Vol. 63 INTERMEDIATES IN HAEM BIOSYNTHESIS 395Henriques, 0. B., Henriques, S. B. & Neuberger, A. (1955).

Biochem. J. 60, 409.Muir, H. M. & Neuberger, A. (1950). Biochem. J. 47, 97.Neuberger, A. & Scott, J. J. (1953). Nature, Lond., 172,

1093.Nicholas, R. E. H. (1951). Biochem. J. 48, 309.Prunty, F. T. G. (1945). Biochem. J. 39, 446.Rimington, C. (1954). Rep. Progr. Chem. 51, 311.Rimington, C. & Sveinsson, S. L. (1950). Scand. J. clin.

Lab. Invest. 2, 209.

Shemin, D. (1955). Rep. 3rd Int. Congr. Biochem., Bruseels,p. 170.

Shemin, D. & Russell, C. S. (1953). J. Amer. chem. Soc. 75,4873.

Shemin, D. & Wittenberg, J. (1951). J. biol. Chem. 192,315.Stern, A. & Wenderlein, H. (1934). Z. phy8. Chem. A, 170,

345.Weatherall, M. (1954). Pharmacol. Rev. 6, 133.Westall, R. G. (1952). Nature, Lond., 170, 614.Wittenberg, J. & Shemin, D. (1950). J. biol. Chem. 185, 103.

Studies in the Biochemistry of Micro-organisms97. FLAVIPIN, A CRYSTALLINE METABOLITE OF ASPERGILLUS FLAVIPES(BAINIER & SARTORY) THOM & CHURCH AND ASPERGILLUS TERREUS THOM*

BY H. RAISTRICK AND P. RUDMANDepartment of Biochemi8try, London School of Hygiene and Tropical Medicine, University of London

(Received 2 January 1956)

The Aspergilli are divided by Thom & Raper (1945),mainly on morphological characters, into fourteengroups, two of which are of interest in the presentcommunication. The AspergiUua flavipe8 groupconsists of a single species, A. flavipe8 (Bainier &Sartory) Thom & Church. The closely related A.terreu8 group includes three species, of which thecommonest and most important is A. terreu8 Thomand its three varieties, boedijni (Bloch.) n.var.,flocco8ua Shih and aureus n.var. It is interesting tonote that old cultures of A. flavipes often resembleA. terreu8, the two species being readily distin-guished, however, when young cultures are ex-amined.We have found that culture filtrates of one strain

each of A. flavipe8 grown on Raulin-Thom solutionand of A. terreu78 grown on Czapek-Dox solutiongive an intense blue-black ferric reaction and animmediate heavy red precipitate with Brady'sreagent (2:4-dinitrophenylhydrazine in aqueous2N hydrochloric acid). We have isolated in a stateof purity the metabolic product responsible forthese reactions and propose for it the trivial nameflavipin, since it does not appear to have beendescribed previously.

There appears to be no published record of theisolation of any metabolic product of A. ftavipe8,although there are a number of references in theliterature to the formation by this species ofculture filtrates having an antibacterial spectrumsimilar to that of penicillin and containing a sub-stance which is destroyed by penicillinaae. Inmarked contrast, different strains of A. terreu8have been shown to produce a large variety of

metabolites, amongwhich are: itaconic acid (Calam,Oxford & Raistrick, 1939); itatartaric acidHO2C.C (OH)(CH2 * OH) . CH2 .C02H (Stodola, Fried-kin, Moyer & Coghill, 1945); expansine, C7H804(Kent & Heatley, 1945); terrein, C8H,003, andcitrinin, C18H1405 (Raistrick & Smith, 1935);geodin, C17H1207C12, and erdin, C16H1007C12 (Rais-trick & Smith, 1936); and terreic acid, C7HE04,from A. terreu8 var. aureus n.var. (Pansacker,Philpot, Jennings & Florey, 1947).

Production of flavipinThe influence of cultural conditions, and particu-

larly of the composition of the culture mediumused, on the course of mould metabolism is wellillustrated by a comparison of the metabolism ofA. flavipes with that of A. terreu8. Thus we haveshown that, although good yields of flavipin areproduced by A. flavipes when grown on Raulin-Thom solution, no flavipin could be detected whenthis species was grown on Czapek-Dox solution.A. terreus, on the contrary, failed to produce anyflavipin on Raulin-Thom solution, though it gavemoderate yields on Czapek-Dox solution. It wasfurther shown that, whereas the optimum incu-bation period at 24° for maximum production offlavipin is 12-14 days with A. flavipes grown onRaulin-Thom medium, the corresponding time is7-9 days for A. terreus grown on Czapek-Doxmedium. The results obtained showed clearly thatflavipin cannot be regarded as an 'end' product ofthe metabolism of either species, since, after theconcentration of flavipin in the culture fluid hadreached a maximum, flavipin was decomposed atabout the same rate as the glucose in the medium* Part 96: Galarraga, Neill & Raistrick (1955).

H. RAISTRICK AND P. RUDMANwas metabolized, and, in the case of A. terreuegrown on Czapek-Dox solution, almost all theflavipin present at the peak of production hadcompletely disappeared after 21 days' incuba-tion.For the bulk preparation of flavipin, cultures of

A. flavipes on Raulin-Thom solution were harvestedafter 12 days' incubation at 240. The culture fluidwas separated by filtration from the mould myce-lium, lightly acidified with hydrochloric acid andexhaustively extracted with ether. The yellow-orange ether extract was dried and evaporated,giving crops of crude flavipin which were purifiedeither by crystallization or, preferably, by subli-mation at 1400 in high vacuum. The yields offlavipin which were obtained were 5-6 g. from 100flask cultures of A. flavipes, each containing350 ml. of Raulin-Thom solution, and about 2 g.from the same number of flask cultures of A.terreus on Czapek-Dox solution.

Properties and structure offlavipin

Flavipin, C.H805, sublimes as small pale-yellowrods, m.p. 233-234° (decomp.). It has no opticalactivity. It contains three active hydrogen atomsand one methyl group attached to carbon but nomethoxyl group. It dissolves in aqueous sodiumbicarbonate giving a yellow solution and in sodiumhydroxide forming a solution which is initiallycherry-red but quickly becomes brown. It hasstrong antifungal properties but is only weaklyantibacterial.The following reactions of flavipin have some

diagnostic value: an intense but unstable blue-black ferric reaction in aqueous solution; an im-mediate dark-red precipitate with Brady's re-agent; reduction of ammoniacal silver nitrate atroom temperature and of Fehling's solution onboiling; a green coloration and a green precipitatewith sodium-mercury amalgam in ethanolicsolution, indicative of three vicinal phenolic groups(Bargellini, 1919).The following crystalline functional derivatives

of flavipin have been prepared and characterized:(a) pentaacetylflavipin hydrate, C19H20011, colour-less rods, m.p. 1800, insoluble in cold aqueoussodium hydroxide and giving no ferric colour andno precipitate with Brady's reagent; (b) mono-methyl ether, C1oH1005, yellow needles, m.p.196-1970; (c) diflavipin hexamethyl ether mono-hydrate, C24H30011, colourless needles, m.p. 138-5-1390, dissolves slowly in aqueous sodium hydr-oxide, gives no ferric reaction, but, on treatmentwith Brady's reagent, gives (d) trimethylflavipin bis-2:4-dinitrophenylhydrazone, C24H22011N8, orangeneedles, m.p. 216-2170; (e) flavipin anhydro-2:4-dinitrophenylhydrazone, C15H1007N4, deep-red rods,

m.p. above 300°; (f ) flavipin 2:4-dinitrophenyl-hydrazine derivative,

C21HL8012N8 = C9H805 + 2C6H604N4- H20,red rods, m.p. 242-243° (decomp.); (g) hydrazinederivative of flavipin,

CqH803N2 = CqH805 + N2H4- 2H20,

by treatment of flavipin with hydrazine hydrate,yellow rods, m.p. above 3000, gives a blue-blackferric reaction but no precipitate with Brady'sreagent.The experimental evidence so far presented

justifies the working hypothesis that the moleculeof flavipin contains the following groups: oneC-CH3 group actually estimated; three hydroxylgroups, possibly vicinal; two carbonyl groups,probably aldehydic and adjacent to each other toexplain the formation of the derivative CH8,03N2on treatment with hydrazine hydrate. The em-pirical formula for flavipin, C9H805, may thereforebe expanded to C6(CH3)(OH)3(CHO)2, and theprobability emerges that flavipin is a fully sub-stituted benzene derivative, i.e. a diformyltri-hydroxymonomethylbenzene. Further, if the in-ferences made from the Bargellini test and from thehydrazine product of flavipin are correct, i.e. thatthe three hydroxyl groups and the two formylgroups are vicinal respectively to each other, onlyone benzenoid structural formula for flavipin ispossible, i.e. structure (I). It will be shown laterthat this structure explains satisfactorily the forma -tion of the functional derivatives described above.

CH3

(I)Flavipin

In order to establish structure (I) on a muchsounder basis, a number of reaction and oxidationproducts of flavipin and its derivatives were in-vestigated.When flavipin was oxidized with alkaline hydro-

gen peroxide a very vigorous and deep-seatedreaction took place, leading to the complete dis-ruption of the molecule. The oxidation productsidentified were: (i) carbon dioxide equivalent toabout 1-5 molecules per molecule of flavipin; (ii)formic acid equivalent to about 75% of 2 molecules.This finding gives strong support to the viewthat flavipin contains two formyl groups; (iii)methylmalonic acid, HO2C. CH(CH3) . C02H, con-firming the presence of a C-CH3 group in flavipin;

396 I956

FLAVIPIN FROM ASPERGILLUS FLAVIPESand (iv) hydroxymalonic acid (tartronic acid),HO2C . CH(OH) . CO2H.The crude, oily, methylated flavipin, obtained by

exhaustive methylation with dimethyl sulphateand anhydrous potassium carbonate in acetone,was refluxed with 40% aqueous sodium hydroxide.A good yield of 4:5:6-trimethoxy-7-methylphtha-lide (II, R=CH3) was isolated from the reactionproducts. The identity of the phthalide, which hasnot been described previously, was established bycomparison with a synthetic specimen prepared bycatalytic reduction with palladium and hydrogenof 7-chloromethyl-4:5:6-trimethoxyphthalide (II,R = CH2Cl) prepared by chloromethylation ofthe trimethyl ether of gallic acid with formalde-hyde and concentrated hydrochloric acid (Edwards,Perkin & Stoyle, 1925; King & King, 1942).

R CH3

CH3.0 7 C0 CH3 ° co

520/CH3.0 - H2 CH3.0 co

.CH3 .CH3

(II)

done by oxidizing with hot alkaline permanganatethe phthalazine derivative of flavipin, CqH803N2(VI), which was prepared by treating flavipin withhydrazine hydrate. The resulting oxidation pro-duct (V) was identified as pyridazine-4:5-dicarboxy-lic acid by comparison with an authentic specimenprepared as described by Gabriel (1903) by hotpermanganate oxidation of phthalazine (4:5-benzopyridazine) (VII), which was synthesized bytreating phthalaldehyde with hydrazine hydrate(Gabriel & Pinkus, 1893).

CH3

HO2C

HO2C N

(V) (VI)

CR

(IIIj

CO2H

CH3. 0 CO2H

CH3.0C.2H.CH3

(IV)

The crude, oily, methylated flavipin was oxidizedwith cold alkaline aqueous potassium permanga-nate. Two of the resulting oxidation products were

identified as 4:5:6-trimethoxy-3-methylphthalicanhydride (III) and 4:5:6-trimethoxybenzene-1:2:3-tricarboxylic acid (IV), the identity of each ofwhich was established by comparison with authen-tic specimens prepared by alkaline potassiumpermanganate oxidation respectively of 4:5:6-trimethoxy-7-methylphthalide in the cold and 7-chloromethyl-4:5:6-trimethoxyphthalide in boilingsolution.

Finally, pure crystalline diflavipin hexamethylether hydrate was oxidized with cold alkalinepotassium permanganate and gave 4:5:6-trimeth-oxy-3-methylphthalic anhydride (III).

Hence, the relative positions of the methyl groupand the three hydroxyl groups in the flavipinmolecule are established, and it now remains toconfirm that the two remaining positions in thebenzene ring are occupied by -CHO groups, as ispostulated in structure (I) and is indicated in thereaction products (II, R = CH3) and (III). This was

(VII)

The conclusion that flavipin is a substituted o-phthalaldehyde is supported by the experimentalfinding mentioned previously that methylatedflavipin is readily converted by boiling aqueoussodium hydroxide into 4:5:6-trimethoxy-7-methyl-phthalide (II, R = CH3), since o-phthalaldehydeitself is well known to undergo rearrangementunder alkaline conditions to'give o-hydroxymethyl-benzoic acid, from which its lactone, phthalide, isreadily formed on acidification.

Flavipin may therefore be described systematic-ally as 1:2-diformyl-4:5:6-trihydroxy-3-methylben-zene (I). This unusual formulation, containing as itdoes five reactive substituents, is reflected in theunusual nature ofsome of its functional derivatives.Thus the product of the acetylation of flavipin withacetic anhydride and either sodium acetate or con-centrated sulphuric acid as catalyst is not a simpletriacetyl derivative. It has the formula C19H20011,contains five acetyl groups and an extra moleculeof water, and since it does not react with 2:4-dinitrophenylhydrazine, except on long standingand therefore probably because of hydrolysis, wesuggest structure (VIII) for it.

Diflavipin hexamethyl ether monohydrate,C24H30011, is, as a molecular-weight estimationshowed, a double molecule and its properties areillustrated in structure (IX). This structure is,however, not to be regarded as definitive since anumber of isomers are possible.

Vol. 63 397

H. RAISTRICK AND P. RUDMAN

(VIII)

The products formed by the interaction of 2:4-dinitrophenylhydrazine with flavipin and itsderivatives are somewhat confusing. When thereaction is carried out in hot glacial acetic acidflavipin gives a product, C21HI8012N8, crystallizingin red rods; this, however, does not appear to be a

bis-2:4-dinitrophenylhydrazone, which would re-

quire the formula C21H,6011N8. It may be the 2:4-dinitrophenylhydrazine salt of a mono-2:4-dinitro-phenylhydrazone, since o-phthalaldehyde, whentreated similarly, gives only a mono-2:4-dinitro-phenylhydrazone. In contradistinction, diflavipinhexamethyl ether monohydrate in ethanol solutiongave, with 2:4-dinitrophenylhydrazine in aqueous2N hydrochloric acid, trimethylflavipin bis-2:4-dinitrophenylhydrazone, C24H22O11N8, as orangeneedles. Finally, when flavipin was heated inbutanol solution with 2:4-dinitrophenylhydrazinethe resulting product was flavipin anhydro-2:4-dinitrophenylhydrazone, Cl5H1007N4, in deep-redrods.

Fungistatic propertie8 of flavipinTwo other derivatives of o-phthalaldehyde have

been described as mould metabolic products, i.e.gladiolic acid (X) from Penicillium gladioliMachacek (Brian, Curtis & Hemming, 1948;Grove, 1952; Raistrick & Ross, 1952) and cyclo-paldic acid (XI) from P. cyclopium Westling(Birkinshaw, Raistrick, Ross & Stickings, 1952).

CO2H CO2H

(X) (XI)

It is interesting therefore to find that all threesubstances have pronounced fungistatic properties.When tested here against the conidia of Botrytisallii Munn in a germination medium at pH 3*5 bythe technique described by Brian & Hemming(1945), 95-100% inhibition of germination was

produced by flavipin at a concentration of 10'0 jug./ml., by gladiolic acid at 10.0 ,ug./ml. and by cyclo-paldic acid at 255g./ml. Under the same condi-tions, and at a concentration of 125 ,ug./ml.,

(iX)

flavipin produced 95-100% inhibition of germina-tion of the conidia of the same strain of A8pergillu8flavipes as was used in its preparation.

Metabolic significance offlavipinDerivatives of 1:2:3-trihydroxybenzene (pyro-

gallol), of which flavipin is obviously one, occur

only rarely as fungal metabolic products. Bern-hard & Albrecht (1947) isolated gallic acid (3:4:5-trihydroxybenzoic acid) as a metabolite of Phy-comyces blakesleeanus Burgeff grown on a solutioncontaining glucose, asparagine, yeast extract andaneurin in addition to mineral salts. Fuscin anddihydrofuscin were first described as antibacterialmetabolites of the mould Oidiodendron fuscumRobak by Michael (1948), and Birkinshaw,Bracken, Michael & Raistrick (1951) described a

number of their derivatives and breakdownproducts, one of which was shown to be 3:4:5-trihydroxyphenylacetic acid. Barton & Hendrick-son (1956) have correlated this experimental workin terms of constitutional formulae for fuscin (XII)and dihydrofuscin (XIII) and have confirmed theirdeductions by synthesis.

(XII)Fuscin

10

(XIII)Dihydrofuscin

398

CH3. CO.(

CH3.CO.O

CH3.CO.

I956

I. CH3

. CH3

FLAVIPIN FROM ASPERGILLUS FLAVIPES

The origin of the 3:4:5-trihydroxyphenylaceticacid breakdown product of dihydrofuscin is indi-cated by the broken line in structure (XIII).Further, it is clear that flavipin, fuscin and dihydro-fuscin are closely related structurally, since theyall potentially contain the basic structure (XIV).

C-

HO C

HO CE

H

(XIV)

EXPERIMENTAL

Cultures(i) Aspergillus flavipes (Bainier & Sartory) Thom &

Church, strain London School of Hygiene and TropicalMedicine (L.S.H.T.M.), catalogue no. S.M. 884, was isolatedhere by Mrs S. Marcus in 1952 from soil collected by her atSidmouth.

(ii) Aspergillus terreus Thom, strain L.S.H.T.M., cata-logue no. A. 465, was isolated here by Mrs Marcus in 1951from an Australian soil sample no. 52.1, kindly madeavailable to us by Professor E. G. Hallsworth, University ofNottingham.Both these cultures were identified by Mr George Smith

of this Department.

Cultural conditions and characteristics(i) Raulin-Thom culture solution (glucose, 75-0g.; tar-

taric acid, 4-0 g.; ammonium tartrate, 4-0 g.; (NH4)2HP04,0-6 g.; (NH4)2SO4, 0-25 g.; K2C03, 0-6 g.; MgCO3, 0-4 g.;FeSO4,7H20, 0-07 g.; ZnSO,, ,7H20, 0-07 g.; distilled water,1500 ml.) was used exclusively for the cultivation of A.flavipes and for the bulk preparation of flavipin.

(ii) Czapek-Dox culture solution (glucose, 50-0 g.;NaNO3, 2-0 g.; KH2PO4, 1-0 g.; KCI, 0-5 g.; MgSO4,7H20,0-5 g.; FeSO4,7H20, 0-01 g.; distilled water, 1000 ml.) wasused for the cultivation of A. terreus.

Batches of 1 1. conical flasks, plugged with cotton wooland each containing 350 ml. of either solution (i) or (ii), wereprepared and sterilized by steaming at 1000 for 0-5 hr. oneach of 3 consecutive days. The contents of the flasks were

then inoculated with a spore suspension in sterile water ofthe appropriate mould grown for 14 days on malt-agarslopes, three flasks being sown from each slope. The inocu-lated flasks were incubated in the dark at 240.

After 7 days' incubation the flask cultures ofA.flavipes onRaulin-Thom solution contained a rather thin, granular,brown-purple mycelium with a purple-brown reverse and adeep-brown, clear culture fluid. After 14 days the myceliumhad become folded and almost black and the dark-brownculture fluid contained much debris. The culture fluid ineach case gave an intense purple-black or blue-black colourwith aqueous FeCl3, slowly fading to purple-brown, and animmediate, very heavy, dark-red, amorphous precipitatewith Brady's reagent (0-32 % 2:4-dinitrophenylhydrazine inaqueous 2N-HCI).The well-formed mycelium in the 7-day cultures of A.

terreus on Czapek-Dox solution was dull pale-yellow withan orange-yellow reverse and showed numerous orange-coloured drops after 14 days' incubation. The culture fluidwas orange-brown in colour and gave reactions with FeCl3and with Brady's reagent which were very similar to but lessintense than those given by the corresponding culturefluids of A. flavipes grown on Raulin-Thom solution.When A. flavipes was grown on Czapek-Dox solution,

although the mould grew normally, no indication of theformation of flavipin could be obtained since the culturefluid after either 7 or 14 day's incubation gave negativeferric and Brady reactions. Contrariwise, when A. terreuswas grown on Raulin-Thom solution, no reaction indicativeof flavipin in the culture fluid could be obtained.

Course of metabolism(i) A.flavipes. In order to follow the course ofmetabolism

of A. flavipes and to determine the optimum time forharvesting, fifty flasks of Raulin-Thom medium wereinoculated with this strain and incubated at 240. Five sets often flasks, chosen at random, were harvested after incubat-ing for 7, 9, 12, 14 and 16 days respectively. The contents ofeach set of ten flasks were combined and filtered, and thefollowing estimations were carried out on the filtrates.(1) pH. (2) Residual glucose, estimated polarimetrically, theinitial concentration being 5-0%. (3) Crude flavipin. Theculture filtrate was acidified with conc. HCl (3 ml./l.) andextracted three times with ether, i vol. of ether being usedeach time. The yellow-orange ethereal extract was driedover anhydrous Na2SOg and filtered and the solvent re-moved. The dried residue of crude flavipin was weighed. Itsweight x 10 is recorded in Table 1, column 4. (4) An anti-fungal assaywas carried out byusing spores of Botryti allii as

Table 1. Course of metabolism of Aspergillus flavipes strain S.M. 884grown on Raulin-ThoM solution at 240

In columns 5-11, O=no germination, 1=less than 1% germination, 3=50-90% germination, and 4=90-100%germination.

Crude Antifungal assay against Botrytis alliiIncubation pH of Residual flavipin Dilution of culture filtrate

glucose(%)

3-503-453-383-103-00

- -r--(g./100flasks)3-055-367-207.394-50

1/4 1/80 0

0 0

0 0

0 0

0 0

1/16 1/32 1/64 1/128 1/2560 4 100% germination0 1 100% germination0 1 3 100% germination0 1 4 100% germination0 1 4 100% germination

period(days)

79121416

culturefiltrate3-1-3-43-1-3-43-1-3-42-9-3412-9-3-1

Vol. 63 399

H. RAISTRICK AND P. RUDMANthe test organism and the germination medium ofpH 3.5 asdescribed by Brian & Hemming (1945). All the five culturefiltrates gave the characteristic intense blue-black ferriccolour and a heavy deep-red precipitate with Brady'sreagent. At no time did the culture filtrates show appreci-able antibacterial activity against either Staphylococcu8aureus or E8cherichia coli. Table 1 indicates clearly that theoptimum incubation period is 12-14 days.

(ii) A. terreus. The course of metabolism of A. terreus wasfollowed in a similar fashion to that of A. flavipea, with theexception that an antifungal assay was not carried out. Theresults obtained are given in Table 2. The pH of the culturefiltrate was between 6.1 and 6-4 in all nine tests, in all ofwhich the characteristic ferric colour and red precipitatewith Brady's reagent were also observed.

Table 2 indicates clearly that, in this case, the optimumincubation period is 7-9 days, after which time flavipin andglucose are metabolized at approximately the same rate.

I8olation offlavipinThe culture fluid from each batch offlask cultures (usually

50-100) of A. flavipes on Raulin-Thom solution, harvestedafter 12 days' incubation at 24°, or of A. terreus on Czapek-Dox solution after 9 days' incubation, was separated fromthe mycelium by straining through muslin and was thenclarified by filtration through a thin layer of kieselguhr.The A.flavipes culture filtrates, pH 3-1-3-4, were acidified

with conc. HCI (1 ml./300 ml. of filtrate) and extracted withether in a large countercurrent extraction apparatus witha bath temperature of 550, four such extractions beingnecessary. The A. terreu,s filtrates, pH 6-1-6-4, gave acleaner product when ether extracted without prior acidifica-tion, five extractions being necessary. The ether extractsfrom either mould were combined, dried over anhydrousNa2SO4 and evaporated to low bulk (50-100 ml.). The solidcrude flavipin, m.p. 190-215', which separated was col-lected, together with a second small crop obtained on furtherconcentration. Complete removal of the ether from thefinal mother liquors gave an intractable gum. Details of anumber of batches ofA. flavipes are given in Table 3, and ofA. terreus in Table 4.

It will be shown later that the most satisfactory method ofpurification of crude flavipin is by sublimation in a highvacuum at a bath temperature of 1400. Hence it becamenecessary to prove that this treatment does not lead to anydecomposition of flavipin. This was done as follows. Theculture filtrate (1500 ml.) from five flasks of A. flavipesgrown on Raulin-Thom solution for 12 days was treatedwith Brady's reagent (310 ml.). After standing for 0-5 hr.the resulting red precipitate was separated by centrifugingand was washed on the centrifuge twice with water, twicewith ethanol and once with ether. The resulting solid wasrepeatedly crystallized from dimethylformamide, givingfinally dark-red needles (49 mg.), m.p. 242-243° (decomp.)not depressed on admixture with the 2:4-dinitrophenyl-hydrazine derivative of the same m.p. prepared fromflavipin purified by vacuum sublimation (see p. 401). Thetwo specimens had also the same colour, crystalline form andanalysis and gave the same purplish-red Neuberg reaction.(Found, on sample prepared from culture 6altrate: C, 44-2;H, 3-2; N, 19-3. C2lHl80L2N8, i.e.

CqH804: N.NH C6H3(N02)2 + C6H3(NO2)2 .NH .NH2requires C, 43-9; H, 3-2; N, 19-5%.)

Table 2. Course of metabolism of Aspergillus terreusstrain A. 465 grown on Czapek-Dox solution at 240

Incubationperiod(days)

789101113141721

Residualglucose(%)3-002-802-492-381-741-190-950-580-20

Crudeflavipin

(g./100 flasks)1-581-301-841-271-200-630370-380-13

Table 3. YieldB of flavipin from Aspergillusflavipes grown on Raulin-Thom 80luttion for12 day8

Residual Yield of No. ofBatch glucose flavipin flasksno. (%) (g.) in batch1 3-48 5-37 982 3-46 3-65 843 3-71 4-57 1004 3.55 5-33 975 3-50 5-87 1006 3-28 4-58 1007 3-10 5-60 978 3-25 6-30 979 3-46 5-60 100

Table 4. Yield8 offlavipin from Aspergillus terreusgrown on Czapek-Dox 8olution for 9 day8

Batchno.123

Residualglucose(%)2-182-802-28

Yield offlavipin

(g.)1-401-872-14

No. offlasks

in batch59100100

Purifi-cation of flavipinCrude flavipin may be crystallized from ethyl acetate,

benzene, CHCl3 or acetic acid, or from mixtures of these.This method of purification is not very satisfactory, sincemany crystallizations are necessary to reach a constantmelting point, probably because of the marked thermo-lability offlavipin (especially in hydroxylic solvents). How-ever, a single sublimation in high vacuum at a bath temper-ature of 1400 raised the m.p. from 180-2150 to 2270 and gavematerial suitable for preparative and degradative work.Three further sublimations gave a product with a constantm.p. of 233-234° and of analytical purity. Specimenspurified by crystallization or by sublimation gave com-parable analyses.

General properties offlavipinFlavipin sublimes as small pale-yellow rods, m.p. 233-

2340 (decomp.) and crystallizes from solvents in yellowneedles of the same m.p. (Found (a) on a recrystallizedspecimen from A. terreus: C, 55-2; H, 4-1; (b) on a sublimedspecimen from A. flavipes: C, 54-9; H, 4-1; N, S, Cl, nil;

400 I956

FLAVIPIN FROM ASPERGILLUS FLAVIPESC-CH3, 7-3; active H, 1-45 %; mol.wt. (cryoseopic indioxan), 194, 197. C9H805 requires C, 55 1; H, 4 1; 1C-CH3,7-7; 3 active H, 1-54%; mol.wt. 196.) Flavipin shows nooptical activity, is thermolabile and photosensitive. It issoluble in water, methanol, ethanol, ethyl acetate andacetone; slightly soluble in CHC13 and benzene, and in-soluble in light petroleum (b.p. 40-50°). It dissolves slowlyin cold aqueous NaHCO3 to give a yellow solution fromwhich it is precipitated on acidification. Its solution inaqueous NaOH is initially cherry-red in colour and rapidlychanges through orange to brown. It gives an orangesolution in cold conc. H2SO4. An ethanolic solution offlavipin gives with ethanolic FeCl3 an unstable steel-bluecolour, increasing momentarily in depth of colour on dilu-tion with water and changing rapidly through reddishpurple and orange to brown. An aqueous solution gives thefollowing reactions: with aqueous FeCl3, an intense buttransient blue-black colour, rapidly becoming brown; withCuS04, a yellow precipitate insoluble in CHCl3; with SnCl2,a heavy, pale-yellow precipitate; with neutral lead acetatea red precipitate; and with Brady's reagent an immediatered precipitate. It immediately reduces ammoniacalAgNO3, and Fehling's solution is turned to green in the coldand is reduced to Cu2O at 1000. It is readily oxidized byDoeuvre's reagent with the formation of Hg, and by alkalinesodium hypoiodite with the formation of CHI3. Flavipingives a positive result in the Bargellini (1919) test, i.e. agreen colour and a green precipitate with sodium amalgam inethanol, indicative of three vicinal hydroxyl groups. It givesa negative result in the following reactions: (i) Schiff'sreagent, the colour is not restored. (ii) The Gibbs (1927) testwith 2:6-dichloroquinone chloroimide at pH 9-2. (iii) Thenitrochromic acid test for primary and secondary alcoholic-OH groups (Fearon & Mitchell, 1932). (iv) No coupling withdiazotized sulphanilic acid. When solid flavipin is exposed tothe vapour of conc. NH3 (d, 0.880) it quickly changes colourto dark brown. If then dissolved in aqueous conc. NH3 itgives a deep olive-brown solution, slowly changing tobrownish orange.

Flavipin exhibits strong antifungal properties in in-hibiting the germination of Botrytis allii spores and, to aneven greater extent, in inhibiting the germination of thespores of the strain of A. flavipes which produces it. Itpossesses only very weak antibacterial properties againsteither Staphylococcus aureus or Escherichia coli.

Functional derivatives of flavipinPentaacetylflavipin hydrate. A mixture of flavipin (0-2 g.),

anhydrous sodium acetate (0.4 g.) and acetic anhydride(2 ml.) was kept at room temp. for 16 hr., since heatingcaused charring. The crude acetate (0 4 g.) recovered as acolourless, amorphous solid by the addition of ice, wascrystallized from methanol (5 ml.) and cooled in acetone-solid C02, giving colourless crystals (65 mg.) which wererecrystallized from methanol. The acetate is dimorphic, slowand undisturbed cooling giving rods, m.p. 180-180-5°, whilenormal crystallization gives a mixture of needles and rods,m.p. 179-180° after rearrangement to the rod form at163-165°. (Found: C, 53 9; H, 4-85- C19H20011, ieC9H305,H20,5Ac, requires C, 53-8; H, 4-75 %.)A mixtureof flavipin (0-1 g.), acetic anhydride (1 ml.) and

conc. H2SO4 (1 drop) was warmed for a few seconds, cooledand treated with ice. The recovered colourless, crystallineproduct (0-19 g.), on crystallization from ethanol, gave an

26

acetate identical with that prepared by the previous method.[Found: C, 53-8; H, 4-7; C-CH3, 18.2 %; mol.wt. (cryoscopicin dioxan), 411. C19H20O11requires C, 53-8; H, 4-75; 6 C-CH3,21.25%; mol.wt. 424.]The acetate is insoluble in cold aqueous 2N-NaOH, gives

no colour with FeCl3 and no precipitate with Brady'sreagent, except on long standing, probably as a result ofpartial acid hydrolysis.

2:4-Dinitrophenylhydrazine derivative of flavipin. Asolution of 2:4-dinitrophenylhydrazine (0-45 g.) in almostboiling acetic acid (8 ml.) was added to a suspension offlavipin (0-2 g.) in cold acetic acid (2 ml.). A red crystallineprecipitate formed almost immediately. The mixture wascooled, shaken intermittently and filtered after 0 5 hr. Thecollected crystals were washed with acetic acid, then withethanol and finally with ether. The damp crystals werecrystallized from dimethylformamide, giving red rods(84 mg.), and addition of an equal volume of methanol to theacetic acid mother liquors gave a second crop (130 mg.).Recrystallization from dimethylformamide gave the 2:4-dinitrophenylhydrazine derivative as red rods, m.p. 242-243°(decomp.), which gave a purplish-red Neuberg reaction, i.e.colour of solution in ethanolic NaOH. (Found: C, 44-1; H,3-2; N, 19-5. C21H15O13N8requires C,43-9; H, 3-2; N, 19.5%.)

o-Phthalaldehyde mono-2:4-dinitrophenylhydrazone. Thisderivative was prepared by treating phthalaldehyde (0.1 g.)in cold acetic acid (1 ml.) with 2:4-dinitrophenylhydrazine(0-22 g.) in hot acetic acid (4 ml.) exactly as describedabove. Crystallization of the crude product from dimethyl-formamide gave o-phthalaldehyde mono-2:4-dinitrophenyl-htydrazone as slender orange-yellow needles (0-1 g.), m.p.198-199' (decomp.), which gave a purplish-red Neubergreaction. (Found: C, 53-7; H, 3-25; N, 17-6. C14HI005N4requires C, 53-5; H, 3-2; N, 17-8%.)

Flavipin anhydro-2:4-dinitrophenylhydrazone. A solutionof 2:4-dinitrophenylhydrazine (0.4 g.) in hot n-butanol(50 ml.) was added to a solution of flavipin (0.2 g.) in hotbutanol (10 ml.), and the mixture was held at 800 for 5 min.The deep-red rods (0-26 g.) which separated were purified bychromatographing in benzene solution on a talc column andeluting with ether-benzene (50:50), followed by recrystalli-zation from benzene. Flavipin anhydro-2:4-dinitrophenyl-hydrazone was thus obtained as deep-red rods, m.p. >300°,which give a pure-blue colour with Neuberg's reagent.(Found: C, 50-6; H, 3 0; N, 15-7. C15H1007N4 requires C,50 3; H, 2-8; N, 15-6%.)

Flavipin monomethyl ether. An excess of a solution ofdiazomethane in ether was added to a suspension of flavipin(0.2 g.) in ether. After standing at room temp. for 0.5 hr. theether and residual diazomethane were removed. The result-ing oil, dissolved in benzene (20 ml.), was chromatographedon acid-washed alumina and was eluted with CHC13. Theresidue obtained on removal of CHC13 was crystallized fromCHC13 and sublimed in high vacuum at 1300, giving flavipinmonomethyl ether (20 mg.) as stumpy yellow needles, m.p.196-197°. (Found: C, 56*8; H, 4-8; OMe, 15-2. C1oH1O05requires C, 57*1; H, 4-8; 1 OMe, 14-8%.) An ethanolicsolution of the product gives with ethanolic FeCI3 a reddishcolour which quickly turns brown and finally green. Itgives an orange precipitate with Brady's reagent. Allattempts to obtain a crystalline derivative by longermethylation with diazomethane, even in the presence ofmethanol, failed.

Methylation of flavipin with dimethyl sulphate. (a) Brief

Bioch. 1956. 63

Vol. 63 401

H. RAISTRICK AND P. RUDMANmethylation. A mixture of fiavipin (1 g.), dry acetone(75 ml.), anhydrous K2CO3 (4 g.) and dimethyl sulphate(3 ml.) was refiuxed for 1 hr., when the initial orange colourhad changed to pale yellow. The mixture was filtered and theacetone was removed from the filtrate. The residual oil wasdissolved in ether (30 ml.), and the solution, on longstanding, ultimately gave long, colourless needles (0.61 g.),m.p. 94-106°, which were purified by continuous extractionin a Soxhlet apparatus with ether, in which the substance isnot very soluble. Repetition of this process finally gave thesubstance as colourless needles with a constant m.p. of138.5-139'. (Found: C, 58-3, 57-9; H, 6-1, 6-3; OMe, 37-2,37-6; mol.wt. cryoscopic in dioxan, 441. CnH,oOll, i.e.[C,H.02(OMe),],, H,O requires C, 58-2; H, 6-1; 6 OMe,37.7%; mol.wt. 494.) This substance, which, for con-

venience, may be called diflavipin hexamethyl ether mono-

hydrate, gives no ferric colour but gives a crystalline orangeprecipitate with Brady's reagent. It dissolves slowly onshaking with aqueous 2x-NaOH.A solution of the methyl ether (0.2 g.) in ethanol (10 ml.)

was treated with Brady's reagent (15 ml.). The resultingorange precipitate (0.12 g.) was collected and recrystallizedfrom ethanol, giving trimethylflavipin bis-2:4-dinitrophenyl-hydrazone as slender orange needles, m.p. 216-217'(decomp.), which gave an orange-red colour with Neuberg'sreagent. (Found: C, 48-1; H, 3-7; N, 19-1; OMe, 14-9.CuH2O211N, requires C, 48-15; H, 3-75; N, 18-8; 3 OMe,15-6 %.)A mixture of fiavipin (0.1 g.), m.p. 229-230° (decomp.),

dry acetone (20 ml.) and anhydrous K,CO3 (1-0 g.) was

refiluxed for 0-5 hr. The portion undissolved in acetone wasseparated by filtration, dissolved in water (5 ml.) and acidi-fied with conc. HCL The resulting yellow precipitate A wascollected, washed and dried. The acetone filtrate was con-

centrated and acidified with HCO, giving a yellow precipitateB, which was combined with precipitate A, total wt. 78 mg.,

m.p. 230-2310 (decomp.). A mixture with the startingmaterial melted at 229-230o (decomp.). Hence the possi-bility may be ruled out that the structure of flavipin mighthave undergone rearrangement during methylation withdimethyl sulphate and K,CO, in acetone.

(b) Prolonged methylation. Methylation for a longer timeappeared to give a different methyl ether. A mixture offlavipin (1-0 g.), anhydrous K2CO, (4-0 g.), dry acetone(50 ml.) and dimethyl sulphate (6-0 ml.) was refluxed for6 hr. Water (100 ml.) was added, and the acetone andresidual dimethyl sulphate were removed by evaporation in

vacuo. The methyl ether was extracted with ether, theextract was dried (Na2SO) and evaporated. The residual oil(1-03 g.) was distilled in vacuo at 0-1 mm. and 111-116°,giving a colourless oil with a methoxyl content of 48-3 and48-7 %, which set to a mass of colourless needles on longstanding. C26H,,HO1, i.e.thedimethyl ether ofC,4H30011, thehydrated form of diflavipin hexamethyl ether, requires47-5% of OMe for 8 OMe groups; C,IH32010, i.e. the di-methyl ether of CuH28O10,the anhydrous form of diflavipinhexamethyl ether, requires49 2% ofOMe for 8 OMe groups.

However, this methyl ether, on treatment in ethanolicsolution with Brady's reagent as described above, gave a

2:4-dinitrophenylhydrazone in slender orange needles,giving an orange-red Neuberg reaction and having m.p. andmixed m.p. 216-217' on admixture with the trimethyl-flavipin bis-2:4-dinitrophenylhydrazone of the same m.p.

mentioned above.

Phthalazine derivative of flavipin. (a) From hydrazinehydrate. A solution of flavipin (0.2 g.) and 50% hydrazinehydrate (0 1 ml.) in 50% (v/v) aqueous ethanol (10 ml.) waskept at room temp. for some hours. The golden-yellow rodswhich separated were collected (0.15 g.) and recrystallizedthrice from ethanol, giving the phthalazine derivative offlavipin as yellow rods, m.p. >3000. (Found: C, 56-05; H,3-9; N, 14-3. C5H803N2 requires C, 56-0; H, 4-15; N, 14-5 %.)In ethanolic solution it gives with ethanolic FeCl3 a blue-black colour which becomes orange on the addition ofwater.It gives no precipitate with Brady's reagent. Its solution in10% aqueous NH3 becomes red in colour on acidificationwith acetic acid.

(b) From 8emicarbazide hydrochloride. A solution offlavipin (0.4 g.) in ethanol (5 ml.) was added to a solution ofsemicarbazide hydrochloride (0-8 g.) and sodium acetate(1.2 g.) in distilled water (20 ml.). The mixture was held at900 for 5 min. The crystals which separated on cooling werecollected (0.225 g.), combined with a second crop (0.032 g.)from the concentrated mother liquors and crystallized frommuch hot water. The product, golden-yeilow rods, m.p.>300°, gave similar reactions to those described in (a)above and the ultraviolet absorption spectra in ethanol ofthetwo specimens were almost identical (see p. 404). (Found:C, 53-6; H, 4-4; N, 13-5. C,H80,N2,IH20 requires C, 53-7;H, 45; N, 13.9%.)

REACTION AND OXIDATION PRODUCTS OFFLAvIPIN AND ITS DERIVATIVES

(a) Action of NaOH on me*hylated flavipin. Isolation of4:5:6-trimethoxy-7-methylphthalide (II). The crude oilymethyl ether of flavipin (0.58 g.) obtained by methylationwith dimethyl sulphate and K2CO in acetone (see above)was refluxed for 15 min. with aqueous NaOH (10 ml. of40 %). The cooled alkaline solutionwas extracted with ether.Removal of the solvent from the ether extract gave 314 mg.of unchanged starting material. The extracted alkalinesolution was acidified to Congo red with HCI and kept over-night. The colourlesscrystalswhich separatedwerecollected,washed with water and dried (227 mg.), m.p. 60-78'. Theywerepurified by crystallization from ethanol + charcoal,thenlight petroleum (b.p. 80-100') and finally by sublimation inhigh vacuum. The colourless crystalline sublimate melted at87-5-88.50, not depressed on admixture with an authenticsynthetic specimen of 4:5:6-trimethoxy-7-methylphthalideof the same m.p. (see p. 404). (Found: C, 60-4; H, 5-8; OMe,39 0. C12HL40, requires C, 60-5; H, 5B9; 3 OMe, 39.0%.)

(b) Oxidation of flavipin with alkine H,02. Formationof C02, formic, methylnalonic and tartronic acids. H202(9-3 ml. of 90-100 vol.), followed by aqueous NaOH(20.0 ml. of N) were introduced through a tap funnel into aflask containing flavipin (1 g.) and through which a currentofN2 freed from 02 and CO. was passed. The issuing gas waspassed through bubblers, the first of which containedBrady's reagent for the detection of volatile aldehydes orketones, while the later ones contained standard Ba(OH)2.The flask was immersed in ice water to moderate theinitially very vigorous reaction. After 0-5 hr. the reactionmixture had become acid and was further acidified-with HCI(20-0 ml. of N) to neutralize the NaOH initially added. N,was passed for a further 1.5 hr. No volatile aldehydes orketones were detected. CO, (0-32 g. and 0 34 g. in duplicateexperiments) equivalent to about 1-5 mol. was estimated.

A40-2 I95)6

FLAVIPIN FROM ASPERGILLUS FLAVIPESThe reaction solution was then distilled in vacuo to

remove volatile acids completely, and its volume was main-tained by the addition of water. The distillate was titratedagainst N-NaOH and insix separate experimentsthe averageamountof 10-3 ml. ofN acidwasestimated; calc. for 2 mol. ofa monobasic acid = 10-2ml. ofN. Formicacidwas shownto bepresent in the distillate by the chromotropic acid test afterreductionto formaldehyde, and in a quantitative estimationby the reduction of HgCl2 to Hg2Cl2 (Fincke, 1913) wasshown to constitute about 75% of the total volatile acid.The reaction solution, from which volatile acids had been

removed, was evaporated to dryness in vacuo. The resultingcolourless crystalline solid was thoroughly dried in vacuoover conc. H2SO4 and was then continuously extracted withNa-dried ether for 8 hr. The ether extract was evaporated todryness, giving a colourless mass of gummy crystals, m.p.90-130' and decomposing at 1400. It had an average wt. insix experiments of 0-435 g. In aqueous solution it wasstrongly acid to Congo red and was obviously a mixturewhich could not, however,be separatedeither bycrystalliza-tion or sublimation. Oxalic acid was present only in traces.A portion of the mixed non-volatile acids (0-185 g.) was

neutralized to pH 6-7-7-0 with NaOH, and a solution of p-bromophenacyl bromide (0.74 g.) in ethanol (10 ml.) wasadded. The mixture was refluxed for 3 hr. and the colourlesssolid (0-26 g.), m.p. 137-139°, which separated on coolingwas collected and crystallized from ethanol, giving colour-less needles, m.p. 161-161.5', not depressed on admixturewith the authentic di-p-bromophenacyl ester of methyl-malonic acid, m.p. 163-164.5' (see p. 404). (Found: C, 46-8;H, 3-3; Br, 31-2. CwH1*O,Br2 requires C, 46-9; H, 3-15; Br,31-2 %.) A second portion of the mixed non-volatile acids(107 mg.) was converted in a similar manner into the di-p-phenylphenacyl ester by heating with p-phenylphenacylbromide (0-735 g.). The crude ester obtained (0-155 g.),m.p. 155-157°, wascrystallized from ethanol,giving the di-p-phenylphenacyl ester of methylmalonic acid as colourlessplates, m.p. 172-173.5'. A mixture of it with an authenticsynthetic specimen, m.p. 176-177-5° (see p. 404), melted at175-176.50.

Tartronic acid was identified by paper chromatography asthe second major constituent ofthe mixednon-volatile acids.Whatman's no. 1 paper was used, water being the stationaryphase and n-butanol containing 1% formic acid the mobilephase. Bromocresol purple was used for development. Thefollowing RF values were obtained: (i) methylmalonic acid,0-88; (ii) tartronic acid, 0-62; (iii) equimolecular mixture ofmethylmalonic and tartronic acids, 0-875 and 0-60; themixed non-volatile acids, 0-87 and 0-61. The presence oftartronic acid was confirmed by the fact that both authentictartronic acid and the mixed non-volatile acids gave a deepcherry-red colour on heating with conc. H2SO4 and re-sorcinol, and a red-purple colour rapidly changing to blue-purple on heating with conc. H3SO4 and pyrogallol. Un-successful attempts to prepare either the p-bromophenacylor p-phenylphenacyl esters of authentic tartronic acidexplain the failure to detect the esters of this acid in themixtures from which the corresponding esters of methyl-malonic acid were successfully isolated.

(c) Oxidation of mehylated flavipin with cold alkalineKMnO4. Isolation of 4:5:6-trimethoxy-3-methylphthalic an-hydride (III) and 4:5:6-trimethoxybenzene-1:2:3-tricarboxylicacid (IV). A mixture of the crude oily methyl ether offlavipin (1-45 g.) made by methylation with dimethyl

sulphate and K2CO3 in acetone (see p. 402), aqueousKMnO4 (60 ml. of 5%) and saturated aqueous Na2CO3(27 ml. of 20%) was stirred at room temp. for 55 hr. Themixture was then acidified with dil. H2S04, and SO2 waspassed to dissolve MnO,. The solution was again madealkaline with Na2CO3 and extracted with ether to removeunoxidized flavipin methyl ether (0-21 g.). The extractedsolution was reacidified with H2SO4 and continuouslyextracted with ethyl acetate for 8 hr. The dried (Na2SO4)extract was freed from solvent and the crystalline residue(0-75 g.), m.p. 165-170°, was fractionally crystallized fromhot water, giving fractions A, separating as colourlessneedles, and B, obtained as colourless rods on evaporation ofthe aqueous mother liquors. Fraction A. 0-15 g.; m.p. 90-1080, was purified by repeated sublimation in high vacuumto give colourless needles (0-13 g.), m.p. 110-1lIP, notdepressed on admixture with authentic synthetic 4:5:6-trimethoxy-3-methylphthalic anhydride, m.p. 111.5-1120(see p.405). (Found: C, 56-9; H, 5-1; OMe, 36-7; C-CH3, 5-7.C12H120, requires C, 57-15; H, 4-8; 3 OMe, 36-9; 1 C-CH3,6-0%.) The substance is not acid to litmus, does not dis-solve in cold aqueous NaHCO3 or Na2CO,, but dissolvesreadily in hot aqueous NaOH. Fraction B. 0-34 g.; m.p.167-1680, was purified by crystallization from ethyl acetate-benzene, giving colourless rods, m.p. 172.5-1730, not de-pressed on admixture with authentic 4:5:6-trimethoxy-benzene-1:2:3-tricarboxylic acid, m.p. 174.5-175-50 (seep. 405). (Found: C, 48-1; H, 4-0; OMe, 30-7. C12HI20,requires C, 48-0; H, 4-0; 3 OMe, 31-0%.)

(d) Oxidation of crystalline diflavipin hexamethyl etherhydrate with cold alkaline KMnO4. Isolation of 4:5:6-tri-methoxy-3-methylphthalic anhydride (III). This crystallinemethyl ether (0-2 g.) was shaken for 48 hr. with a mixture ofaqueous KMnO4 (7 ml. of 5%) and aqueous Na,CO0 (4 ml. of20%). The weight of unchanged methyl ether recovered was40 mg. The oxidation product was worked up as described insection (c) above and gave a crystalline ethyl acetateextract (82 mg.) which was stirred with dilute aqueousNa2CO, (4 ml.). The portion insoluble in Na2CO8 wascollected, dried and heated above its m.p. (109-111°) for2 min. Itwasthen sublimed in high vacuum at 1000, givingcolourless needles (50 mg.), m.p. 111.5-112', not depressedon admixture with authentic 4:5:6-trimethoxy-3-methyl-phthalic anhydride of the same m.p.

(e) Oxidation of the phthalazine derivative of flavipin withalkaline KMnO4. Isolation of pyridazine-4:5-dicarboxylicacid (V). Aqueous KMnO4 (3-7 g. in 185 ml. of water) wasadded at intervals over a period of 20 min. to a stirredmixture of the phthalazine derivative, C9,H80,N2, (0-84 g.)(see p. 402) in boiling water (80 ml.) and aqueous KOH(2-5 ml. of 2N). Excess of KMnO4 was decomposed withethanol and SO0 was passed through the cooled solution toremove Mn02. The solution was acidified with H2SO4 andextracted for 8 hr. with ethyl acetate. Removal of thesolvent from the extract gave a sandy, crystalline oxidationproduct (0-47 g.), which was purified by three crystalliza-tions from boiling water + charcoal. Pyridazine-4:5-dicar-boxylic acidwas thus obtained as colourless plates (0-16g.),m.p. 212.5-213' (decomp.) alone or in admixture with anauthentic synthetic specimen m.p. 209-210' (see p. 405).Both specimens began to decompose at 2000. Gabriel (1903)gives the m.p. as 212-213-5° with decomposition from 2050.(Found: C, 43-0; H, 2-7; N, 16-25. Calc. for C*H404N2:C, 42-9; H, 2-4; N, 16-7%.)

26-2

Vol. 63 403

H. RAISTRICK AND P. RUDMAN

Table 5. Fungistatic activity offlavipin against Botrytis allii and Aspergillus flavipesat 240 and pH 3-5 or 6-0

0 =no germination; (1) = <1% germination; 1= 1-5% germination; 2 =5-50% germination; 3 =50-90% germination;4=90-100%, i.e. complete germination; - =not tested.

Conen. of flavipin (,ug./ml.)Ax _---

OrganismBotrytis alliiAspergiUus flavipesA. flavip&s

pH 80 40 203.5 - - 03.5 - - 06-0 0 (1) (1)

15 10 7-5 50 1- 0

(1)

2 30

(1)

4 2-5 1-254 4- 0

- (1)11

Antifungal tests

The fungistatic activity of flavipin was determined by themethod described by Brian & Hemming (1945). A series ofdilutions of flavipin in a sterile germination medium of thefollowing composition were made up: glucose, 12-5 g.;

ammonium tartrate, 1-00g.; KH2PO4, 1-00g.; MgSO4,7H2O,0-50 g.; distilled water 1000 ml.; pH adjusted to either 3-5 or6-0. Each dilution was inoculated with the conidia of thefungus under test and then incubated for 24 hr. at 240. Theproportion of conidia which had germinated was deter-mined and the results obtained are given in Table 5, thedegrees of inhibition being those adopted by Brian &Hemming (1945).The two test fungi used were the strain of Botrytis allii

Munn used and kindly supplied by Dr P. W. Brian, andAspergillus fiavipes (Bainier & Sartory) Thom & Church,strain L.S.H.T.M. catalogue no. S.M. 884, i.e. the same strainas was used in this work for the preparation of flavipin.

Ultraviolet absorption spectra

The spectra numbered 2, 3, 4, 6 and 7 given below were

carried out in this Department in collaboration withDr J. H. Birkinshaw. They were determined by means of aHilger and Watts Uvispek spectrophotometer. We are

indebted to Dr Gunhild Aulin-Erdtman for the spectranumbered 1 and 5.Numbers 1, 2, 3 and 4 were determined in dioxan solution,

5, 6 and 7 in ethanol solution. The following results were

obtained:(1) Flavipin, A 261 miu. (loge 4.37); 264 m,u., 4-35;

330 my., 3-94 (broad band).(2) Pentaacetylflavipin hydrate. Inflexion at 256-

257 m/A., 2-23; 264 mju., 2-35.(3) Crystalline diflavipin hexamethyl ether hydrate.

243-246m,t., 3-03; 275-277m,4., 3-18; inflexion at 310-318 mus., 2-17.

(4) Oily methyl ether, C26H340,1. 279 mb., 3-21.(5) 4:5:6-Trimethoxy-3-methylphthalic acid. 212 mut.,

4-45; inflexion at 240-250 minz., 3-7-3-8; 287 m 3-17.(6) Phthalazine derivative via hydrazine hydrate.

220 mi/., 4-13; 283 m,u., 4-37; 385-386 miu., 3.74.(7) Phthalazine derivative via semicarbazide hydro-

chloride. 217 miu., 4-03; 283 mi/., 4-27; 384-387 m,u., 3-66.

Infrared absorption 8pectrum

The infrared absorption spectrum of flavipin was kindlydetermined for us by Dr G. Aulin-Erdtman and Dr K. E.Almin. Theyused a Perkin-Elmersingle-beam (double-pass)

instrument and determined the spectrum of freshly sub-limed flavipin in a solid mixture with KBr. They found thefollowing absorption bands, the figures in brackets aftereach band indicating the percentage absorption: 3226 cm.-'(60 %); 3067 (57); 1658 (79); 1639 (65); 1615 (61); 1489 (48);1454 (78); 1390 (57); 1349 (79); 1330 (75); 1303 (56); 1264(86); 1217 (69); 1145 (60); 1115 (81); 1060 (41); 1039 (31);1016 (35); 1002 (33); 972 (75); 932 (55); 765 (48).

SYNTHESES

Methylmalonic acid, HO2C. CH(CH3) .CO2H, was preparedby the hydrolysis of its diethyl ester, which was synthesizedfrom diethyl malonate substantially as described by Conrad& Bischoff (1880). It forms colourless needles from ethylacetate, m.p. 130-133-5° (decomp.). The m.p. given in theliterature varies between 1200 and 1350 (decomp.).

Di-p-bromophenacyl ester of methylmalonic acid was pre-pared from methylmalonic acid and p-bromophenacylbromide as described on p. 403. The ester forms colourlessplates, m.p. 163-164-5°. (Found: C, 46-8; H, 3-3; Br, 32-1.C20H,6O6Br2requires C, 46-9; H, 3-15; Br, 31-2%.)

Di-p-phenylphenacyl ester of methylmalonic acid was pre-pared from methylmalonic acid and p-phenylphenacylbromide as described on p. 403. The ester forms colourlessplates, m.p. 176-177.5'. (Found: C, 75-7; H, 5-3. C32H2606requires, C, 75-9; H, 5.2%.)

7-Chloromethyl-4:5:6-trimethoxyphthalide was preparedaccording to the methodofKing & King (1942). A mixture ofthe trimethyl ether of gallic acid (Mauthner, 1932; 10 g.),aqueous formaldehyde (25 ml. of 40%) and conc. HCI(40 ml.) was refluxed for 20 min. in an oil bath at 1400. Oncooling and addition of water, the oily reaction productsolidified and was collected. It was fractionally crystallizedfrom ethanol, giving, as the more soluble fraction, 7-chloromethyl-4:5:6-trimethoxyphthalide (3-88 g.) in colour-less needles, m.p. 84-85°. King & King (1942) give the m.p.as 850.

4:5:6-Trimethoxy-7-methylphthalide. A solution in ethanolof 7-chloromethyl-4:5:6-trimethoxyphthalide (1.0 g.) wasshaken with a 5% palladium oxide on carbon catalyst(1-0 g.) in hydrogen at atmospheric pressure. The uptake ofH2 (95 ml.) was complete in 10 min. The catalyst wasremoved by filtration and, on removal of the solvent fromthe filtrate, there remained long colourless needles (0-84 g.),m.p. 80-850. Crystallization from ethanol gave 4:5:6-trimethoxy-7-methylphthalide as colourless needles, m.p.87-88.50, not depressed on admixture with the product ofthe action of NaOH on methylated flavipin, m.p. 87-5-88.50 [see p. 402, section (a)]. (Found: C, 60-5; H, 5-9;C-CH3? 8-1. C12H140r requires C, 60-5; H, 5-9; 1 C-CH3,

404 I956

Vol. 63 FLAVIPIN FROM ASPERGILLUS FLAVIPES 40563 %.) The substance is very soluble in ethanol, CHC13,ethyl acetate, and moderately soluble in light petroleum(b.p. 80-100°). It can be readily sublimed in high vacuum.

4:5:6-Trimethoxybenzene-1:2:3-tricarboxylicacid. 7-Chloro-methyl-4:5:6-trimethoxyphthalide (1-0 g.) was refluxedfor 3 hr. with aqueous KOH (40 ml. of 0-03N), and KMnO4(6-0 g.) was added at intervals. The residual KMnO4 wasthen destroyed by adding methanol (1 ml.). PrecipitatedMnO2 was separated by filtration and oxalic acid was re-moved from the filtrate by adjusting the pH to 2-0 with HCIand addition of CaCl2. Precipitated calcium oxalate wasremoved by centrifuging, and the clear supernatant, afterfurther acidification with conc. HCI (3-0 ml.), was con-tinuously extracted with ethyl acetate for 8 hr. The extractwas dried (Na2SO4) and the solvent removed to give slightlygummy needles (0-42 g.) which were purified by crystalliza-tion from dry ethyl acetate + charcoal, giving 4:5:6-trimethoxybenzene-1:2:3-tricarboxylic acid as colourless rods,m.p. 174-5-175-5°, which did not depress the m.p. 172-5-1730 of the KMnO4 oxidation product of the oily methylether of flavipin [see section (c), fraction B, p. 403]. (Found:C, 48-25; H, 4-2; OCH3, 31-0. C,2H12O9 requires C, 48-0;H, 4 0; 3 OMe, 31-0%.)

4:5:6-Trimethoxy-3-methylphthalic anhydride. 4:5:6-Tri-methoxy-7-methylphthalide (0-23 g.) was refluxed for10 min. with aqueous KOH (37 ml. of 0.03N). KMnO4(1-0 g. in H20, 25 ml.) was added to the cooled solution whichwas kept at room temp. for 24 hr. Excess of KMnO4 andMnO2 were removed as described above. The residualsolution was strongly acidified with HCI, saturated withNaCl and thoroughly extracted with ethyl acetate. Removalof the solvent from the extract gave a colourless product(0-17 g.) from which free acid was removed by shaking itssolution in ether with aqueous NaHCO3. The extractedether solution was evaporated to dryness and the residuewas purified by repeated sublimation in high vacuum at100° and crystallization from light petroleum (b.p. 80-100°),giving 4:5:6-trimethoxy-3-methylphthalic anhydride as colour-less needles, m.p. 111-5-112°, not depressing the m.p. 111-5-1120 of the KMnO4 oxidation product of diflavipin hexa-methyl ether monohydrate [see section (d), p. 403]. (Found:C, 57-2; H, 4 9; OMe, 36-9. C,2H1206 requires C, 57-1;H, 4-8; 3 OMe, 36.9%.)

Pyridazine-4:5-dicarboxylic acid (Gabriel, 1903). Phthal-azine (Gabriel & Pinkus, 1893) was synthesized by treatinga mixture of phthalaldehyde (3.0 g.), NaOH (33 ml. of 2N)and water (100 ml.) with hydrazine sulphate (3.4 g.). Themixture was well shaken, concentrated to 20 ml. andextracted with benzene. The benzene solution was ex-tracted with HCI and the extract was made alkaline and wasre-extracted with benzene. Removal of the benzene gavecrude phthalazine (1-5 g.).Aqueous KMnO4 (100 ml. of 2-5 %) was added gradually

to a mixture, kept boiling, of phthalazine (0.4 g.) and NaOH(1 ml. of 2N). Excess of KMnO4 and precipitated MnO2were removed as described above. The filtrate was concen-trated to 5 ml., acidified with H2SO4 and extracted withethyl acetate. After removal of the solvent the pale-brownsolid residue (0.10 g.) was purified by sublimation in highvacuum at 1200 and crystallization from ethyl acetate.Pyridazine-4:5-dicarboxylic acid was thus obtained ascolourless plates, m.p. 209-210° (decomp.), which did notdepress the m.p. of the KMnO4 oxidation product of thephthalazine derivative of flavipin [see section (e), p. 403].

SUMMARY

1. A hitherto undescribed mould metabolite,now named flavipin, has been isolated from culturefiltrates of Aspergillus flavipes (Bainier & Sartory)Thom & Church grown on Raulin-Thom solutionand of A. terreus Thom grown on Czapek-Doxsolution.

2. Flavipin, CgH,05, forms pale-yellow rods,m.p. 233-234° (decomp.).

3. A number of its functional derivatives anddegradation products are described, from thestructures of which it is deduced that flavipin is1:2-diformyl-4:5:6-trihydroxy-3-methylbenzene.

4. Flavipin is the third mould metabolite to bedescribed which is a substituted o-phthalaldehyde,the other two being gladiolic acid from Penicilliumgladioli Machacek and cyclopaldic acid from P.cyclopium Westling. All three substances arepowerful antifungal agents.

5. Flavipin is also a substituted pyrogallol, aclass of compounds which occur only rarely asfungal metabolic products.

One of us (P.R.) wishes to thank the Agricultural Re-search Council for an Agricultural Research Studentshipwhich has enabled him to take part in this work. We are alsograteful to Professor and Mrs Holger Erdtman of the RoyalTechnicalHigh School, Stockholm, and to Professor D. H. R.Barton, F.R.S., for many helpful discussions on the structureof flavipin.

REFERENCES

Bargellini, G. (1919). Gazz. chim. ital. 49, 47.Barton, D. H. R. & Hendrickson, J. B. (1956). J. chem. Soc.

p. 1028.Bernhard, K. & Albrecht, H. (1947). Helv. chim. acta, 30,

627.Birkinshaw, J. H., Bracken, A., Michael, S. E. & Raistrick,H. (1951). Biochem. J. 48, 67.

Birkinshaw, J. H., Raistrick, H., Ross, D. J. & Stickings,C. E. (1952). Biochem. J. 50, 610.

Brian, P. W., Curtis, P. J. & Hemming, H. G. (1948). J. gen.Microbiol. 2, 341.

Brian, P. W. & Hemming, H. G. (1945). Ann. appl. Biol. 32,214.

Calam, C. T., Oxford, A. E. & Raistrick, H. (1939).Biochem. J. 33, 1488.

Conrad, M. & Bischoff, C. A. (1880). Liebigs Ann. 204,146.

Edwards, G. A., Perkin, W. H. jun. & Stoyle, F. W. (1925).J. chem. Soc. 127, 195.

Fearon, W. R. & Mitchell, D. M. (1932). Analyst, 57, 372.Fincke, H. (1913). Biochem. Z. 51, 253.Gabriel, S. (1903). Ber. dtsch. chem. Ges. 36, 3373.Gabriel, S. & Pinkus, G. (1893). Ber. dtsch. chem. Ges. 26,.

2210.Galarraga, J. A., Neill, K. G. & Raistrick, H. (1955).

Biochem. J. 61, 456.Gibbs, H. D. (1927). J. biol. Chem. 72, 649.Grove, J. F. (1952). Biochem. J. 50, 648.

406 H. RAISTRIOK AND P. RUDMAN I956Kent, J. & Heatley, N. G. (1945). Nature, Lond., 156,295.King, F. E. & King, T. J. (1942). J. chem. Soc. p. 726.Mauthner, F. (1932). Organic Synthe8es, Collective vol. 1,

522. New York: John Wiley and Sons Inc. London:Chapman and Hall Ltd.

Michael, S. E. (1948). Biochem. J. 43, 528.Pansacker, B. F., Philpot, F. J., Jennings, M. A. & Florey,H. W. (1947). Quoted in Antibiotice, Florey et al. (1949),1, 337. London: Oxford University Press.

Raistrick, H. & Ross, D. J. (1952). Biochem. J. 50, 635.Raistrick, H. & Smith, G. (1935). Biochem. J. 29,

606.Raistrick, H. & Smith, G. (1936). Biochem. J. 30,

1315.Stodola, F. H., Friedkin, M., Moyer, A. J. & Coghill, R. D.

(1945). J. biol. Chem. 161, 739.Thom, C. & Raper, K. B. (1945). A Manual of the Aspergilli.

Baltimore: Williams and Wilkins.

The Identification of the Major Diazotizable Metaboliteof 4:4'-Diaminodiphenyl Sulphone in Rabbit Urine

BY S. R. M. BUSHBY AND A. J. WOIWODWelkome Re8earch Laboratoriea, Beckenham, Kent

(Received 16 September 1955)

Titus & Bernstein (1949), while studying the phar-macology ofwater-soluble disubstituted derivativesof 4:4'-diaminodiphenyl sulphone (DADPS) in miceand dogs, found that DADPS itself was excretedmainly as a water-soluble substance which wasreadily hydrolysed by dilute acid to free DADPS.Lowe (1952) also reported a similar change in thedrug given to man for the treatment of leprosy andshowed that as much as 80% of the diazotizablematerial excreted in the urine was present in thisform.DADPS is regarded as the most effective drug

available for the treatment of leprosy and in viewof the low proportion of the drug excreted in thefree form it seemed probable, especially ifthe water-soluble derivative were also the major constituentin the blood, that the antileprotic activity of thedrug was due to this acid-labile metabolite. Wetherefore examined by paper chromatography andpaper ionophoresis the excretion products ofDADPS, and in a preliminary communication(Bushby & Woiwod, 1955) we reported the isolationof the major diazotizable excretion product fromrabbit urine and its identification as a monoconju-gate with glucuronic acid. We also reported thesynthesis of the conjugate. In this paper the syn-thesis of this conjugate is described, and theevidence for its presence in urine and blood ispresented.

EXPERIMENTALPaper chromatography. The apparatus and procedures

used were those of Woiwod (1949). The solvents used weren-butanol saturated with water, n-butanol-acetic acid-water (12-5:3: 125, by vol.), phenol saturated with waterand n-propanol-n-butanol-water (2:3:5, by vol.). No. 4Whatman papers were washed with several changes ofdistilled water in an enamelled tray and dried at 370before use.

lonophores8i. Samples were run on strips ofno. 4Whatmanpaper with 0 05m-Na2HPO4 as electrolyte and a potentialgradient of 7-0v/cm.

Detection ofprimary aromatic amino group8. Two reagentsprays were used. (1) A 1% (w/v) solution of p-dimethyl-aminobenzaldehyde in 20% (v/v) conc. HCl-ethanol,diluted with 19 vol. of ethanol just before use. Colour wasdeveloped at room temp. To avoid background colorationthe ethanol was redistilled from KOH and KMnO4. (2) Asolution of NaNO3 [05% (w/v) in 20% (v/v) N-HCl-ethanol], followed, after the paper had nearly dried, by0-1% (w/v) N-(l-naphthyl)ethylenediamine dihydro-chloride in the same solvent.

Detedion of glucuronic acid. Aniline hydrogen phthalate,benzidine and naphthoresorcinol sprays were used. Thesensitivity of the last two reagents was markedly improvedby using phosphoric acid in the spray as suggested byBryson & Mitchell (1951).

E8timation of glucuronic acid. Glucuronic acid in materialeluted from paper was determined either by the copper-reduction method of Shaffer & Somogyi (1933) or the colori-metric procedure of Jones (1954). The latter gave much lesscolour with glucuronic acid than with glucose and otherreducing sugars owing to the SnCl2 in the reagent. When thiswas replaced by Na3SO3 the colour obtained with glu-curonic acid equalled that given by glucose. The modifiedreagent consisted of 0-2% (w/v) benzidine in acetic acidcontaining 0 05% (w/v) Na,SO8. The fine precipitate whichformedwas filtered offafter 30 min. and the reagent was usedimmediately.

Synthesi8 of the sodium salt of the acid-labileconjugate ofDADPS with glucuronic acid

Sodium glucuronate (4.3 g.) in 15 ml. ofwater adjusted topH 5*5 was added to diaminodiphenyl sulphone (5 g.) in15 ml. ofacetone. The mixture was refluxed for 1 hr., 100 ml.of water added, the precipitated DADPS removed by filtra-tion and the filtrate concentrated in vacuo to about 50 ml.Afteradjustment to pH 7-0, the solutionwas extracted threetimes with equal volumes of phenol equilibrated with0-067M phosphate buffer (pH 7.4). This extracted a large