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

Department of Chemistry and Pharmacy

Emil Fischer Center

Friedrich-Alexander-University

Schuhstraße 19, D-91052 Erlangen

www.medchem.uni-erlangen.de

CXCR3

CXCL9

AMG487

GPCR

40.7 kDa

Chemokine

14.0 kDa

Small molecule

0.6 kDa

Mechanism of action: Endogenous ligands (CXCL9, CXCL10 and CXCL11) bind to N-terminus of CXCR3 and activate it with their own flexible N-terminus via transmembrane binding pocket. Synthetic allosteric modulators bind directly to the TM domain without interaction with receptor’s N-terminus.

Involvement of CXCR3 – chemokine signaling system in pathological conditions: Multiple sclerosis Psoriasis Rheumatoid arthritis Breast and colon cancer Transplant rejection

Biological function of chemokine system: Migration and response of immune cells

Chemokine-based binding assays are inconvenient Allosteric radioligand is needed

a) N-Boc-D-alanine, IBCF, NMM, then ANA, DCM, - 20°C … RT, 20 h; b) p-phenetidine, DCM, RT, 24 h; c) IBCF, NMM, DCM, RT, 2 h; d) TFA, DCM, 4–8 h; e) (±)-N-Boc-2-aminobutyric acid, IBCF, NMM, then ANA, DCM, - 20°C … RT, 20 h; f) N-Boc-L-phenylalanine, IBCF, NMM, then ANA, DCM, - 20°C … RT, 20 h; g) 4-methoxybenzylamine, DCM, RT, 20 h; h) N-Boc-β-alanine, IBCF, NMM, then ANA, DCM, - 20°C … RT, 20 h; i) pyridine-3-carbaldehyde, NaBH(OAc)3, DCE, RT, 20 h; j) corresponding substituted phenylacetic acid, HATU, DIPEA, DMF, 45°C, 20 h; k) Ammonium Cerium (IV) Nitrate, CH3CN/H2O, RT, 20 h, 85%.

Binding Characteristics of RAMX3

at the human CXCR3 receptor expressed in HEK-293 cells

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]

[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

Binding data and functional activities

of the 8-azaquinazolinone derivatives

at the human CXCR3 receptor

expressed in HEK-293 cells

Compd pKi[a] pIC50

[b]

CXCL11 7.23±0.09 8.45±0.11[c]

rac-AMG487 7.85±0.10 6.05±0.09

rac-NBI-74330 9.02±0.16 6.97±0.08

“cold” RAMX3 8.85±0.11 7.40±0.13

6b 8.20±0.20 4.99±0.13

6d <5.00 5.54±0.16

6e 6.36±0.14 5.72±0.12

7b 5.05±0.42 5.46±0.09

7c 6.38±0.27 5.68±0.20

7e 6.33±0.17 4.89±0.21

8d <5.00 n.d.

NVP-BDZ824[d] 8.93±0.14 n.d.

RAMX3

Comparison of binding and functional data revealed some unusual correlation between affinity and activity of compounds: compound 6b has higher affinity than AMG487 but lower efficacy than its own truncated analogue 7b. On the contrary, compound 6d is not able to completely displace RAMX3 at a concentration of 10 μM but still inhibits activation of CXCR3 with efficiency similar to other tested compounds. These observations are in accord with the notion that allosteric modulator SAR needs to differentiate the influence of chemical modifications on compound affinity from the cooperativity exhibited toward orthosteric ligands, as the two properties are not correlated. Data for compound 8d imply that a phenyl substituent attached to 8-azaquinazolinone core is essential for affinity and efficacy of this class of CXCR3 receptor negative allosteric modulators. Binding experiments with ergoline derivative NVP-BDZ824 demonstrated that RAMX3 can be used as a tool to interrogate allosteric binding sites. The obtained binding affinity of this compound was found to be five-fold higher than Ki value reported for the assay using [125I]CXCL11 (1.17 nM vs. 6 nM respectively)[2].

NVP-BDZ824

Conclusions

[a] Radioligand binding: determined from the displacement of the radioligand RAMX3 (c = 1 nM) from HEK-293 cell membranes expressing CXCR3. IC50 values were converted into pKi values according to Cheng-Prussoff equation; values reflect the mean ± SEM of two to three experiments performed in triplicate .

[b] Inhibition of CXCL11-mediated activation of CXCR3: determined by the gene reporter (luciferase) assay; values reflect the mean ± SEM of three to five experiments performed in triplicate. All derivatives fully inhibited CXCL11 (20 nM)-mediated activation of the CXCR3 receptor. [c] pEC50 value for the stimulation of CXCR3 by CXCL11; the value reflects the mean ± SEM of three experiments performed in triplicate [d] An ergoline derivative developed by Novartis[2].

References

1. V. Bernat et al. ChemMedChem, 7 (2012), 1481-1489

2. G. Thoma et al. Bioorg. Med. Chem. Lett, 19 (2009), 6185-6188

CHEMBL1077827