Synthesis and evaluation of fluorine-18 labeled glyburide analogs as beta-cell imaging agents.

Glyburide is a prescribed hypoglycemic drug for the treatment of type 2 diabetic patients. We have synthesized two of its analogs, namely N-[4-[beta-(2-(2'-fluoroethoxy)-5-chlorobenzenecarboxamido)ethyl]benzenesulfonyl]-N'-cyclohexylurea (2-fluoroethoxyglyburide, 8b) and N-[4-[beta-(2-(2'-fluoroethoxy)-5-iodobenzenecarboxamido)ethyl]benzenesulfonyl]-N'-cyclohexylurea (2-fluoroethoxy-5-deschloro-5-iodoglyburide, 8a), and their fluorine-18 labeled analogs as beta-cell imaging agents. Both F-18 labeled compound 8a and compound 8b were synthesized by alkylation of the corresponding multistep synthesized hydroxy precursor 4a and 4b with 2-[(18)F]fluoroethyl tosylate in DMSO at 120 degrees C for 20 minutes followed by HPLC purification in an overall radiochemical yield of 5-10% with a synthesis time of 100 minutes from EOB. The octanol/water partition coefficients of compounds 8a and 8b were 141.21 +/- 27.77 (n = 8) and 124.33 +/- 21.61 (n = 8), respectively. Insulin secretion experiments of compounds 8a and 8b on rat islets showed that both compounds have a similar stimulating effect on insulin secretion as that of glyburide. In vitro binding studies showed that approximately 2% of compounds 8a and 8b bound to beta TC3 and Min6 cells and that the binding was saturable. Preliminary biodistribution studies in mice showed that the uptake of both compounds 8a and 8b in liver and small intestine were high, whereas the uptake in other organs studied including pancreas were low. Additionally, the uptake of compound 8b in vivo was nonsaturable. These results tend to suggest that compounds 8a and 8b may not be the ideal beta-cell imaging agents.

A simplified one-pot synthesis of 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine([18F]FHPG) and 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) for gene therapy.

9-[(3-[18F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG, 2) has been synthesized by nucleophilic substitution of N(2)-(p-anisyldiphenylmethyl)-9-[[1-(p-anisyldiphenylmethoxy)-3-toluenesulfonyloxy-2-propoxy]methyl]guanine (1) with potassium [18F]fluoride/Kryptofix 2.2.2 followed by deprotection with 1 N HCl and purification with different methods in variable yields. When both the nucleophilic substitution and deprotection were carried out at 90 degrees C and the product was purified by HPLC (method A), the yield of compound 2 was 5-10% and the synthesis time was 90 min from EOB. However, if both the nucleophilic substitution and deprotection were carried out at 120 degrees C and the product was purified by HPLC, the yield of compound 2 decreased to 2%. When compound 2 was synthesized at 90 degrees C and purified by Silica Sep-Pak (method B), the yield increased to 10-15% and the synthesis time was 60 min from EOB. Similarly, 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG, 4) was synthesized with method A and method B in 9% and 10-15% yield, respectively, in a synthesis time of 90 and 60 min, respectively, from EOB. Compound 2 was relatively unstable in acidic medium at 120 degrees C while compound 4 was stable under the same condition. Both compound 2 and compound 4 had low lipid/water partition coefficient (0.126 +/- 0.022, n=5 and 0.165 +/- 0.023, n=5, respectively). Although it contains non-radioactive ganciclovir ( approximately 5-30 microg) as a chemical by-product, compound 2 synthesized by method B has a similar uptake in 9L glioma cells as that synthesized by method A, and is a potential tracer for imaging herpes simplex virus thymidine kinase gene expression in tumors using PET. Similarly, compound 4 synthesized by method B contains approximately 10-25 microg of penciclovir as a chemical by-product. Thus, the simplified one pot synthesis (method B) is a useful method for synthesizing both compound 2 and compound 4 in good yield for routine clinical use, and the method is readily amenable for automation.

Imaging in vivo herpes simplex virus thymidine kinase gene transfer to tumour-bearing rodents using positron emission tomography and.

Radiolabelled ganciclovir analogues have shown promise as imaging agents to detect herpes simplex virus thymidine kinase (HSVtk) expression. This study evaluated the use of positron emission tomography (PET) imaging with 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG) to assess gene transfer into tumours. HSVtk-positive and HSVtk-negative cell lines were first treated in vitro with [18F]FHPG. To assess the efficacy of PET in detecting HSVtk expression following in vivo gene transfer, mice were injected intravenously with an adenovirus encoding HSVtk (Ad.HSVtk), a control vector (Ad.Bgl2) or saline. Subcutaneous human glioma xenografts were grown in mice and treated by direct injection of Ad.HSVtk or Ad.Bgl2. Imaging was performed 48 h after transduction. Similar experiments were performed using Fischer rats implanted with syngeneic tumours. The presence of the HSVtk protein was confirmed by immunohistochemistry. Biodistribution studies were also obtained in 14 naive mice. In vitro studies showed high and specific uptake of [18F]FHPG in HSVtk-positive cell lines, with an uptake ratio of up to 27:1. PET imaging and direct counting of major organs demonstrated HSVtk-specific tracer retention. In mice, HSVtk-positive tumours retained 3.4% dose/gram as compared to 0.6% for control tumours (P=0.03). They were clearly seen on the PET images as early as 100 min post injection. Similar results were obtained with syngeneic rat tumours. Biodistribution studies demonstrated the rapid distribution and clearance of the tracer in all major organs. Our results demonstrate that PET imaging of HSVtk gene transfer to tumours is feasible and is highly specific for HSVtk expression.

N-(n-Benzylpiperidin-4-yl)-2-[18F]fluorobenzamide: a potential ligand for PET imaging of breast cancer.

N-(N-Benzylpiperidin-4-yl)-2-[(18)F]fluorobenzamide (2), a potential ligand for PET imaging of sigma receptor, has been found to be a potential agent for detection of breast cancer. In vivo studies in severe combined immunodeficient (SCID) mice bearing MDA-MB231 tumors showed that the uptake of compound 2 in these tumors was high (3.8%/g); the ratios of tumor/muscle and tumor/blood were 6.2 and 7.0, respectively, at 1 h postinjection. Pretreatment of SCID mice with haldol increased the uptake of compound 2 in blood, muscle, and other well-perfused organs while decreasing its uptake in tumors. The ratios of tumor/muscle and tumor/blood decreased from 6.2 and 7.0 to 1.3 and 1.1, respectively, at 1 h postinjection. At 2 h postinjection, the ratios of tumor/muscle and tumor/blood decreased from 4.9 and 7.8 to 1.4 and 1.4, respectively. The tumor uptake of compound 2 in SCID mice bearing primary tumor explants from a human breast cancer patient was lower than that in MDA-MB231 tumors (1.66%/g versus 3.78%/g), and the ratios of tumor/muscle and tumor/blood were 3.5 and 3.7, respectively, at 1 h postinjection. These results suggest that compound 2 may be a potential ligand for PET imaging of breast cancer.

Synthesis of new hypoxia markers EF1 and [18F]-EF1.

We report on the preparation of a hypoxia marker 2-(2-nitroimidazol-1[H]-yl)-N-(3-fluoropropyl)acetamide (EF1) and its 18F analog, 2-(2-nitroimidazol-1[H]-yl)-N- (3-[18F]fluoropropyl)acetamide ([18F]-EF1). Two methods for the preparation of 3-fluoropropylamine, the EF1 side chain, are described. [18F]-EF1 was prepared with a radiochemical yield of 2% by nucleophilic substitution of bromine in 2-(2-nitroimidazol-1[H]-yl)-N-(3-bromopropyl)acetamide (EBr1) by carrier-added 18F in DMSO at 120 degrees C. Our results demonstrate the preparation of clinically relevant amounts of [18F]-EF1 for use as a non-invasive hypoxia marker with detection using positron emission tomography (PET).

Synthesis and biological evaluation of [11C]MK-912 as an alpha2-adrenergic receptor radioligand for PET studies.

In vitro studies showed that MK-912 ((2S, 12bS)1',3'-dimethylspiro(1,3,4,5',6,6',7,12b-octahydro -2H-benzo[b]furo[2,3-a]quinolizine)-2,4'-pyrimidin-2'-one) is a potent alpha2-adrenergic receptor antagonist with high affinity (Ki = 0.42, 0.26 and 0.03 nM to alpha2A, alpha2B and alpha2C, respectively) and high selectivity (alpha2A/alpha1A = 240; alpha2A/D-1 = 3600; alpha2A/D-2 = 3500; alpha2A/5-HT1 = 700; alpha2A/5-HT2 = 4100). The compound was labeled with 11C and evaluated in rodents and monkey as a specific radioligand for studying alpha2-adrenergic receptors using PET. [11C]MK-912 was synthesized by methylation of its desmethyl precursor, L-668,929, with [11C]CH3I in (Bu3O)P=O at 85 degrees C for 8 min followed by purification with HPLC in 18% yield in a synthesis time of 45 min from end of bombardment (EOB). The specific activity was 0.83-0.93 Ci/micromol and the radiochemical purity was 97%. The initial uptake of [11C]MK-912 in mouse brain, heart, lung, liver and kidney was high (5%, 4%, 5%, 17% and 8% per gram of organ, respectively, at 5 min postinjection) and the activities were then slowly cleared from these organs. The uptake of [11C]MK-912 in rat olfactory tubercle, a brain region with high density of alpha2-adrenergic receptors, was reduced by 30%, and the ratio of radioactivity in olfactory tubercle/cerebellum was reduced from 2:1 to 1:1 by coinjection of [11C]MK-912 with a potent alpha2-adrenergic receptor antagonist, atipamezole (3 mg/kg), indicating that compound 2 binds to alpha2-adrenergic receptors. However, a PET study in a rhesus monkey revealed that the initial influx of [11C]MK-912 into various brain regions (cerebellum, cortex, olfactory tubercle and striatum) was high (0.02%/cc), and the radioactivity was then washed out slowly and without significantly differential retention in these brain regions. This, coupled with the fact that none of the high-density alpha2-adrenergic receptor brain regions exceeds a few millimeters in diameter, suggests that [11C]MK-912 is probably not an ideal radioligand for studying alpha2-adrenergic receptors in humans using commercially available PET.

N-(N-benzylpiperidin-4-yl)-2-[18F]fluorobenzamide: a potential ligand for PET imaging of sigma receptors.

Four nitro- and fluorobenzamides (1-4) have been synthesized in good yields from nitro- and fluoro-substituted benzoyl chloride with 4-amino-1-benzylpiperidine. In vitro studies showed that these compounds have high affinities to sigma receptors. N-(N-Benzylpiperidin-4-yl)-2-fluorobenzamide (3), in particular, bound to sigma receptors with high affinity (Ki = 3.4 nM, guinea pig brain membranes) and high selectivity (sigma-2/sigma-1 = 120). It was, therefore, labeled with 18F and evaluated as a sigma receptor radioligand. N-(N-Benzylpiperidin-4-yl)-2-[18F]fluorobenzamide (3a) was synthesized in one step by nucleophile substitution of the 2-nitro precursor (1) with [18F]fluoride in DMSO at 140 degrees C for 20 min followed by purification with HPLC in 4-10% yield (decay corrected). The synthesis time was 90 min and the specific activity was 0.4-1.0 Ci/mumol. Tissue distribution in mice revealed that the uptakes of 3a in the brain, heart, liver, lungs, spleen, kidneys and small intestine were high, and the radioactivity in these organs remained constant from 60 to 120 min post-injection. The radioactivity in the bone did not significantly increase, suggesting in vivo defluorination may not be the major route of metabolism of 3a in mice. Blocking studies with haloperidol in rats indicated that the uptake of compound 3a in the rat brain was selective to haloperidol-sensitive sigma sites. These results suggest that compound 3a is a potent sigma receptor radioligand and may be a potential ligand for PET imaging of sigma receptors in humans.

P-[18F]-MPPF: a potential radioligand for PET studies of 5-HT1A receptors in humans.

The purpose of this study was to develop a radiopharmaceutical that could be used to selectively image 5-HT1A receptors with positron emission tomography (PET). No-carrier-added 4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p-[18F] fluorobenzamido]ethylpiperazine (p-[18F]-MPPF, 2) was synthesized by the nucleophilic substitution of the corresponding nitro precursor 1 with K[18F]/Kryptofix 2.2.2. in dimethyl sulfoxide (DMSO) at 140 degrees C for 20 min followed by purification with high-performance liquid chromatography (HPLC) in 10% yield in a synthesis time of 90 min from end of bombardment (EOB). Specific activity was 1-4 Ci/microM. Biodistribution studies in rats showed that the initial uptake of 2 in the brain was high (0.7% dose/g tissue at 2 min). It was then rapidly eliminated. Rates of elimination were significantly slower in brain regions with high concentrations of 5-HT1A receptors (hippocampus, cortex, and hypothalamus) than in control regions. The maximum hippocampal/cerebellar ratio was 5.6:1 at 30 min postinjection. Uptake values in serotonergic, but not in control, regions were significantly reduced by prior treatment with either (+/-)-8-OH-DPAT (2 mg/kg, i.v., 5 min prior) or WAY 100635 (1 mg/kg, i.v., 5 min prior). Radioactivity in the femur did not increase with time, suggesting that in vivo defluorination may not be the major route of metabol sm. PET studies of 2 in a monkey demonstrated selective uptake and retention of 2 in the hippocampus. The hippocampal/cerebellar ratio was 3:1 at 30 min postinjection. The ratio was reduced to 1:1 by administering (+/-)-8-OH-DPAT (2 mg/kg, i.v.) 23 min postinjection of 2. Analyses of arterial plasma by HPLC revealed that 20% of radioactivity in the plasma remained as the parent compound 2 at 30 min postinjection. The results suggest that p-[18F]-MPPF may be a useful radioligand for studying cerebral 5-HT1A receptors in humans with PET techniques.

PET study of the distribution of [11C]fluoxetine in a monkey brain.

No-carrier-added [11C]fluoxetine (2) was synthesized by methylation of norfluoxetine (1) with [11C]H3I in 20% radiochemical yield in a synthesis time of 40 min from EOB with a specific activity of 0.48 Ci/microM (EOB). In vivo study in mouse indicated that the uptake of 2 in mouse tissues was high and the radioactivity remained constant throughout the study. The uptake of 2 in mouse brain was 4%/g. PET study in a Rhesus monkey also showed that the uptakes of 2 in different brain regions were similar and the retention of radioactivity in these regions remained constant throughout the study (80 min). Analysis of arterial plasma by HPLC showed that only 20% of radioactivity in the plasma remained as 2 at 30 min post-injection. These results suggest that the uptake of fluoxetine in monkey brain is probably not receptor mediated. Rather, blood flow, lipophilicity or other transport mechanisms may play a role in its uptake.

Comparative PET studies of the distribution of (-)-3,4-methylenedioxy-N-[11C]methamphetamine and (-)-[11C]methamphetamine in a monkey brain.

Carbon-11 labeled (-)-methamphetamine and (-)-3,4-methylenedioxy-N-methamphetamine were synthesized by methylation of the corresponding desmethyl precursors with [11C]H3I in 40-60% yield in a synthesis time of 30 min from EOB with a specific activity of 0.5-1.2 Ci/microM. PET studies in a Rhesus monkey revealed that the uptakes of both compounds in different brain regions were similar, and the retention of radioactivity in these brain regions remained constant throughout the study for the former while it was washed out slowly for the latter. The half-life of (-)-3,4-methylenedioxy-N-methamphetamine in monkey brain was approximately 70 min. Analyses of arterial plasma by HPLC revealed that 50% of radioactivity in the plasma remained as (-)-methamphetamine while only 3% remained as (-)-3,4-methylenedioxy-N-methamphetamine at 60 min post-injection. These results suggest that the uptakes of both compounds in monkey brain are probably not receptor mediated. Rather, blood flow, lipophilicity of the compounds or other transport mechanisms may play a role in their uptakes.

Fluorine-18 and carbon-11 labeled amphetamine analogs--synthesis, distribution, binding characteristics in mice and rats and a PET study in monkey.

No-carrier-added (NCA) (+-)-p-[18F]fluoroamphetamine (2a) and (+-)-6-[18F]fluoro-3,4-methylene-dioxy-amphetamine (2b) were synthesized through a multistep synthesis by nucleophilic substitution of the appropriate precursors (p-nitrobenzaldehyde, 1a and 6-nitropiperonal 1b, respectively) with [18F]fluoride followed by condensation with nitroethane and reduction with LAH in 20-30% yield (EOB) in a synthesis time of 90-109 min from EOB. NCA (-)-[11C]methamphetamine (4a) and (+-)-3,4-methylene-dioxy-N-[11C]methamphetamine (4b) were synthesized by methylation of the appropriate desmethyl precursors 3a and 3b with [11C]H3I in 40-60% yield (EOB) in a synthesis time of 30 min from EOB. Animal studies in mouse and rat revealed that the relative tissue uptake of these radiotracers was kidneys > lungs > liver > spleen > brain > heart > blood. The uptakes of these radiotracers in mouse brain were high and similar at 5 min post-injection (approx. 5%/g) but radioactivity then declined rapidly (approx. 1%/g at 60 min post-injection). For compounds 2a and 2b, the activity in the femur did not increase with time indicating in vivo defluorination may not be the major route of metabolism. Monoamine uptake inhibitors (nomifensine, fluoxetine and nisoxetine) did not inhibit but enhance the uptake of (-)-[11C]methamphetamine (4a) in the rat brain by greater than 50%. A PET study in a Rhesus monkey revealed that the uptakes of (-)-[11C]methamphetamine in different brain regions were similar and the retention of the radioactivity in these regions remained constant throughout the study. Analysis of arterial plasma by HPLC showed that 50% of radioactivity remained as 4a at 60 min post-injection.

Synthesis of (+/-)-[18F]BMY 14802, its enantiomers and their anatomical distributions in rodents.

A potential antipsychotic drug, BMY 14802 was labeled with 18F and its distribution in rodents was studied. No-carrier-added (NCA) (+/-)-[18F]BMY 14802 (5) was synthesized by two methods in 5-10% radiochemical yield in a synthesis time of 130-140 min from EOB with a specific activity of 0.5-1.5 Ci/microM. (+)- and (-)-[18F]BMY 14802 was synthesized by the chiral reduction of alpha-(4-[18F]fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine-b utanone (4) with chiral reducing agent, (+)- and (-)-beta-chlorodiisopinocampheylborane [(+)- and (-)-DIP chloride] in 6-10% radiochemical yield in a synthesis time of 150 min from EOB. Animal studies in mouse and in rat revealed that the distribution of 5 in each tissue was high at 5 min, the radioactivity then declined rapidly in all tissues studied except in the liver and in the small intestine. The radioactivity in the femur did not increase with time indicating in vivo defluorination may not occur. The uptakes of (+/-)-[18F]BMY 14802 and its enantiomers, (+)- and (-)-[18F]BMY 14802 in rat cerebellum, brain stem, hippocampus and spinal cord were similar and were significantly reduced by prior treatment of rat with haldol. This suggests that (+/-)-[18F]BMY 14802 and its enantiomers bind to sigma-receptors in a similar fashion.

Synthesis and biodistribution of the alpha 2-adrenergic receptor antagonist (11C)WY26703. Use as a radioligand for positron emission tomography.

The purpose of these experiments was to label an alpha 2-adrenergic receptor ligand with a positron emitting isotope and then test this radioligand in vivo. No-carrier-added [11C]WY26703 was synthesized by methylation of its desmethyl precursor, WY27050 with [11C]H3I followed by purification with HPLC in 14% yield in a synthesis time of 35 min from EOB. Ki values for unlabeled WY26703, ranged from 0.52-1.55 nM in tissues that express a single alpha 2-adrenergic receptor subtype. Tail vein injections of [11C]WY26703 in mice revealed that the compound was distributed in the brain, heart, lungs, spleen, and kidneys. In the brains of rats treated with atipamezole, an alpha 2-adrenergic receptor antagonist, there was no decrease in [11C] accumulation indicating a lack of observable specific binding of the radioligand. When brain tissue was homogenized and filtered, however, atipamezole decreased [11C] activity by 53%. Therefore, [11C]WY26703 crosses the blood-brain barrier and specifically binds to alpha 2-adrenergic receptors with high affinity. Atipamezole treatment decreased only the area of the locus coeruleus [11C] value of the various regions of the brain. The affinity, however, of [11C]WY26703 does not appear to distinguish alpha 2-receptors from nonspecific binding sites. PET study of [11C]WY26703 in a Rhesus monkey showed that influx of [11C]WY26703 into the brain was high for the first few minutes but radioactivity then declined rapidly and did not retain in a specific brain region. This suggests that [11C]WY26703 may not be a useful ligand for imaging human alpha 2-adrenergic receptors by positron emission tomography.

Reaction rates of superoxide radicals with the essential amino acids.

Upper limits for the rates of reaction of amino acids with superoxide radicals have been determined spectrophotometrically by the stopped flow method. Rate measurements at 23 degrees C for the reaction of HO2 with amino acids were made in the pH range between 1 and 2; similar measurements for O2- were taken near pH 10. The results show that, overall, amino acids are relatively unreactive toward both HO2 and O2-. Computed second-order rate constants for their interaction with HO2 range from 101 mol-1s-1 for aliphatic amino acids to about 600 1 mol-1s-1. The second-order rate constants for the interaction of amino acids with O2- are smaller and range from 0.1 to about 20 1 mol-1s-1.