New reagents for the introduction of the thiomethyl group at sulfhydryl residues of proteins with concomitant spectrophotometric titration of the sulfhydryls: Methyl 3-nitro-2-pyridyl disulfide and methyl 2-pyridyl disulfide

Two new reagents for the titration of sulfhydryl groups in peptides and proteins and for their temporary blocking with the thiomethyl group have been developed. The sulfhydryl groups in cysteine, glutathione, and papain react quantiatively under mild conditions with these reagents, methyl 3-nitro-2-pyridyl disulfide and methyl 2-pyridyl disulfide, with concomitant methanethiolation and without the need to employ a large excess of reagent. Because of the chromophoric properties of the 3-nitro-2-thiopyridone and 2-thiopyridone products, spectrophotometric titration of the sulfhydryl groups can be carried out, accompanying their methanethiolation. The modification of the sulfhydryl groups in peptides and proteins with thiomethyl is rapidly and completely reversible upon addition of thiols such as l-cysteine.     

"Marking" the nitrogen atoms of phenyl-(2-pyridyl)-(3-pyridyl)-(4-pyridyl)-methane. Synthesis and absolute configuration of the corresponding tris(pyridine N-oxide)

To "mark" the nitrogen atoms in phenyl-(2-pyridyl)-(3-pyridyl)-(4-pyridyl)methane (1), we have synthesized the corresponding tris(pyridine N-oxide) 2 by oxidation of 1 with m-chloroperbenzoic acid. The nitrogen atoms of 2 are unequivocally determined by the X-ray crystal analysis of a single crystal of rac-2 whereas the nitrogen atoms cannot be assigned at all in the case of rac-1. N-Oxide 2 can be resolved by chiral high-performance liquid chromatography under similar conditions to those used for the resolution of 1. The calculated circular dichroism (CD) curve for (R)-2 on the basis of time-dependent density functional theory reproduces the experimental spectra very well to suggest that the second-eluted fraction ([CD(+)283]-2) is the R isomer, namely (R)-[CD(+)283]-2. The independent absolute configuration determinations for 1 and 2 are in keeping with the chemical correlation between the two compounds by oxidation of (R)-1 into (R)-2.     

QSAR studies of N(1)-(5-chloro-2-pyridyl)-2-{[4-(alkyl methyl)benzoyl]amino}-5-chlorobenzamide analogs

Factor X(a) has materialized as a key enzyme for the intervention of blood coagulation cascade and for the development of new antithrombotic agents. It is the lone enzyme that is responsible for the production of thrombin and is therefore an attractive target for the control of thrombus formation. The biological activities (log1/IC(50)) of anthranilamide-based factor X(a) inhibitors were quantitatively analyzed in terms of physicochemical parameters by the regression analysis. Structural requirements for maximal potency were derived from the results of a quantitative structure activity relationship analysis. The leave-one-out cross-validation method was used to judge the predictive power of final equations.     

Synthesis of methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside and methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside

Treatment of methyl 3-O-benzyl-2-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha-D- mannopyranoside (1) with tert-butyldiphenylsilyl chloride in N,N-dimethylformamide afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-mannopyranoside (2). Oxidation of 2 with pyridinium chlorochromate, followed by reduction of the carbonyl group, and subsequent O-deacetylation afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-alpha-D-mannopyranosyl- alpha-D- talopyranoside (5). Cleavage of the tert-butyldiphenylsilyl group of 5 with tetrabutylammonium fluoride in oxolane, followed by hydrogenolysis, gave methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside (7). O-Deacetylation of 1 gave methyl 3-O-benzyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (8). Treatment of 8 with tert-butyldiphenylsilyl chloride afforded a 6,6'-disilyl derivative, which was converted into a 2',3'-O-isopropylidene derivative, and then further oxidized with pyridinium chlorochromate. The resulting diketone was reduced and removal of the protecting groups gave methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside (15). The structures of both 7 and 15 were established by 13C-n.m.r. spectroscopy.     

Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Br?nsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.     

Synthesis of the 3- and 4-methyl, 3,4-dimethyl, and 3,4,6-trimethyl ethers of methyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside

The methyl ethers of 2-amino-2-deoxy-D-mannose are reference compounds in studies, by the methylation procedure, of the chemical structure of polysaccharides containing 2-amino-2-deoxy-D-mannose and 2-amino-2-deoxy-D-mannuronic acid residues. Methylation of methyl 2-acetamido-2-deoxy-α-D-mannopyranoside (1) gave the 3,4,6-trimethyl ether. Methylation of the 6-trityl ether of 1, followed by detritylation, gave the 3,4-dimethyl ether of 1. Methylation of the 4,6-O-benzylidene derivative (6) of 1, followed by removal of the benzylidene group, gave the 3-methyl ether of 1. Benzoylation of 6, followed by removal of the benzylidene group and monobenzoylation, gave the 3,6-dibenzoate of 1, which was methylated, and the product saponified, to give the 4-methyl ether of 1; the latter compound was also obtained by a similar route via the 3-O-acetyl-6-O-benzoyl derivative.     

Identification of 2-(4-pyridyl)thienopyridinones as GSK-3β inhibitors

The discovery of a novel series of 2-(4-pyridyl)thienopyridinone GSK-3β inhibitors is reported. X-ray crystallography reveals its binding mode and enables rationalization of the SAR. The initial optimization of the template for improved cellular activity and predicted CNS penetration is also presented.     

Synthesis of [26,27-2H6]brassinosteroids from 23,24-bisnorcholenic acid methyl ester

A number of hexadeuterated brassinosteroids (BS) containing a hydroxy group at C-22 or a 22R,23R-diol function were prepared starting from 23,24-bisnorcholenic acid methyl ester for biosynthetic studies. Synthesis of the cyclic part was accomplished via the initial hydroboration-oxidation of Delta(5)-double bond. The key step in the synthesis of the side chain involved addition of (2S)-[3,4-(2)H(6)]2,3-dimethylbutylphenyl sulfone to the corresponding C-22 aldehydes.     

Synthesis and biological activities of 2-[(heteroaryl)methyl]imidazolines

A series of 2-[(heteroaryl)methyl]imidazolines was synthesized and tested for their activities at α(1)- and α(2)-adrenoceptors and imidazoline I(1) and I(2) receptors. The most active 2-[(indazol-1-yl)methyl]imidazolines showed high or moderate affinities for α(1)- and α(2)-adrenoceptors. However, their intrinsic activities at α(2A)-adrenoceptors proved to be negligible. A selected 7-chloro derivative behaved as a potent α(1)-adrenoceptor antagonist and exhibited peripherally mediated hypotensive effects in rats.     

Re(I)(tricarbonyl) complexes with the 2-(2-pyridyl)-N-methyl-benzimidazole, 2-(2-pyridyl)benzoxazole and 2-(2-pyridyl)benzothiazole ligands - syntheses, structures, electrochemical and spectroscopic studies

The reactions of Re(CO)₅Cl with the chelating ligands 2-(2-pyridyl)-N-methylbenzimidazole, 2-(2-pyridyl)benzoxazole and 2-(2-pyridyl)benzothiazole afforded neutral fac-Re(CO)₃(L)Cl and ionic complexes with structures confirmed by means of X-ray measurements. UV-vis absorption and emission properties have been studied at room and 77 K temperatures in order to determine the nature of the lowest electronically excited states. Electrochemical behaviour of the investigated fac-Re(CO)₃(L)Cl and complexes has been studied using cyclic and square-wave voltammetry. Preliminary results from the electrogenerated chemiluminescence studies of the ionic and the neutral fac-Re(CO)₃(MPBI)Cl complexes are briefly presented.     

Synthesis and chemical characterization of Cu, Ni and Zn complexes of 3,5-bis(2′-pyridyl)-1,2,4-oxadiazole and 3-(2′-pyridyl)5-(phenyl)-1,2,4-oxadiazole ligands

The synthesis and structural characterization of Ni^II, Cu^II and Zn^II complexes of two chelating 1,2,4-oxadiazole ligands, namely 3,5-bis(2′-pyridyl)-1,2,4-oxadiazole (bipyOXA) and 3-(2′-pyridyl)5-(phenyl)-1,2,4-oxadiazole (pyOXA), is here reported. The formed hexacoordinated metal complexes are [M(bipyOXA)₂(H₂O)₂](ClO₄)₂ and [M(pyOXA)₂(ClO₄)₂], respectively (M = Ni, Cu, Zn). X-ray crystallography, ¹H and ¹³C NMR spectroscopy and C, N, H elemental analysis data concord in attributing them an octahedral coordination geometry. The two coordinated pyOXA ligands assume a trans coplanar disposition, while the two bipyOXA ligands are not. The latter result is a possible consequence of the formation of H-bonds between the coordinated water molecules and the nitrogen atom of the pyridine in position 5 of the oxadiazole ring. The expected splitting of the d metal orbitals in an octahedral ligand field explains the observed paramagnetism of the d⁸ and d⁹ electron configuration of the nickel(II) and copper(II) complexes, respectively, as determined by the broadening of their NMR spectra.     

Two forms of a copper(II) complex of 3-phenyl-5-(2-pyridyl)-4-(4-pyridyl)-4H-1,2,4-triazole: a six-coordinate monomer versus a five-coordinate polymer

The reaction of the new bidirectional ligand 3-phenyl-5-(2-pyridyl)-4-(4-pyridyl)-4H-1,2,4-triazole (pyppt) with Cu(ClO₄)₂ · 6H₂O in a 2:1 molar ratio in EtOH affords the complex [Cu^II(pyppt)₂(ClO₄)₂] · H₂O (1) as a microcrystalline turquoise solid. Recrystallisation of complex 1 from MeCN by vapour diffusion of Et₂O gives blue crystals of the monomeric octahedral complex [Cu^II(pyppt)₂(ClO₄)₂] · MeCN (2). In contrast, addition of EtOH to a solution of complex 1 in MeCN followed by slow evaporation yields blue crystals of the five-coordinate polymeric complex {[Cu^II(pyppt)₂](ClO₄)₂ · EtOH}_∞ (3). The structures of both complexes have been determined by single crystal X-ray diffraction.     

Synthesis and biological evaluation of 2- and 4-hydroxymestranol

The synthesis of 2-hydroxy and 4-hydroxymestranol by oxidation of mestranol with m-chloroperbenzoic acid is described. The oral estrogenicity and contragestational activity of these and related catechol estrogen derivatives in the rat is also presented.     

Synthesis and Antiviral Evaluation of Novel 2,3-Dihydroxypropyl Nucleosides from 2- and 4-Thiouracils

Regioselective alkylation of 2-thiouracils 1a–c and 4-thiouracils 7a,b with 2,3-O-isopropylidene-2,3-dihydroxypropyl chloride (2) afforded 2-{[(2,2-Dimethyl-1,3-dioxolan-4-yl) methyl]thio}pyrimidin-4(1H)-ones 3a–c and 4-{[(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl]thio} pyrimidin-2(1H)-ones 8a,b, respectively. Further alkylation with 2 and/or 2,3-O-isopropylidine-1-O-(4-toluenesulfonyl)-glycerol (4) gave the acyclo N-nucleosides 5a–c and 9a,b whose deprotection afforded 6a–c and 10a,b. 2-(Methylthio)pyrimidin-4(1H)-ones 11a–c and 4-(methylthio)pyrimidin-2(1H)-ones 14a,b were treated with 2 and/or 4 to give 12a–c and 15a,b which were deprotected to give 13a–c and 16a,b. Pyrimidine-2,4(1H,3H)-dithiones 17a–c were treated with two equivalents of 2 to give 2,4-bis{[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]thio}pyrimidines 18a–c. Deprotection of compounds 18a–c gave 2,4-bis[(2,3-dihydroxypropyl)thio]pyrimidines 19a-c. The activity of the deprotected nucleosides against Hepatitis B virus was evaluated and showed moderate inhibition activity against HBV with mild cytotoxicity.     

4-(2-pyridyl)piperazine-1-carboxamides: potent vanilloid receptor 1 antagonists

A series of 4-(2-pyridyl)piperazine-1-carboxamide analogues based on the lead compound 1 was synthesized and evaluated for VR1 antagonist activity in capsaicin-induced (CAP) and pH (5.5)-induced (pH) FLIPR assays in a rat VR1-expressing HEK293 cell line. Potent VR1 antagonists were identified through SAR studies. From these studies, 18 was found to be very potent in the in vitro assay [IC(50)=4.8 nM (pH) and 35 nM (CAP)] and orally available in rat (F%=15.1).