Synthesis and biological assay of erlotinib analogues and BSA-conjugated erlotinib analogue

Article abstract of DOI:10.1016/j.bmcl.2017.02.059

A series of erlotinib analogues that have structural modification at 6,7-alkoxyl positions is efficiently synthesized. The in vitro anti-tumor activity of synthesized compounds is studied in two non-small cell lung cancer (NSCLC) cell lines (A549 and H1975). Among the synthesized compounds, the iodo compound 6 (ETN-6) exhibits higher anti-cancer activity compared to erlotinib. An efficient method is developed for the conjugation of erlotinib analogue-4, alcohol compound, with protein, bovine serum albumin (BSA), via succinic acid linker. The in vitro anti-tumor activity of the protein attached erlotinib analogue, 8 (ETN-4-Suc-BSA), showed stronger inhibitory activity in both A549 and H1975 NSCLC cell lines.

Full text of DOI:10.1016/j.bmcl.2017.02.059

Accepted Manuscript
Synthesis and Biological Assay of Erlotinib Analogues and BSA-Conjugated
Erlotinib Analogue
Ramalingam Boobalan, Kuang-Kai Liu, Jui-I Chao, Chinpiao Chen
PII:
S0960-894X(17)30202-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.059
BMCL 24731
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 December 2016
14 February 2017
23 February 2017
Please cite this article as: Boobalan, R., Liu, K-K., Chao, J-I., Chen, C., Synthesis and Biological Assay of Erlotinib
Analogues and BSA-Conjugated Erlotinib Analogue, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://
dx.doi.org/10.1016/j.bmcl.2017.02.059
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Synthesis and Biological Assay of Erlotinib
analogues and BSA-Conjugated Erlotinib
analogue
Leave this area blank for abstract info.
Ramalingam Boobalana,b, Kuang-Kai Liuc, Jui-I Chaoc,*, Chinpiao Chena,b,*
HN
O
N
OMe
R
CHO
N
O
R= OH, OMs, I, NH2
OH
OMe
HN
O
Isovanillin
O
N
OMe
O
N
R
N
O
n
O
8 (ETN-4-Suc-BSA)

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Synthesis and Biological Assay of Erlotinib Analogues and BSA-Conjugated
Erlotinib Analogue
Ramalingam Boobalana,b, Kuang-Kai Liuc, Jui-I Chaoc, ∗ and Chinpiao Chena,b ∗
a Department of Chemistry, National Dong Hwa University, Soufeng, Hualien 974, Taiwan
b Department of Nursing, Tzu Chi University of Science and Technology, Hualien 970, Taiwan
c Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan
ARTICLE INFO
ABSTRACT
Article history:
Received
A series of erlotinib analogues that have structural modification at 6,7-alkoxyl positions is
efficiently synthesized. The in vitro anti-tumor activity of synthesized compounds is studied in
two non-small cell lung cancer (NSCLC) cell lines (A549 and H1975). Among the synthesized
compounds, the iodo compound 6 (ETN-6) exhibits higher anti-cancer activity compared to
erlotinib. An efficient method is developed for the conjugation of erlotinib analogue-4, alcohol
compound, with protein, bovine serum albumin (BSA), via succinic acid linker. The in vitro
anti-tumor activity of the protein attached erlotinib analogue, 8 (ETN-4-Suc-BSA), showed
stronger inhibitory activity in both A549 and H1975 NSCLC cell lines.
Revised
Accepted
Available online
Keywords:
Anti-cancer
BSA
2009 Elsevier Ltd. All rights reserved.
Erlotinib
EGFR
NSCLC
The epidermal growth factor receptor (EGFR) is one of an
important anti-cancer drug target due to the fact that it involves
in various cell regulatory processes through tyrosine kinase
activity such as cell division, cell growth, motility, adhesion, and
apoptosis. Among the several EGFR-targeted drugs, 4-
anilinoquinazoline based molecules have more success in anti-
cancer treatment.1 Erlotinib (Tarceva®), one of the 4-
anilinoquinazoline based molecule, is well known small molecule
epidermal growth factor receptor-tyrosine kinase inhibitor
(EGFR-TKI) and used as an oral drug for non-small cell lung
cancer (NSCLC).2 However, the efficacy of erlotinib, also of
other EGFR-TKI such as gefitinib, is decreased due to acquired
point mutations in the kinase domain of EGFR.3 As there is need
of new strategy to overcome this problem, chemist have been
involved in various researches that include synthesizing and
and we envisioned that modifications in side chain would afford
higher anti-cancer potent molecules.
F
F
O
OR
HN
Cl
O
O
HN
Cl
O
N
N
N
O
N
N
N
O
N
O
Our previous
work
Gefitinib
Side chain modified Gefitinib analogue
more potent than Gefitinib
HN
HN
O
O
This work
N
R'
N
O
O
R''
N
O
N
O
synthesis and biological activity of
side chain modified analogues
Erlotinib
studying anti-cancer activity of
structurally modified
erlotinib/gefitinib derivatives,4 conjugating/combining multi-
targeting drugs with erlotinib,5 and also synthesizing new anti-
cancer small molecules to replace erlotinib/gefitinib.6
Figure 1. Structure of gefitinib, erlotinib and its structurally modified
analogues.
The conjugation of multi-drugs together through covalent
spacer and applying them to target cancer cells in multiple
pathways is one of the effectively growing area.5 Although few
reports were published on combinational anti-cancer study of
erlotinib and cetuximab (antibody, i.e., protein)5c or bovine serum
albumin (BSA, as a carrier protein),5e the direct conjugation of
erlotinib with protein through linker and its anti-cancer activity
In our earlier work, we have done structural modification in
the morpholine side chain part of gefitinib and that highly
enhanced the anti-cancer activity against several cell lines
compared to parent compound gefitinib (Figure 1).7a Although
there are several reports having similarly synthesized structurally
modified erlotinib analogues and studied their biological
activity,4 there is still a lot of space for the structural modification
———
∗ Corresponding author. Tel.: +886 3 863 3597; fax: +886 3 863 0475; e-mail: chinpiao@mail.ndhu.edu.tw (C. Chen)
∗ Corresponding author. Tel.: +886 3 571 2121x56965; fax: +886 3 555 6219; e-mail: jichao@faculty.nctu.edu.tw (J.-I Chao)

has never been studied. Currently, albumin-bound drugs (eg.,
Abraxane, an albumin-bound Paclitaxel) have developed to
enhance drug efficacy that can be applied in treating human
cancers.5j Albumin as drug carrier provides the advantages for
drug stability, solubility and delivery efficiency. We envisioned
that the conjugation of erlotinib analogues with these proteins
would increase anti-cancer potency. As a result, herein, we report
the efficient synthesis of seven erlotinib analogues with
modification at 6,7-alkoxy side chain, developed new synthetic
route for the conjugation of erlotinib analogue with protein BSA
through succinic acid linker, and studied their biological activity
against various cancer cell lines.
The deprotection of benzyl group of 1 by various reaction
conditions, such as hydrogenation with Pd/C, and reaction with
TFA, were unsuccessful for the conversion of 1 to 2. Therefore,
the synthesis of erlotinib analogue-2 was done by sequential
reactions starting from isovanillin 9 (Scheme 2). Protection of
hydroxyl group of 9 by treating it with 2-methoxyethylchloride
that is synthesized from 2-chloroethanol,9 followed by nitrile
synthesis via one pot oxime formation-dehydration, then
chemoselective nitration, reduction, formamidine formation and
finally reaction with 3-ethynylaniline produced desired ETN-2 in
six steps. Although synthesis of 2 is reported in the literature,10
the current synthetic route is more concise and efficient.
The structure of newly synthesized erlotinib analogues are
shown in Figure 2. The synthesis of 1 (ETN-1), Scheme 1, is
commenced with conversion of commercially available
isovanillin 9 to benzonitrile compound 10 through one pot oxime
formation-dehydration reaction.7b Base promoted benzylation of
hydroxyl compound 10 by treating with benzyl bromide afford
11 in 99% yield. The compound 11 was reacted with nitric acid-
acetic acid mixture for chemoselective nitration at less hindered
position that produced nitro compound 12 in 80% yield.
Reduction of nitro compound 12 by reaction with SnCl2 in acid
medium resulted aniline compound 13 in 73% yield. The aniline
compound 13 was then refluxed with neat N,N-
CHO
CHO
CN
a
O
O
O
O
b
c
OH
CN
OMe
9
OMe
OMe
18
17
CN
HN
H2N
O
O
O2N
O
O
e, f
O
d
N
O
N
OMe
OMe
OMe
2 (ETN-2)
20
19
dimethylformamide dimethylacetal for
1 h that yielded
g
formamidine intermediate 14 which was immediately treated
with 3-ethynylaniline 15 in acetic acid at 130 ºC for 1 h. The
above reaction believed to proceed via Dimroth rearrangement8
and that successfully afford desired erlotinib analogue 1 in 93%.
O
HO
Cl
Cl
16
MOECl
Scheme 2. Synthesis of erlotinb analogue-2 (ETN-2). Reagents and
conditions: (a) MOECl,16, K2CO3, DMF, 85 ºC, 20 h, 97%; (b) NH2OH·HCl,
NaCHO2, HCO2H, 85 ºC, 12 h, 97%; (c) conc. HNO3, AcOH, RT, 12 h, 82%;
(d) SnCl2, conc. HCl, AcOH/DCM, reflux, 5h, 80%; (e) DMF·DMA, reflux, 1
h; (f) 3-NH2PhCCH 15, AcOH, 130 ºC, 2h, 50% (over two steps); (g) CaCO3,
neat Me2SO4, 120 ºC, 12 h, 43%.
HN
HN
HN
OBn
OMe
O
O
O
N
N
O
N
OMe
R
N
N
OMe
N
1 (ETN-1)
2 (ETN-2)
3 (ETN-3): R=OAc;
4 (ETN-4): R=OH
5 (ETN-5): R=OMs;
6 (ETN-6): R=I
The synthesis of erlotinib analogue-3 is commenced with O-
demethylation of anisole compound 19 by reacting with AlCl3,
DCM at 70 ºC in seal tube (Scheme 3) that furnished phenol
compound 21 in good yield (75%).11 In order to synthesize
aniline compound from 21 like earlier schemes, the hydroxyl
group was protected either with acid chloride or with 2-
bromoethanol (see supporting information for elaborated scheme)
and then proceeded to reduction using SnCl2, conc. HCl/AcOH
condition. However, the above reactions were not afford the
desired aniline compound instead afford acetyl deprotected
aniline or unisolable material. Then, the phenol compound 21
was then protected with 2-bromoethyl acetate 2212 and that
yielded compound 23 in 88% yield. When 23 was applied to
SnCl2, conc. HCl/AcOH reduction condition, again the reaction
was sluggish and produced mixture of products. Therefore, the
reduction of 23 was carried out with mild acidic condition,
Fe/NH4Cl in H2O/MeOH. Although single product in good yield
(83%) was obtained by above mild condition, it was ortho-
aminobenzamide 24 rather than ortho-aminobenzonitrile.
According to reported literature,13 this might be due to sequential
reactions of reduction, hetereocyclization, and reductive ring
opening. However, the conversion of ortho-aminobenzamide 24
to 4-quinazolinone 25 was easily achieved in high yield (79%) by
reaction with N,N-dimethylformamide dimethylacetal followed
by acetic acid at reflux temperature. 4-Quinazolinone 25 was
then successfully transformed into erlotinib analogue 3 (ENT-3)
through reaction with thionyl chloride followed by reaction with
3-ethynylaniline in DMF at 110 ºC.
HN
N
O
O
7 (ETN-7): R=NH2
O
OMe
O
N
R
N
n
O
8 (ETN-4-Suc-BSA)
Figure 2. Structure of synthesized side chain modified erlotinib analogues.
NC
OBn
OMe
OHC
OH
NC
OH
a
b
c
OMe
OMe
9 (Isovanilin)
11
10
NC
OBn
OMe
NC
H2N
OBn
OMe
NC
N
OBn
OMe
e
d
O2N
N
12
13
14
Formamidine
NH
N
H2N
OBn
OMe
15
N
f
1 (ETN-1)
Scheme 1. Synthesis of erlotinib analogue-1 (ETN-1). Reagents and
conditions: (a) NH2OH·HCl, NaCHO2, HCO2H, 85 ºC, 12 h, 85%; (b) BnBr,
K2CO3, CH3CN, reflux, 1.5 h, 99%; (c) conc. HNO3, AcOH, RT, 1 h, 80%;
(d) conc. HCl, SnCl2, AcOH/DCM, 60 ºC, 1 h, 73%; (e) DMF·DMA, reflux,
2 h; (f) 15, AcOH, 130 ºC, 1 h, 93% (over two steps).

Later, erlotinib acetate 3 was submitted to ester hydrolysis by
reacting with 5M KOH that gave alcohol compound 4, ETN-4
(Scheme 4). Mesylation reaction of ETN-4 was easily produced
ETN-5 in 84% yield. The synthesis of iodo compound 6 was
effectively achieved with 73% yield by reaction of 5 with NaI in
acetone at 80 ºC under seal tube reaction condition. The seal tube
reflux reaction of iodo compound 6 with 30% ammonia in
methanol at 85 ºC efficiently yielded amino compound 7, ETN-7.
and 60 µM). The methoxy compound, ETN-2, the alcohol
compound, ETN-4, and the mesylate compound, ETN-5 were
also found to have higher anti-cancer activity compared to
erlotinib in A549 cancer cell line at 10 µM and 20 µM
concentrations. In H1975 cancer cell line (Figure 4) also, the
methoxy compound, ETN-2, the alcohol compound, ETN-4, the
mesylate compound, ETN-5 and, the iodo compound, ETN-6
were shown higher anti-cancer activity compared to erlotinib,
ETN, at 5 µM and 10 µM concentrations, however, the
differences were small. Moreover, compound 6 (ETN-6) exhibits
higher anti-cancer activity compared to erlotinib is studied in two
EGFR-TKI drug resistant non-small cell lung cancer (NSCLC)
cell lines (A549 and H1975). Interestingly, A549 cells were more
sensitive than those H1975 in inducing cell death by ETN-6. We
suggest that different potency between A549 and H1975 may be
from various mutation types (eg., K-Ras, EGFR) in lung cancer
cells. The precise mechanism of ETN-6 induced cell death in
various lung cancer types should be further investigated.
CN
CN
NC
O
O
O2N
O
O
O2N
O
O
a
b
O
OAc
O2N
OH
OMe
19
23
21
O
N
O
O
O
O
O
HN
OMe
OAc
f
d, e
c
H2N
O
OAc
H2N
25
24
OH
Br
Br
Cl
HN
h
Erlotinib
O
g
O
N
OMe
OAc
ETN-1
100
N
OMe
OAc
ETN-2
ETN-3
ETN-4
OAc
N
O
N
O
22
26
3 (ETN-3)
80
ETN-5
ETN-6
ETN-7
60
40
20
0
Scheme 3. Synthesis of elrotinib analogues-3 (ETN-3). Reagents and
conditions: (a) AlCl3, DCM, seal tube, 70 ºC , 2 h, 75%; (b) AcO(CH2)2Br 22,
K2CO3, DMF, 80 ºC, 12 h, 88%; (c) Fe, NH4Cl, H2O/MeOH, 70 ºC, 1 h, 83%;
(d) DMF·DMA, reflux, 2 h; (e) AcOH, 130 ºC, 1 h, 79% (over two steps); (f)
SOCl2, cat. DMF, reflux, 2 h; (g) 3-NH2PhCCH 15, DMF, 110 ºC, 2 h, 80%
(over two steps); (h) Ac2O, DMAP, DCM, rt, 15 h, 82%.
0
5
10
20
concentration (µM)
40
60
HN
HN
O
O
O
a
N
OMe
OAc
N
OMe
OH
Figure 3. Comparison of cell viability of erlotinib analogues and erlotinib in
A549 cell line.
N
N
O
3 (ETN-3)
4 (ETN-4)
100
80
HN
b
O
O
c
N
OMe
OMs
N
5 (ETN-5)
Erlotinib
60
ETN-1
ETN-2
ETN-3
A
40
ETN-4
HN
HN
N
ETN-5
ETN-6
O
O
O
N
OMe
I
OMe
NH2
d
ETN-7
20
N
N
O
6 (ETN-6)
7 (ETN-7)
0
0
0.5
concentration (µM)
1
5
10
20
Scheme 4. Synthesis of ETN analogues 4, 5, 6, and 7. Reagents and
conditions: (a) 5M KOH, MeOH, RT, 1.5 h, 97%; (b) MsCl, DMAP,
Pyridine, RT, 20 h, 84%; (c) NaI, acetone, seal tube, 80 ºC, 24 h, 73%; (d)
30% NH3 in MeOH, seal tube, 85 ºC, 6 h, 91%.
Figure 4. Comparison of cell viability of erlotinib analogues and erlotinib in
H1975 NSCLC cell lines.
Later, we intend to conjugate protein with erlotinib analogue-
4, which was chosen due to as it has complete core structure of
erlotinib, and study its anti-cancer activity. In order to conjugate
protein to ETN-4, we chosen bovine serum albumin (BSA)
protein as a standard for this particular work mainly because of
its characterization of stability and easy handling. As direct
attachment of erlotinib or its derivatives to BSA never been
reported, there was need of model study for confirmation of
The above synthesized compounds, ETN-1 to ETN-7, were
tested for in vitro anti-cancer activity and the results of MTT
assays of cell viabilities at different concentration after 72 h in
A549 (human lung adenocarcinoma cell line) and H1975
(NSCLC cell line) are shown in Figure 3 and 4. In A549 cell line
(Figure 3), newly synthesized the iodo compound ETN-6 is
proved to be a highly potent anti-cancer small molecule
compared to erlotinib (ETN) at all concentrations (5, 10, 20, 40,

attachment of small molecule to BSA. For this model study
(Scheme 5) 4-((4-cyanobenzyl)oxy)-4-oxobutanoic acid 29 was
used as it possess infrared spectroscopy detectable cyano group
and its synthesis accomplished by two simple steps that are
reduction of 4-cyanobenzaldehyde 27 followed by reaction with
succinic anhydride.14 Then the attachment of monosuccinate 29
to BSA was achieved via active ester 30 by first EDC·HCl
coupling reaction of 29 with HOBt in slightly basic buffer
solution followed by reaction with BSA and then protein
purification processes such as dialysis using Spectrum Pro 7
membrane (molecular cut off of 25000) at room temperature, and
lyophilization. The obtained compound 31 showed peak at 2231
cm-1 in infrared spectrum corresponds to cyano group and also
showed increased mass in MALDI mass spectrum that
corresponds to attachment of 16 molecules (approximately).
HN
HN
O
O
a
N
OMe
OH
N
OMe
O
O
N
O
N
O
OH
4 (ETN-4)
O
Monosuccinate 32
HN
N
O
O
b
c, d, e
O
OMe
N
O
N
N
O
N
O
Activated ester 33
HN
O
O
O
N
OMe
O
MALDI
CN
CN
CN
∼ 7 molecules
N
R n
For IR spectrum
N
O
b
a
8 (ETN-4-Suc-BSA)
O
Scheme 6. Synthesis of BSA conjugated compound 8 (ETN-4-Suc-BSA).
Reagents and conditions: (a) Succinic anhydride, pyridine, DMAP (catalytic),
RT, 20 h, 99%; (b) Na2CO3, phosphate buffer saline (pH = 7.54), EDC·HCl,
RT, 1.5 h; (c) BSA, RT, 15 h; (d) Dialysis using Spectra pro 7 membrane
(MWCO:25000), RT, 2 days; (e) Lyophilization.
OH
CH2OH
CHO
O
27
Monosuccinate 29 O
28
NC
d, e, f
N
c
O
Then, the synthesized BSA protein conjugated compound 8
was tested for anti-cancer activity in two NSCLC cell lines
(A549 and H1975) and results are shown in Figure 5 and 6. MTT
assay of cell viability results showed that compound 8 have high
anti-cancer potency than erlotinib at various concentrations in
both A549 and H1975 cell lines.
O
O
N
N
O
Activated ester 30
IR peak
2231 cm-1
NC
O
N
O
MALDI
R n
n = ∼ 16 molecules
O
31
100
80
Scheme 5. Model study for attachment of small molecule to BSA. Reagents
and conditions: (a) NaBH4, Dowex 50, THF, RT, 15 min, 99%; (b) Succinic
anhydride, DMAP, DCM, RT, 15 h, 80%; (c) Na2CO3, phosphate buffer
saline (pH = 7.54), EDC·HCl, RT, 1.5 h; (d) BSA, RT, 15 h; (e) Dialysis
using Spectra pro
Lyophilization.
7
membrane
(MWCO:25000), RT, 2 days; (f)
60
Erlotinib
40
8 (ETN-4-Suc-BSA)
After confirming the success of above process for the
attachment of small molecule into BSA, the above optimized
condition was applied for the conjugation of ETN-4 with BSA.
The attachment was commenced by the reaction of ETN-4 with
succinic anhydride in pyridine (Scheme 6) that afford the
monosuccinate 32 in very high yield (99%). Then, the
monosuccinate 32 was attached to BSA via activated ester 33 by
following the same optimized experimental procedure of the
attachment of 29 to BSA. The obtained compound 8 (ETN-4-
Suc-BSA) showed increased mass that corresponds to attachment
of 7 molecules (approximately) in MALDI mass spectrum.
20
0
0
0.5 1
concentration (µM)
5
Figure 5. Comparison of cell viability of compound 8 (ETN-4-Suc-BSA) and
erlotinib in A549 cell line.
100
80
60
Erlotinib
40
8 (ETN-4-Suc-BSA)
20
0
0
0.5 1
concentration (µM)
5

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Figure 6. Comparison of cell viability of compound 8 (ETN-4-Suc-BSA) and
erlotinib in H1975 NSCLC cell lines.
In conclusion, this letter represents the efficient synthetic
routes for synthesis of side chain modified erlotinib analogues.
The key reactions that involved in synthesis are one-pot oxime
formation-dehydration for the formation of nitrile, quinazoline
ring formation reaction between aniline and o-cyanoaniline via
formamidine intermediate, Fe/NH4Cl catalyzed reduction-
hetereocyclization-reductive ring opening reaction for the
formation of o-aminobenzamide, high yielding seal tube
reactions for O-demethylation, sodium iodide substitution,
ammonia substitution. The anti-cancer activity of synthesized
compounds is tested and among them, iodo compound 6 (ETN-6)
exhibits as a high potent anti-cancer compound for NSCLC cell
line A549. We have developed new and efficient method for
direct conjugation of erlotinib analogue to protein BSA and this
will be a useful synthetic route for the attachment of various
protein drugs to erlotinib analogues. The anti-cancer activity of
ETN-4-Suc-BSA showed that 8 is much higher activity than the
simple erlotinib itself in both A549 and H1975 NSCLC cell lines.
A549 and H1975 cell lines have been shown to resist EGFR-TKI
including erlotinib and gefitinib. We demonstrated novel
erlotinib analogues and its BSA-conjugates to increase the
efficacy and overcoming drug resistance in lung cancer cells,
which can develop for NSCLC therapy.
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The authors thank Ministry of Science and Technology,
Taiwan for financially supporting this research under the Grants
MOST 103-2113-M-277 -001 -MY3 and MOST 103-2811-M-
259-005.
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Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/xxxx
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