Discovery and preclinical evaluation of [4-[[1-(3-fluorophenyl)methyl]-1H- indazol-5-ylamino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]carbamic acid, (3S)-3-morpholinylmethyl ester (BMS-599626), a selective and orally efficacious inhibitor of human epide

Article abstract of DOI:10.1021/jm9010065

Structure-activity relationships in a series of 4-[1H-indazol-5-ylamino] pyrrolo[2,1-f][1,2,4]triazine-6-carbamates identified dual human epidermal growth factor receptor (HER)1/HER2 kinase inhibitors with excellent biochemical potency and kinase selectiv

Full text of DOI:10.1021/jm9010065

J. Med. Chem. 2009, 52, 6527–6530 6527
DOI: 10.1021/jm9010065
such as breast, ovarian, and bladder tumors, as well as in
squamous cell carcinomasofthe headandneck.3 Heterodimer
signaling is believed to play a significant role in the pathobio-
logy of these tumors. The ability of HER2 to transactivate
other receptors of the HER family by heterodimerization
suggests that simultaneous inhibition of HER1 and HER2
may provide superior efficacy than single receptor targeting
via different and potentially nonoverlapping mechanisms.1 A
high degree of sequence homology (83% identity) between the
two kinase domains supports the feasibility of identifying
compounds that inhibit both receptors with comparable
potency. Lapatinib, an oral dual inhibitor of HER1 and
HER2, was recently approved in combination with capecita-
bine for the treatment of patients with HER2 overexpressing
advanced or metastatic breast cancer.4
We have previously reported the discovery of pyrrolo[2,1-f ]-
[1,2,4]triazine nucleus as a versatile scaffold for ATP-compe-
titive kinase inhibitors.5 Subsequently, we demonstrated that
dual inhibitors of HER1 and HER2 kinases can be prepared
via introduction of a lipophilic 5-amino-1-benzylindazole
substituent at C4 and a carbamate linker at the C6 position
of the pyrrolotriazine.6 The extended aniline motif conferred
improved potency against HER2 while retaining HER1 in-
hibition. As depicted in Table 1, 1 was the initial lead that
exhibited potent inhibition of HER2/HER1 enzymes and
submicromolar potency in antiproliferative assays using
HER2/HER1-dependent cells. Compound 1 did not show
significant activity against HER2/HER1 independent A2780
tumor cells, suggesting thereby that the cellular effects are
specific to inhibition of HER kinases. This paper describes
further optimization of in vitro potency and pharmacokinetic
properties leading up to the identification of 13, a HER1/
HER2 kinase inhibitor that has advanced into clinical devel-
opment.7
Discovery and Preclinical Evaluation of
[4-[[1-(3-fluorophenyl)methyl]-1H-indazol-
5-ylamino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-
6-yl]carbamic Acid, (3S)-3-Morpholinylmethyl
Ester (BMS-599626), a Selective and Orally
Efficacious Inhibitor of Human Epidermal
Growth Factor Receptor 1 and 2 Kinases
Ashvinikumar V. Gavai,*,§ Brian E. Fink,§
David J. Fairfax,‡ Gregory S. Martin,‡ Lana M. Rossiter,‡
Christian L. Holst,‡ Soong-Hoon Kim,§ Kenneth J. Leavitt,§
Harold Mastalerz,§ Wen-Ching Han,§ Derek Norris,§
Bindu Goyal,§ Shankar Swaminathan,§ Bharat Patel,§
Arvind Mathur,§ Dolatrai M. Vyas,§ John S. Tokarski,§
Chiang Yu,§ Simone Oppenheimer,§ Hongjian Zhang,§
Punit Marathe,§ Joseph Fargnoli,§ Francis Y. Lee,§
Tai W. Wong,§ and Gregory D. Vite§
§Bristol-Myers Squibb Research and Development,
P.O. Box 4000, Princeton, New Jersey 08543, and
‡Albany Molecular Research, 21 Corporate Circle,
P.O. Box 15098, Albany, New York 12212
Received July 8, 2009
Abstract: Structure-activity relationships in a series of 4-[1H-
indazol-5-ylamino]pyrrolo[2,1-f][1,2,4]triazine-6-carbamates identi-
fied dual human epidermal growth factor receptor (HER)1/HER2
kinase inhibitors with excellent biochemical potency and kinase
selectivity. On the basis of its favorable pharmacokinetic profile and
robust in vivo activity in HER1 and HER2 driven tumor models, 13
(BMS-599626) was selected as a clinical candidate for treatment of
solid tumors.
Previous studies had established that biochemical potency
could be improved via optimization of the linker group at C6
position.6 In the proposed binding model for the pyrrolotria-
zine nucleus in the ATP binding site of HER1 kinase, the C6
position points toward a specificity pocket that leads to sur-
face exposed protein.6 It was deemed to be an appropriate
position to incorporate a polar functionality, such as an
aliphatic amine, through the C6 carbamate. This change
was predicted to favorably influence physical properties and
the pharmacokinetic profile of the kinase inhibitors. To
evaluate this hypothesis, a variety of cyclic amines were
appended to the pyrrolotriazine using the C6 carbamate
linker. As shown in Table 1, the N-piperidinylethyl and
N-piperazinylethyl analogues 2 and 3 displayed the desired
increase in potency in HER2 overexpressing BT474 and N87
cell proliferation assays. The corresponding morpholine deri-
vative 4 was less potent in the enzymatic assays. It was
expected that sustained inhibition of HER1 and HER2 for a
prolonged period of time would be necessary for robust in
vivo activity in tumor models. To that end, plasma exposures
of lead candidates were investigated in mice for 4 h following
oral administration. The observed low systemic exposures of2
and 3 (Table 1) were attributed to a combination of poor
absorption and insufficient metabolic stability in isolated liver
microsomes.8 It has been suggested that the greater polarity
Receptor tyrosine kinases play a crucial role as signal
transducers in dysregulated cell proliferation, differentiation,
and evasion from apoptosis. Two members of the epidermal
growth factor receptor family, HER1 (EGFR) and HER2
(ErbB2), have been clinically validated as rational targets for
cancer therapy.1 Activation of the HERa receptors via homo-
orheterodimerizationis followed by receptor autophosphory-
lation, recruitment of adaptor proteins, and the activation of
downstream signaling, such as those mediated by Ras/Raf/
MAPK and the PI3K/Akt pathways.2 Overexpression or
constitutiveactivation of HER1 and HER2 has been observed
in many tumor types, including colon, breast, ovarian, head
and neck, and non-small cell lung cancer, and frequently
correlates with poor clinical prognosis.
A number of pharmacologic agents have demonstrated
clinical benefit via modulation of HER1 or HER2 receptor.1
These include HER1-specific antibody cetuximab, HER2-
specific antibody trastuzumab, and small molecule HER1
kinase inhibitors gefitinib and erlotinib. Coexpression of
HER1 and HER2 kinases has been shown in many tumors,
*To whom correspondence should be addressed. Phone: 609-252-
5091. Fax: 609-252-3993. E-mail: ashvinikumar.gavai@bms.com.
a Abbreviations: HER, human epidermal growth factor receptor;
MRT, mean residence time; TGI, tumor growth inhibition.
r
2009 American Chemical Society
Published on Web 10/12/2009
pubs.acs.org/jmc

6528 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Gavai et al.
Table 1. Enzymatic and Cellular Activitiesa and 4 h Plasma Exposures of Pyrrolotriazine C6 Carbamate Analogues
a See ref 7b for description of the assay conditions. b IC50 values are reported as the mean of three independent determinations. c Compounds were formulated as
solutions in PEG400/Tween80/water (40:10:50) and administered orally at 50 mg/kg to three Balb/C mice. d BT474 is a breast tumor cell line (HER2þþþ/HER1þ).
e N87 is a gastric tumor cell line (HER2þþþ/HER1þ). f Sal2 is a salivary gland tumor cell line with a constitutively active HER2. g A2780 is an ovarian tumor cell
line (HER2-/HER1-). h NT = not tested.
and basicity of primary and secondary amines relative to the
tertiary aminereduce their rate of metabolism by the CYP3A4
isozyme.9 Indeed, 5 and 6 showed a significant improvement
in metabolic stability8 and retained comparable HER1/HER2
inhibitory potency in the in vitro assays. Low Cmax attained
during the exposure study in mice indicated that some of these
compounds suffered from poor oral absorption. In the
4-substituted piperidine series, a number of compounds with
varying chain length (for example, 7 and 8) afforded the
desired level of potency in the biochemical and cell-based
assays. This observation provided further support to the
proposed binding hypothesis that C6 substitution extends
up to the surface exposed region of the protein.6 A one-carbon
spacer between piperidine and the carbamate was determined
to be optimal to achieve a combination of good in vitro
potency and satisfactory absorption characteristics. Within
the isomeric piperidinylmethyl carbamates 8-10, 2-substi-
tuted analogue 10 provided the highest plasma concentration
at the end of the 4 h oral exposure study. Previously, we had
observed that replacement of the piperidine functionality in 2
with a less basic morpholine(4) provided a significant boost to
oral absorption, as evidenced by a 6-fold higher Cmax in the
mouse PK study. Indeed, a similar change from 10 to 11 had
the intended impact on Cmax, although magnitude of the
increase was less remarkable. Introduction of a fluorine atom
at meta-position on the phenyl ring gave 12 with a further
boost in the oral exposure in mice via significantly enhanced
metabolic stability8 and oral absorption. Replacement of C5
ethyl with a methyl group provided 13 with a similar in vitro
potency and pharmacokinetic profile. At 10 μM, 13 did not

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6529
Table 2. Pharmacokinetic Parameters for 13
Scheme 1. Synthesis of 13a
parameter
mousea
rata
dogb
monkeyb
po dose (mg kg-1
)
120c
25c
18
10d
2.5d
0.6
0.7
2.9
2.8
2.8
41.6
5.7
40
25d
1.5d
1.2
5d
iv dose (mg kg-1
Cmax (μM), po
Tmax (h), po
)
5d
0.5
1.5
3.6
5.7
6.4
10.5
4.0
31
1.0
147
2.7
3.7
21.3
4.7
83
2.3
AUCe (μM.h), po
plasma t1/2 (h), po
MRT (h), iv
14.0
7.2
6.9
Cl, (mL min-1 kg-1), iv
24.8
10.1
49
V
ss (L kg-1), iv
po (%)
F
a Data reported as an average of three animals. b Data reported as an
average of two animals. c Vehicle: PG/ Tween80/ water (40:10:50).
d Vehicle: PEG400/Tween80/water (50:0.1:50). e AUC(0-24h)
.
a Reagents and conditions: (a) (1) POCl3, i-Pr2NEt, toluene, 110 °C;
(2) K2HPO4, H2O; (3) 15, i-Pr2NEt, 70-80 °C; (4) i-PrOH, 55-22 °C,
84% overall; (b) (1) aq NaOH (50%), THF-MeOH (8:3), 60 °C;
(2) conc HCl, THF, room temperature, 88% overall; (c) 17, diphenyl-
phosphoryl azide, Et3N, toluene, 87 °C, 82%; (d) conc HCl, MeOH-
H2O (1:7), 77%.
Table 3. In Vivo Antitumor Activity of 13 against L2987, GEO, KPL4,
and N87 Tumor Xenografts
dosea
(mg kg-1
TGIb
(%)
tumor model
L2987
)
schedule
P c
30
60
q.d. ꢀ 14
q.d. ꢀ 14
q.d. ꢀ 14
q.d. ꢀ 14
q.d. ꢀ 14
q.d. ꢀ 14
q.d. ꢀ 21
q.d. ꢀ 21
q.d. ꢀ 21
q.d. ꢀ 21
q.d. ꢀ 21
,50
58
0.1480
0.0001
0.0001
0.2043
0.0001
0.0001
0.0001
0.0019
0.2043
0.0003
0.0005
exhibit significant off-target effects as reflected in its activity
on the control A2780 tumor cells. The 4 h plasma levels of 13
in the oral exposure study were more than 100-fold higher
than its IC50 for inhibition of HER2/HER1 kinases. Com-
pounds 11-13 possess S-configuration at the asymmetric
center on the morpholine. The corresponding R-enantiomers
exhibited reduced potency in the cellular assays. Despite its
initial promise, further evaluation of 12 was discontinued
because of its low bioavailability in higher species (data not
shown). On the basis of its promising in vitro potency and
pharmacokinetic profile, 13 was selected for further charac-
terization in selectivity, safety, PK, and in vivo efficacy
studies.
Compound 13 was synthesized according to the sequence
illustrated in Scheme 1.10 Thus, treatment of intermediate 146
with phosphorus oxychloride in the presence of diisopropyl-
ethylamine afforded the corresponding chloroimidate that
was condensed with aminoindazole 156 to give carboxylic acid
16 in 74% overall yield after base-mediated hydrolysis. Cur-
tius rearrangement was accomplished by heating with diphe-
nylphosphoryl azide, and the resulting isocyanate intermediate
was trapped with enantiopure alcohol 17.11 Deprotection of
the Boc group under acidic conditions and subsequent crystal-
lization from ethyl acetate afforded 13 in 63% yield from
intermediate 16.
The pharmacokinetic parameters obtained for 13 in four
species are summarized in Table 2. The compound has high
intrinsic permeability in Caco-2 cells (214 nm/s) and was well
absorbed following oral administration from solution formu-
lations at the doses denoted in Table 2. Favorable half-lives
(t1/2) and mean residence times (MRT) were observed across
species with this compound. Systemic clearance12 was mode-
rate to low compared to the hepatic blood flow for the
respective species, and volume of distribution (Vss) was larger
than total body water, indicating extensive extravascular
distribution. The measured oral bioavailability (Fpo) ranged
from 31% in cynomolgus monkey to 83% in mice.
In addition to potent inhibition of HER1 and HER2, 13
weakly inhibited the related receptor HER4 with an IC50 of
190 nM and exhibited greater than 100-fold selectivity against
a panel of diverse protein kinases.7b In N87, Sal2, and GEO
tumor cells with activated HER2 and/or HER1 receptors, 13
180
60
99
39
GEO
120
180
60
95
88
KPL4
N87
61
180
60
129
39
120
180
81
71
a Vehicle: propylene glycol/Tween80/water (40:10:50). b Percent tumor
growth inhibition (TGI) during treatment. c Probability of mean tumor
weight at the end of drug treatment.
elicited dose-dependent inhibition of receptor phosphoryla-
tion and downstream signaling through Akt and MAPK
pathways. The potency of this inhibition was consistent with
the potency observed for inhibition of cell proliferation.
Furthermore, 1 μM 13 inhibited epidermal growth factor
dependent HER1/HER2 heterodimer formation in AU565
breast cancer cells.7b
Results for the in vivo activity of 13 in L2987 human lung,
GEO human colon, KPL4 human breast, and N87 human
gastric carcinoma xenograft models are summarized in Table 3.
In the L2987 tumors that are dependent on HER1 signaling,
once daily oral administration of 13 resulted in a dose-
dependent inhibition of tumor growth (Figure 1).13 At
60 mg/kg, the compound achieved a significant delay in tumor
growth during the course of treatment. Nearly complete
tumor stasis was observed throughout the dosing regimen at
the 180 mg/kg dose level, but tumor growth resumed follow-
ing the cessation of treatment. 13 exhibited similar antitumor
activity in HER1-overexpressing GEO tumors and HER2-
amplified xenograft models, including KPL4 breast and N87
gastric tumors. No overt toxicity as measured by significant
weight loss or morbidity was observed at any of the dose levels
tested in these experiments.
In conclusion, optimization of the in vitro potency and
pharmacokinetic profile of a series of substituted 4-[1H-
indazol-5-ylamino]-pyrrolo[2,1-f][1,2,4]triazine-6-carbamates
led to the identification of 13, a selective dual inhibitor of
HER1 and HER2 kinases, which was orally efficacious in
human HER1 (GEO, L2987) or HER2 (KPL4, N87) depen-
dent tumor xenograft models at multiple dose levels. On the

6530 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Gavai et al.
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(7) (a) Presented in part at the “First Time Disclosure of Clinical
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Chemical Society, San Diego, CA, March 2005; MEDI 21. (b) Wong,
T. W.; Lee, F. Y.; Yu, C.; Luo, F. R.; Oppenheimer, S.; Zhang, H.;
Smykla, R. A.; Mastalerz, H.; Fink, B. E.; Hunt, J. T.; Gavai, A. V.; Vite,
G. D. Preclinical antitumor activity of BMS-599626, a pan-HER kinase
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(8) The rates of metabolism of 2, 3, 5, 6, 11, 12, and 13 in mouse liver
microsomes were 0.27, 0.10, 0.04, 0.02, 0.14, 0.01, and 0.02 nmol
min-1 mg protein-1, respectively, after a 10 min incubation at 10 μM.
(9) Smith, D. A.; Jones, C. J.; Walker, D. K. Design of drugs involving
the concepts and theories of drug metabolism and pharmaco-
kinetics. Med. Res. Rev. 1996, 16, 243–266.
(10) Swaminathan, S.; Gavai, A. V.; Fan, J.; Patel, B. P.; Norris, D. J.;
Corbett, R. M.; Zheng, B. WO Patent Application 058245 A2,
2005.
Figure 1. In vivo efficacy of 13 (q.d. ꢀ 14, po) versus L2987 human
tumor xenografts in athymic mice.
basis of its favorable in vitro pharmacology, broad spectrum
in vivo efficacy in multiple tumor models, and a satisfactory
pharmacokinetic profile, 13 was advanced into clinical trials.
(11) Brown, G. R.; Foubister, A. J.; Wright, B. Chiral synthesis of
3-substituted morpholines via serine enantiomers and reductions of
5-oxomorpholine-3-carboxylates. J. Chem. Soc., Perkin Trans. 1
1985, 2577–2580.
Acknowledgment. We thank Bristol-Myers Squibb Dis-
covery Analytical Sciences Department for compound char-
acterization efforts.
(12) Observed systemic clearance of 13 correlated with in vitro clearance
in hepatocytes. The predicted hepatocyte clearance values in
mouse, rat, monkey, dog, and human were 62.6, 35.6, 18.3, 17.9,
and 6.3 mL/min/kg, respectively.
Supporting Information Available: Full experimental proce-
dures and characterization data for 13; characterization data for
1-12. This material is available free of charge via the Internet at
http://pubs.acs.org.
(13) Tumors were implanted subcutaneously in athymic mice and
staged to approximately 100-150 mm3 prior to the initiation of
drug treatment. The compound was evaluated on a once daily (q.d.)
oral dosing regimen for 14 consecutive days. An active result in
these studies is defined as >50% TGI over at least one tumor
volume doubling time. For additional experimental details, see the
following: Borzilleri, R. M.; Zheng, X.; Qian, L.; Ellis, C.; Cai,
Z.-w.; Wautlet, B. S.; Mortillo, S.; Jeyaseelan, R., Sr.; Kukral,
D. W.; Fura, A.; Kamath, A.; Vyas, V.; Tokarski, J. S.; Barrish,
J. C.; Hunt, J. T.; Lombardo, L. J.; Fargnoli, J.; Bhide, R. S.
Design, synthesis, and evaluation of orally active 4-(2,4-dofluoro-
5-(methoxycarbamoyl)phenylamino)pyrrolo[2,1-f][1,2,4]triazines
as dual vascular endothelial growth factor receptor-2 and fibro-
blast growth factor receptor-1 inhibitors. J. Med. Chem. 2005, 48,
3991–4008.
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