Radiosynthesis of [18F]DPA‐714, a selective radioligand for imaging the translocator protein (18 kDa) with PET

Recently, a novel series of 2‐phenylpyrazolo[1,5‐a]pyrimidineacetamides has been reported as selective ligands of the translocator protein (18 kDa). Within this series, DPA‐714 (N,N‐diethyl‐2‐(2‐(4‐(2‐fluoroethoxy)phenyl)‐5,7‐dimethylpyrazolo[1,5‐a]pyrimidin‐3‐yl)acetamide, K_i=7.0 nM) is a compound, which had been designed with a fluorine atom in its structure, allowing labelling with fluorine‐18 (half‐life: 109.8 min) and in vivo imaging using positron emission tomography. DPA‐714 and its tosyloxy derivative (N,N‐diethyl‐2‐(2‐(4‐(2‐toluenesulfonyloxyethoxy)phenyl)‐5,7‐dimethylpyrazolo[1,5‐a]pyrimidin‐3‐yl)acetamide) as precursor for the labelling with fluorine‐18 were synthesized in two steps from DPA‐713 (N,N‐diethyl‐2‐(2‐(4‐methoxyphenyl)‐5,7‐dimethylpyrazolo[1,5‐a]pyrimidin‐3‐yl)acetamide) and obtained in 32 and 42% yields, respectively. [¹⁸F]DPA‐714 was synthesized using a simple one‐step process (a tosyloxy‐for‐fluorine nucleophilic aliphatic substitution), which has been fully automated on our Zymate‐XP robotic system. It involves: (A) reaction of K[¹⁸F]F‐Kryptofix^®222 with the tosyloxy precursor (4.5–5.0 mg, 8.2–9.1 µmol) at 165°C for 5 min in dimethyl sufloxide (0.6 mL) followed by (B) C18 PrepSep cartridge pre‐purification and finally (C) semi‐preparative high‐performance liquid chromatography (HPLC) purification on a Waters X‐Terra? RP18. Typically, 5.6–7.4 GBq of [¹⁸F]DPA‐714 (>95% chemically and radiochemically pure) could be obtained with specific radioactivities ranging from 37 to 111 GBq/µmol within 85–90 min (HPLC purification and SepPak^®‐based formulation included), starting from a 37 GBq [¹⁸F]fluoride batch (overall non‐decay‐corrected and isolated radiochemical yield: 15–20%). Copyright © 2008 John Wiley & Sons, Ltd.     

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 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.     

Synthesis and characterization of [N‐methyl‐3H]loperamide

Loperamide is a piperidine butyramide mu‐opiate receptor agonist and currently employed to treat diarrhea. Because a single past report of tritiating loperamide was limited to only a very low specific activity product without technical details or extensive analysis, the synthesis of [N‐methyl‐³H]loperamide at high specific activity is now described in detail. An imine precursor was alkylated with [³H]methyl iodide to obtain a quaternary intermediate, which was then reacted with 4‐(4‐chlorophenyl)‐4‐hydroxypiperidine to afford the desired product [N‐methyl‐³H]loperamide, characterized by thin layer chromatography (TLC), HPLC, MS, UV, and proton‐decoupled tritium NMR.     

Enzymatic synthesis of l‐tryptophan and 5prime‐hydroxy‐l‐tryptophan labeled with deuterium and tritium at the alpha‐carbon position

The enzymatic synthesis of l‐tryptophan and its derivative 5′‐hydroxy‐l‐tryptophan labeled with deuterium and tritium at the α‐carbon position is reported. The mixture containing S‐methyl‐l‐cysteine, indole or 5‐hydroxyindole dissolved in deuteriated or tritiated water has been converted to [2‐²H]‐l‐tryptophan, [2‐³H]‐l‐tryptophan, 5′‐hydroxy‐[2‐²H]‐l‐tryptophan, and 5′‐hydroxy‐[2‐³H]‐l‐tryptophan, respectively, in a one‐pot reaction using the enzyme tryptophanase. The same reaction carried out in heavy water with THO added yielded either doubly labeled [2‐²H/³H]‐l‐tryptophan or 5′‐hydroxy‐[2‐²H/³H]‐l‐tryptophan. Copyright © 2003 John Wiley & Sons, Ltd.     

Syntheses of halogen derivatives of L‐tryptophan,L‐tyrosine and L‐phenylalanine labeled with hydrogen isotopes

Halogenated, labeled with tritium and doubly with deuterium and tritium, derivatives of L ‐tryptophan, i.e. 5′‐bromo‐[2‐³H]‐, 5′‐bromo‐[2‐²H/³H]‐, 5′‐fluoro‐[2‐³H]‐5′‐fluoro‐[2‐²H/³H]‐, 6′‐fluoro‐[2‐³H]‐, 6′‐fluoro‐[2‐²H/³H]‐L ‐tryptophan, as well as, L ‐tyrosine, i.e. 3′‐fluoro‐[2‐³H]‐, 3′‐fluoro‐[2‐²H/³H]‐, 3′‐chloro‐[2‐³H]‐, and 3′‐chloro‐[2‐²H/³H]‐L ‐tyrosine, and also L ‐phenylalanine, i.e. 2′‐fluoro‐[(3S)‐³H]‐, 2′‐fluoro‐[(3S)‐²H/³H]‐, 2′‐chloro‐[(3S)‐³H]‐, 2′‐chloro‐[(3S)‐²H/³H]‐, 4′‐chloro‐[(3S)‐³H]‐, and 4′‐chloro‐[(3S)‐²H/³H]‐L ‐phenylalanine were synthesized using enzymatic methods. Isotopomers of L ‐tryptophan were synthesized by coupling of halogenated indoles with S‐methyl‐L ‐cysteine carried out in deuteriated or tritiated incubation media. Labeled halogenated derivatives of L ‐tyrosine were obtained by the enzymatically supported exchange between halogenated L ‐tyrosine and isotopic water. Labeled halogenated isotopologues of L ‐Phe were synthesized by the enzymatic addition of ammonia to halogenated cinnamic acid. As a source of hydrogen tritiated water (HTO) and heavy water (D₂O) with addition of HTO were used.     

Synthesis of 3H‐, 14C‐, and stable‐isotope‐labelled galantamine

Reminyl^® is a newly approved drug, used in the treatment of mild to moderate Alzheimer disease. The active compound, galantamine, was initially isolated from the bulbs of certain Narcissus species, but is at the moment also produced synthetically. In the process leading to the final approval, the synthesis of tritium‐, carbon‐14‐ and stable‐isotope‐labelled galantamine for pharmacokinetic studies was required. Racemic (±)‐1‐bromonarwedine, a compound available as intermediate from the commercial synthesis, was transformed to racemic 1‐bromo‐galantamine. Catalytic bromo‐tritium exchange, followed by HPLC purification and resolution afforded tritium‐labelled galantamine. The [¹⁴C]‐label was introduced on the nitrogen as well as on the oxygen‐methyl position. This was achieved by N‐ and O‐demethylation of galantamine and reaction of the thoroughly purified intermediate with [¹⁴C]‐methyl iodide. Stable‐isotope‐labelled galantamine was obtained likewise by ¹³CD₃OD‐methylation of O‐demethylated galantamine under Mitsunobu conditions. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of tritium‐labelled 3′‐azido‐3′‐deoxythymidine

New approaches to the synthesis of 3′‐azido‐3′‐deoxythymidine labelled with tritium in the heterocyclic base have been developed. With this aim, enzymatic transribosylation with [³H]thymine using the enzyme preparation from rat liver and a three‐step chemical synthesis with use of the tritium labelled precursor were studies. The enzyme preparation did not catalyse the transfer of the 3′‐azido‐3′‐deoxyribosyl fragment to the [³H]thymine residue. 5′‐O‐Benzoyl‐2,3′‐anhydrothymidine was taken as a precursor for the tritium labelling by the chemical methods. The resulting [³H]3′‐azido‐3′‐deoxythymidine was obtained with a specific radioactivity of 18.3 Ci/mmol, the tritium is located in the C‐6 position of the thymine residue. Copyright © 2003 John Wiley & Sons, Ltd.     

Labelling of anilines,benzylamines and some N‐heterocyclics using cycloocta‐1,5‐dienyliridium(I)‐1,1,1,5,5,5‐hexafluoro‐pentan‐2,4‐dionate and isotopic hydrogen gas in DMF or DMA

A wide range of anilines, benzylamines and some N‐heterocyclics can be ortho‐deuterated at room temperature using deuterium gas and cycloocta‐1,5‐dienyliridium(I)‐1,1,1,5,5,5‐hexafluoropentan‐2,4‐dionate in DMF or DMA. The method is applicable to labelling with tritium. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis and evaluation of 5,7‐dichloro‐4‐(3‐{4‐[4‐(2‐[18F]fluoroethyl)‐piperazin‐1‐yl]‐phenyl}‐ureido)‐1,2,3,4‐tetrahydroquinoline‐2‐carboxylic acid as a potential NMDA ligand to study glutamatergic neurotransmission in vivo

The neurotransmitter glutamate is thought to be crucially involved in a huge number of neurological and psychiatric disorders, such as Morbus Parkinson, Alzheimer's disease and schizophrenia. Aiming at an improved diagnostic tool for PET a new [¹⁸F]fluorine labelled NMDA receptor ligand was developed that may potentially allow the in vivo visualization of glutama‐tergic neurotransmission. The ¹⁹F‐analogue trans‐5,7‐dichloro‐4‐(3‐{4‐[4‐(2‐fluoroethyl)‐piperazin‐1‐yl]‐phenyl}‐ureido)‐1,2,3,4‐tetrahydro quinoline‐2‐carboxylic acid was synthesised to determine the binding affinity, lipophilicity and biodistribution of the ligand. This substance exhibits a K_i of 12 nM for the glycine binding site using [³H]MDL‐105,519 assays on pig cortical membranes. A logD of 1.3 was determined for this compound according to the OECD guidelines employing the HPLC method. Radiosynthesis of this ligand was achieved by labelling the precursor trans‐5,7‐dichloro‐4‐[3‐(4‐piperazin‐1‐yl‐phenyl)‐ureido]‐1,2,3,4‐tetrahydroquinoline‐2‐carboxylic acid methyl ester with 2‐[¹⁸F]fluoroethyltosylate and subsequent cleaving of the methyl ester moiety, resulting in an overall decay corrected yield of 35% of the final product trans‐5,7‐dichloro‐4‐(3‐{4‐[4‐(2‐[¹⁸F]fluoroethyl)‐piperazin‐1‐yl]‐phenyl}‐ureido)‐1,2,3,4‐tetrahydroquinoline‐2‐carboxylic acid. The biodistribution kinetics of this compound were determined with Sprague Dawley rats ex vivo for brain, liver, kidney, and bone. The ligand showed a maximum brain uptake 30 min.p.i. of about 0.1% ID/g. Copyright © 2003 John Wiley & Sons, Ltd.     

Preparation of tritium‐labeled PF‐622, a novel fatty acid amide hydrolase inhibitor

To make a detailed characterization of the mechanism of inhibition and selectivity of a novel fatty acid amide hydrolase inhibitor PF‐622 , 3 tritium isotopomers were prepared. [³H] PF‐622a labeled at the piperazine ring B and [³H] PF‐622b labeled at both the ring B and phenyl ring A were synthesized via catalytic H(hydrogen)‐T(tritium) exchange, utilizing 1 equiv and excess of Crabtree's catalyst, respectively. The preparation of [³H] PF‐622c labeled only at the phenyl ring A was achieved via tritiodebromination of the bromide precursor, using Pd(PPh₃)₄ as a catalyst. The observations from these tritiation reactions might open a new perspective in the labeling for the targets having a similar moiety.     

[18F]DPA‐C5yne,a novel fluorine‐18‐labelled analogue of DPA‐714: radiosynthesis and preliminary evaluation as a radiotracer for imaging neuroinflammation with PET

DPA‐C5yne, the lead compound of a novel series of DPA‐714 derivatives in which the fluoroethoxy chain linked to the phenylpyrazolopyrimidine scaffold has been replaced by a fluoroalkyn‐1‐yl moiety, is a high affinity (K_i: 0.35 nM) and selective ligand targeting the translocator protein 18 kDa. In the present work, DPA‐C5yne was labelled with no‐carrier‐added [¹⁸F]fluoride based on a one‐step tosyloxy‐for‐fluorine nucleophilic substitution reaction, purified by cartridge and HPLC, and formulated as an i.v. injectable solution using a TRACERLab FX N Pro synthesizer. Typically, 4.3–5.2 GBq of [¹⁸F]DPA‐C5yne, ready‐to‐use, chemically and radiochemically pure (> 95%), was obtained with specific radioactivities ranging from 55 to 110 GBq/µmol within 50–60 min, starting from a 30 GBq [¹⁸F]fluoride batch (14–17%). LogP and LogD of [¹⁸F]DPA‐C5yne were measured using the shake‐flask method and values of 2.39 and 2.51 were found, respectively. Autoradiography studies performed on slices of ((R,S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolopropionique (AMPA)‐lesioned rat brains showed a high target‐to‐background ratio (1.9 ± 0.3). Selectivity and specificity of the binding for the translocator protein was demonstrated using DPA‐C5yne (unlabelled), PK11195 and Flumazenil (central benzodiazepine receptor ligand) as competitors. Furthermore, DPA‐C5yne proved to be stable in plasma at 37°C for at least 90 min.     

Labelling of the guanylate cyclase activator cinaciguat (BAY 58‐2667) with carbon‐14, tritium and stable isotopes

For studies of pharmacokinetics and drug metabolism of the new soluble guanylate cyclase activator cinaciguat (BAY 58‐2667) the ¹⁴C‐labelled compound was synthesized. The tritiated compound was required to elucidate the mode of action and the stable labelled compound was required for bio‐analytical studies by quantitative mass spectrometry as well. Two radiosyntheses are described with different formation of the labelled intermediate 1‐(chloro[¹⁴C]methyl)‐4‐(2‐phenylethyl)benzene. The first one started with ¹⁴C‐carboxylation of 1‐bromo‐4‐(2‐phenylethyl)benzene yielding the desired product in 5 steps. In the second synthesis intermediate 1‐(chloro[¹⁴C]methyl)‐4‐(2‐phenylethyl)benzene was formed by chloromethylation of bibenzyl with [¹⁴C]paraformaldehyde/hydrochloric acid subsequently resulting in the final product in three steps. Tritium labelling was performed by tritium exchange of the diester intermediate using an organo‐iridium catalyst and subsequent saponification. The stable labelled compound was synthesized via a convergent synthesis starting with ¹³C,¹⁵N‐cyanation of 1‐(chloromethyl)‐2‐{[4‐(2‐phenylethyl)benzyl]oxy}benzene and ¹³C‐cyanation of methyl 4‐bromobenzoate, respectively. The labelled product was obtained after 7 chemical steps. Copyright © 2010 John Wiley & Sons, Ltd.     

N‐2‐(4‐N‐(4‐[18F]Fluorobenzamido)phenyl)‐propyl‐2‐propanesulphonamide: synthesis and radiofluorination of a putative AMPA receptor ligand

Arylpropylsulphonamides are in the focus of research as α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolpropionic acid (AMPA) receptor ligands. A new fluorine‐18‐labelled potentiator of AMPA receptors was synthesized as a potential radiotracer for cerebral imaging with positron emission tomography. Using N‐2‐(4‐N‐(4‐nitrobenzamido)phenyl)‐propyl‐2‐propanesulphonamide ( 7 ) as labelling precursor for a Kryptofix 2.2.2^®/K₂CO₃‐activated nucleophilic radiofluorination, the putative AMPA receptor ligand N‐2‐(4‐N‐(4‐[¹⁸F]fluorobenzamido)phenyl)‐propyl‐2‐propanesulphonamide [¹⁸F] 8 was obtained in one step. Optimization of the reaction parameters time, temperature, solvent and concentration gave a radiochemical yield of 38±8% at 180°C in dimethylsulphoxide within 30‐min reaction time. After a solid‐phase extraction followed by a high‐performance liquid chromatography separation, the product could be obtained in radiochemical yields of 5±1.5%. Radiochemical purity was higher than 95% and the specific activity amounted to 77±40 GBq/µmol. First in vitro assays with rat brain slices revealed a high non‐specific binding and a uniform distribution of [¹⁸F] 8 not lending it for in vivo imaging purposes. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis of [3α‐3H] 17α‐Hydroxy pregnenolone and [3α‐3H] Pregnenolone

For the first time, [3α‐³H] 17α‐hydroxy pregnenolone (1) was synthesized through a multiple step sequence. The presence of [3β‐³H] isomer in RP‐HPLC purified product was identified by tritium NMR. The [3β‐³H] isomer was then separated from [3α‐³H] 17α‐hydroxy pregnenolone with chiralPAK AD‐H column. [3α‐³H] pregnenolone (2) was synthesized from commercial available 5‐pregnen‐3,20‐dione in one step with an improved procedure.     

A convenient synthesis of the muscarinic cholinergic antagonist (R)‐[N‐methyl‐3H]quinuclidinyl benzilate methiodide

A useful synthesis of (R)‐[N‐methyl‐³H]quinuclidinyl benzilate methiodide is described with the product characterized by thin‐layer chromatography (TLC), high‐performance liquid chromatography (HPLC), tritium nuclear magnetic resonance (NMR), and mass spectrometry (MS). Several methods are provided to purify the radioligand, and its storage and stability are also discussed.     

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 and in vitro evaluation of 18F‐β‐carboline alkaloids as PET ligands

A one‐step ¹⁸F‐labelling strategy was used to prepare four ¹⁸F‐labelled analogues of 7‐methoxy‐1‐methyl‐9H‐β‐carboline (harmine): 7‐(2‐[¹⁸F]fluoroethoxy)‐1‐methyl‐9H‐β‐carboline (5), 7‐(3‐[¹⁸F]fluoro‐propoxy)‐1‐methyl‐9H‐β‐carboline (6), 7‐[2‐(2‐[¹⁸F]fluoroethoxy)ethoxy]‐1‐methyl‐9H‐β‐carboline (7), and 7‐{2‐[2‐(2‐[¹⁸F]fluoroethoxy)ethoxy]‐ethoxy}‐1‐methyl‐9H‐β‐carboline (8). These were synthesized as potential positron emission tomography ligands for monoamine oxidase A (MAO‐A). A solution of pure labelled compound in buffer was obtained in <70 min from end of radionuclide production, with a decay‐corrected yield of up to 23%. The average specific binding to MAO‐A in rat brain, determined by autoradiography experiments, was highest for compounds 7 and 8 (89±2 and 96±1%, respectively), which was obtained at <1 nM radioligand concentration. Copyright © 2008 John Wiley & Sons, Ltd.     

Carbon‐11 labelling of an N‐sulfonylamino acid derivative: a potential tracer for MMP‐2 and MMP‐9 imaging

Matrix metalloproteinases (MMPs) are a family of proteinases that play an important role in cancer. Non invasive imaging of MMPs would allow the evaluation of MMP activity in cancer and the assessment of the response of MMP inhibitor based therapies. In this paper, we investigated the synthesis and labelling by methylation with [¹¹C]CH₃I of an N‐sulfonylamino acid derivative, the (2R)‐3‐methyl‐2‐[[4‐[(4‐methoxybenzoyl)amino]benzenesulfonyl]amino] butanoic acid, a selective and high potent MMP‐2 and MMP‐9 inhibitor, for cancer imaging with positron emission tomography. Labelling of (2R)‐3‐methyl‐2‐[[4‐[(4‐hydroxybenzoyl)amino]benzenesulfonyl] amino] butanoic acid was carried out in a radiochemical yield of 50%–60% within 40 min with a specific activity of 11–26 GBq/µmol (EOS). In vitro inhibitory activity studies, biodistribution and in vivo serum stability in normal mice are also described. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis of isotopically labeled 1,3‐dithiane

The 1,3‐dithiane is a protected formaldehyde anion equivalent that could serve as a useful labeled synthon. We report a facile synthesis of 1,3‐[2‐¹³C]‐ and 1,3‐[2‐¹³C, 2‐²H₂]dithiane in two steps from [¹³C]‐ or [¹³C, ²H₃]methyl phenyl sulfoxide. We have previously reported the high yield synthesis of [¹³C]methyl phenyl sulfide from [¹³C]MEOH and the oxidation of [¹³C]methyl phenyl sulfide to [¹³C]methyl phenyl sulfoxide. Here, we describe the facile exchange of deuterium from ²H₂O into [¹³C]methyl phenyl sulfoxide to yield [¹³C, ²H₃]methyl phenyl sulfoxide. Thus, from [¹³C]MEOH and ²H₂O, all possible C2 stable isotopomers of 1,3‐dithiane are available. Our synthetic route is also amenable to preparation of radiolabeled 1,3‐dithianes. Copyright © 2014 John Wiley & Sons, Ltd.