A short and efficient synthesis of 4-[2,2-dimethyl-4(tol-4-yl)benzochrom-3-en-7-yl]benzoic acid - a potent retinoic acid receptor antagonist.

An efficient synthesis of a potent RAR antagonist is described starting from disubstituted beta-naphthol. The functional groups on 2 and 3 positions of the beta-naphthol 5 were elaborated into benzochromanone 8. The title compound was prepared by Suzuki coupling of the left and right hand pieces.     

A reverse phase HPLC method for the separation of two stereo isomers of 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocyclopentyl)propanoic acid

This study describes successful method development and separation of two stereo isomers of 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocyclopentyl)propanoic acid by reverse phase high-performance liquid chromatography. Baseline resolution was achieved on a J'sphere-ODS-H80 (150 mm × 4.6 mm, 4 μm) column using mobile phase consisting of 0.05% triflouroacetic acid in water-acetonitrile (85:15, v/v) at a flow rate of 1.0 ml/min. The detection was carried out at 228 nm. The title compound, in turn, can be obtained by C-alkylation of methyl 2-[4-(methylthio)phenyl]acetate with 2(S)-iodomethyl-8,8-dimethyl-6,10-dioxaspiro[4.5]decane followed by consecutive hydrolysis and oxidation. The partially validated analytical method (system suitability, peak homogeneity, linearity, precision, robustness, and solution stability) has limit of detection and limit of quantification, 0.15 and 0.50 μg/ml respectively. Alternatively, the new method is being routinely utilized to monitor epimerization of α-carbon of the propanoic acid in the title compound by crystallization-induced dynamic resolution.     

Genotoxic activity of 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid and its peptidyl derivatives determined by Ames and SOS response tests

Compounds derived from 1,2,4-oxadiazole have being reported for their anti-inflamatory activity. However, those compounds should be devoid of any genotoxic side effect. In this work, the genotoxic activity of peptidomimetic moiety-containing 1,2,4-oxadiazoles derivatives was tested based on the Ames and SOS Chromotest. The results showed no mutagenic activity on the Ames test for 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid (POPA) parental drug, but a weak SOS response induction on Chromotest. The chemical modifications reduced that response to a non-significative level, with l-phenylalanine peptidomimetic derivative being showing the lowest induction response. The results pointed out for the effectiveness of promoting chemical modifications of biological active compounds to increase its mode of action, showed in previous work, without increasing and even decreasing its DNA damage effect.     

Effects of 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one on the synthesis of cholesterol and ubiquinone in rat intestinal epithelial cell cultures

The relationship between cholesterol and ubiquinone synthesis in rat intestinal epithelial cell cultures was examined by using 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one hydrochloride (U18666A). Addition of U18666A to cells caused a greater than 90% inhibition of incorporation of [3H]acetate into cholesterol and an apparent large increase in the incorporation of [3H]acetate and [3H]mevalonate into ubiquinone. However, the incorporation of 4-hydroxy[U-14C]benzoate, a ring precursor of ubiquinone, was unchanged. The apparent increase of 3H incorporation into ubiquinone was found to be due to the formation of a contaminant that has been identified as squalene 2,3:22,23-dioxide. Following incubation of cells with U18666A, its removal from the medium resulted in a decrease in squalene 2,3:22,23-dioxide labeling and a corresponding increase in the polar sterol fraction. These results demonstrate that U18666A inhibits the reaction catalyzed by 2,3-oxidosqualene cyclase (EC 5.4.99.7). As a result, the isoprenoid precursors are diverted not to ubiquinone as has been suggested but to squalene 2,3:22,23-dioxide, a metabolite not on the cholesterol biosynthetic pathway. Removal of the drug allows cyclization of squalene 2,3:22,23-dioxide, leading to formation of compounds with chromatographic properties of polar sterols.     

The synthesis and biological evaluation of 2-(3-methyl or 3-phenylisoxazol-5-yl)-3-aryl-8-thiabicyclo[3.2.1]octanes

Cocaine, a potent stimulant of the central nervous system, owes its reinforcing and stimulant properties to its ability to inhibit monoamine uptake systems such as the Dopamine Transporter (DAT), and the Serotonin Transporter (SERT) located on presynaptic neurons in the striatum. The search for pharmacotherapies for cocaine addiction has focused on the design of compounds that bind selectively to the DAT and manifest slow onset of stimulatory action with long duration of action. We had reported that 3-aryl-2-carbomethoxy-8-thiabicyclo[3.2.1]octanes are potent and selective inhibitors of the DAT. In this Letter we report on the effects of replacement of the 2-carbomethoy group by a 2-isoxazole. This new class of 8-thiabicyclo[3.2.1]octanes provides potent and selective inhibitors of the DAT. The 3β-aryl compounds are particularly potent inhibitors of DAT (IC₅₀ = 7-43 nM) with substantial selectivity versus inhibition of SERT.     

Determination of free N-acetylneuraminic acid in urine by high-performance liquid chromatography using 3-[(1-[[4-(5,6-dimethoxy-1-oxoisoindolin-2-yl)-2-methoxyphenyl]sulfonyl]pyrrolidin-2-yl)carbonylamino]phenylboronic acid as a fluorescent labeling reagent

A highly sensitive high-performance liquid chromatography (HPLC) method for the determination of urinary N-acetylneuraminic acid (NeuAc) using 3-[(1-[[4-(5,6-dimethoxy-1-oxoisoindolin-2-yl)-2-methoxyphenyl]sulfonyl]pyrrolidin-2-yl)carbonylamino]phenylboronic acid as a fluorescent labeling reagent was developed. The labeling reaction was carried out at 30 degrees C for 30 min in the presence of pyridine. The derivative was monitored at Ex 314 nm and Em 388 nm. The detection limit of NeuAc was about 48 fmol per injection. The relative standard deviations of within-day and between-day precisions were 2.6-3.3 and 1.7-3.3%, respectively. Urine diluted 10 times with distilled water was analyzed by employing the standard-addition method. The concentrations were 8-89 nmol/mg creatinine (30+/-28 nmol/mg creatinine, n=9).     

Genotoxic activity of 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid and its peptidyl derivatives determined by Ames and SOS response tests

Compounds derived from 1,2,4-oxadiazole have being reported for their anti-inflammatory activity. However, those compounds should be devoid of any genotoxic side effect. In this work, the genotoxic activity of peptidomimetic moiety-containing 1,2,4-oxadiazoles derivatives was tested based on the Ames and SOS Chromotest. The results showed no mutagenic activity on the Ames test for 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid (POPA) parental drug, but a weak SOS response induction on Chromotest. The chemical modifications reduced that response to a non-significative level, with l-phenylalanine peptidomimetic derivative being showing the lowest induction response. The results pointed out for the effectiveness of promoting chemical modifications of biological active compounds to increase its mode of action, showed in previous work, without increasing and even decreasing its DNA damage effect.     

Inhibitors of sterol synthesis. The effects of dietary 5 alpha-cholest-8(14)-en-3 beta-ol-15-one on the fate of [4-14C]cholesterol and [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one after intragastric administration to rats

The effect of dietary administration (0.1% in a rat chow diet) of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent inhibitor of cholesterol biosynthesis with marked hypocholesterolemic activity, on the fate of [4-14C]cholesterol and [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one has been studied after intragastric administration of the labeled sterols to rats. In general, the distribution of 3H in major tissues paralleled that of 14C with no unusual concentration of 3H in any of the organs. Only trace amounts of 3H and 14C were recovered in urine. Administration of the 15-ketosterol was associated with decreased absorption of the labeled cholesterol as indicated by decreased levels of 14C in the various tissues and organs of the 15-ketosterol-treated rats (relative to ad libitum and pair-fed control animals) and increased levels of 14C in feces and intestinal contents at 12 and 48 h after the administration of the labeled cholesterol. Studies of the distribution of 3H in liver indicated rapid conversion of the 15-ketosterol to cholesterol and cholesteryl esters. The amounts of 3H recovered in the various tissues and organs at both 12 and 48 h after the administration of the labeled sterols were considerably less than the corresponding values for 14C, a finding which suggests a lower absorption of the 15-ketosterol (relative to cholesterol) and/or a more rapid clearance and biliary excretion of the 15-ketosterol and its metabolites.     

Determination of a new fluoroquinolone antimicrobial agent, (S)-10-[(S)-(8-amino-6-azaspiro[3,4]octan-6-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de[1,4] benzoxazine-6-carboxylic acid hemihydrate,DV-7751a,in human serum and urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of a new fluoroquinolone antimicrobial agent, (S)-10-[(S)-(8-amino-6-azaspiro[3,4]octan-6-yl)]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido [1,2,3-de][1,4]benzoxazine-6-carboxylic acid hemihydrate (DV-7751a, I) in human serum and urine has been developed. Compound I and the internal standard were extracted from serum and urine by means of Bond Elut C₈ LRC column. The extracts were chromatographed on a reversed-phase Inertsil ODS-2 column using tetrahydrofuran-50 mM KH₂PO₄ (pH 2)-1 M ammonium acetate (19:81:1, v/v) as the mobile phase at a flow-rate of 1.0 ml/min. Fluorescence detection at an excitation wavelength of 305 nm and an emission wavelength of 530 nm resulted in a limit of quantitation of 0.0098 μg/ml for serum and 0.098 μg/ml for urine. The method showed satisfactory sensitivity, precision, accuracy, recovery and selectivity. Stability studies showed that I was stable in serum and urine for at least 1 month at −20°C and for at least 48 h at room temperature.     

3-(4-Piperidinyl)- and 3-(8-aza-bicyclo[3.2.1]oct-3-yl)-2-phenyl-1H-indoles as bioavailable h5-HT2A antagonists

A series of 3-(4-piperidinyl)- and 3-(8-aza-bicyclo[3.2.l]oct-3-yl)-2-phenyl-1H-indoles have been prepared and evaluated as ligands for the h5-HT_2A receptor. 3-(8-Phenethyl-8-aza-bicyclo[3.2.l]oct-3-yI)-2-phenyl-1H-indole is a high-affinity (1.2 nM), selective (>800 fold over h5-HT_2C and hD₂ receptors) antagonist at the h5-HT_2A receptor with oral bioavailability in rats.     

Validation of liquid chromatography assay for the quantitation of (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]propionic acid (SU006668) in human plasma and its application to a phase I clinical trial

The validation of an analytical method to quantify the antiangiogenic, (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]propionic acid (SU006668) for pharmacokinetic determination in a phase I clinical trial, is described. HPLC, with a gradient mobile phase and UV detection at 440 nm, was used. SU006668 was extracted from plasma by precipitation of proteins with acetonitrile. The assay was linear from 25 to 2000 ng/ml (r(2)=0.997); sensitive (limit of quantification 25 ng/ml), accurate (RE 2.6-11.9%) and reproducible (inter-batch precision C.V. 3.2%). Pharmacokinetic data for six patients are presented. They show linear pharmacokinetics with a low volume of distribution and induction at doses of 50, 100 and 200 mg/m(2).     

{2-[1-(3-Methoxycarbonylmethyl-1H-indol-2-yl)-1-methyl-ethyl]-1H-indol-3-yl}-acetic acid methyl ester (MIAM): its anti-cancer efficacy and intercalation mechanism identified via multi-model systems

{2-[1-(3-Methoxycarbonylmethyl-1H-indol-2-yl)-1-methyl-ethyl]-1H-indol-3-yl}-acetic acid methyl ester (MIAM) was provided as a DNA-intercalator. For the comprehensive evaluation of this new intercalator, an assay system consisting of cell, S180 mouse, healthy mouse, spectrum, non-spectrum, and gel electrophoresis models was constructed. On the cell (S180, K562, MCF-7, HeLa and HepG2) models, MIAM selectively inhibited the viability of HeLa. On the S180 mouse model, 0.89, 8.9, 89 and 890 μmol kg(-1) of MIAM dose-dependently inhibited the tumor growth. Even at a dose of 890 μmol kg(-1), MIAM did not damage the treated S180 mice. The safety of MIAM was supported by a high spleen index and an obvious increase of body weight of the treated S180 mice. On the healthy mouse model the LD(50) value of MIAM is higher than 890 μmol kg(-1). The ultraviolet (UV), fluorescence, circular dichroism (CD), relative viscosity, melting curve, and gel electrophoresis assays of DNA with or without MIAM consistently supported an intercalation mechanism for MIAM.     

A sensitive and specific enzyme assay for elastase activity using α-[3H]elastin as substrate

A method for the determination of elastase activity is described which uses soluble α-[³H]elastin as substrate. Soluble α-elastin was shown to have the same substrate specificity as natural insoluble elastin. At a substrate concentration of 1 mg/ml, approximately three times half-saturating substrate concentration, the assay is rapid, 1 h, sensitive, 10 ng/ml elastase, and linear up to an enzyme concentration of 250 ng/ml. The addition of 1000 μ/ml Trasylol or 10^?4mN-α-tosyl-l-lysyl chloromethane and 10^?4m tosyl-l-phenylalanyl chloromethane allowed the specific measurement of elastase activity in the presence of trypsin and chymotrypsin activity.     

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities.

The entomopathogenic fungus Neozygites parvispora (Entomophthorales: Zygomycetes) grows in vitro as irregularly rod-shaped hyphal bodies in a complex medium. In order to simplify the medium composition and determine growth-promoting compounds for the cultivation of this fungus, we were looking for a rapid and quantitative method to estimate the number of living cells in small volumes of liquid culture. A colorimetric method for the determination of cell densities using MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] proved to be more accurate and timesaving than conventional hemocytometer counting.     

Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.     

Demonstration of two exchangeable non-catalytic and two cooperative catalytic sites in isolated bovine heart mitochondrial F1, using the photoaffinity labels [2-3H]8-azido-ATP and [2-3H]8-azido-ADP

The photoreactive nucleotides [2-3H]8-azido-ATP and [2-3H]8-azido-ADP could be used to label the nucleotide binding sites on isolated mitochondrial F1-ATPase to a maximum of 4 mol of nucleotide per mol F1, also when the F1 was depleted of tightly bound nucleotides. At a photolabel concentration of 300-1000 microM, label was found on both alpha and beta subunits in a typically 1:3 ratio, independent of the total amount bound. Under these conditions the covalent binding of two nucleotides is needed for full inactivation (Wagenvoord, R.J., Van der Kraan, I. and Kemp, A. (1977) Biochim. Biophys. Acta 460, 17-24). At lower concentrations of [2-3H]8-azido-ATP (20 microM), it was found that covalent binding of only 1 mol of nucleotide per mole F1 was required for complete inactivation to take place indicating catalytic site cooperativity in the mechanism of ATP hydrolysis. Under those conditions, radioactivity was only found on the beta subunits, which would indicate that the catalytic site is located on a beta subunit and that a second site is located on the alpha/beta interface. It is found that four out of the six nucleotide binding sites are exchangeable and can be labelled with 8-azido-AT(D)P, i.e., two catalytic sites and two non-catalytic sites.     

3-(4-Piperidinyl)- and 3-(8-aza-bicyclo[3.2.1]oct-3-yl)-2-phenyl-1H-indoles as bioavailable h5-HT2A antagonists

A series of 3-(4-piperidinyl)- and 3-(8-aza-bicyclo[3.2.l]oct-3-yl)-2-phenyl-1H-indoles have been prepared and evaluated as ligands for the h5-HT_2A receptor. 3-(8-Phenethyl-8-aza-bicyclo[3.2.l]oct-3-yI)-2-phenyl-1H-indole is a high-affinity (1.2 nM), selective (>800 fold over h5-HT_2C and hD₂ receptors) antagonist at the h5-HT_2A receptor with oral bioavailability in rats.     

In vitro and in vivo pharmacological profile of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562), a novel and putative atypical antipsychotic

In vitro and in vivo pharmacological properties of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562), a novel atypical antipsychotic, were investigated. NRA0562 showed high affinities for human cloned dopamine D(1), D(2), D(3) and D(4) receptors with Ki values of 7.09, 2.49, 3.48 and 1.79 nM. In addition, NRA0562 had high affinities for the 5-HT(2A) receptor and the alpha(1) adrenoceptor with Ki values of 1.5 and 0.56 nM, and moderate affinity for the histamine H(1) receptor. Using in vivo and ex vivo receptor binding studies in rats, we showed NRA0562 occupied frontal cortical 5-HT(2A) receptors and alpha(1) adrenoceptor potently, while occupancy of striatal dopamine D(2) receptor was moderate as were other atypical antipsychotics. NRA0562 dose-dependently inhibited methamphetamine (MAP)-induced locomotor hyperactivity in rats. At higher dosage, NRA0562 dose-dependently antagonized MAP-induced stereotyped behavior and induced catalepsy dose-dependently and significantly in rats. But, the ED(50) value in inhibiting MAP-induced locomotion hyperactivity was 10 times lower than that in inhibiting MAP-induced stereotyped behavior, and 30 times lower than that in inducing catalepsy. In addition, the potency of NRA0562 in antagonizing MAP-induced hyperactivity in rats was higher than that of other antipsychotics, clozapine, risperidone and olanzapine. NRA0562 had favorable properties in view of prediction of extrapyramidal side effects. As this antipsychotic has a unique profile with affinity and occupancy for receptors, we propose that NRA0652 may have unique atypical antipsychotic activities, and a moderate liability of extrapyramidal motor side effects seen in the treatment with classical antipsychotics.     

3-(Cyclopropylmethyl)-7-((4-(4-[11C]methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine: Synthesis and preliminary evaluation for PET imaging of metabotropic glutamate receptor subtype 2

Selective metabotropic glutamate receptor 2 (mGluR2) inhibitors have been demonstrated to show therapeutic effects by improving alleviating symptoms of schizophrenic patients in clinical studies. Herein we report the synthesis and preliminary evaluation of a ¹¹C-labeled positron emission tomography (PET) tracer originating from a mGluR2 inhibitor, 3-(cyclopropylmethyl)-7-((4-(4-methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (CMTP, 1a). [¹¹C]CMTP ([¹¹C]1a) was synthesized by O-[¹¹C]methylation of desmethyl precursor 1b with [¹¹C]methyl iodide in 19.7 ± 8.9% (n = 10) radiochemical yield (based on [¹¹C]CO₂) with >98% radiochemical purity and >74 GBq/μmol molar activity. Autoradiography study showed that [¹¹C]1a possessed moderate in vitro specific binding to mGluR2 in the rat brain, with a heterogeneous distribution of radioactive accumulation in the mGluR2-rich brain tissue sections, such as the cerebral cortex and striatum. PET study indicated that [¹¹C]1a was able to cross the blood–brain barrier and enter the brain, but had very low specific binding in the rat brain. Further optimization for the chemical structure of 1a is necessary to increase binding affinity to mGluR2 and then improve in vivo specific binding in brain.