Design and synthesis of novel fluorescently labelled nucleosides

A series of novel 5′‐labelled uridine with fluorophores: N‐alkylcarbazole, alkyloxynaphthalene and alkylfluorene were synthesized through H‐phosphonate by Arbuzov reaction, and were characterized by using ¹H NMR, ³¹P NMR and ESI‐MS analysis. The fluorescence spectra of all the compounds in solution and solid powder were described. The experimental results showed that the fluorescence emission intensity of 5′‐labelled uridine is very sensitive to solvents. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis of deuterated 5‐n‐alkylresorcinols

Four alternative strategies for the preparation of deuterium poly‐labelled 5‐n‐alkylresorcinols are explored. Ring‐labelled ²H₃‐alkylresorcinols synthesized by acidic H/D exchange are stable under electrospray ionization MS conditions but scrambling occurs in electron bombardment ionization MS. Side chain‐labelled ²H₄‐derivatives prepared by two different total synthesis approaches are contaminated by isotopologues with varying number of deuterium labels due to H/D redistribution and exchange during D₂ gas deuterogenation. The derivative carrying an ω‐²H₃ label is isotopically pure and completely stable under all relevant analytical conditions encountered in quantitation work. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of [2H5]‐ebastine fumarate and [2H5]‐hydroxyebastine

This study describes the synthesis of deuterium‐labelled ebastine fumarate and its deuterium‐labelled metabolite hydroxyebastine. The synthesis of the two desired compounds both used [²H₅]‐bromodiphenylmethane as deuterium‐labelled reagent, which was synthesized beforehand in three steps. [²H₅]‐ebastine was synthesized in further three steps with a 27% overall yield and [²H₅]‐hydroxyebastine was synthesized in further seven steps with a 13% overall yield. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis of deuterium‐labelled paclitaxel and its hydroxyl metabolite

This paper describes the synthesis of deuterium‐labelled paclitaxel and its hydroxyl metabolite. Paclitaxel labelled with ²H was obtained in four steps using the commercially available [²H₅]benzoic chloride as the stable labelled reagent with a 40% overall yield. The hydroxyl metabolite labelled with ²H was prepared starting from deuterium‐labelled paclitaxel in six steps with a 42% overall yield based on unrecovered starting material. Copyright © 2011 John Wiley & Sons, Ltd.     

Unusual base‐catalyzed exchange in the synthesis of deuterated PF‐2413873a

The preparation of deuterated PF‐2413873 (4‐[3‐cyclopropyl‐1‐(methanesulfonylmethyl)‐5‐methyl‐1H‐pyrazol‐4‐yl]oxy‐2,6‐dimethylbenzonitrile, 1) is described for use as a bioanalytical standard in clinical trials. Two strategies were investigated. The sulfone‐containing substituent was labelled by base‐catalyzed exchange, but unacceptable deuterium loss was noted under assay conditions. Alternatively, labelling 4‐cyano‐3,5‐dimethylphenol was achieved by heating with deuterium oxide over platinum oxide. After building up the pyrazole ring we discovered that, during the subsequent alkylation to attach the methylthiomethyl group, the base, potassium t‐butoxide, caused unwanted scrambling of deuteriums on the aromatic portion and the methylthiomethyl group. Thus, it was necessary to remove all base‐labile hydrogens to prevent their exchange. This was accomplished by alkylating the pyrazole with per‐deuterated chloromethyl methylsulfide, oxidation to the sulfone, and selective removal of its deuteriums by treatment with sodium hydroxide. The unusual sensitivity and selectivity of these base‐promoted exchange reactions are discussed. Thus, 4‐[3‐cyclopropyl‐1‐(methanesulfonylmethyl)‐5‐methyl‐1H‐pyrazol‐4‐yl]oxy‐[²H₆]2,6‐dimethyl‐[3,5‐²H]benzonitrile (17) was obtained, labelled with eight deuterium atoms and an acceptable D₀/D₈ ratio. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of deuterium labelled diclofenac with improved isotopic enrichment

A five‐step synthesis of deuterium‐labelled diclofenac starting from 2‐phenyl[²H₅] acetic acid is described. The synthesis prevents deuterium from scrambling during the reaction. It offers the labelled compound with over 99% isotopic enrichment. It also provides a possible alternative route for the synthesis of deuterium‐labelled 4′‐hydroxydiclofenac, which is the principal human metabolite of diclofenac. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of Brostallicin (PNU‐166196A) Labelled with 2H and 14C

Brostallicin (PNU‐166196A), a DNA minor groove binder, has been labelled with ²H and ¹⁴C. The preparation of the deuterium specifically labelled [²H₄]brostallicin was achieved according to a nine‐step sequence starting from 1,2‐diamino[1,1,2,2‐²H₄]ethane (1) . [¹⁴C]Brostallicin was obtained via a four‐step procedure in 31% overall radiochemical yield starting from 1‐methyl‐4‐nitropyrrole‐2‐[¹⁴C]carboxylic acid (9) . Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of deuterium‐labelled (−)‐galanthamine

The synthesis of deuterium‐labelled galanthamine is reported. 6‐[²H₃]methoxy‐N‐[²H₃]methyl‐(?)‐galanthamine was obtained in seven steps from galanthamine. The synthesis was carried out by selective O‐ and N‐demethylations. The [²H₃]‐N‐methyl and [²H₃]‐O‐methyl‐groups were introduced by selective aminoreduction and O‐methylation. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of 15N‐labelled 3,5‐dimethylpyridine

¹⁵N‐labelled pyridines are liquid‐ and solid‐state nuclear magnetic resonance (NMR) probes for chemical and biological environments because their ¹⁵N chemical shifts are sensitive to hydrogen‐bond and protonation states. By variation of the type and number of substituents, different target pyridines can be synthesized exhibiting different pK_a values and molecular volumes. Various synthetic routes have been described in the literature, starting from different precursors or modification of other ¹⁵N‐labelled pyridines. In this work, we have explored the synthesis of ¹⁵N ¹⁵N‐labelled pyridines using a two‐step process via the synthesis of alkoxy‐3,4‐dihydro‐2H‐pyran as precursor exhibiting already the desired pyridine substitution pattern. As an example, we have synthesized 3,5‐dimethylpyridine‐¹⁵N (lutidine‐¹⁵N) as demonstrated by ¹⁵N‐NMR spectroscopy. That synthesis starts from methacrolein, propenyl ether, and ¹⁵N‐labelled NH₄Cl as nitrogen source.     

Synthesis of four racemic nicotine isotopomers doubly labelled with stable isotope

Four racemic nicotine isotopomers, doubly labelled with stable isotope, were prepared for use in studies of plant metabolism. First, starting from halogeno nicotinates, one deuterium was introduced into the pyridine moiety by reductive dehalogenation with zinc, and choosing deuterated acetic acid as the acidic medium. Pyridine ²H‐labelled nornicotine derivatives were then formed in two reaction steps, a condensation reaction with N‐vinylpyrrolidinone followed by catalytic hydrogenation. The deuteromethylation of these monodeuterated nornicotines was achieved by reduction of the corresponding ethyl carbamates with deuterated lithium aluminium hydride. The four regioisotopomers obtained were at the least 90% labelled on each site, sufficient for the metabolic studies envisaged. Copyright © 2009 John Wiley & Sons, Ltd.     

Efficient and facile synthesis of novel stable monodeuterium labeled ractopamine

A novel synthetic route to stable deuterium labeled ractopamine was disclosed with 6.49% total yield and 97.7% isotopic abundance. Its structure and the isotope‐abundance were confirmed according to ¹H‐NMR and high‐resolution mass spectrometry.     

Synthesis of [2H7]indatraline

Deuterium‐labelled indatraline was synthesized in high efficiency employing a Friedel–Crafts alkylation of [²H₆]benzene with (E)‐3‐(3,4‐dichlorophenyl)acrylic acid as a key step. The desired labelling of the final compound was ascertained in two ways, by incorporation of [²H₆]benzene in the target molecule and additionally by deuterium transfer to the non‐deuterated aryl moiety of the Friedel–Crafts alkylation product from [²H₆]benzene, the latter thus serving as reagent and solvent.     

Synthesis of highly pure 14C‐labelled DL‐allantoin and 13C NMR analysis of labelling integrity

A number of synthetic approaches are assessed to prepare allantoin labelled with ¹⁴C given certain requirements and technical limitations. A method that fulfils these criteria is described to achieve the synthesis of highly pure ¹⁴C‐labelled allantoin with the label introduced to the ureido carbonyl group in the final step by reaction of 5‐chlorohydantoin with [¹⁴C]urea. The chosen method favours high purity at the expense of radiochemical yield, which is achieved at a level of 8%. The integrity of the label is then investigated by performing an NMR analysis of ¹³C‐labelled allantoin synthesized by the same method. The ¹³C NMR spectrum confirms partial scrambling of the label to the C‐2 position by equilibration of the product via a putative bicyclic intermediate, which had been suggested by other workers. The ¹⁴C‐labelled allantoin synthesized by this method is therefore assigned as DL‐[H₂N¹⁴CO/¹⁴C‐2]allantoin. This study also includes the first full characterization of a side product, 5‐hydroxy‐5‐methoxyhydantoin, obtained by the reaction of a 5‐hydroxyhydantoin intermediate with the methanol solvent. Copyright © 2009 John Wiley & Sons, Ltd.     

Efficient tritiation of the translocator protein (18 kDa) selective ligand DPA‐714

DPA‐714 (N,N‐diethyl‐2‐(2‐(4‐(2‐fluoroethoxy)phenyl)‐5,7‐dimethylpyrazolo[1,5‐a]pyrimidin‐3‐yl)acetamide) is a recently discovered fluorinated ligand of the translocator protein 18 kDa (TSPO). Labelled with the short‐lived positron emitter fluorine‐18, this structure is today the radioligand of reference for in vivo imaging of microglia activation and neuroinflammatory processes with positron emission tomography. In the present work, an isotopically tritium‐labelled version was developed ([³H]DPA‐714), in order to access high resolution in vitro and ex vivo microscopic autoradiography studies, repeated and long‐lasting receptor binding studies and in vivo pharmacokinetic determination at late time points. Briefly, DPA‐714 as reference, and its 3,5‐dibrominated derivative as precursor for labelling, were both prepared from DPA‐713 in nonoptimized 32% (two steps) and 10% (three steps) yields, respectively. Reductive debromination using deuterium gas and Pd/C as catalyst in methanol, performed at the micromolar scale, confirmed the regioselective introduction of two deuterium atoms at the meta positions of the phenyl ring. Tritiodebromination was analogously performed using no‐carrier tritium gas. HPLC purification provided >96% radiochemically pure [³H]DPA‐714 (7 GBq) with a 2.1 TBq/mmol specific radioactivity. Interestingly, additional hydrogen‐for‐tritium exchanges were also observed at the 5‐methyl and 7‐methyl positions of the pyrazolo[1,5‐a]pyrimidine, opening novel perspectives in the labelling of compounds featuring this heterocyclic core.     

Synthesis of deuterium‐labelled 5′‐O‐[N‐(Salicyl)sulfamoyl]adenosine (Sal‐AMS‐d4) as an internal standard for quantitation of Sal‐AMS

5′‐O‐[N‐(Salicyl)sulfamoyl]adenosine (Sal‐AMS, 1) is a potent inhibitor of the bifunctional enzyme salicyl‐AMP ligase in Mycobacterium tuberculosis. This inhibitor acts by disrupting the biosynthesis of the mycobactin siderophores that are essential for the process of iron acquisition. To aid with in vitro metabolism and in vivo pharmacokinetic studies of Sal‐AMS, a stable deuterium‐labelled Sal‐AMS analog (Sal‐AMS‐d₄) was synthesized. This deuterium‐labelled analog was used as an internal standard to conduct in vitro plasma and microsomal stability studies. Sal‐AMS was found to be stable for 24 h in human plasma and 1 h in human liver microsomes at 37°C. Copyright © 2008 John Wiley & Sons, Ltd.     

Dye Labelling of Nucleosides

Dye‐labelled nucleosides were obtained in 30–79% (average 45%) yields by treating N‐(4‐arylazobenzoyl)‐1H‐benzotriazoles 3a–b with appropriate nucleosides. Similarly, 3a–b afforded dye‐labelled threoninol conjugates in 55–89% (average 67%) yields. All novel products were characterized by NMR and elemental analysis.     

A simple and efficient synthesis of [2H10]deuterated bromfenvinphos by the Perkow reaction

(E,Z)‐2‐bromo‐1‐(2,4‐dichlorophenyl)vinyl bis[ethyl‐²H₅] phosphate ([²H₁₀]bromfenvinphos), a regiospecifically deuterium‐labelled pesticide, was synthesized in two steps starting from [²H₆]ethanol, phosphorus trichloride and 2,4‐dichlorophenacylidene bromide, and fully characterized. The deuterated biologically active bromfenvinphos is an important compound for the advancement of environmental degradation testing and some mass spectrometric studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Expedient synthesis of deuterium‐labelled amides within micro‐reactors

The pharmaceutical industry relies heavily on the synthesis of small quantities (10–500 mg) of stable, isotopically labelled compounds in the evaluation of new drug candidates for metabolism studies. As a result of the phenomenal cost of labelled materials even the preparation of small quantities can be extremely expensive. In this paper, for the first time, we report that micro‐reactor technology may be used to prepare stable deuterium‐labelled compounds by conducting all optimization experiments using unlabelled precursors and simply substituting the labelled derivatives once the optimization is complete. Here, we wish to present a simple, general procedure for the synthesis of amides containing isotopic labels demonstrated using [C‐²H₃]acetyl chloride 1 . The reaction is carried out within a micro‐reactor set‐up which we believe offers superiority over other reported methods viz requiring stoichiometric quantities of reagents, high containment of the system and generality of the technique, obtaining products in high yields. Copyright © 2007 John Wiley & Sons, Ltd.     

The synthesis of a benzamidine‐containing NR2B‐selective NMDA receptor ligand labelled with tritium or fluorine‐18

A novel tritium or flourine‐18‐labelled benzamidine‐containing NR2B‐selective NMDA receptor ligand has been synthesized. This compound was designed to contain the fluoromethoxy group to allow for the synthesis of a high specific activity, fluorine‐18‐labelled PET tracer for imaging studies of the NR2B receptor. In addition to the fluorine‐18‐labelled compound, this compound was also tritium labelled. The tritiated ligand (11 Ci/mmol) was synthesized by a gas tritiation reaction of an aryl bromide precursor. The fluorine‐18 ligand (2916 Ci/mmol), which was deuterated in the fluoromethoxy group to aid in metabolic stability, was synthesized by alkylating a phenolic precursor with [¹⁸F]fluoromethylbromide‐d₂. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis of tertiary 14C‐labelled nonylphenol isomers

The ring‐¹⁴C‐labelled p‐nonylphenol (NP) isomers 4(3′,5′‐dimethyl‐3′‐heptyl)‐phenol (p353NP), 4(3′,6′‐dimethyl‐3′‐heptyl)‐phenol (p363NP) and 4(2′,6′‐dimethyl‐2′‐heptyl)‐phenol (p262NP) were synthesized for application in metabolism and sorption studies. Friedel–Crafts alkylation of ¹⁴C‐labelled phenol and the corresponding tertiary nonylalcohol with BF₃ as catalyst was used. After clean‐up of p262NP and p363NP by preparative thin‐layer chromatography radiochemical yields amounted to 62.8 and 64.6%, specific radioactivities were 332 and 88.2 MBq/mmol, and radiochemical purities 97.6 and 99.0%. For both isomers, a large‐scale synthesis with non‐labelled phenol was additionally developed, which led to pure products (96 and 99%, respectively) without further purification steps. In the case of p353NP, which was formed as a diastereomeric mixture, the crude synthetic product had a radiochemical purity of 96.9% (radiochemical yield: 76.0%; specific activity: 298 MBq/mmol); thus, purification was not necessary. All products were characterized by means of gas chromatography‐mass spectroscopy, ¹H‐ and ¹³C‐NMR, as well as IR. Copyright © 2002 John Wiley & Sons, Ltd.