- Email: [email protected]
Isolation and identification of the major urinary metabolite of metronidazole
Life Sciences Vol . 6, pp . 1811-1819, 1967 . Printed in Great Britain .
Pergamon Press Ltd.
L90LATION AND IDENTIFICATION OF THE MAJOR URINARY METABOLITE OF METRONII~ZOLE J. E. 3tambaugh, L. G. Feo and R. RI . Manthei Departments od Pharmacology and Urology Jefferson Medical College, Philadelphia, Pa. (Received 1 March 1967 ; in final form 29 May 1967) Metronídazole (1-(2-i~ydraa~ett~yl)-2-methyl-5-nítroimidazole) is an orally effective trichomonacidal agent. In previous reports (1, 8, 3) we showed that chra~matograms a~f urines from patients receiving this drug contained six bands, not found in normal urine, that absorb in the ultraviolet region . Two of these bands comprise 70~ ad the total nitro~ontaining compounds excreted and consist of approximately equal amounts of unchanged metronidazole and a major metabolite . In this report, we present the isolation and identification ad the major metabolite of metronidazole, 1-(2-hydroxyetl~yl)-2-hydroxymethyl-5-nitroimidazole, found in the urine ad all patients investigated. This compound is formed by the oxidation of the 2-methyl group of metronidazole to the corresponding alcohol . \ H,3 C
(Fig. 1) C;2CHZOH N
N\ ~C-N02
ÇH20H `C
;HaCHaOH N
; ~-NOZ
1812
METRONIDAZOLE METABOLITE
Vol . 6, No . 17
Experimental Twenty-four hour urines from patients receiving 250 mg . of metronidazole orally three times daily were collected and frozen for storage . The urines were thawed at room temperature, desalted, and the urea destroyed with urease . The urines were then deproteinized, concentrated, and spotted on Whatman lkl paper strips .
The strips were de -
veloped in several solvent systems by descending chromatography, and the nitro-containing metabolites were located on the strips as absorption bands under ultra-violet light.
Characteristic color reactions of
the metabolites were observed on the strips by reducing the nitro group with 1. 5~ titanous chloride in 10~ acetic acid and overspraying with var ious color reagents . The major urinary metabolite of metronidazole was isolated by the uee á an ion exchange resin. Twenty-five cc . of the concentrated urine was poured onto Amberlite XAII-2 resin in a 300 mm column, 24 mm in diameter, formed in water . The column was developed with 5~ ammonia in water, and the fraction containing the major metabolite was identified by paper chromatography .
The crude metabolite was purüied by refrac-
tionatian on smaller columns od the Amberlite resin and was crystallized as pale yellow needles after standing several days in absolute ethanol at room temperature, (m . p. 80-84°). 1-(2-hydroxyethyl)-5-nitroimida~ole was prepared by refluxing 40 grams of 4(5)-nitroimidazole with 800 cc of ethylene chlorhydrin at 127°C far 24 hours. The excess ethylene chlorhydrin was evaporated off; the residue was taken up in water, filtered to remove the insoluble nitroimidazole, and extracted at pA 8 with ethyl acetate. A clear yellow
Vol . 6, No . 17
METRONIDAZOLE METABOLITE
1813
oil was obtained from the ethyl acetate extract which crystallized to pale yellow needles on standing in the cold overnight. Recrystallization from acetone/water yielded 17 .3 gm (33~ yield) of 1-(2-hydroxyetigl)-5-nitroimidazole, (m .p . 95-97°) (lit . 97-98°) (4). 1-(2-hydra~xyethyl)-2-hydroxymethyl-5-nitroimidazole was prepared b9 refluxing 14 .3 grams a~f 2-hydroxymethyl-4(5)-nitroimidazole with 80 cc of ethylene chlorhydrin at 127°C for twenty-four hours. The excess ethylene chlorhydrin was evaporated a~ff, and the brown oil residue was taken up in 2N NaOH .
The basic solution was extracted with eti~yl acetate,
and the extract was taken to dryness . The resultant yellow oil was taken up in absolute ethanol, charcoaled, filtered and the desired product crystallized out after standing several days at room temperature as pale yellow needles, (m .p. 81-83°) (Calcd. for CgHgN304 : C, 38 .29; H, 4.82; N, 22 .33 .
Found: C, 38 .32 ; H, 4 .85; N, 22 .18) .
Infrared spectra of this isolated products were recorded from KHr pellets or from thin films with a Perkin Elmer Model 337 spectrophoto meter. Results The ultra-violet absorption, the Rf values in three solvent systems, and the characteristic color reactions of metronidazole, the isolated urinary metabolite (Metabolite A) and some synthesized nitroimidazoles are listed in Table 1 . Identical color reactions were observed with metabolite A, 1- (2 hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole, and 1-(2-hydroxyett~yl)5-nitroimidazole . After reduction of the nitro group, a characteristic intermediary orange color was seen on reacting these compounds with
1814
METRONIDAZOLE METABOLITE
Ó .., d Fa
M
N
d
.r ~ .~ D+ .~ D~ ~
41
Vol . 6, No . 17
O
O
Ó aD
M
O U
w
0 0
U
I
~4
w a co
F
q
ó m S
d w
H w Ó N O f=1 O a
>x a
v
O
I
N >, a
z
w
I
U W
N
ó ó
a, a
~, a
a
N ó
N ó
ó ó
ó
ti ó
~ m ó ó
.-I~
ó
N ti ó
d
ó
o o dl ó ó
~
~.
d
..1 .c
~,
d
~n N
...
~~
I .,
F q
,. Y
0
m
33
r; 3 â 3 .ö 3 ~ ... ~ F ~ O-1 ~ .yW +O+ Cd ~ ".Û C) N m v b ~ :~ m~ c~. O ~ " óó ~~
~
r~ .~ ~ ~á .-=1
.0 3
O
N y
m~~~~ ~a~ q á m
N
r.l
I r I v~ v 1 1 I N~ N
m
QU
I If!
Ó
~
~
`~ x 0 ó :: ~n -, o qc ~ ~ o rl y
c
c 1 N .,
.,O
O á
ó
4l ir
0
a
,.
~ó H
POi v
~
m
M "~ 1 "r1 .~ q iF
m y iF
FOI Û ~ V v ~c,~c.c. ~á~~á
NN O ~n~~ O wO Ó O . . N O .r NM iF iF
Yol . 6, No . 17
MEI'RONIDAZOLE METABOLITE
1815
p-dimethylaminobenzaldehyde (Ehrlich's reagent) that was not observed with metronidazole or 1-acetic acid-2-methyl-5-nitroimidazole . A purple color was obtained with diazotized sulfanilic acid (Pau),y's reagent) tom pared to a tan color seen with metronidazole and the 1 acetic acid derivative. These three compounds also gave a yellow color with ninhydrin reagent as opposed to a characteristic green color obtained with metronidazole and the 1-acetic acid homologue . Comparison of the Rf values in several solvent systems indicated that 1-(2-hydroxyethyl)-5-nitroixnidazole is slightly more acidic in nature than metabolite A. 1-(2-hydroxyethyl)-2-hydroxymeti~yl-5-nitroimidazole and metabolite A were chromatographlcally identical in the solvent systems used .
Two-way chromatograms od urine from patients containing synthetic
1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole were run on thin layer Avicel plates, and the synthetic material and metabolite A were observed as a single spot with the same Rf value in four different solvent systems. Chromatograms of normal urine to which metronidazole was added indicated that the compound was stable in urine over a period a~f several months . The I. R . spectra of 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroímidazole and of metabolite A were observed to be identical in all absorbing regions. The OH stretching vibration appeared at 3375 cm -1 in these compounds in contrast to the 3200 cm nidazole .
-1
band seen with metro-
The presence of the hydroxymetl~yl group was indicated by
the C-0 stretching vibration of twin peaks at 1058 and 1070 cm -1 com-1 pared to a single peak at 1070 cm noted with metronidazole. The I. R. spectra o~f 1-(2-hydroxyethyl)-5-nitroimidazole and metabolite A showed several significant differences in ring substitution in the 800 to 1200 cm
-1
1816
I~TRONIDAZOLE METABOLITE
Yol . 6, No . 17
region . A strong 1120 cm -l band was considered to be indicative of a 1, 5-ring substitution pattern and was sat present in the spectra of metabolite A. Discussion Both the hydra~xyethyl group and the methyl group of metronidazole are available for oxidative metabolism . Oxidation at the one position would result in a relatively stable 1 acetic acid substituted ímidazole. Oxidation at the two position would result in the formation of either an alcohol, an aldehyde, or as acid, depending upon the conditions of oxidation.
The car-
bo~xylic acid resulting from complete oxidation of the methyl group would tend to be unstable due to the influence of the adjacent ring nitrogens (5), and decarboxylation would occur to form 1-(2-hydrga~etl{yl)-5-nitroimidazole .
It is also possible that intramolecular cyclization could occur
between the 2-acid and the 1-l~ydrm~yetl~yl group to form a relatively stable Y
-lactose (6). In an attempt to prepare the major urinary metabolite, metronidazole
was subjected to oxidation by various known methods.
Hydrogen peroxiàe
oxidation resulted in the destruction of the imidazole ring, and potassium permanganate oxidation lead to numerous oxidation and breakdown products which were not readily isolated . When metronidazole was reacted with a chromic acid medium, several products were obtained . One of these products was 1-acetic acid-2-methyl-5-nitroimidazole and corresponded to a urinary metabolite of metronidazole. Ings et al (7), working with a limited sample size, reported this compound to be the major metabolite formed from metronidazole a.nd concluded, on the basis oá their studies, that unchanged metronidazole, the lucid derivative, and a glucuronide conjugate
Vol. 6, No . 17
MET1tONIDAZOLE METABOLITE
1817
were the primary urinary excretion products of the drug . However, is our studies the 1 acid derivative comprised less than 10~ aá the total nitrocontaining urinary metabolites of metronidazole. Oxidation of metronidazole with dilute nitric acid produced several oxidation products, one of which had the same chromatographic properties as the major metabolite isolated from urine. This oxidation pro duct and the metabolite, after reduction of the vitro group, reacted purple with diazotized sulfanilic acid, sad failed to produce a green color with ainhydrin reagent. 31nce the green ninhydrin reaction was observed only with compounds containing a 2-methyl group and the purple diazo reaction was not observed when the 2-methyl group was present, it was concluded that the major metabolite was a nitroimiàazole derivative in which the 2-methyl group had been either oxidized or removed. In a preliminary study, 1-(2-hydroxyeti~yl)-5-nitroimidazole was found to be similar to the major metabolite on the basis of paper chromatographic separation and colorimetric reactions (8). Subsequent isolation of the metabolite from an alumina column and comparison of its I. R. spectra with lmown 1-(E-hydroxyett~yl)-5-nitroimidazole indicated that the compounds were identical. However, simple eluates of the metabolite ba~i from paper chromatograms of freshly collected urines gave I. R. spectra that were different than either of the above two compounds.
Fur-
ther studies revealed that the metabolite was a relatively labile substance and was probably oxidized to 1-(2-hydroxyethyl)-5-nitroimidazole during the isolation procedures originally employed . The LR . spectra of the major metabolite obtained from freshly collected urines indicated that the metabolite was a 1, 2, 5-substituted im-
1818
METRONIDAZOLE METAHOLITE
Vol. 6, No . 17
idazole. Various substituted nitroimidazoles were synthesized and compared to the major metabolite obtained from paper strip eluates. It was found that 1-(2-hydroxyethyl)-2-hydroacymethyl-5-nitroimidazole possewed the same chromatographic properties as the urinary unlrnown.
In
addition the urinary unlmown isolated from an Amberlite column was found to have the same chemical properties and gave an I. R. spectra that was iàentical in all respects with synthetic 1-(2-hgdra~xyethyl)-2-hydroxymethgl5-nítroimidazole . Conclusions The urinary metabolites of metronidazole were studied in man, and the major metabolite was isolated by chromatographic methods. Comparisan of the isolated metabolite with several synthetic nitroimidazoles re vealed that 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole is the major urinary metabolite of metronidazole. It is concluded that the metabolism of metranidazole involves mainly an oxidative pathway in which the oxidation of the 2 -methyl radical takes place in preference to that of the hydroxyetlgl group.
The pharmacolagic properties of this metabolite re-
main to be investigated . Acknowledgem enta The authors wish to thank Mrs. Lydia Hangs for excellent technical assistance and Rohm and Haas for a generous supply of Amberlite XAD-2 resin used in this study.
This work was supported in part by a grant frown
G. D. Searle and Comparry . References I. R. W. Manthei, R. S. Horn and L. G. Feo, The Pharmacologist (1982 ).
4
170
Vol . 6, No . 17
1819
METRONIDAZOLE METABOLITE
2 . R. W. Manthai, W. To and L. G. Feo, The Pharmacologist
5
235
(1983) . 3. R. W. Manthai and L. G. Feo, Wiadomosci Parazytologiczne T-X No. 2 -3, 177 (1984) . 4. D. R. Hoff and J. K. Barmat, U . S. Patent 3, 107, 201 (Oct . 15, 1983), Chem . Abstracts 59, 15878e (1983) . 5. K. Hofman, The Chemistry of Heterocyclic Compounds -- Imídazole and Its Derivatives Part 1, p. 175, Interscience Publishers, Inc ., New York (1953) . 6. Merck and Co. , Neth . Patent 8, 409, 117 (Feb. 8, 1985), Chem . Abstracts 83, ß07c (1985) . 7. R. M. J. Ings, G. L. Law and E . W. Parnell, Biochem . Pharmac. 15, 515-519 (1968) . 8 . R. W. Manthei, J. E. Stambaugh and L. G. F~o, Federation Proc. 24, 1848 (1985) .
Pergamon Press Ltd.
L90LATION AND IDENTIFICATION OF THE MAJOR URINARY METABOLITE OF METRONII~ZOLE J. E. 3tambaugh, L. G. Feo and R. RI . Manthei Departments od Pharmacology and Urology Jefferson Medical College, Philadelphia, Pa. (Received 1 March 1967 ; in final form 29 May 1967) Metronídazole (1-(2-i~ydraa~ett~yl)-2-methyl-5-nítroimidazole) is an orally effective trichomonacidal agent. In previous reports (1, 8, 3) we showed that chra~matograms a~f urines from patients receiving this drug contained six bands, not found in normal urine, that absorb in the ultraviolet region . Two of these bands comprise 70~ ad the total nitro~ontaining compounds excreted and consist of approximately equal amounts of unchanged metronidazole and a major metabolite . In this report, we present the isolation and identification ad the major metabolite of metronidazole, 1-(2-hydroxyetl~yl)-2-hydroxymethyl-5-nitroimidazole, found in the urine ad all patients investigated. This compound is formed by the oxidation of the 2-methyl group of metronidazole to the corresponding alcohol . \ H,3 C
(Fig. 1) C;2CHZOH N
N\ ~C-N02
ÇH20H `C
;HaCHaOH N
; ~-NOZ
1812
METRONIDAZOLE METABOLITE
Vol . 6, No . 17
Experimental Twenty-four hour urines from patients receiving 250 mg . of metronidazole orally three times daily were collected and frozen for storage . The urines were thawed at room temperature, desalted, and the urea destroyed with urease . The urines were then deproteinized, concentrated, and spotted on Whatman lkl paper strips .
The strips were de -
veloped in several solvent systems by descending chromatography, and the nitro-containing metabolites were located on the strips as absorption bands under ultra-violet light.
Characteristic color reactions of
the metabolites were observed on the strips by reducing the nitro group with 1. 5~ titanous chloride in 10~ acetic acid and overspraying with var ious color reagents . The major urinary metabolite of metronidazole was isolated by the uee á an ion exchange resin. Twenty-five cc . of the concentrated urine was poured onto Amberlite XAII-2 resin in a 300 mm column, 24 mm in diameter, formed in water . The column was developed with 5~ ammonia in water, and the fraction containing the major metabolite was identified by paper chromatography .
The crude metabolite was purüied by refrac-
tionatian on smaller columns od the Amberlite resin and was crystallized as pale yellow needles after standing several days in absolute ethanol at room temperature, (m . p. 80-84°). 1-(2-hydroxyethyl)-5-nitroimida~ole was prepared by refluxing 40 grams of 4(5)-nitroimidazole with 800 cc of ethylene chlorhydrin at 127°C far 24 hours. The excess ethylene chlorhydrin was evaporated off; the residue was taken up in water, filtered to remove the insoluble nitroimidazole, and extracted at pA 8 with ethyl acetate. A clear yellow
Vol . 6, No . 17
METRONIDAZOLE METABOLITE
1813
oil was obtained from the ethyl acetate extract which crystallized to pale yellow needles on standing in the cold overnight. Recrystallization from acetone/water yielded 17 .3 gm (33~ yield) of 1-(2-hydroxyetigl)-5-nitroimidazole, (m .p . 95-97°) (lit . 97-98°) (4). 1-(2-hydra~xyethyl)-2-hydroxymethyl-5-nitroimidazole was prepared b9 refluxing 14 .3 grams a~f 2-hydroxymethyl-4(5)-nitroimidazole with 80 cc of ethylene chlorhydrin at 127°C for twenty-four hours. The excess ethylene chlorhydrin was evaporated a~ff, and the brown oil residue was taken up in 2N NaOH .
The basic solution was extracted with eti~yl acetate,
and the extract was taken to dryness . The resultant yellow oil was taken up in absolute ethanol, charcoaled, filtered and the desired product crystallized out after standing several days at room temperature as pale yellow needles, (m .p. 81-83°) (Calcd. for CgHgN304 : C, 38 .29; H, 4.82; N, 22 .33 .
Found: C, 38 .32 ; H, 4 .85; N, 22 .18) .
Infrared spectra of this isolated products were recorded from KHr pellets or from thin films with a Perkin Elmer Model 337 spectrophoto meter. Results The ultra-violet absorption, the Rf values in three solvent systems, and the characteristic color reactions of metronidazole, the isolated urinary metabolite (Metabolite A) and some synthesized nitroimidazoles are listed in Table 1 . Identical color reactions were observed with metabolite A, 1- (2 hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole, and 1-(2-hydroxyett~yl)5-nitroimidazole . After reduction of the nitro group, a characteristic intermediary orange color was seen on reacting these compounds with
1814
METRONIDAZOLE METABOLITE
Ó .., d Fa
M
N
d
.r ~ .~ D+ .~ D~ ~
41
Vol . 6, No . 17
O
O
Ó aD
M
O U
w
0 0
U
I
~4
w a co
F
q
ó m S
d w
H w Ó N O f=1 O a
>x a
v
O
I
N >, a
z
w
I
U W
N
ó ó
a, a
~, a
a
N ó
N ó
ó ó
ó
ti ó
~ m ó ó
.-I~
ó
N ti ó
d
ó
o o dl ó ó
~
~.
d
..1 .c
~,
d
~n N
...
~~
I .,
F q
,. Y
0
m
33
r; 3 â 3 .ö 3 ~ ... ~ F ~ O-1 ~ .yW +O+ Cd ~ ".Û C) N m v b ~ :~ m~ c~. O ~ " óó ~~
~
r~ .~ ~ ~á .-=1
.0 3
O
N y
m~~~~ ~a~ q á m
N
r.l
I r I v~ v 1 1 I N~ N
m
QU
I If!
Ó
~
~
`~ x 0 ó :: ~n -, o qc ~ ~ o rl y
c
c 1 N .,
.,O
O á
ó
4l ir
0
a
,.
~ó H
POi v
~
m
M "~ 1 "r1 .~ q iF
m y iF
FOI Û ~ V v ~c,~c.c. ~á~~á
NN O ~n~~ O wO Ó O . . N O .r NM iF iF
Yol . 6, No . 17
MEI'RONIDAZOLE METABOLITE
1815
p-dimethylaminobenzaldehyde (Ehrlich's reagent) that was not observed with metronidazole or 1-acetic acid-2-methyl-5-nitroimidazole . A purple color was obtained with diazotized sulfanilic acid (Pau),y's reagent) tom pared to a tan color seen with metronidazole and the 1 acetic acid derivative. These three compounds also gave a yellow color with ninhydrin reagent as opposed to a characteristic green color obtained with metronidazole and the 1-acetic acid homologue . Comparison of the Rf values in several solvent systems indicated that 1-(2-hydroxyethyl)-5-nitroixnidazole is slightly more acidic in nature than metabolite A. 1-(2-hydroxyethyl)-2-hydroxymeti~yl-5-nitroimidazole and metabolite A were chromatographlcally identical in the solvent systems used .
Two-way chromatograms od urine from patients containing synthetic
1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole were run on thin layer Avicel plates, and the synthetic material and metabolite A were observed as a single spot with the same Rf value in four different solvent systems. Chromatograms of normal urine to which metronidazole was added indicated that the compound was stable in urine over a period a~f several months . The I. R . spectra of 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroímidazole and of metabolite A were observed to be identical in all absorbing regions. The OH stretching vibration appeared at 3375 cm -1 in these compounds in contrast to the 3200 cm nidazole .
-1
band seen with metro-
The presence of the hydroxymetl~yl group was indicated by
the C-0 stretching vibration of twin peaks at 1058 and 1070 cm -1 com-1 pared to a single peak at 1070 cm noted with metronidazole. The I. R. spectra o~f 1-(2-hydroxyethyl)-5-nitroimidazole and metabolite A showed several significant differences in ring substitution in the 800 to 1200 cm
-1
1816
I~TRONIDAZOLE METABOLITE
Yol . 6, No . 17
region . A strong 1120 cm -l band was considered to be indicative of a 1, 5-ring substitution pattern and was sat present in the spectra of metabolite A. Discussion Both the hydra~xyethyl group and the methyl group of metronidazole are available for oxidative metabolism . Oxidation at the one position would result in a relatively stable 1 acetic acid substituted ímidazole. Oxidation at the two position would result in the formation of either an alcohol, an aldehyde, or as acid, depending upon the conditions of oxidation.
The car-
bo~xylic acid resulting from complete oxidation of the methyl group would tend to be unstable due to the influence of the adjacent ring nitrogens (5), and decarboxylation would occur to form 1-(2-hydrga~etl{yl)-5-nitroimidazole .
It is also possible that intramolecular cyclization could occur
between the 2-acid and the 1-l~ydrm~yetl~yl group to form a relatively stable Y
-lactose (6). In an attempt to prepare the major urinary metabolite, metronidazole
was subjected to oxidation by various known methods.
Hydrogen peroxiàe
oxidation resulted in the destruction of the imidazole ring, and potassium permanganate oxidation lead to numerous oxidation and breakdown products which were not readily isolated . When metronidazole was reacted with a chromic acid medium, several products were obtained . One of these products was 1-acetic acid-2-methyl-5-nitroimidazole and corresponded to a urinary metabolite of metronidazole. Ings et al (7), working with a limited sample size, reported this compound to be the major metabolite formed from metronidazole a.nd concluded, on the basis oá their studies, that unchanged metronidazole, the lucid derivative, and a glucuronide conjugate
Vol. 6, No . 17
MET1tONIDAZOLE METABOLITE
1817
were the primary urinary excretion products of the drug . However, is our studies the 1 acid derivative comprised less than 10~ aá the total nitrocontaining urinary metabolites of metronidazole. Oxidation of metronidazole with dilute nitric acid produced several oxidation products, one of which had the same chromatographic properties as the major metabolite isolated from urine. This oxidation pro duct and the metabolite, after reduction of the vitro group, reacted purple with diazotized sulfanilic acid, sad failed to produce a green color with ainhydrin reagent. 31nce the green ninhydrin reaction was observed only with compounds containing a 2-methyl group and the purple diazo reaction was not observed when the 2-methyl group was present, it was concluded that the major metabolite was a nitroimiàazole derivative in which the 2-methyl group had been either oxidized or removed. In a preliminary study, 1-(2-hydroxyeti~yl)-5-nitroimidazole was found to be similar to the major metabolite on the basis of paper chromatographic separation and colorimetric reactions (8). Subsequent isolation of the metabolite from an alumina column and comparison of its I. R. spectra with lmown 1-(E-hydroxyett~yl)-5-nitroimidazole indicated that the compounds were identical. However, simple eluates of the metabolite ba~i from paper chromatograms of freshly collected urines gave I. R. spectra that were different than either of the above two compounds.
Fur-
ther studies revealed that the metabolite was a relatively labile substance and was probably oxidized to 1-(2-hydroxyethyl)-5-nitroimidazole during the isolation procedures originally employed . The LR . spectra of the major metabolite obtained from freshly collected urines indicated that the metabolite was a 1, 2, 5-substituted im-
1818
METRONIDAZOLE METAHOLITE
Vol. 6, No . 17
idazole. Various substituted nitroimidazoles were synthesized and compared to the major metabolite obtained from paper strip eluates. It was found that 1-(2-hydroxyethyl)-2-hydroacymethyl-5-nitroimidazole possewed the same chromatographic properties as the urinary unlrnown.
In
addition the urinary unlmown isolated from an Amberlite column was found to have the same chemical properties and gave an I. R. spectra that was iàentical in all respects with synthetic 1-(2-hgdra~xyethyl)-2-hydroxymethgl5-nítroimidazole . Conclusions The urinary metabolites of metronidazole were studied in man, and the major metabolite was isolated by chromatographic methods. Comparisan of the isolated metabolite with several synthetic nitroimidazoles re vealed that 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole is the major urinary metabolite of metronidazole. It is concluded that the metabolism of metranidazole involves mainly an oxidative pathway in which the oxidation of the 2 -methyl radical takes place in preference to that of the hydroxyetlgl group.
The pharmacolagic properties of this metabolite re-
main to be investigated . Acknowledgem enta The authors wish to thank Mrs. Lydia Hangs for excellent technical assistance and Rohm and Haas for a generous supply of Amberlite XAD-2 resin used in this study.
This work was supported in part by a grant frown
G. D. Searle and Comparry . References I. R. W. Manthei, R. S. Horn and L. G. Feo, The Pharmacologist (1982 ).
4
170
Vol . 6, No . 17
1819
METRONIDAZOLE METABOLITE
2 . R. W. Manthai, W. To and L. G. Feo, The Pharmacologist
5
235
(1983) . 3. R. W. Manthai and L. G. Feo, Wiadomosci Parazytologiczne T-X No. 2 -3, 177 (1984) . 4. D. R. Hoff and J. K. Barmat, U . S. Patent 3, 107, 201 (Oct . 15, 1983), Chem . Abstracts 59, 15878e (1983) . 5. K. Hofman, The Chemistry of Heterocyclic Compounds -- Imídazole and Its Derivatives Part 1, p. 175, Interscience Publishers, Inc ., New York (1953) . 6. Merck and Co. , Neth . Patent 8, 409, 117 (Feb. 8, 1985), Chem . Abstracts 83, ß07c (1985) . 7. R. M. J. Ings, G. L. Law and E . W. Parnell, Biochem . Pharmac. 15, 515-519 (1968) . 8 . R. W. Manthei, J. E. Stambaugh and L. G. F~o, Federation Proc. 24, 1848 (1985) .