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A simple, rapid one-pot synthesis of heterocyclic (risedronate, zoledronate) and aminoalkyl (pamidronate, alendronate, neridronate) bisphosphonate drugs using microwave irradiation is presented. Good yields of product are achieved within 20 min, in contrast to conventional synthesis, which typically requires a day or longer.

Kinetics studies of dNTP analogues having pyrophosphate-mimicking β,γ-pCXYp leaving groups with variable X and Y substitution reveal striking differences in the chemical transition-state energy for DNA polymerase β that depend on all aspects of base-pairing configurations, including whether the incoming dNTP is a purine or pyrimidine and if base-pairings are right (T•A and G•C) or wrong (T•G and G•T). Brønsted plots of the catalytic rate constant (log(kpol)) versus pKa4 for the leaving group exhibit linear free energy relationships (LFERs) with negative slopes ranging from −0.6 to −2.0, consistent with chemical rate-determining transition-states in which the active-site adjusts to charge-stabilization demand during chemistry depending on base-pair configuration. The Brønsted slopes as well as the intercepts differ dramatically and provide the first direct evidence that dNTP base recognition by the enzyme–primer–template complex triggers a conformational change in the catalytic region of the active-site that significantly modifies the rate-determining chemical step.

We report synthesis of the first fluorescently labeled conjugates of risedronate (1), using an epoxide linker strategy enabling conjugation of 1 via its pyridyl nitrogen with the label (carboxyfluorescein). Unlike prior approaches to create fluorescent bisphosphonate probes, the new linking chemistry did not abolish the ability to inhibit protein prenylation in vitro, while significantly retaining hydroxyapatite affinity. The utility of a fluorescent 1 conjugate in visualizing osteoclast resorption in vitro was demonstrated.

Some current and potential therapeutic uses of bisphosphonates and phosphonocarboxylates are summarized. The feasibility of synthesizing α-hydroxy α-alkyl/aryl methyl-enebisphos-phonates via Grignard addition to tetraalkyl carbonylbisphosphonates 6 is demonstrated, and a new synthesis of 6 under very mild conditions is described.

An improved method for enantioseparation of racemic 1-(α-aminobenzyl)-2-naphthols has been developed by the reaction in situ of Betti base product mixtures with L-(+)-tartaric acid taken in a 1:1 ratio. The products of this reaction are (-)- 1-(α-aminobenzyl)-2-naphthol tartrate, the acetal of benzaldehyde and tartaric acid as well as (+)-l,3-diaryl-2,3-dihydro-1H-naphth[1,2-e][1,3]oxazine, which can be easily separated by crystallization.

Invasive fungal infections cause significant morbidity and mortality among immunocompromised individuals, posing an urgent need for new antifungal therapeutic strategies. Here we investigate a chromatin-interacting module, the bromodomain (BD) from the BET family of proteins, as a potential antifungal target in Candida albicans, a major human fungal pathogen. We show that the BET protein Bdf1 is essential in C. albicans and that mutations inactivating its two BDs result in a loss of viability in vitro and decreased virulence in mice. We report small-molecule compounds that inhibit C. albicans Bdf1 with high selectivity over human BDs. Crystal structures of the Bdf1 BDs reveal binding modes for these inhibitors that are sterically incompatible with the human BET-binding pockets. Furthermore, we report a dibenzothiazepinone compound that phenocopies the effects of a Bdf1 BD-inactivating mutation on C. albicans viability. These findings establish BET inhibition as a promising antifungal therapeutic strategy and identify Bdf1 as an antifungal drug target that can be selectively inhibited without antagonizing human BET function.

In contrast to both its parent “troika” acid ( E - 1 , a phosphorylating agent at pH 7 and 25 °C) and its C-methyl isomer ( E - 2 , which is stable at both acidic and neutral pH), ( E )-(hydroxyimino)(hydroxymethoxyphosphinyl)acetic acid E - 3 was unreactive at pH 7 and 25 °C but at pH 1.5 fragmented to methyl phosphate 10 (15%) and methyl phosphorocyanidate 11 (85%). The minor product is consistent with solvent phosphorylalion, the reaction exclusively observed with E - 1 . The non-phosphorylating fragmentation pathway is proposed to involve a preliminary E → Z isomerizalion of 3 prior to C α -C β cleavage. Dual fragmentation pathways were also detected ( 31 P NMR) when the DCHA + salt of E - 3 ( E - 9 ) was heated in acetonitrile or EtOH; in addition to phosphorylation products (16–19%), 11 was formed (81–84%). Reaction of E - 9 in refluxing EtOH: t -BuOH (1:1) showed low stereoselectivity in product formation (~3:1 ethyl methyl phosphate: t -butyl methyl phosphate), supporting a dissociative phosphorylation process.

Recently, we synthesized the first individual β,γ-CHX-dGTP diastereomers [(R)- or (S)-CHX, where X is F or Cl] and determined their structures in ternary complexes with DNA polymerase β (pol β). We now report stereospecificity by pol β on the mixed β,γ-CHX diastereomer pairs using nuclear magnetic resonance and on the separate diastereomers using transient kinetics. For both the F and Cl diastereomers, the R isomer is favored over the S isomer for G·C correct incorporation, with stereospecificities [(k(pol)/K(d))(R)/(k(pol)/K(d))(S)] of 3.8 and 6.3, respectively, and also for G·T misincorporation, with stereospecificities of 11 and 7.8, respectively. Stereopreference for the (R)-CHF-dGTP diastereomer was abolished for k(pol) but not K(d) with mutant pol β (R183A). These compounds constitute a new class of stereochemical probes for active site interactions involving halogen atoms. As Arg183 is unique in family X pols, the design of CXY deoxyribonucleotide analogues to enhance interaction is a possible strategy for inhibiting BER selectively in cancer cells.

DNA polymerase (pol) β is a member of the X-family of DNA polymerases and plays an important role in base excision repair that cleanses the genome of simple base lesions.[1] It has been the subject of extensive studies examining its key roles in repair[2] and cancer.[3] In an effort to identify features of the pol β active site that modulate nucleotide binding, we have made modifications in the triphosphate moiety and studied the effect on enzyme binding, specificity, and chemistry.[4] Replacement of the Pα-O-Pβ bridging oxygen with a carbon atom prevents catalysis,[4b] whereas modification of the Pβ-O-Pγ bridging oxygen alters leaving group properties.[4a] The introduction of these substituents may also enable entirely new bonding (or repulsive) active site interactions, not present with the natural nucleoside triphosphate, and thus could inform mechanistic insight as well as inhibitor design seeking to exploit pol β as a drug target.[4a] Previously we studied several α,β-methylene-dNTP analogues as probes for the capture of pre-insertion substrate complexes with pol β and found that α,β-CF2-analogues have an apparent equilibrium dissociation constant (Kd) that is two orders of magnitude greater than the corresponding α,β-CH2-analogues and the β,γ-dGTP-CF2-analogue.[4a, 4b] To further explore the molecular basis of pol β nucleotide binding, the dimethylated (α,β-C(Me)2-, (5)) and the monofluorinated (R/S)-(α,β-CHF-, (6a/b)) dATP analogues have been synthesized and their Kd values determined. In addition, the X-ray crystallographic structures of ternary substrate complexes of pol β and DNA primer template formed from α,β-CF2-dATP[4b] (7) and 6a/b have been solved. Interestingly, only one diastereomer of 6 is found in the active site (Figure 1). Figure 1 X-ray crystal structure of the DNA pol β active site containing (S)-α,β-CHF-dATP. The solvent-excluded pol β active site surface is colored by atom (carbon, gray; nitrogen, light blue; oxygen, pink; magnesium, dark blue ... 5 and 6a/b were prepared by reaction of 2′-deoxyadenosine 5′-tosylate with the tris(tetrabutylammonium) salt of the corresponding bisphosphonic acid followed by enzymatic phosphorylation of the resulting dA 5′-bisphosphonate[4b] (Scheme 1). In a previously reported synthesis of 6a/b in which (R/S)-α,β-CHF-dADP (4a/b) was phosphorylated with p-nitrobenzyl phosphormorpholidate,[5] an approximately 1:1 mixture of α,β-CHF-dATP diastereomers was observed in the 19F NMR spectrum. Similarly, phosphorylation of 4a/b using nucleoside diphosphate kinase and a catalytic amount of ATP generates both diastereomers as observed in the 19F and 31P (Pα) NMR spectra. Although precise quantification is not possible due to signal overlap, by 19F NMR analysis the isomers are in roughly similar abundance, indicating that the enzymatic phosphorylation did not significantly enrich a particular isomer. Scheme 1 Synthesis of α,β-CXY-dATP analogues. As expected, 5 and 6a/b are not pol β substrates (SI, Figure S25) and inhibit gap-filling DNA synthesis by pol β in a concentration dependent manner (SI, Figures S26 and S28). The apparent equilibrium binding constant for 6a/b is approximately 10 μM, similar to that of the natural substrate and significantly lower than that of the α,β-CF2-dATP analogue (Figure 2).[4b] The α,β-CH2-dATP analogue binds with about a 10-fold higher affinity[4b, 6] than 6a/b consistent with a correlation between the basicity of the phosphonate moiety and the strength of inhibitor binding.[4b] However, α,β-C(Me)2-dATP (5) which has two electron-donating substituents on the α,β-methylene, has a Kd of approximately 1100 μM and does not conform to this trend. Figure 2 DNA pol β dissociation constants of dATP and α,β-methylene analogues. The data for dATP, α,β-CH2-dATP, and α,β-CF2-dATP were previously published.[4b, 9] The Ki (i.e., Kd) values were determined ... The crystallographic structures of ternary complexes of pol β with incoming α,β-CF2-dATP (7) (PDB ID 3TFR) or α,β-CHF-dATP (6) (PDB ID 3TFS) opposite a templating thymidine, resolved at 2.00 A (SI Table S1) were next determined. All corresponding atoms in the active site of these structures superimpose well with previously determined ternary complex structures of pol β where the reaction was trapped by deletion of the nucleophilic 3′-OH on the primer terminus or by using a nitrogen in place of the α,β-bridging oxygen (Figure 3A).[8] Thus, α,β-CHF-dATP and 7 are well accommodated in the polymerase active site as has been shown previously for α,β-CF2-dTTP.[4b] Figure 3 A) The ternary complex crystallographic structures of pol β with an incoming α,β-CF2-dATP (7) (gray carbons) and α,β-NH-dUTP (light blue carbons; PDB ID 2FMS)[8] were superimposed using all 326 Cα (rmsd ... Molecular docking of 5 and 6 with pol β-DNA binary complex using Autodock Vina[7] suggests that the unexpectedly low binding affinity of 5 can be attributed to an unfavorable steric interaction, specifically a clash of the methyl groups of 5 with a structural water bound to Asp276 (PDB ID 3TFS water 3). When docked in the absence of this structural water, the lowest energy conformations for both 5 and 6 overlap well with the coordinates of the 6 found in the crystal structure, whose conformation is congruent with those of DNA pol β-bound α,β-CF2-dTTP[4b] and α,β-NH-dUTP.[8] However, when docking using a macromolecule file prepared to include the water bound to Asp276, 5 is unable to achieve the experimentally observed conformation while the geometry of 6 is unperturbed (SI, Figure S27). Incubation of crystals of a binary DNA complex of pol β with the α,β-CHF-dATP diastereomer mixture (6a/b) resulted in a complex in which only the (S)-CHF-stereoisomer (6b) could be found by X-ray analysis (Figures 1 and ​and3B).3B). Modeling (R)-α,β-CFH-dATP (6a) into the electron density failed to account for the observed density (Figure 3C). The close overlap of the CF2- and the (S)-CHF- conformations indicates that exclusion of 6a analogue is not the result of an unattainable binding conformation or destabilizing steric interaction (Figure 3B). As we reasoned previously,[4d, 4e] stabilization of as little as ~1 kcal/mol could be sufficient to generate the selectivity observed in the crystal structures. In an earlier study, we reported a series of stereospecifically formed pol β-DNA-dGTP ternary complexes that provided evidence for a stabilizing polar interaction between the active site Arg183 and the chiral phosphonate C-F in CHF, CMeF, and CClF dGTP-β,γ analogues.[4d, 4e] Unlike the β,γ-CXF-dGTP-DNA-pol β ternary complexes, in this structure no direct stabilizing interactions with active site residues can be identified. The structure does, however, reveal that the (S)-isomer fluorine is within 2.8 A (B-factor: 15.4 A2) of an oxygen in a structural water molecule, present in both structures (for CF2 distance = 2.9 A, B-factor: 15.1 A2), that is within hydrogen bonding distance of several key atoms (a carboxylate anion oxygen and carboxyamide nitrogen of Asp276, phosphoryl oxygen of Pβ; Figure 1). Observing no similar interactions with the pro-(R)-fluorine in the CF2-structure, one might consider a weak interaction of the (S)-fluorine in 6b with structural water as the source of its stabilization in the complex relative to 6a, however other factors might be invoked[10] and further work will be necessary to clarify the origin of the observed stereospecificity. In summary, our results show the influence of a structural water bound to Asp276 on the nucleotide binding properties of DNA pol β. Kd determinations for a series of α,β-bridging substituted dATP analogues display a correlation with the basicity of the bisphosphonate moiety and the binding affinity to pol β. This trend, however, does not extend to the α,β-C(Me)2-dATP analogue which displays a larger than expected Kd attributed to an unfavorable steric interaction of the methyl substituents with the structural water bound to Asp276. The X-ray crystal structure of α,β-CHF-dATP in complex with pol β and DNA contains only one diastereomer despite the presence of both diastereomers in the crystallization mixture. Comparison of this structure with that of α,β-CF2-dATP raises the possibility of a non-covalent weak interaction between the ligand and the active site structural water proximal to Asp276. Our structural observations may be worth taking into account in PDB studies[11] searching for fluorine/protein interactions.

The configuration at phosphorus in cyclic (S)-HPMPC (1, cidofovir) and (S)-HPMPA (2) phenyl ester (5 and 6, respectively) diastereomers ((R(p))-5, (R(p))-6, (S(p))-6) was determined by X-ray crystallography and correlated to their (1)H and (31)P NMR spectra in solution. (R(p))-5 and (R(p))-6 have chair conformations with the nucleobase substituent equatorial and the P-OPh axial. Perhaps surprisingly, (S(p))-6 is (a, a) in the crystal and exists largely as an equilibrium of (a, a)/(e, e) conformers in chloroform or acetonitrile.

A series of novel β,γ-methylene-, monofluoromethylene-, and difluoromethylene-bisphosphonophosphate alkyl monoesters was synthesized. The compounds were conveniently detected during preparative HPLC using post-column derivatization with a phosphate-specific chemosensor.

The first examples of α-azido bisphosphonates [(RO)(2)P(O)](2)CXN(3) (1, R = i-Pr, X = Me; 2, R = i-Pr, X = H; 3, R = H, X = Me; 4, R = H, X = H) and corresponding β,γ-CXN(3) dGTP (5-6) and α,β-CXN(3) dATP (7-8) analogues are described. The individual diastereomers of 7 (7a/b) were obtained by HPLC separation of the dADP synthetic precursor (14a/b).

Progress in the synthesis of novel fluorescent conjugates of N-heterocyclic bisphosphonate drugs and related analogues, together with some recent applications of these compounds as imaging probes, are briefly discussed.

Phosphonocarboxylate (PC) analogues of bisphosphonates are of interest due to their selective inhibition of a key enzyme in the mevalonate pathway, Rab geranylgeranyl transferase (RGGT). The dextrarotatory enantiomer of 2-hydroxy-3-(imidazo[1,2-a]pyridin-3-yl)-2-phosphonopropanoic acid (3-IPEHPC, 1) is the most potent PC-type RGGT inhibitor thus far identified. The absolute configuration of (+)-1 in the active site complex has remained unknown due to difficulties in obtaining RGGT inhibitor complex crystals suitable for X-ray diffraction analysis. However, we have now succeeded in crystallizing (−)-1 and here report its absolute configuration (AC) obtained by X-ray crystallography, thus also defining the AC of (+)-1. An Autodock Vina 1.1 computer modeling study of (+)-1 in the active site of modified RGGT binding GGPP (3DSV) identifies stereochemistry-dependent interactions that could account for the potency of (+)-1 and supports the hypothesis that this type of inhibitor binds at the TAG tunnel, inhibiting the second geranylgeranylation step. We also report a convenient 31P NMR method to determine enantiomeric excess of 1 and its pyridyl analogue 2, using α- and β-cyclodextrins as chiral solvating agents, and describe the synthesis of a small series of 1 α-X (X = H, F, Cl, Br; 7a–d) analogues to assess the contribution of the α-OH group to activity at enzyme and cellular levels. The IC50 of 1 was 5–10× lower than 7a–d, and the LED for inhibition of Rab11 prenylation in vitro was 2–8× lower than for 7a–d. However, in a viability reduction assay with J774 cells, 1 and 7b had similar IC50 values, ∼10× lower than those of 7a and 7c–d.

Cidofovir (HPMPC), a broad spectrum antiviral agent, cannot be administered orally due to ionization of its phosphonic acid group at physiological pH. One prodrug approach involves conversion to the cyclic form (cHPMPC, 1) and esterification by the side chain hydroxyl group of a peptidomimetic serine. Transport studies in a rat model have shown enhanced levels of total cidofovir species in the plasma after oral dosing with L-Val-L-Ser-OMe cHPMPC, 2a. To explore the possibility that 2a and its three L/D stereoisomers 2b-d undergo active transport mediated by the peptide-specific intestinal transporter PEPT1, we performed radiotracer uptake and electrophysiology experiments applying the two-electrode voltage clamp technique in Xenopus laevis oocytes overexpressing human PEPT1 (hPEPT1, SLC15A1). 2a-d did not induce inward currents, indicating that they are not transported, but the stereoisomers with an L-configuration at the N-terminal valine (2a and 2b) potently inhibited transport of the hPEPT1 substrate glycylsarcosine (Gly-Sar). A "reversed" dipeptide conjugate, L-Ser-L-Ala-OiPr cHPMPC (4), also did not exhibit detectable transport, but completely abolished the Gly-Sar signal, suggesting that affinity of the transporter for these prodrugs is not impaired by a proximate linkage to the drug in the N-terminal amino acid of the dipeptide. Single amino acid conjugates of cHPMPC (3a and 3b) or cHPMPA (5, 6a and 6b) were not transported and only weakly inhibited Gly-Sar transport. The known hPEPT1 prodrug substrate valacyclovir (7) and its L-Val-L-Val dipeptide analogue (8) were used to verify coupled transport by the oocyte model. The results indicate that the previously observed enhanced oral bioavailability of 2a relative to the parent drug is unlikely to be due to active transport by hPEPT1. Syntheses of the novel compounds 2b-d and 3-6 are described, including a convenient solid-phase method to prepare 5, 6a and 6b.

It is difficult to overestimate the importance of nucleoside triphosphates in cellular chemistry: They are the building blocks for DNA and RNA and important sources of energy. Modifications of biologically important organic molecules with fluorine are of great interest to chemists and biologists because the size and electronegativity of the fluorine atom can be used to make defined structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been around for some time, the progress in the area of modified triphosphates was limited by the lack of synthetic methods allowing to access bisCF 2 -substituted nucleotide analogs—one of the most interesting classes of nonhydrolyzable nucleotides. These compounds have “correct” polarity and the smallest possible steric perturbation compared to natural nucleotides. No other known nucleotides have these advantages, making bisCF 2 -substituted analogs unique. Herein, we report a concise route for the preparation of hitherto unknown highly acidic and polybasic bis(difluoromethylene)triphosphoric acid 1 using a phosphorous(III)/phosphorous(V) interconversion approach. The analog 1 compared to triphosphoric acid is enzymatically nonhydrolyzable due to substitution of two bridging oxygen atoms with CF 2 groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the triphosphate polyanion. The fluorinated triphosphoric acid 1 was used for the preparation of the corresponding fluorinated deoxynucleotides (dNTPs). One of these dNTP analogs (dT) was demonstrated to fit into DNA polymerase beta (DNA pol β) binding pocket by obtaining a 2.5 Å resolution crystal structure of a ternary complex with the enzyme. Unexpected dominating effect of triphosphate/Mg 2+ interaction over Watson–Crick hydrogen bonding was found and discussed.

In contrast to both its parent “troika” acid ( E - 1 , a phosphorylating agent at pH 7 and 25 °C) and its C-methyl isomer ( E - 2 , which is stable at both acidic and neutral pH), ( E )-(hydroxyimino)(hydroxymethoxyphosphinyl)acetic acid E - 3 was unreactive at pH 7 and 25 °C but at pH 1.5 fragmented to methyl phosphate 10 (15%) and methyl phosphorocyanidate 11 (85%). The minor product is consistent with solvent phosphorylalion, the reaction exclusively observed with E - 1 . The non-phosphorylating fragmentation pathway is proposed to involve a preliminary E → Z isomerizalion of 3 prior to C α -C β cleavage. Dual fragmentation pathways were also detected ( 31 P NMR) when the DCHA + salt of E - 3 ( E - 9 ) was heated in acetonitrile or EtOH; in addition to phosphorylation products (16–19%), 11 was formed (81–84%). Reaction of E - 9 in refluxing EtOH: t -BuOH (1:1) showed low stereoselectivity in product formation (~3:1 ethyl methyl phosphate: t -butyl methyl phosphate), supporting a dissociative phosphorylation process.

Carbonylphosphonates are defined for the purposes of this Review as phosphonates containing a proximal C=O function. In this review we focus on α-keto and β-ketophosphonates, including two classes of trifunctional carbonyl phosphonates, α-ketophosphonoglyoxylates and carbonylbisphosphonates. These compounds (and analogous derivatives) exhibit distinctive, multiple chemical properties reflecting in part the mutual intereaction of their neighboring carbonyl and phosphonate groups. In some cases, they present special problems in synthesis, and they have useful applications ranging from metal chelation to medicinal chemistry. Here they are considered from the points of view of synthesis, reactivity, value as synthons, and actual or potential bioactivity. Recent (>1995) advances are emphasized.

Abnormal replication of DNA is associated with many important human diseases, most notably viral infections and neoplasms. Existing approaches to chemotherapeutics for diseases associated with dysfunctional DNA replication classically involve nucleoside analogues that inhibit polymerase activity due to modification in the nucleobase and/or ribose moieties. These compounds must undergo multiple phosphorylation steps in vivo, converting them into triphosphosphates, in order to inhibit their targeted DNA polymerase. Nucleotide monophosphonates enable bypassing the initial phosphorylation step at the cost of decreased bioavailability. Relatively little attention has been paid to higher nucleotides (corresponding to the natural di- and triphosphate DNA polymerase substrates) as drug platforms due to their expected poor deliverability. However, a better understanding of DNA polymerase mechanism and fidelity dependence on the triphosphate moiety is beginning to emerge, aided by systematic incorporation into this group of substituted methylenebisphosphonate probes. Meanwhile, other bridging, as well as non-bridging, modifications have revealed intriguing possibilities for new drug design. We briefly survey some of this recent work, and argue that the potential of nucleotide-based drugs, and intriguing preliminary progress in this area, warrant acceptance of the challenges that they present with respect to bioavailability and metabolic stability.

Rab geranylgeranyl transferase (RGGT) catalyzes the posttranslational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki. (+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and RabCXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases. publishersversion published

The paper describes a simple, direct synthesis of enantiopure cyclic α-aminophosphonic acids on the basis of silyldealkylation and followed by hydrolysis of the parent diastereoisomeric cyclic 1,4,2-oxazaphosphorines, which have been obtained by intramolecular stereospecific nucleophilic addition of phosphites to imines. In this approach, the high diastereoselectivity of the stereocontrolling penultimate step is preserved by conversion of the intermediate ester to the final phosphonic acid under very mild, nonracemizing conditions. © 2008 Wiley Periodicals, Inc. Heteroatom Chem 19:575–582, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.20480

β,γ-Fluoromethylene analogues of nucleotides are generally considered to be useful mimics of the natural substrates for DNA polymerases, but direct structural evidence defining their active site interactions has not been available. In addition, the effect of introducing a new chiral center (the CHF carbon) has been unexplored. We report here structural studies of the diastereomeric β,γ-CHF analogues (R, 3; S, 4) of dGTP interacting with the active site of DNA pol β, a repair enzyme that plays an important role in base excision repair (BER) and oncogenesis. The conjugation of dGMP 5‘-morpholidate with a tetrabutylammonium salt of (fluoromethylene)bisphosphonic acid (6b, prepared like its difluoro analogue 7b via fluorination of tetraisopropyl methylenebisphosphonate carbanion with Selectfluor) gives a 1:1 mixture of 3 and 4 (by 19F NMR, pH 10). The β,γ-CF2 (2) and β,γ-CH2 (1) dGTP analogues were also synthesized. Crystallization from a solution containing 3 + 4 together with a preformed DNA pol β complex o...

Phosphoryl-transfer reactions have long been of interest due to their importance in maintaining numerous cellular functions. A phosphoryl-transfer reaction results in two possible stereochemical outcomes: either retention or inversion of configuration at the transferred phosphorus atom. When the product is phosphate, isotopically-labeled [ 16 O, 17 O, 18 O]-phosphate derivatives can be used to distinguish these outcomes; one oxygen must be replaced by sulfur or esterified to achieve isotopic chirality. Conventionally, stereochemical analysis of isotopically chiral phosphate has been based on 31 P NMR spectroscopy and involves complex chemical or enzymatic transformations. An attractive alternative would be direct determination of the enantiomeric excess using chiroptical spectroscopy. ( S )-Methyl-[ 16 O, 17 O, 18 O]-phosphate (MePi ∗ ), 7 and enantiomeric [ 16 O, 17 O, 18 O]-thiophosphate (TPi ∗ ), 10 , were previously reported to exhibit weak electronic circular dichroism (ECD), although with 10 the result was considered to be uncertain. We have now re-examined the possibility that excesses of 7 and 10 enantiomers can be detected by ECD spectrometry, using both experimental and theoretical approaches. 7 and both the ( R ) and ( S ) enantiomers of 10 ( 10a , 10b ) were synthesized by the ‘Oxford route’ and characterized by 1 H, 31 P and 17 O NMR, and by MS analysis. Weak ECD could be found for 7 , with suboptimal S/N. No significant ECD could be detected for the 10 enantiomers. Time-dependent DFT (TDDFT) calculations of the electronic excitation energies and rotational strengths of the same three enantiomers were carried out using the functional B3LYP and the basis set 6-311G ∗∗ . The isotopically-perturbed geometries were predicted using the anharmonic vibrational frequency calculational code in GAUSSIAN 03. In the case of 10 , calculations were also carried out for the hexahydrated complex to investigate the influence of the aqueous solvent. The predicted excitation wavelengths are greater than the observed wavelengths, a not unusual result of TDDFT calculations. The predicted anisotropy ratios are 2.9 × 10 −5 for 7 , −5.3 × 10 −6 for 10a/b , and 1.7 × 10 −6 for 10a/b ⋅(H 2 O) 6 . For 7 the predicted anisotropy ratio approximates that observed in this work, 4.5 × 10 −5 at 208 nm. For 10a/b , the upper limits of the experimental anisotropy ratios ( −6 at 225 nm, pH 9; −6 at 236 nm, pH 12) are comparable to the predicted magnitude of the value for 10a/b . The lower predicted value for 10a/b · (H 2 O) 6 suggests that the aqueous environment affects the ECD significantly. Altogether, the TDDFT calculations together with a stereochemical analysis based on NMR and the MS data support the conclusion that the experimental ECD results for MePi ∗ and TPi ∗ may be reliable in order of magnitude.

Cidofovir (HPMPC) is a broad-spectrum anti-viral agent whose potential, particularly in biodefense scenarios, is limited by its low oral bioavailability. Two prodrugs (3 and 4) created by conjugating ethylene glycol-linked amino acids (L-Val, L-Phe) with the cyclic form of cidofovir (cHPMPC) via a P-O ester bond were synthesized and their pH-dependent stability (3 and 4), potential for in vivo reconversion to drug (3), and oral bioavailability (3) were evaluated. The prodrugs were stable in buffer between pH 3 and 5, but underwent rapid hydrolysis in liver (t(1/2) = 3.7 min), intestinal (t(1/2) = 12.5 min), and Caco-2 cell homogenates (t(1/2) = 20.2 min). In vivo (rat), prodrug 3 was >90% reconverted to cHPMPC. The prodrug was 4x more active than ganciclovir (IC50 value, 0.68 microM vs 3.0 microM) in a HCMV plaque reduction assay. However, its oral bioavailability in a rat model was similar to the parent drug. The contrast between the promising activation properties and unenhanced transport of the prodrug is briefly discussed.

Triethyl fluorophosphonoacetate and triethyl difluorophosphonoacetate are directly synthesized from triethyl phosphonoacetate by treatment with NaH and 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (Selectfluor®). Contrary to a recent report [C.J. Hamilton, S.M. Roberts, J. Chem. Soc., Perkin Trans. 1 (1999) 1051–1056], the reaction proceeded in THF without the need for DMF as a co-solvent. This method is more selective and provides greater convenience and safety than fluorination of the same substrate by treatment with t-BuOK and perchloryl fluoride (FClO3) in toluene while offering advantages over a number of previously described methods employing alternative electrophilic fluorinating reagents or other approaches. Either the monofluoro or the difluoro product can be obtained predominantly by adjusting the molar ratio of base and Selectfluor®. Triethyl 2-fluoro-2-phosphonopropionate (ethyl 2-(diethoxyphosphinyl)-2-fluoropropanoate) is also more conveniently made from triethyl 2-phosphonopropanoate using NaH/Selectfluor® in THF than with FClO3/t-BuOK in toluene. Detailed procedures are given for obtaining the corresponding triacids in quantitative yield from the fluorinated triesters by P,P-silyldealkylation with bromotrimethylsilane followed by one-pot double hydrolysis with H2O, and isolation as stable dicyclohexylammonium (DCHA) or pyridinium (Py) salts. Substitution of EtOH for H2O in the latter procedure provides the CO-ester phosphonic diacids, isolated as DCHA salts, in one step. 1H, 13C, 31P and 19F NMR data are given for the compounds prepared.

Prodrugs of phosphonoformic acid (PFA), an anti-viral agent used clinically as the trisodium salt (foscarnet), are of interest due to the low bioavailability of the parent drug, which severely limits its utility. Neutral PFA triesters are known to be susceptible to P-C bond cleavage under hydrolytic de-esterification conditions, and it was previously found that P,C-dimethyl PFA P-N conjugates with amino acid ethyl esters did not release PFA at pH 7, and could not be fully deprotected under either acid or basic conditions, which led, respectively, to premature cleavage of the P-N linkage (with incomplete deprotection of the PFA ester moiety), or to P-C cleavage. Here we report that novel, fully deprotected PFA-amino acid P-N conjugates 4 can be prepared via coupling of C-methyl PFA dianion 2 with C-ethyl-protected amino acids using aqueous EDC, which gives a stable monoanionic intermediate 3 that resists P-C cleavage during subsequent alkaline deprotection of the two carboxylate ester groups. At 37 degrees C, the resulting new PFA-amino acid (Val, Leu, Phe) conjugates (4a-c) undergo P-N cleavage near neutral pH, cleanly releasing PFA. A kinetic investigation of 4a hydrolysis at pH values 6.7, 7.2, and 8.5 showed that PFA release was first-order in [4a] with respective t(1/2) values of 1.4, 3.8, and 10.6 h.

A novel nucleotide analogue is described, in which the alpha,beta-phosphoric anhydride oxygen of a nucleoside 5(')-diphosphate is replaced by a carbonyl group: the carbonylbisphosphonate analogue 5 of 2('),3(')-dideoxy-3(')-azidothymidine 5(')-diphosphate (AZT 5(')-diphosphate). 5 was synthesized from tetramethyl (diazomethylene)bisphosphonate 1 via the trimethyl ester 4 of the corresponding AZT 5(')-(diazomethylene)bisphosphonate 6, which is also a new type of nucleotide analogue. The ultimate product 5 was isolated by reverse-phase HPLC, and characterized by 31P, 13C, and 1H NMR; and by high-resolution mass spectrometry. The ketone group of 5 is a visible chromophore (yellow) and reversibly forms a colorless hydrate. The ketone hydrate 'pK' is about 4.2 when excess of magnesium ion is present. The potential of such analogues as novel inhibitors of enzymes mediating nucleotide-dependent biochemical processes is discussed.

Carbonylphosphonates are defined for the purposes of this Review as phosphonates containing a proximal C=O function. In this review we focus on α-keto and β-ketophosphonates, including two classes of trifunctional carbonyl phosphonates, α-ketophosphonoglyoxylates and carbonylbisphosphonates. These compounds (and analogous derivatives) exhibit distinctive, multiple chemical properties reflecting in part the mutual intereaction of their neighboring carbonyl and phosphonate groups. In some cases, they present special problems in synthesis, and they have useful applications ranging from metal chelation to medicinal chemistry. Here they are considered from the points of view of synthesis, reactivity, value as synthons, and actual or potential bioactivity. Recent (>1995) advances are emphasized.

Synthetic approaches to a new class of tyrosine-linked prodrugs of two 3-hydroxy-2-(phosphonomethoxypropyl) (HPMP) nucleotide analogues ((S)-HPMPC and (S)-HPMPA) are outlined.

A new and efficient route for the synthesis of cyclic aminophosphonic acids by the reaction of dialkylchlorophosphites with β-aldiminoalcohols has been described.

The possibility of using of easily available 1-(α-aminobenzyl)-2-naphthols as chiral auxiliaries in the synthesis of non-racemic α-aminophosphonates has been shown.

The catechol moiety in a chromophore was used in an indicator displacement assay for the chemosensing of the antiviral drug foscarnet (trisodium phosphonoformate, abbreviated as PFA). Applications of two methods were investigated, namely UV-Vis absorption and fluorescence spectroscopy measuring coordination of a metal to a catechol-based indicator. Yb3+ complexation with chromogenic pyrocatechol violet in 10 mM HEPES buffer at pH 7.0 yields a blue chemosensor that responds to the presence of PFA with the release of yellow pyrocatechol violet (PV). The YbPV coordination complex responds linearly to the PFA concentration with a 2 μM detection limit. Metal ion complexation of a range of metal ions (trivalent Al, Ga, In, Sc, La, Gd, Er, Yb, and Fe, and divalent Cu) to the fluorescent sensor 6,7-dihydroxy-4-methylcoumarin (also referred to as 4-methylesculetin and abbreviated ME) resulted in fluorescence quenching in 10 mM HEPES buffer at pH 7.0. Addition of foscarnet to the quenched coordination complex liberated the ligand fluorophore which could be observed by its fluorescence. The coordinating complex was optimized for determining foscarnet by varying the metal ion, resulting in increased sensitivity to the analyte and selectivity against phosphate. Cu2+ was selected as the most effective ion and its performance in this assay was further investigated. The effect of the co-ligand in the ternary coordination complex, Cu2+–6,7-dihydroxy-4-methylcoumarin–co-ligand, was examined, and 2-picolylamine was found to be the optimal co-ligand. This ternary complex improves the detection limit of PFA to 0.5 μM and is stable for at least 72 hours, rendering it a potential sensor for PFA in chromatographic analysis.