Strychnobaillonine, an unsymmetrical bisindole alkaloid with an unprecedented skeleton from Strychnos icaja roots.

A reinvestigation of the roots of Strychnos icaja resulted in the isolation of a new bisindole alkaloid named strychnobaillonine (1) with original C-17-N-1' and C-23-C-17' junctions, in addition to sungucine, bisnordihydrotoxiferine, and strychnohexamine (2). Compound 1 showed potent activity against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum in vitro with an IC50 value of 1.1 µM. The structures of the compounds were defined by detailed spectroscopic analyses, especially 1H and 13C NMR, DEPT, HSQC, COSY, NOESY, HMBC, and HRESIMS. The proposed absolute configuration was based on biosynthetic considerations and spectroscopic data (CD, NMR) supported by molecular modeling.

Macrocycle-embedded ß-lactams as novel inhibitors of the Penicillin Binding Protein PBP2a from MRSA.

Assuming that bicyclic ß-lactams endowed with high conformational adaptability should more easily form acyl-enzyme complexes with PBP2a than the traditional antibiotics, we have prepared a series of bis-2-oxo-azetidinyl macrocycles as potential inhibitors. The compounds are formally "head-head" (HH) cyclodimers of 1-(ω-alkenoyl)-3-(S)-(ω'-alkenoylamino)-2-azetidinones, with various lengths of the alkene chains, obtained by two successive metathesis reactions using the Grubbs catalyst. All compounds behave as acylating inhibitors of PBP2a and one ß-lactam (5c), embedded into the largest ring (32 atoms), features an activity close to that of Ceftobiprole. Conformational analyses, theoretical reactivity models and docking experiments in PBP2a cavity allow to propose a novel pharmacophore, i.e. the 3-(S)-acylamino-1-acyl-2-azetidinone ring, with the syn-conformation of the imide function, associated to a flexible macrocycle favoring the opening of the active site.

Dimeric bisindole alkaloids from the stem bark of Strychnos nux-vomica L.

Strychnos nux-vomica L. (Loganiaceae) is famous for its monomeric alkaloid content, such as strychnine, a convulsant poison. The stem bark of the tree is traditionally used to treat intermittent fever in South East Asia. In various studies, it appeared that dimeric indolo-monoterpenic alkaloids possess a promising activity on Plasmodium falciparum. Three bisindolomonoterpenic alkaloids together with strychnochrysine, previously identified in the root bark of S. nux-vomica, were isolated from the stem bark. The structures of these compounds were established using NMR spectroscopy and mass spectrometry. Stereochemistry of the compounds was confirmed by molecular modelling. This then allowed the structural determination of strychnoflavine, a coloured bisindole alkaloid previously isolated from the root bark of the tree. Moreover, the conformational inversion in alkaloids possessing an ether bond in the strychnane moiety could be easily predicted by specific δ (13)C NMR values. These longicaudatine-type alkaloids were found to display in vitro antiplasmodial activity against a chloroquine resistant strain and a chloroquine sensitive strain. The most interesting was strychnochrysine showing an IC(50) value at around 10 µM.

Governing Parameters of Long-Range Intramolecular S-to-N Acyl Transfers within (S)-Acyl Isopeptides.

The governing parameters for the long-range intramolecular S-to-N acyl transfer in (S)-acyl isopeptides are shown by computational and statistical methods (principal component analysis and cluster analysis) to be driven by enthalpic and geometric effects over the range n = 5-20. The results emphasize the dependency of ΔG(‡) on the geometrical parameters governing the approach of the reactive termini and the importance of stabilizing intramolecular hydrogen bonds in the transition states (TSs), rather than the effects of TS ring-size. The competition between the intra- (uni-) and inter- (bi)molecular acyl transfers were studied for representative examples.

Novel fragments of clavulanate observed in the structure of the class A ß-lactamase from Bacillus licheniformis BS3.

OBJECTIVES: Our aim was to unravel the inactivation pathway of the class A ß-lactamase produced by Bacillus licheniformis BS3 (BS3) by clavulanate. METHODS: The interaction between clavulanate and BS3 was studied by X-ray crystallography, pre-steady-state kinetics and mass spectrometry. RESULTS: The analysis of the X-ray structure of the complex yielded by the reaction between clavulanate and BS3 indicates that the transient inactivated form, namely the cis-trans enamine complex, is hydrolysed to an ethane-imine ester covalently linked to the active site serine and a pentan-3-one-5-ol acid. It is the first time that this mechanism has been observed in an inactivated ß-lactamase. Furthermore, the ionic interactions made by the carboxylic group of pentan-3-one-5-ol may provide an understanding of the decarboxylation process of the trans-enamine observed in the non-productive complex observed for the interaction between clavulanate and SHV-1 and Mycobacterium tuberculosis ß-lactamase (Mtu). CONCLUSIONS: This work provides a comprehensive clavulanate hydrolysis pathway accounting for the observed acyl-enzyme structures of class A ß-lactamase/clavulanate adducts.

Non-symmetrically substituted phenoxazinones from laccase-mediated oxidative cross-coupling of aminophenols: an experimental and theoretical insight.

Oxidative cross-coupling reactions of substituted o-aminophenols were catalyzed by a commercial laccase to produce non-symmetrically substituted phenoxazinones for the first time. Identification by (1)H-, (13)C- and (31)P-NMR, and by HPLC-PDA and HPLC-MS/MS of exclusively two kinds of substituted phenoxazinones out of four potential heterocyclic frameworks was confirmed by a DFT study. The redox-properties of the substrates, their relative rates of conversion and the rigid docking of selected substrates led to a revisited mechanistic pathway for phenoxazinones biosynthesis. Our suggestions concern both the first formal two-electron oxidation by laccase and the first intermolecular 1,4-conjugated addition which secures the observed regioselectivity.

12- to 22-membered bridged ß-lactams as potential penicillin-binding protein inhibitors.

As potential inhibitors of penicillin-binding proteins (PBPs), we focused our research on the synthesis of non-traditional 1,3-bridged ß-lactams embedded into macrocycles. We synthesized 12- to 22-membered bicyclic ß-lactams by the ring-closing metathesis (RCM) of bis-ω-alkenyl-3(S)-aminoazetidinone precursors. The reactivity of 1,3-bridged ß-lactams was estimated by the determination of the energy barrier of a concerted nucleophilic attack and lactam ring-opening process by using ab initio calculations. The results predicted that 16-membered cycles should be more reactive. Biochemical evaluations against R39 DD-peptidase and two resistant PBPs, namely, PBP2a and PBP5, revealed the inhibition effect of compound 4d, which featured a 16-membered bridge and the N-tert-butyloxycarbonyl chain at the C3 position of the ß-lactam ring. Surprisingly, the corresponding bicycle, 12d, with the PhOCH(2)CO side chain at C3 was inactive. Reaction models of the R39 active site gave a new insight into the geometric requirements of the conformation of potential ligands and their steric hindrance; this could help in the design of new compounds.

Combination of capillary electrophoresis, molecular modelling and nuclear magnetic resonance to study the interaction mechanisms between single-isomer anionic cyclodextrin derivatives and basic drug enantiomers in a methanolic background electrolyte.

In order to improve our knowledge of the mechanisms of enantiomer recognition pattern in nonaqueous systems, an approach combining nonaqueous CE (NACE), molecular modelling and NMR was undertaken. Bupivacaine and propranolol were selected as model compounds and their interactions with two single-isomer highly charged ß-CD derivatives, namely heptakis(2,3-di-O-methyl-6-O-sulfo)-ß-CD (HDMS-ß-CD) and heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD (HDAS-ß-CD), were studied. The CD-bupivacaine complexes were evaluated by 2-D Rotating-frame Overhauser Effect SpectroscopY (ROESY) experiments. From these experiments, it can be assumed that inclusion complexes are not formed, whatever the CD derivative used. Molecular modelling was performed at the RHF/MINI-1 or B3LYP/6-31G(d) level. External as well as inclusion type complexes with the alkyl chain of propranolol into both CD cavities were located. Interaction energies calculated for bupivacaine and propranolol correlated with the enantiomer migration order observed in the NACE experiments using both anionic CD derivatives. The interaction of propranolol with HDMS-ß-CD or HDAS-ß-CD gives rise to a family of external and inclusion complexes in which some are more probably obtained.

Spectroscopic studies and molecular modeling for understanding the interactions between cholesterol and cyclodextrins.

PURPOSE: Cholesterol is a major lipid constituent of biological membranes which could be included in cyclodextrin (CD) cavities. Solubilization and cell extraction of cholesterol have been previously performed in order to study its interaction with beta-CD and methylated beta-derivatives notably. The present work aims at confirming the formation of inclusion complexes between these CDs and cholesterol in order to understand their solubilization and cell extraction capacities. METHODS: In this context, liquid-state NMR spectroscopy (1H NMR studies and ROESY experiments) as well as theoretical studies (molecular modeling) have been performed. RESULTS: Rather than preferential conformations, the spectroscopic studies showed us the possible interactions between cholesterol and dimethyl-beta-CD, trimethyl-beta-CD, randomly methylated beta-CD or Crysmeb®. Weak interactions were detected using the latter one, confirming the advantage of the low substitution to decrease membrane loss of integrity and cytotoxicity. Molecular modeling studies should be used to determine which stoichiometry and conformations are energetically more favorable. The semi-empirical AM1 level was used to investigate both 1:1 and 1:2 complexes whereas 1:1 complexes were also studied using minimal or double ζ basis sets. Four conformations for each 1:2 complexes have been envisaged and studied for the methylated CDs. CONCLUSIONS: These studies allowed us to confirm the interactions between cholesterol and beta-CDs especially the methylated derivatives.

Design of new potent and selective secretory phospholipase A(2) inhibitors. 6-Synthesis, structure-activity relationships and molecular modelling of 1-substituted-4-[4,5-dihydro-1,2,4-(4H)-oxadiazol-5-one-3-yl(methyl)]-functionalized aryl piperazin/one/dione derivatives.

The group IIA human non-pancreatic secretory phospholipase A(2) (hnp-sPLA(2)) is one of the enzymes implied in the inflammatory process. In the course of our work on inhibitors of this enzyme we investigated the influence of rigidity of the piperazine region on the biological activity. Several modifications were explored. Various linkers, such as amide, urea, carbamate, or alkoxyphenyl were inserted between the piperazine and the lipophilic chain. Also, modification of the piperazine core to incorporate carbonyl groups was studied. In an in vitro fluorimetric assay using the human GIIA (HPLA(2)) and porcine pancreatic GIB enzymes, compound 60a (Y=phenoxy, R=C(18)H(37), Z=CH(2)) had the optimal activity with an IC(50)=30nM on HPLA(2). By means of molecular modelling we attempted to get informations towards comprehension of differences in activity.

Structural basis of the inhibition of class A beta-lactamases and penicillin-binding proteins by 6-beta-iodopenicillanate.

6-Beta-halogenopenicillanates are powerful, irreversible inhibitors of various beta-lactamases and penicillin-binding proteins. Upon acylation of these enzymes, the inhibitors are thought to undergo a structural rearrangement associated with the departure of the iodide and formation of a dihydrothiazine ring, but, to date, no structural evidence has proven this. 6-Beta-iodopenicillanic acid (BIP) is shown here to be an active antibiotic against various bacterial strains and an effective inhibitor of the class A beta-lactamase of Bacillus subtilis BS3 (BS3) and the D,D-peptidase of Actinomadura R39 (R39). Crystals of BS3 and of R39 were soaked with a solution of BIP and their structures solved at 1.65 and 2.2 A, respectively. The beta-lactam and the thiazolidine rings of BIP are indeed found to be fused into a dihydrothiazine ring that can adopt two stable conformations at these active sites. The rearranged BIP is observed in one conformation in the BS3 active site and in two monomers of the asymmetric unit of R39, and is observed in the other conformation in the other two monomers of the asymmetric unit of R39. The BS3 structure reveals a new mode of carboxylate interaction with a class A beta-lactamase active site that should be of interest in future inhibitor design.

alphavbeta3 Integrin-targeting Arg-Gly-Asp (RGD) peptidomimetics containing oligoethylene glycol (OEG) spacers.

RGD peptides are used in biomaterials science for surface modifications with a view to elicit selective cellular responses. Our objective is to replace peptides by small peptidomimetics acting similarly. We designed novel molecules targeting alpha(v)beta(3) integrin and featuring spacer-arms (for surface grafting), which do not disturb the biological activity, from (l) N-(3-(trifluoromethyl)benzenesulfonyl) tyrosine used as scaffold. Various Arg-mimics were fixed on the phenol function, and the ortho position was used for the coupling of OEG spacers. All peptidomimetics were active in the nM range in a binding test toward human alpha(v)beta(3) integrin (IC(50) = 0.1 to 1.7 nM) and selective versus platelet integrin alpha(IIb)beta(3). Selected compounds revealed excellent ability to inhibit bone cells adhesion on vitronectin. Modeling and docking studies were performed for comparing the most active RGD peptidomimetic to cilengitide, i.e., cyclo-[RGDfN(Me)V]-. Lastly, the adhesion of endothelial cells on a cultivation support grafted with RGD peptidomimetics was significantly improved.

Thiaminylated adenine nucleotides. Chemical synthesis, structural characterization and natural occurrence.

Thiamine and its three phosphorylated derivatives (mono-, di- and triphosphate) occur naturally in most cells. Recently, we reported the presence of a fourth thiamine derivative, adenosine thiamine triphosphate, produced in Escherichia coli in response to carbon starvation. Here, we show that the chemical synthesis of adenosine thiamine triphosphate leads to another new compound, adenosine thiamine diphosphate, as a side product. The structure of both compounds was confirmed by MS analysis and 1H-, 13C- and 31P-NMR, and some of their chemical properties were determined. Our results show an upfield shifting of the C-2 proton of the thiazolium ring in adenosine thiamine derivatives compared with conventional thiamine phosphate derivatives. This modification of the electronic environment of the C-2 proton might be explained by a through-space interaction with the adenosine moiety, suggesting U-shaped folding of adenosine thiamine derivatives. Such a structure in which the C-2 proton is embedded in a closed conformation can be located using molecular modeling as an energy minimum. In E. coli, adenosine thiamine triphosphate may account for 15% of the total thiamine under energy stress. It is less abundant in eukaryotic organisms, but is consistently found in mammalian tissues and some cell lines. Using HPLC, we show for the first time that adenosine thiamine diphosphate may also occur in small amounts in E. coli and in vertebrate liver. The discovery of two natural thiamine adenine compounds further highlights the complexity and diversity of thiamine biochemistry, which is not restricted to the cofactor role of thiamine diphosphate.

Large ring 1,3-bridged 2-azetidinones: experimental and theoretical studies.

The relationship between angular strain and (re)activity of bicyclic 2-azetidinones is still an open question of major concern in the field of penicillin antibiotics. Our study deals with original 13-membered-ring 1,3-bridged 2-azetidinones related to the carbapenem family, and featuring a "planar amide" instead of the "twisted amide" typical of penam derivatives. The bicycles 11 and 12 were obtained from acetoxy-azetidinone 7, via the key-intermediate 10, by using the RCM (ring closing metathesis) strategy. Theoretical predictions and experimental results of hydrolysis showed that the large bicycle 12, endowed with high conformational flexibility, is more reactive than the bicycle 11, including a CC bond of E configuration, and the monocyclic 2-azetidinone precursor 10. The processing of 2-azetidinones 10-12 in the active site of serine enzymes has been computed by ab initio methods, considering three models. Due to geometrical parameters of the enzymic cavity (nucleophilic attack from the alpha-face), precursor 10 was predicted more active than 11 and 12 in the acylation step by Ser-OH. Indeed, bicycles 11 and 12 are modest inhibitors of PBP(2a), while 10 is a good to excellent inhibitor of PBP(2a) and R39 bacterial enzymes.

Fagraldehyde, a secoiridoid isolated from Fagraea fragrans.

A secoiridoid aglycone with an atypical skeleton, named fagraldehyde (1), together with several known secoiridoids (gentiopicroside (2), sweroside (3), and swertiamarin (4)) were isolated from the bark and leaves of Fagraea fragrans collected in Cambodia. The conformations of 1 were evaluated on the basis of molecular modeling and NOESY correlations. A hypothetical biogenesis of fagraldehyde was proposed to explain the unusual skeleton. Compound 1 was weakly active in vitro against Plasmodium falciparum.

Theoretical and experimental vibrational study of miconazole and its dimers with organic acids: application to the IR characterization of its inclusion complexes with cyclodextrins.

The geometry, frequency and intensity of the vibrational bands of miconazole were derived from the density functional theory (DFT) calculations with the hybrid functional B3LYP and the 6-31G(d) basis set. Starting from the fully AM1 optimized geometries of miconazole/betaCD/acids complexes, the miconazole/acid dimers were reoptimized at the B3LYP/6-31G(d) level. Three acids were studied: maleic, fumaric and l-tartaric acids. To begin with the vibrational spectral data obtained from solid phase in mid FT-IR spectrum of miconazole and its dimers are assigned based on the results of the normal modes calculations. All the observed spectra and the calculated ones are found to be in good agreement. In a second step, theoretical results allowed the assignment of FT-IR spectrum for the miconazole/HPgammaCD inclusion complex produced by supercritical carbon dioxide treatment and confirmed the inclusion of miconazole. The experimental spectra for the miconazole/HPgammaCD/acids complexes prepared by supercritical carbon dioxide processing were also assigned using theoretical results. The results confirmed the presence of a genuine inclusion complex and also the interaction between miconazole and the acid.

Theoretical and experimental investigations of organic acids/cyclodextrin complexes and their consequences upon the formation of miconazole/cyclodextrin/acid ternary inclusion complexes.

(1)H NMR spectrometry, FT-IR spectroscopy, as well as molecular modeling at the AM1 level and normal mode analysis were used to characterise the interactions and the formation of inclusion complexes between three organic acids: maleic, fumaric, L-tartaric acids and betaCD. In aqueous medium, the complexation was confirmed by (1)H NMR spectroscopy using two-dimensional technique. The stable geometries of the complexes were determined by molecular modeling. Experimental infrared frequencies were assigned on the base of the vibrational normal mode calculation at the fully optimized geometry for the inclusion complexes. All the results point out the presence of stable inclusion complexes between acids and betaCD at the solid state. These results show the double role of the acid. Correlated with the theoretical and experimental data previously obtained for the miconazole/CD/acids complexes, in function of both acids and CDs structures, the acids can either stabilize the complexes by formation of a multicomponent complex or form acid/CD inclusion complexes, hindering the guest inclusion.

Theoretical and experimental investigations on miconazole/cyclodextrin/acid complexes: molecular modeling studies.

The inclusion of miconazole into cyclodextrin cavity has been demonstrated by different authors. Preliminary studies have shown which fragment of the molecule is involved in the inclusion. In the present study, AM1 approximate molecular orbital calculations have been performed on several cyclodextrins complexes (betaCD, HPbetaCD and HPgammaCD) with miconazole and acidic compounds (maleic, fumaric and L-tartaric acids) as partners. For all the binary complexes, the inclusion of the dichlorobenzene-CH(2)-O-group leads to the most stable complex. For the ternary complexes, depending on their conformation and/or their structures, the acids can either stabilize or destabilize the complex. All the theoretical results were in good agreement with experimental data of miconazole inclusion yields into cyclodextrins. This work clearly demonstrates that the structure of both cyclodextrin and acid plays a key-role in the formation of inclusion complexes.

Charge transfer study through the determination of the ionization energies of tetrapeptides X3-Tyr, X = Gly, Ala, or Leu. Influence of the inclusion of one glycine in alanine and leucine containing peptides.

The energies of the fundamental and several excited states of tetrapeptide radical cations were determined at the outer valence Green's function (OVGF) level, at three geometries corresponding to the lowest energy conformations: two for the neutral and one for the cation. The conformations were optimized at the density functional theory level within the B3LYP framework. It was found that, from a purely energetic point of view, a charge initially created on the tyrosine chromophore could migrate without any geometrical change and without further activation once the excited electronic state of the ionized chromophore was formed. This migration could reach the NH(2) terminus for the neutral conformations but should stop at the adjacent peptide link for the cation conformation. These results stress the probable influence of the electronic coupling between the states rather than the existence of a barrier on the charge pathway to explain the difference between the peptides in the charge-transfer process leading to the loss of an iminium [NH(2)=CHR](+) cation. The dissociation energy of the asymptote related to the formation of this NH(2) terminus iminium cation was calculated for few species and it appears that the excess energy available for dissociation is significant when starting from the lowest energy conformations of the neutral or the cation, provided that the charge transfer is effective. It was also found that the amino acids did not conserve their energetic properties and their zero order energy levels turned to a complete new energetic scheme corresponding to the conformation of the peptide.

Structure-activity relationships in platelet-activating factor. Part 14: synthesis and biological evaluation of piperazine derivatives with dual anti-PAF and anti-HIV-1 activity.

As HIV-associated dementia prevalence has risen with the lifespan of HIV-infected individuals, there is an important need for antiretroviral and anti-inflammatory drugs targeting the central nervous system. Platelet-activating factor, a mediator of inflammation, is an HIV-induced neurotoxin secreted in the infected brain. In this work, we developed piperazine derivatives bearing a heterocyclic moiety as PAF-antagonists and HIV-1 replication inhibitors with micromolar potency.