Quantitative structure-activity relationship study of ATP-sensitive potassium channel openers: derivatives of 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide

The inhibitory activity of glucose-induced insulin secretion on isolated rat pancreatic islets and the contractile activity of KCl-depolarized rat aorta rings of the derivatives of 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide are quantitatively analyzed using multiple regression analysis. The study has helped to ascertain the role of different substituents in explaining these observed inhibitory activities. From a derived most significant correlation equation, it was concluded that a less hydrophobic 3-substituent and a less bulky 7-substituent in addition to a 3-aminoisopropyl and a 6-chloro substituent are advantageous to enhance the inhibitory action of a compound towards rat pancreatic islets. On the other hand, the more hydrophobic 6- and 7-substituents augment the contractile activity. The analysis, in this way, provided the grounds for rationalizing the substituent selection in designing the improved potency compounds in the series.     

Similarities and differences between organic cation inhibition of the Na,K-ATPase and PMCA

The effects of three classes of organic cations on the inhibition of the plasma membrane Ca pump (PMCA) were determined and compared to inhibition of the Na pump. Quaternary amines (tetramethylammonium, tetraethylammonium, and tetrapropylammonium, TMA, TEA, and TPA, respectively) did not inhibit PMCA. This is not to imply that PMCA is inherently selective against monovalent cations because guanidine and tetramethylguanidine inhibited PMCA by competing with Ca(2+). The divalent organic cation, ethyl diamine, inhibited PMCA but was not competitive with Ca(2+). In contrast, propyl diamine did compete with Ca(2+) and was about 10-fold more potent than butyl diamine in inhibiting PMCA. For the Na pump, both TEA and TPA inhibited, but TMA did not. TEA, guanidine, and tetramethylguanidine inhibition was competitive with Na(+) for ATPase activation and with K(+) for pNPPase activation, both of which are cytoplasmic substrate cation effects. Thus, these findings are consistent with TEA, guanidine, and tetramethylguanidine inhibiting from the cytoplasmic side of the Na pump; in contrast, we have previously shown that TPA did not inhibit from the cytoplasmic side. The divalent alkane diamines ethyl, propyl, and butyl diamine all inhibited the Na pump and all competed at the intracellular surface. The order of potency was ED > PD > BD consistent with an optimal size for binding; similarly, for the quaternary amines TMA is apparently too small to make appropriate contacts, and TPA is too large. Homology models based upon the high-resolution SERCA structure are included to contextualize the kinetic observations.     

Substrate specificity of the 3-methylaspartate ammonia-lyase reaction: observation of differential relative reaction rates for substrate-product pairs

A range of substituted fumaric and aspartic acid substrates for the enzyme 3-methylaspartate ammonia-lyase (EC 4.3.1.2) have been synthesized and used to study the kinetics of the catalyzed reaction in both the forward (deamination) and reverse (conjugative amination) reaction directions. The rates of amination for all of the alpha, beta-unsaturated substrates studied (bearing substituents the size of an ethyl group or smaller) were similar under [s] much greater than KM conditions although KM values for the substrates varied by a factor of 25. The rates of deamination for the corresponding 3-substituted amino acid substrates varied widely with structure under [s] much greater than KM conditions, and thus for substrate-product pairs the ratio for V(forward)/V(reverse) also varied. These differential reaction rates indicate that there is a step in the deamination direction that is especially sensitive to the size of the 3-substituent of the substrate and that a relatively large group (methyl to ethyl in size) is required for binding in order to reduce the activation energy for this step. Given that it is proposed that the enzyme operates via an E1cb-type mechanism where C-N bond cleavage is rate limiting, it is likely that binding of the C-3 substituent of aspartic acid substrates affects the alignment of the nascent carbanion with the C-N bond for elimination.     

Design, synthesis, and evaluation of oxazole transthyretin amyloidogenesis inhibitors

Ten oxazoles bearing a C(4) carboxyl group were synthesized and evaluated as transthyretin (TTR) amyloid fibril inhibitors. Substituting aryls at the C(2) position of the oxazole ring reveals that a 3,5-dichlorophenyl substituent significantly reduced amyloidogenesis. The efficacy of these inhibitors was enhanced further by installing an ethyl, a propyl, or a CF(3) group at the C(5) position. The CF(3) substitution at C(5) also improves the TTR binding selectivity over all the other proteins in human blood.     

Glucose-6-phosphatase catalytic enzyme inhibitors: synthesis and in vitro evaluation of novel 4,5,6,7-tetrahydrothieno[3,2-c]- and -[2,3-c]pyridines

The discovery of the first class of potent glucose-6-phosphatase catalytic site inhibitors, substituted 4,5,6,7-tetrahydrothieno[3,2-c]- and -[2,3-c]pyridines, is described. Optimisation of this series involved solution phase combinatorial synthesis and very potent compounds were prepared with IC50 values down to 140 nM. The structure activity relationship (SAR) of these compounds indicates that: a tetrahydrothieno[3,2-c]pyridine core ring system and the isomeric [2,3-c] system are equipotent and much better than the corresponding benzo analogue, 1,2,3,4-tetrahydro-isoquinoline. The 4-substituent of the tetrahydrothieno[3,2-c]pyridine ring has to be a phenyl group, optionally substituted with a lipophilic 4-substituent, such as trifluoromethoxy or chloro. The 5-substituent of the tetrahydrothieno[3,2-c]pyridine ring has to be a substituted benzoyl; anisoyl and (E)-3-furan-3-ylacryloyl are the best of the investigated groups. Substitution in the benzoyl ortho position seems to be forbidden, whereas substitution in the meta position is tolerated only if a methoxy para substituent is present. These SAR findings were parallel to those obtained in the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine system. Enantioselectivity in enzyme recognition was observed and the activity resided in all cases only in one of the enantiomers.     

Some biochemical properties of alkyl phosphotriesters of cyclic AMP.

The biochemical properties of several alkyl phosphotriesters of cyclic AMP were studied with respect to their interactions with beef heart protein kinase and cyclic nucleotide phosphodiesterase. Ethyl and propyl triesters did not enhance the phosphorylation of histone by protein kinase and methyl, ethyl, propyl and butyl triesters were poor competitors for the cyclic AMP binding site of the enzyme. However, these alkyl phosphotriesters were effective inhibitors of cyclic nucleotide phosphodiesterase with the K$\text{i}$'s arrayed in the following order: methyl > ethyl > propyl > butyl > cetyl triester. Metabolic studies with mice indicated that intraperitoneal injection of low doses of propyl triester for one week significantly increased cyclic AMP concentration.     

The Search for a Volatile Human Specific Marker in the Decomposition Process

In this study, a validated method using a thermal desorber combined with a gas chromatograph coupled to mass spectrometry was used to identify the volatile organic compounds released during decomposition of 6 human and 26 animal remains in a laboratory environment during a period of 6 months. 452 compounds were identified. Among them a human specific marker was sought using principle component analysis. We found a combination of 8 compounds (ethyl propionate, propyl propionate, propyl butyrate, ethyl pentanoate, pyridine, diethyl disulfide, methyl(methylthio)ethyl disulfide and 3-methylthio-1-propanol) that led to the distinction of human and pig remains from other animal remains. Furthermore, it was possible to separate the pig remains from human remains based on 5 esters (3-methylbutyl pentanoate, 3-methylbutyl 3-methylbutyrate, 3-methylbutyl 2-methylbutyrate, butyl pentanoate and propyl hexanoate). Further research in the field with full bodies has to corroborate these results and search for one or more human specific markers. These markers would allow a more efficiently training of cadaver dogs or portable detection devices could be developed.     

Influence of substituent ribose on transition state affinity in reactions catalyzed by adenosine deaminase

Adenosine deaminase from calf intestine hydrolyzes adenine at a limiting rate four orders of magnitude lower than that for adenosine, while Km values for these substrates are about the same (Wolfenden, R., et al. (1969), Biochemistry 8, 2412-2415). Reactivity of 6-substituents, toward nucleophilic displacement, is found to be affected only slightly by removal of ribose as a 9-substituent, in model reactions. Substituent ribose thus appears to stabilize, selectively, the transition state for enzymatic deamination. In contrast with the small influence of substituent ribose on the apparent binding affinity of substrates, removal of substituent ribose from a potential transition state analogue, 1,6-dihydro-6-hydroxy-methylpurine ribonucleoside, results in a lowering of its affinity for the enzyme by several orders of magnitude. The synthesis of the analogue and related compounds is described, and their properties compared with those of other photoadducts and of the naturally occurring inhibitors covidarabine and coformycin. Binding of these inhibitors is found to result in the appearance of ultraviolet-absorbing bands in the neighborhood of 323 nm.     

Rational design of 6-methylsulfonylindoles as selective cyclooxygenase-2 inhibitors

The introduction of 3-arylmethyl, 3-aryloxy and 3-arylthio moieties into a 6-methylsulfonylindole framework using rational drug design led to potent, selective COX-2 inhibitors having efficacy in a rat carrageenan air pouch model. Incorporation of a conformationally more rigid 3-aroyloxy substituent onto the 6-methylsulfonylindole scaffold led to selective, but considerably less potent COX-2 inhibitors. Variation of the hydrophilicity and size of the indole 2-substituent of 3-arylthio-6-methylsulfonylindole inhibitors led to modulation of the COX-2 human whole blood (HWB) potency and selectivity.     

Chemistry of opium alkaloids. Part 44: synthesis and opioid receptor binding profile of substituted ethenoisomorphinans and ethenomorphinans

7- and 8-substituted 6alpha,14alpha-ethenoisomorphinans were synthesized by reaction of properly substituted morphinan-6,8-dienes (analogues of thebaine) with methyl vinyl ketone or ethyl acrylate. Reaction with the appropriate Grignard reagent gave the 7- and 8-dialkylmethanols, respectively. Cleavage of the 3-methyl ether with KOH/glycol or boron tribromide afforded the 3-hydroxyl derivatives. In general, the compounds with the ethoxycarbonyl or dimethylmethanol substituent at the 8alpha-position showed lower affinity for the mu, kappa, and delta opioid receptor subtypes than the corresponding 7alpha- and 7beta-substituted compounds. Introduction of a chloro substituent in position 18 increased the potency significantly. The 7-substituent could be connected to the 18-position without loss of affinity. 5Beta-alkyl substitution of 6alpha,14alpha-ethenoisomorphinans led to a decrease in affinity for the three opioid receptor subtypes. In the 5beta-methyl series the affinity for the mu and delta receptors increased from 7alpha-dimethylmethanol to 7alpha-methylhexylmethanol. In the 5beta-alkyl series, the affinity for the mu-receptor could be increased by connecting the 5- and 7-substituents, yielding a compound with high mu-selectivity. The new 6beta,14beta-ethenomorphinans did not show affinity for any of the opioid receptors, in accordance with the inactivity earlier found in in vivo experiments.     

Molecular docking studies on quinazoline antifolate derivatives as human thymidylate synthase inhibitors

We have performed molecular docking on quinazoline antifolates complexed with human thymidylate synthase to gain insight into the structural preferences of these inhibitors. The study was conducted on a selected set of one hundred six compounds with variation in structure and activity. The structural analyses indicate that the coordinate bond interactions, the hydrogen bond interactions, the van der Waals interactions as well as the hydrophobic interactions between ligand and receptor are responsible simultaneously for the preference of inhibition and potency. In this study, fast flexible docking simulations were performed on quinazoline antifolates derivatives as human thymidylate synthase inhibitors. The results indicated that the quinazoline ring of the inhibitors forms hydrophobic contacts with Leu192, Leu221 and Tyr258 and stacking interaction is conserved in complex with the inhibitor and cofactor.     

5H-Dibenzo[c,h]1,6-naphthyridin-6-ones: novel topoisomerase I-targeting anticancer agents with potent cytotoxic activity

5H-Dibenzo[c,h]1,6-naphthyridine-6-ones can exhibit potent antitumor activity. The effect of varied substituents at the 5-position of 5H-8,9-dimethoxy-2,3-methylenedioxydibenzo[c,h]1,6-naphthyridine on relative cytotoxicity and topoisomerase I-targeting activity was evaluated. Potent TOP-1-targeting activity is observed when the 5-position is substituted with either a 2-(N,N-dimethylamino)ethyl group, as in 3a, or a 2-(pyrrolidin-1-yl)ethyl substituent, 3c. In contrast, the addition of a beta-methyl group or a beta-hydroxymethyl group to compound 3a, as in 3b and 3j, results in a loss of significant TOP1-targeting activity. While the presence of a 3-(N,N-dimethylamino)propyl substituent at the 5-position or a methyl(2-tetrahydrofuranyl) group allows for retention of TOP1-targeting activity, the 2-(4-methyl-1-piperazinyl)ethyl analogue, 3d, did not exhibit significant activity. Replacement of the N,N-dimethylamino group of 3a with either C(2)H(5) or OH, as in 3f and 3h, respectively, also had a negative impact on both cytotoxicity and TOP1-targeting activity. Treatment of 3a with LAH gave the 5,6-dihydrodibenzo[c,h]naphthyridine, 4a. This dihydro derivative has approximately 2/3 the potency of 3a as a TOP1-targeting agent. Compounds 3a, 3b, 3h, 3i, and 4a were evaluated for antitumor activity in the human tumor xenograft model using athymic nude mice. The non-estrogen responsive breast tumor cell line, MDA-MB-435, was used in these assays. Compound 3a proved to be effective in regressing tumor growth in vivo when administered either by ip injection or orally 3x week at a dose of 2.0mg/kg. Compound 4a when administered orally 5x weekly at a dose of 40 mg/kg also suppressed tumor growth.     

Primary alcohols and di-alcohols as growth substrates for the purple nonsulfur bacterium Rhodobacter capsulatus

Growth experiments were performed with the purple nonsulfur bacterium Rhodobacter capsulatus to test its ability to use aliphatic, methyl-substituted, and unsaturated alcohols, as well as di-alcohols, as carbon sources for growth. Both phototrophic and chemotrophic growth was observed on a wide variety of such alcohols. By contrast, secondary or tertiary alcohols, or primary alcohols containing an ethyl or propyl substituent, did not support growth. In addition, preculture history and serial subculturing were found to be important factors for obtaining reliable growth of R. capsulatus on alcohols. Collectively, these results suggest that the carbon nutritional diversity of Rhodobacter capsulatus is even greater than previously suspected and that besides metabolizing organic acids and fatty acids in nature, this species may also be a major consumer of alcohols.     

The 3D-QSAR analysis of 4(3H)-quinazolinone derivatives with dithiocarbamate side chains on thymidylate synthase

Thymidylate synthase (TS) is a critical enzyme for DNA biosynthesis and many nonclassical lipophilic antifolates targeting this enzyme are quite efficient and encouraging as antitumor drug. In this paper, the binding model of 14 antifolates of 4(3H)-quinazolinone derivatives with dithiocarbamate side chains was examined using molecular simulation methods--FlexiDock and SCORE2.0. The resulted conformation and orientation of these antifolates were directly applied to CoMFA study. A good correlation between the calculated binding energies of these antifolates complexed with TS and their inhibitory activities was derived. The robust QSAR model, its three-dimensional contour map, and binding score for these antifolates derived from SCORE2.0 provided guidelines for structural optimization of current antifolates.     

Trifunctional agents as a design strategy for tailoring ligand properties: irreversible inhibitors of A1 adenosine receptors

The 1,3-phenylene diisothiocyanate conjugate of XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]- oxy]phenyl]-1,3-dipropylxanthine, a potent A1 selective adenosine antagonist) has been characterized as an irreversible inhibitor of A1 adenosine receptors. To further extend this work, a series of analogues were prepared containing a third substituent in the phenyl isothiocyanate ring, incorporated to modify the physiochemical or spectroscopic properties of the conjugate. Symmetrical trifunctional cross-linking reagents bearing two isothiocyanate groups were prepared as general intermediates for cross-linking functionalized congeners and receptors. Xanthine isothiocyanate derivatives containing hydrophilic, fluorescent, or reactive substituents, linked via an amide, thiourea, or methylene group in the 5-position, were synthesized and found to be irreversible inhibitors of A1 adenosine receptors. The effects of the 5-substituent on water solubility and on the A1/A2 selectivity ratio derived from binding assays in rat brain membranes were examined. Inhibition of binding of [3H]-N6-(2-phenylisopropyl)-adenosine and [3H] CGS21680 (2-[2-[4-carboxyethyl)phenyl]ethyl]amino] adenosine-5'-N-ethylcarboxamide) at central A1 and A2 adenosine receptors, respectively, was measured. A conjugate of XAC and 1,3,5-triisothiocyanatobenzene was 894-fold selective for A1 receptors. Reporter groups, such as fluorescent dyes and a spin-label, were included as chain substituents in the irreversible binding analogues, which were designed for spectroscopic assays, histochemical characterization, and biochemical characterization of the receptor protein.     

Non-additivity of Functional Group Contributions in Protein-Ligand Binding: A Comprehensive Study by Crystallography and Isothermal Titration Calorimetry

Additivity of functional group contributions to protein-ligand binding is a very popular concept in medicinal chemistry as the basis of rational design and optimized lead structures. Most of the currently applied scoring functions for docking build on such additivity models. Even though the limitation of this concept is well known, case studies examining in detail why additivity fails at the molecular level are still very scarce. The present study shows, by use of crystal structure analysis and isothermal titration calorimetry for a congeneric series of thrombin inhibitors, that extensive cooperative effects between hydrophobic contacts and hydrogen bond formation are intimately coupled via dynamic properties of the formed complexes. The formation of optimal lipophilic contacts with the surface of the thrombin S3 pocket and the full desolvation of this pocket can conflict with the formation of an optimal hydrogen bond between ligand and protein. The mutual contributions of the competing interactions depend on the size of the ligand hydrophobic substituent and influence the residual mobility of ligand portions at the binding site. Analysis of the individual crystal structures and factorizing the free energy into enthalpy and entropy demonstrates that binding affinity of the ligands results from a mixture of enthalpic contributions from hydrogen bonding and hydrophobic contacts, and entropic considerations involving an increasing loss of residual mobility of the bound ligands. This complex picture of mutually competing and partially compensating enthalpic and entropic effects determines the non-additivity of free energy contributions to ligand binding at the molecular level.     

Chemical Structure and Acaricidal Activity of 3-Alkylthiophenyl 4-Nitrophenyl Ethers

Many 4-nitro diphenylether derivatives with R–S (O)_x-substituents were systematically synthesized and their pesticidal activities were investigated. 3-n-Propylthiophenyl 4-nitro-phenyl ether and its sulfoxide analog exhibited remarkable acaricidal and chlorosis-inducing activities on the young leaves of plants. For all compounds with the substituent at the 4 position in the 3-n-propylthio (and -sulfinyl) phenyl group, chlorosis-inducing activity vanished and an acaricidal activity stronger than that of the parent compound appeared. Evidence that the acaricidal and chlorosis-inducing activities are greatly influenced by the size of the 4-sub-stituent and the hydrophobicity of R in the 3-R-S (O)_x-substituent is presented.     

Alkyl substituent in place of the thymine methyl group controls the A-X conformational bimorphism in poly(dA-dT).

Circular dichroism studies of a family of poly(dA-y5dU) polynucleotides (y = H, methyl, ethyl, propyl, butyl or pentyl) were conducted in water-alcohol solutions containing sodium or cesium counterions. The polynucleotides denatured or adopted A- or X-DNA double helices depending on the concentration and type of alcohol, type of counterions and the length of the aliphatic substituent in place of the thymine methyl group. Short aliphatic substituents and sodium cations favored A-DNA while long aliphatic substituents and cesium cations promoted X-DNA. This study demonstrates delicacy of the conformational equilibrium of poly(dA-dT) between the A- and X-DNA double helices which depends on both intramolecular and intermolecular factors.