Synthesis of bis-(methyl 3,4,6-tri-O-acetyl-D-glucopyranosid-2-yl)-oxamides

The synthesis of a new bis-(D-glucopyranosid-2-yl)oxamides via the key intermediate, N-acetyl N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl) oxamic acid chloride (2alpha) is described. Treatment of compound 2alpha with methyl 3,4,6-tri-O-acetyl-2-amino-2-deoxy-beta-D-glucopyranoside afforded N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl)-N'-(methyl 3,4,6-tri-O-acetyl-beta-D-glucopyranosid-2-yl)-oxamide. Reaction of 2alpha with 1,2-diaminoethane afforded 1,2-bis-[N,N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)]ethyloxamide as a main product, while 2-N-[N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)oxamide]-ethyl acetamide was formed as a side product. Reaction of 2alpha with 1,3-diamino-2-hydroxypropane gave only 1,3-bis-N,N-[N'-(methyl 3',4',6'-tri-O-acetyl-2'-deoxy-alpha-D-glucopyranosid-2'-yl)-oxamido]-2-propanol.     

Binding of ethylenediamine to phosphatidylserine is inhibitory to Na+/K(+)-ATPase.

Covalent linkage of ethylenediamine with the Na+/K(+)-ATPase complex from rabbit kidney outer medulla by the use of the water-soluble carbodiimide, N-ethyl,N'-(3-dimethylaminopropyl)carbodiimide, resulted in a 73% reaction with phosphatidylserine and only 27% with carboxylic groups in the proteic component of the enzyme. Condensation products from the reaction between phosphatidylserine and ethylenediamine, N-(O-phosphatidylseryl)ethylenediamine, N,N'-bis(O-phosphatidylseryl)ethylenediamine and its intermediary product O-phosphatidyl-[N,N'-bis(seryl)]ethylenediamine, were synthesised. Symmetrically substituted ethylenediamine was the most likely condensation product of ethylenediamine with endogenous phosphatidylserine. The synthesised lipids were incorporated in proteoliposomes containing Na+/K(+)-ATPase and only the addition of the phospholipid phosphatidylcholine. The ratio of phospholipid to protein was 52 (w/w). These proteoliposomes were perforated by the addition of 0.5% cholate and both the Na(+)-dependent phosphorylation level and its dependence on Na+, Mg2+ and ATP were measured. Phosphatidylcholine alone increased the half-maximal activation concentration for Na+ ([Na+]0.5) from 0.2 to 1-2 mM, for Mg2+ from 0.1 to 0.8 microM and for ATP from 0.02 to 0.3 microM. The Ki for K+ (in the absence of Na+) was unaffected: 12.8 microM vs. 12.5 microM in the non-reconstituted system. Replacing 10 mol% of phosphatidylcholine by phosphatidylethanolamine: or phosphatidylserine had no significant effect on [Na+]0.5: 1.1 and 0.7 mM, respectively. Replacing 5 mol% phosphatidylcholine by the bis(phosphatidylseryl) substituent of ethylenediamine further increased [Na+]0.5 to 13.7 mM, while half-maximal activation concentrations for Mg2+ and ATP were unaltered. The mono-phosphatidylseryl derivatives of ethylenediamine, each 5 mol%, also increased [Na+]0.5, but to a lesser extent (3.2-3.8 mM). In addition to their competitive effects, the phosphatidylseryl-substituted ethylenediamine compounds exerted a slowly-increasing non-competitive inhibition, not only in phosphorylation, but also in overall ATPase activity, which was reduced, although not abolished, by exogenous protein (bovine serum albumin). A detergent-like action in the usual sense is unlikely since liposomes containing these lipids remained intact. These studies prove that phospholipids are not only required for optimal activity of this transport enzyme, but in excess or in compositions deviating from the normal, may also be inhibitory.     

Dual neurokinin NK(1)/NK(2) antagonists: N-[(R,R)-(E)-1-arylmethyl-3-(2-oxo-azepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamides and 3-[N'-3,5-bis(trifluoromethyl)benzoyl-N-arylmethyl-N'-methylhydrazino]-N-[(R)-2-oxo-azepan-3-yl]propionamides.

Based on the structure of N-[(R,R)-(E)-1-(4-chlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (1), attempts to improve the NK(2) affinity have resulted in the discovery of N-[(R,R)-(E)-1-(3,4-dichlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (9, DNK333) exhibiting a 5-fold improved affinity to the NK(2) receptor in comparison to 1. Simplification of the structure via elimination of a chiral centre led to 3-[N'-3,5-bis(trifluoromethyl)benzoyl-N-(3,4-dichlorobenzyl)-N'-methylhydrazino]-N-[(R)-2-oxo-azepan-3-yl]propionamide (22), a potent and fairly balanced NK(1)/NK(2) antagonist.     

Synthesis and spectral studies of 2-[(N-ethyl carbazole)-3-sulfonyl ethylenediamine]-1-N,N-2-(2-methypyridy) as a fluorescence probe for Zn²⁺

A novel Zn(2+) fluorescence probe, 2-[(N-ethyl carbazole)-3-sulfonyl ethylenediamine]-1-N,N-bis(2-methypyrbidy), was designed and synthesized via simple steps, and its structure was confirmed by IR and (1)H NMR. The probe gives significant fluorescence enhancement immediately following Zn(2+) addition at neutral pH and exhibits improved selectivity for Zn(2+) compared to the other metal ions in aqueous solution. The spectra and fluorescence quantum yield of the synthesized compound were carefully investigated by UV-vis absorption and fluorescence spectra in various solvents.     

Synthesis properties of the naturally occurring N-[(9-beta-D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl]-L-threonine (mt-6A) and other related synthetic analogs.

The naturally occurring modified nucleoside, N-[(9-beta-D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl]-L-threonine (mt6A), and the corresponding glycine analog mg6A were synthesized from N6-methyl-2',3',5'-tri-O-acetyladenosine and the appropriately blocked isocyanates derived from threonine and glycine. The natural mt6A isolated from Escherichia coli tRNA (F. Kimura-Harada et al. (1972), Biochemistry 11, 3910), from wheat embryo tRNA (R. Cunningham and M. W. Gray (1974), Biochemistry 13, 543), and from rat liver tRNA (Rogg et al. (1975), Eur. J. Biochem. 53, 115) was found to be identical with the synthetic mt6A in paper and thin-layer chromatography and electrophoresis. Several analogs of the parent 6-ureidopurine ribonucleoside, N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]-L-thronine (t6A), were also prepared. Starting from 2',3',5'-tri-O-acetylguanosine and 2',3',5'-tri-O-acetylcytidine and the above isocyanates, the t6A analogs, N-[(9-beta-D-ribofuranosyl-6-oxo-1H-purin-2-yl)carbamoyl]-L-threonine (t2G) and N-[(1-beta-D-ribofuranosyl-2-oxypyrimidin-4-yl)carbamoyl]-L-threonine (t4C), were prepared. Also synthesized were the corresponding glycine analogs, g2G and g4C, from guanosine and cytidine, respectively. The 2'-deoxyribosyl analog, N-[(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)carbamoyl]-L-threonine (2'-deoxy-t6A), and the arabinosyl derivative, N-[(9-beta-D-arabinofuranosylpurin-6-yl)carbamoyl]-L-threonine (t6AraA), were synthesized from the appropriate urethane and the requisite amino acid. The ureido group in mt6A could not be hydrolyzed by the enzymes urease, peptidase, and protease. Various chemical and biological properties of the naturally occurring mt6A and the related analogs are discussed.     

Spectroscopic investigations on DNA binding profile of two new naphthyridine carboxamides and their application as turn-on fluorescent DNA staining probes

Abstract

Two new 10-methoxydibenzo[b,h][1,6]naphthyridine-2-carboxamide derivatives (R1 and R2) have been synthesized and characterized using different spectral techniques. The binding of these probes with DNA was investigated using spectral (Electronic, fluorescence, ¹H NMR and circular dichroism) and molecular docking studies. These probes exhibited a strong fluorescence around 440?nm upon excitation around 380?nm. Electronic and competitive fluorescence titration studies, in HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] buffer/dimethyl sulfoxide (pH 7.4) medium, suggest that these probes bind strongly to DNA, which is substantiated by ¹H NMR study. The binding constants are calculated to be 5.3?×?10⁷ and 6.8?×?10⁶ M^?1 for R1 and R2, respectively. From the results of spectral studies, it is proposed that the mechanism of binding of these probes with DNA is through minor groove binding mode, which is further confirmed by circular dichroism and molecular docking studies. Initial cell viability screening using MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay shows that normal Vero cells are viable towards these probes at nano molar concentration, which is the concentration range employed in the present study for DNA staining (IC₅₀ in the order of 0.023?mM). The enhancement in fluorescence intensity of these probes upon binding with DNA enables the staining of DNA in agarose gel in gel electrophoresis experiment. The sensitivity of these probes is comparable with that of ethidium bromide and DNA amounts as low as 4 nano gram are detectable.

Communicated by Ramaswamy H. Sarma     

Synthesis and reactions of O-acetylated benzyl alpha-glycosides of 6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-N-acetylmuramoyl-L- alanyl-D-isoglutamine esters: the base-catalysed isoglutamine in equilibrium glutamine rearrangement in peptidoglycan-related structures

Condensation of benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2- deoxy-3-O-[(R)-1-carboxyethyl]-alpha-D-glucopyranoside (2) and its 4-acetate (4) with L-alanyl-D-isoglutamine benzyl ester via the mixed anhydride method yielded N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-lacto yl)-L- alanyl-D-isoglutamine benzyl ester (5) and its 4-acetate (6), respectively. Condensation by the dicyclohexylcarbodi-imide-N-hydroxysuccinimide method converted 2 into benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl- 2-deoxy-beta-D-glucopyranosyl)-3-O-[(R)-1-carboxyethyl]-2-deoxy-alpha-D- glucopyranoside 1',4-lactone (7). In the presence of activating agents, 7 underwent aminolysis with the dipeptide ester to give 5. Zemplén O-deacetylation of 5 and 6 led to transesterification and alpha----gamma transamidation of the isoglutaminyl residue to give N-(2-O-[benzyl 2-acetamido-6-O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyr anosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (8) and -glutamine methyl ester (9). Treatment of 6 with MgO-methanol caused deacetylation at the GlcNAc residue to give a mixture of N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2- deoxy-beta-D-glucopyranosyl)-4-O-acetyl-2,3-dideoxy-alpha-D-glucopyra nosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (11) and -glutamine methyl ester (12). Benzyl or methyl ester-protection of peptidoglycan-related structures is not compatible with any of the reactions requiring alkaline media. Condensation of 2 with L-alanyl-D-isoglutamine tert-butyl ester gave N-(2-O-[benzyl 2-acetamido- 6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2,3-d ideoxy- alpha-D-glucopyranosid-3-yl]-(R)-lactoyl-L-alanyl-D-isoglutamine tert-butyl ester (16), deacetylation of which, under Zemplén conditions, proceeded without side-reactions to afford N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-la cotyl)-L- alanyl-D-isoglutamine tert-butyl ester (17).     

Polyazacyclophanes incorporating two pyridine units and a heteroaromatic pendant group as potential cleaving agents of mRNA 5'-cap structure

Four hexaazacyclophanes, 16a-d, incorporating two pyridine units and a (pyridin-2-yl)methyl or (quinolin-2-yl)methyl pendant group at one of the ring N-atoms have been prepared. The key step of the synthesis is an intermolecular cyclization of N,N-bis{[6-(tosyloxymethyl)pyridin-2-yl]methyl}-2-nitrobenzenesulfonamide (7) with either tert-butyl bis{2-[(2-nitrophenylsulfonyl)amino]ethyl}carbamate (2a) or tert-butyl bis{3-[(2-nitrophenylsulfonyl)amino]propyl}carbamate (2b) in the presence of anhydrous Cs(2)CO(3). Removal of the acid-labile tert-butoxycarbonyl protection then allows attachment of the pendant group by reductive alkylation to the exposed secondary amino group, and deprotection of the remaining aliphatic ring N-atoms completes the synthesis. The ability of the cyclophanes and their dinuclear Cu(2+) and Zn(2+) complexes to cleave the mRNA cap structure, m(7)G(5')pppG(5') (1), has been studied.     

Thiolation and 2-methylthio- modification of Bacillus subtilis transfer ribonucleic acids.

Six thionucleosides found in Bacillus subtilis transfer ribonucleic acids were investigated: N6-(delta 2-isopentenyl)-2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, 2-methylthioadenosine, N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, and one unknown (X1). The presence of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine was demonstrated based on the affinity of the transfer ribonucleic acid containing it for an immunoadsorbent made with the antibody directed toward N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine. The existance of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine in two species of lysine transfer ribonucleic acids was also confirmed by high-resolution mass spectrometry. Four of these thionucleosides--N6-(delta 2-isopenenyl)-2-methylthioadenosine, 2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, and the unknown designated X1--occurred only in specific areas in the elution profile of an RPC-5 column and probably affect the chromatographic properties of the transfer ribonucleic acids containing them. In contrast with Escherichia coli, where 4-thiouridine is the most frequent type of sulfur-containing modification, approximately one-third of the sulfur groups in B. subtilis transfer ribonucleic acid are present as thiomethyl groups on the 2 position of an adenosine or modified adenosine residue.     

DNA polymerase action blocked by adenine adducts induced by 5-hydroxymethylchrysene sulfate.

Modification of M13mp10 single-stranded DNA with 5-hydroxymethylchrysene (5HCR) sulfate, the ultimate carcinogenic metabolite of 5-methylchrysene, resulted in formation of N6[(chrysen-5-yl)methyl]adenine and N2[(chrysen-5-yl)methyl]-guanine at the ratio of 2.7:1. Measurement of DNA synthesis using this modified template and E.coli DNA polymerase I (Klenow fragment) demonstrated that increasing levels of adducts caused a progressive decline in replication. Analysis of reaction products on DNA-sequence gels revealed DNA elongation to be arrested exclusively at adenine adducts in -AAAGGA- and -AACA- sequences.     

A convenient synthesis of bifunctional chelating agents based on diethylenetriaminepentaacetic acid and their coordination chemistry with yttrium(III)

The bifunctional chelating agents N,N,N',N',N'-pentakis(carboxymethyl)-1- [(4-aminophenyl)methyl]-diethylenetriamine and N,N,N',N',N'-pentakis(carboxymethyl)-1-[(4-aminophenyl)methyl]-4- methyldiethylenetriamine were prepared in six-step syntheses in overall yields of 38% and 31%, respectively. The use of bromoacetate esters in the synthesis allowed large-scale flash chromatographic purification of reaction products. The synthesis of N,N,N',N',N'-pentakis(carboxymethyl)-1- [(4-aminophenyl)-methyl]-4-methyldiethylenetriamine resulted in a mixture of two diastereomers. Chelation of yttrium-(III) with these bifunctional chelating agents resulted in 1:1 chelates. In the case of N,N,N',N',N'-pentakis(carboxymethyl)-1-[4- aminophenyl)methyl]diethylenetriamine, two diastereomers were observed upon chelation, as expected. In the case of N,N,N',N',N'- pentakis(carboxymethyl)-1-[(4-aminophenyl)-methyl]-4- methyldiethylenetriamine, only three of the four anticipated diastereomers were observed.     

Synthesis and spectroscopic properties of reconstituted zinc-myoglobin appending a DNA-binding platinum(II) complex

A reconstituted zinc-myoglobin (ZnMb) dyad, ZnMb-[Pt(bpy)(en)]2+, has been prepared by incorporating chemically-modified zinc-porphyrin, being capable of DNA-binding of the Pt complex, [Pt(bpy)(en)]2+, where bpy and en are 2,2'-bipyridine and ethylenediamine, respectively. The steady-state fluorescence of the cofactor, [Pt(bpy)(mu-enPP)Zn]Cl2, in MeOH indicates that the excited singlet state of zinc--porphyrin was almost quenched, probably because of the strong hydrophobic and pi-pi stacking interactions between the [Pt(bpy)(mu-enPP)Zn]2+ ions. In the reconstituted ZnMb-[Pt(bpy)(en)]2+, the quenching reaction of 1(ZnMb)* with the [Pt(bpy)(en)]2+ moiety does not occur, indicating apo-Mb matrix is essential. On the other hand, when the [Pt(bpy)(en)]2+ moiety was excited, the enhancement of the fluorescence from ZnMb unit was observed. It is suggested that the energy transfer from (1)([Pt(bpy)(en)]2+)* to ZnMb occurs. The spectroscopic changes of ZnMb-[Pt(bpy)(en)]2+ in the presence of calf-thymus DNA were also provided. Soret band at 428 nm gradually decreased, and isosbestic points at 321, 414, and 432 nm were observed with increasing the DNA concentration. When the Pt(II) moiety was excited at lambda(ex) 321 nm, the fluorescence signal around 600 nm similarly decreased. The synthetic manipulation of ZnMb by using a DNA-binding Pt(II) complex demonstrates sensitive fluorescent signal for DNA and valuable information to study photoinduced electron transfer within a Mb-DNA complex.     

Cadmium-109 as a probe of the metal binding sites in horse liver alcohol dehydrogenase.

The noncatalytic and catalytic zinc atoms of horse liver alcohol dehydrogenase, [(LADH)Zn2Zn2] or LADH, have been replaced differentially with 109Cd by equilibrium dialysis, resulting in two new enzymatically active species, [(LADH)109Cd2Zn2] and [(LADH)109Cd2109Cd2]. The UV difference spectra of the cadmium enzymes vs. native [(LADH)Zn2Zn2] reveal maxima at 240 nm with molar absorptivities, delta epsilon 240, of 1.6 X 10(4) M-1 cm-1 per noncatalytic 109Cd atom and 0.9 X 10(4) M-1 cm-1 per catalytic 109Cd atom, consistent with coordination of the metals by four and two thiolate ligands, respectively, strikingly similar to the 250-nm charge-transfer absorbance in metallothionein. Carboxymethylation of the Cys-46 ligand to the catalytic metal in LADH presumably lowers the overall stability constant of the coordination complex and results in loss of catalytic 109Cd or catalytic cobalt but not catalytic zinc from the enzyme.     

Characterization of indo-1 and quin-2 as spectroscopic probes for Zn2(+)-protein interactions

1-[2-Amino-5-(6-carboxyindol-2-yl)phenoxyl]-2-(2'- amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (indo-1) and 2-[2-(bis(carboxymethyl)amino-5-methylphenoxy) methyl]-6- methyl-8-[bis-(carboxymethyl)amino]quinoline (quin-2) are sensitive, spectral indicators for Zn2+. Additions of subsaturating Zn2+ to 10-80 microM indo-1 or quin-2 at pH 7.0 produce uv difference spectra with isosbestic wavelengths at 342 and 282 nm or at 342, 317, and 252 nm, respectively. Formation of 1:1 Zn2+:indicator complexes at pH 7.0 and 20 degrees C in the absence (presence) of 100 mM KCl gives delta epsilon max = -2.4 +/- 0.2 X 10(4) M-1 cm-1 at 367 nm (-2.1 +/- 0.2 X 10(4) M-1 cm-1 at 365 nm) for indo-1 and delta epsilon max = -2.7 +/- 0.1 X 10(4) M-1 cm-1 at 266 nm (-2.6 +/- 0.1 X 10(4) M-1 cm-1 at 265 nm) for quin-2. Competition experiments at pH 7.0 and 20 degrees C with indo-1 and quin-2 and also 4-(2-pyridylazo)resorcinol (PAR) as the second chelator in the absence (presence) of 100 mM KCl yield apparent affinity constants: K'A = 2.5 +/- 1.0 X 10(10) M-1 (6.2 +/- 0.5 X 10(9) M-1) for indo-1 binding Zn2+ and K'A = 9.4 +/- 3.3 X 10(11) M-1 (2.7 +/- 0.1 X 10(11) M-1) for quin-2 binding Zn2+. The above constants provide the basis for rapid steady-state spectrophotometric determinations of the affinity of a protein for Zn2+ with K'A approximately 10(10) - 10(13) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

The thulium complex of 1,4,7,10-tetrakis{[N-(1H-imidazol-2-yl)carbamoyl]methyl}-1,4,7,10-tetraazacyclododecane (dotami) as a ParaCEST contrast agent

1,4,7,10-Tetrakis{[N-(1H-imidazol-2-yl)carbamoyl]methyl}-1,4,7,10-tetraazacyclododecane (dotami), a tetra(1H-imidazol-2-yl) derivative of the well-studied octadentate 1,4,7,10-tetrakis[(carbamoyl)methyl]-1,4,7,10-tetraazacyclododecane (dotam) ligand, was synthesized by reaction of 1,4,7,10-tetraazacyclododecane with N-(1H-imidazol-2-yl)chloroacetamide in high yield. Its tricationic thulium complex was isolated as a water-soluble chloride salt. The detection of the mildly acidic amide and amine protons by direct proton NMR in aqueous solution was unsuccessful, but such exchangeable protons could be detected via their chemical exchange-dependent saturation transfer (CEST) effect. The observed CEST effect was distinctly different from that found for respective dotam complexes and is, therefore, ascribed to exchangeable protons associated with the imidazole substituent.     

Synthesis and structure-activity relationships of N-{1-[(6-fluoro-2-naphthyl)methyl]piperidin-4-yl}benzamide derivatives as novel CCR3 antagonists

A novel class of potent CCR3 receptor antagonists were designed and synthesized starting from N-{1-[(6-fluoro-2-naphthyl)methyl]piperidin-4-yl}benzamide (1),which was found by subjecting our chemical library to high throughput screening (HTS). The CCR3 inhibitory activity of the synthesized compounds against eotaxin-induced Ca(2+) influx was evaluated using CCR3-expressing preB cells. Systematic chemical modifications of 1 revealed that the 6-fluoro-2-naphthylmethyl moiety was essential for CCR3 inhibitory activity in this new series of CCR3 antagonists. Further structural modifications of the benzamide and piperidine moieties of 1 led to the identification of exo-N-{8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3- yl}biphenyl-2-carboxamide [corrected] (31) as a potent CCR3 antagonist with an IC(50) value of 0.020 microM.     

Determination of endogenous melatonin in the individual pineal glands of inbred mice using precolumn oxidation reversed-phase micro-high-performance liquid chromatography

The amount of endogenous melatonin in the individual pineal glands of inbred mice has been determined using reversed-phase micro-high-performance liquid chromatography after precolumn oxidation of melatonin to a compound having strong fluorescence. The fluorescent compound was identified as N-[(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide. The excitation and emission wavelengths of this compound are 245 and 380 nm, respectively, and the fluorescence intensity is 6.8 times greater than that of melatonin. Molar absorptivity and fluorescence quantum yield of this compound are 46,300[L mol(-1)cm(-1)] and 0.31 (245 nm), respectively. The lower quantification limit of melatonin in biological samples using this precolumn oxidation method is 200 amol, and the calibration curve of spiked melatonin is linear from 200 amol to 50 fmol (r>0.999). The sensitivity of the present method is almost 10 times higher than that of the previous method. The values of endogenous melatonin obtained for ICR, C57BL, BALB/c, and AKR mice are 4.7, 6.1, 7.4, and 18.8 fmol/pineal gland, respectively. The amounts of endogenous pineal melatonin of these strains had not been clearly reported due to the poor enzymatic activities for melatonin biosynthesis; this is the first report that clearly demonstrates the existence of endogenous melatonin in these inbred mice.     

Regioselective enzymatic aminoacylation of lobucavir to give an intermediate for lobucavir prodrug

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N′-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC™ BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield).     

In Silico Analog Design for Terbinafine Against Trichophyton rubrum: A Preliminary Study

The diseases caused by dermatophytes are common among several other infections which cause serious threat to human health. It is evident that enzyme squalene epoxidase is responsible for prolonged dermatophyte infection and it is appealing to note that this enzyme is also responsible for fatty acid synthesis in these groups of fungi. In the present study, terbinafine drug which targets enzyme squalene epoxidase has been explored to design its various novel analogues. The present study suggests that many more prominent drug analogues could be constituted which may be crucial towards designing new drug candidates. In the present study, we have designed a series of such analogues viz. [(2E)-6,6-dimethylhept-2-en-4-yn-1-yl](methyl)(naphthalen-1-ylmethyl)amine, N-[8-({[(2E)-6,6-dimethylhept-2-en-4-yn-1-yl](methyl)amino}methyl)naphthalen-1-yl]-2-(sulfoamino) acetamide, {[4-(dihydroxyamino)-8-({[(2E)-6,6-dimethylhept-2-en-4-yn-1-yl](methyl)amino}methyl)naphthalen-1-yl]sulfanyl}methanol and (R)-{[4-({[(2E,6R)-6,7-dimethyloct-2-en-4-yn-1-yl](methyl)amino}methyl)-5-[(hydroxysulfamoyl)amino]naphthalen-1-yl]amino}sulfinic acid. Moreover, further by molecular docking approach the binding between enzyme and designed analogues was further analysed. The present preliminary report suggested a considerably good docking interaction score of −338.75 kcal/mol between terbinafine and squalene epoxidase from Trichophyton rubrum. This preliminary study implies that few designed candidate ligands can be effectual towards the activity of this enzyme and can play crucial role in pathogenesis control of T. rubrum.     

Pyridinium-carbaldehyde: active Maillard reaction product from the reaction of hexoses with lysine residues

Besides the formation of the aminotriazine N6-[4-(3-amino-1,2,4-triazin-5-yl)-2,3-dihydroxybutyl]-L-lysine, the reaction of [1-13C]D-glucose with lysine and aminoguanidine leads to the generation of 6-[2-([[amino(imino)methyl]hydrazono]methyl)pyridinium-1-yl]-L-norleucine (14-13C1). The dideoxyosone N6-(2,3-dihydroxy-5,6-dioxohexyl)-L-lysine was shown to be a precursor in the formation of 14-13C1, which proceeds via the reactive carbonyl intermediate 6-(2-formylpyridinium-1-yl)-L-norleucine (13-13C1). In order to study the reactivity of 13-13C1, the model compound 1-butyl-2-formylpyridinium (18) was prepared in a two-step procedure starting from 2-pyridinemethanol. The reaction of the pyridinium-carbaldehyde 18 with L-lysine yielded the Strecker analogous degradation product 2-(aminomethyl)-1-butylpyridinium and another compound, which was shown to be as 1-butyl-2-[(2-oxopiperidin-3-ylidene)methyl]pyridinium. Reaction of 18 with the C-H acidic 4-hydroxy-5-methylfuran-3(2H)-one leads to the formation of the condensation product 1-butyl-2-[hydroxy-(4-hydroxy-5-methyl-3-oxofuran-2(3H)-ylidene)methyl]-pyridinium.