Efficient syntheses of benzyl and n‐octyl [1,3‐13C2]acetoacetates,and their application to syntheses of 13C‐labelled pyrrole and 13C‐labelled hymecromone

Benzyl [1‐¹³C]acetate (2a) was prepared via esterification of sodium [1‐¹³C]acetate (1) with benzyl bromide in the presence of 18‐crown‐6‐ether in 97% yield. n‐Octyl [1‐¹³C]acetate (2b) was rapidly obtained by microwave irradiation of 1‐bromooctane and potassium [1‐¹³C]acetate (obtained by salt exchange of 1) absorbed on Al₂O₃ in 82% yield. Solvent‐free Claisen condensation of benzyl or n‐octyl [1‐¹³C]acetate (2a or 2b) in the presence of potassium tert‐butoxide efficiently gave benzyl or n‐octyl [1,3‐¹³C₂]acetoacetate (3a or 3b) in 51 or 68% yield, respectively. Dibenzyl 2,4‐dimethyl[2,4‐¹³C₂]pyrrole‐3,5‐di[¹³C]carboxylate (4) was synthesized from benzyl [1,3‐¹³C₂]acetoacetate (3a) in 54% yield. [2,4‐¹³C₂]Hymecromone (6) (7‐hydroxy‐4‐methyl[2,4‐¹³C₂]coumarin) was obtained from n‐octyl [1,3‐¹³C₂]acetoacetate (3b) and 1,3‐benzenediol (5) in 73% yield. Copyright © 2008 John Wiley & Sons, Ltd.     

Biosynthesis of glucose‐d‐13C6 from Hansenula polymorpha and methanol‐13C

A simple and effective method for synthesis of glucose‐d ‐¹³C₆ by fermentation using the methylotrophic yeast Hansenula polymorpha with 99% abundance methanol‐¹³C is described. Using methanol‐¹³C as a sole source of carbon, H. polymorpha can accumulate large amounts of α,α‐trehalose‐¹³C₁₂ under unfavourable growth conditions; the trehalose can then be hydrolysed to give glucose‐d ‐¹³C₆ with 98.5% abundance ¹³C. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis of C‐14 and C‐13, H‐2‐labeled IKK inhibitor: [14C] and [13C4,D3]‐N‐(6‐chloro‐7‐methoxy‐9H‐pyrido[3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide

[¹⁴C]‐N‐(6‐Chloro‐7‐methoxy‐9H‐pyrido [3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide (5B ), an IKK inhibitor, was synthesized from [¹⁴C]‐barium carbonate in two steps in an overall radiochemical yield of 41%. The intermediate, [carboxyl‐¹⁴C]‐2‐methylnicotinic acid, was prepared by the lithiation and carbonation of 3‐bromo‐2‐methylpyridine. [¹³C₄,D₃]‐N‐(6‐chloro‐7‐methoxy‐9H‐pyrido [3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide (5C ) was synthesized from [1,2,3,4‐¹³C₄]‐ethyl acetoacetate and [D₄]‐methanol in six steps in an overall yield of 2%. [¹³C₄]‐2‐methylnicotic acid, was prepared by condensation of [¹³C₄]‐ethyl 3‐aminocrotonate and acrolein, followed by hydrolysis with lithium hydroxide. Copyright © 2006 John Wiley & Sons, Ltd.     

13C‐ and 14C‐labelling of N‐[1‐(4‐chlorophenyl)‐1H‐pyrrol‐2‐yl‐methyleneamino] guanidinium acetate

N‐[1‐(4‐chlorophenyl)‐1H‐pyrrol‐2‐yl‐¹³C₄‐methyleneamino]guanidinium acetate has been synthesized by a four‐step procedure. This involved reduction of the Weinreb amide N,N′‐dimethyl‐N,N′‐dimethyloxybutane‐1,4‐diamide‐1,2,3,4‐¹³C₄ by Dibal‐H to give the corresponding unstable dialdehyde which is reacted in situ with 4‐chloroaniline to form 1‐(4‐chlorophenyl)‐1H‐pyrrole‐¹³C₄. This pyrrole analogue underwent a Vilsmeyer acylation with POCl₃/DMF followed by final reaction with aminoguanidine bicarbonate to produce the desired labelled compound with 99% atom ¹³C. By using DMF [α‐¹⁴C] a radio‐labelled analogue was synthesized with a specific activity of 60 mCi/mmol. Copyright © 2005 John Wiley & Sons, Ltd.     

Synthesis of 13C‐labelled sulfated N‐acetyl‐d‐lactosamines to aid in the diagnosis of mucopolysaccharidosis diseases

Morquio A syndrome is an autosomal mucopolysaccharide storage disorder that leads to accumulation of keratan sulfate. Diagnosis of this disease can be aided by measuring the levels of keratan sulfate in the urine. This requires the liquid chromatography tandem mass spectrometry (LCMS/MS) measurement of sulfated N‐acetyl‐d ‐lactosamines in the urine after cleavage of the keratan sulfate with keratanase II. Quantification requires isotopically‐labelled internal standards. The synthesis of these ¹³C₆‐labelled standards from ¹³C₆‐galactose and N‐acetylglucosamine is described. The required protected disaccharide is prepared utilising a regioselective, high yielding β‐galactosylation of a partially protected glucosamine acceptor and an inverse addition protocol. Subsequent synthesis of the ¹³C₆‐labelled mono and disulfated N‐acetyllactosamines was achieved in five and eight steps, respectively, from this intermediate to provide internal standards for the LCMS/MS quantification of keratan sulfate in urine.     

A method for the preparation of [13C6]‐labelled 2‐substituted naphthalenes

A reliable route is described for the preparation of various 2‐substituted derivatives of [1,2,3,4,4a,8a‐¹³C₆]‐naphthalene via the bromide 10. The approach is used to prepare [naphthalene‐1,2,3,4,4a,8a‐¹³C₆]‐2‐(2‐bromoethyl)naphthalene (1), a key intermediate in the synthesis of labelled SR57746A, Xaliproden (2). Copyright © 2010 John Wiley & Sons, Ltd.     

An innovative and efficient synthesis of stable isotope labelled 1‐methyl‐5‐(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)‐1H‐pyrazole via [13C42H3] N‐methylpyrazole

In support of a programme to develop a treatment for cancer, a stable isotope labelled version of the drug candidate was required. The key labelled intermediate was [¹³C₄²H₃] N‐methylpyrazole prepared by a novel bisacetal cyclisation. This was prepared from commercially available diethyl [¹³C₃] malonate and [¹³C²H₃] iodomethane. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis of di‐docosahexaenoyl (C22:6)‐bis(monoacylglycerol) phosphate in unlabelled and C‐13 labelled forms for use as a biomarker of drug induced phospholipidosis

Di‐docosahexaenoyl (C22:6)‐bis(monoacylglycerol) phosphate (BMP) has been identified as a promising biomarker for drug‐induced phospholipidosis (DIPL). Both unlabelled and stable isotope labelled versions of BMP were desired for use as internal standards. Isopropylideneglycerol was converted to 4‐methoxyphenyldiphenylmethyl‐3‐PMB‐glycerol in three steps. Initially, the 2‐postion of the glycerol was protected as a t‐butyldiphenylsilyl ether, which proved to be a mistake; deprotection of the ether resulted in the decomposition of the compound. A switch to a t‐butyldimethylsilyl ether protecting group resulted in an intermediate that could be deprotected to the alcohol to give the target compound after salt exchange. The same procedure was used to prepare [¹³C₆]BMP from [¹³C₃]glycerol.     

Synthesis of isotopically labelled glycyl‐L‐prolyl‐L‐glutamic acid (Glypromate®) and derivatives

The related tripeptides glycyl‐L ‐prolyl‐L ‐glutamic acid (GPE) and glycyl‐L ‐2‐methylprolyl‐L ‐glutamic acid (G‐2‐MePE) were labelled with commercially available [1,2,3,4,5‐¹³C₅, 2‐¹⁵N₁]‐L ‐glutamic acid in 3 steps in excellent overall yield with high isotope incorporation. A related cyclic dipeptide was labelled with [2,2‐²H₂, 2‐¹⁵N₁]glycine giving a mixture of compounds resulting from deuterium scrambling. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis of 13N‐labelled radiotracers by using microfluidic technology

Microfluidics has recently emerged as a useful alternative to traditional methods for the preparation of radiotracers labelled with positron emitters. Up to date, microfluidics technology has been applied to the radiosynthesis of ¹⁸F‐labelled and ¹¹C‐labelled compounds; however, application to other shorter‐lived positron emitters has not been investigated. The radiosynthesis of S‐[¹³N]nitrosothiols and N‐[¹³N]nitrosamines was approached in microfluidic system by reaction of the corresponding thiol or secondary amine, respectively, with [¹³N]NO₂^? in the presence of mineral acid. The radiosynthesis of azo compounds was carried out by reaction of the same labelling agent with primary aromatic amines in acidic media to yield the corresponding diazonium salts, which were further reacted with aromatic amines and alcohols to yield the corresponding ¹³N‐labelled azo compounds. Radiochemical conversion values for S‐[¹³N]nitrosothiols and ¹³N‐labelled azo compounds calculated from chromatographic profiles improved our previous results by using conventional methods. The formation of N‐[¹³N]nitrosamines could not be detected, independently of experimental conditions. In conclusion, the preparation of S‐[¹³N]nitrosothiols and ¹³N‐labelled azo compounds was successfully achieved by using microfluidics technology. Higher radiochemical conversions than those previously reported using conventional synthetic strategies have been obtained. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis of 14C‐labeled and 13C‐,15N‐labeled dasatinib and its piperazine N‐dealkyl metabolite

Dasatinib (SPRYCEL^®) is a multiple kinase inhibitor approved for the treatment of chronic myelogenous leukemia and Philadelphia chromosome‐positive acute lymphoblastic leukemia in patients with resistance to prior therapy, including imatinib mesylate (Gleevec^®). Radiolabeled dasatinib and its piperazine N‐dealkyl metabolite were synthesized to investigate absorption, distribution, metabolism, and elimination of the compounds in humans and animals. These compounds were prepared following a three‐step sequence, which included thiazole carboxamide formation via cyclization of labeled thiourea with a brominated oxyacrylamide precursor. In the final step a common intermediate was converted to either [¹⁴C]dasatinib or the radiolabeled piperazine N‐dealkyl metabolite with labeling in the aminothiazole ring. Syntheses of both compounds were achieved with radiochemical purities in excess of 98%. Stable‐labeled dasatinib and the piperazine N‐dealkyl metabolite were also needed for use as mass spectral internal standards in support of bioanalytical assays. By following the same route used for the carbon‐14 synthesis, [¹³C₄, ¹⁵N₂]dasatinib and the [¹³C₄, ¹⁵N₂]metabolite were prepared with labeling in both the dichloropyrimidine and thiazole ring systems. This convergent process introduced stable isotope labeling through (1, 2, 3‐¹³C₃) diethyl malonate and [¹³C,¹⁵N₂]thiourea. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of 5‐[4,5‐13C2]‐ and 5‐[1,5‐13C2]aminolevulinic acid

5‐[4,5‐¹³C₂]‐ and 5‐[1,5‐¹³C₂]Aminolevulinic acid (ALA) have been synthesized by the Gabriel condensation of potassium phthalimide with ethyl bromo[1,2‐¹³C₂]acetate (derived from [1,2‐¹³C₂]acetic acid) or ethyl bromo[2‐¹³C]‐acetate (derived from sodium [2‐¹³C]acetate), followed by conversion to the chloride, coupling reaction with 2‐ethoxycarbonylethylzinc iodide derived from ethyl 3‐iodopropionate or 2‐methoxy[¹³C]carbonylethylzinc iodide derived from methyl 3‐iodo[1‐¹³C]propionate (generated from potassium [¹³C]cyanide), and hydrolysis. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of N1‐tritylethane‐1,1,2,2‐d4‐1,2‐diamine: a novel mono‐protected C‐deuterated ethylenediamine synthon

A convenient and high‐yield synthesis for N¹‐tritylethane‐1,1,2,2‐d₄‐1,2‐diamine, a novel mono‐protected ethylenediamine‐C‐d₄, is reported. N¹‐tritylethane‐1,1,2,2‐d₄‐1,2‐diamine was prepared in three steps from ethylene oxide‐d₄ in a combined yield in the range 68–76%. Also reported is a synthesis of ethylenediamine‐C‐d₄ in two steps from 1,2‐dibromoethane‐d₄ in a combined yield in the range 61–65%.     

Synthesis of 13C and 15N labeled 2,4‐dinitroanisole

Syntheses of [¹³C₆]‐2,4‐dinitroanisole (ring‐¹³C₆) from [¹³C₆]‐anisole (ring‐¹³C₆) and [¹⁵N₂]‐2,4‐dinitroanisole from anisole using in situ generated acetyl nitrate and [¹⁵N]‐acetyl nitrate, respectively, are described. Treatment of [¹³C₆]‐anisole (ring‐¹³C₆) with acetyl nitrate generated in 100% HNO₃ gave [¹³C₆]‐2,4‐dinitroanisole (ring‐¹³C₆) in 83% yield. Treatment of anisole with [¹⁵N]‐acetyl nitrate generated in 10 N [¹⁵N]‐HNO₃ gave [¹⁵N₂]‐2,4‐dinitroanisole in 44% yield after two cycles of nitration. Byproducts in the latter reaction included [¹⁵N]‐2‐nitroanisole and [¹⁵N]‐4‐nitroanisole.     

Controlled synthesis of labelled 3‐L‐chlorotyrosine‐[ring‐13C6] and of 3,5‐L‐dichlorotyrosine‐[ring‐13C6]

Pure 3‐L ‐chlorotyrosine‐[ring‐¹³C₆] is prepared by chlorination of the 5‐oxazolidinone of L ‐tyrosine‐[ring‐¹³C₆] with SO₂Cl₂ in CH₃COOH‐Et₂O and successive one‐pot regeneration of the protected aminoacidic functions by BCl₃ in dichloromethane. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis of 13C3‐hydroxyacetone

¹³C₃‐Hydroxyacetone is prepared in three steps from ¹³C₂‐2‐bromoacetic acid. Bromide displacement by sodium p‐methoxybenzyl alkoxide followed by treatment of the carboxylic acid with ¹³C‐methyl lithium furnishes PMB‐protected hydroxyacetone. Deprotection using DDQ delivers the title compound. Copyright © 2003 John Wiley & Sons, Ltd.     

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

Synthesis of N‐[1‐13C]caproyl‐N′‐phenylthiourea

An optimal synthesis of N‐[1‐¹³C]caproyl‐N′‐phenylthiourea with isotopic enrichment 82% is described, starting from barium [¹³C]carbonate, using five synthetic steps. Yields were 95% relative to caproyl chloride and 46% relative to barium carbonate. Oxidation of the title compound with manganese dioxide yields the corresponding ureide. Structural similarities with anticonvulsants such as phenacemide make N‐caproyl‐N′‐phenylthiourea an interesting model compound. Copyright © 2004 John Wiley & Sons, Ltd.     

X‐ray and 13C solid‐state NMR studies of N‐benzoyl‐phenylalanine

Abstract: A crystalline sample of N‐benzoyl‐dl ‐phenylalanine 1 and a polycrystalline sample of N‐benzoyl‐l ‐phenylalanine 2 were studied using ¹³C high‐resolution solid‐state NMR spectroscopy. The X‐ray structure of the dl form was established. Sample 1 crystallizes in a monoclinic form with a P2₁/c space group, a = 11.338(1) Å, b = 9.185(1) Å, c = 14.096(2) Å, β = 107.53(3)°, V = 1400(3) ų, Z = 4 and R = 0.053. The principal elements of the ¹³C chemical shift tensors δ_ii for 1 and 2 , selectively ¹³C (99%) labeled at the carboxyl groups were calculated. On the basis of ¹³C δ_ii analysis the hydrogen bonding pattern for sample 2 was deduced. Enriched samples were used to establish the intermolecular distance between chemically equivalent nuclei for 1 and spatial proximity in heterogeneous domain for 2 , employing the ODESSA pulse sequence. The consistence of the complementary approach covering X‐ray data, analysis of the ¹³C δ_ii parameters and ODESSA results is revealed.     

Large‐scale preparation of 13C‐labeled 2‐(phenylthio)acetic acid and the corresponding labeled sulfoxides and sulfones

We have developed large‐scale efficient procedures for the conversion of commercially available [¹³C]‐ or [²H₃,¹³C]methanol and ¹³CO₂ or ¹³C‐labeled bromoacetic acid to 2‐(phenylthio)[1,2‐¹³C₂]‐, [1‐¹³C]‐, and [2‐¹³C]acetic acid. The resulting derivatives are versatile, chemically stable, and nonvolatile two‐carbon labeling precursors. We have used the ¹³C‐isotopomers of 2‐(phenylthio)acetic acid in the synthesis of ¹³C‐labeled acrylic acid, methacrylic acid, and trans‐crotonic acid.