Alkynylpyrimidine amide derivatives as potent, selective, and orally active inhibitors of Tie-2 kinase

Article abstract of DOI:10.1021/jm061112p

The recognition that aberrant angiogenesis contributes to the pathology of inflammatory diseases, cancer, and myocardial ischemia has generated considerable interest in the molecular mechanisms that regulate blood vessel growth. The receptor tyrosine kinase Tie-2 is expressed primarily by vascular endothelial cells and is critical for embryonic vasculogenesis. Interference with the Tie-2 pathway by diverse blocking agents such as soluble Tie-2 receptors, anti-Tie-2 intrabodies, anti-Ang-2 antibodies, and peptide-F _c conjugates has been shown to suppress tumor growth in xenograft studies. An alternative strategy for interfering with the Tie-2 signaling pathway involves direct inhibition of the kinase functions of the Tie-2 receptor. Herein we describe the development of alkynylpyrimidine amide derivatives as potent, selective, and orally available ATP-competitive inhibitors of Tie-2 autophosphorylation.

Full text of DOI:10.1021/jm061112p

J. Med. Chem. 2007, 50, 627-640
627
Alkynylpyrimidine Amide Derivatives as Potent, Selective, and Orally Active Inhibitors of Tie-2
Kinase
Victor J. Cee,*,†,‡ Brian K. Albrecht,*,† Stephanie Geuns-Meyer,† Paul Hughes,§ Steve Bellon,| James Bready,§ Sean Caenepeel,§
Stuart C. Chaffee,† Angela Coxon,§ Maurice Emery, Jenne Fretland, Paul Gallant,∞ Yan Gu,| Brian L. Hodous,†
Doug Hoffman,# Rebecca E. Johnson,† Richard Kendall,§ Joseph L. Kim,| Alexander M. Long,| David McGowan,†
Michael Morrison,∞ Philip R. Olivieri,† Vinod F. Patel,† Anthony Polverino,§ David Powers,∞ Paul Rose,| Ling Wang,§ and
Huilin Zhao|
Departments of Medicinal Chemistry, Molecular Pharmacology, and Molecular Structure, Amgen Inc., One Kendall Square, Bldg. 1000,
Cambridge, Massachusetts 02139; Departments of Molecular Pharmacology, Oncology Research, Pharmaceutics, and Pharmacokinetics and
Drug Metabolism, Amgen, Inc., One Amgen Center DriVe, Thousand Oaks, California 91320
ReceiVed September 25, 2006
The recognition that aberrant angiogenesis contributes to the pathology of inflammatory diseases, cancer,
and myocardial ischemia has generated considerable interest in the molecular mechanisms that regulate
blood vessel growth. The receptor tyrosine kinase Tie-2 is expressed primarily by vascular endothelial cells
and is critical for embryonic vasculogenesis. Interference with the Tie-2 pathway by diverse blocking agents
such as soluble Tie-2 receptors, anti-Tie-2 intrabodies, anti-Ang-2 antibodies, and peptide-Fc conjugates
has been shown to suppress tumor growth in xenograft studies. An alternative strategy for interfering with
the Tie-2 signaling pathway involves direct inhibition of the kinase functions of the Tie-2 receptor. Herein
we describe the development of alkynylpyrimidine amide derivatives as potent, selective, and orally available
ATP-competitive inhibitors of Tie-2 autophosphorylation.
Introduction
Angiogenesis, the formation of new blood vessels from
preexisting vasculature, is essential for embryonic development
as well as normal physiological processes such as wound healing
and the menstrual cycle.1 The recognition that aberrant angio-
genesis contributes to the pathology of inflammatory diseases,2
cancer,3 and myocardial ischemia4 has generated considerable
interest in the molecular mechanisms that regulate blood vessel
growth.5 Receptor tyrosine kinases expressed primarily by
vascular endothelial cells have emerged as important mediators
of angiogenesis.6 It has been well-established that activation of
KDR (VEGFR2) by vascular endothelial growth factor (VEGF)
strongly promotes angiogenesis by enhancing endothelial cell
proliferation and survival.7 While critical for embryonic vas-
culogenesis, the determination of the precise role of the Tie-2
receptor (tyrosine kinase with immunoglobulin and epidermal
growth factor homology domains-2) in pathological angiogenesis
has been complicated by the presence of numerous endogenous
ligands, including angiopoeitin-1 (Ang-1) and angiopoeitin-2
(Ang-2).8 While Ang-1 is known to activate Tie-2 by inducing
Figure 1. Reported Tie-2 inhibitors.
Tie-2 phosphorylation,9 Ang-2 has been shown to antagonize
the effects of Ang-1,10 although in some contexts Ang-2 has
been observed to induce Tie-2 phosphorylation.10,11 Interference
with the Tie-2 pathway by diverse blocking agents such as
soluble Tie-2 receptors,12 anti-Tie-2 intrabodies,13 and anti-
Ang-2 antibodies and peptide-Fc conjugates14 has been shown
to suppress tumor growth in xenograft studies. An alternative
strategy for interfering with the Tie-2 signaling pathway involves
direct inhibition of the kinase functions of the Tie-2 receptor.
To this end, we wished to develop an ATP-competitive small
molecule inhibitor of Tie-2. In order to establish the effect of
Tie-2 kinase inhibition on pathological angiogenesis, it was
critical that the small molecule possess selectivity against kinases
known to be involved in angiogenesis, particularly KDR.
In contrast to the many reports of small molecule inhibitors
of KDR with selectivity against Tie-2,15 there have been few
reports of Tie-2 inhibitors with selectivity against KDR. In 2001,
Abbott Laboratories reported the 5-arylpyrrolo[2,3-d]pyrimidin-
4-amine derivative 1 (Figure 1) as a potent and selective (>100-
fold over KDR enzyme) inhibitor of Tie-2.16 More recently,
compound 2, based on a structurally related 3-arylpyrazolo-
* To whom correspondence should be addressed. V.J.C.: tel, 805-313-
5500; fax, 805-480-1337; e-mail, vcee@amgen.com. B.K.A.: tel, 617-444-
5166; fax, 617-577-9822; e-mail, brian.albrecht@amgen.com.
† Department of Medicinal Chemistry.
‡ Present address: Department of Medicinal Chemistry, Amgen, Inc.,
One Amgen Center Drive, Thousand Oaks, CA 91320.
§ Department of Oncology Research.
|| Department of Molecular Structure.
Department of Pharmacokinetic and Drug Metabolism.
∞ Department of Molecular Pharmacology.
# Department of Pharmaceutics.
10.1021/jm061112p CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/25/2007

628 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Cee et al.
Figure 3. Development of the alkynylpyrimidine amide series.
with the goal of improving selectivity while maintaining the
impressive potency against Tie-2 kinase.
Chemistry
The mild and versatile Sonogashira reaction22 was useful in
the synthesis of this class of kinase inhibitor. In an effort to
quickly develop SAR, two synthetic schemes were employed.
Conversion of benzoic acid derivatives 7 to the corresponding
acid chloride followed by amide bond formation with a variety
of anilines 8 afforded amides 9 (Scheme 1). Commercially
available 5-iodo-2-aminopyrimidine 10 was treated with TMS-
acetylene under Sonogashira conditions followed by cleavage
of the terminal silane, yielding alkynylpyrimidine 11. A final
Sonogashira coupling of alkynylpyrimidine 11 and aryl iodide
9 produced the requisite core structure 6. The conversion of
aryl fluoride 6f to 6g under methanolysis conditions provided
additional diversity. In conjunction with the chemistry developed
in Scheme 1, it was also feasible to install a variety of
heterocycles as the final step (Scheme 2). Aryl iodide 9 was
converted to the corresponding terminal acetylene 12 by utilizing
conditions similar to those described in Scheme 1. With the
acetylene in hand a variety of heteroaromatic halides (R4X) were
coupled to yield the desired analogues 13.
The majority of our efforts to afford a kinase inhibitor with
the desired profile was focused on modifying the arylbenzamide
portion. While some of the requisite anilines are commercially
available, the majority were synthesized. Treatment of 4-fluoro-
3-nitrobenzotrifluoride 14 with a variety of commercially
available amines [piperidine, N1,N1,N3,N3-tetramethylpropane-
1,3-diamine, 4-methylpiperazine, and (R)/(S)-3-dimethylami-
nopyrrolidine] and base in THF at elevated temperatures
afforded substituted nitroanilines 15 (Scheme 3). Reduction of
the nitro group to the corresponding aniline under catalytic
hydrogenation conditions provided aniline 8 for coupling in
Scheme 1.
The synthesis of piperidine-substituted anilines for analogues
6j-l required additional functionalization (Scheme 4). Aniline
8j, containing a 4-N,N-dimethylamino substituent, was prepared
from commercially available 1-benzylpiperidin-4-amine, which
upon reductive amination, hydrogenolysis, and N-arylation with
4-fluoro-3-nitrobenzotrifluoride 14 gave 15j. Reduction of the
nitro group under standard conditions delivered the correspond-
ing aniline 8j. Enantiomeric anilines 8k and 8l containing a
3-N,N-dimethylamino-substituted piperidine were prepared sepa-
rately starting from commercially available (R)- and (S)-tert-
butyl piperidin-3-ylcarbamate. Arylation with 14 gave 15k and
15l. Removal of the Boc-carbamate with HCl followed by
reductive amination with formaldehyde resulted in the corre-
sponding tertiary amines, which were subsequently reduced by
hydrogenation to afford anilines 8k and 8l.
Figure 2. X-ray crystal structure of 3a bound in the ATP binding site
of Tie-2 kinase.
[3,4-d]pyrimidine-6-amine core, was reported by GlaxoSmith-
Kline as a potent and selective (>140-fold over KDR enzyme)
Tie-2 inhibitor.17 The in vivo antiangiogenic activity of both 1
and 2 was demonstrated in murine models of angiogenesis. We
recently disclosed the pyridyltriazine amide derivative 3 as a
potent (IC50 ) 29 nM) and selective (270-fold over KDR
enzyme) inhibitor of Tie-2 with oral bioavailability in rats.18
Key to the development of 3 was the finding that a 2,5-
disubstituted anilide moiety is critical for achieving selectivity
over KDR and other kinases. While 3 is an acceptable molecule
for the in vivo study of Tie-2 inhibition, we recognized that
considerable improvements could be made by excising nones-
sential elements of this molecule. Here we describe the
development of alkynylpyrimidine amide derivatives as potent,
selective, and orally available Tie-2 inhibitors.
Structural information from cocrystal structures of molecules
analogous to 3 (3a, Figure 2) and the kinase domain of Tie-218
indicated that these molecules bind the inactive “DFG-out”
conformation of the protein.19,20 The methylaminopyrimidine
ring binds to the linker residue Ala905 via two hydrogen bonds,
with distances of 3.0 Å (MeNH) and 2.8 Å (N). The aryl amide
moiety directs the terminal CF3-substituted aromatic ring into
the extended hydrophobic pocket, and the carbonyl oxygen
makes a hydrogen bond with the backbone NH of Asp982 (DFG
motif). The pyridine ring plays an important structural role in
positioning the pyrimidine ring within H-bonding distance to
the linker residues and placing the central aryl ring in the first
hydrophobic pocket. While the pyridine ring is observed to
participate in an edge-to-face π-stacking interaction with Phe983
of the DFG motif, we questioned whether this interaction was
necessary for high binding affinity. This question prompted the
series of successive modifications illustrated in Figure 3.
Beginning with the nonselective but highly potent 4, replacement
of the pyridylpyrimidine ether with a 2-aminoquinazoline
resulted in 5, with comparable potency.21 Analogue 5 could be
further simplified by replacement of the quinazoline with an
acetylene linkage between the pyrimidine and benzamide
portions of the molecule to give 6a. As this compound was an
effective inhibitor of Tie-2 and of considerably reduced mo-
lecular weight, an effort to explore the SAR of 6a was initiated

Orally ActiVe Inhibitors of Tie-2 Kinase
Scheme 1. General Method Aa
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 629
a Reagents and conditions: (a) SOCl2, ∆; (b) iPr2NEt, CH2Cl2; (c) (i) PdCl2(PPh3)2, CuI, TMS-acetylene, Et3N, MeCN; (ii) MeOH, K2CO3; (d) PdCl2(PPh3)2,
CuI, Et3N, MeCN, ∆; (e) NaOMe, MeOH, ∆.
Scheme 2. General Method Ba
a Reagents and conditions: (a) PdCl2(PPh3)2, TMS-acetylene, Et3N, CuI, MeCN, ∆; (b) R4X (Br, I) PdCl2(PPh3)2, CuI, Et3N, MeCN, ∆.
Scheme 3. General Method Ca
Scheme 5. Synthesis of Demethylated 3-Aminopiperidine
Analoguesa
a Reagents and conditions: (a) RR′NH, NaHCO3, THF, ∆; (b) Pd/C,
H2, MeOH.
Scheme 4. Synthesis of
N,N-Dimethylaminopiperidine-Containing Anilinesa
a Reagents and conditions: (a) NaH, MeI, DMF; (b) (i) Pd/C, H2, MeOH;
(ii) 2-fluoro-5-iodobenzoyl chloride, TEA, CH2Cl2. (c) for 9n, (i) 4 M HCl
in dioxane; (ii) 11, PdCl2(PPh3)2, CuI, Et3N, MeCN, ∆. (d) for 9m, (i) 11,
PdCl2(PPh3)2, CuI, Et3N, MeCN, ∆; (ii) TFA, CH2Cl2.
Discussion
The 2-aminopyrimidine moiety of 6a was envisioned to
interact with the linker region of Tie-2 in the ATP-binding cleft
via hydrogen bonds between the CdO and N-H of Ala905
and the 2-NH2 and N of the aminopyrimidine, respectively. The
2-aminopyrimidine could be replaced with other heterocycles
containing a similar donor-acceptor motif with no discernible
change in inhibitory potency (Table 1, 13a,b). Although the
picolinamide moiety has been employed successfully in other
kinase inhibitors,23 in the case of 13c, this proved to be an
inferior linker-binding element for Tie-2. Quinoline (13d) and
quinoxaline (13e) heterocycles were tolerated, but these mol-
ecules were less potent than 6a. We concluded that modification
of the linker-binding element offered little in the way of
a Reagents and conditions: (a) (i) HCHO, NaBH3CN, AcOH/MeOH;
(ii) HCl/dioxane/Et2O; (iii) Pd/C, H2, MeOH; (iv) 14, TEA, THF, ∆; (b)
Pd/C, H2, MeOH; (c) 14, NaHCO3, THF, ∆; (d) (i) 4 M HCl in dioxane;
(ii) HCHO, NaBH3CN, HOAC, MeOH; (iii) Pd/C, H2, MeOH.
Routes to demethylated analogues 6m/n were useful in order
to produce these potential metabolites of 6l. Aniline 15l (Scheme
5) was alkylated with methyl iodide to give 15m (Scheme 5).
Both 15l and 15m were processed by a similar sequence
according to Scheme 5 to give potential metabolites 6m/n.

630 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Table 1. SAR of Heterocyclic Alkynyl Amide Derivativesa
Cee et al.
potent of 6a-c. Incorporation of a bulky, hydrophobic sub-
stituent such as piperidine at R3 provided selectivity over KDR
(6d-f), as anticipated on the basis of the SAR of 3 (Figure 1).
Surprisingly, in this series of compounds, the fluorine-substituted
benzamide 6f is the most potent of 6d-f. This trend was also
reflected in an Ang-1-stimulated Tie-2 phosphorylation assay
in the EA.hy 926 endothelial cell line. Apparently, the presence
of the o-fluorine is beneficial only in conjunction with a bulky
N-aryl group (R3). As the bulky N-aryl group (R3) prevents a
hydrogen-bonding interaction between the amide N-H and
Glu872 of Tie-2 (Figure 2),18 we speculated that the fluorine
substituent provides a favorable electrostatic interaction24 with
the free amide N-H, reducing the desolvation penalty upon
binding to Tie-2. The plausibility of this theory was tested with
6g, which contains an o-methoxy substituent to serve as a
hydrogen-bond acceptor for the amide N-H. Indeed, this
compound has approximately the same inhibitory activity as
6f, which supports the hypothesis that engagement of the amide
N-H results in more potent Tie-2 inhibitors. Moreover,
selectivity vs KDR and Lck was maintained; however, the
compound exhibited reduced cellular activity, which may be
due to poor permeability. The optimal molecule in the series
6a-g was 6f, due to its potency (Tie-2 IC50 ) 17 nM) and
selectivity (28-fold over KDR, 21-fold over Lck). 6f was further
shown to inhibit Ang-1-stimulated Tie-2 autophosphorylation
in the Ea.hy 926 endothelial cell line with an IC50 of 22 nM.
Despite the favorable in vitro activity of compound 6f, it was
found to be a poor candidate for in vivo studies due to minimal
aqueous solubility (<1 µg/mL, 0.01 N HCl). Our attention
focused on the incorporation of anilides containing basic amines
to improve the physical properties of this series (Table 3).
Incorporation of a tetramethylpropane-1,3-diamine moiety,
which had previously provided a high level of selectivity in a
related series (3, Figure 1), resulted in a relatively nonselective
compound (6h). It was hypothesized that the lack of selectivity
was due to the conformational flexibility of the tetramethyl-
propane-1,3-diamine substituent; therefore, more rigid ortho-
substituents were explored (6i-p). A 4-N-methylpiperazine
substituent (6i) proved to be an effective means of incorporating
a basic amine while selectivity (84-fold over KDR, 16-fold over
Lck) was maintained, although the cellular activity of the
compound was somewhat lower than expected. Substitution of
N,N-dimethylamines onto a piperidine framework was also
explored, with 4-substitution (6j) being markedly inferior to
3-substitution (6k/l). The (S) stereochemistry was found to be
a Final [ATP]: 5.0 µM (Tie-2), 1.0 µM (KDR), 0.5 µM (Lck).
selectivity, and that the 2-aminopyrimidine was suitable for
further SAR studies.
Given the similarity of the N-arylbenzamide portion of 6a
and 3 (Figure 1), modification of this region of the molecule
was expected to be an effective means of achieving selectivity
against KDR.18 While substitution of the benzamide ring did
not provide useful kinase selectivity, para (R1) and ortho (R2)
substitution was found to be important in conjunction with
substitution of the N-aryl group (R3), as illustrated in Table 2.
The desmethyl analogue 6b exhibited reduced potency relative
to 6a, presumably due to loss of favorable hydrophobic
interactions. An o-fluoro substituent (6c) resulted in a further
degradation of Tie-2 inhibitory activity. In this series of
compounds, the fluorine-substituted benzamide 6c is the least
Table 2. SAR of the N-Arylbenzamide Portion of Pyrimidine Alkynyl Amidesa
IC50 (nM)
inhibn of cellular
Tie-2 phosphoryl-
enzyme
compd
R1
R2
R3
Tie-2
KDR
Lck
ation (EA.hy 926)
6a
6b
6c
6d
6e
6f
Me
H
H
Me
H
H
H
H
F
H
H
F
OMe
H
H
H
3
11
47
30
63
17
21
2
<1
6
1
19
65
42
410
NT
NT
NT
39
250
22
piperidine
piperidine
piperidine
piperidine
>1000
>1000
480
370
6g
H
390
>25000
1200
a Final [ATP]: 5.0 µM (Tie-2); 1.0 µM (KDR); 0.5 µM (Lck).

Orally ActiVe Inhibitors of Tie-2 Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 631
Table 3. SAR of the N-Arylbenzamide Portion of Pyrimidine Alkynyl Amidesa
IC50 (nM)
inhibn of cellular
Tie-2 phosphoryl-
enzyme
compd
R
Tie-2
KDR
Lck
ation (EA.hy 926)
6f
6h
6i
6j
6k
6l
6m
6n
6o
6p
piperidine
17
8
13
620
44
5
14
13
220
140
480
11
1100
170
370
21
210
150
780
130
620
1480
1920
1100
22
NT
84
NT
98
8
26
33
NT
NT
N1,N1,N3,N3-tetramethylpropane-1,3-diamine
4-methylpiperazine
4-dimethylaminopiperidine
(R)-3-dimethylaminopiperidine
(S)-3-dimethylaminopiperidine
(S)-3-methylaminopiperidine
(S)-3-aminopiperidine
810
1030
1010
800
890
3000
(R)-3-dimethylaminopyrrolidine
(S)-3-dimethylaminopyrrolidine
a Final [ATP]: 5.0 µM (Tie-2); 1.0 µM (KDR); 0.5 µM (Lck).
Table 4. Kinase Selectivity of 6l
hypothesis was provided by in vitro incubations of 6l and rat
liver microsomes (RLM). 6l exhibited high clearance (677 µL/
min/mg), and compounds 6m and 6n were produced.27 In
contrast to the high clearance exhibited in rat liver microsomes,
compound 6l was cleared at a much slower rate (<50 µL/min/
mg) in mouse liver microsomes (MLM). Compounds 6m and
6n were much more consistent across these species with respect
to liver microsomal clearance. The mouse microsomal clearance
data suggested that all three compounds 6l-n were good
candidates for our mouse-based pharmacodynamic assay (vide
infra). It was also shown that 6l-n could be dosed orally in
rats with acceptable bioavailability (F ) 39-54%), supporting
po administration. Compounds 6l and 6m were ultimately
chosen for further in vivo studies.
The ability of 6l to modulate Tie-2 phosphorylation levels in
vivo was assessed in a mouse pharmacodynamic model (Figure
4). A single dose of compound was administered to female CD-1
NU/NU mice po at 10, 30, and 100 mg/kg at time zero. Human
angiopoietin-1 (h-Ang-1) was administered iv after 2.75 h to
stimulate Tie-2 phosphorylation. Mouse lungs were harvested
at 3 h and phosphorylated Tie-2 levels were measured by
Western blot analysis. A dose-dependent inhibition of stimulated
Tie-2 phosphorylation was observed, with Tie-2 phosphorylation
reduced to sub-basal levels at the 30 and 100 mg/kg doses. The
10 mg/kg dose provided an approximately 50% reduction in
stimulated Tie-2 phosphorylation, with a plasma concentration
of 6l at 1660 nM. This concentration is 208-fold greater than
the cellular IC50 of 6l (8 nM). However, taking into account
nonspecific binding to mouse plasma proteins (6l ) 97.9%
bound in mouse plasma), the free unbound fraction of 6l is 32
nM and this correlates fairly well with the IC50 determined in
the cell-based assay (8 nM). Monomethyl derivative 6m
produced a qualitatively similar result. The robust inhibition of
Tie-2 phosphorylation upon po administration of 6l and 6m
indicates that these molecules are suitable for the in vivo study
of Tie-2 signaling in angiogenesis.
ideal, with compound 6l exhibiting potent inhibition of the Tie-2
kinase domain (IC50 ) 5 nM) and Ang-1-stimulated Tie-2
phosphorylation in endothelial cells (IC50 ) 8 nM) along with
high selectivity (205-fold over KDR, 25-fold over Lck). In
addition, the aqueous solubility of 6l (>200 µg/mL, 0.01 N HCl)
was considerably improved over that of 6f (<1 µg/mL, 0.01 N
HCl). We recognized the potential for the dealkylative metabo-
lism of 6l, and the sequentially demethylated analogues 6m and
6n were prepared and found to be potent and selective Tie-2
inhibitors. Finally, we established that N,N-dimethylamino-
substituted pyrrolidines (6o,p) were inferior to N,N-dimethy-
lamino-substituted piperidines (6k,l).
Compound 6l was screened against a panel of tyrosine and
serine/threonine kinases (Table 4) and was found to be >20-
fold selective over many kinases, with the exception of the nerve
growth factor receptor kinase TrkA25 (4-fold) and Src family
member FGR26 (9-fold). Importantly, 6l was selective against
kinases known to be involved in angiogenesis (58-fold over
EphB4, 205-fold over KDR, and >1000-fold over PDGFR).
The selectivity profile of 6m and 6n was found to be very
similar to that of 6l.
The in vitro and in vivo pharmacokinetic properties of
compounds 6l-n are summarized in Table 5. Compound 6l,
bearing the N,N-dimethylamino substituent, exhibited relatively
high clearance (2.4 L/h/kg) when dosed intravenously in male
rats. It was speculated that facile N-demethylation contributed
to the high clearance of 6l, and the low clearance exhibited by
sequentially demethylated compounds 6m and 6n is consistent
with this hypothesis. Additional evidence in support of this
Conclusions
An effort to reduce the molecular weight of pyridyl-triazine
Tie-2 inhibitor 3 by excising less important elements of the
molecule led to a series of alkynylpyrimidine amide derivatives.
Modification of the N-arylbenzamide portion of these molecules
provided selectivity over other kinases. The combination of an

632 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Table 5. Pharmacokinetic Properties of 6l-n
Cee et al.
Cl (µL/min/mg)
b
b
c
Clb
Vss
T1/2
AUC0-∞
Fc
compd
R
RLM
MLM
(L/h/kg)
(L/kg)
(h)
(ng h/mL)
(%)
6l
6m
6n
NMe2
NHMe
NH2
677
<50
<100
<50
<50
<100
2.4
0.15
0.32
11.8
2.2
4.6
7.9
10.6
11.4
800
18,600
9,300
39
54
51
a Male Sprague-Dawley rats (n ) 3). b iv, 2.0 mg/kg. (DMSO). c po, 5.0 mg/kg (pH 2.2 OraPlus).
chromatography was performed using either glass columns packed
with silica gel (200-400 mesh, Aldrich Chemical) or medium-
pressure liquid chromatography (MPLC) on a CombiFlash Com-
panion (Teledyne Isco) with RediSep normal-phase silica gel (35-
60 µm) columns and UV detection at 254 nm. Preparative reversed-
phase HPLC was performed on a Gilson (215 liquid handler), YMC-
Pack Pro C18, 150 × 30 mm i.d. column, eluting with a binary
solvent system using a gradient elution (A, H2O with 0.1% TFA;
B, CH3CN with 0.1% TFA) with UV detection at 254 nm. All final
compounds were purified to >95% purity as determined by high
performance liquid chromatography (HPLC) with an Agilent 1100
Series insrument and UV detection at 254 nm (system A, Zorbax
SB-C8, 4.6 × 150 mm, 15 min, 1.5 mL/min flow rate, from 5 to
95% 0.1% TFA in CH3CN/from 95 to 5% 0.1% TFA in H2O;
system B, Phenomenex Synergi, 2 × 50 mm, 3 min, 1.0 mL/min
flow rate, from 5 to 95% 0.1% formic acid in CH3CN/from 95 to
5% 0.1% formic acid in H2O; system C, Zorbax SB-C8, 4.6 × 75
mm, 12 min, 1.0 mL/min flow rate, from10 to 90% 0.1% formic
acid in CH3CN/from 90 to 10% 0.1% formic acid in H2O). NMR
spectra were determined with either a Bruker AV-400 (400 MHz)
spectrometer or a Varian 400 or 300 MHz spectrometer at ambient
temperature. Low-resolution mass spectral (MS) data were deter-
mined on an Agilent 1100 Series LC-MS with UV detection at
254 nm and a low resonance electrospray mode (ESI). Chemical
shifts are reported in ppm from the solvent resonance (DMSO-d6,
2.50 ppm). Data are reported as follows: chemical shift, multiplicity
(s ) singlet, d ) doublet, t ) triplet, q ) quartet, br ) broad,
m ) multiplet), coupling constants, and number of protons.
High-resolution mass spectra (HRMS) were obtained on a high
resonance electrospray time-of-flight mass spectrometer (Agilent).
Combustion analysis was performed by Galbraith Laboratories, Inc.,
Knoxville, TN.
General Method A. 3-Halobenzoic acid 7 (10 mmol) was taken
up in SOCl2 (4 mL). The resulting slurry was heated to reflux for
2 h, at which time the reaction was cooled and concentrated under
reduced pressure to afford the corresponding acid chloride, which
was used without further purification.
The acid chloride (1 equiv) was taken up in CH2Cl2 (0.1 M)
followed by the addition of iPr2NEt (1.2 equiv) and the appropriate
aniline 8 (1 equiv). The mixture was allowed to stir at room
temperature for 3 h. The reaction mixture was diluted with CH2-
Cl2 and washed with water, saturated aqueous NaHCO3, and brine.
The organic layer was dried over anhydrous sodium sulfate, filtered,
and concentrated under reduced pressure to afford benzamide 9,
which was used without further purification.
Figure 4. Compound 6l inhibits h-Ang-1-stimulated Tie-2 phospho-
rylation in mouse lungs. Data points represent the mean ( SE, n ) 3:
(*) p e 0.05 vs vehicle + h-Ang-1 by one-way ANOVA with Dunett’s
posthoc test. Bars and p-Tie-2 represent phosphorylated Tie-2, and
circles represent plasma concentration.
o-fluorine substituent on the aryl carboxamide with an o-
piperidine substituent on the aniline fragment was found to be
ideal in terms of potency and selectivity. Installation of basic
amines onto the piperidine framework provided compounds with
greatly improved physical properties. Compounds 6l and 6m
exhibited favorable pharmacokinetic properties in rat and are
suitable for oral dosing (F ) 39 and 54%, respectively). In a
mouse pharmacodynamic model, 6l exhibited a dose-dependent
inhibition of Ang-1-stimulated Tie-2 phosphorylation in lung
tissue, with suppression of Tie-2 phosphorylation below basal
levels after a single dose of 30 mg/kg (po). Monomethyl
derivative 6m produced a qualitatively similar result. These data
indicate that 6l and 6m are suitable small molecules for the in
vivo study of Tie-2 signaling in angiogenesis.
Experimental Section
5-Ethynylpyrimidin-2-amine 11 (2 equiv), benzamide 9 (1 equiv),
and PdCl2(PPh3)2 (0.05 equiv) were taken up in MeCN:Et3N (3:1,
0.1 M) in a sealable tube. To the resulting mixture was added CuI
(0.05 equiv) and the reaction vessel was sealed and heated to
80 °C until complete consumption of benzamide 9 was observed
by LC-MS analysis (typically 2h). The reaction mixture was
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. An-
hydrous solvents were obtained from Aldrich and used directly.
All reactions involving air- or moisture-sensitive reagents were
performed under a nitrogen or argon atmosphere. Silica gel

Orally ActiVe Inhibitors of Tie-2 Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 633
concentrated under reduced pressure and purified by silica gel
chromatography or reverse-phase HPLC to afford the title com-
pounds.
according to general method A starting from 3-iodo-4-methyl-N-
(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)benzamide (0.13 g,
0.26 mmol) to give 6d (0.12 g, 99%): 1H NMR (400 MHz, DMSO-
d6) δ ppm 9.65 (s, 1 H), 8.46 (s, 2 H), 8.30-8.40 (m, 1 H), 7.99-
8.14 (m, 1 H), 7.86 (dd, J ) 8.0, 1.6 Hz, 1 H), 7.46-7.55 (m, 2
H), 7.37 (d, J ) 8.3 Hz, 1 H), 7.21 (s, 2 H), 2.85-2.94 (m, 4 H),
2.53 (s, 3 H), 1.66-1.76 (m, 4 H), 1.49-1.62 (m, 2 H); HRMS
General Method B. Benzamide 9 (1 equiv) and PdCl2(PPh3)2
(0.05 equiv) were taken up in MeCN:Et3N (3:1, 0.1 M) followed
by the addition of TMS-acetylene (5 equiv) and CuI (0.05 equiv).
The reaction mixture was allowed to stir at ambient temperature
until complete consumption of the benzamide was observed. To
this mixture was added excess K2CO3 and the mixture was
allowed to stir for approximately 1.5 h. The mixture was filtered
through a pad of Celite. To the filtrate was added silica gel, and
the mixture was concentrated under reduced pressure and purified
via automated flash chromatography (silica gel, from 10% to 50%
EtOAc in hexanes, gradient elution) to afford the desired terminal
acetylene 12.
To a solution of heteroaromatic halide (2 equiv), alkyne 12 (1
equiv), and PdCl2(PPh3)2 (1 equiv) in MeCN:Et3N (3:1, 0.1 M)
was added CuI (0.05 equiv). The tube was sealed and heated to
90 °C for 1 h. The mixture was cooled to ambient temperature,
concentrated under reduced pressure, and purified either by silica
gel chromatography or reverse-phase HPLC to afford title com-
pounds 13.
General Method C. To a round-bottom flask was added RR′NH
(1 equiv), sodium bicarbonate (2.5 equiv), and 1-fluoro-2-nitro-4-
(trifluoromethyl)benzene 14 (1 equiv). The mixture was diluted with
THF (0.1 M), fitted with a water-cooled reflux condenser, and was
heated to 70 °C. After 14 h, the orange mixture was cooled to
ambient temperature, and filtered through a glass frit, rinsing with
EtOAc. The filtrate was concentrated under reduced pressure and
was purified by recrystallization from hexanes or silica gel
chromatography. The resulting nitrobenzene was taken up in MeOH
(0.1 M), purged with argon, and treated with Pd/C (10%, 0.1 equiv).
The flask was fitted with an H2 balloon and allowed to stir at
ambient temperature. Upon completion of the reaction, the vessel
was purged with argon, the reaction was filtered through a bed of
Celite, and the filtrate was concentrated under reduced pressure to
afford title anilines 8, which were used without further purification.
3-(2-(2-Aminopyrimidin-5-yl)ethynyl)-4-methyl-N-(3-(trifluo-
romethyl)phenyl)benzamide (6a) was prepared according to
general method A starting from 3-iodo-4-methyl-N-(3-(trifluoro-
methyl)phenyl)benzamide (0.17 g, 0.42 mmol) to give 6a as an
off-white solid (0.046 g, 27%): 1H NMR (400 MHz, DMSO-d6) δ
ppm 10.57 (s, 1 H), 8.48 (s, 2 H), 8.25 (s, 1 H), 8.11 (s, 1 H), 8.08
(d, J ) 8.1 Hz, 1 H), 7.85-7.93 (m, 1 H), 7.61 (t, J ) 8.1 Hz, 1
H), 7.50 (d, J ) 8.1 Hz, 1 H), 7.46 (d, J ) 7.7 Hz, 1 H), 7.22 (s,
2 H), 2.52 (s, 3 H); HRMS m/z ) 397.1276 [M + H]+, calcd
[C21H15F3N4O + H]+ ) 397.1271; HPLC tR A, 8.86 min; C,
7.68 min.
m/z ) 502.1834 [M + Na]+, calcd [C26H24F3N5O + Na]+
502.1825; HPLC tR A, 9.79 min; B, 2.75 min.
)
3-(2-(2-Aminopyrimidin-5-yl)ethynyl)-N-(2-(piperidin-1-yl)-
5-(trifluoromethyl)phenyl)benzamide (6e) was prepared according
to general method A starting from 3-iodo-N-(2-(piperidin-1-yl)-5-
(trifluoromethyl)phenyl)benzamide (0.13 g, 0.26 mmol) to give 6e
(0.12 g, quant.) as a light yellow solid: 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.72 (s, 1 H), 8.45 (s, 2 H), 8.32 (s, 1 H), 8.07
(s, 1 H), 7.95 (d, J ) 8.0 Hz, 1 H), 7.74 (d, J ) 7.7 Hz, 1 H), 7.63
(dd, J ) 7.7 Hz, 1 H), 7.51 (dd, J ) 8.5, 1.3 Hz, 1 H), 7.37 (d, J
) 8.2 Hz, 1 H), 7.21 (s, 2 H), 2.84-3.00 (m, 4 H), 1.64-1.77 (m,
4 H), 1.50-1.61 (m, 2 H); HRMS m/z ) 488.1674 [M + Na]+,
calcd [C25H22F3N5O + Na]+ ) 488.1669; HPLC tR A, 9.50 min;
B, 2.70 min.
5-(2-(2-Aminopyrimidin-5-yl)ethynyl)-2-fluoro-N-(2-(piperi-
din-1-yl)-5-(trifluoromethyl)phenyl)benzamide (6f) was prepared
according to general method A starting from 2-fluoro-5-iodo-N-
(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)benzamide (0.20 g,
0.41 mmol) to give 6f (0.21 g, quant.) as an off-white solid: 1H
NMR (400 MHz, DMSO-d6) δ ppm 9.94 (d, J ) 9.9 Hz, 1 H),
1
1
8.65 (s, H), 8.47 (s, 2H), 7.78 (m, H), 7.45-7.56 (m, 4H), 7.22
(s, 2H), 2.86-2.88 (m, 4H), 1.71 (m, 4 H), 1.56 (m, 2H); HRMS
m/z ) 506.1573 [M + Na]+, calcd [C25H21F4N5O + Na]+
506.1574; HPLC tR A, 10.35 min; B, 2.79 min.
)
5-(2-(2-Aminopyrimidin-5-yl)ethynyl)-2-methoxy-N-(2-(pip-
eridin-1-yl)-5-(trifluoromethyl)phenyl)benzamide (6g). A mixture
of 5-(2-(2-aminopyrimidin-5-yl)ethynyl)-2-fluoro-N-(2-(piperidin-
1-yl)-5-(trifluoromethyl)phenyl)benzamide (0.14 g, 0.29 mmol) and
NaOMe (0.5 M solution in methanol, 2.0 mL, 1.0 mmol) in a sealed
tube was heated to reflux. After 16 h, the reaction was cooled and
partitioned between EtOAc and brine. The organic layer was dried
over anhydrous sodium sulfate, filtered, and concentrated. The
material was purified by preparative TLC, eluting with 30% acetone/
dichloromethane to give 6g as a white solid (0.037 g, 26%): 1H
NMR (400 MHz, DMSO-d6) δ ppm 10.49 (s, 1 H), 8.72-8.80 (m,
1 H), 8.45 (s, 2 H), 8.17 (d, J ) 2.1 Hz, 1 H), 7.74 (dd, J ) 8.7,
2.3 Hz, 1 H), 7.33-7.51 (m, 3 H), 7.15 (s, 2 H), 4.13 (s, 3 H),
2.76-2.95 (m, 4 H), 1.65-1.80 (m, 4 H), 1.53-1.64 (m, 2 H);
HRMS m/z ) 518.1775 [M + Na]+, calcd [C26H24F3N5O2+Na]+
) 518.1774. Anal. (C26H24F3N5O2) C, H, N.
5-(2-(2-Aminopyrimidin-5-yl)ethynyl)-N-(2-((3-(dimethylami-
no)propyl)(methyl)amino)-5-(trifluoromethyl)phenyl)-2-fluo-
robenzamide (6h) was prepared according to general method A
starting from N-(2-((3-(dimethylamino)propyl)(methyl)amino)-5-
(trifluoromethyl)phenyl)-2-fluoro-5-iodobenzamide (0.23 g, 0.41
mmol) ,affording 6h as an off-white solid (0.20 g, 95%): 1H NMR
(400 MHz, DMSO-d6) ppm 9.96 (d, J ) 7.7 Hz, 1 H), 8.50 (s, 1
H), 8.46 (s, 2 H), 8.02 (d, J ) 5.5 Hz, 1 H), 7.70-7.84 (m, 1 H),
7.37-7.59 (m, 3 H), 7.22 (s, 2 H), 2.90-3.09 (m, 2 H), 2.68 (s, 3
H), 2.15 (t, J ) 7.0 Hz, 2 H), 1.99 (s, 6 H), 1.47-1.65 (m, 2 H);
3-(2-(2-Aminopyrimidin-5-yl)ethynyl)-N-(3-(trifluoromethyl)-
phenyl)benzamide (6b) was prepared according to general method
A starting from 3-iodo-N-(3-(trifluoromethyl)phenyl)benzamide
(0.10 g, 0.26 mmol) to give 6b (0.067 g, 69%) as a white solid:
1H NMR (400 MHz, DMSO-d6) δ ppm 10.64 (s, 1 H), 8.47 (s, 2
H), 8.26 (s, 1 H), 8.13 (s, 1 H), 8.08 (d, J ) 8.0 Hz, 1 H), 7.97 (d,
J ) 7.8 Hz, 1 H), 7.74 (d, J ) 8.0 Hz, 1 H), 7.57-7.67 (m, 2 H),
7.48 (d, J ) 7.5 Hz, 1 H), 7.20 (s, 2 H); HRMS m/z ) 383.1121
[M + H]+, calcd [C20H13F3N4O + H]+ ) 383.1114. Anal.
(C20H13F3N4O·0.25H2O) C, H, N.
5-(2-(2-Aminopyrimidin-5-yl)ethynyl)-2-fluoro-N-(3-(trifluo-
romethyl)phenyl)benzamide (6c) was prepared according to
general method A starting from 2-fluoro-5-iodo-N-(3-(trifluoro-
methyl)phenyl)benzamide (0.30 g, 0.73 mmol) to give 6c as an off
white solid (0.045 g, 15%): 1H NMR (400 MHz, acetone-d6) δ
ppm 9.81 (s, 1 H), 8.43 (s, 2 H), 8.30 (s, 1 H), 8.04 (d, J ) 8.4 Hz,
1 H), 7.95 (dd, J ) 6.6, 2.2 Hz, 1 H), 7.73 (ddd, J ) 8.4, 4.8, 2.2
Hz, 1 H), 7.63 (t, J ) 8.1 Hz, 1 H), 7.50 (d, J ) 7.7 Hz, 1 H), 7.36
(dd, J ) 10.4, 8.6 Hz, 1 H), 6.49 (s, 2 H); HRMS m/z ) 401.1030
[M + H]+, calcd [C20H12F4N4O + H]+ ) 401.1020. Anal.
(C20H12F4N4O‚H2O) C, H, N.
HRMS m/z ) 515.2186 [M + H]+, calcd [C26H26F4N6O + H]+
515.2177. Anal. (C26H26F4N6O‚0.25H2O) C, H, N.
)
3-(2-(2-Aminopyrimidin-5-yl)ethynyl)-N-(2-(4-methylpiperazin-
1-yl)-5-(trifluoromethyl)phenyl)benzamide (6i) was prepared
according to general method A starting from 3-iodo-N-(2-(4-
methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)benzamide (0.20 g,
0.39 mmol) to give 6i (0.064 g, 33%) as a white solid: 1H NMR
(400 MHz, DMSO-d6) δ ppm 9.89 (d, J ) 8.3 Hz, 1 H), 8.59 (s,
1 H), 8.46 (s, 2 H), 8.01-8.07 (m, 1 H), 7.72-7.82 (m, 1 H), 7.41-
7.60 (m, 3 H), 7.21 (s, 2 H), 3.09 (s, 3 H), 2.97 (s, 4 H), 2.60 (s,
3 H), 2.30 (s, 3 H), 1.08-1.27 (m, 5 H); HRMS m/z ) 499.1872
[M + H]+, calcd [C25H22F4N6O + H]+ ) 499.1864; HPLC tR A,
6.32 min; B, 1.56 min.
3-(2-(2-Aminopyrimidin-5-yl)ethynyl)-4-methyl-N-(2-(piperi-
din-1-yl)-5-(trifluoromethyl)phenyl)benzamide (6d) was prepared

634 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Cee et al.
5-(2-(2-Aminopyrimidin-5-yl)ethynyl)-N-(2-(4-(dimethylami-
no)piperidin-1-yl)-5-(trifluoromethyl)phenyl)-2-fluorobenz-
amide (6j) was prepared according to general method A starting
from N-(2-(4-(dimethylamino)piperidin-1-yl)-5-(trifluoromethyl)-
phenyl)-2-fluoro-5-iodobenzamide (0.22 g, 0.40 mmol) to give 6j
filtered, and concentrated in vacuo to give 6m (0.81 g, 65%) as a
white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.57-8.63
(m, 1 H), 8.45 (s, 2 H), 7.97-8.07 (m, 1 H), 7.69-7.81 (m, 1 H),
7.46-7.57 (m, 2 H), 7.40 (d, J ) 8.3 Hz, 1 H), 7.20 (s, 2 H),
3.01-3.13 (m, 1 H), 2.92-3.00 (m, 1 H), 2.69-2.81 (m, 1 H),
2.53-2.64 (m, 2 H), 2.09-2.22 (m, 3 H), 1.75-1.91 (m, 2 H),
1.59-1.71 (m, 1 H); HRMS m/z ) 535.1839 [M + Na]+, calcd
[C26H24F4N6O + Na]+ ) 535.1840. Anal. (C26H24F4N6O·0.75H2O)
C, H, N.
1
(0.038 g, 18%) as a light yellow solid: H NMR (400 MHz, DMSO-
d6) δ ppm 9.92 (d, J ) 8.5 Hz, 1 H), 8.60 (s, 1 H), 8.46 (s, 2 H),
8.04 (d, J ) 5.9 Hz, 1 H), 7.73-7.81 (m, 1 H), 7.42-7.56 (m, 3
H), 7.20 (s, 2 H), 3.09-3.18 (m, 2 H), 2.73 (t, J ) 10.9 Hz, 2 H),
2.21 (s, 7 H), 1.85-1.93 (m, 2 H), 1.57-1.70 (m, 2 H); HRMS
m/z ) 527.2188 [M + H]+, calcd [C27H26F4N6O + H]+ ) 527.2177.
Anal. (C27H26F4N6O·0.7H2O) C, H, N.
(S)-N-(2-(3-Aminopiperidin-1-yl)-5-(trifluoromethyl)phenyl)-
5-(2-(2-aminopyrimidin-5-yl)ethynyl)-2-fluorobenzamide (6n).
To a slurry of 9n (1.6 g, 2.6 mmol) in 5 mL of dioxane at 0 °C
was added a solution of HCl (4.0 M in dioxane, 6.5 mL, 26 mmol).
The clear yellow solution was allowed to warm to ambient
temperature and was stirred for 1 h. The reaction was concentrated
in vacuo to give (S)-N-(2-(3-aminopiperidin-1-yl)-5-(trifluoro-
methyl)phenyl)-2-fluoro-5-iodobenzamide dihydrochloride (1.6 g,
quant.) as a peach-colored solid: 1H NMR (400 MHz, DMSO-d6)
δ ppm 9.96 (d, J ) 3.2 Hz, 1 H), 8.48 (s, 1 H), 8.20 (s, 3 H), 8.12
(dd, J ) 6.6, 2.2 Hz, 1 H), 7.91-8.00 (m, 1 H), 7.54 (dd, J ) 8.3,
1.6 Hz, 1 H), 7.35 (d, J ) 8.5 Hz, 1 H), 7.26 (dd, J ) 10.3, 10.3
Hz, 1 H), 3.50 (s, 1 H), 3.22-3.32 (m, 1 H), 3.02-3.13 (m, 1 H),
2.91-3.00 (m, 1 H), 2.64-2.76 (m, 1 H), 1.61-2.01 (m, 4 H);
MS m/z ) 508 (M + H)+, calcd for C19H18F4IN3O ) 507.
In a 16 × 120 mm resealable pyrex tube, (S)-N-(2-(3-amino-
piperidin-1-yl)-5-(trifluoromethyl)phenyl)-2-fluoro-5-iodobenzamide
dihydrochloride (0.25 g, 0.43 mmol), 5-ethynylpyrimidin-2-amine
(0.10 g, 0.86 mmol), bis(triphenylphosphine)palladium(II) dichloride
(0.015 g, 0.022 mmol), and copper(I) iodide (0.0041 g, 0.022 mmol)
were taken up in CH3CN (5 mL) and triethylamine (0.91 mL), and
the tube was flushed with nitrogen. The tube was sealed and the
reaction heated to 70 °C for 3 h. The reaction was cooled, diluted
with EtOAc, and filtered through a pad of Celite with rinsing with
100 mL of EtOAc. The resulting clear yellow solution was extracted
twice with 1 N NaOH, dried over anhydrous sodium sulfate, filtered,
and concentrated to an orange oil. The material was dissolved in a
minimal amount of CH2Cl2 and purified by silica gel chromatog-
raphy (0-100% 90/10/1 CH2Cl2/MeOH/concentrated NH4OH in
CH2Cl2). The product-containing fractions were combined and
concentrated to afford 6n (0.13 g, 61%) as a pale yellow solid: 1H
NMR (400 MHz, DMSO-d6) δ ppm 8.62 (s, 1 H), 8.46 (s, 2 H),
7.98-8.08 (m, 1 H), 7.73-7.80 (m, 1 H), 7.46-7.56 (m, 2 H),
7.40 (d, J ) 8.0 Hz, 1 H), 7.20 (s, 2 H), 3.17 (d, J ) 2.9 Hz, 1 H),
2.99-3.06 (m, 1 H), 2.85-2.96 (m, 2 H), 2.64-2.77 (m, 1 H),
2.39-2.48 (m, 1 H), 1.76-1.90 (m, 2 H), 1.58-1.72 (m, 1 H),
1.12-1.27 (m, 1 H); HRMS m/z ) 499.1866 [M + H]+, calcd
[C26H24F4N6O + H]+ ) 499.1864. Anal. (C25H22F4N6O·1.7H2O)
C, H, N.
(R)-5-(2-(2-Aminopyrimidin-5-yl)ethynyl)-N-(2-(3-(dimethyl-
amino)piperidin-1-yl)-5-(trifluoromethyl)phenyl)-2-fluorobenzamide
(6k) was prepared according to general method A starting from
(R)-N-(2-(3-(dimethylamino)piperidin-1-yl)-5-(trifluoromethyl)phen-
yl)-2-fluoro-5-iodobenzamide (0.22 g, 0.42 mmol), to give 6k (0.18
g, 80%) as a off-white solid: 1H NMR (400 MHz, DMSO-d6) δ
ppm 9.90 (d, J ) 8.0 Hz, 1 H), 8.60 (s, 1 H), 8.46 (s, 2 H), 8.01-
8.06 (m, 1 H), 7.72-7.84 (m, 1 H), 7.42-7.60 (m, 3 H), 7.21 (s,
2 H), 3.12 (d, J ) 11.1 Hz, 1 H), 3.02 (d, J ) 11.0 Hz, 1 H),
2.54-2.74 (m, 2 H), 2.38-2.48 (m, 1 H), 2.14 (s, 6 H), 1.86-
1.97 (m, 1 H), 1.78-1.87 (m, 1 H), 1.61-1.75 (m, 1 H), 1.27-
1.42 (m, 1 H); HRMS m/z ) 549.1999 [M + Na]+, calcd
[C27H26F4N6O + Na]+ ) 549.1996. Anal. (C27H26F4N6O) C, H, N.
(S)-5-(2-(2-Aminopyrimidin-5-yl)ethynyl)-N-(2-(3-(dimethyl-
amino)piperidin-1-yl)-5-(trifluoromethyl)phenyl)-2-fluorobenzamide
(6l) was prepared according to general method A starting from (S)-
N-(2-(3-(dimethylamino)piperidin-1-yl)-5-(trifluoromethyl)phenyl)-
2-fluoro-5-iodobenzamide (0.15 g, 0.28 mmol), to give 6l (0.096
g, 66%) as a off-white solid: 1H NMR (400 MHz, DMSO-d6) δ
ppm 9.90 (d, J ) 8.0 Hz, 1 H), 8.60 (s, 1 H), 8.46 (s, 2 H), 8.01-
8.06 (m, 1 H), 7.72-7.84 (m, 1 H), 7.42-7.60 (m, 3 H), 7.21 (s,
2 H), 3.12 (d, J ) 11.1 Hz, 1 H), 3.02 (d, J ) 11.0 Hz, 1 H),
2.54-2.74 (m, 2 H), 2.38-2.48 (m, 1 H), 2.14 (s, 6 H), 1.86-
1.97 (m, 1 H), 1.78-1.87 (m, 1 H), 1.61-1.75 (m, 1 H), 1.27-
1.42 (m, 1 H); HRMS m/z ) 527.2191 [M + H]+, calcd
[C27H26F4N6O + H]+ ) 527.2177. Anal. (C27H26F4N6O·0.25H2O)
C, H, N.
(S)-5-(2-(2-Aminopyrimidin-5-yl)ethynyl)-2-fluoro-N-(2-(3-
(methylamino)piperidin-1-yl)-5-(trifluoromethyl)phenyl)benz-
amide (6m). In a 25-mL round-bottom flask, 9m (0.496 g, 0.80
mmol), 5-ethynylpyrimidin-2-amine (0.19 g, 1.6 mmol), bis-
(triphenylphosphine)palladium(II) dichloride (0.028 g, 0.040 mmol),
and copper(I) iodide (0.0076 g, 0.040 mmol) were taken up in
CH3CN (10 mL) and triethylamine (1.7 mL). The reaction was
flushed with nitrogen, sealed, and heated to 70 °C for 16 h. The
reaction was cooled and transferred to a larger flask with EtOAc
and concentrated under reduced pressure. The solid was adsorbed
onto 4 g of silica gel from 10% MeOH/CH2Cl2, dried, and purified
by silica gel chromatography (0-75% EtOAc/hexanes). Product-
containing fractions were combined and concentrated to afford (S)-
tert-butyl 1-(2-(5-(2-(2-aminopyrimidin-5-yl)ethynyl)-2-fluorobenz-
amido)-4-(trifluoromethyl )phenyl)piperidin-3-yl(methyl)carbamate
(0.40 g, 81%) as a orange foam: 1H NMR (400 MHz, DMSO-d6)
δ ppm 9.82-10.22 (m, 1 H), 8.61 (s, 1 H), 8.46 (s, 2 H), 8.07 (d,
J ) 6.1 Hz, 1 H), 7.72-7.83 (m, 1 H), 7.44-7.58 (m, 3 H), 7.21
(s, 2 H), 4.06-4.23 (m, 1 H), 2.97-3.13 (m, 1 H), 2.90 (d, J )
7.7 Hz, 2 H), 2.75 (s, 3 H), 2.54-2.67 (m, 1 H), 1.58-1.91 (m, 4
H), 1.33 (s, 9 H); MS m/z ) 613 [M + H]+, calcd for C31H32F4N6O3
) 612.
(R)-5-(2-(2-Aminopyrimidin-5-yl)ethynyl)-N-(2-(3-(dimethyl-
amino)pyrrolidin-1-yl)-5-(trifluoromethyl)phenyl)-2-fluorobenzamide
(6o) was prepared according to general method A starting from
(R)-N-(2-(3-(dimethylamino)pyrrolidin-1-yl)-5-(trifluoromethyl)phen-
yl)-2-fluoro-5-iodobenzamide (0.25 g, 0.45 mmol) to give 6o (0.041
g, 18%) as an off white solid: 1H NMR (400 MHz, DMSO-d6) δ
ppm 10.10 (s, 1 H), 8.44 (s, 2 H), 7.88 (dd, J ) 7.0, 2.2 Hz, 1 H),
7.66-7.74 (m, 1 H), 7.49 (d, J ) 2.2 Hz, 1 H), 7.38-7.46 (m, 2
H), 7.21 (s, 2 H), 6.88 (d, J ) 8.8 Hz, 1 H), 3.47-3.56 (m, 1 H),
3.37-3.47 (m, 2 H), 3.25-3.33 (m, 1 H), 2.59-2.72 (m, 1 H),
2.15 (s, 6 H), 2.04-2.13 (m, 1 H), 1.62-1.76 (m, 1 H); HRMS
m/z ) 535.1842 [M + Na]+, calcd [C26H24F4N6O + Na]+
535.1840; HPLC tR A, 6.80 min; C, 4.69 min.
)
To a yellow solution of (S)-tert-butyl 1-(2-(5-(2-(2-aminopyri-
midin-5-yl)ethynyl)-2-fluorobenzamido)-4-(trifluoromethyl)phenyl)-
piperidin-3-yl(methyl)carbamate (1.5 g, 2.5 mmol) in 15 mL of
CH2Cl2 at 0 °C was added TFA (0.94 mL, 12 mmol). The reaction
was allowed to warm to ambient temperature and was stirred
overnight. An additional 5 equiv of TFA was added, and after 3
days the reaction was cooled to 0 °C, and water was added, followed
by basification with 1 N NaOH. The organic layer was separated,
and the aqueous layer was extracted once with CH2Cl2. The
combined organic layers were dried over anhydrous sodium sulfate,
(S)-5-(2-(2-Aminopyrimidin-5-yl)ethynyl)-N-(2-(3-(dimethyl-
amino)pyrrolidin-1-yl)-5-(trifluoromethyl)phenyl)-2-fluorobenzamide
(6p) was prepared according to general method A starting from
(S)-N-(2-(3-(dimethylamino)pyrrolidin-1-yl)-5-(trifluoromethyl)phen-
yl)-2-fluoro-5-iodobenzamide (0.15 g, 0.288 mmol) to give 6p
(0.092 g, 61%) as an off-white solid: 1H NMR (400 MHz, DMSO-
d6) δ ppm 10.10 (s, 1 H), 8.44 (s, 2 H), 7.88 (dd, J ) 7.0, 2.2 Hz,
1 H), 7.66-7.74 (m, 1 H), 7.49 (d, J ) 2.2 Hz, 1 H), 7.38-7.46
(m, 2 H), 7.21 (s, 2 H), 6.88 (d, J ) 8.8 Hz, 1 H), 3.47-3.56 (m,
1 H), 3.37-3.47 (m, 2 H), 3.25-3.33 (m, 1 H), 2.59-2.72 (m, 1

Orally ActiVe Inhibitors of Tie-2 Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 635
H), 2.15 (s, 6 H), 2.04-2.13 (m, 1 H), 1.62-1.76 (m, 1 H); HRMS
volume was approximately 100 mL. The vessel was placed in a
Parr shaker, treated with 2 atm H2, and shaken overnight. The
reaction was flushed with nitrogen, and filtered through a pad of
Celite rinsing with 1.3 L of methanol, and the filtrate was
concentrated under reduced pressure. The oil was taken up in CH2-
Cl2, dried over Na2SO4, filtered, and concentrated under reduced
pressure to give 8l (12.1 g, 98%) as a red oil, which was used
without further purification: 1H NMR (400 MHz, DMSO-d6) δ
ppm 7.01 (d, J ) 8.1 Hz, 1 H), 6.95 (d, J ) 1.9 Hz, 1 H), 6.83 (d,
J ) 8.1 Hz, 1 H), 5.08 (s, 2 H), 3.21 (d, J ) 10.7 Hz, 1 H), 3.02
(d, J ) 11.5 Hz, 1 H), 2.29-2.47 (m, 3 H), 2.21 (s, 6 H), 1.85-
1.95 (m, 1 H), 1.72-1.81 (m, 1 H), 1.58-1.72 (m, 1 H), 1.17-
1.36 (m, 1 H); MS m/z ) 288 [M + H]+, calcd for C14H20F3N3 )
287.
(S)-tert-Butyl 1-(2-(2-Fluoro-5-iodobenzamido)-4-(trifluoro-
methyl)phenyl)piperidin-3-ylcarbamate (9n). To a 100-mL round-
bottom flask was added 15l (1.0 g, 2.6 mmol) and palladium, 10
wt % on activated carbon, 50% water (0.55 g) under nitrogen.
Methanol (10 mL) was added via syringe, and the atmosphere was
purged with hydrogen from a balloon. The reaction was allowed
to stir rapidly under hydrogen for 24 h. The flask was purged with
nitrogen, filtered through Celite, rinsing with 100 mL of MeOH,
and concentrated to give (S)-tert-butyl 1-(2-amino-4-(trifluoro-
methyl)phenyl)piperidin-3-ylcarbamate (0.92 g, 99%) as a gray
solid, which was used without further purification: 1H NMR (400
MHz, DMSO-d6) δ ppm 8.03-8.15 (m, 3 H), 7.93 (d, J ) 9.6 Hz,
1 H), 6.32 (s, 2 H), 4.68-4.86 (m, 1 H), 4.16 (d, J ) 13.1 Hz, 1
H), 3.86-3.96 (m, 1 H), 3.65-3.74 (m, 1 H), 3.47-3.57 (m, 1 H),
2.81-3.04 (m, 2 H), 2.66-2.77 (m, 1 H), 2.49-2.50 (s, 9 H), 2.37-
2.60 (m, 1 H); MS m/z ) 360 [M + H]+, calcd for C17H24F3N3O2
) 359.
In a screw-cap vial, (S)-tert-butyl 1-(2-amino-4-(trifluoromethyl)-
phenyl)piperidin-3-ylcarbamate (0.915 g, 2.5 mmol) was taken up
in dichloromethane (5 mL). Triethylamine (0.47 mL, 3.3 mmol)
and 2-fluoro-5-iodobenzoyl chloride (0.80 g, 2.8 mmol) were then
added. The tube was sealed and the reaction stirred overnight. The
reaction mixture was poured into EtOAc/saturated aqueous sodium
bicarbonate. The organic layer was dried over anhydrous sodium
sulfate and concentrated in vacuo. The resulting foam 9n (1.59 g,
quant.) was used without further purification: 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.90 (d, J ) 6.4 Hz, 1 H), 8.48 (s, 1 H), 8.19 (d,
J ) 5.4 Hz, 1 H), 7.93-8.03 (m, 1 H), 7.51 (d, J ) 8.3 Hz, 1 H),
7.36 (d, J ) 8.6 Hz, 1 H), 7.28 (dd, J ) 10.5, 9.2 Hz, 1 H), 6.97
(d, J ) 7.8 Hz, 1 H), 3.57-3.73 (m, 1 H), 3.04-3.20 (m, 1 H),
2.84-2.97 (m, 1 H), 2.61-2.76 (m, 1 H), 2.51-2.60 (m, 1 H),
1.74-1.87 (m, 2 H), 1.60-1.72 (m, 1 H), 1.34 (s, 9 H), 1.22-
1.47 (m, 1 H); MS m/z ) 608 [M + H]+, calcd for C24H26F4IN3O3
) 607.
(S)-tert-Butyl 1-(2-(2-Fluoro-5-iodobenzamido)-4-(trifluoro-
methyl)phenyl)piperidine-3-carboxylate (9m). To a 100-mL
round-bottom flask were added 15m (1.8 g, 4.3 mmol) and
palladium, 10 wt. % on activated carbon, 50% water (0.92 g) under
nitrogen. MeOH (12 mL) was added via syringe, and the atmosphere
was purged with hydrogen from a balloon. The reaction was allowed
to stir rapidly under hydrogen for 8 h. The flask was purged with
nitrogen, filtered through Celite, with rinsing with 100 mL of
MeOH, and concentrated to give (S)-tert-butyl 1-(2-amino-4-
(trifluoromethyl)phenyl)piperidin-3-yl(methyl)carbamate (1.44 g,
89%) as a gray solid, which was used without further purification:
1H NMR (400 MHz, DMSO-d6) δ ppm 7.02 (d, J ) 8.1 Hz, 1 H)
6.96 (d, J ) 1.8 Hz, 1 H) 6.83 (d, J ) 8.0 Hz, 1 H) 5.11 (s, 2 H)
3.92-4.19 (m, 1 H) 2.86-3.15 (m, 2 H) 2.74 (s, 3 H) 2.57-2.69
(m, 1 H) 2.28-2.47 (m, 1 H) 1.50-1.85 (m, 4 H) 1.40 (s, 9 H);
MS m/z ) 374 [M + H]+, calcd for C18H26F3N3O2 ) 373.
In a vial, (S)-tert-butyl 1-(2-amino-4-(trifluoromethyl)phenyl)-
piperidin-3-yl(methyl)carbamate (0.500 g, 1.3 mmol) was taken up
in CH2Cl2 (5 mL). The solution was cooled to 0 °C and
triethylamine (0.24 mL, 1.7 mmol) and 2-fluoro-5-iodobenzoyl
chloride (0.42 g, 1.5 mmol) were then added. The tube was sealed
and the reaction stirred for 2 h. The reaction mixture was poured
into EtOAc/1 N NaOH. The organic layer was dried over anhydrous
m/z ) 535.1838 [M + Na]+, calcd [C26H24F4N6O + Na]+
535.1840. Anal. (C26H24F4N6O‚0.1CH2Cl2) C, H, N.
)
1-(2-Amino-4-(trifluoromethyl)phenyl)-N,N-dimethylpiperi-
din-4-amine (8j). To 15j (3.4 g, 11 mmol) was added Pd/C (10%,
0.57 g) under nitrogen. Methanol (25 mL) was added via syringe,
H2 gas was introduced, and the mixture was stirred vigorously under
an atmosphere of H2. After 96 h, the mixture was flushed with
nitrogen, filtered through Celite, and concentrated. The residue was
resubjected to the reaction conditions. After 12 h, the reaction was
flushed with nitrogen, filtered through Celite and concentrated. The
resulting solid was triturated with methanol 10 times to give 8j
(0.67 g, 22%) as a pink solid: 1H NMR (400 MHz, DMSO-d6) δ
ppm 8.12 (d, J ) 1.8 Hz, 1 H), 7.82 (dd J ) 2.2, 8.8 Hz, 1 H),
7.41 (d, J ) 8.8 Hz, 1 H), 3.33-3.36 (m, 2 H), 2.94 (dd, J ) 11.7
Hz, 2 H), 2.28-2.30 (m, 1 H), 1.80-1.83 (m, 2 H), 1.47 (ddd, J
) 3.7, 12.5, 12.5 Hz, 2 H); MS m/z ) 288.2 (M + H)+, calcd for
C14H20F3N3 ) 287.3.
(R)-1-(2-Amino-4-(trifluoromethyl)phenyl)-N,N-dimethyl-
piperidin-3-amine (8k) was synthesized in a manner analogous
to 8l but starting from 15k (1.0 g, 2.6 mmol) to give 8k (0.80 g,
quant., three steps) as a red oil, which was used without further
purification: 1H NMR (400 MHz, DMSO-d6) δ ppm 7.01 (d, J )
8.1 Hz, 1 H), 6.95 (d, J ) 1.9 Hz, 1 H), 6.83 (d, J ) 8.1 Hz, 1 H),
5.08 (s, 2 H), 3.21 (d, J ) 10.7 Hz, 1 H), 3.02 (d, J ) 11.5 Hz, 1
H), 2.29-2.47 (m, 3 H), 2.21 (s, 6 H), 1.85-1.95 (m, 1 H), 1.72-
1.81 (m, 1 H), 1.58-1.72 (m, 1 H), 1.17-1.36 (m, 1 H); MS m/z
) 288 [M + H]+, calcd for C14H20F3N3 ) 287.
(S)-1-(2-Amino-4-(trifluoromethyl)phenyl)-N,N-dimethylpip-
eridin-3-amine (8l). A solution of 15l (15.6 g, 40 mmol) was cooled
to 0 °C, hydrochloric acid (4.0 M in dioxane, 80 mL, 321 mmol)
was added, and the mixture was allowed to warm to ambient
temperature. The orange solution was allowed to stir for 14 h, at
which point it was concentrated in vacuo to give (S)-1-(2-nitro-4-
(trifluoromethyl)phenyl)piperidin-3-amine dihydrochloride (13.4 g,
92%) as a yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm
9.96 (d, J ) 3.2 Hz, 1 H), 8.47 (s, 1 H), 8.20 (s, 3 H), 8.12 (dd, J
) 6.6, 2.2 Hz, 1 H), 7.90-8.00 (m, 1 H), 7.53 (dd, J ) 8.4, 1.8
Hz, 1 H), 7.34 (d, J ) 8.5 Hz, 1 H), 7.20-7.30 (m, 1 H), 3.43-
3.54 (m, 1 H), 3.26 (dd, J ) 11.6, 2.4 Hz, 1 H), 3.00-3.14 (m, 1
H), 2.94 (dd, J ) 11.5, 5.3 Hz, 1 H), 2.64-2.76 (m, 1 H), 1.81-
2.00 (m, 2 H), 1.61-1.80 (m, 2 H); MS m/z ) 290.0 [M + H]+,
calcd for C12H14F3N3O2 ) 289.
To a yellow solution of (S)-1-(2-nitro-4-(trifluoromethyl)phenyl)-
piperidin-3-amine dihydrochloride (13.4 g, 37 mmol) in 123 mL
of MeOH under nitrogen at 0 °C were added formaldehyde (37%
solution) (14 mL, 185 mmol), acetic acid (11 mL, 185 mmol), and
sodium cyanoborohydride (4.6 g, 74 mmol) in portions over 5 min.
The cloudy mixture was warmed to ambient temperature. After 10
min, the reaction became quite hot and was cooled with an ice
bath. The reaction was allowed to warm to ambient temperature,
and after 1.5 h, the reaction was judged complete by LC-MS. The
solvent was removed under reduced pressure, and the flask cooled
to 0 °C. Water was added, and the mixture was basified with 1 N
NaOH and 6 N NaOH. The mixture was extracted with 1 × 200
mL EtOAc and 1 × 100 mL EtOAc, and the combined organics
were dried over anhydrous Na2SO4 and concentrated under reduced
pressure to give (S)-N,N-dimethyl-1-(2-nitro-4-(trifluoromethyl)-
phenyl)piperidin-3-amine (13.6 g, quant.) as an orange oil, which
was used without further purification: 1H NMR (400 MHz, DMSO-
d6) δ ppm 8.59 (s, 1 H), 8.45 (s, 2 H), 7.98-8.05 (m, 1 H), 7.71-
7.80 (m, 1 H), 7.44-7.54 (m, 2 H), 7.40 (d, J ) 8.5 Hz, 1 H), 3.07
(d, J ) 10.1 Hz, 1 H), 2.87-2.98 (m, 1 H), 2.70-2.80 (m, 1 H),
2.53-2.65 (m, 2 H), 2.14 (s, 3 H), 1.76-1.89 (m, 2 H), 1.58-
1.71 (m, 1 H); MS m/z ) 318 [M + H]+, calcd for C14H18F3N3O2
) 317.
A 500-mL Parr pressure bottle was charged with palladium, 10
wt % on activated carbon, 50% water (9.1 g) under nitrogen. (S)-
N,N-Dimethyl-1-(2-nitro-4-(trifluoromethyl)phenyl)piperidin-3-
amine (13.6 g, 43 mmol) was added as a solution in methanol via
syringe, rinsing in with multiple methanol washes until the final

636 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Cee et al.
sodium sulfate and concentrated in vacuo. The resulting off-white
foam 9m (0.91 g, quant.) was used without further purification:
1H NMR (400 MHz, DMSO-d6) δ ppm 9.70-10.19 (m, 1 H), 8.57
(s, 1 H), 8.23 (d, J ) 2.1 Hz, 1 H), 7.90-8.08 (m, 1 H), 7.42-
7.60 (m, 2 H), 7.28 (dd, J ) 11.2, 8.8 Hz, 1 H), 4.04-4.22 (m, 1
H), 2.97-3.14 (m, 1 H), 2.89 (d, J ) 6.8 Hz, 2 H), 2.74 (s, 3 H),
2.52-2.65 (m, 1 H), 1.55-1.89 (m, 4 H), 1.33 (s, 9 H); MS m/z )
622 [M + H]+, calcd for C25H28F4IN3O3 ) 621.
1.0 mmol) to give 13d as white solid (0.18 g, 51%): 1H NMR
(400 MHz, acetone-d6) δ ppm 9.90 (s, 1 H), 9.04 (s, 1 H), 8.57 (s,
1 H), 8.34 (s, 1 H), 8.24 (s, 1 H), 8.13 (d, J ) 8.4 Hz, 1 H), 8.09
(d, J ) 8.4 Hz, 1 H), 8.00 (t, J ) 7.3 Hz, 2 H), 7.82 (t, J ) 7.1 Hz,
1 H), 7.68 (t, J ) 7.5 Hz, 1 H), 7.61 (t, J ) 7.9 Hz, 1 H), 7.52 (d,
J ) 8.1 Hz, 1 H), 7.47 (d, J ) 7.7 Hz, 1 H), 2.59-2.69 (m, 3 H);
HRMS m/z ) 431.1376 [M + H]+, calcd [C26H17F3N2O + H]+
)
431.1366. Anal. (C26H17F3N2O) C, H, N.
5-Ethynylpyrimidin-2-amine (11). Into a 1-L round-bottom
flask were placed 2-amino-5-iodopyrimidine (8.0 g, 36 mmol),
acetonitrile (300 mL), triethylamine (30 mL), TMS-acetylene (7.7
g, 78 mmol), PdCl2(PPh3)2 (1.3 g, 1.8 mmol), and CuI (0.34 g, 1.8
mmol). The vessel was filled with argon and allowed to stir at room
temperature for 3 h. The solvent was evaporated and the material
was taken up in methanol (400 mL). Excess potassium carbonate
(10 equiv) was added, and the mixture was stirred at room
temperature for 1.5 h. Activated charcoal was added and the mixture
was filtered through Celite. The filtrate was concentrated under
reduced pressure to afford a tan solid, which was added to a solution
of 10% methanol in water (200 mL). The resulting precipitate was
isolated by filtration and dried in a vacuum oven to constant mass
to afford 11 (3.5 g, 81%) as a tan solid: 1H NMR (400 MHz,
4-Methyl-3-(2-(quinoxalin-2-yl)ethynyl)-N-(3-(trifluorometh-
yl)phenyl)benzamide (13e) was prepared according to general
method B starting from 3-ethynyl-4-methyl-N-(3-(trifluoromethyl)-
phenyl)benzamide (0.17 g, 0.57 mmol) and 2-bromoquinoxaline31
(0.080 g, 0.38 mmol) to afford 13e as a white solid (0.15 g, 92%):
1H NMR (300 MHz, acetone-d6) ppm 9.94 (s, 1 H), 9.00-9.19
(m, 1 H), 8.34-8.37 (m, 1 H), 8.33 (d, J ) 2.2 Hz, 1 H), 8.13-
8.16 (m, 1 H), 8.10-8.13 (m, 1 H), 8.07-8.10 (m, 1 H), 8.05 (d,
J ) 1.9 Hz, 1 H), 7.87-7.97 (m, 2 H), 7.54-7.66 (m, 2 H), 7.47
(d, J ) 7.7 Hz, 1 H), 2.69 (s, 3 H); HRMS m/z ) 432.1319 [M +
H]+, calcd [C26H17F3N2O + H]+ ) 432.1318; HPLC tR A, 10.11
min; B, 2.30 min.
N,N-Dimethyl-1-(2-nitro-4-(trifluoromethyl)phenyl)piperidin-
4-amine (15j). To a mixture of 4-amino-1-benzylpiperidine (5.0
g, 26 mmol), NaBH3CN (3.3 g, 53 mmol), AcOH (7.5 mL, 132
mmol) in 130 mL of MeOH at 0 °C under nitrogen was added
formaldehyde (37 wt % in water, 5.3 mL) as a solution in 15 mL
of MeOH slowly dropwise via a pressure-equalized addition funnel
over 15 min. The resulting clear solution was allowed to warm to
ambient temperature and was allowed to stir for approximately 60
h. The reaction was quenched by the addition of 20 mL of saturated
aqueous potassium carbonate. The mixture was concentrated under
reduced pressure, and water and EtOAc were added. The organic
layer was removed, and the aqueous layer was extracted twice with
EtOAc. The combined organic layers were dried with Na2SO4,
filtered, and concentrated to give a cloudy oil, which was dissolved
in methylene chloride and filtered through a fritted funnel. The
solvent was removed to give a waxy solid, which was purified by
silica gel chromatography (DCM/MeOH/concentrated NH4OH). The
resulting material was dissolved in diethyl ether, cooled to 0 °C,
and treated with 20 mL of 4 N HCl in dioxane. The solvent was
removed in vacuo to give 1-benzyl-N,N-dimethylpiperidin-4-amine
dihydrochloride (7.02 g, 92%) as a white solid: MS m/z ) 219.1
(M + H)+, calcd for C14H22N2 ) 218.3.
1
CDCl3) δ ppm 8.43 (s, 2H), 5.21 (br s, 2H), 3.20 (s, H); MS m/z
) 120 [M + H]+, calcd for C6H5N3 ) 119.
3-Ethynyl-4-methyl-N-(3-(trifluoromethyl)phenyl)benz-
amide (12) was prepared according to general method B starting
with 3-iodo-4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide (1.5
g, 3.7 mmol) to afford 12 (0.44 g, 91%) as an off white solid: 1H
NMR (400 MHz, DMSO-d6) δ ppm: 10.55 (s, 1H), 8.24 (br s, 1H),
8.10 (d, J ) 1.90 Hz), 8.06 (d, J ) 9.06 Hz), 7.91 (dd, J ) 8.04,
1
1.90 Hz), 7.61 (t, J ) 7.97 Hz), 7.43-7.51 (m, 2H), 4.55 (s, H),
2.47 (s, 3H); MS m/z ) 302 [M - H]-, calcd for C17H12NO )
303.
3-(2-(6-Aminopyridin-3-yl)ethynyl)-4-methyl-N-(3-(trifluoro-
methyl)phenyl)benzamide (13a) was prepared according to general
method B starting from 3-ethynyl-4-methyl-N-(3-(trifluoromethyl)-
phenyl)benzamide (0.050 g, 0.33 mmol) to give 13a (0.009 g, 7%)
as an off white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 10.55
(s, 1 H), 8.25 (s, 1 H), 8.17 (s, 1 H), 8.02-8.12 (m, 2 H), 7.86 (d,
J ) 8.0 Hz, 1 H), 7.60 (t, J ) 6.9 Hz, 1 H), 7.55 (d, J ) 8.0 Hz,
1 H), 7.41-7.51 (m, 2 H), 6.48 (s, 2 H), 3.33 (s, 3 H); HRMS m/z
) 396.1326 [M + H]+, calcd [C22H16F3N3O + H]+ ) 396.1318;
HPLC tR A, 7.39 min; B, 1.84 min.
To 1-benzyl-N,N-dimethylpiperidin-4-amine dihydrochloride (6.7
g, 23 mmol) was added Pd/C (10%, 2.4 g) under argon. Methanol
(100 mL) was added via syringe, H2 gas was introduced, and the
mixture was stirred vigorously under an atmosphere of H2. After
48 h, the mixture was flushed with nitrogen, filtered through Celite,
and concentrated to afford a mixture of starting material and N,N-
dimethylpiperidin-4-amine dihydrochloride as a white solid. This
solid was treated with 1-fluoro-2-nitro-4-trifluoromethylbenzene (3.2
mL, 23 mmol), triethylamine (13 mL, 92 mmol), and 50 mL of
THF. The reaction was fitted with a water-cooled reflux condenser
and the mixture was heated to 75 °C for 12 h. The mixture was
allowed to cool to ambient temperature, filtered through a fritted
funnel, and concentrated to an orange oil. The residue was purified
by silica gel chromatography (DCM/MeOH/concentrated NH4OH)
to give 15j (3.4 g, 47%) as an orange oil: 1H NMR (400 MHz,
DMSO-d6) δ ppm 6.98 (d, J ) 7.7 Hz, 1 H), 6.94 (s, 1 H), 6.82 (d,
J ) 8.1 Hz, 1 H), 5.11 (s, 2 H), 3.15 (d, J ) 11.0 Hz, 2 H), 2.41-
2.57 (m, 2 H), 2.20 (s, 6 H), 2.10-2.29 (m, 1 H), 1.82 (d, J )
11.0 Hz, 2 H), 1.52-1.68 (m, 2 H); MS m/z ) 318.1 (M + H)+,
calcd for C14H18F3N3O2 ) 317.3.
(R)-tert-Butyl 1-(2-nitro-4-(trifluoromethyl)phenyl)piperidin-
3-ylcarbamate (15k) was prepared in an analogous manner to 15l
starting with (R)-3-N-Boc-aminopiperidine (0.97 g, 4.5 mmol) to
give 15k (1.8 g, 94%) as an orange solid: 1H NMR (400 MHz,
DMSO-d6) δ ppm 8.11 (s, 1 H), 7.83 (d, J ) 8.3 Hz, 1 H), 7.41 (d,
J ) 8.8 Hz, 1 H), 6.94 (d, J ) 6.6 Hz, 1 H), 3.40-3.51 (m, 1 H),
3.28-3.34 (m, 1 H), 3.13-3.22 (m, 1 H), 2.92 (dd, J ) 10.4 Hz,
1 H), 2.79 (dd, J ) 10.6, 10.6 Hz, 1 H), 1.80-1.91 (m, 1 H), 1.71-
3-(2-(3H-Imidazo[4,5-b]pyridin-6-yl)ethynyl)-4-methyl-N-(3-
(trifluoromethyl)phenyl)benzamide (13b) was prepared according
to general method B starting from 3-ethynyl-4-methyl-N-(3-
(trifluoromethyl)phenyl)benzamide (0.15 g, 0.48 mmol) and 6-bromo-
3H-imidazo[4,5-b]pyridine28 (0.070 g, 0.37 mmol) to give 13b as
a tan solid (0.031 g, 20%): 1H NMR (400 MHz, DMSO-d6) δ ppm
10.60 (s, 1 H), 8.61 (s, 1 H), 8.57 (s, 1 H), 8.23-8.33 (m, 2 H),
8.20 (s, 1 H), 8.09 (d, J ) 7.7 Hz, 1 H), 7.93 (d, J ) 8.1 Hz, 1 H),
7.61 (t, J ) 8.1 Hz, 1 H), 7.54 (d, 1 H), 7.47 (d, J ) 8.1 Hz, 1 H),
2.59 (s, 3 H); HRMS m/z ) 421.1275 [M + H]+, calcd
[C23H15F3N4O + H]+ ) 421.1271; HPLC tR A, 8.14 min; B, 2.10
min.
N-Methyl-5-(2-(2-methyl-5-((3-(trifluoromethyl)phenyl)car-
bamoyl)phenyl)ethynyl)picolinamide (13c) was prepared accord-
ing to general method B starting from 3-ethynyl-4-methyl-N-(3-
(trifluoromethyl)phenyl)benzamide (0.16 g, 0.54 mmol) and 5-bromo-
N-methylpicolinamide29 (0.089 g, 0.41 mmol) to give 13c as a white
solid (0.086 g, 48%): 1H NMR (400 MHz, acetone-d6) δ ppm 9.89
(s, 1 H), 8.79 (s, 1 H), 8.38 (s, 1 H), 8.33 (s, 1 H), 8.21 (d, J ) 1.5
Hz, 1 H), 8.17 (s, 2 H), 8.12 (d, J ) 8.1 Hz, 1 H), 8.01 (dd, J )
7.9, 1.6 Hz, 1 H), 7.61 (t, J ) 7.9 Hz, 1 H), 7.52 (d, J ) 8.1 Hz,
1 H), 7.47 (d, J ) 7.7 Hz, 1 H), 2.98 (d, J ) 4.8 Hz, 3 H), 2.61 (s,
3H);HRMSm/z)460.1247[M+Na]+,calcd[C24H18F3N3O2+Na]+
) 460.1243. Anal. (C24H18F3N3O2·0.75H2O) C, H, N.
4-Methyl-3-(2-(quinolin-3-yl)ethynyl)-N-(3-(trifluoromethyl)-
phenyl)benzamide (13d) was prepared according to general method
A starting from 3-iodo-4-methyl-N-(3-(trifluoromethyl)phenyl)-
benzamide (0.32 g, 0.80 mmol) and 3-ethynylquinoline30 (0.16 g,

Orally ActiVe Inhibitors of Tie-2 Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 637
1.80 (m, 1 H), 1.47-1.63 (m, 1 H), 1.37 (s, 9 H), 1.25-1.46 (m,
1 H); MS m/z ) 390 [M + H]+, calcd for C17H20F3N3 ) 389.
0.01 mM ATP (0.02 µL; commercially available from Sigma-
Aldrich Co.), and water (15.84 µL) to bring the total volume to 20
µL/well.
(S)-tert-Butyl 1-(2-Nitro-4-(trifluoromethyl)phenyl)piperidin-
3-ylcarbamate (15l). To a 200-mL round-bottom flask were added
(S)-3-N-Boc-aminopiperidine (10.9 g, 54.4 mmol), sodium bicar-
bonate (11.4 g, 136 mmol), THF (100 mL), and 1-fluoro-2-nitro-
4-(trifluoromethyl)benzene (7.62 mL, 54.4 mmol). The reaction was
fitted with a water-cooled reflux condensor and the yellow mixture
was heated to 70 °C. After 14 h, the mixture was cooled to ambient
temperature and filtered through a glass frit, with rinsing with
EtOAc. Concentration under reduced pressure afforded an orange
oil, which crystallized on standing to an orange solid. The material
was treated with 250 mL of hexanes and heated to 60 °C. Small
amounts of EtOAc were added until all solid dissolved, with a total
volume of 10 mL of EtOAc. The solution was allowed to cool
overnight, resulting in the formation of orange crystals. The liquid
was decanted, and the crystals were rinsed twice with 50 mL of
hexanes. The crystals were collected, and the filtrate was concen-
trated to an orange solid, which was recrystallized to give a total
of 15l (18.3 g, 86%) as orange crystals: 1H NMR (400 MHz,
DMSO-d6) δ ppm 8.11 (s, 1 H), 7.83 (d, J ) 8.3 Hz, 1 H), 7.41 (d,
J ) 8.8 Hz, 1 H), 6.94 (d, J ) 6.6 Hz, 1 H), 3.40-3.51 (m, 1 H),
3.28-3.34 (m, 1 H), 3.13-3.22 (m, 1 H), 2.92 (dd, J ) 10.4 Hz,
1 H), 2.79 (dd, J ) 10.6, 10.6 Hz, 1 H), 1.80-1.91 (m, 1 H), 1.71-
1.80 (m, 1 H), 1.47-1.63 (m, 1 H), 1.37 (s, 9 H), 1.25-1.46 (m,
1 H); MS m/z ) 390 [M + H]+, calcd for C17H20F3N3 ) 389.
(S)-tert-Butyl Methyl(1-(2-nitro-4-(trifluoromethyl)phenyl)-
piperidin-3-yl)carbamate (15m). To an orange solution of (S)-
tert-butyl 1-(2-nitro-4-(trifluoromethyl)phenyl)piperidin-3-ylcar-
bamate (1.5 g, 3.9 mmol) in DMF (13 mL) at 0 °C was added
sodium hydride as a 60% dispersion in mineral oil (0.19 g, 4.8
mmol). Bubbling was observed, and the solution became darker
orange. After 20 min, iodomethane (0.30 mL, 4.8 mmol) was added
dropwise via syringe. The orange mixture was allowed to warm to
room temperature over 30 min. Water was added, followed by
diethyl ether. The organics were washed once with water and with
brine, dried over anhydrous sodium sulfate, filtered, and concen-
trated under reduced pressure to give 15m (1.8 g, quant.) as an
orange semisolid which was used without further purification: 1H
NMR (400 MHz, DMSO-d6) δ ppm 8.14 (s, 1 H), 7.84 (d, J ) 8.2
Hz, 1 H), 7.45 (d, J ) 9.0 Hz, 1 H), 3.82-3.99 (m, 1 H), 3.02-
3.30 (m, 3 H), 2.87-2.95 (m, 1 H), 2.74 (s, 3 H), 1.51-1.85 (m,
4 H), 1.40 (s, 9 H); MS m/z ) 404 [M + H]+, calcd for
C18H24F3N3O4 ) 403.
The reaction was initiated in each well by adding 20 µL per
well of an enzyme preparation consisting of 50 mM Hepes (1.0
µL; from a 1000 mM stock solution commercially available from
Gibco Co.), 0.05% BSA (0.1 µL), 4 mM DTT (0.08 µL; from a
1000 mM stock solution available from Sigma-Aldrich Co.), a
2.4 × 10-7 M Tie-2 (0.02 µL, from a 4 mM concentration stock),
and water (18.8 µL) to dilute the enzyme preparation to a total
volume of 20 µL. The plate was incubated for about 90 min at
room temperature. After incubation, 160 µL of a filtered detection
mixture [prepared from 0.001 mg/mL of SA-APC (0.0765 µL;
available as a 2.09 mg/mL stock solution from Gibco), 0.03125
nM Eu-Ab (0.1597 µL; available in a 31.3 nM stock solution from
Gibco), and water to bring the total volume to 160 µL (159.73
µL)] was added to each well to stop the reaction therein. The plate
was then allowed to equilibrate for about 3 h and read on a Ruby
Star fluorescent reader (available from BMG Technologies, Inc.)
using a four-parameter fit using activity base to calculate the
corresponding IC50’s for the test and standard compounds in each
well.
IC50’s for the inhibition of the KDR kinase enzyme for individual
compounds were measured using an HTRF assay, utilizing the
procedure described above for the Tie-2 kinase enzyme with the
following changes: 1 µM gastrin (Biotin long chain EEEEAYG-
WLDF), 1 µM ATP, 50 pM His-tagged bisphosphorylated KDR
kinase domain, 90 min reaction, 30 min after quench equilibrium;
buffers, 50 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MgCl2, 5
mM MnCl2, 0.05% BSA, 2 mM DTT.
Conditions for All Other Enzyme Assays. Peptide substrate
sequences (all substrates at 1 µM unless stated otherwise) were as
follows: gastrin, biotin (long chain) EEEEAYGWLDF free acid;
HS-1B, biotin (long chain) EQEDEPEGIYGVLF free acid; HER-
2, biotin (long chain) GGMEDIYFEFMGGKKK free acid; ATF2,
biotinylated natural protein substrate, kinase dead, N-term Avitag;
PLK, biotin (long chain) AGAGRRRSLLELHKR free acid;
CDK_1, biotin (long chain) VIPINGSPRTPRRGQNR free acid;
GFAP, biotin (long chain) RRRITSAARRS free acid; poly EY,
from Sigma (P7244), poly(Glu, Tyr) sodium salt (4:1). The
following peptides/enzyme constructs were used: PDGF-poly EY/
GST KD, TrkA-gastrin/GST KD, EpHB4-HS-1B/GST KD, Lck-
gastrin/GST KD, Lyn-gastrin/GST FL, CSK-HS-1B/GST FL,
Fgr-gastrin/GST KD, Fes-gastrin/GST KD, INSR-gastrin/GST
KD, cMet-gastrin/GST KD, Jak3-HER-2/GST KD, Jak2-HER-
2/GST KD, Jak1-Tyk2 peptide/GST KD, cKit-HER-2/His KD,
Itk-HS-1B/GST KD, p38R-ATF2 (100 nM)/His FL, CDK5-
CDK_1/(100 nM)/His FL (KD ) kinase domain; FL ) full length;
GST ) glutathione-S-transferase tagged; His ) His tagged). All
assays were quenched within the linearity of the enzyme. All assays
were run at the Km for ATP. Buffer conditions for all assays are
based on the Tie-2 example with minor variations. PDGF was
determined by a radioactive filtration assay. MAPH filter plates
and 1 Ci/mmol 33P were used.
Cell-Based Tie-2 Autophosphorylation Assay. The IC50 of the
test compounds for inhibition of Tie-2 autophosphorylation was
determined using a plate-based immunoassay using the Delfia
detection platform. EA.hy926 cells were cultured in a growth
medium solution containing DMEM supplemented with 10% FBS
serum and penicillin-streptomycin-glutamine. The cells were
plated in 24-well tissue culture plates at a final cell density of 2 ×
105 cells/well. The cells were incubated for 5 h at 37 °C. Medium
was then removed, and the cells were washed twice with 500 µL
of PBS at room temperature. A 500-µL portion of F12 nutrient
mixture supplemented with 0.5% FBS was then added to each well,
and the cells were incubated at 37 °C overnight. Immediately prior
to the addition of test compound, medium was replaced with a
preparation of 500 µL of DMEM plus 1% BSA.
Solubility Determination. Aqueous solubility was determined
according to an automated procedure.32
Plasma Protein Binding. Mouse plasma protein binding was
determined in filtered mouse plasma by ultrafiltration33 at 37 °C
with a 10 µM concentration of 6l.
Microsomal Incubations. Rat and mouse liver microsomes were
incubated at 0.1 mg/mL protein,1.0 µM substrate, and 1 mM
NADPH in pH 7.4 phosphate buffer at 37 °C. The reaction was
stopped with 0.5% formic acid in acetonitrile at 0, 5, 15, 30, and
45 min and analyzed by LC-MS/MS.
Homogeneous Time-Resolved Fluorescence Enzyme Assays.
All kinase assays were run at the Km for ATP and quenched within
the linearity of the enzyme. All enzyme data described were an
average of two determinations, and the Tie-2 data were commonly
an average of four determinations. IC50’s for the inhibition of the
Tie-2 kinase enzyme for individual compounds were measured
using an HTRF assay, utilizing the following procedure: In a 96-
well plate (available from Costar Co.) was placed 1 µL of each
test and standard compound per well in 100% DMSO having a 25
µM final compound concentration (3-fold, 10-point dilution). To
each well was added 20 µL of a reaction mixture formed from
Tie-2 (4.0 µL; of a 10 mM stock solution available from Gibco),
0.05% BSA (0.1 µL; from a 10% stock solution available from
Sigma-Aldrich Co.), 0.002 mM BLC HER-2 KKK (biotinylated
long chain peptide; 0.04 µL; from a 0.002 mM stock solution),
Anti-hTie2 antibody (100 µg, R & D Systems, Inc., AF313) was
diluted with 10 mL of ice-cold PBS to prepare a 10 µg/mL antibody
concentration stock. A 96-well microplate (Perkin-Elmer Wallac,

638 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Cee et al.
AAAND-0001) was coated with 100 µL of the anti-Tie-2 antibody
stock and the plate was stored at 4 °C overnight. The plates were
washed three times with 200 µL of PBS 0.1% Tween20, and
blocked for 1 h by 5% BSA. The plates were then stored at room
temperature for about 1 h.
500-0121) and the concentration adjusted to 10 mg/mL by dilution
in complete RIPA lysis buffer.
Tie-2 was immunoprecipitated from 2 mg of total protein
overnight at 4 °C by 4 µg anti-murine Tie-2 antibody (R&D,
AF762) in the presence of 50 µL of protein A/G beads (Pierce,
20421). The protein A/G beads were recovered by centrifugation
and washed four times in RIPA buffer containing 300 µM NaVO4
and a protease inhibitor cocktail (Sigma-Aldrich, P8340). The beads
were resuspended in 50 µL of 2× sample buffer and resolved on
a 10% polyacrylamide gel under denaturing and reduced conditions.
The proteins were then transferred to a nitrocellulose membrane.
The membrane was then blocked overnight in blocking buffer
(0.25% gelatin in TBST, 20 mM Tris-HCl pH 7.4, 150 mM NaCl,
0.1% Tween 20). The nitrocellulose filter was then probed with
anti-phosphotyrosine 4G10 antibody at a concentration of 0.5 µg/
mL in blocking buffer for 2 h at room temperature. The filter was
washed three times in blocking buffer and probed with horseradish
peroxidase conjugated anti-mouse IgG (Amersham, NA931V,
1:3000 dilution) for 1 h. The filter was washed three times in
blocking buffer. Phosphorylated Tie-2 was detected using a
chemiluminescent method and quantified using a chemiluminescent
imaging system (Bio-Rad). Images were recorded on Kodak
scientific imaging film.
A 20-µL aliquot of a selected Tie-2 reference compound was
placed in a selected well of a 96-well plate, diluted 1:4 with 100%
DMSO from an initial concentration of 10 mM to a final
concentration of 2.5 mM, and then diluted 1:3 with 100% DMSO
for a 10-point dilution to a final concentration of 0.128 µM. Test
compounds (10 µL of a 10 mM concentration) were similarly
diluted 1:4 with 100% DMSO to obtain a sample concentration of
2.5 mM and then diluted 1:3 for a 10-point dilution to finally obtain
a concentration of 0.128 µM for each test compound. DMSO (20
µL) served as a positive control, and 10 µL of the 2.5 mM reference
compound served as the negative control. A 2-µL aliquot from each
well (test compounds and positive and negative controls) in the
96-well plate was added to designated wells in the 24-well cell
culture plate (1:250 dilution). The culture plate was incubated for
2.5 h at 37 °C. Tie-2 autophosphorylation was then stimulated by
the addition of angiopoietin 1. Recombinant human Ang1 was
diluted to a final concentration of 10 µg/mL in 1% BSA/DMEM
and stored on ice. hAng1 (5 µL of 10 µg/mL) was added to each
well of the 24-well plate containing the EA.hy926 cells. The plate
was then shaken at 700 rpm at 37 °C for about 2.5 min. After
shaking, the wells were incubated for a further 7.5 min at 37 °C.
The medium was removed and 400 µL of ice cold PBS + 300 µM
NaVO4 was added. The wells were kept on ice for at least 5 min
and washed one time with ice-cold PBS containing 300 µM NaVO4.
The cells were lysed with 150 µL of RIPA, containing 300 µM
NaVO4 and a protease inhibitor cocktail (Sigma-Aldrich, P8340).
The cells were lysed on ice for 30 min.
Cell lysate (140 µL) was added to the antibody-coated plate and
the plate was incubated at 4 °C for 2 h. The lysate was removed
and the plate was washed three times each with 400 µL of Delfia
wash buffer. The plate was tap-dried with a paper towel. The anti-
phosphotyrosine antibody 4G10 (Upstate, 05-321) was diluted with
Delfia assay buffer to make a solution of about 1 µg/mL. Diluted
antibody (100 µL) was added to the plate and the plate was
incubated at room temperature for 1 h. The plate was again washed
three times with 400 µL of wash buffer. Eu-N1-labeled anti-mouse
antibody (Perkin-Elmer Wallac, AD0124) was diluted with Delfia
assay buffer to a final concentration of 0.1 µg/mL. Diluted antibody
(100 µL) was added to each well of the plate and the plate was
incubated at room temperature for 1 h. The plate was washed three
times as described above. Delfia enhancement solution (100 µL,
Perkin-Elmer Wallac, 1244-105) was added to each well and the
plate was incubated at room temperature for 5 min in the dark.
The europium signal was measured with a Victor multilabel counter
(Wallac Model 1420). Raw data were analyzed using a fit equation
in XLFit. IC50 values were then determined using Grafit software.
Tie-2 Pharmacodynamic Assay. The effect of 6l on Tie-2
phosphorylation was evaluated in the lungs of female CD-1 NU/
NU mice (n)3/group). 6l was administered by oral gavage at 10,
30, and 100 mg/kg at the 0 hour time point. Tie-2 phosphorylation
was induced by recombinant human Angiopoietin-1 (R&D Systems
Inc. Minneapolis, MN). 12 µg of Angiopoietin-1 was administered
i.v. 15 minutes prior to the 3 hour time point. Then the lungs were
immediately dissected and snap frozen in liquid nitrogen. Blood
samples were taken by cardiac puncture to determine compound
concentrations in the plasma.
Individual mouse lungs were homogenized in 0.75 mL of RIPA
buffer containing 300 µM NaVO4 and a protease inhibitor cocktail
(Sigma-Aldrich, P8340). The insoluble debris was removed fol-
lowing centrifugation at 4 °C for 15 min at 14 000 rpm. The
supernatants were then precleared with 50 µL of protein A/G
agarose beads (Sigma) for 1 h at 4 °C. The supernatants were then
centrifuged at 3000 rpm for 2 min and the supernatants decanted
into a fresh Eppendorf. The protein concentration of each lysate
was determined using the colorimetric RC DC system (Bio-Rad,
The filter was then stripped and the filter was then probed with
the anti-murine Tie-2 antibody (R&D, AF762) for 2 h at room
temperature. The blot was washed three times in wash buffer and
incubated with horseradish peroxidase linked anti-goat Ig (Dako-
Cytomation, P0160, 1:3000 dilution) for 1 h at room temperature.
The filter was washed three times in blocking buffer. Total Tie-2
was detected using a chemiluminescent method and quantified using
a chemiluminescent imaging system (Bio-Rad). Images were
recorded on Kodak scientific imaging film.
Pharmacokinetic Studies. Male Sprague-Dawley rats with
surgically implanted femoral vein and jugular vein cannulae were
obtained from Charles River Laboratories (Boston, MA). Animals
were fasted overnight, and the following day compounds were
administered either by oral gavage or by intravenous bolus injection
(N ) 3 animals per study). Oral and iv formulations were made
24-48 h prior to dosing, while intravenous formulations were made
on the day of dosing. Blood samples were collected over 24 h via
jugular cannula into a heparinized tube. Following centrifugation,
plasma samples were stored in a freezer to maintain -70 °C until
analysis. Lithium-heparinized plasma samples (50 µL) were
precipitated with 100% acetonitrile containing the internal standard
(IS). The supernatant was transferred into a 96-well plate, and an
aliquot of 20 µL was injected onto an LC-MS/MS system. The
analytes were separated by a reversed-phase on a C-18 analytical
column. The analyte ions were generated by an electrospray
ionization (ESI) source and detected by a Sciex API3000 triple
quadrupole mass spectrometer operated in the multiple reaction
monitoring (MRM) mode. Study sample concentrations were
determined from a weighted (1/x2) linear regression of peak area
ratios (analyte peak area/IS peak area) versus the theoretical
concentrations of the calibration standards. Pharmacokinetic pa-
rameters were calculated using a small molecules discovery assay
(SMDA) within the computer program Watson (InnaPhase).
Acknowledgment. We thank Julie Belzile and Perry Novak
for a large-scale synthesis of 11, and Jay Larrow for a large-
scale synthesis of 15l.
Supporting Information Available: Elemental analysis, HPLC
data, and metabolite identification from microsomal incubation of
6l. This material is available free of charge via the Internet at http://
pubs.acs.org.

Orally ActiVe Inhibitors of Tie-2 Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 639
F.; Sun, J.-R.; Zhang, N.; Lu, J.; Estrada, J.; Kumar, R.; Coxon, A.;
Kaufman, S.; Pretorius, J.; Scully, S.; Cattley, R.; Payton, M.; Coats,
S.; Nguyen, L.; Desilva, B.; Ndifor, A.; Hayward, I.; Radinsky, R.;
Boone, T.; Kendall, R. Suppression of angiogenesis and tumor growth
by selective inhibition of angiopoeitin-2. Cancer Cell 2004, 6, 507-
516.
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