Enzymatic synthesis of phenylpyruvic acid labeled with deuterium,tritium, and carbon‐14

The synthesis of isotopomers of phenylpyruvic acid, PPA, selectively labeled with hydrogen isotopes in the 3‐position of the side‐chain is reported. Three deuterium or tritium labeled isotopomers of L‐phenylalanine, L‐Phe, i.e. [(3S)‐²H]‐L‐, [(3S)‐³H]‐L‐, and doubly labeled [(3S)‐²H/³H]‐L‐Phe were synthesized using the enzyme phenylalanine ammonia lyase (EC 4.3.1.5). In the second step these isotopomers of L‐Phe were converted into [(3S)‐²H], [(3S)‐³H]‐, and [(3S)‐²H/³H]‐isotopomers of PPA using the enzyme L‐phenylalanine dedydrogenase (EC 1.4.1.20). The isotopomer of PPA labeled with ¹⁴C in carboxylic group, [1‐¹⁴C]‐PPA, was obtained in a two‐step enzymatic reaction using [1‐¹⁴C]‐cinnamic acid as the starting substrate. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of selectively labeled histidine and its methylderivatives with deuterium,tritium, and carbon‐14

Isotopologues of l ‐histidine and its N‐methylderivatives labeled with deuterium and tritium at the 5‐position in the imidazole ring were obtained using the isotope exchange method. The deuterium‐labeled isotopologues [5‐²H]‐l ‐histidine, [5‐²H]‐N^τ‐methyl‐l ‐histidine, [5‐²H]‐N^π‐methyl‐l ‐histidine, and [2,5‐²H₂]‐l ‐histidine were synthesized by isotope exchange method carried out in a fully deuterated medium with. The same reaction conditions were applied to synthesize [5‐³H]‐N^τ‐methyl‐l ‐histidine, [5‐³H]‐N^π‐methyl‐l ‐histidine, and [5‐³H]‐l ‐histidine with specific activity of 2.0, 5.0, and 2.6 MBq/mmol, respectively. The N^π‐[methyl‐¹⁴C]‐histamine was obtained with specific activity of 0.23 MBq/mmol in a one‐step reaction by the direct methylation of histamine by [¹⁴C]iodomethane.     

A possible mechanism for hepatotoxicity induced by BIRB-796, an orally active p38 mitogen-activated protein kinase inhibitor

BIRB‐796, a selective inhibitor of p38 mitogen‐activated protein kinase, has entered clinical trials for the treatment of autoimmune diseases. Levels of alanine transaminase, a biomarker of hepatic toxicity in clinical pathology, were found to be increased in Crohn's disease patients treated with BIRB‐796. The purpose of the present study was to clarify the molecular mechanism(s) of this hepatotoxicity. A toxicogenomic analysis using a highly sensitive DNA chip, 3D‐Gene? Mouse Oligo chip 24k, indicated that BIRB‐796 treatment activated the nuclear factor (erythroid‐derived 2)‐like 2 signaling pathway, which plays a key role in the response to oxidative stress. A reactive intermediate of BIRB‐796 was detected by the glutathione‐trapping method using mouse and human liver microsomes. The production of this reactive metabolite in the liver may be one of the causes of BIRB‐796's hepatotoxicity. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis of deuterium‐, tritium‐, and carbon‐14‐labeled BILN2061, a potent hepatitis C virus protease inhibitor

Hepatitis C virus (HCV) serine protease is a target for antiviral therapy against HCV infection, a leading cause of liver transplantation in the US. BILN2061, (1S, 4R, 6S, 7Z, 14S, 18R)‐14‐cyclopentyloxycarbonylamino‐18‐[2‐(2‐isopropylamino‐thiazol‐4‐yl)‐7‐methoxyquinolin‐4‐yloxy]‐2,15‐dioxo‐3,16‐diazatricyclo[14.3.0.0^4,6]nonadec‐7‐ene‐4‐carboxylic acid, is a potent inhibitor of HCV and the first compound in this class of cyclic peptides in human trials. Here, we report the synthesis of deuterium‐labeled BILN2061 with isotopic enrichment of 99%, tritium‐labeled BILN2061 with a specific activity of 17.1 GBq/mmol, and carbon‐14‐labeled BILN2061 with a specific activity of 1.83 GBq/mmol. The isotopes were incorporated via a Hantzsch thiazole synthesis of labeled isopropyl thiourea and α‐bromoketone intermediate. The preparation of labeled isopropyl thiourea is reported. Copyright © 2005 John Wiley & Sons, Ltd.     

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‐1‐¹⁴C bromide, and n‐butyl‐²H₉ 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.     

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 [¹⁴C]‐(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‐²H₆‐acetamide was prepared and condensed with a penultimate intermediate to give [²H₆]‐(1) in very high yield and in more than 99% isotopic enrichment.     

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.     

The synthesis of a tritium,carbon‐14, and stable isotope‐labeled cathepsin C inhibitors

As part of a medicinal chemistry program aimed at developing a highly potent and selective cathepsin C inhibitor, tritium, carbon‐14, and stable isotope‐labeled materials were required. The synthesis of tritium‐labeled methanesulfonate 5 was achieved via catalytic tritiolysis of a chloro precursor, albeit at a low radiochemical purity of 67%. Tritium‐labeled AZD5248 was prepared via a 3‐stage synthesis, utilizing amide‐directed hydrogen isotope exchange. Carbon‐14 and stable isotope‐labeled AZD5248 were successfully prepared through modifications of the medicinal chemistry synthetic route, enabling the use of available labeled intermediates.     

Synthesis of herbicidal ZJ0273 labeled with tritium and carbon‐14

ZJ0273, propyl 4‐(2‐(4,6‐dimethoxypyrimidin‐2‐yloxy)benzylamino)benzoate, is a broad‐spectrum herbicidal ingredient used for weed control in oilseed rape in China. Two mono‐labeled ZJ0273, propyl 4‐(2‐(4,6‐dimethoxypyrimidin‐2‐yloxy)[phenyl‐3,4,5,6‐³H₄]benzylamino)benzoate (7) and propyl 4‐(2‐(4,6‐dimethoxy[4,6‐¹⁴C₂]pyrimidin‐2‐yloxy)benzylamino)benzoate (12), were synthesized separately from [2,3,4,5,6‐³H₅]phenol in a four‐step yield of 27% and from 4,6‐dichloro‐2‐(methylthio)[4,6‐¹⁴C₂]pyrimidine in a three‐step yield of 54%. In addition, two dual‐labeled analogues of ZJ0273 were prepared by homogeneously mixing tritium‐labeled ZJ0273 (7) in the benzyl ring separately with two carbon‐14‐labeled ZJ0273 (2, 12) in the benzoate ring and the pyrimidine ring. These labeled ZJ0273 could be used as radiotracers in the studies on the metabolism, mode of action, environmental behavior, and fate of ZJ0273. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of potent lymphocyte function‐associated antigen‐1 inhibitors labeled with carbon‐14 and deuterium,part 1

The lymphocyte function‐associated antigen‐1 (LFA‐1) is an essential component in normal immune system function and is a target for drug discovery for its broad therapeutic potential in treating inflammatory diseases. Here, we report the synthesis of three potent antagonists of LFA‐1 labeled with carbon‐14 and deuterium to support drug metabolism and pharmacokinetics studies. Carbon‐14 labeled (R)‐1‐acetyl‐5‐(4‐bromobenzyl)‐3‐(3,5‐dichlorophenyl)‐5‐methyl‐imidazolidine‐2,4‐dione (1) was prepared in 27% radiochemical yield in two steps and with a specific activity of 2.1 GBq/mmol by using [¹⁴C]‐phosgene. Carbon‐14 labeled 5‐bromopyrimidine was used to prepare (R)‐5‐(1‐piperazinylsulfonyl)‐1‐(3,5‐dichlorophenyl)‐3‐[4‐(5‐pyrimidinyl)benzyl]‐3‐methyl‐1‐H‐imidazo[1,2a]imidazol‐2‐one (2) and (R)‐1‐[7‐(3,5‐dichlorophenyl)‐5‐methyl‐6‐oxo‐5‐(4‐pyrimidin‐5‐yl‐benzyl)‐6,7‐dihydro‐5H‐imidazo[1,2‐a]imidazole‐3‐sulfonyl]piperidin‐4‐carboxylic acid amide (3) via a Suzuki reaction with the corresponding boronic acid esters in 42% and 67% radiochemical yield and specific activities of 1.85 GBq/mmol and 1.95 GBq/mmol, respectively. Deuterium labeled piperazine was reacted with the sulfonyl chloride derivative (7), followed by a Suzuki coupling to the pyrimidine boronic ester to give deuterium labeled (2) in 47% yield. Deuterium labeled isonipecotamide was reacted in a similar way with the sulfonyl chloride derivative (14) to furnish deuterium labeled (3) in one step and in 94% yield. Copyright © 2011 John Wiley & Sons, Ltd.     

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. The key transformations were the modified Guareschi–Thorpe condensation of 2‐cyano‐¹⁴C‐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 [¹⁴C]‐ (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‐2,2,3,3,4,5,5,6,6‐²H₉. 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‐[2‐¹⁴C]pyrimidine [¹⁴C]‐(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 [¹⁴C]‐(13), which was synthesized in one pot from enolization of trifluoromethylketone (12), followed by bromine–magnesium exchange and then electrophile trapping reaction with [¹⁴C]‐carbon dioxide. A chiral auxiliary (S)‐1‐(4‐methoxyphenyl)ethylamine was then coupled to this acid to give [¹⁴C]‐(15). Propargylation and separation of diastereoisomers by crystallizations gave the desired diastereomer [¹⁴C]‐(17) in 34% yield. Sonogashira coupling to iodopyridine (10) followed by cyclization to the azaindole [¹⁴C]‐(18) and finally removal of the chiral auxiliary gave [¹⁴C]‐(2) in 7% overall yield. For stable isotope syntheses, [¹³C₆]‐(1) was obtained in three steps using [¹³C₄]‐(6) and trimethylsilylacetylene‐[¹³C₂] in 26% yield, while [²H₅]‐(2) was obtained by first preparing the iodopyridine [²H₅]‐(10) in five steps. Then, Sonogashira coupling to chiral alkyne (24) and cyclization gave [²H₅]‐(2) in 42% overall yield.     

A novel asymmetric synthesis of tritium and carbon‐14 labeled (R)‐ibuprofen

An efficient asymmetric synthesis of tritium and carbon‐14 labeled R‐ibuprofen was achieved in good overall yield (15% and 47%, respectively) and excellent enantiomerical excess (>98% e.e.), using (4R, 5S)‐4‐methyl‐5‐phenyl‐2‐oxazolidinone as a chiral auxiliary. Copyright © 2006 John Wiley & Sons, Ltd.     

In vitro metabolite identification of ML3403, a 4-pyridinylimidazole-type p38 MAP kinase inhibitor by LC-Qq-TOF-MS and LC-SPE-cryo-NMR/MS

Prediction of the metabolic profile of a potential new drug is recommended at an early stage in industrial drug discovery process to determine whether or not any potentially reactive or toxic metabolites are formed. In the present study, we investigated the in vitro metabolism of ML3403 ({4-[5-(4-Fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine), a potent and selective p38 MAP kinase inhibitor using mouse liver microsomes. The combination of LC-ESI-Qq-TOF (tandem quadrupole time-of-flight)-MS (mass spectrometer) and LC-SPE (solid phase extraction)-cryo-NMR (nuclear magnetic resonance)/MS at 600?MHz has been applied for comprehensive and straightforward structural elucidation of ML3403 metabolites. It was possible to determine the metabolic profile of ML3403, revealing eight different metabolites formed by N-desalkylation, S-mono- and di-oxidation, aliphatic hydroxylation and pyridine-N-oxidation. The ESI-Qq-TOF-MS data yielded elemental compositions of all metabolites and their fragments by evaluation of the accurate mass and isotopic pattern information using the sigma-fit algorithm. Evaluation of 2D NMR spectra obtained from pure ML3403 an its major metabolite ML3603 allowed the unequivocal assignment of the resonances in 1D NMR spectra obtained directly from the microsomal incubation by LC-SPE-cryo-NMR/MS. The presented method significantly decreases the time required for a complete structural assignment of metabolites from microsomal in vitro assays.     

Enzymatic synthesis of N‐methylhistamine labeled with deuterium and tritium

The isotopomers of N^π‐methylhistamine (πMeHA) and N^τ‐methylhistamine (τMeHA) labeled with deuterium and tritium at the α‐carbon atom of the side chain were obtained using the enzyme histidine decarboxylase (HDC, EC 4.1.1.22) from Lactobacillus 30a. The deuterium labeled isotopomers [(αR)‐²H]‐πMeHA and [(αR)‐²H]‐τMeHA were synthesized by enzymatic decarboxylation of N^π‐methyl‐, and N^τ‐methyl‐L ‐histidines (respectively) in a fully deuteriated incubation medium. The same decarboxylation carried out in a tritiated medium resulted in tritiated [αR‐³H]‐πMeHA and [αR‐³H]‐τMeHA isotopomers of N‐methylhistamine. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of the olanzapine labeled by carbon‐14

Olanzapine is one of the most widely used antipsychotic drugs, which acts as an antagonist for multiple neurotransmitter receptor sites. 2‐Methyl‐4‐(4‐methyl‐1‐piperazinyl)‐10H‐thieno [2,3‐b][1,5] benzodiazepine (Olanzapine) labeled with carbon‐14 in the four positions has been synthesized as part of a three‐step sequence from 2‐amino‐5‐methylthiophene‐3‐carbonitrile‐[carbonitrile‐¹⁴C].     

The synthesis of tritium,carbon‐14 and stable isotope labelled selective estrogen receptor degraders

As part of a Medicinal Chemistry program aimed at developing an orally bioavailable selective estrogen receptor degrader, a number of tritium, carbon‐14, and stable isotope labelled (E)‐3‐[4‐(2,3,4,9‐tetrahydro‐1H‐pyrido[3,4‐b]indol‐1‐yl)phenyl]prop‐2‐enoic acids were required. This paper discusses 5 synthetic approaches to this compound class.     

Synthesis of carbon‐14, carbon‐13 and deuterium labeled forms of thioacetamide and thioacetamide S‐oxide

Thioacetamide (TA) is a model hepatotoxin that undergoes metabolic activation via two successive S‐oxidations. The ultimate toxic metabolite thioacetamide S,S‐dioxide, or its tautomer acetimidoyl sulfinic acid CH₃C(NH)SO₂H, then acylates lysine side chains on cellular proteins leading to cellular dysfunction or death. To identify individual target proteins, quantitate the extent of their modification and elucidate the structural details of their modification, we required both radio‐labeled and stable‐labeled forms of TA and its intermediate metabolite thioacetamide S‐oxide (TASO). The latter is stable when purified but can be difficult to isolate. Considering currently available isotopic precursors, we devised and report here methods for the synthesis and isolation of TA and TASO labeled with C‐14, C‐13, and/or deuterium. The methods are straightforward, utilize readily available precursors, and are amenable to small scale. Copyright © 2011 John Wiley & Sons, Ltd.     

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‐[¹³C₆]. 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 [¹⁴C]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%.     

Synthesis of carbon‐14 labeled dibutyltin dichloride

This report describes the preparation of carbon‐14 labeled dibutyltin dichloride from carbon‐14 labeled barium carbonate in 30% overall radiochemical yield. The key steps were (a) the preparation of carbon‐14 labeled butyl bromide from carbon‐14 labeled barium carbonate via carbon‐14 labeled butyl mesylate, and (b) the direct preparation of tetrabutyltin by the lithium‐mediated reaction of dibutyltin dichloride with carbon‐14 labeled butyl bromide. Copyright © 2007 John Wiley & Sons, Ltd.