Isoxazolone based inhibitors of p38 MAP kinases

Article abstract of DOI:10.1021/jm701343f

SAR of N-alkylated isoxazolones as p38 MAP kinase inhibitors was realized. The data herein show the possibility of transferring the SAR study and evaluation from N-1-substituted imidazole to isoxazolones. Optimization of substituent was realized.

Full text of DOI:10.1021/jm701343f

2580
J. Med. Chem. 2008, 51, 2580–2584
Isoxazolone Based Inhibitors of p38 MAP Kinases
Stefan A. Laufer* and Simona Margutti
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Eberhard Karls UniVersity of Tuebingen,
Auf der Morgenstelle 8, 72076 Tuebingen, Germany
ReceiVed October 26, 2007
SAR of N-alkylated isoxazolones as p38 MAP kinase inhibitors was realized. The data herein show the
possibility of transferring the SAR study and evaluation from N-1-substituted imidazole to isoxazolones.
Optimization of substituent was realized.
Introduction
In a continuous effort to develop improved p38 MAPa kinase
inhibitors, we focused our attention on the suitability of the
isoxazolone ring as bioisosteric replacement for the imidazole
ring of SB203580 (Figure 1).
A large body of evidence indicates that p38 activity is critical
for the normal immune and inflammatory responses.1 The
requirement for p38 activation in cellular responses has been
defined largely through the use of experimental pyridinylimi-
dazole anti-inflammatory drugs, the cytokine-suppressive anti-
inflammatory drugs (CSAIDs). The most extensively charac-
terized is the compound SB203580.
p38 inhibitors are potent inhibitors of LPS-mediated TNF-R
production in macrophages.2 The ability of p38 inhibitors to
Figure 1. Prototypical pyridinylimidazole p38 inhibitor SB203580 and
related vicinal bis-aryl p38 MAP kinase inhibitors. IC50 ( SEM (µM)
block TNF-R synthesis can be exploited in the treatment of
inflammatory diseases.
p38R: 1, 0.813 ( 0.04; 2, 14.7% inhibition at 10 µM; 3, 1.15 ( 0.03.
IC50 SB203580, 0.045 ( 0.01.
Previous work from our laboratories described the synthesis
and SAR of N-1-substituted imidazole p38 inhibitors, exempli-
Scheme 1a
fied by 1 (Figure 1),3 which contain the prototypical vicinal
bis-aryl pharmacophore. In the cited publication, we reported
optimization of a novel structural class of tetrasubstituted
imidazoles inhibitors of p38 MAP kinase4,5 by imidazole N-1
substitutions. The work has demonstrated that by utilization of
an in vitro enzymatic assay as a primary screening tool, N-1
substitution can successfully lead to potent and efficacious p38
MAP kinase inhibitors.
The development of the imidazole based p38 MAP kinase
inhibitors into anti-inflammatory drugs was obstructed by their
severe liver toxicity, as the pyridinylimidazoles were found to
a Reagents: (a) hydroxylamine chloride, NaOH 50%, H2O/ice/ethanol,
<10 °C then room temp, 1 h; (b) N-chlorosuccinimide, DMF, room temp;
(c) NaHDMS, ethyl 4-pyridylacetate, THF, room temp, 6 h; (d) conc HCl;
(e) alkyl halide, Et3N, CH2Cl2.
interact with the hepatic cytochrome P450 enzymes involved
in drug metabolism.6 In this prospective we investigated the
potential of the isoxazole as bioisosteric replacement of the
imidazole heterocycle.7 This led to the development of isox-
azoles and then consequentially to isoxazolones as an emerging
leading series in our project. Herein, we report the synthesis
and SAR of 5-alkoxyisoxazoles (2, Figure 1) and of N-
substituted 5-isoxazolones (3, Figure 1). Some of the reported
structures are claimed as anticytokine agents by Procter &
Gamble Pharmaceuticals;8 nevertheless, to date, no p38 MAP
kinase inhibition data have been published.9 Our investigation
shows that the structure–activity study realized on the N-1-
substituted imidazoles3 could be transferred to the isoxazolone
series but not to the isoxazoles.
Chemistry
The synthesis of the 5-alkoxyisoxazoles was based on
O-alkylation of the pyridinylisoxazolone 6 synthesized from
commercially available starting materials (Scheme 1).
According to Scheme 1, 6 was synthesized via 1,3-dipolar
cycloaddition,10 where the 1,3-dipole is 4-fluorophenylnitrile oxide
generated from the corresponding hydroxamic acid chloride 5,
which was obtained by a high yield two-step procedure starting
from 4-fluorobenzaldehyde. Ethyl 4-pyridylacetate is deprotonated
* To whom correspondence should be addressed. Phone: +49(0)7071-
2978788. Fax: +49(0)7071-295961. E-mail: stefan.laufer@uni-tuebingen.de.
a Abbreviations: p38 MAP, p38 mitogen activated protein; CSAIDs,
cytokine-suppressive anti-inflammatory drugs; LPS, lipopolysaccaride; TNF-
R, tumor necrosis factor-R; SAR, structure–activity relationship.
10.1021/jm701343f CCC: $40.75
2008 American Chemical Society
Published on Web 03/29/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2581
Scheme 2a
Figure 4. 1H-1H NMR NOESY (bold line) and 1H-13C NMR HMBC
(dashed line) correlations in 7e (chemical shift in ppm).
a Reagents: (a) THF, room temp, 6 h; (b) conc HCl.
Table 2. Inhibition of p38 MAP Kinasea
Figure 2. Tautomerism of 5-hydroxyisoxazole, 6.
Table 1. 13C NMR Chemical Shifts for 6 in DMSO and Aqueous
Systems
a In aqueous solutions, shifts calculated from DMSO)39.52 ppm relative
to Me4Si.
a IC50 SB203580 ) 0.045 ( 0.011 µM. b Percentage of inhibition at 10
µM (concentration of test compound). Inhibition data from p38 kinase assay,
ref 20.
Scheme 3a
Figure 3. 1H-1H NMR NOESY (bold line) and 1H-13C NMR HMBC
(dashed line) correlations in 6 (chemical shift in ppm).
by sodium hexamethyldisilazide (NaHDMS) and added to an
equimolar amount of 4-fluorophenyl chloride oxime. The in situ
generated nitrile oxide acts on the deprotonated ester to give the
conjugated base (O- form) of 6. Column chromatography over
SiO2 allows removal of starting material and side products by the
elution with ethyl acetate. Elution with methanol allows isolation
of the conjugated base. The sodium salt of the base is then
converted to the protonated OH and crystallized. The ring closure
yielding the O- form of isoxazolone is mapped in Scheme 2.
It is well-known11,12 that 5-hydroxyisoxazoles are in equi-
librium with the other two tautomeric forms: CH and NH forms
(Figure 2). According to literature11,13–16 for 3-substituted
isoxazolin-5-ones in a solvent of low dielectric constant, the
a Reagents: (a) CDI, DMF, room temp; (b) (4-fluorophenyl)acetic acid
ethyl ester, NaH, room temp; (c) NH2OH·HCl, H2O, MeOH, 80 °C.
predominant form is the CH. The NH form becomes more
favored when substituents on C-4 are introduced and in more
polar solvents. For 5-hydroxyisoxazoles, contrary to what was
reported for 3-hydroxyisoxazolones,17 the OH form is rarely
observed; it was detected only when chelation took place
between the hydroxyl group and the 4-substituent.11
Analytic on 6 seems to indicate that in solution it assumes
an oxo form,11,13,14 as most of the compounds with a hydroxyl

2582 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Table 3. Inhibition of p38 MAP Kinasea
Brief Articles
Figure 5. Tautomeric equilibrium for 9.
Figure 6. 1H-1H NMR NOESY (bold line) and 1H-13C NMR HMBC
(dashed line) correlations in 10e (chemical shift in ppm).
Scheme 4. Synthesis of Ureas and Thioureas Derivatives
Table 4. p38 Inhibition
The data are in close agreement with literature,13 and they
indicate that 6, also in basic conditions, assumes the NH form.
A ring sp2 carbon bonded to one heteroatom (C-3) lies upfield
from a second sp2 carbon bonded to two heteroatom (C-5).
Furthermore, 2D NMR experiments confirmed those data
(Figure 3).
Derivatives of 6 were prepared by alkylation. As pointed out,
6 exists, in polar solvents such as DMSO, in the NH form. This
experimental evidence, nevertheless, does not imply absence
of tautomeric equilibrium undetectable by NMR. Different
tautomers could potentially lead to different alkylation products:
N-alkylation for the NH form and O-alkylation for the OH form.
In the literature it is possible to find examples of alkylation of
3-hydroxyisoxazoles18 as well as 3-hydroxyisothiazoles19 that
show that alkylation depends on the relative rates of reaction
of the two tautomers rather than on their relative proportions.
In the case of 6, alkylation reactions had been realized in
different solvent systems (DMF and CHCl3) to investigate
a IC50 SB203580 ) 0.045 ( 0.011 µM. b Percentage of inhibition at 10
µM (concentration of test compound). Inhibition data from p38 kinase assay,
ref 20.
function in the position R or γ to a nitrogen atom. Chemical
shifts from 13C NMR experiments in DMSO-d6, 90% D2O/10%
DMSO-d6, and 89.98% D2O/10% DMSO-d6/0.02% NaOH for
6 are listed in Table 1.

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2583
Figure 7. Suggested binding modus of 6 in the ATP pocket of p38 MAPK.
possible dependence of reaction product from solvent polarity,
but in all examined cases no dependency was observed and in
all cases the O-alkylation product was obtained versus the
N-alkylation one (Scheme 1).
obtained by N-hydro-C-alkylamino addition reactions according
to Scheme 4. Compound 9 was added to the isocyanate (or
isothiocyanate) in a polar solvent such as DMF in the presence
of sodium hydride as a base.
The alkylation products were identified by 1H-1H NMR
1
NOESY and by H-13C NMR HMBC (Figure 4).
Biological Results and Discussion
In Table 2 the 5-alkoxyisoxazole series is listed. The nature
of the introduced substituents in the 5 position at the isoxazole
core was chosen on the basis of an N-1-substituted imidazole
study undertaken in our research group.3 According to the cited
study, systematic optimization of the imidazole N-1 substituent
resulted in the identification of rests able to confer high
inhibitory activity to the molecule. For those compounds the
range of the experimental IC50 was (6–9) × 10-2 µM. The
undertaken study showed that alkoxy substitution in the 5
position of the isoxazole core is not tolerated, and it was not
possible to calculate any IC50 for any of the series members.
Compound 9, regioisomer of 6, was synthesized according
to the procedure outlined in Scheme 3. The first step of the
synthesis is a Claisen condensation of 2-aryl acetate ester and
pyridylimidazolide to give the ꢀ-ketoester 8 by using NaH as
the base system. The reaction had been also realized by using
harpoon bases such as LDA, NaHDMS, and sodium tert-
butoxide and by using, according to a classical Claisen
condensation scheme, sodium ethanolate. In these cases, no
reaction (LDA, sodium ethanolate) or just traces of product
(NaHDMS, tert-butoxide) were observed. The low conversions
result primarily from the fact that the product 1,3-dicarbonyl
compound is a carboxylic acid that quenches unreacted bases
at a rate that exceeds acylation.
Similar to what is described for 6, derivatives of 9 were
prepared (Table 3). The same considerations about tautomery
and reactivity toward alkylation done previously can be applied
to compound 9, in solution the different tautomeric forms could
theoretically interconvert according to the equilibrium in Figure
5. Alkylation reactions were realized by treating 9 with the
appropriate alkyl halide using triethylamine as base. Also in
this case, as it was observed for 6, experiments realized in CHCl3
and DMF showed absence of dependency of reaction products
by solvent nature. A 2D NMR study allowed identification of
the products as N-alkyl-5-isoxazolones, underlying a difference
in reactivity for the two tautomers (Figure 6). The presence of
the 4-fluorophenyl moiety in the 4 position on the isoxazolone
ring may affect the reactivity of the molecule toward nucleo-
philic substitution, leading to N-alkyl-5-isoxazolones. The series
of analogues was then completed by reacting 9 with isocyanates
and isothiocyanates, introducing carboxamides functions in the
2 position of the ring. Substituted ureas and thioureas were
The biological activity for the analogues in Table 2 shows that
alkoxy substitution in 5 position of the isoxazole core is not
tolerated; the unsatisfactory inhibition activity may be related to
sterical effects. The biological activity of 6 and 9 are reported in
Table 4. Of note is that the hydrogen bond acceptor for 9, as shown
by cocrystallization in mutated p38 active side,9 is the carbonyl
oxygen, and accepting the binding mode suggested in Figure 7 is
the ring nitrogen for 6. Different studies on the hydrogen bonding
properties of oxygen and nitrogen acceptors in aromatic hetero-
cycles have been published21,22 showing that nitrogen is a
significantly better H-bond acceptor than oxygen. In particular,
studies of Boehm et al.22 of the hydrogen bond capabilities of
oxygen atoms covalently bonded to an sp2 hybridized atom showed
that these were often considerably weaker acceptors than equivalent
nitrogen atoms. This is in contrast to the common expectations,
based solely on electronegativity, that both are good acceptors.
Nevertheless, for the two regioisomers 6 and 9, it must be taken
into account that the nitrogen is endocyclic while the carbonyl
oxygen exocyclic, this fact, influencing the length of the H-bond,
and the part of the molecules exposed to the ribose binding site, is
different in the two cases.
As the data in Table 4 show, the alkylated derivatives exert
relatively good activity, but they did not represent a real optimiza-
tion of the inhibitory potency of 9. The most potent alkyl substituent
was the propanol 10i and the ethoxyethyl 10f. These data are in
accordance with the already cited optimization study of the
imidazole N-1 substituents undertaken in our research group and
recently published (Figure 1).3 This evidence is relevant because
it allows application of the SAR of isoxazolones to imidazole and
vice versa. The most promising compounds were the urea and the
amide derivatives 10p-u. The good activity of the urea derivatives
is in agreement with the findings of researchers at Procter &
Gamble.9 Laughlin nevertheless reported significant metabolism
in an in vitro rat hepatocyte metabolic stability assay. For this
reason, the good inhibitory activity of amide-type 10p and 10q
can be considered a satisfying result.
Experimental Section. Typical procedures
Synthesis of 5-Alkoxyisoxazoles. General Procedure I. To a
suspension of 6 in DMF (2 mL/1mmol of 6) was added 3 equiv
of Et3N, and the mixture was refluxed for 2 h. The limpid
solution was then combined with halogen halide (1.8 equiv) and

2584 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Brief Articles
the mixture stirred under reflux for another 2 h and subsequently
at room temperature overnight. The mixture was poured into
water, and the two phases were partitioned. The water phase
was extracted twice with DCM. The collected organic phases
were washed with H2O, brine, and H2O, dried over Na2SO4, and
concentrated under vacuum. The final product was purified by
column chromatography on SiO2.
References
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4-[3-(4-Fluorophenyl)-5-methoxyisoxazol-4-yl]pyridine (7a).
7a was obtained according to general procedure I by reacting 111
µL (1.8 mmol) of methyl iodide and 256 mg (1 mmol) of 6 and
subsequent purification by column chromatography on SiO2 (eluent
THF). Yield 160 mg (22%); C15H11FN2O2 (MW 270.26); mp 229
1
°C; H NMR (DMSO-d6) δ (ppm) 3.84 (s, 3H, -CH3), 7.29–7.51
(m, 6H), 8.03–8.06 (d, 2H); 13C NMR (DMSO-d6) δ (ppm) 45.0,
85.4 (C4 Isox), 115.7, 116.3 (J)22.0, C4 4FPh), 128.9 (J ) 2.9,
C2 4FPh), 129.3 (J ) 8.5, C3/C5 4FPh), 142.1, 149.1, 161.4 (C3
Isox), 163.0 (J ) 229.0 Hz, C1 4FPh), 174.0 (C5 Isox); FTIR 3049,
2965, 1676, 1619, 1517, 1475, 1443, 1415, 1366, 1219, 1202, 956,
883, 844 cm-1; MS 271 (M + 1), 254, 243, 227, 212, 134, 94.
Anal. (C15H11FN2O2) C, H, N, O.
(6) Adams, J. L.; Boehm, J. C.; Kassis, S.; Gorycki, P. D.; Webb, E. F.;
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Gallagher, T. F. Pyrimidinylimidazole inhibitors of CSBP/p38 kinase
demonstrating decreased inhibition of hepatic cytochrome P450
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N-Alkylation of 9. General Procedure II. To a suspension of
9 (1 equiv) in DMF (2 mL/1mmol 9) was added 1.8 equiv of Et3N,
and the mixture was refluxed for 2 h. The cooled mixture (room
temperature) was combined with alkyl halide (1.5 equiv) (if not
specified otherwise, the chloride was used) and stirred for 3 h at
room temperature. The reaction was worked up following method
a, b, or c depending on the nature of the residue.
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4-(4-Fluorophenyl)-3-(pyridin-4-yl)-2-tosylisoxazol-5(2H)-
one (10p). 10p was synthesized according to general procedure II
by reacting 1.36 g (7.2mmol) of 4-methylbenzenesulfonyl chloride
with 1 g (3.9 mmol) of 9. Purification following method c
(crystallization from acetone) afforded the title compound. Yield
1
50 mg (3%); C21H15FN2O4S (MW 410.07); mp 198 °C; H NMR
(DMSO-d6) δ (ppm) 2.51 (s, 3H, -CH3), 6.96–7.26 (m, 4H),
7.37–7.43 (m, 4H), 7.64 (dd, J ) 0.5/1.4 Hz; 2H, Py), 8.78 (dd, J
) 0.5/1.4 Hz; 2H, Py); 13C NMR (DMSO-d6) δ (ppm) 21.8, 114.9
(C4 isoxazolone), 116.1 (J ) 19.1 Hz, C2/C6 4FPh), 121.4 (J )
3.5, C4 4FPh), 121.9, 125.8, 126.9, 127.6, 130.3 (J ) 8.4 Hz, C3/
C5 4FPh), 140.5, 147.6, 149.9, 155.6 (C3 isoxazolone), 163.0 (J )
251.0 Hz, C1 4FPh), 167.0 (C5 isoxazolone); FTIR 3053, 1770,
1638, 1590, 1509, 1384, 1228, 1173, 1158, 954, 848, 812, 690
cm-1. Anal. (C21H15FN2O4S) C, H, N, O.
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tautomerism of isoxazolones. J. Am. Chem. Soc. 1928, 50, 504–515.
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General Procedure III. To a solution of 9 (1 equiv) in DMF (1
mL/1mmol of 9), sodium hydride (1 equiv) and the appropriate
isocyanate (1.5 eq) were added. The mixture was stirred for 12 h
at room temperature and then combined with water. Filtration of
the formed precipitate afforded the pure title compound. A further
purification step was not necessary.
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N-Ethyl-4-(4-fluorophenyl)-5-oxo-3-(pyridin-4-yl)isoxazole-
2(5H)-carboxamide (10r). 10rwas synthesized according to the
general procedure III starting from 1 g (3.9mmol) of 9 and 0.4 g
(5.8 mmol) of ethyl isocyanate. Yield 293 mg (23%);
1
C17H14FN3O3 (MW 327.1); mp 200 °C; H NMR (DMSO-d6) δ
(ppm) 1.03 (t, 3H), 3.08 (m, 2H), 7.08–7.25(m, 4H, 4FPh), 7.45
(d, 2H, J ) 1.6 Hz, Py), 8.45 (t, 1H, exch.), 8.65 (d, 2H, J )
1.6 Hz, Py); 13C NMR (DMSO-d6) δ (ppm) 14.9, 22.5, 105.5
(C4 isoxazolone), 115.9 (J ) 21.3 Hz, C2/C6 4FPh), 123.0 (J
)3.1, C4 4FPh), 124.2, 131.0 (J ) 7.9 Hz, C3/C5 4FPh), 136.9,
148.2, 149.9, 154.4 (C3 isoxazolone), 161.9 (J ) 240.0 Hz, C1
4FPh), 166.2 (C5 isoxazolone); FTIR 3072, 2923, 2854, 1750,
(20) Laufer, S.; Thuma, S.; Peifer, C.; Greim, C.; Herweh, Y.; Albrecht,
A.; Dehner, F. An immunosorbent, nonradioactive p38 MAP kinase
assay comparable to standard radioactive liquid-phase assays. Anal.
Biochem. 2005, 344, 135–137.
1723, 1579, 1508, 1219, 954, 841, 826 cm-1
(C17H14FN3O3) C, H, N, O.
. Anal.
Acknowledgment. EU-Craft Program, Project Macrocept
(FP6) and Andy Liedtke for helpful discussions and proofreading.
(21) Nobeli, I.; Price, S. L.; Lommerse, J. P. M.; Taylor, R. Hydrogen
bonding properties of oxygen and nitrogen acceptors in aromatic
heterocycles. J. Comput. Chem. 1997, 18, 2060–2074.
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in competitive situations: which is the hydrogen-bond acceptor?
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Supporting Information Available: General synthetic proce-
dures, spectral and analytical and biochemical experiments. This
material is available free of charge via the Internet at http://
pubs.acs.org.
JM701343F