Discovery, total synthesis, HRV 3C-protease inhibitory activity, and structure-activity relationships of 2-methoxystypandrone and its analogues.

2-Methoxystypandrone, a naphthoquinone, was isolated from a Chinese herb Polygonum cuspidatum by bioassay guided fractionation using HRV 3C-protease assay. It showed an IC(50) value of 4.6 microM and is moderately selective. A new 10-step, total synthesis of 2-methoxystypandrone was accomplished in 45% overall yield using a Diels-Alder approach. Several analogues of this compound were prepared. Isolation, synthesis and HRV 3C-protease structure-activity relationships of these compounds have been described.

Synthesis of a muscarinic receptor antagonist via a diastereoselective Michael reaction, selective deoxyfluorination and aromatic metal-halogen exchange reaction.

An efficient synthesis of a structurally unique, novel M(3) antagonist 1 is described. Compound 1 is conveniently disconnected retrosynthetically at the amide bond to reveal the acid portion 2 and the amine fragment 3. The synthesis of key intermediate 2 is highlighted by a ZnCl(2)-MAEP complex 19 catalyzed diastereoselective Michael reaction of dioxolane 7 with 2-cyclopenten-1-one (5) to establish the contiguous quaternary-tertiary chiral centers and a subsequent geminal difluorination of ketone 17 using Deoxofluor in the presence of catalytic BF(3).OEt(2). The synthesis of the amine moiety 3 is highlighted by the discovery of a novel n-Bu(3)MgLi magnesium-halogen exchange reaction for selective functionalization of 2,6-dibromopyridine. This new and practical metalation protocol obviated cryogenic conditions and upon quenching with DMF gave 6-bromo-2-formylpyridine (26) in excellent yield. Further transformations afforded the amine fragment 3 via reductive amination with 35, Pd-catalyzed aromatic amination, and deprotection. Finally, the highly convergent synthesis of 1 was accomplished by coupling of the two fragments. This synthesis has been used to prepare multi-kilogram quantities of the bulk drug.

Use of a dual monoclonal solid phase and a polyclonal detector to create an immunoassay for the detection of human cardiac troponin I.

OBJECTIVES: We report the development of a fully automated, random access, chemiluminescent immunoassay, for the detection of human cardiac Troponin I (cTnI) in serum and plasma for use on the ACS:180(R) System. DESIGN AND METHODS: This assay format uses a combination of two monoclonal antibodies covalently coupled to paramagnetic (PMP) particles as a solid phase and an affinity purified polyclonal antibody, specific to the N-terminal domain of cTnI (peptide-3 region) labeled with a chemiluminescent compound as the detector antibody. The assay offers excellent low-end sensitivity and precision. RESULTS: No interferences are observed from by blood components such as HAMA and drugs used in cardiac therapy. Patient samples tested on the ACS:180 cTnI assay showed good correlation with the Stratus cTnI assay (ACS: cTnI = 1. 02*Stratus + 0.05 g/L, r = 0.96, n = 1170). CONCLUSION: Paired with the other ACS:180 cardiac assays, myoglobin and CKMBII, the ACS:180 system now offers an excellent panel of cardiac assay for use in rapid and accurate diagnosis of a myocardial event.

Phosphorylated morpholine acetal human neurokinin-1 receptor antagonists as water-soluble prodrugs.

The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3-oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans.

Efficient enantioselective synthesis of sertraline, a potent antidepressant, via a novel intramolecular nucleophilic addition to imine.

[formula: see text] An efficient enantioselective synthesis of sertraline, an antidepressant, utilizing anionic imine ring closure is described.

Comparison of monolayer and bilayer plates used in antibiotic assay.

Standard curves of 5 antibiotics were determined in an antibiotic assay using bilayer and monolayer agar plates and AOAC-specified test organisms and agar media. Micrococcus luteus ATCC 9341a and antibiotic medium No. 2 were used to prepare the penicillin G standard curve. The same organism and antibiotic medium No. 11 were used to prepare the erythromycin standard curve. Standard curves for streptomycin, tetracycline, and gentamicin were prepared, respectively, with antibiotic medium No. 5 and Bacillus subtilis ATCC 6633, antibiotic medium No. 8 and B. cereus ATCC 11778, and antibiotic medium No. 11 and Staphylococcus epidermidis ATCC 12228. Assays of inhibition by meat fortified with penicillin, streptomycin, gentamicin, tetracycline, erythromycin also were performed on monolayer and bilayer plates. Differences in standard curves and inhibitory responses obtained with monolayer and bilayer plates were < 10%. Thus, monolayer plates are acceptable for use in analyses of meat and poultry for antibiotics residues, with savings in laboratory resources and time.

Cryopreservation of bacterial vegetative cells used in antibiotic assay.

A long-term cryopreservation study of vegetative cells of Micrococcus lutea ATCC 9341a, Micrococcus lutea ATCC 15957, and Staphylococcus epidermidis ATCC 12228 cells, used in our antibiotic bioassay procedure, was conducted. The cryoprotective abilities of 1% methylcellulose solution and a 15% glycerol solution at -14 degrees C were determined. More organisms remained viable in 1% methylcellulose than in 15% glycerol. Overall survival of Staphylococcus epidermidis ATCC 12228 after 365 days was 1.5 logs lower than the other 2 organisms. The sensitivity and the resistance of the preserved organisms to various antibiotics did not change. The methodology is simple and inexpensive, saves analytical time, and avoids risk of contamination and sudden loss of a well-characterized culture.

Isolation and characterization of the major equilibrium product of FK-520.

The existence of an isomeric form in equilibrium with the major component of FK-520 in polar solutions has been demonstrated. This minor component has been isolated in high yield and purity by a novel crystallization strategy and preparative HPLC. The equilibrium product was characterized by NMR and MS.

Isolation and identification of impurities in L-696, 229 drug substance.

Two low level impurities in 3-[2-(2-benzoxazolyl)ethyl]-5-ethyl-6-methyl-2(1H)-pyridinone drug substance (L-696,229) have been isolated by a combination of preparative HPLC, solid-phase extraction and liquid-liquid extraction. They were identified as 3-[2-(2-benzoxazolyl)ethyl]-5-ethyl-6-(2-phenylethyl)-2(1H)-pyridinone (I) and 6,6'-(2-phenyl-1,3-propanediyl)bis[3-[2-(2-benzoxazolyl)ethyl]-5-ethyl-2(1H)-pyridinone] (II) by mass spectrometry and by their (13)C and (1)H-NMR spectra.

Cochinmicins, novel and potent cyclodepsipeptide endothelin antagonists from a Microbispora sp. II. Structure determination.

Cochinmicins I, II, and III are competitive endothelin antagonists produced by Microbispora sp. ATCC 55140. The cochinmicins are cyclic depsipeptides containing six alpha-amino acids and a pyrrolecarboxylic acid. Based upon MS, 1D and 2D NMR, and LC data, the structures and absolute stereochemistries of the cochinmicins have been assigned. All three components have the same basic sequence and contain one equivalent each of D-allo-threonine, D-alanine, L-phenylalanine, D-phenylalanine, 5-chloropyrrole-2-carboxylic acid (or pyrrole-2-carboxylic acid in cochinmicin I), plus two equivalents of 3,5-dihydroxyphenylglycine (DHPG). The phenylalanine residues were differentiated via a methanolysis product which contained only one of the phenylalanine residues. Both DHPG residues have the D configuration in the more active cochinmicins I and III. Cochinmicin II contains both D- and L-DHPG and these residues have been differentiated in the sequence based upon 1H NMR data.

1-(substituted)benzyl-5-aminoimidazole-4-carboxamides are potent orally active inhibitors of Trypanosoma cruzi in mice.

1-(Substituted)benzyl-5-aminoimidazole-4-carboxamides are potent orally active inhibitors of Trypanosoma cruzi infections in mice. The most active compounds are the 1-(4-chlorobenzyl)- and 1-(3,4-dichlorobenzyl)-analogs (L-153,094 [2] and L-153,153 [4], resp.) which are approximately 7-fold more potent upon oral administration than nifurtimox (Lampit) in suppressing parasite levels in the blood of mice with acute Trypanosoma cruzi infections.

Origin of monacolin L from Aspergillus terreus cultures.

In freshly harvested Aspergillus terreus cultures grown for the production of lovastatin (formerly called mevinolin), no monacolin L could be detected. However, during the isolation of lovastatin, significant quantities of monacolin L appeared. It has been discovered that a new metabolite structurally related to the members of the monacolin series is present. This metabolite is unstable and under mildly acidic conditions and elevated temperature, it converts to monacolin L. The subject metabolite is proven to be a hydroxylated derivative of dihydromonacolin L identified as 3 alpha-hydroxy-3,5-dihydromonacolin L. It seems that all monacolin L found later during various treatments of the broth and broth extracts is formed from that precursor via a dehydration reaction. The new metabolite was converted to its phenacyl ester, by means of extractive alkylation, for isolation and structure elucidation by chemical, chromatographic and spectroscopic methods. This ester, on standing, gradually formed the corresponding lactone.

Determination of the stenotic aortic valve area in adults using Doppler echocardiography.

The severity of aortic stenosis was evaluated by Doppler echocardiography in 48 adults (mean age 67 years) undergoing cardiac catheterization. Maximal Doppler systolic gradient correlated with peak to peak pressure gradient (r = 0.79, y = 0.63x + 25.2 mm Hg) and mean Doppler gradient correlated with mean pressure gradient (r = 0.77, y = 0.59x + 10.0 mm Hg) by manometry. The transvalvular pressure gradient is flow dependent, however, and associated left ventricular dysfunction was common in our patients (33%). Thus, of the 32 patients with an aortic valve area less than or equal to 1.0 cm2 at catheterization, 6 (19%) had a peak Doppler gradient less than 50 mm Hg. To take into account the influence of volume flow, aortic valve area was calculated as stroke volume, measured simultaneously by thermodilution, divided by the Doppler systolic velocity integral in the aortic jet. Aortic valve areas calculated by this method were compared with results at catheterization in the total group (r = 0.71). Significant aortic insufficiency was present in 71% of the population. In the subgroup without significant coexisting aortic insufficiency, closer agreement of valve area with catheterization was noted (n = 14, r = 0.91, y = 0.83x + 0.24 cm2). Transaortic stroke volume can be determined noninvasively by Doppler echocardiographic measures in the left ventricular outflow tract, just proximal to the stenotic valve. Aortic valve area can then be calculated as left ventricular outflow tract cross-sectional area times the systolic velocity integral of outflow tract flow, divided by the systolic velocity integral in the aortic jet.(ABSTRACT TRUNCATED AT 250 WORDS)

Selenium dioxide oxidation of vitamin D3 acetate to a dimer of 1-oxotransvitamin D3 acetate.

The treatment of vitamin D3 acetate with selenium dioxide and t-butyl hydroperoxide leads to a mixture from which a Diels-Alder dimer of 1-oxotransvitamin D3 acetate was isolated.

Neocarzinostatin chromophore: presence of a highly strained ether ring and its reaction with mercaptan and sodium borohydride.

Spectroscopic evidence suggests the presence of a highly strained ether ring (Fig. 1) (possibly an epoxide) in the C12-subunit of the previously determined partial structure 2a (Fig. 2) of the major neocarzinostatin chromophore (NCS-Chrom A) which completes assignment of all the oxygens in the molecule. The main product from mercaptan treatment suggests opening of the ether ring involving the addition of one molecule of mercaptan as well as reduction of the C12-substructure, whereas a parallel two-step reduction occurs on NaBH4 treatment. Both reactions occur with rearrangement of the C12-substructure and the implication for the mechanism of action of NCS-Chrom A in DNA strand scission activity is discussed. The evidence suggests a downward revision of the molecular formula for NCS-Chrom A as well as minor components B and C by two protons.

Isomeric phenylthioimidazo[1,2-alpha]pyridines as anthelmintics.

A series of isomeric imidazo[1,2-alpha]pyridine-2-carbamates was prepared for testing as anthelmintics. The analogues were synthesized by reacting the appropriate 2-aminopyridine and methyl chloroacetylcarbamate. Steric hindrance in the 2,6-disubstituted derivative resulted in the formation of the isomeric 3-substituted analogue as the major product. Carbon-13 NMR proved useful in the structural assignments in this series. None of the analogues exhibited the potency of methyl 6-(phenylsulfinyl)imidazo[1,2-alpha]pyridine-2-carbamate when tested against Nematospiroides dubius in mice.

Biological and physicochemical characteristics of four serotypes of Salmonella enteritidis.

Four serotypes of Salmonella enteritidis, Anatum ATCC 9270, Newbrunswick ATCC 1608, Oranienburg 200 E, and Pullorum RM, were studied to determine biological, chemical, or physical differences which might explain variations in Salmonella virulence as previously reported by McCullough and Eisele (J. Infect. Dis. 88:278-289, 1951; 89:259-265, 1951). These investigators found that serotype Pullorum was significantly less virulent than serotypes Newport, Derby, Barielly, Meleagridis and Anatum when fed to healthy humans. Results of our own experiments showed that serotype Pullorum RM had a generation time approximately twice that of serotype Anatum 9270. The volume of serotype Pullorum was approximately one-half the volume of the other serotypes used (Anatum 9270, Newbrunswick 1608, Oranienburg 200 E, Cubana 12007, and Meleagridis DR). The number of cells required to yield 1 g dry weight was substantially higher for serotype Pullorum RM than for serotypes Anatum 9270, Newbrunswick 1608, and Oranienburg 200 E. The yield of endotoxin per gram dry weight for serotype Pullorum RM averaged 22 mg/g, whereas yields of endotoxin for serotypes Anatum 9270, Newbrunswick 1608, and Oranienburg 200 E averaged 32 to 35 mg/g. The relative abundance of the four major fatty acids (measured by gas chromatography) also showed distinct differences among the serotypes. Pullorum RM contained less lauric and 3-hydroxymyristic acids and more myristic and palmitic acids than the other three serotypes. The identity of 3-hydroxymyristate was confirmed by mass spectroscopy. Serotype Pullorum RM required 10 times more lipopolysaccharides (endotoxin) to obtain a 50% lethal dose in mice than the other three serotypes. When the lipid part was separated from the polysaccharide and solubilized with bovine serum, the 50% lethal dose of serotype Pullorum RM was equal to that of the other three.

Twenty-four-hour immunofluorescence technique for the detection of salmonellae in nonfat dry milk.

A detection procedure was developed in which a newly devised lysine-iron medium was used as a one-step selective and enrichment medium for detection of salmonellae by the fluorescent-antibody technique. Incubation was conducted in two steps: initially at 30 C for 5 hr to resuscitate sublethally stressed cells, followed by incubation at 39 C for 17 hr. Twenty-seven strains of salmonellae from groups A-I were utilized in the development of this procedure which was sensitive enough to detect one Salmonella bacterium in 100 g of nonfat dry milk.

Increased sensitivity of immunofluorescent assay for Salmonella in nonfat dry milk.

The necessity of developing a quick, sensitive, and reliable test for Salmonella in nonfat dry milk (NDM) is evident from the recent tracing of Salmonella outbreaks to this product. Normally, coagulation of casein occurs when assaying NDM under regular cultural conditions, raising the possibility of trapped bacteria. After 20 hr of incubation of NDM in preenrichment lactose broth, enrichment was achieved by using Selenite-Cystine Broth. Smears from the enrichment broth were examined by the fluorescent-antibody technique (FAT) with a commercially available polyvalent O globulin conjugated with fluorescein. Standard cultural methods (SCM) were performed for comparison with FAT. Sensitivity of FAT was definitely improved by the use of trypsin. Casein coagulation of NDM can be avoided by addition of trypsin to samples during initial preenrichment in lactose broth. Samples containing approximately one Salmonella per 10 g were easily detected by FAT with the use of trypsin-treated samples. The method required only 42 hr to complete. Additionally, the use of trypsin enhanced recovery of Salmonella by use of SCM, as evidenced by alteration in the observed coliform to Salmonella ratios.