XENOBIOTICA,1975, VOL.
5, NO. 7, 413-420
Metabolism of Papaverine I. Identification of Metabolites in Rat Bile F. M. BELPAIRE, M. G. BOGAERT, M. T. ROSSEEL J. F. and C . Heymans Institute of Pharmacology
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and M. A N T E U N I S Department of Organic Chemistry, University of Gent, B-9000 Gent, Belgium
(Received 25 October 1974) 1. After hydrolysis with glusulase of bile from rats treated with papaverine, four metabolites (A, B, C and D) were separated. 2. The structures of A, B and C were established as the monodemethylated compounds, 4‘-desmethyl-, 7-desmethyl-, and 6-desmethylpapaverine, respectively. 3. D was formed from papaverine by bis-demethylation. Since it was found in cat bile after administering either A or C, but not B, it was identified as 4’,6desmethylpapaverine.
Introduction Papaverine [1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline], an alkaloid with a 1-benzylisoquinoline structure, is used clinically as an antispasmodic agent. Only scanty data on its metabolism are available : it is metabolized to conjugated phenolic compounds, and Axelrod et al. (1958) identified the major urinary metabolite as conjugated 4’-desmethylpapaverine in man and the guinea-pig. I n rat bile after hydrolysis of conjugates four metabolites were detected by t.1.c. (Belpaire & Bogaert, 1975 a). The present paper describes the separation, purification and identification of these metabolites.
Materials and methods Materials [6’-3H]Papaverine (sp. activity 1.6 Ci/mmol) was obtained as previously described (Belpaire & Bogaert, 1973). Radiochemical purity was more than 95%. l-(3,4-Dimethoxybenzyl)-6-hydroxy-7-methoxyisoquinolinewas obtained from Professor F. Brochmann-Hanssen, University of California, San Francisco. 1-(3-Hydroxy-4-methoxybenzyl)-6,7-dimethoxyisoquinoline (palaudine) was obtained from Dr. A. Brossi, Hoffmann-La Roche, U.S.A. The following materials were used for analysis : Kieselgel GF,,., (E.Merck) ; M N Kieselgel, 70-325 Mesh (Macherey Nagel and Co.) ; Gas-Chrom Q,60-80 mesh (Supelco Inc.) ; QF-1, XE-60 and OV-1 (Applied Science Laboratories Inc.) ; SE-30 (Varian) ; glusulase (Serva). Animals and dosing A first series of experiments was performed with male Wistar rats (body wt. 300 g) to obtain sufficient material for purification and identification of the
F. M . Belpaire et al.
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metabolites. T h e common bile duct was catheterized under ether anaesthesia. T h e animals were then kept in restraining cages for 5 days and the bile was collected. Papaverine was given intraperitoneally (40 mg/kg/day). I n some experiments [3H]papaverine (sp. activity 0.25 pCi/mg) was administered. Metabolites were administered intravenously to anaesthetized rats (urethane, 1250 mg/kg intraperitoneally) and cats (sodium pentobarbital, 40 mg/kg intraperitoneally). T h e bile was collected for 6 h, while the body temperature was maintained at 37". Column chromatography T h e bile of groups of 20 rats injected with papaverine during 5 days, was pooled. After incubation with glusulase (1000 units/ml, at p H 5) for 24 h, the bile was extracted 3 times with 5 volumes of chloroform. T h e chloroform layers were combined, evaporated and the residue redissolved in the solvent (SF. gr. 0-91) (95 : 5 : 5). This mixture ethyl acetate-methanol-ammonia extract was put on a silica gel column (50 x 3 cm), the column eluted with the same solvent mixture at a flow rate of 11 drops per minute, and fractions of 10 ml were collected with a fraction collector. I n experiments with [3H]papaverine, 100 pl of each column fraction was counted for radioactivity ; in the other experiments, the fractions were examined by t.1.c. Thin-layer chromatography T h e silica gel plates were developed in various solvent systems and the spots viewed in U.V. light at 254 nm. R, values of papaverine, its metabolites and palaudine, a potential metabolite, are shown in Table 1. Table 1. Thin-layer chromatography on silica gel of papaverine and metabolites
I Papaverine Metabolite A
B C D Palaudine
0.88 0.75 0.40 0.23 0.11 0.67
RE.values in solvent I1 I11 IV 0.80 0.69 0.50 0.32
-
-
0.93 0.87 0.87 0.80
0.71
0.62
0.87
0.87 0.76 0.56 0.35
-
Solvent systems (by volume) : I : ethyl acetate-methanol-ammonia (sp. gr. 0.91) (2576,) (95 : 5 : 5) I1 : chloroform-dioxane-ethyl acetateammonia (sp. gr. 0.91) (25%) (25 : 60 : 10 : 5 ) 111 : ethanol-dioxane-benzene-ammonia (sp. gr. 0.91) (25:;) (5 : 40 : 50 : 2) IV : chloroform-methanol (90 : 10).
Gas-liquid chromatography A Varian Aerograph Model 2100 gas chromatograph with a flame ionization detector was used. T h e metabolites were separated on Gas Chrom Q coated with QF-1 (3.5y0), SE-30 (2%) or XE-60 (3y0), packed in a glass column (1.8 m x 2.5 mm i.d.). T h e column temperature was 190" and the carrier-gas
415
Metabolites of Papaverine
flow rate (nitrogen) was 30 mllmin. T h e injector and detector temperature was 250". T h e retention times of the metabolites relative to that of papaverine are given in Table 2. Table 2. Relative retention times of papaverine metabolites on gas chromatography
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Gas chromatography with flame ionization detection was used. Retention times, relative to papaverine = 1.0
XE-60 3Yo Metabolite A
B C D
1.19 2.02 1.18 1.53
Liquid phase SE-30
2%
QF-1 3.5%
0.98 1.29 0.94 0.99
1*07 1.57 1.02 1*01
Radioisotopic methods Radioactivity was measured in a Packard Tri-Carb Scintillation Spectrometer 3380, with external standardization. Quench correction was made with the Tri-Carb Absolute Activity Analyser model no. 544. Bray Scintillator solution was used for measurement of radioactivity in bile or in column eluates. Radioactivity on thin-layer plates was detected using a Packard Model 7201 Radiochromatogram Scanner. Mass spectrometry Mass spectra were obtained with a Gnom, Mat 111, mass spectrometer coupled to a Varian Aerograph 1400-10 gas chromatograph. A 1.5 m x 2 mm i.d. glass column of 1% OV-1 on Gaschrom Q was used at a temperature of 275" and a helium flow rate of 15 ml per min. T h e temperature of the injector, separator and inlet line was 300". Nuclear magnetic resonance spectrometry Nuclear magnetic resonance spectra were recorded on a Varian HA-100 N M R spectrometer (100 MHz) in CDCl, or D,O-CH,OD-NaOD (50 : 50 f 3 eq.), using tetramethylsilane as standard (internal and external respectively).
Results and discussion Preparation of biliary metabolites Bile from papaverine-treated rats was chromatographed silica gel after hydrolysis and extraction. Measurement of t.1.c. of the different fractions showed that four products were which were designated metabolites A, B, C and D. None of
on a column of radioactivity and clearly separated, these metabolites
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F. M . Belpaire et al.
had an R , value corresponding to that of authentic 3'-desmethylpapaverine (palaudine). T h e fractions containing one product were combined, evaporated and dissolved in 1 ml of ethanol. Cooling the solutions of A and of B to -30" caused crystallization ; each product was recrystallized several times. I n this way about 100 mg of metabolite A and 30 mg of metabolite B were prepared. Crystallization was not possible for metabolites C and D, since the amounts of these products were too small, and for identification of C and D, the column eluates were used. T h e purities of the crystallized metabolites A and B, and of the solutions of metabolites C and D were checked by g.1.c. and t.1.c. For metabolites A and B, injection of 10 pg on a QF-1 column for g.1.c. showed only one peak on the chromatogram. For metabolites C and D, g.1.c. analysis showed peaks in addition to the main one ; these impurities are due to endogenous substances present in the bile and not to other known metabolites, as shown by using the different g.1.c. columns described. Thin-layer chromatography of the metabolites A and B in various solvent systems (Table 1) revealed only one spot for each whereas for metabolites C and I>, apart from the main spot, a brown spot was always visible at the origin. These results show that metabolites A and B were pure and C and D were contaminated with bile substances. Mass spectrometry T h e m/e values of the most important fragment ions of papaverine and the four metabolites are summarized in Table 3. T h e mass spectra of the five compounds all showed molecular ion peaks. T h e m/e values of the molecular ions of metabolites A, B and C are 14 mass units lower than that of papaverine, suggesting that metabolites A, B and C are monodemethylated products. T h e m/e of the molecular ion peak of metabolite D however, is 28 mass units lower than that of the parent compound, showing that metabolite D is a bis-demethylated product. For all compounds the base peak corresponds to the (M-H)+ ion. Table 3.
Mass spectra of papaverine and its metabolites : the most important fragments
Fragment ion
M+ (M - H)+ (M - CH,)+ (M - OCH3)+ (M - H - OCH3)+ (M - OCH3 - CH,)+
m/e values of Papaverine Metabolites
339 338 324 308 307 293
A
B
C
D
325 324 310 294 293 279
325 324 310 294 293 279
325 324 310 294 293 279
311 310 296 280 279 265
I t is possible to deduce from these spectra whether the hydroxyl group is located on the benzyl moiety or on the isoquinoline moiety of the molecule though not its exact position, since each molecule may be cleaved in two positions
Metabolites of Papaverine
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as shown in Fig. 1 : one giving a CH,-isoquinoline fragment (fragment I) and the other a benzyl fragment (fragment 11). T h e mje values of these fragment ions in the different metabolites are shown in Table 4. These peaks are of low intensity, but are characteristic. A hydroxyl group is present on the CH,-isoquinoline moiety in metabolites B, C and D, and a hydroxyl group is present on the benzyl moiety in metabolites A and D. Since on chromatographic evidence A is known not to be 3'-desmethylpapaverine, it must therefore be 4'-desmethylpapaverine.
Fig. 1. Benzyl fragment (11) and CH,-isoquinoline fragment ( I ) from benzylisoquinolines on mass spectrometry.
Table 4. Fragment ions I and 11 from papaverine and its metabolites Substituents*
Papaverine MetaboliteA B C D
R1
R,
R,
R4
CH, CH, CH, H H
CH, CH, H CH, CH,
CH, CH, CHB CH, CH,
CH, H CH, CH, H
mle value of fragment ion 1 I1
202 202 188 188 188
* For letter symbols and structure, see Fig.
151 137 151 151 137
1.
Nuclear magnetic resonance T h e position of the hydroxyl groups in metabolites A and B has been determined by lH-n.m.r. spectrometry (Table 5) ; the amounts of metabolites C and D available were insufficient for n.m.r. analysis. T h e general structure of papaverine and its metabolites is shown in Fig. 2. T h e signals of protons a, b and c were assigned unambiguously by means of their splitting patterns. Protons a and c form an AB system with 35=5*6 Hz, revealing their vicinal character (ortho-coupling in pyridine ring). Because c is also long-range coupled with b, which shows no other coupling, they must be situated in a planar zig-zag pattern. T h e signal of proton f has fine structure (doublet of doublets) due to coupling with g and e, with values corresponding to meta- and para-couplings. Protons g and e form a highly degenerate AB spin system, with broader lines for one of the partners due to meta-coupling. T h e degeneracy disappears when
G(CDC1,) G(D,O-CD,OD-NaOD (+)A
G(CDC13) G(D,O-CD,OD-NaOD (+)A
)
)
7.35 7.50 -0.15
7.55 7.13 0.42
8.35
8.20 0.15
8.30 7.92 0.38
7.51 7.39 0.12
7.55 -0.14
7.41 6.55 0.27
6.82
7.06 7.00 0.06
6.70 6.79 0.10
Metabolite B
7.23 -0.19
7.04
Metabolite A
6.81 6.90 -0.09
6.65 0.12
6.77
Chemical shift (p.p.m.) of protons C d e f
6.71 6.78 -0.05
6.56 0.25
6.81
g
A = S(CDC1,) - S(D,O-CD,OD-NaOD). For letter symbols and structure, see Fig. 2.
b
4.01
3.99
R,O
Relative chemical shifts of protons in metabolites A and BX
a
Table 5.
-
3.89
R,O
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3.76
3.72
R,O
3.61
-
R,O
4.45
4.50
CH,+
5
4
m
Y'
2
is
3
Metabolites of Papaverine
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d
419
c
OR4
Fig. 2.
Symbols used for protons in nuclear magnetic resonance analysis of the metabolites of papaverine.
the spectra are run in CDC1,-C,D, (50 : S O ) , where J(g, e)- 8.2 Hz (orthocoupling in a benzene nucleus). Also 4J(g, f) is observed in the latter solvent (4J(g, f) = 1.5/2.0 Hz ; a typical value for a meta-coupling). The signal for proton d is a sharp singlet. By comparison of the chemical shifts of the non-dissociated product with those of the anion structure, the exact positions of the hydroxyl groups were determined. An increase in electron density causes a screening effect for the aromatic ring protons, especially when the ring involved also carries the negative charge. For metabolite A, such an effect is seen for protons a, e, f and g. Since the effect is smaller for f than for e, it can be concluded that OR, is an hydroxyl group, and that OR, is a methoxy group. For metabolite B however, there is deshielding except for a, b, c and d, with a large effect on a and b, indicating that OR, is an hydroxyl group. The &values of the different methoxy groups are in agreement with the values found by Brochmann-Hanssen and Hirai (1968). Identification of metabolite C On t.1.c. the R, value of the purified metabolite C , shown by mass spectrometry to be monodemethylated on the isoquinoline moiety (see Fig. 3), corresponds to that of the reference substance 6-desmethylpapaverine. Identification of metabolite D Metabolite D was identified by studying the biotransformation of metabolites A, B and C. When metabolite A was injected intravenously into the rat (5 mg/kg), the bile contained only metabolite A in conjugated form. Therefore, cats were used in these studies, and t.1.c. after hydrolysis of the bile with glusulase showed the presence of metabolite D after administering metabolites A or C to the cats but not after giving B. These experiments confirm the mass spectrometric findings that metabolite D is a bis-demethylated product and show that one hydroxyl group is located in the para position on the benzyl moiety of the molecule and the other in position 6 on the isoquinoline moiety (Fig. 3). Our results show that in the rat papaverine is demethylated in various positions of the molecule. T h e following papers (Belpaire & Bogaert, 1975 a, b)
420
Metabolites of Papaoerine
6
OCH3
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OCH3
\
OH
Fig. 3. Structures of papaverine and its metabolites.
deal with species differences in papaverine metabolism and with the breakdown of the substance in nitro by liver microsomal enzymes.
Acknowledgments T h e authors thank Professor Dr. A. F. D e Schaepdryver and Dr. A. Herman for their helpful comments and discussions. Professor F. Brochmann-Hanssen, University of California, San Francisco and Dr. A. Brossi from Hoffmann-La Roche, U.S.A. kindly supplied some benzylisoquinoline derivatives. T h e technical assistance of Misses M. Muylaert and C. Van Nieuwenhuyse is acknowledged. References AXELROD, J., SHOFER, R., INSCOE, J. K., KING,W. M. & SJOERDSMA, A. [1958]. J. Pharmac. exp. Ther., 124, 9. BELPAIRE, F. M. & BOGAERT, M. G. [1973]. Biochem. Pharmac., 22, 59. BELPAIRE, F. M. & BOGAERT, M. G. [1975 a]. Xenobiotica, 5, 421. BELPAIRE, F. M. & BOGAERT, M. G. [1975 b]. Xenobiotica, 5, 431. BROCHMANN-HANSSEN, E. & HIRAI,K. [1968]. J. pharm. Sci., 57, 940.
5, NO. 7, 413-420
Metabolism of Papaverine I. Identification of Metabolites in Rat Bile F. M. BELPAIRE, M. G. BOGAERT, M. T. ROSSEEL J. F. and C . Heymans Institute of Pharmacology
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and M. A N T E U N I S Department of Organic Chemistry, University of Gent, B-9000 Gent, Belgium
(Received 25 October 1974) 1. After hydrolysis with glusulase of bile from rats treated with papaverine, four metabolites (A, B, C and D) were separated. 2. The structures of A, B and C were established as the monodemethylated compounds, 4‘-desmethyl-, 7-desmethyl-, and 6-desmethylpapaverine, respectively. 3. D was formed from papaverine by bis-demethylation. Since it was found in cat bile after administering either A or C, but not B, it was identified as 4’,6desmethylpapaverine.
Introduction Papaverine [1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline], an alkaloid with a 1-benzylisoquinoline structure, is used clinically as an antispasmodic agent. Only scanty data on its metabolism are available : it is metabolized to conjugated phenolic compounds, and Axelrod et al. (1958) identified the major urinary metabolite as conjugated 4’-desmethylpapaverine in man and the guinea-pig. I n rat bile after hydrolysis of conjugates four metabolites were detected by t.1.c. (Belpaire & Bogaert, 1975 a). The present paper describes the separation, purification and identification of these metabolites.
Materials and methods Materials [6’-3H]Papaverine (sp. activity 1.6 Ci/mmol) was obtained as previously described (Belpaire & Bogaert, 1973). Radiochemical purity was more than 95%. l-(3,4-Dimethoxybenzyl)-6-hydroxy-7-methoxyisoquinolinewas obtained from Professor F. Brochmann-Hanssen, University of California, San Francisco. 1-(3-Hydroxy-4-methoxybenzyl)-6,7-dimethoxyisoquinoline (palaudine) was obtained from Dr. A. Brossi, Hoffmann-La Roche, U.S.A. The following materials were used for analysis : Kieselgel GF,,., (E.Merck) ; M N Kieselgel, 70-325 Mesh (Macherey Nagel and Co.) ; Gas-Chrom Q,60-80 mesh (Supelco Inc.) ; QF-1, XE-60 and OV-1 (Applied Science Laboratories Inc.) ; SE-30 (Varian) ; glusulase (Serva). Animals and dosing A first series of experiments was performed with male Wistar rats (body wt. 300 g) to obtain sufficient material for purification and identification of the
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metabolites. T h e common bile duct was catheterized under ether anaesthesia. T h e animals were then kept in restraining cages for 5 days and the bile was collected. Papaverine was given intraperitoneally (40 mg/kg/day). I n some experiments [3H]papaverine (sp. activity 0.25 pCi/mg) was administered. Metabolites were administered intravenously to anaesthetized rats (urethane, 1250 mg/kg intraperitoneally) and cats (sodium pentobarbital, 40 mg/kg intraperitoneally). T h e bile was collected for 6 h, while the body temperature was maintained at 37". Column chromatography T h e bile of groups of 20 rats injected with papaverine during 5 days, was pooled. After incubation with glusulase (1000 units/ml, at p H 5) for 24 h, the bile was extracted 3 times with 5 volumes of chloroform. T h e chloroform layers were combined, evaporated and the residue redissolved in the solvent (SF. gr. 0-91) (95 : 5 : 5). This mixture ethyl acetate-methanol-ammonia extract was put on a silica gel column (50 x 3 cm), the column eluted with the same solvent mixture at a flow rate of 11 drops per minute, and fractions of 10 ml were collected with a fraction collector. I n experiments with [3H]papaverine, 100 pl of each column fraction was counted for radioactivity ; in the other experiments, the fractions were examined by t.1.c. Thin-layer chromatography T h e silica gel plates were developed in various solvent systems and the spots viewed in U.V. light at 254 nm. R, values of papaverine, its metabolites and palaudine, a potential metabolite, are shown in Table 1. Table 1. Thin-layer chromatography on silica gel of papaverine and metabolites
I Papaverine Metabolite A
B C D Palaudine
0.88 0.75 0.40 0.23 0.11 0.67
RE.values in solvent I1 I11 IV 0.80 0.69 0.50 0.32
-
-
0.93 0.87 0.87 0.80
0.71
0.62
0.87
0.87 0.76 0.56 0.35
-
Solvent systems (by volume) : I : ethyl acetate-methanol-ammonia (sp. gr. 0.91) (2576,) (95 : 5 : 5) I1 : chloroform-dioxane-ethyl acetateammonia (sp. gr. 0.91) (25%) (25 : 60 : 10 : 5 ) 111 : ethanol-dioxane-benzene-ammonia (sp. gr. 0.91) (25:;) (5 : 40 : 50 : 2) IV : chloroform-methanol (90 : 10).
Gas-liquid chromatography A Varian Aerograph Model 2100 gas chromatograph with a flame ionization detector was used. T h e metabolites were separated on Gas Chrom Q coated with QF-1 (3.5y0), SE-30 (2%) or XE-60 (3y0), packed in a glass column (1.8 m x 2.5 mm i.d.). T h e column temperature was 190" and the carrier-gas
415
Metabolites of Papaverine
flow rate (nitrogen) was 30 mllmin. T h e injector and detector temperature was 250". T h e retention times of the metabolites relative to that of papaverine are given in Table 2. Table 2. Relative retention times of papaverine metabolites on gas chromatography
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Gas chromatography with flame ionization detection was used. Retention times, relative to papaverine = 1.0
XE-60 3Yo Metabolite A
B C D
1.19 2.02 1.18 1.53
Liquid phase SE-30
2%
QF-1 3.5%
0.98 1.29 0.94 0.99
1*07 1.57 1.02 1*01
Radioisotopic methods Radioactivity was measured in a Packard Tri-Carb Scintillation Spectrometer 3380, with external standardization. Quench correction was made with the Tri-Carb Absolute Activity Analyser model no. 544. Bray Scintillator solution was used for measurement of radioactivity in bile or in column eluates. Radioactivity on thin-layer plates was detected using a Packard Model 7201 Radiochromatogram Scanner. Mass spectrometry Mass spectra were obtained with a Gnom, Mat 111, mass spectrometer coupled to a Varian Aerograph 1400-10 gas chromatograph. A 1.5 m x 2 mm i.d. glass column of 1% OV-1 on Gaschrom Q was used at a temperature of 275" and a helium flow rate of 15 ml per min. T h e temperature of the injector, separator and inlet line was 300". Nuclear magnetic resonance spectrometry Nuclear magnetic resonance spectra were recorded on a Varian HA-100 N M R spectrometer (100 MHz) in CDCl, or D,O-CH,OD-NaOD (50 : 50 f 3 eq.), using tetramethylsilane as standard (internal and external respectively).
Results and discussion Preparation of biliary metabolites Bile from papaverine-treated rats was chromatographed silica gel after hydrolysis and extraction. Measurement of t.1.c. of the different fractions showed that four products were which were designated metabolites A, B, C and D. None of
on a column of radioactivity and clearly separated, these metabolites
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F. M . Belpaire et al.
had an R , value corresponding to that of authentic 3'-desmethylpapaverine (palaudine). T h e fractions containing one product were combined, evaporated and dissolved in 1 ml of ethanol. Cooling the solutions of A and of B to -30" caused crystallization ; each product was recrystallized several times. I n this way about 100 mg of metabolite A and 30 mg of metabolite B were prepared. Crystallization was not possible for metabolites C and D, since the amounts of these products were too small, and for identification of C and D, the column eluates were used. T h e purities of the crystallized metabolites A and B, and of the solutions of metabolites C and D were checked by g.1.c. and t.1.c. For metabolites A and B, injection of 10 pg on a QF-1 column for g.1.c. showed only one peak on the chromatogram. For metabolites C and D, g.1.c. analysis showed peaks in addition to the main one ; these impurities are due to endogenous substances present in the bile and not to other known metabolites, as shown by using the different g.1.c. columns described. Thin-layer chromatography of the metabolites A and B in various solvent systems (Table 1) revealed only one spot for each whereas for metabolites C and I>, apart from the main spot, a brown spot was always visible at the origin. These results show that metabolites A and B were pure and C and D were contaminated with bile substances. Mass spectrometry T h e m/e values of the most important fragment ions of papaverine and the four metabolites are summarized in Table 3. T h e mass spectra of the five compounds all showed molecular ion peaks. T h e m/e values of the molecular ions of metabolites A, B and C are 14 mass units lower than that of papaverine, suggesting that metabolites A, B and C are monodemethylated products. T h e m/e of the molecular ion peak of metabolite D however, is 28 mass units lower than that of the parent compound, showing that metabolite D is a bis-demethylated product. For all compounds the base peak corresponds to the (M-H)+ ion. Table 3.
Mass spectra of papaverine and its metabolites : the most important fragments
Fragment ion
M+ (M - H)+ (M - CH,)+ (M - OCH3)+ (M - H - OCH3)+ (M - OCH3 - CH,)+
m/e values of Papaverine Metabolites
339 338 324 308 307 293
A
B
C
D
325 324 310 294 293 279
325 324 310 294 293 279
325 324 310 294 293 279
311 310 296 280 279 265
I t is possible to deduce from these spectra whether the hydroxyl group is located on the benzyl moiety or on the isoquinoline moiety of the molecule though not its exact position, since each molecule may be cleaved in two positions
Metabolites of Papaverine
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as shown in Fig. 1 : one giving a CH,-isoquinoline fragment (fragment I) and the other a benzyl fragment (fragment 11). T h e mje values of these fragment ions in the different metabolites are shown in Table 4. These peaks are of low intensity, but are characteristic. A hydroxyl group is present on the CH,-isoquinoline moiety in metabolites B, C and D, and a hydroxyl group is present on the benzyl moiety in metabolites A and D. Since on chromatographic evidence A is known not to be 3'-desmethylpapaverine, it must therefore be 4'-desmethylpapaverine.
Fig. 1. Benzyl fragment (11) and CH,-isoquinoline fragment ( I ) from benzylisoquinolines on mass spectrometry.
Table 4. Fragment ions I and 11 from papaverine and its metabolites Substituents*
Papaverine MetaboliteA B C D
R1
R,
R,
R4
CH, CH, CH, H H
CH, CH, H CH, CH,
CH, CH, CHB CH, CH,
CH, H CH, CH, H
mle value of fragment ion 1 I1
202 202 188 188 188
* For letter symbols and structure, see Fig.
151 137 151 151 137
1.
Nuclear magnetic resonance T h e position of the hydroxyl groups in metabolites A and B has been determined by lH-n.m.r. spectrometry (Table 5) ; the amounts of metabolites C and D available were insufficient for n.m.r. analysis. T h e general structure of papaverine and its metabolites is shown in Fig. 2. T h e signals of protons a, b and c were assigned unambiguously by means of their splitting patterns. Protons a and c form an AB system with 35=5*6 Hz, revealing their vicinal character (ortho-coupling in pyridine ring). Because c is also long-range coupled with b, which shows no other coupling, they must be situated in a planar zig-zag pattern. T h e signal of proton f has fine structure (doublet of doublets) due to coupling with g and e, with values corresponding to meta- and para-couplings. Protons g and e form a highly degenerate AB spin system, with broader lines for one of the partners due to meta-coupling. T h e degeneracy disappears when
G(CDC1,) G(D,O-CD,OD-NaOD (+)A
G(CDC13) G(D,O-CD,OD-NaOD (+)A
)
)
7.35 7.50 -0.15
7.55 7.13 0.42
8.35
8.20 0.15
8.30 7.92 0.38
7.51 7.39 0.12
7.55 -0.14
7.41 6.55 0.27
6.82
7.06 7.00 0.06
6.70 6.79 0.10
Metabolite B
7.23 -0.19
7.04
Metabolite A
6.81 6.90 -0.09
6.65 0.12
6.77
Chemical shift (p.p.m.) of protons C d e f
6.71 6.78 -0.05
6.56 0.25
6.81
g
A = S(CDC1,) - S(D,O-CD,OD-NaOD). For letter symbols and structure, see Fig. 2.
b
4.01
3.99
R,O
Relative chemical shifts of protons in metabolites A and BX
a
Table 5.
-
3.89
R,O
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3.76
3.72
R,O
3.61
-
R,O
4.45
4.50
CH,+
5
4
m
Y'
2
is
3
Metabolites of Papaverine
Xenobiotica Downloaded from informahealthcare.com by Kungliga Tekniska Hogskolan on 08/31/15 For personal use only.
d
419
c
OR4
Fig. 2.
Symbols used for protons in nuclear magnetic resonance analysis of the metabolites of papaverine.
the spectra are run in CDC1,-C,D, (50 : S O ) , where J(g, e)- 8.2 Hz (orthocoupling in a benzene nucleus). Also 4J(g, f) is observed in the latter solvent (4J(g, f) = 1.5/2.0 Hz ; a typical value for a meta-coupling). The signal for proton d is a sharp singlet. By comparison of the chemical shifts of the non-dissociated product with those of the anion structure, the exact positions of the hydroxyl groups were determined. An increase in electron density causes a screening effect for the aromatic ring protons, especially when the ring involved also carries the negative charge. For metabolite A, such an effect is seen for protons a, e, f and g. Since the effect is smaller for f than for e, it can be concluded that OR, is an hydroxyl group, and that OR, is a methoxy group. For metabolite B however, there is deshielding except for a, b, c and d, with a large effect on a and b, indicating that OR, is an hydroxyl group. The &values of the different methoxy groups are in agreement with the values found by Brochmann-Hanssen and Hirai (1968). Identification of metabolite C On t.1.c. the R, value of the purified metabolite C , shown by mass spectrometry to be monodemethylated on the isoquinoline moiety (see Fig. 3), corresponds to that of the reference substance 6-desmethylpapaverine. Identification of metabolite D Metabolite D was identified by studying the biotransformation of metabolites A, B and C. When metabolite A was injected intravenously into the rat (5 mg/kg), the bile contained only metabolite A in conjugated form. Therefore, cats were used in these studies, and t.1.c. after hydrolysis of the bile with glusulase showed the presence of metabolite D after administering metabolites A or C to the cats but not after giving B. These experiments confirm the mass spectrometric findings that metabolite D is a bis-demethylated product and show that one hydroxyl group is located in the para position on the benzyl moiety of the molecule and the other in position 6 on the isoquinoline moiety (Fig. 3). Our results show that in the rat papaverine is demethylated in various positions of the molecule. T h e following papers (Belpaire & Bogaert, 1975 a, b)
420
Metabolites of Papaoerine
6
OCH3
Xenobiotica Downloaded from informahealthcare.com by Kungliga Tekniska Hogskolan on 08/31/15 For personal use only.
OCH3
\
OH
Fig. 3. Structures of papaverine and its metabolites.
deal with species differences in papaverine metabolism and with the breakdown of the substance in nitro by liver microsomal enzymes.
Acknowledgments T h e authors thank Professor Dr. A. F. D e Schaepdryver and Dr. A. Herman for their helpful comments and discussions. Professor F. Brochmann-Hanssen, University of California, San Francisco and Dr. A. Brossi from Hoffmann-La Roche, U.S.A. kindly supplied some benzylisoquinoline derivatives. T h e technical assistance of Misses M. Muylaert and C. Van Nieuwenhuyse is acknowledged. References AXELROD, J., SHOFER, R., INSCOE, J. K., KING,W. M. & SJOERDSMA, A. [1958]. J. Pharmac. exp. Ther., 124, 9. BELPAIRE, F. M. & BOGAERT, M. G. [1973]. Biochem. Pharmac., 22, 59. BELPAIRE, F. M. & BOGAERT, M. G. [1975 a]. Xenobiotica, 5, 421. BELPAIRE, F. M. & BOGAERT, M. G. [1975 b]. Xenobiotica, 5, 431. BROCHMANN-HANSSEN, E. & HIRAI,K. [1968]. J. pharm. Sci., 57, 940.
