Further evaluation of 5-alkyl-5-deaza antifolates. 5-propyl- and 5-butyl-5-deaza analogues of aminopterin and methotrexate

5-Propyl-5-deaza and 5-butyl-5-deaza analogues of classical antifolates were synthesized by extensions of a previously reported general route which proceeds through 2,4-diamino-5-alkylpyrido[2,3-d]pyrimidine-6-carbonitrile intermediates followed by reductive condensation with diethyl N-4-(aminobenzoyl)-L-glutarnate to give diethyl esters of 5-alkyl-5-deazaaminopterin types. N¹⁰-Methyl derivatives, i.e., derivatives of 5-alkyl-5-deazamethotrexate, were also prepared by reductive methylation of the N¹⁰-H compounds. 5-Ethyl-5-deazamethotrexate was prepared using an alternative route through 6-(bromomethyl)-2,4-diamino-5-ethylpyrido[2,3-d]pyrimidine. These antifolates were evaluated for inhibition of dihydrofolate reductase (DHFR) from L1210 cells, their effect on L1210 and S180 tumor cell growth in culture, and carrier-mediated transport through L1210 cell membranes. Inhibitory effect on DHFR was lowered relative to methotrexate in 5-propyl-5-deazaaminopterin and 5-propyl-5-deazamethotrexate by 2- to 3-fold (K_i = 9.3 and 11.7 pM, respectively, vs. 4.3 pM for methotrexate) and by 17- to 18-fold in 5-butyl-5-deaza-aminopterin and 5-butyl-5-deazamethotrexate (K_i = 74 and 78 pM, respectively). Molecular modeling using graphics derived from human DHFR show the propyl and butyl compounds interacting with the enzyme in conformations that account for these slight decreases in binding.     

Common circulant homogeneous factorisations of the complete digraph

In this paper we determine the positive integers n and k for which there exists a homogeneous factorisation of a complete digraph on n vertices with k ‘common circulant’ factors. This means a partition of the arc set of the complete digraph K_n into k circulant factor digraphs, such that a cyclic group of order n acts regularly on the vertices of each factor digraph whilst preserving the edges, and in addition, an overgroup of this permutes the factor digraphs transitively amongst themselves. This determination generalises a previous result for self-complementary circulants.     

A high‐throughput bioanalytical platform using automated infusion for tandem mass spectrometric method optimization and its application in a metabolic stability screen

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the bioanalytical method of choice to support plate‐based, in vitro early ADME (Absorption, Distribution, Metabolism and Excretion) screens such as metabolic stability (Metstab) assessment. MS/MS method optimization has historically been the bottleneck in this environment, where samples from thousands of discrete compounds are analyzed on a monthly basis, mainly due to the lack of a high‐quality commercially available platform to handle the necessary MS/MS method optimization steps for sample analysis by selected reaction monitoring (SRM) on triple quadrupole mass spectrometers. To address this challenge, we recently developed a highly automated bioanalytical platform by successfully integrating QuickQuan? 2.0, a unique high‐throughput solution featuring MS/MS method optimization by automated infusion, with a customized in‐house software tool in support of a Metstab screen. In this platform, a dual‐column setup running parallel chromatography was also implemented to reduce the bioanalytical cycle time for LC/MS/MS sample analysis. A set of 45 validation compounds was used to demonstrate the speed, quality and reproducibility of MS/MS method optimization, sample analysis, and data processing using this automated platform. Metstab results for the validation compounds in microsomes from multiple species (human, rat, mouse) showed good consistency within each batch, and also between batches conducted on different days. We have achieved and maintained a monthly throughput of 1300 compound assays representing 500 discrete compounds per instrument per month on this platform, and it has been used to generate metabolic stability data for more than 25 000 compounds to date with an overall success rate of more than 95%. Copyright © 2009 John Wiley & Sons, Ltd.     

Repetitive Phonatory Tasks for Identifying Vocal Fold Paresis

Vocal fold paresis may be present in patients with voice complaints. Identification of paresis is important so that appropriate neurolaryngologic evaluation can be ordered and the appropriate treatment can be offered. Repetitive phonatory tasks (RPTs) fatigue patients vocally and may elicit signs of subtle paresis. In this study, four laryngologists independently reviewed the RPT portions of routine fiberoptic voice examinations of 100 patients in a blinded fashion. All patients had presented with voice complaints, were suspected of having a movement disorder of the larynx, and had been referred for laryngeal electromyography (LEMG). Predictions were compared with LEMG results and with predictions made at the time of each initial evaluation. Although RPTs are useful to laryngologists, predictions based on the entire examination are more accurate. LEMG can confirm clinical suspicions or identify paresis missed on fiberoptic laryngeal examination.     

Reactions involving di-trans-[12]annulenes

The low temperature complete dehydrohalogenation of pentabromocyclododecene (C12H17Br5) with potassium tert-butoxide in THF followed by exposure to potassium metal leads to the formation of the anion radical of 1,5-di-trans-[12]annulene, which loses hydrogen and undergoes ring closure to form the anion radical of 11,12-dihydro-[8]annuleno-[6]annulene. This product can, in turn, be isolated as its neutral molecule via reoxidation with iodine. A [12]annulene obtained via the dimerization of 1,5-hexadiyne in the presence of 18-crown-6 and potassium tert-butoxide undergoes ring closure, with concomitant loss of hydrogen, to yield the heptalene anion radical. It follows that the heptalene anion radical precursor was the 1,7-di-trans isomer of [12]annulene.     

Luminescence temperature sensing using poly(vinyl alcohol)-encapsulated Ru(bpy)3 2+ films

The ruthenium(II) diimine complexes, such as ruthenium(II) tris(bipyridyl), Ru(bpy)3 2+, possess highly luminescent excited states that are not only readily quenched by oxygen but also by an increase in temperature. The former effect can be rendered insignificant by encapsulating the complex in an oxygen impermeable polymer, although encapsulation often leads also to a loss of temperature sensitivity. The luminescence properties of Ru(bpy)3 2+ encapsulated in PVA were studied as a function of oxygen concentration and temperature and found to be independent of the former, but still very sensitive towards the latter. The results were fitted to an established Arrhenius-type equation, based on thermal quenching of the emitting state by a slightly higher (DeltaE= 3100 cm(-1))3d-d state that deactivates very rapidly (10(-13) s)via a non-radiative process.     

Stimulus Dependent Redistribution of Membrane Raft Cholesterol in Human Platelets

Cell membranes provide a requisite dynamic interface to facilitate communication between the extracellular environment and the intracellular milieu. These membranes contain proteins that span and/or are loosely associated with the lipid bilayer. The organization of lipids and proteins components into membrane micro-domains provides a temporal and spatial signaling platform for communication. Recently, cholesterol and sphingomyelin enriched membrane micro-domains known as lipid rafts have been implicated in cell signaling events. In these studies we have advanced our hypothesis that stimulus dependent rearrangement of cholesterol into and out of membrane rafts provides a unique lipid–mediated regulatory mechanism. Using fluorescent derivatives of cholesterol, we have shown that membrane raft associated cholesterol was altered in response to collagen-induced platelet aggregatory stimulation. Collagen stimulation resulted in a rapid redistribution of cholesterol from the outer to the inner membrane monolayer. The reorganization of the outer membrane monolayer resulted in a concomitant increase in outer monolayer fluidity. These studies are the first to show that membrane cholesterol was released from the exchangeable membrane raft pool in response to physiological stimuli.     

Simultaneous butyltin determinations in the microlayer,water column and sediment of a northern Chesapeake Bay marina and receiving system

Butyltins were determined in the microlayer, water column and sediment of a northern Chesapeake Bay marina and its receiving system. Concentrations of the toxicant species tributyltin (TBT) ranged from 60 to 4130 ng dm⁻³ in the microlayer, from 34 to 367 ng dm⁻³ in the water column and from <0.05 to 1.4 m̈g g⁻¹ (dry weight) in sediment. TBT concentrations in all three environmental compartments were higher in the marinas than in the receiving system. Concentrations of TBT in the microlayer and water column of the study area were potentially toxic to sensitive aquatic biota. The microlayer appears to be depleted in dibutyltin relative to tributyltin compared to both water column and sediment.