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Ethyl 8-fluoro-5-[11C]methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate

, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894, vog.hin.mln.ibcn@dacim

Created: ; Last Update: June 10, 2008.

Chemical name:Ethyl 8-fluoro-5-[11C]methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylateimage 49701077 in the ncbi pubchem database
Abbreviated name:[11C]FMZ
Synonym:[11C]Flumazenil; Romazicon®; Ro 15-5528
Agent Category:Compound
Target:Gamma-aminobutyric acid type A receptors (GABAA receptors)
Target Category:Ligand-receptor binding
Method of detection:Positron emission tomography (PET)
Source of Signal/Contrast:11C
  • Checkbox In vitro
  • Checkbox Rodents
  • Checkbox Non-human primates
  • Checkbox Humans
Click on the above structure for additional information in PubChem.



The development of epilepsy has been associated with the impairment of gamma-aminobutyric acid (GABA) neurotransmission in the central nervous system because a reduced GABA release has been observed in the mesial temporal lobe during epilepsy episodes in animals and a GABA-mediated inhibition loss was reported in human epileptic hippocampal sclerosis (1, 2). The GABA receptors (GABAR) comprise several different pharmacological subtypes depending on the type of subunits constituting the receptor complex, and GABA mediates its effects primarily through the GABAA receptors (3). Also, individuals with the Angleman syndrome (AS) have a neurodevelopmental disorder that results in severe mental retardation, delayed motor development, and epilepsy (4). It has been shown that surviving gabrag3-knockout mice are epileptic and have a phenotype that is similar to AS patients, indicating that the GABRβ3 gene in humans could have a role in the development of AS (5). Interestingly, genes coding for the various GABAR subunits (β3, α5, and γ3) are located within the same 15q11-q13 region of the human chromosome that is believed to have a function in the development of AS (5, 6), suggesting that the GABARβ3 gene may have a role in the development of AS in humans. In addition, the GABAAknockout mice were shown to have reduced levels of the GABAA receptor.

A benzodiazepine (BDZ) site antagonist labeled with radioactive carbon ([11C]), ethyl 8-fluoro-5-[11C]methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([11C]flumazenil ([11C]FMZ)), which has a high affinity for the GABAA receptors, has been used widely with positron emission tomography (PET) for the investigation of the these receptors (7). Flumazenil is available commercially in the United States and has been approved by the United States Food and Drug Administration for use in various clinical trials.



The synthesis of [11C]FMZ has been described by Nagren and Halldin (8). Briefly, 8-fluoro-5,6-dihydro-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid (also known as desmethylflumazenil or desmethyl-FMZ or Ro 15-5528) was mixed with [11C]methyl triflate in a sealed reaction vessel at 0°C containing sodium hydroxide and acetone. The vessel was heated to 60°C for 1 min, and a mixture of acetonitrile:0.01 M phosphoric acid was injected into the vessel. The mixture was separated on a Waters reverse-phase high-performance liquid chromatography μ-Bondapak-C18 column, and the purified product was collected in a propylene glycol/ethanol mixture. The mobile phase was evaporated, and the residue was dissolved in propylene glycol/ethanol contained in physiological phosphate buffer (pH 7.4) and sterile filtered. The radiochemical yield (decay corrected) of this reaction was reported to be 65–75%. The radiochemical purity and specific activity of the product were not provided (8).

A robotic method for the synthesis of [11C]FMZ has also been published (9). The synthesis was performed on an Anatech RB 86 robotic system in a vial containing Ro 15-5528, potassium fluoride, and aluminum oxide in acetonitrile (9). [11C]Methyl iodide was trapped for 90 s in the vial, and the reaction mixture was kept at room temperature for another 120 s. A solid-phase extraction technique was used to separate [11C]FMZ from its precursor (Ro 15-5528). This was followed by removal of the solvent in a stream of nitrogen at 130°C. The purified [11C]FMZ was dissolved in an ethanol:saline mixture, and the solution was sterile filtered. The synthesis time was reported to be 30 min from the time of [11C]methyl iodide trapping. The radiochemical purity of [11C]FMZ was reported to be >99% with a specific activity of 11.1–59.2 GBq/μmol (0.3–1.6 Ci/μmol). The stability of the radiochemical was not reported (9).

In Vitro Studies: Testing in Cells and Tissues


The in vitro characteristics of [11C]Ro 15-1788 were studied in membranes isolated from the rat forebrain (10). The various kinetic constants were determined with two different techniques involving a phosphorimager and γ-counter measurements. The dissociation constants (KD) were reported to be 2.0 ± 0.9 and 2.1 ± 0.5 nM, respectively, by the two methods, and the number of binding sites (Bmax) was determined to be 1.3 ± 0.5 and 1.7 ± 0.3 pmol/mg protein by the respective methods. In competition experiments the 50% inhibitory concentration (IC50) of unlabelled Ro 15-1788 was reported to be 4.03 ± 0.75 nM.

Animal Studies



Using [11C]FMZ, investigators have shown that the binding potential (Bmax/KD) of the GABAA receptors is reduced significantly over a 12-h period in the trauma-site cortex of rats subjected to a moderate lateral fluid percussion injury (11). This demonstrated that the GABA receptors were involved in cellular dysfunction as a result of the fluid percussion injury in these animals.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


Matsunaga et al. investigated the effect of yohimbine administration on BDZ receptor binding in the central nervous system of non-human primates (rhesus monkeys) (12). Using [11C]FMZ, the investigators reported that yohimbine, an α2-adrenoceptor antagonist, increased the binding potential for the BDZ receptors in the monkey brain, indicating that a state of anxiety potentiated the effects of anxiolytics.

Human Studies


Using [11C]FMZ with PET in 11 patients with acute hemispheric ischemic stroke, Heiss et al. showed that this radiopharmaceutical is a suitable agent to distinguish between irreversibly damaged and viable neuronal tissue during early onset of the condition (13). In another study in 12 patients, the probability of predicting the cortical infarction in early ischemic stroke using PET versus diffusion-weighted magnetic resonance imaging (based on movement of water molecules, which is the method of choice to detect ischemic lesions) for the central BDZ receptors with [11C]FMZ was determined (14). With results obtained from this study, the investigators concluded that, although the two methods were comparable, the probability of false-positive predictions were lower when PET with [11C]FMZ was used.

Koepp et al. used [11C]FMZ to demonstrate a reduction in the number of GABA/central BDZ receptors in the hippocampus of patients with mesial temporal lobe epilepsy caused by unilateral hippocampal sclerosis (15). These observations were later confirmed by comparing in vivo [11C]FMZ and ex vivo [3H]FMZ binding in patients with hippocampal sclerosis with anterior temporal lobe resections (7).

[11C]FMZ has also been used to estimate the synaptic density of BZD in five individuals who became blind at an early age (16). Compared with sighted control subjects, a significantly lower (P < 0.01) BDZ density was reported in the cerebellum of the blind subjects. However, the BZD density was comparable in the visual areas and other cortical regions of the blind patients and the controls, indicating a modification of the cerebellar neural circuitry in the blind individuals.

The binding of [11C]FMZ was studied in four AS patients (17). Among these patients, three had a maternal deletion of 15q11-q13 leading to a loss of the β3 subunit of the GABAR, and the fourth patient had a mutation in the ubiquitin protein ligase (UBE3A) but had a normal β3 subunit gene. Compared to the fourth patient, a lower binding of [11C]FMZ was observed in the frontal, parietal, hippocampal, and cerebellar regions of the brain of the first three patients, indicating that the 15q11-q13 deletion leads to a reduced number of GABAR in the AS patients. However, using [11C]FMZ, Asahina et al. reported an increase in total expression of GABAA receptors in the cerebral cortex and the cerebellum of five AS patients with a deletion and one patient with a UBE3A mutation (18).

Supplemental Information



During M.J., Ryder K.M., Spencer D.D. Hippocampal GABA transporter function in temporal-lobe epilepsy. Nature. 1995;376(6536):174–7. [PubMed: 7603569]
Knowles W.D., Awad I.A., Nayel M.H. Differences of in vitro electrophysiology of hippocampal neurons from epileptic patients with mesiotemporal sclerosis versus structural lesions. Epilepsia. 1992;33(4):601–9. [PubMed: 1628573]
Luddens H., Korpi E.R., Seeburg P.H. GABAA/benzodiazepine receptor heterogeneity: neurophysiological implications. Neuropharmacology. 1995;34(3):245–54. [PubMed: 7630479]
Moncla A., Malzac P., Voelckel M.A., Auquier P., Girardot L., Mattei M.G., Philip N., Mattei J.F., Lalande M., Livet M.O. Phenotype-genotype correlation in 20 deletion and 20 non-deletion Angelman syndrome patients. Eur J Hum Genet. 1999;7(2):131–9. [PubMed: 10196695]
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Glatt K., Glatt H., Lalande M. Structure and organization of GABRB3 and GABRA5. Genomics. 1997;41(1):63–9. [PubMed: 9126483]
Koepp M.J., Hand K.S., Labbe C., Richardson M.P., Van Paesschen W., Baird V.H., Cunningham V.J., Bowery N.G., Brooks D.J., Duncan J.S. In vivo [11C]flumazenil-PET correlates with ex vivo [3H]flumazenil autoradiography in hippocampal sclerosis. Ann Neurol. 1998;43(5):618–26. [PubMed: 9585356]
Nagren K., Halldin C. Methylation of amide and thiol functions with [11C]methyltriflate, as examplified by [11C]NMSP, [11C]flumazenil and [11C]methionine. Journal of Labelled Compounds and radiopharmaceuticals. 1998;41:831–841.
Krasikova R., Fedorova O., Korsakov M., Nagren K., Maziere B., Halldin C. A fast and convenient method for robotic preparation of [11C]flumazenil avoiding HPLC purification. Journal of Labelled Compounds and Radiopharmaceuticals. 2000;43:613–621.
Sihver W., Sihver S., Bergstrom M., Murata T., Matsumura K., Onoe H., Andersson Y., Bjurling P., Fasth K.J., Westerberg G., Ogren M., Jacobsson G., Lundqvist H., Oreland L., Watanabe Y., Langstrom B. Methodological aspects for in vitro characterization of receptor binding using 11C-labeled receptor ligands: a detailed study with the benzodiazepine receptor antagonist [11C]Ro 15-1788. Nucl Med Biol. 1997;24(8):723–31. [PubMed: 9428597]
Sihver S., Marklund N., Hillered L., Langstrom B., Watanabe Y., Bergstrom M. Changes in mACh, NMDA and GABA(A) receptor binding after lateral fluid-percussion injury: in vitro autoradiography of rat brain frozen sections. J Neurochem. 2001;78(3):417–23. [PubMed: 11483644]
Matsunaga T., Tsukada H., Nishiyama S., Sekine Y., Kakiuchi T., Iyo M., Mori N. Yohimbine increases the binding potential for [11C]flumazenil in the monkey brain. J Neural Transm. 2001;108(12):1375–82. [PubMed: 11810402]
Heiss W.D., Kracht L., Grond M., Rudolf J., Bauer B., Wienhard K., Pawlik G. Early [(11)C]Flumazenil/H(2)O positron emission tomography predicts irreversible ischemic cortical damage in stroke patients receiving acute thrombolytic therapy. Stroke. 2000;31(2):366–9. [PubMed: 10657407]
Heiss W.D., Sobesky J., Smekal U., Kracht L.W., Lehnhardt F.G., Thiel A., Jacobs A.H., Lackner K. Probability of cortical infarction predicted by flumazenil binding and diffusion-weighted imaging signal intensity: a comparative positron emission tomography/magnetic resonance imaging study in early ischemic stroke. Stroke. 2004;35(8):1892–8. [PubMed: 15218157]
Koepp M.J., Richardson M.P., Brooks D.J., Poline J.B., Van Paesschen W., Friston K.J., Duncan J.S. Cerebral benzodiazepine receptors in hippocampal sclerosis. An objective in vivo analysis. Brain. 1996;119(Pt 5):1677–87. [PubMed: 8931589]
Sanabria-Bohorquez S.M., De Volder A.G., Arno P., Sibomana M., Coppens A., Michel C., Veraart C. Decreased benzodiazepine receptor density in the cerebellum of early blind human subjects. Brain Res. 2001;888(2):203–211. [PubMed: 11150476]
Holopainen I.E., Metsahonkala E.L., Kokkonen H., Parkkola R.K., Manner T.E., Nagren K., Korpi E.R. Decreased binding of [11C]flumazenil in Angelman syndrome patients with GABA(A) receptor beta3 subunit deletions. Ann Neurol. 2001;49(1):110–3. [PubMed: 11198279]
  • 18. Asahina, N., T. Shiga, K. Egawa, H. Shiraishi, S. Kohsaka, and S. Saitoh, [(11)C]flumazenil positron emission tomography analyses of brain gamma-aminobutyric acid type A receptors in Angelman syndrome. J Pediatr, 2008. 152(4): p. 546-9, 549 e1-3. [PubMed: 18346513]
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