Formation of aminoxyl radicals in the reaction between penicillins and hydrogen peroxide

FreeRadical Biology & Medicine, Vol. 13, pp. 455-457, 1992

0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.

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Brief Communication FORMATION OF AMINOXYL RADICALS IN THE REACTION BETWEEN PENICILLINS AND HYDROGEN PEROXIDE

CARL LAGERCRANTZ Department of Medical Physics, University of GOteborg, Medicinaregatan 11, S-413 90 GOteborg, Sweden

(Received 20 February 1992; Revised 28 April 1992; Accepted 29 April 1992) Abstract--Aminoxyl radicals are formed in high yield in the reaction between penicillins and hydrogen peroxide in water solutions in the pH range between 7 and 8. The nine-line EPR spectrum, 3 × 3 ( 1:2: l), indicated an interaction of the unpaired electron with one ~4N nucleus (a~ = 1.44 roT) and two equivalent hydrogen nuclei (all = 2.00 mT). The reaction involves an oxidative cleavage of the beta-lactam ring of the penicillins with the formation of a cyclic aminoxyl radical, in which the thiazolidine ring carries the nitroxide group ( ~ N-O" ). It is suggested that the reaction with the formation ofaminoxyl radicals can also take place in vivo in the deactivation of penicillins by metabolically formed hydrogen peroxide. Keywords--Penicillins, Beta-lactam, Aminoxyl radicals, EPR spectroscopy, Hydrogen peroxide, Free radicals

(S6dert/ilje). 6-Aminopenicillanic acid was from Sigma Chemical Co. (St. Louis, MO), clavulanic acid (Li-salt) from SmithKline Beecham (Brentford, UK), and cephadroxil from Bristol-Myers Squibb Co (New York, NY). The substances were used as supplied.

INTRODUCTION

The beta-lactam ring of antibiotics such as the penicillins is a prerequisite for the activity of these substances. Deactivation of penicillins by cleavage of the beta-lactam ring takes place in a number of reactions, including reaction with beta-lactamases, dilute alkali, or oxidative reagents. 1-4 This study found that aminoxyl radicals are formed in high yield in the reaction between some penicillins dissolved in H20 and hydrogen peroxide in the pH range between about 7 and 8.

RESULTS AND DISCUSSION

Figure 1 shows the EPR spectrum of the radicals formed in the reaction between equimolar amounts of ampicillin and hydrogen peroxide (0.05 M) in a water solution with an initial pH value adjusted to about 7.6 by the addition of NaOH. The radicals appeared almost immediately and could be observed for several hours at room temperature. The nine-line spectrum, 3 × 3 (1:2:1), indicated an interaction of the unpaired electron with one 14N nucleus (aN = 1.44 mT) and two equivalent hydrogen nuclei (all = 2.00 mT). An identical spectrum was obtained when the reaction was performed in D20, which indicates that the two hydrogen atoms are not exchangeable. Almost identical spectra were obtained with phenoxymethylpenicillin, benzylpenicillin, and 6-aminopenicillanic acid. It is considered that the radicals are aminoxyl radicals 3 formed by oxidation in the cleavage of the betalactam ring of the penicillins 1 (Eq. 1).

EXPERIMENTAL PROCEDURES

Electron paramagnetic resonance (EPR) spectra were recorded using a Varian E-9 spectrometer at 20°C with a microwave power of 1-5 mW and a 100 kHz modulation amplitude of 0.05 mT. The samples were contained in a flat aqueous solution cell. Hyperfine splittings constants were measured by comparison with the splittings of Fremy's radical (aN =

1.3 mT). The penicillins were obtained from AB Astra

Address correspondence to: Carl Lagercrantz. 455

456

C. LAGERCRANTZ formed according to Eq. 1 and that the nitroxide group (~---N-O') is located on the thiazolidine ring. The hydrogen atoms attached to carbon atoms 2 and 5 of 3 are structurally nonequivalent. However, the coupling constants aH are equal, very probably due to accidentally equal angles between their C-H bonds and the nodal plane of the nitrogen pn orbital leading to identical or nearly identical overlap of orbitals. The formation of an additional carboxylic group in the cleavage of the beta-lactam ring leads to a decrease of the pH value of the reaction mixture during the reaction with hydrogen peroxide. Therefore, it was necessary to add a certain amount of alkali to maintain the pH in the rather narrow range (i.e., about 7 to 8) compatible with the stability of the aminoxyl radicals 3. The formation of aminoxyl radicals by oxidation with H:O2 in alkaline water solution is a general reaction of secondary amines. The reaction is rather slow without the addition of substances such as sodium wolframate.6-8 The experiments performed with the penicillins indicated that the aminoxyl radicals were formed rather rapidly. In the presence of H20 z, no radicals could be detected in reaction mixtures to which a wolframate or iron ions had been added. It is not clear whether the reaction mechanism involves a concerted cleavage of the beta-lactam ring and formation of the aminoxyl radicals, or whether the reaction takes place in two separate steps with oxidation of the secondary amine 2 formed in a hydrolytic cleavage of the ring. The latter alternative might be the more probable one, since the radical concentration increased after the addition of alkali to counteract the initial decrease of pH. On the other hand, the rapid formation of the radicals might be in favor of the former mechanism. Evidently, hydrogen peroxide constitutes the active reagent in both alternatives. The stability of peniciUins has a maximum in the pH range between 6 and 7 (Ref. 1). Therefore, the cleavage of the ring seems not to be promoted by the actual pH. The pH value is rather critical for the stability of the aminoxyl radicals (3) (i.e., pH 7 to 8). The restricted pH range and the failure to observe any radicals in the presence of a wolframate or iron ions are

f+ 2.6

mT

Fig. 1. EPR spectrumofthe radicalsformedin the reactionbetween equimolaramountsof ampicillinand hydrogenperoxide(0.05 M) dissolvedin H20. pH adjustedwith NaOH to about 7.6.

R = ~ - C H 2 - C O N H - : benzylpenicillin R = ~ - C H ( N H 2 ) - C O N H - : ampicillin R = ~ - O - C H z - C O N H - : phenoxymethylpenicillin R = H2N-: 6-aminopenicillanic acid The nitrogen atom in position 1 of the penicillins constitutes the nitrogen atom of the nitroxide group of the aminoxyl radical 3 formed in the oxidative cleavage of the beta-lactam ring (Eq. l). The triplet splitting 1:2"1 of the EPR spectrum originates from the interaction of the unpaired electron with the hydrogen atoms attached to carbon atoms 2 and 5 on the thiazolidine ring. The large coupling constants, a n = 2.00 mT, of the hydrogen atoms, in this case equivalent, is a characteristic of cyclic aliphatic aminoxyl radicals. Thus, the radicals formed by oxidation of pyrrolidine exhibit a coupling constant of the four equivalent beta-hydrogen atoms of 2.32 mT. 5 In this case, the large coupling constant is consistent with a planar or nearly planar structure of the aminoxyl radical with the C-H bonds at an angle of approximately 60 ° to the nodal plane of the nitrogen pn orbital) These findings support the suggestion that the radicals derived from the penicillins are aminoxyl radicals

H '

H '

"

H

CH

H ~l

S

4 3"~CHa 1

2 H 1

a

- COO(H)

•

O-~-C'

I

(H)O

H iJ

-CH3

.

H202 •

R~

H , I

~

j O ~

/

4 3x~

o=c

/

H

2

Equation 1

(H)O

[ Oy

/ 3

H

CH 3

Radicals and penicillins

probably connected with the unstability of the thiazolidine ring of the radical 3. Some experiments were also performed with the beta-lactam substances clavulanic acid ((Z)-(2R, 5 R)-3-(2-hydroxyethyliden)-7-oxo-4-oxa- 1-azabicyclo[3.2.0]heptan-2-carboxylic acid) and cephadroxil((6R,7R)-7-[(R)-2-amino-2-(p-hydroxyphenyl)acetamido]-3-methyl-8-oxo- 5-thia- 1-azabicyclo[4.2.0]oct2-en-2-carboxylic acid). However, no radicals could be detected in the reaction between these substances and H202, probably due to the instability of the ring structure carrying the nitroxide group. An alternative interpretation of the EPR spectrum (Fig. 1) might involve the formation of an aminoxyl radical by oxidation of the nitrogen atom of the peptide bond, or the amino group, in the side chain attached to carbon atom 6 of the penicillins. However, this structure seems to be ruled out, since aminoxyl radicals produced by oxidation of the nitrogen atom of open-chain substances do not exhibit coupling constants of the beta-hydrogen atoms larger than about 1.5 mT. Furthermore, aminoxyl radicals of the type -CH2N(O')-C(~---O)- exhibit relatively small coupling constants aN (i.e., between 0.7 to 0.8 mT; Ref. 9). In view of the fact that hydrogen peroxide is continuously produced in living cells, ~° it seems possible that aminoxyl radicals of the type described here may also be formed in connection with the deactivation of penicillins in vivo.

457

Acknowledgements--The author is indebted to Dr. R. E. Caner, Professor L. Eberson, and Dr. B. EkstrOm for valuable discussions. This work was supported by grants from The Royal Society of Arts and Sciences in Gothenburg.

REFERENCES 1. Cheney, L. C. Antibiotics. In: Gilman, H., ed. Organic chemistry.. An advanced treatise. Vol. III. New York: John Willey; 1953:533-580. 2. Kaiser, G. V.; Kukolja, S. Modifications of the Beta-lactam System. In: Flynn, E. H., ed. Cephalosporins and penicillins. Chap. 3. New York: Academic Press; 1972:74-133. 3. Diirckheimer, W.; Blumbach, J.; Lattrell, R.; Scheunemann, K. H. Recent developments in the field of beta-lactam antibiotics. Angew. Chem. Ed. Engl. 24:180-201; 1985. 4. Donewitz, G. R.; Mandell, G. L. Beta-lactam antibiotics. N. Eng. J. Med. 318:419-426; 1988. 5. Hudson, A.; Hussein, H. A. Electron spin resonance ofaliphatic nitroxides. Part II. The cyclic radicals from pyrrolidine, piperidine, morpholine, and hexamethyleneimine. J. Chem. Soc. B:251-253; 1968. 6. Rozantsev, E. G. In: Ulrich, H., ed. Free nitroxyl radicals. New York: Plenum Press; 1970;67-92. 7. Forrester, A. R.; Hay, J. M.; Thomson, R. H. Organic chemistry of stable free radicals. London: Academic Press; 1968. 8. Lagercrantz, C. The pica and pH dependence of the formation of nitroxide radicals from some drug substances with an aliphatic secondary amino group by oxidation with hydrogen peroxide. An electron spin resonance (ESR) study. Acta Chem. Scand. B41:526-535; 1987. 9. Mackor, A.; Wajer, Th. A. J. W.; de Boer, Th. J. C-nitroso compounds--VI. Acyl-alkyl-nitroxidesfrom acyl radicals and nitroso compounds as studied by ESR. Tetrahedron 24:16231631; 1968. 10. Chance, B.; Sies, H.; Boveris, A. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59:527-605; 1979.