Synthesis and activity of new aryl- and heteroaryl-substituted pyrazole inhibitors of the transforming growth factor-β type I receptor kinase domain

Article abstract of DOI:10.1021/jm0205705

Pyrazole-based inhibitors of the transforming growth factor-β type I receptor kinase domain (TβR-I) are described. Examination of the SAR in both enzyme- and cell-based in vitro assays resulted in the emergence of two subseries featuring differing selectivity versus p38 MAP kinase. A common binding mode at the active site has been established by successful cocrystallization and X-ray analysis of potent inhibitors with the TβR-I receptor kinase domain.

Full text of DOI:10.1021/jm0205705

© Copyright 2003 by the American Chemical Society
Volume 46, Number 19
September 11, 2003
Letters
opment. Cell surface binding of TGF-â ligand leads to
Syn th esis a n d Activity of New Ar yl- a n d
Heter oa r yl-Su bstitu ted P yr a zole
In h ibitor s of th e Tr a n sfor m in g Gr ow th
F a ctor -â Typ e I Recep tor Kin a se Dom a in
the formation of a heteromeric complex involving type
I (TâR-I) and type II (TâR-II) receptors, each of which
are transmembrane-spanning proteins featuring a serine/
threonine kinase domain. Downstream signal transduc-
tion is mediated by the TâR-I kinase domain through
the phosphorylation of Smad proteins, which as oligo-
meric complexes translocate to the nucleus and regulate
gene response via association with DNA transcription
factors.1 The TGF-â signaling pathway may play a role
in a number of disease states involving inflammation,
angiogenesis, and immune function, including fibrosis,2
wound healing,3 Alzheimer’s disease,4 atherosclerosis,5
hypertension,6 restenosis,7 and cancer.8
J . Scott Sawyer,*,† Bryan D. Anderson,#
Douglas W. Beight,† Robert M. Campbell,#
Michael L. J ones,§ David K. Herron,†
J ohn W. Lampe,§ J efferson R. McCowan,†
William T. McMillen,† Nicholas Mort,†
Stephen Parsons,† Edward C. R. Smith,†
Michal Vieth,† Leonard C. Weir,† Lei Yan,‡
Faming Zhang,† and J onathan M. Yingling‡
Discovery Chemistry Research and Technology,
Cancer Research, and Lead Optimization Biology,
The Lilly Research Laboratories, A Division of
Eli Lilly and Company, Lilly Corporate Center,
Indianapolis, Indiana 46285, and Lead Generation
Chemistry, The Lilly Research Laboratories, A Division of
Eli Lilly and Company, 20 T. W. Alexander Drive,
Research Triangle Park, North Carolina 27709
Our efforts in this area began with the in vitro
screening of a large library of compounds as potential
inhibitors in a TGF-â-dependent cell-based assay. Prom-
ising hits were then evaluated as inhibitors of auto-
phosphorylation of the isolated human TâR-I kinase
domain in the form of a constitutively active construct
(T204D mutation)9 produced in Sf9 insect cells and
purified by nickel-affinity chromatography. Diheteroaryl-
substituted pyrazole 1 was quickly identified as a potent
inhibitor (IC50 ) 51 nM, Table 1) and was chosen as a
platform for SAR development. Compounds were further
evaluated as inhibitors of TGF-â-dependent luciferase
production in mink lung cells (p3TP Lux)10 and growth
in mouse fibroblasts (NIH 3T3).11
Received December 18, 2002
Abstr act: Pyrazole-based inhibitors of the transforming growth
factor-â type I receptor kinase domain (TâR-I) are described.
Examination of the SAR in both enzyme- and cell-based in
vitro assays resulted in the emergence of two subseries
featuring differing selectivity versus p38 MAP kinase. A
common binding mode at the active site has been established
by successful cocrystallization and X-ray analysis of potent
inhibitors with the TâR-I receptor kinase domain.
The 4-(quinolin-4-yl)-substituted pyrazole analogues
(Table 1) were prepared as illustrated for the syntheses
of compounds 2 and 10 (Scheme 1). Lepidine was
deprotonated and condensed with an appropriate pi-
colinic ester to provide the intermediate ketone, which
generally was not purified but treated directly with
DMF dimethyl acetal and hydrazine to provide final
product 2. Alternatively, 10 was obtained through
treatment of the intermediate ketone with the appropri-
ate hydrazide followed by thermal cyclization of the
resulting hydrazone hydrochloride salt. Treatment of
the ketone intermediate with benzylhydrazine hydrate
in the presence of 2 equiv of base followed by DMF
dimethyl acetal provided the N-benzyl analogue 12
The multifunctional cytokine transforming growth
factor-â (TGF-â) is a member of a large family of growth
factors involved in the regulation of a diverse array of
biological processes including cell growth and dif-
ferentiation, matrix modulation, and embryonic devel-
* To whom correspondence should be addressed. Phone: 317-276-
7923. Fax: 317-276-1417. E-mail: jss@lilly.com.
† Discovery Chemistry Research and Technology, The Lilly Research
Laboratories.
#
Lead Optimization Biology, The Lilly Research Laboratories.
§ Lead Generation Chemistry, The Lilly Research Laboratories.
‡ Cancer Research, The Lilly Research Laboratories.
10.1021/jm0205705 CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/12/2003

3954 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Ta ble 1. 4-Heteroaryl-Substituted Pyrazole Inhibitors
Letters
Sch em e 1. Representative Synthesis of
4-(Quinolin-4-yl)-Substituted Pyrazoles
Sch em e 2. Synthesis of 4-Aryl-Substituted Pyrazoles
A limited exploration of the SAR of the 4-(quinolin-
4-yl)-substituted pyrazoles (Table 2) revealed a number
of interesting observations. Replacement of the pyridin-
2-yl group with phenyl (3) or thiophene-2-yl (11) led to
a loss of activity in both the enzyme and cellular assays.
However, the addition of a trifluoromethyl group to 3
(4) restored some of this loss. Substitution of fluorine
at the 5-position of pyridine (5) maintained good activ-
ity, but chloro analogue 6 exhibited a noticeable drop
in potency. In addition, substitution of methyl (2) or
bromo (7) at the 6-position of pyridine improved activity,
particularly in the NIH 3T3 assay, relative to 1. Bromo-
substituted 7 was the most potent inhibitor tested with
an IC50 in the cellular assays of about 2 nM. Taken
together, these results appear to implicate the pyridine
nitrogen in providing a key interaction at the TâR-I
(Table 1). In the case of the 4-phenyl-substituted series
(Table 3), the intermediate ketones were obtained after
Claisen condensation and decarboxylation of the corre-
sponding â-ketoesters or â-ketonitriles, which were then
cyclized as described above (e.g., 16, Scheme 2). For 9
and 20, the corresponding aryl- or heteroaryl-substi-
tuted acetone was used in an initial aldol condensation
with 2-formyl-6-methylpyridine, followed by Lewis acid
mediated addition of a polymer-supported sulfonyl-
hydrazide to affect cyclization and provide the 5-methyl-
substituted pyrazole ring system.
Ta ble 2. In Vitro Activity of 4-Heteroaryl-Substituted Pyrazole TGF-â RI Kinase Inhibitors
a
a
a
compd
TGF-â RIK, IC50
,
µM (n)
p3TP Lux, IC50
,
µM (n)
NIH 3T3, IC50
,
µM (n)
p38 MAPK, inhibition,b µM
1
2
3
4
5
6
7
8
9
0.051 ( 0.0015 (258)
0.034 ( 0.003 (4)
0.382 ( 0.045 (4)
0.217 ( 0.011 (6)
0.091 ( 0.016 (5)
1.76 ( 0.160 (5)
0.031 ( 0.0035 (3)
0.150 ( 0.018 (3)
0.082 ( 0.0058 (3)
0.203 ( 0.046 (3)
0.353 ( 0.098 (4)
0.369 ( 0.044 (3)
0.047 ( 0.003 (55)
0.0029 ( 0.0004 (3)
1.20 ( 0.25 (7)
0.355 ( 0.041 (10)
0.094 ( 0.022 (6)
>10 (2)
0.089 ( 0.010 (68)
0.0071 ( 0.003 (4)
2.92 ( 0.70 (4)
0.524 ( 0.103 (6)
0.076 ( 0.011 (6)
4.67 ( 1.22 (5)
0.74
0.078
0.025
0.0068
1.3
2.3
0.11
7.7
0.25
0.28
0.40
0.51
0.0029 ( 0.0013 (3)
0.683 ( 0.164 (9)
0.024 ( 0.004 (5)
0.094 ( 0.013 (3)
1.66 ( 0.94 (4)
0.0012 ( 0.0001 (3)
1.68 ( 0.39 (4)
0.148 ( 0.046 (3)
0.538 ( 0.248 (3)
2.81 ( 0.48 (3)
10
11
12
0.399 ( 0.055 (3)
1.34 ( 0.09 (4)
a
b
Mean values ( SEM for a minimum of three determination. Single point determination.

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 3955
Ta ble 3. 4-Aryl-Substituted Pyrazole Inhibitors
compd
R1
R2
R3
R4
13
14
15
16
17
18
19
20
H
H
H
H
H
H
H
CH3
H
F
H
F
Cl
OH
F
H
H
H
H
H
H
F
H
H
CH3
CH3
CH3
CH3
CH3
CH3
OCH3
H
F igu r e 1. X-ray crystal structures of 1 and 18 bound to the
ATP-binding site of the TâR-I kinase domain.
kinase domain pharmacophore. The electronic disposi-
tion of the pyrazole ring also effects activity because the
“reversed” substitution pattern results in a 5-fold drop
in potency (8). Activity was somewhat maintained when
methyl or cyclopropyl was substituted for hydrogen at
the 5-position of the pyrazole ring (9 and 10), while
substitution of benzyl on the 1-position (12) led to lower
activity.
Assessing the SAR from each series suggests that the
minimum requirements for tight binding at the active
site consist of the presence of a 2-pyridyl ring on the
3-position of pyrazole and an aryl or heteroaryl sub-
stituent at the 4-position featuring a hydrogen bond
acceptor. This hypothesis is supported further by the
X-ray crystal structures of 1 and 18 bound to the ATP-
binding site of the TâR-I kinase domain (Figure 1). In
each structure, the pyridyl nitrogen is committed to a
hydrogen bond to a water molecule held in place by
additional hydrogen bonds to Asp-351, Glu-245, and
Tyr-249. For quinolin-4-yl 1, the quinoline nitrogen
atom also acts as a hydrogen bond acceptor from the
backbone N-H of residue His-283. This same inter-
action is also observed in the case of 18, where the
hydrogen bond acceptor is the oxygen of the hydroxyl
substituent. Also, there is enough flexibility at the active
site Asp-351 and Lys-232 side chains to allow the shift
needed to accommodate the additional spacing require-
ment demanded by the 4-substituted aryl substructure.
This hydrogen-bonding interaction corresponds to
that observed for the pyridyl nitrogen of SB203580 with
the N-H residue of Met-109 in the active site of p38
MAP kinase,14 while the pyrazol-3-yl substituent from
each of our series occupies the hydrophobic binding
pocket corresponding to that filled by the 4-fluorophenyl
group of known p38 inhibitors. The difference in TâR-
I/p38 selectivity observed for our two series is best
explained by the His-283 hydrogen-bonding interaction.
Owing to differences in the hydrogen-bonding environ-
ment attributed to sequence/structure variations, the
TâR-I hinge tolerates weaker acceptors, such as aro-
matic fluorine, while the more flexible p38 hinge
requires a stronger acceptor such as quinoline nitrogen.
Compounds in the quinolin-4-yl series exhibited a
range of activity as inhibitors of p38 MAP kinase, with
the most potent examples having phenyl in place of
pyridine (3 and 4), a result we found unsurprising given
the activity of known structurally related p38 inhibi-
tors.12 We addressed this issue of selectivity by substi-
tuting the quinolin-4-yl group with aryl while keeping
the pyridin-2-yl moiety intact (Table 4). While phenyl
analogue 13 exhibited a significant loss of activity in
kinase inhibition, the substitution of a 4-fluorophenyl
group provided 14, which possessed moderate activity
in the enzyme assay.13 Compound 15, the simple
6-methyl-substituted pyrindin-2-yl analogue of 13, ex-
hibited similar activity relative to 14. Combining the
observed SAR into 16 gave a potent inhibitor with an
IC50 in the enzyme assay of 70 nM and activity against
p38 of greater than 20 µM. Since incorporation of fluoro
at the 4-position of the phenyl group had such a
pronounced effect on activity, further substitutions were
examined. Although a chloro substituent (17) failed to
improve cellular activity relative to 16, hydroxyl-
substituted 18 proved to be extremely active in all
assays examined, while still maintaining moderate
selectivity against p38. Placement of an additional
fluoro at the 3-position of 16 (19) was detrimental, but
excellent activity and selectivity were observed with 20,
which incorporates a 4-methoxy group at phenyl and a
5-methyl group on the pyrazole ring.
Ta ble 4. In Vitro Activity of 4-Aryl-Substituted Pyrazole TGF-â RI Kinase Inhibitors
a
a
a
compd
TGF-â RIK, IC50
,
µM (n)
p3TP Lux, IC50
,
µM (n)
NIH 3T3, IC50
,
µM (n)
p38 MAPK, inhibition,b µM
13
14
15
16
17
18
19
20
3.99 ( 0.322 (5)
0.275 ( 0.022 (6)
0.543 ( 0.033 (4)
0.070 ( 0.005 (6)
0.057 ( 0.007 (6)
0.031 ( 0.004 (6)
0.151 ( 0.025 (5)
0.044 ( 0.004 (6)
>9.0 (3)
9.54 ( 3.6 (3)
>20
6.8
3.53 ( 1.69 (5)
2.59 ( 0.57 (4)
3.37 ( 0.474 (4)
0.139 ( 0.029 (5)
0.551 ( 0.145 (4)
0.044 ( 0.016 (4)
0.788 ( 0.246 (5)
0.047 ( 0.018 (5)
2.16 ( 0.037 (3)
0.041 ( 0.010 (4)
0.421 ( 0.104 (4)
0.032 ( 0.007 (4)
0.683 ( 0.163 (5)
0.061 ( 0.011 (3)
>20
>20
>20
0.56
>20
5.4
a
b
Mean values ( SEM for a minimum of three determination. Single point determination.

3956 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Letters
Transforming Growth Factor-â1 in Essential Hypertension. Am.
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Furthermore, the interactions of the pyridine nitrogen
present in both series with active site water molecules,
together with the presence of different hydrophobic
pocket gate-keeper residues (Ser-280 in TâR-I versus
Thr-106 in p38), would also be expected to affect
selectivity depending on how deeply the pyrazole-3-yl
group is embedded. This in turn may depend on whether
the backbone warhead is quinoline-4-yl or the more
sterically demanding 4-substituted phenyl group.
In summary, we have developed an interesting series
of pyrazole-based inhibitors of the TâR-I kinase domain.
Two subclasses of inhibitors, involving different sub-
stituents at the 4-position of the pyrazole ring, were
identified. The quinolin-4-yl series was found to have
good to excellent activity at the active site and in
relevant cellular assays but shown to possess only
moderate selectivity against p38 MAP kinase. Alterna-
tively, the aryl-substituted series exhibited equally good
in vitro activity with generally high selectivity against
p38 MAP kinase. The SAR of these compounds, together
with an active site model derived from selective enzyme/
inhibitor cocrystallization and X-ray crystal structure
analysis, will be used to further elucidate the pharma-
cophore of the TâR-I kinase domain.
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Su p p or tin g In for m a tion Ava ila ble: Representative ex-
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