Discovery of potent and orally bioavailable N,N′-diarylurea antagonists for the CXCR2 chemokine receptor

Article abstract of DOI:10.1016/j.bmcl.2004.06.097

A series of 3-substituted N,N′-diarylureas was prepared and CXCR2 receptor affinities as well as pharmacokinetic properties were examined. A series of 3-substituted N,N′-diarylureas was prepared and the structure-activity relationship relative to CXCR2 re

Full text of DOI:10.1016/j.bmcl.2004.06.097

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4375–4378
Discovery of potent and orally bioavailable
N,N0-diarylurea antagonists for the CXCR2 chemokine receptor
Qi Jin,* Hong Nie, Brent W. McCleland, Katherine L. Widdowson, Michael R. Palovich,
John D. Elliott, Richard M. Goodman, Miriam Burman, Henry M. Sarau, Keith W. Ward,
Melanie Nord, Bonnie M. Orr, Peter D. Gorycki and Jakob Busch-Petersen*
GlaxoSmithKline, PO Box 1539, King of Prussia, PA 19406-0939, USA
Received 12 May 2004; revised 24 June 2004; accepted 26 June 2004
Abstract—A series of 3-substituted N,N0-diarylureas was prepared and the structure–activity relationship relative to CXCR2 recep-
tor affinity as well as their pharmacokinetic properties were examined. In vitro microsomal metabolism studies indicated that the
lower clearance rates of the 3-sulfonamido-substituted compounds were most likely due to the suppression of glucuronidation.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Interleukin-8 (IL-8) and related CXC chemokines
(ENA-78, GCP-2, GROa,b and c) play an important
role in the trafficking of neutrophils to sites of inflamma-
tion, which is consistent with their potential involvement
in pathophysiological processes such as arthritis, reper-
fusion injury, and asthma. Indeed, elevated plasma lev-
els of IL-8 and GROa have been associated with these
conditions in humans.1 Thus far, two seven transmem-
brane G-protein coupled receptors have been identified,
which are activated by IL-8 (CXCR1 and CXCR2).
CXCR1 binds IL-8 and GCP-2 with high affinity while
CXCR2 binds several ELR containing chemokines
including IL-8, GCP-2, ENA-78, GROa, GROb and
GROc with high affinity.2 The potential therapeutic va-
lue for small-molecule antagonists of the IL-8 receptors
is further supported by studies done with CXCR2
mouse gene knockouts, which show elevated lympho-
cytes without apparent pathogenic consequences indi-
cating that these receptors are not required for normal
physiology.3
and 2 are representative structures.5 However, while
these compounds displayed high affinity and selectivity
for the CXCR2 receptor, poor oral bioavailability and
high rates of in vivo clearance were observed. It was
thought that this might be due to rapid glucuronidation
and/or sulfation of the phenolic function. Therefore, a
series of compounds with varying substitution at the
adjacent 3-position was prepared to examine its influ-
ence on pharmacokinetic properties as well as on recep-
tor affinity.
As a part of this effort, the 3-carboxamide- (3), 3-amino-
methyl-(4), 3-sulfonamide-(5a) and 3-N,N-dimethylsul-
fonamide-(5b)
synthesized.
substituted
diphenylureas
were
The synthesis of 3-carboxamide substituted diphenyl-
urea 3 is presented in Scheme 1. Commercially available
nitrile 6 was debenzylated to yield the phenol 7. Nitra-
tion with NaNO3 and sulfuric acid resulted in the nitro
compound 8, which after hydrolysis of the nitrile with
H2SO4 yielded the primary amide 9. Reduction of the ni-
tro group with SnCl2 in EtOH followed by coupling
with 2-bromophenyl isocyanate produced the desired
urea 3. As presented in Scheme 2, nitration of 2,6-di-
chlorobenzyl bromide 10 resulted in the nitro product
11, which underwent the classical Gabriel procedure to
give benzylamine 12. Following Boc protection of the
amine, selective displacement of the chlorine ortho to
the nitro group was effected by exposure to potassium
Our laboratory has previously disclosed a series of N,N0-
phenylureas, which act as potent and selective CXCR2
antagonists.4 The early SAR evaluation indicated that
substitution at both the 3-position and 4-position on
the phenolic-bearing ring was well tolerated. Urea 1
Keywords: CXCR2; IL-8; Diarylureas.
*
Corresponding authors. Tel.: +1-6102707831; fax: +1-6102704451;
e-mail: jakob.2.busch-petersen@gsk.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.097

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Q. Jin et al. / Bioorg. & Med. Chem. Lett. 14 (2004) 4375–4378
was reacted with either ammonium hydroxide or di-
CN
CN
CN
Cl
OH
methyl amine to give the sulfonamides 15, which upon
hydrolysis of the oxazole moiety gave the amino-phen-
ols 16. Treatment with 2-bromophenyl isocyanates re-
sulted in the formation of the final ureas 5a and 5b.
Cl
OBn
Cl
Cl
OH
a
b
NO2
8
6
7
CONH2
CONH2
OH
Cl
O
OH
c
d,e
The CXCR1 and CXCR2 affinities for the six com-
pounds were determined in SPA-binding assays using
N
H
N
H
NO2
125I]-IL-8, and their in vivo pharmacokinetic properties
Br
[
9
3
were examined in Sprague–Dawley rats. The results are
summarized in Table 1. As seen previously with com-
pounds of this class, a high degree of selectivity for
CXCR2 over CXCR1 was observed.4 Also, electron-
withdrawing substituents at the 3-position appeared to
be favored, presumably due to the increased acidity of
the phenol.7 However, the fact that the 3-carboxamide
3 is approximately 6-fold more potent than the 3-chloro
compound 2 indicates that the acidity of the phenol is
not the only determinant of the affinity, since these com-
pounds are predicted to have very similar pKa values
(Table 1). Thus, amide or sulfonamide substituents at
the 3-position appear to have additional interactions,
which contribute to the binding affinity of these com-
pounds. As the tertiary sulfonamide 5b displays similar
potency to the primary sulfonamide 5a and the carbox-
amide 3, it does not seem to matter whether or not the 3-
substituent contains a hydrogen bond donating moiety.
Consequently, the observed increase in affinity is most
likely due to the amide or sulfonamide acting as hydro-
gen bond acceptors interacting with one or more resi-
dues in the receptor binding site.
Scheme 1. Reagents and conditions: (a) TFA, CH2Cl2, 66%;
(b) NaNO3, NaNO2, H2SO4, CH2Cl2, 27%; (c) H2SO4, 60ꢁC, 89%;
(d) SnCl2, EtOH, 72%; (e) 2-bromophenyl isocyanate, DMF, 73%.
NH2
Cl
Br
Br
Cl
Cl
Cl
Cl
Cl
a
b,c
Cl
NO2
NO2
10
11
12
NH2
NHBoc
OH
OH
d,e,f
g,h
Cl
O
N
H
N
NH2
H
Br
13
4
Scheme 2. Reagents and conditions: (a) H2SO4, HNO3, 86%; (b)
potassium phthalimide, DMF, rt, 79%; (c) hydrazine hydrate, EtOH,
rt, 81%; (d) (Boc)2O, CH2Cl2, rt, 95%; (e) (i) KOAc, 18-crown-6,
DMSO, 100ꢁC, (ii) NaOH 31%; (f) H2 (g), 10% Pd/C, AcOEt, 92%; (g)
2-bromophenyl isocyanate, DMF, 90%; (h) 4N HCl in 1,4-dioxane, rt,
34%.
The pharmacokinetic properties of the compounds were
tested in Sprague–Dawley rats. Despite their common
phenolic moiety, these compounds exhibited substantial
differences in both clearance and oral bioavailability.
Both the unsubstituted urea 1 as well as the 3-chloro
substituted compound 2 were rapidly cleared and
showed little oral bioavailability. The 3-carboxamide-
substituted urea 3 was prepared to examine the influence
of a polar moiety adjacent to the 2-phenol. However,
this compound only showed a slight decrease in clear-
ance when compared to the unsubstituted analog 1. Sim-
ilarly, placement of a charged group at the 3-position, in
the form of a aminomethyl moiety (4), did little to re-
duce clearance, but did improve the oral bioavailability.
In contrast, introduction of sulfonamide substituents
at the 3-position substantially decrease clearance and in-
creased oral bioavailability. Thus, urea 5a was cleared
approximately 13 times slower than that of unsubsti-
tuted compound 1, and 6 times slower than the 3-car-
box-amide 3.
acetate in the presence of 18-crown-6, which after a
basic work-up yielded the corresponding phenol.
Hydrogenation of the nitro group produced the 2-hyd-
roxy-aniline 13, which was then coupled with 2-bromo-
phenyl isocyanate. Deprotection of the amine under
acidic conditions afforded the desired urea 4. The prep-
aration of the sulfonamide substituted ureas 5a and 5b is
outlined in Scheme 3. The synthesis started from the sul-
fonyl chloride 14, which was prepared in high yields
according to a previously published procedure.6 This
R'
R''
O
R'
R''
O
N
S
Cl
S
N
S
O
O
O
O
Cl
Cl
Cl
OH
O
N
O
N
b
a
tBu
tBu
NH2
14
15
16
R'
R''
c
N
S
Further in vitro metabolism studies using rat and hu-
man hepatic microsomes were carried out in the pres-
ence and absence of uridine diphosphate glucuronic
acid (UDPGA), to discern the role of glucuronidation
in metabolism. As outlined in Table 2, clearance of the
carboxamide 3 was low, but was significantly enhanced
in the presence of UDPGA, demonstrating the impor-
tant role of glucuronidation in the clearance of this
amide. However, the intrinsic clearance of sulfonamide
5a was only minimally enhanced in the presence of
O
O
OH
Br
Cl
O
N
H
N
H
5
Scheme 3. Reagents and conditions: (a) R0R00NH, Et3N, CH2Cl2, rt;
(R0, R00 =H: 78%; R0, R00 =Me: 74%) (b) 10% aq H2SO4, reflux ; (R0,
R00 =H: 75%; R0, R00 =Me: 63%) (c) 2-bromophenyl isocyanate, DMF;
(R0, R00 =H: 62%; R0, R00 =Me: 84%).

Q. Jin et al. / Bioorg. & Med. Chem. Lett. 14 (2004) 4375–4378
4377
Table 1. CXCR1 and CXCR2 binding affinities, pharmacokinetic properties for ureas 1–5b
R
OH
Br
Cl
O
N
N
H
H
Compound
R
CXCR2a (IC50, nM)
CXCR1a (IC50, nM)
CLb (mL/min/kg)
Oral F (%)
cLogDc
pKa (calcd)d
1
H
Cl
906
63
10
114
7
<30,000
13,360
6037
323
73.6
105
104
16.1
23.4
6.6
13
3.85
4.02
2.43
0.33
2.05
3.23
8.24
7.24
7.11
6.85
6.12
6.19
2
3
CONH2
CH2NH2ÆHCl
SO2NH2
SO2NMe2
3.6
67
4
30,000
1366
4317
5a
5b
86
100
12
Abbreviations: CL: systemic plasma clearance; F: percent bioavailability.
a Binding assays were performed on membranes of Chinese hamster ovary (CHO) cell lines expressing either CXCR1 or CXCR2. The CHO-CXCR1
and CHO-CXCR2 membranes were prepared according to Kraft and Anderson.8 All assays were performed in 96-well microtiter plates using
radio-labeled [125I]-IL-8 (human recombinant; concentration: 0.23nM). The binding results are expressed as a mean of three individual experi-
ments.
b Pharmacokinetic experiments were conducted using an iv·po crossover design in male Sprague–Dawley rats (n=3 per study). Compounds were
administered in an aqueous solution containing 1% DMSO and up to 20% hydroxypropyl-beta-cyclodextrin at dosages of 4 and 8lmol/kg for iv
and po doses. Blood samples were obtained from a lateral tail vein and plasma analyzed for drug content using LC/MS/MS methodologies, with a
lower limit of quantification of >10ng/mL for each analyte.
c cLogD values were calculated using MANTIS 2.1.0 software based on the Daylight/PCModels 4.81 package supplied by Daylight Chemical
Information Systems, Los Altos.
d pKa values were calculated using ACD/pKa software supplied by Advanced Chemical Development, Toronto.
Table 2. In vitro intrinsic clearance of ureas 3 and 5a in rat and human
liver microsomes
however, in this case it seems to be less significant as
judged by the calculated logDs (Table 1).
Compound
Clint
(mL/min/gliver)a
Clint
(mL/min/gliver)a
In summary, placement of a 3-sulfonamido substituent
on the previously described diaryl urea scaffold resulted
in the discovery of a new class of potent and selective
CXCR2 receptor antagonists with greatly improved
pharmacokinetic properties. In vitro studies performed
with hepatic microsomes indicate that the enhanced
pharmacokinetic properties of these compounds may
be a result of decreased susceptibility to phenolic glucu-
ronidation. Compounds of this type should prove highly
useful for the investigation of the pharmacology related
to the CXCR2 receptor in vivo.
Rat
Human
(ꢀ)
(+)
UDPGA
(ꢀ)
(+)
UDPGA
UDPGA
UDPGA
3
5a
1.5
Stable
5.3
1.6
0.74
Stable
15
2.7
a In vitro intrinsic clearance experiments were conducted in commer-
cially purchased rat or human liver microsomes at 0.5mg microsomal
protein/mL incubation and final compound concentrations of 0.5–
1lM. Disappearance of substrate was monitored over a 30-min
incubation by LC/MS/MS and the intrinsic clearance (CLint) calcu-
lated from the slope of the substrate disappearance versus time curve.
References and notes
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