Ian R. Pottie

Synthesis of 6H-dibenzo[b,d]pyran-6-ones using the inverse electron demand Diels-Alder reaction.

A set of coumarin-fused electron-deficient 1,3-dienes was synthesized, which differ in the nature of the electron-withdrawing group (EWG) at the terminus of the diene unit and (when EWG = CO(2)Me) the nature and position of substituents. These dienes reacted with the enamine derived from cyclopentanone and pyrrolidine to afford the corresponding cyclopenteno-fused 6H-dibenzo[b,d]pyran-6-ones, most likely via a domino inverse electron demand Diels-Alder (IEDDA)/elimination/transfer hydrogenation sequence. The parent diene (EWG = CO(2)Me, no substituents) was reacted with a range of electron-rich dienophiles (mostly enamines) to afford the corresponding 6H-dibenzo[b,d]pyran-6-ones or their nondehydrogenated precursors, which were aromatized upon treatment with a suitable oxidant. The enamines could either be synthesized prior to the reaction or generated in situ. The syntheses of 30 dibenzopyranones are reported.

Differential binding of phenothiazine urea derivatives to wild-type human cholinesterases and butyrylcholinesterase mutants.

A series of N-10 urea derivatives of phenothiazine was synthesized and each compound was evaluated for its ability to inhibit human cholinesterases. Most were specific inhibitors of BuChE. However, the potent inhibitory effects on both cholinesterases of one sub-class, the cationic aminoureas, provide an additional binding mechanism to cholinesterases for these compounds. The comparative effects of aminoureas on wild-type BuChE and several BuChE mutants indicate a binding process involving salt linkage with the aspartate of the cholinesterase peripheral anionic site. The effect of such compounds on cholinesterase activity at high substrate concentration supports ionic interaction of aminoureas at the peripheral anionic site.

Crystal structures of two 1,2-bis-(1-aryl-3-methyltriazene-3-yl-)ethanes with aryl substituents of opposite polarity

The crystal and molecular structure of 1,2-bis-{1-(2-cyanophenyl)-3-methyltriazen-3-yl-}ethane (1) and 1,2-bis-{1-(2-methoxyphenyl)-3-methyltriazen-3-yl-}ethane (2) have been determined by single crystal X-ray diffraction analysis. Bis-triazene (1) exists as the “staggered” conformation in the solid state, with an anti-anti configuration around the N2–N3 bond of the triazene units, whereas 2 assumes a “gauche” conformation with the syn-syn configuration in the triazene units. Crystal data: 1 C18H18N8, triclinic, space group P −1, a = 6.108(2), b = 8.118(3), c = 9.600(4)Å, α = 101.37(7)°, β = 96.47(7)°, γ = 102.71(5)°, V = 449.1(3)Å3, Z = 2; 2 C18H24N6O2, monoclinic, space group P 21/n, a = 13.119(5), b = 7.745(2), c = 19.201(2)Å, β = 96.47(7)°, V = 1910.3(9)Å3, Z = 4.

Synthesis and Preliminary Evaluation of Piperidinyl and Pyrrolidinyl Iodobenzoates as Imaging Agents for Butyrylcholinesterase

PurposeThe purpose of this study is to synthesize and evaluate specific agents for molecular imaging of butyrylcholinesterase (BuChE), known to be associated with neuritic plaques and neurofibrillary tangles in Alzheimer’s disease (AD). In this study, these agents were tested in a normal rat model. The distribution of radiolabel was compared with known BuChE histochemical distribution in the rat brain.ProceduresIodobenzoate esters were synthesized and tested, through spectrophotometric analysis, as specific substrates for BuChE. These compounds were converted to the corresponding 123I esters from tributyltin intermediates and purified for studies in the rat model. Whole body dynamic scintigraphic images were obtained for biodistribution studies. Autoradiograms of brain sections were obtained and compared to histochemical distribution of the enzyme in this model system.ResultsThe three iodobenzoate esters studied were specific substrates for BuChE. Whole body biodistribution studies with 123I-labeled compounds showed rapid disappearance from the body while radioactivity was retained in the head region. Brain section autoradiography of animals injected with these labeled compounds indicated that most areas known to contain BuChE corresponded to areas of radioactivity accumulation.ConclusionBuChE-specific radiolabeled iodobenzoates enter the brain and, in general, label areas known to exhibit BuChE activity in histochemical studies. Such molecules may represent a new direction for the development of agents for the molecular imaging of BuChE in the living brain, especially in regions where BuChE-containing neuropathological structures appear in AD.

Cysteine Thioesters as Myelin Proteolipid Protein Analogues to Examine the Role of Butyrylcholinesterase in Myelin Decompaction

Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disorder involving demyelination, axonal transection, and neuronal loss in the brain. Recent studies have indicated that active MS lesions express elevated levels of butyrylcholinesterase (BuChE). BuChE can hydrolyze a wide variety of esters, including fatty acid esters of protein. Proteolipid protein (PLP), an important transmembrane protein component of myelin, has six cysteine residues acylated, via thioester linkages, with fatty acids, usually palmitic, that contribute to the stability of myelin. Experimental chemical deacylation of PLP has been shown to lead to decompaction of myelin. Because of elevated levels of BuChE in active MS lesions and its propensity to catalyze the hydrolysis of acylated protein, we hypothesized that this enzyme may contribute to deacylation of PLP in MS, leading to decompaction of myelin and contributing to demyelination. To test this hypothesis, a series of increasing chain length (C2-C16) acyl thioester derivatives of N-acetyl-l-cysteine methyl ester were synthesized and examined for hydrolysis by human cholinesterases. All N-acetyl-l-cysteine fatty acyl thioester derivatives were hydrolyzed by BuChE but not by the related enzyme acetylcholinesterase. In addition, it was observed that the affinity of BuChE for the compound increased the longer the fatty acid chain, with the highest affinity for cysteine bound to palmitic acid. This suggests that the elevated levels of BuChE observed in active MS lesions could be related to the decompaction of myelin characteristic of the disorder.

Synthesis and Preliminary Evaluation of Phenyl 4-123I-Iodophenylcarbamate for Visualization of Cholinesterases Associated with Alzheimer Disease Pathology

Acetylcholinesterase and butyrylcholinesterase accumulate with brain β-amyloid (Aβ) plaques in Alzheimer disease (AD). The overall activity of acetylcholinesterase is found to decline in AD, whereas butyrylcholinesterase has been found to either increase or remain the same. Although some cognitively normal older adults also have Aβ plaques within the brain, cholinesterase-associated plaques are generally less abundant in such individuals. Thus, brain imaging of cholinesterase activity associated with Aβ plaques has the potential to distinguish AD from cognitively normal older adults, with or without Aβ accumulation, during life. Current Aβ imaging agents are not able to provide this distinction. To address this unmet need, synthesis and evaluation of a cholinesterase-binding ligand, phenyl 4-123I-iodophenylcarbamate (123I-PIP), is described. Methods: Phenyl 4-iodophenylcarbamate was synthesized and evaluated for binding potency toward acetylcholinesterase and butyrylcholinesterase using enzyme kinetic analysis. This compound was subsequently rapidly radiolabeled with 123I and purified by high-performance liquid chromatography. Autoradiographic analyses were performed with 123I-PIP using postmortem orbitofrontal cortex from cognitively normal and AD human brains. Comparisons were made with an Aβ imaging agent, 2-(4′-dimethylaminophenyl)-6-123I-iodo-imidazo[1,2-a]pyridine (123I-IMPY), in adjacent brain sections. Tissues were also stained for Aβ and cholinesterase activity to visualize Aβ plaque load for comparison with radioligand uptake. Results: Synthesized and purified PIP exhibited binding to cholinesterases. 123I was successfully incorporated into this ligand. 123I-PIP autoradiography with human tissue revealed accumulation of radioactivity only in AD brain tissues in which Aβ plaques had cholinesterase activity. 123I-IMPY accumulated in brain tissues with Aβ plaques from both AD and cognitively normal individuals. Conclusion: Radiolabeled ligands specific for cholinesterases have potential for use in neuroimaging AD plaques during life. The compound herein described, 123I-PIP, can detect cholinesterases associated with Aβ plaques and can distinguish AD brain tissues from those of cognitively normal older adults with Aβ plaques. Imaging cholinesterase activity associated with Aβ plaques in the living brain may contribute to the definitive diagnosis of AD during life.

A molecular recognition event in the reaction of a diazonium salt with N, N-dimethylethylenediamine

Diazotization of p-aminobenzonitrile and coupling of the resulting diazonium salt with N,N-dimethylethylenediamine affords a novel material that has been shown by X-ray crystallography to be a cocrystal (3) of the pentaazadiene (1) and the diaryltriazene (2). Crystal data: monoclinic, space group P21/c, a = 9.363(5), b = 21.509(3), c = 15.862(5) Å, β = 98.86(5)°, V = 3156.3(18) Å3, Z = 4, Rw = 0.035, Rint = 0.037. The pairing of the two molecules 1 and 2 appears to represent a unique molecular recognition event involving a combination of hydrogen bonding and π–π stacking. The crystal structure shows that the N–H of the triazene is within hydrogen-bonding distance of the tertiary nitrogen atom in the alkyl side-chain of the pentazadiene; furthermore, the benzene rings of the triazene and the pentazadiene are stacked off-register and separated by 3.53 Å. The formation of the components of the cocrystal is rationalised in terms of the formation and decomposition of the 1-aryl-3-alkyltriazene, Ar–N=N–NH–CH2CH2NH2.

Targeting butyrylcholinesterase for preclinical single photon emission computed tomography (SPECT) imaging of Alzheimer's disease

Diagnosis of Alzheimers disease (AD) in vivo, by molecular imaging of amyloid or tau, is constrained because similar changes can be found in brains of cognitively normal individuals. Butyrylcholinesterase (BChE), which becomes associated with these structures in AD, could elevate the accuracy of AD diagnosis by focusing on BChE pathology in the cerebral cortex, a region of scant BChE activity in healthy brain.

Geminal acylation of α-heterosubstituted cyclohexanones and their ketals

Geminal acylation of derivatives of cyclohexanone with Br, Cl, F, and OCH3 in the α position, and of their corresponding dimethyl ketals, could not be accomplished to a significant extent following...

SYNTHESIS AND IN VIVO SPECT EVALUATION OF A NOVEL BUTYRYLCHOLINESTERASE DIAGNOSTIC RADIOLIGAND FOR ALZHEIMER’S DISEASE

artifact was rejected through independent component analysis (ICA) of MEG channels. Sensor level analysis was performed by filtering (0.3-3Hz) and averaging across trials. Source localization was performed using the Minimum Norm technique in Statistical Parametric Mapping (SPM8). Results: Preliminary results at the sensor level show that there is increased magnetic field activity in the delta frequency range (0.5-3Hz) occurring 300-400ms postblink, consistent with prior studies using EEG. Source location identified increased activity in areas of the parietal, temporal, and frontal lobes consistent with the DMN, occurring in the 500ms post-blink interval relative to pre-blink (FWE p<0.001). Conclusions:To our knowledge, this is the first study of BRO activity using MEG. Our preliminary results suggest BROs originate from regions within the DMN. Continuing work examines activity in other frequency bands, and involves time-frequency, region-of-interest, as well as functional connectivity analyses. BROs may provide an exciting new avenue for accessing the DMN as a clinical marker for AD.