New dual inhibitors of EGFR and HER2 protein tyrosine kinases

Article abstract of DOI:10.1016/j.bmcl.2005.07.027

A novel series of dual EGFR and HER2 inhibitors based on the pyrrolo[2,1-f][1,2,4]triazine nucleus is described. A general route toward their synthesis, which enables functionalization at multiple sites, has been developed. Biological evaluation in enzyma

Full text of DOI:10.1016/j.bmcl.2005.07.027

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4774–4779
New dual inhibitors of EGFR and HER2 protein tyrosine kinases
Brian E. Fink,* Gregory D. Vite, Harold Mastalerz, John F. Kadow, Soong-Hoon Kim,
Kenneth J. Leavitt, Karen Du, Donald Crews, Toomas Mitt, Tai W. Wong,
John T. Hunt, Dolatrai M. Vyas and John S. Tokarski
Departments of Oncology Chemistry, Discovery Biology, and Computer Aided Drug Design,
Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543-4000, USA
Received 20 May 2005; revised 21 July 2005; accepted 21 July 2005
Available online 18 August 2005
Abstract—A novel series of dual EGFR and HER2 inhibitors based on the pyrrolo[2,1-f][1,2,4]triazine nucleus is described. A gen-
eral route toward their synthesis, which enables functionalization at multiple sites, has been developed. Biological evaluation in
enzymatic and cell-based assays has identified a series of C-6 carbamates with potent biochemical and cellular activities.
Ó 2005 Elsevier Ltd. All rights reserved.
The role of receptor tyrosine kinases (RTK) as key reg-
ulators of the cellular processes governing proliferation
and differentiation has led to intensive efforts focused
on identifying selective inhibitors for use in cancer treat-
ments.1 Among the known RTKs, the ErbB protein ki-
nases are one of the most studied cell signaling families.
The ErbB family consists of four receptors: epidermal
growth factor receptor (EGFR/ErbB-1), HER2 (ErbB-
2/neu), ErbB-3, and ErbB-4. There is considerable evi-
dence, both preclinical and clinical, associating EGFR
and HER2 with the occurrence and progression of cer-
tain cancers.2 Current therapies using either monoclonal
antibodies (cetuximab and trastuzumab) or small mole-
cules (erlotinib) to selectively target EGFR or HER2
have demonstrated success in the clinic.3 However, both
EGFR and HER2 may act in a synergistic manner
through the formation of heterodimers to produce mito-
genic signaling in excess of that produced from either
receptor alone.4 Additionally, overexpression of either
or both EGFR and HER2 is often correlated with poor
response to chemotherapeutic agents and overall poor
patient prognosis.5 Targeting both EGFR and HER2
has therefore become a promising approach to cancer
treatment.
inhibitors.6 We demonstrated that variation of the C-4
aniline resulted in compounds with potent and selective
biochemical inhibition of VEGFR-2 or EGFR (Fig. 1).
In addition, earlier SAR indicated that the C-5 and C-6
positions were tolerant of substitution and might be
promising sites for introduction of functionality to mod-
ulate the physicochemical properties of the core. Here,
we report on our efforts to extend further the use of
the pyrrolotriazine core to the development of a com-
pound with dual EGFR and HER2 activities.
The goal of the present work was to improve on the
EGFR biochemical activity originally found in 1, while
incorporating HER2 activity and improving the overall
physicochemical properties of the molecule. A proposed
binding mode for 1, modeled after the erlotinib/EGFR
X-ray structure,7 is shown in Figure 2.
F
Cl
HN
Me
4
5
N
3
6
Recently, we identified the pyrrolo[2,1-f][1,2,4]triazine
nucleus as a novel scaffold for ATP-competitive kinase
N
2
N
7
1
1
EGFR IC50: 0.100 µM
VEGFR-2 IC50: >10 µM
Keywords: panHER; EGFR; Kinase inhibitor.
*
Corresponding author. Tel.: +1 6092523420; fax: +1 6092526601;
e-mail: brian.fink@bms.com
Figure 1. Novel kinase inhibitor template.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.027

B. E. Fink et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4774–4779
4775
To simultaneously functionalize the 4-, 5-, and 6-posi-
tions, a general synthetic method was developed that
would allow for incorporation of additional functionality
at these positions (Scheme 1). The reaction of two equiv-
alents of ethylisocyanoacetate with a variety of aldehydes
in the presence of DBU afforded, in a single step, moder-
ate to good yield of pyrrolodiesters 2.8 Deprotonation of
2 and N-amination with O-(diphenylphosphinyl)-hy-
droxylamine9 afforded a series of N-aminopyrroles 3,
that were readily cyclized in formamide to afford the
pyrrolotriazinone cores 4.
Activation of the 4-position of intermediate 4 via the
corresponding chloroimidate using neat POCl3 was fol-
lowed by addition of an amine in acetonitrile to afford
pyrrolotriazines 5. Various functional groups were
investigated at the 6-position to determine their effect
on activity and their viability as linkers to append addi-
tional polar/solubilizing groups. Hydrolysis of 5 with
LiOH afforded the carboxylic acid 6, which was coupled
to amines or alcohols via the corresponding acid chlo-
ride to afford a series of amides 7 or esters 8 (Scheme
2). Alternatively, Curtius rearrangement of 6 with
DPPA and reaction of the intermediate isocyanate with
an alcohol afforded a series of carbamates 9. Reduction
of 5e with DIBAL-H to alcohol 10, followed by MnO2
oxidation and reductive amination, afforded amine 11
(Scheme 3). Alkylation of 10 with ethyl iodide afforded
an ether analog 12. Activation of 10 with neat SOCl2,
followed by treatment with NaCN and acidic hydrolysis,
afforded a separable mixture of 13 and 14, correspond-
ing to one carbon homologated analogs of 6 and 7.
Figure 2. Predicted binding mode of compound 1 in the X-ray
structure of HER1 based on co-crystal structures of similar kinase
inhibitors. A few select residues are shown for orientation.7
In this model, the pyrrolotriazine core is oriented in the
ATP-binding site such that N-1 makes a hydrogen bond
with the hinge region M769 carbonyl in a manner simi-
lar to the N-1 of the quinazoline core. The aniline por-
tion of the molecule is then oriented into
a
hydrophobic pocket deep within the binding site. Pre-
sumably the size and functionality of this pocket are
important contributors to the selectivity of an agent
for a specific kinase. Substitution at the C-5 position is
directed into a smaller hydrophobic pocket flanked by
V702 and L820, and is roughly equivalent to the ribose
binding region of ATP. In this binding mode, the C-6
position points toward surface-exposed protein and
was deemed to be an appropriate position to incorpo-
rate polar/solubilizing functionality.
In pursuit of our goal to improve both EGFR and
HER2 activities in this series, biochemical activities were
R1
DBU, THF, 50 oC
30 - 60%
O
CO2Et
CN
CO2Et
R1
H
EtO2C
NH
2
R1 = See Table 2
R1
formamide, 165 oC
87%
Ph2PO2NH2, NaH
CO2Et
NH2
EtO2C
N
DMF
41%
3
O
NHR2
R1
R1
1. POCl3, 100 oC
NH
N
EtO2C
R3O2C
2. NH2-R2, Et3N, CH3CN
77%
N
N
N
N
4
5 R3 = Et
6 R3 = H
LiOH, 65 oC
THF/MeOH/H2O
89%
R2 = See Table 1
Scheme 1.

4776
B. E. Fink et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4774–4779
NHAr
N
Et
O
1. (COCl)2, DMF(cat), CH2Cl2
N
RX
2. RNH2 or ROH, DIEA, CH2Cl2
NHAr
N
Et
10 - 64%
7 X = NH
8 X = O
N
HO2C
N
NHAr
N
Et
1. DPPA, Et3N, 50 oC, 1,4-dioxane
6
N
HN
O
N
2. ROH, 1,4-dioxane, 80 oC
25 - 40%
RO
N
9
Ar =
N
N
Scheme 2.
NHAr
N
Et
EtHN
N
N
11
1. MnO2, CH2Cl2, 87%
2. EtNH2, NaCNBH3
CH3CN, 39%
NHAr
NHAr
N
Et
Et
DIBAL-H
HO
EtO
NaHMDS, EtI
16%
N
5e
N
THF, 23 oC
91%
N
N
N
10
12
1. SOCl2, NaCN, DMF, 53%
2. AcOH, H2SO4/H2O, reflux, 77%
NHAr
NHAr
Et
Et
O
O
AcOH, H2SO4/H2O
HO
H2N
N
N
reflux
61%
N
N
N
N
13
14
Ar =
N
N
Scheme 3.
assessed using in vitro enzyme assay methods previously
reported.6,10
5,6-bicyclic system (5a, 5b or 5c). However, this activity
was somewhat attenuated if the aromatic ring was dis-
placed by one carbon (5d). Additional lipophilic aryl
groups were tolerated (5e), but the nature and orienta-
tion of the added aryl group appear to be important
for potency (5f). The SAR, relative to HER2, is some-
what more straightforward. As the size increased, inhib-
itory potency versus HER2 followed in parallel. While
5a–5d showed modest activity against HER2, increasing
Based on our proposed binding mode, substitution at
the C-4 position was seen as an area likely to influence
both the potency and selectivity of this series (Table
1). The SAR at position 4, relative to EGFR, indicates
that enzyme activity is effectively inhibited when the
C-4 substituent is a relatively small aniline or fused

B. E. Fink et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4774–4779
4777
Table 1. Structure–activity relationship for the C-4 position
Table 2. Structure–activity relationship for the C-5 and C-6 positions
NHR
Et
N
N
N
EtO2C
N
N
R1
HN
Compound
R
HER2a,b
EGFRb
N
R2
IC50 (lM)
IC50 (lM)
N
N
F
5a
5b
5c
2.8
3.0
3.3
0.09
0.06
0.05
Compound R1
R2
HER2
a
EGFR
a
Cl
IC50 (lM) IC50 (lM)
5e
5g
5h
5i
Et
H
CO2Et
CO2Et
CO2Et
CO2Et
0.20
0.39
0.20
0.21
1.2
0.20
0.12
0.20
0.20
0.95
Br
H
Me
iPr
N
N
5j
nPr CO2Et
5k
6
Bn
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
CO2Et
CO2H
>25
>25
0.04
0.11
0.13
0.50
0.17
0.73
9.7
0.02
0.12
0.07
0.20
0.21
0.49
7.4
5d
7.2
0.20
7a
7b
7c
9a
10
11
12
13
14
C(O)NH2
C(O)NHMe
C(O)NMe2
NHC(O)OBn
CH2OH
5e
5f
0.18
0.82
0.12
0.61
N
O
CH2NHEt
CH2OEt
N
1.4
2.4
0.95
1.1
CH2C(O)NH2
CH2CO2H
0.68
1.6
a IC50 values are reported as the mean of at least three determinations.
Variability around the mean value was <15%.
a Recombinant HER2 cytoplasmic sequence is expressed in Sf9 insect
cells as an untagged protein and purified by ion-exchange chroma-
tography. HER2 kinase activity is measured under the same condi-
tions as for EGFR. See Refs. 6 and 10 for assay conditions.
b IC50 values are reported as the mean of at least three determinations.
Variability around the mean value was <15%.
small alkyl groups, such as methyl, ethyl, and isopropyl
(Table 2).
Both HER2 and EGFR activity eroded with increasing
size/shape of the C-5 substituent, indicating a shape
preference of this pocket (compare 5i vs. 5j). While sub-
stitution at the C-4 and C-5 positions resulted in gains in
potency, overall they did not lead to the levels of activity
necessary for good cellular potency (data not shown).
Since our binding model indicated that the C-6 position
is directed toward solvent-exposed regions of the bind-
ing pocket, substantial gains in biochemical potency
were not expected. To our surprise, carboxylic acid 6
showed a ꢀ10-fold improvement in activity against each
receptor. This result indicates that the nature of the link-
er group at C-6 dramatically impacts binding. Primary
and secondary amides 7a or 7b showed similar inhibito-
ry activity compared to the corresponding esters; how-
ever, the dimethylamide 7c resulted in a loss of activity
within this series compared to 7a or 7b. Interestingly,
the benzyl carbamate 9a showed activity similar to both
esters and amides. Substituents lacking the ester or
amide carbonyl group, for example, the ethyl ether 12
or ethylamine 11, provided significantly reduced potency
as did one atom homologation in the amide 13 or car-
boxylic acid 14. These results taken together indicate a
distinct requirement for the immediate C-6 linker to be
planar or nearly planar (i.e., first atom attached at C-6
is preferentially sp2 hybridized) with the pyrrolotriazine
ring and to contain a hydrogen bond acceptor group.
These findings are reinforced by our modeling studies,
which suggest that the linker atoms at the C-6 position
occupy a constricted space between the N-terminal
steric bulk and overall lipophilicity, as with N-benzyl
indazole 5e or biaryl ether 5f, greatly improved activity.
A similar trend was recently reported in a quinazoline
series where increasing size at the C-4 aniline position
substantially reduced EGFR activity and moderately
improved HER2 activity.11
The origins of this effect are unclear, given the sequence
similarities between EGFR and HER2 in this region, the
only difference being Cys775 (EGFR) versus Ser783
(HER2). It has been suggested that these residues, along
with two threonine residues, form a water-mediated
hydrogen bonding network that contributes to the shape
and polarity of the hydrophobic pocket.11 Perhaps inter-
action with this network, in the Ser-containing HER2,
requires additional binding affinity that can only be at-
tained through incorporation of a second distal ring in
HER2. Given the overall plasticity of receptor tyrosine
kinases, sequence differences in regions distal to the
ATP-binding site may influence the size of this binding
pocket. Further explanation may have to await more
complete HER2 structural information.
Since good biochemical potency was achieved against
both receptors when the N-benzylindazole was used at
the C-4 position, further SAR was developed within that
series. A survey of substitution patterns at the C-5 posi-
tions showed that both receptors tolerate a variety of

4778
B. E. Fink et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4774–4779
Table 3. Effect of linker on enzymatic and cellular activity12
N
N
HN
Et
N
R
N
N
a
Compound
R
IC50 (lM)
HER2
0.03
EGFR
0.04
BT474b
7.0
Sal2c
5.8
H
N
N
N
N
N
7d
8a
9b
O
O
O
O
0.24
0.04
0.17
0.04
3.1
1.6
N
N
0.86
0.46
N
H
O
a IC50 values are reported as the mean of at least three determinations. Variability around the mean value was <15%.
b Cell line BT474 (breast carcinoma) overexpresses HER2.13
c Cell line Sal2 (salivary gland carcinoma) contains a constitutively active HER2.14
and C-terminal lobes. While this position is directed
toward the solvent, substituents can therefore still inter-
act with the protein.
Table 4. Kinase selectivity profile for compound 9b
a
Kinase
IC50 (lM)
EGFR
HER2
0.04
0.04
>10
Since simple amides, esters, and carbamates showed
equivalent activity, several small libraries were prepared
using these groups to append polar/solubilizing groups
to the core. Table 3 shows a representative series of
homologous imidazole-substituted compounds contain-
ing each of the three linkers. While the imidazole group
had modest effects when linked through an ester (8a),
improved biochemical potency was observed in both
the amide 7d and the carbamate 9b cases. In addition,
9b showed good anti-proliferative activity in both the
HER2-driven BT474 (breast carcinoma) and Sal2 (sali-
vary gland carcinoma) cell lines,14 with 10-fold better
potency over the analogous amide 7d. Given the similar-
ity of the two compounds, in terms of structure, physical
properties, and biochemical activity, the reason for the
improved cellular activity is unclear. Western blot anal-
ysis of phosphorylated HER2 protein in BT474 cells
showed significantly lower levels of phosphorylation
upon treatment with 9b, relative to 7d (data not shown).
Therefore, the improvement in cellular activity for 9b
may simply reflect improved cell penetration.
Met
FAK
>25
>25
p38
MAPKAP kinase 2
IGF-1R
>25
>25
a IC50 values are reported as the mean of at least three determinations.
Variability around the mean value was <15%.
cellular activity may be modulated through the C-6 po-
sition. Use of a carbamate linker at the C-6 position
was found to be optimal for appending polar function-
ality, a combination of which contributed favorably to
the biochemical activity, as well as cellular activity in
the BT474 and Sal2 cell lines.
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tyrosine kinase inhibitors. We have demonstrated that
potent and selective dual EGFR/HER2 inhibitors may
be prepared through modulation of the C-4 position,
while additional potency, physical properties, and

B. E. Fink et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4774–4779
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