Trolox, Troxy! or Troxyl + 200 pM GSH (a); scans b and c - 10 s and 10 rain after the start of reactions. ThereaRer consecutive scans were recorded every 60 s. C.
Vol. 40, No. 6, December 1996 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Pages 1211-1219
TEMPACE AND TROXYL-NOVEL SYNTHESIZED 2,2,6,6-TETRAMETHYLPIPERIDEVE DERIVATIVES AS ANTIOXIDANTS A N D RADIOPROTECTORS
Diana Metodiewa1", Janusz Skolimowski2 and Stefan Karolczak I l Institute of Applied Radiation Chemistry, Technical University of L6d$, Wrtblewsldego 15, 93-590 Ltd2, Poland; 2Department of Organic Chemistry, University of Ltd,, Narutowicza 68, 90-136 Ltd$., Poland. Received October 18, 1996
SUMMARY: Two novel 2,2,6,6-tetramethylpiperidine derivatives (Tempace and Troxyl) were synthesized and their capacity to act as scavengers of superoxide, inhibitors of iron and ascorbate-driven Fenton reaction and radioprotectors was tested. The possibility for one-electron oxidation of novel compounds by heme-ferryl species was also examined. The results clearly indicate that Tempace and Troxyl are acting as promising antioxidants and radioprotectors thus providing a base for further investigations and pharmacological applications. Keywords: antioxidants, radioprotectors, Fenton reaction, tetramethyl- piperidine
INTRODUCTION Piperidine nitroxides appear to be a new class of non-thiol protectors against a variety of oxidative stress including ionizing radiation (1-3). Possible mechanisms can involve their superoxide dismutase-type activity (4), catalase-mimic activity (5) and scavenging of oxy- and carbon radicals (6,7). Contrary to most antioxidants which are reducing agents, nitroxides and their derivatives act predominantly as mild oxidants (1,8). These properties can include oxidation of reduced transition metals and thereby inhibition of their involvement in Fenton reactions (1,6).
ABBREVIATIONS: TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl); TOLH, 1,4dihydroxy-2,2,6,6-tetramethylpiperidin e;Tempace, 4-aeetamide-2,2,6,6-tetramethylpi-peridineN-oxyl; Troxyl, salt: 6-hydroxy-2,2,5,7,8-penthamethyl-chroman carboxylate; NBT, Nitro-BT, p-nitrotetrazolium blue; DR, Deoxyribose; XO, xanthine oxidase; TBARS, thiobarbituric acid reactive substances; X, xanthine; SOD, superoxide dismutase. * To whom correspondence should be adressed. Fax: (48-42) 36-02-46 1039--9712/96/06121 i-09505.00/0 1211
Copyright © 1996 by Academic Press Australia. All rights of reprrmluction in any filrm reserved.
Vol. 40, No. 6, 1996
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
Recent use ofni~oxides and their derivatives as therapeutically promising substances attract substantial research interest (2,9-14). The published results demonstrate both beneficial and undesirable effects and clearly indicate the need to search for and to design new, more versatile and less toxic analogues. Such analogues may find broad pharmacological appfications in protection against oxidative stress and/or radiation injury. As a part of our studies on the chemical and biochemical charactefist~ of new synthesized anfioxidants of potential pharmacological applications, we designed two novel 2,2,6,6tetramethylpipefidine derivatives, named Tempace and Troxyl, shown in the following probe structures and compared them with TEMPOL: 0 3 H
3
Tempace
H~O/~Y~- OH H~"~'~'~O"0~
3
HO A - - -
Troxyl
3
TEMPOL
We tested their scavenging activity on reactive oxygen species which were generated enzymatically or radiolyticaUy. The inhibiting effect of Tempace and Troxyl on iron and ascorba~-driven Fenton reaction was also examined. The antioxidant properties of the new synthesized compounds as a function of time and concentration were compared with those of Trolox (water soluble vit E analogue) or TEMPOL - the first and the most investigated member ofni¢oxide antioxidants and radioprotectors (1-14).
MATERIALS AND METHODS TEMYOL and TOLH were synthesized as previously described (15). Tempace was synthesized using TOLH (16). Troxyl was prepared by low presm~e reduction of TElVIPOL using hydrogen and palladium on activated carbon as catalyst, followed by addition of Trolox (16). To confirm the presented structures of Tempace, Troxyl and Tempol NMR IH and 13C spectra were performed in deutereted methanol on the Varian Gemini instrument (200 MHz). ESR spectra were recorded using an X-band spectrometer Varian El09 (modulation ~equency 9.4 GHz, amplitude 1.0 G, microwave power I0 roW). Spectrophotometric measurements were performed on a Hewlett-Packard diode array instrument (HP-8452).
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BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL
All chemicals of analytical grade were purchased from S i v a Chem. Co., except hydrogen peroxide and ferrous sulfate (AnalalL BDH) or Trolox~ (Aldrich Chem.). All experiments were performed in a potassium phosphate buffer (50 mM, pH 7.0) at room temperature. Superoxide was generated from 0.2 mM X and 0.02 U/ml XO(17). Scavenging of superoxide was monitored at 560 nm using the NBT method (18).The urate formation in X/XO systems was investigated kinetically at 295 nm (19). Comparative measurements of DR oxidation were monitored by reaction with thiobarbituric acid and expressed as TBARS (20,21). DR solutions were irradiated in 6°Co source at constant dose equal to 11 Gy. min-I in the presence of air. Each result represents the mean of three separate triplicate experiments; the SD (not shown for clarity) was below 5% of the mean.
RESULTS AND DISCUSSION The X/XO system generated superoxide as measured by the reduction of NBT: this reduction was inhibited by TEMPOL, Tempace and Troxyl in a concentration - dependent manner (Fig. 1A-C). The extent of the observed inhibitory effect was investigated in the concentration range between 50 ~
- 0.2 mM (TEMPOL and Tempace) and 50 gM - 0.6 mM
(Troxyl). Trolox at a concentration lower than 0.5 mM did not markedly affect the NBT reduction. At the concentration of 50 pM the above mentioned substances inhibited the reduction of NBT (100 gM) by 52% (TEMPOL), 30% (Tempace) and 40% (Troxyl). The rate of xanthine oxidation to uric acid by XO in the absence or presence of investigated compounds was not changed, indicating that TEMPOL, Tempace and Troxyl are neither substrates or inhibitors of XO. Therefore, it is obvious that the tested compounds are active as superoxide scavengers. It is of interest if this is a superoxide dismutase - type activity as demonstrated before for Tempol (1,4) or a simple one-electron reaction. Research in our laboratories is now aiming to answer this question. One-electron oxidation of antioxidants by heme-ferryl species of peroxidases may provide some insight into the mechanism of their action in situ (17). Comparative peroxidation of Trolox and Troxyl (Fig.2) clearly indicates that chromane moiety of both substancies is a substrate for one-electron oxidation. The evident increase of absorbance at 270 nm seen in Fig. 2A and B is assigned to formation of o-quinone through self-dismutation of formed chromanoxyl radicals (22). One novel feature of Troxyl oxidation was revealed: uncommon
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The concentration-dependent influence of Tempol (A), Tempace 03) and
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320
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effect of GSH presence (C). The reactions were started by addition of 60 nM horse radish peroxidase and 50 I~M H202 to 100 ~ Trolox, Troxy! or Troxyl + 200 pM GSH (a); scans b and c - 10 s and 10 rain after the start of reactions. ThereaRer consecutive scans were recorded every 60 s.
Comparison of one-electron oxidation of Trolox (A) and Troxyl (B) and
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