Biosynthesis of pterocarpan,isoflavan and coumestan metabolites of Medicago sativa: chalcone,isoflavone and isoflavanone precursors

Comparative feeding experiments in CuCl₂,- and UV-treated lucerne (Medicago sativa) seedlings have shown that 2′,4,4′-trihydroxychalcone-[carbonyl-¹⁴C] and formononetin-[Me-¹⁴C] but not 2′,4′-dihydroxy-4-methoxychalcone-[carbonyl- ¹⁴C] or daidzein-[4-¹⁴C] were incorporated into the phytoalexins demethylhomopterocarpin, sativan and vestitol, and also into 9-O-methylcoumestrol. The synthesis of 9-O-methylcoumestrol is greatly stimulated by this abiotic treatment but coumestrol production is not noticeably affected. Daidzein and the trihydroxychalcone were precursors of coumestrol. The results are interpreted in favour of a mechanism in which methylation is an integral part of the aryl migration process associated with the biosynthesisof 4′-methoxyisoflavonoids. Formononetin, 2′,7-dihydroxy-4′-methoxyisoflavone-[Me-¹⁴C], 7-hydroxy-4′-methoxyisoflavanone-[Me-¹⁴C] and 2′,7-dihydroxy-4′-methoxyisoflavanone-[Me-¹⁴C] were all excellent precursors of demethylhomopterocarpin, sativan, vestitol and 9-O-methylcoumestrol, and thus a metabolic grid may be involved in their biosynthetic origin.     

Synthesis and in vitro biological evaluation of carbon-11-labeled quinoline derivatives as new candidate PET radioligands for cannabinoid CB2 receptor imaging

Cannabinoids have been recently proposed as a new family of potential antitumor agents, and cannabinoid receptor 2 (CB2) is believed to be over-expressed in tumor cells. This study was designed to develop new radioligands for imaging of CB2 receptor in cancer using biomedical imaging technique positron emission tomography (PET). Carbon-11-labeled 2-oxoquinoline and 2-chloroquinoline derivatives, [¹¹C]6a–d and [¹¹C]9a–d, were prepared by O-[¹¹C]methylation of their corresponding precursors using [¹¹C]CH₃OTf under basic conditions and isolated by a simplified solid-phase extraction (SPE) method in 40–50% radiochemical yields based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 15–20 min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 111–185 GBq/μmol. Radioligand binding assays indicated compounds 6f, 6b, and 9f display potent in vitro binding affinities with nanomolar K_i values and at least 100–2000-fold selectivity for CB2.     

The synthesis and N.M.R. spectroscopy of derivatives of 6-amino-6-deoxy-D-galactose-6-15N

Derivatives of 6-amino-6-deoxy-D-galactose-6-¹⁵N have been synthesized by reaction of the 6-deoxy-6-iodo (1) or 6-O-p-tolylsulfonyl derivative of 1,2:3,4-di-O-isopropylidene-α-D-galactopyranose with potassium phthalimide-¹⁵N. The reaction of 1 also yielded an elimination product, 6-deoxy-1,2:3,4-di-O-isopropylidene-β-L-arabino-hex-5-enopyranose. The structures of the 6-amino-6-deoxy-D-galactose derivatives and their precursors were characterized by proton- and ¹³C-n.m.r. spectroscopy, with confirmation of the ¹³C assignments by selective proton decoupling. Selective broadening of the C-1, C-4, C-5, and C-6 resonances of 6-amino-6-deoxy-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose by low concentrations of cupric ion was observed, and studied by computerized measurements of the ¹³C linewidths. The application of this broadening to ¹³C-spectral assignments of amino sugar derivatives is indicated.     

Ureide Synthesis in a Cell-Free System from Cowpea (Vigna unguiculata [L.] Walp.) Nodules : STUDIES WITH O(2), pH, AND PURINE METABOLITES

The effect of O₂ and pH on the in vitro synthesis of ¹⁴C-labeled ureides from [8-¹⁴C]hypoxanthine in a cell-free system from cowpea nodules was investigated. Under conditions which suppressed uricase (EC 1.7.3.3) activity, namely low O₂ concentrations and low pH, ureide synthesis was inhibited and the ¹⁴C label incorporated into uric acid was increased. Conversely, conditions which increased uricase activity, namely high O₂ concentrations and high pH, also stimulated ureide synthesis, and the ¹⁴C label was incorporated principally into allantoin. The overall response of the system to O₂ concentration and pH indicated that the per cent distribution of total ¹⁴C label incorporated into uric acid was inversely related to that into allantoin. In the present study there was evidence that uricase (EC 1.7.3.3) controlled the in vitro rate of ureide synthesis in the cell-free system. Adenine and guanine inhibited xanthine dehydrogenase (EC 1.2.1.37) and as a consequence ureide synthesis from [8-¹⁴C]hypoxanthine was also inhibited.     

Synthesis of carbon-11-labeled 4-(phenylamino)-pyrrolo[2,1-f][1,2,4]triazine derivatives as new potential PET tracers for imaging of p38α mitogen-activated protein kinase

The reference standards methyl 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (10a), methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (10b) and corresponding precursors 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylic acid (11a), methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylic acid (11b) were synthesized from methyl crotonate and 3-amino-4-methylbenzoic acid in multiple steps with moderate to excellent yields. The target tracer [¹¹C]methyl 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate ([¹¹C]10a) and [¹¹C]methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate ([¹¹C]10b) were prepared from their corresponding precursors with [¹¹C]CH₃OTf under basic condition through O-[¹¹C]methylation and isolated by a simplified solid-phase extraction (SPE) method in 50–60% radiochemical yields at end of bombardment (EOB) with 185–555 GBq/μmol specific activity at end of synthesis (EOS).     

Nonspecific cytotoxic effects of antigen-transformed lymphocytes: II. Inhibition by drugs

High concentrations of prednisolone (10^?5M) failed to inhibit the nonspecific cytotoxic effects of human lymphocytes that had already transformed in response to PPD. In contrast, prednisolone added at the beginning of lymphocyte culture caused a significant inhibition of subsequent cytotoxicity at concentrations as low as 10^?8M. A single concentration of prednisolone (10^?6M) caused progressively less inhibition the later it was added in the lymphocyte culture period, and it is suggested that there is a steroid-sensitive phase in the early stages of development of nonspecific cytotoxicity after stimulation of lymphocytes with antigen. This steroid-sensitive phase could not be attributed to a difference in lysosome activity, since chloroquine caused the same degree of inhibition at the beginning as at the end of culture. In addition, studies with cycloheximide, actinomycin D, and mitomycin C indicated that cytotoxicity by transformed lymphocytes depended on protein synthesis but not on short-term RNA or DNA synthesis.     

A Missense Mutation in Rev7 Disrupts Formation of Polζ, Impairing Mouse Development and Repair of Genotoxic Agent-induced DNA Lesions

Repro22 is a mutant mouse produced via N-ethyl-N-nitrosourea-induced mutagenesis that shows sterility with germ cell depletion caused by defective proliferation of primordial germ cells, decreased body weight, and partial lethality during embryonic development. Using a positional cloning strategy, we identified a missense mutation in Rev7/Mad2l2 (Rev7^C70R) and confirmed that the mutation is the cause of the defects in repro22 mice through transgenic rescue with normal Rev7. Rev7/Mad2l2 encodes a subunit of DNA polymerase ζ (Polζ), 1 of 10 translesion DNA synthesis polymerases known in mammals. The mutant REV7 did not interact with REV3, the catalytic subunit of Polζ. Rev7^C70R/C70R cells showed decreased proliferation, increased apoptosis, and arrest in S phase with extensive γH2AX foci in nuclei that indicated accumulation of DNA damage after treatment with the genotoxic agent mitomycin C. The Rev7^C70R mutation does not affect the mitotic spindle assembly checkpoint. These results demonstrated that Rev7 is essential in resolving the replication stalls caused by DNA damage during S phase. We concluded that Rev7 is required for primordial germ cell proliferation and embryonic viability and development through the translesion DNA synthesis activity of Polζ preserving DNA integrity during cell proliferation, which is required in highly proliferating embryonic cells.     

Involvement of Protein Kinase C Activation in L-Leucine-Induced Stimulation of Protein Synthesis in L6 Myotubes

Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [³H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, α-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A₂ and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C.     

Estimating glutathione synthesis with deuterated water: a model for peptide biosynthesis

Glutathione (GSH), an intracellular tripeptide that combats oxidative stress, must be continually replaced due to loss through conjugation and destruction. Previous methods, estimating the synthesis of GSH in vivo, used constant infusions of labeled amino acid precursors. We developed a new method based on incorporation of ²H from orally supplied ²H₂O into stable C-H bonds on the tripeptide. The incorporation of ²H₂O into GSH was studied in rabbits over a 2-week period. The method estimated N, the maximum number of C-H bonds in GSH that equilibrate with ²H₂O as amino acids. GSH was analyzed by liquid chromatography/mass spectrometry after derivatization to yield GSH-N-ethylmaleimide (GSNEM). A model, which simulated the expected abundance at each mass isotopomer for the GSNEM ion at various values for N, was used to find the best fit to the data. The plateau labeling fit best a model with N = 6 of a possible 10 C-H bonds. Thus, the amino acid precursors do not completely equilibrate with ²H₂O prior to GSH synthesis. Advantages of this new method include replacing costly amino acid infusions with the oral administration of ²H₂O and a statistical basis for estimating N.     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

Studies on Mitomycins,Carcinostatic Antibiotics

Mitomycin A (C₁₆H₁₉O₆N₃) and mitomycin C (C₁₅H₁₈O₅N₄) are pigments which have the quinoid structure. When treated with aqueous ammonia, mitomycin A is converted to mitomycin C. Acid hydrolysis of mitomycin C gave three degradation products, namely, C₁₄H₁₅O₅N₄, C₁₄H₁₅O₆N₃ and C₁₃H₁₄O₅N₂. Acetylation with acetic anhydride and pyridine and methylation with methyl iodide gave monoacetyl and monomethyl derivatives of mitomycin C respectively, though diacetate of demethyl derivatives were obtained when boiled with acetic anhydride.     

Cytochrome P450 3A Enzymes Catalyze the O6-Demethylation of Thebaine,a Key Step in Endogenous Mammalian Morphine Biosynthesis

Morphine, first characterized in opium from the poppy Papaver somniferum, is one of the strongest known analgesics. Endogenous morphine has been identified in several mammalian cells and tissues. The synthetic pathway of morphine in the opium poppy has been elucidated. The presence of common intermediates in plants and mammals suggests that biosynthesis occurs through similar pathways (beginning with the amino acid l-tyrosine), and the pathway has been completely delineated in plants. Some of the enzymes in the mammalian pathway have been identified and characterized. Two of the latter steps in the morphine biosynthesis pathway are demethylation of thebaine at the O³- and the O⁶-positions, the latter of which has been difficult to demonstrate. The plant enzymes responsible for both the O³-demethylation and the O⁶-demethylation are members of the Fe^II/α-ketoglutarate-dependent dioxygenase family. Previous studies showed that human cytochrome P450 (P450) 2D6 can catalyze thebaine O³-demethylation. We report that demethylation of thebaine at the O⁶-position is selectively catalyzed by human P450s 3A4 and 3A5, with the latter being more efficient, and rat P450 3A2. Our results do not support O⁶-demethylation of thebaine by an Fe^II/α-ketoglutarate-dependent dioxygenase. In rat brain microsomes, O⁶-demethylation was inhibited by ketoconazole, but not sulfaphenazole, suggesting that P450 3A enzymes are responsible for this activity in the brain. An alternate pathway to morphine, oripavine O⁶-demethylation, was not detected. The major enzymatic steps in mammalian morphine synthesis have now been identified.     

The effect of mitomycin C treatment of infected erythrocytes on infection with Babesia microti and Babesia rodhaini in mice

In vitro treatment of Babesia microti infected erythrocytes with mitomycin C before their injection into mice prolonged the prepatent period of infection, reduced the levels of the infection in the ‘breakthrough’ parasitaemia and induced protection against reinfection. Treatment of B. microti with mitomycin C at a concentration of 25 μg ml^?1 resulted in a mean peak parasitaemia of 6.2% in the infected mice compared with 46.5% in control mice injected with untreated B. microti parasites. In addition, mice survived a normally fatal B. rodhaini infection if injected with 6.2 × 10⁷ infected erythrocytes treated with 25 μg ml^?1 mitomycin C and four of five mice survived infection with 6.2 × 10⁵ similarly treated infected erythrocytes. However, the degree of protection against B. rodhaini was dependent on the concentration of mitomycin C used to treat the parasites and treatment of 5 × 10⁷ infected erythrocytes with 50 μg ml^?1 resulted in survival of only four of the five infected mice. In addition, when 100 μg ml^?1 of mitomycin C was used to treat B. rodhaini parasites, the course of infection, although delayed, was indistinguishable from that seen in the control mice and all the mice died. The latter results and the lack of efficacy of comparable numbers of heat killed parasites suggested the necessity for sufficient, non-replicating, mitomycin C treated parasites to metabolize and produce and/or present protective antigens to the host.     

Utilization of exogenously supplied primary precursors for essential oil synthesis in cymbopogon species

The relative efficiency of incorporation of the exogenously supplied primary precursors [2-¹⁴C]acetate, [U-¹⁴C]glucose and [U-¹⁴C]sucrose into essential oil was determined in the immature leaves of threeCymbopogon species(C. martinii,C. winterianus andC. flexuosus). Acetate was most efficiently incorporated into essential oil inC. winterianus andC. flexuosus, whereas glucose was the best precursor inC. martinii. The observations are consistent when expressed as radioactivity [Bq] of essential oil per leaf, as percent incorporation or moles of precursors utilized for essential oil synthesis. Thus, there is selectivity in the efficiency of precursor utilization for the oil synthesis inCymbopogon species.     

Formation of vicine and convicine by Vicia faba

Methods are described for the quantitative extraction and separation of the pyrimidine glucosides, vicine and convicine. The contents of these two substances in germinating seeds and young seedlings of Vicia faba remain constant for the first 2 weeks. Net synthesis and accumulation of vicine and convicine occurs in developing seeds. That the synthesis occurs within the pod and the pyrimidine glucosides are not translocated into them, was shown by injection of ¹⁴C-labelled precursors into the pods. [1-¹⁴C]- and [2-¹⁴C]-acetate were weakly incorporated but much greater incorporation was observed with [U-¹⁴C]-aspartic acid and [6-¹⁴C]-orotic acid. The results indicate that the orotic acid pathway is involved in the formation of the pyrimidine ring of both vicine and convicine.     

A simple method of the preparation of 2′-O-Methyladenosine Methylation of adenosine with methyl iodide in anhydrous alkaline medium

A simple and effective method of the methylation on the 2′-O position of adenosine is described. Adenosine is treated with CH₃I in an anhydrous alkaline medium at 0°C for 4 h. The major products of this reaction are monomethylated adenosine at either the 2′-O or 3′-O position (total of 64%) and the side products are dimethylated adenosine (2′,3′-O-dimethyladenosi, 21%, and N⁶-2′-O-dimethyladenosine, 11%). The ratio of 2′-O- and 3′-O-methyladenosine has been found to be 8 to 1. Therefore, this reaction preferentially favors the synthesis of 2′-O-methyladenosine. The monomethylated adenosine is isolated from reaction mixture by a silica gel column chromatography. Then the pure 2′-O-methyladenosine can be separated by crystallization in ethanol from the mixture of 2′-O and 3′-O-methylated isomers. The overall yield of 2′-O-methyladenosine is 42%.     

Replication of Coliphage M-13 II. Intracellular Deoxyribonucleic Acid Forms Associated with M-13 Infection of Mitomycin C-treated Cells

Intracellular deoxyribonucleic acid (DNA) forms associated with bacteriophage M-13 infection have been isolated and characterized. Escherichia coli HF4704 (F⁺, hcr⁻, thy⁻) cells were treated with mitomycin C to inhibit host-cell DNA synthesis and were then infected with phage M-13. This treatment permitted radioactive labeling of phage-specific DNA forms with ³H-thymine. These labeled DNA components were characterized by sucrose density sedimentation and equilibrium density gradient centrifugation in neutral and ethidium bromide CsCl gradient. Two double-stranded circular forms were found with properties analogous to the replicative form I and replicative form II of X174. A third component, identified as single-stranded DNA, was isolated in some samples removed 45 min after phage synthesis was initiated.     

Synthesis of carbon-11-labeled casimiroin analogues as new potential PET agents for imaging of quinone reductase 2 and aromatase expression in breast cancer

Carbon-11-labeled casimiroin analogues were first designed and synthesized as new potential PET agents for imaging of quinone reductase (QR) 2 and aromatase expression in breast cancer. [¹¹C]casimiroin (6-[¹¹C]methoxy-9-methyl-[1,3]dioxolo[4,5-h]quinolin-8(9H)-one, [¹¹C]11) and its carbon-11-labeled analogues 5,6,8-trimethoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]17), 8-methoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]21a), 6,8-dimethoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]21b), and 5,8-dimethoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]21c), were prepared from their corresponding precursors with [¹¹C]methyl triflate ([¹¹C]CH₃OTf) under basic conditions (NaH) through either O- or N-[¹¹C]methylation and isolated by semi-preparative HPLC method in 40-50% radiochemical yields decay corrected to end of bombardment (EOB), based on [¹¹C]CO₂, and 111-185 GBq/μmol specific activity at the end of synthesis (EOS).     

Studies on the inhibition of hepatic lipogenesis by N6,O2′-dibutyryl adenosine 3′,5′-monophosphate

Fatty acid synthesis by isolated liver cells is dependent upon the availability of lactate and pyruvate. A lag in fatty acid synthesis is explained by time being required for lactate and pyruvate to accumulate to maximum concentrations in the incubation medium. The initial rate of fatty acid synthesis is not linear with cell concentration, being disproportionately greater at higher cell concentrations because optimal lactate and pyruvate concentrations are established in the medium more rapidly. The accumulation of lactate and pyruvate is inhibited markedly by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. This accounts in part for the inhibition of fatty acid synthesis caused by this cyclic nucleotide. Other sites of action are apparent, however, because exogenous lactate plus pyruvate only partially relieves the inhibition. The profile of metabolic intermediates suggests that N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate inhibits the conversion of glycogen to pyruvate and lactate by decreasing the effectiveness of phosphofructokinase and pyruvate kinase.     

Addition of 1-nitroalkanes to methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside and N6-benzoyl-2′,3′-O-isopropylideneadenosine-5′-aldehyde

Various 1-nitroalkanes reacted with methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside to yield methyl 6-alkyl-6-deoxy-2,3-O-isopropylidene-6-nitro-β-d-ribofuranosides in 64–79% yield. Similarly, nitromethane and 1-nitropentane reacted with N⁶-benzoyl-2′,3′-O-isopropylideneadenosine-5′-aldehyde, to yield the corresponding 9-[6-alkyl-6-deoxy-2,3-O-isopropylidene-6-nitro-α-l-talo(β-d-allo)furanosyl]-N⁶-benzoyladenines in 74 and 44% yield, respectively. The potential utility of this nitroalkane addition for the synthesis of nucleosides having a C-5′C-6′ bond is discussed.