Synthesis, crystal structure, and activity of pyrazole-based inhibitors of p38 kinase

Article abstract of DOI:10.1021/jm0611915

A series of pyrazole inhibitors of p38 mitogen-activated protein (MAP) kinase were designed using a binding model based on the crystal structure of 1 (SC-102) bound to p38 enzyme. New chemistry using dithietanes was developed to assemble nitrogen-linked s

Full text of DOI:10.1021/jm0611915

5712
J. Med. Chem. 2007, 50, 5712-5719
Synthesis, Crystal Structure, and Activity of Pyrazole-Based Inhibitors of p38 Kinase
Matthew J. Graneto,* Ravi G. Kurumbail,† Michael L. Vazquez, Huey-Sheng Shieh,† Jennifer L. Pawlitz,†,§
Jennifer M. Williams,† William C. Stallings,†,§ Lifeng Geng,§ Ashok S. Naraian,§ Francis J. Koszyk,§ Michael A. Stealey,§
Xiangdong D. Xu,§ Richard M. Weier,§ Gunnar J. Hanson,§ Robert J. Mourey,‡ Robert P. Compton,‡ Stephen J. Mnich,‡
Gary D. Anderson,‡ Joseph B. Monahan,‡ and Rajesh Devraj
Pfizer Global Research & DeVelopment, St. Louis Laboratories, 700 Chesterfield Village Parkway, Chesterfield, Missouri 63107
ReceiVed October 12, 2006
A series of pyrazole inhibitors of p38 mitogen-activated protein (MAP) kinase were designed using a binding
model based on the crystal structure of 1 (SC-102) bound to p38 enzyme. New chemistry using dithietanes
was developed to assemble nitrogen-linked substituents at the 5-position of pyrazoles. Calculated log D
was used in tandem with structure-based design to guide medicinal chemistry strategy and improve the in
ViVo activity of a series of molecules. The crystal structure of an optimized inhibitor, 4 (SC-806), in complex
with p38 enzyme was obtained to confirm the hypothesis that the addition of a basic nitrogen to the molecule
induces an interaction with Asp112 of p38R. A compound identified from this series was efficacious in an
animal model of rheumatic disease.
Introduction
p38 kinase is a member of the mammalian mitogen-activated
protein (MAP) kinase family which comprises important signal-
ing molecules that mediate cellular response to extracellular
signals such as environmental stress, growth factors, cytokines,
and bacterial endotoxins.1-4 Members of the MAP kinase family
are proline-directed serine/threonine kinases that share sequence
similarity and conserved structural domains. In addition to the
p38 kinase pathway, the MAP kinase superfamily also includes
two other parallel pathways: c-Jun amino-terminal kinases
(JNKs) and extracellular signal-regulated kinases (ERKs). Four
isoforms of p38 have been identified to date: p38R, p38â, p38γ,
and p38δ. On the basis of the size of the buried lipophilic pocket
at the ATP site, p38R and p38â kinases form one subgroup
while p38γ and p38δ kinases segregate as another subgroup.
Ever since the discovery of p38 kinase in mid-90s, the
pharmaceutical industry has aggressively pursued it as a target
for the development of disease-modifying antirheumatic drugs
(DMARDs).5-9 Cell-based experiments and preclinical studies
in animals have clearly demonstrated that inhibition of p38R
kinase results in significant attenuation of the production of
tumor necrosis factor alpha (TNFR) in response to bacterial
lipopolysaccharide (LPS) or other stimulants. Moreover, data
from early clinical trials in humans have corroborated these
Figure 1. Ribbon diagram of p38R complexed with 1. The inhibitor
preclinical findings.10
is bound at the ATP binding site located at the hinge region between
the two lobes of the enzyme. Crystals of p38R complex were obtained
In this paper, we describe a program to investigate structure-
activity relationships of the 5-position of an early lead molecule,
1 (SC-102),11 with nitrogen-linked substituents. Much of the
rationale for these efforts is based on the crystal structure of 1
bound to p38 enzyme (Figure 1). The binding mode of 1 (Figure
2a) was used to determine which of the positions on the
molecule might be amenable for further optimization of binding.
Our early work employed a 4-fluorophenyl group as shown in
1; however, structure acitivity relationships unrelated to this
manuscript12 led to our later work employing a 4-chlorophenyl
group to explore the structure-activity relationships of the
by hanging drop vapor diffusion methods. Diffraction data were
measured at the IMCA-CAT beamline at the Advanced Photon Source
to 1.65 Å resolution, and the structure has been refined with good
agreement between the data and the model (Rfree of 27.9% and Rwork
:
25.8%).
5-position of the pyrazole ring. In the process of this exploration,
we developed novel chemical methods to assemble 5-aminopy-
razoles.
Chemistry
* To whom correspondence should be addressed. Phone: 636-247-7355.
Fax: 636-247-2086. E-mail: matthew.j.graneto@pfizer.com.
† Structural and Computational Chemistry.
‡ Biological Sciences.
The pyrazole core structures in this paper were constructed
by two methods. In our early work, a thiosemicarbazide route13
was used. When not available commercially, thiosemicarbazides
214 were prepared by reacting an amine with thiophosgene and
§ No longer employed by Pfizer.
10.1021/jm0611915 CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/19/2007

Pyrazole-Based Inhibitors of p38 Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5713
We then sought a way to reliably prepare thioamide com-
pounds of our template. Several attempts to modify the
conditions of step b in Scheme 2 to produce more of the
thioamide resulted in complex mixtures and ketene aminal
formation. Finally, a new method that could cleanly prepare
thioamides of our template was found. We discovered that by
tying the thiomethyl groups of compound 6 together to form a
dithietane17,18 ring, the product 10 would cleanly react with 1
equiv of a secondary amine to produce the necessary thioamides
11 (Scheme 3). Further, not only could dithietanes be cleanly
prepared on our template, but purified thioamide compounds
would condense with hydrazine to produce the desired pyrazoles
in good yield (Scheme 3). This new procedure was scalable
and could be routinely carried out without chromatography.
Biological Results and Discussion
Our earlier work had identified 1 as a moderately potent
inhibitor of p38 MAP kinase (p38 enzyme IC50 ) 600 nM, log
D @ pH 7 ) 1.4, LPS-induced TNF production in rat (rLPS)
% inhibition @ 20 mpk ) 90).12 With an inhibitor-bound X-ray
structure of p38 enzyme in hand, we used 1 as a new lead for
further improvement. The inhibitor 1 has two aromatic groups
attached to a central pyrazole template. Analysis of our crystal
structure (Figures 1 and 2a) showed that the 4-fluorophenyl
group is bound in a deep lipophilic pocket of the enzyme,
completely shielded from the solvent. The pyridine ring of 1 is
enclosed in a hydrophobic pocket of the enzyme formed by
residues Val 38, Ala 51, His 107, Leu 108, Met 109, and Leu
167. Moreover, the nitrogen at the 4-position of the pyridine
ring accepts a hydrogen bond from the amide nitrogen of Met
109 (3.0 Å). The pyrazole group of 1 serves as a central
framework that helps to optimally position the two aromatic
groups in the ATP binding site of p38 kinase. One edge of the
pyrazole moiety is stabilized by interactions with the flexible
glycine flap (residues 31-36) that contains the consensus motif,
Gly-X-Gly-X-X-Gly where X is any amino acid.19 In addition,
one of the nitrogen atoms of the pyrazole ring directly interacts
(3.5 Å) with the carboxylate of Asp 168. However, the
5-position of the pyrazole ring is not involved in any key
interactions. Substitution of this 5-position should have the
correct trajectory to place substituents in the unoccupied solvent
region that could help to improve the physical properties of the
inhibitors. Moreover, the molecular electrostatic surface (Figure
3) indicates that a basic substituent appended to the 5-position
might also contribute to enhancing the potency by directly
engaging with the side chain of Asp 112.
While carbon-, oxygen-, and sulfur-linked substituents were
also investigated, this paper is focused on nitrogen-linked
substituents at the 5-position. First, we wanted to choose groups
for inclusion at the 5-position that were large enough to fill the
available space. We felt that by sweeping water out of the
pocket, a tighter binding inhibitor could be found.20-22 A variety
of lipophilic groups were prepared as shown in Table 1.
Compounds 12-16 demonstrate that large lipophilic groups are
tolerated by the enzyme. While these compounds show a modest
increase in potency over the initial lead 1, this did not translate
to antiinflammatory efficacy in an animal model, rLPS. We
postulated that this may be due to poor physical properties, such
as a high log D and low aqueous solubility.23-25
Figure 2. a. Close-up view of the ATP site of 1/p38R complex.
Interactions of 1 at the ATP site of p38R. Some of the key side chains
of p38R are displayed (C: green, N: dark blue, O: red, S: yellow,
and F: orange). The carbon atoms of the inhibitor are shown in gold
color. Some of the key hydrogen bonds are shown as dotted white lines.
Some of the bound solvent molecules near the ligand are shown as
pink spheres. The pyridyl nitrogen forms a hydrogen bond with the
peptide backbone of Met 109 while the central pyrazole interacts with
Asp 168. The carbon at C-5 of pyrazole is ∼6.5 Å away from the side
chain of Asp 112. b. Chemical structure of 1.
hydrazine (Scheme 1). Once prepared, the thiosemicarbazides
are then condensed with 2-chloro-1-(4-fluorophenyl)-2-(pyridin-
4-yl)ethanone 3,15 to produce a pyrazole via cyclocondensation
and desulfurization using methods similar to those described
by Pfeiffer et al.16
Although this method was productively used to make many
analogues, it suffers from several faults. First, it uses thiophos-
gene, which requires special handling. Second, it necessitates
the preparation of a thiosemicarbazide of each new desired
amine. And more significantly, it often provided poor yields
and intractable mixtures. We therefore sought a method that
would alleviate some of these problems.
In our initial attempts, a dithioketeneacetal group17 was
installed on 1-(4-fluorophenyl)-2-(4-pyridyl)ethanone, 5. The
thiomethyl groups were then displaced by thiomorpholine to
produce a ketene aminal, 7. With this intermediate in hand, we
expected nucleophilic hydrazine to condense with the keto group
of the ketene aminal compound 7 and then do an intramolecular
displacement of one of the amines to complete the cyclization
to the pyrazole (Scheme 2, upper path). Much to our surprise,
the ketene aminal compound 7 was inert to hydrazine under a
variety of conditions including high temperatures, extended
reaction times, or refluxing in neat hydrazine hydrate and did
not produce the desired pyrazole.
In the course of our experiments, we discovered some desired
product was formed in very low yield. We postulated that the
pyrazole product arose from incomplete formation of the ketene
aminal compound 7. The thioamide compound 8, carried on as
an impurity, was responsible for all of the pyrazole formation
(Scheme 2, lower path).
We then prepared a series of compounds with improved
physical properties. First, we tried to lower the log D, and second
we hoped to further increase potency by engaging nearby amino
acid residues with hydrogen-bonding potential. A careful
examination of the 1 crystal structure (Figure 2a) showed that

5714 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Scheme 1a Synthesis of Pyrazoles Using Thiosemicarbazide
Graneto et al.
a Reagents and conditions: (a) N-methylpiperazine, ether, -10°; (b) N2H4, dropwise in ether; (c) DMF, 100°
Scheme 2a Synthesis of Pyrazoles Using Dithioketeneacetals.
a Reagents and conditions: (a) NaH, CS2, CH3I; (b) thiomorpholine, toluene; (c) N2H4.
Scheme 3a Synthesis of Pyrazoles Using Dithietanes.
a Reagents and conditions: (a) CS2, CH2Br2, acetone, K2CO3; (b) N-methylpiperazine, toluene; (c) N2H4.
potent), they both show a better correlation between their
enzyme activity and antiinflammatory efficacy in the animal
model rLPS. Entries 19-21 also have a lower log D and
improved physical properties. In addition, all three compounds
contain a moiety with a basic nitrogen that could potentially
interact with Asp 112 of p38R. A direct comparison of
compounds 12 and 13 to compounds 21 and 4, respectively,
(nitrogen for carbon substitution in the piperazine vs piperidine
analogues) showed that the piperazine analogues provided an
additional increase in enzyme potency accompanied by an
increase in in ViVo antiinflammatory activity. Thus, compounds
19-21 and 4 offer both improved physical properties and greater
potency, which is confirmed by the high levels of activity in
the in ViVo antiinflammatory assay.
Our presumption that the basic nitrogen of compounds 19-
21 and 4 interacts with the side chain of Asp 112 was confirmed
when we solved the crystal structure of 4 complexed with p38R.
As expected, the pyridyl nitrogen interacts with the adenine
recognition element in the hinge region while the 4-chlorophenyl
group binds in the unique lipophilic pocket of p38R (Figure 4).
The two nitrogen atoms of the central pyrazole form solvent-
mediated interactions (2.5-2.8 Å) with the carboxylate of Asp
168. The piperazine ring at the 5-position of the pyrazole extends
toward the solvent surface displacing a bound water molecule
seen in the crystal structure of 1 (Figure 2a) and forms van der
Waals interactions with residues from the glycine-rich loop,
hinge region, and the C-lobe of p38R. The piperazine ring and
the pyrazole ring have a torsion angle of 36°. The terminal,
basic nitrogen of the piperazine ring forms a hydrogen bond to
Figure 3. Molecular electrostatic surface of 1/p38R complex. The
ligand is shown as a stick figure (C: pink, N: dark blue, H: white,
and F: orange). The surface (partially transparent) is color-coded based
on the electrostatic charge distribution. Blue represents positive charge
and red indicates negative charge. Some of the amino acid residues of
p38 are visible through the surface and are labeled. One of the striking
features is the presence of an intense, negative charge due to Asp 112
on the C-lobe of the enzyme, ∼6.5 Å away from the C-5 position of
the pyrazole of the inhibitor.
the side-chain carboxylate of Asp 112 is only 6.5 Å away from
the 5-position of the pyrazole. We also observed that the main-
chain carbonyl oxygen of Ser 154 is pointed toward the
5-position of the pyrazole (6.4 Å).
Compounds 17 and 18 (Table 2) are examples of compounds
with a reduced log D. While these two compounds are only
slightly better than 1 in the in Vitro assay (3-5 times more

Pyrazole-Based Inhibitors of p38 Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5715
Table 2. 5-Position Substituents with Lower Log Da
Table 1. 5-Position Lipophilic Substituentsa
a All compounds analyzed C, H, N. b Calculated using ACD software.
c Compounds dosed orally 4 h prior to LPS challenge.
a bound solvent molecule (2.8 Å) which in turn is engaged in
an interaction with the Asp 112 side chain (2.8 Å) and main-
chain oxygen of Ser 154 (2.6 Å).
a All compounds analyzed C, H, N. b Calculated using ACD software.
c Compounds dosed orally 4 h prior to LPS challenge.
Comparison of the crystal structure of 4 with similar
imidazole analogues reveals subtle differences in the orientation
and location of the substituents from the central heterocyclic
rings. For example, Wilson et al.26 have reported the crystal
structure of p38R kinase complexed with 23 (VK-19911, Kd )
15 nM) that contains a piperidyl moiety from a central imidazole
ring in an analogous position to the piperazyl group of 4 (Figure
5a). They observed that the piperidyl group forms a salt bridge
with the carboxylate group of Asp 168 and forms van der Waals
interactions with the side chains of Met 109 and Leu 167.
Similarly, Wang et al.27 have reported that the piperidine ring
in 22 (SB-220025, p38 enzyme IC50 ) 19 nM) forms a hydrogen
bond with one of the oxygen atoms of the side-chain carboxylate
of Asp 168 in its complex with murine p38R kinase. 22 is an
imidazole-based inhibitor that contains 2-aminopyrimidine as
the hinge-region recognition element (Figure 5a). Similar to 23,
the piperidine ring in 22 also forms an edge-to-face interaction
with the side chain of Tyr 35 from the flexible glycine loop.
Superposition of the crystal structures of p38 complexed with
4 and 22 (PDB accession code 1BL7) clearly show the different
locations of the piperazyl and piperidyl substituents in the two
compounds (Figure 5a). The two structures were overlaid using
a cluster of 13 CR atoms distributed around the ligand binding
site to minimize artifacts that arise during usual superposition
of kinase structures. The aromatic rings of the two inhibitors
do not superimpose exactly on each other because of subtle
conformational differences in the ATP site of the two complexes.
As a result, the piperazine group of 4 points in a slightly different
trajectory compared with the piperidyl substituent of 22. Also,
there are subtle changes in the conformation of the saturated
heterocyclic rings in the two compounds. Consequently, the
piperazyl nitrogen substituted by the methyl group of 4 is located
significantly closer to the side-chain carboxylate of Asp 112
(3.5 Å) than to that of Asp 168 (7.7 Å). In contrast, the
piperidine nitrogen of 22 forms a hydrogen-bonding interaction
with the carboxylate of Asp 168, similar to that observed in
the structure of 23.26 These structural differences illustrate that
although the imidazole-based inhibitors (22 and 23) and the
pyrazole-based compounds discussed in this manuscript have
similar chemical structures, there are significant differences in
their molecular interactions with the target enzyme.

5716 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Graneto et al.
Figure 4. Close-up view of 4/p38R complex. Interactions of 4 at the
ATP site of p38R. Atoms are colored as in Figure 2a and key hydrogen
bonds are shown as dotted white lines. Diffraction data were measured
at the IMCA-CAT beamline at the Advanced Photon Source to 1.8 Å
resolution, and the structure has been refined with good agreement
between the data and the model (Rfree of 27.5% and Rwork: 23.2%).
The central pyrazole ring interacts with Asp 168 via two bound solvent
molecules. The terminal piperazyl group forms water-mediated interac-
tions with Ser 154 and the side chain of Asp 112.
In ViWo Efficacy Models. Compounds with high levels of
activity in the p38 MAP kinase enzyme assay15 were routinely
advanced to in ViVo assays. The rLPS assay data shown in
Tables 1 and 2 was generated by ELISA analysis of TNFR from
serum samples collected 1.5 h after intravenous LPS challenge
of male Harlen Lewis rats. Compound administration was by
oral gavage 4 h prior to LPS challenge.15
Compound 4, with an efficacy of 98% @ 5 mpk in the rLPS
assay, was advanced to an animal model of rheumatic disease,
mouse collagen-induced arthritis (mCIA).28,29 The mCIA animal
model was chosen since the histopathology of joint inflammation
is similar to that in human RA, as is the pathogenic role of
TNFR and IL-1.30,31 Arthritis was induced in male DBA/1 mice
by subcutaneous injection of type II chick collagen in Freunds
adjuvant on days 1 and 21. Compound 4 was administered in
Methocel/Tween by oral gavage bid from days 21-56. Anti-
TNF antibody32 was administered ip once a week as a
comparator. As shown in Figure 6, both anti-TNF antibody and
4 at 15 and 30 mg/kg significantly decreased incidence of
arthritis in animals compared to vehicle treatment with p < 0.05
(Logistical Regression Model). Statistically, there were no
differences between animals treated with 4 (30 mg/kg) or anti-
TNF antibody.
Figure 5. a. Overlap of the crystal structures of p38R complexes with
4 and 22 from ref 27. Some of the key side chains of p38R are
displayed. The carbon atoms of the protein and ligand in the 4 structure
are shown in green while those in the 22 structure are shown in gold.
Nitrogen atoms in both structures are colored as dark blue, oxygen as
red, sulfur as white, chlorine as light blue, and fluorine as pink. The
piperidyl group in the 22 structure forms hydrogen-bonding interactions
with the carboxylate side chain of Asp 168. In contrast, the piperazyl
group in the 4 structure points toward the side chain of Asp 112 and
forms water-mediated interactions with the carboxylate side chain of
Asp 112 (See Figure 4). b. Chemical structures of 23, 4, and 22.
Figure 6. Inhibition of collagen-induced arthritis by 4. Mice treated
between days 21 and 56 with 4 (po bid) or anti-TNF antibody32 (ip
once/week) were evaluated for signs of arthritis. Groups marked with
an asterisk were significantly different from vehicle, p < 0.05, 8-10
animals per group.
Conclusion
A series of compounds were made to explore the structure-
activity relationships at the 5-position of pyrazole inhibitors of
p38 MAP kinase. New chemistry using dithietanes was discov-
ered to facilitate the exploration of the 5-position with nitrogen-
linked substituents. Crystal structure data for 1 was used to
determine the binding mode which helped to guide synthesis
and elucidation of structure-activity relationships. The com-
pounds disclosed show the progression of improved physical
properties and potency leading to 4, a potent inhibitor of p38
MAP kinase. The binding mode of 4 was confirmed by its
crystal structure bound to p38 enzyme, and 4 was shown to
suppress disease incidence at 30 mpk in an eight week model
of mouse collagen-induced arthritis.
Experimental Section
General. All reagents and solvents were of commercial quality
1
and used without further purification. H spectra were recorded
using a 300 or 400 MHz Varian NMR spectrometer. Chemical shifts
are reported in parts per million relative to an internal standard.
Melting points were determined using a Mettler FP81HT automated
melting point apparatus and are uncorrected. Microanalyses were
performed by Atlantic Microlabs Norcross, GA. Column chroma-
tography was performed on a Biotage preparative liquid chroma-
tography instrument using silica gel columns or on a Gilson HPLC
system using a 15 um 100A, C18 column (25 mm ID × 100 mm

Pyrazole-Based Inhibitors of p38 Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5717
L). Reported yields are not optimized, with emphasis on purity of
products rather than quantity.
stirred at room temperature for 24 h. An additional 2 equiv of
potassium carbonate and 1 equiv of carbon disulfide was added,
and the stirring was continued for another 24 h. Solvent was
removed, and the residue was partitioned between dichloromethane
and water. The organic layer was washed with brine, dried (MgSO4),
and filtered. The filtrate was concentrated, and the crude product
was stirred with 1 L of a mixture of ethyl acetate and ether (1:9)
to give pure product (78.4 g, 82%) as a yellow solid: mp 185.3-
185.4 °C; 1H NMR (acetone-d6/300 MHz) 8.49 (m, 2H), 7.31 (m,
4H), 7.09 (m, 2H), 4.39 (s, 2H). Anal. (C15H10ClNOS2) C, H, N.
1-(4-Chlorophenyl)-3-(4-methylpiperazin-1-yl)-2-pyridin-4-yl-
3-thioxopropan-1-one (11). A mixture of 10 (78.3 g, 0.24 mol)
and 1-methylpiperazine (75.0 g, 0.73 mol) in toluene (800 mL)
was heated to reflux for 2 h. Solvent and excess 1-methylpiperazine
was removed under vacuum, and the residue was triturated with a
mixture of ethyl acetate and ether (1:3) to give the product as yellow
4-[3-(4-Fluorophenyl)-1H-pyrazol-4-yl]pyridine (1). To a sus-
pension of 1-(4-fluorophenyl)-2-(4-pyridyl)ethanone15 (30 g, 0.14
mol) in THF (50 mL) was added dimethylformamide dimethylacetal
(50 mL). The mixture was stirred at room temperature for 48 h,
concentrated, and triturated with hexanes (150 mL) to produce a
yellow solid which was dissolved in ethanol (125 mL) and cooled
to 0 °C. Hydrazine hydrate (12.5 g, 0.25 mol) was added, and the
mixture was stirred at room temperature for 3 h. The mixture was
concentrated, dissolved in chloroform (200 mL), washed with
water (100 mL), and extracted with hydrochloric acid solution
(10%, 150 mL). The aqueous extract was treated with activated
charcoal (0.5 g) at 70 °C for 10 min, filtered through celite, and
neutralized with stirring and external cooling to pH 7-8 with
sodium hydroxide (20%). A fine white precipitate was filtered and
1
1
dried (27.3 g, 91%): mp 207.7-207.8 °C; H NMR (DMSO-d6/
crystals (53.0 g, 60%): mp 149-151 °C; H NMR (acetone-d6/
300 MHz) 13.4 (bs, 1H), 8.49 (d, J ) 2 Hz, 2H), 7.9 (bs, 1H),
7.34 (d, J ) 4.4 Hz, 2H), 7.27 (m, 2H), 7.16 (m, 2H). Anal. (C14H10-
FN3) C, H, N.
300 MHz) 8.58 (m, 2H), 7.95 (m, 2H), 7.54 (m, 4H), 6.54 (s, 1H),
4.37 (m, 2H), 4.1 (m, 2H), 3.92 (m, 1H), 2.49 (m, 2H), 2.33 (m,
1H), 2.20 (s, 3H), 1.99 (m, 1H). Anal. (C19H20ClN3OS) C, H, N.
4-[3-(4-Chlorophenyl)-5-(3,5-dimethylpiperidin-1-yl)-1H-pyra-
zol-4-yl]pyridine (12). This compound was prepared using the
methods described in the synthesis of 4, 10, and 11. mp 245.7-
1-[5-(4-Chlorophenyl)-4-(4-pyridinyl)-1H-pyrazol-3-yl]-4-me-
thylpiperazine (4). To a suspension of 11 (52.0 g, 0.14 mol) in
500 mL of dry tetrahydrofuran was added anhydrous hydrazine
(8.9 g, 0.28 mol) dropwise. The reaction mixture was stirred
at room temperature for 16 h. A pale yellow precipitate was
filtered and recrystallized from hot methanol to produce a white
1
247.3 °C; H NMR (DMSO-d6/400 MHz) 8.43 (d, J ) 6.0 Hz,
2H), 7.43 (d, J ) 8.8 Hz, 2H), 7.25 (d, J ) 8.4 Hz, 2H), 7.23 (d,
J ) 6.0 Hz, 2H), 3.08 (br d, J ) 10.0 Hz, 2H), 2.08 (dd, J ) 11.6
Hz and J ) 11.2 Hz, 2H), 1.67 (m, 2H), 0.71 (d, J ) 6.4 Hz, 6H).
ESHRMS m/z 367.1689 (M + H, C21H23ClN4 requires 367.1693).
Anal. (C21H23ClN4) C, H, N.
4-[3-(4-Chlorophenyl)-5-(4-methylpiperidin-1-yl)-1H-pyrazol-
4-yl]pyridine (13). This compound was prepared using the methods
described in the synthesis of 4, 10, and 11. mp 261.1-261.8 °C;
1H NMR (DMSO-d6/300 MHz) 11.6 (br s, 1H), 8.47 (m, 2H), 7.46
(m, 2H), 7.28 (m, 4H), 3.16 (d, J ) 12.0 Hz, 2H), 2.55 (br m, 2H),
1.58 (d, J ) 11.7 Hz, 2H), 1.43 (br m, 1H), 1.20 (m, 2H), 0.92 (d,
J ) 6.3 Hz, 3H). Anal. (C20H21ClN4) C, H, N.
2-[3-(4-Chlorophenyl)-4-pyridin-4-yl-1H-pyrazol-5-yl]decahy-
droisoquinoline (14). This compound was prepared using the
methods described in the synthesis of 4, 10, and 11. mp 231.4-
232.7 °C; 1H NMR (DMSO-d6/300 MHz) 12.7 (br s, 1H), 8.48 (d,
J ) 5.6 Hz, 2H), 7.45 (d, J ) 8.4 Hz, 2H), 7.27 (m, 4H), 3.08 (m,
2H), 2.65 (m, 2H), 1.2-1.7 (br m, 10H). ESHRMS m/z 393.1851
(M + H, C23H25ClN4 requires 393.1846). Anal. (C23H25ClN4) C,
H, N.
1
powder (30.2 g, 60%): mp 267-268 °C; H NMR (acetone-d6/
300 MHz) 11.8 (br s, 1H), 8.46 (m, 2H), 7.41 (m, 2H), 7.33 (br m,
4H), 2.96 (m, 4H), 2.41 (m, 4H), 2.22 (s, 3H). Anal. (C19H20ClN5)
C, H, N.
1-(4-Fluorophenyl)-3,3-bis(methylthio)-2-pyridin-4-ylprop-2-
en-1-one (6). To 1-(4-fluorophenyl)-2-(4-pyridyl)ethanone15 (5) (1.0
g, 4.7 mmol) in anhydrous THF (10 mL) was added a solution of
1 M potassium tert-butoxide in THF (10 mL, 10 mmol). The
reaction mixture was stirred for 15 min at room temperature, and
then carbon disulfide (0.31 mL, 5.1 mmol) was added. After several
minutes, methyl iodide (0.64 mL, 10.3 mmol) was added and the
reaction allowed to stir for 4 h. The reaction mixture was diluted
with saturated sodium bicarbonate solution (25 mL) and extracted
twice with ethyl acetate (35 mL). The combined ethyl acetate layers
were washed with water (25 mL) and brine (25 mL). The organic
solution was dried (MgSO4), filtered, and concentrated to an orange
oil. The oil solidified upon standing (1.4 g, 94%): mp 80.2-82.1
1
°C; H NMR (CDCl3/300 MHz) 8.59 (d, 2H), 7.96 (m, 2H), 7.38
(m, 2H), 7.14 (m, 2H), 2.33 (s, 3H), 2.23 (s, 3H). Anal. (C16H14-
FNOS2) C, H, N.
2-[3-(4-Chlorophenyl)-4-pyridin-4-yl-1H-pyrazol-5-yl]-1,2,3,4-
tetrahydroisoquinoline (15). This compound was prepared using
the methods described in the synthesis of 4, 10, and 11. mp 234.9-
235.8 °C; 1H NMR (DMSO-d6/300 MHz) 12.7 (br s, 1H), 8.50 (d,
J ) 4.5 Hz, 2H), 7.50 (d, J ) 8.4 Hz, 2H), 7.32 (m, 4H), 7.10 (m,
4H), 4.23 (s, 2H), 3.08 (m, 2H), 2.79 (m, 2H). ESHRMS m/z
387.1417 (M + H, C23H19ClN4 requires 387.1376). Anal. (C23H19-
ClN4) C, H, N.
1-[3-(4-Chlorophenyl)-4-pyridin-4-yl-1H-pyrazol-5-yl]decahy-
droquinoline (16). This compound was prepared using the methods
described in the synthesis of 4, 10, and 11. mp 226.7-228.0 °C;
1H NMR (DMSO-d6/300 MHz) 12.6 (br s, 1H), 8.44 (dd, J ) 4.8
Hz and J ) 1.6 Hz, 2H), 7.4-7.2 (br m, 4H), 2.96 (m, 3H), 0.7-
1.8 (br m, 13H). ESHRMS m/z 393.1842 (M + H, C23H25ClN4
requires 393.1846). Anal. (C23H25ClN4) C, H, N.
1-[3-(4-Chlorophenyl)-4-pyridin-4-yl-1H-pyrazol-5-yl]piperi-
din-3-ol (17). This compound was prepared using the methods
described in the synthesis of 4, 10, and 11. mp 233.9-234.4 °C;
1H NMR (DMSO-d6/300 MHz) 12.6 (br s, 1H), 8.45 (dd, J ) 4.5
Hz and J ) 1.5 Hz, 2H), 7.47 (d, J ) 7.8 Hz, 2H), 7.28 (m, 4H),
4.68 (d, J ) 4.5 Hz, 1H), 3.55 (m, 1H), 3.22 (br d, J ) 8.7 Hz,
1H), 2.99 (br d, J ) 11.7 Hz, 1H), 2.37 (m, 1H), 1.85 (m, 1H),
1.60 (m, 1H), 1.47 (m, 1H), 1.18 (m, 1H). ESHRMS m/z 355.1324
(M + H, C19H19N4ClO requires 355.1326). Anal. (C19H19ClN4O)
C, H, N.
4-[5-(4-Fluorophenyl)-4-pyridin-4-yl-1H-pyrazol-3-yl]thio-
morpholine (9). To 6 in toluene (6 mL) was added thiomorpholine
(502 µL, 5 mmol). The reaction mixture was heated between 80
and 110 °C. After about 3 h, 1-(4-fluorophenyl)-2-pyridin-4-yl-
3,3-dithiomorpholin-4-ylprop-2-en-1-one (7) began to precipitate
from the reaction mixture. When the starting material was con-
sumed, the reaction mixture was cooled to room temperature, and
the remaining 7 was removed by filtration. To the toluene solution
were added hydrazine hydrate (1 mL) and sufficient ethanol to
create a homogeneous solution. The reaction mixture was then
stirred at room temperature for 3 d. The reaction mixture was diluted
with ethyl acetate (50 mL) and extracted twice with water and once
with brine (25 mL). The organic solution was dried (MgSO4),
filtered, and concentrated to a reddish solid. The solid was triturated
with acetonitrile, collected by filtration, and dried in vacuo. The
dried material was recrystallized from ethanol, acetonitrile, and ethyl
acetate to produce a white solid (63 mg, 7%): 1H NMR (DMSO-
d6/300 MHz) 12.65 (br s, 1H), 8.45 (m, 2H), 7.27 (M, 6H), 3.14
(m, 4H), 2.63 (m, 4H). ESLRMS m/z 341 (M + H). ESHRMS m/z
341.1241 (M + H, C18H18FN4S requires 341.1236).
1-(4-Chlorophenyl)-2-(1,3-dithietan-2-ylidene)-2-pyridin-4-
ylethanone (10). To a solution of 1-(4-chlorophenyl)-2-(4-pyridyl)-
ethanone15 (70.0 g, 0.3 mol), dibromomethane (200 mL) and carbon
disulfide (25.9 g, 0.34 mol) in acetone (800 mL) was added
potassium carbonate (83.0 g, 0.6 mol). The reaction mixture was
1-[3-(4-Chlorophenyl)-4-pyridin-4-yl-1H-pyrazol-5-yl]piperi-
din-4-ol (18). This compound was prepared using the methods

5718 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Graneto et al.
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described in the synthesis of 4, 10, and 11. mp 268.8-268.9 °C;
1H NMR (DMSO-d6/300 MHz) 12.6 (br s, 1H), 8.45 (d, J ) 6.0
Hz, 2H), 7.40 (d, J ) 8.4 Hz, 2H), 7.30 (m, 4H), 4.65 (br d, J )
4.2 Hz, 1H), 3.55 (m, 1H), 3.11 (br d, J ) 12.6 Hz, 2H), 2.65 (dd,
J ) 9.9 Hz and J ) 10.5 Hz, 2H), 1.73 (d, J ) 10.2 Hz, 2H), 1.44
(m, 2H). ESHRMS m/z 355.1329 (M + H, C19H19N4ClO requires
355.1326). Anal. (C19H19ClN4O) C, H, N.
(8aS)-2-[3-(4-Chlorophenyl)-4-pyridin-4-yl-1H-pyrazol-5-yl]-
octahydropyrrolo[1,2-a]pyrazine (19). This compound was pre-
pared using the methods described in the synthesis of 4, 10, and
1
11. mp 226.1-228.8 °C; H NMR (DMSO-d6/400 MHz) 12.6 (br
s, 1H), 8.44 (dd, J ) 5.6 Hz and J ) 1.6 Hz, 2H), 7.43 (br d, J )
7.2 Hz, 2H), 7.25 (d, J ) 8.4 Hz, 2H), 7.22 (d, J ) 5.6 Hz, 2H),
3.21 (d, J ) 11.2 Hz, 1H), 3.02 (d, J ) 11.6 Hz, 1H), 2.92 (t, J )
7.6 Hz, 1H), 2.85 (d, J ) 10.8 Hz, 1H), 2.69 (m, 1H), 2.40 (m,
1H), 2.14 (m, 1H), 2.02 (m, 2H), 1.61 (m, 3H), 1.21 (m, 1H).
ESHRMS m/z 380.1589 (M + H, C21H22ClN5 requires 380.1642).
Anal. (C21H22ClN5) C, H, N.
1-[5-(4-Chlorophenyl)-4-pyridin-4-yl-1H-pyrazol-3-yl]-3-me-
thylpiperazine (20). This compound was prepared using the
methods described in the synthesis of 4, 10, and 11. mp 233.4-
1
234.7 °C; H NMR (DMSO-d6/400 MHz) 8.4 (d, J ) 8 Hz, 2H),
7.4 (d, J ) 11 Hz, 2H), 7.23-7.27 (m, 4H), 3.3 (m, 2H), 2.9 (m,
1H), 2.7 (m, 2H), 2.5 (m, 1H), 2.2 (m, 1H), 0.85 (d, J ) 8.8 Hz,
3H). Anal. (C19H20ClN5+1.3%H2O) C, H: calcd, 6.04; found, 5.52,
N.
1-[5-(4-Chlorophenyl)-4-pyridin-4-yl-1H-pyrazol-3-yl]-3,5-
dimethylpiperazine (21). This compound was prepared using the
methods described in the synthesis of 4, 10, and 11. mp 226.5-
228.1 °C; 1H NMR (DMSO-d6/400 MHz) 8.4 (d, J ) 7.6 Hz, 2H),
7.4 (m, 2H), 7.3 (m, 4H), 3.3 (m, 2H), 2.9 (m, 2H), 2.8 (m, 2H),
0.8 (d, J ) 8.4 Hz, 6H). Anal. (C20H22ClN5) C, H, N.
p38 Enzyme Assay. p38R kinase activity was determined by
monitoring the phosphorylation of epidermal growth factor peptide
(EGFRP) in the presence of [γ-33P]ATP as described previously.33
Reaction mixtures included 25 mM HEPES pH 7.5, 200 µM
EGFRP, and 50 µM ATP (this concentration represents Km levels
for ATP). Reactions were initiated by the addition of 10-20 nM
p38R previously activated with GST_MKK6 (p38R:MKK6, 100:
1) for 1 h at 30 °C in the presence of 50 µM ATP. Compounds
were tested over the range of 0.001 µM to 100 µM in 10% DMSO.
Reactions proceeded for 60 min and were terminated using Dowex
resin.
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Acknowledgment. Diffraction data for the inhibitor com-
plexes were collected at IMCA-CAT beamline 17-ID at the
Advanced Photon Source. Use of the IMCA-CAT beamline 17-
ID at the Advanced Photon Source was supported by the
companies of the Industrial Macromolecular Crystallography
Association through a contract with the Center for Advanced
Radiation Sources at the University of Chicago. Use of the
Advanced Photon Source was supported by the U.S. Department
of Energy, Office of Science, Office of Basic Energy Sciences,
under Contract No. W-31-109-Eng-38.
Supporting Information Available: Combustion analysis data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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