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The ribonucleases H (RNases H) of HIV and hepatitis B virus are type 1 RNases H that are promising drug targets because inhibiting their activity blocks viral replication. Eukaryotic ribonuclease H1 (RNase H1) is an essential protein and a probable off-target enzyme for viral RNase H inhibitors. α-hydroxytropolones (αHTs) are a class of anti-RNase H inhibitors that can inhibit the HIV, hepatitis B virus, and human RNases H1; however, it is unclear how these inhibitors could be developed to distinguish between these enzymes. To accelerate the development of selective RNase H inhibitors, we performed biochemical and kinetic studies on the human enzyme, which was recombinantly expressed in Escherichia coli. Size-exclusion chromatography showed that free RNase H1 is monomeric and forms a 2:1 complex with a substrate of 12 bp. FRET heteroduplex cleavage assays were used to test inhibition of RNase H1 in steady-state kinetics by two structurally diverse αHTs, 110 and 404. We determined that turnover rate was reduced, but inhibition was not competitive with substrate, despite inhibitor binding to the active site. Given the compounds' reversible binding to the active site, we concluded that traditional noncompetitive and mixed inhibition mechanisms are unlikely. Instead, we propose a model in which, by binding to the active site, αHTs stabilize an inactive enzyme-substrate-inhibitor complex. This new model clarifies the mechanism of action of αHTs against RNase H1 and will aid the development of RNase H inhibitors selective for the viral enzymes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Developing new medications for the treatment of cocaine dependence continues to be a research priority. Compelling evidence indicates that mixed opioid receptor agonists, particularly bifunctional compounds that target nociceptin/orphanin FQ peptide (NOP) and mu opioid receptors, may be useful for the treatment of cocaine addiction. Here, we verify that potent and selective pharmacological activation of NOP receptors is sufficient to reduce relevant facets of cocaine addiction in animal models. Accordingly, we determined whether systemic injections of the small molecule AT-312 (0, 1, 3 mg/kg) could reduce operant cocaine self-administration, motivation for cocaine, and vulnerability to cocaine relapse in rats. Results indicate that a potent and selective NOP receptor agonist was equally efficacious in reducing the number of cocaine infusions in short (1-hour), as well as long (6-hour) access sessions. When tested on an economic-demand reinforcement schedule, AT-312 reduced Q 0 , the parameter that describes the amount of drug consumed at zero price, while leaving the parameter α, a measure of motivation for drug consumption, unaltered. Furthermore, AT-312 successfully reduced conditioned reinstatement of cocaine seeking. In contrast, the NOP receptor agonist did not modify food self-administration. Blockade of the NOP receptor with the antagonist SB-612111 prevented the effect of AT-312 in decreasing cocaine-reinforced responding under a 2-hour fixed ratio 1 schedule, suggesting a NOP receptor-mediated mechanism. This work demonstrates that potent and selective activation of NOP receptors is sufficient to decrease cocaine taking and seeking behaviors in rats., (© 2019 Society for the Study of Addiction.)

Mitochondrial free calcium is critically linked to the regulation of cellular metabolism. Free ionic calcium concentration within these organelles is determined by the interplay between two processes: exchange across the mitochondrial inner membrane and calcium-buffering within the matrix. During stimulated calcium uptake, calcium is primarily buffered by orthophosphate, preventing calcium toxicity while allowing for well-regulated yet elevated calcium loads. However, if limited to orthophosphates only, this buffering system is expected to lead to the irreversible formation of insoluble precipitates, which are not observed in living cells, under physiological conditions. Here, we demonstrate that the regulation of free mitochondrial calcium requires the presence of free inorganic polyphosphate (polyP) within the organelle. We found that the overexpression of a mitochondrial-targeted enzyme hydrolyzing polyP leads to the loss of the cellular ability to maintain elevated calcium concentrations within the organelle, following stimulated cytoplasmic signal. We hypothesize that the presence of polyP prevents the formation of calcium-phosphate insoluble clusters, allowing for the maintenance of elevated free calcium levels, during stimulated calcium uptake., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Nociceptin/Orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP). In this study novel nonpeptide NOP ligands were characterized in vitro in receptor binding and [ 35 S]GTPγS stimulated binding in membranes of cells expressing human NOP and classical opioid receptors, calcium mobilization assay in cells coexpressing the receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, the electrically stimulated mouse vas deferens and the mouse colon bioassays. The action of the AT compounds were compared with standard NOP agonists (N/OFQ and Ro 65-6570) and the NOP selective antagonist SB-612111. AT compounds displayed high NOP affinity and behaved as NOP agonists in all the functional assays consistently showing the following rank order of potency AT-127≥AT-090≥AT-035>AT-004= AT-001. AT compounds behaved as NOP full agonists in the calcium mobilization and mouse colon assays and as partial agonists in the [ 35 S]GTPγS and BRET assays. Interestingly AT-090 and AT-127, contrary to standard nonpeptide agonists that display G protein biased agonism, behaved as an unbiased agonists. AT-090 and AT-127 displayed higher NOP selectivity than Ro 65-6570 at native mouse receptors. AT-090 and AT-127 might be useful pharmacological tools for investigating the therapeutic potential of NOP partial agonists., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The natural product ferutinin was shown to act as an agonist to estrogen receptor ERα and agonist/antagonist to ERβ featuring a weak antiproliferative activity toward breast cancer cells. To enhance this activity, ferutinin analogues were synthesized by esterification of jaeschkenadiol with different acids. These compounds were assayed for their in vitro antiproliferative activity against estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) breast cancer cell lines. Among the compounds, 3c' exhibited a potent inhibitory selective activity against MCF-7 with IC50 value of 1 μM. Docking simulation of 3c' in the ligand binding domain of the ERs indicated a potential antagonism interaction with both ER subtypes. Functional assay showed that 3c' binds as an antagonist to ERα protein while ferutinin acts as an agonist., (© 2015 John Wiley & Sons A/S.)

The phenolic constituents of lahpet-so, a traditional postfermented tea of Myanmar produced under anaerobic conditions, were examined. The major polyphenols were identified to be pyrogallol and 4'-hydroxyphenyl-3-(2'',4'',6''-trihydroxyphenyl)-propan-2-ol, 3',4'-dihydroxyphenyl-3-(2'',4'',6''-trihydroxyphenyl)-propan-2-ol, and 3',4',5'-trihydroxyphenyl-3-(2'',4'',6''-trihydroxyphenyl)-propan-2-ol. The hydroxydiphenylpropan-2-ols were identical to the initial metabolites produced from green tea catechins by mammalian intestinal bacteria. In addition, an anaerobic mixed-fermentation experiment using lahpet-so and Japanese commercial green tea afforded two new catechin degradation products together with known compound bruguierol B and the above-mentioned catechin metabolites. Based on spectroscopic evidence, the structures of the new compounds were concluded to be 4-(2,5-dihydroxyhexyl)benzene-1,2-diol and (5S,8R)-6,7,8,9-tetrahydro-5-methyl-5·8-epoxy-5H-benzocycloheptene-2,3,4-triol. Interestingly, the production mechanism was deduced to be the inverse of the biosynthesis of the flavan-3-ol A ring.

Activation of brain nociceptin/orphanin FQ (NOP) receptors leads to attenuation of mu-opioid receptor (MOP receptor)-mediated antinociception. Buprenorphine, a high-affinity partial MOP receptor agonist also binds to NOP receptors with 80 nM affinity. The buprenorphine-induced inverted U-shaped dose-response curve for antinociception may be due to NOP receptor activation, given that, in the presence of the NOP receptor antagonist, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J113397), or in NOP receptor knockout mice, buprenorphine has a steeper dose-response curve and acts as a full agonist. To further explore the involvement of the direct activation of NOP receptors by buprenorphine and other compounds that activate both NOP and MOP receptors, the antinociceptive effects of 1-(1-(2,3,3alpha,4,5,6-hexahydro-1H-phenalen-1-yl)piperidin-4-yl)-indolin-2-one. (SR16435), 3-ethyl-1-(1-(4-isopropylcyclohexyl)piperidin-4-yl)-indolin-2-one (SR16507), buprenorphine, pentazocine, and morphine, compounds with varying levels of MOP and NOP receptor affinity and efficacy, were assessed in mice using the tail-flick assay. The ability of the selective NOP receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111) to potentiate antinociception induced by the above compounds was examined to investigate whether activation of NOP receptors leads to attenuation of MOP receptor-mediated antinociception. SB-612111 potentiated antinociception induced by buprenorphine and the other mixed NOP/MOP receptor agonists SR16435 and SR16507. However, SB-612111 had no effect on pentazocine or morphine antinociception, two compounds with no NOP receptor-binding affinity. These results further support the hypothesis that activation of NOP receptors can lead to attenuation of MOP receptor-mediated antinociception elicited by mixed NOP/MOP receptor compounds such as buprenorphine, SR16435, and SR16507 and that, although buprenorphine has low efficacy in vitro, it has significant NOP receptor agonist activity in vivo.

It is essential to understand the molecular processes underlying long-term memory to provide therapeutic targets of aberrant memory that produce pathological behaviour in humans. Under conditions of recall, fully-consolidated memories can undergo reconsolidation or extinction. These retrieval-mediated memory processes may rely on distinct molecular processes. The cellular mechanisms initiating the signature molecular events are not known. Using infusions of protein synthesis inhibitors, antisense oligonucleotide targeting brain-derived neurotrophic factor (BDNF) mRNA or tPA-STOP (an inhibitor of the proteolysis of BDNF protein) into the hippocampus of the awake rat, we show that acquisition and extinction of contextual fear memory depended on the increased and decreased proteolysis of proBDNF (precursor BDNF) in the hippocampus, respectively. Conditions of retrieval that are known to initiate the reconsolidation of contextual fear memory, a BDNF-independent memory process, were not correlated with altered proBDNF cleavage. Thus, the processing of BDNF was associated with the acquisition of new information and the updating of information about a salient stimulus. Furthermore, the differential requirement for the processing of proBDNF by tPA in distinct memory processes suggest that the molecular events actively engaged to support the storage and/or the successful retrieval of memory depends on the integration of ongoing experience with past learning.

Ferutinin is a potent phytoestrogen extracted from plants of the genus Ferula. The biological activity of this sesquiterpene is associated with the esterification of p-hydroxybenzoic acid with the daucane alcohol, jaeschkeanadiol. A HPLC method was developed to investigate the stability of ferutinin in acidic and basic solutions (pH 1.5 and 9.0, respectively), in buffer (pH 7.4) as well as in serial dilutions of albumin and in human plasma. The degradation of ferutinin was relatively slow at physiological pH 7.4 compared with low or high pH. Ferutinin was fully stable in human plasma as well as in albumin solution and the stability increased with albumin concentration. The binding of ferutinin to albumin was investigated by fluorescence spectroscopy. Ferutinin decreased the fluorescence of HSA and that of the only tryptophan residue located in domain IIA. As a result of the interaction between ferutinin and albumin, the binding of bilirubin decreased. The stability of ferutinin in plasma is attributable to ferutinin-albumin binding., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Metabotropic glutamate receptor 1 (mGlu(1) receptor) has been suggested to play an important role in pain transmission. In this study, the effects of a newly-synthesized mGlu(1) receptor antagonist, (R)-N-cycloheptyl-6-({[(tetrahydro-2-furyl)methyl]amino}methyl)thieno[2,3-d]pyrimidin-4-ylamine (YM-230888), were examined in a variety of rodent chronic pain models in order to characterize the potential analgesic profile of mGlu(1) receptor blockade. YM-230888 bound an allosteric site of mGlu(1) receptor with a K(i) value of 13+/-2.5 nM and inhibited mGlu(1)-mediated inositol phosphate production in rat cerebellar granule cells with an IC(50) value of 13+/-2.4 nM. It showed selectivity for mGlu(1) versus mGlu(2)-mGlu(7) subtypes and ionotropic glutamate receptors. YM-230888 recovered mechanical allodynia with an ED(50) value of 8.4 mg/kg p.o. in L5/L6 spinal nerve ligation models. It also showed antinociceptive response at doses of 10 and 30 mg/kg p.o. in streptozotocin-induced hyperalgesia models. In addition, it significantly reduced pain parameters at a dose of 30 mg/kg p.o. in complete Freund's adjuvant-induced arthritic pain models. Although YM-230888 showed no significant effect on rotarod performance time at doses of 10 or 30 mg/kg p.o., it significantly decreased it at a dose of 100 mg/kg p.o. On the other hand, YM-230888 showed no significant sedative effect in locomotor activity measurement up to 100 mg/kg p.o. These results suggest that the blockade of mGlu(1) receptors is an attractive target for analgesics. YM-230888 has potential as a new analgesic agent for the treatment of various chronic pain conditions. In addition, YM-230888 may be a useful tool for the investigation of mGlu(1) receptors.

The compound SB-612111 [(-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol] was recently identified as a selective antagonist for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). In the present study, the in vitro pharmacological profile of SB-612111 at human recombinant NOP receptors expressed in Chinese hamster ovary (CHO) cells [receptor binding, guanosine 5'-O-(3-[(35)S]thio)triphosphate (GTPgamma[(35)S]) binding, and cAMP level experiments] as well as at native NOP receptors expressed in peripheral (mouse and rat vas deferens, guinea pig ileum) and central (mouse cerebral cortex synaptosomes releasing [(3)H]5-HT) preparations was evaluated and compared with that of the standard nonpeptide antagonist (+/-)J-113397 [(+/-)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one]. SB-612111 produced a concentration-dependent displacement of [(3)H]N/OFQ binding to CHO(hNOP) cell membranes, showing higher affinity and NOP selectivity over classical opioid receptors than (+/-)J-113397. SB-612111 and (+/-)J-113397 competitively antagonized the effects of N/OFQ on GTPgamma[(35)S] binding in CHO(hNOP) cell membranes (pK(B), 9.70 and 8.71, respectively) and on cAMP accumulation in CHO(hNOP) cells (pK(B), 8.63 and 7.95, respectively), being per se inactive. In isolated peripheral tissues of mice, rats, and guinea pigs and in mouse cerebral cortex synaptosomes preloaded with [(3)H]5-HT, SB-612111 competitively antagonized the inhibitory effects of N/OFQ, with pA(2) values in the range of 8.20 to 8.50. In parallel experiments, (+/-)J-113397 was found to be 2- to 9-fold less potent than SB-612111. In the electrically stimulated tissues, 1 microM SB-612111 did not modify the effects of classical opioid receptor agonists. In conclusion, the results of the present study demonstrated that SB-612111 is among the most potent and NOP-selective nonpeptide antagonists identified to date.

The paper presents the results of studies concerning the effects of four cyclic ketones, i.e. cyclopentanone (Pen), cyclohexanone (Hex), cycloheptanone (Hep) and cyclooctanone (Oct) on metabolism of ethanol (EtOH) in vitro. The fraction S9 (supernatant with the removed mitochondria) was used obtained from homogenized rat livers. An increase in reduced nicotinamide adenine dinucleotide (NADH) was examined spectrophotometrically (at 340 nm) while the substrate disappearance and metabolite increase were determined using head-space gas chromatography. The addition of cyclic ketones statistically significantly affected a decrease in the A(340) level, particularly during co-metabolism of EtOH and Hex. The EtOH loss was significantly higher (than the loss observed during metabolism of EtOH alone) only in EtOH-Hex and EtOH-Hep systems, which may be explained by the fact that reoxidation of NADH to NAD+ is quicker in these systems than dissociation of the alcohol dehydrogenase (ADH)-NADH complex.

Norcarane is a valuable mechanistic probe for enzyme-catalyzed hydrocarbon oxidation reactions because different products or product distributions result from concerted, radical, and cation based reactions. Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b catalyzes the oxidation of norcarane to afford 3-hydroxymethylcyclohexene and 3-cycloheptenol, compounds characteristic of radical and cationic intermediates, respectively, in addition to 2- and 3-norcaranols. Past single turnover transient kinetic studies have identified several optically distinct intermediates from the catalytic cycle of the hydroxylase component of sMMO. Thus, the reaction between norcarane and key reaction intermediates can be directly monitored. The presence of norcarane increases the rate of decay of only one intermediate, the high-valent bis-mu-oxo Fe(IV)(2) cluster-containing species compound Q, showing that it is responsible for the majority of the oxidation chemistry. The observation of products from both radical and cationic intermediates from norcarane oxidation catalyzed by sMMO is consistent with a mechanism in which an initial substrate radical intermediate is formed by hydrogen atom abstraction. This intermediate then undergoes either oxygen rebound, intramolecular rearrangement followed by oxygen rebound, or loss of a second electron to yield a cationic intermediate to which OH(-) is transferred. The estimated lower limit of 20 ps for the lifetime of the putative radical intermediate is in accord with values determined from previous studies of sterically hindered sMMO probes.

Parazoanthoxanthin A is a fluorescent yellow nitrogenous pigment of the group of zoanthoxanthins, which show a broad range of biological activity. These include, among others, the ability to bind to DNA. In this study we have used a variety of spectroscopic (intrinsic fluorescence emission and UV-spectroscopy) and hydrodynamic techniques (viscometry) to characterize in more detail the binding of parazoanthoxanthin A to a variety of natural and synthetic DNA duplexes in different buffer conditions. Our results reveal the following five significant features: (i) Parazoanthoxanthin A exhibits two modes of DNA binding: One binding mode exhibits properties of intercalation, while the second binding mode is predominantly electrostatic in origin. (ii) The apparent binding "site size" for parazoanthoxanthin A near physiological salt concentration (100 mM NaCl) is in the range of 7 +/- 1 base pairs for natural genomic DNA duplexes (calf thymus and salmon testes DNA) and alternating synthetic polynucleotides (poly[d(AT)]. poly[d(AT)] and poly[d(GC)]. poly[d(GC)]). A slightly larger apparent binding site size of 9 +/- 1 bp was obtained for parazoanthoxanthin A binding to the synthetic homopolymer poly[d(A)]. poly[d(T)]. (iii) Near physiological salt concentration (100 mM NaCl) parazoanthoxanthin A binds with the same approximate binding affinity of 2-5 x 10(5) M(-1) to all DNA polymers studied. (iv) At low salt concentration, parazoanthoxanthin A preferentially binds alternating poly[d(AT)]. poly[d(AT)] and poly[d(GC)]. poly[d(GC)] host duplexes. (v) Parazoanthoxanthin A inhibits DNA polymerase in vitro., (Copyright 2001 Academic Press.)

A series of 5-hydroxy- and 5,6-dihydroxy-1,2,3,7,12,12a-hexahydrobenzo[5,6]cyclohepta[1,2,3-ij]isoquinoline derivatives (5a--e and 6a--e) were synthesized as conformationally rigid analogues of 1-benzyltetrahydroisoquinoline and evaluated for their affinity at D(1) and D(2) dopamine receptors. All compounds showed lower D(1) and D(2) affinities than dopamine. The 5-hydroxy-1-methyl-2,3,12,12a-hexahydrobenzo[5,6]cyclohepta[1,2,3-ij]isoquinoline 5a and the 5,6-dihydroxy analogue 6a showed D(2) agonist activity. This was proved by their effects on prolactin release from primary cultures of rat anterior pituitary cells. Molecular modeling studies showed that the geometric parameters (namely the distances from meta and para hydroxyl oxygens to the nitrogen and the height of nitrogen from the hydroxylated phenyl ring plane) of the dopaminergic pharmacophore embedded in our compounds have lower values in comparison with those observed in D(1) and D(2) selective ligands.

Neodictyoprolenol [(-)-(3S)-(1,5Z,8Z)-undecatrien-3-ol] and dictyoprolenol [(-)-(3S)-(1,5Z,8Z)-undecadien-3-ol]), which had been proposed as possible biosynthetic intermediates of the sex pheromones of marine brown algae such as dictyopterene B [(-)-trans-1-((1'E,3'Z)-hexadienyl)-2-vinylcyclopropane], D' [(+)-6-((1'Z)-butenyl)-1,4-cycloheptadiene] and C' [(+)-6-butyl-1,4-cycloheptadiene], were again identified in the essential oils from Dictyopteris prolifera, D. latiscula, and in D. undulata, together with the C11-related volatile compounds such as neodictyoprolene, dictyoprolene and dictyopterenes. Incubation of D. prolifela preparation with racemic neodictyoprolenol and dictyoprolenol as substrates showed (S)-enantioselective decreases of the added substrates and increases in dictyopterenes. From these results, a possible pathway to form dictyopterenes is discussed.

Enantiopure epoxy cycloheptenyl sulfones syn-7b and anti-7b are prepared in five high-yielding and stereospecific operations from 1, 3-cycloheptadiene. These substrates serve as effective precursors for cis- and trans-substituted tetrahydrofurans (12, 10) which are segments of the antineoplastic agent IKD-8344.

A concise asymmetric total synthesis of (+)-aphanamol I is described, based on the transition metal catalyzed [5 + 2] allenyl-vinylcyclopropane cycloaddition. The key cycloaddition precursor is convergently assembled from (R)-(+)-limonene and cyclopropane diester through a novel decarboxylative dehydration reaction. The metal-catalyzed [5 + 2] cycloaddition of this precursor proceeds with complete chemo, endo/exo, and diastereoselectivity in 93% yield, representing an effective general route to bicyclo[5.3.0]decane derivatives.

2-Methoxy-5-(2',3',4'-trimethoxy)-2,4,6-cycloheptatrien-1-one (MTC) is a colchicine analogue that lacks the B ring. 2-Methoxy-5-(2',4'-dimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MD) is an A-ring analogue of MTC, in which one methoxy group is replaced by a hydrogen atom. This paper describes the kinetic features of MDC binding to tubulin, and compares its behaviour with MTC to analyse the effect of the A-ring modification on the recognition process by tubulin. Binding is accompanied by a strong enhancement of MDC fluorescence and quenching of protein fluorescence. The kinetic and thermodynamic parameters were obtained from fluorescence stopped-flow measurements. The kinetics are described by a single exponential, indicating that this drug does not discriminate between the different tubulin isotypes. The observed pseudo-first-order rate constant of the fluorescence increase upon binding increases in a non-linear way, indicating that this ligand binds with a similar overall mechanism as colchicine and MTC, consisting of a fast initial binding of low affinity followed by a slower isomerisation step leading to full affinity. The K1 and k2 values for MDC at 25 degrees C were 540 +/- 65 M(-1) and 70 +/- 6 s(-1) respectively. From the temperature dependence, a reaction enthalpy change (deltaH(o)1) of the initial binding of 49 +/- 11 kJ/mol(-1) and an activation energy for the second step of 28 +/- 9 kJ/mol(-1) were calculated. Displacement experiments of bound MDC by MTC allowed the determination of a rate constant of reverse isomerisation of 0.60 +/- 0.07 s(-1) at 25 degrees C and the activation energy of 81 +/- 6 kJ/mol(-1). The overall binding constant was (6.3 +/- 0.2) x 10(4) M(-1) at 25 degrees C. Combination of these results with the kinetic parameters for association gives a full characterisation of the enthalpy pathway for the binding of MDC. The pathway of MDC is shown to differ considerably from that of MTC binding. Since its structural difference is located in ring A, this result indicates the use of ring A in the first step. The kinetics of the binding of MDC in the presence of some A-ring colchicine analogues (podophyllotoxin, 3',4',5'-trimethoxyacetophenone and N-acetylmescaline) and a C-ring analogue (tropolone methyl ether) suggest that the A and C rings are involved in the binding of MDC.

In order to examine the correlation between activity and hydrophilicity of the side chain of sodium 3-[4-(4-chlorobenzenesulfonylamino)butyl]-6-isopropylazulene -1-sulfonate (KT2-962), a non-prostanoid TXA2/PGH2 receptor antagonist, one or two hydroxyl groups were introduced into the isopropyl moiety. A series of 6-hydroxylated-isopropylazulenes were synthesized by regioselective oxidation of 6-isopropylazulenes and their in vitro and in vivo antagonistic activities were studied. Both the primary and tertiary alcohols, monohydroxylated derivatives, exhibited potent biological activities comparable to unmodified 6-isopropylazulenes both in vitro and in vivo. In contrast, the activities of 1,2- and 1,3-diols of 6-substituted derivatives, markedly decreased, but recovered by O-isopropylidenation of the dihydroxyl moiety. These findings indicate that the moderate hydrophobicity of substituent at the 6-position of the azulene ring might be required for the activity and the size of the substituent at this position, not so rigid for keeping potent biological activity.

The UV-VIS absorption data of four bacteriorhodopsin (BR) analogs formed from azulene-retinals of varying polyene chain length show that the one-bond-shortened to one-bond-lengthened analogs possess comparable opsin shift values to that of BR. A two-bond-shortened analog exhibited a much smaller opsin shift. These data, combined with those reported for the C-22 retinal analog (Tokunaga et al., 1977, Biophys. J. 19, 191-198) were analyzed by molecular modelling and computer graphics in terms of a model where conformational flexibility of the appended butyl is the controlling factor in determining ease of pigment formation and protein/substrate interaction.

Unsaturated C8 and C11 hydrocarbons act as chemical signals (chemotaxis) during sexual reproduction of many marine brown algae. One of these compounds, namely (+)-(6S)-6-(1Z-butenyl)cyclohepta-1,4-diene (= ectocarpene) is also formed as a major hydrocarbon by the flowering plant Senecio isatideus (Asteraceae). On administration of enantiospecifically labelled (8R)- or (8S)-[7,8-2H2]trideca-3,6,9-trienoic acid instead of the natural precursor dodeca-3,6,9-trienoic acid to this plant, the artificial C12 analogue of ectocarpene is formed. Mass spectroscopic analysis of the metabolites revealed this process to be enantiospecific for the C(8)-HRe atom of the precursor acid. The stereochemical course of the overall reaction is in agreement with a precise, U-shaped embedding of the trienoic acid into the active center of the enzyme(s) and Re-attack onto a hydrogen at the C(8) methylene group of this precursor. The reactive intermediate cyclizes by pi-orbital interaction between C(4) and C(6) of the acid and is accompanied by decarboxylation. The first product is a thermally unstable (1S,2R)-cis-1-(1E,3Z-hexadienyl)-2-vinylcyclopropane, which immediately rearranges to (+)-(6S)-ectocarpene (homo-Cope rearrangement). The present work provides first experimental evidence for a homo-Cope rearrangement as a naturally occurring electrocyclic reaction.

1. Azulene was rapidly metabolized in the rat and excreted in the urine as the sulphate conjugate of 1-hydroxyazulene; 45% of the dose was recovered as pure metabolite from the 0-24 h urine. 2. [1,3(-2)H2]Azulene was converted in vivo to 1-hydroxyazulene sulphate which was shown by 1H-n.m.r. spectroscopy to contain one deuterium at C-3 and essentially no detectable deuterium at C-2. This result suggests that, if a 1,2-oxide of azulene was an intermediate in the 1-hydroxylation, it rearranged with direct loss of deuterium from C-1 rather than an NIH shift to C-2.

The pharmacokinetics of bencyclane were studied in 4 healthy volunteers. In a randomized cross-over design, 35 and 70 mg of bencyclane were infused intravenously and 71 and 143 mg were given orally as a solution in water. After intravenous application bencyclane is distributed very rapidly into tissues (t1/2 approx. 7 min). The volume of distribution is about 600 l. In almost every subject the plasma levels rose again after this distribution phase. Bencyclane is eliminated mainly by metabolism. At the most 3% of the dose is excreted as unchanged drug in the urine. Total clearance is appr. 44 l/h, the elimination half-life approx. 12 h. Oral bioavailability ranges between 18 and 84%.

At 26 h after inoculation of rats with Diplococcus pneumoniae, the serum concentrations of 10 and 20 individual amino acids were lower than corresponding values observed in pair-fed controls. In contrast, only 2 of 20 serum amino acids were similarly decreased in rats inoculated with Salmonella typhimurium. Despite these serum differences, a greater accumulation of labeled non-metabolizable amino acids occurred in the livers of rats infected with S. typhimurium. These data suggested a greater increase in the flux of amino acids from muscle to liver in the rats infected with S. typhimurium as compared to those infected with D. pneumoniae. A similar increase in serum protein synthesis was observed in rats infected with D. pneumoniae or S. typhimurium. However, with the latter infection, a larger percentage of the amino acids appeared to be utilized as a source of energy in addition to their role as precursors of proteins.

The role of microtubules in 3H-labeled protein secretion in rat lacrimal glands was probed by the use of colchicine and two of its analogues that reversibly bind to tubulin. These analogues were 2-methoxy-5-(2,3,4,4'-trimethoxyphenyl)-2,4,6-cycloheptatriene-1-o ne and 2,3,4,4'-tetramethoxy-1,1'-biphenyl, the latter having been synthesized for these studies. Immunofluorescence revealed that untreated exocrine acinar cells contained an intact microtubule network, which was totally abolished by drug addition. Subsequent drug removal restored the network for the two reversibly binding drugs--more rapidly so for the biphenyl, but this was not the case with colchicine. The protein-secretory process was examined by adding the three drugs at various stages--prepulse incubation, pulse, maturation, apical storage of granules, and discharge under cholinergic stimulation. Comparison with the kinetics of microtubular network restoration, which differed for the two reversibly binding drugs, led to the conclusion that the microtubular system is critical to the maturation phase of secretion.

Pheromone secretion in living female gametes of Ectocarpus siliculosus was examined by closed-loop extraction and gas chromatography. The pheromone (ectocarpene) recovery efficiency was calibrated with synthetic ectocarpene and was found to depend strongly on the volume of the closed-loop system. Maximum extraction efficiency of ectocarpene (17.4%) was obtained with a small-volume system. In addition to the main compound ectocarpene, the pheromone bouquet of E. siliculosus contained minor amounts of hormosirene, multifidene and dictyotene, which until now are known only as sperm-attractants in other brown algae. The pheromone bouquet was identical in 4 clones of different geographic origins. Settled female gametes of Ectocarpus siliculosus continued to secrete pheromone and to attract male gametes for up to 7 h after their release with an ectocarpene secretion rate of 1 x 10(5) molecules s-1 cell-1.

The colchicine analog 2-methoxy-5-(2',3',4'-trimethoxyphenol) tropone (AC) was used as a fluorescent probe to study the binding kinetics of podophyllotoxin at high concentrations. The observed pseudo-first order rate constant showed a linear concentration dependence up to 1 mM. The bimolecular rate constant (195 M-1 s-1 at 15 degrees C) and the activation energy (57 kJ/mol) correspond perfectly with those previously determined in the submicromolar range (Cortese, F., Bhattacharyya, B., and Wolf, J. (1977) J. Biol. Chem. 252, 1134-1140). Displacement kinetics of bound AC by podophyllotoxin, allow the determination of the dissociation rate constants for AC. By studying the temperature dependence, and combining with the binding rate constants previously determined (Engelborghs, Y., and Fitzgerald, T.J. (1986) Ann. N.Y. Acad. Sci. 466, 709-717) a full characterization of the kinetic pathway is possible. This is shown to differ considerably from the pathway of colchicine binding.

The conformational effects of colchicine, podophyllotoxin, and tropolone methyl ether binding to tubulin have been studied. Conditions for the stability of the purified calf brain protein were established, and the effects of binding were examined by means of difference absorption spectroscopy, circular dichroism, fluorescence, activation of tubulin GTPase, and tubulin self-association reactions. The tubulin-colchicine complex was isolated and characterized. It displays quenched intrinsic protein fluorescence, ligand fluorescence, and GTPase activity, probably accompanied by minor perturbations in the secondary structure. The conformation of the tubulin-colchicine complex appears to be nonidentical with that of the unliganded protein. Podophyllotoxin was not found to induce any of the mentioned changes. This ligand seems to bind through a hydrophobic interaction of its trimethoxybenzene ring with tubulin, as does colchicine. Binding of tropolone methyl ether, which is the analogue of the other part of the colchicine molecule that binds to tubulin, produced effects consistent with a ligand-linked conformational change. The small perturbation by tropolone methyl ether of the circular dichroism spectrum of tubulin resembles changes induced by colchicine.

The stabilized carbonium ion salt, tropylium tetrafluoroborate, was oxidized to tropone (cycloheptatrienone) by rabbit liver aldehyde oxidase but not by the closely related molybdenum hydroxylase, xanthine oxidase. The tropylium cation is an aromatic hydrocarbon which lacks the aldehyde, imine, or iminium functional groups present in other substrates of aldehyde oxidase. The unique structural features of the tropylium ion should make it a useful tool for mechanistic studies of aldehyde oxidase.

The thermodynamics and kinetics of the binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl) tropone (termed AC because it lacks the B-ring of colchicine) have been characterized by fluorescence techniques. The fluorescence of AC is weak in aqueous solution and is enhanced 250-fold upon binding to tubulin. The following thermodynamic values were obtained for the interaction at 37 degrees C: K = 3.5 X 10(5) M-1; delta G0 = -7.9 kcal/mol; delta H0 = -6.8 kcal/mol; delta S0 = 3.6 entropy units. The AC-tubulin complex is 1-2 kcal/mol less stable than the colchicine-tubulin complex. The change in fluorescence of AC was employed to measure the kinetics of the association process, and quenching of protein fluorescence was used to measure both association and dissociation. The association process, like that of colchicine, could be resolved into a major fast phase and a minor slow phase. The apparent second order rate constant for the fast phase was found to be 5.2 X 10(4) M-1 S-1 at 37 degrees C, and the activation energy was 13 kcal/mol. This activation energy is 7-11 kcal/mol less than that for the binding of colchicine to tubulin. The difference in activation energies can most easily be rationalized by a mechanism involving a tubulin-induced conformational change in the ligand ( Detrich , H. W., III, Williams, R. C., Jr., Macdonald, T. L., Wilson, L., and Puett , D. (1981) Biochemistry 20, 5999-6005). Such a change would be expected to have a small activation energy in AC because it possesses a freely rotating single bond in place of the B-ring of colchicine.

1. Two metabolites, isolated from the urine of rats dosed with bencyclane fumarate, were characterized as cis-1-benzyl-1-(3-N,N-dimethylaminopropoxy)-4-hydroxycycloheptane (metabolite I) and 1-benzyl-1-(3-N,N-dimethylaminopropoxy)-4-oxocycloheptane (metabolite II). 2. Bencyclane and the two metabolites were determined in the urine of rats and volunteers by g.l.c. Metabolite I was a major metabolite in men, being excreted in urine to the extent of 23.5% dose in the first 24 h.