyrrolidino moiety. Then, the 3D
structures of the developed compounds were energetically
minimized to be explored as kinase inhibitors by docking into
the crystal structure of HER2 kinase (PDB code: 3PP0),48 in
complex with its cocrystallized kinase inhibitor. The GOLD
software package version 5.2.2 (Cambridge Crystallographic
Data Centre, Cambridge, U.K.)49 was used in this study, which
is described in detail under the Molecular Docking part. The
newly synthesized candidates were biologically evaluated in
enzyme inhibitor-based assays and cell antiproliferative activity
E
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Figure 5. Proposed mechanism of Dimroth rearrangement for the preparation of 6-nitroquinazoline derivative (7).
a
Scheme 3. Synthesis of Target Compounds 15a−l and 17a−d
a
Reagents and conditions: (a) triethylorthoformate, (Ac)2O, reflux, 30 h; (b) glacial HOAc, reflux, 7 h; (c) SnCl2, MeOH, reflux, under N2, 1.5 h;
(d) ClCH2COCl, NaHCO3, acetone, 0 °C, under N2, 1 h; (e) appropriate secondary amine (50 equiv), reflux, 2 h or ethanolamine (5 equiv),
MeOH, TEA, reflux, 4 h; (f) using 13a, 4-(chloromethyl)benzoyl chloride, NaHCO3, acetone, 0 °C, under N2, 0.5 h; (g) excess appropriate
secondary amine (50 equiv), reflux, 2 h or ethanolamine (5 equiv), MeOH, TEA, reflux, 4 h.
F
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ones. The following are the biological screening tests that had
been carried out in the present work; in vitro protein kinase
profiling was conducted to figure out the degree of selectivity
obtained against the target HER2 using 20 different types of
kinases, IC50 determination against wild and HER2 L755S
mutant kinases, screening against National Cancer Institute-
NCI 60 human cell panel, and the WST-8 antiproliferative
assay using BT-474 and AU-565 BC cell lines (HER2+), as
well as MCF7 and MDA-MB-231 BC cell lines (HER2−),
were included. In addition, we also investigated the apoptotic
inducing effect in BT-474 cells; western blot analysis was also
established to study the effect of some selected candidates on
inhibition of the HER2 downstream targets (ERK and AKT).
In vivo antitumor efficacy and microsomal stability assay were
also established for the most promising candidates.
The target compounds 10a−c were prepared through a
nucleophilic substitution on the chloroacetamido moiety of
compound 9 using ethanolamine or the appropriate cyclic
secondary amines (morpholine and N-methyl piperazine)
according to the procedure described in Scheme 2 to afford
pure compounds in 57−87% yields. Compounds 10a−c were
1
confirmed by multiple spectroscopic data including their H
NMR spectra which exhibited two singlet signals at δ 5.25 and
δ 3.19−3.35 for benzyloxy CH2 protons and acetamido CH2
protons, respectively. Besides, a quartet signal at δ 3.54−3.49
and a triplet signal at δ 2.69−2.66 were representing the four
protons of the ethanolamine terminal in the target compound
(10a), and two triplet signals at δ 2.55−3.69 were detected for
the eight protons of the morpholino ring in compound (10b).
Another two broad singlet signals at δ 2.41−2.57 were assigned
for the eight protons of the piperazino ring in compound
(10c). An additional panel of novel target compounds (15a−l
and 17a−d) was prepared to study the structure−activity
relationship (SAR) and to enhance the selectivity, as shown in
Scheme 3. The lapatinib anilino moiety at position 4, namely,
3-chloro-4-((3-fluorobenzyl)oxy) aniline in Scheme 2, was
replaced by substituted phenoxyaniline fragments to enhance
the selectivity against HER2 kinase, whereas the 6-acetamido
linker was kept in compounds (15a−l) or replaced by a bulkier
6-methyl benzamido linker in compounds (17a−d). Reaction
of 2-amino-5-nitrobenzonitrile (5) with triethylorthoformate in
the presence of acetic anhydride produced the formimidate
derivative (11), which was then cyclized upon reflux with the
appropriately substituted phenoxyaniline (4b−d) to afford 6-
nitro-4-anilinoquinazoline derivatives (12a−c) through Dim-
roth rearrangement. It was noteworthy that reduction of the 6-
nitro-quinazolione derivatives (12a−c) to the corresponding
6-amino analogues (13a−c) was adequately accomplished
using SnCl2/MeOH, where they were obtained within short
reaction time (1.5 h), in high purities and excellent yields (86−
94%). In comparison, the other reported conditions using
iron/CaCl2/EtOH afforded these compounds within a longer
reaction time (12 h) and in inferior purities and lower yields
(52−65%). Also, a longer reaction time (24 h) is needed on
using Na2S/dioxane.
Chemistry. In this part, we described the preparation of
novel 6-substituted-4-anilinoquinazolines (10a−c, 15a−I,
17a−d, and 25a−h) which were purified by flash column
chromatography. The structures of the newly synthesized
compounds were elucidated by different spectroscopic
1
methods, including H NMR, 13C DEPT, 13C NMR, and
high-resolution mass spectrometry (HRMS).
The synthesis of the different substituted aniline derivatives
(4a−d) is described in Scheme 1. Reaction of the
commercially available 2-chloro-4-nitrophenol (1a) or 2-
chloro-1-fluoro-4-nitrobenzene (1b) with 1-(bromomethyl)-
3-fluorobenzene (2a) or the appropriate phenol derivative
(2b−d) was carried out in dimethylformamide (DMF) or
acetonitrile in the presence of potassium carbonate to afford
the corresponding nitro derivatives (3a−d) in good yields of
73−89%.50,51 Reduction of the nitro derivatives (3a−d) into
their amino analogues (4a−d) were achieved by either heating
with Fe/CaCl2 in aqueous ethanolic solution to prepare (4a)
intermediate or via reflux with SnCl2 for 2.5 h in methanol
under nitrogen to afford intermediates (4b−d). The reduction
methods gave the desired intermediates in very good yields of
77−82%, in shorter reaction times, and with higher purity than
the reported methods using H2/Pt−C,50,51 SnCl2/EtOAc/
reflux/24 h,52,53 Na2S/dioxane/70−80 °C/24 h (Zinin
reaction),54 or Fe/NH4Cl/EtOH/reflux/2 h.44 The H NMR
1
Compounds (13a−c) were subsequently acylated with 2-
chloroacetyl chloride in acetone to yield the corresponding 2-
chloroacetamido derivatives (14a−c), while compound (16)
was prepared by acylation of intermediate (13a) with 4-
(chloromethyl)benzoyl chloride to afford the corresponding 4-
(chloromethyl)benzamido derivative (16). Two series of the
target compounds (15a−l, 43−92% yields) and (17a−d, 48−
78% yields) were synthesized through nucleophilic substitution
of compounds (14a−c) and (16), respectively, with the
appropriate secondary amines or ethanolamine, as described in
Scheme 3. Partial hydrolysis was detected in compounds
(17a−d) bearing a benzamido linker to their corresponding 6-
amino quinazoline analogues as a result of the nucleophilic
attack of the reacting amines on the reactive carbonyl group of
the benzamido moiety. Therefore, the hydrolytic products
were removed by flash column chromatography to get the
target compounds in a pure form. Compounds (15a−l) were
spectrum of 4c revealed a broad signal at δ 5.42, exchanged
with D2O, representing the two protons of the NH2 group.
The impact of the acetamido linker at position 6 was
investigated by applying Scheme 2, and the 3-chloro-4-((3-
fluorobenzyl)oxy)aniline (4a) was incorporated into position 4
of the quinazoline nucleus through Dimroth rearrangement, as
depicted in Scheme 2. The reaction of 2-amino-5-nitro-
benzonitrile (5) with DMF-DMA (dimethylformamide-dime-
thylacetal) afforded the formimidamide intermediate (6) in a
90% yield, which was then cyclized upon reflux with
compound 4a in glacial acetic acid into 6-nitro-4-anilinoqui-
nazoline derivative (7) through Dimroth rearrangement
(Figure 5) as reported for similar derivatives.55
Reduction of (7) could be attained by a sodium dithionite/
ammonia mixture or SnCl2/methanol, but the yield was very
low (30%) and the product requires purification by flash
column chromatography. In the present study, reflux of iron in
an alcoholic/aqueous solution of calcium chloride was
implemented to efficiently afford a 6-amino-4-anilinoquinazo-
line derivative (8) in an excellent yield of 97% with high purity
without further purification. Subsequently, compound 8 was
acylated by 2-chloroacetyl chloride in acetone to afford the
corresponding 2-chloroacetamido derivative (9) in 61% yield.
1
confirmed by H NMR spectra exhibiting a singlet signal at δ
3.15−3.38 for the acetamido CH2 protons, in addition to, 13C
DEPT revealed an inverted signal at δ 53.17−63.69,
representing the acetamido CH2 carbon. On the other hand,
1H NMR spectra of (17a−d) revealed the characteristic AB
doublet spin system signals of the phenylene residue at δ 7.46−
G
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Figure 6. Two pathways conducted for the preparation of 6-nitroquinazoline derivatives.
a
Scheme 4. Synthesis of the Target Compounds 25a−h
a
Reagents and conditions: (a) NH2CHO, 190 °C, 1 h; (b) AC2O, pyridine, 100 °C, 2 h; (c) SOCl2, DMF, 95 °C, 5 h; (d) acetonitrile, K2CO3, rt, 8
h (with 4a); acetonitrile, K2CO3, reflux, 12 h (with 4b or 4c); (e) NH4OH, MeOH, rt, 2 h, (f) Cl(CH2)3Br, K2CO3, TBAB, acetonitrile, reflux, 2 h;
(g) appropriate amine (100 equiv), reflux, 2 h or ethanolamine (10 equiv), MeOH, TEA, reflux, 8 h.
8.03 and a characteristic 13C DEPT inverted signal for benzyl
CH2 at δ 52.97−63.42.
The synthesis of 4-substituted phenoxy/benzyloxy anilino
quinazolin-6-ol and their 6-substituted propoxy analogues
(25a−h) is depicted in detail in Scheme 4. The quinazoline-
4,6-diol (19) was prepared using the Niementowski syn-
thesis,56 by heating the commercially available 5-hydroxy
anthranilic acid (18) in formamide.
It is noteworthy that the success of Dimroth rearrangement
for the preparation of 6-nitroquinazoline derivatives (7) and
(12a−c) depends on the used precursors of formimidamide
(6) or the formimidate (11), as shown in Schemes 2 and 3,
respectively. We have observed that intermediate (6) could be
used properly to afford the desired 6-nitroquinazoline
derivatives (7) and (12a−c). However, the intermediate
(11) was not the appropriate precursor for the preparation
of 6-nitroquinazoline derivative (7), as shown in Figure 6.
1
The H NMR spectrum of (19) showed a characteristic
singlet signal of the C-2 proton of the quinazoline nucleus at δ
7.90, whereas the two OH protons were detected at δ 10.05
and 12.02. In addition, the melting point was consistent with
the reported one.57,58 The phenolic OH at the C-6 position of
(19) was selectively protected by acetylation via heating with
H
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Figure 7. Protein kinase profiling for compounds (10a, 10b, 15a, 15e, 15l, 17d, and 25f) at 10 μM single dose concentration, represented by %
activity or % inhibition, against a panel of 20 kinases in comparison with two reference compounds lapatinib and sorafenib using the [γ-32P]ATP
radiometric assay method. (−) values: inhibition of target activity; (+) values: activation of target activity; >−25% changes in activity compared to
the control were considered to be significant inhibition.
acetic anhydride at 100 °C for 2 h in the presence of pyridine
as a basic catalyst to afford 4-hydroxy-6-quinazolinyl acetate
(20).57 The 1H NMR spectrum of compound (20) exhibited a
singlet signal at δ 2.31 attributed to the three protons of the
CH3 group accompanied by the disappearance of the C-6
phenolic OH signal at δ 10.05. Chlorination of (20) was
carried out by heating with thionyl chloride.57,59 The freshly
prepared 4-chloro derivative (21) was unstable; therefore, it
was immediately coupled with the appropriate aniline
derivative (4a−c) by long-term reflux in isopropanol, in the
presence of trimethylamine (TEA),44,57 or in N-methyl
pyrrolidinone.42 However, using the reported methods, the
desired compounds (22a−c) were obtained in very low yields
(20−42%) with multispot side products. Thus, we significantly
optimized the coupling reaction using acetonitrile/K2CO3,
either at room temperature (for 22a) or under reflux (for
22b,c), to afford the desired intermediates in good purity and
with higher yields (60−90%). The melting point of compound
22a was consistent with the reported one (290−292 °C).57
1
Compounds 22b and 22c were confirmed by their H NMR
spectral data with the appearance of the signals of eleven
aromatic protons at δ 6.74−8.69. Hydrolysis of the acetylated
intermediates (22a−c) to the corresponding quinazolin-6-ol
derivatives (23a−c) was conducted using aqueous ammonia in
methanol (1:3) at room temperature up to 12 h. The melting
point of the compound (23a) was consistent with the reported
one (>300 °C).57 The new compounds (23b and 23c) were
confirmed by their 1H NMR spectra showing a singlet
exchangeable signal at δ 10.14 of the 6-OH proton with a
concurrent disappearance of the singlet signal at δ 2.38 and the
13C DEPT signal at δ 21.21−21.22 of the CH3 group.
Alkylation of compounds (23a−c) was carried out by reflux
with 1-bromo-3-chloropropane in the presence of K2CO3 and a
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catalytic amount of tetrabutylammonium bromide
(TBAB).57,60
selected because the JAK2-STAT3 signaling pathway is
known as a crucial signaling pathway for regulating cancer
progression and metastasis.67 C-kit, c-Met, FLT1,4, KDR, and
PDGFRα kinases are involved in angiogenesis and were
reported as effective targets in the treatment of solid cancers.
MAPKAPK2, ROCK2, and p38α kinases were used as
examples of different target serine/threonine kinases to explore
selectivity to our target tyrosine kinase.
Selection Criteria for the Test Compounds for Kinase
Profiling. Seven compounds were screened for kinase profiling
including the lead compound (10a) and its morpholino
analogue (10b). Other different compounds bearing an
acetamido spacer with a 4-phenoxyaniline moiety (15a, 15e,
and 15l), compound 17d bearing a benzamido spacer and a 4-
phenoxyaniline moiety, and compound 25f with a propoxy
spacer and a 4-phenoxyaniline moiety. The selected candidates
are representative of the optimized analogues of the lead
compound (10a; generated by CADD).
Throughout this study, the inhibition of target activity by the
compound was given negative (−) values while activation of
target activity was given positive (+) values, and only >25%
changes in activity compared to the control were considered to
be significant. Six out of 20 kinases tested by compound 17d
exhibited moderate-to-high inhibition scores ranging from −40
to −98% including HER4 (−98%), HER2 (−92%), EGFR
(HER1) (−70%), ROCK2 (−47%), JAK2 (−47%), and ABL1
(−40%), whereas two kinases showed an activation, including
c-Kit (31%) and FLT4 (57%), as shown in Figure 7.
The new compounds (24b and 24c) were elucidated
spectroscopically through the appearance of two triplets at δ
4.26−4.28 and δ 3.85 and one pentet at δ 2.25 corresponding
to the six protons of the chloropropoxy moiety, along with the
disappearance of a singlet signal at δ 10.14 of the 6-OH proton.
In addition, their 13C DEPT spectra revealed three inverted
signals at δ 32.13−32.15, δ 42.43−42.46, and δ 65.59−65.61
which represent three CH2 groups of the chloropropoxy
moiety. The target compounds (25a−g) could be prepared
through nucleophilic substitutions on (24a−c) with the
appropriate secondary amines in dry acetonitrile, by heating
under reflux in the presence of anhydrous K2CO3 a catalytic
amount of potassium iodide.61 In the current study, the target
compounds (25a−g) were synthesized by refluxing the mixture
of compounds (24a−c) with 100 equiv of appropriate
secondary amine, including N-methyl piperazine, morpholine,
and diethylamine. Compounds (25a−g) were afforded in
excellent yields (76−94%) and high purity. On the other hand,
because of the high nucleophilic reactivity of ethanolamine,
compound 25h could not be prepared under the same reaction
condition adopted for (25a−g), where multispot degradation
byproducts were obtained. Therefore, compound 25h was
prepared by heating under reflux a mixture of intermediate 24c
and excess ethanolamine (10 equiv) in MeOH, rather than the
solvent-free reaction, in the presence of a catalytic amount of
1
TEA to afford compound (25h) with 42% yield. H NMR
spectra of compounds 25a and 25b exhibited singlet signals at
δ 5.14 and δ 5.25, respectively, for their characteristic benzylic
protons (OCH2C6H4F) with a concurrent appearance of two
broad singlets at δ 2.48 and δ 3.74 for the eight protons of the
morpholino moiety or at δ 2.30 and δ 2.38 for the eight
protons of the piperazino moiety, respectively. In addition, the
six protons of the propoxy linker were detected as two triplets
and one pentet at δ 1.90−4.18. Moreover, 13C DEPT spectra
revealed six signals for the CH2 groups, whereas compounds
25c−h (missing benzylic protons) were confirmed by their
aliphatic signals of the corresponding secondary or primary
amines.
In Vitro Protein Kinase Inhibition Assay. The protein
kinase profiles of seven compounds (10a, 10b, 15a, 15e, 15l,
17d, and 25f) against a panel of 20 protein kinases using the
[γ-32P]ATP radiometric assay method were evaluated by
Kinexus Bioinformatics Corporation, Vancouver, British
Columbia, Canada. Two reference kinase inhibitors on the
phosphotransferase activity were used including lapatinib
(EGFR and HER2 dual selective TKI) and sorafenib
(nonselective potent multikinase inhibitor). This assay was
performed at 10 μM concentration in a single measurement by
employing the standardized assay methodology outlined in the
Experimental Section part.
Rationale for Selection of the 20 Kinase Panels. The
EGFR (HER1), HER2, and HER4 kinases were selected as the
most common targets in therapy of aggressive BC. ABL kinase
was selected as an antitarget kinase in BC which was reported
to negatively modulate HER2+ BC progression in vivo.62,63
Meanwhile, BRAF, ERK1, and ERK2 kinases were picked out,
being a downstream signal transducer in the EGFR family. The
SRC activity was reported to be the main mode of resistance to
Herceptin as the first-line therapy for HER2+ BC.64 The FAK
is overexpressed and activated in several advanced-stage solid
cancers including multidrug-resistance BC.65,66 JAK2 was
For compound 15a, four kinases revealed moderate-to-high
inhibition scores ranging from −33 to −97% including HER4
(−97%), HER2 (−92%), EGFR (−67%), and JAK2 (−33%).
Similar to 17d, c-Kit (46%) and FLT4 (47%) were moderately
activated in the presence of the compound 15a as well as
ERK1 (32%). In the presence of 15e, five kinases were
inhibited ranging from −37 to −94% including HER4 (−94%),
HER2 (−91%), EGFR (−71%), JAK2 (−58%), and ABL1
(−37%). The other five kinases were activated, including KDR
(38%), SRC (38%), FLT1 (51%), PDGFR alpha (82%), and c-
Kit (115%). Compound 15l inhibited five kinases from
moderate-to-high level ranging from −49 to −99% inhibition,
including HER4 (−99%), HER2 (−93%), EGFR (−78%),
JAK2 (−70%), and ABL1 (−49%), while two kinases were
activated, including c-Kit (43%) and PDGFR-α (86%). Testing
of compound 25f revealed moderate-to-high inhibitions
ranging from −65 to −98% against 5 kinases including
HER4 (−99%), HER2 (−92%), JAK2 (−87%), EGFR
(−65%), and ALB1 (−65%), with activations noted with c-
Kit (39%), KDR (40%), ERK1 (41%), and PDGFR-α (115%).
Compound 10a exhibited high inhibition against HER4
(−98%), HER2 (−93%), ABL1 (−86%), JAK2 (−86%), and
EGFR (−78%), whereas it activated KDR (32%) and PDGFR-
α (54%). Similarly, compound 10b showed high inhibition of
HER4 (−97%), HER2 (−92%), ABL1 (−86%), EGFR
(−81%), and JAK2 (−74%) and moderate activation toward
KDR (46%), FLT1 (49%), and PDGFR-α (55%). Regarding
the reference compounds used in this study, sorafenib as a
potent nonselective multikinase inhibitor was tested at 10 μM
against the same panel of 20 protein kinases. It showed strong
inhibitions against ABL1 (−97%), FLT4 (−97%), JAK2
(−94%), and B-RAF (−92%), whereas it revealed moderate
inhibitions against FLT1 (−78%), EGFR (−74%), c-Kit
(−63%), HER4 (−60%), PDGFR-α (−55%), and P38-α
(−46%). Lapatinib was used as a selective dual kinase
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Figure 8. Docking of compound 17d (ball and stick, colored by element) into human HER2 kinase (PDB code: 3PP0) within an rmsd of 3.1 Å
from the native cocrystallized ligand (03Q). It exhibited two hydrogen bonds (green-dotted lines) with M801 and S728 in addition to multiple
hydrophobic interactions. The color codes are hydrophobic (π−alkyl and alkyl−alkyl): ---; hydrophobic (π−π): ---; hydrophobic (π−σ): ---;
halogen acceptor bonding: --- m-chloro atom interacts hydrophobically by a halogen acceptor with L796 and K753 residues.
inhibitor, which interrupts the HER2/neu and EGFR path-
ways. Five out of 20 tested kinases were moderately to strongly
inhibited including HER4 (−99%), JAK2 (−97%), ABL1
(−90%), EGFR (−80%), and HER2 (−52%), while c-Kit,
FLT1, KDR, and PDGFR-α kinases were moderately to highly
activated within 37−86%, and these activations increase the
possibility that in some cancer patients, lapatinib could in
principle exacerbate their cancer.
native bound ligand. Compound 17d was docked into HER2
kinase by two hydrogen bonds between its quinazoline-N1H
and terminal ethanolamine-OH and NH moiety of M801 and
NH of S728 amino acids, respectively. Besides, hydrophobic
interaction plays a crucial role in the binding of compound 17d
into the intensively hydrophobic HER2 kinase domain, where
its planar quinazoline ring interacts hydrophobically with L726,
L852, Ala751, and M801 by π−σ and π−alkyl interactions, as
clearly depicted in Figure 8. Moreover, its m-chloro-6-aniline
moiety interacts comprehensively by π−alkyl interactions with
the V734 and K753 bond, whereas the m-chloro atom interacts
hydrophobically by the halogen acceptor with L796 and K753
residues. Noticeably, the terminal phenoxy moiety was deeply
harbored into the hydrophobic HER2 selectivity pocket
through four π−π and π−alkyl interactions with F864,
M774, L785, and L796. Accordingly, compound 17d
demonstrated a GoldScore (external vdw) of 62.94.
In Vitro HER2 Kinase Inhibitory Activity (IC50) against
Wild and L755S Mutant Kinases. The ADP-Glo Assay
method was performed for kinase profiling of five compounds
(10a, 10c, 15i, 17d, and 25b) to evaluate their IC50 using
concentrations 0.5 nM to 25.0 μM. Two protein kinases
(HER2 wild-type and HER2 L755S mutant) were used in a
single measurement. Lapatinib was used as a positive control at
the same used concentrations.
The abovementioned results indicated that our newly
synthesized compounds revealed a significant selective
inhibition against HER2 and HER4 kinases within −92 to
−99% and to a moderate extent against the subfamily closely
related to RTK EGFR (HER1) within −67 to −81%. These
results highly support our design for selective HER2 kinase
inhibitors for the treatment of aggressive BC. Furthermore, the
test compounds exhibited moderate-to-substantial kinase
inhibitory effects against ABL1 and JAK2 kinases within −25
to −86% and −33 to −87%, respectively. In comparison with
lapatinib as a dual EGFR/HER2 kinase inhibitor, compounds
10a, 15a, and 17d and to a lower extent 10b are considered as
more safe compounds in activating c-KIT, FLT1, KDR, and
PDGFR-α kinases. Various studies reported that PDGFR
expression (including PDGFR-α and β and both show similar
kind of functions) is associated with poor prognosis of BC
patients via activating of Stat3 to support BC initiation and
progression.68−71 Accordingly and in comparison with
lapatinib (activating PDGFR-α by 56%), compounds 15a
and 17d (affecting PDGFR-α by 18% and −4, respectively) are
considered as the safest and potent compounds for the
treatment of aggressive HER2+ BC.
In Vitro HER2 Kinase Inhibitory Activity against Wild
Type. Compounds 10c, 15i, 17d, and 25b revealed the most
potent activities against HER2+ BC cell lines (BT474 and
AU565; IC50: 0.08−0.20 μM), in comparison with the lead
compound 10a (IC50: 0.22−0.24 μM), respectively.
The potent inhibitory activity of compound 17d against
HER2 kinase (−92%) is highly correlated with its robust
binding affinity into human HER2 kinase (PDB code: 3PP0)
with a GoldScore fitness of 92.63 compared to 84.59 for the
Out of these compounds, 15i, 17d, and 25b were further
selected by NCI for 5-dose assay against NCI60 cancer cell
lines. Therefore, these compounds were selected for further
evaluation of their IC50 against wild-type HER2 kinase. At the
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Figure 9. % IC50 graph and calculations for the five compounds (10a, 10c, 15i, 17d, and 25b) and lapatinib tested against HER2 wild-type kinase
using the ADP-Glo assay method at (A) concentrations 0.01−25.0 μM and (B) concentrations 0.5−100 nM.
initially used concentrations (0.01−25 μM), all the test
compounds exhibited high inhibitions (50−91%) greater
than lapatinib (14−88%; IC50 = 95.5 nM) against HER2
wild-type kinase, as shown in Figure 9A. Therefore,
concentrations of 0.5−100 nM were further used to calculate
the IC50 of the test compounds. At 100 nM, compound 25b
revealed the highest inhibition (91%) and at 0.5 nM, all the
compounds exhibited inhibition of HER2 wild-type kinase at
3−25%, as shown in Figure 9B. All five test compounds (10a,
10c, 15i, 17d, and 25b) have IC50 scores ranging from 5.4 to
12.0 nM. In all cases, IC50 values for the test compounds were
far lower than the IC50 value for lapatinib 95.5 nM, as listed in
Table 1.
tested with HER2 L755S mutant kinase as the highest
inhibition score was only 39%.
It is known that the L755S mutation correlates with
activated HER2 signaling and transformation, but this may
require complementary mutations.76 It has also been
previously reported that HER2 T733I and L755S mutations
are associated with resistance to inhibition by lapatinib in
vitro.77−79
Antiproliferative Activity Assay. The antiproliferative
activity of the newly synthesized compounds was conducted
by WST-8 assay against a panel of four different HER2-
expressing BC cell lines. The selected panel has different
genetic backgrounds including wild-type luminal AU565 cell
line with abnormally high expressing HER2,80 TPBC
(HER2+/ER+/PR+) BT474 cells, estrogen and progesterone
receptor-positive (ER+/PR+) MCF7 cells, and triple-negative
(MDA-MB-231) cells. These four BC cell lines represent two
different genetic backgrounds of tumor suppressor p53,
including AU565/p53 wild type (wt), BT474/p53 mutant
(mt), MCF-7/p53 (wt), and MDA-MB-231/p53 (mt).
Phenotyping and molecular features of the cell lines used in
our study are listed in Table 3.
Table 1. IC50 of Compounds 10a, 10c, 15i, 17d, and 25b in
Comparison with Lapatinib Tested against HER2 Wild-
Type Kinase at Concentrations Ranging from 0.5 nM to 25
μM Using the ADP-Glo Assay Method
IC50
(nM)
95% confidence
SEM
(nM)
intervals
R2
DOF
10a
10c
15i
17d
25b
lapatinib
10.4
6.1
5.4
12.0
8.3
95.5
5.512−19.430
4.679−7.969
4.291−6.666
8.412−17.114
3.757−18.404
43.97−207.40
1.159
1.064
1.052
1.085
1.202
1.276
0.9958
0.9993
0.9995
0.9989
0.9922
0.9939
5
5
5
5
5
5
Lapatinib is an FDA-approved dual EGFR/HER2 TKI for
the treatment of HER2+ metastatic BC was used as a positive
control.
Our compounds revealed statistically significant selective
and potent antiproliferative activities against HER2+ BC cells
(AU565 and BT474) of mean SEM (0.295 0.03207 μM)
compared to HER2− BC cells (MCF7 and MDA-MB-231) of
In Vitro HER2 Kinase Inhibitory Activity against L755S
Mutant Type. HER2 pathway reactivation by the HER2/
L755S mutation was found to be the common somatic
mutation which constitutes a mechanism of acquired resistance
to lapatinib-containing HER2-targeted therapies in preclinical
HER2+ BC models.72 The most frequently altered codon in
HER2 exon 19 that encodes the tyrosine kinase intracellular
domain was L755 (10 mutations, L755S);73 therefore, the
kinase domain mutant HER2 L755S was used in this study in
which HER2 is mutated in 14.05% of breast carcinoma patients
with ERBB2 L755S present in 0.74% of all breast carcinoma
patients,74,75 as depicted in Figure 10. However, none of the
compounds showed a strong inhibition with the HER2-L755S
mutant kinase, and the inhibitions ranged from 12% at the
lowest concentration of 0.1 μM of the test compounds up to
the highest inhibition of 39% at a concentration of 25 μM
(Table 2). The lapatinib followed the same pattern of
inhibition as the test compounds. According to these results,
there were no IC50 values determined for the compounds
mean
SEM (5.817
0.9121 μM). Notably, all the
synthesized compounds were tested against human metastatic
high expressing HER2 BC cells (AU565) and revealed potent
antiproliferative activities with an IC50 of 0.099−0.923 μM
range. Compounds 10b, 15h, 15i, 15k, 25a, and 25b are highly
potent with IC50 less than 0.2 μM, as shown in Table 4.
Remarkably, compound 25b (IC50 = 0.099) showed the most
potent activity and most comparable one to lapatinib (IC50
=
0.064 μM). To a lower extent, 75% of other compounds
exhibited antiproliferative activity less than 0.5 μM in the range
of 0.205−0.456 μM against the same cell line.
These results indicate that our designed compounds
significantly target HER2 kinase, which is highly expressed in
AU565 BC cells. Interestingly, 23 out of 27 (85%) of the
designed compounds showed an improved IC50 of 0.081−
0.540 μM against triple-positive BT474 BC cells with more
potency than HER2 BC cells (AU565). These results also
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Figure 10. Top disease cases with HER2 L755S (data retrieved from refs 66 and 67).
and comparable activities to lapatinib with an IC50 of 0.091,
0.081, 0.081, and 0.083 μM, respectively. The potent
antiproliferative activity of compound 25b against HER2+
BC cells is a reflection of its high binding affinity (GoldScore
fitness 90.70) into human HER2 kinase compared to 84.59 for
the native bound ligand. Compound 25b was docked
proximately superimposed on the native cocrystallized 03Q
ligand within a root-mean-square deviation (rmsd) of 1.1 Å. It
bound to the binding site of the titled kinase by one hydrogen
bond between its quinazoline-N1H and HN moiety of M801 as
a key amino acid, as shown in Figure 12. Despite compounds
15h, 15i, 25a, and 25b that showed the lowest IC50,
compound 15d displayed the highest augmentation of
antiproliferative activity by targeting triple-positive BT474
BC cells (IC50 = 0.107 μM) of ∼6.0 times more potency than
AU565 (0.663 μM). The antiproliferative activities of the
tested compounds were noticeably diminished using other BC
cells lacking HER2 kinase such as estrogen and progesterone
receptor-positive (ER+/PR+) MCF7 cells and triple-negative
(MDA-MB-231) cells (IC50 ranges from 0.321 to 18.119 and
0.737 to 30.162 μM, respectively). In contrast, the IC50 ranges
of the HER2+ BC cells (AU565 and BT474) are from 0.099 to
0.923 and 0.081 to 0.540 μM, respectively. This means that
MCF7 and MDA-MB-231 cells are less sensitive to the
inhibitory effects of the tested compounds than the BC cells
harboring HER2 kinase. Nevertheless, the majority (74−89%)
of our designed quinazoline derivatives exhibited superior
Table 2. % Activity Change in Compounds 10c, 15i, 17d,
and 25b in Comparison with Lapatinib Tested against
HER2 L755S Mutant Kinase at Concentrations Ranging
from 0.1 to 25 μM Using the ADP-Glo Assay Method
0.1 μM 0.5 μM 1.0 μM 5.0 μM 10 μM 25 μM
10c
15i
17d
25b
−16
−12
−15
−12
−14
−21
−20
−19
−19
−18
−26
−20
−20
−22
−23
−29
−24
−22
−25
−31
−32
−33
−27
−31
−35
−33
−28
−29
−39
−37
lapatinib
indicate that the quinazoline derivatives selectively target
HER2 kinase in addition to other oncologic pathways, which
have been investigated and discussed under apoptosis assay
and western blotting. Noticeably, multitargeting of the triple-
positive BT474 BC cell was detected by 85% of the tested
compounds. This augmented effect was noticed to a higher
extent for compounds 15d, 15l, 17a, 25d, 25f, and 25h up to 6
times antiproliferative activity compared to their IC50 toward
AU565 BC cells (HER2+), whereas compounds 10b,c, 15a−c,
15e,f,h,i,k, 17b−d, 25a−c, 25e, and 25g exhibited a lower
boost (up to 2.0 times) of antiproliferative activity by targeting
other receptors/pathways. Likewise to their effects against
AU565 cells, compounds 10b−c, 15d,h,i,k,l, and 25a,b,f
exhibited high antiproliferative activity toward BT474 cells
with IC50 ≤ 0.2 μM, as depicted in Figure 11. Strikingly,
compounds 15h, 15i, 25a, and 25b revealed the most potent
Table 3. Characterization, Molecular Information, Culture Conditions, and Clinical Features for the Four BC Cell Lines Used
a
80
in Our Study; the Information was Retrieved from Ref 73
cell line
AU565
BT474
MCF7
MDA-MB-231
HER2
ER
PR
P53
origin
subtype
tumor
culture medium
references
81,83
+
+
−
−
−
+
+
−
+
+
NA
mt
wt
metastasis
primary
metastasis
metastasis
H
LB
LA
TNB
AC
DMEM/F12
DMEM/F12
RPMI, DMEM
RPMI, DMEM
81,82,84−87
7,9,81,83
IDC
IDC
AC
3,4,7,9,86
−
−
mt
a
HER2: human epidermal growth factor receptor 2; ER: estrogen receptor; PR: progesterone receptor; NA: not available; wt: wild type; mt:
mutant type; H: HER2 positive; LA: luminal A; LB: luminal B; TNB: triple-negative B; RPMI: Roswell Park Memorial Institute (RPMI) 1640
medium; DMEM: Dulbecco’s modified Eagle medium; AC: adenocarcinoma; and IDC: invasive ductal carcinoma.
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Table 4. Antiproliferative Activity (IC50; μM) of Compounds 10a−25h against Four BC AU565, BT474, MCF7, and MDA-
MB-231 Cell Lines
inhibition of MCF7 and MDA-MB231 cells than lapatinib.
Compounds 10c, 15d, 25b, 17b, 25d, and 25f−h inhibited cell
proliferation of MCF7 cells with IC50: 0.321−1.933 μM,
implying their greater potency (up to 20 times) more than
lapatinib (IC50: 6.301 μM), and specifically, compounds 15d
(IC50: 0.321 μM) and 25d (IC50: 0.913 μM) exhibited the
lowest IC50 values. Remarkably, the antiproliferative activities
of compounds 15d, 15f, 15i, 15k, 17b, 25b, 25d, and 25f−h
against MCF7 cells (IC50: 0.321−2.647 μM) are highly
consistent with their augmented antiproliferative effects
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Figure 11. Dose−response curves for cell viability by WST-8 assay of AU565 and BT474 cells and their corresponding IC50 values (standard error
are given). Cells were treated with compounds 10c, 15d, 15i, 17d, and 25b at concentrations 0.01−100 μM for 72 h. Compounds were tested in
three independent experiments.
shown toward AU565 and BT474 breast cell lines. These
results support the suggestion that our compounds are
selectively targeting HER2+ (in AU565 cells) and get boosted
by targeting other receptors as shown in triple-positive
(HER2+/ER+/PR+) BT474 cells, and to a lower extent,
they can influence the estrogen and progesterone receptors
(ER+/PR+) as depicted in MCF7 cells in the absence of
HER2 as a main target. On the other hand, the majority of the
tested compounds (∼67%) displayed poor antiproliferative
activities against triple-negative (MDA-MB-231) BC cell lines
with IC50: 4.503−30.162 μM, which represents 15−100%
times higher IC50 than that against HER2-expressing cells
(AU565 and BT474). This is clearly explained by the absence
of HER2 and other boosting targets such as ER/PR expressed
in these cells. To a lower extent, compounds 10c, 15c, 15f,
15g, 15j, 17b, 25b, 25d, and 25f−h exhibited an acceptable
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Figure 12. Docking of compound 25b (ball and stick, colored by element) into human HER2 kinase (PDB code: 3PP0). It exhibited one hydrogen
bond (green dotted lines) with M801. It was docked within an rmsd of 1.10 Å from the native cocrystallized 03Q ligand (shown as yellow sticks).
IC50 in the range of 0.737−3.924 μM, that is, up to 13 times
less active than their corresponding activities against AU565
and BT474 breast cell lines. The reasonable explanation of this
slight activity against triple-negative (MDA-MB-231) BC cell
lines is the expression of 3% of HER2 kinase in comparison
with 100% in triple-positive (HER2+/ER+/PR+) BT474 cells,
whereas MDA-MB-231 cells expressed about 9 times EGFR
(also known as HER1) more than BT474 cells.88 Furthermore,
HER1 which is overexpressed in 15−30% of breast
carcinoma89,90 is frequently associated with poor prognosis in
triple-negative BC.91−93 The aforementioned facts might
explain the slight activities of some compounds toward
triple-negative (MDA-MB-231) BC cell lines.
In Vitro Primary Single-Dose (10 μM Concentration)
Screening against the NCI 60 Human Cell Panel. The
detailed discussion of single-dose and 5-dose screening toward
NCI 60 human cancer cell lines and the cytotoxicity mean
graphs from NCI is presented in the Supporting Information.
Single-dose testing was performed at 10 μM concentration
against 60 cell lines, representing nine tumor subpanels. As
depicted in Figure S1, many of the test-substituted quinazoline
compounds exhibited distinctive patterns of selectivity and
sensitivity against different NCI cell panels. Remarkably,
compounds 15f, 15i, 17c, 17d, 25b, 25d, and 25f−h exhibited
mean cell growth and lethality in the range of 11.85−19.30%
and −1.30 to −31.03%, respectively. Therefore, these potent
compounds in addition to 15g, 15k, 17a, and 25e were
selected by the DTP program in NCI for further 5-dose
screening using the mean growth percent of 47.7% as a
minimum criterion for inhibition. Compounds 15f, 15i, 17c,
17d, 25b, 25d, and 25f−h exhibited broad spectrum cell
growth inhibition and lethality against BC up to −78.7%. All
test compounds exhibited significant inhibition and lethality in
the range of 18.8 to −66.0% against MDA-MB-468 cells.
Compounds 15f, 17c, 25b, and 25g exhibited significant
lethality in the range of −11.7 to −78.7% against MCF7,
MDA-MB-231, HS578T, and MDA-MB-468 cells, whereas
compounds 15i, 17d, 25f, and 25h showed lethality in the
range of −18 to −74.6% against MCF7, MDA-MB-231, and
MDA-MB-468 cells. Unfortunately, none of the six human BC
cells used by the DTP in NCI was the HER2+ cell. Among
these six cancer cells, four cells, namely, MDA-MB-231,
HS578T, BT549, and MDA-MB-468, are characterized by the
lack of expression of the three main molecular markers (ER,
PR, and HER2) and referred as triple-negative,84,94 whereas
MCF-7 and T-47D breast cell lines are ER+/PR+, making
them an ideal model to study hormone response.84
In Vitro Five-Dose Full NCI 60 Cell Panel Screening.
Surprisingly, out of 20 compounds tested initially at a single
high dose (10−5 M), 13 compounds were selected to be
screened by five-dose concentration against the full NCI 60
cell panel. These compounds, namely, 15f, 15g, 15i, 15k, 17a,
17c, 17d, 25b, 25d, 25e, 25f, 25g, and 25h, exhibited
significant growth inhibitions in the single-dose screen. The
criteria for efficiently capturing these thirteen compounds and
progressing them to the 5-dose antiproliferative activity were
based on careful analysis of historical DTP screening data. In
this assay, the test compounds were incubated at five different
concentrations (0.01, 0.1, 1.0, 10, and 100 μM). Three dose−
response parameters (GI50, TGI, and LC50) were calculated for
each compound against each cell line (Table S2). Compounds
15i, 25b, and 25g demonstrated the most potent antiprolifer-
ative activities against the tested NCI 60 cell panel.
Compounds 15i and 25b exhibited the same pattern of
potency against MDA-MB-468, the triple-negative BC cells
with GI50 = 0.4 μM (Figure S2).
Sensitivity of the Test Compounds toward 9 Subpanels of
NCI 60 Cell Lines. To a higher extent, the test compounds 15i
and 25b exhibited impressive anticancer activity against most
of the NCI 60 cell lines representing nine different subpanels
with GI50 values between 0.4 and 2.1 μM. The best inhibitions
were revealed against BC cell lines with average GI50 values of
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Figure 13. Effect of optimized compounds on the HER2 signaling pathway in BC cells. AU565 (A) and BT474 (B) cells were treated with
compounds 15d, 15i, and 25b at concentrations of their IC50, 0.5 IC50, and 0.1 IC50 for each compound at 24 and 72 h. Lapatinib was used as an
authenticated control for the test compounds. Western blot analysis was performed and GAPDH was used as a reference loading control. Data
represented results collected from at least two independent experiments.
1.21 and 1.37 μM, respectively. Noticeably, BC MDA-MB-468
cells were inhibited selectively by many compounds including
15i, 15k, 17d, 25b, 25e, 25g and 25h with GI50 equal to 0.4,
0.2, 0.6, 0.4, 0.5, 0.9, and 0.6 μM, respectively. The selectivity
ratio (S) of the screening results of the selected test 13
compounds against NCI-60 human tumor cell lines at the five-
dose level was calculated as reported95 by dividing its average
GI50 toward a full panel of NCI 60 cell lines (MID-all) by its
average GI50 toward cell lines of a particular subpanel (MID),
that is, higher value indicates better selectivity. As per this
criterion, the majority (77−85%) of the test compounds
exhibited a remarkable selectivity profile toward BC (S: 1.05−
1.44), as shown in Table S3 and depicted in Figure S3.
Regarding the selectivity against a particular subpanel of NCI-
60 cell lines, compounds 15k, 17a, 17d, 25e, and 25f were
found to be the most significantly selective toward BC cells
with a selectivity ratio (S) of 1.24, 1.27, 1.44, 1.24, and 1.26,
respectively, as obviously shown in Figure S3.
Annexin V PI/FITC Apoptosis Assay. Based on the previous
reports29,96,97 that show that lapatinib induces cell cycle arrest
and apoptosis in HER2-positive BC cells, Annexin V
fluorescein isothiocyanate/propidium iodide (FITC/PI) apop-
tosis assay was conducted for the most potent compounds
revealing augmented effects in BT474 cells (10c, 15d, 15i,
17d, and 25b). These compounds were chosen for the
apoptosis assay based on their powerful antiproliferative
activities and/or augmented effect on targeting triple+
BT474 cells in comparison with their effects on HER2+
AU565 cells. The BT474 cells were treated with the selected
compounds and labeled by Annexin V PI/FITC, followed by
flow cytometric analysis. Compounds 10c, 15d, 15i, 17d, and
25b induced early-phase apoptosis of BT474 in a dose-
dependent manner by 3−10-fold compared to the control cells,
as shown in Figure S4. Remarkably, compound 10c at the
lowest concentration (0.2 μM) showed significant early-phase
apoptosis of about sevenfold induction compared to the
control cells. This activity was increased to 10-fold compared
to the control at 0.5, 1.0, and 5 μM. To a lower extent,
compounds 25b, 15i, 15d, and 17d exhibited early-phase
apoptosis up to sixfold at 0.5−5.0 μM concentration compared
to the control. Regarding the late apoptotic changes as
identified by PI staining, compound 10c showed a notable
increase (2−5-folds) in late apoptosis at 0.5−5.0 μM
concentrations compared to their counterparts. At the same
concentration, compounds 25b and 15i induced late-phase
apoptosis to a lower level (≤3-fold) compared to the control
cells, whereas compounds 15d and 17d have insignificant
changes in late-phase apoptosis of the BT474 cell lines. To
some extent, these results can partially explain the preferential
effects, particularly for compounds 15d and 15i on HER2+
AU565 cells (IC50: 0.663 and 0.135 μM) in comparison with
their effects on the triple-positive BT474 cells (IC50: 0.107 and
0.081 μM, respectively). Despite some of these apoptotic
results are partially consistent with the WST-8 and NCI 60 cell
line antiproliferative assays, we cannot speculate to correlate
these assays to each other. Additionally, these compounds may
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exhibit their antitumor effects by downregulating other
different hallmarks of cancer98,99 without affecting apoptosis.
Hence, further assays were conducted to investigate the
mechanism by which our compounds reveal their antitumor
effects through other pathways than the apoptosis. Further
Annexin V apoptosis assays using MCF7 and MDA-MB-231
cells were conducted with the potent compounds 10c, 15i, and
25b which revealed antiproliferative activities of IC50 ranges of
1.78−2.65 and 0.74−4.50 μM, respectively, as shown in Figure
S5. In contrast to their apoptotic results using BT474 cells, the
tested compounds (10c, 15i, and 25b at 5.0 μM) exhibited
insignificant effects on the early- and late-phase apoptosis of
MCF7 and MDA-MB-231 cell lines. These results anticipate
the specificity of our optimized compounds toward HER+
harboring cells.
At the same concentration and time point, compound 25b
inhibited HER2 expression comparable to lapatinib, as
depicted in Figure 13A.
The effect of the three test compounds and lapatinib on
HER2 expression as well as its downstream targets was further
investigated using the BT474 triple-positive BC cell line. Our
results showed that at IC50 concentrations, all compounds,
specifically compound 15i, and lapatinib revealed a substantial
reduction in HER2 expression after 24 h of treatment. In the
same way, compounds 15i, 25b, and lapatinib significantly
inhibited the activity of Akt as indicated by the notable
reduction of phosphorylated AKT after 72 h (Figure 13B). At
0.5 IC50 and 0.1 IC50 concentrations, most of the compounds
particularly compound 25b and lapatinib revealed a dramatic
inhibition in the activity of both Akt and ERK1/2 after 72 h
(Figure 13B). A close look at the overall immunoblotting
analysis using AU565 and BT474 cells indicates that all the
three tested compounds were able to inhibit the expression/
activity of HER2 and/or at least one of its downstream targets
in at least at a one-time point or three used concentrations.
Furthermore, the HER2 signaling pathway in BC BT-474
cells was evaluated using western blot analysis for compound
17d. The cells were treated at IC50 (0.204 μM), 0.5 IC50, and
0.1 IC50 concentrations within two-time points (24 and 48 h).
GAPDH was used as a loading control. Using BT474 BC cells,
our results indicated that at 0.1 and 0.5 IC50 concentrations,
compound 17d has particularly reduced HER2 and pAKT
expressions compared to the control 24 h, whereas this effect
was exhibited at IC50 at 48 h. To a lower extent, compound
17d exhibited a mild inhibitory effect against Akt and pERK1/
2 after 24 and 48 h, as shown in Figure 14.
Cell Growth Inhibition by Modulation of HER2
Downstream Signaling Pathways. Compounds 15d, 15i,
and 25b were selected to investigate their effects on the
expression of HER2 kinase and its downstream signaling
proteins. These three compounds were selected for further
assessment at the molecular level because they exhibited the
most potent antiproliferative activities against the HER2+ BC
cells, in addition to the significant augmented activity of
compound 15d in the triple (+) BT-474 cells. Moreover, the
apoptosis induction effect of these compounds was proved to
be enhanced. Furthermore, these compounds were the best
representatives for the derivatives bearing a 4-substituted
phenoxy anilino moiety (15d and 15i) and those bearing a 4-
substituted benzyloxy anilino moiety (25b). It is well-known
that the activation of HER signaling triggers several down-
stream signaling pathways, mainly mitogen-activated protein
kinase/extracellular-related kinase (MAPK/ERK) and phos-
phatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt path-
ways.100 The effects of the three test compounds as well as
lapatinib, as a positive control, on HER2 and its main
downstream signaling pathways were assessed by immunoblot-
ting. The expression level of HER2 and the activities of ERK1/
2 and Akt were compared in cellular lysates prepared from
HER2+ BC cell lines (AU565 and BT474) at different time
points and concentrations. The AU565 and BT474 cells were
treated with IC50, 0.5 IC50, and 0.1 IC50 concentrations for
each compound including lapatinib. The treated cells were
harvested from each concentration after 24 and 72 h, and HER
expression and activities of ERK1/2 and Akt were determined
by western blot analysis as described.101 Using AU565 BC
cells, our results indicated that IC50 concentrations of
compound 25b and lapatinib dramatically reduced the Akt
activity after 24 and 72 h of treatment. This inhibitory effect of
ERK kinase was significantly exhibited by lapatinib after 24 h
and to a lower extent after 72 h. Compound 25b and lapatinib
demonstrated mild inhibition of HER2 expression after 24 h,
whereas compounds 15d and 15i did not show any detectable
inhibitions on these kinases (Figure 13A). At a lower
concentration of 0.5 IC50, compound 15i showed a significant
reduction in HER2 expression compared to lapatinib at 24 h
but not at 72 h. Peculiarly, the tested compounds, as well as
lapatinib, inhibited Akt expression (total Akt) after 72 h.
However, only compound 25b and lapatinib were able to
demonstrate the inhibitory effect on Akt activity. At a
concentration of 0.5 IC50, compound 15i showed inhibition
of pERK expression almost similar to that of lapatinib after 24
and 72 h. Remarkably, compounds 15i and 25b reduced pAKT
competitively to lapatinib at 0.1 IC50 concentration after 24 h.
Figure 14. Western blot analysis for compound 17d to evaluate the
HER2 signaling pathway in BC cells (BT-474). The cells were treated
with different concentrations at IC50 (0.204 μM), 0.5 IC50, and 0.1
IC50 at 24 and 48 h. GAPDH was used as a loading control.
In Vivo Antitumor Efficacy for the Most Potent
Optimized Scaffolds Using the BT474 Athymic Nude
Mice (Foxn1nu/Foxn1+) Xenograft Mouse Model. Encour-
aged by the potent inhibitory activity of compounds 17d and
25b regarding their low IC50 against BT474 cell lines as
demonstrated by the WST-8 assay data, we were excited to
know whether this inhibition can be reproduced in the
preclinical BC model.
Rationale for the Selection of Compounds 17d and 25b
for the In Vivo Study. The selection criteria of compounds
17d and 25b for evaluation of their in vivo anticancer activity
depended on the overall activity of these compounds and
NOT limited to the antiproliferative activities. Regarding the
selection of compound 25b for in vivo testing, compounds 15i,
25a, and 25b revealed COMPARABLE or almost SAME
potency against BT474 cells, IC50 values (0.08 μM; Table 4).
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Figure 15. Effect of compounds 17d and 25b on tumor growth of BT474 cells in the preclinical mouse model. (A) Represented images of mice
implanted with 2 × 106 BT474 cells on the dorsal flank. After growing of the nodules, mice were randomized 5 mice per group and received the
vehicle and compound 17d and 25b, respectively; then, all mice were sacrificed 6 weeks after the initial treatment. The remarkable metastases in
the vehicle group are pointed out by red arrows. At the end of the experiment, tumor tissues were resected and preserved. (B) Tumor growth was
measured twice a week for 6 weeks after treatments. (C,D) Whole-body weight per each mouse was recorded twice a week and the resected tumors
were weighed per each animal. (E) Formalin-fixed paraffin-embedded xenograft tissues were stained with hematoxylin (H) and eosin (E), and
proliferative index Ki67 for the three groups as indicated. Significance considered at p < 0.05 regarding mice that received vehicle. The
magnification for H&E and Ki67 staining was 200× and for inserts was 400×.
S
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a
Table 5. Mouse Microsomal Stability Assay Result of the Test Compounds
test compound
R2
T1/2 (min) CLint(mic) (μL/min/mg) CLint(liver) (mL/min/kg) remaining (T = 60 min) (%) remaining (NCF = 60 min) (%)
17d
25b
testosterone
diclofenac
propafenone
0.9822
0.9749
0.9991
0.9978
0.9968
>145
57.4
5.0
60.0
2.1
<9.6
24.2
279.1
23.1
<38.0
95.7
1105.4
91.5
102.7
46.4
0.0
50.2
0.0
100.2
93.5
103.7
101.8
105.5
668.1
2645.6
a
NCF: no cofactor. No NADPH regenerating system is added to NCF samples (replaced by buffer) during the 60 min incubation. If the NCF
remaining is less than 60%, then non-NADPH-dependent metabolism occurs; R2: correlation coefficient; T1/2: half-life; CLint(mic): intrinsic
clearance; CLint(mic) = 0.693/T1/2/mg microsome protein per mL; CLint(liver) = CLint(mic) × mg microsomal protein/g liver weight × g liver weight/
kg body weight mg microsomal protein/g liver weight: 45 mg/g for all species; and liver weights: 88 g/kg for a mouse.
Compound 25b is considered as the best candidate in this
series (1) with similar activities, (2) being previously selected
by NCI as a promising candidate for 5-dose assay, (3) with its
potent inhibitory activity against HER2 kinase (IC50 of 8.3
nM), and (4) it shares the 4-benzyloxyaniline moiety of
lapatinib and could be considered as a well-optimized analogue
by a comparative in vivo study.
On the other hand, compound 17d was selected for in vivo
testing being; (1) revealing superior kinase profiling selectivity
over the targeted HER2 kinase, with a particular insignificant
effect on the off-targeted PDGFRα kinase (−4.0%), which was
overactivated by lapatinib (56%) and other test compounds
(18−115%), (2) 17d was selected by NCI for 5-dose assay and
revealed the highest sensitivity toward BC cell lines within
NCI60 cell lines, and (3) it has the 4-phenoxyaniline moiety
and could be considered as the best example for studying the
beneficial effect of this moiety on the in vivo efficacy, compared
to the 4-benzyloxyaniline moiety of lapatinib.
both groups were compared (p = 0.155, ANOVA/Tukey), as
depicted in Figure 15D. Histologically, the H&E stain of the
xenograft tumor tissues indicated that the cells are originated
from the xenograft and had the typical morphology of cancer
cells. This was unequivocally observed in the vehicle-treated
tissues with round cells and enlarged nuclei. Interestingly, large
areas of necrosis were observed in compounds 17d- and 25b-
sensitive tumors, as depicted by red arrows in Figure 15E
(upper panel), the higher magnification showing fibrosis and
inflammation in compounds 17d- and 25b-treated tumors. On
the other hand, the xenograft tissues stained by the ki67
antibody indicated that the proliferative cells are most
abundant in the vehicle control group, whereas they are
dramatically reduced on treatment by compound 17d and to a
less extent by compound 25b, as clearly depicted in Figure 15E
(lower panel). Interestingly, a metastasis case was observed in
the vehicle group but not detected in the mice treated by the
test compounds, as obviously pointed out with red arrows in
Figure 15A, whereas no metastasis was detected in the other
groups treated by the compounds 17d and 25b. This study
anticipates that compound 17d is a potential replacement
therapy for lapatinib to sensitize HER2-overexpressing BCs
and significantly reduce the tumorigenesis and metastasis of
the HER2+ BC. Nevertheless, the in vivo efficacy of compound
25b is less active as compared to compound 17d.
Fortunately, our findings are fit in-line with the reported
postulations103,104 that the superior in vitro activity of the
lipophilic 3-fluoro benzyl derivatives (as 25b) would not
translate into a better in vivo efficacy because of their higher
protein binding.
On the other hand, Mohamed et al. reported the O-
dealkylation on the 3-fluorobenzyl moiety of lapatinib as an
abundant metabolic pathway.105 Similarly, the metabolic study
of pyrotinib indicated that the O-dealkylated metabolite
exhibited weak activity against HER2 kinase because of the
loss of interaction with the HER2 back pocket.106 Accordingly,
to investigate the metabolic stability of compounds 17d
(bearing 3-fluorophenyl moiety) and 25b (bearing 3-
fluorobenzyl moiety), a microsomal stability assay was
conducted.
In this study, female athymic nude mice (Foxn1nu/Foxn1+)
were used to examine these two compounds 17d and 25b.
When the volume of the engrafted tumors reached around
100−150 mm3, the tested compounds were administered at 50
mg/kg, by oral gavage 5×/week, and continued over 55
days.80,81 To evaluate the treatment response of the BT474
xenograft growth in mice, the tumor growth curves of the three
tested groups were analyzed and compared among each other
using the R package TumGrowth according to Enot et al.
(2018).102 The tumor growth curves of the three groups were
subjected to type II ANOVA and pairwise comparisons across
groups using this statistical package. According to the statistical
data and as depicted in Figure 15A, oral treatment of the nude
female mice with 50 mg/kg of compound 17d substantially
inhibited the BT474 tumor growth compared to either the
vehicle control (p = 0.0013) or compound 25b treated group
(p = 0.01). Mice treated with compound 25b demonstrated
mild growth inhibition at the same dose; however, this
reduction did not reach a significant value when compared to
the vehicle control group (p = 0.5352), as shown in Figure
15B. More details were illustrated in Figure S6 illustrating the
average tumor growth rate (tumor size with error bars vs time)
for the three mice groups that received the vehicle, compound
17d and 25b, respectively. These data were further confirmed
when using the average tumor weight of the three treatment
groups after animals were sacrificed. The average tumor
weights for the 17d group (0.572 g) were significantly less than
that of the vehicle control group (2.046 g) and the p-value of
the comparison was 0.024 (ANOVA/Tukey test). The average
tumor weight of the vehicle control group (2.046 g) is still
higher than that of the 25b compound group (1.223 g).
However, there was no significant difference recorded when
CD-1 Mouse Microsomal Stability Assay. Compounds 17d
and 25b, 5 μL at a final concentration of 100 μM, were
incubated with pooled mouse liver microsomes (final
concentration 0.56 mg/mL) in phosphate buffer 0.1 M (pH
7.4) in a 37 °C water bath, and the reaction was initiated by
the addition of an NADPH cofactor. The reaction was allowed
for different time points, 5, 15, 30, 45, and 60 min and after
quenching of the reactions, each bioanalysis plate was sealed
and shaken for 10 min prior to LC−MS/MS analysis. The
equation of first-order kinetics was used to calculate T1/2 and
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Table 6. Optimization of the Docking into HER2 Kinase (PDB: 3PP0)
hydrogen bonds between compounds and
HER2 kinase
a
b
c
compd
trial 1
GoldScore
87.29
S(hb-ext)
S(vdw-ext)
flexible residues
atom of compd
quinazoline-N1H
terminal CH2O
amino acid
rmsd (Å)
3.8604
63.3455
M801
D863
T798
M801
D863
HN of M801
HN of C805
3.11
3.17
2.44
1.54
trial 2
trial 3
trial 4
94.26
89.75
91.40
3.9375
2.0682
6.4519
68.4816
66.9045
64.8467
quinazoline-N1H
pyridine-N
terminal CH2O
quinazoline-N1H
pyridine-N
HN of M801
HN of D863
HN of C805
HN of M801
HO of T862
HN of D863
HN of M801
HN of D863
HO of T862
OC of D863
HN of M801
OC of D863
HN of M801
HN of D863
M801
M801
pyridine-N
quinazoline-N1H
pyridine-N
pyridine-N
terminal CH2OH
quinazoline-N1H
terminal CH2OH
quinazoline-N1H
pyridine-N
trial 5
85.52
6.8702
61.0202
rigid
0.99
d
native ligand
a
a
b
S(hb_ext) GoldScore protein−ligand hydrogen bonding. S(vdw_ext): GoldScore external Vdw (van der Waals interactions between the protein
c
d
and ligand). rmsd: Root-mean-square deviation. 03Q: 2-(2-[4-((5-chloro-6-[3-(trifluoromethyl)phenoxy]pyridin-3-ylamino)-5H-pyrrolo[3,2-
d]pyrimidin-5-yl]ethoxy)ethanol).
Figure 16. Optimization of the docking protocol of the cocrystallized ligand (03Q) into HER2 kinase (PDB: 3PP0). The five docked trials are
labeled (1−5) in comparison with the native bound ligand (03Q, shown as yellow sticks) in which trial 4 (shown as ball and sticks) utilizing M801
as a flexible residue with 50,000 operations exhibited the best-optimized protocol with an rmsd of 1.54 Å and four hydrogen bonds.
CLint(mic) (μL/min/mg). Testosterone, propafenone, and
diclofenac were used as positive controls as reference
compounds with high and medium clearance, respectively.
As shown in Table 5, compound 17d that revealed higher
Molecular Docking. Docking Validation and Protocol
Optimization. The scoring function of the GOLD program
was validated by checking the extent of the resemblance of the
best-docked conformation of the cocrystallized ligand with the
reported biological method.107 Therefore, we initiated this
study by redocking of the cocrystallized ligand (03Q: 2-(2-[4-
((5-chloro-6-[3-(trifluoromethyl)phenoxy]pyridin-3-ylamino)-
5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethoxy)ethanol)) into chain
B (75% overall quality) of the HER2 kinase domain (PDB:
3PP0). The docking protocol was optimized by running five
trials of docking including rigid and flexible docking, as listed
in Table 6. Trial 4, involving a flexible docking with M801
T1/2 = >145 min compared to compound 25b T1/2 = 57.4 min
along with lower intrinsic clearance was observed with
compound 17d, CLint(mic) = <9.6 mL/min/kg compared to
25b, CLint(mic) = 24.2 mL/min/kg. Accordingly, compound
25b is likely to be rapidly cleared in vivo resulting in a short
duration of action. This underpins the less sufficient antitumor
activity of 25b compared to 17d in the in vivo animal model.
U
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Figure 17. SAR studies of the newly synthesized 4,6 disubstituted quinazolines.
residue and 50,000 operations, exhibited the best-optimized
protocol with an rmsd of 1.54 Å and four hydrogen bonds, as
shown in Figure 16. The binding modes for the 03Q native
ligand obtained in trial 4 were greatly consistent with its
reported cocrystal structure bound to HER2 kinase.
Compounds having acyclic polar residues (ethanolamine,
dimethylamine, and diethylamine) along with an acetamido
or propoxy spacer exhibited inferior activity of 2−3 times less
than compounds bearing a terminal morpholine or N-methyl
piperazine moiety. However, the reverse was observed in the
presence of a bulkier benzamide linker, where compounds with
acyclic polar residues exhibited 2−5 times more potential than
compounds bearing cyclic terminal residues.
GoldScore Fitness of the Designed Quinazoline Deriva-
tives into HER2 Kinase. The GoldScore algorithmic function
was applied in this molecular docking because it affords
superior docking results than the ChemScore function in
GOLD as a powerful molecular docking program.49 All the
docking results, including GoldScore fitness, S(hb-ext), S(vdw-
ext), hydrogen bonds between the ligand and HER2 kinase,
and rmsd between the docked pose and the native cocrystal-
lized ligand, are listed in the Supporting Information, Table S4.
Our compounds were docked into the human HER2 kinase
domain revealing Gold fitness scores ranging from 77.38 to
93.29. Compounds 10c, 15f, 15i, 15j, 17d, 25b, and 25h
exhibited the most noteworthy GoldScore fitness of 90.14−
93.29 in comparison with the cocrystallized compound 03Q of
91.40. The majority of the test compounds (17 out of 27) were
docked proximately superimposed on the native ligand within
≤2.50 Å. They docked into the ATP-binding site through up
to three hydrogen bonds to a higher extent with M801 (NH)
and C805 (NH) amino acids.
Structure−Activity Relationship. In this study, three key
structural features of the designed compounds were discussed.
These include the polar terminal residues at position 6, the
anilino substitution at position 4, and the flexible spacer
between the quinazoline ring and the terminal hydrophilic
moieties, as depicted in Figure 17. Regarding the antiprolifer-
ative assay, compounds 10b, 15a, 15e, 15h, 17a, 25a, 25c, and
25e bearing a morpholine terminal residue revealed potent and
selective activity against HER2+ BC cells (BT-474 and AU-
565). These compounds exhibited IC50 in sub-micromolar
concentrations (IC50 = 0.08−0.9 μM) with a noticeably
diminished activity against HER2− BC cells MCF7 and MDA-
MB-231 (IC50 = 5.5−30 μM). Meanwhile, compounds having
an N-methylpiperazine residue (10c, 15f, 15i, 17b, 25b, 25d,
and 25f) exhibited a broad spectrum of activity against BC cell
lines. Their antiproliferative strengths vary ranging from strong
(IC50 = 0.08−0.7 μM) against HER2+ BC cells to moderate
activity (IC50 = 0.7−2.5 μM) against HER2− BC cells.
Thus, the steric effect of the substituents at position 6 of the
quinazoline scaffold has a significant impact on the
antiproliferative activity, where these substituents will accom-
modate into the solvent-accessible region of HER2 kinase. It is
noteworthy that the methyl substitution on the phenoxyaniline
moiety at position 4 dramatically enhanced the cytotoxic
activity of compounds within 4 to 6 times higher than
substitution by a trifluoromethyl or fluoro moiety. Considering
that compounds have the same spacer and terminal residues,
compounds 15h, 15i, and 15k with a methyl substituent on the
phenoxyaniline ring at position 4 exhibited IC50 with an
average of 0.1 μM against AU565 BC cells. Meanwhile,
compounds 15e−15g and compounds 15a, 15b, and 15d
bearing a trifluoromethyl and fluoro group, respectively,
revealed an average of IC50 = 0.4 and 0.6 μM, respectively.
Regarding the protein kinase profiling assay, compounds
10a, 10b, 15a, 15e, 15l, 17d, and 25f demonstrated the same
pattern of inhibitory activity and selectivity against the HER
kinase family (EGFR, HER2, and HER4) with higher
particular potency against the target HER2 kinase (−91 to
93%) and with moderate-to-weak inhibition on other non-
target 17 kinases. Regardless of the type of the spacer or the
polar terminal residue, the test quinazoline derivatives bearing
4-substituted phenoxyaniline moieties (15a, 15e, 15l, 17d, and
25f) showed remarkable selectivity against the target HER2
kinase (−91 to −92%) and moderate inhibition against EGFR
(−65 to −78%). Compound 17d having a benzamide spacer
and ethanolamine terminal residue demonstrated the best
selectivity among the test compounds with −92% inhibition of
HER2, −70% for EGFR, −47% for ROCK2, −47% for JAK-2,
and −40% for ABL1 kinase. Nevertheless, the test quinazoline
derivatives (10a and 10b) substituted with a benzyloxyaniline
moiety at position 4 exhibited inferior selectivity against HER2
of −92 to −93%, EGFR of −78 to −81%, JAK-2 of −74 to
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−86%, and ABL of −86%. However, the tested compounds
10a, 10c, and 25b or 15i and 17d bearing benzyloxyaniline or
phenoxyaniline moieties at position 4 revealed superior HER2
inhibition with an IC50 of 10.4, 6.1, and 8.3 nM or 5.4 and 12
nM, respectively, compared to lapatinib IC50 of 95.5 nM. It is
noteworthy that the benzyloxyaniline moiety is metabolically
labile to O-dealkylation which may result in high clearance and
low in vivo activity, as observed in the in vivo efficacy of 25b.
Accordingly, we can conclude that the substitution at position
4 with a phenoxyaniline or benzyloxyaniline moiety mainly
affects the selectivity against HER2 kinase and the metabolic
stability in vivo. Additionally, the polar substitution at position
6 can also modulate the cellular activity of the tested
compounds.
IC50, and 0.1 IC50 for each compound. The apoptosis assay for
compounds 10c, 15i, 17d, and 25b revealed induction of the
early-phase apoptosis in BT-474 cells 3−10-fold compared to
the negative control. Furthermore, out of 20 compounds tested
at 10 μM single dose in the full NCI 60 cell panel, 13
compounds (15f, 15g, 15i, 15k, 17a, 17c, 17d, 25b, 25d, 25e,
25f, 25g, and 25h) were selected to be evaluated at five
concentration levels, where compound 17d demonstrated the
highest selectivity against BC. The SAR study expounded that
the terminal polar moieties not only affect the drug-likeness
and pharmacokinetic properties of the compound but also
modulate the cellular activity. We have noticed that
compounds bearing a morpholino moiety or its acyclic
analogue ethanolamine revealed potent and selective anti-
proliferative activities against HER2(+) BC cell lines (BT-474
and AU-565). Meanwhile, the N-methylpiperazino-bearing
compounds exhibited extended moderate activity against the
other HER2(−ve) BC cell lines. Inspired by the potent
antiproliferative and enzymatic activities of compounds 17d
and 25b bearing the 4-substituted phenoxyanilino and 4-
substituted benzyloxyanilino moiety, respectively, were se-
lected to be tested for their in vivo antitumor efficacy using the
BT-474 athymic nude mice (Foxn1nu/Foxn1+) xenograft model.
Even though compound 25b exhibited higher antiproliferative
activity in WST-8 assay (IC50 = 0.083 μM) than compound
17d (IC50 = 0.204 μM), both compounds behaved differently
in in vivo evaluation. Compound 17d substantially inhibited
the BT-474 tumor growth compared to either the vehicle
control or compound 25b-treated groups. This was attributed
to the higher T1/2 and lower intrinsic clearance of compound
17d (T1/2 = >145, CLint(mic) = <9.6 mL/min/kg), compared to
compound 25b (T1/2 = 57.4 min, CLint(mic) = 24.2 mL/min/
kg). Interestingly, metastasis was observed in the nude mice
treated by the vehicle which was not detected in the other
groups treated by compounds 17d and 25b. Altogether, we
eventually identified compound 17d as a promising candidate
for HER2+ BC therapy deserving further investigations.
CONCLUSIONS
■
In summary, novel 27 4,6-disubstituted quinazoline derivatives
were designed and synthesized guided by the binding mode of
the current dual EGFR/HER2 inhibitor, lapatinib. This design
was conducted through multistep reactions to incorporate
different flexible spacers at position 6 of the quinazoline ring,
namely, acetamido, methylbenzamido, or propoxy. This was
followed by a nucleophilic substitution with several polar
fragments. At position 4, the N-(4-(3-chloro-4-(3-
fluorobenzyl)oxy)phenyl) amino moiety of lapatinib was
replaced by the N-(4-(3-chloro-4-(3-substituted-phenoxy)-
phenyl)amino) moiety to properly fit into the lipophilic back
pocket with an enhanced selectivity against the target HER2
kinase. Increasing the selectivity against HER2 kinase was
assumed to minimize the EGFR inhibition-associated side
effects such as severe rash and diarrhea. The synthetic
methodologies involved in this project have been optimized
to report the most appropriate reaction conditions affording
the targeted and intermediate compounds at a higher yield and
purity. The in vitro protein kinase profiling for the lead
compound (10a) and the other representative analogues (10b,
15a, 15e, 15l, 17d, and 25f) at 10 μM single-dose
concentration against 20 kinases demonstrated a superior
significant selectivity than lapatinib. Our compounds revealed
inhibitions within a range of −92 to −99% against HER2 and
HER4 kinases and within −67 to −81% against EGFR
(HER1). Remarkably, the screened compounds specifically
17d exhibited negligible activation or even mild inhibition of
other nontarget kinases which were activated by lapatinib.
These results anticipate lower side effects and toxicity of
compound 17d. This preferential selectivity of the test
compounds was an encouraging result to evaluate the cytotoxic
activity of the newly synthesized quinazolines against HER2
BC cell lines and HER2-negative cells for comparison. The
majority of synthesized compounds demonstrated potent and
selective antiproliferative activities toward HER2+ BC cell line
AU-565. Furthermore, many compounds revealed augmented
activity against triple-positive cells (BT-474) with noticeably
diminished activity against HER2-negative BC cells (MCF7
and MDA-MB-231). Against wild-type HER2 kinase, the
privileged compounds (10a, 10c, 15i, 17d, and 25b)
demonstrated higher inhibitory activity against HER2 kinase
with IC50 values in the range of 5.4−12 nM compared to
lapatinib (95.5 nM). On the other hand, the western blot
analysis indicated that the phosphorylation of HER2 down-
stream signaling through AKT and ERK pathways was
impaired in BT-474 and AU-565 cells treated with compounds
15d, 15i, and 25b at different concentration points of IC50, 0.5
EXPERIMENTAL SECTION
■
Chemistry. This part is concerned with the synthetic schemes
adopted for the synthesis of the designed compounds and the
description of the synthetic chemical steps used, aided by a literature
survey for each route. All the synthesized compounds were purified
using flash column chromatography and then subjected to a
comprehensive spectrometric analysis including 1H NMR, 13C
DEPT, and 13C NMR. In addition, all 27 key target compounds
have been further analyzed by HRMS. Using integrated analytical
methods, the purity of the synthesized compounds was confirmed at
≥95% by ultrahigh-performance liquid chromatography/MS
(UHPLC/MS) and NMR methods. All different spectra of our
compounds have been uploaded in the Supporting Information for
publication. Thin-layer chromatography (TLC) analysis of reaction
mixtures was performed using Sigma-Aldrich silica gel 60 Å, F254
TLC plates, and visualized under UV. Flash column chromatography
was carried out on RediSep Rf-Teledyne ISCO cartilages, 230−450
mesh using CombiFlash Rf 200-Teledyne ISCO. The reaction
progress was monitored using an Advion Expression-L Mass
spectrophotometer using TLC plate extraction or ASAP techniques.
1H, 13C DEPT, and 13C NMR spectra were recorded in CDCl3 or
dimethyl sulfoxide (DMSO) at ambient temperature using a Bruker
300 MHz spectrometer. Coupling constants were expressed in Hz and
the abbreviations used for multiplicity are as follows: s = singlet, d =
doublet, t = triplet, q = quartet, p = pentet, m = multiplet, and br =
broad. 1H and 13C NMR chemical shifts are reported in ppm
downfield of tetramethylsilane and are, respectively, referenced to the
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residual solvent (δ = 7.26 and 77.36 ppm for CDCl3, δ = 2.54 and
40.45 ppm for DMSO-d6). High-resolution mass spectra (HRMS)
were obtained on LC/MS-TOFAgilent 6210 using electrospray
ionization (ESI). Melting points were recorded on a Stuart melting
point smp20 apparatus using Kimax Glass Capillary Tubes, 100 mm.
2-Chloro-1-((3-fluorobenzyl)oxy)-4-nitrobenzene (3a). To a
mixture of 2-chloro-4-nitrophenol (1a) (3.47 g, 20.0 mmol) and
potassium carbonate (2.76 g, 20.0 mmol) in DMF (20 mL), 3-
fluorobenzyl bromide (2a) (3.81 g, 2.47 mL, 20.2 mmol) was added.
After being stirred at 85 °C for 3 h, water (40 mL) was added and the
mixture was extracted with ethyl acetate (EtOAc). The organic layer
was washed with saturated brine (30 mL), dried over anhydrous
sodium sulfate (Na2SO4), and concentrated in vacuum to yield 3a
(4.10 g, 73% yield) as a light-yellow solid.108,109
2-Chloro-4-nitro-1-(3-(substituted)phenoxy)benzene (3b−
d). To a mixture of 2-chloro-1-fluoro-4-nitrobenzene (1b) (3.50 g,
20.0 mmol) and potassium carbonate (2.76 g, 20.0 mmol) in
acetonitrile (20 mL) was added appropriate phenol 2b−d (20.0
mmol). After being stirred at 85 °C for 5 h, the reaction mixture was
filtered while hot and the filtrate was concentrated under reduced
pressure to afford 3b−d.110,111
2-Chloro-1-(3-fluorophenoxy)-4-nitrobenzene (3b). (4.7 g, yield
89%), yellow solid.
2-Chloro-4-nitro-1-(3-(trifluoromethyl)phenoxy)benzene (3c).
(5.2 g, yield 83%), yellow solid.
2-Chloro-1-(3-methylphenoxy)-4-nitrobenzene (3d). (4.1 g, yield
78%), faint yellow oil.
3-Chloro-4-((3-fluorobenzyl)oxy)aniline (4a). To a suspension
of (3a) (3.6 g, 12.8 mmol) and reduced iron [2.3 g, 38.4 mmol (3 mol
equiv)] in ethanol (50 mL), a solution of calcium chloride (1.5 g, 12.8
mmol, 5 mL water) was added. The mixture was stirred at 85 °C for 8
h. The formed black solid was removed by filtration.
Then, the filtrate was centrifuged for 30 min and then filtered again.
The clear filtrate was concentrated under vacuum. The residue was
washed with water, filtered, dried, and purified by silica gel column
chromatography (eluent, EtOAc/hexane = 1:3) to yield 4a (2.5 g,
77% yield) as yellowish white crystals.44
(2-cyano-4-nitro-phenyl)-N,N-dimethyl-formamidine (6) as a yellow
solid (3 g, 90% yield, mp 153−155 °C, as reported).112
Synthesis of 4-(3-Chloro-4-(3-fluorobenzyloxyl)-
phenylamino)-6-nitroquinazoline (7). Compound (6; 2 g, 9.17
mmol) was held at reflux for 3 h with 3-chloro-4-((3-fluorobenzyl)-
oxy)aniline (4a) (2.3 g, 9.17 mmol) in glacial acetic acid (10 mL).
The formed precipitate was filtered while hot and washed with
diethylether (3 × 10 mL) to afford the corresponding nitroquinazo-
line derivative (7) as a yellow to orange solid (3.6 g, 92.5% yield, mp;
280 °C, as reported).110,113
Synthesis of 4-(3-Chloro-4-(3-fluorobenzyloxyl)-
phenylamino)-6-aminoquinazoline (8). To a suspension of the
6-nitro derivative (15; 2.12 g, 5 mmol) and reduced iron (0.85 g, 15
mmol) in ethanol (50 mL), a solution of calcium chloride (0.6 g, 5
mmol, 5 mL water) was added. The mixture was stirred at 90 °C for
12 h. The formed black solid was removed by filtration, and the
filtrate was centrifuged for 30 min and then was filtered. The clear
filtrate was concentrated under vacuum to afford 8 as a pure yellow
solid (97% yield, mp; 190 °C, as reported).100,110
2-Chloro-N-(4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-
amino)quinazolin-6-yl) Acetamide (9). To a solution of 6-
aminoquinazoline (8) (1.3 g, 3.2 mmol) in acetone (20 mL),
NaHCO3 (0.3 g, 3.5 mmol) was added. The reaction mixture was
stirred under nitrogen at 0 °C. This was followed by the dropwise
addition of solution of 2-chloroacetylchloride (0.4 g, 3.4 mmol) in
acetone (2 mL). The reaction mixture was stirred for 1 h at 0 °C.
The excess solvent was removed under reduced pressure. Cold
water (20 mL) was added to the remaining residue and the resulting
suspension was neutralized with a saturated solution of NaHCO3, if
necessary. The formed solid was then filtered, dried, and washed with
diethylether (3 × 15 mL) to afford compound 9 as a pure yellow
solid. (0.9 g, yield 61%, mp >300 °C); 1H NMR (300 MHz, DMSO):
δ 11.31 (s, 1H, NH, exchanged with D2O), 11.21 (s, 1H, NH,
exchanged with D2O), 8.97 (s, 1H, Ar-2-H), 8.82 (s, 1H, Ar-5-H),
8.06−8.03 (d, J = 8.6 Hz, 1H, Ar-8-H), 7.96−7.93 (d, J = 8.8 Hz, 1H,
Ar-7-H), 7.86 (s, 1H, Ar-2′-H), 7.61−7.59 (d, J = 7.9 Hz, 1H, Ar-6″-
H), 7.49−7.47 (d, J = 7.2 Hz, 1H, Ar-6′-H), 7.34−7.31 (m 3H, Ar-
5′,2″, 4″-H), 7.22−7.16 (t, J = 7.8 Hz, 1H, Ar-5″-H), 5.30 (s, 2H,
CH2, OCH2C6H4F), 4.44 (s, 2H, CH2, acetamido). 13C DEPT (75
MHz, DMSO): δ 150.81 (1C, Ar-2-CH), 131.04 (1C, Ar-5″-CH),
129.50 (1C, Ar-8-CH), 126.69 (1C, Ar-7-CH), 124.95 (1C, Ar-6″-
CH), 123.82 (1C, Ar-6′-CH), 122.53 (1C, Ar-2′-CH), 115.40 (1C,
Ar-5′-CH), 114.59 (1C, Ar-4″-CH), 114.39 (1C, Ar-2″-CH), 113.45
(1C, Ar-5-CH), 69.83 (1C, CH2, OCH2C6H4F), 43.89 (1C, CH2,
acetamido); MS (APCI+): m/z 471.1 [M + H]+.
Synthesis of N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)-
phenyl)amino)quinazolin-6-yl)-2-((2-hydroxyethyl)amino)-
acetamide (10a). A mixture of the chloroacetamido intermediate
(9) (0.47 g, 1 mmol) and 2-aminoethanol (0.25 g, 0.3 mL, 5 mmol)
in methanol (20 mL) was held at reflux for 4 h in the presence of
triethylamine (15 drops). Methanol was then removed under reduced
pressure, and the remaining residue was dissolved in dichloromethane
(DCM) and purified by flash column chromatography with
ethylacetate/MeOH (90:10%) as an eluent to yield compound 10a.
White solid, mp 140−145 °C, (0.28 g, yield; 57%); 1H NMR (300
MHz, DMSO-d6): δ 10.15 (s, 1H, NH, exchanged with D2O), 9.76 (s,
1H, NH, exchanged with D2O), 8.60 (s, 1H, Ar-2-H), 8.51 (s, 1H, Ar-
5-H), 8.07−8.04 (d, J = 9 Hz, 1H, Ar-8-H), 7.99 (d, J = 2.5 Hz (m-
coupling), 1H, Ar-2′-H), 7.78−7.75 (d, J = 9 Hz, 1H, Ar-7-H), 7.73−
7.69 (dd, J = 9.0, 2.5 Hz, 1H, Ar-6′-H), 7.51−7.44 (m, 1H, Ar-4″-H),
7.34−7.29 (m, 2H, Ar-5′,2″-H), 7.27−7.24 (d, J = 9, Ar-6″-H), 7.21−
7.16 (t, J = 9 Hz, 1H, Ar-5″-H), 5.25 (s, 2H, CH2, OCH2C6H4F),
4.68−4.65 (t, J = 4.9 Hz, 1H, OH, exchanged with D2O), 3.54−4.49
(q, J = 5 Hz, 1H, CH2, OHCH2CH2NH), 3.37 (s, 2H, CH2,
acetamido), 2.69−2.66 (t, J = 5.3 Hz, 2H, CH2, OHCH2CH2NH);
13C DEPT (75 MHz, DMSO-d6): δ 153.67 (1C, Ar-2-CH), 131.07
General Procedure for the Synthesis of 3-Chloro-4-(3-
(substituted)phenoxy)aniline (4b−d). A mixture of the appro-
priate nitro derivative (3b−d) (5 mmol) and stannous chloride (4.7 g,
25 mmol) in methanol (20 mL) was stirred at reflux for 2.5 h under a
nitrogen atmosphere. Excess methanol was removed under reduced
pressure, the remaining residue was dissolved in ethyl acetate (200
mL), and the obtained solution was neutralized with an aqueous
saturated solution of sodium bicarbonate and stirred for 1 h. The
resulting gelatinous precipitate was centrifuged for 15 min and then
discarded by filtration. The clear filtrate was transferred into a
separating funnel and the organic phase was separated. The aqueous
phase was extracted with ethyl acetate (2 × 20 mL), and the organic
fractions were combined, dried over anhydrous sodium sulfate, and
concentrated under vacuum to afford the corresponding aniline
derivatives 4b−d.
3-Chloro-4-(3-fluorophenoxy)aniline (4b). (0.97 g, yield 82%),
yellow oil solidifies on cooling.44
3-Chloro-4-(3-(trifluoromethyl)phenoxy)aniline (4c). (1.13 g,
yield 79%) brown oil solidifies on cooling.108,109 1H NMR (300
MHz, DMSO-d6): δ 7.57−7.52 (t, J = 7.9 Hz, 1H, Ar-5′-H), 7.38−
7.36 (d, J = 7.8 Hz, 1H, Ar-4′-H), 7.10−7.06 (m, 2H, Ar-2′,6′-H),
7.00−6.97 (d, J = 8.6 Hz, 1H, Ar-5-H), 6.75−6.76 (d (m-coupling), J
= 1.6 Hz, 1H, Ar-2-H), 6.61−6.57 (dd, J = 8.6, 1.7 Hz, 1H, Ar-6-H),
5.42 (s, 2H, NH2, exchanged with D2O).
3-Chloro-4-(3-methylphenoxy)aniline (4d). (0.87 g, 75%), color-
less oil.44
Synthesis of N′-(2-Cyano-4-nitrophenyl)-N,N-dimethylfor-
mimidamide (6). A stirred mixture of 2-amino-5-nitrobenzonitrile
(5) (2.5 g, 15.3 mmol) and N,N-dimethylformamide dimethylacetal
(5 mL, 37.6 mmol, 2.4 equiv) was heated under reflux for 2 h and
then cooled to room temperature. The resulting precipitate was
filtered, washed with ether (3 × 25 mL), and then dried to afford N′-
(1C, Ar-5″-CH), 130.96 (1C, Ar-8-CH), 128.84 (1C, Ar-7-CH),
127.24 (1C, Ar-6″-CH), 124.54 (1C, Ar-6′-CH), 123.77 (1C, Ar-2′-
CH), 115.00 (1C, Ar-5′-CH), 114.69 (1C, Ar-4″-CH), 114.34 (1C,
Ar-2″-CH), 112.13 (1C, Ar-5-CH), 69.83 (1C, CH2, OCH2C6H4F),
X
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60.83 (1C, CH2, OHCH2CH2NH), 53.16 (1C, CH2, acetamido),
52.18 (1C, CH2, OHCH2CH2NH); 13C NMR (75 MHz, DMSO): δ
171.18 (1C, CO), 167.28 (1C, Ar-4-C), 161.05 (1C, Ar-3″-C)
157.77 (1C, Ar-4′-C), 153.67 (1C, Ar-2-CH), 150.01 (1C, Ar-6-C),
146.91 (1C, Ar-8a-C), 140.11 (1C, Ar-1″-C), 136.81 (1C, Ar-1′-C),
133.86 (1C, Ar-3′-C), 131.06 (1C, Ar-5″-CH), 130.96 (1C, Ar-8-
CH), 128.84 (1C, Ar-7-CH), 127.24 (1C, Ar-6″-CH), 124.54 (1C,
Ar-6′-CH), 123.76 (1C, Ar-2′-CH), 115.72 (1C, Ar-4a-C), 115.28
(1C, Ar-5′-CH), 114.72 (1C, Ar-4″-CH), 114.33 (1C, Ar-2″-CH),
112.13 (1C, Ar-5-CH), 69.85 (1C, CH2, OCH2C6H4F), 60.84 (1C,
CH2, OHCH2CH2NH), 53.16 (1C, CH2, acetamido), 52.17 (1C,
CH2, OHCH2CH2NH); HRMS (ES+): m/z calcd for
C25H24ClFN5O3 [M + 1]+, 496.1552; found, 496.1547.
General Procedure for the Synthesis of Compounds 10b
and 10c. A mixture of compound 9 (0.47 g, 1 mmol) and the
appropriate secondary amine, 1-methyl piperazine or morpholine (50
mmol), was heated under reflux temperature for 2 h. The reaction
mixture was then cooled to room temperature, poured onto iced-
water (30 mL) portion wise while stirring, and 0.2 g of NaCl was
added, where a precipitate was formed. The resultant precipitate was
filtered, washed with water, and dried. Trituration of the dried
precipitate with diethylether (3 × 15 mL) afforded the desired
products (10b and 10c) as pure compounds with no need for further
purification.
OCH2C6H4F), 62.14 (1C, CH2, acetamido), 54.98 (2C, 2CH2,
piperazino), 53.28 (2C, 2CH2, piperazino), 46.20 (1C, CH3); 13C
NMR (75 MHz, DMSO): δ 169.01 (1C, CO), 164.28 (1C, Ar-4-
C), 161.05 (1C, Ar-3″-C), 157.81 (1C, Ar-4′-C), 153.76 (1C, Ar-2-
CH), 150.06 (1C, Ar-6-C), 147.02 (1C, Ar-8a-C), 140.19 (1C, Ar-1″-
C), 136.64 (1C, Ar-1′-C), 133.78 (1C, Ar-3′-C), 131.07 (1C, Ar-5″-
CH), 130.96 (1C, Ar-8-CH), 128.77 (1C, Ar-7-CH), 127.65 (1C, Ar-
6″-CH), 124.83 (1C, Ar-6′-CH), 123.79 (1C, Ar-2′-CH), 115.65
(1C, Ar-4a-C), 115.28 (1C, Ar-5′-CH), 114.70 (1C, Ar-4″-CH),
114.33 (1C, Ar-2″-CH), 112.63 (1C, Ar-5-CH), 69.83 (1C, CH2,
OCH2C6H4F), 62.14 (1C, CH2, acetamido), 54.99 (2C, 2CH2,
piperazino), 53.28 (2C, 2CH2, piperazino), 46.20 (1C, CH3); HRMS
(ES+): m/z calcd for C28H29ClFN6O2 [M + 1]+, 535.2025; found,
535.2024.
Ethyl-N-(2-cyano-4-nitrophenyl)formimidate (11). 2-Amino-
5-nitrobenzonitrile (5; 5 g, 30.6 mmol) was held at reflux in triethyl
orthoformate (50 mL) for 30 h in the presence of acetic anhydride
(10 drops). The reaction mixture was cooled to room temperature,
poured onto ice water, and stirred for half an hour. The formed
precipitate was filtered under vacuum and left to dry to yield
compound 11. Yellow solid, 5.6 g, yield 88%.114
General Procedure for the Synthesis of Compounds (12a−
c). Compound (11; 2.19 g, 10 mmol) was held at reflux for 7 h with
the appropriate aniline derivative (4b−d) (10.2 mmol) in glacial
acetic acid (20 mL). The reaction mixture was then poured onto the
ice/water mixture and stirred for 30 min with the addition of 0.2 g of
NaCl, and the precipitate formed was filtered, washed with
diethylether, and dried. The obtained solid was recrystallized from
methanol to afford the corresponding nitroquinazoline derivative
(12a−c) as a fluffy yellow solid.
N-(3-Chloro-4-(3-fluorophenoxy)phenyl)-6-nitroquinazolin-4-
amine (12a). (3.1 g, yield 76%); 1H NMR (300 MHz, DMSO-d6): δ
10.47 (s, 1H, NH, exchanged with D2O), 9.61 (s, 1H, Ar-5-H), 8.75
(s, 1H, Ar-2-H), 8.55−8.52 (d, J = 9 Hz, 1H, Ar-7-H), 8.23 (s, 1H,
Ar-2′-H), 7.94−7.88 (m, 2H, Ar-2″, 8-H), 7.46−7.38 (m, 1H, Ar-6′-
H), 7.31−7.28 (d, J = 8.5 Hz, 1H, Ar-4″-H), 6.99−6.93 (t, J = 8.4 Hz,
1H, Ar-5″-H), 6.87−6.83 (d, J = 10.5 Hz, 1H, Ar-5′-H), 6.79−6.76
(d, J = 8.5 Hz, 1H, Ar-6″-H); 13C DEPT (75 MHz, DMSO-d6): δ
157.90 (1C, Ar-2-CH), 131.89 (1C, Ar-8-CH), 130.06 (1C, Ar-5″-
CH), 127.15 (1C, Ar-7-CH), 124.5 6 (1C, Ar-5′-CH), 123.15 (1C,
Ar-6′-CH), 122.60 (1C, Ar-5-CH), 121.15 (1C, Ar-2′-CH), 113.02
(1C, Ar-6″-CH), 110.19 (1C, Ar-4″-CH), 104.72 (1C, Ar-2″-CH);
MS (APCI−): m/z 411.5[M + 1]+.
N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-
quinazolin-6-yl)-2 Morpholino Acetamide (10b). Yellowish white
1
solid, mp 175−180 °C, (0.4 g, yield; 87%); H NMR (300 MHz,
DMSO-d6): δ 10.01 (s, 1H, NH, exchanged with D2O), 9.77 (s, 1H,
NH, exchanged with D2O), 8.62 (s, 1H, Ar-2-H), 8.51 (s, 1H, Ar-5-
H), 8.01−7.97 (dd, J = 8.7, 1.9 Hz, 2H, Ar-8, 2′-H) 7.77−7.74 (d, J =
8.8 Hz, 1H, Ar-7-H), 7.72−7.68 (dd, J = 8.9, 2.3 Hz, 1H, Ar-6′-H),
7.50−7.43 (m, 1H, Ar-4″-H), 7.34−7.30 (m, 2H, Ar-5′, 2″-H), 7.27−
7.24 (d, J = 9, Ar-6″-H), 7.21−7.15 (t, J = 9 Hz, 1H, Ar-5″-H), 5.25
(s, 2H, CH2, OCH2C6H4F), 3.69−3.66 (t, J = 6, 4H, 2CH2,
morpholino), 3.21 (s, 2H, CH2, acetamido), 2.58−2.55 (t, J = 6, 4H,
2CH2, morpholino); 13C DEPT (75 MHz, DMSO): δ 153.77 (1C,
Ar-2-CH), 131.05 (1C, Ar-5″-CH), 130.95 (1C, Ar-8-CH), 128.73
(1C, Ar-7-CH), 127.81 (1C, Ar-6″-CH), 124.67 (1C, Ar-6′-CH),
123.76 (1C, Ar-2′-CH), 115.27 (1C, Ar-5′-CH), 114.74 (1C, Ar-4″-
CH), 114.33 (1C, Ar-2″-CH), 112.85 (1C, Ar-5-CH), 69.89 (1C,
CH2, OCH2C6H4F), 66.57 (2C, 2CH2, morpholino), 62.38 (1C,
CH2, acetamido), 53.72 (2C, 2CH2, morpholino); 13C NMR (75
MHz, DMSO): δ 168.80 (1C, CO), 164.29 (1C, Ar-4-C), 161.06
(1C, Ar-3″-C), 157.83 (1C, Ar-4′-C), 153.76 (1C, Ar-2-CH), 150.09
(1C, Ar-6-C), 147.06 (1C, Ar-8a-C), 140.21 (1C, Ar-1″-C), 136.63
(1C, Ar-1′-C), 133.84 (1C, Ar-3′-C), 131.05 (1C, Ar-5″-CH), 130.94
(1C, Ar-8-CH), 128.73 (1C, Ar-7-CH), 127.80 (1C, Ar-6″-CH),
124.67 (1C, Ar-6′-CH), 123.74 (1C, Ar-2′-CH), 115.65 (1C, Ar-4a-
C), 115.27 (1C, Ar-5′-CH), 114.78 (1C, Ar-4″-CH), 114.33 (1C, Ar-
2″-CH), 112.85 (1C, Ar-5-CH), 69.92 (1C, CH2, OCH2C6H4F),
66.57 (2C, 2CH2, morpholino), 62.38 (1C, CH2, acetamido), 53.73
(2C, 2CH2, morpholino); HRMS (ES+): m/z calcd for
C27H26ClFN5O3 [M + 1]+, 522.1708; found, 522.1705.
N-(3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)-6-nitroqui-
nazolin-4-amine (12b). (3.5 g, yield; 78%), mp 185−187 °C, as
reported.50
N-(3-Chloro-4-(m-tolyloxy)phenyl)-6-nitroquinazolin-4-amine
1
(12c). (3.3 g, yield: 82%), H NMR (300 MHz, DMSO-d6): δ 10.48
(s, 1H, NH, exchanged with D2O), 9.63 (s, 1H, Ar-5-H), 8.75 (s, 1H,
Ar-2-H), 8.57−8.54 (dd, J = 9.1, 1.9 Hz, 1H, Ar-7-H), 8.19 (s, 1H, Ar-
2′-H), 7.95−7.92 (d, J = 9.1 Hz, 1H, Ar-8-H), 7.79−7.76 (d, J = 9.1
Hz, 1H, Ar-6′-H), 7.35−7.32 (d, J = 9.1 Hz, 1H, Ar-5′-H), 7.24−7.19
(m, 1H, Ar-2″), 7.12−7.07 (t, J = 7.4 Hz, 1H, Ar-5″-H), 7.00−6.97
(d, J = 7.7 Hz, 1H, Ar-6″-H), 6.80−6.78 (d, J = 7.5 Hz, 1H, Ar-4″-H),
2.25 (s, 3H, CH3). 13C DEPT (75 MHz, DMSO-d6): δ 158.03 (1C,
Ar-2-CH), 132.02 (1C, Ar-8-CH), 130.03 (1C, Ar-5″-CH), 127.93
(1C, Ar-7-CH), 127.17 (1C, Ar-4″-CH), 124.88 (1C, Ar-5′-CH),
124.38 (1C, Ar-6′-CH), 123.24 (1C, Ar-5-CH), 121.20 (1C, Ar-2′-
CH), 119.92 (1C, Ar-2″-CH), 117.83 (1C, Ar-6″-CH). MS (ESI+):
m/z 407.1 [M + 1]+.
General Procedure for the Synthesis of Compounds (13a−
c). A mixture of the 6-nitro quinazoline derivatives (12a−c) (5
mmol) and stannous chloride (25 mmol) in methanol (20 mL) was
stirred at reflux for 1.5 h, under a nitrogen atmosphere. Excess
methanol was removed under reduced pressure, the remaining residue
was dissolved in ethyl acetate (200 mL), and the resulting solution
was neutralized with an aqueous saturated solution of NaHCO3 and
stirred for 1 h. The resulting gelatinous precipitate was centrifuged for
45 min and then discarded by filtration. The clear filtrate was
transferred into a separating funnel, and the organic layer was
N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-
quinazolin-6-yl)-2-(4-methylpiperazin-1-yl)acetamide (10c). Gray-
ish brown solid, mp 155−158 °C, (0.4 g, yield; 78%); 1H NMR (300
MHz, DMSO-d6): δ 9.93 (s, 1H, NH, exchanged with D2O), 9.77 (s,
1H, NH, exchanged with D2O), 8.59 (s, 1H, Ar-2-H), 8.51 (s, 1H, Ar-
5-H), 8.02−7.99 (d, J = 9 Hz, 1H, Ar-8-H), 7.98−7.97 (d, J = 3 Hz
(m-coupling) 1H, Ar-2′-H), 7.77−7.74 (d, J = 9 Hz, 1H, Ar-7-H),
7.72−7.68 (dd, J = 9, 3 Hz, 1H, Ar-6′-H), 7.50−7.43 (m, 1H, Ar-4″-
H), 7.34−7.29 (m, 2H, Ar-5′, 2″-H), 7.27−7.24 (d, J = 9, Ar-6″-H),
7.21−7.15 (t, J = 9 Hz, 1H, Ar-5″-H), 5.25 (s, 2H, CH2,
OCH2C6H4F), 3.19 (s, 2H, CH2, acetamido), 2.57 (br s, 4H,
2CH2, piperazino), 2.41 (br s, 4H, 2CH2 piperazino), 2.18 (s, 3H,
CH3); 13C DEPT (75 MHz, DMSO): δ 153.76 (1C, Ar-2-CH),
131.07 (1C, Ar-5″-CH), 130.96 (1C, Ar-8-CH), 128.77 (1C, Ar-7-
CH), 127.65 (1C, Ar-6″-CH), 124.67 (1C, Ar-6′-CH), 123.79 (1C,
Ar-2′-CH), 115.29 (1C, Ar-5′-CH), 114.69 (1C, Ar-4″-CH), 114.34
(1C, Ar-2″-CH), 112.63 (1C, Ar-5-CH), 69.83 (1C, CH2,
Y
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separated from the aqueous one. The aqueous phase was extracted
with ethyl acetate (2 × 20 mL), and the organic fractions were
combined, dried over anhydrous sodium sulfate, and concentrated
under vacuum to afford the corresponding 6-amino quinazoline
derivatives 13a−c.
2-Chloro-N-(4-((3-chloro-4-(m-tolyloxy)phenyl)amino)-
1
quinazolin-6-yl) Acetamide (14c). (2.01 g, yield; 89%); H NMR
(300 MHz, DMSO-d6): δ 11.22 (s, 2H, 2NH, exchanged with D2O),
8.97 (s, 1H, Ar-2-H), 8.83 (s, 1H, Ar-5-H), 8.08−7.95 (m, 3H, Ar-Ar-
2′,8,7-H), 7.65−7.62 (d, J = 8.8 Hz, 1H, Ar-6′-H), 7.36−7.34 (d, J =
7.8 Hz, 1H, Ar-6″-CH), 7.26−7.21 (m, 1H, Ar-2″-CH), 7.14−7.09 (t,
J = 7.8 Hz, 1H, Ar-5″-CH), 6.97−6.94 (d, J = 8.8 Hz, 1H, Ar-5′-H),
6.86−6.83 (d, J = 7.9 Hz, 1H, Ar-4″-CH), 4.44 (s, 2H, CH2), 2.24 (s,
3H, CH3); 13C DEPT (75 MHz, DMSO-d6): δ 150.93 (1C, Ar-2-
CH), 132.10 (1C, Ar-8-CH), 129.47 (1C, Ar-5″-CH), 128.01 (1C,
Ar-7-CH), 126.71 (1C, Ar-4″-CH), 124.98 (1C, Ar-5′-CH), 124.7
(1C, Ar-6′-CH)3, 122.94 (1C, Ar-2′-CH), 119.29 (1C, Ar-2″-CH),
118.40 (1C, Ar-6″-CH), 113.44 (1C, Ar-5-CH), 43.86 (1C, CH2),
16.12 (1C, CH3); MS (APCI+): m/z 453.6 [M + 1]+.
General Procedure for the Synthesis of Compounds (15a−
l). A mixture of appropriate 6-chloro-acetamido-quinazoline derivative
(14a−c) (1 mmol) and the appropriate secondary amine, 1-methyl
piperazine or morpholine (50 mmol), was placed into a 25 mL round-
bottomed flask or into a capped vial in the case of low boiling point
secondary amine including dimethylamine or diethylamine (50
mmol). The reaction mixture was heated under reflux temperature
and monitored by TLC. After 2 h, the TLC of reaction mass indicated
the absence of intermediate (14a−c). The reaction mixture was
cooled to room temperature and poured onto iced-water (30 mL)
portion wise while stirring, and the resultant precipitate was collected
by filtration, washed with water, dried, and purified by flash column
chromatography using DCM/methanol (95:5%) as an eluent to afford
the desired products (15a-1).
N-4-(3-Chloro-4-(3-fluorophenoxy)phenyl)quinazoline-4,6-dia-
1
mine (13a). Brown oil, (1.63 g, yield; 86%), H NMR (300 MHz,
DMSO-d6): δ 9.54 (s, 1H, NH, exchanged with D2O), 8.38 (s, 1H,
Ar-2-H), 8.27 (s, 1H, Ar-2″-H), 7.91−7.88 (d, J = 8.7 Hz, 1H, Ar-8-
H), 7.57−7.54 (d, J = 8.8 Hz, 1H, Ar-7-H), 7.43−7.38 (m, 1H, AR-5-
H), 7.34 (s, 1H, Ar-2′-H), 7.28−7.25 (d, J = 8.5 Hz, 2H, Ar-4″, 6′-H),
6.96−6.91 (t, J = 8.1 Hz, 1H, Ar-5″-H), 6.82−6.79 (d, J = 10.5 Hz,
1H, Ar-5′-H), 6.75−6.73 (d, J = 8.2 Hz, 1H, Ar-6″-H), 5.65 (s, 2H,
NH2, exchanged with D2O). MS (APCI+): m/z 381.2 [M + 1]+.
N-4-(3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)-
quinazoline-4,6-diamine (13b). Yellow solid, 1.97 g, yield; 86%, mp
104−106 °C, as reported.110
N-4-(3-Chloro-4-(m-tolyloxy)phenyl)quinazoline-4,6-diamine
(13c). Brown solid, (1.77 g, yield; 94%), 1H NMR (300 MHz,
DMSO-d6):δ 9.46 (s, 1H, NH, exchanged with D2O), 8.37 (s, 1H, Ar-
2-H), 8.23 (s, 1H, Ar-2′-H), 7.81−7.78 (d, J = 8.6 Hz, 1H, Ar-8-H),
7.57−7.54 (d, J = 8.8 Hz, 1H, Ar-7-H), 7.34 (s, 1H, Ar-5-H), 7.31−
7.25 (m, 2H, Ar-6″, 6′), 7.20−7.15 (m, 1H, Ar-2″-H), 7.07−7.02 (t, J
= 7.3 Hz, 1H, Ar-5″-H), 7.00−6.97 (d, J = 8.9 Hz, 1H, Ar-5′-H),
6.73−6.70 (d, J = 7.5 Hz, 1H, Ar-4″-H), 5.61 (s, 2H, NH2, exchanged
with D2O), 2.26 (s, 3H, CH3). 13C DEPT (75 MHz, DMSO-d6): δ
150.10 (1C, Ar-2-CH), 131.89 (1C, Ar-8-CH), 129.17 (1C, Ar-5″-
CH), 127.78 (1C, Ar-7-CH), 124.23 (1C, Ar-4″-CH), 123.90 (1C,
Ar-5′-CH), 123.49 (1C, Ar-6′-CH), 122.05 (1C, Ar-2′-CH), 120.53
(1C, Ar-2″-CH), 117.07 (1C, Ar-6″-CH), 101.34 (1C, Ar-5-CH),
16.21 (1C, CH3). MS (APCI+): m/z 377.1 [M + 1]+.
N-(4-((3-Chloro-4-(3-fluorophenoxy)phenyl)amino)quinazolin-6-
yl)-2-morpholino Acetamide (15a). Yellow solid, 0.39 g, yield; 77%,
1
mp 122−125 °C; H NMR (300 MHz, DMSO-d6): δ 10.05 (s, 1H,
NH, exchanged with D2O), 9.95 (s, 1H, NH, exchanged with D2O),
8.68 (s, 1H, Ar-2-H), 8.59 (s, 1H, Ar-5-H), 8.22 (s, 1H, Ar-2″-H),
8.03−8.00 (d, J = 9 Hz, 1H, Ar-8-H), 7.90−7.87 (d, 1H, J = 8.5, Ar-
6′-H), 7.81−7.78 (d, J = 9 Hz, 1H, Ar-7-H), 7.44, 7.37 (dd, 1H, Ar-
4″-H), 7.30−7.27 (d, 1H, J = 8.7, Ar-5′-H), 6.97−6.92 (t, J = 7.1 Hz,
1H, Ar-5″-H), 6.84−6.74 (m, 2H, Ar-2′, 6″-H), 3.67 (br s, 4H, 2CH2,
morpholino), 3.22 (s, 2H, CH2, acetamido), 2.57 (br s, 4H, 2CH2,
morpholino); 13C DEPT (75 MHz, DMSO-d6): δ 153.58 (1C, Ar-2-
CH), 131.86 (1C, Ar-5″-CH), 128.84 (1C, Ar-8-CH), 128.00 (1C,
Ar-7-CH), 124.08 (1C, Ar-5′-CH), 122.83 (1C, Ar-6′-CH), 112.78
(1C, Ar-2′-CH), 110.28 (1C, Ar-6″-CH), 110.00 (1C, Ar-4″-CH),
104.80 (1C, Ar-2″-CH), 104.47 (1C, Ar-5-CH), 66.57 (2C, 2CH2,
morpholine), 62.40 (1C, CH2, acetamido), 53.72 (2C, 2CH2,
morpholine); 13C NMR (75 MHz, DMSO-d6): δ 173.77 (1C, C
O), 163.85 (1C, Ar-4-C), 162.42 (1C, Ar-3″-C), 158.34 (1C, Ar-1″-
C), 151.81 (1C, Ar-2-CH), 150.97 (1C, Ar-6-C), 142.58 (1C, Ar-8a-
C), 141.52 (1C, Ar-4′-C), 136.62 (1C, Ar-1′-C), 136.49 (1C, Ar-5″-
CH), 133.54 (1C, Ar-8-CH), 132.73 (1C, Ar-3′-C), 130.09 (1C, Ar-
7-CH), 128.90 (1C, Ar-5′-CH), 127.66 (1C, Ar-6′-CH), 120.45 (1C,
Ar-2′-C), 117.52 (1C, Ar-4a-C), 115.03 (1C, Ar-6″-CH), 114.75 (1C,
Ar-4″-CH), 109.49 (1C, Ar-2″-CH), 109.16 (1C, Ar-5-CH), 71.29
(2C, 2CH2, morpholino), 67.10 (2C, 2CH2, morpholino), 58.42 (2C,
2CH2, morpholino); HRMS (ES+): m/z calcd for C26H24ClFN5O3
[M + 1]+, 508.1552; found, 508.1559.
General Procedure for the Synthesis of Compounds 14a−c.
To the solution of intermediate 13a−c (5 mmol) in acetone (20 mL),
NaHCO3 (0.44 g, 5.2 mmol) was added. The reaction mixture was
stirred under nitrogen at 0 °C, followed by the dropwise addition of 2-
choroacetyl chloride (0.4 mL, 5.2 mmol) and stirring was continued
for 1 h at 0 °C. Excess solvent was then removed under reduced
pressure.
Cold water was added (20 mL) to the remaining residue and the
resulting suspension was neutralized with a solution of NaHCO3, if
necessary (if pH still acidic). The formed solid was then filtered and
purified by column chromatography with EtOAc as an eluent to afford
compounds 14a−c as a yellow solid.
2-Chloro-N-(4-((3-chloro-4-(3-fluorophenoxy)phenyl)amino)-
quinazolin-6-yl)acetamide (14a). (1.41 g, yield; 62%), 1H NMR
(300 MHz, DMSO-d6): δ 10.93 (s, 2H, 2NH, exchanged with D2O),
8.83 (s, 1H, Ar-2-H), 8.69 (s, 1H, Ar-5-H), 8.14 (s, 1H, Ar-2″-H),
7.9−7.94 (d, J = 8.9 Hz, 1H, Ar-8-H), 7.87−7.80 (m, 2H, Ar-7, 6′-H),
7.45−7.38 (dd, 1H, Ar-4″-H), 7.30−7.28 (d, J = 8.8 Hz, 1H, Ar-5′-
H), 6.99−6.93 (t, J = 7.7 Hz, 1H, Ar-5″-H), 6.86−6.76 (m, 2H, Ar-
2′,6″-H), 4.40 (s, 2H, CH2). 13C DEPT (75 MHz, DMSO-d6): δ
152.59 (1C, Ar-2-CH), 131.90 (1C, Ar-5″-CH), 128.40 (1C, Ar-8-
CH), 126.65 (1C, Ar-7-CH), 125.12 (1C, Ar-5′-CH), 123.76 (1C,
Ar-6′-CH), 122.55 (1C, Ar-2′-CH), 113.11 (1C, Ar-6″-CH), 112.99
(1C, Ar-4″-CH), 110.19 (1C, Ar-2″-CH), 104.71 (1C, Ar-5-CH),
43.90 (1C, CH2). MS (APCI+): m/z 457.2 [M + 1]+.
2-Chloro-N-(4-((3-chloro-4-(3-(trifluoromethyl) phenoxy)-
phenyl)amino)quinazolin-6-yl)acetamide (14b). 1.36 g, yield;
54%, 1H NMR (300 MHz, DMSO-d6): δ 10.94 (s, 2H, NH,
exchanged with D2O), 8.87 (s, 1H, Ar-2-H), 8.73 (s, 1H, Ar-5-H),
8.17−8.18 (d (m-coupling), J = 1.2 Hz, 1H, Ar-2′-H), 7.97−7.94 (d, J
= 9 Hz, 1H, Ar-8-H), 7.89−7.82 (m, 2H, Ar-7, 6′-H), 7.66−7.61 (t, J
= 7.8 Hz, 1H, Ar-5″-H), 7.51−7.48 (d, J = 7.7 Hz, 1H, Ar-4″-H),
7.36−7.33 (d, J = 7.7 Hz, 1H, Ar-6″-H), 7.28−7.24 (m, 2H, Ar-2″,
5′), 4.41 (s, 2H, CH2); 13C DEPT (75 MHz, DMSO-d6): δ 152.40
(1C, Ar-2-CH), 131.98 (1C, Ar-5″-CH), 128.60 (1C, Ar-8-CH),
126.16 (1C, Ar-7-CH), 125.40 (1C, Ar-6″-CH), 124.10 (1C, Ar-5′-
CH), 122.74 (1C, Ar-6′-CH), 120.96 (1C, Ar-4″-CH), 120.16 (1C,
Ar-2′-CH), 113.56 (1C, Ar-2″-CH), 113.12 (1C, Ar-5-CH), 43.90
(1C, CH2); MS (ESI+): m/z 507.1 [M + 1]+.
N-(4-((3-Chloro-4-(3-fluorophenoxy)phenyl)amino)quinazolin-6-
yl)-2-(4-methylpiperazin-1-yl)acetamide (15b). Light-brown solid,
1
0.35 g, yield; 68%, mp 163−165 °C; H NMR (300 MHz, DMSO-
d6): δ 9.97 (s, 1H, NH, exchanged with D2O), 9.95 (s, 1H, NH,
exchanged with D2O), 8.65 (s, 1H, Ar-2-H), 8.59 (s, 1H, Ar-5-H),
8.22 (s, 1H, Ar-2′-H), 8.05−8.02 (d, J = 8.7 Hz, 1H, Ar-8-H), 7.90−
7.89, 7.87−7.86 (dd, J = 8.8, 1.07 Hz, 1H, Ar-7-H), 7.81−7.78 (d, 1H,
J = 9, Ar-6′-H), 7.45−7.37 (m, 1H, Ar-2″-H), 7.30−7.28 (d, 1H, J =
8.9, Ar-5′-H), 6.98−6.92 (t, J = 8.3 Hz, 1H, Ar-5″-H), 6.84−6.81 (d, J
= 8.5 Hz, 1H, Ar-4″-H), 6.78−6.75 (d, J = 8.2 Hz, 1H, Ar-6″-H), 3.20
(s, 2H, CH2, acetamido), 2.57 (br s, 4H, 2CH2, piperazino), 2.41 (br
s, 4H, 2CH2, piperazino), 2.18 (s, 3H, CH3); 13C DEPT (75 MHz,
DMSO-d6): δ 153.54 (1C, Ar-2-CH), 131.75 (1C, Ar-5″-CH), 128.86
(1C, Ar-8-CH), 127.84 (1C, Ar-7-CH), 125.01 (1C, Ar-5′-CH),
124.07 (1C, Ar-6′-CH), 122.79 (1C, Ar-2′-CH), 112.75 (1C, Ar-6″-
Z
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CH), 112.58 (1C, Ar-4″-CH), 110.01 (1C, Ar-2″-CH), 104.46 (1C,
Ar-5-CH), 62.17 (1C, CH2, acetamido), 54.99 (2C, 2CH2,
piperazine), 53.29 (2C, 2CH2, piperazino), 46.23 (1C, CH3); 13C
NMR (75 MHz, DMSO-d6): δ 169.09 (1C, CO), 159.12 (1C, Ar-
4-C), 157.63 (1C, Ar-1″-C), 153.57 (1C, Ar-2-CH), 147.12 (1C, Ar-
6-C), 146.17 (1C, Ar-8a-C), 137.91 (1C, Ar-4′-C), 136.84 (2C, Ar-1′,
3″-C), 131.74 (1C, Ar-5″-CH), 128.87 (1C, Ar-8-CH), 127.86 (1C,
Ar-7-CH), 125.29 (2C, Ar-3′-C, Ar-6″-CH), 125.00 (1C, Ar-5′-CH),
124.10 (2C, Ar-6′, 4″-CH), 122.86 (1C, Ar-2′-CH), 115.76 (1C, Ar-
4a-C), 110.00 (1C, Ar-2″-CH), 104.46 (1C, Ar-5-CH), 62.15 (1C,
CH2, acetamido), 54.98 (2C, 2CH2, piperazino), 53.28 (2C, 2CH2,
piperazino), 46.21 (1C, CH3); HRMS (ES+): m/z calcd for
C27H27ClFN6O2 [M + 1]+, 521.1868; found, 521.1868.
H), 8.03−8.00 (d, J = 9 Hz, 1H, Ar-8-H), 7.92−7.89 (d, 1H, J = 9 Hz,
Ar-7-H), 7.81−7.78 (d, 1H, J = 9, Ar-6′-H), 7.65−7.59 (t, J = 9 Hz,
1H, Ar-5″-H), 7.49−7.46 (d, 1H, J = 9 Hz, Ar-4″-H), 7.35−7.32 (d,
1H, J = 9 Hz, Ar-6″-H), 7.25−7.21 (m, 2H, Ar-2″, 5′), 3.67 (br s, 4H,
2CH2, morpholino) 3.22 (s, 2H, CH2), 2.57 (br s, 4H, 2CH2,
morpholino); 13C DEPT (75 MHz, DMSO-d6): δ 153.58 (1C, Ar-2-
CH), 131.91 (1C, Ar-5″-CH), 128.87 (1C, Ar-8-CH), 128.02 (1C,
Ar-7-CH), 124.12 (1C, Ar-6″-CH), 122.83 (2C, Ar-5′, 6′-CH),
120.67 (1C, Ar-4″-CH), 119.90 (1C, Ar-2′-CH), 113.27 (1C, Ar-2″-
CH), 112.74 (1C, Ar-5-CH), 66.57 (2C, 2CH2, morpholino), 62.41
(1C, CH2), 53.72 (2C, 2CH2, morpholino); 13C NMR (75 MHz,
DMSO-d6): δ 168.90 (1C, CO), 158.12 (1C, Ar-4-C), 157.63 (1C,
Ar-1″-C), 153.57 (1C, Ar-2-CH), 147.16 (1C, Ar-6-C), 145.84 (1C,
Ar-8a-C), 138.15 (1C, Ar-4′-C), 136.83 (2C, Ar-1′, 3″-C), 131.91
(1C, Ar-5″-CH), 128.85 (1C, Ar-8-CH), 128.01 (1C, Ar-7-CH),
125.37 (1C, Ar-3′-C), 125.22 (1C, CF3), 124.12 (1C, Ar-6″-CH),
122.95 (2C, Ar-5′, 6′-CH), 120.69 (1C, Ar-4″-CH), 119.91 (1C, Ar-
2′-CH), 115.76 (1C, Ar-4a-C), 113.26 (1C, Ar-2″-CH), 112.75 (1C,
Ar-5-CH), 66.56 (2C, 2CH2, morpholino), 62.40 (1C, CH2), 53.72
(2C, 2CH2, morpholino); HRMS (ES+): m/z calcd for
C27H24ClF3N5O3 [M + 1]+, 558.1520; found, 558.1528.
N-(4-((3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)-
quinazolin-6-yl)-2-(4-methylpiperazin-1-yl)acetamide (15f). Brown
oil solidifies when kept in the fridge overnight, 0.27 g, yield; 48%: 1H
NMR (300 MHz, DMSO-d6): δ 9.96 (s, 2H, 2NH, exchanged with
D2O), 8.65 (s, 1H, Ar-2-H), 8.59 (s, 1H, Ar-5-H), 8.25 (s, 1H, Ar-2′-
H), 8.06−8.03 (d, J = 9 Hz, 1H, Ar-8-H), 7.92−7.89 (d, 1H, J = 9 Hz,
Ar-7-H), 7.81−7.78 (d, 1H, J = 9, Ar-6′-H), 7.64−7.59 (t, J = 6 Hz,
1H, Ar-5″-H), 7.48−7.46 (d, 1H, J = 6 Hz, Ar-4″-H), 7.34−7.32 (d,
1H, J = 6 Hz, Ar-6″-H), 7.25−7.21 (m, 2H, Ar-2″, 5′), 3.20 (s, 2H,
CH2, acetamido), 2.57 (br s, 4H, 2CH2, piperazino), 2.41 (br s, 4H,
2CH2, piperazine), 2.18 (s, 3H, CH3); 13C DEPT (75 MHz, DMSO-
d6): δ 153.54 (1C, Ar-2-CH), 131.90 (1C, Ar-5″-CH), 128.87 (1C,
Ar-8-CH), 127.85 (1C, Ar-7-CH), 124.12 (1C, Ar-6″-CH), 122.94
(2C, Ar-5′, 6′-CH), 120.66 (1C, Ar-4″-CH), 119.90 (1C, Ar-2′-CH),
113.31 (1C, Ar-2″-CH), 112.54 (1C, Ar-5-CH), 62.15 (1C, CH2,
acetamido), 54.97 (2C, 2CH2, piperazino), 53.26 (2C, 2CH2,
piperazino), 46.18 (1C, CH3); 13C NMR (75 MHz, DMSO-d6): δ
169.08 (1C, CO), 158.12 (1C, Ar-4-C), 157.62 (1C, Ar-1″-C),
153.54 (1C, Ar-2-CH), 147.14 (1C, Ar-6-C), 145.84 (1C, Ar-8a-C),
138.15 (1C, Ar-4′-C), 136.85 (2C, Ar-1′, 3″-C), 131.90 (1C, Ar-5″-
CH), 128.87 (1C, Ar-8-CH), 127.85 (1C, Ar-7-CH), 125.81 (1C, Ar-
3′-C), 125.40 (1C, CF3), 124.12 (1C, Ar-6″-CH), 122.94 (2C, Ar-5′,
6′-CH), 120.67 (1C, Ar-4″-CH), 119.95 (1C, Ar-2′-CH), 115.78
(1C, Ar-4a-C), 113.32 (1C, Ar-2″-CH), 112.54 (1C, Ar-5-CH), 62.15
(1C, CH2, acetamido), 54.97 (2C, 2CH2, piperazino), 53.26 (2C,
2CH2, piperazino), 46.18 (1C, CH3); HRMS (ES+): m/z calcd for
C28H27ClF3N6O2 [M + 1]+, 571.1836; found, 571.1837.
N-(4-((3-Chloro-4-(3-fluorophenoxy)phenyl)amino)quinazolin-6-
yl)-2-(diethylamino) Acetamide (15c). Brown solid, 0.4 g, yield; 86%,
1
mp 110−115 °C; H NMR (300 MHz, DMSO-d6): δ 9.95 (s, 2H,
2NH, exchanged with D2O), 8.62 (s, 1H, Ar-2-H), 8.59 (s, 1H, Ar-5-
H), 8.24 (s, 1H, Ar-2″-H), 8.21−8.18 (d, J = 9 Hz, 1H, Ar-8-H),
7.92−7.89 (d, 1H, J = 9 Hz, Ar-7-H), 7.81−7.78 (d, 1H, J = 9, Ar-5′-
H), 7.45−7.37 (dd, 1H, Ar-6′-H), 7.30−7.27 (d, 1H, J = 8.8, Ar-2′-
H), 6.98−6.92 (t, J = 9 Hz, 1H, Ar-5″-H), 6.84−6.81 (d, J = 9 Hz,
1H, Ar-4″-H), 6.77−6.74 (d, 1H, J = 9 Hz, Ar-6″-H), 3.23 (s, 2H,
CH2, acetamido), 2.68−2.61 (q, J = 7 Hz, 4H, 2CH2,
(CH3CH2)2N−), 1.09−1.04 (t,
J = 7 Hz, 6H, 2CH3,
(CH3CH2)2N−); 13C DEPT (75 MHz, DMSO-d6): δ 153.53 (1C,
Ar-2-CH), 131.85 (1C, Ar-8-CH), 128.92 (1C, Ar-5″-CH), 127.38
(1C, Ar-7-CH), 124.04 (1C, Ar-5′-CH), 122.79 (1C, Ar-6′-CH),
112.77 (1C, Ar-2′-CH), 112.18 (1C, Ar-6″-CH), 110.28 (1C, Ar-4″-
CH), 104.81 (1C, Ar-2″-CH), 104.46 (1C, Ar-5-CH), 57.78 (1C,
CH2, acetamido), 48.48 (2C, 2CH2, (CH3CH2)2N−), 12.44 (2C,
2CH3, (CH3CH2)2N−); 13C NMR (75 MHz, DMSO-d6): δ 170.98
(1C, CO), 159.06 (1C, Ar-4-C), 158.92 (1C, Ar-3″-C), 157.65
(1C, Ar-1″-C), 153.55 (1C, Ar-2-CH), 146.86 (1C, Ar-6-C), 146.29
(1C, Ar-8a-C), 137.69 (1C, Ar-4′-C), 136.69 (1C, Ar-1′-C), 131.89
(1C, Ar-8-CH), 128.80 (1C, Ar-5″-CH), 127.51 (1C, Ar-7-CH),
125.38 (1C, Ar-3′-C), 124.20 (1C, Ar-5′-CH), 122.83 (1C, Ar-6′-
CH), 115.68 (1C, Ar-4a-C), 112.72 (1C, Ar-2′-CH), 112.05 (1C, Ar-
6″-CH), 110.29 (1C, Ar-4″-CH), 104.67 (1C, Ar-2″-CH), 104.34
(1C, Ar-5-CH), 57.68 (1C, CH2, acetamido), 48.47 (2C, 2CH2,
(CH3CH2)2N−), 12.35 (2C, 2CH3, (CH3CH2)2N−); HRMS (ES+):
m/z calcd for C26H26ClFN5O2 [M + 1]+, 494.1759; found, 494.1756.
N-(4-((3-Chloro-4-(3-fluorophenoxy)phenyl)amino)quinazolin-6-
yl)-2-(dimethylamino)acetamide (15d). Brown solid, 0.3 g, yield:
72%; 1H NMR (300 MHz, DMSO-d6): δ 10.00 (s, 1H, NH,
exchanged with D2O), 9.91 (s, 1H, NH, exchanged with D2O), 8.65
(s, 1H, Ar-2-H), 8.58 (s, 1H, Ar-5-H), 8.23 (s, 1H, Ar-2″-H), 8.13−
8.10 (d, J = 9 Hz, 1H, Ar-8-H), 7.90−7.87 (d, 1H, J = 8.6, Ar-6′-H),
7.80−7.77 (d, J = 9 Hz, 1H, Ar-7-H), 7.44−7.37 (dd, 1H, Ar-4″-H),
7.30−7.27 (d, 1H, J = 8.8, Ar-5′-H), 6.98−6.92 (t, J = 8.5 Hz, 1H, Ar-
5″-H), 6.85−6.75 (m, 2H, Ar-2′,6″-H), 3.16 (s, 2H, CH2, acetamido),
2.33 (s, 6H, 2CH3); 13C DEPT (75 MHz, DMSO-d6): δ 153.46 (1C,
Ar-2-CH), 131.73 (1C, Ar-5″-CH), 128.82 (1C, Ar-8-CH), 127.64
(1C, Ar-7-CH), 123.93 (1C, Ar-5′-CH), 122.79 (1C, Ar-6′-CH),
112.77 (1C, Ar-2′-CH), 110.27 (1C, Ar-6″-CH), 109.98 (1C, Ar-4″-
CH), 104.80 (1C, Ar-2″-CH), 104.46 (1C, Ar-5-CH), 63.69 (1C,
CH2), 45.95 (2C, 2CH3); 13C NMR (75 MHz, DMSO-d6): δ 169.44
(1C, CO), 165.00 (1C, Ar-4-C), 161.76 (1C, Ar-3″-C), 159.14
(1C, Ar-1″-C), 153.50 (1C, Ar-2-CH), 146.11 (1C, Ar-6-C), 136.96
(1C, Ar-8a-C), 131.86 (1C, Ar-4′-C), 131.73 (1C, Ar-5″-CH), 128.82
(1C, Ar-8-CH), 127.65 (1C, Ar-7-CH), 125.31 (1C, Ar-1′-C), 123.94
(1C, Ar-5′-CH), 122.80 (1C, Ar-6′-CH), 122.69 (1C, Ar-3′-C),
112.75 (1C, Ar-2′-CH), 112.45 (1C, Ar-4a-C), 110.27 (1C, Ar-6″-
CH), 109.99 (1C, Ar-4″-CH), 104.79 (1C, Ar-2″-CH), 104.46 (1C,
Ar-5-CH), 63.68 (1C, CH2), 45.94 (2C, 2CH3); HRMS (ES+): m/z
calcd for C24H22ClFN5O2 [M + 1]+, 466.1446; found, 466.1441.
N-(4-((3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)-
quinazolin-6-yl)-2-morpholino-acetamide (15e). Yellow solid, 0.42
g, yield; 77%, mp 105−108; 1H NMR (300 MHz, DMSO-d6): δ 10.05
(s, H, NH, exchanged with D2O), 9.96 (s, H, NH, exchanged with
D2O), 8.67 (s, 1H, Ar-2-H), 8.60 (s, 1H, Ar-5-H), 8.25 (s, 1H, Ar-2′-
N-(4-((3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)-
quinazolin-6-yl)-2-(dimethylamino)acetamide (15g). Brown solid,
1
0.42 g, yield; 83%; H NMR (300 MHz, DMSO-d6): δ 9.99 (s, 2H,
2NH, exchanged with D2O), 8.65 (s, 1H, Ar-2-H), 8.59 (s, 1H, Ar-5-
H), 8.25 (s, 1H, Ar-2′-H), 8.13−8.10 (d, J = 9 Hz, 1H, Ar-8-H),
7.92−7.89 (d, 1H, J = 9 Hz, Ar-7-H), 7.80−7.77 (d, 1H, J = 9 Hz, Ar-
6′-H), 7.64−7.59 (t, J = 6 Hz, 1H, Ar-5″-H), 7.48−7.46 (d, 1H, J = 6
Hz, Ar-4″-H), 7.34−7.32 (d, 1H, J = 6 Hz, Ar-6″-H), 7.25−7.21 (m,
2H, Ar-2″, 5′), 3.15 (s, 2H, CH2), 2.33 (s, 6H, 2CH3); 13C DEPT (75
MHz, DMSO-d6): δ 153.45 (1C, Ar-2-CH), 131.89 (1C, Ar-5″-CH),
128.82 (1C, Ar-8-CH), 127.66 (1C, Ar-7-CH), 123.97 (1C, Ar-6″-
CH), 122.95 (1C, Ar-5′-CH), 122.77 (1C, Ar-6′-CH), 120.69 (1C,
Ar-4″-CH), 119.94 (1C, Ar-2′-CH), 113.29 (1C, Ar-2″-CH), 112.44
(1C, Ar-5-CH), 63.68 (1C, CH2), 45.93 (2C, 2CH3); 13C NMR (75
MHz, DMSO): δ 169.41 (1C, CO), 158.13 (1C, Ar-4-C), 157.51
(1C, Ar-1″-C), 153.44 (1C, Ar-2-CH), 147.02 (1C, Ar-6-C), 145.82
(1C, Ar-8a-C), 138.27 (1C, Ar-4′-C), 136.98 (1C, Ar-1′-C), 131.88
(1C, Ar-5″-CH), 131.40 (1C, Ar-3″-C), 130.99 (1C, Ar-3′-C), 128.82
(1C, Ar-8-CH), 127.64 (1C, Ar-7-CH), 125.41 (1C, CF3), 123.97
(1C, Ar-6″-CH), 122.92 (1C, Ar-5′-CH), 122.77 (1C, Ar-6′-CH),
120.69 (1C, Ar-4″-CH), 119.88 (1C, Ar-2′-CH), 115.82 (1C, Ar-4a-
C), 113.28 (1C, Ar-2″-CH), 112.44 (1C, Ar-5-CH), 63.68 (1C,
AA
https://dx.doi.org/10.1021/acs.jmedchem.0c01647
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
CH2), 45.93 (2C, 2CH3); HRMS (ES+): m/z calcd for
C25H22ClF3N5O2 [M + 1]+, 516.1414; found, 516.1424.
(t, J = 7.1 Hz, 6H, 2-CH3, (CH3CH2)2N−); 13C DEPT (75 MHz,
DMSO-d6): δ 153.60 (1C, Ar-2-CH), 131.95 (1C, Ar-8-CH), 128.89
(1C, Ar-5″-CH), 127.84 (1C, Ar-7-CH), 127.32 (1C, Ar-4″-CH),
124.34 (1C, Ar-5′-CH), 124.10 (1C, Ar-6′-CH), 122.82 (1C, Ar-2′-
CH), 120.24 (1C, Ar-2″-CH), 117.40 (1C, Ar-6″-CH), 112.16 (1C,
Ar-5-CH), 57.82 (1C, CH2, acetamido), 48.50 (2C, 2-CH2,
(CH3CH2)2N−) 16.20 (1C, CH3, tolyl), 12.44 (2C, 2-CH3,
(CH3CH2)2N−); 13C NMR (75 MHz, DMSO-d6): δ 170.60 (1C,
CO), 157.64 (1C, Ar-4-C), 155.03 (1C, Ar-1″-C), 153.59 (1C, Ar-
2-CH), 148.05 (1C, Ar-6-C), 147.01 (1C, Ar-8a-C), 136.74 (1C, Ar-
4′-C), 136.41 (1C, Ar-3″-C), 131.94 (1C, Ar-8-CH), 128.88 (1C, Ar-
5″-CH), 128.26 (1C, Ar-1′-C), 127.84 (1C, Ar-7-CH), 127.31 (1C,
Ar-4″-CH), 124.34 (1C, Ar-5′-CH), 124.10 (1C, Ar-6′-CH), 123.80
(1C, Ar-3′-C), 122.83 (1C, Ar-2′-CH), 120.23 (1C, Ar-2″-CH),
117.41 (1C, Ar-6″-CH), 115.79 (1C, Ar-4a-C), 112.17 (1C, Ar-5-
CH), 57.83 (1C, CH2, acetamido), 48.51 (2C, 2-CH2,
(CH3CH2)2N−), 16.19 (1C, CH3, tolyl), 12.44 (2C, 2-CH3,
(CH3CH2)2N); HRMS (ES+): m/z calcd for C27H29ClN5O2 [M +
1]+, 490.2010; found, 490.2015.
N-(4-((3-Chloro-4-(m-tolyloxy)phenyl)amino)quinazolin-6-yl)-2-
morpholino Acetamide (15h). Off-white solid, 0.42 g, yield; 84%; 1H
NMR (300 MHz, DMSO-d6): δ 10.04 (s, 1H, NH, exchanged with
D2O), 9.89 (s, 1H, NH, exchanged with D2O), 8.64 (s, 1H, Ar-2-H),
8.55 (s, 1H, Ar-5-H), 8.16 (s, 1H, Ar-2′-H), 8.03−8.00 (d, J = 8.7 Hz,
1H, Ar-8-H), 7.79−7.76 (d, J = 8.8 Hz, 1H, Ar-7-H), 7.75 (s, 1H, Ar-
2″-H), 7.32−7.30 (d, 1H, J = 7.1 Hz, Ar-6′-H), 7.21−7.19 (d, 1H, J =
7.3 Hz, Ar-5′-H), 7.09−7.04 (t, J = 8.8 Hz, 1H, Ar-5″-H), 6.99−6.96
(d, J = 8.9 Hz, 1H, Ar-6″-H), 6.76−6.73 (d, J = 8.8 Hz, 1H, Ar-4″-H),
3.67 (br s, 4H, 2CH2, morpholino), 3.21 (s, 2H, CH2, acetamido),
2.56 (br s, 4H, 2CH2, morpholino), 2.26 (s, 3H, CH3); 13C DEPT
(75 MHz, DMSO-d6): δ 153.66 (1C, Ar-2-CH), 131.95 (1C, Ar-8-
CH), 128.78 (1C, Ar-5″-CH), 127.85 (2C, Ar-7, 4″-CH), 124.40
(1C, Ar-5′-CH), 124.12 (1C, Ar-6′-CH), 122.90 (1C, Ar-2′-CH),
120.22 (1C, Ar-2″-CH), 117.39 (1C, Ar-6″-CH), 112.78 (1C, Ar-5-
CH), 66.57 (2C, 2CH2, morpholino), 62.38 (1C, CH2, acetamido),
53.70 (2C, 2CH2, morpholino), 16.19 (1C, CH3); 13C NMR (75
MHz, DMSO-d6): δ 168.92 (1C, CO), 157.73 (1C, Ar-4-C),
155.01 (1C, Ar-1″-C), 153.66 (1C, Ar-2-CH), 148.08 (1C, Ar-6-C),
147.09 (1C, Ar-8a-C), 136.73 (1C, Ar-4′-C), 136.37 (1C, Ar-3″-C),
131.95 (1C, Ar-8-CH), 128.77 (1C, Ar-5″-CH), 128.26 (1C, Ar-1′-
C), 127.84 (2C, Ar-7, 4″-CH), 124.41 (1C, Ar-5′-CH), 124.11 (1C,
Ar-6′-CH), 123.79 (1C, Ar-3′-C), 122.90 (1C, Ar-2′-CH), 120.20
(1C, Ar-2″-CH), 117.41 (1C, Ar-6″-CH), 115.70 (1C, Ar-4a-C),
112.78 (1C, Ar-5-CH), 66.57 (2C, 2CH2, morpholino), 62.38 (1C,
CH2, acetamido), 53.71 (2C, 2CH2, morpholino), 16.18 (1C, CH3);
HRMS (ES+): m/z calcd for C27H27ClN5O3 [M + 1]+, 504.1802;
found, 504.1799.
N-(4-((3-Chloro-4-(m-tolyloxy)phenyl)amino)quinazolin-6-yl)-2-
(dimethylamino) Acetamide (15k). Yellow solid, 0.22 g, yield; 84%;
1H NMR (300 MHz, DMSO-d6): δ 9.96 (s, 2H, 2NH, exchanged
with D2O), 8.62 (s, 1H, Ar-2-H), 8.55 (s, 1H, Ar-5-H), 8.17 (s, 1H,
Ar-2′-H), 8.13−8.10 (d, J = 8.9 Hz, 1H, Ar-8-H), 7.79−7.76 (d, J =
8.9 Hz, 2H, Ar-7, 6′-H), 7.33−7.30 (d, J = 7.1 Hz, 1H, Ar-6″-H),
7.21−7.16 (m, 1H, Ar-2″-H), 7.09−7.04 (t, J = 7.1 Hz, 1H, Ar-5″-H),
7.00−6.97 (d, J = 8.9 Hz, 1H, Ar-5′-H), 6.77−6.74 (d, J = 7.2 Hz, 1H,
Ar-4″-H), 3.15 (s, 2H, CH2), 2.33 (s, 6H, 2CH3), 2.26 (s, 3H, CH3,
tolyl); 13C DEPT 135 (75 MHz, DMSO-d6): δ 153.52 (1C, Ar-2-
CH), 131.93 (1C, Ar-8-CH), 128.78 (1C, Ar-5″-CH), 127.83 (1C,
Ar-7-CH), 127.56 (1C, Ar-4″-CH), 124.21 (1C, Ar-5′-CH), 124.08
(1C, Ar-6′-CH), 122.69 (1C, Ar-2′-CH), 120.24 (1C, Ar-2″-CH),
117.39 (1C, Ar-6″-CH), 112.49 (1C, Ar-5-CH), 63.68 (1C, CH2),
45.93 (2C, 2-CH3), 16.19 (1C, CH3, tolyl); 13C NMR (75 MHz,
DMSO-d6): δ 169.39 (1C, CO), 157.59 (1C, Ar-4-C), 155.05 (1C,
Ar-1″-C), 153.52 (1C, Ar-2-CH), 148.00 (1C, Ar-6-C), 146.99 (1C,
Ar-8a-C), 136.89 (1C, Ar-4′-C), 136.55 (1C, Ar-3″-C), 131.93 (1C,
Ar-8-CH), 128.78 (1C, Ar-5″-CH), 128.26 (1C, Ar-1′-C), 127.82
(1C, Ar-7-CH), 127.56 (1C, Ar-4″-CH), 124.22 (1C, Ar-5′-CH),
124.08 (1C, Ar-6′-CH), 123.82 (1C, Ar-3′-C), 122.69 (1C, Ar-2′-
CH), 120.23 (1C, Ar-2″-CH), 117.40 (1C, Ar-6″-CH), 115.80 (1C,
Ar-4a-C), 112.49 (1C, Ar-5-CH), 63.69 (1C, CH2), 45.93 (2C, 2-
CH3), 16.18 (1C, CH3, tolyl); HRMS (ES+): m/z calcd for
C25H25ClN5O2 [M + 1]+, 462.1697; found, 462.1693.
N-(4-((3-Chloro-4-(m-tolyloxy)phenyl)amino)quinazolin-6-yl)-2-
(4-methylpiperazin-1-yl)acetamide (15i). Light-brown solid, 0.47 g,
1
yield; 92%; H NMR (300 MHz, DMSO-d6): δ 9.94 (s, 1H, 1NH,
exchanged with D2O), 9.87 (s, 1H, 1NH, exchanged with D2O), 8.62
(s, 1H, Ar-2-H), 8.56 (s, 1H, Ar-5-H), 8.17−8.16 (d (m-coupling), J
= 2.1 Hz, 1H, Ar-2′-H), 8.04−8.01 (d, J = 9 Hz, 1H, Ar-8-H), 7.79−
7.76 (d, J = 9 Hz, 1H, Ar-7-H), 7.78−7.74 (dd, J = 9 Hz, 2.1 Hz, 1H,
Ar-6′-H), 7.34−7.31 (d, J = 7.3 Hz, 1H, Ar-6″-H), 7.22−7.17 (m, 1H,
Ar-2″-H), 7.10−7.05 (t, J = 7.3 Hz, 1H, Ar-5″-H), 7.00−6.97 (d, J =
8.9 Hz, 1H, Ar-5′-H), 6.77−6.74 (d, J = 7.4 Hz, 1H, Ar-4″-H), 3.19
(s, 2H, CH2, acetamido), 2.57 (br s, 4H, 2-CH2, piperazino), 2.41 (br
s, 4H, 2-CH2, piperazino), 2.26 (s, 3H, CH3, piperazino), 2.18 (s, 3H,
CH3, tolyl); 13C DEPT (75 MHz, DMSO-d6): δ 153.63 (1C, Ar-2-
CH), 131.94 (1C, Ar-8-CH), 128.82 (1C, Ar-5″-CH), 127.82 (2C,
Ar-7, 4″-CH), 124.39 (1C, Ar-5′-CH), 124.10 (1C, Ar-6′-CH),
122.89 (1C, Ar-2′-CH), 120.23 (1C, Ar-2″-CH), 117.39 (1C, Ar-6″-
CH), 112.61 (1C, Ar-5-CH), 62.15 (1C, CH2, acetamido), 54.99
(2C, 2-CH2, piperazino), 53.29 (2C, 2-CH2, piperazino), 46.21 (1C,
CH3, piperazino), 16.21 (1C, CH3, tolyl); 13C NMR (75 MHz,
DMSO-d6): δ 169.05 (1C, CO), 157.72 (1C, Ar-4-C), 155.03 (1C,
Ar-1″-C), 153.63 (1C, Ar-2-CH), 148.06 (1C, Ar-6-C), 147.12 (1C,
Ar-8a-C), 136.77 (1C, Ar-4′-C), 136.41 (1C, Ar-3″-C), 131.95 (1C,
Ar-8-CH), 128.83 (1C, Ar-5″-CH), 128.27 (1C, Ar-1′-C), 127.84
(2C, Ar-7, 4″-CH), 124.40 (1C, Ar-5′-CH), 124.11 (1C, Ar-6′-CH),
123.78 (1C, Ar-3′-C), 122.90 (1C, Ar-2′-CH), 120.21 (1C, Ar-2″-
CH), 117.42 (1C, Ar-6″-CH), 115.73 (1C, Ar-4a-C), 112.62 (1C, Ar-
5-CH), 62.15 (1C, CH2, acetamido), 55.00 (2C, 2-CH2, piperazino),
53.29 (2C, 2-CH2, piperazino), 46.20 (1C, CH3, piperazino), 16.18
(1C, CH3, tolyl); HRMS (ES+): m/z calcd for C28H30ClN6O2 [M +
1]+, 517.2119; found, 517.2120.
N-(4-((3-Chloro-4-(m-tolyloxy)phenyl)amino)quinazolin-6-yl)-2-
((2-hydroxyethyl)amino)acetamide (15l). A mixture of intermediate
14c (0.45 g, 1 mmol) and 2-aminoethanol (0.25 g, 0.3 mL, 5 mmol)
in methanol (20 mL) was held at reflux for 6 h in the presence of
triethylamine (15 drops). Methanol was then removed under reduced
pressure, and the remaining residue was recrystallized from acetone to
yield compound 15l.
White solid, 0.2 g, yield: 43%; 1H NMR (300 MHz, DMSO-d6): δ
9.88 (s, 2H, 2NH, exchanged with D2O), 8.63 (s, 1H, Ar-2-H), 8.56
(s, 1H, Ar-5-H), 8.18 (s, 1H, Ar-2′-H), 8.09−8.05 (d, J = 9.1 Hz, 1H,
Ar-8-H), 7.80−7.77 (dd, J = 9.1 Hz, 3.1 Hz, 2H, Ar-7, 6′-H), 7.33−
7.31 (d, J = 6 Hz, 1H, Ar-6″-H), 7.19−7.17 (d, J = 6 Hz, 1H, Ar-4″-
H), 7.10−7.05 (t, J = 6 Hz, 1H, Ar-5″), 7.00−6.97 (d, J = 9 Hz, 1H,
Ar-5′-H), 6.76−6.74 (s, 1H, Ar-2″-H), 4.67 (s, 1H, OH, exchanged
with D2O), 4.66 (s, 1H, 1NH, exchanged with D2O), 3.51 (br s, 2H,
CH2, OHCH2CH2NH), 3.38 (s, 2H, CH2, acetamido), 2.68 (br s, 2H,
CH2, OHCH2CH2NH), 2.27 (s, 3H, CH3); 13C DEPT (75 MHz,
DMSO-d6): δ 153.55 (1C, Ar-2-CH), 131.94 (1C, Ar-8-CH), 128.89
(1C, Ar-5″-CH), 127.85 (1C, Ar-7-CH), 127.36 (1C, Ar-4″-CH),
124.26 (1C, Ar-5′-CH), 124.09 (1C, Ar-6′-CH), 122.76 (1C, Ar-2′-
CH), 120.26 (1C, Ar-2″-CH), 117.37 (1C, Ar-6″-CH), 112.11 (1C,
Ar-5-CH), 60.85 (1C, CH2, OHCH2CH2NH), 53.17 (1C, CH2,
acetamido), 52.18 (1C, CH2, OHCH2CH2NH), 16.20 (1C, CH3);
13C NMR (75 MHz, DMSO-d6): δ 171.18 (1C, CO), 157.66 (1C,
N-(4-((3-Chloro-4-(m-tolyloxy)phenyl)amino)quinazolin-6-yl)-2-
(diethylamino) Acetamide (15j). Yellow solid, 0.37 g, yield; 77%; 1H
NMR (300 MHz, DMSO-d6): δ 9.92 (s, 1H, NH, exchanged with
D2O), 9.86 (s, 1H, NH, exchanged with D2O), 8.57 (s, 1H, Ar-2-H),
8.56 (s, 1H, Ar-5-H), 8.20−8.17 (m, 2H, Ar-8, 2′-H), 7.80−7.77 (d, J
= 9 Hz, 2H, Ar-7, 2″-H), 7.33−7.31 (d, 1H, J = 7.4, Ar-6′-H), 7.19−
7.17 (d, 1H, J = 7.3, Ar-5′-H), 7.09−7.05 (t, J = 8.9 Hz, 1H, Ar-5″-
H), 7.00−6.97 (d, J = 8.9 Hz, 1H, Ar-6″-H), 6.77−6.74 (d, J = 8.8 Hz,
1H, Ar-4″-H), 3.23 (s, 2H, CH2, acetamido), 2.68−2.61 (q, J = 7.1
Hz, 4H, 2-CH2, (CH3CH2)2N−), 2.26 (s, 3H, CH3, tolyl), 1.08−1.04
Ar-4-C), 155.04 (1C, Ar-1″-C), 153.55 (1C, Ar-2-CH), 147.97 (1C,
Ar-6-C), 146.98 (1C, Ar-8a-C), 136.91 (1C, Ar-4′-C), 136.47 (1C,
AB
https://dx.doi.org/10.1021/acs.jmedchem.0c01647
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Ar-3″-C), 131.94 (1C, Ar-8-CH), 128.90 (1C, Ar-5″-CH), 128.24
(1C, Ar-1′-C), 127.84 (1C, Ar-7-CH), 127.37 (1C, Ar-4″-CH),
124.27 (1C, Ar-5′-CH), 124.09 (1C, Ar-6′-CH), 122.76 (1C, Ar-2′-
CH), 120.26 (1C, Ar-2″-CH), 117.37 (1C, Ar-6″-CH), 115.80 (1C,
Ar-4a-C), 112.11 (1C, Ar-5-CH), 60.81 (1C, CH2, OHCH2CH2NH),
53.14 (1C, CH2, acetamido), 52.15 (1C, CH2, OHCH2CH2NH),
16.21 (1C, CH3); HRMS (ES+): m/z calcd for C25H25ClN5O3 [M +
1]+, 478.1646; found, 478.1642.
N-(4-((3-Chloro-4-(3-fluorophenoxy)phenyl)amino)-
quinazolin-6-yl)-4-(chloromethyl)benzamide (16). To the sol-
ution of intermediate 13a (1.5 g, 4 mmol) in acetone (20 mL),
NaHCO3 (0.35 g, 4.2 mmol) was added. The reaction mixture was
stirred under nitrogen at 0 °C, followed by the dropwise addition of a
solution of 4(choromethyl) benzoyl chloride (0.8 g, 4.2 mmol) in
acetone (2 mL). The reaction mixture was stirred for 30 min at 0 °C.
Excess solvent was removed under reduced pressure. Cold water (20
mL) was added to the remaining residue and the resulting suspension
was neutralized with a saturated solution of NaHCO3, if necessary.
The solid obtained was filtered, dried, and washed with DCM and
diethylether to afford compound 16 as pure greenish-yellow solid. 1.1
g, yield: 51%.
C), 159.00 (1C, Ar-4″-C), 157.73 (1C, Ar-8′a-C), 153.68 (1C, Ar-2′-
CH), 147.34 (1C, Ar-4-C), 146.12 (1C, Ar-6′-C), 142.57 (1C, Ar-1″-
C), 138.02 (1C, Ar-1-C), 137.36 (1C, Ar-4′a-C), 131.84 (1C, Ar-5‴-
CH), 129.34 (2C, Ar-2, 6-CH), 128.84 (1C, Ar-8′-CH), 128.14 (2C,
Ar-3,5-CH), 125.29 (1C, Ar-3″-C), 124.00 (1C, Ar-7′-CH), 122.74
(1C, Ar-5″-CH), 113.91 (1C, Ar-6″-CH), 112.76 (1C, Ar-2″-CH),
110.26 (1C, Ar-6‴-CH), 109.98 (1C, Ar-4‴-CH), 104.81 (1C, Ar-2‴-
CH), 104.47 (1C, Ar-5′-CH), 66.65 (2C, 2CH2, morpholino), 62.43
(1C, CH2, −CH2C6H4), 53.66 (2C, 2CH2, morpholino); HRMS
(ES+): m/z calcd for C32H28ClFN5O3 [M + 1]+, 584.1865; found,
584.1869.
N-(4-((3-Chloro-4-(3-fluorophenoxy)phenyl)amino)quinazolin-6-
yl)-4-((4-methylpip erazin-1-yl)methyl)benzamide (17b). Brown
1
solid, 0.39 g, yield; 67%; H NMR (300 MHz, DMSO-d6): δ 10.58
(s, 1H, NH, exchanged with D2O), 10.00 (s, 1H, NH, exchanged with
D2O), 8.93 (s, 1H, Ar-2′-H), 8.61 (s, 1H, Ar-5′-H), 8.25 (s, 1H, Ar-
2‴-H), 8.02−8.00 (ds, J = 7.1 Hz, 3H, Ar-2″,2,6-H), 7.92−7.90 (d, J
= 8.4 Hz, 1H, Ar-8′-H), 7.84−7.81 (d, J = 8.5 Hz, 1H, Ar-7′-H),
7.48−7.46 (d, J = 7.0 Hz, 2H, Ar-3,5-H), 7.41−7.36 (m, 1H, Ar-6″-
H), 7.29−7.26 (d, J = 7.6 Hz, 1H, Ar-4‴-H), 6.97−6.92 (t, J = 7.7 Hz,
1H, Ar-5‴-H), 6.84−6.80 (d, J = 10.4 Hz, 1H, Ar-5″-H), 6.77−6.75
(d, J = 7.7 Hz, 1H, Ar-6‴-H), 3.54 (s, 2H, CH2, −CH2C6H4), 2.38
(br s, 8H, 4-CH2, piperazino), 2.17 (s, 3H, CH3); 13C DEPT (75
MHz, DMSO-d6): δ 153.66 (1C, Ar-2′-CH), 131.84 (1C, Ar-5‴-CH),
129.22 (2C, Ar-2, 6-CH), 128.73 (1C, Ar-8′-CH), 128.13 (2C, Ar-
3,5-CH), 124.00 (1C, Ar-7′-CH), 122.74 (1C, Ar-5″-CH), 113.93
(1C, Ar-6″-CH), 112.79 (1C, Ar-2″-CH), 110.27 (1C, Ar-6‴-CH),
109.99 (1C, Ar-4‴-CH), 104.81 (1C, Ar-2‴-CH), 104.47 (1C, Ar-5′-
CH), 62.00 (1C, CH2, −CH2C6H4), 55.06 (2C, 2CH2, piperazino),
52.89 (2C, 2CH2, piperazino), 46.01 (1C, CH3); 13C NMR (75 MHz,
DMSO-d6): δ 165.96 (1C, Ar-4′-C), 165.01 (1C, CO), 161.77
(1C, Ar-3‴-C), 159.14 (1C, Ar-1‴-C), 159.00 (1C, Ar-4″-C), 157.74
(1C, Ar-8′a-C), 153.65 (1C, Ar-2′-CH), 147.32 (1C, Ar-4-C), 146.15
(1C, Ar-6′-C), 142.99 (1C, Ar-1″-C), 138.02 (1C, Ar-1-C), 137.39
(1C, Ar-4′a-C), 131.71 (1C, Ar-5‴-CH), 129.22 (2C, Ar-2, 6-CH),
128.72 (1C, Ar-8′-CH), 128.12 (2C, Ar-3,5-CH), 125.29 (1C, Ar-3″-
C), 124.01 (1C, Ar-7′-CH), 122.73 (1C, Ar-5″-CH), 113.93 (1C, Ar-
6″-CH), 112.79 (1C, Ar-2″-CH), 110.27 (1C, Ar-6‴-CH), 109.99
(1C, Ar-4‴-CH), 104.81 (1C, Ar-2‴-CH), 104.48 (1C, Ar-5′-CH),
62.01 (1C, CH2, −CH2C6H4), 55.06 (2C, 2CH2, piperazino), 52.89
(2C, 2CH2, piperazino), 46.01 (1C, CH3); HRMS (ES+): m/z calcd
for C33H31ClFN6O2 [M + 1]+, 597.2181; found, 597.2184.
1H NMR (300 MHz, DMSO-d6): δ 10.81 (s, 2H, 2NH, exchanged
with D2O), 9.05 (s, 1H, Ar-2′-H), 8.74 (s, 1H, Ar-5′-H), 8.18 (s, 1H,
Ar-2‴-H), 8.15−8.12 (d, J = 9 Hz, 1H, Ar-8′-H), 8.10−8.07 (d, J =
7.9 Hz, 2H, Ar-2,6-H), 7.92−7.89 (d, J = 9 Hz, 1H, Ar-7′-H), 7.86−
7.83 (d, J = 9 Hz, 1H, Ar-6″-H), 7.65−7.62 (d, J = 7.8 Hz, 2H, Ar-3,5-
H), 7.46−7.38 (m, 1H, Ar-2″-H), 7.32−7.29 (d, J = 9 Hz, 1H, Ar-5″-
H), 7.00−6.94 (t, J = 8.4 Hz, 1H, Ar-5‴-H), 6.88−6.85 (d, J = 8.4,
1H, Ar-4‴-H), 6.80−6.77 (d, J = 8.3 Hz, 1H, Ar-6‴-H), 4.87 (s, 2H,
CH2); 13C DEPT (75 MHz, DMSO-d6): δ 152.37 (1C, Ar-2′-CH),
131.92 (1C, Ar-5‴-CH), 129.76 (1C, Ar-8′-CH), 129.37 (2C, Ar-2,
6-CH), 128.63 (2C, Ar-3,5-CH), 125.92 (1C, Ar-7′-CH), 125.20
(1C, Ar-5″-CH), 123.81 (1C, Ar-6″-CH), 122.54 (1C, Ar-2″-CH),
114.35 (1C, Ar-6‴-CH), 113.07 (1C, Ar-4‴-CH), 110.24 (1C, Ar-2‴-
CH), 104.78 (1C, Ar-5′-CH), 45.83 (1C, CH2). MS (APCI+): m/z
533.2 [M + 1]+.
General Procedure for the Synthesis of Compounds (17a−
c). A mixture of intermediate 16 (0.53 g, 1 mmol) and the appropriate
secondary amine, 1-methyl piperazine or morpholine (50 mmol), was
placed into a 25 mL round-bottomed flask or into a capped vial in the
case of low boiling point secondary amine, dimethylamine (100
mmol). The reaction mixture was heated under reflux and monitored
by TLC. After 2 h, the TLC of the reaction mass indicated the
absence of intermediate 16. The reaction mixture was cooled to room
temperature and poured onto iced-water (30 mL) portion wise with
stirring. The resultant precipitate was filtered, washed with water, and
dried. The obtained product was purified by flash column
chromatography using hexane/ethylacetate (90:10%) as an eluent
to afford the desired products (17a−c).
N-(4-((3-Chloro-4-(3-fluorophenoxy)phenyl)amino)quinazolin-6-
yl)-4-((dimethyl amino)methyl) Benzamide (17c). Yellow solid, 0.42
1
g, yield; 78%, mp 135−138 °C (dec); H NMR (300 MHz, DMSO-
d6): δ 10.60 (s, 1H, NH, exchanged with D2O), 10.01 (s, 1H, NH,
exchanged with D2O), 8.94 (s, 1H, Ar-2′-H), 8.62 (s, 1H, Ar-5′-H),
8.25 (s, 1H, Ar-2‴-H), 8.03−8.00 (ds, J = 7.4 Hz, 3H, Ar-2″,2,6H),
7.93−7.90 (d, J = 8.8 Hz, 1H, Ar-8′-H), 7.85−7.82 (d, J = 8.9 Hz, 1H,
Ar-7′-H), 7.50−7.47 (d, J = 7.6 Hz, 2H, Ar-3,5-H), 7.42−7.39 (d, J =
9 Hz, 1H, Ar-6″-H), 7.31−7.28 (d, J = 8.5 Hz, 1H, Ar-4‴-H), 6.98−
6.92 (t, J = 8.4 Hz, 1H, Ar-5‴-H), 6.85−6.82 (d, J = 9 Hz, 1H, Ar-5″-
H), 6.78−6.75 (d, J = 8.4 Hz, 1H, Ar-6‴-H), 3.48 (s, 2H, CH2), 2.17
(s, 6H, 2CH3); 13C DEPT (75 MHz, DMSO-d6): δ 153.68 (1C, Ar-
2′-CH), 131.86 (1C, Ar-5‴-CH), 129.17 (2C, Ar-2, 6-CH), 128.74
(1C, Ar-8′-CH), 128.12 (2C, Ar-3,5-CH), 124.01 (1C, Ar-7′-CH),
122.77 (1C, Ar-5″-CH), 113.91 (1C, Ar-6″-CH), 112.77 (1C, Ar-2″-
CH), 110.27 (1C, Ar-6‴-CH), 109.99 (1C, Ar-4‴-CH), 104.79 (1C,
Ar-2‴-CH), 104.46 (1C, Ar-5′-CH), 63.42 (1C, CH2), 45.48 (2C,
2CH3); 13C NMR (75 MHz, DMSO-d6): δ 165.97 (1C, Ar-4′-C),
165.00 (1C, CO), 161.76 (1C, Ar-3‴-C), 159.14 (1C, Ar-1‴-C),
158.99 (1C, Ar-4″-C), 157.73 (1C, Ar-8′a-C), 153.68 (1C, Ar-2′-
CH), 147.30 (1C, Ar-4-C), 146.13 (1C, Ar-6′-C), 143.65 (1C, Ar-1″-
C), 138.00 (1C, Ar-1-C), 137.37 (1C, Ar-4′a-C), 131.86 (1C, Ar-5‴-
CH), 129.17 (2C, Ar-2, 6-CH), 128.74 (1C, Ar-8′-CH), 128.11 (2C,
Ar-3,5-CH), 125.30 (1C, Ar-3″-C), 124.01 (1C, Ar-7′-CH), 122.77
(1C, Ar-5″-CH), 113.92 (1C, Ar-6″-CH), 112.77 (1C, Ar-2″-CH),
110.27 (1C, Ar-6‴-CH), 110.00 (1C, Ar-4‴-CH), 104.80 (1C, Ar-2‴-
CH), 104.46 (1C, Ar-5′-CH), 63.43 (1C, CH2), 45.48 (2C, 2CH3);
N-(4-((3-Chloro-4-(3-fluorophenoxy)phenyl)amino)quinazolin-6-
yl)-4-(morpholinomethyl)benzamide (17a). Light-yellow solid, 0.27
1
g, yield; 48%, mp 134−137 °C; H NMR (300 MHz, DMSO-d6): δ
10.59 (s, 1H, NH, exchanged with D2O), 10.01 (s, 1H, NH,
exchanged with D2O), 8.94 (s, 1H, Ar-2′-H), 8.62 (s, 1H, Ar-5′-H),
8.26 (s, 1H, Ar-2‴-H), 8.03−8.01 (ds, J = 7.5 Hz, 3H, Ar-2″,2,6-H),
7.93−7.90 (d, J = 9 Hz, 1H, Ar-8′-H), 7.85−7.82 (d, J = 9 Hz, 1H,
Ar-7′-H), 7.51−7.49 (d, J = 7.8 Hz, 2H, Ar-3,5-H), 7.44−7.36 (dd, J
= 8.1 Hz, 1.2 Hz, 1H, Ar-6″-H), 7.30−7.27 (d, J = 9 Hz, 1H, Ar-4‴-
H), 6.97−6.92 (t, J = 9 Hz, 1H, Ar-5‴-H), 6.85−6.81 (d, J = 8.4 Hz,
1H, Ar-5″-H), 6.78−6.75 (d, J = 9 Hz, 1H, Ar-6‴-H), 3.59 (br s, 4H,
2CH2, morpholino), 3.55 (s, 2H, CH2, −CH2C6H4), 2.38 (br s, 4H,
2CH2, morpholino); 13C DEPT (75 MHz, DMSO-d6): δ 153.67 (1C,
Ar-2′-CH), 131.84 (1C, Ar-5‴-CH), 129.34 (2C, Ar-2, 6-CH),
128.76 (1C, Ar-8′-CH), 128.15 (2C, Ar-3,5-CH), 123.99 (1C, Ar-7′-
CH), 122.74 (1C, Ar-5″-CH), 113.91 (1C, Ar-6″-CH), 112.76 (1C,
Ar-2″-CH), 110.26 (1C, Ar-6‴-CH), 109.98 (1C, Ar-4‴-CH), 104.80
(1C, Ar-2‴-CH), 104.47 (1C, Ar-5′-CH), 66.65 (2C, 2CH2,
morpholino), 62.43 (1C, CH2, −CH2C6H4), 53.66 (2C, 2CH2,
morpholino); 13C NMR (75 MHz, DMSO-d6): δ 165.93 (1C, Ar-4′-
C), 165.01 (1C, CO), 161.77 (1C, Ar-3‴-C), 159.15 (1C, Ar-1‴-
AC
https://dx.doi.org/10.1021/acs.jmedchem.0c01647
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HRMS (ES+): m/z calcd for C30H26ClFN5O2 [M + 1]+, 542.1759;
found, 542.1755.
mL), K2CO3 (1.4 g, 9.8 mmol) was added. For compound 22a, the
reaction mixture was stirred at room temperature under a nitrogen
atmosphere for 8 h, and the formed precipitate was filtered and
washed with acetonitrile, water, and ether in order and dried to
provide product (22a) in a pure form. For compounds 22b,c, the
reaction mixture was refluxed under a nitrogen atmosphere for 12 h,
cooled to room temperature, and filtered. The filtrate was evaporated
under reduced pressure, and the produced oily mass was dissolved in
DCM and purified by flash column chromatography using hexane/
ethyl acetate (80:20%) as an eluent to afford compound 22b or using
DCM/methanol (90:10%) to afford compound 22c in a pure form.
4-(3-Chloro-4-(3-fluorobenzyloxy)phenylamino)quinazolin-6-yl
Acetate (22a). Yellow solid, 3.8 g, yield: 90%, mp 290−292 °C, as
reported.115
N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-
quinazolin-6-yl)-2-((2-hydroxyethyl)amino) Acetamide (17d). A
mixture of intermediate 17 (0.53 g, 1 mmol) and 2-aminoethanol
(0.25 g, 0.3 mL, 5 mmol) in methanol (20 mL) was held at reflux for
4 h in the presence of triethylamine (15 drops). Methanol was
removed under reduced pressure, and the remaining residue was
dissolved in DCM and purified by flash column chromatography with
ethylacetate/MeOH (90:10%) as an eluent to afford compound 17d.
1
Brown solid, 0.37 g, yield: 68%; H NMR (300 MHz, DMSO): δ
10.59 (s, 2H, 2NH, exchanged with D2O), 10.03 (s, 1H, NH,
exchanged with D2O), 8.95 (s, 1H, Ar-2′-H), 8.61 (s, 1H, Ar-5′-H),
8.26 (s, 1H, Ar-2‴-H), 8.06 (s, 1H, Ar-2″-H), 8.03−8.00 (d, J = 8 Hz,
2H, Ar-2,6-H), 7.93−7.90 (d, J = 8.4 Hz, 1H Ar-4‴-H), 7.85−7.82 (d,
J = 8.9 Hz, 1H, Ar-8′-H), 7.54−7.51 (d, J = 7.9 Hz, 2H, Ar-3,5-H),
7.45−7.37 (m, 1H, Ar-6″-H), 7.30−7.28 (d, J = 8.9 Hz, 1H, Ar-7′-H),
6.98−6.92 (t, J = 8.4 Hz,, 1H, Ar-5‴), 6.85−6.82 (d, J = 9 Hz, 1H, Ar-
5″-H), 6.78−6.75 (d, J = 8.3 Hz, 1H, Ar-6‴-H), 4.50 (s, 1H, OH,
exchanged with D2O), 3.81 (s, 2H, CH2, −NHCH2C6H4), 3.51−3.48
(t, J = 5.6 Hz, 2H, CH2, OHCH2CH2NH−), 2.61−2.58 (t, J = 5.7 Hz,
2H, CH2, OHCH2CH2NH−); 13C DEPT (75 MHz, DMSO-d6): δ
153.65 (1C, Ar-2′-CH), 131.86 (1C, Ar-5‴-CH), 128.85 (1C, Ar-8′-
CH), 128.32 (2C, Ar-2,6-CH), 128.07 (2C, Ar-3,5-CH), 123.98 (1C,
Ar-7′-CH), 122.72 (1C, Ar-5″-CH), 113.97 (1C, Ar-6″-CH), 112.77
(1C, Ar-2″-CH), 110.27 (1C, Ar-6‴-CH), 109.99 (1C, Ar-4‴-CH),
104.81 (1C, Ar-2‴-CH), 104.48 (1C, Ar-5′-CH), 60.87 (1C, CH2,
OHCH2CH2NH−), 52.97 (1C, CH2, NHCH2C6H4), 51.57 (1C,
CH2, OHCH2CH2NH−); 13C NMR (75 MHz, DMSO-d6): δ 165.96
(1C, Ar-4′-C), 165.01 (1C, CO), 161.77 (1C, Ar-3‴-C), 159.15
(1C, Ar-1‴-C), 159.01 (1C, Ar-4″-C), 157.69 (1C, Ar-8′a-C), 153.64
(1C, Ar-2′-CH), 147.27 (1C, Ar-4-C), 146.10 (1C, Ar-6′-C), 145.82
(1C, Ar-1″-C), 137.42 (1C, Ar-1-C), 133.07 (1C, Ar-4′a-C), 131.85
(1C, Ar-5‴-CH), 128.87 (1C, Ar-8′-CH), 128.32 (2C, Ar-2, 6-CH),
128.06 (2C, Ar-3,5-CH), 125.30 (1C, Ar-3″-C), 123.98 (1C, Ar-7′-
CH), 122.72 (1C, Ar-5″-CH), 113.98 (1C, Ar-6″-CH), 112.80 (1C,
Ar-2″-CH), 110.27 (1C, Ar-6‴-CH), 109.99 (1C, Ar-4‴-CH), 104.81
(1C, Ar-2‴-CH),,104.48 (1C, Ar-5′-CH), 60.87 (1C, CH2,
OHCH2CH2NH−), 52.97 (1C, CH2, NHCH2C6H4), 51.57 (1C,
CH2, OHCH2CH2NH−). HRMS (ES+): m/z calcd for
C30H26ClFN5O3 [M + 1]+, 558.1708; found, 558.1703.
4-((3-Chloro-4-(3-fluorophenoxy)phenyl)amino)quinazolin-6-yl
1
Acetate (22b). Brown solid, 3.1 g, yield: 76%; H NMR (300 MHz,
DMSO-d6): δ 9.88 (s, 1H, NH, exchanged with D2O), 8.68 (s, 1H,
Ar-2-H), 8.35−8.36 (d (m-coupling), J = 2.2 Hz, 1H, Ar-5-H), 8.30−
8.29 (d (m-coupling), J = 2.5 Hz, 1H, Ar-2′-H), 7.93−7.90 (dd, J =
8.9, 2.5 Hz, 1H, Ar-6′-H), 7.89−7.86 (d, J = 9 Hz, 1H, Ar-8-H),
7.72−7.69 (dd, J = 9.0, 2.2 Hz, 1H, Ar-7-H), 7.44−7.36 (m, 1H, Ar-
2″-H), 7.31−7.28 (d, J = 8.9 Hz, 1H, Ar-5′-H), 6.97−6.92 (t, J = 8.3
Hz, 1H, Ar-5″-H), 6.85−6.81 (dd, J = 8.3, 2.1 Hz, 1H, Ar-4″-H),
6.77−6.74 (dd, J = 8.3 Hz, 1.9 Hz, 1H, Ar-6″-H), 2.38 (s, 3H, CH3);
13C DEPT (75 MHz, DMSO-d6): δ 154.59 (1C, Ar-2-CH), 131.85
(1C, Ar-8-CH), 129.88 (1C, Ar-7-CH), 129.06 (1C, Ar-5″-CH),
123.83 (1C, Ar-5′-CH), 122.80 (1C, Ar-6′-CH), 122.50 (1C, Ar-2′-
CH), 115.10 (1C, Ar-6″-CH), 112.85 (1C, Ar-5-CH), 110.32 (1C,
Ar-4″-CH), 104.87 (1C, Ar-2″-CH), 21.22 (1C, CH3); MS (APCI+):
m/z 424.1 [M + 1]+.
4-((3-Chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)-
quinazolin-6-ylacetate (22c). Yellow solid, 2.7 g, yield: 60%; 1H
NMR (300 MHz, DMSO-d6): δ 9.89 (s, 1H, NH, exchanged with
D2O), 8.69 (s, 1H, Ar-2-H), 8.35−8.32 (d, J = 8.3 Hz, 2H, Ar-5,8-H),
7.95−7.92 (d, J = 8.5 Hz, 1H, Ar-7-H), 7.89−7.86 (d, J = 8.9 Hz, 1H,
Ar-6′-H), 7.72−7.69 (d, J = 8.9 Hz, 1H, Ar-5′-H), 7.64−7.59 (t, J =
7.9 Hz, 1H, Ar-5″-H), 7.48−7.46 (d, J = 7.8 Hz, 1H, Ar-4″-H), 7.35−
7.32 (d, J = 8 Hz, 1H, Ar-6″-H), 7.25−7.2 (m, 2H, Ar-2′,2″-H), 2.38
(s, 3H, CH3); 13C DEPT (75 MHz, DMSO-d6): δ 154.57 (1C, Ar-2-
CH), 131.89 (1C, Ar-8-CH), 129.87 (1C, Ar-7-CH), 129.07 (1C, Ar-
5″-CH), 123.86 (1C, Ar-6″-CH), 122.97 (1C, Ar-5′-CH), 122.59
(1C, Ar-6′-CH), 120.71 (1C, Ar-4″-CH), 119.93 (1C, Ar-2′-CH),
115.10 (1C, Ar-2″-CH), 113.35 (1C, Ar-5-CH), 21.2 (1C, CH3); MS
(APCI+): m/z 474.1 [M + 1]+.
Quinazoline-4,6-diol (19). 2-Amino-5-hydroxybenzoic acid (18;
2.00 g, 13.1 mmol) and formamide (9.37 mL, 235 mmol) were mixed
and stirred for 1 h at 190 °C, a precipitate was formed while hot, and
then, the reaction mixture was cooled to room temperature. The solid
was filtered, triturated with H2O, and washed with diethyl ether to
afford quinazoline-4,6-diol (19).
General Procedure for the Synthesis of Compounds (23a−
c). A mixture of compound 22a, 22b, or 22c (5 mmol) and
ammonium hydroxide (20 mL) in methanol (60 mL) was stirred at
room temperature for 3−12 h. The clear solution formed was
evaporated under reduced pressure, and the residue was diluted with
water (30 mL) and stirred for 30 min. The resulting solid was filtered,
washed with water, and dried at 50 °C to afford compounds 23a−c.
4-(3-Chloro-4-(3-fluorobenzyloxy)phenylamino)quinazolin-6-ol
(23a). Reaction time: 12 h, yellow solid, 1.9 g, yield 71%; mp >300
°C, as reported.115
Brown solid, mp >300 (dec),109,115 1.5 g, yield: 71%; H NMR
1
(300 MHz, DMSO-d6): δ 12.02 (s, 1H, OH, exchanged with D2O),
10.05 (s, 1H, OH, exchanged with D2O), 7.90 (s, 1H, Ar-2-H), 7.54−
7.51 (d, J = 8.8 Hz, 1H, Ar-8-H), 7.41−7.40 (d (m-coupling), J = 2.3
Hz, 1H, Ar-5-H), 7.27−7.23 (dd, J = 8.7, 2.5 Hz, 1H, Ar-7-H).
4-Hydroxyquinazolin-6-yl Acetate (20). A mixture of (19)
(2.00 g, 12.3 mmol) and pyridine (2.5 mL) in acetic anhydride (18
mL) was stirred at 100 °C for 2 h. The reaction mixture was poured
into ice-cold water (100 mL). The formed precipitate was filtered,
washed with water, and dried to obtain the product (20).109,115
4-((3-Chloro-4-(3-fluorophenoxy)phenyl)amino)quinazolin-6-ol
1
(23b). Reaction time: 4 h, yellow solid, 1.75 g, yield: 92%; H NMR
1
(300 MHz, DMSO-d6): δ 10.14 (s, 1H, OH, exchanged with D2O),
9.65 (s, 1H, NH, exchanged with D2O), 8.5 (s, 1H, Ar-2-H), 8.31 (s,
1H, Ar-2″-H), 7.95−7.93 (d, J = 8.2 Hz, 1H, Ar-8-H), 7.80 (s, 1H, Ar-
2′-H), 7.72−7.69 (d, J = 8.9 Hz, 1H, Ar-6′-H), 7.47−7.43 (m, 1H, Ar-
6″-H), 7.41−7.38 (d, J = 7.5 Hz, 1H, Ar-4″-H), 7.29−7.26 (d, J = 8.8
Hz, 1H, Ar-5′-H), 6.96−6.91 (t, J = 7.7 Hz, 1H, Ar-5″-H), 6.82−6.81
(d (m-coupling), J = 2.3 Hz, 1H, Ar-5-H), 6.76−6.73 (d, J = 8.3 Hz,
1H, Ar-7-H); 13C DEPT (75 MHz, DMSO-d6): δ 151.74 (1C, Ar-2-
CH), 131.69 (1C, Ar-8-CH), 129.87 (1C, Ar-5″-CH), 124.81 (1C,
Ar-7-CH), 123.49 (1C, Ar-5′-CH), 122.86 (1C, Ar-6′-CH), 122.28
(1C, Ar-2′-CH), 112.68 (1C, Ar-6″-CH), 110.19 (1C, Ar-4″-CH),
105.43 (1C, Ar-2″-CH), 104.71 (1C, Ar-5-CH); MS (APCI+): m/z
382.1 [M + 1]+.
Off-white solid, 1.5 g, yield: 60%, mp >300 °C; H NMR (300
MHz, DMSO-d6): δ 12.34 (s, 1H, OH, exchanged with D2O), 8.10 (s,
1H, Ar-2-H), 7.83−7.82 (d (m-coupling), J = 1.8 Hz, 1H, Ar-5-H),
7.74−7.71 (d, J = 8.8 Hz, 1H, Ar-8-H), 7.60−7.57(dd, J = 8.8, 1.8 Hz,
1H, Ar-7-H), 2.31 (s, 3H, CH3).
General Procedure for the Synthesis of Compounds (22a−
c). A mixture of (20) (2.00 g, 9.8 mmol), SOCl2 (15 mL), and DMF
(0.5 mL) was heated under reflux (95 °C) for 5 h. Excess SOCl2 was
removed under reduced pressure. The residue was treated with
toluene (10 mL) and the solvent was evaporated under reduced
pressure to yield 4-chloroquinazolin-6-yl acetate (21) in a pure form.
Immediately, to a mixture of intermediate 21 and the appropriate
aniline derivative (4a−c) (9.8 mmol) dissolved in acetonitrile (25
AD
https://dx.doi.org/10.1021/acs.jmedchem.0c01647
J. Med. Chem. XXXX, XXX, XXX−XXX