Metabolism of prazosin in rat and characterization of metabolites in plasma, urine, faeces, brain and bile using liquid chromatography/mass spectrometry (LC/MS).

1. Prazosin, 2-[4-(2-furanoyl)-piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, is an antihypertensive agent that has been used safely since 1976 and is currently being investigated for the treatment of post-traumatic stress disorder. The in vivo metabolism of prazosin in rat was first reported in 1977, although at the time analytical techniques were not as sophisticated, nor were the mass spectrometers as sensitive, as today. Recently, the in vitro metabolism of prazosin in rat liver microsomes and cryopreserved hepatocytes was investigated using liquid chromatography/mass spectrometry (LC/MS), which revealed new metabolic pathways. 2. In the present work, rat in vivo metabolism was reinvestigated using a quadrupole time-of-flight mass spectrometer coupled with ultra-performance liquid chromatography, or chip-based nanoflow electrospray ionization, with the aim of identifying metabolites revealed by the in vitro studies and any new metabolites. 3. It is reported that prazosin was metabolized in rats to produce the metabolites observed in vitro. In addition, new phase I metabolites, M18, M20 and M21, were formed and conjugation with glucose or taurine formed the new phase II metabolites, M16 and M19, respectively. 4. Evidence for bioactivation of prazosin included detection of ring-opened metabolites (M4 and M7) and a cysteinyl-glycine conjugate (M17). Further support to the structure of the ring-opened metabolite M7 was obtained by nuclear magnetic resonance (NMR) experiments on M7 isolated from urine.

Anticonvulsants containing the N-(3-aryl-2-propenoyl) amido pharmacophore.

A series of 1-(3-aryl-2-propenoyl)-4-oxopiperidines (1) as well as some related semicarbazones (2) and thiosemicarbazones (3) were prepared in order to determine whether the relative locations of aryl rings and amidic groups would lead to novel anticonvulsant agents. Initially the compounds were administered intraperitoneally to mice and examined in the maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) and neurotoxicity (NT) screens. The biodata revealed that anticonvulsant properties were displayed by most of the compounds in series (1), in half of the semicarbazones (2) while protection was absent by members of series (3). Molecular modeling was utilized in order to compare the positions of a phenyl ring in relation to amidic groups in representative compounds in series (1-3) with previously reported anticonvulsant agents. Molecular simplification of 4-oxo-1-(3-phenyl-2-propenoyl)piperidine (la) led to 1-(3-phenyl-2-propenoyl)piperidine (7) and N,N-diethylcinnamamide (8) with retention of anticonvulsant properties. Both (la) and (8) afforded protection in the hippocampal kindling screen in rats. When administered orally to rats, (la) and (8) demonstrated activity in the MES screen and in the case of (8), a huge protection index was observed revealing it to be an important lead compound. The IC50 values of all of the compounds towards murine P388 cells were in excess of 50 microM while several compounds displayed cytotoxicity towards Mycobacterium tuberculosis.

A toxicological evaluation of 2-dimethylaminomethyl-1-phenyl-2-propen-1-one hydrochloride.

2-Dimethylaminomethyl-1-phenyl-2-propen-1-one hydrochloride (3) is a novel cytotoxic and anticancer agent. The objective of this study was to obtain information pertaining to possible toxic symptoms detected by in vivo evaluations in mice and an in vitro test for mutagenicity. The data obtained revealed that 3 had no effect on alanine transaminase, aspartate transaminase, HDL cholesterol and protein concentrations in sera nor were variations in the numbers of red and white blood cells detected. Furthermore autopsies of treated mice revealed no pathological symptoms in the heart, kidney, brain, spleen and testes. However elevation of the concentrations of total cholesterol, triglycerides, creatinine and urea were noted in treated mice as well as inflammation of the liver and lungs. Chromosomal aberrations were detected in a micronuclei test. In the Ames test, compound 3 was converted into one or more mutagens in the presence (but not the absence) of a murine liver homogenate. Thus future molecular modifications of 3 should bear in mind approaches to reduce or minimize unwanted side effects.

Formation of the quaternary ammonium-linked glucuronide of nicotine in human liver microsomes: identification and stereoselectivity in the kinetics.

The formation of the N1-glucuronide metabolite of each nicotine enantiomer was studied in pooled human liver microsomes (n = 6). The metabolite formed from natural S(-)-nicotine was identified by comparison of the high-pressure liquid chromatography (HPLC) retention time and positive ion electrospray ionization-mass spectral characteristics with a synthetic reference standard. A radiometric HPLC method was used to quantify the metabolite. The specificity of the assay method was demonstrated by experiments in which beta-glucuronidase treatment of incubated assay samples resulted in elimination of the peak due to the N1-glucuronide metabolite. The glucuronides of S(-)- and R(+)-nicotine were formed by one-enzyme kinetics, with K(m) values of 0.11 and 0.23 mM and V(max) values of 132 and 70 pmol/min/mg of protein, respectively. There is marked stereoselectivity in the apparent intrinsic clearance values (V(max)/K(m)) in that the value for S(-)-nicotine is 4 times greater than for the R(+)-isomer (1.2 versus 0.31 microl/min/mg of protein).

Quaternary ammonium-linked glucuronidation of 1-substituted imidazoles: studies of human UDP-glucuronosyltransferases involved and substrate specificities.

A series of eight 1-substituted imidazoles was investigated as model substrates for glucuronidation at an aromatic tertiary amine of polyaza heterocyclic ring systems. The human UDP-glucuronosyltransferases (UGTs) involved and substrate specificities were investigated. Nine expressed enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) were examined, but only UGT1A4 catalyzed the formation of a quaternary ammonium-linked glucuronide metabolite for six of the substrates. UGT1A3 also catalyzed the glucuronidation of the previously investigated 1-phenylimidazole but none of the newly investigated compounds. No glucuronidation was observed with 1-(4-nitrophenyl)imidazole, the compound with the 4-phenyl substituent with the largest electron withdrawing effect. The incubation conditions for the determination of the kinetic constants for UGT1A4 catalysis of six substrates were optimized and included incubation at pH 7.4 with alamethicin at 10 microg/mg of protein. Latency disrupting agents, including alamethicin and sonication, enhanced glucuronidation 1.25-fold at most. There were 17.5- and 2.2-fold variations in the apparent K(m) (range, 0.18-3.15 mM) and V(max) values (range, 0.16-0.35 nmol/min/mg of protein). Linear correlation analyses between UGT1A4 kinetics and substrate physicochemical parameters showed significant correlation between V(max) and both the partition coefficient (log P, n-octanol/water) and pK(a) and between K(m) and pK(a), thereby indicating that the lipophilicity and the ease of availability of the tertiary amine lone pair of electrons of the substrate are important with respect to enzyme catalysis.

Cytotoxic and anticancer properties of some 4-aryl-3-arylcarbonyl-1-ethyl-4-piperidinols and related compounds.

A previous investigation revealed that various 4-aryl-3-arylcarbonyl-1-ethyl-4-piperidinols and related vinylogs were cytotoxic to both murine and human tumour cell lines. In particular, 1a and 2a were identified as useful prototypic molecules. Structural modifications of 1a and 2a were accomplished leading to 1b-e and 2b-d which displayed cytotoxicity towards murine P388 and L1210 leukemic cells as well as human Molt 4/C8 and CEM T-lymphocytes. Among the new compounds, the greatest average potencies against these four cell lines were displayed by 1b and 2b, having approximately one quarter and one half of the potency of the reference drug melphalan, respectively. The synthesis and bioevaluation of three open chain analogues of 1b-d, namely 3a-c, did not reveal unequivocally whether this molecular modification led to increases in cytotoxicity or not. Compounds 2a-d were substantially more active than melphalan using a panel of human tumour cell lines. In addition, several compounds displayed selective toxicity to both colon and leukemic cancer cells. The 4-piperidinol 2d was active in the in vivo hollow fibre assay. This study revealed compounds with greater potency than 1a and 2a and it has confirmed that 1,3,4-trisubstituted-4-piperidinols and related compounds are novel groups of candidate antineoplastic and anticancer agents.

Inhibition of H(+)-ATPase-mediated proton pumping in Cryptococcus neoformans by a novel conjugated styryl ketone.

We investigated the in vitro susceptibility of clinical isolates of Cryptococcus neoformans to the novel conjugated styryl ketone NC1175 by broth microdilution. The MIC(90) and the MFC of NC1175 for C. neoformans were 1 and 2 mg/L, respectively. NC1175 at low concentrations (1-4 mg/L) completely inhibited the glucose-induced acidification of the external medium caused by the extrusion of intracellular protons mediated by the plasma membrane located H(+)-ATPase. These data suggest that NC1175 is a fungicidal agent for C. neoformans and its possible cellular target(s) include the H(+)-ATPase.

Synthesis and identification of the quaternary ammonium-linked glucuronide of 1-phenylimidazole in human liver microsomes and investigation of the human UDP-glucuronosyltransferases involved.

1-Phenylimidazole was investigated as a potential model substrate with respect to formation of a quaternary ammonium-linked glucuronide (N(+)-glucuronide) at an aromatic type tertiary amine. A reference sample of the potential N(+)-glucuronide metabolite of 1-phenylimidazole was obtained by organic synthesis. The structural identity of the metabolite formed by incubation of 1-phenylimidazole with human liver microsomes was proven to be the N(+)-glucuronide by exhibiting the same HPLC retention time and electrospray ionization mass spectrum as the reference sample. The screening of 1-phenylimidazole against a panel of nine expressed human UDP-glucuronosyltransferases indicated the involvement of UGT1A3 and UGT1A4 in the formation of the N(+)-glucuronide metabolite.

Ureylene anticonvulsants and related compounds.

The results from a previous study led to the postulate that a number of aryl semicarbazones displaying anticonvulsant activity in the maximal electroshock (MES) screen interacted at both a hydrophobic and a hydrogen bonding areas on a specific binding site. These two parts of the binding site may be referred to as areas A and B, respectively. In order to circumvent the possible problems of the carbimino group in semicarbazones, such as toxicity and acid lability, some related ureylenes were considered. Initial evidence suggested that a second lipophilic group in the molecule was advantageous; this group may interact at area C on the proposed binding site. Most of the compounds prepared with a view to interacting at areas A, B and C showed protection in mice against MES induced seizures. Of particular interest were the compounds 1d, j which contained an alpha-methylbenzyl group attached to the N1 atom of the ureylenes which afforded good protection in the MES screen. The areas A and C at which lipophilic moieties were considered to interact were capable of accommodating groups of different sizes as measured by their solvent accessible surface areas. A number of compounds were active when given orally to rats and devoid of neurotoxicity at the doses utilized. Several compounds including 1d, f, j, 2d are useful prototypic molecules for subsequent development of further novel anticonvulsants.

Anticonvulsant properties of various acetylhydrazones, oxamoylhydrazones and semicarbazones derived from aromatic and unsaturated carbonyl compounds.

Various acetylhydrazones, oxamoylhydrazones and semicarbazones were prepared as candidate anticonvulsants with a view to examining the viability of a putative binding site hypothesis. Atomic charge calculations were undertaken to determine the hydrogen bonding capacities of various molecules. The biological results obtained revealed that in general the acetylhydrazones and semicarbazones afforded good protection against convulsions while the oxamoylhydrazones were significantly less active. These data suggest that terminal electron-donating groups enhanced the hydrogen bonding capabilities and anticonvulsant properties of these molecules.

Proton-pumping-ATPase-targeted antifungal activity of a novel conjugated styryl ketone.

NC1175 (3-[3-(4-chlorophenyl)-2-propenoyl]-4-[2-(4-chlorophenyl)vinyle ne]-1- ethyl-4-piperidinol hydrochloride) is a novel thiol-blocking conjugated styryl ketone that exhibits activity against a wide spectrum of pathogenic fungi. Incubation of NC1175 with various concentrations of cysteine and glutathione eliminated its antifungal activity in a concentration-dependent fashion. Since NC1175 is a lipophilic compound that has the potential to interact with cytoplasmic membrane components, we examined its effect on the membrane-located proton-translocating ATPase (H(+)-ATPase) of yeast (Candida albicans, Candida krusei, Candida guilliermondii, Candida glabrata, and Saccharomyces cerevisiae) and Aspergillus (Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, and Aspergillus nidulans) species. The glucose-induced acidification of external medium due to H(+)-ATPase-mediated expulsion of intracellular protons by these fungi was measured in the presence of several concentrations of the drug. NC1175 (12.5 to 50 microM) inhibited acidification of external medium by Candida, Saccharomyces, and Aspergillus species in a concentration-dependent manner. Vanadate-inhibited hydrolysis of ATP by membrane fractions of C. albicans was completely inhibited by 50 microM NC1175, suggesting that the target of action of NC1175 in these fungi may include H(+)-ATPase.

In-vitro and in-vivo susceptibility of Aspergillus fumigatus to a novel conjugated styryl ketone.

We investigated the in-vitro and in-vivo susceptibility of Aspergillus fumigatus to the novel conjugated styryl ketone NC1175 and the results were compared with those obtained for amphotericin B and itraconazole. All 20 clinical isolates of A. fumigatus examined were susceptible to NC1175 (MIC = 5.54 +/- 2.48 mg/L; range 2.92-11.68 mg/L), and the minimum lethal concentration (MLC) was only twice the MIC, suggesting that NC1175 is fungicidal. The mean MIC values of amphotericin B (1.22 +/- 0.58 mg/L; range 0.5-4 mg/L) and itraconazole (0.37 +/- 0.11 mg/L; range 0.125-0.5 mg/L) were approximately nine- and 22-fold, respectively, lower than that of NC1175. Both amphotericin B-resistant (n = 18) and itraconazole-resistant (n = 28) isolates of A. fumigatus were as susceptible to NC1175 as amphotericin B-, and itraconazole-susceptible isolates. Kill curve experiments revealed that NC1175 at 23.35 mg/L (approximately four times the MIC) killed > or = 99% of conidia within 24 h of exposure to the drug. The in-vivo susceptibility of A. fumigatus to NC1175 was investigated using a murine pulmonary aspergillosis model. Treatment of infected mice with amphotericin B or NC1175 did not result in significant improvement of the mean survival (amphotericin B, 7.05 +/- 0.07 days; NC1175, 6.65 +/- 1.25 days) of the animals compared with that of the placebo group (7.21 +/- 1.20 days). However, semiquantitative organ culture revealed that clearance of A. fumigatus occurred in 16.6%, 50% and 66.6% of the mice treated with placebo, NC1175 and amphotericin B, respectively (P value for the control and the treated groups <0.01). These results suggest that NC1175 has in-vivo and in-vitro activity against A. fumigatus and can be used as a prototypic molecule for further development as an antifungal agent.

Cytotoxic and anticancer activities of some 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides.

A series of 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides demonstrated marked cytotoxicity towards approximately 55 human tumour cell lines from different neoplastic diseases. In general they were more potent than melphalan and displayed selective toxicity towards human leukemic cells. A representative compound, 1-phenyl-2-dimethyl-aminomethyl-2-propen-1-one hydrochloride (2a), had similar cytotoxicity as melphalan towards murine P388 and L1210 leukemic cells. In addition, 2a reduced the sizes of a number of human tumour xenografts including colon, prostatic and melanotic cancers passaged in athymic mice. Compound 2a showed excellent activity towards Ehrlich ascites carcinoma and B16F1 melanoma in mice which was enhanced using niosomes. One may conclude from the data generated that 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides are a novel series of cytotoxic and anticancer agents.

4-(beta-Arylvinyl)-3-(beta-arylvinylketo)-1-ethyl-4-piperidinols and related compounds: a novel class of cytotoxic and anticancer agents.

The syntheses of a series of 1-aryl-5-diethylamino-1-penten-3-one hydrochlorides 1 and 1-aryl-3-diethylamino-1-propanone hydrochlorides 4 were accomplished. Attempts to prepare the corresponding bis(5-aryl-3-oxo-4-pentenyl)ethylamine hydrochlorides 2 and bis(3-aryl-3-oxopropyl)ethylamine hydrochlorides 5 led to the formation of a series of 4-(beta-arylvinyl)-3-(beta-arylvinylketo)-1-ethyl-4-piperidi nol hydrochlorides 9 and 4-aryl-3-arylketo-1-ethyl-4-piperidinol hydrochlorides 11, most of which were converted subsequently into the corresponding quaternary ammonium salts 10 and 12, respectively. The structures of these compounds were determined by 1H NMR spectroscopy and confirmed by X-ray crystallography of representative molecules. Most compounds displayed significant cytotoxicity toward murine P388 and L1210 cells as well as human tumors. In general, Mannich bases containing olefinic bonds were more cytotoxic than the analogues without this functional group, while the piperidines 9 and 11 were more potent than the acyclic analogues 1 and 4, respectively. Correlations were noted between various physicochemical constants in the aryl rings and cytotoxicity. Compound 9d displayed promising in vivo activity against colon cancers. This study has revealed that the piperidines 9 and 11 constitute new classses of cytotoxic agents.

Application of fluorescence microscopy to measure apoptosis in Jurkat T cells after treatment with a new investigational anticancer agent (N.C.1213).

1. Apoptosis as the mechanism of cell death induced by a new cytotoxic and anticancer agent (N.C.1213) was investigated by morphological and biochemical criteria in human Jurkat T leukemia cells. 2. The effect of N.C.1213 on the survival of Jurkat T, LV-50, H-9, and Molt-3 cells was measured. Jurkat T cells exhibited the highest response, with less than 10% of the cells remaining viable after exposure to 10 microM N.C.1213 for a 24 hr period. All other cell cultures were also affected but to a lesser extent. 3. With the use of a fluorescence microscope, several morphological features characteristic of apoptosis such as condensed chromatin and apoptotic bodies were identified in Jurkat T cells after exposure to N.C.1213 and melphalan. The results indicated that melphalan was more cytotoxic than N.C.1213 as shown by the dye exclusion test. However, N.C.1213 showed a greater apoptotic index than melphalan. The IC50 of N.C.1213 in Jurkat T cells was determined to be 3.5 microM. 4. A DNA ladder (fragmentation of DNA into multimers of approximately 200 base pairs), which is one characteristic feature of apoptosis, was not detected when Jurkat T cells were exposed to N.C.1213. Hence it is probable that the key morphological events in apoptosis observed in the present experimental conditions precede the internucleosomal cleavage of DNA.

In vitro antifungal activity of some Mannich bases of conjugated styryl ketones.

Four Mannich bases of some conjugated styryl ketones IIa-IId were examined for antifungal activity. These compounds were designed as thiol-alkylators and had two centers for attack by cellular thiols. The most potent compounds IIa and IIb possessed hydrophobic, electron-attracting substituents in the aryl rings and in general had minimum inhibitory concentration (MIC) values of 0.2-25 microM against a variety of fungi. None of the four compounds inhibited the growth of a number of bacteria (MIC > 100 microM). The minimum fungicidal concentration (MFC) values for IIa and IIb were generally either similar or twofold higher than the MIC figures for fungi. Compound IIa demonstrated rapid, concentration-dependent inhibition of the growth of Candida albicans B311. The toxicity of IIa to normal human cells was much lower than the concentrations of this compound required to inhibit fungal growth. In summary, this study of four prototypic molecules has revealed that this class of compounds may have potential for further development as candidate antifungal agents.

Biliobiliary fistula: preoperative diagnosis and management implications.

Experience with cholecystohepaticodochal and cholecystocholedochal fistulas as a result of an erosion of gallstones from the gallbladder into the adjacent common duct in five patients is presented. The incidence was 1.4% in a population of 350 patients undergoing cholecystectomy. The condition was indicated clinically on the basis of a symptom triad of jaundice, fever, and pain with cholelithiasis in a small contracted gallbladder. In addition, proximal intra- and extrahepatic ductal dilatation, calculus in the common duct, and normal-caliber (or unprofiled) distal common duct on ultrasound scan were present in all the patients. Endoscopic retrograde cholangiopancreatography proved to be the most useful means of investigation, and it confirmed the diagnosis in four patients before surgery. A modified antegrade cholecystectomy was performed with the gallbladder opened inferiorly at the fundus, and the stones were evacuated. A partial cholecystectomy and choledochoplasty were accomplished with gallbladder flaps whenever feasible. Other useful operative procedures are side-to-side hepaticodochojejunostomy and hepaticodochoduodenostomy. In the presence of high benign bile duct stricture, an approach to the left hepatic duct is now preferred for biliary bypass.