Synthetic Methods

Along with the growth of contract research organizations for preparative organic synthesis and process development over the past decades, a subset of these enterprises have developed capabilities for the preparation of isotopically labeled compounds. This includes stable and radioactive isotopes, and in many cases some commonly used reagents and intermediates are offered for sale in the company’s catalog. Before that time, even relatively simple isotopically labeled materials were only accessible on an ad hoc basis, and depended on the capability and determination of individual researchers. These days, all but the most esoteric substances can be obtained from CROs, and this avenue can be explored before committing to an in-house program, especially for radiolabeled compounds.

Hydrogen isotopes (2H, 3H)

Tritium labeling is one of the subjects of a recent monograph.14)

At the present time, deuterium and tritium have become readily available as an offshoot from the production for nuclear weapons and heavy water reactors, and these isotopes in the form of labeled precursors and reagents are marketed by many general and specialized fine chemicals suppliers. Deuterium and tritium can be incorporated into organic molecules using a catalytic reduction with D2 or T2 gas. The stable isotope D2 is available commercially in cylinders, and can be used in conjunction with standard atmospheric or pressurized hydrogenation reactors. More precautions are necessary with the radioactive T2, and the current best practice is to employ a tritium manifold in which the T2 is stored as uranium(III) tritide (UT3), from which the T2 can be generated reversibly and quantitatively in a closed system by controlled heating of a UT3 bed. The pressure of the system is then used to quantify the amount of tritium admitted to the reaction vessel that is connected to the manifold, and unused tritium then is taken up by the uranium bed (or U-bed) when the reaction is complete.

An example of palladium-catalyzed halogen exchange is shown below for the synthesis of tritium-labeled mephenytoin. Here the parent molecule is iodinated with N-iodosuccinimide (NIS) in triflic acid, followed by Pd-catalyzed iodine exchange to give the labeled compound Here the parent molecule is iodinated with N-iodosuccinimide (NIS) in triflic acid, followed by Pd-catalyzed iodine exchange to give the labeled compound [3H3](S)-mephenytoin.

labeled mephenytoin

The catalytic addition of 2H2 or 3H2 to carbon-carbon double or triple bonds is possible, but the position of the label often becomes scrambled through migration processes when heterogeneous catalysts are employed. Such reactions, along with similarly catalyzed aromatic exchange reactions, are generally useful only when site-specific labeling is not required. Deactivated or homogenous catalysts tend to be more specific, as does the use of isotopic diimide.

Transfer deuterogenation has been demonstrated using aqueous-soluble iridium and rhodium catalysts in D2O as solvent and deuterium source through exchange with formic acid. Mesaconic acid underwent reaction in high chemical yield (>99%) with high levels of deuterium incorporation (>96%).6)

labeled mesaconic acid

A particularly elegant example of hydrogen isotopic labeling is a synthesis of chiral acetic acid (TDH*CCO2H), which successfully utilized tritiation of a lithiated acetylide followed by a controlled catalytic deuterogenation. The intermediate then was induced to undergo a [1,3] hydride shift, after which ozonolysis afforded the target labeled acetic acid, as shown below:11)

Synthesis of chiral T- and D-labeled acetic acid

Reagents & conditions
a: n-BuLi, THF; b: T2O; c: D2 (1 bar)/Pd-BaSO4, MeOH, quinoline; d: 2 eq NaH, DMF, 80°C, 15 h (58% for 3 steps); e: O3, H2O2, CH2Cl2 (87%)

Hydrogen isotopes can also be introduced via reduction with isotopic hydride reagents, transition metal-mediated exchange. An example with NaBD4 is shown in the following reduction of an iminium salt:9)

Use of Sodium Borodeuteride

Another option is to incorporate a suitably labeled commercially available reagent such as methyl iodide (CD3I, CT3I), or the derived organometallic reagents such as organolithiums and organomagnesium halides.

Carbon isotopes (11C, 13C, 14C)

14C labeling is one of the subjects of a recent monograph.14)

The ultimate source for the radioactive carbon isotopes (11C & 14C) is isotopic carbon monoxide or dioxide produced through nuclear reaction. Labeled CO2 can then be reduced to methanol, for example, and treatment of the labeled methanol with HI affords labeled methyl iodide. The rapidity of the procedure is especially important in the case of the short-lived 11C, and a preparation is shown below in which [11C]methyl iodide was routinely prepared in 70%-95% radiochemical yield from [11C]carbon dioxide within 3-5 min after its generation.18) This reagent can then be used in the preparation of L-[methyl-11C]methionine.

labeled methionine

The use of HI adsorbed on alumina is another rapid and high-yielding (97%) procedure.13) Labeled methanol can be converted into the triflate to provide a more reactive methylating agent, and labeled methyl triflate can also be generated by the reaction of labeled methyl iodide in the gas phase with silver triflate on a solid support.

Doubly labeled (13C, 2H) methyl sulfate is commercially available as a methylating agent, and its efficient use in the preparation of a drug metabolite intermediate is shown below:45)

Use of labeled methyl sulfate

A preparation of doubly 14C-labeled glycerin has been published starting from Ba[14C]CO3. The overall yield is quite low (RY = 6% from Ba[14C]CO3), although this might be due to the fact that the intermediates and final product are quite difficult to handle and isolate without material losses.37)

labeled glycerin

[14C]Paraformaldehyde is available commercially, and can be used in heterocycle condensations such as the Pictet-Spengler cyclization with tryptamine and its analogs. Two examples were published in which 52-67% yields were obtained for the cyclization.43)

Radiolabeled nitro[14C]methane can be prepared via the reaction of [14C]methyl iodide with silver nitrite in a vacuum line system in 84% yield.40)

Labeled CO2 can also be reacted with an organometallic reagent to generate a labeled organic carboxylate. An example of this is the synthesis of [carboxyl-14C]3,4-dehydro-DL-proline, in which metalated pyrrole is carboxylated with [14C]CO2 (generated from barium [14C]carbonate and concentrate sulfuric acid) as the limiting reagent. This sequence afforded the 14C-labeled amino acid derivative in 37% overall:27)

Use of labeled carbon dioxide

11C-labeled diazomethane has been produced from 11C-methane that is generated in a cyclotron and then converted into 11C-chloroform by chlorination on pumice stone impregnated with CuCl2 at 310 °C. This is next reacted with hydrazine and potassium hydroxide in ethanol, followed by diazotization. This synthesis of [11C]CH2N2 gave a 30% yield from [11C]CH4 and was complete 10 min after the end of bombardment.15)

14C-labeled diazomethane can be generated from a suitably labeled precursor. [14C]Methyl amine was prepared by the Gabriel synthesis with [14C]methyl iodide, and this was used to produce the precursor as shown below. This material was used in Arndt-Eistert reactions to prepare labeled adriamycin analogs for ADME studies.34)

labeled diazomethane

Sodium [14C]cyanide is commercially available, and can be utilized to prepare copper [14C]cyanide, which was used in aryl halide displacements as shown below at left.41) The corresponding copper [13C]cyanide is also commercially available, and an example of an aryl halide displacement is shown at below right.45)

Use of labeled sodium cyanide

C-2 labeled malonic acid ([14C]CH2(CO2)2) is commercially available, and has been employed in the Knoevenagel reaction to generate cinnamic acid derivatives that were utilized in the synthesis of H3 receptor antagonists. In addition to the example shown, p-trifluoromethoxybenzaldehyde gave the condensation product in 89% yield:44)

Use of labeled malonic acid

14C-labeled CO2 can be stored by adsorbing into molecular sieves (analogous to the tritium manifold) and then used quantitatively by warming to release and cooling to readsorb.

The natural abundance of 13C is 1.1%, and this stable isotope can be “harvested” by fractionation methods, for example the fractional distillation of carbon monoxide, to obtain a labeled precursor.5) Once it has been fractionated, the CO can be converted into various carbon synthons for further use. 13C can be also prepared radiochemically, typically through proton bombardment of nitrogen, as in the attack by protons at 10 mA upon N2/5% H2 over 1 min.12)

One of the simplest forms of carbon isotopic labeling of a compound is through the introduction of a methyl group, and this subject has been reviewed.12) In cases where the parent compound is readily available, the conversion can be effected through demethylation and remethylation with a labeled reagent.

The [11C]carbon monoxide can be utilized in a wide variety of palladium-catalyzed aryl and vinyl coupling reactions using the Långström high-pressure micro-autoclave, with the products determined by the coreactants.1) For example, amines and hydrazine produces amides and hydrazides:23)

Use of labeled carbon monoxide

A similar procedure with an aqueous base quench yields the corresponding arylcarboxylic acids.28) Application to 2-bromobenzamides produced the corresponding phthalimides. In the case shown, the same reaction was carried out with [11C]CO and later with [13C]CO, the latter to serve for verifying the position of the label using 13C NMR. The yield in the latter case with 13C was 48%:31)

labeled phthalimide

Suzuki coupling with [11C]CO or [13C]CO insertion has been demonstrated, as shown in the 11C example below:33)

Insertion of labeled carbon monoxide

A complete protocol has been described in detail for the reaction of [11C]CO with an alkyl halide and water under UV radiation to produce carboxylic acids.22)

labeled carbon acids

In addition to the use of labeled CO2 in the synthesis of marked compounds, there are other one-carbon synthons such as [11C]methyl iodide (which can be transformed into a labeled methyl organometallic) and [11C]cyanide. This latter can be utilized in a variety of reactions including palladium-catalyzed aryl halide displacement, aziridines opening, and hydrocyanation.1)


Nitrogen (13N)

13N is a relatively short-lived isotope (half-life: 9.97 min) that is used in PET studies. It is generated in the form of [13N]NH4+ by bombarding mixtures of water/ethanol (H2O) with high energy protons, where the presence of ethanol serves to decrease the amount of [13N]NO2- and [13N]NO3- generated as nuclear side products. These other radiochemical products can also be employed in synthesis, as shown below in the case of the diazo compound below, with a total synthesis time of 13 min.25)

labeled nitrogen

Phosphorus (32P)

32P is used in tumor imaging and radiotherapy. Radiolabeling with 32P is predominantly applied to the incorporation of labeled phosphate into nucleic acids and phospholipids. This is done with [32P]ATP, [32P]cyclic AMP, or [32P]cPc (cytidine 3',5'-bis(phosphate)), or inorganic [32P]orthophosphate in biochemical preparations. There are some reagents for doing 32P labeling in a manner more familiar to organic chemists, such as the use of dibenzyl-[32P]phosphonate to conduct a reductive phosphorylation of plastoquinone as shown below:46)

Use of labeled dibenzylphosphonate

Due to the scarcity of commercial 32P-labeled reagents, some workers have developed their own preparations as shown below for a procedure to obtain ethyl[32P]phosphonic acid to be used in microbial metabolism studies:29)

Use of labeled phosphonic acid


Oxygen (15O)

The extremely short half-life of 15O (122 s) means that is cannot be used to radiolabel a substrate. It is used in medical diagnosis in the form of [15O]O2 and [15O]H2O for inhalation studies.

Sulfur (33S, 34S, 35S, 36S)

A preparation of the low-abundance, stable isotope-labeled [34S]dibenzothiophene was developed so that it could be used as a standard in the GC-ICP-IDMS analysis of petroleum products, using a standard condensation with 34S-enriched sulfur. The yield of 67% was based on sulfur, and accounting for the 34SH2 produced as a side product:24)

labeled dibenzothiophene

Methane[35S]sulfonyl chloride was prepared to radiolabel an orally active spiropiperidine-based GH secretagogue, using the sequence depicted below:39)

labeled spiropiperidine

Methane[35S]sulfonic acid is now available commercially, making labeled mesyl chloride more accessible.

Radiolabeled elemental sulfur can be employed in the synthesis of aryl[35S]sulfonyl chlorides. An aryl halide is converted to the corresponding Grignard reagent, which is reacted with [35S]S8 to give the aryl [35S]mercaptan, which is oxidized to the target aryl[35S]sulfonyl chloride:42)

labeled sulfonyl chloride


18F is possibly the most widely used radiolabel in PET imaging. 36Cl is used in the form of chloramine, NH2[36Cl]Cl, to monitor the metabolic fate of chloramine. Its extremely long half-life (3.01 × 105 y) makes it useful in the monitoring of groundwater on geologic time scales, and it has also been used to monitor diffusion related to nuclear weapons detonations. 123I is especially preferred for use in tumor imaging, 125I is utilized for the short-range detection of tumor margins, and 131I is used in tumor therapy.

The topic of radiohalogen incorporation into organic systems has been reviewed.30) Isotopic halogens are relatively easily incorporated through displacement reactions using the halide form, and alternatively suitable forms can be used in electrophilic reactions.

Fluorine (18F)

18F-fluorinating agents include [18F]F2 and CH3COO[18F]F can be reacted with a nucleophilic substrate, as shown below for the use of radiolabeled molecular fluorine in the synthesis of 6-[18F]fluoro-L-DOPA. The total synthesis time, including cooling, trapping of [18F]F2 and HPLC purification, was 45 min., of which approximately 15 min. was required for quality control. This preparation has been implemented as a one-pot procedure in a fully automated synthesis module:26)

labeled fluoro-L-dopa

18F is used for PET imaging in the form of the ligand [18F]fludeoxyglucose or [18F]fluorodeoxyglucose, commonly abbreviated 18F-FDG or FDG. This compound is useful in imaging tumors based on enhanced glucose consumption in the affected sites, but also more generally to image metabolism. It is produced by nucleophilic displacement of triflate in the mannose derivative shown below by [18F]KF in the presence of the Kryptofix 222.16) The base hydrolysis step can be carried out in a reverse-phase chromatography system using NaOH(aq) as the mobile phase, thus combining this final step with purification. A modification of this conversion that employs an ionic liquid as solvent for the displacement step has also been developed; this makes solvent evaporation unnecessary and gives an overall yield of 60%.17)

labeled fluoro-mannose

Other workers compared 18F-fluorinating agents for the iododestannylation reaction to make 18F-FDG, and [18F]F2 gave a 25-33% yields while CH3COO[18F]F gave 8%.36)

Displacement by [18F]F- on aromatic substrates has also been employed, including diaryliodonium salts.32)

Chlorine (36Cl)

36Cl has been used in a mechanistic study of butadiene polymerization, where Ni(36Cl)O2CCF3 was used to initiate the polymerization in the same manner as a nickel π-allyl complex, and the 36Cl radiolabel was detected in the polymer.21)

36Cl-labeled vinyl chloride has been prepared for use in polymer degradation studies.38) This particular pathway is wasteful of label in that half the radiolabeled chlorine is discarded.

labeled vinyl chloride

Bromine (76Br)

Halide-organotin displacement serves to introduce 76Br into the furfural molecule.7)

labeled furfural

Iodine (123I, 125I, 131I)

An excellent review is available on the topic of radioiodination techniques for small organic molecules.8) These techniques include a variety of electropositive iodine sources, such as molecular iodine, iodine monochloride, NaI or KI with chloramine-T or iodogen, electrolytic methods, and enzymatic methods; nucleophilic exchange in solution or melt, iododediazonization, iododeboronation, iododestannylation, iododesilation, and iododethallation.

An example of the introduction of 125I via iododestannylation is shown below in the preparation of a 5-pyrrolidinylsulfonyl isatin derivative used as an imaging agent for caspases in apoptosis.10)

labeled 5 pyrrolidinylsulfonyl isatin derivative