Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Metabolism of α-Methylfluorene-2-acetic acid (Cicloprofen): Isolation and Identification of Metabolites from Rat Urine S. J. Lan, A. V. Dean, K. J. Kripalani & A. I. Cohen To cite this article: S. J. Lan, A. V. Dean, K. J. Kripalani & A. I. Cohen (1978) Metabolism of αMethylfluorene-2-acetic acid (Cicloprofen): Isolation and Identification of Metabolites from Rat Urine, Xenobiotica, 8:2, 121-131 To link to this article: http://dx.doi.org/10.3109/00498257809060391
Published online: 30 Sep 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [University of California Santa Barbara]
Date: 26 October 2015, At: 20:02
XENOBIOTICA,
1978, VOL. 8,
NO.
2, 121-131
Metabolism of a-Methylfluorene-%aceticacid (Cicloprofen): Isolation and Identification of Metabolites from Rat Urine S. 3. LAN*, A. V. DEAN and K. J. KRIPALANI
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
Drug Metabolism Department, The Squibb Institute for Medical Research, New Brunswick, N.J. 08903, U.S.A.
and A. I. COHEN Analytical R&D Department, The Squibb Institute for Medical Research, Princeton, N.J. 08540, U.S.A.
(Received 14 April 1977) 1. Four metabolites of K-methylfluorene-2-acetic acid (cicloprofen) have been isolated from rat urine and identified as the 7-hydroxy, 9-hydroxy, 7,9-dihydroxy and 9-hydroxy-9-methoxy derivatives of cicloprofen. 2. 7-Hydroxy cicloprofen was the major metabolite, contributing 47% of the total radioactivity excreted in rat urine. The other three metabolites each contributed approx. 10% of the radioactivity in urine. There was little unchanged drug excreted in urine (2.6%); at least three other minor metabolites have not been identified. 3. A metabolic pathway for the formation of the 9-hydroxy-9-methoxy metabolite of cicloprofen is proposed.
Introduction T h e metabolic fate of a-methylfluorene-2-acetic acid (cicloprofen), an anti-inflammatory agent (Millonig et al., 1972), has been studied in several species including rats, dogs, monkeys, and humans. Preliminary results of these studies have been reported (Kripalani, Weliky & Schreiber, 1972; Kripalani et al., 1973). Cicloprofen undergoes the following metabolic biotransformations : (1) stereospecific inversion of the ( - )-enantiomer of cicloprofen to its (+)-antipode (Kripalani et al., 1976; Lan et al., 1976); (2) hydroxylation of the fluorene rings; and (3) conjugation with glucuronic acid or sulphate. Several hydroxylated metabolites of cicloprofen have been isolated from rat urine and identified as the 7-hydroxy, 9-hydroxy, 7,9-dihydroxy, and 9-hydroxy9-methoxy derivatives of cicloprofen by mass and n.m.r. spectrometric analyses. This paper describes the isolation and identification procedures. Materials and methods Materials K-Methylfluorene-2-acetic acid (cicloprofen) and cc-methyl-(7-hydroxyfluorene)-2-acetic were synthesized by the methods of Stiller et al. (1972). Labelled cicloprofen (a-["C]methylfluorene-2-acetic acid, sp. radioactivity 23 pCi/mg) was synthesized by the same procedures. The m.p. and i.r. spectra, for labelled and unlabelled cicloprofen were the same, as were the R, values in the three solvent systems: (1) benzene-diethyl ethermethanol, 8 : 3 : 1; (2) chloroform-methanol-ammonia soln, (sp. gr. 0.88), 11 : 6 : 1 ; and (3) benzene-methanol- ammonia soln. (sp. gr. 0*88),60 : 30 : 1. The radiochemical purity of the labelled compound, as determined by t.1.c. in these three solvent systems and by radioautography, was 99*80/,.
* T o whom communications and reprint requests should be addressed.
122
S . J . Lan et al.
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
Charles River outbred C D albino rats were purchased from Charles River Breeding Laboratories (Wilmington, Mass., U.S.A.). Silica gel G F or Quanta-gram Q1F precoated thin-layer plates (250 pm in thickness) were obtained from Analtech, Inc., (Newark, Del., U.S.A.), or from Quantum Industries (Fairfield, N.J., U.S.A.), respectively. Helix pomatia P-glucuronidase extract (type H-3, 9000 Fishman units per 0.1 ml, also containing sulphatase activity) was purchased from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). All other chemicals were of reagent grade.
Methods Drug administration and sample collections: [14C]Cicloprofen was administered orally as an aq. soln. of the sodium salt. Eight male rats, 350-380 g, were each given a 100 mg/kg daily dose of the drug for 4 days. Rats were then housed in individual metabolic cages, and food and water were supplied ad lib. Samples of urine and faeces, collected daily for 6 days, were frozen until analysed. Isolation and identification of metabolites: The urine samples collected during all six days were pooled and lyophilized to dryness. The residue was dissolved in 200 ml of water and extracted twice with 600ml of ethyl acetate. The aq. fraction was then brought to pH 1 with conc. HCI and further extracted twice with 600 ml of ethyl acetate. The extractions recovered 90% of the radioactivity from urine. The ethyl acetate extracts were combined and evaporated to dryness at 45" in vacuo. The residue was dissolved in water (100 ml), adjusted to pH 1 with HCI, and extracted with benzene (2 x 250 ml) followed by ethyl acetate (2 x 250 ml). The benzene extracts were combined as were the ethyl acetate extracts. The pooled benzene extract was concentrated to 100 ml by evaporation, and extracted with an equal vol. of 0.1 M NaOH. After acidification of the NaOH s o h . to p H 1 with HCl, the radioactive materials were extracted back into benzene (200ml). As the last benzene extraction only removed approx. 70y0 of the radioactivity from the acidic aq. soln., the remainder was extracted with ethyl acetate (200 ml), which was combined with the ethyl acetate extract obtained previously. The benzene and ethyl acetate extracts were evaporated to dryness separately and the residues dissolved in a small volume of ethyl acetate. The extracted radioactive materials were separated and purified by repeated chromatography on silica gel t.1.c. plates with solvent systems: (A) chloroform-acetic acid, 95 : 5 ; (B) benzene-diethyl ether-methanol, 8 : 3 : 1 ; (C) chloroform-acetone-acetic acid, 70 : 20 : 10; and (D) benzene-diethyl ether-acetic acid, 80 : 30 : 2. Compounds were visualized under short wavelength U.V. light, and radioactivity was located by scanning the plates with an Actigraph I11 Scanner (Nuclear Chicago, Des Plains, Ill., U.S.A.). Silica gel in the area corresponding to each radioactive spot was scraped from the plate, packed into a small column, and the radioactivity was eluted with methanol. Methanol was then removed by evaporation, and the residues redissolved in a small volume of methanol. Metabolites isolated and purified by t.1.c. were subjected to mass and n.m.r. spectrometric analyses with an Associated Electrical Industries Model MS-902 double-focusing mass spectrometer and a Varian Associates SL-100 n.m.r. spectrometer. Quantification of urinary metabolites :To determine the percentage distribution of radioactivity among the metabolites excreted in urine, a portion (4 ml) of the pooled 0-48 h urine from three rats that received a dose (50 mg/kg) of [14C]cicloprofenwas mixed with an equal vol. of 0.2 M sodium acetate buffer (pH 5.2). P-Glucuronidase (45 000 Fishman units) was added, and the mixture was incubated at 37" for 24 h with constant shaking. The incubation mixture was extracted with 30 ml of ethyl acetate, and the aqueous fraction was adjusted to pH 1 with 6 M HCI and further extracted with an additional 30 ml of ethyl acetate. The ethyl acetate extracts were combined and evaporated to dryness, and the residues dissolved in a small volume of ethyl acetate. The extractions were more than 95% complete. Aliquots of the ethyl acetate soh. were chromatographed on silica gel t.1.c. plates in solvent systems A and B. Compounds were visualized under U.V. light, and radioactivity located by scanning the plates with an Actigraph I11 Scanner. Silica gel in the area corresponding to each radioactive spot was scraped from the plate, mixed with 1 5 ml of Bray's solution (Bray, 1960), and counted in a Packard Tri-Carb liquid scintillation spectrometer, Model 3380. Automatic external standardization was used to determine the counting efficiency.
Metabolism of a-MethylJuorene-2-acetic Acid
123
Results Isolation and purification of metabolites Eight radioactive metabolites were obtained from the pooled urine of rats that received multiple doses of [14C]cicloprofen by extraction with organic solvents and t.1.c. in different solvent systems. Table 1 shows the R, values of cicloprofen and its metabolites after t.1.c. in four solvent systems. Table 1. RF values of cicloprofen and its metabolites after thin-layer chromatography
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
R, values and solvent system (see text) Compound
A
B
I Cicloprofen I1 9-Hydroxy9-methoxy I11 7-Hydroxy IV 9-Hydroxy V 7,9-Dihydroxy VI Unknown 1* VII Unknown 2 VIII Others
0.63
0-75
0.53
0.57 0.57 0.44 0.26 0-33 0.19 0
0.40 0.40 0.10 0.10 0.10 0
C
D
0.48 0.37
0.63 0.73 0.52
0.22 0.06
* Mass spectrometry indicated that this compound could be another dihydroxylated metabolite other than 7,9-dihydroxy-cicloprofen. Thin-layer chromatography of the radioactive materials in the benzene extract in solvent system A produced three radioactive fractions ( R , 0.63, 0.53 and 0.40). Fraction 1 (RF0.63) contained a single radioactive compound (Compound I) that had the same R, value as authentic cicloprofen in solvent systems A and B. Fraction 2 ( R , 0-53) also contained a single radioactive compound after chromatography in solvent systems B and D. This compound was further purified from non-radioactive impurities by repeated chromatography in systems A, B and D. When the purified compound was dissolved in methanol, concentrated, and stored at 0-5", a white radioactive precipitate was obtained. The purified compound (Compound 11) did not melt, but gradually decomposed at temperatures above 235". Fraction 3 had the same R , value (0.40) as authentic a-methyl-(7-hydroxyfluorene)-2-acetic acid in solvent system A. By chromatography in solvent system B, Fraction 3 was separated into two radioactive compounds (RF0*57 and 0.44). One (compound 111) was associated with the major portion of the radioactivity, and had the same R, value (0.57) as authentic ac-methyl-(7-hydroxyfluorene)-2-acetic acid. T h e other compound (Compound IV) was further purified in solvent system D. T h e ethyl acetate extract was separated into five radioactive fractions by t.1.c. in solvent system A. Fractions 1, 2 and 3 contained the same radioactive metabolites found in the benzene extract. Fraction 4 had an R, value of 0.1 and Fraction 5 consisted of compounds that remained at the origin, presumably conjugates or more polar metabolites. Fraction 4 was separated by chromatography in solvent system B into three radioactive compounds: V(R, 0.26), VI (R, 0.33), and V I I (R, 0.19). These compounds were further purified in solvent system C before subjecting them to mass and n.m.r. spectrometric analyses. Radioactive materials in Fraction 5 were not studied further because of the small amounts available,
124
S. J . Lan et al.
Mass spectrometry of cicloprofen and its metabolites T h e mass spectrum of cicloprofen (Fig. 1) serves as the model for elucidation of the structures of the metabolites. The mass spectrum of cicloprofen yields a prominent molecular ion (M+) at mle238. T h e loss of the carboxyl group yields a prominent fragment ion at m / e 193. The latter further loses the methyl group to form the ion at m / e 178 and the methine group to form the ion at mle 165. T h e ion at mle 223 is derived from the loss of the methyl group from M+.
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
100
I
I
?3
Compound I that had the same R , value as authentic cicloprofen in two different solvent systems also had a mass spectrum that was identical with that of cicloprofen. T h e mass spectrum of Compound I1 (Fig. 2) shows the M+ at m / e 284 and a prominent ion at m / e 239 that are 46 amu (atomic mass unit) greater than the corresponding ions in cicloprofen. T h e increase of 46 amu in the cicloprofen molecule indicates the introduction of two oxygen, one carbon, and two hydrogen atoms onto the cicloprofen molecule. The fragment ions at mle269, 224 and 211 correspond to the respective fragment ions at mle223, 178 and 165 of the cicloprofen molecule. These resuIts suggest that Compound I1 probably is a hydroxylated and methoxylated derivative of cicloprofen. T h e positional assignments of the hydroxy and the methoxy groups on the cicloprofen molecule, based on n.m.r. spectrometry, will be discussed later. T h e mass spectrum of Compound 111 (Fig. 3 ) shows the M+ at m / e 254 and fragment ions at m / e 239 (M+ - CH,), 209 (M+ - COOH), 194 (M+- CH, and COOH) and 181 (M+- CHCH, and COOH) that are 16 amu greater than the correponding ions in cicloprofen. Thus, it indicates the introduction of an additional oxygen onto the cicloprofen molecule. Comparison of the fragmentation patterns of Compound I11 and cicloprofen also indicates that the additional oxygen atom was introduced onto the fluorene moiety rather than
Metabolism of
a-Methy&uorene-Z-acetic
-~
100r----7---
i
90
1,
Acid
I
@g$fm"
n 211 239
M+ -CH*269
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
M + - C 4 -COOH=224
269
MRSS/CHRRGE
Fig. 2. Mass spectrum of Compound I I . cc-Methyl-(9-hydroxy-9-rnethoxyfluorene)-2-aceticacid.
1
W
>
H
MRSS/CHRRGE
Fig. 3. Mass spectrum of Compound I I I . cc-Methyl-(7-hydroxyfluorene)-2-acetic acid.
125
126
S. J: Lan et al.
I1
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
!
MRSS/CHRRGE
Fig. 4. Muss spectrum of Compound IV. cr-Methyl-(9-hydroxy-9H-fluorene)-2-aceticacid.
u+ 270 197
I
MRSS/CHQRGE
Fig. 5. Muss spectrum of Compound V . cc-Methyl-(7,9-dihydroxy-9H-fluorene)-2-acetic acid.
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
Metabolism of a- MethylJEuorene-2-acetic Acid
127
onto the side chain of cicloprofen. The mass spectra of Compound 111 and of an authentic sample of a-methyl-(7-hydroxyfluorene)-2-acetic acid were identical. T h e mass spectrum of Compound I V (Fig. 4) shows it to be a mixture of two compounds, one of the two having an Mf of mle284. T h e principal ions of the major component are the M+ at mle254 and the fragment ions at mje 209-207, 181 (base peak), 165 and 152, suggesting that the metabolite had an additional oxygen atom introduced onto the fluorene moiety. The fragmentation pathways of the two monohydroxy metabolites differ considerably (compare Figs. 3 and 4). It is likely that, while Compound 111 is indeed 7-hydroxylated or aryl hydroxylated, Compound IV is 9-hydroxylated. Furthermore, the mass spectrum of Compound V (Fig. 5 ) shows the Mf at mle270 and the diagnostic fragment ions at mle225-223, 207 and 197, 16 amu greater than the corresponding ions in Compound IV. Thus, the mass spectral data indicates that Compound V is the aryl monohydroxylated metabolite of 9-hydroxy cicloprofen. T h e mass spectrum of Compound VI (not shown) also shows the M+ at m/e 270 and fragment ions at mle225 and 197, suggesting that this compound could be another dihydroxylated metabolite. Compound VII did not yield a suitable mass spectrum. N.m.r. spectrometry of cicloprofen and its metabolites T h e positional assignments of the functional groups in the metabolites of cicloprofen by n.m.r. spectrometry depends in large measure on making consistent assignments in model compounds. T h e complete assignments of the n.m.r. spectra of fluorene (Bartle, Jones & Bavin, 1971; Jones, Matthews & Bartle, 1972; Douris & Mathieu, 1971), its simple aryl derivatives (Mathieu, Milano & Douris, 1974), and its 9-substituted derivatives (Mathieu, 1971 a, b, c) have been reported. Using the published data, the expected changes in chemical shifts on introduction of hydroxyl or methoxyl groups at the C-1 and C-3 to C-9 positions were evaluated. Assuming that these additive effects hold for the n.m.r. spectrum of cicloprofen too, a list of expected chemical shifts of the metabolites was calculated (Table 2) and compared to the chemical shifts and proton coupling patterns of the metabolites. The n.m.r. spectrum of Compound I1 indicates the loss of the C-9 methylene protons without appearance of the appropriate C-9 methine proton. Because the aryl proton resonances have not been shifted sufficiently for aryl hydroxylation or methoxylation, it is possible that Compound I1 is a methyl hemiketal of the C-9 0x0 derivative, which agrees with both the n.m.r. and mass spectra. Thus, the structure of Compound I1 is tentatively assigned as a-methyl-(9-hydroxy-9-methoxyfluorene)-2-acetic acid. T h e structure of Compound I11 is assigned as a-methyl-(7-hydroxyfluorene)2-acetic acid based on the n.m.r. and mass spectra. Typical of aromatic compounds, protons that are ortho to each other on the fluorene ring have coupling constants of 8 to 9 Hz while protons that are meta to each other have coupling constants of 2 to 3 Hz. Of significance for the n.m.r. spectrum of Compound I11 is the doublet near 6 7 of 2 Hz coupling for the C-8 proton, and the quartet of lines near 6 6.8 with both ortho and meta proton couplings. The methyl doublet at 6 1.48 confirms the retention of the side-chain, although the methine proton is not readily observed, which is presumed to coincide with the C-9 methylene resonance at 6 3.80. The observed spectrum of Compound I11 agrees very well with the calculated spectrum of the 7-hydroxy derivative of cicloprofen (Table 2).
7.48 7.45 7.52 7.41
Observed Compound I1 Compound I11 Compound IV Compound V
a
7.48 7.17 7.1 5 7.55 7.53 7.53 7.38 7.40 7.55 7.44 7.50 7.61 7.41 7-33 7.25 7.34 7.25
6.43 6.70 7.40 7.45 7.35 7.25 7.27 7.36 7.36 7.33 7.36 7.26
-
7.27 7.36
7.51 7.58 7.56 7.5
8.14 8.17 7-73 7.53 7.57 7.75 7.74 7.54 7.62 7.54
-
7.48 7.53 7.34
7.57 7.58 7.56 7.5
7.34 7.49 7.57 7.53 7.48 7.54 7-62 7.33
-
-
7.74 7.75 7.73 8.17 8.14
7.70
C-5H
__
7.16 6.81d 7.24 6.76d
6.74 6.82 7.30 7.21 7.27 7.30 6.73
7.30 7.30 7.29 7.31 7.34 6.64 6.37
7.26
C-6H
-
7.28
-
7.16
-
6.66 6.59 7.16 7.20
-
7.19 7.19 7.18 7.20 7.23 7.03 7.08 6.73
7.1 5
C-7H
-
7.28 6.99e 7.56 7.04e
7.53 7.64 7.01
-
7.47 7.46 7.56 7.56 7.23 6.91 7.20 7.51 6.98 7.08
7.50
C-8H
3.80 5.50 5.42
-
3.86 3.88 3.82 3.83 3.78 3.78 3.83 3.82 3.77 3.76 3.88 3.86 5.49 5.49 5-46
3.80
C-9H
CH
OCH3
1.47(d) 3.90, 3.94 1.48(d) 3.76(m) 1.50(d) -3.7 (m) 1.48(d)
CH,
Observed spectrum in deuteromethanol containing tetramethylsilane as an internal reference. For substituted cicloprofen derivatives assuming that the effects are additive. In deuteromethanol containing tetramethylsilane as an internal reference. J,=8 HZJm=2 Hz. Jm=2 Hz.
-
-
7.70
7.32
7.47
Cicloprofena
Calculatedb 1 -OH 1 -OCH, 3-OCHs 4-OCH, 4-OH 5-OH 5-OCH, 6-OCH, 7-OH 7-OCH3 8-OCH3 8-OH 9-OH 9-OCH3 7,9-diOH
C-4H
C-3H
C-1H
Compound
Chemical shifts in 6 values
Table 2. Calculated and observed chemical shifts of derivatives of cicloprofen
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
t Q Dc
c
Metabolism of ol-Methyljluorene-2-acetic Acid
129
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
T h e n.m.r. spectrum of Compound IV indicated the structural assignment to be ol-methyl-(9-hydroxy-9H-fluorene)-2-acetic acid with a C-9 proton resonance at 6 5.50 and aryl proton resonances predicted from the calculated spectra (Table 2). The n.m.r. spectrum of Compound V demonstrates it to be an aryl hydroxylated derivative of 9-hydroxy cicloprofen. A reasonable assignment of the structure of Compound V would be a-methyl-(7,9-dihydroxy9H-fluorene)-Z-acetic acid, for the aryl proton resonances are quite similar to those assigned to the 7-hydroxy derivative of cicloprofen; it would be expected that the introduction of a 9-hydroxyl group would have only a minimal influence on the chemical shifts (Table 2). The poor quality of the n.m.r. spectra of Compounds VI and VII did not permit a structural assignment for these two compounds. Metabolism of cicloprofen by rats Cicloprofen was readily metabolized by rats, and, along with its metabolites, was eliminated mainly via biliary excretion. T h e ratio of the amount of radioactivity excreted in faeces to that in urine ranged from 2 to 3.2 (Dean et al., 1977). When multiple oral doses (100 mg/kg per day) of the drug were administered to rats, the drug was excreted in urine largely as unconjugated metabolites. However, when a single 50 mg/kg dose of [14C]ciclopr~fenwas administered to rats, a major portion ( > 7 0 % ) of the radioactive materials in urine was excreted as conjugates. Excretion of greater amounts of unconjugated metabolites in urine by rats after receiving multiple oral doses (100 mg/kg per day) than after receiving a single 50 mg/kg dose might have been due to saturation of the conjugating-enzyme system, or to depletion of the cofactor (i.e. UDP-glucuronic acid) required for the conjugation reaction. Hydrolysis of the conjugates was achieved either by heating with 4 M HC1 or by incubation with a mixture of P-glucuronidase and sulphatase. Table 3 shows the percentage of each of the metabolites in the hydrolysed urine of rats that received a 50 mg/kg oral dose. There was little unchanged cicloprofen (2.6%) in urine, demonstrating its extensive biotransformation. The 7-hydroxy derivative of cicloprofen was the major metabolite (47%) excreted in urine. T h e other three metabolites, the 9-hydroxy, 7,9-dihydroxy and 9-hydroxy-9-methoxy derivatives of Table 3. Amounts of cicloprofen and its metabolites present in urine after administration of [W]cicloprofento rats
Compound I Cicloprofen I I 9-Hydroxy-9-methoxy 111 7-Hydroxy IV 9-Hydroxy V 7,9-Dihydroxy VI Unknown 1 VII Unknown 2 VIII Others
Amount of radioactivity in urine (yo) 2.6 10.1 47.1 10.4 10.1 n.d. 11.6 8.1
Figures are for pooled 0 to 48 h urine, from three rats given a single 50 mg/kg oral dose, which contained 17.2% dose. More than 70% of the metabolites were excreted in urine as glucuronide and/or sulphate conjugates. Data were obtained after hydrolysis with /3-glucuronidase and sulphatase. Distribution of radioactivity among metabolites was calculated from data obtained after t.1.c. in solvent systems A and B. n.d., could not be determined.
S. J . Lan et al.
130
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
cicloprofen, were each present to the extent of approximately 10% of the total radioactivity in urine. There were at least three minor metabolites that have not been identified.
Discussion Rat urinary metabolites of cirloprofen have been separated into eight fractions by thin-layer chromatography in different solvent systems. Four major metabolites have been tentatively identified, by mass spectrometry and nuclear magnetic resonance, as the 7-hydroxy, 9-hydroxy, 7,9-dihydroxy, and 9-hydroxy-9-methoxy derivatives of cicloprofen. These four metabolites together with unchanged cicloprofen accounted for 80% of the radioactivity excreted in urine. Hydroxylation and conjugation with glucuronic acid or sulphate are the major metabolic reactions. Isolation and identification of the 9-hydroxy-9-methoxy derivative of cicloprofen is of interest. Metabolic formation of a methyl hemiketal has not been previously reported. A logical pathway for the formation of the 9-hydroxy-9-methoxy derivative of cicloprofen may involve the oxidation of the carbon atom in the 9-position of cicloprofen to the 9-ox0 derivative, followed by hydration of the %ox0 to the 9,9-dihydroxy derivative and subsequent 0-methylation (Fig. 6). I t was considered that the 9-hydroxy-9-methoxy derivative might not be a true metabolite, but an artifact derived from the reaction of the %ox0 derivative of cicloprofen with methanol during isolation and purification procedures. Accordingly, the %ox0 derivative of cicloprofen was synthesized by treating cicloprofen with chromic acid. T h e synthesized %ox0 derivative is a yellowish compound, which was not converted to the 9-hydroxy-9-methoxy derivative of cicloprofen when treated with methanol by the same isolation and purification procedures applied to the latter. Thus, it is concluded that the 9-hydroxy-9-methoxy derivative of cicloprofen is a genuine metabolite.
W i
C P
3
- COOH
n
I C -COOH
I H
/
\
c-
H
HO-
~
.OH
0
.
cH3
a
Fig. 6. Proposed metabolic pathways of cicloprofen in rats. Numbered compounds are metabolites isolated and identified. Metabolites were excreted mainly as glucuronide or sulphate conjugates.
Metabolism of ~-~ethyZ~uorene-Z-acetic Acid
131
Fig. 6 shows the proposed metabolic pathways of cicloprofen in the rat. Among the proposed intermediate metabolites, the 9-0x0 and the 9,9-dihydroxy derivatives of cicloprofen have not been detected in the urine of rats dosed with cicloprofen. Assuming that cicloprofen is metabolized to its 9-hydroxy-9methoxy derivative via the proposed metabolic pathway, then either the conversion of cicloprofen to its 9-0x0 derivative or the subsequent formation of the 9,9-dihydroxy derivative is the rate-limiting reaction. T h e formation of 9-hydroxy-9-methoxy cicloprofen from %ox0 cicloprofen in rats is being investigated.
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
References BARTLE,K. D., JONES,D. W. & BAVIN,P. M. G. (1971). J’. chem. SOC., (B), p. 388. BRAY,G. A. (1960). Analyt. Biochem., 1, 279. DEAN,A. V., LAN,S. J., KRIPALANI, K. J., DIFAZIO,L. T. & SCHREIBER, E. C. (1977). Xenobiotica, 7,549. DOURIS, J. & MATHIEU, A. (1971). Bull. SOC.Chim. France, p. 3365. JONES,D. W., MATTHEWS, R. S. & BARTLE, K. D. (1972). Spectrochim. Acta, 28A,2053. KRIPALANI, K. J., WELIKY,I. & SCHREIBER, E. C. (1972). Abstract, Med. Chem. 39, 164th ACS Nat’l Meeting, New York, N.Y., August 27 to September 1, 1972. KRIPALANI, K. J., VAHIDI,A., MISCHLER, T. W., NEISS,E. S. & SCHREIBER, E. C. (1973). The Pharmacologist, 15, 228. KRIPALANI, K. J., ZEINEL-ABDIN, A., DEAN,A. V. & SCHREIBER, E. C. (1976). Xenobiotica, 6, 159. LAN,S. J., KRIPALANI, K. J., DEAN,A. V., EGLI,P., DIFAZIO,L. T. & SCHREIBER, E. C. (1976). Drug Metab. Disp., 4, 330. MATHIEU, A. (1971 a). Bull. SOC. Chim. France, p. 1526. MATHIEU, A. (1971 b). Bull. SOC. Chinz. France, p. 1533. MATHIEU, A. (1971 c). Bull. SOC. Chim. France, p. 1540. MATHIEU, A., MILANO, J. C. & DOURIS, J. (1974). Bull. SOC.Chim. France, p. 299. MILLONIG, R. C., GOLDLUST, M. B., RUBIN,B., TURKHEIMER, A. R., SCHREIBER, W. F. & BELL,C. (1972). Abstract, Med. Chem. 38, 164th ACS Nat’l Meeting, New York, N.Y., August 27 to September 1, 1972. STILLER, E. T., DIASSI,P. A., GERSCHUTZ, D., MEIKLE,D., MOETZ,J., PRINCIPE, P. A. & LEVINE,S. D. (1972). J. Med. Chem., 15, 1029.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Metabolism of α-Methylfluorene-2-acetic acid (Cicloprofen): Isolation and Identification of Metabolites from Rat Urine S. J. Lan, A. V. Dean, K. J. Kripalani & A. I. Cohen To cite this article: S. J. Lan, A. V. Dean, K. J. Kripalani & A. I. Cohen (1978) Metabolism of αMethylfluorene-2-acetic acid (Cicloprofen): Isolation and Identification of Metabolites from Rat Urine, Xenobiotica, 8:2, 121-131 To link to this article: http://dx.doi.org/10.3109/00498257809060391
Published online: 30 Sep 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [University of California Santa Barbara]
Date: 26 October 2015, At: 20:02
XENOBIOTICA,
1978, VOL. 8,
NO.
2, 121-131
Metabolism of a-Methylfluorene-%aceticacid (Cicloprofen): Isolation and Identification of Metabolites from Rat Urine S. 3. LAN*, A. V. DEAN and K. J. KRIPALANI
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
Drug Metabolism Department, The Squibb Institute for Medical Research, New Brunswick, N.J. 08903, U.S.A.
and A. I. COHEN Analytical R&D Department, The Squibb Institute for Medical Research, Princeton, N.J. 08540, U.S.A.
(Received 14 April 1977) 1. Four metabolites of K-methylfluorene-2-acetic acid (cicloprofen) have been isolated from rat urine and identified as the 7-hydroxy, 9-hydroxy, 7,9-dihydroxy and 9-hydroxy-9-methoxy derivatives of cicloprofen. 2. 7-Hydroxy cicloprofen was the major metabolite, contributing 47% of the total radioactivity excreted in rat urine. The other three metabolites each contributed approx. 10% of the radioactivity in urine. There was little unchanged drug excreted in urine (2.6%); at least three other minor metabolites have not been identified. 3. A metabolic pathway for the formation of the 9-hydroxy-9-methoxy metabolite of cicloprofen is proposed.
Introduction T h e metabolic fate of a-methylfluorene-2-acetic acid (cicloprofen), an anti-inflammatory agent (Millonig et al., 1972), has been studied in several species including rats, dogs, monkeys, and humans. Preliminary results of these studies have been reported (Kripalani, Weliky & Schreiber, 1972; Kripalani et al., 1973). Cicloprofen undergoes the following metabolic biotransformations : (1) stereospecific inversion of the ( - )-enantiomer of cicloprofen to its (+)-antipode (Kripalani et al., 1976; Lan et al., 1976); (2) hydroxylation of the fluorene rings; and (3) conjugation with glucuronic acid or sulphate. Several hydroxylated metabolites of cicloprofen have been isolated from rat urine and identified as the 7-hydroxy, 9-hydroxy, 7,9-dihydroxy, and 9-hydroxy9-methoxy derivatives of cicloprofen by mass and n.m.r. spectrometric analyses. This paper describes the isolation and identification procedures. Materials and methods Materials K-Methylfluorene-2-acetic acid (cicloprofen) and cc-methyl-(7-hydroxyfluorene)-2-acetic were synthesized by the methods of Stiller et al. (1972). Labelled cicloprofen (a-["C]methylfluorene-2-acetic acid, sp. radioactivity 23 pCi/mg) was synthesized by the same procedures. The m.p. and i.r. spectra, for labelled and unlabelled cicloprofen were the same, as were the R, values in the three solvent systems: (1) benzene-diethyl ethermethanol, 8 : 3 : 1; (2) chloroform-methanol-ammonia soln, (sp. gr. 0.88), 11 : 6 : 1 ; and (3) benzene-methanol- ammonia soln. (sp. gr. 0*88),60 : 30 : 1. The radiochemical purity of the labelled compound, as determined by t.1.c. in these three solvent systems and by radioautography, was 99*80/,.
* T o whom communications and reprint requests should be addressed.
122
S . J . Lan et al.
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
Charles River outbred C D albino rats were purchased from Charles River Breeding Laboratories (Wilmington, Mass., U.S.A.). Silica gel G F or Quanta-gram Q1F precoated thin-layer plates (250 pm in thickness) were obtained from Analtech, Inc., (Newark, Del., U.S.A.), or from Quantum Industries (Fairfield, N.J., U.S.A.), respectively. Helix pomatia P-glucuronidase extract (type H-3, 9000 Fishman units per 0.1 ml, also containing sulphatase activity) was purchased from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). All other chemicals were of reagent grade.
Methods Drug administration and sample collections: [14C]Cicloprofen was administered orally as an aq. soln. of the sodium salt. Eight male rats, 350-380 g, were each given a 100 mg/kg daily dose of the drug for 4 days. Rats were then housed in individual metabolic cages, and food and water were supplied ad lib. Samples of urine and faeces, collected daily for 6 days, were frozen until analysed. Isolation and identification of metabolites: The urine samples collected during all six days were pooled and lyophilized to dryness. The residue was dissolved in 200 ml of water and extracted twice with 600ml of ethyl acetate. The aq. fraction was then brought to pH 1 with conc. HCI and further extracted twice with 600 ml of ethyl acetate. The extractions recovered 90% of the radioactivity from urine. The ethyl acetate extracts were combined and evaporated to dryness at 45" in vacuo. The residue was dissolved in water (100 ml), adjusted to pH 1 with HCI, and extracted with benzene (2 x 250 ml) followed by ethyl acetate (2 x 250 ml). The benzene extracts were combined as were the ethyl acetate extracts. The pooled benzene extract was concentrated to 100 ml by evaporation, and extracted with an equal vol. of 0.1 M NaOH. After acidification of the NaOH s o h . to p H 1 with HCl, the radioactive materials were extracted back into benzene (200ml). As the last benzene extraction only removed approx. 70y0 of the radioactivity from the acidic aq. soln., the remainder was extracted with ethyl acetate (200 ml), which was combined with the ethyl acetate extract obtained previously. The benzene and ethyl acetate extracts were evaporated to dryness separately and the residues dissolved in a small volume of ethyl acetate. The extracted radioactive materials were separated and purified by repeated chromatography on silica gel t.1.c. plates with solvent systems: (A) chloroform-acetic acid, 95 : 5 ; (B) benzene-diethyl ether-methanol, 8 : 3 : 1 ; (C) chloroform-acetone-acetic acid, 70 : 20 : 10; and (D) benzene-diethyl ether-acetic acid, 80 : 30 : 2. Compounds were visualized under short wavelength U.V. light, and radioactivity was located by scanning the plates with an Actigraph I11 Scanner (Nuclear Chicago, Des Plains, Ill., U.S.A.). Silica gel in the area corresponding to each radioactive spot was scraped from the plate, packed into a small column, and the radioactivity was eluted with methanol. Methanol was then removed by evaporation, and the residues redissolved in a small volume of methanol. Metabolites isolated and purified by t.1.c. were subjected to mass and n.m.r. spectrometric analyses with an Associated Electrical Industries Model MS-902 double-focusing mass spectrometer and a Varian Associates SL-100 n.m.r. spectrometer. Quantification of urinary metabolites :To determine the percentage distribution of radioactivity among the metabolites excreted in urine, a portion (4 ml) of the pooled 0-48 h urine from three rats that received a dose (50 mg/kg) of [14C]cicloprofenwas mixed with an equal vol. of 0.2 M sodium acetate buffer (pH 5.2). P-Glucuronidase (45 000 Fishman units) was added, and the mixture was incubated at 37" for 24 h with constant shaking. The incubation mixture was extracted with 30 ml of ethyl acetate, and the aqueous fraction was adjusted to pH 1 with 6 M HCI and further extracted with an additional 30 ml of ethyl acetate. The ethyl acetate extracts were combined and evaporated to dryness, and the residues dissolved in a small volume of ethyl acetate. The extractions were more than 95% complete. Aliquots of the ethyl acetate soh. were chromatographed on silica gel t.1.c. plates in solvent systems A and B. Compounds were visualized under U.V. light, and radioactivity located by scanning the plates with an Actigraph I11 Scanner. Silica gel in the area corresponding to each radioactive spot was scraped from the plate, mixed with 1 5 ml of Bray's solution (Bray, 1960), and counted in a Packard Tri-Carb liquid scintillation spectrometer, Model 3380. Automatic external standardization was used to determine the counting efficiency.
Metabolism of a-MethylJuorene-2-acetic Acid
123
Results Isolation and purification of metabolites Eight radioactive metabolites were obtained from the pooled urine of rats that received multiple doses of [14C]cicloprofen by extraction with organic solvents and t.1.c. in different solvent systems. Table 1 shows the R, values of cicloprofen and its metabolites after t.1.c. in four solvent systems. Table 1. RF values of cicloprofen and its metabolites after thin-layer chromatography
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
R, values and solvent system (see text) Compound
A
B
I Cicloprofen I1 9-Hydroxy9-methoxy I11 7-Hydroxy IV 9-Hydroxy V 7,9-Dihydroxy VI Unknown 1* VII Unknown 2 VIII Others
0.63
0-75
0.53
0.57 0.57 0.44 0.26 0-33 0.19 0
0.40 0.40 0.10 0.10 0.10 0
C
D
0.48 0.37
0.63 0.73 0.52
0.22 0.06
* Mass spectrometry indicated that this compound could be another dihydroxylated metabolite other than 7,9-dihydroxy-cicloprofen. Thin-layer chromatography of the radioactive materials in the benzene extract in solvent system A produced three radioactive fractions ( R , 0.63, 0.53 and 0.40). Fraction 1 (RF0.63) contained a single radioactive compound (Compound I) that had the same R, value as authentic cicloprofen in solvent systems A and B. Fraction 2 ( R , 0-53) also contained a single radioactive compound after chromatography in solvent systems B and D. This compound was further purified from non-radioactive impurities by repeated chromatography in systems A, B and D. When the purified compound was dissolved in methanol, concentrated, and stored at 0-5", a white radioactive precipitate was obtained. The purified compound (Compound 11) did not melt, but gradually decomposed at temperatures above 235". Fraction 3 had the same R , value (0.40) as authentic a-methyl-(7-hydroxyfluorene)-2-acetic acid in solvent system A. By chromatography in solvent system B, Fraction 3 was separated into two radioactive compounds (RF0*57 and 0.44). One (compound 111) was associated with the major portion of the radioactivity, and had the same R, value (0.57) as authentic ac-methyl-(7-hydroxyfluorene)-2-acetic acid. T h e other compound (Compound IV) was further purified in solvent system D. T h e ethyl acetate extract was separated into five radioactive fractions by t.1.c. in solvent system A. Fractions 1, 2 and 3 contained the same radioactive metabolites found in the benzene extract. Fraction 4 had an R, value of 0.1 and Fraction 5 consisted of compounds that remained at the origin, presumably conjugates or more polar metabolites. Fraction 4 was separated by chromatography in solvent system B into three radioactive compounds: V(R, 0.26), VI (R, 0.33), and V I I (R, 0.19). These compounds were further purified in solvent system C before subjecting them to mass and n.m.r. spectrometric analyses. Radioactive materials in Fraction 5 were not studied further because of the small amounts available,
124
S. J . Lan et al.
Mass spectrometry of cicloprofen and its metabolites T h e mass spectrum of cicloprofen (Fig. 1) serves as the model for elucidation of the structures of the metabolites. The mass spectrum of cicloprofen yields a prominent molecular ion (M+) at mle238. T h e loss of the carboxyl group yields a prominent fragment ion at m / e 193. The latter further loses the methyl group to form the ion at m / e 178 and the methine group to form the ion at mle 165. T h e ion at mle 223 is derived from the loss of the methyl group from M+.
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
100
I
I
?3
Compound I that had the same R , value as authentic cicloprofen in two different solvent systems also had a mass spectrum that was identical with that of cicloprofen. T h e mass spectrum of Compound I1 (Fig. 2) shows the M+ at m / e 284 and a prominent ion at m / e 239 that are 46 amu (atomic mass unit) greater than the corresponding ions in cicloprofen. T h e increase of 46 amu in the cicloprofen molecule indicates the introduction of two oxygen, one carbon, and two hydrogen atoms onto the cicloprofen molecule. The fragment ions at mle269, 224 and 211 correspond to the respective fragment ions at mle223, 178 and 165 of the cicloprofen molecule. These resuIts suggest that Compound I1 probably is a hydroxylated and methoxylated derivative of cicloprofen. T h e positional assignments of the hydroxy and the methoxy groups on the cicloprofen molecule, based on n.m.r. spectrometry, will be discussed later. T h e mass spectrum of Compound 111 (Fig. 3 ) shows the M+ at m / e 254 and fragment ions at m / e 239 (M+ - CH,), 209 (M+ - COOH), 194 (M+- CH, and COOH) and 181 (M+- CHCH, and COOH) that are 16 amu greater than the correponding ions in cicloprofen. Thus, it indicates the introduction of an additional oxygen onto the cicloprofen molecule. Comparison of the fragmentation patterns of Compound I11 and cicloprofen also indicates that the additional oxygen atom was introduced onto the fluorene moiety rather than
Metabolism of
a-Methy&uorene-Z-acetic
-~
100r----7---
i
90
1,
Acid
I
@g$fm"
n 211 239
M+ -CH*269
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
M + - C 4 -COOH=224
269
MRSS/CHRRGE
Fig. 2. Mass spectrum of Compound I I . cc-Methyl-(9-hydroxy-9-rnethoxyfluorene)-2-aceticacid.
1
W
>
H
MRSS/CHRRGE
Fig. 3. Mass spectrum of Compound I I I . cc-Methyl-(7-hydroxyfluorene)-2-acetic acid.
125
126
S. J: Lan et al.
I1
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
!
MRSS/CHRRGE
Fig. 4. Muss spectrum of Compound IV. cr-Methyl-(9-hydroxy-9H-fluorene)-2-aceticacid.
u+ 270 197
I
MRSS/CHQRGE
Fig. 5. Muss spectrum of Compound V . cc-Methyl-(7,9-dihydroxy-9H-fluorene)-2-acetic acid.
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
Metabolism of a- MethylJEuorene-2-acetic Acid
127
onto the side chain of cicloprofen. The mass spectra of Compound 111 and of an authentic sample of a-methyl-(7-hydroxyfluorene)-2-acetic acid were identical. T h e mass spectrum of Compound I V (Fig. 4) shows it to be a mixture of two compounds, one of the two having an Mf of mle284. T h e principal ions of the major component are the M+ at mle254 and the fragment ions at mje 209-207, 181 (base peak), 165 and 152, suggesting that the metabolite had an additional oxygen atom introduced onto the fluorene moiety. The fragmentation pathways of the two monohydroxy metabolites differ considerably (compare Figs. 3 and 4). It is likely that, while Compound 111 is indeed 7-hydroxylated or aryl hydroxylated, Compound IV is 9-hydroxylated. Furthermore, the mass spectrum of Compound V (Fig. 5 ) shows the Mf at mle270 and the diagnostic fragment ions at mle225-223, 207 and 197, 16 amu greater than the corresponding ions in Compound IV. Thus, the mass spectral data indicates that Compound V is the aryl monohydroxylated metabolite of 9-hydroxy cicloprofen. T h e mass spectrum of Compound VI (not shown) also shows the M+ at m/e 270 and fragment ions at mle225 and 197, suggesting that this compound could be another dihydroxylated metabolite. Compound VII did not yield a suitable mass spectrum. N.m.r. spectrometry of cicloprofen and its metabolites T h e positional assignments of the functional groups in the metabolites of cicloprofen by n.m.r. spectrometry depends in large measure on making consistent assignments in model compounds. T h e complete assignments of the n.m.r. spectra of fluorene (Bartle, Jones & Bavin, 1971; Jones, Matthews & Bartle, 1972; Douris & Mathieu, 1971), its simple aryl derivatives (Mathieu, Milano & Douris, 1974), and its 9-substituted derivatives (Mathieu, 1971 a, b, c) have been reported. Using the published data, the expected changes in chemical shifts on introduction of hydroxyl or methoxyl groups at the C-1 and C-3 to C-9 positions were evaluated. Assuming that these additive effects hold for the n.m.r. spectrum of cicloprofen too, a list of expected chemical shifts of the metabolites was calculated (Table 2) and compared to the chemical shifts and proton coupling patterns of the metabolites. The n.m.r. spectrum of Compound I1 indicates the loss of the C-9 methylene protons without appearance of the appropriate C-9 methine proton. Because the aryl proton resonances have not been shifted sufficiently for aryl hydroxylation or methoxylation, it is possible that Compound I1 is a methyl hemiketal of the C-9 0x0 derivative, which agrees with both the n.m.r. and mass spectra. Thus, the structure of Compound I1 is tentatively assigned as a-methyl-(9-hydroxy-9-methoxyfluorene)-2-acetic acid. T h e structure of Compound I11 is assigned as a-methyl-(7-hydroxyfluorene)2-acetic acid based on the n.m.r. and mass spectra. Typical of aromatic compounds, protons that are ortho to each other on the fluorene ring have coupling constants of 8 to 9 Hz while protons that are meta to each other have coupling constants of 2 to 3 Hz. Of significance for the n.m.r. spectrum of Compound I11 is the doublet near 6 7 of 2 Hz coupling for the C-8 proton, and the quartet of lines near 6 6.8 with both ortho and meta proton couplings. The methyl doublet at 6 1.48 confirms the retention of the side-chain, although the methine proton is not readily observed, which is presumed to coincide with the C-9 methylene resonance at 6 3.80. The observed spectrum of Compound I11 agrees very well with the calculated spectrum of the 7-hydroxy derivative of cicloprofen (Table 2).
7.48 7.45 7.52 7.41
Observed Compound I1 Compound I11 Compound IV Compound V
a
7.48 7.17 7.1 5 7.55 7.53 7.53 7.38 7.40 7.55 7.44 7.50 7.61 7.41 7-33 7.25 7.34 7.25
6.43 6.70 7.40 7.45 7.35 7.25 7.27 7.36 7.36 7.33 7.36 7.26
-
7.27 7.36
7.51 7.58 7.56 7.5
8.14 8.17 7-73 7.53 7.57 7.75 7.74 7.54 7.62 7.54
-
7.48 7.53 7.34
7.57 7.58 7.56 7.5
7.34 7.49 7.57 7.53 7.48 7.54 7-62 7.33
-
-
7.74 7.75 7.73 8.17 8.14
7.70
C-5H
__
7.16 6.81d 7.24 6.76d
6.74 6.82 7.30 7.21 7.27 7.30 6.73
7.30 7.30 7.29 7.31 7.34 6.64 6.37
7.26
C-6H
-
7.28
-
7.16
-
6.66 6.59 7.16 7.20
-
7.19 7.19 7.18 7.20 7.23 7.03 7.08 6.73
7.1 5
C-7H
-
7.28 6.99e 7.56 7.04e
7.53 7.64 7.01
-
7.47 7.46 7.56 7.56 7.23 6.91 7.20 7.51 6.98 7.08
7.50
C-8H
3.80 5.50 5.42
-
3.86 3.88 3.82 3.83 3.78 3.78 3.83 3.82 3.77 3.76 3.88 3.86 5.49 5.49 5-46
3.80
C-9H
CH
OCH3
1.47(d) 3.90, 3.94 1.48(d) 3.76(m) 1.50(d) -3.7 (m) 1.48(d)
CH,
Observed spectrum in deuteromethanol containing tetramethylsilane as an internal reference. For substituted cicloprofen derivatives assuming that the effects are additive. In deuteromethanol containing tetramethylsilane as an internal reference. J,=8 HZJm=2 Hz. Jm=2 Hz.
-
-
7.70
7.32
7.47
Cicloprofena
Calculatedb 1 -OH 1 -OCH, 3-OCHs 4-OCH, 4-OH 5-OH 5-OCH, 6-OCH, 7-OH 7-OCH3 8-OCH3 8-OH 9-OH 9-OCH3 7,9-diOH
C-4H
C-3H
C-1H
Compound
Chemical shifts in 6 values
Table 2. Calculated and observed chemical shifts of derivatives of cicloprofen
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
t Q Dc
c
Metabolism of ol-Methyljluorene-2-acetic Acid
129
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
T h e n.m.r. spectrum of Compound IV indicated the structural assignment to be ol-methyl-(9-hydroxy-9H-fluorene)-2-acetic acid with a C-9 proton resonance at 6 5.50 and aryl proton resonances predicted from the calculated spectra (Table 2). The n.m.r. spectrum of Compound V demonstrates it to be an aryl hydroxylated derivative of 9-hydroxy cicloprofen. A reasonable assignment of the structure of Compound V would be a-methyl-(7,9-dihydroxy9H-fluorene)-Z-acetic acid, for the aryl proton resonances are quite similar to those assigned to the 7-hydroxy derivative of cicloprofen; it would be expected that the introduction of a 9-hydroxyl group would have only a minimal influence on the chemical shifts (Table 2). The poor quality of the n.m.r. spectra of Compounds VI and VII did not permit a structural assignment for these two compounds. Metabolism of cicloprofen by rats Cicloprofen was readily metabolized by rats, and, along with its metabolites, was eliminated mainly via biliary excretion. T h e ratio of the amount of radioactivity excreted in faeces to that in urine ranged from 2 to 3.2 (Dean et al., 1977). When multiple oral doses (100 mg/kg per day) of the drug were administered to rats, the drug was excreted in urine largely as unconjugated metabolites. However, when a single 50 mg/kg dose of [14C]ciclopr~fenwas administered to rats, a major portion ( > 7 0 % ) of the radioactive materials in urine was excreted as conjugates. Excretion of greater amounts of unconjugated metabolites in urine by rats after receiving multiple oral doses (100 mg/kg per day) than after receiving a single 50 mg/kg dose might have been due to saturation of the conjugating-enzyme system, or to depletion of the cofactor (i.e. UDP-glucuronic acid) required for the conjugation reaction. Hydrolysis of the conjugates was achieved either by heating with 4 M HC1 or by incubation with a mixture of P-glucuronidase and sulphatase. Table 3 shows the percentage of each of the metabolites in the hydrolysed urine of rats that received a 50 mg/kg oral dose. There was little unchanged cicloprofen (2.6%) in urine, demonstrating its extensive biotransformation. The 7-hydroxy derivative of cicloprofen was the major metabolite (47%) excreted in urine. T h e other three metabolites, the 9-hydroxy, 7,9-dihydroxy and 9-hydroxy-9-methoxy derivatives of Table 3. Amounts of cicloprofen and its metabolites present in urine after administration of [W]cicloprofento rats
Compound I Cicloprofen I I 9-Hydroxy-9-methoxy 111 7-Hydroxy IV 9-Hydroxy V 7,9-Dihydroxy VI Unknown 1 VII Unknown 2 VIII Others
Amount of radioactivity in urine (yo) 2.6 10.1 47.1 10.4 10.1 n.d. 11.6 8.1
Figures are for pooled 0 to 48 h urine, from three rats given a single 50 mg/kg oral dose, which contained 17.2% dose. More than 70% of the metabolites were excreted in urine as glucuronide and/or sulphate conjugates. Data were obtained after hydrolysis with /3-glucuronidase and sulphatase. Distribution of radioactivity among metabolites was calculated from data obtained after t.1.c. in solvent systems A and B. n.d., could not be determined.
S. J . Lan et al.
130
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
cicloprofen, were each present to the extent of approximately 10% of the total radioactivity in urine. There were at least three minor metabolites that have not been identified.
Discussion Rat urinary metabolites of cirloprofen have been separated into eight fractions by thin-layer chromatography in different solvent systems. Four major metabolites have been tentatively identified, by mass spectrometry and nuclear magnetic resonance, as the 7-hydroxy, 9-hydroxy, 7,9-dihydroxy, and 9-hydroxy-9-methoxy derivatives of cicloprofen. These four metabolites together with unchanged cicloprofen accounted for 80% of the radioactivity excreted in urine. Hydroxylation and conjugation with glucuronic acid or sulphate are the major metabolic reactions. Isolation and identification of the 9-hydroxy-9-methoxy derivative of cicloprofen is of interest. Metabolic formation of a methyl hemiketal has not been previously reported. A logical pathway for the formation of the 9-hydroxy-9-methoxy derivative of cicloprofen may involve the oxidation of the carbon atom in the 9-position of cicloprofen to the 9-ox0 derivative, followed by hydration of the %ox0 to the 9,9-dihydroxy derivative and subsequent 0-methylation (Fig. 6). I t was considered that the 9-hydroxy-9-methoxy derivative might not be a true metabolite, but an artifact derived from the reaction of the %ox0 derivative of cicloprofen with methanol during isolation and purification procedures. Accordingly, the %ox0 derivative of cicloprofen was synthesized by treating cicloprofen with chromic acid. T h e synthesized %ox0 derivative is a yellowish compound, which was not converted to the 9-hydroxy-9-methoxy derivative of cicloprofen when treated with methanol by the same isolation and purification procedures applied to the latter. Thus, it is concluded that the 9-hydroxy-9-methoxy derivative of cicloprofen is a genuine metabolite.
W i
C P
3
- COOH
n
I C -COOH
I H
/
\
c-
H
HO-
~
.OH
0
.
cH3
a
Fig. 6. Proposed metabolic pathways of cicloprofen in rats. Numbered compounds are metabolites isolated and identified. Metabolites were excreted mainly as glucuronide or sulphate conjugates.
Metabolism of ~-~ethyZ~uorene-Z-acetic Acid
131
Fig. 6 shows the proposed metabolic pathways of cicloprofen in the rat. Among the proposed intermediate metabolites, the 9-0x0 and the 9,9-dihydroxy derivatives of cicloprofen have not been detected in the urine of rats dosed with cicloprofen. Assuming that cicloprofen is metabolized to its 9-hydroxy-9methoxy derivative via the proposed metabolic pathway, then either the conversion of cicloprofen to its 9-0x0 derivative or the subsequent formation of the 9,9-dihydroxy derivative is the rate-limiting reaction. T h e formation of 9-hydroxy-9-methoxy cicloprofen from %ox0 cicloprofen in rats is being investigated.
Downloaded by [University of California Santa Barbara] at 20:02 26 October 2015
References BARTLE,K. D., JONES,D. W. & BAVIN,P. M. G. (1971). J’. chem. SOC., (B), p. 388. BRAY,G. A. (1960). Analyt. Biochem., 1, 279. DEAN,A. V., LAN,S. J., KRIPALANI, K. J., DIFAZIO,L. T. & SCHREIBER, E. C. (1977). Xenobiotica, 7,549. DOURIS, J. & MATHIEU, A. (1971). Bull. SOC.Chim. France, p. 3365. JONES,D. W., MATTHEWS, R. S. & BARTLE, K. D. (1972). Spectrochim. Acta, 28A,2053. KRIPALANI, K. J., WELIKY,I. & SCHREIBER, E. C. (1972). Abstract, Med. Chem. 39, 164th ACS Nat’l Meeting, New York, N.Y., August 27 to September 1, 1972. KRIPALANI, K. J., VAHIDI,A., MISCHLER, T. W., NEISS,E. S. & SCHREIBER, E. C. (1973). The Pharmacologist, 15, 228. KRIPALANI, K. J., ZEINEL-ABDIN, A., DEAN,A. V. & SCHREIBER, E. C. (1976). Xenobiotica, 6, 159. LAN,S. J., KRIPALANI, K. J., DEAN,A. V., EGLI,P., DIFAZIO,L. T. & SCHREIBER, E. C. (1976). Drug Metab. Disp., 4, 330. MATHIEU, A. (1971 a). Bull. SOC. Chim. France, p. 1526. MATHIEU, A. (1971 b). Bull. SOC. Chinz. France, p. 1533. MATHIEU, A. (1971 c). Bull. SOC. Chim. France, p. 1540. MATHIEU, A., MILANO, J. C. & DOURIS, J. (1974). Bull. SOC.Chim. France, p. 299. MILLONIG, R. C., GOLDLUST, M. B., RUBIN,B., TURKHEIMER, A. R., SCHREIBER, W. F. & BELL,C. (1972). Abstract, Med. Chem. 38, 164th ACS Nat’l Meeting, New York, N.Y., August 27 to September 1, 1972. STILLER, E. T., DIASSI,P. A., GERSCHUTZ, D., MEIKLE,D., MOETZ,J., PRINCIPE, P. A. & LEVINE,S. D. (1972). J. Med. Chem., 15, 1029.
