Fluorocitrate inhibition of aconitase. Reversibility of the inactivation

Fluoride ion is released nearly stoichiometrically when (?)-erythro-fluorocitrate is incubated with aconitase. The release of F^? parallels the loss in activity and could arise from direct displacement of F^? by a base on the enzyme or from dehydration to fluoro-cis-aconitate and attack of an enzymic base to release F^?. Aconitase inactivated by ¹⁴C-fluorocitrate does not retain radioactivity when passed through G-50 Sephadex or precipitated by ammonium sulfate. Fullenzymicactivity can be regained after either of these treatments by activation by cysteine and ferrous salts. These data are consistent with the report of fluorocitrate being a competitive (and non-competitive) inhibitor of aconitase (Villafranca, J.J. (1972) Intra-Science Chem. Rept. 6 (4), 1–11) which rapidly inactivates the enzyme. This inactivated enzyme may be a very labile covalent complex, a very tight complex between enzyme and fluoro-cis-aconitate or a tight complex between a defluorinated deravitive of fluorocitrate.In the course of Peters (1957) extensive work on the toxic effects of fluoroacetate, he determined that fluoroacetate was metabolically converted to fluorocitrate. This finding and the fact that citrate levels rise soon after ingestion of fluoroacetate led to the suggestion that fluorocitrate inactivates aconitase (E.C. 4.2.1.2).Recently, conflicting reports concerning the site of inactivation in mitochondria by the inhibiting isomer of fluorocitrate, (?)-erythro-fluorocitrate (1R:2R, 1-fluoro-2-hydroxy-1,2,3-propanetricarboxylate) have appeared (Eanes et al. 1972; Brand et al, 1973). Eanes et al. (1972) contends that the tricarboxylate carrier is the site of inhibition, while Brand et al. (1973) has compelling evidence that aconitase is the site of inhibition. This controversy is a matter of intrepretation of the results and a greater knowledge of the inactivation of aconitase by fluorocitrate may be useful in these interpretations. The results reported herein are concerned with the mechanism of inactivation of purified mitochondrial aconitase by fluorocitrate and demonstrate that this reaction is readily reversible.     

Determination of absolute configuration and binding efficacy of benzimidazole-based FabI inhibitors through the support of electronic circular dichroism and MM-GBSA techniques

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC₅₀ of benzimidazole (?)-1 is ～100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (?)/(+)-1 and (?)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (?)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (?) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (?) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.     

Enantiomeric trans β-aryl-δ-iodo-γ-lactones derived from 2,5-dimethylbenzaldehyde induce apoptosis in canine lymphoma cell lines by downregulation of anti-apoptotic Bcl-2 family members Bcl-xL and Bcl-2

For many years, studies focused on developing new natural or synthetic compounds with antineoplastic activity have attracted the attention of researchers. An interesting group of such compounds seem to be those with both lactone moiety and an aromatic ring which, in addition to antimicrobial or antiviral activity, also exhibit antitumor properties. The study shows antitumor activity of two enantiomeric trans isomers of 5-(1-iodoethyl)-4-(2′,5′-dimethylphenyl)dihydrofuran-2-one. Our aim was to determine their antitumor activity manifested as an ability to induce apoptosis in selected canine cancer cell lines as well as to evaluate differences in their strength depending on the configuration of their stereogenic centers. The enantiomers (+)-(4R,5S,6R)-1 and (?)-(4S,5R,6S)-2 were found to induce classical caspase-dependent apoptosis through downregulation of the expression of anti-apoptotic proteins Bcl-xL and Bcl-2. Although the mechanism of apoptosis induction was the same for both enantiomers, they differed in their strength, as stronger antineoplastic activity in vitro was exhibited by isomer (+)-(4R,5S,6R)-1.     

The synthesis and degradation of benzyl 4,6-O-benzylidene-2,3-dideoxy-3-C-ethyl-2-C-hydroxymethyl-α-d-glucopyranoside and -mannopyranoside

The use of carbohydrates for establishing, by synthesis, the absolute configuration of branched aliphatic alcohols is demonstrated by the synthesis and degradation of carbohydrate derivatives that contain two branch points. Benzyl 4,6-O-benzylidene-2,3-dideoxy-3-C-ethyl-2-C-hydroxymethyl-α-d-glucopyranoside (23) and -mannopyranoside (24) were formed from benzyl 2,3-anhydro-4,6-O-benzylidene-α-d-mannopyranoside (17) by a reaction sequence that involved ring-opening with ethylmagnesium chloride, oxidation, epimerisation, methylenation, and hydroboronation. The gluco isomer 23 was converted into (+)-(R)-2,3-bisacetoxymethylpentyl acetate (1) by sequential hydrogenolysis, borohydride reduction, periodate oxidation, borohydride reduction, and acetylation. The synthesis of 1 provides confirmatory evidence for the absolute configuration of the alkaloid pilocarpine (2). Unidentified products, and not the expected free-sugars, were obtained by acidic hydrolysis of methyl 4,6-O-benzylidene-2,3-dideoxy-3-C-ethyl-2-C-hydroxymethyl-α-d-glucopyranoside (8) and -mannopyranoside (9). Convenient syntheses of benzyl α-d-glucopyranoside derivatives are described.     

(−)-(S,S)-12-hydroxy-13-octadec-cis-9-enolide,a 14-membered lactone from Crepis conyzaefolia seed oil

Crepis conyzaefolia (Gouan) Dalle Torre seed oil contains about 3% of (?)-(S,S)-12-hydroxy-13-octadec-cis-9-enolide (1), a lactone of (?)-threo-12,13-dihydroxyoleic acid. The absolute configuration of the acid has been established as D-12, L-13 (12-S, 13-S) and the lactone has the same absolute configuration.     

Synthesis and Antimicrobial Activity of Chiral cis-Cycloheximide Isomers

Such (+)- and (?)-cis-cycloheximide isomers as isocyclohcximide (1a, 1b), α-epiisocycloheximide (2a, 2b) and neocycloheximide (3a, 3b) were synthesized by aldol condensation of (?)-(2R, 4R)- and (+)-(2S, 4S)-cis-2,4-dimethyl-1-cyclohexanone (5a, 5b). obtained by microbial resolution, with 4-(2-oxoethyl)-2,6-piperidinedione (7). The absolute configuration of the (?)-cis-ketone 5a was confirmed by chemical correlation with natural (2S, 4S, 6S, αR)-cycloheximide (4). The newly synthesized isomer, (?)-α-epiisocycloheximide (2b), showed strong antimicrobial activity against S. cerevisiae andP. oryzae close to that of natural cycloheximide (4).     

Leucoflavonine,a new bioactive racemic flavoalkaloid from the leaves of Leucosceptrum canum

A new flavoalkaloid racemate, leucoflavonine (1), together with its flavonoid precursor pectolinarigenin (2), was isolated from the leaves of Leucosceptrum canum collected from Tibet. Its structure was established by comprehensive spectroscopic analysis. Chrial separation of the enantiomers of 1 was achieved, and their absolute configurations were determined as S-(+)- and R-(?)-leucoflavonines ((+)-1a and (?)-1b) by comparison of their computational and experimental optical rotations. Biological assays indicated that both (+)-1a and (?)-1b exhibited inhibitory activity against acetylchlorinesterase (AChE) in vitro (IC₅₀?=?68.0?±?8.6 and 18.3?±?1.8?μM, respectively). Moreover, (?)-1b displayed cytotoxicity against human hepatoma cells HepG2 (IC₅₀?=?52.9?±?3.6?μM), and inhibited the production of interleukelin-2 (IL-2) in Jurkat cells (IC₅₀?=?16.5?±?0.9?μM), while (+)-1a showed no obvious activity in these assays.     

Withanolides from Withania aristata and their cytotoxic activity

Seven new withanolides (1-7), along with three known ones (8-10), were isolated from the leaves of Withania aristata. Their structures were elucidated on the basis of spectroscopic analysis, including 2D NMR experiments and spectrometric techniques, and the absolute configuration of 1 and 2 was established by CD analysis. In the search for new cytotoxic compounds from Withania species, the isolated compounds 1-9, along with two derivatives, were assayed for their cytotoxicity against HeLa, MCF-7 and A-549 human tumor cell lines. Derivative (4S,20R,22R)-27-acetoxy-4-p-bromobenzoyloxy-1-oxo-witha-2,5,16,24-tetraenolide (13) showed cytotoxicity against all the cell lines assayed with IC₅₀ values ranging from 2.8 to 3.6 μM, and (4S,20R,22R)-4,27-diacetoxy-4-hydroxy-1-oxo-witha-2,5,16,24-tetraenolide (12) exhibited an IC₅₀ value of 5.4 μM on the MCF-7 cell line.     

(−)-Epilamprolobine and its N-oxide,lupin alkaloids from Sophora tomentosa

From the fresh leaves of Sophora tomentosa, three new lupin alkaloids, (?)-epilamprolobine, (+)-epilamprolobine N-oxide and 5-(3′-methoxycarbonylbutyroyl)aminomethyl-trans-quinolizidine N-oxide, have further been isolated along with (+)-matrine, (+)-matrine N-oxide, (+)-sophocarpine N-oxide, (?)-anagyrine, (?)- baptifoline, (?)-cytisine, (?)-N-methylcytisine, (?)-N-formylcytisine, (?)-N-acetylcytisine and (±)-ammodendrine. The absolute configurations of (+)-epilamprolobine N-oxide (1R:5R:6S) and (?)-epilamprolobine (5R:6S) have also been established by spectroscopic data and by comparison with synthetic (+)-epilamprolobine (5S:6R)derived from (?)-lupinine (5R:6R). (?)-Epilamprolobine is a diastereomer of (+)-lamprolobine (5R:6R) in Lamprolobium fruticosum and 5-(3′-methoxycarbonylbutyroyl) aminomethyl-trans-quinolizidine N-oxide is presumed to be an artefact. A biosynthetic pathway for the formation of (?)-epilamprolobine is also proposed.     

Aporphine alkaloids and cytotoxic lignans from the roots of Illigera luzonensis

Six aporphine alkaloids, (+)-(S)-N-butyrylcaaverine (1), (+)-(S)-N-propionylcaaverine (2), (+)-(S)-N-acetylcaaverine (3), (+)-(6aR,7R)-N-butyrylnorushinsunine (4), (+)-(6aR,7R,E)-N-(but-2-enoyl)norushinsunine (5), and N-formyldehydrocaaverine (6) were isolated from the roots of Illigera luzonensis, together with 16 known compounds. Their structures were determined through spectroscopic and MS analyses. Among the isolates, (−)-deoxypodophyllotoxin (13) was the most cytotoxic, with IC₅₀ values of 0.0057, 0.0067, 0.00004, and 0.0035 μg/mL, respectively, against DLD-1, CCRF-CEM, HL-60, and IMR-32 cell lines. In addition, (−)-yatein (12) exhibited cytotoxic effects, with IC₅₀ values of 0.81, 0.20, and 0.59 μg/mL, respectively, against DLD-1, CCRF-CEM, and HL-60 cell lines.     

Bevantolol hydrochloride (nc-1400): Absolute configuration and direct separation of the enantiomers

Synthesis of (?)-bevantolol hydrochloride from 3,4-dimethoxyphenethylamine and (S)-(+)-m-tolyl glycidyl ether derived from (R)-(?)-epichlorohydrin established the absolute configuration of the (+) and (?) enantiomer as R and S, respectively. The purity of the enantiomers was determines using a chiral cellulose column (CHIRALCEL OD®) which allowed direct separation of the enantiomers. A separation factor (α) of 4.20 and a resolution factor (Rs) of 9.21 were obtained. © 1995 Wiley-Liss, Inc.     

The isolation of (−)-E-1-chlorotridec-1-ene-6,8-diol from a marine cyanophyte

(?)-E-1-Chlorotridec-1-ene-6,8-diol is a major constituent in a mixture of cyanophytes, tentatively identified as primarily Schizothrix calcicola and Oscillatoria nigroviridis, from Enewetak Atoll. The absolute configuration of the diol is probably 6R,8R.     

Absolute configuration and stereochemical analysis of 3α,6β-dibenzoyloxytropane

Both enantiomers of 3α,6β-dibenzoyloxytropane (1) have been prepared from optical active 6β-hydroxyhyoscyamines establishing their absolute configurations as (?)-(3R,6R) and (+)-(3S,6S)-dibenzoyloxytropane. Independent stereochemical confirmation was obtained by vibrational circular dichroism measurements, since bands characteristic of (3R,6R) and (3S,6S) configurations of tropanediols derivatives were observed. In addition, a chiral HPLC method was developed for determining absolute configurations of tropane-related natural substances at the microgram (μg) level. The complete ¹H NMR characterization of the scaffold of 1 is also reported.     

Inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 macrophage cells by lignans isolated from Euonymus alatus leaves and twigs

The 80% methanolic extract of Euonymus alatus leaves and twigs afforded three new lignans, (−)-threo-4,9,4′,9′-tetrahydroxy-3,7,3′,5′-tetramethoxy-8-O-8′-neolignan (1), (−)-threo-4,9,4′,9′-tetrahydroxy-3,5,7,3′-tetramethoxy-8-O-8′-neolignan (2), (7R,8R,7′R)-(+)-lyoniresinol (3), together with seventeen known lignans (4-20). The structures of 1-20 were elucidated by extensive 1D and 2D spectroscopic methods including ¹H NMR, ¹³C NMR, ¹H-¹H COSY, HMQC, HMBC and NOESY. All the isolated compounds except for dilignans (19 and 20) significantly inhibited nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells.     

An approach to branched-chain amino sugars,particularly derivatives of l-vancosamine (3-amino-2,3,6-trideoxy-3-C-methyl-l-lyxo-hexose) and its d enantiomer,via the cyanohydrin route

Methyl 4,6-O-benzylidene-2-deoxy-α-d-erythro-hexopyranosid-3-ulose reacted with potassium cyanide under equilibrating conditions to give, initially, methyl 4,6-O-benzylidene-3-C-cyano-2-deoxy-α-d-ribo-hexopyranoside (7), which, because it reverted slowly to the thermodynamically stable d-arabino isomer, could be crystallised directly from the reaction mixture. The mesylate derived from the kinetic product 7 could be converted by published procedures into methyl 3-acetamido-2,3,6-trideoxy-3-C-methyl-α-d-arabino-hexopyranoside, which was transformed into methyl N-acetyl-α-d-vancosaminide on inversion of the configuration at C-4. A related approach employing methyl 2,6-dideoxy-4-O-methoxymethyl-α-l-erythro-hexopyranosid-3-ulose gave the kinetic cyanohydrin and thence, via the spiro-aziridine 27, methyl 3-acetamido-2,3,6-trideoxy-3-C-methyl-α-l-arabino-hexopyranoside, a known precursor of methyl N-acetyl-α-l-vancosaminide.     

Alkaloids and styryllactones from the leaves of Goniothalamus tamirensis

Three new compounds, goniotamirine (1), goniotamiric acid (2), and 3,5-demethoxypiperolide (3) were isolated from the leaves of Goniothalamus tamirensis (Annonaceae), together with sixteen known compounds, (?)-N-nornuciferine (4), (?)-norisocorydine (5), (?)-isocorydine (6), (?)-3-hydroxynornuceferine (7), (?)-O-methylisopiline (8), (?)-anonaine (9), (?)-roemerine (10), (?)-roemeroline (11), (?)-boldine (12), glaunine (13), liriodenine (14), 9-deoxygoniopypyrone (15), 8-epi-9-deoxygoniopypyrone (16), 8-epi-9-deoxygoniopypyrone acetate (17), goniodiol (18) and goniothalamin (19). The structures were established from spectral analysis, including mass spectrometry and 2D-NMR. The absolute configuration of 1 was determined from analysis of its MTPA amide derivatives. The cytotoxicity of all isolates was evaluated against KB cells. Only compound 4 (N-nornuciferine) showed a moderate activity with an IC₅₀ value of 12 μg/mL.     

Asymmetric total synthesis and antiproliferative activity of goniothalamin oxide isomers

Goniothalamin oxide (1) is a styryl lactone which was isolated from bark and leaves of several Goniothalamus species. This natural product has some interesting biological properties such as larvicidal and tripanocidal activities. However, no studies on the antiproliferative profile of goniothalamin oxide (1) and its stereoisomers have been reported yet. Here, goniothalamin epoxide (1), isogoniothalamin epoxide (2) and their enantiomers were prepared via epoxidation of (R)-and (S)-goniothalamin (4). A 3:2 molar ratio in favor of goniothalamin oxide (1) and ent-1 was observed from (R)- and (S)-4, respectively, when 3-chloroperbenzoic acid (mCPBA) was employed while an increase to 6:1 molar ratio was achieved with (S,S)-Jacobsen’s catalyst. Antiproliferative activity of these epoxides revealed that ent-isogoniothalamin oxide (ent-2) was the most active against the eight cancer cell lines studied. These results indicate that 6S, 7R and 8R absolute configurations are beneficial for the activity of these epoxides.     

Diamidato-bis(diphenylphosphino) platinum(II) complexes: Synthesis, characterization, and reactivity in the presence of acid

The reaction of Pt(COD)Cl₂, where COD is 1,5-cyclooctadiene, with one equivalent of a diamidato-bis(phosphino) Trost ligand ((R,R)-2 = N,N′-bis(2-diphenylphosphino-1-benzoyl)-(1R,2R)-1,2-diaminocyclohexane, (R,R)-3 = N,N′-bis(2-diphenylphosphino-1-naphthoyl)-(1R,2R)-1,2-diaminocyclohexane, or (±)-4 = N,N′-bis(2-diphenylphosphino-1-benzoyl)-1,2-bis(aminobenzene)) in the presence of base afforded square planar diamidato-bis(phosphino) platinum(II) complexes (R,R)-2-Pt, (R,R)-3-Pt, (±)-4-Pt. Characterization of all complexes included the solution and solid state structure determination of each complex based on multinuclear NMR and X-ray analyses, respectively. Stability of the complexes in acid was examined on addition of HCl to (R,R)-2-Pt in chloroform and compared to the unreactive nature of the similar diamidato-bis(phosphino) complex 1-Pt (1 = 1,2-bis-N-[2′-(diphenylphosphino)benzoyl]diamino-benzene) in the presence of acid. Protonation of the bound amidato nitrogen atoms of (R,R)-2-Pt was observed along with decoordination of the nitrogen atoms from the platinum(II) center producing (R,R)-2-PtCl₂ in quantitative yield by NMR analysis. Confirmation of the product was made on comparison of the NMR spectra to that of authentic (R,R)-2-PtCl₂ prepared on reaction of Pt(COD)Cl₂ with (R,R)-2 in CH₂Cl₂ and characterized by single-crystal X-ray diffraction analysis and NMR spectroscopy. Results add to the knowledge of rich coordination chemistry of bis(phosphino) ligands with late transition metals, metal-amidato chemistry, and has implications in catalysis.     

Absolute configuration of the pyrrolizidine alkaloid acetylgynuramine

The absolute configuration of the pyrrolizidine alkaloid acetylgynuramine was determined by X-ray diffraction to be (?)-(1aR,6bR,10R,11S)-9,14-dioxo-10-hydroxy-13-cis-ethylidene-11-methoxyacetyl-10-methyl-1a,2,3,6b-tetrahydro-5H-pyrrolizino-(1a,6b,6a,b,c)-1,8-dioxa-cyclododecane.