Optimization of gefitinib analogues with potent anticancer activity

Article abstract of DOI:10.1016/j.bmcl.2014.09.056

The interactions of gefitinib (Iressa) in EGFR are hydrogen bonding and van der Waals forces through quinazoline and aniline rings. However the morpholino group of gefitinib is poorly ordered due to its weak electron density. A series of novel piperazino analogues of gefitinib where morpholino group substituted with various piperazino groups were designed and synthesized. Most of them indicated significant anti-cancer activities against human cancer cell lines. In particular, compounds 52-54 showed excellent potency against cancer cells. Convergent synthetic approach has been developed for the synthesis of gefitinib intermediate which can lead to gefitinib as well as numerous analogues.

Full text of DOI:10.1016/j.bmcl.2014.09.056

Accepted Manuscript
Optimization of Gefitinib Analogues with Potent Anticancer Activity
Kai-Hao Yin, Yi-Han Hsieh, Rohidas S. Sulake, Su-Pei Wang, Jui-I Chao,
Chinpiao Chen
PII:
S0960-894X(14)01009-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.09.056
BMCL 22022
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 June 2014
31 August 2014
19 September 2014
Please cite this article as: Yin, K-H., Hsieh, Y-H., Sulake, R.S., Wang, S-P., Chao, J-I., Chen, C., Optimization of
Gefitinib Analogues with Potent Anticancer Activity, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://
dx.doi.org/10.1016/j.bmcl.2014.09.056
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Optimization of Gefitinib Analogues with
Potent Anticancer Activity
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Kai-Hao Yin, Yi-Han Hsieh, Rohidas S. Sulake, Su-Pei Wang, Jui-I Chao* and Chinpiao Chen*
Department of Chemistry, National Dong Hwa University, Soufeng, Hualien 974, Taiwan
F
F
O
O
HN
Cl
HN
Cl
O
N
O
nCH3
m
OH
N
N
N
OMe
N
N
OCH3
7
m = 2-7, n = 0-19
1

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Optimization of Gefitinib Analogues with Potent Anticancer Activity
Kai-Hao Yina, Yi-Han Hsieha, Rohidas S. Sulakea, Su-Pei Wangb, Jui-I Chaob,  and Chinpiao Chen a, 
a Department of Chemistry, National Dong Hwa University, Soufeng, Hualien 974, Taiwan
b Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The interactions of Gefitinib (Iressa) in EGFR are hydrogen bonding and van der Waals forces
through quinazoline and aniline rings. However the morpholino group of gefitinib is poorly
ordered due to its weak electron density. A series of novel piperazino analogues of gefitinib
where morpholino group substituted with various piperazino groups were designed and
synthesized. Most of them indicated significant anti-cancer activities against human cancer cell
lines. In particular, compounds 52–54 showed excellent potency against cancer cells.
Convergent synthetic approach has been developed for the synthesis of gefitinib intermediate
which can lead to gefitinib as well as numerous analogues.
Available online
Keywords:
Gefitinib
Iressa
Anti-cancer
A549
2014 Elsevier Ltd. All rights reserved.
EGFR
The epidermal growth factor receptor (EGFR) belongs to the
family of protein-tyrosine kinases receptor (RTK), which plays
an essential role in the regulation of cell growth, differentiation,
and survival.1 Because of their multidimensional role in the
progression of cancer, EGFR and its family members have
emerged as attractive targets for anti-cancer therapy. Several
EGFR kinase inhibitors achieved significant benefits for cancer
patients and have been approved for clinical use. Despite the
clear clinical benefits, their efficacy is eventually decreased
because of acquired point mutations in the kinase domain of
EGFR.2 Considering the increasing number of reports3 of
resistance among patients as well as the need of new drug
development for different types of cancers; small molecule
kinase inhibitors with new scaffolds are actively being sought.
EGFR-TK inhibitors represent an important new class of
anticancer drugs. Gefitinib (Figure 1) is an orally active and
selective EGFR-TK inhibitor, which has been approved for the
treatment of advanced non-small cell lung cancer. The
interactions of gefitinib in the active site of the wild-type EGFR
consists of hydrogen bonding between quinazoline ring with the
main chain amide of Met793.4 The chloro group of aniline is
surrounded by side chains of Lys745, Leu788 and Thr790. The
methoxy group of quinazoline has van der Waals interactions
with Gly796. However, the morpholino part is not involved in
any interaction and is randomly ordered due to its lower electron
density. Consequently, it was considered that modification of
morpholine part in gefitinib with group bearing active site for
potency. Numerous gefitinib derivatives with modifications and
different substitution on aromatic rings have been synthesized
and evaluated for biological application but modification at
morpholino side chain is rarely reported. 5–7
In this paper, we present convergent synthetic approach for
the synthesis of gefitinib as well as the synthesis of novel series
of gefitinib analogues, with modification at morpholino part by
substituted piperazino derivative and their potent anti-cancer
activity against cancer cell line.
Aniline part
F
Morpholine part
O
O
HN
Cl
O
O
N
m
N
O
n
N
N
N
OCH3
Cl
Substituted Piperazines
Quinazoline part
Gefitinib (Iressa)
Figure 1. Modification of morpholino part of Gefitinib with substituted
piperazino derivative.
Although several approaches dealing with the synthesis of
gefitinib have been reported,8–18 there are certain steps which
gives poor yields or difficult to repeat. Most of the synthetic
attraction with EGFR could provide inhibitors with greater
———
 Corresponding author. Tel.: +886-3-863-3597; fax: +886-3-863-0475; e-mail: chinpiao@mail.ndhu.edu.tw
 Corresponding author. Tel.: +886-3-571-2121ext 56965; fax: +886-3-555-6219; e-mail: jichao@faculty.nctu.edu.tw
1

approaches described in the literature required large excess of
methanesulfonic acid for selective demethylation and an excess
of acetic anhydride for acetylation to give intermediate
quinaqzolones. Synthetic procedures comprise unstable
intermediate and usage of corrosive chemicals like thionyl
chloride/phosphoryl chloride. Consequently, we have modified
the approach that eventually resulted in a high-yielding synthesis
of gefitinib analogues and avoids unstable intermediates (Scheme
1).
In order to try to find different structural types of tyrosine
kinase inhibitors we have systematically replaced the morpholine
moiety of the gefitinib with substituted piperazino derivatives to
enhance electron density of this part as well as to modify
lipophilicity. This method of varying the physical properties of
the compounds was predicted to favorably influence their
pharmacokinetic properties. Synthesis of substituted piperazino
derivatives begins with commercially available N-benzyl-4-
piperazine (Scheme 2). Secondary amine of 8 was reacted with
methyl 3-chlorocarbonylpropionate to give the amide 9. Benzyl
group of amide 9 was deprotected by hydrogenation and the
resulting piperazine was coupled with 1-bromo-3-chloropropane
to yield the requisite substituted piperazino part 10. Coupling of
the piperazino moiety 10 with gefitinib intermediate 7 was
achieved with K2CO3 in DMF at ambient temperature to provide
gefitinib analogue 11 in good yield which after saponification
gave the acid compound 12.
The synthesis of key gefitinib intermediate 7 has been
accomplished with commercially available 3-hydroxy-4-
methoxybenzaldehyde. Transformation of benzaldehyde to
benzonitrile has been achieved by reacting benzaldehyde 1 with
sodium formate/formic acid and hydroxylamine sulfate to afford
3-hydroxy-4-methoxybenzinitrile 2. The hydroxyl group was
protected as benzyl ether by treating benzonitrile 2 with benzyl
bromide and potassium carbonate in acetonitrile at room
temperature to afford 3 in 83% yield. Chemoselective nitration of
3 at less hindered position was achieved with the mixture of
nitric acid-acetic acid solution to afford the nitro compound 4.
Reduction of the nitro group was carried out in hydrochloric
acid-acetic acid medium using SnCl2 at 60 °C to produce the
aniline 5 in 73% yield. Synthesis of quinazoline ring was
achieved by heating aniline 5 with the dimethylformamide
dimethylacetal (DMF–DMA) in benzene to produce the N,N-
dimethyl formamidine intermediate. This intermediate was then
coupled with 3-chloro-4-fluoroaniline in acetic acid at 130 °C to
produce the crude material 6, which was further purified by
recrystallization in EtOAc to give pure material in 54% yield.
The benzyl group was deprotected by hydrogenation to afford the
crucial gefitinib intermediate 7 which can be coupled with
different side chains to provide numerous gefitinib analogues.
Cl
Bn
N
Bn
N
N
a
b
N
C
N
H
N
O
CO2CH3
10
O
CO2CH3
8
9
F
O
c
OR
HN
Cl
O
N
N
O
N
CN
CHO
CN
N
OCH3
a
b
11: R = CH3
12: R = H
OBn
OH
OH
d
OCH3
OCH3
OCH3
Scheme 2. Synthesis of piperazino derivative. Reagents and conditions: (a)
ClCO(CH2)2COOCH3, pyridine, CH2Cl2, RT, 12 h; (b) (i) H2, Pd/C, MeOH,
12 h; (ii) Br(CH2)3Cl, TEA, THF, RT, 14 h; (c) 7, K2CO3, DMF, 90 °C, 14 h;
(d) LiOH, MeOH, 60 °C, 14 h.
3
1
2
CN
CN
O2N
H2N
c
d
These synthetic analogues were screened for the anti-cancer
activity and it was observed that the ester compound 11 exhibited
more potent activity than gefitinib against RKO colorectal cancer
and BFTC905 bladder cancer cell lines; whereas acid compound
12 was less potent against these cancer cell lines than gefitinib
(Fig. 1, (a) and (b)). As the ester compound 11, in which the
morpholine group is replaced by substituted piperazine ring
indicated enhanced potency against cancer cell relative to
gefitinib, we wished to determine whether the different
substituents on the piperazine ring affect the potency against
cancer cell lines. Therefore, we extendedly designed and
synthesized several gefitinib analogues esters in order to evaluate
their anticancer activities.
OBn
OBn
OCH3
OCH3
5
4
F
F
HN
Cl
HN
Cl
e
f
OBn
OH
N
N
N
OCH3
N
OCH3
To determine the effect of the length of carbon chain in
between the two carboxylic groups, different length carbon chain
of piperazino moieties were synthesized (Scheme 3).
Deprotection in N-Boc-piperazine 13 was achieved with
hydrochloric acid15 in ethyl acetate to give the salt of substituted
piperazine 14, which then was coupled with different acyl
chlorides prepared from the corresponding acid with thionyl
chloride in benzene to give piperazino compounds 15–19.
Coupling of piperazino derivatives with gefitinib intermediate 7
was achieved with K2CO3 in DMF to provide the desired
6
7
Scheme 1. Synthesis of gefitinib intermediate 4-(3′-chloro-4′-fluoroanilino)-
6-hydroxy-7-methoxyquinazoline. Reagents and conditions: (a) HCOONa,
HCOOH, (NH2OH)2·H2SO4, 85 °C, 5 h; (b) BnBr, K2CO3, CH3CN, RT, 1.5 h;
(c) AcOH, HNO3; (d) SnCl2, conc HCl, AcOH, 1 h; (e) (i) DMF-DMA,
AcOH, benzene, 105 °C; (ii) 3-chloro-4-fluoroaniline, AcOH, 130 °C, 3 h; (f)
H2, Pd/C, MeOH, 24 h.
2

different carbon-chain length in between carbonyl groups
analogues 20–24. Several carboxylic esters of acid 12 were
synthesized in order to evaluate the effect of different
substitution on piperazine on the anti-cancer activity. Various
monocarboxylic acids were converted to the corresponding acyl
chlorides with thionyl chloride in benzene, and then the resultant
acyl chlorides were coupled with piperazine 14 to produce
piperazino compounds 25–43. Coupling of these piperazino
derivatives with gefitinib intermediate 7 afforded different ester
analogues 44–62.
Figure 2. Inhibitory cell viabilities of the synthesized compounds and
gefitinib in (a) RKO, (b) BFTC905 and (c) A549 cell lines. Cell viabilities
were determined by MTT assays after 24 h at different concentrations.
It was assumed that introduction of lactone group instead of
ether in the morpholine moiety of gefitinib will enhance electron
density as well as provide extra interaction site with EGFR-TK
inhibitor. Such type of lactone 66 was synthesized starting from
the commercially available ethanolamine. The amino group of
ethanolamine was protected by benzyl group by reductive
alkylation to afford the protected aminoalcohol 63. The
alkylation of the aminoalcohol 63 with ethyl bromoacetate
followed by lactonization of intermediate ester was achieved by
refluxing with p-toluenesulfonic acid to provide the lactone 64.
Deprotection of benzyl group of lactone 64 by hydrogenation
provided free amine which was then coupled with 1-bromo-3-
chloropropane to give lactone side chain 65. This lactone side
chain was coupled with gefitinib intermediate 7 to furnish
requisite lactone compound 66 (Scheme 4).
(a) RKO cell line
Gefitinib analogue-11 (C27H31ClFN5O5)
Gefitinib analogue-12 (C26H29ClFN5O5)
180
Gefitinib (C22H24ClFN4O3)
160
140
120
100
80
a
HN
BocN
2HCl
N
Cl
N
Cl
c
60
14
13
40
20
b
Cl
O
HO
Cl
O
N
n
n
m
0
m
control
20
40
60
80
O
O
O
O
Treatment (M, 24h)
(b) BFTC905 cell line
d
N
O
n
Gefitinib analogue-11 (C27H31ClFN5O5)
Gefitinib analogue-12 (C26H29ClFN5O5)
Gefitinib (C22H24ClFN4O3)
m
120
100
80
60
40
20
0
O
O
15-19 n = 3-7, m = 0
25-43 n = 2, m = 1-19
F
O
O
HN
Cl
N
O
m
n
O
N
N
N
OCH3
20-24 n = 3-7, m = 0
44-62 n = 2, m = 1-19
control
20
40
60
80
Treatment (M, 24h)
(c) A549 cell line
Scheme 3. Synthesis of gefitinib derivatives. Reagents and conditions: (a)
HCl(g), EtOAc; (b) SOCl2, benzene; (c) Pyridine, CH2Cl2, RT, 14 h; (d) 7,
K2CO3, DMF, 80 °C, 12 h.
Gefitinib analogue-52
Gefitinib analogue-53
Gefitinib analogue-54
Gefitinib analogue-66
Gefitinib
Ph
100
80
60
40
20
0
b
a
N
O
OH
OH
Ph
N
H
H2N
89%
96%
O
63
64
F
O
Cl
c
d
O
N
HN
Cl
O
51%
41%
N
N
O
O
N
OCH3
0
10
20
40
60
80
65
66
Treatment (M, 24 h)
3

Supplementary data
Scheme 4. Synthesis of lactone analogue. Reagents and conditions: (a) (i)
PhCHO, MeOH, MS, 0 °C to RT, 72 h; (ii) NaBH4, EtOH, 0 °C to RT, 24 h;
(b) (i) BrCH2COOEt, TEA, benzene, 0 °C to RT, 24 h; (ii) p-TSA, reflux, 2 h;
(c) (i) H2, Pd/C, MeOH, RT, 12 h; (ii) Br(CH2)3Cl, K2CO3, CH3CN, 80 °C, 12
h; (d) 7, K2CO3, DMF, 80 °C, 12 h.
Supplementary data associated with this article can be found,
in the online version, at http://
References and notes
We compared the anticancer activity of the synthesized
analogues and gefitinib by the cell viability of MTT assay against
A549, a human lung adenocarcinoma cell line. Figure 2-(c)
presented the cell viabilities of the most prominent compounds
against A549 cells in various concentrations (0, 10, 20, 40, 60
and 80 μM). Compounds 20–24 are the homologation derivatives
of ester compound 11; their cell viabilities (supporting
information) indicates that extension in the length of carbon
chain [from -(CH2)3- to -(CH2)7-] between two carbonyl groups
does not improve the anti-cancer potency against A549 cells.
Compounds 44–62 are another series of homologation
derivatives of compound 11, their ester group was extended from
methyl group (-CH3) to icosyl group (-(CH2)19CH3). All the
compounds in this series show similar or higher activity than
gefitinib. The highest potency against the A549 cell line was
observed for compounds 52–54. When the concentration of
compounds increased above 40 μM these analogues shows 15–20
fold more potency than gefitinib against A549 cells.
Considerable enhancement of potency against A549 cells of these
analogues highlights the importance of substituted piperazino
side chain in gefitinib. Notwithstanding, the EGFR mutations
have been found and associated with acquired resistance to
gefitinib. The mutation of EGFR, which substitutes methionine
for threonine at position 790 (T790M) in exon 20 of EGFR, is
one of the important mutation that was acquired resistance to
gefitinib20. Although this study does not provide the structure
modifications of drug resistance issues on the gefitinib analogues,
these novel synthetic compounds will be further investigated on
the overcoming drug resistance.
Interestingly, replacing ether linkage with lactone moiety in
the morpholine part such as compound 66 does not show any
enhancement in the activity rather it gives very poor results. This
was somewhat surprising given that the lactone would be
expected to have more polarity and additional center for
hydrogen-bonding with EGFR-TK inhibitor.
In conclusion, we have developed convergent synthetic
approach for the synthesis of gefitinib intermediate which
enabled us to easily synthesize numerous analogues of gefitinib
with modification in morpholine part. Our synthetic method for
gefitinib involves stable intermediates and procedures can be
scale-up more easily and may also be less expensive. The impact
of substituted piperazine on anti-cancer activity was very large
and compounds 52–54 exhibited good activity against A-549
cells. These compounds are promising candidates for studies in
animal models to determine whether they represent lead
compounds for development of drugs that could replace gefitinib.
This initial study has set the stage for the generation of novel
anti-cancer compounds with modification in morpholine part of
gefitinib.
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Acknowledgement
The authors thank Ms. L. M. Hsu, at the Instruments Center,
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4

Graphical Abstract
Optimization of Gefitinib Analogues with Potent Anticancer Activity
Kai-Hao Yin, Yi-Han Hsieh, Rohidas S. Sulake, Su-Pei Wang, Jui-I Chao* and
Chinpiao Chen*
F
F
O
O
HN
Cl
n
HN
Cl
O
N
O
CH3
m
OH
N
N
N
N
OMe
OCH3
N
7
m = 2-7, n = 0-19