Synthesis of [H]- and -[C]-labelled sulprostone (CP-34,089/ZK-57,671)

The synthesis of N-methanesulfonyl 16-phenoxy-ω-tetranor PGE₂ carboxamide (sulprostone — CP-34,089/ZK-57,671) labeled with tritium and carbon-14 is described. Sulprostone labeled with tritium in the phenoxy moiety by means of catalytic hydrogenolysis was obtained in a 17% radiochemical yield with a specific activity of 1.0 Ci/mmol. The methanesulfonamide-¹⁴C derivative of sulprostone was prepared from methyl-¹⁴C iodide in an 11.8% radiochemical yield having a specific activity of 18.8 mCi/mmol.     

Synthesis of tritium-labeled N-acetyl PGE2 carboxamide (CP-27,987)

The preparation of N-acetyl PGE₂ carboxamide (CP-27,987) regioselectively labeled at C-18 and C-19 with tritium is described. The overall radiochemical yield was 8.0% at a specific activity of 0.49 Ci/mmole. The synthesis employed is applicable to the preparation of tritium labeled natural prostaglandins and a variety of analogs.     

Radiosynthesis of [11C]Vandetanib and [11C]chloro-Vandetanib as new potential PET agents for imaging of VEGFR in cancer

Vandetanib (ZD6474) and its chlorine analogue chloro-Vandetanib are potent and selective vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors with low nanomolar IC₅₀ values. [¹¹C]Vandetanib and [¹¹C]chloro-Vandetanib, new potential PET agents for imaging of VEGFR in cancer, were first designed, synthesized and labeled at nitrogen and oxygen positions from their corresponding N- and O-des-methylated precursors, in 40-50% decay corrected radiochemical yield and 370-555 GBq/μmol specific activity at end of bombardment (EOB).     

The synthesis of a radioactive cytokinin with high specific activity

6-benzylaminopurine-[p-¹H-benzyl] at a specific activity of 10 Ci/mmol was synthesized by reacting p-bromo-6-benzylaminopurine with carrier-free tritium gas in the presence of 10% Pd/C. A radiochemical purity of 97% was obtained by a one-step purification of the tritiated reaction product using cellulose TLC. This simple procedure yields the highly active cytokinin, 6-benzylaminopurine, with tritium at near maximum specific activity in a known, stable position.     

Synthesis and in vivo characterization of d-(+)-(N1-[11C]methyl)-2-Br-LSD: a radioligand for positron emission tomographic studies of serotonin 5-HT2 receptors

d-(+)-Nl-Methyl-2-Br-LSD (MBL), which displays high affinity and selectivity for serotonin receptors in vitro, has been labeled with carbon-11 for localization of cerebral serotonin 5-HT₂ receptors by positron emission tomography. [¹¹C]MBL was prepared from [¹¹C]iodomethane and d-(+)-2-Br-LSD within 20 min from end of bombardment. The average specific activity of [¹¹C]MBL was 2300 mCi/μ mol and the average radiochemical yield was 17%, both at end of synthesis. The in vivo regional distribution of radioactivity in brain after i.v. administration of [¹¹C]MBL to mice paralleled the known density of serotonin 5-HT₂ receptors. The maximum specific binding, defined by a frontal cortex to cerebellum radioactivity concentration ratio of 5.4 to 1, was reached 30 min postinjection. Administration of ketanserin, a potent serotonin 5-HT₂ receptor antagonist, markedly blocked radioligand binding in all brain regions examined except cerebellum.     

Synthesis of 6-[18F]fluoropyridoxal and radioactivity distribution in rat at 60 min

6-[¹⁸F]Fluoropyridoxal was synthesized by the flourination of a propylamine derivative of pyridoxal (pyridoxal Schiff base) with ¹⁸F-labelled acetylhypofluorite. Two different fluorinating agents, 5% F₂ in N₂ and acetylhypofluorite, were investigated with nonradioactive material. The evaluation of reactions in CH₃CN and chloroform showed CH₃CN to be the better solvent and CH₃COOF to be the better fluorinating reagent. The synthesis gave a radiochemical yield of about 18% (expressed at the end of synthesis) and required 35–40 min to complete. The specific activity of the final radiopharmaceutical at the end of the synthesis was about 25.9 GBq/mmol (700 mCi/mmol).The tissue distribution of 6-fluoropyridoxal in rat at 60 min is also reported. A large concentration in liver and kidney indicates that this radiopharmaceutical could be of special interest in the imaging of liver functions. The concentration in the brain might also allow in vivo PET imaging of the 6-(fluoropyridoxal) uptake if a high efficiency PET scanner is used.     

Synthesis of a fluorinated fatty acid,dl-erythro-9,10-[18F]difluoropalmitic acid,and biodistribution studies in rats

9,10-Difluoropalmitic acid (DFPA) labeled with the cyclotron produced, positron emitting radionuclide ¹⁸F has been synthesized as a potential analogue of 9,10-[³H]palmitic acid, a fatty acid which has been used to study lipid metabolism in rat brain and pituitary. [¹⁸F]DFPA was prepared by the direct and stereoselective addition of [¹⁸F]F₂ to the double bond of cis-9,10-palmitoleic acid. The fluorination was carried out in FCCl₃ at −70 °C using a low concentration of F₂ (0.5%) in neon. [¹⁸F]DFPA has been obtained in radiochemical yields of 12–16% from end-of-bombardment (EOB) in approx. 2.5 h. Chemical and radiochemical purity exceeded 95%, and specific activities calculated to EOB ranged from 500 to 1000 mCi/mmol. [¹⁸F]DFPA crosses the blood-brain barrier and is incorporated into rat brain at about twice the level of that of 9,10-[³H]palmitic acid. The synthesis of [¹⁸F]DFPA permits us to study the biological disposition and metabolism of a vicinal-difluoro fatty acid.     

A Rapid,High-Yield Method of Producing M 0-[12]a14 Iodoinsujlin

Abstract

We have developed a rapid method for producing homogeneous mono-[¹²]A₁₄ iodoinsulin with high specific activity and yield. After iodination by the lactoperoxidase method, the labeled peptides were applied to a C₁₈ Porasil^R prs-column, washed with aqueous buffer to eliminate the free [¹²]-iodide and placed “in-line” with a C-18 HPLC column; mono-[12]A₁₄ iodoinsulin was then eluted isocratically with 29% aceto-nitrile in 16 minutes. The labeled hormone was extremely stable, and proved suitable for various biological studies.     

Synthesis and preliminary biological evaluation of [11C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate for the fractalkine receptor (CX3CR1)

The reference standard methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate (5) and its precursor 2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucine (6) were synthesized from 6-amino-2-mercaptopyrimidin-4-ol and BnBr with overall chemical yield 7% in five steps and 4% in six steps, respectively. The target tracer [¹¹C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate ([¹¹C]5) was prepared from the acid precursor with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–50% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the specific activity (SA) at EOB was 370–1110 GBq/μmol with a total synthesis time of ～40-min from EOB. The radioligand depletion experiment of [¹¹C]5 did not display specific binding to CX₃CR1, and the competitive binding assay of ligand 5 found much lower CX₃CR1 binding affinity.     

Synthesis of [¹¹C]PBR06 and [¹⁸F]PBR06 as agents for positron emission tomographic (PET) imaging of the translocator protein (TSPO)

The translocator protein 18 kDa (TSPO) is an attractive target for molecular imaging of neuroinflammation and tumor progression. [¹⁸F]PBR06, a fluorine-18 labeled form of PBR06, is a promising PET TSPO radioligand originally developed at NIMH. [¹¹C]PBR06, a carbon-11 labeled form of PBR06, was designed and synthesized for the first time. The standard PBR06 was synthesized from 2,5-dimethoxybenzaldehyde in three steps with 71% overall chemical yield. The radiolabeling precursor desmethyl-PBR06 was synthesized from 2-hydroxy-5-methoxybenzaldehyde in five steps with 12% overall chemical yield. The target tracer [¹¹C]PBR06 was prepared by O-[¹¹C]methylation of desmethyl-PBR06 with [¹¹C]CH₃OTf in CH₃CN at 80 °C under basic condition and isolated by HPLC combined with SPE purification with 40–60% decay corrected radiochemical yield and 222–740 GBq/μmol specific activity at EOB. On the similar grounds, [¹⁸F]PBR06 was also designed and synthesized. The previously described Br-PBR06 precursor was synthesized from 2,5-dimethoxybenzaldehyde in two steps with 78% overall chemical yield. A new radiolabeling precursor tosyloxy-PBR06, previously undescribed tosylate congener of PBR06, was designed and synthesized from ethyl 2-hydroxyacetate, 4-methylbenzene-1-sulfonyl chloride, and N-(2,5-dimethoxybenzyl)-2-phenoxyaniline in four steps with 50% overall chemical yield. [¹⁸F]PBR06 was prepared by the nucleophilic substitution of either new tosyloxy-PBR06 precursor or known Br-PBR06 precursor in DMSO at 140 °C with K[¹⁸F]F/Kryptofix 2.2.2 for 15 min and HPLC combined with SPE purification in 20–60% decay corrected radiochemical yield, >99% radiochemical purity, 87–95% chemical purity, and 37–222 GBq/μmol specific activity at EOB. Radiosynthesis of [¹⁸F]PBR06 using new tosylated precursor gave similar radiochemical purity, and higher specific activity, radiochemical yield and chemical purity in comparison with radiosynthesis using bromine precursor.     

The first synthesis of [11C]J147, a new potential PET agent for imaging of Alzheimer’s disease

J147 was synthesized from 2,4-dimethylphenylhydrazine hydrochloride and 3-methoxybenzaldehyde in 2 steps with 71% overall yield. The precursor desmethyl-J147 was synthesized from 3-hydroxybenzaldehyde and 2,4-dimethylphenylhydrazine hydrochloride in 4 steps with 63% overall yield. [¹¹C]J147 was prepared from desmethyl-J147 with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 35–50% radiochemical yield based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB), with 370–740 GBq/μmol specific activity at EOB.     

Stearoyl[1-14C]sulfogalactosylsphingosine ([14C]sulfatide) as substrate for cerebroside sulfatase assay

A convenient method for large scale preparation of stearoyl[1-¹⁴C]sulfogalactosylsphingosine has been developed. The first step consists in preparing the lysosulfatide intermediate with a good yield, which we have successfully performed. In the second step, the lysoderivative is coupled to [1-¹⁴C]stearic acid through the acyl chloride procedure. The labeled substrate thus synthetized appears to be particularly convenient for cerebroside sulfatase determination, because of the stability of the ¹⁴C isotope, compared to the other isotopes (tritium or ³⁵S) which have been previously used for the same purpose.     

3-(Cyclopropylmethyl)-7-((4-(4-[11C]methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine: Synthesis and preliminary evaluation for PET imaging of metabotropic glutamate receptor subtype 2

Selective metabotropic glutamate receptor 2 (mGluR2) inhibitors have been demonstrated to show therapeutic effects by improving alleviating symptoms of schizophrenic patients in clinical studies. Herein we report the synthesis and preliminary evaluation of a ¹¹C-labeled positron emission tomography (PET) tracer originating from a mGluR2 inhibitor, 3-(cyclopropylmethyl)-7-((4-(4-methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (CMTP, 1a). [¹¹C]CMTP ([¹¹C]1a) was synthesized by O-[¹¹C]methylation of desmethyl precursor 1b with [¹¹C]methyl iodide in 19.7 ± 8.9% (n = 10) radiochemical yield (based on [¹¹C]CO₂) with >98% radiochemical purity and >74 GBq/μmol molar activity. Autoradiography study showed that [¹¹C]1a possessed moderate in vitro specific binding to mGluR2 in the rat brain, with a heterogeneous distribution of radioactive accumulation in the mGluR2-rich brain tissue sections, such as the cerebral cortex and striatum. PET study indicated that [¹¹C]1a was able to cross the blood–brain barrier and enter the brain, but had very low specific binding in the rat brain. Further optimization for the chemical structure of 1a is necessary to increase binding affinity to mGluR2 and then improve in vivo specific binding in brain.     

A high-yield route to synthesize the P-glycoprotein radioligand [11C]N-desmethyl-loperamide and its parent radioligand [11C]loperamide

N-Desmethyl-loperamide and loperamide were synthesized from α,α-diphenyl-γ-butyrolactone and 4-(4-chlorophenyl)-4-hydroxypiperidine in five and four steps with 8% and 16% overall yield, respectively. The amide precursor was synthesized from 4-bromo-2,2-diphenylbutyronitrile and 4-(4-chlorophenyl)-4-hydroxypiperidine in 2 steps with 21–57% overall yield. [¹¹C]N-Desmethyl-loperamide and [¹¹C]loperamide were prepared from their corresponding amide precursor and N-desmethyl-loperamide with [¹¹C]CH₃OTf through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 20–30% and 10–15% radiochemical yields, respectively, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB), with 370–740 GBq/μmol specific activity at EOB.     

Preparation and characterization of biologically active iodo- and [3H]secretin

Synthetic secretin has been iodinated at the N-terminal histidine, leading to an almost 100% yield of mono- and diiodo-secretin (“lodo-secretin”). The catalytic exchange of iodine against tritium results in the preparation of secretin labeled with tritium mainly at the histidine residue (7 Ci/mmol). Iodo-secretin and [³H]secretin have the same potency in stimulating pancreatic adenylate cyclase as secretin, but the apparent affinity of [³H]secretin for this enzyme is twice as high as for iodo-secretin. [³H]Secretin binds rapidly to pancreatic plasma membranes. Adding excess unlabeled secretin reduces the tracer binding by about 70%.     

A convenient method for regional monoamine oxidase-a determination by [14C]clorgyline autoradiography

The availability of clorgyline for regional monoamine oxidase-A (MAO-A) determination was examined using [¹⁴C]clorgyline in rat. [¹⁴C]Clorgyline was synthesized by the methylation reaction of N-desmethylclorgyline and [¹⁴C]methyliodide in dimethylformamide with high radiochemical yield. The MAO-A distribution map by autoradiography correlated with that by histochemical technique and its quantity was consistent with the calculated MAO-A amount based on previous reports. The combination of labeled clorgyline and autoradiographic technique will promise the quantitative measurement of regional MAO-A distribution.     

[3H] 5,7-Dichlorokynurenic Acid,a High Affinity Ligand for the NMDA Receptor Glycine Regulatory Site

Abstract

The NMDA subtype of glutamate receptors is allosterically linked to a strychnine-insensitive glycine regulatory site. Kynurenic acid and its halogenated derivatives are non-competitive NMDA antagonists acting at the glycine site. We have prepared [³H] 5,7-dichlorokyrurenic acid (DCKA) as an antagonist radioligand and have characterized its binding. 3-Bromo-5,7-DCKA was catalytically dehalogenated in the presence of tritium gas and HPLC purified to yield [³H] 5,7-DCKA with a specific activity of 17.6 Ci/mmol. [³H] 5,7-DCKA bound to rat brain synaptosomes with a K_d of 69 ± 23 nM and B_max = 14.5 ± 3.2 pmoles/mg protein. Binding was 65–70% specific at 10 nM [³H] 5,7-DCKA. This ligand is thus more selective and has higher affinity than [³H] glycine, in addition to being an antagonist.     

Synthesis of 2,6-difluoro-N-(3-[11C]methoxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfonamidio)benzamide as a new potential PET agent for imaging of B-RafV600E in cancers

The authentic standard 2,6-difluoro-N-(3-methoxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfonamidio)benzamide was synthesized from 2,6-difluorobenzoic acid and 3-amino-5-hydroxypyrazole in 9 steps with 1% overall chemical yield. Direct desmethylation of the reference standard with TMSCl/NaI gave the precursor 2,6-difluoro-N-(3-hydroxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfonamidio)benzamide for radiolabeling in 70% yield. The target tracer 2,6-difluoro-N-(3-[¹¹C]methoxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfonamidio)benzamide was prepared from the precursor with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–50% decay corrected radiochemical yields with 370–740 GBq/μmol specific activity at end of bombardment (EOB).     

A new radiochemical assay for argininosuccinase with purified [14C]argininosuccinate

A sensitive and simple radiochemical assay is described to measure argininosuccinase activity in crude tissue homogenates and cultured cells. The method depends on the use of argininosuccinate labeled uniformly with ¹⁴C in the six carbons of the arginine moiety. On incubation in the presence of excess arginase, the [U-¹⁴C]arginine formed is measured as the sum of radioactivity in [U-¹⁴C]ornithine and [¹⁴C]urea. Separation from the substrate is accomplished on a small Domex 1-acetate column eluted with 25 mm acetic acid; ornithine and urea emerge in the first few milliliters while unutilized substrate remains on the column. [¹⁴C]Argininosuccinate was synthesized enzymatically from l-[U-¹⁴C]arginine and fumarate and isolated and purified as the barium salt. Development of a new purification step has brought the amino acid to a purity of 97% as judged by chromatographic and barium analysis. With the present specific radioactivity, as little as 5 to 10 nmol of product can be accurately measured under kinetically optimum conditions.     

Synthesis of tritium-labeled N-acetyl PGE2 carboxamide (CP-27,987)

The preparation of N-acetyl PGE2 carboxamide (CP-27,987) regioselectively labeled at C-18 and C-19 with tritium is described. The overall radiochemical yield was 8.0% at a specific activity of 0.49 Ci/mmole. The synthesis employed is applicable to the preparation of tritium labeled natural prostaglandins and a variety of analogs.