Synthesis and structure-activity relationships of 3,5-diarylisoxazoles and 3,5-diaryl-1,2,4-oxadiazoles, novel classes of small molecule interleukin-8 (IL-8) receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.05.080

A novel series of 3,5-diarylisoxazole and 3,5-diaryl-1,2,4-oxadiazole IL-8 inhibitors has been identified. These compounds exhibit activity in an IL-8 binding assay as well as in a functional assay of IL-8 induced elastase release from neutrophils. In add

Full text of DOI:10.1016/j.bmcl.2004.05.080

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4307–4311
Synthesis and structure–activity relationships of
3,5-diarylisoxazoles and 3,5-diaryl-1,2,4-oxadiazoles, novel
classes of small molecule interleukin-8 (IL-8) receptor antagonists
Michele A. Weidner-Wells,* Todd C. Henninger, Stephanie A. Fraga-Spano,
Christine M. Boggs, Michele Matheis, David M. Ritchie, Dennis C. Argentieri,
Michael P. Wachter and Dennis J. Hlasta
Johnson and Johnson Pharmaceutical Research and Development, LLC 1000 Route 202, Raritan, NJ 08869, USA
Received 14 April 2004; revised 27 May 2004; accepted 27 May 2004
Available online
Abstract—A novel series of 3,5-diarylisoxazole and 3,5-diaryl-1,2,4-oxadiazole IL-8 antagonists has been identified. These com-
pounds exhibit activity in an IL-8 binding assay as well as in a functional assay of IL-8 induced elastase release from neutrophils. In
addition, one of the compounds exhibits oral activity in a rat adjuvant arthritis model.
Ó 2004 Elsevier Ltd. All rights reserved.
Interleukin-8 (IL-8), a 72-amino acid peptide, is a
member of the C–X–C family of chemokines. It is pro-
duced primarily by monocytes and macrophages.1 Its
production can be triggered by inflammatory stimuli
such as IL-1, TNF-a, LPS, and IL-4.2 IL-8 is a potent
chemotactic agent for neutrophils, and can stimulate
neutrophil degranulation and induce basophil histamine
release.1 Neutrophils are prime players in the inflam-
matory response and are responsible for extensive tissue
damage due to the release of lysosomal enzymes such as
elastase, myeloperoxidase, cathepsins, etc. High levels of
IL-8 have been detected in sites of artherosclerosis and
ischemic reperfusion injury, in the synovial fluid and
cells from rheumatoid arthritis patients and also in
psoriatic plaques.1 The IL-8 receptor is a seven trans-
membrane domain G-protein coupled receptor (GPCR)
that is found in abundant quantities on neutrophils.3
Inhibition of the actions of IL-8 on neutrophils should
limit their migration to the sites of inflammation, pre-
vent activation, and thus inhibit the subsequent release
of lysosomal enzymes. Therefore, IL-8 receptor antago-
nists may lead to unique anti-inflammatory agents.
Relatively few classes of small molecule IL-8 receptor
antagonists have been reported, making the identifica-
tion of new classes of antagonists an important objec-
tive. Widdowson and co-workers described the first
potent nonpeptide antagonist of IL-8.4;5 This com-
pound, a substituted diphenyl urea 1, was shown to
inhibit IL-8 induced neutrophil migration with an
IC50 ¼ 22 nM. Recently, Li et al. has disclosed a series of
2-amino-3-heteroarylquinoxalines 2 that inhibited IL-8
binding with potencies in the range from 0.11 to 33 lM.6
Baxter et al. reported on a series of orally bioavailable
triazolethiol inhibitors, such as compound 3.7
HO
N
N
Het
O
N
N
NH(CH2)nNRR1
H
H
OH
Br
1
2
Cl
Cl
N
N
N
SH
3
We have identified a series of regioisomeric 3,5-diaryl-
isoxazoles 4 and 5 along with a series of 3,5-diaryl-1,2,4-
oxadiazoles 6, which act as IL-8 receptor antagonists.8;9
Keywords: IL-8; Inflammation; Diarylisoxazoles.
* Corresponding author. Tel.: +1-908-704-5933; fax: +1-908-203-8109;
e-mail: mwells@prdus.jnj.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.080

4308
M. A. Weidner-Wells et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4307–4311
R
R
O
3
5
3
5
O
R2R1N
N
O
NR1R2
N
O
4
5
R
3
N
5
O
NR1R2
N
O
6
Herein we describe the synthesis and structure–activity
relationships of these novel series.
1,2,4-Oxadiazoles 6 were synthesized according to
Scheme 2. Amide oxime 10, prepared from the desired
benzonitrile derivative, was condensed with 4-methoxy
benzoyl chloride in pyridine to afford oxadiazole nucleus
11.11 Phenol 12, generated by deprotection of 11 with
boron tribromide, was alkylated with the appropriately
substituted chloro alkyl amine to yield ether 6.
The key reaction for the preparation of the isoxazoles 4
and 5 is a nitrile oxide [3þ2] dipolar cycloaddition
reaction between an appropriately substituted chloro
oxime and a phenyl acetylene derivative. Scheme 1
exemplifies the synthesis of isoxazoles 4. Anisaldehyde
was converted in two steps into the chloro oxime 7
utilizing N-chlorosuccinimide as the chlorinating
agent.10 This was reacted with the appropriately
substituted phenylacetylene derivative to afford isoxaz-
ole 8. Deprotection of the aryl methyl ether with boron
tribromide afforded phenol 9 in good overall yield.
Alkylation with the appropriately substituted chloro
alkylamine produced isoxazole targets 4. Isoxazoles 5
were generated in an analogous manner utilizing the
appropriately substituted chloro oxime and methoxy-
phenylacetylene in the cycloaddition reaction.
The compounds were screened in a receptor-binding
assay in human neutrophils using 125I-IL-8 as ligand
with the results being reported as an IC50.12
Early on it was clear that a basic side chain was required
for activity since the methoxy intermediates 8 and 11
and phenol intermediates 9 and 12 were inactive in the
assay. In addition, several analogs of 4 in which the
amine was replaced with a carboxylic acid were inactive
(data not shown). The 5-aryl group of the isoxazole 4
was essential for activity since replacement with either a
a, b
c
MeO
MeO
MeO
R
Cl
CHO
N
O
N
OH
7
8
HO
d
e
R
4
N
O
9
Scheme 1. Reagents and conditions: (a) hydroxylamine hydrochloride, pyridine, methylene chloride, 95%; (b) N-chlorosuccinimide, DMF, 0 °C,
93%; (c) phenylacetylene, triethylamine, ethyl acetate, 80–90%; (d) BBr3, methylene chloride, 0 °C, 75–90%; (e) chloroalkyl amine, potassium
carbonate, DMF, 90 °C, 60–85%.
a
b
R
R
R
N
O
OMe
NH2
OH
CN
N
N
10
11
R
c
d
OH
N
O
6
N
12
Scheme 2. Reagents and conditions: (a) aq hydroxylamine, ethanol, reflux, 78–89%; (b) methoxy benzoyl chloride, pyridine, reflux, 70–85%; (c) BBr3,
methylene chloride, 0 °C, 75–90%; (e) chloroalkyl amine, potassium carbonate, DMF, 90 °C, 60–85%.

M. A. Weidner-Wells et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4307–4311
Table 1. IL-8 receptor antagonist activity of isoxazoles 4
4309
3
4
2
Z
3
2
X
O
m
R
4
N
O
4
Compound
Z
m
Position
X
R
IC50 (lM)
4a
4b
4c
4d
4e
4f
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
1
1
1
1
1
1
4
4
4
4
3
2
CH
CH
CH
CH
CH
CH
H
10.6
2.7
4-Cl
4-F
3.1
4-NO2
4-Cl
4-Cl
12.1
8.1
4.4
4g
4h
4i
1
1
1
1
2
1
4
4
4
4
4
4
CH
CH
CH
CH
CH
N
H
5.4
6.2
N
H
N
N
N
N
N
N
H
7.6
4j
4-Cl
4-Cl
H
1.6
N
N
N
4k
4l
8.9
11.5
benzyl or n-butyl group led to complete loss of activity.
In addition, replacement of the ether linkage of isox-
azole 4 or oxadiazole 6 with an amide gave compounds
2–5-fold less potent (data not shown).
The structure–activity relationships of the regioisomeric
isoxazoles 5 were also determined. As is evident from the
data in Table 2, the N-methylpiperazinyl group is
slightly better than the dimethylamino group (5a vs 5b
and 5c vs 5d).
The relationship between substitution of the 5-phenyl
ring of 4 and binding activity was investigated by varying
the dipolarophile utilized in the cycloaddition reaction.
From the data in Table 1, a variety of functional
groups were tolerated, though halogens seem to be
optimal with 4b and 4c being more potent than 4a and
4d.
A wide variety of substituents on the 3-phenyl ring were
investigated since numerous benzaldehyde derivatives
were commercially available. The 4-phenoxy compound
5g as well as the 3-chloro analog 5e showed decreased
potency. The 4-chloro analog 5d and the 4-fluoro analog
5f are equipotent, similar to the trend found with isox-
azoles 4.
The effect of regiochemistry of the aminoalkoxy chain
on activity was examined. The ortho and para substi-
tuted analogs, 4f and 4b, respectively, were slightly more
potent than meta analog 4e.
With respect to the central heteroaryl nucleus, generally,
isoxazoles 5 were equipotent to isoxazoles 4. The 1,2,4-
oxadiazoles 6 exhibit the same trends as isoxazoles 5
(some data not shown for the oxadiazoles), except that
now there is a difference in activity between the 4-fluoro
(6a) and 4-chloro (6b) analogs with the 4-chloro analog
6b being more potent. Replacement of the N-methyl-
piperazinyl group with a morpholine (6c) resulted in a
complete loss of activity, most likely due to the reduced
basicity of the ring nitrogen.
The aminoalkoxy side chain was modified in two ways:
by varying the nature of the amine and by changing the
length of the chain. Comparison of amines 4a,g,h, and 4i
indicates that there is not a significant difference in
activity as the amine is varied; therefore, we decided to
utilize the N-methylpiperazinyl group to flesh out the
structure–activity relationships for this series of isox-
azoles. From the limited number of chain lengths
examined, the three-carbon length 4j was approximately
5-fold more potent than the four-carbon chain 4k. The
two-carbon analog with a dimethylamino group was less
potent than the three carbon analog 4a (data not shown)
possibly due to the lower pKa of the dimethylamino
group of the former. In addition, the 5-(4-chlorophenyl)
group was replaced with a 2-pyridyl ring leading to a 10-
fold reduction in potency (4j vs 4l).
Several of the more potent compounds were tested in a
functional assay to determine the inhibition of IL-8
induced elastase release from neutrophils13 (Table 3).
These compounds displayed inhibitory activity in the
functional assay with similar potency as in the IL-8
receptor binding assay.
Compound 5f was tested in vivo in a rat adjuvant
arthritis model.14 In this assay, Mycobacteria butyricum

4310
M. A. Weidner-Wells et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4307–4311
Table 2. IL-8 receptor antagonist activity of isoxazoles
oxadiazoles 6
5 and
3
2.5
2
R
Y
O
N
O
1.5
1
Z
5, 6
0.5
0
Compound
R
H
Z
Y
IC50 (lM)
5a
NMe2
CH
6.7
Vehicle
I
ndoa
10.0
Compound 5f (mg/kg, p.o.)
1.0
3.0
30.0
5b
5c
5d
H
CH
CH
CH
3.0
3.9
2.1
N
N
aIndomethacin; 0.25 mg/kg
4-Cl
4-Cl
NMe2
Figure 1. Rat adjuvant arthritis model.
N
N
N
N
N
N
N
N
5e
5f
3-Cl
4-F
CH
CH
CH
CH
N
9.6
2.0
may serve as a starting point for the design of more
potent analogs.
5g
5h
6a
6b
6c
4-PhO
4-CF3
4-F
22.7
6.0
References and notes
N
N
N
N
N
1. Kemeny, L.; Ruzicka, T.; Dobozy, A.; Michel, G. Intern.
Arch. Allergy Inflammation 1994, 104, 317–322.
2. Strieter, R. M.; Koch, A. E.; Antony, V. B.; Fick, R. B.,
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1994, 123, 183–197.
10.5
2.5
4-Cl
4-Cl
N
N
N
>25
O
N
3. Wu, D.; LaRosa, G. J.; Simon, M. I. Science 1993, 261,
101–103.
4. (a) White, J.; Lee, J.; Young, P.; Hertzberg, R.; Jureqicz,
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E.; Martin, L.; Lee, J. M.; White, J. R.; Sarau, H. M.
J. Med. Chem. 2004, 47, 1319–1321.
Table 3. Inhibition of IL-8 induced elastase release from neutrophils
Compound
Elastase
IC50 (lM)
IL-8
IC50 (lM)
4c
5d
5f
4.0
3.6
3.8
4.5
3.1
2.1
2.0
2.5
6b
5. Widdowson, K. L.; Veber, D. F.; Jurewicz, A. J.;
Hertzberg, R. P.; Rutledge, M. C., Jr. U.S. Patent
5780483, 1998.
was injected into the footpad of rats. Adjuvant-induced
arthritis was allowed to develop for 10 days. At this
time, a baseline paw size was determined. Indomethacin
was used as a positive control and vehicle as a negative
control. Compound 5f was administered orally once a
day from day 10 through day 14. The amount of
swelling in the paw on day 14 was compared to that of
day 10. A reduction in paw swelling indicates that the
compound is exhibiting therapeutic anti-inflammatory
effects. Isoxazole 5f afforded a 30% reduction in paw
volume at 10 mg/kg. Minimal effect was seen at lower
doses of 5f (Fig. 1).
6. Li, J. J.; Carson, K. G.; Trivedi, B. K.; Yue, W. S.; Ye, Q.;
Glynn, R. A.; Miller, S. R.; Connor, D. T.; Roth, B. D.;
Luly, J. R.; Low, J. E.; Heilig, D. J.; Yang, W.; Qin, S.;
Hunt, S. Bioorg. Med. Chem. 2003, 3777–3790.
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Ritchie, D. M.; Matheis, M.; Argentieri, D.; Hlasta, D. J.;
Wachter, M. Abstract of Papers, Nat. Med. Chem. Sym.,
Kansas City, MO, June 2000, Abstract B-18.
9. Weidner-Wells, M. A.; Henninger, T. C.; Hlasta, D. J.
U.S. Patent 20020049213, 2002.
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11. Chiou, S.; Shine, H. J. J. Heterocycl. Chem. 1989, 26, 125–
128.
12. IL-8 receptor binding assay: This is a radiolabeled
receptor binding assay conducted in human neutrophils
which have been purified from fresh human blood by
dextran sedimentation. The experiment is conducted by
adding reaction buffer to each well, followed by the
addition of drug, 20 lL I125-IL-8 (final concentra-
tion ¼ 0.125 nM) ligand and cell suspension. After the
We have identified a series of 3,5-diarylisoxazole and
3,5-diaryl-1,2,4-oxadiazole IL-8 receptor antagonists
with low micromolar potency. Several of these com-
pounds demonstrate activity in a functional assay
involving the inhibition of IL-8 induced release of elas-
tase from neutrophils. Oral activity was observed for
compound 5f in a rat adjuvant arthritis model.
Although the desired nanomolar potency was not
achieved with this series of inhibitors, these compounds

M. A. Weidner-Wells et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4307–4311
4311
plates are incubated at 37 °C for 90 min they are aspirated
and counted for 1 min in a Packard TopCount. Drug
treated wells are compared to vehicle wells for inhibition
of binding. Unlabeled IL-8 serves as the standard anta-
gonist and has an IC50 of approximately 1 nM.
reaction is proportional to the amount of elastase released
by IL-8 challenge since the substrate is in molar excess.
Inhibition of the reaction by drug treatment is determined
by comparing the drug treated wells with those treated
with vehicle.
13. Elastase assay: Neutrophils are purified from human
blood by dextran sedimentation. The procedure is con-
ducted by adding 100 lL IL-8 and 47 lL PBS to each well
of a 96-well plate, followed by the addition of 3 lL drug
(2.5 mM in 25% methanol) or vehicle. Neutrophils stim-
ulated with cytochalasin B (30 lg/mL in 3% DMSO) are
then added and the plate is incubated for 30 min at 37 °C.
The plate is then centrifuged and a fraction of the
supernatant is transferred to a 96-well black plate to
which 50 lL elastase substrate is added. The fluorescence
of the wells is read at 0 time and at 90 min to determine the
change over time (velocity) in a spectrofluorometer with
an excitation @ 360 and emission @ 460. Velocity of the
14. Adjuvant-induced Arthritis model: Male Lewis rats are
injected in the footpad with 0.1 mL of Mycobacteria
butyricum suspended in light mineral oil at a concentration
of 7.5 mg/mL. Adjuvant-induced arthritis is allowed to
develop and on day 10 a baseline paw size reading is made
on the uninjected paw with a mercury displacement edema
computer. Dosing with test compound begins on day 10
and continues through day 14, with the paws again dipped
on day 14 to measure paw swelling. Compound is
administered by oral gavage and the effects of test
compounds are determined by comparing the paw swelling
in drug treated animals with that of vehicle treated
controls.