The poster was presented during International Symposium on Medicinal Chemistry in Berlin, September 2012. It was awarded with one of the Poster Prizes.

1. Department of Chemistry and Pharmacy
Emil Fischer Center
Friedrich-Alexander-University
Schuhstraße 19, D-91052 Erlangen
www.medchem.uni-erlangen.de
SYNTHESIS AND APPLICATION
OF THE FIRST SMALL-MOLECULE RADIOLIGAND
TARGETING THE HUMAN CHEMOKINE RECEPTOR CXCR3
V. Bernat†, Prof. Dr. M. Heinrich†, P. Baumeister‡, Prof. Dr. A. Buschauer‡, Dr. N. Tschammer†
†University of Erlangen-Nuremberg, ‡University of Regensburg
Biological function of chemokine system:
Migration and response of immune cells
Involvement of CXCR3 – chemokine signaling system in
pathological conditions:
AMG487
 Multiple sclerosis
 Psoriasis
 Rheumatoid arthritis
CXCL9
 Breast and colon cancer
 Transplant rejection
Mechanism of action:
Endogenous ligands (CXCL9, CXCL10 and CXCL11) bind to N-
terminus of CXCR3 and activate it with their own flexible N-terminus CXCR3
via transmembrane binding pocket. GPCR Chemokine Small molecule
Synthetic allosteric modulators bind directly to the TM domain 40.7 kDa 14.0 kDa 0.6 kDa
without interaction with receptor’s N-terminus.
Binding Characteristics of RAMX3
Chemokine-based binding assays are inconvenient at the human CXCR3 receptor expressed in HEK-293 cells
Allosteric radioligand is needed
Saturation Binding Binding Kinetics
Kd , nM Kd , nM kon , min-1nM-1 koff , min-1 t1/2 , min
1.08±0.18[a] 0.93[b] 0.045±0.003[c] 0.041±0.007[d] 16.9[e]
RAMX3 [a] Equilibrium dissociation constant; values reflect the means ± SEM of 5
independent experiments performed in triplicate. [b] Kinetically derived
dissociation constant. [c] Association rate constant ± SEM. [d] Dissociation rate
constant ± SEM from linear regression analysis. [e] Half-life, calculated as ln2/koff
Conclusions
Binding data and functional activities
CHEMBL1077827
Comparison of binding and functional
of the 8-azaquinazolinone derivatives data revealed some unusual correlation
at the human CXCR3 receptor between affinity and activity of
expressed in HEK-293 cells compounds: compound 6b has higher
Compd pKi[a] pIC50[b] affinity than AMG487 but lower efficacy
7.23±0.09 8.45±0.11[c] than its own truncated analogue 7b. On
CXCL11
the contrary, compound 6d is not able to
rac-AMG487 7.85±0.10 6.05±0.09
completely displace RAMX3 at a
rac-NBI-74330 9.02±0.16 6.97±0.08 concentration of 10 μM but still inhibits
“cold” RAMX3 8.85±0.11 7.40±0.13 activation of CXCR3 with efficiency
8.20±0.20 4.99±0.13 similar to other tested compounds.
6b
6d <5.00 5.54±0.16
These observations are in accord with the
6e 6.36±0.14 5.72±0.12 notion that allosteric modulator SAR
7b 5.05±0.42 5.46±0.09 needs to differentiate the influence of
6.38±0.27 5.68±0.20 chemical modifications on compound
7c
affinity from the cooperativity exhibited
7e 6.33±0.17 4.89±0.21
toward orthosteric ligands, as the two
8d <5.00 n.d. properties are not correlated.
NVP-BDZ824[d] 8.93±0.14 n.d.
Data for compound 8d imply that a
[a] Radioligand binding: determined from the phenyl substituent attached to 8-
displacement of the radioligand RAMX3 (c = 1 nM) from azaquinazolinone core is essential for
HEK-293 cell membranes expressing CXCR3. IC50 values affinity and efficacy of this class of CXCR3
were converted into pKi values according to Cheng- receptor negative allosteric modulators.
Prussoff equation; values reflect the mean ± SEM of two to
three experiments performed in triplicate . Binding experiments with ergoline
[b] Inhibition of CXCL11-mediated derivative NVP-BDZ824 demonstrated
activation of CXCR3: determined by that RAMX3 can be used as a tool to
the gene reporter (luciferase) assay; interrogate allosteric binding sites. The
values reflect the mean ± SEM of obtained binding affinity of this
three to five experiments performed compound was found to be five-fold
in triplicate. All derivatives fully higher than Ki value reported for the
a) N-Boc-D-alanine, IBCF, NMM, then ANA, DCM, - 20°C … RT, 20 h; b) p- inhibited CXCL11 (20 nM)-mediated assay using [125I]CXCL11 (1.17 nM vs. 6
phenetidine, DCM, RT, 24 h; c) IBCF, NMM, DCM, RT, 2 h; d) TFA, DCM, 4–8 h; e) activation of the CXCR3 receptor. nM respectively)[2].
(±)-N-Boc-2-aminobutyric acid, IBCF, NMM, then ANA, DCM, - 20°C … RT, 20 h; [c] pEC50 value for the stimulation of
NVP-BDZ824
f) N-Boc-L-phenylalanine, IBCF, NMM, then ANA, DCM, - 20°C … RT, 20 h; g) 4- CXCR3 by CXCL11; the value reflects
methoxybenzylamine, DCM, RT, 20 h; h) N-Boc-β-alanine, IBCF, NMM, then the mean ± SEM of three experiments
ANA, DCM, - 20°C … RT, 20 h; i) pyridine-3-carbaldehyde, NaBH(OAc)3, DCE, RT, performed in triplicate References
20 h; j) corresponding substituted phenylacetic acid, HATU, DIPEA, DMF, 45°C, [d] An ergoline derivative developed
1. V. Bernat et al. ChemMedChem, 7 (2012),
20 h; k) Ammonium Cerium (IV) Nitrate, CH3CN/H2O, RT, 20 h, 85%. by Novartis[2]. 1481-1489
2. G. Thoma et al. Bioorg. Med. Chem. Lett, 19
(2009), 6185-6188