Interaction of Angiotensin I-Converting Enzyme with Two Potent Tricyclic Inhibitors

Abstract

Inhibition of rabbit lung angiotensin I-converting enzyme was studied with two inhibitors that combined tricyclic mimics of a substrate C-terminal dipeptide recognition unit with a 4-phenylbutanoic acid fragment. The overall inhibition constant for [4S-[4α,7α(R),12bB]]-7–[S-(l-carboxy-3-phenylpropyl)amino]-1,2,3,4,6,7,8,12b-octahydro-6-oxopyrido[2,1-a][2]benzazepine-4-carboxylic acid (MDL 27,088) was approximately 4pM, whereas that for [4R-[4α,7α(S),12β]]-7–[S-(1-carboxy-3-phenylpropyl)amino]-3,4,6,7,8,12b-hexahydro-6-oxo-1H-[1,4]thiazino[3,4-a][2]benzazepine-4-carboxylic acid (MDL 27,788) was estimated to be 46 pM. The formation of an initial complex of target enzyme and MDL 27,088 and its slower isomerization to a second complex were characterized kinetically. Both compounds appear to be among the most potent inhibitors known for this enzyme.     

CI-906 and CI-907: new orally active nonsulfhydryl angiotensin-converting enzyme inhibitors

CI-906, [3S-[2[R*(R*)]], 3R*]-2-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]-amino]-1-oxopropyl] 1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid, monohydrochloride, and CI-907, [2S-[1[R*(R*)]], 2 alpha, 3a beta, 7a 7a beta]-1-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]amino] 1-oxopropyl]octahydro-1H-indole-2-carboxylic acid, monohydrochloride, are two new nonsulfhydryl-type angiotensin-converting enzyme (ACE) inhibitors. Monoester (prodrug) and diacid forms produced concentration-related ACE inhibition in guinea pig serum (IC50 for CI-906 = 8.3 X 10(-9) M, diacid = 2.8 X 10(-9) M; CI-907 = 1.0 X 10(-7) M, diacid = 2.6 X 10(-9) M). In isolated rabbit aortic rings and in in vivo rat and dog autonomic studies, both compounds were highly specific in suppressing the contractile or pressor responses to angiotensin I. In two-kidney, one-clip Goldblatt (renin-dependent) hypertensive rats there was a good correlation between the inhibition of vascular converting enzyme and blood pressure lowering and a poor correlation between blood pressure lowering and plasma and brain converting enzyme inhibition. Cardiovascular, pulmonary, and central nervous system performance evaluations showed no side effects or gross toxicity. The preclinical profile shows CI-906 and CI-907 to be specific, potent, orally active ACE inhibitors. They are expected to have therapeutic utility in hypertension and in any other condition where converting enzyme inhibition would be useful.     

6-Substituted 3,4-dihydro-naphthalene-2-carboxylic acids: synthesis and structure-activity studies in a novel class of human 5alpha reductase inhibitors

Novel 3,4-dihydro-naphthalene-2-carboxylic acids were synthesized and evaluated for 5alpha reductase inhibitory activity. This enzyme exists in two isoforms and is a pharmacological target for the treatment of benign prostatic hyperplasia, male pattern baldness and acne. In the present study non-steroidal compounds capable of mimicking the transition state of the steroidal substrates were prepared. The synthetic strategy for the preparation of compounds 1-6 consisted of triflation followed by subsequent Heck-type carboxylation or methoxy carbonylation for 6-phenyl-3,4-dihydronaphthalen-2(1H)-one 1c. A Negishi-type coupling reaction between 6-(trifluoro-methanesulfonyloxy)-3,4-dihydro-naphthalene-2-carboxylic acid methyl ester 7b and various aryl bromides led, after further transformations, to 6-substituted 3,4-dihydro-naphthalene-2-carboxylic acids 7-15. In a similar way the corresponding naphthalene-2-carboxylic acids 16 and 17 were obtained. The DU 145 cell line and prostate homogenates served as enzyme sources for the human type 1 and type 2 isozymes, whereas ventral prostate was employed to evaluate rat isozyme inhibitory potency. The most active inhibitors identified in this study were 6-[4-(N,N-dicyclohexylaminocarbonyl)phenyl]-3,4-dihydro-naphthalene-2-carboxylic acid (3) (IC50 = 0.09 microM, rat type 1), 6-[3-(N,N-dicyclohexylaminocarbonyl)phenyl]-3,4-dihydro-naphthalene-2-carboxylic acid (13) (IC50 = 0.75 microM, human type 2; IC50 = 0.81 microM, human type 1) and 6-[4-(N,N-diisopropylamino-carbonyl)phenyl]naphthalene-2-carboxylic acid (16) (IC50 = 0.2 microM, human type 2). The latter compound was shown to deactivate the enzyme in an uncompetitive manner (Ki = 90 nM; Km, Testosterone = 0.8-1.0 microM) similar to the steroidal inhibitor Epristeride. Select inhibitors (13 and 16) were tested in vivo using testosterone propionate-treated, juvenile, orchiectomized SD-rats. None of the compounds was active at a dose of 25 mg/kg. This result might in part be ascribed to the relatively poor in vitro rat isozyme inhibitory potency.     

The response to thromboxane A2 analogues in human platelets. Discrimination of two binding sites linked to distinct effector systems

Thromboxane A2 (TXA2) induces platelet shape change, secretion, and aggregation. Using a novel TXA2/prostaglandin endoperoxide receptor antagonist, [1r-[1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-[[(1,1'- biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl) cyclopentyl]-4-heptenoic acid hydrochloride (GR32191), we demonstrate that these responses are mediated by at least two receptor-effector systems. GR32191 non-competitively inhibited platelet aggregation to the TXA2 mimetics, (15S)-hydroxy-11,9-(epoxymethano) prostadienoic acid (U46619) and [1S-(1 alpha,2 beta(5Z),3 alpha (1E,-3S), 4 alpha)]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)7- oxabicyclo[2.2.1]hept-2yl]-5-heptenoic acid by binding irreversibly to a TXA2/prostaglandin endoperoxide receptor. Dissociation of [3H]GR32191 from human platelets demonstrated two specific binding sites, one which was rapidly dissociating and a site to which binding was essentially irreversible. Stimulation by U46619 of platelets incubated with GR32191 and subsequently washed to expose the reversible binding site failed to aggregate or to secrete [3H]5-hydroxy-tryptamine; formation of inositol phosphates and activation of protein kinase C were markedly suppressed. In contrast, platelet shape change and calcium stimulation remained at 90% of control. Furthermore, stimulation of the reversible binding site with U46619 induced aggregation in the presence of ADP, demonstrating its functional importance in amplifying the response to other agonists. These data suggest that TXA2 mediates platelet activation through at least two receptor-effector systems; one linked to phospholipase C activation, resulting in platelet aggregation and secretion and a second site mediating an increase in cytosolic calcium and platelet shape change.     

Thromboxane A2 receptors are influenced by cell density in cultured rat aortic smooth muscle cells.

The influence of cell density on the binding characteristics of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors in rat aortic vascular smooth muscle cells in culture were determined using [1S- (1 alpha, 2 beta (5Z), 3a (1E, 3R*), 4 alpha)]- 7 -[3- (3-hydroxy -4- (4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan- 2yl]-5-heptenoic acid (125I-BOP). The Bmax for 125I-BOP was 5,430 +/- 139 sites/cell (26.9 +/- 5.7 fmoles/mg protein) for cells cultured in 1% fetal calf serum and 2809 +/- 830 sites/cell (13.1 +/- 2.2 fmoles/mg protein) for cells cultured in 10% fetal calf serum. Cells were allowed to grow to varying densities and then harvested for assay. There was a negative correlation between the Bmax and the cell density per flask. The Kd for I-BOP did not significantly vary in any of the studies. The results demonstrate that cell density plays an important role in influencing the expression of vascular TXA2/PGH2 receptors.     

6-Substituted 3,4-Dihydro-naphthalene-2-carboxylic Acids: Synthesis and Structure-Activity Studies in a Novel Class of Human 5α Reductase Inhibitors

Novel 3,4-dihydro-naphthalene-2-carboxylic acids were synthesized and evaluated for 5 α reductase inhibitory activity. This enzyme exists in two isoforms and is a pharmacological target for the treatment of benign prostatic hyperplasia, male pattern baldness and acne. In the present study non-steroidal compounds capable of mimicking the transition state of the steroidal substrates were prepared. The synthetic strategy for the preparation of compounds 1 - 6 consisted of triflation followed by subsequent Heck-type carboxylation or methoxy carbonylation for 6-phenyl-3,4-dihydro-naphthalen-2(1H)-one 1c. A Negishi-type coupling reaction between 6-(trifluoro-methanesulfonyloxy)-3,4-dihydro-naphthalene-2-carboxylic acid methyl ester 7b and various aryl bromides led, after further transformations, to 6-substituted 3,4-dihydro-naphthalene-2-carboxylic acids 7 - 15. In a similar way the corresponding naphthalene-2-carboxylic acids 16 and 17 were obtained. The DU 145 cell line and prostate homogenates served as enzyme sources for the human type 1 and type 2 isozymes, whereas ventral prostate was employed to evaluate rat isozyme inhibitory potency. The most active inhibitors identified in this study were 6-[4- (N, N -dicyclohexylaminocarbonyl)phenyl]-3,4-dihydro-naphthalene-2-carboxylic acid (3) (IC 50 =0.09 μM, rat type 1), 6-[3- (N, N -dicyclohexylaminocarbonyl)phenyl]-3,4-dihydro-naphthalene-2-carboxylic acid (13) (IC 50 =0.75 μM, human type 2; IC 50 =0.81 μM, human type 1) and 6-[4- (N, N -diisopropylamino-carbonyl)phenyl]naphthalene-2-carboxylic acid (16) (IC 50 =0.2 μM, human type 2). The latter compound was shown to deactivate the enzyme in an uncompetitive manner (Ki=90 nM; Km, Testosterone=0.8-1.0 μM) similar to the steroidal inhibitor Epristeride. Select inhibitors (13 and 16) were tested in vivo using testosterone propionate-treated, juvenile, orchiectomized SD-rats. None of the compounds was active at a dose of 25 mg/kg. This result might in part be ascribed to the relatively poor in vitro rat isozyme inhibitory potency.     

Structure-activity relationship studies of 3-dodecanoylindole-2-carboxylic acid inhibitors of cytosolic phospholipase A2alpha-mediated arachidonic acid release in intact platelets: variation of the keto moiety

Recently we found that 1-methyldodecanoylindole-2-carboxylic acid (1) and 1-[2-(4-carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (4) were inhibitors of the cytosolic phospholipase A2alpha (cPLA2alpha)-mediated arachidonic acid release in calcium ionophore A23187-stimulated human platelets with IC50-values of 4.8 microM (1) and 0.86 microM (4). We have now replaced the 3-acyl residue of these compounds by alkylated sulfinyl-, sulfony-, sulfinamoyl-, sulfamoyl-, carbonylamino-, or carbonylaminomethyl-substituents. Structure-activity relationship studies revealed that the pronounced cellular activity of 4 strongly depends on the presence of the 3-acyl moiety. Surprisingly, when testing 4 and its derivatives in an assay with the isolated cPLA2, none of these compounds showed an inhibitory potency at 10 microM indicating that they do not inhibit cPLA2alpha in the cells by a direct interaction with the active site of the enzyme.     

Further characterization of allergenically active oligosaccharitols isolated from a sea squirt H-antigen.

Complete primary structures of five allergenically active oligosaccharitols (HPG-beta 2-N5a, -N6, -N7a, -N7b, and -N9) derived from a sea squirt H-antigen were studied. Structural characterization was carried out by a new method in which products of limited periodate oxidation, followed by derivatization with p-aminobenzoic acid ethyl ester, were analyzed by a combination of HPLC, fast atom-bombardment mass spectrometry, sequential glycosidase digestion, methylation analysis, and 500-MHz 1H NMR. Established structures of GalNAc beta 1-4 (GalNAc alpha 1-2Fuc alpha 1-3) GlcNAc beta 1-3GalNAc-ol, GalNAc beta 1-4GlcNAc beta 1-3 (GalNAc beta 1-4GlcNAc beta 1-6) GalNAc-ol, GalNAc beta 1-4GlcNAc beta 1-3[GalNAc beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1-6] GalNAc-ol, GalNAc beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1-3[GalNAc beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1-6] GalNAc-ol, and GalNAc beta 1-4 (GalNAc alpha 1-2Fuc alpha 1-3)GlcNAc beta 1-3 [GalNAc beta 1-4 (GalNAc alpha 1-2Fuc alpha 1-3)GlcNAc beta 1-6]GalNAc-ol are represented by HPG-beta 2-N5a, -N6, -N7a, -N7b, and -N9, respectively. These structures have not been encountered previously. Oligosaccharide units GalNAc alpha 1-2Fuc alpha 1-, GalNAc beta 1-4GlcNAc beta 1-, and Fuc alpha 1-3GlcNAc beta 1- are considered to be the allergenically specific epitopes. Partial assignments of 500-MHz 1H NMR spectra of these novel O-linked oligosaccharitols were attempted.     

Iridoid glucosides and flavones from the aerial parts of Avicennia marina

Two new iridoid glucosides, namely, 2'-O-[(2E,4E)-5-phenylpenta-2,4-dienoyl]mussaenosidic acid (1; mussaenosidic acid = [1S-(1alpha,4aalpha,7alpha,7aalpha)]-1-(beta-D-glucopyranosyloxy)-1,4a,5,6,7,7a-hexahydro-7-hydroxy-7-methylcyclopenta[c]pyran-4-carboxylic acid) and 2'-O-(4-methoxycinnamoyl)mussaenosidic acid (2), were isolated from the aerial parts of the mangrove plant Avicennia marina. Beside that, one known iridoid glucoside, 2'-O-coumaroylmussaenosidic acid (3) and four known flavones (flavone = 2-phenyl-4H-1-benzopyran-4-one) including 4',5-dihydroxy-3',7-dimethoxyflavone (4), 4',5-dihydroxy-3',5',7-trimethoxyflavone (5), 4',5,7-trihydroxyflavone (6), and 3',4',5-trihydroxy-7-methoxyflavone (7) were also isolated and identified. The structures of these compounds were elucidated by NMR spectroscopy and by low- and high-resolution mass spectrometry. The chemotaxonomic significance of these findings was discussed. In addition, each isolated compound was evaluated for the ability of alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical-scavenging activity.     

8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors, such as alpha7, alpha3beta4 and alpha4beta2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (100 nM), a linoleic acid derivative, potentiated responses of alpha7, alpha3beta4 and alpha4beta2 ACh receptors expressed in Xenopus oocytes that are blocked by 3-(1-[dimethylaminopropyl] indol-3-yl)-4-[indol-3-yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for alpha3beta4 ACh receptors. DCP-LA enhanced the nicotine-triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell-shaped dose-dependent manner at concentrations ranging from 10 nM to 10 microM, although DCP-LA by itself had no effect on GABA release. The DCP-LA action was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors, but not by mecamylamine or dihydro-beta-erithroidine, an inhibitor of alpha3beta4 and alpha4beta2 ACh receptors. A similar effect on GABA release was obtained with 12-O-tetradecanoylphorbol 13-acetate, a PKC activator. DCP-LA (100 nM) also enhanced GABA release triggered by choline, an agonist of alpha7 ACh receptors, but not 3-[2(s)-azetidinylmethoxy] pyridine, an agonist of alpha4beta2 ACh receptors. In addition, DCP-LA (100 nM) increased the rate of nicotine-triggered GABA(A) receptor-mediated miniature inhibitory post-synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or alpha-bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP-LA stimulates GABA release by enhancing activity of pre-synaptic alpha7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.     

Enzymatic synthesis of chiral intermediates for Omapatrilat, an antihypertensive drug

Biocatalytic processes were used to prepare chiral intermediates required for the synthesis of Omapatrilat 1 by three different routes. The synthesis and enzymatic conversion of 2-keto-6-hydroxyhexanoic acid 3 to L-6-hydroxynorleucine 2 was demonstrated by reductive amination using beef liver glutamate dehydrogenase. To avoid the lengthy chemical synthesis of the ketoacid 3, a second route was developed to prepare the ketoacid by treatment of racemic 6-hydroxy norleucine [readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin 4] with D-amino acid oxidase from porcine kidney or Trigonopsis variabilis followed by reductive amination to convert the mixture completely to L-6-hydroxynorleucine in 98% yield and 99% enantiomeric excess (e.e.). The enzymatic synthesis of (S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (allysine ethylene acetal, 5) was demonstrated using phenylalanine dehydrogenase (PDH) from T. intermedius. Phenylalanine dehydrogenase was cloned and overexpressed in Escherichia coli and Pichia pastoris. Using PDH from E. coli or P. pastoris, the enzymatic process was scale-up to prepare kg quantity of allysine ethylene acetal 5. The reaction yields of >94% and e.e. of >98% were obtained for allysine ethylene acetal 5. An enzymatic process was developed for the synthesis of [4S-(4a,7a,10ab)]1-octahydro-5-oxo-4 [[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid [BMS-199541-01]. The enzymatic oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-[N[[(phenyl-methoxy)carbonyl] L-homocysteinyl] L-lysine)-1,1-disulphide [BMS-201391-01] to produce BMS-199541-01 using a novel L-lysine epsilon-aminotransferase (LAT) from Sphingomonas paucimobilis SC 16113 was demonstrated. This enzyme was overexpressed in E. coli and a process was developed using the recombinant enzyme.     

Synthesis and SAR of 3-arylsulfonyl-pyrazolo[1,5-a]pyrimidines as potent serotonin 5-HT6 receptor antagonists

Syntheses of a series of novel 3-sulfonyl-pyrazolo[1,5-a]pyrimidines and their 5-HT(6) receptor antagonistic structure-activity relationship are disclosed. The nature and position of substituents, which affect their receptor antagonistic activity, are analyzed. Among all synthesized derivatives, {3-(3-chlorophenylsulfonyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-2-yl}-methyl-amine 33 (K(i)=190 pM), (3-phenylsulfonyl-7-methyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine 44 (K(i)=240 pM), (3-phenylsulfonyl-5-metoxymethyl-7-methyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine 50 (K(i)=270 pM), and (3-phenylsulfonyl-5-methyl-7-metoxymethyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine 52 (K(i)=280 pM) are the most potent antagonists of the 5-HT(6) receptors.     

Synthesis of dopamine transporter selective 3-diarylmethoxymethyl-8-arylalkyl-8-azabicyclo[3.2.1]octane derivatives

A series of diarylmethoxymethyltropane-GBR hybrid analogues with all three possible stereochemical orientations at C3 were synthesized and evaluated at dopamine and serotonin transporters. The 3alpha derivatives were found to be the most potent compounds with the 3alpha-di(4-fluorophenyl)methoxymethyl-8-(3-phenylpropyl)-8-azabicyclo[3.2.1]octane 15b (Ki = 5 nM) being the most potent compound of the series. The corresponding 3-di(4-fluorophenyl)-methoxymethyl-8-(3-phenylpropyl)-8-azabicyclo[3.2.1]oct-2-ene 12b (Ki = 12 nM) was slightly less potent than the 3alpha-analogue, while the 3beta-di(4-fluorophenyl)methoxymethyl-8-(3-phenylpropyl)-8-azabicyclo[3.2.1]octane 23b (Ki = 78 nM) exhibited only modest affinity for the dopamine transporter. Only the 3alpha-analogue 15b (SERT/DAT = 48) exhibited higher SERT/DAT selectivity than GBR 12909. These results indicate that the dopamine transporter can tolerate some variability in proximity of the benzhydryl ether to the basic nitrogen atom of the tropane without loss in potency. In addition, the structure-activity data for these tropane-GBR 12909 hybrid analogues support previous findings that the stereochemical and conformational effects imparted by unsaturation at C3 are important for dopamine transporter selectivity over the serotonin transporter.     

Cis-trans isomerization of an angiotensin I-converting enzyme inhibitor. An enzyme kinetic and nuclear magnetic resonance study

The angiotensin I-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) inhibitor, ramiprilat (2-[N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-Ala]-(1S,3S,5S)-2- azabicyclo[3.3.0]octane-3-carboxylic acid), is shown to exist in tow conformational isomers, cis and trans, which interconvert around the amide bond. The two conformers were separated by reversed-phase high-performance liquid chromatography. The conformers were identified by nuclear Overhauser effect measurements. From line shape analysis the isomerization rate constants were determined to be kcis----trans = 15 s-1 and ktrans----cis = 5 s-1 at 368 K in [2H]phosphate buffer (p2H 7.5). By enzyme kinetic studies using 3-(2-furylacryloyl)-L-Phe-Gly-Gly as substrate, the trans conformer was found to be the most potent enzyme inhibitor, whereas the cis conformer had a very low inhibitory effect. A new inhibition mechanism is presented for this type of slow, tight-binding inhibitors that contain an amide bond. This mechanism involves an equilibrium between the two conformers and the enzyme-bound inhibitor complex.     

Novel ofloxacin derivatives: synthesis, antimycobacterial and toxicological evaluation

Thirty novel 9-fluoro-2,3-dihydro-8,10-(mono/di-sub)-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acids were synthesized from 2,3,4,5-tetrafluoro benzoic acid and evaluated for in vitro and in vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium smegmatis (MC(2)) and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from mycobacteria. Among the synthesized compounds, 10-[2-carboxy-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl]-9-fluoro-2,3-dihydro-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid was found to be the most active compound in vitro with MIC99 of 0.19 microM and 0.09 microM against MTB and MTR-TB, respectively. In the in vivo animal model also the same compound decreased the bacterial load in lung and spleen tissues with 1.91 and 2.91--log10 protections, respectively, at the dose of 50mg/kg body weight. Compound 10-[(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)]-9-fluoro-2,3-dihydro-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid was found to be the most active in the inhibition of the supercoiling activity of DNA gyrase with an IC(50) of 10.0 microg/mL. The results demonstrate the potential and importance of developing new oxazino quinolone derivatives against mycobacterial infections.     

Design,synthesis, and biological evaluation of a series of beta-lactam-based prodrugs

By use of pro-dual-drug concept the synthesis of 6-beta-[(R)-2-(clavaminio-9-N-yl)-2-(4-hydroxyphenylacetamido)]penicillanic acid (10), 6-beta-[(R)-2-(amino)-2-(4-(clavulano-9-O-yl)phenylacetamido)]penicillanic acid (13), (Z)-4-[2-(amoxycillin-4-O-yl)ethylidene]-2-(clavulano-9-O-yl)-3-methoxy-Delta(alpha,beta)-butenolide (19), and 3-[(amoxicillin-4-O-yl)methyl]-7-(phenoxyacetamido)-(1-oxo)-3-cephem-4-carboxylic acid (23) was accomplished. Unlike penicillin G, ampicillin, or amoxicillin, these four heretofore undescribed compounds 10, 13, 19, and 23 showed notable activity against beta-lactamase (betaL) producing microorganisms, Staphylococcus aureus A9606, S. aureus A15091, S. aureus A20309, S. aureus 95, Escherichia coli A9675, E. coli A21223, E. coli 27C7, Pseudomonas aeruginosa 18S-H, and Klebsiella pneumoniae A20634 TEM. In comparison with amoxicillin (9), alpha-amino-substituted compound 10 and butenolide derivative 19 showed a broadened spectrum of antibacterial activity; yet they were found to be less active than 13 and 23. Like clavulanic acid (7) or cephalosporin-1-oxide (21), the newly synthesized compounds 10, 13, 15, 16, 19, or 23 functioned as potent inhibitors of various bacterial betaLs.     

Thiol modification as a probe of conformational forms of the F1 ATPase of Escherichia coli and of the structural asymmetry of its beta subunits

The sulfhydryl groups of soluble and membrane-bound F1 adenosine triphosphatase of Escherichia coli were modified by reaction with the fluorescent thiol reagents 5-iodoacetamidofluorescein, 2-[(4'-iodoacetamido)anilino]naphthalene-6-sulfonic acid 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-d iaz ole and 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid. Whereas gamma and delta subunits were always labeled by these reagents, the beta subunit reacted preferentially in the soluble enzyme, and the alpha subunit in the membrane-bound enzyme. This suggests that the soluble enzyme undergoes a conformational change on binding to the membrane. The three beta subunits of the soluble ATPase did not react with chemical reagents in a similar manner. One beta subunit was cross-linked to the epsilon subunit on treatment of the ATPase with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide, as observed previously by L?tscher et al. [Biochemistry (1984) 23, 4134-4140]. A second beta subunit, which did not cross-link to the epsilon subunit, was modified preferentially by the fluorescent thiol reagents and by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The third beta subunit was less chemically reactive than the others. Both alpha and beta subunits of the soluble 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole-modified enzyme were labeled by the fluorescent thiol reagents. Thus, the modified enzyme, which is inactive, probably has a different conformation from the native soluble ATPase.     

Microbiological degradation of bile acids. Metabolites formed from 3-(3a alpha-hexahydro-7a beta-methyl-1,5-dioxoindan-4 alpha-yl) propionic acid by Streptomyces rubescens.

1. The metabolism of 3-(3a alpha-hexahydro-7a beta-methyl-1,5-dioxoindan-4 alpha-yl)propionic acid (III), which is a possible precursor of 2,3,4,6,6a beta, 7,8,9,9a alpha,9b beta-decahydro-6a beta-methyl-1H-cyclopenta[f]quinoline-3,7-dione (II) formed from cholic acid (I) by streptomyces rubescens, was investigated by using the same organism. 2. This organism effected amide bond formation, reduction of the carbonyl groups, trans alpha beta-desaturation and R-oriented beta-hydroxylation of the propionic acid side chain and skeleton cleavage, and the following metabolites were isolated as these forms or their derivatives: compound (II), 1,2,3,4 a beta,-5,6,6a beta,7,8,9a alpha,9b beta-dodecahydro-6a beta -methylcyclopental[f][1]benzopyran-3,7-dione (IVa), (1R)-1,2,3,4a beta,5,6,6a beta,7,8,9.9a alpha,9b beta-dodecahydro-1-hydroxy-6a beta-methylcyclopenta[f][1]benzopyran-3,7-dione (IVb), (E)-3-(3aalpha-hexahydro-5 alpha-hydroxy-7a beta-methyl-l-oxo-indan-4 alpha-yl)prop-2-enoic acid (V), (+)-(5R)-5-methyl-4-oxo-octane-1,8-dioic acid (VI), 3-(4-hydroxy-5-methyl-2-oxo-2H-pyran-6-yl)propionic acid (VII) and 3-(3a alpha-hexahydro-1 beta-hydroxy-7a beta-methyl-5-oxoindan-4 alpha-yl)propionic acid (VIII). The metabolites (IVb), (V), (VI) and (VII) were new compounds, and their structures were established by chemical synthesis. 3. The question of whether these metabolites are true degradative intermediates is discussed, and a degradative pathway of compound (III) to the possible precursor of compound (VII), 7-carboxy-4-methyl-3,5-dioxoheptanoyl-CoA (IX), is tentatively proposed. The further degradation of compound (IX) to small fragments is also considered.     

Steps in the conversion of alpha-ketosuberate to 7-mercaptoheptanoic acid in methanogenic bacteria

The biosynthetic steps involved in the conversion of alpha-ketosuberate to 7-mercaptoheptanoic acid were studied in cell-free extracts of methanogenic bacteria. The pathway was established by measuring the incorporation of stable isotopically labeled precursors into the S-methyl ether methyl ester derivative of the enzymatically generated 7-mercaptoheptanoic acid by using gas chromatography-mass spectrometry (GC-MS). Quantitation of the 7-mercaptoheptanoic acid produced in the incubations with the substrates was accomplished by using an internal standard of 6-mercaptohexanoic acid. [4,4,6,6-2H4]-2-Oxosuberic acid, [7-2H]-7-oxoheptanoic acid, [2-2H]-2(RS)-(5-carboxypentyl)thiazolidine-4(R)-carboxylic acid, and S-(6-carboxyhexyl)cysteine were each shown to be converted to 7-mercaptoheptanoic acid. Incubation of cell extracts with a mixture of 2(RS)-(5-carboxypentyl)thiazolidine-4(R)-carboxylic acid and [2-2H]-2-(RS)-(5-carboxypentyl)-[34S]thiazolidine-4(R)-carboxylic acid showed that both 34S and 2H are incorporated into the 7-mercaptoheptanoic acid but only after separation of the cysteine from the [7-2H]-7-oxyheptanoic acid portion of the molecule. Furthermore, the sulfur from the cysteine was incorporated into the thiol only after its elimination from the cysteine and subsequent mixing with an unlabeled sulfur source which had a molecular weight of sufficient size that it was excluded from Sephadex G-25. Hydrogen sulfide was found to supply the sulfur for the production of the 7-mercaptoheptanoic acid in a reaction that was shown to obtain its reducing equivalents from hydrogen via an F420-dependent hydrogenase.     

Discovery and pharmacological characterization of N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide hydrochloride (anagliptin hydrochloride salt) as a potent and selective DPP-IV inhibitor

In the course of our program for discovery of novel DPP-IV inhibitors, a series of pyrazolo[1,5-a]pyrimidines were found to be novel DPP-IV inhibitors. We identified N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide hydrochloride (4a) and described its pharmacological profiles.