In the central nervous system, both stroke and neurotrauma have been proposed to initiate a sequelae of oxidative events, which ultimately lead to neuronal cell death(1) . Studies have reported that the nitrone spin trap -phenyl-tert-butyl nitrone (PBN) ()can significantly ameliorate neuronal cell loss and neurologic deficits induced by stroke in a gerbil model of global ischemia(2, 3, 4) . Furthermore, PBN was shown by ESR spectroscopy to trap lipid-derived radicals in cortical tissue of these animals. Recent work has shown remarkable protective effects afforded by PBN in focal ischemia employing both transient (5) and permanent (6) ischemia.

There are a number of additional situations of oxidative stress in which PBN has demonstrated beneficial effects. In animal models of septic shock, where activation of inflammatory cells leads to an oxidative burst, PBN has been reported to reduce endotoxin-associated mortality(7) . Bolli et al.(8) have utilized PBN to demonstrate radical formation following reperfusion in a dog model of myocardial dysfunction referred to as ``stunning.'' Of interest was the finding that administration of the spin trap significantly improved post-ischemic contractile recovery. In Langendorf heart preparations subjected to doxorubicin-mediated formation of superoxide and hydrogen peroxide, treatment with PBN prevented the cardiotoxicity associated with the redox cycling of doxorubicin(9) .

Historically, nitrone spin traps such as PBN have been utilized to trap short-lived reactive radicals like OH as the resultant nitroxide is a more stable radical and can be detected by ESR spectroscopy. Nitrones react more rapidly with carbon-centered radicals than the oxygen-centered radicals that are thought to be the primary radicals generated in vivo. Reactions of these initial radicals with cellular biomolecules such as lipids lead to the formation of secondary carbon and oxygen-centered radicals. PBN has been used recently in a number of in vivo or ex vivo studies as an analytical tool to demonstrate radical formation as evidenced by trapping of the secondary radicals. Mason and colleagues have demonstrated radical adducts of PBN in bile of vitamin E and selenium-deficient rats challenged with either copper (10) or carbon tetrachloride(11) . Human patients undergoing elective cardioplegia were shown to have radicals present in coronary sinus blood using PBN(12) . More recently, investigators have demonstrated that nitrones like PBN can inhibit the oxidation of lipids including low density lipoproteins (13) and proteins such as glutamine synthetase(14) .

The ability of nitrones such as PBN to trap radicals to form more stable adducts, coupled with its apparent beneficial pharmacologic activity in animal models, suggests that nitrones may represent a valuable therapy for treatment of oxidative injury. However, PBN suffers from a requirement for relatively high doses to achieve protective activity. Furthermore, many radical adducts of PBN, particularly the OH adduct, are unstable and undergo decomposition reactions, which limit its use in ESR spectroscopy. Efforts to synthesize phenyl substituted analogs of PBN with improved adduct stability have met with limited success(15, 16) . We have recently prepared a series of novel cyclic variants of PBN and examined their utility as therapeutic agents in animal models of ischemia/reperfusion injury (17) and endotoxic shock(18, 19) . In each case, the cyclic nitrones were much more active than PBN. Recently, we have also reported on the ability of these compounds to function as effective inhibitors of Cu-dependent oxidation of low density lipoproteins(20) . Of great interest was the demonstration of both lipid and protein radical trapping. Herein, we describe the synthesis of the cyclic nitrones and characterization of their ability to function as radical traps in a number of in vitro assays and by ESR spectroscopy.

MATERIALS AND METHODS

The isoquinoline-based nitrones were synthesized from the appropriate formamide by a multistep procedure(21) . First, the formamide was cyclized to a 3,3-disubstituted 3,4-dihydroisoquinoline by a modified Bischler-Napieralski reaction essentially following the literature procedure(22) . Where regioisomers resulted from cyclization, the mixture of dihydroisoquinolines was readily separated by silica gel chromatography. For the six-membered ring nitrones, the dihydroisoquinoline was first reduced with sodium borohydride in methanol and the resulting 3,4-disubstituted tetrahydroisoquinoline was oxidized using catalytic sodium tungstate dihydrate and 30% aqueous hydrogen peroxide in ethanol:water affording the nitrone(23) . Alternatively, the dihydroisoquinoline could be oxidized directly to the nitrone using the same conditions, but this required multiple additions of 30% hydrogen peroxide and several days to approach completion. The benzazepine-based nitrones were made in a similar fashion, but the yield in the modified Bischler-Napieralski cyclization was much lower. The resulting imines had to be immediately reduced to the secondary amines before oxidation to the 6,7-nitrones. All final products gave satisfactory elemental and spectral data. Complete details of the synthesis have been submitted for publication elsewhere. ()

Chemicals

Molecular Modeling

Several of the structures were subjected to an estimated logP (octanol/water partition coefficient) via a fragment-based approach (28) . The methods are implemented in the program PCMODELS(29) , which comprises an estimation of logP based on structural considerations (represented as cLogP) and an estimation of the molar refractivity, which is not utilized here.

Radical Trapping and Antioxidant Activity

In Table 1-III, the values represent the IC as determined from a single experiment employing at least five concentrations. In most instances, the numbers reflect the mean of duplicate experiments and MDL 101,002 was generally run as a ``positive'' control to ensure reproducibility of the methodology. When evaluating the effect of structural variation and ring substitution on activity, for any values that appeared to be unusual relative to other analogs, the assay was repeated and the average of two experiments with similar values are reported.

Inhibition of Lipid Peroxidation

The liposomes were added to 25-ml beakers in a Dubnoff metabolic shaker at 37 °C. To the liposomes were added the test compound (in buffer or ethanol), histidine-FeCl (250:50 µM final), FeCl (50 µM final, prepared in N-purged water) and sufficient buffer to achieve a final lipid concentration of 0.5 mM. Oxidations were initiated by the addition of Fe and carried out under an air atmosphere with shaking. One ml aliquots were removed at 0,2,4,6,8,10,12 and 15 min and added to 2 ml of 0.67% thiobarbituric acid (TBA): 10% trichloroacetic acid (2:1) in 0.25 N HCl, containing 0.05 ml of 2% BHT to terminate oxidation (30) followed by heating at 100 °C for 20 min. After cooling, the tubes were centrifuged at 3,000 rpm for 10 min and the absorbance of the resultant supernatant read at 532 nm - 580 nm. Quantitation of TBARS was determined by comparison to a standard curve of malondialdehyde equivalents generated by acid catalyzed hydrolysis of 1,1,3,3-tetraethoxypropane. The IC was determined with the 15 min time point using GraphPad INPLOT 4. The data reported represent the value determined from a curve generated with at least 5 concentrations of nitrone. MDL 101,002 was routinely run to verify the reproducibility of the data over time.

Assessment of Cyclic Nitrones as OH Traps Using p-NDA

Assay mixtures in glass cuvettes contained 0.02 ml of HO, 0.02 ml of test compound, 0.10 ml of p-NDA, and 50 mM NaCl, pH 7.0, to a final volume of 0.98 ml. The oxidation was initiated by the addition of 0.02 ml of Fe, and the bleaching of p-NDA was monitored as the loss in absorbance at 440 nm for 100 s. To generate concentration curves, serial dilutions of the test compounds were made such that a constant volume of 0.02 ml was added to the reaction mixture. Ethanol itself is an OH trap; thus, controls contained an equal volume of ethanol for any test compound requiring this vehicle. The IC values for the nitrones were determined by GraphPad InPlot 4 and represent the amount of spin trap required to inhibit the bleaching of p-NDA by 50%. The values presented are from a minimum of two determinations for each concentration of nitrone.

ESR Spin Trapping

Trapping of OH by the cyclic nitrones was confirmed using [C]ethanol and [C]formate. The reaction of OH with these substrates yields the -hydroxyethyl and CO radicals, respectively, which are subsequently trapped by the nitrones. The C nucleus further splits the signal. Ethanol or formate, as well as their C isotopes, was included at 5% final concentration and Fenton's reagent (Fe/HO) was used as described above. The parameters were magnetic field strength 3480 G, microwave power 10 milliwatts, modulation amplitude 1.025 G, scan range 100 G, scan time 168 s, and receiver gain 1 10.

The ability of MDL 101,002 and PBN to trap superoxide anion was also assessed using ESR spectroscopy. The radical was generated using either FMN/NADPH or xanthine/xanthine oxidase. The iron chelator DETAPAC was included to minimize formation of OH. The reaction mixtures contained 0.375 ml of spin trap at 50 mM in phosphate-buffered saline, 0.083 ml of 3 mM FMN, 0.017 ml of 30 mM NADPH, and 0.025 ml of 1 mM DETAPAC. For enzymatic generation of O, xanthine oxidase was included at 0.05 units and xanthine was 0.33 mM. Immediately after the addition of NADPH or xanthine oxidase, the mixtures were transferred to a quartz flat cell and the spectra recorded. The spectrometer parameters were as described above for the OH studies.

Ferrous Iron-induced Oxidative Stress in Mice

RESULTS

Molecular Modeling

Figure 1: Structures of the cyclic nitrones.

Figure 2: The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of MDL 101,002 and PBN (syn and anti forms). The structures were subjected to a full energy optimization using the AM1 Hamiltonian in MOPAC, and the HOMO and LUMO for PBN and MDL 101,002 are shown.

Effect of Nitrones on Iron-induced Liposomal Peroxidation

Figure 3: Effect of MDL 101,002 on iron-induced peroxidation of soybean phosphatidylcholine liposomes. Liposomes were treated with a mixture of Fe (50 µM) and histidine-Fe (250:50 µM) in the presence or absence of varying concentrations of MDL 101,002. At the times indicated, 1-ml aliquots were removed and the degree of lipid oxidation determined by TBARS as described under ``Materials and Methods.'' The data represent a single experiment; the IC of MDL 101,002 has been confirmed in at least five independent experiments.

The effect of halogen substitution on inhibition of lipid peroxidation was also investigated. A fluoro substituent in the 6- or 7-position increased potency approximately 2-fold. Monochloro substitution at any position on the phenyl ring increased activity 7-10-fold, relative to MDL 101,002. The 7-Cl substituted spirocyclohexyl compound (MDL 101,694), the 6,8-dichloro derivative of MDL 101,002 (MDL 100,630), and the 7,9-dichloro compound in the seven-membered ring series (MDL 104,342) were the most active inhibitors of lipid peroxidation with IC values of 26-30 µM. Addition of a phenyl group to the nitrone carbon (MDL 100,818) eliminated activity as expected. The log 1/IC values for all of the cyclic nitrones were plotted against their cLogP values (from Table 2). In Fig. 4it is apparent that there is a linear relationship with the exception of PBN.

Figure 4: Correlation of cLogP values versus IC values for inhibition of lipid peroxidation for selected cyclic nitrones. The IC values were determined in the liposome experiments, and the LogP values are computer-estimated. The data are those presented in Table 1. The data represent one to three separate determinations. While the overall degree of peroxidation varies slightly between experiments, when calculated as a percentage of control (no nitrone), the variation is less than 15% between experiments. For all compounds, MDL 101,002 was run in parallel as a positive control.

OH Trapping Ability of Cyclic Nitrones

When the effect of halogen substitution on OH trapping was studied, no clear structure activity relationship was evident. In general, fluoro or chloro substituents tended to decrease OH trapping activity in all of the three series of structures. Interestingly, the dichloro compound MDL 100,630 had comparable activity relative to the corresponding monochloro compounds and, in the seven-membered ring series, the 7,9-dichloro analog (MDL 104,342) was nearly 10 times more active compared to the corresponding unsubstituted compound MDL 102,389 (IC values of 1.76 mMversus 18.1 mM). Determination of rate constants (k) for reaction with OH using the 2-deoxyribose assay (34, 35) revealed that all cyclic nitrones were more reactive than PBN with k increased 3.5-fold (MDL 101,882) to 15-fold (MDL 102,073) (data not shown).

ESR Spectra of Spin Adducts of PBN and MDL 101,002

Figure 5: ESR spectra demonstrating trapping of OH by PBN and MDL 101,002. Hydroxyl radical was generated using HO (0.3 mM) and Fe (0.15 mM), and the nitrones were present at 100 mM. The reaction was started by the addition of Fe and the spectra recorded immediately. The spectrometer conditions were as stated under ``Materials and Methods.''

Figure 6: ESR spectra of the OH adduct of MDL 101,002 recorded at various time points. The OH adduct of MDL 101,002 was generated using Fe and HO and the spectra recorded at 0, 4, 10, and 20 min after the addition of Fe. Spectrometer conditions were as specified under ``Materials and Methods.''

The trapping of OH by MDL 101,002 was confirmed by using [C]formate and [C]ethanol. Trapping of the CO radical generated by the action of OH on 5% formate yields a six-line spectrum (a = 16.0 G and a = 10.2 G) (Fig. 7). When [C]formate is used, the signal is further split into 12 lines (a = 16.2 G, a = 10.3 G, and a

Figure 7: ESR spectra demonstrating trapping of CO and -hydroxyethyl (C) radicals by MDL 101,002. The CO and -hydroxyethyl radicals were generated from the reaction of OH with formate and ethanol, respectively. Spectra were recorded immediately after the addition of Fe using both C and C of formate and ethanol.

Trapping of O by MDL 101,002 was also tested using: 1) the reduction and autoxidation of FMN or 2) xanthine and xanthine oxidase to generate the radical. A six-line spectrum was observed for MDL 101,002 with splitting constants of a = 14.2 G and a = 4.3 G (Fig. 8). Inclusion of 200 units of SOD nearly totally abolished the signal, thus insuring the adduct arose from trapping of O. Irrespective of the radical generating system, the adduct intensity with MDL 101,002 was approximately twice that of PBN. A compilation of splitting constants for PBN, POBN, and MDL 101,002 with various radical species is shown in Table 4.

Figure 8: ESR spectra demonstrating trapping of O by PBN and MDL 101,002. Superoxide anion was generated by: 1) the activity of xanthine oxidase (0.05 units) on xanthine (0.33 mM) or 2) autoxidation of reduced FMN (125 µM) in the presence of NADPH (250 µM). The spectra were recorded immediately following addition of enzyme or FMN and trapping of O was verified by the inclusion of superoxide dismutase (200 units). The ESR conditions were as defined under ``Materials and Methods.''

Effect of Nitrones on Iron-induced Mortality

Figure 9: Effect of cyclic nitrones on ferrous iron-induced mortality in mice. Ferrous iron (5 µl of 50 mM) injected into the brains of mice induced convulsions followed by death. The nitrones were administered 20 min prior to iron and the time to death monitored as an index of in vivo radical trapping activity. The compounds were dosed at the following levels: Tirilazad, 10 mg/kg (a high dose based on literature data); PBN, 150 mg/kg; MDL 101,002, 100 mg/kg.

DISCUSSION

Structure-Activity Relationships for Radical Trapping by Cyclic Nitrones

The data in Fig. 3demonstrate that MDL 101,002 decreases the level of TBARS in liposomes without markedly altering the shape of the curve as do chain terminating phenolic antioxidants such as -tocopherol. The curves in Fig. 3suggest that that the nitrones function to trap lipid-derived radicals resulting in an overall decreased level of TBA-reactive aldehydes. Trapping of a radical is a less efficient means to inhibit lipid peroxidation than H atom or electron transfer as characteristic of phenolic antioxidants but, nonetheless, the cyclic nitrones can still completely prevent peroxidation in iron-challenged liposomes.

It was determined that all of the cyclic nitrones were considerably more active inhibitors of lipid peroxidation than was PBN in this system. The unsubstituted cyclic variant MDL 101,002 was approximately 8-fold more potent than PBN. An analysis of the variants of MDL 101,002 revealed a correlation of activity with lipophilicity (Fig. 4). This is not unexpected, as it is known that the ability of antioxidants to inhibit lipid oxidation is determined to an extent by the accessibility of the antioxidant to the lipid radicals. However, it is clear that lipophilicity does not solely account for the difference between MDL 101,002 and PBN, in as much as the cLogP for MDL 101,002 is 1.01 as compared to 1.23 for PBN. This indicates that the cyclic nitrones are inherently more effective traps in a membrane system. This is supported by the methoxy variants, which have even greater water solubility but are nonetheless more active inhibitors of lipid peroxidation than is PBN. This may reflect the more favorable HOMO and LUMO values for the cyclic nitrones as compared to PBN.

The utility of the nitrones to ameliorate in vivo oxidative damage was demonstrated in the iron-injected mouse model. Pre-administration with the nitrones minimized convulsions and delayed the time to death. The anticonvulsant valproic acid was without effect, suggesting that the pharmacologic action of the nitrones derived from radical trapping. Interestingly, the potent lipid peroxidation inhibitor U74006F also had no effect. These data imply that the efficacy of the nitrones stems, at least in part, from trapping of primary radicals. We have previously shown the nitrones to prevent protein oxidation(20) . It would be of great interest to compare the nitrones and U74006F for effects on both lipid and protein oxidation in this model system; such data could provides significant new insight into the mechanism of iron-induced, radical-dependent damage in biological systems.

The LUMO for MDL 101,002 is lower than for PBN, which should increase its reactivity with O-centered radicals(32) . An evaluation of this was performed by study of reaction of the nitrones with OH and O, the two radicals of most relevance to biological systems. The nitrones were tested to ascertain whether they could compete for OH with p-NDA, which reacts rapidly and stoichiometrically with OH(31) . This assay is quite stringent in that it is difficult to compete with p-NDA for OH. Nonetheless, in a biological milieu, the diffusion distance of OH is miniscule, and thus we chose this assay to aid in determining whether the nitrones could trap OH in the presence of a highly competitive substrate. As expected, the nitrones react with OH, but much less readily than does p-NDA, as evidenced by the requirement for millimolar concentrations to compete with 100 µMp-NDA. The reactivity of PBN was poor, as shown by an estimated IC value of 72 mM. Conversely, the cyclic nitrones were able to inhibit p-NDA bleaching in the low millimolar to high micromolar range. In the 101,002 series, the addition of methoxy substituents had little effect on OH trapping when assayed by the p-NDA or 2-deoxyribose method. The presence of chloro substituents on the phenyl ring tended to decrease reactivity with OH with the exception of MDL 100,630.

When the nitrone-containing ring was expanded to seven atoms by an additional methylene (MDL 102,389), a significant loss of OH trapping activity relative to MDL 101,002 was observed. There was a slight additional loss of activity with chloro substitution, although the 7,9-Cl analog MDL 104,342 exhibited good activity with an IC of 1.76 mM. Coupled with an IC value of 26 µM against lipid peroxidation, this compound represents the most active nitrone radical trap that has been synthesized in this series and, to the best of our knowledge, exceeds the activity of any other nitrone. Linking of the gem-dimethyls with a three-methylene chain to produce the spirocyclohexyl compound MDL 102,832 decreased OH trapping activity as assessed by the p-NDA assay. As in the other two classes of cyclic nitrones, methoxy substitution improved activity slightly while chloro substitution decreased activity. A dichloro analog in this series was not synthesized.

Utility of Cyclic Nitrones as Spin Traps for OH and O

MDL 101,002 and PBN were also examined for their ability to trap O using ESR spectroscopy. The reaction of superoxide with MDL 101,002 produced an adduct with a more intense six-line spectrum with a hydrogen coupling constant which was approximately half that of the OH adduct. This is similar to the differences in hydrogen coupling for the OH and O adducts of 5,5-dimethylpyrroline-1-oxide, where it is speculated that trapping of O induces a conformational change such that the altered bond angles bring the hydrogen in closer proximity to the nitrogen nucleus. These data suggest that the cyclic nature of our nitrones leads to a significant difference in trapping and adduct conformation relative to PBN as predicted by molecular modeling studies. Thus, MDL 101,002 is much better suited to distinguishing between OH and O than is PBN, but offers the advantage over 5,5-dimethylpyrroline-1-oxide of readily trapping C-centered radicals as suggested by the studies in liposomes. The greater trapping efficiency of the cyclic compounds, coupled with the difference in hydrogen splitting between OH and O adducts, suggests MDL 101,002 to be a superior spin trap to PBN for in vitro studies when a continuous flux of radicals is being generated.

In summary, the data presented herein suggest that cyclic nitrones represent valuable tools for the study of oxidative injury. Correlations between molecular modeling and experimental data allowed us to confirm previous calculations with PBN(32) , which implied that electron transfer from the HOMO of the spin trap to the LUMO is critical for trapping of O-centered radicals. In vivo, the cyclic nitrones are capable of decreasing the effects of iron-induced oxidative stress, apparently by virtue of radical trapping. Of great significance is the fact that the nitroxides generated via radical trapping are much more stable than those of PBN and the adducts with OH and O are clearly distinguishable. Furthermore, we have prepared a series of spin traps which encompass a wide range of lipophilicity. Together, these properties suggest the compounds could be extremely useful in the study of radical trapping in biological systems and could be used to probe radical formation at the cellular level. To this end, we are currently using the nitrones in a cell culture model of ischemia/reperfusion injury to explore the site and type of radical(s) generated. Such information will greatly enhance our understanding of ischemia-induced cell injury.

FOOTNOTES

()

The abbreviations used are: PBN, -phenyl-tert-butyl nitrone; BHT, butylated hydroxytoluene; cLogP, computer-determined octanol:water partition coefficient; DETAPAC, diethylene-triaminepentaacetic anhydride; HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital; p-NDA, para-nitrosodimethylaniline; TBA, thiobarbituric acid.

()

R. C. Bernotas, D. A. Hay, A. A. Carr, T. R. Nieduzak, G. Adams, D. F. Ohlweiler, and C. E. Thomas submitted for publication.

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Hoechst Marion Roussel, Inc. 2110 E. Galbraith Rd., Cincinnati, OH 45215. Tel.: 513-948-7491; Fax: 513-948-6439.

ACKNOWLEDGEMENTS

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