William J. D. Whish

2-Nitroimidazol-5-ylmethyl as a potential bioreductively activated prodrug system: reductively triggered release of the parp inhibitor 5-bromoisoquinolinone

5-Chloromethyl-1-methyl-2-nitroimidazole reacted efficiently with the anion derived from 5-bromoisoquinolin-1-one to give 5-bromo-2-((1-methyl-2-nitroimidazol-5-yl)methyl)isoquinolin -1-one. Biomimetic reduction effected release of the 5-bromoisoquinolin-1-one. The 2-nitroimidazol-5-ylmethyl unit thus has potential for development as a general prodrug system for selective drug delivery to hypoxic tissues.

Synthesis of thiophenecarboxamides, thieno[3,4-c]pyridin-4(5H)-ones and thieno[3,4-d]pyrimidin-4(3H)-ones and preliminary evaluation as inhibitors of poly(ADP-ribose)polymerase (PARP)

Inhibitors of poly(ADP-ribose)polymerase (PARP) inhibit repair of damaged DNA and thus potentiate radiotherapy and chemotherapy of cancer. Treatment of 3-cyanothiophene with potassium nitrate and concentrated sulphuric acid gave 5-nitrothiophene-3-carboxamide. 4-Nitrothiophene-2-carboxamide and 5-nitrothiophene-2-carboxamide were formed similarly from 2-cyanothiophene. Reduction with tin(II) chloride gave the corresponding aminothiophenecarboxamide salts which were isolated via their N-Cbz derivatives. Lithiation of 3,4-dibromothiophene at -116 degrees C and quenching with alkyl chloroformates gave 4-bromothiophene-3-carboxylates, which were hydrolysed to 4-bromothiophene-3-carboxylic acid. Hurtley reactions with the enolates of pentane-2,4-dione and of 1-phenylbutane-1,3-dione, followed by acyl cleavage, led to 4-(2-oxopropyl)thiophene-3-carboxylic acid and 4-phenacylthiophene-3-carboxylic acid, respectively. Condensation with ammonia in acetic acid gave 6-methyl- and 6-phenylthieno[3,4-c]pyridin-4-ones, which were selectively nitrated at the 1- and 7-positions or were dinitrated. Ethyl 4-acetamido- and 4-benzamido-thiophene-3-carboxylates were cyclised to 2-methyl- and 2-phenyl-thieno[3,4-d][1,3]oxazin-4-ones, respectively. Ring-opening with ammonia and recyclisation led to 2-substituted thieno[3,4-d]pyrimidin-4-ones. The aminothiophenecarboxamides are analogues of 3-aminobenzamide, a selective inhibitor of poly(ADP-ribose)polymerase (PARP); the thienopyridinones and the thienopyrimidinones are analogues of isoquinolin-1-ones and quinazolin-4-ones, respectively, which inhibit this enzyme. In preliminary assays, several thienopyridinones and thienopyrimidinones showed potent inhibitory activity against PARP.

Estimation of parameters for cell-surface interactions : Maximum binding force and detachment constant

A convenient model is presented which can be used to quantify the relationship between applied shear and attached cell fraction in cell/surface interaction studies. The model uses two parameters (the shear stress required to detach the total attached cell population and a detachment constant) based on the estimated strength of the cell/bead interaction force. Use of these parameters allows results obtained on different systems to be compared. The model has been applied to data from three systems. 1) The effects of shear on the interaction between anti-goat IgG-coated beads and surface immobilized goat IgG;1 2) The effect of applying fluid shear stress to a stable fraction of attached 3T3 fibroblast cells on glass;2 and 3) The interaction of suspended yeast cells with surface-immobilized concanavalin A which is reported here. In the yeast system, the model provided a convenient aid for quantifying the effect of competing glucose on the interaction strength where it was found that the detachment constant for yeast interaction with surface-bound conA increases with the glucose concentration while the maximum shear stress and the binding force between the yeast cells and conA decreases.

Effect of polyamines and Mg++ on poly(ADP-ribose) synthesis and ADP-ribosylation of histones in wheat.

Summary Polyamines in the presence or absence of Mg ++ have been shown to stimulate 2–4 fold the total synthesis of poly(ADP-ribose) in isolated wheat nuclei. The stimulation by Mg ++ and polyamines is dose dependent and is an affect on actual synthesis of new chains of polymer since neither Mg ++ nor polyamines increase the average chain length of the polymer synthesised or inhibit the degradation of the polymer in isolated nuclei. Mg ++ and polyamine treated nuclei showed an increased ADP-ribosylation of total histones to the same extent as did total polymer synthesised. Furthermore the distribution of poly(ADP-ribose) between histone H1 and the other core histones remained the same in control, Mg ++ and polyamine treated nuclei.

The kinetics of affinity-mediated cell-surface attachment

Data and a semi-empirical model are presented that describe the affinity interaction of yeast cells with a Concanavalin A derivatised surface. The model uses 3 parameters to describe the time course of cell attachment from a flowing suspension of yeast cells, over a range of flow rates, and gives an effective global fit to the data obtained. Further modifications allow the effects of a soluble competitor (glucose) on binding to be quantified in terms of a saturation effect, and an effective global fit is obtained. A comparison was made between the relationship between steady-state attached fraction and applied shear with similar data reported earlier (Ming, F. et al, 1998) for the detachment of pre-adsorbed cells. This shows that there is an order of magnitude difference between the forces required to effect complete detachment in the two systems, and that the nature of the relationship between shear and attached fraction is profoundly different. The magnitude of this time-dependent stabilization might be explained in terms of a progressive reorientation of cell relative to the surface such that the number of bonds is maximized.

5-Nitrofuran-2-ylmethyl group as a potential bioreductively activatedpro-drug system

5-Substituted isoquinolin-1-ones have been synthesised by one-pot Curtius rearrangement of the corresponding substituted 3-phenylpropenoyl azides and cyclisation. Arylmethylation of the anions of the isoquinolinones with benzyl halides [4-methoxybenzyl chloride, 2-(chloromethyl)furan and 5-nitro-2-(tosyloxymethyl)furan] takes place exclusively at nitrogen. Nitration of 2-(furan-2-ylmethyl)isoquinolin-1-one in strongly acidic medium gives 2-(5-nitrofuran-2-ylmethyl)isoquinolin-1-one, whereas weaker acidic conditions lead to dinitration. Curtius rearrangement of 3-carboranylbutanoyl azide and trapping with 5-nitrofuran-2-ylmethanol gives 5-nitrofuran-2-ylmethyl N-(3-carboranylpropyl)carbamate. Biomimetic reduction of these nitrofuranylmethyl derivatives of anticancer drugs triggers release of the parent drugs. Thus, these nitrofurans have potential applications as pro-drugs for selective release of therapeutic drugs in hypoxic solid tumours.

A method for analyzing the ADP-ribosylation of nuclear proteins on polyacrylamide gels

A method is described for analyzing the extent of mono- and oligo-ADP-ribosylation of specific nuclear proteins directly from the protein bands on polyacrylamide gels. Ethylamine treatment of the gel slices containing the [3H]ADP-ribosylated protein results in the release of the protein associated [3H]mono- and oligo-ADP-ribose which are recovered following lyophilization of the ethylamine extract. Subsequent aminoethyl cellulose chromatography of this released material permits a quantitative separation of mono-ADP-ribose and its derivatives from oligo-ADP-ribose as evidenced by thin-layer chromatographic analysis of the separated fractions. This methodology and the determination of the extent of mono- and oligo-ADP-ribosylation is demonstrated here using [3H]ADP-ribosylated H1 synthesized in pig thymus nuclei incubated in the presence of [3H]NAD. The method is shown to be applicable to both sodium dodecyl sulfate and acid-urea polyacrylamide gels. Furthermore, by performing such analyses on purified and crude H1 preparations the method is shown to allow accurate determinations of the extent of mono- and oligo-ADP-ribosylation without having to first purify the acceptor protein to homogeneity.

Specificity of Poly(ADP-Ribose) Synthetase Inhibitors

Poly(ADP-ribose) synthetase inhibitors, and in particular 3-aminobenzamide, have been used extensively in recent years as probes to elucidate the function of poly(ADP-ribose) in the cell. Our initial report [1] on substituted benzamides as physiologically specific inhibitors was based on the observation that cells grew at an unchanged rate in the presence of 2 mM 3-aminobenzamide, a concentration 1,000 times the Ki value. In general, high concentrations are needed to elicit a cellular response compared to assays in vitro. Interpretation of results is complicated by the possibility of affecting a target other than poly(ADP-ribose) synthetase. Recently, processes other than ADP-ribosylation have been reported to be altered by benzamides. A major criticism of most work is that the studies entail the use of only one inhibitor, usually 3-aminobenzamide.

In vitro poly-(ADP-ribosyl)ation of chromatin proteins in the rat tapeworm, Hymenolepis diminuta

1. (ADP-ribose)-transferase activity in crude chromatin of H. diminuta was demonstrated. 2. Chromatin proteins were ADP-ribosylated in vitro and selectively extracted. 60, 12 and 18% of the (ADP-ribose)n of chromatin proteins was associated with total histones, histone H1 and histone H2B, respectively. 3. The extent of oligo-(ADP-ribose) compared to total (ADP-ribose)n in the chromatin fraction, in the histone fraction, the histone H1 fraction and the histone H2B fraction was 45, 60, 26 and 49%, with an average chain length of 2.8, 2.1, 1.8 and 2.6, respectively. 4. Analysis of (ADP-ribosyl)n-ated proteins by acetic acid/urea polyacrylamide gel electrophoresis demonstrated that histone H1, histone H2B and a 35 kDa non-histone protein were major (ADP-ribose)n acceptors.

The distribution of calmodulin/calmodulin binding proteins in the rat tapeworm, Hymenolepis diminuta.

Live tapeworms have been fixed to retain antigenicity of their proteins, and subsequently prepared for electron microscopy. Thin sections of tapeworms were prepared from resin blocks. Sections were immunocytochemically labelled using a colloidal gold probe and viewed using transmission electron microscopy. Calmodulin was detected associated with cellular structures to which calmodulin has previously been linked in other higher eukaryotes. Calmodulin would appear to have a similar role of importance in tapeworms, as it does in higher eukaryotes although tapeworms are prevalently a syncitium.