Novel pyrrolo[2,1-f][1,2,4]triazin-4-amines: Dual inhibitors of EGFR and HER2 protein tyrosine kinases

Article abstract of DOI:10.1016/j.bmcl.2010.11.100

A novel series of 5-((4-aminopiperidin-1-yl)methyl)-pyrrolo[2,1-f][1,2,4] triazin-4-amines with small aniline substituents at the C4 position were optimized for dual EGFR and HER2 protein tyrosine kinase inhibition. Compound 8l exhibited promising oral efficacy in both EGFR and HER2-driven human tumor xenograft models.

Full text of DOI:10.1016/j.bmcl.2010.11.100

Bioorganic & Medicinal Chemistry Letters 21 (2011) 781–785
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel pyrrolo[2,1-f][1,2,4]triazin-4-amines: Dual inhibitors of EGFR
and HER2 protein tyrosine kinases
⇑
Brian E. Fink , Derek Norris, Harold Mastalerz, Ping Chen, Bindu Goyal, Yufen Zhao,
Soong-Hoon Kim, Gregory D. Vite, Francis Y. Lee, Hongjian Zhang, Simone Oppenheimer,
John S. Tokarski, Tai W. Wong, Ashvinikumar V. Gavai
Department of Oncology Chemistry, Discovery Biology, Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, NJ 08543-4000, United States
Department of Computer Aided Drug Design, Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, NJ 08543-4000, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 5-((4-aminopiperidin-1-yl)methyl)-pyrrolo[2,1-f][1,2,4]triazin-4-amines with small ani-
line substituents at the C4 position were optimized for dual EGFR and HER2 protein tyrosine kinase inhi-
bition. Compound 8l exhibited promising oral efficacy in both EGFR and HER2-driven human tumor
xenograft models.
Received 30 September 2010
Revised 18 November 2010
Accepted 22 November 2010
Available online 30 November 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
EGFR
HER2
Kinase inhibitor
Receptor tyrosine kinases (RTK) play key roles in the processes
governing cellular proliferation, differentiation and evasion from
apoptosis. Among the known RTKs, the ErbB family, in particular
EGFR and HER2 have been extensively studied and clinically
validated as targets for cancer therapies.1 The ErbB family consists
of four receptors: epidermal growth factor receptor (EGFR/ErbB-1/
HER1), HER2 (ErbB-2/neu), HER-3 and HER-4. Over-expression
and/or constitutive activation of EGFR and HER2 have been observed
in numerous tumor types, including colon, breast, ovarian, head and
neck, and non-small cell lung cancers.2 Current therapies using
either monoclonal antibodies (cetuximab and trastuzumab) or small
molecules (erlotinib) to selectively target EGFR or HER2, or more
recently, small molecules that target both receptors (lapatinib), have
demonstrated success in the clinic.3
ment in cellular potency in the HER2-overexpressing N87 gastric
carcinoma cell line (Table 1). However, the increase in cellular
activity did not result in substantially improved in vivo potency.
Even though a five- to ten-fold improvement in anti-proliferative
activity was seen, all three compounds were active in EGFR or
HER2 driven in vivo tumor models in doses ranging from 60 to
180 mg/kg.4–6 Insufficient plasma exposure for 2 and 3 was
thought to be the likely cause for the apparent lack of improve-
ment in in vivo potency. Rapid disappearance of drug during phar-
macokinetic profiling was observed in a mouse pharmacokinetic
study for 2 and 3 relative to 1 (Table 1). It is likely that 2 and 3
do not maintain sufficient plasma concentrations to provide
sustained inhibition of EGFR and HER2 for the period of time
necessary for robust in vivo activity in tumor models.
We have previously reported the discovery and preclinical
evaluation of [4-[[1-(3-fluorophenyl)methyl]-1H-indazol-5-ylami-
no]-5-methyl-pyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamic acid, (3S)-
3-morpholinylmethyl ester (BMS-599626, 1), as a selective and
orally efficacious inhibitor of EGFR and HER2 kinases, culminating
in our first entry into the clinic with a dual EGFR/HER2 inhibitor.4
Subsequently, the discovery and preclinical profiles of the C5-
substituted pyrrolotriazines 25 and 36 have been disclosed. While
2 and 3 exhibited EGFR/HER2 biochemical potency comparable to
that of 1, the C5-substituted analogs showed dramatic improve-
Here we describe our efforts to optimize the C5-substituted
pyrrolotriazine series, focusing on reducing the molecular weight
and overall lipophilicity as a means to improve the ADME proper-
ties and thereby improve exposure and in vivo potency.
A general synthetic approach was developed to allow for the ra-
pid generation of C4 aniline SAR with a 4-aminopiperidinylmethyl
or a morpholin-2-yl methoxymethyl group at the C5 position of
the pyrrolotriazine (Scheme 1). 5-Methyl-4-(methylthio)pyrrol-
o[2,1-f][1,2,4]triazine 46,7 was reacted with N-bromosuccinimide
(NBS) and catalytic benzoyl peroxide in carbon tetrachloride and
the resulting 5-bromomethylpyrrolotriazine was treated with
either (S)-tert-butyl 2-(hydroxymethyl)morpholine-4-carboxylate
or tert-butyl piperidin-4-ylcarbamate to afford 5 or 6, respectively.
Oxidation of the C4 thiomethyl ether with m-CPBA, followed by
⇑
Corresponding author. Tel.: +1 609 252 3420.
E-mail address: brian.fink@bms.com (B.E. Fink).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.100

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B. E. Fink et al. / Bioorg. Med. Chem. Lett. 21 (2011) 781–785
Table 1
Table 2
Pyrrolotriazine EGFR/HER2 inhibitors
Comparison of C5 amine substitutions
R
O
H2N
R
N
R2
N
HN
HN
7 R1 =
Me
6
N
O
R1
HN
O
HN
5
4
N
NH
O
HN
H2N
N
N
O
N
1
8 R1
=
N
N
N
N
2
1
O
R2
IC50
(lM)
a
HN
R
N
HER2
EGFR
N87
A2780b
NA
HN
O
R =
N
F
N
N
N
7a
8a
>1.0
0.95
>1.0
NA
NA
N
N
N
3
0.36
NA
a
N87 MEDc (mg/kg)
Mouse exposured
Cmax/C4 h M)
N
IC50
(
lM)
(
l
EGFR
HER2
N87b
1
2
3
0.032
0.035
0.061
0.023
0.022
0.055
0.450
0.035
0.083
120
60
60
4.1/4.1
4.5/2.7
9.5/5.8
7b
8b
0.67
0.01
0.02
4.4
3.9
2.3
a
IC50 values are reported as the mean of at least three determinations. Variability
0.007
0.12
around the mean value was <15%.
N87 is a gastric tumor cell line (HER2+++/EGFR+).
b
c
MED–minimum efficacious dose when dosed Q1D Â 14, po. An active dose is
8c
0.04
>1.0
0.009
0.88
0.14
NA
3.7
NA
defined as %tumor growth inhibition (TGI) >50%. Tumor cells were implanted
subcutaneously in athymic mice and staged to approximately 100 mg prior to the
initiation of drug therapy.
8d
d
Average 4 h exposure in three male Balb/C mice for compounds administered
orally at 50 mg/kg in Tween 80/PEG400/water (10/40/50).
a
IC50 values are reported as the mean of at least three determinations. Variability
around the mean value was <15%.
b
A2780 is an ovarian tumor cell line (HER2À/EGFRÀ).
SMe
N
R
SMe
N
R
NHAr
N
Me
a
b
on an extended hydrophobic motif at C4 and even a small aniline
group at C4 is tolerated when optimal compensating interactions
are identified at the C5 position.
A potential binding mode for 8b in the ATP binding site was
modeled using the published X-ray structure of the complex be-
tween erlotinib and the EGFR (Fig. 1).12 In the model, the pyrrolo-
triazine core is oriented in the ATP binding site such that there is a
hydrogen bond between N1 and the hinge region Met769 NH, serv-
ing to anchor and orient the pyrrolotriazine core in the ATP binding
pocket. In this orientation, the C4 anilino group extends back into a
N
N
N
N
N
N
4
O
O
5 R =
6 R =
7 R =
BocN
O
HN
O
BocHN
H2N
8 R =
N
N
Scheme 1. Reagents and conditions: (a) NBS (1.05 equiv), benzoyl peroxide
(catalytic), CCl4, N2, reflux, 10 min, then (S)-tert-butyl 2-(hydroxymethyl)morpho-
line-4-carboxylate or tert-butyl piperidin-4-ylcarbamate (1.5 equiv), toluene,
reflux, 55%; (b) m-CPBA (2.1 equiv), CH2Cl2, 0 °C to rt, 30 min; ArNH2 (1.0 equiv),
rt, 1–2 h; 15% TFA/CH2Cl2, 20–50%.
hydrophobic pocket formed partially by the
keeper residue Thr766.
aC-helix beyond gate-
In our binding model, the C5 substituent is predicted to extend
into the ribose-phosphate pocket where the piperidinyl amino
group is available to participate in an extensive network of hydro-
gen bonds with the side chains of Asp831 and Asn818, and also
with the backbone carbonyl oxygen of Arg817. Accessing this net-
work of hydrogen bonding interactions within the ribose-phos-
phate pocket is hypothesized to more than compensate for any
loss in binding energy associated with the reduction of hydropho-
bic non-bonded interactions when the C4 N-benzylindazole of 1 is
replaced by a small aniline ring. Molecular modeling suggests the
existence of an intramolecular hydrogen bond between the C4 ani-
line NH and the tertiary piperidinyl nitrogen, stabilizing the bioac-
tive conformation depicted in Figure 1. Conformational analysis
and molecular dynamics simulations with explicit waters indicate
that the global energy minimum for 8b is identical to the presumed
bioactive conformation shown in Figure 1. These results are sup-
ported by small molecule X-ray structures for related analogs (data
not shown) and through 1H NMR studies where a significant down-
field shift was observed for the C4 aniline NH, indicating a likely
hydrogen bonding interaction with the tertiary piperidinyl nitrogen
in solution.13 In contrast, while the morpholine nitrogen of 7b may
addition of an aniline and subsequent removal of the amine
protecting group provided 7 or 8 in 20–50% yield.
Initial efforts to reduce the molecular weight and lipophilicity
in the series focused on truncating the C4 N-benzylindazole
(Table 2). Removal of the phenyl ring (7a and 8a) resulted in signif-
icantly reduced biochemical potency against both HER2 and EGFR.
These results were expected since previous SAR had suggested that
HER2 potency in particular required the burying of a hydrophobic
group deep into the ATP binding pocket. This had been suggested
in a number of publications and corroborated by the published
crystal structure of lapatinib bound to EGFR.8–11 Replacement of
the entire N-benzylindazole of 3 with a m-acetylenic aniline (7b)
resulted in the loss of HER2 activity while EGFR activity was
maintained, again consistent with previous SAR. Surprisingly,
when the same modification was made to compound 2, the result-
ing analog (8b) demonstrated biochemical potency superior to
either extended analog against both EGFR and HER2. Our results
indicate that potent dual EGFR/HER2 inhibition is not dependent

B. E. Fink et al. / Bioorg. Med. Chem. Lett. 21 (2011) 781–785
783
Table 3
C4 Aniline SAR
(R)n
H2N
N
HN
N
N
N
a
R
IC50
(l
M)
N87
HER2
EGFR
A2780
8e
8f
8g
8h
8i
8j
8k
8l
2-Me
3-Me
4-Me
3-F
3-Cl
3-Br
3-Cl, 5-Cl
3-Cl, 4-F
3-Cl, 4-OMe
0.66
0.02
0.05
0.032
0.009
0.006
0.13
0.01
0.21
0.02
3.30
0.14
0.79
0.28
0.06
0.05
3.36
0.12
1.21
>5
>5
>5
>5
>5
2.6
4.0
>5
>5
0.005
0.006
0.006
0.004
0.004
0.034
0.006
0.07
8m
a
IC50 values are reported as the mean of at least three determinations. Variability
around the mean value was <15%.
Figure 1. Predicted binding mode of 8b in the X-ray structure of EGFR.12
versatile intermediate for further SAR development since it is air
stable and can be stored for extended periods of time without
appreciable decomposition. Aromatic amines were added selec-
tively to the C4 position allowing for the subsequent introduction
of aliphatic amines at the C5 position, either stepwise or in a one-
pot procedure.
Introduction of a methyl group around the C4 aniline ring
(Table 3) identified the m-, and p-positions as most tolerant of sub-
stitution with respect to biochemical potency, while o-substitution
resulted in a significant loss of activity (8e). This likely arises from
increased steric interactions between the o-substituent and the
pyrrolotriazine core forcing the aniline ring to adopt a less than opti-
mal geometry in the binding pocket. That the loss in activity is more
pronounced relative to HER2 than HER1 may be the result of a single
residue difference between HER1 (Cys775) and HER2 (Ser783) in
this region of the ATP binding pocket. It has been suggested that a
strong network of water-mediated hydrogen bonds involving
Ser783 in HER2 substantially impacts the structural requirements
for inhibitor binding.14 Among the m- and p-substitutions (8f vs
8g), the m-substituted analogs were favored by a factor of 2–3 which
also be positioned to take advantage of the network of hydrogen
bonds in the ribose-pocket5, conformational analysis of 7b shows
a larger pool of low energy conformations other than the presumed
bioactive conformation and the 1H NMR chemical shift for the C4
aniline NH does not indicate an intramolecular hydrogen bond
between the C4 aniline NH and the C5 alkoxymethyl group.13 These
results suggest 7b is not pre-organized to adopt the required
bioactive conformation resulting in lower binding affinity for the
morpholine-substituted analogs and establishing the aminopiperi-
dine as an optimal C5 substituent for dual EGFR/HER2 activity.
Further modification of the C4 aniline demonstrated that an
unsubstituted phenyl ring (8c) retained most of the biochemical
activity in this series. Again, the strong network of interactions
made between the 4-aminopiperidine group and the protein target
serve to anchor the inhibitor in the binding pocket. Substitution of
the C4 phenyl group with an aliphatic group (8d) was not tolerated
and served to define the limits for binding to EGFR and HER2 at this
position. Potent anti-proliferative activity was maintained in the
HER2-driven N87 gastric carcinoma cell line for these truncated
analogs. Importantly, minimal activity was observed in non-HER
dependent tumor cell lines such as A2780, indicating that anti-pro-
liferative effects of this series are dependent on EGFR/HER2
inhibition.
also translated into better cellular activity (0.14 vs 0.79 lM). Further
Table 4
Profile of lead dual HER2/EGFR inhibitors
a
IC50
(l
M)
GEO MED (mg/kg)
Having defined the minimum requirements for potent inhibi-
tion of EGFR and HER2 kinases in this series, further optimization
of the aniline ring was investigated. Toward that end, an improved
synthetic route was developed and is described in Scheme 2.5,6
Pyrrolotriazine 9 was treated with NBS and catalytic benzoyl
peroxide at reflux followed by reaction with triethylamine to
afford the triethylammonium chloride 10. Compound 10 was a
HER2
EGFR
N87
1
0.03
0.01
0.02
0.01
0.01
0.020
0.007
0.005
0.004
0.006
0.42
0.12
0.14
0.06
0.12
120
15
60
60
15
8b
8f
8i
8l
a
IC50 values are reported as the mean of at least three determinations. Variability
around the mean value was <15%.
Et3+N
Cl
N
Cl
N
Me
b, c
a
8
N
N
Table 5
N
N
Kinase selectivity profile for compound 8l
9
Kinase
Fold selectivity
versus HER2
10
Scheme 2. Reagents and conditions: (a) NBS (1.05 equiv), benzoyl peroxide
(catalytic), CCl4, N2, reflux, 10 min; Et3N; 80%; (b) ArNH2 (1.0 equiv), rt, 75–90%;
(c) tert-butyl piperidin-4-ylcarbamate (1.5 equiv), toluene, reflux; 15% TFA/CH2Cl2;
63–95%.
BTK, JAK2, JAK3, KDR, Lck, PKCd, PKCh, Flt3
>10
>100
PKCa, CDK2, FGFR, Met, GSK3b, CAMKII, IKK2, IGF-1R,
IRAK4, AurA, MK2, p38
a, CK1A1, IKK2, InsR, PLK1, SRC

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B. E. Fink et al. / Bioorg. Med. Chem. Lett. 21 (2011) 781–785
Table 6
Profile of lead compounds in N87 tumor model
Compound
N87 MED (mg/kg)
24 h exposurea
C24 h
Dose (mg/kg)
C3 h
(l
M)
(lM)
AUC0–24 h (lM h)
3
8l
60
<15
240
15
5.8
7.2
0.1
6.8
100
160
a
Mean 24 h serum exposure in three male Balb/C mice for compounds administered orally in Tween 80/PEG400/water (10/40/50).
improved in vivo potency. A comparison of drug concentration at
the 3 and 24 h time points shows that while both compounds show
similar plasma concentrations at 3 h, at 24 h the concentration of
8l remains significantly higher.
In addition, analysis of N87 tumor lysates using a phospho-HER2
endpoint demonstrated that 8l suppressed HER2 signaling for a
much longer period of time relative to 3 (Fig. 2), consistent with
the observed improvements in pharmacokinetics and in vivo
potency.
120
100
80
60
40
20
0
3 Hours
24 Hours
Compound 8l showed a good level of kinase selectivity in a pa-
nel of diverse protein kinases (Table 5). Among the more potent
off-target activities were Lck (IC50 = 0.37
lM) and KDR (IC50 =
0.11 M), however, a 10-fold window was achieved against these
l
important kinases. Greater than 100-fold selectivity was achieved
versus a number of other receptor tyrosine kinases such as IGF-
1R as well as serine/threonine kinases such as CDK2.
In summary, we have identified the C5 aminopiperidine as an
important contributor to intrinsic potency of pyrrolotriazines
against both HER2 and EGFR kinases. This substitution allows
modification of the C4 position to reduce the overall size and
lipophilicity leading to higher exposures and greatly improved
in vivo potency in both HER2 (N87) and EGFR (GEO)-driven tumor
models.
Control
8l
3
Figure 2. Pharmacodynamic analysis of N87 tumors at 3 and 24 h after 21 days of
dosing (8l = 15 mg/kg, 3 = 240 mg/kg). Each sample represents the pooling of three
individual tumor samples.16
exploration of the m-position (8h–8j) showed that small hydropho-
bic substitutions were more potent and demonstrated correspond-
ing improvements in cellular anti-proliferative activity. Compound
8i was of particular interest, demonstrating extremely potent activ-
ity in N87 cells (IC50 = 60 nM) while showing no observable potency
in HER2/EGFR independent A2780 tumor cells (IC50 >5000 nM).
Di-substitution on the C4 aniline ring was found to parallel the
SAR developed for mono-substitution. The m- and p-positions were
tolerant of substitution (8l) although increases in either steric bulk
or polarity at the p-position resulted in decreased biochemical po-
tency (8m). Other substitution patterns, such as 3,5-disubstitution,
resulted in a substantial decrease in biochemical potency (8k).
Potential lead candidates were evaluated in vivo in the EGFR-
driven GEO colon tumor xenograft model (Table 4). The rapid
growth kinetics associated with the GEO xenografts allowed for
rapid screening of compounds using an abbreviated 10-days
dosing schedule. All compounds showed robust in vivo activity in
the GEO model at minimum efficacious doses ranging from 15 to
60 mg/kg. Initial lead 8b demonstrated good efficacy at doses as
low as 15 mg/kg, however it was accompanied by significant
weight loss, perhaps related to an undetermined off-target activity.
Compound 8i, among the most potent in the N87 cellular assay,
provided lower than expected potency in vivo that was attributed
to poor serum exposure. Compound 8l showed in vivo potency
superior to the previous lead 1, and was selected for further
evaluation.
References and notes
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Anti-tumor activity for compound 8l in the N87 gastric cancer
xenograft model was even more impressive (Table 6). Compound
8l (MED <15 mg/kg) was over four-fold more potent than 3
(MED = 60 mg/kg). Pharmacokinetic analysis of drug exposure in
serum over 24 h in Balb/C mice showed that 8l had comparable
drug exposure levels to 3, but at a fraction of the dose (15 mg/kg
vs 240 mg/kg).15 Clearly, improving the pharmacokinetic profile
through reducing molecular size and lipophilicity resulted in

B. E. Fink et al. / Bioorg. Med. Chem. Lett. 21 (2011) 781–785
785
11. Wood, E. R.; Truesdale, A. T.; McDonald, O. B.; Yuan, D.; Hassel, A.; Dickerson, S.
H.; Ellis, B.; Pennisi, C.; Horne, E.; Lackey, K.; Alligood, K. J.; Rusnak, D. W.;
Gilmer, T. M.; Shewchuk, L. Cancer Res. 2004, 64, 6652.
16. Tumors were excised and homogenized in a glass homogenizer using a buffer
that contained 1% Triton X-100, 40 mM Tris–HCl (pH 7.7), 10% glycerol, 1 mM
EDTA, 0.15 M NaCl, 1 mM sodium vanadate, and 40 lM ammonium molybdate.
12. Stamos, J.; Slikowski, C.; Eigenbrot, C. J. Biol. Chem. 2002, 277, 46,265. Molsoft
modeling software was used to produce this figure. Molsoft LLC, 3366 No.
Torrey Pines Ct., Ste. 300, La Jolla, CA 92037.
Tumor lysates were centrifuged at 12,000Âg for 10 min and the supernatants
were saved. Since the HER2 gene is amplified in N87 tumors, receptor
phosphorylation was readily quantitated by western blot analyzes using tumor
13. Compound 8b: 1H NMR (400 MHz, DMSO-d6) d 11.9 (s, 1H, NH); 7b: 1H NMR
(400 MHz, DMSO-d6) d 9.6 (s, 1H, NH).
lysates directly. Tumor lysates (50 lg proteins) were fractionated by gel
electrophoresis and western blot analysis was carried out using anti-
phosphotyrosine antibody (PY20, Invitrogen), and anti-HER2 antibody (see
Ref. 4b). HER2 receptor phosphorylation was quantified by densitometry and
normalized against HER2 receptor levels.
14. Bhattacharya, S. K.; Cox, E. D.; Kath, J. C.; Mathiowetz, A. M.; Morris, J.; Moyer, J.
D.; Pustilnik, L. R.; Rafidi, D. T.; Su, C.; Wessel, M. D. Biochem. Biophys. Res.
Commun. 2003, 307, 267.
15. The free fraction (fu) for 8l and
3
were 1.2% and 2.6%, respectively, as
M.
determined by equilibrium dialysis at 10
l