Synthesis and structure-activity relationships of new disubstituted phenyl-containing 3,4-diamino-3-cyclobutene-1,2-diones as CXCR2 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.02.010

A series of 3,4- and 3,5-disubstituted phenyl-containing cyclobutenedione analogues were synthesized and evaluated as CXCR2 receptor antagonists. Variations in the disubstitution pattern of the phenyl ring afforded new compounds with potent CXCR2 binding

Full text of DOI:10.1016/j.bmcl.2008.02.010

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 1864–1868
Synthesis and structure–activity relationships of new disubstituted
phenyl-containing 3,4-diamino-3-cyclobutene-1,2-diones
as CXCR2 receptor antagonists
Gaifa Lai,a,* J. Robert Merritt,a Zhenmin He,a Daming Feng,a Jianhua Chao,b
Michael F. Czarniecki,b Laura L. Rokosz,a Tara M. Stauffer,a
Diane Rindgenb and Arthur G. Taverasb
aPharmacopeia, Inc., 3000 Eastpark Boulevard, Cranbury, NJ 08512, USA
bSchering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
Received 27 December 2007; revised 6 February 2008; accepted 7 February 2008
Available online 10 February 2008
Abstract—A series of 3,4- and 3,5-disubstituted phenyl-containing cyclobutenedione analogues were synthesized and evaluated as
CXCR2 receptor antagonists. Variations in the disubstitution pattern of the phenyl ring afforded new compounds with potent
CXCR2 binding affinity in the low nanomolar ranges. Moreover, two potent compounds 19 and 26 exhibited good oral pharma-
cokinetic profiles.
Ó 2008 Elsevier Ltd. All rights reserved.
The chemokine receptor CXCR2, a seven-transmem-
brane G-protein-coupled receptor, was cloned and iden-
tified in the early 1990s.1 Its natural ligands, interleukin-
8 (IL-8), granulocyte chemotactic protein-2 (GCP-2),
and other related CXC chemokines, bind with it to exert
a number of pathophysiological effects such as attrac-
tion and accumulation of neutrophils toward the sites
of inflammation.2 CXCR2 mouse gene knockout studies
show that there are elevated leukocytes and lymphocytes
without apparent pathogenic consequences, indicating
that CXCR2 is not required for normal physiology.3 In-
creased levels of CXCR2 and its ligand IL-8 have been
observed in humans with diseases such as arthritis, asth-
ma, and chronic obstructive pulmonary disease
(COPD).4 This suggests that the CXCR2 receptor and
IL-8 may play a pivotal role in these inflammatory dis-
orders. Therefore, antagonists of CXCR2 receptor could
be in principle used in the treatment of inflammatory
and related diseases.
last decade.5 Several structural classes have been identi-
fied to be potent inhibitors of the CXCR2 receptor
(Fig. 1), including quinoxaline 1,6 triazolethiol 2,7
N,N0-diarylureas 3 and 4,8 cyanoguanidine 5,9 imidazol-
ylpyrimidine 6,10 and diaminocyclobutenedione 7.11
Among these, CXCR2 antagonists 3 and 48a disclosed
by GSK and antagonist 711a identified through our joint
research program have been progressed into the clinical
trials for COPD.
During the course of lead optimization leading to the
discovery of compound 7, it was observed that replace-
ment of the 5-methylfuryl group in 7 with phenyl and 3-
fluorophenyl in cyclobutenediones (8 and 9)11a also
showed quite potent CXCR2 receptor affinity in the
nanomolar ranges (Fig. 2). Based upon this observation,
we decided to explore the impact of disubstitution of the
phenyl ring on CXCR2 receptor binding and pharmaco-
kinetic properties in hopes of finding a non-furanyl can-
didate with development potential. Herein we report the
synthesis and preliminary structure–activity relation-
ships of a series of 3,4- and 3,5-disubstituted phenyl-
containing cyclobutenediones (10–29) as novel CXCR2
receptor antagonists.
CXCR2 antagonists have indeed attracted much atten-
tion as targets for small-molecule drug discovery in the
Keywords: CXCR2 receptor; Antagonists; Cyclobutenediones; Synthe-
sis; Structure–activity relationships.
Scheme 1 shows the synthesis of target compounds 26
and 27 from commercially available 3-bromo-5-fluoro-
toluene (30). Thus, lithiation of 30 with n-BuLi in
*
Corresponding author. Tel.: +1 609 452 3729; fax: +1 609 655
4187; e-mail: galai@pcop.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.010

G. Lai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1864–1868
1865
Cl
Cl
O
Cl
Cl
N
N
Cl
S
S
O
H2N
N N
N
S
O
N
H
N
H
Cl
NEt2
SH
N
H
OH
Cl
3
1
2
N
NC
Cl
CN
O
N
N
N
NHR
F3CO
O
Me2N
N
N
S
N
H
N
H
N
N
H
H
NH
Br
O
OH
Cl
N
6
4
5
O
O
Me2N
O
N
H
N
H
O
OH
7
Figure 1. CXCR2 receptor antagonists.
O
O
O
O
Me2N
Me2N
F
N
N
N
N
H
H
H
H
O
OH
O
OH
9
8
CXCR2 IC50 = 4.9 nM, Ki =9 nM
CXCR2 IC50 = 6.8 nM, Ki = 4.7 nM
O
O
O
O
R3
R3
Me2N
R1
R2
Me2N
R1
N
N
N
N
H
H
H
H
O
OH
O
OH
R2
10-18
Figure 2. Cyclobutenedione CXCR2 receptor antagonists.
19-29
THF, followed by addition of DMF, readily furnished
the desired aldehyde 31 in 66% yield. Treatment of 31
with (R)-(ꢀ)-2-phenylglycinol in the presence of MgSO4
in THF and subsequent silylation with TMSCl gave rise
to the protected imine 32. Diastereoselective addition of
ethylmagnesium chloride or isopropylmagnesium chlo-
ride to 32 in THF and desilylation of the corresponding
adducts with 2.5 M H2SO4 solution provided the desired
amino alcohols 33a or 33b.12,13 Oxidative cleavage of
33a and 33b was accomplished with periodic acid in
the presence of MeNH2 in aqueous methanol to give
the chiral amines 34a and 34b, respectively. Finally,
reaction of 34a and 34b with the previously reported
cyclobutenedione intermediate 3511a afforded the target
compounds 26 and 27. Compounds 10–25 disclosed in
Tables 1 and 2 were synthesized from the corresponding
bromides or aldehydes in a similar manner as described
in Scheme 1.
The target compound 28, having a 3-cyano-5-methyl-
phenyl moiety, was synthesized as outlined in Scheme
2. Sequential monolithiation of 3,5-dibromotoluene
(36) with t-BuLi and formylation with DMF, followed
by protection, provided the acetal 37, which was sub-
jected to another lithiation and treatment with DMF
to give the monoprotected dialdehyde 38. Reaction of
38 with (R)-(ꢀ)-2-phenylglycinol and subsequent silyla-
tion smoothly afforded the intermediate 39. Diastereose-
lective nucleophilic addition of ethylmagnesium chloride
to 39 was followed by removal of both the acetal and the
silyl groups in the resulting adduct with 2.5 M H2SO4
solution to furnish the desired amino alcohol 40.12,13 Di-
rect conversion of the formyl group in 40 to the cyano
group was accomplished using ammonia and MnO2 in
the presence of MgSO4 in THF14 to give the desired ni-
trile 41 in 83% yield. Oxidative cleavage of 41 yielded the
chiral amine 42, which was reacted with the intermediate

1866
G. Lai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1864–1868
Ph
H
OTMS
N
Me
Br
Me
CHO
a
b
Me
F
30
F
F
31
32
Ph
OH
NH2
HN
c
d
Me
R3
Me
R3
F
F
33a R3 = Et
34a R3 = Et
33b R3 = i-Pr
34b R3 = i-Pr
O
O
Me2N
O
O
N
OEt
R3
H
O
OH
Me2N
Me
35
N
H
N
H
e
O
OH
F
26 R3 = Et
27 R3 = i-Pr
Scheme 1. Reagents and conditions: (a) n-BuLi, THF, ꢀ78 °C; then DMF; (b) (R)-(ꢀ)-2-phenylglycinol, MgSO4, THF; then TMSCl, Et3N, CH2C12.
(c) EtMgCl, THF, ꢀ20 °C, or i-PrMgCl, THF, ꢀ20 °C; then 2.5 M H2SO4; (d) H5IO6, MeNH2, MeOH, H2O, rt; (e) MeOH, DIEA, 65 °C, overnight.
Table 1. CXCR2 binding data of 3,4-disubstitutedphenyl analogues
Table 2. CXCR2 binding data of 3,5-disubstituted phenyl analogues
O
O
O
O
R3
R3
R1
R1
N
N
N
H
OH
N
H
OH
H
H
R2
O
O
R2
N
N
Compound R1
R2
R3
Et
Kia (nM) Rat AUCb (lM h)
Compound R1 R2
R3
Kia (nM) Rat AUCb (lM h)
10
11
12
13
14
15
16
17
18
F
F
28c
5.2
NT
6.37
0.3
19
20
21
22
23
24
25
26
27
28
29
F
F
F
F
F
F
F
F
Et
i-Pr
cy-Pr
1.7
1.9
18.52
1.28
NT
NT
NT
NT
NT
3.52
2.29
NT
NT
F
OMe Et
F
OMe i-Pr 1.5
4.5
7.9
F
CF3
CF3
F
Et
i-Pr 13.9
19.4
NT
NT
1.1
CF3 Et
CF3 i-Pr
F
Me
13.3
11.8
4.4
Et
Et
6.1c
11.9
Me CF3 Et
Me CF3 i-Pr
OMe
OMe
CF3
F
NT
NT
NT
F
i-Pr 16.0
Et 38.5
F
F
Me Et
Me i-Pr
2.8
1.0
F
Me CN Et
CN Et
1.9
1.7
a Receptor binding was conducted as described in Ref. 16. Data are
means of at least two independent determinations.
b Data were generated based on a 6-h study, po dosing (10 mg/kg),
n = 2. NT, not tested.
F
a Receptor binding was conducted as described in Ref. 16. Data are
means of at least two independent determinations.
b Data were generated based on a 6-h study, po dosing (10 mg/kg),
n = 2. NT, not tested.
c The tested compounds were racemic.
3511a to provide the target compound 28. Compound 29
was synthesized in a similar fashion from 3-bromo-5-
fluorobenzaldehyde.15
taining cyclobutenedione derivatives and the results
are summarized in Table 1. Attachment of the second
fluorine atom at the C4 position of the phenyl ring
(10) led to less binding potency as compared to com-
pound 9. Addition of methoxyl group at the C4 position
moderately improved CXCR2 binding affinity (11 and
The CXCR2 binding activity was determined using a
Ba/F3-hCXCR2 overexpressing membrane binding as-
say.16 We first examined 3,4-disubstituted phenyl-con-

G. Lai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1864–1868
1867
O
O
Br
Br
Br
CHO
O
a
b
O
Me
36
Me
37
Me
38
Ph
H
Ph
OTMS
OH
O
N
HN
d
c
OHC
O
Me
Me
40
39
Ph
NH2
OH
HN
e
NC
f
NC
Me
42
Me
41
O
O
O
O
Me2N
N
OEt
H
O
OH
Me2N
CN
N
N
35
H
H
O
OH
g
Me
28
Scheme 2. Reagents and conditions: (a) (i) t-BuLi, THF, ꢀ78 °C; then DMF; (ii) ethylene glycol, p-TsOH H2O, C6H6, reflux; (b) t-BuLi, THF,
ꢀ78 °C; then DMF; (c) (R)-(ꢀ)-2-phenylglycinol, MgSO4, THF; then TMSCl, Et3N, CH2Cl2; (d) EtMgCl, THF, ꢀ20 °C; then 2.5 M H2SO4; (e)
MgSO4, MnO2, 2 M NH3 in i-PrOH, THF; (f) H5IO6, MeNH2, MeOH, H2O, rt; (g) MeOH, DIEA, 65 °C, overnight.
12), whereas addition of the trifluoromethyl group at
that position led to a threefold loss in CXCR2 affinity
(13 and 14). When fluorine was switched to the C4 posi-
tion of the phenyl ring, attaching methyl at the C3 posi-
tion slightly improved CXCR2 affinity (15), while
introduction of methoxyl or trifluoromethyl substituent
at that position (16–18) markedly decreased the binding
potency. The 3,4-disubstitution pattern did not provide
a significant affinity increase from data presented in Ta-
ble 1.
the right-side amine in 19 with isopropyl or cyclopropyl
did not improve potency (20 and 21). Analogues 26 and
27 with a 3-fluoro-5-methylphenyl moiety showed excel-
lent affinity. However, introduction of the trifluoro-
methyl group (22 to 25) decreased CXCR2 binding
activity. Furthermore, the incorporation of the cyano
and methyl groups or the cyano group and fluorine at
the C3 and C5 positions of the phenyl ring (28 and 29)
yielded potent affinity for CXCR2 receptor.
Pharmacokinetic studies have been conducted with se-
lected compounds. As shown in Table 1, compound 12
displayed much lower oral exposure (AUC, 0.3 lM h)
in rapid rat PK tests than the clinical trial compound
7 (AUC, 49.0 lM h).11a Table 3 lists some PK data for
compounds 19, 26, and 7. Both new compounds 19
Next, we turned to assess the effect of 3,5-disubstitution
of the phenyl ring on CXCR2 receptor binding activity.
As shown in Table 2, the 3,5-difluorophenyl group-con-
taining compound 19 displayed high binding affinity.
Replacement of the ethyl group at the benzylic site of
Table 3. Pharmacokinetic data of compounds 19, 26 and 7
Parameter
19
26
7
Rat
Monkey
3
Rat
Monkey
3
Rat
Monkey
Dose, po (mg/kg)
Oral bioavailability (%)
t1/2 (h)
10
10
10
33
3
19.1
13
34.1
5.5
18
23.3
2
7.8
3.4
22
Mean residence time (h)
Oral AUC (0–24 h) (lM h)
3.8
8.8
1.8
18.1
3.1
5.6
2

1868
G. Lai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1864–1868
Foley, J. J.; Sarau, H. M.; Busch-Petersen, J. Bioorg. Med.
Chem. Lett. 2006, 16, 5513.
and 26 exhibited comparable or better oral bioavailabil-
ity and exposure (AUC) in full rat and rapid monkey
PK tests as compared to 7.
10. Ho, K.-K.; Auld, D. S.; Bohnstedt, A. C.; Conti, P.;
Dokter, W.; Erickson, S.; Feng, D.; Inglese, J.; Kingsbury,
C.; Kultgen, S. G.; Liu, R.-Q.; Masterson, C. M.;
Samama, P.; Smit, M.-J.; Son, E.; van der Louw, J.;
Vogel, G.; Webb, M.; Wijkman, J.; You, M. Bioorg. Med.
Chem. Lett. 2006, 16, 2724.
In summary, a series of 3,4- and 3,5-disubstituted phe-
nyl-containing cyclobutenedione analogues have been
synthesized and evaluated as CXCR2 receptor antago-
nists. Several new compounds have been identified to
possess high CXCR2 binding affinity with the low nano-
molar ranges. Two potent compounds 19 and 26 exhib-
ited good oral pharmacokinetic profiles. Their further
evaluation in animal pulmonary studies and other tests
will be reported in due course.
11. (a) Dwyer, M. P.; Yu, Y.; Chao, J.; Aki, C.; Chao, J.; Biju,
P.; Girijavallabhan, V.; Rindgen, D.; Bond, R.; Mayer-
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G.; Wang, W.; Stauffer, T. M.; Ozgur, L.; Baldwin, J.;
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J. R.; Rokosz, L. L.; Nelson, K. H., Jr.; Kaiser, B.; Wang,
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Bioorg. Med. Chem. Lett. 2006, 16, 4107; (c) Chao, J.;
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Biju, P.; Rindgen, D.; Jakway, J.; Hipkin, R. W.; Fossetta,
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13. The addition of Grignard reagents to the imines and
subsequent desilylation produced one desired diastereo-
mer only or predominantly in all cases, which was detected
by 1H NMR analysis. Column chromatography yielded
the pure sample.
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15. 3-Bromo-5-fluorobenzaldehyde was prepared by reduction
of 3-bromo-5-fluorobenzoic acid with DIBAL-H and
subsequent oxidation of the resultant benzyl alcohol with
MnO2.
16. CXCR2 SPA assay. Compound samples or no compound
controls were first prepared in neat DMSO, followed by
intermediate dilution to 5% DMSO (1% DMSO final
concentration) in assay buffer (50 mM Tris, pH7.6, 1 mM
CaCl2, 50 mM NaCl, 5 mM MgCl2, 0.002% NaN3, 0.1%
BSA). For each well of a 96-well plate, a reaction mixture
containing 1.5 lg of Ba/F3-hCXCR2 cell membranes
(generously provided by Schering-Plough Research Insti-
tute) and 150 lg/well WGA–SPA beads (GE Healthcare)
was prepared in assay buffer and added to the compound
samples or controls. The compound + membrane/SPA
mixture was pre-incubated for 6 h at room temperature. A
250 pM stock of [125I]-IL-8 (Perkin-Elmer, 50 pM final)
was prepared in assay buffer and added to the com-
pound + membrane/SPA bead mixture following the 6 h
incubation time. The total assay volume was 100 lL. The
complete mixture (compound, membrane/SPA, radio-
labeled [125I]-IL-8) was incubated for 6 h before cpm/well
was determined in a Microbeta Trilux counter (Perkin-
Elmer). Data were fit to the Michaelis–Menten equation to
generate binding IC50 values, followed by conversion to Ki
values using the Cheng–Prusoff equation. All Ki data
represent the average of two or more determinations.
9. Nie, H.; Widdowson, K. L.; Palovich, M. R.; Fu, W.;
Elliott, J. D.; Bryan, D. L.; Burman, M.; Schmidt, J. J.;