8-THP-DHI analogs as potent Type I dual TIE-2/VEGF-R2 receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.04.021

A novel series of 8-(2-tetrahydropyranyl)-12,13-dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazoles (THP-DHI) was synthesized and evaluated as dual TIE-2 and VEGF-R2 receptor tyrosine kinase inhibitors. Development of the structure-activity relationships (SAR) with the support of X-ray crystallography led to identification of 7f and 7g as potent, selective dual TIE-2/VEGF-R2 inhibitors with excellent cellular potency and acceptable pharmacokinetic properties. Compounds 7f and 7g were orally active in tumor models with no observed toxicity.

Full text of DOI:10.1016/j.bmcl.2010.04.021

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3356–3360
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
8-THP-DHI analogs as potent Type I dual TIE-2/VEGF-R2 receptor tyrosine
kinase inhibitors
a
a
a
a
Robert L. Hudkins a, , Allison L. Zulli , Ted L. Underiner , Thelma S. Angeles , Lisa D. Aimone ,
Sheryl L. Meyer a, Daniel Pauletti a, Hong Chang a, Elena V. Fedorov b, Steven C. Almo b,
Alexander A. Fedorov b, Bruce A. Ruggeri a
*
a Discovery Research, Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, United States
b Albert Einstein College of Medicine, Bronx, NY 10461, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 8-(2-tetrahydropyranyl)-12,13-dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazoles (THP-
DHI) was synthesized and evaluated as dual TIE-2 and VEGF-R2 receptor tyrosine kinase inhibitors.
Development of the structure–activity relationships (SAR) with the support of X-ray crystallography
led to identification of 7f and 7g as potent, selective dual TIE-2/VEGF-R2 inhibitors with excellent cellular
potency and acceptable pharmacokinetic properties. Compounds 7f and 7g were orally active in tumor
models with no observed toxicity.
Received 2 March 2010
Revised 7 April 2010
Accepted 8 April 2010
Available online 13 April 2010
Keywords:
VEGF-R2
Ó 2010 Elsevier Ltd. All rights reserved.
TIE-2
Kinase inhibitor
Angiogenesis
Angiogenesis, the development of new blood vessels from the
endothelium of a pre-existing vasculature, is a critical process re-
quired by the majority of solid tumors to maintain localized
growth and metastatic dissemination within the host.1 The clinical
application of kinase inhibitors and anti-VEGF antibodies to halt
angiogenesis in tumors has been validated as a therapeutic strat-
egy by positive clinical results with bevacizumab (Avastin),2
sorafenib 1 (Nexavar),3 and sunitinib 2 (Sutent) (Fig. 1).4 However,
tumor angiogenesis is a heterogeneous process, with organ- and
tumor-specific requirements for different angiogenic cytokines
and growth factors and it is therefore likely that optimal anti-
angiogenic therapy may require inhibition of multiple angiogenic
targets and signaling pathways.5 Inhibition of a single factor or li-
gand-receptor axis (e.g., VEGF-VEGF-R) may not be sufficient for
robust inhibition of angiogenesis or tumor growth.6
pathway alone.8 Inhibition of multiple angiogenic targets such as
VEGF-R2 and TIE-2 are currently being evaluated clinically9 and
preclinically.10
Previously we reported on our first generation pan-VEGF-R
candidate CEP-5214 (3a), and its prodrug CEP-7055 (3b) that
advanced into phase 1 clinical trials (Fig. 2).11 Our second gener-
ation strategy was to build in TIE-2 activity and improve upon the
profile relative to CEP-7055 in terms of biochemical, pharmacoki-
netic, pharmacodynamic and in vivo anti-tumor efficacy. Dual
TIE-2/VEGF-R2 inhibitors were advanced with IC50 values less
than 25 nM that demonstrated good cell potency and pharmaco-
kinetic properties for in vivo evaluation. As outlined in past
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
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 mole-
cule-based approaches against a variety of human tumors.7 Appli-
cation 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
H
N
N
O
CONHMe
O
F
H
N
Me
H
Cl
CF3
N
Me
N
H
NEt2
N
H
O
O
2 Sunitinib
1 Sorafenib
* Corresponding author. Tel.: +1 610 738 6283; fax: +1 610 738 6558.
E-mail address: rhudkins@cephalon.com (R.L. Hudkins).
Figure 1. Structures of approved angiogenesis inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.021

R. L. Hudkins et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3356–3360
3357
H
N
gave 9 followed by reductive cyclization (RaNi/H2, DMF, MeOH) to
produce the N11-substituted lactam intermediates 10.15 Friedel–
Craft acylation using methyl 5-chloro-5-oxovalerate (AlCl3, DCM/
MeNO2) produced keto-ester intermediates 11, that were reduced
to diol 12 with lithium borohydride. Acid cyclization of 12 (TFA,
DCE) produced racemic THP analogs 7.16
The N-1 methyl pyrazole analogs 13a–k were synthesized for
comparison with the N-2 methyl analogs using the route de-
scribed in Scheme 1, starting with the N-1 methyl cyano-ester
intermediate.13,15,17
H
N
O
4
6
O
O
H
O
8
3
O
7
N
N
N
N
1
N
OR
11
10
3a R = H
3b R = COCH2NMe2
4
H
N
O
The THP-DHI analogs (7 and 13) were screened against recom-
binant human VEGF-R2 and TIE-2 using a heterogeneous time
resolved fluorescence (TRF) readout and recombinant human
H
R8
N
N
N
O
N
phospholipase C-c/glutathione S-transferase (GST) as sub-
N
N
strate.12–14 The SAR is shown in Tables 1 and 2. As reported pre-
viously, VEGF-R2 inhibitory activity could be maintained for a
variety of R11 alkyl substituent. However, the optimal balance
for dual potency was achieved with increasing alkyl chain length
up to C3 or C4. The general SAR trend revealed TIE-2 potency was
optimal with a propyl (7d, 7e) or butyl (7f, 7g) group, and de-
creased up to two orders of magnitude with increasing alkyl size
(e.g., see 7i–l). For VEGF-R2 potency, R11 H (7a), methyl (7b) and
ethyl (7c) were tolerated, but did not shown favorable pharmaco-
kinetic properties. In the saturated alkyl series, optimum dual po-
tency and pharmacokinetic properties were achieved with R11
being n-propyl (7d: TIE-2 IC50 = 3 nM, VEGF-R2 IC50 = 8 nM), i-
propyl (7e: TIE-2 IC50 = 10 nM, VEGF-R2 IC50 = 21 nM), n-butyl
(7f: TIE-2 IC50 = 10 nM, VEGF-R2 IC50 = 24 nM) and i-butyl (7g:
TIE-2 IC50 = 3 nM, VEGF-R2 IC50 = 11 nM). To assess the selectivity
of the THP isomer, 7e was separated by chiral HPLC and showed
no stereoselectivity between the R and S isomers for TIE-2 or
VEGF-R2 (data not shown).
O
N
R11
O
Cl
O
R8
N
=
/
7
/
S
5
6
Figure 2. Structures of VEGF-R2 and dual Tie-2/VEGF-R2 inhibitors.
PK properties for further lead optimization.12 Early SAR develop-
ment produced a series of carbamate (e.g., 4) and urea dual TIE-2/
VEGF-R2 inhibitors meeting enzyme and cellular potency criteria,
but with suboptimal pharmacokinetic properties. Also, 4 dis-
played in vivo toxicity in tumor models.12 More advanced series
in the optimization process produced several potential lead can-
didates with impressive pharmacokinetic and in vivo anti-tumor
efficacy profiles, such as oximes13 (CEP-11393, 5) and thienyl ke-
tones14 (CEP-11709, 6). In this Letter we disclose the synthesis,
R11 structure–activity relationships (SAR), TIE-2 X-ray crystallog-
raphy and profile for a new series of 2-tetrahydropyran (THP)
dual inhibitors 7 with significant oral in vivo anti-tumor efficacy.
The THP-DHI analogs were synthesized from the common cya-
no-ester intermediate 8 (Scheme 1).15 As described previously,
The cellular activity was assessed using a VEGF-R autophospho-
rylation assay as described previously.12–14 In general, the series
demonstrated potent cellular activity. Compounds 7a–e showed
complete inhibition of VEGF stimulated VEGF-R2 autophosphory-
N
11-alkylation could be carried out with better yields and product
Table 1
isolation on the cyano-ester intermediate 8 prior to lactam forma-
TIE-2 and VEGF-R2 SAR for THP-N2-methyl-DHI analogs
tion.12–14 Alkylation of 8 (10 N NaOH, alkyl halide, acetone reflux)
H
N
O
NC
CO2Et
Me
N
N
NC
N
CO2Et
O
N
N
N
N
a
b
N
R11
N
H
R11
8
9
Entry
R11
TIE-2a
VEGF-R2a
VEGF-R2
Cell IC50 nM
H
N
H
N
O
O
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
H
Me
Et
Pr
i-Pr
n-Bu
i-Bu
i-Pent
44
41
14
3
10
10
3
21
78
41
102
469
9
17
27
16
142
5
15
6
<10
<10
<10
<10
<10
10–50
10–50
ND
O
N
N
N
N
c
d
8
N
N
R11
21
24
11
77
52
55
44
149
18
22
3
R11
CO2Me
11
10
H
H
N
N
O
O
n-Pent
c-Pentyl
c-Hexyl
CH2(c-Hex)
CH2CH@CH
CH2CH2OEt
(CH2)2OH
(CH2)3OH
CH2CH2NEt2
ND
<50
ND
OH
N
N
N
N
HO(CH2)4
O
e
ND
N
N
<50
<50
<50
<50
<50
R11
7
R11
12
16
16
Scheme 1. Reagents and conditions: (a) R11–X, 10 N NaOH, acetone, reflux, >90%;
(b) Raney-Ni, DMF, MeOH, 50 psi, 75–85%; (c) MeO2C(CH2)3COCl, AlCl3, CH2Cl2,
CH3NO2, rt, 50–75%; (d) LiBH4, THF, 0 °C?60 °C, 50–55%; (e) DCE, TFA 0 °C?rt,
>90%.
a
IC50 values in nM reported as the average of at least two separate determina-
tions; ND = not determined.

3358
R. L. Hudkins et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3356–3360
Table 2
ATP donor–acceptor interactions at the hinge region with the lac-
TIE-2 and VEGF-R2 SAR for THP-N1-methyl-DHI analogs
tam N–H sharing a hydrogen bond with Glu917 carbonyl, and
the lactam C@O accepting a hydrogen bond with the backbone
amide of Cys919. The 8-THP occupied a hydrophobic pocket with
the ether oxygen forming a significant hydrogen bond with
Asp1046, analogous as reported with 3a.11a
H
N
O
N
N
O
To understand the binding pose for TIE-2, the cytoplasmic ki-
nase domain of TIE-2 (residues 808–1124) was co-crystallized with
S-7e and an X-ray structure of the complex obtained at 2.4 Å reso-
lution. This structure was subsequently used for docking experi-
ments with new analogs. The final 2.4 Å data set for the crystal
of the complex was 99.2% complete with an Rmerge = 0.061. The
structure of the S-7e complex was solved by molecular replace-
ment using program EPMR.18a The APO-TIE-2 monomer structure
(PDB ID 1FVR) was used as the search model. The solution as one
clear monomer was found in a search using all data between
15 Å and 4 Å. The model was subsequently refined with CNS.18b
The bound S-7e could be seen clearly in the electron density maps
immediately after the first cycle of rigid body refinement of the
protein molecule alone. Iterative cycles of manual rebuilding with
TOM18c and refinement with CNS resulted in a model of the
complex at Rcryst = 0.238, Rfree = 0.265. The final structure contains
2349 protein atoms, 1 inhibitor molecule, and 39 water molecules
for single monomer of the complex in the asymmetric unit. The
chain segments 857–869 and 995–1001 had weak electron density
and was not included in the final model. None of the non-glycine
residues lie in the disallowed regions of the Ramachandran plot.
The X-ray data collection and crystallographic refinement statistics
are represented in the table in supplementary information. The
coordinates for S-7e complex were deposited in the Protein Data
Bank (PDB code 3L8P).
Figure 3 shows a ribbon diagram of S-7e bound in the ATP pock-
et of TIE-2. The S-7e–TIE-2 binding complex represents a DFG-in
Type I kinase inhibitor binding mode. The lactam NH/CO form a
bidentate donor/acceptor interaction with Glu903 (gk + 1)/Ala905
(gk + 3) at the hinge region. The THP ether oxygen serves as an
acceptor for Asp982 (DFG) backbone amide and the THP occupies
a hydrophobic pocket flanked by gatekeeper Ile902 and Phe983
(DFG-in), with the cavity defined by Leu903, Leu888, Ile886,
Leu876 and Leu985 (Fig. 4). The size of the hydrophobic pocket
may explain the potency of both isomers, as modeling showed that
both isomers can satisfy the Asp982 THP-oxygen hydrogen bond
interaction.
Me
N
R11
Entry
R11
TIE-2a
VEGF-R2a
13a
13b
13c
13d
13e
13f
13g
13j
H
Me
Et
Pr
715
554
88
24
103
53
18
300
350
8
22
9
20
44
37
36
65
7
i-Pr
n-Bu
i-Bu
c-Pentyl
(CH2)2OH
13k
a
IC50 values in nM reported as the average of at least two separate
determinations.
lation in HUVECs at 10 nM, and 7f–g had estimated IC50 values of
10–50 nM.11
In general, the N-1 methyl series displayed TIE-2 IC50 values
that were 5–15-fold weaker compared with the corresponding
N-2 analog, whereas potency for VEGF-R2 was typically within
twofold (Table 2). Compound 13d (TIE-2 IC50 = 24 nM, VEGF-R2
IC50 = 20 nM), achieved the dual enzyme potency criteria and
showed complete inhibition of VEGF stimulated VEGF-R2 auto-
phosphorylation in cells at 10 nM. However, in rat pharmacoki-
netic experiments it suffered from high clearance (CL = 855 mL/
min/kg) and short half-life (iv t1/2 = 0.3 h) after iv administration
and was not further progressed.
Based on their dual enzyme and cellular potencies, compounds
7d, 7f and 7g were evaluated for pharmacokinetic properties in the
rat. Compounds 7f and 7g showed acceptable oral exposure and
intrinsic pharmacokinetic parameters in the rat, while the oral
exposure for 7d was lower than desired. Pharmacokinetic parame-
ters for 7f and 7g are shown in Table 3. The oral bioavailability for
7f was 16% after determining the plasma level exposure after iv
(1 mg/kg) and po (10 mg/kg) administration over a 24 h period.
The iv terminal half-life was 1.5 h with a volume of distribution
of 2 L/kg and a clearance rate of 15 mL/min/kg. The oral Cmax was
255 ng/mL. Compound 7g also showed acceptable oral exposure
and intrinsic pharmacokinetic properties (t1/2 = 1.9 h, CL = 20 ml/
min/kg) in the rat. The isomers of 7g and 7f did not display signif-
icant differences in intrinsic iv t1/2, CL and oral bioavailability in
rat. Compounds 7f and 7g displayed excellent in vitro metabolic
stability in liver S9 fractions across species and had IC50 values
greater than 10 lM for the cytochrome P450 isoforms.
To assist in the design of inhibitors and support the SAR, a DFG-
in inhibitor binding model of VEGF-R2 described previously was
used for docking experiments.11a,14 The proposed binding mode
for S-7e was consistent with the lactam moiety mimicking the
Table 3
Rat pharmacokinetic properties for 7f and 7g
1 mg/kg iv
7f
7g
10 mg/kg po
7f
7g
t1/2 (h)
1.5
15
1159
2.0
1.9
20
862
3.2
%F
t1/2 (h)
AUC0–1 (ng h/mL)
Cmax (ng/mL)
16
2.8
1889
255
15
3.9
1264
154
CL (mL/min/kg)
AUC0–1 (ng h/mL)
Vd (L/kg)
Figure 3. Ribbon diagram and ATP-active site of the TIE-2–S-7e X-ray crystal
structure.

R. L. Hudkins et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3356–3360
3359
was achieved at 3 mg/kg BID. Plasma levels 2 h post the final dose
were 900 nM and 950 nM (3 mg/kg) and 158 nM and 321 nM
(1 mg/kg) for 7f and 7g, respectively. Compound 7g demonstrated
a 10% increase in the incidence of partial and complete regressions.
The angiosarcoma model was used in a screening format, and de-
tailed follow-up studies were not conducted in this model.
In conclusion, the synthesis and in vitro optimization of a no-
vel series of potent THP-DHI dual TIE-2/VEGF-R2 inhibitors iden-
tified 7f and 7g with dual enzyme and cellular potency, and
acceptable pharmacokinetic properties. These analogs demon-
strated functional activity in vitro and oral in vivo anti-tumor
activity consistent with an anti-angiogenic mechanism. In addi-
tion, a DFG-in X-ray co-crystal structure of S-7e with TIE-2
was solved to assist in future design of Type I inhibitors. Further
details and profile of the THP series will be published in due
course.
Figure 4. TIE-2–S-7e complex showing key interactions.
Compounds 7f and 7g were profiled for selectivity against a
broader panel of 60 tyrosine and serine/threonine kinases at
Supplementary data
3 lM concentration (Millipore). Compounds 7f and 7g showed po-
tent inhibition of PDGFRb (>90% inhibition) and FGFR-3 (100% inhi-
bition) and the src family (lyn, lck, fyn, yes and blk; >90% inhibition
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.021.
at 3
lM). In contrast, 7f and 7g did not inhibit EGFR or IR (IC50 val-
ues >1
l
M). Also selectivity against a number of serine/threonine
References and notes
kinases was observed with weak inhibition for CDKs, CHK1, GSK-
3b, JNKs, MAPK, MEK1 and the PKC isoforms. Compounds 7f and
7g were found to potently inhibit VEGF-R1 (IC50 values of 28 nM
and 16 nM, respectively) and VEGF-R3 family members (IC50 val-
ues of 5 nM and 9 nM, respectively).
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pharmacokinetic properties, 7f and 7g were evaluated in two func-
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were evaluated in the rat aortic ring explant model where micro-
vessel growth could be stimulated by a variety of endogenous
pro-angiogenic factors including VEGF and Ang-1.11a,13,14 Com-
pounds 7f and 7f significantly inhibited the VEGF stimulated
growth of microvessels in a dose-dependent manner with esti-
mated EC50 values of 1.2 and 0.9 nM, respectively. Compounds 7f
and 7g also displayed significant concentration-related inhibition
of complete HUVEC capillary tube formation relative to control in
the absence of apparent HUVEC cytotoxicity with EC50 values of
0.6 nM and 2 nM, respectively (Table 4).
The in vivo anti-tumor efficacy of the THP-DHI analogs were
routinely evaluated using the SVR murine angiosarcoma xenograft
model following 10-days of oral BID administration in nude mi-
ce.11a,13,14 A significant anti-tumor efficacy with a flat dose–re-
sponse was observed at oral doses of 1 mg/kg and 3 mg/kg BID,
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Table 4
Ex vivo and in vivo profile for 7f and 7g
Assay
7f
7g
Rat aortic ring (EC50, nM)
HUVEC capillary tube formation (EC50, nM)
1.2
0.6
0.9
2
SVR angiosarcoma xenograft in nude mice
1 mg/kg po BID
Max% inhibition of tumor volume versus vehicle
% Regressions
52%
—
59%
PRa 10%
CRa 10%
3 mg/kg po BID
Max% inhibition of tumor volume versus vehicle (%)
63%
10%
61%
10%
% Regressionsa
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a
PR = partial regressions, CR = complete regressions.

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