Protein kinase C modulators bearing dicarba-CLOSO-dodecaborane as a hydrophobic pharmacophore.

The size and position of a hydrophobic moiety on a benzolactam skeleton, which reproduces the active conformation and biological activity of teleocidins, play an important role in the appearance of the activity. We have designed and synthesized benzolactams bearing dicarba-closo-dodecaborane. These compounds showed potent binding affinity to protein kinase C, providing a further example of the application of carborane as the hydrophobic pharmacophore of biologically active molecules.     

Dehydrocostuslactone and plant growth activity of derived guaianolides

The stereostructures of two new guaianolides, isodehydrocostuslactone and isozaluzanin C, isolated previously from Saussurea lappa, have been confirmed by their correlation with dehydrocostuslactone. Twenty new derivatives have been synthesized from these guaianolides and these have been tested as plant growth regulators. The conjugated lactones which have an exocyclic methylene group at C-4 conjugated with a C-3 ketone, show distinct enhancement in their root-forming potential, as compared with their 3-deoxy derivatives. Of further significance is the fact that these ketones display maximum activity only at lower concentrations. Other compounds show the expected structure-biological activity relationships displayed in general by guaianolides. However, the presence of an epoxide at the C-3, C-4 position does not affect the biological activity, which is indeed the case when the epoxide group occupies the C-4, C-14 position in guaianolides. The major biological parameter studied was rooting in-stem cuttings of Phaseolus aureus.     

A common pharmacophore for Taxol and the epothilones based on the biological activity of a taxane molecule lacking a C-13 side chain

Extensive structure-activity studies done with Taxol have identified the side chain at C-13 as one of the requirements for biological activity. Baccatin III, an analogue of Taxol lacking the C-13 side chain, has none of the biological characteristics of Taxol. Since 2-m-azido Taxol, a Taxol derivative with a m-azido substituent in the C-2 benzoyl ring, has greater activity than Taxol, we questioned whether 2-m-azido baccatin III might be active. 2-m-Azido baccatin III inhibited the proliferation of human cancer cells at nanomolar concentrations, blocked cells at mitosis, and reorganized the interphase microtubules into distinct bundles, a typical morphological change induced by Taxol. In contrast to 2-m-azido baccatin III, 2-p-azido baccatin III was similar to baccatin III, having no Taxol-like activity, further indicating the specificity and significance of the 2-meta position substituent. Molecular modeling studies done with the C-2 benzoyl ring of Taxol indicated that it fits into a pocket formed by His227 and Asp224 on beta-tubulin and that the 2-m-azido, in contrast to the 2-p-azido substituent, is capable of enhancing the interaction between the benzoyl group and the side chain of Asp224. The observation that the C-13 side chain is not an absolute requirement for biological activity in a taxane molecule has enabled the development of a new common pharmacophore model between Taxol and the epothilones.     

Cytostatic effects of structurally different ginsenosides on yeast cells with altered sterol biosynthesis and transport

Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene glycosides show membranolytic activity, and, therefore, are considered to be promising antimicrobial drugs. However, the interrelation between their structure, biological activities, and target membrane lipid composition remains elusive. Here we studied the antifungal effects of four Panax triterpene glycosides (ginsenosides) with sugar moieties at the C-3 (ginsenosides Rg3, Rh2), C-20 (compound K), and both (ginsenoside F2) positions in Saccharomyces cerevisiae mutants with altered sterol plasma membrane composition. We observed reduced cytostatic activity of the Rg3 and compound K in the UPC2-1 strain with high membrane sterol content. Moreover, LAM gene deletion reduced yeast resistance to Rg3 and digitonin, another saponin with glycosylated aglycon in the C-3 position. LAM genes encode plasma membrane-anchored StARkin superfamily-member sterol transporters. We also showed that the deletion of the ERG6 gene that inhibits ergosterol biosynthesis at the stage of zymosterol increased the cytostatic effects of Rg3 and Rh2, but not the other two tested ginsenosides. At the same time, in silico simulation revealed that the substitution of ergosterol with zymosterol in the membrane changes the spatial orientation of Rg3 and Rh2 in the membranes. These results imply that the plasma membrane sterol composition defines its interaction with triterpene glycoside depending on their glycoside group position. Our results also suggest that the biological role of membrane-anchored StARkin family protein is to protect eukaryotic cells from triterpenes glycosylated at the C-3 position.     

Synthesis,anticancer activity,and SAR analyses of compounds containing the 5:7-fused 4,6,8-triaminoimidazo[4,5-e][1,3]diazepine ring system

Described herein are our limited structure–activity relationship (SAR) studies on a 5:7-fused heterocycle (1), containing the 4,6,8-triaminoimidazo[4,5-e][1,3]diazepine ring system, whose synthesis and potent broad-spectrum anticancer activity we reported a few years ago. Our SAR efforts in this study are mainly focused on judicial attachment of substituents at N-1 and N⁶-positions of the heterocyclic ring. Our results suggest that there is some subtle correlation between the substituents attached at the N-1 position and those attached at the N⁶-position of the heterocycle. It is likely that there is a common hydrophobic binding pocket on the target protein that is occupied by the substituents attached at the N-1 and N⁶-positions of the heterocyclic ligand. This pocket appears to be large enough to hold either a C-18 alkyl chain of N⁶ and no attachment at N-1, or a combined C-10 at N⁶ and a CH₂Ph at N-1. Any alkyl chain shorter or longer than C-10 at N⁶ with a CH₂Ph attached at N-1, would result in decrease of biological activity.     

Studies of genotoxicity and mutagenicity of nitroimidazoles: demystifying this critical relationship with the nitro group

Nitroimidazoles exhibit high microbicidal activity, but mutagenic, genotoxic and cytotoxic properties have been attributed to the presence of the nitro group. However, we synthesised nitroimidazoles with activity against the trypomastigotes of Trypanosoma cruzi, but that were not genotoxic. Herein, nitroimidazoles (11-19) bearing different substituent groups were investigated for their potential induction of genotoxicity (comet assay) and mutagenicity (Salmonella/Microsome assay) and the correlations of these effects with their trypanocidal effect and with megazol were investigated. The compounds were designed to analyse the role played by the position of the nitro group in the imidazole nucleus (C-4 or C-5) and the presence of oxidisable groups at N-1 as an anion receptor group and the role of a methyl group at C-2. Nitroimidazoles bearing NO2 at C-4 and CH3 at C-2 were not genotoxic compared to those bearing NO₂ at C-5. However, when there was a CH3 at C-2, the position of the NO2 group had no influence on the genotoxic activity. Fluorinated compounds exhibited higher genotoxicity regardless of the presence of CH3 at C-2 or NO2 at C-4 or C-5. However, in compounds 11 (2-CH3; 4-NO2; N-CH2OHCH2Cl) and 12 (2-CH3; 4-NO2; N-CH2OHCH2F), the fluorine atom had no influence on genotoxicity. This study contributes to the future search for new and safer prototypes and provide.     

Substituted 2-(3′,4′,5′-trimethoxybenzoyl)-benzo[b]thiophene derivatives as potent tubulin polymerization inhibitors

The central role of microtubules in cell division and mitosis makes them a particularly important target for anticancer agents. On our early publication, we found that a series of 2-(3′,4′,5′-trimethoxybenzoyl)-3-aminobenzo[b]thiophenes exhibited strong antiproliferative activity in the submicromolar range and significantly arrested cells in the G2–M phase of the cell cycle and induced apoptosis.In order to investigate the importance of the amino group at the 3-position of the benzo[b]thiophene skeleton, the corresponding 3-unsubstituted and methyl derivatives were prepared. A novel series of inhibitors of tubulin polymerization, based on the 2-(3,4,5-trimethoxybenzoyl)-benzo[b]thiophene molecular skeleton with a methoxy substituent at the C-4, C-5, C-6 or C-7 position on the benzene ring, was evaluated for antiproliferative activity against a panel of five cancer cell lines, for inhibition of tubulin polymerization and for cell cycle effects. Replacing the methyl group at the C-3 position resulted in increased activity compared with the corresponding 3-unsubstituted counterpart. The structure–activity relationship established that the best activities were obtained with the methoxy group placed at the C-4, C-6 or C-7 position. Most of these compounds exhibited good growth inhibition activity and arrest K562 cells in the G2–M phase via microtubule depolymerization.     

Specificities of Calreticulin Transacetylase to acetoxy derivatives of 3-alkyl-4-methylcoumarins: Effect on the activation of nitric oxide synthase

Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl groups from polyphenolic acetates (PAs) to the receptor proteins and modulates their biological activities. CRTAase was conveniently assayed by the irreversible inhibition of cytosolic glutathione S-transferase (GST) by the model acetoxycoumarin, 7,8-diacetoxy-4-methylcoumarin (DAMC). We have studied earlier, the influence of acetoxy groups on the benzenoid ring, the effect of reduction of double bond at C-3 and C-4 position, the effect of methyl/phenyl group at C-4, and the influence of position of carbonyl group with respect to oxygen heteroatom in the benzopyran nucleus, for the catalytic activity of CRTAase. In this communication, we have extended our previous work; wherein we studied the influence of an alkyl group (ethyl, hexyl and decyl) at the C-3 position of the acetoxy coumarins on the CRTAase activity. The substitution at C-3 position of coumarin nucleus resulted in the reduction of CRTAase activity and related effects. Accordingly the formation of NO in platelets by C-3 alkyl substituted acetoxy coumarins was found to be much less compared to the unsubstituted analogs. In addition the alkyl substitution at C-3 position exhibited the tendency to form radicals other than NO.     

8-Hydroxyguanosine and 8-methoxyguanosine possess immunostimulating activity for B lymphocytes

The present paper extends previous observations of Goodman and Weigle (M.G. Goodman and W.O. Weigle, J. Immunol. 128, 2399, 1982) and describes the activation of B lymphocytes by a number of C-8-substituted guanine ribonucleosides. 8-Hydroxyguanosine stimulates both proliferation and differentiation of murine B cells while 8-methoxyguanosine stimulates only differentiation and 8-aminoguanosine has no discernible effect on B-cell activation. The former two compounds also increase the magnitude of the antibody response to the type 2 antigen trinitrophenyl-AECM-Ficoll. These data demonstrate that guanosine, which is itself inhibitory to murine B cells, is converted into an immunostimulatory molecule after substitution at its C-8 position with methoxy or hydroxy groups and the bromo or mercapto group not essential for conferring biological activity to this nucleoside. However, our data also suggest that substitution of different groups at the C-8 position does influence the biological activity of this molecule.     

Synthesis and AMPA receptor antagonistic activity of a novel 7-imidazolyl-6-trifluoromethyl quinoxalinecarboxylic acid with a substituted phenyl group and improved its good physicochemical properties by introduced CF3 group

We describe the synthesis, physicochemical, and biological properties of a novel series of 7-imidazolyl-6-trifluoromethyl quinoxalinecarboxylic acids with a substituted phenyl group attached through a urethane linkage at the C-7 position. We found that the introduction of trifluoromethyl group at the C-6 position brought about good biological activity and physicochemical properties. Among them, compound 9k (KRP-199), which has a 4-carboxyphenyl group, was found to possess high potency and selectivity for the AMPA-R in vitro and to exhibit good neuroprotective effects in vivo. Furthermore, the compound showed good physicochemical properties such as stability to light and good solubility in aqueous solutions.     

Inhibitory effect of methyl jasmonate and its related compounds on kinetin-induced retardation of oat leaf senescence

The senescence-promoting activities of methyl jasmonate and its related compounds were compared with respect to structure-activity relationships. The activities were assayed by using oat ( A vena saliva L. cv. Victor) leaf segments in the presence of 2 μg/ml kinetin. Dextrorotatory methyl jasmonate prepared from an authentic sample of the racemate mixture was less active than the naturally occurring levorotatory form especially at its low concentrations (0.1 to 2.5 μg/ml). The activity of jasmonic acid, the free acid form of methyl jasmonate, was much less than the methyl ester, and this relationship was true for the other compounds tested. The reduction of the unsaturated bond in the substituent at the C-2 position and the keto group at the C-3 position greatly reduced the activity. The length of the n -alkyl substituents at the C-2 position had also a significant effect on the activity. From these results, it is concluded that the important functional groups for the high senescence-promoting activity of the methyl jasmonate related compounds are the methyl acetate substituent at the C-l position, the 2' cis -pentenyl or n -pentyl group at the C-2, position and the keto group at the C-3 position in methyl jasmonate.     

Binding of N-dansylgalactosamine to winged-bean tuber lectin: studies by fluorescence quenching titrations

The winged-bean tuber lectin binds to N-dansyl(5-dimethylaminonaphthalene-1-sulphonic acid)galactosamine, leading to a 12.5-fold increase in dansyl fluorescence with a concomitant 25 nm blue-shift in the emission maximum. The enhancement of fluorescence intensity was completely reversed by the addition of methyl alpha-galactopyranoside. The lectin has two binding sites per molecule for this fluorescent sugar and an association constant of 2.59.10(5) M-1 at 25 degrees C. The binding of N-dansylgalactosamine to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent with alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are also critical for sugar binding to this lectin.     

Design, efficient synthesis, and anti-HIV activity of 4'-C-cyano- and 4'-C-ethynyl-2'-deoxy purine nucleosides

Some 4'-C-ethynyl-2'-deoxy purine nucleosides showed the most potent anti-HIV activity among the series of 4'-C-substituted 2'-deoxynucleosides whose 4'-C-substituents were methyl, ethyl, ethynyl and so on. Our hypothesis is that the smaller the substituent at the C-4' position they have, the more acceptable biological activity they show. Thus, 4'-C-cyano-2'-deoxy purine nucleosides, whose substituent is smaller than the ethynyl group, will have more potent antiviral activity. To prove our hypothesis, we planned to develop an efficient synthesis of 4'-C-cyano-2'-deoxy purine nucleosides (4'-CNdNs) and 4'-C-ethynyl-2'-deoxy purine nucleosides (4'-EdNs). Consequently, we succeeded in developing an efficient synthesis of six 2'-deoxy purine nucleosides bearing either a cyano or an ethynyl group at the C-4' position of the sugar moiety from 2'-deoxyadenosine and 2,6-diaminopurine 2'-deoxyriboside. Unfortunately, 4'-C-cyano derivatives showed lower activity against HIV-1, and two 4'-C-ethynyl derivatives suggested high toxicity in vivo.     

Syntheses and biological activity of C-3'-difluoromethyl-taxoids

A series of new taxoids bearing difluoromethyl group at the C-3' position and modifications at the C-10 and C-14 positions has been synthesized and their biological activities studied. The in vitro cytotoxicity assay results indicate that these newly developed taxoids exhibit comparable to several times better activity against drug-sensitive cell line LCC6-WT, and 40-70 times better activity against the corresponding drug-resistant cancer cell line LCC6-MDR as compared to that of paclitaxel. Apoptosis analysis has revealed the exceptional activity of SB-T-12843 (1e) in inducing apoptosis in both MDR-bearing and MDR-negative cancer cells.     

11-(Ethylthio)undecanoic acid. A myristic acid analogue of altered hydrophobicity which is functional for peptide N-myristoylation with wheat germ and yeast acyltransferase

The covalent attachment of myristic acid to the NH2-terminal glycine residue of proteins is catalyzed by the enzyme myristoyl CoA:protein N-myristoyltransferase (NMT). Using synthetic octapeptide substrates we have identified and characterized an NMT activity in wheat germ lysates used for cell-free translation of exogenous mRNAs. C-12 and C-14 fatty acids are efficiently transferred to the peptides by this plant NMT, but C-10 and C-16 fatty acids are not. Glycine is required as the NH2-terminal residue: peptides with an NH2-terminal alanine were not substrates. Peptides with proline, aspartic acid, or tyrosine residues adjacent to the NH2-terminal glycine were also not myristoylated. Serine in the fifth position reduced the peptide's Km up to 4000-fold. We have chemically synthesized a sulfur analogue of myristate, 11-(ethylthio)undecanoic acid. Its CoA ester is as good a substrate as myristoyl-CoA for both wheat germ and yeast NMT. Peptides linked to 11-(ethylthio)undecanoic acid are less hydrophobic than the corresponding myristoylpeptides. 11-(Ethylthio)-undecanoic acid may, therefore, help define the role of myristic acid in targeting of acyl proteins within cells.     

Design,synthesis and evaluation of 2,4-disubstituted pyrimidines as cholinesterase inhibitors

A group of 2,4-disubstituted pyrimidine derivatives (7a–e, 8a–e and 9a–d) that possess a variety of C-2 aliphatic five- and six-membered heterocycloalkyl ring in conjunction with a C-4 arylalkylamino substituent were designed, synthesized and evaluated as cholinesterase (ChE) inhibitors. The steric and electronic properties at C-2 and C-4 positions of the pyrimidine ring were varied to investigate their effect on ChE inhibitory potency and selectivity. The structure–activity relationship (SAR) studies identified N-benzyl-2-thiomorpholinopyrimidin-4-amine (7c) as the most potent cholinesterase inhibitor (ChEI) with an IC₅₀ = 0.33 μM (acetylcholinesterase, AChE) and 2.30 μM (butyrylcholinesterase, BuChE). The molecular modeling studies indicate that within the AChE active site, the C-2 thiomorpholine substituent was oriented toward the cationic active site region (Trp84 and Phe330) whereas within the BuChE active site, it was oriented toward a hydrophobic region closer to the active site gorge entrance (Ala277). Accordingly, steric and electronic properties at the C-2 position of the pyrimidine ring play a critical role in ChE inhibition.     

Substrate recognition at the cytoplasmic and extracellular binding site of the lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus catalyzes the uptake of lactose in an exchange reaction with intracellularly formed galactose. The interactions between the substrate and the cytoplasmic and extracellular binding site of LacS have been characterized by assaying binding and transport of a range of sugars in proteoliposomes, in which the purified protein was reconstituted with a unidirectional orientation. Specificity for galactoside binding is given by the spatial configuration of the C-2, C-3, C-4, and C-6 hydroxyl groups of the galactose moiety. Except for a C-4 methoxy substitution, replacement of the hydroxyl groups for bulkier groups is not tolerated at these positions. Large hydrophobic or hydrophilic substitutions on the galactose C-1 alpha or beta position did not impair transport. In fact, the hydrophobic groups increased the binding affinity but decreased transport rates compared with galactose. Binding and transport characteristics of deoxygalactosides from either side of the membrane showed that the cytoplasmic and extracellular binding site interact differently with galactose. Compared with galactose, the IC(50) values for 2-deoxy- and 6-deoxygalactose at the cytoplasmic binding site were increased 150- and 20-fold, respectively, whereas they were the same at the extracellular binding site. From these and other experiments, we conclude that the binding sites and translocation pathway of LacS are spacious along the C-1 to C-4 axis of the galactose moiety and are restricted along the C-2 to C-6 axis. The differences in affinity at the cytoplasmic and extracellular binding site ensure that the transport via LacS is highly asymmetrical for the two opposing directions of translocation.     

Synthesis and biological evaluation of novel 1 beta-methylcarbapenems with isothiazoloethenyl side chains

The synthesis of novel 1 beta-methylcarbapenems 1a,b bearing isothiazoloethenyl moieties at C-5 position of pyrrolidine ring and their biological evaluation are described. Both compounds showed potent and well-balanced antibacterial activity as well as high stability to DHP-I. Especially, 5-isothiazole derivative 1a exhibited excellent DHP-I stability and advanced pharmacokinetics profiles, compared to 5-isoxazole derivative 2, imipenem, and meropenem.     

Chemical synthesis,microbial transformation and biological evaluation of tetrahydroprotoberberines as dopamine D1/D2 receptor ligands

Dopamine D1/D2 receptors are important targets for drug discovery in the treatment of central nervous system diseases. To discover new and potential D1/D2 ligands, 17 derivatives of tetrahydroprotoberberine (THPB) with various substituents were prepared by chemical synthesis or microbial transformation using Streptomyces griseus ATCC 13273. Their functional activities on D1 and D2 receptors were determined by cAMP assay and calcium flux assay. Seven compounds showed high activity on D1/D2 receptor with low IC₅₀ values less than 1?µM. Especially, top compound 5 showed strong antagonistic activity on both D1 and D2 receptor with an IC₅₀ of 0.391 and 0.0757?µM, respectively. Five compounds displayed selective antagonistic activity on D1 and D2 receptor. The SAR studies revealed that (1) the hydroxyl group at C-9 position plays an important role in keeping a good activity and small or fewer substituents on ring D of THPBs may also stimulate their effects, (2) the absence of substituents at C-9 position tends to be more selective for D2 receptor, and (3) hydroxyl substitution at C-2 position and the substitution at C-9 position may facilitate the conversion of D1 receptor from antagonist to agonist. Molecular docking simulations found that Asp 103/Asp 114, Ser 107/Cys 118, and Trp 285/ Trp 386 of D1/ D2 receptors are the key residues, which have strong interactions with the active D1/D2 compounds and may influence their functional profiles.