Synthesis and antinociceptive activity of pyrrolidinylnaphthalenes

In this paper the synthesis of the racemates (2R,3S/2S,3R)-1,2-dimethyl-3-[2-(6-substituted naphthyl)]-3-hydroxypyrrolidine 1b-d [(2R,3S/2S,3R)-1b-d] are reported. Compounds 1b-d were prepared by reaction of the racemic 1,2-dimethyl-3-pyrrolidone 2 with the lithiation product obtained from 2-bromo-6-substituted naphthalene 3b-d. Pharmacological properties of (2R,3S/2S,3R)-1a-d are also described. Analgesic activity was investigated by the hot plate test and binding affinities towards mu, delta and kappa opioid receptors were evaluated. A preliminary evaluation of the in vivo side-effects was also accomplished using the rota-rod test. Interesting antinociceptive activity was shown by all compounds and in particular by 1d, which is the most active compound, since it is six-fold more potent than morphine and has lower side effects on the locomotory activity.     

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates as conformationally constrained inhibitors of acetylcholinesterase

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates (1-15) are synthesized as the conformationally constrained inhibitors of acetylcholinesterase and mimic gauche, eclipsed, and anti-conformations of acetylcholine, respectively. All carbamates 1-15 are characterized as the pseudo substrate inhibitors of acetylcholinesterase. For a series of geometric isomers, the inhibitory potencies are as follows: benzene-1,4-di-N-substituted carbamate (para compound) > benzene-1,3-di-N-substituted carbamate (meta compound) > benzene-1,2-di-N-substituted carbamate (ortho compound). Therefore, benzene-1,4-di-N-substituted carbamates (para compounds), with the angle of 180 degrees between two C(benzene)-O bonds, mimic the preferable anti C-O/C-N conformers of acetylcholine for the choline ethylene backbone in the acetylcholinesterase catalysis.     

Acid-catalyzed hydrolysis of α-ketoglutaramic acid : A proposed mechanism involving decarboxylation and amide hydrolysis of the γ-lactam, 2-pyrrolidone-5-hydroxy-5-carboxylic acid

The rates of the acid-catalyzed decarboxylation and amide hydrolysis of α-ketoglutaramic acid, the keto analog of glutamine, were investigated and the products of the reactions were characterized. In strong acid at 100°C, amide hydrolysis and decarboxylation occur with about equal facility, yielding α-ketoglutaric acid and 5-hydroxy-2-pyrrolidone, respectively. 5-Hydroxy-2-pyrrolidone undergoes further amide hydrolysis so that the products of complete acid hydrolysis of α-ketoglutaramic acid are ammonia (100%), carbon dioxide (50%), and approximately equal yields (50%) of α-ketoglutaric acid and succinic semialdehyde (β-formylpropionic acid). At increasing pH values, the relative rate of decarboxylation to amide hydrolysis of α-ketoglutaramic acid increases, such that, at pH values of 2 or greater, decarboxylation occurs almost exclusively. The decarboxylation product 5-hydroxy-2-pyrrolidone, was characterized chromatographically and by its infrared and pmr spectra; the compound may be regarded as the cyclized form of succinamic semialdehyde. A mechanism for the competing amide hydrolysis and decarboxylation reactions is proposed, and the potential biological significance of the decarboxylation pathway is discussed.     

Correlation between protein kinase C alpha activity and membrane phase behavior.

Lipid activation of protein kinase C alpha (PKC alpha) was studied by using a model mixture containing 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1, 2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS), and 1, 2-dimyristoyl-sn-glycerol (1,2-DMG). This lipid mixture was physically characterized by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and 31P-nuclear magnetic resonance (31P-NMR). Based on these techniques, a phase diagram was constructed by keeping a constant DMPC/DMPS molar ratio of 4:1 and changing the concentration of 1,2-DMG. This phase diagram displayed three regions and two compounds: compound 1 (C1), with 45 mol% 1,2-DMG, and compound 2 (C2), with 60 mol% 1,2-DMG. When the phase diagram was elaborated in the presence of Ca2+ and Mg2+, at concentrations similar to those used in the PKC alpha activity assay, the boundaries between the regions changed slightly and C1 had 35 mol% 1,2-DMG. The activity of PKC alpha was studied at several temperatures and at different concentrations of 1,2-DMG, with a maximum of activity reached at 30 mol% 1,2-DMG and lower values at higher concentrations. In the presence of Ca2+ and Mg2+, maximum PKC alpha activity occurred at concentrations of 1,2-DMG that were close to the boundary in the phase diagram between region 1, where compound C1 and the pure phospholipid coexisted in the gel phase, and region 2, where compounds C1 and C2 coexisted. These results suggest that the membrane structure corresponding to a mixture of 1,2-DMG/phospholipid complex and free phospholipid is better able to support the activity of PKC alpha than the 1,2-DMG/phospholipid complex alone.     

Study of the characterization and crystallization of 4-hydroxy-2-pyrrolidone

A systematic study of the characterization for racemic species of 4-hydroxy-2-pyrrolidone was undertaken. The melting point phase diagram of (R)- and (S)-4-hydroxy-2-pyrrolidone was determined by differential scanning calorimetry. The ternary phase diagram of (R)- and (S)-4-hydroxy-2-pyrrolidone with isopropanol was constructed at 15, 20, 25, and 35 degrees C. The crystalline nature of 4-hydroxy-2-pyrrolidone racemate was also characterized by means of comparison of solid-state FTIR spectra and powder X-ray diffraction patterns of the racemic mixture with those of one of the enantiomers. It is shown that (+/-)-4-hydroxy-2-pyrrolidone is a racemic conglomerate. The enthalpies of fusion of (R)-4-hydroxy-2-pyrrolidone and (+/-)-4-hydroxy-2-pyrrolidone and entropy of mixing of (R)- and (S)-4-hydroxy-2-pyrrolidone were calculated using the thermodynamic data. The solubility and supersolubility diagrams of (R)- and (S)-4-hydroxy-2-pyrrolidone in isopropanol were determined over a temperature range of 4-35 degrees C. The optical resolution of (+/-)-4-hydroxy-2-pyrrolidone was successfully achieved by preferential crystallization.     

Schizosaccharomyces pombe produces novel Gal0-2Man1-3 O-linked oligosaccharides

Schizosaccharomyces pombe whole-cell glycoproteins, previously depleted of N-linked glycans by sequential treatment with endo-ss-N-acetylglucosaminidase H and peptide-N4-asparagine amidohydrolase F, were ss-eliminated with 0.1 M NaOH/1 M NaBH4 to release the O-linked oligosaccharides. The saccharide-alditols were separated by gel-exclusion chromatography into pools from Hexitol to Hex4Hexitol in size. Analysis of the Hexitol pool indicated Man to be the only sugar linked to Ser or Thr residues. The Hex1Hexitol pool contained two components, Galalpha1,2Man-ol (2A) and Manalpha1, 2Man-ol (2B). The Hex2Hexitol pool contained two components, Galalpha1,2Manalpha1,2Man-ol (3A) and Manalpha1,2Manalpha1,2Man-ol (3B). The two Hex3Hexitol components were Galalpha1,2(Galalpha1, 3)Manalpha1,2Man-ol (4A) and Manalpha1,2(Galalpha1,3)Manalpha1, 2Man-ol (4B). The Hex4Hexitol component was found to be a single isomer with the composition of Galalpha1,2(Galalpha1,3)Manalpha1, 2Manalpha1,2Man-ol (5AB). Surprisingly, galactobiose was not detected in any of these oligosaccharides. The gma12 (T. G. Chappell and G. Warren (1989) J. Cell Biol., 109, 2693-2707) and gth1 (T. G. Chappell personal communication) alpha1, 2-galactosyltransferase-deficient mutants and the gma12/gth1 double mutant S.pombe strains were similarly examined. The results indicated that gma12p is solely responsible for the addition of terminal alpha1,2-linked Gal in compound 2A, while one or both of gma12p and gth1p are required for the alpha1,2-linked Gal in 4A. Both transferases are largely responsible for terminal Gal in isomer 5AB. Neither gma12 nor gth1 had any discernible effect on the structure of the large N-linked galactomannans as determined by 1H NMR spectroscopy. Thus, while gth1p and gma12p appear responsible for adding alpha1,2-linked Gal to terminal Man, neither adds galactose side chains to the N-linked poly alpha1,6-Man outerchain, nor the O-linked branch-forming alpha1,3-linked Gal. Furthermore, the presence of Hexalpha1,2(Galalpha1,3)Manalpha1,2- structures in the O-linked glycans implies the presence of a novel branch-forming alpha1,3-galactosyltransferase in S.pombe.     

Metabolism of alpha-D-[1,2-13C]glucose pentaacetate and alpha-D-glucose penta[2-13C]acetate in rat hepatocytes

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.     

Multiply 13C-substituted monosaccharides: synthesis of D-(1,5,6-13C3)glucose and D-(2,5,6-13C3)glucose.

D-(1,5,6-13C3)Glucose (7) has been synthesized by a six-step chemical method. D-(1,2-13C2)Mannose (1) was converted to methyl D-(1,2-13C2)mannopyranosides (2), and 2 was oxidized with Pt-C and O2 to give methyl D-(1,2-13C2)mannopyranuronides (3). After purification by anion-exchange chromatography, 3 was hydrolyzed to give D-(1,2-13C2)mannuronic acid (4), and 4 was converted to D-(5,6-13C2)mannonic acid (5) with NaBH4. Ruff degradation of 5 gave D-(4,5-13C2)arabinose (6), and 6 was converted to D-(1,5,6-13C3)glucose (7) and D-(1,5,6-13C3)mannose (8) by cyanohydrin reduction. D-(2,5,6-13C3)Glucose (9) was prepared from 8 by molybdate-catalyzed epimerization.     

Antimycobacterial activity: synthesis of novel 3-(substituted phenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]isoxazole analogues

In this study, a series of novel 3-(substituted phenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]isoxazole analogues were synthesized and evaluated for antimycobacterial activity against Mycobacterium tuberculosis (MTB) H(37)Rv and isoniazid resistant M. tuberculosis (INHR-MTB). All the newly synthesized compounds were showing moderate to high inhibitory activities. The compound 6,7-dimethoxy-3-(4-chloro phenyl)-4H-indeno[1,2-c]isoxazole (4b) was found to be the most promising compound, active against MTB H(37)Rv and INHR-MTB with minimum inhibitory concentrations of 0.22 and 0.34 μM.     

Thermotropic phase behaviour of the pseudobinary mixtures of DPPC/C12E5 and DMPC/C12E5 determined by differential scanning calorimetry and ultrasonic velocimetry

The present paper reports on the phase behaviour of the pseudobinary aqueous mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/pentaethylene glycol monododecyl ether (C12E5) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine monohydrate (DMPC)/C12E5. Both systems exhibit a variety of mesophases, such as lamellar gel, liquid crystalline and micellar phases. The phase diagrams show peritectic and eutectic behaviours. The existence of a compound complex is established. From the phase diagrams, the temperature dependence of the solubilisation parameters is obtained. The phase diagrams, especially with respect to the solubilisation process were qualitatively explained assuming that the packing of the constituents plays a dominating role. Finally, differential scanning calorimetry and ultrasonic velocimetry are compared concerning their potentials to determine characteristics of phase transitions in pseudobinary phospholipid/surfactant mixtures.     

Δ′-Pyrroline: An intermediary metabolite in the conversion of putrescine to 2-pyrrolidone

Δ′-Pyrroline, an oxidative product of putrescine metabolism, was chemically synthesized and incubated with a rat liver homogenate. The incubation mixture was fractionated on an amino acid analyzer before and after acid hydrolysis. The hydrolyzed sample, in contrast to the unhydrolyzed sample, contained a ninhydrin positive compound that cochromatographed with γ-aminobutyric acid, the product of 2-pyrrolidone acid hydrolysis. Authentic 2-pyrrolidone had the same retention time as the Δ′-pyrroline metabolite when analyzed by high-pressure liquid chromatography. It is concluded that Δ′-pyrroline is an intermediary metabolite in the pathway from putrescine to 5-hydroxy-2-pyrrolidone.     

The chemical and enzymatic interaction of glutathione with the fungal metabolite,fusarin C

Glutathione (GSH) interacts both chemically and enzymatically with fusarin C, a mutagenic metabolite produced by Fusarium moniliforme. The chemical reaction, which is pH-dependent, results in the formation of both fusarin A and a compound that lacks the 2-pyrrolidone moiety thereby suggesting an interaction at the C-13–C-14 epoxide. Enzymatic interaction of fusarin C with GSH also appears to occur at this site as fusarins A and D, which lack the epoxide, do not serve as substrates for GSH-S-transferases. The interaction of GSH with fusarin C appears to be an important deactivation step which could explain the lack of carcinogenicity observed for fusarin C in rats.     

Discovery of novel antitubercular 1,5-dimethyl-2-phenyl-4-([5-(arylamino)-1,3,4-oxadiazol-2-yl]methylamino)-1,2-dihydro-3H-pyrazol-3-one analogues

In search of potential therapeutics for tuberculosis, we describe herewith the synthesis, characterization and antimycobacterial activity of 1,5-dimethyl-2-phenyl-4-([5-(arylamino)-1,3,4-oxadiazol-2-yl]methylamino)-1,2-dihydro-3H-pyrazol-3-one analogues. Among the synthesized compounds, 4-[(5-[(4-fluorophenylamino]-1,3,4-oxadiazol-2-yl)methylamino]-1,2-dihydro-1,5-dimethyl-2-phenylpyrazol-3-one (4a) was found to be the most promising compound active against Mycobacterium tuberculosis H(37)Rv and isoniazid resistant M. tuberculosis with minimum inhibitory concentrations, 0.78 and 3.12μg/mL, respectively, free from any cytotoxicity (>62.5μg/mL).     

Incorporation of (2-3H)glycerol into rat brain 1,2-diacyl-sn-glycero-3-phosphorylcholine and 1,2-diacyl-sn-glycerol molecular species in vivo

Rat brain 1,2-diacyl-sn-glycerols (diglycerides) and 1,2-diacyl-sn-glycerols obtained from 1,2-diacyl-sn-glycero-3-phosphorylcholine after treatment with phospholipase C differ markedly in carbon number distribution. 70% of the 1,2-diacyl-sn-glycerols had a total of 38 fatty acid carbon atoms, and there was no detectable change in the 1,2-diacyl-sn-glycerol mass pattern between 7 and 23 days of age. In contrast, 1,2-diacyl-sn-glycero-3-phosphorylcholine contained at most 10% of this molecular species in the brains of rats of comparable age. A small increase in the C(36) species of 1,2-diacyl-sn-glycero-3-phosphorylcholine, which is associated with myelination, was noted between 10 and 17 days. The incorporation of intracranially injected [2-(3)H]glycerol into 1,2-diacyl-sn-glycero-3-phosphoryl-choline species with polyunsaturated fatty acids containing 20 or 22 carbon atoms was greater than into the species containing only saturated and/or monoenoic fatty acids between 30 min and 24 hr. The 1,2-diacyl-sn-glycerol fractions containing polyunsaturated fatty acids had the lowest specific activity at 30 min. The specific activity of the particular 1,2-diacyl-sn-glycerol fraction containing the stearate-arachidonate pair is the lowest for 4 hr after intracranial injection of the isotope. Thus, molecular species of 1,2-diacyl-sn-glycerol and 1,2-diacyl-sn-glycero-3-phosphorylcholine differed considerably in their labeling patterns, and a direct precursor-product relationship could not be demonstrated during the time period studied.     

Substituent effects on the decomposition of chemiluminescent tricyclic aromatic dioxetanes

Three tricyclic 1,2‐dioxetane derivatives, 1a, 2a and 3a were synthesized from their corresponding 1,4‐dioxin acenaphthylene compounds, 1, 2 and 3, by reaction with singlet‐oxygen (¹O₂) in dichloromethane. Evidence for the formation of the dioxetanes 1a, 2a and 3a is provided by the chemiluminescence (CL) that corresponds to the emission from the electronically excited diesters 1b*, 2b* and 3b*, which are decomposed thermally from the dioxetanes 1a, 2a and 3a, respectively. The highly strained 1,2‐dioxetane ring decomposes from a twisted geometry by simultaneous cleavages of the O–O and C–C bonds, producing the electronically excited diester that emits CL. It was observed that the CL from compound 2a is red‐shifted relative to that of compounds 1a and 3a suggesting a higher degree of stabilization for the excited state by the electron‐donating methoxy group. Also, a study of the solvent effect on fluorescence shows a significant red‐shift in compound 2b, indicating a more polar excited state. The kinetics of the thermal decomposition of the 1,2‐dioxetanes clearly demonstrate that the CL characteristics of compound 2a are quite different from those of compounds, 1a and 3a. These results are consistent with the proposed intramolecular chemically initiated electron exchange luminescence (CIEEL) mechanism which is triggered by the electron‐donating group of compound 2a. Copyright © 2013 John Wiley & Sons, Ltd.     

Loss of tritium from coprostanone derived from [1,2(n)-3H]cholesterol or [7(n)-3H]cholesterol

After oral administration of a mixture of [1,2(n)-3H]cholesterol and [4-14C]cholesterol to a baboon, fecal coprostanone had a 46% lower 3H/14C ratio than the dose administered. Loss of 3H by enolization of the 3-ketone could account for the decrease in 3H/14C. If [7(n)-3H]cholesterol was administered instead of [1,2(n)-3H]cholesterol a 23% loss of 3H from coprostanone was found. Procedures requiring measurement of 3H-coprostanone derived from [1,2(n)-3H]- or [7(n)-3H]cholesterol could be seriously in error unless an appropriate correction for loss of 3H is made.     

Conversion of 5-(1,2-epoxy-2,6,6-trimethylcyclohexyl) -3-methylpenta-cis-2-trans-4-dienoic acid into abscisic acid in plants

(±)-5-(1,2-Epoxy-2,6,6-trimethylcyclohexyl) -3-methyl[2-¹⁴C]penta-cis-2-trans-4-dienoic acid is converted into abscisic acid by tomato fruit in 1.8% yield (or 3.6% of one enantiomer if only one is utilized) and 15% of the abscisic acid is derived from the precursor. The 2-trans-isomer is not converted. The amounts of [2-³H]mevalonate incorporated into abscisic acid have shown that the 40-times higher concentration of (+)-abscisic acid in wilted wheat leaves in comparison with unwilted ones reported by Wright & Hiron (1969) arises by synthesis. The conversion of (±)-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl) -3-methyl-[2-¹⁴C]penta-cis-2-trans-4-dienoic acid into abscisic acid by wheat leaves is also affected in the same way by wilting and it is concluded from this that the epoxide or a closely related compound derived from it is on the biosynthetic pathway leading to abscisic acid. The oxygen of the epoxy group was shown, by ¹⁸O-labelling, to become the oxygen of the tertiary hydroxyl group of abscisic acid.     

Oxidative fragmentation of collagen and prolyl peptide by Cu(II)/H2O2. Conversion of proline residue to 2-pyrrolidone.

Oxidative degradation of collagen and the model peptides by Cu(II)/H2O2 has been studied. The depolymerization of collagen was predominantly observed by use of gel filtration chromatography. Polyproline was used as a model for collagen, and the oxidative modification was examined by amino acid analysis. Glutamic acid and gamma-aminobutyric acid were identified in the hydrolysates of oxidized polyproline. The formation of glutamic acid was reduced by treatment with NaBH4. The model peptide, (Pro-Pro-Gly)10, was also degraded by Cu(II)/H2O2, and a new N-terminal glycine was generated in proportion to the reaction time. Hydroxyl radical scavengers show only partial inhibition of the degradation of (Pro-Pro-Gly)10. In order to estimate the fragmentation mechanism, we used N-tert-butoxycarbonyl (Boc)-L-prolylglycine as a model for collagen and (Pro-Pro-Gly)10. The degradation products were isolated and characterized. Then N-tert-Boc-2-pyrrolidone, which provides gamma-aminobutyric acid by acid hydrolysis, was identified. The formation of a 2-pyrrolidone compound from oxidized Boc-L-prolylglycine is direct evidence for the scission of the peptide bond. The time-dependent formation of N-tert-Boc-2-pyrrolidone and liberation of glycine from N-tert-Boc-L-prolylglycine exposed to Cu(II)/H2O2 was observed. These results suggest that the cleavage of the peptide bond (Pro-Gly) was caused by oxidation of the proline residue, which led to the formation of the 2-pyrrolidone compound. We confirmed that proline oxidation leads to the fragmentation of proteins, accompanied by the formation of a 2-pyrrolidone structure.     

Synthesis and evaluation of glucocerebrosidase inhibitory activity of anhydro deoxyinositols from (+)-epi- and (-)-vibo-quercitols.

Twelve 1,2- and 2,3-anhydro-1,2,3,4,5-cyclohexanepentols were synthesized from (+)-epi- and (-)-vibo-quercitols, readily available by bioconversion of myo-inositol, and assayed for inhibitory activity against glucocerebrosidase (mouse liver). Among them 1L-1,2-anhydro-1,2,4/3,5-cyclohexanepentol, the 3-deoxy derivative of the irreversible inhibitor conduritol B epoxide (CBE), has been demonstrated to be a highly potent and specific inhibitor, almost comparable to the parent compound.     

Cyclodextrin-bound 6-(4-methoxyphenyl)imidazo[1,2-alpha+/-]pyrazin-3(7H)-ones with fluorescein as green chemiluminescent probes for superoxide anions

In providing chemiluminescent probes that have high chemiluminescence intensity and high specificity to superoxide anions, novel chemiluminescent probes involving cyclodextrins covalently bound to 6-(4-methoxyphenyl)imidazo[1,2-alpha]pyrazin-3(7H)-one with fluorescein were synthesized and characterized. Using the hypoxanthine-xanthine oxidase system for the generation of the superoxide anions, these novel chemiluminescent probes showed higher superoxide-induced chemiluminescence intensity than that of 6-[4-[2-[N(')-(5-fluoresceinyl)thioureido]-ethoxy]phenyl]-2-methylimidazo[1,2-alpha]pyrazin-3(7H)-one (FCLA). When tested at a probe concentration of 1.0 microM, compound 6, in which 6-(4-methoxyphenyl)imidazo[1,2-alpha]pyrazin-3(7H)-one and fluorescein are covalently attached on the secondary and primary hydroxyl faces of gamma-cyclodextrin, respectively, showed green luminescence intensity that was 26 times that of FCLA, which was also the highest luminescence intensity in this present study. At probe concentrations of less than 1.0 microM, the ratio of the superoxide-dependent chemiluminescence intensity to the background chemiluminescence intensity for compound 6 was higher than that of FCLA. This high superoxide-induced chemiluminescence intensity and superoxide specificity in low probe concentrations indicates that 6 can be more effective than FCLA toward the measurement of superoxide anions.