S T C D I EWITH ~ DEUTERATED ZOXAZOLAJIISE
Jaiiuary 1970
establish that a D atom was present in the crucial C-6 position of the 3-chlorobenzoxazole nucleus, the dideuteriozoxazolamine was deaminated h>-drolytically a t C-2 with DjO under acid catalysis to give a dideuterated 3-chloro-2-hydroxybenzoxazole, H
I
H
YH
D 3
6.92 2
6 6.67
1
?
P
which can be assigned to a C-7 proton and is in accord with the assignment of structure 5. Pharmacological Studies.-The durations of pharmacological action of zoxazolamine and the deuterated analog were compared, using male Sprague-Dan-ley rats iveighing 215-290 g. To minimize the deviation of the mean due to individual variation among rats, t'he rats were separated into two groups, long sleepers and short sleepers. This procedure is based on the duration of sleep exhibited by each rat after the iritraperitorieal administration of hexobarbital (100 mg > l ~ g ) Both . ~ of these groups of rats were used the day after hexobarbital treatment to examine the duration of paralysis caused by zoxazolamine and deuteriozoxwzolamirie. can be seen in Table I, in neither group ~ - R Sthe duration T.itjLi:
c,: 4
I
Zos.izoi..~msi: IX.JI.:CTIOS
I
H
31
92
5
Chlorination of 5-chloro-2-hydroxybenzoxazole(1) with S02Cl, i.: hnowri to produce 3,6-dichloro-2hydroxybenzoxazole (2). When the dideuterated 5chloro-2-hydroxybenzoxazole was iubjected to these chlorinating conditions in deuterioacetic acid as solvent, a sample was obtained that was identical in all respects with that of the reported 5,6-dichloro-2-hydroxybenzoxazole ; combust ion analysis indicated the loss of exactly one D atom. This series of experiments firmly secure5 C-6 as the position of one of the D atoms in the benzoxazole nucleus of zoxazolamine. This leaves either structure 3 or 4 to be considered for the dideuterated derivative. S m r data of 5-chloro-2-hydroxj berizoxazole (1) were used to assign the location of the second D preient 111 the dideuteriozoxazolamine. The aromatic protoris of 1 appear as a multiplet in the 6 6.6-6.8 region of the nmr ipectrum. A relativelj- uncomplicated singlet appear., at 6 6.S which can be azsigned to the C-4 proton Ggnal, iiiice thi. proton, ortho to C1, would be deshielded arid absorbed at the lowest field position and be verj weakly coupled to the irieta and pa1.a hydrogens. The nmr spectrum of the dideuterio derivative of 1 diows the disappearance of the sharp kiglet peak and only L: broad peak a t 6 6.79. MT/2 = 1.5 Hz. The broadneis of the band is only compatible with the replacement of the C-4 proton by D. The broadened C-7 proton signal in 4 can be attributed to coupling with the adjacent C-6 D atom: J D - H values of 1-1.5 Hz have been ~ b s e r v e d . ~This nmr evidence thuy permits the choice of 4 over 3 for the dideuteriozoxazolamine. E'urther evidence for the placement of the second D a t C-4 in the berizoxazole nucleus comes from examination of the nmr spectra of the deuterated 5,6-dichloro-2h?*droxybenzoxazole ( 5 ) arid 3-chloro-2-hydroxybenzoxazole (1). The nmr of 1 shoni the C-4 proton at 6 6.75 arid a multiplet for the C-6 and C-7 protonq centered a t 6 6.67. On conversion to the dichloro derivative 2 (the C-4 proton) remains a t 6 6.75, whereas the C-7 proton i> shifted to 6 6.92. The nmr of the deuterio derivative of 2 \hot\-< a single peak a t 6 6.92, ( 5 ) H. Spiesecke and IT. G . Sclineider, J . C'hem. I ' h g s . , Sli, i 3 1 (1561).
Iluration of
Group
Compound"
paralysis, min
Zosazolaniine 4%; +C 162 (4)" l)eiiteiiozosazolamilie-4,~-(~~ 417 i 78 ( 3 ) Short sleepers Zoxazolamiiie 28x7 +C 80 14) l)eiite~iozo~azolai~iitie-4,6-tiu 242 i :28 (4) Each rat vias injected iiitraperitoiieally with GO mg kg of zoxazolamiiie or deiit,eiiozoxazolamiiie-4,6-~/~,dissolved ili a 1: 1: 1 mixtiue of climethylacetamide-propyleiie glyuil-505 aqueous glycerol. b Figures ill parenthese.5 refer t'o the iiumber of rats iii each group. Each value in a mean i .taiidard deviation. Long sleepers
of actio11 of zoxazolamine significantly different from that of its deuterated analog. The data were analyzed by Students' t test. E'rom these experiments i)z uiuo, it would appear that there was no isotope effect on the biological hydroxylation of the aromatic ring. 1u I'itro Metabolic Studies.-The metabolism of zoxazolamirie and the deuterated analog by liver microsomes was next studied in order to compare the result,s in uitro ivith those obtained in uiuo. This study serves t o augment the it1 vivo sleeping time data, which indicate t'hat zoxazolamiiie-4,6-c12is metabolized at an essentially unaltered rat e compared to zoxazolamine. Liver microsomes were prepared from male SpragueDawley rats which were injected intraperitoneally with 20 nig/lig of 3,4-benzpyrene in corn oil 24 hr before sacrifice. The liver \vas homogenized in three volumes ( v f w ) of 1.137, IiCl and was centrifuged at 10,OOOy for 10 min. The supernatant fraction was used for incubation. The comparative metabolic data are presented in Table 11. The in vz'tw data indicated that regardless of the method of analysis, the deuteriozoxazolamirie \vas metabolized considerably more slon-1)- t hail zoxazolamine. Whether this reduced rate of metabolism of deuteriozoxazolamine is due to decrease in the affinity t,o the degradative enzyme or to the slower cleavage rate of C-D bond than that of C-H bond camlot be amwered from these experiments. The reason is not clear for the absence of' difference in the duration of pharmcologic action between the two forms of zoxazolnmirie despite their difference in the irr v i t i ~ degradation rates by the liver supernatant fraction. A loss in the potency of a drug by deutera(6) C. Mitoma, S. E. Neubauer, K. L. Badger, a n d T. J. Sorich, 11, Proc. SOC.Ezptl. B i d . M e d . , 125, 284 (1567).
\'Ill,
reported by Elison, et d.,; for deuteriomorphine, c m be ruled out iii the present study. 1)eterminatioii of EDjo values and tmhe95% confidence limits bj. the method of Litchfielti niid IT~lcoxoii~ revealed no difference between the two coriipouiidd, .is mg kg (53--61) for zoxazolamiiie xiid 59 mg kg ( - X - N ) for deuteriozosazolRniiiie. Our apparent inability to detect this expected pharmncologic difference c a n be ratioiialixed by the app:irent insensitivity of the i l l uico sleep time method employed to distinguish bet\veen the compounds. The present' iri vitro data differ from those of acet:ii~ilide,~ phenobarbita1,"J m d dirnethylphenol!" where substitution of H by D or ;I T a t o m at the site of aiwnatic ring hjdroxylation had little effect on the rate of hydroxyl,'I t 1011. '
Experimental Section 2-Amino-4-chlorophenol-3,5-d~.--In :i 50-rn1, thick-wall glass 1 iilie,
2.!434 g of fL-amiiio-4-chlorophenol was dissolved in a solu-
( T ) C. Elison, II. Rapoport, R. Latimen, arid 11. IV. Elliott. Science, 134, 1078 (1961).
(8) J. T . LitcIifield, J r . , and F. W. \Tileoxon. ,I. Phm~niic~11. Ciptl. Therap., 96, 99 (19413). (9) h1. Tanabe, 1). 'iasuda, .J. Tngg. a n d C . Mitoma. Biocliem. P l m ? m , i r o ! . , 16, 2230 (1967). (10) .J. A I , I'esel. 1). C:. D a y t i i n . ( ' . 1,. Ta\.rir:llv, 1,. I%raiid,and I,. ( ' . l l a r h , .1. . \ I d . C h e m . . 10, 3 7 1 (1967). (11) I{. .I. 13. \Vood and L. L. Ingrsliain. .Iri,h. Biochcni. B i o p h ~ a .98, . ii!J (1962).
I:!
