Glutathione conjugates of misonidazole

Vol. 112, No. 3, 1983 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 16, 1983 Pages 1013-1020

GLUTATHIONE CONJUGATES OF HISC?iIDAZOLE

A.J. VARGHESE

Physics Division, Ontario Cancer Institute,

500 Sherbourne Street, Toronto, Ontario M4X lK9, Canada

Rf!ceived March 30, 1983

SUMMARY: The hydroxylamine derivative of misonidazole reacts with qlutathione -- under physiological conditions to form two isomeric conjugates. Eased on physical and chemical properties, the two conjugates have been identified as l-~[2-amino-(4-glutathion-S-yl)-l-imidazolyl]-3-methoxypropanol and l--[2-amino-(5-glutathion-~-vl)-l-imidazolyl]-3-methoxypropano1. The formation of the glutathione conjugates of reduced misonidazole offers a molecular mechanism for the depletion of GSH in mammalian cells after exposure to misonidazole under hypoxic conditions.

Misonidazole (Fig. 1) is currently undergoing clinical trials (l-5)

as a radiation sensitizer in the treatment of certain types of human tumors.

Neurotoxicity is a limiting factor in the clinical use of the drug. Another

inportant property that is of potential clinical significance in cancer

chemotherapy of the drug is that it is more toxic to hypoxic cells than to

aerobic cells (6-12). A number of studies (9, 11-16) indicate that reductive

metabolism of the drug has a role in the hypoxic cell toxicity of the drug.

E\en though there is evidence for the reductive metabolism of the drug in

man, its role in neurotoxicity is not known. Since reduction of nitro

ccmpounds proceeds through reactive intermediates such as the nitroso and

hldroxylamine derivatives, one possible mechanism for the toxic nroperty of

misonidazole is the modification of cell constituents by these reactive

reduction intermediates. Results of a number of recent studies (18-22)

suggest that intracellular thiols play a significant role in the hypoxic

cell toxicity of misonidazole. In this paper, iJe present evidence that

misonidazole, after reduction to the hydroxylamine state, reacts with

glutathione under physiological conditions to form stable qlutathione

ccnjuqates.

0006-291X/83 $1.50

Copyright 0 I983 by Academic Press, Inc. 1013 Ail rights of reproduction in an), form reserved.


H-C =C-H

I I N\Q CH,-E- CH2- OCH3

I NO2

I

Fig. 1 Structure of misonidazole. The asterisk shows the [14Cl label.

MATERIALS

Misonidazole was obtained from Dr. Carey Snithen of Roche Products, Ltd., Welwyn City, Hertfordshire, England. GSH and ascorbic acid were purchased from Sigma Chemical Cc., St. Louis, MO. [2-14C]-Misonidazole was synthesized as described previously (20 uCi/n mole) (131. [Glycine-2-3H] GSH was purchased from New England Nuclear, Boston, MA and diluted to 0.05 mCi/m mole. The amino and hydrazo derivatives of misonidazole were prepared as described previously (15). Raney Nickel (activated) was purchased from Aldrich Chemical co., Milwaukee, WI, U.S.A.

METHODS

Reaction with GSH

Misonidazole (2 mM) was reduced with zinc dust in the presence of ammonium chloride as described previously (15). When the drug was fully reduced as indicated by the absence of absorbance above 240 nm, the suspension was filtered. Unless otherwise stated, one volume of the filtrate was added to an equal volume of a solution (10 n-&l) of GSH in 0.05M phosphate buffer (pH 7) and the reaction was allowed to proceed at 37O in the absence of air for one hour.

HPLC Analysis

A Waters liquid chromtograph equipped with a Waters Model 440 absorbance detector operated at 254 nrn and a Waters u Bondapak ClS column (0.9 x 30 cm) with water:methanol:acetic acid (94:5:1) as solvent were used, the flow rate was 2 ml/min, and 200 ~1 samples were injected. When radioactivity measurements were required, one ml fractions were collected and counted. For the large scale preparation of GS-M, the fractions containing the product were pooled and lyophilized. This was done successively until milligram quantities were obtained. The lyophilized sample was used for all subsequent studies.

Spectroscopic Studies

Proton NMR analysis was performed by Dr. Arthur Gray, University of Toronto on the 360-MHz FT systems. Fast Atom Bombardment (FAB) spectra were obtained by Dr. J.L. Holmes, Department of Chemistry, University of Ottawa, Ontario, Canada.

RESULTS

Results of preliminary studies indicated that misonidazole, after

reduction with zinc dust to the hydroxylamine derivative reacted with GSH in

phosphate buffer (pH 7) and the reaction product (GS-MIS) could be separated

1014


r I I 1

9

b)

I

I-

,_

e r,

1

a)

IO 0 - 30 20 IO 0 Fraction number

Fig. 2 HPLC separation of GS-MIS. Reduced misoniaazole was reacted with -- GSH and the reaction mixture was analyzed as described in Methods. Fig. 2a shows the absorbance monitored at 254 nm. The retention volume in ml is the x-axis. Fig. 2b shows the distribution of [14C] activity in one ml fractions when [2-l*C] misonidazole was reduced and reacted with GSH. The distribution of [3H] activity in one ml fractions when [3H] GSH was reacted with reduced misonidazble is shown in Fig. 2c.

b> the HPLC separation procedure described above. A typical separation of

tt.e reaction mixture is shown in Fig. 2a. GS-MIS has a retention volume of

2:. ml. Plisonidazole had a retention volume of 60 ml under the same

conditions. The optimum pH for the formation of GS-KIS was pH 8. In

solutions of pH > 4.5, detectable amounts of GS-MIS were not detected and

in solutions of pH < 9 the yield decreased markedly. The optimum pH for the

fclrmation of GS-MIS was pH 8 - 8.5. When GSH was reacted with either the

amine or hydrazo derivative of misonidazole, there was no evidence for the

formation of the glutathione product. Both the amine and the hydrazo

dt:rivatives have been reported to be formed when misonidazole is reduced with

zinc dust (15).

When the experiment was repeated using reduced [2- .4C] misonidazole, the

d>stribution of radioactivity in the HPLC fractions is shown in Figure 2b.

Pz-ior to the addition of GSH, radioactivity was not detected in fractions

beyond the 20th. The results of a similar study using [3H] GSH for the

1015


Ml t -1

FAB spectrum I in glycerol 1 t

peoks from glycerol *

60 -

* Lu L. I

Doped wth NaCL

Doped with KCL

60

300 400 500 600 700 M/Z

Fig. 3 FAB spectra of GS-MIS. M represents the Molecular Weight.

reaction are shown in Figure 2c. Again, when GSH without the addition of

reduced misonidazole was analyzed, radioactivity was not detected in fractions

after the 20th. The presence of both [l*C] and [3H] activities in the

fractions (nos. 22-24) containing GS-MIS suggests the presence of both the

GSH and misonidazole residues in the product.

Additional information on the structure of GS-EIIS was obtained by mass

spectrometry by Fast Atom Bombardment ionization technique. From the spectra

shown in Figure 3, it is apparently clear that GS-MIS has a molecular weight


of 476, which could result by the elimination of water (M.W. 18) from an

addition product of qlutathione (M.W. 307) and the h;.droxylamine derivative

of misonidazole (?I.\<. 187).

Valuable information on the nature of lir:<age between the misonidazole

an'1 glutathione residues was obtained from studies on the acid-stability of

the product. Compounds having N-S linkages are known to be unstable in

acidic solutions (23). The acid-stability was studied by heating the adduct

prepared from [2-14C] misonidazole and GSH in O.lN HCl for one hour and

analyzing the product by HPLC. The result did not show any detectable change

from that of the untreated adduct indicating the stability of the adduct in

acid and hence the absence of a N-S linkage.

Results of desulfurization studies provided confirmatory evidence on the

nature of linkage between the two residues. The adduct, prepared from

[2-14c] misonidazole and GSH, was refluxed with Raney Nickel in 70% alcohol

for one hour (24). The suspension was filtered and an aliquot was injected

for HPLC analysis. Radioactivity measurements of the HPLC fractions indicated

th,a presence of only one radioactive peak which had the same retention volume

as the amine derivative of misonidazole. The identity of the amine was

further confirmed by the preparati'on of the dansyl derivative (17). Since

desulfurization with Raney Nickel is known to cleave C-S bonds to form -CH

grl2ups, it is to be concluded that the adduct has a C-S linkage (18).

The data presentedare consistent with either of the two isomeric

structures shown in Figure 4 for the qlutathione-misonidazole adduct, The

NMR spectrum (Figure 51, in addition to confirming the structure, indicated

the presence of both isomers. Signals for all the non-exchangeable protons

of both the glutathione and misonidazole residues are seen in the spectrum.

The assignments are given in Table 1. The two singlets in the aromatic

region and the doubling of the signals oL + the protons of the side chain

group of the misonidazole residue provide confirmatory evidence for the

presence of both isomers.

1017


,CO- NH - CHTCOOH R= CH2-CH, NH-CO-CH2-CH2-CH-COOH

I NH2

Rt=CH2-CH - CH20CH3

AH

4 Fig. Proposed structures for the two isomers of GS-MIS.

Incubation of mammalian cells under hypoxic conditions in the presence

of misonidazole has been shown to reduce the intracellular concentration of

GSH (25). The formation of GS-MIS from GSH and reduced misonidazole under

physiological conditions described in the present study provides a molecular

mechanism for the misonidazole-induced loss of GSH in the cells. Whether the

h A

Fig. 5 Proton NMR spectrum of GS-MIS. The spec:rum was taken in D20 on a 360 MHz FT NMR system.

Vol. 112, No. 3, 1983 8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1

360 MHZ 'H-NMR Spectral Properties of MIS-GS

Assignment Chemical Shift (in ppm from TMS)

Glutathione residue

C*S-lY cys-6 9lY-a

qlu-cX 91-y qlu-B

Misonidazole residue

= N - CH = CH - N - -

= N - CH = CH - N - -

'- N - 5 - CHOH - CH2 - OCH3

'- N - CH2 - CHOH - CH2 - OCH3 -

N - CH2 - CHOH - CH2 - OCH3

CH2 - 0 - CH 3

4.52 (1) 3.02; 3.22

3.74 (2)

3.56 (1) 2.12 (2) 2.56 (2)

6.96 (f)

7.15 (%)

4.02; 3.76

4.2 (1)

3.38 (2)

3.36 (3)

(2)

(2)

removal of GSH as GS-MIS contributes to the hypoxic cell toxicity of

misonidazole is a topic for further investigation. Sowever, it is of

interest to note that the intracellular concentration of [2-14C] misonidazole

leading to 50% toxicity in EM-6 tumor cells is estimated to be about 3.7 mM,

wh:ch is equivalent to the concentration of GSH in the cell 126). The

formation of GS-MIS0 is also of importance as a possible pathway for the

removal of the toxic metabolites resulting from the reduction of misonidazole.

In either case, additional studies are required to determine the biological

imljortance of GS-MIS.

ACKNOWLEDGEMENTS --

This work was supported by the Ontario Cancer Treatment and Research Foundation and the National Cancer Institute of Canada.

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Glutathione conjugates of misonidazole