Synthesis and evaluation of the anti-proliferative and NF-κB activities of a library of simplified tylophorine analogs

Tylophorine and many related phenanthropiperidine alkaloids are extraordinarily potent anti-proliferative agents. Despite their impressive anti-cancer activity, clinical development of these alkaloids has been hampered by their poor solubility and neurological side effects. Although it has been suggested that developing polar phenanthropiperidines will mitigate these undesired properties, the lack of practical methods for the synthesis of such analogues has limited this effort. Here, we present a concise synthetic approach to N-substituted phenanthropiperidines, which enabled a systematic investigation of structure-activity relationships at an underexplored region of the tylophorine scaffold. This work suggests that ring E of tylophorine is essential for the anti-proliferative activity of the 6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline core scaffold.     

Synthesis and analgesic effects of novel β2-tryptophan hexapeptide analogs

Aiming to develop more potent analgesic substances a new series of hexapeptides containing β²-tryptophan analogues was synthesized. The Trp in position 4 and 5, respectively in Ac-Arg-Phe-Met-Trp-Met-Lys-NH₂ (opioid receptor antagonist) and Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH₂ (highly potent and selective NOP-receptor agonist) was substituted by the (S)-2-(1-methyl-1H-indol-3-yl)propionic residue or the (S)-2-(5-methoxy-1H-indol-3-yl)propionic residue. The analgesic effect of the four newly synthesized compounds has been evaluated in male Wistar rats by PP- and HP tests and compared to the native templates. Further estimation of the mechanisms of action of each compound was achieved using specific antagonists—naloxone for opioid and JTC801 for the NOP receptor. Replacement of Trp with β²-tryptophan analogues in 4th position (Ac-Arg-Phe-Met-Trp-Met-Lys-NH₂) led to increased and longer lasting analgesic effect. The results obtained permit us to assume that both opioid and NOP receptors take part in the newly synthesized compounds analgesic effects.     

Synthesis and structure–activity relationship of p-carborane-based non-secosteroidal vitamin D analogs

1α,25-Dihydroxyvitamin D₃ [1α,25(OH)₂D₃: 1] is a specific modulator of nuclear vitamin D receptor (VDR), and novel vitamin D analogs are therapeutic candidates for multiple clinical applications. We recently developed non-secosteroidal VDR agonists bearing a p-carborane cage (a carbon-containing boron cluster) as a hydrophobic core structure. These carborane derivatives are structurally quite different from classical secosteroidal vitamin D analogs. Here, we report systematic synthesis and activity evaluation of carborane-based non-secosteroidal vitamin D analogs. The structure–activity relationships of carborane derivatives are different from those of secosteroidal vitamin D derivatives, and in particular, the length and the substituent position of the dihydroxylated side chain are rather flexible in carborane derivatives. The structure–activity relationships presented here should be helpful in development of non-secosteroidal vitamin D analogs for clinical applications.     

Synthesis,electrochemical and spectral properties of some ω-N-quinonyl amino acids

Summary. Four series of ω-N-quinonyl amino acids were synthesized by Michael-like additions. The quinones include 2-phenylthio-1,4-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone and 2,3-dichloro-1,4-naphthoquinone. These modified amino acids can be used for post chain assembly modifications of biologically active peptides, which target the quinonic drug to a cancer damaged area. The electron-transfer capabilities of the modified amino acids were probed by cyclic voltammetry measurements. The results described in this paper show that the novel N-quinonyl amino acids are effective in producing semiquinone radicals similarly to the unconjugated quinones themselves. A direct relation was found between the first reduction potentials of the quinones and their reactivity towards the ω-amino acids. The successful generation of stable semiquinone radicals by the novel quinone derivatives is a prerequisite for the manifestation of site-directed antitumor activity of corresponding quinone-peptide conjugates. Received January 3, 2001 Accepted March 28, 2001     

Conversion of ω-terminal-acetylenic sugar derivatives into acetylenic glycosides and nucleosides,and formation of “double-headed nucleoside” analogs

3,5-Di-O-acetyl-6,7-dideoxy-1,2-O-isopropylidene-β-L-ido- and α-d-gluco-hept-6-ynofuranose were separately deacetonated, and the products acetylated, to give the 1,2,3,5-tetra-O-acetyl analogs (2 and 6). Fusion of compounds 2 and 6 with 2,6-dichloropurine under acid catalysis produced 2,6-dichloro-9-(2,3,5-tri-O-acetyl-6,7-dideoxy-α-L-ido-hept-6-ynofuranosyl)-9H-purine (3) and its β-d-gluco analog 7, respectively. Methanolic ammonia converted 3 in good yield into 2-chloro-9-(6,7-dideoxy-α-L-ido-hept-6-ynofuranosyl)-6-methoxy-9H-purine. Treatment of compound 3 with mesityl nitrile oxide gave a “double-headed nucleoside” analog. Upon treatment with phenyl azide, the d-gluco derivative 7 produced another “double-headed nucleoside”. Fusion of 2 and 6 with p-nitrophenol yielded the respective p-nitrophenyl glycosides. The stereochemistry and regiospecificity of the reactions were verified spectroscopically.     

Synthesis and physical properties of phosphatidylcholines containing ω-cyclohexyl fatty acyl chains

Phosphatidylcholines (PCs) with cyclohexyl fatty acids as acyl chains were synthesized from 11-cyclohexyl and 13-cyclohexyl fatty acids and their physical properties were examined. The thermotropic behavior and barrier function of liposomal membranes formed from these PCs were studied. These PCs showed about 10°C lower gel-to-liquid crystalline phase transition temperatures (T_c) than the corresponding straight-chain PCs. The properties of mixtures of these cyclohexyl acyl PCs with straight-chain PCs were rather different from those observed with mixtures of straight-chain PCs. Cyclohexyl fatty acyl PCs showed barrier functions even above the T_c unlike the corresponding straight-chain PCs. These results indicate significant differences between the overall packing of cyclohexyl fatty acyl PCs and of the corresponding straight-chain PCs both in the gel state and in the liquid crystalline state. The significance of these cyclohexyl acyl chains in polar lipids, which are abundant in the thermophilic acidophilic bacterium, Bacillus acidocaldarius, is discussed.     

Synthesis and evaluation of C2 functionalized analogs of the α-tubulin-binding natural product pironetin

Pironetin is an α-tubulin-binding natural product with potent antiproliferative activity against several cancer cell lines that inhibits cell division by forming a covalent adduct with α-tubulin via a Michael addition into the natural product’s α,β-unsaturated lactone. We designed and prepared analogs carrying electron-withdrawing groups at the α-position (C2) of the α,β-unsaturated lactone with the goal to generate potent and selective binding analogs. We prepared derivatives containing halogens, a phenyl, and a methyl group at the C2 position to evaluate the structure-activity relationship at this position. Testing of the analogs in ovarian cancer cell lines demonstrated 100–1000-fold decreased antiproliferative activity.     

Synthesis and binding properties of endomorphin-2analogs containing α-hydroxymethyl amino acids

Novel endomorphin-2 analogs containing the unusual amphiphilic amino acid (R)- and (S)--hydroxymethyltyrosine in position 1 and (R)- and (S)--hydroxymethylphenylalanine in the positions 3 and 4 were synthesized via the solid-phase method. The binding characteristics of the synthetic analogs may suggest that -hydroxymethyl substitution of aminoacid residues influences the conformation of a peptide much more than simply increasing the local amphiphilic character of the peptide.     

Synthesis, β-glucuronidase inhibition and molecular docking studies of hybrid bisindole-thiosemicarbazides analogs

Hybrid bisindole-thiosemicarbazides analogs (1–18) were synthesized and screened for β-glucuronidase activity. All compounds showed varied degree of β-glucuronidase inhibitory potential when compared with standard d-saccharic acid 1,4-lactone (IC₅₀ = 48.4 ± 1.25 μM). Compounds 4, 7, 9, 6, 5, 12, 17 and 18 showed exceptional β-glucuronidase inhibition with IC₅₀ values ranging from 0.1 to 5.7 μM. Compounds 1, 3, 8, 16, 13, 2 and 14 also showed better activities than standard with IC₅₀ values ranging from 7.12 to 15.0 μM. The remaining compounds 10, 11, and 15 showed good inhibitory potential with IC₅₀ values 33.2 ± 0.75, 21.4 ± 0.30 and 28.12 ± 0.25 μM respectively. Molecular docking studies were carried out to confirm the binding interaction of the compounds.     

Synthesis of indole analogs as potent β-glucuronidase inhibitors

Natural products are the main source of motivation to design and synthesize new molecules for drug development. Designing new molecules against β-glucuronidase inhibitory is utmost essential. In this study indole analogs (1–35) were synthesized, characterized using various spectroscopic techniques including ¹H NMR and EI-MS and evaluated for their β-glucuronidase inhibitory activity. Most compounds were identified as potent inhibitors for the enzyme with IC₅₀ values ranging between 0.50 and 53.40 μM, with reference to standard d-saccharic acid 1,4-lactone (IC₅₀ = 48.4 ± 1.25 μM). Structure-activity relationship had been also established. The results obtained from docking studies for the most active compound 10 showed that hydrogen bond donor features as well as hydrogen bonding with (Oε1) of nucleophilic residue Glu540 is believed to be the most importance interaction in the inhibition activity. It was also observed that hydroxyl at fourth position of benzylidene ring acts as a hydrogen bond donor and interacts with hydroxyl (OH) on the side chain of catalysis residue Tyr508. The enzyme-ligand complexed were being stabilized through electrostatic π-anion interaction with acid-base catalyst Glu451 (3.96 Å) and thus preventing Glu451 from functioning as proton donor residue.     

Synthesis and bioevaluation of new tacrine-cinnamic acid hybrids as cholinesterase inhibitors against Alzheimer’s disease

Small molecule cholinesterases inhibitor (ChEI) provides an effective therapeutic strategy to treat Alzheimer’s disease (AD). Currently, the discovery of new ChEI with multi-target effect is still of great importance. Herein, we report the synthesis, structure–activity relationship study and biological evaluation of a series of tacrine-cinnamic acid hybrids as new ChEIs. All target compounds are evaluated for their in vitro cholinesterase inhibitory activities. The representatives which show potent activity on cholinesterase, are evaluated for the amyloid β-protein self-aggregation inhibition and in vivo assays. The optimal compound 19, 27, and 30 (human AChE IC₅₀?=?10.2?±?1.2, 16.5?±?1.7, and 15.3?±?1.8?nM, respectively) show good performance in ameliorating the scopolamine-induced cognition impairment and preliminary safety in hepatotoxicity evaluation. These compounds deserve further evaluation for the development of new therapeutic agents against AD.     

Synthesis of 4′-ester analogs of resveratrol and their evaluation in malignant melanoma and pancreatic cell lines

4′-Ester analogs of the disease preventative agent resveratrol were synthesized and evaluated for their potential as anti-melanoma and pancreatic cancer agents. A decarbonylative Heck coupling was used to assemble the protected stilbene core structure. The 4′-acetate and the palmitoate analogs demonstrated selective activity with DM443 and DM738 cells over normal NHDF cells.     

Cloning-Independent Expression and Analysis of ω-Transaminases by Use of a Cell-Free Protein Synthesis System

Herewith we report the expression and screening of microbial enzymes without involving cloning procedures. Computationally predicted putative ω-transaminase (ω-TA) genes were PCR amplified from the bacterial colonies and expressed in a cell-free protein synthesis system for subsequent analysis of their enzymatic activity and substrate specificity. Through the cell-free expression analysis of the putative ω-TA genes, a number of enzyme-substrate pairs were identified in a matter of hours. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function to accelerate the discovery of novel enzymes.Recent advances in genome sequencing technology have accumulated enormous amounts of sequence information (12). Although protein function encoded in nucleotide sequences can be annotated using computational alignment tools, in many cases, significant similarity to proteins with known function is hard to establish (5, 18). To understand the biological function of these unknown proteins, as well as to validate the computer-annotated results, efficient methods that enable rapid translation of genetic information into protein function are in high demand. The availability of high-throughput method for protein generation is also essential for accelerating the discovery and evolution of biocatalysts (3, 4, 6, 14, 22, 23) used in industry. While gene cloning and cultivation of transformed cells have long been used as standard methods for production of recombinant proteins, the vast amount of sequence information from various genome sequencing projects is now demanding a throughput of protein expression that exceeds that of the present in vivo expression techniques.Compared to cell-based gene expression, cell-free protein synthesis offers substantial advantages in speed and flexibility for the simultaneous expression of multiple proteins (7, 9, 13, 16, 19, 21). As a part of our efforts to extend the application of cell-free protein synthesis into the field of enzyme technology, we report in this paper an integrated methodology for fast expression screening of enzymes using ω-transaminases (ω-TAs) as a model target. Transaminases are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze reversible transfer of amine groups to keto acids, producing diverse proteogenic or nonproteogenic amino acids (1).In this work, ω-TA genes from microbial colonies were amplified by PCR and directly expressed in a cell-free protein synthesis system. Expressed enzymes were then screened for their activity toward different amine donors by colorimetric measurement of the changes in the concentration of pyruvate, which was used as a common amine acceptor. As a result, analysis of the substrate specificities of the enzymes encoded by 11 ω-TA genes toward 16 amine-donating compounds were completed within a matter of hours, identifying a number of enzyme-substrate matches.We started by examining whether sufficient amount of proteins could be generated for enzymatic analysis of ω-TAs when the PCR products amplified from the bacterial colonies were used as the template for cell-free synthesis reactions. The efficiency of protein synthesis was compared for reactions programmed with a plasmid-cloned ω-TA gene from Vibrio fluvialis (Vfω-TA) (pIVEX2.3d ω-TA Vf) and reactions programmed with the same gene prepared by two-step PCR from a bacterial colony (Vibrio fluvialis JS17 [20]), as depicted in Fig. ​Fig.1.1. The ω-TA genes examined in this study are listed in Table S1 in the supplemental material along with their bacterial sources.Open in a separate windowFIG. 1.Experimental scheme for cloning-independent cell-free expression screening of ω-transaminases. The expression templates for cell-free synthesis were prepared through two-step amplification of the target open reading frame (ORF) from bacterial genomes. PCR products were translated into corresponding enzymes in microtiter plates as described in the text. Upon completion of the synthesis reaction, the reaction mixture was sequentially supplied with the assay mixture and chromogenic compound to determine the residual pyruvate concentration after the amine transfer reaction. Abbreviations: For and Rev Primer, forward and reverse primers, respectively; 5′-UTR, 5′ UTR; T7P, T7 promoter; RBS, ribosome binding site; T7T, T7 terminator; ω-TAp, putative ω-transaminase; ω-TA Rs, ω-transaminase from Rhodobacter sphaeroides; ω-TA At, ω-transaminase from Agrobacterium tumefaciens.The templates for cell-free synthesis of ω-TA were prepared by colony PCR and subsequent second-round PCR using the MEGA primers flanking the T7 promoter, ribosome binding site, polyhistidine tag, and the T7 terminator. All of the PCRs were carried out using LA Taq DNA polymerase (Takara Bio Inc., Otsu, Japan). PCR products were directly used as the template for protein synthesis without purification. The standard cell-free reaction mixture consisted of the following components in a final volume of 50 μl: 57 mM HEPES-KOH (pH 7.5); 1.2 mM ATP; 0.85 mM (each) CTP, GTP, and UTP; 1.7 mM dithiothreitol; 0.17 mg/ml Escherichia coli total tRNA mixture (from strain MRE600); 90 mM potassium glutamate; 80 mM ammonium acetate; 12 mM magnesium acetate; 34 μg/ml l-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid); 1.5 mM (each) 20 amino acids; 2% polyethylene glycol 8000 (PEG 8000); 67 mM creatine phosphate; 3.2 μg/ml creatine kinase; 10 μM l-[U-¹⁴C]leucine (11.3 GBq/mmol); 0.5 μg/ml PCR-amplified DNA; and 14 μl of the S12 extract (11). Cell-free synthesized proteins were quantified by measuring trichloroacetic acid (TCA)-insoluble radioactivity (10), and the size and relative solubility of the synthesized protein were determined by Western blot analysis on a 12% Tricine-SDS-polyacrylamide gel (17). Mouse anti-histidine-tagged IgG antibody (Merck KGaA, Darmstadt, Germany) and rabbit anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Sigma, St. Louis, MO) were used as the primary and secondary antibodies, respectively. The PCR products served as translation substrates appropriate for producing as much protein as the corresponding plasmid-cloned gene when expressed in the reaction mixture (see Fig. S1 in the supplemental material).We next proceeded to amplify 11 ω-TA genes, including 9 putative ones from the colonies of bacterial origins (see Table S2 in the supplemental material for the list of primers used in this study), and express them in the reaction mixture prepared in two microtiter plates. Each of the 11 target genes was added to the plates by column on the plate (columns 2 through 12). Column 1 was used for negative-control reactions without any template DNAs (Fig. ​(Fig.1).1). After the PCR-amplified template DNAs were added to the plates, the plates were sealed with a plastic film to prevent evaporation and incubated at 37°C. From the measurements of TCA-insoluble radioactivity, it was estimated that 301 (±13) to 501 (±9) μg/ml of the encoded enzymes were produced after 90 min of incubation (see Fig. S2A in the supplemental material). Although most of the cell-free synthesized ω-TAs was highly insoluble in the initial experiments conducted under standard reaction conditions, the relative amounts of the soluble enzymes were markedly improved by using the GroEL/ES-enriched S12 extract (8) (Fig. S2B).Instead of the conventional high-performance liquid chromatography (HPLC) methods which have limited throughput for handling many reaction samples from different enzyme-substrate combinations, in this study, we used a simple colorimetric method for combinatorial analysis of the cell-free synthesized ω-TAs with different amine-donating substrates. Using Vfω-TA as a model ω-TA, it was first examined whether the progress of amine transfer reaction can be assayed quantitatively by colorimetric measurement of the residual pyruvate concentration. On the basis of our previous finding that Vfω-TA takes amine donors containing aryl groups as effective substrates (20), cell-free synthesized Vfω-TA was incubated with α-methylbenzylamine or benzylamine in the presence of pyruvate, and the residual pyruvate concentration in the assay mixture was determined. In brief, the amine transfer reaction was initiated by adding 50 μl of assay mixture (50 mM Tris-HCl buffer [pH 7.2], 10 mM sodium pyruvate, 10 mM [each] amine donors, and 20 μM pyridoxal-5′-phosphate) to the completed cell-free synthesis reaction mixtures (50 μl) in a 96-well plate. After 3 h of incubation at 37°C, the assay mixture was diluted with an equal volume of distilled water. When 100 μl of the diluted solution was transferred to a fresh plate containing 50 μl of 0.5 mM 2,4-dinitrophenylhydrazine (DNP), a yellow precipitate of pyruvate-dinitrophenylhydrazone (PA-DNPH) derivative was formed instantly. The absorbance at 450 nm was measured in a microplate reader (Bio-Rad, Hercules, CA) and compared with a standard curve to determine the amount of residual pyruvate in each well. Although the sensitivity of the residual pyruvate assay was as low as 0.01 mM, considering the error range, it was determined that a sensitivity of 0.1 mM should be used instead. When the optical density at 450 nm (OD₄₅₀) was measured after the addition of DNP and referred to a standard curve, the conversion yield based on the amount of residual pyruvate concentration showed good correlation with the results from standard HPLC assay (Table ​(Table11 and Fig. ​Fig.2A,2A, insets) where acetophenone or benzaldehyde generated from the corresponding substrates was separated in the Discovery HS F5 (5-μm particle size; 150- by 4.6-mm inner diameter [i.d.]; Supelco, Bellefonte, PA) column and measured at 254 nm. In addition, the relative amount of pyruvate was also able to be compared visually by adding 100 μl of 4 N NaOH solution, which turned the color of PA-DNPH to dark red (2, 15).Open in a separate windowFIG. 2.(A) Reactivity of 16 amine donors toward Vibrio fluvialis ω-TA. Reduced amounts of pyruvate concentration after the amine transfer reactions are plotted. Substrate number abbreviations: S01, α-methylbenzylamine; S02, α-ethylbenzylamine; S03, benzylamine; S04, 3-phenyl-1-propylamine; S05, phenylbutylamine; S06, 1-aminoindan; S07, ethylamine; S08, propylamine; S09, butylamine; S10, amylamine; S11, isopropylamine; S12, sec-butylamine; S13, β-alanine; S14, 3-amino-n-butyric acid; S15, phenylalanine; S16, 3-amino-3-phenylpropionic acid. The insets show HPLC traces of acetophenone and benzaldehyde after termination of transamination reaction by use of Vibrio fluvialis ω-TA against α-methylbenzylamine and benzylamine. mAU, milliabsorbance units. (B) Photo image of the assay plate after the addition of DNP and NaOH. ω-TA Vf, ω-TA from Vibrio fluvialis.

TABLE 1.

Activity comparison of Vibrio fluvialis ω-TA by HPLC and colorimetric method

Synthesis and in vitro antiproliferative evaluation of d-secooxime derivatives of 13β- and 13α-estrone

d-Secooximes were synthesized from the d-secoaldehydes in the 13β- and 13α-estrone series. The oximes were modified at three sites in the molecule: the oxime function was transformed into an oxime ether, oxime ester or nitrile group, the propenyl side-chain was saturated and the 3-benzyl ether was removed in order to obtain a phenolic hydroxy function. Triazoles were formed via Cu(I)-catalysed azide–alkyne cycloaddition (CuAAC) from 3-(prop-2-yniloxy)-d-secooximes and benzyl azides. All the products were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7, A2780 and A431). Some of them exhibited activities with submicromolar IC₅₀ values, better than that of the reference agent cisplatin. The structural modifications led to significant differences in the cytostatic properties. Flow cytometry indicated that one of the most potent agents resulted in a cell cycle blockade.     

Synthesis of lantadene analogs with marked in vitro inhibition of lung adenocarcinoma and TNF-α induced nuclear factor-kappa B (NF-κB) activation

The new series of pentacyclic triterpenoids reduced lantadene A (3), B (4), and 22β-hydroxy-3-oxo-olean-12-en-28-oic acid (5) analogs were synthesized and tested in vitro for their NF-κB and IKKβ inhibitory potencies and cytotoxicity against A549 lung cancer cells. The lead analog (11) showed sub-micromolar activity against TNF-α induced activation of NF-κB and exhibited inhibition of IKKβ in a single-digit micromolar dose. At the same time, 11 showed promising cytotoxicity against A549 lung cancer cells with IC₅₀ of 0.98 μM. The Western blot analysis further showed that the suppression of NF-κB activity by the lead analog 11 was due to the inhibition of IκBα degradation, a natural inhibitor of NF-κB. The physicochemical evaluation demonstrated that the lead analog 11 was stable in the simulated gastric fluid of pH 2, while hydrolyzed at a relatively higher rate in the human blood plasma to release the active parent moieties. Molecular docking analysis showed that 11 was hydrogen bonded with the Arg-31 and Gln-110 residues of the IKKβ.     

Synthesis of 2′-Deoxyformycin B and 2′-Deoxyoxoformycin B

Abstract

3-β-D-Ribofuranosylpyazolo[4,3-d]pyrimidines (formycins)¹ modified in the heteroaromatic moiety are of biological interest as analogues of adenosine and guanosine, and have been the objects of intensive synthetic chemical effort by several groups.^2-9 2′-Deoxynucleosides^2c,2d,7b,13 and other analogties of the formycins modified in the sugar moiety^10-12 are also of potential interest, but have been less extensively studied. Examples of the 2′-deoxyribonucleoside type known to date include the 2′-deoxy-6-thioguanosine analogue 1, the 2′-deoxyadenosine (dAdo) analogue 2 (2′-deoxyformycin A),^10,13 and the 2-chloro-2′-deoxyadenosine analogue 3.^7b Compound 2 was found to be 10-15 times more potent than 2′-deoxyadenosine as an inhibitor of the growth of S49 cells, a murine lymphoma line of T-cell origin.¹³ Activity depended on 5′- phosphorylation, since mutants lacking the enzymes adenosine kinase (AK) and deoxycytidine kinase (dCK) were insensitive to the drug. Furthermore, activity was comparable in the presence and absence of an AK inhibitor, suggesting that 2, unlike dAdo, may be a poor substrate for adenosine deaminase. That 5′-phosphorylation of 2 was mediated by AK rather than dCK was indicated by the fact that miitants lacking only dCK retained sensitivity. This contrasted with the behavior of dAdo, which is known to be n substrate for both AK and dCK.¹⁴     

Amide analogs of antifungal dioxane–triazole derivatives: Synthesis and in vitro activities

A new series of triazole compounds possessing an amide-part were efficiently synthesized and their in vitro antifungal activities were investigated. The amide analogs showed excellent in vitro activity against Candida, Cryptococcus and Aspergillus species. The MICs of compound 23d against C. albicans ATCC24433, C. neoformans TIMM1855 and A. fumigatus ATCC26430 were ?0.008, 0.031 and 0.031 μg/mL, respectively, (MICs of fluconazole: 0.5, >4 and >4 μg/mL; MICs of itraconazole: 0.125, 0.25, 0.25 μg/mL). Furthermore, compound 23d was stable under acidic conditions.     

Inheritance of B subunits of glutenin and ω-and γ-gliadins in tetraploid wheats

A double-1RS wheat-rye translocation line lacking all B subunits of glutenin was produced in durum wheat cv ‘Langdon’ for use in backcrosses and testcrosses in the study of the inheritance of low-molecular-weight (LMW) glutenin subunits in tetraploid wheats. The B subunits of glutenin and γ-and ω-gliadin bands present in parents derived from Triticum durum and T. dicoccoides, encoded by Glu-3 and Gli-1 loci, respectively, were found to be inherited mainly as units (blocks), as reported previously. Two rare recombination events between the Glu-A3 and Gli-A1 loci were detected in testcross progeny from ‘Edmore’ x T. dicoccoides landrace 19–27. Several rare recombinants were also detected within the 1BS-controlled B subunits of glutenin blocks, suggesting that there are two separate tightly linked loci (3.07±1.35 cM) within the Glu-B3 ‘locus’. Evidence was also obtained for the presence of an additional locus coding for a B subunit of glutenin in ‘Edmore’ that is loosely linked (20.9±3.18%) with the main Glu-B3 ‘locus’.     

Synthesis and evaluation of the anti-hepatitis B virus activity of 4′-Azido-thymidine analogs and 4′-Azido-2′-deoxy-5-methylcytidine analogs: structural insights for the development of a novel anti-HBV agent

Abstract

Hepatitis B virus (HBV) infection is a major worldwide health problem that requires the development of improved antiviral therapies. Here, a series of 4′-Azido-thymidine/4′-Azido-2′-deoxy-5-methylcytidine derivatives (6, 10–15) were synthesized, and their anti-HBV activities evaluated. Compounds 10–15 were synthesized via an S_NAr reaction of 18, in which the 4-position of the thymine moiety was activated as the 2,4,6-triisopropylbenzenesulfonate. Compounds 11–15 showed no antiviral activity. However, 4′-Azido thymidine (6) and 4′-Azido-2′-deoxy-5-methylcytidine (10) displayed significant anti-HBV activity (EC₅₀ = 0.63 and 5.99?µM, respectively) with no detectable cytotoxicity against MT-2 cells up to 100?µM.