Water-Soluble Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines as Human A 3 Adenosine Receptor Antagonists Pier Giovanni Baraldi,* ,† Giulia Saponaro, † Romeo Romagnoli, † Mojgan Aghazadeh Tabrizi, † Stefania Baraldi, † Allan R. Moorman, §,# Sandro Cosconati, ∥ Salvatore Di Maro, ⊥ Luciana Marinelli, ⊥ Stefania Gessi, ‡ Stefania Merighi, ‡ Katia Varani, ‡ Pier Andrea Borea, ‡ and Delia Preti † † Dipartimento di Scienze Farmaceutiche and ‡ Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia, Universita ̀ di Ferrara, 44100 Ferrara, Italy § King Pharmaceuticals, Inc., Research and Development, 4000 CentreGreen Way, Suite 300, Cary, North Carolina 27513, United States ∥ Dipartimento di Scienze Ambientali, Seconda Universita ́ di Napoli, Via Vivaldi 43, 81100 Caserta, Italy ⊥ Dipartimento di Chimica Farmaceutica e Tossicologica, Universita ̀ “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy * S Supporting Information ABSTRACT: A relevant problem of the pyrazolo[4,3-e]- [1,2,4]triazolo[1,5-c]pyrimidine nucleus, an attractive scaffold for the preparation of adenosine receptor antagonists, is the low water solubility. We originally functionalized the C 5 position with a salifiable 4-pyridylcarbamoyl moiety that conferred good water solubility at low pH (<4.0) but poor solubility at physiologic pH, indicative of the dissociation of the pyridinium species. Here we replaced the pyridin-4-yl moiety with a 1-(substituted)piperidin-4-yl ring to exploit the higher basicity of this nucleus and for the the possibility to generate stable, water-soluble salts. The hydrochloride salt of the 1-(cyclohexylmethyl)piperidin-4-yl derivative (10, K i (hA 3 ) = 9.7 nM, IC 50 (hA 3 ) = 30 nM, K i (hA 1 /hA 3 ) = 351, K i (hA 2A /hA 3 ) > 515, IC 50 (hA 2B) >5 μM) showed a solubility of 8 mg/mL at physiological pH and gave a stable aqueous system suitable for intravenous infusion. Molecular modeling studies were helpful in rationalizing the available structure−activity relationships and the selectivity profile of the new ligands. ■ INTRODUCTION Adenosine, a ubiquitous nucleoside essential for the proper functioning of every cell in mammalian species is directly linked to energy metabolism through ATP, ADP, and AMP, while at the extracellular level it regulates a wide range of biological functions through activation of specific receptors (adenosine receptor, AR). 1−4 These adenosine receptors belong to the superfamily of the G-protein-coupled receptors (GPCRs) and are classified as A 1 ,A 2A ,A 2B , and A 3 . The A 3 AR subtype is the most recently characterized member of the family. 4 Activation of the subtype has been shown to inhibit adenylate cyclase, to increase phosphatidyli- nositol-specific phospholipase C and D activity, to elevate intracellular Ca 2+ and IP 3 (inositol 1,4,5-trisphosphate) levels, and to enhance the release of inflammatory and allergic mediators from mast cells. 5,6 The potential therapeutic applications derived from the modulation of this receptor subtype have been recently reviewed. 7,8 It is becoming increasingly apparent that an antagonist of A 3 AR might be therapeutically useful for the acute treatment of glaucoma. 9,10 Specific antagonists are currently undergoing biological testing for their potential use in the treatment of stroke, neuro- degenerative diseases, allergy, asthma, and COPD (Thomson Reuters Integrity source). Our group provided the first evidence that the A 3 AR plays a role in colon tumorigenesis and, more importantly, can potentially be used as a diagnostic marker or a therapeutic target for colon cancer. 11 The pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (PTP) nucleus has been shown repeatedly to serve as an attractive scaffold for the preparation of adenosine receptor antagonists. Systematic substitution of the C 2 -, C 5 -, N 7 -, N 8 -, or C 9 -positions of PTPs 7,12−16 allowed a structure−activity relationship (SAR) profile to be delineated for this class of molecules, primarily defining receptor subtype selectivity. A 2-furyl or a 2- (substituted)phenyl 17,18 ring appeared important for affinity toward all four AR subtypes. The presence of a free amine at the C 5 -position combined with an arylalkyl moiety at the N 7 - position was found to significantly promote both affinity and selectivity at the A 2A AR subtype. 12 In contrast, introduction of an arylurea moiety at the C 5 -position and small alkyl chains (e.g., methyl or propyl) at the N 8 -position led to the Received: March 6, 2012 Published: May 9, 2012 Article pubs.acs.org/jmc © 2012 American Chemical Society 5380 dx.doi.org/10.1021/jm300323t | J. Med. Chem. 2012, 55, 5380−5390