Preparation and evaluation of soluble epoxide hydrolase inhibitors with improved physical properties and potencies for treating diabetic neuropathic pain

Soluble epoxide hydrolase (sEH), a novel therapeutic target for neuropathic pain, is a largely cytosolic enzyme that degrades epoxy-fatty acids (EpFAs), an important class of lipid signaling molecules. Many inhibitors of sEH have been reported, and to date, the 1,3-disubstituted urea has the highest affinity reported for the sEH among the central pharmacophores evaluated. An earlier somewhat water soluble sEH inhibitor taken to the clinic for blood pressure control had mediocre potency (both affinity and kinetics) and a short in vivo half-life. We undertook a study to overcome these difficulties, but the sEH inhibitors carrying a 1,3-disubstituted urea often suffer poor physical properties that hinder their formulation. In this report, we described new strategies to improve the physical properties of sEH inhibitors with a 1,3-disubstituted urea while maintaining their potency and drug-target residence time (a complementary in vitro parameter) against sEH. To our surprise, we identified two structural modifications that substantially improve the potency and physical properties of sEH inhibitors carrying a 1,3-disubstituted urea pharmacophore. Such improvements will greatly facilitate the movement of sEH inhibitors to the clinic.     

Optimization of 1,3-disubstituted urea-based inhibitors of Zika virus infection

Zika virus (ZIKV) has become a public health concern worldwide due to its association with congenital abnormalities and neurological diseases. To date, no effective vaccines or antiviral drugs have been approved for the treatment of ZIKV infection, and new inexpensive therapeutic options are urgently needed. In this study, we have used an in vitro plaque assay to assess an antiviral activity of the second generation of anti-ZIKV compounds, based on 1,3-disubstituted (thio)urea scaffold. Several compounds in the library were found to possess excellent activity against Zika virus with IC₅₀ values <200 pM. The most active analog, A5 exhibited an exceptional IC₅₀ = 85.1 ± 1.7 pM. Further analysis delineated structural requirements necessary for potent antiviral effects of this class of compounds. Collectively, our findings suggest that 1,3-disubstituted (thio)urea derivatives are excellent preclinical candidates for the development of anti-ZIKV therapeutics.     

Structural requirement of phenylthiourea analogs for their inhibitory activity of melanogenesis and tyrosinase

Effect of a series of 1-phenylthioureas 1a-k and 1,3-disubstituted thioureas 2a-k were evaluated against melanin formation in melanoma B16 cell line and mushroom tyrosinase. Inhibitory activity of tyrosinase of 1-phenylthioureas 1a-k is parallel to their melanogenic inhibition. Thus, the melanogenic inhibition in melanoma B16 cells of 1-phenylthioureas could be the result of inhibition of tyrosinase. However, 1,3-diaryl or 1-phenyl-3-alkylthioureas, 2a-k, appears as melanogenic inhibitor without inhibition of tyrosinase. The molecular docking study of 1e and 2b to binding pocket of tyrosinase provided convincing explanation regarding the necessity of direct connection of planar phenyl to thiourea unit without N'-substitution of phenylthioureas 1 as tyrosinase inhibitor and 2 as non-tyrosinase inhibitor.     

Synthesis of glycerol 1,3-dihexadecyl ether

The synthesis of 1,3-dihexadecyloxy-2-propanol (glycerol 1,3-dihexadecyl ether) is reported. The method is applicable to the preparation of other 1,3-disubstituted glycerols where the substituents are not affected by acid or by catalytic hydrogenolysis conditions.     

Effects of mutations at threonine-654 on the insoluble glucan synthesized by Leuconostoc mesenteroides NRRL B-1118 glucansucrase

Twelve different amino acids were each substituted for threonine-654 in a cloned glucansucrase from Leuconostoc mesenteroides NRRL B-1118. Both the native and the cloned enzyme with threonine at position 654 produced a water-insoluble glucan containing approximately 44 mol% 1,3-disubstituted α-d-glucopyranosyl units and 29 mol% 1,6-disubstituted α-d-glucopyranosyl units. Several substitutions yielded an enzyme that produced an increased percentage of 1,3-disubstituted α-d-glucopyranosyl units, with corresponding decreases in 1,6-disubstituted α-d-glucopyranosyl units. Only one substitution, tyrosine, resulted in a significant increase in the percentage of 1,6-disubstituted α-d-glucopyranosyl units, with a concomitant increase in glucan yield. The mutated enzymes that produced the highest levels of 1,3-disubstituted α-d-glucopyranosyl units were also significantly activated by the addition of dextran, but glucan yields were also lower in these mutants.     

Cloning and tissue distribution of the human B3GALT7 gene, a member of the β1,3-Glycosyltransferase family

We report here the cloning and tissue distribution of the human B3GALT7 gene, a member of the beta1,3-Glycosyltransferase family, structurally related to the beta1,3-Galactosyltransferase family and beta1,3- N -acetylglucosaminyltransferase family, isolated from a human lung cDNA library. B3GALT7 is mapped to chromosome 19q13.2 by browsing the UCSC genomic database. It contains an ORF with length of 1191bp, encoding a protein with a signal peptide sequence and galactosyl-T domain, and its molecular weight and isoelectric point is predicted to be 43.3 kDa and 8.67 respectively. The molecular weight of the protein when expressed in E. coli corresponded to that expected. Northern blotting showed that B3GALT7 was highly expressed in lung, throat and ileum, whereas the expression level was low in tongue, breast, uteri, testis. In addition, it was also demonstrated that B3GALT7 is differentially transcribed in human tumor cell lines.     

1,3-disubstituted 4-aminopiperidines as useful tools in the optimization of the 2-aminobenzo[a]quinolizine dipeptidyl peptidase IV inhibitors

In a search for novel DPP-IV inhibitors, 2-aminobenzo[a]quinolizines were identified as submicromolar HTS hits. Due to the difficult synthetic access to this compound class, 1,3-disubstituted 4-aminopiperidines were used as model compounds for optimization. The developed synthetic methodology and the SAR could be transferred to the 2-aminobenzo[a]quinolizine series, leading to highly active DPP-IV inhibitors.     

A library construction of 2,5-disubstituted pyrrole compounds by using solid/solution-phase syntheses

Using solid- and solution-phase synthesis, a library of 2,5-disubstituted pyrrole compounds was constructed. This is the first report that Stetter reaction was applied to the solid-phase synthesis for C-C bond formation. Some of 2,5-disubstituted pyrrole compounds showed the inhibitory activity of LPS-induced mouse B-lymphocyte proliferation.     

Design and pharmacology of quinuclidine derivatives as M2-selective muscarinic receptor ligands

In our search for M2-selective muscarinic receptor antagonists, we synthesized 1,3-disubstituted indenes. The effects of different basic moieties with regard to binding and selectivity towards the five distinct muscarinic receptor subtypes were investigated. The results show that the quinuclidine series afforded the most promising compounds in terms of both receptor affinity and M2-subtype selectivity.     

Synthesis of new azino fused benzimidazolium salts. a new family of DNA intercalating agents. I

A series of new pyrido[1,2-a]- and pyridazino[1,6-a]benzimidazolium salts have been synthesized from readily available 1,3-disubstituted 2-alkylbenzimidazolium salts. Their affinity to DNA and in vitro cytotoxicity versus HT-29 have been tested. The initial results show that the title compounds are a new family of intercalating agents.     

A facile synthesis of 1,5-disubstituted-2-aminoimidazoles: antibiotic activity of a first generation library

An efficient synthetic route to 1,5-disubstituted 2-aminoimidazoles from readily available amino acids and aldehydes has been developed. A library of simple analogues was synthesized and several compounds were shown to exhibit notable antibiotic activity against a variety of bacterial strains including multi-drug resistant isolates.     

Determination of absolute configuration of conformationally flexible cis-dihydrodiol metabolites: effect of diene substitution pattern on the circular dichroism spectra and optical rotations

Absolute configurations of a number of cis-dihydrodiols (cis-1,2-dihydroxy-3,5-cyclohexadienes), synthetically useful products of TDO-catalyzed dihydroxylations of 1,2- and 1,3-disubstituted benzene derivatives, have been determined by a comparison of calculated and experimental CD spectra and optical rotations and by methods involving X-ray crystallography, 1H NMR spectra of diastereoisomeric derivatives, and by stereochemical correlations. The computations disclosed a significant effect of the substituents on conformational equilibria of cis-dihydrodiols and chiroptical properties of individual conformers. The assigned absolute configurations of cis-dihydrodiols have allowed the validity of a simple predictive model for TDO-catalyzed arene dihydroxylations to be extended.     

Microwave-assisted synthesis of imidazoles: reaction of p-toluenesulfonylmethyl isocyanide and polymer-bound imines

A convenient method for the synthesis of 1,5-disubstituted imidazoles has been developed on a polymeric support using base-promoted 1,3-dipolar cycloaddition reaction of p-toluenesulfonylmethyl isocyanide (TOSMIC) with immobilized imines under microwave irradiation. The immobilized imines were synthesized by the reaction of various primary benzyl amines with 4-formyl-3-methoxyphenoxymethyl polystyrene in the presence of trimethyl orthoformate at room temperature. Cleavage from the polymeric support using trifluoroacetic acid gave the desired 1,5-disubstituted imidazoles with excellent yield and high purity.     

Synthesis of 2'-([1,2,3]triazol-1-yl)-2'-deoxyadenosines

A reliable and efficient protocol for the synthesis of 2 '-([1,2,3]triazol-1-yl)-2 '-deoxyadenosine derivatives from vidarabine is presented. Vidarabine was converted to 2'-azido-2'-deoxy-3',5-O-(tetraisopropyldisiloxane-1,3-diyl)-adenosine. This azide was used as the starting material for the Cu(I)-catalyzed parallel synthesis of 1,2,3-triazoles using a variety of alkynes. The reactions proceeded in good yield and gave almost exclusively the 1,4-disubstituted 1,2,3-triazoles.     

Synthesis and SAR of 1,3-disubstituted cyclohexylmethyl urea and amide derivatives as non-peptidic motilin receptor antagonists

A series of 1,3-disubstituted cyclohexylmethyl urea and amide derivatives were synthesized as motilin receptor antagonists. Starting from known motilin antagonists, 1a and 1b, the cyclopentene scaffold was replaced and the four recognition elements optimized to arrive at a potent novel series.     

Design and Synthesis of 2′-Deoxy-2′-[(1,2,3)Triazol-1-Yl]Uridines Using Click Chemistry Approach

A series of novel nucleosides bearing a 1,2,3-triazole moiety at the 2′-position of the sugar moiety has been synthesized starting from 2′-azidouridine and using the copper (I)-catalyzed Huisgen–Sharpless–Meldal 1,3-dipolar cycloaddition reaction. The reactions proceeded in overall yield of 52–82% and gave almost exclusively the 1,4-disubstituted 1,2,3-triazoles. The 2′-azidouridine was synthesized from uridine in two steps, and reacted with a variety of differently substituted alkynes to give the desired 2′-triazole-substituted uridine derivatives.     

Cloning of two genes from Bacillus circulans WL-12 which encode 1,3-beta-glucanase activity

Two genes encoding distinct 1,3-beta-glucanases have been cloned from Bacillus circulans and expressed in Escherichia coli. A cosmid library of B. circulans WL-12 DNA was constructed in the broad-host-range cosmid pLAFR1 and screened in E. coli for clones which exhibited 1,3-beta-glucanase activity. Two 1,3-beta-glucanase-positive clones were identified which contained genes encoding two independent 1,3-beta-glucanases as shown by biochemical, physical and molecular analyses. The cosmids, designated pMON5401 (27.1 kb insert) and pMON5402 (24.7 kb insert), encoded 68 kDa and 40 kDa 1,3-beta-glucanases, respectively. Both 1,3-beta-glucanases were purified from their respective E. coli strains, characterized biochemically, and were shown to exhibit lytic activity against purified yeast cell wall preparations.     

Non-nucleoside inhibitors of the hepatitis C virus NS5B polymerase: discovery and preliminary SAR of benzimidazole derivatives

Benzimidazole 5-carboxamide derivatives from a combinatorial screening library were discovered as specific inhibitors of the NS5B polymerase of the hepatitis C virus (HCV). Initial hit-to-lead activities taking advantage of high-throughput parallel synthetic techniques, identified a 1,2-disubstituted benzimidazole 5-carboxylic acid scaffold as the minimum core for biological activity. Potent analogues in this series inhibit the polymerase at low micromolar concentrations and provide an attractive "drug-like" lead structure for further optimization and the development of potential HCV therapeutics.