Discovery and preclinical studies of (R)-1-(4-(4-fluoro-2-methyl-1H-indol- 5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-ol (BMS-540215), an in vivo active potent VEGFR-2 inhibitor

Article abstract of DOI:10.1021/jm051106d

A series of substituted 4-(4-fluoro-1H-indol-5-yloxy)pyrrolo-[2.1-f][1,2,4] triazine-based inhibitors of vascular endothelial growth factor receptor-2 kinase is reported. Structure-activity relationship studies revealed that a methyl group at the 5-position and a substituted alkoxy group at the 6-position of the pyrrolo[2,1-f][1,2,4]triazine core gave potent compounds. Biochemical potency, kinase selectivity, and pharmacokinetics of the series were optimized and in vitro safety liabilities were minimized to afford BMS-540215 (12), which demonstrated robust preclinical in vivo activity in human tumor xenograft models. The L-alanine prodrug of 12, BMS-582664 (21), is currently under evaluation in clinical trials for the treatment of solid tumors.

Full text of DOI:10.1021/jm051106d

© Copyright 2006 by the American Chemical Society
Volume 49, Number 7
April 6, 2006
Letters
Table 1. SAR at the R5, R2′, and R4′ Positions of
4-(1H-Indol-5-yloxy)pyrrolo[2,1-f][1,2,4]triazinea
Discovery and Preclinical Studies of
(R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-
methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-
2-ol (BMS-540215), an In Vivo Active Potent
VEGFR-2 Inhibitor
Rajeev S. Bhide,* Zhen-Wei Cai, Yong-Zheng Zhang,
Ligang Qian, Donna Wei, Stephanie Barbosa,
compd
R1
R2′
R4′
R5
VEGFR-2, IC50, µM
1
2
3
4
5
6
Me
Et
Et
Et
Me
Me
H
H
H
H
Me
Me
H
H
H
H
H
F
H
Me
Et
i-Pr
Me
Me
0.21b
0.087
0.31
0.47
0.078
0.017
Louis J. Lombardo, Robert M. Borzilleri, Xiaoping Zheng,
Laurence I. Wu, Joel C. Barrish, Soong-Hoon Kim,
Kenneth Leavitt, Arvind Mathur, Leslie Leith, Sam Chao,
Barri Wautlet, Steven Mortillo, Robert Jeyaseelan Sr.,
Daniel Kukral, John T. Hunt, Amrita Kamath, Aberra Fura,
Viral Vyas, Punit Marathe, Celia D’Arienzo,
a IC50 values are reported as the mean of at least three individual
determinations. Variability around the mean value was <50%. b value
reported from one experiment.
George Derbin, and Joseph Fargnoli
Bristol-Myers Squibb, Pharmaceutical Research Institute,
Princeton, New Jersey 08543-4000
tion, and survival.4 As a result, VEGFR-2 has become a
compelling target for small molecule kinase inhibitors.5 Re-
cently, clinical validation of the VEGF signaling pathway has
been provided by the demonstration of an overall survival benefit
in colorectal cancer patients treated with bevacizumab, a
monoclonal antibody to the ligand VEGF-A, in combination
with standard regimens (IFL or Saltz).6 Many small molecule
inhibitors of the VEGFR-2 kinase domain have shown promising
results in preclinical studies and are currently under clinical
evaluation.7 Herein, we describe the identification of a selective
inhibitor of VEGFR-2 kinase with robust preclinical in vivo
activity and a safety profile that is suitable for chronic dosing.
Recently, we reported that phenol-substituted pyrrolo[2,1-f]-
[1,2,4]triazines afforded potent inhibitors of VEGFR-2 kinase
but lacked good oral bioavailability.8 Our search for compounds
with better pharmacokinetic properties led to the discovery of
4-(4-fluoro-1H-indol-5-yloxy)pyrrolo[2,1-f][1,2,4]triazines shown
in Table 1. This report describes the synthesis, structure-activity
relationships, and antitumor activity of this indole-based series
of compounds. Initial structure-activity relationship studies
(SAR) in the series were investigated using analogues with an
ester group at the 6-position. Similar to earlier observations,8
incorporation of a methyl group at the 5-position (R5), as in
compound 2, gave optimal potency against the VEGFR-2
enzyme. Although the introduction of a methyl group at the
ReceiVed NoVember 2, 2005
Abstract: A series of substituted 4-(4-fluoro-1H-indol-5-yloxy)pyrrolo-
[2,1-f][1,2,4]triazine-based inhibitors of vascular endothelial growth
factor receptor-2 kinase is reported. Structure-activity relationship
studies revealed that a methyl group at the 5-position and a substituted
alkoxy group at the 6-position of the pyrrolo[2,1-f][1,2,4]triazine core
gave potent compounds. Biochemical potency, kinase selectivity, and
pharmacokinetics of the series were optimized and in vitro safety
liabilities were minimized to afford BMS-540215 (12), which demon-
strated robust preclinical in vivo activity in human tumor xenograft
models. The L-alanine prodrug of 12, BMS-582664 (21), is currently
under evaluation in clinical trials for the treatment of solid tumors.
Inhibition of tumor angiogenesis as a means of tumor growth
arrest has been an area of intense research interest over the past
decade.1 Vascular endothelial growth factor (VEGF) isoforms
and their cognate tyrosine kinase receptors (VEGFRs) have been
especially attractive targets for the inhibition of angiogenesis.2,3
VEGFR-2 is of particular interest because it is the principal
kinase involved in multiple processes of angiogenesis, including
vascular permeability, endothelial cell (EC) proliferation, migra-
* To whom the correspondence should be addressed. Phone: 609-252-
4395, Fax: 609-252-3993. E-mail: rajeev.bhide@bms.com
10.1021/jm051106d CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/16/2006

2144 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Table 2. SAR at R6 Linker
Letters
a IC50 values are reported as the mean of at least three individual determinations or as individual IC50 values in the case of less than three measurements.
Variability around the mean value was <50%. For assay conditions see ref 9. b Compounds were evaluated in 4 h exposure study in mice at 50 mg/kg and
formulated as solutions in 7:1:2 poly(ethylene glycol)400:ethanol:water. c For CYP inhibition assay details see ref 9. d ND, data not determined; e See
Supporting Information.
2-position of the indole ring (R2′) did not affect the potency
against VEGFR-2 (2, 5), introduction of a fluorine group at the
4-position of indole ring (R4′) gave a 4-fold increase in potency
against VEGFR-2 (6).
activity in hERG patch-clamp and CYP3A4 liability assays.
Compounds with an amino group on the side chain (8, 10)
exhibited potent hERG ion channel activity and CYP3A4
inhibition, which was reduced in the 3-hydroxypyrrolidine
derivative 11. Compounds without an amino group on the side
chain (12, 14, and 15) possessed reduced potency against the
hERG ion channel and a range of activity against a panel of
cytochrome P450 (CYP) isozymes.11 Among the compounds
tested, 11, 12, and 15 showed reduced inhibition of CYP3A4
activity compared to compounds 13 and 14. On the basis of its
superior enzyme potency against VEGFR-2, acceptable plasma
protein binding (human 98.1%), and good exposure profile in
the mouse, compound 12 was chosen for additional kinase
selectivity, pharmacokinetic, and in vivo efficacy studies.
The reaction sequence for the preparation of 12 is detailed
in Scheme 1. The 6-position phenol 178 was first protected as
a benzyl ether by treatment with benzyl bromide and K2CO3 in
acetonitrile. The resulting intermediate was heated in aqueous
HCl at 80 °C to remove the phenyl ether protecting group at
the 4-position to obtain 18 in 64% yield over two steps. The
4-oxo group of 18 was converted to chloroimidate 19 by
treatment with phosphorus oxychloride at 110 °C. Reaction of
4-fluoro-5-hydroxy-2-methylindole10 20 with crude 19 in DMF
in the presence of NaH and hydrogenolysis of the resulting
6-benzylated intermediate with ammonium formate and 10%
palladium on carbon afforded the key compound 7 in 76%
overall yield. Treatment of 7 with (R)-(+)-propylene oxide in
ethanol at 70 °C in the presence of triethylamine afforded 12
with high optical purity (99.5% ee).
On the basis of these data, we chose to continue our SAR
studies using analogues with methyl groups at R5 and R2′ and
with fluorine at R4′. It was observed that the compounds with
a 6-hydroxy substituent showed good enzyme potency. There-
fore, our subsequent SAR studies were conducted using
analogues with a 6-position ether. The ester at the 6-position
was converted to a hydroxyl group (7) via a reduction, oxidation,
and Baeyer-Villiger rearrangement sequence.8 Although phenol
7 was a potent kinase inhibitor, it showed poor plasma exposure
upon oral administration. Therefore, the phenol was alkylated
with different groups to afford the series of 6-position ethers
described in Table 2. Molecular modeling suggested that these
compounds would bind in the ATP binding site8,9 and that the
linkers at the 6-position would be solvent exposed. Consistent
with this hypothesis, substitution at R6 affected kinase activity
only marginally, but allowed modulation of aqueous solubility,
pharmacokinetic properties, and the in vitro safety profile.
Compounds containing an amine, alcohol, or ether group on
the R6 tether (Table 2) were potent against the VEGFR-2
enzyme, whereas analogues containing a sulfone (15) or a
sulfonamide (16) exhibited moderate potency. In 4 h oral
exposure studies in mice, amines generally had modest in vivo
exposures (8-11), whereas compounds with an alcohol, sulfone,
or sulfonamide group (12 to 16) showed high oral exposures.
Compounds in Table 2 were further differentiated by their

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2145
Scheme 1. Synthesis of 12a
a Reagents and reaction conditions: (a) benzyl bromide, K2CO3, 70 °C;
(b) 1 N HCl, 80 °C, 64%, two steps; (c) POCl3, 110 °C; (d) DMF, NaH,
76%, two steps; (e) 10% Pd/C, HCOONH4, DMF, 86%; (f) (R)-(+)-
propylene oxide, Et3N, EtOH, 70 °C, 53%.
Figure 1. In vivo antitumor activity of 12 versus H3396 xenografts
in athymic mice with dosing once a day for 10 days from day 14 to
day 23 postimplant.
Table 5. Rat 24 Hour PK Study of 12a
Table 3. Kinase Selectivity Profile of 12
solution
25
micronized suspension
100 200
enzyme
IC50 (nM)
Ki (nM)
dose (mg/kg)
25
VEGFR-2 (human)
Flk-1 (mouse)
VEGFR-1
FGFR-1
PDGFR-â
EGFR
25
89
380
26
28
60
Cmax (µM)
6.4
13.4
0.55
3.35
2.05
8.43
1.71
7.61
AUCtot (µM‚h)
148
a Vehicle for solution study was PEG400:ethanol:water (7:1:2) and for
suspension study was water with 0.1% tween 80.
>6000
>1900
>2500
>25000
>50000
LCK
PKCR
JAK-3
water (3:2). The compound was rapidly absorbed with Tmax of
1 h, a favorable half-life (t1/2) of 2.7 h, and mean residence
time (MRT) of 3.6 h. The measured oral bioavailability (Fpo)
in this experiment was 88%. The steady-state volume of
distribution (Vss) was high, indicating extensive extravascular
distribution, and systemic clearance (Cl) was much lower than
the hepatic blood flow.
Table 4. Parameters for12 in a 24 Hour PK Study in Micea
dose,
mg/kg
AUCtot
µM‚h
,
t1/2
h
,
MRT,
h
Cl,
Vss,
L/kg
Fpo,
%
mL/min/kg
iv, 10
po, 60
49
224
2.7
3.6
3.6
9.3
2.0
88
The in vivo efficacy of 12 was evaluated against H3396
xenografts in athymic mice (Figure 1). Once daily oral
administration of 12 as a solution in PEG 400:water (7:3) for
10 days (days 14 to 23 post implant) inhibited the growth of
established tumors (staged to 80-120 mm3 size) in a dose
dependent manner. At 60 and 90 mg/kg doses, nearly complete
inhibition of growth was observed throughout the dosing period,
with tumor growth inhibition11 of 85% and 97%, respectively.
No overt toxicity as measured by weight loss and morbidity
was observed in either cohort of animals, suggesting good safety.
Solid dosage formulations of compound 12 were assessed in
the rat to support advancement of this compound as a develop-
ment candidate. Rats were dosed with micronized 12 as a
suspension in water to mimic the solid dosage formulation which
would be used in the clinic. At 25 mg/kg, micronized suspen-
sions afforded significantly lower systemic exposures than those
obtained from solution formulations (Table 5). Additionally,
drug exposure did not increase proportionally with dose,
suggesting that nonlinear pharmacokinetics in humans could
present a development issue.
Low aqueous solubility at equilibrium (<1 µg/mL at pH 6.5)
and low pKa (2.8) were believed to be contributing to the
observed poor absorption from solid dosage form. Prodrug
formation was recognized as an effective means to improve the
pharmaceutic and pharmacokinetic properties of this otherwise
attractive development candidate. The successful outcome of
this strategy will be reported on shortly in a subsequent
manuscript, which will describe the prodrug 21 (Figure 2) which
is currently in clinical development.
In summary, a novel series of 4-(4-fluoro-1H-indol-5-yloxy)-
pyrrolo[2,1-f][1,2,4]triazines with potent VEGFR-2 activity was
identified. Structure-activity relationship studies led to the
discovery of (R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-
methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-ol (12) as
a Data taken from an average of three animals. For assay details see
ref 9.
The enzyme kinetics and kinase selectivity profile of 12 were
examined (Table 3). Compound 12 was found to be an ATP
competitive inhibitor of human VEGFR-2, with a Ki of 26 nM.
It had moderate potency against two of three other kinases
implicated in angiogenesis, VEGFR-1 and FGFR-1, but it
exhibited good selectivity against PDGFR-â.11 Since compound
12 targets the mouse endothelial cells rather than the human
tumor cells in the preclinical in vivo mouse models, it was also
tested and found to be good inhibitor of Flk-1, the mouse
homologue of VEGFR-2. Compound 12 showed good selectivity
against a variety of other receptor and nonreceptor kinases as
shown in Table 3. It should be noted that AZD21717f which
also contains fluoroindole group 20 on a quinazoline nucleus,
exhibits potent activity against PDGFR-â and c-Kit kinases.
Inhibition of proliferation of human umbilical vein endothelial
cells (HUVEC) by 12 was investigated. Following stimulation
by growth factors VEGF-A or FGF, the inhibition of prolifera-
tion of HUVEC cells was measured. A good correlation was
observed between cellular potency and activity against the
isolated enzymes with potency against VEGF-stimulated HU-
VECs (40 nM) being higher than against FGF-stimulated
HUVECs (276 nM).11 When tested against a panel of human
tumor cell lines, 12 showed lower antiproliferative potency (IC50
> 2 µM). In particular, activity was low against the cell line
used in the in vivo tumor xenograft mouse efficacy model
(human breast carcinoma H3396), supporting the concept that
the efficacy of the compound is driven by its antiangiogenic
properties.
In a mouse (male Balb-c) pharmacokinetic study11 (Table 4)
12 was administered at 60 mg/kg orally as a solution in PEG400:
Tween80 (75:25) and at 10 mg/kg intravenously in PEG400:

2146 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Letters
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with C-7 basic side chains: design and structure activity relationship
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Jani, J. P.; Knauth, E.; Goodwin, P.; Higdon, C.; Rossi, A. M.;
Emerson, E.; Finkelstein, M.; Floyd, E.; Harriman, S.; Atherton, J.;
Hillerman, S.; Soderstrom, C.; Kou, K.; Gant, T.; Noe, M. C.; Foster,
B.; Rastinejad, F.; Marx, M. A.; Schaeffer, T.; Whalen, P. M.;
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Robinson, C.; Ruggeri, B. A.; Dionne, C. A.; Vaught, J. L.; Mallamo,
J. P.; Hudkins, R. L. A new class of potent vascular endothelial
growth factor receptor tyrosine kinase inhibitors: structure-activity
relationships for a series of 9-alkoxymethyl-12-(3-hydroxypropyl)-
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CEP-5214 and its dimethylglycine ester prodrug clinical candidate
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Kendrew, J.; Hennequin, L. F.; Valentine, P. J.; Barry, S. T.; Brave,
S. R.; Smith, N. R.; James, N. H.; Dukes, M.; Curwen, J. O.; Chester,
R.; Jackson, J. A.; Boffey, S. J.; Kilburn, L. L.; Barnett, S.; Richmond,
G. H. P.; Wadsworth, P. F.; Walker, M.; Bigley, A. L.; Taylor, S.
T.; Cooper, L.; Beck, S.; Jurgensmeier, J. M.; Ogilvie, D. J.
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Figure 2. Structure of 21.
an orally active compound in the H3396 xenograft model of
human breast carcinoma, demonstrating almost complete tumor
growth stasis during the dosing period. On the basis of its
excellent enzymatic potency against VEGFR-2, good kinase
selectivity profile, and acceptable safety profile, compound 12
was considered as a candidate for further development and its
prodrug formation was considered to improve pharmaceutics
properties.
Acknowledgment. We thank George Trainor and Robert
Kramer for scientific advice, and the Discovery analytical
sciences for compound characterization.
Supporting Information Available: Characterization data for
compounds 1-16. Full experimental details and the characterization
data for compound 12. This material is available free of charge on
the web at http://pubs.acs.org.
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JM051106D