Synthesis and biological evaluation of novel photo-clickable adenosine and cyclic ADP-ribose analogs: 8-N3-2'-O-propargyladenosine and 8-N3-2'-O-propargyl-cADPR.

A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2'-O-propargyl-ether) were compactly combined within the structure of the adenosine nucleoside itself. We synthesized 8-N3-2'-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5'-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N3-2'-O-propargyl-NAD. This NAD analog was recognized by Aplysia californica ADP-ribosyl cyclase and enzymatically cyclized producing 8-N3-2'-O-propargyl cyclic ADP-ribose. Photo-clickable cyclic-ADP-ribose analog was envisioned as a probe to label cyclic ADP-ribose binding proteins. The monofunctional 8-N3-cADPR has previously been shown to be an antagonist of cADPR-induced calcium release [T.F. Walseth et. al., J. Biol. Chem (1993) 268, 26686-26691]. 2'-O-propargyl-cADPR was recognized as an agonist which elicited Ca2+ release when added at low concentration to sea urchin egg homogenates. The bifunctional 8-N3-2'-O-propargyl cyclic ADP-ribose did not elicit Ca2+ release at low concentration or impact cyclic ADP-ribose mediated Ca2+ release either when added to sea urchin egg homogenates or when microinjected into cultured human U2OS cells. The photo-clickable adenosine will none-the-less be a useful scaffold for synthesizing photo-clickable probes for identifying proteins that interact with a variety of adenosine nucleotides.

Synthesis and Evaluation of PPARδ Agonists That Promote Osteogenesis in a Human Mesenchymal Stem Cell Culture and in a Mouse Model of Human Osteoporosis.

We synthesized a directed library of compounds to explore the structure-activity relationships of peroxisome proliferator-activated receptor δ (PPARδ) activation relative to mesenchymal stem cell (MSC) osteogenesis. Our scaffold used para-substituted cinnamic acids as a polar headgroup, a heteroatom and heterocycle core connecting units, and substituted phenyl groups for the lipophilic tail. Compounds were screened for their ability to increase osteogenesis in MSCs, and the most promising were examined for subunit specificity using a quantitative PPAR transactivation assay. Six compounds were selected for in vivo studies in an ovariectomized mouse model of human postmenopausal osteoporosis. Four compounds improved bone density in vivo, with two (12d and 31a) having activity comparable to that of GW0742, a well-studied PPARδ-selective agonist. 31a (2-methyl-4-[N-methyl-N-[5-methylene-4-methyl-2-[4-(trifluoromethyl)phenyl]thiazole]]aminocinnamic acid) had the highest selectivity for PPARδ compared to other subtypes, its selectivity far exceeding that of GW0742. Our results confirm that PPARδ is a new drug target for possible treatment of osteoporosis via in situ manipulation of MSCs.

Highly sensitive UHPLC-MS/MS method for the simultaneous estimation of propafenone and its metabolites 5-hydroxypropafenone and N-depropylpropafenone on human dried blood spots technique and application to a pharmacokinetic study.

A highly sensitive, rapid and selective UHPLC-MS/MS method has been developed and validated for quantification of the propafenone (PF), 5-hydroxypropafenone (5-OHPF) and N-depropylpropafenone (N-DPF) on human dried blood spot (DBS). The assay procedure involves a solid-liquid extraction of PF, 5-OHPF and N-DPF and amlodipine (internal standard, I.S.) from dried human DBS cards using water and acetonitrile. The chromatographic resolution was achieved on a BEH C18 column using a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile with 0.1% formic acid at flow rate of 0.6mL/min. The UHPLC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Total run time of analysis was 1.1min and elution of PF, 5-OHPF, N-DPF and I.S. occurred at 0.69, 0.6, 0.68 and 0.73min, respectively. A detailed method validation was performed as per the regulatory guidelines and the standard curves found to be linear in the range of 5.11-1000ng/mL for PF and 5-OHPF and 0.51-100ng/mL for N-DPF with a correlation coefficient of ≥0.99 for all the drugs. The intra- and inter-day accuracies were in the range of 95.6-107 and 93.5-103; 93.4-106 and 96.3-107 and 87.9-103 and 96.5-102%, for PF, 5-OHPF and N-DPF, respectively. The intra- and inter-day precisions were in the range of 2.50-5.52 and 3.38-5.18; 2.16-6.34 and 3.23-4.94 and 2.63-7.55 and 1.56-10.2%, for PF, 5-OHPF and N-DPF, respectively. The validated assay method was successfully applied to a pharmacokinetic study in humans. The key pharmacokinetic parameters AUC0-∞ and Cmax were 6057±1526, 2002±515 and 525±202 ng*h/mL and 653±183, 295±37.5 and 68.4±13.6ng/mL for PF, 5-OHPF and N-DPF, respectively.