Toward discovery of mutant EGFR inhibitors; Design, synthesis and in vitro biological evaluation of potent 4-arylamino-6-ureido and thioureido-quinazoline derivatives

Article abstract of DOI:10.1016/j.bmc.2016.05.063

A new series of 4-anilinoquinazolines with C-6 ureido and thioureido side chains and various substituents at the C-4 anilino moiety was designed, synthesized and evaluated as wild type (WT) and mutant EGFR inhibitors. Most of the compounds inhibited EGFR kinase wild type (EGFR WT) with IC₅₀values in the low nanomolar range (<0.495–9.05?nM) and displayed more potent cytotoxic effect in BaF/3 expressing EGFR WT than reference compound gefitinib. The anti-proliferative effect of all synthesized compounds against gefitinib insensitive double mutant cell lines Ba/F3 expressing Del19/T790M and Ba/F3 expressing L858R/T790M were assayed. Compounds 4d, 6f, 7e showed significant inhibition (IC₅₀?=?1.76–2.38?μM) in these mutant lines and significant Her2 enzyme inhibition (IC₅₀?=?19.2–40.6?nM) compared to lapatinib (60.1?nM). The Binding mode of compounds 6d, 6f, 7a, 7b and 8b were demonstrated. Furthermore, growth inhibition against gefitinib insensitive cell lines PC9-GR4 (Del19/T790M) were tested, compounds 6f and 7e showed about eight and three folds respectively greater potency than gefitinib. Our structure–activity relationships (SAR) studies suggested that presence of ethyl piperidino urea/thiourea at 6-position and bulky group of (3-chloro-4-(3-fluorobenzyloxy)phenyl)amino at 4-position of quinazoline may serve as promising scaffold for developing inhibitors against wild type and mutant EGFR.

Full text of DOI:10.1016/j.bmc.2016.05.063

Accepted Manuscript
Toward discovery of mutant EGFR inhibitors; Design, synthesis and in vitro
biological evaluation of potent 4-arylamino-6-ureido and thioureido-quinazo-
line derivatives
Samar Mowafy, A. Galanis, Zainab M Doctor, Raymond M. Paranal, Deena S.
Lasheen, Nahla A. Farag, Pasi A. Jänne, Khaled A.M. Abouzid
PII:
S0968-0896(16)30407-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.063
BMC 13047
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 March 2016
23 May 2016
28 May 2016
Please cite this article as: Mowafy, S., Galanis, A., Doctor, Z.M., Paranal, R.M., Lasheen, D.S., Farag, N.A., Jänne,
P.A., Abouzid, K.A.M., Toward discovery of mutant EGFR inhibitors; Design, synthesis and in vitro biological
evaluation of potent 4-arylamino-6-ureido and thioureido-quinazoline derivatives, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.05.063
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Toward discovery of mutant EGFR inhibitors; Design, synthesis and in
vitro biological evaluation of potent 4-arylamino-6-ureido and thioureido-
quinazoline derivatives
Samar Mowafya, b, c, A. Galanisd, Zainab M Doctora,b, Raymond M. Paranald, Deena S.
Lasheene, Nahla A. Farag c, Pasi A. Jänned and Khaled A.M. Abouzide
a Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
b Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, 250 Longwood
Ave., SGM 628, Boston, MA 02115, USA
c Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, Km28 Cairo-
Ismailia Road (Ahmed Orabi District), Cairo-Egypt
d Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
e Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, 11566
Cairo, Egypt
Correspondence to: samarmowafy@gmail.com (S. Mowafy), khaled.abouzid@pharma.asu.edu.eg
Abstract:
A new series of 4-anilinoquinazolines with C-6 ureido and thioureido side chains and various
substituents at the C-4 anilino moiety was designed, synthesized and evaluated as wild type
(WT) and mutant EGFR inhibitors. Most of the compounds inhibited EGFR kinase wild type
(EGFR WT) with IC50 values in the low nanomolar range (<0.495-9.05 nM) and displayed more
potent cytotoxic effect in BaF/3 expressing EGFR WT than reference compound gefitinib. The
anti-proliferative effect of all synthesized compounds against gefitinib insensitive double mutant
cell lines Ba/F3 expressing Del19/T790M and Ba/F3 expressing L858R/T790M were assayed.
Compounds 4d, 6f, 7e showed significant inhibition (IC50= 1.76-2.38 μM) in these mutant lines
and significant Her2 enzyme inhibition (IC50= 19.2-40.6 nM) compared to lapatinib (60.1 nM).
The Binding mode of compounds 6d, 6f, 7a, 7b and 8b were demonstrated. Furthermore, growth
inhibition against gefitinib insensitive cell lines PC9-GR4 (Del19/T790M) were tested,
compounds 6f and 7e showed about eight and three folds respectively greater potency than
gefitinib. Our structure-activity relationships (SAR) studies suggested that presence of ethyl
piperidino
urea/thiourea at
6-position and
bulky group of (3-chloro-4-(3-
fluorobenzyloxy)phenyl)amino at 4-position of quinazoline may serve as promising scaffold for
developing inhibitors against wild type and mutant EGFR.
1

Keywords:
EGFR inhibitors; Quinazolines; Urea derivatives; Synthesis; EGFR-Her2 Dual inhibitors; EGFR
L858R/T790 mutant; EGFR Del19/T790M mutant.
1. Introduction:
The epidermal growth factor receptor (EGFR, Her1, ErbB1) is a member of four homologous
members of ErbB family, along with Her2/ ErbB2, Her3/ ErbB3 and Her4/ ErbB4. They play an
important role in many of the signaling processes that regulate numerous cellular functions of
cell growth, differentiation, and angiogenesis [1, 2]. This signaling cascade is initiated upon
ligand binding, through formation of hetero- and homo-dimers where hetero-dimerization with
ErbB2 is the preferred partner for maximum signaling [3]. Overexpression and mutations of
EGFR and ErbB2 have been observed in various human cancers including breast, lung, colon,
and prostate cancers and are associated with poor prognosis [4]. Therefore, inhibition of EGFR
and erbB2 kinase activity has emerged as a promising approach to cancer therapy [3]
Several EGFR inhibitors have been approved by FDA for treatment of cancer [5, 6]. Gefitinib
and erlotinib are first generation EGFR inhibitors that are used for treatment of non-small lung
cancer. Lapatinib is dual EGFR/ Her2 dual inhibitor approved for the treatment of Her2 positive
breast cancer and also is investigated in phase П clinical trials for lung cancer treatment [5, 7].
T790M mutation has caused resistance to the first generation and hence second generation
irreversible inhibitors were generated to overcome resistance, these inhibitors, as Afatinib and
Dacomitinib, have a Michael acceptor group at the 6-position of quinazoline that bind to a
unique cysteine residue (cys 797) to overcome drug resistance and increase selectivity towards
ErbB family [8], (figure 1). A drawback of these irreversible covalent inhibitors is that they
might react with related off-target proteins, giving rise to unexpected and increased toxicity [9].
Therefore, it is important to develop inhibitors with reduced toxicity while retaining activity
towards resistant cell lines.
2

Erlotinib
Gefitinib
Lapatinib
Afatinib
Dacomitinib
Figure 1: EGFR TK inhibitors
EGFR L858R and EGFR Del19 are activating mutations of EGFR that function by
increasing the affinity with ATP. These mutations represent a common mechanism of tumor
development [23, 29]. On the other hand, the T790M mutation is a secondary deactivating
mutation of a key amino-acid. This mutation induces resistance to the available first generation
TKIs (reversible inhibitors), causes loss of binding affinity to the active site and has been
detected in 50% of NSCLC patients that acquired resistance [7, 10]. Therefore, it is a major
challenge against EGFR driven cancers to develop inhibitors against the double mutants
L858R/T790M and Del19/T790M that activates EGFR and prevents the inhibition [7].
.
2. Rationale:
In our previous publication, Mowafy et al [2], a novel series of 6- urea anilinoquinazoline
derivatives was synthesized and showed promising potent and selective inhibition towards
EGFR-TK both biochemically as enzyme inhibitors and in-vitro cytotoxic effect against high
EGFR expressing cell lines. Molecular modeling supported those findings by diplaying extra
hydrogen bonding of 6-urea substitution to unique Cysteine (cys 797) of EGFR.
3

In addition, it has been reported that Her2 amplification is a potential mechanism of acquired
resistance to EGFR inhibition in EGFR mutant lung cancers [11]. Thus, combining dual EGFR-
Her2 inhibiton and designing lapatinib analogues could be an effective strategy for treatment of
EGFR mutant tumors.
Based on these findings, this urea series was further investigated and optimized. Novel 6-
ureido- and 6-thioureido-4-arylaminoquinazoline derivatives were designed and synthesized
based on structure–activity relationship of the previously reported EGFR inhibitors[12]; the
anilinoquinazoline core was retained with several modifications at the 6-position side chain by
attaching different urea and thiourea derivatives as additional hydrogen bond acceptor functions.
Also, different substitution pattern have been introduced at 4-anilino position with electron
withdrawing group at 3’/and 4’ position in a fashion similar to gefitinib, erlotinib and lapatinib,
The effect of these changes on selectivity, potency against EGFR kinase as well as wild and
mutant EGFR expressing cells lines was investigated, (figure 2).
Figure 2: Rationale and Design
Gefitinib and structure A reported in our previous publication were both used as lead compounds
[2].Several optimizations have been introduced to urea at the 6-position to give the substituted ureas and
4

thioureas using the following strategies: (i) substitution of the N-terminal of the urea with ethyl
piperidino and/ or morpholino moieties; (ii) replacement of ureas with thioureas and substitution
of the N-terminal of the thioureas with ethyl or propyl side chain linked to piperidino and/ or
morpholino moieties; (iii) different substitution pattern at the 4-anilino position to mimic gefitinib,
erlotinib and lapatinib.
3. Chemistry
The synthesis of the new 6-ureido- and 6-thioureido-4-arylaminoquinazolines derivatives
(target compounds) is shown in Schemes 1 and 2. The 6-nitroquinazolines (1a–f) were generated
following published literature methods from 5-nitroanthranilo nitrile by refluxing with
dimethylformamide dimethylacetal (DMF–DMA) to give the corresponding formamidine, which
was then reacted with substituted anilines in acetic acid [2, 13]. The nitro derivatives (1a-f) were
reduced to their corresponding the 6-aminoquinazolines (2a-f) by refluxing with SnCl2/acetic
acid under nitrogen. The corresponding ureas (3a-d) and acetamido derivatives (4a-d) were
synthesized out of the same reaction by stirring of key intermediates (2a-e) with potassium
cyanate in glacial acetic acid, the acetamido derivatives (4a-d) were generated as side products
of this reaction, they were isolated, purified and characterized through NMR (1HNMR, 13CNMR,
1
1H–15N HSQC and H–13C HSQC) and MS. The phenyl carbamate derivatives (5a-c) were
prepared by reacting the respective 6-aminoquinazolines with phenyl chloroformate, they further
underwent nucleophilic reaction with various amines to afford the corresponding compounds
(6a-f). In scheme 2, the thiourea derivatives (7a-g, 8a-c) were obtained via the reaction of key
intermediates (2a, c-f) with the appropriate isothiocyanate.
5

Scheme 1. Reagents and conditions: (i) SnCl2/MeOH, reflux; (ii) KCNO/ glacial Acetic acid, stir
at rt; (iii) Phenyl chloroformate, DIPEA, THF, 5ºC, 3h; (iv) 2-Morpholinoethylamine/ 2-
Piperidinylethylamine, THF, 70 ºC, 4 h.
Scheme 2. Reagents and conditions: (i) 2-(4-Morpholinyl)ethyl-isothiocyanate/ 2-(1-
º
Piperidinyl)ethyl isothiocyanate, THF, 0 C /rt; (ii) 3-(4-Morpholinyl)propyl isothiocyanate,
THF, 0 ºC /rt
6

4. Results and discussion:
The synthesized compounds were evaluated in enzyme-based and cell-based assays for their
ability to inhibit EGFR wild type (EGFR WT), and the proliferation against Ba/F3 cell lines
expressing wild type EGFR, transformed Ba/F3 cells harboring L858R (Table 1, 2) and Ba/F3
cells harboring double mutants either L858R/T790M or Del19/T790M and (figure 3, 4).
a. Enzyme and cellular WT/L858R BaF/3 inhibition of EGFR
As shown in table 1, 2, compounds (3a-d, 4a-d, 6a–d, 7a–d, f, g and 8b, c) revealed
inhibition of WT EGFR activity with an IC50 = <0.495–9.05 nM
First, the influence of the substitution of the N-terminal of the urea on EGFR inhibition was
investigated, table 1. The acetyl derivatives (4a-c); which are classical isosteres to the urea
derivatives (3a-c), showed almost similar enzyme affinity and EGFR inhibition to their
corresponding urea derivatives (3a-c), however, they showed better cytotoxicity in WT and
L858R Ba/F3 cell lines; where the 3-bromo acetamido derivative (4a), showed an IC50 of 0.041
µM in Ba/F3 cells expressing WT EGFR and an IC50 of 0.032 µM, in Ba/F3 L858R EGFR cells
compared to compound (3a), which showed an IC50 of 0.273 µM in Ba/F3 WT EGFR cells and
IC50 of 0.315 µM in Ba/F3 cells expressing L858R EGFR. This may be attributed to the higher
lipophilicity of acetamido group compared to urea moiety. However, for the benzyloxy
analogues, the urea derivative (3d) showed better affinity and cytotoxicity profile than the
corresponding acetamido derivative (4d). As for substituting the N-terminal with ethyl piperidino
and/ or morpholino moieties (6a-f), the structure activity relationship (SAR) for enzyme
inhibition seems to be rather flat while interestingly substitution of urea with ethyl piperidine
showed dramatic increase in growth inhibition in Ba/F3 cells expressing WT EGFR, where the
3-bromo derivative, (6b) and the 3-chloro, 4-fluoro derivative, (6d) showed IC50s of 0.021 µM
and 0.062 µM respectively compared to the 3-bromo derivative ( 3a) and the 3-chloro, 4-fluoro
derivative, (3c) which showed IC50 of 0.273 µM and 0.315 µM, respectively. In contrast, for
benzyloxy derivatives; the unsubstituted urea derivative 3d showed slightly better inhibition in
the cell based assay than substituted ones 6e and 6f.
7

Second, we investigated the influence of the replacement of the linker at the six position of
quinazoline from urea linked compounds (6b, 6c, 6d) to thiourea analogues (7c, 7a, 7b),
respectively, table 2; In general, On comparing EGFR inhibition and cell based assay for both
cases, the urea linker showed better EGFR inhibition and cytotoxic effect than the corresponding
thiourea analogues; where compound (6d) inhibited EGFR enzyme with IC50 of 1.06 nM and
showed antiproliferative IC50 of Ba/F3 cells expressing WT EGFR of 0.062 μM versus (3.70
nM and 0.080 μM) for compound (7b), respectively.
Third, the effect of increasing carbon chain length from two to three carbons was also studied,
three carbon derivatives demonstrated better inhibition for EGFR and Her2 on comparing (7a,
8b) and (7d, 8c)
Fourth, as for 4-anilino substitution, all substitution showed nanomolar EGFR inhibition, where
as the 3-Cl, 4-F anilino in compound (8b) showed highest EGFR inhibition of (<0.495 nM)
which is more potent than gefitinib (IC50 = 0.509 nM). As for 3,4 dichloroanilino substitution in
compound (7f), showed the least inhibition among the tested compound series (IC50 = 9 nM).
The bulky substitution of (3-chloro-4-(3-fluorobenzyloxy)phenyl)amino similar to that of
Lapatinib allowed dual inhibition of Her2 and EGFR; where Her2 inhibition was verified for the
following compounds (3d, IC50 = 26.8 nM), (4d, IC50 32.3= nM), (6f, IC50 = 19.2 nM ), (7d, IC50
33.1= nM), (7e, IC50 =40.6 nM ) and (8c, IC50 = 12.1 nM)
8

(3, 4)
(6)
Table 1: The effect of newly synthesized compounds (3a-d, 4a-d, 6a-f) on EGFR/Her2
enzymatic activity and antiproliferation against Ba/F3expressing EGFR WT and L858R
R
Y
EGFR/Her2
IC50 (nM)
Ba/F3 EGFR (IC50,
µM)
WT
L858R
0.315
0.032
0.364
0.068
0.401
0.371
0.407
NT
3-Br
NH2
CH3
NH2
CH3
NH2
CH3
0.803
1.01
0.273
0.041
0.194
0.039
0.315
0.264
0.186
0.41
3a
3-Br
4a
3-Ethynyl
3-Ethynyl
3-Cl,4-F
3-Cl,4-F
2.22
3b
2.04
4b
0.854
3c
0.662
4c
3-Cl,4-OCH2-Ph-3F NH2
3-Cl,4-OCH2-Ph-3F CH3
2.03 / Her2: 26.8
3.96/Her2: 32.3
1.43
3d
4d
3-Br
3-Br
O
CH2
O
0.269
0.021
0.087
0.062
0.259
>0.5
NT
6a
6b
0.772
NT
3-Cl,4-F
2.92
0.053
0.077
>0.5
6c
3-Cl,4-F
CH2
O
1.06
6d
3-Cl,4-OCH2-Ph-3F
NT
6e
3-Cl,4-OCH2-Ph-3F CH2
Her2: 19.2
0.509
>0.5
6f
0.127
0.022
gefitinib
lapatinib
Her2: 60.1
9

(7)
(8)
Table 2: The effect of compounds (7a-g, 8a-c) on EGFR/Her2 enzymatic activity and
antiproliferation against Ba/F3expressing EGFR WT and L858R
R
Y
EGFR /Her2
IC50 (nM)
Ba/F3 EGFR (IC50, µM)
WT
L858R
0.316
0.154
0.069
0.267
>0.5
3-Cl,4-F
3-Cl,4-F
3-Br
O
2.25
3.70
0.150
0.080
0.076
0.332
0.296
0.772
0.066
0.247
0.390
0.271
0.127
7a
CH2
CH2
7b
1.81
7c
3-Cl,4-OCH2-Ph-3F O
2.26 / Her2: 33.1
Her2: 40.6
9.05
7d
3-Cl,4-OCH2-Ph-3F CH2
7e
7f
3-Cl,4-Cl
4-Cl
O
O
-
0.653
0.065
0.347
0.513
>0.5
5.06
7g
3-Br
NT
8a
3-Cl,4-F
-
<0.495
8b
3-Cl,4-OCH2-Ph-3F -
1.50 / Her2: 12.1
0.509
8c
0.022
gefitinib
lapatinib
Her2: 60.1
10

b. Testing cytotoxic effect of synthesized compounds on double mutants Ba/F3
L858R/T790M and Ba/F3 Del19/T790M:
All the synthesized compounds were screened against the double mutant Ba/F3
L858R/T790M and Del19/T790M cell lines at single high concentration of 10 μM to identify the
most interesting compounds, as shown in figure 3 and 4.
Among all tested compounds, compounds (4d, 6f and 7e), having the bulky substitution of (3-
chloro-4-(3-fluorobenzyloxy)phenyl)amino, showed the highest inhibition against both double
mutants. To determine potency, the IC50 of those compounds were determined, (table 3). The
compounds showed significant inhibition of both double mutants of Ba/F3 harboring
L858R/T790M or Del T790M compared to gefitinib whose IC50 could not be attained at the
maximum tested concentration of 10 uM.
Table 3: Antiproliferative activity of 4d, 6f, 7e on transformed Ba/F3 cells
Compound
Ba/F3 EGFR (IC50, µM)
L858R/T790M
Del19/T790M
2.10
2.03
1.76
>10
2.28
2.38
2.31
>10
4d
6f
7e
gefitinib
11

Figure 3: Assay of growth percent of double mutant del19/T790M Ba/F3 cells after adding 10
uM of compounds, and plotted relative to DMSO
Figure 4: Assay percent growth after treating Ba/F3 double mutant L858R/T790M with 10 uM
of compounds and plotted relative to DMSO
12

c. Testing cytotoxic effect on PC9 and mutant PC9-GR4 cells
To further investigate the cytotoxic effect of the new series, the most potent compounds, (4d,
6f, 7e) in addition to (3c, 4c) (gefitinib analogues) for comparison purposes were tested against
one NSCLC cell line, PC9 cells; that was reported to be of high dependency on the EGF
receptor/ERK1/2 and Akt pathway for its survival and proliferation compared to other NSCLC
and it is sensitive towards gefitinib [14] and also compounds were tested against mutant PC9-
GR4 that has T790M mutation (Del19/T790M) cells that is gefitinib insensitive [10], (table 4, figure
5a and 5b).
Most compounds tested retained activity against mutant cell lines except 3c and showed better
cytotoxic profiles especially for compounds 6f and 7e which showed about eight and three times
greater potency respectively when compared to gefitinib.
Table 4: Antiproliferative activity of compounds on transformed PC9 and PC9-GR4
Compound
PC9
PC9-GR4
(IC50, µM)
(IC50, µM)
0.9611
0.2247
2.156
21.7
12.62
5.701
8.313
2.157
16.33
3c
4c
7e
0.6616
1.583
4d
6f
0.063
gefitinib
13

Figure 5a: PC9 cells were treated with compounds (3c, 4c, 7e, 4d, 6f and gefitinib) at the indicated
concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to
untreated controls
Figure 5b: PC9-GR4 cells were treated with compounds (3c, 4c, 7e, 4d, 6f and gefitinib) at the
indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative
to untreated controls
14

Molecular docking and Binding mode of compounds 6d, 6f, 7a, 7b and 8b
The results of in vitro activity which showed that urea derivative (6d), displayed lower
IC50 than corresponding thiourea derivatives (7b). Also three carbon linker (8b) showed lower
IC50 than corresponding two carbon linker derivative (7a). Moreover, compound (6f) was found
to be the most active compound against double mutant cell lines Ba/F3 expressing Del19/T790M
and Ba/F3 expressing L858R/T790M. Therefore, a docking study was performed to explain
differences in activity and predict the binding modes and orientation of the synthesized
compounds (6d, 7a, 7b, 8b and 6f) at the active site of the ATP binding site of EGFR-TK.
The coordinates of the EGFR-TK structure were obtained from the crystal structure of
EGFR (PDB code 1XKK) with its inhibitor, the 3D proposed binding mode of compounds 6d,
7a, 7b and 8b were generated by docking simulation, (figure 6, 7). Compounds showed a
docking pattern model showed similar to lapatinib [15]; (i) The N-1 nitrogen of the quinazoline
scaffold was hydrogen-bonded to the main chain NH of the hinge region Met793, (ii) The 4-
anilino moiety occupied a deep back hydrophobic pocket (iii) The C-6 urea-linked side chains
were positioned at the solvent interface of EGFR where the (O/S) atom of the urea/thiourea
derivatives form a hydrogen bond with Cys797, which was absent in reference compound
gefitinib which could enhance the binding affinity of these compounds.
For urea derivatives 6d with IC50 = 1.06 nM forms four hydrogen bond; N-1 of
quinazoline with Met793, oxygen of urea with Cys797, NH of the piperridine forms a hydrogen
bond with Asp800 and fluorine atom makes hydrogen bonding to Lys745 with a docking score
of (cDocker energy = -29.32), figure 6A. On the other hand, compound 7b with IC50 = 3.70 nM,
forms only two hydrogen bonds with N-1 of quinazoline with Met793 and the sulphur of
thiourea forms hydrogen bond with Cys797 with lower docking score (Cdocker energy= -24.08),
see Figure 6B. This may be attributed to the lower electronegativity of Sulphur atom compared
to oxygen, thus making weaker hydrogen bonding.
15

Figure 6: Proposed binding modes of compound 6d (A) and compound 7b (B); compound 6d (Cdocker
energy= -29.32) form four hydrogen bonding with Met793, Cys797, Asp800 and Lys745, while
compound 7b (Cdocker energy= -24.08)forms only two hydrogen bonding Met793, Cys797
Moreover, on comparing three carbon linker derivative (8b, IC50=<0.495) with two
carbon linker derivative (7a, IC50= 2.25nM), the higher inhibition of three carbon linker
compound (8b) may be attributed to forming of two extra interactions (carbon- hydrogen bond)
between morpholine ring and Arg841 and Ser720 where both bonds are absent in two carbon
linker (7a), (figure 7A, 7B)
16

Figure 7: Proposed binding mode of compounds 7a (A) and 8b (B); both compounds makes two
hydrogen bonds with Met793 and Cys797, while compound 8b forms other two weak interactions (carbon
hydrogen bond) with Arg841 and Ser720
Next, we explored the binding mode of (6f); the most promising compound against
double mutant cell lines Ba/F3 expressing Del19/T790M and Ba/F3 expressing L858R/T790M,
The crystal structure of double mutant (PDB 3W2O) was used, figure 8. Compound (6f) forms
hydrogen bond with Met793, Cys797 and Phe858 in addition to another interaction (salt bridge)
with Asp800, figure 8.
17

Figure 8: Proposed binding mode of compounds 6f on crystal structure (PDB 3W2O); it makes three
hydrogen bonds with Met793, Cys797 and Phe856 and salt bridge with Asp800
Western Blot Analysis:
In addition to the kinase and cellular inhibitory activities, we further investigated if the
compounds inhibit the phosphorylation of EGFR and the activation of downstream signaling
Pathways in PC9 and PC9-GR4 cell lines. Compounds (3c, 4c, 7e, 4d, 6f and gefitinib) were
selected for further experiments. PC9 and PC9-GR4 cells were incubated with 10 uM of selected
compounds for 6 h, see Figure 9a, 9b. Potent inhibition of EGFR phosphorylation was observed
with almost complete inhibition observed at PC9 cells and much less inhibition was observed at
resistant PC9-GR4. Inhibition of EGFR phosphorylation was accompanied by a less concomitant
inhibition of Akt phosphorylation.
18

Figure 9a: Effect of inhibition of phosphorylation of EGFR and AKT after incubation for 6h with 10uM
of corresponding compounds (3c, 4c, 7e, 4d, 6f and gefitinib) against PC9 cell lines
Figure 9b: Effect of inhibition of phosphorylation of EGFR and AKT after incubation for 6h with 10uM
of corresponding compounds (3c, 4c, 4d, 6f, 7e and gefitinib) against PC9-GR4 cell lines
d. Testing cytotoxic effect on BT-474 cells
In addition, to investigating the effect of on Her2 enzyme inhibition table 1, 2, we investigated
cytotoxic effect of most promising lapatinib analogues on Her2 overexpressing cell lines BT-474, figure
10 and table 5.
All Compounds tested for Her2 enzyme inhibition were more active than reference compound lapatinib,
table 1, 2. While on testing compounds 4d, 6f, 7e for their cytotoxic effect on BT-474, lapatinib had
the highest cell growth inhibition followed by 4d and then 7e and 6f, respectively.
19

Figure 10: BT-474 cells were treated with compounds (4d, 6f, 7e, gefitinib and lapatinib) at the
indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted
relative to untreated controls
Table 5: Cytotoxic effect of compounds on BT-474
Compound
BT-474
(IC50, µM)
0.057
4d
6f
0.76
0.34
7e
lapatinib
gefitinib
0.031
1.24
20

5. Final Conclusion
A new series of 4-anilinoquinazolines with C-6 ureido and thioureido linked side chains was
designed, synthesized and evaluated as WT and mutant EGFR inhibitors. Most of the compounds
showed IC50 values of WT EGFR kinase in the low nanomolar range (<0.495-9.05 nM) and
displayed more potent cytotoxic effect in Ba/F3 expressing WT EGFR than reference compound
gefitinib and inhibited EGFR activity and EGFR autophosphorylation in the PC9 NSCLC cell
line. Among those, compounds 4d, 6f, 7e showed significant inhibition in both double mutant
Ba/F3 cell lines expressing Del19/T790M and Ba/F3 expressing L858R/T790M and showed
significant inhibition of Her2 enzyme activity (IC50= 19.2-40.6 nM) compared to lapatinib (60.1
nM). Furthermore, compounds 6f and 7e successfully retained activity in PC9-GR4 cells
(Del19/T790M), these are the most promising compounds. Our SAR studies reveal that presence
of ethyl piperidino urea/thiourea at 6-position and bulky group of (3-chloro-4-(3-
fluorobenzyloxy)phenyl)amino at 4-position of quinazoline is the most potent against resistant
double mutants cell lines which can be utilized in future studies.
In addition, compound 6f showed higher sensitivity to resistant cell lines compared to
gefitinib; 6f was 8 times more potent in PC9-GR4 cells and more than 5 times potent against
double mutant Ba/F3 expressing Del19/T790M and Ba/F3 expressing L858R/T790M than
gefitinib, while 6f was 25 times less potent in PC9 cells and had an IC50 greater than 500 nM in
Ba/F3 WT EGFR cells.
Based on results of this study, we concluded that compounds targeting dual inhibition of
EGFR and ErbB2 afforded superior inhibition and better targeting of mutant cell lines over
agents that target only EGFR such as gefitinib. Dual inhibitors retained considerable activity
towards double mutant del19/T790M and L858R/T790M without the cytotoxicity of covalent
inhibitors that can bind off targets. In addition, they inhibit Her2 overexpression which may
occur as a potential mechanism of acquired resistance of EGFR inhibition [11].
21

6. Experimental:
6.1 Chemistry
Unless otherwise noted, reagents and solvents were obtained from commercial suppliers and
were used without further purification. 1H NMR spectra were recorded on a 400 MHz (Varian
7600 AS) or 500 MHz (Bruker Avance III) instrument, and chemical shifts are reported in parts
per million (ppm, δ) downfield from tetramethylsilane (TMS). Coupling constants (J) are
reported in Hz. Spin multiplicities are described as s (singlet), brs (broad singlet), t (triplet), q
(quartet), and m (multiplet). Mass spectra were obtained on a Waters Micromass ZQ instrument.
Preparative HPLC was performed on a Waters Symmetry C18 column (19 mm × 50 mm, 5μm)
using a gradient of 5−95% acetonitrile in water containing 0.05% trifluoroacetic acid (TFA) over
22 min (28 min run time) at a flow rate of 20 mL/min. Purification of compounds was performed
with either a Teledyne ISCO CombiFlash Rf system or a Waters Micromass ZQ preparative
system. Purities of assayed compounds were in all cases greater than 95%, as determined by
reverse-phase HPLC analysis.
General method for preparation of 6-nitro-4-arylaminoquinazoline 1a-f
According to reported procedures:
A
mixture of N’-(2-cyano-4-nitrophenyl)-N,N-
dimethylformamide (3.0 g, 13.7 mmol) and appropriate aniline (15.1 mmol) in glacial acetic acid
was stirred and refluxed for about 2 h, reactions were monitored with TLC. After the reaction
was completed the mixture was filtered while hot. The solid was washed with diethyl ether and
dried to yield 3a-f [16, 17]
4-(3,4-Chlorofluorophenylamino)-6-nitroquinazoline 1a: Yield 93% as reported [13, 18]
4-(3,4-Dichlorophenylamino)-6-nitroquinazoline 1b: yield (86%); mp 297–298oC , as reported
[13]
4-(4-Chlorophenylamino)-6-nitroquinazoline 1c :Yield 87% as yellow crystals, mp 264oC , (as
reported) [17]
4-(3-Bromophenylamino)-6-nitroquinazoline 1d: Yield 76.5% as yellow crystals, mp 267-270
0C, (as reported) [17]
4-(3-Ethynylphenylamino)-6-nitroquinazoline 1e: Yield 95% as yellow crystals, mp 271-272
0C, (as reported) [13]
22

4-[3-Chloro-4-(3-fluorobenzyloxy)phenylamino]-6-nitroquinazoline 1f: as reported [19]
Amino-4-arylaminoquinazoline 2a-f:[16]
Following a published procedure [16], a mixture of the nitroquinazoline derivative (5
mmol) and stannous chloride (25 mmol) in MeOH (20 mL) was stirred for 1 h at reflux. Excess
MeOH was removed under reduced pressure; the remaining residue was dissolved in EtOAc
(200 mL) and made alkaline with an aqueous solution of NaHCO3 and stirred for about one h.
The resulting mixture was filtrated under vacuum through celite. The organic phase was
separated from the aqueous phase and the aqueous phase was extracted with EtOAc (2x 20 mL),
the organic fractions were combined, washed with brine, and concentrated under reduced
pressure. The crude product was purified with column chromatography
(Dichloromethane/Methanol =9:1) to obtain the corresponding aminoquinazoline derivative 2a-f.
4
N -(3,4-Chloroflorophenyl)-4,6-quinazolinediamine 2a: yield 82%; as reported [18]
4
o
N -(3-Bromophenyl)-4,6-quinazolinediamine 2b: Yield 78%, mp 267-270 C , as reported [17,
18]
N4-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)quinazoline-4,6-diamine 2C: yield 70%, as
reported [19]
o
N4-(4-Ethynylphenyl)quinazoline-4,6-diamine (2d): Yield 62%, mp 110–111 C , (as reported)
[13]
4
N -(4-Chlorophenyl)quinazoline-4,6-diamine (2e): Yield 88% as yellow crystals, mp 237-
o
239 C (as reported) [16]
3,4-Dichlorophenyl)quinazolin-4,6-diamine (2f): yield 5.5 g (86%); mp 243–244oC, as
reported [13]
General Method for preparation of 4-(arylamino)-6-ureidoquinazoline: [2]
A mixture of appropriate amine (0.01 mol) and potassium cyanate (0.011 mol, 0.89 g) in glacial
acetic acid was stirred at RT for 1 h. The product was poured over cold water and either filtered
off or extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulphate,
filtered, and concentrated. The crude product was concentrated and purified with reversed
preparative HPLC to give desired product. On purification of the product, we found two products urea
23

and acetamido, both were separated, characterized and tested, Acetamido product comes out as result of
side acetylating reaction
1
1-(4-((4-Bromophenyl)amino)quinazolin-6-yl)urea 3a : yield 30%, H NMR (500 MHz,
DMSO-d6) δ ppm 10.98 (br. s., 1 H), 9.21 (s, 1 H), 8.74 (s, 1 H), 8.58 (d, J=1.88 Hz, 1H), 7.96-
7.93 (m, 1 H), 7.92 (dd, J=8.89, 1.88 Hz, 1 H), 7.75 (d, J=9.16 Hz, 1 H), 7.67 (d, J=8.93 Hz, 1
13
H), 7.46-7.32 (m, 2 H), 6.21 (br. s., 2 H), C NMR (126 MHz, DMSO-d6) δ ppm 160.89,
159.36, 156.22, 150.07, 141.12, 139.74, 131.07, 128.78, 128.64, 127.08, 123.52, 122.82, 121.64,
115.11, 110.18, MS (ESI) m/z 458.29 (M + H)+.
1
1-(4-((3-Ethynylphenyl)amino)quinazolin-6-yl)urea 3b : yield 35%, H NMR (400 MHz,
DMSO-d6) δ ppm 10.94 (br. s., 1 H), 9.07 (s, 1 H), 8.71 (s, 1 H), 8.55 (d, J=1.96 Hz, 1 H), 7.92
(dd, J=9.19, 2.15 Hz, 1 H), 7.85-7.78 (m, 1 H), 7.73 (d, J=9.00 Hz, 1 H) 7.70 - 7.63 (m, 1 H),
7.47-7.35 (m, 2 H), 6.15 (br. s., 2 H), 4.19 (s, 1 H), MS (ESI) m/z 304.34 (M + H)+.
1
1-(4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)urea 3c : yield 49%, H NMR (400
MHz, DMSO-d6) δ ppm 10.82 (br. s., 1 H, exchangeable by D2O), 9.04 (s, 1 H, exchangeable by
D2O), 8.68 (s, 1 H), 8.52 (d, J=1.96 Hz, 1 H), 7.97 (dd, J=6.85, 2.54 Hz, 1 H), 7.88 (dd, J=9.00,
2.35 Hz, 1 H), 7.72 (d, J=9.00 Hz, 1 H), 7.68-7.59 (m, 1 H), 7.45 (t, J=9.00 Hz, 1 H), 6.13 (br. s.,
2 H, exchangeable by D2O), MS (ESI) m/z 332.39 (M + H)+
1-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)urea 3d [20]: yield
1
32%, H NMR (400 MHz, DMSO-d6) δ ppm 11.03 (br. s., 1 H, exchangeable by D2O), 9.11 (s, 1
H, exchangeable by D2O), 8.74 (s, 1 H), 8.58 (d, J=1.96 Hz, 1 H), 7.95 (dd, J=9.19, 2.15 Hz, 1
H), 7.85 (d, J=2.44 Hz, 1 H), 7.76 (d, J=9.00 Hz, 1 H), 7.58 (dd, J=9.20, 2.44 Hz, 1 H 1 H), 7.47
- 7.44 (m, 1 H), 7.38 - 7.24 (m, 3 H), 7.18 (td, J= 9.2, 2.4, Hz, 1 H), 6.20 (br. s., 2 H,
13
exchangeable by D2O), 5.28 (s, 2 H), C NMR (126 MHz, DMSO-d6) δ ppm 163.64, 161.71,
159.35, 156.18, 151.75, 149.99, 141.05, 139.91, 131.62 , 131.13, 131.06, 128.53, 126.57, 124.81,
123.84 , 121.59, 115.33, 115.17, 114.65, 114.44 , 110.22, 69.85, MS (ESI) m/z 438.44 (M +
H)+.
N-(4-((4-Bromophenyl)amino)quinazolin-6-yl)acetamide 4a : yield 29%, 1H NMR (500 MHz,
DMSO-d6) δ ppm 11.07 (br. s, 1 H, exchangeable by D2O), 10.50 (s, 1 H, exchangeable by D2O),
24

8.87 (d, J=1.88 Hz, 1 H), 8.78 (s, 1 H), 7.96 (t, J=1.88 Hz, 1 H), 7.93 (dd, J=9.16, 2.00 Hz, 1 H),
13
7.81 (d, J=8.88 Hz, 1 H), 7.67 (dd, J=8.83, 1.88 Hz, 1 H), 7.45-7.32 (m, 2 H), 2.09 (s, 3 H), C
NMR (126 MHz, DMSO-d6) δ ppm 169.32, 159.57, 150.88, 139.65, 139.21, 131.08, 129.39,
128.89, 127.14, 123.55, 123.08, 121.64, 114.92, 112.64, 24.39, MS (ESI) m/z 457.30 (M + H)+.
1
N-(4-((3-Ethynylphenyl)amino)quinazolin-6-yl)acetamide 4b : yield 32%, H NMR (400
MHz, Solvent) δ ppm 10.96 (br. s., 1 H), 10.42 (s, 1 H), 8.83 (d, J=2.00 Hz, 1 H), 8.73 (s, 1 H),
7.89 (dd, J=8.90, 2.05 Hz, 1 H), 7.84-7.79 (m, 1 H), 7.76 (d, J=9.00 Hz, 1 H), 7.65 - 7.71 (m, 1
H), 7.41 (t, J=8.0 Hz, 1 H), 7.31 (d, J=8.0 Hz, 1 H), 4.19 (s, 1 H), 2.09 (s, 3 H), MS (ESI) m/z
303.35 (M + H)+.
1
N-(4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)acetamide 4c : yield 38%, H NMR
(400 MHz, DMSO-d6) δ ppm 10.80 (br. s., 1 H, exchangeable by D2O), 10.39 (s, 1 H,
exchangeable by D2O), 8.79 (d, J=2.4Hz, 1 H), 8.69 (s, 1 H), 7.97 (dd, J=6.85, 2.54 Hz, 1 H),
7.86 (dd, J=9.00, 1.96 Hz, 1 H), 7.77 (d, J=9.00 Hz, 1 H), 7.66 - 7.63 (m, 1 H), 7.44 (t, J=9.00
Hz, 1 H), 2.08 (s, 3 H), MS (ESI) m/z 331.4 (M + H)+.
N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)acetamide 4d : yield
1
30%, H NMR (400 MHz, Solvent) δ ppm 11.07 (br. s., 1 H), 10.49 (s, 1 H), 8.87 (d, J=2Hz, 1
H), 8.79 (s, 1 H), 7.94 (dd, J=9.00, 2.15 Hz, 1 H), 7.87 (d, J=2.54 Hz, 1 H), 7.83 (d, J=9.00 Hz, 1
H), 7.60 (dd, J=8.90, 2.54 Hz, 1 H), 7.48 - 7.45 (m, 1 H), 7.38 - 7.27 (m, 3 H), 7.17 (td, J=8.8,
2.4 Hz, 1 H), 5.30 (s, 2 H), 2.15 (s, 3 H), 13C NMR (126 MHz, DMSO-d6) δ ppm 169.25, 163.65,
161.71, 159.34, 156.49, 151.59, 151.12, 139.93, 138.94, 131.12, 131.06, 126.38, 124.60, 123.84,
123.82, 121.28, 115.33, 115.16, 114.65, 114.44, 112.62, 69.85, 24.37, MS (ESI) m/z 437.45 (M
+ H)+.
General Method for preparation of phenyl (4-(arylamino)quinazolin-6-yl)carbamate 5a-c
[8]
To a solution of corresponding amine (0.21 mmol) in THF (2 mL) containing DIPEA (75 µL,
0
0.43 mmol), phenyl chloroformate (30 µL, 0.24 mmol) was added in dropwise at 5 C for 1 h
under nitrogen. The reaction was monitored by TLC, the reaction mixture was diluted with ethyl
acetate and washed with saturated sodium carbonate solution, dried with anhydrous Na2SO4,
25

filtered and concentrated. The crude solid was purified by column chromatography
(dichloromethane: ethyl acetate = 1:1)
Phenyl (4-((3-bromophenyl)amino)quinazolin-6-yl)carbamate 5a : yield 43% as light yellow
solid, 1H NMR (400 MHz, DMSO-d6) δ ppm 10.58 (br. s., 1 H, exchangeable by D2O), 9.90 (s, 1
H), 8.62 (s, 1 H), 8.59- 8.55 (m, 1 H), 8.14 (t, J=1.96 Hz, 1 H), 7.87- 7.71 (m, 3 H), 7.52- 7.41
(m, 2 H), 7.38- 7.20 (m, 5H), MS (ESI) m/z 435.35 (M + H)+.
Phenyl (4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)carbamate 5b: yield 41% as
yellow solid1H NMR (500 MHz, DMSO-d6) δ ppm 10.61 (br. s., 1 H, exchangeable by D2O), 9.95
(s, 1 H, exchangeable by D2O), 8.62 (d, J=2.20 Hz, 1 H) 8.57 (s, 1 H), 8.12 (dd, J=6.87, 2.50 Hz,
1 H), 7.86- 7.81 (m, 2 H), 7.81-7.78(m,1H), 7.54-7.38 (m, 3 H), 7.35- 7.16 (m, 3 H), MS (ESI)
m/z 409.52 (M + H)+.
Phenyl (4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)carbamate 5c :
1
yield 43% as light yellow solid, H NMR (400 MHz, DMSO-d6) δ ppm 10.53 (br. s, 1 H, ,
exchangeable by D2O), 9.77 (s, 1 H, , exchangeable by D2O), 8.57 (d, J=1.17 Hz, 1 H), 8.51 (s, 1
H), 7.93 (d, J=2.74 Hz, 1 H), 7.82 -7.74 (m, 2 H), 7.66 (dd, J=9.00, 2.35 Hz, 1 H), 7.49 - 7.39
(m, 3 H), 7.34 -7.12 (m, 7 H), 5.23 (s, 2 H), MS (ESI) m/z 515.19 (M + H)+.
General Method for preparation of 1-(4-(Arylamino)quinazolin-6-yl)-3-(2-(piperidin-1-
yl)ethyl)urea 6b,d,f
A mixture of corresponding carbamate (0.5 mmol) and 2-Piperidinylethylamine (131.3 µl, 1
mmol) was stirred in THF (3 mL). The reaction mixture was heated to reflux for 3 h. The
reaction was cooled to room temperature; the crude reaction mixture was extracted three times
with ethyl acetate (10 mL x 3). The combined organic phase was wash with saturated sodium
bicarbonate, dried with Na2SO4 and concentrated. The residue was purified by preparative HPLC
to provide the desired product
1-(4-((4-Bromophenyl)amino)quinazolin-6-yl)-3-(2-(piperidin-1-yl)ethyl)urea 6b : yield 87%
as yellow solid, 1H NMR (400 MHz, DMSO-d6) δ ppm 10.45 (br. s, 1 H, exchangeable by D2O),
9.41 (s, 1 H, exchangeable by D2O), 9.22 (br. s., 1 H, exchangeable by D2O), 8.67 (s, 1H), 8.52
(d, J= 2Hz, 1 H), 8.12- 8.08 (m, 1 H), 7.96 (dd, J=9.20, 2.15 Hz, 1 H), 7.77 (d, J= 9.00 Hz, 2 H),
26

7.48- 7.30 (m, 2 H), 3.71- 3.49 (m, 6 H), 3.17- 3.07 (m, 2 H), 2.95- 2.91 (m, 2 H), 1.87- 1.55 (m,
4 H), MS (ESI) m/z 469.35 (M + H)+.
1-(4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)-3-(2-(piperidin-1-yl)ethyl)urea 6d :
1
yield 85% as yellow solid, H NMR (400 MHz, DMSO-d6) δ ppm 10.83 (br. s, 1 H,
exchangeable by D2O), 9.49 (s, 1 H, exchangeable by D2O), 9.20 (br. s., 1 H, exchangeable by
D2O), 8.73 (s, 1 H), 8.57 (d, J=2.35 Hz, 1 H), 7.97 (dd, J=9.00, J=2 Hz, 1 H), 7.79 (d, J=9.00 Hz,
1 H), 7.49-7.47 (m, 1H), 7.45 (t, J=9.00 Hz, 1 H), 7.29 (dd, J=9 Hz, J=2 Hz, 1 H), 3.56- 3.44
(m, 4 H), 3.22- 3.09 (m, 2 H), 3.03- 2.79 (m, 2 H), 1.81 (m, 2 H), 1.56 - 1.74 (m, 4 H), MS (ESI)
m/z 443.57 (M + H)+.
1-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)-3-(2-(piperidin-1-
yl)ethyl)urea 6f : yield 89% as yellow solid, 1H NMR (400 MHz, DMSO-d6) δ ppm 11.00 (br. s,
1 H, exchangeable by D2O), 9.55 (s, 1 H, exchangeable by D2O), 9.22 (br. s, 1 H, exchangeable
by D2O), 8.75 (s, 1 H), 8.60 (d, J=1.96 Hz, 1 H), 7.99 (dd, J=9.00, 2.00 Hz, 1 H), 7.85 (d, J=2.00
Hz, 1 H), 7.79 (d, J=9.00 Hz, 1 H), 7.59 (dd, J=8.61, 2.35 Hz, 1 H), 7.52-7.39 (m, 1 H), 7.24 -
7.36 (m, 2 H), 7.17 (td, J=8.8, 2.00 Hz, 1 H), 7.07 (t, J=5.60 Hz, 1 H), 5.28 (s, 2 H), 3.64 - 3.39
(m, 4 H), 3.19-3.15 (m, 2 H), 3.03-2.79 (m, 2 H), 1.79 - 1.83 (m, 2 H), 1.54 - 1.75 (m, 4 H), MS
(ESI) m/z 452.97 (M + H)+.
General
Method
for
preparation
of
1-(4-(arylamino)quinazolin-6-yl)-3-(2-
morpholinoethyl)urea 6a,c,e [8]
A mixture of appropriate carbamate (0.5 mmol) and 2-Morpholinoethylamine (131.3 µl, 1 mmol)
was stirred in THF (3 mL). The reaction mixture was heated to reflux for 3 h. The reaction was
cooled to room temperature; the crude reaction mixture was extracted three times with ethyl
acetate (10 mL x 3). The combined organic phase was wash with saturated sodium bicarbonate,
dried with Na2SO4 and concentrated. The residue was purified by preparative HPLC to provide
the desired product
1-(4-((4-Bromophenyl)amino)quinazolin-6-yl)-3-(2-morpholinoethyl)urea 6a: Yield 87% as
yellowish solid, 1H NMR (400 MHz, DMSO-d6) δ ppm 10.89 (br. s, 1 H, exchangeable by D2O),
9.87 (br. s, 1 H, exchangeable by D2O), 9.52 (s, 1 H, exchangeable by D2O), 8.77 (s, 1 H), 8.60
27

(d, J=1.96 Hz, 1 H), 8.03-7.99 (m, 1 H), 7.81-7.79 (d, J=9 Hz, 1H), 7.75-7.73 (d, J=8Hz, 1H),
7.44-7.39 (m, 2 H), 7.01 (t, J= 5.48 Hz, 1 H), 3.98 (s, 2 H), 3.56- 3.50 (m, 6 H), 3.25 (t, J= 6.06
Hz, 2 H), 3.16 (br. s, 2 H), MS (ESI) m/z 471.33 (M + H)+.
1-(4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)-3-(2-morpholinoethyl)urea 6c
:
1
Yield 91 % as yellow solid, H NMR (400 MHz, DMSO-d6) δ ppm 10.53 (br. s., 1 H,
exchangeable by D2O), 10.18 (br. s., 1 H, exchangeable by D2O), 9.42 (s, 1 H, exchangeable by
D2O), 8.74 (s, 1 H), 8.59 (d, J=2 Hz, 1 H), 8.08- 7.94 (m, 1 H), 7.81 (d, J=9.00 Hz, 1 H), 7.78-
7.64 (m, 1 H), 7.51 (t, J=9.00 Hz, 1 H), 7.12- 6.96 (m, 1H), 3.82- 3.97 (m, 4 H), 3.56- 3.51 (m, 4
H), 3.26 (t, J=5.8 Hz, 2 H), 3.16 (s, 2 H), MS (ESI) m/z 445.33 (M + H)+.
1-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)-3-(2-
1
morpholinoethyl)urea 6e : Yield 85% as yellow solid, H NMR (400 MHz, DMSO-d6) δ ppm
10.99 (br. s., 1 H, exchangeable by D2O), 9.86 (br. s., 1 H, exchangeable by D2O), 9.54 (s, 1 H,
exchangeable by D2O), 8.75 (s, 1 H), 8.59 (d, J=1.96 Hz, 1 H), 7.99 (dd, J=9.00, 1.96 Hz, 1 H),
7.85 (d, J=2.44 Hz, 1 H), 7.79 (d, J=9.00 Hz, 1 H), 7.59 (dd, J=9.00, 2.35 Hz, 1 H), 7.49-7.44
(m, 1 H), 7.35- 7.26 (m, 2 H), 7.18 (td, J=8.8, 1.88 Hz, 1 H), 7.04 (t, J=8.8 Hz, 1 H), 5.28 (s, 2
H), 4.07- 3.60 (m, 6 H), 3.58 - 3.40 (m, 2 H), 3.26 -3.22 (m, 2 H), 3.13 (br. s, 2 H), MS (ESI)
m/z 551.28 (M + H)+.
General
Method
for
preparation
of
1-(4-(arylamino)quinazolin-6-yl)-3-(2-
morpholinoethyl)thiourea 7a, d, f, g
A mixture of appropriate amine (0.11 mmol) and 2-(4-Morpholinyl)ethyl isothiocyanate (31.2
µL , 0.22 mmol) was stirred in 2 ml THF, the reaction mixture was heated at 600C. The reaction
was monitored with TLC. The reaction mixture was then diluted with ethyl acetate and washed
with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was
concentrated and purified with reversed preparative HPLC to give desired product
1-(4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)-3-(2-morpholinoethyl)thiourea 7a :
yield 75%, 1H NMR (500 MHz, Acetone) δ ppm 10.38 (br. s., 1 H, exchangeable by D2O), 10.28
(br. s., 1 H, exchangeable by D2O), 8.55 ( s, 1 H, exchangeable by D2O), 8.37 (s,1H), 8.14 (d,
J=2 Hz, 1 H), 7.82 (dd, J=8.8 Hz, J= 2Hz,1 H), 7.78 (m, 2 H), 7.71 (d, J=8.8Hz, 1H), 7.32 (t,
28

J=9.2 Hz,1 H), 3.62-3.55 (m, 2 H), 3.44 (t, 4 H, J=4 Hz), 2.57 (s, 2 H), 2.42 (s, 4 H), MS (ESI)
m/z 461.31 (M + H)+.
1-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)-3-(2-
morpholinoethyl)thiourea 7d: yield 77% , 1H NMR (500 MHz, DMSO-d6) δ ppm 10.85 (br. s.,
1 H, exchangeable by D2O), 10.52 (br. s., 1 H, exchangeable by D2O), 10.03 (br. s., 1 H,
exchangeable by D2O), 8.83 (s, 1 H), 8.51(d, J=2 Hz,1 H), 8.43-8.41 (m, 1 H), 8.01 (dd,
J=9.00, 2 Hz, 1 H), 7.93 (d, J=2.44 Hz, 1 H), 7.82 (d, J=8.85 Hz, 1 H), 7.66 (dd, J=9.00, 2.55
Hz, 1 H), 7.54 - 7.45 (m, 1 H), 7.38 - 7.29 (m, 2 H), 7.25 - 7.15 (m, 1 H), 5.35 (s, 2 H), 4.11-
3.86 (m, 4 H), 3.80 (br. s., 2H), 3.52 (br. s., 2 H), 3.36 (t, J=6.26 Hz, 2 H), 3.17 (br. s, 2H), MS
(ESI) m/z 567.58 (M + H)+.
1-(4-((3,4-Dichlorophenyl)amino)quinazolin-6-yl)-3-(2-morpholinoethyl)thiourea 7f : yield
1
79%, H NMR (400 MHz, CD3OD) δ ppm 10.34 (s, 1H, exchangeable by D2O), 10.27 (s, 1H,
exchangeable by D2O), 9.78 (s, 1H, exchangeable by D2O), 8.72 (s,1H), 8.45 (d, 1H, J = 2 Hz),
8.27 (dd, J = 8.8 Hz, J= 2Hz, 1 H), 8.16 (d, J=2 Hz, 1 H), 7.89 (d, J = 8.8 Hz, 1H), 7.82 (dd,
J=8.8 Hz, J=2Hz, 1 H), 7.51 (d, J=8.8Hz, 1H), 4.08 (t, J=5.6 Hz, 2H), 3.92-3.89 (m, 4H), 3.53 (t,
J=5.6 Hz, 4H), 3.27-3.24 (m, 2H), MS (ESI) m/z 477.3 (M + H)+.
1-(4-((4-Chlorophenyl)amino)quinazolin-6-yl)-3-(2-morpholinoethyl)thiourea 7g : yield
1
80%, H NMR (400 MHz, acetone) δ ppm 10.80 (s, 1 H, exchangeable by D2O), 10.2 (s, 1H,
exchangeable by D2O), 9.15 (s, 1 H, exchangeable by D2O), 8.90 (s,1H), 8.80 (d, , J=2 Hz, 1H),
8.42 - 8.39 (dd, J = 9 Hz, J=2Hz, 1 H), 8.05-8.03 (d, J=8.8 Hz, 1 H), 7.93-7.91 (d, J=8.8 Hz, 2
H), 7.50-7.48 (d, J=8.8 Hz, 2 H), 4.22- 4.15 (s, 2 H), 4.03 (s, 4 H), 3.68 (t, J=5.6 Hz, 2 H), 3.63-
3.57 (m, 2 H), 3.41- 3.28 (m, 2 H), MS (ESI) m/z 443.38 (M + H)+.
General Method for preparation of 1-(4-(arylamino)quinazolin-6-yl)-3-(2-(piperidin-1-
yl)ethyl)thiourea 7b, c, e
To a solution of appropriate amine (0.11 mmol) in 2 ml THF, 2-(1-piperidinyl)ethyl
isothiocyanate (36.1 µL , 0.22 mmol) was added, the reaction mixture was heated at 600C. The
reaction was monitored with TLC. The reaction mixture was then diluted with ethyl acetate and
29