Tapan Ray

Physiological and Molecular Characterization of Aristolochic Acid Transport by the Kidney

Consumption of herbal medicines derived from Aristolochia plants is associated with a progressive tubulointerstitial disease known as aristolochic acid (AA) nephropathy. The nephrotoxin produced naturally by these plants is AA-I, a nitrophenanthrene carboxylic acid that selectively targets the proximal tubule. This nephron segment is prone to toxic injury because of its role in secretory elimination of drugs and other xenobiotics. Here, we characterize the handling of AA-I by membrane transporters involved in renal organic anion transport. Uptake assays in heterologous expression systems identified murine organic anion transporters (mOat1, mOat2, and mOat3) as capable of mediating transport of AA-I. Kinetic analyses showed that all three transporters have an affinity for AA-I in the submicromolar range and thus are likely to operate at toxicologically relevant concentrations in vivo. Structure-activity relationships revealed that the carboxyl group is critical for high-affinity interaction of AA-I with mOat1, mOat2, and mOat3, whereas the nitro group is required only by mOat1. Furthermore, the 8-methoxy group, although essential for toxicity, was not requisite for transport. Mouse renal cortical slices avidly accumulated AA-I, achieving slice-to-medium concentration ratios >10. Uptake by slices was sensitive to known mOat1 and mOat3 substrates and the organic anion transport inhibitor probenecid, which also blocked the production of DNA adducts formed with reactive intracellular metabolites of AA-I. Taken together, these findings indicate that OAT family members mediate high-affinity transport of AA-I and may be involved in the site-selective toxicity and renal elimination of this nephrotoxin.

Palladium-catalyzed aryl cyanations with [14C]KCN: Synthesis of 14C-labelled fadrozole, a potent aromatase inhibitor

The potent aromatase inhibitor [14C]Fadrozole (1), was prepared in a single radiosynthetic step via a palladium(0)-catalyzed cyanation of the imidazole-containing aryl iodide 2b with [14C]KCN. Attempted preparation of 2b by metal-halogen interchange of the corresponding aryl bromide 2a with tert-butyl lithium followed by quenching with iodine afforded only the imidazole iodide 5 via disproportionation of the intermediate anion. The desired precursor was finally synthesized through a three-step sequence beginning with the alkylation of known imidazole derivative 8 with 4-iodobenzylbromide. This alkylation product was treated with thionyl chloride to convert a side chain hydroxyl to its corresponding primary chloride 10. Cyclization of chloride 10 using LDA/TMEDA gave the desired aryl iodide 2b. While initial attempts at the palladium(0)-catalyzed cyanation of 2b with unlabelled KCN in THF at reflux gave modest yields of Fadrozole, the reaction with [14C]KCN afforded only trace amounts of [14C]Fadrozole. By including Copper(I) iodide as a co-catalyst and using deoxygenated THF, the palladium(0)-catalyzed cyanation of 2b gave [14C]Fadrozole in 39% radiochemical yield with >99% radiochemical purity. Copyright

Utilization of stable isotope labeling to facilitate the identification of polar metabolites of KAF156, an antimalarial agent

Identification of polar metabolites of drug candidates during development is often challenging. Several prominent polar metabolites of 2-amino-1-(2-(4-fluorophenyl)-3-((4-fluorophenyl)amino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)ethanone ([14C]KAF156), an antimalarial agent, were detected in rat urine from an absorption, distribution, metabolism, and excretion study but could not be characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) because of low ionization efficiency. In such instances, a strategy often chosen by investigators is to use a radiolabeled compound with high specific activity, having an isotopic mass ratio (i.e., [12C]/[14C]) and mass difference that serve as the basis for a mass filter using accurate mass spectrometry. Unfortunately, [14C]KAF156-1 was uniformly labeled (n = 1–6) with the mass ratio of ∼0.1. This ratio was insufficient to be useful as a mass filter despite the high specific activity (120 μCi/mg). At this stage in development, stable isotope labeled [13C6]KAF156-1 was available as the internal standard for the quantification of KAF156. We were thus able to design an oral dose as a mixture of [14C]KAF156-1 (specific activity 3.65 μCi/mg) and [13C6]KAF156-1 with a mass ratio of [12C]/[13C6] as 0.9 and the mass difference as 6.0202. By using this mass filter strategy, four polar metabolites were successfully identified in rat urine. Subsequently, using a similar dual labeling approach, [14C]KAF156-2 and [13C2]KAF156-2 were synthesized to allow the detection of any putative polar metabolites that may have lost labeling during biotransformations using the previous [14C]KAF156-1. Three polar metabolites were thereby identified and M43, a less polar metabolite, was proposed as the key intermediate metabolite leading to the formation of a total of seven polar metabolites. Overall this dual labeling approach proved practical and valuable for the identification of polar metabolites by LC-MS/MS.

Proton exchange reactions in isotope chemistry (II) synthesis of stable isotope‐labeled LCQ908

The proton exchange reaction was applied to the preparation of stable isotope-labeled LCQ908. For this synthesis, a suitable intermediate with protons alpha to a carbonyl group was first subjected to the H-D exchange reaction; subsequent coupling of a carbonyl group with [(13)C2 ]triethyl phosphonoacetate, followed by hydrogenation and hydrolysis, led to the stable labeled compound. Incorporation of two carbon-13 atoms in the molecule eliminated the presence of undesired M+0.