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Antineoplastic ether lipids have entered phase I clinical trial and, although their mechanism of action remains unclear, it is widely believed that the plasma membrane is the primary cellular drug target. In the present study the hypothesis was tested that metabolism of ether lipids acts as a detoxification process. [31P]-nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of the ether lipid SRI 62-834 (SRI) and the phosphate ester hexadecylphosphocholine (HPC) in the presence of both isolated phospholipases C and D and post-mitochondrial rat liver homogenate. Both SRI and HPC were slowly metabolised by phospholipase D to their alkyl phosphates and choline, and the alkyl phosphates were subsequently metabolised by phosphatase to yield the alcohols and inorganic phosphate. These studies failed to detect any metabolism of either SRI or HPC by phospholipase C, and the metabolism of platelet-activating factor (PAF) by this enzyme was not inhibited by the addition of either compound. The cytotoxicity of SRI, the related compound HPC and their metabolites was determined in vitro using three cell lines. Cytotoxicity was measured by analysis of cell growth kinetics, MTT assay and lactate dehydrogenase release. Closely similar results were obtained in the JB1 rat hepatoma cell line, in the non-transformed BL8 rat hepatocyte cell line, and in A549 human lung adenocarcinoma cells. SRI was the most toxic of the compounds analysed, the concentration required to produce 50% toxicity or growth inhibition (IC50) being 6-9 microM. The putative metabolite of SRI, 2,2'-bis(hydroxymethyl)tetrahydrofuran, and the known metabolites [2'-(octadecyloxymethyl)tetrahydrofuran-2'-yl]methyl phosphate and 2-hydroxymethyl-2-octadecyloxymethyltetrahydrofuran exhibited IC50 values of > 200, > 100 and 40-70 microM, respectively, consistent with metabolic detoxification. HPC was more cytotoxic (IC50, 37 microM) than its phosphate metabolite (IC50, 140 microM), but its toxicity was similar to that of its metabolite hexadecanol (IC50, 28 microM), suggesting that only the former metabolic route leads to detoxification.

The autoinducer (4S)-4,5-dihydroxypentane-2,3-dione ((S)-DPD, AI-2) facilitates chemical communication, termed 'quorum sensing', amongst a wide range of bacteria, The synthesis of (S)-DPD is challenging in part due to its instability. Herein we report a novel synthesis of (S)-DPD via (2S)-2,3-O-isopropylidene glyceraldehyde, through Wittig, dihydroxylation and oxidation reactions, with a complimentary asymmetric synthesis to a key precursor. Its enantiomer (R)-DPD, was prepared from d-mannitol via (2R)-2,3-O-isopropylideneglyceraldehyde. The synthesized enantiomers of DPD have AI-2 bioluminescence-inducing properties in the Vibrio harveyi BB170 strain.

The psychoactive properties of N,N-dimethyltryptamine (DMT) are known to induce altered states of consciousness in humans. These properties attract great interest from clinical, neuroscientific, clandestine and forensic communities. The Breath of Hope Synthesis was reported on an internet website as a convenient two-step methodology for the preparation of DMT. The analytical characterisation of the first stage was the subject of previous publications by the authors and involved the thermal decarboxylation of tryptophan and the formation of tryptamine. The present study reports on the characterisation of the second step of this procedure which was based on the methylation of tryptamine. This employed methyl iodide and benzyltriethylammonium chloride/sodium hydroxide as a phase transfer catalyst. The reaction product was characterised by liquid chromatography/electrospray ionisation tandem mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry. Quantitative evaluation was carried out in positive multiple reaction monitoring mode (MRM), which included synthesis of the identified reaction products. MRM screening of the product did not lead to the detection of DMT. Instead, 11.1% tryptamine starting material, 21.0% N,N,N-trimethyltryptammonium iodide (TMT) and 47.4% 1-N-methyl-TMT were detected. A 0.5% trace of the monomethylated N-methyltryptamine was also detected. This study demonstrated the impact on product purity of doubtful synthetic methodologies discussed on the internet.

The crystal structure of (2R,4S,5R)-3,4-dimethyl-2-phenoxy-5-phenyl-1,3,2-oxazaphospholidin-2-one (7) exhibits a C(5) envelope in which this atom is below the O(1)–P(2)–N(3)–C(4) plane. The diastereoisomer, (2S,4S,5R)8 adopts a half-chair conformation with C(4) above and C(5) below the O(1)–P(2)–N(3) plane. Both (2S,4S,5R)- and (2R,4S,5R)-3,4-dimethyl-2,5-diphenyl-1,3,2-oxazaphospholidin-2-one (9 and 10), and (2S,4S,5R)-3,4-dimethyl-2,5-diphenyl-1,3,2-oxazaphospholidin-2-thione (12) adopt C(4) envelopes in the crystal structures, with C(4) above the C(5)–O(1)–P(2)–N(3) plane for 9 and below for 10 and 12, (2R,4S,5R)-3,4-Dimethyl-2,5-diphenyi-1,3,2-oxazaphospholidine-2-thione (11) exists as a half-chair with O(1) above and C(5) below the P(2)–N(3)–C(4) plane. The C(4) and C(5) envelope conformations for 7, 9, 10 and 12 are consistent with the P–O–C–H vicinal coupling constants derived from 1H NMR spectra in solution.

Natural myo-inositol phosphate antioxidants containing the 1,2,3-trisphosphate motif bind Fe(3+) in the unstable penta-axial conformation.

Many oral care products incorporate an antibacterial compound to prevent the formation of dental plaque which predisposes teeth to dental caries or periodontal disease. Triclosan (TCN) is a commonly used antiplaque agent in toothpastes. Strategies to increase the delivery efficiency of antibacterials using formulation aids such as polyamidoamine (PAMAM) dendrimers are of interest. Solubilisation studies over the pH range 5-12 demonstrated an increase in the level of TCN solubilised with increasing dendrimer concentration (1 mM-5 mM). However, the dendrimer was unable to enhance TCN solubility at lower pH values and the solubilising effect observed was attributed to the ionization of TCN (pKa 8.14) resulting from dendrimer induced pH changes. End group modification of G3 PAMAM dendrimer with phenylalanine in order to promote solubility through pi-pi stacking between TCN and the amino acid has been carried out. Phenylalanine:G3 PAMAM conjugates of different ratios (32:1, 21:1, 16:1) were synthesized. The fully conjugated dendrimer (32:1) had poor aqueous solubility, whereas the 21:1 and 16:1 dendrimer conjugates were water soluble. The 21:1 conjugate was tested for its ability to solubilise TCN, however, again there was no increase over control buffer solutions of the same pH. An alternative approach under investigation is to directly conjugate TCN to PAMAM dendrimers via a hydrolysable linkage.

2-O-tert-Butyldimethylsilyl-4,6-bis-O-pyrenoyl-myo-inositol-1,3,5-orthoformate (6) and 2-O-tert-butyldimethylsilyl-4-O-[4-(dimethylamino)benzoyl]-6-O-pyrenoyl-myo-inositol-1,3,5-orthoacetate (10) adopt conformationally restricted unstable chairs with five axial substituents. In the symmetrical diester 6, the two pi-stacked pyrenoyl groups are electron acceptor-donor partners, giving a strong intramolecular excimer emission. In the mixed ester 10, the pyrenoyl group is the electron acceptor and the 4-(dimethylamino)benzoyl ester is the electron donor, giving a strong intramolecular exciplex emission. The conformation of the mixed ester 10 was assessed using 1H NMR spectroscopy (1H-NOESY) and computational studies. which showed the minimum inter-centroid distance between the two aromatic systems to be approximately 3.9 A. Upon addition of acid, the orthoformate/orthoacetate trigger in 6 and 10 was cleaved, which caused a switch of the conformation of the myo-inositol ring to the more stable penta-equatorial chair, leading to separation of the aromatic ester groups and loss of excimer and exciplex fluorescence, respectively. This study provides proof of principle for the development of novel fluorescent molecular probes.

The psychoactive properties of N , N -dimethyltryptamine (DMT) 1a are known to induce altered states of consciousness in humans. This particular attribute attracts great interest from a variety of scientific and also clandestine communities. Our recent research has confirmed that DMT reacts with dichloromethane (DCM), either as a result of work-up or storage to give a quaternary N -chloromethyl ammonium salt 2a . Furthermore, this was observed to undergo rearrangement during analysis using gas chromatography–mass spectrometry (GC–MS) with products including 3-(2-chloroethyl)indole 3 and 2-methyltetrahydro-β-carboline 4 (2-Me-THBC). This study further investigates this so far unexplored area of solvent interactions by the exposure of DMT to other halogenated solvents including dibromomethane and 1,2-dichloroethane (DCE). The N -bromomethyl- and N -chloroethyl quaternary ammonium derivatives were subsequently characterised by ion trap GC–MS in electron and chemical ionisation tandem MS mode and by NMR spectroscopy. The DCE-derived derivative formed at least six rearrangement products in the total ion chromatogram. Identification of mass spectrometry generated by-products was verified by conventional or microwave-accelerated synthesis. The use of deuterated DCM and deuterated DMT 1b provided insights into the mechanism of the rearrangements. The presence of potentially characteristic marker molecules may allow the identification of solvents used during the manufacture of controlled substances, which is often neglected since these are considered inert.

UCS 15A, an antibiotic produced by Streptomyces sp., has been reported to specifically disrupt SH3 domain-mediated interactions in eukaryotic cells. Interestingly, in the case of the non-receptor tyrosine kinase Src, UCS15A was effective in suppressing the SH3 domain-mediated intermolecular rather than intramolecular interactions, and thus prevented Src interactions with certain downstream effectors without affecting Src kinase activity. Here the synthesis of a novel difluoro analogue of UCS15A is described. The effects of this compound (8) on Src activity were tested in HCT 116 colorectal carcinoma cells engineered for inducible expression of c-Src. The presence of compound (8) resulted in the increased activity of the induced c-Src implicating that (8) acts as a c-Src activator in vivo. These observations are supported by computer modelling studies which suggest that the aldehyde group of (8) may covalently bind to a lysine residue in the SH2-kinase linker region situated in the proximity of the SH3 domain, which could promote a conformational change resulting in increased Src activity.

N,N-Dimethyltryptamine (DMT) 1 is a simple tryptamine derivative with powerful psychoactive properties. It is abundant in nature and easily accessible through a variety of synthetic routes. Most work-up procedures require the use of organic solvents and halogenated representatives are often employed. DMT was found to be reactive towards dichloromethane, either during work-up or long term storage therein, which led to the formation of the quaternary ammonium salt N-chloromethyl-DMT chloride 2. Analysis of this side-product by gas chromatography ion trap mass spectrometry (GC-MS), both in electron and chemical ionisation tandem MS modes, gave only degradation products. For example, 2 could not be detected but appeared to have rearranged to 3-(2-chloroethyl)indole 3 and 2-methyltetrahydro-beta-carboline 4, whereas HPLC analysis enabled the detection of 2. GC-MS is a standard tool for the fingerprinting of drug products. The identification of a particular synthetic route is based on the analysis of impurities, provided these side products can be established to be route-specific. The in situ detection of both 3 and 4 within a DMT sample may have led to erroneous conclusions with regards to the identification of the synthetic route.

Src signalling and transduction are directly involved in cell growth, cell cycle, malignant transformation and cell migration, providing therapeutic opportunities through inhibition of Src. Here we report virtual screening for novel compounds that inhibit the Src-SH3 protein–protein interaction with a proline-rich peptide ligand. Computational docking of the ZINC compound database was performed using GOLD. Top-scoring compounds were assayed using a fluorescence polarization-based assay. A benzoquinoline derivative showed micromolar inhibition of binding between Src-SH3 and the proline-rich peptide. Several analogues were subsequently assayed showing the requirement of a linker between the benzoquinoline and phenyl rings, and electron donating substituents on the phenyl ring.

A series of novel imidazolyluracil conjugates were rationally designed and synthesised to probe the active site constraints of the angiogenic enzyme, thymidine phosphorylase (TP, E.C. 2.4.2.4). The lead compound in the series, 15d, showed good binding in the active site of human TP with an inhibition in the low μM range. The absence of a methylene bridge between the uracil and the imidazolyl sub-units (series 16) decreased potency (up to 3-fold). Modelling suggested that active site residues Arg202, Ser217 and His116 are important for inhibitor binding.

Thymidine phosphorylase (EC 2.4.2.4) catalyses the reversible phosphorolysis of pyrimidine 2'-deoxynucleosides, forming 2-deoxyribose-1-phosphate and pyrimidine. 5-Chloro-6-(2-imino-pyrrolidin-1-yl)methyl-uracil hydrochloride (TPI, 1) and its 5-bromo analogue (2), 6-(2-amino-imidazol-1-yl)methyl-5-bromo-uracil (3) and its 5-chloro analogue (4) act as tight-binding stoichiometric inhibitors of recombinant E. coli thymidine phosphorylase, and thus can be used as the first active-site titrants for it using either thymidine or 5-nitro-2'-deoxyuridine as substrate.

N-Alkylated tryptamines have complex psychoactive properties. Routes for clandestine synthesis are described on Internet websites one of which involves the thermolytic decarboxylation of tryptophan to tryptamine as a precursor to psychoactive compounds. High boiling solvents and ketone catalysts have been employed to facilitate the decarboxylation of tryptophan. The present study has revealed that there is formation of tetrahydro--carboline (THBC) derivatives which may originate from reaction with both the solvent and the ketone catalyst. The application of gas chromatography electron- and chemical-ionisation ion trap tandem mass spectrometry (GC–IT-MS–MS), in combination with nuclear magnetic resonance (NMR), led to the isolation and identification of 1,1disubstituted-tetrahydro--carbolines formed as major impurities in the tryptamine. Confirmation was by synthesis of the THBC derivatives from tryptamine using Pictet-Spengler cyclisation. Under EI-conditions, mass spectral characterisation of the THBCs suggests predominance of alkyl cleavage. These impurities will yield a useful profile for identification of the synthetic pathway and likely reagents employed, particularly a “fingerprint” of the ketone catalyst and an insight into the influence of solvents and catalysts on the formation of by-products. © 2006 Elsevier B.V. All rights reserved.

Twelve symmetrically and 13 asymmetrically N,N-disubstituted glyoxalylamide precursors and their corresponding tryptamine derivatives have been characterised by gas chromatography low-pressure chemical ionisation ion trap tandem mass spectrometry (CI-IT-MS-MS) with internal (in situ) ionisation using methanol as the chemical ionisation reagent. Mass spectral differences and similarities between the investigated compounds are discussed and put into context with previous investigations. In tryptamines the formation of [CH2=N+R2R3] iminium ions after beta-cleavage appears to be the dominating process. Dissociation of the protonated molecule into [3-vinylindole]+ for example, appears to be a minor pathway when compared with electrospray triple quadrupole tandem mass spectrometry (ESI-TQ-MS-MS) where this ion transition was found to be of distinctive importance. CI-IT-MS-MS is also found to enable the differentiation between most isomeric derivatives studied.

Thymidine phosphorylase (TP) is an important target enzyme for cancer chemotherapy because it is expressed at high levels in the hypoxic regions of many tumors and inhibitors of TP have been shown in animal model studies to inhibit angiogenesis and metastasis, and to promote tumor cell apoptosis. The 5-halo-6-[(2'-aminoimidazol-1'-yl)methyl]uracils (3, X = Cl, Br) are very potent inhibitors of E. coli and human TP with IC(50) values of approximately 20 nM when the enzyme concentration is approximately 40 nM. Their 4'-aminoimidazol-1'-yl analogues (4, X = Cl, Br) are >350-fold less active with IC(50) values of approximately 7 microM. The 5-unsubstituted analogues (3 and 4, X = H) were both less active than their 5-halo derivatives. Determination of pK(a) values and molecular modeling studies of these compounds in the active site of human TP was used to rationalize their activities. The finding that 3, X = Br has a poor pharmacokinetic (PK) profile in mice, coupled with the desire for tumor selectivity, led us to design prodrugs. The corresponding 2'-nitroimidazol-1'-ylmethyluracils (5, X = Cl, Br) are >1000-fold less active (IC(50) 22-24 microM) than their 2'-amino analogues and are reduced to the 2'-amino inhibitors (3, X = Cl, Br) by xanthine oxidase (XO). As XO is also highly expressed in many tumors, the 2'-nitro prodrugs have the potential to selectively deliver the potent 2'-aminoimidazol-1'-yl TP inhibitors into hypoxic solid tumors.

5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), a new psychoactive tryptamine derivative, has been synthesised by the Speeter and Anthony procedure. This synthetic route was characterised by ESI-MS-MS, ESI-TOF-MS and NMR. Side products have been identified as 3-(2-N,N-diisopropylamino-ethyl)-1H-indol-5-ol (5), 2-N,N-diisopropylamino-1-(5-methoxy-1H-indol-3-yl)-ethanol (6), 2-(5-methoxy-1H-indol-3-yl)-ethanol (7) and 2-N,N-diisopropylamino-1-(5-methoxy-1H-indol-3-yl)-ethanone (8).

Structure-based computational screening of the National Cancer Institute database of anticancer compounds identified novel non-nucleobase-derived inhibitors of human thymidine phosphorylase as candidates for lead optimization. The hierarchical in silico screening strategy predicted potentially strong low molecular weight ligands exhibiting a range of molecular scaffolds. Of the thirteen ligands assayed for activity, all displayed inhibitory activity against Escherichia coli thymidine phosphorylase. One compound, hydrazine carboxamide 2-[(1-methyl-2,5-dioxo-4-pentyl-4-imidazolidinyl)methylene], was found to inhibit E. coli thymidine phosphorylase with an IC(50) value of 20 microM and an IC(50) value of 77 microM against human thymidine phosphorylase. As this hydantoin derivative lacks the undesirable ionic sites of existing tight-binding nucleobase-derived inhibitors, such as 5-chloro-6-[(2-iminopyrrolidin-1-yl)methyl]uracil hydrochloride, it provides an opportunity for the design of potent thymidine phosphorylase inhibitors with improved pharmacokinetic properties.

Thymidine phosphorylase (TP) is an angiogenic growth factor and a target for anticancer drug design. Molecular modeling suggested that 2'-aminoimidazolylmethyluracils would be potent inhibitors of TP. The novel 5-halo-2-aminoimidazolylmethyluracils (4b/4c) were very potent inhibitors of E. coli TP (IC50 approximately 20 nM). Contrastingly, the corresponding 2'-nitroimidazolylmethyluracil (as bioreductively activated) prodrugs (3b/3c) were 1000-fold less active (IC50 22-24 microM). This approach may be used to selectively deliver TP inhibitors into hypoxic regions of solid tumors where TP is overexpressed.

The arylethylamines substituted in the aryl ring, side-chain carbons and on the terminal amine, comprise a large number of human mood and behaviour altering chemicals. Some of these psychotropic drugs have been used since pre-history, but in many states are proscribed and are consequently subject to clandestine synthesis and illegal traffic world-wide in the forms particularly of amphetamines and to a lesser extent tryptamines. The chemistry employed in the synthesis of these compounds is dictated often by the available precursors and relies usually on relatively simple, unsophisticated conversion reactions to a suitable product. The internet web sites and documentation of the recreational drug culture have been studied alongside the professional scientific and regulatory literature. The review demonstrates the great complexity of the chemistry and neuro-pharmacology of these chemicals and the challenge faced by legislative bodies to control their traffic and use for the sake of social welfare.

2-Amino-2-carboxyethylphosphinic acid exists as a zwitterion (2-ammonio-2-carboxyethylphosphinate) with anionic phosphinic, cationic alkylammonium and neutral carboxyl groups. The P-C-C-C and P-C-C-N conformations are gauche and trans, respectively. In contrast to 2-amino-2-carboxyethylphosphonic acid, intramolecular hydrogen bonding from ammonium to phosphinic moieties does not occur. Each phosphinic O atom accepts two intermolecular hydrogen bonds, as does the water molecule; every ammonium, carboxyl and water H atom participates

Background: NRH: quinone oxidoreductase 2 (NQO2), is a cytosolic flavoprotein that catalyses the two-electron reduction of quinones into hydroquinones. The physiological role of NQO2 in cancer biology is still not well understood. Some studies have suggested that inhibiting NQO2 could have a great potential in chemoprevention. This is based on the observation that NQO2 gene knockdown promotes protection against the toxicity of quinones such as menadione. Aim: To evaluate the biological role of novel NQO2 inhibitors in cancer therapeutics. Novel inhibitors will be used as pharmacological tools to study the physiological role of NQO2 in cancer cells, and to identify the consequences of inhibiting NQO2 on the tumor microenvironment. Methods: Novel compounds with diverse structures have been tested for their inhibition of NQO2 enzymatic activity in a cell-free system using a pure enzyme assay. The functional activity of these compounds as NQO2 inhibitors in the MDA-MB 231 breast cancer cells was also assessed. The effect of these inhibitors on the cellular proliferation of MDA-MB 231 breast cancer cells was tested using an MTT assay. Off-target (DNA binding) effects were analyzed by measuring the DNA thermal temperature. Moreover, the effect of these inhibitors on modulating TNFα-mediated NF-kB transcriptional activity was tested and the mechanism behind this modulation was also examined. Results: The novel inhibitors tested resulted in the inhibition of NQO2 activity in the cell free and the cellular system at nano-molar concentrations .Inhibition of MDA-MB 231 cell proliferation was also witnessed at low micro-molar concentrations. The inhibitors also displayed no DNA binding ability. In addition, the inhibitors were able to attenuate TNFα-mediated NF-kB transcriptional activity by blocking the phosphorylation of IkBα. This has resulted in the suppression of cell growth and promotion of cell apoptosis. Conclusions: This study has demonstrated novel potent inhibitors of NQO2 that are functionally active in the cells at nano-molar concentrations. The effect of the inhibitors on TNFα-mediated NF-kB transcriptional activity raises the possibility that inhibiting NQO2 could modulate the tumor micro-environmental factors, such as metastasis and angiogenesis. Citation Format: Elham Santina, Amy Chadwick, Soraya Al Nabulsi, Sally Freeman, Constantinos Demonacos, Ian Stratford. Biological role of novel NQO2 inhibitors in cancer therapeutics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1388. doi:10.1158/1538-7445.AM2014-1388

Inositol monophosphatase (IMPase) catalyses the hydrolysis of myo-inositol monophosphates to myo-inositol. which is required in the phosphatidyl inositol cell signalling pathway. Here the enzyme structure, mechanism and inhibition of IMPase are reviewed. Lithium, an effective therapy for manic depression. is an uncompetitive inhibitor. In the search for alternative inhibitors to lithium, substrate-based inhibitors, bisphosphonates, terpenoid and tropolone analogues are described.

2-O-tert-Butyldimethylsilyl-4,6-bispyrenoyl-myo-inositol-1,3,5-orthoformate (1) and 2-O-tert-butyldimethylsilyl-4-[(4-di-methylamino)benzoyl]-6-pyrenoyl-myo-inositol-1,3,5-orthoacetate (2) adopt unstable chair conformations with five substituents axial, in which the aromatic esters participate in π-stacking, and give excimer and exciplex fluorescence, respectively. Upon addition of acid, the orthoformate/orthoacetate lock is cleaved, which allows the inositol ring to switch to the more stable pen-ta-equatorial chair conformation, with loss of exciplex/excimer fluorescence.

Publisher Summary This chapter discusses prodrug design for phosphates and phosphonates. The phosphate group has an important role in virtually all of the major metabolic pathways in nature. There is little therapeutic scope for underivatized phosphate and phosphonate analogues because they are doubly ionized at physiological pH and are unable to cross cell membranes by passive diffusion. Only a few compounds can utilize active transporter pathways. These include the antiviral phosphonoformate (1), which shows some affinity for inorganic phosphate transporters [1, 2], the herbicide bialaphos (2) , which is transported by a peptide carrier, and the antibiotic fosfomycin (3) , which utilizes the 3-phosphoglycerate transport pathway. Phosphate monoesters are also prone to rapid hydrolysis by phosphatases, another property, which makes them unsuitable drugs. This instability coupled with their high aqueous solubility makes phosphate monoesters ideal prodrug modifications of drugs, which have low aqueous solubility. A wide range of prodrug modifications have been utilized in the design of lipophilic triesters of phosphates or diesters of phosphonates. Cleavage to the parent drug can occur by chemical hydrolysis or be catalyzed by a range of enzymes, including carboxyesterases and phosphodiesterases.

Acyclovir is an effective drug for the treatment of HSV and VZV infections, which after phosphorylation to the triphosphate, inhibits viral DNA polymerase. Acyclovir has low oral bioavailability, therefore prodrugs have been developed, and the L-valyl ester, valaciclovir, recently has been licensed for the treatment of shingles. Ganciclovir is used against CMV, and famciclovir, a lipophilic prodrug of penciclovir, is marketed for shingles. The acyclic nucleoside phosphonates are active against thymidine kinase-resistant viral strains. Promising analogs are PMEA (in clinical trial for the treatment of AIDS) and (S)- HPMPC (good in vivo activity against HSV, VZV, CMV, and EBV). Oligonucleotides incorporating acyclic nucleosides at the 3’t and 5’t-ends, or constituted of amino acyclic nucleosides, are resistant to cleavage by nucleases and may be useful in antisense and/or antigene therapy. HEPT is active against HIV-1: It binds in a hydrophobic pocket on reverse transcriptase, rather than in the polymerase active site. Some acyclic nucleosides are potent inhibitors of purine and pyrimidine nucleoside phosphorylase. These compounds may have a therapeutic niche in combination therapy with antiviral and anticancer nucleosides, and in the treatment of diseases involving the T-cell.

3-Arsonopyruvate was prepared in four steps from glycine. The arsenic-carbon bond was formed by a Meyer reaction between alkaline arsenite and 2-bromo-3-hydroxy-2-(hydroxymethyl)propionic acid; the 3-arsono-2-hydroxy-2-(hydroxymethyl) propionic acid formed was oxidized with periodate to give 3-arsonopyruvate. This proves to be an alternative substrate for phosphoenolpyruvate mutase, giving pyruvate, which was assayed using lactate dehydrogenase. The K(m) is 20 microM, similar to that observed for the natural substrate phosphonopyruvate (17 microM), whereas the kcat. of 0.01 s-1 was much lower than that for phosphonopyruvate (58 s-1). Arsonopyruvate competitively inhibited the action of the mutase on phosphonopyruvate.

Using biomimetic chemical reduction or Clostridium perfringens cell extract containing azoreductase, the dimer-fluorescent probe 2,4-O-bisdansyl-6,7-diazabicyclooct-6-ene, which possesses a conformationally constrained cis-azo bridge, is reduced to the tetra-equatorial 2,4-O-bisdansyl-cyclohexyl-3,5-bisammonium salt which exhibits fluorescence indicative of a dansyl monomer.

The intramolecular heterodimer probe 4-O-dabsyl-2-O-dansyl-myo-inositol-1,3,5-orthoformate undergoes biomimetic reduction of the azo bond in the dabsyl group to give a fluorescence emission band at 560 nm. This is attributed to the loss of energy transfer between the fluorophore (dansyl) and quencher (dabsyl) groups as a result of the formation of 4-O-(4-aminobenzene sulfonyl)-2-O-dansyl-myo-inositol-1,3,5-orthoformate.

myo-Inositol phosphates possessing the 1,2,3-trisphosphate motif share the remarkable ability to completely inhibit iron-catalysed hydroxyl radical formation. The simplest derivative, myo-inositol 1,2,3-trisphosphate [Ins(1,2,3)P(3)], has been proposed as an intracellular iron chelator involved in iron transport. The binding conformation of Ins(1,2,3)P(3) is considered to be important to complex Fe(3+) in a 'safe' manner. Here, a pyrene-based fluorescent probe, 4,6-bispyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-bispyrenoyl Ins(1,2,3,5)P(4)], has been synthesised and used to monitor the conformation of the 1,2,3-trisphosphate motif using excimer fluorescence emission. Ring-flip of the cyclohexane chair to the penta-axial conformation occurs upon association with Fe(3+), evident from excimer fluorescence induced by pi-pi stacking of the pyrene reporter groups, accompanied by excimer formation by excitation at 351 nm. This effect is unique amongst biologically relevant metal cations, except for Ca(2+) cations exceeding a 1 : 1 molar ratio. In addition, the thermodynamic constants for the interaction of the fluorescent probe with Fe(3+) have been determined. The complexes formed between Fe(3+) and 4,6-bispyrenoyl Ins(1,2,3,5)P(4) display similar stability to those formed with Ins(1,2,3)P(3), indicating that the fluorescent probe acts as a good model for the 1,2,3-trisphosphate motif. This is further supported by the antioxidant properties of 4,6-bispyrenoyl Ins(1,2,3,5)P(4), which closely resemble those obtained for Ins(1,2,3)P(3). The data presented confirms that Fe(3+) binds tightly to the unstable penta-axial conformation of myo-inositol phosphates possessing the 1,2,3-trisphosphate motif.

The enzyme carboxyphosphonoenolpyruvate mutase catalyses the formation of one of the two C-P bonds in bialaphos, a potent herbicide isolated from Streptomyces hygroscopicus. The gene encoding the enzyme has been cloned from a subgenomic library from S. hygroscopicus by colony hybridisation using an exact nucleotide probe. An open reading frame has been identified that encodes a protein of molecular mass 32700 Da, in good agreement with the subunit molecular mass of the carboxyphosphonoenolpyruvate mutase recently isolated from this source [Hidaka, T., Imai, S., Hara, O., Anzai, H., Murakami, T., Nagaoka, K.Seto, H. (1990) J. Bacteriol. 172, 3066-3072]. The gene shares significant sequence similarity with that of phosphoenolpyruvate mutase, an enzyme that catalyses the related interconversion of phosphoenolpyruvate and phosphonopyruvate. When the carboxyphosphonoenolpyruvate-mutase gene was subcloned into the vector pET11a, the mutase was expressed as about 20% of the total soluble cellular protein in Escherichia coli. The mutase has been purified to homogeneity in three steps in 40% yield. With malate dehydrogenase/NADH, (hydroxyphosphinyl)pyruvate gives (hydroxyphosphinyl)lactate (kcat 164 s-1 and Km 680 microM) and this spectrophotometric assay for the product of the mutase reaction has been employed in the mechanistic studies. The kinetics for the mutase reaction have been evaluated for the substrate, carboxyphosphonoenolpyruvate, and for the putative reaction intermediate carboxyphosphinopyruvate, both of which have been prepared by chemical synthesis. Carboxyphosphonoenolpyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 0.020 s-1 and a Km of 270 microM, and carboxyphosphinopyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 7.6 x 10(-4) s-1 and a Km of 2.2 microM. Although the exogenously added intermediate is not kinetically competent, these results suggest that the mechanism for the mutase reaction involves an initial rearrangement to the intermediate carboxyphosphinopyruvate, followed by decarboxylation to yield the product (hydroxyphosphinyl)pyruvate.

The degree of alkylation of the side chain nitrogen in tryptamines is one important factor that affects psychoactivity. The method of Speeter and Anthony is considered to be one of the most important synthetic preparative methods. The final step in this reaction is based on the reduction of a (substituted) indole-3-yl-glyoxalylamide to the desired tryptamine with metal hydride. Twelve symmetrically and 13 asymmetrically N,N-disubstituted glyoxalylamides and their corresponding tryptamine derivatives have been synthesised and characterised by gas chromatography EI-ion trap mass spectrometry, electrospray-triple quadrupole-tandem mass spectrometry and NMR spectroscopy. Mass spectral and NMR similarities and differences between the investigated compounds are discussed. A solvent dependency is observed that has to be taken into consideration for the unambiguous assignment of (1)H- and (13)C-NMR chemical shifts. The (1)H-NMR study demonstrated that one can evaluate the rotamer populations of the asymmetrical glyoxalylamides. In a forensic or clinical scenario where single or multiple reaction monitoring approaches are contemplated, the appropriate ion transitions of choice may then focus on the two main fragmentations, namely beta-cleavage ([M+H](+)-->CH(2)N(+)R(2)R(3)) and/or alpha-cleavage ([M+H](+)-->[3-vinylindole](+)), respectively. The synthesis, NMR and MS analytical data presented provide the forensic analyst and clinical biochemist with a detailed and self-consistent body of information and mechanisms for the spectral identification of the more likely psychoactive tryptamines that may be met.

DL-1,2-Dideoxy-1,2-difluoro-myo-inositol was prepared from DL-3,4,5,6-tetra-O-benzyl-myo-inositol in five steps in an overall yield of 36%. The fluoro substituents were introduced with DAST in separate steps by displacement of hydroxy substituents with inversion of stereochemistry. Difluorination could not be achieved in one step because of competing formation of a 1,4-anhydro derivative. DL-1,2-Dideoxy-1,2-difluoro-scyllo-inositol was prepared in 42% overall yield using similar chemistry, with the required inversion of one stereocentre being accomplished by displacement of a tosyl group with caesium propionate. Both difluoroinositol analogues increased the levels of phytic acid (InsP6) in a skeletal muscle cell line. Each compound was tetraphosphorylated with dibenzyl N,N-diisopropylphosphoramidite in the presence of 1H-tetrazole, with subsequent oxidation of the phosphite with MCPBA. The P–OBn groups were removed by H2/Pd–C, and the sodium salts of the tetrakisphosphates of the difluoroinositol analogues were obtained by cation exchange.

The bis[2-(trimethylsilyl)ethyl]N,N-dialkylphosphoramidites 7a and b and bis[2-(methyldiphenylsilyl)ethyl]N,N-dialkylphosphoramidites 6a and b have been prepared by reaction of the N,N-dialkylphosphorochloridites 5a and b with the appropriate 2-(trialkylsilyl)ethanol. In the presence of 1H-tetrazole, the phosphoramidites 6a, b and 7a, b phosphorylated MeOH, PhCH2OH, PhCH2CH2OH, Me(PhCH2CH2)CHOH, 2,3,4,5,6-penta-o-benzyl-myo-inositol and ButOH, to give the phosphites 8a–h. Without isolation, these were oxidised to the corresponding phosphate triesters 9a–h with m-chloroperoxybenzoic acid. Treatment of the triesters 9a–h with tetrabutylammonium fluoride removes only one 2-(trialkylsilyl)ethyl group to give the diesters 10a–h, whereas treatment with a solution of hydrofluoric acid in acetonitrile–water gives the phosphate monoesters 11a–e.

The crystal structure of sodium tetra(cyclohexylammonium)myo-inositol 1,2,3-trisphosphate is found in the chair form with the three phosphate groups adopting the more stable equatorial-axial-equatorial conformation.

The bis(aroyloxymethyl) esters of benzylphosphonate 8(XPh, ArPh, 2-MeC6H4 or 2,4,6-Me3C6H2) and methoxycarbonylmethylphosphonate 8(XMeO2C, ArPh, 2-MeC6H4 or 2,4,6-Me3C6H2) have been prepared by reaction of 2 equiv. of the appropriate aroyloxymethyl iodide with the disilver salt of either benzylphosphonate or methoxycarbonylmethylphosphonate. The cyclohexylammonium salts of the mono(aroyloxymethyl) esters of benzylphosphonate 12(XPh, ArPh, 2-MeC6H4 or 2,4,6-Me3C6H2, M+C6H11NH3+) were prepared by reaction of silver benzyl benzylphosphonate with the appropriate aroyloxymethyl iodide, with subsequent hydrogenolysis to remove the P-O-benzyl group. The bis(aroyloxymethyl) esters 8(XPh or MeO2C, ArPh, 2-MeC6H4 or 2,4,6-Me3C6H2) and the mono(aroyloxymethyl) salts 12(XPh, ArPh, 2MeC6H4 or 2,4,6-Me3C6H2, M+C6H11NH3+) were stable towards chemical hydrolysis at 37 °C at physiological pH. In the presence of porcine liver carboxyesterase, the bis(aroyloxymethyl) esters of benzylphosphonate 8(XPh, ArPh or 2-MeC6H4) degraded to the mono(aroyloxymethyl) esters 12(XPh, ArPh or 2-MeC6H4,), which showed slow hydrolysis to benzylphosphonate. For the bis(aroyloxymethyl) esters of methoxycarbonylmethylphosphonate 8(XMeO2C, ArPh or 2MeC6H4) there was competition between the esterase-catalysed hydrolyses of the aroyloxymethyl and methoxycarbonyl groups. For the triester 8(XMeO2C, Ar2,4,6-Me3C6H2) cleavage of the methoxycarbonyl group was observed and hydrolysis of the sterically hindered 2,4,6-trimethylbenzoyl group (Ar2,4,6-Me3C6H2) was not detected for any compound.

The di(4-acetoxybenzyl) ester of methylphosphonate 4(X = H, R = Me) and the di(4-acyloxybenzyl) esters of methoxycarbonylmethylphosphonate 4(X = MeO2C, R = Me, Et, Pr, Pri, Bu or But) were prepared from the appropriate benzyl alcohol and phosphonic dichloride. At pD 8.0 and 37 °C, both series of compounds hydrolyse with half-lives greater than 24 h to the corresponding mono(4-acyloxybenzyl) esters 5(X = H or MeO2C, R = Me, Et, Pr, Pri Bu or But) which were prepared by treatment of the di(4-acyloxybenzyl) esters 4 with sodium or lithium iodide. The mono(4-acyloxybenzyl) esters 5(X = H, R = Me) and 5(X = MeO2C, R = Me, Et, Pr, Pri or But) undergo chemical hydrolysis to methylphosphonate 6(X = H), and methoxycarbonylmethylphosphonate 6(X = MeO2C) respectively, together with 4-hydroxybenzyl alcohol and the appropriate acylate anion. The rates of hydrolysis of the mono(4-acyloxybenzyl) esters decrease as the length and steric bulk of the acyl group increases, with half-lives ranging from ∼ 150 h for the acetyl analogues to 2240 h for the pivaloyl derivative. The hydrolyses of the di- and mono-(4-acyloxybenzyl) esters were catalysed by porcine liver carboxyesterase (PLCE), and in all cases the acylate anion was formed. The rate of enzymatic hydrolysis was most rapid for the 4-butanoyloxybenzyl and 4-isobutanoyloxybenzyl analogues. The methoxycarbonyl ester of the phosphonoacetate analogues was not cleaved by PLCE. The methylphosphonate generated from the reaction of 4(X = H, R = Me) in the presence of esterase and H218O, did not contain 18O attached directly to phosphorus. These results suggest that both the chemical and enzymatic hydrolyses of the mono(4-acyloxybenzyl) esters and the PLCE-catalysed hydrolyses of the di(4-acyloxybenzyl) esters proceed via hydrolysis of the acyl group to give the acylate anion and the unstable 4-hydroxybenzyl esters. The electron-donating 4-hydroxy group facilitates the cleavage of the benzyl-oxygen bond with the formation of the 4-hydroxybenzyl carbonium ion 9, which readily reacts either with water or the phosphate buffer. The 4-acyloxybenzyl phosphoesters provide the first example of a protecting group which will enable the bioactivation of phosphonate prodrugs at rates appropriate to biological systems.

The para-substituted analogues of dibenzyl methoxycarbonylphosphonate (1; X = N3, NO2, CF3) have been prepared in ca. 60% yield and their product and kinetic profiles of hydrolysis have been evaluated. Relative to the unsubstituted analogue 1(X = H) which has a half-life of 64 min, the para-substituted triesters of phosphonoformate undergo rapid hydrolysis at pH 7.4 and 36.4°C with half-lives of 2.5 min (X = N3), 5.6 min (X = NO2) and 15 min (X = CF3). In contrast to 1(X = H), which on hydrolysis gives ca. 30% C–P bond cleavage with dibenzyl phosphite formation, the para-substituted analogues hydrolyse predominantly to the para-substituted benzyl methoxycarbonyl-phosphonate diesters (2). Triester 1(X = NO2) hydrolyses to the diester 2(X = NO2) with P–O bond cleavage, consistent with nucleophilic attack of water at phosphorus and identical to that observed for the unsubstitued analogue. In contrast, triester 1(X = N3) hydrolyses to the diester 2(X = N3) with C–O bond cleavage presumably via an intermediate with benzyl carbonium ion character.

Dibenzyl (methoxycarbonyl)phosphonate (1) has been prepared by the reaction of benzyl alcohol with (methoxycarbonyl)phosphonic dichloride. The hydrolysis of 1 proceeded rapidly, with a half-life of 60 min at 36.4 °C and pH 7.4, by two main pathways. The dominant pathway (k1, 6.56 × 10–3 min–1) yielded the diester, benzyl (methoxycarbonyl)phosphonate (2) and benzyl alcohol (3), with P–O cleavage. The second (k2, 3.55 × 10–3 min–1) gave dibenzyl phosphite (4), possibly arising from hydrolysis of the carboxyl ester followed by decarboxylation. Benzyl phosphite (5) was also observed, which arises from the hydrolysis of 4 with P–O cleavage (k5, 9.04 × 10–4 min–1). Other products formed in small amounts were, benzyl (benzyloxycarbonyl)phosphonate (6)(k3, 3.59 × 10–4 min–1) and dibenzyl phosphate (7)(k4, 4.24 × 10–4 min–1). The rapid and complicated hydrolysis of 1, involving four competitive reactions, two of which involve C–P bond cleavage, suggests that triesters of phosphonoformate are unlikely to be suitable prodrug forms.