Ligand-biased and probe-dependent modulation of chemokine receptor CXCR3 signaling by negative allosteric modulators

Article abstract of DOI:10.1002/cmdc.201402507

Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein-coupled receptors (GPCRs). The CXC chemokine receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8-azaquinazolinone derivative (N-{1-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl]butanamide, 1b) that can inhibit CXC chemokine 11 (CXCL11)-dependent G protein activation over β-arrestin recruitment with 187-fold selectivity. This compound also demonstrates probe-dependent activity, that is, it inhibits CXCL11- over CXCL10-mediated G protein activation with 12-fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.

Full text of DOI:10.1002/cmdc.201402507

DOI: 10.1002/cmdc.201402507
Full Papers
Ligand-Biased and Probe-Dependent Modulation of
Chemokine Receptor CXCR3 Signaling by Negative
Allosteric Modulators
Viachaslau Bernat,[a, c] Regine Brox,[a] Markus R. Heinrich,[a] Yves P. Auberson,[b] and
Nuska Tschammer*[a]
Over the last decade, functional selectivity (or ligand bias) has
evolved from being a peculiar phenomenon to being recog-
nized as an essential feature of synthetic ligands that target
G protein-coupled receptors (GPCRs). The CXC chemokine re-
ceptor 3 (CXCR3) is an outstanding platform to study various
aspects of biased signaling, because nature itself uses function-
al selectivity to manipulate receptor signaling. At the same
time, CXCR3 is an attractive therapeutic target in the treatment
of autoimmune diseases and cancer. Herein we report the dis-
covery of an 8-azaquinazolinone derivative (N-{1-[3-(4-ethoxy-
phenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-4-
(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl]butanamide,
1b) that can inhibit CXC chemokine 11 (CXCL11)-dependent
G protein activation over b-arrestin recruitment with 187-fold
selectivity. This compound also demonstrates probe-depen-
dent activity, that is, it inhibits CXCL11- over CXCL10-mediated
G protein activation with 12-fold selectivity. Together with
a previously reported biased negative allosteric modulator
from our group, the present study provides additional informa-
tion on the molecular requirements for allosteric modulation
of CXCR3.
Introduction
Agonists of G protein-coupled receptors (GPCRs) do not uni-
formly activate all cellular signaling pathways linked to a given
receptor (a phenomenon termed ligand or agonist bias).[1–3]
The CXC chemokine receptor 3 (CXCR3), a group A GPCR, is an
excellent example: its functions are regulated by at least three
endogenous protein ligands, CXC chemokines CXCL9, CXCL10,
and CXCL11. These chemokines are mainly induced by interfer-
on (IFN)-g.[4] Each of them evokes different cellular responses
upon binding to the receptor.[4–7] Thus, ligand-biased signaling
is employed by nature itself to regulate the functions of
CXCR3. Dysregulation of the CXCR3 network is associated with
a number of autoimmune diseases such as rheumatoid arthri-
tis, psoriasis, and multiple sclerosis.[8,9] Several animal models
showed beneficial effects of down-regulation of CXCR3 signal-
ing in rheumatoid arthritis,[10] allograft rejection,[11] and in some
types of cancer.[12,13] At the same time the role of CXCR3 is
much more complex and controversial in neuroinflamma-
tion[14–16] and immune responses to bacterial infection.[17] The
only clinical candidate that targets CXCR3, AMG487, failed in
phase IIa clinical trials for rheumatoid arthritis and psoriasis
due to lack of efficacy.[18,19]
The complex properties of allosteric modulation of GPCRs,
which include probe dependence and biased signaling, can
present both challenges and opportunities for preclinical drug
discovery.[2,20–27] By blocking harmful and maintaining beneficial
stimuli of the same endogenous signal transmitter, allosteric
modulators provide finely tuned instruments for restoring
physiological conditions affected by pathology. For CXCR3,
two nonpeptidic small-molecule ligands were reported as
biased allosteric agonists of the receptor, as they recruit b-ar-
restin with greater efficacy than the endogenous agonist.[28,29]
One of them, PS372424, demonstrated beneficial therapeutic
effects in a humanized mouse model of arthritis.[30]
[a] Dr. V. Bernat,+ R. Brox,+ Prof. Dr. M. R. Heinrich, Dr. N. Tschammer
Department of Chemistry and Pharmacy, Medicinal Chemistry
Emil Fischer Center, Friedrich Alexander University
Schuhstraße 19, 91052 Erlangen (Germany)
From a pharmacological perspective, an agonist can not
only elicit biased signaling, but antagonists can also act per-
missively.[31] Our research group recently reported the discov-
ery of the first biased negative allosteric modulator of CXCR3
(boronic acid 14, see Scheme 2 below), which preferentially
blocks CXCL11-mediated b-arrestin recruitment over G protein
activation.[32]
E-mail: nuska.tschammer@fau.de
[b] Dr. Y. P. Auberson
Novartis Institutes for BioMedical Research
Global Discovery Chemistry, WKL-136.6.82
Klybeckstrasse 141, Basel (Switzerland)
[c] Dr. V. Bernat+
Current affiliation: Department of Chemistry
The Scripps Research Institute
130 Scripps Way, 3A1, 33458 Jupiter, FL (USA)
Another important aspect of allosteric modulation of chemo-
kine receptors is probe dependence. Probe-dependent allos-
tery provides a manner for fine tuning the chemokine re-
sponse. For example, the antagonist of the chemokine recep-
tor CXCR4, AMD-3100, blocks CXCL12 binding to CXCR4 but
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402507.
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enhances the binding of CXCL12 to its other natural
receptor, CXCR7.[33] Aplaviroc, an allosteric modulator
of the chemokine receptor CCR5, alters its conforma-
tion, rendering it unable to bind and support HIV-
1 viral fusion and the binding of [125I]CCL3. At the
same time it does not interfere with the binding and
function of CCL5.[34,35] For CXCR3, probe dependence
was reported for nonpeptidic small-molecule alloste-
ric agonists.[28,29] These ligands are able to suppress
the binding of CXCL10 to CXCR3, but not that of
CXCL11.[29] Probe dependence can therefore lead to
major therapeutic advantages in the pharmacologi-
cal manipulation of chemokine systems.
Herein we report the discovery of small-molecule
negative allosteric modulators of CXCR3 that show
a bias for the inhibition of a given signaling path-
way, and probe-dependent behavior. Among them,
1b is a unique negative allosteric modulator that
demonstrates probe-dependent biased signaling at CXCR3; it
has the propensity to inhibit a specific pathway depending on
the endogenous agonist used.
Scheme 1. Design of 8-azaquinazolinone chemotype CXCR3 ligands with improved phys-
icochemical properties.
[37]
necessary for antagonistic effects is C8–9
.
To increase the hy-
drophilicity of the molecule while keeping its polar surface
area (PSA) below the commonly used cutoff for good oral
availability (<90 ꢁ2), we introduced a single oxygen atom into
the alkyl chain (compounds 1a and 1b, Scheme 1). Addition of
the second oxygen atom to the alkyl chain would further im-
prove hydrophilicity (from clogP 3.0 to 2.62), but would lead
to higher PSA (96.80 ꢁ2). Further elongation of the alkyl chain
has been shown to have a minor influence on the compounds’
bioactivity.[37] It would also negatively affect lipophilicity, in-
creasing clogP to 3.98 for the closest higher homologue of
1b. Therefore, we focused ligand design onto the identifica-
tion of the shortest alkyl chain that would elicit activity in
binding and functional assays at CXCR3.
Results and Discussion
Ligand design
Alkyl-substituted 8-azaquinazolinones were originally identified
as antagonists of the chemokine receptor CXCR3 by high-
throughput screening.[36,37] Subsequent structure–activity rela-
tionship (SAR) studies have shifted the focus of medicinal
chemists toward lead compounds with aromatic rings in place
of alkyl chains.[37,38] Although this modification led to increases
in binding affinity, the accompanying rigidification of the scaf-
fold did not improve the physi-
cochemical parameters of the li-
gands, which suffer high lipo-
philicity.
To facilitate the in vivo testing
of these negative allosteric mod-
ulators we attempted to opti-
mize
their
physicochemical
properties by increasing their
flexibility and the hydrophilicity
of the carboxamide side chain.
The presence of rigid hydropho-
bic aromatic rings is known to
have a negative effect on the
ADME properties of drug-like
compounds.[39,40] Hence, we re-
placed the fluorinated aromatic
ring in the structure of non-triti-
ated (’cold’) allosteric radioli-
gand RAMX3[41] (cRAMX3) with
a flexible fluorooxoalkyl chain
(Scheme 1). According to avail-
able SAR data, the optimal
length of the alkyl side chain
Scheme 2. Design of ‘chimeric’ compound 14b through the combination of structural features of ligands biased
either toward b-arrestin (14) or toward G protein (1b).
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The functionally selective boronic acid 14[32] (Scheme 2) ex-
hibits 24-fold selectivity for inhibiting CXCL11-mediated b-ar-
restin over G protein recruitment. After synthesizing 1a and
1b and confirming the high functional bias and probe de-
pendence for the latter (see below) we became interested in
testing the combination of structural features of these two
molecules. The side chain of 1b was thus introduced into the
structure of 14, leading to the design of the ‘chimeric’ ligand
14b (Scheme 2).
by propane-1,3-diol or butane-1,4-diol to afford 5a and 5b, re-
spectively. The free hydroxy group of 5a was replaced by fluo-
rine via treatment with diethylaminosulfur trifluoride, leading
to 6a. In contrast, application of this method to the synthesis
of 6b resulted in poor conversion of the starting material, and
quenching of the reaction mixture with methanol yielded
a high content of the corresponding methyl ether. Therefore,
6b was instead prepared by a combination of tosylation and
nucleophilic substitution. Finally, the benzyl protecting groups
were removed by catalytic hydrogenation, and the resulting
primary alcohols were converted into carboxylic acids 7a and
7b via Jones oxidation.
Synthesis
Aldehyde 3 (Scheme 3) was synthesized according to a pub-
lished two-step sequence:[42] a Wittig reaction between 1-
methylpiperidin-4-one and (methoxymethyl)triphenylphospho-
nium chloride, followed by acidic hydrolysis of the resulting
vinyl ether 2.
The fluoroalkyloxycarboxylic acids 7a and 7b were synthe-
sized through a five-step sequence: first, commercially avail-
able 4-(benzyloxy)butan-1-ol was converted into the corre-
sponding bromide 4. The bromine atom was then substituted
The synthesis of the racemic primary amine 8 was reported
previously.[38,41] Reductive alkylation of 8 with piperidinealde-
hyde 3 was accomplished with sodium triacetoxyborohydride,
and the resulting secondary amine 9 was coupled with in-situ-
activated carboxylic acids 7a and 7b. The tertiary amides 1a
and 1b obtained from this sequence had improved drug-like
properties, with lower molecular weight (Mr 567.7 and
581.7 Da, respectively, vs. 625.7 Da) and higher polarity (tR =
15.7 and 16.2 min, respectively, vs. 17.5 min in RP-HPLC) than
cRAMX3.
For the synthesis of com-
pound 14b, primary amine 8
was reductively alkylated with
aldehyde 10[42] by using a pyri-
dine–borane complex as reduc-
ing agent.[32] The resulting sec-
ondary amine 11[32] was acylated
with 4-(4-fluorobutoxy)butanoic
acid (7b), and the trifluorobo-
rate moiety was finally convert-
ed into a boronic acid by treat-
ment with trimethylsilyl chloride
in aqueous acetonitrile, yielding
14b.
Biological properties
To determine the binding affini-
ties of novel allosteric modula-
tors to the receptor, we used
the
allosteric
radioligand
RAMX3[41] displacement assay.
The ability of novel allosteric
modulators to inhibit the che-
mokine-mediated activation of
CXCR3 was measured by
a [35S]GTPgS incorporation assay
and a b-arrestin 2 recruitment
assay (PathDetect, DiscoverX).
The activation of CXCR3 was
achieved by chemokines CXCL11
and CXCL10. The data were ana-
lyzed by nonlinear regression
Scheme 3. Synthesis of building blocks and fluorooxoalkyl derivatives of 8-azaquinazolinones. Reagents and condi-
tions: a) Ph3P+CH2OMeClꢀ, tBuOK, THF, ꢀ208C!RT, 18 h, 64%; b) 5n HCl, H2O/THF, 2 h, 54%; c) CBr4, PPh3, CH2Cl2,
08C!RT, 16 h, 91%; d) propane-1,3-diol or butane-1,4-diol, KOH, DMSO, 08C!RT, 2 h, 65–75%; e) DAST, CH2Cl2,
ꢀ308C!RT, 5 h, 39% for n=2, 0% for n=1; f) TsCl, NEt3, DMAP, CH2Cl2, 08C!RT, 3 h; then nBu4N+Fꢀ, THF, reflux,
20 h, 65% for n=1; g) H2, Pd/C, MeOH, 6 h; h) CrO3, H2SO4, acetone, 30–40 min, 80–90% over two steps; i) 3, NaB-
H(OAc)3, AcOH, 1,2-dichloroethane, 20 h, 77%; j) 7a or 7b, HATU, DIPEA, DMF, 458C, 20 h, 65–72%; k) 10, pyri-
dine–borane complex, KHF2, THF, RT, 16 h, 92%; l) Me3SiCl, H2O/MeCN, 2 h, 34% over two steps.
using
the
algorithms
in
Prism 5.0 (GraphPad Software,
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San Diego, CA, USA), fitting experimental curves to the param-
eters of the tertiary complex model to determine the equilibri-
um dissociation constant pKb and cooperativity factors a and
ab [Equations (1)–(6)].[27,32]
Table 2. Ability of 8-azaquinazolinone derivatives to inhibit CXCL10- and
CXCL11-mediated activation of CXCR3 as measured in [35S]GTPgS incorpo-
ration assays.[a]
Compd
CXCL10
CXCL11
Compound 1b exhibited good affinity in the radioligand dis-
placement assay (Kb =81 nm) and noncompetitive displace-
ment of RAMX3 (a=0.14) (Table 1). In functional assay 1b in-
pKb (95% C.I.)
ab
pKb (95% C.I.)
ab
cRAMX3
1a
1b
14
14b
8.11 (8.44–7.77)
<5.0
7.64* (8.06–6.86)
7.24* (7.58–6.90)
6.97 (7.40–6.55)
0.01
–
0.30
0.05
0.08
7.84 (8.06–7.62)
8.26 (8.93–7.59)
8.73 (9.38–8.09)
6.05 (6.61–5.49)
7.62 (8.22–7.03)
0
0.44
0.14
0.10
0.31
Table 1. Affinity of 8-azaquinazoline derivatives as determined by alloste-
ric radioligand RAMX3 displacement assay.[a]
[a] Inhibition of CXCL10- (50 nm) and CXCL11-mediated (5 nm) activation
of CXCR3 was determined by [35S]GTPgS incorporation assay with the
membrane preparations of HEK293T cells transiently expressing CXCR3.
Values reflect the mean pKb with 95% confidence interval (C.I.) of three to
five experiments performed in triplicate. Two-tail unpaired t-test was per-
formed to estimate the significance between pKb and ab values observed
between both chemokines: *p<0.05.
Compd
pKb (95% C.I.)
Kb
a
cRAMX3
1a
1b
14
14b
7.92 (8.10–7.75)
6.01 (6.30–5.71)
7.09 (7.37–7.05)
7.42 (7.79–7.05)
5.67 (5.82–5.52)
11.9
980
81.1
38.1
2120
0
0.21
0.14
0.29
0
[a] Allosteric radioligand displacement assays were carried out with
RAMX3 (1 nm) and membrane preparations of HEK293T cells transiently
expressing CXCR3; values are the mean with 95% confidence interval
(C.I.) of three experiments performed in triplicate.
We have previously reported 14 as a negative allosteric
modulator that preferentially inhibits CXCL11-mediated b-ar-
restin 2 recruitment over G protein activation.[32] After discover-
ing that allosteric modulator 1b preferably blocks the CXCL11-
mediated activation of G protein rather than b-arrestin recruit-
ment, we designed boronic acid derivative 14b by combining
hibited CXCL11-induced activation of G protein at a lower con-
centration than b-arrestin 2 recruitment (187-fold difference in
Kb: 1.9 vs. 355 nm, with similar
cooperativity factors ab: 0.09
vs. 0.07, respectively; Tables 2
and 3, Figure 1). Additionally,
for 1b strong probe depend-
ence was observed. Compound
1b inhibited CXCL10-induced
activation of G protein at a sig-
nificantly higher concentration
(Kb value of 22.9 nm) relative to
inhibition of the CXCL11-in-
duced activation (two-tail un-
paired t-test, p<0.05). The co-
operativity between 1b and
CXCL10 was also decreased
(ab=0.30). In the b-arrestin 2
recruitment assay 1b showed
no probe dependence (Table 3,
Figure 1); 1b is thus a unique
negative allosteric modulator
that demonstrates probe-de-
pendent biased signaling.
While 1b, with nine heavy
atoms in the carboxamide side
chain, had good affinity and ac-
tivity in the G protein activation
assay, ligand 1a, which is one
methylene group shorter, exhib-
ited greatly diminished binding
and functional activity. This ob-
servation is in agreement with
previous SAR data.[37]
Figure 1. Ligand-biased and probe-dependent signaling in CXCR3. A),B): The ability of 8-azaquinazolinone deriva-
tives to inhibit the CXCL10- (50 nm) and CXCL11-mediated (5 nm) activation of CXCR3 was determined by
[35S]GTPgS incorporation assay with membrane preparations of HEK293T cells transiently expressing CXCR3.
C),D): The ability of 8-azaquinazolinone derivatives to inhibit the CXCL10- (400 nm) and CXCL11-mediated
(100 nm) recruitment of b-arrestin 2 to CXCR3 was determined with HEK293 cells expressing all the components
of PathHunter (DiscoverX) and CXCR3-PK2. Black: cRAMX3, green: 1a, orange: 1b, magenta: 14, blue: 14b.
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rification. Anhydrous solvents were of the highest commercially
available grade and were stored over molecular sieves under a ni-
trogen atmosphere. Flash chromatography was performed on
Merck silica gel 60 (40–63 mm) as stationary phase under positive
pressure of dry nitrogen gas. HR-ESIMS analyses were conducted
on a Bruker Daltonik microTOF II (M/DM>16500). HPLC–MS and
HPLC purity analyses were performed with an Agilent Binary Gradi-
ent system using UV detection (l=254 nm) in combination with
ChemStation software (Rev. A. 10.02 [1757]). A Zorbax Eclipse XDB-
C8 (4.6 mmꢂ150 mm, 5 mm) column was used at a flow rate of
0.5 mLminꢀ1 in reversed-phase mode (eluent: MeOH/H2O+0.1%
HCOOH, 10!100% in 21 min, 100% for 3 min). Mass detection
was conducted with a Bruker Esquire 2000 ion-trap mass spectrom-
Table 3. Ability of 8-azaquinazolinone derivatives to inhibit CXCL10- and
CXCL11-mediated activation of CXCR3 as measured in b-arrestin 2 recruit-
ment assays.[a]
Compd
CXCL10
pKb (95% C.I.)
CXCL11
pKb (95% C.I.)
ab
ab
cRAMX3
1a
1b
14
14b
7.66 (7.99–7.33)
0
7.68 (8.00–7.35)
0
–
–
–
–
0.07
0
6.41 (6.96–5.87)
7.68 (8.13–7.23)
–
0.02
0.04
–
6.45 (6.74–6.16)
7.12 (7.33–6.91)
–
–
[a] Inhibition of CXCL10- (400 nm) and CXCL11-mediated (100 nm) recruit-
ment of b-arrestin 2 to CXCR3 was determined with HEK293 cells express-
ing all the components of PathHunter (DiscoverX) and CXCR3-PK2. Values
reflect the mean pKb with 95% confidence interval (C.I.) of three experi-
ments performed in triplicate. Two-tail unpaired t-test was performed to
estimate the significance between pKb and ab values observed between
both chemokines.
1
eter using an APCI or ESI ionization source. H and 13C NMR spectra
were recorded on Bruker Avance 360 or 600 FT-NMR spectrome-
ters. Chemical shifts (ppm) were calculated relative to TMS (1H) or
solvent signal (13C) as internal standards. Synthesis of previously re-
ported intermediates are provided in the Supporting Information.
3-[4-(Benzyloxy)butoxy]propan-1-ol (5a): To a slurry of propane-
1,3-diol (1.62 g, 21.3 mmol) and KOH (1.61 g, 28.7 mmol) in DMSO
(10 mL) [(4-bromobutoxy)methyl]benzene (4) (1.03 g, 4.2 mmol)
was added dropwise at 08C under argon. The mixture was stirred
for 20 h at 08C. The reaction mixture was then diluted with H2O
(20 mL), the layers were separated, and the organic phase was di-
luted with CH2Cl2 (10 mL) and washed with saturated aqueous
NH4Cl, NaHCO3, and NaCl. The organic layer was dried over anhy-
drous Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was purified by flash chromatography (1!3%
MeOH in CH2Cl2) and obtained as a colorless oil (0.66 g, 65%):
HPLC–MS: tR =23.3 min, m/z (ESI) found 261.5, calcd 261.3 ([M+
structural features of 1b and 14. The ‘chimeric’ ligand 14b dis-
played greatly decreased affinity for CXCR3 in RAMX3 displace-
ment and both functional assays (Table 1–3, Figure 1). At the
same time, double replacement of substituents at tertiary
amide core of cRAMX3 restored the competitive character of
the radioligand displacement (a=0; Figure 1, Table 1).
These observations support our hypothesis that structurally
similar allosteric modulators can bind to CXCR3 in different ori-
entations.[32] Based on the present data we hypothesize further
that these orientations elicit different functional responses,
leading to biased signal transduction. It is plausible that by op-
timization of the high-affinity ligand cRAMX3 we forced 1b
(and earlier 14)[32] to adopt functionally selective orientation,
whereas by combining structural modifications we lost this
binding preference, erasing the signaling bias and impairing
affinity (14b).
1
Na]+), purity: n/a (254 nm, 220 nm); H NMR (360 MHz, CDCl3): d=
7.26–7.37 (m, 5H), 4.50 (s, 2H), 3.75 (q, J=5.4 Hz, 2H), 3.59 (t, J=
5.7 Hz, 2H), 3.39–3.52 (m, 4H), 2.47 (t, J=5.3 Hz, 1H), 1.81 (quint.,
J=5.6 Hz, 2H), 1.67 ppm (m, J=2.9 Hz, 4H); 13C NMR (91 MHz,
CDCl3): d=138.6, 128.3 (2C), 127.6 (2C), 127.5, 72.8, 71.0, 70.1,
70.0, 62.1, 32.0, 26.4, 26.4 ppm; HR-ESIMS: m/z found 261.1462,
calcd 261.1461 for C14H22NaO3 [M+Na]+.
4-[4-(Benzyloxy)butoxy]butan-1-ol (5b): To a slurry of butane-1,4-
diol (2.87 g, 31.9 mmol) and KOH (2.68 g, 47.8 mmol) in DMSO
(25 mL) [(4-bromobutoxy)methyl]benzene (4) (1.55 g, 6.37 mmol)
was slowly added under nitrogen atmosphere under gentle cool-
ing in an ice bath (maintaining DMSO liquid). The mixture was al-
lowed to warm to ambient temperature and stirred for 2 h. Then
the reaction was quenched with H2O (30 mL) and the two layers
were separated. The organic phase was diluted with CH2Cl2 (15 mL)
and washed with saturated aqueous NH4Cl and NaHCO3. The or-
ganic layer was dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by flash chromatogra-
phy (2.5% MeOH in CH2Cl2), yielding 5b as a colorless liquid (1.2 g,
75%): TLC (2.5% MeOH in CH2Cl2): Rf =0.22; HPLC–MS: tR =
23.8 min, m/z (ESI) found 275.6, calcd 275.3 ([M+Na]+), purity:
Conclusions
Regulation of CXCR3 signaling is an attractive therapeutic strat-
egy in autoimmune diseases. However, the complex pharma-
cology of the endogenous chemokine signaling system poses
a nontrivial challenge for the design of compounds with tail-
ored functional activity. Herein we describe the first negative
allosteric modulator of CXCR3 with functional selectivity to in-
hibit the G protein pathway (1b). Additionally, the compound
demonstrated probe-dependent functional bias and better
physicochemical properties (molecular weight and polarity)
than known ligands of the same chemotype. Such information
is crucial for further structure-based development of allosteric
ligands that target CXCR3, particularly of those intended for in
vivo applications.
1
n/a (254 nm, 220 nm); H NMR (600 MHz, CDCl3): d=7.30–7.36 (m,
4H), 7.26–7.30 (m, 1H), 4.50 (s, 2H), 3.63 (brs, 2H), 3.47–3.52 (m,
2H), 3.42–3.47 (m, 4H), 2.52 (brs, 1H), 1.61–1.72 ppm (m, 8H);
13C NMR (91 MHz, CDCl3): d=138.6, 128.3 (2C), 127.6 (2C), 127.5,
72.8, 70.8, 70.8, 70.1, 62.7, 30.4, 26.9, 26.5, 26.4 ppm; HR-ESIMS:
m/z found 253.1799, calcd 253.1798 for C15H25O3 [M+H]+.
Experimental Section
{[4-(3-Fluoropropoxy)butoxy]methyl}benzene (6a):
Schlenk flask was charged with 5a (0.34 g, 1.42 mmol) in CH2Cl2
(20 mL) to give colorless solution. Triethylamine (0.99 mL,
7.1 mmol) and 4-dimethylaminopyridine (DMAP; 18 mg,
A
50 mL
Chemistry
a
General: All chemicals and solvents were purchased from Sigma–
Aldrich, Acros, or Alfa Aesar and were used without additional pu-
0.15 mmol) were added, and the mixture was cooled to 08C. Tolue-
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nesulfonyl (tosyl) chloride (0.41 g, 2.1 mmol) was added. The mix-
ture was stirred for 10 min at 08C and was then allowed to warm
to ambient temperature over 3 h. Consumption of starting material
was monitored by TLC (40% Et2O in hexane). Then the reaction
mixture was diluted to 50 mL with CH2Cl2 and washed with H2O
(2ꢂ20 mL) and brine (30 mL). The organic layer was dried over an-
hydrous Na2SO4, filtered and concentrated under reduced pressure.
The resulting residue was dried under high vacuum. The resulting
crude tosylate was dissolved in anhydrous MeCN (15 mL), and
yielding 7a as a pale-yellow oil (134 mg, 90% over two steps):
HPLC–MS: tR =15.3 min, m/z (ESI) found 187.3, calcd 187.2 ([M+
1
Na]+), purity: n/a (254 nm, 220 nm); H NMR (600 MHz, CDCl3): d=
11.20 (brs, 1H), 4.53 (dt, J=47.2, 5.9 Hz, 2H), 3.54 (t, J=6.1 Hz,
2H), 3.49 (t, J=6.0 Hz, 2H), 2.46 (t, J=7.3 Hz, 2H), 1.94 (dquint.,
J=25.7, 6.0 Hz, 2H), 1.91 ppm (tt, J=7.2, 6.2 Hz, 2H); 13C NMR
(151 MHz, CDCl3): d=179.3, 81.0 (d, J=163.6 Hz), 69.7, 66.4 (d, J=
5.5 Hz), 30.9, 30.8 (d, J=19.8 Hz), 24.7 ppm; HR-ESIMS: m/z found
187.0750, calcd 187.0741 for C7H13FNaO3 [M+Na]+.
a
1m solution of tetra-n-butylammonium fluoride (4.25 mL,
4.25 mmol) was added. The resulting mixture was held at reflux for
20 h, then cooled to ambient temperature and concentrated under
reduced pressure. The residue was partitioned between Et2O
(25 mL) and H2O (25 mL). The aqueous layer was discarded and the
organic layer was washed with saturated aqueous NaHCO3 (20 mL)
and brine (20 mL), then dried over Na2SO4, filtered and concentrat-
ed under reduced pressure. The residue was purified by flash chro-
matography (10% Et2O in hexane), yielding 6a as a colorless liquid
(218 mg, 64%): TLC (35% Et2O in hexane): Rf =0.54; HPLC–MS: tR =
24.4 min, m/z (ESI) found 263.5, calcd 263.3 ([M+Na]+), purity:
4-(4-Fluorobutoxy)butanoic acid (7b): A solution of 6b (110 mg,
0.43 mmol) and palladium on carbon (10 wt.%, 11 mg, 10.34 mmol)
in MeOH (2 mL) was stirred under hydrogen atmosphere for 20 h.
The reaction mixture was filtered over a Celite plug and concen-
trated under reduced pressure. The crude fluoroalcohol (62 mg,
88%) was obtained as a pale-yellow oil. The crude product was dis-
solved in acetone (2 mL), and Jones reagent was added at 08C
until a dark-orange color persisted. The reaction mixture was al-
lowed to warm to ambient temperature over 40 min and was then
quenched with iPrOH (10 mL). The mixture was additionally stirred
for 25 min at ambient temperature, was then diluted with H2O
(10 mL) and extracted with EtOAc (3ꢂ10 mL). The combined or-
ganic layers were washed with brine (10 mL), dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure. The
crude product was obtained as a colorless oil (62 mg, 80% over
two steps) after drying under high vacuum. The product 7b was
used for acylation of the secondary amines without further purifi-
cation. HPLC–MS: tR =18.5 min, m/z (ESI) found 201.3, calcd 201.2
([M+Na]+), purity: n/a (254 nm, 220 nm); 1H NMR (600 MHz,
CDCl3): d=11.07 (brs, 1H), 4.46 (dt, J=47.4, 6.0 Hz, 2H), 3.46 (t, J=
6.4 Hz, 2H), 3.47 (t, J=6.2 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 1.90 (tt,
J=7.4, 6.4 Hz, 2H), 1.72–1.83 (m, 2H), 1.64–1.72 ppm (m, 2H);
13C NMR (151 MHz, CDCl3): d=179.2, 83.9 (d, J=163.6 Hz), 70.3,
69.5, 31.0, 27.3 (d, J=19.8 Hz), 25.5 (d, J=5.5 Hz), 24.8 ppm; HR-
ESIMS: m/z found 201.0903, calcd 201.0897 for C8H15FNaO3 [M+
Na]+.
1
n/a (254 nm, 220 nm); H NMR (600 MHz, CDCl3): d=7.30–7.37 (m,
4H), 7.25–7.30 (m, 1H), 4.50 (s, 2H), 4.07 (ddt, J=56.5, 10.2, 6.2 Hz,
2H), 3.45–3.52 (m, 4H), 3.42 (t, J=6.1 Hz, 2H), 1.91 (quint., J=
6.2 Hz, 2H), 1.60–1.71 ppm (m, 4H); 13C NMR (91 MHz, CDCl3): d=
138.6, 128.3 (2C), 127.6 (2C), 127.5, 81.3 (d, J=164.2 Hz), 72.8, 70.8,
70.1, 66.3 (d, J=5.6 Hz), 30.9 (d, J=19.8 Hz), 26.5, 26.4 ppm; HR-
ESIMS: m/z found 241.1600, calcd 241.1598 for C14H22FO2 [M+H]+.
{[4-(4-Fluorobutoxy)butoxy]methyl}benzene (6b): A solution of
5b (0.30 g, 1.19 mmol) in anhydrous CH2Cl2 (10 mL) was purged
with dry nitrogen gas while cooling to ꢀ308C followed by addition
of diethylaminosulfur trifluoride (DAST; 0.31 mL, 2.4 mmol). The re-
action mixture was stirred for 5 h and was then quenched with
MeOH (20 mL). The mixture was allowed to warm to ambient tem-
perature. The solvent was removed under reduced pressure and
the pale-yellow liquid residue was dried in high vacuum. The resi-
due was purified by flash chromatography (15!20% Et2O in
hexane), yielding 6b as colorless oil (119 mg, 39%): HPLC–MS: tR =
25.1 min, m/z (ESI) found 277.5, calcd 277.3 ([M+Na]+), purity:
3-(4-Ethoxyphenyl)-2-(1-{[(1-methylpiperidin-4-yl)methyl]ami-
no}ethyl)pyrido[2,3-d]pyrimidin-4(3H)-one (9): To a suspension of
primary amine 8[41] (1.09 g, 3.5 mmol) in anhydrous 1,2-dichloro-
ethane (10 mL) aldehyde 3 (0.45 g, 3.5 mmol) was added followed
by sodium triacetoxyborohydride (1.1 g, 5.2 mmol) and acetic acid
(0.2 mL, 3.5 mmol). The solution was stirred for 18 h at ambient
temperature. Then the reaction mixture was diluted with CH2Cl2
(20 mL) and washed with saturated aqueous NaHCO3. The organic
layer was extracted with 1n HCl (3ꢂ15 mL), the combined aque-
ous layers were basified with 25% aqueous NH3 and back-extract-
ed with EtOAc (3ꢂ20 mL). The combined organic layers were dried
over anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The crude product was purified by flash chromatography
(eluent: NH3(aq)/MeOH/CH2Cl2 1:9:90). Yield: 1.14 g (77%) as
a pale-yellow foam: HPLC–MS: tR =10.7 min, m/z (APCI) found
422.4, calcd 422.2 ([M+H]+), purity: 95% (254 nm); 1H NMR
(600 MHz, CDCl3): d=8.99 (dd, J=4.6, 2.2 Hz, 1H), 8.61 (dd, J=7.8,
2.2 Hz, 1H), 7.43 (dd, J=7.8, 4.6 Hz, 1H), 7.14 (s, 2H), 7.03–7.09 (m,
2H), 4.11 (q, J=7.0 Hz, 2H), 3.49 (q, J=6.6 Hz, 1H), 2.83 (brd, J=
11.2 Hz, 2H), 2.41 (dd, J=11.2, 6.7 Hz, 1H), 2.25 (s, 3H), 2.20 (dd,
J=11.2, 6.7 Hz, 1H), 1.88 (t, J=11.7 Hz, 2H), 1.65–1.77 (m, 2H), 1.47
(t, J=7.0 Hz, 3H), 1.30 (d, J=6.7 Hz, 3H), 1.27–1.34 (m, 1H), 1.16–
1.27 ppm (m, 2H); 13C NMR (91 MHz, CDCl3): d=165.5, 162.9, 159.8,
157.8, 156.2, 136.8, 129.1, 129.1, 128.1, 122.2, 116.1, 115.9, 115.7,
63.9, 55.7, 55.6, 55.6, 53.9, 46.3, 36.1, 30.7, 30.5, 21.2, 14.7 ppm; HR-
ESI-MS: m/z found 422.2560, calcd 422.2551 for C24H32N5O2 [M+
H]+.
1
n/a (254 nm, 220 nm); H NMR (360 MHz, CDCl3): d=7.25–7.42 (m,
5H), 4.50 (s, 2H), 4.45 (dt, J=47.3, 6.0 Hz, 2H), 3.49 (t, J=6.1 Hz,
2H), 3.34–3.46 (m, 4H), 1.59–1.86 ppm (m, 8H); 13C NMR (91 MHz,
CDCl3): d=138.6, 128.3 (2C), 127.6 (2C), 127.5, 84.0 (d, J=
164.2 Hz), 72.8, 70.6, 70.1, 70.1, 27.3 (d, J=19.8 Hz), 26.5, 26.5,
25.6 ppm (d, J=5.3 Hz); HR-ESIMS: m/z found 277.1579, calcd
277.1574 for C15H23FNaO2 [M+Na]+.
4-(3-Fluoropropoxy)butanoic acid (7a): To a slurry of palladium
on carbon (10 wt%, 25 mg, 0.024 mmol) in anhydrous THF (5 mL)
was added 6a (0.22 g, 0.91 mmol) followed by a rinse with THF
(1.5 mL). The slurry was stirred under hydrogen atmosphere for
20 h. The reaction mixture was filtered over a Celite plug and the
filter cake was washed with EtOAc (15 mL). The filtrate was concen-
trated under reduced pressure and dried under high vacuum yield-
ing the crude fluoroalcohol as a colorless oil. This was dissolved in
acetone (3 mL), and Jones reagent was added until a brown–
orange color persisted (total amount ~1.5 mL). The mixture was
stirred at ambient temperature for 3 h. Then the reaction was
quenched with iPrOH (10 mL) and diluted with 1n HCl to dissolve
precipitated salts (25 mL). The aqueous layer was extracted with
EtOAc (3ꢂ25 mL). The combined organic layers were washed with
brine (15 mL), dried over anhydrous Na2SO4, filtered and concen-
trated under reduced pressure. The residue was purified by flash
chromatography (AcOH/EtOAc/EtOH/hexane 0.25:9.25:3:87.5),
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N-{1-[3-(4-Ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimi-
din-2-yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)me-
thyl]butanamide (1b): In a 50 mL Schlenk flask crude 7b (32 mg,
0.18 mmol), HATU (75 mg, 0.19 mmol) and DIPEA (35 mL,
0.20 mmol) were dissolved in anhydrous DMF (1 mL) under dry ni-
trogen atmosphere. The mixture was stirred for 10 min followed by
addition of 9 (49 mg, 0.116 mmol) as solution in anhydrous DMF
(1 mL). The mixture was stirred for 20 h at 458C. The reaction mix-
ture was diluted to 30 mL with CH2Cl2 and washed with saturated
aqueous NaHCO3 (2ꢂ15 mL) and brine (15 mL). The organic layer
was dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure. The crude product was purified by flash chroma-
tography (25% NH3(aq)/MeOH/CH2Cl2 1:9:90), yielding 1b as
a white foam (44 mg, 65%): HPLC–MS: tR =16.2 min, m/z (APCI)
{5-[(N-{1-[3-(4-Ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyri-
midin-2-yl]ethyl}-4-(4-fluorobutoxy)butanamido)methyl]-2-fluo-
rophenyl}boronic acid (14b):
A solution of HATU (189 mg,
0.50 mmol), 7b (84 mg, 0.47 mmol), and DIPEA (95 mL, 0.54 mmol)
in anhydrous DMF (1 mL) was stirred for 10 min at ambient tem-
perature under nitrogen atmosphere. A solution of potassium
(5-[({1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-
2-yl]ethyl}amino)methyl]-2-fluorophenyl}trifluoroborate
(11)[32]
(124 mg, 0.24 mmol) in anhydrous DMF (1 mL) was added and the
reaction mixture was stirred for 20 h at 458C under nitrogen at-
mosphere. The same amount of activated 4-(4-fluorobutoxy)buta-
noic acid was added and stirring was continued for another 24 h.
Then the reaction mixture was diluted with EtOAc (15 mL) and
washed with saturated NaHCO3 (2ꢂ10 mL) and brine (10 mL). The
organic layer was dried over anhydrous Na2SO4, filtered and con-
centrated under reduced pressure. The residue was redissolved in
the mixture of MeCN and H2O (3:1, 10 mL) and treated with chloro-
trimethylsilane (0.30 mL, 2.36 mmol). The mixture was stirred at
ambient temperature for 2 h. Then the solvent was removed under
reduced pressure and the residue was purified by flash chromatog-
raphy (2.5% MeOH in CH2Cl2), yielding 14b as a pale-yellow oil
(50 mg, 34%): HPLC–MS: tR =19.7 min, m/z (APCI) found 623.5,
637.4, calcd 623.5 ([M+H]+), 637.5 ([M+CH3]+), purity: 95%
1
found 582.6, calcd 582.7 ([M+H]+), purity: 98% (254 nm); H NMR
(360 MHz, CDCl3, two rotamers in ratio 1:0.35, shifts of the major
rotamer are listed): d=8.97 (dd, J=4.7, 2.0 Hz, 1H), 8.59 (dd, J=
7.8, 2.0 Hz, 1H), 7.53 (dd, J=8.6, 2.5 Hz, 1H), 7.42 (dd, J=7.9,
4.6 Hz, 1H), 7.12–7.18 (m, 1H), 6.98–7.09 (m, 2H), 5.22 (q, J=
7.1 Hz, 1H), 4.47 (dt, J=47.2, 6.1 Hz, 2H), 4.10 (q, J=7.0 Hz, 2H),
3.33–3.59 (m, 6H), 2.86 (s, 2H), 2.32–2.51 (m, 2H), 2.27 (s, 3H),
1.76–1.87 (m, 4H), 1.61–1.75 (m, 7H), 1.46 (t, J=7.0 Hz, 3H), 1.46
(d, J=7.0 Hz, 3H), 1.21–1.38 ppm (m, 2H); 13C NMR (151 MHz,
CDCl3, two rotamers in ratio 1:0.35, signals of the major rotamer are
listed): d=174.3, 163.0, 162.7, 159.7, 157.4, 156.1, 136.8, 129.7,
129.0, 127.9, 122.3, 116.4, 115.7, 115.3, 83.9 (d, J=164.1 Hz), 70.1,
69.8, 63.8, 55.6 (2C), 52.9, 50.7, 46.2, 37.1, 30.6, 30.3, 30.2, 27.4 (d,
J=19.8 Hz), 25.6 (d, J=5.5 Hz), 25.5, 16.8, 14.8 ppm; HR-ESIMS:
m/z found 582.3456; calcd 582.3450 for C32H45FN5O4 [M+H]+.
1
(254 nm); H NMR (600 MHz, CDCl3, two rotamers in ratio 1:0.07, sig-
nals of the major rotamer are listed): d=9.00 (dd, J=4.6, 1.9 Hz,
1H), 8.66 (dd, J=7.8, 1.8 Hz, 1H), 8.08 (d, J=3.9 Hz, 1H), 7.98 (brs,
2H), 7.79 (dd, J=8.7, 2.6 Hz, 1H), 7.49 (dd, J=7.8, 4.7 Hz, 1H), 7.15
(ddd, J=11.3, 8.7, 2.8 Hz, 2H), 7.06–7.11 (m, 1H), 6.98–7.05 (m, 2H),
5.21 (d, J=18.8 Hz, 1H), 5.11 (q, J=7.2 Hz, 1H), 5.12 (d, J=18.9 Hz,
1H), 4.34 (dtd, J=47.5, 6.1, 6.1, 1.7 Hz, 2H), 4.13 (q, J=6.9 Hz, 2H),
3.29–3.35 (m, 4H), 2.50 (dt, J=16.0, 7.3 Hz, 1H), 2.29 (dt, J=16.1,
7.4 Hz, 1H), 1.83 (quint., J=6.6 Hz, 2H), 1.50–1.70 (m, 4H), 1.48 (t,
J=6.9 Hz, 3H), 1.38 ppm (d, J=7.3 Hz, 3H); 13C NMR (151 MHz,
CDCl3, two rotamers, signals of the major rotamer are listed): d=
174.9, 166.8 (d, J=245.9 Hz), 165.3, 162.4, 159.9, 157.1, 155.3,
137.8, 134.5 (d, J=8.2 Hz), 134.1 (d, J=3.0 Hz), 130.3, 129.1, 129.0,
127.7, 122.4, 116.7, 115.9, 115.5 (d, J=25.8 Hz), 115.3, 83.8 (d, J=
164.1 Hz), 69.9, 69.5, 63.8, 53.5, 48.1, 30.3, 27.2 (d, J=19.8 Hz), 25.4
(d, J=5.2 Hz), 24.9, 16.2, 14.8 ppm; HR-ESIMS: m/z found 659.2812;
calcd 659.2823 for C33H39BF2N4O6 [M+CH2 +Na]+.
N-{1-[3-(4-Ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimi-
din-2-yl]ethyl}-4-(3-fluoropropoxy)-N-[(1-methylpiperidin-4-yl)-
methyl]butanamide (1a): A solution of 7a (96 mg, 0.59 mmol),
HATU (238 mg, 0.63 mmol) and DIPEA (112 mL, 0.64 mmol) in anhy-
drous DMF (2 mL) was stirred for 10 min at ambient temperature
under nitrogen atmosphere. A solution of 9 (165 mg, 0.39 mmol)
in anhydrous DMF (1 mL) was added. The reaction mixture was
stirred for 20 h at 458C. The reaction mixture was diluted with
EtOAc to 30 mL, washed with saturated aqueous NaHCO3 (15 mL)
and brine (15 mL). The organic layer was dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure. Residual
DMF was removed under high vacuum. The crude product was pu-
rified by flash chromatography (25% NH3(aq)/MeOH/CH2Cl21:9:90).
Yield: 160 mg, 72%; HPLC–MS: tR =19.5 min, m/z(ESI) found 569.0,
calcd 568.7 ([M+H]+), purity: 99% (254 nm, 220 nm); 1H NMR
(360 MHz, CDCl3, two rotamers in ratio 1:0.6, signals of the major
rotamer are listed): d=8.92 (dd, J=4.6, 2.0 Hz, 1H), 8.59 (dd, J=
7.8, 2.0 Hz, 1H), 7.61 (dd, J=8.7, 2.6 Hz, 1H), 7.43 (dd, J=7.8,
4.7 Hz, 1H), 7.16 (dd, J=8.6, 2.6 Hz, 1H), 7.00–7.11 (m, 2H), 5.10 (q,
J=7.1 Hz, 1H), 4.52 (dt, J=47.2, 6.0 Hz, 2H), 4.10 (q, J=7.0 Hz,
2H), 3.44–3.56 (m, 3H), 3.40 (t, J=6.0 Hz, 2H), 3.27–3.36 (m, 1H),
2.76–2.90 (m, 1H), 2.70 (s, 3H), 2.50–2.61 (m, 1H), 2.42 (t, J=7.3 Hz,
2H), 1.92 (dquint., J=26.1, 6.0 Hz, 2H), 1.82 (quint., J=6.5 Hz, 2H),
1.76–1.87 (m, 2H), 1.65–1.74 (m, 2H), 1.51–1.59 (m, 1H), 1.46 (t, J=
6.9 Hz, 3H), 1.44 (d, J=7.1 Hz, 3H), 1.30–1.41 (m, 1H), 0.79–
1.05 ppm (m, 1H); 13C NMR (151 MHz, CDCl3, two rotamers, signals
of the major rotamer are listed): d=174.1, 162.9, 162.9, 159.8, 157.3,
156.0, 137.0, 129.9, 129.1, 127.8, 122.4, 116.5, 115.7, 115.3, 81.4 (d,
J=163.0 Hz), 69.7, 66.3 (d, J=5.5 Hz), 63.8, 55.7, 55.5, 53.6, 49.3,
45.0 (brs), 35.6, 30.8 (d, J=19.8 Hz), 30.3, 28.6 (brs), 27.9 (brs),
25.4, 16.5, 14.8 ppm; HR-ESIMS: m/z found 568.3289; calcd
568.3294 for C31H43FN5O4 [M+H]+.
Biological characterization
Cell culture and transfection: Human embryonic kidney (HEK) cells
were cultured in 150 mm cell culture plates in DMEM/F-12 supple-
mented with 10% fetal bovine serum (FBS), 2 mm l-glutamine, 1%
penicillin–streptomycin and incubated at 378C in a humid atmos-
phere with 5% CO2. At 50–70% confluency, the cells were transi-
ently transfected with 20 mg of the CXCR3 cDNA using TransIT-293
transfection reagent (Mirus Corporation) and harvested 48 h after
transfection.
Membrane preparation: 48 h post-transfection, cells were washed
with PBS twice and harvested with a scraper. Afterward cells were
treated with Tris-EDTA buffer (10 mm Tris, 0.5 mm EDTA, 5 mm KCl,
140 mm NaCl, pH 7.4), and harvested using a cell scraper. Cells
were pelleted at 1100 g for 8 min at 48C, re-suspended in Tris-
EDTA-MgCl2 buffer (50 mm Tris, 5 mm EDTA, 1.5 mm CaCl2, 5 mm
MgCl2, 5 mm KCl, 120 mm NaCl, pH 7.4) and followed by lysis with
an Ultra-Turrax instrument. After centrifugation at 50000 g at 48C
for 18 min, the membranes were re-suspended in binding buffer
(50 mm Tris, 1 mm EDTA, 5 mm MgCl2) and subsequently homogen-
ized with a glass–Teflon homogenizer (20 strokes). The homogen-
ized membranes were shock-frozen in liquid nitrogen and stored
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Full Papers
at ꢀ808C. The protein concentration was determined by the Lowry
Binding studies: The data from the allosteric radioligand displace-
ment assays were analyzed by two different approaches. The data
obtained in the homologous competition assays were analyzed by
nonlinear regression:
method with bovine serum albumin (BSA) as a standard.[43]
Allosteric radioligand displacement assays: Receptor binding studies
were performed on membrane preparations of HEK cells express-
ing the corresponding receptor. Tritium-labeled RAMX3 (specific
activity: 80.4 Cimmolꢀ1) at 1 nm was used for the assays. To deter-
mine unspecific binding, NBI-74330 (5 mm) was used. The assays
were carried out in 96-well plates at a protein concentration of
30 mgmLꢀ1 in a total volume of 200 mL. The incubation buffer con-
tained 20 mm HEPES, 10 mm MgCl2, 100 mm NaCl, and 0.1% BSA
(pH 7.4). After incubating for 1 h at 378C, the binding was stopped
by filtration through Whatman GF/B filters using a 96-channel cell
harvester (Brandel, Unterfçhring). The filters were rinsed five times
with ice-cold Tris-NaCl buffer. After drying for 3 h at 608C, filters
were sealed with melt-on scintillator sheets (Melti-Lex G/HS) and
the trapped radioactivity was measured in a microplate scintillation
counter (Micro Beta Trilux scintillator). Three to five experiments
per compound were performed with each concentration in tripli-
cate. The Kd and Bmax values were determined in the homologous
competition assay and yielded following results: the pKd and Bmax
Bottom þ ðTopꢀBottomÞ
ð1Þ
Y ¼
50ꢀXÞꢂHillslopeÞ
½1 þ 10ððlog IC
ꢃ
Because we often observed incomplete displacement of the radio-
ligand by our novel allosteric ligands, we applied the ternary com-
plex model of allosterism to analyze these data. The data from allo-
steric radioligand binding studies were fitted to a equation[27]
using Prism 5.0:
ꢀ
ꢁ
½Bꢃ
KA 1 þ K
B
ꢀ
ꢁ
Kapp
¼
ð2Þ
ð3Þ
a½Bꢃ
1 þ
KB
Y0 ð1 þ KAÞ
Y ¼
values for the CXCR3 were 7.84ꢁ0.09 and 5710ꢁ700 fmolmgꢀ1
.
ð½cꢃ þ Kapp
Þ
The pKd value determined in these experiments was used in the
analysis of the data obtained in the allosteric radioligand binding
studies to determine the parameters according to the allosteric ter-
nary complex model.
in which Kapp describes the occupancy of the orthosteric site, KA is
the KD value of RAMX3 for the investigated receptor, [c] the radioli-
gand concentration, [B] the concentration of novel allosteric modu-
lator, KB the equilibrium dissociation constant of modulator bind-
ing, and a the ternary complex constant, which denotes the coop-
erativity factor.[27] The KA value for the CXCR3 wild-type was set at
14 nm. The concentration of the allosteric radioligand RAMX3 was
set to 1 nm. Values of a>1 denote positive cooperativity, whereas
a<1 denotes negative cooperativity. Values of a approaching zero
are indistinguishable from competitive antagonism. When a ap-
[35S]GTPgS incorporation assays: The [35S]GTPgS incorporation assay
was performed on membrane preparations of transiently transfect-
ed HEK293 cells expressing the CXCR3 receptor. The assay was car-
ried out in 96-well plates at a final volume of 200 mL. The incuba-
tion buffer contained 20 mm HEPES, 10 mm MgCl2·6H20, 100 mm
NaCl, and 100 mgLꢀ1 saponin (pH 7.4). Membranes (30 mgmLꢀ1 of
membrane protein), with 5 nm CXCL11 and various concentrations
of test compound and 1 mm GDP were pre-incubated in the ab-
sence of [35S]GTPgS for 30 min at 378C. After the addition of
0.10 nm [35S]GTPgS membranes were incubated for additional
30 min at 378C. Incubation was terminated by filtration through
Whatman GF/B filters soaked with ice-cold PBS. The filter-bound ra-
dioactivity was measured as described above. Three to four experi-
ments per compound were performed with each concentration in
triplicate.
proaches zero, the Kb value approaches the Ki value[27]
.
Functional studies: To obtain the EC50 values for CXCL11 in wild-
type CXCR3 and its mutants, dose–response curves were fitted by
nonlinear regression:
Bottom þ ðTopꢀBottomÞ
ð4Þ
Y ¼
50ꢀXÞꢂHillslopeÞ
½1 þ 10ððlog IC
ꢃ
To characterize the allosteric profile of novel ligands, we applied
the ternary complex model of allosterism to analyze the data ob-
tained from the functional assays as described before.[32] The as-
sumptions were that the allosteric modulators do not cause the
depression of maximal response or the suppression of the basal ac-
tivity. The depression of maximal response or the suppression of
the basal activity are not accounted for in an allosteric ternary
complex model (ATCM). Importantly, even if these assumptions do
not hold entirely true for all the novel allosteric modulators, this
analysis enables an initial approximation and a semi-empirical esti-
mate of cooperativity. The data from functional studies in which
discrete concentrations of agonist CXCL11 and CXCL10 were used,
were fitted to following equations using Prism 5.0:
b-arrestin 2 recruitment assays: The measurement of b-arrestin re-
cruitment was performed using the PathHunter assay purchased
from DiscoverX (DiscoverX, Birmingham, UK) according to the man-
ufacturer’s protocol. HEK293 cells stably expressing b-gal enzyme
acceptor were transiently transfected with the ProLink tagged
wild-type CXCR3 receptor using the TransIT-293 transfection re-
agent from Mirus (purchased from MoBiTec, Gçttingen, Germany).
The assay was carried out in 384-well plates containing 2000 cells
in 20 mL. After incubation at 378C (5% CO2, 95% relative humidity)
overnight, 100 nm CXCL11 and various concentrations of test com-
pounds in PBS, pH 7.4 and 0.20% BSA were added and the incuba-
tion continued for 4 h. Detection mix was then added and incubat-
ed for a further 60 min at ambient temperature. Chemilumines-
cence was determined with a microplate reader Victor3V (Perkin-
Elmer, Rodgau, Germany).
ꢀ
ꢁ
ꢁ
½Bꢃ
KA 1 þ K
B
ꢀ
Kapp
¼
ð5Þ
Data analysis: Data were analyzed by nonlinear regressions using
the algorithms in Prism 5.0 (GraphPad Software, San Diego, CA,
USA). The equations and models used are in detail described
below. Obtained parameters were compared by two-way unpaired
t-test to estimate the significance in observed differences between
the pKB and a values.
ab½Bꢃ
1 þ
KB
Y0 ð1 þ KAÞ
Y ¼
ð6Þ
ð½cꢃ þ Kapp
Þ
&
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Full Papers
[18] K. Berry, M. Friedrich, K. Kersey, M. J. Stempien, F. Wagner, J. J. van Lier,
R. Sabat, K. Wolk, Inflammation Res. 2004, 53, S222.
[19] M. Wijtmans, D. Verzijl, R. Leurs, I. J. P. de Esch, M. J. Smit, ChemMed-
Chem 2008, 3, 861–872.
[20] B. T. Andresen, Endocr. Metab. Immune Disord. Drug Targets 2011, 11,
92–98.
[21] E. J. Whalen, S. Rajagopal, R. J. Lefkowitz, Trends Mol. Med. 2011, 17,
126–139.
[22] S. Rajagopal, S. Ahn, D. H. Rominger, W. Gowen-macdonald, C. M. Lam,
S. M. Dewire, J. D. Violin, R. J. Lefkowitz, Mol. Pharmacol. 2011, 80, 367–
377.
in which Kapp describes the occupancy of the orthosteric site, KA is
the EC50 value of CXCL11 or CXCL10 for the investigated receptor,
[c] the concentration of CXCL11 or CXCL10 used, [B] the concentra-
tion of novel allosteric modulator, KB the equilibrium dissociation
constant of modulator binding, and ab the ternary complex con-
stant, which denotes the cooperativity factor.[27] In the [35S]GTPgS
accumulation assay the KA value of CXCL11 was set at 1.5 nm and
for CXCL10 at 3.3 nm. The concentration of CXCL11 was set to
5 nm and of CXCL10 to 50 nm. In the b-arrestin 2 assay the KA
value CXCL11 was set at 41 nm and for CXCL10 at 7.3 nm. The con-
centration of CXCL11 was set to 100 nm and of CXCL10 at 40 nm.
[23] D. Wootten, A. Christopoulos, P. M. Sexton, Nat. Rev. Drug Discovery
2013, 12, 630–644.
[24] T. Kenakin, C. Watson, V. Muniz-Medina, A. Christopoulos, S. Novick, ACS
Chem. Neurosci. 2012, 3, 193–203.
[25] S. Rajagopal, K. Rajagopal, R. J. Lefkowitz, Nat. Rev. Drug Discovery 2010,
9, 373–386.
Acknowledgements
N.T., V.B., and R.B. thank the German Research Foundation for fi-
nancial support (DFG; Graduate Training School GRK1910 and
grant TS287/2-1). N.T. participates in the European COST Action
CM1207 (GLISTEN: GPCR–Ligand Interactions, Structures, and
Transmembrane Signalling: a European Research Network).
[26] Z.-G. Gao, D. Verzijl, A. Zweemer, K. Ye, A. Gçblyçs, A. P. IJzerman, K. A.
Jacobson, Biochem. Pharmacol. 2011, 82, 658–668.
[27] A. Christopoulos, T. Kenakin, Pharmacol. Rev. 2002, 54, 323–374.
[28] D. J. Scholten, M. Canals, M. Wijtmans, S. de Munnik, P. Nguyen, D. Ver-
zijl, I. J. P. de Esch, H. F. Vischer, M. J. Smit, R. Leurs, Br. J. Pharmacol.
2012, 166, 898–911.
[29] B. Nedjai, H. Li, I. L. Stroke, E. L. Wise, M. L. Webb, J. R. Merritt, I. Hender-
son, A. E. Klon, A. G. Cole, R. Horuk, N. Vaidehi, J. E. Pease, Br. J. Pharma-
col. 2012, 166, 912–923.
Keywords: allosteric modulation
·
chemokine receptor
CXCR3 · ligand-biased signaling · probe dependence
[30] G. O’Boyle, C. R. J. Fox, H. R. Walden, J. D. P. Willet, E. R. Mavin, D. W.
Hine, J. M. Palmer, C. E. Barker, C. A. Lamb, S. Ali, J. A. Kirbya, Proc. Natl.
Acad. Sci. USA 2012, 109, 4598–4603.
[31] T. Kenakin, Nat. Rev. Drug Discovery 2005, 4, 919–927
[32] V. Bernat, T. H. Admas, R. Brox, F. W. Heinemann, N. Tschammer, ACS
Chem. Biol. 2014, 9, 2664–2677.
[33] I. Kalatskaya, Y. A. Berchiche, S. Gravel, B. J. Limberg, J. S. Rosenbaum, N.
Heveker, Mol. Pharmacol. 2009, 75, 1240–1247.
[34] C. Watson, S. Jenkinson, W. Kazmierski, T. Kenakin, Mol. Pharmacol.
2005, 67, 1268–1282.
[1] T. Kenakin, Trends Pharm. Sci. 20035, 24, 346–354.
[2] T. P. Kenakin, A. Christopoulos, Nat. Rev. Drug Discovery 2013, 12, 205–
216.
[3] T. Kenakin, J. Pharmacol. Exp. Ther. 2011, 336, 296–302.
[4] J. R. Groom, A. D. Luster, Immunol. Cell Biol. 2011, 89, 207–215.
[5] A. Kouroumalis, R. J. B. Nibbs, H. Aptel, K. L. Wright, G. Kolios, S. G.
Ward, J. Immunol. 2005, 175, 5403–5411.
[6] R. A. Colvin, G. S. V. Campanella, J. Sun, A. D. Luster, J. Biol. Chem. 2004,
279, 30219–30227.
[7] S. Rajagopal, D. L. Bassoni, J. J. Campbell, N. P. Gerard, C. Gerard, T. S.
Wehrman, J. Biol. Chem. 2013, 288, 35039–35048.
[35] K. Maeda, H. Nakata, Y. Koh, T. Miyakawa, H. Ogata, Y. Takaoka, S. Shi-
bayama, K. Sagawa, D. Fukushima, J. Moravek, Y. Koyanagi, H. Mitsuya,
J. Virol. 2004, 78, 8654–8662.
[36] J. C. Medina, M. G. Johnson, A.-R. Li, J. Liu, A. X. Huang, L. Zhu, A. P.
Marcus (Tularik Inc.), US Pat. No. US2002/0169159 A1, 2002.
[37] M. Johnson, A.-R. Li, J. Liu, Z. Fu, L. Zhu, S. Miao, X. Wang, Q. Xu, A.
Huang, A. Marcus, F. Xu, K. Ebsworth, E. Sablan, J. Danao, J. Kumer, D.
Dairaghi, C. Lawrence, T. Sullivan, G. Tonn, T. Schall, T. Collins, J. Medina,
Bioorg. Med. Chem. Lett. 2007, 17, 3339–3343.
[38] S. Storelli, D. Verzijl, J. Al-Badie, N. Elders, L. Bosch, H. Timmerman, M. J.
Smit, I. J. P. de Esch, R. Leurs, Arch. Pharm. 2007, 340, 281–291.
[39] T. J. Ritchie, S. J. F. Macdonald, Drug Discovery Today 2009, 14, 1011–
1020.
[8] S. Lacotte, S. Brun, S. Muller, H. Dumortier, Ann. N. Y. Acad. Sci. 2009,
1173, 310–317.
[9] T. L. Sørensen, M. Tani, J. Jensen, V. Pierce, C. Lucchinetti, V. A. Folcik, S.
Qin, J. Rottman, F. Sellebjerg, R. M. Strieter, J. L. Frederiksen, R. M. Ran-
sohoff, J. Clin. Invest. 1999, 103, 807–815.
[10] I. Salomon, N. Netzer, G. Wildbaum, S. Schif-Zuck, G. Maor, N. Karin, J.
Immunol. 2002, 169, 2685–2693.
[11] W. W. Hancock, B. Lu, W. Gao, V. Csizmadia, K. Faia, J. a. King, S. T.
Smiley, M. Ling, N. P. Gerard, C. Gerard, J. Exp. Med. 2000, 192, 1515–
1520.
[40] S. E. Ward, P. Beswick, Expert Opin. Drug Discovery 2014, 9, 995–1003.
[41] V. Bernat, M. R. Heinrich, P. Baumeister, A. Buschauer, N. Tschammer,
ChemMedChem 2012, 7, 1481–1489.
[42] J. Frçhlich, F. Sauter, C. Hametner, M. Pfalz, ARKIVOC 2009, 2009, 298–
308.
[12] J. Pan, M. D. Burdick, J. a. Belperio, Y. Y. Xue, C. Gerard, S. Sharma, S. M.
Dubinett, R. M. Strieter, J. Immunol. 2006, 176, 1456–1464.
[13] T. C. Walser, S. Rifat, X. Ma, N. Kundu, C. Ward, O. Goloubeva, M. G. John-
son, J. C. Medina, T. L. Collins, A. M. Fulton, Cancer Res. 2006, 66, 7701–
7707.
[43] O. H. Lowry, N. J. Rosebrough, L. Farr, R. J. Randall, J. Biol. Chem. 1951,
193, 265–275.
[14] M. Krauthausen, S. L. Ellis, J. Zimmermann, M. Sarris, D. Wakefield, M. T.
Heneka, I. L. Campbell, M. Mꢃller, Am. J. Pathol. 2011, 179, 2346–2359.
[15] M. Krauthausen, S. Saxe, J. Zimmermann, M. Emrich, M. T. Heneka, M.
Mꢃller, J. Neuroinflammation 2014, 11, 109.
[16] M. Mꢃller, S. Carter, M. J. Hofer, I. L. Campbell, Neuropathol. Appl. Neuro-
biol. 2010, 36, 368–387.
[17] D. P. Widney, Y. Hu, A. K. Foreman-Wykert, K. C. Bui, T. T. Nguyen, B. Lu,
C. Gerard, J. F. Miller, J. B. Smith, Infect. Immun. 2005, 73, 485–493.
Received: November 25, 2014
Revised: January 14, 2015
Published online on && &&, 0000
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&
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FULL PAPERS
V. Bernat, R. Brox, M. R. Heinrich,
Y. P. Auberson, N. Tschammer*
How amazing can 1b? The chemokine
receptor CXCR3 is an outstanding plat-
form to study various aspects of alloste-
ric modulation. Herein we report the
discovery of a small molecule (com-
pound 1b) that can inhibit CXCL11-de-
pendent G protein activation over b-ar-
restin recruitment with 187-fold selectiv-
ity. Compound 1b also demonstrates
probe-dependent activity: it inhibits
CXCL11- over CXCL10-mediated G pro-
tein activation with 12-fold selectivity.
&& – &&
Ligand-Biased and Probe-Dependent
Modulation of Chemokine Receptor
CXCR3 Signaling by Negative
Allosteric Modulators
&
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ÝÝ These are not the final page numbers!