120 Biochem. J. (1961) 78, 120

Condensed Tannins 7. ISOLATION OF (-)-7:3':4'-TRIHYDROXYFLAVAN-3-OL [(-)-FISETINIDOL], A NATURALLY OCCURRING FROM BLACK-WATTLE HEARTWOOD*

By D. G. ROUX AND E. PAULUS Leather Industries Research Institute, Rhodes University, Grahamstown, South Africa (Received 9 May 1960) The interrelated compounds (+ )- aqueous phase extracted with ethyl acetate in each in- 7:3':4'-trihydroxyflavan-3:4-diol (Keppler, 1957) stance. The combined organic and ethyl acetate extracts of 2:3-trans:3:4-cis configuration (Clark-Lewis & from each fraction were evaporated to 50 ml. and applied Roux, 1959), (+ )-fustin (2:3-trans) and in bands to sheets (5 ml./sheet) of 221 in. x 18j in. What- 1958, man no. 3 chromatographic paper. The chromatograms (Roux & Paulus, 1960) have been isolated were developed by upward migration in 2 % acetic acid for from black-wattle-wood tannins. In the present 12 hr. The fustin (RF 0-43 on Whatman no. 3 paper), the study a new catechin, (-)-fisetinidol, correspond- polymeric leuco-fisetinidin and the ( +)-mollisacacidin ing to these substances has been isolated, and their (traces) (RF 0 57) bands were located with toluene-p- biogenesis is discussed. sulphonic acid. Bands in the region Rp 050 which appeared yellowish in ordinary light were cut out and eluted with 70% ethanol. Concentrate of the eluents gave 0-113 g. of EXPERIMENTAL AND RESULTS pale-yellow crystals. The mother liquors were purified by All melting points are uncorrected. Mixed melting running on five sheets, as above, yielding a further 0-05 g. points were on equimolecular mixtures of the substances Pale-yellow crystals of (- )-fisetinidol, m.p. 211-214°, (Roux & Maihs, 1960). Analyses of C, H, methoxyl and were obtained on recrystallizing from water. They differed acetyl are by Weiler and Strauss, Oxford. Infrared com- in shape from those of ( +)-fustin (m.p. 209-211°), of which parisons were by Dr J. R. Nunn, National Chemical 1-7 g. was obtained by cutting and eluting the sheets in Research Laboratory, C.S.I.R., Pretoria. the region Rp 0*43. The crystals of ( - )-fisetinidol did not lose their colour on recrystallizing with charcoal and the Isolation of m.p. remained unchanged. The substance gave the same (-)-fisetinidol colour reactions as (+ )-fustin with ammoniacal silver Compounds soluble in ethyl acetate from black-wattle nitrate (grey-black), with bis-diazotized benzidine (yellow) heartwood (120 g.) were obtained as described by Roux & and with ferric alum (green), but treatment with the Paulus (1960) and redissolved in ethyl acetate (800 ml.). toluene-p-sulphonic acid reagent gave a yellow fluorescence Chloroform (400 ml.) was added slowly with vigorous (u.v. light) with ( +)-fustin and a yellow colour with the stirring and the buff-coloured tannin precipitate sucked new substance. Microfusion under anhydrous conditions off and discarded. The filtrate was dried under vacuum in (Roux, 1958a) afforded resorcinol and protocatechuic a rotary evaporator. The solid product (41 g.) was ex- acid only. Infrared-absorption curves over the range 25- tracted with light petroleum to remove residual wax. The 15 u showed the absence of carbonyl groups. This sug- wax-free solids (40 g.) were dissolved in 400 ml. of the gested that the substance was a catechin (flavan-3-ol) with lower phase of a water-butan-2-ol-petroleum (b.p. 40- the same pattern of hydroxylation as (+)-fustin. After 1500) (5:3:2) mixture and introduced into the first eight drying for 2 hr. at 1100 the substance became anhydrous tubes of a 160-tube, 50 ml. underphase, automatic Craig (Found: C, 65-4; H, 5-3. C15H1405 requires C, 65-7; H, machine (Glasapparatebau G6ttingen, Helmut Rettberg). 5.2%) [X]25 - 8-6±0.5° in acetone-water (1:1) (c, 0-7). The upper and lower phases of the above mixture were (-) - 7: 3': 4' - Trimethoxyflavan- 3 - ol. (- ) -Fisetinidol used for countercurrent separation, and after 160 transfers (100 mg.) in methanol (20 ml.) was treated with excess of the upper layer of every fifth tube was examined by two- anhydrous diazomethane in ether for 24 hr. at - 50. The dimensional chromatography on Whatman no. 1 paper solids were recovered by distillation under vacuum in a with water-saturated butan-2-ol and then aq. 2% acetic rotary evaporator, and crystallized from ethanol as fine acid. needles, m.p. 121-1230. [,z]25-31.9±0.60 in tetrachloro- (+ )-Fustin (RF 0-37 in 2 % acetic acid on Whatman no. 1 ethane (c, 0 8) (Found: C, 67-6; H, 6-2; OMe, 30 5. C18H2005 paper) was present in tubes 106-136, and in tubes 106-121 requires C, 68-3; H, 6-4; OMe, 29.4%). it was accompanied by a second substance of R. 0 42. The second substance was present also in tubes 96-105 where Synthetic (-)-fisettinidol and derivatives it was accompanied by what appeared to be a polymeric leuco-fisetinidin of the same RF as (+ )-fustin. (- )-Fisetinidol. ( +)-7:3':4'-Trihydroxyflavan-3:4-diol The contents of tubes 96-105 and 106-121 were run off [(+ )-mollisacacidin] (0 7 g.) was separated from tubes separately, the organic phase was separated, and the 36-66 of the Craig machine by the same method as above and 550 mg. was hydrogenated in dioxan with palladium * Part 6: Roux & Paulus (1960). catalyst as described by Weinges (1958, 1959). The dioxan Vol. 78 ISOLATION OF (-)-FISETINIDOL 121 solution of the flavan-3:4-diol was introduced hot and the the enantiomer of (- )-fisetinidol from wattle heartwood apparent absorption, which included contraction on and from the hydrogenation of (+ )-7:3':4'-trihydroxy- cooling, of 104-2 ml. took place over lII hr. The catalyst flavan-3:4-diol (cf. Clark-Lewis & Roux, 1959; Roux & was removed by filtration, and the solvent removed under Paulus, 1960). These fisetinidols have identical RF values in vacuum. The syrup remaining was treated with 15 ml. of partitioning mixtures [0-82 in water-saturated butan-2-ol, water and concentrated to 5 ml., expelling the remaining and 0-81 in butan-l-ol-acetic acid-water (4:1:5)]. In 2% dioxan. From the solution crystals formed immediately acetic acid with reference substance (+)-fustin (0 37 on (300 mg.). These were recrystallized from water to give Whatman no. 1 paper) the Rp values were: (+ )-fisetinidol buff transparent crystals, m.p. 211-2140; [a]23- 8.8+0.80 synthetic, 043; (-)-fisetinidol synthetic, 0-48; (-)-fise- in acetone-water (1:1) (c, 1.1) (Found: C, 65-6; H, 5-3. tinidol from black wattle, 0-48. This confirms the optical C15H1405 requires C, 65 7; H, 5-2%). A mixed m.p. of the identity of synthetic and natural (-)-fisetinidols. The natural and the synthetic compounds showed no depres- higher Rp of (-)-fisetinidol in water or 2% acetic acid sion. Their infrared-absorption curves were superimposable [compared with ( +)-fisetinidol] apparently confirms its over the range 2-5s15j. stereochemical relationship with ( +)-fustin, and (-+ )- ( and O-Trimethyl-( - )-ftetinidol. (- )-Fisetinidol (400 mg.) 7:3':4'-trihydroxyflavan-3:4-diol [also +)-catechin in 80 ml. of methanol was methylated with diazomethane (+ )-gallocatechin], which similarly have higher Rp values as above. The product crystallized from ethanol (260 mg.) in 2% acetic acid than their corresponding enantiomers in fine needles, m.p. 121-1230; [z]23 - 32-1 +070 in tetra- (Roux & Paulus, 1960). chloroethane (c, 1-2) (Found: C, 68-3; H, 6-4; OMe, 28-8. Cj8H2005 requires C, 68-2; H, 6-4; OMe, 29.4%). A mixed Estimation of (-)-fisetinidol in black-wattle wood from black-wattle m.p. with the corresponding derivative Samples prepared for the estimation of the radial no Their infrared curves were wood showed depression. distribution of (+ )-mollisacacidin and (+ )-fustin (Roux & identical over the range 2-5-15ju. Paulus, 1960) were also used for the estimation of the - O-Trimethyl-( - )-fise- Acetyl-O-trimethyl-( )-fisetinidol. radial distribution of (-)-fisetinidol by means of paper was with ml. of pyridine tinidol (50 mg.) acetylated 0-2 chromatography and densitometry (Roux & Maihs, 1960). and 0-2 ml. of acetic anhydride at room temperature for The ( - )-fisetinidol was dissolved in methanol (0-1% soln.) was worked up as for tetra-acetyl-( 3 hr. The product -)- and applied to chromatograms in 10 ug. increments over 1960) and it crystallized as long fustin (Roux & Paulus, the range 10-90 g. to obtain a standard curve for estima- m.p. 19-1 i 1.50 in fine needles from ethanol, 95_97O; [a]D nitrate reagent was used for C, 67-1; H, 6-1; OMe, tion. Ammoniacal silver spray tetrachloroethane (c, 0 6) (Found: detection. Results are shown in Table 1. 26-5; CO-CH3, 10-9. C20H2206 requires C, 67-0; H, 6-2; OMe, 26-0; CO*CH3, 12-0%). Tetra-acetyl-( - )-fisetinidol. (- )-Fisetinidol (50 mg.) was DISCUSSION acetylated with acetic anhydride (0 3 ml.) and pyridine (0-2 ml.) for 3 and for 24 hr. at room temperature. In each (-)-Fisetinidol from black-wattle heartwood is instance the product failed to crystallize from methanol, a new, naturally occurring catechin and the second ethanol, ethanol-water and other solvents. The white catechin of the 'resorcinol series' to be isolated. powder obtained on pouring into water melted over the The first catechin of this group, (-)-7:3':4':5' range 50-60'; []2D3 + 0-9 ±0-20 in tetrachloroethane (c, 1 1) (Found: C, 62-0; H, 4-9; COCH3, 36-7. C23H2209 requires tetrahydroxyflavan-3-ol [(-)-], was C, 62-4; H, 5-0; COCH3, 38.2%). isolated by Roux & Maihs (1959, 1960) from the Chromatographic comparison of natural and synthetic bark of the same tree, Acacia mollissima. These fisetinidols. (+ )-Fisetinidol was prepared, as above, by the substances correspond also in their absolute con- hydrogenation of (- )-7:3':4'-trihydroxyflavan-3:4-diol figuration (see below) to (+ )-catechin and (+ )- (Weinges, 1958) from Schinopsis quebracho-colorado, and is gallocatechin of the 'phloroglucinol series' and may alternatively be designated as 5-deoxycatechin and Table 1. Radial distribution of (-)-fisetinidol in 5-deoxygallocatechin respectively. 50-year-old specimens of Acacia mollissima The structure of (-)-7:3':4'-trihydroxyflavan-3- ol was established as for (-)-robinetinidol by Values are expressed as percentage of dry extract. For hydrogenation of (+ )-7:3':4'-trihydroxyflavan-3:4- methods of extraction see Roux & Paulus (1960). diol in the 4 position under conditions established Location of sample Tree A Tree B by Weinges (1958, 1959). The natural and syn- Methanol extract thetic proved to be identical. The (+ )- Heartwood periphery 1.1 0-9 7:3':4'-trihydroxyflavan-3:4-diol, occurring natur- Middle heartwood 0-6 0-9 ally in the wood of A. mollissima (Keppler, 1957), Central heartwood 0-5 0-7 may be formed from (+ )-fustin present in the Combined methanol and acetone- same wood (Roux & Paulus, 1960) by hydrogen- water extracts ation under conditions established by Freudenberg Heartwood periphery 1-1 Middle heartwood 0-8 & Roux (1954) and Roux & Freudenberg (1958). Central heartwood 0-7 These are therefore stereochemically interrelated. Ethyl acetate extract of whole 1-1 As the stereochemically interrelated substances heartwood (-)-7: 3': 4'-trihydroxyflavan-3: 4-diol, (-)-fustin 122 D. G. ROUX AND E. PAULUS 1961 and (+)-fisetinidol have the same absolute con- single precursor explains the extreme complexity figuration as (-)-catechin (Weinges, 1958, 1959; of condensed tannin mixtures usually found in Freudenberg & Weinges, 1959), it follows that Nature. The present work also links up with the their above enantiomers (Clark-Lewis & Roux, suggestion by Robinson (1936), supported by 1958, 1959; Roux & Paulus, 1960) have the same Harborne (1960), of a single C1E; precursor for absolute configuration as (+)-catechin. The new based on a flavan-2:3:4-triol catechin may therefore tentatively be designated structure. By comparison, the present work as (2R:3S)-7:3':4'-trihydroxyflavan-3-ol (cf. Cahn, suggests that catechins (flavan-3-ols), dihydro- Ingold & Prelog, 1956; Freudenberg, 1956; Birch, flavonols (flavan-4-one-3-ols) and flavonols as well Clark-Lewis & Robertson, 1957). as tannins originate from a single flavan-3:4-diol The stereochemically interrelated (+ )-7:3':4'- precursor. Anthocyanidins could similarly originate trihydroxyflavan-3:4-diol, (+ )-fustin and (-)- from flavan-3:4-diols by interconversion. fisetinidol (also fisetin) occur in black-wattle wood The interconversion apparent in black-wattle forming 2-8-13-8 %, 1.3-28o/ and 0.5-11 % re- wood finds a number of parallels in Nature. For spectively of the solids in the acetone-water example in Schinopi8s spp. (quebracho), (-)- extract. The (+ )-7:3':4'-trihydroxyflavan-3:4-diol 7:3':4'-trihydroxyflavan-3:4-diol the main pre- is not only by far the most prominent component, cursor of quebracho tannins (Roux, 1958b, c) is but declines in concentration from the peripheral apparently accompanied by (-)-fustin (chromato- to the central heartwood. It is difficult to escape graphic evidence) and fisetin. Also in Acacia the conclusion that the flavan-3:4-diol, (+ )- melanoxylon, melacacidin (King & Bottomley, mollisacacidin, is first formed and subsequently 1954) consisting of two stereoisomeric 7:8:3':4'- undergoes conversion into both (-)-fisetinidol and tetrahydroxyflavan-3:4-diols (Roux, 1958b), is (+ )-fustin, and that the latter is finally also con- accompanied by the corresponding flavonol (King verted into fisetin (Fig. 1). In addition to these & Bottomley, 1954) and 2:3-dihydroflavonol interconversions the main transformation in the (chromatographic evidence). In (otinu8 coggygrza wood is undoubtedly the conversion of (+ )-7:3':4'- (syn. Rhus cotinu8) (-)-7:3':4'-trihydroxyflavan- trihydroxyflavan-3:4-diol into a number of leuco- 3:4-diol is accompanied by (-)-fustin and fisetin fisetinidin tannins, as suggested by Roux (1958b). (Freudenberg & Weinges, 1959), and in Robinia The concept of interconversion and also of con- p8eudacacia (+ )-7:3':4':5'-tetrahydroxyflavan-3:4- densation into a number of related tannins from a diol is accompanied by (+ )-dihydrorobinetin and

n

(IV) (V) Fig. 1. Suggested formation of tannins, catechins, 2:3-dihydroflavonols and flavonols from the flavan-3:4-diol of wattle wood. (I) (+)-7:3':4'-Trihydroxyflavan-3:4-diol (2:3-trans:3:4-cis); (II) (+)-fustin (2:3-trans); (III) (- )-7:3':4'-trihydroxyflavan-3-ol (2:3-tran8); (IV) fisetin; (V) leuco-fisetinidin tannins. Vol. 78 ISOLATION OF (-)-FISETINIDOL 123 robinetin (Weinges, 1958). In all these instances arise by interconversion, and the accompanying the suggested conversion of the flavan-3:4-diol into leuco-fisetinidin condensed tanninsby condensation. the corresponding 2:3-dihydroflavonol appears to predominate. This work is financed by the annual grant of the South In some woods two flavan-3:4-diol precursors are African Wattle Growers' Union to the Leather Industries present, for example 7:4'-dihydroxyflavan-3:4-diol Research Institute. and 7:3':4'-trihydroxyflavan-3:4-diol in certain Guibourtia spp. (Roux, 1959). In black-wattle-bark REFERENCES tannins similarly, two interrelated groups of com- ponents are present, namely (a) (-)-robinetinidol, Birch, A. J., Clark-Lewis, J. W. & Robertson, A. V. (1957). robinetin (Kirby & White, 1955), leuco-robinet- J. chem. Soc. p. 3586. inidin (traces) and complex leuco- Cahn, R. S., Ingold, C. K. & Prelog, V. (1956). Experientia, tannins and (b) fisetin, leuco-fisetinidin (traces) 12, 81. Clark-Lewis, J. W. & Roux, D. G. (1958). Chem. & Ind. and complex leuco-fisetinidin tannins. These two p. 1475. groups of substances may arise from the mono- Clark-Lewis, J. W. & Roux, D. G. (1959). J. chem. Soc. meric flavan-3:4-diols by the same paths of inter- p. 1402. conversion and condensation as is evident for Freudenberg, K. (1956). Sci. Proc. R. Dublin Soc. 27, 153. wattle-wood tannins. Freudenberg, K. & Roux, D. G. (1954). Naturwissen- 8chaften, 41, 450. Freudenberg, K. & Weinges, K. (1959). Chem. & lnd. SUMMARY p. 486. 1. (-)-7:3':4'-Trihydroxyflavan-3-ol [(-)-fiset- Harborne, J. B. (1960). Biochem. J. 74, 262. Keppler, H. H. (1957). J. chem. Soc. p. 2721. inidol], a new catechin, has been isolated from the King, F. E. & Bottomley, W. (1954). J. chem. Soc. p. 1399. heartwood of Acacia mollis8ima. The radial distri- Kirby, K. S. & White, T. (1955). Biochem. J. 60, 582. bution of the substance in the wood has been Robinson, R. (1936). Nature, Lond., 137, 172. estimated. Roux, D. G. (1958a). J. Amer. Leath. Chem. A88. 53, 384. 2. (-)-Fisetinidol has also been prepared syn- Roux, D. G. (1958b). Nature, Lond., 181, 1454. thetically by the hydrogenation of (+)-7:3':4'-tri- Roux, D. G. (1958c). Chem. & Ind. p. 161. hydroxyflavan-3:4-diol. Roux, D. G. (1959). Nature, Lond., 183, 890. 3. The new catechin has the same configuration Roux, D. G. & Freudenberg, K. (1958). Liebigs Ann. 613, at C-2 and C-3 as (+ )-7:3':4'-trihydroxyflavan-3:4- 56. Roux, D. G. & Maihs, E. A. (1959). Nature, Lond., 182, diol and as (+ )-fustin, all of which occur in the 1798. wood. Roux, D. G. & Maihs, E. A. (1960). Biochem. J. 74, 44. 4. The suggestion is made that the flavan-3:4- Roux, D. G. & Paulus, E. (1960). Biochem. J. 77, 315. diol is the single precursor from which the corre- Weinges, K. (1958). Liebig8 Ann. 615, 203. sponding catechin, 2:3-dihydroflavonol and flavonol Weinges, K. (1959). Liebige Ann. 627, 229.

Biochem. J. (1961) 78, 123 The Effect of Riboflavin and Mepacrine on the Metabolism of 5-Hydroxytryptamine

BY M. H. WISEMAN AND T. L. SOURKES Allan Memorial In8titute of Psychiatry, McGill Univer8ity, Montreal, Canada (Received 20 May 1960) The monoamine-oxidase pathway has been yellow enzyme (Swedin, 1943; Kapeller-Adler, identified as an important route of degradation of 1949; Goryachenkova, 1956). Hawkins (1952b) 5-hydroxytryptamine (serotonin) in many species. considered that the decreased activity of mono- Because of the similarity of its action to that of amine oxidase in the liver of rats, which is a con- D-amino acid oxidase, the coenzyme of which is comitant of riboflavin deficiency, is due to im- flavinadenine dinucleotide, Richter (1937) sug- paired protein utilization and postulated that the gested that monoamine oxidase may be a flavo- vitamin is involved in synthesis of the apoenzyme protein. Diamine oxidase has been shown to be a of monoamine oxidase. Her findings have been