The Synthesis of Diphenyl Phosphonate Analogues of α-Amino Acids as Enzyme Inhibitors

Abstract

α-Aminoalkylphosphonic acids are analogues of natural aminoacids and as such have been the subject of much research effort over past years¹. The diphenyl esters of α- aminoalkylphosphonic acids are particularly potent and show high selectivity as irreversible inhibitors of serine proteinases. Thus far, α-aminoalkylphosphonic acid ester analogues of a number of aliphatic- and aromatic aminoacids have been prepared including valine, phenylalanine, tryptophan, and tyrosine², and the basic aminoacids ornithine, lysine. etc.³. We have now also prepared the a-diphenyl phosphonate analogues of the acidic aminoacids, aspartic and glutamic⁴. These have been examined as potential inactivators of serine proteinases exhibiting a P₁ specificity for aspartate and glutamate, e.g. S. aureus V8 protease and granzyme B.     

A Hapten for the Generation of Antibody Phosphatases

Abstract

A series of hydroxymethylene and α,β-unsaturated aminophosphinic acids was prepared, modelled on the ‘exploded’ transition state proposed (Herschlag [1]) for the chemical hydrolysis of phosphate monoesters. They exhibit the dual utility of being transition state analogues for the chemical hydrolysis of phosphate monoesters and having the potential for inhibition of phosphatase enzymes. (1) was chosen to be utilised as a hapten molecule for the generation of catalytic antibodies.     

Application of phosphonate and thiophosphate analogues of nucleotides to studies of some enzyme reactions

The enzymatic cleavage of a scissile P O bond can be blocked by recourse to phosphonate analogues of biological phosphate esters. α-Fluorophosphonates have an enhanced electronegativity at the bridging carbon, which, in many cases, makes them superior to simple methylene phosphonates for the study of enzyme reactions. Thus, the β,γ-difluoro-methylene analogue of ATP is a good substrate for the interferon-induced (2→5)A_n synthetase, which converts it into a (2→5)A₄ species having a 5′-β,γ-difluoromethylenetriphosphate. This binds strongly to RNase L but does not activate it. The unsymmetrical Ap₄Aases from Artemia and Lupin are strongly inhibited by P^1,P⁴-dithiophosphate analogues of diadenosyl-5′,5‴-P¹,P⁴-tetraphosphate although nonregiospecific cleavage of certain P²,P³-methylene analogues can be observed. Some of these analogues are remarkably effective inhibitors of platelet aggregation and are effective inhibitors in vivo of arterial blood-clotting in rabbits. Separation of all diastereoisomers of P¹,P⁴-dithiophosphate analogues of Ap₄A is achieved cleanly using reverse-phase hplc chromatography and this provides direct access to β,γ-CHF-bridged analogues of ATP with resolved stereochemistry at the CHF center. Lastly, growing cells of Dictyostelium discoideum not only tolerate a range of substituted methylene bisphosphonates in their growth medium but actually incorporate them into nucleotide analogues of ATP and Ap₄A.     

A Versatile Method for the Preparation of Enantiomeric Benzene-1,2-bis(alanine) Derivatives

Abstract

A versatile method for the preparation of dicarba analogues of cystine as substituted benzene-, dihydrobenzene-, and tetrahydrobenzene-1,2-bis(alanine) derivatives is described. The partially saturated products resulted from Diels-Alder adduct formation with stereoselectively prepared (2R,7R)-2,7-diacetamido-4,5-bis (methylene)octane-1,8-dioic acid dimethyl ester. Aromatization of the dihydro adducts by manganese dioxide provided the parent benzene-1,2-bis(alanine) derivatives.     

Synthesis of Carbohydrate Formacetal Dimers Under Non-Acidic Conditions 1

Abstract

The linking mode between sugar units in biopolymers such as polysaccharides or oligonucleotides has recently gained increased attention. For example, neutral analogues of phosphodiester linkages have been designed to produce modified DNA fragments² or nucleic acid mimics with promising properties in the control of gene expression.³ A recent focus has been directed towards neutral and achiral linkers like ethylene glycol, propoxy⁴ or longer alkyl chains⁵ but also the simple methylene group.⁶ In the latter case, the methylene linker was part of a formacetal moiety.

     

Anisochrony of the O-methylene protons of the C-2-ethyl ester function in triethyl meso,cis-1,3-dimethylcyclohexane-1,2,3-tricarboxylate—Atropisomerism of an ester group

The methylene protons of the ethyl ester function at C-2 in triethyl meso, cis-1,3-dimethylcyclohexane-1,2,3-tricarboxylate (A) are anisochronous, despite the apparent symmetry of structure A. The necessary diastereotopy is thought to be engendered by the central ester being stabilized in a rotamerization in which the O:C·O plane is held parallel to the general plane of the 6-membered ring while fast exchanges take place among rotamers about the alkyl–oxygen bond; other rotamers about the ester axis are thought to encounter high order repulsive steric/polar interactions with the flanking ester functions and C-methyl groups. Evidence for the axial orientation of the C-1 and C-2 ester functions in A, based on comparisons of its ¹H NMR spectral characteristics with those of its RS-cis, trans and meso, trans analogues, is presented. The role of ion-pairing in the stereoselectivity of methylation of precursor enolates, leading to the formation of these three systems, is briefly discussed. A comment on the appropriateness, or otherwise, of drawing conformational conclusions from the magnitudes of the anisochrony in comparable systems is included. Triester A is thought to be the first instance where atropisomerism about an sp³? sp² bond involving an ordinary ester function has been detected employing a prochiral sensor group.     

The Methoxycarbonylation of Vinyl Acetate Catalyzed by Palladium Complexes of [1,2‐Phenylenebis(methylene)]bis[di(tert‐butyl)phosphine]

Palladium complexes of [1,2‐phenylenebis(methylene)]bis[di(tert‐butyl)phosphine] ( 1 ) catalyze the methoxycarbonylation of vinyl acetate (= ethenyl acetate) in the presence of methanesulfonic acid (Scheme 1). High selectivities to ester products can be obtained if free phosphine ligand is in excess over the amount of added acid (Table 1). Selectivities to methyl 2‐acetoxypropanoate, a precursor to lactate esters, can be as high as 3.6 : 1 at low temperature and pressure (Table 2). Replacing ^tBu by ⁱPr groups leads to less‐active catalysts and lower selectivities to the branched product. Replacing the phenylene moiety by a naphthalenediyl moiety also gives lower activity, but with similar selectivity to the phenylene‐based analogues. Linear hydrocarbon‐chain linkers as the backbone instead of the phenylenebis(methylene) linker leads to poor catalysis, except for a propane‐1,3‐diyl linker, which gives good rates but poor branched selectivity (Table 5). The effect of different reaction conditions on the catalysis is discussed. The syntheses of the new xylene‐based diphosphines 2 – 5 with one to four ⁱPr groups replacing the ^tBu groups at the P‐atoms of 1 and of the ligands 6 and 7 based on 1,2‐ and 2,3‐dimethylnaphthalene are also described (Schemes 2 and 3).     

Synthesis of methylene- and difluoromethylenephosphonate analogues of uridine-4-phosphate and 3-deazauridine-4-phosphate

Cytidine triphosphate synthetase (CTPS) catalyzes the formation of cytidine triphosphate from glutamine, uridine-5'-triphosphate (UTP), and adenosine-5'-triphosphate. Inhibitors of CTPS are of interest because of their potential as therapeutic agents. One approach to potent enzyme inhibitors is to use analogues of high energy intermediates formed during the reaction. The CTPS reaction proceeds via the high energy intermediate UTP-4-phosphate (UTP-4-P). Four novel analogues of uridine-4-phosphate (U-4-P) and 3-deazauridine-4-phosphate (3-deazaU-4-P) were synthesized in which the labile phosphate ester oxygen was replaced with a methylene and difluoromethylene group. The methylene analogue of U-4-P, compound 1, was prepared by a reaction of the sodium salt of tert-butyl diethylphosphonoacetate with protected, 4-O-activated uridine followed by acetate deprotection and decarboxylation. It was found that this compound undergoes relatively facile dephosphonylation presumably via a metaphosphate intermediate. The difluoromethylene derivative, compound 2, was prepared by electrophilic fluorination of protected 1. This compound was stable and did not undergo dephosphonylation. Synthesis of the methylene analogue of 3-deazaU-4-P, compound 3, was achieved by ribosylation of protected 4-(phosphonomethyl)-2-hydroxypyridine. Electrophilic fluorination was also employed in the preparation of protected 4-(phosphonodifluoromethyl)-2-hydroxypyridine which was used as the key building block in the synthesis of difluoro derivative 4. These compounds represent the first examples of a nucleoside in which the base has been chemically modified with a methylene or difluormethylenephosphonate group.     

A neutralization-reionization and reactivity mass spectrometry study of the generation of neutral hydroxymethylene

Neutral hydroxymethylene HCOH is an important intermediate in several chemical reactions; however, it is difficult to observe due to its high reactivity. In this work, neutral hydroxymethylene and formaldehyde were generated by charge exchange neutralization of their respective ionic counterparts and then were reionized and detected as positive‐ion recovery signals in neutralization–reionization mass spectrometry in a magnetic sector instrument of BEE geometry. The reionized species were characterized by their subsequent collision‐induced dissociation mass spectra. The transient hydroxymethylene neutral was observed to isomerize to formaldehyde with an experimental time span exceeding 13.9 µs. The vertical neutralization energy of the HCOH^+? ion has also been assayed using charge transfer reactions between the fast ions and stationary target gases of differing ionization energy. The measured values match the result of ab initio calculations at the QCISD/6‐311 + G(d,p) and CCSD(T)/6‐311 + + G(3df,2p) levels of theory. Neutral hydroxymethylene was also produced by proton transfer from CH₂OH⁺ to a strong base such as pyridine, confirmed by appropriate isotopic labeling. There is a kinetic isotope effect (KIE) for H⁺ versus D⁺ transfer from the C atom of the hydroxymethyl cation of ～3, consistent with a primary KIE of a nearly thermoneutral reaction. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis of C‐Glycosyl Phosphate and Phosphonate,Analogues of N‐Acetyl‐α‐d‐Glucosamine 1‐Phosphate

Synthesis of α‐C‐ethylene phosphate and phosphonate as well as α‐C‐methylene phosphate analogues of N‐acetyl‐α‐d‐glucosamine 1‐phosphate is reported starting from the common perbenzylated 2‐acetamido‐2‐deoxy‐α‐C‐allyl glucoside. Anomerisation of the corresponding amino α‐C‐glucosyl aldehyde to the β‐aldehyde was observed. Thus, both amino α‐ and β‐C‐glucosyl methanol were obtained after reduction.     

Methotrexate analogs. 7. Synthesis of two higher homologs and a positional isomer of methotrexate diethyl ester as potential antitumor agents

Analogs of methotrexate diethyl ester ( 1 ) were prepared, in which the distances separating the ester functions from each other and from the carboxamide function of the p-aminobenzoate moiety were varied via the use of methylene groups as “spacers”. The diethyl esters 3 and 4 , with D,L-α-aminoadipate and D,L-α-aminopimelate side chains in place of L-glutamate, displayed approximately the same order of activity as compound 1 against bacterial and mammalian cells in culture, and were inhibitors of the enzyme dihydrofolate reductase. When given intraperitoneally to L1210 1eukemic mice at a dose of 120 mg./kg. q3d 1,4,7, compound 4 produced a 67% increase in survival and no evidence of toxicity, whereas methotrexate diethyl ester ( 1 ) gave a 44% increase in survival at a dose of 45 mg./kg. q3d 1,4,7 but was toxic at higher doses. The positional isomer 2 was inactive.     

NOVEL UNSYMMETRICAL N,N-BIS(METHYLENE)BISPHOSPHONIC ACIDS OF α,ω-DIAMINES. PREPARATION AND CHARACTERIZATION OF [[(2-AMINOETHYL)IMINO]BIS(METHYLENE)]BISPHOSPHONIC ACID AND [[(6-AMINOHEXYL)IMINO]BIS(METHYLENE)]BISPHOSPHONIC ACID

Abstract

Reaction of ethylenediamine with phosphorous acid and formaldehyde in molar ratio 1:2:2 gives [[(2-aminoethyl)imino]bis(methylene)]bisphosphonic acid (2a) as the major product. Similarly, reaction of hexamethylenediamine with phosphorous acid and formaldehyde in molar ratio 1:2:2 yields [[(6-aminohexyl)imino]bis(methylene)]bisphosphonic acid (2b) which is isolated either as [[(6-carbobenzoxyaminohexyl)imino]bis](methylene)]bisphosphonic acid (3b) or as [[6-(N-benzoylamino)hexyl)imino]bis(methylene)]bisphosphonic acid (4b). Removal of the carbobenzoxy group with HBr from 3b or the benzoyl group with HCl from 4b gives pure [[(6-aminohexyl)imino]bis(methylene)]bisphosphonic acid (2b). All compounds were characterized by ¹³C NMR, ³¹P NMR and elemental analysis.     

Chemistry of Inositol Lipid Mediated Cellular Signaling

It is now slightly more than a decade since Michael Berridge and his collaborators reported in Nature “…micromolar concentrations of Ins(1,4,5)P₃ (1D -myo-inositol 1,4,5-trisphosphate) release Ca²⁺ from a non-mitochondrial intracellular Ca²⁺ store in pancreatic acinar cells. Our results strongly suggest that this is the same Ca²⁺ store that is released by acetylcholine”. This observation ushered in an a new era in the field of signal transduction with the discovery of a small-molecule second messenger liniking the spatially separated events of cell surface receptor activation and intracellular Ca²⁺ mobilization. This event, which has spawned what is now one of the most active fields of current biology, also stimulated a renaissance in inositol and inositol phosphate chemistry. The synthesis of inositol polyphosphates presents a number of problems: the regiospecific protection of inositol and the optical resolution of the resulting precursors, the phosphorylation of the polyol, removal of all phosphate protecting groups without phosphate migration, and finally the purification of the water-soluble target polyanion. With the solution of these problems over the last few years it is now possible to look beyond the synthesis of naturally occurring inositol polyphosphates, whose number has been steadily increasing, to the design of chemically modified inositol phosphate analogues with the prospect of developing enzyme inhibitors, rationally modified receptor ligands and antagonists, and perhaps, through pharmacological intervention in signal transduction pathways, even the therapeutical agents of the future.     

Study of the Phosphoryl‐Transfer Mechanism of Shikimate Kinase by NMR Spectroscopy

The phosphoryl‐transfer mechanism of shikimate kinase from Mycobacterium tuberculosis and Helicobacter pylori, which is an attractive target for antibiotic drug discovery, has been studied by 1D ¹H and ³¹P NMR spectroscopy. Metaphosphoric acid proved to be a good mimetic of the metaphosphate intermediate and facilitated the ready and rapid evaluation by NMR spectroscopic analysis of a dissociative mechanism. The required closed form of the active site for catalysis was achieved by the use of ADP (product) or two synthetic ADP analogues (AMPNP, AMPCP). Molecular dynamics simulation studies reported here also revealed that the essential arginine (Arg116/Arg117 in H. pylori and M. tuberculosis, respectively), which activates the γ‐phosphate group of ATP for catalysis and triggers the release of the product for turnover, would also be involved in the stabilisation of the metaphosphate intermediate during catalysis. We believe that the studies reported here will be helpful for future structure‐based design of inhibitors of this attractive target. The approach is also expected be useful for studies on the possible dissociative mechanism of other kinase enzymes.     

Synthesis of novel donor mimetics of UDP-Gal, UDP-GlcNAc, and UDP-GalNAc as potential transferase inhibitors

For the enzymatic transfer of galactose, N-acetylglucosamine, and N-acetylgalactosamine, UDP-Gal (1), UDP-GlcNAc (2), and UDP-GalNAc (3) are employed, and UDP serves as a feedback inhibitor. In this paper the synthesis of the novel UDP-sugar analogues 4, 5, and 6 as potential transferase inhibitors is described. Compounds 4-6 feature C-glycosidic hydroxymethylene linkages between the sugar and nucleoside moieties in contrast to the anomeric oxygens in the natural derivatives 1-3.     

Synthesis,photophysical and NMR evaluations of thiourea-based anion receptors possessing an acetamide moiety

The synthesis and photophysical evaluation of three diaryl thiourea-based anion receptors (4–6) for comparison with their urea counterparts (1–3) is outlined. These anion receptors posses an acetamide functionality on one of the aryl groups and an electron-withdrawing CF₃ group on the other. By varying the position of the acetamide group, in the o-, m- and p-positions of 4–6, respectively, the anion binding ability was both tuneable and found to be, in some cases, significantly different from that seen for the urea analogues 1–3. The binding affinities of the receptors 4–6, as well as the binding stoichiometries, were evaluated using UV–vis absorption spectroscopy in MeCN. However, these receptors were not sufficiently emissive to quantify the anion recognition using fluorescence. The results confirmed strong binding of these receptors to anions such as fluoride, acetate, phosphate, pyrophosphate and chloride. Nevertheless, the overall results obtained did not conform to the anticipated trends seen for 1–3, which is most likely due to the enhanced binding affinity of the thiourea analogues 4–6. The binding interactions were also investigated by using ¹H NMR which confirmed that these receptors interacted with the anions in a stepwise manner, where the primary anion binding interaction occurred at the thiourea side, which led to an activation of the acetamide moiety towards the second anion binding interaction, an example of an allosteric activation mode.     

Enzyme mediated-transesterification of verbascoside and evaluation of antifungal activity of synthesised compounds

Enzymatic acylation of verbascoside, a polyhydroxylated natural product, has been reported in this study using five different commercial lipases and taking p-nitrophenyl alkanoates as acyl donors. Out of these enzymes, the immobilised Candida antarctica lipase B was found as the enzyme of choice. Mono- and di-acylated products were formed, with mono as major product indicating high regioselective nature of such transformations. A series of acyl esters of verbascoside have been synthesised by this enzymatic transesterification methodology. The lipophilicity of the synthesised analogues was also checked. The analogues were further subjected to synergistic antifungal activity with amphotericin B (AmB) against Candida albicans. Fourfold reduction in minimum inhibitory concentration of AmB was observed with few synthesised analogues such as verbascoside 4″-octanoate (3b), verbascoside 4″-palmitate (3d) and verbascoside 4″,4′-dipalmitate (4d) at a concentration of 0.5 μg/mL.     

Antifungal activity of cinnamic acid and benzoic acid esters against Candida albicans strains

Candida albicans is an important opportunistic fungal pathogen capable of provoking infection in humans. In the present study, we evaluated the antifungal effect of 23 ester derivatives of the cinnamic and benzoic acids against 3 C. albicans strains (ATCC-76645, LM-106 and LM-23), as well as discuss their Structure–Activity Relationship (SAR). The antifungal assay results revealed that the screened compounds exhibited different levels of activity depending on structural variation. Among the ester analogues, methyl caffeate (5) and methyl 2-nitrocinnamate (10) were the analogues that presented the best antifungal effect against all C. albicans strains, presenting the same MIC values (MIC = 128 μg/mL), followed by methyl biphenyl-2-carboxylate (21) (MIC = 128, 128 and 256 μg/mL for C. albicans LM-106, LM-23, and ATCC-76645, respectively). Our results suggest that certain molecular characteristics are important for the antifungal action.     

Synthetic Modification of 1,3-Thiazolidin-4-One 1,1-Dioxides

Abstract

The reactivity of EWG (electron withdrawing group)-activated methylene group in 1,3-thiazolidin-4-one 1,1-dioxides were investigated. Novel derivatives of 1,3-thiazolidin-4-one 1,1-dioxides were prepared (the heterocyclic core was modified with carboxamide and carboxthioamide moieties as carbonyl group analogues). The products, which have functional groups for possible future modifications, are described and characterized.     

Biginelli-like reaction with dialkyl acetone-1,3-dicarboxylates: a remarkable case of steric control

A Biginelli-type condensation is described using dialkyl acetone-1,3-dicarboxylates as active methylene compounds for the preparation of monastrol analogues. Unexpectedly, the reaction with salicylaldehyde formed two different products depending on the ester alkyl group. This product dichotomy was found to be caused by the steric effects exerted by the alcohol terminus of the ester group in the active methylene component. Previous controversial results as to the structure of the Biginelli product 3 are also discussed.