Thienopyrimidine-based dual EGFR/ErbB-2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.12.011

Two new series of potent and selective dual EGFR/ErbB-2 kinase inhibitors derived from novel thienopyrimidine cores have been identified. Isomeric thienopyrimidine cores were evaluated as isosteres for a 4-anilinoquinazoline core and several analogs conta

Full text of DOI:10.1016/j.bmcl.2008.12.011

Bioorganic & Medicinal Chemistry Letters 19 (2009) 817–820
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thienopyrimidine-based dual EGFR/ErbB-2 inhibitors
a
b
a
a
Tara R. Rheault a, , Thomas R. Caferro , Scott H. Dickerson , Kelly H. Donaldson , Michael D. Gaul ,
Aaron S. Goetz c, Robert J. Mullin d, Octerloney B. McDonald e, Kimberly G. Petrov a, David W. Rusnak,
Lisa M. Shewchuk f, Glenn M. Spehar d, Anne T. Truesdale e, Dana E. Vanderwall b, Edgar R. Wood e,
David E. Uehling a
*
a Department of Oncology Medicinal Chemistry, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709-3398, USA
b Department of Computational and Structural Chemistry, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709-3398, USA
c Department of Screening and Compound Profiling, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709-3398, USA
d Department of Oncology Biology, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709-3398, USA
e Department of Molecular Biochemistry, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709-3398, USA
f Department of Structural Biology, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709-3398, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new series of potent and selective dual EGFR/ErbB-2 kinase inhibitors derived from novel thienopyr-
imidine cores have been identified. Isomeric thienopyrimidine cores were evaluated as isosteres for a
4-anilinoquinazoline core and several analogs containing the thieno[3,2-d]pyrimidine core showed
Received 7 November 2008
Revised 1 December 2008
Accepted 3 December 2008
Available online 7 December 2008
anti-proliferative activity with IC50 values less than 1
lM against human tumor cells in vitro.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Kinase
Inhibitor
Epidermal growth factor receptor
EGFR
ErbB-2
Thieno[3,2-d]pyrimidine
Thieno[2,3-d]pyrimidine
Lapatinib
ErbB family type I receptor tyrosine kinases (EGFR, ErbB-2,
ErbB-3, and ErbB-4) play a critical role in mediating growth factor
signaling.1 After ligand binding, these RTKs propagate signaling
through formation of hetero- and homo-dimers with hetero-
dimerization of ErbB-2 preferred for maximum signaling.2 Over-
expression EGFR and ErbB-2 has been associated with oncogenic
activity such as unregulated cell growth, proliferation, differentia-
tion and survival.3 In fact, elevated levels of these receptors have
been observed in a number of human cancers including breast,
lung, colon, and prostate cancer.4 Disruption of ErbB family signal-
ing by treatment with monoclonal antibodies (either anti-EGFR or
anti ErbB-2 MAbs) or small molecule inhibitors of EGFR has led to
significant advances in cancer treatment and has largely validated
this targeted therapy approach.2,3,5
EGFR/ErbB-2 inhibitor also recently approved by the FDA.9 It was
postulated during the development of TykerbTM that targeting both
F
O
NH
N
Cl
NH
N
N
O
O
N
O
O
N
O
O
1 IressaTM
2 Tarceva TM
O
F
NH
N
Cl
Two small molecule EGFR-selective inhibitors, IressaTM (gefiti-
nib,6 1) and TarcevaTM (erlotinib,7 2) have recently been approved
by the FDA for treatment of certain cancers over-expressing EGFR
(Fig. 1).8 TykerbTM (lapatinib, 3) is the first example of a dual
O
N
N
H
SO2Me
3 TykerbTM
Figure 1. Examples of ErbB family inhibitors. IressaTM and TarcevaTM are FDA-
approved potent EGFR inhibitors. TykerbTM is a potent inhibitor of both EGFR and
ErbB-2, also recently approved by the FDA.
* Corresponding author. Tel.: +1 919 483 3339; fax: +1 919 483 6053.
E-mail address: tara.r.rheault@gsk.com (T.R. Rheault).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.011

818
T. R. Rheault et al. / Bioorg. Med. Chem. Lett. 19 (2009) 817–820
Ar
ErbB-2 and EGFR with a dual inhibitor may offer advantages rela-
tive to selective inhibition of either enzyme.8b,9
BOC
N
NH
N
a
S
IressaTM (1), TarcevaTM (2), and TykerbTM (3) are selective, ATP-
competitive inhibitors that contain a 4-anilinoquinazoline scaffold
as a common structural feature. Inhibition of ErbB-2 activity with
TykerbTM is approximately 100-fold greater than that of either
IressaTM or TarcevaTM. It is thought that the 4-benzyloxy aniline moi-
ety present in TykerbTM is the structural feature responsible for the
potent ErbB-2 inhibitory activity observed with that molecule.10
In an effort to develop novel non-quinazoline dual inhibitors of
EGFR and ErbB-2, thieno[3,2-d]pyrimidine and thieno[2,3-
d]pyrimidine cores were evaluated (Core A and Core B, Scheme
1). The 4-benzyloxyaniline portion of TykerbTM was conserved in or-
der to maintain sufficient ErbB-2 activity, and a survey of both thi-
enopyrimidine cores combined with a variety of heterocycle
attachments was performed. The structure–activity relationships
and biological evaluation of these compounds are described
herein.11
N
HO2B
Br +
6
11
Ar
Ar
Ar
BOC
N
NH
NH
N
NH
N
H
H
CHO
S
N
S
N
S
c
N
N
b
N
N
12
13
14
e
Ar
Ar
NH
N
NH
H
H
d
R1
R1
S
N
N
N
N
S
amine
N
15 Core A
16 Core B
Thieno[3,2-d]pyrimidine (Core A) and thieno[2,3-d]pyrimidine
(Core B) analogs with furan and thiophene linkers were synthe-
sized according to the sequence shown in Scheme 1. Nucleophilic
displacement of known 6-bromo-4-chlorothieno[3,2-d]pyrimidine
(4) with aniline 5 provided bromothienopyrimidine intermediate
6.12 Suzuki coupling of bromide 6 with boronic acids 7 gave hetero-
aryl (R = H) or heteroaryl aldehyde (R = CHO) products 8. Finally,
reduction or reductive amination of the aldehyde functionality
yielded the desired analogs 9 (Core A). The corresponding thie-
no[2,3-d]pyrimidine analogs 10 (Core B), could be made from the
same reaction sequence starting with 6-bromo-4-chlorothie-
no[2,3-d]pyrimidine.
R1 = alkyl amine
Scheme 2. General synthetic route. Reagents and conditions: (a) Pd(dppf)Cl2,
DMF:toluene 1:1 or DMA, 2 M Na2CO3, 64–96%; (b) DMA, 150 °C, 96%;
(c) i—Vilsmeier reagent, DMF; ii—10% NaOH, 19–46%; (d) 1° amine, AcOH,
DCM:MeOH 2:1, NaBH(OAc)3, 46–79%; (e) formaldehyde, 2° amine, AcOH, 99%.
forts on the thienopyrimidine core isomer, heteroaryl linker and
hydrophilic side chain. Table 1 shows enzyme inhibition data for
thieno[3,2-d]pyrimidine (A) with furan, thiophene and pyrrole
linkers and a variety of hydrophilic side chains.14 Compounds con-
taining Core A were potent inhibitors of EGFR, despite changes in
the hydrophilic side chain, heteroaryl functionality (see com-
pounds 17–23, 25, and 29) and orientation (compare 22 and 23).
Compounds containing a furan linker with a hydrophilic side chain
(18–23) were more potent inhibitors of ErbB-2 than unsubstituted
furan 17. Interestingly, changing the heteroaryl linker from a furan
or thiophene, containing an H-bond acceptor atom (17 and 24), to a
pyrrole, containing an H-bond donor (26), resulted in reduced po-
tency at EGFR but not for ErbB-2. Also, addition of hydrophilic side
chains to a pyrrole linker did not improve potency at ErbB-2 to the
same degree that the addition enhanced potency for both furan
and thiophene analogs (see 17, 22, 24, 25, 26, and 29).
Pyrrole-linked thienopyrimidines were prepared according to
Scheme 2. Suzuki coupling followed by thermal BOC-deprotection
gave pyrrole 13. Subsequent reaction with Vilsmeier reagent gave
the desired pyrrole-2-aldehyde 14, which was further transformed
into secondary amines 15 by reductive amination.13 Alternatively,
tertiary amines were obtained in one step from pyrrole 13 by a
Vilsmeier-type reaction followed by in situ reductive amination
with a secondary amine. An analogous sequence was carried out
to arrive at compounds 16 (Core B).
By choosing to maintain the TykerbTM head group, optimized for
inhibition of both ErbB-2 and EGFR, we were able to focus SAR ef-
Table 2 shows enzyme data for a similar series of analogs con-
taining thieno[2,3-d]pyrimidine (Core B). Compounds in this series
were generally less potent inhibitors of EGFR compared to analogs
containing Core A (compare 22 and 32, 25 and 35, 29 and 39). Ben-
zyl amine substitution (31) was not tolerated in ErbB-2 and thio-
phene linker orientation had a major impact on both EGFR and
ErbB-2 potency (compare 34 and 35). Compounds 30 and 32 were
significantly more effective dual inhibitors of EGFR and ErbB-2
compared to their Core A counterparts 17 and 22. This level of en-
zyme inhibition is similar to that reported for TykerbTM (3), 11 nM
and 9 nM, respectively.9d
Figures 2 and 3 show molecular models of both thienopyrimi-
dine isomers 22 and 32, containing the furan linker and pendant
amine side chain of 3, overlayed with a crystal structure of 3 in
EGFR. The benzyloxy aniline head group of molecules 3, 22 and
32 neatly fills the back pocket of the ATP binding region of EGFR.11
The quinazoline (3) or thienopyrimidine (22 and 32) functions as
point-binder to the hinge region and the heteroaryl linker and
hydrophilic side chain extends into the solvent exposed region.
The trajectory of the amine-containing side chain appears to be a
key difference when comparing both thienopyrimidine core iso-
mers A and B. Core B (32, Fig. 3) has a very high degree of overlap
with 3. This is in support of the observation that 32 has similar
EGFR and ErbB-2 enzyme potencies to that obtained with 3.
Cl
S
N
Br
Ar
NH
N
N
R
X
a
S
4
N
+
Br +
HO2B
O
6
7
F
X = S or O
R = H or CHO
NH2
Cl
5
Ar
N
Ar
NH
N
NH
N
R
R1
c
S
X
S
X
b
N
8
9
Core A
R1 = alkyl amine or OH
Ar
NH
N
R1
X
N
S
10 Core B
Scheme 1. General synthetic route. Reagents and conditions: (a) i-PrOH, 60 °C, 80–
100%; (b) Pd(dppf)Cl2, DMF:toluene 1:1 or DMA, 2 M Na2CO3, 27–64%; (c) AcOH,
DCM:MeOH 2:1, NaBH(OAc)3, 37–67%.

T. R. Rheault et al. / Bioorg. Med. Chem. Lett. 19 (2009) 817–820
819
Table 1
Table 2
Enzyme activities of compounds containing core A
Enzyme activities of compounds containing core B
O
F
O
Cl
F
Cl
NH
N
NH
N
R
S
N
S
R
N
B
A
a
a
Compound
R
EGFR IC50
ErbB-2 IC50
a
a
Compound
R
EGFR IC50
10
ErbB-2 IC50
30
15
14
898
6
O
17
709
O
O
H
N
31
32
33
34
35
36
37
38
54
12
18
19
20
21
22
23
24
25
26
27
28
2
5
146
250
122
68
O
O
N
H
N
N
S
O
O
N
O
O
9
43
S
S
3
OH
H
N
96
95
H
N
S
S
3
O
O
CN
O
O
O
S
H
N
H
N
304
115
68b
121
138
299
203
246b
111
153
1
71
S
O
O
O
O
O
S
H
N
N
H
4
86
S
O
N
N
H
8
113
31
S
H
N
H
N
N
H
3
S
O
O
H
N
39
S
O
N
H
68
4
121
76
O
N
H
a
Mean values in nanomolar, >1 determination.
One determination.
b
N
N
H
H
N
6
68
N
H
H
N
29
7
113
S
O
N
H
O
a
Mean values in nanomolar, >1 determination.
Another key conformational difference between thienopyrimi-
dine molecules and quinazolines such as 3 is the presence of di-
pole–dipole interactions within the molecule that favor the furan
oxygen atom orienting in the opposite direction to the sulfur atom
in the thienopyrimidine core. Figure 2 shows that not only does
Core A (22) orient the hydrophilic tail in a different trajectory than
3, but it also forces the furan oxygen atom in the opposite direc-
tion. Additionally, pyrroles (26–29 and 36–39) may prefer an intra-
molecular hydrogen bond to the sulfur of the thienopyrimidine and
orient opposite their furan or thiophene counterparts
Figure 2. Overlay of 22 (green, Core A) model with crystal structure of 3 (gray) in
EGFR.
Representative compounds from both the Core A and Core B
series were also evaluated in cellular proliferation assays. (Table
3)15 Unsubstituted furan and thiophene analogs 17, 24, 30, and
33, from both the Core A and B series showed minimal potency

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T. R. Rheault et al. / Bioorg. Med. Chem. Lett. 19 (2009) 817–820
cancer cell proliferation in the series containing thienopyrimidine
Core A. Finally, compounds in both series, 22 and 32, are 10- to
1000-fold selective for EGFR and ErbB-2 over a variety of other ki-
nases including SRC, CDK2, P38, VEGFR2, and Aurora A and B.
References and notes
1. (a) Supuran, C. T.; Scozzafava, A. Expert Opin. Ther. Patents 2004, 141, 35; (b)
Yarden, Y.; Sliwkowski, M. X. Nat. Rev. Mol. Biol. 2001, 2, 127.
2. Cockerill, G. S.; Lackey, K. E. Curr. Top. Med. Chem. 2002, 2, 1001. and references
therein.
3. Johnston, J. B.; Navaratnam, S.; Pitz, M. W.; Maniate, J. M.; Wiechec, E.; Baust,
H.; Gingerich, J.; Skliris, G. P.; Murphy, L. C.; Los, M. Curr. Med. Chem. 2006, 13,
3483.
4. (a) Salomon, D. S.; Brandt, R.; Ciardiello, F.; Normanno, N. Crit. Rev. Oncol.
Hematol. 1995, 19, 183; (b) Woodburn, J. R. Pharmacol. Ther. 1999, 82, 241.
5. Kamath, S.; Buolamwini, J. K. Med. Res. Rev. 2006, 26, 569.
6. Wakeling, A. E.; Guy, S. P.; Woodburn, J. R.; Ashton, S. E.; Curry, B. J.; Barker, A.
J.; Gibson, K. H. Cancer Res. 2002, 62, 5749.
Figure 3. Overlay of 32 (green, Core B) model with crystal structure of 3 (gray) in
EGFR.
7. Moyer, J. D.; Barbacci, E. G.; Iwata, K. K.; Arnold, L.; Boman, B.; Cunningham, A.;
Diorio, C.; Doty, J.; Morin, M. J.; Moyer, M. P.; Neveu, M.; Pollack, V. A.;
Pustilnik, L. R.; Reynolds, M. M.; Slaon, D.; Theleman, A.; Miller, P. Cancer Res.
1997, 57, 4838.
8. (a) Hirsch, F. R.; Witta, S. Curr. Opin. Oncol. 2005, 17, 118. and references
therein; (b) Burris, H. A. Oncologist 2004, 9, 10.
Table 3
Inhibition of cellular proliferation
a
a
a
Compound (Core)
HN5 IC50
(l
M)
BT474 IC50
(lM)
HFF IC50 (lM)
17 (A)
18 (A)
20 (A)
22 (A)
23 (A)
24 (A)
25 (A)
26 (A)
29 (A)
30 (B)
32 (B)
33 (B)
36 (B)
8.09
1.22
21.25
1.88
>30.00
4.46
9. (a) Gomez, H. L. et al. A Phase II, Randomized Trial Using the Small Molecule
Tyrosine Kinase Inhibitor Lapatinib as a First-Line Treatment in Patients with
FISH Positive Advanced or Metastatic Breast Cancer. In 41st Annual Meeting of
the American Society for Clinical Oncologists, Orlando, FL, May 14–17, 2005;
Abstract 3046. See also Abstracts 4594 and 3583.; (b) Johnston, S. R. D.; Leary,
A. Drugs Today 2006, 7, 441; (c) Spector, N.; Raefsky, E.; Hurwitz, H.; Hensing,
T.; Dowlati, A.; Dees, C.; O’Neil, B.; Koch, K.; Smith, D. A.; Mangum, S.; Burris, H.
A. Proc. Am. Assoc. Clin. Oncol. 2003, 193. Abstract 772; (d) Rusnak, D. W.;
Lackey, K.; Affleck, K.; Wood, Edgar R.; Alligood, Krystal J.; Rhodes, N.; Keith, B.
R.; Murray, D. M.; Knight, W. B.; Mullin, R. J.; Gilmer, T. M. Mol. Cancer Ther.
2001, 1, 85.
10. (a) Rusnak, D. W.; Affleck, K.; Cockerill, S. G.; Stubberfield, C.; Harris, R.; Page,
M.; Smith, K. J.; Guntrip, S. B.; Carter, M. C.; Shaw, R. J.; Jowett, A.; Stables, J.;
Topley, P.; Wood, E. R.; Brignola, P. S.; Kadwell, S. H.; Reep, B. R.; Mullin, R. J.;
Alligood, K. J.; Keith, B. R.; Crosby, R. M.; Murray, D. M.; Knight, W. B.; Gilmer, T.
M.; Lackey, K. E. Cancer Res. 2001, 61, 7196; (b) Zhang, Y. -M.; Cockerill, S.;
Guntrip, S. B.; Rusnak, D.; Smith, K.; Vanderwall, D.; Wood, E.; Lackey, K. Bioorg.
Med. Chem. Lett. 2004, 14, 111.
0.10c
0.41
0.72c
0.42
9.39c
>30.00
0.14c
2.19
0.73c
5.77
7.95c
10.74
>30.00c
2.37c
0.10c
0.57c
0.77
0.60c
0.54c
0.63
3.73
>30.00
0.42
>30.00
0.42
>30.00
19.54
9.72b
>3.00c
10.08
3.22
2.71c
1.98c
a
Values are means of two experiments, CTG assay format.
n = 1.
MEB assay format.
b
c
11. For analogous SAR with
a
series of compounds containing
a
4-
in proliferation assays in cancer cell lines driven by over-expres-
sion of EGFR (HN5) and ErbB-2 (BT474) even though they all had
reasonable levels of target potency at the enzyme level. For com-
parison, 3 has a reported IC50 of 120 nM and 100 nM on HN5 and
BT474 cancer cell lines, respectively.9d Conversely, unsubstituted
pyrrole 26 demonstrated sub-micromolar potency despite having
similar or less potent enzyme inhibition compared to 17, 24, and
30. Pyrroles 26 and 29 did show more potent inhibition of normal
cell proliferation (HFF) compared to furan analogs 17 and 22. How-
ever, inhibition of intracellular autophosphorylation with pyrroles
26 and 29 probed by an ErbB-2 DELFIA assay16 did show IC50s con-
sistent with the BT474 anti-proliferative activity observed, 826 nM
and 431 nM, respectively. The presence of an H-bond donor in the
hydrophilic tail appeared to improve potency on cancer cell prolif-
eration (compare 18 to 20 and 22). Finally, compounds 22 (Core A)
and 32 (Core B) were found to be equipotent inhibitors of HN5 and
BT474 proliferation, despite the observation that 32 is approxi-
mately 12 times more potent on ErbB-2 at the enzyme level.
In conclusion, we have been able to develop dual inhibitors of
EGFR and ErbB-2 containing a novel thienopyrimidine core. We
have also been able to demonstrate sub-micromolar inhibition of
anilinoquinazoline core, see: Petrov, K. G.; Zhang, Y. -M.; Carter, M.;
Cockerill, G. S.; Dickerson, S.; Gauthier, C. A.; Guo, Y.; Mook, R. A., Jr.; Rusnak,
D. W.; Walker, A. L.; Wood, E. R.; Lackey, K. E. Bioorg. Med. Chem. Lett. 2006, 16,
4686.
12. Wood, E. R.; Shewchuk, L. M.; Ellis, B.; Brignola, P.; Brashear, R. L.; Caferro, T. R.;
Dickerson, S. H.; Dickson, H. D.; Donaldson, K. H.; Gaul, M.; Griffin, R. J.; Hassell,
A. M.; Keith, B.; Mullin, R.; Petrov, K. G.; Reno, M. J.; Rusnak, D. W.; Tadepalli, S.
M.; Ulrich, J. C.; Wagner, C. D.; Vanderwall, D. E.; Waterson, A. G.; Williams, J.
D.; White, W. L.; Uehling, D. E. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2773.
13. Sun, L.; Cui, J.; Liang, C.; Zhou, Y.; Nematalla, A.; Wang, X.; Chen, H.; Tang, C.;
Wei, J. Bioorg. Med. Chem. Lett. 2002, 12, 2153.
14. Brignola, P. S.; Lackey, K.; Kadwell, S. H.; Hoffman, C.; Horne, E.; Carter, H. L.;
Stuart, J. D.; Blackburn, K.; Moyer, M. B.; Alligood, K. J.; Knight, W. B.; Wood, E.
R. J. Biol. Chem. 2002, 277, 1576.
15. The HN5 cell line is a head and neck carcinoma line that over-expresses EGFR.
The BT474 line is a breast carcinoma that over-expresses ErbB-2. The HFF cell
line is a human foreskin fibroblast line that represents normal cells. The MEB
proliferation assay was previously described in Ref. 9d. The CTG proliferation
assay protocol was as follows: cells were treated with compounds in 0.1%
DMSO and incubated for 72 h at 37 °C, 5% CO2. Viable cells were quantified
using CellTiter-Glo reagent (Promega, Madison, WI) and luminescence
detection on a Victor 2V plate reader (Perkin-Elmer, Turku, Finland). Either
proliferation assay format gives an IC50 within a 2-fold range for this chemical
series.
16. Dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), see:
Braunwalder, A. F.; Yarwood, D. R.; Sills, M. A.; Lipson, K. E. Anal. Biochem. 1996,
238, 159. BT474 cells and their culture conditions are described in Ref. 15.