Part 3: synthesis and biological evaluation of some analogs of the antitumor agents, 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid, and 2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic acid

2-{4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (X469) and 2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic Acid (SH80) are among the most highly and broadly active antitumor agents to have been developed in our laboratories. However, the mechanism(s) of action of these agents remain to be elucidated, which prompted our continued endeavor to delineate a pharmacophoric pattern, from which a putative target might be deduced. Herein, we provide additional evidence that intact quinoxaline and quinoline rings in XK469 and SH80, respectively, are fundamental to the activities of these structures against transplanted tumors in mice. The consequence of further modification of the heterocyclic ring system in XK469 and SH80, leading to [1,8]naphthyridine; pyrrolo[1,2-a]; imidazo[1,2-a]; and imidazo[1,5-a] derivatives, all deprive the parent structures of antitumor activity. Introduction of CH3, CF3, CH3O, CO2H, or C6H5 substituents at C4 of the quinoline ring of SH80 led to weakly active antitumor agents. Similarly, the phenanthridine analog of SH80 manifested only modest cytotoxicity. Lastly, XK469 and SH80 are both significantly more active than the corresponding regioisomeric structures, 2-{4-[(7-halo-4-quinolinyl)oxy]phenoxy)propionic acids.     

{alpha}-Galactosyl (Gal{alpha}1-3Gal{beta}1-4GlcNAc-R) epitopes on human cells: synthesis of the epitope on human red cells by recombinant primate {alpha}1,3galactosyltransferase expressed in E.coli

Developing methods for in vitro synthesis of the carbohydratestructure Gal     

Understanding the unexpected linearity of the trans-{Mn(NO)2}8 unit in a phthalocyanine complex: some thoughts on dinitrosylheme intermediates in biology

Simple MO arguments provide a qualitative explanation for the near-linear ON-Mn-NO arrangement observed for the trans-{Mn(NO)2}8 anion [Mn(Pc)(NO)2]-, which is unexpected for an Enemark-Feltham electron count n>6. The metal center in this species may be described as low-spin d6("t2g6") and the two unpaired electrons occupy a pair of eu orbitals composed of NO(pi*) components, giving rise to a triplet ground state. In a certain sense, these eu SOMOs may be likened to the SOMO (singly occupied molecular orbital) of the allyl radical. The electronic structure of this species is quite different from that of diamagnetic dinitrosylheme intermediates, which have been spectroscopically characterized in synthetic studies as well as proposed for soluble guanylate cyclase and cytochrome c'. Some speculative remarks are offered as to why this proposal is not an unreasonable one from an electronic-structural perspective.     

3-(2-Ethoxy-4-{4-[3-hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy]butoxy}phenyl)propanoic acid: a brain penetrant allosteric potentiator at the metabotropic glutamate receptor 2 (mGluR2)

We have identified and synthesized a brain penetrant propanoic acid as an allosteric potentiator of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward improving the potency, level of potentiation and brain penetration led to the discovery of 8 (EC50=1200 nM, 77% potentiation, 119% brain/plasma in rat, 20 mpk i.p., brain level of 5700 nM).     

The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide

In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.     

(3R)-3-amino-4-(2,4,5-trifluorophenyl)-N-{4-[6-(2-methoxyethoxy)benzothiazol-2-yl]tetrahydropyran-4-yl}butanamide as a potent dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Novel series of 3-amino-N-(4-aryl-1,1-dioxothian-4-yl)butanamides and 3-amino-N-(4-aryltetrahydropyran-4-yl)butanamides were synthesized and evaluated as dipeptidyl peptidase IV (DPP-IV) inhibitors. Derivatives incorporating the 6-substituted benzothiazole group showed highly potent DPP-IV inhibitory activity. Oral administration of (3R)-3-amino-4-(2,4,5-trifluorophenyl)-N-{4-[6-(2-methoxyethoxy)benzothiazol-2-yl]tetrahydropyran-4-yl}butanamide (12u) reduced blood glucose excursion in an oral glucose tolerance test.     

Discovery of 4-{1-[({1-[4-(trifluoromethyl)benzyl]-1H-indol-7-yl}carbonyl)amino]cyclopropyl}benzoic acid (MF-766), a highly potent and selective EP4 antagonist for treating inflammatory pain

The discovery of a highly potent and selective EP₄ antagonist MF-766 is discussed. This N-benzyl indole derivative exhibits good pharmacokinetic profile and unprecedented in vivo potency in the rat AIA model.     

Synthetic modification of the 2-oxypropionic acid moiety in 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469), and consequent antitumor effects. Part 4

The criteria for the activity of 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469) and 2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic acid (SH80) against transplanted tumors in mice established in previous studies, require a (7-halo-2-quinoxalinoxy)- or a (7-halo-2-quinolinoxyl)-residue, respectively, bridged via a 1,4-OC(6)H(4)O-linker to C(2) of propionic acid. The present work demonstrates that substitution of fluorine at the 3-position of the 1,4-OC(6)H(4)O-linker of XK469 leads to a 10-fold reduction in activity, whereas the corresponding 2-fluoro analog proved to be 100-fold less active than XK469. Moreover, the latter tolerated substitution of but a single, additional methyl group to the 2-position of the propionic acid moiety, that is, the isobutyric acid analog, without loss of significant in vivo activity. Indeed, an intact 2-oxypropionic acid moiety is a prerequisite for maximum antitumor activity of 1a.     

Synthesis and activity of di- or trisubstituted N-(phenoxyalkyl)- or N-{2-[2-(phenoxy)ethoxy]ethyl}piperazine derivatives on the central nervous system

Aim of the study was evaluation of anxiolytic, antidepressant, anticonvulsant and analgesic activity in a series of a consistent group of compounds. A series of eleven new N-(phenoxyalkyl)- or N-{2-[2-(phenoxy)ethoxy]ethyl}piperazine derivatives has been obtained. Their affinity towards 5-HT_1A, 5-HT_2A, 5-HT₆, 5-HT₇, D₂ and α₁ receptors has been assessed, and then functional assays were performed. The compounds were evaluated in mice, i.p. for their antidepressant-like (forced swim test), locomotor, anxiolytic-like (four-plate test) activities as well as – at higher doses – for anticonvulsant potential (MES) and neurotoxicity (rotarod). Two compounds (3, 6) were also evaluated for their analgesic activity in neuropathic pain models (streptozocin test, oxaliplatin test) and they were found active against allodynia in diabetic neuropathic pain at 30?mg/kg. Among the compounds, anxiolytic-like, anticonvulsant or analgesic activity was observed but antidepressant-like activity was not. One of the two most interesting compounds is 1-{2-[2-(2,4,6-trimethylphenoxy)ethoxy]ethyl}-4-(2-methoxyphenyl)piperazine dihydrochloride (9), exhibiting anxiolytic and anticonvulsant activity in mice, i.p. 30 min after administration (at 2.5?mg/kg and ED₅₀?=?26.33?mg/kg, respectively), which can be justified by the receptor profile: 5-HT_1A K_i?=?5?nM (antagonist), 5-HT₇ K_i?=?70?nM, α₁ K_i?=?15?nM, D₂ K_i?=?189?nM (antagonist). Another interesting compound is 1-[3-(2,4,6-trimethylphenoxy)propyl]-4-(4-methoxyphenyl)piperazine dihydrochloride (3), exhibiting anxiolytic, anticonvulsant and antiallodynic activity in mice, i.p., 30?min after administration (at 10?mg/kg, ED₅₀?=?23.50?mg/kg, at 30?mg/kg, respectively), which can be related with 5-HT_1A weak antagonism (K_i?=?146?nM), or other possible mechanism of action, not evaluated within presented study. Additionally, for the most active compound in the four-plate test (7), molecular modeling was performed (docking to receptors 5-HT_1A, 5-HT_2A, 5-HT₇, D₂ and α_1A).     

Iodo bridged lead(II) compounds of azoimidazoles: Single crystal X-ray structures of [di-iodo-{1-methyl-2-(p-tolylazo)imidazole}lead(II)]n and {1-methyl-3-benzyl-2-(p-tolylazo)imidazolium}m-{tri-iodoplumbate(II)}m

PbI₂ forms iodo-bridged neutral polymer upon reaction with 1-alkyl-2-(arylazo)imidazoles (RaaiR′). The reaction of PbI₂ and dialkyl imidazolium iodides [RaaiR′R″]⁺I⁻ has synthesized {1,3-dialkyl-2-(arylazo)imidazolium}_m-{tri-iodoplumbate(II)}_m. The complexes are characterized by different spectroscopic studies. Iodobridged chelated polymer, [Pb(RaaiR′)I₂]_n, has been established by single crystal X-ray diffraction measurements in one case. Tri-iodoplumbates form iodo bridged anion polymer, which connects [RaaiR′R″]⁺ by hydrogen bonding and are placed in between the pillars of [Pb(μ-I)₆]_n motif.     

2-O-(beta-D-glucuronyl)-7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose: a constituent of the Bordetella pertussis endotoxin.

The presence of bound D-glucuronic acid in the endotoxin of Bordetella pertussis was demonstrated. The branched chain trisaccharide named in the title was isolated after hydrolysis of the endotoxin with 3 M HCl for 2 h at 100 degrees C. Its structure was established by chemical and enzymic degradation.     

Rational design, synthesis and evaluation of (6aR( *),11bS( *))-1-(4-fluorophenyl)-4-{7-[4-(4-fluorophenyl)-4-oxobutyl]1,2,3,4,6,6a,7,11b,12,12a(RS)-decahydropyrazino[2',1':6,1]pyrido[3,4-b]indol-2-yl}-butan-1-one as a potential neuroleptic agent

In our pursuit to prepare a potent antipsychotic compound, a novel 1,2,3,4,6,6a,7,11b,12,12a-decahydropyrazino[2',1':6,1]pyrido[3,4-b]indole derivative was synthesized which incorporates the butyrophenone substructure twice. This molecule has shown D(1), D(2) and 5-HT(2A) receptor blocking activity where the ratio pK(i) (5-HT(2A)) to pK(i) (D(2)) is 1.42 better than risperidone (1.15). It blocks amphetamine induced hyperactivity/stereotypy and secondary conditioned avoidance responses in rodents at lower doses than those required for the neuroleptic drugs haloperidol and centbutindole (biriperone).     

{beta}1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/GA2 synthase) forms homodimers in the endoplasmic reticulum: a strategy to test for dimerization of Golgi membrane proteins

Many Golgi membrane-bound glycosyltransferases exist as intermoleculardisulfide bonded species, some of which have been demonstratedto be homodimers. Evidence for homodimer formation has comeprimarily from radiation inactivation experiments. We utilizedan alternative strategy to test for homodimer formation of thecloned ß1,4 N-acetylgalactosaminyltransferase (GalNAcT)responsible for synthesis of the glycosphingolipids GM2, GD2,and GA2. We stably transfected CHO cells with. myc epitope-taggedGalNAcT, which localizes primarily to the Golgi, and a hemagglutinin(HA) epitope-tagged GalNAcT fusion protein in which the cytoplasmicdomain of GalNAcT was replaced by an ER retention signal. Wethen sought evidence for dimer formation between the two formsof GalNAcT. Immunoprecipitation with anti-myc or anti-HA co-immunoprecipitatedthe HA-tagged form or the myc-tagged form, respectively, providingevidence for the physical association of the two forms of GalNAcT.As a result of this association, GalNAcT/myc increased in theER as demonstrated by Western blots and immunofluorescence.The rapid formation of dimers provided further evidence fordimer formation occurring in the ER. In summary, these resultsdemonstrate that GalNAcT forms homodimers as a result of intermoleculardisulfide bond formation in the ER. Furthermore, this ER motifstrategy is potentially useful for demonstrating homodimer formationof other Golgi enzymes. ganglioside glycosyltransferase     

Bond elongation in the anion radical of coordinated tetrazine ligands: A crystallographic, spectroscopic and computational study of a reduced {Re(CO)3Cl} complex

The binuclear complex [(μ-Me₂BPTZ)(Re(CO)₃Cl)₂] (1), where Me₂BPTZ = 3,6-(5-methyl-pyridyl)-1,2,4,5-tetrazine, can be reduced by addition of bis(η⁵-pentamethylcyclopentadienyl) iron(II) (decamethylferrocene, Fc^∗), to obtain a stable radical anion form 1⁻. A single-crystal X-ray diffraction study of the radical anion (1⁻)(Fc^∗+) was conducted and compared with a computational model of the same compound in the neutral and reduced states. As such, this work presents the first structural analysis of a reduced diimine ligand that is coordinated to {Re(CO)₃Cl} moieties. Bond-length changes within the tetrazine ring system were consistent with previously reported examples of tetrazine radicals and with calculated structures that show clear elongation of the azo-type NN bond. Consistently atomic charge calculations indicate that the extra electron in the radical anion resides largely at the tetrazine core. A negligible change in the Re-Cl bond length is observed and computed.     

The unusual tetrasaccharide sequence GlcA{beta}-3GalNAc(4-sulfate)({beta}1-4GlcA(2-sulfate){beta}1-3GalNAc(6-sulfate) found in the hexasaccharides prepared by testicular hyaluronidase digestion of shark cartilage chondroitin sulfate D

Eight hexasaccharide fractions were isolated from commercialshark cartilage chondroitin sulfate D by means of gel nitrationchromatography and HPLC on an amine-bound silica column afterexhaustive digestion with sheep testicular hyaluronidase. Capillaryelectrophoresis of the enzymatic digests as well as one- andtwo-dimensional 500 MHz ¹H-NMR spectroscopy demonstrated thatthese hexasaccharides share the common core saccharide structureGlcAß1-3GalNAcß1-4GlcAß1-3GalNAcß1-4GlcAß1-3GalNAcwith three, four, or five sulfate groups in different combinations.Six structures had the same sulfation profiles as those of theunsaturated hexasaccharides isolated from the same source afterdigestion with chondroitinase ABC (Sugahara et al., Eur. J.Biochem., 293, 871–880, 1996) and the other two have notbeen reported so far. In the new components, a D disaccharideunit, GlcA(2-sulfate)ß1-3GalNAc(6-sulfate), characteristicof chondroitin sulfate D was arranged on the reducing side ofan A disaccharide unit, GlcAß1-3GalNAc(4-sulfate),forming an unusual A-D tetrasaccharide sequence, GlcAß1-3GalNAc(4-sulfate)-4GlcA(2-sulfate)ß1-3GaINAc(6-sulfate)which is known to be recognized by the monoclonal antibody MO225.These findings support the notion that the tetrasaccharide sequence,GlcAß1-3GalNAc(4-sulfate)ß1-4GlcAß1-3GalNAc(6-sulfate)is included in the acceptor site of a hitherto unreported 2-O-sulfotransferaseresponsible for its synthesis. The sulfated hexasaccharidesisolated in this study will be useful as authentic oligosaccharideprobes and enzyme substrates in studies of sulfated glycosaminogly-cans. sulfated hexasaccharides chondroitin sulfate D hyaluronidase ¹ H-NMR     

Synthesis of 7-O-(2-deoxy-2-sulfamido-alpha-D-glucopyranosyl)-4-methylcoumarin sodium salt: a fluorogenic substrate for sulfamidase

The title compound, useful for testing the efficacy of heparin sulfamidase, was synthesized in good yield starting from 2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-glucopyranosyl fluoride, reducing the azido group efficiently with SnCl(2)-PhSH-Et(3)N reagent and finally crystallizing the N-sulfated product from methanol after deacetylation.     

Mouse bestrophin-2 is a bona fide Cl(-) channel: identification of a residue important in anion binding and conduction

Bestrophins have recently been proposed to comprise a new family of Cl(-) channels. Our goal was to test whether mouse bestrophin-2 (mBest2) is a bona fide Cl(-) channel. We expressed mBest2 in three different mammalian cell lines. mBest2 was trafficked to the plasma membrane as shown by biotinylation and immunoprecipitation, and induced a Ca(2+)-activated Cl(-) current in all three cell lines (EC(50) for Ca(2+) = 230 nM). The permeability sequence was SCN(-): I(-): Br(-): Cl(-): F(-) (8.2: 1.9: 1.4: 1: 0.5). Although SCN(-) was highly permeant, its conductance was approximately 10% that of Cl(-) and SCN(-) blocked Cl(-) conductance (IC(50) = 12 mM). Therefore, SCN(-) entered the pore more easily than Cl(-), but bound more tightly than Cl(-). Mutations in S79 altered the relative permeability and conductance for SCN(-) as expected if S79 contributed to an anion binding site in the channel. P(SCN)/P(Cl) = 8.2 +/- 1.3 for wild-type and 3.9 +/- 0.4 for S79C. G(SCN)/G(Cl) = 0.14 +/- 0.03 for wild-type and 0.94 +/- 0.04 for S79C. In the S79 mutants, SCN(-) did not block Cl(-) conductance. This suggested that the S79C mutation altered the affinity of an anion binding site for SCN(-). Additional evidence that S79 was located in the conduction pathway was provided by the finding that modification of the sulfhydryl group in S79C with MTSET(+) or MTSES(-) increased conductance significantly. Because the effect of positively and negatively charged MTS reagents was similar, electrostatic interactions between the permeant anion and the channel at this residue were probably not critical in anion selectivity. These data provide strong evidence that mBest2 forms part of the novel Cl(-) conduction pathway in mBest2-transfected cells and that S79 plays an important role in anion binding in the pore of the channel.     

Coordination characteristics of di-O-picolyl derivative of 1,1′-methylene-bis(2-naphthol): First crystal structure of a monomeric Cu(II) complex of bis{2-[(pyridin-2-yl)methoxy] naphthalen-1-yl}methane

Monomeric Cu(II) complex of bis{2-[(pyridin-2-yl)methoxy] naphthalen-1-yl}methane (L) having a composition, {[Cu(L)(H₂O)(ClO₄)](ClO₄)(CH₃OH)₂} (1) has been synthesized and characterized by analytical, spectral and magnetic methods and the structure has been established for the first time based on single crystal XRD. The Cu²⁺ center shows axially elongated octahedron with a bound water and perchlorate moieties, where two of the coordinations were long but within the van der Waals distance. The bound water, perchlorate ions and the methanol of crystallization are primarily responsible for the formation of extended lattice structure resulted from the intra-helical interactions. Complex 1 exhibit catecholase activity as studied using 3,5-di-tert-butyl catechol.     

(1->3),(1->4)-{beta}-d-Glucans in the cell walls of the Poales (sensu lato): an immunogold labeling study using a monoclonal antibody

(1→3),(1→4)-?-Glucans had previously been detected in nonlignified cell wall preparations of only the Poaceae and five other families in the graminoid clade of the Poales (s.l.). Cell walls of vegetative organs of 12 species in nine families of the Poales (s.l.) were examined by immunogold labeling using a monoclonal antibody to (1→3),(1→4)-?-glucans. Three types of wall-labeling patterns were identified depending on the density of labeling of the nonlignified walls of epidermal and parenchyma cells and the lignified walls of sclerenchyma fibers and xylem tracheary elements: type 1 in Poaceae and Flagellariaceae, type 2 in Restionaceae and Xyridaceae, and type 3 in Cyperaceae and Juncaceae. Type 1 had the heaviest labeling of nonlignified walls and type 2 the heaviest labeling of lignified walls. Type 3 had the least wall labeling, with only very light labeling of nonlignified and lignified walls. No labeling was found over walls of Typhaceae, Sparganiaceae, or Bromeliaceae. The results are discussed in relation to Poales phylogeny.