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• W2080829228 abstract "A wide variety of N-methylpyridinium and quinolinium cationic inhibitors of mitochondrial complex I was synthesized to develop potent and specific inhibitors acting selectively at one of the two proposed ubiquinone binding sites of this enzyme (Gluck, M. R., Krueger, M. J., Ramsay, R. R., Sablin, S. O., Singer, T. P., and Nicklas, W. J. (1994) J. Biol. Chem. 269, 3167–3174).N-Methyl-2-n-dodecyl-3-methylquinolinium (MQ18) inhibited electron transfer of complex I at under μmorder regardless of whether exogenous or endogenous ubiquinone was used as an electron acceptor. The presence of tetraphenylboron (TPB−) potentiated the inhibition by MQ18 in a different way depending upon the molar ratio of TPB− to MQ18. In the presence of a catalytic amount of TPB−, the inhibitory potency of MQ18 was remarkably enhanced, and the extent of inhibition was almost complete. The presence of equimolar TPB−partially reactivated the enzyme activity, and the inhibition was saturated at an incomplete level (∼50%). These results are explained by the proposed dual binding sites model for ubiquinone (cited above). The inhibition behavior of MQ18 for proton pumping activity was similar to that for electron transfer activity. The good correlation of the inhibition behavior for the two activities indicates that both ubiquinone binding sites contribute to redox-driven proton pumping. On the other hand,N-methyl-4-[2-methyl-3-(p-tert-butylphenyl)]propylpyridinium (MP6) without TPB− brought about approximately 50% inhibition at 5 μm, but the inhibition reached a plateau at this level over a wide range of concentrations. Almost complete inhibition was readily obtained at low concentrations of MP6 in the presence of TPB−. Thus MP6 appears to be a selective inhibitor of one of the two ubiquinone binding sites. With a combined use of MP6 and 2,3-diethoxy-5-methyl-6-geranyl-1,4-benzoquinone, we also provided kinetic evidence for the existence of two ubiquinone binding sites. A wide variety of N-methylpyridinium and quinolinium cationic inhibitors of mitochondrial complex I was synthesized to develop potent and specific inhibitors acting selectively at one of the two proposed ubiquinone binding sites of this enzyme (Gluck, M. R., Krueger, M. J., Ramsay, R. R., Sablin, S. O., Singer, T. P., and Nicklas, W. J. (1994) J. Biol. Chem. 269, 3167–3174).N-Methyl-2-n-dodecyl-3-methylquinolinium (MQ18) inhibited electron transfer of complex I at under μmorder regardless of whether exogenous or endogenous ubiquinone was used as an electron acceptor. The presence of tetraphenylboron (TPB−) potentiated the inhibition by MQ18 in a different way depending upon the molar ratio of TPB− to MQ18. In the presence of a catalytic amount of TPB−, the inhibitory potency of MQ18 was remarkably enhanced, and the extent of inhibition was almost complete. The presence of equimolar TPB−partially reactivated the enzyme activity, and the inhibition was saturated at an incomplete level (∼50%). These results are explained by the proposed dual binding sites model for ubiquinone (cited above). The inhibition behavior of MQ18 for proton pumping activity was similar to that for electron transfer activity. The good correlation of the inhibition behavior for the two activities indicates that both ubiquinone binding sites contribute to redox-driven proton pumping. On the other hand,N-methyl-4-[2-methyl-3-(p-tert-butylphenyl)]propylpyridinium (MP6) without TPB− brought about approximately 50% inhibition at 5 μm, but the inhibition reached a plateau at this level over a wide range of concentrations. Almost complete inhibition was readily obtained at low concentrations of MP6 in the presence of TPB−. Thus MP6 appears to be a selective inhibitor of one of the two ubiquinone binding sites. With a combined use of MP6 and 2,3-diethoxy-5-methyl-6-geranyl-1,4-benzoquinone, we also provided kinetic evidence for the existence of two ubiquinone binding sites. Mitochondrial NADH-ubiquinone oxidoreductase (complex I) 1The abbreviations used are: complex I, mitochondrial NADH-ubiquinone oxidoreductase; diethoxy-Q2, 2,3-diethoxy-5-methyl-6-geranyl-1,4-benzoquinone; MPP+,N-methylphenylpyridinium; Q1, ubiquinone-1; Q2, ubiquinone-2; SMP, submitochondrial particles; TPB−, tetraphenylboron; MQ18,N-methyl-2-n-dodecyl-3-methylquinolinium; MP6,N-methyl-4-[2-methyl-3-(p-tert-butylphenyl)]propylpyridinium; THF, tetrahydrofuran. is a large enzyme that catalyzes the oxidation of NADH by ubiquinone coupled to proton translation across the inner membrane (1Hatefi Y. Annu. Rev. Biochem. 1985; 54: 1015-1069Crossref PubMed Google Scholar, 2Walker J.E. Q. Rev. Biophys. 1992; 25: 253-324Crossref PubMed Scopus (681) Google Scholar). Due to the enormous complexity of this enzyme, little is known about the pathway of the electron(s) and the mechanism of proton pumping. There is a wide variety of inhibitors of mitochondrial complex I (3Friedrich T. Van Heck P. Leif H. Ohnishi T. Forche E. Kunze B. Jansen R. Trowitzsch-Kienast Höfle G. Reichenbach H. Weiss H. Eur. J. Biochem. 1994; 219: 691-698Crossref PubMed Scopus (251) Google Scholar). Except rhein (4Kean E.A. Gutman M. Singer T.P. J. Biol. Chem. 1971; 246: 2346-2353Abstract Full Text PDF PubMed Google Scholar) and diphenyleneiodonium (5Majander A. Finel M. Wikström M. J. Biol. Chem. 1994; 269: 21037-21042Abstract Full Text PDF PubMed Google Scholar) which inhibit electron input into the enzyme, all inhibitors act at or close to the ubiquinone reduction site (i.e. so-called “rotenone site”) (3Friedrich T. Van Heck P. Leif H. Ohnishi T. Forche E. Kunze B. Jansen R. Trowitzsch-Kienast Höfle G. Reichenbach H. Weiss H. Eur. J. Biochem. 1994; 219: 691-698Crossref PubMed Scopus (251) Google Scholar). Among the inhibitors, positively charged neurotoxic N-methylphenylpyridinium (MPP+) and its analogues exhibit unique inhibitory action (6Gluck M.R. Krueger M.J. Ramsay R.R. Sablin S.O. Singer T.P. Nicklas W.J. J. Biol. Chem. 1994; 269: 3167-3174Abstract Full Text PDF PubMed Google Scholar), although their inhibitory potencies are much poorer than those of classical potent inhibitors like piericidin A and rotenone. A series of studies on the inhibition mechanism of MPP+ analogues by Singer and colleagues (6Gluck M.R. Krueger M.J. Ramsay R.R. Sablin S.O. Singer T.P. Nicklas W.J. J. Biol. Chem. 1994; 269: 3167-3174Abstract Full Text PDF PubMed Google Scholar, 7Ramsay R.R. Youngster S.K. Nicklas W.J. McKeown K.A. Jin Y.Z. Heikkila R.E. Singer T.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9168-9172Crossref PubMed Scopus (58) Google Scholar, 8Ramsay R.R. Krueger M.J. Youngster S.K. Singer T.P. Biochem. J. 1991; 273: 481-484Crossref PubMed Scopus (43) Google Scholar, 9Ramsay R.R. Singer T.P. Biochem. Biophys. Res. Commun. 1992; 189: 47-52Crossref PubMed Scopus (76) Google Scholar) have suggested that MPP+ analogues are bound at two sites, one being accessible to the relatively hydrophilic inhibitors (termed the “hydrophilic site”) and one shielded by a hydrophobic barrier on the enzyme (the “hydrophobic site”), and that occupation of both sites is required for complete inhibition. This notion is supported by the existence of two EPR-detectable species of complex I-associated ubisemiquinones (10Vinogradov A.D. Sled V.D. Burbaev D.S. Grivennikova V.G. Moroz J.A. Ohnishi T. FEBS Lett. 1995; 370: 83-87Crossref PubMed Scopus (109) Google Scholar) and by circumstantial evidence derived from studies on other types of complex I inhibitors (11Gutman M. Mersmann M. Luthy J. Singer T.P. Biochemistry. 1970; 9: 2678-2687Crossref PubMed Scopus (46) Google Scholar, 12Gutman M. Singer T.P. Casida J.E. J. Biol. Chem. 1970; 245: 1992-1997Abstract Full Text PDF PubMed Google Scholar, 13Yagi T. Arch. Biochem. Biophys. 1990; 281: 305-311Crossref PubMed Scopus (109) Google Scholar, 14Ueno H. Miyoshi H. Ebisui K. Iwamura H. Eur. J. Biochem. 1994; 225: 411-417Crossref PubMed Scopus (53) Google Scholar). Thus MPP+ analogues are useful probes with which to characterize the structural and mechanistic features of the ubiquinone reduction site of complex I. However, MPP+ analogues synthesized to date have certain limitations when they are used as a complex I inhibitor. For instance, the inhibition by MPP+ analogues requires very high concentrations (approximate mm order) compared with classical inhibitors, and there has been no specific inhibitor that acts selectively at one of the two proposed binding sites. In particular, the latter point seems to be unusual since if indeed there are two distinct binding sites, it is unlikely that their structural properties are completely identical. In addition, considering that proton pumping machinery would be close to the ubiquinone binding site which is a part of the membrane-embedded segment of the enzyme (15Yagi T. Biochemistry. 1987; 26: 2822-2828Crossref PubMed Scopus (70) Google Scholar, 16Yagi T. Hatefi Y. J. Biol. Chem. 1988; 263: 16150-16155Abstract Full Text PDF PubMed Google Scholar, 17Hofhaous G. Weiss H. Leonard K. J. Mol. Biol. 1991; 221: 1027-1043Crossref PubMed Scopus (168) Google Scholar), it remained to be defined whether the two MPP+(inevitably ubiquinone) binding sites contribute to proton pumping. To overcome these problems and advance the usefulness of MPP+analogues, potent and specific inhibitors acting selectively at one of the two MPP+ binding sites are earnestly required. In the present study, we synthesized a wide variety ofN-methylpyridinium and quinolinium cationic inhibitors (Fig. 1) to develop such a candidate. In searching through our compound sets, we found some very potent and unique inhibitors. Analysis of the inhibition behaviors of these compounds provided strong support for the existence of the two ubiquinone binding sites in complex I and revealed that both sites contribute to redox-driven proton pumping. Moreover, this study identified for the first time a potent inhibitor that exhibits highly selective inhibition against one of the two binding sites.Figure 1Structure of N-methylpyridinium and quinolinium analogues synthesized in this study.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Antimycin A and oligomycin were purchased from Sigma. Pyranine (8-hydroxy-1,3,6-pyrene trisulfonate) was obtained from Molecular Probes. MOA-stilbene was provided by Aburahi Laboratories, Shionogi Co., Ltd. (Shiga, Japan). Q1 and Q2 were a generous gift from Eisai Co. (Tokyo, Japan). Piericidin A was generously provided by Dr. Shigeo Yoshida (RIKEN, Japan). Diethoxy-Q2(2,3-diethoxy-5-methyl-6-geranyl-1,4-benzoquinone) was from a previous sample (18Sakamoto K. Miyoshi H. Takegami K. Mogi T. Anraku Y. Iwamura H. J. Biol. Chem. 1996; 271: 29897-29902Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Other chemicals were commercial products of analytical grade. All synthetic compounds were characterized by 1H NMR spectra (Bruker ARX-300) and elemental analyses for C, H, and N, within an error of ±0.3%. To a solution of 4-chloropyridine (3.0 g, 17.5 mmol) in 60 ml of a mixture of Me2SO/THF (4:6), phenol (1.6 g, 17.5 mmol), KOH (1.5 g, 17.5 mmol), and a catalytic amount of CuI were added and refluxed for 12 h. The reaction mixture was extracted by Et2O and washed with brine. The organic phase was dried over MgSO4 and evaporated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane = 3:7) to give MP1 in a 60% yield: m.p. 222–223 °C, 1H NMR (CDCl3, 300 MHz) δ 4.21 (s, 3H,N-CH3), 7.34 (d, J = 6.3, 2H), 7.45 (t, J = 7.2, 2H), 7.53 (d, J = 7.2, 2H), 7.61 (t, J = 7.2, 2H), and 8.81 (d,J = 6.3, 2H). C12H12O1N1/I Calculated:C46.03H3.86N4.47Found:C46.03H3.80N4.37MP2, MP3, MP4, MP5, and MQ11 were synthesized by the same method using 4-chloropyridine (or 4-chloroquinoline) and commercially available corresponding alcohols. MP2: m.p. 182–183 °C,1H NMR (CDCl3, 300 MHz) δ 4.52 (s, 3H,N-CH3), 7.04 (d, J = 6.3, 1H), 7.46 (t, J = 7.6, 1H), 7.47 (d, J = 7.6, 2H), 7.57 (t, J = 7.6, 2H), 7.65 (t,J = 7.6, 2H), 7.65 (dd, J = 6.4, 8.7, 1H), 8.25 (t, J = 8.7, 1H), 9.22 (d, J= 6.3, 1H). C12H12O1N1/I Calculated:C46.03H3.86N4.47Found:C45.75H3.85N4.28MP3: m.p. 205–206 °C, 1H NMR (CDCl3, 300 MHz) δ 4.57 (s, 3H, N-CH3), 7.31 (d,J = 6.9, 2H), 7.32 (d, J = 7.4, 2H), 7.54 (dd, J = 6.8, 8.1, 1H), 7.56 (dd,J = 7.0, 7.5, 1H), 7.61 (dd, J = 7.0, 8.3, 1H), 7.69 (d, J = 8.1, 1H), 7.92 (d,J = 8.3, 1H), 7.98 (d, J = 7.5, 1H). C16H14O1N1/I Calculated:C52.91H3.89N3.86Found:C52.87H3.88N3.80MP4: m.p. 90–91 °C, 1H NMR (CDCl3, 300 MHz) δ 4.03 (s, 2H), 4.31 (s, 3H, N-CH3), 7.03 (d, J = 7.5, 2H), 7.20 (m, 2H), 7.24 (m, 5H), 7.33 (m, 2H), 8.67 (d, J = 7.5, 2H). C19H18O1N1/CF3SO3 Calculated:C56.4H4.27N3.29Found:C56.31H4.31N3.26MP5: m.p. 88–89 °C, 1H NMR (CDCl3, 300 MHz) δ 3.89 (s, 2H), 4.27 (s, 3H, N-CH3), 6.97 (d, J = 7.4, 2H), 6.99 (m, 1H), 7.04 (m, 3H), 7.10 (m, 2H), 7.39 (m, 3H), 8.39 (d, J = 7.4, 2H). C19H18O1N1/CF3SO3 Calculated:C56.47H4.27N3.29Found:C56.36H4.30N3.10MQ11: m.p. 179 °C, 1H NMR (CDCl3, 300 MHz) δ 4.78 (s, 3H, N-CH3), 7.02 (d,J = 7.0, 1H), 7.25 (d, J = 6.2, 2H), 7.50 (t, J = 6.2, 1H), 7.62 (t, J = 6.2, 2H), 8.00 (dd, J = 6.2, 10.0, 1H), 8.26 (d,J = 6.2, 1H), 8.26 (dd, J = 8.2, 10.1, 1H), 8.73 (d, J = 8.2, 1H), 10.28 (d, J= 7.0, 1H). C16H14O1N1/I Calculated:C48.85H3.72N3.45Found:C48.92H3.81N3.44 To a solution of commercially available 2-methyl-3-(p-tert-butylphenyl)propionaldehyde (2.2 g, 10.8 mmol) in 15 ml of methanol, NaBH4 (0.5 g, 13.2 mmol) was added, and the reaction mixture was stirred at room temperature for 30 min. After the solvent was removed in vacuo, the residue was extracted by Et2O and washed with brine to give 2-methyl-3-(p-tert-butylphenyl)propionaldehyde in a quantitative yield. To a solution of 2-methyl-3-(p-tert-butylphenyl)propionaldehyde in 15 ml of Et2O, PBr3 was added dropwise at 0 °C, and the reaction mixture was stirred at room temperature for 2 h. Purification by silica gel column chromatography (ethyl acetate/hexane = 1:9) gave 1-brome-2-methyl-3-(p-tert-butylphenyl)propane in a 25% yield. In 20 ml of THF under N2, 1-bromo-2-methyl-3-(p-tert-butylphenyl)propane (0.7 g, 2.6 mmol) was stirred with metal Mg to give a Grignard reagent. To a 5-ml solution of pyridine (0.24 g, 2.6 mmol) in CH2Cl2, tert-butyldimethylsilyl trifluoromethanesulfonate (0.69 g, 2.6 mmol) (19Akiba K. Iseki Y. Wada M. Bull. Chem. Soc. Jpn. 1984; 57: 1984-1999Google Scholar) was added, and the mixture was stirred at room temperature for 5 min. After removal of CH2Cl2 in vacuo, the residue was dissolved in 3 ml of THF. To this solution, the above Grignard reagent was added and stirred at room temperature for 24 h. The reaction mixture was extracted by Et2O and washed with brine. The organic phase was dried over MgSO4 and evaporated. The crude product was autooxidized by stirring at room temperature for 24 h. The product was isolated by silica gel column chromatography (ethyl acetate/hexane = 3:7) in a 14% yield; m.p. 125–126 °C.1H NMR (CDCl3, 300 MHz) δ 0.89 (d,J = 6.6, 3H), 1.31 (s, 9H,tert-butyl), 2.19 (m, 1H), 2.58 (m, 3H), 2.91 (m, 1H), 4.41 (s, 3H, N-CH3), 7.07 (d, J= 8.2, 2H), 7.31 (d, J = 8.2, 2H), 7.66 (d,J = 6.6, 2H), 8.70 (d, J = 6.6, 2H). C20H28N1/CF3SO3 Calculated:C58.45H6.54N3.25Found:C58.17H6.41N3.19 4-Chloropyridine (3.0 g, 20.0 mmol) and metal Na (0.5 g, 22.0 mmol) was stirred in undecanol (10.0 g, 60.0 mmol) at 100 °C for 8 h. The reaction mixture was extracted by Et2O and washed with brine. The organic phase was dried over MgSO4 and evaporated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane = 7:3) to give MP7 in a 60% yield. MP7: m.p. 99–100 °C, 1H NMR (CDCl3, 300 MHz) δ 0.88 (t, J = 6.9, 3H), 1.27–1.47 (m, 14H), 1,66 (m, 2H), 1.85 (m, 2H), 4.29 (t, 2H), 4.50 (s, 3H, N-CH3), 7.39 (d, J= 7.3, 2H), 9.01 (d, J = 7.3, 2H). C17H30O1N1/I Calculated:C52.18H7.73N3.58Found:C52.13H7.57N3.52MP10: m.p. 65 °C, 1H NMR (CDCl3, 300 MHz) δ 0.88 (t, J = 6.5, 3H), 1.27–1.54 (m, 16H), 1.67 (t, J = 7.4, 3H,N-CH2CH 3), 1.88 (q,J = 6.5, 2H), 4.29 (t, J = 6.5, 2H), 4.79 (q, 2H, N-CH 2CH3), 7.42 (d, J = 7.5, 2H), 9.05 (d, J = 7.5, 2H). C18H32O1N1/I Calculated:C53.33H7.96N3.46Found:C53.05H7.88N3.45MP9 and MQ17 were synthesized by the same method using 2-chloropyridine and 2-chloroquinoline, respectively. MP9: m.p. 58–59 °C, 1H NMR (CDCl3, 300 MHz) δ 0.88 (t, J = 6.5, 3H), 1.27–1.53 (m, 14H), 1.50 (m, 2H), 1.97 (q, 2H), 4.59 (t, J = 6.5, 2H), 4.23 (s, 3H,N-CH3), 7.56 (dd, J = 7.7, 8.7, 1H), 7.70 (d, J = 8.7, 1H), 8.51 (dd, J= 6.4, 7.7, 1H), 9.09 (d, J = 6.4, 1H). C17H30O1N1/I Calculated:C52.18H7.73N3.58Found:C51.94H7.60N3.55MQ17: m.p. 116 °C, 1H NMR (CDCl3, 300 MHz) δ 0.88 (t, J = 6.5, 3H), 1.28–1.42 (m, 16H), 2.03 (m, 2H), 4.53 (t, J = 6.5, 2H), 4.66 (s, 3H,N-CH3), 7.51 (d, J = 7.1, 1H), 7.88 (t, J = 8.8, 1H), 8.10 (d, J = 8.8, 1H), 8.16 (t, J = 8.8, 1H), 8.49 (d,J = 8.8, 1H), 10.24 (d, J = 7.0, 1H). C21H32O1N1/I Calculated:C57.14H7.30N3.17Found:C57.04H7.21N3.13 To a solution of 3-hydroxypyridine (3.0 g, 31.6 mmol) in 60 ml of THF, NaH (0.8 g, 34.8 mmol) and undecyl bromide (8.2 g, 34.8 mmol) were added, and the mixture was refluxed for 8 h. The reaction mixture was extracted by Et2O and washed with brine. The organic phase was dried over MgSO4 and evaporated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane = 3:7) to give MP8 in a 60% yield; m.p. 90–91 °C. 1H NMR (CDCl3, 300 MHz) δ 0.88 (t, J = 6.3, 3H), 1.27–1.49 (m, 14H), 1.45 (m, 2H), 1.84 (q, 2H), 4.36 (t, J = 6.3, 2H), 4.73 (s, 3H, N-CH3), 7.64 (m, 1H), 7.96 (m, 1H), 8.83 (m, 1H), 9.03 (s, 1H). C17H30O1N1/I Calculated:C52.18H7.73N3.58Found:C52.13H7.60N3.54 To a solution of diisopropylamine (3.5 g, 35 mmol) in 100 ml of THF, 21.9 ml of 1.6 m n-butyl lithium (35 mmol) and hexamethylphosphoric triamide (6.1 g, 35.0 mmol) were added slowly at −78 °C under N2, and the mixture was stirred for 30 min (20Kaiser, E. M., and Petty, J. O. (1975) Synthesis705–706.Google Scholar). This reaction mixture was supplemented with commercially available 3-methylquinoline (5.0 g, 35.0 mmol) in 30 ml of THF and stirred for 1 h. After adding ethyl bromide (3.8 g, 35.0 mol), the mixture was slowly warmed to room temperature, and the solvent was removed in vacuo. The product (3-n-propylquinoline) was isolated by silica gel column chromatography (ethyl acetate/hexane = 3:17) in a 58% yield. To metal lithium (0.1 g, 13.6 mmol) suspended in 20 ml of THF, n-pentyl bromide (1.0 g, 6.8 mmol) was added dropwise at 0 °C under N2, and the mixture was stirred for 1 h. After adding 3-n-propylquinoline (0.9 g, 5.3 mmol) in 10 ml of THF dropwise, this mixture was stirred at room temperature for 30 min. The reaction mixture was extracted by Et2O and washed with brine. The organic phase was dried over MgSO4 and evaporated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane = 3:17) to give 1,2-dihydro-2-n-pentyl-3-n-propylquinoline in a 62% yield. 1,2-Dihydro-2-n-pentyl-3-n-propylquinoline was oxidized by stirring with 10% palladium on carbon in methanol with a catalytic amount of HCl at room temperature for 3 h. The reaction mixture was extracted by Et2O and washed with brine. The organic phase was dried over MgSO4 and evaporated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane = 3:17) to give MQ14 in a 43% yield; m.p. 99 °C. 1H NMR (CDCl3, 300 MHz) δ 0.96 (t,J = 7.3, 3H), 1.11 (t, J = 7.3, 3H), 1.46 (m, 2H), 1.58 (m, 2H), 1.77 (m, 2H), 1.83 (m, 2H), 2.93 (t,J = 7.7, 2H), 3.40 (m, 2H), 4.65 (s, 3H,N-CH3), 7.85 (t, J = 7.3, 1H), 8.08 (dd, J = 7.3, 9.5, 1H), 8.10 (d, J= 7.3, 1H), 8.39 (d, J = 9.5, 1H), 8.60 (s, 1H). C18H26N1/CF3SO3 Calculated:C56.28H6.46N3.45Found:C56.17H6.38N3.22MQ13 and MQ18 were synthesized by the same method used for MQ14. 3-Methylquinoline was reacted with n-pentyl bromide andn-dodecyl bromide for the preparation of MQ13 and MQ18, respectively. MQ13: m.p. 125–126 °C, 1H NMR (CDCl3, 300 MHz) δ 0.93 (t, J = 7.0, 3H), 1.44 (m, 2H) 1.56 (m, 2H), 1.71 (m, 2H), 1.74 (m, 2H), 2.97 (m, 2H), 3.10 (s, 3H), 4.60 (s, 3H, N-CH3), 7.84 (t,J = 7.5, 1H), 8.07 (dd, J = 7.5, 9.0, 1H), 8.10 (d, J = 7.5, 1H), 8.33 (d, J= 9.0, 1H), 8.56 (s, 1H). C16H22N1/CF3SO3 Calculated:C54.10H5.88N3.71Found:C54.03H5.90N3.42MQ18: m.p. 140–141 °C, 1H NMR (CDCl3, 300 MHz) δ 0.88 (t, J = 6.5, 3H), 1.27–1.39 (m, 16H), 1.61 (m, 4H), 2.71 (s, 3H), 3.40 (t,J = 7.9, 2H), 4.62 (s, 3H,N-CH3), 7.83 (t, J = 9.4, 1H), 8.06 (m, 2H), 8.35 (d, J = 9.4, 1H), 8.62 (s, 1H). C23H36N1/CF3SO3 Calculated:C60.61H7.63N2.94Found:C60.48H7.66N2.92MQ 12 was prepared by reacting methyl lithium and 3-n-pentylquinoline which was synthesized from 3-methylquinoline and n-butyl bromide by the same method as that for MQ14. 1H NMR (CDCl3, 300 MHz) δ 0.94 (t, J = 7.0, 3H), 1.45 (m, 2H), 1.55 (m, 2H), 1.71 (m, 2H), 2.69 (s, 3H), 3.37 (m, 2H), 4.59 (s, 3H,N-CH3), 7.81 (t, J = 7.6, 1H), 8.04 (dd, J = 7.6, 9.0, 1H), 8.09 (d, J= 7.6, 1H), 8.34 (d, J = 9.0, 1H), 8.68 (s, 1H). C16H22N1/CF3SO3 Calculated:C54.10H5.88N3.71Found:C53.83H5.86N3.67MQ19 was prepared by reacting methyl lithium with 3-dodecylquinoline that was driven from a reaction of 3-methylquinoline and n-undecyl bromide according to the method for MQ14.1H NMR (CDCl3, 300 MHz) δ 0.88 (t,J = 6.5, 3H), 1.27–1.43 (m, 18H), 1.73 (m, 2H), 2.96 (t, J = 7.8, 2H), 3.11 (s, 3H), 4.61 (s, 3H,N-CH3), 7.84 (t, J = 8.8, 1H), 8.06 (m, 2H), 8.33 (d, J = 8.8, 1H), 8.54 (s, 1H). C23H36N1/CF3SO3 Calculated:C60.61H7.63N2.94Found:C60.33H7.75N2.89 To a solution of 5-nonanone (21 g, 148 mmol) in 15 ml of methanol, NaBH4 (5.6 g, 148 mmol) was added slowly at 0 °C, and the mixture was stirred for 1 h. After the reaction mixture was extracted by Et2O and washed with brine, removal of Et2O gave 5-nonanol in a quantitative yield. To a solution of 5-nonanol (5.0 g, 34.7 mmol) in 15 ml of pyridine, p-toluenesulfonyl chloride (7.3 g, 38.4 mmol) was added dropwise, and the mixture was stirred at room temperature for 24 h. The reaction mixture was extracted by Et2O and washed with brine. The organic phase was dried over MgSO4 and evaporated. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane = 1:20) to give 1-n-butylpentyl p-toluenesulfonate. To a solution of NaBr (3.1 g, 2.6 mmol) in 10 ml of water, 1-n-butylpentyl p-toluenesulfonate (7.8 g, 2.6 mmol) and a catalytic amount of tetra-n-decylammonium bromide were added, and the mixture was refluxed at 100 °C for 24 h (21Landini, D., Onici, S., and Rolla, F. (1975) Synthesis430–431.Google Scholar). The reaction mixture was extracted by Et2O and washed with brine to give 5-bromononane in a 66% yield. To a solution of commercially available 2-methylquinoline (1.0 g, 7.0 mmol) in 10 ml of THF, 4.8 ml of 1.6 m n-butyl lithium (7.7 mmol) was added dropwise at −20 °C, and the mixture was stirred for 1 h. To the reaction mixture, 5-bromononane (1.45 g, 7.0 mmol) was added, and the mixture was stirred at room temperature for 24 h. After the reaction mixture was extracted by Et2O and washed with brine, the organic phase was dried over MgSO4 and evaporated. The crude final product was isolated by silica gel column chromatography (ethyl acetate/hexane = 3:7) in a 10% yield; m.p. 131 °C. 1H NMR (CDCl3, 300 MHz) δ 0.88 (t, J = 6.6, 6H), 1.26–1.51 (m, 12H), 1.95 (m, 1H), 3.37 (d, J = 7.2, 2H), 4.61 (s, 3H, N-CH3), 7.77 (d,J = 8.5, 1H), 7.91 (t, J = 8.5, 1H), 8.18 (m, 2H), 8.47 (d, J = 8.5, 1H), 8.77 (d,J = 8.5, 1H). C19H28N1/CF3SO3 Calculated:C58.18H6.97N3.23Found:C58.30H7.12N3.14MQ16 was synthesized by reacting commercially available 4-methylquinoline and 5-bromononane prepared by the above method.1H NMR (CDCl3, 300 MHz) δ 0.88 (t,J = 6.6, 6H), 1.33–1.43 (m, 12H), 1.87 (m, 1H), 3.22 (d, J = 7.2, 2H), 4.75 (s, 3H,N-CH3), 7.80 (d, J = 8.5, 1H), 7.98 (t, J = 8.5, 1H), 8.21 (t, J= 8.5, 1H), 8.35 (d, J = 8.5, 2H), 9.52 (d,J = 8.5, 1H). C19H28N1/CF3SO3 Calculated:C58.18H6.97N3.23Found:C57.90H6.93N3.11 N-Methylation of the above compounds was carried out by either of the following methods: the reacting of pyridine or quinoline analogues with methyl iodide (5.0 eq) in acetone at room temperature for 2 h, or reacting pyridine or quinoline analogues with methyl trifluoromethanesulfonate (1.1 eq) in dry CH2Cl2 under N2 at room temperature for 1 h. MP10 was prepared using ethyl iodide in place of methyl iodide. All of the pyridinium and quinolinium salts were purified by recrystallization from CH2Cl2/Et2O at least three times. Bovine heart submitochondrial particles (SMP) were prepared by the method of Matsuno-Yagi and Hatefi (22Matsuno-Yagi A. Hatefi Y. J. Biol. Chem. 1985; 260: 14424-14427Abstract Full Text PDF Google Scholar) using a sonication medium containing 0.25 m sucrose, 1 mm succinate, 1.5 mm ATP, 10 mm MgCl2, 10 mm MnCl2, and 10 mm Tris-HCl (pH 7.4) and stored in a buffer containing 0.25 m sucrose and 10 mm Tris-HCl (pH 7.4) at −78 °C. SMP containing a pH probe pyranine in the internal compartment were prepared as for ordinary SMP, except that the sonication medium contained 1 mm pyranine. After sonicated particles were collected by centrifugation in a Beckman type 50Ti rotor for 45 min at 43,000 rpm, the pellet was washed twice in buffer A containing 0.25 msucrose, 50 mm KCl, and 10 mm Tris-HCl (pH 7.4) and suspended by homogenization in the same buffer at 30–40 mg of protein/ml. Untrapped pyranine was removed by gel filtration on Sephadex G-25 equilibrated with buffer A. The filtration was done at 4 °C, and the particles were collected by centrifugation as described above. The sediments were finally suspended in buffer A. The NADH oxidase activity was followed spectrometrically with a Shimadzu UV-3000 at 340 nm (ε = 6.2 mm−1cm−1) at 25 °C. The reaction medium contained 0.25m sucrose, 1 mm MgCl2, and 50 mm phosphate buffer (pH 7.4). The final mitochondrial protein concentration was 30 μg of protein/ml. The reaction was started by adding 50 μm NADH. The NADH-Q1oxidoreductase activity was determined following NADH oxidation at 25 °C in the same reaction medium in the presence of 50 μm Q1, 0.2 μm antimycin A, 0.2 μm MOA-stilbene, and 2 mm KCN. For the assays with SMP containing pyranine, the reaction medium contained 0.25m sucrose, 50 mm KCl, 10 mmMgSO4, 50 μm Q1, 0.2 μm antimycin A, 0.2 μm MOA-stilbene, 2 mm KCN, and 30 mm Tris-HCl (pH 7.4). Unless otherwise noted, SMP were incubated with inhibitors for 5 min before starting the reaction. When the incubation time was extended to 30 or 120 min, SMP and a definite concentration of inhibitor were preincubated in test tubes at 3 ml of total volume with 30 μg of protein/ml on ice. The test tubes were put into a thermostatic water bath (25 °C) 5 min before starting the reaction to revert the temperature to 25 °C, and a 2.5-ml aliquot was placed in a spectrometer cuvette. The proton pumping activity of complex I was determined with SMP containing pyranine by monitoring changes in the fluorescence intensity of pyranine, which reflects acidification in the internal compartment of SMP, at 510 nm (excited at 460 nm) with a Shimadzu RF-5000 at 25 °C. The reaction medium and final mitochondrial protein concentration were the same as those for the electron transfer experiments, except that the reaction medium contained 0.2 μm oligomycin and 0.2 μm valinomycin. The inhibition of NADH-Q1 oxidoreductase activity by newly synthesized pyridinium and quinolinium derivatives was examined using SMP. Throughout this study, Q1 was mainly used as an electron acceptor since this quinone is considered to be an ideal exogenous substrate for complex I assay (23Fato R. Estornell E. Bernardo S.D. Pallotti F. Castelli G.P. Lenaz G. Biochemistry. 1996; 35: 2705-2716Crossref PubMed Scopus (151) Google Scholar). The I50 values obtained by inhibitor alone are listed in TableI with that of MPP+ as control. The inhibitory potencies varied widely depending upon structure. TheN-methylpyridiniums possessing an undecyloxy group (MP7, MP8, and MP9) appeared to be potent inhibitors regardless of substitution positions on the pyridinium ring. Replacement of a methyl group of MP7 by an ethyl group retained the activity (MP7versus MP10). The inhibitory potencies of these compounds were much stronger than those of original MPP+ and its simple alkyl analogues (6Gluck M.R. Krueger M.J. Ramsay R.R. Sablin S.O. Singer T.P. Nicklas W.J. J. Biol. Chem. 1994; 269: 3167-3174Abstract Full Text PDF PubMed Google Scholar, 8Ramsay R.R. Krueger M.J. Youngster S.K. Singer T.P. Biochem. J. 1991; 273: 481-484Crossref PubMed Scopus (43) Google Scholar). Replacing the pyridinium ring by the quinolinium ring slightly enhanced the activity (MP1 versusMQ11, and MP7 versus MQ17). In this case as well, variation in substitution positions did not significantly affect the activity (MQ18 versus MQ19). These results along with the fact thatN-benzylpyridiniums retain activity (24Murphy M.P. Krueger M.J. Sablin S.O. Ramsay R.R. Singer T.P. Biochem. J. 1995; 306: 359-365Crossref PubMed Scopus (25) Google Scholar) indicated that the steric requirement for the binding of MPP+ analogues to complex I is not so severe. This conclusion is consistent with the notion that the ubiquinone reduction site is spacious enough to accommodate a variety of structurally different inhibitors in a dissimilar manner, as claimed from the studies on other types of complex I inhibitors (3Friedrich T. Van Heck P. Leif H. Ohnishi T. Forche E. Kunze B. Jansen R. Trowitzsch-Kienast Höfle G. Reichenbach H. Weiss H. Eur. J. Biochem. 1994; 219: 691-698Crossref PubMed Scopus (251) Google Scholar, 25Sakamoto K. Miyoshi H. Matsushita K. Nakagawa M. Ikeda J. Ohshima M. Adachi O. Iwamura H. Eur. J. Biochem. 1996; 237:" @default.
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