Aminopyridinecarboxamide-based inhibitors: Structure-activity relationship

Article abstract of DOI:10.1016/j.bmc.2009.10.040

Series of aminopyridinecarboxamide-based inhibitors were synthesized and tested against human recombinant IKK-2 and in IL-1β stimulated synovial fibroblasts. The 2-amino-5-chloropyridine-4-carboxamides were identified as the most potent inhibitors with im

Full text of DOI:10.1016/j.bmc.2009.10.040

Bioorganic & Medicinal Chemistry 18 (2010) 403–414
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Aminopyridinecarboxamide-based inhibitors: Structure–activity relationship
b
a
b
a
Dominique F. Bonafoux a, , Sheri L. Bonar , Michael Clare , Ann M. Donnelly , Jeanette L. Glaenzer ,
Julia A. Guzova b, He Huang a, Nandidni N. Kishore b, Francis J. Koszyk a, Patrick J. Lennon a, Adam Libby a,
Sumathy Mathialagan b, David S. Oburn a, Sharon A Rouw b, Cynthia D. Sommers b, Catherine S. Tripp b,
*
Lori J. Vanella b, Richard Weier a, Serge G. Wolfson a, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of aminopyridinecarboxamide-based inhibitors were synthesized and tested against human
recombinant IKK-2 and in IL-1b stimulated synovial fibroblasts. The 2-amino-5-chloropyridine-4-carbox-
amides were identified as the most potent inhibitors with improved cellular activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 25 September 2009
Revised 19 October 2009
Accepted 23 October 2009
Available online 27 October 2009
Keywords:
IKK2
NFkB
Inhibitor
1. Introduction
tive inhibitor, which binds IKK-2 tightly with a relatively slow off
rate and is efficacious in a chronic model of arthritis with no ad-
Nuclear factor kappa B (NF-
j
B) is an inducible transcriptional
verse effects at maximally efficacious doses (Fig. 1).12,13
activator that is inappropriately activated and/or disregulated in
a number of inflammatory disorders such as arthritis.1 NF-
B is re-
tained in the cytoplasm in an inactive form by association with
regulatory proteins called inhibitors of B (I B). Upon stimulation
with proinflammatory cytokines such as interleukin 1 beta (IL-1b)
or tumor necrosis factor alpha (TNF ), IKK-2 phosphorylates I B,
leading to its degradation and the release of NF- B in the cyto-
plasm. The NF- B subsequently translocates to the nucleus where
it binds target genes and regulates their transcription.2 IKK-2,
being the converging point for the activation of NF- B in response
The 2-chloropyridine-3-carboxamide derivatives 1 and 2,14 two
early precursor to PHA-408, had been found to be potent inhibitors
of IKK-2 (Fig. 2) with respective IC50’s of 34 nM and 47 nM, but
only showed modest to low activity in our cell based assay (SF-
j
j
j
IL-8 IC50 = 5 lM and 12 lM, respectively). Thus, we launched a
a
j
synthetic program to explore the SAR of this template and improve
the cellular activity. Our effort relied on the introduction of a vari-
ety of solubilizing amines into the 2-chloropyridine-3-carboxam-
ide template, which led us to investigate three chemical series:
the 2-aminopyridine-3-carboxamides (3, 4), 6-aminopyridine-2-
carboxamides (5, 6) and 2-aminopyridine-4-carboxamides (7, 8).
j
j
j
to a variety of proinflammatory stimuli, is therefore an attractive
target for the pharmaceutical industry. Various natural products
as well as anti-inflammatory agents such as aspirin3 have been
reported to be weak, non-selective inhibitors of IKK-2. More
recently, thiophenecarboxamides,4 indolecarboxamides,5 benzam-
ides,6 2,4-diarylpyridines,7 aminopyrimidines,8 b-carbolines,9 pyr-
rolopyridines10 and imidazo(1,2-a)thieno(3,2-e)pyrazines11 were
shown to be potent IKK-2 inhibitors.
F
N
N
H
N
O
We recently reported that the 8-(5-chloro-2-(4-methylpipera-
zin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo-
[g]indazole-3-carboxamide (PHA-408) is a selective, ATP-competi-
CONH2
Cl
N
N
N
* Corresponding authors.
E-mail addresses: dominique.bonafoux@abbott.com (D.F. Bonafoux), horng-chih.
huang@pfizer.com (H.-C. Huang).
Figure 1. Structure of PHA-408.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.040

404
D. F. Bonafoux et al. / Bioorg. Med. Chem. 18 (2010) 403–414
Ar
N
N
H
N
O
N
CONH2
Cl
1: Ar = 3,4-(OCH2O)C6H3
2: Ar = 4-F-C6H4
Ar
Ar
Ar
N
N
N
N
N
N
H
N
H
N
H
N
O
N
CONH2
O
CONH2
O
CONH2
R1R2N
Cl
Cl
N
NR1R2
NR1R2
N
7: Ar = 3,4-(OCH2O)C6H3
3: Ar =3,4-(OCH2O)C6H3
4: Ar = 4-F-C6H4
5: Ar = 3,4-(OCH2O)C6H3
6: Ar = 4-F-C6H4
8: Ar = 4-F-C6H4
Figure 2. Pyridine analogs of prototype compounds 1 and 2.
with acid15 11 (Scheme 3). Subsequent amination of 12, under
2. Chemistry
conditions similar to those of Scheme 2, led predominantly to
the desired 5a and 5b. It is worth noting that the amination of
12 required long reaction times and resulted in significant
amounts of byproducts leading to troublesome purifications and
modest yields for 5a (28%) and 5b (52%).
As a result of the low selectivity and the long reaction times
associated with the direct amination of the 3,6-dichloropyridine-
2-carboxamide 12 (Scheme 3), an alternative synthesis was
developed for the preparation of additional 3-chloro-6-aminopyri-
dine-2-carboxamides. This route relied on the synthesis of the
3-chloro-6-morpholino-2-pyridine carboxylic acid hydrochloride
(14) and the 3-chloro-6-(4-methylpiperazinyl)-2-pyridine carbox-
ylate (15) followed by coupling with amine 9 or 10 and afforded
the desired products in yields ranging from 61% to 88%
(Scheme 4).
2.1. 2-Aminopyridine-3-carboxamides
The 2-aminopyridine-3-carboxamides (3, 4) were obtained, with
yields ranging from 46% to 75%, by reacting the previously described
2-chloropyridine-3-carboxamides (1, 2)14 with primary and second-
ary amines in ethanol at 100 °C in a sealed tube (Scheme 1).
The free amino derivatives 3e and 4e were obtained, in 32 and
46% yield, respectively, by deprotection of p-methoxybenzylami-
nopyridines 3a and 4a (Scheme 2).
2.2. 6-Aminopyridine-2-carboxamides
3,6-Dichloropyridine-2-carboxamides 12 and 13 were synthe-
sized by coupling the amino dihydrobenzoindazoles 9 and 1014
Ar
Ar
N N
H
N N
H
O
N
N
a
O
N
N
CONH2
CONH2
R1R2N
Cl
3: Ar =3,4-(OCH2O)C6H3
1: Ar = 3,4-(OCH2O)C6H3
4: Ar = 4-F-C6H4
2: Ar = 4-F-C6H4
R1R2N
R1R2N
p-MeOC6H4CH2NH
O
N
N
N
3a, 4a
3b, 4b
3c, 4c
3d, 4d
MeHN
Scheme 1. Reagents and conditions: (a) R1R2NH (6 equiv), EtOH, 100 °C.
Ar
Ar
N N
N N
H
N
H
N
H3CO
O
N
O
N
CONH2
CONH2
a
H
N
H2N
3a: Ar = 3,4-(OCH2O)C6H3
4a: Ar = 4-F-C6H4
3e: Ar = 3,4-(OCH2O)C6H3
4e: Ar = 4-F-C6H4
Scheme 2. Reagents and conditions: (a) TFA, CH2Cl2, rt.

D. F. Bonafoux et al. / Bioorg. Med. Chem. 18 (2010) 403–414
405
Ar
N N
Ar
H
CO2H
N
O
N
N N
CONH2
Cl
H2N
a
CONH2
Cl
N
+
Cl
Cl
9: Ar = 3,4-(OCH2O)C6H3
10: Ar = 4-F-C6H4
12: Ar = 3,4-(OCH2O)C6H3
11
13: Ar = 4-F-C6H4
b
Ar
N N
H
N
O
CONH2
Cl
N
NR1R2
5a: NR1R2 = NHCH3
5b: NR1R2 = morpholino
Scheme 3. Reagents and conditions: (a) HATU, Et3N, DMF, rt; (b) R1R2NH (>20 equiv), 82 °C, 5 days.
Ar
N N
H
N
O
O
OH
CONH2
b
Cl
Cl
N
N
N
N
O
O
a
14
6b: Ar = 4-F-C6H4
O
OH
Cl
N
Cl
Ar
11
c
N N
H
O
N
CONH2
Cl
O
OH
N
d
Cl
N
N
N
N
N
5c: Ar = 3,4-(OCH2O)C6H3
6c: Ar = 4-F-C6H4
15
Scheme 4. Reagents and conditions: (a) morpholine (excess), DMA, 80 °C, 64 h; (b) 10, HBTU, Et3N, DMF, rt; (c) N-methylpiperazine (excess), DMF, 95 °C, 3 days, (d) 9 or 10,
HBTU, Et3N, DMF, rt.
2.3. 2-Aminopyridine-4-carboxamides
less than about 0.5 lM were subsequently tested in a cellular assay
using synovial fibroblasts from rheumatoid arthritis patients.
These cells were stimulated with interleukin-1b (IL-1b) and the re-
sponse to this stimulation was measured as the percentage of inhi-
bition of interleukin-8 (IL-8) production (SF IL-8 IC50).18
2,5-Dichloropyridine-4-carboxamides 17 and 18 were synthe-
sized by coupling acid 1614 with amines 9 and 10. In contrast to
the 3-chloro-6-aminopyridine-2-carboxamides, the 2-amino-5-
chloropyridine-4-carboxamides
7 and 8 could be efficiently
synthesized by direct amination of the 2,5-dichloropyridine-4-car-
boxamides 17 and 18 with satisfactory yields (Scheme 5). This
route was thus used for the synthesis of most of the inhibitors in
this series. Coupling a 2-amino-5-chloropyridine-4-carboxylic acid
to amine 9 or 10 also provided entries to analogs 8a (PHA-408), 7p
and 8p (Scheme 6).
2-Aminopyridine-4-carboxamides 23a and 23b, the des-chloro
analogs of 8a and 8p, were synthesized according to Scheme 7
by direct amination of intermediate 22.
4. Results and discussion
4.1. 2-Aminopyridine-3-carboxamides
As shown in Table 1, the replacement of the chlorine at the 2-po-
sition of the pyridine ring in 1 and 2 with a small amino group (3e,
4e) or sterically more demanding cyclic amino group (3c, 3d and
4c, 4d) led, in all cases, to a dramatic loss of potency against IKK-2
compared to prototype compounds 1 and 2. The most active 2-ami-
nopyridine-3-carboxamide synthesized in this series, 3b (IKK-2
3. Biology
IC50 = 0.210 lM, SF IL-8 IC50 = 3.37 lM), was six times less potent
on the enzyme than precursor 1, suggesting that the chloro ortho
to the carboxamide on the pyridine ring is an important structural
feature of this template to achieve good activity on IKK-2.
These inhibitors were initially evaluated for inhibition of re-
combinant human IKK-2.16,17 Compounds with an IC50 on IKK-2

406
D. F. Bonafoux et al. / Bioorg. Med. Chem. 18 (2010) 403–414
Ar
N N
H
N
Ar
O
CONH2
CO2H
N N
H2N
Cl
Cl
CONH2
a
+
N
N
Cl
Cl
9: Ar = 3,4-(OCH2O)C6H3
10: Ar = 4-F-C6H4
16
17: Ar = 3,4-(OCH2O)C6H3
18: Ar = 4-F-C6H4
b
Ar
N N
H
N
O
CONH2
Cl
NR1R2
N
7: Ar = 3,4-(OCH2O)C6H3
8: Ar = 4-F-C6H4
NR1R2
NR1R2
NR1R2
7a
8g
N
NH
8l
N
N
N
N
N
7b, 8b
NH
N
N
N
8m
8n
N
8h
8i
NH
8c
N
N
N
N
Et
NMe(CH2)2NMe2
NH(CH2)2NMe2
NH
8d
8e
8f
8o
8j
N
N
N
N
N
N
N
(CH2)2OMe
NH
8k
Scheme 5. Reagents and conditions: (a) HATU, Et3N, DMF, rt; (b) R1R2NH (excess), 100 °C, 24 h.
4.2. 6-Aminopyridine-2-carboxamides
4.3. 2-Amino-5-chloro-pyridine-4-carboxamides
Having shown that the ortho chloro substituent in the proto-
type carboxamide 1 was key for potency, we decided to focus on
related pyridine systems where the chloro is maintained ortho to
the carboxyamide moiety. We first studied 3-chloro-6-aminopyri-
dine-2-carboxamides (Table 2), such as 5 and 6, which were gen-
erally more potent IKK-2 inhibitors than the 2-aminopyridine-3-
carboxamides (Table 1), especially when the amino substituents
were morpholine and N-methylpiperazine. The most potent inhib-
itors in this series, 5c and 6c, exhibited modest activity on the en-
zyme (about 10 times less potent than 1 and 2) and were found to
have modest activity in the SF IL-8 assay. In spite of enhancement
of enzyme activity in some analogs, these compounds did not ap-
proach the activity of the prototype compounds 1 and 2 and their
cellular potency did not show any differentiation. We hypothe-
sized that the pyridyl nitrogen ortho to the amide might have
formed an internal hydrogen-bond with the amide NH, and unfa-
vorably flipped the trajectory of the substituents on the pyridine
ring.
To prevent the hypothesized internal hydrogen-bond forma-
tion, we moved the pyridine nitrogen further away from the amide
moiety with the 2-amino-5-chloro-pyridine-4-carboxamides
which were, in most cases, very potent IKK-2 inhibitors compara-
ble to prototype compounds 1 and 2 with IC50’s ranging from
0.010 lM (8b) to 0.146 lM (8p) (Table 3). Moreover many showed
better potency in the cell assay (Table 3), in particular compounds
7a, 8a (PHA-408), 8e, and 7p featuring N-substituted piperazines
or an unsubstituted morpholine, with SF IL-8 IC50 values of about
1 lM or less. Further studies to understand the nature of this
cell-potency improvement will be reported in due time.
Most of the current series, with an ortho chloro, had IKK-2 IC50
values below 0.100 lM. As a means to confirm the importance of
that ortho chloro, the des-chloro analogs of 8a (PHA-408) and 8p
(23a and 23b, respectively) were tested in our in vitro assays.
The IKK-2 IC50 values for 23a and 23b were 0.125
0.589 M, respectively, thus 4–5-fold lower than those of the cor-
responding chloro containing inhibitors 8a (PHA-408) and 8p, and
lM and
l

D. F. Bonafoux et al. / Bioorg. Med. Chem. 18 (2010) 403–414
407
Ar
N
N N
H
N
O
CONH2
Cl
O
OH
b
Cl
N
N
N
N
N
8a (PHA-408) : Ar = 4-F-C6H4
a
19
O
OH
Cl
Cl
N
Ar
16
N N
H
c
O
N
O
OH
CONH2
Cl
Cl
d
N
N
20
N
N
O
O
7p: Ar = 3,4-(OCH2O)C6H3
8p: Ar = 4-F-C6H4
Scheme 6. Reagents and conditions: (a) N-methylpiperazine (excess), DMA, 100 °C, 8 days; (b) 10, HBTU, Et3N, DMF, rt; (c) morpholine (excess), DMA, 80 °C, 4 days; (d) 9 or
10, HBTU, Et3N, DMF, rt.
F
F
N
N
N
H
N
N
H
N
O
CONH2
O
CONH2
CO2H
b
a
10
+
NR1R2
NR1R2
N
Cl
N
N
Cl
23
21
22
23a
N
N
N
23b
O
Scheme 7. Reagents and conditions: (a) HATU, Et3N, DMF, rt; (b) R1R2NH (excess), 100 °C.
Table 1
Table 2
Inhibition of IKK-2 by 2-aminopyridine-3-carboxamides
Inhibition of IKK-2 by 3-chloro-6-aminopyridine-2-carboxamides
Ar
Ar
N
N
N
N
H
H
O
N
O
N
N
CONH2
CONH2
5: Ar = 3,4-(OCH2O)C6H3
6: Ar = 4-F-C6H4
Cl
X
N
NR1R2
X, Ar = 3,4-
(OCH2O)C6H3
IC50
(
l
M)
X, Ar = 4-F–C6H4 IC50 (lM)
R1R2N
MeNH
Morpholino
Morpholino
N-Methylpiperazinyl
N-Methylpiperazinyl
IKK-2 IC50
(
lM)
SF IL-8 IC50 (lM)
5a
5b
6b
5c
6c
0.477
0.612
1.25
0.297
0.348
5.74
>100
3.04
4.42
4.83
1
3e
Cl
NH2
0.034
1.34
0.210
5.91
2
4e
Cl
NH2
4b NHMe
0.047
2.32
3.21
3b NHMe
3c Morpholino
3d N-Methylpiperazinyl 17.3
4c
Morpholino
4d N-Methylpiper-
14.2
16.2
azinyl
5. Conclusion
showed a lack of activity in cells with an SF IL-8 IC50 greater than
M. It is worth noting that compound 8a (PHA-408), which
selectivity profile against 36 kinases was previously reported,12
5
l
In summary, we showed that the 2-amino-5-chloropyridine-4-
carboxamides were potent IKK-2 inhibitors with improved inhibi-
tion of IL-8 production in stimulated synovial fibroblasts for sev-
eral compounds compared to our earlier prototypes 1 and 2. The
did not inhibit IKK-1 (IKK-1 IC50 = 14
on closely related TBK1 or iKKi.
lM), nor did it show activity

408
D. F. Bonafoux et al. / Bioorg. Med. Chem. 18 (2010) 403–414
Table 3
Inc./Sigma–Aldrich) in 900 mM sodium format buffer, pH 3 (the re-
sin is in a slurry of 1 volume resin to 2 volumes of sodium formate
buffer) was added to each well to stop the reaction. The resin was
Inhibition of IKK-2 by 2-amino-5-chloro-pyridine-4-carboxamides
Ar
N
N
H
N
mixed and allowed to settle for one hour and 50
was transferred to a top count plate followed by the addition of
150 L of scintillation fluid (Microscint 40) (Packard) to each well.
Incorporation of [
33P] ATP was measured using a Top-Count NXT
lL of supernatant
O
CONH2
Cl
l
7: Ar = 3,4-(O2CH2)C6H3
8: Ar = 4-F-C6H4
c-
N
X
(Packard Instrument Co.).
X
IKK-2 IC50
M)
SF IL-8 IC50 (lM)
(
l
6.1.2. Cell assay
Cytokine production and toxicity determination in IL-1b in-
duced synovial fibroblast cells have been described previously.15
In brief; synovial fibroblast cells were plated in 96-well plates at
1.5 ꢀ 104 cells/well in DMEM (Invitrogen) containing 15% fetal bo-
vine serum (Hyclone) and allowed to attach overnight. The growth
media were replaced with DMEM containing 1% fetal bovine ser-
um, and the cells were pre-treated with increasing concentrations
of compound or media in (0.2% Me2SO final) for 1 h prior to an 18 h
stimulation with 1 ng/mL IL-1b R&D Systems). Supernatants were
collected and analyzed for secreted IL-8 by ELISA. IL-8 concentra-
tion was extrapolated from standard curves using a 4 parameter
logistic model by Softmax Pro 3.1.2(2) (Molecular Devices). Cyto-
toxicity was assessed using an Alamar Blue assay (Promega) and
was below 20% at the highest concentration of inhibitors tested.
1
2
H, Ar = 3,4-(OCH2O)C6H4
H, Ar = 4-F-C6H4
N-Methylpiperazinyl
N-Methylpiperazinyl
Piperazinyl
Piperazinyl
N-Ethylpiperazinyl
N-iso-Propylpiperazinyl
0.034
0.047
0.033
0.025
0.040
0.010
0.052
0.107
0.059
0.098
5
12
7a
8a
7b
8b
8c
8d
8e
8f
0.63
0.94
1.86
2.47
1.53
1.49
1.02
5.5
N-(Methoxyethyl)piperazinyl
3,5-Dimethyl-N-
methylpiperazinyl
8g
8h
3,5-Dimethylpiperazinyl
(R)-3-Methyl-N-
methylpiperazinyl
0.046
0.048
8.6
2.18
8i
8j
(R)-3-Methylpiperazinyl
(S)-3-Methyl-N-
0.054
0.045
3.98
1.31
methylpiperazinyl
6.2. General procedure for the synthesis of 2-aminopyridine-3-
carboxamides
8k
8l
(S)-3-Methylpiperazinyl
N-Methylhomopiperazinyl
0.051
0.029
0.027
1.32
2.36
1.94
4.18
8m Homopiperazinyl
8n
N,N,N0-Trimethylethylenediamine 0.019
6.2.1. 1-(1,3-Benzodioxol-5-yl)-8-[({2-[(4-methoxybenzyl)amino]-
pyridin-3-yl}carbonyl-)amino]-4,5-dihydro-1H-benzo[g]indazole-
3-carboxamide (3a)
8o
7p
8p
N,N-Dimethylethylenediamine
Morpholino
Morpholino
0.070
0.090
0.146
4.72
0.849
2.14
A mixture of 1 (2 g, 4 mmol) and p-methoxybenzylamine (2.8 g,
20 mmol) in 10 mL EtOH was stirred at 100 °C for 48 h in a sealed
tube. The off-white precipitate that formed in the crude reaction
mixture upon cooling down to room temperature was filtered,
washed with EtOH and Et2O and dried under vacuum. Yield 60%.
1H NMR (DMSO-d6, 300 MHz): d 2.88–2.94 (m, 4H), 3.71 (s, 3H),
4.56 (d, 2 H, J = 5.6 Hz), 5.98 (s, 2H), 6.60–6.64 (dd, 1H, J = 7.5 Hz,
4.7 Hz), 6.88 (d, 2H, J = 8.4 Hz), 6.96 (s, 2H), 7.11 (s, 1H), 7.23–7.35
(m, 5 H), 7.39 (s, 1H), 7.49 (s, 1H), 7.95 (d, 1H, J = 7.45 Hz), 8.17 (d,
1H, J = 4.7 Hz), 8.23 (t, 1H, J = 5.6 Hz), 10.06 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz): d 20.3, 29.6, 43.9, 55.7, 102.5, 108.1, 109.0,
110.8, 111.4, 114.4, 115.8, 120.5, 120.7, 120.8, 126.6, 129.2, 129.3,
132.7, 133.1, 134.4, 137.7, 139.8, 142.6, 148.4, 148.6, 152.0, 157.8,
158.8, 164.8, 167.1; Anal. Calcd for C33H28N6O5: C, 67.34; H, 4.79;
N, 14.28. Found: C, 67.08; H, 4.78; N, 14.19.
chloro ortho to the carboxamide, as well as the piperazinyl groups
at the 2-position of the pyridine ring, were identified as key struc-
tural features in this series to achieve good selectivity, good cellu-
lar activity and good in vivo efficacy as previously reported for 8a
(PHA-408).12
6. Experimental section
6.1. General methods and material
Column chromatography was performed using 200–400 mesh
silica gel. 1H NMR and 13C NMR were obtained on Varian Inova-
300 and Varian Inova-400 spectrometers using CDCl3, DMSO-d6,
D2O or MeOH-d4 as internal standards. Mass spectra (MS) were re-
corded with a Micromass ZMD spectrometer. High resolution mass
spectra were recorded using a Perseptive Biosystems Mariner TOF
mass spectrometer. Elemental analysis were performed by Atlantic
MicroLab, GA. The (2R,6S)-1,2,6-trimethylpiperazine, (2R)-1,2-
dimethylpiperazine and (2S)-1,2-dimethylpiperazine were synthe-
sized according to a procedure described elsewhere.9
6.2.2. 1-(4-Fluorophenyl)-8-[({2-[(4-methoxybenzyl)-amino]py-
ridin-3-yl}carbonyl)amino]-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (4a)
Same procedure as 3a. Yield 35%. 1H NMR (DMSO-d6, 300 MHz):
d 2.89–2.95 (m, 4H), 3.72 (s, 3H), 4.53 (d, 2H, J = 5.5 Hz), 6.61 (dd,
1H, J = 7.7 Hz, 4.8 Hz), 6.9 (d, 2H, J = 8.6 Hz), 7.24–7.36 (m, 8H),
7.55–7.60 (m, 3H), 7.94 (dd, 1H, J = 7.7 Hz, 1.7 Hz), 8.17 (dd, 1H,
J = 4.8 Hz, 1.7 Hz), 8.21 (t, 1H, J = 5.5 Hz), 10.0 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz): d 20.3, 29.6, 44.0, 55.7, 110.7, 111.4, 114.4,
115.8, 117.1 (d, JC–F = 23.0 Hz), 120.9, 121.1, 126.4, 128.8 (d,
JC–F = 8.7 Hz), 129.2, 129.3, 132.6, 133.1, 136.9 (d, JC–F = 2.8 Hz),
137.6, 137.7, 139.8, 143.1, 152.0, 157.8, 158.8, 162.6 (d,
JC–F = 245.7 Hz), 164.4, 167.0; MS m/z (M+1+) 563.
6.1.1. IKK-2. enzyme assay
Kinase activity was measured using a biotinylated I
jBa peptide
(Gly-Leu-Lys-Lys-Glu-Arg-Leu-Leu-Asp-Asp-Arg-His-Asp-Ser32
-
Gly-Leu-Asp-Ser36-Met-Lys-Asp-Glu-Glu) (American Peptide Co.).
Twenty microliters of the standard reaction mixture contained
5
l
M
biotinylated
I
j
B
a
peptide, 0.1
l
Ci/reaction
[c-
33P] ATP
(Amersham) (about 1 ꢀ 105 cpm), 1
lM ATP (Sigma), 1 mM DTT
(Sigma), 2 mM MgCl2 (Sigma), 2 mM MnCl2 (Sigma), 10 mM NaF
6.2.3. 1-(1,3-Benzodioxol-5-yl)-8-({[2-(methylamino)pyridin-3-
yl]carbonyl}amino)-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (3b)
(Sigma), 25 mM Hepes (Sigma) buffer, pH. 7.6 and 20
solution (46 nM/rxn or 200 ng/rxn) and 10 L inhibitor in a final
volume of 50 L. After incubation at 25 °C for 30 min, the reaction
was stopped by the addition of 150 L of AG1XB resin (Supelco
lL enzyme
l
Same procedure as 3a. Yield 75%. 1H NMR (DMSO-d6, 300 MHz):
d 2.88–2.95 (m, 4H + 3H), 6.12 (s, 2H), 6.57–6.61 (dd, 1H, J = 7.6 Hz,
l
l

D. F. Bonafoux et al. / Bioorg. Med. Chem. 18 (2010) 403–414
409
4.7 Hz), 6.95–6.98 (dd, 1H, J = 8 Hz, 2 Hz), 7.07 (d, 1H, J = 8 Hz),
7.13 (d, 1H, J = 2 Hz), 7.25–7.34 (m, 3H), 7.45–7.50 (m, 2H), 7.82–
7.83 (m, 1H), 7.91–7.95 (dd, 1H, J = 7.6 Hz, 1.7 Hz), 8.18–8.20 (dd,
1H, J = 4.8 Hz, 1.8 Hz), 10.02 (s, 1H).%); 13C NMR (DMSO-d6,
75 MHz): d 20.3, 28.3, 29.6, 102.6, 108.2, 109.2, 110.9, 111.0,
115.8, 120.6, 126.5, 129.2, 132.9, 134.5, 137.4, 137.8, 139.9,
142.7, 148.5, 148.6, 152.1, 158.6, 164.6, 167.0; HRMS calcd for
C26H23N6O4 483.1775, found 483.1787.
6.2.8. 1-(4-Fluorophenyl)-8-({[2-(4-methylpiperazin-1-yl)pyridin-
3-yl]carbonyl}amino)-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (4d)
Same procedure as 3a. The crude mixture was cooled down to
room temperature and partitioned between CHCl3 and H2O. The or-
ganic layer was dried over MgSO4, the solvents removed under vac-
uum and the resulting solid triturated with Et2O. Yield 49%. 1H
NMR (DMSO-d6, 300 MHz): d 2.1 (s, 3H), 2.23 (t, 4H, J = 4.6 Hz),
2.43–2.45 (m, 4H), 3.15 (t, 3H, J = 4.6 Hz), 6.85 (dd, 1H,
J = 7.38 Hz, 4.83 Hz), 7.24–7.39 (m, 6H), 7.50–7.60 (m, 4H), 8.19
(dd, 1H, J = 4.83 Hz, 1.88 Hz), 10.2 (s, 1H); 13C NMR (DMSO-d6,
75 MHz): d 20.4, 29.7, 46.4, 49.0, 55.1, 114.6, 115.8, 117.2 (d,
JC–F = 23.1 Hz), 119.8, 121.3, 121.4, 126.6, 128.7 (d, JC–F = 9.1 Hz),
129.6, 132.9, 136.9 (d, JC–F = 2.8 Hz), 138.0, 139.0, 139.8, 143.2,
149.7, 158.2, 162.6 (d, JC–F = 245.9 Hz), 164.5, 166.4; Anal. Calcd
for C30H29N7O4: C, 66.27; H, 5.37; N, 18.66. Found: C, 66.30; H,
5.36; N, 18.74.
6.2.4. 1-(4-Fluorophenyl)-8-({[2-(methylamino)pyridin-3-yl]-
carbonyl}amino)-4,5-dihydro-1H-benzo[g]indazole-3-carboxa-
mide (4b)
Same procedure as 3a at 98 °C for 48 h. After cooling down to
room temperature, the crude suspension was dissolved in EtOAc
and washed twice with H2O. The organic layer was dried over MgSO4
and the solvent removed under vacuum. The resulting yellow solid
was triturated with Et2O. Yield 61%. 1H NMR (DMSO-d6, 300 MHz):
d 2.89–2.95 (m, 4H+3H), 6.57 (dd, 1H, J = 7.7 Hz, 4.8 Hz), 7.28–7.40
(m, 4H), 7.43 (t, 2H, J = 7.7 Hz), 7.55–7.62 (m, 3H), 7.7–7.78 (m,
1H), 7.90 (dd, 1H, J = 7.7 Hz, 1.8 Hz), 8.18 (dd, 1H, J = 4.8 Hz,
1.8 Hz), 10.01 (s, 1H); 13C NMR (DMSO-d6, 75 MHz): d 20.4, 28.3,
29.6, 110.9, 111.1, 115.8, 117.2 (d, JC–F = 23.4 Hz), 120.8, 121.1,
126.4, 129.0 (d, JC–F = 8.9 Hz), 129.3, 133.0, 136.9, 137.2 (d,
JC–F = 3.6 Hz), 137.8, 139.9, 143.1, 152.1, 158.6, 162.8 (d,
JC–F = 245.5 Hz), 164.5, 167.0; HRMS calcd for C25H22FN6O2
457.1783, found 457.1752.
6.2.9. 8-{[(2-Aminopyridin-3-yl)carbonyl]amino}-1-(1,3-benzo-
dioxol-5-yl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide
(3e)
Compound 3a (1.96 g, 3.3 mmol) was dissolved in 6 mL CH2Cl2
and reacted with 5 mL TFA at room temperature for 36 h. The crude
reaction mixture was diluted with CH2Cl2 and basified with a sat-
urated aqueous solution of Na2CO3. The layers were separated and
the organic layer was dried over MgSO4. The solvent was removed
under vacuum and the resulting residue was triturated with EtOH.
Yield 32%. 1H NMR (DMSO-d6, 300 MHz): d 2.88–2.95 (m, 4H), 6.12
(s, 2H), 5.58–6.62 (dd, 1H, J = 7.6 Hz, 4.7 Hz), 6.97–7.08 (m, 4H),
7.17 (d, 1H, J = 1.9 Hz), 7.28 (s, 1H), 7.32 (s, 2H), 7.55 (s, 2H),
7.95–7.98 (dd, 1H, J = 7.7 Hz, 1.7 Hz), 8.11–8.13 (dd, 1H,
J = 4.7 Hz, 1.7 Hz), 9.99 (s, 1H); 13C NMR (DMSO-d6, 75 MHz): d
20.4, 29.6, 102.6, 108.2, 109.1, 110.5, 111.9, 115.8, 120.6, 120.7,
126.5, 129.2, 132.9, 134.5, 137.6, 137.9, 139.9, 142.6, 148.5,
148.7, 152.3, 159.6, 164.6, 166.9; HRMS calcd for C25H21N6O4
469.1619, found 496.1644.
6.2.5. 1-(1,3-Benzodioxol-5-yl)-8-{[(2-morpholin-4-ylpyridin-
3-yl)carbonyl]amino}-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (3c)
Same procedure as 3a. Yield 64%. 1H NMR (DMSO-d6, 300 MHz):
d 2.86–2.92 (m, 4H), 3.17 (t, 4H, J = 4.3 Hz), 3.53 (t, 4H, J = 4.29 Hz),
6.10 (s, 2H), 6.1 (s, 2H), 6.9–7.01 (m, 3H), 7.11 (d, 1H, J = 1.88 Hz),
7.26–7.31 (m, 3H), 7.50–7.52 (m, 2H), 7.68 (dd, 1H, J = 7.5 Hz,
1.88 Hz), 8.25 (dd, 1H, J = 4.7 Hz, 1.88 Hz), 10.28 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz): d 20.4, 29.7, 49.7, 66.6, 102.7, 107.9, 109.0,
114.6, 116.2, 119.6, 120.4, 121.0, 121.5, 126.8, 129.5, 132.9, 134.4,
137.9, 139.2, 139.7, 142.7, 148.4, 148.6, 149.5, 158.1, 164.5, 166.4;
HRMS calcd for C29H27N6O5 539.2037, found 539.2009.
6.2.10. 8-{[(2-Aminopyridin-3-yl)carbonyl]amino}-1-(4-
fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (4e)
6.2.6. 1-(4-Fluorophenyl)-8-{[(2-morpholin-4-ylpyridin-3-
yl)carbonyl]amino}-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (4c)
Same procedure as 3e yield 46%. 1H NMR (DMSO-d6, 400 MHz):
d 2.85–2.91 (m, 4H), 6.57 (dd, 1H, J = 7.7 Hz, 4.8 Hz), 6.92 (s, br,
2H), 7.24–7.38 (m, 6H), 7.51–7.57 (m, 3H), 7.87 (dd, 1H),
J = 7.7 Hz, 1.4 Hz), 8.04 (dd, 1HH, J = 3.4 Hz, 1.4 Hz), 9.95 (s, 1H);
13C NMR (DMSO-d6, 100 MHz): d 20.3, 29.6, 110.7, 111.9, 115.7,
117.2 (d, JC–F = 23.0 Hz), 120.8, 121.2, 126.4, 128.8 (d, JC–F = 8.9 Hz),
129.3, 133.0, 136.9 (d, JC–F = 2.8 Hz), 137.8, 138.0, 139.8, 143.1,
151.6, 159.1, 162.6 (d, JC–F = 245.7 Hz), 164.5, 166.7; HRMS calcd
for C24H20FN6O2 443.1626, found 443.1618.
Same procedure as 3a. Yield 52%. 1H NMR (DMSO-d6, 300 MHz):
d 2.89–2.94 (m, 4H), 3.17 (t, 4H, J = 4.4 Hz), 3.54 (t, 4H, J = 4.4 Hz),
6.92 (dd, 1H, J = 7.4 Hz, 4.8 Hz), 7.24–7.41 (m, 5H), 7.54–7.62 (m,
4H), 7.65 (dd, 1H, J = 7.4 Hz, 1.9 Hz), 8.26 (dd, 1H, J = 4.7 Hz,
1.9 Hz), 10.26 (s, 1H); 13C NMR (DMSO-d6, 75 MHz): d 20.4, 29.7,
49.7, 66.6, 114.5, 116.1, 117.2 (d, JC–F = 22.8 Hz), 119.7, 121.4,
121.5, 126.7, 128.7 (d, JC–F = 9.1 Hz), 129.7, 133.0, 136.9 (d,
JC–F = 2.9 Hz), 138.0, 139.2, 139.8, 143.2, 149.5, 158.0, 162.6 (d,
JC–F = 245.6 Hz), 164.5, 166.5; Anal. Calcd for C28H25N6O5: C, 65.6;
H, 4.9; N, 16.4. Found: C, 65.61; H, 4.92; N, 16.58.
6.3. Synthesis of 3,6-dichloropyridine-2-carboxamides
6.3.1. 1-(1,3-Benzodioxol-5-yl)-8-{[(3,6-dichloropyridin-2-yl)car-
bonyl]amino}-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide
(12)
6.2.7. 1-(1,3-Benzodioxol-5-yl)-8-({[2-(4-methylpiperazin-1-
yl)pyridin-3-yl]carbonyl}-amino)-4,5-dihydro-1H-benzo[g]-
indazole-3-carboxamide (3d)
3,6-Dichloro-2-pyridine carboxylic acid (11) (1.654 g,
8.14 mmol), HATU (3.27 g, 8.6 mmol) and finally Et3N (1.68 g,
2.32 mL, 16.6 mmol) were added to a solution of 9 (2 g, 5.74 mol)
in 29 mL of DMF. The reaction mixture was stirred at room temper-
ature for 3 h. The crude reaction mixture was concentrated to
approximately 1/3 of the initial volume. Upon addition of water to
this DMF residue, a solid was formed. This solid was triturated with
water, filtered, and dried under reduced pressure. Yield 80%. 1H NMR
(DMSO-d6, 300 MHz): d 2.82–3.01 (m, 4H), 6.11 (s, 2H), 6.97–7.07
(m, 2H), 7.38 (d, 1H, J = 1 Hz), 7.81 (s, 1H), 7.33–7.42 (m, 3H), 7.52
(s, 1H), 7.51 (d, 1H, J = 9 Hz), 8.15 (d, 1H, J = 9 Hz), 10.57 (s, 1H);
Same procedure as 3a. Yield 65%. 1H NMR (DMSO-d6, 300 MHz):
d 2.13 (s, 3H), 2.28 (t, 4H, br), 2.88–2.94 (m, 4H), 3.20 (t, 4H, br),
6.11 (s, 2H), 6.87–7.03 (m, 3H), 7.11 (d, 1H, J = 1.81 Hz), 7.24–
7.49 (m, 5H), 7.66 (dd, 1H, J = 7.45 Hz, 1.81 Hz), 8.25 (dd, 1H,
J = 4.83 Hz, 1.81 Hz), 10.26 (s, 1H); 13C NMR (DMSO-d6, 75 MHz):
d 20.4, 29.7, 46.4, 49.1, 55.1, 102.6, 107.9, 109.1, 114.7, 115.9,
119.7, 120.4, 120.9, 121.4, 126.8, 129.5, 132.8, 134.4, 137.9,
139.1, 139.7, 142.7, 148.5, 148.7, 149.5, 158.3, 164.6, 166.3; HRMS
calcd for C30H30N7O4 552.2354, found 552.2365.

410
D. F. Bonafoux et al. / Bioorg. Med. Chem. 18 (2010) 403–414
13C NMR (DMSO-d6, 300 MHz): d 20.4, 29.7, 102.6, 107.9, 109.2,
115.2, 120.1, 120.5, 121.0, 126.9, 127.5, 128.1, 129.7, 133.7, 134.3,
137.1, 139.6, 142.4, 142.7, 148.4, 148.5, 148.7, 152.4, 162.3, 164.6;
HRMS calcd for C25H18Cl2N5O4 522.0730, found 522.0709.
6.5.2. Potassium 3-chloro-6-(4-methylpiperazin-1-yl)pyridine-
2-carboxylate (15)
A
mixture of 3,6-dichloro-2-pyridine carboxylic acid (11)
(0.60 g, 3.125 mmol) and N-methylpiperazine (7.2 g, 7.98 mL,
72 mmol) in 1 mL DMA was stirred at 95 °C for 3 days. The volatiles
were removed under vacuum. The resulting residue was washed
with a saturated solution of K2CO3 and with CH2Cl2. Three layers
were formed. The middle layer was separated and the solvent re-
moved under reduced pressure. The resulting solid was dried un-
der reduced pressure in the presence of P4O10. Yield 96%. 1H
NMR (D2O, 300 MHz): d 2.18 (s, 3H), 2.43 (s, br, 4H), 3.37 (s, br,
4H), 6.77 (d, 1H, J = 9 Hz), 7.58 (d, 1H, J = 9 Hz); MS m/z (M+1+) 256.
6.3.2. 8-{[(3,6-Dichloropyridin-2-yl)carbonyl]amino}-1-(4-fluo-
rophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (13)
Same procedure as 12, replacing HATU with HBTU. Yield 89%.
1H NMR (DMSO-d6, 300 MHz): d 2.82–3.02 (m, 4H), 7.24–7.43
(m, 6H), 7.51–7.63 (m, 3H), 7.71 (d, 1H, J = 9 Hz), 10.52 (s, 1H);
13C NMR (DMSO-d6, 300 MHz): d 20.5, 29.2, 115.0, 117.3 (d,
JC–F = 22.9 Hz), 120.1, 121.5, 126.8, 127.5, 128.0, 128.8 (d,
JC–F = 8.9 Hz), 129.8, 133.8, 136.8 (d, JC–F = 3.3 Hz), 137.2, 139.7,
142.4, 143.2, 148.4, 152.5, 162.3, 162.7 (d, JC–F = 243.4 Hz), 164.5;
HRMS calcd for C24H17Cl2FN5O2 496.0738, found 496.0701.
6.5.3. 8-{[(3-Chloro-6-morpholin-4-ylpyridin-2-yl)carbonyl]-
amino}-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-
3-carboxamide (6b)
3-Chloro-6-morpholinyl-2-pyridine carboxylic acid (14)
(0.294 g, 1.21 mmol), HBTU 0.385 g (1.2 mmol), and finally Et3N
(0.4 mL, 2.9 mmol) were added to a solution of 10 (0.258 g,
0.8 mmol) in 4 mL of DMF. The reaction mixture was stirred at
room temperature for 3 h. The crude reaction mixture was concen-
trated to approximately 1/3 of the initial volume of the reaction
mixture. Upon addition of water to this DMF residue, a solid was
formed. This solid was triturated with water, filtered, and dried un-
der reduced pressure. Yield 84%. 1H NMR (CDCl3, 300 MHz): d 2.89–
3.01 (m, 2H), 3.06–3.18 (m, 2H), 3.43–3.54 (m, 4H), 3.82–3.94 (m,
4H), 5.39 (s, 1H), 6.65–6.82 (m, 3H), 7.18–7.23 (m, 3H), 7.43–7.56
(m, 3H), 7.62 (m, 1H), 9.18 (s, 1H); 13C NMR (DMSO-d6, 300 MHz):
d 20.1, 29.8, 45.6, 66.6, 111.5, 114.4, 116.7 (JC–F = 23.3 Hz), 119.7,
122.4, 126.5, 128.1 (JC–F = 9.2 Hz), 129.6, 133.7, 136.2, 136.5
(JC–F = 1.9 Hz), 142.4, 142.7, 156.0, 158.0, 161.4, 161.5, 162.5,
162.6 (JC–F = 246.2 Hz), 164.7; HRMS calcd for C28H25ClFN6O3
547.1655, found 547.1574.
6.4. Synthesis of 6-amino-3-chloropyridine-2-carboxamides by
direct amination
6.4.1. 1-(1,3-Benzodioxol-5-yl)-8-({[3-chloro-6-(methylamino)-
pyridin-2-yl]-carbonyl}-amino)-4,5-dihydro-1H-benzo[g]-
indazole-3-carboxamide (5a)
A mixture of 12 (1.042 g, 2.0 mmol) and N-methylamine (6 mL
of 33 w% solution in EtOH) in 1 mL DMF was stirred for 5 days at
82 °C in a sealed tube. The volatiles were removed under reduced
pressure and the resulting residue was purified by reverse phase
preparative HPLC. Yield 28%. 1H NMR (DMSO-d6, 300 MHz,): d
2.76 (d, 3H, J = 5.5 Hz), 2.84–3.99 (m, 4H), 6.16 (s, 2H), 6.57 (d,
1H, J = 9 Hz), 6.91–7.04 (m, 3H), 7.12 (d, 1H, J = 1 Hz), 7.22–7.34
(m, 3H), 7.46–7.54 (m, 2H), 10.19 (s, 1H); 13C NMR (DMSO-d6,
400 MHz): d 20.4, 28.5, 29.7, 102.6, 108.0, 109.1, 111.8, 114.8,
114.9, 119.7, 120.5, 120.9, 126.8, 129.4, 129.5, 133.0, 134.4,
137.6, 139.4, 139.7, 142.7, 148.5, 148.6, 157.8, 163.9, 164.6; HRMS
calcd for C26H22ClN6O4 517.1386, found 517.1397.
6.5.4. 1-(1,3-Benzodioxol-5-yl)-8-({[3-chloro-6-(4-methylpipera-
zin-1-yl)pyridin-2-yl]-carbonyl}amino)-4,5-dihydro-1H-
benzo[g]indazole-3-carboxamide (5c)
6.4.2. 1-(1,3-Benzodioxol-5-yl)-8-{[(3-chloro-6-morpholin-4-ylp-
yridin-2-yl)carbonyl]-amino}-4,5-dihydro-1H-benzo[g]ind-
azole-3-carboxamide (5b)
Compound 5c was obtained by the same procedure as 6b after
acidification of 15. Yield 61% yield. 1H NMR (DMSO-d6, 400 MHz): d
2.70 (s, 3H), 2.72–4.30 (m, 12H), 6.02 (s, 2H), 6.92–7.12 (m, 4H),
7.22–7.36 (m, 3H), 7.42–7.53 (m, 2H), 7.77 (d, 1H, J = 9 Hz), 10.32
(s, 1H); 13C NMR (DMSO-d6, 400 MHz): d 20.4, 29.5, 43.5, 43.9,
53.3, 103.2, 108.6, 109.7, 111.8, 115.8, 118.0, 120.7, 121.2, 121.7,
127.6, 130.3, 134.0, 135.2, 138.4, 140.5, 141.4, 143.6, 149.4,
149.6, 149.8, 157.4, 164.7, 165.7; HRMS calcd for C30H29ClN7O4
586.1964, found 586.1984.
Same procedure as 5a. Yield 52%. 1H NMR (CDCl3, 300 MHz) d
2.91–3.00 (m, 2H), 3.11–3.20 (m, 2H), 3.44–3.53 (m, 4H), 3.82–
3.91 (m, 4H), 5.41 (s, 1H), 6.74–6.86 (m, 2H), 6.90–7.04 (m, 2H),
7.16 (d, 1H, J = 1 Hz), 7.28–7.35 (m, 2H), 7.54–7.22 (m, 2H), 9.32
(s, 1H); 13C NMR (CDCl3, 300 MHz): d 20.2, 29.8, 45.7, 66.6, 102.4,
107.7, 108.5, 111.4, 114.5, 119.3, 120.2, 121.2, 121.8, 126.8,
129.4, 133.6, 134.3, 136.2, 139.9, 141.7, 142.3, 148.7, 148.8,
156.1, 161.2, 164.7; HRMS calcd for C29H26ClN6O5 573.1648, found
573.1646.
6.5.5. 8-({[3-Chloro-6-(4-methylpiperazin-1-yl)pyridin-2-yl]car-
bonyl}amino)-1-(4-fluoro-phenyl)-4,5-dihydro-1H-benzo[g]-
indazole-3-carboxamide (6c)
6.5. Synthesis of 6-amino-3-chloropyridine-2-carboxamides by
coupling
Same procedure as 5c. Yield 88%. 1H NMR (400 MHz, DMSO-d6):
d 2.19 (s, 3H), 2.31–2.39 (m, 4H), 2.84–2.88 (m, 4H), 3.41–3.51 (m,
4H), 6.94 (d, 1H, J = 9 Hz), 7.20 (d, 1H, J = 2 Hz), 7.25–7.40 (m, 4H),
7.48–7.60 (m, 4H), 7.62 (d, 1H, J = 9 Hz), 10.20 (s, 1H); 13C NMR
(400 MHz, DMSO-d6): d 20.2, 29.9, 42.8, 43.4, 44.4, 52.9,110.6,
115.0, 116.1, 117.2 (JC–F = 23.5 Hz), 120.0, 121.4, 126.6, 128.7
(JC–F = 8.8 Hz), 129.6, 133.2, 136.8 (JC–F = 2.8 Hz), 136.9, 137.6,
139.9, 140.1, 143.2, 148.9, 157.1,162.6 (JC–F = 244.3 Hz), 163.9,
164.5; HRMS calcd for C29H27ClFN7O2 560.1972, found 560.1960.
6.5.1. 3-Chloro-6-morpholinyl-2-pyridine carboxylic acid
hydrochloride (14)
A
mixture of 3,6-dichloropyridine-2-carboxylic acid (11)
(0.55 g, 2.86 mmol) and morpholine (1.37 g, 1.37 mL, 15.7 mmol)
in 1.37 mL of N,N-dimethylacetamide (DMA) was stirred for 24 h
at 80 °C. An additional volume of morpholine (1.36 g, 1.36 mL,
15.7 mmol) was added and the reaction mixture stirred at
80 °C for an additional 40 h. After cooling to room temperature,
the volatiles were removed under vacuum in the presence of tol-
uene. The residue was dissolved in water and washed with ether
to remove the excess morpholine. The aqueous layer was acidi-
fied to pH 2 and the product extracted with ether. Crystallization
from water afforded a white solid. Yield 53%. MS m/z (M+1+)
243.
6.6. Synthesis of 2,5-dichloropyridine-4-carboxamides
6.6.1. 1-(1,3-Benzodioxol-5-yl)-8-[(2,5-dichloroisonicoti-noyl)-
amino]-4,5-dihydro-1H-benzo-[g]indazole-3-carboxamide (17)
2,5-Dichloroisonicotinic acid (16) (1.65 g, 8.6 mmol), HATU
(3.27 g, 8.6 mmol) and finally Et3N (2.32 mL, 16.6 mmol) were

D. F. Bonafoux et al. / Bioorg. Med. Chem. 18 (2010) 403–414
411
added to a solution of 9 (2 g, 5.74 mmol) in 29 mL of DMF. The
6.7.3. 8-[(5-Chloro-2-piperazin-1-ylisonicotinoyl)amino]-1-(4-
reaction mixture was stirred at room temperature for 3 h. The
crude reaction mixture was concentrated to about 10 mL of DMF.
Upon addition of water to this DMF residue, a white solid was
formed, which was triturated with water for 20 min and filtered.
The solid was collected, dissolved in THF and dried over MgSO4. Re-
moval of the solvent afforded a brown solid, which was triturated
in warm CH3CN. Yield 73%. 1H NMR (DMSO-d6, 300 MHz): d 2.90–
2.92 (m, 4H), 6.09 (s, 2H), 6.94–7.04 (m, 2H), 7.12 (d, 1H, J = 2 Hz),
7.26–7.38 (m, 4H), 7.51 (s, 1H), 7.81 (s, 1H), 8.61 (s, 1H), 10.54 (s,
1H); 13C NMR (DMSO-d6, 75 MHz): d 20.3, 29.7, 102.6, 107.9, 109.1,
115.0, 120.0, 120.4, 121.0, 123.9, 126.9, 127.7, 129.6, 133.8, 134.3,
137.0, 139.5, 142.7, 146.7, 148.5, 148.6, 149.5, 150.3, 161.7, 164.5;
HRMS calcd for C25H18Cl2FN5O4 522.0730, found 522.0725.
fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (8b)
Same procedure as 7b at 100 °C for 24 h. The off-white precip-
itate that formed in the crude reaction mixture was filtered and
washed with EtOH. Yield: 53%. 1H NMR (DMSO-d6, 300 MHz): d
2.67 (t, 4H, J = 4.9 Hz), 2.85–2.90 (m, 4H), 3.36 (t, 4H, J = 4.9 Hz),
6.80 (s, 1H), 7.18–7.19 (m, 1H), 7.25 (s, 1H), 7.27–7.35 (m, 3H),
7.37–7.39 (dd, 1H, J = 8 Hz, 2 Hz), 7.51–7.55 (m, 3H), 8.09 (s, 1H),
10.28 (s, 1H); 13C NMR (DMSO-d6, 75 MHz): d 20.3, 29.7,46.0,
46.5, 106.3, 115.0, 117.2 (d, JC–F = 23.0 Hz), 120.0, 121.4, 126.6,
128.7 (d, JC–F = 9.1 Hz), 129.6, 133.4, 136.8 (d, JC–F = 2.8 Hz), 137.5,
139.6, 143.2, 145.1, 147.5, 158.4, 162.2 (d, JC–F = 246 Hz), 163.9,
164.4; Anal. Calcd for C28H25ClFN7O2: C, 61.59; H, 4.62; N, 17.96.
Found: C, 61.39; H, 4.69; N, 17.97.
6.6.2. 8-[(2,5-Dichloroisonicotinoyl)amino]-1-(4-fluoro-phenyl)-
4,5-dihydro-1H-benzo-[g]indazole-3-carboxamide (18)
6.7.4. 8-{[5-Chloro-2-(4-ethylpiperazin-1-yl)isonicotinoyl]amino}-
1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carbox-
amide (8c)
Same procedure as 17. Crystallized from CH3CN. Yield 73%. 1H
NMR (DMSO-d6, 400 MHz): 2.86–2.91 (m, 4H), 7.18 (d, 1H,
J = 0.9 Hz), 7.25 (s, 1H), 7.32–7.36 (m, 4H), 7.52–7.56 (m, 3H),
7.75 (s, 1H), 8.57 (s, 1H), 10.49 (s, 1H); 13C NMR (DMSO-d6,
100 MHz): d 20.3, 29.7, 114.9, 117.2 (d, JC–F = 23.0 Hz), 120.0,
121.4, 123.8, 126.7, 127.6, 128.6 (d, JC–F = 9.1 Hz), 129.7, 133.9,
136.7 (d, JC–F = 2.6 Hz), 137.0, 139.5, 143.2, 146.7, 149.5, 150.2,
161.7, 162.6 (d, JC–F = 245.9 Hz), 164.4; HRMS calcd for
C24H17Cl2FN5O2 496.0738, found 496.0759.
A mixture of 18 (0.89 g, 1.81 mmol) and 1-ethylpiperazine (2 g,
2.3 mL, 18.1 mmol) in 4.5 mL of DMA was stirred at 100 °C for 21 h.
The mixture was poured into water, causing a precipitate to form,
which was filtered and washed with water. This solid was dis-
solved in acetonitrile, dried over MgSO4, and dried under vacuum.
The product was slurried in acetonitrile, filtered, and washed with
acetonitrile. Yield 75%. 1H NMR (DMSO-d6, 400 MHz): d 0.99 (t, 3H,
J = 7.2 Hz), 2.32 (q, 2H, J = 7.2 Hz), 2.38 (t, 4H, J = 4.9 Hz), 2.84–2.97
(m, 4H), 3.47 (t, 4H, J = 4.9 Hz), 6.87 (s, 1H), 7.20–7.21 (m, 1H), 7.27
(s, br, 1H), 7.30–7.38 (m, 3H), 7.40 (dd, 1H, J = 8.1 Hz, 2.0 Hz), 7.53–
7.58 (m, 3H), 8.13 (s, 1H), 10.32 (s, 1H); 13C NMR (DMSO-d6,
100 MHz): d 12.6, 20.3, 29.7, 45.4, 52.3, 52.7, 106.4, 115.0, 115.3,
117.2 (d, JC–F = 23.0 Hz), 120.0, 121.4, 126.6, 128.6 (d, JC–F = 9.1 Hz),
129.6, 133.4, 136.8 (d, JC–F = 2.8 Hz), 137.5, 139.6, 143.2, 145.2,
147.5, 158.1, 162.6 (d, JC–F = 245.7 Hz), 163.9, 164.4; HRMS calcd
for C30H30ClFN7O2 574.2128, found 574.2153.
6.7. Synthesis of 2-amino-5-chloro-pyridine-4-carboxamides
by direct amination
6.7.1. 1-(1,3-Benzodioxol-5-yl)-8-{[5-chloro-2-(4-methylpipera-
zin-1-yl)isonicotinoyl]-amino}-4,5-dihydro-1H-benzo[g]-
indazole-3-carboxamide (7a)
A mixture of 17 (1.2 g, 2.3 mmol) and N-methylpiperazine
(4.6 mL, 46 mmol) was heated at 100 °C in a sealed tube for 24 h.
After removal of the volatiles under vacuum, the residue was par-
titioned between water and CH2Cl2. The organic layer was washed
an additional time with water and dried over MgSO4. The crude
product mixture was purified by chromatography on silica gel
using CH2Cl2/MeOH = 12:1 to 10:2. Yield 46%. 1H NMR (DMSO-d6,
400 MHz): d 2.18 (s, 3H), 2.34–2.35 (d, 2H, J = 5 Hz), 2.89–2.91
(m, 4H), 3.49 (d, 2H, J = 5 Hz), 6.08 (s, 2H), 6.91–7.03 (m, 3H), 7.1
(d, 1H, J = 2 Hz), 7.24–7.3 (m, 3H), 7.38–7.41 (dd, 1H, J = 8.3 Hz,
2 Hz), 7.49 (s, 1H), 8.13 (s, 1H), 10.33 (s, 1H); 13C NMR (DMSO-d6,
100 MHz): d 20.3, 29.7, 45.2, 46.4, 54.9, 102.6, 106.6, 107.8, 109.1,
115.1, 115.4, 119.9, 120.3, 120.9, 126.8, 129.5, 133.4, 134.3, 137.5,
139.6, 142.7, 145.2, 147.5, 148.4, 148.6, 158.1, 163.9, 164.5; HRMS
calcd for C30H29ClN7O4 586.1964, found 586.1988.
6.7.5. 8-{[5-Chloro-2-(4-isopropylpiperazin-1-yl)isonicotinoyl]-
amino}-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-
3-carboxamide (8d)
Same procedure as 8c. Yield 72%. 1H NMR (DMSO-d6, 400 MHz):
d 0.95 (d, 6H, J = 6.4 Hz), 2.44–4.47 (m, 4H overlapping with
DMSO), 2.64 (sep, 1H, J = 6.4 Hz), 2.84–2.97 (m, 4H), 3.46 (t, 4H,
J = 4.8 Hz), 6.86 (s, 1H), 7.20–7.21 (m, 1H), 7.27 (s, br, 1H), 7.30–
7.38 (m, 4H), 7.41 (dd, 1H, J = 8.12 Hz, 2.0 Hz), 7.53–7.58 (m, 3H),
8.12 (s, 1H); 10.32 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): d 18.8,
20.3, 29.7, 45.7, 48.4, 54.4, 106.4, 114.9, 115.2, 117.2 (d,
JC–F = 23.0 Hz), 120.0, 121.4, 126.6, 128.6 (d, JC–F = 9.1 Hz), 129.6,
133.4, 136.8 (d, JC–F = 2.8 Hz), 137.5, 139.6, 143.2, 145.2, 147.5,
158.1, 162.6 (d, JC–F = 246.5 Hz), 163.9, 164.4; HRMS calcd for
C31H32ClFN7O2 588.2285, found 588.2287.
6.7.2. 1-(1,3-Benzodioxol-5-yl)-8-[(5-chloro-2-piperazin-1-yli-
sonicotinoyl)amino]-4,5-dihydro-1H-benzo-[g]indazole-3-
carboxamide (7b)
6.7.6. 8-({5-Chloro-2-[4-(2-methoxyethyl)piperazin-1-yl]isonic-
otinoyl}amino)-1-(4-fluoro-phenyl)-4,5-dihydro-1H-benzo[g]-
indazole-3-carboxamide (8e)
A mixture of 17 (1 g, 1.8 mmol) and piperazine (3 g, 36 mmol)
in 4 mL EtOH was stirred at 95 °C for 24 h. After allowing the reac-
tion mixture to cool down to room temperature, the volatiles were
removed under vacuum. The residue was triturated with H2O and
finally with EtOH. Yield: 55%. 1H NMR (DMSO-d6, 300 MHz): d 2.71
(s, br, 4H), 2.86–3.30 (m, 4H), 3.39 (s, br, 4H), 6.07 (s, 2H), 6.85 (s,
1H), 6.93–6.95 (dd, 1H, J = 8.2 Hz, 1.9 Hz), 7.00 (d, 1H, J = 8.2 Hz),
7.09 (d, 1H, J = 1.9 Hz), 7.24–7.30 (m, 3H), 7.38–7.41 (dd, 1H,
J = 8.2 Hz, 1.74 Hz), 7.49 (s, 1H), 8.12 (s, 1H), 10.32 (s, 1H); 13C
NMR (DMSO-d6, 75 MHz): d 20.3, 29.7, 45.9, 46.5, 102.6, 106.4,
107.8, 109.1, 115.0, 115.1, 119.9, 120.3, 120.9, 126.8, 129.5,
133.4, 134.3, 137.5, 139.6, 142.7, 145.2, 147.5, 148.4, 148.6,
158.4, 163.9, 164.5; HRMS calcd for C29H27ClN7O4 572.1808, found
572.1782.
Same procedure as 8c. The mixture was added to water causing
a precipitate to form. This precipitate was filtered and washed with
water. The solid was dissolved in THF, dried over MgSO4, and dried
under vacuum. The resulting solid was dissolved in EtOH, precipi-
tated by addition of CH3CN and finally triturated it in this solvent
mixture. Yield 54%. 1H NMR (DMSO-d6, 400 MHz): d 2.43–2.48 (m,
4H+3H overlapping with DMSO), 2.87–2.93 (m, 4H), 3.42 (t, 2H,
J = 5.8 Hz), 3.46 (t, 4H, J = 4.7 Hz), 6.87 (s, 1H), 7.20–7.22 (m, 1H),
7.27 (s, br, 1H), 7.29–7.38 (m, 3H), 7.40 (dd, 1H, J = 8.2 Hz,
1.94 Hz), 7.53–7.58 (m, 3H), 8.12 (s, 1H), 10.31 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): d 20.3, 29.7, 45.3, 53.4, 57.7, 58.6, 70.6,
106.4, 114.9, 115.3, 117.2 (d, JC–F = 23.2 Hz), 120.0, 121.4, 126.6,

412
D. F. Bonafoux et al. / Bioorg. Med. Chem. 18 (2010) 403–414
128.6 (d, JC–F = 8.9 Hz), 129.6, 133.4, 136.8 (d, JC–F = 2.8 Hz), 137.5,
139.4, 143.2, 145.2, 147.5, 158.1, 162.6 (d, JC–F = 245.7 Hz), 163.9,
164.4; Anal. Calcd for C31H31ClFN7O3: C, 61.64; H, 5.17; N, 16.23.
Found: C, 61.65; H, 5.22; N, 16.04.
JC–F = 23.0 Hz), 120.0, 121.4, 126.6, 128.6 (d, JC–F = 8.9 Hz), 129.6,
133.4, 136.8 (d, JC–F = 3.0 Hz), 137.5, 139.6, 143.2, 145.2, 147.5,
158.1, 162.2 (d, JC–F = 245 Hz), 164.0, 164.4; HRMS calcd for
C29H28ClFN7O2 560.1972, found 560.1969.
6.7.7. 8-({5-Chloro-2-[(3R,5S)-3,4,5-trimethylpiperazin-1-yl]-
isonicotinoyl}amino)-1-(4-fluoro-phenyl)-4,5-dihydro-1H-
benzo[g]indazole-3-carboxamide (8f)
6.7.11. 8-({5-Chloro-2-[(3S)-3,4-dimethylpiperazin-1-yl]isoni-
cotinoyl}amino)-1-(4-fluoro-phenyl)-4,5-dihydro-1H-benzo[g]-
indazole-3-carboxamide (8j)
Same procedure as 7b at 100 °C for 5 days. The off-white precip-
itate that formed in the crude reaction mixture was filtered and
washed with EtOH. Yield 77%. 1H NMR (DMSO-d6, 400 MHz): d
1.01 (d, 6H, J = 12.3 Hz), 2.05 (m, br, 2H), 2.13 (s, 3H), 2.47–2.53
(2H, m, overlaps with DMSO), 2.88–2.93 (m, 4H), 4.11 (d, 2H,
J = 12.3 Hz), 6.92 (s, 1 H), 7.21 (d, 1H, J = 2.0 Hz), 7.27 (s, 1H),
7.3–7.38 (m, 3H), 7.41 (dd, 1H, J = 8.2 Hz, 2.0 Hz), 7.54–7.58 (m,
3H), 8.11 (s, 1H), 10.32 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): d
19.3, 20.3, 29.7, 38.0, 51.9, 57.6, 106.3, 114.9, 115.0, 117.2 (d,
JC–F = 23.0 Hz), 120.0, 121.4, 126.6, 128.6, (d, JC–F = 8.9 Hz), 129.6,
133.4, 136.8 (d, JC–F = 2.8 Hz), 137.5, 139.6, 143.2, 145.3, 147.4,
157.5, 162.6 (d, JC–F = 246.0 Hz), 163.9, 164.4; HRMS calcd for
C31H32ClFN7O2 588.2285, found 588.2212.
Same procedure as 7b at 100 °C for 3 days. The off-white precip-
itate that formed in the crude reaction mixture was filtered and
washed with EtOH. Yield 55%. 1H NMR (DMSO-d6, 400 MHz): d
1.0 (d, 3H, J = 6.17 Hz), 1.96–1.98 (m, 1H), 2.02–2.09 (m, 1H),
2.16 (s, 3H), 2.48–2.54 (m, 1H), 2.74 (d, br, 1H, J = 11.7 Hz), 2.85–
2.93 (m, 4H), 4.02–4.09 (m, 2H), 6.89 (s, 1H), 7.21 (d, 1H,
J = 2.0 Hz), 7.27 (s, 1H), 7.29–7.38 (m, 2H), 7.41 (dd, 1H,
J = 8.2 Hz, 2.0 Hz), 7.54–7.58 (m, 2H), 8.11 (s, 1H), 10.33 (s, 1H);
13C NMR (DMSO-d6, 100 MHz): d 17.2, 20.3, 29.7, 42.8, 45.4, 51.8,
55.2, 57.6, 106.4, 115.0, 115.1, 117.2 (d, JC–F = 23.2 Hz), 120.0,
121.4, 126.6, 128.7 (d, JC–F = 9.1 Hz), 129.6, 133.4, 136.8 (d,
JC–F = 3.0 Hz), 137.5, 139.6, 143.2, 145.2, 147.5, 157.9, 162.6 (d,
JC–F = 245.8 Hz), 163.9, 164.4; HRMS calcd for C30H30ClFN7O2
574.2128, found 574.2098.
6.7.8. 8-({5-Chloro-2-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-
isonicotinoyl}amino)-1-(4-fluorophenyl)-4,5-dihydro-1H-
benzo[g]indazole-3-carboxamide (8g)
6.7.12. 8-({5-Chloro-2-[(3S)-3-methylpiperazin-1-yl]-isonicotin-
oyl}amino)-1-(4-fluoro-phenyl)-4,5-dihydro-1H-benzo[g]-
indazole-3-carboxamide (8k)
Same procedure as 8c. The solid was dissolved in CH3CN, dried
over MgSO4, and the solvent stripped. Yield 88%. 1H NMR (DMSO-
d6, 400 MHz): d 0.97 (d, 6H, J = 6.3 Hz), 2.16–2.26 (m, 3H), 2.61–
2.70 (m, 2H), 2.83–2.97 (m, 4H), 4.12 (dd, 2H, J = 1.9 Hz, 12.1 Hz),
6.87 (s, 1H); 7.21–7.22 (m, 1H), 7.28 (s, br, 1H), 7.30–7.39 (m,
3H); 7.41 (dd, 1H, J = 8.19 Hz, 2.01 Hz), 7.54–7.58 (m, 3H), 8.10
(s, 1H); 10.31 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): d 19.8,
20.3, 29.7, 50.7, 51.9, 106.3, 114.6, 114.9, 117.2 (d, JC–F = 23.0 Hz),
120.0, 121.4, 126.6, 128.6 (d, JC–F = 9.3 Hz), 129.6, 133.4, 136.8,
137.5, 139.6, 143.2, 145.2, 147.4, 157.8, 162.6 (d, JC–F = 245.7 Hz),
164.0, 164.4; HRMS calcd for C30H30ClFN7O2 574.2128, found
574.2117.
Same procedure as 8c. The solid was dissolved in acetonitrile,
dried over MgSO4, and the solvent stripped. Yield 66%. 1H NMR
(DMSO-d6, 400 MHz): d 0.98 (d, 3H, J = 6.3 Hz), 2.29–2.35 (m,
1H), 2.57–2.71 (m, 3H), 2.87–2.93 (m, 5H), 3.28 (s, br, 1H), 4.04–
4.13 (m, 2H), 6.85 (s, 1H), 7.21 (d, 1H, J = 2 Hz), 7.27 (s, br, 1H),
7.29–7.38 (m, 3H), 7.41 (dd, 1H, J = 8.3 Hz, 2.0 Hz), 7.54–7.58 (m,
3H), 8.11 (s, 1H), 10.31 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): d
19.8, 20.3, 29.7, 45.5, 45.7, 50.6, 52.5, 106.3, 114.8, 114.9, 117.2
(d, JC–F = 23.2 Hz), 120.0, 121.4, 126.6, 128.6 (d, JC–F = 9.1 Hz),
129.6, 133.4, 136.8 (d, JC–F = 2.8 Hz), 137.5, 139.6, 143.2, 145.2,
147.4, 158.1, 162.6 (d, JC–F = 245.9 Hz), 163.9, 164.4; HRMS calcd
for C29H28ClFN7O2 560.1972, found 560.1958.
6.7.9. 8-({5-Chloro-2-[(3R)-3,4-dimethylpiperazin-1-yl]isonico-
tinoyl}amino)-1-(4-fluoro-phenyl)-4,5-dihydro-1H-benzo[g]-
indazole-3-carboxamide (8h)
6.7.13. 8-{[5-Chloro-2-(4-methyl-1,4-diazepan-1-yl)isonicotinoyl]-
amino}-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-
3-carboxamide (8l)
Same procedure as 7b at 100 °C for 4 days. The off-white precip-
itate that formed in the crude reaction mixture was filtered and
washed with EtOH. Yield 55%. 1H NMR (DMSO-d6, 400 MHz): d
1.0 (d, 3H, J = 6.17 Hz), 1.96–2.0 (m, 1H), 2.05–2.06 (m, 1H), 2.15
(s, 3H), 2.48–2.54 (m, 1H), 2.74 (d, br, 1H, J = 11.5 Hz), 2.85–2.93
(m, 4H), 4.01–4.09 (m, 2H), 6.89 (s, 1H), 7.21 (d, 1H, J = 2.0 Hz),
7.27 (s, 1H), 7.29–7.38 (m, 2H), 7.41 (dd, 1H, J = 8.2 Hz, 2.0 Hz),
7.53–7.58 (m, 2H), 8.11 (s, 1H), 10.33 (s, 1H); 13C NMR (DMSO-
d6, 100 MHz): d 17.2, 20.3, 29.7, 42.8, 45.4, 51.8, 55.2, 57.6, 106.4,
115.0, 115.2, 117.2 (d, JC–F = 23.2 Hz), 120.0, 121.4, 126.6, 128.7
(d, JC–F = 9.1 Hz), 129.6, 133.4, 136.8 (d, JC–F = 2.8 Hz), 137.5,
139.6, 143.2, 145.2, 147.5, 157.9, 162.6 (d, JC–F = 246.0 Hz), 163.9,
164.4; HRMS calcd for C30H30ClFN7O2 574.2128, found 574.2094.
Same procedure as 7b at 95 °C for 24 h. After allowing the reac-
tion mixture to cool, the volatiles were removed under vacuum
and the residue triturated with H2O. Yield 71%. 1H NMR (DMSO-
d6, 300 MHz): d 1.81–1.83 (m, 2H), 2.21 (s, 3H), 2.41 (t, 2H,
J = 5.5 Hz), 2.53 (t, 2H, J = 4.56 Hz), 2.87–2.93 (m, 4H), 3.53 (t, 2H,
J = 5.5 Hz), 3.63–3.67 (m, 2H), 6.62 (s, 1H), 7.19 (d, 1H, J = 1.9 Hz),
7.27–7.38 (m, 4H), 7.42–7.45 (dd, 1H, J = 8.12 Hz, 1.88 Hz), 7.53–
7.58 (m, 3H), 8.07 (s, 1H), 10.31 (s, 1H); 13C NMR (DMSO-d6,
75 MHz): d 20.3, 27.4, 29.7, 46.6, 46.7, 47.1, 57.2, 57.8, 104.9,
113.7, 115.0, 117.2 (d, JC–F = 23.0 Hz), 120.0, 121.4, 126.6, 128.6
(d, JC–F = 9.1 Hz), 129.6, 133.4, 136.8 (d, JC–F = 2.8 Hz), 137.5,
139.6, 143.2, 145.0, 147.5, 157.1, 162.6 (d, JC–F = 245 Hz), 164.1,
164.4. Anal. Calcd for C30H29ClFN7O2: C, 62.77; H, 5.09; N, 17.08.
Found: C, 62.52; H, 5.11; N, 17.11.
6.7.10. 8-({5-Chloro-2-[(3R)-3-methylpiperazin-1-yl]isonicotinoyl}
amino)-1-(4-fluoro-phenyl)-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (8i)
6.7.14. 8-{[5-Chloro-2-(1,4-diazepan-1-yl)isonicotinoyl]amino}-
1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxa-
mide (8m)
Same procedure as 7b at 100 °C for 24 h. The white precipitate
that formed in the crude reaction mixture was filtered and washed
with EtOH. Yield 82%. 1H NMR (DMSO-d6, 400 MHz): d 1.68–1.72
(m, 2H), 2.63 (t, 2H, J = 5.84 Hz), 2.79 (t, 2H, J = 5 Hz), 2.91–2.93
(m, 4H), 3.59 (t, 2H, J = 5 Hz), 3.64 (t, 2H, J = 5.84 Hz), 6.62 (s,
1H), 7.2 (d, 1H, J = 2 Hz), 7.28–7.4 (m, 4H), 7.44 (dd, 1H,
Same procedure as 8c. The solid was dissolved in acetonitrile,
dried over MgSO4, and the solvent stripped. Yield 72%. 1H NMR
(DMSO-d6, 400 MHz): d 0.97 (d, 3H, J = 6.2 Hz); 2.28–2.34 (m,
1H), 2.57–2.70 (m, 3H), 2.87–2.93 (m, 5 H), 3.29 (s, br, 1H), 4.05–
4.09 (m, 2H), 6.85 (s, 1H), 7.20 (d, 1H, J = 1.8 Hz), 7.27–7.38 (m,
4H), 7.41 (dd, 1H, J = 8.2 Hz, 1.8 Hz), 7.53–7.58 (m, 3H), 8.11 (s,
1H), 10.31 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): d 19.9, 20.3,
29.7, 45.6, 45.7, 50.6, 52.6, 106.3, 114.8, 114.9, 117.2 (d,

D. F. Bonafoux et al. / Bioorg. Med. Chem. 18 (2010) 403–414
413
J = 8.2 Hz, 2 Hz), 7.55–7.60 (m, 3H), 8.08 (s, 1H), 10.32 (s, 1H); 13C
NMR (DMSO-d6, 100 MHz): d 20.3, 29.5, 29.7, 46.7, 48.3, 48.9, 51.0,
105.0, 113.6, 115.0, 117.2 (d, JC–F = 23.2 Hz), 120.0, 121.4, 126.6,
128.7 (d, JC–F = 8.9 Hz), 129.6, 133.4, 136.8 (d, JC–F = 3.0 Hz), 137.5,
139.6, 143.2, 145.0, 147.6, 157.1, 162.6 (d, JC–F = 245.8 Hz), 164.1,
164.4; HRMS calcd for C29H28ClFN7O3 560.1972, found 560.1936.
vacuum and the resulting yellow solid partitioned between water
and Et2O. The aqueous layer was acidified to pH 1.5 using an aque-
ous solution of HCl and extracted once with Et2O and three times
with CH2Cl2. The organic extracts were combined and the solvents
removed under vacuum. The resulting yellow solid was crystal-
lized from MeOH. Yield: 34%. 1H NMR (DMSO-d6, 300 MHz): d
3.45 (t, 4H, J = 4.8 Hz), 3.67 (t, 4H, J = 4.8 Hz), 7.06 (s, 1H), 8.21 (s,
1H), 13.79 (s (br), 1H); 13C HMR (DMSO-d6, 75 MHz): d 45.6,
66.4, 107.3, 116.1, 141.3, 148.3, 158.5, 166.8; MS m/z (M+1+) 243.
6.7.15. 8-({5-Chloro-2-[[2-(dimethylamino)ethyl]-(methyl)amino]
isonicotinoyl}amino)-1-(4-fluorophenyl)-4,5-dihydro-1H-
benzo[g]indazole-3-carboxamide (8n)
Same procedure as 7b at 100 °C for 24 h. After removal of the
volatiles under vacuum, the residue was partitioned between
water and CH2Cl2. The organic layer was washed an additional time
with water and dried over MgSO4. The crude product mixture was
purified by preparative HPLC. Yield 47%. 1H NMR (DMSO-d6,
400 MHz): 2.13 (s, 6H), 2.34 (t, 2H, J = 6.7 Hz), 2.87–2.95 (m, 4H),
2.96 (s, 3H), 3.59 (t, 2H, J = 6.7 Hz), 6.58 (s, 1H), 7.2 (d, 1H,
J = 2 Hz), 7.27 (s, 1H), 7.29–7.37 (m, 3H), 7.41–7.44 (dd, 1H,
J = 8 Hz, 2 Hz), 7.53–7.58 (m, 3H), 8.13 (s, 1H), 10.36 (s, 1H); 13C
NMR (DMSO-d6, 100 MHz): d 20.3, 29.7, 37.1, 46.1, 47.8, 56.8,
105.0, 113.8, 114.5, 117.2 (d, JC–F = 23.3 Hz), 120.0, 121.4, 126.6,
128.6, 128.7 (d, JC–F = 8.9 Hz), 129.6, 133.4, 136.8 (d, JC–F = 2.8 Hz),
137.5, 139.6, 143.2, 144.9, 147.5, 157.4, 162.6 (d, JC–F = 245 Hz),
164.1, 164.4; HRMS calcd for C29H30ClFN7O2 562.2128, found
562.2120.
6.8.3. 8-{[5-Chloro-2-(4-methylpiperazin-1-yl)isonicotinoyl]-
amino}-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-
3-carboxamide (8a)
5-Chloro-2-(N-methyl-piperazinyl)isonicotinic acid hydrochlo-
ride (20) (0.59 g, 2.02 mmol), HATU (0.755 g, 0.00198 mol) and fi-
nally Et3N (1.09 mL, 0.0078 mol) were added to a solution of 10
(0.432 g, 0.00134 mol) in DMF (8 mL). The reaction mixture was
stirred at room temperature for 3 h. The crude reaction mixture
was concentrated to about 3 mL of DMF. Upon addition of water
to this DMF residue, a white solid was formed, which was tritu-
rated with water for 20 min and filtered. The solid was collected,
dissolved in THF and dried over MgSO4. The crude material was
purified by chromatography on a silica gel column (MeOH/
CH2Cl2 = 1:12). Yield 40%. 1H NMR (DMSO-d6, 400 MHz): d 2.17
(s, 3H), 2.33 (t, 4H, J = 4.8 Hz), 2.87–2.93 (m, 4H), 3.47 (t, 4H,
J = 4.8 Hz), 6.87 (s, 1H), 7.2 (d, 1H, J = 1.9 Hz), 7.27 (s, 1H), 7.29–
7.41 (m, 5H), 7.53–758 (m, 3H), 8.13 (s, 1H), 10.31 (s, 1H); 13C
NMR (DMSO-d6, 100 MHz): d 20.3, 29.7, 45.2, 46.4, 54.9, 106.5,
115.0, 115.3, 117.2 (d, JC–F = 23.3 Hz), 120.0, 121.4, 126.6, 128.7
(d, JC–F = 8.8 Hz), 129.6, 133.4, 136.8 (d, JC–F = 3.0 Hz), 137.5,
139.6, 143.2, 145.2, 147.5, 158.1, 162.6 (d, JC–F = 245 Hz), 163.9,
164.4; HRMS calcd for C29H28ClFN7O2 560.1972, found 560.1968.
6.7.16. 8-[(5-Chloro-2-{[2-(dimethylamino)ethyl]amino}-
isonicotinoyl)amino]-1-(4-fluoro-phenyl)-4,5-dihydro-1H-
benzo[g]indazole-3-carboxamide (8o)
Same procedure as 7b at 100 °C for 24 h. The white precipitate
that formed in the crude reaction mixture was filtered and washed
with EtOH and finally purified by reverse phase preparative HPLC.
Yield 14%. 1H NMR (DMSO-d6, 400 MHz): d 2.13 (s, 6H), 2.34 (t, 2H,
J = 6.5 Hz), 2.87–2.92 (m, 4H), 3.29–3.30 (m, 2H), 6.49 (s, 1H), 6.78
(t, 1H, J = 5.3 Hz), 7.2 (d, 1H, J = 2 Hz), 7.27 (s, 1H), 7.29–7.40 (m,
4H), 7.53–7.58 (m, 3H), 7.99 (s, 1H), 10.3 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): d 20.3, 29.7, 39.4, 45.9, 58.7, 107.6, 113.7,
115.0, 117.2 (d, JC–F = 22.8 Hz), 120.0, 121.4, 126.7, 128.7 (d,
JC–F = 9.3 Hz), 129.6, 133.4, 136.8 (d, JC–F = 3.0 Hz), 137.5, 139.6,
143.2, 144.4, 147.5, 158.1, 162.6 (d, JC–F = 245.7 Hz), 164.0, 164.4;
HRMS calcd for C28H28ClFN7O2 548.1972, found 548.1934.
6.8.4. 1-(1,3-Benzodioxol-5-yl)-8-[(5-chloro-2-morpholin-4-
ylisonicotinoyl)amino]-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (7p)
Same procedure as 8a. Purified by trituration with Et2O. Yield:
88%. 1H NMR (DMSO-d6, 400 MHz): d 2.88–2.93 (m, 4H), 3.46 (t,
4H, J = 4.6 Hz), 3.66 (t, 4H, J = 4.6 Hz), 6.09 (s, 2H), 6.94 (s, 1H),
6.97 (d, 1H, J = 2 Hz), 7.02 (d, 1H, J = 8.2 Hz), 7.11 (d, 1H,
J = 1.9 Hz), 7.25 (s, 1H), 7.29–7.32 (m, 2H), 7.39–7.42 (dd, 1H,
J = 8.2 Hz, 2 Hz), 7.5 (s, 1H), 8.19 (s, 1H), 10.36 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): d 20.3, 29.7, 45.7, 66.5, 102.6, 106.6, 107.9,
109.1, 115.1, 115.9, 119.9, 120.4, 121.0, 126.8, 129.5, 133.4,
134.3, 137.5, 139.6, 142.7, 145.3, 147.5, 148.5, 148.6, 158.3,
163.8, 164.5, HRMS calcd for C29H26ClN6O5 573.1648, found
573.1641.
6.8. 2-Amino-5-chloropyridine-4-carboxamides by coupling
with 2-amino-5-chlorocarboxylic acids
6.8.1. 5-Chloro-2-(4-methylpiperazin-1-yl)isonicotinic acid
hydrochloride (19)
A mixture of 2,5-dichloropyridine-4-carboxylic acid (16) (3 g,
0.0156 mol) and N-methylpiperazine (30.7 g, 0.30 mol) in 10 mL
of DMA was stirred at 100 °C for 8 days. The volatiles were re-
moved under vacuum. The resulting residue was washed with a
saturated solution of K2CO3 and with CH2Cl2. The solvent was re-
moved under vacuum and the resulting residue dissolved in the
minimum amount of water, acidified to pH 1 with an aqueous solu-
tion of HCl (1 N) and washed with CH2Cl2. The product crystallized
from the acidic aqueous layer upon standing at room temperature.
Yield: 44%. 1H NMR (D2O, 300 MHz): d 2.85 (s, 3H), 3.13 (t, 2H,
J = 12.28 Hz), 3.34 (t, 2H, J = 14.3 Hz), 3.56 (d, 2H, J = 12.28 Hz),
4.18 (d, 2H, J = 14.3 Hz), 7.08 (s, 1H), 8.00 (s, 1H); 13C NMR (D2O,
75 MHz): d 43.1, 43.4, 52.54, 109.5, 117.6, 142.3, 147.9, 154.4,
169.5.
6.8.5. 8-[(5-Chloro-2-morpholin-4-ylisonicotinoyl)amino]-1-
(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (8p)
Same procedure as 8a. The crude solid was triturated with Et2O,
EtOH and finally CH3CN, filtered and dried under vacuum. Yield
82%. 1H NMR (DMSO-d6, 400 MHz): d 2.80–2.95 (m, 4H), 3.42 (t,
4H, J = 5 Hz), 3.62 (t, 4H, J = 5 Hz), 6.88 (s, 1H), 7.17–7.21 (m, 1H),
7.23–7.41 (m, 5H), 7.49–7.58 (m, 3H), 8.14 (s, 1H), 10.31 (s, 1H);
13C NMR (DMSO-d6, 100 MHz): d 20.3, 29.7, 45.7, 66.5, 106.5,
114.9, 115.8, 117.2 (d, JC–F = 23.0 Hz), 120.0, 121.4, 126.6, 128.6
(d, JC–F = 8.9 Hz), 129.6, 133.4, 136.8 (d, JC–F = 2.8 Hz), 137.4,
139.6, 143.2, 145.3, 147.5, 158.3, 162.6 (d, JC–F = 247.6 Hz),164.4;
HRMS calcd for C28H25ClFN6O3 547.1655, found 547.1683.
6.8.2. 5-Chloro-2-morpholin-4-ylisonicotinic acid (20)
6.8.6. 8-[(2-Chloroisonicotinoyl)amino]-1-(4-fluorophenyl)-
4,5-dihydro-1H-benzo-[g]indazole-3-carboxamide (22)
2-Chloroisonicotinic acid (21) (2 g, 12.7 mmol), HATU (4.8 g,
12.63 mmol), and finally Et3N (3 g, 4.2 mL, 30 mmol), were added
A mixture of 2,5-dichloropyridine-4-carboxylic acid (16) (1.6 g,
0.0083 mol) and morpholine (10.9 g, 0.125 mol) in 4 mL of DMA
was stirred at 80 °C for 4 days. The volatiles were removed under

414
D. F. Bonafoux et al. / Bioorg. Med. Chem. 18 (2010) 403–414
2. Swenftleben, U.; Karin, M. Crit. Care Med. 2002, 30, S18.
3. Yin, M.-J.; Yamamoto, Y.; Gaynor, R. B. Nature 1998, 396, 77.
to a solution of 10 (2.72 g, 8.46 mmol) in 42 mL of DMF. The mix-
ture was stirred at room temperature for 4 h. The crude reaction
mixture was concentrated under vacuum. Upon addition of water
to this DMF residue, a white solid was formed, which was tritu-
rated with water and filtered. The resulting solid was dissolved
in CH3CN, decolorized with activated carbon, dried over MgSO4,
and crystallized from CH3CN. Yield 45%. 1H NMR (DMSO-d6,
400 MHz): 2.83–2.95 (m, 4H), 7.26 (s, 1H), 7.30–7.42 (m, 5H),
7.50–7.58 (m, 3H), 7.66 (dd, 1H, J = 5.1 Hz, 1.3 Hz), 7.78 (s, 1H),
8.53 (d, 1H, J = 5.1 Hz), 10.36 (s, 1H); 13C NMR (DMSO-d6,
100 MHz): d 20.3, 29.7, 115.3, 117.2 (d, JC–F = 23.2 Hz), 120.6,
121.4, 121.8, 122.8, 126.6, 128.6 (d, JC–F = 9.1 Hz), 129.6, 133.8,
136.8 (d, JC–F = 2.8 Hz), 137.4, 139.6, 143.2, 146.0, 151.3, 151.4,
162.6 (d, JC–F = 245.9 Hz), 162.9, 164.4; HRMS calcd for
C24H18ClFN5O2 462.1128, found 462.1115.
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6.8.7. 1-(4-Fluorophenyl)-8-{[2-(4-methylpiperazin-1-yl)-
isonicotinoyl]amino}-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide (23a)
A mixture of 22 (1 g, 2.17 mmol) and N-methylpiperazine (3.3 g,
3.64 mL, 32.9 mmol) in 5 mL DMA was stirred at 100 °C for 88 h.
The crude reaction mixture was concentrated. Upon addition of
water to this DMA residue, a solid was formed, which was tritu-
rated with water. The solid was dissolved in CH3CN, decolorized
with activated carbon, and dried over MgSO4 before being crystal-
lized from CH3CN. Yield 42%. 1H NMR (DMSO-d6, 400 MHz): d 2.17
(s, 3H), 2.35 (t, 4H, J = 4.8 Hz), 2.82–2.95 (m, 4H), 3.47 (t, 4H,
J = 4.8 Hz), 6.85 (d, 1H, J = 5.2 Hz), 7.01 (s, 1H), 7.25 (s, 1H), 7.25–
7.30 (m, 2H), 7.33–7.38 (m, 2H), 7.40 (dd, 1H, J = 8.2 Hz, 1.7 Hz),
7.51–7.56 (m, 3H), 8.16 (d, 1H, J = 5.1 Hz), 10.10 (s, 1H); 13C
NMR (DMSO-d6, 100 MHz): d 20.3, 29.7, 45.2, 46.4, 55.0, 105.4,
11.2, 115.5, 117.1 (d, JC–F = 23.0 Hz), 120.6, 121.3, 126.5, 128.6 (d,
JC–F = 9.1 Hz), 129.4, 133.3, 136.8 (d, JC–F = 2.8 Hz), 137.7, 139.7,
143.2, 144.3, 148.9, 159.9, 162.6 (d, JC–F = 245.7 Hz), 164.4, 165.1;
HRMS calcd for C29H29FN7O2 526.2361, found 526.2322.
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6.8.8. 1-(4-Fluorophenyl)-8-[(2-morpholin-4-ylisonicotinoyl)-
amino]-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (23b)
Same procedure as for 23a at 80 °C for 160 h followed by 100 °C
for 172 h. The solid was crystallized from CH3CN. Yield 38%. 1H
NMR (DMSO-d6): d 2.56–2.59 (m, 4H), 3.44 (t, 4H, J = 4.8 Hz),
3.66 (t, 4H, J = 4.8 Hz), 6.89 (d, 1H, J = 1.07 Hz), 6.9 (s, 1H), 7.01
(s, br, 1H), 7.25–7.30 (m, 2H), 7.33–7.38 (m, 2H), 7.4 (dd, 1H,
J = 8.25 Hz, 1.9 Hz), 7.51–7.56 (m, 3H), 8.19 (d, 1H, J = 5.1 Hz),
10.12 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): d 20.3, 29.7, 45.7,
66.6, 105.4, 111.7, 115.5, 117.2 (d, JC–F = 23.0 Hz), 120.6, 121.4,
126.5, 128.6 (d, JC–F = 9.1 Hz), 129.5, 133.4, 136.8 (d, JC–F = 2.8 Hz),
137.7, 139.7, 143.2, 144.4, 148.9, 160.0, 162.6 (d, JC–F = 245.5 Hz),
164.4, 165.1; HRMS calcd for C28H26FN6O3 513.2045, found
513.2054.
12. Mbalaviele, Gabriel; Sommers, Cynthia D.; Bonar, Sheri L.; Mathialagan,
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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.
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Tripp, C. S. J. Biol. Chem. 2002, 277, 13840.
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References and notes
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