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Synthesis, characterization, and in vitro evaluation of the selective P2Y2 receptor antagonist AR-C118925

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Abstract

The Gq protein-coupled, ATP- and UTP-activated P2Y2 receptor is a potential drug target for a range of different disorders, including tumor metastasis, inflammation, atherosclerosis, kidney disorders, and osteoporosis, but pharmacological studies are impeded by the limited availability of suitable antagonists. One of the most potent and selective antagonists is the thiouracil derivative AR-C118925. However, this compound was until recently not commercially available and little is known about its properties. We therefore developed an improved procedure for the synthesis of AR-C118925 and two derivatives to allow up-scaling and assessed their potency in calcium mobilization assays on the human and rat P2Y2 receptors recombinantly expressed in 1321N1 astrocytoma cells. The compound was further evaluated for inhibition of P2Y2 receptor-induced β-arrestin translocation. AR-C118925 behaved as a competitive antagonist with pA 2 values of 37.2 nM (calcium assay) and 51.3 nM (β-arrestin assay). Selectivity was assessed vs. related receptors including P2X, P2Y, and adenosine receptor subtypes, as well as ectonucleotidases. AR-C118925 showed at least 50-fold selectivity against the other investigated targets, except for the P2X1 and P2X3 receptors which were blocked by AR-C118925 at concentrations of about 1 μM. AR-C118925 is soluble in buffer at pH 7.4 (124 μM) and was found to be metabolically highly stable in human and mouse liver microsomes. In Caco2 cell experiments, the compound displayed moderate permeability indicating that it may show limited peroral bioavailability. AR-C118925 appears to be a useful pharmacological tool for in vitro and in vivo studies.

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Abbreviations

AIBN:

Azobisisobutyronitrile

AP4A:

Diadenosine tetraphosphate

AR-C118925:

5-{[5-(2,8-Dimethyl-5H-dibenzo[a,d]cyclohepten-5-yl)-3,4-dihydro-2-oxo-4-thioxo-1(2H)-pyrimidinyl]methyl}-N-(1H-tetrazol-5-yl)-2-furancarboxamide

ATP:

Adenosine-5′-triphosphate

CHO:

Chinese hamster ovary

CYP:

Cytochrome P450

DMEM:

Dulbecco’s modified Eagle’s medium

DMF:

Dimethylformamide

DMF-DEA:

Dimethylformamide-diethylacetal

E5′-NT:

Ecto-5′-nucleotidase

EDTA:

Ethylenediaminetetraacetic acid

Fluo-4AM:

Fluo-4 acetoxymethyl ester

MRS2768:

Uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt

HBSS:

Hank’s balanced salt solution

HMDS:

Hexamethyldisilazane

hNPP1:

Human nucleotide pyrophosphatase subtype 1

hNTPDase2:

Human nucleoside triphosphate diphosphohydrolase subtype 2

HOBt:

Hydroxybenzotriazole

LDA:

Lithium diisopropylamide

NBS:

N-Bromosuccinimide

rt:

Room temperature

SEM:

Standard error of the mean

THF:

Tetrahydrofuran

UTP:

Uridine-5′-triphosphate

vs:

Versus

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Acknowledgments

This study was supported by the Ministry for Innovation, Science, Research and Technology of the State of North Rhine-Westphalia through the NRW International Graduate Research School Biotech-Pharma (M.R., C.E.M.). We thank Amelie Fiene, Angelika Fischer, Marianne Freundlieb, Sonja Hinz, Sangyong Lee, Anika Püsche, Anja B. Scheiff, Clara B. Schoeder, and Stefanie Weyer for selectivity testing.

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Authors and Affiliations

  1. PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Sciences Bonn (PSB), Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany

    Muhammad Rafehi, Joachim C. Burbiel, Isaac Y. Attah, Aliaa Abdelrahman & Christa E. Müller

  2. Pharmazeutisches Institut, Pharmazeutische Chemie I, An der Immenburg 4, D-53121, Bonn, Germany

    Muhammad Rafehi, Isaac Y. Attah, Aliaa Abdelrahman & Christa E. Müller

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  1. Muhammad Rafehi
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Correspondence to Christa E. Müller.

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Electronic supplementary material

ESM. 1

Supporting information includes the descriptions of in vitro assays used for selectivity testing, data on targets not directly related to the P2Y2 receptor, potency and efficacy results for the concentration-effect curves of UTP following pre-incubation with different, fixed concentrations of AR-C118925, and additional data on the physicochemical and pharmacokinetic properties of AR-C118925. (PDF 303 kb)

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Rafehi, M., Burbiel, J.C., Attah, I.Y. et al. Synthesis, characterization, and in vitro evaluation of the selective P2Y2 receptor antagonist AR-C118925. Purinergic Signalling 13, 89–103 (2017). https://doi.org/10.1007/s11302-016-9542-3

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