Comparison of N,N′-diarylsquaramides and N,N′-diarylureas as antagonists of the CXCR2 chemokine receptor

Article abstract of DOI:10.1016/j.bmcl.2006.12.067

N,N′-diarylsquaramides were prepared and evaluated as antagonists of CXCR2. The compounds were found to be potent and selective antagonists of CXCR2. Significant differences in SAR was observed relative to the previously described N,N′-diarylurea series. As was the case in the N,N′-diarylurea series, placing sulfonamide substituent adjacent to the acidic phenol significantly reduced the clearance in rat pharmacokinetic studies.

Full text of DOI:10.1016/j.bmcl.2006.12.067

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1713–1717
Comparison of N,N0-diarylsquaramides and N,N0-diarylureas as
antagonists of the CXCR2 chemokine receptor
Brent W. McCleland, Roderick S. Davis, Michael R. Palovich, Katherine L. Widdowson,
Michelle L. Werner, Miriam Burman, James J. Foley, Dulcie B. Schmidt, Henry M. Sarau,
Martin Rogers, Kevin L. Salyers,à Peter D. Gorycki, Theresa J. Roethke, Gary J. Stelman,
Leonard M. Azzarano, Keith W. Ward§ and Jakob Busch-Petersen*
GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
Received 7 December 2006; accepted 20 December 2006
Available online 23 December 2006
Abstract—N,N0-diarylsquaramides were prepared and evaluated as antagonists of CXCR2. The compounds were found to be
potent and selective antagonists of CXCR2. Significant differences in SAR was observed relative to the previously described
N,N0-diarylurea series. As was the case in the N,N0-diarylurea series, placing sulfonamide substituent adjacent to the acidic phenol
significantly reduced the clearance in rat pharmacokinetic studies.
Ó 2007 Elsevier Ltd. All rights reserved.
Interleukin-8 (IL-8, CXCL8) and related CXC chemo-
kines (ENA-78 (CXCL5), GCP-2 (CXCL6), GROa
CXCL1), GROb (CXCL2) and GROc (CXCL3) play
an important role in the trafficking of immune cells to
sites of inflammation which is consistent with their
potential involvement in pathophysiological processes
such as arthritis, reperfusion injury and asthma. Indeed,
elevated plasma levels of IL-8 and GROa have been
associated with these conditions in humans.1 Thus far,
two seven transmembrane 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 all of the
above-mentioned ELR + chemokines with high affinity.2
The potential therapeutic value for small-molecule
antagonists of the IL-8 receptors is further supported
by studies done with CXCR2 mouse gene knockouts
which show elevated leukocytes and lymphocytes
without apparent pathogenic consequences indicating
that these receptors are not required for normal
physiology.3
As previously disclosed,4–6 a series of N,N0-diarylureas
has been identified as potent, selective CXCR2
antagonists. This series was later expanded to include
N,N0-diarylguanidines and N,N0-diarylcyanoguanidines
(Fig. 1).7 Squaramides (3,4-diamino-1,2-dioxocyclobut-
enes) have previously been described as bioisosteres of
ureas and cyanoguanidines8 and it was decided to incor-
porate this structural feature into the above-mentioned
compound series.
Chemistry. Compounds to elucidate the basic SAR of
the phenol-bearing left-hand side were prepared as
O2N
O
N
H
N
H
Br
Br
OH
OH
Cl
O
N
O
S
N
H
N
H
Me2N
O
CN
Cl
O
*
Corresponding author. Tel.: +1 601 270 7831; fax: +1 610 270
4451; e-mail: jakob.2.busch-petersen@gsk.com
Co-op student, 2000.
S
O
N
H
N
H
Me2N
Ph
OH
à
Figure 1. Examples of urea and cyanoguanidine-type CXCR2
antagonists.
Present address: Kalypsys Inc., San Diego, CA, USA.
Present address: Bauch and Lomb, Rochester, NY, USA.
§
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.067

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B. W. McCleland et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1713–1717
O
O
O
O
O
OH
OH
a
R
+
NH2
Et
N
H
Ph
N
N
H
Ph
H
R
2
3
1
Scheme 1. Preparation of diarylsquaramides for basic SAR. Reagents: (a) AlMe3, CH2Cl2.
outlined in Scheme 1. Aminophenols 1, either commer-
cially available or obtained using standard methodology
as previously described,5 were treated with commercially
available 3-(ethyloxy)-4-(phenylamino)-3-cyclobutene-
1,2-dione 2 in the presence of trimethylaluminum to give
the diarylsquaramides 3. Sulfonamide and amide-con-
taining diarylsquaramides were prepared as outlined in
Scheme 2. For the sulfonamide-containing diarylsquara-
mides 10a, the synthesis started from the commercially
available 2,6-dichlorophenylsulfonyl chloride 4a which
was hydrolysed, selectively nitrated and re-chlorinated
to yield the sulfonyl chloride 6a which was reacted with
a variety of amines to give sulfonamides 7a. Selective
displacement of the 2-chloro substituent with hydroxide
produced the nitrophenols 8a which were subsequently
reduced to the aminophenols 9a. Treatment with 3,4-
dichlorocyclobut-3-ene-1,2-dione in THF at ambient
temperature followed by exposure to a variety of ani-
lines in DMF at 40 °C produced the diarylsquaramides
10a. As for the amide-containing diarylsquaramides
10b, the synthesis began with commercially available
2,6-dichloro-3-nitrobenzoic acid or 2-chloro-3-nitroben-
zoic acid which following acid chloride formation under
standard conditions (6b) were treated with a variety of
amines to yield amides 7b. Chloride displacement,
reduction and squaramide formation were carried out
under the same reaction conditions used for the sulfon-
amide containing diarylsquaramides.
The SAR of the diarylsquaramide series9 is substantially
different from that of the urea series as illustrated in
Table 1. While both series require the presence of two
urea/squaramide NHs as well as the phenolic group,
the ureas are highly sensitive to the ionization state of
the phenol10 while the diarylsquaramides seem to be
affected less. Thus, the squaramide 3a (R,R1 = H),
which had a pKa of 8.611 equal to 5.9% ionization under
the assay conditions (pH 7.4), maintained reasonable
affinity for the CXCR2 receptor with an IC50 of
235 nM. However, the corresponding urea (pKa: 9.1;
2.0% ionization at pH 7.4) was a weak inhibitor of
IL-8 binding with an IC50 > 30 lM. When an electron-
withdrawing group such as nitro or cyano was added
in the 3- or 4-position (entries b, c, e and h), the pKa
of the phenolic group was lowered which led to the com-
pounds being predominantly ionized under the assay
conditions (squaramide 3c: pKa: 7.09; %ionization, 67).
For the diarylsquaramides, this resulted in an approx.
10-fold enhancement in receptor affinity while the effect
on the corresponding ureas was a substantially greater
increase of >100-fold. This suggests that while the estab-
lishment of a salt bridge between the phenoxide and the
SO2Cl
Cl
SO2OH
Cl
SO2Cl
Cl
Cl
Cl
Cl
b
d
a
NO2
NO2
4a
5a
6a
XCl
XNR1R2
Cl
XNR1R2
OH
Cl
R
R
c
R
NO2
NO2
NO2
6a/b
7a/b
8a/b
e
XNR1R2
O
O
R1R2NX
R
OH
f
R
OH
N
N
H
NH2
H
R3
9a/b
10a/b
a: R= Cl; X= SO2. b: R= Cl,H; X= CO.
Scheme 2. Preparation of sulfonamide and amide-containing diarylsquaramides 10. Reagents and conditions: (a) i—LiOH, MeOH; ii—H2SO4,
HNO3, 85%; (b) i—KOH; ii—POCl3, PCl5; (c) HNR1R2, Et3N, DCM, 74–89%; (d) NaH, H2O, THF, 50–68%; (e) H2, Pt/C, 65–85%; (f) i—3,4-
dichlorocyclobut-3-ene-1,2-dione, THF, rt; ii—ArNH2, DMF, 40 °C, 19–89%.

B. W. McCleland et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1713–1717
1715
Table 1. CXCR2 receptor affinities of N,N0-diarylsquaramides and their corresponding ureasa
O
O
3
6
4'
4
5
OH
Squaramide: X=
2
R
3'
X
O
1'
1
N
2'
N
Urea: X=
R1
H
H
3
Compound 3
R
R1
Squaramide CXCR2a,b,c [IC50 (nM)]
Urea CXCR2a,b [IC50 (nM)]
a
b
c
d
e
f
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
235
32
>30,000
276
200
3-CN
4-CN
4-Me
4-NO2
4-NO2, 5-Cl
5-Me
5-Cl
15
205
22
25,000
320
950
52
g
h
i
308
54
>30,000
>30,000
>30,000
>30,000
>9000
25
6-Me
6-NO2
H
1720
358
51
j
k
l
4-CN
4-NO2
36
37
m
22
a Binding assays were performed on Chinese hamster ovary (CHO) cell lines expressing either CXCR2. The CHO-CXCR2 membranes were prepared
according to Kraft and Anderson.12 All assays were performed in 96-well microtitre plates using radiolabelled [125I]-IL-8 (human recombinant,
concn: 0.23 nM). The binding results are expressed as a mean of three individual experiments.
b All active compounds were at least 20-fold selective versus CXCR1.
c The functional activities of the diarylsquaramides 3a, 3c, 3e, 3f, 3h, 3k and 3m were tested in a calcium mobilization assay performed with human
neutrophils using GROa as the ligand. The resulting IC50’s were within 5-fold of the binding IC50’s reported in this table.
receptor is very important in the urea series, the polar
phenol/receptor interaction may be somewhat different
in nature for the squaramide series. The SAR of the
non-phenol-bearing phenyl ring was also quite different:
whereas introduction of a 2-bromo substituent in the
urea series led to a substantial increase (approx. 10-fold)
in receptor affinity (entries l and m), the effect in the
squaramide series appeared to be negligible. These dif-
ferences are perhaps not surprising when you consider
that the dioxocyclobutene core of the squaramides clear-
ly is a larger group than the urea carbonyl, thus pushing
tice energy thus counteracting the solubilizing effect of
the phenoxide ion. Regarding the 3-position substituent,
the receptor was able to tolerate remarkably large
groups such as the 4-piperidylpiperidinesulfonamide
group (10k) without loss of receptor potency. With
respect to solubility, amides appear to have slightly
higher solubility than sulfonamides. Regarding the
amide/sulfonamide substitution, methylhomopiperazine
(10i, 10j and 10p) seems to be the substituent that most
effectively solubilizes compounds of this type. The phar-
macokinetic properties of several compounds were as-
sessed in the rat. One interesting question was whether
or not the presence of a 3-sulfonamide substituent would
be required for suppression of rapid phenol glucuronida-
tion leading to high clearance as was the case in the dia-
rylurea series.6 Certainly the sulfonamide-containing
compounds (10e–k) appear to have low to very low
clearance while non-sulfonamide-containing diarylsqu-
aramides have higher clearance. It is important to note,
however, that the clearance of the squaramide series as a
whole appears to be lower than that of the diarylurea
series. This, combined with the fact that the volumes
of distribution observed with the diarylsquaramide series
are very low, suggests that this series may be more pro-
tein bound than the diarylurea series. Some diarylsqu-
aramides such as 10h, however, do display a desirable
pharmacokinetic profile with low clearance, not too
low volume of distribution and acceptable oral bioavail-
ability. Non-sulfonamide-containing diarylsquaramides
by and large displayed poor oral bioavailability.
˚
the anilino moieties approximately 1 A further apart.
Hence, it is reasonable to expect that the two series will
experience slightly different binding environments. The
fact that both series are able to attain high-binding affin-
ity may be seen as evidence that the receptor binding
pocket is quite flexible. As was the case the urea and
cyanoguanidines series,7 the 3-position adjacent to the
phenol is very tolerant to substitution and is an ideal
location for the introduction of groups aimed at modu-
lating physicochemical parameters such as solubility.
The aqueous solubility of the diarylsquaramide template
is inherently rather low and thus solubilizing sulfon-
amide and amide groups were introduced at this posi-
tion. The results are summarized in Table 2. As was
the case with the simple diarylsquaramides (Table 1),
the nature of the substituents at the 4- and 2-positions
appears to have a very limited effect on the CXCR2
potency of the amide and sulfonamide-substituted com-
pounds 10. In addition, the effect of these substituents
on solubility is also rather limited. In the case of the
4-substituent, this is interesting since one might have
expected that a higher degree of phenol ionization might
result in higher solubility. One possible explanation
would be that more acidic phenol also increases the lat-
In summary, diarylsquaramides have been shown to be
potent inhibitors of the CXCR2 receptor. The SAR of
this series was found to be distinctly different from that
of the closely related diarylurea series. While the volume

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B. W. McCleland et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1713–1717
Table 2. C-3 sulfonamide and amide-substituted diarylsquaramides: CXCR2 affinity, solubility and pharmacokinetics
O
OH
O
R1
3
R
N
N
H
H
R2
Compound 10
R
R1
R2
CXCR2
[IC50 (nM)]a,b,c
Aqueous
solubility (lM)b,d
Rat PK
(CL: mL/min/kg;
Vdss: L/kg; %F)b,e
c
d
e
f
Cl
CN
Me
Cl
F
H
Br
Br
Me
H
32
15
15
8
3
14
—
H
15; 0.15; 1
4; 0.25; 83
0.6; 0.14; 23
—
SO2NMe2
10
SO2NH(CH2)2OMe
73
O
N
g
h
i
Br
n-Pr
Br
Cl
13
8
44
S
O
O
S
N
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
H
78
7; 0.89; 43
4; 0.24; 23
4; 0.31; 58
1.2; 0.24; 5
—
O
O
S
10
20
18
14
138
160
7
N
O
O
S
j
N
O
O
S
N
k
l
H
O
CONH2
H
88
O
N
m
n
o
p
CF3
Br
H
86
45; 0.66; 13
—
O
N
Cl
H
28
27
44
143
121
178
O
N
—
N
O
N
H
H
15; 0.33; 0.6
N
a Binding assays were performed as described in Table 1.
b All active compounds were at least 20-fold selective versus CXCR1.
c Compounds 10g, 10h, 10o and 10p were tested as trifluoroacetic acid salts whereas compounds 10i–k were tested as hydrochloric acid salts.
d Aqueous solubility was measured in 0.05 M, pH 7.4, phosphate buffer-containing 2% DMSO using standard high-throughput techniques.
e Pharmacokinetic experiments were conducted using an iv x po crossover design in male Sprague–Dawley rats (n = 3 per study). With the exception
of compound 10h, which was tested as a single compound, all compounds were tested in cassette experiments where four compounds are dosed
simultaneously to each animal. Compounds were administered in an aqueous solution containing 1% DMSO and up to 20% hydroxypropyl-b-
cyclodextrin at dosages of 4 and 8 lmol/kg for iv and po doses, respectively. 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 <10 ng/mL for each analyte.
of distribution generally was low in this series, some
3-sulfonamide-containing diarylsquaramides did display
a desirable pharmacokinetic profile with low clearance
and moderate to high oral bioavailability.

B. W. McCleland et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1713–1717
1717
R. In Peptides 1996; Ramage, R., Ed.; Mayflower Scien-
tific Ltd.: Birmingham, UK, 1998; p 87.
Acknowledgments
5. Widdowson, K. L.; Elliott, J. D.; Veber, D. F.; Nie, H.;
Rutledge, M. C.; McCleland, B. W.; Xiang, J.-N.;
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D. E.; Martin, L.; Lee, J. M.; White, J. R.; Sarau, H. M.
J. Med. Chem. 2004, 47, 1319.
6. Jin, Q.; Nie, H.; McCleland, B. W.; Widdowson, K. L.;
Palovich, M. R.; Elliott, J. D.; Goodman, R. M.; Burman,
M.; Sarau, H. M.; Ward, K. W.; Nord, M.; Orr, B. M.;
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7. Nie, H.; Widdowson, K. L.; Palovich, M. R.; Fu, W.;
Elliott, J. D.; Bryan, D. L.; Burman, M.; Schmidt, D. B.;
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The authors thank Xiaoliu Geng and Alan P. Hill for
solubility and pKa measurements, respectively.
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K. H.; Kaiser, B.; Wang, W.; Stauffer, T. M.; Ozgur, L. E.;
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11. pKa values were determined spectroscopically on a Sirius
Glppka-Dpas system.