High-performance liquid chromatography of 2,4-disubstituted-5(4H)-oxazolones, anhydrides and other activated forms of N-acyl- and N-alkoxycarbonylamino acids

Normal phase high-performance liquid chromatography has been achieved of the common activated forms of valine on a LiChrosorb-CN or a muPorasil (underivatized silica) column using hexane containing 1.5 or 5% tert.-butanol as solvent. Compounds examined include the 2-alkoxy-5(4H)-oxazolones and symmetrical and mixed anhydrides of N-tert.-butoxycarbonyl-, N-benzyloxycarbonyl-, and N-9-fluorenylmethoxycarbonylvaline, the chloride of the latter, a p-nitrophenyl ester, 2-methyl-4-isopropyl-5(4H)-oxazolone, valine-N-carboxyanhydride, and the N- and O-ethoxycarbonyl adducts of l-hydroxybenzotriazole. The 5(4H)-oxazolones from N-tert.-butoxycarbonylvaline and N-benzyloxycarbonylglycylvaline decomposed during chromatography on the muPorasil but not the LiChrosorb-CN column. The method allows direct monitoring of reactions involving generation or consumption of activated forms of amino acids.     

Stereoselective acylation of a racemic amine with C(alpha)-methyl phenylglycine-based dipeptide 5(4H)-oxazolones

Reactions of a racemic amine with chiral, N(alpha)-acetylated, C(alpha)-methyl l-phenylglycine-based dipeptide 5(4H)-oxazolones proceed diastereoselectively to give predominantly dipeptide alkylamides comprising d-alpha-phenylethylamine. Diastereoselectivity is remarkably sensitive to solvent polarity and reaction temperature but not significantly to the nature of the C(alpha)-tetrasubstituted alpha-amino acid at position 1 of the dipeptide. The beta-turn 3D structures of the aminolysis products were established in CDCl(3) solution by FT-IR absorption and in one case in the crystal state by X-ray diffraction as well.     

Synthesis of 3-alkyl(aryl)-4-alkylidenamino-4,5-dihydro-1H-1,2,4-triazol-5-ones and 3-alkyl-4-alkylamino-4,5-dihydro-1H-1,2,4-triazol-5-ones as antitumor agents

A series of 3-alkyl-4-phenylethylidenamino- (8) and 3-alkyl-4-(3-phenylallylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (9) was synthesized from the reaction of the corresponding 3-alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones (1), with phenylacetaldehyde and cinnamaldehyde. 3-Alkyl-4-(2-phenylethylamino)- (10) and 3-alkyl-4-(3-phenylpropylamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (11) were obtained from the selective reduction of compounds (8) and (9) with NaBH₄. The in vitro antitumor activity of the novel compounds was screened and the highest inhibition of tree tumor cell lines was observed for the compounds containing phenylethylenamino and phenylethylamino groups at position 4 of 1,2,4-triazol ring.     

Unsaturated 4,4′-bis-[5(4H)-oxazolones]: Synthesis and evaluation of their ortho-palladation through C-H bond activation

Aromatic unsaturated 4,4′-bis-[5(4H)-oxazolones] have been prepared and their structural properties discussed. Metallation studies of these substrates towards palladium(II) acetate were also investigated.     

Novel side reactions accompanying activation and aminolysis of N-benzoyl-2-alkylserines

2-Phenyl-4-alkyl-4-hydroxymethyl-1,3-oxazolones (2a-d) have been identified as side products accompanying activation of N-benzoyl-2-alkylserines (1a-d). Oxazolones 2a-d in the presence of amine rearrange subsequently to corresponding 4-alkyl-2-phenyl-4,5-dihydro-1,3-oxazole-5 carboxylic acids (4a-d) at a 20-68% yield.     

Molecular cloning of two genes encoding cinnamate 4-hydroxylase (C4H) from oilseed rape (Brassica napus)

Cinnamate 4-hydroxylase (C4H) is a key enzyme of phenylpropanoid pathway, which synthesizes numerous secondary metabolites to participate in development and adaption. Two C4H isoforms, the 2192-bp BnC4H-1 and 2108-bp BnC4H-2, were cloned from oilseed rape (Brassica napus). They both have two introns and a 1518-bp open reading frame encoding a 505-amino-acid polypeptide. BnC4H-1 is 57.73 kDa with an isoelectric point of 9.11, while 57.75 kDa and 9.13 for BnC4H-2. They share only 80.6% identities on nucleotide level but 96.6% identities and 98.4% positives on protein level. Showing highest homologies to Arabidopsis thaliana C4H, they possess a conserved p450 domain and all P450-featured motifs, and are identical to typical C4Hs at substrate-recognition sites and active site residues. They are most probably associated with endoplasmic reticulum by one or both of the N- and C-terminal transmembrane helices. Phosphorylation may be a necessary post-translational modification. Their secondary structures are dominated by alpha helices and random coils. Most helices locate in the central region, while extended strands mainly distribute before and after this region. Southern blot indicated about 9 or more C4H paralogs in B. napus. In hypocotyl, cotyledon, stem, flower, bud, young- and middle-stage seed, they are co-dominantly expressed. In root and old seed, BnC4H-2 is dominant over BnC4H-1, with a reverse trend in leaf and pericarp. Paralogous C4H numbers in Brassicaceae genomes and possible roles of conserved motifs in 5' UTR and the 2nd intron are discussed.     

Synthesis of 5-(4-hydroxy-3-methylphenyl) -5- (substituted phenyl)-4, 5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone derivatives with anti-viral activity

A series of N1-nicotinoyl-3- (4-hydroxy-3-methyl phenyl)-5-(substituted phenyl)-2-pyrazolines were synthesized by the reaction between isoniazid (INH) and chalcones and were tested for their in vitro anti-viral activity. Among the compounds, the electron withdrawing group substituted analogues 5-(4-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4, 5-dihydro-1H-1- pyrazolyl-4-pyridylmethanone (4b), 5-(2-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyri- dylmethanone (4i), 5-(4-fluorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro- 1H-1-pyrazolyl-4-pyridylmethanone (4h) and 5-(2,6-dichlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro- 1H-1-pyrazolyl-4-pyridyl methanone (4j) were the most promising and the halogeno function appeared to be essential for antiviral activity.     

Synthesis of 5-(4-hydroxy-3-methylphenyl)-5-(substituted phenyl)-4, 5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone derivatives with anti-viral activity

A series of N¹-nicotinoyl-3- (4-hydroxy-3-methyl phenyl)-5-(substituted phenyl)-2-pyrazolines were synthesized by the reaction between isoniazid (INH) and chalcones and were tested for their in vitro anti-viral activity. Among the compounds, the electron withdrawing group substituted analogues 5-(4-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4, 5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone (4b), 5-(2-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone (4i), 5-(4-fluorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone (4h) and 5-(2,6-dichlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyridyl methanone (4j) were the most promising and the halogeno function appeared to be essential for antiviral activity.     

5-Alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one derivatives inhibit growth of lung cancer A549 cell by inducing apoptosis

we found that 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one derivatives 8d, 8e and 8f could effectively induce apoptosis in A549 lung cancer cells and elevate the levels of integrin beta4 and ROS. The data suggested that these compounds might be promising agents for the cancer therapy, and these compounds would be useful tools for further investigate the functions of integrin beta4 in regulation of the cancer cell apoptosis.     

4-Hydroxy-3-nitrophenyl (NP) acetyl-hapten specific lymphocyte proliferation. I. Mice bearing Igh-1b allotype can cross-react with 4-hydroxy-5-iodo-3-nitrophenyl (NIP) acetyl hapten

Hapten specific T cell proliferation was induced in several strains of mice. When lymph node T cells from 4-hydroxy-3-nitrophenyl acetyl-keyhole lympet hemocyanin (NP-KLH)-primed mice were stimulated in vitro with NP-polymer glutamic acid-lysine-phenyl alanine (NP-GL phi) or NP-ovalbumin (NP-OVA), they displayed a good level of proliferative responses. It was observed that NP-GL phi could induce NP-hapten specific proliferation even with NP-KLH lymphocytes from GL phi nonresponder strains. NP-KLH primed lymphocytes from C57BL/6 (H-2b, Igh-1b), CKB (H-2k, Igh-1b), CWB (H-2b, Igh-1b), and B10.BR (H-2k, Igh-1b) mice showed good proliferative responses to both 4-hydroxy-5-iodo-3-nitrophenyl (NIP) acetyl-GL phi and NIP-OVA antigens. However, NP-KLH primed lymphocytes from C3H/He (H-2k, Igh-1j) and C3H. SW (H-2b, Igh-1j) mice displayed poor proliferative responses to NIP-GL phi and NIP-OVA antigen. These results suggested that the gene coding for the NIP-cross-reaction might be mapped in the Ig heavy-chain linked locus.     

Studies in vitro on the effects of 1H,2H,4H(5H)-octafluorocyclohexane and 1H,4H(2H)-nonafluorocyclohexane on enzymes and organelles

A comparison has been made of the effect of 1H,2H,4H(5H)-octafluorocyclohexane, which is highly toxic (LD(50) 17mg./kg. in rats), and of 1H,4H(2H)-nonafluorocyclohexane, which is relatively non-toxic (LD(50)>440mg./kg. in rats), on the respiration of rat liver homogenates and mitochondria in vitro. 1H,2H,4H(5H)-Octafluorocyclohexane strongly inhibited the respiration of both homogenates and mitochondria, but neither compound had any significant effect on glycolysis or on glutamate dehydrogenase or NADH-cytochrome c reductase activity. 1H,2H,4H(5H)-Octafluorocyclohexane, however, caused a very marked inhibition of cytochrome oxidase activity, causing an almost complete lesion in this region of the respiratory chain. 1H,4H(2H)-Nonafluorocyclohexane was without effect in this respect. A marked decrease in turbidity of mitochondrial suspensions at 520nm. was caused by addition of both compounds, the effect being greater with 1H,2H,4H(5H)-octafluorocyclohexane. ATP, Mg(2+) and bovine serum albumin did not reverse these changes. Mitochondrial adenosine triphosphatase activity was increased twofold by the toxic compound, but only slightly by the non-toxic compound. Electron-microscopic examination of mitochondria treated with 1H,2H,4H(5H)-octafluorocyclohexane revealed gross morphological damage, whereas the effect of 1H,4H(2H)-nonafluorocyclohexane appeared to be merely to cause swelling. The results obtained account, to some extent at any rate, for the toxic effects of 1H,2H,4H(5H)-octafluorocyclohexane.     

2''-Deoxy-3,7-dideazaguanosine and related compounds. Synthesis of 6-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl) and 1-beta-D-arabinofuranosyl-1H-pyrrolo[3,2-c]pyridin-4(5H)-one via direct glycosylation of a pyrrole precursor.

The synthesis of two new analogs of 2'-deoxyguanosine, 6-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-1H-pyrrolo[3,2-c] pyridin-4(5H)-one (8) and 6-amino-1-beta-D-arabinofuranosyl-1H-pyrrolo[3,2-c]-pyridin-4(5H)-one (13) has been accomplished by glycosylation of the sodium salt of ethyl 2-cyanomethyl-1H-pyrrole-3-carboxylate (4c) using 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose( 5) and 1-chloro-2,3,5-tri-O-benzyl-alpha-D-arabinofuranose (9), respectively. The resulting blocked nucleosides, ethyl 2-cyanomethyl-1-(2-deoxy-3,5-di-O-p-toluoyl-beta-D-erythro- pentofuranosyl)-1H-pyrrole-3-carboxylate (6) and ethyl 2-cyanomethyl-1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl)- 1H-pyrrole-3-carboxylate, were ring closed with hydrazine to form 5-amino-6-hydrazino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-1H- pyrrolo[3,2-c]-pyridin-4(5H)-one (7) and 5,6-diamino-1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl)-1H- pyrrolo[3,2-c]pyridin-4(5H)-one (11), respectively. Treatment of 7 with Raney nickel provided the 2'-deoxyguanosine analog 8 while reaction of 11 with Raney nickel followed by palladium hydroxide/cyclohexene treatment gave the 2'-deoxyguanosine analog 13. The anomeric configuration of 8 was assigned as beta by proton NMR, while that of 13 was confirmed as beta by single-crystal X-ray analysis of the deblocked precursor ethyl 2-cyanomethyl-1-beta-D-arabinofuranosyl-1H-pyrrole-3-carboxylate (10a).     

6-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones: dual-acting 5-HT1 receptor antagonists and serotonin reuptake inhibitors

Investigation of a series 6-[2-(4-aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones has led to the discovery of potent 5-HT(1A/1B/1D) receptor antagonists with and without additional SerT affinity. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.     

Synthesis and biological evaluation of novel 5-alkyl-2-arylthio-6-((3,4-dihydroquinolin-1(2H)-yl)methyl)pyrimidin-4(3H)-ones as potent non-nucleoside HIV-1 reverse transcriptase inhibitors

A series of novel S-DABO analogues of 5-alkyl-2-arylthio-6-((3,4-dihydroquinolin-1(2H)-yl)methyl)pyrimidin-4(3H)-ones were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were compounds 6c1,6c6, and 6b1 (EC(50)=0.24 ± 0.05, 0.38 ± 0.13, 0.39 ± 0.05 μM, respectively), which possess improved or similar HIV-1 inhibitory activity compared with nevirapine (NVP) (EC(50)=0.21 μM) and delavirdine (DLV) (EC(50)=0.32 μM). None of these compounds were active against HIV-2 replication. Furthermore, enzyme inhibitory assays were performed with selected derivatives against HIV-1 wtRT, confirming that the main target of these compounds is the HIV-1 RT and these new S-DABOs are acting as NNRTIs. The preliminary structure-activity relationship (SAR) of these new congeners is discussed briefly and rationalized by docking studies.     

Experimental and DFT computational studies on 5-benzyl-4-(3,4-dimethoxyphenethyl)-2H-1,2,4-triazol-3(4H)-one

The triazole compound, 5-benzyl-4-(3,4-dimethoxyphenethyl)-2H-1,2,4-triazol-3(4H)-one, has been synthesized and characterized by ¹H-NMR, ¹³C-NMR, IR, and X-ray single-crystal determination. The compound crystallizes in the monoclinic space group P2₁ with a?=?11.8844(3) Å, b?=?17.5087(4) Å, c?=?17.3648(6) Å, β?=?99.990(2)? and Z?=?8. In addition to the molecular geometry from X-ray experiment, the molecular geometry, vibrational frequencies and gauge including atomic orbital (GIAO) ¹H- and ¹³C-NMR chemical shift values of the title compound in the ground state have been calculated using the density functional method (B3LYP) with 6-31G(d,p) basis set. The calculated results show that the optimized geometries can well reproduce the crystal structure and the theoretical vibrational frequencies and chemical shift values show good agreement with experimental ones. Besides, molecular electrostatic potential (MEP), natural bond orbital (NBO), and frontier molecular orbitals (FMO) analysis of the title compound were performed by the B3LYP/6-31G(d,p) method.     

Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides

An effective in vitro enzymatic synthesis is described for the production of nucleoside triphosphates (NTPs) which are stereo-specifically deuterated on the H5" position with high selectivity (>98%), and which can have a variety of different labels (13C, 15N, 2H) in other positions. The NTPs can subsequently be employed in the enzymatic synthesis of RNAs using T7 polymerase from a DNA template. The stereo-specific deuteration of the H5" immediately provides the stereo-specific assignment of H5' resonances in NMR spectra, giving access to important structural parameters. Stereo-chemical H-exchange was used to convert commercially available 1,2,3,4,5,6,6-2H-1,2,3,4,5,6-13C-D-glucose (d7-13C6-D-glucose) into [1,2,3,4,5,6(R)-2H-1,2,3,4,5,6-13C]-D-glucose (d6-13C6-D-glucose). [1',3',4',5"-2H-1',2',3',4',5'-13C]GTP (d4-13C5-GTP) was then produced from d6-13C6-D-glucose and guanine base via in vitro enzymatic synthesis employing enzymes from the pentose-phosphate, nucleotide biosynthesis and salvage pathways. The overall yield was approximately 60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d4-13C5-GTP, together with in vitro synthesised d5-UTP, d5-CTP and non-labelled ATP, were used in the synthesis of a 31 nt RNA derived from the primer binding site of hepatitis B virus genomic RNA. (13C,1H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly 13C/15N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with 13C-labelled non-deuterated RNA.     

Synthesis and antibacterial activities of 5-hydroxy-4-amino-2(5H)-furanones

Starting from the mucohalogen acids 1a and b 5-hydroxy-2(5H)-furanones 2a-h have been prepared and tested. These novel 4-amino-5-hydroxy 2(5H)-furananones have shown a broad antibiotic activity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 in the micromolar range. A one step synthesis from mucohalogen acids towards the antibacterials 2a-h was developed, in which the target was obtained from 1a and b under reflux in toluene in presence of a catalytic amount of sulfuric acid. The derivatives 2b and c displayed a MIC and MBC of 4/8mug/ml, against Staphylococcus aureus with a selectivity towards the resistant strains.     

Novel 5-HT(1A/1B/1D) receptors antagonists with potent 5-HT reuptake inhibitory activity

Novel 2-methyl-5-quinolinyl-1-piperazinylalkyl-3,4-dihydro-2H-1,4-benzoxazin-3-ones showing high affinities for the 5-HT(1A/1B/1D) receptors coupled with potent 5-HT reuptake inhibitory activity have been discovered. This is the first report describing docking of the lead compound 6-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-2H-1,4-benzoxazin-3(4H)-one 1, into a model of the 5-HT transporter and the 5-HT(1A) receptor model.     

Microwave-assisted preparation of the quorum-sensing molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and structurally related analogs

An optimized procedure for the efficient preparation of 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal or PQS) and a diverse range of structurally related 2-alkyl-4-quinolones with biological activity is presented. The two-step synthesis begins with the formation of α-chloro ketones by the coupling of a Weinreb amide (2-chloro-N-methoxy-N-methylacetamide) and an appropriate Grignard reagent. The resulting α-chloro ketones can be reacted with commercially available anthranilic acids under microwave irradiation conditions to furnish the desired 2-alkyl-4-quinolone products. As a typical example, the synthesis of PQS, a molecule involved in quorum sensing in the pathogenic bacterium Pseudomonas aeruginosa, is described in detail. The first step of this process (α-chloro ketone formation) takes ～10 h in total to complete from commercially available bromoheptane and 2-chloro-N-methoxy-N-methylacetamide. The second step (microwave-assisted reaction with anthranilic acid) takes ～14 h in total to complete (the reaction typically proceeds in ～30 min, with work-up and purification requiring ～13 h).     

Synthesis and anti-HIV activity of 1,3,4,5-tetrahydro-2H-1,4-benzodiazepin-2-one (TBO) derivatives. Truncated 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2( 1H)-on es (TIBO) analogues

4,5,6,7-Tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1 H)-ones (TIBO), 1, have been shown to significantly inhibit HIV-1 replication, as reported in detail in our prior publications. Since our earlier reports, we have modified the TIBO structures 1 by removing the 5-membered ring of 1, generating 1,3,4,5-tetrahydro-2H-1,4-benzodiazepin-2-ones (TBO), 4, a bicyclic series of compounds. Although compounds 4 possess modest activity when compared to TIBO analogues 1, they clearly demonstrated significant anti-HIV-1 activity.