Synthesis and biological profile of the pan-vascular endothelial growth factor receptor/tyrosine kinase with immunoglobulin and epidermal growth factor-like homology domains 2 (VEGF-R/TIE-2) inhibitor 11-(2-Methylpropyl)-12, 13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4 H -indazolo[5,4-a]pyrrolo[3,4-c] carbazol-4-one (CEP-11981): A novel oncology therapeutic agent

Article abstract of DOI:10.1021/jm201449n

A substantial body of evidence supports the utility of antiangiogenesis inhibitors as a strategy to block or attenuate tumor-induced angiogenesis and inhibition of primary and metastatic tumor growth in a variety of solid and hematopoietic tumors. Given the requirement of tumors for different cytokine and growth factors at distinct stages of their growth and dissemination, optimal antiangiogenic therapy necessitates inhibition of multiple, complementary, and nonredundant angiogenic targets. 11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8- (pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (11b, CEP-11981) is a potent orally active inhibitor of multiple targets (TIE-2, VEGF-R1, 2, and 3, and FGF-R1) having essential and nonredundant roles in tumor angiogenesis and vascular maintenance. Outlined in this article are the design strategy, synthesis, and biochemical and pharmacological profile for 11b, which completed Phase I clinical assessing safety and pharmacokinetics allowing for the initiation of proof of concept studies.

Full text of DOI:10.1021/jm201449n

Article
pubs.acs.org/jmc
Synthesis and Biological Profile of the pan-Vascular Endothelial
Growth Factor Receptor/Tyrosine Kinase with Immunoglobulin and
Epidermal Growth Factor-Like Homology Domains 2 (VEGF-R/TIE-2)
Inhibitor 11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-
2
-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one
(
CEP-11981): A Novel Oncology Therapeutic Agent
Robert L. Hudkins,* Nadine C. Becknell, Allison L. Zulli, Ted L. Underiner, Thelma S. Angeles,
Lisa D. Aimone, Mark S. Albom, Hong Chang, Sheila J. Miknyoczki, Kathryn Hunter, Susan Jones-Bolin,
Hugh Zhao, Edward R. Bacon, John P. Mallamo, Mark A. Ator, and Bruce A. Ruggeri*
Discovery Research, Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380, United States
ABSTRACT: A substantial body of evidence supports the utility of antiangiogenesis inhibitors
as a strategy to block or attenuate tumor-induced angiogenesis and inhibition of primary and
metastatic tumor growth in a variety of solid and hematopoietic tumors. Given the requirement
of tumors for different cytokine and growth factors at distinct stages of their growth and
dissemination, optimal antiangiogenic therapy necessitates inhibition of multiple, comple-
mentary, and nonredundant angiogenic targets. 11-(2-Methylpropyl)-12,13-dihydro-2-methyl-
8
-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (11b, CEP-11981)
is a potent orally active inhibitor of multiple targets (TIE-2, VEGF-R1, 2, and 3, and FGF-R1)
having essential and nonredundant roles in tumor angiogenesis and vascular maintenance.
Outlined in this article are the design strategy, synthesis, and biochemical and pharmacological
profile for 11b, which completed Phase I clinical assessing safety and pharmacokinetics allowing for the initiation of proof of
concept studies.
INTRODUCTION
■
Angiogenesis, the development of new blood vessels from the
endothelium of a pre-existing vasculature, is a critical process
required by the majority of solid tumors to maintain localized
1
growth and metastatic dissemination within the host. The
clinical application of kinase inhibitors and antivascular
endothelial growth factor (VEGF) antibodies to halt angio-
genesis in tumors has been validated as a therapeutic strategy
2
3
by positive clinical results with bevacizumab, sorafenib 1,
4
5
sunitinib 2, and pazopanib 3 (Figure 1). The dual vascular
endothelial growth factor receptor kinase 2/fibroblast growth
factor receptor 1 (VEGF-R2/FGF-R1) inhibitor 4 (brivanib,
6
BMS-582664) advanced to phase III. There is a substantial
body of literature detailing the utility of antiangiogenesis
inhibitors as a strategy to block or attenuate tumor-induced
angiogenesis and inhibit primary and metastatic tumor growth
in a variety of solid and hematopoietic tumor models. Given the
heterogeneity and organ-specificity of the tumor-associated
vasculature and the requirement of tumors for different
cytokine and growth factors at distinct stages of their growth
and dissemination, optimal antiangiogenic therapies may
necessitate the inhibition of multiple, complementary, and
Figure 1. Structures of angiogenesis inhibitors.
phases spatially and temporally in the angiogenic process in
coordinated and complementary roles to that of the VEGF−
VEGF-R kinase axes. Principal among these is the angiopoietin
7
nonredundant angiogenic targets. Several angiogenic ligands
and their corresponding receptor tyrosine kinases have been
implicated in angiogenesis and vasculogenesis, acting at distinct
Received: October 26, 2011
Published: December 12, 2011
©
2011 American Chemical Society
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Figure 2. First and second generation angiogenesis inhibitors.
family of angiogenic growth factors (Ang-1−4) and their
endothelial cell-specific receptor tyrosine kinases, tyrosine
kinase with immunoglobulin and EGF-like domains 1 and 2
CHEMISTRY
■
The 8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-
c]carbazol-4-one analogues were synthesized from 5-cyano-4-
ethoxycarbonyl-12,13-dihydro-2-methyl-indazolo[5,4-a]-
carbazole intermediate 7. The DHI core and intermediate 7
were constructed via a regioselective Diels−Alder reaction as
described in detail previously. The key transformation to
11
(
TIE-1 and TIE-2), which have been implicated in vessel
1
3
stabilization, maturation, and remodeling, and the proper
8
hierarchical organization of the rudimentary vasculature.
14
The inhibition of tumor angiogenesis and vascular
remodeling achieved in nonclinical studies by modulating the
angiopoietin-TIE-2 axis alone, and in concert with the VEGF−
VEGF-R2 axis, has been demonstrated using biochemical,
molecular, and small molecule-based approaches against a
elaborate the N position for SAR development was the efficient
N-alkylation of 7 to cyano-esters 8a−c using NaOH in acetone,
without hydrolysis of the ester or nitrile. Alkylation at the lactam
stage produced multiple inseparable mixtures including the
mono and N,N-dialkylated products. Intermediates 8a−c were
selectively nitrated at the C-8 position to give 9a−c using exactly
two equivalents of HNO in AcOH at 80 °C for 1 h. A one-
pot, reductive cyclization procedure was developed to convert
the nitro-cyano-esters 9a−c (Raney-Ni/H in DMF-MeOH) to
the amino-lactam intermediates 10a−c. Anilines 10a−c were
reacted with 2-chloropyrimidines, 4-chloro-pyridine, or 2-
chloropyridazine at n-butanol or t-butanol reflux to efficiently
produce the target amino-heteroaryls 11a−j (Scheme 1). The
9
variety of human tumors. Application of adenoviral delivered
anti-TIE-2 and anti-VEGF-R2 have also demonstrated
improved activity when both the TIE-2 and VEGF-R2 signaling
pathways are targeted relative to targeting each pathway
14a
3
1
0
alone. Inhibition of multiple angiogenic targets such as
VEGF-R2 and TIE-2 are currently being evaluated clinically
2
14d
1
1
and preclinically.
Previously, we reported on our first generation VEGF-R
12
candidate 5b (CEP-7055), a prodrug of 5a (CEP-5214).
Indenocarbazole 5a had potent activity for VEGF-R1, R2, and
R3 (Figure 2) but had weak activity for TIE-2 (IC > 10 μM)
a
5
0
Scheme 1.
12a
as well as several other angiogenesis targets. The objective
for a second generation follow-on compound was to design in
TIE-2 activity and advance a broad-based angiogenesis inhibitor
with superior biochemical, pharmacokinetic (PK), pharmaco-
dynamic and in vivo antitumor efficacy compared to that of 5b.
As outlined in a series of publications, structural modification to
the indenocarbazole core identified the N2-methyl-12,13-
dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazole scaffold (DHI)
with improved dual activity and PK properties for further
1
3
lead optimization. Modification to the 8-position identi-
fied several series and leads with cellular potency, PK, and in
1
3d
vivo antitumor efficacy such as tetrahydropyrans (THP),
1
3c
13b
13a
oximes, thienyl ketones, and carbamates. The isopropyl
carbamate 6 met the dual TIE-2/VEGF-R2 enzyme (VEGF-R2
IC50 = 5 nM; TIE-2 = 11 nM) and cellular potency criteria
(
VEGF-R2 cell IC50 < 10 nM) and showed significant dose-
a
Reagents and conditions: (a) alkyl iodide, NaOH, acetone, reflux,
related antitumor efficacy at oral doses as low as 0.3 mg/kg
1
8 h, 87−93% ; (b) HNO , AcOH, 80 °C, 1 h 70−87%; (c) H , Raney-
3
2
1
3a
bid. However, 6 suffered from suboptimal pharmacokinetic
properties and showed dose-limited toxicity that prevented
further advancement. An additional concern for 6 and the
carbamate/urea series was the theoretical toxicity associated
with the potential aniline metabolite. Therefore, motivated to
remove the aniline red flag and improve the pharmacokinetic
properties, a series of constrained pyrimidinyl-2-amines were
designed as bioisosteric replacements for the carbamate/urea
moiety. In this article, we report the synthesis and structure−
activity relationships (SAR) for the 2-pyrimidin-2-ylamine-DHI
series and the identification of the clinical candidate 11b.
Ni, DMF-MeOH, 81−97%; (d) heteroaryl-chloride, 1-butanol, reflux,
40−65%.
1-methyl regioisomer 15 was synthesized for comparison in an
analogous manner starting with 5-(1H-indol-2-yl)-1-methyl-6,7-
dihydro-1H-indazole 12 (Scheme 2).
13c,d,14a−c
RESULTS AND DISCUSSION
■
In Vitro VEGF-R2 and TIE-2 Structure−Activity
Relationships. The pyrimidin-2-ylamino DHI analogues were
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a
Scheme 2.
was 95%, markedly lower than that of approved kinase
inhibitors such as dasatinib, sorafenib, lapatanib, or nilotinib.
15
Next, the effect of substitution around the pyrimidine ring was
explored with selected alkyl, methoxy, and CF groups. The 5-
3
ethylpyrimidinyl showed improved TIE-2 potency about 6-fold
with an n-propyl (11d: TIE-2 IC = 4 nM) and 2-fold with an
50
isobutyl (11e: TIE-2 IC50 = 10 nM). However, both 11d and
1e showed poor metabolic stability and poor rat pharmacoki-
1
netic properties and were not further advanced. The SAR further
revealed that substitutions in the 4-position or combined 4,6-
positions were not tolerated. 4-Methoxy 11g and 4-trifluo-
romethyl 11h reduced VEGF-R2 and abolished TIE-2 activity,
while 4,6-dimethyl 11f abolished both VEGF-R2 and TIE-2
activity. The requirement for the pyrimidinyl group for TIE-2
activity was further demonstrated by changing it to a 3-
pyridazinyl 11i or 4-pyridyl 11j, both of which were weak to
inactive for TIE-2. The position of the methyl group was
important as the 1-methyl isomer 15 displayed IC50 values that
were 11- and 16-fold weaker compared with the corresponding
N-2 methyl 11b.
a
Reagents and conditions: (a) 1-iodo-2-methyl-propane, NaOH,
acetone, reflux, 18 h, 90%; (b) HNO , AcOH, 80 °C, 1 h, 85%; (c)
H , Raney-Ni, DMF-MeOH, 80% ; (d) 2-chloropyrimidine, 1-butanol,
reflux, 45%.
3
2
The VEGF-R2 cellular activity was evaluated using a porcine
endothelial cell line transfected with a chimeric receptor
consisting of the extracellular portion of rat TRK-A (rTRK-A)
and the intracellular domain of human vascular endothelial
growth factor receptor (VEGF-R2/KDR) as described
screened against recombinant human VEGF-R2 and TIE-2 using
a heterogeneous time-resolved fluorescence (TRF) readout and
recombinant human phospholipase C-γ/glutathione S-transferase
12a,13
(
GST) as substrate.
The enzyme inhibitory data is shown in
13
Table 1. Dual TIE-2/VEGF-R2 inhibitors with IC50 values less
than 25 nM that demonstrated acceptable cell potency, and
pharmacokinetic properties were advanced for in vivo evaluation.
Previous dual TIE-2/VEGF-R2 SAR studies on the core revealed
previously. In general, the series demonstrated very potent
cellular activity. Ligand induced stimulation of VEGF-R2
phosphorylation was blocked by 11a−e with IC50 values of
less than 10 nM. Further, 11b inhibited ligand-induced human
TIE-2 phosphorylation in an analogous rTRK-A-human TIE-2
chimeric endothelial cell line with an IC50 value of
approximately 50 nM, in acceptable agreement with the
1
1
that a three or four carbon alkyl unit at N-R was optimum for
13a−c
balanced dual potency and PK.
Ideal dual target potency
and PK properties were achieved with an n-propyl, i-propyl,
and i-butyl group and further increasing or decreasing the
size of the R11 alkyl reduced the balanced dual profile. In the
pyrimidinyl-amine series, the focus was therefore on optimizing 8-
pyrimidinyl-amine region while maintaining the best R11 alkyl.
The constrained pyrimidinyl n-propyl 11a retained dual potency
with VEGF-R2 and TIE-2 IC values of 6 nM and 27 nM,
enzyme activity on isolated recombinant human TIE-2 (IC50
=
22 nM). On the basis of the enzyme and cellular dual target
potency and metabolic and pharmacokinetic profiles, 11b was
selected for advanced discovery flow profiling,
Selectivity Profile for 11b. Compound 11b was screened
for kinase selectivity against a panel of 126 kinases at 1 μM
screening concentration and 100 μM ATP concentration
(Upstate KinaseProfiler). The S(90)126 value was 0.18 (fraction
of kinases inhibited greater than 90%). In a separate screen of
101 selected kinases using an ATP concentration close to the
Km of the enzyme showed a comparable and favorable
selectivity S(90)101 value of 0.15. Subsequent IC50 values
were generated for 23 kinases that demonstrated greater than
80% inhibition. In addition to TIE-2 (22 ± 6 nM) and VEGF-
R2 (4 ± 1 nM), compound 11b demonstrated inhibitory
activity against angiogenesis targets VEGF-R1 (3 ± 1 nM) and
FGF-R1 (13 ± 2) with IC50 values within the potency range
observed for inhibition of the primary targets. Compound 11b
also inhibited TRK-A (IC50 = 3 nM), TRK-B (IC50 = 5 nM),
Ret (IC = 5 nM), BLK (IC = 8 nM), TAK1 (IC = 14 nM),
5
0
1
1
respectively. Varying the N-R alkyl to i-butyl 11b showed
comparable potency (VEGF-R2 IC50 = 4 nM; TIE-2 IC50
2 nM), while i-propyl 11c had a 3-fold drop in TIE-2 potency
VEGF-R2 IC50 = 3 nM; TIE-2 IC50 = 76 nM) and did not
=
2
(
meet discovery criteria. Metabolic stability studies were conducted
on 11a and 11b using hepatic S9 fractions from mouse, rat, dog,
monkey, and human. Both compounds showed 65−77% of the
parent remaining after 2 h in mouse, rat, and dog liver S9, while
monkey was lower (11a = 44%; 11b = 54%). In human S9, 11b
showed 79% remaining after 2 h compared to 11a, which was
5
4%. Compounds 11a and 11b were further evaluated for
pharmacokinetic properties in the rat. Compound 11b showed
acceptable oral exposure and intrinsic pharmacokinetic param-
eters in the rat (Table 2), while the oral exposure for 11a (% F =
50
50
50
22) was lower. The oral bioavailability for 11b was 44% after
and MST2 (21 nM) with IC values less than 25 nM (Table 3).
50
determining the plasma level exposure after i.v. (0.8 mg/kg) and
p.o. (5 mg/kg) administration over a 24 h period. The i.v.
terminal half-life was 1.9 h (oral half-life = 3 h), with a volume of
distribution of 1.3 L/kg and a low clearance rate of 4.3 mL/min/
kg. Compound 11b also showed acceptable selectivity against
CYP isoforms 2D6, 3A4, and 2C19 (IC > 30 μM for 3A4 and
Compound 11b showed weak activity for ABL, ALK, CDKs,
CHK2, cMET, EGFR, GSK3β, IGF-R1, IRK, JAK2, JNKs,
MEK, MKKs, PDGFR, PKBs, and PKCs. A broad profile
screen of 11b in binding assays for 75 receptors, ion channels,
and transporters (Cerep screen) revealed 7 targets (adenosine
A , A ; angiotensin AT1; muscarinic M and M ; neurokinin
5
0
2A
3
2
4
2
D6; 9 μM for 2C19) and no issues with CYP3A4 induction.
NK and NK ) that displayed ≥80% inhibition at a screening
1
2
The permeability was evaluated in the Caco-2 intestinal epithelial
concentration of 10 μM. Follow-up K values revealed
i
cell line where it was classified as high permeability (Papp = > 4 ×
generally weak activity for these targets, with the adenosine
A2A receptor displaying the most potent activity with a Ki
−6
1
0 cm/sec). Compound 11b binding to human plasma proteins
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Table 1. In Vitro TIE-2 and VEGF-R2 Data
a
b
c
IC50 in nM ± SEM. NT = not tested. N1 methyl analogue.
Table 2. Pharmacokinetic Properties of 11b
acceptable selectivity for the hERG channel with an IC value
5
0
of 20 μM in Chinese Hamster Ovary cells and did not
produce any significant effects on QTc interval or on action
potential duration in vitro in the dog Purkinje fiber assay.
Molecular Modeling. A TIE-2 cocrystal structure with a
THP-DHI analogue (PDB code 3L8P) was solved at 2.4 Å
a
b
rat
monkey
i.v.
t1/2 (h)
1.9
1.3
4.3
8559
1330
3.0
2
1.7
2.6
18
Vd (L/kg)
CL (mL/min/kg)
AUC (ng·h/mL)
Cmax (ng/mL)
t1/2 (h)
p.o.
2482
491
2.4
2
resolution was subsequently used for docking experiments with
13d,16a
new analogues.
Compound 11b docked in the TIE-2
model represents a DFG-in Type I kinase inhibitor binding
tmax (h)
16b,c
mode.
Key interactions identified in the predicted model
F (%)
44
93
were the lactam NH/CO bidentate donor/acceptor with
Glu903 (gk +1)/Ala905 (gk +3) at the hinge region, the N1
pyrimidine nitrogen H-bonding with Asp982 (DFG) backbone
amide, and the amino potential NH donor with the Asp982
side chain (Figure 3). The 8-pyrimidine moiety occupies a
hydrophobic pocket flanked by gatekeeper Ile902 and Phe983
(DFG-in), with the cavity defined by Leu903, Leu888, Ile886,
Leu876, and Leu985.
a
Male rat administered at 0.8 mg/kg i.v. and 5 mg/kg p.o. Parameters
were calculated from composite mean plasma concentration−time
data (n = 12). Administered at 0.5 mg/kg i.v. and 3 mg/kg p.o. for
monkey. Parameters were calculated using plasma concentration−time
data for individual animals (monkey n = 4).
b
value of 0.5 μM and the muscarinic M subtype the weakest
activity with a K of 2.9 μM. In addition, 11b showed
4
i
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Table 3. Kinase Selectivity Data for 11b
showed dose-dependence near proportional exposure up to 55
mg/kg, with further dose related exposure to 100 mg/kg p.o. In
monkeys (5, 25, and 100 mg/kg p.o.), a similar experiment
showed dose dependent exposure to 100 mg/kg.
kinase
IC50 (nM)
ALK (h)
132
42
8
AURK-A (h)
BLK (m)
In Vitro and Ex Vivo Antiangiogenic Profile of 11b.
The antiangiogenic activity of 11b was evaluated in both
functional (ex vivo rat aortic ring explant cultures) and target-
directed (in vitro HUVEC capillary-tube formation) bioassays
in order to assess their antiangiogenic activities and potential
cytotoxic profiles in primary cell and organ cultures upon
BTK (h)
93
140
48
37
13
89
3
CHK2 (h)
CK1
cSRC (h)
FGF-R1 (h)
FGF-R3 (h)
VEGF-R1 (h)
FLT3 (h)
FMS (h)
17
prolonged exposure. Figure 4A,B summarizes the results
obtained with 11b in the ex vivo rat aortic ring collagen gel
explants in serum-free conditions and in vitro HUVEC capillary
tube formation assays. Compound 11b exhibited dose-related
inhibitory effects on peak phase microvessel growth (day 8−10
in culture) in rat aortic ring explants in 3D-collagen gels under
serum-free conditions with an EC of approximately 4 nM, and
71
78
21
246
5
MST2 (h)
PDGF-Rα (h)
RET (h)
50
inhibition of complete capillary-tube formation by HUVECs on
Matrigel with an EC50 of approximately 2 nM. These effects
were observed in the absence of apparent cytotoxicity. In
addition to the potential synergistic effects of TIE-2 and pan-
VEGF-R inhibition on the readout of these bioassays, the
contributions of additional kinase inhibitory properties by
TAK1(h)
14
3
TRK-A (h)
TRK-B (h)
5
11b (for e.g. FGF-R1) may contribute to the significant
antiangiogenic activities observed. To evaluate inhibition of
TIE-2-mediated biological activity independent of VEGF-R, the
dose-related inhibition of recombinant human angiopoietin
1
-stimulated HUVEC chemotaxis was evaluated in a modified
Boyden chamber assay. Compound 11b exhibited significant
inhibitory activity against Ang-1-stimulated HUVEC chemo-
taxis at concentrations ≥50 nM in absence of HUVEC
cytotoxicity (data not shown). This result is consistent with
the human TIE-2 IC50 for 11b (Table 1). Similarly, in vitro
bioassays evaluating VEGF- and FGF-induced effects on the
proliferation, migration, and survival of HUVEC, HMVEC, and
BAEC demonstrated EC50 values of 8−38 nM (data not
shown), consistent with the biological activities highlighted
above. Collectively, these data confirm the robust, dose-related
antiangiogenic activity of 11b across a panel of target-directed
bioassays.
Figure 3. Predicted binding mode of 11b with TIE-2.
A similar exercise was carried out to explore putative binding
In Vivo Profile for 11b. The in vivo antitumor efficacy of
11b was assessed on the A375 human melanoma and U251MG
human glioblastomas, two highly angiogenic VEGF-R2 and
TIE-2 dependent solid tumors. Oral administration of 11b (1.0
to 30 mg/kg bid; Tween: methylcellulose) to nude mice
bearing established U251MG human glioblastoma xenografts
resulted in a sustained dose-related (3 mg/kg, 61% (p < 0.01);
interactions of 11b with a DFG-in inhibitor binding model of
12a,13b−d
VEGF-R2 described previously.
The proposed binding
mode for 11b was consistent with the lactam moiety mimicking
the ATP donor−acceptor interactions at the hinge region with
the lactam N−H sharing a hydrogen bond with the Glu917
carbonyl and the lactam CO accepting a hydrogen bond with
the backbone amide of Cys919. The pyrimidine occupied the
hydrophobic pocket analogous, as reported, with 5a with the
N1 nitrogen forming a significant hydrogen bond with Asp1046
and the N3 nitrogen potentially interacting with a Lys868 (not
1
0 mg/kg, 75% (p < 0.001); and 30 mg/kg, 88% (p < 0.001))
maximum inhibition of tumor xenograft growth relative to that
of vehicle-treated controls after 29 days with a minimum
effective dose (MED) of 3 mg/kg bid (Figure 5 and Table 4). A
1
2a,13d
shown).
2
0% and 44% incidence of partial tumor regressions was
Pharmacokinetic Properties of 11b in Rats and
Monkeys. Compound 11b was absorbed with higher oral
bioavailability in monkeys (F = 93%) and slightly lower in rats
achieved with 10 and 30 mg/kg bid, respectively. At 30 mg/kg
bid Cmax plasma and tumor levels of 11b were 1200 ± 79
ng/mL and 401 ± 62 ng/g, respectively. Similarly, oral
administration of 11b to nude mice bearing established A375
human melanoma xenografts resulted in a MED of 0.3−1 mg/
kg bid for sustained antitumor efficacy. Significant inhibition of
A375 tumor xenograft growth (maximally 98% at 10 mg/kg
bid; p < 0.001) and sustained partial and complete tumor
regressions (maximally 50% and 40%, respectively, at 10 mg/kg
bid) were achieved and were directly related to tumor and
plasma exposure (Figure 6 and Table 4). At 10 mg/kg bid, Cmax
(
F = 44%). In rats, the systemic clearance was low relative to
the estimated hepatic plasma flow, and the volume of
distribution was approximately equal to total body water. In
monkeys, the i.v. half-life (t1/2 = 1.7 h) was comparable to that
in rat, but the clearance (18 mL/min/kg) and the volume of
distribution (2.6 L/kg) were larger (Table 2). The oral Tmax
was 2 h in both species, consistent with the oral half-life. An
oral dose escalation study in rat (10, 30, 55, and 100 mg/kg po)
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Figure 4. Antiangiogenic activity of 11b ex vivo rat aortic ring explant cultures and in vitro HUVEC capillary-tube formation bioassays. Ex vivo rat
aortic ring explant cultures and target-directed in vitro HUVEC capillary-tube formation to assess their antiangiogenic activities and potential
cytotoxic profiles in primary cell and organ cultures. Compound 11b exhibited dose-related inhibitory effects on peak phase microvessel growth (day
8
−10 in culture) in rat aortic ring explants in 3D-collagen gels under serum-free conditions with an EC50 of approximately 4 nM, and inhibition of
complete capillary-tube formation by HUVECs on Matrigel with an EC50 of approximately 2 nM. P < 0.05**, p < 0.01* relative to control cultures.
Figure 5. Oral antitumor activity of 11b in the U251 MG human
Figure 6. Oral antitumor activity of 11b in the A375 human melanoma
glioblastoma xenograft. Tumor cells were injected subcutaneously in
xenograft model. Tumor cells were injected subcutaneously in serum-free
serum-free media into the right flank of athymic female nude mice.
media into the right flank of athymic female nude mice. Ten days post-
Twelve days post-implantation, mice with established tumors were
implantation, mice with established tumors were randomized into
randomized into treatment groups (n = 10 mice/group) and
treatment groups (n = 10 mice/group) and administered 11b orally
administered 11b orally (100 μL/dose) at the doses indicated and
(
100 μL/dose) at the doses indicated and relative tumor volume to those
tumor volumes measured as defined in the Experimental Section.
Statistical analyses for efficacy of 11b are detailed in Table 4.
at day 1 of treatment with 11b measured as defined in the Experimental
Section. Statistical analyses for efficacy of 11b are detailed in Table 5.
Table 4. Effects of 11 b in the U251 MG Human
Glioblastoma Model
Table 5. Effects of 11b in the A375 Human Melanoma
Model
%
incidence of
tumor regressions
% incidence of
tumor regressions
partial/
% maximum tumor growth
treatment
1b
dose
stasis complete inhibition vs vehicle control
partial/
dose
0.3 mg/kg 20%
% maximum tumor growth
treatment
stasis complete inhibition vs vehicle control
1
1 mg/kg
0
0
43% NS
(p.o. bid)
11b
(p.o. bid)
10%
71% p < 0.05
3
1
3
mg/kg
0
0
0
0
61% p < 0.01
75% p < 0.001
88% p < 0.001
0 mg/kg 20%
0 mg/kg 44%
1 mg/kg
3 mg/kg
10%
11%
50%
20%
22%
40%
73% p < 0.01
90% p < 0.001
98% p < 0.001
1
0 mg/kg
plasma and tumor levels of 11b were 456 ± 44 ng/mL and
40 ± 20 ng/g, respectively. Administration of 11b was well-
tolerated, with no apparent toxicity or significant body weight
Compound 11b was further evaluated in the P388 murine
leukemia model to assess effects of varying dose schedules on
survival in a systemic hematological tumor model. An
improvement in median survival was observed at 30 mg/kg
1
loss or morbidity in solid tumor-bearing animals.
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Figure 7. Survival benefit of continuous and intermittent oral administration of 11b in the P388 systemic murine leukemia model in mice. Kaplan−
Meier survival plot of continuous versus intermittent once and twice daily oral dosing regimens of 11b (30 mg/kg po) on median survival of DBA/2
mice implanted with murine P388 leukemia cells. Details of analyses are defined in the Experimental Section and the text.
bid monotherapy in P388 leukemic DBA/2 mice relative to
vehicle-treated tumor-bearing mice. Both continuous bid and
qd oral administration and all intermittent oral dosing regimens
bid and qd evaluated at 30 mg/kg in leukemic mice conferred
sustained improvements in median survival (p < 0.05 or
greater) relative to that of vehicle-treated leukemic animals.
Survival data generated by Kaplan−Meier analyses for
continuous and intermittent bid and qd administration of
target-directed biological activity of 11b in humans (data not
shown). Phase 2 proof-of-concept studies with 11b are planned
in select genotyped cancer patient populations to fully assess
the therapeutic potential of this novel kinase inhibitor.
EXPERIMENTAL SECTION
Chemistry. All reagents and anhydrous solvents were obtained
from commercial sources and used as received. H NMRs were
■
1
11b in the murine P388 leukemia ascite model in DBA/2 mice
obtained at 400 MHz in the solvent indicated with tetramethylsilane as
an internal standard. Coupling constants (J) are in Hertz (Hz).
Compounds were purified by normal phase flash silica gel
chromatography or automated silica gel chromatography on a
Combi-Flash Companion (ISCO, Inc.) monitored at 254 and 290 nm
detection. Preparative TLC was run using Silica Gel GF 20 ×
are shown in Figure 7.
CONCLUSIONS
■
Compound 11b was identified as a potent, orally active
inhibitor of human TIE-2, VEGF-R1 and 2, and FGF-R1.
Collectively, it exhibits excellent permeability, metabolic
stability and pharmacokinetic properties across multiple species
and was advanced into full development. The in vitro, ex vivo,
and in vivo pharmacologic activities conducted across a panel of
angiogenesis assays and subcutaneous and orthotopic rodent
and human tumor models demonstrated sustained and
concentration (dose) related antiangiogenic and antitumor
efficacy (tumor growth inhibition, tumor regressions, and
survival benefits) consistent with a potent TIE-2/VEGF-R1, R2
inhibitor with a broader kinase inhibitory profile. Clinical phase
I studies assessing the safety and pharmacokinetics of 11b have
been completed and are the subject of a separate publication.
Nonetheless, these clinical studies indicate linear, dose related
increases in plasma pharmacokinetic exposure (mean Cmax and
AUC) with oral administration of 11b at doses of 3.0 to 126
2
0 cm × 1000 μm plates (Analtech). Liquid chromatography−mass
spectrometry (LC/MS) were obtained on a Bruker Esquire 2000 ion
trap LCMS with the Agilent 1100 HPLC equipped with an Agilent
Eclipse XDB-C8 2 × 30 cm 3.5 μm column. All final compounds
tested in Table 1 had purity >96% determined by High Pressure
Liquid Chromatography (HPLC) using a Zorbax RX-C8, 5 × 150 mm
column eluting with a mixture of acetonitrile and water containing
0.1% trifluoroacetic acid with a gradient of 10−100%.
5-Cyano-11,12-dihydro-2-methyl-10-propyl-indazolo[5,4-a]-
carbazole-4-carboxylic Acid Ethyl Ester (8a). 5-Cyano-2,10,11,12-
tetrahydro-2-methyl-indazolo[5,4-a]carbazole-4-carboxylic acid ethyl
ester 7 (1.0 g, 2.7 mmol), 10 N NaOH (10 mL), and 1-iodopropane
(
2.7 mL, 27.0 mmol) in acetone (35 mL) was stirred at reflux
overnight. The reaction was concentrated, triturated with water, and
collected. The product was purified by flash chromatography (1%
methanol in DCM) to afford 1.0 g (90%) as a tan solid. H NMR
1
(DMSO-d ) δ: 8.44 (m, 1H), 7.80 (m, 1H), 7.64 (s, 1H), 7.62 (m,
6
1H), 7.36 (m, 1H), 4.59 (m, 2H), 4.52 (m, 2H), 3.87 (s, 3H), 3.53 (m,
2H), 2.88 (m, 2H), 1.83 (m, 2H), 1.37 (m, 3H), 0.91 (m, 3H). LCMS
m/z: 413 (M + 1).
The intermediates 8b and 8c were synthesized from 7 using the
method for 8a.
2
mg/m and an oral half-life of 8−15 h on a 28 day once daily
treatment cycle, with a 14 day treatment free period. Further,
exposure-related increases in VEGF-R2 inhibition ex vivo were
demonstrated with 11b in a human plasma-based cellular
bioassay using the rTRK-A-human/VEGF-R2 chimeric endo-
thelial cells described in this article. Normalized cellular VEGF-
R2 inhibition greater than 90% in select patient plasma samples
from the phase 1 study was observed, especially in the 73, 97.4,
5
-Cyano-11,12-dihydro-2-methyl-10-(2-methylpropyl)-
indazolo[5,4-a]carbazole-4-carboxylic Acid Ethyl Ester (8b).
1
HNMR (DMSO-d ) δ: 8.45 (m, 1H), 7.84 (m, 1H), 7.64 (s, 1H),
6
7
.62 (m, 1H), 7.35 (m, 1H), 4.52 (m, 4H), 3.87 (s, 3H), 3.51 (m, 2H),
2.87 (m, 2H), 1.37 (m, 4H), 0.80 (d, 6H, J = 7 Hz); LCMS m/z: 427
2
and 126.6 mg/m dosing cohorts, suggestive of molecular
(M + 1).
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5
-Cyano-11,12-dihydro-2-methyl-10-(1-methylethyl)-
2.12 (m, 1H), 1.19 (m, 3H), 0.80 (d, 6H, J = 7 Hz). LCMS m/z: 506
(M + 1).
1
indazolo[5,4-a]carbazole-4-carboxylic Acid Ethyl Ester (8c). H
NMR (DMSO-d ) δ: 8.44 (d, 1H, J = 8 Hz), 7.92 (d, 1H, J = 9 Hz),
.66 (s, 1H), 7.57 (m, 1H), 7.36 (m, 1H), 5.27 (m, 1H), 4.51 (m, 2H),
.87 (s, 3H), 3.48 (m, 2H), 2.82 (m, 2H), 1.63 (d, 6H, J = 7 Hz), 1.39
11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(4,6-dime-
6
7
3
(
thylpyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]-
1
carbazol-4-one (11f). H NMR (DMSO-d ) δ: 9.50 (s, 1H), 8.86 (s,
6
1
1
H), 8.69 (m, 1H), 8.37 (s, 1H), 7.70 (m, 1H), 7.61 (m, 1H), 6.62 (s,
H), 4.77 (s, 2H), 4.43 (m, 2H), 3.86 (s, 3H), 3.40 (m, 2H), 2.82 (m,
m, 3H). LCMS m/z: 413 (M + 1).
1-Propyl-11,12-dihydro-2-methyl-8-amino-4H-indazolo-
5,4-a]pyrrolo[3,4-c]carbazol-4-one (10a). To 8a (800 mg, 1.94
mmol) in acetic acid (70 mL) was added dropwise 70% nitric acid
248 μL, 3.88 mmol). The reaction was heated at 80 °C for 1 h and
1
[
2H), 2.36 (s, 6H), 2.12 (m, 1H), 0.80 (d, 6H, J = 7 Hz). LCMS m/z:
506 (M + 1).
1
1-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(4-methoxy-
(
pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]-
then cooled to rt. The resulting precipitate was collected by filtration
and washed with water, ether, and then dried. The crude nitro
intermediate was dissolved in DMF−MeOH (30 mL, 1:1) and
hydrogenated with excess Raney-Ni at 50 psi for 16 h on a Parr
apparatus. The catalyst was removed by filtration through a bed of
Celite, and the filtrate was concentrated under reduced pressure and
dried to give 10a (529 mg, 71%) as a tan solid. H NMR (DMSO-d )
δ: 8.83 (s, 1H), 8.30 (s, 1H), 7.37 (d, 1H, J = 9 Hz), 7.07 (s, 1H), 6.84
d, 1H, J = 9 Hz), 4.82 (s, 2H), 4.67 (s, 2H), 4.43 (m, 2H), 3.85 (s,
H), 3.41 (m, 2H), 2.82 (m, 2H), 1.77 (m, 2H), 0.88 (m, 3H). LCMS
1
carbazol-4-one (11g). H NMR (DMSO-d ) δ: 9.50 (s, 1H), 8.86
6
(
(
(
s, 1H), 8.35 (m, 2H), 8.20 (m, 1H), 7.80 (m, 1H), 7.65 (m, 1H), 6.25
m, 1H), 4.75 (s, 2H), 4.43 (m, 2H), 3.98 (s, 3H), 3.86 (s, 3H), 3.43
m, 2H), 2.83 (m, 2H), 2.57 (m, 1H), 0.80 (d, 6H, J = 7 Hz). LCMS
m/z: 508 (M + 1).
1
11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(4-trifluoro-
6
methyl-pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-
1
c]carbazol-4-one (11h). H NMR (DMSO-d ) δ: 10.23 (s, 1H),
6
(
3
8
4
2
.87 (s, 1H), 8.81 (m, 1H), 8.45 (m, 2H), 7.70 (s, 2H), 7.23 (m, 1H),
.74 (s, 2H), 4.45 (m, 2H), 3.87 (s, 3H), 3.43 (m, 2H), 2.83 (m, 2H),
.13 (m, 1H), 0.81 (d, 6H, J = 7 Hz). LCMS m/z: 546 (M + 1).
m/z: 386 (M + 1).
The intermediates 10b and 10c were synthesized from 8b and 8c
using the method for 10a.
11-(2-Methylethyl)-12,13-dihydro-2-methyl-8-(3-pyridaziny-
lamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (11i).
1
1
1-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-amino-4H-
H NMR (DMSO-d ) δ: 9.26 (s, 1H), 8.78 (s, 1H), 8.63 (m, 1H),
6
1
indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (10b). H NMR
DMSO-d ) δ: 8.84 (s, 1H), 8.31 (s, 1H), 7.39 (m, 1H), 7.06 (m,
8
.38 (m, 1H), 8.30 (m, 1H), 7.77 (m, 1H), 7.70 (m, 1H), 7.45 (m,
1H), 7.15 (m, 1H), 5.23 (m, 1H), 4.73 (s, 2H), 3.93 (s, 3H), 3.39 (m,
H), 2.81 (m, 2H), 1.62 (d, 6H, J = 7 Hz); LCMS m/z: 464 (M + 1).
(
6
1
3
7
H), 6.82 (m, 1H), 4.82 (s, 2H), 4.68 (s, 2H), 4.34 (m, 2H), 3.85 (s,
H), 3.38 (m, 2H), 2.80 (m, 2H), 2.07 (m, 1H), 0.77 (d, 6H, J =
Hz). LCMS m/z: 400 (M + 1).
2
1
1-(2-Methylethyl)-12,13-dihydro-2-methyl-8-(4-pyridinyla-
1
mino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (11j). H
1
1-(1-Methylethyl)-12,13-dihydro-2-methyl-8-amino-4H-
NMR (DMSO-d ) δ: 9.26 (s, 1H), 8.83 (s, 1H), 8.78 (s, 1H), 8.37 (s,
6
1
indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (10c). H NMR
DMSO-d ) δ: 8.81 (s, 1H), 8.30 (s, 1H), 7.49 (d, 1H), 7.19 (s,
1
H), 8.16 (m, 2H), 7.82 (d, 1H, J = 8 Hz), 7.68 (m, 1H), 7.29 (m,
(
6
1H), 6.89 (m, 2H), 5.30 (m, 1H), 4.75 (s, 2H), 3.86 (s, 3H), 3.41 (m,
2H), 2.79 (m, 2H), 1.62 (d, 6H, J = 7 Hz). LCMS m/z: 463 (M + 1).
5-Cyano-11,12-dihydro-1-methyl-10-(2-methylpropyl)-
indazolo[5,4-a]carbazole-4-carboxylic acid ethyl ester (13).
This compound was synthesized from intermediate 12 using the
method for 8a. LCMS m/z: 427 (M + 1).
1
3
3
H), 6.78 (d, 1H), 5.12 (m, 1H), 4.84 (s, 2H), 4.71 (s, 2H), 3.85 (s,
H), 3.36 (m, 2H), 2.78 (m, 2H), 1.54 (d, 6H, J = 7 Hz); LCMS m/z:
86 (M + 1).
1
1-Propyl-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-
4
H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (11a). Inter-
mediate 9a (1.0 g, 2.6 mmol) and 2-chloropyrimidine (390 mg, 3.4
mmol) in 1-butanol (35 mL) were heated at reflux for 24 h. The
mixture was then cooled to rt and concentrated under vacuum. The
product was purified using silica gel column chromatography (9%
11-(2-Methylpropyl)-12,13-dihydro-1-methyl-8-amino-4H-
indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (14). This com-
pound was synthesized from 13 using the method for 10a; tan solid.
LCMS m/z: 400 (M + H).
MeOH in DCM) to give 11a as a tan solid (633 mg, 53%). Purity
11-(2-Methylpropyl)-12,13-dihydro-1-methyl-8-(pyrimidin-
1
2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one
9
2
1
2
9%. H NMR (DMSO-d ) δ: 9.55 (s, 1H), 8.86 (s, 1H), 8.47 (m,
6
(
1
15). This compound was synthesized from 14 using the method for
1a. H NMR (DMSO-d ) δ: 9.55 (s, 1H), 8.75 (s, 1H), 8.47 (d, 2H,
6
H), 8.36 (s, 1H), 8.29 (m, 1H), 7.81 (m, 1H), 7.61 (m, 1H), 6.79 (m,
H), 4.74 (s, 2H), 4.53 (m, 2H), 3.87 (s, 3H), 3.46 (m, 2H), 2.84 (m,
H), 1.82 (m, 2H), 0.90 (m, 3H). LCMS m/z: 464 (M + H).
Compounds 11b−11j were synthesized using the method for 11a.
1
J = 5 Hz), 8.32 (s, 1H), 8.30 (s, 1H), 7.81 (m, 1H), 7.63 (d, 1H, J = 9
Hz), 6.81 (t, 1H, J = 5 Hz), 4.75 (s, 2H), 4.44 (d, 2H, J = 7 Hz), 3.81
(s, 3H), 3.48 (t, 2H, J = 7 Hz), 2.97 (t, 2H, J = 7 Hz), 2.12 (m, 1H),
1
1-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-
0
.81 (d, 6H, J = 7 Hz). LCMS m/z: 478 (M + 1).
2
(
-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one
1
VEGF-R2 and TIE-2 Kinase Assays. Compounds were tested for
11b). H NMR (DMSO-d ) δ: 9.57 (s, 1H), 8.87 (s, 1H), 8.47 (m,
6
their ability to inhibit the kinase activity of baculovirus-expressed
cytoplasmic domain of VEGF-R2 or TIE-2 using the TRF detection
system. Briefly, each 96-well Costar high binding plate (Corning
Costar #3922, Corning, NY) was coated with 100 μL/well of 10 μg/
mL substrate solution (recombinant GST-PLCγ) in Tris-buffered
saline (TBS). The kinase reaction mixture (total volume = 100 μL/
well) consisting of 20 mM HEPES, pH 7.2, 40 μM ATP, 10 mM
2
1
2
H), 8.40 (s, 1H), 8.29 (m, 1H), 7.81 (m, 1H), 7.64 (m, 1H), 6.80 (m,
H), 4.75 (s, 2H), 4.43 (m, 2H), 3.87 (s, 3H), 3.43 (m, 2H), 2.83 (m,
H), 2.12 (m, 1H), 0.80 (d, 6H, J = 7 Hz). LCMS m/z 478 (M + 1).
1
1-(1-Methylethyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-
ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one
1
(
11c). H NMR (DMSO-d ) δ: 9.56 (s, 1H), 8.78 (s, 1H), 8.47 (d,
6
2
H, J = 5 Hz), 8.36 (s, 1H), 8.28 (s, 1H), 7.78 (m, 2H), 6.81 (t, 1H,
J = 5 Hz), 5.23 (m, 1H), 4.73 (s, 2H), 3.93 (s, 3H), 3.39 (m, 2H), 2.81
m, 2H), 1.61 (d, 6H, J = 7 Hz). LCMS m/z: 464 (M + 1).
1-Propyl-12,13-dihydro-2-methyl-8-(5-ethyl-pyrimidin-2-
ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one
MnCl , 0.1% bovine serum albumin (BSA), and test compound
2
(
diluted in DMSO; 2.5% DMSO final in assay) was then added to the
(
assay plate. Enzyme (30 ng/mL VEGF-R2 or 200 ng/mL TIE-2) was
added, and the reaction was allowed to proceed at 37 °C for 15 min.
Detection of the phosphorylated product was performed by adding
100 μL/well of Eu−N1 labeled PY100 antibody (PerkinElmer #
AD0160, Boston, MA) diluted 1:5,000 in TBS-T containing 0.25%
BSA). Incubation at 37 °C then proceeded for 1 h, followed by the
addition of 100 μL of enhancement solution (PerkinElmer #1244-
105). The plate was gently agitated, and after 30 min, the fluorescence
of the resulting solution was measured using the PerkinElmer
EnVision 2100 (or 2102) multilabel plate reader. Inhibition curves
for compounds were generated by plotting percent control activity
1
1
(
11d). H NMR (DMSO-d ) δ: 9.44 (s, 1H), 8.85 (s, 1H), 8.37 (s,
6
3
2
1
H), 8.31 (m, 1H), 7.80 (m, 1H), 7.59 (m, 1H), 4.74 (s, 2H), 4.54 (m,
H), 3.87 (s, 3H), 3.46 (m, 3H), 2.84 (m, 2H), 2.50 (m, 2H), 1.83 (m,
H), 1.19 (m, 3H), 0.90 (m, 3H). LCMS m/z: 492 (M + 1).
11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(5-ethylpyri-
midin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-
one (11e). H NMR (DMSO-d ) δ: 9.43 (s, 1H), 8.86 (s, 1H), 8.37
1
6
(
s, 3H), 8.31 (s, 1H), 7.79 (m, 1H), 7.62 (m, 1H), 4.75 (s, 2H), 4.43
(
m, 2H), 3.87 (s, 3H), 3.43 (m, 2H), 2.83 (m, 2H), 2.50 (m, 2H),
9
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Article
versus log10 of the concentration of compound. IC50 values were
pharmacokinetic parameters were calculated by a noncompartmental
method using WinNonlin software (Professional Version 4.1,
Pharsight Corporation, Palo Alto, CA, 1997). For experiments to
determine detailed rat PK parameters, rats were administered 1 mg/kg
i.v. and 3 mg/kg p.o. in saline and parameters calculated from
composite mean plasma concentration−time data (n = 12). Monkey
PK (0.5 mg/kg i.v.; 47.4% polyethylene glycol (PEG) 400; 31.1%
water; 20.7% hydroxyl-β-cyclodextrin (HPBCD); and 0.8% Pluronic
F-68 and p.o. Three mg/kg; Ora Plus) parameters calculated using
plasma concentration−time data for individual animals (monkey n = 4).
In Vitro Capillary Tube Formation Assay with HUVECs on
Matrigel and Ex Vivo Rat Aortic Ring Explant Assay in
Collagen Gel Matrices. The ability of 11b to inhibit angiogenesis
in vitro in a capillary tube formation assay utilizing human umbilical
vein endothelial cells (HUVECs) cultured on a synthetic basement
membrane matrix and ex vivo in rat aortic ring explant cultures was
calculated by nonlinear regression using the sigmoidal dose−response
(
variable slope) equation in GraphPad Prism as follows:
y = bottom + (top − bottom)/(1 + 10(log IC50 − x)
×
Hill slope)
where y is the % kinase activity at a given concentration of compound,
x is the logarithm of the concentration of compound, bottom is the %
of control kinase activity at the highest compound concentration
tested, and top is the % of control kinase activity at the lowest
compound concentration examined. The values for bottom and top
were fixed at 0 and 100, respectively. IC50 values were reported as the
average of two or more separate determinations.
VEGF-R2 Cellular Assay. The porcine aortic endothelial cell line
PAE 145) stably transfected with chimeric rTRK-A/KDR receptor
(
17
conducted using published methods.
containing the extracellular domain of rat TRK-A and the intracellular
domain of human VEGF-R2 was used for this study. Subconfluent
cells were serum-starved by replacing media with 5 mL of serum-free
DMEM (Cellgro 10-013-CM; Mediatech, Inc., Herndon, VA)
containing 0.05% BSA for 1 h at 37 °C. At the same time, test
compound (various concentrations) or DMSO (control) was added to
the cells (0.1% final DMSO in assay). To induce phosphorylation, cells
were treated with nerve growth factor (NGF; Harlan Bioproducts for
Science, Inc. BT-5017; Indianapolis, IN) at a concentration of 10 ng/
mL for 5 min. Cells were lysed in FRAK buffer (10 mM Tris at pH 7.5,
A375 Melanoma and U251 MG Glioblastoma Sc Xenograft
Models. Tumor cells were obtained from the American Type Culture
Collection (Manassas, VA) and cultured to subconfluency in
6
RPMI1620 medium with 10% FBS. Harvested cells (5 × 10 ) were
injected subcutaneously in serum-free media into the right flank of
athymic female nude mice (Charles River). Twelve days post-
implantation, mice with established tumors (approximately 60−100
3
mm ) were randomized into treatment groups (n = 10 mice/group)
and administered 11b bid (100 μL/dose) at doses of 0.3 mg/kg, 1 mg/
kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg depending upon the specific
model in an oral suspension of methylcellulose−Tween (Ora-Plus).
Tumor volumes were determined every 3−5 days using vernier
5
0 mM NaCl, 1% Triton X-100, 20 mM NaF, 2 mM sodium
pyrophosphate, 0.1% BSA, and 25 mM β-glycerophosphate)
containing 1 mM activated sodium vanadate and protease inhibitors
3
calipers and the formula: V (mm ) = 0.5236 × length (mm) × width
(
Protease Inhibitor Cocktail Set III Calbiochem # 539134; San Diego,
CA) sheared with a 27-gauge syringe, then centrifuged at 12,000g for
5 min at 4 °C. Clarified cell lysates were normalized to protein using
(
mm) [length (mm + width (mm)/2]. Relative tumor volumes to that
at the start of treatment were calculated for the A375 melanoma
model. Statistical analyses of changes in tumor volume (or relative
tumor volume) with 11b treatment were done using the Mann−
Whitney Rank Sum test, with p < 0.05 deemed significant.
1
the bicinchoninic acid (BCA; Pierce # 23235; Rockford, IL) method,
immunoprecipitated with anti-VEGF-R2 antibody (CEP-133) for 1 h,
followed by incubation with Protein A Sepharose (Sigma # P3391; St.
Louis, MO) for another hour at 4 °C. Immunoprecipitates were
washed three times with FRAK buffer containing protease/
phosphatase inhibitors, 4× sample buffer was added, and then heated
for 5 min at 85 °C. Samples were processed using the standard
protocol involving gel separation (NuPAGE 3−8% Tris-acetate gel;
NOVEX # EA-03585; Invitrogen, Carlsbad, CA) and transfer to
nitrocellulose membrane. The membranes were blocked with Blocking
Buffer (LiCor #927−40000; Lincoln, NE) overnight, then immuno-
blotted with 4G10 antiphosphotyrosine antibody, followed by
incubation with Alexa Fluor 680 goat antimouse IgG (Molecular
Probes #A-21058; Eugene, OR) at RT for 1 h. Phosphorylated
proteins were visualized by scanning on the Odyssey Infrared Imaging
System (LiCor). Inhibition of phosphorylation was calculated with the
help of the Gel-Pro Analyzer 3.1 software (Fotodyne, Inc.; Hartland,
WI).
P388 Murine Leukemia Model. P388 murine leukemia cells were
obtained from the American Type Culture Collection (Manassas, VA)
and cultured in suspension in RPMI1620 medium with 10% FBS. At
approximately 6 weeks of age, female DBA/2 mice (Charles River)
were implanted intraperitoneally with leukemic cells and the resultant
ascites isolated and serially passaged in DBA/2 mice for three cycles to
generate highly malignant leukemic clones for subsequent evaluation
in efficacy studies. Upon intraperitoneal implantation of ascitic P388
leukemia cells into DBA/2 mice, animals were randomized into
treatment groups (n = 12 mice/group) to receive 11b orally at a
3
0 mg/kg dose in methylcellulose: Tween vehicle on four distinct
continuous and alternate dosing schedules, bid continuous; qd
continuous; bid and qd × 7 days followed by drug holiday ×
7
days; bid and qd × 14 days followed by drug holiday × 7 days; bid
and qd × 7 days followed by drug holiday × 14 days. Mice were
weighed twice weekly and monitored daily. The effects of 11b on the
survival of tumor bearing mice were analyzed by the Kaplan−Meier
method as required for data sets using SAS (SAS 8.2, SAS Institute,
Inc. Cary, NC). Mann−Whitney Rank Sum test analyses were used to
compare mean and median survival times and body weights between
treatment groups.
TIE-2 Cellular Assay. The TIE-2 cell-based assay was performed as
described above for VEGF-R2 except that the PAE 145 cell line stably
transfected with chimeric rTRK-A/TIE-2 receptor containing the
extracellular domain of rat TRK-A, and the intracellular domain of
human TIE-2 was used.
Rat Pharmacokinetics. Adult male Sprague−Dawley rats (275−
50 g; Charles River, Kingston, New York) and male cynomolgus
3
AUTHOR INFORMATION
monkeys (2−4 kg, Covance Laboratories, Alice TX) were used in the
experiments. All animal usage was approved by the Cephalon IACAC.
For routine compound screening, rats were dosed via the lateral tail
vein at the indicated dose for i.v. administration (3% DMSO, 30%
solutol, 67% phosphate buffered saline) or via oral gavage (0.6%
methylcellulose/Tween 80 (99.5:0.5) or Ora Plus) at the indicated
dose. Rats were fasted overnight prior to p.o. administration. Serial
blood samples were collected from the lateral tail vein into heparinized
collection tubes (approximately 0.25 mL) at 7 sampling times over a
■
Corresponding Author
*
Phone: 610-738-6283. Fax: 610-738-6558. E-mail: rhudkins@
cephalon.com or Robert.Hudkins@tevapharm.com.
ABBREVIATIONS
■
VEGF-R, vascular endothelial growth factor receptor kinase;
TIE-2, tyrosine kinase with immunoglobulin and EGF-like
domains 2; FGF-R, fibroblast growth factor receptor; DHI, N2-
methyl-12,13-dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazole;
HUVEC, primary human umbilical vein endothelial cells; KDR,
6
h or a 24 h period as indicated. The plasma was separated by
centrifugation, and the sample was prepared for HPLC/MS analysis by
protein precipitation with acetonitrile. The plasma samples were
analyzed for drug and internal standard via LC-MS/MS protocol. The
9
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Journal of Medicinal Chemistry
Article
kinase insert domain receptor; RTK, receptor tyrosine kinase;
PDGF-R, platelet-derived growth factor receptor; TRF,
heterogeneous time-resolved fluorescence; GST, human
phospholipase C-γ/glutathione S-transferase; PK, pharmacoki-
netics; TRK-A, neurotrophic tyrosine kinase receptor type 1;
BAEC, bovine aortic endothelial cells; HMVEC, human dermal
microvascular endothelial cells
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growth factor (VEGF). Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 8219−
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