Structure-activity relationship of pyrrole based S-nitrosoglutathione reductase inhibitors: carboxamide modification

The enzyme S-nitrosoglutathione reductase (GSNOR) is a member of the alcohol dehydrogenase family (ADH) that regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). GSNO and SNOs are implicated in the pathogenesis of many diseases including those in respiratory, gastrointestinal, and cardiovascular systems. The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious GSNOR inhibitor which is currently in clinical development for acute asthma. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogs of N6022 focusing on carboxamide modifications on the pendant N-phenyl moiety. We have identified potent and novel GSNOR inhibitors that demonstrate efficacy in an ovalbumin (OVA) induced asthma model in mice.     

Discovery of potent and novel S-nitrosoglutathione reductase inhibitors devoid of cytochrome P450 activities

The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious S-nitrosoglutathione reductase (GSNOR) inhibitor and is currently undergoing clinical development for the treatment of acute asthma. GSNOR is a member of the alcohol dehydrogenase family (ADH) and regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). Reduced levels of GSNO, as well as other nitrosothiols (SNOs), have been implicated in the pathogenesis of many diseases including those of the respiratory, cardiovascular, and gastrointestinal systems. Preservation of endogenous SNOs through GSNOR inhibition presents a novel therapeutic approach with broad applicability. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on removal of cytochrome P450 inhibition activities. We identified potent and novel GSNOR inhibitors having reduced CYP inhibition activities and demonstrated efficacy in a mouse ovalbumin (OVA) model of asthma.     

Synthesis and biological evaluation of novel dihydro-aryl/alkylsulfanyl-cyclohexylmethyl-oxopyrimidines (S-DACOs) as high active anti-HIV agents

A novel dihydro-aryl/alkylsulfanyl-cyclohexylmethyl-oxopyrimidines (S-DACOs) combinatory library was synthesized and evaluated with C8166 cells infected by the HIV-1_IIIB in vitro, using Nevirapine (NVP) and Zidovudine (AZT) as positive control. The anti-HIV screening results revealed that C-6-cyclohexylmethyl substituted pyrimidinones possessed higher selective index than its 6-arylmethyl counterparts. Compounds 1g, 1c, 1e and 1b showed potent anti-HIV activities with EC₅₀ values of 0.012, 0.025, 0.088 and 0.162 nM, respectively.     

Structural and functional characterization of a plant S-nitrosoglutathione reductase from Solanum lycopersicum

S-nitrosoglutathione reductase (GSNOR), also known as S-(hydroxymethyl)glutathione (HMGSH) dehydrogenase, belongs to the large alcohol dehydrogenase superfamily, namely to the class III ADHs. GSNOR catalyses the oxidation of HMGSH to S-formylglutathione using a catalytic zinc and NAD⁺ as a coenzyme. The enzyme also catalyses the NADH-dependent reduction of S-nitrosoglutathione (GSNO). In plants, GSNO has been suggested to serve as a nitric oxide (NO) reservoir locally or possibly as NO donor in distant cells and tissues. NO and NO-related molecules such as S-nitrosothiols (S-NOs) play a central role in the regulation of normal plant physiological processes and host defence. The enzyme thus participates in the cellular homeostasis of S-NOs and in the metabolism of reactive nitrogen species. Although GSNOR has recently been characterized from several organisms, this study represents the first detailed biochemical and structural characterization of a plant GSNOR, that from tomato (Solanum lycopersicum). SlGSNOR gene expression is higher in roots and stems compared to leaves of young plants. It is highly expressed in the pistil and stamens and in fruits during ripening. The enzyme is a dimer and preferentially catalyses reduction of GSNO while glutathione and S-methylglutathione behave as non-competitive inhibitors. Using NAD⁺, the enzyme oxidizes HMGSH and other alcohols such as cinnamylalcohol, geraniol and ω-hydroxyfatty acids. The crystal structures of the apoenzyme, of the enzyme in complex with NAD⁺ and in complex with NADH, solved up to 1.9 Å resolution, represent the first structures of a plant GSNOR. They confirm that the binding of the coenzyme is associated with the active site zinc movement and changes in its coordination. In comparison to the well characterized human GSNOR, plant GSNORs exhibit a difference in the composition of the anion-binding pocket, which negatively influences the affinity for the carboxyl group of ω-hydroxyfatty acids.     

Synthesis and characterization of platinum(IV) complexes with N,S and S,S heterocyclic ligands

The reactions of [PtMe₃(OAc)(bpy)] (4) with the N,S and S,S containing heterocycles, pyrimidine-2-thione (pymtH), pyridine-2-thione (pytH), thiazoline-2-thione (tztH) and thiophene-2-thiol (tptH), resulted in the formation of the monomeric complexes [PtMe₃(-κS)(bpy)] ( = pymt, 5; pyt, 6; tzt, 7; tpt, 8), where the heterocyclic ligand is coordinated via the exocyclic sulfur atom. In contrast, in the reactions of [PtMe₃(OAc)(Me₂CO)_x] (3, x = 1 or 2) with pymtH, pytH, tztH and tptH dimeric complexes [{PtMe₃(μ-)}₂] (μ- = pymt, 9; pyt, 10; tzt, 11) and the tetrameric complex [{PtMe₃(μ₃-tpt-κS)}₄] (12), respectively, were formed. The complexes were characterized by microanalyses, ¹H and ¹³C NMR spectroscopy and negative ESI-MS (12) measurements. Single-crystal X-ray diffraction analysis of [PtMe₃(pymt-κS)(bpy)] (5) exhibited a conformation where the pymt ligand lies nearly perpendicular to the complex plane above the bpy ligand that was also confirmed by quantum chemical calculations on the DFT level of theory.     

Oxygenated verticillene derivatives from Bursera suntui

Medium polarity fractions of the hexane extracts of the stems of Bursera suntui afforded six previously known (1-6) and four hitherto unknown verticillane derivatives: (1S,3Z,7S,8S,11S,12S)-(+)-7,8-epoxyverticill-3-en-12,20-diol (7), (1S,3Z,7S,8S,11S,12S)-(+)-7,8-epoxyverticill-3-en-12,20-diol 20-acetate (8), (1S,3Z,7S,11S,12S)-(+)-verticilla-3,8(19)-dien-7,12,20-triol (9), and (1S,3Z,7S,11S,12S)-(+)-verticilla-3,8(19)-dien-7,12,20-triol 20-acetate (10). Acetylation of 9 and 10 yielded (1S,3Z,7S,11S,12S)-(+)-verticilla-3,8(19)-dien-7,12,20-triol 7,20-diacetate (11), while hydrolysis of 8 gave 7. The structures and stereochemistry of 7-11 were established by spectroscopic analyses, particularly by 1D and 2D NMR spectra and HRESIMS. The conformational preferences of 7-11 were studied by molecular mechanics modelling employing the Monte Carlo protocol followed by B3LYP/DGDZVP DFT calculation, thus supporting the observed ¹H NMR NOESY cross peaks.     

Novel 12-membered non-antibiotic macrolides from erythromycin A; EM900 series as novel leads for anti-inflammatory and/or immunomodulatory agents

Herein, we report the design and synthesis of the novel 12-membered non-antibiotic macrolide (8R,9S)-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A (EM900), which was found to be a potent anti-inflammatory and/or immunomodulatory agent, capable of promoting monocyte to macrophage differentiation. This molecule shows improved acid stability, does not exhibit any anti-bacterial activity and has relatively low cytotoxicity against THP-1 cells. In addition, one of its analogues, (8R,9S)-4″,13-O-diacetyl-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A (EM911), was found to be twice as effective as EM900.     

Distant Neighbor Base Sequence Context Effects in Human Nucleotide Excision Repair of a Benzo[a]pyrene-derived DNA Lesion

The effects of non-nearest base sequences, beyond the nucleotides flanking a DNA lesion on either side, on nucleotide excision repair (NER) in extracts from human cells were investigated. We constructed two duplexes containing the same minor groove-aligned 10S (+)-trans-anti-B[a]P-N²-dG (G?) DNA adduct, derived from the environmental carcinogen benzo[a]pyrene (B[a]P): 5′-C-C-A-T-C-G?-C-T-A-C-C-3′ (CG?C-I), and 5′-C-A-C3-A4-C5-G?-C-A-C-A-C-3′ (CG?C-II). We used polyacrylamide gel electrophoresis to compare the extent of DNA bending, and molecular dynamics simulations to analyze the structural characteristics of these two DNA duplexes. The NER efficiencies are 1.6(± 0.2)-fold greater in the case of the CG?C-II than the CG?C-I sequence context in 135-mer duplexes. Gel electrophoresis and self-ligation circularization experiments revealed that the CG?C-II duplex is more bent than the CG?C-I duplex, while molecular dynamics simulations showed that the unique -C3-A4-C5- segment in the CG?C-II duplex plays a key role. The presence of a minor groove-positioned guanine amino group, the Watson-Crick partner to C3, acts as a wedge; facilitated by a highly deformable local -C3-A4- base step, this amino group allows the B[a]P ring system to produce a more enlarged minor groove in CG?C-II than in CG?C-I, as well as a local untwisting and enlarged and flexible Roll only in the CG?C-II sequence. These structural properties fit well with our earlier findings that in the case of the family of minor groove 10S (+)-trans-anti-B[a]P-N²-dG lesions, flexible bends and enlarged minor groove widths constitute NER recognition signals, and extend our understanding of sequence context effects on NER to the neighbors that are distant to the lesion.     

Structural comparison of alkylated derivatives of (bmmp-dmed)Ni and (bmmp-dmed)Zn

The sulfur-alkylation of the nickel (1) and zinc (2) complexes of the dithiolate N₂S₂ ligand N,N′-bis-2-methyl-mercaptopropyl-N,N′-dimethylethylenediamine, H₂(bmmp-dmed), have been investigated. Reactions with iodomethane yield [(Me-bmmp-dmed)Ni]PF₆ (3), [(Me₂-bmmp-dmed)NiI₂] (4), and [(Me₂-bmmp-dmed)ZnI]₂[ZnI₄] (5). Addition of iodoacetamide yields [(AA₂-bmmp-dmed)Ni]I₂ (6) and [(AA₂-bmmp-dmed)Zn]I₂ (7). Each of the metal-thioether products (3-7) have been characterized spectroscopically and by X-ray crystallography. Structural data is compared with that of the previously reported thiolato precursors 1 and 2. Sulfur-alkylation of 1 results in small relative changes in the nickel-sulfur bond distance, whereas for 2, the zinc-sulfur bond distance increases significantly, but is not cleaved. The difference between nickel and zinc is attributed to the release of a π*-bonding interaction between the metal and sulfur upon alkylation that compensates for the decreased σ-donor ability of the thioether in the case of nickel, but not for zinc.     

Synthesis, structures and ligating properties of 2,6-bis(phosphino)thiophenol derivatives

New t-butyl-aryl thioethers where the aryl group is 2,6-bis(phosphino)phenyl have been synthesized. The syntheses were completed via sequential ortho-lithiations of t-butylphenylsulfide, followed by chlorophosphine (ClPR₂) quenches; symmetric (2,6-bis(diphenylphosphino)phenyl, (4a)) and unsymmetric (2-diisopropylphosphino-6-diphenylphosphino)phenyl, (4b) aryl groups were obtained. Treatment of 4a with Li or Na naphthalenide yielded 2,6-bis(diphenylphosphino)thiophenol 5. Reactions of 4a or 5 with NiCl₂ · 6H₂O yielded nickel bis(phosphinothiophenolate) 6. Compounds 4a,b, 5 and 6 were characterized by ¹H and ³¹P NMR, and by mass-spectrometry. In addition, 4a, 5 and 6 were characterized by single crystal X-ray diffraction methods.     

(25S)-Cholesten-26-oic acid derivatives from an Indonesian soft coral Minabea sp.

(25S)-3-Oxocholesta-1,4-dien-26-oic acid (1) and a new (25S)-18-acetoxy-3-oxocholesta-1,4-dien-26-oic acid (2) were isolated from a soft coral Minabea sp. (cf. aldersladei) collected in North Sulawesi, Indonesia, together with two known cholic-acid-type compounds, 3-oxochol-1,4-dien-24-oic acid (3) and 3-oxochol-4-en-24-oic acid (4). The structures of these compounds were determined on the basis of their spectroscopic data. The absolute stereochemistry at C-25 of 2 was determined by comparative ¹H NMR study using chiral anisotropic reagents [(S)- and (R)-phenylglycine methyl esters]. This is the first to report compound 1 as a natural product.     

Steroids from the leaves of Chinese Melia azedarach and their cytotoxic effects on human cancer cell lines

Three new (1-3) and several known (4-6) steroids were isolated from the leaves of Chinese Melia azedarach. The structures of the new compounds were elucidated by means of spectroscopic methods including 2D NMR techniques and mass spectrometry to be (20S)-5,24(28)-ergostadiene-3β,7α,16β,20-tetrol (1), (20S)-5-ergostene-3β,7α,16β,20-tetrol (2), and 2α,3β-dihydro-5-pregnen-16-one (3). The cytotoxicities of the isolated compounds against three human cancer cell lines (A549, H460, U251) were evaluated; only compounds 1, 2, and (20S)-5-stigmastene-3β,7α,20-triol (4) were found to show significant cyctotoxic effects with IC₅₀s from 12.0 to 30.1 μg/mL.     

Monoterpenoids from the aerial parts of Aruncus dioicus var. kamtschaticus and their antioxidant and cytotoxic activities

The aerial parts of Aruncus dioicus var. kamtschaticus afforded five new monoterpenoids (1-5): 4-(erythro-6,7-dihydroxy-9-methylpent-8-enyl)furan-2(5H)-one (1, aruncin A), 2-(8-ethoxy-8-methylpropylidene)-5-hydroxy-3,6-dihydro-2H-pyran-4-carboxylic acid (2, aruncin B), 4-(hydroxymethyl)-6-(8-methylprop-7-enyl)-5,6-dihydro-2H-pyran-2-one-11-O-β-d-glucopyranoside (3, aruncide A), (3S,4S,5R,10R)-3-(10-ethoxy-11-hydroxyethyl)-4-(5-hydroxy-7-methylbut-6-enyl)oxetan-2-one-11-O-β-d-glucopyranoside (4, aruncide B), and (3S,4S,5R,7R)-5-(9-methylprop-8-enyl)-1,6-dioxabicyclo[3,2,0]heptan-2-one-7-(hydroxymethyl)-12-O-β-d-glucopyranoside (5, aruncide C). Compound 2 showed potent cytotoxicity against Jurkat T cells with an IC₅₀ value of 17.15 μg/mL. In addition, compounds 7 and 10 exhibited moderate antioxidant activity with IC₅₀ values of 46.3 and 11.7 μM, respectively.     

Pharmacologic inhibition of S-nitrosoglutathione reductase protects against experimental asthma in BALB/c mice through attenuation of both bronchoconstriction and inflammation

Background

S-nitrosoglutathione (GSNO) serves as a reservoir for nitric oxide (NO) and thus is a key homeostatic regulator of airway smooth muscle tone and inflammation. Decreased levels of GSNO in the lungs of asthmatics have been attributed to increased GSNO catabolism via GSNO reductase (GSNOR) leading to loss of GSNO- and NO- mediated bronchodilatory and anti-inflammatory actions. GSNOR inhibition with the novel small molecule, N6022, was explored as a therapeutic approach in an experimental model of asthma.

Methods

Female BALB/c mice were sensitized and subsequently challenged with ovalbumin (OVA). Efficacy was determined by measuring both airway hyper-responsiveness (AHR) upon methacholine (MCh) challenge using whole body plethysmography and pulmonary eosinophilia by quantifying the numbers of these cells in the bronchoalveolar lavage fluid (BALF). Several other potential biomarkers of GSNOR inhibition were measured including levels of nitrite, cyclic guanosine monophosphate (cGMP), and inflammatory cytokines, as well as DNA binding activity of nuclear factor kappa B (NFκB). The dose response, onset of action, and duration of action of a single intravenous dose of N6022 given from 30 min to 48 h prior to MCh challenge were determined and compared to effects in mice not sensitized to OVA. The direct effect of N6022 on airway smooth muscle tone also was assessed in isolated rat tracheal rings.

Results

N6022 attenuated AHR (ED₅₀ of 0.015?±?0.002 mg/kg; Mean?±?SEM) and eosinophilia. Effects were observed from 30 min to 48 h after treatment and were comparable to those achieved with three inhaled doses of ipratropium plus albuterol used as the positive control. N6022 increased BALF nitrite and plasma cGMP, while restoring BALF and plasma inflammatory markers toward baseline values. N6022 treatment also attenuated the OVA-induced increase in NFκB activation. In rat tracheal rings, N6022 decreased contractile responses to MCh.

Conclusions

The significant bronchodilatory and anti-inflammatory actions of N6022 in the airways are consistent with restoration of GSNO levels through GSNOR inhibition. GSNOR inhibition may offer a therapeutic approach for the treatment of asthma and other inflammatory lung diseases. N6022 is currently being evaluated in clinical trials for the treatment of inflammatory lung disease.     

Novel dammarane-type sapogenins from Panax ginseng berry and their biological activities

Three new dammarane-type sapogenins (1, 3, and 5) together with two known ones (2 and 4) were isolated from the total hydrolyzed saponins extracted from Panax ginseng berry. Their structures were elucidated using a combination of 1D and 2D ¹H and ¹³C NMR spectra and mass spectroscopy as 20(R)-25-methoxyl-dammarane-3β,12β,20-triol (1), 20(R)-25-methoxyl-dammarane-3β,6α,12β,20-tetrol (2), 20(R)-20-methoxyl-dammarane-3β,12β,25-triol (3), 20(R)-20,25-dimethoxyl-dammarane-3β,12β-diol (4), and (12R,20S,24S)-20,24-; 12,24-diepoxy-dammarane-3β-ol (5). Their antitumor activities were evaluated in six human cancer cell lines. The novel compounds 1 and 3 showed significant cytotoxic activity against the six cell lines. The IC₅₀ values of 3 against HepG2, Colon205, and HL-60 were the lowest (8.78, 8.64, and 3.98 μM, respectively). Compounds 1 and 20(S)-25-OCH₃-PPD, which are a pair of configuration isomers, showed a 10- to 100-fold greater growth inhibition than ginsenoside-Rg₃ (an anti-cancer clinical agent in China). The data presented here may be useful for the development of novel anti-cancer agents.     

Sesquiterpene constituents from the essential oils of the liverworts Mylia taylorii and Mylia nuda

The essential oils and extracts of Mylia taylorii and M. nuda were investigated by gas chromatography, mass spectrometry, NMR spectroscopy and chemical correlations. Beside several known compounds 13 new constituents including three new carbon skeletons could be identified. Four hydrocarbons with a molecular formula of C₁₅H₂₂ (m/z 202) were identified as myli-4(15)-ene (1), aromadendra-1(10),4(15)-diene (19), aromadendra-4,10(14)-diene (20) and aromadendra-4,9-diene (21). Three oxaspiro-compounds were identified as 7-epi-bourbon-3-en-5,11-oxide (22), guai-3,10(14)-dien-5,11-oxide (23) and guai-3,9-dien-5,11-oxide (24). The absolute configuration of myli-4(15)-en-3-one (5) could be established by chemical correlation. Together with α-taylorione (7) the corresponding 6,11-seco-compound taylopyran (25) with a new carbon skeleton was identified which serves as a precursor to taylocyclane (26) and taylofuran (27). Taynudol (28) contains a new carbon skeleton with a cyclobutenyl structure.     

Aspergilones A and B, two benzylazaphilones with an unprecedented carbon skeleton from the gorgonian-derived fungus Aspergillus sp

Two novel benzylazaphilone derivatives with an unprecedented carbon skeleton, aspergilone A (1), and its symmetrical dimer with a unique methylene bridge, aspergilone B (2), have been isolated from the culture broth of a marine-derived fungus Aspergillus sp. from a gorgonian Dichotella gemmacea. Their structures and relative stereochemistries of 1 and 2 were elucidated using a combination of NMR spectroscopy and X-ray crystallography. Compound 1 not only exhibited in vitro selective cytotoxicity but also showed potent antifouling activity.     

Identification and in vitro anti-esophageal cancer activity of a series of halogenated monoterpenes isolated from the South African seaweeds Plocamium suhrii and Plocamium cornutum

Five known (1, 2, 4, 6 and 7) halogenated monoterpenes together with 1Z,3R^∗,4S^∗,5E,7Z)-1-bromo-3,4,8-trichloro-7-(dichloromethyl)-3-methylocta-1,5,7-triene (3) and (3R^∗,4S^∗)-3,4,6,7-tetrachloro-3,7-dimethyl-octen-1-ene (5) were isolated from the red macroalga Plocamium suhrii and their structures deduced from their spectroscopic data. The seven compounds from P. suhrii together with five related compounds from Plocamium cornutum have been evaluated for their cytotoxic effects on an esophageal cancer cell line (WHCO1). Compounds 1-6 showed greater cytotoxicity in this assay as compared to the known anticancer drug cisplatin.     

Two dimeric lignans with an unusual α,β-unsaturated ketone motif from Zanthoxylum podocarpum and their inhibitory effects on nitric oxide production

Two new dimeric lignans, zanthpodocarpins A (1) and B (2), and five known lignans, eudesmin (3), (1R,2R,5R,6S)-2-(3,4-dimethoxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (4), dimethoxysamin (5), rel-(1R,5R,6S)-6-(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octan-2-one (6), and magnone A (7), were isolated from the barks of Zanthoxylum podocarpum. Their structures were identified by using spectroscopic methods. Compounds 1 and 2 are rare dilignans bearing an unusual α,β-unsaturated ketone group from a natural source. Bioassay showed that compounds 1 and 2 could inhibit nitric oxide (NO) production in LPS stimulated RAW 264.7 cells with IC₅₀ values of 5.31 μM and 12.15 μM, respectively.