5-Amino-pyrazoles as potent and selective p38α inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.10.034

The synthesis and structure-activity relationships (SAR) of p38α MAP kinase inhibitors based on a 5-amino-pyrazole scaffold are described. These studies led to the identification of compound 2j as a potent and selective inhibitor of p38α MAP kinase with excellent cellular potency toward the inhibition of TNFα production. Compound 2j was highly efficacious in vivo in inhibiting TNFα production in an acute murine model of TNFα production. X-ray co-crystallography of a 5-amino-pyrazole analog 2f bound to unphosphorylated p38α is also disclosed.

Full text of DOI:10.1016/j.bmcl.2010.10.034

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6886–6889
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Amino-pyrazoles as potent and selective p38a inhibitors
⇑
Jagabandhu Das , Robert V. Moquin, Alaric J. Dyckman, Tianle Li, Sidney Pitt, Rosemary Zhang,
Ding Ren Shen, Kim W. McIntyre, Kathleen Gillooly, Arthur M. Doweyko, John A. Newitt, John S. Sack,
Hongjian Zhang, Susan E. Kiefer, Kevin Kish, Murray McKinnon, Joel C. Barrish, John H. Dodd,
Gary L. Schieven, Katerina Leftheris
Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, NJ 08543-4000, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and structure–activity relationships (SAR) of p38
a MAP kinase inhibitors based on a
Received 10 August 2010
Revised 4 October 2010
Accepted 6 October 2010
Available online 13 October 2010
5-amino-pyrazole scaffold are described. These studies led to the identification of compound 2j as a
potent and selective inhibitor of p38 MAP kinase with excellent cellular potency toward the inhibition
of TNF production. Compound 2j was highly efficacious in vivo in inhibiting TNF production in an
acute murine model of TNF production. X-ray co-crystallography of a 5-amino-pyrazole analog 2f bound
to unphosphorylated p38 is also disclosed.
a
a
a
a
a
Keywords:
p38 Inhibitors
Amino-pyrazoles
Ó 2010 Elsevier Ltd. All rights reserved.
Identification of potent and selective p38
a
inhibitors as clinical
converted to its acid chloride which was subsequently treated with
aniline 7 to afford 2 in excellent yield.4a
candidates for the treatment of inflammatory disorders has been
an area of continued interest in the pharmaceutical industry.1 We
recently disclosed a novel series of pyrazolo-pyrimidines as potent
The synthesis of amino-pyrazole analogs 2 with a secondary ami-
no substituent at C5 followed a similar route (Scheme 2). Accord-
ingly, alkylation of amino-pyrazole 6 with an alkyl bromide in THF
in the presence of sodium hydride formed amino-pyrazole 8.
Alternatively, diazotization of 5 with tert-butyl nitrite in acetonitrile
followed by addition of Cu(II) bromide formed a bromo derivative
which is treated with an alkyl amine in ethanol at elevated temper-
ature to form 8. Saponification, followed by coupling of acid 9 with
aniline 7 under similar conditions as described in Scheme 1 afforded
analogs 2.
and selective inhibitors of the p38
graphic studies of pyrazolo-pyrimidine 1 complexed with p38
a
MAP kinase.2 X-ray co-crystallo-
a
revealed some of the key binding interactions (Fig. 1). One of the
pyrazole nitrogens is engaged in a H-bond interaction with the back-
bone of hinge residue Met109. The aniline group occupies the hydro-
phobic selectivity pocket while the aniline NH forms a H-bond with
gate keeper Thr106 and the carboxamide NH with Glu71. Lack of any
productive interaction of the pyrimidine ring with the enzyme sug-
gested that the bicyclic core may be truncated to an amino-pyrazole
core 2 which could maintain all the key H-bond interactions.
In this Letter we describe the synthesis and structure–activity
relationship (SAR) studies in the 5-amino-pyrazole series 2 that
led to the identification of compound 2j as a potent and selective
The preliminary structure-activity optimization studies of the
amino-pyrazoles (2) are outlined in Tables 1–3. Compounds were
evaluated for their ability to inhibit phosphorylation of substrate
(myelin basic protein) by recombinant human p38
blood mononuclear cell based assay (hPBMC) was used to measure
a.
4b A peripheral
p38
a
inhibitor. Analog 2j was also highly efficacious in vivo in an
production in mice.
the ability of compounds to inhibit LPS-induced TNF
a production
acute pharmacodynamic model of TNF
a
in human primary cells.8
The synthesis of amino-pyrazole analogs 2 with a primary
amino substituent at C5 followed a general route as outlined in
Scheme 1. Condensation of hydrazine 3 with 2-ethoxymethylene-
malononitrile 4 in ethanol in the presence of triethylamine3
formed 5-aminopyrazole 5. Saponification of 5 with aq sodium
hydroxide in ethanol afforded carboxylic acid 6 which was coupled
with aniline 7 under standard peptide coupling conditions (HATU,
i-Pr2NEt, DMF) to form 2. Alternatively, the carboxylic acid 6 was
Table 1 summarizes some of the key SAR findings for carboxam-
ide modification with the N-1-phenyl-5-amino-pyrazole core. Both
the methyl and ethyl amide analogs 2a and 2b were found to be
equipotent to the pyrazolo-pyrimidine analog 1 in the p38a enzyme
and cellular assays. The corresponding cyclopropyl carboxamide 2c
also retained activity in both enzyme and cellular assays. A signifi-
cant improvement in biochemical potency and more importantly,
the cellular potency was observed with the N-methyl and N-ethyl-
pyrazole analogs 2d, and 2e, respectively. Finally, the isoxazole
amide 2f was found to be one of the most potent inhibitorwithin this
series.
⇑
Corresponding author. Tel.: +1 609 252 5068; fax: +1 609 252 6804.
E-mail address: jagabandhu.das@bms.com (J. Das).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.034

J. Das et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6886–6889
6887
Hydrophobic Pocket
Hydrophobic Pocket
Glu71
Glu71
Me
HN
Me
HN
Thr106
Thr106
H
N
H
N
R3
Me
Met109
O
O
Met109
N
O
N
N
N
1
N
2
5
N
1
NH
R2
R1
p38α IC50 = 10 nM
hPBMC TNFα IC50 = 67 nM
Figure 1. Binding mode of pyrazolo-pyrimidine 1 based on X-ray data. Proposed binding mode of amino-pyrazoles 2.
Based on the improved cellular potencies of the N-methyl-pyra-
zole (2d), N-ethyl-pyrazole (2e), and isoxazole carboxamides (2f),
further SAR optimization was focused on these analogs. Some of
the key SAR observations for N1 substitution are summarized in Ta-
ble 2. Replacement of the phenyl group in isoxazole carboxamide 2f
with a pyridine ring (2g) retained the biochemical potency in vitro.
However, analog 2g is eightfold less potent than 2f in the cellular as-
say. Substitution at either 2- or 4-position on the phenyl ring was
well tolerated. The 2-F-phenyl derivative 2j was identified to be
mixed results. The methylamine analog 2o was less potent in the
cellular assay despite retaining the biochemical activity relative
to the amino derivative 2f. A similar trend was observed with
the 5-aminoethyl-N1-pyridyl analog 2q compared to 2g. In
contrast, ethyl carboxamide analog 2p retained both enzyme and
cellular potencies of the corresponding 5-amino derivative 2b.
The 5-aminoethyl analog 2r was identified as one of the most
potent p38
a inhibitors in this series.
Based on its both enzyme and cellular p38
a
inhibitory poten-
the most potent analog in the cellular assay (TNF
a
IC50 = 1 nM). Fi-
cies, compound 2j was selected for further evaluation. The kinase
selectivity profile of 2j was determined against several receptor,
non-receptor tyrosine kinases, and serine/threonine kinases (Table
4). Compound 2j was shown to be at least 2500-fold selective over
these different kinases.
nally, N1 phenyl can be replaced with an alkyl group (2m, n) without
significant loss in potency in both the biochemical and cellular
assays.
We next proceeded to SAR studies focused on 5-amino substitu-
tion5 (Table 3). Alkylation of the 5-amino group showed some
Compound 2j was further profiled as a representative example
of this novel chemotype (Table 5). In addition to its highly potent
p38a inhibitory potencies in both biochemical and cellular assays,
and exquisite selectivity over other kinases, compound 2j was de-
void of any significant Herg and CYP liability issues (low levels of
COOCH2CH3
NH2
NC
COOEt
OEt
R1-NHNH2
a
+
N
N
R1
5
4
3
Table 1
SAR for 4-carboxamide modification
NHR3
H3C
O
Me
Me
HN
COOH
NH2
O
NH
b
H
N
H
N
c or d
N
HN
R3
N
N
Me
CH3
NH2
N
R1
R1
NHR3
O
O
H2N
N
O
7
2
O
6
N
N
N
N
N
1
NH2
Scheme 1. Reagents and conditions: (a) EtOH, Et3N, 60 °C, 1 h, 60%; (b) EtOH, aq
NaOH; (c) HATU, i-Pr2NEt, DMF; (d) SOCl2, CH2Cl2; CH2Cl2, i-Pr2NEt.
2a-f
COOCH2CH3
NH2
COOCH2CH3
NHR2
Compds
R3
p38
a
IC50, nMa
hPBMC TNFa
IC50, nMb
a
d
N
1
2a
2b
—
Me
Et
10
14
8
67
48
16
N
N
R1
or b, c
N
R1
5
8
2c
7
26
NHR3
H3C
O
Me
Me
N
N
2d
7
5
O
COOH
NHR2
NH
e or f
N
N
N
N
NHR2
2e
2f
5
3
8
1
N
CH3
N
R1
NHR3
R1
H2N
O
N
2
7
O
9
Scheme 2. Reagents and conditions: (a) NaH, THF, R2Br;(b) AcCN, t-BuONO, CuBr2;
(c) R2NH2, EtOH, 150 °C; (d) EtOH, aq NaOH; (e) HATU, i-Pr2NEt, DMF; (f) SOCl2,
CH2Cl2; CH2Cl2, i-Pr2NEt.
a
n = 4, variation in individual values, <20%.
n = 3, variation in individual values, <25%.
b

6888
J. Das et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6886–6889
Table 2
Table 4
SAR for N-1 substitution
Kinase selectivity profile of 2j
NHR3
O
Kinase
p38
KDR
Btk
CaMKII
MK2
Itk
STAT3
FGFR1
GSK3
Syk
IC50
(
l
M)
Kinase
IC50 (lM)
H3C
O
a
0.002
>50
>15
>50
>30
>50
>10
>50
>5
Lck
Lckf
7
>50
>50
>5
NH
VEGF
Tyk2
PLK1
DGAT
PKAb
N
>15
>20
>40
>40
>40
>13
>20
9.2
NH2
N
R1
R3
PKC
PKCd
PKC
a
Compds
R1
p38
a
IC50, nMa
hPBMC TNF
a
IC50, nMb
>50
>50
>10
s
IGF-1R
Jak3
PKCf
PDGFb
O
N
2g
2h
2-Pyridyl
2
3
8
5
N
N
Me
Me
2-F-phenyl
2-F-phenyl
2-F-phenyl
4-F-phenyl
4-F-phenyl
Table 5
In vitro profiling data for 5-amino-pyrazole analog 2j
N
N
2i
2j
2k
2l
4
2
2
2
2
1
2
2
Parameter
p38 IC60
Result
O
N
a
1.97 nM
1.2 nM
hPBMC LPS-TNF
a IC50
hERGa IC50
>160
HLM 0.001, RLM 0.000, MLM 0.000c
> 40 M 1A2, 2D6, 3A4; >13 M 2C19; 23
33 g/mL
lM
N
N
Me
Metabolic stabilityb
CYPd inhibition IC50
Aq Soln, pH 6.5
l
l
lM 2C9
l
O
N
Caco-2 permeability
Murine LPS-TNF
231 nm/s
>75% inhibition
a
Predose (5 mg/kg; 24 h)
Me
N
N
a
b
c
Flux assay.
nmol/min/mg protein.
HLM, human liver microsome; RLM, rat liver microsome; MLM, mouse liver
2m
2n
n-Bu
n-Bu
7
2
17
2
O
N
microsome.
d
CYP, cytochrome P450.
a
n = 4, variation in individual values, <20%.
n = 3, variation in individual values, <25%.
b
ability studies. The in vivo efficacy of 2j was demonstrated in an
acute pharmacodynamic model of LPS-induced TNF production
in mice. Mice were dosed orally with 2j at 5 mg/kg, 24 h prior to
LPS administration and the plasma TNF level was measured
a
Table 3
SAR for 5-amino substitution
a
90 min later. Compound 2j inhibited circulating TNF
a level by
NHR3
O
>75%.4
H3C
O
To understand the binding mode of this novel class of p38
inhibitors, compound 2f was co-crystallized with purified, unphos-
phorylated p38 MAP kinase and the X-ray structure of the com-
plex determined.2,7,8 The key binding interactions between
compound 2f and the p38 enzyme are illustrated in Figure 2.
a
NH
a
R2
N
N
N
H
a
R1
Compds R1
R2
R3
p38a
IC50, nMa hPBMC TNF
IC50, nMb
a
O
O
N
N
2f
Ph
H
3
1
2o
2p
Ph
Ph
Me
Et
5
6
7
Et
15
O
O
N
N
2q
2r
2-Pyridyl
Et
4
5
30
1
2-Me-phenyl Et
a
n = 4, variation in individual values, <20%.
n = 3, variation in individual values, <25%.
b
inhibitions). In addition, compound 2j demonstrated excellent sta-
bility in rat, mouse, and human liver microsome assays6 in vitro.
Compound 2j also had good aqueous solubility at physiologic pH
and excellent permeability as determined by Caco-2 cell perme-
Figure 2. Binding interactions between 2f and unphosphorylated p38
X-ray crystallographic analysis. Hydrogen bond distances are given in angstroms
with key protein residues labeled.
a based on

J. Das et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6886–6889
6889
53, 2345; (f) Wagner, G.; Laufer, S. Med. Res. Rev. 2006, 26, 1; (g) Westra, J.;
Limburg, P. C. Mini-Rev. Med. Chem. 2006, 6, 867.
2. Das, J.; Moquin, R. V. M.; Pitt, S.; Zhang, R.; Shen, D. R.; McIntyre, K. W.; Gillooly,
K.; Doweyko, A. M.; Sack, J. S.; Zhang, H.; Kiefer, S. E.; Kish, K.; McKinnon, M.;
Barrish, J. C.; Dodd, J. H.; Schieven, G. L.; Leftheris, K. Bioorg. Med. Chem. Lett.
2008, 18, 2652.
The X-ray structure of 2f reveals a combination of H-bonding and
hydrophobic interactions. Consistent with our previous findings
with other p38a inhibitors, the pendant carboxamido-aniline ring
occupies an angular hydrophobic pocket and makes key hydrogen
bond interactions. Specifically, the aniline NH is H-bonded with
Thr106 (1.97 Å). The hinge region residue, Met109 forms an H-
bond with one of the pyrazole nitrogens (2.14 Å). The phenyl ring
attached to the pyrazolo-pyrimidine ring is skewed out of plane.
This orientation may explain some of the SAR observed regarding
substitution at the ortho-position of the phenyl ring.
3. Schmidt, P.; Druey, J. Helv. Chim. Acta 1956, 39, 986.
4. (a) For experimental details, see: Dyckman, A. J.; Das, J.; Leftheris, K.; Liu, C.;
Moquin, R. V.; Wrobleski, S. U.S. Patent 7,390,810 B2, 2008; For a description of
the biological assays, and X-ray crystal structure determination protocol, see:
(b) Hynes, J., Jr.; Dyckman, A. J.; Lin, S.; Wrobleski, S. T.; Wu, H.; Gillooly, K. M.;
Lonial, H.; Loo, D.; McIntyre, K. W.; Pitt, S.; Shen, D. R.; Shuster, D. J.; Zhang, X.;
Behnia, K.; Marathe, P. H.; Doweyko, A.; Barrish, J.; Dodd, J.; Schieven, G.;
Leftheris, K. J. Med. Chem. 2008, 51, 4.
5. For additional 5-substitution SAR in this series, see: Dyckman, A. J.; Li, T.; Das, J.;
Moquin, R. V. M.; Pitt, S.; Zhang, R.; Shen, D. R.; McKinnon, M.; McIntyre, K. W.;
Gillooly, K.; Shuster, D.; Taylor, T.; Doweyko, A. M.; Sack, J. S.; Zhang, H.;
Marathe, P.; Barrish, J. C.; Dodd, J. H.; Leftheris, K., manuscript in preparation.
6. Rat, Mouse and Human Liver Microsome Stability Assays: Rat and mouse
Microsomes were purchased from XenoTech (Kansas City, KS). The human
microsomes were purchased from In Vitro Technology (Baltimore, MD) and
were pooled from 10 individual donors. The rates of oxidative metabolism were
In conclusion, we utilized structure based drug design to iden-
tify a series of exquisitely potent and selective p38
a inhibitors
using a novel 5-amino-pyrazole scaffold. SAR optimization at mul-
tiple positions led to the identification of analog 2j as one of the
most potent p38
for inhibition of TNF-
clear cells (PBMCs). Oral efficacy was also demonstrated with this
analog in an acute murine model of TNF inhibition. In addition,
the molecular basis for p38 inhibition of this inhibitor class was
a
inhibitor with single digit nanomolar potency
a
release in human peripheral blood mononu-
measured in triplicate under the following conditions: compound 2j, 3 lM final
a
concentration; final microsomal protein concentration approximately 1 mg/mL;
NADPH 1 mM; pH 7.4 potassium phosphate buffer, 56 mM. Incubations were
performed at 37 °C and were initiated by the addition of the substrate. Aliquot
a
established via X-ray crystal structure determination of an en-
zyme–inhibitor complex of a related analog 2f.
(100 lL) of the incubation were quenched at 0, and 10 min by the addition of 1
volume of acetonitrile. Results from 10-min incubations were used for the rate
calculations. Sample were analyzed by the LC/MS/MS assay for the amount of
the drug remaining at any time point. The percent metabolized was calculated
based on the disappearance of the parent compound.
References and notes
7. Sack, J. S.; Kish, K. F.; Pokross, M.; Xie, D.; Duke, G. J.; Tredup, J. A.; Kiefer, S. A.;
Newitt, J. A. Acta Crystallogr., Sect. D 2008, 64, 705.
8. The X-ray coordinates have been deposited with the RCSB Protein Data Bank
(RCSB ID Code: rcsb060940 and PDB ID Code: 30CG).
1. For some recent reviews, see: (a) Sweeney, S. E. Nat. Rev. Rheumatol. 2009, 5,
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Opin. Pharm. 2004, 4, 372; (d) Kumar, S.; Boehm, J.; Lee, J. C. Nat. Rev. Drug Disc.
2003, 2, 717; (e) Goldstein, D. M.; Kuglstatter, Y. L.; Soth, M. J. J. Med. Chem. 2009,