Synthesis and structure-activity relationship of 3-phenyl-3H-quinazolin-4- one derivatives as CXCR3 chemokine receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2005.03.070

A series of 3-phenyl-3H-quinazolin-4-ones have been synthesized and tested for affinity and activity at the chemokine CXCR3 receptor. The most potent compound (1d) has been evaluated using radioligand binding and calcium mobilization assays and is considered a useful tool for further characterization of the CXCR3 receptor.

Full text of DOI:10.1016/j.bmcl.2005.03.070

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2910–2913
Synthesis and structure–activity relationship of
3-phenyl-3H-quinazolin-4-one derivatives as CXCR3
chemokine receptor antagonists
Stefania Storelli,a Pauline Verdijk,b Dennis Verzijl,a Henk Timmerman,a
Andrea C. van de Stolpe,a Cornelis P. Tensen,b Martine J. Smit,a
Iwan J. P. De Escha and Rob Leursa,*
aLeiden/Amsterdam Center for Drug Research (LACDR), Division of Medicinal Chemistry, Faculty of Sciences,
Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
bDepartment of Dermatology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands
Received 22 December 2004; revised 14 March 2005; accepted 17 March 2005
Available online 19 April 2005
Abstract—A series of 3-phenyl-3H-quinazolin-4-ones have been synthesized and tested for affinity and activity at the chemokine
CXCR3 receptor. The most potent compound (1d) has been evaluated using radioligand binding and calcium mobilization assays
and is considered a useful tool for further characterization of the CXCR3 receptor.
Ó 2005 Elsevier Ltd. All rights reserved.
Chemokines constitute a superfamily of small secreted
proteins that attract and activate a variety of cell types.
They are classified by structure according to the number
and spacing of conserved cysteine residues into four
major groups (CC, CXC, CX3C and XC).1,2 Chemokine
receptors are defined by their ability to signal on binding
of one or more members of the chemokine superfamily
of chemotactic cytokines. All of them belong to the fam-
ily of G protein-coupled receptors (GPCRs).3,4
reactions10,16,17) and is associated with the infiltration
of CXCR3-positive T-cells.
The involvement of CXCR3 in the pathophysiology of
the Th1-type diseases suggests that CXCR3 may serve
as a new molecular target for anti-inflammatory
therapies.18,19
For further characterization of the receptor and for val-
idating CXCR3 as a viable drug target, the development
of small, nonpeptidergic CXCR3 ligands is essential. In
patent literature, it has been suggested that decanoic acid
(2-dimethylamino-ethyl)-{1-[3-(4-fluoro-phenyl)-4-oxo-
3,4-dihydro-quinazolin-2-y]-ethyl}-amide (1c) (Fig. 1)
The chemokine receptor CXCR3 is mainly expressed on
activated T cells and natural killer cells and binds
CXCL9 (monokine induced by IFN-c; Mig), CXCL10
(IFN-c-inducible protein 10; IP10) and CXCL11
(IFN-c-inducible T cell a-chemoattractant; I-TAC/
IP9).5–9 Exposure of cells expressing CXCR3 to
CXCL9, CXCL10 and CXCL11 leads to an increase
of intracellular calcium via activation of Gi proteins.10
Increased expression of CXCR3-targeting chemokines
is observed in chronic inflammatory diseases (rheuma-
toid arthritis,11,12 multiple sclerosis,13 hepatitis C-in-
fected liver,14 atherosclerosis15 and chronic skin
Keywords: Chemokine; CXCR3; Synthesis; SAR.
*
Figure 1. Structure of decanoic acid (2-dimethylamino-ethyl)-{1-[3-(4-
fluoro-phenyl)-4-oxo-3,4-dihydro-quinazolin-2-y]-ethyl}-amide (1c).
Corresponding author. Tel.: +31 20 5987600; fax: +31 20 5987610;
e-mail: leurs@few.vu.nl
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.070

S. Storelli et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2910–2913
2911
is a CXCR3 ligand with micromolar affinity.20 We set
out to explore the SAR of this compound. More specif-
ically, the role of the nonyl chain of lead structure (1c)
was investigated. In addition, the effect of the substituent
at the 3-phenyl ring on the biological activity was
evaluated.21
pounds 5i and 1i, respectively, in low isolated yields
(10–15%).
Compounds 1a–n, 5a–f and 5i were tested for their abil-
ity to displace [125I]CXCL10 from HEK-293 cells
expressing the human CXCR3 receptor.24 HEK293–
CXCR3 cells were grown at 5% CO2 at 37 °C in Dul-
beccoÕs modified EagleÕs medium supplemented with
10% foetal bovine serum, 250 mg/mL G-418, 50 IU of
penicillin per mL, and 50 mg of streptomycin per mL.
These cells were seeded in poly-L-lysine-coated 48-well
plates. After 24 h, binding was performed on whole cells
for 4 h at 4 °C using approximately 70 pM 125I-labelled
CXCL10 (Perkin–Elmer Life Science, Boston, USA) in
binding buffer (50 mM HEPES [pH 7.4], 1 mM CaCl2,
5 mM MgCl2, 0.5% BSA) containing increasing concen-
trations of the indicated compounds. After incubation,
cells were washed three times with ice-cold binding buf-
fer supplemented with 0.5 M NaCl. Subsequently, cells
were lysed and counted in a Wallac Compugamma
counter.
The
3-phenyl-3-H-quinazolin-4-ones
1a–n
were
prepared according to Scheme 1. Reaction of 2-amino-
benzoic acid with propionyl chloride gave 2-propionyl-
amino-benzoic acid (2) (76% yield). Treatment of this
intermediate with phosphorus trichloride and the appro-
priate substituted aniline gave compounds 3a–h in mod-
erate to good yields (35–75%). Reaction with bromine
gave the intermediates 4a–h (60–85% yields) that were
used for the subsequent reaction with the commercially
available N,N-dimethyl-ethylene-diamine. The crude
oils were readily purified by flash chromatography giv-
ing the corresponding key derivatives 5a–h (5–65%
yields). Reaction with decanoyl, cyclohexanecarbonyl,
benzoyl or naphthalene-1-carbonyl chloride afforded
compound 1a–h and 1j–n (10–55% yields). The methoxy
group of 5b and 1b was converted to a hydroxy moiety
by treatment with boron tribromide22,23 to give com-
In patent literature, compound 1c has been reported as
CXCR3 antagonist in the micromolar range.20 We con-
Scheme 1. Reagents: (i) CH3CH2COCl, DMF; (ii) PCl3, substituted anilines, toluene; (iii) Br2, CH3COOH, CH3COONa; (iv) H2N(CH2)2N(CH3)2,
EtOH, reflux; (v) RCOCl, Et3N, 1,4-dioxane; (vi) BBr3, MeOH.

2912
S. Storelli et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2910–2913
Replacement of the 4-fluoro atom of 1c by a 4-CF3
(1f) or 4-OH (1i) group is not allowed for activity.
Compound 1d has the highest affinity for CXCR3 in the
[
125I]CXCL10 binding assay and was therefore tested in
a [Ca2+] mobilization assay. In this assay, CXCL11-in-
duced increases in [Ca2+] were quantified by monitoring
the fluorescence of Fluo-4 AM-loaded HEK293–
CXCR3 cells, using an automated NOVOstar micro-
plate reader. Cells were loaded in HanksÕ balanced salt
solution containing 20 mM HEPES, 2.5 mM Probene-
cid, 2 lM Fluo-4 AM and 0.02% Pluronic F-127
[pH = 7.4]. Thereafter they were washed three times
and fluorescence was measured (1 data point/s, excita-
tion 485 nm, emission 520 nm) for 10 s to calculate the
mean basal value. Cells were preincubated for 10 min
with the indicated compounds (10 lM). After injection
of the agonist CXCL11 (10 nM) fluorescence was re-
corded for 50 s. To determine the maximal fluorescence
of the system, cells were exposed to Triton X-100 (0.25%
[v/v]). Basal and maximal values determined for each
well were used to normalize the data. Cells preincubated
with compounds 1d and 5d show comparable baseline
fluorescence to nontreated cells. Results were expressed
as percentage of the maximal fluorescence induced by
Triton X-100.
Figure 2. Displacement of [125I]-CXCL10 binding to CXCR3. The
binding experiments were carried out on HEK-293 cells expressing the
human CXCR3 receptor. The compounds 1a–n, 5a–f and 5i (10 lM)
were tested for their ability to displace [125I]CXCL10. Error bars show
standard error of the mean.
firmed this result in our binding assay where 1c showed
an IC50 value of 3.2 lM. The importance of the nonyl
chain of 1c was investigated by its removal (5a–i) or sub-
stitution with different hydrophobic moieties (1j–n).
These modifications result in a total loss of affinity
(Fig. 2), suggesting an important role of the flexible ali-
phatic chain.
Stimulation of the human CXCR3 receptor with 10 nM
CXCL11 resulted in a rapid increase of intracellular
[Ca2+], which returned thereafter to baseline levels
(Fig. 3). Preincubation of the cells with 10 lM of 1d,
10 min prior to chemokine stimulation completely abro-
gated the CXCL11-induced [Ca2+] increase, while 5d
had no effect (Fig. 3).These data demonstrate that com-
pound 1d can efficiently block CXCR3-mediated cal-
cium release.
The role of the para-fluoro atom of the phenyl ring in
the lead compound 1c was also studied (Table 1). The
presence of appropriate substituents is required for affin-
ity at CXCR3, since the unsubstituted analogue (1a) is
five times less potent (IC50 = 15 lM) than the lead com-
pound (1c). Substitutions with different electronic and
lipophilic properties resulted in ligands with altered
affinities for the CXCR3 receptor. The para-methoxy
and para-methyl derivatives (1b and 1e) showed the
same degrees of affinity as the prototype, 1c, whereas
the para-cyano derivative, 1d, was threefold more potent
than 1c. In fact within this series 1d is the only com-
pound to show submicromolar affinity. Shifting the
cyano substituent from the para- to the ortho- or meta-
position caused a large decrease in CXCR3 affinity
(IC50 = 10 lM for 1g and 5.8 lM for 1h, respectively).
Table 1. IC50 values of the compounds 1a–i for their displacement of
125I]CXCL10 binding to CXCR3
[
a
Compounds
R
X
IC50 (lM)
1a
1b
1c
1d
1e
1f
Nonyl
Nonyl
Nonyl
Nonyl
Nonyl
Nonyl
Nonyl
Nonyl
Nonyl
H
15 (10–20)
3.3 (2.2–4.4)
3.2 (2.3–4.2)
0.93 (0.74–1.1)
3.1 (2.0–4.1)
>100
4-OCH3
4-F
4-CN
4-CH3
4-CF3
2-CN
3-CN
4-OH
1g
1h
1i
10 (8.6–12)
5.8 (5.1–6.5)
>100
Figure 3. Compound 1d abrogates CXCR3-mediated [Ca2+] mobili-
zation. Effect of compounds 1d and 5d (10 lM) on the CXCL11
(10 nM) induced Ca2+ mobilization in HEK-293–CXCR3 cells. Com-
pound were added 10 min prior to chemokine stimulation (indicated
by the arrow). The maximal response is defined as maximal Ca2+
concentration induced by CXCL11.The experiment is representative of
two independent experiments performed in triplicate. Error bars show
standard error of the mean.
a The binding experiments were carried out on HEK-293 cells
expressing the human CXCR3 receptor. The compounds were tested
for their ability to displace [125I]CXCL10. The values are represented
as the mean and the interval of the IC50 (lM) values of at least three
independent experiments.

S. Storelli et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2910–2913
2913
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In conclusion, we have described the SAR of a series of
3-phenyl-3H-quinazolin-4-one derivatives for their ac-
tion as CXCR3 antagonist. Compound 1d has the high-
est affinity for the human CXCR3 receptor and is able to
effectively inhibit CXCR3 mediated calcium mobiliza-
tion in transfected HEK-293 cells. Currently we are
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The authors thank Dr. C. Fitzsimons for support with
the calcium experiments and Drs. K. Biber and H. W.
Boddeke (University of Groningen) for providing the
HEK-293 cells expressing CXCR3 receptor. M.J.S.
was supported by the Royal Netherlands Academy of
Arts and Sciences. S.S. was supported by a NWO-Pio-
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