Novel EGFR inhibitors prepared by combination of dithiocarbamic acid esters and 4-anilinoquinazolines

Article abstract of DOI:10.1016/j.bmcl.2011.04.096

On the basis of combination strategy, a novel series of EGFR inhibitors were designed and synthesized by combination of dithiocarbamic acid esters and 4-anilinoquinazolines. The effect of the synthesized compounds on cell proliferation was evaluated by MTT assay in three human cancer cell lines: MDA-MB-468, SK-BR-3 and HCT-116. Two compounds (11d and 11f) were found more potent against all three cell lines and five compounds (11a, 11d-11g) were found more potent against both MDA-MB-468 and SK-BR-3 than Lapatinib. SAR studies revealed that the substituents on C6 and C7 positions of quinazoline, the amine component of dithiocarbamate moiety and the linker greatly affected the activity. This work provides a promising new strategy for the preparation of potent tyrosine kinase inhibitors.

Full text of DOI:10.1016/j.bmcl.2011.04.096

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3637–3640
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel EGFR inhibitors prepared by combination of dithiocarbamic acid
esters and 4-anilinoquinazolines
⇑
⇑
Ri-Dong Li, Xin Zhang, Qiao-Yan Li, Ze-Mei Ge , Run-Tao Li
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
On the basis of combination strategy, a novel series of EGFR inhibitors were designed and synthesized by
combination of dithiocarbamic acid esters and 4-anilinoquinazolines. The effect of the synthesized com-
pounds on cell proliferation was evaluated by MTT assay in three human cancer cell lines: MDA-MB-468,
SK-BR-3 and HCT-116. Two compounds (11d and 11f) were found more potent against all three cell lines
and five compounds (11a, 11d–11g) were found more potent against both MDA-MB-468 and SK-BR-3
than Lapatinib. SAR studies revealed that the substituents on C6 and C7 positions of quinazoline, the
amine component of dithiocarbamate moiety and the linker greatly affected the activity. This work pro-
vides a promising new strategy for the preparation of potent tyrosine kinase inhibitors.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 4 March 2011
Revised 17 April 2011
Accepted 21 April 2011
Available online 28 April 2011
Keywords:
Combination strategy
Dithiocarbamic acid ester
4-Anilinoquinazoline
EGFR inhibitor
Synthesis
The epidermal growth factor receptors (EGFR) are a member of
the ErbB receptor family of tyrosine kinases (TK) that are overex-
pressed in several human solid tumors. They are often associated
with more aggressive disease and poorer clinical outcome.1,2
Therefore, EGFR as a target of anticancer drugs has received much
attention in the past decade. A series of EGFR inhibitors with differ-
ent molecular scaffolds have been discovered. Among them, 4-ani-
linoquinazolines plays a key role and have been extensively
studied.3 As shown in Figure 1, Gefitinib (1) and Erlotinib (2) have
been approved for the treatment of non-small-cell lung cancer
(NSCLC).4–11 Lapatinib (3), a potent dual inhibitor of ErbB-1 and
ErbB-2, was approved in 2007 for treating breast cancer.12 Many
other 4-anilinoquinazoline derivatives are still under evaluation
in clinical trials for the treatment of cancer (4–6).13 Nevertheless,
it is still necessary to develop novel EGFR targeting therapeutic
agents with enhanced potency that can overcome drug resistance.
During the past decade, the study on the SAR of quinazoline
EGFR inhibitors mainly focused on the modification of the C4 aryla-
mino group. In recent years, the substituents at C6 position of qui-
nazoline have received increasing attention in the development of
more potent dual or multi-target inhibitors. The representative
substituents at C6 position of quinazoline are listed in Figure 2. Al-
most all substituents were designed to bind with the –SH of
Cys773 in EGFR through hydrogen bonding (Fig. 2, I)14–16 or cova-
lent bonding (Fig. 2, II).17–19
Recently, dithiocarbamic acid esters, a common class of organic
molecules, have also attracted great attention due to their cancer
chemopreventive and anticancer action.20,21 It was reported that
when a suitable molecular scaffold was incorporated into dithioc-
arbamic acid esters as a key pharmacophore, the molecule showed
significant anticancer activity, such as thalidomide dithiocarba-
mates,22 chromones dithiocarbamates,23 and quinazolinone dithio-
carbamates (Fig. 3 and entries 7–9).24 In our effort to seek for new
kinds of anticancer drugs, we have identified a novel dithiocarba-
mate compound 10 (990207) with potent anticancer activity
(Fig. 3 and entry 10).25
Considering the SAR of quinazoline EGFR inhibitors and the re-
search progress on dithiocarbamic acid esters, we designed and
synthesized a new kind of EGFR inhibitors 11 by combining differ-
ent dithiocarbamic acid esters and selected quinazoline scaffolds
with two kinds of linkers (Fig. 4).
The synthesis of compounds 11a–11c is shown in Scheme 1.
The quinazoline-4,6-diol 13 was prepared via Neimentowaki syn-
thesis by heating 12 in formamide at 150 °C. Acetyl protection of
the phenolic hydroxyl group of 13 with acetic anhydride gave 14.
Chlorination of 14 using thionyl chloride afforded the intermediate
15. Coupling 15 with appropriate anilines yielded compounds
16a–16c. Subsequent hydrolysis of the acetyl group using aqueous
ammonia in methanol gave the corresponding C6-OH quinazolines
17a–17c. Alkylation of the C6-OH of 17a–17c with 1-bromo-3-
chloropropane gave the corresponding ethers 18a–18c. Finally,
the target compounds 11a–11c were prepared according to our
established method by the reaction of compounds 18a–18c with
carbon disulfide and 1-methylpiperazine in the presence of anhy-
drous potassium phosphate.25
⇑
Corresponding authors.
E-mail address: lirt@mail.bjmu.edu.cn (R.-T. Li).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.096

3638
R.-D. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3637–3640
OH
F
OH
HO
COOH
NH2
O
HO
b
HN
N
a
N
O
HN
N
Cl
N
O
O
N
O
O
O
O
N
O
N
N
N
13
14
12
Gefitinb (1)
R1
Erlotinib (2)
Cl
HN
N
R2
O
O
c
O
d
O
F
N
N
HN
N
Br
H
HN
N
Cl
H
O
N
N
N
N
S
O
15
16a: R1 = 3-F-C6H4CH2O-, R2 = Cl
16b: R1 = F, R2 = Cl
O
O
O
N
PD168393 (4)
Lapatinib (3)
16c: R1 = H, R2 = Br
R1
R2
R1
R2
F
F
HN
HN
e
f
Cl
O
HN
N
Cl
HO
H
HN
N
Cl
H
N
N
N
N
N
N
O
N
N
O
O
N
O
18a: R1 = 3-F-C6H4CH2O-, , R2 = Cl
18b: R1 = F, R2 = Cl
18c: R1 = H, R2 = Br
R1
17a: R1 = 3-F-C6H4CH2O-, , R2 = Cl
17b: R1 = F, R2 = Cl
17c: R1 = H, R2 = Br
N
O
BIBW2992 (6)
Canertinib (5)
O
Figure 1. Representative inhibitors of EGFR tyrosine kinase.
N
HN
N
R2
g
N
S
O
N
NHAr
S
R
N
11a: R1 = 3-F-C6H4CH2O-, , R2 = Cl
X
N
11b: R1 = F, R2 = Cl
11c: R1 = H, R2 = Br
RO(CH2)nO-;
HONHCO(CH2)nO-;
(CH2)nO-;
I
R =
O
N
Scheme 1. Synthesis of compounds 11a–11c. Reagents and conditions: (a)
H2NCHO, 150 °C; (b) Ac2O, pyridine; (c) SOCl2, DMF, reflux; (d) i-PrOH, aniline,
Et3N; (e) ammonia, MeOH; (f) ClCH2CH2CH2Br, K2CO3, TBAB, CH3CN, reflux; (g) CS2,
1-methylpiperazine, K3PO4, DMF.
H
N
CONH-;
S
MeO2S
O
S
n
R1C
II R = R1CH=CHCONH-; R1C CCONH-;
C(CH2)nO-;
CONH-
n
CH2=CHCON
CONH-;
CH2=CHCON
m
HO
COOH
HO
COOMe
Cl
O
COOMe
b
a
Figure 2. Representative substituents at C6 position of quinazoline EGFR inhibitors.
MeO
MeO
MeO
21
19
20
Cl
O
COOMe
Cl
O
COOMe
d
c
O
S
MeO
MeO
NO2
OH
NH2
Cl
O
O
NR1R2
22
23
S
N
O
S
X
R
Cl
O
O
O
Cl
O
f
e
N
N
N
N
Amine
S
8
7
MeO
MeO
(Thalidomide dithiocarbamates)
24
R1
R2
(Chromones dithiocarbamates)
25
HN
N
26a: R1 =3-F-C6H4CH2O-, R2 = Cl
26b: R1 = F, R2 = Cl
26c: R1 = H, R2 = Br
S
CN
Cl
O
O
N
S
g
N
N
S
HN
Me
S
NR1R2
MeO
N
R1
9
10
(990207)
R3
N
HN
N
R2
(Quinazolinone dithiocarbamates)
h
S
O
R4
N
Figure 3. Structures of some dithiocarbamates with anticancer activities.
S
MeO
11d: R1 =3-F-C6H4CH2O-, R2 = Cl, R3R4NH = 1-methylpiperazine
11e: R1 = F, R2 = Cl, R3R4NH = 1-methylpiperazine
11f: R1 = H, R2 = Br, R3R4NH = 1-methylpiperazine
R1
: R1 = 3-F-C6H4CH2O-, R2 = Cl, R3R4NH = piperazine
11g
S
HN
N
R2
: R1 =3-F-C6H4CH2O-, R2 = Cl, R3R4NH = morpholine
: R1 = 3-F-C6H4CH2O-, , R2 = Cl, R3R4NH = thiomorpholine
11h
11i
R3
X
X = CH2O, CONH
5 = H, OMe
N
R4
S
N
11j: R1 = 3-F-C6H4CH2O-, R2 = Cl, R3R4NH = 1-(bis(4-fluorophenyl)methyl)piperazine
R
R5
Scheme 2. Synthesis of compounds 11d–11j. Reagents and conditions: (a) MeOH,
SOCl2; (b) ClCH2CH2CH2Br, K2CO3, TBAB, CH3CN, reflux; (c) HNO3, H2SO4, CHCl3; (d)
Fe, NH4Cl, MeOH/H2O, reflux; (e) H2NCHO, POCl3, 90 °C; (f) SOCl2, DMF, reflux; (g) i-
PrOH, aniline, Et3N; (h) CS2, R3R4NH, K3PO4, DMF.
11
Figure 4. The structure of compounds 11.
The target compounds 11d–11j were prepared as shown in
Scheme 2. Esterification of the starting material 3-hydroxy-4-
methoxybenzoic acid 19 in the presence of thionyl chloride and
methanol at ambient temperature gave 20. Compound 20 was

R.-D. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3637–3640
3639
F
MDA-MB-468 which overexpresses EGFR; SK-BR-3 which overex-
presses ErbB-2 and to a lesser extent EGFR; HCT-116 which is be-
lieved not to express either EGFR or ErbB-2 to a significant extent.
The marketed drug Lapatinib, a dual EGFR/ErbB-2 inhibitor, was
used as a positive control for the assay. The biologic results are
shown in Table 1.
F
HN
N
Cl
HN
N
Cl
H
N
a
Br
H2N
R5
N
N
O
R5
27a: R5 = H
27b: R5 = OMe
28a:
28b:
R
R
5 = H
5 = OMe
The results indicated that most compounds showed moderate
to good activity with IC50 values in the
lM range, except
compounds 11h–11j. Two compounds (11d and 11f) were found
more potent against all three cell lines and five compounds (11a,
11d–11g) were found more potent against both MDA-MB-468
and SK-BR-3 cell lines than the positive control Lapatinib. The com-
pounds 11a and 11e are EGFR/ErbB-2 dual inhibitors due to their
selective inhibition of MDA-MB-468 and SK-BR-3.
F
b
N
HN
N
Cl
H
N
N
S
N
S
O
R5
We designed compounds 11a–11f in order to identify a suitable
4-arylamino group of quinazoline and to test the effect of C7
substituted methoxyl group on the activities. Three kinds of
4-arylamino quinoline scaffold were selected based on the known
EGFR inhibitors. Although all the compounds (11a–11f) exhibited
excellent activities, compounds 11a and 11d with 4-(3-chloro-4-
(3-fluorobenzyloxy))phenylaminoquinoline scaffold showed the
best activity among them. The biologic results of compounds
11a–11c (C7-H) and 11d–11f (C7-OMe) demonstrated that the
C7 methoxy group significantly improved the potency.
Replacing the 1-methylpiperazine of compound 11d with vari-
ous cyclic amines, including piperazine (11g), morpholine (11h),
thiomorpholine (11i), and 1-(bis(4-fluorophenyl)methyl)-pipera-
zine (11j), caused severe drop or complete loss of activity. Thus,
the amino moiety in the dithiocarbamate is very important for
the inhibitory activity. It was suggested that the tertiary amino
group of the 1-methylpiperazine in compound 11d might serve
as a receptor of hydrogen bond and form hydrogen bonding with
the –SH of Cys773 in EGFR.
5 = H
5 = OMe
11k:
11l:
R
R
Scheme 3. Synthesis of compounds 11k and 11l. Reagents and conditions: (a) 3-
bromopropanoyl chloride, THF, Et3N; (b) CS2, 1-methylpiperazine, K3PO4, DMF.
reacted with 1-bromo-3-chloropropane to afford 21 in 93% yield.
Intermediate 21 was nitrated affording compound 22, which was
then reduced by powdered iron and ammonium chloride in aque-
ous methanol to give 23. Compound 23 was treated with formam-
ide to give the key quinazoline intermediate 24. Chlorination of 24
using thionyl chloride yielded the desired intermediate 25. The
coupling of 25 with anilines gave the corresponding compounds
26a–26c. Finally, reaction of 26a–26c and carbon disulfide with
different secondary amines in the presence of anhydrous K3PO4
generated the target compounds 11d–11j.
The synthesis of target compounds 11k and 11l was shown in
Scheme 3. The key intermediates 27a and 27b were prepared
according to the literature.19 Acylation of 27a–27b with 3-bromo-
propanoyl chloride gave the corresponding intermediates
28a–28b. The synthesis of the final products 11k–11l from
28a–28b was similar to the preparation of compounds 11a–11c
from 18a–18c.
Interestingly, the linker between the quinazoline scaffold and
the dithiocarbamic acid ester moiety also greatly affected the
activity. Upon replacement of the ether linker in compounds 11b
and 11e with the amide linker, the activity was decreased and
the selectivity was increased. The corresponding compounds 11k
and 11l bearing the amide linker only selectively inhibited the
The effect of the target compounds on cell proliferation was
evaluated by MTT assay in three human cancer cell lines, that is,
Table 1
Inhibitory effect of target compounds 11a–11l on the growth of tumor cell lines
R1
R1
R2
F
R3
Me
N
HN
N
R2
HN
N
N
HN
N
Cl
H
N
N
S
O
N
S
O
N
S
N
N
N
R4
S
S
S
O
MeO
R5
11k-11l
11a-11c
11d-11j
Compd
Structure
Tumor cell IC50 (l
M)a
R1
R2
R3R4N
R5
MDA-MB-468b
SK-BR-3b
HCT-116b
11a
11b
11c
11d
11e
11f
11g
11h
11i
3-FC6H4CH2O
F
H
3-FC6H4CH2O
F
H
3-FC6H4CH2O
3-FC6H4CH2O
3-FC6H4CH2O
3-FC6H4CH2O
F
Cl
Cl
Br
Cl
Cl
Br
Cl
Cl
Cl
Cl
Cl
Cl
1-Me-Pip
1-Me-Pip
1-Me-Pip
1-Me-Pip
1-Me-Pip
1-Me-Pip
Pip
Morpholine
Thiomorpholine
1-(4-FC6H4)2CH2-Pip
1-Me-Pip
H
H
H
7.46
2.40
8.13
8.52
3.57
2.23
2.99
3.33
>100
>100
>100
9.74
>100
4.35
60.44
39.20
12.84
20.20
83.28
18.04
22.99
>100
>100
>100
>100
>100
42.36
52.01
69.72
3.84
6.56
6.35
16.92
>100
>100
>100
>100
14.19
23.46
MeO
MeO
MeO
MeO
MeO
MeO
MeO
H
11j
11k
11l
F
1-Me-Pip
MeO
Lapatinib
a
Cell line MDA-MB-468 (breast carcinoma) overexpresses EGFR; cell line SK-BR-3 (breast carcinoma) overexpresses ErbB-2; HCT-116 (colon carcinoma) is believed not to
express EGFR or ErbB-2 to a significant extent.
b
Dose-response curves were determined at five concentrations. The IC50 values are the concentrations in micromolar needed to inhibit cell growth by 50% as determined
from these curves.

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5. Chan, S. K.; Gullick, W. J.; Hill, M. E. Eur. J. Cancer 2006, 42, 17.
growth of SK-BR-3 cell line (IC50 = 9.74 lM) and MDA-MB-468
(IC50 = 14.19 lM). These results indicated that it would be impor-
tant to pay more attention on the modification of linker in future
study.
6. Ji, H.; Zhao, X.; Yuza, Y.; Shimamura, T.; Li, D.; Protopopov, A.; Jung, B. L.;
McNamara, K.; Xia, H.; Glatt, K. A.; Thomas, R. K.; Sasaki, H.; Horner, J. W.; Eck,
M. J.; Discafani, C. M.; Maher, E.; Shapiro, G. I.; Meyerson, M.; Wong, K.-K. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 7817.
7. Bikker, J. K.; Brooijmans, N.; Wissner, A.; Mansour, T. S. J. Med. Chem. 2009, 52,
1493.
8. Vansteenkiste, J. F. Expert Rev. Anticancer Ther. 2004, 4, 5.
9. Tiseo, M.; Loprevite, M.; Ardizzoni, A. Curr. Med. Chem.: Anticancer Agents 2004,
4, 139.
10. Bonomi, P. Expert Opin. Invest. Drugs 2003, 12, 1395.
11. Rusnak, D. W.; Lackey, K.; Affleck, K.; Wood, E. R.; Alligood, K. J.; Rhodes, N.;
Keith, B. R.; Murray, D. M.; Knight, W. B.; Mullin, R. J.; Gilmer, T. M. Mol. Cancer
Ther. 2001, 1, 85.
12. Petrov, K. G.; Zhang, Y.-M.; Carter, M.; Cockerill, G. S.; Dickerson, S.; Gauthier, C.
A.; Guo, Y.; Mook, R. A.; Rusnak, D. W.; Walker, A. L.; Wood, E. R.; Lackey, K. E.
Bioorg. Med. Chem. Lett. 2006, 16, 4686.
13. Garofalo, S.; Rosa, R.; Bianco, R.; Tortora, G. Expert Opin. Ther. Patents 2008, 18,
889.
14. Antonello, A.; Tarozzi, A.; Morroni, F.; Cavalli, A.; Rosini, M.; Hrelia, P.;
Bolognesi, M. L.; Melchiorre, C. J. Med. Chem. 2006, 49, 6642.
15. Cai, X.; Zhai, H. X.; Wang, J.; Forrester, J.; Qu, H.; Yin, L.; Lai, C. J.; Bao, R.; Qian,
C. J. Med. Chem. 2010, 53, 2000.
In conclusion, a novel series of 4-anilinoquinazoline derivatives
with dithiocarbamic acid ester moiety at C6 position were designed
and synthesized, and their biologic activities were evaluated by
MTT assay in three human cancer cell lines, MDA-MB-468, SK-BR-
3 and HCT-116. It was found that the compounds 11a and 11e,
being dual inhibitors of EGFR and ErbB-2 kinases, were more potent
than Lapatinib, and compounds 11d, 11f and 11g were interesting
multi-target kinase inhibitors for further studies. Some valuable
structure activity relationships (SAR) were revealed. These results
supported this promising new approach for the preparation of po-
tent tyrosine kinase inhibitors with dual or multi-targets.
Acknowledgments
16. Pawar, V. G.; Sos, M. L.; Rode, H. B.; Rabiller, M.; Heynck, S.; Otterlo, W. A.;
Thomas, R. K.; Rauh, D. J. Med. Chem. 2010, 53, 2892.
The authors thank the National Natural Science Foundation of
China (No. 20672009) and the State Key New Drug Development
Program (contract grant No. 2009ZX09103-131).
17. Tsou, H. R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.;
Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.;
Greenberger, L. M.; Wang, Y. F.; Wissner, A. J. Med. Chem. 2001, 44, 2719.
18. Ban, H. S.; Tanaka, Y.; Nabeyama, W.; Hatori, M.; Nakamura, H. Bioorg. Med.
Chem. 2010, 18, 870.
Supplementary data
19. Cha, M. Y.; Lee, K. O.; Kim, J. W.; Lee, C. G.; Song, J. Y.; Kim, Y. H.; Lee, G. S.; Park,
S. B.; Kim, M. S. J. Med. Chem. 2009, 52, 6880.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.096.
20. Gerhauser, C.; You, M.; Pezzuto, J. M. Cancer Res. 1997, 57, 272.
21. Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2000,
10, 1887.
22. Zahran, M. A. H.; Salemb, T. A. R.; Samaka, R. M.; Agwa, H. S.; Awad, A. R. Bioorg.
Med. Chem. 2008, 16, 9708.
References and notes
23. Huang, W.; Ding, Y.; Miao, Y.; Liu, M. Z.; Yang, G. F. Eur. J. Med. Chem. 2009, 44,
3687.
24. Cao, S. L.; Feng, Y. P.; Jiang, Y. Y.; Liu, S. Y.; Ding, G. Y.; Li, R. T. Bioorg. Med. Chem.
Lett. 2005, 15, 1915.
1. Yarden, Y.; Sliwknowski, M. X. Nat. Rev. Mol. Cell. Biol. 2001, 2, 127.
2. Teman, S.; Kawaguchi, H.; El-Naggar, A. K.; Jelinek, J.; Tang, H.; Liu, D. D.; Lang,
W.; Issa, J. P.; Lee, J. J.; Mao, L. J. Clin. Oncol. 2007, 25, 2164.
25. Hou, X. L.; Ge, Z. M.; Wang, T. M.; Guo, W.; Cui, J. R.; Cheng, T. M.; Lai, C. S.; Li, R.
T. Bioorg. Med. Chem. Lett. 2006, 16, 4214.
3. Denny, W. A. Farmaco 2001, 56, 51.
4. Kobayashi, S.; Boggon, T. J.; Dayaram, T.; Janne, P.; Kocher, O.; Meyerson, M.;
Johnson, B. E.; Eck, M. J.; Tenen, D. G.; Halmos, B. N. Engl. J. Med. 2005, 352, 786.