Design, structure-activity relationships and in vivo characterization of 4-Amino-3-benzimidazol-2-ylhydroquinolin-2-ones: Novel class of receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1021/jm800790t

The inhibition of key receptor tyrosine kinases (RTKs) that are implicated in tumor vasculature formation and maintenance, as well as tumor progression and metastasis, has been a major focus in oncology research over the last several years. Many potent small molecule inhibitors of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) kinases have been evaluated. More recently, compounds that act through the complex inhibition of multiple kinase targets have been reported and may exhibit improved clinical efficacy. We report herein a series of potent, orally efficacious 4-amino- 3-benzimidazol-2-ylhydroquinolin-2-one analogues as inhibitors of VEGF, PDGF, and fibroblast growth factor (FGF) receptor tyrosine kinases. Compounds in this class, such as 5 (TKI258), are reversible ATP- competitive inhibitors of VEGFR-2, FGFR-1, and PDGFRβ with IC₅₀ values <0.1 μM. On the basis of its favorable in vitro and in vivo properties, compound 5 was selected for clinical evaluation and is currently in phase I clinical trials.

Full text of DOI:10.1021/jm800790t

278
J. Med. Chem. 2009, 52, 278–292
Design, Structure-Activity Relationships and in Vivo Characterization of
4-Amino-3-benzimidazol-2-ylhydroquinolin-2-ones: A Novel Class of Receptor Tyrosine Kinase
Inhibitors
Paul A. Renhowe,* Sabina Pecchi, Cynthia M. Shafer, Timothy D. Machajewski, Elisa M. Jazan, Clarke Taylor,
William Antonios-McCrea, Christopher M. McBride, Kelly Frazier, Marion Wiesmann, Gena R. Lapointe, Paul H. Feucht,
Robert L. Warne, Carla C. Heise, Daniel Menezes, Kimberly Aardalen, Helen Ye, Molly He, Vincent Le, Jayesh Vora,
Johanna M. Jansen, Mary Ellen Wernette-Hammond, and Alex L. Harris
NoVartis Institutes for Biomedical Research, 4560 Horton Street, EmeryVille, California 94608-2419
ReceiVed June 30, 2008
The inhibition of key receptor tyrosine kinases (RTKs) that are implicated in tumor vasculature formation
and maintenance, as well as tumor progression and metastasis, has been a major focus in oncology research
over the last several years. Many potent small molecule inhibitors of vascular endothelial growth factor
receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) kinases have been evaluated. More
recently, compounds that act through the complex inhibition of multiple kinase targets have been reported
and may exhibit improved clinical efficacy. We report herein a series of potent, orally efficacious 4-amino-
3-benzimidazol-2-ylhydroquinolin-2-one analogues as inhibitors of VEGF, PDGF, and fibroblast growth
factor (FGF) receptor tyrosine kinases. Compounds in this class, such as 5 (TKI258), are reversible ATP-
competitive inhibitors of VEGFR-2, FGFR-1, and PDGFRꢀ with IC50 values <0.1 µM. On the basis of its
favorable in vitro and in vivo properties, compound 5 was selected for clinical evaluation and is currently
in phase I clinical trials.
Introduction
stimulating factor-1 receptor), PDGFRꢀ, VEGFR-1, VEGFR-
2, and FLT-3 and is in phase I clinical trials for acute
myelogenous leukemia (AML) and myelodysplastic syndrome
(MDS).8 In addition, Axitinib (AG-013736, 4) is an inhibitor
of CSF-1R, VEGFR, and PDGFR and is in phase II clinical
studies for solid tumors, including metastatic renal cell carci-
noma (RCC).9
While embarking on an effort to develop RTK inhibitors, we
discovered a class of molecules based on a 3-benzimidazol-2-
ylhydroquinolin-2-one scaffold (Figure 2) that potently inhibit
VEGF, FGF, and PDGF receptor kinases with IC50 values <0.1
µM. These compounds were also found to possess attractive
pharmacokinetic characteristics and efficacy in several human
tumor xenograft models. Herein, we describe our progress from
lead discovery and optimization of this new class of compounds
through the selection of a molecule, 5 (TKI258, Figure 2), for
clinical development.10-12
Angiogenesis and maintenance of newly formed blood vessels
are important normal physiological processes that are also
utilized by tumors to promote their survival, growth, and
metastasis.1 A subset of growth factors (VEGFa, FGF, and
PDGF) and their receptor tyrosine kinases are key signaling
molecules during angiogenesis and vascular maintenance. Not
surprisingly, many cancer types secrete these growth factors,
which can, in turn, stimulate the surrounding tissues and
encourage new blood vessel formation. Among the three growth
factors, VEGF is considered most important for both normal
and pathological angiogenesis.2 In addition, some tumors have
been found to overexpress the receptors to VEGF, PDGF, and
FGF, which allows them to attain additional growth advantages
and promote tumor survival.3-5 Therefore, direct antitumor
effects may be achievable in these tumors if signaling through
the receptors is blocked by ATP competitive small molecules
that inhibit tyrosine kinase activity. Recent approvals of
VEGFR-2 and PDGFRꢀ inhibiting agents, such as small
molecules Sunitinib (Figure 1, 1, SU11248) and Sorafenib
(Figure 1, 2, BAY43-9006), confirm the attractiveness of
targeting these receptors for cancer therapy.1,6 In addition,
several multitargeted small molecule kinase inhibitors have been
developed and are being evaluated in clinical trials.7 For
example, 3 (ABT-869) is an inhibitor of CSF-1R (colony
Chemistry
Multiple synthetic routes for the synthesis of the 3-benzimi-
dazol-2-ylhydroquinolin-2-one scaffold were explored before
optimal conditions were developed. The initial method involved
the reaction of anthranilic acid esters (6a and 6b) with methyl
malonyl chloride. The products (7a and 7b) were then condensed
with phenylenediamine at elevated temperatures in the absence
of solvent13-15 to yield the 4-hydroxy-3-benzimidazol-2-ylhy-
droquinolin-2-ones (8a and 8b)16 in low to moderate yields
(Scheme 1, a). The synthesis of 8c started from anthranilonitrile
and used the same conditions as for the preparation of 8a and
8b to yield the 4-amino-3-benzimidazol-2-ylhydroquinolin-2-
one 8c in poor yield (Scheme 1, a). The formation of the N1,N1′-
dimethylated analogue 10 was achieved by the reaction of
N-methylisatoic anhydride 9 and ethyl 2-(1-methyl)benzimida-
zol-2-ylacetate in the presence of LHMDS in THF (Scheme 1,
b), while the 4-unsubstituted analogue (12) could be obtained
* To whom correspondence should be addressed. Phone: +1-510-923-
4055. Fax: +1-510-923-3360. E-mail: paul.renhowe@novartis.com.
a Abbreviations: RTKs, receptor tyrosine kinases; VEGFR, vascular
endothelial growth factor receptor; PDGFR, platelet-derived growth factor
receptor; FGFR, fibroblast growth factor receptor; CSF-1R, colony stimulat-
ing factor-1 receptor; AML, acute myelogenous leukemia; MDS, myelo-
dysplastic syndrome; RCC, renal cell carcinoma; HTS, high throughput
screening; HMVEC, human vascular endothelial cells; VDW, Van Der
Waals; CL, plasma clearance; Vss, volume of distribution; MED, minimum
effective dose; CR, complete response; PR, partial response; ITD, internal
tandem duplication.
10.1021/jm800790t CCC: $40.75
2009 American Chemical Society
Published on Web 12/29/2008

4-Amino-3-benzimidazol-2-ylhydroquinolin-2-ones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 279
discovered. This reaction resulted in the formation of the desired
4-amino-3-benzimidazol-2-ylhydroquinolin-2-ones in good yields
(Scheme 3),19 was amenable to multigram scale, and was able
to accommodate the presence of many diverse functional groups
such as free amines, nitriles, and esters on either the benzimi-
dazole acetate or anthranilonitrile. Worth noting is that the
position and nature of R1 affect the yield of the condensation
reaction. When R1 is ortho to the nitrile and is bulkier than H
or F, the formation of the quinolinone ring is much less efficient.
For example, the yields of compounds 5 and 19a, which are
substituted at C5, respectively, with a fluorine and hydrogen
were 60 and 50%. Compounds 19k and 19l, however, which
are substituted with a chlorine and methyl at C5, both had
significantly lower isolated yields (8%).
For novel benzimidazoles, the required phenylenediamine
could be prepared from commercially available starting materi-
als. For instance, introduction of amino-linked substituents at
the 5′ site on the benzimidazole ring was achieved by reaction
of 2-nitro-5-fluoroaniline with amines at elevated temperatures
to yield the 5-amino-2-nitroanilines 21 in high yield and purity
(Scheme 4). Ether linked moieties were accessed by reaction
of 3-amino-4-nitrophenol with alcohols under Mitsunobu condi-
tions (Scheme 4, b) to give 24. Reduction of 21 or 24 in the
presence of Pd/C and H2 in EtOH provided the phenylenedi-
amines 22 and 25, which were used immediately to avoid
oxidative decomposition (Scheme 4). The condensation of
phenylenediamines with ethyl 3-ethoxy-3-iminopropanoate hy-
drochloride 20 in EtOH then provided the requisite benzimi-
dazole acetates in good yield (Scheme 4, 23a-d and 26).20
The benzimidazole acetates were then reacted with anthra-
nilonitriles in the presence of LHMDS to provide the desired
4-amino-3-benzimidazol-2-ylhydroquinoloin-2-ones (Scheme 3).
Further modifications could then be carried out as outlined in
Scheme 5. For instance, methyl 2-(4-amino-2-oxo-1,2-dihyd-
roquinolin-3-yl)-1H-benzo[d]imidazole-5-carboxylate 19ad could
be converted to the piperazinyl amide 19h or the piperazinyl-
methyl analogue 19i under standard conditions (Scheme 5, a).
Furthermore, acylation of the secondary amine 19ah with
bromoacetyl chloride, followed by displacement of the bromine
with dimethylamine, gave amide 19j.
Figure 1. Multikinase Inhibitors.
Figure 2. 3-Benzimidazol-2-ylhydroquinolin-2-one Scaffold and 5.
Scheme 1. Syntheses of
3-Benzimidazol-2-ylhydroquinolin-2-one Scaffold
A variety of moieties could also be incorporated into the
A-ring of the hydroquinolin-2-one either on the anthranilonitrile
starting material or on an advanced 4-amino-3-benzimidazol-
2-ylhydroquinoloin-2-one intermediate. For instance, amines
such as N-methylpiperazine could be introduced into the 5- and
6-positions of the hydroquinolin-2-one by synthesizing the
appropriate piperazinyl anthranilonitriles 27a and 27c according
to reported methods (Scheme 6, a and c).21,22 The introduction
of an ether such as N-methylpiperidin-3-yloxy at the 5-position
of ring A was achieved by reaction of 3-hydroxy-N-methylpi-
peridine with 2-amino-6-fluorobenzonitrile in the presence of
NaH in NMP at 100 °C (Scheme 6, b, 27b). These modified
anthranilonitriles could be carried forward as described in
Scheme 3. Other compounds could be synthesized by a late-
stage modification, as in the formation of the amide (Scheme
6, reaction d, 19t). In this case, 4-aminoisophthalonitrile reacted
with ethyl 2-[5-(4-methylpiperazinyl)benzimidazol-2-yl]acetate
23b under the standard LHMDS reaction conditions to give the
advanced intermediate 19ai. The nitrile on the A ring was then
hydrolyzed to the carboxylic acid and coupled to benzylamine
to give 19t.
by condensation of 2-aminobenzaldehyde 11 and ethyl 2-ben-
zimidazol-2-ylacetate under Knoevenagel conditions (Scheme
1, c).17
Examples introducing basic amines at C4 were synthesized
as shown in Scheme 2 and previously described.18 The length
of this route and the harsh conditions employed for the removal
of the protecting group in the last step made it impractical for
compounds bearing a simple NH2 at C4, in particular when the
introduction of substituents on either the A ring or the D ring
was required.
Results and Discussion
A new synthetic route was thus required that was short,
amenable to scale up, and tolerant of a variety of functional
groups. To this end, the condensation of benzimidazole acetates
with anthranilonitriles in the presence of LHMDS in THF was
Identification of Lead Scaffold and Structure-Activity
Relationships. Compound 8a, a commercially available com-
pound, was identified through high throughput screening (HTS)

280 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Renhowe et al.
Scheme 2. Synthesis of 4-Amino-3-benzimidazol-2-ylhydroquinolin-2-one Scaffold via the Isatoic Anhydride Routea
a Reagents and conditions: (a) LHMDS, THF (b) Tf2O, Pyr (c) R′R′′NH2, DIEA; (d) triflic acid, HCl, 80 °C.
Scheme 3. Synthesis of 4-Amino-3-benzimidazol-2-ylhydroquinolin-2-one Scaffold via the LHMDS Route
Scheme 4. Synthesis of Novel Benzimidazole Acetatesa
a Reagents and conditions: (a) R2NH, Et3N, NMP, 90 °C; (b)H2, Pd/c, EtOH; (c)EtOH, 80 °C, (d) ROH, PPh3, DIAD, THF.
as an inhibitor of VEGFR-2, FGFR-1, and PDGFRꢀ. While the
submicromolar activity and low molecular weight of the
molecule led us to consider this a good initial hit, it had
undesirable physicochemical properties (e.g., solubility) that
required further optimization to yield a drug-like compound.
Initially, the key structural features required for potent kinase
inhibition needed to be determined. Molecular modeling sug-
gested that the NH in position 1 of the hydroquinolin-2-one,
the quinolinone carbonyl and the benzimidazole NH formed a
donor-acceptor-donor motif that would bind to the hinge
region of the RTKs. To test this hypothesis, we systematically
masked the hydrogen bond donors by methylation as in 8b, 8d,
and 10. These changes led to significant potency loss against
all three RTKs, with the dimethylated analogue 10 showing no
kinase activity up to 25 µM (Table 1). Interestingly, monom-
ethylation seemed to affect the kinase selectivity profile as well.
Introduction of a methyl on the benzimidazole NH (8d) had a
more dramatic effect on VEGFR-2 affinity than removal of the
contact from the NH in position 1 of the hydroquinoloin-2-one
(8b). The effect is opposite for FGFR-1, which indicates that
despite the high homology of the two ATP binding sites,
selectivity opportunities still exist that are likely due to small
changes in the binding site shape, influencing the accessibility
of alternate binding poses of the methylated ligands.
group (8c) significantly improved RTK affinity and also led to
improved cell potency with an EC50 of 0.078 µM. The potency
decrease observed (NH2 > OH > H) suggests an important role
for a hydrogen bond donating group in the C4 position (8c, 8a,
and 12). This observation is further supported by a 4-fold
decrease in potency when a methylamino group (16a) is replaced
with a dimethylamino group (16b). However, this loss in RTK
affinity is not as dramatic as the loss caused by masking one of
the hydrogen bonds in the donor-acceptor-donor motif, which
interacts with the hinge region. One hypothesis to explain this
more subtle effect is that the hydrogen on the C4 substituent is
needed to stabilize the benzimidazole tautomer such that the
hydrogen bond donating moiety points toward the hinge and
presents the full donor-acceptor-donor motif.
Once the basic structural components needed for affinity to
our targets of interest were understood, a study of the
structure-activity relationship around the periphery of the
scaffold was undertaken together with the incorporation of
moieties that might impart favorable physicochemical properties.
Thus, incorporation of larger substituents on the C4 NH of the
hydroquinolin-2-one was explored and found to be tolerated
(16a-d). When the substituent carried an additional basic
nitrogen, solubility was improved.23 More importantly, it was
discovered that this position was useful in modulating the
selectivity profile of this class of compounds among the RTKs.
For instance, both 16c and 16d are more potent against PDGFR
than VEGFR-1 (3000-fold) and FGFR (>1500-fold), with
similar trends seen against other kinases as well. In particular,
incorporation of a large basic amine such as an aminoquinu-
clidine as in 16c led to very potent CHK-1 and GSK-3
inhibitors.18 For our RTK inhibitor program, however, the NH2
Having established the necessity of the donor-acceptor-donor
binding motif, the contribution of the C4 hydroxy to the RTK
affinity was explored. Removal of the hydroxyl group (12) led
to significant loss in biochemical potency against VEGFR-2 and
FGFR-1; however, cell-based activity in VEGF-driven prolifera-
tion of human vascular endothelial cells (HMVEC) was
maintained. Replacing the hydroxyl with an unsubstituted amino

4-Amino-3-benzimidazol-2-ylhydroquinolin-2-ones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 281
Scheme 5. Late Stage Modifications to the
at C6 such as chlorine (19o) and a primary amine (19p)
preserves activity, however, a dimethylamino group at this
position (19q) shows a slight drop in affinity with a complete
loss of cell-based potency. At C6, some aromatic substituents
caused no change in affinity (19s and 19t) while others did (19r)
depending on the length and flexibility of the linker to the
scaffold. In spite of the fact that the biochemical potency for
compounds 19s and 19t is similar to the unsubstituted example
(19d), the cell potency loss is significant. This may be a function
of a difference in cell permeability and access to the relevant
subcellular compartments; however, no data were collected to
determine the above parameters.25 At C7, any substituent larger
than a fluorine led to significant decrease in potency against all
targets (19v and 19z).
4-Amino-3-benzimidazol-2-ylhydroquinolin-2-one Corea
To finish exploring the SAR of the A ring, a number of
solubilizing groups at the C5 and C6 positions were incorporated
(Table 4). At C5, groups such as piperazine (19aa) and
1-methylpiperidin-3-ol (19ab) yielded highly potent analogues.
The cellular potency for 19aa decreased, but for 19ab, the
cellular potency was retained. Finally, adding a piperazine at
C6 (19ac) led to significant loss of affinity for VEGFR-2 and
FGFR-1.
Many of the compounds highlighted in this paper show
greater in vitro potency against PDGFRꢀ than VEGFR-2 or
FGFR-1. However, for 5, biochemical inhibition of VEGFR-2,
FGFR-1, and PDGFRꢀ translated into comparable EC50s for
inhibition of VEGFR-2 and PDGFRꢀ phosphorylation (0.046
and 0.051 µM) and 3-fold less potent inhibition of FGFR-1
phosphorylation in cells (0.166 µM).10,12
The culmination of the medicinal chemistry effort led to the
selection of compound 5 as a candidate for further development.
Compound 5 has high potency not only against VEGFR-2,
FGFR-1, and PDGFRꢀ but also against VEGFR-1/3, FGFR-3,
c-Kit, CSF-1R, and FLT-3 enzymes (IC50 values between 3 and
27 nM), which translates to inhibition of VEGF-, FGF-, SCF-,
CSF-, and PDGF-mediated cell proliferation and induction of
apoptosis in specific cancer cell lines.10-12 The antiproliferative
effect of 5 corresponded with reduction of VEGF-mediated
KDR/VEGFR-2 and ERK phosphorylation in endothelial cells.26
In summary, 5 inhibits multiple growth factor receptor tyrosine
kinases that are important for tumor angiogenesis and tumor
growth.
a Reagents and conditions: (a) EtOH, KOH; (b) N-methylpiperazine,
EDCI, HOAT, Et3N, DMF; (c) DIBAL-H, PhMe; (d) MnO2, DMA; (e)
N-methylpiperazine, Na(OAc)3BH, DMA; (f) bromoacetyl chloride, DMA,
Et3N; (g) dimethylamine, EtOH.
group was the optimal substituent at C4 as it avoided inhibition
of these additional serine threonine kinases, which could
complicate the pharmacological application of these agents. The
NH2 substitution at C4 was thus maintained, and subsequent
efforts focused on modifying the A and D rings to attain
additional enzymatic affinity and to improve physicochemical
properties.
Initially, small groups such as methyl were appended to the
D ring and were generally tolerated (19a-19b, Table 2). The
major goal for this series was to improve solubility. In fact,
the poor physicochemical properties of the early compounds
prevented us from obtaining a meaningful pharmacokinetic
profile as a suitable formulation for in vivo dosing could not
be found. To this end, a number of solubilizing groups was
explored (19c-19j). It was found that C5′ tolerated a variety
of substituents including amines (19c-19f), ethers (19g), amides
(19h and 19j), and methylene extended amines (19i) without
significant variations in in vitro potency. Carboxylic acid, ester,
and nitrile C5′ substitutions were also tolerated.24 Varying the
substituent in this position did not affect the selectivity profile,
suggesting that this substituent is most likely solvent exposed.
A basic amine with a pKa in the range of 7-9 on the C5′
substituent (as in 19d-19j) improved the cellular potency 2-fold.
As the C5′ 1-methylpiperazine moiety afforded a potent
compound (19d) with adequate solubility and in vivo properties,
1-methylpiperazine was selected as the default D ring substituent
for A ring SAR (Table 3). Small, electronegative substituents
such as fluorine are tolerated at C5 (5), C6 (19n), and C7 (19u)
of ring A. However, a fluorine substituent at C8 (19w) as well
as difluoro substitution (19x and 19y) resulted in a degradation
of affinity. Introducing a chlorine (19k) or methyl (19l) at C5
slightly improves affinity, whereas C5-methylamino (19m)
retains affinity compared to 19d. Incorporation of substituents
Molecular Modeling. VEGFR-2 binding site models for the
4-amino-3-benzimidazol-2-ylhydroquinolin-2-one based inhibi-
tors described herein were developed using a homology model
based on the FGFR-1 crystal structure (PDB access code
2FGI)27 and optimized using the observed SAR as well as crystal
structure data for a compound with the same core in CHK-1.18
The conformation of the activation loop in the FGFR-1 template
has the highly conserved Asp-Phe-Gly (or DFG) motif in the
active and so-called “in” conformation in which the phenyla-
lanine packs under helix C and fits in an internal hydrophobic
pocket. Inactive kinase structures have been crystallized where
the DFG motif adopts a radically different conformation, which
perturbs the catalytically active orientation of the activation loop
and helix C and moves the phenylalanine of the DFG-motif
“out” of its pocket, leaving it available for occupancy by
inhibitors.28
The choice for the DFG-in conformation as a template was
based on the fact that the current series of inhibitors does not
seem to extend far enough into the phosphate binding region
to induce the DFG-out conformational shift. It is quite possible
that the protein exhibits a conformational equilibrium between
DFG-in and DFG-out conformations while bound to these

282 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Renhowe et al.
Scheme 6. Synthesis of Ring A Substituted 4-Amino-3-benzimidazol-2-ylhydroquinolin-2-onesa
a Reagents and conditions: (a) N-methylpiperazine, DMF, 90°C; (b) H2, Pd/C, EtOH; (c) ethyl 2-benzimidazol-2-ylacetate, LHMDS, THF; (d) NaH,
NMP, 100°C; (e) N-methylpiperazine, Et3N, NMP, 50°C; (f) 23b, LHMDS, THF; (g) NaOH, EtOH, 100°C; (h) benzylamine, EDCI, HOAT, Et3N, DMF.
Table 1. Structure-Activity Relationships at C4
Table 2. Structure-Activity Relationships Around Ring D
inhibitors,29 but for the purposes of understanding SAR from a
structural perspective, the DFG-in conformation seems most
appropriate.
The model derived for 5 is shown in Figure 3. The interactions
with the modeled active site include an optimal set of three
hydrogen bonds to the hinge domain (Glu917 and Cys919). The
A-ring makes good Van Der Waals (VDW) contacts to the
gatekeeper residue, Val916, and is in addition well-positioned

4-Amino-3-benzimidazol-2-ylhydroquinolin-2-ones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 283
Table 3. Structure-Activity Relationships around Ring A
compd
R5
R6
R7
R8
VEGFR-2 IC50 (µM)
FGFR-1 IC50 (µM)
PDGFRꢀ IC50 (µM)
HMVEC EC50 (µM)
19d
5
19k
19l
H
F
Cl
Me
MeNH
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
F
Cl
NH2
Me2N
PhC(O)NH
BnNH
BnNHC(O)
H
H
H
F
H
F
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
0.042
0.065
0.014
0.012
0.053
0.078
0.043
0.019
0.16
0.009
0.011
0.002
0.003
0.015
0.040
0.024
0.007
0.083
0.90
0.008
0.010
0.003
0.37
0.19
0.11
0.002
0.005
0.0003
0.0007
0.002
0.001
0.008
0.007
0.007
0.039
0.0006
0.0001
0.0004
0.009
0.014
0.003
0.005
0.030
0.019
0.013
0.012
0.005
0.017
0.010
0.039
0.017
2
19m
19n
19o
19p
19q
19r
19s
19t
19u
19v
19w
19x
19y
19z
0.98
-
1.1
0.021
0.050
0.014
1.0
0.56
0.19
0.12
0.034
0.27
0.42
0.48
0.080
3.3
MeNH
H
F
F
H
H
H
0.13
5.3
0.030
1.8
Me2N
Table 4. Incorporation of Solubilizing Groups on Ring A
6-substituted analogues like 19t. The C6-vector does point
toward the catalytic Lys868, which may be why the rigid C6-
piperazine analogue (19ac) has highly reduced affinity. The
reduced affinity for the C6-dimethyl-amino compound (19q) is
hypothesized to be due to a clash of one of the methyl groups
with the Lys868/ Glu885 salt-bridge. The benzyl-amide analogue
19t would experience a similar steric clash, but the CdO
functionality in the ligand might induce a conformational change
in the protein to allow a hydrogen bond between the CdO and
the side chain of Lys868. The vector available for substitution
on C5 is pointing into the biggest area of available space, which
is supported by the good affinity for all 5-substituted analogues.
Finally, substituting the C5′ position has little effect on potency
or selectivity, which is in agreement with this model where
substituents on that position are solvent-exposed and the kinases
in our panel are expected to have comparable solvent access
from the ATP binding site.
Pharmacokinetic Profile. Once solubility was improved, the
attractive pharmacokinetic properties of the scaffold were
apparent. The single dose pharmacokinetics of 5 were studied
in mice and rats, and the summary of PK parameters is presented
in Table 5.31 In both species, oral bioavailability was high
(>70%). After intravenous injection, 5 exhibited moderate to
high plasma clearance (CL) relative to liver plasma flow, a large
volume of distribution (Vss) and was eliminated with a terminal
half-life (t1/2) of approximately 3 h.
to engage in an S-H/π interaction with Cys1045. This is a
similar interaction as described for a newly designed FLT-3
kinase inhibitor30 and involves the residue preceding the DFG-
motif. Residues engaged in favorable VDW contacts with the
ligand are Leu840, Val848 (both in the P-loop and ceiling of
the purine pocket), Ala866 (ceiling of the purine pocket), Val
899 (floor of the purine pocket), Phe918 (part of the hinge),
Lys920, Gly922 (both in the lower hinge region), and Leu1035
(floor of the purine pocket).
To validate this binding mode model, the SAR described
above is evaluated in context of the model. The 7- and
8-positions of the core are in close contact with the gatekeeper
residue (Val916), while the 8-position is also in VDW contact
with the hinge (Glu917). This snug fit explains why not even
F-substitution is allowed at the 8-position, and at the 7-position,
nothing bigger than F seems to be tolerated without effecting
affinity. The 6-position has a vector pointing into available
space, supported by the observation of various high affinity
In Vivo Pharmacology of 5 in Preclinical Models.
Dose-response effects of 5 were examined for potency in
preclinical models of angiogenesis and specific target-driven
cancers. The murine Matrigel model of bFGF-induced neovas-
cularization was used to evaluate the effects of 5 on in vivo
angiogenesis. Supplementation with bFGF induces blood vessel
formation in Matrigel implants that was quantified by measuring
the amount of hemoglobin in Matrigel plugs following removal
from animals (Figure 4). Compound 5 administered orally at
doses from 3 to 300 mg/kg for 8 days demonstrated a dose-

284 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Renhowe et al.
Figure 3. Binding site model for compound 5 in the VEGFR-2 homology model. (a) Cartoon representation of the kinase with residues interacting
with the ligand in stick model. (b) Surface representation, with the surface colored by atom type (red ) oxygen, blue ) nitrogen, yellow ) sulfur,
gray ) carbon/hydrogen). The donor-acceptor-donor motif is shown to interact with the hinge region while the 1-methylpiperazine substituent on
C5′ points into solution.
Table 5. Summary of Plasma Pharmacokinetic Parameters of 5a
species
dose (mg/kg)
%Fb
Cmax (ng/mL)b
AUC (ng ·min/mL)b
CL (mL/min/kg)c
Vss (mL/kg)c
t1/2 (h)c
mouse
rat
5 (IV)/20 (PO)
5 (IV)/20 (PO)
77
73
670
480
360000
340000
42
43
12000
13000
2.7
3.5
a CL, clearance; F, oral bioavailability; t1/2, half-life; Vss, volume of distribution. b Determined from the oral data. c Determined from the intravenous data.
The efficacy of 5 was examined in three subcutaneous human
xenograft tumor models, KM12L4A (colon), DU145 (prostate),
and MV4;11 (AML) (Table 6). In efficacy studies, mice with
subcutaneous tumors were randomized at a mean size of 200-500
mm3, and treatments of vehicle or 5 were administered daily by
oral gavage. Dose response effects and statistically significant tumor
growth inhibition were observed in all three models tested, with
disease stabilization and some tumor regressions occurring at 100
mg/kg/d dose level (Table 6). The minimum effective dose (MED)
for KM12L4A (expressing VEGFR and PDGFRꢀ) and DU145
(expressing VEGFR, FGFR, and PDGFRꢀ) were 10 mg/kg/d and
30 md/kg/d, respectively, and the corresponding calculated ED50’s
were 17 and 23 mg/kg/d, respectively. In comparison, in the
MV4;11 xenograft tumor model, driven by FLT-3 ITD mutations,
potent tumor growth inhibition was observed at doses of g1 mg/
Figure 4. Treatment with 5 inhibits angiogenesis in a bFGF-induced
kg/d with a MED of 1 mg/kg/d. Pertinently, in this target-driven
Matrigel model in vivo. Matrigel containing bFGF was injected
FLT-3 ITD MV4;11 model, significantly lower ED50s (3 mg/kg/d
subcutaneously into mice, and 5 was administrated orally for 8 days
(0-200 mg/kg/d). The Matrigel plugs were removed, and hemoglobin
concentration was measured (n ) 8 mice/group).
vs 17-23 mg/kg/d in KM12L4A or DU145) and significantly
higher numbers of responses (both CR and PR) were observed at
doses g10 mg/kg/d.
dependent inhibition of angiogenesis with a calculated ED50 of
3 mg/kg (Figure 4). Substantial antiangiogenic activity with 5
was observed at 3 mg/kg/d (54%, p ) 0.01), indicating potent
inhibition of bFGF-driven angiogenesis in vivo.
Conclusion
In conclusion, the 4-amino-3-benzimidazol-2-ylhydroquino-
lin-2-one series represents a novel scaffold with multikinase
Table 6. Efficacy of 5 in Tumor Xenograft Models in Vivoa
tumor model (cancer type)
KM12L4A (colon)
target RTKs expressed
TGIb (dose, mg/kg/d; tumor responsesc)
MED (mg/kg/d)e
10
ED50 (mg/kg/d)d
17
VEGFR
PDGFRꢀ
44% (10; 0 CR/PR)
55% (30; 0 CR/PR)
95% (100; 1CR, 3PR)
39% (10; 0 CR/PR)
70% (30; 0 CR/PR)
86% (100; 1PR)
23% (1; 0 CR/PR)
65% (5; 0 CR/PR)
92% (10; 1CR, 5PR)
96% (30; 1CR; 8PR)
DU145 (prostate)
MV4;11 (AML)
VEGFR
PDGFRꢀ
FGFR
FLT-3 ITD
VEGFR
cKIT
30
1
23
3
PDGFRꢀ
FGFR
a Subcutaneous KM12L4A, HCT-116, DU145, or MV4;11 tumors in nude or SCID-NOD mice (size of 200-500 mm3, 10/group) were treated with daily
oral administration of either vehicle or 5. RTK, receptor tyrosine kinase; ITD, internal tandem duplication. b Tumor growth inhibition (TGI) was calculated
as [1 - (mean tumor volume of treated group/mean tumor volume of control group) × 100], when mean of vehicle tumors were ∼1000-2000 mm3,
KM12L4A (day 9); DU145 (day 22); MV4;11 (day 15). c Tumor responses were defined as either complete (no measurable tumor; CR), or partial (50-99%
tumor volume reduction; PR) compared to tumor volume for each animal at treatment initiation. d MED ) minimum effective dose; lowest dose that is
statistically different vs vehicle treatment. e ED50 ) dose of test agent producing 50% TGI.

4-Amino-3-benzimidazol-2-ylhydroquinolin-2-ones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 285
MHz, DMSO-d6) 160.8, 147.7, 142.8, 138.1, 138.7, 134.4, 131.6,
129.0, 122.6, 122.1 (2 Cs), 120.0, 119.1, 118.3, 115.2, 112.8. LCMS
(m/z) 262.2 (MH+), Rt 1.88 min. HRMS found m/z 262.0974;
C16H12N3O requires 262.0981. Anal. calcd for C16 H11N3O: C 73.55
H 4.24, N 16.08; found: C 70.79, H 3.95, N 15.28.
affinity. These compounds inhibit RTKs such as VEGFR-2,
FGFR-1, PDGFRꢀ, and FLT-3, possess good pharmacokinetic
and pharmacological characteristics, and are well tolerated. They
may find application in the treatment of solid and hematological
malignancies, acting not only as antiangiogenic agents but also
affecting tumors in which the above pathways are deregulated
and contribute to tumorigenesis. Among all compounds sur-
veyed, 5 was selected to advance to development, based on its
favorable in vitro potency, drug-like properties, and attractive
pharmacological and toxicological profiles. Compound 5 is
currently in phase I clinical trials.
4-amino-3-(1H-benzo[d]imidazol-2-yl)quinolin-2(1H)-one (8c).
To 2-aminobenzenecarbonitrile 6c (0.1 g, 0.85 mmol, 1.0 equiv)
in CH2Cl2 (5 mL) was added sequentially Et3N (0.36 mL, 2.55
mmol, 3.0 equiv) and methyl malonylchloride (0.11 mL, 1.02 mmol,
1.2 equiv). The reaction was stirred at room temperature for 30
min, upon which starting materials had been consumed as monitored
by LCMS. The reaction was quenched with NaHCO3 (aq., satd.)
and extracted with CH2Cl2 (3x). The combined organics were dried
(MgSO4), filtered, and concentrated to a yellow oil (106 mg), which
was purified by silica gel chromatography to provide 7c as a white
solid (77 mg, 0.35 mmol, 41% yield). Compound 7c was then
combined with phenylenediamine (0.038 g, 0.35 mmol) in a vial.
The two solids were ground together with a glass rod, and the vial
was capped and heated in an oil bath with stirring at 150 °C for
4 h. The reaction was then cooled to room temperature, and DMF
was added. The reaction was subjected to sonication, and the
resulting white solid was collected and dried to provide 8c (8.8
mg, 9% yield).
Experimental Section
1
Chemistry. H and 13C NMR spectra of all compounds were
recorded at 300 and 75 MHz, respectively. 1H shifts are referenced
to the residual protonated solvent signal (δ 2.50 for DMSO-d6),
and 13C shifts are referenced to the deuterated solvent signal (δ
39.5 for DMSO-d6). 13C multiplicities were deduced from DEPT
or APT experiments. Mass spectrometric analysis was performed
on one of two LCMS instruments: a Waters system (Alliance HT
HPLC and a Micromass ZQ mass spectrometer; column: Eclipse
XDB-C18, 2.1 mm × 50 mm; solvent system: 5-95% acetonitrile
in water with 0.1% TFA; flow rate: 0.8 mL/min; molecular weight
range: 150-850; cone voltage: 20 V; column temperature 40 °C)
or a Hewlett-Packard system (Series 1100 HPLC; column: Eclipse
XDB-C18, 2.1 mm × 50 mm; solvent system: 5-95% acetonitrile
in water with 0.1% TFA; flow rate: 0.8 mL/min; molecular weight
range: 150-850; cone voltage: 50 V; column temperature 30 °C).
All masses were reported as those of the protonated parent ions.
The following compounds were commercially available: 2-ami-
nobenzonitrile (Aldrich), 2-amino-5-chlorobenzonitrile (Aldrich),
2-amino-5-fluorobenzonitrile (Aldrich), 2-amino-6-chlorobenzoni-
trile (Alfa Aesar), 2,5-diaminobenzonitrile (Apin), 2-amino-6-
methylbenzonitrile (Fluka), 8a and 8b (Aurora Screening Library).
The synthesis of 2-amino-5-(dimethylamino)benzonitrile,32 2-amino-
General Method for LHMDS Cyclization (Method A). LH-
MDS (1.65 mL, 1 M in THF, 1.65 mmol, 5.0 equiv) was added
dropwise to a solution of ethyl 2-(1H-benzo[d]imidazol-2-yl)acetate
(67.6 mg, 0.33 mmol, 1.0 equiv), 2-aminobenzenecarbonitrile 6c
(60 mg, 0.5 mmol, 1.5 equiv), and anhydrous THF (15 mL). The
resulting solution was stirred at room temperature for 5 h and then
quenched with NH4Cl (aq, sat.). The mixture was diluted with H2O
and extracted with EtOAc (3×). The combined organic layers were
washed once with water, dried with MgSO4, and concentrated to
approximately 10 mL. A yellow solid precipitated, which was
collected via filtration and dried under reduced pressure to provide
8c (28 mg). Additional material (16 mg) was obtained by
concentrating the filtrate and purifying the resulting solid by silica
gel chromatography (5% MeOH/CH2Cl2). Overall 44 mg (48%
4-fluorobenzonitrile,33 have previously been described. 1H and 13
C
yield) of 8c was obtained. H NMR (300 MHz, DMSO-d6) 11.38
1
NMR spectra of the 4-amino-3-benzymidazol-2-yl hydroquinoli-
nones were recorded on the hydrochloride salts, except for 12, which
was recorded as the free base and 5, which was recorded as the
sulfate salt. Elemental analyses were performed on free base.
4-Hydroxy-1-methyl-3-(1-methyl-1H-benzo[d]imidazol-2-
yl)quinolin-2(1H)-one (10). To a solution of ethyl 2-(1-methyl)-
benzimidazol-2-ylacetate (122 mg, 0.56 mmol, 1.0 equiv) in a
mixture of anhydrous THF at -78 °C under an atmosphere of
nitrogen was added LHMDS (1 M in THF, 0.85 mL, 1.5 equiv).
After 1 h, a solution of 1-methylbenzo[d]1,3-oxazaperhydroine-
2,4-dione 9 (100 mg, 0.56 mmol, 1.0 equiv) in anhydrous THF
was added dropwise, and the resulting mixture was allowed to warm
to room temperature and stirred overnight. The reaction was then
quenched with NH4Cl (aq, sat.), and the aqueous layer was extracted
with CH2Cl2 (4×). The combined organic layers were dried over
Na2SO4 and concentrated in vacuo, and the resulting crude material
was purified by column chromatography on silica gel to afford 10
(1 H, s), 8.19 (1 H, d, J ) 8.3 Hz), 7.65 (2 H, bs), 7.56 (1 H, dd,
J ) 8.3, 1.0 Hz), 7.33 (1 H, d, J ) 8.3 Hz), 7.23 (1 H, dd, J ) 8.3,
1.0 Hz), 7.15 (2 H, m). 13C NMR (75 MHz, DMSO-d6) 161.6,
155.2, 139.6, 133.4, 128.7, 126.7, 126.2, 125.0, 124.4, 122.5, 116.8,
114.5, 113.3, 89.1. LCMS (m/z) 276.8 (MH+), Rt 1.68 min. HRMS
found m/z 277.1087; C16H13N4O requires 277.1083.
Ethyl 2-(6-Methylbenzimidazol-2-yl)acetate (18a).17 Synthe-
sized as in example 18c. (80% yield). 1H NMR (300 MHz, DMSO-
d6) 7.37 (1 H, d, J ) 8.1 Hz), 7.27 (1 H, bs), 6.95 (1 H, dd, J )
8.1, 1.5 Hz), 4.11 (2 H, q, J ) 7.2 Hz), 3.91 (2 H s), 3.37 (3 H, s),
1.18 (3 H, t, J ) 7.2 Hz). 13C NMR (75 MHz, DMSO-d6) 168.8,
147.3, 138.0, 136.6, 130.6, 122.9, 114.7, 114.0, 60.7, 35.1, 21.2,
14.0. LCMS (m/z) 219.1 (MH+), Rt 1.42 min. HPLC Rt 14.90 min.
Anal. calcd for C12H14N2O2: C 66.04, H 6.47, N 12.48; found C
66.18, H 6.34, N 12.55.
4-Amino-3-(6-methylbenzimidazol-2-yl)hydroquinolin-2-
1
one (19a). Method A (50% yield). H NMR (300 MHz, DMSO-
1
d6) 11.53 (1 H, s, NH), 8.36 (1 H, d, J ) 8.1 Hz), 7.68 (1 H, d, J
) 8.7 Hz), 7.62 (1 H, d, J ) 8.1 Hz), 7.59 (1 H, bs), 7.41 (1 H, d,
J ) 7.5 Hz), 7.27 (2 H, m), 2.50 (3 H, s). 13C NMR (75 MHz,
DMSO-d6) 160.9, 154.4, 146.3, 139.0, 134.0, 132.5, 131.0, 125.7,
124.1, 121.6, 116.1, 113.5, 113.3, 112.7, 88.4, 21.2. LCMS (m/z):
291.1 (MH+), Rt 1.89 min. HPLC Rt 19.66 min. HRMS found m/z
291.1255; C17H15N4O requires 291.1240.
(30 mg, 20% yield) as an off-white solid. H NMR (300 MHz,
DMSO-d6) 8.16 (1 H, dd, J ) 7.8, 1.5 Hz), 7.96 (1 H, m), 7.81 (1
H, m), 7.73 (1 H, m), 7.57 (3H, m), 7.30 (1 H, dd, J ) 8.1, 7.2
Hz), 3.85 (3 H, s), 3.60 (3 H, s). LCMS (m/z) 306 (MH+), Rt 1.95
min.
3-Benzimidazol-2-ylhydroquinolin-2-one (12). 2-Aminoben-
zaldehyde 11 (180 mg, 1.47 mmol, 1.0 equiv) and ethyl 2-benz-
imidazol-2-ylacetate (300 mg, 1.47 mmol, 1.0 equiv) were dissolved
in EtOH (6 mL). Piperidine (0.150 mL, 129 mg, 1.47 mmol, 1.0
equiv) was added, and the solution was heated at 70 °C overnight.
A precipitate formed, which was filtered. The solid was washed
with EtOH and dried to obtain 12 (300 mg, 78% yield) as bright-
yellow needles. 1H NMR (300 MHz, DMSO-d6) 12.65 (1 H, s, NH),
12.47 (1 H, s, NH), 9.11 (1 H, s), 7.95 (1 H, d, J ) 7.8 Hz), 7.70
(2 H, bs), 7.61 (1 H, dd, J ) 8.1, 7.2 Hz), 7.44 (1 H, d, J ) 8.1
Hz), 7.27 (1 H, dd, J ) 7.8, 7.2 Hz), 7.20 (2 H, m). 13C NMR (75
Ethyl 2-(5,6-Dimethylbenzimidazol-2-yl)acetate (18b).34 Syn-
thesized as in example 18c (86% yield). 1H NMR (300 MHz,
DMSO-d6) 12.0 (1 H, bs, NH), 7.24 (2 H, s), 4.10 (2 H, q, 7.5
Hz), 3.87 (2 H, s), 2.27 (6H, s), 1.17 (3 H, t, J ) 7.5 Hz). LCMS
(m/z) 233.1 (MH+), Rt 1.76 min.
4-Amino-3-(5,6-dimethylbenzimidazol-2-yl)hydroquinolin-2-
1
one (19b). Method A (25% yield). H NMR (300 MHz, DMSO-
d6) 11.48 (1H, s, NH), 8.29 (1 H, d, J ) 8.1 Hz), 8.20 (1 H, bs,
NH), 7.61 (2 H, dd, J ) 8.1, 7.5 Hz), 7.54 (2 H, s), 7.35 (1 H, d,

286 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Renhowe et al.
J ) 8.1 Hz), 7.23 (1 H, dd, J ) 8.1, 7.5 Hz), 2.38 (6 H, bs). 13C
NMR (75 MHz, DMSO-d6) 161.5, 154.9, 146.2, 139.7, 134.3, 133.2,
131.4, 124.6, 122.3, 116.7, 114.3, 113.3, 89.1, 20.6. LCMS (m/z)
305.3 (MH+), Rt 2.31 min. HPLC Rt 20.53 min.
4-Amino-3-[6-(4-methylpiperazinyl)benzimidazol-2-yl]hydro-
quinolin-2-one (19d). Method A (60% yield). 1H NMR (300 MHz,
DMSO-d6) 11.47 (1 H, s, NH), 11.32 (1 H, bs, NH), 8.26 (1 H, d,
J ) 8.1 Hz), 7.63 (2 H, dd, J ) 8.4, 8.1 Hz), 7.35 (1 H, d, J ) 8.4
Hz), 7.27 (2 H, bs), 7.23 (1 H, d, J ) 8.1 Hz), 3.82 (2 H, m), 3.53
(2 H, m), 3.24 (4 H, m), 2.82 (3 H, s). 13C NMR (75 MHz, DMSO-
d6) 161.2, 154.0, 147.3, 146.9, 138.7, 134.1, 132.3, 128.4, 123.9,
121.6, 116.0, 115.5, 114.6, 112.8, 100.5, 89.0, 52.1, 47.0, 41.9.
LCMS (m/z) 375.2 (MH+), Rt 1.38 min. HPLC Rt 14.09 min. HRMS
found m/z 375.1895; C21H23N6O requires 375.1927. Anal. calcd for
C21H22N6O: C67.36, H 5.92, N 22.44; found C67.15, H 5.84, N
22.27.
5-(4-Methyl(1,4-diazaperhydroepinyl))-2-nitrophenylamine
(21c). Method B (20% yield). 1H NMR (300 MHz, DMSO-d6) 7.81
(1 H, d, J ) 9.9 Hz), 7.23 (2 H, bs, NH2), 6.25 (1 H, dd, J ) 9.9,
2.7 Hz), 6.05 (1 H, d, J ) 2.7 Hz), 3.56 (2 H, m), 3.49 (2 H, m),
3.62 (2 H, m), 2.48 (2 H, m), 2.28 (3 H, s), 1.88 (2 H, m). 13C
NMR (75 MHz, DMSO-d6) 153.7, 148.4, 127.5, 122.1, 104.0, 94.6,
56.7, 55.9, 48.1, 47.9, 45.7, 26.4. LCMS (m/z) 251.3 (MH+), Rt
1.57 min. Anal. calcd for C12H18N4O2: C 57.58, H 7.25, N 22.38;
found C 57.03, H 7.11, N 22.31.
5-Morpholin-4-yl-2-nitrophenylamine (21a): General Method
for SNAr Reaction (Method B). 5-Fluoro-2-nitrophenylamine (30.0
g, 0.19 mol) was dissolved in anhydrous 1-methyl-2-pyrrolidinone
(NMP, 40 mL). Triethylamine (58 mL, 0.42 mol, 2.2 equiv) and
morpholine (18 mL, 0.21 mol, 1.1 equiv) were added. The reaction
vessel was fitted with a reflux condenser and heated at 100 °C for
16 h. The reaction was cooled to room temperature and then poured
into 1 L of water. A yellow precipitate formed, which was collected
by filtration through a sintered glass funnel, washed with ether,
and dried under vacuum to yield 21a (38.0 g, 91%) as a yellow
solid.
Ethyl 2-(5-Morpholin-4-ylbenzimidazol-2-yl)acetate (23a):
General Method for Formation of the Benzimidazole Acetate
(Method C). A 2 L round-bottom flask was charged with
5-morpholin-4-yl-2-nitrophenylamine (20.0 g, 90 mmol, 1.0 equiv)
and anhydrous ethanol (800 mL). Palladium on carbon (10% w/w,
6.6 g) was added, and the reaction vessel was fitted with a 3-way
stopcock. The flask was evacuated under vacuum and then filled
with hydrogen. The reaction was stirred overnight under a 1 atm
of hydrogen gas, with additional hydrogen added as necessary. The
reaction was flushed with nitrogen, and ethyl 3-ethoxy-3-imino-
propanoate hydrochloride (44.1 g, 220 mmol, 2.4 equiv) was added.
The reaction vessel was fitted with a reflux condenser and heated
at 90 °C overnight under a positive pressure of nitrogen. The
reaction was then cooled to room temperature and filtered through
a pad of celite. The filtrate was concentrated under reduced pressure.
The resulting residue was dissolved in CH2Cl2 (500 mL), neutralized
with ammonium hydroxide, and washed with water (2 × 100 mL).
The organic phase was concentrated and filtered through a plug of
silica gel using EtOAc. The filtrate was concentrated in vacuo to
afford 23a (21.2 g, 81% yield) as a brown solid (product is very
Ethyl 2-[6-(4-Methyl-1,4-diazaperhydroepinyl)benzimidazol-
1
2-yl]acetate (23c). Method C (53% yield). H NMR (300 MHz,
DMSO-d6) 7.26 (1 H, m), 6.62 (2 H, m), 4.09 (2 H, q, J ) 7.2
Hz), 3.83 (2 H, s), 3.50 (2 H, m), 3.41 (2 H, m), 2.62 (2 H, m),
2.44 (2 H, m), 2.25 (3 H, s), 1.90 (2 H, m), 1.18 (3 H, t, J ) 7.2
Hz). LCMS (m/z) 317.4 (MH+), Rt 1.47 min.
4-Amino-3-[6-(4-methyl(1,4-diazaperhydroepinyl))benzimida-
zol-2-yl]hydroquinolin-2-one (19e). Method A (5% yield). 1H
NMR (300 MHz, DMSO-d6) 11.67 (1 H, bs, NH), 11.48 (1 H, s,
NH), 8.31 (1 H, d, J ) 8.1 Hz), 7.64 (1 H, d, J ) 9.3 Hz), 7.58 (1
H, dd, J ) 8.4, 7.2 Hz), 7.36 (1 H, d, J ) 8.4 Hz), 7.27 (2 H, bs),
7.20 (1 H, dd, J ) 8.1, 7.2 Hz), 3.96 (2 H, m), 3.60 (3 H, m), 3.44
(2 H, m), 3.23 (1 H, m), 2.78 (3 H, s), 2.41 (1 H, m), 2.30 (1 H,
m). 13C NMR (75 MHz, DMSO-d6) 161.7, 154.9, 146.8, 145.5,
139.6, 134.7, 133.2, 127.0, 124.8, 122.3, 116.7, 115.0, 113.7, 113.4,
98.3, 89.2, 55.3, 50.7, 47.1, 43.7, 23.5. LCMS (m/z) 389.4 (MH+),
Rt 1.72 min.
1
hygroscopic). H NMR (300 MHz, DMSO-d6) 7.35 (1 H, d, J )
8.7 Hz), 6.94 (1 H, d, J ) 1.8 Hz), 6.89 (1 H, dd, J ) 8.7, 1.8 Hz),
4.1 (2 H, q, J ) 7.2 Hz), 3.88 (2 H, s), 3.74 (4 H, m), 3.04 (4 H,
m), 1.18 (3 H, t, J ) 7.2 Hz). 13C NMR (75 MHz, DMSO-d6)
168.8, 147.4, 146.8, 138.2, 134.3, 115.8, 112,9, 99.9, 66.2, 60.7,
50.4, 35.1, 14.0. LCMS (m/z): 290.1 (MH+), Rt 1.36 min. HPLC
Rt 13.38 min. Anal. calcd for C15H19N3O3: C 62.27, H 6.62, N 14.51;
found C 62.30, H 6.49, N 14.04.
(3S)-1-(3-Amino-4-nitrophenyl)pyrrolidin-3-yl]dimethy-
1
lamine (21d). Method B (88% yield); mp 148-149 °C. H NMR
(300 MHz, acetone-d6) 7.90 (1 H, d, J ) 9.5 Hz), 6.88 (2 H, bs,
NH2), 6.09 (1 H, dd, J ) 9.5, 2.7), 5.90 (1H, d, J ) 2.7 Hz), 3.56
(2 H, m), 3.36 (1 H, m), 3.15 (1 H, app. t, J ) 8.4 Hz), 2.81 (1 H,
m), 2.24 (6 H, s), 2.22 (1 H, m), 1.88 (1 H, m). 13C NMR (75
MHz, DMSO-d6) 151.8, 148.3, 127.5, 122.4, 104.3, 94.5, 64.5, 51.7,
46.6, 40.3, 29.3. LCMS (m/z): 251.2 (MH+), Rt 1.34 min. HPLC:
4-Amino-3-(6-morpholin-4-ylbenzimidazol-2-yl)hydroquino-
lin-2-one (19c). Method A (61% yield). 1H NMR (300 MHz,
DMSO-d6) 11.48 (1 H, s), 9.20 (1 H, bs), 8.31 (1 H, d, J ) 8.4
Hz), 8.11 (1 H, bs), 7.81 (1 H, d, J ) 8.7 Hz), 7.72 (1 H, d, J )
8.7 Hz), 7.59 (1 H, dd, J ) 8.4, 7.2), 7.38 (1 H, d, J ) 8.4 Hz),
7.24 (1 H, dd, J ) 8.1, 7.2), 4.11 (4H, bs), 3.59 (4H, bs). 13C NMR
(75 MHz, DMSO-d6) 162.4, 155.0, 152.6, 139.9, 139.1, 137.0,
133.8, 132.9, 124.6, 122.3, 116.7, 116.2, 115.2, 113.5, 107.5, 90.0,
64.6, 54.7. LCMS (m/z) 362.1 (MH+), Rt 1.65 min. HPLC Rt 18.16
min; Anal. calcd for C20H19N5O2 + 0.6 H2O: C 64.33, H 5.49, N
18.76; found C 64.21, H 5.37, N 18.37.
22
Rt 13.48 min; [R]D ) -16.3 (c ) 0.57, DMSO).
Ethyl 2-{5-[(3S)-3-(dimethylamino)pyrrolidinyl]benzimidazol-
2-yl}acetate (23d). Method C (76% yield); mp 135 °C (decomposi-
1
tion). H NMR (300 MHz, DMSO-d6) 7.30 (1 H, d, J ) 9.3 Hz),
6.50 (1 H, s), 6.41 (1 H, d, J ) 9.3 Hz), 4.12 (2 H, q, J ) 7.2 Hz),
3.85 (2 H, s), 3.41 (1 H, m), 3.29 (2 H, m), 3.04 (1 H, m), 2.80 (1
H, m), 2.21 (6 H, s), 2.15 (1 H, m), 1.82 (1 H, m), 1.20 (3 H, t, J
) 7.2 Hz). LCMS m/z 317.1 (MH+), Rt 1.45 min. HPLC Rt 11.6
5-(4-Methylpiperazinyl)-2-nitrophenylamine (21b).35 Method
B (96% yield). 1H NMR (300 MHz, DMSO-d6) 7.78 (1 H, dd, J )
9.6, 0.9 Hz), 7.24 (2 H, bs), 6.34 (1 H, dd, J ) 9.6, 2.2 Hz), 6.19
(1 H, d, J ) 2.2 Hz), 3.27 (4 H, t, J ) 4.8 Hz), 2.36 (4H, t, J )
4.8 Hz), 2.17 (3 H, s). 13C NMR (75 MHz, DMSO-d6) 155.0 (C),
148.3 (C), 127.1 (CH), 122.9 (C), 105.4 (CH), 97.5 (CH), 54.1
(CH2), 46.1 (CH2), 45.6 (CH3). LCMS m/z 237.3 (MH+), Rt 1.23
min. Anal. calcd for C11H16N4O2: C, 55.92; H, 6.83; N, 23.71.
Found: C, 55.96; H, 6.62, N, 23.68.
Ethyl 2-[6-(4-Methylpiperazinyl)benzimidazol-2-yl]acetate
(23b). Method C (83% yield). 1H NMR (300 MHz, DMSO-d6) 7.33
(1 H, d, J ) 8.7 Hz), 6.92 (1 H, bs), 6.87 (1 H, d, J ) 8.7 Hz),
4.10 (2 H, q, J ) 7.2 Hz), 3.87 (2 H, s), 3.06 (4 H, m), 2.45 (4 H,
m), 2.20 (3 H, s), 1.18 (3 H, t, J ) 7.2 Hz). 13C NMR (75 MHz,
DMSO-d6) 168.9, 147.5, 146.6, 136.2, 136.0, 118.5, 113.3, 98.1,
60.7, 54.9, 50.06, 45.7, 35.1, 14.0. LCMS (m/z) 303.1 (MH+), Rt
1.15 min. HPLC Rt 9.85 min.
22
min; [R]D ) -25.3 (c ) 1, DMSO).
3-{5-[(3S)-3-(Dimethylamino)pyrrolidinyl]benzimidazol-2-yl}-
4-aminohydroquinolin-2-one (19f). Method A (1.5 g, 43% yield);
mp 280 °C (decomposition). 1H NMR (300 MHz, DMSO-d6) 11.79
(1 H, bs, NH), 11.46 (1 H, s, NH), 8.27 (1 H, d, J ) 7.8 Hz), 8.1
(1 H, bs, NH), 7.61 (1 H, d, J ) 9.0 Hz) 7.60 (1 H, app. t, J ) 5.4
Hz), 7.34 (1 H, d, J ) 7.8 Hz), 7.23 (1 H, app t, J ) 7.8 Hz), 6.88
(1 H, d, J ) 9.0 Hz), 6.80 (1 H, s), 4.04 (1 H, m), 3.70 (2 H, m),
3.57 (1 H, m), 3.35 (1 H, m), 2.81 (6 H, s), 2.44 (2 H, m). 13C
NMR (75 MHz, DMSO-d6) 160.83, 154.02, 144.97, 138.95, 132.44,
123.94, 121.64, 116.03, 114.39, 112.69, 111.61, 94.37, 88.66, 63.82,
49.43, 47.02, 41.26, 40.70, 26.51. LCMS m/z 389.1 (MH+), Rt 1.72
min. HRMS found m/z 389.2050; C22H25N6O requires 389.2084;
22
[R]D ) -21.5 (c ) 0.5, DMSO). Anal. calcd for C22H24N6O +
0.5H2O: C 66.48, H 6.34, N 21.14; found C 66.15, H 6.01, N 21.05.
5-(2-Morpholin-4-ylethoxy)-2-nitrophenylamine (24). Diiso-
propyl azodicarboxylate (14 mL, 71.4 mmol) was added dropwise

4-Amino-3-benzimidazol-2-ylhydroquinolin-2-ones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 287
to a stirred solution of 3-amino-4-nitrophenol (10.1 g, 64.9 mmol),
triphenylphosphine (18.7 g, 71.4 mmol), and N-(2-hydroxyethyl)-
morpholine (7.9 mL, 64.9 mmol) in THF (100 mL) at 0 °C. The
mixture was allowed to warm to room temperature and left to stir
for 18 h. The solvent was then evaporated, and the product was
purified by flash chromotography (98:2 CH2Cl2:MeOH), yielding
24 as a dark reddish-brown syrup (5.62 g, 32%). LCMS (m/z) 268.0
(MH+), Rt 1.01 min.
4-(2-Morpholin-4-ylethoxy)benzene-1,2-diamine (25). To a
solution of 5-(2-morpholin-4-ylethoxy)-2-nitrophenylamine (5.62
g) in EtOH (100 mL), Pd/C (200 mg) was added. The reaction
vessel was purged (3×) with nitrogen and then stirred under a H2
atmosphere for 18 h. The reaction was filtered through a celite plug,
and the plug was washed with EtOH. The solution was used
immediately in the next step. LC/MS (m/z) 238.3 (MH+) Rt 0.295
min.
NH), 9.40 (1 H, bs, NH), 8.30 (1 H, d, J ) 1.5 Hz), 8.28 (1 H, d,
J ) 8.1 Hz), 7.88 (1 H, dd, J ) 8.7. 1.5 Hz), 7.76 (1 H, d, J ) 8.7
Hz), 7.60 (1 H, dd, J ) 8.1, 7.5 Hz), 7.37 (1 H, d, J ) 8.4 Hz),
7.25 (1 H, dd, J ) 8.4, 7.5 Hz). 13C NMR (75 MHz, DMSO-d6)
168.4, 162.5, 155.1, 153.4, 139.5, 139.1, 135.5, 132.9, 125.5, 124.5,
122.3, 116.7, 114.7, 113.5, 90.1. LCMS (m/z) 321.3 (MH+), Rt 2.25
min.
4-Amino-3-{6-[(4-methylpiperazinyl)carbonyl]benzimidazol-
2-yl}hydroquinolin-2-one (19h). A mixture of 19ae (70 mg, 0.22
mmol, 1.0 equiv), 1-methyl piperazine (0.024 mL, 0.22 mmol, 1.0
equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (EDC 50 mg, 0.26 mmol, 1.2 equiv), 1-hydroxy-7-azabenzo-
triazole (HOAT, 35 mg, 0.26 mmol, 1.2 equiv), and Et3N (0.06
mL, 0.44 mmol, 2.5 equiv) in DMF (3 mL) was stirred at 23 °C
for 20 h. The reaction mixture was partitioned between H2O and
EtOAc. The combined organic layers were dried with Na2SO4 and
concentrated. Water was added, and the precipitate was filtered
and dried. The crude solid was purified by reverse phase HPLC to
Ethyl 2-[5-(2-Morpholin-4-ylethoxy)benzimidazol-2-yl]acetate
(26). To the solution of 25 in EtOH described above (125 mL),
ethyl 3-ethoxy-3-iminopropanoate hydrochloride (4.06 g, 20.8
mmol) was added. This reaction mixture was heated for 18 h at 70
°C, at which time the solvent was evaporated and the residue was
taken up in CH2Cl2. The organic layer was washed once with H2O
and then purified by flash chromatography (10:1:2 CH2Cl2:MeOH:
EtOAc) to yield 26 as a dark reddish-brown syrup (4.94 g, 70%
yield over two steps). 1H NMR (300 MHz, CDCl3) 7.4 (1 H, broad
s), 6.9 (1 H, broad s), 6.83 (1 H, dd, J ) 9.0 Hz), 4.18 (2 H, q),
4.1 (2 H, t, J ) 5.4 Hz), 3.96 (2 H, s), 3.71 (4 H, t, J ) 5.1 Hz),
2.77 (2 H, t, J ) 5.7 Hz), 2.54 (4 H, t, J ) 4.8 Hz), 1.25 (3 H, t,
J ) 7.2 Hz). LCMS (m/z) 334.4 (MH+) Rt 1.08 min.
4-Amino-3-[5-(2-morpholin-4-ylethoxy)-benzimidazol-2-yl]hy-
droquinolin-2-one (19g). Method A (239 mg, 14% yield). 1H NMR
(300 MHz, D2O) 7.83 (d, J ) 8.7, 1H), 7.59 (t, J ) 7.2, 1H), 7.52
(d, J ) 8.7, 1H), 7.24 (t, J ) 6.9, 1H), 7.19 (d, J ) 7.8, 1H), 7.10
(s, 1H), 6.98 (dd, J ) 9, 1H), 4.32 (t, J ) 5.1, 2H), 4.11 (2H,
broad m), 3.88 (broad m, 2H), 3.64 (t, J ) 5.1, 2H), 3.60 (broad
m, 2H), 3.33 (broad m, 2H). Note: At 50 °C, the broad multiplets
at 3.33, 3.60, 3.88, and 4.11 resolved into two broad multiplets of
4 H each (δ 3.73, 4.28). LCMS (m/z) 406.4 (MH+) Rt 1.56 min.
HRMS found m/z 406.1850; C22H24N5O3 requires 406.1873.
2-[5-(Methoxycarbonyl)benzimidazol-2-yl]acetate (18c). Meth-
yl 3,4-diaminobenzoate (14.85 g, 89.4 mmol, 1 equiv) was stirred
with ethyl-3-ethoxy-3-iminopropanoate hydrochloride (26.3 g, 134.9
mmol, 1.5 equiv) in EtOH (300 mL) at 70 °C overnight. The
reaction mixture was cooled to room temperature, and the excess
unreacted ethyl-3-ethoxy-3-iminopropanoate hydrochloride was
removed by filtration. The EtOH was removed under reduced
pressure, and the residue was dissolved in water and extracted with
CH2Cl2 (3×). The organic extracts were dried over Na2SO4, and
the solvent removed under reduced pressure. The solid was triturated
with Et2O to yield 18c as an off-white solid (18.4 g, 78%). 1H NMR
(300 MHz, DMSO-d6) 8.15 (1 H, d, J ) 1.5 Hz), 7.81 (1 H, dd, J
) 8.4, 1.5 Hz), 7.61 (1 H, d, J ) 8.4 Hz), 4.14 (2 H, q, J ) 7.2
Hz), 4.04 (2 H, s), 3.86 (3 H, s), 1.20 (3 H, t, J ) 7.2). 13C NMR
(75 MHz, DMSO-d6) 169.2, 167.5, 151.3, 143.1, 139.3, 123.7,
123.5, 117.7, 115.3, 61.6, 52.6, 35.8, 14.7. LCMS (m/z) 263.3
(MH+), Rt 1.82 min. Anal. calcd for C13H14N2O4: C 59.41, H 5.12,
N 10.51; found C 59.54, H 5.38, N 10.68.
1
afford 19h (29.8 mg, 34%). H NMR (300 MHz, DMSO-d6) 8.20
(1 H, d, J ) 8.1 Hz), 7.81 (1 H, d, J ) 1.5 Hz), 7.77 (1 H, d, J )
8.4 Hz), 7.59 (1 H, dd, J ) 8.1, 7.2 Hz), 7.42 (1 H, dd, J ) 8.4,
1.2 Hz), 7.33 (1 H, d, J ) 8.4 Hz), 7.23 (1 H, dd, J ) 8.4, 7.2 Hz),
4.12 (2 H, m), 3.40 (4 H, m), 3.11 (2 H, m). 13C NMR (75 MHz,
DMSO-d6) 169.6, 161.6, 154.4, 151.2, 138.5, 135.7, 134.3, 132.3,
129.1, 124.0, 122.3, 121.6, 116.0, 114.2, 113.6, 112.8, 89.1, 51.9,
42.0, 40.1. LCMS (m/z) 403.3 (MH+), Rt 1.80 min. Anal. calcd for
C22H22N6O2 + 2H2O: C 60.26, H 5.98, N 19.17; found C 60.38, H
5.48, N 18.84.
4-amino-3-(6-(hydroxymethyl)-1H-benzo[d]imidazol-2-yl)quin-
olin-2(1H)-one (19af). A suspension of methyl 2-(4-amino-2-oxo-
3-hydroquinolyl) benzimidazole-6-carboxylate 19ad (1.0 g, 3.1
mmol, 1.0 equiv) in toluene (100 mL) was treated with DIBAL-H
(10.3 mL of 1.5 M soln in toluene, 15.5 mmol, 5.0 equiv) at room
temperature. The reaction mixture was stirred for 10 h and was
then treated with NaF (2.6 g, 62 mmol, 20 equiv) followed by water
(0.27 mL, 15 mmol, 4.8 equiv). The reaction was stirred at room
temperature for an additional 1.5 h, and then DMF was added. The
resulting mixture was heated at 100 °C for 1 h and then filtered.
The filtrate was evaporated to one-quarter of the original volume
and treated with 50 mL of water. The resulting solid was collected
1
and dried to give 19af (475 mg, 50% yield). H NMR (300 MHz,
DMSO-d6) 13.28 (1 H, s), 11.42 (1 H, s), 8.19 (1 H, d, J ) 8.8
Hz), 7.58 (3 H, m), 7.34 (1 H, d, J ) 8.8 Hz), 7.26 (2 H, m), 4.58
(2 H, s), 4.5-4.9 (1 H, bs). LCMS (m/z) 307.1 (MH+), Rt 1.47
min.
2-(4-Amino-2-oxo-1,2-dihydroquinolin-3-yl)-1H-benzo[d]imi-
dazole-6-carbaldehyde (19ag). 19af (320 mg, 1.04 mmol, 1.0
equiv) was dissolved in DMA (4 mL), and MnO2 (450 mg, 5.2
mmol, 5.0 equiv) was added. The reaction mixture was heated under
microwave irradiation at 140 °C for 10 min. The reaction mixture
was then filtered through celite, and water was added to the filtrate.
The resulting solid was collected and dried to give 19ag (310 mg,
97% yield). 1H NMR (300 MHz, DMSO-d6) 13.28 (1 H, s), 11.42
(1 H, s), 10.00 (1 H, s), 8.42 (1 H, bs), 8.24 (1 H, d, J ) 6.3 Hz),
8.19 (1 H, d, J ) 4.6 Hz), 7.86 (1 H, d, J ) 8.0 Hz), 7.74 (1 H, dd,
J ) 8.0, 4.6 Hz), 7.58 (1 H, dd, J ) 8.0, 1.0 Hz), 7.34 (1 H, d, J
) 8.0 Hz), 7.23 (1 H, dd, J ) 8.0, 1.0 Hz). LCMS (m/z) 305.1
(MH+), Rt 2.47 min.
4-Amino-3-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]im-
idazol-2-yl)quinolin-2(1H)-one (19i). 19ag (250 mg, 0.82 mmol,
1.0 equiv) was suspended in MeOH (5 mL). Piperazine (123 mg,
1.23 mmol, 1.5 equiv) and HOAc (0.15 mL, 2.46 mmol, 3.0 equiv)
were added followed by NaBH3CN (257 mg, 4.1 mmol, 5.0 equiv).
The reaction was heated at 90 °C for 6 h in a glass bomb. The
solution was cooled and then poured into water and extracted with
EtOAc. The organic layer was dried and concentrated, and the
resulting oil was purified by reverse phase HPLC. The purified
fractions were combined, neutralized with NaHCO3 (aq, sat.), and
extracted with EtOAc (2×). The organic extracts were combined,
dried, and concentrated to give 19i (160 mg, 50% yield). LCMS
(m/z) 389.1 (MH+), Rt 1.46 min.
Methyl 2-(4-Amino-2-oxo-3-hydroquinolyl) benzimidazole-
6-carboxylate (19ad). Method A (21% yield). 1H NMR (300 MHz,
DMSO-d6) 13.28 (1 H, s), 11.42 (1 H, s), 8.37 (1 H, bs), 8.24 (1
H, d, J ) 6.3 Hz), 8.20 (1 H, d, J ) 4.6 Hz), 7.80 (1 H, d, J ) 8.0
Hz), 7.74 (1 H, dd, J ) 8.0, 4.6 Hz), 7.56 (1 H, dd, J ) 8.0, 1.0
Hz), 7.34 (1 H, d, J ) 8.0 Hz), 7.24 (1 H, dd, J ) 8.0, 1.0 Hz),
3.85 (3 H, s).
2-(4-Amino-2-oxo-3-hydroquinolyl)benzimidazole-6-carboxy-
lic acid (19ae). The crude material obtained in the previous step
was dissolved in a 1:1 mixture of EtOH and 30% aq KOH (10
mL) and stirred overnight at 70 °C. The reaction mixture was cooled
and acidified with 1N HCl. A precipitate formed. The solid was
filtered, washed with water, and dried to obtain 19ae (190 mg, 40%)
1
as a brown solid. H NMR (300 MHz, DMSO-d6) 11.46 (1 H, s,

288 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Renhowe et al.
4-Amino-3-(6-(methylamino)-1H-benzo[d]imidazol-2-yl)quin-
olin-2(1H)-one (19ah). Method A (63% yield). HRMS found m/z
306.1354; C17H15N5O requires 306.1349.
393.3 (MH+), Rt 1.84 min. HRMS found m/z 393.1846; C21H22N6OF
requires 393.1833. Anal. calcd for C21H21FN6O: C 64.24, H 5.39,
N 21.42; found C 64.87, H 5.24, N 20.91.
N-(2-(4-Amino-2-oxo-1,2-dihydroquinolin-3-yl)-1H-benzo[d]im-
idazol-6-yl)-2-(dimethylamino)-N-methylacetamide (19j). To a
solution of 19ah (75 mg, 0.25 mmol, 1.0 equiv) in DMA (6 mL)
was added 2-(dimethylamino)acetic acid (38 mg, 0.37mmol, 1.5
equiv), EDC (57.4, 0.37 mmol, 1.5 equiv), HOBT (50 mg, 0.37
mmol,1.5 equiv), and Et3N (0.104 mL, 0.74 mmol, 3 equiv). The
reaction mixture was stirred at room temperature for 12 h. The
reaction mixture was then concentrated to a thick oil and triturated
with water. The resulting solid was collected and purified by reverse
phase HPLC. The purified fractions were combined, neutralized
with NaHCO3 (aq, sat.), and extracted with EtOAc (2×). The
organic extracts were combined, dried, and concentrated to provide
19j (70 mg, 72% yield). LCMS (m/z) 391.2 (MH+), Rt 1.68 min.
4-Amino-5-fluoro-3-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]im-
4-Amino-6-chloro-3-[6-(4-methylpiperazinyl)benzimidazol-2-
yl]hydroquinolin-2-one (19o). Method A (46% yield). H NMR
1
(300 MHz, DMSO-d6) 11.5 (1 H, s), 9.6 (1 H, bs), 8.33 (1 H, d, J
) 2.1 Hz), 7.60 (2 H, dd, J ) 8.7, 2.1 Hz), 7.34 (1 H, d, J ) 8.7
Hz), 7.28 (1 H, s), 6.97 (1 H, dd, J ) 9.0, 2.1 Hz), 3.75 (1 H, m),
3.55 (1 H, m), 3.24 (1 H, m), 3.0 (1 H, m), 2.9 (3 H, s). 13C NMR
(75 MHz, DMSO-d6) 161.5, 153.8, 147.6, 147.3, 138.4, 134.4,
132.9, 126.6, 124.0, 118.7, 116.6, 115.2, 114.6, 101.1, 89.9, 52.6,
47.6, 42.7. HRMS found m/z 409.1550; C21H22N6OCl requires
409.1538.
4,6-Diamino-3-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imida-
zol-2-yl)quinolin-2(1H)-one (19p). Method A (45% yield). 1H
NMR (300 MHz, DMSO-d6) 11.35 (1 H, s), 9.91 (1 H, bs), 7.71
(1 H, s), 7.55 (1 H, d, J ) 8.8 Hz), 7.29 (3 H, m), 6.96 (1 H, dd,
J ) 8.8, 1.8 Hz), 3.75 (1 H, m), 3.55 (1 H, m), 3.24 (1 H, m), 3.0
(1 H, m), 2.9 (3 H, s). 13C NMR (75 MHz, DMSO-d6) 162.6,159.3,
158.8, 153.1, 151.8, 146.6, 136.1, 130.5, 125.9, 119.1, 117.8, 115.7,
115.2, 114.7, 114.3, 91.7, 53.2, 48.1, 42.8.
1
idazol-2-yl)quinolin-2(1H)-one (5). Method A (60% yield). H
NMR (300 MHz, DMSO-d6) 11.66 (1H, s, NH), 9.73 (1H, bs, NH),
7.66 (1H, d, J ) 9.1 Hz), 7.59 (1H, ddd, J ) 8.5, 8.0, 6.0 Hz),
7.29 (1H, dd, J ) 9.1, 1.5 Hz), 7.23 (1H, s), 7.15 (1H, d, J ) 8.0
Hz), 7.04 (1H, dd, J ) 13.8, 8.5 Hz), 3.86 (2H, d, J ) 12.7 Hz),
3.56 (2H, d, J ) 11.4 Hz), 3.23 (2H, m), 3.07 (2H, m), 2.88 (3H,
s). 13C NMR (75 MHz, DMSO-d6) 162.5, 159.2, 151.9 (d, J )
42.3 Hz), 147.0, 140.2, 136.5, 133.2, 132.9 (d, J ) 14.1 Hz), 118.1,
114.7, 113.9, 113.0, 108.6 (d, J ) 18.9 Hz), 104.9, 103.3, 99.4,
91.5, 48.4, 43.6. Anal. calcd for C21H21N6OF·1.5H2O: C 60.13, H
5.77, N 20.04, F 4.53; found C 60.74, H 5.98, N 18.50, F 4.13.
HRMS found m/z 393.1812; C21H22N6OF requires 393.1833.
4-Amino-5-chloro-3-[6-(4-methylpiperazinyl)benzimidazol-2-
yl]hydroquinolin-2-one (19k). Method A (8% yield). 13C NMR
(75 MHz, DMSO-d6) 161.2, 154.0, 147.3, 146.9, 138.7, 134.1,
132.3, 127.4, 123.9, 120.4, 116.6, 115.5, 114.6, 100.5, 89.0, 52.1,
47.0, 41.9. HRMS found m/z 409.1550; C21H22N6OCl requires
409.1538.
4-Amino-5-methyl-3-[6-(4-methylpiperazinyl)benzimidazol-2-
yl]hydroquinolin-2-one (19l). Method A (8% yield). 1H NMR (300
MHz, DMSO-d6) 11.56 (1 H, bs, NH), 11.37 (1 H, s, NH), 7.62 (1
H, d, J ) 9.0 Hz), 7.41 (1 H, dd, J ) 8.4, 7.5 Hz), 7.26 (1 H, d,
J ) 2.1 Hz), 7.21 (1 H, d, J ) 8.4 Hz), 7.20 (1 H, dd, J ) 9.0, 2.1
Hz), 7.00 (1 H, d, J ) 7.5 Hz), 3.78 (2 H, m), 3.51 (2 H, m), 3.25
(4 H, m), 2.85 (3 H, s), 2.80 (3 H, s). 13C NMR (75 MHz, DMSO-
d6) 160.6, 157.1, 147.6, 146.9, 139.9, 136.0, 133.7, 131.5, 127.9,
125.8, 115.6, 114.4, 112.8, 100.3, 89.4, 52.0, 47.0, 41.9, 23.2.
LCMS (m/z) 389.5 (MH+), Rt 1.88 min. HPLC Rt 15.03 min. HRMS
found m/z 389.2053; C22H25N6O requires 389.2084; Anal. calcd for
C22H24N6O + 0.5H2O: C 66.64, H 6.71, N 20.73; found C 66.36,
H 6.14, N 20.00.
4-Amino-6-(dimethylamino)-3-[6-(4-methylpiperazinyl)ben-
zimidazol-2-yl]hydroquinolin-2-one (19q). Method A (48% yield).
1H NMR (300 MHz, DMSO-d6) 11.17 (1 H, s), 9.90 (1 H, bs),
7.58 (1 H, d, J ) 8.7 Hz), 7.42 (1 H, s), 7.25 (3 H, m), 7.0 (1 H,
dd, J ) 9.0, 2.4 Hz), 3.75 (1 H, m), 3.55 (1 H, m), 3.24 (1 H, m),
3.0 (1 H, m), 2.9 (3 H, s). 13C NMR (75 MHz, DMSO-d6) 162.1,
159.6, 159.1, 153.8, 151.4, 146.9, 144.9, 132.4, 122.7, 121.4, 118.8,
117.5, 115.5, 115.0, 114.1, 111.0, 107.9, 101.9, 90.9, 53.2, 48.0,
42.8. HRMS found m/z 418.2360; C23H28N7O requires 418.2349.
N-(4-Amino-3-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imida-
zol-2-yl)-2-oxo-1,2-dihydroquinolin-6-yl)benzamide (19r). To a
solution of 19p (20 mg, 0.05 mmol, 1.0 equiv) in 1.0 mL of N,N-
dimethylacetamide (1.0 mL) was added benzoyl chloride (14 mg,
0.10 mmol, 2.0 equiv) followed by Et3N (0.23 mL, 1.65 mmol, 5
equiv). The resulting solution was stirred at room temperature for
8 h. The reaction was concentrated and purified by reverse phase
1
HPLC to provide 19r (21 mg, 79% yield) as a yellow solid. H
NMR (300 MHz, DMSO-d6) 11.4 (1 H, s), 10.4 (1 H, s), 9.85 (1
H, bs), 8.5 (1 H, s), 8.0 (2 H, m), 7.73 (2 H, dd, J ) 2.4, 9.3 Hz),
7.54 (4 H, m), 7.33 (1 H, d, J ) 8.7 Hz), 7.27 (1 H, d, J ) 1.8
Hz), 7.01 (1 H, dd, J ) 2.1, 8.7 Hz), 3.75 (1 H, m), 3.55 (1 H, m),
3.24 (1 H, m), 3.0 (1 H, m), 2.9 (3 H, s). HRMS found m/z
494.2330; C28H28N7O2 requires 494.2298.
4-Amino-6-(benzylamino)-3-(6-(4-methylpiperazin-1-yl)-1H-
benzo[d]imidazol-2-yl)quinolin-2(1H)-one (19s). To a solution of
19p (20 mg, 0.05 mmol, 1.0 equiv) in 1.0 mL of MeOH/CH2Cl2/
AcOH (2:2:1) was added benzaldehyde (11 mg, 0.10 mmol, 2.0
equiv) followed by BH3/Pyr (0.3 mL of ∼8 M). The resulting
solution was stirred at room temperature for 8 h. The reaction
mixture was quenched with H2O, and the solid was purified by
reverse phase HPLC to provide 19s (19 mg, 82% yield) as a yellow
4-Amino-5-(methylamino)-3-(6-(4-methylpiperazin-1-yl)-1H-
benzo[d]imidazol-2-yl)quinolin-2(1H)-one (19m). Compound 5
(50 mg, 0.13 mmol, 1.0 equiv) was dissolved in a solution of
MeNH2 (6 M in EtOH, 3 mL, 18 mmol, 140 equiv) and heated at
180 °C in a sealed vessel for 48 h. The reaction mixture was cooled,
concentrated, and purified via reverse phase HPLC to provide 19m
(21 mg, 41% yield) as a yellow solid. 1H NMR (300 MHz, DMSO-
d6) 11.12 (1 H, s), 9.8 (1 H, bs), 9.75 (1 H, bs), 7.51 (1 H, d, J )
8.7 Hz), 7.35 (1 H, t, J ) 8.1 Hz), 7.24 (1 H, bs), 6.94 (1 H, dd,
J ) 8.1, 2.4 Hz), 6.78 (1 H, d, J ) 8.1 Hz), 6.57 (1 H, d, J ) 7.8
Hz), 3.75 (1 H, m), 3.55 (1 H, m), 3.24 (1 H, m), 3.0 (1 H, m), 2.9
(3 H, s), 2.71 (3 H, s).
1
solid. H NMR (300 MHz, DMSO-d6) 11.09 (1 H, s), 9.9 (1 H,
bs), 7.75 (1 H, d, J ) 8.6 Hz), 7.44 (2 H, m), 7.33 (2 H, m), 7.25
(3 H, m), 7.04 (3 H, m), 4.40 (2 H, s), 3.75 (1 H, m), 3.55 (1 H,
m), 3.24 (1 H, m), 3.0 (1 H, m), 2.9 (3 H, s). 13C NMR (75 MHz,
DMSO-d6) 161.9, 159.2, 158.8, 153.7, 146.7, 143.9, 140.1, 130.9,
128.9, 128.5, 127.6, 122.0, 117.5, 114.7, 114.2, 103.2, 90.8, 53.2,
48.1, 48.0, 42.8.
4-Aminobenzene-1,3-dicarbonitrile (27d).36 A dry round-
bottom flask was charged with 2-amino-5-bromo benzonitrile (1.5
g, 7.5 mmol, 1.0 equiv) and zinc cyanide (880 mg, 7.5 mmol, 1.0
equiv) and DMF (15 mL). Nitrogen was bubbled through the
solution for 5 min, and then Pd[P(Ph)3]4 (693 mg, 0.6 mmol, 8
mol%) was added in one portion. The reaction mixture was stirred
at 90 °C overnight. After cooling to room temperature, NaHCO3
(aq, sat.) was added, and the mixture was extracted with EtOAc.
The organic extracts were collected and dried with Na2SO4.
Evaporation of the solvent under reduced pressure and purification
by column chromatography on silica gel (2% MeOH/CH2Cl2)
4-Amino-6-fluoro-3-[6-(4-methylpiperazinyl)benzimidazol-2-
1
yl]hydroquinolin-2-one (19n). Method A (25% yield). H NMR
(300 MHz, DMSO-d6) 11.51 (1 H, s, NH), 11.30 (1 H, bs, NH),
8.17 (1 H, dd, JH-F ) 10.5, JH-H ) 2.1 Hz), 7.62 (1 H, d, J ) 9.0
Hz), 7.58 (1 H, dd, JH-F ) 11.1, JH-H ) 8.7 Hz), 7.37 (1 H, dd,
JH-H ) 8.7, JH-F ) 7.4 Hz), 7.26 (1 H, bs), 7.20 (1 H, bd, J ) 9.0
Hz), 3.78 (2 H, m), 3.50 (2 H, m), 3.20 (4 H, m), 2.81 (3 H, s). 13
C
NMR (75 MHz, DMSO-d6) 161.6, 157.8 (d, JC-F ) 236 Hz, C
ipso), 154.2, 147.8, 147.3, 136.4, 134.4, 128.5, 121.3 (d, JC-F
24.5 Hz), 118.8, 116.7, 115.3, 114.2 (d, JC-F ) 8.0 Hz), 110.2 (d,
JC-F ) 27.0 Hz), 101.1, 90.1 52.7, 47.7, 42.7, 23.5. LCMS (m/z)
)
1
afforded 27d (900 mg, 84%) as a white solid. H NMR (CDCl3,

4-Amino-3-benzimidazol-2-ylhydroquinolin-2-ones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 289
300 MHz) 7.70 (1 H, d, J ) 1.8 Hz), 7.55 (1 H, d, J ) 8.7, 1.8
Hz), 6.78 (1 H, d, J ) 8.7 Hz), 4.93 (1 H, bs). GC/MS (m/z %)
143 (M+, 100%), 116 (M+-HCN, 30%), Rt 14.4 min.
of the residue by chromatography (3:1 EtOAc:hexanes) on silica
gel afforded 17a (1.0 g, 51%). GC/MS (m/z %) 136 (M+, 100%),
109 (M+-HCN, 30%) Rt 7.93 min.
4-Amino-3-[6-(4-methylpiperazinyl)benzimidazol-2-yl]-2-oxo-
hydroquinoline-6-carbonitrile (19ai). Method A (24% yield). 1H
NMR (300 MHz, DMSO-d6) 11.61 (1 H, bs), 11.09 (1 H, s), 8.88
(1 H, d, J ) 1.5 Hz), 7.95 (1 H. dd, J ) 8.7, 1.5 Hz), 7.66 (1 H,
d, J ) 8.7 Hz), 7.47 (1 H, d, J ) 9.0 Hz), 7.29 (1 H, s), 7.28 (1 H,
d, J ) 9.0 Hz), 3.82 (2 H, m), 3.52 (2 H, m), 3.29 (4 H, m), 2.80,
(3 H, s). 13C NMR (75 MHz, DMSO-d6) 161.6, 153.8, 147.6, 146.7,
142.5, 135.3, 134.4, 130.4, 128.6, 119.5, 117.9, 116.9, 115.3, 113.7,
104.4, 101.2, 90.2, 52.5, 47.7, 42.5. LCMS (m/z) 400.4 (MH+), Rt
1.89 min.
4-Amino-8-fluoro-3-[6-(4-methylpiperazinyl)benzimidazol-2-
yl]hydroquinolin-2-one (19w). Method A (45% yield). H NMR
1
(300 MHz, DMSO-d6) 11.40 (1 H, s, NH) 8.61 (1 H, bs, NH), 8.1
(1 H, d, JH-H ) 8.4 Hz), 7.64 (1 H, d, JH-H ) 9.0 Hz), 7.51 (1 H,
dd, JH-F ) 10.7, JH-H ) 7.8 Hz), 7.26 (1 H, d, JH-H ) 2.1 Hz),
7.21 (1 H, dd, JH-H ) 9.0, 2.1 Hz), 7.20 (1 H, ddd, JH-H ) 8.4,
7.8, JH-F ) 3.0 Hz), 3.80 (2 H, m), 3.51 (2 H, m), 3.22 (4 H, m),
2.8 (3 H, s). 13C NMR (75 MHz, DMSO-d6) 161.7, 154.1, 149.9
(d, JC-F ) 244.0 Hz, C ipso), 147.7, 135.8, 135.0, 128.4 (d, JC-F
) 14.5 Hz), 121.9, 120.3, 117.8, 116.3, 115.7, 115.3, 100.8, 90.4,
52.8, 47.6, 42.5. LCMS (m/z) 393.1 (MH+), Rt 1.48 min. HPLC Rt
14.96 min. HRMS found m/z 393.1802; C21H22N6OF requires
393.1833.
4-Amino-3-[6-(4-methylpiperazinyl) benzimidazol-2-yl]-2-oxo-
hydroquinoline-6-carboxylic acid (19aj). Compound 19ai (24 mg,
0.06 mmol, 1.0 equiv) was dissolved in a 1:1 mixture of EtOH
and 30% aq NaOH (2 mL). The solution was heated to 100 °C for
2 h. The mixture was cooled to room temperature, concentrated,
and neutralized with 1 N HCl. The precipitate was filtered and
washed with H2O twice and dried to afford the desired 19aj in
quantitative yield. LCMS (m/z) 419.2 (MH+), Rt 1.35 min.
{4-Amino-3-[6-(4-methylpiperazinyl)benzimidazol-2-yl]-2-
oxo(6-hydroquinolyl)}-N-benzylcarboxamide (19t). Compound
19aj (25 mg, 0.06 mmol, 1.0 equiv) was suspended in DMF (2
mL). Triethylamine (0.016 mL, 0.12 mmol, 2.0 equiv) and
benzylamine (0.009 mL, 0.08 mmol, 1.2 equiv) were added,
followed by EDC (16 mg, 0.08 mmol, 1.2 equiv) and HOAT (11
mg, 0.08 mmol, 1.2 equiv). The reaction mixture was stirred at
room temperature for 2 days. Water was then added, and the mixture
was extracted with EtOAc. The residue was purified by reverse
4-Amino-6,7-difluoro-3-(6-(4-methylpiperazin-1-yl)-1H-ben-
zo[d]imidazol-2-yl)quinolin-2(1H)-one (19x). Synthesized via
modified isatoic anhydride route as previously described.18 (28%
yield for 3 steps). LCMS (m/z) 411.3 (MH+), Rt 1.82 min.
6-Amino-2,4-difluorobenzenecarbonitrile (17b).38 2,4,6-Trif-
luorobenzonitrile (160 mg, 1 mmol, 1.0 equiv) was dissolved in
CH3CN and conc NH4OH (2:1, 6 mL). The reaction mixture was
stirred at room temperature for 3 days. The reaction mixture was
then diluted with CH2Cl2. The organic phase was washed with H2O,
brine, and then dried with Na2SO4. Evaporation of the solvent under
reduced pressure afforded a mixture of 2-amino-4,6-difluoroben-
zonitrile (17b) and 4-amino-2,6-difluorobenzonitrile. The two
isomers were separated by flash chromatography on silica gel (1:2
EtOAc:hexanes) and the desired 2-amino-4,6-difluorobenzonitrile
1
1
(17b) in 47% yield (72 mg). H NMR (300 MHz, CDCl3) 6.25
phase HPLC to give 19t (13.8 mg, 45%). H NMR (300 MHz,
(1H, ddd, JH-F ) 10.8, 9.9, JH-H ) 2.1 Hz) 6.23 (1H, dd, JH-F
)
DMSO-d6) 11.67 (1 H, s), 11.49 (1 H, bs), 9.42 (1 H, app. t, J )
5.4 Hz), 9.27 (1 H, s), 8.50 (1 H, bs), 8.11 (1 H, dd, J ) 8.4, 1.2
Hz), 7.65 (1 H, d, J ) 9.6 Hz), 7.4-7.1 (7 H, m), 4.49 (2 H, app
d, J ) 5.7 Hz), 3.82 (2 H, m), 3.51 (2 H, m), 3.24 (4 H, m), 2.80,
(3 H, s). 13C NMR (75 MHz, DMSO-d6) 165.1, 161.1, 154.3, 147.2,
146.4, 140.8, 139.8, 133.5, 131.4, 128.2, 127.4, 127.2, 126.7, 124.1,
116.0, 115.8, 114.5, 112.1, 100.2, 88.9, 52.0, 46.9, 42.6, 41.9.
LCMS (m/z) 508.5 (MH+), Rt 2.11 min.
10.7, JH-H ) 2.1 Hz); GC/MS (m/z %) 154 (M+, 100%).
4-Amino-5,7-difluoro-3-[6-(4-methylpiperazinyl)benzimida-
zol-2-yl]hydroquinolin-2-one (19y). Method A (45% yield). H
1
NMR (CDCl3, 300 MHz): 11.75 (1 H, s, NH), 11.50 (1 H, bs, NH),
7.56 (1 H, d, JH-H ) 9.0 Hz), 7.37 (1 H, dd, JH-F ) 11.4, JH-H
2.1 Hz), 7.26 (1 H, d, JH-H ) 1.8 Hz), 7.14 (1 H, ddd, JH-F
)
)
11.4, 9.0, JH-H ) 2.1 Hz), 7.03 (1 H, dd, JH-H ) 9.0, 1.8 Hz), 3.73
(2 H, m), 3.48 (2 H, m), 3.20 (4 H, m), 2.78 (3 H, s). LCMS (m/z)
411.2 (MH+), Rt 1.94 min.
4-Amino-7-fluoro-3-[6-(4-methylpiperazinyl)benzimidazol-2-
1
yl]hydroquinolin-2-one (19u). Method A (46% yield). H NMR
(300 MHz, DMSO-d6) 11.45 (1 H, s), 9.9 (1 H, bs), 8.25 (1 H, dd,
J ) 8.8, 5.8 Hz), 7.55 (1 H, d, J ) 8.8 Hz), 7.38 (1 H, s), 7.1 (2
H, m), 6.95 (1 H, dd, J ) 2.1, 8.8 Hz), 3.75 (1 H, m), 3.55 (1 H,
m), 3.24 (1 H, m) 3.0 (1 H, m), 2.9 (3 H, s). 13C NMR (75 MHz,
DMSO-d6) 166.5,163.2, 161.8, 154.5, 147.8, 147.5, 141.5, 134.3,
127.9, 127.7, 116.6, 115.1. HRMS found m/z 393.1811;
C21H22N6OF requires 393.1833.
4-Amino-7-(dimethylamino)-6-fluoro-3-(6-(4-methylpiperazin-
1-yl)-1H-benzo[d]imidazol-2-yl)quinolin-2(1H)-one (19z). Com-
pound 19x (0.0569 mg, 0.14 mmol, 1.0 equiv) and Me2NH (2 M
in THF, 1.0 mL, 2.0 mmol, 13.3 equiv) were heated in NMP (2
mL) at 80 °C overnight. After cooling, the reaction mixture was
purified by reverse phase HPLC to afford 19z (38.7 mg, 47% yield).
LCMS (m/z) 436.2 (MH+), Rt 1.85 min. HRMS found m/z 436.2274;
C23H27N7OF requires 436.2255.
4-Amino-7-(methylamino)-3-[6-(4-methylpiperazinyl)benzimi-
dazol-2-yl]hydroquinolin-2-one (19v). Compound 19u (28 mg,
0.07 mmol, 1.0 equiv) was combined with MeNH2 (8 M) and 1:1
EtOH:NMP (2 mL) and then heated under microwave irradiation
4 times for 5 min at 220 °C. After cooling, water was added and
the mixture extracted with EtOAc. The organic extracts were
collected and dried over Na2SO4. Evaporation of the solvent under
reduced pressure, and purification of the residue by reverse phase
2-(4-Methylpiperazinyl)-6-nitrobenzenecarbonitrile.21 Yield
1
69%. H NMR (300 MHz, DMSO-d6) 7.83 (1 H, dd, J ) 8.1, 1.2
Hz), 7.78 (1 H, t, J ) 8.1 Hz), 7.58 (1 H, dd, J ) 8.1, 1.2 Hz),
3.23 (4 H, t, J ) 4.8 Hz), 2.49 (4 H, t, J ) 4.8 Hz), 2.22 (3 H, s).
13C NMR (75 MHz, DMSO-d6) 157.7 (C), 150.4 (C), 134.3 (CH),
125.3 (CH), 117.8 (CH), 114.2 (C), 98.7 (C), 54.4 (CH2), 51.2
(CH2), 45.6 (CH3). LCMS m/z 247.2 (MH+), Rt 1.64 min. Anal.
calcd for C12H14N4O2: C, 58.53; H, 5.73; N, 22.75; found: C, 58.60;
H, 5.74, N, 22.62.
1
HPLC afforded 19v (7.1 mg, 21%). H NMR (300 MHz, DMSO-
d6) 11.64 (1 H, bs, NH), 11.11 (1 H, s, NH), 7.99 (1 H, d, J ) 9.0
Hz), 7.62 (1 H, d, J ) 9.6 Hz), 7.23 (1 H, d, J ) 9.6 Hz), 7.22 (1
H, s), 6.56 (1 H, dd, J ) 9.0, 1.8 Hz), 6.32 (1 H, d, J ) 8.1, 1.8
Hz), 3.79 (2 H, m), 3.50 (2 H, m), 3.24 (4 H, m), 2.79 (3 H, bs),
2.72 (3 H, s). 13C NMR (75 MHz, DMSO-d6) 162.9, 155.3, 153.5,
148.1, 147.1, 142.2, 133.1, 126.8, 125.8, 116.7, 110.8, 103.2, 100.5,
94.9, 85.7, 52.6, 47.4, 42.5, 30.1. LCMS (m/z) 404.4 (MH+), Rt
1.72 min. HRMS found m/z 404.2189; C22H26N7O requires 404.2193.
2-Amino-3-fluorobenzonitrile (17a).37 2,3-Difluorobenzonitrile
(2.0 g, 14.4 mmol, 1 equiv) was dissolved in anhydrous EtOH (25
mL) in a glass pressure vessel. The solution was saturated with
gaseous NH3, and the vessel was sealed. The reaction mixture was
stirred at 140 °C for 3 days and then cooled to room temperature.
Evaporation of the solvent under reduced pressure and purification
6-Amino-2-(4-methylpiperazinyl)benzenecarbonitrile (27a).21
1
Yield 28%. H NMR (300 MHz, DMSO-d6) 7.14 (1 H, t, J ) 8.2
Hz), 6.38 (1 H, dd, J ) 8.2, 0.9 Hz), 6.19 (1 H, dd, J ) 8.2, 0.9
Hz), 3.02 (4 H, t, J ) 4.5 Hz), 2.46 (4 H, t, J ) 4.5 Hz), 2.22 (3
H, s). 13C NMR (75 MHz, DMSO-d6) 156.1 (C), 153.2 (C), 134.0
(CH), 117.3 (C), 108.8 (CH), 105.2 (CH), 88.5 (C), 54.7 (CH2),
51.2 (CH2), 45.7 (CH3). LCMS m/z 217.4 (MH+), Rt 0.99 min. Anal.
calcd for C12H16N4: C, 66.64; H, 7.46; N, 25.90; found: C, 66.68;
H, 7.40, N, 25.90.
4-Amino-3-benzimidazol-2-yl-5-(4-methylpiperazinyl)hydro-
quinolin-2-one (19aa). Method A (15% yield). 1H NMR (300 MHz,
DMSO-d6) 11.47 (2 H, s), 9.70 (2 H, bs), 7.72 (2 H, dd, J ) 6.0,
3.3 Hz), 7.55 (1 H, t, J ) 8.0 Hz), 7.34 (2 H, dd, J ) 6.0, 3.3 Hz),

290 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Renhowe et al.
7.20 (1 H, d, J ) 8.0 Hz), 7.06 (1 H, d, J ) 8.0 Hz), 4.80 (6 H,
bs), 3.41 (8 H, m,), 2.83 (3 H, d, J ) 4.2 Hz). 13C NMR (75 MHz,
DMSO-d6) 160.7 (C), 156.1 (C), 150.4 (C), 149.0 (C), 140.5 (C),
133.8 (C), 132.5 (CH), 123.3 (CH), 113.7 (CH), 113.5 (CH), 106.5
(C), 88.9 (C), 52.2 (CH2), 50.2 (CH2), 42.2 (CH3). LCMS m/z 375.4
(MH+), Rt 1.74 min. HRMS found m/z 375.1897; C21H23N6O
requires 375.1927.
gens, optimizing hydrogen bonds, optimizing hydrogens). A Prime
optimization (Prime version 1.6) was performed on the core and
the residues in a 6 Å sphere around it, resulting in a binding pose
of the core with 3 hydrogen bonds to the hinge region (to Glu917-
CO, Cys919-NH, Cys919-CO). A Glide grid was subsequently
created using default settings (Glide version 45208) around the
optimized core, and used to dock ligands like 5, after creating their
input conformations through LigPrep (version 21207) and using
SP precision (GlideScore version SP4.5).
In Vitro Kinase Assays. FGFR-1, PDGFRꢀ, and VEGFR-2
receptor tyrosine kinases used in isolated enzyme assays were
purified from insect cell lysates that were infected with recombinant
baculovirus containing the respective human kinase domains. All
kinases contained the wild type amino acid sequence.
The IC50 values for the inhibition of RTKs were determined in
the Alpha Screen format by measuring the inhibition by compound
of phosphate transfer to a peptide substrate by the respective
enzyme. Briefly, the respective RTK cytosolic domain was pur-
chased as human recombinant protein (VEGFR-2, Invitrogen no.
PV3660, PDGFRꢀ Invitrogen no. P3082, and FGFR-1 Upstate 14-
582) were incubated with serial dilutions of compound in the
presence of peptide substrate and ATP at concentrations at or below
the Km(app) of ATP.
The kinase domain of FGFR-1 was assayed in 50 mM Hepes,
pH 7.5, 2 mM MgCl2, 5 mM MnCl2, 1 mM DTT, 0.1% BSA with
0.2 µM biotinylated peptide substrate (b-GGGGQDGKDYIVLPI-
NH2), VEGFR-2 in 50 mM Hepes, pH 7.5, 5 mM MnCl2,
0.1%BSA, 0.01%Tween-20, 1 mM DTT, and 1 µM of the same
biotinylated substrate and PDGFRꢀ in 50 mM Hepes, pH 7.5, 20
mM MgCl2, 1 mM DTT, 0.1% BSA with 0.1 µM biotinylated
peptide substrate (b-GGLFDDPSYVNVQNL-NH2) at ATP con-
centrations of 5, 1, and 10 µM, respectively, depending on the ATP
Km(app) for the respective enzyme. Reactions were incubated at room
temperature for 2.5 h and then stopped with buffer (70 mM Tris,
pH 7.5, 17.1 mM EDTA, 0.011% Tween-20 for PDGFRꢀ and
FGFR-1; 50 mM Hepes, pH 7.5, 60 mM EDTA, 0.1% BSA, 0.01%
Tween-20 for VEGFR-2). Next the stopped reactions were incu-
bated with PY20 Ab/Alpha Screen IgG beads overnight and then
read on an Alpha Screen reader. The concentration of compound
for 50% inhibition (IC50) was calculated by employing nonlinear
regression using XL-Fit data analysis software and represents the
average of at least 2 experiments. As a control compound,
staurosporine is run on every assay plate. Additionally, a Z′ > 0.5
is required to validate results for each plate.
6-Amino-2-(1-methyl(3-piperidyloxy))benzenecarbonitrile (27b).
3-Hydroxy-1-methylpiperidine (4.1 mL, 35.6 mmol) was added
dropwise to hexane-washed NaH (1.03 g, 43.0 mmol) in NMP (30
mL). After 15 min, 2-amino-6-fluorobenzonitrile (4.03 g, 29.6
mmol) was added in portions. The reaction mixture was heated to
100 °C for 2 h. The reaction mixture was poured into H2O and
extracted with EtOAc (2×). The combined organic layers were
washed with H2O (3×) and brine. The organic layer was dried with
Na2SO4, filtered, and concentrated. The crude material was purified
by column chromatography on silica gel (50% EtOAc/hex to EtOAc
to 5% MeOH/1% Et3N/94% CH2Cl2) to give 27b (3.31 g, 49%
1
yield) as a brown solid. H NMR (300 MHz, DMSO-d6) 7.13 (1
H, t, J ) 8.4 Hz), 6.31 (1 H, d, J ) 8.4 Hz), 6.24 (1 H, d, J ) 8.4
Hz), 5.93 (2 H, s), 4.36 (1 H, sept, J ) 4.5 Hz), 2.80 (1 H, m),
2.49 (1 H, m), 2.14 (3 H, s), 1.98 (3 H, m), 1.69 (1 H, m), 1.51 (1
H, m), 1.25 (1 H, m). 13C NMR (75 MHz, DMSO-d6) 159.6 (C),
153.1 (C), 134.3 (CH), 115.6 (C), 107.3 (CH), 99.8 (CH), 85.4
(C), 73.0 (CH), 59.0 (CH2), 54.7 (CH2), 45.9 (CH3), 29.0 (CH2),
22.4 (CH2). LCMS m/z 232.3 (MH+), Rt 1.47 min.
4-Amino-3-benzimidazol-2-yl-5-(1-methyl(3-piperidyloxy))hy-
1
droquinolin-2-one (19ab). Method A (8% yield). H NMR (300
MHz, DMSO-d6) 11.55 (1H, bs), 11.50 (1 H, s), 11.43 (1 H, s),
10.57 (1 H, bs), 8.90 (2 H, bs), 7.73 (2 H, p, J ) 3.3 Hz), 7.51 (1
H, dt, J ) 8.4, 1.8 Hz), 7.36 (2 H, p, J ) 3.3 Hz), 7.04 (1 H, d, J
) 8.4 Hz), 6.97 (1 H, dd, J ) 24.3, 8.4 Hz), 5.15 (1 H, bs), 4.90
(4 H, bs), 3.79 (1 H, m), 3.21 (3 H, m), 2.80 (3 H, s), 2.07 (4 H,
m). 13C NMR of HCl salt (75 MHz, DMSO-d6) 160.8 (C), 156.1/
156.0 (C), 155.7/155.5 (C), 148.5/148.4 (C), 141.0/140.8 (C), 133.8/
133.1 (CH), 123.5 (CH), 113.9 (CH), 109.9/109.5 (CH), 106.2/
105.9 (CH), 103.9/103.3 (C), 88.5/87.6 (C), 71.7 (CH), 54.1/52.6
(CH2), 43.1/42.6 (CH3), 27.3/25.4 (CH2), 20.3/18.2 (CH2). LCMS
m/z 390.4 (MH+), Rt 1.98 min. HRMS found m/z 390.1900;
C22H24N5O2 requires 390.1924. Anal. calcd for C22H23N5O2: C,
67.85; H, 5.95; N, 17.98; found: C, 67.48; H, 6.01, N, 17.78.
5-(4-Methylpiperazinyl)-2-nitrobenzenecarbonitrile.22 Yield
1
95%. H NMR (300 MHz, DMSO-d6) 8.15 (1 H, d, J ) 8.2 Hz),
7.52 (1 H, d, J ) 2.1 Hz), 7.24 (1 H, dd, J ) 8.2, 2.1 Hz), 3.53 (4
H, m), 2.41 (4 H, m), 2.21 (3 H, s). 13C NMR (75 MHz, DMSO-
d6) 153.4 (C), 135.5 (C), 127.9 (CH), 118.9 (CH), 116.2 (C), 115.5
(CH), 109.1 (C), 54.0 (CH2), 46.2 (CH2), 45.5 (CH3). LCMS m/z
247.3 (MH+), Rt 1.62 min.
Cell Proliferation Assays. HMVECs (human microvascular
endothelial cells) were purchased from Clonetics (San Diego, CA).
Cells were grown as monolayers in media (EBM-2MV from
Clonetics) and maintained at 37 °C in a humidified atmosphere
with 5% CO2. To perform the VEGF-mediated proliferation assay,
cells were seeded in 96-well plates at 2000 cells per well in assay
medium (EBM-2 with 5% FBS). The following day, serial dilutions
of compound at 0.2% final DMSO concentration in assay medium
with 2.5 ng/mL of VEGF were added to the cells. Control wells
contained cells in assay medium without the VEGF. After 72 h,
the number of viable cells left was determined using the CellTiter-
Glo assay (Promega, Madison, WI) as described by the manufac-
turer. Plates were read on a luminometer. The IC50 values were
calculated using nonlinear regression using XL Fit data analysis
software and represent the average of two experiments.
2-Amino-5-(4-methylpiperazinyl)benzenecarbonitrile (27c).22
1
Yield 91%. H NMR (300 MHz, DMSO-d6) 7.07 (1 H, dd, J )
9.0, 2.7 Hz), 6.84 (1 H, d, J ) 2.7 Hz), 6.75 (1 H, d, J ) 9.0 Hz),
2.92 (4 H, t, J ) 4.8 Hz), 2.40 (4 H, t, J ) 4.8 Hz), 2.18 (3 H, s).
13C NMR (75 MHz, DMSO-d6) 145.7 (C), 142.1 (C), 125.1 (CH),
118.5 (C), 117.7 (CH), 116.7 (CH), 93.7 (C), 54.7 (CH2), 49.5
(CH2), 45.7 (CH3). LCMS m/z 217.4 (MH+), Rt 0.70 min.
4-Amino-3-benzimidazol-2-yl-6-(4-methylpiperazinyl)hydro-
quinolin-2-one (19ac). Method A (1% Yield). LCMS m/z 375.4
(MH+), Rt 1.87 min. HRMS found m/z 375.1909; C21H23N6O
requires 375.1927.
Molecular Modeling. The VEGFR-2 homology model was built
using Chemical Computing Group’s MOE software. Default settings
were used in the alignment and homology modeling module and
the FGFR-1 crystal structure 2FGI from the Brookhaven Protein
Data Bank was used as a template. The model was brought into
Maestro (version 8.0.308, Schro¨dinger LLC) and aligned with the
CHK-1 crystal structure of the 6-Cl analogue of 16c (PDB code
2GDO).18 The 4-amino-3-benzimidazol-2-ylhydroquinolin-2-
one core was extracted from the CHK-1 structure and placed into
the aligned active site of the VEGFR-2 homology model. The
complex was prepared for Prime optimization using the Protein
Preparation Wizard in Maestro (fixing bond orders, adding hydro-
Cell Lines. Human colon tumor cell lines (KM12L4A), prostate
(DU145), and acute myelogenous leukemia (AML) FLT-3 ITD cell
line (MV4;11) were obtained from American Tissue Culture
Collection (Rockville, MD). KM12L4A, or DU145, cells were
cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD)
supplemented with 10% fetal bovine serum (FBS) and 1% L-
glutamine. MV4;11 cells were grown in Iscoves modified Dulbecco
medium (IMBM) supplemented with 10% fetal bovine serum (FBS,
Gibco Life Technologies, Gaithersburg, MD) containing 4 mM
L-glutamine, 5 ng/mL granulocyte-macrophage colony stimulating
factor (GM-CSF, R&D Systems, Minneapolis, MN), and 1%
penicillin and streptomycin. Cells were grown as adherent or

4-Amino-3-benzimidazol-2-ylhydroquinolin-2-ones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 291
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suspension cultures and maintained in a humidified atmosphere at
37 °C and 5% CO2. Cells were used in exponential growth phase,
with viability >98% (assessed using trypan blue staining) and
determined free of mycoplasma.
In Vivo bFGF-Mediated Matrigel Angiogenesis Assays. A
mixture of 0.5 mL of Matrigel (Becton Dickinson, Bedford, MA)
with 2 µg of bFGF (Chiron Corporation; Emeryville, CA) was
implanted subcutaneously into female BDF1 mice (Charles River,
Wilmington, MA). Vehicle or doses of 5 (HCl salt, formulated in
5 mM citrate) ranging from 0 to 200 mg/kg/d was given daily via
oral gavage for 8 days. On day 8, 4 h post dose, Matrigel plugs
were removed and homogenized in 0.5 mL deionized water using
a Polytron homogenizer (Kinematica Littau, Switzerland). Hemo-
globin content in Matrigel plugs was quantified by a colorimetric
assay using Drabkin’s procedure (Hemoglobin Test Kit, Sigma
Diagnostics, St. Louis, MO).
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In Vivo Tumor Efficacy Studies. Nude (nu/nu) or SCID-NOD
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flank of female nude mice. DU145 tumor pieces (2 mm × 2 mm)
from a passaged DU145 tumor (500 mm3) were implanted
subcutaneously using a trocar into male nude mice. Tumors were
allowed to grow to a size of approximately 200-500 mm3 prior to
initiation of drug treatment. Mice were randomly assigned into
cohorts (typically 10 mice/group) and treated with either vehicle
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1
(mm3) using the formula:
/
2
(length (mm) × [width (mm)]2).
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Acknowledgment. We thank Lara Nordhal and Yan Tang
for their excellent technical expertise with the in vivo studies,
and Weiping Jia, Keshi Wang, and Dazhi Tang for their
analytical support.
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Supporting Information Available: Elemental analysis data or
high resolution mass spectrometry data for key compounds, MS,
HPLC traces, and 1H NMR for 5. This material is available free of
charge via the Internet at http://pubs.acs.org.
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JM800790T