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Correlation of redox potentials and inhibitory effects on Epstein-Barr virus activation of 2-azaanthraquinones
Cancer Letters 212 (2004) 1–6 www.elsevier.com/locate/canlet
Correlation of redox potentials and inhibitory effects on Epstein-Barr virus activation of 2-azaanthraquinones Junko Koyamaa,*, Izumi Moritaa, Norihiro Kobayashia, Toshiyuki Osakaib, Hiroki Hottab, Junko Takayasuc, Hoyoku Nishinoc, Harukuni Tokudac a
Faculty of Pharmaceutical Sciences, Kobe Pharmaceutical University, Higashinada, Kobe 658-8558, Japan b Department of Chemistry, Faculty of Science, Kobe University, Nada, Kobe 657-8501, Japan c Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan Received 25 December 2003; received in revised form 27 February 2004; accepted 8 March 2004
Abstract As a continuation of our studies using natural and synthetic products as cancer chemopreventive agents, we examined the standard redox potentials of some 2-azaanthraquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the redox potentials and the inhibitory effects of the 2-azaanthraquinones on Epstein-Barr virus early antigen (EBV-EA) activation. It has been further shown that the correlation can be enhanced by introducing an electronic properties, i.e. the atomic charges at the C5 and O12 atoms in the quinone skeleton ring and the HOMO energy as additional parameters. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Epstein-Barr virus; 2-Azaanthraquinone; Redox potential; Cyclic voltammetry
1. Introduction Quinones (anthraquinones, naphthoquinones, and heteronaphthoquinones) are important naturally occurring pigments that are widely distributed in nature and are known to demonstrate various physiological activities as antibiotics and anti-cancer agents. In our previous studies, we reported the inhibitory effects of a number of anthraquinones, naphthoquinones and 1-azaanthraquinones on 12-Otetradecanoylphorbol-13-acetate (TPA)-induced * Corresponding author. Tel.: þ81-78-441-7549; fax: þ 81-78441-7550. E-mail address: [email protected] (J. Koyama).
EBV-EA activation [1 – 3]. We have also found inhibitory activities of the mono- and di-substituted anthraquinones and bianthraquinones on EBV-EA activation, and have studied their connections with the electronic properties of the anthraquinones [3,4]. In studies of the structure-activity relationships for drugs, the standard redox potential is one of the most important parameters to determine the physiological activities. We employed cyclic voltammetry to determine the standard redox potentials of 9 anthraquinones, 9 naphthoquinones and 9 1-azaanthraquinones at the physiological pH 7.2, and found definite correlations between the standard redox potentials and the inhibitory effects (log IC50) on EBV-EA activation [5 –7].
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.03.005
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J. Koyama et al. / Cancer Letters 212 (2004) 1–6
In the present study, we report the redox potentials of 10 2-azaanthraquinones and the structure-activity relationship between the inhibitory effects and the redox potentials of the 2-azaanthraquinones. Furthermore, we have calculated some electronic properties of the 2-azaanthraquinones by the PM3 method using the CAChe MOPAC program [8]. It has been revealed that the atomic charges at C5 and O12 could be used as other useful parameters to characterize the inhibitory effect on EBV-EA activation. Moreover, it was found that the 1-azaanthraquinones and 2-azaanthraquinones had similar correlations of log IC50 on EBV-EA activation with the redox potential and the charges of the carbonyl group.
2. Material and methods 2.1. Reagents and materials Benzo[g ]isoquinoline-5,10-dione (1) was purchased from Aldrich (Wiscosin, USA). 6-Methoxybenzo[g ]isoquinoline-5,10-dione (2) and 9-methoxybenzo[g ]isoquinoline-5,10-dione (3) were synthesized from isoquinoline-5,8-dione and 1-methoxy-1,3-cyclohexadiene [9]. 6,8-Dimethoxybenzo [g ]isoquinoline-5,10-dione (4), 7,9-dimethoxybenzo [g ]isoquinoline-5, 10-dione (6), and 9-bromo-6,8dimethoxybenzo[g ]isoquinoline-5,10-dione (10) were synthesized from isoquinoline-5, 8-dione and 1,1-dimethoxyethene [10]. 6,9-Dihydroxybenzo[g ] isoquinoline-5,10-dione (8) was synthesized from cinchomeronic anhydride and 1,4-dimethoxybenzene [11]. 6-Hydroxy-8-methoxybenzo[g ]isoquinoline5,10-dione (5), 9-hydroxy-7-methoxybenzo[g ]isoquinoline-5,10-dione (7), and 9-hydroxybenzo[g ] isoquinoline-5,10-dione (9) were synthesized by demethylation with boron trifluoride from (4), (6) and (3), respectively (Fig. 1). 6-Hydroxy-8-methoxybenzo[g ]isoquinoline-5,10dione (5) IR (CHCl3) cm21: 1688, 1667, 1575. 1HNMR (CDCl3) d: 3.97 (3H, s, OCH3), 6.74 (1H, d, J ¼ 2.2 Hz, 7-H), 7.39 (1H, d, J ¼ 2.2 Hz, 9-H), 8.07 (1H, dd, J ¼ 5 Hz, 4-H), 9.10 (1H, d, J ¼ 5 Hz, 3-H), 9.51 (1H, s, 1-H). HR-MS m/z: 255.0544 (Mþ, Calcd for C14H9NO4: 255.0531). 9-Hydroxy-7-methoxybenzo[g ]isoquinoline-5,10dione (7) IR (CHCl3) cm21: 1682, 1633, 1579.
1
H-NMR (CDCl3) d: 3.96 (3H, s, OCH3), 6.78 (1H, d, J ¼ 2.5 Hz, 8-H), 7.38 (1H, d, J ¼ 2.5 Hz 6-H), 8.03 (1H, d, J ¼ 5 Hz 4-H), 9.08 (1H, d, J ¼ 5 Hz, 3-H), 9.56 (1H, s, 1-H). HR-MS m/z: 255.0558 (Mþ, Calcd for C14H9NO4: 255.0531). 9-Hydroxybenzo[g ]isoquinoline-5,10-dione (9) IR (CHCl3) cm21: 3400, 1680, 1640, 1579. 1H-NMR (CDCl3) d: 7.38 (1H, d, J ¼ 8 Hz 8-H), 7.77 (1H, t, J ¼ 8 Hz 7-H), 7.85 (1H, d, J ¼ 8 Hz 6-H), 8.06 (1H, d, J ¼ 5 Hz, 4-H), 9.12 (1H, d, J ¼ 5 Hz, 3-H), 9.59 (1H, s, 1-H), 12.44 (1H, s, OH). HR-MS m/z: 225.0408 (Mþ, Calcd for C13H7NO3: 225.0425). The tissue culture reagents, 12-O-tetradecanoylphorbol-13-acetate (TPA), n-butyric acid and other reagents, were from Nacalai Tesque. The EBVgenome-carrying lymphoblastoid cells (Raji cells derived from Burkitt’s lymphoma) were cultured in RPMI 1640 medium (Nissui), as described elsewhere [12]. Spontaneous activation of EBV-EA in our subline of Raji cells was less than 0.1%. 2.2. Procedure of EBV-EA activation The inhibition of the EBV-EA activation was assayed using Raji cells (virus non-producer), which were cultivated in 10% FBS RPMI 1640 medium. The indicator cells (Raji) (1 £ 106/ml) were incubated at 37 8C for 48 h in 1 ml of the medium containing n-butyric acid (4 mM, inducer), 2 ml of TPA (20 ng/ml in DMSO), and a known amount of test compound dissolved in DMSO. Smears were made
Fig. 1. Structures of Compounds 1–10.
J. Koyama et al. / Cancer Letters 212 (2004) 1–6
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Table 1 Inhibitory effects of 2-azaanthraquinones on EBV-EA activation Compound
% to control (% viability) log IC50a
Molar ratio (to 32 pmol TPA)
1 2 3 4 5 6 7 8 9 10 a
1000
500
100
10
63.6 ^ 2.2 (60) 43.7 ^ 1.3 (60) 44.9 ^ 1.5 (60) 41.3 ^ 1.1 (60) 16.8 ^ 0.8 (70) 40.5 ^ 1.6 (60) 37.5 ^ 1.5 (60) 2.5 ^ 0.4 (50) 57.2 ^ 2.3 (60) 49.6 ^ 1.9 (60)
84.9 ^ 1.9 69.2 ^ 2.3 71.6 ^ 2.1 68.5 ^ 2.4 61.5 ^ 2.4 67.2 ^ 2.3 63.4 ^ 2.5 20.3 ^ 2.7 (60) 76.3 ^ 2.0 79.3 ^ 2.1
100 ^ 0.2 91.3 ^ 0.5 92.4 ^ 0.3 89.1 ^ 1.0 79.0 ^ 1.2 88.0 ^ 1.0 85.2 ^ 1.1 67.5 ^ 1.4 81.4 ^ 1.0 94.5 ^ 0.5
100 ^ 0.0 100 ^ 0.0 100 ^ 0.0 100 ^ 0.2 100 ^ 0.4 100 ^ 0.3 100 ^ 0.2 100 ^ 0.5 100 ^ 0.2 100 ^ 0.0
3.127 2.937 2.951 2.922 2.765 2.916 2.880 2.579 3.081 3.008
The molar ratio of test compound to TPA giving 50% inhibition against a positive control (100%) was defined as IC50.
from the cell suspension. The activated cells were stained by high-titer EBV-EA positive sera from nasopharyngeal carcinoma (NPC) patients and detected by a conventional indirect immunofluorescence technique [13]. In each assay, at least 500 cells were counted, and the experiments were repeated three times. The average EA induction was compared with that of the positive control experiments with n-butyric acid (4 mM) plus TPA (32 pmol), in which EA induction was usually around 40%. 2.3. Electrochemical measurements Cyclic voltammetry was performed by a conventional three-electrode system using a laboratoryconstructed microcomputer-controlled system, in which a potentiostat (Hokuto Denko, HA-301) was used for controlling the working electrode potential. A plastic-formed-carbon (PFC) electrode of a surface area of 0.071 cm2 (BAS, PFCE-3), an Ag/AgCl (saturated KCl) electrode, and a platinum coil electrode were used as the working, reference, and counter electrodes, respectively. For each voltammogram, pretreatment of the working electrode was carried out as previously described [5 – 7]. The test solutions were degassed with prepurified N2 gas prior to the voltammetric measurements. The electrolytic cell was water-jacketed to maintain the temperature at 25 ^ 0.1 8C.
3. Results and discussion Ten 2-azaanthraquinones were tested for their inhibitory activities using a short-term in vitro assay of the EBV-EA activation induced by TPA in Raji cells. Their inhibitory effects on activation of the EBV-EA and the log IC50 values are shown in Table 1. 6,9-Dihydroxy-2-azaanthraquinone (8) showed the lowest viability and the highest inhibitory effect on Raji cells. Fig. 2 shows a typical cyclic voltammogram which was recorded in the presence of 0.1 mM 2-azaanthraquinone (1) in 0.1 M phosphate buffer (pH 7.2). The voltage scan rate was usually set
Fig. 2. Cyclic voltammogram of 2-azaanthraquione (1) at a PFC electrode in 0.1 M phosphate buffer (pH 7.2).
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Table 2 Epa ; Ecp ; E0 values of 2-azaanthraquinones Compound
Epa (V)
Epc (V)
E 0 (V)a
DEp (mV)
1 2 3 4 5 6 7 8 9 10
20.346 20.316 20.324 20.338 20.402 20.329 20.328 20.400 20.326 20.358
20.371 20.332 20.340 20.355 20.431 20.347 20.343 20.422 20.340 20.382
20.359 20.324 20.332 20.347 20.416 20.338 20.336 20.411 20.333 20.370
25 16 16 17 29 18 15 22 14 24
a
Assumed to be Emid :
at 100 mV s21. As seen in the figure, a well-defined wave with cathodic and anodic peaks was obtained. Although the wave seems to be somewhat affected by the adsorption of the redox species on the electrode surface, the peak separation, i.e. the difference between the cathodic and anodic peak potentials ðDEp ¼ Epa 2 Epc Þ is 29 mV, being close to the theoretical value of ca. 30 mV for a two-electron reversible wave [14]. The cathodic peak is ascribed to the two-electron reduction of the carbonyl groups, whereas the anodic peak is due to the reoxidation of the resultant hydroxyl groups. Similar reversible waves were obtained for the other 2-azaanthraquinones, though the DEp values for some 2-azaanthraquinones (2) – (4), (6) – (9) were somewhat low (see Table 2), possibly due to the effect of the adsorption of redox species on the electrode surface. However, when the concentration of the redox species was increased up to 0.5 mM, the wave shape was a little affected, but the midpoint potential ðEmid ¼ ðEpc þ Epa Þ=2Þ between the cathodic and the anodic peaks was hardly changed. Accordingly, the ‘adsorption wave’ for the adsorbed species on the electrode, if any, seems to be overlapped with the ‘diffusion wave’ for the redox species diffusing from the bulk solution onto the electrode and vice versa. Thus, Emid might be adequately assumed to be the apparent standard redox potential, E0 ; under the given conditions. It is generally known that the redox potentials of quinones are affected by pH. In this study, however, the E0 values for the 2-azaanthraquinones were determined at the physiological pH 7.2.
We examined their connection with the anti-tumor promoting effect. In Fig. 3, the values of log IC50 are plotted versus E0 (in V). The plot shows that there is an apparent correlation between the values of log IC50 and E0 : The log IC50 has been found to be represented by a regression line: log IC50 ¼ 4:021 þ 3:098E0 ðn ¼ 10; r ¼ 0:654Þ
ð1Þ
where n and r are, respectively, the number of test compounds and the correlation coefficient. Thus it has been revealed that the redox potential at pH 7.2 is an important parameter determining the EBV-EA activation of the 2-azaanthraquinones. The more negative the E0 of the 2-azaanthraquinone, the stronger the anti-tumor promoting effect. This tendency was similar to that of 1-azaanthraquinones [7]. There was some definite correlation between the log IC50 and the redox potential of the 19 1-aza- and 2-azaanthraquinones, the regression equation being log IC50 ¼ 4:259 þ 3:795E0 ðn ¼ 19; r ¼ 0:819Þ: Furthermore, we examined the correlation of log IC50 with the electronic properties of the 2-azaanthraquinones. Table 3 shows the electronic properties, including the HOMO energy, the LUMO energy, the difference between the HOMO and LUMO energies, and the charges at the C5, C10, O11, and O12 atoms. Among these electronic properties, the charges at C10 and O11 and the LUMO energy show no correlations with log IC50. On the other hand, the HOMO energy, the difference between the HOMO and LUMO energies, and the charges at C5 and O12 demonstrate some definite correlations. Since the charges at the C5
Fig. 3. Regression plot of log IC50 and standard redox potential at pH 7.2.
J. Koyama et al. / Cancer Letters 212 (2004) 1–6
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Table 3 Electronic properties of 2-azaanthraquinones
Compound
HOMO (eV)
LUMO (eV)
HOMO–LUMO
Charge of C5
Charge of C10
Charge of O11
Charge of O12
1 2 3 4 5 6 7 8 9 10
210.339 29.593 29.579 29.391 29.515 29.373 29.485 29.142 29.622 29.358
21.634 21.370 21.533 21.319 21.735 21.469 21.734 21.921 21.799 21.524
28.705 28.223 28.046 28.072 27.780 27.904 27.751 27.221 27.823 27.834
0.366 0.368 0.364 0.374 0.408 0.361 0.358 0.396 0.361 0.372
0.375 0.373 0.376 0.370 0.369 0.381 0.413 0.404 0.409 0.365
20.304 20.305 20.289 20.301 20.295 20.298 20.364 20.351 20.354 20.282
20.300 20.285 20.301 20.293 20.356 20.297 20.293 20.349 20.296 20.290
0.692
0.400
0.779
0.713
0.222
0.261
0.758
r* (to log IC50)
r* ; correlation coefficient.
and O12 atoms are compensated, respectively. The carbonyl group might be an important functional group. As additional parameters, we introduced the HOMO energy, denoted by d (HOMO), and performed the regression analyses. It was then found that log IC50 can be better expressed by the following equation than Eq. (1): log IC50 ¼ 1:108 þ 2:449E0 2 0:281dðHOMOÞ ðn ¼ 10; r ¼ 0:855Þ
ð2Þ
In conclusion, the standard redox potentials of the 2-azaanthraquinones determined at the physiological pH 7.2 and the charges at C5 and O12 and the HOMO energy are quite useful parameters for the estimation of the inhibitory effects of the 2-azaanthraquinones on EBV-EA activation. Thus, we postulate that it would be a common feature not only for the 1-azaanthraquinones [7], but also the 2-azaanthraquinones that the redox potential and the charges of the carbonyl group have a definite correlation with log IC50 on EBV-EA activation.
Acknowledgements This study was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health and Welfare, Japan, and also from the National Cancer Institute (CA 17625), USA.
References [1] T. Konoshima, M. Kozuka, J. Koyama, T. Okatani, K. Tagahara, H. Tokuda, Studies on inhibitors of skin tumor promotion, VI. Inhibitory effects of quinones on Epstein-Barr virus activation, J. Nat. Prod. 52 (1989) 987– 995. [2] G.J. Kapadia, V. Balasubramanian, H. Tokuda, T. Konoshima, M. Takasaki, J. Koyama, et al., Anti-tumor promoting effects of naphthoquinone derivatives on short term Epstein-Barr early antigen activation assay and in mouse skin carcinogenesis, Cancer Lett. 113 (1997) 47–53. [3] K. Tagahara, J. Koyama, T. Ogura, T. Konoshima, M. Kozuka, H. Tokuda, et al., Electronic properties and inhibitory effects on Epstein-Barr virus activation of mono- and di-substituted anthraquinones, Chem. Express 7 (1992) 557 –560.
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[4] J. Koyama, I. Morita, K. Tagahara, M. Ogata, T. Mukainaka, H. Tokuda, H. Nishino, Inhibitory effects of anthraquinones and bianthraquinones on Epstein-Barr virus activation, Cancer Lett. 170 (2001) 15– 18. [5] J. Koyama, K. Tagahara, T. Osakai, Y. Tsujino, S. Tsurumi, H. Nishino, H. Tokuda, Inhibitory effects on Epstein-Barr virus activation of anthraquinones: correlation with redox potentials, Cancer Lett. 115 (1997) 179 –183. [6] J. Koyama, I. Morita, N. Kobayashi, T. Osakai, H. Hotta, J. Takayasu, et al., Correlation of redox potentials and inhibitory effects on Epstein-Barr virus activation of naphthoquinones, Cancer Lett. 201 (2003) 25–30. [7] J. Koyama, I. Morita, K. Tagahara, T. Osakai, H. Hotta, M.X. Yang, et al., Correlation with Redox potentials and inhibitory effects on Epstein-Barr virus activation of azaanthraquinones, Chem. Pharm. Bull. 49 (2001) 1214–1216. [8] J.J.P. Stewart, Optimization of parameters for semi-empirical methods. I. Method (CAChe scientific, Computer-aided chemistry from the Oxford Molecular Group), J. Comp. Chem. 10 (1989) 209– 220.
[9] K.T. Potts, D. Bhattacharjee, E.B. Walsh, Cycloaddition routes to azaanthraquinone derivatives. 1. Use of azadienophiles, J. Org. Chem. 51 (1986) 2011–2021. [10] D.W. Cameron, K.R. Deutscher, G.I. Feutrill, Nucleo-philic alkenes. IX addition of 1,2-dimethoxyethene to azanaphthoquinones: synthesis of bostrycoidin and 8-O-methylbostrycoidin, Aust. J. Chem. 35 (1982) 1439–1450. [11] D.W. Cameron, K.R. Deutscher, G.I. Feutrill, D.E. Hunt, Synthesis of azaanthraquinones: homolytic substitution of pyridines, Aust. J. Chem. 35 (1982) 1451–1468. [12] Y. Ito, S. Yanase, J. Fujita, T. Harayama, M. Takashima, H. Imanaka, A short-term in vitro assay for promoter substances using human lymphoblastoid cells latently infected with Epstein-Barr virus, Cancer Lett. 13 (1981) 29–37. [13] G. Henle, W. Henle, Immunofluorescence in cells derived from Brukitt’s lymphoma, J. Bacteriol. 91 (1966) 1248–1256. [14] R.S. Nicholson, I. Shain, Theory of stationary electrode polarography. Single scan and cyclic methods applied to reversible, irreversible, and kinetic systems, Anal. Chem. 36 (1964) 706 –723.
Correlation of redox potentials and inhibitory effects on Epstein-Barr virus activation of 2-azaanthraquinones Junko Koyamaa,*, Izumi Moritaa, Norihiro Kobayashia, Toshiyuki Osakaib, Hiroki Hottab, Junko Takayasuc, Hoyoku Nishinoc, Harukuni Tokudac a
Faculty of Pharmaceutical Sciences, Kobe Pharmaceutical University, Higashinada, Kobe 658-8558, Japan b Department of Chemistry, Faculty of Science, Kobe University, Nada, Kobe 657-8501, Japan c Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan Received 25 December 2003; received in revised form 27 February 2004; accepted 8 March 2004
Abstract As a continuation of our studies using natural and synthetic products as cancer chemopreventive agents, we examined the standard redox potentials of some 2-azaanthraquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the redox potentials and the inhibitory effects of the 2-azaanthraquinones on Epstein-Barr virus early antigen (EBV-EA) activation. It has been further shown that the correlation can be enhanced by introducing an electronic properties, i.e. the atomic charges at the C5 and O12 atoms in the quinone skeleton ring and the HOMO energy as additional parameters. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Epstein-Barr virus; 2-Azaanthraquinone; Redox potential; Cyclic voltammetry
1. Introduction Quinones (anthraquinones, naphthoquinones, and heteronaphthoquinones) are important naturally occurring pigments that are widely distributed in nature and are known to demonstrate various physiological activities as antibiotics and anti-cancer agents. In our previous studies, we reported the inhibitory effects of a number of anthraquinones, naphthoquinones and 1-azaanthraquinones on 12-Otetradecanoylphorbol-13-acetate (TPA)-induced * Corresponding author. Tel.: þ81-78-441-7549; fax: þ 81-78441-7550. E-mail address: [email protected] (J. Koyama).
EBV-EA activation [1 – 3]. We have also found inhibitory activities of the mono- and di-substituted anthraquinones and bianthraquinones on EBV-EA activation, and have studied their connections with the electronic properties of the anthraquinones [3,4]. In studies of the structure-activity relationships for drugs, the standard redox potential is one of the most important parameters to determine the physiological activities. We employed cyclic voltammetry to determine the standard redox potentials of 9 anthraquinones, 9 naphthoquinones and 9 1-azaanthraquinones at the physiological pH 7.2, and found definite correlations between the standard redox potentials and the inhibitory effects (log IC50) on EBV-EA activation [5 –7].
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.03.005
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J. Koyama et al. / Cancer Letters 212 (2004) 1–6
In the present study, we report the redox potentials of 10 2-azaanthraquinones and the structure-activity relationship between the inhibitory effects and the redox potentials of the 2-azaanthraquinones. Furthermore, we have calculated some electronic properties of the 2-azaanthraquinones by the PM3 method using the CAChe MOPAC program [8]. It has been revealed that the atomic charges at C5 and O12 could be used as other useful parameters to characterize the inhibitory effect on EBV-EA activation. Moreover, it was found that the 1-azaanthraquinones and 2-azaanthraquinones had similar correlations of log IC50 on EBV-EA activation with the redox potential and the charges of the carbonyl group.
2. Material and methods 2.1. Reagents and materials Benzo[g ]isoquinoline-5,10-dione (1) was purchased from Aldrich (Wiscosin, USA). 6-Methoxybenzo[g ]isoquinoline-5,10-dione (2) and 9-methoxybenzo[g ]isoquinoline-5,10-dione (3) were synthesized from isoquinoline-5,8-dione and 1-methoxy-1,3-cyclohexadiene [9]. 6,8-Dimethoxybenzo [g ]isoquinoline-5,10-dione (4), 7,9-dimethoxybenzo [g ]isoquinoline-5, 10-dione (6), and 9-bromo-6,8dimethoxybenzo[g ]isoquinoline-5,10-dione (10) were synthesized from isoquinoline-5, 8-dione and 1,1-dimethoxyethene [10]. 6,9-Dihydroxybenzo[g ] isoquinoline-5,10-dione (8) was synthesized from cinchomeronic anhydride and 1,4-dimethoxybenzene [11]. 6-Hydroxy-8-methoxybenzo[g ]isoquinoline5,10-dione (5), 9-hydroxy-7-methoxybenzo[g ]isoquinoline-5,10-dione (7), and 9-hydroxybenzo[g ] isoquinoline-5,10-dione (9) were synthesized by demethylation with boron trifluoride from (4), (6) and (3), respectively (Fig. 1). 6-Hydroxy-8-methoxybenzo[g ]isoquinoline-5,10dione (5) IR (CHCl3) cm21: 1688, 1667, 1575. 1HNMR (CDCl3) d: 3.97 (3H, s, OCH3), 6.74 (1H, d, J ¼ 2.2 Hz, 7-H), 7.39 (1H, d, J ¼ 2.2 Hz, 9-H), 8.07 (1H, dd, J ¼ 5 Hz, 4-H), 9.10 (1H, d, J ¼ 5 Hz, 3-H), 9.51 (1H, s, 1-H). HR-MS m/z: 255.0544 (Mþ, Calcd for C14H9NO4: 255.0531). 9-Hydroxy-7-methoxybenzo[g ]isoquinoline-5,10dione (7) IR (CHCl3) cm21: 1682, 1633, 1579.
1
H-NMR (CDCl3) d: 3.96 (3H, s, OCH3), 6.78 (1H, d, J ¼ 2.5 Hz, 8-H), 7.38 (1H, d, J ¼ 2.5 Hz 6-H), 8.03 (1H, d, J ¼ 5 Hz 4-H), 9.08 (1H, d, J ¼ 5 Hz, 3-H), 9.56 (1H, s, 1-H). HR-MS m/z: 255.0558 (Mþ, Calcd for C14H9NO4: 255.0531). 9-Hydroxybenzo[g ]isoquinoline-5,10-dione (9) IR (CHCl3) cm21: 3400, 1680, 1640, 1579. 1H-NMR (CDCl3) d: 7.38 (1H, d, J ¼ 8 Hz 8-H), 7.77 (1H, t, J ¼ 8 Hz 7-H), 7.85 (1H, d, J ¼ 8 Hz 6-H), 8.06 (1H, d, J ¼ 5 Hz, 4-H), 9.12 (1H, d, J ¼ 5 Hz, 3-H), 9.59 (1H, s, 1-H), 12.44 (1H, s, OH). HR-MS m/z: 225.0408 (Mþ, Calcd for C13H7NO3: 225.0425). The tissue culture reagents, 12-O-tetradecanoylphorbol-13-acetate (TPA), n-butyric acid and other reagents, were from Nacalai Tesque. The EBVgenome-carrying lymphoblastoid cells (Raji cells derived from Burkitt’s lymphoma) were cultured in RPMI 1640 medium (Nissui), as described elsewhere [12]. Spontaneous activation of EBV-EA in our subline of Raji cells was less than 0.1%. 2.2. Procedure of EBV-EA activation The inhibition of the EBV-EA activation was assayed using Raji cells (virus non-producer), which were cultivated in 10% FBS RPMI 1640 medium. The indicator cells (Raji) (1 £ 106/ml) were incubated at 37 8C for 48 h in 1 ml of the medium containing n-butyric acid (4 mM, inducer), 2 ml of TPA (20 ng/ml in DMSO), and a known amount of test compound dissolved in DMSO. Smears were made
Fig. 1. Structures of Compounds 1–10.
J. Koyama et al. / Cancer Letters 212 (2004) 1–6
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Table 1 Inhibitory effects of 2-azaanthraquinones on EBV-EA activation Compound
% to control (% viability) log IC50a
Molar ratio (to 32 pmol TPA)
1 2 3 4 5 6 7 8 9 10 a
1000
500
100
10
63.6 ^ 2.2 (60) 43.7 ^ 1.3 (60) 44.9 ^ 1.5 (60) 41.3 ^ 1.1 (60) 16.8 ^ 0.8 (70) 40.5 ^ 1.6 (60) 37.5 ^ 1.5 (60) 2.5 ^ 0.4 (50) 57.2 ^ 2.3 (60) 49.6 ^ 1.9 (60)
84.9 ^ 1.9 69.2 ^ 2.3 71.6 ^ 2.1 68.5 ^ 2.4 61.5 ^ 2.4 67.2 ^ 2.3 63.4 ^ 2.5 20.3 ^ 2.7 (60) 76.3 ^ 2.0 79.3 ^ 2.1
100 ^ 0.2 91.3 ^ 0.5 92.4 ^ 0.3 89.1 ^ 1.0 79.0 ^ 1.2 88.0 ^ 1.0 85.2 ^ 1.1 67.5 ^ 1.4 81.4 ^ 1.0 94.5 ^ 0.5
100 ^ 0.0 100 ^ 0.0 100 ^ 0.0 100 ^ 0.2 100 ^ 0.4 100 ^ 0.3 100 ^ 0.2 100 ^ 0.5 100 ^ 0.2 100 ^ 0.0
3.127 2.937 2.951 2.922 2.765 2.916 2.880 2.579 3.081 3.008
The molar ratio of test compound to TPA giving 50% inhibition against a positive control (100%) was defined as IC50.
from the cell suspension. The activated cells were stained by high-titer EBV-EA positive sera from nasopharyngeal carcinoma (NPC) patients and detected by a conventional indirect immunofluorescence technique [13]. In each assay, at least 500 cells were counted, and the experiments were repeated three times. The average EA induction was compared with that of the positive control experiments with n-butyric acid (4 mM) plus TPA (32 pmol), in which EA induction was usually around 40%. 2.3. Electrochemical measurements Cyclic voltammetry was performed by a conventional three-electrode system using a laboratoryconstructed microcomputer-controlled system, in which a potentiostat (Hokuto Denko, HA-301) was used for controlling the working electrode potential. A plastic-formed-carbon (PFC) electrode of a surface area of 0.071 cm2 (BAS, PFCE-3), an Ag/AgCl (saturated KCl) electrode, and a platinum coil electrode were used as the working, reference, and counter electrodes, respectively. For each voltammogram, pretreatment of the working electrode was carried out as previously described [5 – 7]. The test solutions were degassed with prepurified N2 gas prior to the voltammetric measurements. The electrolytic cell was water-jacketed to maintain the temperature at 25 ^ 0.1 8C.
3. Results and discussion Ten 2-azaanthraquinones were tested for their inhibitory activities using a short-term in vitro assay of the EBV-EA activation induced by TPA in Raji cells. Their inhibitory effects on activation of the EBV-EA and the log IC50 values are shown in Table 1. 6,9-Dihydroxy-2-azaanthraquinone (8) showed the lowest viability and the highest inhibitory effect on Raji cells. Fig. 2 shows a typical cyclic voltammogram which was recorded in the presence of 0.1 mM 2-azaanthraquinone (1) in 0.1 M phosphate buffer (pH 7.2). The voltage scan rate was usually set
Fig. 2. Cyclic voltammogram of 2-azaanthraquione (1) at a PFC electrode in 0.1 M phosphate buffer (pH 7.2).
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Table 2 Epa ; Ecp ; E0 values of 2-azaanthraquinones Compound
Epa (V)
Epc (V)
E 0 (V)a
DEp (mV)
1 2 3 4 5 6 7 8 9 10
20.346 20.316 20.324 20.338 20.402 20.329 20.328 20.400 20.326 20.358
20.371 20.332 20.340 20.355 20.431 20.347 20.343 20.422 20.340 20.382
20.359 20.324 20.332 20.347 20.416 20.338 20.336 20.411 20.333 20.370
25 16 16 17 29 18 15 22 14 24
a
Assumed to be Emid :
at 100 mV s21. As seen in the figure, a well-defined wave with cathodic and anodic peaks was obtained. Although the wave seems to be somewhat affected by the adsorption of the redox species on the electrode surface, the peak separation, i.e. the difference between the cathodic and anodic peak potentials ðDEp ¼ Epa 2 Epc Þ is 29 mV, being close to the theoretical value of ca. 30 mV for a two-electron reversible wave [14]. The cathodic peak is ascribed to the two-electron reduction of the carbonyl groups, whereas the anodic peak is due to the reoxidation of the resultant hydroxyl groups. Similar reversible waves were obtained for the other 2-azaanthraquinones, though the DEp values for some 2-azaanthraquinones (2) – (4), (6) – (9) were somewhat low (see Table 2), possibly due to the effect of the adsorption of redox species on the electrode surface. However, when the concentration of the redox species was increased up to 0.5 mM, the wave shape was a little affected, but the midpoint potential ðEmid ¼ ðEpc þ Epa Þ=2Þ between the cathodic and the anodic peaks was hardly changed. Accordingly, the ‘adsorption wave’ for the adsorbed species on the electrode, if any, seems to be overlapped with the ‘diffusion wave’ for the redox species diffusing from the bulk solution onto the electrode and vice versa. Thus, Emid might be adequately assumed to be the apparent standard redox potential, E0 ; under the given conditions. It is generally known that the redox potentials of quinones are affected by pH. In this study, however, the E0 values for the 2-azaanthraquinones were determined at the physiological pH 7.2.
We examined their connection with the anti-tumor promoting effect. In Fig. 3, the values of log IC50 are plotted versus E0 (in V). The plot shows that there is an apparent correlation between the values of log IC50 and E0 : The log IC50 has been found to be represented by a regression line: log IC50 ¼ 4:021 þ 3:098E0 ðn ¼ 10; r ¼ 0:654Þ
ð1Þ
where n and r are, respectively, the number of test compounds and the correlation coefficient. Thus it has been revealed that the redox potential at pH 7.2 is an important parameter determining the EBV-EA activation of the 2-azaanthraquinones. The more negative the E0 of the 2-azaanthraquinone, the stronger the anti-tumor promoting effect. This tendency was similar to that of 1-azaanthraquinones [7]. There was some definite correlation between the log IC50 and the redox potential of the 19 1-aza- and 2-azaanthraquinones, the regression equation being log IC50 ¼ 4:259 þ 3:795E0 ðn ¼ 19; r ¼ 0:819Þ: Furthermore, we examined the correlation of log IC50 with the electronic properties of the 2-azaanthraquinones. Table 3 shows the electronic properties, including the HOMO energy, the LUMO energy, the difference between the HOMO and LUMO energies, and the charges at the C5, C10, O11, and O12 atoms. Among these electronic properties, the charges at C10 and O11 and the LUMO energy show no correlations with log IC50. On the other hand, the HOMO energy, the difference between the HOMO and LUMO energies, and the charges at C5 and O12 demonstrate some definite correlations. Since the charges at the C5
Fig. 3. Regression plot of log IC50 and standard redox potential at pH 7.2.
J. Koyama et al. / Cancer Letters 212 (2004) 1–6
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Table 3 Electronic properties of 2-azaanthraquinones
Compound
HOMO (eV)
LUMO (eV)
HOMO–LUMO
Charge of C5
Charge of C10
Charge of O11
Charge of O12
1 2 3 4 5 6 7 8 9 10
210.339 29.593 29.579 29.391 29.515 29.373 29.485 29.142 29.622 29.358
21.634 21.370 21.533 21.319 21.735 21.469 21.734 21.921 21.799 21.524
28.705 28.223 28.046 28.072 27.780 27.904 27.751 27.221 27.823 27.834
0.366 0.368 0.364 0.374 0.408 0.361 0.358 0.396 0.361 0.372
0.375 0.373 0.376 0.370 0.369 0.381 0.413 0.404 0.409 0.365
20.304 20.305 20.289 20.301 20.295 20.298 20.364 20.351 20.354 20.282
20.300 20.285 20.301 20.293 20.356 20.297 20.293 20.349 20.296 20.290
0.692
0.400
0.779
0.713
0.222
0.261
0.758
r* (to log IC50)
r* ; correlation coefficient.
and O12 atoms are compensated, respectively. The carbonyl group might be an important functional group. As additional parameters, we introduced the HOMO energy, denoted by d (HOMO), and performed the regression analyses. It was then found that log IC50 can be better expressed by the following equation than Eq. (1): log IC50 ¼ 1:108 þ 2:449E0 2 0:281dðHOMOÞ ðn ¼ 10; r ¼ 0:855Þ
ð2Þ
In conclusion, the standard redox potentials of the 2-azaanthraquinones determined at the physiological pH 7.2 and the charges at C5 and O12 and the HOMO energy are quite useful parameters for the estimation of the inhibitory effects of the 2-azaanthraquinones on EBV-EA activation. Thus, we postulate that it would be a common feature not only for the 1-azaanthraquinones [7], but also the 2-azaanthraquinones that the redox potential and the charges of the carbonyl group have a definite correlation with log IC50 on EBV-EA activation.
Acknowledgements This study was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health and Welfare, Japan, and also from the National Cancer Institute (CA 17625), USA.
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