Rutoxyl [rutin/4-acetamide-1-hydroxy-2,2,6,6-tetramethylpiperidinium] is a new member of the class of semi-natural products of high pharmacological potency

Vol. 42, No. 6, September 1997 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Poges 1261-1270

Rutoxyl [rutin/4-acetamide-l-hydroxy-2,2,6,6-tetramethylpiperidinium] is a new member of the class of semi-natural products of high pharmacological potency Diana Metodiewa 1., Janusz Skolimowski 2, Agata Kochman3 and Stefan Karolczak 1

/Institute of Applied Radiation Chemistry, Technical University of Ldd~, Wrdblewskiego 15, 93-590 L6d2, Poland; 2Departmentof Organic Chemistry, Universityof L6d2, Narutowicza 68, 90-136 L6d~, Poland; 3Departmentof Pathological Anatomy, Medical University of Wroctaw, Marcinkowskiego1, 50-368 Wroctaw, Poland. ReceivedMay 27, 1997 Summary: A novel complex, Rutoxyl [rutin/4-acetamide-l-hydroxy-2,2,6,6-tetramethylpiperidinium] was synthesized and its structure and anticancer activity were investigated. The results reported here are consistent with our idea, that the formation of such a complex of two biologically active molecules: polyphenolic flavonoid antioxidant (Rutin) and nitroxylamine ofnitroxide antioxidant (Tempace), stabilized by hydrogen bond(s) can result in a supra-additive properties. INTRODUCTION Previous work of our laboratories resulted in the design of novel, promising antioxidants and radioprotectors of nitroxide/nitroxylamine class (1) or flavonoid derivatives - flavanone oximes (2). In the course of our search for nontoxic, newly synthesized supra-additive substances of potential pharmacological application, a novel complex, named Rutoxyl [rutin/4-acetamide- 1-hydroxy-2,2,6,6-tetramethylpiperidinium] was synthesized. Its structure, anticancer and antioxidant properties were examined and compared with these of the parent molecules: Rutin and Tempace. It was of particular interest to create a bifunctional, antioxidative substance containing these two different biologically active parts in correct stoichiometry (1:1), without marked changes of their chemical structure, so as to achieve the supra-additive effects. Our strategy for the synthesis of such a novel type of biologically active compound-antioxidant consists of the trapping of a Tempace nitroxylamine into the Rutin molecule in order to prevent its autoxidation by stable complex formation. Abbreviations: R, rutin, 3,3',4',5,7-pentahydroxyflavone-3-rhamnoglucoside; Tempace, 4-acetamide-2,2,6,6-tetramethylpiperidine-N-oxyl; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl; TOLH, 1,4-dihydroxy-2,2,6,6-tetramethylpiperidine; ROS, reactive oxygen species; TMS, tetramethylsilane; N,N-DEAEFo, N,N-diethylaminoethers of flavanone oxime; FeO 3+ and FeO2+, compound I and compound II of horseradish peroxidase 0-IRP); respectively; m.e.d., minimal effective dose. * To whomcorrespondenceshouldbe addressed.Fax: (48-42) 36-02-46; E-mail: [email protected] 1039-9712/97/061261- 12505.00/0 1261

Copyright 9 1997 by Academic Press Australia. All rights of reproduction in any fi~rm reserved.

Vol. 42, No. 6, 1997

BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL

Low molecular weight nitroxides ( ~ N ~ O )

are a new class of membrane permeable

non-thiol protectors against a variety of oxidative stress (3). Nitroxides are subject to steric hindrance, but the fully substitution in the orthoposition inhibits their dismutation thus rendering them stable free radicals which can terminate other radical(s) chain reactions (4,5). It is known recently (5) that nitroxide undergoes chemical and/or cellular reduction to the corresponding one-electron reduced nitroxylamine ()N----OH), which is oxidized back to the nitroxide by various oxidants (chemical of cellular). However, cellular oxidation of nitroxylamine will depend on many factors which include the oxygen tension, the level of ROS and/or the redox status of the cell (3-5). In biological systems, in vivo, there probably exists an equilibrium between nitroxide and its corresponding nitroxylamine derivative (3-5). The acid-base and redox properties of Rutin, the most investigated and representive member of natural, plant polyphenolic (flavonoid) antioxidants class, renders it convenient pharmacological agent, taking place during cells oxidative processes (6-10). It can act as a metal-chelating or - oxidizing agent (11), scavenger of ROS and inhibitor of lipid peroxidation (10-12) and potentially important chemopreventive and anticancer agent (10,13,14). Alternatively, the stability of the Rutin phenoxyl radicals - the product of oneelectron oxidation, and their reactions with cell antioxidants (7-9,15,16) could reflect a possible pro-oxidant effects of this antioxidant, administrated alone in a number of pathological conditions. A reason for this reminder of the complexities of nitroxides and/or flavonoid (rutin) properties is file fact, that some strong structural requirements exist to reveal their antioxidant and therapeutic functions (1-I6). Fundamental to these functions are the respective oxidation-reduction and metal-interactions capacity. In this connection, the design of the complexes [nitroxide/nitroxylamine)-flavonoid] should be in compliance with structure, adequate for complete biological activity of novel synthesized substance. Our concern over the observed recently undesired toxicity of biologically active nitroxides (17) showned clearly that the chosen method for Rutoxyl design based on steric, inductive and hydrogen-bonding properties of parent molecules was a logical and convenient way for its lowering. This strategy was also confirmed by the results of pharmacological tests and comparative study of the activity ofRutoxyl, Tempace (1) and flavanone oximes (2) against Yoshida sarcoma - a very convenient model of tumor for studies on chemotherapy of cancer in vivo (18). Our findings extend the in vitro results (1,2,19) and demonstrate that Rutoxyl at m.e.d. (minimal effective dose) could act as inhibitor of tumor promotion. The exact mechanisms and concentration - dependent effects in vivo using the complete Yoshida screening tests (t8) are in progress.

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MATERIALS AND METHODS Materials. 4-amino-2,2,6,6-tetramethylpiperidine and R were obtained from Sigma. All other chemicals used were also of highest quality comercially available. Synthesis. The synthesis of Rutoxyl was performed as described in Scheme 1 (steps 1-5). The final product was recovered by evaporation of the solvent, recrystalized from methanol and used for identification and assays. Spectroscopy. 1H NMR(250 MHz) and 13C NMR (65 MHz, TMS as reference; DEPT analysis) 1-dimensional measurements were performed on Bruker Avans DPX 250 spectrometer and expressed in 5 scale. The samples were dissolved in CD3OD and taken in 5-ram tubes for analysis. ESR measurements of the investigated substances and solutions, respectively, were performed using X-band spectrometer Bruker 200D-SRC. Acquisition parameters were as follows: microwave frequency 9.58 GHz, modulation frequency 100 kHz, modulation amplitude 1.013 G, sweep time 41 s, The ESR spin-stabilization method (19) using 2n2§ to detect aryloxyl radicals formed in R or Rutoxyl oxidation under air (20) was applied. Spectrophotometric measurements of R or Rutoxyl solutions (UV-visible absorption spectra) were performed on a Hewlett-Packard diode array instrument (HP-8452). Elementar microanalysis (C, H, N) of Rutoxyl samples was also performed. Pharmacological tests. Male rats (Wistar strain, average weight 180-200 g, maintained on this same water and food conditions or day/night cycle) placed in groups of 10 were used Investigated compounds or control solution (physiological saline) were injected intraperitoneally, daily for 5 days, starting 2 hrs after the intraperitoneal transplantation of Yoshida sarcoma (106 cells). 0

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Tempace

Vol. 42, No. 6, 1997

BIOCHEMISTRYc/nd MOLECULAR BIOLOGY INTERNATIONAL

Computer-assisted Molecular Modelling. The molecular replacement, structure refinement and map calculations of Rutoxyl were carried out using the programme HyperChem (Hypercube Training Center MITR PL). Three dimensional model of Rutoxyl molecule was performed on the base of energy calculated (optimilizated) structures of parent molecule (R) related to known empirical results (22). RESULTS AND DISCUSSION The general structure of the newly synthesized complex Rutoxyl (Sch. 1) is shown on Fig. 1. The following experimental evidence: (i) lack of chemical shifts of the 13C and sugar - 13C of Rutoxyl compared to these of Rutin and its Zn 2+ - complexes (Table 1-3) (22); (ii) the results ofelementar microanalysis (Table 4); (iii) lack of bathochromic shifts of Band I of Rutoxyl compared to R (300-380 nm) (23), usually observed after ionization of C-3' and C-4' OH groups o f R catechol ring B (data not shown); (iiii) lack of ESR signal from a possible oxidation of nitroxylamine part of Rutoxyl and stability of its solutions in air, leads us to consider the hydrogen bonds stabilization of the complex (Fig. 1). The hydrogen bond formation between C-3' or C-4' - OH groups of

A

The chemical structure of R is shown:

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O

R= D-Glucose-L-Rhamnose Fig. 1

Stereo view of Rutoxyl A - The doted lines indicate the hydrogen bonds formation between C-4'OH group of R(ring B) and OH group of nitroxylamine (left) and between OH group of the rutinose part of R and oxygen from carbonyl group (right). B - The surfaces shown are about 0.7 of Wan der Waals's radii: Black spheres represent N, deep grey - O and grey - C.

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Vol. 42, No. 6, 1997

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BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

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Vol. 42, No. 6, 1997

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Vol. 42, No. 6, 1997

BIOCHEMISTRY and MOLECULAR BIOLOGYINTERNATIONAL

catechol part of R and OH group of nitroxylamine results in the conformation, which further gives rise to other hydrogen bond at 4-acetamide position of nitroxylamine and rutinose.part of R. Having in mind all experimental observations we hypothesize that there may be interactions of intramolecular communication (supra-additive type) between the two parts of Rutoxyl molecule, which can be an important factor determining its biological activity. However, the question that we should answer is which conformation is the active as there are many energetically accessible conformations for a molecule containing over 100 atoms. In the present communication we consider only the conformer shown in Fig. 1. Since the molecular details about the mechanisms of Rutoxyl action are not known, there is currently no possibility of more detailed understanding and discussion of this point. The relative antitumor properties of Rutoxyl, flavanone oximes (2) and Tempace (1) against Virus Sarcoma (Yoshida) are expressed as % ILS values (Table 5). Compounds with percent ILS greater than 25 at m.e.d, are considered to be active. R (daily dose 2 mg/kg) or TEMPOL (till 60 mg/kg) were not active. Oximes (Y or W) (2) were used in the highest concentrations (2 mg/kg) available in relation to their low solubility. The central finding of this study is that Rutoxyl at five times lower concentration (at m.e.d.) provided about 25% increase of ILS in the initiation phase of tumor, compared to those of Tempace (Table 5). In promoted phase of Yoshida Sarcoma, the effect of Tempace increased twice. These results prompted us to continue the concentration and phase-dependent effects of Rutoxyl: the investigations following the complete Yoshida protocol for promoted disease (18) are in progress. In conclusion it was shown that the nitroxylamine moiety and/or saturation at orthoposition had a decisive effect on observed antitumor activity. Our results (Table 5) also confirmed the suggested role of antioxidants and free radicals in the pathogenesis of Yoshida sarcoma (24). It is worth noting that within cells the equilibrium [nitroxide/nitroxylamine] (4,5) can serve not only as radical chain reaction terminator, but also as sensitive probe for free radical processes (3-5). One electron oxidation of antioxidants (chemical or by heme-ferryl species, FeO 3+ and FeO 2+) may provide some insight into their structure and mechanisms of action in situ (7,15,21). Chemical oxidation of R (Fig. 2a) (Table 2) and peroxidation of R (Fig. 2b) or Rutoxyl (Fig. 2c) Zn2+-complexes, resulted in stable semiquinone radicals in R (Fig. 2a and b) (20,21,25) and nitroxide free radical in Rutoxyl (Fig. 2c) (3). It is evident that the catechol part (ring B) or R was a substrate for oxidation as suggested before (6,7). The nitroxylamine part of Rutoxyl was oxidated by heme-ferryl ion species (Fig. 2c). It is known that peroxidation of nitroxylamines like TOLH by cell peroxidases is sluggish (26). Thus, the intramolecular facilitation of this process by the R-catechol moiety of Rutoxyl cannot be excluded.

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BIOCHEMISTRYo,,"/d MOLECULAR BIOLOGY INTERNATIONAL

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ESR spectrum recorded 1 min after the chemical oxidation of R(a) or enzymatical oxidation of Zn2+- complexed R (b) and Rutoxyl (c). a - 3.3 mg of R in 05 ml of H20 were dissolved in 0.5 mt of a 0.t N NaOH solution25; 0.35 gM HRP were added to an incubation mixture containing 0.30 mM H202, 0.25 M ZnC12 and 0.5 mM R (b) or Rutoxyl (c), respectively (0.1 M acetate buffer, pH 5.6; air).

The results obtained in this work are encouraging as we did find a limited correlation between the molecular properties and the antitumor activity of newly synthesized complex Rutoxyl compared to these of R and Tempace. The antioxidant and antiradical properties of Rutoxyl were also investigated and compared with these of its parent molecules (19). Acknowledgment. We thank Mr. Cz. Ko~ka (Comp. Sci. Lab., MITR) for his excellent assistance and valuable discussions. The support of KBN (Poland) Grant PO4A 04212 is gratefully acknowledged.

REFERENCES 1. Metodiewa, D., Skolimowski, J. and Karolczak, S. (1996) Biochem. MoL BioL lntern. 40, 1211-1219. 2. Metodiewa, D., Kochman, A. and Karolczak, S. (1997) Biochem. Mol. Biol. lntern. (in press). 3. Krishna, M.C. and Samuni, A. (!994)Meth. Enzymol. 234, 580-589. 4. Rachmilewitz, D., Karmeli, F. and Samuni, A. (1994) Gut 35, 1181-1188. 5. Sosnovsky, G. and Paul, B.D. (1984),/. Med. Chem. 27, 782-788.

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6. Bors, W., Michel, C. and Schikora, S. (1995) Free Rad. Biol. Med. 19, 45-52. 7. Jovanovic, S.V., Steenken, S., Tosic, M., Marjanovic, B. and Simic, M.G. (1994) J. Amer. Chem. Soc. 116, 4846-4851. 8. Negre-Salvayre, A., Affany, A., Hariton, C. and Salvayre, R. (1991) Pharmacology 42, 266-272. 9. Hanasaki, Y., Ogawa, S. and Fukui, S. (1994) Free Rad. Biol. Med. 16, 845-850. 10. Aruoma, O.J. and Hallivell, B. (1993) in Food and Cancer Prevention: Chemical and Biological Aspects (K.W.Waldron, J.T.Johnson and G.R.Fenwick, eds.) pp. 119-124, Royal Soc. Chem., Cambridge. 11. Afanasiev, J.B., Dorozko, A.J., Brodski, A.V., Kostyzik, A. and Potapovitch, A.J. (1989) Biochem. Pharmacol. 38, 1763-1769. 12. Salah, N. Miller, N.J., Paganga, G., Tijburg, L., Bolwell G.P. and Rice-Evans, C. (1995) Arch. Biochem. Biophys. 322, 339-346. 13. Miller, J.A. (1994) Chem.-Biol. Interact. 92, 329-341. 14. Webster, R.P., Gawde, M.D. and Bhattacharya, R.K. (1996) Cancer Lett. 109, 185191. 15. Bors, W., Michel, C. and Saran, M. (1994)Meth. Enzymol. 234, 420-429. 16. Skaper, S.D., Fabris, M., Ferrari, V. and Carbonare, M.D. (1997) Free Rad. Biol. Med. 22, 669-678. 17. Hahn, S., Lepinski, D.L., DeLuca, A.M., Mitchell, J.B. and Pellmar, T.C. (1995) Can. J. Physiol. Pharmacol. 73, 399-403. 18. Yoshida, T. (1951) J. Natl. Cancer lnst. (Japan) 12, 947-968. 19. Metodiewa, D., Skolimowski, J., Rutka, J., Kochman, A., Karolczak, S. and Gwoidzifiski, K. (1997), submitted. 20. Kalyanaraman, B., Felix, C.C. and Scaly, R.C. (1984)J. Biol. Chem. 259, 254-358. 21. Metodiewa, D. and Dunford, H.B. (1993) in Atmospheric Oxidation and Antioxidants, vo III (G.Scott, ed.)Elsevier, Amsterdam-London-New York-Tokyo, pp. 287-232. 22. Markham, K.R. and Ternai, B. (1976) Tetrahedron 32, 565-569. 23. Mabry, T.J., Markham, K.R. and Thomas, M.B. (1970) In the Systematic Identification ofFlavonoids, Springer-Verlag, NewYork - Heidelberg - Berlin,. pp. 41-61. 24. Bozzi, A., Mavelli, J., Mondovi, B., Strom, R. and Rotilio, G. (1981)Biochem. J. 194, 369-372. 25. Yoshioka, H., Sigiura, K., Kawahara, R., Fujita, T., Nakino, M., Kamiya, M. and Tsuyumu, S. (1991)Agric. Biol. Chem. 55, 2723-2728. 26. Rosen, G.M., Finkelstein, E. and Rauckman, E.J. (1982) Arch. Biochem. Biophys. 215, 367-378.

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