Synthesis of stable isotope‐labeled metabolites of asenapine

The stable isotope‐labeled synthesis of four of the major metabolites of asenapine is described. The synthesis of [¹³CD₃]‐asenapine N‐oxide proceeded in two synthetic steps. Preparation of [¹³CD₃]‐asenapine 11‐hydroxysulfate and [¹³C₆]‐N‐desmethylasenapine paralleled established synthetic protocols with effective utilization of labeled precursors. The synthesis of [¹³CD₃]‐asenapine N⁺‐glucuronide was achieved in three chemical steps followed by purification.     

Syntheses of three stable isotope‐labeled ethylcyclopropanes

Three stable isotope‐labeled ethylcyclopropanes have been synthesized in preparation for a mechanistic study of its fragmentation to methane and butadiene. Two tactical innovations have been used to deal with practical synthetic challenges posed by the very limited solubility of methylmagnesium iodide in tetrahydrofuran and the high volatility and high tendency to form aerosols characteristic of ethylcyclopropane. Copyright © 2009 John Wiley & Sons, Ltd.     

Expeditious syntheses of stable and radioactive isotope‐labeled anticonvulsant agent,JNJ‐26990990, and its metabolites

Syntheses of stable and radioactive isotope‐labeled anticonvulsant agent, JNJ‐26990990, that is, N‐(benzo[b]thien‐3‐ylmethyl)‐sulfamide and its metabolites are described. [¹³C¹⁵N]Benzo[b]thiophene‐3‐carbonitrile was first prepared by coupling of 3‐bromo‐benzo[b]thiophene with [¹³C¹⁵N]‐copper cyanide. The resultant [¹³C¹⁵N]benzo[b]thiophene‐3‐carbonitrile was reduced with lithium aluminum deuteride to give [¹³CD₂¹⁵N]benzo[b]thiophen‐3‐yl‐methylamine; which was then coupled with sulfamide to afford [¹³CD₂¹⁵N]‐N‐(benzo[b]thien‐3‐ylmethyl)‐sulfamide, the stable isotope‐labeled compound with four stable isotope atoms. Direct oxidation of [¹³CD₂¹⁵N]‐N‐(benzo[b]thien‐3‐ylmethyl)‐sulfamide with hydrogen peroxide and peracetic acid gave the stable isotope‐labeled sulfoxide and sulfone metabolites. On the other hand, radioactive ¹⁴C‐labeled N‐(benzo[b]thien‐3‐ylmethyl)‐sulfamide was prepared conveniently by sequential coupling of 3‐bromo‐benzo[b]thiophene with [¹⁴C]‐copper cyanide, reduction of the carbonitrile to carboxaldehyde, and reductive amination with sulfamide.     

Synthesis of stable isotope–labeled chloroquine and amodiaquine and their metabolites

Anti‐malaria drugs chloroquine and amodiaquine and their metabolites were synthesized to incorporate ¹³C and ¹⁵N starting from U‐¹³C–labeled benzene to give M + 7 isotopomers. Chloroquine and its metabolites were prepared from 7‐chloro‐1,2,3,4‐tetrahydroquinolin‐4‐one through an aryl substitution with the corresponding amines; and the amodiaquine and its metabolites were prepared from 4,7‐dichloroquinoline in a similar fashion.     

Asymmetric syntheses of radioactive and stable isotope‐labeled β‐amino acids

β‐amino acids 1 and 2 are α2δ agonists, which were developed for the treatment of generalized anxiety disorder and insomnia. The stable and radioactive isotope‐labeled β‐amino acids were required to support pre‐clinical and clinical studies. Asymmetric syntheses of deuterium and carbon‐14 labeled β‐amino acids were achieved via chiral auxiliary and diastereoselective hydrogenation methodologies, respectively. The details of these syntheses are reported. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis of carbon‐14 labeled ZJ0712, a novel strobilurin fungicide

ZJ0712, a broad‐spectrum fungicidal ingredient of strobilurin, exhibits a high protective and curative activity against plant pathogenic fungi. To support the study on its metabolism, residue, environmental behavior, and fate for safety evaluation, two versions of carbon‐14 labeled ZJ0712, methyl (E)‐2‐(2‐((2,5‐dimethylphenoxy)methyl)phenyl)‐3‐methoxy[2‐¹⁴C]acrylate ( 2 ) and methyl (E)‐2‐(2‐((2,5‐dimethyl[phenyl‐U‐¹⁴C₆]phenoxy)methyl)phenyl)‐3‐methoxyacrylate ( 3 ), were synthesized from barium [¹⁴C]carbonate in 6‐step yield of 47% and from 2,5‐dimethyl[phenyl‐U‐¹⁴C₆]phenol in the yield of 91%, respectively.     

Facile synthesis of stable isotope‐labeled antibacterial agent RWJ‐416457 and its metabolite

Synthesis of multiple stable isotope‐labeled antibacterial agent RWJ‐416457, (N‐{3‐[3‐fluoro‐4‐(2‐methyl‐2,6‐dihydro‐4H‐pyrrolo[3,4‐c]pyrazol‐5‐yl)‐phenyl]‐2‐oxo‐oxazolidin‐5‐ylmethyl}‐acetamide), and its major metabolite, N‐{3‐[4‐(2,6‐dihydro‐4H‐pyrrolo[3,4‐c]pyrazol‐5‐yl)‐3‐fluoro‐phenyl]‐2‐oxo‐oxazolidin‐5‐ylmethyl}‐acetamide, is described. The stable isotope‐labeled [¹³CD₃]RWJ‐416457 was prepared readily by acetylation of the precursor amine, 5‐aminomethyl‐3‐[3‐fluoro‐4‐(2‐methyl‐2,6‐dihydro‐4H‐pyrrolo[3,4‐c]pyrazol‐5‐yl)‐phenyl]‐oxazolidin‐2‐one with CD₃¹³COCl in pyridine. Synthesis of the stable isotope‐labeled metabolite involved a construction of multiple isotope‐labeled pyrazole ring. N,N‐dimethyl(formyl‐¹³C,D)amide dimethyl acetal was first prepared by treating N,N‐dimethyl(formyl‐¹³C,D)amide with dimethyl sulfate, followed by sodium methoxide. Then, N‐{3‐[3‐fluoro‐4‐(3‐oxo‐pyrrolidin‐1‐yl)‐phenyl]‐2‐oxo‐oxazolidin‐5‐ylmethyl}‐acetamide was condensed with N,N‐dimethyl(formyl‐¹³C,D)amide dimethyl acetal, and the resultant β‐ketoenamine intermediate underwent pyrazole ring formation with hydrazine‐¹⁵N₂, to give the [¹³C¹⁵N₂D]‐labeled metabolite. Copyright © 2012 John Wiley & Sons, Ltd.     

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. Synthetic efforts were focused on the common substituted thiazole moiety, which is easily accessible via a Hantzsch's reaction of α‐bromoketones and mono‐substituted thioureas. In the radioactive synthesis, commercially available carbon‐14 thiourea was utilized to prepare mono‐substituted thioureas, which upon condensation with α‐bromoketones in isopropanol followed by ester hydrolysis gave the desired carbon‐14‐labeled protease inhibitors. The same strategy was used to prepare these inhibitors labeled with stable isotopes. Mono‐substituted thioureas were obtained from commercially available deuterium‐labeled intermediates and then condensed with α‐bromoketones followed by ester hydrolysis to give the deuterium‐labeled inhibitors.     

Synthesis of stable isotope labeled PNU‐95666

Studies towards the synthesis of stable isotope labeled PNU‐95666, a CNS agent currently under development for the treatment of Parkinson's disease, is described. The synthesis of non‐labeled PNU‐95666 starts using a large excess of methylamine. While the non‐labeled methylamine is inexpensive and in plentiful supply, the stable isotope labeled form is expensive and limited availability. Different synthetic routes and conditions, including the use of different aminating agents and various amounts of methylamine were explored and new synthetic conditions were established. Copyright © 2004 John Wiley & Sons, Ltd.     

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 isotope‐labeled HSP90 inhibitor: [13CD3] and [14C]‐TAK‐459

[¹³CD₃]‐TAK‐459 (1A), an HSP90 inhibitor, was synthesized from [¹³CD₃]‐sodium methoxide in three steps in an overall yield of 29%. The key intermediate [¹³CD₃]‐2‐methoxy‐6‐(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)pyridine was synthesized in two steps from 2,6‐dibromopyridine and stable isotope‐labeled sodium methoxide. [¹⁴C]‐TAK‐459 (1B) was synthesized from [¹⁴C(U)]‐guanidine hydrochloride in five steps in an overall radiochemical yield of 5.4%. The key intermediate, [¹⁴C]‐(R)‐2‐amino‐7‐(2‐bromo‐4‐fluorophenyl)‐4‐methyl‐7,8‐dihydropyrido[4,3‐d]pyrimidin‐5(6H)‐one, was prepared by microwave‐assisted condensation.     

Syntheses of isotope‐labeled tobacco‐specific nitrosamines and their metabolites

We report here on the syntheses of three deuterium‐labeled tobacco‐specific nitrosamines namely [2,4,5,6‐d₄]nitrosonornicotine([2,4,5,6‐d₄]NNN), 4‐(methylnitrosamino)‐1‐(3‐[2,4,5,6‐d₄]pyridyl)‐1‐butanone ([2,4,5,6‐d₄]NNK), and 4‐(methylnitrosamino)‐1‐(3‐[2,4,5,6‐d₄]pyridyl)‐1‐butanol ([2,4,5,6‐d₄]NNAL). A metabolite of NNK and myosmine, 4‐hydroxy‐1‐(3‐[2,4,5,6‐d₄]pyridyl)‐1‐butanone, was also synthesized. The synthetic strategy reported here is similar to that reported in the literature for the preparation of corresponding unlabeled compounds. The commercially available [2,4,5,6‐d₄]ethylnicotinate was used as starting material. During the course of these syntheses [2,4,5,6‐d₄]myosmine and [2,4,5,6‐d₄]nornicotine were obtained as stable intermediates. These isotope‐labeled compounds are useful internal standards for quantification of TSNA and their metabolites in smokers in molecular epidemiological studies. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of (S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐ 3‐[(1Z)‐2‐chloro‐3,3,3‐trifluoro‐1‐propenyl]‐2,2‐dimethylcyclopropanecarboxylate‐1‐14C

γ‐Cyhalothrin ( 1a ), (S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐3‐[(1Z)‐2‐chloro‐3,3,3‐trifluoro‐1‐propenyl]‐2,2‐dimethylcyclopropanecarboxylate, is a single‐isomer, synthetic pyrethroid insecticide marketed by Pytech Chemicals GmbH, a joint venture between Dow AgroSciences and Cheminova A/S. As a part of the registration process there was a need to incorporate a carbon‐14 label into the cyclopropyl ring of this molecule. A high yielding radiochemical synthesis of γ‐cyhalothrin was developed from readily available carbon‐14 labeled N‐t‐Boc protected glycine. This seven step synthesis, followed by a preparative normal phase HPLC separation of diastereomers, provided 21.8 mCi of γ‐cyhalothrin‐1‐¹⁴C ( 1b ) with >98% radiochemical purity. Copyright © 2007 John Wiley & Sons, Ltd.     

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.     

Production and application of high quality stable isotope‐labeled human immunoglobulin G1 for mass spectrometry analysis

Here, we describe the production of stable isotope‐labeled human immunoglobulin G1 ([¹³C]‐hIgG1) using [¹³C]‐L‐lysine/arginine–labeled hIgG1. The fermentation process was run in shake flasks containing labeled arginine and lysinethat were incorporated into the produced recombinant hIgG1. The [¹³C]‐hIgG1 was purified, and label incorporation was determined to be >99% at all lysine and arginine moieties. Sequence coverage was confirmed by peptide mapping. [¹³C]‐hIgG1 was then used as an internal standard (IS) for the development of a liquid chromatography–tandem mass spectrometry method applicable to the quantitative analysis of all human types of hIgG1 in rat serum. Four conserved peptides, namely, GPSVFPLAPSSK, TTPPVLDSDGSFFLYSK, VVSVLTVLHQDWLNGK, and FNWYVDGVEVHNAK, originating from different parts of the fraction crystallizable region of hIgG1, were used for quantitation of hIgG1 in rat serum. The calibration curves with a coefficient of determination (r²) between 0.9950 and 0.9962 resulting from the peak area ratio of each peptide to its respective labeled IS were reproducible. A mean bias within ±20.0% of the nominal values and a precision of ≤20.0 % were obtained for the calibration standards and quality control samples for each peptide. [¹³C]‐hIgG1 was shown as a suitable IS for quantitative hIgG1 analysis in preclinical species by LC‐MS/MS.     

Synthesis of stable isotopically labelled 3‐methylfuran‐2(5H)‐one and the corresponding strigolactones

Conventional synthetic procedures of strigolactones (SLs) involve the independent synthesis of ring ABC and ring D, followed by a coupling of the two fragments. Here we prepared three kinds of stable, isotopically labelled D‐ring analogues productively using a facile protocol. Then, a coupling of the D‐rings to ring ABC produced three isotope‐labelled SL derivatives. Moreover, (+)‐D₃‐2′‐epi‐ 1A and (?)‐ent‐D₃‐2′‐epi‐ 1A with high enantiomeric purity were obtained via chiral resolution.     

Synthesis of the lipid peroxidation product 4‐hydroxy‐2(E)‐nonenal with 13C stable isotope incorporation

The aim of this work was to synthesize ¹³C internal standards for the quantification of 4‐hydroxy‐2(E)‐nonenal (HNE), a lipid peroxidation product, and of the etheno‐adducts possibly formed by HNE damage to DNA nucleobases. We designed an eight‐step synthesis starting from ethyl 2‐bromoacetate and giving access to 4‐[(tetrahydro‐2H‐pyran‐2‐yl)oxy]‐2(E)‐nonenal. This compound is a precursor of HNE. The scheme was then used to produce the ¹³C precursor [1,2‐¹³C₂]‐4‐[(tetrahydro‐2H‐pyran‐2‐yl)oxy]‐2(E)‐nonenal. [1,2‐¹³C₂]‐HNE was obtained by acid deprotection. All the intermediary and final compounds were fully characterized by IR, HRMS, ¹H and ¹³C NMR. It is the first synthesis of HNE which enables the incorporation of two ¹³C labels at determined positions. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of 32P‐labeled intermediates on the purine biosynthetic pathway

A fast and efficient route to ³²P‐labeled intermediates on the purine biosynthetic pathway is described. Adenosine kinase catalyzes the phosphorylation of 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICARs) to give [³²P]‐5‐aminoimidazole‐4‐carboxamide ribonucleotide (AICAR). This was then hydrolyzed to [³²P]‐4‐carboxy‐5‐aminoimidazole ribonucleotide (CAIR). Treatment of CAIR with N5‐CAIR mutase gave [³²P]‐5‐aminoimidazole ribonucleotide (AIR) in milligram quantities. Copyright © 2002 John Wiley & Sons, Ltd.     

Site‐specific 2H‐labeled oleic acid and derived esters for use as tracers of ethyl oleate metabolism in honey bees

The inventory of labeled fatty acids and protocols for their syntheses are constantly increasing, but site‐specific labeled precursors in biosynthetic studies are still needed. Ethyl oleate (EO) is an important primer pheromone in honeybees, which is responsible for the regulation of behavioral maturation. During our biosynthetic studies on EO, a site‐specific labeled oleic acid precursor was required. In this report, a synthetic route adaptable to the preparation of [9,10,11,11‐D₄] oleic acid and its derived esters for use as tracers of EO metabolism in honey bees is presented.