XENOBIOTICA,1976,VOL.
6,NO. 3, 137-150
Metabolism of Alkenebenzene Derivatives in the Rat. 11. Eugenol and Isoeugenol Methyl Ethers* EINAR SOLHEIM and RONALD R. SCHELINE Department of Pharmacology, School of Medicine, University of Bergen, MFH-Bygget, 5000 Bergen, Norway Xenobiotica Downloaded from informahealthcare.com by University of Queensland on 10/12/14 For personal use only.
(Received 16 July 1975)
1. The metabolites of 3,4-dimethoxyallylbenzene(eugenol methyl ether) and 3,4-dimethoxypropenylbenzene (isoeugenol methyl ether) in the rat were identified and quantitatively determined by g.1.c. and g.1.c.-mass spectrometry. 2. The major metabolic reactions of 3,4-dimethoxyallylbenzene were oxidation of the allylic side chain to 2-hydroxy-3-(3,4-dimethoxyphenyl)propionic acid, 3,4-dimethoxybenzoic acid and 3,4-dimethoxyci~amic acid, the two latter being largely excreted as their glycine conjugates. The formation of the hydroxy acid presumably involved epoxidation of the double bond and subsequent hydration to the diol whereas the formation of 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid involved migration of the double bond and the formation of cinnamoyl intermediates. Other reactions were 0-demethylation to 4-hydroxy-3-methoxyallylbenzene(eugenol) and 3-hydroxy4-methoxyallylbenzene in equal amounts, oxidation to 1-(3,4-dimethoxyphenyl)2-propen-1 -01, hydroxylation of the benzene ring to a hydroxy-3,4-dimethoxyallylbenzene and oxidation to 3,4-dimethoxyphenylaceticacid. The formation of 1-(3,4-dihydroxyphenyl)propane was found to be carried out by the rat intestinal micro-organisms. A total of at least 63% but as much as 95% dose was accounted for. 3. The major metabolic pathway of 3,4-dimethoxypropenylbenzenewas via the cinnamoyl derivatives, leading to the formation of 4-hydroxy-3-methoxycinnamic acid (ferulic acid), 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid, the two latter being excreted largely as their glycine conjugates. Other reactions were 0-demethylation to 4-hydroxy-3-methoxypropenylbenzene (isoeugenol) and 3-hydroxy-4-methoxypropenylbenzenein equal amounts, and oxidation to 3,4-dimethoxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetone. Epoxidation of the side chain appeared to be a minor metabolic reaction with the propenyl derivative. 4. The biliary metabolites of 3,4-dimethoxyallylbenzeneand 3,4-dimethoxypropenylbenzene were identified and most of the urinary metabolites were also found in the bile.
Introduction Derivatives of allylbenzene and propenylbenzene are naturally occurring plant constituents. Some of these compounds have an effect on the central nervous system (Cesario de Mello et al., 1973), one example being myristicin (3-methoxy-4,5-methylenedioxyallylbenzene),which is generally believed to be responsible for the hallucinogenic effect of nutmeg (see Farnsworth, 1968; Oswald et al., 1971 b). Safrole (3,4-methylenedioxyallylbenzene)has carcinogenic properties (Long et al., 1963),and Borchert et al. (1973a, 1973 b) reported *Part 1 : Xembioticn, 3, 493 (1973). X.B.
L
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138
E. Solheim and R. R. Scheline
that the allyl alcohol metabolite of safrole, namely 1-(3,4-methylenedioxyphenyl)2-propen-1-01, is more carcinogenic in rats and mice than is safrole itself. We have shown (Solheim & Scheline, 1973) that p-methoxyallylbenzene is converted to the corresponding allyl alcohol derivative. However, we also showed that an epoxide was formed and Stillwell et al. (1974) have reported that safrole is also converted to an epoxide. Inasmuch as some epoxides are known to have carcinogenic activity, it is possible that this epoxide could be responsible for some of the carcinogenic activity of safrole (Stillwell et al., 1974). This paper deals with the metabolism of 3,4-dimethoxyallylbenzene(eugenol methyl ether) (I) and 3,4-dimethoxypropenylbenzene (isoeugenol methyl ether) (11) which are found in many plants including banana (Tress1 & Drawert, 1973), nutmeg (Shulgin, Sargent & Naranjo, 1967) and pimenta (Stahl, 1973).
Experimental Chemicals The mass spectral data and gas chromatographic retention times of both purchased and synthesized chemicals are listed in Table 1. The following compounds were obtained from commercial sources : 3,4-dimethoxyallylbenzene(I), 3,4~dimethoxypropenylbenzene(11), 4-hydroxy3-methoxyallylbenzene (IV), 4-hydroxy-3-methoxypropenylbenzene (VI), 3,4-dimethoxybenzoic acid (XXII), 4-hydroxy-3-methoxybenzoic acid (XXI I I), 4- hydroxy- 3-meth oxycinnamic acid (XXVI), 3,4- dimethoxy cinnamic acid (XXVIII), 3,4-dimethoxybenzaldehyde (XX), 4-hydroxy-3-methoxyphenylacetone (XIX), 3,4-dimethoxyphenylaceticacid (XXIV) and 4-hydroxy-3methoxyphenyl methyl ketone (XVII). 3-Hydroxy-4-methoxyallylbmzene(111) and 3-hydroxy-4-methoxypropenylbenzene (V) were both prepared by refluxing 3,4-dimethoxyallylbenzene (I) or 3,4-dimethoxypropenylbenzene (11) (1 g) with pyridine hydrochloride ( 5 g) for approx. 5 min. G.1.c.-mass spectrometry of the two reaction mixtures showed that in both cases the two monodemethylated isomers were formed together with the dihydroxy derivative. The two monodemethylated isomers were formed in equal amounts, but it was not possible to separate them by distillation or preparative g.1.c. In both reaction mixtures the isomer with the greatest retention time was the 4-hydroxy-3-methoxy derivative (IV and VI) (see Table 1). The other isomers must therefore be the 4-methoxy-3-hydroxy derivatives (111 and V). 3,4-Dimethoxyphenyl methyl ketone (XVI) was prepared by methylating 4-hydroxy-3-methoxyphenyl methyl ketone (XVII) with dimethylsulphate according to the method described in Organicum (1970). The product distilled at 112"/0.2 mm Hg. 1-(3,4-Dimethoxyphenyl)-2-propen-l-ol(IX) was prepared by the method described by Borchert et al. (1973 a), for the synthesis of 1-(3,4-methylenedioxypheny1)-Zpropen-1-01 but with veratraldehyde as the starting material. The product distilled at 140"/0-2mm Hg under dry N,. 3-(3,4-Dimethoxyphenyl)-2-propen1-01 (X) was prepared by the method described by Borchert et al. (1973 a) for the synthesis of 3-(3,4-methylenedioxyphenyl)-2-propen-l-o1 but with 1-(3,4-dimethoxypheny1)-2-propen-1-o1(IX) as a starting material. Some of the reaction mixture was subjected to rapid vacuum
Eugenol and Isoeugenol Methyl Ethers
139
Table 1. Retention times on 3% OV-17 and relative abundance of M+ and prominent fragments of 3,4-dimethoxyallylbenzene and 3,4-dimethoxypropenylbenzene, their metabolites and related compounds Compound No.
1 I1 I11
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IV V VI VI I VIII IX X XI XI1 XI11 XIV
xv XVI XVII XVIII XIX
XX XXI XXII
Chemical name
Trivial name
3,4-Dimethoxyallylbenzene Eugenol methyl ether 3,4-DimethoxypropenyIbenzene 3-Hydroxy-4methoxyallylbenzene 4-H ydroxy- 3methoxyallylbenzene 3-Hydroxy-4methoxypropenylbenzene 4-Hydroxy-3methoxypropenylbenzene 1-(3,4-Dihydroxyphenyl)propane1 2-Hydroxy-4,Sdimethoxyallylbenzene* 1-(3,4-Dimethoxyphenyl)-2propen-1-01 3-( 3,4-Dimethoxyphenyl)-2propen-1-01 3-(4-Hydroxy-3-methoxyphenyl)-2-propen-l-01$ 3-(3,4-Dimethoxyphenyl)propan-1-01 3-( 3,4-Dimethoxyphenyl)propylene 1,2-oxide 3-( 3,4-Dimethoxyphenyl)propane-l,2-diol 1-(3,4-Dimethoxyphenyl)propane-l,2-diol*f 3,4-Dimethoxyphenyl methyl ketone 4-Hydroxy-3-methoxyphenyl methyl ketone 1-(3,4-Dimethoxyphenyl)2-propen-1 -one* 4-Hydroxy-3methoxyphenylacetone 3,4-Dimethoxybenzaldehyde
Isoeugenolmethyl ether
Eugenol
#
8.1
6.2 7.3
Isoeugenol
7.7 5.1 11.2
3,4-DimethoxyphenylaIlyl- 8.3 alcohol 13.2 3,4-DimethoxycinnamyIalcohol 8.2 Coniferyl alcohol
12.7 11-7 13.7 10.6 3,4-Dimethoxyacetophenone Acetovanillone
10.3
3,4-Dimethoxyphenyl vinyl ketone
12.2
8.9
10.2 Veratraldehyde
Vanillic acid 4-Hydroxy-3methoxybenzoic acid+ 3,4-Dimethoxyphenylacetic Homoveratric acid XXIV acidt 3-( 3,4-Dimethoxyphenyl)XXV propionic acidt Ferulic acid 4-H ydroxy- 3 XXVI methoxvcinnamic acid+ Dihydroferulic acid XXVII 3-(4-Hyd;oxy- 3-met hoxypheny1)propionic acid+ XXVIII 3,4-Dimethoxycinnamic acid+ XXIX 2-Hydroxy-3-(3,4-dimethoxypheny1)propionic acid*+ XXX 3-Hydroxy-3-(3,4-dimethoxypheny1)propionic acidt XXXI 2-Keto-3-(3,4-dimethoxypheny1)propionic acid*+ XXXII 3,4-Dimethoxybenzoyl3,4-Dimetboxyhippuric glycinet acid XXXIII 3,4-Dimethoxycinnamoylglycinet
-
6.6
6.0
3,4-Dimethoxyphenylacetaldehyde* 3,4-Dimethoxybenzoic acid? Veratric acid
XXIII
Retention Prominent time M + fragments m / e (mid relative abundance
9.1 7.0 10.6 9.3 11.7 12.7 14.2 11.6 14.7 14-0 14.9 145 21.0 39.5
178 163 151 147 107 100 38 12 38 31 178 163 151 147 107 100 50 2 16 50 164 149 137 131 121 100 41 25 30 11 164 149 137 131 121 100 40 24 33 13 164 149 137 131 121 100 70 5 20 10 164 149 137 131 121 100 35 8 29 10 296 267 206 179 73 32 38 10 19 100 194 179 151 123 107 90 100 29 41 18 194 176 161 151 139 80 34 37 48 100 194 176 161 151 138 76 32 47 100 53 324 309 293 204 73 6 22 19 100 27 196 178 165 152 151 3 5 47 100 37 194 177 165 151 138 2 10 100 6 50 212 194 151 107 91 6 100 23 13 13 356 324 293 147 73 4 100 19 70 0.5 180 165 137 122 107 58 100 30 8 10 166 151 123 108 77 59 100 35 12 20 192 165 137 107 91 56 ioo -33 -25 is 180 151 137 122 94 18 4 100 19 7 166 165 151 137 95 100 64 25 14 20 180 165 151 123 107 39 28 100 25 6 196 181 165 137 121 79 10 100 18 8 182 168 151 123 108 46 6 100 27 3 210 179 151 107 91 30 2 100 13 6 224 164 151 121 91 10 100 10 11 35 208 177 145 137 117 100 77 66 6 19 210 165 137 123 107 8 100 11 10 31 222 207 191 163 147 100 25 56 12 16 240 222 151 107 91 9 2 100 7 10 240 222 191 167 109 9 16 100 4 25 238 191 178 151 107 14 9 13 100 15 253 221 165 137 107 26 6 100 15 10 279 248 220 191 163 39 3 4 100 14
No reference compound available, see text. t Carboxylic acids converted to their methyl esters. 1 Hydroxy groups converted to their trimethylsilyl ethers.
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E. Solheim and R. R.Scheline
distillation under dry N, which allowed collection of about 1 g of the cinnamyl alcohol before polymerization of the mixture. The product distilled at 164"/0.2 mm Hg under dry N,. 3-Hydroxy-3-(3~4-dimethoxyphenyl)propionic acid methyl ester was prepared from 3,4-dimethoxybenzaldehyde and methyl bromoacetate according to the method given by Vogel (1956) for the synthesis of 3-hydroxy-3-phenylpropionic acid. The product distilled at 180"/1 mm Hg, and contained small amounts of 3,4-dimethoxycinnamic acid methyl ester, probably formed by dehydration during distillation. High resolution mass spectroscopy of the product gave M+ at m/e 240-1008 which corresponds to the formula of 3-hydroxy-3-(3,4dimethoxypheny1)propionic acid methyl ester (Cl2Hl6O5, calculated mass 240.0997) and a base peak at m/e 167.0705 which corresponds to the formula of the hydroxy-3,4-dimethoxybenzyl fragment (C9Hl1O3, calculated mass 167-0708). 3,4-Dimethoxybenxoylglycine (XXXII) and 3,4-dimethoxycinnamoylglycine (XXXIII) were prepared from 3,4-dimethoxybenzoic acid (XXII) and 3,4-dimethoxycinnamic acid (XXVIII) respectively by the method described by Sheehan & Hess (1955) for the synthesis of peptides and gave m.p. 185" and 192", respectively. The mass spectrum of 3,4-dimethoxybenzoylglycine gave the same fragmentation pattern .as p-methoxybenzoylglycine (Solheim & Scheline, 1973) but with fragments 30 m/e units higher corresponding to the extra methoxy group. High resolution mass spectroscopy of 3,4-dimethoxycinnamoylglycine gave M+ at m/e 265.0950 which corresponds to the formula of 3,4-dimethoxycinnamoylglycine(C,,H1505N, calculated mass 265.0949) and a base peak at m/e 191.0713 w-hichcorresponds to the formula of the 3,4-dimethoxycinnamoyl fragment (C,,H,,03, calculated mass 191.0707). 1-(3,4-Dihydroxyphenyl)propane (VII) was prepared by reduction of 4-hydroxy-3-methoxyallylbenzene(IV) using H, and a 10% Pd on charcoal catalyst. The product was demethylated by refluxing with pyridine hydrochloride for approx. 20 min. The product distilled at 160"/1 mm Hg. The high resolution mass spectrum of the product showed M+ at mle 152.0844 which corresponds to the formula of 3,4-dihydroxypropylbenzene(C,H,,O,, calculated mass 152.0836) and a base peak at mle 123,0432 which corresponds to the formula of the 3,4-dihydroxybenzyl fragment (C&O,, calculated mass 123-0445). 3-(3,4-Dimethoxyphenyl)propylene 1,2-oxide (XIII) was prepared by the general method described by Fieser & Fieser (1967) for the epoxidation of alkenes. A solution of m-chloroperbenzoic acid (8 g) in dichloromethane (100 ml) was added with stiriing to a solution of 3,4-dimethoxyallylbenzene (8 g) in dichloromethane (20 ml). Following stirring for 24 h at room temperature the mixture was filtered and the solution washed with 5% aq. NaHCO,. 3-(3,4-Dimethoxyphenyl)-propylene 1,2-0xide distilled at 110-1 15"/0.5 mm Hg. The product polymerized after some weeks. 3-(3,4-DimethoxyphenyZ)propane-1,2-diol(XIV) was prepared by stirring a small amount of the oxide in 2 M HC1 at approx. 50" for 72 h. G.1.c.-mass spectrometry of the aqueous phase showed that the diol was present. T h e reaction mixture was used as a reference standard. 3-(3,4-Dimethoxyphenyl)p~opun-l-01 (XII) was prepared by reduction of 3,4dimethoxyphenylpropionic acid methyl ester. 3,4-Dimethoxyphenylpropionic
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Eugenol and Isoeugenol Methyl Ethers
141
acid (0.5 g) was methylated with diazomethane in diethyl ether. The reaction mixture was treated with LiAlH, (1 g) in small portions and refluxed for approx. 2 h. The solution was acidified with 2 M HC1 and extracted with ether. The ether extract was dried and the ether evaporated. G.1.c.-mass spectrometry showed that the product was chromatographically pure. 3-(4-Hydroxy-3-methoxyphenyl)-2-propen1-01 [XI) was prepared by reduction of 4-hydroxy-3-methoxycinnamicacid methyl ester with LiAlH,. 4-Hydroxy3-methoxycinnamic acid (1.5 g) was methylated with diazomethane in ether. The reaction mixture was treated cautiously with a suspension of LiAlH, in ether at approx. 0" and then refluxed for 12 h. The solution was hydrolysed with 2 M HCl and extracted with ether. The ether extract was dried and the ether evaporated. G.1.c.-mass spectrometry showed the product (approx. 50%) together with the methyl ester and the saturated alcohol. The reaction mixture was used as a reference standard. 3-f4-Hydroxy-3-methoxyphenyl)propionic acid (XXVII) was available from an earlier investigation (Scheline, 1968). 3-(3,4-DimethoxyphenyZ)propionic acid (XXV) (m.p. 97-98', lit. 98-99') was prepared from 3,4-dimethoxycinnamic acid by the same method as used for XXVII.
Animal experiments Male albino rats (Wistar strain derived) weighing 250-300 g were used. The animals were treated as described previously (Solheim & Scheline, 1973) except that 200 mg/kg doses were used in the quantitative experhents. Extraction of metabolites The urine and bile samples were treated with Glusulase (Endo Laboratories, Inc., Garden City, N.Y.) and extracted with ether according to the method described previously (Solheim & Scheline, 1973). Quantitative determination of metabolites p-Methoxybenzoic acid (2 mg) was used as internal standard and was added to the thawed urine samples. The urines were treated as described previously (Solheim & Scheline, 1973). Experiments were also carried out to assess the degree of recovery of the major metabolites in comparison with that of the internal standard when employing the extraction method described (Solheim & Scheline, 1973). The major metabolites, where reference compound were available, gave the same recovery as the internal standard. I n the cases where no reference compound was available we assumed that the recovery was equal to that of the internal standard. Major metabolites are arbitrarily defined as those accounting for 1oh or more of the dose and no attempts were made to quantitatively determine the remaining metabolites. Incubation with caecal extracts Incubation of the test compounds under anaerobic conditions with rat caecal niicro-organisms was carried out as described previously (Scheline, 1966, 1968).
142
E. Solheim and R . R. Scheline
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Gas chromatography An F & M Model 402 gas chromatograph equipped with flame ionization detector was employed for analytical and quantitative analysis. The glass columns were 0.3 (int. diam.) x 145 cm and were packed with 3% OV-17 on Gas Chrom Q 80-100 mesh. The column temperature was programmed from 110 to 260" at 5"/min and the injector and detector temperature were maintained at 260". Argon (45 ml/min) was employed as carrier gas. Mass spectrometry The mass spectra were obtained with a Varian MAT 111 g.1.c.-mass spectrometi y system. All compounds were introduced into the ionization chamber through the g.1.c. inlet and spectra were taken at 80 eV at a scan speed of 100 masses/sec. The glass column used was 0.2 (int. diam.) x 150 cm packed with 3% OV-17 on Gas Chrom Q 100-120 mesh. The column temperature was programmed from 110 to 260" at 6"/min and the injector and separator temperature were maintained at 260". Helium (15 ml/min) was employed as carrier gas. The high resolution mass spectra were obtained with an AEI MS902 mass spectrometer equipped with a DS 30/64/HL data system.
Results The retention times and the relative abundance of M+ and the most prominent fragments of the reference compounds and metabolites are listed in Table 1. With metabolites for which no reference compound was available, identification is based upon the fragmentation pattern in the mass spectrum and also upon comparisons with mass spectt a of authentic compounds containing similar substituents. Urinary metabolites The metabolites detected in the urine of rats given 3,4-dimethoxyallylbenzene (I), 3,4-dimethoxypropenylbenzene (11) and 3,4-dimethoxycinnamic acid (XXVIII) are shown in Table 2. No unchanged starting compound was found in the urine samples of rats given I or 11. 3,4-Dimethoxyacetophenone (XVI) was detected in urine from animals dosed with 3 4-dimethoxyallylbenzene (I), 3,4-dimethoxypropenylbenzene(11) and 3,4-dimethoxycinnamic acid (XVIII). In a previous paper (Solheim & Scheline, 1973) we found that the related monomethoxy ketone is an artefact and we assumed that the precursor was the corresponding 8-keto acid. This appears to be the case with the dimethoxy compounds as well. As with the p-methoxy derivatives (Solheim & Scheline, 1973) we were not able to detect the tertiary amino-3,4-dimethoxyphenylpropiophenone derivatives described by Oswald et al. (1971 a) although the decomposition product of these compounds, 3,4-dimethoxyphenyl vinyl ketone (XVIII) was detected in both the basic and acidic fractions. Table 3 lists the major urinary metabolites of 3,4-dimethoxyallylbenzene (I) and 3,4-dimethoxypropenylbenzene (11) and the amounts excreted within 24 h. Some of these metabolites were detected in trace amounts only, after 24 h.
Table 2. Urinary metabolites of 3,Cdimethoxyallylbenzene, 3,Cdimethoxypropenylbenzene and 3,4dimethoxycinnamic acid in the rat Compounds given orally or by intraperitonealinjection at doses of 100 or 400 mg/kg. Compound administered
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3,4-Dimethoxyallylbenzene(I)
3,4-Dimethoxypropenylbenzene (11)
3,4Dimethoxycinnamic acid (XXVIII)
3-Hydroxy-4-methoxyallylbenzene(I I I) 4-Hydroxy-3-methoxyallylbenzene(IV) 3-Hydroxy-4-methoxypropenylbenzene (V) 4-Hydroxy-3-methoxypropenylbenzene (VI) 1-(3,4-Dihydroxyphenyl)propane* (VII) VI I 2-Hydroxy-4,5-dimethoxyallylbenzene (VIII) 1-(3,4-Dimethoxyphenyl)-2-propen-1-01 (IX) 3-(3,4-Dimethoxyphenyl)-2-propen-1-01 X (XI 3-(4-Hydroxy-3-methoxyphenyl)-2propen-l-ol*t(XI) 3-(3,4-Dimethoxyphenyl)propan-l-ol*t (tr) (XII) . 3-(3,4-Dimethoxyphenyl)propane-1,2diol* (tr) (XIV) 1-(3,4-Dimethoxyphenyl)propane-l , 2-diol*(tr) (XV) 3,4-Dimethoxyphenylmethyl ketone XVI XVI (XVU 4-Hydroxy-3-methoxyphenylmethyl ketone*t (tr) (XVII) 1-(3,4-Dimethoxyphenyl)-2-propen-1one* (tr) (XVIII) 4-Hydroxy-3-methoxyphenylacetone*~ (tr) (XIX) 3,4-Dimethoxybenzaldehyde* (tr) (XX) 3-4-Dimethoxyphenylacetaldehyde* (tr) M I ) XXII XXI I 3,4-Dimethoxybenzoicacid (XXII) 4-Hydroxy-3-methoxybenzoic acid*? (XXIII) 3,4-Dimethoxyphenylaceticacid XXIV XXIV WIV) 3-(3,4-Dimethoxyphenyl)propionic XXV XXV acid (XXV) 4-Hydroxy-3-methoxycinnamic acid XXVI XXVI (XXVI) 4-Hydroxy-3-methoxyphenylpropionic XXVII acid* (tr) (XXVII) XXVIII 3,4-Dimethoxycinnamicacid (XXVIII) XXVIII 2-Hydroxy-3-(3,4-dimethoxyphenyl)propionic acid (XXIX) 3-Hydroxy-3-(3,4-dimethoxyphenyl)xxx propionic acid (XXX) xxx 2-Keto-3-(3,4-dimethoxyphenyl)propionic acid* (tr) (XXXI) 3,4-Dimethoxybenzoylglycine -11) XXXI I XXXI I 3,4-Dimethoxycinnamoylglycine XXXIII XXXIII (XXXIII)
* Detected only when 400mg/kg was given. t Detected only after intraperitoneal dose. (tr) Trace.
Hydrolysed None
0 0 0
4(3.1-5.3) 7 (4.7-9.3) 0
0
0
0
~~~
0 0 0
0
0 0-5 ( 0 . 4 4 6 )
0 0 0
8 (5.0-10.0)
0
5 (34-7.0)
0.5 (0.3-0.6)
0
0
Hydrolysed
Intraperitoneal
3,4-Dimethoxypropenylbenzene
Hydrolysed None
Oral
3,4-DimethoxypropenyIbenzene
Hydrolysed None
Intraperitoneal
3,4-Dimethoxyallylbenzene
*
84
95
63
88
53
T h e amounts found in the hydrolysed urines were 11% and 1 0 % respectively.
Recovery as major urinary metabolites
~I
55
For further details see text.
79
90
. o 0.5 ( 0 . 3 4 6 ) 0 0.5 (0.3-0.6) bdnzene (VI) 0 0 0 0 3,4-Dimethoxybenzoic acid (XXII) 2 (0.5-3.1) 2 (1.0-3.0) 2 (0.6-3.0) 1 (0-8-2.0) 1 (0.5-1.1) 2 (1.5-2.5) 1 (0.9-2.2) 3 (2.3-3.2) 2-Hvdroxv-4.5-dimethoxvallvlbekzend (VIII) 2 (0.8-2.1) 1 (0.5-1.3) 2 (1.0-3.8) 0 0 0 0 0 3,4-Dimethoxyphenylacetic acid 0 0 0 0 3 (1.6-3.3) 2 (1.7-2'4) 2 (0.6-2.4) 3 (1.2-3.8) (XXIV) 3 4 3,4-Dimethoxyphenyl)propionic acid (XXV) 0 0 1 (0.5-1.9) 1 (0.5-1.8) 2 (0.8-3.9) 1 (0.8-1.9) 0 0 4-Hydroxy-3-methoxycinnamic 0 7 (4.6-10.6) 27 (22.6-34.8) 10 (6.0-15.0) 34 (26-0-38.0) 0 acid (XXVI) 0 0 2-Hydroxy-3-(3,4-dimethoxyphenyl)0 0 0 0 propionic acid (XXIX) 20 (12'7-25.8) 20' 14 (124-21-0) 14' 3,4-Dimethoxycinnamic. acid (XXVI I I) 2 (0.9-3.6) 2 (0.7-3.2) 3 (2.8-5.8) 5 (3'64.8) 1 (04-1.6) 4 (3.145) 2 (1.5-2.2) 5 (3.8-5.1) 3-Hydroxy-3-( 3,4-dimethoxyphenyl)propionic acid (XXX) 2 (0'6-2.8) 2 (1.0-2.5) 5 (34-7.1) 4 (2.0-5.7) 4 (24-5.4) 4 (2.7-9'0) 6 (3.5-9.4) 9 (5.0-12.0) 3,4-Dimethoxybenzoylglycine (-11) 32 (27.0-35.7) 30 (18-6-37.6) 7 (5.9-10.0) 15 (10.0-22.0) 15 (12.0-17.9) 13 (8-0-16.7) 13 (10.0-24.0) 15 (9.1-20.0) 3,4-Dmethoxycinnamoylglycine (XXXIII) 22 (15.0-26.0) 24 (19.6-27.4) 28.1 33 (27.0-44.0) 24 (17.5-29.0) 26 (18.0-32.0) 21 (140-26.0) 21 (18-0-22.0) (23.0-30.0)
4-Hvdroxv-3-methnxvnmn~nvl, - ~ ~ - - - - - -_- .-C - - r - - - , -
Metabolite : 3-Hydroxy-4-methoxyallylbenzene (111) 4-Hydroxy-3-methoxyallylbenzene ( W 3-Hydroxy-4-methoxypropenylbenzene (V)
None
Oral
Route of administration
Treatment of urine
3,4- Dimethoxyallylbenzene
Compound administered
Table 3. Major urinary metabolites of 3,44imethoxyallylbenzene and 3,4-dimethoxypropenylbenzene in the rat Each animal received a dose of 200 mg/kg. Urine samples were collected over a period of 24 h. Results are average values from four or five animals, as % dose with ranges in parentheses.
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CI
P P
145
Eugenol and Isoeutenol Methyl Ethers
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-
Some of the metabolites or their conjugates were unstable to acid. Extraction at p H 1 resulted in reductions in the detectable amounts of' these metabolites and destruction was especially pronounced with l-(3,4-dimethoxyphenyl)allyl alcohol (IX). Maximal amounts of I X were obtained when extraction was carried out at approx. p H 5 . We estimated the amount excreted in the urine to Acid lability was also seen with the conjugates of 4-hydroxybe approx. 1-2%. 3-methoxy- and 4-methoxy-3-hydroxyallyl and -propenyl benzenes. Extraction at approx. p H 5 was carried out to determine these phenol derivatives in the unhydrolysed urines. One of the major metabolites, 2-hydroxy-3-(3,4-dimethoxyphenyl)propionic acid (XXIX) was unstable to p-glucuronidase .hydrolysis. I n the hydrolysed urines the amount of this acid was less than in the unhydrolysed urines. However, the amount in the hydrolysed urine samples must obviously be at least as large as that in the unhydrolysed samples and the latter value is therefore used in Table 3. Furthermore, the quantitative determination of the other hydroxy acid, 3-hydroxy-3-(3,4-dimethoxyphenyl)propionicacid (XXX), indicates that Table 4. Biliary metabolites of 3,4-dimethoxyallylbenzeneand 3,4-dimethoxypropenylbenzene in the rat Compound given orally or by intraperitoneal injection at a dose of 400 mg/kg. Bile samples
+
treated with 8-glucuronidase sulphatase before analysis. Compound administered 3,4-Dimethoxyallylbenzene(I)
3,4-Dimethoxypropenylbenzene(11)
3-Hydroxy-4-methoxyallylbenzene(I)* (111) 4-Hydroxy-3-methoxyallylbenzene (I)(IV) 3-Hydroxy-4-methoxypropenylbenzene
($1(v)
4-Hydroxy-3 -methoxypropenylbenzene (1)(VI)
2-Hydroxy-4,5-Dimethoxyallylbenzene(*) (VI 11) 1-(3,4-Dimethoxyphenyl)-2-propen-I -01 ($) (IX) 3-(3,4-Dimethoxyphenyl)-2-propen-l-ol(t)(X) 3-( 3,4-Dimethoxyphenyl)propane-1,2-diol(t ) (XIV) 3,4-Dimethoxybenzaldehyde(*) (XX) 3,4-Dimethoxybenzaldehyde(*) (XX) 3,4-Dimethoxyphenylacetaldehyde (*) (XXI) 1-(3,4-Dimethoxyphenyl)-2-propen1-one (t) (XVI I I) 3,4-Dimethoxybenzoic acid (*) (XXII) 3,4-Dimethoxybenzoic acid (*) (XXII) 3-(3,4-Dimethoxyphenyl)propionicacid (*) (XXV) (3,4-Dimethoxyphenyl)propionicacid(*) (XXV) 3,4-Dimethoxycinnamic acid (t)(XXVIII) 3,4-Dimethoxycinnamic acid (5) (XXVIII) 2-Hydroxy-3-( 3,4-dimethoxyphenyl)propionic acid (I)(XXIX) 2-Keto-3-(3,4-dimethoxyphenyl)propionic acid (*) (XXXI) 3,4-Dimethoxybenzoylglycine(*) (XXXII) 3,4-Dimethoxycinnamoylglycine(*) (XXXII I) 3,4-Dimethoxycinnamoylglycine ($1 W X W Relative amounts of metabolites detected: (*) small, (t)moderate,
(I)large, ($) very large.
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it is excreted unchanged in the urine and it is therefore likely that this is also the case with the other hydroxy acid. When the rats were switched to a purified diet containg 1% neomycin before dosing, 1-(3,4-dihydroxyphenyl)propane (VII) could not be detected in the urine as a metabolite of 3,4-dimethoxyallyl or -propenyl benzene. No other significant alteration in the pattern of metabolism of 3,4-dimethoxyallylbenzene or 3,4-dimethoxypropenylbenzene was found. 1-(3,4-Dihydroxyphenyl)propane (VII) was detected in the extracts of incubates of 3,4-dimethoxyallyl or -propenyl benzene with rat caecal microorganisms.
Biliary metabolites The metabolites detected in the bile of rats given 3,4-dimethoxyallylbenzene (I) and 3,4-dimethoxypropenylbenzene (11) are shown in Table 4 and are the metabolites consistently found in 4 experiments. The designations of the relative amounts of the biliary metabolites refer to the results obtained in a single experiment with each compound. However, both the numbers of metabolites and the total amount excreted were greater with the ally1 derivative than with the propenyl derivative. .
Discussion The proposed metabolic pathways of 3,4-dimethoxyallylbenzene (I) and 3,4-dimethoxypropenylbenzene(11) in the rat are shown in Figs. 1 and 2. As expected the major metabolic reactions of 3,4-dimethoxyallyl- (I) and 3,4-dimethoxypropenyl-benzene(11) follow the same pathways as we proposed for the corresponding monomethoxy derivatives [Solheim 8z Scheline, 1973)
Fig. 1 . Proposed metabolic pathways of 3,4-dimethoxyallylbezenein the rat. The numbers refer to the compounds listed in Table 1. Compounds in brackets have not been detected. Major and minor metabolic routes are indicated by thick and thin arrows, respectively, and artefact formation is indicated by broken arrows.
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coon
C
Fig. 2. Proposed metabolic pathways of 3,4-dimethoxypropenylbenzenein the rat. The numbers refer to the compounds listed in Table 1. Compounds in brackets have not been detected. Major and minor metabolic routes are indicated by thick and thin arrows, respectively, and artefact formation is indicated by broken arrows.
with some exceptions. The quantitative results show that 0-demethylation is not as important with the dimethoxy derivatives as with the monomethoxy derivatives. Urine of rats dosed with 3,4-dimethoxyallylbenzene (I) contained a metabolite with a mass spectrum indicating that hydroxplation of the benzene ring had occur1ed. When elemicin (3,4,5-trimethoxyallylbenzene)was partially demethylated by treatment with pyridine hydrochloride, none of the reaction products showed the same mass spectrum and retention time as given by the metabolite (unpublished results). This indicates that the 2- or 6-position rather than the 5-position, has been hydroxylated. 2-Hydroxylation will give 2-hydroxy-3,4-dimethoxyallylbenzenewhile 6-hydroxylation will give 2-hydroxy4,5-dimethoxyallylbenzene (VIII). Steric considerations suggest that VIII is more favourable. 3,4-Dihydroxypropylbenzene (VII) is a urinary metabolite of both 3,4-dimethoxyallyl- and propenyl-benzene. However, in animals given neomycin before dosing, VII was not detected in the urine. This compound was also found in incubates of rat caecal micro-organisms with 3,4-dimethoxyallyl- or propenyl-benzene. These findings indicate that VII is a metabolite formed by the rat intestinal microflora. The route via the cinnamoyl derivatives is a major metabolic pathway for both compounds. However, compared with the two monomethoxy allyl- and propenyl-benzenes (Solheim & Scheline, 1973) we detected several new types of metabolites. As with the monomethoxy derivatives, the cinnamic acid (XXVIII) was partly converted to the p-hydroxy acid (XXX) and further to the dimethoxybenzoylglycine. However, the 3,4-dimethoxycinnamic acid was
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also converted to 4-hydroxy-3-methoxycinnamicacid (XXVI), 3-(3,4-dimethoxypheny1)propionic acid (XXV) and 3,4-dimethoxycinnamoylglycine(XXXIII). Cinnamic acid-glycine conjugates have been described earlier both by Dakin (1909) who found that cinnamoylglycine is a minor metabolite of cinnamic acid in dogs and Armstrong, Shaw & Wall (1956) who found that feruloylglycine is a metabolite of ferulic acid (4-hydroxy-3-methoxycinnamic acid) in man. Furthermore, we found that 4-hydroxy-3-methoxycinnamylalcohol (XI) is a metabolite of 3,4-dimethoxypropenylbenzene (11) and that 3-(3,4-dimethoxypheny1)propan-1-01 (XII) is a metabolite of 3,4-dimethoxyallylbenzene(I), both formed via the cinnamoyl pathway. 3-@-Methoxyphenyl)propylene 1,Z-oxide is a biliary metabolite of R-methoxyallylbenzene and the related 1,2-diol is excreted in both the urine and bile (Solheim & Scheline, 1973). The corresponding epoxide (XIII) was not detected in the present experiments with 3,4-dimethoxyallylbenzene(I) but a diol (XIV) was again found. Stillwell et al. (1974) found that both safrole epoxide and diol are metabolites of safrole (3,4-methylenedioxyallylbenzene). Furthermore, we found in the urine and bile a compound which gave a mass spectrum indicating an a-keto acid (XXXI). This acid could arise by further oxidation of the a-hydroxy acid (XXIX) formed via the epoxide-diol pathway. As with p-methoxyallylbenzene we found that hydroxylation of the allyl group occurred, leading to the formation of 1-(3,4-dirnethoxyphenyl)-Z-propen-l-ol (IX). Borchert et al. (1973 a) showed that 1-(3,4-methylenedioxypheny1)-2propen-1-01 is a metabolite of safrole in the rat, and that pre-treatment of rats with phenobarbital increased about 10-fold the excretion of this allyl alcohol after a dose of safrole. We estimated the amount of allyl alcohol (IX) excreted in the urine to be about 1-2y0 of the dose. Oxidation of I X will give rise to the corresponding ketone (XVIII) and this was detected in both bile and urine. However, we were not able to detect the tertiary aminopropiophenone metabolites corresponding to those found by Oswald et al. (1971 b) as metabolites from safrole f3,4-methylenedioxyallylbenzene) or myristicin (3-methoxy-4,Smethylenedioxyallylbenzene). Braun & Kalbhen (1973) have shown that in the isolated perfused liver or during incubation with liver homogenates myristicin is converted to the corresponding amphetamine derivative. Examination of the basic urine fraction from rats given 3,4-dimethoxyallylbenzene revealed no compound which gave a mass spectrum indicating the presence of the dimethoxyamphetamine derivative. As in our previous paper (Solheim & Scheline, 1973), the 3,4-dimethoxyacetophenone (XVI) must be an artefact from decarboxylation of the corresponding j5-keto acid, which was not detected. Small amounts of two other ketones were detected in the urine of rats given 3,4-dimethoxypropenylbenzene (11). 4-Hydroxy-3-methoxyacetophenone (XVII) could similarly be an artefact from decarboxylation of a 4-hydroxy-3-methoxy-/%ketoacid. 4-Hydroxy-3-methoxyphenylacetone (XIX) is a metabolite and could be formed by oxidation and demethylation of the starting compound. As with the monomethoxy derivative (Solheim & Scheline, 1973), 3,4-dimethoxypropenylbenzene [11) is predominantly metabolized via the cinnamoyl pathway. Evidence for this is the finding that the major metabolites are ferulic acid (XXVI) and the two glycine conjugates 3,4-dimethoxybenzoyl-
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glycine (XXXII) and 3,4-dimethoxycinnamoylglycine (XXXIII), which together account for about 70% of the dose. Only small amounts of the diol were detected in urine and bile of rats dosed with 3,4-dimethoxypropenylbenzene(11) indicating that the epoxy-diol pathway is a minor one. However. 3,4dimethoxyallybenzene (I) is extensively metabolized by both the cinnamoyl pathway and the epoxy-diol pathway. The metabolic reaction from 3,4-dimethoxyallylbenzene to 3,4-dimethoxycinnamyl alcohol (X) was not investigated in detail. As shown in Fig. 1 the reaction can go via the allyl alcohol (IX) and Borchert et al. (1973 a) have shown that 1-(3,4-methylenedioxyphenyl)-2-propen-l-o1 can rearrange to 3-(3,4methylenedioxyphenyl)-2-propen-l-ol. Anoth.er possibility is simultaneous o-oxidation and migration of the double bond. The double bond in the allyl benzenes is relatively labile. Migration of this bond in the allyl benzenes has been reported (Mauthner, 1916; Trikojus & White, 1949), and it was also seen during the chemical demethylation of both mono- and dimethoxyallylbenzene in our studies. In the reaction mixture we also found all of the possible propenyl derivatives. But it is unlikely that migration of the double bond alone is the first step because one would then expect to find the demethylated propenyl derivatives in the urine of rats given the allyl benzenes. These were, however, not found either with the mono- or with the dimethoxyallylbenzene. However, in the urine of rats given p-methoxyallylbenzene, approx. 5-10y0 of the dose is converted to the allyl alcohol (Solheim & Scheline, 1973). If the cinnamoyl pathway goes via allyl alcohols only, one would expect this pathway to be more important with the monomethoxy derivative than with dimethoxyallylbenzene where approx. 1-2% of the dose is excreted as the allyl alcohol (IX). But as shown in our previous paper (Solheim & Scheline, 1973) the metabolites of p-methoxyallylbenzene in the cinnamoyl pathway account for only appxox. 14% of the dose while the corresponding figure for dimethoxyallylbenzene (I) is approx. 60%. The present results appear to be best explained by postulating that 3,4-dimethoxycinnamyl alcohol (X) arises mainly via o-oxidation but that some may also be formed by isomerization of the allyl alcohol (IX).
Acknowledgments The authors are indebted to Norges Almenvitenskapelige Forskningsrid and to Norsk Medisinaldepot for grants for the purchase of the gas chromatograph/mass spectrometer used in this study and to the members of the staff of the Chemical Institute, University of Oslo who assisted with the high resolution mass spectrometry. The technical assistance of Mrs. Eli Tepstad, Mrs. Astrid Hetle and Mr. Olav E. Fjellbirkeland is greatly appreciated.
References ARMSTRONG,M. D., SHAW,K. N. F. &WALL,P. E. (1956).
J. biol. Chem., 218, 293. BoRCHERr, P., ~VISLOCKI, P. G., MILLER,J. A. & MILLER,E. C . (1973 a). Cancer Res., 33, 575. BORCHERT, P.,MILLER,J. A., MILLER,E. C. & SHIRES,T. K. (1973 b). Cancer Res., 33, 590. BRAUN,V. & KALBHEN, D. A. (1973). Pharmacology (Basel), 9, 312. CESARIO DE MELLO,A., CARLINI,E. A., DRESSLER, K., GREEN,J. P., W G , S. & MARGOLIS, S. (1973). Psychopharmacologia ( B e d . ) , 31, 349.
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DAKIN,H. D. (1909). J. biol. Chem., 6, 225. FARNSWORTH, N. R. (1968). Science, N.Y., 162, 1086. FIESER, L. F. & FIESER, M. (1967). Reagentsfor Organic Synthesis, p. 136. New York: John Wiley & Sons, Inc. LONG,E. A., NELSON, A. A., FITZHUGH, 0. G. & HANSEN, W. H. (1963). Arch. Pathol., 75, 595. MAUTHNER, F. (1916). Justus Liebigs Annln Chem., 413, 250. ORGANICUM (1970), 9th ed., p. 222. Berlin: VEB Deutscher Verlag der Wissenschaften. OSWALD, E. O.,FISHBEIN, L., CORBETT, B. J. &WALKER, M. P. (1971 a). Biochim. biophys. Acta, 230, 237. OSWALD, E.O., FISHBEIN, L., CORBETT, B. J. & WALKER, M. P. (1971 b). Biochim. biophys. Actu, 244, 322. SCHELINE, R. R. (1966). J. Pharm. Pharmac., 18, 664. SCHELINE, R. R. (1968). Actaphurmac. tox., 26, 189. SHEEHAN, J. C. & HESS,G. P. (1955). J. Am. chem. SOC.,77, 1067. SHULGIN, A. T., SARGENT. T. & NARANJO,C. (1967). U.S. Public Health S e w . Publ. No. 1645,202. SOLHEIM, E. & SCHELINE, R. R. (1973). Xenobiotica, 3, 493. STAHL, E. (1973). Drug Analysis by Chromatography and Microscopy, p. 166. Ann Arbor, Michigan: Ann Arbor Science Publishers, Inc. STILLWELL, W. G., CARMAN, M. J., BELL, L. & HORNING, M. G. (1974). Drug Metab. Disp., 2, 486. TRESSL, R. & DRAWERT, F. (1973). J. agric. Fd Chem., 21, 560. TRIKOJUS, V. M. & WHITE,D. E. (1949j. J. chem. SOC. Part I, 436. VOGEL, A. I. (1956). A Text-Book of Practical Organic Chemistry, 3rd ed. p. 874. London: Longmans, Green & Co. Ltd.
6,NO. 3, 137-150
Metabolism of Alkenebenzene Derivatives in the Rat. 11. Eugenol and Isoeugenol Methyl Ethers* EINAR SOLHEIM and RONALD R. SCHELINE Department of Pharmacology, School of Medicine, University of Bergen, MFH-Bygget, 5000 Bergen, Norway Xenobiotica Downloaded from informahealthcare.com by University of Queensland on 10/12/14 For personal use only.
(Received 16 July 1975)
1. The metabolites of 3,4-dimethoxyallylbenzene(eugenol methyl ether) and 3,4-dimethoxypropenylbenzene (isoeugenol methyl ether) in the rat were identified and quantitatively determined by g.1.c. and g.1.c.-mass spectrometry. 2. The major metabolic reactions of 3,4-dimethoxyallylbenzene were oxidation of the allylic side chain to 2-hydroxy-3-(3,4-dimethoxyphenyl)propionic acid, 3,4-dimethoxybenzoic acid and 3,4-dimethoxyci~amic acid, the two latter being largely excreted as their glycine conjugates. The formation of the hydroxy acid presumably involved epoxidation of the double bond and subsequent hydration to the diol whereas the formation of 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid involved migration of the double bond and the formation of cinnamoyl intermediates. Other reactions were 0-demethylation to 4-hydroxy-3-methoxyallylbenzene(eugenol) and 3-hydroxy4-methoxyallylbenzene in equal amounts, oxidation to 1-(3,4-dimethoxyphenyl)2-propen-1 -01, hydroxylation of the benzene ring to a hydroxy-3,4-dimethoxyallylbenzene and oxidation to 3,4-dimethoxyphenylaceticacid. The formation of 1-(3,4-dihydroxyphenyl)propane was found to be carried out by the rat intestinal micro-organisms. A total of at least 63% but as much as 95% dose was accounted for. 3. The major metabolic pathway of 3,4-dimethoxypropenylbenzenewas via the cinnamoyl derivatives, leading to the formation of 4-hydroxy-3-methoxycinnamic acid (ferulic acid), 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid, the two latter being excreted largely as their glycine conjugates. Other reactions were 0-demethylation to 4-hydroxy-3-methoxypropenylbenzene (isoeugenol) and 3-hydroxy-4-methoxypropenylbenzenein equal amounts, and oxidation to 3,4-dimethoxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetone. Epoxidation of the side chain appeared to be a minor metabolic reaction with the propenyl derivative. 4. The biliary metabolites of 3,4-dimethoxyallylbenzeneand 3,4-dimethoxypropenylbenzene were identified and most of the urinary metabolites were also found in the bile.
Introduction Derivatives of allylbenzene and propenylbenzene are naturally occurring plant constituents. Some of these compounds have an effect on the central nervous system (Cesario de Mello et al., 1973), one example being myristicin (3-methoxy-4,5-methylenedioxyallylbenzene),which is generally believed to be responsible for the hallucinogenic effect of nutmeg (see Farnsworth, 1968; Oswald et al., 1971 b). Safrole (3,4-methylenedioxyallylbenzene)has carcinogenic properties (Long et al., 1963),and Borchert et al. (1973a, 1973 b) reported *Part 1 : Xembioticn, 3, 493 (1973). X.B.
L
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that the allyl alcohol metabolite of safrole, namely 1-(3,4-methylenedioxyphenyl)2-propen-1-01, is more carcinogenic in rats and mice than is safrole itself. We have shown (Solheim & Scheline, 1973) that p-methoxyallylbenzene is converted to the corresponding allyl alcohol derivative. However, we also showed that an epoxide was formed and Stillwell et al. (1974) have reported that safrole is also converted to an epoxide. Inasmuch as some epoxides are known to have carcinogenic activity, it is possible that this epoxide could be responsible for some of the carcinogenic activity of safrole (Stillwell et al., 1974). This paper deals with the metabolism of 3,4-dimethoxyallylbenzene(eugenol methyl ether) (I) and 3,4-dimethoxypropenylbenzene (isoeugenol methyl ether) (11) which are found in many plants including banana (Tress1 & Drawert, 1973), nutmeg (Shulgin, Sargent & Naranjo, 1967) and pimenta (Stahl, 1973).
Experimental Chemicals The mass spectral data and gas chromatographic retention times of both purchased and synthesized chemicals are listed in Table 1. The following compounds were obtained from commercial sources : 3,4-dimethoxyallylbenzene(I), 3,4~dimethoxypropenylbenzene(11), 4-hydroxy3-methoxyallylbenzene (IV), 4-hydroxy-3-methoxypropenylbenzene (VI), 3,4-dimethoxybenzoic acid (XXII), 4-hydroxy-3-methoxybenzoic acid (XXI I I), 4- hydroxy- 3-meth oxycinnamic acid (XXVI), 3,4- dimethoxy cinnamic acid (XXVIII), 3,4-dimethoxybenzaldehyde (XX), 4-hydroxy-3-methoxyphenylacetone (XIX), 3,4-dimethoxyphenylaceticacid (XXIV) and 4-hydroxy-3methoxyphenyl methyl ketone (XVII). 3-Hydroxy-4-methoxyallylbmzene(111) and 3-hydroxy-4-methoxypropenylbenzene (V) were both prepared by refluxing 3,4-dimethoxyallylbenzene (I) or 3,4-dimethoxypropenylbenzene (11) (1 g) with pyridine hydrochloride ( 5 g) for approx. 5 min. G.1.c.-mass spectrometry of the two reaction mixtures showed that in both cases the two monodemethylated isomers were formed together with the dihydroxy derivative. The two monodemethylated isomers were formed in equal amounts, but it was not possible to separate them by distillation or preparative g.1.c. In both reaction mixtures the isomer with the greatest retention time was the 4-hydroxy-3-methoxy derivative (IV and VI) (see Table 1). The other isomers must therefore be the 4-methoxy-3-hydroxy derivatives (111 and V). 3,4-Dimethoxyphenyl methyl ketone (XVI) was prepared by methylating 4-hydroxy-3-methoxyphenyl methyl ketone (XVII) with dimethylsulphate according to the method described in Organicum (1970). The product distilled at 112"/0.2 mm Hg. 1-(3,4-Dimethoxyphenyl)-2-propen-l-ol(IX) was prepared by the method described by Borchert et al. (1973 a), for the synthesis of 1-(3,4-methylenedioxypheny1)-Zpropen-1-01 but with veratraldehyde as the starting material. The product distilled at 140"/0-2mm Hg under dry N,. 3-(3,4-Dimethoxyphenyl)-2-propen1-01 (X) was prepared by the method described by Borchert et al. (1973 a) for the synthesis of 3-(3,4-methylenedioxyphenyl)-2-propen-l-o1 but with 1-(3,4-dimethoxypheny1)-2-propen-1-o1(IX) as a starting material. Some of the reaction mixture was subjected to rapid vacuum
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Table 1. Retention times on 3% OV-17 and relative abundance of M+ and prominent fragments of 3,4-dimethoxyallylbenzene and 3,4-dimethoxypropenylbenzene, their metabolites and related compounds Compound No.
1 I1 I11
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IV V VI VI I VIII IX X XI XI1 XI11 XIV
xv XVI XVII XVIII XIX
XX XXI XXII
Chemical name
Trivial name
3,4-Dimethoxyallylbenzene Eugenol methyl ether 3,4-DimethoxypropenyIbenzene 3-Hydroxy-4methoxyallylbenzene 4-H ydroxy- 3methoxyallylbenzene 3-Hydroxy-4methoxypropenylbenzene 4-Hydroxy-3methoxypropenylbenzene 1-(3,4-Dihydroxyphenyl)propane1 2-Hydroxy-4,Sdimethoxyallylbenzene* 1-(3,4-Dimethoxyphenyl)-2propen-1-01 3-( 3,4-Dimethoxyphenyl)-2propen-1-01 3-(4-Hydroxy-3-methoxyphenyl)-2-propen-l-01$ 3-(3,4-Dimethoxyphenyl)propan-1-01 3-( 3,4-Dimethoxyphenyl)propylene 1,2-oxide 3-( 3,4-Dimethoxyphenyl)propane-l,2-diol 1-(3,4-Dimethoxyphenyl)propane-l,2-diol*f 3,4-Dimethoxyphenyl methyl ketone 4-Hydroxy-3-methoxyphenyl methyl ketone 1-(3,4-Dimethoxyphenyl)2-propen-1 -one* 4-Hydroxy-3methoxyphenylacetone 3,4-Dimethoxybenzaldehyde
Isoeugenolmethyl ether
Eugenol
#
8.1
6.2 7.3
Isoeugenol
7.7 5.1 11.2
3,4-DimethoxyphenylaIlyl- 8.3 alcohol 13.2 3,4-DimethoxycinnamyIalcohol 8.2 Coniferyl alcohol
12.7 11-7 13.7 10.6 3,4-Dimethoxyacetophenone Acetovanillone
10.3
3,4-Dimethoxyphenyl vinyl ketone
12.2
8.9
10.2 Veratraldehyde
Vanillic acid 4-Hydroxy-3methoxybenzoic acid+ 3,4-Dimethoxyphenylacetic Homoveratric acid XXIV acidt 3-( 3,4-Dimethoxyphenyl)XXV propionic acidt Ferulic acid 4-H ydroxy- 3 XXVI methoxvcinnamic acid+ Dihydroferulic acid XXVII 3-(4-Hyd;oxy- 3-met hoxypheny1)propionic acid+ XXVIII 3,4-Dimethoxycinnamic acid+ XXIX 2-Hydroxy-3-(3,4-dimethoxypheny1)propionic acid*+ XXX 3-Hydroxy-3-(3,4-dimethoxypheny1)propionic acidt XXXI 2-Keto-3-(3,4-dimethoxypheny1)propionic acid*+ XXXII 3,4-Dimethoxybenzoyl3,4-Dimetboxyhippuric glycinet acid XXXIII 3,4-Dimethoxycinnamoylglycinet
-
6.6
6.0
3,4-Dimethoxyphenylacetaldehyde* 3,4-Dimethoxybenzoic acid? Veratric acid
XXIII
Retention Prominent time M + fragments m / e (mid relative abundance
9.1 7.0 10.6 9.3 11.7 12.7 14.2 11.6 14.7 14-0 14.9 145 21.0 39.5
178 163 151 147 107 100 38 12 38 31 178 163 151 147 107 100 50 2 16 50 164 149 137 131 121 100 41 25 30 11 164 149 137 131 121 100 40 24 33 13 164 149 137 131 121 100 70 5 20 10 164 149 137 131 121 100 35 8 29 10 296 267 206 179 73 32 38 10 19 100 194 179 151 123 107 90 100 29 41 18 194 176 161 151 139 80 34 37 48 100 194 176 161 151 138 76 32 47 100 53 324 309 293 204 73 6 22 19 100 27 196 178 165 152 151 3 5 47 100 37 194 177 165 151 138 2 10 100 6 50 212 194 151 107 91 6 100 23 13 13 356 324 293 147 73 4 100 19 70 0.5 180 165 137 122 107 58 100 30 8 10 166 151 123 108 77 59 100 35 12 20 192 165 137 107 91 56 ioo -33 -25 is 180 151 137 122 94 18 4 100 19 7 166 165 151 137 95 100 64 25 14 20 180 165 151 123 107 39 28 100 25 6 196 181 165 137 121 79 10 100 18 8 182 168 151 123 108 46 6 100 27 3 210 179 151 107 91 30 2 100 13 6 224 164 151 121 91 10 100 10 11 35 208 177 145 137 117 100 77 66 6 19 210 165 137 123 107 8 100 11 10 31 222 207 191 163 147 100 25 56 12 16 240 222 151 107 91 9 2 100 7 10 240 222 191 167 109 9 16 100 4 25 238 191 178 151 107 14 9 13 100 15 253 221 165 137 107 26 6 100 15 10 279 248 220 191 163 39 3 4 100 14
No reference compound available, see text. t Carboxylic acids converted to their methyl esters. 1 Hydroxy groups converted to their trimethylsilyl ethers.
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distillation under dry N, which allowed collection of about 1 g of the cinnamyl alcohol before polymerization of the mixture. The product distilled at 164"/0.2 mm Hg under dry N,. 3-Hydroxy-3-(3~4-dimethoxyphenyl)propionic acid methyl ester was prepared from 3,4-dimethoxybenzaldehyde and methyl bromoacetate according to the method given by Vogel (1956) for the synthesis of 3-hydroxy-3-phenylpropionic acid. The product distilled at 180"/1 mm Hg, and contained small amounts of 3,4-dimethoxycinnamic acid methyl ester, probably formed by dehydration during distillation. High resolution mass spectroscopy of the product gave M+ at m/e 240-1008 which corresponds to the formula of 3-hydroxy-3-(3,4dimethoxypheny1)propionic acid methyl ester (Cl2Hl6O5, calculated mass 240.0997) and a base peak at m/e 167.0705 which corresponds to the formula of the hydroxy-3,4-dimethoxybenzyl fragment (C9Hl1O3, calculated mass 167-0708). 3,4-Dimethoxybenxoylglycine (XXXII) and 3,4-dimethoxycinnamoylglycine (XXXIII) were prepared from 3,4-dimethoxybenzoic acid (XXII) and 3,4-dimethoxycinnamic acid (XXVIII) respectively by the method described by Sheehan & Hess (1955) for the synthesis of peptides and gave m.p. 185" and 192", respectively. The mass spectrum of 3,4-dimethoxybenzoylglycine gave the same fragmentation pattern .as p-methoxybenzoylglycine (Solheim & Scheline, 1973) but with fragments 30 m/e units higher corresponding to the extra methoxy group. High resolution mass spectroscopy of 3,4-dimethoxycinnamoylglycine gave M+ at m/e 265.0950 which corresponds to the formula of 3,4-dimethoxycinnamoylglycine(C,,H1505N, calculated mass 265.0949) and a base peak at m/e 191.0713 w-hichcorresponds to the formula of the 3,4-dimethoxycinnamoyl fragment (C,,H,,03, calculated mass 191.0707). 1-(3,4-Dihydroxyphenyl)propane (VII) was prepared by reduction of 4-hydroxy-3-methoxyallylbenzene(IV) using H, and a 10% Pd on charcoal catalyst. The product was demethylated by refluxing with pyridine hydrochloride for approx. 20 min. The product distilled at 160"/1 mm Hg. The high resolution mass spectrum of the product showed M+ at mle 152.0844 which corresponds to the formula of 3,4-dihydroxypropylbenzene(C,H,,O,, calculated mass 152.0836) and a base peak at mle 123,0432 which corresponds to the formula of the 3,4-dihydroxybenzyl fragment (C&O,, calculated mass 123-0445). 3-(3,4-Dimethoxyphenyl)propylene 1,2-oxide (XIII) was prepared by the general method described by Fieser & Fieser (1967) for the epoxidation of alkenes. A solution of m-chloroperbenzoic acid (8 g) in dichloromethane (100 ml) was added with stiriing to a solution of 3,4-dimethoxyallylbenzene (8 g) in dichloromethane (20 ml). Following stirring for 24 h at room temperature the mixture was filtered and the solution washed with 5% aq. NaHCO,. 3-(3,4-Dimethoxyphenyl)-propylene 1,2-0xide distilled at 110-1 15"/0.5 mm Hg. The product polymerized after some weeks. 3-(3,4-DimethoxyphenyZ)propane-1,2-diol(XIV) was prepared by stirring a small amount of the oxide in 2 M HC1 at approx. 50" for 72 h. G.1.c.-mass spectrometry of the aqueous phase showed that the diol was present. T h e reaction mixture was used as a reference standard. 3-(3,4-Dimethoxyphenyl)p~opun-l-01 (XII) was prepared by reduction of 3,4dimethoxyphenylpropionic acid methyl ester. 3,4-Dimethoxyphenylpropionic
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acid (0.5 g) was methylated with diazomethane in diethyl ether. The reaction mixture was treated with LiAlH, (1 g) in small portions and refluxed for approx. 2 h. The solution was acidified with 2 M HC1 and extracted with ether. The ether extract was dried and the ether evaporated. G.1.c.-mass spectrometry showed that the product was chromatographically pure. 3-(4-Hydroxy-3-methoxyphenyl)-2-propen1-01 [XI) was prepared by reduction of 4-hydroxy-3-methoxycinnamicacid methyl ester with LiAlH,. 4-Hydroxy3-methoxycinnamic acid (1.5 g) was methylated with diazomethane in ether. The reaction mixture was treated cautiously with a suspension of LiAlH, in ether at approx. 0" and then refluxed for 12 h. The solution was hydrolysed with 2 M HCl and extracted with ether. The ether extract was dried and the ether evaporated. G.1.c.-mass spectrometry showed the product (approx. 50%) together with the methyl ester and the saturated alcohol. The reaction mixture was used as a reference standard. 3-f4-Hydroxy-3-methoxyphenyl)propionic acid (XXVII) was available from an earlier investigation (Scheline, 1968). 3-(3,4-DimethoxyphenyZ)propionic acid (XXV) (m.p. 97-98', lit. 98-99') was prepared from 3,4-dimethoxycinnamic acid by the same method as used for XXVII.
Animal experiments Male albino rats (Wistar strain derived) weighing 250-300 g were used. The animals were treated as described previously (Solheim & Scheline, 1973) except that 200 mg/kg doses were used in the quantitative experhents. Extraction of metabolites The urine and bile samples were treated with Glusulase (Endo Laboratories, Inc., Garden City, N.Y.) and extracted with ether according to the method described previously (Solheim & Scheline, 1973). Quantitative determination of metabolites p-Methoxybenzoic acid (2 mg) was used as internal standard and was added to the thawed urine samples. The urines were treated as described previously (Solheim & Scheline, 1973). Experiments were also carried out to assess the degree of recovery of the major metabolites in comparison with that of the internal standard when employing the extraction method described (Solheim & Scheline, 1973). The major metabolites, where reference compound were available, gave the same recovery as the internal standard. I n the cases where no reference compound was available we assumed that the recovery was equal to that of the internal standard. Major metabolites are arbitrarily defined as those accounting for 1oh or more of the dose and no attempts were made to quantitatively determine the remaining metabolites. Incubation with caecal extracts Incubation of the test compounds under anaerobic conditions with rat caecal niicro-organisms was carried out as described previously (Scheline, 1966, 1968).
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Gas chromatography An F & M Model 402 gas chromatograph equipped with flame ionization detector was employed for analytical and quantitative analysis. The glass columns were 0.3 (int. diam.) x 145 cm and were packed with 3% OV-17 on Gas Chrom Q 80-100 mesh. The column temperature was programmed from 110 to 260" at 5"/min and the injector and detector temperature were maintained at 260". Argon (45 ml/min) was employed as carrier gas. Mass spectrometry The mass spectra were obtained with a Varian MAT 111 g.1.c.-mass spectrometi y system. All compounds were introduced into the ionization chamber through the g.1.c. inlet and spectra were taken at 80 eV at a scan speed of 100 masses/sec. The glass column used was 0.2 (int. diam.) x 150 cm packed with 3% OV-17 on Gas Chrom Q 100-120 mesh. The column temperature was programmed from 110 to 260" at 6"/min and the injector and separator temperature were maintained at 260". Helium (15 ml/min) was employed as carrier gas. The high resolution mass spectra were obtained with an AEI MS902 mass spectrometer equipped with a DS 30/64/HL data system.
Results The retention times and the relative abundance of M+ and the most prominent fragments of the reference compounds and metabolites are listed in Table 1. With metabolites for which no reference compound was available, identification is based upon the fragmentation pattern in the mass spectrum and also upon comparisons with mass spectt a of authentic compounds containing similar substituents. Urinary metabolites The metabolites detected in the urine of rats given 3,4-dimethoxyallylbenzene (I), 3,4-dimethoxypropenylbenzene (11) and 3,4-dimethoxycinnamic acid (XXVIII) are shown in Table 2. No unchanged starting compound was found in the urine samples of rats given I or 11. 3,4-Dimethoxyacetophenone (XVI) was detected in urine from animals dosed with 3 4-dimethoxyallylbenzene (I), 3,4-dimethoxypropenylbenzene(11) and 3,4-dimethoxycinnamic acid (XVIII). In a previous paper (Solheim & Scheline, 1973) we found that the related monomethoxy ketone is an artefact and we assumed that the precursor was the corresponding 8-keto acid. This appears to be the case with the dimethoxy compounds as well. As with the p-methoxy derivatives (Solheim & Scheline, 1973) we were not able to detect the tertiary amino-3,4-dimethoxyphenylpropiophenone derivatives described by Oswald et al. (1971 a) although the decomposition product of these compounds, 3,4-dimethoxyphenyl vinyl ketone (XVIII) was detected in both the basic and acidic fractions. Table 3 lists the major urinary metabolites of 3,4-dimethoxyallylbenzene (I) and 3,4-dimethoxypropenylbenzene (11) and the amounts excreted within 24 h. Some of these metabolites were detected in trace amounts only, after 24 h.
Table 2. Urinary metabolites of 3,Cdimethoxyallylbenzene, 3,Cdimethoxypropenylbenzene and 3,4dimethoxycinnamic acid in the rat Compounds given orally or by intraperitonealinjection at doses of 100 or 400 mg/kg. Compound administered
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3,4-Dimethoxyallylbenzene(I)
3,4-Dimethoxypropenylbenzene (11)
3,4Dimethoxycinnamic acid (XXVIII)
3-Hydroxy-4-methoxyallylbenzene(I I I) 4-Hydroxy-3-methoxyallylbenzene(IV) 3-Hydroxy-4-methoxypropenylbenzene (V) 4-Hydroxy-3-methoxypropenylbenzene (VI) 1-(3,4-Dihydroxyphenyl)propane* (VII) VI I 2-Hydroxy-4,5-dimethoxyallylbenzene (VIII) 1-(3,4-Dimethoxyphenyl)-2-propen-1-01 (IX) 3-(3,4-Dimethoxyphenyl)-2-propen-1-01 X (XI 3-(4-Hydroxy-3-methoxyphenyl)-2propen-l-ol*t(XI) 3-(3,4-Dimethoxyphenyl)propan-l-ol*t (tr) (XII) . 3-(3,4-Dimethoxyphenyl)propane-1,2diol* (tr) (XIV) 1-(3,4-Dimethoxyphenyl)propane-l , 2-diol*(tr) (XV) 3,4-Dimethoxyphenylmethyl ketone XVI XVI (XVU 4-Hydroxy-3-methoxyphenylmethyl ketone*t (tr) (XVII) 1-(3,4-Dimethoxyphenyl)-2-propen-1one* (tr) (XVIII) 4-Hydroxy-3-methoxyphenylacetone*~ (tr) (XIX) 3,4-Dimethoxybenzaldehyde* (tr) (XX) 3-4-Dimethoxyphenylacetaldehyde* (tr) M I ) XXII XXI I 3,4-Dimethoxybenzoicacid (XXII) 4-Hydroxy-3-methoxybenzoic acid*? (XXIII) 3,4-Dimethoxyphenylaceticacid XXIV XXIV WIV) 3-(3,4-Dimethoxyphenyl)propionic XXV XXV acid (XXV) 4-Hydroxy-3-methoxycinnamic acid XXVI XXVI (XXVI) 4-Hydroxy-3-methoxyphenylpropionic XXVII acid* (tr) (XXVII) XXVIII 3,4-Dimethoxycinnamicacid (XXVIII) XXVIII 2-Hydroxy-3-(3,4-dimethoxyphenyl)propionic acid (XXIX) 3-Hydroxy-3-(3,4-dimethoxyphenyl)xxx propionic acid (XXX) xxx 2-Keto-3-(3,4-dimethoxyphenyl)propionic acid* (tr) (XXXI) 3,4-Dimethoxybenzoylglycine -11) XXXI I XXXI I 3,4-Dimethoxycinnamoylglycine XXXIII XXXIII (XXXIII)
* Detected only when 400mg/kg was given. t Detected only after intraperitoneal dose. (tr) Trace.
Hydrolysed None
0 0 0
4(3.1-5.3) 7 (4.7-9.3) 0
0
0
0
~~~
0 0 0
0
0 0-5 ( 0 . 4 4 6 )
0 0 0
8 (5.0-10.0)
0
5 (34-7.0)
0.5 (0.3-0.6)
0
0
Hydrolysed
Intraperitoneal
3,4-Dimethoxypropenylbenzene
Hydrolysed None
Oral
3,4-DimethoxypropenyIbenzene
Hydrolysed None
Intraperitoneal
3,4-Dimethoxyallylbenzene
*
84
95
63
88
53
T h e amounts found in the hydrolysed urines were 11% and 1 0 % respectively.
Recovery as major urinary metabolites
~I
55
For further details see text.
79
90
. o 0.5 ( 0 . 3 4 6 ) 0 0.5 (0.3-0.6) bdnzene (VI) 0 0 0 0 3,4-Dimethoxybenzoic acid (XXII) 2 (0.5-3.1) 2 (1.0-3.0) 2 (0.6-3.0) 1 (0-8-2.0) 1 (0.5-1.1) 2 (1.5-2.5) 1 (0.9-2.2) 3 (2.3-3.2) 2-Hvdroxv-4.5-dimethoxvallvlbekzend (VIII) 2 (0.8-2.1) 1 (0.5-1.3) 2 (1.0-3.8) 0 0 0 0 0 3,4-Dimethoxyphenylacetic acid 0 0 0 0 3 (1.6-3.3) 2 (1.7-2'4) 2 (0.6-2.4) 3 (1.2-3.8) (XXIV) 3 4 3,4-Dimethoxyphenyl)propionic acid (XXV) 0 0 1 (0.5-1.9) 1 (0.5-1.8) 2 (0.8-3.9) 1 (0.8-1.9) 0 0 4-Hydroxy-3-methoxycinnamic 0 7 (4.6-10.6) 27 (22.6-34.8) 10 (6.0-15.0) 34 (26-0-38.0) 0 acid (XXVI) 0 0 2-Hydroxy-3-(3,4-dimethoxyphenyl)0 0 0 0 propionic acid (XXIX) 20 (12'7-25.8) 20' 14 (124-21-0) 14' 3,4-Dimethoxycinnamic. acid (XXVI I I) 2 (0.9-3.6) 2 (0.7-3.2) 3 (2.8-5.8) 5 (3'64.8) 1 (04-1.6) 4 (3.145) 2 (1.5-2.2) 5 (3.8-5.1) 3-Hydroxy-3-( 3,4-dimethoxyphenyl)propionic acid (XXX) 2 (0'6-2.8) 2 (1.0-2.5) 5 (34-7.1) 4 (2.0-5.7) 4 (24-5.4) 4 (2.7-9'0) 6 (3.5-9.4) 9 (5.0-12.0) 3,4-Dimethoxybenzoylglycine (-11) 32 (27.0-35.7) 30 (18-6-37.6) 7 (5.9-10.0) 15 (10.0-22.0) 15 (12.0-17.9) 13 (8-0-16.7) 13 (10.0-24.0) 15 (9.1-20.0) 3,4-Dmethoxycinnamoylglycine (XXXIII) 22 (15.0-26.0) 24 (19.6-27.4) 28.1 33 (27.0-44.0) 24 (17.5-29.0) 26 (18.0-32.0) 21 (140-26.0) 21 (18-0-22.0) (23.0-30.0)
4-Hvdroxv-3-methnxvnmn~nvl, - ~ ~ - - - - - -_- .-C - - r - - - , -
Metabolite : 3-Hydroxy-4-methoxyallylbenzene (111) 4-Hydroxy-3-methoxyallylbenzene ( W 3-Hydroxy-4-methoxypropenylbenzene (V)
None
Oral
Route of administration
Treatment of urine
3,4- Dimethoxyallylbenzene
Compound administered
Table 3. Major urinary metabolites of 3,44imethoxyallylbenzene and 3,4-dimethoxypropenylbenzene in the rat Each animal received a dose of 200 mg/kg. Urine samples were collected over a period of 24 h. Results are average values from four or five animals, as % dose with ranges in parentheses.
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CI
P P
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-
Some of the metabolites or their conjugates were unstable to acid. Extraction at p H 1 resulted in reductions in the detectable amounts of' these metabolites and destruction was especially pronounced with l-(3,4-dimethoxyphenyl)allyl alcohol (IX). Maximal amounts of I X were obtained when extraction was carried out at approx. p H 5 . We estimated the amount excreted in the urine to Acid lability was also seen with the conjugates of 4-hydroxybe approx. 1-2%. 3-methoxy- and 4-methoxy-3-hydroxyallyl and -propenyl benzenes. Extraction at approx. p H 5 was carried out to determine these phenol derivatives in the unhydrolysed urines. One of the major metabolites, 2-hydroxy-3-(3,4-dimethoxyphenyl)propionic acid (XXIX) was unstable to p-glucuronidase .hydrolysis. I n the hydrolysed urines the amount of this acid was less than in the unhydrolysed urines. However, the amount in the hydrolysed urine samples must obviously be at least as large as that in the unhydrolysed samples and the latter value is therefore used in Table 3. Furthermore, the quantitative determination of the other hydroxy acid, 3-hydroxy-3-(3,4-dimethoxyphenyl)propionicacid (XXX), indicates that Table 4. Biliary metabolites of 3,4-dimethoxyallylbenzeneand 3,4-dimethoxypropenylbenzene in the rat Compound given orally or by intraperitoneal injection at a dose of 400 mg/kg. Bile samples
+
treated with 8-glucuronidase sulphatase before analysis. Compound administered 3,4-Dimethoxyallylbenzene(I)
3,4-Dimethoxypropenylbenzene(11)
3-Hydroxy-4-methoxyallylbenzene(I)* (111) 4-Hydroxy-3-methoxyallylbenzene (I)(IV) 3-Hydroxy-4-methoxypropenylbenzene
($1(v)
4-Hydroxy-3 -methoxypropenylbenzene (1)(VI)
2-Hydroxy-4,5-Dimethoxyallylbenzene(*) (VI 11) 1-(3,4-Dimethoxyphenyl)-2-propen-I -01 ($) (IX) 3-(3,4-Dimethoxyphenyl)-2-propen-l-ol(t)(X) 3-( 3,4-Dimethoxyphenyl)propane-1,2-diol(t ) (XIV) 3,4-Dimethoxybenzaldehyde(*) (XX) 3,4-Dimethoxybenzaldehyde(*) (XX) 3,4-Dimethoxyphenylacetaldehyde (*) (XXI) 1-(3,4-Dimethoxyphenyl)-2-propen1-one (t) (XVI I I) 3,4-Dimethoxybenzoic acid (*) (XXII) 3,4-Dimethoxybenzoic acid (*) (XXII) 3-(3,4-Dimethoxyphenyl)propionicacid (*) (XXV) (3,4-Dimethoxyphenyl)propionicacid(*) (XXV) 3,4-Dimethoxycinnamic acid (t)(XXVIII) 3,4-Dimethoxycinnamic acid (5) (XXVIII) 2-Hydroxy-3-( 3,4-dimethoxyphenyl)propionic acid (I)(XXIX) 2-Keto-3-(3,4-dimethoxyphenyl)propionic acid (*) (XXXI) 3,4-Dimethoxybenzoylglycine(*) (XXXII) 3,4-Dimethoxycinnamoylglycine(*) (XXXII I) 3,4-Dimethoxycinnamoylglycine ($1 W X W Relative amounts of metabolites detected: (*) small, (t)moderate,
(I)large, ($) very large.
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it is excreted unchanged in the urine and it is therefore likely that this is also the case with the other hydroxy acid. When the rats were switched to a purified diet containg 1% neomycin before dosing, 1-(3,4-dihydroxyphenyl)propane (VII) could not be detected in the urine as a metabolite of 3,4-dimethoxyallyl or -propenyl benzene. No other significant alteration in the pattern of metabolism of 3,4-dimethoxyallylbenzene or 3,4-dimethoxypropenylbenzene was found. 1-(3,4-Dihydroxyphenyl)propane (VII) was detected in the extracts of incubates of 3,4-dimethoxyallyl or -propenyl benzene with rat caecal microorganisms.
Biliary metabolites The metabolites detected in the bile of rats given 3,4-dimethoxyallylbenzene (I) and 3,4-dimethoxypropenylbenzene (11) are shown in Table 4 and are the metabolites consistently found in 4 experiments. The designations of the relative amounts of the biliary metabolites refer to the results obtained in a single experiment with each compound. However, both the numbers of metabolites and the total amount excreted were greater with the ally1 derivative than with the propenyl derivative. .
Discussion The proposed metabolic pathways of 3,4-dimethoxyallylbenzene (I) and 3,4-dimethoxypropenylbenzene(11) in the rat are shown in Figs. 1 and 2. As expected the major metabolic reactions of 3,4-dimethoxyallyl- (I) and 3,4-dimethoxypropenyl-benzene(11) follow the same pathways as we proposed for the corresponding monomethoxy derivatives [Solheim 8z Scheline, 1973)
Fig. 1 . Proposed metabolic pathways of 3,4-dimethoxyallylbezenein the rat. The numbers refer to the compounds listed in Table 1. Compounds in brackets have not been detected. Major and minor metabolic routes are indicated by thick and thin arrows, respectively, and artefact formation is indicated by broken arrows.
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coon
C
Fig. 2. Proposed metabolic pathways of 3,4-dimethoxypropenylbenzenein the rat. The numbers refer to the compounds listed in Table 1. Compounds in brackets have not been detected. Major and minor metabolic routes are indicated by thick and thin arrows, respectively, and artefact formation is indicated by broken arrows.
with some exceptions. The quantitative results show that 0-demethylation is not as important with the dimethoxy derivatives as with the monomethoxy derivatives. Urine of rats dosed with 3,4-dimethoxyallylbenzene (I) contained a metabolite with a mass spectrum indicating that hydroxplation of the benzene ring had occur1ed. When elemicin (3,4,5-trimethoxyallylbenzene)was partially demethylated by treatment with pyridine hydrochloride, none of the reaction products showed the same mass spectrum and retention time as given by the metabolite (unpublished results). This indicates that the 2- or 6-position rather than the 5-position, has been hydroxylated. 2-Hydroxylation will give 2-hydroxy-3,4-dimethoxyallylbenzenewhile 6-hydroxylation will give 2-hydroxy4,5-dimethoxyallylbenzene (VIII). Steric considerations suggest that VIII is more favourable. 3,4-Dihydroxypropylbenzene (VII) is a urinary metabolite of both 3,4-dimethoxyallyl- and propenyl-benzene. However, in animals given neomycin before dosing, VII was not detected in the urine. This compound was also found in incubates of rat caecal micro-organisms with 3,4-dimethoxyallyl- or propenyl-benzene. These findings indicate that VII is a metabolite formed by the rat intestinal microflora. The route via the cinnamoyl derivatives is a major metabolic pathway for both compounds. However, compared with the two monomethoxy allyl- and propenyl-benzenes (Solheim & Scheline, 1973) we detected several new types of metabolites. As with the monomethoxy derivatives, the cinnamic acid (XXVIII) was partly converted to the p-hydroxy acid (XXX) and further to the dimethoxybenzoylglycine. However, the 3,4-dimethoxycinnamic acid was
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also converted to 4-hydroxy-3-methoxycinnamicacid (XXVI), 3-(3,4-dimethoxypheny1)propionic acid (XXV) and 3,4-dimethoxycinnamoylglycine(XXXIII). Cinnamic acid-glycine conjugates have been described earlier both by Dakin (1909) who found that cinnamoylglycine is a minor metabolite of cinnamic acid in dogs and Armstrong, Shaw & Wall (1956) who found that feruloylglycine is a metabolite of ferulic acid (4-hydroxy-3-methoxycinnamic acid) in man. Furthermore, we found that 4-hydroxy-3-methoxycinnamylalcohol (XI) is a metabolite of 3,4-dimethoxypropenylbenzene (11) and that 3-(3,4-dimethoxypheny1)propan-1-01 (XII) is a metabolite of 3,4-dimethoxyallylbenzene(I), both formed via the cinnamoyl pathway. 3-@-Methoxyphenyl)propylene 1,Z-oxide is a biliary metabolite of R-methoxyallylbenzene and the related 1,2-diol is excreted in both the urine and bile (Solheim & Scheline, 1973). The corresponding epoxide (XIII) was not detected in the present experiments with 3,4-dimethoxyallylbenzene(I) but a diol (XIV) was again found. Stillwell et al. (1974) found that both safrole epoxide and diol are metabolites of safrole (3,4-methylenedioxyallylbenzene). Furthermore, we found in the urine and bile a compound which gave a mass spectrum indicating an a-keto acid (XXXI). This acid could arise by further oxidation of the a-hydroxy acid (XXIX) formed via the epoxide-diol pathway. As with p-methoxyallylbenzene we found that hydroxylation of the allyl group occurred, leading to the formation of 1-(3,4-dirnethoxyphenyl)-Z-propen-l-ol (IX). Borchert et al. (1973 a) showed that 1-(3,4-methylenedioxypheny1)-2propen-1-01 is a metabolite of safrole in the rat, and that pre-treatment of rats with phenobarbital increased about 10-fold the excretion of this allyl alcohol after a dose of safrole. We estimated the amount of allyl alcohol (IX) excreted in the urine to be about 1-2y0 of the dose. Oxidation of I X will give rise to the corresponding ketone (XVIII) and this was detected in both bile and urine. However, we were not able to detect the tertiary aminopropiophenone metabolites corresponding to those found by Oswald et al. (1971 b) as metabolites from safrole f3,4-methylenedioxyallylbenzene) or myristicin (3-methoxy-4,Smethylenedioxyallylbenzene). Braun & Kalbhen (1973) have shown that in the isolated perfused liver or during incubation with liver homogenates myristicin is converted to the corresponding amphetamine derivative. Examination of the basic urine fraction from rats given 3,4-dimethoxyallylbenzene revealed no compound which gave a mass spectrum indicating the presence of the dimethoxyamphetamine derivative. As in our previous paper (Solheim & Scheline, 1973), the 3,4-dimethoxyacetophenone (XVI) must be an artefact from decarboxylation of the corresponding j5-keto acid, which was not detected. Small amounts of two other ketones were detected in the urine of rats given 3,4-dimethoxypropenylbenzene (11). 4-Hydroxy-3-methoxyacetophenone (XVII) could similarly be an artefact from decarboxylation of a 4-hydroxy-3-methoxy-/%ketoacid. 4-Hydroxy-3-methoxyphenylacetone (XIX) is a metabolite and could be formed by oxidation and demethylation of the starting compound. As with the monomethoxy derivative (Solheim & Scheline, 1973), 3,4-dimethoxypropenylbenzene [11) is predominantly metabolized via the cinnamoyl pathway. Evidence for this is the finding that the major metabolites are ferulic acid (XXVI) and the two glycine conjugates 3,4-dimethoxybenzoyl-
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glycine (XXXII) and 3,4-dimethoxycinnamoylglycine (XXXIII), which together account for about 70% of the dose. Only small amounts of the diol were detected in urine and bile of rats dosed with 3,4-dimethoxypropenylbenzene(11) indicating that the epoxy-diol pathway is a minor one. However. 3,4dimethoxyallybenzene (I) is extensively metabolized by both the cinnamoyl pathway and the epoxy-diol pathway. The metabolic reaction from 3,4-dimethoxyallylbenzene to 3,4-dimethoxycinnamyl alcohol (X) was not investigated in detail. As shown in Fig. 1 the reaction can go via the allyl alcohol (IX) and Borchert et al. (1973 a) have shown that 1-(3,4-methylenedioxyphenyl)-2-propen-l-o1 can rearrange to 3-(3,4methylenedioxyphenyl)-2-propen-l-ol. Anoth.er possibility is simultaneous o-oxidation and migration of the double bond. The double bond in the allyl benzenes is relatively labile. Migration of this bond in the allyl benzenes has been reported (Mauthner, 1916; Trikojus & White, 1949), and it was also seen during the chemical demethylation of both mono- and dimethoxyallylbenzene in our studies. In the reaction mixture we also found all of the possible propenyl derivatives. But it is unlikely that migration of the double bond alone is the first step because one would then expect to find the demethylated propenyl derivatives in the urine of rats given the allyl benzenes. These were, however, not found either with the mono- or with the dimethoxyallylbenzene. However, in the urine of rats given p-methoxyallylbenzene, approx. 5-10y0 of the dose is converted to the allyl alcohol (Solheim & Scheline, 1973). If the cinnamoyl pathway goes via allyl alcohols only, one would expect this pathway to be more important with the monomethoxy derivative than with dimethoxyallylbenzene where approx. 1-2% of the dose is excreted as the allyl alcohol (IX). But as shown in our previous paper (Solheim & Scheline, 1973) the metabolites of p-methoxyallylbenzene in the cinnamoyl pathway account for only appxox. 14% of the dose while the corresponding figure for dimethoxyallylbenzene (I) is approx. 60%. The present results appear to be best explained by postulating that 3,4-dimethoxycinnamyl alcohol (X) arises mainly via o-oxidation but that some may also be formed by isomerization of the allyl alcohol (IX).
Acknowledgments The authors are indebted to Norges Almenvitenskapelige Forskningsrid and to Norsk Medisinaldepot for grants for the purchase of the gas chromatograph/mass spectrometer used in this study and to the members of the staff of the Chemical Institute, University of Oslo who assisted with the high resolution mass spectrometry. The technical assistance of Mrs. Eli Tepstad, Mrs. Astrid Hetle and Mr. Olav E. Fjellbirkeland is greatly appreciated.
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E. Solheim and R. R. Scheline
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