Radiosynthesis and Preclinical Evaluation of an α2A-Adrenoceptor Tracer Candidate, 6-[18F]Fluoro-marsanidine.

PURPOSE: The α2-adrenoceptors mediate many effects of norepinephrine and epinephrine, and participate in the regulation of neuronal, endocrine, cardiovascular, vegetative, and metabolic functions. Of the three receptor subtypes, only α2A and α2C are found in the brain in significant amounts. Subtype-selective positron emission tomography (PET) imaging of α2-adrenoceptors has been limited to the α2C subtype. Here, we report the synthesis of 6-[18F]fluoro-marsanidine, a subtype-selective PET tracer candidate for α2A-adrenoceptors, and its preclinical evaluation in rats and mice. PROCEDURES: 6-[18F]Fluoro-marsanidine was synthesized using electrophilic F-18 fluorination with [18F]Selectfluor bis(triflate). The tracer was evaluated in Sprague Dawley rats and in α2A-knockout (KO) and wild-type (WT) mice for subtype selectivity. In vivo PET imaging and ex vivo brain autoradiography were performed to determine the tracer distribution in the brain. The specificity of the tracer for the target was determined by pretreatment with the subtype-non-selective α2-agonist medetomidine. The peripheral biodistribution and extent of metabolism of 6-[18F]fluoro-marsanidine were also analyzed. RESULTS: 6-[18F]Fluoro-marsanidine was synthesized with [18F]Selectfluor bis(triflate) in a radiochemical yield of 6.4 ± 1.7 %. The molar activity was 3.1 to 26.6 GBq/µmol, and the radiochemical purity was > 99 %. In vivo studies in mice revealed lower uptake in the brains of α2A-KO mice compared to WT mice. The results for selectivity were confirmed by ex vivo brain autoradiography. Blocking studies revealed reduced uptake in α2A-adrenoceptor-rich brain regions in pretreated animals, demonstrating the specificity of the tracer. Metabolite analyses revealed very rapid metabolism of 6-[18F]fluoro-marsanidine with blood-brain barrier-permeable metabolites in both rats and mice. CONCLUSION: 6-[18F]Fluoro-marsanidine was synthesized and evaluated as a PET tracer candidate for brain α2A-adrenoceptors. However, rapid metabolism, extensive presence of labeled metabolites in the brain, and high non-specific uptake in mouse and rat brain make 6-[18F]fluoro-marsanidine unsuitable for α2A-adrenoceptor targeting in rodents in vivo.

COMPARISON OF THE EFFECTS OF MARSANIDINE DERIVATIVES ON RAT CARDIOVASCULAR SYSTEM.

Since clonidine was introduced in clinical practice, attempts are still made to obtain substances capable of centrally controlling blood pressure, however with pharmacological profile better than currently avail- able, such as moxonidine and rilmenidine. Recently synthesized indazole derivatives exert promising action on blood pressure and heart rate in Wistar rats. In the present study, our aim was to check which of tested substituted compound exerts the best effect on basic circulatory parameters. Effects of marsanidine (M), 7-Me- marsanidine (7-Me-M), 7-Cl-marsanidine (7-Cl-M) and 7-F-marsanidine (7-F-M) on blood pressure, heart rate and diuresis were compared. Male Wistar rats were receiving iv. tested compounds in two doses: 10 or 100 pg/kg b.w. Mean arterial pressure (MAP), heart rate (HR) and ECG were recorded continuously. Urine samples were collected before and after administration of tested imidazolines. Obtained data were filtered and subjected to statistical analysis. All tested compounds caused a profound decrease of MAP. 7-M-M reduced blood pressure to the highest extent when used in 10 µg/kg b.w. dose. 7-F-M in dose of 100 µg/kg b.w. caused the strongest drop of MAP. The weakest and the shortest effect in duration was observed after M administration. HR was reduced after administration of each compound while the strongest effect was observed after 7-M-M administration in dose of 10 µg/kg b.w. and after 7-Cl-M administered in dose of 100 µg/kg b.w. Again, the weakest and the shortest in duration effect was observed after M administration. The highest increase of diuresis was observed after 7-M-M administration. These data suggest that methyl substituent in 7 position of indazole ring is the most effective in improving hypotensive effects of newly synthesized imidazolidine derivatives.

1-[(Imidazolidin-2-yl)imino]-1H-indoles as new hypotensive agents: synthesis and in vitro and in vivo biological studies.

A series of 1-[(imidazolidin-2-yl)imino]-1H-indole analogues of hypotensive α2 -AR agonists, 1-[(imidazolidin-2-yl)imino]-1H-indazoles, was synthesized and tested in vitro for their activities at α1 - and α2 -adrenoceptors as well as imidazoline I1 and I2 receptors. The most active 1-[(imidazolidin-2-yl)imino]-1H-indoles displayed high or moderate affinities for α1 - and α2 -adrenoceptors and substantial selectivity for α2 -adrenoceptors over imidazoline-I1 binding sites. The in vivo cardiovascular properties of indole derivatives 3 revealed that substitution at C-7 position of the indole ring may result in compounds with high cardiovascular activity. Among them, 7-fluoro congener 3g showed the most pronounced hypotensive and bradycardic activities in this experiment at a dose as low as 10 µg/kg i.v. Metabolic stability of the selected compounds of type 3 was determined using both in vitro and in silico approaches. The results indicated that these compounds are not vulnerable to rapid first-phase oxidative metabolism.

Transfer of SAR information from hypotensive indazole to indole derivatives acting at α-adrenergic receptors: In vitro and in vivo studies.

In a search for novel antihypertensive drugs we applied scaffold hopping from the previously described α1-adrenergic receptor antagonists, 1-[(imidazolin-2-yl)methyl]indazoles. The aim was to investigate whether the α-adrenergic properties of the indazole core were transferable to the indole core. The newly obtained 1-[(imidazolin-2-yl)methyl]indole analogues were screened in vitro for their binding affinities for α1-and α2-adrenoceptors, which allowed the identification of the target-based SAR transfer (T_SAR transfer) as well as structure-based SAR transfer (S_SAR transfer) events. However, when screened in vivo with use of anaesthetized male Wistar rats, the new indole ligands showed a different hemodynamic profile than expected. Instead of the immediate hypotensive effect characteristic of peripheral vasodilatator α1 blockers, a biphasic effect was observed, reminiscent of clonidine-like centrally acting antihypertensive agents. This was supported by subsequent in vitro functional studies in [(35)S]GTPγS binding assay, where the indole analogues displayed partial agonist properties at α2-adrenergic receptors. Since no correlation was found between the in vitro binding to α-adrenoceptors and the in vivo hemodynamic effects of the two series of indazole and indole bioisosteric compounds, in a search for new imidazoline-containing adrenergic drugs, the structure-based SAR transfer information obtained from in vitro binding studies should be treated with caution.

1-[(IMIDAZOLIN-2-YL)AMINO]INDOLINE AND 1-[(IMIDAZOLIN-2 YL)AMINO] 1,2,3,4-TETRAHYDROQUINOLINE DERIVATIVES: NEW INSIGHTS INTO THEIR CIRCULATORY ACTIVITIES.

N-[(Imidazolin-2-yl)amino]indolines and N-[(imidazolin-2-yl)amino]-1,2,3,4-tetrahydroquinolines, previously described in patent literature as hypertensive agents, were synthesized and tested in viny for their affinities to α1- and α2-adrenoceptors as well as imidazoline I, and I2 receptors. The compounds most potent at either α1- or α2-adrenoceptors were administered intravenously to normotensive Wistar rats to determine their effects on mean arterial blood pressure and heart rate. Upon intravenous administration at dose of 0.1 mg/kg to normotensive male Wistar rats, the initial transient pressor effect was followed by long-lasting hypotension and bradycardia. In view of the above results the 1-[(imidazolin-2-yl)amino]indolines and [(imidazolin-2-yl)amino]-1,2,3,4-tetrahydroquinolines are now found to possess circulatory profile characteristic of the centrally acting clonidine-like hypotensive imidazolines.

The effect of 7-fluoro-marsanidine, a novel α2-adrenoceptor agonist, on extracellular noradrenaline in rat frontal cortex: a microdialysis study.

Previous in vitro binding studies identified a 7-fluoro derivative of marsanidine, a partial α2-adrenoceptor (α2-AR) agonist, to display high affinity and selectivity for α2-AR over α1-AR, imidazoline-1 and imidazoline-2 binding sites. In the present study 7-fluoro-marsanidine is further characterised in vivo to investigate its pharmacological effects on extracellular noradrenaline (NA) levels at frontal cortex in conscious freely moving rats using the technique of in vivo brain microdialysis. Peripheral administration of 7-fluoro-marsanidine via intraperitoneal (i.p.) route at the dose of 0.1mg/kg slightly, but non-significantly, decreased extracellular NA level (maximum by 17% at 20 min) in rat frontal cortex compared to basal level. At a higher dose of 1mg/kg, 7-fluoro-marsanidine reduced cortical NA level (maximum by 73% at 40 min) significantly as compared to basal level between 20 and 80 min. In addition, systemic administration of 7-fluoro-marsanidine at both the doses produced rapid onset of sedation in rats. These data suggest that 7-fluoro-marsanidine is able to cross the blood-brain barrier and, by acting as an α2-AR agonist, reduces extracellular NA levels in rat frontal cortex in a dose related manner. Thus, initial studies indicate 7-fluoro-marsanidine to possess favourable functional properties at α2-AR.

Fluorinated analogues of marsanidine, a highly α2-AR/imidazoline I1 binding site-selective hypotensive agent. Synthesis and biological activities.

The aim of these studies was to establish the influence of fluorination of the indazole ring on the pharmacological properties of two selective α2-adrenoceptor (α2-AR) agonists: 1-[(imidazolidin-2-yl)imino]-1H-indazole (marsanidine, A) and its methylene analogue 1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indazole (B). Introduction of fluorine into the indazole ring of A and B reduced both binding affinity and α2-AR/I1 imidazoline binding site selectivity. The most α2-AR-selective ligands were 6-fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6c) and 7-fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6d). The in vivo cardiovascular properties of fluorinated derivatives of A and B revealed that in both cases the C-7 fluorination leads to compounds with the highest hypotensive and bradycardic activities. The α2-AR partial agonist 6c was prepared as a potential lead compound for development of a radiotracer for PET imaging of brain α2-ARs.

Induction of apoptosis in HL-60 cells through the ROS-mediated mitochondrial pathway by ramentaceone from Drosera aliciae.

Ramentaceone (1) is a naphthoquinone constituent of Drosera aliciae that exhibits potent cytotoxic activity against various tumor cell lines. However, its molecular mechanism of cell death induction has still not been determined. The present study demonstrates that 1 induces apoptosis in human leukemia HL-60 cells. Typical morphological and biochemical features of apoptosis were observed in 1-treated cells. Compound 1 induced a concentration-dependent increase in the sub-G1 fraction of the cell cycle. A decrease in the mitochondrial transmembrane potential (ΔΨm) was also observed. Furthermore, 1 reduced the ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax and Bak, induced cytochrome c release, and increased the activity of caspase 3. The generation of reactive oxygen species (ROS) was detected in 1-treated HL-60 cells, which was attenuated by the pretreatment of cells with a free radical scavenger, N-acetylcysteine (NAC). NAC also prevented the increase of the sub-G1 fraction induced by 1. These results indicate that ramentaceone induces cell death through the ROS-mediated mitochondrial pathway.

5-Methyl-3,6,7,8a-tetra-hydro-2H-diimidazo[1,2-c:1',2'-e]pyrido[1,2-a][1,3,5]triazin-5-ium iodide.

The structure of the title compound, C(12)H(16)N(5) (+)·I(-), shows that the methyl-ation reaction with CH(3)I occurred at the imine N atom at position 5 of the 3,6,7,8a-tetra-hydro-2H-diimidazo[1,2-c:1',2'-e]pyrido[1,2-a][1,3,5]triazine system. In the cation, the sp(3)-hybridized C atom belonging to the fused dihydro-pyrine and dihydro-1,3,5-triazine rings deviates by 0.514 (3) Šfrom the best plane defined by the remaining cationic non-H atoms. The fused dihydro-pyridine and dihydro-1,3,5-triazine rings are each in a half-chair conformation with the sp(3)-hybridized C atom as a flap. The iodide anion is 3.573 (2) Šfrom the methyl-ated N atom and exhibits five short C-H⋯I(-) contacts with distances less than 3.16 Å. The structure has been determined from a non-merohedral twin with twin law [-1 0 0 0 - 1 0 0.115 0 1], minor domain = 0.1559 (12).

The guard cell as a single-cell model towards understanding drought tolerance and abscisic acid action.

Stomatal guard cells are functionally specialized epidermal cells usually arranged in pairs surrounding a pore. Changes in ion fluxes, and more specifically osmolytes, within the guard cells drive opening/closing of the pore, allowing gas exchange while limiting water loss through evapo-transpiration. Adjustments of the pore aperture to optimize these conflicting needs are thus centrally important for land plants to survive, especially with the rise in CO(2) associated with global warming and increasing water scarcity this century. The basic biophysical events in modulating membrane transport have been gradually delineated over two decades. Genetics and molecular approaches in recent years have complemented and extended these earlier studies to identify major regulatory nodes. In Arabidopsis, the reference for guard cell genetics, stomatal opening driven by K(+) entry is mainly through KAT1 and KAT2, two voltage-gated K(+) inward-rectifying channels that are activated on hyperpolarization of the plasma membrane principally by the OST2 H(+)-ATPase (proton pump coupled to ATP hydrolysis). By contrast, stomatal closing is caused by K(+) efflux mainly through GORK, the outward-rectifying channel activated by membrane depolarization. The depolarization is most likely initiated by SLAC1, an anion channel distantly related to the dicarboxylate/malic acid transport protein found in fungi and bacteria. Beyond this established framework, there is also burgeoning evidence for the involvement of additional transporters, such as homologues to the multi-drug resistance proteins (or ABC transporters) as intimated by several pharmacological and reverse genetics studies. General inhibitors of protein kinases and protein phosphatases have been shown to profoundly affect guard cell membrane transport properties. Indeed, the first regulatory enzymes underpinning these transport processes revealed genetically were several protein phosphatases of the 2C class and the OST1 kinase, a member of the SnRK2 family. Taken together, these results are providing the first glimpses of an emerging signalling complex critical for modulating the stomatal aperture in response to environmental stimuli.

An update on abscisic acid signaling in plants and more...

The mode of abscisic acid (ABA) action, and its relations to drought adaptive responses in particular, has been a captivating area of plant hormone research for much over a decade. The hormone triggers stomatal closure to limit water loss through transpiration, as well as mobilizes a battery of genes that presumably serve to protect the cells from the ensuing oxidative damage in prolonged stress. The signaling network orchestrating these various responses is, however, highly complex. This review summarizes several significant advances made within the last few years. The biosynthetic pathway of the hormone is now almost completely elucidated, with the latest identification of the ABA4 gene encoding a neoxanthin synthase, which seems essential for de novo ABA biosynthesis during water stress. This leads to the interesting question on how ABA is then delivered to perception sites. In this respect, regulated transport has attracted renewed focus by the unexpected finding of a shoot-to-root translocation of ABA during drought response, and at the cellular level, by the identification of a beta-galactosidase that releases biologically active ABA from inactive ABA-glucose ester. Surprising candidate ABA receptors were also identified in the form of the Flowering Time Control Protein A (FCA) and the Chloroplastic Magnesium Protoporphyrin-IX Chelatase H subunit (CHLH) in chloroplast-nucleus communication, both of which have been shown to bind ABA in vitro. On the other hand, the protein(s) corresponding to the physiologically detectable cell-surface ABA receptor(s) is (are) still not known with certainty. Genetic and physiological studies based on the guard cell have reinforced the central importance of reversible phosphorylation in modulating rapid ABA responses. Sucrose Non-Fermenting Related Kinases (SnRK), Calcium-Dependent Protein Kinases (CDPK), Protein Phosphatases (PP) of the 2C and 2A classes figure as prominent regulators in this single-cell model. Identifying their direct in vivo targets of regulation, which may include H(+)-ATPases, ion channels, 14-3-3 proteins and transcription factors, will logically be the next major challenge. Emerging evidence also implicates ABA as a repressor of innate immune response, as hinted by the highly similar roster of genes elicited by certain pathogens and ABA. Undoubtedly, the most astonishing revelation is that ABA is not restricted to plants and mosses, but overwhelming evidence now indicates that it also exists in metazoans ranging from the most primitive to the most advance on the evolution scale (sponges to humans). In metazoans, ABA has healing properties, and plays protective roles against both environmental and pathogen related injuries. These cross-kingdom comparisons have shed light on the surprising ancient origin of ABA and its attendant mechanisms of signal transduction.