Dibasic benzo[b]thiophene derivatives as a novel class of active site directed thrombin inhibitors. 2. Exploring interactions at the proximal (S2) binding site

In an effort to increase the thrombin inhibitory activity of a novel series of inhibitors (i.e., 1a), substituents were incorporated at the C-3" position of the C-3 aryl ring (2). Consistent with the X-ray crystallography studies, small hydrophobic groups at the C-3" site (Br and Me) enhanced thrombin inhibitory activity by 8-fold. However, a few more hydrophilic substituents (NO2 and OMe) also enhanced the potency of the series. The biological results are discussed in terms of molecular modeling studies.     

Synthesis and elastase inhibitory activity of 6 alpha-chloro-2,2-dimethyl-3 alpha-(pivaloyloxy)methylpenam sulfone, 6 alpha-chloro-2,2-dimethyl-3-exo-methylenepenam sulfone, benzyl and methyl 6 alpha-substituted penicillanate sulfones

The triflates and pivalates of 3 alpha-hydroxymethyl-6-substituted-2,2-dimethylpenam sulfones 3, 5; methyl and benzyl 6-substituted penicillanates 6-9 and 3-exo-methylene-6-substituted-2,2-dimethylpenam sulfone 4 were synthesized. These novel compounds were evaluated as elastase inhibitors using porcine pancreatic elastase. The effects that structural modifications of substituents on C-3 and C-6 in the penam nucleus have on elastase activity were examined and several similarities and distinctions were identified when compared to the reported penicillin esters and amides elastase inhibitors.     

Regulation of rat hepatic 3beta-hydroxysterol delta7-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation

The mechanism for the catalytic reduction of the double bond at C-7, 8 in 7-dehydrocholesterol by 3beta-hydroxysterol Delta7-reductase was investigated by testing structurally related sterols as substrates and potential inhibitors. The hepatic smooth endoplasmic reticulum was identified as the site of enzyme activity. All putative substrates contained 27 carbons, but differed from 7-dehydrocholesterol by the addition of either an ethyl substituent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl substituent at C-24 (ergosterol), epimerization of the hydroxyl from the 3beta- to 3alpha-configuration (7-dehydroepicholesterol), or a saturated double bond at C-5,6 (lathosterol). Two non-steroidal compounds that inhibit 3beta-hydroxysterol Delta7-reductase in vivo (AY 9944 and BM 15.766) were also tested. Ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol were reduced at C-7, 8 to form brassicasterol, sitosterol, and epicholesterol, respectively, but 75% less efficiently than 7-dehydrocholesterol. Increasing concentrations of these sterols competitively inhibited 3beta-hydroxysterol Delta7-reductase activity. The double bond at C-7,8 in lathosterol was not reduced. AY 9944 and BM 15.766 inhibited 3beta-hydroxysterol Delta7-reductase activity non-competitively. 3beta-Hydroxysterol-Delta7-reductase activity declined after microsomes were exposed to alkaline phosphatase, and enzyme activity was increased by phosphorylation with Mg2+, and ATP. These results demonstrate that the reduction of the double bond at C-7,8 requires binding of the enzyme protein with the B-ring of the sterol substrate that contains a double bond at C-5,6. The reaction is hindered by substituents located on the apolar side-chain and epimerization of the hydroxyl group in ring A to a 3alpha-configuration. 3beta-Hydroxysterol Delta7-reductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form.     

Synthesis and antitumor evaluation of new thiazolo[5,4-b]quinoline derivatives

A new synthesis of 9-hydroxy- and 9-(alkylamino)thiazolo[5,4-b]quinolines by cyclization of 4-(ethoxycarbonyl)-5-(arylamino)thiazoles and 5-(arylamino)-4-carbamoylthiazoles, respectively, is described. In vitro cytotoxicity of a large number of derivatives of these compounds has been tested against several cell lines. The highest activities observed are associated with the presence of a 2-[[(N,N-diethylamino)ethyl]amino] substituent at C-2 and a fluorine atom at the C-7 position of the tricyclic planar heteroaromatic framework. Three structural features seem to be essential for antitumor activities: a positive charge density at carbon C-7, a side chain at position C-2 or C-9 of the thiazoloquinoline skeleton with two basic nitrogens and a pKa value of 7.5-10 in the most basic center, and a conformational flexibility of this basic side chain. These structural requirements must be simultaneously satisfied in order to ensure a significant antitumor activity.     

Structure-activity requirements for flavone cytotoxicity and binding to tubulin

A series of 79 flavones related to centaureidin (3,6,4'-trimethoxy-5, 7,3'-trihydroxyflavone, 1) was screened for cytotoxicity in the NCI in vitro 60-cell line human tumor screen. The resulting cytotoxicity profiles of these flavones were compared for degree of similarity to the profile of 1. Selected compounds were further evaluated with in vitro assays of tubulin polymerization and [3H]colchicine binding to tubulin. Maximum potencies for tubulin interaction and production of differential cytotoxicity profiles characteristic of 1 were observed only with compounds containing hydroxyl substituents at C-3' and C-5 and methoxyl groups at C-3 and C-4'.     

Structure of cytochalasins and cytochalasin B binding sites in human erythrocyte membranes

Twenty cytochalasins were tested for binding to and for inhibition of glucose transport in human erythrocyte membrane. In this membrane three cytochalasin B (CB) binding sites have been identified. All but three of the cytochalasins bind at site II. On the other hand, only nine of them, which are structurally closely related, bind at site I and inhibit glucose transport. For site I (and site III) binding and glucose transport inhibitory activities (a) the macrocyclic ring in the cytochalasin molecule must be at least 13-membered, (b) the nature of the aromatic ring at C-10 is not important, (c) the C-20-C-23 region makes a major contribution, and (d) the C-5-C-7 segment has a relatively minor influence. These findings do not support a proposed mechanism which involves 24, C-23, C-20, and C-1 oxygen atoms for interaction of CB with glucose carrier. The structural requirements for site II activity are less stringent. The size and the structure of the macrocyclic ring and the nature of the aromatic residue at C-10 modulate this activity only slightly, if at all. Modifications in the C-5-C-7 region of the molecule, however, result in substantial changes in this activity.     

Development of a binding model to protein tyrosine kinases for substituted pyrido[2,3-d]pyrimidine inhibitors

Previously, our laboratories have reported on a new class of highly potent tyrosine kinase inhibitors based on the pyrido[2, 3-d]pyrimidine core template. To understand the structural basis for the potency and specificity, a model for the binding mode of this class of inhibitors to the tyrosine kinase domains of c-Src, PDGFr, FGFr, and EGFr tyrosine kinases was developed from structural information (principally utilizing the catalytic domain of c-AMP-dependent protein kinase as template) and structure-activity relationship (SAR) information. In the resulting docking mode, the pyrido[2,3-d]pyrimidine template shows a hydrogen-bonding pattern identical to that of olomoucine. The 6-aryl substituent of the heterocycle is located deep in the binding cleft in a pocket not used by ATP, which helps to confer high-affinity binding as well as specificity. The 2-anilino and 2-(dialkylamino)alkylamino substituents as well as the 7-urea substituent of inhibitors within this class are located at the entrance of the binding cleft and make contact with residues in the hinge region between the two kinase lobes. This allows considerable variability and bulk tolerance for C-2 and N-7 substituents. The models presented here are consistent with the SAR seen for the inhibition of a number of isolated enzymes and provide a structural basis to explain their specificity. They have been used successfully to design new highly potent protein kinase inhibitors.     

Clarification of the binding mode of teleocidin and benzolactams to the Cys2 domain of protein kinase Cdelta by synthesis of hydrophobically modified, teleocidin-mimicking benzolactams and computational docking simulation

Phorbol esters (12-O-tetradecanoylphorbol 13-acetate; TPA) and teleocidins are known to be potent tumor promoters and to activate protein kinase C (PKC) by binding competitively to the enzyme. The relationship between the chemical structures and the activities of these compounds has attracted much attention because of the marked structural dissimilarities. The benzolactam 5, with an eight-membered lactam ring and benzene ring instead of the nine-membered lactam ring and indole ring of teleocidins, reproduces the active ring conformation and biological activities of teleocidins. Herein we describe the synthesis of benzolactams with hydrophobic substituents at various positions. Structure-activity data indicate that the existence of a hydrophobic region between C-2 and C-9 and the steric factor at C-8 play critical roles in the appearance of biological activities. We also computationally simulated the docking of teleocidin and the modified benzolactam molecules to the Cys2 domain structure observed in the crystalline complex of PKCdelta with phorbol 13-acetate. Teleocidin and benzolactams fitted well into the same cavity as phorbol 13-acetate. Of the three functional groups hydrogen-bonding to the protein, two hydrogen-bonded with protein atoms in common with phorbol 13-acetate, but the third one hydrogen-bonded with a different protein atom from that in the case of phorbol 13-acetate. The model explains well the remarkable difference in activity between 5 and its analogue having a bulky substituent at C-8.     

Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides

Electrospray ionization-ion trap mass spectrometry, with its capacity to perform multiple stages of fragmentation (MSn), is demonstrated as an effective method for the structural characterization of permethylated N-linked complex glycoprotein oligosaccharides. Complex glycan structural features, such as N-acetyllactosamine antenane, neuraminic acids, and nonreducing terminal GlcNAc monosaccharides, commonly suppress cross-ring and core saccharide cleavages in traditional MS/MS experiments. Using ion trap mass spectrometry, removal of these substituents permits determination of branching patterns and intersaccharide linkages by MS3 and MS4. Both sequence and linkage data are obtained for N-acetyllactosamine and sialyl-N-acetyllactosamine oligosaccharide antennae from biantennary glycans using MS3, and the location of a bisecting GlcNAc residue is also established after exposing the core pentasaccharide. Higher-order experiments further illustrate the potential of electrospray ionization-quadrupole ion trap mass spectrometry for carbohydrate analysis, as MS8 is used to produce significant and otherwise unobtainable branching information for an oligosaccharide from chicken ovalbumin. These studies constitute further evidence of the unique role that ion trap mass spectrometry can assume in the area of oligosaccharide analysis.     

Fungitoxic activity of some cytochalasins and their derivatives on Phytophthora species

The activity of cytochalasin B was tested on 8 Phytophthora species, while the same toxin, some of its derivatives and natural analogues, namely cytochalasin F and deoxaphomin, were assayed at 2 x 10(-5) - 2 x 10(-4) M on the most sensitive species, P. cactorum. A significant inhibitory activity on P. cactorum was shown by cytochalasin B, its 7-monoacetyl derivative, and deoxaphomin. The hydroxy group at C-20 and the conformational freedom of the macrocyclic ring proved to be important structural features for this activity. The 7-hydroxy group at C-7 appeared to have no influence on this toxicity, while a size reduction associated with the carbocyclic nature of the macrocycle seems to lightly increase the activity. The 7-O-acetylcytochalasin B showed selective toxic activity on P. cactorum at the tested concentration, thus suggesting a possible use as a fungicide for this compound.     

Catalytic mechanism and specificity for hydrolysis and transglycosylation reactions of cytosolic beta-glucosidase from guinea pig liver

Cytosolic beta-glucosidase (CBG) from mammalian liver is known for its broad substrate specificity and has been implicated in the transformation of xenobiotic glycosides. CBG also catalyzes a variety of transglycosylation reactions, which have been been shown with other glycosylhydrolases to function in synthetic and genetic regulatory pathways. We investigated the catalytic mechanism, substrate specificity, and transglycosylation acceptor specificity of guinea pig liver CBG by several methods. These studies indicate that CBG employs a two-step catalytic mechanism with the formation of a covalent enzyme-sugar intermediate and that CBG will transfer sugar residues to primary hydroxyls and equatorial but not axial C-4 hydroxyls of aldopyranosyl sugars. Kinetic studies revealed that correction for transglycosylation reactions is necessary to derive correct kinetic parameters for CBG. Further analyses revealed that for aldopyranosyl substrates, the activation energy barrier is affected most by the presence of a C-6 carbon and by the configuration of the C-2 hydroxyl, whereas the binding energy is affected modestly by the configuration and substituents at C-2, C-4, and C-5. These data indicate that the transglycosylation activity of CBG derives from the formation of a covalently linked enzyme-sugar intermediate and that the specificity of CBG for transglycosylation reactions is different from its specificity for hydrolysis reactions.     

Two novel oleanolic acid saponins having a sialyl Lewis X mimetic structure from Achyranthes fauriei root

Two novel triterpene glycosides, achyranthosides E and F, were isolated as methyl esters from the root of Achyranthes fauriei, an antiinflammatory medicinal plant. Their structures were characterized as oleanolic acid glucuronides having unique substituents composed of C6H9O5 and C9H15O7, respectively, at the C-3 position of glucuronic acid. These compounds are active components which can inhibit the excess recruiting of neutrophiles to injured tissues 1,000 times more potently than sialyl Lewis X.     

Cytofluorometric analysis of chondrotoxicity of fluoroquinolone antimicrobial agents

Betulinic acid [1] and platanic acid [2], isolated from the leaves of Syzigium claviforum, were found to be inhibitors of HIV replication in H9 lymphocyte cells. Evaluation of anti-HIV activity with eight derivatives of 1 revealed that dihydrobetulinic acid [3] was also a potent inhibitor of HIV replication. The C-3 hydroxy group and C-17 carboxylic acid group, as well as the C-19 substituents, contribute to enhanced anti-HIV activity. The inhibitory activity of these compounds against protein kinase C (PKC) was also examined, since a correlation between anti-HIV and anti-PKC activities has been suggested. However, there was no apparent correlation between anti-HIV activity and the inhibition of PKC among these compounds.     

Classical/nonclassical hybrid cannabinoids: southern aliphatic chain-functionalized C-6beta methyl, ethyl, and propyl analogues

The stereoelectronic requirements for interaction of the southern aliphatic hydroxyl of cannabimimetic pharmacophores with the CB1 and CB2 receptors are explored. The stereoselective syntheses of three series of classical/nonclassical hybrid cannabinoids are described. These compounds were designed to investigate the importance of the southern aliphatic hydroxyl (SAH) pharmacophore for cannabimimetic activity. Variation in the chain length of the SAH moiety in these 6beta-(hydroxyalkyl)dihydrobenzopyran analogues, from 6beta-hydroxymethyl to 6beta-(omega-hydroxyethyl) and 6beta-(omega-hydroxypropyl), and the effects of replacing the hydroxyl functionality by hydride and iodide are reported. Our results indicate that the SAH pharmacophore has less pronounced effects than the C-3 aliphatic chain on cannabinoid activity. Furthermore, it appears that this southern molecular component is capable of interacting with two different subsites on the receptor and that the nature of this interaction is determined by the terminal substituent on the C-6beta alkyl group. One of the subsites can accommodate the relatively polar SAH pharmacophore, while the second subsite interacts with more hydrophobic C-6beta substituents and can accommodate large spherical pharmacophores separated by three methylene carbons from the tricyclic cannabinoid template.     

Novel disaccharide inhibitors of human glioma cell division

Several alpha-L-Fuc-(1-->3)-alpha-D-GlcNAcOC8H17 disaccharide derivatives bearing different hydroxylated alkyl chains, with or without sulfate groups at C-4 and/or C-6 positions of the GlcNAc unit, have been synthesized and tested as inhibitors of human astrocytoma lines U-373 and U-118. The antimitotic activity was dependent on the structure and position of the hydroxylated chain linked to the disaccharide. The compounds with a pentaerythritol or L-glyceryl chain at the C-6 position showed the best inhibitory properties, with an ID50 value of ca. 200 microM. On the contrary, sulfated disaccharide derivatives were inactive. The antimitotic activities of the compounds tested were essentially independent of the mitogen used to stimulate cell division.     

Relative binding affinity of androstane and C-19-nor-androstane-steroids for the estradiol-receptor in human myometrial and mammary cancer tissue

The relative binding affinity of several androstane- and C-19-nor-androstane-compounds for the estradiol (E2)-receptor in human myometrial and mammary cancer tissue was studied. High speed cytosol was incubated with tritiated E2 alone as well as in the presence of increasing amounts of the compound to be tested. The highest affinity is found for steroids with two hydroxyl-groups at C-3 and C-17 in the beta-position and a double bond at C-4-5 or C-5-6. Saturation of the A-ring decreases the affinity: 5alpha-compounds have less affinity, 5-beta-compounds have less affinity; 5beta-compounds hardly any affinity. The presence of a hydroxyl-group in the 3alpha, 11beta or 16beta-position decreases the affinity, as dose a 3-oxo or 17-oxo-group. Removal of the C-19-methyl-group facilitates the binding. This data led to the concept that flatness of the A-ring in respect to the B-ring of the steroid molecule is a principal requirement for binding to the E2-receptor. The rank order of RBA is identical in myometrium and in mammary cancer tissue, indicating that estrogen-receptors are at least highly similar in both target tissues.     

Comparative molecular field analysis of a series of paclitaxel analogues

A series of 94 paclitaxel analogues exhibiting antitumor activity by promoting the assembly of microtubules and inhibiting the disassembly process of microtubules to tubulin were investigated using the comparative molecular field analysis (CoMFA) method. These compounds belonging to 10 structural classes were randomly divided into a training set of 80 compounds and a test set of 14 compounds. Since the three-dimension structure of ligand--receptor complex is unknown, from X-ray and NMR data we rationally selected the three-dimension structure of paclitaxel in a polar solution as the active conformation and starting structure for molecule modeling, the other molecules were aligned using this molecule model as the template. The most optimal CoMFA yielded a two-components model, with significant cross-validation r2cv of 0.640 and conventional r2 of 0.868. The predictive ability of training set model was tested on the test set of 14 compounds. The tests not only revealed the robustness of the CoMFA model but demonstrated that for our model r2pred based on the mean activity of test set compounds can accurately estimate external predictivity but r2pred based on the mean activity of training set compounds overestimated the model. The CoMFA model explained why the activity of taxoid is sensitive to the stereochemistry of the atoms at C-2' and C-3' positions and the presence of hydroxyl group at C-2' position. The other factors affecting activity were also elucidated according to standard coefficient contour maps of steric and electrostatic fields derived from the CoMFA model.     

Studies of structure-activity relationship of nitroxide free radicals and their precursors as modifiers against oxidative damage

The protective effects of stable nitroxides, as well as their hydroxylamine and amine precursors, have been tested in Chinese hamster V79 cells subjected to H2O2 exposure at fixed concentration or exposure to ionizing radiation. Cytotoxicity was evaluated by monitoring the viability of the cells assessed by the clonogenic assay. The compounds tested at fixed concentration varied in terms of ring size, oxidation state, and ring substituents. Electrochemical studies were carried out to measure the redox midpoint potentials. The studies show that in the case of protection against H2O2 exposure, the protection was determined by the ring size, oxidation state, and redox midpoint potentials. In general the protection factors followed the order nitroxides > hydroxylamines > amines. Both the six-membered ring nitroxides and substituted five-membered ring nitroxides were efficient protectors. For six-membered ring nitroxides, the compounds exhibiting the lowest midpoint potentials exhibited maximal protection. In the case of X-radiation, nitroxides were the most protective though some hydroxylamines were also efficient. The amines were in some cases found to sensitize the toxicity of aerobic radiation exposure. The protection observed by the nitroxides was not dependent on the ring size. However, the ring substituents had significant influence on the protection. Compounds containing a basic side chain were found to provide enhanced protection. The results in this study suggest that these compounds are novel antioxidants which can provide cytoprotection in mammalian cells against diverse types of oxidative insult and identify structural determinants optimal for protection against individual types of damage.     

Isolation, structure elucidation, and biological activity of the steroid oligoglycosides and polyhydroxysteroids from the Antarctic starfish Acodontaster conspicuus

A total of 19 steroids, of which 13 steroidal oligoglycosides (nine new and four known) and six polyhydroxylated steroids (four new and two known), has been isolated from the Antarctic starfish Acodontaster conspicuus. The mixture is dominated by glycosides composed of steroidal aglycons having the hydroxyl groups typically disposed on one side of the tetracyclic nucleus, i.e., 3 beta,4 beta,6 alpha,8,15 beta-, with some having a sulfate at C-6, and differing in the side chains and/or in the disaccharide moieties that are usually attached at C-26, with some at C-28 and C-29. Those compounds are accompanied by minute amounts of glycosides with a delta 8(14)-double bond in the steroid, which is a structural feature not previously found among polyhydroxysteroids derived from starfish. Small amounts of six related unglycosidated polyhydroxysteroids and three higher-molecular-weight asterosaponins complete the composition of the mixture. The structures of the new compounds were determined by interpretation of their spectral data and by comparison with spectral data of known compounds. Eighteen of these compounds were evaluated for their ability to inhibit growth in Antarctic marine bacteria isolated from either the water column or the surfaces of benthic marine invertebrates. Of these compounds, 50% were active against at least one Antarctic marine bacterium. This suggests that these compounds may play an important role in deterring microbial fouling.