Synthesis and biological evaluation of 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide stereoisomers as novel positive allosteric modulators of sigma-1 receptor

Novel positive allosteric modulators of sigma-1 receptor represented by 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide enantiomers were synthesised using an asymmetric Michael addition of 2-nitroprop-1-enylbenzene to diethyl malonate. Following the chromatographic separation of the methyl erythro- and threo-4-nitro-3R- and 3S-phenylpentanoate diastereoisomers, target compounds were obtained by their reductive cyclisation into 5-methyl-4-phenylpyrrolidin-2-one enantiomers and the attachment of the acetamide group to the heterocyclic nitrogen. Experiments with electrically stimulated rat vas deference contractions induced by the PRE-084, an agonist of sigma-1 receptor, showed that (4R,5S)- and (4R,5R)-2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamides with an R-configuration at the C-4 chiral centre in the 2-pyrrolidone ring were more effective positive allosteric modulators of sigma-1 receptor than were their optical antipodes.     

Synthesis and Herbicidal Activities of 1,2-Benzisoxazole-3-acetamide Derivatives

Many 1,2-benzisoxazole-3-acetamides were synthesized and their herbicidal activities in the paddy field were studied. Of the compounds tested, N-α,α-dimethylbenzyl-2-bromo-(1,2-benzisoxazol-3-yl)acetamide 10a was the most effective. Details of the synthesis and the results of herbicidal evaluations are given.     

Synthesis and biological evaluation of 5-benzylidenerhodanine-3-acetic acid derivatives as AChE and 15-LOX inhibitors

Abstract

A series of 5-benzylidenerhodanine-3-acetamides bearing morpholino-, 4-arylpiperazinyl-, or 4-benzylpiperidinyl- moieties were synthesized and their inhibitory activities against acetylcholinesterase (AChE) were evaluated. Alteration of amide part and substitution on the benzylidene moiety resulted in change of anti-AChE activity. The most active compound was the 1-benzylpiperidinyl derivative containing 4-(dimethylamino)benzylidene scaffold. Notably, the intermediate compounds, namely 5-arylidene-rhodanine-3-acetic acids (3), showed mild inhibitory activity against 15-lipoxygenase (15-LOX), while the final compound 4 showed no activity against 15-LOX.     

Synthesis and stereochemistry of 7β- and 7α-amino-, acetamido-, hemisuccinamido- and terephthalamido derivatives of testosterone

The reduction of 3-ethylenedioxy-7-oximino-5-androsten-17β-yl acetate and of its 17β-tetrahydropyranyl ether analog with sodium in ethanol, followed by thin-layer chromatography, allowed the isolation of the corresponding 17β-hydroxy- and 17β-tetrahydropyranyioxy-5-en-7β- and 7α-amines which were also characte-rized as 7-acetamides. The acylation of the two epimeric 17β-hydroxy-5-en-7-amines with succinic anhydride followed by selective saponification of the 17β-hemisuccinate group and diazomethane esterification, gave the corresponding 17β-hydroxy-5-en-7β- and 7α-hemisuccinamido methyl esters characterized also as 17β-acetates. On the other hand, the acylation of the two 17β-tetrahydropyranyl-oxy-5-en-7-amines with the acid chloride of terephthalic acid monomethyi ester led to the more rigid 7β- and 7α-terephthalamido methyl ester side-chains. The acidolysis of the 3-ethyleneketal protecting group of the preceding 5-en-7-N-acyl derivatives regenerated the 4-en-3-oxo function while the 17β-tetrahydropyranyl ether group was cleaved simultaneously into the 17β-alcohol. The four desired 7β- and 7α-hemisuccinamido- and terephthalamido carboxylic side-chain derivatives of 17β-hydroxy-4-androsten-3-one (testosterone) were finally obtained by saponification of the corresponding methyl esters.     

Theophylline-7-acetic acid derivatives with amino acids as anti-tuberculosis agents

A series of amides were synthesized by condensation of theophylline-7-acetic acid and eight commercially available amino acid methyl ester hydrochlorides. Consecutive hydrolysis of six of the amido-esters resulted in the formation of corresponding amido-acids. The newly synthesized compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv. The activity varied depending on the amino acid fragments and in seven cases exerted excellent values with MICs 0.46–0.26 μM. Assessment of the cytotoxicity revealed that the compounds were not cytotoxic against the human embryonal kidney cell line HEK-293T. The theophylline-7-acetamides containing amino acid moieties appear to be promising lead compounds for the development of antimycobacterial agents.     

Structural Basis for Resistance of the Genotype 2b Hepatitis C Virus NS5B Polymerase to Site A Non-Nucleoside Inhibitors

Hepatitis C virus (HCV) exists in six major genotypes. Compared with the 1b enzyme, genotype 2b HCV polymerase exhibits a more than 100-fold reduction in sensitivity to the indole-N-acetamide class of non-nucleoside inhibitors. These compounds have been shown to bind in a pocket occupied by helix A of the mobile Λ1 loop in the apoenzyme. The three-dimensional structure of the HCV polymerase from genotype 2b was determined to 1.9-Å resolution and compared with the genotype 1b enzyme. This structural analysis suggests that genotypic variants result in a different shape of the inhibitor binding site. Mutants of the inhibitor binding pocket were generated in a 1b enzyme and evaluated for their binding affinity and sensitivity to inhibition by indole-N-acetamides. Most of the point mutants showed little variation in activity and IC₅₀, with the exception of 15- and 7-fold increases in IC₅₀ for Leu392Ile and Val494Ala mutants (1b→2b), respectively. Furthermore, a 1b replicon with 20-fold resistance to this class of inhibitors was selected and shown to contain the Leu392Ile mutation. Chimeric enzymes, where the 2b fingertip Λ1 loop, pocket or both replaced the corresponding regions of the 1b enzyme, were also generated. The fingertip chimera retained 1b-like inhibitor binding affinity, whereas the other two chimeric constructs and the 2b enzyme displayed between 50- and 100-fold reduction in binding affinity. Together, these data suggest that differences in the amino acid composition and shape of the indole-N-acetamide binding pocket are responsible for the resistance of the 2b polymerase to this class of inhibitors.     

VARIATION IN APPLE CYTOCHIMERAS

Diploid-tetraploid cytochimeras of apple were investigated by cytological examination of buds from branches selected by the characteristics of the fruits, flowers or pollen. Eight types of cytochimeras were identified on the basis of relative size of cells, nuclei and metaphase mitoses in the apical meristem (protomeristem) and mitoses in meristematic primary tissues. There appeared to be five apical layers which contributed to the stem and four which contributed to the leaves and flower parts. Buds of various cytochimeral patterns (designated by a formula giving the ploidy of the apical layers) were found on some bearing trees propagated from known cytochimeral sources. The most frequently associated types were (a) 2–4–2–2–2 and 2–4–4–4–4 (and 2–4–2–4–4 in one cultivar), or (b) 2–2–2–4–4 and 2x. Some sports were uniformly 2–4–4–4–4. The stability of apple cytochimeras under normal conditions appeared greater in some eultivars than in others. Sprouts from severely pruned 2–2–4–4–4 trees were more variable than unpruned branches. Buds of shoots which grew from radiation-damaged buds were more variable than those from non-irradiated buds and included types not yet found by branch selection. Cytochimeral variation was interpreted to be due to layer replacement resulting from infrequent periclinal divisions in apical or axillary meristems, or from wounding of meristems by ionizing radiation.     

Metabolism of exogenous 4- and 2-hydroxyestradiol in the human male

The metabolic fate of the isomeric catecholestrogens 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) was studied to elucidate possible differences in their metabolism as an explanation for their different bioactivities. Healthy young men (n = 3 each) were infused (90 min) with 4-OHE2 (60 micrograms/h) or 2-OHE2 (100 micrograms/h). The main metabolites were determined in plasma and urine before, during and after infusion. Unconjugated and conjugated steroids, the latter after hot acid hydrolysis, were subjected to chromatography on LH-20 columns and measured by specific RIAs. During the infusion 4-OHE2 reached significant plasma concentrations whereas 2-OHE2 was so rapidly metabolised that its plasma levels remained virtually undetectable in spite of a higher infusion rate. The metabolism of 4-OHE2 was dominated by direct conjugation, that of 2-OHE2 by methyl ether formation. These findings were corroborated by the urinary excretion rates: during the infusion and the first hours afterwards, 4-OHE2 was mainly excreted as 4-OHE2 and 4-hydroxyestrone, while 2-OHE2 was predominantly excreted as 2-hydroxyestradiol 2-methyl ether and 2-hydroxyestrone 2-methyl ether.     

Optimization of medium compositions favoring butanol and 1,3-propanediol production from glycerol by Clostridium pasteurianum

Medium compositions favoring butanol and 1,3-propanediol (1,3-PDO) production from glycerol by Clostridium pasteurianum DSM525 were investigated using statistical experimental designs. Medium components affecting butanol and 1,3-PDO production were screened using a fractional factorial experimental design. Among the six tested variables (phosphate buffer, MnSO4·H2O, MgSO4·7H2O, FeSO4·7H2O, (NH4)2SO4, and yeast extract), FeSO4·7H2O, (NH4)2SO4, and yeast extract were found to be significant variables for further optimization of medium using a Box-Behnken design. Optimal butanol (0.98 g/L/h) and 1,3-PDO (1.19 g/L/h) productivities were predicted by the corresponding quadratic model for each product and the models were validated experimentally under optimized conditions. The optimal medium composition for butanol production was significantly different from that for 1,3-PDO production (0.06 vs. 0 g/L for FeSO4·7H2O, 7.35 vs. 0 g/L for (NH4)2SO4, and 5.08 vs. 8.0 g/L for yeast extract), suggesting that the product formation from glycerol by C. pasteurianum DSM525 can be controlled by changing medium compositions.     

Syntheses and spectroscopic characterization of some phosphoramidates as reversible inhibitors of human acetylcholinesterase and determination of their potency

The ability of phosphoramidates Me2NP(O)(Cl)(p-NHC6H4NO2) 1, Me2NP(O)(p-NHC6H4NO2)2 2, (CH3C6H4O-p)P(O)(p-NHC6H4NO2)2 3 and (CH3C6H40-p)2P(O)(p-NHC6H4NO2) 4 to inhibit human acetylcholinesterase (hAChE) has been evaluated by a modified Ellman's method and spectrophotometric measurements. Results showed that compounds 1 and 2 do not have any inhibitory potency, whereas compounds 3 and 4 were reversible mixed inhibitors. The IC50 values for inhibitors 3 and 4 were 0.143 and 0.581 mM, respectively. The previously unknown compounds 3 and 4 were synthesized and characterized by 1H, 13C, 31P NMR and IR spectroscopy and elemental analysis.     

Studies on some specific Ap4A-degrading enzymes with the use of various methylene analogues of P1P4-bis-(5'',5''''''-adenosyl) tetraphosphate.

Six new methylenephosphonate analogues of P1P4-bis-(5',5'-adenosyl) tetraphosphate, Ap4A, having P2-P3 carbon bridges CF2, CCl2 and CH2CH2 or P1-P2 and P3-P4 carbon bridges CF2, CCl2 and CH2CH2 in the tetraphosphate chain, were examined as substrates or inhibitors for two specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow-lupin seeds and (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from Escherichia coli. All analogues in which the central oxygen atom was replaced by a stable carbon bridge were hydrolysed by the asymmetrical hydrolase (CF2 greater than CCl2 greater than O greater than CHBr greater than CH2 greater than CH2CH2). As expected, these analogues were not hydrolysed by the symmetrical hydrolase, which was also unable to act on analogues having P1-P2 and P3-P4 carbon bridges.     

(4E)-dehydrocitrals [(2E,4E)- and (2Z,4E )-3,7-dimethyl-2,4,6-octatrienals] from acarid mite Histiogaster sp. A096 (Acari: Acaridae)

A mixture of two monoterpenes was obtained as the opisthonotal gland secretion from unidentified Histiogaster sp. A096 (Acari: Acaridae), and their structures were elucidated to be (4E)-dehydrocitrals [(2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienals] by GC/MS, GC/FT-IR, UV and 1H-NMR spectra. Both isomers of (4E)-dehydrocitral prepared by syntheses in 4 steps from 3-methyl-2-butenal with 34.2% yields (based on the ylide) were separated by column chromatography into the (2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienal. Mass spectra together with GC retention times of the purified natural (4E)-dehydrocitrals were identical with those of synthetic (2E,4E)-3,7-dimethyl-2,4,6-octatrienal and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienal. The geometry at the 2-C position of both synthetic (4E)-dehydrocitrals was confirmed by NOESY analyses. This is the first identification of (4E)-dehydrocitrals from the animal kingdom.     

柯拉斯那沉香中2-(2-苯乙基)色酮类化学成分研究

为研究柯拉斯那(Aquilaria crassna Pierre ex Lecomte)沉香的化学成分。实验采用多种柱色谱方法从该沉香中分离得到9个2-(2-苯乙基)色酮类化合物,通过现代波谱学技术分别鉴定为6-甲氧基-2-[2-(3′-羟基-4′-甲氧基苯基)乙基]色酮(1)、5-羟基-6-甲氧基-2-[2-(3′-羟基-4′-甲氧基苯基)乙基]色酮(2)、tetrahydrochromone F(3)、6-甲氧基-2-[2-(3′-甲氧基-4′-羟基苯基)乙基]色酮(4)、6-甲氧基-7-羟基-2-[2-(4′-甲氧基苯基)乙基]色酮(5)、6,7-二甲氧基-2-[2-(3′-羟基-4′-甲氧基苯基)乙基]色酮(6)、6,7-二甲氧基-2-[2-(4′-甲氧基苯基)乙基]色酮(7)、6-羟基-2-[2-(4′-羟基苯基)乙基]色酮(8)、5-羟基-2-[2-(2′-羟基苯基)乙基]色酮(9)。化合物2、3和5～9均为首次从柯拉斯那所得沉香中分离得到。采用MTT法对单体化合物的细胞毒活性进行测试,测试结果表明,化合物1,2和4具有微弱的细胞毒活性。     

Effect of LTB4 on the inhibition of natural cytotoxic activity by PGE2.

NK activity is regulated by arachidonic acid metabolites. More precisely PGE2 and LTB4 decreases and increases respectively non-MHC-restricted cytotoxicity in humans. We have observed similar data in mice since NK activity was inhibited by PGE2 (10(-6) to 10(-8) M) and enhanced by LTB4 (10(-8) to 10(-12) M). On the other hand when PGE2 and LTB4 were combined during the same assay the lysis percentage was smaller than the one which was induced by PGE2 alone. Because PGE2 increases intracellular cyclic AMP and that LTB4 augments cyclic GMP we used a cAMP inducer (forskolin) and a cGMP analogue (8 Br-cGMP) instead of eicosanoids and we observed similar data (i.e., a decrease of natural killing) as when PGE2 was combined with LTB4. When splenocytes are cultured for 1-4 days alone, cytotoxic activity decreases unless they are cultured in the presence of indomethacin. Cytotoxic activity of spleen cells cultured in the presence of PGE2 or LTB4 is respectively decreased or increased. However, splenocytes that were cultured alone for at least 24 hr were no longer sensitive to inhibition by PGE2 but were still PGE2-sensitive when cultured in the presence of LTB4.     

Stimulation of RNA polymerases I and II from mouse L1210 leukemia and Ehrlich ascites carcinoma cells by spermine and spermidine and its modification by ammonium sulfate

Nuclear RNA polymerases from murine L1210 leukemia and Ehrlich carcinoma cells were stimulated more effectively by spermine than by spermidine. Optimal stimulatory concentrations of spermine and spermidine for Ehrlich polymerases Ia and Ib decreased to physiological values and maximal stimulation increased as the concentration of (NH4)2SO4 was reduced from 0.08 to 0 M. In the presence of 0.062-0.074 M (NH4)2SO4 L1210 polymerases Ia, IIa and IIb were stimulated significantly by both polyamines, whereas, at (NH4)2SO4 concentrations of 0.11-0.17 M, stimulation was suppressed and high concentrations of the polyamines were inhibitory. Similarly, stimulation of Ehrlich solubilized polymerase by polyamines was inhibited by 0.064 M (NH4)2SO4.     

Biotransformation of 4-chloro-2-nitrophenol into 5-chloro-2-methylbenzoxazole by a marine Bacillus sp. strain MW-1

Decolourization, detoxification and biotransformation of 4-chloro-2-nitrophenol (4C2NP) by Bacillus sp. strain MW-1 were studied. This strain decolorized 4C2NP only in the presence of an additional carbon source. On the basis of thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), 4-chloro-2-aminophenol, 4-chloro-2-acetaminophenol and 5-chloro-2-methylbenzoxazole were identified as metabolites. Resting cells depleted 4C2NP with stoichiometric formation of 5-chloro-2-methyl benzoxazole. This is the first report of the formation of 5-chloro-2-methylbenzoxazole from 4C2NP by any bacterial strain.     

Comparison of several ELISA tests for detecting the presence of IgG and IgM against herpes simplex viruses

Four enzyme-linked immunosorbent assays designated test 1 (ETI-HSVK-G 1/2); test 2 (ETI-HSVK-M 1/2); test 3 (ETI-HSVK-G 2), and test 4 (BioElisa HSV2 IgG) were studied to evaluate different stages of herpes simplex virus (HSV) infection. Samples (50 sera and 14 cerebrospinal fluid) were included in four groups. Group 1 consisted of samples from patients with primary HSV infections; group 2 comprised samples from patients with recurrent HSV infections; group 3 were samples nonreactive to HSV; and group 4 were samples from patients with infections by other herpes viruses (4a, chickenpox; 4b, herpes zoster; and 4c, infectious mononucleosis by Epstein-Barr virus). The percentages of agreement between tests 1 and 2 were 100 and 72.1%, respectively. The total diagnostic values of tests 1 and 2 were: 100 and 50% sensitivity, respectively; and 100 and 89% specificity, respectively. Few positive results for HSV-2 infection were found, and so, tests 3 and 4 were not evaluated. The results of tests 3 and 4 for a chickenpox patient, and a herpes zoster patient were not in agreement.     

Dimers of beta 2-glycoprotein I mimic the in vitro effects of beta 2-glycoprotein I-anti-beta 2-glycoprotein I antibody complexes

Anti-beta(2)-glycoprotein I antibodies are thought to cause lupus anticoagulant activity by forming bivalent complexes with beta(2)-glycoprotein I (beta(2)GPI). To test this hypothesis, chimeric fusion proteins were constructed of the dimerization domain (apple 4) of factor XI and beta(2)GPI. Both a covalent (apple 4-beta(2)GPI) and a noncovalent (apple 4-C321S-beta(2)GPI) chimer were constructed. As controls, apple 2-beta(2)GPI and apple 4-C321S-beta(2)GPI-W316S, in which beta(2)GPI-W316S is not able to bind to phospholipids, were made. In a phospholipid binding assay, apple 4-beta(2)GPI and apple 4-C321S-beta(2)GPI were able to bind to phospholipids with an affinity 35 times higher than that of plasma-derived beta(2)GPI and apple 2-beta(2)GPI. Apple 4-C321S-beta(2)GPI-W316S did not bind at all. Only apple 4-beta(2)GPI and apple 4-C321S-beta(2)GPI were able to bind to adhered platelets as shown by immunofluorescence. Using the prothrombin time, which was the most responsive coagulation assay, the clotting time was approximately doubled when 200 microg/ml apple 4-beta(2)GPI or apple 4-C321S-beta(2)GPI was added. Addition of 200 microg/ml plasma-derived beta(2)GPI, apple 2-beta(2)GPI, or apple 4-C321S-beta(2)GPI-W316S did not affect clotting time. Clotting time could be corrected with the addition of extra phospholipids, which is indicative for lupus anticoagulant activity. An additional increase in clotting times for apple 4-beta(2)GPI or apple 4-C321S-beta(2)GPI was achieved by the addition of monoclonal antibodies against beta(2)GPI. In conclusion, dimerization of beta(2)GPI explains the in vitro observed effects of beta(2)GPI-anti-beta(2)GPI antibody complexes.     

Study on synthesis,characterization and biological activity of some new nitrogen heterocycle porphyrins

Seven new nitrogen heterocycle porphyrins, 5,10,15,20-tetra[4-(N-pyrrolidinyl)phenyl]porphine (TBPPH(2)), 5,10,15,20-tetra[4-(4'-ethylpiperazinyl)phenyl]porphine (TEPPH(2)), 5,10,15,20-tetra [4-(4'-butylpiperazinyl)phenyl]porphine (TUPPH(2)), 5,10,15,20-tetra[4-(4'-heptylpiperazinyl) phenyl]porphine (THPPH(2)), 5-[4-(4'-ethylpiperazinyl)phenyl]-10,15,20-triphenylporphine (MEPPH(2)), 5-[4-(4'-buthylpiperazinyl)phenyl]-10,15,20-triphenylporphine (MUPPH(2)) and piperazine bridge porphine dimer N,N'-di(5,10,15,20-tetraphenylporphinato)piperazine (DiPPH(2)) have been synthesized by the direct condensation of nitrogen heterocycle substituted benzaldehydes with pyrrole. Each porphine bears one or four substituted pyrrolidine or piperazine moieties that have been used as drugs. Their structures were characterized by elementary analysis, MS, 1H NMR, IR and UV-vis. These nitrogen heterocycle porphyrins aggregates in water and THF solution were studied by the spectrophotofluorimetry. The anticancer activity of these porphines for the liver cancer cells, the stomach tumor cells and the nasopharyngeal carcinoma cancer cells were tested by the MTT assay. Compared with cis-platinum (cis-Pt) and 5-Fluorouracil (5-Fu), the nitrogen heterocycle porphyrins have the better biological activity and might have potential application in medicine.     

3,4-Disubstituted oxazolidin-2-ones as constrained ceramide analogs with anticancer activities

Heterocyclic analogs of ceramide as 3-alkanoyl or benzoyl-4-(1-hydroxy-2-enyl)-oxazolidin-2-ones were designed by binding of primary alcohol and amide in sphinogosine backbone as a carbamate. They were synthesized by addition of acyl halide to the common ring 4-(1-t-butyldimethylsilyloxyhexadec-2-enyl)-oxazolidin-2-one which was elaborated from chiral aziridine-2-carboxylate including stereoselective reduction and ring opening reactions as key steps. Other analogs with different carbon frame at C4 position which is corresponding to the sphingoid backbone were prepared from 3-cyclopentanecarbonyl-4-(1-t-butyldimethylsilyloxybut-2-enyl)-oxazolidin-2-one and straight and cyclic alkenes by cross metathesis. All compounds were tested as antileukemic drugs against human leukemia HL-60 cells. Many of them including propionyl, cyclopentanoyl and p-nitrobenzoyl-4-(1-hydroxyhexadec-2-enyl)-oxazolidin-2-ones showed better antileukemic activities than natural C2-ceramide with good correlation between cell death and DNA fragmentation. There is a drastic change of the activities by the carbon chain lengths at C4 position. Cytotoxicity was induced by caspase activation without significant accumulation of endogenous ceramide concentration or any perturbation of ceramide metabolism.