N-Alkenyl and cycloalkyl carbamates as dual acting histamine H3 and H4 receptor ligands

Previous studies have shown that several imidazole derivatives possess affinity to histamine H(3) and H(4) receptors. Continuing our study on structural requirements responsible for affinity and selectivity for H(3)/H(4) receptor subtypes, two series of 3-(1H-imidazol-4-yl)propyl carbamates were prepared: a series of unsaturated alkyl derivatives (1-9) and a series possessing a cycloalkyl group different distances to the carbamate moiety (10-13). The compounds were tested for their affinities at the human histamine H(3) receptor, stably expressed in CHO-K1 cells. Compounds 1, 2, 5-7, 10-13 were investigated for their affinities at the human histamine H(4) receptor co-expressed with Gα(i2) and Gβ(1)γ(2) subunits in Sf9 cells. To expand the pharmacological profile, compounds were further tested for their H(3) receptor antagonist activity on guinea pig ileum and in vivo after oral administration to mice. All tested compounds exhibited good affinity for the human histamine H(3) receptor with K(i) values in the range from 14 to 194nM. All compounds were active in vivo after peroral administration (p.o.) to Swiss mice, thus demonstrating their ability to cross the blood-brain barrier. The most potent H(3) receptor ligand of these series was compound 5, 3-(1H-imidazol-4-yl)propyl pent-4-enylcarbamate with the highest affinity (K(i)=14nM). Additionally, compound 3 showed remarkable central nervous system (CNS) H(3)R activity, increasing the N(τ)-methylhistamine levels in mice with an ED(50) value of 0.55mg/kg, p.o. evidencing therefore, a twofold increase of inverse agonist/antagonist potency compared to the reference inverse agonist/antagonist thioperamide. In this study, the imidazole propyloxy carbamate moiety was kept constant. The different lipophilic moieties connected to the carbamate functionality in the eastern part of the molecule had a range of influences on the human H(4) receptor affinity (154-1326nM).     

Synthesis and cytotoxicity of 3,4-diaryl-2(5H)-furanones

A series of 3,4-diaryl-2(5H)-furanone derivatives were synthesized and evaluated for their cytotoxicity in a small panel of cancer cell lines. Four out of 10 compounds in this series, for example 3-(3,4,5-trimethoxyphenyl)-4-(4-methoxyphenyl)-, 3-(3,4,5-trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-, 3-(3,4,5-trimethoxyphenyl)-4-(3-amino-4-methoxyphenyl)-, and 3-(3,4,5-trimethoxyphenyl)-4-(2-naphthyl)-2(5H)-furanones, were found to have potent cytotoxic activities with ED50 values of less than 20 nM in most of the cell lines tested.     

New high affinity H3 receptor agonists without a basic side chain

In this study, we replaced the basic amine function of the known histamine H(3) receptor agonists imbutamine or immepip with non-basic alcohol or hydrocarbon moieties. All compounds in this study show a moderate to high affinity for the cloned human H(3) receptor and, unexpectedly, almost all of them act as potent agonists. Moreover, in the alcohol series, we consistently observed an increased selectivity for the human H(3) receptor over the human H(4) receptor, but none of the compounds in this series possess increased affinity and functional activity compared to their alkylamine congeners. In this new series of compounds VUF5657, 5-(1H-imidazol-4-yl)-pentan-1-ol, is the most potent histamine H(3) receptor agonist (pK(i) = 8.0 and pEC(50) = 8.1) with a 320-fold selectivity at the human H(3) receptor over the human H(4) receptor.     

Piperidine variations in search for non-imidazole histamine H(3) receptor ligands

Synthesis and biological evaluation of the novel histamine H(3) receptor ligands is described. Two series of ethers (aliphatic and aromatic) have been prepared by four different methods. Compounds were evaluated for their affinities at recombinant human H(3) receptor stably expressed in CHO cells. The ethers show from low to moderate in vitro affinities in nanomolar concentration range. The most potent compound was the 1-[3-(4-tert-butylphenoxy)propyl]-4-piperidino-piperidine 16 (hH(3)R K(i)=100 nM). Several members of the new series investigated under in vivo conditions, proved to be inactive.     

Kinetics and mechanism of the oxidation of D-fructose by vanadium(V) in H2SO4 medium

The oxidative degradation of D-fructose by vanadium(V) in the presence of H(2)SO(4) has an induction period followed by autoacceleration. The kinetics and mechanism of the induction period have been studied at constant ionic strength. The reaction was followed spectrophotometrically by measuring the changes in absorbance at 350 nm. Evidence of induced polymerization of acrylonitrile and of reduction of mercuric chloride indicates that a free-radical mechanism operates during the course of reaction. Vanadium(V) is only reduced to vanadium(IV). The reaction is first and fractional order in [V(V)] and [D-fructose], respectively; but dependence on [H+] is complex, that is, [equation: see text]. At constant [H2SO4], sodium hydrogensulfate accelerates the reaction. The effect of added sodium sulfate on the H2SO4 and HSO4-catalyzed reaction is also reported. The activation parameters Ea=118 kJ mol(-1), DeltaH#=116 kJ mol(-1), DeltaS#=-301 J K(-1) mol(-1), and DeltaG#=213 kJ mol(-1) are calculated and discussed. Reaction products are also examined, and it is concluded that oxidation of D-fructose by vanadium(V) involves consecutive one-electron abstraction steps.     

2,2-Dimethyl-4,5-diaryl-3(2H)furanone derivatives as selective cyclo-oxygenase-2 inhibitors

A series of 2,2-dimethyl-5-[4-(methylsulfonyl)phenyl]-4-phenyl-3(2H)furanones was prepared and evaluated for their ability to inhibit cyclo-oxygenase-2 (COX-2).     

Praseodymium methanesulfonate catalyzed one-pot synthesis of 3,4-dihydropyrimidin-2-(1H)-ones

A series of 3,4-dihydropyrimidin-2-(1H)-ones compounds was synthesized efficiently by a one-pot cyclocondensation of an aldehyde, 1,3-dicarbonyl compound, and urea in absolute ethanol under refluxing temperature using praseodymium methanesulfonate as catalyst. After the reaction, the catalyst can be easily recovered and reused several times without distinct decrease in reaction yields.     

Prostaglandin H synthase-mediated reaction of carcinogenic arylamines with tRNA and homopolyribonucleotides

Prostaglandin H synthase mediates the reaction of an extensive series of carcinogenic arylamines with tRNA. Structure-activity relationships suggest that benzidine is especially reactive due to extended conjugation between the 4,4'-diamino groups. In trapping experiments with homopolyribonucleotides, benzidine reacts with polyguanylic acid but 4-aminobiphenyl reacts with polycytidylic acid. The nitrenium ion of 4-aminobiphenyl (formed by N,O-acyltransferase activation of N-hydroxy-4-acetylaminobiphenyl) reacts primarily with polyguanylic acid and to a lesser extent with polyadenylic acid. The results suggest that arylamine activation by prostaglandin H synthase does not involve nitrenium ion formation.     

Tetrahydrobiopterin biosynthesis. Studies with specifically labeled (2H)NAD(P)H and 2H2O and of the enzymes involved

The biosynthesis of tetrahydrobiopterin from either dihydroneopterin triphosphate, sepiapterin, dihydrosepiapterin or dihydrobiopterin was investigated using extracts from human liver, dihydrofolate reductase and purified sepiapterin reductase from human liver and rat erythrocytes. The incorporation of hydrogen in tetrahydrobiopterin was studied in either 2H2O or in H2O using unlabeled NAD(P)H or (R)-(4-2H)NAD(P)H or (S)-(4-2H)NAD(P)H. Dihydrofolate reductase catalyzed the transfer of the pro-R hydrogen of NAD(P)H during the reduction of 7,8-dihydrobiopterin to tetrahydrobiopterin. Sepiapterin reductase catalyzed the transfer of the pro-S hydrogen of NADPH during the reduction of sepiapterin to 7,8-dihydrobiopterin. In the presence of partially purified human liver extracts one hydrogen from the solvent is introduced at position C(6) and the 4-pro-S hydrogen from NADPH is incorporated at each of the C(1') and C(2') position of BH4. Label from the solvent is also introduced into position C(3'). These results suggest that dihydrofolate reductase is not involved in the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. They are consistent with the assumption of the occurrence of a 6-pyruvoyl-tetrahydropterin intermediate, which is proposed to be formed upon triphosphate elimination from dihyroneopterin triphosphate, and via an intramolecular redox reaction. Our results suggest that the reduction of 6-pyruvoyl-tetrahydropterin might be catalyzed by sepiapterin reductase.     

Furoxan analogues of the histamine H3-receptor antagonist imoproxifan and related furazan derivatives

Synthesis and pharmacological characterisation of a series of compounds in which the oxime substructure present in imoproxifan was constrained in the pentatomic NO-donor furoxan ring, as well as their structurally related furazan analogues devoid of NO-donating properties, are described. The whole series of products displayed reversible histamine H3-antagonistic activity on guinea-pig ileum. 4-(4-(3-(1H-Imidazol-4-yl)propoxy)phenyl)furoxan-3-carbonitrile 16 was also able to induce partial relaxation when added to the bath after electrical contraction of the guinea-pig ileum during the study of its H3-antagonistic properties. This phenomenon seems to be dependent on NO-mediated sGC activation. The lipophilic-hydrophilic balance of all the products was investigated.     

Synthesis and structure-activity relationships of indole and benzimidazole piperazines as histamine H(4) receptor antagonists

We describe the structure-activity relationships for a series of ligands structurally related to the recently identified (5-chloro-1H-indol-2-yl)-(4-methyl-piperazin-1-yl)-methanone (1) as histamine H(4) receptor (H(4)R) antagonists. Furthermore, we identified related benzimidazoles as novel lead compounds for the H(4)R. The ligands have been evaluated by radioligand displacement studies and functional assays for their interaction with both the human histamine H(3) and H(4) receptors and exhibit pK(i) values up to 7.5 at the human H(4)R.     

Mode of formation of quinoxaline versus 2[1H]-quinoxalinone rings from dehydro-D-erythorbic acid.

The mode of formation of the quinoxaline versus 2[1H]-quinoxalinone rings by the reaction of o-diamines with dehydro-D-erythorbic acid has been investigated. The study was carried out by using one and two molar equivalents of 1,2-diamino-4,5-dimethylbenzene (3b) to give 6,7-dimethyl-3-(1-oxo-D-erythro-2,3,4-trihydroxybutyl)-2[1H]-quino xalinone (4b) and 2-(2-amino-4,5-dimethylphenylcarbamoyl)-3-(D-erythro-glycerol-1-yl )- 6,7-dimethylquinoxaline (6), respectively. The former product exists predominantly as the two furanosyl anomers. Sequential reaction of 4a with 3b has been studied, and the location of each diamine in the product was deduced by using 1H-n.m.r. spectroscopy. A mechanism for the reaction is proposed. Acetate and acetal derivatives of the compound are prepared.     

Role of histone pairs H2A,H2B and H3,H4 in the self-assembly of nucleosome core particles

The role of the histone pairs H2A,H2B and H3,H4 in the kinetics of core particle formation was investigated by using N-(1-pyrene)maleimide-labeled histone H3. The excimer emission intensity of a DNA-core histone complex prepared by direct mixing of DNA and histones in 0.2 m-NaCl is reduced by half when H2A,H2B is omitted. Fluorescence quenching studies and lifetime measurements indicate that the emission differences are probably due to static quenching. In a correctly folded nucleosome or a DNA-(H3,H4) complex, the two pyrene rings are buried and are held very close. DNA-(H3,H4) can interact with additional copies of H3,H4, but only when two dimers of H2A,H2B are correctly bound is there a specific twofold increase in excimer emission.The kinetics of the reaction of H3,H4 with DNA in 0.2 m-NaCl were followed by measuring the increase in 460 nm fluorescence. The apparent rate constant of the dominant kinetic component is ~ 2 × 10^?1 s^?1. If histones H2A,H2B are added immediately after the preparation of the DNA-(H3,H4) complex, an increase in excimer fluorescence is observed, with an apparent rate constant of ~ 6 × 10^?3 s^?1. However, if histones H2A,H2B are added one hour after DNA-(H3,H4) complex formation, there is no increase in excimer fluorescence. These results suggest that an intermediate involving the H3,H4 tetramer is formed first in nucleosome assembly. In the presence of H2A,H2B, this intermediate evolves to the final folded nucleosome, but in the absence of H2A,H2B it rearranges to an unmaturable dead-end complex. Additional experiments show that a very fast transfer of histone pairs (probably H2A,H2B) can take place between partially reconstituted nucleosomes.     

4-(1H-indazol-5-yl)-6-phenylpyrimidin-2(1H)-one analogs as potent CDC7 inhibitors

A series of 4-(4-hydroxyphenyl)-6-phenylpyrimidin-2(1H)-ones were identified by HTS as inhibitors of CDC7. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus on removing potential metabolic liabilities and improving cellular potency.     

Synthesis of Cyclobutane Nucleosides 2-Preparation of Thymine and Uracil Analogues

1-(2-Oxocyclobutyl-4-benzoyloxymethyl)-2,4(1H,3H)-pyrimidinedione and 1-(2-oxocyclobutyl-4-benzoyloxymethyl)-5-methyl-2,4(1H,3H)-pyrimidinedione can be prepared by reaction of uracil and thymine, respectively, with 3-benzoyloxymethyl-2-bromocyclobutanone. The N-alkylation gave both cis and trans isomers with the trans isomer predominating for uracil whereas the trans isomer was the only product which could be isolated for thymine. Both series were subjected to borohydride reduction followed by transesterification with methoxide giving the corresponding uracil and thymine nucleoside analogues. The uracil derivative 1-(2-oxocyclobutyl-4-benzoyloxymethyl)-2,4(1H,3H)-pyrimidinedione was irradiated in aqueous acetonitrile to generate isonucleoside analogues.     

Localization of two antigenic determinants in histone H4

Four overlapping synthetic peptides corresponding to the carboxy-terminal region 80-102 of histone H4 were prepared by solid-phase peptide synthesis. Their antigenic activity was analysed by inhibition of the H4-anti-H4 reaction in complement fixation and enzyme-linked immunosorbent assay. One antigenic determinant was localized in residues 88-96 of the H4 molecule. No antigenic activity was found in peptides 80-89 and 97-102. Antibodies induced by peptide 85-102 were found to bind to free H4 in solution but not to chromatin subunits, suggesting a lack of accessibility of the C-terminal region of H4 in nucleosomes. A second epitope was found to be situated in the N-terminal region 1-53 of histone H4.     

H/2H isotope effect in redox reactions of cytochrome c.

The rate of reaction of ferro- and ferricytochrome c (C(II) and C(III) with ferri- and ferrocyanide and of C(III) with 02- and CO2- was determined in H2O and in 2H2O in the temperature range 5-35 degrees C. No isotope effect was evident in any of the reductions of C(III); the apparent energy of activation was identical in H2O and 2H2O. An isotope effect with kH2O/k2H2O = 1.25 to 1.85, depending on pH for instance was observed in the oxidation of C(II), in the slow phase of oxidation which involves conformational changes. An interpretation (supported by evidence from previous work) involving water molecules in the close vicinity of the reaction site on the protein is discussed.     

Microwave-assisted synthesis of 2(1H)-pyridones and their glucosides as cell proliferation inhibitors

A new series of substituted 2(1H)-pyridones (4a-i) and their glucosides (5, 6a-e) were prepared as potential agents against leukemia (HL-60) cells. Glucosides (5,6a-e) were synthesized using three independent methods. Microwave protocol as an ecologically new method was used to synthesize the target compounds. Structures of the new products were confirmed using one- and two-dimensional NMR spectroscopy. In vitro exposure of pyridones substituted at position 4 with a 2-thienyl or 2-(trifluoromethyl)phenyl were found to exhibit high antiproliferation activities; in particular, 3-cyano-4-(thien-2'-yl)-6-(4'-chlorophenyl)-2(1H)-pyridone (4c) and its glucoside analogue (6c) had the highest activity.     

Synthesis and evaluation of pyridone-phenoxypropyl-R-2-methylpyrrolidine analogues as histamine H3 receptor antagonists

6-{4-[3-(R)-2-Methylpyrrolidin-1-yl)propoxy]-phenyl}-2H-pyridazin-3-one 6 (Irdabisant; CEP-26401) was recently reported as a potent H(3)R antagonist with excellent drug-like properties and in vivo activity that advanced into clinical evaluation. A series of pyridone analogs of 6 was synthesized and evaluated as H(3)R antagonists. Structure-activity relationships revealed that the 5-pyridone regiomer was optimal for H(3)R affinity. N-Methyl 9b showed excellent H(3)R affinity, acceptable pharmacokinetics and pharmaceutical properties. In vivo evaluation of 9b showed potent activity in the rat dipsogenia model and robust wake-promoting activity in the rat EEG model.     

The [4B-3H] NADH-H2O exchange reaction of the mitochondrial NADH dehydrogenase

The purified mitochondrial NADH dehydrogenase enzyme has been shown to catalyze a rapid [4B-3H] NADH-H2O exchange reaction. When the enzyme is subjected to a single freeze-thaw cycle there is a complete loss of NADH dehydrogenation without a measurable decrease in the [4B-3H] NADH-H2O exchange. Complete loss of the [4B-3H] NADH-H2O exchange follows brief exposure to ultraviolet photoirradiation. The differential sensitivity of the water exchange reaction and the dehydrogenase activity suggests a direct involvement of the enzymes flavin cofactor in the catalysis of the [4B-3H] NADH-H2O exchange. Arylazido-beta-alanyl NAD+ (A3'-0-[3-[N-4-azido-2-nitrophenyl)amino] propionyl]NAD+) is shown to be a potent photodependent inhibitor of the [4B-3H] NADH-H2O exchange activity following photoirradiation with visible light. This is consistent with the observed photodependent inhibition of the dehydrogenase activity by this photoprobe (Chen, S. and Guillory, R.J. (1981) J. Biol. Chem. 256, 8318-8323).