METHYLATION AND DEMETHYLATION I N RELATION TO THE I N VITRO METABOLISM OF MESCALINE

John Daly, Julius Axelrod, Bernhard Witkop National Institute of Arthritis and Metabolic Diseases and the National Institute of

Mental Health, Public Health Service, Bethesda, Md.

The actions of the hallucinogenic drug mescaline have been explained as due to the formation of an active metabolite, or to the induction of a formation of a hallucinogenic substance, or to be caused by interference with the detoxification of naturally formed hallucinogen^.^-^ In vivo studies on the metabolism of mescaline have shown that a large part of the mescaline combines with liver protein3 and that varying amounts are excreted unchanged or as the oxidatively deaminated product, 3,4,5-trimethoxyphenylacetic acid.2 z4-7 Certain minor metabolites have also been identified. Harley-Mason2 has isolated 3,4- dihydroxy-5-methoxyphenylacetic acid from human urine after the ingestion of mescaline, while Ratcliffe and Smith8 have found 3,4-dimethoxy-S-hydroxy- phenethylamine (111) as a minor metabolite of mescaline in humans. Gold- stein et uL9 have reported 3 ,4 , 5-trimethoxyphenylethanol as a mescaline metabolite in rats.

In vitro studies with rabbit-liver preparations'O have shown that mescaline is oxidatively deaminated to 3,4,5-trimethoxyphenylacetic acid. Axelrod'l has reported that mescaline undergoes 0-demethylation with rabbit-liver prepara- tions.

Since a thorough study of the metabolism of mescaline and closely related compounds could be expected to yield information that might help to elucidate the mechanism of its action, the in vitro demethylation of mescaline and studies on the in vitro methylation of its demethylated congeners were undertaken.

Materials Mescaline hydrochloride,* me~caline-8-C~~ hydrochloride, f and 3,4,5-tri-

methoxyphenylacetic acid1 were used. Other materials were: 3 ,Pdihydroxy- 5-methoxyphenethylamine hydrochloride (V), prepared as described by Ben- ington et al.,'2 3,5-dimethoxy-4-hydroxyphenethylamine hydrochloride (11) and 3,4-dimethoxy-5-hydroxyphenethylamine (111) , prepared as described by Ratcliffe and Smith,8 3,5-dihydroxy-4-methoxyphenethylamine hydrochloride (IV) 5 and 3,4,5-trihydroxyphenethylamine hydrochloride (VI) , prepared from mescaline hydrochloride or sulfate as described by Hahn,13 as was 3,4,5- trihydroxyphenethylamine-8-C14 hydrochloride from mescaline-8-C14.

Resulhs Enzymatic 0-methylation

by the action of catechol-O-methyltransferasel* of 3 ,4 , S-trihydroxyphenethyla- Enzymatic 0-methylation of mescaline congeners.

* Obtained from Hoffman-La Roche Inc., Nutley, N.J. t Obtained from New England Nuclear Corporation, Boston, Mass.

Obtained from Aldrich Chemical Corporation, New York, N.Y. Synthesized at Sterling-Winthrop Research Institute, Rensselaer, N.Y., and made avail-

able t o us through the courtesy of Sidney Archer.

37

38 Annals New York Academy of Sciences mine (VI), 3,4-dihydroxy-5-methoxyphenethylamine (V) 3 ) 5-dihydroxy-4- methoxyphenethylamine (IV), 3 I 4-dimethoxy-5-hydroxyphenethylamine (III), and 3 ) 5-dimethoxy-4-hydroxyphenethylamine (TI) was carried out by in- cubating at 37" C. for 1.5 hours under nitrogen) the hydrochloride of the amine (5 pmole) dissolved in 0.5 ml. of water with 2 ml. of the soluble supernatant fraction of rat liver, 1 ml. of 0.5 M phosphate buffer, pH 7.9, 5 pmole of S-

Yellow Brown Purple Brown

Brown

Brown

SOLVENT FRONT ->

0 Blue

Blue

0 I 2 3 4

Yellow Brown Purple Green Brown Blue

5 6 Blue Pole Red

FIGURE 1. Paper chromatography (BuOH-HOAC-H~O, 4: 1 : 5 , color with Gibb's reagent) ol products from enzymatic methylation of ( b ) 3,4,5-trihydroxy-phenethylamine and (d) 3 ,Pdihydroxy-5-rnethoxyphenethylamine; (a) and (c) are controls run without S-adenosyl methionine; and ( e ) is reference mixture containing (1) 3,4,5-?'riOH, (2) 3 ,4-DiOH-5-OCHs , (3) 3,5-DiOH-4-OCHx, (4) 3,5-DiOCH3-4-OH, (5) 3 ,4-DiOCH3-5-OH, and (6) 3,4,5- TriOCH3 (mescaline).

adenosylmethioninc, and 0.1 ml. of 0.5 M magnesium chloride. Control iiicubations were carried out in which no S-adenosylmethionine was present.

After incubation] the reaction mixture was acidified with 1 ml. of 2 N hy- drochloric acid and centrifuged to remove the precipitated protein. The decanted solution was lyophilized and the residue triturated with 2.5 ml. of hot ethanol. The ethanol was removed from the extract in vucuo and the residue was chromatographed after trituration with a minimal amount of methanol. A variety of solvent systems was employed. However, only butanol-acetic acid- water (4:l:S) on Whatman No. 1 filter paper allowed separation of all the mescaline congeners (I to VI, see FIGURE 1). Recovery of the compounds T to VT after incubation was ascertained to be satisfactory through an experiment

Daly et al.: In Vitro Metabolism of Mescaline 39

in which all the amine hydrochlorides I to V I were added to an incubation mixture containing no S-adenosylmethionine.

Enzymatic 0-methylation of 3,4,5-trihydroxyphenethylamine (VI) was shown by paper chromatography to result in the formation of three products that were not formed in the absence of S-adenosylmethionine (FIGURE 1). The three products were identified by Rf values and color reactions as 3,4-di- methoxy-5-hydroxyphenethylamine (111, Gibb’s reagent [dichloroquinone- chlorimide] --+ blue15), 3 , 4-dinydroxy-5-methoxyphenethylamine (V, Gibb’s reagent -+ brown, yellow fluorescence under ultraviolet light) and the major product 3 ,S-dihydroxy-4-methoxyphenethylamine (IV, Gibb’s reagent -+ purple).

The catecholamine, 3,4-dihydroxy-S-methoxyphenethylarnine (V) on in- cubation with 0-methyltransferase gave rise to 3,4-dimethoxy-5-hydroxy- phenethylamine (111) that was identified by Rf values and color reactions

I

+SOLVENT

FIGURE 2. Radioactivity scan of paper chromatogram of amines from methylation of C14-3,4,5-trihydroxyphenethylamine in v i l ra (solvent BuOH-HOAC-H~O 4: 1 : S ) .

(Gibb’s reagent + blue). The other possible product of 0-methylation, 3 ,s- dimethoxy-4-hydroxyphenethylamine (11) could not be detected (see FIGURE 2).

The phenolic amines 11, 111, and IV, as expected, did not serve as substrates for catechol-0-methyltransferase.

The enzymatic 0-methylation was also carried out with 3,4, S-trihydroxy- phenethylamine-8-CI4 (VI, 5 Fmole, 160,000 cpm) by the same technique. The final extract was cochromatographed with a mixture of mescaline and con- geners ( I to VI). Development of the chromatograms and scanning for radio- activity in a strip counter revealed three radioactive peaks in addition to the peak due to 3 ,4,5-trihydroxyphenethylamine-8-C1* (see FIGURE 2). The positions of the radioactive peaks coincided with the position of cochromato- graphed 3,4-dihydroxy- 5-methoxyphenethylamine (V), 3,5-dihydroxy-4- methoxyphenethylamine (IV) , and 3,4-dimethoxy-5-hydroxyphenethylamine (111). From the radioactivity scan (FIGURE 2 ) the major 0-methylation product is seen to be the 4-0-methylated amine (IV).

Preliminary investigation of the 0-demethylation of mescaline was carried out as described by Axelrod.’l The formaldehyde formed by oxidative demethylation served with Nash reagentI6 as a measure of enzymatic 0-demethylation. The greater enzymatic activity

Enzymatic 0-demethylation of mescaline.

40 Annals New York Academy of Sciences observed with rabbit-liver preparations compared to those from rat or guinea pig liver prompted the use of a preparation of rabbit-liver microsomes and supernatant fraction" for studies on the 0-demethylation of mescaline-8-CI4 hydrochloride.

Enzymatic 0-demethylation of mescaline-8-C14 hydrochloride was carried out by incubating the mescaline hydrochloride (2.5 pmole, 53,000 cpm) in 0.5 ml. of water with 1 milliliter of a rabbit-liver preparation (300 mg. of liver per milliliter of isotonic potassium chloride) of microsomes and supernatant frac- tion,ll 1.0 ml. of phosphate buffer, 0.1 M pH 7.4, 0.1 ml. of 0.5 M magnesium chloride, 0.2 ml. of a solution containing 2 mg. of glucose-6-phosphate and 2 mg. of triphosphopyridine nucleotide (TPN) and 0.1 ml. of 1 M nicotinamide at 37" C. for 1.5 hours.

After incubation, 0.5 ml. of 2 N hydrochloric acid was added, the precipitated protein removed by centrifugation, and the decanted solution lyophilized.

TABLE 1 RELATIVE AMOUNTS 0-DEMETIIYLATION AND OXIDATIVE DEAMINATION OF

MESCALINE-8-C'4 HYDROCHLORIDE*

,4,5-Trimethoxy- phenylacetic acid

0

Conditions

(1) Enzyme denatured

(2) No cofactors

(3) Nicotinamide and TPN

(3) +Saturated n-Octanol

(3) + Iproniazid (3) + l O P Semicarbizide

__- - ____

0-Demethylation products

0

Mescaline

87

46

35

10

12

100

13

46

61

86

83

0

8

4

4

8

* Results expressed as per cent of total radioactive products isolated.

The residue was extracted with 2.5 ml. of hot ethanol, and the extract taken to dryness in vacuo. The residue was taken up in 2 ml. of 0.1 N hydrochloric acid and extracted 3 times with 1-ml. portions of ethyl acetate. The 0.1 N hy- drochloric acid will be referred to as the amine fraction and the ethyl acetate extract as the acidic fraction. Both fractions were taken to dryness in vucuo and the residues triturated with a minimal amount of methanol. The amine fraction was then cochromatographed with a mixture of authentic amines I to VT, and the acid fraction was cochromatographed with 3 14 ,S-trimethoxy- phenylacetic acid, both on Whatman No. 1 filter paper with butanol-acetic acid-water (4: 1 : 5).

After denaturation of the enzyme by heating for 10 min. in a boiling water bath (before the introduction of the substrate) only one radioactive peak, which corresponded in position to mescaline, was detected (amine fraction). The acidic fraction showed no radioactivity.

In the absence of the cofactors, nicotinamide, glucosed-phosphate and TPN, dmost all of the mescaline was metabolized (see TABLE l), but no other peaks

Daly et al.: In Vitru Metabolism of Mescaline 41 were detected in the amine fraction. The acidic fraction contained a large radioactive peak (Rf 0.80) that was identical in position to the cochromato- graphed 3,4,5-trimethoxyphenylacetic acid.

When the incubation mixture contained all cofactors as described above, the amine fraction was found to contain three radioactive peaks, which coincided with the positions of mescaline (I), 3,4-dimethoxy-5-hydroxyphenethylamine (111) and 3,5-dimethoxy-4-hydroxyphenethylamine (11). Both I1 and I11 were formed in small amounts and their relative activity is presented together in TABLE 1. The 5-hydroxy isomer (111) predominated in a ratio of 1.5-2 to 1 (FIGURE 3). The acidic fraction contained 3,4,5-trimethoxyphenylacetic acid, but its formation appeared to be inhibited by the addition of nicotinamide and TPN. The formation of 3,4,5-trimethoxyphenylacetic acid was strongly inhibited by the presence of either lop3 M iproniazid (monoamine- and diamine- oxidase inhibitor) or M semicarbazide (diamineoxidase inhibitor). The saturation of the reaction mixture with n-octanol (monoamineoxidase inhibitor)

FIGURE 3. Radioactivity scan of paper chromatogram of amine fraction from demethyla- tion of Clc-mescaline irt vitro (solvent BuOH-HOAc.H20 4: 1: 5 ) .

had a lesser effect on the formation of 3,4,5-trimethoxyphenylacetic acid.” Iproniazid and n-octanol also inhibited the demethylation of mescaline. The relative amounts of products formed under various conditions are presented in TABLE 1.

Discussion The results of enzymatic 0-methylation of the 0-demethyl congeners of

mescaline show that only 3,4,5-trihydroxyphenethylamine (VI) and 3,4- dihydroxy-5-methoxyphenethylamine (V) serve as substrates for the enzyme. As shown in FIGURE 4, VI is converted to IV, V and 111, while V is converted only to 111. These results may be compared to those of Booth et U Z . ~ ~ who showed that gallic acid was converted in vivo to the 4-0-methyl isomer and that 3-0-methylgallic acid was methylated in vivo to 3,4-di-O-methylgallic acid. That the middle phenolic group is not always favored for methylatioii was shown by Archer,lY who found that in vitro pyrogallol was converted to the 1-0-methyl- and then to the 1,2-di-O-methyl ether.

The oxidative deamination of mescaline by the diamine oxidase of rabbit liver-preparations as described by ZelleP was confirmed using radioactive mescaline and the product identified as 3,4,5-trihydroxyphenylacetic acid by

42 paper chromatography. In addition the enzyme was shown to be inhibited by the addition of nicotinamide and TPN.

With the microsomal enzyme preparation from rabbit liver, the demethyla- tion of mescaline was found to occur with the formation of small amounts of 3,4-dimethoxy-5-hydroxyphenethylamine (111), previously reported by Kat-

Annals New York Academy of Sciences

C H ~ O ~ C H ~ H Z N H Z

CH3O

OCH3

OCH3

0-METHY L- TRANSFERASE

OH FIGURE 4. Enzymatic methylation and demethylation of mescaline and related com-

pounds.

cliffe and SmithX as a metabolite of mescaline in humans and 3,5-dimethoxy-4- hydroxyphenethylamine.

Further transformations of mescaline and its congeners are under investiga- tion.

Summary (1) The enzymatic 0-methylation of 3 , 4 ,S-trihydroxyphenethylamine with

catechol 0-methyltransferase (rat) gives rise to 3,5-dihydroxy-4-methoxy-

Daly et nl. : In Vitro Metabolism of Mescaline 43 phenethylamine and small amounts of 3,4-dihydroxy-5-methoxyphenethyl- amine and 3 4-dimethoxy-5-hydroxyphenethylamine. Enzymatic O-methyla- tion of 3 4-dihydroxy-5-methoxyphenethylamine forms 3,4-dimethoxy-5-hy- droxyphenethylamine.

(2) Enzymatic 0-methylation of me~caline-8-C~~ hydrochloride using a preparation of microsomes and supernatant fraction (rabbit) forms small amounts of 3 4-dimethoxy-5-hydroxyphenethylamine and 3,5-dimethoxy-4- hydroxyphenethylamine.

(3) The preparation of microsomes and supernatant fraction (rabbit) oxida- tively deaminated mescaline to 3 4,5-trimethoxyphenylacetic acid. The formation of 3 4,5-trimethoxyphenylacetic acid was inhibited by iproniazid, semicarbazide and by nicotinamide and TPN. Lesser inhibition was observed with n-octanol.

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