Low stimulation of NADH oxidation and oxygen consumption by 5-iminodaunorubicin and its derivatives

NADH oxidation and oxygen consumption mediated by 5-iminodaunorubicin and its derivatives have been studied. Experiments were carried out using two enzyme system: microsomes isolated from rat liver and cytochrome c-reductase. All 5-iminoanthracyclines examined were relatively poor electron transfer mediators. In addition, 5-imino-derivatives of daunorubicin modified at sugar moiety were less effective in stimulating NADH oxidation and oxygen radical production than 5-iminodaunorubicin itself. This may be due to the additive effect of 5-imino structure and the presence of bulky substituents at sugar moiety, which might make the compounds poor enzyme substrates.     

Synthesis and biological evaluation of ether bridged bicyclic iminosugar derivatives

Bicyclic iminosugar derivatives with an ether bridge bearing different substituents on C-2 and the nitrogen atom have been synthesized from a C-glycoside bearing an isopropylidene acetal. The activities of these compounds were investigated against several glycosidase enzymes and showed moderate inhibition and activation.     

Acetaminophen stimulates the peroxidative metabolism of anthracyclines

Acetaminophen, a common analgesic and antipyretic drug, is frequently administered to individuals undergoing anthracycline chemotherapy. Here, the effect of acetaminophen on the metabolism of daunorubicin and doxorubicin by isolated enzymes lactoperoxidase and myeloperoxidase, and by myeloperoxidase-containing human leukemia HL-60 cells was investigated using spectrophotometric and EPR techniques. We report that at pharmacological concentrations acetaminophen strongly stimulates oxidation of the anthracyclines by lactoperoxidase and myeloperoxidase systems, which results in irreversibly altered (colorless) products. The initial rate and efficacy of daunorubicin oxidation depends on pH. While at pH approximately 7 the oxidation is rapid and extensive, almost no oxidation occurs at pH approximately 5. In the absence of daunorubicin, oxidation of acetaminophen by lactoperoxidase/hydrogen peroxide is only weakly dependent on pH, however, at pH 7.4 it strongly depends on [daunorubicin]. Ascorbate and reduced glutathione strongly inhibited oxidation of anthracyclines by lactoperoxidase and HL-60 systems. Using EPR, a daunorubicin-derived radical was detected in a daunorubicin/acetaminophen/peroxidase/hydrogen peroxide system as a narrow single line (0.175 mT) with g = 2.0047. When daunorubicin was omitted, only an acetaminophen-melanin EPR signal was detected (g = 2.0043, line width approximately 0.5 mT). Similar results were obtained with doxorubicin. We suggest that the stimulation by acetaminophen is primarily due to its preferential oxidation by peroxidases to the corresponding phenoxyl radical, which subsequently reacts with daunorubicin (doxorubicin). Because biological properties of oxidatively transformed anthracyclines will certainly be different from those of their parent compounds, the possible acetaminophen-enhanced degradation of the anthracyclines in vivo is likely to interfere with anticancer and/or cardiotoxic activities of these agents.     

Derivatives of antineoplastic antibiotics of the daunorubicin series containing methylurea or nitrosomethylurea residues

Derivatives of antitumour anthracycline antibiotics containing N-methylurea moiety in the carbohydrate ring were obtained by the interaction of methyl isocyanate with daunorubicin, doxorubicin, carminomycin and daunorubicin derivatives, substituted at C-13 or C-14 positions. N-Nitrosation of these compounds yielded modified anthracycline antibiotics containing the N-methyl-N-nitrosourea substituent at C-3' position. Alkaline degradation of these derivatives produced, through corresponding isocyanates cyclic 3'-N,4'-carbonylderivatives. In these anthracycline derivatives with sugar cycles conjugated with oxazoline-2-ones the predominant conformations of sugar ring has changed from 1C4 to 4C1, 2,5B, or B0,3 (shown by 1H NMR spectroscopy). It was demonstrated, both in vitro and in vivo, that introduction of methylurea or cytotoxic methylnitrosourea moieties does not potentiate antimicrobial, cytotoxic or antitumour properties of these compounds.     

N-substituted derivatives of 4-piperidinyl benzilate: affinities for brain muscarinic acetylcholine receptors

N-Substituted derivatives of 4-piperidinyl benzilate were synthesized and their affinities for central muscarinic cholinergic receptors determined using an in vitro radioligand binding assay. 4-Piperidinyl benzilate exhibited a Ki value of 2.0 nM. N-Substitution with a methyl or an ethyl group increased the affinity to 0.2 nM, whereas substitution with a n-propyl or isopropyl group decreased the binding affinity over 100 fold. Compounds with aralkyl substitutions at the nitrogen atom of piperidinyl benzilate were also synthesized and evaluated. The Ki values (nM) obtained for these compounds were: benzyl, 0.2; p-nitrobenzyl, 13.0; p-fluorobenzyl, 3.0; phenethyl, 8.0; p-nitrophenethyl, 15.0. These data suggest that a binding region near the piperidinyl nitrogen may tolerate bulky aromatic substitutions (e.g., benzyl or phenethyl) as well or better than straight chain or branched alkyl substitutions (e.g., n-propyl or isopropyl).     

DNA cleaving modes in minor groove of DNA helix by esperamicin and calicheamicin antitumor antibiotics.

This study examines and compares DNA cleavage modes by several esperamicin derivatives and calicheamicin. We found that the deoxyfucose-anthranilate moiety is a key factor to determine their DNA cutting modes. Probably, the bulky moiety hinders the abstraction of hydrogen atom from deoxyribose by the C-1 carbon radical of phenylene diradical. On the basis of the experimental results, detailed DNA cleaving modes in DNA minor groove by esperamicin and calicheamicin have been discussed.     

Synthesis of 2-amido, 2-amino, and 2-azido derivatives of the nitrogen analogue of the naturally occurring glycosidase inhibitor salacinol and their inhibitory activities against O-GlcNAcase and NagZ enzymes

Seven 2-substituted derivatives of the nitrogen analogue of salacinol, a naturally occurring glycosidase inhibitor, were synthesized for structure-activity studies with hexosaminidase enzymes. The target zwitterionic compounds were synthesized by means of nucleophilic attack of the 2-azido-1,4-dideoxy-1,4-imino-D-arabinitol at the least hindered carbon atom of 2,4-O-benzylidene-L-erythritol-1,3-cyclic sulfate. Hydrogenation of the azido zwitterionic compound in methanol resulted in the reduction of the azide and subsequent methylation of the resulting amine in one pot. A similar reaction, with ethanol as the solvent, gave the N-ethyl derivative. The 2-amino analogues were finally obtained by the reduction of the azide function using triphenylphosphine. Acylation of the amine using acetic, propionic, or valeric anhydride afforded the corresponding 2-amido derivatives. Deprotection of the acylated, coupled products using 80% trifluoroacetic acid proceeded smoothly. Unlike their sulfonium ion counterparts, these compounds were stable and did not undergo ring opening. We also report the synthesis of the parent nitrogen heterocycles, N-Boc-1,2,4-trideoxy-2-amino-1,4-imino-D-arabinitol, and 1,2,4-trideoxy-2-acetamido-1,4-imino-D-arabinitol and its corresponding N-Boc protected compound. The 2-substituted analogues and the parent iminoalditol showed marginal activity (<33% at 250 microM) against human O-GlcNAcase and Vibrio cholerae NagZ enzymes.     

Use of dextrans as long and hydrophilic spacer arms to improve the performance of immobilized proteins acting on macromolecules

New dextran-agarose supports, suitable for covalent immobilization of enzymes and proteins acting on macromolecular substrates, were prepared. The thick internal fibers of agarose gels were covered by a low-density layer of long, flexible, hydrophilic, and inert dextran molecules. Rennin and protein A were immobilized on these novel supports and the resulting derivatives exhibited a very high capacity for biological recognition of soluble macromolecular substrates. Caseinolytic activity of this immobilized enzyme was 15-fold higher than activity of directly immobilized rennin, through short spacer arms, on agarose gels. Similarly, the new derivatives of immobilized protein A were able to adsorb up to 2 molecules of immunoglobulin per each molecule of immobilized protein A. When the immobilized proteins were secluded away from the support surface by using these new long and hydrophilic spacer arms, they exhibit minimal steric hindrances that could be promoted by the proximity of the support surface.     

Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating arylsulfonylureido and 5-dibenzo-suberenyl/suberyl moieties

Novel matrix metalloproteinase (MMP)/bacterial collagenase inhibitors are reported, considering the sulfonylated amino acid hydroxamates as lead molecules. A series of compounds was prepared by reaction of arylsulfonyl isocyanates with N-(5H-dibenzo[a,d]cyclohepten-5-yl)- and N-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl) methyl glycocolate, respectively, followed by the conversion of the COOMe to the carboxylate/hydroxamate moieties. The corresponding derivatives with methylene and ethylene spacers between the polycyclic moiety and the amino acid functionality were also obtained by related synthetic strategies. These new compounds were assayed as inhibitors of MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from Clostridium histolyticum (ChC). Some of the new derivatives reported here proved to be powerful inhibitors of the four MMPs mentioned above and of ChC, with activities in the low nanomolar range for some of the target enzymes, depending on the substitution pattern at the sulfonylureido moiety and on the length of the spacer through which the dibenzosuberenyl/suberyl group is connected with the rest of the molecule. Several of these inhibitors also showed selectivity for the deep pocket enzymes (MMP-2, MMP-8 and MMP-9) over the shallow pocket ones MMP-1 and ChC.     

Novel benzoyl nitrogen mustard derivatives of pyrazole analogues of distamycin A: synthesis and antileukemic activity

The design and synthesis of novel benzoic acid mustard (BAM) derivatives of distamycin A bearing one or more pyrazole rings replacing the pyrrole rings of the latter are described. In vitro and in vivo activities against L1210 leukemia are reported and discussed. Some of these compounds show an activity profile comparable to tallimustine 1. All the compounds bearing the pyrazole ring close to the BAM moiety show reduced cytotoxicity in comparison to derivatives characterized by the BAM linked to a pyrrole: the same effect has not been observed when occurring at the amidine terminus of the oligopeptidic frame.     

Specificity of thrombin: I. Esterolytic properties of thrombin,plasma, trypsin,and chymotrypsin with Nβ-substituted guanidino derivatives of p-nitrophenyl-p′-guanidinobenzoate

Thrombin exhibits a restricted specificity, relative to plasmin, trypsin, and chymotrypsin, for a series of derivatives of the titrant substrate p-nitrophenyl-p′-guanidinobenzoate (NPGB). Substitution on the beta guanidino nitrogen of NPGB with an n-butyl, n-hexyl, cyclo-hexyl, or benzyl residue does not prevent the esterolytic cleavage of these derivatives but does markedly alter their substrate properties with the four enzymes investigated.All four enzymes cleave NPGB at equivalent concentrations by releasing p-nitrophenol as pre-steady-state burst reactions followed by its steady-state production. Both chymotrypsin and trypsin similarly display burst reactions with the derivatives at corresponding concentrations. The acyl-enzyme intermediates formed with chymotrypsin, however, are more stable for the derivatives than for NPGB, and those formed with trypsin are less stable. In contrast, plasmin and thrombin exhibit incomplete burst reactions with the derivatives at these concentrations. Except for the cyclo-hexyl derivative, with which plasmin does not react, the derivatives relative to NPGB were cleaved faster by plasmin than by thrombin. These cleavages with thrombin, moreover, were competitively inhibited by benzamidine. Kinetic data obtained for thrombin further indicated that the substituent groups of derivatives hindered the initial formation of enzyme-substrate complexes. These results suggest that thrombin and, most likely, plasmin have restricted primary binding-site regions for small molecule substrates which do not readily accommodate bulky substituent groups.In addition, increasing concentrations of glycerol were found to greatly alter the esterolytic properties of thrombin for the compounds studied. This effect was demonstrated by increased deacylation rates with NPGB and by decreased cleavage rates with the n-butyl derivative.     

Inhibition of thioredoxin reductase but not of glutathione reductase by the major classes of alkylating and platinum-containing anticancer compounds

Mammalian thioredoxin reductase (TrxR) is important for cell proliferation, antioxidant defense, and redox signaling. Together with glutathione reductase (GR) it is the main enzyme providing reducing equivalents to many cellular processes. GR and TrxR are flavoproteins of the same enzyme family, but only the latter is a selenoprotein. With the active site containing selenocysteine, TrxR may catalyze reduction of a wide range of substrates, but can at the same time easily be targeted by electrophilic compounds due to the extraordinarily high reactivity of a selenolate moiety. Here we addressed the inhibition of the enzyme by major anticancer alkylating agents and platinum-containing compounds and we compared it to that of GR. We confirmed prior studies suggesting that the nitrosourea carmustine can inhibit both GR and TrxR. We next found, however, that nitrogen mustards (chlorambucil and melphalan) and alkyl sulfonates (busulfan) efficiently inhibited TrxR while these compounds, surprisingly, did not inhibit GR. Inhibitions were concentration and time dependent and apparently irreversible. Anticancer anthracyclines (daunorubicin and doxorubicin) were, in contrast to the alkylating agents, not inhibitors but poor substrates of TrxR. We also found that TrxR, but not GR, was efficiently inhibited by both cisplatin, its monohydrated complex, and oxaliplatin. Carboplatin, in contrast, could not inhibit any of the two enzymes. These findings lead us to conclude that representative compounds of the major classes of clinically used anticancer alkylating agents and most platinum compounds may easily target TrxR, but not GR. The TrxR inhibition should thereby be considered as a factor that may contribute to the cytotoxicity seen upon clinical use of these drugs.     

1-Methyl-DL-tryptophan, beta-(3-benzofuranyl)-DL-alanine (the oxygen analog of tryptophan), and beta-[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of tryptophan) are competitive inhibitors for indoleamine 2,3-dioxygenase

1-methyl-DL-Trp, beta-(3-benzofuranyl)-DL-alanine (the oxygen analog of Trp), and beta-[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of Trp), each of which has a substitution at the indole nitrogen atom, were found to be the first examples of potent substrate analog competitive inhibitors (Ki 7-70 microM) with respect to the substrates D-Trp and L-Trp for rabbit small intestinal indoleamine 2,3-dioxygenase. Binding studies using optical absorption and CD spectroscopy demonstrated that these three inhibitors cause spectral changes upon binding to the native ferric, ferrous, ferrous-CO, and ferrous-NO enzymes. Such spectral effects of 1-methyl-DL-Trp on all of these enzyme derivatives were similar to those caused by L-Trp, while the sulfur and the oxygen analogs of Trp exhibit relatively small effects except for those observed for the sulfur analog with CD spectroscopy. Each of these three Trp analog inhibitors competes with L-Trp for the ferrous-CO enzyme, a model for the ferrous-O2 enzyme. The present findings demonstrate that, although substitution of a methyl group for the hydrogen atom on the indole nitrogen or of a more electron-inductive sulfur or oxygen atom for the indole nitrogen atom does not prevent the binding of the resulting Trp analog to indoleamine 2,3-dioxygenase, the free form of the indole nitrogen base is an important physical and/or electronic structural requirement for Trp to be metabolized by the enzyme. The inability of 1-methyl-Trp to serve as a substrate for the dioxygenase supports a view that singlet oxygen is not the reactive oxygen species involved in the dioxygenation of Trp by the enzyme.     

Rearrangement of L-2-hydroxyglutarate to L-threo-3-methylmalate catalyzed by adenosylcobalamin-dependent glutamate mutase

Adenosylcobalamin-dependent enzymes catalyze a variety of chemically difficult isomerizations in which a nonacidic hydrogen on one carbon is interchanged with an electron-withdrawing group on an adjacent carbon. We describe a new isomerization, that of L-2-hydroxyglutarate to L-threo-3-methylmalate, involving the migration of the carbinol carbon. This reaction is catalyzed by glutamate mutase, but k(cat) = 0.05 s(-)(1) is much lower than that for the natural substrate, L-glutamate (k(cat) = 5.6 s(-)(1)). EPR spectroscopy confirms that the major organic radical that accumulates on the enzyme is the C-4 radical of L-2-hydroxyglutarate. Pre-steady-state kinetic measurements revealed that L-2-hydroxyglutarate-induced homolysis of AdoCbl occurs very rapidly, with a rate constant approaching those measured previously with glutamate and methylaspartate as substrates. These observations are consistent with the rearrangement of the 2-hydroxyglutaryl radical being the rate-determining step in the reaction. The slow rearrangement of the 2-hydroxyglutaryl radical can be attributed to the poor stabilization by the hydroxyl group of the migrating glycolyl moiety of the radical transiently formed on the migrating carbon. In contrast, with the normal substrate the migrating carbon atom bears a nitrogen substituent that better stabilizes the analogous glycyl moiety. These studies point to the importance of the functional groups attached to the migrating carbon in facilitating the carbon skeleton rearrangement.     

Theoretical investigation of the radical scavenging activity of shikonin and acylshikonin derivatives

The radical scavenging activity of shikonin and acylshikonin derivatives was studied by using density functional theory. The hydrogen bond property of the studied structures was investigated using the atoms in molecules (AIM) theory. It turned out that the hydrogen bond is important for good radical scavenging activity. The hydrogen atom transfer for shikonin and acylshikonin derivatives is difficult to obtain because of the high bond dissociation energy (BDE). However, shikonin and acylshikonin derivatives appear to be good candidates for the one-electron-transfer. The introduction of acyl groups for shikonin decreases the ionization potential (IP) values compared with that of shikonin. The acylshikonin derivatives with 1H-pyrrole, furan, and thiophene groups are expected to be of the highest radical scavenging activity among the compounds investigated in this study. Taking this system as an example, we present an efficient method for the investigation of radical scavenging activity from theoretical point of view.     

Influence of the spacer length on the activity of enzymes immobilised on nylon/polyGMA membranes: Part 1. Isothermal conditions

β-Galactosidase was immobilized on nylon/poly(glycidyl methacrylate) membranes through spacers of different length: hexamethylenediamine, ethylenediamine or hydrazine. The effect of the spacer length on the catalytic behavior of the three membranes was studied in isothermal bioreactors. The behavior of the soluble and insoluble enzymes was compared to know the effects of the immobilization process and of the spacer length.

The enzyme derivatives in comparison with the soluble enzyme exhibited shifts of the optimum pH values towards more acidic solutions. These shifts were found decreasing with the spacer length; while an opposite trend was observed when the optimum temperature values were considered. Also the values of the apparent K_m were found to decrease with the spacer length.

All these results indicated that a soluble enzyme could be considered as an enzyme immobilized on a solid support through a spacer of infinite length.     

Novel 2-substituted nitronyl nitroxides as free radical scavengers: synthesis, biological evaluation and structure-activity relationship

To develop more potent small molecules with enhanced free radical scavenger properties, we designed and synthesized a series of nitronyl nitroxide derivatives 4a-h. A lead compound 4f was discovered based on Ach-induced vascorelaxation assay. Further chemical modification based on this scaffold provided a new series of 2-substituted phenylnitronyl nitroxide derivatives 6a-s. The newly synthesized compounds 6a-s possess improved radical scavenger's activity based on PC12 cell survival assay. Compounds 6g,n,o, and s are some of the most potent compounds in terms of NO, H(2)O(2), and OH scavenging ability. 2-Substitued phenylnitronyl nitroxides had a higher radical scavenging activity with the electron-donating group (EDG). In contrast, the introduction of electron-withdrawing group (EWG) to the aromatic ring led to a dramatic decrease in its radical scavenging activity. These results suggest that the electron-donating group (EDG) of the aromatic ring may be an important factor influencing the radical scavenging behavior of these compounds, and the potency of free radical scavenging activity largely depended on the position and electronic properties of the phenyl ring substituents. The enhanced radical scavenging capacities of the novel 2-substituted nitronyl nitroxides may be potential drug leads against the deleterious action of ROS (reactive oxygen species)/RNS (reactive nitrogen species).     

Cardioprotective activity of melatonin and its novel synthesized derivatives on doxorubicin-induced cardiotoxicity

This study aimed at evaluation of melatonin and some of its novel synthesized derivatives 3, 4, 9 and 10b as cardioprotective agents against doxorubicin-induced acute cardiac toxicity in rats. Also, this work was extended to study the potential role of each synthesized derivative in comparison with the parent compound melatonin. Intraperitoneal injection of a single dose (15mg/kg B.W.) of doxorubicin resulted in significant increase in serum troponin I, leptin, triglycerides, cholesterol and LDL-cholesterol levels with concomitant decrease in serum T(3), T(4) and IL-1alpha levels. In contrast, intraperitoneal injection of melatonin or its synthesized derivatives especially compounds 3 and 10b in a dose of 5mg/kg B.W./day for 10days reversed doxorubicin-induced changes in the above mentioned parameters towards the normal values. The present data revealed that doxorubicin exerts its action mainly through the oxidative stress. This appeared from the significant elevation in serum nitric oxide level and cardiac lipid peroxidation level with concomitant decrease in cardiac antioxidative enzymes activity. Treatment with melatonin and its derivatives 3 and 10b could reduce the markers of oxidative stress and restore the activity of the antioxidative enzymes in the heart tissue. In conclusion, the cardioprotective effect of melatonin and its derivatives may be mediated through the antioxidant and free radical scavenging activity of these compounds.     

Toxicological evaluation of zidovudine and novel chalcogen derivatives in Drosophila melanogaster

Zidovudine (AZT) is the most commonly prescribed antiviral drug for the treatment of human immunodeficiency virus (HIV) infection. However, its chronic administration causes toxic side effects limiting its use. This study aimed to evaluate the toxicity of different concentrations of AZT and novel chalcogen derivatives (7A, 7D, 7G, 7K, 7M) on locomotion, mitochondrial dysfunction, acetylcholinesterase (AChE) activity, and production of reactive oxygen species (ROS) in adult Drosophila melanogaster. Our results show that AZT and its derivative 7K at a concentration of 10 μM impaired flies' locomotor behavior. Furthermore, AZT and the derivatives 7K, 7A, and 7M induced mitochondrial dysfunction observed by a decrease in oxygen flux through mitochondrial complexes I and II. Neither of the compounds tested affected AChE activity or ROS production in flies. According to these data, AZT derivatives presented the following decreasing order of toxicity: 7K > AZT > 7G > 7A > 7M > 7D. Based on the chemical structure, it is possible to infer that the presence of the seleno-phenyl group in 7A and 7G increases their toxicity compared to compounds 7D and 7M. In addition, compounds 7G, 7M, and 7K with three carbon atoms as spacer were more toxic than analogs containing one carbon atom (7A and 7D). Finally, the insertion of a p-methoxyl group enhances toxicity (7K). Based on these results, excepting 7K, all other chalcogen derivatives presented lower toxicity than AZT and are potential drug candidates.     

Benzoyl and cinnamoyl nitrogen mustard derivatives of benzoheterocyclic analogues of the tallimustine: synthesis and antitumour activity

A series of benzoyl and cinnamoyl nitrogen mustards tethered to different benzoheterocycles and to oligopyrroles structurally related to netropsin consisting of two pyrrole-amide units and terminating with an amidine moiety have been synthesised and a structure--activity relationship determined. Derivatives 3--10 have been evaluated for their sequence selective alkylating properties and cytotoxicity against human K562 leukaemia cells. They are 2- to 50-fold less cytotoxic than tallimustine, with compound 8 being the most potent member of this series. Among tallimustine isosters, the compounds with an indole 3 or benzothiophene 6 are 4-fold less cytotoxic than tallimustine, while the compounds with an N-methyl indole or benzofuran showed a 7- and 14-fold reduced cytotoxic potency, respectively. Our preliminary results indicate that these derivatives preferentially bind to AT-rich sequence with a sequence selectivity similar to tallimustine.