An integrated methodology for data processing in dynamic force spectroscopy of ligand-receptor binding.

Dynamic force spectroscopy (DFS), using atomic force microscopy (AFM), is a powerful tool to study ligand-receptor binding. The interaction mode of two binding partners is investigated by exploring stochastic behaviors of bond rupture events. However, to define a rupture event from force-distance measurements is not conclusive or unique in literature. To reveal the influence of event identification methods, we have developed an efficient protocol to manage tremendous amount of data by implementing different choices of peak selection from the force-distance curve. This data processing software simplifies routinely experimental procedures such as cantilever spring constant and force-distance curve calibrations, statistical treatments of data, and analysis distributions of rupture events. In the present work, we took available experimental data from a complex between a chelate metal compound and a monoclonal antibody as a study system.

Novel 3-(4-piperidinylthio)-1H-indoles as potent nonopioid orally active central analgesics.

A series of 3-(4-piperidinylthio)-1H-indoles was synthesized and evaluated in mice in the phenylbenzoquinone(PBQ)-induced writhing and hot plate tests. Most of these compounds are good analgesics with activities comparable to that of morphine. Among them compound 1i (UP 237-61), which has a strong serotonin binding profile, has an interesting antinociceptive activity which is not reversed by naloxone.

Microtiter plate immunoenzymometric assay for estrogen receptor.

The estrogen receptor (ER) status of breast cancer is used both as a prognostic factor and as a predictor of response to endocrine therapy. An immunoenzymometric assay for ER was developed on 96-well microtiter plates (EIA96). This technique involves two monoclonal antibodies directed against different epitopes in the B domain of ER. The two-step protocol (16-18 h and 3 h at 4 degrees C) requires 100 microL of cytosol. This assay showed a detection limit of 0.58 pmol/L. Intra- and interassay CVs of clinical specimens were < or = 5% except for the least concentrated sample (6.5 pmol/L, CV = 6.7%). In a comparison study involving cytosols of breast adenocarcinoma tissue biopsies, we compared the EIA96 with the radioligand assay (RLA) and the Abbott ER-EIA, widely used techniques for determining ER concentration in cytosols of breast cancer tumors. The two EIAs showed excellent agreement; however, two samples showed discrepant results by EIA96 and RLA.

N6-substituted adenosine receptor agonists. Synthesis and pharmacological activity as potent antinociceptive agents.

Novel N6-(indol-3-yl)alkyl derivatives of adenosine were synthesized. The adenosine receptor affinity and the antinociceptive activity of these compounds were assessed in binding studies and the phenylbenzoquinone-induced writhing test. Most of these analogues exhibited a potent analgesic activity without side effects. Among them, compound 3c (UP 202-32) bound to A1 (Ki = 110 nM) and A2 (Ki = 350 nM) adenosine receptors in a specific manner since it did not interact with many other receptors, especially opioid binding sites. The antinociceptive activity in the phenylbenzoquinone assay (ED50 = 3.3 mg/kg po) was antagonized by 8-cyclopentyltheophylline, suggesting that an adenosinergic mechanism underlies the analgesic activity observed with this compound. The data obtained with these new N6-substituted adenosine receptor agonists emphasize the interest of such compounds in the treatment of pain.

Synthesis and angiotensin II receptor antagonist activity of C-linked pyrazole derivatives.

The synthesis and pharmacological activity of new nonpeptide angiotensin II (AII) receptor antagonists are presented. These 5-O-substituted and 5-C-substituted 3-alkylpyrazole derivatives represent a new series of antagonists and have led to the discovery of compounds with potent oral antihypertensive activity in a renal artery-ligated rat model. In vitro, they displayed a high affinity for rat adrenal AII receptors. In vivo structure-activity relationship study has shown the importance of the 4-[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl moiety for oral activity and the critical role of alkyl substituents at the 1- or 2-position. In the case of oral administration, 5-C derivatives were found to be, on the whole, more potent than 5-O derivatives. UP 221-78, 5-hydroxymethyl-3-n-propyl-1-(2,2,2-trifluoroethyl)-4- [[2'-(1H-tetrazol-5-yl)biphenyl-4-]methyl]-1H-pyrazole (79), displayed equivalent antihypertensive activity to the well known antagonist Losartan at 3 mg/kg p.o. in renal artery-ligated rats, with maximal decreases in mean arterial pressure of 60 and 63 mmHg for Losartan and UP 221-78, respectively.

Synthesis and SAR studies of novel triazolopyrimidine derivatives as potent, orally active angiotensin II receptor antagonists.

The synthesis and pharmacological activity of new nonpeptide angiotensin II (AII) receptor antagonists are presented. These [1,2,4]-triazolo[1,5-c]pyrimidine and 1,2,4-triazolo[4,3-c]pyrimidine derivatives represent a new class of bicyclic antagonists that produced a potent, oral antihypertensive activity in the renal artery-ligated rat model. In vitro, they displayed a high affinity for rat adrenal AII receptors and were found to be specific for the AT1 receptor subtype. A SAR study has shown the importance of the 8-[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl for oral activity and the critical role of alkyl substituents at 5- and 7-positions. No significant differences were found between the [1,5-c] and [4,3-c]series. UP 269-6 (5-methyl-7-n-propyl-8-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl ]- [1,2,4]-triazolo[1,5-c]pyrimidin-2(3H)-one, derivative 29) was selected as the lead compound. It was shown to be a highly potent antihypertensive derivative (decrease in mean arterial pressure of 39.6 +/- 7.2 mmHg at 1 mg/kg po in renal artery-ligated rat) with a long duration of action which displayed a high affinity for adrenal AII receptors with a marked selectivity for the AT1 receptor subtype (Ki AT1 = 24 nM; Ki AT2 = 79,200 nM). This compound is currently undergoing extensive pharmacological and clinical development.

Synthesis and structure-activity relationships of novel benzimidazole and imidazo[4,5-b]pyridine acid derivatives as thromboxane A2 receptor antagonists.

A series of 1-benzylbenzimidazole and 3-benzylimidazo[4,5-b]pyridine substituted in the 2-position by an alkanoic or mercaptoalkanoic acid chain was synthesized for evaluation as potential thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor antagonists. The affinity of each compound for washed human platelet TXA2/PGH2 receptors was determined by radioligand binding studies using [125I]PTA-OH. Structure-activity relationships led to the conclusions that 2-alkanoic acid derivatives were slightly more potent than 2-mercaptoalkanoic acids and that compounds possessing a 3,3-dimethylbutanoic acid in the 2-position were definitely the most potent with Ki values of 4-39 nM (11a, 11g-x, 37a, 37f-o, 23a-c). The replacement of this 3,3-dimethylbutanoic acid side chain by a shorter one led to a marked decrease of affinity (11b and 11c; Ki = 5600 and 1700 nM, respectively). Compounds of benzimidazole and imidazo[4,5-b]pyridine series displayed similar potencies (11q and 23c have Ki values of 6 and 7 nM, respectively). The interesting pharmacological profile of compound 23a (UP 116-77: 4-[3-[(4-chlorophenyl)methyl]-6-chloroimidazo[4,5-b]pyridin-2-yl]- 3,3-dimethylbutanoic acid) and its excellent tolerance led us to select this derivative for further development.

Cardiotonic agents. Synthesis and cardiovascular properties of novel 2-arylbenzimidazoles and azabenzimidazoles.

Novel 2-arylbenzimidazoles and azabenzimidazoles were synthesized, and their inotropic action was evaluated. Changes in left ventricular pressure, dP/dt max, were measured as an index of cardiac contractility. The structural features that impart optimal inotropic activity are presented. The most potent compounds were evaluated orally in conscious dogs with implanted Konigsberg pressure transducers. To investigate the mechanism of action, the most potent compounds were tested for their calcium-sensitizing properties and their potential for the inhibition of phosphodiesterase. Two compounds, 1 and 41, showed interesting in vitro and oral activity without side effects. They have a more potent calcium-sensitizing effect than MCI-154 and are under further investigation.

Triterpenoid glycosides from the bark of Mimosa tenuiflora.

Two new saponins were isolated from Mimosa tenuiflora and their structures established as 3-O-[alpha-L-rhamnopyranosyl(1----2)-beta-D-glucopyranosyl-(1----3]-(alp ha-L- arabinopyranosyl-(1----4]-beta-D-xylopyranosyl-(1----2)]-[beta-D- xylopyranosyl-(1----4)]-beta-D-glucopyranosyl)-28-O-alpha-L-rhamnopyrano syl oleanolic acid and 3-O-[alpha-L-rhamnopyranosyl-(1----2)-beta-D-glucopyranosyl-(1----3]-(al pha- L-arabinopyranosyl-(1----4]beta-D-xylopyranosyl-(1----2)]-[beta-D- xylopyranosyl-(1----4)]-beta-D-glucopyranosyl) oleanolic acid.

Analytical profile of the new diuretic beta-blocking agent tienoxolol hydrochloride.

Physicochemical properties, i.e. elemental analysis, spectra (NMR, MS, IR, UV), solubilities, pKa, partition coefficient, melting point, HPLC, and data on purity and stability of ethyl 2-(3-[(1,1-dimethylethyl)amino]-2-hydroxy-propoxy)-5-[(2- thienylcarbonyl)amino]benzoate hydrochloride (tienoxolol), a new drug with antihypertensive properties are reported.

[p-[(Thienylcarbonyl)amino]phenoxy]propanolamines derivatives as diuretic and beta-adrenergic receptor blocking agents.

The synthesis of [[(thienylcarbonyl)amino]phenoxy]propanolamines and their beta-adrenergic blocking and diuretic activity are described. Structure-activity relationships demonstrated that ortho substitution of the phenoxy ring with an hydrogen or an ester function leads to compounds possessing both activities. Ethyl 2-[3-[(1,1-dimethylethyl) amino]-2-hydroxypropoxy]-5-[(2-thienylcarbonyl)amino]benzoate (3d) was selected as the most active compound for further investigation.

Antiinflammatory and analgesic diastereoisomeric derivatives of indan-5-acetic acid.

Various 2-alkyl-alpha-methyl- and 2-alkylindan-5-acetic acids have been prepared. The acids, which can exist in two diastereoisomeric forms that cannot be separated by crystallization or chromatography, can be analyzed in their mixture by NMR in the presence of Eu(dpm)3. It has been possible to reconstitute the two pure racemic 2-isopropyl-alpha-methylindan-5-acetic acids from their enantiomers obtained after resolution of the mixtures through salts with various active bases. The relative configuration of the two asymmetric centers of one of the diastereoisomers salts with various active bases. The relative configuration of the two asymmetric centers of one of the diastereoisomers has been determined by X-ray crystallography. The absolute configurations of the resolved acids have been established by a comparative study of their CD curves. The antiinflammatory and analgesic properties of these compounds as functions of their structure and stereochemistry are discussed.