The development of new isoxazolone based inhibitors of tumor necrosis factor-alpha (TNF-α) production

Article abstract of DOI:10.1016/j.bmcl.2005.02.066

4-Aryl-3-pyridyl and 4-aryl-3-pyrimidinyl based tumor necrosis factor-alpha (TNF-α) inhibitors, which contain a novel isoxazolone five-membered heterocyclic core are described. Many showed sub-micromolar activity against lipopolysaccharide-induced TNF-α production.

Full text of DOI:10.1016/j.bmcl.2005.02.066

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2399–2403
The development of new isoxazolone based inhibitors of
tumor necrosis factor-alpha (TNF-a) production
Steven K. Laughlin, Michael P. Clark,* Jane F. Djung, Adam Golebiowski,
Todd A. Brugel, Mark Sabat, Roger G. Bookland, Matthew J. Laufersweiler,
John C. VanRens, Jennifer A. Townes, Biswanath De, Lily C. Hsieh, Susan C. Xu,
Richard L. Walter, Marlene J. Mekel and Michael J. Janusz
Health Care Research Center, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, Mason, OH 45040, USA
Received 27 July 2004; revised 18 February 2005; accepted 22 February 2005
Available online 25 March 2005
Abstract—4-Aryl-3-pyridyl and 4-aryl-3-pyrimidinyl based tumor necrosis factor-alpha (TNF-a) inhibitors, which contain a novel
isoxazolone five-membered heterocyclic core are described. Many showed sub-micromolar activity against lipopolysaccharide-
induced TNF-a production.
ꢀ 2005 Published by Elsevier Ltd.
In our continuing efforts toward the development of dis-
ease modifying treatments for inflammatory diseases, we
are targeting the development of novel mitogen-acti-
vated protein (MAP) kinase inhibitors. MAP kinases
are part of a signal transduction pathway responsible
for the relay of information from the cell surface to
the nucleus. Some of these MAP kinase families, such
as p38 and JNK (c-Jun amino terminal kinase), are acti-
vated in response to infection, cellular stress and the
proinflammatory cytokines TNF-a (tumor necrosis fac-
tor) and IL-1b (interleukin-1b).1 The overexpression of
these cytokines has been implicated in the pathogenesis
of a number of serious inflammatory disorders such as
rheumatoid arthritis (RA),2 osteoarthritis (OA),3 and
CrohnÕs disease. It has been established that agents that
inhibit p38 MAP kinase can decrease levels of these pro-
inflammatory cytokines,1 and thereby reduce inflamma-
tion and prevent further tissue destruction.
by utilizing a cellular assay as a primary screening tool,
can successfully lead to potent and efficacious cytokine
inhibitors.4 Therefore, our primary assay entails screen-
ing analogues for inhibition of lipopolysaccharide
(LPS)-stimulated TNF-a production in the THP-1 cellu-
lar assay.5 Herein, we report a new structural class of 4-
aryl-3-pyrimidinyl (pyridinyl) based TNF-a inhibitors
that contain a novel isoxazolone core. Our investiga-
tions have yielded potent TNF-a production inhibitors
such as pyridinyl isoxazolone
2
and phenoxy-
pyrimidineisoxazolone 3 (Fig. 1).
Synthesis of the pyridinyl isoxazolone 5 was accom-
plished in only three steps, from commercially available
F
F
F
O
O
O
O
N
O
Previous work from our laboratories described the syn-
thesis and SAR of a series of bicyclic pyrazolone TNF-a
inhibitors, exemplified by compound 1,4 that contain the
prototypical vicinal bis-aryl pharmacophore found in
many p38 inhibitors. The pyrazolone work has demon-
strated that optimizing the TNF-a inhibitory activity,
N
N
N
O
N
N
N
O
N
N
N
NH
O
N
1
2
3
HO
OH
TNF-
α
TNF-
α
TNF-
α
Keywords: p38 Inhibitor; Isoxazolone; TNF-a; Cytokine inhibitor.
Corresponding author. Tel.: +513 622 0012; fax: +513 622 3681;
e-mail: clark.mp@pg.com
IC50 = 252 nM
IC50 = 141 nM
IC50 = 22 nM
*
Figure 1. Vicinal bis-aryl TNF-a production inhibitors.
0960-894X/$ - see front matter ꢀ 2005 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2005.02.066

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S. K. Laughlin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2399–2403
F
substituents performed on the pyridinyl series,9 the eth-
O
O
F
oxymethyl substituent was held constant while a series
of substituents on the pyrimidine ring was examined.
N-Alkylation of isoxazolone 21 with 2-chloromethyl
ethyl ether was followed by oxidation of the thiomethyl
group with Oxoneꢁ. Reaction of the resulting sulfone 22
with either an alcohol under basic conditions (NaH,
THF) or neat amine at elevated temperature, respec-
tively, yielded the desired substituted derivative 23.
a, b
O
OMe
OMe
N
4
F
F
O
O
d
c
2
O
O
N
N
H
N
Table 2 summarizes a survey of phenoxypyrimidine isox-
azolone analogues, while Table 3 summarizes results
for a series of alkylaminopyrimidine isoxazolone ana-
logues. In general, both groups produced several com-
pounds at or below 1 lM potency level. In the
phenoxy series, both the phenoxy substituted 26 and
the fluorinated phenoxides (27–29) yielded compounds
in the 1 lM range. It is possible that the more potent
phenoxy substituents (24–26) may be picking up
N
N
O
N
5
Scheme 1. Reagents and conditions: (a) LDA, THF, 4-pyridine
carboxaldehyde À78 ꢂC, 91%; (b) CrO3, pyridine, 43%; (c) hydroxyl-
amine–HCl, pyridine, 90 ꢂC, 95%; (d) 4-methyl-1-piperazine carbonyl
chloride, Et3N, CH2Cl2, 51%.
starting materials (Scheme 1). Treatment of 4-pyridine
carboxaldehyde with the anion of 4-fluorophenylacetate,
followed by chromium(VI) oxidation provided the
known b-ketoester 4.6 Cyclocondensation of hydroxyl-
amine with b-ketoester 4 provided the core isoxazolone
ring system 5. While the unsubstituted parent isoxazo-
Table 1. TNF-a inhibition data for pyridyl isoxazolone derivatives
F
O
lone
5 possessed only modest activity (TNF-a,
IC50 = 5.8 lM), numerous opportunities existed for fur-
ther functionalization to optimize the potency of these
molecules (Table 1). Acylation with the corresponding
carbamoyl chlorides afforded compounds 2 and 9–10.
Alkylation of the ring nitrogen yielded compounds 11–
17. Alternatively, substituted isoxazolone analogues
(R = methyl (7) and R = cyclohexyl (8)) can be accessed
via a similar route with the appropriate N-alkylated
hydroxylamine. This allowed for analogues to be made
which were difficult to synthesize via the alkylation
method shown in Scheme 1, due to the weak nucleo-
philic nature of the isoxazolone ring nitrogen.
O
N
R
N
a
Compound
R
TNF-a IC50 (lM)
5
7
5.9
H
>10
CH3
8
2
9
>10
0.2
O
N
N
Examination of the results in Table 1 shows that both
the N-methyl (7) and N-cyclohexyl (8) analogues showed
little potency in the whole cell assay. One of the most
promising compounds was the N-methylpiperazine urea
derivative 2. This compound was significantly more po-
tent than the morpholine (9) and piperidine (10) ana-
logues. Unfortunately, the urea derivatives showed
significant metabolism in our in vitro rat hepatocyte
metabolic stability assay.7 We then turned our efforts to-
ward the alkyl derivatives. Alkylated derivatives (11–16)
showed some modest activity. The most potent alkyl
substituent (2.5 lM) was the ethoxymethyl compound
11, which had shown a positive effect as a substituent
on one of our previous TNF-a inhibitor scaffolds.8
The other ether containing substituents were not toler-
ated as well. The benzyl derivative (16) showed little po-
tency (>10 lM) and other benzyl analogues (data not
shown) were likewise inactive. Another promising alkyl-
ated product was the N-triazin-2-yl derivative (17) with
an IC50 = 200 nM.
5.0
O
O
N
N
O
10
11
12
8.0
2.5
4.4
O
O
13
14
6.9
8.6
O
O
15
16
>10
>10
O
N
NH2
N
17
0.2
N
H2N
The related pyrimidinyl isoxazolone series, which was
made analogously to the pyridinyl isoxazolones (Scheme
2), afforded a second site of diversity at the 2-position of
the pyrimidine ring. Based on the survey of isoxazolone
a IC50 of LPS-stimulated TNF-a production in human monocytic cells
(THP-1). Standard deviation for assays typically 30% of the mean
or less.

S. K. Laughlin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2399–2403
2401
F
O
F
a, b
O
c
OMe
OMe
O
O
H
N
N
20
N
N
18
SMe
SMe
F
F
F
O
O
O
f
d, e
O
O
N
O
N
N
H
O
N
N
O
N
N
N
N
SMe
SO2Me
23
X = NH or O
X
21
22
R
Scheme 2. Reagents and conditions: (a) LDA, THF, À78 ꢂC, aldehyde 18 85%; (b) CrO3, pyridine, 43%; (c) H2NOH, pyridine, 90 ꢂC, 80%; (d)
chloromethyl-ethyl ether, Et3N, CH2Cl2, 45%; (e) Oxoneꢁ, THF/MeOH/H2O; (f) R–NH2, toluene 120 ꢂC or R–OH, NaH, THF.
Table 2. TNF-a inhibition values of the phenoxy-pyrimidine bicyclic
pyrazolone analogues
A compound of note from this series is isoxazolone 43,
which incorporates an N-acetaldehyde substituent on
the isoxazolone ring nitrogen. This compound contains
F
O
O
N
Table 3. TNF-a inhibition values of the alkylamino-pyrimidine
isoxazolone analogues
O
N
N
F
O
Ar
O
a
Compound
O–Ar
TNF-a IC50 (lM)
O
N
3
2-Hydroxyphenoxy
3-Acetamidophenoxy
3-Hydroxyphenoxy
Phenoxy
0.14
0.46
0.53
1.00
1.00
1.01
1.08
2.00
6.30
7.50
>10
O
24
25
26
27
28
29
30
31
32
33
34
N
N
NH
R
4-Fluorophenoxy
2-Fluorophenoxy
2,6-Difluorophenoxy
3-Dimethylaminophenoxy
4-Methoxyphenoxy
2-Acetamidophenoxy
2,4-Dimethylphenoxy
4-Ethylphenoxy
a
Compound
R–NH
TNF-a IC50 (lM)
HO
35
0.18
N
H
36
37
0.20
0.21
N
H
>10
a IC50 of LPS-stimulated TNF-a production in human monocytic cells
(THP-1). Standard deviation for assays typically 30% of the mean
or less.
N
H
O
N
38
39
0.94
1.00
N
H
additional hydrogen bonding interactions with other
side chains in the enzyme active site, such as Gly-110
(Figs. 2 and 3).
N
H
40
1.08
N
H
In the alkylamino series (Table 3) the pyrimidine substit-
uents that possessed a secondary a-methyl amine
showed greater potency than the corresponding primary
amines. The three most potent analogues (35–37) con-
tain substituents at the 2-position of the pyrimidine ring
that have increased potency in the previously disclosed
bicyclic pyrazolone series.4 Interestingly, the S-enantio-
mer of the a-methyl benzyl amine analogue 36 is five
times more potent than the corresponding R-enantiomer
40.
HN
41
42
7.40
>10
N
N
H
O
N
N
H
a IC50 of LPS-stimulated TNF-a production in human monocytic cells
(THP-1). Standard deviation for assays typically 30% of the mean
or less.

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S. K. Laughlin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2399–2403
Table 4. Comparison of IC50 values/inhibition data for 3, 25, 36, 40,
and 43
F
O
Compound 3 (nM) 25 (nM) 36 (nM) 40 (nM) 43 (nM)
O
TNF-aa
p38a
140
460
530
520
200
84
1080
1700
117
48
N
H
N
N
O
a IC50 of LPS-stimulated TNF-a production in human THP-1 cells.
Standard deviation for assays typically 30% of the mean or less.
O
43
α
TNF-
IC50 = 117 nM
(140 nM). The possibility of isoxazolone 3 inhibiting
TNF-a production via multiple mechanisms may ex-
plain this difference in potency.
Figure 2. N-Acetaldehyde phenoxy-pyrimidine isoxazolone 43.
In order to elucidate binding site interactions in the isox-
azolone series, X-ray data was collected for the highly
active isoxazolone inhibitor 3 co-crystallized with mu-
tated p38a10 (Fig. 3). We observed the typical vicinal
bis-aryl p38 inhibitor interactions, including binding of
the fluorophenyl ring into the Thr-106 hydrophobic
pocket and a hydrogen bonding interaction between
the Met-109 amide NH and the nitrogen of the pyrim-
idine ring. We also saw a second hydrogen bonding
interaction between the Gly-110 amide NH and the oxy-
gen of the phenoxy group. One of our goals when
designing the isoxazolone series was to pick up the
important Lys-53 interaction with a strong hydrogen-
bond acceptor, in this case the carbonyl oxygen. A
strong hydrogen bonding interaction between the car-
bonyl oxygen of the isoxazolone ring and Lys-53 in
the binding site is confirmed by the X-ray data.
In conclusion, we have reported a novel series of pyrid-
inyl and pyrimidinyl isoxazolone scaffold that maintains
the important binding motif of the vicinal bis-aryl ring
systems in the p38 ATP binding site. Two of the pyrid-
inyl isoxazolones showed sub-micromolar potency
against LPS-stimulated human monocytic cells (THP-
1). In general, the pyrimidinyl isoxazolones were more
potent, yielding several compounds at or below the
1 lM potency level.
Acknowledgements
We are grateful to Richard L. Bohne for providing
some of the initial TNF-a cellular assay data. We would
also like to acknowledge that the X-ray data were col-
lected at Southeast Regional Collaborative Access Team
(SER-CAT) 22-ID beamline at the Advanced Photon
Source, Argonne National Laboratory. Supporting
institutions may be found at www.ser-cat.org/mem-
bers.html. Use of the Advanced Photon Source was sup-
ported by the US Department of Energy, Office of
Science, Office of Basic Energy Sciences, under Contract
No. W-31-109-Eng-38.
Figure 3. Isoxazolone 3 co-crystallized in mutated p38 active site.
a phenoxy-substituted pyrimidine and was made by
alkylating the isoxazolone nitrogen with allyl bromide,
then oxidatively cleaving the olefin with OsO4/NaIO4.
The resulting isoxazolone proved to be our most potent
compound in this series (IC50 = 117 nM).
Compounds 3, 25, 36, 40, and 43 were tested in a human
p38a kinase assay (Table 4). Compounds 25 and 40
showed comparable values to the THP-1 whole cell data
(520 and 1700 nM, respectively). Compounds 36 and 43
showed increased potency in the p38a kinase assay (84
and 48 nM, respectively). Surprisingly, the 2-hydroxy-
phenoxy analogue (3) showed much weaker potency in
the kinase assay (460 nM) than the cellular assay
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