Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2) as an antiinflammatory target: Discovery and in vivo activity of selective pyrazolo[1,5- a ]pyrimidine inhibitors using a focused library and structure-based optimization approach

Article abstract of DOI:10.1021/jm300411k

A novel class of mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2) inhibitors was discovered through screening a kinase-focused library. A homology model of MAPKAP-K2 was generated and used to guide the initial SAR studies and to rationalize the observed selectivity over CDK2. An X-ray crystal structure of a compound from the active series bound to crystalline MAPKAP-K2 confirmed the predicted binding mode. This has enabled the discovery of a series of pyrazolo[1,5-a]pyrimidine derivatives showing good in vitro cellular potency as anti-TNF-α agents and in vivo efficacy in a mouse model of endotoxin shock.

Full text of DOI:10.1021/jm300411k

Article
pubs.acs.org/jmc
Mitogen-Activated Protein Kinase-Activated Protein Kinase 2
(MAPKAP-K2) as an Antiinflammatory Target: Discovery and in Vivo
Activity of Selective Pyrazolo[1,5-a]pyrimidine Inhibitors Using
a Focused Library and Structure-Based Optimization Approach†
Tomomi Kosugi,‡ Dale R. Mitchell,*,§ Aiko Fujino,‡ Minoru Imai,‡ Mika Kambe,‡ Shinji Kobayashi,‡
Hiroaki Makino,‡ Yohei Matsueda,‡ Yasuhiro Oue,‡ Kanji Komatsu,‡ Keiichiro Imaizumi,‡ Yuri Sakai,‡
Satoshi Sugiura,‡ Osami Takenouchi,‡ Gen Unoki,*,‡ Yuko Yamakoshi,‡ Vicky Cunliffe,§ Julie Frearson,§
Richard Gordon,§ C. John Harris,§ Heidi Kalloo-Hosein,§,⊥ Joelle Le,§,# Gita Patel,§ Donald J. Simpson,§
Brad Sherborne,§,▽ Peter S. Thomas,§ Naotaka Suzuki,‡ Midori Takimoto-Kamimura,‡
and Ken-ichiro Kataoka‡
‡Teijin Institute for Bio-medical Research, Teijin Pharma Ltd., Hino, Tokyo 191-8512, Japan
§BioFocus, Chesterford Research Park, Saffron Walden, Essex, CB10 1XL, U.K.
S
* Supporting Information
ABSTRACT: A novel class of mitogen-activated protein kinase-
activated protein kinase 2 (MAPKAP-K2) inhibitors was discovered
through screening a kinase-focused library. A homology model of
MAPKAP-K2 was generated and used to guide the initial SAR
studies and to rationalize the observed selectivity over CDK2. An
X-ray crystal structure of a compound from the active series bound
to crystalline MAPKAP-K2 confirmed the predicted binding mode.
This has enabled the discovery of a series of pyrazolo[1,5-a]
pyrimidine derivatives showing good in vitro cellular potency as anti-
TNF-α agents and in vivo efficacy in a mouse model of endotoxin shock.
pathway at the level of MAPKAP-K2. MAPKAP-K2 is a Ser/
Thr kinase located downstream of p38 MAP kinase and is
known to be a direct substrate of p38 MAP kinase.12−14 A role
for MAPKAP-K2 in mediating the inflammatory response has
been strongly implicated in the observed phenotype of the
MAPKAP-K2-deficient mouse (MAPKAP-K2−/−).13,15 This
mutant mouse is viable and seems normal except for a sig-
nificantly reduced inflammatory response. Mice deficient in
MAPKAP-K2 produce less TNF-α and IL-6 than control litter-
mates in response to lipopolysaccharide. In addition, MAPKAP-
K2 (−/−) mice are protected against lipopolysaccharide-
induced septic shock and collagen-induced arthritis.14−16 Their
size, weight, and lifespan are similar to wild-type littermates
when kept under germ-free conditions. In contrast to p38 MAP
kinase, MAPKAP-K2 is not known to participate in feedback
control loops that limit the production of proinflammatory
cytokines or control the production of antiinflammatory
cytokines.17
INTRODUCTION
■
Tumor necrosis factor-α (TNF-α) is implicated in several
inflammatory diseases such as rheumatoid arthritis,1 and bio-
logical agents such as infliximab, an anti-TNF-α antibody, have
proved to be very effective medications for this disabling
disease.2−5 Therefore, inhibition of TNF-α activity represents a
most promising target for antiinflammatory therapy. The p38
mitogen-activated protein (MAP) kinase pathway has been
shown to play an important role in the production of TNF-α
and other cytokines.6,7 This pathway has therefore been of parti-
cular interest for the discovery of new antiinflammatory agents.
Previous strategies to intervene in this pathway have been based
largely on the development of selective inhibitors of p38 MAP
kinase.8 Such inhibitors have proved to be effective inhibitors of
proinflammatory cytokine production in cell-based assays and
animal models of inflammatory disease. However, the p38 MAP
kinase gene knockout mouse has proven to be embryonically
lethal.9,10 Moreover, cells derived from such embryos have been
demonstrated to have a number of abnormalities in fundamental
cell responses. These observations may indicate that caution
should be paid to long-term therapy with p38 MAP kinase
inhibitors.11
As a molecular mechanism to explain its involvement in
inflammatory pathways, MAPKAP-K2 has been shown to
directly regulate TNF-α by binding to AU-rich elements and
Given these caveats, an attractive alternative strategy for the
development of antiinflammatory agents is the inhibition of this
Received: July 5, 2011
Published: July 2, 2012
© 2012 American Chemical Society
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Figure 1. Reported MAPKAP-K2 inhibitors.
Figure 2. MAPKAP-K2 inhibitors demonstrating in vivo efficacy.
thereby affecting its RNA stability and/or translation.18 Therefore,
MAPKAP-K2 could be a potentially more attractive target for
antiinflammatory therapy than p38 MAP kinase.
derivative (1), part of a small cluster of related hits that had
modest inhibitory activity against MAPKAP-K2. In this paper,
we report the discovery of this new class of MAPKAP-K2
inhibitors and describe the optimization of these pyrazolo
[1,5-a] pyrimidine derivatives to produce potent and selective
compounds.
The lack of clinical efficacy of all p38 MAP kinase inhibitors
to date is most likely a result of biologic adaptation which
results in the rapid reversal of early indications of benefit such
as CRP levels.19,20 Because p38 MAP kinase is highly pleio-
tropic in its ability to intersect and regulate both proinflam-
matory and antiinflammatory pathways including JNK, MAP
kinase phosphatase, IL-10, and negative feedback on TAK-1,
there are multiple routes and mechanisms to allow for reversal
of antiinflammatory effects. One could speculate that MAPKAP-K2,
by the nature of its more downstream and less pathway convergent
position in the network, could escape the majority of the com-
pensatory mechanisms proposed for p38 MAP kinase inhibitors.
This hypothesis however can only be proven by the development of
well propertied molecules for clinical testing.
RESULTS AND DISCUSSION
■
Screening against MAPKAP-K2 using a 100 000-member diverse
library gave only a very low hit-rate of ATP-competitive com-
pounds, and none of these were considered suitable for further
development. Thus, a second campaign was conducted using a
focused set of compounds designed to inhibit kinases;34 5075
compounds from 8 libraries, comprising 11 scaffold chemotypes,
were used in this second screen, and 32 active compounds were
identified and subjected to full IC50 determinations. The active
compounds were clustered around four distinct library scaffolds,
and these clusters showed clear evidence of SAR. Representative
compounds were subjected to kinetic determinations to ascertain
their competitivity with respect to ATP. The most promising hit
compound based on its activity, kinetics, and the potential for
follow up was the pyrazolo[1,5-a]pyrimidine 1.
Several groups have subsequently reported programs also
designed to develop antiinflammatory therapies through the
generation of inhibitors of MAPKAP-K2.21−30 Example com-
pounds illustrating the range of structure types reported as
inhibitors of MAPKAP-K2 are shown in Figure 1.
Recent reports have detailed the in vivo activity of MAPKAP-
K2 inhibitors, and three examples are shown in Figure 2.
Novartis has reported compounds that inhibited LPS-induced
TNFα release in mice and reduced paw swelling in a collagen-
induced arthritis study in both rats and mice.31,32 Pfizer has
reported oral antiinflammatory efficacy for PF-3644022 in both
acute and chronic models of inflammation in rats.33
At the inception of this project, there were no reported small
molecule inhibitors of MAPKAP-K2 on which to initiate a
program, and so it was decided to undertake a high-through-
put screening campaign using a diverse set of 100 000 com-
pounds. As this resulted in a very low hit rate, we next screened
a focused kinase library34 and identified a pyrazolo[1,5-a]pyrimidine
The pyrazolo[1,5-a]pyrimidine 1 exhibited inhibition of
MAPKAP-K2 (Figure 3), being marginally less potent than
staurosporine (Figure 4) but clearly competitive with respect
to ATP (Figure 5). The ATP concentrations of both the
MAPKAP-K2 and CDK2 assays were set at the Km value of
MAPKAP-K2, i.e., at 10 μM. In a focused panel of kinases,
compound 1 was found to inhibit CDK2 more potently than
MAPKAP-K2 and with either moderate or no activity against
the other kinases screened (Figure 3). It was therefore decided
to select CDK2 as a surrogate kinase for monitoring selectivity
during the optimization of the hit compound. The observed
activity against CDK2 was not unexpected given that the library
SFK03 was designed to target the CDK family of kinases.
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substituents at the 7-position, condensation or coupling reac-
tions were carried out before removal of the Boc groups as
shown in Schemes 3−5. This route was successfully applied to a
variety of 3-, 5-, and 7- substituted pyrazolo[1,5-a]pyrimidines.
Despite the 24-fold inverse selectivity with respect to CDK2,
and given the very low screening hit-rate for MAPKAP-K2,
compound 1 was considered to offer promising scope for opti-
mization. An evaluation of the ADME properties of compound
1 showed the compound to have good properties and to be
a suitable scaffold on which to undertake an optimization
program. To facilitate the medicinal chemistry efforts, it was
considered important to predict the likely binding modes of
compound 1 to MAPKAP-K2 and CDK2. However, at this
stage a crystal structure of MAPKAP-K2 was not available and
it was therefore necessary to build a homology model of MAPK
AP-K2 to enable docking studies to be carried out.
A homology model of MAPKAP-K2 was constructed based
on the reported X-ray structures of phosphorylase kinase35 and
cAMP-dependent protein kinase,36 each of which show ca. 30%
sequence identity with MAPKAP-K2 using the Biopolymer
Composer module in Sybyl, v6.8.37 As the central scaffold of
compound 1 is structurally very similar to purvalanol B, a
known inhibitor of CDK2 (Figure 6), compound 1 was docked
in the MAPKAP-K2 model such that the overall geometry was
as close as possible to that of purvalanol B in CDK2.38 In the
resulting docking mode, compound 1 appeared to form hydro-
gen bonds with the main chain NH and CO groups of Leu141
in the hinge region, and the terminal positively charged amine
of the trans-4-aminocyclohexylamino side chain was able to
form a hydrogen bond to Asn191 and a salt bridge to Asp207, as
shown in Figure 7.
Figure 3. Structure and kinase selectivity of hit compound 1.
As expected, the predicted binding mode of compound 1
with MAPKAP-K2 was very similar to that seen with CDK2.
However, the detailed differences in the residues within the
ATP-binding site of the two kinases suggested that the required
MAPKAP-K2 selectivity might be achieved by varying the side
chains while seeking to retain this overall binding mode. We
therefore embarked on a detailed SAR study of a series of
pyrazolo[1,5-a]pyrimidine analogues to identify the key
functionality responsible for activity against MAPKAP-K2 and
selectivity over CDK2.
Initial investigations of the effect of 2-position substitution of
the scaffold were carried out to test the predicted binding mode
of compound 1 with MAPKAP-K2. This mode suggested that
there was little available space to accommodate substituents in
this position, as there would be a steric clash with the ATP-site
backbone. Indeed, it was found that the introduction of sub-
stituents at this position resulted in significant losses in potency
against MAPKAP-K2 (Table 1: compounds 20 and 21).
The binding mode of compound 1 suggested that
substitution at the 3-position would offer a means of access-
ing the hydrophobic subpocket of the ATP site. We considered
that suitable substitutents at this position might lead both to
an improvement of MAPKAP-K2 activity and selectivity over
other kinases, based on the variability of the shape of the
hydrophobic subpocket compared with other more conserved
regions of the active site. However, all 3-substituted analogues
prepared in this study resulted in a reduction of activity against
MAPKAP-K2 (Table 1: compounds 22−24). These results
suggested that the hydrophobic subpocket in MAPKAP-K2 was
too small to accommodate any substituent at this position of
the ligand. Moreover, contrary to our expectations, we found
that compounds with halogens (Cl, Br) or a cyano group at this
Figure 4. IC50 analysis of staurosporine and compound 1. MAPKAP-
K2 assay performed at 30 μM peptide, 10 μM ATP, 0.5 μCi 33P-γ-
ATP/well for 10 min at room temperature. Data shown are mean
SEM of duplicate determinations.
Interference with the cell cycle via inhibition of cell cycle
kinases is, in any event, likely to be an undesirable feature in
antiinflammatory compounds.
Some minor modifications to the synthetic route used to
prepare the initial hit library (SFK03) from which 1 emerged
were undertaken to allow further substitution of this scaffold
and the inclusion of a wider range of substituents. The pre-
paration of the pyrazolo[1,5-a]pyrimidine derivatives described
is outlined in Scheme 1. Synthesis began with the condensa-
tion of commercially available 3-aminopyrazole (2) and
various malonate derivatives to construct the pyrazolo[1,5-a]-
pyrimidine core. The resulting dihydroxy derivative 3 was
converted to 5,7-dichloropyrazolo[1,5-a]pyrimidine (4) by
treatment with phosphorus oxychloride. For the syntheses of
the 3-halogenated compounds, the halogen was introduced at
this stage using N-chlorosuccinimide or N-bromosuccinimide
(Scheme 2). Selective introduction of various anilines at the
7-position to give 5 was achieved by heating with the respective
anilines in the presence of base. Following Boc protection to
give 6, the subsequent introduction of amines at the 5-position
was achieved by heating with an excess of the amine 7. For the
syntheses of the ether-linked derivatives at the 5-position, the
alcohol derivative 10 was introduced as the alkoxide (generated
from the alcohol and sodium hydride). Finally, deprotection
of the Boc-protected compounds (8 and 11) gave the tar-
get compounds 9 and 12. For further transformations of the
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Figure 5. Mechanism of action/Ki determination for compound 1. (a) Primary plot: Rate vs [ATP]. (b) Secondary Lineweaver−Burke plot of 1/V
vs 1/[ATP]. (c) Secondary plot of 1/Vmapp vs [I]. (d) Secondary plot of Kmapp/Vmapp vs [I]. MAPKAP-K2 assay was performed at 30 μM peptide
under a matrix range of concentrations of ATP (4−150 μM) and compound (0−20 μM) in the presence of 1 μCi 33P-γ-ATP/well for 10 min.
Reaction rates are expressed as femtomoles of peptide phosphorylated per minute per mU MAPKAP-K2. Data shown are mean of duplicate data
points; experiments were conducted across three independent determinations yielding Ki values of 7 μM, 7 μM, and 6 μM, respectively.
a
Scheme 1. General Synthetic Route
a
Reagents and conditions: (a) R6-CH(COOEt)2, EtONa, EtOH, reflux; (b) HCl aq; (c) POCl3, Me2NPh, reflux; (d) H2N-R7, Et3N, iPrOH, reflux;
(e) H2N-R7, NaH, DMF/THF, room temp to −50 °C; (f) Boc2O, DMAP (cat.), 1,4-dioxane, room temp; (g) excess H2N-R5 (7), 90 °C; (h) HO-R5
(10), NaH, THF, 0 °C to room temp; (i) TFA, CH2Cl2, room temp.
position showed more potent inhibitory activity against CDK2.
Recently we have confirmed that a cyano group at the 3-
position of such ligands interacts with the phenyl ring of the
Phe80 gatekeeper residue of CDK2 by solving the crystal
structure of compound 22 in complex with CDK2 (unpub-
lished data). Our observations of the improved potency against
CDK2 with substituents at this position are consistent with the
data published by other groups,39 whereby 3-substituents such
as CN, Pr, and Br improve potency against CDK2.
A significant breakthrough in selectivity for MAPKAP-K2 over
CDK2 was achieved when substituents were introduced at the 6-
position. As seen in Table 2, the addition of a small alkyl group
i
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Following up on these findings, we undertook a further
round of optimization of the substituents at the 7-position.
According to the predicted binding mode, it appeared that the
NH group in this position is involved in hydrogen bonding to
the carbonyl group of Leu141. Indeed, it was found that
methylation of the 7-position NH group led to a complete loss
in potency, which supports the involvement of this group in a
key binding interaction (Table 5: compound 50). Replac-
ing a phenyl group in the 7-position by alkyl or benzyl
groups led to a significant loss of potency (Table 5: com-
pounds 51−53), indicating the necessity for an aromatic ring
at the 7-position. The SAR of the 7-aryl group was explored
to further increase the activity against MAPKAP-K2. A positional
scan of the substituents on the 7-phenyl group indicated that para-
substitution was preferred and that a relatively large substit-
uent, whether electron-withdrawing (compound 54) or electron-
donating (compound 55) was tolerated. This is consistent with
the predicted binding mode which indicated that substituents at
the para-position of the phenyl group would be directed toward
the external surface out of the ATP binding site. Taking this into
account, we synthesized further analogues possessing hydrophilic
groups in the para-position but little improvement was achieved
(Table 6, compounds 59 and 60).
At this stage, we succeeded in solving the X-ray cocrystal
structure of the complex of (S)-44 with MAPKAP-K240 and
confirmed that the binding mode predicted from the homology
model was essentially correct. However, as a result of a con-
formational change in the flexible glycine-rich loop (aka P-
loop), the hydrophobic site in the region adjacent to the ligand
was somewhat larger than was previously predicted. On the
basis of this observation from the X-ray crystal structure, we
synthesized further derivatives intended to pick up additional
hydrogen bond interactions within this site. It was found to
be difficult to improve the potency dramatically by the intro-
duction of hydrogen bond donors and acceptors in the
7-position substituent. This is likely to be a consequence of
the site being not just a hydrophobic site but also being
exposed to the solvent surface. Analogues with bicyclic hetero-
aryl or biaryl groups in the 7-position were designed to expand
the contact surface with this hydrophobic site (Table 6). We
were thus able to identify compound 64 as the most potent
analogue in our series, and this compound showed a MAPKAP-
K2 inhibition activity of less than 100 nM with greater than
400-fold selectivity over CDK2.
Scheme 2
Reagents and conditions: (a) N-chlorosuccinimide or N-bromosucci-
nimide, CHCl3, room temp.
at the 6-position led to a dramatic drop in potency against
CDK2 even though the inhibitory activity against MAPKAP-K2
was significantly increased.
Although a range of smaller substituents at the 6-position are
tolerated, increased steric bulk at this position led to a pro-
gressive reduction of the inhibitory activity against MAPKAP-
K2. In conclusion, small alkyl groups such as methyl or ethyl
were found to be preferred.
Encouraged by the beneficial effects of substitution at the 6-
position, we investigated the importance of the trans-4-amino-
cyclohexylamino group at the 5-position. It was found that the
presence of the terminal amino group and its orientation and
distance from the scaffold were very critical for potency; in
particular, retaining the postulated salt bridge to Asp207 seemed
to be critical. Replacement of the linking -NH- group with the
-O- group was well tolerated (Table 3: compound 47). In
summary, replacement of the trans-4-aminocyclohexylamino group
with the ( )-3-piperidylamino group resulted in similar levels of
potency (Table 3: compound 38 vs compound 44) or a modest
improvement in potency (Table 3: compound 35 vs compound 1).
We subsequently showed that a methyl group at the 6-
position in combination with the ( )-3-piperidylamino group
at the 5-position was preferred over the ethyl or unsubstitu-
ted analogues. As shown in Table 4, compound 48 showed
MAPKAP-K2 inhibition with an IC50 of 0.51 μM and, more
importantly, improved selectivity over CDK2 compared to the
other compounds (35 and 49). Finally, for compound 44, we
showed that the (S)-enantiomer of the 3-piperidylamino group
exhibited better potency than the (R)-enantiomer at the 5-
position (Figure 8). An increase in MAPKAP-K2 potency of up
to 10-fold was achieved by the initial derivatization of the 5-,
6-, and 7-position substituents in compound 1 to provide
(S)-44. The compounds with a 3-piperidinylamino group at the
5-position were observed to have better selectivity against CDK-2
compared to those with a 4-aminocyclohexylamino group (com-
pound 35 vs 1 and compound 48 vs 25). As a result of this
improved selectivity, the 3-piperidinylamino group was selected as
the preferred 5-position substituent for further optimization studies.
As indicated above, we solved the X-ray cocrystal structure of
the complex of (S)-44 with MAPKAP-K2 at 2.9 Å resolution40
(PDB ID 3A2C). MAPKAP-K2: (S)-44 crystals belonged to
the space group of P212121, and there were 12 molecules in
each asymmetric unit. Surprisingly, it was found that each
Scheme 3
Reagents and conditions: (a) Pd(OH)2/C, H2, MeOH; (b) 2-methoxyethanol, PPh3, diisopropyl azodicarboxylate in toluene, THF, room temp;
(c) TFA, CH2Cl2, room temp.
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Scheme 4
Reagents and conditions: (a) NaOH aq, MeOH, room temp then HCl aq; (b) carbonyldiimidazole, cyclopentylamine, DMF, room temp; (c) TFA,
CH2Cl2, room temp.
Scheme 5
Table 1. IC50 Values for 2- or 3-Position Derivatives
MAPKAP-K2
IC50 (μM)
CDK2
IC50 μM)
selectivity CDK2/
MAPKAP-K2
compd
R2
R3
1
H
H
1.3
>300
>300
3.1
0.055
−
−
0.0040
0.0030
0.0034
0.042
−
−
0.0013
0.0004
0.0004
Reagents and conditions: (a) 5-acetyl-2-thiopheneboronic acid, Pd(OAc)2,
Na2CO3, PPh3, 1,4-dioxane/H2O; (b) TFA, CH2Cl2, room temp.
20
21
22
23
24
Me
tBu
H
H
H
CN
CI
Br
H
8.1
H
9.2
the (S)-44 molecule bound to the ATP-binding site is pre-
sumably the key interaction for the MAPKAP-K2 inhibitory
activity as the analogous pyrazolo[1,5-a]pyrimidine derivative 1
exhibited clear ATP-competitive inhibitory activity. On the other
hand, an assay to examine substrate competitivity failed to detect
any binding of (S)-44 in the substrate binding pocket. Therefore,
the binding of (S)-44 near the substrate-binding site is very weak
and does not appear to contribute to the inhibitory activity against
MAPKAP-K2. Interestingly, two recent reports indicate similar
behavior in p38α MAP kinase, where lipid ligands41 or small-
molecule p38α inhibitors42 bind to the C-terminal cap region
formed from the conserved MAP kinase insert, in the latter case in
Figure 6. Structure of purvalanol B.
enzyme complex bound two molecules of (S)-44. One mole-
cule of (S)-44 was bound to the ATP-binding site between the
N-terminal and C-terminal domains as expected, and the
other molecule of (S)-44 was bound near the substrate-
binding site of the C-terminal domain as shown in Figure 9.
Although there were two (S)-44 molecules in each complex,
Figure 7. Predicted binding mode of compound (1) in the MAPKAP-K2 homology model. Equivalent residue numbers in CDK2 are shown in
parentheses.
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Table 2. IC50 Values for 6-Position Derivatives
Table 4. IC50 Values for 6-Position Derivatives with
(rac)-3-Piperidylamino Group at the 5-Position
MAPKAP-K2 IC50
CDK2
IC50(μM)
selectivity CDK2/
MAPKAP-K2
compd
R6
(μM)
MAPKAP-K2
IC50 (μM)
CDK2 IC50
(μM)
selectivity CDK2/
MAPKAP-K2
compd
R6
1
H
l.3
0.055
11
0.042
27
35
48
49
H
0.40
0.51
0.70
0.70
33
1.8
65
25
26
27
28
Me
0.40
0.40
0.6
Me
Et
Et
3.6
9.0
nPr
Ph
2.1
3.5
24
34
1.6
6.0
3.8
addition to the ATP site as seen in this work; the binding affinity
at this second site is suggested to be greater than observed herein
for MAPKAP K2.42
We were able to use the binding mode of (S)-44 in the
ATP-binding site to rationalize the previously observed SAR
of this series of analogues. A schematic representation of the
observed interactions is shown in Figure 10, these being largely
consistent with those predicted from the homology model.
Compound (S)-44 makes two hydrogen bonds with the main
chain NH and carbonyl oxygen of Leu141 at the hinge region as
expected. The charged amino group of the 5-(3S)-piperidyl
moiety forms a salt bridge with the side-chain carboxyl group of
Figure 8. Determination of preferred enantiomer.
Table 3. IC50 Values for 5-Position Derivatives
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Table 5. IC50 Values for 7-Position Derivatives
Asp207 together with bidentate hydrogen bonds to the main chain
carbonyl oxygen of Glu190 (2.84 Å) and the carbonyl oxygen on
the side chain of Asn191 (3.10 Å). The other significant interaction
is the van der Waals contact between the 7-p-ethoxyphenyl group
and the sulfur atom of Cys140. According to this X-ray cocrystal
structure, there is little available space around the 3-position, and
this is consistent with the SAR observations at this position.
The cocrystal structure of MAPKAP-K2 and (S)-44 also
provided useful information for understanding the improve-
ment of selectivity for MAPKAP-K2 over CDK2. We found that
the Gly-rich loop took the α-helical conformation (α-form),
while other reported MAPKAP-K2 complexes with ADP,
staurosporine, or various small molecule inhibitors showed the
Gly-rich loop in a β-sheet conformation (β-form). As can be seen
in Figure 11, it was found that the α-form had a slightly larger
hydrophobic site compared to that of the β-form around the
7-position substituents. (S)-44 exhibits a nonplanar conforma-
tion due to the repulsion of the 7-substituent and the methyl
group at the 6-position, and this feature distinguishes it from
other MAPKAP-K2 inhibitors. The interaction with the
nonplanar form of (S)-44 induces the secondary structural change
of the Gly-rich loop from a β-sheet form to the α-helical form to
avoid the conflict of the substituent at the 7-position with Leu70.
As a result of this structural change, a new pocket composed of
Asp142, Cys140, Gln80, and Leu79 emerged and the p-ethoxyphenyl
group at the 7-position of (S)-44 bound in this new pocket.
Notably, a significant improvement in selectivity over CDK2
was observed in the compounds with an ortho-substituted
phenyl group at the 7-position; this trend became greater as
the group at the 6-position became larger (Table 7). The com-
bined effect of these two substituents is to reinforce the non-
planarity of the 7-substituent and, owing to the larger hydro-
phobic pocket in MAPKAP-K2 compared to CDK2, this leads to
an additive effect in terms of selectivity; however, the MAPKAP-
K2 potency of these analogues was generally reduced.
Recently, a structure of a CDK2−(S)-44 complex was
obtained in our laboratory. It was observed that the Gly-rich
loop of CDK2 adopted the β-sheet conformation, with (S)-
44 taking a distorted planar conformation to avoid a clash of
the 7-substituent with the Gly-rich loop as anticipated. More-
over, the 3-piperidylamino group at the 5-position did not make
a favorable interaction similar to that observed in the MAPKAP-
K2:(S)-44 complex between the charged amino group and
Asp207, Glu190, and Asn191 as shown in Figure 10.43
The kinase selectivity of (S)-44 was evaluated in a single
point assay at 10 μM against more than 100 kinases
(Supporting Information Table S1). The majority of kinases
(95) were inhibited by less than 30% at this concentration. Five
kinases were inhibited by >75%, MAPKAP-K2, CHK2, Yes,
Fes, and Flt3, with MAPKAP-K2 showing the highest levels of
inhibition. The inhibition data against a subset of related
kinases are summarized in Table 8. It was observed that (S)-44
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Table 6. IC50 Values for 7-Position Derivatives
Figure 10. The binding interaction of compound (S)-44 and
MAPKAP-K2.
Figure 9. Overlay of crystal structure of MAPKAP-K2 with (S)-44
(gray and red) and MAPKAP-K2 with ADP (blue and green).
There are two molecules of (S)-44 in each monomer complex. One
molecule of (S)-44 was bound to the ATP-binding site between the
N-terminal and C-terminal domains, and the other molecule of (S)-
44 was bound near the substrate-binding site of the C-terminal
domain. The Gly-rich loop of MAPKAP-K2 with (S)-44 took the
α-helix conformation (α-form: shown in red), while MAPKAP-K2
with ADP showed the Gly-rich loop in a β-sheet conformation
(β-form: shown in green).
exhibited very good levels of selectivity over this set of related
kinases. Three of the kinases inhibited modestly by the initial
hit compound 1, i.e., MEK1, PRAK, and ROKII (Figure 3),
were not inhibited to any significant extent by (S)-44.
The initial profiling round indicated that (S)-44 had
significant inhibitory activity (IC50 1.7 μM) toward CHK2
using a radiometric assay. The screening of further analogues
(compounds 26, 38, and 65) and the use of an orthogonal
assay platform confirmed that these compounds showed no
Figure 11. The ATP-binding pocket of (a) MAPKAP-K2 with (S)-44
(α-form) PDB ID 3A2C and (b) MAPKAP-K2 with ADP (β-form)
(PDB ID 1NY3) viewed from the same angle.
inhibition of CHK2 at 30 μM. While we were unable to
determine the reason for the different activities demonstrated
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Table 7. IC50 Values for 7-Position Ortho-Substituted
Derivatives
Table 10. In Vitro Cellular Potency and Pharmacokinetic
Profile
a
MAPKAP-K2
IC50(μM)
CDK2
IC50(μM)
selectivity CDK2/
MAPKAP-K2
compd
R6
65
66
67
H
0.2
1.7
1.5
0.39
6.4
2
3.8
Me
Et
>300
>200
a
Table 8. Kinase Selectivity of Compound (S)-44
kinase
inhibition (%)
kinase
inhibition (%)
CDK1/cyclinB(h)
CDK2/cyclinE(h)
ERK2(h)
12
13
7
AMPK(r)
CaMK2(r)
CaMK4(h)
CHK1(h)
PKBα(h)
p70S6K(h)
PDK1(h)
PKA(h)
35
41
−38
30
3
IKKβ(h)
7
JNK1α1(h)
JNK2α2(h)
p38α(h)
0
a
−33
4
−1
−9
0
Dose: 1 mg/kg iv (0.5% Tween 80 in saline) and 1 mg/kg po (0.5%
b
methyl cellulose) in male mice (three mice per group). Four-fold
p38ß(h)
−14
−2
3
diluted blood was used.
p38γ(h)
PKCα(h)
PKCγ(h)
ROCK-II(h)
Rsk2(h)
5
p38d(h)
−5
23
9
whole blood assays is typical of that seen in other similar
programmes and can be attributed to the higher ATP con-
centration in the cells (typically 1−10 mM) compared to that
in the enzymatic assay system (ATP concentration = 10 μM). It
was also confirmed by Western blot (Figure 12) that HSP27,
MKK4(m)
MKK6 (h)
MKK7ß(h)
MEK1(h)
15
24
26
19
PRAK(h)
62
a
10 μM, single-point assay; (h) = human, (m) = mouse, (r) = rat.
Inhibition of CHK2 by (S)-44 was measured using a Cyclex assay
(IC50 >30 μM).
by (S)-44 in the two assay formats, we concluded that the series
was not significantly active against CHK2.
In addition to demonstrating excellent selectivity over a panel
of kinases, the series retained the good ADME properties of
the initial hit compound 1 (i.e., high stability in human and
mouse liver microsomes, good permeability in Caco-2 cells). A
comparison of some ADME properties for 1 and (S)-44 are
shown in Table 9.
Figure 12. (S)-44 inhibits LPS-induced phosphorylation of HSP27 in
cells. THP-1 cells were preincubated with vehicle or test compound
prior to addition of LPS (10 μg/mL). Cell lysates were subjected to
SDS-PAGE analysis (equivalent protein load in each lane: 20 μg) and
transferred proteins subjected to immunoblotting with an antiphospho
HSP27 antibody.
Table 9. ADME Properties for 1 and (S)-44
1
(S)-44
solubility pH 7.4 (μg/mL)
75
<10
59
70
<10
11
human microsomes (CLint μL/min/mg)
mouse microsomes (CLint μL/min/mg)
Caco-2 permeability (Papp A to B × 10−6 cm/s)
mouse PPB (fraction bound)
the downstream substrate of MAPKAP-K2, was inhibited in
THP-1 cells treated with (S)-44 at 3 and 30 μM. These data are
primarily qualitative but do show indications of a concen-
tration-related effect of (S)-44 and provide evidence that this
compound was most likely acting ‘on-target’ on MAPKAP-K2
in cells. The pharmacokinetic properties of these compounds
were also evaluated in mice and showed good absorption after
oral administration (Table 10).
Compound (S)-44 was used to evaluate the effect of
MAPKAP-K2 inhibitors on the in vivo serum concentration
of TNF-α in a murine model of endotoxin shock. Vehicle
(5 mM HCl/saline) or compound (S)-44 at each dose (mg/kg
body weight) was administered orally into C57BL/6 mice
(200 μL/mouse). LPS (0.5 mg/kg) was administered
7.1
−
28.3
88.6%
Compounds (S)-44, 59, and 64 were further tested in a
cellular assay that measures LPS-stimulated TNF-α production
from THP-1 cells (human acute monocytic leukemia cell line)
and demonstrated good in vitro cellular potency as anti-TNF-α
agents with no cytotoxic effects evident (Table 10) (see
Supporting Information Figures S1−3). (S)-44 also demon-
strated inhibition of LPS-stimulated TNF-α production in
human and mouse whole blood assays. The observed drop-off
in potency between the enzymatic assay and the cell-based or
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intraperitoneally 30 min after the compound was injected. Sixty
minutes after the LPS injection, blood was collected through
the abdominal vein under barbiturate anesthesia. Serum was
collected, and the TNF-α concentration was measured by an
ELISA method. A dose-dependent decrease in serum TNF-α
concentration was observed in the mice treated with (S)-44 and
was similar to the response seen with BIRB 796 as standard
(Figure 13). In the mice dosed orally at 100 mg (S)-44/kg
provides confidence that this compound elicits its effect on
TNF-α secretion through inhibition of MAPKAP-K2.
EXPERIMENTAL SECTION
■
Commercial chemicals and solvents were of reagent grade and used
without further purification. Reactions sensitive to moisture or air were
performed under nitrogen using anhydrous solvents and reagents. The
progress of reactions was determined by either analytical thin layer
chromatography (TLC), performed with TLC Silica gel 60 F254, or
liquid chromatography−mass spectrometry (LC-MS). Silica gel
column chromatography was performed with the indicated solvent
and using silica gel 60. Chemical yields are not optimized. The purity
of all compounds screened in biological assays was determined to be
>95% by HPLC/MS analysis. NMR experiments were recorded on a
400 MHz machine and are referenced to residual solvent signals:
CDCl3 (δ 7.26) or DMSO-d6 (δ 2.49). Chemical shifts are reported in
δ units (parts per million) and are assigned as singlet (s), doublet (d),
doublet of doublets (dd), triplet (t), multiplet (m), broad signal (br),
or very broad signal (vbr). Coupling constants (J) are reported in hertz
(Hz). High-resolution mass spectrometry (HRMS) measurement was
performed on a LCMS-IT-TOF.
General Procedure A for the Synthesis of Compound 3. To a
stirred solution of sodium ethoxide (50 mmol) in EtOH (100 mL)
were added the appropriately 2-substituted malonic acid diester (20
mmol) and appropriately substituted 3-aminopyrazole (20 mmol).
The mixture was heated at reflux for 18 h, during which a precipitate
was formed. (In several cases where the substituent was an alkyl chain,
little or no precipitate was formed. In these situations, EtOH was
removed under reduced pressure. The residue was partitioned between
water and EtOAc, and the phases were separated. The aqueous phase
was acidified (pH 2) with 12 N HCl and back-extracted with EtOAc
(×2). The organic phase was washed with water and brine, dried over
anhydrous Na2SO4, and concentrated to give crude compound 3.) The
reaction mixture was cooled to room temperature, and the precipitate
was collected by filtration and washed with EtOH. The solid was dried
under vacuum. The dried solid was dissolved in water (100 mL), and
the resulting solution was acidified at pH 2 with 12 N HCl. The solid
was collected by filtration and washed with water to give a crude
compound 3. This crude product was used in the next reaction with-
out further purification.
General Procedure B for the Synthesis of Compound 4. The
crude compound 3 (10 mmol) was dissolved in phosphorus
oxychloride (20 mL), and N,N-dimethylaniline (1.0 mL) was added.
The mixture was heated at reflux for 18 h. Excess phosphorus
oxychloride was removed in vacuo. The residue was poured onto ice
(ca. 100 g) and extracted with EtOAc (×2). The combined organic
phase was washed with 1 N HCl several times, until N,N-dimethyl-
aniline was completely removed, and then washed with water and
brine. The organic phase was dried over anhydrous Na2SO4, concen-
trated in vacuo, and purified by silica gel column chromatography to
give compound 4.
Figure 13. The effect of compound (S)-44 on serum TNF-α
concentration in mouse endotoxin shock model. Statistical analysis was
carried out with the Dunnet’s multiple comparison test, with P < 0.05
considered as significant. *P < 0.05 and ***P < 0.001 (vs control).
body weight, the level of serum TNF-α was approximately 14%
compared with that of the vehicle-administered group. The
serum concentration of compound in the (S)-44-administered
group dose-dependently increased from 2, 5, 8, to 10 μM
following dosing at 10, 30, 60, and 100 mg/kg (Figure 12),
these concentrations being comparable to the IC50 values
obtained in the whole blood assay (mouse: IC50 = 5 μM). We
conclude that orally administered (S)-44 was absorbed and was
able to reach the concentrations necessary to effectively inhibit
TNF-α production in a manner similar to that observed in the
whole blood assay. Considering both the observed kinase
selectivity profile of (S)-44 and the observed repression of
HSP27 phosphorylation in the cell-based assay, the inhibition
of MAPKAP-K2 activity contributed to a decrease in the con-
centration of serum TNF-α. Further results of detailed preclinical
studies will be disclosed in a forthcoming publication.
CONCLUSIONS
■
We have demonstrated that novel pyrazolo[1,5-a]pyrimidine
compounds are useful as MAPKAP-K2 inhibitors. SAR studies
were conducted to optimize this series of pyrazolo[1,5-a]
pyrimidine analogues, resulting in the identification of the
highly potent and selective compound 64. The MAPKAP-K2
inhibitory activity of 64 was increased by more than 20-fold
over 1, and selectivity over CDK2 was improved by more than
10 000-fold. According to the structural analysis of the
MAPKAP-K2/(S)-44 complex, the effect of the 6-substituent
on the improvement of selectivity over CDK2 can be ratio-
nalized. Members of the pyrazolo[1,5-a]pyrimidine series exhi-
bited good in vitro cellular potency in LPS-induced TNF-α
secretion cell models and a favorable PK profile. Furthermore,
we have also demonstrated the efficacy of (S)-44 in reducing
TNF-α production in the murine endotoxin shock model. The
excellent in vitro kinase selectivity of (S)-44 (see Supporting
Information Table S1) plus its demonstrated inhibition of
HSP27 phosphorylation, a direct substrate of MAPKAP-K2,
5,7-Dichloropyrazolo[1,5-a]pyrimidine (13). 3-Aminopyrazole
(4.2 g, 50 mmol) was reacted with diethyl malonate (8.1 g, 50 mmol)
according to the general procedures A and B. The crude product was
purified by silica gel column chromatography (hexane/EtOAc = 10:1)
to give the title compound (3.0 g, 16 mmol, yield: 32% for two steps).
1H NMR (CDCl3) δ: 8.23 (1H, d, J = 2.4 Hz), 7.00 (1H, s), 6.75 (1H, d,
J = 2.4 Hz). HRMS calcd for C6H4Cl2N3 (M + H)+, 187.9777; found,
187.9768.
5,7-Dichloro-6-methylpyrazolo[1,5-a]pyrimidine (4e). 3-Ami-
nopyrazole (2.2 g, 26 mmol) was reacted with diethyl methylmalonate
(4.6 g, 26 mmol) according to the general procedure A and B. The
crude product was purified by silica gel column chromatography
(hexane/EtOAc = 10:1) to give the title compound (2.2 g, 11 mmol,
1
yield: 42% for two steps). H NMR (CDCl3) δ: 8.16 (1H, d, J =
2.4 Hz), 6.71 (1H, d, J = 2.4 Hz), 2.56 (3H, s). HRMS calcd for
C7H6Cl2N3 (M + H)+, 201.9933; found, 201.9935.
General Procedure C for the Synthesis of Compound 5. To a
stirred solution of a compound 4 (5.0 mmol) in 2-propanol (50 mL)
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containing triethylamine (10 mmol) was added the appropriate amine
(6.0 mmol). The reaction was heated for 15 h at 80 °C and cooled to
room temperature. In the case that a precipitate was formed, this was
collected by filtration and washed with MeOH. The solid was dried
under vacuum to give compound 5. This product was used in the next
reaction without further purification. In the case that a precipitate did
not form, the reaction mixture was cooled to room temperature, the
residue was partitioned between water and EtOAc, and the organic
phase was separated. The aqueous phase was extracted with EtOAc
(×2). The combined organic phase was washed with water and brine,
dried over anhydrous Na2SO4, concentrated in vacuo, and purified by
silica gel column chromatography to give compound 5.
General Procedure D for the Synthesis of Compound 5. To a
stirred suspension of sodium hydride (3.0 mmol) in DMF (10 mL)
were added the appropriate amine (2.4 mmol) and then a compound 4
(2.0 mmol) in THF (10 mL). The resulting mixture was stirred at
room temperature to 50 °C for 2 h. The reaction was quenched with a
saturated aqueous solution of NH4Cl. After extraction with EtOAc, the
combined organic phase was washed with water and brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The crude product was
purified by washing with MeOH or by silica gel column chromato-
graphy to give a compound 5.
was reacted with di-tert-butyl dicarbonate (0.44 g, 2.0 mmol)
according to the general procedure E. The crude product was purified
by silica gel column chromatography (hexane/EtOAc = 8/1) to give the
title compound (0.33 g, 0.83 mmol, yield: 83%). 1H NMR (CDCl3) δ: 8.16
(1H, d, J = 2.4 Hz), 7.45 (1H, dd, J = 6.3, 2.4 Hz), 7.26−7.22 (1H, m),
7.13 (1H, t, J = 8.5 Hz), 6.72−6.69 (2H, m), 1.37 (9H, s).
tert-Butyl N-{5-Chloro-6-methylpyrazolo[1,5-a]pyrimidin-7-
yl}-N-(4-ethoxyphenyl)carbamate (6m). 5-Chloro-N-(4-ethoxy-
phenyl)-6-methylpyrazolo[1,5-a]pyrimidin-7-amine (5n) (1.0 g, 3.3 mmol)
was reacted with di-tert-butyl dicarbonate (1.4 g, 6.6 mmol) according
to the general procedure E. The crude product was purified by silica
gel column chromatography (hexane/EtOAc = 8/1) to give the title
compound (1.1 g, 2.8 mmol, yield: 85%). 1H NMR (CDCl3) δ:
8.10 (1H, s), 7.25−7.20 (2H, m), 6.82 (2H, d, J = 8.8 Hz), 6.65
(1H, d, J = 2.0 Hz), 3.99 (2H, q, J = 6.9 Hz), 2.31 (3H, s), 1.39−1.32
(12H, m).
tert-Butyl N-[4-(Benzyloxy)phenyl]-N-{5-chloro-6-
methylpyrazolo[1,5-a]pyrimidin-7-yl}carbamate (6o). N-[4-
(Benzyloxy)phenyl]-5-chloro-6-methylpyrazolo[1,5-a]pyrimidin-7-
amine (5p) (0.36 g, 0.98 mmol) was reacted with di-tert-butyl
dicarbonate (0.43 g, 2.0 mmol) according to the general procedure E.
The crude product was purified by silica gel column chromatography
(hexane/EtOAc = 8/1) to give the title compound (0.37 g, 0.80 mmol,
yield: 82%). 1H NMR (CDCl3) δ: 8.10 (1H, d, J = 2.2 Hz), 7.41−7.31
(5H, m), 7.25−7.21 (2H, m), 6.90 (2H, d, J = 8.8 Hz), 6.65 (1H, d, J =
2.2 Hz), 5.02 (2H, s), 2.31 (3H, s), 1.30 (9H, s).
tert-Butyl N-{5-Chloro-6-methylpyrazolo[1,5-a]pyrimidin-7-
yl}-N-(2-methyl-1,3-benzothiazol-6-yl)carbamate (6u). N-{5-
Chloro-6-methylpyrazolo[1,5-a]pyrimidin-7-yl}-2-methyl-1,3-benzo-
thiazol-6-amine (5v) (0.32 g, 0.98 mmol) was reacted with di-tert-butyl
dicarbonate (0.43 g, 2.0 mmol) according to the general procedure E.
The crude product was purified by silica gel column chromatography
(hexane/EtOAc = 8/1) to give the title compound (0.29 g, 0.67 mmol,
yield: 68%). 1H NMR (CDCl3) δ: 8.12 (1H, d, J = 2.2 Hz), 7.88 (1H,
d, J = 8.8 Hz), 7.78 (1H, s), 7.35 (1H, d, J = 8.8 Hz), 6.69 (1H, d, J =
2.2 Hz), 2.82 (3H, s), 2.32 (3H, s), 1.35 (9H, s).
General Procedure F for the Synthesis of Compound 9. A
mixture of the compound 6 (1.0 mmol) and appropriate amine (1.0 g)
was heated at 80−85 °C for 18 h. If the amine was not liquid at
this reaction temperature, MeCN was added as a solvent. The crude
material was then partitioned between EtOAc and water. The organic
phase was then separated and washed with water and brine, dried
over anhydrous Na2SO4, and concentrated in vacuo. The residue
was dissolved in CH2Cl2 (5.0 mL) and TFA (3.0 mL). The reaction
mixture was stirred for 1 h at room temperature and then evaporated
in vacuo. The residue was partitioned between EtOAc and a saturated
aqueous solution of NaHCO3. The organic phase was separated,
washed with water and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by preparative HPLC.
To get a salt-free product, the purified product was dissolved in EtOAc
and washed with saturated aqueous solution of NaHCO3 and brine,
dried over anhydrous Na2SO4, and concentrated in vacuo to give the
title compound.
5-Chloro-N-(3-chloro-4-fluorophenyl)pyrazolo[1,5-a]-
pyrimidin-7-amine (5a). 5,7-Dichloropyrazolo[1,5-a]pyrimidine
(13) (0.51 g, 2.7 mmol) was reacted with 3-chloro-4-fluoroaniline
(0.47 g, 3.3 mmol) according to the general procedure C to give the
1
title compound (0.69 g, 2.3 mmol, yield: 85%). H NMR (CDCl3) δ:
8.07−8.04 (2H, m), 7.48−7.45 (1H, m), 7.32−7.28 (2H, m), 6.54−
6.53 (1H, m), 6.18 (1H, s). HRMS calcd for C12H8Cl2FN4 (M + H)+,
297.0105; found, 297.0107.
N-[4-(Benzyloxy)phenyl]-5-chloro-6-methylpyrazolo[1,5-a]-
pyrimidin-7-amine (5p). 5,7-Dichloro-6-methylpyrazolo[1,5-a]-
pyrimidine (4e) (0.25 g, 1.2 mmol) was reacted with 4-benzyloxyani-
line (0.30 g, 1.5 mmol) according to the general procedure C to give
the title compound (0.36 g, 0.98 mmol, yield: 82%). 1H NMR
(CDCl3) δ: 8.07 (1H, s), 8.00 (1H, d, J = 2.4 Hz), 7.46−7.35 (5H, m),
7.13−7.09 (2H, m), 7.02−6.98 (2H, m), 6.49 (1H, d, J = 2.4 Hz), 5.09
(2H, s), 1.90 (3H, s). HRMS calcd for C20H18ClN4O (M + H)+,
365.1164; found, 365.1153.
Ethyl 3-({5-Chloro-6-methylpyrazolo[1,5-a]pyrimidin-7-yl}-
amino)benzoate (5r). 5,7-Dichloro-6-methylpyrazolo[1,5-a]-
pyrimidine (4e) (0.25 g, 1.2 mmol) was reacted with ethyl 3-amino-
benzoate (0.25 g, 1.5 mmol) according to the general procedure C to
1
give the title compound (0.32 g, 0.98 mmol, yield: 82%). H NMR
(CDCl3) δ: 8.10 (1H, s), 8.02 (1H, d, J = 2.2 Hz), 7.92−7.90 (1H, m),
7.77−7.75 (1H, m), 7.48 (1H, t, J = 7.9 Hz), 7.28 (1H, dd, J = 7.9,
2.2 Hz), 6.55 (1H, d, J = 2.2 Hz), 4.40 (2H, q, J = 7.2 Hz), 1.95 (3H,
s), 1.41 (3H, t, J = 7.2 Hz). HRMS calcd for C16H16ClN4O2 (M + H)+,
331.0956; found, 331.0949.
5-Chloro-N-(2-chlorophenyl)-6-methylpyrazolo[1,5-a]-
pyrimidin-7-amine (5x). 5,7-Dichloro-6-methylpyrazolo[1,5-a]-
pyrimidine (4e) (0.25 g, 1.2 mmol) was reacted with 2-chloroaniline
(0.19 g, 1.5 mmol) according to the general procedure D to give the
title compound (0.22 g, 0.77 mmol, yield: 64%). 1H NMR (CDCl3) δ:
8.04 (1H, d, J = 2.4 Hz), 7.95 (1H, s), 7.51 (1H, dd, J = 7.9, 1.5 Hz),
7.30 (1H, td, J = 7.9, 1.5 Hz), 7.18 (1H, td, J = 7.9, 1.5 Hz), 7.03 (1H,
dd, J = 7.9, 1.5 Hz), 6.56 (1H, d, J = 2.4 Hz), 1.95 (3H, s). HRMS
calcd for C13H11Cl2N4 (M + H)+, 293.0355; found, 293.0342.
General Procedure E for the Synthesis of Compound 6. To a
stirred solution of compound 5 (1.0 mmol) in 1,4-dioxane (5.0 mL)
was added di-tert-butyl dicarbonate (2.0 mmol) followed by a
catalytic amount of 4-dimethylaminopyridine. The reaction mixture
was stirred at room temperature for 3 h. If starting material was
detected by TLC, the reaction mixture was warmed to 60 °C and
stirred for further 3 h. The reaction mixture was concentrated in
vacuo. The residue was purified by silica gel column chromatography
to give compound 6.
5-N-(trans-4-Aminocyclohexyl)-7-N-(3-chloro-4-
fluorophenyl)pyrazolo[1,5-a]pyrimidine-5,7-diamine (1). tert-
Butyl N-(3-chloro-4-fluorophenyl)-N-{5-chloropyrazolo[1,5-a]-
pyrimidin-7-yl}carbamate (6a) (0.40 g, 1.0 mmol) was reacted
with trans-1,4-cyclohexanediamine (1.0 g, 8.8 mmol) according to the
general procedure F to get the title compound (0.20 g, 0.53 mmol,
1
yield: 53%). H NMR (DMSO-d6) δ: 7.80 (1H, d, J = 2.0 Hz), 7.60
(1H, dd, J = 6.8, 2.4 Hz), 7.48 (1H, t, J = 9.0 Hz), 7.42−7.38 (1H,
m), 6.77 (1H, d, J = 7.8 Hz), 5.94 (1H, d, J = 2.0 Hz), 5.57 (1H, s),
3.68 (1H, s), 2.85−2.80 (1H, m), 1.96−1.85 (4H, m), 1.34−1.11
(4H, m). HRMS calcd for C18H21ClFN6 (M + H)+, 375.1495; found,
375.1497.
7-N-(4-Ethoxyphenyl)-6-methyl-5-N-(S-piperidin-3-yl)-
pyrazolo[1,5-a]pyrimidine-5,7-diamine (44). tert-Butyl N-{5-
chloro-6-methylpyrazolo[1,5-a]pyrimidin-7-yl}-N-(4-ethoxyphenyl)-
carbamate (6m) (0.50 g, 1.2 mmol) was reacted with 3-amino-1-Boc-
piperidine (1.0 g, 5.0 mmol) according to the general procedure
tert-Butyl N-(3-Chloro-4-fluorophenyl)-N-{5-chloropyrazolo-
[1,5-a]pyrimidin-7-yl} carbamate (6a). 5-Chloro-N-(3-chloro-4-
fluorophenyl)pyrazolo[1,5-a]pyrimidin-7-amine (5a) (0.30 g, 1.0 mmol)
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F to get the title compound. For the syntheses of (rac)-44, (S)-44, and
(R)-44, ( )-3-amino-1-Boc-piperidine, (S)-3-amino-1-Boc-piperidine,
]pyrimidin-7-yl}and (R)-3-amino-1-Boc-piperidine were used respec-
tively (0.25 g, 0.68 mmol, yield: 57% for (S)-44. (rac)-44 and (R)-44
gave similar yields). 1H NMR (DMSO-d6) δ: 8.54 (1H, s), 7.75 (1H,
d, J = 2.0 Hz), 6.90−6.83 (4H, m), 6.05 (1H, d, J = 7.8 Hz), 5.98
(1H, d, J = 2.0 Hz), 4.17 (1H, s), 3.96 (2H, q, J = 6.8 Hz), 3.17 (1H,
d, J = 7.8 Hz), 2.96−2.93 (1H, m), 2.58−2.52 (2H, m), 1.91−1.86
(1H, m), 1.74−1.70 (1H, m), 1.65 (3H, s), 1.57−1.52 (2H, m), 1.30
(3H, t, J = 6.8 Hz). HRMS calcd for C20H27N6O (M + H)+,
367.2241; found, 367.2243.
vacuo. The residue was dissolved in CH2Cl2 (2.0 mL). To this solution
was added TFA (1.0 mL), and the reaction mixture was stirred for 3 h.
The solvent was removed in vacuo. The residue was purified by
preparative HPLC. To get a salt-free product, the purified product was
dissolved in EtOAc, washed with a saturated aqueous solution of
NaHCO3 and brine, dried over anhydrous Na2SO4, and concentrated
in vacuo to give the title compound (13 mg, 0.032 mmol, yield: 20%
1
for two steps from 16). H NMR (DMSO-d6) δ: 8.50 (1H, s), 7.73
(1H, d, J = 2.0 Hz), 6.91−6.84 (4H, m), 5.97 (1H, d, J = 2.0 Hz), 5.95
(1H, d, J = 7.8 Hz), 4.10−4.01 (3H, m), 3.64−3.62 (2H, m), 3.29
(3H, s), 3.07 (1H, dd, J = 11.7, 3.2 Hz), 2.85−2.80 (1H, m), 2.49−
2.42 (2H, m), 1.89−1.82 (1H, m), 1.67−1.64 (1H, m), 1.66 (3H, s),
1.59−1.41 (2H, m). HRMS calcd for C21H29N6O2 (M + H)+,
397.2347; found, 397.2338.
General Procedure G for Measurement of Enzyme Activity
Inhibition. Compound Preparation. Compounds were dissolved
in DMSO at a concentration of 10 mM and stored in aliquots at
−20 °C. Compounds in DMSO from these stock aliquots were
diluted in DMSO to produce the required range of 30× stock
solutions. These stock solutions were then subjected to 1:3
dilutions to prepare the required range of 10× stock solutions,
and 5 μL of each solution was used in each 50 μL reaction. A final
DMSO concentration of 3% was maintained throughout all
compound dilution series to maximize compound solubility.
Compounds were routinely tested at final concentrations ranging
from 300 μM to 0.001 μM but may have been tested at lower
concentrations depending upon their activity.
MAPKAP-K2 Assay. The kinase reaction was conducted in a round-
bottomed polypropylene 96-well plate. MAPKAP-kinase 2 was diluted
to 0.5 mU/μL in diluent buffer (50 mM Tris/HCl. pH7.5, 0.1 mM
EGTA, 0.1% (v/v) β-mercaptoethanol, 1 mg/mL BSA). Five
microliters of compound or 30% DMSO was added to each well
followed by 25 μL of substrate cocktail (final concentration: 10 μM
ATP, 30 μM peptide (KKLNRTLSVA), 0.5 μCi of 33P-γ-ATP (or
1 μCi for kinetic determinations) in 50 mM Tris pH7.5, 0.1 mM
EGTA, 10 mM Mg-acetate, and 0.1% β-mercaptoethanol). The
reaction was initiated with the addition of 20 μL of enzyme solution
per well or 20 μL of diluent buffer without enzyme. The plate was
shaken for 10 s and then left at room temperature for 30 min. The
reaction was terminated with 50 μL of 150 mM phosphoric acid. An
amount of 90 μL of the reaction mixture was then transferred into a
96-well P81 filter plate (Whatmann) and incubated at room
temperature for 5 min. The filter plate was then washed four times
with 200 μL of 75 mM phosphoric acid per well on a plate vacuum
manifold (Millipore) and dried in an oven for 2−3 h. Packard
MicroScint ‘0’ (30 μL) was then added to each well, and the plate was
mixed for 30 min and subjected to liquid scintillation counting on a
Packard TopCount.
CDK2 Assay. The kinase reaction was conducted in a round-
bottomed polypropylene 96-well plate. CDK2 was diluted to
0.5 ng/μL in diluent buffer (50 mM Tris/HCl. pH 7.5, 0.1 mM EGTA,
0.1% (v/v) β-mercaptoethanol, 1 mg/mL BSA). Five microliters of
compound or 30% DMSO was added to each well followed by 25 μL
of substrate cocktail (final 10 μM ATP, 0.1 mg/mL histone type III-S,
0.2 μCi 33P-γ-ATP in 50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 2 mM
DTT, 10 mM MgCl2, 0.01% Brij-35). The reaction was initiated with
the addition of 20 μL of enzyme solution per well or 20 μL of diluent
buffer without enzyme. The plate was shaken for 10 s and then left at
room temperature for 60 min. The reaction was terminated with 50 μL
of 150 mM phosphoric acid. An amount of 90 μL of the reaction
mixture was then transferred into a 96-well P81 filter plate (Whatman)
and incubated at room temperature for 5 min. The filter plate was then
washed four times with 200 μL of 75 mM phosphoric acid per well on a
plate vacuum manifold (Millipore) and dried in an oven for 2−3 h.
Packard MicroScint ‘0’ (30 μL) was then added to each well, the plate
was mixed for 30 min and subjected to liquid scintillation counting on a
Packard TopCount.
6-Methyl-7-N-(2-methyl-1,3-benzothiazol-6-yl)-5-N-(S-piper-
idin-3-yl)pyrazolo[1,5-a]pyrimidine-5,7-diamine (64). tert-Butyl
N-{5-chloro-6-methylpyrazolo[1,5-a]pyrimidin-7-yl}-N-(2-methyl-1,
3-benzothiazol-6-yl)carbamate (6u) (0.15 g, 0.35 mmol) was reacted
with (S)-3-amino-1-Boc-piperidine (1.0 g, 5.0 mmol) according to the
general procedure F to get the title compound (66 mg, 0.17 mmol,
1
yield: 49%). H NMR (DMSO-d6) δ: 8.93 (1H, s), 7.76 (1H, d, J =
8.8 Hz), 7.74 (1H, d, J = 2.2 Hz), 7.37 (1H, d, J = 2.2 Hz), 7.10 (1H,
dd, J = 8.8, 2.2 Hz), 6.10 (1H, d, J = 7.8 Hz), 6.02 (1H, d, J = 2.2 Hz),
4.15−4.07 (1H, m), 3.12 (1H, dd, J = 12.0, 3.4 Hz), 2.88−2.85 (1H,
m), 2.72 (3H, s), 2.53−2.47 (2H, m), 1.88 (1H, s), 1.76 (3H, s),
1.72−1.65 (1H, m), 1.61−1.45 (2H, m). HRMS calcd for C20H24N7S
(M + H)+, 394.1808; found, 394.1807.
tert-Butyl (S)-3-[(7-{[(tert-Butoxy)carbonyl](4-ethoxyphenyl)-
amino}-6-methylpyrazolo[1,5-a]pyrimidin-5-yl)amino]-
piperidine-1-carboxylate (8a). A mixture of tert-butyl N-{5-chloro-
6-methylpyrazolo[1,5-a]pyrimidin-7-yl}-N-(4-ethoxyphenyl)carbamate
(6m) (0.40 g, 0.99 mmol) and (S)-3-amino-1-Boc-piperidine (2.0 g,
10 mmol) was heated together at 80−85 °C for 18 h. The reaction
mixture was cooled to room temperature and was directly purified by
silica gel column chromatography (hexane/EtOAc = 8/1) to give the
title compound (0.43 g, 0.76 mmol, yield: 77%). 1H NMR (CDCl3) δ:
7.84 (1H, d, J = 2.0 Hz), 7.29−7.26 (2H, m), 6.81−6.79 (2H, m), 6.16
(1H, d, J = 2.0 Hz), 4.22 (1H, s), 3.98 (2H, q, J = 7.0 Hz), 3.78−3.22
(4H, m), 2.02 (3H, s), 1.91−1.83 (2H, m), 1.73−1.61 (2H, m), 1.50−
1.30 (21H, m).
tert-Butyl (S)-3-[(7-{[4-(Benzyloxy)phenyl][(tert-butoxy)-
carbonyl]amino}-6-methylpyrazolo[1,5-a]pyrimidin-5-yl)-
amino]piperidine-1-carboxylate (15). tert-Butyl N-[4-(benzyloxy)-
phenyl]-N-{5-chloro-6-methylpyrazolo[1,5-a]pyrimidin-7-yl}-
carbamate (6o) (0.47 g, 1.0 mmol) was reacted with (S)-3-amino-1-
Boc-piperidine (2.0 g, 10 mmol) according to an analogous fashion
to 8a. The reaction mixture was purified by silica gel column
chromatography (hexane/EtOAc = 8/1) to give the title compound
(0.43 g, 0.68 mmol, yield: 68%). 1H NMR (CDCl3) δ: 7.84 (1H, d,
J = 2.0 Hz), 7.40−7.29 (7H, m), 6.88 (2H, dd, J = 8.8, 2.0 Hz),
6.16 (1H, d, J = 2.0 Hz), 5.01 (2H, s), 4.22 (1H, s), 3.82−3.16
(4H, m), 2.02 (3H, s), 1.88 (1H, s), 1.77−1.56 (3H, m), 1.48−1.29
(18H, m).
7-N-[4-(2-Methoxyethoxy)phenyl]-6-methyl-5-N-[(S)-piperi-
din-3-yl]pyrazolo[1,5-a]pyrimidine-5,7-diamine (59). To a
stirred solution of tert-butyl (S)-3-[(7-{[4-(benzyloxy)phenyl][(tert-
butoxy)carbonyl]amino}-6-methylpyrazolo[1,5-a]pyrimidin-5-yl)-
amino]piperidine-1-carboxylate (15) (1.0 g, 1.6 mmol) in MeOH
(50 mL) was added Pd(OH)2/C (50 mg, 10% on carbon) under
nitrogen atmosphere. The reaction mixture was stirred at room tem-
perature for 16 h under hydrogen atmosphere, and Pd(OH)2/C was
filtered off. The solvent was removed in vacuo to give the crude
alcohol product 16 (0.81 g, 1.5 mmol, yield: 94%). This crude product
was used in the next reaction without further purification.
To a stirred solution of crude product 16 (81 mg, 0.15 mmol), 2-
methoxyethanol (23 mg, 0.30 mmol), and triphenylphosphine (52 mg,
0.20 mmol) in THF (5.0 mL) was added a solution of diisopropyl
azodicarboxylate (43 mg, 0.21 mmol) in toluene (0.11 mL) at 0 °C.
The reaction mixture was stirred at room temperature for 10 h. The
reaction was quenched by addition of water. EtOAc was added, and
the organic phase was separated. The aqueous phase was extracted
with EtOAc (×2), and the combined organic phase was washed with
water and brine, dried over anhydrous Na2SO4, and concentrated in
LPS-Induced TNF-α Production in THP-1 Cells. THP-1 cells
(ATCC: TIB-202) were suspended in THP-1 media (90% RPMI 1640
containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L
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D-glucose, 10 mM HEPES, 1 mM sodium pyrubate, 0.05 mM 2-ME,
10% heat-inactivated FBS) at a density of 1 × 105 cells/mL. An
amount of 100 μL of THP-1 cell suspension was then plated in a
96-well tissue culture plate and incubated in a CO2 incubator for 2 h.
Cells were pretreated for 1 h in a CO2 incubator in the presence of a
further 40 μL of THP-1 media and 50 μL of test compound (serial
dilutions in DMSO). LPS was then added (10 μg/mL final), and the
cells were further incubated for 18 h. The cell supernatants were
harvested and measured for TNF-α concentration using a HTRF
Human TNF-α kit (CisBio). Compound effects on cell growth during
the assay period were assessed by assaying residual cells with the
CellTiter 96 Aqueous One Solution Reagent (Promega).
Present Addresses
⊥GlaxoSmithKline Medicines Research Centre, Gunnels Wood
Road, Stevenage, Hertfordshire, SG1 2NY, U.K.
#Computational and Structural Chemistry, GlaxoSmithKline
Medicines Research Centre, Gunnels Wood Rd., Stevenage,
Hertfordshire, SG1 2NY, U.K.
▽Merck Research Laboratories, 2000 Galloping Hill Rd.,
Kenilworth, NJ 07033.
Notes
The authors declare no competing financial interest.
Whole Blood Assays. Mouse whole blood or human whole blood
(from two healthy and nonmedicated volunteers in the latter case) was
drawn and diluted four times in RPMI-1640. Diluted blood (360 μL
per well) was dispensed into 48-well plates and test compound
(0.4−100 μM) added in DMSO (20 μL). LPS (10 μg/mL) was added
prior to an overnight incubation. The concentrations of TNF-α in the
cell supernatants were measured by using an ELISA (R&D Systems,
Inc.).
LPS-Induced HSP27 Phosphorylation in THP-1 Cells. THP-1 cells
(5 × 105 cells/dish) were incubated in a CO2 incubator for 1 h. The
cells were then preincubated in the presence of test compound ((S)-
44: 3 μM and 30 μM; BIRB796: 0.1 μM) for 1 h. After addition of
LPS (10 μg/mL), the cells were further incubated for 1 h and then
washed with PBS. Following lysis at 4 °C for 15 min, lysates were
centrifuged and then subjected to SDS-PAGE analysis (20 μg protein/
lane) followed by protein transfer and immunoblotting with an
antiphospho-specific antibody (Cell Signaling Technology).
Pharmacokinetics. The iv and po pharmacokinetic profile were
investigated at a dose of 1 mg/kg in male mice. Formulations were
0.5% Tween 80 in saline for iv dosing and 0.5% methyl cellulose
aqueous solution for po dosing. The plasma concentration was mea-
sured by LC/MS/MS after serial blood sampling. The individual
pharmacokinetic parameters were calculated by noncompartmental
analysis and the mean parameters calculated. AUC0−24 is the area
under plasma concentration−time curve from time 0 to 24 h after oral
administration. CLtot is the plasma total clearance obtained from iv
administration.
Effect of (S)-44 on TNF-α Production in Murine Endotoxin Shock
Model. Vehicle (5 mM HCl/saline) or 10, 30, 60, or 100 mg/kg body
weight of (S)-44 (5 mM HCl/saline) (n = 6) was administered orally
to C57BL/6 mice (male, 8 week old; Charles River Japan). Thirty
minutes later, 0.5 mg/kg body weight of lipopolysaccharide (LPS;
E. coli. 055:B5, Sigma-Aldrich Co.) was administered intraperitoneally.
Sixty minutes after the LPS administration, blood was collected through
the abdominal vein under barbiturate anesthesia. Serum was prepared for
measurement of TNF-α concentration by the ELISA method (R&D
Systems, Inc.). Statistical analysis was carried out with the Dunnet’s
multiple comparison test, with P < 0.05 being considered as significant.
ABBREVIATIONS USED
■
MAPKAP-K2, mitogen-activated protein kinase-activated pro-
tein kinase 2; CDK2, cyclin dependent kinase 2; TNF-α, tumor
necrosis factor-α; MAP, mitogen-activated protein; SAR,
structure−activity relationship; Ser, serine; Thr, threonine;
mRNA, messenger ribonucleic acid; SFK, SoftFocus kinase;
Boc, butyloxycarbonyl; ATP, adenosine triphosphate; cAMP,
cyclic adenosine monophosphate; Leu, leucine; Asn, aspar-
agine; Asp, aspartic acid; Phe, phenylalanine; ADP, adenosine
diphosphate; Glu, glutamic acid; Cys, cysteine; CDK1, cyclin
dependent kinase 1; ERK2, extracellular signal-regulated kinase
2; IKKβ, inhibitor of nuclear factor kappa-B kinase subunit
beta; JNK1α1, c-Jun N-terminal kinase 1 alpha 1; JNK2α2,
c-Jun N-terminal kinase 2 alpha 2; p38α, mitogen-activated
protein kinase p38 alpha; p38β, mitogen-activated protein
kinase p38 beta; p38γ, mitogen-activated protein kinase p38
gamma; p38δ, mitogen-activated protein kinase p38 delta;
MKK4, mitogen-activated protein kinase kinase 4; MKK6,
mitogen-activated protein kinase kinase 6; MKK7β, mitogen-
activated protein kinase kinase 7 beta; MEK1, dual specificity
mitogen-activated protein kinase kinase 1; AMPK, 5′ adenosine
monophosphate-activated protein kinase; CaMK2, calmodulin-
dependent protein kinase 2; CaMK4, calmodulin-dependent
protein kinase 4; CHK1, checkpoint kinase 1; CHK2,
checkpoint kinase 2; PKBα, protein kinase B alpha; p70S6K,
70 kDa ribosomal protein S6 kinase 1; PDK1, 3-phosphoinosi-
tide-dependent protein kinase 1; PKA, protein kinase A; PKCα,
protein kinase C alpha; PKCγ, protein kinase C gamma;
ROCK-II, Rho-associated kinase 2; Rsk2, ribosomal protein S6
kinase 2; PRAK, p38-regulated and activated kinase; LPS,
lipopolysaccharide
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ASSOCIATED CONTENT
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(7) Lee, J. C.; Laydon, J. T.; McDonnell, P. C.; Gallagher, T. F.;
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