Synthesis and activity of new aryl- and heteroaryl-substituted 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole inhibitors of the transforming growth factor-β type I receptor kinase domain

Article abstract of DOI:10.1016/j.bmcl.2004.04.007

We have expanded our previously reported series of pyrazole-based inhibitors of the TGF-β type I receptor kinase domain (TβR-I) to now include new 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole analogues. Limited examination of the SAR of this new series in both e

Full text of DOI:10.1016/j.bmcl.2004.04.007

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3581–3584
Synthesis and activity of new aryl- and heteroaryl-substituted
5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole inhibitors of the
transforming growth factor-b type I receptor kinase domain
J. Scott Sawyer,a,* Douglas W. Beight,a Karen S. Britt,b Bryan D. Anderson,b
Robert M. Campbell,b Theodore Goodson, Jr.,a David K. Herron,a Hong-Yu Li,a
William T. McMillen,a Nicholas Mort,a Stephen Parsons,a Edward C. R. Smith,a
Jill R. Wagner,b Lei Yan,c Faming Zhanga and Jonathan M. Yinglingc
aDiscovery Chemistry Research and Technology, The Lilly Research Laboratories, Lilly Corporate Center,
Indianapolis, IN 46285, USA
bLead Optimization Biology, The Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA
cCancer Research, The Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 24 December 2003; accepted 2 April 2004
Abstract—We have expanded our previously reported series of pyrazole-based inhibitors of the TGF-b type I receptor kinase
domain (TbR-I) to now include new 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole analogues. Limited examination of the SAR of this new
series in both enzyme and cell based in vitro assays has revealed selectivity differences with respect to p38 MAP kinase (p38 MAPK)
depending on the nature of the ‘warhead’ group on the dihydropyrrolopyrazole ring. As with our original pyrazole series, phenyl
substituents tended to show greater selectivity against p38 MAPK than those comprised of the quinoline-4-yl moiety. We have also
achieved co-crystallization and X-ray analysis of compounds 3 and 15, two potent examples of this new series, with the TbR-I
receptor kinase domain.
Ó 2004 Elsevier Ltd. All rights reserved.
Transforming growth factor-b (TGF-b) has been pos-
tulated to be involved in a number of disease states,
including inflammation, fibrosis, cancer, asthma, and
cardiovascular disease.1 The complex function of this
cytokine, which mediates pathways involving the regu-
lation of gene response and DNA transcription factors,
is dependent upon the activation of the type I (TbR-I)
and type II (TbR-II) receptors, transmembrane-span-
ning proteins containing serine/threonine kinase
domains.2 Inhibition of these domains with small mol-
ecule inhibitors would be expected to interrupt down-
stream signal transduction and potentially provide
palliative effects on diseases modulated via TGF-b. Only
a few such inhibitors have been described in the litera-
ture, including a series of triaryl-substituted imidazole-
based compounds, members of which are selective
against p38 MAPK.3
Recently, we reported on a series of pyrazole-based
inhibitors of the TGF-b type I receptor kinase domain
(TbR-I) featuring either phenyl- or quinoline-4-yl war-
heads that were shown to hydrogen bond with the
peptide backbone in the ATP binding pocket.
Limited examination of the SAR of this series in both
enzyme and cell based in vitro assays resulted in the
emergence of two sub-series featuring differing selectiv-
ity versus p38 MAP kinase. Specifically, our group
was the first to report the co-crystallization and
X-ray analysis of 1 (LY364947, IC50 ¼ 51 nM) and 2
H
N
H
N
N
N
N
N
N
HO
* Corresponding author. Tel.: +1-317-276-7923; fax: +1-317-276-1417;
e-mail: jss.dcrt@lilly.com
1
2
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.007

3582
J. Scott Sawyer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3581–3584
(IC50 ¼ 31 nM) from each of these sub-series with the
TbR-I receptor kinase domain.4 Recently, the X-ray co-
crystallization analysis of 1 with the TbR-I receptor
kinase domain was independently confirmed.5
O
N
a,b
N
N
We now disclose a new platform based on the 5,6-di-
hydro-4H-pyrrolo[1,2-b]pyrazole scaffold that features
additional substitutions at the warhead ring systems as
well as the 2-pyridyl group. As with our earlier work,
compounds were evaluated as inhibitors of autophos-
phorylation of the isolated human TbR-I kinase domain
in the form of a constitutively active construct (T204D
mutation)6 produced in Sf9 insect cells and purified by
nickel-affinity chromatography. The synthesis and
evaluation of di-heteroaryl-substituted 5,6-dihydro-4H-
pyrrolo[1,2-b]pyrazole 3 revealed a potent inhibitor
(IC50 ¼ 104 nM, Table 1) and the platform was therefore
chosen for further SAR development. The resulting
compounds were also evaluated as inhibitors of TGF-b-
dependent luciferase production in mink lung cells
(p3TP Lux)7 and growth in mouse fibroblasts (NIH
3T3).8 Compounds were also evaluated as inhibitors of
p38 MAP kinase as described previously.4
3a
c
N
N
N
N
N
O
N
d
N
N
3
3b
Scheme 1. Representative synthesis of 4-(quinolin-4-yl)-substi-
tuted 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazoles: (a) LiHMDS, THF;
(b) methyl picolinate, 75% (for two steps); (c) 1-amino-2-pyrrolodi-
none, EtOH, py, 50%; (d) NaH, DMF, 50%.
The 4-(quinoline-4-yl)-substituted dihydropyrrolopy-
razole analogues (Table 1) were prepared as illustrated
for the synthesis of 3 (Scheme 1). Lepidine was deprot-
onated and condensed with an appropriate picolinic
ester to provide the intermediate ketone 3a, which was
subsequently condensed with 1-amino-2-pyrrolidinone
to provide the corresponding acyl hydrazone 3b. Base-
mediated cyclization of 3b provided final compound 3.
Sulfone 12 was prepared via Oxoneâ-mediated oxida-
tion of thioether 11, which was made in turn by treat-
ment of the corresponding 7-bromo-substituted
quinoline9 with ethanethiol. For the phenyl-substituted
examples (Table 2), the corresponding ketones were
made by deprotonation of the requisite benzyl nitriles
followed by condensation with picolinic ester and sub-
sequent decarboxylation (e.g., 15a, Scheme 2). Similar to
the quinolin-4-yl examples above, hydrazone formation
and cyclization provided the final target compounds.
The methoxy group in 21 was introduced via treatment
of 15 with methoxide, which could then be demethylated
with boron tribromide to provide 22.
Exploration of the SAR of the quinolin-4-yl compounds
(Table 1) revealed a number of interesting observations.
While good enzyme activity was observed with the
unsubstituted pyridyl-2-yl analogue 3, better overall
cellular activity was observed after the addition of a
6-methyl or 6-ethyl group (4 and 5). However, larger
branched groups at this position (6) led to reduced
activity. As in the original pyrazole series, substitution
Table 1. 4-(Quinolin-4-yl)-substituted 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole inhibitors
N
N
R1
N
R2
N
R3
Compd
R1
R2
R3
TGF-bRIK
IC50, lMa (n)
p3TP Lux
IC50, lMa (n)
NIH 3T3
IC50, lMa (n)
p38 MAPK
IC50, lMb
3
H
H
H
H
H
H
F
0.104 0.033 (4)
0.078 0.017 (5)
0.071 0.048 (3)
1.3
0.345 0.316 (11)
0.094 0.048 (8)
0.143 0.082 (3)
2.0
5.1 8.2 (4)
7.9
0.68
0.62
15
4
H
Me
Et
Pri
H
0.280 0.176 (3)
0.500 0.310 (3)
2.0
5
H
6
H
7
H
Cl
0.151 0.032 (4)
0.054 0.018 (4)
0.048 0.024 (3)
0.088 0.061 (3)
0.110 0.017 (3)
9.29
0.475 0.148 (3)
0.196 0.078 (4)
0.093 0.039 (5)
0.086 0.0017 (4)
0.285 0.127 (4)
>20
0.664 0.232 (3)
0.282 0.067 (3)
0.727 0.500 (4)
0.152 0.063 (3)
0.326 0.058 (3)
––
11
8
H
H
H
H
H
H
1.8
9
Cl
Me
Me
H
0.44
0.059
1.6
10
11
12
OEt
SEt
SO2Et
H
9.8
a Mean values SEM for a minimum of three determinations.
b 10-point IC50 determination.

J. Scott Sawyer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3581–3584
Table 2. 4-Phenyl-substituted 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole inhibitors
3583
N
N
N
R3
R1
R2
Compd
R1
R2
R3
TGF-bRIK
IC50, lMa (n)
p3TP Lux
IC50, lMa (n)
NIH 3T3
IC50, lMa (n)
p38 MAPK
IC50, lM
13
14
15
16
17
18
19
20
21
22
Me
F
H
H
H
F
Me
H
4.0
>20
>20
>20b
>20b
>20b
>20b
>20b
>20b
>20b
>20b
4.6c
1.32 0.291 (3)
0.175 0.088 (4)
0.343 0.185(4)
0.877 0.445 (4)
2.62 0.991 (4)
19.5
3.86 2.57 (4)
0.096 0.016 (3)
1.51 0.697(4)
2.55 1.15 (4)
11.8 3.73 (3)
>20
3.33 0.294 (3)
0.339 0.349 (4)
1.04 0.108 (3)
1.20 0.528 (3)
5.62 0.778 (3)
>20
F
Me
Me
CF3
CH2OH
Me
H
F
F
H
H
H
H
H
H
F
SO2Me
OMe
OMe
OH
0.646 0.288 (3)
0.121 0.074 (3)
0.047 0.006 (3)
6.83 2.74 (4)
0.304 0.069 (4)
0.022 0.0036 (4)
4.71 2.76 (3)
0.397 0.123 (3)
0.105 0.089 (4)
Me
Me
4.3c
a Mean values SEM for a minimum of three determinations.
b Single point determination.
c 10-point IC50 determination.
1 lM, with the 7-ethoxy-substituted analogue 10 dis-
playing an IC50 of 59 nM. Nitrogen substitutions at the
7-position have also been examined.10
O
CN
N
a,b,c
As discussed above, compounds in the quinoline-4-yl
series exhibited a range of activity as inhibitors of p38
MAPK. We addressed the issue of selectivity by
substituting the quinolin-4-yl group with phenyl while
keeping the pyridin-2-yl moiety intact (Table 2). While
the 4-methylphenyl analogue 13 exhibited a significant
loss of activity in kinase inhibition, the substitution of a
4-fluorophenyl group provided 15, which possessed
good activity in the enzyme assay. In addition, greatly
decreased activity against p38 was noted with these
compounds. Des-methyl 14 lost significant TbR-I
activity relative to 15. Lower cell activity was observed
with the addition of a second fluorine (16), and overall
activity was decreased when the 6-pyridyl substituent
was modified (17 and 18).
F
F
15a
d
N
N
N
N
O
N
N
e
R
F
15, R =F
15b
f
21, R = OMe
22, R = OH
g
As the above results were very similar to the original
pyrazole series, we decided to once again focus on
substitutions at the 4-position of the phenyl group.
While the strongly electron-withdrawing sulfonyl group
led to greatly decreased activity (19), substitution with
methoxy gave good activity (21), while the hydroxy
analogue 22 proved to be highly potent and selective
against p38 MAP kinase. Relative to 6-methyl-substi-
tuted 21, 6-hydrido-substituted 20 displayed decreased
activity, as predicted.
Scheme 2. Representative synthesis of 4-phenyl-substituted 5,6-dihy-
dro-4H-pyrrolo[1,2-b]pyrazole inhibitors: (a) NaH, THF; (b) 6-meth-
ylpicolinic methyl ester; (c) aq HCl, 50% for three steps; (d) 1-amino-
2-pyrrolodinone, AcOH, py; (e) NaH, DMF, 25% for two steps;
(f) NaOMe, 74%; (g) BBr3, CH2Cl2, 71%.
of fluorine at the 5-position of pyridine (7) maintained
good activity. The best overall activity was observed for
compounds having electron-donating substituents at the
7-position of the quinolin-4-yl moiety (8–11), while
oxidation of thioether 11 to the corresponding sulfone
12 led to a significant loss of activity. The general trend
observed with the quinolin-4-yl substituted compounds
was that greater activity against the TGF-b enzyme also
correlated with greater p38 MAP kinase activity. With
the exception of 8, activity below 100 nM against TGF-b
RI kinase corresponded to p38 activity of less than
Assessment of our previous pyrazole series suggested
that the minimum requirements for tight binding at the
active site consist of the presence of a 2-pyridyl group on
the 3-position of the pyrazole ring and an aryl or het-
eroaryl substituent at the 4-position featuring a hydro-
gen bond acceptor. This hypothesis is supported further
by the X-ray crystal structures of the 5,6-dihydro-4H-
pyrrolo[1,2-b]pyrazoles 3 and 15 bound to the ATP site

3584
J. Scott Sawyer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3581–3584
References and notes
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Figure 1. X-ray crystal structure of 3 (thin bonds) and 15 (thick bonds)
bound to the ATP-binding site of the TbR-I kinase domain.
4. Sawyer, J. S.; Anderson, B. D.; Beight, D. W.; Campbell,
R. M.; Jones, M. L.; Herron, D. K.; Lampe, J. W.;
McCowan, J. R.; McMillen, W. T.; Mort, N.; Parsons,
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of the TbR-I kinase domain (Fig. 1).11 In each structure,
the pyridyl nitrogen is committed to a hydrogen bond at
a water molecule held in place by additional hydrogen
bonds to Asp-351, Glu-245, and Tyr-249. For quinoline-
4-yl 3, the quinoline nitrogen also acts as a hydrogen
bond acceptor from the backbone N–H of residue His-
283. This same interaction is also observed in the case of
15, where the hydrogen bond acceptor is the fluorine
atom. There is enough flexibility at the active site to
allow the shift needed to accommodate the additional
spacing requirement demanded by the 4-substituted aryl
substructure. The selectivity observed for the 4-phenyl-
substituted series over p38 MAPK follows from the
argument we advanced in our earlier study of the pyr-
azole scaffold.4 Variations in the structure of the TbR-I
hinge region allow for the key His-283 hydrogen bond to
be formed with weaker acceptors, such as fluorine, while
the hinge region of p38 requires a stronger acceptor, in
this case quinoline nitrogen. There are also differences in
the hydrophobic pocket gate-keeper residues between
the two enzyme active sites that facilitate binding of the
4-phenyl-substituted series in the TbR-I domain in
preference to that of p38.
8. Leof, E. B.; Proper, J. A.; Goustin, A. S.; Shipley, G. D.;
DiCorleto, P. E.; Moses, H. L. Proc. Natl. Acad. Sci.
U.S.A. 1986, 83, 2453.
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G.; Goodson, T.; Herron, D. K.; Li, H.-Y.; Liao, J.;
McMillen, W. T.; Miller, S. C.; Mort, N. A.; Sawyer,
J. S.; Smith, E. C. R.; Yingling, J. M. PCT Int.
Appl. WO 0294833 A1, 2002; Chem. Abstr. 2002, 138, 4598.
10. Li, H.-Y.; Wang, Y.; Yan, L.; Campbell, R. M.; Ander-
son, B. D.; Wagner, J. R.; Yingling, J. M. Bioorg. Med.
Chem. Lett. 2004, 14, following paper in this issue.
doi:10.1016/j.bmcl.2004.04.065.
11. Crystals were grown in a hanging drop plate by mixing
4.5 mg/mL TbR-I kinase at a 1:1 (v/v) ratio with a solution
containing 30% (w/v) PEG-4000, 100 mM Tris–HCL,
pH 8.5, 200 mM Li2SO4, 5 mM DTT at room temperature.
Compounds were soaked into the crystals at 10 M excess
for 48 h. X-ray diffraction data were collected at )170 °C
with a Mar CCD detector at the IMCA beam line ID-17 at
Advanced Photon Source in Argonne National Labora-
tories. The structure was solved by molecular replacement
and refined by program CNX.
Acknowledgements
The authors thank Phil Iversen for statistical analysis
and Dave Clawson for Figure 1.