Couuer . =1 Complexes of l,lO-Phenanthroline and Related Compounds as Superoxide Dismutase Mimetics G. J. Bijloo, H. van der Coot, A. Bast, and H. Timmerman Department of Pharmacochemistry, Vrije Universiteit, Amstemlam, The Netherlands

ABSTRACT In a preliminq study we tested CuS045Hz0, (~(II))z[3,Jdiisopropylsalicylate]salicylatel4~2H20 and a number of copper complexes of substituted l,lO-pbenanthrolines for superoxide anion dismutase activity. It appeared that this activity depends on the ligands involved and might be governed by the redox potential of the C!u(I) complex/C!u(II)complex couple. The strong superoxide anion dismutase activity of Cu(II)lDMPh complex can be expected considering its high redox potential. gather surprisingly is the superoxide anion diimutase activity of the Cu(l)[DMP]~complex since it involves oxidation to Cu(II)[DMP]2complex. Prom regression analysis it wss established that steric and field effects of the substituentsof the investigatedphenantluolinesplay an important role in SOD activity and therefore it is ciXiCiii&d

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INTRODUCTION Oxygen radicals play a role in many pathophysiological conditions. In conditions such as ischemia, arthetiosclerosis, irradiation, and inflammation the dismutation of superoxide anion radicals (02 --) is important in order to avoid serious damage to living organisms. Many ways are persued to control or prevent tissue damage caused by free radicals. Wide pot&ally therapeutic implications emerge when adequate pharmacological regulation of free radical processes become possible. One of the enzymes hv0ivd in the endogenous protection against oxygen radical toxicity is superoxide dismutase (SOD). It has often been reported that copper complexes can exhibit SOD activity. Authors like Czapski [ 1, 21, Huber [3], Sorenson [4], Leuthauser [5], and many others have investigated several copper complexes in this respect. Most of these authors however, did not work on SOD mimetics with the aim to predict SOD activity on the basis of physico-chemical properties. Since in SOD activity a one electron transfer is involved,

Address reprint requests to: Ing. G. J. Bijloo, Dept. of pharmaco&emistry, Vrije Universiteit, De Boelelaan 1083, lOS1HV Amsterdam, The Netherlands. Journal of Inotganic Biochemistry, 40,23X244 (1990) @ 1990 Ehvicr Science Publishing Co., Inc., 655 Avenue

ofthe Americas.

NY, NY 10010

0162-O134/90/$3~

230

G. J. Bijroo et al.

the redox potential of a copper complex might be an important parameter for its activity. Since regression analysis reveals that steric and field effects play a role, we conclude that the complex formation is important for the superoxide dismutase-like activity. EXPERIMENTAL Chemicals Xantbine oxidase (grade I), Fb(II1) cytochrome c (type III) and catalase were obtained from Sigma Chemical Co. (U. S .). Xanthine was purchased from Merck (FRG), CuZn-superoxide dismutase from Boehringer-Mannbeim (FRG), mannitol, NazHPOh, NaI-I#04, CuSO45H20, DMSO, and Cu(N0&.3H20 from Baker (the Netberlands); -,1.1- &Phenanthrnline & &s)-rl_&&y!-l) ~@nhenanthmline ~_-_--- -_-- ___-__[DMQ _ _ --_-_---- ___-__[Phenl L- -----a were obtained from Loba Chemie (Austria). Bis(2,9dimetbyl-l,lO-phenanthroline)Cu(I)nitrate (Cu(I)[DMP]z) and other 2,9-disubstituted-l , lO-phenantbrolines (Table 1) were from laboratory stock [6, 71. (Cu(II))z [3,5-diisopropylsali$ylate]4.2H20 TABLE 1. SOD Activity of Copper Complexesof 1, lo-Phenanthroline Derivatives - log EC,(M)

E,W) 0.40 vs NHE’13’ O.lP’

8.08 f 0.11 6.16 f 0.04

Cu-Zn-SOD

CuSO, .5H 2O

6.58 f 0.04 CyDIPS,.l&O

4.63 f 0.10 @I

Cd7

C3H7

CH,O CH,O C,H,O C,H,O Cl

H CH,O H C,H,O Cl

E,

- 0.055”5’

EC, not reached 5.20 f 0.13 5.77 f 0.15 5.57 f 0.04 5.76 f 0.17 4.54Lto.11 4.72 f 0.03 4.45 f 0.01 4.68 f 0.06 4.73 f 0.03

0.0 0.0 0.0 0.0 0.3 0.6 0.28 0.56 0.82

- 1.31 -2.62 -1.60 -3.20 -0.55 -1.10 - 0.55 - 1.10 -1.94

5.87 f 0.01

0.0

- 2.48

4.75 f 0.01 EC, not reached becauseof hydrolysis

0.59”@

(CurDIPS4.2H20) was kindly provided by J. R. J. Sorenson, University of Arlcmsas, Little Rock, U.S. Methods Superoxide dismutase activity of copper complexes was determined according to the method of McCord and Pridovich [8]. Superoxide anion radicals (02 -*) were generated in sodium phosphate buffer (pH 7.8) using xanthine (5.10m5 M) and 0.6 U xanthine oxidase to properly measure the formation of Ib(II) cytochrome c. Reduction of Fe(III) cytochrome c (1.1.1O-5 M) to T;e(II.)cytochrome c was followed spectrophotometrically ( Aminco DW2A spectrophotometer , dual wavelength mode 550/540 run). Contrary to McCord and Pridovich we did not use EDTA in order to avoid the formation of Cu-EDTA complexes. Cu(II)[phen]p complex was prepared by dissolving CU(NO~)~-~H~O in water and adding two equivalents of l,lO-phenanthroline dissolved in DMSO, or used directly from laboratory stock in a 10% DMSO/water solution. The other [2,9di~ub&uted-1 _l&nhenanthrolinel~ CufID r~~-~~-~~-~~ _~~~~_~_ \~~, complexes were prepared in the same way -L--L______ _,__ using cus04~5&0 instead of CU(NO~)~-~H#. Cu(I)[DMP]2 complex WASdissolved in 10% DMSO/water, Cu2DIPS4.5HrO was dissolved in water. Inhibition of the reduction of Fb(III) cytochrome c by supcroxide dismutase (SOD) was used for comparative purposes. The amount of copper complex which gives 50% inhibition of the formation of I;e(II) cytochrome c has been determined. RESULTS AND DISCUSSION Xanthine and xanthine oxidase are used to generate 02 -* radicals. These radicals reduce Pe(III) cytochrome c to Pe(I1) cytochrome c according to the following reaction: Pe(II1) cytochrome c + 0,.

+ Fe(H) cytochrome c + 02.

(1)

At physiological pH the following decomposition of &-* also occurs, leading to the formation of H202: 20,’ + 2H+ ---)H202 + 02.

(2)

Hz& can also reduce the Fe(III) cytochrome c: Pe(II1) cytochrome c + Hz02 + Fe(H) cytochrome c + 0;. + 2H+.

(3)

Purthermore the Hz02 can give a Fenton-type reaction in which the highly reactive OH radical can arise: Fe(I1) cytochrome c + Hz02 + Iie(III) cytochrome c + OH + OH-,

(4)

Fe(I1) cytochrome c + OH. + Fe(III) cytochrome c + OH-.

(5)

In general the occurrence of reactions (3) and (4) can be shown by adding catalase which acts as an Hz& scavenger and the occurrence of reaction (5) canbe shown by

240

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Bijroo et al.

adding mamtitol which acts as an OW scavenger. RnQmately, no intluence of either catalase or mannitol could be established in our experimental setup. Cu-Zn-superoxide dismutase (SOD) is a major naturally occurring superoxide anion scavenger. The following reactions take place: Cu(II)-SOD + 0,.

+ Cu(I)-SOD + 9,

CuWSOD + 0;. + 2H+ + Cu(II)-SOD + HzOz.

(6) (7)

The net reaction is the same as in reaction (2): 20;’ +2H+ -+H2% +a.

(8)

When the SOD is added to the assay superoxide anion radicals will be scavenged by the enzyme, ieaving less superoxide anion radicals availabie for the reduction of R(III) cytochrome c. This could be measured spectrophotometrically as in that case less Fb(II) cytochrome c per time is formed. It is known that free Cu(II) ions and a number of Cu(II) complexes can be used as scavengers of 0;. [9, lo], in which case the reaction will be comparable to that mentioned in reaction (6). As the SOD mimetic effect involves a redox cycle of Cu(I1) and Cu(I) it is to be expected that the redox potential of a copper(D) complex/copper(I) complex couple influences the SOD activity. Because it is well known that the redox potentials of copper complexes depend on their ligands a number of complexes have been tested. The ECm values are given in Table 1. Additionally, all these complexes have been tested for a direct redox reaction between k(III) cytochrome c and Cu(I) complex. R(II1) cytochrome c + &(I) complex + l+(H) cytochrome c + Cu(II) complex. (9) This reaction did not occur with the complexes described in this paper (not shown). From Figure 1 it can be seen that SOD inhibits the a-* induced formation of l+(D) cytochrome c up to about So%. CUSO~~~H~Oshowed about 90% inhibition of the formation of R(II) cytochrome c (Fig. 2), CuzDIP& -2H20 gave a maximal inhibition of about So% (Fig. 3), and Cu(I)[DMP]2 complex about 70% (Fig. 4). F;or the Cu(II)[Phen]2 complex the maximal inhibition was not more than about 50% FIGURE 1. Inhibition of the forma-

NHI%inhibilm

tion 80 .

.

preselw

;; 60. 40’ 20 .

0:. . !

I .

l

0

8

of Fe0 cytochn#neCinthe of superoxide disxmltase.

SUPEROXIDE DISMUTASE MIMETICS

100'

FIGURE 2. Inhibition of the formation of Fe(II) cytochrome c in the presence of CuSO4.SH20.

9E inhibition

tt

80'

241

60'

QO Do

40'

20'

0 3.5



,i0 ,15 510 51, ,i0

6.5

7.0

• | 8.0

7.5

-log conc CuSO4.SH20

(Fig. 5); therefore a proper ECso could not be established. The reason for this is not known, but it is clear that the ligands used to coordinate copper play a role in this phenomenon. The other complexes tested all gave a maximum of about 90% inhibition of the formation of Fe(II) cytochrome c. From the experiments it is obvious that ECs0 values depend strongly on the ligands (see Table l). No copper complex has been found that has a log EC50 value in the same range as the supemxide dismutase itself ( - l o g ECso -- 8.08 4-0.11). The best log ECso values were measured for Cu2DIPS4-2H20 and for free Cu(II) ( log ECso = 6.58 4- 0.04 and 6.16 4- 0.04, respectively). From stability constants of these complexes it can be calculated that at complex concentration the amount of free copper ions is very low. This amour~ of free copper ions cannot be the cause of the SOD-like activity. To exert its SOD mimetic activity a Cu(I) species has to convert into its Cu(II) form. In the case of Cu(I)(DMP)2 with its redox potential of + 590 mV rather unphysiological circumstances are required to achieve oxidation to the Cu(II)(DMP)2 complex, which is very unlikely to be realized by reaction (10). -

2H + + O~-" + e

-,

H202

(lO)

Figure 6 shows that under the conditions of our assay the absorbance of Cu(H)[DMP]2 decreases in time, which has to be ascribed to the reduction of Cu(II)[DMP]2 into Cu(I)[DMP]2 by O2-" radicals ( • ). During this period, reducI~"

FIGURE 3. Inhibition of the formation of Fe01) cytochrome c in the presence of Cu2DI~'2H20.

% inhibilio~ 80'

0. 60"

..# e 4o • 40"

20"

D 0 3.5

410 -log conc Cu2DIPS4.2H20

242

G. J. Bijloo et al. too

FIGURE 4. (0) Inhibition of the formarion of Fe(ID cytochrome c in the presence of Cu(I)[DMP]2., (/X) inhibition of the formation of Fe0I) cytochrome c in the presence of Cu(ID[DMP]2.

% inhibition 8o $

$

s

6O

"t $o

4o

t 2o

0 3.5

4.0

4.5

i

i



5.0

5.5

6.0

J 6.5

710

, 7.5

i 8.0

-log conc Cu(I)[DMP] 2

tion of Fe(III) cytochrome c to Fe(II) cytochrome c does not occur at all ( + ) , probably due to the fact that all 02-" radicals are consumed in the reduction of Cu(II)[DMP]2 complex. This can be concluded from the parallel part of the lines ( • ) and (+). At the moment the concentration of Cu(II)[DMP]2 complex is in equilibrium with the concentration of Cu(I)[DMP]2 complex (compare ( • ) and ( + ) ) , Fe(III) cytochrome c is reduced to Fe(II) cytochrome c (+). However the rate of reduction of Fe(III) cytochrome c is still lower than the rate of reduction of Fe(III) cytochrome c without the presence of copper complex ( A ). We therefore assume that the Cu(I)[DMP]2 complex shows SOD-like activity too. This is confirmed in the experiment in which Cu(I)[DMP]2 complex is added to the assay and shows an inhibition of the formation of Fe(II) cytochrome c (see Figs. 4 and 6 (x)). Table 1 shows a - log ECs0 of 5.87 for the Cu(I)[DMP]2 complex. It seems that in this case a reaction comparable to reaction (7) followed by reaction (6) takes place. It is obvious that only 02-" radicals were involved because neither catalase (H202 scavenger) nor mannltol (OH" scavenger) showed any influence on the reactions taking place (not shown). Huber et al. [3] have demonstrated that the various copper complexes they tested have the ability to decompose H202 via the oxidation of Cu(I) complex to Cu(II) complex (Fenton reaction). In case of the Cu(I)[DMP]2 complex we found no reaction with catalase, meaning that in this example there probably is no Fentou type reaction. For a number of substituted phenanthrolines (Table 1) the following relation between - l o g ECs0 and the steric parameter Es of Taft [11] was obtained: - log ECs0 = -0.539( 4- 0.117)F_~ + 4.240( 4- 0.217)

(11)

n = 10, r = 0.852, s = 0.316. FIGURE 5. Inhibition of the formarion of Fe(H) cytochrome c in the presence of Cu(H)[Phen]2.

100% inhibition 80.~

60-~

It I 40-

0

! •

20 "~

3.5

4.0

4.5

5.0

5.5

6.0

$

615 7'0 715 8;0 -log conc Cu(II)[Phen]2

SUPEROXIDE DISMUTASE MIMETICS

243

0.25" 0.20 ~

~

,

,

.

,

~

.

& .I.

0.15 0.10 -

0.05" 0.00

FIGURE 6. ( A ) Reduction of Fe(HI) cytochrome c to 1~(11) cytochrome c by O2-'; (+) reduction of Fe(IH) cytochromc c to Fe(ID cytochromc c by Oz-" in the presence of Cu(II)[DMP]2; ( • ) reduction of Cu(H)[DMP]2 to Cu(I)[DMP]2 by O2-" ( × ) reduction of Fe(IIl) cytochmme c to Fe(ID cytochrome c by 02-- in the presence of Cu(I)[DMP]2; all other information in text.

The negative values of Es together with the negative coefficient of this parameter suggest that bulkiness favors the inhibitory activity of the complex. This is explained by the fact that the bulkiness of the substituent favors the formation of the Cu(I) complexes preferring the tetrahedral coordination. In contrast, Cu(II) complexes have a square planar coordination which is negatively influenced by the bulkiness of the substituent. Further improvement of the statistics of Eq. (11) could be achieved by adding the electronic inductive parameter ax of Charton [12] as an independent paralneter. - log EC~ = -0.411( + 0.070)~ - 0.909( -4-0.205)o~ + 4.684( d=0.155) (12) n = 10, r = 0.963, s = 0.173. The positive values of the OI parameter combined with the negative sign of the coefficient of this parameter indicate that the field effect should be as small as possible. Since the - log ECs0 of these compounds correlates with the parameters E, and OI whidl ar~ involved in copper complex formation, and since the redox potential depends on the ratio Cu(I) complex/Cu(II) complex the relation between - l o g EC50 and the redox potential is obvious. Unfortunately, in literature until now we were not able to find redox potential values of any appropriate complex which would enable us to study a direct relationship between the ECs0 and Eo values of the pbenanthrolines. CONCLUSION Using the xanthine/xanthine oxidase system to generate O2-" we have shown that copper complexes of a series of compounds show superoxide dismutase-libe activity; these complexes do not have an efficiency comparable to SOD. This finding seems in good agreement with the findings of Czapski and Goldstein [10]. From the experiments it is clear that ligands used to coordinate copper determine the extent of the dismutase activity as measured via the inhibition of the formation of Fe(II) cytochrome c. The expectation that redox potentials play a role was confirmed

244

G. J. Bijloo et al.

by the very strong SOD-like activity of the Cu(II)[DMP]2 complex. The results of the regression analysis which shows a good correlation between the ECs0 values and the steric parameter F~ are also an indication that the redox potentials of these copper complexes play a role. We had expected that Cu(1)DI~&P2 complex would be devoid of any SOD activity. However, as this complex shows a SOD-like activity and because the field effect plays a role in the regression analysis, one has to assume that other physico-chemical effects are also involved. The authors wish to thank the students, Miss A. C. LangedUk and Mr. A. Baarslag for valuable technical assistance.

REFERENCES 1. G. Czapski, S. Goldstein, and D. Meyerstein, Free Pad. Res. Comm. 4, 221 (1988). 2. G. Czapski and S. Goldstein, Free Pad. Res. Comm. 1, 157 (1986). 3. K, L. Huber, R. Sridhar, E. H. Griflith, E. L. Amma, and J. Roberts, Biochim. Biophys. Acta 915, 267 (1987). 4. J. R. J. Sorenson, J. Med. Chem. 27, 1747 (1984). 5. S. W. C. Leuthauser, L. W. Oberley, T. D. Oberley, J. R. J. Sorenson, and G. R. Beuttner, in Oxygen and Oxy-radicals in Chemistry and Biology, M. A. J. Rogers and E. L. Powers, Eds., Acad. Press, 1981, p. 679. 6. P. J. Pijper, H. van der Goot, H. Timmerman, and W. Th. Nanta, Eur. J. Med. Chem. 19, 399 (1984). 7. L. Gmelin Gmelins Handbuch der Anorg. Chemie, Auflage 8, Kupfer, Teil B-Lieferung 4, Verlag Chemie GmbH, Weinheim, 1966, pp. 1456-1467. 8. J. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). 9. J. R. J. Sorenson, Chemistry in Brittain, p. 169 (1989). 10. G. Czapski and S. Goldstein, Free Rad. Res. Comm. 4, 225 (1988). 11. S. H. Unger and C. Hansch, Progr. Phys. Org. Chem. 12, 91 (1976). 12. M. Charton, Progr. Phys. Org. Chem. 13, 119 (1981). 13. I. Kirnura, T. Koike, Y. Shimizu, and M. Kodama, Inorg. Chem. 25, 2242 (1986). 14. D. A. Phipps, in Methods and Metabolism, P. W. Atkins, J. S. E. Holker, and A. K. HoUiday Eds., Clarendon Press, Oxford (1976). 15. S. Goldstein and G. Czapski, J. Amer. Chem. Soc. 105, 7276 (1983). 16. T. M. Florence, J. L. Stauber, K. K. Mann, J. Inorg. Biochem. 24, 243 (1985). Received December 18, 1989; accepted March 7, 1990

Copper complexes of 1,10-phenanthroline and related compounds as superoxide dismutase mimetics.

In a preliminary study we tested CuSO4.5H2O, (Cu(II]2[3,5-diisopropylsalicylate]4.2H2O and a number of copper complexes of substituted 1,10-phenanthro...
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