Synthesis of a novel series of artemisinin dimers with potent anticancer activity involving Sonogashira cross-coupling reaction

A series of C-10 acetal artemisinin dimers were synthesized using Sonogashira cross-coupling reaction. All these novel semisynthetic artemisinin dimers exhibited excellent growth inhibitory activity against Lung A-549 human cancer cell line.     

Synthesis and biological activity of new melatonin dimeric derivatives

A new series of melatonin (MLT) dimers were obtained by linking together two melatonin units with a linear alkyl chain through the MLT acetamido group or through a C-2 carboxyalkyl function. The binding properties of these ligands were evaluated in in vivo experiments on cloned human MT(1) and MT(2) receptors expressed in NIH3T3 rat fibroblast cells. The class of 2-carboxyalkyl dimers was the most interesting one with compounds having good MT(1)/MT(2) nanomolar affinity. The data obtained suggest that the spacer length is crucial for optimal interaction at both receptor subtypes as well as to determine functional activity of the resulting dimers.     

Crystal structure of head-to-head dimers of cholic and deoxycholic acid derivatives with different symmetric bridges

The crystal structure of three head-to-head dimers (having two cholic acid or deoxycholic acid units) linked at carbon atoms C3 by aromatic or alkyl bridges is studied. An internal coordinates system is necessary for describing the relative orientation in the space of the two bile acid residues. Five angles (three torsion and two common ones) are necessary for defining the relative position of both steroid residues in space. Carbon atoms C3 (which always carries a α-hydroxy group in natural bile acids), and C10 and C13 (which always carry β-methyl groups) of each steroid residue are suitable for this purpose. Furthermore, the distance between each C3 carbon atoms of both steroid residues will allow one to locate the steroids in space. The three dimers selected provide a large range of values for these angles. The packing, hydrogen bond network, and location of guest in the three crystals are discussed.     

Biosynthetic intermediates in the conversion of fucosterol to oogoniol

When grown in the presence of [CD₃]-methionine Achlya heterosexual produces oogoniols containing two deuterium atoms which are located at C-28     

Facile synthesis and anticancer activity of C-10 non-acetal deoxoartemisinin dimers

In this research, N-(2-aminoethyl)glycine-linked C-10 non-acetal deoxoartemisinin dimers were synthesized by using solution phase peptide synthesis approach. In addition, chemical modification of the C-10 non-acetal deoxoartemisinin monomers and dimers by adding a lysine unit to the N-terminus has been performed. The biological activities of all synthesized compounds were evaluated against the colon cancer cell line (Caco-2). The non-acetal deoxoartemisinin monomers 12a, 15a–c and dimers 13a, 16a–c were active against Caco-2 cells and more potent than dihydroartemisinin.     

Boron neutron capture therapy using 10B entrapped anti-CEA immunoliposome

A new murine monoclonal antibody (2C-8) was prepared by immunizing mice ip with CEA producing human pancreatic cancer cell line, AsPC-1.SDS-PAGE and Western blot analysis showed that 2C-8 monoclonal antibody recognized CEA and NCA. This anti-CEA monoclonal antibody was conjugated with large multilamellar liposomes incorporated 10B compound (Cs2 10B12H11SH). This immunoliposomes applicated to boron neutron capture therapy. AsPC-1 cells were incubated with the 10B-Lip-MoAb(CEA) for 8 hours. After the irradiation with thermal neutron (1 x 10(11)-1 x 10(13) n/cm2), boronated AsPC-1 cells were showed decreasing uptake of 3H-TdR compared with control group. The numbers of 10B atoms in liposomes bound to an antibody were in proportion to the dose of 10B compounds added and maximum number of 10B atoms was approximatory 1.2 x 10(4)/Ab. These data indicated that the immunoliposomes could deliver highly amount of 10B atoms to the tumor cells and exert cytotoxic effect by thermal neutron. BNCT with immunoliposome may be useful to the non resectable malignant tumors in clinical application.     

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.     

The mechanism of the C-13(3) esterification step in the biosynthesis of bacteriochlorophyll a

5-Aminolaevulinate labelled with 18O at its C-1 carboxy oxygen atoms was prepared and incorporated into bacteriochlorophyll aphytyl of Rhodopseudomonas sphaeroides and bacteriochlorophyll ageranylgeranyl of Rhodospirillum rubrum. The biosynthetic samples of the bacteriochlorophylls were separately processed to obtain their isoprenyl alcohol components from the C-17(3) ester linkages and methanol from the C-13(3) methoxycarbonyl group. Methods were developed for the quantification of the isotopic composition of the various alcohols (methanol, phytol, geranylgeraniol). It was shown that the hydroxyl oxygen atoms of all the three alcohols originated from one of the C-1 oxygen atoms of the precursor 5-aminolaevulinate. In the light of these results the in vivo mechanism for the O-methylation reaction at C-13(3) during the biosynthesis of the two species of bacteriochlorophylls is discussed.     

Novel sulfur and selenium containing bis-α-amino acids from 4-hydroxyproline

The synthesis of new substituted prolines carrying at C-4 a second α-amino acid residue is reported. The amino acid, l-cysteine or l-selenocysteine, is linked to the proline ring through the sulfur or the selenium atom, respectively. The products were prepared with different stereochemistry at C-4, in few and clean high-yielding steps, with suitable protections for solid phase applications. The introduction of both sulfur and selenium atoms at C-4 of the proline ring seems to enhance significantly the cis geometry at the prolyl amide bond.     

On the Metabolic Relationship between the Calvin Cycle and the Tricarboxylic Acid Cycle: IV. A Plant Survey for Anomalous Acetyl Coenzyme A

Leaves of 10 randomly selected plants representing eight dicotyledonous families were exposed to ¹⁴CO₂ for a 10-minute period in the light. Citrate and alanine were isolated, purified isotopically, and degraded to obtain the ¹⁴C-isotope distribution of corresponding carbon atoms, i.e. citrate (C-1,2) and alanine (C-2,3). The cited carbon atoms of alanine were equally labeled as is typical of a 3-carbon intermediate derived from photosynthetic 3-phosphoglycerate. The carbon atoms of citrate, equivalent to acetyl-CoA, were unequally labeled. The citrate (C-1,2) isotope ratio ranged from 1.20 to 1.78 for the various plants compared to a ratio of unity in the uniformly labeled control. The results infer that 3-phosphoglycerate produced in the chloroplast is not the singular precursor of mitochondrial citrate.     

Carbon-13 nuclear magnetic resonance studies of adenosylcobalamin and alkylcorrinoids, selectively enriched with carbon-13.

The carbon-13 nuclear magnetic resonance spectra of a series of alkylcorrinoids, selectively enriched with 13C in the alkyl ligand, were recorded at 25.2 MHz and 25 degrees. The nature of the axial ligands markedly affects the chemical shift of the labeled alkyl moiety (trans effect) as well as the 13C resonances of selected carbon atoms of the corrin ring (cis effect). Although a number of factors appear to influence the trans effect on the chemical shift of the alkyl ligand (important among them being electric field effects), the cis effect appears to be dominated by changes in charge density (at the methine bridge carbon atoms, C-5, C-10, C-15) and by steric effects (at the methyl groups at C-1, C-5, and C-15) accompanying axial ligation. Spin-latice relaxation times of several organocorrinoids, selectively labeled with 13C in the ligands attached to cobalt, were also measured. The T1 values of the methylene carbons of [5'-13C]adenosylcobalamin and [2-13C]carboxymethylcobalamin are very similar to that of the methine bridge carbon atom C-10 of the corrin ring, indicating that rotation about the carbon-cobalt bond of these two corrinoids is severely restricted. On the other hand, internal rotation about the carbon-cobalt bond of methylcobalamin is rapid.     

Structures of Dimers Produced from Methyl Linoleate during Initial Stage of Autoxidation

A structural investigation on the main fraction of dimers formed during the induction period of autoxidation in methyl linoleate (ML) was carried out. The dimeric fraction (A₂), which was isolated from the autoxidized ML (POV= 18) by various Chromatographic techniques and gave a single spot on TLC, was further separated into four major components (components 1–4) by high performance liquid chromatography (HPLC). The mean molecular weights of these components were found to be 643–655 and component 4 gave the parent peak 652 on an FD-mass spectrum which corresponded to 2 × ML + 4O. The reduced products of each component with stannous chloride were identified in common as methyl 9- or 13-hydroxy octadecadienoate and methyl 9,13-dihydroxy octadecenoate by GC-MS. These results show that all of these dimers contained a peroxide bridge linking between a pair of MLs across C-9 or C-13 on one of the MLs and C-9 or C-13 on the other, with a hydroperoxy group.     

Effect of 1-n-Decylimidazole on Gibberellin Biosynthesis in Tan-ginbozu,a Dwarf Variety of Rice

Previous structural studies of less-polar dimers in autoxidized methyl linoleate (ML) have been extended to polar dimers. After isolation by successive silicic acid and gel permeation chromatography, the dimeric fraction of linoleate was separated into two major fractions, A₁ and A₂, according to their polarities. The polar dimers (A₁) were further fractionated by HPLC either directly or after reduction with triphenyl phosphine on a micro silica column. Isolated subfractions were characterized by UV, IR, GC-MS and FD-MS after suitable derivatizations. FD-MS of all these dimers showed a molecular ion peak which corresponds to 2 × ML + 6 × O and the reduction of each subfraction with stannous chloride gave equimolar amounts of 9 and 13-hydroxy octadecadienoate, and 9, 10, 13 and/or 9, 12, 13-trihydroxy octadecenoate. These results combined with others show that the A₁ dimers are composed of isomeric mixtures containing a peroxide bridge linking a methyl octadecadienoate and a 9, 12 and/or 10, 13-dihydroperoxy octadecenoate across C-9 and/or 13 on each of them.     

New assumptions about oxidative processes involved in steroid hormone biosynthesis: is the role of cytochrome P-450-activated dioxygen limited to hydroxylation reactions or are dioxygen insertion reactions also possible?

The traditional conception of the chemical pathways leading to the formation of the steroid hormones is derived by piecing together the results of several independent in vitro incubation experiments. The results of these experiments have led to the assumption that some relevant cytochrome P-450's (P-450_scc, P-450_arom, P-450_aldo, etc.) are “polyfunctional” and catalyze several successive hydroxylation reactions, which lead to the formation of the hormonal products. This essay offers an alternative view. It advances the suggestion that the oxygenated intermediates in the relevant biosynthetic conversions are reactive species that are formed by addition of both atoms of dioxygen onto two neighboring carbon atoms of steroidal precursors. Space-filled Stuart molecular models, generated by a computer program, suggest that the oxidized intermediates resemble hydroperoxides or cyclic peroxides (1,2-dioxanes). For the aromatization process required for estrogen biosynthesis, the atoms of dioxygen are bonded to C-2 and C-19 of the C₁₉-precursor. For aldosterone formation, dioxygen is bonded to C-11 and C-18 of an appropriate precursor. Moreover, the results obtained from a computer program that provides information about “molecular mechanics” (bond angles and bond distances as well as total potential energies for each conformation of a molecule) suggest that consideration be given to the possibility that cortisol also can be biosynthesized by P-450-activated dioxygen addition to C-11 and C-17 of an appropriate precursor. Neither the traditional view of steroidogenic pathways nor the suggestions advanced here have been established by compelling experimental findings. Both hypotheses are saddled with untested assumptions, which are necessary because the dynamic processes can only be discerned by indirect means.

The origins of some naturally occurring steroids hydroxylated at C-17, C-18 and C-19 are examined in the light of the suggestions made in this essay.     

Novel fungal metabolites, demethylsorbicillin and oxosorbicillinol, isolated from Trichoderma sp. USF-2690

The novel compounds, demethylsorbicillin (1) and oxosorbicillinol (2), were isolated from a fermentation broth of Trichoderma sp. USF-2690. The structures of these compounds, which were determined from spectroscopic evidence, suggest the possibility that methylation at C-6 and oxidation at C-1 and C-6 of sorbicillin were controlled in the early polyketide stage before the formation of oxidized sorbicillin dimers. In a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay, 2 gave an ED50 value of 87.7 microM.     

Mechanism and stereochemistry of the 5-aminolaevulinate synthetase reaction

1. Two mechanisms for the biosynthesis of 5-aminolaevulinate from glycine and succinyl-CoA (3-carboxypropionyl-CoA) are considered. One of the mechanisms involves the retention of both the C-2 H atoms of glycine during the synthesis of 5-aminolaevulinate, whereas the other predicts the retention of only one of the C-2 H atoms of glycine. 2. Highly purified 5-aminolaevulinate synthetase from Rhodopseudomonas spheroides was used to show that the C-2 H atom of glycine with R configuration is specifically removed during the biosynthesis of 5-aminolaevulinate. 3. The mechanism of the condensation therefore differs from the analogous reaction of the biosynthesis of sphinganine from palmitoyl-CoA and serine, in which the C-2 H of serine is retained (Wiess, 1963).     

New Nystatin-Related Antifungal Polyene Macrolides with Altered Polyol Region Generated via Biosynthetic Engineering of Streptomyces noursei

Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.     

Identification of the cuticular lipid composition of the Western Flower Thrips Frankliniella occidentalis

The Western Flower Thrips Frankliniella occidentalis effectively resists many insecticides, but it can be controlled by the use of bioinsecticides such as entomopathogenic fungi. The epicuticular chemistry of these insects is therefore of great interest, and accordingly, the cuticular lipid composition of F. occidentalis was analysed. It was found that the cuticular lipids of both the adult and larval stages of F. occidentalis consist of two groups of compounds--hydrocarbons and free fatty acids. The same hydrocarbon pattern was found in both adults and larvae, with the exception of n-hentriacontane, which was detected only in adult insects. The following homologous series were identified: n-alkanes from C-25 to C-29 (31) with the marked dominance of odd numbers of carbon atoms, 3-methylalkanes with 26 and 28 carbon atoms, and branched monomethylalkanes (branched at C-9, -11, -13 and -15) with 26, 28 and 30 carbon atoms. The chemical composition of the free fatty acids consists of two homologous series: saturated (C(14:0), C(16:0), C(18:0)) and unsaturated fatty acids (C(16:1) and C(18:1)). This analysis confirmed the lack of potential inhibitors of entomopathogenic fungi in the cuticular lipids of this insect species.     

Multiple molecular forms of human plasma butyrylcholinesterase. I. Apparent molecular parameters and broad pattern of the quaternary structure (author's transl)]

Apparent molecular parameters (molecular weights, sedimentation constants, partial specific volumes, free electrophoretic mobilities and isoelectric points) of the four molecular forms C-1, C-2, C-3 and C-4 of human plasma butyrylcholinesterase (EC 3.1.1.8) have been demonstrated by polyacrylamide gel electrophoresis methods and centrifugation in sucrose gradient. The C-1 component is the monomeric form of the enzyme )Mr = 84 800 +/- 5800). All the forms are partially interconvertible and C-1, C-3, C-4 are size isomers corresponding to the monomer, dimer and tetramer of the enzyme. An estimation of the general shape of these forms attempted from electrophoretic and hydrodynamic parameters suggests that they are prolate ellipsoids. The C-4 component in which the axial ratio is at least equal to 8 appears to be arranged as a dimer of dimers (C-3)2 in which the two units are associated in a quasi-linear fashion. The C-2 component is composed of C-1 associated with an inactive smaller subunit, which is responsible for its specific electrical properties (mobility and isoelectric point).