Thiobarbiturates as sirtuin inhibitors: virtual screening, free-energy calculations, and biological testing

NAD+-dependent histone deacetylases (sirtuins) are enzymes that cleave acetyl groups from lysine residues in histones and other proteins. Potent selective sirtuin inhibitors are interesting tools for the investigation of the biological functions of these enzymes and may be future drugs for the treatment of cancer or neurodegenerative diseases. Herein we present the results from a protein-based virtual screen of a commercial database with subsequent biological testing of the most promising compounds. The combination of docking and in vitro experimental testing resulted in the identification of novel sirtuin inhibitors with thiobarbiturate structure. To rationalize the experimental results, free-energy calculations were carried out by molecular mechanics Poisson-Boltzmann/surface area (MM-PBSA) calculations. A significant correlation between calculated binding free energies and measured Sirt2 inhibitory activities was observed. The analyses suggested a molecular basis for the interaction of the identified thiobarbiturate derivatives with human Sirt2. Based on the docking and MM-PBSA calculations we synthesized and tested five further thiobarbiturates. The MM-PBSA method correctly predicted the activity of the novel thiobarbiturates. The identified compounds will be used to further explore the therapeutic potential of sirtuin inhibitors.     

Ureidosulfocoumarin Derivatives As Selective and Potent Carbonic Anhydrase IX and XII Inhibitors

Owing to severe allergic reactions (anaphylaxis) and resistance exhibited by sulfonamide-based carbonic anhydrase (CA) inhibitors, non-classical or non-sulfonamide CA inhibitors are gaining increased attention by medicinal chemists. In this context, we report the design and synthesis of 30 new non-sulfonamide sulfocoumarin derivatives as CA inhibitors. They were investigated against hCA I and II (cytosolic isozymes) as well as hCA IX and XII (transmembrane, tumor-associated enzymes). All compounds showed prominent selectivity for the tumor-associated isoenzymes hCA IX and XII over the cytosolic isoenzymes hCA I and II. Among all synthesized compounds, 1-(2,2-dioxidobenzo[e][1,2]oxathiin-6-yl)-3-(o-tolyl)urea( 5 j )and1-(3-fluorophenyl)-3-(8-methoxy-2,2-dioxidobenzo[e][1,2]oxathiin-6-yl)urea( 5 q )were found to be more potent and to have better inhibition constant values against hCA IX than the standard acetazolamide (AAZ), with K_i values of 23.6 and 23.3 nM, respectively. All other compounds were found to be active under K_i=920 nM against hCA IX and XII.This study provides a new perspective for the future development of non-sulfonamide derivatives as selective CA inhibitors.     

Inhibitory pheromonal activity promoted by sulfur analogs of the sex pheromone of the female processionary mothThaumetopoea pityocampa (Denis and schiff)

New sulfur analogs of the sex pheromone of the female processionary mothThaumetopoea pityocampa have been found to be effective inhibitors of the natural pheromone activity both in EAG bioassays and field tests. The structures of these analogs have been derived from replacement of the oxygen atom(s) of the acetate group by sulfur (compounds 3-5) and the olefinic moiety of the enyne function by the isosteric SCH₂ group (compounds 6 and 7). The synthesis and biological activity of 3-[(Z)-12-pentadecen-10-ynylthio]-1,1,1-trifluoropropan-2-one (8), a closely related structure to the pheromone is also described.     

Substituting N��-thioacetyl-lysine for N��-acetyl-lysine in Peptide Substrates as a General Approach to Inhibiting Human NAD+-dependent Protein Deacetylases

Inhibitors of human NAD⁺-dependent protein deacetylases possess great value for deciphering the biology of these enzymes and as potential therapeutics for metabolic and age-related diseases and cancer. In the current study, we have experimentally demonstrated that, the potent inhibition we obtained previously for one of these enzymes (i.e. sirtuin type 1 (SIRT1)) by simply replacing N^ε-thioacetyl-lysine for N^ε-acetyl-lysine in its peptide substrate, represented a general and efficient strategy to develop potent and selective inhibitors of human NAD⁺-dependent protein deacetylase enzymes. Indeed, by using this simple inhibition strategy, potent (low-micromolar) and selective (≤40-fold) SIRT2 and SIRT3 inhibitors, which were either comparable or superior to currently existing inhibitors, have also been quickly identified in the current study. These inhibitors could be used as chemical biological tools or as lead compounds for further focused structure-activity optimization.     

Total Synthesis and Antileishmanial Activity of the Natural Occurring Acetylenic Fatty Acids 6-Heptadecynoic Acid and 6-Icosynoic Acid

The first total syntheses of the naturally occurring acetylenic fatty acids—6-heptadecynoic acid (59% overall yield) and 6-icosynoic acid (34% overall yield)—was accomplished in four steps. Using the same synthetic sequence the naturally occurring fatty acids (6Z)-heptadecenoic acid (46% overall yield) and (6Z)-icosenoic acid (27% overall yield) were also synthesized. The Δ⁶ acetylenic fatty acids displayed good antiprotozoal activity towards Leishmania donovani promastigotes (EC₅₀ = 1–6 μg/mL), but the 6-icosynoic acid was the most effective in the series. In addition, the (6Z)-icosenoic acid was a much better antiprotozoal compound (EC₅₀ = 5–6 μg/mL) than the (6Z)-heptadecenoic acid (EC₅₀ > 25 μg/mL). The saturated fatty acids n-heptadecanoic acid and n-eicosanoic acid were not effective towards L. donovani, indicating that the Δ⁶ unsaturation in these fatty acids is necessary for leishmanicidal activity. In addition, both the 6-icosynoic acid and the (6Z)-icosenoic acid were inhibitors of the Leishmania DNA topoisomerase IB enzyme (EC_50’s = 36–49 μM), a possible intracellular target for these compounds. This is the first study assessing fatty acids as inhibitors of the Leishmania DNA topoisomerase IB enzyme.     

Virtual Screening against p50 NF‐κB Transcription Factor for the Identification of Inhibitors of the NF‐κB–DNA Interaction and Expression of NF‐κB Upregulated Genes

Virtual screening against NF‐κB p50 using docking simulations was applied by starting from a three‐dimensional (3D) database containing more than 4.6 million commercially available structures. This database was filtered by specifying a subset of commercially available compounds sharing a (2E,Z)‐3‐(2‐hydroxyphenyl)‐2‐propenoate substructure and relevant druglike properties. Docking to p50 NF‐κB was performed with a test set of six known inhibitors of NF‐κB–DNA interactions. In agreement with docking results, the highest‐scored compound displayed a high level of inhibitory activity in electrophoretic mobility shift assay (EMSA) experiments (inhibition of NF‐κB–DNA interactions) and on biological functions dependent on NF‐κB activity (inhibition of IL‐8 gene expression in cystic fibrosis IB3‐1 cells). We found that this in silico screening approach is suitable for the identification of low‐molecular‐weight compounds that inhibit NF‐κB–DNA interactions and NF‐κB‐dependent functions. Information deduced from the discovery of the new lead compound and its binding mode could result in further lead optimization resulting in more potent NF‐κB inhibitors.     

Structure-activity relationships between stimulus molecule and response of a pheromone receptor cell in turnip moth,Agrotis segetum

The response of an antennal receptor cell of the turnip moth,Agrotis segetum, was recorded during stimulation with a series of (Z)-7-dodecenyl acetate analogs with structural variations of the acetate group. The investigated receptor cell is known to be highly selective to (Z)-7-dodecenyl acetate. All parts of the acetate group were found to be of great importance for full biological activity. The results indicate very strict requirements on the shape of the polar functional group, as well as on its electron distribution for a successful interaction with the antennal receptor cell.     

Synthesis of N‐Hydroxycinnamides Capped with a Naturally Occurring Moiety as Inhibitors of Histone Deacetylase

Histone deacetylase (HDAC) inhibitors are regarded as promising therapeutics for the treatment of cancer. All reported HDAC inhibitors contain three pharmacophoric features: a zinc‐chelating group, a hydrophobic linker, and a hydrophobic cap for surface recognition. In this study we investigated the effectiveness of osthole, a hydrophobic Chinese herbal compound, as the surface recognition cap in hydroxamate‐based compounds as inhibitors of HDAC. Nine novel osthole‐based N‐hydroxycinnamides were synthesized and screened for enzyme inhibition activity. Compounds 9 d , 9 e , 9 g exhibited inhibitory activities (IC₅₀=24.5, 20.0, 19.6 nM ) against nuclear HDACs in HeLa cells comparable to that of suberoylanilide hydroxamic acid (SAHA; IC₅₀=24.5 nM ), a potent inhibitor clinically used for the treatment of cutaneous T‐cell lymphoma (CTCL). While compounds 9 d and 9 e showed SAHA‐like activity towards HDAC1 and HDAC6, compound 9 g was more selective for HDAC1. Compound 9 d exhibited the best cellular effect, which was comparable to that of SAHA, of enhancing acetylation of either α‐tubulin or histone H3. Molecular docking analysis showed that the osthole moiety of compound 9 d may interact with the same hydrophobic surface pocket exploited by SAHA and it may be modified to provide class‐specific selectivity. These results suggest that osthole is an effective hydrophobic cap when incorporated into N‐hydroxycinnamide‐derived HDAC inhibitors.     

Rational Design and Synthesis of Selective PRMT4 Inhibitors: A New Chemotype for Development of Cancer Therapeutics**

Protein arginine N-methyl transferase 4 (PRMT4) asymmetrically dimethylates the arginine residues of histone H3 and nonhistone proteins. The overexpression of PRMT4 in several cancers has stimulated interest in the discovery of inhibitors as biological tools and, potentially, therapeutics. Although several PRMT4 inhibitors have been reported, most display poor selectivity against other members of the PRMT family of methyl transferases. Herein, we report the structure-based design of a new class of alanine-containing 3-arylindoles as potent and selective PRMT4 inhibitors, and describe key structure–activity relationships for this class of compounds.     

In Vitro and In Silico Evaluation of New 1,3,4-Oxadiazole Derivatives of Pyrrolo[3,4-d]pyridazinone as Promising Cyclooxygenase Inhibitors

Since long-term use of classic NSAIDs can cause severe side effects related mainly to the gastroduodenal tract, discovery of novel cyclooxygenase inhibitors with a safe gastric profile still remains a crucial challenge. Based on the most recent literature data and previous own studies, we decided to modify the structure of already reported 1,3,4-oxadiazole based derivatives of pyrrolo[3,4-d]pyridazinone in order to obtain effective COX inhibitors. Herein we present the synthesis, biological evaluation and molecular docking studies of 12 novel compounds with disubstituted arylpiperazine pharmacophore linked in a different way with 1,3,4-oxadiazole ring. None of the obtained molecules show cytotoxicity on NHDF and THP-1 cell lines and, therefore, all were qualified for further investigation. In vitro cyclooxygenase inhibition assay revealed almost equal activity of new derivatives towards both COX-1 and COX-2 isoenzymes. Moreover, all compounds inhibit COX-2 isoform better than Meloxicam which was used as reference. Anti-inflammatory activity was confirmed in biological assays according to which title molecules are able to reduce induced inflammation within cells. Molecular docking studies were performed to describe the binding mode of new structures to cyclooxygenase. Investigated derivatives take place in the active site of COX, very similar to Meloxicam. For some compounds, promising druglikeness was calculated using in silico predictions.     

Evidence of pheromonal constancy among sexual and asexual females in a population of fall cankerworm,Alsophila pometaria (Lepidoptera: Geometridae)

The compounds (Z,Z,Z)-3,6,9-nonadecatriene (I), (Z,Z,Z,E)-(II), and (Z,Z,Z,Z)-3,6,9,11-nonadecatetraene (III) have been implicated as components of the female sex pheromone of the fall cankerworm. Chromatographie determination of the proportions of these compounds in individual females of sympatric asexual and sexual reproductive forms of the species, with concurrent analysis of the electrophoretic profiles of the same females, showed that the I: II: III proportion of compounds was constant across electrophoretically differing asexual genotypes and between these and the sexual form. Life-history characters, in contrast, typically show great variation among these genetic groups. The results indicate that pheromonal constancy is maintained in a reproductive system that is theoretically vulnerable to selective pressures that would lead to heterogeneity in the species' pheromonal communication channel.     

Pyrazolonderivate. I. Synthese,Stereochemie und photochemisches Verhalten von Azomethinen des 1,5-Dimethyl-2-phenyl-4-formyl-Δ4-pyrazolinon-(3) (Antipyrinaldehyd)

Pyrazolone Derivatives. I. Synthesis, Stereochemistry, and Photochemical Behaviour of Azomethines of 1,5-Dimethyl-2-phenyl-4-formyl-Δ⁴-pyrazolinone-(3) (Antipyraldehyde) Condensation of Antipyraldehyde with primary amines yielded a series of anils. By ¹H-n.m.r.-spectroscopy and ¹⁵N-isotope technique is shown that the azomethines exist in the E-configuration. By a photochemical reaction the Z-isomer is produced which could be detected at low temperatures.     

Development of Novel Amides as Noncovalent Inhibitors of Immunoproteasomes

The development of immunoproteasome-selective inhibitors is a promising strategy for treating hematologic malignancies, autoimmune and inflammatory diseases. In this context, we report the design, synthesis, and biological evaluation of a new series of amide derivatives as immunoproteasome inhibitors. Notably, the designed compounds act as noncovalent inhibitors, which might be a promising therapeutic option because of the lack of drawbacks and side effects associated with irreversible inhibition. Among the synthesized compounds, we identified a panel of active inhibitors with K_i values in the low micromolar or sub-micromolar ranges toward the β5i and/or β1i subunits of immunoproteasomes. One of the active compounds was shown to be the most potent and selective inhibitor with a K_i value of 21 nm against the single β1i subunit. Docking studies allowed us to determine the mode of binding of the molecules in the catalytic site of immunoproteasome subunits.     

Comprehensive Study of Isobutane Selective Oxidation Over Group I and II Phosphomolybdates: Structural and Kinetic Factors

Abstract  

Various phosphomolybdates were synthesized using cations from Groups 1 and 2 of the periodic table. These compounds were of the form M _x H_3−xn [PMo₁₂O₄₀], with n being the cationic charge (+1 or +2). XRD analysis shows pure phosphomolybdic acid has a triclinic structure. A body centered cubic (BCC) structure gradually develops with addition of Group 1 cations, and the triclinic phase is completely replaced by the BCC phase once metal cations occupy a volume greater than 9–11 ?³ per phosphomolybdate anion. The Group 2 compounds do not form a cubic phase, however the triclinic phase distorts once cationic volume is greater about 5 or 6 ?³ and appears to become somewhat amorphous. Isobutane selective oxidation over the compounds yielded methacrolein (primary product), 3-methyl-2-oxetanone (lactone), acetic acid, propene, methacrylic acid, carbon dioxide and water as products. Propene was formed over the Group 1 compounds exclusively and methacrylic acid formation was observed with BaH[PMo₁₂O₄₀] only. Products form via two distinct processes: Category 1 product has an exponential profile and coverage is consistent with a Langmuir model, Category 2 formations are consistent with desorptions from within the bulk of the substrates. Methacrolein forms via both Category 1 and 2 processes, whilst all other products are formed by Category 2 exclusively. A rigorous kinetic analysis yielded accurate activation parameters. Category 1 methacrolein formation apparent activation energies ranged from 34.7 ± 1.3 to 119 ± 4 kJ mol⁻¹. Category 2 formations ranged from 34.3 ± 0.4 to 726 ± 172 kJ mol⁻¹. No relationship between activity and composition or structure could be ascertained, despite investigation into correlations using several different models.     

N‐Benzyl‐4‐((heteroaryl)methyl)benzamides: A New Class of Direct NADH‐Dependent 2‐trans Enoyl–Acyl Carrier Protein Reductase (InhA) Inhibitors with Antitubercular Activity

Isoniazid (INH) remains one of the cornerstones of antitubercular chemotherapy for drug‐sensitive strains of M. tuberculosis bacteria. However, the increasing prevalence of multidrug‐resistant (MDR) and extensively drug‐resistant (XDR) strains containing mutations in the KatG enzyme, which is responsible for the activation of INH into its antitubercular form, have rendered this drug of little or no use in many cases of drug‐resistant tuberculosis. Presented herein is a novel family of antitubercular direct NADH‐dependent 2‐trans enoyl–acyl carrier protein reductase (InhA) inhibitors based on an N‐benzyl‐4‐((heteroaryl)methyl)benzamide template; unlike INH, these do not require prior activation by KatG. Given their direct InhA target engagement, these compounds should be able to circumvent KatG‐related resistance in the clinic. The lead molecules were shown to be potent inhibitors of InhA and showed activity against M. tuberculosis bacteria. This new family of inhibitors was found to be chemically tractable, as exemplified by the facile synthesis of analogues and the establishment of structure–activity relationships. Furthermore, a co‐crystal structure of the initial hit with the enzyme is disclosed, providing valuable information toward the design of new InhA inhibitors for the treatment of MDR/XDR tuberculosis.     

Synthesis of the sex pheromone of the Japanese beetle

The stereospecific synthesis of the sex pheromone produced by the female Japanese beetle (Popillia japonica Newman), (R,Z)-5-1-decenyl)-dihydro-2(3H)-furanone, is described. The pure syntheticR,Z form is a powerful attractant for males of the species in field bioassays and was competitive in attractiveness with live females, whereas the racemicZ isomer and theS,Z enantiomer were strong inhibitors of male response. The saturated analogs of both enantiomers were also synthesized stereospecifically.Mention of a commercial or proprietary product in this paper does not constitute an endorsement of that product by the USDA.     

Metabolically blocked analogs of housefly sex pheromone: II. Metabolism studies

Analogs of (Z)-9-tricosene (Z9–23: Hy) bearing methyl substituents, cyclopropyl groups, fluorine substituents, and additional double bonds were used to probe the substrate requirements for the monooxygenase system that converts Z9–23: Hy to the corresponding epoxide and ketone. Three of the seven analogs tested, 10-fluoro-(Z)-14-tricosene, 10,10-difluoro-(Z)-14-tricosene, and 14-methyl-(Z)-9-tricosene, were metabolized to the corresponding epoxide. Compounds with two methyl groups, a cyclopropane group, a hydroxy group, or an additional double bond at the 14 position were not epoxidized at the 9,10 position. This suggests that only minimal structural change at the 14-position of Z9–23: Hy is allowed with retention of metabolic activity. None of the analogs tested were hydroxylated at the position equivalent to the 14 position of Z9–23:Hy. Of the 13 analogs tested as inhibitors of Z9–23:Hy metabolism, the two compounds that were the most effective inhibitors in both male and female houseflies were (Z)-14-tricosen-10-one and 1-nonyl-1-[(Z)-4-tetradecen-1-yl]-cyclopropane. These data show that the poly substrate monooxy genase that metabolizes Z9–23: Hy in the housefly has very strict structural requirements for the substrate.     

A Facile and Efficient Method for the Synthesis of Labeled and Unlabeled Very Long Chain Polyunsaturated Fatty Acids

Several methods are available for elongation of fatty acid acyl chains. The present paper describes adaptation to the fatty acid field of a previously published protocol for manganese-based Wurtz type coupling of alkyl bromides. 22-Bromo-3(Z),6(Z),9(Z),12(Z),15(Z),18(Z)-docosahexaene, easily prepared from 4(Z),7(Z),10(Z),13(Z),16(Z),19(Z)-docosahexaenoic acid, was coupled to homologous ω-bromoesters by stirring for 4 hours at 40°C in the presence of manganese powder, a nickel catalyst and terpyridine. This afforded in yields of 70–75% a series of ω3-hexaenoates of chain lengths of 32–40 carbons. The corresponding fatty acids of >98% purity were obtained following saponification and final purification. By using methyl [2,2,3,3,4,4-²H₆]10-bromodecanoate as coupling partner it was possible to prepare a very long chain fatty acid in isotopically labeled form, i.e., [2,2,3,3,4,4-²H₆]14(Z),17(Z),20(Z),23(Z),26(Z),29(Z)-dotriacontahexaenoic acid. Also prepared were the monounsaturated long chain fatty acids 15(Z)-octadecenoic acid and 15(Z)-tetracosenoic acid. Very long chain fatty acids have been isolated from retina and other tissues and are of biological relevance. The methodology described will assist in further analytical and biological studies in this field.     

Azetidinones as Zinc‐Binding Groups to Design Selective HDAC8 Inhibitors

2‐Azetidinones, commonly known as β‐lactams, are well‐known heterocyclic compounds. Herein we described the synthesis and biological evaluation of a series of novel β‐lactams. In vitro inhibition assays against HDAC isoforms showed an interesting isoform‐selectivity of these compounds towards HDAC6 and HDAC8. The isoform selectivity changed in response to modification of the azetidinone‐ring nitrogen atom substituent. The presence of an N‐thiomethyl group is a prerequisite for the activity of these compounds in the micromolar range towards HDAC8.     

Photoinduced Isomerization and Hepatoxicities of Semaxanib,Sunitinib and Related 3‐Substituted Indolin‐2‐ones

3‐Substituted indolin‐2‐ones are an important class of compounds that display a wide range of biological activities. Sunitinib is an orally available multiple tyrosine kinase inhibitor that has been approved by the US Food and Drug Administration (FDA) for the treatment of renal cell cancer. Sunitinib and a related compound, semaxanib, exist as thermodynamically stable Z isomers, which photoisomerize to E isomers in solution. In this study, 17 3‐substituted indolin‐2‐ones were synthesized, and the kinetics of their photoisomerization were studied by ¹H NMR spectroscopy. The rate constants for photoisomerization ranged from 0.009 to 0.048 h^?1. Selected compounds were tested for cytotoxicity in the TAMH liver cell line. E/Z mixtures of four compounds were also assessed for toxicity in the TAMH and HepG2 cell lines. In some cases, the stereochemically pure drug was more toxic than the E/Z mixtures, but a general statement cannot be made. Our studies show that each stereoisomer could contribute differently to toxicity, suggesting that stereochemical purity issues that could arise from isomerization cannot be ignored.