A Radioactivity-mass Spectrometry Calibration Method Coupled with Biosynthesis to Generate a Metabolite Standard for Enzyme Kinetics Studies.

In early stages of drug development, the absence of authentic metabolite standards often results in semi-quantitative measurements of metabolite formation in reaction phenotyping studies using mass spectrometry (MS), leading to inaccuracies in the determination of enzyme kinetic parameters, such as the Michaelis constant (K). Moreover, it is impossible to ascertain the maximum rate of enzyme-catalyzed reactions (k or V). The use of radiolabeled parent compounds can circumvent this problem.

Carbon 14 and Carbon 13 Syntheses of Velagliflozin.

Velagliflozin is the active ingredient of the first oral liquid medication approved by the Food and Drug Administration for the treatment of diabetes in cats. This compound belongs to the known class of sodium-glucose cotransporter 2 inhibitors approved to treat diabetes in human. Here, we report the detailed synthesis of velagliflozin labeled with carbon 14 and carbon 13.

Stable isotope synthesis of glycine transporter 1 inhibitor Iclepertin (BI 425809) and its major metabolites.

Stable isotope labeled Iclepertin (BI 425809, 1) and its major metabolites are needed as internal standards in bioanalytical studies. BI 425809 consists of two main building blocks, 5-methylsulfonyl-2-[(1R)-2,2,2-trifluoro-1-methyl-ethoxy]benzoic acid (2) and 3-[(1R,5R)-3-azabicyclo[3.1.

Carbon 14 and stable isotope synthesis of two potent and selective phosphodiesterase type 4 inhibitors.

(R)-2-(4-(Benzo[d]oxazol-2-yl)piperazin-1-yl)-4-((tetrahydro-2H-pyran-4-yl)amino)-6,7-dihydrothieno[3,2-d]pyrimidine 5-oxide (1) and (R)-2-(4-(4-chlorophenoxy)piperidin-1-yl)-4-((tetrahydro-2H-pyran-4-yl)amino)-6,7-dihydrothieno[3,2-d]pyrimidine 5-oxide (2) are two potent and selective inhibitors of phosphodiesterase type 4 (PDE4). In this manuscript, we report the detailed synthesis of these two compounds labeled with carbon 14 and with stable isotopes. The core (R)-4-((tetrahydro-2H-pyran-4-yl)amino)-6,7-dihydrothieno[3,2-d]pyrimidine 5-oxide is common in both inhibitors.

Carbon 14 synthesis of glycine transporter 1 inhibitor Iclepertin (BI 425809) and its major metabolites.

Carbon 14 labeled Iclepertin (BI 425809, 1) and its major metabolites were needed for ADME and several other studies necessary for the advancement of this drug candidate in clinical trials. Iclepertin is composed of two main chemical blocks, (R)-5-(methylsulfonyl)-2-([1,1,1-trifluoropropan-2-yl]oxy)benzoic acid (2), and 3-[(1R,5R)-3-azabicyclo[3.1.

Characterization of Divergent Metabolic Pathways in Elucidating an Unexpected, Slow-Forming, and Long Half-Life Major Metabolite of Iclepertin.

Purpose: After single oral dosing of the glycine reuptake transporter (GlyT1) inhibitor, iclepertin (BI 425809), a single major circulating metabolite, M530a, was identified. However, upon multiple dosing, a second major metabolite, M232, was observed with exposure levels ~ twofold higher than M530a. Studies were conducted to characterize the metabolic pathways and enzymes responsible for formation of both major human metabolites.

Synthesis of BI 894416 and BI 1342561, two potent and selective spleen tyrosine kinase inhibitors, labeled with carbon 14 and with deuterium.

(R)-4-((R)-1-((6-(1-[tert-butyl]-1H-pyrazol-4-yl)-2-methyl-2H-pyrazolo[3,4-d]pyridin-4-yl)oxy)ethyl)pyrrolidin-2-one (BI 894416, 1) and (R)-4-((R)-1-((6-(1-[tert-butyl]-1H-pyrazol-4-yl)-2,3-dimethyl-2H-indazol-4-yl)oxy)ethyl)pyrrolidin-2-one (BI 1342561, 2) are two new potent and selective spleen tyrosine kinase inhibitors developed to treat severe asthma. Both compounds have similar structures and they differ only in the bicyclic moiety 2-methyl-2H-pyrazolo[4,3-c]pyridine in 1 versus 2,3-dimethyl-2H-indazole in 2. In the carbon 14 synthesis, 1-(1-[tert-butyl]-1H-pyrazol-4-yl)ethan-1-one-1- C ([ C]-8) was prepared from the cyanation of 4-bromopyrazole using zinc [ C]cyanide followed by methyl lithium addition on the nitrile group.

Synthesis of stable isotope-labelled firocoxib.

Firocoxib (ML-1,785,713) is a nonsteroidal, potent, and selective COX-2 inhibitor, approved for the control of pain and inflammation associated with osteoarthritis in dogs and horses, as well as to control postoperative pain and inflammation in dogs. We employed a six-step synthesis to prepare firocoxib-[ C ] in an overall yield of 35% from the commercially available bromobenzene-[ C ]. The synthetic route involved the preparation of the key intermediate phenyl- C -methyl sulfide using cesium carbonate and S-methylthiourea sulfate under transition-metal free conditions.

Synthesis of highly potent lymphocyte function-associated antigen-1 antagonists labeled with carbon-14 and with stable isotopes, part 3.

The drug candidates (2) and (3) are highly potent LFA-1 inhibitors. They were efficiently prepared labeled with carbon-14 using a palladium-catalyzed carboxylation of an iodo-precursor (5) and sodium formate- C to afford acid [ C]-(6), which was coupled via an amide bond to chiral amines (7) and (8) in 52% and 48% overall yield, respectively, and with specific activities higher than 56 mCi/mmol and radiochemical purities of 99%. For stable isotopes synthesis, the amine [ H ]-(7) was synthesized in three steps from 2-cyanopyridine- H using Kulinkovich-Szymonik aminocyclopropanation, followed by coupling to L-alanine-2,3,3,3- H -N-t-BOC, and then removal of the BOC-protecting group.

Potent and selective CC chemokine receptor 1 antagonists labeled with carbon-13, carbon-14, and tritium.

1-(4-Fluorophenyl)-1H-pyrazolo[3,4-c]pyridine-4-carboxylic acid (2-methanesulfonyl-pyridin-4-ylmethyl)-amide (1) and its analogs (2) and (3) are potent CCR1 antagonists intended for the treatment of rheumatoid arthritis. The detailed syntheses of these 3 compounds labeled with carbon-13 as well as the preparation of (1) and (2) labeled with carbon-14, and (1) labeled with tritium, are described.

Potent and selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 labeled with carbon-13 and carbon-14.

(S)-6-(2-Hydroxy-2-methylpropyl)-3-((S)-1-(4-(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl)ethyl)-6-phenyl-1,3-oxazinan-2-one (1) and (4aR,9aS)-1-(1H-benzo[d]midazole-5-carbonyl)-2,3,4,4a,9,9a-hexahydro-1-H-indeno[2,1-b]pyridine-6-carbonitrile hydrochloride (2) are potent and selective inhibitor of 11β-hydroxysteroid dehydrogenase type 1 enzyme. These 2 drug candidates developed for the treatment of type-2 diabetes were prepared labeled with carbon-13 and carbon-14 to enable drug metabolism, pharmacokinetics, bioanalytical, and other studies. In the carbon-13 synthesis, benzoic- C acid was converted in 7 steps and in 16% overall yield to [ C ]-(1).

Buscopan labeled with carbon-14 and deuterium.

Hyosine butyl bromide, the active ingredient in Buscopan, is an anticholinergic and antimuscarinic drug used to treat pain and discomfort caused by abdominal cramps. A straightforward synthesis of carbon-14- and deuterium-labeled Buscopan was developed using scopolamine, n-butyl-1- C bromide, and n-butyl- H bromide, respectively. In a second carbon-14 synthesis, the radioactive carbon was incorporated in the tropic acid moiety to follow its metabolism.

Carbon-13 and carbon-14 labeled dabigatran etexilate and tritium labeled dabigatran.

Dabigatran etexilate or pradaxa, a novel oral anticoagulant, is a reversible, competitive, direct thrombin inhibitor. It is used to prevent strokes in patients with atrial fibrillation and the formation of blood clots in the veins (deep venous thrombosis) in adults who have had an operation to replace a hip or a knee. Pradaxa is the only novel oral anticoagulant available with both proven superiority to warfarin and a specific reversal agent for use in rare emergency situations.

A potent IκB kinase-β inhibitor labeled with carbon-14 and deuterium.

3-Amino-4-(1,1-difluoro-propyl)-6-(4-methanesulfonyl-piperidin-1-yl)-thieno[2,3-b]pyridine-2-carboxylic acid amide (1) is a potent IκB Kinase-β (IKK-β) inhibitor. The efficient preparations of this compound labeled with carbon-14 and deuterium are described. The carbon-14 synthesis was accomplished in six radiochemical steps in 25% overall yield.

Synthesis of two potent glucocorticoid receptor agonists labeled with carbon-14 and stable isotopes.

Two potent glucocorticoid receptor agonists were prepared labeled with carbon-14 and with stable isotopes to perform drug metabolism, pharmacokinetics, and bioanalytical studies. Carbon-14 labeled (1) was obtained from an enantiopure alkyne (5) via a Sonogashira coupling to a previously reported 5-amino-4-iodo-[2-(14)C]pyrimidine [(14)C]-(6), followed by a base-mediated cyclization (1) in 72% overall radiochemical yield. Carbon-14 labeled (2) was prepared in five steps employing a key benzoic acid intermediate [(14)C]-(13), which was synthesized in one pot from enolization of trifluoromethylketone (12), followed by bromine-magnesium exchange and then electrophile trapping reaction with [(14)C]-carbon dioxide.

A novel five-lipoxygenase activity protein inhibitor labeled with carbon-14 and deuterium.

2-[4-(3-{(1R)-1-[4-(2-Aminopyrimidin-5-yl)phenyl]-1-cyclopropylethyl}-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl]-N,N-dimethylacetamide (1), is a novel and selective five-lipoxygenase activity protein (FLAP) inhibitor with excellent pharmacokinetics properties. The availability of a key chiral intermediate allowed the synthesis of [(14) C]-(1) in six radiochemical steps and in 47% overall radiochemical yield with a specific activity of 51 mCi/mmol using carbon-14 zinc cyanide. 2-Chloro-N,N-dimethyl-(2)H6-acetamide was prepared and condensed with a penultimate intermediate to give [(2)H6]-(1) in very high yield and in more than 99% isotopic enrichment.

Synthesis of deleobuvir, a potent hepatitis C virus polymerase inhibitor, and its major metabolites labeled with carbon-13 and carbon-14.

Deleobuvir, (2E)-3-(2-{1-[2-(5-bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl-1H-indole-6-carboxamido]cyclobutyl}-1-methyl-1H-benzimidazol-6-yl)prop-2-enoic acid (1), is a non-nucleoside, potent, and selective inhibitor of hepatitis C virus NS5B polymerase. Herein, we describe the detailed synthesis of this compound labeled with carbon-13 and carbon-14. The synthesis of its three major metabolites, namely, the reduced double bond metabolite (2) and the acyl glucuronide derivatives of (1) and (2), is also reported.

Synthesis of empagliflozin, a novel and selective sodium-glucose co-transporter-2 inhibitor, labeled with carbon-14 and carbon-13.

Empagliflozin, (2S,3R,4R,5S,6R)-2-[4-chloro-3-[[4-[(3S)-oxolan-3-yl]oxyphenyl]methyl]phenyl]-6-(hydroxymethyl)oxane-3,4,5-triol was recently approved by the FDA for the treatment of chronic type 2 diabetes mellitus. Herein, we report the synthesis of carbon-13 and carbon-14 labeled empagliflozin. Carbon-13 labeled empagliflozin was prepared in five steps and in 34% overall chemical yield starting from the commercially available α-D-glucose-[(13)C6].

Mass balance, metabolite profile, and in vitro-in vivo comparison of clearance pathways of deleobuvir, a hepatitis C virus polymerase inhibitor.

The pharmacokinetics, mass balance, and metabolism of deleobuvir, a hepatitis C virus (HCV) polymerase inhibitor, were assessed in healthy subjects following a single oral dose of 800 mg of [(14)C]deleobuvir (100 μCi). The overall recovery of radioactivity was 95.2%, with 95.

Hepatitis C virus serine protease: synthesis of radioactive and stable isotope-labeled potent inhibitors.

Drug candidates labeled with radioactive and stable isotopes are required for absorption, distribution, metabolism, and excretion (ADME) studies, pharmacokinetics, autoradiography, bioanalytical, and other research activities. The findings from these studies are crucial in the development of a drug candidate and its approval for human use. Herein, we report the synthesis of potent and selective hepatitis C virus serine protease inhibitors related to BILN 2061 and BI 201335 labeled with radioactive and stable isotopes.

Mass balance and metabolite profiling of steady-state faldaprevir, a hepatitis C virus NS3/4 protease inhibitor, in healthy male subjects.

The pharmacokinetics, mass balance, and metabolite profiles of faldaprevir, a selective peptide-mimetic hepatitis C virus NS3/NS4 protease inhibitor, were assessed at steady state in 7 healthy male subjects. Subjects received oral doses of 480 mg faldaprevir on day 1, followed by 240 mg faldaprevir on days 2 to 8 and 10 to 15. [14C]faldaprevir (240 mg containing 100 μCi) was administered on day 9.

Sodium-proton exchanger isoform-1: synthesis of a potent inhibitor labeled with deuterium and carbon-14.

Sodium-proton exchangers, NHEs are plasma membrane proteins that are essential in the regulation of intracellular pH of the myocardium. There are nine known variously expressed isoforms of NHEs with NHE-1 being the predominant isoform in the heart. N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine (1) is a potent NHE 1-inhibitor with good pharmacokinetics.

Synthesis of a potent cathepsin s inhibitor labeled with deuterium and carbon-14.

Morpholine-4-carboxylic acid {(S)-1-[4-cyano-1-(3-morpholin-4-yl-propyl)-piperidin-4-ylcarbamoyl]-4,4- dimethyl-hexyl}-amide, (1) is a potent reversible and selective cathepsin S inhibitor. Deuterium labeled (1) was prepared in four steps in 62% overall yield from [2H8]- morpholine and chiral acid (6). Carbon-14 labeled (1) was obtained in two steps using sodium [14C]-cyanide in a modified Strecker reaction followed by amide bond formation with acid (6) in 74% overall radiochemical yield.