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[3.1.0]hexan-5-yl]-5-(trifluoromethyl)isoxazole (3) linked to each other via an amide bond. We used fluoro[13 C6 ]benzene as the starting material in the preparation of [13 C6 ]-2. This intermediate was then employed to access carbon 13 labeled Iclepertin ([13 C6 ]-1) and other metabolites. The major metabolite BI 761036 (6), which resulted from cytochrome P450 oxidation and amide hydrolysis of BI 425809, was prepared labeled with carbon 13 and nitrogen 15 via two synthetic routes. In the first route, diethyl [13 C3 ]malonate, [13 C]methyl iodide, and hydroxyl[15 N]amine were used to provide [13 C4 ,15 N]-BI 761036 ([13 C4 ,15 N]-6a) in 13 steps in 6% overall yield, whereas in the second route, [13 C3 ]propargyl alcohol, potassium [13 C]cyanide, and [15 N]ammonia were used to furnish [13 C4 ,15 N]-BI 761036 ([13 C4 ,15 N]-6b) in 11 steps in 1% overall yield. The detailed stable isotope synthesis of 1 and its major metabolites is described.

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. In the radioactive synthesis, the carbon 14 atom was introduced in the benzoxazole moiety using [14 C]carbon disulfide to obtain [14 C]-1 in five steps at a 55% overall yield. [14 C]Urea was used to incorporate the carbon 14 atom in two steps in the dihydrothieno[3,2-d]pyrimidine intermediate, which was then transformed in four more steps to [14 C]-2 at a 30% overall yield. Both compounds were isolated with specific activities higher than 54 mCi/mmol, radio- and chemical-purities higher than 99%, and with excellent enantiomeric excess. In the stable isotope synthesis, [2 H8 ]piperazine was used to prepare [2 H8 ]-1 in three steps in 72% overall yield, while [13 C6 ]phenol was used to prepare [13 C6 ]-2 in four steps in 18% overall yield.

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[3.1.0]hexan-5-yl]-5-(trifluoromethyl)isoxazole (3) linked to each other via an amide bond. In the first synthesis of carbon 14 labeled 1, 2-fluorobenzoic acid, carboxyl-14 C was converted to [14 C]-2 in three steps and then coupled to 3 to provide [14 C]-1a in 45% overall yield. In the second synthesis, [14 C]-3 was prepared in six radioactive steps and coupled to the acid 2 to furnish [14 C]-1b in 20% overall yield. Both synthetic routes provided [14 C]-1a and [14 C]-1b with specific activities higher than 53 mCi/mmol and radiochemical, chemical, and enantiomeric purities above 98%. Two major metabolites of 1, BI 761036 and BI 758790, were also prepared labeled with carbon 14 using intermediates already available from the synthesis of [14 C]-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. METHODS: In vitro studies were conducted with human and recombinant enzyme sources and enzyme-selective inhibitors. The production of iclepertin metabolites was monitored by LC-MS/MS. RESULTS: Iclepertin undergoes rapid oxidation to a putative carbinolamide that spontaneously opens to an aldehyde, M528, which then undergoes reduction by carbonyl reductase to the primary alcohol, M530a. However, the carbinolamide can also undergo a much slower oxidation by CYP3A to form an unstable imide metabolite, M526, that is subsequently hydrolyzed by a plasma amidase to form M232. This difference in rate of metabolism of the carbinolamine explains why high levels of the M232 metabolite were not observed in vitro and in single dose studies in humans, but were observed in longer-term multiple dose studies. CONCLUSIONS: The long half-life iclepertin metabolite M232 is formed from a common carbinolamine intermediate, that is also a precursor of M530a. However, the formation of M232 occurs much more slowly, likely contributing to its extensive exposure in vivo. These results highlight the need to employ adequate clinical study sampling periods and rigorous characterization of unexpected metabolites, especially when such metabolites are categorized as major, thus requiring safety assessment.

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-1-14 C ([14 C]-8) was prepared from the cyanation of 4-bromopyrazole using zinc [14 C]cyanide followed by methyl lithium addition on the nitrile group. The enolate of [14 C]-8 was then used to access these two bicyclic moieties via pyrano-pyrazoles [14 C]-11 and [14 C]-12, which were further transformed in few more steps to [14 C]-(1) and [14 C]-2. Both inhibitors contain a tert-butyl group. Introducing tert-butyl-d9 will not only provide internal standards for bioanalytical studies, but it is also expected to slow down the metabolism of these two compounds. Most of the metabolites of compound 1, for example, are the result of tert-butyl oxidation, like alcohol 3, acid 4, and the further N-demethylation of 4 to 5. The detailed preparation of these deuterium-labeled metabolites is also described.

Synthesis of beta-site amyloid precursor protein-cleaving enzyme 1 inhibitors BI 1147560 and BI 1181181 labeled with carbon-14 and deuterium.

The generation of amyloid beta peptides that aggregate in the brain is believed to play a major role in Alzheimer's disease. In theory, the inhibition of beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1), which catalyzes the initial rate-limiting step in amyloid beta production, may slow or stop Alzheimer's disease. Herein, we report the preparation of two potent BACE1 inhibitors, BI 1147560 (1) and BI 1181181 (2), labeled with carbon-14 and with deuterium. The use of advanced key chiral intermediates like 3 and 5 shortened the carbon-14 syntheses of these two compounds to five and six steps, respectively, and helped in preparing them with very high chemical purity and enantiomeric excess without deviating from the process chemistry route. For the deuterium synthesis, oxetan-3-ylmethanamine [2 H6 ]-7 and 2-fluoro-2-methylpropan-1-amine [2 H6 ]-9 were prepared then used with the chiral intermediate 5 to furnish deuterium labeled 1 and 2, respectively.

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-[13 C6 ] in an overall yield of 35% from the commercially available bromobenzene-[13 C6 ]. The synthetic route involved the preparation of the key intermediate phenyl-13 C6 -methyl sulfide using cesium carbonate and S-methylthiourea sulfate under transition-metal free conditions. A two-step preparation of firocoxib-[13 C,2 H3 ] via the sulfinic acid derivative of firocoxib and methyl iodide-[13 C,2 H3 ] using the procedure of Gauthier and Yoshikawa was first undertaken. However, the deuterium atoms of the methyl sulfone undergo extensive exchange in aqueous media even at neutral pH. The isotope-labelled firocoxib is intended as an internal standard for bioanalyses of firocoxib from biological matrices.

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-14 C to afford acid [14 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 [2 H8 ]-(7) was synthesized in three steps from 2-cyanopyridine-2 H4 using Kulinkovich-Szymonik aminocyclopropanation, followed by coupling to L-alanine-2,3,3,3-2 H4 -N-t-BOC, and then removal of the BOC-protecting group. Amide bond formation with acid (6) gave [2 H8 ]-(2) in 36% overall yield. The amine [13 C4 ,15 N]-(8) was obtained in two steps using L-threonine-14 C4 ,15 N and then coupled to acid [13 C]-(6) to give [13 C5 ,15 N]-(3) in 56% overall yield.

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 11ß-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 11ß-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-13 C6 acid was converted in 7 steps and in 16% overall yield to [13 C6 ]-(1). Aniline-13 C6 was converted in 7 steps to 1H-benzimidazole-1-2,3,4,5,6-13 C6 -5-carboxylic acid and then coupled to a tricyclic chiral indenopiperidine to afford [13 C6 ]-(2) in 19% overall yield. The carbon-14 labeled (1) was prepared efficiently in 2 radioactive steps in 41% overall yield from an advanced intermediate using carbon-14 labeled methyl magnesium iodide and Suzuki-Miyaura cross coupling via in situ boronate formation. As for the synthesis of [14 C]-(2), 1H-benzimidazole-5-carboxylic-14 C acid was first prepared in 4 steps using potassium cyanide-14 C, then coupled to the chiral indenopiperidine using amide bond formation conditions in 26% overall yield.

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-1-14 C bromide, and n-butyl-2 H9 bromide, respectively. In a second carbon-14 synthesis, the radioactive carbon was incorporated in the tropic acid moiety to follow its metabolism. Herein, we describe the detailed preparations of carbon-14- and deuterium-labeled Buscopan.

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. The detailed description of the synthesis of carbon-13 and carbon-14 labeled dabigatran etexilate, and tritium labeled dabigatran is described. The synthesis of carbon-13 dabigatran etexilate was accomplished in eight steps and in 6% overall yield starting from aniline-13 C6 . Ethyl bromoacetate-1-14 C was the reagent of choice in the synthesis of carbon-14 labeled dabigatran etexilate in six steps and 17% overall yield. Tritium labeled dabigatran was prepared using either direct tritium incorporation under Crabtree's catalytic conditions or tritium-dehalogenation of a diiodo-precursor of dabigatran.

A potent IκB kinase-ß 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-ß (IKK-ß) 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. The key transformations were the modified Guareschi-Thorpe condensation of 2-cyano-(14) C-acetamide and a keto-ester followed by chlorination to 2,6-dichloropyridine derivative in one pot. The isolated dichloropyridine was then converted in three steps in one pot to [(14) C]-(1). The carbon-14 labeled (1) was isolated with a specific activity of 54.3 mCi/mmol and radiochemical purity of 99.8%. The deuterium labeled (1) was obtained in eight steps and in 57% overall chemical yield using 4-hydroxypiperidine-2,2,3,3,4,5,5,6,6-(2) H9 . The final three steps of this synthesis were run in one pot.

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 chiral auxiliary (S)-1-(4-methoxyphenyl)ethylamine was then coupled to this acid to give [(14)C]-(15). Propargylation and separation of diastereoisomers by crystallizations gave the desired diastereomer [(14)C]-(17) in 34% yield. Sonogashira coupling to iodopyridine (10) followed by cyclization to the azaindole [(14)C]-(18) and finally removal of the chiral auxiliary gave [(14)C]-(2) in 7% overall yield. For stable isotope syntheses, [(13)C6]-(1) was obtained in three steps using [(13)C4]-(6) and trimethylsilylacetylene-[(13)C2] in 26% yield, while [(2)H5]-(2) was obtained by first preparing the iodopyridine [(2)H5]-(10) in five steps. Then, Sonogashira coupling to chiral alkyne (24) and cyclization gave [(2)H5]-(2) in 42% overall yield.

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. Aniline-(13) C6 was the starting material to prepare butyl (E)-3-(3-methylamino-4-nitrophenyl-(13) C6 )acrylate [(13) C6 ]-(11) in six steps. This intermediate was then used to obtain [(13) C6 ]-(1) and [(13) C6 ]-(2) in five and four more steps, respectively. For the radioactive synthesis, potassium cyanide-(14) C was used to prepare 1-cylobutylaminoacid [(14) C]-(23) via Buchrer-Bergs reaction. The carbonyl chloride of this acid was then used to access both [(14) C]-(1) and [(14) C]-(2) in four steps. The acyl glucuronide derivatives [(13) C6 ]-(3), [(13) C6 ]-(4) and [(14) C]-(3) were synthesized in three steps from the acids [(13) C6 ]-(1), [(13) C6 ]-(2) and [(14) C]-(1) using known procedures.

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 95.1% recovered from feces. Deleobuvir had moderate to high clearance, and the half-life of deleobuvir and radioactivity in plasma were ∼ 3 h, indicating that there were no metabolites with half-lives significantly longer than that of the parent. The most frequently reported adverse events (in 6 of 12 subjects) were gastrointestinal disorders. Two major metabolites of deleobuvir were identified in plasma: an acyl glucuronide and an alkene reduction metabolite formed in the gastrointestinal (GI) tract by gut bacteria (CD 6168), representing ∼ 20% and 15% of the total drug-related material, respectively. Deleobuvir and CD 6168 were the main components in the fecal samples, each representing ∼ 30 to 35% of the dose. The majority of the remaining radioactivity found in the fecal samples (∼ 21% of the dose) was accounted for by three metabolites in which deleobuvir underwent both alkene reduction and monohydroxylation. In fresh human hepatocytes that form biliary canaliculi in sandwich cultures, the biliary excretion for these excretory metabolites was markedly higher than that for deleobuvir and CD 6168, implying that rapid biliary elimination upon hepatic formation may underlie the absence of these metabolites in circulation. The low in vitro clearance was not predictive of the observed in vivo clearance, likely because major deleobuvir biotransformation occurred by non-CYP450-mediated enzymes that are not well represented in hepatocyte-based in vitro models.

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]. For the radiosynthesis, the carbon-14 atom was introduced in three different positions of the molecule. In the first synthesis, Carbon-14 D-(+)-gluconic acid δ-lactone was used to prepare specifically labeled empagliflozin in carbon-1 of the sugar moiety in four steps and in 19% overall radiochemical yield. Carbon-14 labeled empagliflozin with the radioactive atom in the benzylic position was obtained in eight steps and in 7% overall radiochemical yield. In the last synthesis carbon-14 uniformly labeled phenol was used to give [(14)C]empagliflozin in eight steps and in 18% overall radiochemical yield. In all these radiosyntheses, the specific activities of the final compounds were higher than 53 mCi/mmol, and the radiochemical purities were above 98.5%.