The discovery of highly selective erbB2 (Her2) inhibitors for the treatment of cancer

Article abstract of DOI:10.1016/j.bmcl.2007.03.046

The synthesis and biological evaluation of potent and selective inhibitors of the erbB2 kinase is presented. Based on the 4-anilinoquinazoline chemotype, the syntheses of several new series of erbB2 inhibitors are described with quinazoline and pyrido[4,3

Full text of DOI:10.1016/j.bmcl.2007.03.046

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3081–3086
The discovery of highly selective erbB2 (Her2) inhibitors
for the treatment of cancer
Blaise Lippa,* Goss S. Kauffman, Joel Arcari, Tricia Kwan, Jinshan Chen,
William Hungerford, Samit Bhattacharya, Xumiao Zhao, Courtney Williams,
Jun Xiao, Leslie Pustilnik, Chunyan Su, James D. Moyer, Ling Ma,
Mary Campbell and Stefanus Steyn
Pfizer, Inc., PGRD Groton, MS-8220-2203 Eastern Point Road, Groton, CT 06340, USA
Received 31 January 2007; revised 14 March 2007; accepted 14 March 2007
Available online 19 March 2007
Abstract—The synthesis and biological evaluation of potent and selective inhibitors of the erbB2 kinase is presented. Based on the
4-anilinoquinazoline chemotype, the syntheses of several new series of erbB2 inhibitors are described with quinazoline and
pyrido[4,3-d]pyrimidine cores. The vast majority of these compounds are found to be >100· selective over the closely related EGFR
kinase. Two lead compounds are further shown to have low clearance and moderate bioavailability in rat.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Breast cancer is the second leading cause of cancer
related death in American women. It is estimated that
214,640 new cases of breast cancer were diagnosed and
41,430 deaths occured in the U.S. during 2006.1 Current
statistics suggest that 12.7% of American women born
today (ꢀ1/8) will be diagnosed with breast cancer at
some point in their life. While advances continue to be
made in the treatment of this disease, current chemo-
therapy options are often limited by a lack of efficacy
and toxicity; thus new therapies are urgently needed to
treat this disease.2
At Pfizer, an effort to selectively target erbB2 was initi-
ated. Despite >80% homology between EGFR and
erbB2 in their kinase domains,7,8 a kinase selective ap-
proach was chosen for two reasons: (1) to avoid un-
wanted toxicity from inhibiting EGFR, such as
acneiform skin rashes, and (2) to facilitate the ability
to combine a selective erbB2 agent with other selective
drugs, including an EGFR agent, in a tunable fashion
so that different relative levels of selective inhibition
could be achieved. We believed that this approach is
advantageous from a scientific viewpoint in the long
term when compared to being limited to a fixed selectiv-
ity profile inhibiting multiple kinases from an unselec-
tive single agent.
The erb family of receptors are transmembrane receptor
tyrosine kinases involved in a wide range of signal trans-
duction and cellular functions, and have become a very
fruitful area for the successful development of drugs to
treat cancer.3 erbB2 (Her2) is found to be significantly
overexpressed in 20–30% of human breast cancers and
is associated with a poor prognosis.4 The humanized
antibody herceptin (trastuzumab) from Genentech tar-
gets erbB2 and has been approved for the treatment of
breast cancer.5 EGFR (erbB1) has also been targeted
for the treatment of cancer, and several agents have been
approved with this mode of action.6
Previously, Pfizer reported on a series of anilinoquinazo-
lines exemplified by compounds 1 and 2 (Fig. 1).9–11
CH2OH
N
MeO
OMe
O
O
N
HN
O
HN
N
HN
4
6
N
N
7
N
Keywords: erbB2; Her2; Anilinoquinazoline; Kinase; Cancer.
*
2
1
Corresponding author. Tel.: +1 860 715 4660; fax: +1 860 686
6258; e-mail: blaise.lippa@pfizer.com
Figure 1. erbB2 selective anilinoquinazolines.9–11
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.046

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B. Lippa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3081–3086
These inhibitors were shown to be 11–42 nM erbB2
antagonists with 60–100· selectivity over EGFR. Based
on a homology model and comparisons with the crystal
structure of an inhibitor bound to EGFR,12 it is believed
that the N1 of the quinazoline makes a key hydrogen
bond to the hinge region of the protein, and that the ani-
line portion of the inhibitors is largely responsible for ki-
Table 1 shows the potency of piperidine compounds in
inhibiting the isolated erbB2 kinase,15 and erbB2 in a
cellular system, as well as their selectivity in inhibiting
EGFR in the same cellular system.16,17 Initial unsubsti-
tuted piperidine and piperidine alkyl analogs were found
to be inactive (8a and b). Simple amide derivatives were
also inactive (8c,g), however, bulky amides (8d–f) and
urea (8i) showed surprising potency and selectivity.
Notably, these 4-piperidines were significantly more
potent than 3-piperidines (data not shown). Table 2
contains compounds in the phenyl amide class. These
compounds showed moderate to excellent potency and
remarkable selectivity over EGFR when substituted at
the 30 position with a bulky amide (9c–f). Simple amides
caused little inhibition (9a,b). At the 40 position, the
SAR was considerably more narrow, with only the neo-
pentyl amide displaying significant potency (9k). For all
of these series, the distance between the amide and the
N1 of the quinazoline as well as the need for large
groups on the amide for potency suggests the possibility
that these compounds require a protein conformational
change to a DFG out form to bind, although no defin-
itive kinetic or crystallographic data has been generated
to support this hypothesis.13
nase selectivity because it is buried deep in
a
hydrophobic pocket. The quinazoline 6 position substit-
uents are believed to be pointed toward a solvent acces-
sible portion of the protein, and thus this position may
be an ideal place to introduce modifications designed
to improve ADME properties.
The goal of the effort described herein was to build on
this level of potency, increase selectivity, and further
elaborate SAR within this exciting chemotype, which
is already present in three approved cancer drugs.6 In
particular, a focus was on elaborating the anilino por-
tion of the molecule with more polarity by utilizing mo-
tifs that have been shown to induce a DFG out
conformation in other kinases.13 Thus, amide substitu-
ents were envisioned on the terminal phenyl group. In
addition, replacement of this terminal phenyl group with
substituted piperidines was also sought.
To further explore the SAR of these novel quinazoline
C4 substituents, we sought to combine them with other
quinazoline C6 and C7 substituents, as well as C6 substi-
tuted pyridopyrimidines. As described previously, this
portion of the inhibitors is believed to point toward a
solvent accessible portion of the binding pocket, and
thus polar substituents were envisioned. In particular,
ethers and amines were chosen due to their intrinsic
polarity and based on the precedents of previous erb
inhibitors.6,18,19
The initial analogs in this series were made with the C6
alkyne substituent of the quinazoline. The synthesis be-
gins with a nucleophilic displacement of the fluoro
group of 4 with either 3- or 4-substituted piperidine
alcohols or phenols (3a and 3b, Scheme 1). Reduction
of the nitro group affords 5 in good yield. The resulting
amine is then added to the C6-alkyne substituted 4-chlo-
roquinazoline (6),14 followed by acid catalyzed depro-
tection of either the tBoc group of piperidine
containing intermediates, or the t-Bu ester of the phenol
containing intermediates. The resulting amine and acid
substrates (7) are then subjected to standard high speed
analoging protocols resulting in final analogs 8 and 9.
The readily available 4-chloro-6,7-dimethoxy and
6,7-dimethoxyethoxy ether starting materials were first
utilized to synthesize di-ether analogs in an analogous
a)
MeO
HN
1a) NaH, DMF
O
b)
MeO
HN
Cl
F
OR2
R1OH
(4)
N
OR1
O
O2N
6
HN
N
3
N
2) H2, Pd(OH)2, EtOH
63-85% (2 steps)
H2N
1:1 t-BuOH: DCE, Δ
N
R1 =
HO
5
b) HCl (g)
77-99%
CO2tBu
R2 = a
R3CO2H,
CDI, DMF
NBoc
7
HO
HO
R2 = b
O
(b)
(a)
R4R5NH, HATU,
DMF
NR4R5
O
R3
N
MeO
HN
R2 =
HO
O
MeO
CO2H
O
NH
(a)
O
HN
HN
O
N
HN
(b)
N
N
9
N
8
Scheme 1. The synthesis of amide substituted anilinoquinazoline analogs. DMF = N,N-dimethylformamide; DCE = 1,2-dichloroethane; CDI = 1,10-
carbonyldiimidazole; HATU = O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate.

B. Lippa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3081–3086
3083
Table 1. erbB2 kinase and cell inhibition, and EGFR cell selectivity for the piperidine sub-class of anilinoquinazolines 8a–i
MeO
HN
O
O
N
HN
R
6
N
N1
8
Compound
R
erbB2 kinase inhibition
a
IC50 (nM)
erbB2 cell inhibition
a
IC50 (nM)
Cell selectivity
versus EGFRa
8a
8b
8c
8d
8e
8f
H
Me
>10,000
>10,000
>10,000
150
NT
NT
NT
51
NT
NT
Ac
NT
>200·
92·
400·
NT
CO(c-Bu)
CO(m-ClPh)
COCH2t-Bu
COCH2OMe
CO(2-THF)
CONH(2,6-difluoroPh)
210
94
75
16
8g
8h
8i
>10,000
550
76
NT
2900
190
NT
>53·
NT, not tested.
a Values are means of at least two experiments, assay error is <2·.
Table 2. erbB2 kinase and cell inhibition, and EGFR cell selectivity for phenyl amide analogs 9a–k
MeO
3' O
O
HN
O
4' R
HN
N
N
9
Compound
Amide position
R
erbB2 kinase inhibition
a
IC50 (nM)
erbB2 cell inhibition
a
IC50 (nM)
Cell selectivity
versus EGFR
9a
9b
9c
9d
9e
9f
30
30
30
30
30
30
40
40
40
40
40
NHMe
N(Me)2
NHPh
>10,000
>10,000
140
NT
NT
NT
160
30
NT
NT
NT
NHc-Bu
NHt-Bu
230
18
>64·
250·
91·
NT
NHCH2t-Bu
NHMe
N(Me)2
130
110
NT
NT
NT
NT
23
9g
9h
9i
>10,000
>10,000
>10,000
>10,000
140
NT
NT
NHPh
NHt-Bu
NHCH2t-Bu
9j
9k
NT
200·
NT, not tested.
a Values are means of at least two experiments, assay error is <2·.
fashion as that described in Scheme 1. Subsequently,
C6-monoethers were synthesized (Scheme 2) beginning
with the esterification of anthranilic acid 10 followed
by alkylation of the phenol under standard conditions
to provide 12 where R1 = Me, MeOEt, and morphol-
ine propyl. Cyclization of the anthranilic acid with
OH
R1O
R1Cl, 65-77%
COOCH3
NH2
NaH, DMF
MeOH, H2SO4
47%
HO
COOCH3
NH2
HO
COOH
NH2
R1O
formamide
N
Δ, 70-79%
N
11
12
10
13
O
O
O
R2
Cl
R1O
As in Scheme 1
POCl3, DMF
NR2
N
HN
HN
R1O
N
R1O
DCE, 64-93%
N
N
14
N
N
15
16
R1 =
O
N
H3CO
Me
or
,
Scheme 2. The synthesis of C6 monoether analogs. DMF = N,N-dimethylformamide; DCE = 1,2-dichloroethane.

3084
B. Lippa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3081–3086
formamide to the quinazoline followed by chlorination
afforded the key 4-chloro-6-ether 14. Elaboration of
this intermediate into final analogs took place as de-
scribed in Scheme 1.
show moderate to excellent kinase and cell potency, as
well as EGFR selectivity. Particularly with regard to
erbB2 cell potency, the pyridopyrimidine analogs are
superior. For example, compound 20b was shown to
have a potency of <10 nM in the erbB2 cell assay,
whereas its EGFR cell potency was >10 lM resulting
in >1000· selectivity.
The synthesis of C6-aminopyridopyrimidine inhibitors
is shown in Scheme 3. Beginning with 4-chloro-6-fluoro-
pyrido[3,4-d]pyrimidine (17)19 the 4-chloro group is first
displaced with the aniline intermediates (5) of Scheme 1
to provide 18. Amine nucleophiles are then utilized to
displace the 6-fluoro group with high heat in DMSO
to provide intermediate 19. Analogs are then completed
as described previously.
Two of the most promising pyridopyrimidine com-
pounds were assayed to determine their pharmacoki-
netic properties in rat. Compound 20f as the HCl salt
showed low clearance (Cl) of 2.5 mL/min/kg and a low
volume of distribution (Vd) of 0.2 L/kg resulting in a
half-life of 1 h after a single 2 mg/kg IV dose. When
dosed orally (5 mg/kg) in 0.5% methylcellulose, this
compound was 54% bioavailable (F). When dosed IV,
Tables 3 and 4 show the potency and selectivity of C6
and C7 analogs. In general, all analogs in these series
OR1
OR1
R2R3NH, Et3N
Cl
5, Et3N
DMSO, 120 oC
HN
N
HN
F
DCE/ t-BuOH
F
R2R3N
N
N
N
N
N
N
56-80%
89-99%
R2R3N
N
N
17
18
19
O
O
O
R4
NR4
As in Scheme 1
HN
N
HN
N
R2R3N
N
N
N
N
21
20
Scheme 3. The synthesis of pyrido[4,3-d]pyrimidine analogs. DCE = 1,2-dichloroethane; DMSO = dimethylsulfoxide.
Table 3. erbB2 kinase and cell inhibition, and EGFR cell selectivity for the piperidine sub-class of quinazoline ether and C6-amino substituted
pyrido[4,3-d]pyrimidines 15a–l and 20a–f
O
O
NR3
NR3
HN
N
HN
N
R1
R2
R1
N
N
N
15
R3
20
Compound R1
R2
erbB2 kinase
inhibition IC50 (nM) IC50 (nM)
erbB2 cell inhibition Cell selectivity
versus EGFRa
a
a
15a
15b
15c
15d
15e
20a
20b
15f
15g
15h
15i
OMe
O(CH2)2OMe
OMe
OMe COCH2t-Bu
O(CH2)2OMe COCH2t-Bu
100
260
96
76
530
NT
19
>130·
11·
NT
H
COCH2t-Bu
COCH2t-Bu
COCH2t-Bu
COCH2t-Bu
COCH2t-Bu
O(CH2)2OMe
O(CH2)3 morpholine
NMe2
H
120
75
>500·
>290·
>230·
>1000·
23·
6.5·
11·
>300·
32·
25·
H
—
34
51
23
<10
<10
67
Morpholine
OMe
—
OMe
CONH(2,6-difluoroPh)
O(CH2)2OMe CONH(2,6-difluoroPh) 230
19
O(CH2)2OMe
OMe
840
250
33
H
CONH(2,6-difluoroPh)
CONH(2,6-difluoroPh)
CONH(2,6-difluoroPh)
CONH(2,6-difluoroPh)
CONH(2,6-difluoroPh)
CONH(2,6-difluoroPh)
46
27
55
25
21
7
O(CH2)2OMe
O(CH2)3 morpholine
NHMe
H
15j
H
55
20c
20d
20e
15k
15l
—
—
—
180
20
NMe2
68·
Morpholine
O(CH2)2OMe
O(CH2)2OMe
Morpholine
16
210·
>19·
>350·
>300·
O(CH2)2OMe CO(c-pent)
77
44
18
530
29
H
—
CO(c-pent)
CO(c-pent)
20f
33
NT, not tested.
a Values are means of at least two experiments, assay error is <2·.

B. Lippa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3081–3086
3085
Table 4. erbB2 kinase and cell inhibition, and EGFR cell selectivity for the phenyl amide sub-class of quinazoline ethers and C6-amino substituted
pyrido[4,3-d]pyrimidines 16a and 21a–f
O
O
O
O
HN
N
HN
N
R1
R2
NH
R1
NH
N
N
t
Bu
N
tBu
16
R2
21
Compound
R1
erbB2 kinase inhibition
a
IC50 (nM)
erbB2 cell inhibition
a
IC50 (nM)
Cell selectivity
versus EGFRa
16a
21a
21b
21c
21d
21e
21f
OMe
NHMe
OMe
—
—
77
30
310
13
1·
230·
230·
>770·
>100·
>410·
>660·
NH(CH2)2 morpholine
NMe2
Pyrrolidine
280
48
15
—
—
<10
<10
24
36
21
Morpholine
NMe Piperazine
—
—
130
15
NT, not tested.
a Values are means of at least two experiments, assay error is <2·.
compound 21e was also found to have low Cl of
2.5 mL/min/kg, and a slightly higher Vd of 0.4 L/kg,
resulting in a half-life of 1.9 h. An initial oral formula-
tion of this compound in 0.5% methyl cellulose provided
only 5% F, presumably due to poor absorption. An
in vitro measurement of permeability in the Caco2 sys-
tem predicted good permeability (A ! B: 15.4 1.8 ·
10ꢁ6 cm/s), however the solubility of this compound
was found to be poor, likely resulting in the low bio-
availability. To address this issue, 21e was dosed in a
self-emulsifying drug delivery system (SEDDS) formula-
tion consisting of a mixture of Miglyol 812, Polysorbate
80, and Capmul MCM. This formulation caused bio-
availability to increase to 37%, presumably due to en-
hanced solubility in this formulation.20 Compound 21e
was further screened against 14 diverse kinases and
found to have >10 lM potency against all kinases tested
(EGFR, IRK, IGF-1R, CDK2, CDK5, cSRC, FES,
JAK3, PKCa, PKCh, LCK, PDGFRB, PKA, and
Wee-1) thus demonstrating broad kinase selectivity.
mine an optimal ratio of erbB2/EGFR inhibition for
anticancer activity.
Acknowledgments
John Kath, Joel Morris, Matthew Marx, Richard Con-
nell, Jun Xiao, Xumiao Zhao, Matthew D. Wessel, and
Sukhamaya Bain provided additional chemistry sup-
port. Additional biology support was received from
Ann Marie Rossi, Jitesh Jani, Erling Emerson, Larry
Wagner, Jean Beebe, Kevin Coleman, and Francis
Bourbonais.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.03.046.
In conclusion, several new classes of anilinoquinazoline
inhibitors were synthesized. These inhibitor classes con-
sist of novel piperidine and benzamide functionality on
the northern portion of the molecule combined with a
quinazoline C6-alkyne amide, C6,C7-bisethers, C6
monoethers, and amine substituted pyridopyrimidines.
These compounds were shown to be potent inhibitors
of erbB2 in kinase and cellular assays, and are extremely
selective over EGFR, despite the high homology that ex-
ists between these kinases. Two representative com-
pounds are further shown to have moderate oral
bioavailability with low clearance. It is expected that
agents of this type could be effective in the treatment
of cancer either as single agents, or in combination with
other chemotherapeutic drugs. The exquisite kinase
selectivity of these compounds should offer the scientific
community excellent tools for further understanding the
erbB2 signaling cascade, and the implications for inhib-
iting this pathway. Other future studies should include
combining these agents with EGFR inhibitors to deter-
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14. Ripin, D. H. B.; Bourassa, D. E.; Brandt, T.; Castaldi, M.
J.; Frost, H. N.; Hawkins, J.; Johnson, P. J.; Massett, S.
S.; Neumann, K.; Phillips, J.; Raggon, J. W.; Rose, P. R.;
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15. erbB2 kinase assays were conducted as described in Refs.
9,10. Briefly, recombinant erbB intracellular domain
(residues 675–1255) was expressed in Baculovirus-infected
Sf9 cells as GST fusion proteins and purified. The
phosphorylation of poly(Glu, Tyr) was then measured in
the presence of ꢀ15 ng of this recombinant protein,1 mM
20. Pouton, C. W. Adv. Drug Deliv. Rev. 1997, 25, 47.