Aminopyridinecarboxamide-based inhaled IKK-2 inhibitors for asthma and COPD: Structure-activity relationship

Article abstract of DOI:10.1016/j.bmc.2010.12.027

Installation of sites for metabolism in the lead compound PHA-767408 was the key focus of the IKK-2 inhaled program. This paper reports our efforts to identify a novel series of aminopyridinecarboxamide-based IKK-2 inhibitors, which display low nanomolar potency against IKK-2 with long duration of action (DOA), and metabolically labile to phase I and/or phase II metabolizing enzymes with potential capability for multiple routes of clearance. Several compounds have demonstrated their potential usefulness in the treatment of asthma and chronic obstructive pulmonary disease (COPD).

Full text of DOI:10.1016/j.bmc.2010.12.027

Bioorganic & Medicinal Chemistry 19 (2011) 1242–1255
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Aminopyridinecarboxamide-based inhaled IKK-2 inhibitors for asthma
and COPD: Structure–activity relationship
Jin Xie a, , Gennadiy I. Poda a, Yiding Hu c, Natalie X. Chen a, Richard F. Heier a, Serge G. Wolfson a,
⇑
Matthew T. Reding a, Patrick J. Lennon a, Ravi G. Kurumbail a, Shaun R. Selness a, Xiong Li b,
Nandini N. Kishore b, Cynthia D. Sommers b, Lori Christine b, Sheri L. Bonar b, Neetu Venkatraman b,
Sumathy Mathialagan b, Sarah J. Brustkern b, Horng-Chih Huang a,
⇑
a Department of Medicinal Chemistry, Pfizer Inc., 700 Chesterfield Parkway West, St. Louis, MO 63017, United States
b Department of Biology, Pfizer Inc., 700 Chesterfield Parkway West, St. Louis, MO 63017, United States
c Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., 700 Chesterfield Parkway West, St. Louis, MO 63017, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Installation of sites for metabolism in the lead compound PHA-767408 was the key focus of the IKK-2
inhaled program. This paper reports our efforts to identify a novel series of aminopyridinecarboxa-
mide-based IKK-2 inhibitors, which display low nanomolar potency against IKK-2 with long duration
of action (DOA), and metabolically labile to phase I and/or phase II metabolizing enzymes with potential
capability for multiple routes of clearance. Several compounds have demonstrated their potential useful-
ness in the treatment of asthma and chronic obstructive pulmonary disease (COPD).
Ó 2010 Elsevier Ltd. All rights reserved.
Received 13 October 2010
Revised 7 December 2010
Accepted 13 December 2010
Available online 23 December 2010
Keywords:
Potent
Selective
IKK-2
Inhibitors
Inhaled approach
Asthma
COPD
DOA
1. Introduction
core components of I
(IKK , also known as IKK-1), I
IKK-2) and NF- b essential modulator (NEMO). IKK-2, the pivotal
enzyme in the classical pathway of NF- b activation, which is re-
quired for the expression of a range of inflammatory mediators
associated with chronic lung inflammation characteristic of asth-
ma and COPD, is therefore, an attractive target for pharmaceutical
industry.4 IKK-2 inhibitors have the potential to deliver a profound
anti-inflammatory impact in a range of diseases.
j
b kinase (IKK) complex include I
jb kinase
a
a
jb kinase b (IKKb, also known as
Chronic obstructive pulmonary disease (COPD) is the 4th cause
of death in the U.S. according to the ‘2007 NHLBI Morbidity and
Mortality Chart Book’. An estimated 24 million US adults have
COPD and 23 million have asthma. Protein kinases are believed
to play a crucial role in the expression and activation of inflamma-
tory mediators in the airways of patients with asthma and COPD.1
Nuclear factor kappa b (NF-
vator that is activated in airway epithelium in asthma/COPD pa-
tients.2 Normally, NF-
b is sequestered in the cytoplasm in an
inactive form by the inhibitory I b proteins. However, activation
by oxidative stress (e.g., cigarette smoking) leads to activation of
signaling cascades on the I b kinase (IKK) complex, induces pul-
monary inflammation, which results in a progressive decline in
lung function.3 Inhibition of NF-
b activity might, therefore, be
j
j
jb) is an inducible transcriptional acti-
Because of the key role played by the inhibition of IKK-2 in the
activation of NF-jb, coupled with the druggability of protein ki-
j
j
nases as a target class, a number of pharmaceutical companies
have been pursuing drug discovery programs aimed at identifying
IKK-2 inhibitors. These include thiophenecarboxamides,5
indolecarboxamides,6 substituted pyrazole ureas,7 benzamides,8
2,4-diarylpyridines,9 aminopyrimidines,10 b-carbolines,11 and
pyrrolopyridines12 and imidazo(1,2-a)thieno(3,2-e)pyrazines13
derivatives. Bonafoux and Huang recently reported that the 8-(5-
chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluoroph
j
j
an effective alternative approach to treat asthma and COPD. The
⇑
Corresponding authors. Tel.: +1 636 247 6251; fax: +1 636 247 5204 (J.X.).
E-mail addresses: jin.xie@pfizer.com (J. Xie), hhhuan01@hotmail.com
enyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide
(PHA-
767408 or PHA-408), a selective, ATP-competitive inhibitor, which
(H.-C. Huang).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.027

J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
1243
binds IKK-2 tightly with a relatively slow off rate was efficacious in
a chronic model of arthritis with no adverse effects at maximally
efficacious doses.14
DOA and favorable pharmacokinetic properties required for an in-
haled drug, while maintaining the core structure and key binding
elements of PHA-408 for excellent selectivity. We have explored
modifications on the Southern domain independently by incorpo-
rating functional groups to the terminal piperazinyl to enhance
metabolic liability mediated by the phase I metabolizing enzymes,
mainly cytochromes P450 in the liver. We have also optimized the
Northern domain, with an aim of incorporation of metabolic ‘han-
dles’ for rapid clearance, in particular, phenols for phase II conjuga-
tion. Combination of the phase I and phase II metabolic pathways,
that is, multiple clearance mechanisms, are expected to reduce the
risk of potential drug–drug interactions observed with a single
clearance route (Fig. 2). A variety of combinations in the Northern
domain and the Southern domain led us to investigate several
chemical series.
Figure 1 lists three scaffolds that were the focus of drug discov-
ery efforts over the past several years in our labs. While the fully
elaborated PHA-408 tricyclic pyrazole series show good selectivity
for IKK-2 over other kinases, the smaller ureido-carboxamide scaf-
folds (PF-267 and PHA-495) are not as selective.5d,7 The amide
functionality, chloropyridyl group and the terminal piperazinyl
group all contribute significantly to the overall potency of the
PHA-408 series.14 The core skeleton of PHA-408 exhibits low nM
potency against IKK-2, excellent selectivity against a large panel
of kinases, and encouragingly demonstrates a long duration of ac-
tion (DOA).14c Many compounds in this series, however, have
shown unfavorable pharmacokinetic characteristics, such as poor
oral absorption, high clearance and low systemic exposure in pre-
clinical species, and moderate margin over hERG inhibition.
Even though the optimization of this scaffold for an oral drug
represents a lot of hurdles, including high protein binding and hERG
activity for PHA-408, many of the properties of this scaffold lend
themselves suitable for an inhaled drug to treat inflammatory lung
disease, such as COPD and asthma. The IKK-2 inhibitor, as an anti-
inflammatory agent, will be directly delivered to lung via inhala-
tion, and take action on the pulmonary target. On the other hand,
once the drug passes through lung into systemic circulation, it will
be quickly cleared by hepatic metabolism to minimize the systemic
exposure and subsequent cardiovascular event. The high clearance,
mediated by hepatic metabolizing enzymes, is favorable in inhaled
drug design. Thus, the metabolically unstable compounds are cho-
sen from in vitro assay, for example, human liver microsomal (HLM)
stability assay, as a drug candidate in lead selection and advance-
ment. Ideal inhalation agents possess optimized pharmacodynamic
properties, including target selectivity, potency and duration of
action, and optimized pharmacokinetic properties, including
high pulmonary deposition, prolonged pulmonary residence time,
high systemic clearance and lower oral bioavailability to achieve
high efficacy and minimized adverse effects related to the systemic
exposure.15 Thus we have chosen PHA-408 as a starting point for
pulmonary drug delivery to achieve low nano-molecular potency,
pharmacokinetic properties and safety.
2.1. Southern domain modification: divergent synthetic efforts
Most of the N-substituted pyrazolyl-aminopyridine-3-carbox-
amides 1 were obtained, with yields ranging from 40% to 85%, by
reacting the previously reported PHA-801 with commercially
available pyrazole aldehydes through reductive amination using
sodium triacetoxyborohydride or resin-bound cyanoborohydride
(Scheme 1).
Compound 2a was synthesized by reductive amination of PHA-
801 with ethyl 2-(4-formyl-1H-pyrazol-1-yl) acetate using sodium
triacetoxyborohydride. Subsequent amidation of 2a with various
amines in methanol (Scheme 2) led predominantly to the desired
product 3. It is worth noting that it required long reaction times
and higher temperature for secondary amines and also resulted
in lower yields of products leading to troublesome purifications
and modest yields.
Compound
4 was synthesized by reductive amination of
PHA-801 with 2-(4-formyl-1H-pyrazol-1-yl) acetic acid using so-
dium triacetoxyborohydride. Subsequent esterification of 4 with
various alcohols in the presence of thionyl chloride (Scheme 3)
gave the desired products 5 in yields ranging from 60% to 80%.
Compound
6 was synthesized by reductive amination of
PHA-801 with 1H-pyrazole-4-carbaldehyde using sodium triacet-
oxyborohydride. Subsequent alkylation of 6 with various alkyl bro-
mides in the presence of sodium hydride in DMF gave the desired
products 7 in yields ranging from 60% to 80% (Scheme 4).
This paper reports our efforts to identify drug candidates that
are potent against IKK-2 enzyme, metabolically labile in human li-
ver microsomes (HLM stability <30%14c), no/reduced hERG activity,
long duration of action, and demonstrating the capacity for multi-
ple routes of clearance to minimize systemic exposure and manage
potential drug–drug interaction (DDI) risks.
2.2. Northern domain modification
Synthetic Scheme 5 illustrates a six step procedure used for the
preparation of analogs 13, 14, 15, 16, 17, 18, 19 and 20. Nitrotetr-
alone was converted into the diketo ester enolate 8 following the
literature.14d The diketo ester enolate 8 was refluxed with corre-
sponding R1- and/or R2- substituted phenylhydrazine in acetic
acid to give pyrazole intermediate 9. For 16, 17, 18, 19, substituted
2. Chemistry
We launched a medicinal chemistry program to explore the SAR
around the PHA-408 lead to improve potency, cellular activity,
F
H
HN
N
N
N
H
O
N
N
O
N
CONH2
S
CONH2
CONH2
Cl
H2N
NH
H2N
NH
N
N
O
O
N
PF-267
PHA-495
PHA-408
Figure 1. IKK-2 inhibitors series developed by Pfizer.

1244
J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
zinc dust in ethanol to give corresponding intermediates 10. In
R1
N
the following step, the conversion of ester to amide was achieved
by reacting with liquid ammonia in a pressured tube at high tem-
perature to give amides 11. The resulting amides 11 can react with
2,5-dichloroisonicotinic acid in pyridine to give the desired inter-
mediates 12. Direct amination of intermediates 12 with N-methyl-
piperazine afforded the desired products 13, 14, 15 and 20 with
satisfactory yields. For 16, 17, 18, 19, demethylation of intermedi-
ates 12 was carried out first in the presence of boron tribromide,
then the crude intermediates were mixed with N-methylpipera-
zine and refluxed overnight to give the corresponding final prod-
ucts 16, 17, 18, 19 with moderate yields.
1-(3-Chloro-4-methoxyphenyl)hydrazine 21 (Scheme 6) was
synthesized by treatment of 3-chloro-4-methoxyaniline in concen-
trated hydrochloric acid at 0 °C with a solution of sodium nitrite in
water, then reduced by stannous chloride to give final product 21
(31% yield).
northern domain
N
H
O
N
CONH2
Cl
N
N
southern domain
R3
N
R2
Figure 2. Strategy for exploring the SAR around PHA-408.
methoxyphenyl hydrazines were used to give the corresponding
pyrazoles 9. Then the nitro group was reduced to amine by using
H
O
F
F
N
N
R
N
N
N
N
H
H
O
N
O
N
CONH2
CONH2
Cl
Cl
N
N
N
N
N
NR
NH
N
PHA-801
1
Scheme 1. Reagents and conditions: pyrazole aldehyde (1.5 equiv), sodium triacetoxyborohydride (2.5 equiv), DMSO, rt.
F
F
N
N
N
N
H
H
O
N
CONH2
O
N
R1R2NH
CONH2
Cl
Cl
N
N
N
N
R2
R1
O
N
N
N
N
N
N
N
O
O
2a
3
Scheme 2. Reagents: R1R2NH (10 equiv), CH3OH.
F
F
N
N
N
N
H
H
O
N
CONH2
O
N
R1OH, SOCl2
CONH2
Cl
Cl
N
N
N
N
OH
N
N
N
N
O
N
R1
N
O
O
4
5
Scheme 3. Reagents and conditions: (a) ROH (excess), SOCl2, 80 °C.

J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
1245
F
F
N
N
N
N
H
H
O
N
CONH2
O
N
R1CH2Br, NaH
CONH2
Cl
Cl
N
NH
N
N
N
N
N
N
N
R1
N
6
7
Scheme 4. Reagents and conditions: (a) R1CH2Br (1.2 equiv), NaH (1.5 equiv), rt.
R2
R1
O
OLi
O
NO2
EtO
NO2
b
a
N
N
OEt
O
O2N
O
8
9
R2
R1
R2
R1
d
e
c
N
N
OEt
H2N
N
N
NH2
H2N
O
O
10
11
R2
R1
R2
R1
N
N
H
N
N
O
N
CONH2
H
f
O
N
CONH2
Cl
Cl
N
N
Cl
N
N
13) R1=H; R2=F;
14) R1=F; R2=F;
12
15) R1=CH3; R2=H;
16) R1=OH; R2=H;
17) R1=OH; R2=Cl;
18) R1=Cl; R2=OH
19) R1=H; R2=OH
20) R1=OCH3; R2=H;
Scheme 5. Reagents and conditions: (a) ethyl oxalate (1.2 equiv), lithium bis(trimethylsilyl) amide in ether, rt; (b) substituted phenylhydrazine hydrochloride (1.2 equiv),
acetic acid, 80 °C, 16 h; (c) zinc dust (excess), acetic acid, ethanol; (d) ethanolic ammonia (1:1); (e) 2,5-dichloroisonicotinic acid (1.2 equiv), HBTU, diisopropylethyl amine,
DMF; (f) boron tribromide (10 equiv) 16 h; then N-methylpiperazine, reflux.
3. Biology
Cl
NH2
Cl
NHNH2
The synthesized inhibitors were initially evaluated for inhibition
of recombinant human IKK-2.16,17 Compounds with an IC50 on IKK-
2 less than 5 nM were subsequently tested in the human peripheral
blood mononuclear cells (PBMC) screen.14c The PBMC assay was our
primary cell-based assay, used to measure inhibitory effects of IKK-
2 inhibitors on the functional response of lipopolysaccharide (LPS)
O
O
21
Scheme 6. Reagents: (a) sodium nitrite, then stannous chloride in concentrated
HCl.

1246
J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
stimulated TNF-a release from PBMC. The results of this assay were
5. Results and discussion
used to determine if a compound has reasonable cell permeation in
vitro, therefore, advanced further for in vitro and in vivo testing.
Compounds with an IC50 on human PBMC less than 50 nM subse-
quently progressed for the DOA studies in enzymatic and cell-based
assays to determine the dissociation rate constants for human IKK-
2 and human PBMC cells.14c
Figure 3a and b below show PHA-408 docked into the IKK-2
homology model following the IFD procedure described above.
When docking new synthetic targets and rationalizing SAR some li-
gands were observed to flip 180° allowing the carboxamide portion
of PHA-408 to bind to both the backbone NH and carbonyl of Cys99
or the backbone NH of Cys99 and backbone carbonyl of Gln97. The
identified docked orientation consistent with major SAR but does
not explain all modifications, and we do not have any direct exper-
imental evidence regarding the possible binding mode. Based on
the docking experiments, we believe that the carboxamide moiety
of the inhibitor forms interactions with the hinge region of the IKK-
2 ATP binding site (Fig. 3a and b). 4-Fluorophenyl binds deep in the
pocket towards the methionine gate-keeper Met96. The molecule
occupies the adenine, sugar and phosphate sub-sites of ATP with
the piperazine moiety nearby the conserved catalytic Asp166, the
4. Molecular modeling
IKK-2 is a large protein kinase molecule (756 amino acids) with
several structural modules including an N-terminal protein kinase
domain, a leucine-zipper motif and a coiled–coiled domain.18 To
our knowledge, there is no known crystal structure of IKK-2 re-
ported in the literature. However, we have generated homology
models of IKK-2 based on crystal structures of cAMP-dependent
protein kinase (PKA)19 and CHK-1 kinase.20 The homology model
of IKK-2 was built using a PKA in house crystal structure as a tem-
plate in the complex with a fused pyrrolopyrimidine analog that
was not structurally similar to PHA-408. However, the kinase do-
main of this high resolution protein structure was fully resolved
(without any unresolved residues neither in the Gly-rich loop nor
in the activation loop) and the Phe residue (Phe54 in PKA) at the
very tip of the Gly-rich loop was pointing towards the solvent
allowing more room for the initial placement of the ligands for
the IFD docking. The O14920 sequence of the human IKK-2 was re-
trieved from SwissProt and used to build the homology model. We
performed a BLAST search using the first 300 amino acids that cor-
respond to the kinase domain of IKK-2. Three proteins were iden-
tified with highest sequence identity: death-associated protein
kinase 1 (DAPK1, 32% sequence identity), p21-activated kinase 1
(PAK1, 30% sequence identity) and PKA (30% sequence identity).
All three kinases (DAPK1, PAK1 and PKA) have 7 amino acids in
the hinge region as IKK-2 does. This fact does not speak in favor
of any of these templates. PKA has an advantage over DAPK1 since
it has the same gate-keeper residue (Met, M96 in IKK-2) as IKK-2
does while DAPK1 has Leu (L93). While PAK1 also has Met
(M344) as gate-keeper, it has Arg residue (R299) instead of the sol-
vent-exposed Lys across the hinge (K106 in IKK-2). As a result of
the homology modeling procedure, the Cartesian coordinates for
the structurally-conserved regions have been assigned from PKA
and the loops that had insertions in the sequence alignment were
built, sampled and energy minimized afterwards while keeping the
rest of the protein fixed and the tethering positional constraints
were gradually released. The ATP binding site of IKK-2 was pre-
shaped by placing PHA-408 optimized at Becke3LYP/6-31G⁄ level
with Jaguar (version 6.5, 2005, Schrödinger, LLC, New York, NY)
from its putative IKK-2-bound conformation, and the whole pro-
tein-ligand complex was refined via the multi-step energy minimi-
zation procedure with first the ligand and the protein backbone
fixed, followed by gradually released tethering positional con-
strains on the protein backbone and the ligand (ff91, 4r dielectric
constant, RMS gradients <0.1 kcal/mol A2). The conformation of
the A-loop in the IKK-2 homology model is the weakest part. How-
ever, with the exception of Tyr169, the residue followed the con-
served DFG motif (DLG in IKK-2) in protein kinases, no other
residues were in close contact with PHA-408.
aC-helix and the activation loop. Substituents of the piperazine
ring would be binding right outside of the ATP site phosphate re-
gion towards the activation loop and would be either partially sol-
vent-exposed or engaging the A-loop via weak solvent-exposed
interactions. With PHA-408 demonstrating some DOA and based
on its binding mode we thought that placing groups of the pipera-
zine ring towards the A-loop,
aC-helix or solvent front would
either preserve or improve this effect in most cases. This strategy
has been proven successful.
As previously disclosed,14 PHA-408 was potent in IKK-2 enzyme
and cellular assays and further demonstrated promising duration
of action. As a pulmonary delivered drug candidate for treatment
of COPD, its in vitro pharmacologic profile requires good DOA in
enzyme and cell-based assays, high clearance in liver microsomal
stability assays, rapid in vivo clearance and poor systemic expo-
sure. Multiple routes of elimination are preferred to mitigate po-
tential clinical DDI. To identify new candidates with better DOA
in enzyme and cell-based assays, a small SAR set of analogs was de-
signed and synthesized to explore the Northern portion of the mol-
ecule. As shown in Table 1, most modifications to the phenyl group
resulted in potent IKK-2 inhibitors in both enzyme and cellular as-
says. In addition, UDP-glucuronosyltransferases (UGT) catalyzed
phase II metabolism was involved in the clearance of this scaffold
of compounds when a hydroxy group was introduced. Particularly,
acidic chlorophenol demonstrated rapid glucuronidation clearance
in vitro, when substitution groups are properly arranged, such as
analog 17 (data not shown). However, the loss of DOA in the
We used the Induced Fit Docking (IFD) procedure from Schö-
dinger21 to dock PHA-408 and its analogs. First, the protein and li-
gand van der Waals radii have been scaled down to 0.5 and 20
docking poses were generated using standard precision (SP) Glide
scoring. Second, a 6 Å shell of residues around the Ligand was min-
imized with Prime. Finally, the ligands were docked with extra pre-
cision (XP) scoring followed up with post-docking minimization
for each of 20 poses.
Figure 3a. Top view of PHA-408 docked into IKK-2 homology model following the
Induced Fit Docking procedure from Schödinger, Inc. The Gly-rich loop is hidden for
clarity and the ATP IKK-2 binding site depicted by the Connolly surface color-coded
by the calculated Electrostatic Potential (red color for negative electrostatic
potential and blue color for positive electrostatic potential).

J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
1247
In Table 3 were shown pairs of compounds with variations in
both the pyrazole and the phenyl rings to demonstrate translat-
ability of the DOA effect from simultaneous variations. Similar to
the simple methyl piperazines shown in Table 1 (PHA-408 and
13), 3- and 4-fluoro analogs (22b and 22a) were equipotent in en-
zyme and cells with similar enzyme DOA, albeit slightly shorter t1/2
for the 3-flurophenyl analog. Replacing the methyl with an ethoxy-
acetyl group led to a pair of compounds with great potency and
DOA in both enzyme and cells. These compounds showed short
half life in HLM, were highly metabolized by microsomal enzymes,
including cytochromes P450s and possibly esterases. However,
binding in the competition dofetilide (DOF) assay (a radiolabeled
ligand to the hERG or Kv11.1 potassium ion channel) was also high,
indicating the increased risk of potential adverse cardiovascular
side effects.
Before expanding the SAR around the pyrazolyl nitrogen, we
also took time to explore the effect of substitutions on the pipera-
zine and pyrazole rings. As shown in Table 4, methyl substitution
on either rings in the piperazinyl pyrazole, (41, 42, and 43) resulted
in analogs of similar enzyme potency as the parent lead compound
22a. Even though the enzyme DOA was all >4 h, translation from
the enzyme into cell potency varies. Unsubstituted pyrazoles
(22a and 43) showed only 2–3-folds right shift, while 41 and 42
(enzyme DOA = 8 h) showed much bigger cell-to-enzyme ratio
(20–30-folds). For the two carboxylic acid analogs off the pipera-
zine ring (44 and 45), the analog with distal carboxyl group from
pyrazole was more potent in enzymatic assay and had better en-
zyme DOA. However, the enzyme-to-cell translation was under-
standably much worse for both analogs with >100-fold right shift
due to their poor permeability. At the moment, we do not have
reasonable explanations for the observed right shift in the en-
zyme-to-cell activity translation. Most potent inhibitors have poor
permeability, which might lead to great right shift from enzyme to
cell potency.
Figure 3b. Side-by-side stereo view of PHA-408 docked in IKK-2. H-bonds between
the protein and the ligand are shown by bold dotted lines. Only key residues in
proximity to the ligand are shown for clarity.
enzymatic assay was observed with the chlorophenolic analogs, 17
and 18. Based on the proposed binding mode in the IKK-2 homol-
ogy model, bulky substitution on the phenyl ring could lead to
unfavorable clashes with the protein and the Southern moiety,
which could adopt an alternative conformation with the piperazine
ring turning toward the kinase solvent front, resulting in the loss of
DOA. With that hypothesis in mind, we then redirected our atten-
tion to modifications of the Southern region.
The second set of analogs was designed to probe SAR around
piperazine and their effect on DOA, while maintaining or enhanc-
ing the metabolic liability. A few of those compounds were exem-
plified in Table 2. Replacement of the methylpiperazine ring in
PHA-408 with a hydroxypiperidine or a linear methoxyethylamine,
resulted in analogs (PF-296 and PF-544, respectively) with great
potency in enzyme and cellular assays, but again with no DOA in
the enzymatic assay. A methoxyethyl group on the piperazine
yielded PHA-919, a very potent IKK-2 inhibitor in the cells, how-
ever, it also lost the DOA. Pyrazoyl methyl substitution on the
piperazine has led to a potent compound 22a and with good
DOA. A focused library was thus constructed to fine-tune the SAR
around the pyrazolyl moiety.
Summarizing the results from these brief SAR studies, we con-
cluded that the quickest route to minimize the DOF inhibitory ef-
fect and optimize DOA and cell potency was to focus on
derivatization of N-substitutions on the pyrazole ring.
In Table 5, comparing 22a with the unsubstituted analog 6, the
methyl group does not seem to affect the in vitro properties, such
Table 1
Effect of substituent on the Northern phenyl group on inhibition of IKK-2*
Y
N
N
H
O
N
CONH2
Cl
N
N
N
CPD
Y=
IKK-2 IC50 (nM)
PBMC IC50 (nM)
Enzyme t1/2 (h)
PHA-408
13
14
15
16
17
18
19
20
4-Fluoro
3-Fluoro
3,4-Difluro
4-Methyl
1.6
4.8
10
39
14
65
38
75
46
3
2
14
0
1
1
1
1
0
4-Hydroxy
3.5
2.8
2.2
1.2
6.1
3-Chloro-4-hydroxy
3-Hydroxy-4-chloro
3-Hydroxy
89
33
4-Methoxy
*
Inhibition of kinase activity was assessed as described under Materials and Methods. IC50 data are expressed as a mean of three or more experiments and standard deviations
were within 50% of the reported value. Dissociation kinetics t1/2 from rhIKK-2 and PBMC cell activity were determined as described under Materials and Methods.

1248
J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
Table 2
Effect of the Southern region substitutions on duration of action in enzymatic assay*
F
F
N
N
F
H
N
O
CONH2
N
N
O
N
N
H
H
Cl
O
N
CONH2
O
N
CONH2
Cl
N
N
Cl
N
X
N
N
N
N
H
X = CH2CH2OCH3, PHA-919
OH
PF-9296
PF-544
X = 22a
,
N
N
CPD
hIKK-2 IC50 (nM)
PBMC IC50 (nM)
Enzyme t1/2 (h)
PHA-408
PF-296
PF-544
PHA-919
22a
1.6
0.6
0.6
1.9
3
39
15
134
8
2.8
0.1
0.1
0.6
4
11
*
Inhibition of kinase activity was assessed as described under Materials and Methods. IC50 data are expressed as a mean of three or more experiments and standard deviations
were within 50% of the reported value. Dissociation kinetics t1/2 from rhIKK-2 and PBMC cell activity were determined as described under Materials and Methods.
Table 3
Analogs with variation in both Northern and Southern regions*
F
N
N
H
O
N
CONH2
Cl
N
N
X = CH3: 22
X = CH2CO2Et: 2
N
4-fluorophenyl: a
3-fluorophenyl: b
N
N
X
CPD
IKK-2 IC50 (nM)
PBMC IC50 (nM)
Enzyme t1/2 (h)
Cell t1/2 (h)
HLM t1/2 (min)
DOF (% of effect)
22a
22b
2a
3
2
1
5
11
14
6
4
3
5
5
17
14
3
91
83
95
78
>4
>4
2b
9
3
*
Inhibition of kinase activity was assessed as described under Materials and Methods. IC50 data are expressed as a mean of three or more experiments and standard deviations
were within 50% of the reported value. Dissociation kinetics for both enzyme and cell t1/2, PBMC cell activity, human liver microsomal clearance, and DOF inhibition
experiments were determined as described under Materials and Methods.
as potency, DOA, HLM stability and DOF inhibitory effect. The sim-
ple ethyl analog, 23, has a better DOA, but its cellular potency has
dropped significantly. Further functionalization of the ethyl group
with cyano, ether, alcohol or amine, all leads to very potent inhib-
itors (24, 25, 26, 27 and 28) in both enzyme and cells. With the
exception of longer ether 26 which lost DOA in the cellular assay,
these compounds all maintained the DOA in both enzyme and
cells, and desirable high clearance in HLM, but also showed similar
DOF liability which was alleviated slightly with more hydrophilic
substituents such as alcohols or amines. Alkoxyacetyls (29, 2a, 30
and 31) showed very similar properties in all aspects as analogs
just discussed. The corresponding carboxylic acid 4 is very potent
in enzyme with a big right shift in the cell assay, and good DOA,
but due to its higher hydrophilicity it showed low clearance and
DOF inhibition. This trend was also observed in the amide analogs,
more hydrophilic analogs, such as primary amide 32 and second-
ary amide with a hydroxy tail 38; tend to have low clearance
and DOF inhibition and bigger right shift in the cellular assay. Sur-
prisingly they also have shorter DOA in the cellular assay. The ter-
tiary amide with bis-hydroxy tail 39 and a secondary amide with a
longer amino tail 40 both showed low DOF inhibition.
6. Conclusion
In an effort to rationalize the SAR of DOA in the PHA-408 series,
and to minimize DOF inhibition effect while maintaining desirable
high clearance, we explored two regions in the lead compound
PHA-408 which are amenable to structural modifications with

J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
1249
Table 4
7.2. IKK-2 enzyme assay (hIKK-2)
Effect of pyrazolyl substitution on duration of action in enzyme*
The detailed IKK-2 Enzyme assay was described previously.15,16
In brief, electroplated 96-well plates were coated with anti-phos-
F
F
N
pho-IjBa (Santa Cruz Biochemicals, Santa Cruz, CA) at a concentra-
N
H
N
N
N
O
tion of 5 pmol/well. All kinase assays were performed in a 30-ll
CONH2
H
O
N
CONH2
total volume for 90 min in kinase buffer containing 25 mM HEPES,
10 mM NaF, 0.1% bovine serum, albumin, 0.0005% Triton X-100,
5 mM MnCl2, 5 mM MgCl2, 1 mM dithiothreitol, and 2% Me2SO,
pH 7.5. For rhIKK-2 activity, inhibitor and rhIKK-2 (0.1 nmol/well)
Cl
Cl
N
N
N
N
N
were added simultaneously to plates with substrate (1
lM ATP,
R5
N
2
l
M biotinylated I Ser32-Ala36 peptide). The reaction was
jBa
R6
stopped by washing the plates twice with Tris-buffered saline/
Tween 20. Electrochemiluminescence was generated by adding
R3
R4
N
N
N
N
sulfo-tagged streptavidin (25
ll/well) for 1 h and following the
manufacturer’s (MesoScale Discovery) instructions. Phospho-I
jBa
43: X = CH3; R5 = H; R6 = CH3
44: X = CH3; R5 = CO2H; R6 = H
45: X = CH3; R5 = H; R6 = CO2H
22a: R3 = H; R4 = H
41: R3 = H; R4 = CH3
42: R3 = CH3; R4 = H
levels were quantified with the use of a Sector Imager 6000 (Meso-
Scale Discovery). Specific IKK kinase was determined by subtract-
ing values from a non-peptide control.
CPD
IKK-2 IC50 (nM)
PBMC IC50 (nM)
Enzyme t1/2 (h)
22a
41
42
43
44
45
3.2
4.3
2.8
3.5
0.5
3.9
11
118
67
9
529
432
4
8
8
6
4.2
0.3
7.3. IKK-2 PBMC cellular assay
Primary cell culture has been described previously.15 In brief;
human whole blood was collected from healthy donors in so-
dium-heparinized tubes (BD, Franklin Lakes, NJ). Neutrophils or
PBMC were isolated by Ficoll separation. Cells were resuspended
in RPMI 1640 medium with penicillin–streptomycin (10 U/ml)
and 5% FBS and plated into 96-well culture plates at
2.5 Â 105 cells/well. Compounds were added to the cells and incu-
bated for 1 h before a 16-h LPS (20 ng/ml) stimulation. Superna-
tants were collected and placed in new 96-well plate for analysis
*
Inhibition of kinase activity was assessed as described under Materials and
Methods. IC50 data are expressed as a mean of three or more experiments and
standard deviations were within 50% of the reported value. Dissociation kinetics t1/2
from rhIKK-2 and PBMC cell activity were determined as described under Materials
and Methods.
for TNFa release by ELISA.
little or no penalty on potency. The effort was guided by the IKK-2
homology model and docking experiments that allow partial pro-
tein flexibility at the binding site. Based on the proposed binding
mode of PHA-408 it was hypothesized that substituents of piper-
azine would either maintain or improve the duration of action in
enzymatic and cell-based assays at least for best analogs. This
hypothesis was proven to be successful. Indeed, it was found that
a small heterocycle attached to the piperazine ring can be substi-
tuted with various hydrophilic tails to reduce DOF inhibition and
still maintain cell potency and DOA. We have also demonstrated
that slow offset from the IKK-2 enzyme translates into an ex-
tended DOA in in vitro washout assays. In summary, 27, 28 and
40 have the best overall properties in potency, DOA, HLM, DOF
and no hERG activity. The three compounds were advanced for fur-
ther characterization in the expanded kinase selectivity panel,
in vivo pharmacokinetic profiling and in vivo inhaled COPD effi-
cacy models. The results from these studies will be reported in
due time.
7.4. Human IKK-2 t1/2 off-rate determination
The dissociation rate constants of compounds from human
IKK-2 were determined by following the recovery of the human
IKK-2 kinase activity under the following conditions. Compounds
were pre-incubated (25–100 nM) with 25 nM of human IKK-2 for
1 h at room temperature. The kinase reaction was started with
the addition of 100 mM ATP and 1 mM 5-fluorescein (FAM)-
GRHDSGLDSMK-NH2. The kinase activity was followed over time
using a Caliper Lab Chip 3000. All reactions were carried out in
25 mM Hepes, 5 mM MgCl2, 5 mM MnCl2, 10 mM NaF, 0.1%
BSA, and 1 mM DTT at pH 7.5. All data were best fit using Grafit
4.0.
7.5. IKK-2 PBMC cell DOA
Using frozen cell technology, the PBMCs were thawed and pla-
ted into 96-well plates. Compounds were added to the cells and
incubated for 1 h. Then after 1 h the compounds were washed
off four times, by spinning the 96-well plate and removing all
the supernatant. The control plate was not washed. Then the
plates were either stimulated for 4 h with LPS, or some time
was allowed to pass for DOA purposes (0.5 h or 24 h) before stim-
7. Experimental section
7.1. General methods and materials
Column chromatography was performed using 200–400 mesh
silica gels. 1H NMR and 13C NMR were obtained on Varian Inova-
300 and Varian Inova-400 spectrometers using DMSO-d6 as the
internal standard. For some compounds, peaks near the water sig-
nal are missing, and exchangeable protons may also be missing due
to water in the sample. Mass spectra (MS) were recorded with a
Micromass ZMD spectrometer. High resolution mass spectra were
ulation. The supernatant was then assayed for TNF
MSD technology.
a release via
7.6. Human liver microsomal stability
Analogs (1 lM) were incubated in human liver microsomes
(0.8 mg/ml) with the addition of a NADPH-regenerating system
(1 mM NADP+, 5 mM isocitric acid, 1 unit/ml isocitric acid dehy-
drogenase) in 100 mM potassium phosphate buffer (pH 7.4) for
30 min. Acetonitrile with internal standard was added to stop
recorded using
a Perceptive Biosystems Mariner TOF mass
spectrometer. Elemental analysis was performed by Atlantic
MicroLab, GA.

1250
J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
Table 5
Effect of pyrazole substituents on in vitro properties*
F
N
N
H
O
N
CONH2
Cl
N
N
N
N
N
X
CPD
X=
hIKK2 IC50 (nM)
PBMC IC50 (nM)
Enzyme t1/2 (h)
Cell t1/2 (h)
HLM t (min)
DOF (% of effect)
½
6
22a
23
24
25
26
27
28
29
2a
30
31
4
32
33
34
35
36
37
38
39
40
H
1.9
3.2
4.2
7.6
2.8
6.6
2.2
3.7
1.1
1.3
1.3
4.7
0.5
0.9
1.4
2.0
2.3
1.3
2.5
1.0
10.2
3.3
16
11
102
9
16
21
15
12
54
6
3
4
>8
5
4
4
5
4
6
5
4
6
5
5
6
5
5
6
4
5
4
6
8
17
78
91
Methyl
Ethyl
CH2CH2CN
(CH2)3OCH3
(CH2)3OCH2CH2OCH3
(CH2)3OH
(CH2)3N(CH3)2
CH2CO2CH3
CH2CO2CH2CH3
CH2CO2CH2CH2OCH3
CH2CH2CO2CH2CH3
CH2CO2H
>4
>4
<4
>4
>4
6
3
3
9
11
98
76
69
49
42
24
95
66
90
29
38
57
94
50
58
98
12
À1
34
>4
>4
>4
4
<4
>4
3
3
3
120
58
13
9
14
221
7
15
36
13
10
10
9
CH2CONH2
CH2CONHCH3
CH2CONH(i-propyl)
CH2CON(CH3)2
CH2CONH(CH2)2OCH3
CH2CONH(CH2)2SCH3
CH2CONH(CH2)2OH
CH2CON((CH2)2OH)2
CH2CONH(CH2)3N(CH3)2
<4
>4
4
<4
>4
8
3
3
120
474
26
36
*
Inhibition of kinase activity was assessed as described under Materials and Methods. IC50 data are expressed as a mean of three or more experiments and standard deviations
were within 50% of the reported value. Dissociation kinetics t1/2 for both enzyme and cell, PBMC cell activity, human liver microsomal clearance, and DOF inhibition
experiments were determined as described under Materials and Methods.
the reaction. The reaction mixture was centrifuged, and superna-
tant was analyzed for test compound by liquid chromatography
7.9. General procedure for reductive amination using resin-
bound cyanoborohydride for compound 1
coupled to tandem mass spectrometry, and
reported.
t
(min) was
1/2
Silica-bound cyanoborohydride resin (1.5 equiv) was added to a
solution of PF-2687138 (1 equiv) and an appropriate aldehyde
(1.5 equiv) in 1 mL of DMSO/acetic acid (70/30) and agitated on
an orbit shaker for 18 h. The reaction was filtered through a
7.7. Dofetilide binding
The dofetilide competitive binding assay is performed using a
384-well fluorescence polarization method. The assay is con-
ducted using HEK-293 cell membranes transfected with the hERG
protein. The compounds of interest are allowed to compete for
binding to the potassium channel (IKr) along with Cy3B tagged
N-desmethyl dofetilide. Assay plates are read using a Tecan
Safire2.
0.45 lm PTFE membrane syringe filter and was purified using re-
verse-phase chromatography with acetonitrile/water as the mobile
phase.
7.10. General procedure for amidation for compound 3
Mixture of 0.065 mM of compound 2a and 10 equiv of primary
amines in 3 ml MeOH was stirred at rt overnight. After the solvent
was removed, the residue was re-dissolved in 1 ml DMSO and puri-
fied using reverse-phase chromatography with acetonitrile/water
as the mobile phase.
7.8. General procedure for reductive amination using sodium
triacetoxyborohydride for compound 1
Sodium triacetoxyborohydride (2.5 equiv) was added to a solu-
tion of PF-2687138 (1 equiv) and an appropriate aldehyde
(1.5 equiv) in 0.5 mL DMSO with acetic acid (2 equiv) and stirred
at room temperature for 2 h. The reaction was filtered through a
7.11. General procedure for esterification for compound 5
Mixture of 0.058 mM of compound 4 in 1 mL alcohol was added
2 equiv SOCl2, the mixture was heated to 80 °C and stirred over-
night. After the solvent was removed, the residue was re-dissolved
in 1 ml DMSO and purified using reverse-phase chromatography
with acetonitrile/water as the mobile phase.
0.45 lm PTFE membrane syringe filter and evaporated over night
using a nitrogen stream. The material was purified using reverse-
phase chromatography with acetonitrile/water as the mobile
phase.

J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
1251
7.12. General procedure for N-alkylation for compound 7
1H) 7.22 (m., 3H) 7.33–7.55 (m, 8H) 8.07–8.15 (m, 1H) 10.27
(s, 1H); LRMS (M+H) calcd for C37H39ClFN10O3 725.3, found
725.3.
To a slurry of sodium hydride (1.5 equiv) in 1 mL DMF at 0 °C
was added compound 6 (1 equiv). The mixture was stirred for
30 min; an appropriate alkyl bromide (1.2 equiv) of 0.058 mM of
compound 4 was added. The reaction mixture was warmed up to
rt, and stirred overnight. After the solvent was removed, the resi-
due was re-dissolved in 1 ml DMSO and purified using reverse-
phase chromatography with acetonitrile/water as the mobile
phase.
7.12.7. 8-({5-Chloro-2-[4-({1-[2-(methylamino)-2-oxoethyl]-
1H-pyrazol-4-yl}methyl)piperazin-1-yl]isonicotinoyl}amino)-1-
(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (33)
1H NMR (DMSO-d6, 400 MHz): d ppm 2.56–2.60 (m, 1H) 2.68 (d,
J = 4.39 Hz, 3H) 2.95–3.09 (m, 6H) 3.47 (s, 3H) 3.53–3.64 (m, 5H)
4.78 (s, 2H) 6.96 (s, 1H) 7.28–7.36 (m, 2H) 7.40–7.52 (m, 4H)
7.59 (s, 1H) 7.62 - 7.72 (m, 3H) 7.93–8.02 (m, 1H) 8.22 (s, 1H)
10.38 (s, 1H); LRMS (M+H) calcd for C35H35ClFN10O3 697.3, found
697.3.
7.12.1.8-({5-Chloro-2-[4-(1H-pyrazol-4-ylmethyl)piperazin-1-yl]
isonicotinoyl}amino)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo
[g]indazole-3-carboxamide (6)
Prepared using the general reductive amination procedure. 1H
NMR (DMSO-d6, 400 MHz): d ppm 2.12–2.57 (m, 8H) 2.70–3.07
(m, 4H) 3.34–3.62 (m, 2H) 6.82 (s, 1H) 7.10–7.68 (m, 12H) 8.10
(s, 1H) 10.25 (s, 1H); HRMS (M+H) calcd for C32H30ClFN9O2
626.2117, found 626.2037.
7.12.8. 8-{[5-Chloro-2-(4-{[1-(2-{[3-(dimethylamino)propyl]
amino}-2-oxoethyl)-1H-pyrazol-4-yl]methyl}piperazin-1-
yl)isonicotinoyl]amino}-1-(4-fluorophenyl)-4,5-dihydro-1H-
benzo[g]indazole-3-carboxamide (40)
1H NMR (DMSO-d6, 400 MHz): d ppm 1.53 (t, J = 6.95 Hz, 2H)
2.11 (s, 6H) 2.22 (t, J = 6.77 Hz, 2H) 2.38–2.48 (m, 4H) 2.94 (d,
J = 9.52 Hz, 4H) 3.10 (q, J = 6.59 Hz, 2H) 3.39 (s, 3H) 3.45–3.56
(m, 5H) 4.71 (s, 2H) 6.88 (s, 1H) 7.21–7.28 (m, 2H) 7.32–7.44 (m,
4H) 7.52 (s, 1H) 7.56–7.63 (m, 2H) 7.93–8.01 (m, 1H) 8.15 (s, 1H)
10.30 (s, 1H); LRMS (M+H) calcd for C39H44ClFN11O3 768.3, found
768.3.
7.12.2. 8-[(5-Chloro-2-{4-[(1-methyl-1H-pyrazol-4-yl)methyl]
piperazin-1-yl}isonicotinoyl)amino]-1-(4-fluorophenyl)-4,5-
dihydro-1H-benzo[g]indazole-3-carboxamide (22a)
Prepared using the general reductive amination procedure. 1H
NMR (DMSO-d6, 400 MHz): d ppm 2.22–2.50 (m, 8H) 2.81–2.95
(m, 4H) 3.40–3.49 (m, 2H) 3.74 (s, 3H) 6.84 (s, 1H) 7.19 (d,
J = 1.83 Hz, 1H) 7.23–7.42 (m, 6H) 7.49–7.60 (m, 4H) 8.10 (s, 1H)
10.30 (s, 1H); HRMS (M+H) calcd for C33H32ClFN9O2 640.2273,
found 640.2233.
7.12.9. 8-[(5-Chloro-2-{4-[(1-{2-[(2-hydroxyethyl)amino]-2-
oxoethyl}-1H-pyrazol-4-yl)methyl]piperazin-1-yl}isonicoti-
noyl)amino]-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]
indazole-3-carboxamide (38)
7.12.3. 8-[(5-Chloro-2-{4-[(1-methyl-1H-pyrazol-4-yl)methyl]
piperazin-1-yl}isonicotinoyl)amino]-1-(4-fluorophenyl)-4,,5-
dihydro-1H-benzo[g]indazole-3-carboxamide (4)
1H NMR (DMSO-d6, 400 MHz): d ppm 2.34–2.45 (m, 4H) 2.93 (d,
J = 9.15 Hz, 4H) 3.15 (q, J = 5.98 Hz, 2H) 3.36–3.47 (m, 5H) 3.47–
3.57 (m, 4H) 4.63–4.70 (m, 1H) 4.73 (s, 2H) 6.88 (s, 1H) 7.20–
7.28 (m, 2H) 7.32–7.44 (m, 4H) 7.47–7.54 (m, 1H) 7.54–7.64 (m,
2H) 7.92–8.02 (m, 1H) 8.14 (s, 1H) 10.30 (s, 1H); LRMS (M+H) calcd
for C36H37ClFN10O4 727.2, found 727.3.
Prepared using the general reductive amination procedure. 1H
NMR (400 MHz, DMSO-d6) d ppm 2.25–2.50 (m, 8H) 2.81–2.95
(m, 4H) 3.40–3.49 (m, 2H) 4.86 (s, 2H) 6.86 (s, 1H) 7.19–7.26 (m,
1H) 7.28–7.42 (m, 6H) 7.49–7.60 (m, 4H) 8.13 (s, 1H) 10.28 (s,
1H); HRMS (M+H) calcd for
684.2154.
C34H32ClFN9O4 684.2172, found
7.12.10. 8-[(2-{4-[(1-{2-[Bis(2-hydroxyethyl)amino]-2-oxoeth-
yl}-1H-pyrazol-4-yl)methyl]piperazin-1-yl}-5-chloroisonicoti-
noyl)amino]-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]inda
zole-3-carboxamide (39)
7.12.4. 8-{[2-(4-{[1-(2-Amino-2-oxoethyl)-1H-pyrazol-4-yl]me-
thyl}piperazin-1-yl)-5-chloroisonicotinoyl]amino}-1-(4-fluoro-
phenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (32)
Prepared using the general reductive amination procedure. 1H
NMR (DMSO-d6, 400 MHz): d ppm 2.55–2.68 (m, 4H) 2.94–3.09
(m, 6H) 3.47 (s, 2H) 3.53–3.64 (m, 4H) 4.78 (s, 2H) 6.96 (s, 1H)
7.28–7.36 (m, 1H) 7.40–7.52 (m, 5H) 7.56–7.62 (m, 1H) 7.62–
7.72 (m, 3H) 7.93–8.02 (m, 1H) 8.22 (s, 1H) 10.38 (s, 1H); HRMS
(M+H) calcd for C34H33ClFN10O3 683.2331, found 683.2361.
1H NMR (DMSO-d6, 400 MHz): d ppm 2.42 (s, 4H) 2.93 (d,
J = 9.15 Hz, 4H) 3.15 (q, J = 5.98 Hz, 2H) 3.36–3.47 (m, 7H) 3.47–
3.57 (m, 6H) 4.63–4.70 (m, 1H) 5.08 (s, 2H) 6.88 (s, 1H) 7.20–
7.28 (m, 2H) 7.32–7.44 (m, 4H) 7.47–7.54 (m, 1H) 7.54–7.64 (m,
2H) 7.92–8.02 (m, 1H) 8.14 (s, 1H) 10.30 (s, 1H); LRMS (M+H) calcd
for C38H41ClFN10O5 771.3, found 771.3.
7.12.118. -[(5-Chloro-2-{4-[(1-{2-[(2-methoxyethyl)amino]-2-
oxoethyl}-1H-pyrazol-4-yl)methyl]piperazin-1-yl}isonicotinoyl)
amino]-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g] indazole-
3-carboxamide (36)
7.12.5. 8-({5-Chloro-2-[4-({1-[2-(dimethylamino)-2-oxoethyl]-
1H-pyrazol-4-yl}methyl)piperazin-1-yl]isonicotinoyl}amino)-1-
(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (35)
1H NMR (DMSO-d6, 400 MHz): d ppm 2.34–2.44 (m, 4H) 2.85–
2.99 (m, 4H) 3.38 (br s, 3H) 3.45–3.55 (m, 5H) 4.72 (s, 2H) 6.86
(s, 1H) 7.22 (br s, 2H) 7.27–7.65 (m, 8H) 7.98–8.07 (m, 1H) 8.12
(s, 1H) 10.28 (s, 1H); LRMS (M+H) calcd for C37H39ClFN10O4
741.3, found 741.3.
1H NMR (400 MHz, DMSO-d6) d ppm 2.36–2.46 (m, 4H) 2.82 (s,
3H) 2.85–2.97 (m, 4H) 2.99 (s, 3H) 3.35–3.55 (m, 6H) 5.01 (s, 2H)
6.87 (s, 1H) 7.19–7.64 (m, 10H) 8.13 (s, 1H) 10.29 (s, 1H); LRMS
(M+H) calcd for C36H37ClFN10O3 711.2, found 711.2.
7.12.6. 8-({5-Chloro-2-[4-({1-[2-(isopropylamino)-2-oxoethyl]-
1H-pyrazol-4-yl}methyl)piperazin-1-yl]isonicotinoyl}amino)-1-
(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carbox-
amide (34)
7.12.12. 8-{[5-Chloro-2-(4-{[1-(2-{[2-(methylthio)ethyl]
amino}-2-oxoethyl)-1H-pyrazol-4-yl]methyl}piperazin-1-
yl)isonicotinoyl]amino}-1-(4-fluorophenyl)-4,5-dihydro-1H-
benzo[g]indazole-3-carboxamide (37)
1H NMR (DMSO-d6, 400 MHz): d ppm 1.04 (d, J = 6.59 Hz, 6H)
2.33–2.44 (m, 4H) 2.85–2.92 (m., 4H) 3.37 (s, 2H) 3.43–3.53 (m,
4H) 3.72–3.88 (m, 1H) 4.26–4.34 (m, 1H) 4.65 (s, 2H) 6.86 (s,
1H NMR (DMSO-d6, 400 MHz): d ppm 2.05 (s, 2H) 2.38–2.48 (m,
5H) 2.88–3.00 (m, 5H) 3.40 (s, 3H) 3.51 (s, 6H) 4.74 (s, 2H) 6.88 (s,
1H) 7.22–7.28 (m, 2H) 7.32–7.44 (m, 4H) 7.51–7.54 (m, 1H) 7.56–

1252
J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
7.64 (m, 3H) 8.07–8.18 (m, 2H) 10.30 (s, 1H); LRMS (M+H) calcd for
37H39ClFN10O3S 757.2, found 757.3.
7.12.19. 8-{[5-Chloro-2-(4-{[1-(3-methoxypropyl)-1H-pyrazol-
4-yl]methyl}piperazin-1-yl)isonicotinoyl]amino}-1-(4-fluoro
phenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (25)
1H NMR (DMSO-d6, 400 MHz): d ppm 1.93 (t, J = 6.58 Hz, 2H)
2.36 (t, J = 4.56 Hz, 4H) 2.80–2.97 (m, 4H) 3.18 (s, 3H) 3.22 (t,
J = 6.18 Hz, 2H) 3.34 (s, 2H) 3.46 (d, J = 4.83 Hz, 4H) 4.06 (t,
J = 6.98 Hz, 2H) 6.86 (s, 1H) 7.20 (d, J = 1.88 Hz, 1H) 7.24–7.43
(m, 6H) 7.50–7.60 (m, 4H) 8.12 (s, 1H) 10.31 (s, 1H); HRMS
(M+H) calcd for C36H38ClFN9O3 698.2692, found 698.2799.
C
7.12.13. Methyl [4-({4-[4-({[3-(aminocarbonyl)-1-(4-fluorophe-
nyl)-4,5-dihydro-1H-benzo[g]indazol-8-yl]amino}carbonyl)-5-
chloropyridin-2-yl]piperazin-1-yl}methyl)-1H-pyrazol-1-yl]
acetate (29)
1H NMR (DMSO-d6, 400 MHz): d ppm 2.25–2.50 (m, 4H)
2.81–2.95 (m, 4H) 3.39 (s, 3H) 3.43–3.53 (m, 6H) 4.86 (s, 2H)
6.86 (s, 1H) 7.19–7.26 (m, 2H) 7.28–7.66 (m, 9H) 8.13 (s, 1H)
10.28 (s, 1H); LRMS (M+H) calcd for C35H34ClFN9O4 698.2, found
698.3.
7.12.20. 8-({5-Chloro-2-[4-({1-[3-(2-methoxyethoxy)propyl]-
1H-pyrazol-4-yl}methyl)piperazin-1-yl]isonicotinoyl}amino)-1-
(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxa-
mide (26)
7.12.14. Ethyl [4-({4-[4-({[3-(aminocarbonyl)-1-(4-fluorophe-
nyl)-4,5-dihydro-1H-benzo[g]indazol-8-yl]amino}carbonyl)-5-
chloropyridin-2-yl]piperazin-1-yl}methyl)-1H-pyrazol-1-yl]
acetate (2a)
1H NMR (DMSO-d6 and water, 400 MHz): d ppm 1.88–2.04 (m,
2H) 2.34–2.46 (m, 4H) 2.84–3.06 (m, 4H) 3.25 (s, 1H) 3.31–3.36
(m, 3H) 3.36–3.59 (m, 11H) 4.10 (t, J = 6.77 Hz, 2H) 6.87 (s, 1H)
7.19–7.60 (m, 11H) 8.14 (s, 1H) 10.30 (s, 1H); LRMS (M+H) calcd
for C38H42ClFN9O4 742.3, found 742.3.
1H NMR (DMSO-d6, 400 MHz): d ppm 1.19 (t, J = 7.14 Hz, 2H)
2.41 (s, 4H) 2.88–3.00 (m, 4H) 3.40 (s, 3H) 3.45–3.57 (m, 5H)
4.13 (q, J = 6.95 Hz, 2H) 5.00 (s, 2H) 6.88 (s, 1H) 7.25 (d, 2H)
7.32–7.44 (m, 4H) 7.51 (s, 1H) 7.59 (dd, J = 8.42, 4.76 Hz, 2H)
7.63 (s, 1H) 8.15 (s, 1H) 10.31 (s, 1H); LRMS (M+H) calcd for
7.12.21. 8-[(5-Chloro-2-{4-[(1-methyl-1H-pyrazol-4-yl)methyl]
piperazin-1-yl}isonicotinoyl)amino]-1-(3-fluorophenyl)-4,5-
dihydro-1H-benzo[g]indazole-3-carboxamide (22b)
C36H36ClFN9O4 712.3, found 712.3.
Prepared using the general reductive amination general proce-
dure. 1H NMR (DMSO-d6, 400 MHz): d ppm 2.30–2.42 (m, 8H)
2.83–3.01 (m, 4H) 3.39–3.55 (m, 2H) 3.79 (s, 3H) 6.87 (s, 1H)
7.20–7.67 (m, 12H) 8.12 (s, 1H) 10.31 (s, 1H); LRMS (M+H) calcd
for C33H32ClFN9O2 640.3, found 640.3.
7.12.15. Ethyl 3-[4-({4-[4-({[3-(aminocarbonyl)-1-(4-fluorophe-
nyl)-4,5-dihydro-1H-benzo[g]indazol-8-yl]amino}carbonyl)-5-
chloropyridin-2-yl]piperazin-1-yl}methyl)-1H-pyrazol-1-yl]
propanoate (31)
1H NMR (DMSO-d6, 400 MHz): d ppm 1.12 (t, J = 7.05 Hz, 3H)
2.31–2.43 (m, 4H) 2.80 (t, J = 6.50 Hz, 2H) 2.84–3.01 (m, 4H) 3.34
(s, 2H) 3.41–3.54 (m, 4H) 3.95–4.08 (m, 2H) 4.28 (t, J = 6.59 Hz,
2H) 6.85 (s, 1H) 7.19–7.26 (m, 1H) 7.27–7.43 (m, 7H) 7.48 (s, 1H)
7.52–7.61 (m, 2H) 8.12 (s, 1H) 10.28 (s, 1H); LRMS (M+H) calcd
for C37H38ClFN9O4 726.3, found 726.3.
7.12.22. Ethyl [4-({4-[4-({[3-(aminocarbonyl)-1-(3-fluoro phe-
nyl)-4,5-dihydro-1H-benzo[g]indazol-8-yl]amino} carbonyl)-5-
chloropyridin-2-yl]piperazin-1-yl}methyl)-1H-pyrazol-1-yl]
acetate (2b)
Prepared using the general reductive amination general proce-
dure. 1H NMR (DMSO-d6, 400 MHz): d ppm 1.19 (t, J = 7.14 Hz,
2H) 2.41 (s, 4H) 2.88–3.00 (m, 4H) 3.40 (s, 3H) 3.45–3.57 (m, 5H)
4.13 (q, J = 6.95 Hz, 2H) 5.00 (s, 2H) 6.88 (s, 1H) 7.20–7.67 (m,
12H) 8.12 (s, 1H) 10.31 (s, 1H); LRMS (M+H) calcd for
7.12.16. 8-({5-Chloro-2-[4-({1-[3-(dimethylamino)propyl]-1H-
pyrazol-4-yl}methyl)piperazin-1-yl]isonicotinoyl}amino)-1-(4-
fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (28)
C36H36ClFN9O4 712.3, found 712.3.
1H NMR (DMSO-d6, 400 MHz): d ppm 1.82 (t, J = 6.85 Hz, 2H)
2.02–2.13 (m, 8H) 2.36 (t, J = 4.70 Hz, 4H) 2.83–2.97 (m, 4H) 3.34
(s, 2H) 3.41–3.51 (m, 4H) 4.03 (t, J = 6.98 Hz, 2H) 6.86 (s, 1H)
7.20 (d, J = 2.15 Hz, 1H) 7.25–7.44 (m, 6H) 7.49–7.62 (m, 4H)
8.12 (s, 1H) 10.31 (s, 1H); LRMS (M+H) calcd for C37H41ClFN10O2
711.3, found 711.3
7.12.23. 8-{[5-Chloro-2-(4-methylpiperazin-1-yl)isonicotinoyl]
amino}-1-(3-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-
3-carboxamide (13)
1H NMR (DMSO-d6, 400 MHz): d ppm 2.32 (s, 3H) 2.40–2.45 (m,
4H) 2.92–3.09 (m, 4H) 3.52–3.67 (m, 4H) 6.97 (s, 1H) 7.27–7.72 (m,
9H) 8.22 (s, 1H) 10.39 (s, 1H); LRMS (M+H) calcd for C29H28ClFN7O2
560.2, found 560.2.
7.12.17. 8-{[5-Chloro-2-(4-{[1-(2-cyanoethyl)-1H-pyrazol-4-
yl]methyl}piperazin-1-yl)isonicotinoyl]amino}-1-(4-fluorophe-
nyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (24)
1H NMR (DMSO-d6, 400 MHz): d ppm 2.38 (t, J = 4.56 Hz, 4H)
2.86–2.96 (m, 4H) 3.00 (t, J = 6.31 Hz, 2H) 3.36 (s, 2H) 3.41–3.51
(m, 4H) 4.31 (t, J = 6.31 Hz, 2H) 6.87 (s, 1H) 7.21 (d, J = 1.88 Hz,
1H) 7.25–7.45 (m, 6H) 7.49–7.61 (m, 3H) 7.68 (s, 1H) 8.12 (s, 1H)
10.31 (s, 1H); LRMS (M+H) calcd for C35H33ClFN10O2 679.2, found
679.2
7.12.24. 8-[(5-Chloro-2-{(3S)-3-methyl-4-[(1-methyl-1H-
pyrazol-4-yl)methyl]piperazin-1-yl}isonicotinoyl)amino]-1-(4-
fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (43)
1H NMR (DMSO-d6, 400 MHz): d ppm 1.10 (d, 3H, J = 6.3 Hz),
2.05–2.35 (m, 2H), 2.65–2.79 (m, 2H), 2.87–2.93 (m, 6H), 3.60–
3.74 (m, 1H), 3.76 (s, 3H) 3.90–4.0 (m, 2H), 6.85 (s, 1H), 7.21–
7.65 (m, 11H), 8.11 (s, 1H), 10.31 (s, 1H); LRMS (M+H) calcd for
7.12.18. 8-{[5-Chloro-2-(4-{[1-(3-hydroxypropyl)-1H-pyrazol-4-
yl]methyl}piperazin-1-yl)isonicotinoyl]amino}-1-(4-fluorophe-
nyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (27)
Prepared using the general reductive amination general proce-
dure. 1H NMR (DMSO-d6, 400 MHz): d ppm 2.83–2.97 (m, 4H)
3.30–3.36 (m, 4H) 3.42–3.54 (m, 4H) 4.07 (t, J = 6.98 Hz, 2H) 4.52
(t, J = 5.10 Hz, 1H) 6.86 (s, 1H) 7.21 (d, J = 1.88 Hz, 1H) 7.25–7.43
(m, 6H) 7.50–7.62 (m, 4H) 8.12 (s, 1H) 10.31 (s, 1H); LRMS
(M+H) calcd for C35H36ClFN9O3 684.2, found 684.2.
C34H34ClFN9O2 654.2, found 654.2.
7.12.25. 4-(4-{[3-Carbamoyl-1-(4-fluorophenyl)-4,5-dihydro-
1H-benzo[g]indazol-8-yl]carbamoyl}-5-chloropyridin-2-yl)-1-
[(1-methyl-1H-pyrazol-4-yl)methyl]piperazine-2-carboxylic
acid (45)
Prepared using the general reductive amination general
procedure. 1H NMR (DMSO-d6, 400 MHz): d ppm 2.17 (m, 1H)
2.79–3.00 (m, 9H) 3.75 (s, 3H) 3.80–4.0 (m, 2H), 6.85 (s, 1H),

J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
1253
7.21–7.65 (m, 11H), 8.11 (s, 1H), 10.31 (s, 1H); LRMS (M+H) calcd
for C34H32ClFN9O4 684.2, found 684.2.
evaporated to dryness to obtain compound 11 (13.75 g, >95%) as
a yellow solid. 1H NMR (400 MHz, DMSO-d6): d 2.72 (t, 2H,
J = 6.6 Hz), 2.84–2.88 (m, 2H), 3.84 (s, 3H), 4.80 (br s, 1H), 5.98 (s,
1H), 6.38–6.41(m, 1H), 6.97 (d, 1H, J = 8.1 Hz), 7.1 (d, 2H,
J = 8.8 Hz), 7.24 (s, 1H), 7.42 (d, 2H, J = 8.7 Hz), 7.48 (s, 1H); LRMS
(M+H) calcd for C19H19N4O2 335.1, found 335.1.
7.12.26. 1-(4-{[3-Carbamoyl-1-(4-fluorophenyl)-4,5-dihydro-
1H-benzo[g]indazol-8-yl]carbamoyl}-5-chloropyridin-2-yl)-4-
[(1-methyl-1H-pyrazol-4-yl)methyl]piperazine-2-carboxylic
acid (44)
Prepared using the general reductive amination general proce-
dure. 1H NMR (DMSO-d6, 400 MHz): d ppm 1.60–2.0 (m, 4H)
2.72–2.82 (m, 1H) 2.83–3.02 (m, 6H) 3.45–3.60 (m, 2H) 3.75 (s,
3H) 3.80–3.95 (s, 1H), 4.29–4.38 (s, 1H) 6.55 (s, 1H), 7.15–7.65
(m, 11H), 8.11 (s, 1H), 10.31 (s, 1H); LRMS (M+H) calcd for
7.12.31. 8-(2,5-Dichloroisonicotinamido)-1-(4-methoxy- phenyl) -
4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (12: R1 = OCH3;
R2 = H)
Compound 11 (13.75 g, 0.04 mol) was added to a DMF (48 mL)
solution of 2,5-dichloroisonicotinic acid, (9.47 g, 0.0411 mol),
HBTU (O-benzotriazole-N,N,N’,N’-tetramethyluronium-hexafluoro-
phosphate, 18.7 g, 0.0493 mol) and diisopropylethylamine (8.52
g, 0.0493 mol) at 23 °C. The reaction mixture was stirred at 23 °C
for 3 h. The completion of the reaction was monitored by thin layer
chromatography. At the end of this time, the reaction mixture was
concentrated to 12 ml by distillation under reduced pressure at
60 °C. The reaction mixture was cooled to 23 °C then diluted with
water (1 L) and stirred for 1 h to obtain a solid precipitate. The solid
residue was then stirred for 1 h with 1 L of 9:1 water and triethyl-
amine. The solid was filtered and dried under vacuum to obtain
compound 12 (19.5 g, 93.3%) as a yellow solid. 1H NMR (400
MHz, DMSO-d6): d 2.93 (br s, 4H), 3.81 (s, 3H), 7.08 (d, 2H,
J = 8.3 Hz), 7.18 (s, 1H), 7.29–7.43 (m, 5H), 7.45 (s, 1H), 7.55 (s,
1H), 7.8 (s, 1H), 8.6 (s, 1H); LRMS (M+H) calcd for C25H20Cl2N5O3
508.0, found 508.0.
C34H32ClFN9O4 684.2, found 684.2.
7.12.27. 8-{[5-Chloro-2-(4-methylpiperazin-1-yl)isonicotinoyl]
amino}-1-(3,4-difluorophenyl)-4,5-dihydro-1H-benzo[g]
indazole-3-carboxamide (14)
Prepared using the general reductive amination general proce-
dure. 1H NMR (DMSO-d6, 400 MHz): d ppm 2.34 (s, 3H), 2.40–
2.45 (m, 4H), 2.87–2.93 (m, 4H), 3.47 (m, 4H), 6.87 (s, 1H), 7.20–
7.65 (m, 8H), 8.13 (s, 1H), 10.31 (s, 1H); LRMS (M+H) calcd for
C29H27ClF2N7O2 578.2, found 578.2.
7.12.28. Ethyl 1-(4-methoxyphenyl)-8-nitro-4,5-dihydro-1H-
benzo[g]indazole-3-carboxylate (9: R1 = OCH3; R2 = H)
To a solution of compound 814a (55 g, 0.19 mol) in 830 mL of 1:1
glacial acetic acid and absolute ethanol, 4-methoxyphenylhydra-
zine hydrochloride (35 g, 0.2 mol) was added and stirred at 80 °C
for 16 h under nitrogen. The reaction mixture was then cooled to
0–5 °C and filtered. The residue was washed with cold ether and
dried to afford compound 9 (64 g, 88%) as brownish yellow solid.
1H NMR (400 MHz, DMSO-d6): d 1.30–1.33 (m, 3H), 3.01–3.04
(m, 2H), 3.08–3.12 (m, 2H), 3.85 (s, 3H), 4.32 (q, 2H, J = 7.1 Hz),
7.17 (d, 2H, J = 8.8 Hz), 7.41 (d, 1H, J = 2.1 Hz), 7.52 (d, 2H,
J = 8.8 Hz), 7.67 (d, 1H, J = 8.2 Hz), 8.07 (dd, 1H, J = 2.2, 8.3 Hz);
LRMS (M+H) calcd for C21H20N3O5 394.1, found 394.1.
7.12.32. 8-{[5-Chloro-2-(4-methylpiperazin-1-yl)isonicotinoyl]
amino}-1-(4-hydroxyphenyl)-4,5-dihydro-1H-benzo[g] inda-
zole-3-carboxamide (16: R1 = OH; R2 = H)
Compound 12 (1 g, 0.002 mol) was placed in a 25 mL round bot-
tomed flask, and boron tribromide (1.85 mL, 0.02 mol) was added at
5 °C under a nitrogen atmosphere. The reaction mixture was al-
lowed to stir at 23 °C for 16 h. Thin layer chromatography con-
firmed the absence of starting material. Ice (5 g) was poured into
the reaction mixture with continued stirring, then diluted with
dichloromethane (20 mL), filtered, and the residue obtained was
repeatedly washed with dichloromethane, and was dried under
vacuum. This afforded (1 g) of crude brown intermediate which
was used in the subsequent reaction without purification. The
crude intermediate (0.36 mmol) was mixed with N-methylpipera-
zine (0.8 mL) and refluxed for 8 h, monitoring the reaction by thin
layer chromatography. At the end of the reaction, N-methyl pipera-
zine was removed under reduced pressure. The slurry was tritu-
rated with water (3 Â 20 mL) and filtered each time. The residue
was dried under vacuum. This material was purified with silica
gel column chromatography (Biotage) using ethyl acetate/metha-
nol/ammonia (1:0.05:0.03) as eluent, affording the product 12. 1H
NMR (DMSO-d6, 400 MHz): d ppm 2.20 (s, 3H), 2.33–2.40 (m, 4H),
2.85–2.97 (m, 4H), 6.85–6.92 (m, 2H), 7.21–7.35 (m, 4H), 7.46
(dd, J = 8.2, 2.0 Hz, 1H), 7.51 (s, 1H,), 8.15 (s, 1H), 10.35 (s, 1H);
HRMS (M+H) calcd for C29H29ClN7O3 558.1942, found 558.2000.
7.12.29. Ethyl 8-amino-1-(4-methoxyphenyl)-4,5-dihydro-1H-
benzo[g]indazole-3-carboxylate (10: R1 = OCH3; R2 = H)
Compound 9 (64 g, 0.16 mol) was dissolved in 1.28 L of acetic
acid and 640 mL of ethanol. Zinc dust (85.38 g, 1.31 mol) was added
in portions at 5–10 °C with stirring over a 30 min period. After addi-
tion of the zinc dust was completed, the reaction mixture was al-
lowed to stir at 23 °C for 1 h. The completion of the reaction was
monitored by thin layer chromatography. The reaction mixture
was then filtered through a bed of Celite and washed with ethyl ace-
tate (3 Â 150 mL). The filtrate was diluted with dichloromethane
(2.5 L) then neutralized (pH 7) with saturated sodium bicarbonate
solution. The organic layer was separated and washed with water
and brine successively, dried over anhydrous sodium sulfate and
evaporated to dryness to afford 65 g of crude product. This material
was recrystallized twice with 7:3 dichloromethane: hexane giving
31.5 g (53.3%) of compound 10 as a yellow solid. 1H NMR
(400 MHz, DMSO-d6): d 1H NMR (400 MHz, DMSO-d6): d 1.30–1.33
(m, 3H), 3.01–3.04 (m, 2H), 3.08–3.12 (m, 2H), 3.85 (s, 3H), 4.32
(q, 2H, J = 7.1 Hz), 7.17 (d, 2H, J = 8.8 Hz), 7.41 (d, 1H, J = 2.1 Hz),
7.52 (d, 2H, J = 8.8 Hz), 7.67 (d, 1H, J = 8.2 Hz), 8.07 (dd, 1H, J = 2.2,
8.3 Hz); LRMS (M+H) calcd for C21H22N3O3 364.1, found 364.1.
7.12.33. 1-(3-Chloro-4-methoxyphenyl)hydrazine (21)
3-Chloro-4-methoxyaniline (20 g, 0.13 mol) was mixed with
concentrated hydrochloric acid (23.4 mL) and treated at 0 °C with
a solution of sodium nitrite (8.75 g, 0.13 mol) in (36.9 mL) of water.
A well cooled solution of stannous chloride (85.89 g in 74.1 mL of
concentrated hydrochloric acid) was added to the reaction solution
and allowed to stand for 2 h. After filtration, the reaction mixture
was shaken with cold caustic potash solution (164.5 mL, 25%) pro-
ducing a crystalline solid. The crystalline solid was washed with
water and dried in a vacuum desiccator. Recrystallization of the
crude mass with (1:3 benzene/hexane) afforded 21 (6.74 g,
30.77%) as yellow crystalline solid. 1H NMR (400 MHz, DMSO-d6):
7.12.30. 8-Amino-1-(4-methoxyphenyl)-4,5-dihydro-1H-
benzo[g]indazole-3-carboxamide (11, R1 = OCH3; R2 = H)
Compound 10 (15 g, 0.04 mol) was treated with 750 mL of eth-
anolic ammonia (1:1) solution at À78 °C inside a 2 l autoclave ves-
sel. The reaction mixture was slowly warmed to 23 °C and then
heated to 120 °C for 48 h. The reaction mixture was then cooled
to 23 °C and was transferred to a round bottomed flask and

1254
J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
d 3.71 (s, 3H), 3.94 (s, 2H), 6.53 (s, 1H), 6.68 (dd, 1H, J = 2.4, 8.8 Hz),
6.87 (d, 1H, J = 2.4 Hz), 6.92 (d, 1H, J = 8.8 Hz).
2H) 2.84–3.04 (m, 5H) 3.70 (d, J = 14.27 Hz, 1H) 3.96 (d,
J = 12.08 Hz, 2H) 4.10 (q, J = 7.20 Hz, 2H) 4.99 (s, 2H) 6.87 (s, 1H)
7.18–7.27 (m, 2H) 7.27–7.44 (m, 5H) 7.49 (br s, 1H) 7.52–7.66
(m, 3H) 8.10 (s, 1H) 10.28 (s, 1H); LRMS (M+H) calcd for
7.12.34. 1-(3-Chloro-4-hydroxyphenyl)-8-{[5-chloro-2-(4-
methylpiperazin-1-yl)isonicotinoyl]amino}-4,5-dihydro-1H-
benzo[g]indazole-3-carboxamide (17)
C37H38ClFN9O4 726.3, found 726.3.
Compound 17 was synthesized following the same synthetic
protocol as described for the synthesis of compound 16. 1H NMR
(DMSO-d6, 400 MHz): d ppm 2.22 (3H, s), 2.38 (4H, br s), 2.83–
2.99 (4H, m), 6.92 (1H, s), 7.08 (1H, d, J = 8.8 Hz), 7.23–7.37 (3H,
m), 7.46–7.58 (2H, m), 8.17 (1H, s), 10.40 (1H, s); LRMS (M+H)
calcd for C29H28Cl2N7O3 592.0, found 592.0.
References and notes
1. (a) Adock, I. M.; Chung, K. F.; Caramori, G.; Ito, K. Eur. J. Pharmacol. 2006, 533,
118; (b) Barnes, P. Pharmacol. Ther. 2006, 109, 238.
2. (a) Baldwin, A. S., Jr J. Clin. Invest. 2001, 107, 3; (b) Bonizzi, G.; Karin, M. Treands
Immunol. 2004, 25, 280; (c) Ghosh, S.; Karin, M. Cell 2002, 109, S81.
3. Swenftleben, U.; Karin, M. Crit. Care Med. 2002, 30, S18.
4. (a) Castro, A. C.; Dang, L. C.; Soucy, F.; Grenier, L.; Mazdiyasni, H.; Hottelet, M.,
et al Bioorg. Med. Chem. Lett. 2003, 13, 2419; (b) Karin, M.; Yamamoto, Y.; Wang,
Q. M. Nat. Rev. Drug Disc. 2004, 3, 17; (c) Cosio, B. G.; Mann, B.; Ito, K.; Jazrawi,
E.; Barnes, P. J.; Chung, K. F., et al Am. J. Respir. Crit. Care Med. 2004, 170, 141.
5. (a) Baxter, A.; Brough, S.; Faull, A.; Johnstone, C.; Mcinally, T.; PCT Int. Appl. WO
0158890 A1, 2001.; (b) Griffiths, D.; Johnstone, C., 2003, WO 2003010163 A1
20030206.; (c) Baxter, A.; Brough, S.; Cooper, A.; Floettmann, E.; Foster, S.;
Harding, C.; Kettle, J.; McInally, T.; Martin, C.; Mobbs, M.; Needham, M.;
Newham, P.; Paine, S.; St-Gallay, St.; Salter, S.; Unitt, J.; Xue, T. Bioorg. Med.
Chem. Lett 2004, 14, 2817; (d) Bonafoux, D.; Bonar, S.; Christine, L.; Clare, M.;
Donnelly, A.; Guzova, J.; Kishore, N.; Lennon, P.; Libby, A.; Mathialagan, S.;
McGhee, W.; Rouw, S.; Sommers, C.; Tollefson, M.; Tripp, C.; Weier, R.; Wolfson,
S.; Min, Y. Bioorg. Med. Chem. Lett. 2005, 15, 2870.
6. (a) Kerns, J. K.; PCT Int. Appl. WO 2007076286 A2, 2007.; (b) Kerns, J. K.;
Lindenmuth, M.; Lin, X.; Nie, H.; Thomas, S. M., PCT Int. Appl. WO 034317 A2,
2006.; (c) Deng, J.; Kerns, J. K.; Jin, Q.; Lin, G.; Lin, X.; Lindenmuth, M.; Neipp, C.
E.; Nie, H. T.; Sonia, M.; Widdowson, K. L., PCT Int. Appl. WO 005534 A2, 2007.
7. Clare, M.; Fletcher, T. R.; Hamper, B. C.; Hanson, G. A.; Heier, R. F.; Huang, H.;
Lennon, P. J.; Oburn, D. S.; Reding, M. T.; Stealey, M. A.; Wolfson, S. G.; Xie, J.,
PCT Int. Appl. WO 037797 A1, 2005.
8. (a) Morey, J. V.; Christopher, J. A. PCT Int. Appl. WO 025575 A1, 2007.; (b)
Christopher, J. A.; Avitabile, B. G.; Bamborough, P.; Champigny, A. C.; Cutler, G.
J.; Dyos, S. L.; Grace, K. G.; Kerns, J. K.; Kitson, J. D.; Mellor, G. W.; Morey, J. V.;
Morse, M. A.; O’Malley, C. F.; Patel, C. B.; Probst, N.; Rumsey, W.; Smith, C. A.;
Wilson, M. J. Bioorg. Med. Chem. Lett. 2007, 17, 3972; (c) Waelchli, R.; Bollbuck,
B.; Bruns, C.; Buhl, T.; Eder, J.; Feifel, R.; Hersperger, R.; Janser, P.; Revesz, L.;
Zerwes, H.-G.; Schlapbach, A. Bioorg. Med. Chem. Lett. 2006, 16, 108.
9. (a) Murata, T.; Sakakibara, S.; Yoshino, T.; Ikegami, Y.; Masuda, T.; Shimada, M.;
Shintani, T.; Shimazaki, M.; Lowinger, T. B.; Ziegelbauer, K. B.; Fuchikami, K.;
Umeda, M.; Komura, H.; Yoshida, N. WO 03024935 A2, 2002.; (b) Murata, T.;
Shimada, M.; Sakakibara, S.; Yoshino, T.; Kadono, H.; Masuda, T.; Shimazaki, M.;
Shintani, T.; Fuchikami, K.; Sakai, K.; Inbe, H.; Takeshita, K.; Niki, T.; Umeda, M.;
Bacon, K. B.; Ziegelbauer, K. B.; Lowinger, T. B. Bioorg. Med. Chem. Lett. 2003, 13,
913.
10. (a) Binghama, A. H.; Davenport, R. J.; Fosbeary, R.; Gowersa, L.; Knighta, R. L.;
Lowea, C.; Owena, D. A.; Parrya, D. M.; Pitt, W. R. Bioorg. Med. Chem. Lett. 2008,
18, 3622; (b) Okamoto, Y.; Kubota, H.; Sato, I.; Hattori, K.; Kanayama, T.;
Yokoyama, K.; Terai, Y.; Takeuchi, M. PCT Int. Appl. WO 100341 A1, 2005.; (c)
Clare, M.; Hagen, T. J.; Houdek, S. C.; Lennon, P. J.; Weier, R. M.; Xu, X., PCT Int.
Appl. WO 040133 A1, 2005.; (d) Bingham, A. H.; Davenport, R. J.; Fosbeary, R.;
Gowers, L.; Knight, R. L.; Lowe, C.; Owen, D. A.; Parry, D. M.; Pitt, W. R. Bioorg.
Med. Chem. Lett. 2008, 18, 3622.
7.12.35. 1-(4-Chloro-3-hydroxyphenyl)-8-{[5-chloro-2-(4-meth-
ylpiperazin-1-yl)isonicotinoyl]amino}-4,5-dihydro-1H-benzo[g]
indazole-3-carboxamide (18)
Compound 18 was synthesized following the same synthetic
protocol as described for the synthesis of compound 16. 1H NMR
(DMSO-d6, 400 MHz): d ppm 2.22 (3H, s), 2.38 (4H, br s), 2.83–
2.99 (4H, m), 6.92 (1H, s), 7.08 (1H, d, J = 8.8 Hz), 7.23–7.37 (3H,
m), 7.46–7.58 (2H, m), 8.17 (1H, s), 10.40 (1H, s); LRMS (M+H)
calcd for C29H28Cl2N7O3 592.0, found 592.0.
7.12.36. 8-{[5-Chloro-2-(4-methylpiperazin-1-yl)isonicotinoyl]
amino}-1-(3-hydroxyphenyl)-4,5-dihydro-1H-benzo[g] inda-
zole-3-carboxamide (19)
Compound 19 was synthesized following the same synthetic
protocol as described for the synthesis of compound 16. 1H NMR
(DMSO-d6, 400 MHz): d ppm 2.21 (3H, s), 2.38 (4H, br s), 2.85–
2.98 (4H, m), 6.84–6.93 (3H, m), 7.22–7.36 (3H, m), 7.46–7.58
(2H, m), 8.16 (1H, s), 9.88 (1H, s), 10.37 (1H, s).; LRMS (M+H) calcd
for C29H29ClN7O3 558.0, found 558.1.
7.12.37. 8-{[5-Chloro-2-(4-methylpiperazin-1-yl)isonicotinoyl]
amino}-1-(4-methoxyphenyl)-4,5-dihydro-1H-benzo[g]
indazole-3-carboxamide (20)
Compound 12 (200 mg, 0.393 mmol) was treated with N-meth-
ylpiperazine (0.79 mL, 7.84 mmol). The reaction mixture was
heated at 80 °C for 16 h, when thin layer chromatography indicated
the completion of the reaction. The reaction mixture was diluted
with water (20 mL), extracted with dichloromethane (3 Â 30 mL)
and dried over anhydrous sodium sulfate. Evaporation of the solvent
followed by silica gel column chromatography using methanol/
dichloromethane (40:60) as the eluent afforded the desired product
as a light yellowish solid. 1H NMR (DMSO-d6, 400 MHz): d ppm 2.20
(s, 3H) 2.32–2.40 (m, 1H) 2.84–2.99 (m, 4H) 3.46–3.57 (m, 4H) 3.81
(s, 3H) 6.88 (s, 1H) 7.08 (d, J = 8.79 Hz, 2H) 7.18–7.25 (m, 2H) 7.32 (d,
J = 8.05 Hz, 1H) 7.39–7.49 (m, 3H) 8.15 (s, 1H) 10.27 (s, 1H); HRMS
(M+H) calcd for C30H31ClN7O3 558.1942, found 558.2.
11. Castro, A. C.; Dang, L. C.; Soucy, F.; Grenier, L.; Mazdiyasni, H.; Hottelet, M.; Parent,
L.; Pien, C.; Palombella, V.; Adams, J. Bioorg. Med. Chem. Lett. 2003, 13, 2419.
12. Bamborough, P.; Barker, M. D.; Campos, A. A.; Cousins, R. C.; Faulder, P.; Hobbs,
H.; Holmes, D. S.; Johnston, M. J.; Liddle, J. P., Jeremy J.; Pritchard, J. M.;
Whitworth, C., PCT Int. Appl. WO 2008034860 A1, 2008.
13. Belema, M.; Bunker, A.; Nguyen, V. N.; Beaulieu, F.; Ouellet, C.; Qiu, Y.; Zhang,
Y.; Martel, A.; Burke, J. R.; McIntyre, K. W.; Pattoli, M. A.; Daloisio, C.; Gillooly,
K. M.; Clarke, W. J.; Brassil, P. J.; Zusi, F. C.; Vyas, D. M. Bioorg. Med. Chem. Lett.
2007, 17, 4284.
7.12.38. 8-[(5-Chloro-2-{(3S)-3-methyl-4-[(1-methyl-1H-pyra-
zol-4-yl)methyl]piperazin-1-yl}isonicotinoyl)amino]-1-(4-fluoro
phenyl)-45-dihydro-1H-benzo[g]indazole-3-carboxamide (35)
1H NMR (DMSO-d6, 400 MHz): d ppm 1.10 (d, J = 5.86 Hz, 3H)
2.07–2.17 (m, 1H) 2.19–2.30 (m, 1H) 2.65–2.76 (m, 2H) 2.83–
3.02 (m, 6H) 3.67 (d, J = 14.27 Hz, 1H) 3.76 (s, 3H) 3.95 (d,
J = 10.98 Hz, 2H) 6.85 (s, 1H) 7.15–7.44 (m, 8H) 7.49 (br s, 1H)
7.52–7.60 (m, 2H) 8.09 (s, 1H) 10.28 (s, 1H); LRMS (M+H) calcd
for C34H34ClFN9O2 654.2, found 654.2.
14. (a) Bonafoux, D.; Bonar, S.; Clare, M.; Donnelly, A.; Guzova, J.; Kishore, N.;
Lennon, P.; Libby, A.; Mathialagan, S.; McGhee, W.; Rouw, S.; Sommers, C.;
Tollefson, M.; Tripp, C.; Weier, R.; Wolfson, S.; Huang, H.-C. Bioorg. Med. Chem.
Lett. 2010, 18, 413; (b) Mbalaviele, G.; Sommers, C. D.; Bonar, S. L.;
Mathialagan, S.; Schindler, J. F.; Guzova, J. A.; Shaffer, A. F.; Melton, M. A.;
Christine, L. J.; Tripp, C. S.; Chiang, P.-C.; Thompson, D. C.; Hu, Y.; Kishore, N. J.
Pharmacol. Exp. Ther. 2009, 329, 14; (c) Sommers, C. D.; Thompson, J. M.;
Guzova, J. A.; Bonar, S. L.; Rader, R. K.; Mathialagan, S.; Venkatraman, N.;
Holway, V. W.; Kahn, L. E.; Hu, G.; Garner, D. S.; Huang, H.-C.; Chiang, P.-C.;
Schindler, J. F.; Hu, Y.; Meyer, D. M.; Kishore, N. N. J. Pharmacol. Exp. Ther. 2009,
330, 377; (d) Bergmanis, A. A.; Bonafoux, D.; Clare, M.; Crich, J. Z.; Fletcher, T.
R.; Geng, L.; Hagen, T. J.; Hamper, B. C.; Hanson, G. J.; Houdek, S. C.; Huang, H.;
Iula, D. M.; Koszyk, F. J.; Lennon, P. J.; Liao, S.; Liao, S.; Metz, S.; Mohler, S. B.;
Nguyen, M.; Oburn, D. S.; Owen, T. J.; Partis, R. A.; Scates, A. M.; Stealey, M. A.;
Tollefson, M. B.; Vazquez, M. L.; Weier, R. M.; Wolfson, S. G.; Xu, X. PCT Int. WO
200324935, 2003.
7.12.39. Ethyl [4-({(2S)-4-[4-({[3-(aminocarbonyl)-1-(4-fluoro-
phenyl)-45-dihydro-1H-benzo[g]indazol-8-yl]amino} car-
bonyl)-5-chloropyridin-2-yl]-2-methylpiperazin-1-yl} methyl)-
1H-pyrazol-1-yl]acetate (36)
15. Tayab, Z. R.; Hochhaus, G. Expert Opin. Drug Deliv. 2005, 2, 519.
16. Kishore, N.; Huynh, Q. K.; Mathialagan, S.; Hall, T.; Rouw, R.; Creely, D.; Lange,
G.; Caroll, J.; Reitz, B.; Donnelly, A.; Boddupalli, H.; Combs, R. G.; Kretzmer, K.;
Tripp, C. S. J. Biol. Chem. 2002, 277, 13840.
1H NMR (DMSO-d6, 400 MHz): d ppm 1.04–1.27 (m, 6H) 2.05–
2.19 (m, 1H) 2.21–2.31 (m, 1H) 2.52 (s, 1H) 2.72 (d, J = 11.89 Hz,

J. Xie et al. / Bioorg. Med. Chem. 19 (2011) 1242–1255
1255
17. Huynh, Q. K.; Boddupalli, H.; Rouw, S. A.; Koboldt, C. M.; Hall, T.; Sommers, C.;
Hauser, S. D.; Pierce, J. L.; Combs, R. G.; Reitz, B. A.; Diaz-collier, J. A.; Weinberg,
R. A.; Hodd, B. L.; Kilpatrick, B. F.; Tripp, C. S. J. Biol. Chem. 2000, 275, 25883.
18. (a) Chen, F.; Castranova, V.; Shi, X.; Demers, L. M. Clin. Chem. 1999, 45, 7; (b)
Ghosh, S.; May, M. J.; Kopp, E. B. Annu. Rev. Immunol. 1998, 16, 225; (c) Karin, M.
Oncogene 1999, 18, 6867.
19. Mathialagan, S.; Poda, G. I.; Kurumbail, R. G.; Selness, S. R.; Hall, T.; Reitz, B. A.;
Weinberg, R. A.; Kishore, N.; Mbalaviele, G. Protein Expr. Purif. 2010, 72, 254.
20. Poda, G.; Clare, M.; Kurumbail, R. G., unpublished data.
21. Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R. J. Med. Chem.
2006, 49, 534.