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Reductive fragmentation of 2-nitroimidazoles: Amines and aldehydes
In/ J Radrorron Oncology Bid Phys VoI Pnnlcd I” the U.S.A. All nghls reserved
IO.
PD.
1331-1340
??Session II
REDUtXIVE
FRAGMENTATION OF 2-NITROIMIDAZOLES: AMINES AND ALDEHYDES
J. A. RALEIGH,
PH.D.
AND S. F. LIU, M.Sc.
Radiobiology. Cross Cancer Institute, Edmonton, Alberta T6G 122 Canada Glyoxal has been identified as a product in the fragmentation of reduced 2-nitroimidazole radiosensitizers. Quantitative analysis of glyoxal as its bis (2,4_dinitrophenylhydrazone) derivative shows that it is formed in good yield (9-23%) in a variety of 2-nitroimidazoles. In addition to glyoxal a second as-yet-unidentified carbonyl compound, and a series of amines are formed when reduced 2-nitroimidazoles fragment in the presence of water. One of the amines derived from misonidazole is identified as I-amino-3-methoxypropan-2-01, the product of extensive ring cleavage. Radiation chemical reduction of the 2-nitroimidazoles proceeds with the consumption of 3 electrons for each molecule reduced. This could imply that a radical disproportionation or dimerization step is involved in the reductive degradation of 2-nitroimidazoles. Radiosensitizer, fragmentation.
Nitroimidazole,
Radiation chemical reduction, Glyoxal, I-amino-3-methoxypropan-2-o],
Reductive
graphic analysis based on the bis (2,4dinitrophenylhydrazone) derivative, we have found that in the radiation chemical reduction of 2-nitroimidazoles, 9-23s of the molecular fragments from the degraded drugs can be detected as glyoxal. In addition to glyoxal, a series of amines are produced when reduced 2-nitroimidazoles fragment. In the case of misonidazole, one of these has been identified as ! -amino-3-methoxypropan-2-01 which incorporates one ring nitrogen and the side chain of the original misonidazole molecule.
INTRODUCTION Reductive metabolism is a possible source of 2-nitroimidazole toxicity. In addition to radical intermediates,‘*‘*“.” the formation of reactive compounds from the reductive fragmentation of 2-nitroimidazoles may be involved. The fragmentation is reported to proceed from the hydroxylamine derivative. ‘.6.‘5.‘8The putative 2 electron, nitroso precursor could not be detected under conditions in which the analogous nitrosobenzene intermediate can be trapped.13,14 One of the molecular fragments formed when misonidazole is reduced in vitro is a guanidino derivative which incorporates 3 nitrogen atoms and 1 carbon atom of the original 2-nitroimidazole structure.’ We have shown that the remaining two carbon atoms in the ring can appear as glyoxal. 9.‘2 Glyoxal reacts readily with nucleic acids and proteins.4,‘6 It could contribute to the toxicity of reductively activated 2-nitroimidazoles and to the preincubation effect wherein the shoulder of survival curves of irradiated cells is diminished by hypoxic preincubation with 2-nitroimidazole radiosensitizers.” The relative importance of glyoxal would depend on its yield from 2nitroimidazole fragmentation. By means of a chromato-
METHODS
AND MATERIALS
The 2-amino derivative of misonidazole was prepared by catalytic reduction according to a literature procedunz3 2,4-Dinitrophenylhydrazine was recrystallized from lbutanol before use. 2,4_Dinitrofluorobenzene was used as received. Glyoxal bis (2,4dinitrophenylhydrone) was prepared from a nominal 40% aqueous solution of glyoxal and its identity confirmed by mass spectrometry. Gravimetric analysis of the glyoxal solution by means of the bis (2,4dinitrophenylhydrazone) derivative showed it to contain 4 1% of glyoxal. This standardized solution was
Reprint requests to: J. A. Raleigh. Acknowledgements-This work was supported by the National Cancer Institute of Canada and the Alberta Cancer Board. The 2-nitroimidazoles (Table) were generously supplied by the National Institutes of Health U.S.A. through the good offices of Dr. V. L. Narayanan. 2,4-Dinitrophenylhydrazine and glyoxal were obtained from the Fisher Chemical Company and 2,4-
dinitrofluorobenzene from BDH Chemicals. High pressure liquid chromatography (HPLC) was performed on a Varian 5000 system incorporating a Varichrom variable wavelength detector and a Spectra Physics SP4 100 computing integrator. Irradiations were performed in a Gammacell 220 6oCo irradiator available from Atomic Energy of Canada Limited. Accepted for publication 22 March 1984. 1337
1338
Radiation Oncology 0 Biology 0 Phyws
used to develop calibration curves for glyoxal in aqueous solutions. Radiation chemical reduction of the 2-nitroimidazoles was achieved by means of a procedure similar to that of Whillans and Whitmore.” Typically, a I.0 mmolar solution of the 2-nitroimidazole in doubly distilled water containing 0.1 molar sodium formate buffered at pH 7 or 9 with 0.01 molar NazHPQ, was irradiated in a 6oCogamma irradiation facility. The solutions were bubbled with nitrogen before and then throughout the time of irradiation. Under these conditions of irradiation approximately six microequivalents of electrons are produced for each kilorad of radiation absorbed in the solution. The dose-rate of the 60Co-gamma radiation was 8.1 krad/min as measured by Fricke dosimetry.” Glyoxal was quantified in terms of its bis (2,4-dinitrophenylhydrazone) derivative which was formed by heating 1.0 ml of a glyoxal-containing solution with 20 ~1 of a 2,4_dinitrophenylhydrazine reagent in a sealed test tube for 20 minutes at 70”. The reagent was prepared by dissolving 400 mg of 2.4-dinitrophenylhydrazine in 2.0 ml of concentrated sulfuric acid and adding this solution to 13 ml of 80% aqueous ethyl alcohol. It was found that 20 minutes of heating at 70’ was required to complete the reaction of glyoxal in the concentration range of interest. (A second, as-yet-unidentified aldehyde was observed during the analysis for glyoxal. We are presently attempting to discover its structure). A calibration curve for glyoxal was established under the conditions of our analysis. Prior to HPLC analysis (Partisil ODS-2, 70% aqueous acetonitrile), the test solution was mixed with I .O ml of dimethylsulfoxide in order to solubilize the bis (2,4-dinitrophenylhydrazone) derivative of glyoxal that had formed. We have found this analysis suitable for measuring glyoxal in biological fluids. The loss of 2-nitroimidazoles was measured by HPLC analysis. Amines formed during the reductive fragmentation of the 2-nitroimidazoles were detected and isolated as their 2,4-dinitrophenyl derivatives. Following radiation reduction of 200 mg of misonidazole in I.0 liter of doubly distilled water containing 0.1 molar sodium formate buffered at pH 7.0 with 0.01 molar Naz HPO,, the solution was concentrated in vuczw to 100 ml and 500 mg 2.4dinitrofluorobentene added. The reaction mixture was stirred for 16 hours at which time the amine derivatives had precipitated as an oil. The mixture of products was fractionated by preparative thin layer chromatography (silica gel G; benzene/methanol, 8S/ 15). RESULTS
AND
DISCUSSION
The chromatographic profile of the 2,4-dinitrophenyl derivatives of the amines formed when misonidazole fragments following reduction show the presence of at least seven amino compounds. One of the amines has been identified as the 2,4-dinitrophenyl derivative of misonidazole amine by chromatographic and mass spectro-
August 1984. Volume IO. Number 8
graphic comparison with an authentic sample prepared as described above. Of the remaining components of the mixture only one has been identified to date. It was isolated by preparative TLC and shown to be a single compound by HPLC (picrate mp 272“). Its high resolution mass spectrum (m/e 271.0800 (molecular ion corresponding to an elemental composition of C,,,H,3NJ06) and m/e 196.0354 (base peak corresponding to the loss of the fragment CHOHCHZOCH~ from the parent compound)) and nuclear magnetic resonance spectrum ((CDCl,) 3.45 ppm (singlet 3H, OCH& 3.55 ppm (overlapping triplets, 4H,NHCHZCHOHC’H@CH,), 6.98 ppm (doublet, 1H, J = 0.1 ppm), 8.28 ppm (doublet of doublets, 1H, J = 0.1 ppm and 0.02 ppm) and 9. I5 ppm (doublet. 1H, J = 0.02 ppm), the last three resonances attributable to the 2,4_dinitrophenyl moiety) are consistent with the structural assignment of the 2,4-dinitrophenyl derivative of 1-amino-3-methoxypropan-2-01. The corresponding molecular fragment from I-(2-hydroxyethyl)-2-nitroimidazole: i.e.. ethanolamine, has been isolated in the same way and identified by chromatographic and mass spectrographic comparison with an authentic sample of the 2,4-dinitrophenyl derivative of ethanolamine. Figure I represents a plausible mechanism for the formation of Iamino-3-methoxypropan-2-01 from reductively activated misonidazole. This mechanism expands on that proposed for glyoxal formation’.” in that a water molecule attacks the 2-position rather than the S-position in the hypothetical hydroxylamine intermediate. The figure can also account for the formation of hydroxyurea which has been
NHOH
OH NHOH
N H.C=’
li-
‘NHOH
‘NH HO H, r$HR
iR
\
\ FHzNHGo ‘NHOH CHO
NH, \
FH? R
/+O NHOH R
=
CH,CHOHCH,OCH,
Fig. I. Possible mechanism for the formation of ~-amino-Jmethoxypropan-2-01 and hydroxyurea from reductively activated misonidazole.
Fragmentation of 2-nitroimidazoles 0 J. A. Table I. Radiation
chemical reduction
1-(R)-2-nitroimidazole R = CH$ZHOHCH20CHj CH$ZONHCH2CH20H CH2CHOHCH2F CHzCHzOH Ribose Dideoxyhexenpyranose
Glyoxal yield (percent) 20 20 14 15 23 9 (a) 15 (8)
RALEIGH
AND
S.
F. LIU
1339
of 2-nitroimidazoles
Nitroimidazole loss (rMole/Krad) 1.9 1.8 2.0 1.9 1.9 2.1 2.1
P’M 0
MIS0
LOSS
0
GLYOXAL
FORMATION /
200
reported recently.’ At this stage, the aldehydes depicted in the figure can only be considered possible products. Addition of a nucleophile other than water (eg., protein amine or thiol) to the 2-position would lead to binding of a sensitizer labelled with 14Cat the 2position. Attack at the 5-position could also lead to binding but the stability of the bound intermediate is in question. The yields of glyoxal from a series of reductively-activated 2nitroimidazoles (Table 1) were determined from slopes of dose-yield plots such as in Figure 2 for misonidazole. Glyoxal is clearly an important product of fragmentation and could contribute to the hypoxic cell cytotoxicity and preincubation effect of 2nitroimidazoles. It seems possible that precursors of aldehydes more potent than glyoxal might be built into the ring of 2-nitroimidazoles whereby chemotherapeutics directed at the hypoxic zones in tumors might be developed. We are presently pursuing this possibility. Radiation chemical reduction of the 2-nitroimidazoles consumes 3 electrons per molecule of drug based on the fact that each krad of radiation delivers 6 microequivalents of electrons to the irradiated solution.‘* While a detailed discussion is beyond the scope of this paper, the result is clear (Fig. 2) and implies that reductive fragmentation of 2-nitroimidazoles involves the disproportionation or dimerization of the three electron adduct (eg. ZRNOH’ RNHOONHR; such a dimer could fragment in a way analogous to that for the hydroxylamine intermediate). reAn earlier study” which showed that misonidazole quired 3.5-4.0 electrons per molecule for its radiation
chemical reduction differs from the present study in that the authors took the reaction to completion in which case fragmentation products such as glyoxal will consume additional electrons (Raleigh and Liu, unpublished data,
??
0 100 0/
J_o/o’o
+o-
I
I
1
50
100 Krod
Fig. 2. Radiation reduction of misonidazole. Misonidazole loss (y = 1.9x - 3.6, r = 0.997); Glyoxal formation (y = 0.39x - 0.13, r = 0.992).
1983) and ultraviolet spectroscopy rather than HPLC was used to quantify the loss of misonidazole. SUMMARY We have shown that glyoxal, a potentially toxic product, is found in reasonably good yield when reduced 2-nitroimidazoles fragment. Building molecular components of even more toxic aldehydes into the ring of 2-nitroimidazoles may provide therapeutic gains when the target is the hypoxic zones of tumors. The identification of lamino-3-methoxypropan-2-01 indicates how extensive the fragmentation of reduced 2-nitroimidazoles can be and points to a possible mechanism for the fragmentation. The consumption of 3 electrons for the reductive decomposition of each 2-nitroimidazole molecule raises interesting mechanistic considerations.
REFERENCES Biaglow. J.E.. Jacobson. B.. Greenstock, C.L.. Raleigh, J.A.: Effect of nitrobenzene derivatives on electron transfer in cellular and chemical models. Mol. Pharmacol. 13: 269282. 1977,
Biaglow. J.E.. Jacobson. B.. Koch. C.J.: The catalytic effect of the carcinogen “4-nitroquinoline N-oxide” on the oxidation of vitamin C. Bioph.t*s. Res. C’ummzr~z.70: 13161323. 1976.
3. Born. J.L., Hadley, W.M.. Anderson, S.L.. Yuhas, J.M.: Host and hypoxic cell toxicity studies with the terminal reduction product of misonidazole. In Radiation Sensitizers. Their Use in Clinical Management of Cancer, L.W. Brady (Ed.). New York, Masson Publishing. 1980, pp. 79-82. 4. Carmichael, G.G.. McMaster. G.K. .bferhods in Enzvmolog~‘, Vol. 65. L. Grossman and K. Moldave (Eds.). New York. Academic Press. 1980. pp. 383-389.
I340
Radiation Oncology 0 Biology 0 Physics
.August 1984. Volume IO. Number 8
5. Clarke, E.D.. Wardman.
P.. Goulding, K.H.: Anaerobic reduction of nitroimidazoles by reduced flavin mononucleotide and by xanthine oxidase. Biochem. Pharmacol. 29:
2684-2686. 1980. 6. Josephy, P.D., Mason, R.P.: Chemical and enzymatic nitroreduction: Free radical and diamagnetic products of nitroimidazoles. In Bioactivation o/‘Foreign Compounds. M.W. Anders (Ed.). New York, Academic Press. 1984 (In press). 7. Knox. R.J., Knight, R.C.. Edwards, D.I.: Studies on the action of nitroimidazole drugs. The products of nitroimidazole reduction. Biochem. Pharmacol. 32: 2 149-2 156.
1983. 8. Koch, R.L., Rose, C.. Rich, T.A., Goldman, P.: Comparative misonidazole metabolism in anaerobic bacteria and hypoxic Chinese hamster lung tibroblast (V-79-473) cells. Biochem. Pharmacol. 31: 41 l-414, 1982. 9. Liu, S.F.. Raleigh, J.A.: Reductive fragmentation of misonidazole formation
in the presence of xanthine oxidase. Glyoxal (Abstract). Radiat. Res. 91: 376, 1982. IO. Mason, R.P.: Free radical metabolites of foreign compounds and toxicological significance. In Reviews in Biochemical Toxicology, Vol. I. E. Hodgson, J.R. Bend and R.M. Philpot (Eds). New York, Elsevier North Holland, 1970. pp. I5 I 200. I I. Mason, R.P., Holtzman, J.L.: The mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates. Biochem. 14: 1626-1632, 1975. 12. Raleigh. J.A., Liu. SF.: Reductive fragmentation of 2-ni-
13.
14.
15.
16.
troimidazoles in the presence of nitroreductase. Glyoxal formation from misonidazole. B/oclir~n. Plrarntuc~i 32: 1444-1446. 1983. Raleigh. J.A.. Shum. F.Y.: Oxygen mimetic reactions of nitroaromatic radiosensitizers activated by nitroreductases in the presence of model membranes. In O.\-.t:qen and O-Y!,radicals in Chemistry and Biology. M.A.J. Rodgers and E.L. Powers (Eds.). New York. Academic Press. I98 I. pp. 355361. Raleigh. J.A.. Shum. F.Y.. Liu. SF.: Nitroreductase-induced binding of nitroaromatic radiosensitizers to unsaturated lipids. Nitroxyl adducts. Biochem. Pharmacol. 30: 292 I 2925. 1981. Rauth, A.M.. Battistella. R.. McClelland. R.A.. Fuller. J.R.. Seaman. E.: Possible role of N-l substitution on the mutagenicity of 2nitroimidazoles (Abstract). Radiat. Res. 91: 376, 1982. Schauenstein. E.. Esterbauer. H.. Zollner. H.: .JIdeh~~desin
Biological Systems. Their Natural Occurrence und Bioiogical .-lctivities. London, Pion Limited, 1977. 17. Spinks. J.W.T.. Woods. R.J.: An Introduction to Radiatron Chemistry. 2nd edition. New York, Wiley and Sons. 1976. pp. 93-98. 18. Whillans, D.W.. Whitmore. G.F.: The radiation reduction of misonidazole. Radial. Res. 86: 3 I I-324, I98 I. 19. Whitmore, G.F., Varghese, A.J., Culyas, S.: Glyoxal as a misonidazole-mimetic agent. In Proceedings qf‘the Seventh International Congress qf Radiation Research. J.J. Boerse. G.W. Barendsen, H.B. Kal. A.J. Van Der Kogel (Eds.). Netherlands, Martinus Nijhoff. 1983 (Abstract B6-31).
IO.
PD.
1331-1340
??Session II
REDUtXIVE
FRAGMENTATION OF 2-NITROIMIDAZOLES: AMINES AND ALDEHYDES
J. A. RALEIGH,
PH.D.
AND S. F. LIU, M.Sc.
Radiobiology. Cross Cancer Institute, Edmonton, Alberta T6G 122 Canada Glyoxal has been identified as a product in the fragmentation of reduced 2-nitroimidazole radiosensitizers. Quantitative analysis of glyoxal as its bis (2,4_dinitrophenylhydrazone) derivative shows that it is formed in good yield (9-23%) in a variety of 2-nitroimidazoles. In addition to glyoxal a second as-yet-unidentified carbonyl compound, and a series of amines are formed when reduced 2-nitroimidazoles fragment in the presence of water. One of the amines derived from misonidazole is identified as I-amino-3-methoxypropan-2-01, the product of extensive ring cleavage. Radiation chemical reduction of the 2-nitroimidazoles proceeds with the consumption of 3 electrons for each molecule reduced. This could imply that a radical disproportionation or dimerization step is involved in the reductive degradation of 2-nitroimidazoles. Radiosensitizer, fragmentation.
Nitroimidazole,
Radiation chemical reduction, Glyoxal, I-amino-3-methoxypropan-2-o],
Reductive
graphic analysis based on the bis (2,4dinitrophenylhydrazone) derivative, we have found that in the radiation chemical reduction of 2-nitroimidazoles, 9-23s of the molecular fragments from the degraded drugs can be detected as glyoxal. In addition to glyoxal, a series of amines are produced when reduced 2-nitroimidazoles fragment. In the case of misonidazole, one of these has been identified as ! -amino-3-methoxypropan-2-01 which incorporates one ring nitrogen and the side chain of the original misonidazole molecule.
INTRODUCTION Reductive metabolism is a possible source of 2-nitroimidazole toxicity. In addition to radical intermediates,‘*‘*“.” the formation of reactive compounds from the reductive fragmentation of 2-nitroimidazoles may be involved. The fragmentation is reported to proceed from the hydroxylamine derivative. ‘.6.‘5.‘8The putative 2 electron, nitroso precursor could not be detected under conditions in which the analogous nitrosobenzene intermediate can be trapped.13,14 One of the molecular fragments formed when misonidazole is reduced in vitro is a guanidino derivative which incorporates 3 nitrogen atoms and 1 carbon atom of the original 2-nitroimidazole structure.’ We have shown that the remaining two carbon atoms in the ring can appear as glyoxal. 9.‘2 Glyoxal reacts readily with nucleic acids and proteins.4,‘6 It could contribute to the toxicity of reductively activated 2-nitroimidazoles and to the preincubation effect wherein the shoulder of survival curves of irradiated cells is diminished by hypoxic preincubation with 2-nitroimidazole radiosensitizers.” The relative importance of glyoxal would depend on its yield from 2nitroimidazole fragmentation. By means of a chromato-
METHODS
AND MATERIALS
The 2-amino derivative of misonidazole was prepared by catalytic reduction according to a literature procedunz3 2,4-Dinitrophenylhydrazine was recrystallized from lbutanol before use. 2,4_Dinitrofluorobenzene was used as received. Glyoxal bis (2,4dinitrophenylhydrone) was prepared from a nominal 40% aqueous solution of glyoxal and its identity confirmed by mass spectrometry. Gravimetric analysis of the glyoxal solution by means of the bis (2,4dinitrophenylhydrazone) derivative showed it to contain 4 1% of glyoxal. This standardized solution was
Reprint requests to: J. A. Raleigh. Acknowledgements-This work was supported by the National Cancer Institute of Canada and the Alberta Cancer Board. The 2-nitroimidazoles (Table) were generously supplied by the National Institutes of Health U.S.A. through the good offices of Dr. V. L. Narayanan. 2,4-Dinitrophenylhydrazine and glyoxal were obtained from the Fisher Chemical Company and 2,4-
dinitrofluorobenzene from BDH Chemicals. High pressure liquid chromatography (HPLC) was performed on a Varian 5000 system incorporating a Varichrom variable wavelength detector and a Spectra Physics SP4 100 computing integrator. Irradiations were performed in a Gammacell 220 6oCo irradiator available from Atomic Energy of Canada Limited. Accepted for publication 22 March 1984. 1337
1338
Radiation Oncology 0 Biology 0 Phyws
used to develop calibration curves for glyoxal in aqueous solutions. Radiation chemical reduction of the 2-nitroimidazoles was achieved by means of a procedure similar to that of Whillans and Whitmore.” Typically, a I.0 mmolar solution of the 2-nitroimidazole in doubly distilled water containing 0.1 molar sodium formate buffered at pH 7 or 9 with 0.01 molar NazHPQ, was irradiated in a 6oCogamma irradiation facility. The solutions were bubbled with nitrogen before and then throughout the time of irradiation. Under these conditions of irradiation approximately six microequivalents of electrons are produced for each kilorad of radiation absorbed in the solution. The dose-rate of the 60Co-gamma radiation was 8.1 krad/min as measured by Fricke dosimetry.” Glyoxal was quantified in terms of its bis (2,4-dinitrophenylhydrazone) derivative which was formed by heating 1.0 ml of a glyoxal-containing solution with 20 ~1 of a 2,4_dinitrophenylhydrazine reagent in a sealed test tube for 20 minutes at 70”. The reagent was prepared by dissolving 400 mg of 2.4-dinitrophenylhydrazine in 2.0 ml of concentrated sulfuric acid and adding this solution to 13 ml of 80% aqueous ethyl alcohol. It was found that 20 minutes of heating at 70’ was required to complete the reaction of glyoxal in the concentration range of interest. (A second, as-yet-unidentified aldehyde was observed during the analysis for glyoxal. We are presently attempting to discover its structure). A calibration curve for glyoxal was established under the conditions of our analysis. Prior to HPLC analysis (Partisil ODS-2, 70% aqueous acetonitrile), the test solution was mixed with I .O ml of dimethylsulfoxide in order to solubilize the bis (2,4-dinitrophenylhydrazone) derivative of glyoxal that had formed. We have found this analysis suitable for measuring glyoxal in biological fluids. The loss of 2-nitroimidazoles was measured by HPLC analysis. Amines formed during the reductive fragmentation of the 2-nitroimidazoles were detected and isolated as their 2,4-dinitrophenyl derivatives. Following radiation reduction of 200 mg of misonidazole in I.0 liter of doubly distilled water containing 0.1 molar sodium formate buffered at pH 7.0 with 0.01 molar Naz HPO,, the solution was concentrated in vuczw to 100 ml and 500 mg 2.4dinitrofluorobentene added. The reaction mixture was stirred for 16 hours at which time the amine derivatives had precipitated as an oil. The mixture of products was fractionated by preparative thin layer chromatography (silica gel G; benzene/methanol, 8S/ 15). RESULTS
AND
DISCUSSION
The chromatographic profile of the 2,4-dinitrophenyl derivatives of the amines formed when misonidazole fragments following reduction show the presence of at least seven amino compounds. One of the amines has been identified as the 2,4-dinitrophenyl derivative of misonidazole amine by chromatographic and mass spectro-
August 1984. Volume IO. Number 8
graphic comparison with an authentic sample prepared as described above. Of the remaining components of the mixture only one has been identified to date. It was isolated by preparative TLC and shown to be a single compound by HPLC (picrate mp 272“). Its high resolution mass spectrum (m/e 271.0800 (molecular ion corresponding to an elemental composition of C,,,H,3NJ06) and m/e 196.0354 (base peak corresponding to the loss of the fragment CHOHCHZOCH~ from the parent compound)) and nuclear magnetic resonance spectrum ((CDCl,) 3.45 ppm (singlet 3H, OCH& 3.55 ppm (overlapping triplets, 4H,NHCHZCHOHC’H@CH,), 6.98 ppm (doublet, 1H, J = 0.1 ppm), 8.28 ppm (doublet of doublets, 1H, J = 0.1 ppm and 0.02 ppm) and 9. I5 ppm (doublet. 1H, J = 0.02 ppm), the last three resonances attributable to the 2,4_dinitrophenyl moiety) are consistent with the structural assignment of the 2,4-dinitrophenyl derivative of 1-amino-3-methoxypropan-2-01. The corresponding molecular fragment from I-(2-hydroxyethyl)-2-nitroimidazole: i.e.. ethanolamine, has been isolated in the same way and identified by chromatographic and mass spectrographic comparison with an authentic sample of the 2,4-dinitrophenyl derivative of ethanolamine. Figure I represents a plausible mechanism for the formation of Iamino-3-methoxypropan-2-01 from reductively activated misonidazole. This mechanism expands on that proposed for glyoxal formation’.” in that a water molecule attacks the 2-position rather than the S-position in the hypothetical hydroxylamine intermediate. The figure can also account for the formation of hydroxyurea which has been
NHOH
OH NHOH
N H.C=’
li-
‘NHOH
‘NH HO H, r$HR
iR
\
\ FHzNHGo ‘NHOH CHO
NH, \
FH? R
/+O NHOH R
=
CH,CHOHCH,OCH,
Fig. I. Possible mechanism for the formation of ~-amino-Jmethoxypropan-2-01 and hydroxyurea from reductively activated misonidazole.
Fragmentation of 2-nitroimidazoles 0 J. A. Table I. Radiation
chemical reduction
1-(R)-2-nitroimidazole R = CH$ZHOHCH20CHj CH$ZONHCH2CH20H CH2CHOHCH2F CHzCHzOH Ribose Dideoxyhexenpyranose
Glyoxal yield (percent) 20 20 14 15 23 9 (a) 15 (8)
RALEIGH
AND
S.
F. LIU
1339
of 2-nitroimidazoles
Nitroimidazole loss (rMole/Krad) 1.9 1.8 2.0 1.9 1.9 2.1 2.1
P’M 0
MIS0
LOSS
0
GLYOXAL
FORMATION /
200
reported recently.’ At this stage, the aldehydes depicted in the figure can only be considered possible products. Addition of a nucleophile other than water (eg., protein amine or thiol) to the 2-position would lead to binding of a sensitizer labelled with 14Cat the 2position. Attack at the 5-position could also lead to binding but the stability of the bound intermediate is in question. The yields of glyoxal from a series of reductively-activated 2nitroimidazoles (Table 1) were determined from slopes of dose-yield plots such as in Figure 2 for misonidazole. Glyoxal is clearly an important product of fragmentation and could contribute to the hypoxic cell cytotoxicity and preincubation effect of 2nitroimidazoles. It seems possible that precursors of aldehydes more potent than glyoxal might be built into the ring of 2-nitroimidazoles whereby chemotherapeutics directed at the hypoxic zones in tumors might be developed. We are presently pursuing this possibility. Radiation chemical reduction of the 2-nitroimidazoles consumes 3 electrons per molecule of drug based on the fact that each krad of radiation delivers 6 microequivalents of electrons to the irradiated solution.‘* While a detailed discussion is beyond the scope of this paper, the result is clear (Fig. 2) and implies that reductive fragmentation of 2-nitroimidazoles involves the disproportionation or dimerization of the three electron adduct (eg. ZRNOH’ RNHOONHR; such a dimer could fragment in a way analogous to that for the hydroxylamine intermediate). reAn earlier study” which showed that misonidazole quired 3.5-4.0 electrons per molecule for its radiation
chemical reduction differs from the present study in that the authors took the reaction to completion in which case fragmentation products such as glyoxal will consume additional electrons (Raleigh and Liu, unpublished data,
??
0 100 0/
J_o/o’o
+o-
I
I
1
50
100 Krod
Fig. 2. Radiation reduction of misonidazole. Misonidazole loss (y = 1.9x - 3.6, r = 0.997); Glyoxal formation (y = 0.39x - 0.13, r = 0.992).
1983) and ultraviolet spectroscopy rather than HPLC was used to quantify the loss of misonidazole. SUMMARY We have shown that glyoxal, a potentially toxic product, is found in reasonably good yield when reduced 2-nitroimidazoles fragment. Building molecular components of even more toxic aldehydes into the ring of 2-nitroimidazoles may provide therapeutic gains when the target is the hypoxic zones of tumors. The identification of lamino-3-methoxypropan-2-01 indicates how extensive the fragmentation of reduced 2-nitroimidazoles can be and points to a possible mechanism for the fragmentation. The consumption of 3 electrons for the reductive decomposition of each 2-nitroimidazole molecule raises interesting mechanistic considerations.
REFERENCES Biaglow. J.E.. Jacobson. B.. Greenstock, C.L.. Raleigh, J.A.: Effect of nitrobenzene derivatives on electron transfer in cellular and chemical models. Mol. Pharmacol. 13: 269282. 1977,
Biaglow. J.E.. Jacobson. B.. Koch. C.J.: The catalytic effect of the carcinogen “4-nitroquinoline N-oxide” on the oxidation of vitamin C. Bioph.t*s. Res. C’ummzr~z.70: 13161323. 1976.
3. Born. J.L., Hadley, W.M.. Anderson, S.L.. Yuhas, J.M.: Host and hypoxic cell toxicity studies with the terminal reduction product of misonidazole. In Radiation Sensitizers. Their Use in Clinical Management of Cancer, L.W. Brady (Ed.). New York, Masson Publishing. 1980, pp. 79-82. 4. Carmichael, G.G.. McMaster. G.K. .bferhods in Enzvmolog~‘, Vol. 65. L. Grossman and K. Moldave (Eds.). New York. Academic Press. 1980. pp. 383-389.
I340
Radiation Oncology 0 Biology 0 Physics
.August 1984. Volume IO. Number 8
5. Clarke, E.D.. Wardman.
P.. Goulding, K.H.: Anaerobic reduction of nitroimidazoles by reduced flavin mononucleotide and by xanthine oxidase. Biochem. Pharmacol. 29:
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