Alkylation of tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of d(ATTTTCA)

Alkylation of E. coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphamide of oligonucleotide d(ATTTTCA) complementary to the sequence UGAAms2i6AA psi in the anticodon loop of tRNAPhe was studied. Three guanine residues--G28/29, G24 and G10 were found to be alkylated. Two binding sites for the reagent in the tRNA were assumed to be present. The efficiency of the alkylation of tRNA from these sites as well as an average association constant (Ka 3,8 X 10(3)M-1) for the reagent interaction with tRNA were evaluated.     

Affinity modification of Escherichia coli ribosomes with 2',3'-O-[4-(N-2-chloroethyl)-N-methylamino]-benzylidene derivative of AUGU3 in the 70S initiation complexes

Affinity labelling of the Escherichia coli ribosomes with the 2',3'-O-[4-(N-(2-chloroethyl)-N-methylamino]benzylidene derivative of AUGU3(AUGU3[14C]CHRCl) has been studied within 70S initiation complexes ribosome.AUGU3[14C]CHRCl.fMet-tRNA(Metf) and binary complex ribosome.AUGU3[14C]CHRCl. Various ways of the 70S initiation complex formation resulted in differently labelled products. Proteins S5, S7, S9, L1, L16 were thus identified as cross-linked with AUGU3[14C]CHRCl within an initiation complex obtained in the presence of initiation factors IF-1, IF-2, IF-3, whereas only proteins S5 and S7 were cross-linked within the complex obtained with the sole factor IF-2. Proteins S1, S3, L1 and L33 were labelled within the initiation complex obtained nonenzymatically but only protein S1 within the binary complex. In all complexes formed with use of initiation factors labelling of IF-2 factor was invariably observed.     

Affinity modification of Escherichia coli ribosomes with the non-hydrolyzed GTP analog, gamma-[4-N-(2-chloroethyl)-N-methylaminobenzyl]amide of GTP

Substituted gamma-amides of GTP viz. GTP gamma-[4-N-(2-chloro- and gamma-[4-N-(2-hydroxyethyl)-N-methylaminobenzyl]amide (CIRCH2NHpppG and OHRCH2NHpppG, resp.) were shown to be unhydrolisable GTP analogues in the EF-Tu-dependent GTP-ase reaction of ribosomes. The reactive analogue, CIRCH2NHpppG, was used for affinity labelling within the 70S ribosome.poly(U).tRNAPhe(P-site).Phe-tRNAPhe.EF-Tu.CIR[14C]CH2.NHpppG complex. Both 50S and 30S subunits were thus labelled but 50S subunit was modified considerably more than 30C subunit. Labelled were proteins L17, L21, S16, S21, and rRNA of both subunits, 23C rRNA within 50C subunit being labelled preferentially as compared with 50C proteins. No labelling of EF-Tu within the complex was detected.     

Study of the structural-energy aspects of complementary-addressed modifications of nucleic acid with reactive 4-]N-methyl-N-(2-chloroethyl)-amino]benzyl-3'- or 5'-phosphamide oligonucleotide derivatives by a molecular mechanics method

Calculations of probabilities of the complementary addressed modification of target NA by 3'- or 5'-reactive derivatives of oligonucleotides carrying a 4-[N-(2-chloroethyl)-N-methyl]aminobenzyl group attached to the 3'- or 5'-terminal phosphates through a phosphoroamide linkage have been made. It is shown that the structural basis of the high efficiency and positional specificity depending on the NA target base sequence is the extent of structural correspondence of the energetically optimal conformation of the active group in the complex to the mutual arrangement of the active group and nucleophilic site needed for the chemical reaction. The 3'-derivative has the highest dependence of efficiency and positional specificity of the alkylation on the target NA base sequence. The maximal positional specificity of the alkylation is found for the modification of the cytidine at the first position from the terminal complementary base pair at the 5'-end of the target NA. For the 5'-derivative, the alkylating ability was determined to depend on the insertion of additional methylene bridges into the standard phosphoroamide linker: two methylene groups provide for the maximal increase of the modification ability of the nucleophilic site of the target NA in the double-stranded part of the complex. The efficiency of alkylation of the target NA in a three component complex with oligonucleotide-effector also complementary to the target NA have been studied. It was found that formation of the three-component complex lead to an additional stabilization of the conformation needed for the reaction of the active group, in comparison with two-component complex, by means of the intercalation of the phenyl group of the reagent in the gap between the oligonucleotide derivative and the oligonucleotide effector.     

Chemical transformation of radioactive 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-[32P]phosphamides of oligodeoxyribonucleotides during in vivo experiments

The practical use of reactive oligonucleotide derivatives for complementarily addressed modification of nucleic acids in vivo includes several steps, at which side chemical reactions resulting in a decrease of the modification efficiency may take place. Chemical reactions of 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-[32P]phosphamides of oligodeoxyribonucleotides were studied in vivo. The intermolecular self-alkylation at the reactive residue of the alkylating derivative was found in the precipitate of its lithium salt under acetone at-20 degrees C. The effects of pH, buffer solutions, salts, temperature, phenol, cell culture suspensions, tissue homogenates, etc., on the stability of the derivatives were studied. A sufficient cleavage of the phosphamide bond was observed at pH less than 3. In fresh liver homogenates the nucleolytic degradation of the oligonucleotide part of the reagent was shown to occur. After intraperitoneal injection of mice with radioactive alkylating derivatives up to 50% of the reagent was included into the blood biopolymers within one hour. The covalently linked to the biopolymers oligonucleotide appeared to be highly degraded thereby.     

The 5S rRNA-protein complex of Escherichia coli studied by carbodiimide modification]

5S rRNA-protein complex has been reconstituted from 5S rRNA and total protein of large (L) ribosomal subunit of Escherichia coli. The complex consists of 5S rRNA and 3 proteins only: L5, L18, L25. A water-soluble carbodiimide [N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimide-methyl-p-toluolsulp honate] cross-links L18 to 5S rRNA at pH 7.2 and L25 to 5S rRNA at pH 7.7. This pH-dependence of cross-linked proteins is a consequence of the difference in stability of the initial complex: the complex has all three proteins at pH 7.7 but L18 mainly at pH 7.2. It has been shown that L18 stimulates the chemical modification of U87 and U89 residues of 5S rRNA by carbodiimide. A model of L18-5S rRNA complex has been proposed.     

Synthesis and antibacterial activity of N-[2-(5-bromothiophen-2-yl)-2-oxoethyl] and N-[(2-5-bromothiophen-2-yl)-2-oximinoethyl] derivatives of piperazinyl quinolones

A series of N-[2-(5-bromothiophen-2-yl)-2-oxoethyl] and N-[2-(5-bromothiophen-2-yl)-2-oximinoethyl]derivatives of piperazinyl quinolones were synthesized and evaluated for antimicrobial activity against Gram-positive and Gram-negative microorganisms. Some of these derivatives exhibit comparable or better activity against Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis, than ciprofloxacin, norfloxacin and enoxacine as reference drugs.     

Interaction of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitroso urea hydrochloride with nucleic acids and proteins

The binding of the 14C-labelled-ethylene and -pyrimidine moieties of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU) to the biological macromolecules was studied with the AH-130 hepatoma-bearing rats, suspension of AH-130 cells, and isolated nucleic acids and proteins. In all systems examined, a significant level of the binding of the [14C]ethylene of ACNU to nucleic acids, probably due to alkylation, was observed. In contrast, the extent of the binding of the [14C]-pyrimidine was negligible. When a compound lacking the 4-amino group of ACNU (deamino-ACNU) was used for the binding study, relatively higher binding of this compound than that of ACNU to [14C]lysine was observed. It was revealed, therefore, that the low binding of ACNU to proteins could be due to instantaneous depletion of an isocyanate-intermediate, according to the formation of an intramolecularly carbamoylated product with the amino-group on the pyrimidine ring of ACNU molecule during incubation. This could be the molecular basis for the low carbamoylating activity of ACNU in vivo and in vitro, and the antitumor action of ACNU would be dependent on its alkylating activity only.     

Structure of the O-polysaccharide of Escherichia coli O150 containing 2-acetamido-4-O-[(S)-1-carboxyethyl]-2-deoxy-d-glucose

An acidic O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Escherichia coli O150 and studied by sugar and methylation analyses, triflic acid solvolysis, Smith degradation, (1)H and (13)C NMR spectroscopy, including 2D ROESY, (1)H,(13)C HSQC, HMQC-TOCSY, and HMBC experiments. The polysaccharide was found to contain a regioisomer of N-acetylisomuramic acid, 2-acetamido-4-O-[(S)-1-carboxyethyl]-2-deoxy-d-glucose [d-GlcNAc4(Slac)]. The structure of its hexasaccharide repeating unit was established.     

Study of the mRNA-binding region of ribosomes at different steps of translation. II. Affinity modification of Escherichia coli ribosomes by benzylidene derivative of AUGU6 in the 70S initiation complex

2',3'-O-(4-[N-(2-chloroethyl)-N-methylamino]) benzylidene derivative of AUGU6 was used for identification of the proteins in the region of the mRNA-binding centre of E. coli ribosomes. This derivative alkylated ribosomes (preferentially 30S ribosomal) with high efficiency within the 70S initiation complex. In both 30S and 50S ribosomal subunits proteins and rRNA were modified. Specificity of the alkylation of ribosomal proteins and rRNA with the reagent was proved by the inhibitory action of AUGU6. Using the method of two-dimensional electrophoresis in polyacrylamide gel the proteins S4, S12, S13, S14, S15, S18, S19 and S20/L26 which are labelled by the analog of mRNA were identified.     

Density functional computational studies on (E)-2-[(2-Hydroxy-5-nitrophenyl)-iminiomethyl]-4-nitrophenolate

Density functional calculations of the structure, atomic charges, molecular electrostatic potential and thermodynamic functions have been performed at B3LYP/6-31G(d,p) level of theory for the title compound (E)-2-[(2-hydroxy-5-nitrophenyl)-iminiomethyl]-4-nitrophenolate. The results show that the phenolate oxygen atom and all of the nitro group oxygen atoms have bigger negative charges, and the coordination ability of these atoms differs in different solvents. The energetic behavior of the title compound in solvent media has been examined using B3LYP method with the 6-31G(d,p) basis set by applying the Onsager method and the isodensity polarized continuum model (IPCM). The results obtained with these methods reveal that the IPCM method yielded a more stable structure than Onsager’s method. In addition, natural bond orbital and frontier molecular orbital analysis of the title compound were performed using the B3LYP/6-31G(d,p) method.     

Inhibition of IKK-2 by 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides

A series of 21 novel 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides were synthesized and evaluated for the inhibition of IKK-2. In spite of their often modest activity on the enzyme, six selected analogs showed significant inhibition of the production of inflammatory cytokine IL-8 in IL-1beta stimulated rheumatoid arthritis-derived synovial fibroblasts, demonstrating their potential usefulness as NF-kappaB regulators.     

Affinity labeling of bovine liver glutamate dehydrogenase with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate

Bovine liver glutamate dehydrogenase reacts with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TA-5'-DP) and 5'-triphosphate (8-BDB-TA-5'-TP) to yield enzyme with about 1 mol of reagent incorporated/mol of enzyme subunit. The modified enzyme is catalytically active but has decreased sensitivity to inhibition by GTP, reduced extent of activation by ADP, and diminished inhibition by high concentrations of NADH. Since modified enzyme, like native glutamate dehydrogenase, reversibly binds more than 1 mol each of ADP and GTP, it is unlikely that 8-BDB-TA-5'-TP reacts directly within either the ADP or GTP regulatory sites. The rate constant for reaction of enzyme exhibits a nonlinear dependence on reagent concentration with KD = 89 microM for 8-BDB-TA-5'-TP and 240 microM for 8-BDB-TA-5'-DP. The ligands ADP and GTP alone and NADH alone produce only small decreases in the rate constant for the reaction of enzyme with 8-BDB-TA-5'-TP, but the combined addition of 5 mM NADH + 200 microM GTP reduces the reaction rate constant more than 10-fold and the reagent incorporation to about 0.1 mol/mol of enzyme subunit. These results suggest that 8-BDB-TA-5'-TP reacts as a nucleotide affinity label in the region of the GTP-dependent NADH regulatory site of bovine liver glutamate dehydrogenase.     

3-[2-(Aminomethyl)-5-[(pyridin-4-yl)carbamoyl]phenyl] benzoates as soft ROCK inhibitors

Clinical development of ROCK inhibitors has so far been limited by systemic or local ROCK-associated side effects. A soft drug approach, which involves predictable metabolic inactivation of an active compound to a nontoxic metabolite, could represent an attractive way to obtain ROCK inhibitors with improved tolerability. We herein report the design and synthesis of a new series of soft ROCK inhibitors structurally related to the ROCK inhibitor Y-27632. These inhibitors contain carboxylic ester moieties which allow inactivation by esterases. While the parent esters display strong activity in enzymatic (ROCK2) and cellular (MLC phosphorylation) assays, their corresponding carboxylic acid metabolites have negligible functional activity. Compound 32 combined strong efficacy (ROCK2 IC₅₀ = 2.5 nM) with rapid inactivation in plasma (t_1/2 <5′). Compound 32 also demonstrated in vivo efficacy when evaluated as an IOP-lowering agent in ocular normotensive New-Zealand White rabbits, without ocular side effects.     

Discovery of (2S)-N-[(1R)-2-[4-cyclohexyl-4-[[(1,1-dimethylethyl)amino]carbonyl]-1-piperidinyl]-1-[(4-fluorophenyl)methyl]-2-oxoethyl]-4-methyl-2-piperazinecarboxamide (MB243), a potent and selective melanocortin subtype-4 receptor agonist

We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the melanocortin subtype-4 receptor. Further in vivo development of lead agonist, MB243, is disclosed.     

Affinity labeling of the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase by 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate

Bovine liver glutamate dehydrogenase reacts covalently with the new adenosine analogue 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate with incorporation of about 1 mol of reagent/mol of enzyme subunit. Modified enzyme completely loses its normal ability to be inhibited by high concentrations of reduced diphosphopyridine nucleotide (DPNH) (greater than 100 microM), which binds at a regulatory site distinct from the catalytic site; however, the modified enzyme retains its full activity when assayed at 100 microM DPNH in the absence of allosteric compounds. The enzyme is still activated by ADP, is inhibited by GTP (albeit at higher concentrations), and binds 1.5-2 mol of [14C]GTP/subunit. A plot of initial velocity vs. DPNH concentration for the modified enzyme, in contrast to the native enzyme, followed Michaelis-Menten kinetics. The rate constant (k) for loss of DPNH inhibition (as measured at 0.6 mM DPNH) exhibits a nonlinear dependence on reagent concentration, suggesting a reversible binding of reagent (Kd = 0.19 mM) prior to irreversible modification. At 0.1 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate, k = 0.036 min-1 and is not affected by alpha-ketoglutarate, 100 microM DPNH, or GTP alone but is decreased to 0.0094 min-1 by 5 mM DPNH and essentially to zero by 5 mM DPNH plus 100 microM GTP. Incorporation after incubation with 0.25 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate for 2 h at pH 7.1 is 1.14 mol/mol of subunit in the absence but only 0.24 mol/mol of subunit in the presence of DPNH plus GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and evaluation of N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide: a deuterium-substituted radioligand for peripheral benzodiazepine receptor

N-(5-Fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide ([(18)F]2) is a potent ligand (IC(50): 1.71 nM) for peripheral benzodiazepine receptor (PBR). However, in vivo evaluation on rodents and primates showed that this ligand was unstable and rapidly metabolized to [(18)F]F(-) by defluorination of the [(18)F]fluoromethyl moiety. In this study, we designed a deuterium-substituted analogue, N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide ([(18)F]5) as a radioligand for PBR to reduce the in vivo metabolic rate of the non-deuterated [(18)F]2. The design principle was based on the hypothesis that the deuterium substitution may reduce the rate of defluorination initiated by cleavage of the C-H bond without altering the binding affinity for PBR. The non-radioactive 5 was prepared by reacting diiodomethane-d(2) (CD(2)I(2), 6) with a phenol precursor 7, followed by treatment with tetrabutylammonium fluoride. The ligand [(18)F]5 was synthesized by the alkylation of 7 with [(18)F]fluoromethyl iodide-d(2) ([(18)F]FCD(2)I, [(18)F]9). Compound 5 displayed a similar in vitro affinity to PBR (IC(50): 1.90 nM) with 2. In vivo evaluation demonstrated that [(18)F]5 was metabolized by defluorination to [(18)F]F(-) as a main radioactive component, but its metabolic rate was slower than that of [(18)F]2 in the brain of mice. The deuterium substitution decreased the radioactivity level of [(18)F]5 in the bone of mouse, augmented by the percentage of specific binding to PBR in the rat brain determined by ex vivo autoradiography. However, the PET image of [(18)F]5 for monkey brain showed high radioactivity in the brain and skull, suggesting a possible species difference between rodents and primates.     

Biosynthesis of lipid A in Escherichia coli: identification of UDP-3-O-[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine as a precursor of UDP-N2,O3-bis[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine

The lipid A disaccharide of the Escherichia coli envelope is synthesized from the two fatty acylated glucosamine derivatives UDP-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucosamine (UDP-2,3-diacyl-GlcN) and N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D-glucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) [Ray, B. L., Painter, G., & Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859]. We have previously shown that UDP-2,3-diacyl-GlcN is generated in extracts of E. coli by fatty acylation of UDP-GlcNAc, giving UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc as the first intermediate, which is rapidly converted to UDP-2,3-diacyl-GlcN [Anderson, M. S., Bulawa, C. E., & Raetz, C. R. H. (1985) J. Biol. Chem. 260, 15536-15541; Anderson, M. S., & Raetz, C. R. H. (1987) J. Biol. Chem. 262, 5159-5169]. We now demonstrate a novel enzyme in the cytoplasmic fraction of E. coli, capable of deacetylating UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc to form UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine. The covalent structure of the previously undescribed UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine intermediate was established by 1H NMR spectroscopy and fast atom bombardment mass spectrometry. This material can be made to accumulate in E. coli extracts upon incubation of UDP-3-O-[(R)-3- hydroxymyristoyl]-GlcNAc in the absence of the fatty acyl donor [(R)-3-hydroxymyristoyl]-acyl carrier protein. However, addition of the isolated deacetylation product [UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine] back to membrane-free extracts of E. coli in the presence of [(R)-3-hydroxymyristoyl]-acyl carrier protein results in rapid conversion of this compound into the more hydrophobic products UDP-2,3-diacyl-GlcN, 2,3-diacyl-GlcN-1-P, and O-[2-amino-2-deoxy-N2,O3- bis[(R)-3-hydroxytetradecanoyl]-beta-D-glucopyranosyl]-(1----6)-2-amino- 2-deoxy-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucopyranose 1-phosphate (tetra-acyldisaccharide-1-P), demonstrating its competency as a precursor. In vitro incubations using [acetyl-3H]UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc confirmed release of the acetyl moiety in this system as acetate, not as some other acetyl derivative. The deacetylation reaction was inhibited by 1 mM N-ethylmaleimide, while the subsequent N-acylation reaction was not. Our observations provide strong evidence that UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine is a true intermediate in the biosynthesis of UDP-2,3-diacyl-GlcN and lipid A.     

Synthesis and biological activity of N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid

2-Deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a potent inhibitor of thymidylate synthase. Its analogue, N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid, containing p-aminophenylacetic acid residue substituting p-aminobenzoic acid residue, was synthesized. The new analogue exhibited a moderately potent thymidylate synthase inhibition, of linear mixed type vs. the cofactor, N(5,10)-methylenetetrahydrofolate. The Ki value of 0.34 microM, determined with a purified recombinant rat hepatoma enzyme, was about 30-fold higher than that reported for inhibition of thymidylate synthase from mouse leukemia L1210 cells by ICI 198583 (Hughes et al., 1990, J. Med. Chem. 33, 3060). Growth of mouse leukemia L5178Y cells was inhibited by the analogue (IC50 = 1.26 mM) 180-fold weaker than by ICI 198583 (IC50 = 6.9 microM).