Simultaneous tritium labelling of two potent 5-HT4 ligands

Two potent and selective 5-HT₄ ligands, [³H]-5-[(N-propylpiperidin-4-yl)methoxy]-1,2,3,4-tetrahydrobenzo[h][1,6] naphthyridine (1a) and [³H]-1-methyl-5-[(N-propylpiperidin-4-yl)methoxy]pyrrolo[1,2-a]thieno[2,3-e]pyrazine (2a) were radiolabelled with tritium. Radioactive labelling was achieved by simultaneous tritium reduction of a mixture of both propargylic precursors (1c–2c). The two tritiated ligands thus obtained were radiochemically pure and possessed high radioactive specific activities. These tritiated 5-HT₄ ligands will allow for binding characterization as an essential tool for their further development.     

Synthesis, characterization and antibacterial activity of 2-[1-(5-chloro-2-methoxy-phenyl)-5-methyl-1H-pyrazol-4-yl]-5-(substituted-phenyl)-[1,3,4]oxadiazoles

In the present investigation a series of novel 2-[1-(5-chloro-2-methoxy-phenyl)-5-methyl-1H-pyrazol-4-yl]-5-(substituted-phenyl)-[1,3,4]oxadiazoles (4a–j) were synthesized by cyclization of substituted-benzoic acid N′-[1-(5-chloro-2-methoxy-phenyl)-5-methyl-1H-pyrazole-4-carbonyl]-hydrazide by using phosphorousoxychloride at 120 °C. The chemical structure of the newly synthesized compounds was characterized by analytical and spectral (IR, ¹H NMR, ¹³C NMR and LC–MS) methods. The title compounds were screened for qualitative (zone of inhibition) and quantitative antibacterial activity (MIC) by agar cup plate and microtitration methods, respectively. Among the synthesized compounds in this series compound 2-[1-(5-chloro-2-methoxyphenyl)-5-methyl-1H-pyrazol-4-yl]-5-(4-fluorophenyl)-1,3,4-oxadiazole (4b) was found to exhibit significant antibacterial activity with MICs of 22.4, 29.8, 29.6 and 30.0 μg/mL against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, respectively. The other compounds exhibited moderate activity when compared to standard substance Ampicillin.     

The induction of hepatic microsomal mixed-function oxidase activities in the mouse by mirex, 3, 4, 5, 3', 4', 5'-hexachlorobiphenyl, and equimolar dosages of both

At low oral dosages (15 μmole/kg) neither mirex nor 3,4,5,3′,4′,5′-hexachlorobiphenyl was found to significantly elevate hepatic microsomal cytochrome(s) P-450 in the mouse, 7 days post-treatment. The associated drug metabolic activities, rmaminopyrine-N-demethylase, p-nitroanisole-O-demethylase, and aniline hydroxylase, were also not significantly elevated above control levels. However, aminopyrine-N-demethylase and p-nitroanisole-O-demethylase activities were significantly elevated by a 1:1 molar combination of the two compounds (“Both”), administered at a total molar concentration (15 μmole/kg) equal to that of either of the two constituents. Concomitant administration of the molar sum of Mirex and the hexachlorobiphenyl significantly elevated all three metabolic activities and cytochrome(s) P-450 concentration. The relatively greater effect of “Both” in elevating these activities may be due to the simultaneous elevation of cytochromes P-450 and P-448.     

One-pot synthesis and biological evaluation of 2-pyrrolidinyl-4-amino-5-(3',4',5'-trimethoxybenzoyl)thiazole: a unique, highly active antimicrotubule agent

A wide variety of small molecules with diverse molecular scaffolds inhibit microtubule formation. In this article we report a one-pot procedure for the preparation of a novel 2-(N-pyrrolidinyl)-4-amino-5-(3′,4′,5′-trimethoxybenzoyl)thiazole in which the size of the substituent at the C-2 position of the thiazole ring plays an essential role in compound activity. The most active agent (3f) inhibited at submicromolar concentrations the growth of tumor cell lines. It also inhibited tubulin polymerization with an activity quantitatively similar to that of CA-4, and treatment of HeLa cells resulted in their arrest at the G2-M phase of the cell cycle. Furthermore, 3f was effective against multidrug resistant cancer cells and inhibited the growth of the HT-29 xenograft in a nude mouse model. This indicated that 3f is a promising new antimitotic agent with encouraging preclinical potential.     

Synthesis and biological activity of novel N6-substituted and 2,N6-disubstituted adenine ribo- and 3'-C-methyl-ribonucleosides as antitumor agents

A series of N⁶-aminopurine-9-β-d-ribonucleosides and ribose-modified 3′-C-methyl analogues substituted at N⁶-position with a small group like hydroxy, methoxy or amino group or at C2(N⁶) position have been synthesized and tested against a panel of human leukemia and carcinoma cell lines. N⁶-Hydrazino-9-β-d-ribofuranosyl-purine (5) displayed the best antiproliferative activity in the low micromolar or submicromolar range against all tested tumor cell lines. The activity of this nucleoside is related in part to ribonucleotide reductase inhibition. C2-modification or 3′-C-methylation in N⁶-substituted adenosine analogues leads to a decrease or loss in activity.     

Synthesis and pharmacological properties of novel benzamide derivatives acting as ligands to the 5-hydroxytryptamine 4 (5-HT4) receptor

Aseries of 4-amino-5-chloro-2-methoxy-N-(1-substituted piperidin-4-ylmethyl)benzamides was synthesized as novel gastroprokinetic agents. The affinity of these compounds for the 5-hydroxytryptamine 4 (5-HT₄) receptor was evaluated. Among these compounds, 4-amino-5-chloro-2-methoxy-N-[1-[5-(1-methylindol-3-ylcarbonylamino)pentyl]piperidin-4-ylmethyl]benzamide (3f, Y-34959) showed a higher affinity for the 5-HT₄ receptor (Ki = 0.30 nmol/L) than for other receptors, and was confirmed to be a potent 5-HT₄ receptor agonist having contractile effects in the isolated guinea-pig ascending colon (EC₅₀ = 1.2 nmol/L). In dogs, compound 3f increased gastroprokinetic motility of both the gastric antrum and the ascending colon. In addition, this effect on the colon was inhibited by azasetron, a selective 5-HT₃ receptor antagonist, demonstrating that the effect of gastroprokinetic agents having 5-HT₃ receptor antagonism on the colon were reduced compared with that of selective 5-HT₄ receptor agonists.     

Synthesis and biological evaluation of unsaturated keto and exomethylene D-arabinopyranonucleoside analogs: novel 5-fluorouracil analogs that target thymidylate synthase

The synthesis of pyrimidine unsaturated keto and exomethylene arabinopyranonucleoside analogs as potential antitumor and antiviral agents is described. Commercially available 1,2,3,4-tetra-O-acetyl-d-arabinopyranose (1) was condensed with silylated thymine, uracil, 5-fluorouracil, N⁴-benzoyl cytosine and 5-(trifluoromethyl)uracil, respectively, deacetylated and acetylated to afford 1-(3,4-O-isopropylidene-α-d-arabinopyranosyl)pyrimidine analogs 4. Two different synthetic routes were investigated for the conversion of compounds 4 into the new 1-(2,3,4-trideoxy-2-methylene-α-pent-3-enopyranosyl)nucleoside derivatives of thymine (10a), uracil (10b), 5-fluorouracil (10c) and N⁴-benzoyl cytosine (10d). Only the first approach could afford derivative 10d. Debenzoylation of 10d afforded 1-(2,3,4-trideoxy-2-methylene-α-pent-3-enopyranosyl)cytosine (10f). The first approach resulted also to the 2-keto-3,4-unsaturated analogs 9. The new analogs did not show inhibition of DNA and RNA virus replication in cell culture. The 2′-ketonucleoside derivatives 9 were found to be more cytostatic than the corresponding 2′-exomethylene nucleosides 10. The 5-fluorouracil unsaturated keto derivative 9c and the exomethylene derivatives 10c and 13c showed antiproliferative activity in the lower micromolar range. Experimental evidence revealed that 9c, 10c and 13c may act as novel types of 5-fluorouracil releasing prodrugs, and points to thymidylate synthase as target for their cytostatic action.     

Synthesis, antimicrobial activity and structure-activity relationship study of N,N-dibenzyl-cyclohexane-1,2-diamine derivatives

We report herein synthesis and antimicrobial activity of a series of N,N-dibenzyl-cyclohexane-1,2-diamine derivatives. In order to study the structure-activity relationship of substituted dibenzyl-cyclohexane-1,2-diamine derivatives, 44 structurally diverse compounds were synthesized and tested against Gram-positive and Gram-negative bacterial strains. Among them, compounds 17-20, 26, 37, 38 were found to be more active than tetracycline with MIC value ranging 0.0005-0.032 μg/mL and no hemolysis upto 1024 μg/mL in mammalian erythrocytes was observed. Some of the compounds have also shown very promising antifungal activity against Candida albicans, Candida glabrata and Geotrichum candidium.     

Immunogenicity and safety of a pentavalent acellular pertussis combined vaccine including diphtheria, tetanus, inactivated poliovirus and conjugated Haemophilus Influenzae type b polysaccharide for primary vaccination at 2, 3, 4 or 3, 4, 5 months of age in infants in China

The aim was to demonstrate the immunogenicity and safety of a DTaP-IPV//PRP-T combined vaccine (Pentaxim^®) compared to individual vaccines in infants in the People's Republic of China. Infants (N = 792) were randomly assigned to receive DTaP-IPV//PRP-T at 2, 3 and 4 months of age (Group A) or 3, 4 and 5 months of age (Group B), or DTaP (Wuhan Institute of Biological Products), PRP-T (Act-Hib^®) and IPV (Imovax^® Polio) at 3, 4 and 5 months of age (Group C). Antibody titers were measured pre- and 1 month after the third vaccination; non-inferiority analyses were performed for seroprotection/seroconversion (SP/SC) rates. Safety was assessed 1 month after the primary series. SP/SC rates for the DTaP-IPV//PRP-T vaccine were high and non-inferior to the controls. Reactogenicity was low for each group and no hypotonic hyporesponsive episode or seizure was reported. In conclusion, the DTaP-IPV//PRP-T vaccine was highly immunogenic, non-inferior to the commercially available control vaccines and had a good safety profile for both primary administration schedules.     

Cytotoxic,Antiproliferative and Pro-Apoptotic Effects of 5-Hydroxyl-6,7,3′,4′,5′-Pentamethoxyflavone Isolated from Lantana ukambensis

Lantana ukambensis (Vatke) Verdc. is an African food and medicinal plant. Its red fruits are eaten and highly appreciated by the rural population. This plant was extensively used in African folk medicinal traditions to treat chronic wounds but also as anti-leishmanial or cytotoxic remedies, especially in Burkina Faso, Tanzania, Kenya, or Ethiopia. This study investigates the in vitro bioactivity of polymethoxyflavones extracted from a L. ukambensis as anti-proliferative and pro-apoptotic agents. We isolated two known polymethoxyflavones, 5,6,7,3′,4′,5′-hexamethoxyflavone (1) and 5-hydroxy-6,7,3′,4′,5′-pentamethoxyflavone (2) from the whole plant of L. ukambensis. Their chemical structures were determined by spectroscopic analysis and comparison with published data. These molecules were tested for the anti-proliferative, cytotoxic and pro-apoptotic effects on human cancer cells. Among them, 5-hydroxy-6,7,3′,4′,5′-pentamethoxyflavone (2) was selectively cytotoxic against monocytic lymphoma (U937), acute T cell leukemia (Jurkat), and chronic myelogenous leukemia (K562) cell lines, but not against peripheral blood mononuclear cells (PBMCs) from healthy donors, at all tested concentrations. Moreover, this compound exhibited significant anti-proliferative and pro-apoptotic effects against U937 acute myelogenous leukemia cells. This study highlights the anti-proliferative and pro-apoptotic effects of 5-hydroxy-6,7,3′,4′,5′-pentamethoxyflavone (2) and provides a scientific basis of traditional use of L. ukambensis.     

High intrapatient HIV-1 evolutionary rate is associated with CCR5-to-CXCR4 coreceptor switch

In approximately 70% of individuals infected with HIV-1 subtype B, the virus switches coreceptor use from exclusively CCR5 use (R5 virus) to either inclusion of or exclusively CXCR4 use (X4 virus) during infection. This switch is associated with an accelerated loss of CD4⁺ T-cells and a faster progression to AIDS. Despite intensive research, the mechanisms responsible for coreceptor switch remains elusive. In the present study, we investigated associations between viral evolutionary rate and selection pressure versus viral coreceptor use and rate of disease progression in eight patients with longitudinally sampled HIV-1 env V1–V3 sequences. By employing a Bayesian hierarchical phylogenetic model, we found that the HIV-1 evolutionary rate was more strongly associated with coreceptor switch than with rate of disease progression in terms of CD4⁺T-cell decline. Phylogenetic analyses showed that X4 variants evolved from R5 populations. In addition, coreceptor switch was associated with higher evolutionary rates on both the synonymous and non-synonymous substitution level, but not with dN/dS ratio rates. Our findings suggest that X4 viruses evolved from pre-existing R5 viral populations and that the evolution of coreceptor switch is governed by high replication rates rather than by selective pressure. Furthermore, the association of viral evolutionary rate was more strongly associated with coreceptor switch than disease progression. This adds to the understanding of the complex virus–host interplay that influences the evolutionary dynamics of HIV-1 coreceptor use.     

Synthesis, characterization and pharmacological activity of 4-{[1-substituted aminomethyl-4-arylideneamino-5-sulfanyl-4,5-dihydro-1H-1,2,4-triazol-3-yl]methyl}-2H-1,4-benzothiazin-3(4H)-ones

A new series of Schiff and Mannich bases derivatives (6) of 4-[(4-amino-5-sulfanyl-4H-1,2,4-triazol-3-yl)methyl]-2H-1,4-benzothiazin-3(4H)-one (4), derived from (3-oxo-2,3-dihydro-4H-1,4-benzothiazin-4-yl)acetic acid (3) were synthesized. The structures of all newly synthesized compounds were elucidated by elemental analysis, IR, ¹H NMR and mass spectral data. Synthesized compounds were evaluated for their anti-inflammatory and analgesic activity. Among the tested compounds, the (3-oxo-2,3-dihydro-4H-1,4-benzothiazin-4-yl)acetic acid (3) possess analgesic activity comparable to that of pentazocine; activity decreased on derivatization of the carboxylic acid group. However the anti-inflammatory activity of (3-oxo-2,3-dihydro-4H-1,4-benzothiazin-4-yl)acetic acid (3) increased by derivatization of the carboxylic acid group and some of the compounds showed anti-inflammatory activity comparable to that of indomethacin.     

Synthesis and anti-microbial evaluation of N-[5-(4-tert-amino-2-butynyl)thio-1,3,4-thiadiazol-2yl]-2-phenoxyacetamides

A series of N-[5-(4-tert-amino-2-butynyl)thio-1,3,4-thiadiazol-2-yl]-2-phenoxyacetamides have been prepared. The in vitro anti-microbial activity of these compounds against a variety of Gram-positive, Gram-negative bacteria and Candida species is reported. Some of the prepared derivatives exhibited anti-microbial activity.     

Active hexose correlated compound potentiates the antitumor effects of low-dose 5-fluorouracil through modulation of immune function in hepatoma 22 tumor-bearing mice

BACKGROUND/OBJECTIVES

A variety of immunomodulators can improve the efficacy of low-dose chemotherapeutics. Active hexose correlated compound (AHCC), a mushroom mycelia extract, has been shown to be a strong immunomodulator. Whether AHCC could enhance the antitumor effect of low-dose 5-fluorouracil (5-FU) via regulation of host immunity is unknown.

MATERIALS/METHODS

In the current study Hepatoma 22 (H22) tumor-bearing mice were treated with PBS, 5-FU (10 mg·kg^-1·d^-1, i.p), or AHCC (360 mg·kg^-1·d^-1, i.g) plus 5-FU, respectively, for 5 d. CD3⁺, CD4⁺, CD8⁺, and NK in peripheral blood were detected by flow cytometry. ALT, AST, BUN, and Cr levels were measured by biochemical assay. IL-2 and TNFα in serum were measured using the RIA kit and apoptosis of tumor was detected by TUNEL staining. Bax, Bcl-2, and TS protein levels were measured by immunohistochemical staining and mRNA level was evaluated by RT-PCR.

RESULTS

Diet consumption and body weight showed that AHCC had no apparent toxicity. AHCC could reverse liver injury and myelosuppression induced by 5-FU (P < 0.05). Compared to mice treated with 5-FU, mice treated with AHCC plus 5-FU had higher thymus index, percentages of CD3⁺, CD4⁺, and NK cells (P < 0.01), and ratio of CD4⁺/CD8⁺ (P < 0.01) in peripheral blood. Radioimmunoassay showed that mice treated with AHCC plus 5-FU had the highest serum levels of IL-2 and TNFα compared with the vehicle group and 5-FU group. More importantly, the combination of AHCC and 5-FU produced a more potent antitumor effect (P < 0.05) and caused more severe apoptosis in tumor tissue (P < 0.05) compared with the 5-FU group. In addition, the combination of AHCC and 5-FU further up-regulated the expression of Bcl-2 associated X protein (Bax) (P < 0.01), while it down-regulated the expression of B cell lymphoma 2 (Bcl-2) (P < 0.01).

CONCLUSIONS

These results support the claim that AHCC might be beneficial for cancer patients receiving chemotherapy.     

Safety,reactogenicity and immunogenicity of Francisella tularensis live vaccine strain in humans

We evaluated the safety, reactogenicity and immunogenicity of escalating doses of a new Francisella tularensis Live Vaccine Strain (LVS) lot by scarification (SCAR) or subcutaneously (SQ) in humans. Subjects (N = 10/group) received one dose of LVS via SCAR at 10⁵,10⁷ or 10⁹ cfu/ml or SQ at 10², 10³,10⁴ or 10⁵ cfu/ml; 14 subjects received placebo. All doses/routes were well tolerated. When compared to placebo, vaccination with 10⁷ SCAR and 10⁹ SCAR resulted in significantly higher serologic response frequencies, as measured by ELISA for IgG, IgM, IgA and microagglutination; whereas vaccination with 10⁵ SCAR, 10⁷ SCAR 10⁹ SCAR and 10⁵ SQ elicited a significantly higher interferon-γ response frequency.     

Stability and efficacy of the 3′-UTR A4G-G5A variant of viral hemorrhagic septicemia virus (VHSV) as a live attenuated immersion VHSV vaccine in olive flounder (Paralichthys olivaceus)

Viral hemorrhagic septicemia virus (VHSV) is the causative agent of viral hemorrhagic septicemia in fish, a disease that affects a number of teleost fish species including olive flounder (Paralichthys olivaceus). In this study, we assessed the safety and efficacy of two recombinant attenuated VHSV strains, termed A4G-G5A and ΔNV, with the purpose to select the most suitable vaccine strain. The virus strains were passaged in two commercially available cell lines, EPC and RTG-2, and the strains were also tested for residual virulence in zebrafish (Danio rerio). The A4G-G5A strain showed an attenuated growth profile in both the EPC and RTG-2 cell lines compared to wild-type (WT) VHSV (JF-09, genotype IVa), whereas the growth profile of ΔNV was comparable to the WT strains in RTG-2 cells in contrast to EPC cells. Moreover, ΔNV had higher residual virulence compared to A4G-G5A and was highly pathogenic to zebrafish. The A4G-G5A strain was chosen as vaccine candidate and tested for efficacy in in vivo fish studies in the target species, olive flounder, using an immersion vaccine scheme. Groups of fish were immunized with 10^2.5, 10^3.5, 10^4.5, and 10^5.5 TCID₅₀/ml of A4G-G5A giving 5–13.3 cumulative percent mortality (CPM) post immunization. Immunization was followed by a challenge experiment using VHSV-WT. The relative percent survival (RPS) in immunized groups ranged from 81.6% to 100%, correlating with vaccination dose. This study demonstrates that while strain A4G-G5A has retained some residual virulence it confers high level of protection in immunized olive flounder.     

Synthesis and studies of novel 2-(4-cyano-3-trifluoromethylphenyl amino)-4-(quinoline-4-yloxy)-6-(piperazinyl/piperidinyl)-s-triazines as potential antimicrobial, antimycobacterial and anticancer agents

A series of novel s-triazine analogs were synthesized and characterized by IR, ¹H NMR, ¹³C NMR, ¹⁹F NMR spectroscopy and elemental analysis. Preliminary screening of target compounds against eight bacteria (Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris, Shigella flexneria), four fungi (Aspergillus niger, Aspergillus fumigatus, Aspergillus clavatus, Candida albicans) and Mycobacterium tuberculosis H37Rv indicated that 5d, 5h, 5n, 5p, 5q, 5r, 5s, 5t and 5u were the most active compounds among twenty one studied. Thus, they were further subjected to in vitro biological evaluation against human prostate cancer cell line (DU-145) and the results indicate that two compounds 5n and 5s were markedly active.     

Synthesis, antitumor and antimicrobial activities of 4-(4-chlorophenyl)-3-cyano-2-(β-O-glycosyloxy)-6-(thien-2-yl)-nicotinonitrile

4-(4-Chlorophenyl)-3-cyano-6-(thien-2-yl)-1H-pyridin-2-one (2) was obtained by reaction of 2-acetyl thiophene with 4-chlorobenzaldehyde and ethyl cyanoacetate in presence of ammonium acetate or by the reaction of α,β-unsaturated compound 1 with ethyl cyanoacetate in the presence of ammonium acetate. 4-(4-Chlorophenyl)-2-(2′,3′,4′,6′-tetra-O-acetyl-β-d-gluco/galactopyranosyloxy)-6-(thien-2-yl)nicotinonitrile (5a and 5b), riboside 11, xyloside 12 and lactoside 16 were prepared by the reaction of 2 with glycosyl/galactosyl/xylosyl/lactosyl bromide and peracetylated xylose/ribose under the conventional and microwave irradiation methods. The reaction has regioselectively gave the O-glycosides and not the N-glycosides. The glycosides 5a,b, riboside 11, xyloside 12 and lactoside 16 were deacetylated in the presence of Et₃N/MeOH and few drops of water to give 7a,b, 13, 14 and 17. The structure of the new synthesized compounds was confirmed by using IR, ¹H, ¹³C NMR spectra and microanalysis. Selected members of these compounds were screened for antitumor and antibacterial activity.     

Synthesis, antidepressant and antifungal evaluation of novel 2-chloro-8-methylquinoline amine derivatives

A new series of N-[(2-chloro-8-methylquinolin-3-yl)methyl]-(substituted)-aniline/butylamine/cyclohexylamine/benzylamine derivatives (4a-p) was synthesized by nucleophilic substitution reaction of 2-chloro-3-(chloromethyl)-8-methylquinoline 3 with various aliphatic and aromatic amines in absolute ethanol in the presence of triethylamine (TEA). The newly synthesized secondary amines were characterized by the combined use of IR, ¹H NMR, ¹³C NMR, mass spectral data and microanalyses. The antidepressant activity of the synthesized compounds (4a-p) was evaluated by Forced swim test in rats and their neurotoxicity was evaluated by the rotarod test. Test compounds and clomipramine were administered intraperitoneally at dose of 100 mg/kg and 20 mg/kg respectively. Preliminary antidepressant screening of compounds (4a-p) revealed that compounds 4b, 4c, 4d, 4e, 4i and 4o significantly (P < 0.01) reduces the duration of immobility time. These compounds were also tested in-vitro for MAO inhibitory effect. All the compounds were also screened for antifungal activity against Aspergillus niger MTCC 281, Aspergillus flavus MTCC 277, Monascus purpureus MTCC 369 and Penicillium citrinum NCIM 768 strains.