Search of antitubercular activities in tetrahydroacridines: synthesis and biological evaluation

A series of 9-substituted tetrahydroacridines were synthesized by nucleophilic substitution of chloro group with different nucleophiles in 9-chlorotetrahydroacridine (2). The latter could be obtained by POCl(3) mediated cyclization of the intermediate enamine, which in turn, was prepared by acid catalyzed condensation of anthranilic acid and cyclohexanone. Most of the compounds on antitubercular evaluation against M. tuberculosis H37 Rv and H37 Ra strains exhibited potent activities with MIC 6.125-0.78 microg/mL comparable to the standard drugs.     

Clavulanate inactivation of Staphylococcus aureus beta-lactamase.

The interaction of clavulanic acid with beta-lactamase from Staphylococcus aureus was investigated, particularly with a view to determining whether conformational effects are involved. The inactivation at neutral pH is essentially stoichiometric, leading to an inactive species with an enamine chromophore. Two forms of the enamine were observed, the first-formed having a positive ellipticity with a maximum near 290 nm. This species slowly converted into the stable form of the inactivated enzyme that had a negative ellipticity with a minimum at 275 nm. This change in sign of the ellipticity of the enamine is consistent with the previously proposed cis-trans isomerization of the enamine [Cartwright & Coulson (1979) Nature (London) 278, 360-361). Both the far-u.v.c.d. and the intrinsic viscosity of the inactivated enzyme indicated that negligible change in conformation of the enzyme accompanied inactivation. The rates of inactivation and enamine formation were compared at low temperatures, where the initial rates were slow enough to be monitored. The rate of loss of 95% of the catalytic activity was almost 100-fold faster than the rate of formation of the first-formed enamine species. The remaining 5% activity was lost with a rate comparable with that for formation of the initial enamine. The simplest explanation of these results is that a relatively stable acyl-enzyme intermediate builds up initially and more slowly partitions between turnover (hydrolysis) and enamine formation. The initially formed enamine is in the cis conformation but slowly isomerizes to the more stable trans form.     

The cyanogenic substrate for horseradish peroxidase is a conjugated enamine

We identify the cyanogenic substrate for horseradish peroxidase (HRP) as a conjugated enamine and explore this unusual reaction using alpha-aminocinnamate (RH) as follows. 1) HRP catalyzes the oxidation of RH by O2 (and its peroxidation by H2O2 to form R-R) to produce, simultaneously, CN- and benzaldehyde cyanohydrin. 2) RH is transient and must be generated in situ. The properties of the cyanogenic reaction of HRP are independent of the method of preparation of RH (whether this be condensation of NH3 with phenylpyruvate, enzymatic hydrolysis of glycyldehydrophenylalanine, or oxidation of L-phenylalanine by L-amino acid oxidase). 3) The oxidation of RH is a free radical chain reaction initiated by HRP Compounds I and II (I (or II) + RH----R. + II (or HRP], propagated by RO2. (R. + O2----RO2., RO2. + RH----R. + RO2H), and terminated by recombination reactions such as 2R.----R2 and RO2.----R' + HO2. followed by R. + HO2.----RH + O2. KMnO4 and K3Fe(CN)6 can substitute for HRP. 4) The proximal precursor of CN- and cyanohydrin is postulated to be RO2H (phi-CH(-O2H)-CCO2-(= NH]. These results explain why cyanide is generated from the synergistic action of HRP and L-amino acid oxidase on aromatic L-amino acids and O2 and suggest that the requirement for a beta-aryl substituent on the enamine originates in the reaction of RH with HRP, or of R with O2, rather than the imine/enamine tautomerization of the L-amino acid oxidase product.     

Brewers' yeast pyruvate decarboxylase produces acetoin from acetaldehyde: a novel tool to study the mechanism of steps subsequent to carbon dioxide loss

A gas-liquid chromatographic technique was developed for the determination of both acetaldehyde and the 3-4% acetoin side product that results from the brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) catalyzed reaction of pyruvic acid. Employing this method enabled the demonstration of the catalysis of acetaldehyde condensation to acetoin by the enzyme. It was found that the acetoin produced enzymatically from pyruvic acid or from acetaldehyde was optically active, thus providing stereochemical information about the reaction. Deuterium kinetic isotope effects (employing CH3CHO and CH3CDO) were determined on the steady-state kinetic parameters to be 4.5 (Vmax) and 3.2 (Vmax/Kappm), respectively. This enabled, for the first time, the estimation of relative kinetic barriers for steps past decarboxylation. It could be concluded that (a) C-H bond scission was part of rate limitation in the enzyme-catalyzed condensation of acetaldehyde to acetoin and that (b) among the steps leading to the release of acetaldehyde, protonation of the key enamine intermediate was part of rate limitation. This latter finding is also directly applicable to the mechanism of pyruvate decarboxylation.     

Stereospecific proton transfer by a mobile catalyst in mammalian fructose-1,6-bisphosphate aldolase

Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys(146) --> Met aldolase were solved in complex with dihydroxyacetone phosphate. The structural analysis revealed trapping of the enamine intermediate at Lys(229) in native aldolase. Mutation of conserved active site residue Lys(146) to Met drastically decreased activity and enabled trapping of the putative iminium intermediate in the crystal structure showing active site attachment by C-terminal residues 360-363. Attachment positions the conserved C-terminal Tyr(363) hydroxyl within 2.9A of the C3 carbon in the iminium in an orientation consistent with incipient re face proton transfer. We propose a catalytic mechanism by which the mobile C-terminal Tyr(363) is activated by the iminium phosphate via a structurally conserved water molecule to yield a transient phenate, whose developing negative charge is stabilized by a Lys(146) positive charge, and which abstracts the C3 pro(S) proton forming the enamine. An identical C-terminal binding mode observed in the presence of phosphate in the native structure corroborates Tyr(363) interaction with Lys(146) and is consistent with transient C terminus binding in the enamine. The absence of charge stabilization and of a mobile C-terminal catalyst explains the extraordinary stability of enamine intermediates in transaldolases.     

Penicillanic acid sulfone: interaction with RTEM beta-lactamase from Escherichia coli at different pH values

The interaction of the sulfone of penicillanic acid with the TEM-2 beta-lactamase from Escherichia coli has been investigated as a function of pH between pH 7.0 and 9.6. The first-formed acyl-enzyme suffers one of three fates: deacylation, tautomerization to a bound enamine that transiently inhibited the enzyme, and a process (possibly transimination) that leads to enzyme inactivation. The observed changes in ultraviolet absorbance are consistent with the initially observed product of deacylation being the enamine tautomer (4) of the imine from malonsemialdehyde and penicillamine sulfinate. The same enamine can be generated nonenzymically from the sulfone at high pH. The transiently inhibited enzyme appears to be the same enamine attached to the enzyme by an ester linkage. The rather complex kinetic behavior can be deconvuluted by exploiting the effect of pH on the partitioning of the acyl-enzyme between deacylation and the transiently inhibited form of the enzyme. The pathways followed by penicillanic acid sulfone provide a model for the behavior of a number of other reagents that inactivate the beta-lactamase.     

Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment

The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D --> H exchange from the C2alpha position of HEThDP-d(4), as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 10(7), corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.     

Following the reactions of mechanism-based inhibitors with beta-lactamase by Raman crystallography

The reactions between three clinically relevant inhibitors, tazobactam, sulbactam, and clavulanic acid, and SHV beta-lactamase (EC 3.5.2.6) have been followed in single crystals using a Raman microscope. The data are far superior to those obtained for the enzyme in aqueous solution and allow us to identify species on the reaction pathway and to measure the rates of the accumulation and decay of these species. A key intermediate on the reaction pathway is an acyl enzyme formed between Ser70 and the lactam ring's C=O group. By using the E166A deacylation deficient variant of the enzyme, we were able to focus on the process of acyl enzyme formation. The Raman data show that all three inhibitors form an enamine-type acyl enzyme reaching maximal populations at 10, 22, and 29 min for sulbactam, clavulanic acid, and tazobactam, respectively. The enamine intermediate exhibits a characteristic and relatively intense band near 1595 cm(-1) due to a stretching motion of the O=C-C=C-NH moiety that shifts to lower frequency upon NH <--> ND exchange. This feature was used to follow the kinetics of enamine buildup and decay in the crystal. Quantum mechanical calculations support the assignment of the 1595 cm(-1) band, as well as several other bands, to a trans-enamine species. The Raman data also demonstrate that the lactam ring opens prior to enamine formation since the lactam ring carbonyl (C=O) peak disappears prior to the appearance of the enamine 1595 cm(-1) band. Tazobactam appears to form approximately twice as much enamine intermediate as sulbactam and clavulanic acid, which correlates with its superior performance in the clinic, a finding that may bear on future drug design.     

Synthesis,Antiproliferative and Cytotoxic Activities,DNA Binding Features and Molecular Docking Study of Novel Enamine Derivatives

Novel enamine derivatives were synthesized from the reaction of lactone and chalcones and their antiproliferative and cytotoxic activities against six cancer cell lines (e. g., HeLa, HT29, A549, MCF7, PC3 and Hep3B) and one normal cell lines (e. g., FL) were investigated along with their mode of interactions with CT‐DNA. Most of the enamine derivatives with IC₅₀ values of 86–168 μM demonstrated much stronger antiproliferative activity than the starting molecules against the cancer cells. While, among the enamine derivatives, four compounds displayed higher cytotoxic potency than the control drugs (5‐fluorouracil and cisplatin) against the Hep3B cell lines, these compounds did not exhibit any significant toxicity against normal cells, FL. The UV/VIS spectral data suggest that eight compounds cause hypochromism with a slight bathochromic shift (～6 nm), indicating that they bind to the DNA by way of an intercalative or minor groove binding mode. The binding constants of the compounds are in the range of 0.1×103 M^?1–2.3×104 M^?1. The antiproliferative activity of studied enamine derivatives could possibly be due to their DNA binding as well as their cytotoxic properties. In addition to these assays, the chalcones and enamine derivatives were investigated by molecular docking to calculate the synergistic effect of antiproliferative activities against six human cancer cell lines.     

Slow reversible inhibitions of rabbit muscle aldolase with substrate analogues: synthesis, enzymatic kinetics and UV difference spectroscopy studies

Various dihydroxyacetone-phosphate (DHAP) analogues bearing an aromatic ring or β-dicarbonyl structures were synthesized. Their capacity to form a stabilized iminium ion or conjugated enamine in the reaction catalyzed by rabbit muscle aldolase (EC 4.1.2.13) were investigated by enzymatic kinetics and UV difference spectroscopic techniques. Whereas the aromatic derivative led to competitive inhibition without detectable iminium ion formation, slow reversible inhibitions of aldolase by β-dicarbonyl compounds was shown to have taken place. Conjugated enamine formation at the active site of the enzyme was detected by their specific absorbances close to 317 nm.     

Oxidation of pyruvic acid by flavins in the presence of amines

Pyruvic acid or other enolizable α-keto acids in the presence of primary or cyclic secondary amines in aprotic polar solvents efficiently reduce flavins and isoalloxazines to their 1,5-dihydro derivatives. The other product of this reaction is the diamide of citraconic acid (1). In the absence of flavin, the diamide of methylsuccinic acid (2) is obtained as the major product. The specificity for α-keto acid and amine, stoichiometric requirements, rates and quantities of CO₂ evolution, and the kinetics of the reaction were studied. On the basis of these data a mechanism is proposed that involves an enamine condensation followed by decarboxylation and then either reaction with flavin to give ultimately (1) and dihydroflavin, or reaction with an additional amine to give (2). The possible import of this kind of mechanism in biochemical systems is briefly discussed.     

A novel L-neopentylglycine derivative as auxiliary for copper-catalyzed asymmetric Michael reactions

L-Neopentylglycine diethylamide (4a). was prepared from the new unnatural amino acid L-neopentylglycine (1). The utilization of amide 4a as a chiral auxiliary in the copper(II)-catalyzed asymmetric Michael reaction was investigated in comparison with L-valine diethylamide (4b). Cyclic beta-oxocarboxylates 7 react with 4a and 4b to give the respective enaminoesters 8, which were converted with methyl vinyl ketone (9) in the presence of 10 mol% Cu(OAc)(2). H2O at room temperature in acetone to yield the optically active Michael addition products (R)-10a, b with high selectivity independent of the starting enamine. In the case of the seven-membered beta-oxocarboxylate 7c, however, the valine-derived enamine 8f led to higher enantioselectivity for product 10c. Despite the bulkiness of the neopentyl group, the isopropyl group with an alpha-branch has a better stereoinducing effect.     

Isomerization of delta 1-piperideine-2-carboxylate to delta 2-piperideine-2-carboxylate on complexation with flavoprotein D-amino acid oxidase.

The 1,646 cm-1 band in a resonance Raman spectrum obtained with excitation in the charge-transfer band of the complex of oxidized D-amino acid oxidase (DAO) with the oxidation product of D-lysine catalyzed by DAO shifted to 1,617 cm-1 upon 2-13C substitution of lysine. Thus, the band is assigned to a C(2) = C(3) stretching mode of the enamine, delta 2-piperideine-2-carboxylate (En). In the enzyme-free solution, the product is preferentially in the cyclic imine form, delta 1-piperideine-2-carboxylate (Im). Thus, DAO has a higher affinity for the enamine form than for the imine form. The pH effects on the affinity of DAO for the product and on the molar absorption coefficient at 630 nm in the charge-transfer band, suggest that the enzyme-bound product is En in the neutral form at the N atom. As the value of observed rate constant between DAO and the product was constant at high product concentrations, the binding mechanism can be explained as follows; E + Im in equilibrium with EIm in equilibrium with EEN: rapid bimolecular and slow unimolecular processes. The isomerization of the imine form to the enamine form proceeds in the slow process. The low affinity of Im for DAO may be due to a steric repulsion of the hydrogen atoms of Im at C(3) in the active site. The hydrogen atoms of a substrate D-amino acid at C(3), which correspond to the C(3) hydrogens of Im, may act repulsively in the active site and the repulsive energy may induce strain or distortion of the substrate and the enzyme, accelerating the catalytic reaction.     

Generation and fate of enamines in the microsomal metabolism of cyclic tertiary amines

Microsomal oxidation of 1-benzylpiperidine (1-BP) and its cis-2,6-dimethyl analog was studied to assess the involvement of endocyclic enamines, in equilibrium with the initially formed iminiums, in the metabolic activation of cyclic tertiary amines such as phencyclidine. Since the iminiums can be trapped with cyanide, the selective prevention by cyanide of the metabolic production of 1-benzyl-3-piperidone from 1-BP implicates the iminium in equilibrium with enamine as the source of this metabolite. In cases where iminium-enamine coupling is sterically prevented, the iminium in equilibrium with enamine species can be studied independently and are found to be more potent metabolism-dependent inactivators of cytochrome P-450 than are the corresponding parent amines. Possible mechanisms for biological oxidation of cyclic enamines to reactive intermediates are considered.     

Molecular modeling of penicilloate anions: an RHF-SCF analysis

An ab initio restricted Hartree-Fock self-consistent field (RHF-SCF) analysis of penicilloate anions was performed at the TZV level with GAMESS. Geometry optimization was initialized by the semi-empirical AM1 method followed by optimization at the 6-31++G** level. The total energy obtained was -1116.0997 a.u. for the penicilloate amine, -1115.3164 a.u. for the imine, -1115.2969 a.u. for the enamine and -1115.2017 a.u. for the amine that was deprotonated at the thiazolidine nitrogen. Formation of the free thiolate in the imine and enamine anions by deprotonation of the penicilloate amine is associated with: (1) an increase in total energy (2) an increase in the energy of the highest occupied molecular orbital (HOMO) to that of anti-bonding (3) a decrease in chemical hardness (4) an increase in the chemical potential (5) a more negative Mulliken net charge on the sulfur atom and (6) an increase in the Mulliken atomic population on the former thiazolidine sulfur atom in the HOMO. The RHF-SCF analysis presented here suggests a potential role for the thiolate sulfur of penicilloate anions, especially of the imine, as a chemically reactive soft nucleophile.     

Theoretical elucidation on the functional role of pyrrolidine-type ionic liquids in inducing stereoselectivity of the Michael addition of cyclohexanone with trans-β-nitrostyrene

Density functional theory calculations have been carried out to elucidate the stereoselectivity of the Michael addition of cyclohexanone with trans-β-nitrostyrene, induced by a chiral ionic liquid (CIL) pyrrolidine-imidazolium bromide. By comparison of the C-C bond forming processes in the absence and presence of Br(-) anion, we found that intermolecular H-bonds between the imidazolium cation and the nitro group of trans-β-nitrostyrene and the steric hindrance of the imidazolium cation moiety on the Si-face of enamine dominate the stereoselectivity of the Michael addition. The presence of Br(-) anion obviously reduces the barrier by increasing the polarity of the C4=C5 bond of enamine. The theoretical results rationalize well the early experimental finding, and reveal a valuable clue for the further CIL design with high catalytic efficiency.     

Mechanistic crystallography. Mechanism of inactivation of gamma-aminobutyric acid aminotransferase by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid as elucidated by crystallography

(1R,3S,4S)-3-Amino-4-fluorocyclopentane-1-carboxylic acid (7) was previously shown to be a mechanism-based inactivator of gamma-aminobutyric acid aminotransferase (GABA-AT) [Qiu, J. and Silverman, R. B. (2000) J. Med. Chem. 43, 706-720]. Two mechanisms were considered as reasonable possibilities, a Michael addition mechanism and an enamine mechanism. On the basis of a variety of chemical studies, including tedious radiolabeling experiments, it was concluded that inactivation by 7 proceeds by a Michael addition mechanism. Here, a crystal structure of 7 bound to pig liver GABA-AT is reported, which clearly demonstrates that the adduct formed is derived from an enamine mechanism. This represents another example of how crystallography is an important tool for elucidation of inactivation mechanisms.     

Development of small designer aldolase enzymes: catalytic activity, folding, and substrate specificity

Small (24-35 amino acid residues) peptides that catalyze carbon-carbon bond transformations including aldol, retro-aldol, and Michael reactions in aqueous buffer via an enamine mechanism have been developed. Peptide phage libraries were created by appending six randomized amino acid residues to the C-terminus or to the N-terminus of an 18-mer alpha-helix peptide containing lysine residues. Reaction-based selection with 1,3-diketones was performed to trap the amino groups of reactive lysine residues that were necessary for the catalysis via an enamine mechanism by formation of stable enaminones. The selected 24-mer peptides catalyzed the reactions with improved activities. The improved activities were correlated with improved folded states of the peptides. The catalyst was then improved with respect to substrate specificity by appending a phage display-derived substrate-binding module. The resulting 35-mer peptide functioned with a significant proportion of the catalytic proficiency of larger protein catalysts. These results indicate that small designer enzymes with good rate acceleration and excellent substrate specificity can be created by combination of design and reaction-based selection from libraries.     

Efficient synthesis of carbocyclic nucleoside, (+/-)-homocarbovir via pi-allylpalladium complex formation from the allyl-N,N-ditosylimide substrate

The synthesis of a carbovir analogue, (+/-)-homocarbovir (3) was achieved from norbornadiene (4) in seven steps and 27% overall yield. This route involves a Meinwald-type rearrangement, an acid-hydrolysis of N-tosyl bicyclic enamine 5, and a Pd(0)-catalyzed coupling reaction.