Synthesis of quinazoline analogues with differential activity for HER2-overexpressing breast cancer cells

Full text of DOI:10.1002/bkcs.10358

Note
DOI: 10.1002/bkcs.10358
BULLETIN OF THE
S.-Y. Jang et al.
KOREAN CHEMICAL SOCIETY
Synthesis of Quinazoline Analogues with Differential Activity
for HER2-overexpressing Breast Cancer Cells
Sun-Young Jang,†,‡,§ Seul Lee,‡,§ Mira Kim,†,‡ Young Jin Ham,† Mi Young Cha,† Kwee Hyun Suh,†
†,
Young Hoon Kim,† Ara Kwon,‡ Hae Ju Kang,‡ Kyung Hoon Min,‡, and Kwang-Ok Lee
*
*
†Hanmi Research Center, Hanmi Pharm. Co. Ltd, Gyeonggi-do 445-813, Republic of Korea.
*E-mail: kolee@hanmi.co.kr
‡College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea. *E-mail: khmin@cau.ac.kr
Received March 18, 2015, Accepted March 26, 2015, Published online June 23, 2015
Keywords: HER2, Tyrosine kinase inhibitor, Quinazoline analogues
HER2 (also known as ErbB2) is a member of the epidermal
growth factor receptor (EGFR) family, which also includes
EGFR, HER2, HER3, and HER4. It is a receptor tyrosine
kinase1,2 and plays a pivotal role in oncogenic transformation
and tumorigenesis via homodimerization or heterodimerization
with EGFR family members to activate intracellular
signaling.3,4 Overexpression oramplification of HER2receptor
is seen in a variety of cancers, including breast, gastric,
pancreatic, andbladdercancer.5 Recent cancer genome sequen-
cing has shown that somatic mutations in HER2 are driver
eventsinbreast6 andnon–smallcelllungcancers.7 Drugstarget-
ing HER2 include monoclonal HER2 antibodies, such as tras-
tuzumab8 andpertuzumab,9 andtyrosinekinaseinhibitors,such
as lapatinib.10 These drugs are used clinically in combination
with other anticancer drugs for the treatment of HER2-positive
breastcancer. LapatinibisaninhibitorofbothEGFRandHER2
and exhibits potent antiproliferative activity against EGFR- and
HER2-dependent cancer cells. However, potent inhibition of
EGFR causes side effects, including diarrhea and skin rash,
which diminish patient quality of life during long-term treat-
ment. These adverse events are common with EGFR tyrosine
kinase inhibitors such as gefitinib11 and erlotinib12
(Figure 1). Thus, when using lapatinib, it is important to mini-
mizethesideeffectsbyreducingtheinhibitionofEGFRactivity
and maximizing the drug efficacy.13 Herein, we describe potent
inhibitors with differential activity in HER2-dependent breast
cancer cells and EGFR-dependent cancer cells.
nitroquinazoinone 2 using thionyl chloride in the presence
of phosphoryl chloride and DMF (dimethylformamide)
afforded 4-chloroquinazoline 3, which was converted to ami-
noquinazoline 4 through the addition of 3-chloro-4-(pyridin-
2-ylmethoxy) aniline and subsequent reduction using iron.
Coupling of N-Boc-protected amino acids and aminoquinazo-
line 4 followed by deprotection of the N-Boc group using tri-
fluoroacetic acid provided the desired analogues (6–14).
The synthetic route for C7-modified quinazoline analogues
is illustrated in Scheme 2. 6-Fluoro-7-nitroquinazolinone 15
was synthesized from commercially available 2-amino-4-
fluoro-5-nitrobenzoic acid using the same synthetic method
used for compound 2. Nucleophilic substitution with
alcohols or amine in the presence of potassium trimethylsila-
nolate in DMSO (dimethyl sulfoxide) gave the quinazolinone
16, which was converted to chloroquinazoline 17 and reduced
to 7-aminoquinazline 18. Finally, N-Boc-L-proline was
coupled with 7-aminoquinazoline 18 in the presence of
EDCI
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide),
and N-Boc group was deprotected to provide C6-modified qui-
nazoline analogues (20–23).
Antiproliferation assay15 was performed to determine the
selectivity between A-431, an EGFR-overexpressing cancer
cell line, and SKBr-3, an HER2-overexpressing breast cancer
cell line (Table 1). Gefitinib was used as an EGFR selective
inhibitorandlapatinib wasusedasanEGFR/HER2 dualinhib-
itor. Acyclicamino acid derivative 6showed higher selectivity
for SKBr-3 cells than the corresponding cyclic derivative 8.
(S)-Isomer 7 exhibited ~10-fold greater potency than racemic
compound 8. Ring-expanded compounds (R)-9 and (S)-10
were also synthesized. (S)-Isomer 10,16 which had a GI50 of
8.8 nM, showed more than 200-fold greater potency than
(R)-isomer 9, which had a GI50 of 2148 nM in SKBr-3 cells.
In terms of selectivity, the activity of (S)-isomer 10 in
SKBr-3 cells was ~21-fold greater than that in A-431 cells.
However, (R)-isomer 9 did not show differential activity in
A-431 and SKBr-3 cells. N-methylation of the pyrrolidine
group of compound 10 led to a complete loss of activity. Piper-
idine derivatives 12–14 had similar activity in both A431 and
SKBr-3 cells, but were less active than compound 10.
We commenced with the synthesis of a series of quinazoline
derivatives (Table 1), in which an amide replaced the substi-
tuted furan moiety of lapatinib at the C6 position, as well as
the C7 position (Figure 2). The aniline moiety at the C4 position
was fixed as 3-chloro-4-(pyridin-2-ylmethoxy)aniline, because
it was a useful substituent to increase growth inhibitory activity
against SkBr3 cells according to our previous report.14
The synthesis of C6-modified quinazoline analogues is
outlined in Scheme 1. 7-Nitro-4(3H)-quinazolinone 2 was
obtained from the reaction of 2-amino-5-nitrobenzoic acid
1
with formamide at 170 ꢀC. Chlorination of 7-
§ These authors contributed equally to this work.
Bull. Korean Chem. Soc. 2015, Vol. 36, 1933–1935
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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KOREAN CHEMICAL SOCIETY
Next, we synthesized the derivatives of compound 10 by
and HER2 was explored in vitro.17 Unlike the results from
the cell-based assay, EGFR and HER2 were inhibited by
99% and 90%, respectively, by 1 μM of compound 10. Data
from cell-based assays and in vitro kinase assay were not con-
sistent. Thus, further investigation, including biochemical
experiments suchaswestern blotting, is required toinvestigate
the high differential activity of compound 10.
In summary, we discovered novel quinazoline analogues
that had high potency and selectivity for HER2-
overexpressing cancer cells compared with EGFR-
overexpressing cancer cells. The most potent compound,
10, could be a useful chemical tool to find a therapeutic target
for the treatment of HER2-dependent cancer patients.
introducing various substituents at the C7 position. However,
introduction of fluoro (20), dimethylamino (22), and 1-
methyl-piperidinyl methyloxy (23) groups resulted in a loss
of activity, except for the introduction of methoxy group.
Only methoxy analogue 21 showed moderate activity and
selectivity. Among the derivatives, (S)-proline derivative 10
was the most potent, with high selectivity for SKBr-3 over
A431 cells. The kinase activity of compound 10 for EGFR
O
O
F
S
O
NH
HN
N
Cl
O
N
Acknowledgments. This research was supported by Korea
Drug Development Fund (KDDF) funded by Ministry of Sci-
ence, ICT and Future Planning, Ministry of Trade, Industry &
Energy, and Ministry of Health & Welfare (KDDF-201208-
07), and by the Chung-Ang University Research Scholarship
Grants in 2014.
Lapatinib
F
O
HN
Cl
HN
N
N
O
O
O
O
N
O
O
N
N
Gefitinib
Erlotinib
O
Figure 1. Chemical structures of EGFR tyrosine kinase inhibitors
(gefitinib, erlotinib, and lapatinib).
N
HN
N
Cl
H
N
R1 = aminoethyl or cyclic amines
R1
R2 = H, F, CH3O, Me2N,
N
Table 1. Activity of synthesized quinazoline derivatives in A-431
and SKBr-3 cells.15
N-methylpiperidininy lmethoxy
O
R2
Figure 2. Design of quinazoline derivatives based on lapatinib.
O
N
O
O
N
Cl
N
HN
N
Cl
c, d
a
b
H
N
O2N
O2N
O2N
R1
OH
NH2
NH
N
N
O
R2
3
1
2
(GI50, nM)
O
O
N
N
Compound
A-431
SKBr-3
Fold ratioa
HN
Cl
HN
N
Cl
H
N
e, f
H2N
6
7
4697
471
213
29
22
16
N
R
N
N
8
9
10
11
12
13
14
20
21
22
23
Gefitinib
Lapatinib
1298
1390
184
>1000
208
276
2148
8.8
>5000
58
4.7
0.65
21
5
4
HN
O
O
O
HN
R:
–
H2N
NH
NH
10
O
O
3.6
4.4
2.8
–
6
7
8
9
323
142
73
51
O
HN
HN
>1000
483
>1000
2888
28
>1000
56
>1000
2487
206
29
N
H
N
8.5
–
O
O
O
11
12
13
14
1.2
0.14
3.4
Scheme 1. Reagents and conditions: (a) formamide, 170 ꢀC; (b)
SOCl2, POCl3, DMF (cat.); (c) 3-chloro-4-(pyridin-2-ylmethoxy)
aniline, iPrOH, reflux; (d) Fe, c-HCl (cat.), 50% EtOH, reflux; (e)
N-Boc-amino acid, EDCI, pyridine, rt; (f ) TFA, DCM, rt.
98
a GI50 for A431/GI50 for SKBr3.
Bull. Korean Chem. Soc. 2015, Vol. 36, 1933–1935
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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BULLETIN OF THE
Note
KOREAN CHEMICAL SOCIETY
O
N
O
N
7. R. Bose, S. M. Kavuri, A. C. Searleman, W. Shen, D. Shen,
D. C. Koboldt, J. Monsey, N. Goel, A. B. Aronson, S. Li,
C. X. Ma, L. Ding, E. R. Mardis, M. J. Ellis, Cancer Discov.
2013, 3(2), 224.
Cl
N
a
O2N
F
O2N
R
b
O2N
R
NH
NH
N
15
16
17
8. M. M. Goldenberg, Clin. Ther. 1999, 21, 309.
9. S. J. Malenfant, K. R. Eckmann, C. M. Barnett, Pharmacother-
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other. 2006, 7, 177.
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14. M. Y. Cha, K. O. Lee, J. W. Kim, C. G. Lee, J. Y. Song,
Y. H. Kim, G. S. Lee, S. B. Park, M. S. Kim, J. Med. Chem.
2009, 52, 6880.
O
N
O
N
c, d
HN
Cl
e, f
HN
Cl
H
H2N
R
N
N
N
N
H
N
O
R
N
18
19
O
H3C
N
R =
F
O
N
20
21
22
23
Scheme 2. Reagents and conditions: (a) alcohols or amine, KOTMS,
DMSO, rt; (b) SOCl2, POCl3, DMF (cat.); (c) 3-chloro-4-(pyridin-2-
ylmethoxy)aniline, iPrOH, reflux; (d) Fe, c-HCl (cat.), EtOH/H2O
(1:1), reflux; (e) N-Boc-L-proline, EDCI, pyridine, rt; (f ) TFA,
DCM, rt.
15. Thenumberofviablecells wasdeterminedusing aCell Counting
Kit 8 (Dojindo Co. Japan) according to the manufacturer's
instruction.
16. Spectral data of compound 13: 1H-NMR (CDCl3, 600 MHz):
δ 10.12 (s, 1H), 8.87 (d, 1H, J = 2.4 Hz), 8.66 (1H, s), 8.09
(d, 1H, J = 4.2 Hz), 8.05 (d, 1H, J = 1.2 Hz), 7.82 (d, 1H, J = 9
Hz), 7.75(s, 1H), 7.53 (dd, 1H, J = 9 Hz, 2.4 Hz), 7.48 (dd, 1H,
J = 9 Hz, 2.4 Hz), 7.53 (d, 1H, J = 9 Hz), 7.25–7.29 (m, 1H),
6.94–6.99 (m, 2H), 6.88 (dt 1H, J = 8.4 Hz, 1.8 Hz), 5.58
(s,2H), 3.92 (q, 1H, J = 9.6 Hz, 5.4 Hz), 3.12 (dt, 1H, J = 10.2
Hz, 6.6 Hz), 2.24–2.27 (m, 1H), 2.03–2.09 (m, 1H), 1.76–1.82
(m, 2H), 13C-NMR (CDCl3, 150 MHz): δ 174.1, 163.8, 162.2,
157.8, 154.1, 146.9, 139.3, 139.2, 137.4, 135.8, 133.7, 131.5,
130.3, 130.3, 129.5, 125.5, 124.6, 123.9, 122.7, 122.7, 115.3,
114.9, 114.7, 114.2, 114.1, 109.5, 108.7, 61.01, 52.5, 52.5,
47.4, 30.8, 26.4, HRMS (ESI): exact mass calcd. for C27H24FN7O
[M+H]+, 482.2099, found 482.2086.
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Bull. Korean Chem. Soc. 2015, Vol. 36, 1933–1935
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de 1935