The development of monocyclic pyrazolone based cytokine synthesis inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.03.007

4-Aryl-5-pyrimidyl based cytokine synthesis inhibitors that contain a novel monocyclic, pyrazolone heterocyclic core are described. Many of these inhibitors showed low nanomolar activity against LPS-induced TNF-α production. One of the compounds (6e) was found to be efficacious in the rat iodoacetate (RIA) in vivo model of osteoarthritis. The X-ray crystal structure of a pyrazolone inhibitor cocrystallized with mutated p38 (mp38) is presented.

Full text of DOI:10.1016/j.bmcl.2005.03.007

Bioorganic & Medicinal ChemistryLetters 15 (2005) 2285–2289
The development of monocyclic pyrazolone based
cytokine synthesis inhibitors
Adam Golebiowski,* Jennifer A. Townes, Matthew J. Laufersweiler, Todd A. Brugel,
Michael P. Clark, Cynthia M. Clark, Jane F. Djung, Steven K. Laughlin, Mark P. Sabat,
Roger G. Bookland, John C. VanRens, Biswanath De, LilyC. Hsieh, Michael J. Janusz,
Richard L. Walter, Mark E. Webster and Marlene J. Mekel
Procter and Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Rd, Mason, OH 45040, USA
Received 3 December 2004; revised 1 March 2005; accepted 3 March 2005
Abstract—4-Aryl-5-pyrimidyl based cytokine synthesis inhibitors that contain a novel monocyclic, pyrazolone heterocyclic core are
described. Manyof these inhibitors showed low nanomolar activityagainst LPS-induced TNF- a production. One of the compounds
(6e) was found to be efficacious in the rat iodoacetate (RIA) in vivo model of osteoarthritis. The X-raycrystal structure of a
pyrazolone inhibitor cocrystallized with mutated p38 (mp38) is presented.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Numerous efforts in anti-inflammation research have
been focused on the development of small molecule
inhibitors of cytokine release. The overexpression of
cytokines, such as TNF-a and IL-1b, has been impli-
cated in a number of serious inflammatorydisorders.
Consequently, agents that inhibit the production of
TNF-a can decrease levels of these proinflammatory
cytokines,1 and therebyreduce inflammation and
prevent further tissue destruction in diseases such as
rheumatoid arthritis (RA),2 osteoarthritis (OA),3 and
Crohnꢀs disease.
Manyother small molecule TNF- a production inhibi-
tors have been reported containing a common 4-aryl-
5-pyrimidinyl based motif fused to a 5- or 6-membered
heterocyclic core. SB2035801 (Fig. 1) represents a proto-
typical pyridyl imidazole-based inhibitor, although
numerous structural classes, for example, pyrroles, pyr-
imidines, pyridines, pyrimidones, indoles, heteroindoles,
ureas, and various fused bicyclic heterocycles containing
a varietyof functionalityhave been reported to inhibit
cytokine activity.4
In our previous paper, we reported the development of a
class of triazole based cytokine inhibitors (e.g., 1; Fig.
1).5 Researchers from Merck reported studies on pyr-
roles and other heterocycles as inhibitors of p38 kinase.6
Two monocyclic pyrazolones were presented (e.g., 2;
Fig. 1) with good p38a-kinase inhibition, but weak
whole cell assayactivities. We speculated that alkyl sub-
stitution of both pyrazolone ring nitrogen atoms could
facilitate cell membrane permeabilityand improve cellu-
lar cytokine synthesis inhibition profile. This led to the
development of mono-, bi-, and tricyclic pyrazolones
as an emerging, leading series in our project.7–9 Many
compounds in these scaffolds exhibited good to excellent
activities in the THP-1 cell-based assay, inhibiting the
production of TNF-a with IC50s in the low nanomolar
range. Herein we wish to report the synthesis and struc-
ture–activityrelationships for the series of monocyclic
We are interested in the efficacyof our inhibitors against
the overall cascade that leads to TNF-a production and
have identified p38 MAP kinase as one of the critical tar-
gets of inhibition. However, considering that manyother
kinases (e.g., JNK, MAPKAPK2) participate in the sig-
naling cascade leading to TNF-a synthesis induction and
that p38 kinase itself exists in several conformational
states (inactive and active conformations being the major
ones) we decided to use a whole cell assay(inhibition of
TNF-a release from lipopolysaccharide (LPS) induced
THP-1 cells) as our primaryscreening tool.
Keywords: Cytokine synthesis inhibitors; p38 kinase.
*
Corresponding author. Tel.: +1 513 622 3802; fax: +1 513 622
3681; e-mail: golebiowski.a@pg.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.007

2286
A. Golebiowski et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2285–2289
F
F
F
O
N
N
N
N
R
N
R
R
N
N
H
H
N
N
N
O
R =
R =
S
Cl
R =
O
1
2 - ref 6
SB203580
TNF-α IC50 = 1070 nM
p38 IC50 = 2430 nM
TNF-α IC50 = 4224 nM
p38 IC50 = 35 nM
TNF-α IC50 = 72 nM
p38 IC50 = 136 nM
Figure 1. p38a-kinase/TNFa synthesis inhibitors.
Table 1. Symmetrically substituted pyrazolones
F
pyrazolones. All compounds were tested for the inhibi-
tion of TNF-a production using lipopolysaccharide
(LPS) stimulated human monocytic cells (THP-1).11
O
R1
N
The synthesis of the first compounds in this series was
based on the addition of alkyl hydrazines to b-ketoester
4. Coupling of methyl-4-fluorophenylacetate (3),
(Scheme 1) with 2-thiomethylpyrimidine-4-carboxalde-
hyde as described by Smith et al.10 followed bychro-
mium oxidation provides b-ketoester 4. Addition of
symmetrically substituted hydrazines to b-ketoester 4
gives the core pyrazolone ring system 5. Further func-
tionalization of the pyrimidine ring was achieved via
an oxidation–nucleophilic substitution reaction se-
quence, using sodium phenoxide, leading to pyrazolone
6a.
N
R2
N
N
R3
R3
Compound
R1
R2
TNFa IC50 (nM)
O
6a
Me
Me
288
NH
6b
6c
Me
Me
Me
Me
41
In general, amino substitution at the pyrimidine ring led
to more active compounds than with phenoxyanalogs
(this trend was also observed in the bicyclic pyrazolone
series—see Refs. 7,8). An attempt to increase solubility
byreplacement of the benzly amino substituent with
amino-pyridine ring system led to decrease of activity
(compare 6e vs 6f, 6g, 6h). Increasing the size of the
substitution on the ring nitrogens of the pyrazolone
NH
1431
MeO
HO
NH
6d
Me
Me
3970
NH
NH
6e
6f
Me
Me
Me
Me
13
F
O
F
a, b
O
OMe
1429
OMe
O
N
F
N
4
3
N
SMe
NH
F
6g
6h
Me
Me
Me
Me
647
O
O
c
d, e
N R
N
R
N R
N
R
N
NH
NH
N
N
>5000
N
N
X
SMe
N
5
6
6i
Et
Et
120
Scheme 1. Reagents and conditions: (a) LDA, THF, À78 ꢁC, 2-
methylsulfanyl-pyrimidine-4-carbaldehyde; (b) CrO3, pyridine; (c)
RNHNHRÆHCl, EtOH, 90 ꢁC; (d) Oxoneꢂ, THF/MeOH/H2O, rt;
(e) R1NH2, toluene, 120 ꢁC or ArOH, NaH, THF.
All chiral compounds are pure enantiomers as indicated.

A. Golebiowski et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2285–2289
2287
from methyl to ethyl resulted in a 10-fold loss of potency
(compare 6e vs 6i). Isobutyl analog (6b) showed very
good potencyhowever is potentiallymetabolically
unstable. All our attempts to substitute it with more
metabolicallystable fragments resulted in a surprising
loss of activity(e.g., 6c,d) (see Table 1).
A complementary, regioisomer synthesis starts with
hydrazine 12, which in an analogous synthetic sequence
was transformed into pyrazolone 15 (see Scheme 3).
Syntheses of both 1,1-acyl-methyl hydrazines 7 and 12
were accomplished in one step via a highlyregioselective
monoacylation of methyl hydrazine12 (see Scheme 4).
In order to increase solubilityof our compounds we
decided to explore SAR around non-symmetrically
substituted pyrazolones. For the synthesis of this class
of compounds we applied an alternative synthetic proto-
col that was developed recentlyin our group ( Scheme 2).
Tables 2 and 3 summarize a preliminarysurveyof phen-
oxy- and amino-pyrimidine substituents for the non-
symmetrically substituted, monocyclic pyrazolones of
type 11—with R1 substituent fixed as methyl
(R1 = Me) and monocyclic pyrazolones type 15—with
R2 substituent fixed as methyl (R2 = Me). In general,
substitution of either methyl group with a more polar
functional group increased potency(compare 6d, 11d,
Hydrazone formation between acylated hydrazine 7 and
an aldehyde (R2CHO), followed byhydride reduction
led to trisubstituted hydrazine 8. Acylation using acid
chloride 9 afforded bis-acylated hydrazine 10. Sodium
hydride mediated cyclization followed by sulfoxide
formation and displacement led to the final compound 11.
F
O
O
a
O
7; R =
HN
+
Cl
R
N
NH2
R
F
N
NH2
O
12; R =
F
16
b
O
a
N
S
N
N
HN
O
R2
N
Scheme 4. Reagents and conditions: (a) DCM, À78 ꢁC.
NH2
Cl
8
7
N
Table 2. Non-symmetrically substituted pyrazolones 11 (R1 = Me)
9
S
F
F
O
F
c - e
O
O
N
N
O
N
R2
S
N
N
N
R2
R2
N
N
N
N
N
N
R3
R3
11
10
Compound R2
R3
TNF-a
IC50 (nM)
Scheme 2. Reagents and conditions: (a) R2CHO, EtOH, reflux,
15 min; then NaBH3CN, MeOH, pH 4; (b) acid chloride 9, pyridine;
(c) NaH, DMF, 0 ꢁC; (d) mCPBA, DCM; (e) R4NH2, toluene +120 ꢁC,
or PhOH, NaH, THF.
O
O
O
11a
11b
11c
1428
2652
924
O
O
NH.TFA
H
NH2
a
N
N
R1
N
N
F
N
N
N
O
O
O
N
H
S
S
12
13
11d
75
F
N
H
O
NH.TFA
NH.TFA
O
R1
N
c - e
R1
N
b
O
N
NH
N
11e
11f
155
309
N
N
N
R3
14
15
N
S
NH
O
Scheme 3. Reagents and conditions: (a) R1CHO, EtOH, reflux,
15 min; then NaBH3CN, MeOH, pH 4; (b) 4-fluorophenylacetyl
chloride, pyridine; (c) NaH, DMF, 0 ꢁC; (d) mCPBA, DCM; (e)
R4NH2, toluene +120 ꢁC, or PhOH, NaH, THF.
All chiral compounds are pure enantiomers as indicated.

2288
A. Golebiowski et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2285–2289
Table 3. Non-symmetrically substituted pyrazolones 15 (R2 = Me)
Table 4. Pharmacokinetic properties and in vivo data of selected
compounds (rat)
F
Compound
TNF-a
IC50 (nM)
Metaba
Sol
(mg/mL)
t1/2
F
O
(h)
(%)
R1
N
6e
13
75
44
0
0.123
>30.0
0.429
1.1
2.2
ND
3.2
32
15
N
11d
11f
15d
309
32
30
32
ND
17
N
N
0.650
R3
a Metabolism measured as percent loss at 4 h in rat hepatocytes.
Compound
R1
R3
TNF-a
IC50 (nM)
X-rayanalysis of compound 15d cocrystallized with mu-
tated p38 kinase is presented in Figure 2.13 A hydrogen
bond between the backbone amide N–H of Met-109 and
the pyrimidine ring was observed. A second hydrogen
bond was formed between the N–H of the amine substi-
tuent and the backbone carbonyl of Met-109. The pyr-
azolone carbonyl forms a third hydrogen bond with
the Lys 53 amine group. A favorable interaction be-
tween the piperidine ring and Asp168 carboxylate group
is also observed.
O
15a
1814
NH.TFA
Me
NH
Me
Me
15b
15c
1085
261
NH.TFA
NH.TFA
OH
H
N
O
H
N
One of our earlygoals was to improve the aqueous sol-
ubilityof initial leads. Unfortunatelythis led to com-
pounds with lower bioavailability( Table 4). From the
series of monocyclic pyrazolones only compound 6e
was found to significantly( p < 0.05) inhibit knee joint
degeneration in the rat iodoacetate (RIA) model of
osteoarthritis (dose 25 mg/kg).16,17
Me
Me
Me
15d
15e
15f
32
15
14
NH.TFA
H
N
N
In conclusion, we have reported the synthesis of a series
of cytokine synthesis inhibitors, based on a monocyclic
pyrazolone scaffold. Many of these inhibitors showed
low nanomolar activityin whole cell assay(inhibition
of TNF-a release from lipopolysaccharide (LPS)
induced THP-1 cells). Pyrazolone 6e was found to be
orallyactive in rat iodoacetate (RIA) in vivo model of
osteoarthritis.
H
N
N
O
All chiral compounds are pure enantiomers as indicated.
15c). Presumablythis could result from picking up addi-
tional polar interaction with the Asp168 carboxylate
group (Figure 2).
Acknowledgements
We are grateful to S. Heitmeyer, K. Brown, and K.
Yuellig for rat iodoacetate data; A. L. Roe, D. C. Ack-
ley, A. M. Walter, C. R. Dietsch, and D. M. Bornes for
PK and metabolic stabilitydata, and M. Buchalova for
solubilitydata.
We would also like to acknowledge that the X-raydata
were collected at Southeast Regional Collaborative Ac-
cess Team (SER-CAT) 22-ID beam line at the Advanced
Photon Source, Argonne National Laboratory. Sup-
porting institutions maybe found at www.ser-cat.org/
members.html. Use of the Advanced Photon Source
was supported bythe US Department of Energy, Office
of Science, Office of Basic EnergySciences, under
Contract No. W-31-109-Eng-38.
References and notes
1. For a recent review, see: Adams, J. L.; Badger, A. M.;
Kumar, S.; Lee, J. C. p38 MAP Kinase: Molecular Target
for the Inhibition of Pro-inflammatoryCtoykines
Figure 2. Compound 15d bound in the active site of mutated p38a-
kinase.

A. Golebiowski et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2285–2289
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(Oregon, WI) under anesthesia were injected in the
patellar ligament region of the left leg (flexed 90ꢁ at the
knee) with 20 lL of a 10 mg/mL concentration of mono-
sodium iodoacetate (IA) (Aldrich Chemical, Milwaukee,
WI). Animals (groups of 15) were dosed for 7 days BID
(ꢀevery12 h) with the potential inhibitor (25 mg/kg) or
vehicle (2.5 mL/kg). Animals were sacrificed on day22 and
the left joint was disarticulated and fixed in 10% formalin
for 24–48 h prior to capturing the image. An image of the
tibial cartilage surface was captured using an Optimas
(Optimas, Media Cybernetics LP, Silver Springs, MA)
image analysis system. Three independent observers
assessed the damage in a blinded manner using a scale
of 0–4 of increasing severity(0 = normal; 4 = maximum
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