Syntheses of benzoquinolizidine and benzoindolizidine derivatives as anti-amnesic agents.

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.

Syntheses of 1,2-diamino and 1,2-aminoalcohol derivatives in the piperidine and pyrrolidine series as anti-amnesic agents.

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.

Isolation and identification of a major metabolite of PNU-107859, an MMP inhibitor from the biliary fluid of rats.

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.

Solution structures of stromelysin complexed to thiadiazole inhibitors.

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.

In vitro metabolic transformations of 2,4-dipyrrolidinylpyrimidine: a chemical probe for P450-mediated oxidation of tirilazad mesylate.

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.

Cycloalkylpyranones and cycloalkyldihydropyrones as HIV protease inhibitors: exploring the impact of ring size on structure-activity relationships.

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.

In vitro metabolism of tirilazad mesylate in male and female rats. Contribution of cytochrome P4502C11 and delta 4-5 alpha-reductase.

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.

Synthesis and biological activity of spirocyclic benzopyran imidazolone potassium channel openers.

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.

Naphtho and benzo analogues of the kappa opioid agonist trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide.

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).

Structure elucidation of ficellomycin.

The structure of ficellomycin, an antibiotic previously discovered by Argoudelis et al., is elucidated as valyl-2-[4-guanidyl-1-azabicyclo[3.1.0]hexan-2-yl]glycine (1) by NMR, MS, and derivatization studies. The 1-azabicyclo[3.1.0]hexane moiety in 1 represents an unusual ring system making ficellomycin a unique natural product compound.

Adenylylation of trospectomycin by crude enzyme preparations from Escherichia coli.

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).

O-demethylpaulomycins A and B, U-77,802 and U-77,803, paulomenols A and B, new metabolites produced by Streptomyces paulus.

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.

Structural relationships between senfolomycins and paulomycins.

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.

New paulomycins produced by Streptomyces paulus.

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-related antibiotics: paldimycins and antibiotics 273a2. Isolation and characterization.

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.

Paldimycins A and B and antibiotics 273a2 alpha and 273a2 beta. Synthesis and characterization.

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.

Isolation, identification and synthesis of a metabolite of tazadolene succinate.

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.