Radiosynthesis and in vivo evaluation of a fluorine-18 labeled pyrazine based radioligand for PET imaging of the adenosine A2B receptor

On the basis of a pyrazine core structure, three new adenosine A_2B receptor ligands (7a–c) were synthesized containing a 2-fluoropyridine moiety suitable for ¹⁸F-labeling. Compound 7a was docked into a homology model of the A_2B receptor based on X-ray structures of the related A_2A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A₁ receptor, 7a was radiolabeled as the most suitable candidate (K_i(A_2B)?=?4.24?nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [¹⁸F]7a with a standardized uptake value (SUV) of ≈1 at 5?min post injection followed by a fast wash out. Metabolism studies of [¹⁸F]7a in mice revealed the formation of a blood–brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo.     

Interactions of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas with dopamine D2 and 5-hydroxytryptamine 5HT(1A) receptors

It is suggested that the ratio of dopamine D(2) to 5-hydroxytryptamine 5-HT(1A) activity is an important parameter that determines the efficiency of antipsychotic drugs. Here we present the synthesis of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas and their structure-activity relationship studies on dopamine D(2) and 5-hydrohytryptamine 5-HT(1A) receptors. It was shown that ligand selectivity and affinity strongly depends on their topology and the presence of a pyridyl group in the head of molecules. Molecular modeling studies using homology modeling and docking simulation revealed a rational explanation for the ligand behavior. The observed binding modes and receptor-ligand interactions provided us with a clue for optimizing the optimal selectivity towards 5-HT(1A) receptors.     

Characterization and Inhibition of a Class II Diterpene Cyclase from Mycobacterium tuberculosis: IMPLICATIONS FOR TUBERCULOSIS*

Mycobacterium tuberculosis remains a widespread and devastating human pathogen, whose ability to infiltrate macrophage host cells from the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from M. tuberculosis, edaxadiene, whose ability to arrest phagosomal maturation in isolation presumably contributes to this critical process in M. tuberculosis infections. (Mann, F. M., Xu, M., Chen, X., Fulton, D. B., Russell, D. G., and Peters, R. J. (2009) J. Am. Chem. Soc., in press). Here, we present characterization of the class II diterpene cyclase that catalyzes the committed step in edaxadiene biosynthesis, i.e. the previously identified halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Intriguingly, our kinetic analysis suggests a potential biochemical regulatory mechanism that triggers edaxadiene production upon phagosomal engulfment. Furthermore, we report characterization of potential HPS inhibitors: specifically, two related transition state analogs (15-aza-14,15-dihydrogeranylgeranyl diphosphate (7a) and 15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (7b)) that exhibit very tight binding. Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway. Finally, we provide evidence indicating that this pathway exists only in M. tuberculosis and is not functional in the closely related Mycobacterium bovis because of an inactivating frameshift in the HPS-encoding gene. Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans.Tuberculosis is a prevalent human disease that leads to >1.5 million deaths annually. Over 98% of these fatalities are caused by infections of the eponymous microbe Mycobacterium tuberculosis (1). M. tuberculosis can be extremely infectious, with a dose of as little as a single bacterium sufficient for establishment of a potentially fatal infection (2). Intriguingly, the closely related Mycobacterium bovis appears to be less infectious in humans (3) and is a significantly less common causative agent of tuberculosis (1), despite sharing >99.9% genome sequence identity with M. tuberculosis (4).M. tuberculosis is taken up by and resides in macrophage cells in the mammalian immune system, specifically in phagosome compartments that are arrested at an early stage of endocytic progression (2). The ability of M. tuberculosis to block such phagosomal maturation has been attributed to multiple factors. Although mycobacterial cell-surface lipids have a clear role, that of other effectors remains less definitive, as different genetic screens have indicated roles for non-overlapping sets of genes (5).A genetic screen focused on primary effects very early in the infection process strongly implicated the product of a five-gene isoprenoid biosynthetic operon (6). In particular, inactivating transposon insertion in the two unique (presumably non-redundant) genes in the operon resulted in mutant M. tuberculosis unable to fully block phagosomal maturation. Closely following work demonstrated that the first of these, Rv3377c, encoded a class II diterpene cyclase that catalyzed bicyclization and rearrangement of (E,E,E)-geranylgeranyl diphosphate (GGPP⁴; 1a) to halimadienyl diphosphate (HPP; 3a) (Fig. 1) (7). The corresponding enzyme has been termed halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Recently, we have reported that the second implicated gene, Rv3378c, encodes a subsequently acting class I diterpene cyclase that further cyclizes HPP (3a) to the novel tricyclic diterpene edaxadiene (4), which directly inhibits phagosomal maturation in vitro (8), consistent with the results of the previously reported genetic screen (6). Edaxadiene (4) then presumably contributes, at least at an early stage in the infection process, to the phagosomal arrest that provides M. tuberculosis with its host cell/compartment, with HPS catalyzing the committed step in its biosynthesis.Open in a separate windowFIGURE 1.Reaction catalyzed by HPS and subsequent production of edaxadiene (4). Shown is the acid-catalyzed protonation-initiated bicyclization of GGPP (1) to a copalyl diphosphate carbocation intermediate (2), the subsequent rearrangement via a series of alternating 1,2-hydride and methyl migrations to form the HPP (3a) product after terminating deprotonation, and the following separate additional cyclization of 3a to edaxadiene (4) catalyzed by Rv3378c/M. tuberculosis edaxadiene synthase (MtEDS).Initial functional characterization of HPS was limited by enzymatic instability. Here, we report the development of a construct amendable to kinetic characterization, along with the implications of the observed striking Mg²⁺ cofactor inhibition effect. We further report analysis of potential inhibitors, with two transition state analogs (7a and 7b) (Fig. 2) found to exhibit high affinity. In addition, investigation of the corresponding gene in M. bovis demonstrates the presence of an inactivating frameshift, abrogating the ability of this otherwise closely related mycobacterium to produce edaxadiene (4), which we hypothesize contributes to its reduced infectivity and/or virulence in humans relative to M. tuberculosis.Open in a separate windowFIGURE 2.Outline of chemical syntheses of aza analog inhibitors 15-aza-GGPP (7a) and 15-aza-GGSPP (7b) from 14,15-epoxy-GGOH (5) via a common 15-aza-GGOH intermediate (6a).