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Tacrine, one of the drugs available for Alzheimer's disease based on the cholinergic approach, suffers from considerable toxicity. Many analogues of tacrine has been prepared which retain the pharmacologically rich aminopyridine or aminoquinoline motifs. The current research is a continuation of our efforts in the area of 11-aminobenzoquinolizidines (4) and 10-aminobenzoindolizidines (5) (cf. ref9). A serendipitous discovery led us to the biologically active open chain analogue 9, and we proceeded to elaborate on this molecule. Overall, the compounds we prepared were poor inhibitors of acetylcholinesterase as compared to tacrine. The single exception was compound 20 which exhibited an effect comparable to that of tacrine, but only at a dose in the order of 10(-3) M. However, despite the poor acetylcholinesterase inhibition by 9, this compound was found to be an effective antiamnesic agent.

Tacrine, one of the drugs available for Alzheimer's disease based on the cholinergic approach, suffers from considerable toxicity. Many analogues of tacrine have been prepared which retain the pharmacologically rich aminopyridine or aminoquinoline motifs. The current research was undertaken to produce an acetylcholinesterase inhibitor by employing 11-aminobenzoquinolizidines (4) and 10-aminobenzoindolizidines (5) as templates. Thus, we aimed to achieve three goals relative to tacrine: eliminate the pyridine and quinoline moieties and render the molecule less flat. Overall, the compounds we prepared were poorer inhibitors of acetylcholinesterase compared to tacrine. The single exception was compound 6f which exhibited an effect comparable to that of tacrine, but only at a dose of the order of 10(-3) M. However, despite the poor acetylcholinesterase inhibition by 6b, this compound proved to be an effective anti-amnesic agent at 45 mg/kg dose.

PNU-107859, an important representative structure in a novel class of matrix metalloproteinases (MMP) inhibitors known as thiadiazoles, was found to be quickly eliminated from rats. A major metabolite (approximately 10% of total dose) was found to be present in the bile of rats. The metabolite in question was isolated and purified from the bile fluids collected from six cannulated rats. From a total of approximately 75 mg of PNU-107859 administered to rats, 3.3 mg of the metabolite was recovered. The NMR and mass spectrometry results indicated that the metabolite is a glucuronide conjugate (1-deoxy-1beta-substituted D-glucopyranosiduronic acid) of the intact drug. Furthermore, the UV, MS, and NMR data established that the conjugate is located at the nitrogen alpha to the thiocarbonyl of the thiadiazole ring.

Cyclization of (S,S)-alpha-[(1-phenylethyl)amino]-alpha-(2-iodophenyl)acetonitrile with (COCl)(2) in toluene or chlorobenzene afforded the atropisomeric pyrazinone (aS,S) 6-(2-alpha-iodophenyl)-3,5-dichloro-1-(1-phenylethyl)-2(1H)-pyrazinone in 57% yield. With smaller ortho substituents (F, Cl, CH(3), CF(3), OCH(3)) on the aromatic ring, mixtures of atropisomers were obtained from the cyclization reaction. All of the individual atropisomers prepared were stable at room temperature. All but the o-fluoro-substituted atropisomers were stable at elevated temperatures. This paper describes a stereoselective synthesis of pyrazinones and suggests a mechanism for formation via an interesting transfer of chirality.

Unregulated or overexpressed matrix metalloproteinases (MMPs), including stromelysin, collagenase, and gelatinase. have been implicated in several pathological conditions including arthritis and cancer. Small-molecule MMP inhibitors may have therapeutic value in the treatment of these diseases. In this regard, the solution structures of two stromelysin/ inhibitor complexes have been investigated using 1H, 13C, and 15N NMR spectroscopy. Both-inhibitors are members of a novel class of matrix metalloproteinase inhibitor that contain a thiadiazole group and that interact with stromelysin in a manner distinct from other classes of inhibitors. The inhibitors coordinate the catalytic zinc atom through their exocyclic sulfur atom, with the remainder of the ligand extending into the S1-S3 side of the active site. The binding of inhibitor containing a protonated or fluorinated aromatic ring was investigated using 1H and 19F NMR spectroscopy. The fluorinated ring was found to have a reduced ring-flip rate compared to the protonated version. A strong, coplanar interaction between the fluorinated ring of the inhibitor and the aromatic ring of Tyr155 is proposed to account for the reduced ring-flip rate and for the increase in binding affinity observed for the fluorinated inhibitor compared to the protonated inhibitor. Binding interactions observed for the thiadiazole class of ligands have implications for the design of matrix metalloproteinase inhibitors.

1. We have determined that 2,4-dipyrrolidinylpyrimidine (2,4-DPP), used as a model for studies of the metabolism of therapeutic agents containing this moiety, undergoes three characteristic hydroxylations when incubated with male rat liver microsomes. Analysis of microsomal incubates of stable isotope labelled analogues of 2,4-DPP by particle beam-liquid chromatography-mass spectrometry (LC-PB-MS) has shown that the three metabolites are 4-(3-hydroxypyrrolidinyl)-2-(pyrrolidinyl)-pyrimidine (M1), 4-(2-hydroxypyrrolidinyl)-2-(pyrrolidinyl)-pyrimidine (M2) and 2-(2-hydroxypyrrolidinyl)-4-(pyrrolidinyl)-pyrimidine (M3). 2. We determined that enzymes of the cytochrome P450 family are responsible for the in vitro hydroxylations of 2,4-DPP. 3. We observed that in microsomal incubations carried out in the presence of cyanide, a single cyanide adduct is formed implicating an iminium ion intermediate in the oxidation of the 2-pyrrolidine ring. 4. We also determined the intermolecular deuterium isotope effects for the formation of each of the three products. For M1, kH/kD = 14.55 +/- 0.54; for M2, kH/kD = 6.01 +/- 0.65; and for M3, kH/kD = 5.35 +/- 1.18. 5. We interpret these data as suggesting that M2 and M3 are formed by the same mechanism, probably including the formation of an iminium ion, and that M1 is formed by direct hydrogen abstraction.

A series of N-substituted bridgehead azabicycloalkanes has been prepared and examined as substrates for microbiological oxygenation using the fungi Beauveria bassiana, Rhizopus nigricans, Aspergillus ochraceus, and Rhizopus arrhizus. Oxygenation using B. bassiana of N-tosyl-7-azabicyclo[2.2.1]heptane gave N-[p-(hydroxymethyl)benzenesulfonyl]-7-azabicyclo[2.2.1]heptane (56% yield), of N-(phenyloxycarbonyl)-7-azabicyclo[2.2.1]heptane gave the 2-endo-ol (56% yield, 51% ee), of N-BOC-7-azabicyclo[2.2.1]heptane gave the 2-endo-ol (10% yield), of N-Cbz-7-azabicyclo[2.2.1]heptane gave the 2-endo-ol (28%), of N-(phenyloxycarbonyl)-8-azabicyclo[3.2.1]octane gave the 3-endo-ol, and of N-(phenyloxycarbonyl)-9-azabicyclo[3.3.1]nonane gave the 3-exo-ol (30%) and 3-one (16%). Oxygenation using R. nigricans of N-BOC-7-azabicyclo[2.2.1]heptane gave the 2-endo-ol (62% yield, 28% ee) and the 2-exo-ol (27% yield, 42% ee). Oxidation of the N-BOC-7-azabicyclo[2.2.1]heptan-2-ols gives the 2-ketone, a synthetic intermediate useful for conversion to the natural product, epibatidine. Oxygenation of N-(phenyloxycarbonyl)-7-azabicyclo[2.2.1]heptane using R. arrhizus gives the 2-endo-ol (5% yield, 31% ee) and the 2-exo-ol (18% yield, 22% ee). Oxygenation of N-(phenyloxycarbonyl)-8-azabicyclo[3.2.1]octane using A. ochraceous gives the 3-endo-ol (36%) and the 3-one (4%).

An acyl iminium ion-initiated tandem cyclization gave an unexpected dienone product, a seco-azasteroid (2). The factors governing the formation of 2 were investigated in an attempt to optimize its formation. The reaction was applied to a more elaborate system, resulting in the synthesis of the full steroid skeleton of 13-azaandrosta-1,4-diene-3,17-dione (3), which contains the unusual substitution of a chlorine atom for the axial 19-methyl.

Previously, 3-substituted cycloalkylpyranones, such as 2d, have proven to be effective inhibitors of HIV protease. In an initial series of 3-(1-phenylpropyl) derivatives with various cycloalkyl ring sizes, the cyclooctyl analog was the most potent. We became interested in exploring the influence of other structural changes, such as substitution on the phenyl ring and saturation of the 5,6-double bond, on the cycloalkyl ring size structure-activity relationship (SAR). Saturation of the 5,6-double bond in the pyrone ring significantly impacts the SAR, altering the optimal ring size from eight to six. Substitution of a sulfonamide at the meta position of the phenyl ring dramatically increases the potency of these inhibitors, but it does not change the optimal ring size in either the cycloalkylpyranone or the cycloalkyldihydropyrone series. This work has led to the identification of compounds with superb binding affinity for the HIV protease (Ki values in the 10-50 pM range). In addition, the cycloalkyldihydropyrones showed excellent antiviral activity in cell culture, with ED50 values as low as 1 microM.

An investigation of the scope and mechanism of a new synthesis of cyclopentenes from 3,6-dihydro-2H-thiopyrans is described. Alkyl halides substituted with an electron-withdrawing group in the alpha-position were reacted with sodium thiosulfate, yielding the corresponding Bunte salts, which could be transformed to reactive thiocarbonyl compounds by elimination of the elements of bisulfite with mild base treatment. In situ trapping by 1,3-dienes afforded in good yields a variety of 3,6-dihydro-2H-thiopyrans substituted with electron-withdrawing groups at the 2-position. Exposure of these cycloadducts to strong base at low temperature effected a novel ring contraction, affording 2-(methylthio)-3-cyclopentenes after quenching with methyl iodide. The level of diastereoselectivity exhibited during the generation of these cyclopentenes was found to be dependent on the nature of the electron-withdrawing group at the 2-position of the dihydrothiopyran as well as the substitution pattern originally present in the diene component. In some cases, reducing the temperature during the ring contraction resulted in the isolation of good yields of vinyl cyclopropanes of high isomeric purity. With one substrate, highly diastereoselective rearrangement of a vinyl cyclopropane to a cyclopentene was unambiguously demonstrated, suggesting that this might be a key feature of the overall ring contraction mechanism.

Tirilazad mesylate, a potent inhibitor of membrane lipid peroxidation in vitro, is under clinical development for the treatment of subarachnoid hemorrhage and head injury. In rat, tirilazad seems to be highly extracted and is cleared almost exclusively via hepatic elimination. The biotransformation of tirilazad has been investigated in liver microsomal preparations from adult male and female Sprague-Dawley rats. Tirilazad metabolism in male rat liver microsomes resulted in the formation of two primary metabolites: M1 and M2. In incubations with female rat liver microsomes, M2 was the only primary metabolite detected. Structural characterization of M1 and M2 by mass spectrometry demonstrated that M2 was formed by reduction of the delta 4-double bond in the steroid A-ring, whereas M1 arose from oxidative desaturation of one pyrrolidine ring. Further structural analysis of M2 by proton NMR demonstrated that reduction at C-5 had occurred by addition of hydrogen in the alpha-configuration. Using metabolic probes and antibodies specific to individual hepatic microsomal enzymes, CYP2C11 and 3-oxo-5 alpha-steroid:NADP+ delta 4-oxidoreductase (5 alpha-reductase) were identified as responsible for the formation of M1 and M2, respectively. The formation of M1 was inhibited by testosterone, nicotine, cimetidine, and anti-CYP2C11 IgG. The formation of M2 was inhibited by finasteride, a potent inhibitor of 5 alpha-reductase. Kinetic analysis of CYP2C11-mediated M1 formation in male rat liver microsomal incubations revealed that M1 formation occurred through a low-affinity/low-capacity process (KM = 16.67 microM, Vmax = 0.978 nmol/mg microsomal protein/min); the formation of M2 was mediated by 5 alpha-reductase in a high-affinity/low-capacity process (KM = 3.07 microM, Vmax = 1.06 nmol/mg microsomal protein/min). In contrast, the formation of M2 in female rat liver microsomes was mediated by 5 alpha-reductase in a high-affinity/high-capacity process (KM = 2.72 microM, Vmax = 4.11 nmol/mg microsomal protein/min). Comparison of calculated intrinsic formation clearances (Vmax/KM) for M1 and M2 indicated that the female rat possessed a greater in vitro metabolic capacity for tirilazad biotransformation than the male rat. Therefore, the clearance of tirilazad mesylate in the rat is mediated primarily by rat liver 5 alpha-reductase, and the capacity in the female rat is 5-fold the capacity in the male. These observations correlate with documented differences in 5 alpha-reductase activity and predict a gender difference in tirilazad hepatic clearance in vivo.

A series of novel spirocyclic benzopyran imidazolones were synthesized as rigid analogues of cromakalim. These compounds cause a dose-dependent membrane hyperpolarization of A10 rat aorta cells. This hyperpolarization was blocked by pretreatment with glyburide, indicating that the spirocyclic benzopyran imidazolones were acting by increasing the open probability of ATP-sensitive potassium channels in A10 cells. Representative compounds also showed potent in vivo activity as hypotensive agents in normotensive rats. Many of the compounds described are much more potent than cromakalim both in vitro and in vivo, with one of the most potent compounds being 2,3-dihydro-2,2-dimethyl-6-nitro-2'-(propylamino)spiro[4H-1-benzopyran- 4,4'-[4H]imidazol]-5'(1'H)-one (5r). It is concluded that the N1' nitrogen of the imidazolone is an effective substitute for the carbonyl oxygen of cromakalim. The rigid spirocyclic ring fusion holds this nitrogen in an optimum orientation relative to the benzopyran ring.

Further elaboration on the structure-activity relationships in our U-50,488 series has revealed that benzologation of this cyclohexane-1,2-diamine derivative provides compounds which either maintain the interaction with the kappa receptor (e.g. compounds 3a and 5a in the phenylacetamido series) or eliminate the mu receptor mediated analgesia (e.g. compounds 3-6 in the benzamido series). Naphthologation also caused the elimination of mu receptor mediated analgesia (e.g. compounds 17a and 17b).

The structures of melinacidines II, III and IV were determined by physicochemical methods. Melinacidine IV is considered to be identical to 11alpha,11alpha'-dihydroxychaetocin while melinacidins II and III are isomeric to chaetocin and verticillins A and B.

A new antibiotic U-62162 has been isolated from the fermentations of Streptomyces verdensis Dietz, sp. n. (UC-8157). The compound has been characterized and its gross structure has been elucidated. The antibiotic inhibited the growth of Gram-positive bacteria (particularly Staphylococcus aureus) but was inactive in experimentally infected animals.

Metabolism of tazadolene (1), a novel non-opioid analgesic with antidepressant properties, affords the 4-hydroxy and 3-methoxy-4-hydroxy derivatives (phenyl ring) of the drug, and N-[2-(phenylmethylene)cyclohexyl]-beta-alanine (4). The isolation, identification and synthesis of the latter metabolite is described.

The gorgonian coral Pseudoplexaura porosa contains a lipoxygenase capable of converting exogenous arachidonic acid into (8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid. The (8R)- (or 8-L-) configuration in this product, opposite to that observed in previously reported 8-lipoxygenase products, was determined unambiguously by comparison of oxidative ozonolysis fragments with authentic malic acid-derived standards. Extracts from the coral contained no detectable prostaglandins (PGAs, PGBs, PGEs, or PGFs). Although arachidonic acid represents one of the most abundant of the common fatty acids found in the phospholipid and total lipid fractions of P. porosa, products ascribable to the arachidonic acid 8-lipoxygenase pathway ((8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid, the corresponding alcohol 8-hydroxyeicosatetraenoic acid, further transformation products) have not yet been identified in the coral extracts. The physiological significance of the 8-lipoxygenase in this species remains a matter for speculation.

This report defines the transformations that antitumor antibiotic CC-1065 underwent under basic and acidic conditions. The isolation, purification, characterization, and biological properties of a cyclopropapyrroloindole fragment, and an acidic fragment, PDE-I dimer, from a mild alkaline fragmentation and the phenolic product, AAP, resulting from alkylation of acetic acid by the cyclopropyl function are described.

Addition of clindamycin to whole-cell cultures of Streptomyces coelicolor Müller resulted in the loss of in vitro activity against organisms sensitive to clindamycin. Incubation of such culture filtrates with crude alkaline phosphatase generated a biologically active material identified as clindamycin. Fermentation broths containing inactivated clindamycin yielded clindamycin 3-ribonucleotides and clindamycin 3-phosphate the structure of which was established by physicochemical and enzymatic means. Attempts to transform clindamycin to clindamycin 3-ribonucleotides by lysates or partially purified enzyme preparations from S. coelicolor have failed.

Rancinamycins are secondary metabolites produced by Streptomyces lincolnensis in a sulfur-depleted culture medium. The structures (except stereochemistry) of the main components of the rancinamycin-complex were determined by the use of IR, UV, PMR, and CMR spectra.

Employing osmotically shocked lysate of a spectinomycin resistant strain of Escherichia coli, trospectomycin, a new alkylspectinomycin, was adenylylated in the presence of adenosine 5'-triphosphate and magnesium ion. A highly resistant strain of E. coli was obtained by transforming a laboratory strain with a newly constructed plasmid consisting of pBR322 and a determinant for spectinomycin resistance originally found on a low copy number plasmid in E. coli strain NR79. The biologically inactive adenylylated trospectomycin was found to be trospectomycin 6-(5'-adenylate).

The antibiotic rubradirin, C48H46N4O20 was cleaved at an ester function by aqueous methylamine into rubransarol A, C23H23NO8, and a methyl amide, C26H28N4O12. Rubradirin B, C40H33N3O15, was similarly cleaved in methanolic ammonia into rubransarol B, C23H23NO8, and the primary amide, C17H13N3O7. The rubransarols are shown to be unique ansamycins which are isomeric at a double bond in the large ring.

O-Demethylpaulomycin A (C33H44N2O17S), O-demethylpaulomycin B (C32H42N2O17S), paulomenol A (C29H43NO16), paulomenol B (C28H41NO16), and the hydrogen sulfide adducts of paulomycin A (U-77,802, C34H48N2O17S2), and paulomycin B (U-77,803, C33H46N2O17S2) have been isolated from fermentations of Streptomyces paulus strain 273. The structure of these compounds was determined by 1H and 13C NMR and fast atom bombardment mass spectrum spectroscopic techniques and degradative studies. The antibacterial properties of these new metabolites, which are related to paulomycins A and B (J. Antibiotics 35: 285-294, 1982), are briefly discussed.

Paldimycin (antibiotic 273a1) and antibiotic 273a2 as well as their individual components, paldimycins A (273a1 alpha) and B (273a1 beta) and antibiotics 273a2 alpha and 273a2 beta were synthesized from paulomycin, paulomycin A and paulomycin B, respectively, by reacting with N-acetyl-L-cysteine. The semisynthetic antibiotics had chromatographic behavior (TLC, HPLC) and physical and chemical properties identical to the properties of the corresponding antibiotics produced by Streptomyces paulus.

Desertomycin was isolated from Streptomyces macronensis Dietz sp. nov. UC 8271. Extensive spectroscopic work led us to place desertomycin in the macrocyclic lactone family which contains monazomycin, scopafungin, primycin, azalomycin F4a and niphithricins A and B. The apparent molecular formula was determined by fast atom bombardment mass spectroscopy to be C57H109NO24 (MW = 1,191). Mild acid hydrolysis yielded mannose but contrary to published reports, glutamic acid is not a constituent of desertomycin.

Paulomycin A2 (C34H46N2O17S), paulomycin C (C32H42N2O17S), paulomycin D (C31H40N2O17S), paulomycin E (C29H36N2O16S) and paulomycin F (C29H38N2O16S) have been isolated from fermentations of Streptomyces paulus strain 273. The structure of these compounds was determined using NMR and mass spectroscopic techniques. The new paulomycins, like paulomycins A and B (J. Antibiotics 35: 285-294, 1982) are highly active mainly against Gram-positive organisms.

Paulomycin A, C34H46N2O17S and paulomycin B, C33H44N2O17S are two antibiotics produced by Streptomyces paulus strain 273 (UC 5142). Both antibiotics, which are mainly active against a variety of Gram-positive bacteria, contain an isothiocyanate group and in this respect they are related to senfolomycins A and B and proceomycin.

Senfolomycins A and B (Antimicrob. Agents Chemother.-1965: 828-831, 1966) are two antibacterial agents with physico-chemical and biological properties similar to those of paulomycin. Recent studies indicate that senfolomycin A (C29H36N2O16S, MW 700) has molecular composition and fast atom bombardment MS fragmentation pattern identical to those of paulomycin E. Extensive NMR work indicates that the two antibiotics, which have been separated by HPLC and TLC, differ only in the stereochemistry of the OCH3 group present in their respective sugar moieties. Indirect evident suggests that senfolomycin B is dihydrosenfolomycin A (C29H38N2O16S, MW 702) and in this respect it is related to paulomycin F. The proposed structures for senfolomycins A and B are discussed.

The isolation of paulomycins A and B from fermentations of Streptomyces paulus has been reported earlier [J. Antibiotics 35: 285-294, 1982]. Further work on the antibiotics produced by S. paulus revealed the production of two paulomycin-related compounds, antibiotics 273a1 and 273a2 which were isolated by procedures involving extractions and chromatography over buffered silica gel. Antibiotic 273a1 which has been named paldimycin, was found to be a mixture of two materials, paldimycins A and B (antibiotics 273a1 alpha, and 273a1 beta). Similarly, antibiotic 273a2 was found to consist of antibiotic 273a2 alpha and antibiotic 273a2 beta. Paldimycin and antibiotic 273a2, which are produced by addition of two or one molecules of N-acetyl-L-cysteine, respectively, to paulomycins A and B, are active vs. Gram-positive bacteria.

Feldamycin, a new antibacterial agent, and nojirimycin, a previously described antibiotic have been isolated from cultures of Streptomyces ficellus. Feldamycin, C17H25N7O5, is an amphoteric compound which inhibits a variety of bacteria in vitro but is found to be ineffective in the treatment of experimental bacterial infections in mice. Nojirimycin (5-amino-5-deoxy-D-glucose) has been isolated previously from cultures of several species of streptomycetes.

1-Methylpseudouridine is a new metabolite isolated from culture filtrates of Streptomyces platensis. The structure of this compound was determined from its physical and spectral properties.

Ficellomycin is a new basic antibiotic produced by Streptomyces ficellus. Ficellomycin, C13H24N6O3, inhibits the growth of gram-positive bacteria in vitro and is effective in the treatment of experimental Staphylococcus aureus infections in mice.

A new metabolite N-acetyl-L-phenylalanyl-L-phenylalaninol was isolated from culture filtrates of Emericellopsis salmosynnemata which produces zervamicins I and II. The structure was assigned from spectral properties and degradative studies.

The preparation of 2"-deoxykanamycin B (12) and 2",3',4'-trideoxykanamycin B (14) from neamine (1) is described. Key intermediates in the synthesis of these 2"-deoxyaminoglycoside antibiotics are 3',4'-bis-O-(p-nitrobenzoyl)-1,2',3,6'-tetrakis-N-(trifluoroacetyl)neamine (6) and 3',4'-dideoxy-1,2',3,6'-tetrakis-N-(trifluoroacetyl)neamine (9). The amino groups of these intermediates are blocked by the trifluoroacetyl group, a blocking group not widely used in aminoglycoside chemistry.