Discovery and evaluation of N-cyclopropyl-2,4-difluoro-5-((2-(pyridin-2- ylamino)thiazol-5-ylmethyl)amino)benzamide (BMS-605541), a selective and orally efficacious inhibitor of vascular endothelial growth factor receptor-2

Article abstract of DOI:10.1021/jm060347y

Substituted 3-((2-(pyridin-2-ylamino)thiazol-5-ylmethyl)-amino)benzamides were identified as potent and selective inhibitors of vascular endothelial growth factor receptor-2 (VEGFR-2) kinase activity. The enzyme kinetics associated with the VEGFR-2 inhibi

Full text of DOI:10.1021/jm060347y

3766
J. Med. Chem. 2006, 49, 3766-3769
Squibb compound collection. In general, these compounds
displayed weak to moderate inhibition of VEGFR-2 kinase
activity. For example, N-(5-((5-tert-butyloxazol-2-yl)methyl-
thio)thiazol-2-yl)-3,4-dimethoxybenzamide (2), which demon-
strated an IC50 of 50 nM against CDK2, was subsequently found
to be a 1900 nM inhibitor of VEGFR-2.
Discovery and Evaluation of N-Cyclopropyl-
2,4-difluoro-5-((2-(pyridin-2-ylamino)thiazol-5-
ylmethyl)amino)benzamide (BMS-605541), a
Selective and Orally Efficacious Inhibitor of
Vascular Endothelial Growth Factor Receptor-2
Robert M. Borzilleri,* Rajeev S. Bhide, Joel C. Barrish,
Celia J. D’Arienzo, George M. Derbin, Joseph Fargnoli,
John T. Hunt, Robert Jeyaseelan, Sr., Amrita Kamath,
Daniel W. Kukral, Punit Marathe, Steve Mortillo,
Ligang Qian, John S. Tokarski, Barri S. Wautlet,
Xiaoping Zheng, and Louis J. Lombardo
Bristol-Myers Squibb Pharmaceutical Research Institute,
P.O. Box 4000, Princeton, New Jersey 08543-4000
ReceiVed March 24, 2006
Abstract: Substituted 3-((2-(pyridin-2-ylamino)thiazol-5-ylmethyl)-
amino)benzamides were identified as potent and selective inhibitors
of vascular endothelial growth factor receptor-2 (VEGFR-2) kinase
activity. The enzyme kinetics associated with the VEGFR-2 inhibition
of 14 (Ki ) 49 ( 9 nM) confirmed that the aminothiazole-based
analogues are competitive with ATP. Analogue 14 demonstrated
excellent kinase selectivity, favorable pharmacokinetic properties in
multiple species, and robust in vivo efficacy in human lung and colon
carcinoma xenograft models.
A comparison of the relative binding modes of 1 and 2 in
the VEGFR-2 ATP-binding site is provided in Figure 1.7b,10 In
this model, the pyrrolo[2,1-f][1,2,4]triazine and aminothiazole
cores are anchored to the hinge region of the adenine binding
pocket via a highly conserved hydrogen-bond interaction with
the backbone amide-NH of Cys919. The hydroxamate of 1 is
believed to be engaged in hydrogen-bond interactions with
Asp1046 and Glu885. The overlay of 1 and 2 in this model
suggested that attachment of the N-methoxybenzamide onto the
aminothiazole template may lead to improved potency relative
to the initial lead 2. Herein, we describe the optimization and
preclinical assessment of these selective aminothiazole-based
VEGFR-2 inhibitors.
To evaluate the aforementioned hypothesis, N-acyl-2-
aminothiazole and the N-2-pyridinylaminothiazole moiety used
to prepare potent pan-Src kinase inhibitors11 and structurally
related VEGFR-2 inhibitors12 were appended via a thiomethyl
linker to the N-methoxybenzamide. As depicted in Table 1, the
initial N-2-pyridinylaminothiazole analogue 3 displayed potent
inhibition of VEGFR-2 kinase activity and VEGF-stimulated
human umbilical vein endothelial cell (HUVEC) proliferation.
Contrary to the SAR observed in the pyrrolo[2,1-f][1,2,4]triazine
series,7b the simple N-methylamide analogue 4 retained activity
in the biochemical assay. Because of the potential metabolic
instability associated with the thiomethyl group, variations in
the linker region (X-Y) were pursued. The methylamine
analogue 5 was approximately 3-fold less potent than 4 in the
VEGFR-2 assay. The ethyl- and n-propylbenzamide analogues
provided similar biochemical potency relative to 5 (data not
shown). Consistent with earlier observations,7b incorporation of
a fluorine atom at R1 of the benzamide ring (i.e., 6) provided a
10-fold increase in intrinsic activity relative to 5. Compound 6
also demonstrated significant antiproliferative activity in the
VEGF-driven HUVEC assay. Since the 5-tert-butyloxazolyl-
methylthio moiety was previously shown to be critical for potent
CDK2 inhibition in the aminothiazole series,9 we evaluated and
confirmed the selectivity of the current series for VEGFR-2
versus CDK2 (compound 6, IC50 ) 5100 nM). Replacement of
the methyl group of amide 6 with a cyclopropyl substituent was
well tolerated with the amine (Y ) NH) and ethyl (Y ) CH2)
linkers (i.e., 7 and 8, respectively). Compound 8 was found to
be the most potent analogue in the biochemical and cellular
Tumor-induced angiogenesis, or the formation of new capil-
lary networks between neoplastic cells and endothelial cells of
the host,1 is required for solid tumor growth and metastases.2
VEGF,a a key stimulator of tumor angiogenesis, promotes
endothelial cell proliferation, survival, migration, invasion, and
differentiation.3 The intrinsic activity responsible for these
cellular events is dependent on the highly specific binding of
the VEGF ligands to their respective cell surface expressed
tyrosine kinase receptors VEGFR-1 (Flt-1), VEGFR-2 (KDR,
Flk-1), and VEGFR-3 (Flt-4).4 For more than a decade, the
VEGF signaling pathway has been the target of extensive drug
discovery research aimed at developing therapies that control
the abnormal vascular development operative in cancer progres-
sion.5 To this end, the chimeric anti-VEGF monoclonal antibody
bevacizumab (Genentech) was recently approved to treat patients
with metastatic colorectal cancer when combined with standard
chemotherapy (IFL/Saltz regimen).6 In addition to biological-
based antiangiogenesis approaches, several small-molecule
inhibitors of VEGFR-2 kinase activity have progressed to
various stages of preclinical and clinical development.5
We have recently disclosed the structure-activity relation-
ships (SARs) of various ATP-competitive VEGFR-2 inhibitors
assembled from the pyrrolo[2,1-f][1,2,4]triazine scaffold.7 Four-
point pharmacophore8a and atom pairs8b similarity searches
conducted with the potent N-methoxybenzamide-substituted
pyrrolo[2,1-f][1,2,4]triazine inhibitor 1 (VEGFR-2 IC50 ) 18
nM)7b identified several N-acyl-2-aminothiazole-based cyclin-
dependent kinase (CDK2) inhibitors9 from the Bristol-Myers
* To whom correspondence should be addressed. Phone: 609-252-6236.
Fax: 609-252-7410. E-mail: robert.borzilleri@bms.com.
a Abbreviations: VEGF, vascular endothelial growth factor; IFL, irino-
tecan-5-fluorouracil-leucovorin; HUVEC, human umbilical vein endothelial
cell; PK, pharmacokinetics; TFA, trifluoroacetic acid; AUC, area under the
curve; Vss, steady-state volume of distribution; Cl, clearance; MRT, mean
residence time; TGI, tumor growth inhibition; hERG, human ether-a-go-
go-related gene; HEK, human embryonic kidney cells.
10.1021/jm060347y CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/26/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3767
Scheme 1a
a Reagents and conditions: (a) 2-bromo-3-hydroxypropenal, NaOAc,
acetone, reflux, 2 h, 66%; (b) (1) thionyl chloride, reflux, 3 h; (2) CH2Cl2,
cyclopropylamine, Et3N, -40 °C to room temperature, 80% overall; (c)
H2, 10% Pd/C, EtOAc-EtOH (1:1), 2 h, 97%; (d) Et3SiH, CH2Cl2-TFA
(1:1), 1 h, 74%.
Figure 1. Proposed binding model of 1 and 2 in the ATP-binding site
of VEGFR-2 kinase.
Table 2. Kinase Selectivity Profile of 14
Table 1. Enzymatic and Cellular Activitiesa of
N-2-Pyridinylaminothiazole Analogues
kinase
enzyme IC50, nM
kinase
enzyme IC50, nM
VEGFR-2
Flk-1
VEGFR-1
PDGFR-â
c-kit
FGFR-1
GSK-3
MEK
AKT
p38
Src
CDK2
23
40
400
200
370
IKK
>10000
>10000
>25000
>40000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
SRPK1
IGF-1R
PKC R, δ, τ, ú
Her1
Her2
Lck
Itk
Syk
JAK-3
CaMKII
MAPKAP K2
IC50, nM
>5000
>5000
>5000
>5000
>5000
>10000
>10000
growth
inhibition
VEGFR-2 of HUVECsc
kinase
driven by
compd
X
Y
R1 R2
R3
inhibitionb
VEGFd
3
4
5
6
7
S
S
CH2
CH2
H
H
H
F
F
F
F
F
F
F
F
F
H
H
H
H
H
H
H
H
H
H
F
OCH3
CH3
CH3
74e
88e
280e
30
29
14
110
17
64
85
71
110
NDf
NDf
20
CH2 NH
CH2 NH
CH2 NH
CH2 CH2
CH2 NH
CH2 NH
CH2 NH
CH2 NH
CH2 NH
CH2 NH
CH3
Compound 14 was prepared in a straightforward manner
according to the synthetic sequence outlined in Scheme 1. Thus,
condensation of thiourea 1515 with 2-bromo-3-hydroxyprope-
nal16 in the presence of sodium acetate in refluxing acetone
provided key aldehyde intermediate 16. 5-Amino-N-cyclopropyl-
2,4-difluorobenzamide (18) was obtained from 2,4-difluoro-5-
nitrobenzoic acid 1717 using a two-step reaction sequence
involving cyclopropylamide bond formation and subsequent
reduction of the nitro group. The reductive amination of
aldehyde 16 with 18 was accomplished with triethylsilane to
afford analogue 14 in 74% yield.
To better understand the enzyme inhibitory properties of 14,
the compound was evaluated in an in-house kinase selectivity
panel and the Ki was determined for VEGFR-2 kinase. Analogue
14 was confirmed to be an ATP-competitive inhibitor of
VEGFR-2, with a Ki of 49 ( 9 nM. As illustrated in Table 2,
14 was found to be a potent inhibitor of the mouse homologue
of VEGFR-2 (Flk-1) and demonstrated moderate activity against
VEGFR-1, PDGFR-â, and c-kit. Greater than 200-fold selectiv-
ity was observed for all the other receptor and nonreceptor
kinase targets found in the panel.
The pharmacokinetic parameters obtained for 14 in mouse,
rat, and cynomolgus (cyno) monkey are summarized in Table
3. In each case, 14 demonstrated a moderate steady-state volume
of distribution (Vss) and low systemic clearance (Cl) compared
to the hepatic blood flow for the respective species. The
compound was well absorbed following oral administration of
drug from solution formulations at the doses denoted in Table
3. Favorable half-lifes (t1/2) and mean residence times (MRT)
were observed across species with 14, albeit these values were
slightly higher in cyno. The measured oral bioavailabilities (Fpo)
were 100% for mouse and rat and 52% in monkey.
cyclopropyl
cyclopropyl
cyclopentyl
benzyl
CH2(2-pyridyl)
(CH2)2-N-morpholine
CH3
14
5
8
9
NDf
120
53
10
11
12
13
14
NDf
52
F
cyclopropyl
23
25
a See ref 7b for a description of the assay conditions. b IC50 values are
reported as the mean of at least three independent determinations. Variability
around the mean value was <50%. c Human umbilical vein endothelial cells.
d All compounds evaluated in the HUVEC assay demonstrated an IC50
>
2500 nM in the unstimulated growth of L2987 human lung carcinoma cells.
e Single determination. f ND ) not determined.
proliferation assays. However, 8 exhibited poor in vitro meta-
bolic stability13a and provided relatively low systemic exposures
in mouse 4 h oral PK studies.13b Additional amide analogues,
such as the cyclopentyl derivative 9 and 11-12, which contain
terminal polar groups, were 2- to 4-fold less potent than 7 in
the VEGFR-2 kinase assay. The benzylamide 10 demonstrated
significant enzymatic activity but was nearly one order of
magnitude less potent than 7 in the HUVEC proliferation assay.
Additional profiling revealed that the 4-fluorobenzamide
derivative 7 possessed low chemical stability under aqueous
acidic conditions.14a Thus, a second fluorine atom was intro-
duced on the ring at R2 in an effort to increase the electron-
deficient nature of the benzamide ring. It was postulated that
the resulting decrease in basicity of the aniline nitrogen would
minimize the potential for acid-catalyzed hydrolysis. Although
the corresponding methylamide 13 was slightly less potent in
the biochemical assay, cyclopropylamide 14 (BMS-605541)
retained excellent in vitro potency and displayed enhanced
solution stability at low pH.14b On the basis of its improved
chemical stability and impressive in vitro profile, 14 was selected
for further characterization in kinase selectivity, PK, and in vivo
efficacy studies.
The encouraging pharmacokinetic profile exhibited by 14
supported additional characterization of the compound in in vivo
efficacy studies. Thus, the aminothiazole analogue was evaluated
in the L2987 human lung and HCT-116 human colon carcinoma

3768 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Table 3. Pharmacokinetic Parameters for 14 in Mouse, Rat, and Cyno
Letters
delayed rectifier potassium channel (IKr).19 Since potent inhibi-
tion of the hERG channel and blockade of the IKr current have
been linked to drug-induced (acquired) QT-interval prolongation
and ultimately life threatening ventricular arrhythmias including
torsades de pointes, 14 was evaluated in preliminary in vitro
cardiovascular safety studies. Compound 14 was found to have
an IC50 of 16.6 µM against the hERG channel expressed in
HEK-293 cells.19 The activity observed in the electrophysiology
assay suggests a low to moderate risk for prolongation of the
QT interval in vivo.
parameter
mousea
ratb
cynob
po dosec (mg kg-1
)
90
10
50
10
5
1e
iv dosed (mg kg-1
)
Cmax (µM), po
148
0.5
649f
1.7
3.4
11.8
1.7
100
44.0(6.0)
2.0(0)
8.0(4.0)
0.75(0)
28.5g(13)
7.1(1.5)
7.9(0.3)
3.9(0.6)
1.6(0.3)
52
T
max (h), po
AUC (µM‚h), po
1/2 (h), po
202g(44)
2.2(1.2)
3.5(0.4)
13.6(2.1)
1.1(0.1)
100
t
MRT (h), po
Cl (mL min-1 kg-1), iv
Vss (L kg-1), iv
F
po (%)
In conclusion, novel 3-((2-(pyridin-2-ylamino)thiazol-5-yl-
methyl)amino)benzamides with potent VEGFR-2 kinase inhibi-
tory activity were identified from the N-methoxybenzamide-
substituted pyrrolo[2,1-f][1,2,4]triazines using computer-assisted
drug design techniques. Optimization of the series SAR led to
the identification of 14, which was orally active in the human
lung (L2987) and colon (HCT-116) carcinoma xenograft models
at multiple dose levels. On the basis of its favorable in vitro
pharmacology, in vivo efficacy, and pharmacokinetic profile,
14 was advanced into preclinical in vivo safety studies.
a Composition serum concentration-time profiles were constructed for
the PK analysis. b Data reported as an average of three animals with
associated standard deviations in parentheses. c Vehicle: PEG400/Tween-
80/water (40:10:50). d Vehicle: PEG400/Tween-80/water (20:5:75). e Ve-
hicle: 30:70 PEG400/water. f AUC(0-24h) g AUCtotal
. .
Table 4. In Vivo Antitumor Activity of 14 against Established L2987
and HCT-116 Xenografts Implanted Subcutaneously in Athymic Mice
tumor model
L2987b
dose, mg kg-1
schedule
TGI,a %
60
90
180
25
50
12.5
25
1qd × 14
16
55
67
23
67
21
51
64
L2987c
2qd × 14
2qd × 14
Acknowledgment. We thank Mark Witmer for assistance
with the enzyme kinetics, Danshi Li for conducting the hERG
assay, and Bethanne Warrack and Discovery Analytical Sciences
for compound characterization efforts.
HCT-116c
50
a Maximum percent tumor growth inhibition over one tumor volume
doubling time. b Vehicle: PEG400/water (70:30). c Vehicle: 100% PEG400.
Supporting Information Available: Experimental procedures
and characterization data for 3-14, table of combustion analysis
and HPLC analysis data for key compounds, and detailed descrip-
tion of pharmacokinetic assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
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