Metabolism of fluoranthene by Mycobacterium sp. strain AP1

The pyrene-degrading Mycobacterium strain AP1 was found to utilize fluoranthene as a sole source of carbon and energy. Identification of metabolites formed from fluoranthene (by growing cells and washed-cell suspensions), the kinetics of metabolite accumulation, and metabolite-feeding studies all indicated that strain AP1 oxidizes fluoranthene using three alternative routes. The first route is initiated by dioxygenation at C-7 and C-8 and, following meta cleavage and pyruvate release, produces a hydroxyacenaphthoic acid that is decarboxylated to acenaphthenone (V). Monooxygenation of this ketone to the quinone and subsequent hydrolysis generates naphthalene-1,8-dicarboxylic acid (IV), which is further degraded via benzene-1,2,3-tricarboxylic acid (III). A second route involves dioxygenation at C-1 and C-2, followed by dehydrogenation and meta cleavage of the resulting diol. A two-carbon fragment excision of the meta cleavage product yields 9-fluorenone-1-carboxylic acid (II), which appears to undergo angular dioxygenation and further degradation to produce benzene-1,2,3-tricarboxylic acid (III), merging this route with the 7,8-dioxygenation route. Decarboxylation of benzene-1,2,3-tricarboxylic acid to phthalate (VIII), as well as further oxidation of the latter, would connect both routes with the central metabolism. The identification of Z-9-carboxymethylenefluorene-1-carboxylic acid (I) suggests a third route for fluoranthene degradation involving dioxygenation at C-2, C-3, and ortho cleavage. There is no evidence of any further degradation of this compound.     

Microbial transformations of N-(4-chlorphenyl)-benzoisothiazolone

Summary The microbial degradation of N-(4-chlorphenyl)-benzoisothiazolone (1) was studied by Streptomyces species in analogy to the metabolism of this drug in animals. As main metabolite 2-thiomethyl-N-(4-chlorphenyl)-benzamide (3) was found. The corresponding sulfoxide (4) and the sulfone (5) were obtained as further transformation products. The unmethylated 2-sulfhydryl compound (2) could be isolated in only small amounts. The degradation pathway follows by these results via a reductive cleavage of the S-N-bond of the isothiazolone ring with subsequent methylation of the sulfhydryl group and further oxidation of the resulting thiomethyl substituent.     

Synthesis and in vitro evaluation of novel 2,6,9-trisubstituted purines acting as cyclin-dependent kinase inhibitors.

Novel C-2, C-6, N-9 trisubstituted purines derived from the olomoucine/roscovitine lead structure were synthesized and evaluated for their ability to inhibit starfish oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases in purified extracts. Structure activity relationship studies showed that increased steric bulk at N-9 reduces the inhibitory potential whereas substitution of the aminoethanol C-2 side chain by various groups of different size (methyl, propyl, butyl, phenyl, benzyl) only slightly decreases the activity when compared to (R)-roscovitine. Optimal inhibitory activity against CDK5, CDK1 and CDK2, with IC50 values of 0.16, 0.45 and 0.65 microM, respectively, was obtained with compound 21 containing a (2R)-pyrrolidin-2-yl-methanol substituent at the C-2 and a 3-iodobenzylamino group at the C-6 of the purine. Compound 21 proved cytotoxic against human tumor HeLa cells (LD50-6.7 microM versus 42.7 microM for olomoucine, 24-h contact). Furthermore, unlike olomoucine, compound 21 was effective upon short exposure (LD50= 25.3 microM, 2-h contact). The available data suggest that the affinity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straightforward cell cycle phase specificity of the cytotoxic response to 21 was observed in synchronized HeLa cells. With the noticeable exception of pronounced lengthening of the S-phase transit by 21 applied during early-S in synchronized HeLa cells, and in striking contrast with earlier reports on studies using plant or echinoderm cells. olomoucilnc and compound 21 were unable to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible hlock in GI and G2 phase occurred at high olomoucine concentration, correlated with induced cell death. Moreover, chmronic exposure to lethal doses of compound 21 resulted in massive nuclear fragmentation, evocative of mitotic catastrophe with minour amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the intracellular uptake of nuicleosides with high efficiency.     

Novel Taxa-4(20),12-diene and 2(3→20)Abeotaxane from Needles of Taxus canadensis

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7β,9α,10β-triacetoxytaxa-4(20),12-diene-2α,5α,11β-triol (1) and 2α,7β,10β-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.     

Conversion of 1-benzoylindole by Aspergillus strains

Biotransformation of 1-benzoylindole (BI) by the strains Aspergillus flavus VKM F-1024 and Aspergillus oryzae VKM F-44 was studied. The major metabolites isolated were identified as 4-hydroxyindole (4-HI), 5-hydroxyindole (5-HI), 4-hydroxy-1-benzoylindole, 4-hydroxy-1-(4′-hydroxy)-benzoylindole and indole. The structure of the metabolites was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy. The pathways of BI metabolism via initial monohydroxylation at C-4 and C-5 followed by cleavage of the benzoyl substituent to yield 4-HI and 5-HI were proposed. Indole was formed as a by-product, and its role as a potent inhibitor of BI hydroxylation at C-4 and C-5 is discussed. Received: 22 June 1999 / Received revision: 6 December 1999 / Accepted: 12 December 1999     

Synthesis of 5-beta-D-ribofuranosylcytosine (pseudo-cytidine) and its alpha isomer

The dilithio derivative of 2,4-di-O,N-trimethylsilylcytosine was condensed with 2,4:3,5-di-O-benzylidene-D-ribose to give a mixture of the protected, epimer at C-1′ pentitols 5 and 6; in addition, a compound substituted at N-3 or N-4, whose structure was not elucidated, was also obtained. The epimers were treated with acid to give 4-amino-2-hydroxy-5-(β-and α-D-ribofuranosyl)pyrimidine (10 and 12). The n.m.r. spectrum of 10 corresponds predominantly to the C-2′ endo structure. On the other hand, the n.m.r. spectrum of 12 presents couplings identical with those of the “α pseudo-uridine”. On nitric deamination, each isomer gave in a highly preponderant yield the corresponding pseudo-uridine at C-1′.     

Vieloplains A-G,seven new guaiane-type sesquiterpenoid dimers from Xylopia vielana

Seven new guaiane-type sesquiterpene dimers vieloplains A-G, connecting patterns through three different direct CC bonds compounds 1–5 (C-3 to C-3′, C-4 to C-1′), compound 6 (C-2 to C-3′, C-4 to C-2′) and compound 7 (C-2 to C-1′, C-4 to C-2′) were isolated from the roots of Xylopia vielana. Their absolute configurations were established by NOESY analysis, the Cu Kα X-ray crystallographic the experiment circular dichroism (ECD) and the calculated ECD. Among them, only compound 6 showed a considerable cytotoxicity against DU145 cells with IC₅₀ values of 9.5 μM. Flow cytometry analysis confirmed that 6 caused death of DU145 cells via apoptosis induction.     

Synthesis of (S)-13-Hydroxy (2E,4E,8E)- and (2E,4E,8Z)-Tetradecatrienoic Acids

The syntheses of (S)-13-hydroxy-(2E,4E,8E)-tetradecatrienoic acid (1) and (2E,4E,8Z)-tetradecatrienoic acid (2) were carried out by using the Wittig reaction as the key step. The asymmetric center at C-13 and the double bond between C-8 and C-9 for natural compound 1 were reconfirmed as being of (S) configuration and E, respectively.

The relationship between the structure of the unsaturated hydroxy fatty acids and their inhibitory effect on the growth of lettuce was investigated.     

Mechanistic studies on C-19 demethylation in oestrogen biosynthesis

Mechanistic aspects of the biosynthesis of oestrogen have been studied with a microsomal preparation from full-term human placenta. The overall transformation, termed the aromatization process, involves three steps using O₂ and NADPH, in which the C-19 methyl group of an androgen is oxidised to formic acid with concomitant production of the aromatic ring of oestrogen: [Formula: see text] To study the mechanism of this process in terms of the involvement of the oxygen atoms, a number of labelled precursors were synthesized. Notable amongst these were 19-hydroxy-4-androstene-3,17-dione (II) and 19-oxo-4-androstene-3,17-dione (IV) in which the C-19 was labelled with ²H in addition to ¹⁸O. In order to follow the fate of the labelled atoms at C-19 of (II) and (IV) during the aromatization, the formic acid released from C-19 was benzylated and analysed by mass spectrometry. Experimental procedures were devised to minimize the exchange of oxygen atoms in substrates and product with oxygens of the medium. In the conversion of the 19-[¹⁸O] compounds of types (II) and (IV) into 3-hydroxy-1,3,5-(10)-oestratriene-17-one (V, oestrone), it was found that the formic acid from C-19 retained the original substrate oxygen. When the equivalent ¹⁶O substrates were aromatized under ¹⁸O₂, the formic acid from both substrates contained one atom of ¹⁸O. It is argued that in the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), the C-19 oxygen of the former remains intact and that one atom of oxygen from O₂ is incorporated into formic acid during the conversion of the 19-oxo compound (IV) into oestrogen. This conclusion was further substantiated by demonstrating that in the aromatization of 4-androstene-3,17-dione (I), both the oxygen atoms in the formic acid originated from molecular oxygen. 10β-Hydroxy-4-oestrene-3,17-dione formate, a possible intermediate in the aromatization, was synthesized and shown not to be converted into oestrogen. In the light of the cumulative evidence available to date, stereochemical aspects of the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), and mechanistic features of the C-10–C-19 bond cleavage step during the conversion of the 19-oxo compound (IV) into oestrogen are discussed.     

Tetranorlanostane and Lanostane Triterpenoids with Cytotoxic Activity from the Epidermis of Poria cocos

Two unprecedented tetranorlanostane triterpenoids, poricolides A ( 1 ) and B ( 2 ), and two new lanostane triterpenoids, 3β-acetoxy-24-methyllanosta-8,16,24(31)-trien-21-oic acid ( 3 ) and 3β-acetoxylanosta-7,9(11),16,23-tetraen-21-oic acid ( 4 ), were isolated from the epidermis of Poria cocos. The structures of 1–4 were determined via analysis of ¹H-, ¹³C-, and 2D-NMR, and HR-ESI-MS data, and the absolute configurations of 1 and 3 were established by single-crystal X-ray diffraction analysis. Compounds 1 and 2 were the first report of tetranorlanostane triterpenoid having a δ-lactone ring at C(17). Compounds 3 and 4 were rare lanostane triterpenoids having a double bond between C(16) and C(17). Compounds 1–4 exhibited potent antiproliferative effects against A549, SMMC-7721, MCF-7, and SW480 cancer cell lines with IC₅₀ values from 16.19±0.38 to 27.74±1.12 μM.     

Biotransformation of α-Santonin by Cell Suspension Cultures of Five Plants

Cell suspension cultures of five plants (Catharanthus roseus, Ginkgo biloba, Platycodon grandiflorum, Taxus cuspidata, Phytolacca asinosa) were employed to bioconvert the eudesmanolide compound, α-santonin. Reactions occurring were hydroxylation (C-1, C-11 and C-15), reduction of the double bond [1(2) or 3(4)], rearrangment of the eudesmanolide skeleton to a guaianolide skeleton and lactone-ring hydrolysis. Four new compounds were identified.     

Substituent — Directed Aralkylation and Alkylation Reactions of the Tricyclic Analogues of Acyclovir and Guanosine

Abstract

Aryl or tert-butyl substituent in the 6 position of 3,9-dihydro-3-[(2-hydroxyethoxy)methyl]-9-oxo-6-R-5H-imidazo[1,2-α]purine (6-R-TACV)¹ 1 partly directs aralkylation reactions into unusual positions: N-4 to give 3 and C-7 to give N-5, 7-disubstituted or N-4, 7-disubstituted derivatives. In the case of alkylation the effect is limited to aryl substituent and position N-4. Replacement of acyclic moiety of 1 with a ribosyl one like in 7 prevents N-4 substitution. Cleavage of the third ring of 3b to give 3-benzylacyclovir 10 is an example of a new short route to 3-aralkyl-9-substituted guanines.     

Characterization and molecular cloning of a novel endoglucanase from Trichoderma sp. C-4

A fungal strain, C-4, was isolated from etiolated leaves. Based on taxonomic studies, the fungus C-4 can be classified as a strain of Trichoderma species. When strain C-4 was cultured in Mandels medium at 28°C for 6 days, the enzyme activities detected in the broth corresponded to 8.2 U/ml (28.1 U/mg) carboxymethylcellulase activity. An endoglucanase (EG; F-I-II) was purified from the culture filtrate of the strain through a four-step procedure—chromatography on Sephacryl S-200, DEAE-Sephadex A-50, Con A-Sepharose, and Chromatofocusing on Mono-P (HPLC). The molecular weight of this EG, which was called C4endoII, was determined to be about 51 kDa. The optimum temperature and pH of C4endoII were 50°C and 5.0, respectively. Incubation at 50°C for 24 h did not destroy the cellulose degradation activity. Amino acid sequence analysis revealed the N-terminal sequence of an internal peptide of C4endoII to be Phe-Ala-Gly-Ile-Asn-Ile-Ala-Gly-Phe-Asp-Phe, which is homologous to EGII from Trichoderma reesei. A C4endoII cDNA (C4endoII) was cloned from a cDNA library constructed using the mRNA of the strain cultivated in a cellulase-induction medium. The deduced protein sequence of C4endoII was 417 amino acids long and had a putative signal sequence of 21 amino acids with a predicted cleavage site after Ala-21. A single potential N-glycosylation site was present in the amino acid sequence.     

Structure of 2-acetamido-3-amino-2,3-dideoxy-d-glucofuranurono-6,3-lactam

An X-ray crystallographic analysis of the title compound, an N-acetyl derivative of the 2,3-diamino-2,3-dideoxy-d-glucofuranurono-6,3-lactam found in the hydrolyzate of Pseudomonas aeruginosa P14 lipopolysaccharide, was performed. The crystals are monoclinic, space group P2₁, a = 11.704(2), b = 5.333(1), c = 7.399(2) Å, β = 91.63(2)°, and Z = 2. The structure was solved by direct methods and refined by the block-diagonal least-squares method to a final R value of 0.046 for 796 independent reflections. The dihedral angle between the mean plane through the furanose ring and that through the γ-lactam ring is 106.2(2)°, the furanose ring is ¹T₂, and the C-3, C-4, C-6, and N-3 atoms of the γ-lactam ring are nearly coplanar. The conformation in aqueous solution is discussed, based on the ¹H-n.m.r. data.     

A review of acacic acid-type saponins from Leguminosae-Mimosoideae as potent cytotoxic and apoptosis inducing agents

The aim of this review is to highlight updated results on the biologically active saponins from Leguminosae-Mimosoideae. Acacic acid-type saponins (AATS), is a class of very complex glycosides possessing a common aglycon unit of the oleanane-type (acacic acid = 3β, 16α, 21β trihydroxy-olean-12-en-28 oic acid), having various oligosaccharide moieties at C-3 and C-28 and an acyl group at C-21. About sixty molecules of this type have been actively explored in recent years from Leguminosae family, from a chemical point of view and some fifty were reported to possess cancer related activities. These include cytotoxic/antitumor, immunomodulatory, antimutagenic, and apoptosis inducing properties and appear to depend on the acylation and esterification by different moieties at C-21 and C-28 of the acacic acid-type aglycone. One can observe that the (6S) configuration of the outer monoterpenyl moiety (MT) seems more potent in mediating high cytotoxicity than its (6R) isomer. Furthermore, the trisaccharide moiety {β-d-Xylopyranosyl-(1→2)-β-d-Fucopyranosyl-(1→6)- N-Acetamido 2-β-d-Glucopyranosyl-} at C-3, the tetrasaccharide moiety {β-d-Glucopyranosyl-(1→3)-[α-L-Arabinofuranosyl-(1→4)]-α-l-Rhamnopyranosyl-(1→2)-β-d-Glucopyranosyl} at C-28 of the aglycone, and the inner MT hydroxylated at its C-9, having a (6S) configuration can be important substituent patterns for the induction of apoptosis of AATS. Because of their interesting cytotoxic/apoptosis inducing activity, some AATS can be useful in the search for new potential antitumor agents from Fabaceae. Furthermore, the sequence 28-O-{Glc-(1→3)-[Ara_f-(1→4)]-Rha-(1→2)-Glc-Acacic acid}, often encountered in the genera Acacia, Albizia, Archidendron, and Pithecellobium may represent a chemotaxonomic marker of the Mimosoideae subfamily.     

Acetate and carbon dioxide assimilation by Desulfovibrio vulgaris (Marburg), growing on hydrogen and sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source and acetate plus CO₂ as the sole carbon sources. The incorporation of U-¹⁴C acetate into alanine, aspartate, glutamate, and ribose was studied. The labelling data show that alanine is synthesized from one acetate (C-2 + C-3) and one CO₂ (C-1), aspartate from one acetate (C-2 + C-3) and two CO₂ (C-1 + C-4), glutamate from two acetate (C-1–C-4) and one CO₂ (C-5), and ribose from 1.8 acetate and 1.4 CO₂. These findings indicate that in Desulfovibrio vulgaris (Marburg) pyruvate is formed via reductive carboxylation of acetyl-CoA, oxaloacetate via carboxylation of pyruvate or phosphoenol pyruvate, and -ketoglutarate from oxaloacetate plus acetyl-CoA via citrate and isocitrate. Since C-5 of glutamate is derived from CO₂, citrate must have been formed via a (R)-citrate synthase rather than a(S)-citrate synthase. The synthesis of ribose from 1.8 mol of acetate and 1.4 mol of CO₂ excludes the operation of the Calvin cycle in this chemolithotrophically growing bacterium.     

Inhibition of human parainfluenza virus type 1 sialidase by analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid

Eleven novel analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) modified at the C-4 and C-9 positions were designed and tested for their ability to inhibit sialidase of human parainfluenza virus type 1 (hPIV-1). The analogs modified by the cyanomethyl, amidinomethyl, and thiocarbamoylmethyl groups at the C-4 position exhibited potent inhibition against hPIV-1 sialidase compared with Neu5Ac2en. The most effective compound was thiocarbamoylmethyl analog (4-O-thiocarbamoylmethyl-Neu5Ac2en). The activity of 4-O-thiocarbamoylmethyl-Neu5Ac2en causing 50% enzyme inhibition at a concentration of approximately 1.0×10^–5M was 30-fold larger than Neu5Ac2en. While, the analogs of Neu5Ac2en modified by the azido and N-acetyl groups at the C-9 showed a decrease in inhibition of sialidase compared with the 9-hydroxy analogs. In addition, 4-O-thiocarbamoylmethyl-Neu5Ac2en strongly inhibited hPIV-1 infections of Lewis lung carcinoma-monkey kidney cells in comparison with Neu5Ac2en. The present findings would provide useful information for the development of anti-human parainfluenza virus compounds.     

Lycopodium Alkaloids from Huperzia serrata and Their Anti-acetylcholinesterase Activities

One new fawcettimine-type alkaloid ( 1 ), one new miscellaneous-type alkaloid ( 2 ), four new lycodine-type alkaloids ( 3 – 6 ), and eight known ones ( 7 – 14 ) were isolated from the whole plants of Huperzia serrata. Their structures and absolute configurations were elucidated based on spectroscopic data, X-ray diffraction, ECD calculation and Mosher's method. Compound 1 was a rare C₁₈N₂-type Lycopodium alkaloid, possessing serratinine skeleton with an amide side chain in C-5. The absolute configuration of the 18-OH of compounds 4 – 6 were first determined by Mosher's method. Moreover, compounds 1 – 14 were assayed anti-acetylcholinesterase effect in vitro, and compound 7 showed significant anti-acetylcholinesterase activity with an IC₅₀ value of 16.18±1.64 μM.     

Syntheses of Jasmonic Acid Related Compounds and Their Structure-Activity Relationships on the Growth of Rice Seedings

Jasmonic acid (JA)-related compounds were synthesized, and their inhibitory activities on rice seedling growth were investigated. Three functions (C-1 CH₂COOH or CH₂COOCH₃, C-2 (Z)-2′-pentenyl or n-pentyl and C-3 ketone or hydroxyl) were essential for exhibiting inhibitory activity in this series of compounds. A dihydro-JA-related compound, 4-acetyl-nonanoic acid, also showed inhibitory activity similar to JA.     

X-ray crystal, and molecular, structure of 1,2,3-tri-O-acetyl-4,5-dideoxy-4-C-[(R)-phenylphosphinyl]-α- -lyxofuranose: The first x-ray analysis of a pentofuranose having phosphorus in the hemiacetal ring

X-Ray crystallographic analysis was performed on the compound to which had been assigned the structure of 1,2,3-tri-O-acetyl-4,5-dideoxy-4-C-[(R)-phenylphosphinyl]-α- -lyxofuranose. The results showed that the compound has the proposed configuration, the five-membered ring is in the E₃ conformation, with a tendency towards the ³T₂ form, the substituents at P-5 and C-5 are linked bisectionally, the acetoxyl group at C-2 and the methyl group at C-4 are linked quasiequatorially, and the acetoxyl group at C-3 is linked axially.