Biochemical Pharmacology, 1966, Vol. 15, pp. 645-655. Pergamon Press Ltd., Printed in Great Britain.
`
`METABOLIC STUDIES OF TETRABENAZINE,
`A PSYCHOTROPIC DRUG IN ANIMALS AND MAN
`D. E. SCHWARTZ, H. BRUDERER, J. RIEDER and A. BRossI
`Departments of Experimental Medicine and Chemical Research,
`F. Hoffmann-La Roche and Co. Ltd., Basle, Switzerland.
`(Received 26 July 1965; accepted 7 October 1965)
`
`Abstract-Following administration of tetrabenazine (2-oxo-3-isobutyl-9,10-dimethoxy-
`1,2,3,4,6,7-hexahydro-1 lbH-benzo[a]quinolizine) to animals or man, 9 metabolites of
`the drug were detected in the urine by thin layer chromatography. Five of these com-
`pounds were found unconjugated and 4 conjugated with glucuronic acid.
`The structures of all 5 unconjugated metabolites and of 2 aglucones were established
`either by comparison in thin layer chromatography with synthetic compounds or by
`NMR mass spectroscopy and elemental analysis of metabolites isolated by column
`chromatography on silicic acid.
`The main steps of biological degradation of the drug are:
`reduction of the keto group at C2
`oxidation at position 2' of the isobutyl side chain
`selective ether cleavage at C9, followed by conjugation of the phenols to glucuronic
`acid.
`The same pattern of metabolite formation was observed in the rabbit, in the dog
`and in man. In all three species the glucuronides represent the prevailing form of ex-
`cretion of the drug in the urine.
`A general scheme for the biological degradation of tetrabenazine is suggested.
`A description of the synthesis of several metabolites is given.
`
`1. INTRODUCTION
`THE PHARMACOLOGY of tetrabenazine, 2-oxo-3-isobutyl-9,10-dimethoxy-1,2,3,4,6,
`[a] quinolizine, has been extensively investigated.1 Like
`7-hexahydro-llbH-benzo
`6
`CH30
`
`CH 30
`
`N41
`
`'i' NCz
`4
`
`2
`
`0
`
`2
`CH 2-C
`2'
`JN 11
`
`7 CH3
`
`CH3
`
`3,
`reserpine, tetrabenazine depletes monoamine stores in tissues; unlike this drug,
`however, it acts more specifically on brain stores and does not affect gastric secre-
`tion, intestinal motility or blood pressure.
`The distribution of the drug in animal tissues has been studied in the rabbit2 and,
`using a highly sensitive and specific spectrofluorimetric method 3, in the guinea
`pig4. The present paper deals with the identification and characterization of a number
`of tetrabenazine metabolites; an attempt is made to specify the many biological
`pathways along which these substances are formed.
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`D. E. SCHWARTZ, H. BRUDERER, J. RIEDER and A. BROSS[
`
`2. METHODS
`1. Species investigated and mode of administration
`The drug was administered intraperitoneally to male rabbits (30 mg/kg) and to
`female dogs surgically prepared for bladder catheterization (18 mg/kg) or subcut-
`aneously to man (2 mg/kg) in the form of its methanesulfonate salt. In both animal
`species the urine was collected by catheterization. After urine collection specimens
`were immediately frozen.
`
`2. Extraction of tetrabenazine and its unconjugated metabolites I, 11, III, IV and V
`from urine
`The first 24-hr urine specimen was concentrated to about 1 its volume under
`reduced pressure at +400 and extracted twice with 6 volumes of benzene, an adequate
`amount of sodium sulfate and magnesium oxide mixture (5:1 w/w) being added to
`saturate the aqueous phase and to adjust its pH to 10. The benzene extracts were
`separated, filtered and combined.
`
`3. Isolation of conjugated metabolites of tetrabenazine
`A. Separation of the glucuronides. The aqueous phase left behind by the benzene
`extraction of the free metabolites was stored at +4'. The major portion of sodium
`sulfate which had crystallised was filtered off together with magnesium oxide. The
`pH of the filtrate was adjusted to 4 with glacial acetic acid and the glucuronides were
`isolated according to Kamil, Smith and Williams. 5
`B. Enzymatic cleavage of the glucuronides. The solution containing the glucuronides
`in their acid form was ice-cooled and brought to pH 4-5 with cone. ammonia, and
`buffered with A volume of 0-1 molar sodium acetate. For each 10 ml solution 20,000
`units f-glucuronidase in the form of Glusulase* were added in 3 portions over a
`24-hr period of incubation at 37' .
`C. Extraction of the aglucones VI, VII, VIII and IX. The incubated solution of the
`glucuronides was saturated under agitation with sodium sulfate, magnesium oxide
`was added to raise the pH to -
`10 and the aglucones extracted with 6 volumes of
`benzene.
`
`4. Thin layer chromatographic analysis of tetrabenazine metabolites
`Benzene extracts of the free metabolites (see under 2.2.) or the aglucones (see
`under 2.3.C.) were evaporated to dryness under reduced pressure. For thin layer
`chromatography weighed residues or the reference compounds were dissolved in
`methanol :benzene (1:1) respectively to 0-5 or 0-1 per cent concentration, and 10-20 41
`applied on the chromatogram.
`Chromatography was done on silicic acid chromatoplates (silica gel G, Merck)
`prepared according to Stahl. 6 The following systems were used for development:
`(a) t-amylalcoholf : di-n-butylether : 0-25 % aq. NH 40H (80:7:13)
`(b) chloroform : n-butanol : 2-5 % aq. NH 40H (80:20:0.6)
`(c) toluene : acetone : 25 Yo NH 4OH (50:50:1)
`(d) cyclohexanol saturated with water.
`
`* Glusulase (suc d'Helix pomatia): Industrie biologique franqaise, Gennevilliers, Seine, France.
`t t-Amylalcohol: "Chemische Fabrik Schweizerhalle".
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`Metabolic studies of tetrabenazine
`
`Visualization and identification. Spots were made visible in u.v. light (350-360 mp)
`by spraying the chromatogram with a mercuric acetate reagent (200 mg mercuric
`acetate in 90 ml methanol + 10 ml acetic acid). The chromatoplates were then heated
`at 1100 for 10 min in' a drying oven. Under these conditions tetrabenazine and all
`mentioned metabolites are converted to dehydro-compounds of high molecular
`fluorescence intensity, so that even amounts as small as 0.1 p.g can be detected on the
`chromatogram. Furthermore, the difference in the colour of fluorescence desplayed
`by each compound provides further means of identification (see Table t). While the
`ketonic compounds, tetrabenazine, metabolite III and their corresponding phenolic
`aglucones VI and IX show a clear blue fluorescence, the alcoholic compounds II and
`III and their corresponding phenolic aglucones VII and VIII appear as gray or yellow
`spots. Metabolites TV and V on the other hand are of a darker blue (see Table 1.)
`The phenolic aglucones show in chromatographic system (a) very similar Rf values
`to those of their corresponding methyl ethers: in system (b) however they can easily
`be differentiated from one another. Dibromoquinone chlorimide reagent was also
`used to detect phenols.
`Column chromatography. When larger amounts of the metabolites were needed for
`the purpose of identification, column chromatography on silicic acid was used.
`Columns were packed with silicic acid* according to Marvel and Rands, 7 except that
`instead of chloroform a mixture consisting of equal portions of peroxide frce iso-
`propylethert and chloroform saturated with water were used and the column washed
`with isopropylether saturated with water.
`The benzene extracts from the urine containing the free metabolites the aglu-
`cones respectively (see under 2.2 and 2.3.) were evaporated to dryness. The residue
`left by the extract containing the free metabolites was taken up in the minimum
`quantity of isopropylether-chloroform (1:1):, and put on the top of the column.
`Development of the chromatogram was carried out with isopropylether, isopropyl-
`ether-chloroform, chloroform and chloroform-n-butanol mixtures of increasing
`polarity. All phases were saturated with water. Single fractions were collected, eva-
`porated to dryness and analysed separately by thin layer chromatography (see under
`2.4.). In a typical chromatogram tetrabenazine was eluted with isopropylether-
`chloroform (2:1)
`
`I with isopropylether-chloroform (1:2)
`II with chloroform
`III with chloroform + 2 % n-butanol
`IV with chloroform + 10% n-butanol
`V with chloroform + n-butanol (1:1).
`
`5. Separation of ketonic and non-ketonic material
`The material present in the urine extracts (free metabolites or aglucones) can be
`separated into a ketonic and a non-ketonic fraction, according to Girard8 using
`(Carbazoylmethyl) trimethylammonium chloride.
`* Silicic acid, 100 mesh, Mallinckrodt, anal. reagent.
`t Isopropylether Shell was passed through a column of neutral aluminium oxide activity I the day
`of use.
`In the case of the aglucones the residue was dissolved in chloroform to which was added a few
`drops of methanol.
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`

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`44)
`3-4
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`0~
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`Ru..
`
`4)
`
`4.-
`
`00
`
`D. E. SCHWARTZ, H. BRUDERER, J. Rmio R and A. BROSSI
`
`U
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`Metabolic studies of tetrabenazine
`
`6. Spectra
`(a) Ultra-violet spectra were taken in analytical grade ethanol or ethanol made up
`to contain 0.01 N HCI or NaOH resp.
`(b) NMR spectra were taken in CDCIs using a Varian A-60 NMR-spectrometer.
`(c) Mass-spectra were taken with an MS 9 from AEI, Manchester, England.
`
`7. Oxidation of metabolite IV with aluminium isopropylate
`Eight mg of metabolite IV obtained by column chromatography (see under 2.4.) and
`400 mg aluminium isopropylate were dissolved in 2 ml toluene and 2 ml cyclohex-
`anone and heated in a sealed tube for 36 hr at 140"C. After cooling the reaction
`mixture was extracted with N H 2SO 4. The sulfuric acid extract was then re-extracted
`with benzene after adjusting the pH to 10 with magnesium oxide. The reaction product
`was separated from unchanged material by thin layer chromatography. The proper
`band was eluted by agitation with 2 ml dist. water, 0-5 g of MgO + Na2SO 4 (1:5) and
`20 ml of benzene. The benzene extraction was repeated and the combined benzene
`extracts concentrated under vacuum. The residue (5 mg), a colourless film, was found
`pure when analysed by thin layer chromatography.
`
`3. RESULTS AND DISCUSSION
`1. The direct benzene extract from the urine of tetrabenazine treated animals
`(rabbit, dog or man), when analysed by thin layer chromatography, showed 5 distinct
`fluorescent spots which will be designated here as metabolites I, II, III, IV and V.
`
`Metabolites I and 11
`When this extract was chromatographically analysed, metabolites I and I were
`found to be identical in 4 chromatographic systems with the two synthetic isomers of
`[a] quino-
`2-hydroxy-3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro-1 IbH-benzo
`lizine.
`When I was administered to rabbits or to dogs instead of tetrabenazine, only one
`unconjugated metabolite was formed which corresponded chromatographically to
`metabolite IV of tetrabenazine; similarly following administration of II only one
`unconjugated metabolite was formed which corresponded to metabolite V of tetra-
`benazine (see Fig. I and scheme of tetrabenazine metabolism, on p. 650).
`
`Metabolite IV
`This was isolated, from the urine of dogs which had received I, by column chromato-
`graphy on silicic acid (see under 2.5.). It was found pure when analysed by thin layer
`chromatography. It formed a colourless oil and when dried under high vacuum solidified
`to a slightly coloured film. The substance showed a strong affinity for water. Analysis
`was therefore performed after 48 hr drying at 50' in high vacuum over phosphorus
`pentoxide
`
`C19 H2 9 N 04 + 0.35 mole H2 0
`calc.
`66-77
`8.76
`20.36
`1.84
`
`C
`H
`0
`H 20
`
`(mol. wt. = 341-76)
`found
`67.04
`8.90
`20.07
`2-14
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`D. E. SCHWARTZ, H. BRuDERER, J. REDER and A. BRossi
`
`The correct molecular weight of IV was determined by high resolution mass
`spectroscopy. Due to the high intensity of the peak at m/e 334 (M-H), the measure-
`ment was done on this peak:
`
`found
`calc.
`
`334.2016
`334.2018
`
`-0.0013
`for C19 H28 N 04
`
`The elemental composition of the molecule is
`cular weight = 335.209
`
`therefore C19 H29 N 04 and its mole-
`
`Scheme of tetrabenazine
`metabolism.
`
`V
`
`\II
`
`OH
`
`-
`
`i~ CII~/
`
`KX
`
`-.
`
`zL~ V.l
`6R.
`
`113
`
`ED - unconjugated metabolites
`conjugated metaboltes = aglucones
`of glucuronides
`structure not eslablished
`deduced by analogy
`
`Y
`
`-*'
`
`--
`
`On the basis of these results the presence of two alcoholic groups in the molecule
`must be assumed. One alcoholic group is that already present in metabolite I, the
`metabolic precursor of IV, whereas the position of the second alcoholic group could
`be ascertained by nuclear magnetic resonance. The NMR spectra of metabolite I
`and metabolite II show doublets for the isopropyl groups at 0.85 and 0.92 ppm
`(J = 6"5 c/s), whereas in the case of metabolite IV the doublet is replaced by a single
`band at 1.20 ppm (J = 6.0 cps). One must therefore assume that in metabolite IV
`the second alcoholic group is located in the side chain, as indicated above.
`Similarly metabolite V was isolated by column chromatography from the urine of
`dogs which had received II. Nuclear magnetic resonance of this compound showed a
`single band at 1.30 ppm indicating that in this metabolite also the proton in the iso-
`butyl side chain is replaced by a tertiary alcoholic group.
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`-f
`
`ront
`
`tetrabenazine
`
`-XL
`
`I
`
`start-
`
`A
`
`B
`
`C
`
`D
`
`E
`
`Fio. 1.
`Rabbit urine: unconjugated metabolite following administration of II
`A.
`Rabbit urine: unconjugated metabolite following administration of I
`B.
`C. and D. Rabbit urine: unconjugated metabolites following administration of tetrabenazine.
`Rabbit urine: aglucones of glucuronides following administration of tetrabenazine-
`E.
`Spot at start was not always observcd and may be an artifact.
`
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`Metabolic studies of tetrabenazine
`
`Metabolite III
`Since upon administration of compound I or compound II no formation of meta-
`bolite III was observed one may conclude that III must be metabolically derived from
`tetrabenazine to which it is structurally related in a different way than are I and II.
`In fact when the bulk of the free metabolites was treated with Girard T reagent a
`ketonic fraction was isolated which contained III together with unchanged tetra-
`benazine, while all other free metabolites (I, II, IV and V) remained in the non-
`ketonic fraction. One may therefore conclude that the ketonic group of tetrabenazine
`remains unaltered in metabolite III. This is further substantiated by the fact that
`reduction of HI with lithiumaluminiumhydride gives rise to the formation of IV and
`V. On the other hand when IV was oxidised with aluminium isopropylate under
`drastic conditions (see 2.7) the main reaction product could be separated from the
`unchanged material by thin layer chromatography. Chromatographic analysis in
`3 systems showed it to be identical with metabolite III. Its molecular weight was
`determined by high resolution mass spectroscopy. Due to the high intensity of the
`peak at m/e 332 (M-H), the measurement was done on this peak:
`
`found
`calc.
`
`332.1866 ± 0.0030
`332.1861 for C19H26NO4
`
`and its molecular weight = 333.194.
`The elemental composition of the molecule is therefore C19H27NO4. The cracking
`pattern, similar to that of tetrabenazine, was found to be in agreement with the pro-
`posed structure (see scheme p. 650).
`The same general pattern of metabolite formation was found when the urine of
`rabbits, dogs or humans was examined in thin layer chromatography.
`
`2. The conjugated metabolites (aglucones)
`The bulk of the aglucones obtained after enzymatic hydrolysis of the glucuronides
`isolated from the urine of animals which had received tetrabenazine (see under
`2,3) showed the presence of at least 4 compounds which after treatment with mercuric
`acetate fluoresce intensely (see Fig. 1) These aglucones are not found in control
`urine of the same animals. We shall designate them as rnetabolites VI, VII, VIII and
`IX (formula: see scheme p. 650).
`The bulk of the aglucones as well as the single aglucones, isolated by column
`chromatography, show in the u.v. spectrum a shift of the maximum from 285 rnp in
`acid or neutral ethanol to 300 mit in alkaline ethanol, indicating the presence of a
`phenolic group.
`Administration of I or Il to rabbits or dogs showed that VII is issued from I, VIII
`from II.
`When the aglucones are treated with Girard T reagent compounds VI and IX are
`found predominantly in the ketonic, VII and VIII in the non-ketonic fraction.
`The presence of a ketonic function in VI and IX indicates that they originate from
`tetrabenazine and metabolite III.
`Phenols corresponding to the metabolites IV and V were usually not seen (Fig. I).
`One can assume that these metabolites because of their more polar character are
`excreted as such, and thus escape further metabolic transformation.
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`D. E. SCHWARTZ, H. BRUDERER, J. RIEDER and A. BROSSI
`
`Structure. The aglucones isolated from the urine of tetrabenazine-treated animals
`were partially separated by column chromatography on silicic acid. One ot the main
`fractions obtained deposited crystals upon standing for several weeks in the
`refrigerator. The mother liquor was decanted and the crystals washed several times
`with ice-cold petroleum ether and benzene. Thin layer chromatography of this
`material showed a single component corresponding to the aglucone VII. After
`reaction with diazomethane in ether the alkaline-acid shift was no longer observed
`and the product of the reaction was found to be identical in thin layer chromato-
`graphy to metabolite I.
`The definitive structure of these crystals (=VII) was established by comparison
`in thin layer chromatography: it was found identical in all systems to the synthetic
`compound 2,9-dihydroxy-3-isobutyl-10-methoxy-1,2,3,4,6,7-hexahydro-l lbH-benzo-
`[aiquinolizine with which it melted without depression.
`On the other hand VI was found identical in thin layer chromatography with the
`synthetic compound 2-oxo-3-isobutyl-9-hydroxy-10-methoxy-1,2,3,4,6,7-hexahydro-
`I lbH-benzo[a]quinolizine hydrochloride (see scheme p. 650). One may assume that
`it is metabolically formed directly from tetrabenazine.
`On the basis of the established structure for VI and VII the same phenolic structure
`may be postulated for VIII and IX, aglucones which would derive from II and III
`respectively. We have shown previously9 that Versidyneg (=racemic 1-p-chloro-
`phenethyl-2-methyl-6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline)
`and one of its
`metabolites (N-desmethyl-Versidyne) undergo in the organism a selective ether
`cleavage in position 6 of the isoquinoline ring structure.
`
`'
`
`ICH.0/
`CH3O-
`
`4 ?'CH]
`
`,
`
`C HO"
`
`v
`
`" '--
`
`H0
`
`i CHI-
`
`0
`
`Tetrabenazine0
`
`CH2
`
`H
`
`CL
`
`Versidyne
`
`More recently Koe and Pinson 10 studied the metabolism of Quantril (=2-acetoxy-
`3-diethylcarbamoyl-9,10-dimethoxy- 1,2,3,4,6,7-hexahydro- 11 bH-benzo[a]quinolizine)
`and, on the basis of theoretical considerations, tentatively placed the phenolic OH
`of their aglucones at C-9. In view of our own findings with Versidyne and tetra-
`benazine we feel confident that the same selective ether cleavage may occur in vivo
`in the case of a larger number of isoquinoline compounds.
`According to Axelrod" enzymes which degrade various aromatic ethers are present
`in the microsome fraction of the liver of a number of vertebrates. Such enzymes may
`well be responsible for the selective cleavage of ether linkage in this series of com-
`pounds, as was observed for Versidyne9 and tetrabenazine. These aglucones could
`not be detected free in the urine, one may therefore conclude that they are immediately
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`Metabolic studies of tetrabenazine
`
`conjugated in the organism. A quantitative analysis of each metabolite was not
`attempted in the case of tetrabenazine since labelled material was not available for
`this study. We may say however that while tetrabenazine can be detected only in
`the
`larger amounts
`in somewhat
`its unconjugated metabolites
`trace amount,
`glucuronides represent the prevailing form of excretion of the drug in the urine.
`Metabolites 1, II and V, pharmacologically tested for ethanol potentiation, were
`found to be less active than tetrabenazine.15
`
`SYNTHESIS OF METABOLITES
`
`1. Introduction
`The synthesis of both isomeric alcohols I and II has already been described.' 2
`Both the benzoquinolizines VII and VI were synthetized according to the following
`scheme.' 3
`
`C 1H5300
`
`+
`
`G
`(D
`H2.-N(CH 5 J
`/3 ,
`
`0 (b)
`
`H
`
`CH,
`
`OHS4
`
`The condensation product of 6-benzyloxy-7-methoxy-3,4-dihydroisoquinoline
`(a)14 with the quaternary salt (b) gives the benzoquinolizine-derivative (c), which after
`debenzylation gives the phenol VI. Etherification of this compound with diazomethane
`leads to tetrabenazine. The reduction of the ketone (c) with sodium borohydride
`yields only the trans-alcohol (d) (OH trans to the substituent at C-3), which after
`debenzylation leads to the phenolic compound VII. Alternatively one can obtain the
`phenolic compound VII by reduction of the ketone VI with sodium borohydride.
`Etherification of VII gives the trans-alcohol I.
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`D. E. SCHWARTZ, H. BRUDFRER, J. RiERm and A. BROSSr
`
`2. Experimental
`2-Oxo-3-isobutyl-9-benzyloxy-l0-methoxy- 1,2,3,4,6,7-hexahydro- I I bH-benzo[a]quino-
`lizine (c): 20 g 6-Benzyloxy-7-methoxy-3,4-dihydroisoquinoline
`(a) (m.p. 1010),
`25-8 g 3-Dimethylaminomethyl-5-methyl-hexan-2-one methiodide (b) (m.p. 179-
`180 °) were heated in 500 ml ethanol for 1 hr at 1000 (oilbath).
`After addition of potassium hydroxide (20 g) and water (200 ml) the solution was
`evaporated under reduced pressure. The residue was extracted twice with 500 ml of
`chloroform and the resulting extract was washed with saturated sodium chloride
`solution (2 X 50 ml), dried over sodium sulfate, filtered and distilled. The red-brown
`residual oil (26 g) was purified by column chromatography using 10 times the quantity
`of aluminium oxide (Act. II). 8.5 g of a lightbrown oil was eluted with benzene, it
`crystallized upon addition of a small amount of isopropyl ether. Two further crystal-
`lizations from ethyl acetate gave colourless crystals, m.p. 132.5-133' (5.8 g).
`
`C. 5 H 3 1NO3 (mol. wt. 393.5) calc. C 76-3 H 7-9%
`found C 76.2 H 7-9%
`282 mr, log e = 3.62
`u.v. spectrum (ethanol) Anax
`286 mi, log e = 3-62
`Amax
`i.r. spectrum (KBr pellet): 5"91 p (C
`0)
`
`2-Oxo-3-isobutyl-9-hydroxy-10-methoxy-l,2,3,4,6,7-hexahydro-1lbH-benzo[a]quinoli-
`zine, Hydrochloride (VI). 1 g (c) was dissolved in ethanol (100 ml) and hydrogenated
`in presence of 5 % palladium on charcoal (200 mg) at room temperature. After the
`theoretical amount of hydrogen was absorbed (1j hour) the catalyst was filtered
`and the filtrate evaporated. The hydrochloride was crystallized from ethanol-ether,
`white crystals, m.p. 217-219' (dec.) separated (940 mg).
`
`C18H25NO3 .HCI
`calc. C 63.6 H 7.7 Cl 10.4%
`found C 63.6 H 7-6 C1 10.3%
`(mol. wt. 339.9)
`u.v. spectrum (ethanol): Amax. = 286 mI.,
`log e = 3-60
`
`The above phenolic base (20 mg) was dissolved in ether (20 ml) and treated with
`an excess of diazomethane overnight. After evaporation of the solvent the residue
`was crystallized from a small quantity of isopropyl ether to obtain colourless crystals,
`m.p. 126' which gave no depression with an authentic sample of tetrabenazine.
`100 mg VI was dissolved in ethanol (5 ml) and reduced with sodium borohydride
`(100 mg) for 4 hr at room temperature. After evaporation of the solvent the oily
`residue was taken up in methylenechloride. The methylenechloride extract was
`dried over magnesium sulfate, filtered and evaporated. The crystalline residue, after
`two crystallizations from isopropyl ether, had m.p. 168' (VII).
`2-IHydroxy-3-isobutyl-9-benzyloxy-I 0-methoxy- 1,2,3,4,6,7-hexahydro -11 bH-benzo[a]-
`quinolizine (d). 1.5 g of the benzyloxyketone (c) was dissolved in 100 ml ethanol and
`reduced with 500 mg sodium borohydride for 4 hr at room temperature. After evapora-
`tion of the solvent, the residue was treated with 50 ml water and extracted with chloro-
`form (3 x 50 ml). The chloroform extract was washed with saturated sodium chloride
`solution (2 x 10 ml), dried over sodium sulfate and evaporated. The residue crystal-
`lized on
`the addition of isopropyl ether. The base was recrystalized
`twice
`
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`

`Metabolic studies of tetrabenazine
`
`from a mixture of ethyl acetate and petroleum ether (40-60') to give 1.25 g of light
`yellow crystals, m.p. 175-177*.
`
`C25H33NO3
`(mol. wt. 395"5)
`
`calc. C 75.9 H 8.4
`found C 75-6 H 8.3
`
`u.v. spectrum (ethanol): Amax. = 281 miz, log e = 3"61
`Amax. = 285 mfk, log c = 3"61
`i.r. spectrum (KBr pellet): 3.22 14 (OH)
`
`2,9-Dihydroxy-3-isobutyl-10-methoxy- l,2,3,4,6,7-hexahydro-11 bH-benzo[a]quinolizine
`(VII). I g (d) was dissolved in ethanol (100 ml) and hydrogenated in presence of 5 %0
`palladium on charcoal (200 mg) at room temperature. After the theoretical amount
`of hydrogen was absorbed (1f hr) the catalyst was filtered off and the filtrate
`evaporated. The crystalline residue was crystallized from methanol-ethyl acetate
`to give 745 mg whitish crystals, m.p. 192.5-193'.
`
`C 8H2 7N0 3 .0.5 CHsOH calc. C 69-1 H 9.1 0 174 OCH 3 14"6%
`found C 68.6 H 9-I 0 175 OCH3 146%.
`
`The above phenolic base (20 mg) was dissolved in ether (20 ml) and treated with
`an excess of diazomethane overnight. After evaporation of the solvent the residue
`crystallized upon addition of ethyl acetate to give pale yellow crystals, m.p. 1680
`which showed no depression with a sample of authentic 1, prepared from tetrabenazine.
`
`Acknowledgemens-We are greatly indebted to Dr. W. Vetter and Dr. G. Englert of the physical
`department Hoffmann-La Roche, Basie, for the mass- and MR spectra of our compounds and
`their interpretation.
`
`REFERENCES
`1. A. PLETSCHER, A. BRossi and K. F. GEY, Inf. Rev. Neurobiology 4, 275 (1962). See also footnotes.
`2. G. P. QUINN, P. A. SHORE and B. B. BRODIE, J. Pharmac. exp. Ther. 127, 103 (1959).
`3. D. E. SCHWARTZ and J. RIEDER, Clin. Chim. Acta 6, 453 (1961).
`4. D. E. SCHWARTZ, A. PLETSCHER, K. F. GEY and J. RIEDER, Heiv. Physiologica et Pharmacologica
`Acta 18, 10 (1960).
`5. I. A. KAMIL, J. H. SMITH and R. T. WILLIAMS, Biochem. J. 50, 235 (1952).
`6. E. STAHL, Chem. Zeitung 82, 323 (1958).
`7. C. S. MARVEL and R. D. RANDS JR., J. Am. chem. Soc. 72, 2642 (1950).
`8. A. GIaD and G. SANDULESCO, Hev. Chim. Acta 19, 1095 (1936).
`9. D. E. SCHWARTZ, H. BRUDERER, J. RIEDER and A. BRossi, Biochem. Pharmac. 13, 777 (1964).
`10. B. K. KoE and R. PINSON JR.,J. Med. Chem. 7, 635 (1964).
`11. J. AXELROD, Biochem. J. 63, 634 (1956).
`12. A. BROSSI, L. H. CHOPARD-DIT-JEAN and 0. SCHNIDER, Hev. Chim. Acta 41, 1793 (1958).
`13. H. T. OPENSHAw and N. WHITTAKER, J. chem. Soc. 1449 (1963).
`14. A. R. BATTERSBY et al., Tetrahedron 14, 46 (1961).
`15. A. PLETSCHER, H. BESENDORF and H. P. BXCHTOLD, Archiv exp. Path. u. Pharm. 232,499 (1958).
`
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