Several NADH model compounds, N-alkyl-1,4-dihydronicotinamides, some of them possessing amphiphilic properties, have been synthesized, and the kinetics of their reaction with a biologically active liphophilic quinone, avarone, has been studied in a protic solvent both in the presence and absence of cationic, anionic or non-ionic surfactants. In the absence of micellar agents, the medium- and long-chain N-dodecyl (3) and N-heptadecyl (4) derivatives show a significant increase in the reaction rates compared to other model compounds, due to the stabilization of the semiquinone intermediate. Anionic surfactants retard the reaction, non-ionic surfactants slightly accelerate the reaction with the short-chain derivatives, and retard the reaction with the medium- and long-chain derivatives, and the cationic surfactants increase the reaction rate with all derivatives except the long-chain 4. The results support the e-p-e mechanism of the reduction of lipophilic quinones by NADH models in protic medium

Gasic, MJ

Sladić, Dušan

Zlatović, Mario

Journal of the Serbian Chemical Society

English

MIROSLAV J. GASIC

DUSAN SLADIC

MARIO ZLATOVIC

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The kinetics of the reduction of the lipophilic quinone avarone by n-alkyl-1,4-dihydronicotinamides of various lipophilicities

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J. Serb. Chem. Soc. 64(11)647-654(1999) UDC 547.567:66.094.1:543.867-PPJSCS-2703 Original scientific paperThe kinetics of the reduction of the lipophilic quinone avaroneby n-alkyl-1,4-dihydronicotinamides of various lipophilicitiesMARIO ZLATOVI], DU[AN SLADI] and MIROSLAV J. GA[I]Faculty of Chemistry and Center for Chemistry, ICTM, University of Belgrade,Studentski trg 14, Belgrade, Yugoslavia(Received 16 March 1999)Several NADH model compounds, N-alkyl-1,4-dihydronicotinamides, someof them possessing amphiphilic properties, have been synthesized, and the kinetics oftheir reaction with a biologically active liphophilic quinone, avarone, has been studiedin a protic solvent both in the presence and absence of cationic, anionic or non-ionicsurfactants. In the absence of micellar agents, the medium- and long-chain N-dodecyl(3) and N-heptadecyl (4) derivatives show a significant increase in the reaction ratescompared to other model compounds, due to the stabilization of the semiquinoneintermediate. Anionic surfactants retard the reaction, non-ionic surfactants slightlyaccelerate the reaction with the short-chain derivatives, and retard the reaction withthe medium- and long-chain derivatives, and the cationic surfactants increase thereaction rate with all derivatives except the long-chain 4. The results support the e-p-emechanismof the reductionof lipophilic quinones byNADHmodels in proticmedium.Key words: avarone, quinone, NADH models, surfactants, kinetics.The redox reactions of compounds containing aquinone/hydroquinonemoietyhave been extensively examined1 but still, for particular cases, simplified orinconclusive presentations of the mechanism are occasionally offered. The ambi-guities in the interpretation are usually related to cases in which it was experimen-tally difficult to distinguish between stepwise proton-electron-proton transfers2 andone-step two-electron (hydride transfer) processes.3 Consequently, effective ex-trapolations of the results of chemical experiments to biological systems is ham-pered both by the complexity of these systems and an apparent susceptibility of thereaction course of structurally different quinoid compounds to the reaction condi-tions.4,5Nevertheless, the established importance of pyridine nucleotide coenzymesin enzymatic redox reactions prompted the investigation of the mechanism of theoxidation of NADH and related compounds as electron donors with simple qui-nones. The currently growing interest for this reaction is also instigated by the factthat many physiologically active compounds of natural or synthetic origin containquinoid moieties in a redox equilibrium.647For some of quinoid compounds it has been well established that they undergodirect nucleophilic addition in a biological medium; for others, particularly those withlipid properties, redox activation in the cell membranes seems to be the crucial step forthe subsequent reaction sequence. One such redox couple, the sesquiterpenes avaroland avarone6 (Fig. 1), has ween the subject of intensive investigation by severalgroups.7,8 This work was initiated by our finding of the pronounced antitumor activityof this redoxcouple.9On thebasis of complementary investigations,wehave suggestedthat this couple undergoes reversible, one electron transfer with the formation ofquinone radical anions which, in reaction with molecular oxygen, produce superoxideradicals, the species responsible for the observed bioactivity.10,11 This proposal wassubstantiated by kinetic experiments on the oxidation of BNAH with avarone and aseriesof structurallyrelatedquinones indifferentproticandnon-proticsolvents.12Sincethe avarol/avarone redox couple has lipid properties, it is quite likely that in living cells,the redox activation of this couple takes place in biological membranes. Therefore, toapproximate biological conditions, kinetic and electrochemical experiments werecarried out in cationic, anionic and neutral micellar systems, yielding results whichconfirmed the formation of the proposed quinone anion radical intermediate in theoxidation of BNAH.12,13In this work, this approach was extended by following the course of thereduction of avarone withN-alkyl-1,4-dihydronicotinamide derivatives of differentchain length, some of them possessing amphiphilic properties, in a protic solvent,both in the presence and absence of surfactans. These amphiphilic compounds canbe considered as good NADH models since no significant change in the redoxpotential, resulting from differences in the alkyl chain length, has been observed.14RESULTS ANDDISCUSSIONIn this work, four alkyl derivatives, N-butyl(1), N-octyl(2), N-dodecyl(3) andN-heptadecyl-1,4-dihydronicotinamide (4) (Fig. 2) were synthesized by alkylationof nicotinamide and subsequent sodium dithionite reduction of the N-alkyl-3-car-bamoylpyridinium halide (Scheme 1).Fig. 1. The sesquiterpenoid hydroquinone/quinone couple, avarol and avarone648 ZLATOVI], SLADI] and GA[I]The reaction rates for the reduction of avarone by the NADH models weredetermined in a protic polar solvent (H2O : EtOH, 1:1, v/v), both in the presence andabsence of surfactants (SDS,CTAB,Tween 80). The solutionwasbufferedwith 0.02Msodium phosphate buffer solution (pH 6.98). The concentration of both reactants was1×104M.The reactionwas carried out under an inert atmosphere (argon, < 3ppmO2).The reaction rates were followed by monitoring the changes in the UVabsorption at λ=350 nm (characteristic absorption maximum for the 1,4-dihydroni-cotinamide functionality).The results are given in Table I, as relative second order rate constants in regardto the rate constant of the reaction of BNAH with avarone (k=0.67 mol1 dm3 s1).In the absence of micellar agents, the rate of the lipophilic quinone avaronereduction by the NADHmodel compounds depends on the number of carbon atomsFig. 2. Synthesized derivatives of 1,4-dihydronicotinamideScheme 1.KINETICS OF AVAROL REDUCTION 649in the N-alkyl chain. The reaction was slowest with the N-butyl derivative, andfastest with theN-heptadecyl derivative. The significant increase in the reaction ratewith the N-dodecyl and the N-heptadecyl derivatives is in accordance with ourpreviously proposed mechanism.11As step I (Scheme 2) is the rate determinig step,the formation of a tightly bound ion pair in step II, stabilized by both ionic andhydrophobic interactions, should result in an increase in the reaction rate withincreasing alkyl chain length of the derivatives. In this respect, the long-chainN-alkyl derivatives are taking on the role of CTA+, which accelerates the reactionof avarone with the NADH model compounds by stabilizing the semiquinoneintermediate. However, they are even more effective, since they directly participatein the formation of the ion pair in step II, and also by increasing the localconcentrations of the reacting species. In the proposed e-p-e mechanism, steps III,IVandVare very fast, so that additional acceleration of these steps has no significanteffect on the total reaction rate.The effect of surfactants on the reaction rates is shown is Table I and Fig. 3.In Fig. 3, for each NADH model compound, the reaction rate without addedsurfactant is taken as unity and the rates in the presence of a surfactant are givenrelative to it. As expected, the anionic surfactant SDS deccelerates the reaction, dueto the destabilization of the semiquinone intermediates. The nonionic surfactantTween 80 slightly accelerates the reaction with BNAH and the N-butyl derivativeby bringing together the reacting species, but decreases the reaction rate with theFig. 3. The relative reaction rate constants of the reduction of avarone by various NADH deriva-tives in ethanol-water 1:1 (v/v), pH 6.98, with and without surfactants. The relative reaction rateconstants were calculated with respect to the rate of reaction in the absence of surfactant for a par-ticular derivative.650 ZLATOVI], SLADI] and GA[I]medium- and long-chain derivatives, probably due to their automicellization, so thatthere is a concurrent distribution of the lipophilic quinone between the Tween 80micelles, and the micelles of the lipophilic NADHmodel. In this way, the effectiveconcentration of the quinone is lowered, resulting in a decrease in the reaction rate.As expected, this effect is most pronounced with the N-heptadecyl derivative. Thecationic surfactant CTAB accelerates the reaction with BNAH and short- andmedium-chain N-butyl, N-octyl and N-dodecyl derivatives by stabilization of thesemiquinone intermediate. However, the reaction of the N-heptadecyl derivative isdeccelerated in the presence of the cationic surfactant. In addition, there is a strongacceleration of the reaction in the presence of CTAB, compared to the reaction inthe presence of Tween 80 for all the NADH models except the N-heptadecylderivative. The reason for the different behaviour of the short- and medium-chainderivatives vs. the long-chain N-heptadecyl derivative might lie in the difference intheir solubilization in CTABmicelles. Namely, electrochemical studies have shownthat BNAH is not solubilized inCTABmicelles.13On the other hand, kinetic studiesindicate that the uncharged NADH model 1-hexadecyl-4-cyano-1,4-dihydronicotin-amide is well solubilized in CTAB micelles, contrary to the positively chargedoxidized froms.15 Thus, different effects of CTAB can be observed depending onthe solubilization of the NADH derivative in the micelle. Since avarone is solu-bilized in CTABmicelles, then, if there is no solubilization of theNADHderivative,an increase of the reaction rate is expected, due to the stabilization of the semiqui-none derivative, assuming that the reaction takes place in the Stern region of themicelle. Such an effect is observed with BNAH and the short- and medium-chainderivatives. On the other hand, if the NADH model is solubilized in the CTABI II IIIR–NADH + Q →← [R–NADH,Q] →←  [R–NADH•+, Q•−] →← IV V→←  [R–NAD•, QH•]  →←  [R–NADH+,QH–] →←  R–NAD+ + QH–R = –CH2–Ph, –C4H9, –C8H17, –C12H25, –C17H35Scheme 2.TABLE I. The relative reaction rate constants of reduction of avarone by various NADH derivativesin ethanol-water: 1:1 (v/v), pH 6.98, with and without surfactantsType and conc. ofsurfactantN-alkyl-1,4-dihydronicotinamide(M) BNAH 1 2 3 40 1.00 0.79 1.08 2.02 2.62SDS, 1.5×10-3 0.92 0.73 0.97 1.85 2.19Tween 80, 1.5×10-3 1.05 0.81 1.02 1.98 2.18CTAB, 1.5×10-3 1.45 1.06 1.37 2.54 2.31KINETICS OF AVAROL REDUCTION 651micelles, as expected for the N-heptadecyl derivative, then the positively chargedone-electron oxidation product (radical cation) would be destabilized in the posi-tively charged micelle, resulting in a decrease in the reaction rate.Our results support the e-p-e mechanism of the reduction of lipophilic qui-nones by BNAH and its derivatives in a protic medium. However, the hydridetwo-electron reductionmechanism cannot be excluded in reactions of less lipophilicquinones and under different reaction conditions.EXPERIMENTALPhysical measurements1H-NMR spectra were recorded on a Varian FT-80A instrument using TMS as an internalstandard;UV/VIS spectra were recoreded on a BeckmanD-25 spectrophotometer;melting points weredetermined in a Thiele apparatus and are uncorrected; molar absorption coefficient and λmax weredetermined in ethanol-water mixture at pH 6.98, using 1×10-4 M concentrations.Avarol was isolated16from the marine sponge Dysidea avara and avarone was obtained byoxidation of avarol with silver(I) oxide as described earlier.61-Benzyl-3-carbamoylpyridinium chloride: 0.6 g of nicotinamide and 1.0 g of benzylchloride(Fluka)were refluxed in 12ml ofMeOH.After 3 h theMeOHwas partially evaporated and the solutionwas left in a cold place for the quaternary ammonium salt to crystallize. After filtration, the crudeproduct (1.2 g) was recrystallized from MeOH. The yield of white 1-benzyl-3-carbamoyl-pyridiniumchloride was 0.9 g, (74%), m.p.220 ºC; microanalysis: calcd. for C13H13ClN2O: C 62.78%, H 5.27%,N 11.26%; found: C 62.05%, H 5.45%, N 11.68%.N-alkyl derivatives of nicotinamide (general procedure): 10 mmol of nicotinamide was dis-solved in 15 ml of 1-pentanol and a solution of 12mmol of the n-alkyl halide in 1-pentanol was added.The mixture was refluxed for 3-4 h. After cooling the product solidified and occluded almost all of thesolvent. The content of the flask was transferred onto a Büchner funnel, filtered and washed with asmall amount of cold 1-pentanol and light petroleum. The crystals were dried for several hours invacuo at 60 ºC. Dried salt was used for subsequent reduction without the need for additionalpurification. The derivatives obtained by this procedure are:1-Butyl-3-carbamoylpyridinium bromide: yield 80.5%,m.p. >220 ºC,microanalysis: calcd. forC10H15BrN2O: C 46.35%, H 5.83%, N 10.81%; found: C 46.36%, H 5.99%, N 10.71%.1-Octyl-3-carbamoylpyridinium bromide: yield 65.7%,m.p. >220 ºC,microanalysis: calcd. forC14H23BrN2O: C 53.34%, H 7.35%, N 8.89%; found: C 52.76%, H 7.17%, N 9.44%.1-Dodecyl-3-carbamoylpyridinium bromide: yield 68.2%,m.p. > 220 ºC,microanalysis: calcd.for C18H31BrN2O: C 58.22%, H 8.41%, N 7.54%; found: C 58.77%, H 8.48%, N 7.64%.1-Heptadecyl-3-carbamoylpyridinium bromide: yield 60.3%, m.p. >220 ºC, microanalysis:calcd. for C23H41BrN2O: C 62.57%, H 9.36%, N 6.35%; found: C 61.95%, H 9.36%, N 6.42%.The reduction of the quaternary ammonium salts was carried out according to the classicalWestheimer procedure.17N-butyl-1,4-dihydronicotinamide: 5 mmol of 1-butyl-3-carbamoylpyridinium bromide dis-solved in 10 ml of water was added dropwise into a mixture of 1.38 g anhydrous Na2CO3 and 2.61 gfreshNa2S2O4 (Fluka, 85%) in 15ml ofwater at 40º50 ºC.After 1012min the oily yellowish dihydroderivative separates. The reactionmixturewas allowed to cool for 5minand then extractedwithCHCl3.The extract was washed with water, dried over anh. Na2CO3, and the CHCl3 evaporated. Afterrecrystallisation from ethanol and drying in a vacuum desiccator over P2O5, 65% of the yellowdihydroderivative was obtained; m.p. 135 ºC (decomp.); λmax 352 nm; ε350 6720; 1H-NMR (CDCl3):δ 7.35 (1H, s), 7.00 (1H, s), 5.68 (2H, m), 4.70 (1H, m), 3.10 (2H, t), 1.03 (4H, s), 0.90 (3H,t).652 ZLATOVI], SLADI] and GA[I]N-Alkyl derivatives of 1,4-dihydronicotinamide (general procedure): 5 mmol of the quaternaryammonium salt dissolved in 10 ml of solvent was added dropwise into a mixture of 1.38 g anhydrousNa2CO3 and 2.61 g fresh Na2S2O4 (Fluka, 85%), in 15 ml of solvent kept at 4050 ºC. After 1012min, the yellow dihydro derivative separates. The reaction mixture was allowed to cool for 5 min andthen the solid product was filtered, washed with cold water, recrystallized from ethanol, dried invacuum desiccator over P2O5 and used immediately. The derivatives obtained by this procedure are:N-Benzyl-1,4-dihydronicotinamide: solvent: water; yield 61%; m.p. 110 ºC (decomp.); λmax354 nm; ε350 6950; 1H-NMR (CDCl3): δ 7.12 (5H, m), 5.70 (1H, d), 5.25 (1H, s), 4.70 (1H, m), 4.20(2H,s), 3.10 (2H, s).N-Octyl-1,4-dihiydronicotinamide: solvent: water-ethanol (9:1); yield 70%; m.p. 120 ºC (de-comp.); λmax 352 nm; ε350 6812; 1H-NMR (CDCl3): δ 7.31 (1H, s), 7.00 (1H, s), 5.67 (2H, m), 4.62(1H, m), 3.09 (2H, t), 1.26 (12H, s), 0.90 (3H, t).N-Dodecyl-1,4-dihydronicotinamide: solvent: water-ethanol (9:1); yield 81%; m.p. 100 ºC(decomp.); λmax 350 nm; ε350 6834; 1H-NMR (CDCl3): δ 7.26 (1H, s), 7.03 (1H, d), 5.72 (2H, m),5,.23 (2H, s), 4.72 (1H, q), 3.08 (2H, t), 1.25 (20H, s), 0.90 (3H, t).N-Heptadecyl-1,4-dihydronicotinamide: solvent: water-ethanol (9:1); yield 62%; m.p. 94 ºC(decomp.); λmax 354 nm; ε350 6806-1H-NMR (CDCl3): δ 7.21 (1H, s), 7.00 (1H, d), 5.78 (2H,m), 5.30(2H, s), 4.68 (1H, m), 3.10 (2H, t), 1.20 (30H, s), 0.90 (3H, t).Determination of the reaction rates. The reduction reactions of avarone by the dihydronicotinamidederivatives were performed in quartz cells with hermetic Teflon stoppers. The concentrations of avaroneand 1,4-dihydronicotinamide derivatives were 1.00 × 10-4. M. The solutions were made in ethanol-water1:1 (v/v) with 0.02M sodium phosphate buffer pH 6.98. The CTAB, SDS and Tween 80 surfactants werecommercial products (Serva) and were used in 1.50 × 10-3 M concentrations. All solutions were purgedwith argon (< 3ppmO2) prior to use and all workwas doen under an argon atmosphere. The reactionsweremonitored on a Beckman D-25 spectrophotometer, using the change in absorption at λ=350 nm (charac-teristic absorption maximum for the 1,4-dihydronicotinamide functionality).I Z V O DKINETIKA REDUKCIJE LIPOFILNOG HINONAAVARONA N-ALKIL-1,4-DI-HIDRONIKOTINAMIDIMA RAZLI^ITE LIPOFILNOSTIMARIO ZLATOVI], DU[AN SLADI] i MIROSLAV J. GA[I]Hemijski fakultet, Studentski trg 14, Beograd i Centar za hemiju IHTM-a, Wego{eva 12, BeogradSintetisano je nekoliko N-alkil-1,4-dihidronikotinamida, model-jediwewaNADH, od kojih neki imaju amfifilne osobine, i prou~avana je kinetika wihovereakcije sa biolo{kim aktivnim, lipofilnim hinonom avaronom u proti~nomrastvara~u u prisustvu katjonskih, anjonskih ili nejonskih povr{inski aktivnih sup-stanci ibezwih. Bez dodatihmicelarnih agenasa,N-dodecil-derivat 3 (derivat sredweduine niza) i dugolan~aniN-heptadecil-derivat 4 pokazuju zna~ajno pove}awe brzinereakcije u pore|ewu sa drugim model-jediwewima, usled stabilizacije semihinonskihintermedijera. Anjonski povr{inski aktivni agensi usporavaju reakciju, nejonskiagensi dovode do slabog ubrzawa sa derivatima kratkog alkil-niza, a usporavaju reak-ciju sa derivatima sredweg i dugog niza, dok katjonski agensi ubrzavaju reakciju sa svimderivatima, sem sa dugolan~anim 4. Rezultati ukazuju na mehanizam e-p-eredukcijelipofilnih hinona modelima NADH u proti~nom medijumu.(Primqeno 16. marta 1999).KINETICS OF AVAROL REDUCTION 653REFERENCES1. Special publications Tetrahedron Symposia-in-print, Tetrahedron 42 (1986) 9752. S. Fukuzumi, S. Koumitsu, K. Hironaka. T. Tanaka. J. Am. Chem. Soc. 109 (1987) 305 andreferences therein3. L. L. Miller, J. R. Valentine, J. Am. Chem. Soc. 110 (1988) 3982 and references therein4. P. OBrien, Chem. Biol. Interactions 80 (1991) 15. E. Cadenas, P. Hochstein, L. Ernster, Adv. Enzymol. 65 (1992) 976. L. Minale, R. Riccio, G. Sodano, Tetrahedron lett. 38 (1974) 34017. M. L. Ferrándiz, M. J. Sanz, G. Bustos, M. Payá, M. J. Alcaraz, S. De Rosa, Eur. J. Pharmacology253 (1994) 75 and references therein8. S. Hirsch, A. Rudi, Y. Kashman, Y. Loya, J. Nat. Prod. 54 (1991) 929. W. E. G. Müller, A. Maidhof, R. K. Zahn, H. C. Schöder, M. J. Ga{i}, D. Heideman, A. Bernd, B.Kurelec, E. Eich, G. Seibert, Cancer Res. 45 (1985) 482210. D. Sladi}, M. J. Ga{i}, J. Serb. Chem. Soc. 59 (1994) 91511. M. J. Ga{i}, J. Serb. Chem. Soc. 53 (1988) 229 and references therein12. N. Dogovi}, D. Sladi}, M. J. Ga{i}, I. Tabakovi}, A. Davidovi}, E. Guni}, Gazz. Chim. Ital. 121(1991) 6313. A. Davidovi}, I. Tabakovi}, D. Sladi}, N. Dogovi}, M. J. Ga{i}, Bioelectrochem. Bioenerg. 26(1991) 45714. D. D. Tanner, H. K. Singh, A. Kharrad, A. R. Stern, J. Org. Chem. 52 (1987) 214215. J. Baumucker, M. Calzadilla, M. Centeno, G. Lehrmann, M. Urdanela, P. Lindquist, D. Dunham,M. Price, B. Sears, E. H. Cordes, J. Am. Chem. Soc. 94 (1972) 816416. D. Sladi}, Ph. D. Thesis, Faculty of Chemistry, University of Belgrade, Belgrade (1991)17. D. Mauzerall, F. H. Westheimer, J. Am. Chem. Soc. 77 (1955) 2261.654 ZLATOVI], SLADI] and GA[I]

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The kinetics of the reduction of the lipophilic quinone avarone by n-alkyl-1,4-dihydronicotinamides of various lipophilicities

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