Ashok Chaudhary

Fluoroolefins as amide bond mimics in dipeptidyl peptidase IV inhibitors

The synthesis, selectivity, rat pharmacokinetic profile, and drug metabolism profiles of a series of potent fluoroolefin-derived DPP-4 inhibitors (4) are reported. A radiolabeled fluoroolefin 33 was shown to possess a high propensity to form reactive metabolites, thus revealing a potential liability for this class of DPP-4 inhibitors.

In vitro characterization of [3H]MethoxyPyEP, an mGluR5 selective radioligand

We have characterized the in vitro properties of 3-[3H]methoxy-5-(pyridin-2-ylethynyl)pyridine ([3H]MethoxyPyEP), an analogue of the mGluR(5) receptor subtype antagonist MPEP [2-methyl-6-(phenylethynyl)-pyridine], in rat tissue preparations using tissue homogenates and autoradiography. Binding of [3H]MethoxyPyEP to rat cortex, hippocampus, thalamus and cerebellum membrane preparations revealed saturable, high affinity binding (3.4 +/- 0.4 nM, n = 4 in rat cortex) to a single population of receptors in all regions studied except for cerebellum. Binding was found to be relatively insensitive to pH and insensitive to DTT. High concentrations of NEM both reduce receptor concentration and binding affinity for the radioligand. In time-course studies at room temperature k(on) and k(off) were determined as 2.9 x 10(7) M(-1) min(-1) and 0.11 min(-1) respectively. The rank order of affinities, as assessed by equilibrium competition studies, of a variety of ligands suggested binding of the radioligand selectively to mGluR5 (MPEP > trans-azetidine-2,4-dicarboxylic acid congruent with (S)-4-carboxyphenylglycine congruent with (+)MK801 congruent with CP-101,606 congruent with clozapine congruent with atropine congruent with ketanserin congruent with yohimbine congruent with benoxathian). Autoradiographic studies with [3H]MethoxyPyEP showed that binding was regioselective, with high density of binding in caudate and hippocampus, intermediate binding in thalamus and very low density in the cerebellum. These data show that [3H]MethoxyPyEP is a high affinity radioligand useful for the in vitro study of mGluR5 receptor distribution and pharmacologic properties in brain.

High-Throughput Radiometric CYP2C19 Inhibition Assay Using Tritiated (S)-Mephenytoin

A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C19 in human liver microsomes is described. The new assay, which does not require high-performance liquid chromatography (HPLC) separation or mass spectrometric detection, is based on the release of tritium as tritiated water that occurs upon CYP2C19-mediated 4′-hydroxylation of (S)-mephenytoin labeled with tritium in the 4′ position. Because this reaction is subject to an NIH shift, tritium was also introduced into the 3′- and 5′-positions of the tracer to enhance formation of a tritiated water product. Tritiated water was separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent and sensitive to CYP2C19 inhibitors. IC50 values for 15 diverse drugs differed less than 2.5-fold from those determined by quantification of the unlabeled 4′-hydroxy-(S)-mephenytoin product, using HPLC coupled to mass spectrometric detection. All of the steps of the new assay, namely incubation, product separation, and radioactivity counting, are performed in a 96-well format and can be automated. This assay represents a non-HPLC, high-throughput version of the classic (S)-mephenytoin 4′-hydroxylation assay, which is the most widely used method to assess the potential for CYP2C19 inhibition of new chemical entities.

A nonpeptide oxytocin receptor antagonist radioligand highly selective for human receptors.

A novel, potent nonpeptide oxytocin receptor antagonist (1-(1-(2-(2,2,2-trifluoroethoxy)-4-(1-methylsulfonyl-4-piperidinyloxy) phenylacetyl)-4-piperidinyl)-3,4-dihydro-2(1H)-quinolinone) has been identified that can be labeled to high specific activity with [35S]. In binding studies, this compound exhibits sub-nanomolar affinity and a high degree of selectivity (900-1800-fold) for human oxytocin receptors compared to human vasopressin receptors. This compound appears suitable for studying the pharmacology of oxytocin receptors in human and nonhuman primate tissues, for which there is currently a paucity of highly selective tools. It may also be useful as a nonlabeled competitor or as a radioligand in autoradiographic studies of oxytocin receptor localization in these tissues.

Improved synthesis of stable isotope labeled and carbon-14 labeled (S)-(−)-3-[3-(methylsulfonyl) phenyl]-1-propylpiperidine hydrochloride; (−)-OSU-6162

(−)OSU-6162, ((S)-(−)-3-[3-(methylsulfonyl)phenyl]-1-propylpiperidine hydrochloride), 1, it is dopamine autoreceptor antagonist with potential atypical antipsychotic properties. The synthesis of stable labeled and carbon-14 labeled (−)-OSU-6162 was achieved by an alkylation of the anion of (S)-3-(1-propylpiperdine-3-yl)-benzenethiol, 5, with either [13C, 2H3]methyl iodide or [14C]methyl iodide to provide the corresponding methyl sulfide. Selective oxidation of the methyl sulfide to the sulfone and conversion to its hydrochloride salt gave either stable isotope labeled (−)-OSU-6162 or carbon-14 labeled (−)-OSU-6162. The overall radiochemical yield was 36% with chemical and radiochemical purity exceeding 99% by HPLC analysis. The precursor thiol (5) was provided by a modified synthetic route from commercially available (S)-(−)-3-(3-(hydroxyphenyl)-1-propylpiperidine); ((S)-(−)-PPP), 2, in three steps via the triisopropylsilanethiolate intermediate (7). Copyright