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THE CHEMISTRY OF THE "AMINOCHROMES"*P A R T I. T H E P R E PA R A T IO N A N D P A P E R C H R O M A T O G R A PH Y O F P U R EA D R E N O C H R O M E 1

A B S T R A C TTh e prepara t ion of adre~iochrorn e n a pu re s tab le c rys ta l line form has been car r ied out b yth e s i lvcr ox ide os icla tion of ad rena l ine in ~ ne th an ol i th th e use of a n an ion-exchange res in( D o w e s -l ( C 1 -) ) t o r e m o v e h e a v y m e t a l i o n s f ro m t h e r ea c t io n l n i s t ~ ~ r erior to the isolationof the prod uct . I ts paper chromatographic behavior toge ther wi th tha t o f th ree der iva t ives( a d r e n o l u t in , a d r c n o c h r o m e n ~o n o r e m i c a r b a z o n e ,and adrenochrome monoisonico t in ic ac idhydraz ide) in s ix d i f fe ren t so lvent sys tems has been examined . \Water was found to be theb e s t p a p e r c h r o m a t o g r a p h i c s o l v e n t s o fa r e s a m i n e d f o r t h i s se r ie s of C O I I I ~ O L I I I ~ S .

Adre~lochrome I), the su bsta~ lcemainly responsible for the red colors produced duringthe mild oxidation of ad renaline (11), was hrs t isolated from the p roducts of an enzym aticoxidation by Green and Richter ( I) .Subsequen tly, inorganic oxidizing agent s, particul arly

silver oxide, were successfully employed and i t was observed that adrenochrome could becrystallized from methanol (containing a little formic acid) at -80" (cf. Veer ( 2 ) ; Mac-Carthy (3 ) ; Harley-Mason (4 ) ;Sobo tka and Austin ( 5)). Green and Richter reported th atadrenochrome was unstable both in the solid stat e and in solutio~ l1) however, Sobotkaand Austin claim th at the dry crystalline nlaterial can be liept indefinitely at 0" (5).Th e stabili ty of aqueous solutions of adrenochrome has been shown to bemarliedly affect edby pH (cf. Zambotti and i\Joret (7)); under alkaline conditions and in the presence ofcertain metallic cations, particularll. zinc, adrenochrome readily rearranges into adre-nolutin (N-n1ethyl-3,5,G-i11doletriol,11) (cf. Lund (8) ; Fischer, Derouaux, Lainb ot, andLecomte (9) ; Harley-h11ason and Bu'Locli (10) ; Fischer and Lecomte (11)).

Ma nz~ scri pt eceived Decernber 11 , 1.957.Contr ibzi t iofz fro m the Psychiatr ic Research Un it , U?zivers i ty Hosp i tnl , Saskntoon, Saskatchewan. Th isinvestigation was supported b y grants froriz the Departnzeltt of Healt h and Wel far e, Ott awn, and the RockefellerFoundation. Th is work was carried out under the az~spic es f the Saskatchewan Conzmit tee on SchizophreniaResearch.*T he term "anzinochronzes" was suggested b y Sobotka afzd Au st in (5 ) for the highly colored cyclic oxidationproducts of P-(S,4-dihydroxypheny1)-ctltylaminesnd re lated compo~~?zds .hese szibstances are often ornzulatedas substituted 2,s-dilzydroindole-5,6-qz~iqiones,lthou gh there is some ulzcertainty us to the existence of a trueo-quinonoid strzicture (4 , 6) .Can. J. Chem. Vol. 3G (1958)

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854 C.-\NADIAN JOURNAL OF CHEMISTRY. VOL. 36 . l95SRzlany samples of adrenochrome, prepared by existing methods, th at were available to

the authors appeared to contain varying amounts of adrenolutin (sho\vn paper chromato-graphically by a methocl to be discussed late r) and black water-insoluble melanin-liliecompounds. I t appeared no t improbable th at contamination of the products by metal in acolloidal form or metallic ions, derived from the oxidizing agent, might be respoilsible forthe deteriorat ion of the samples and for the ap parent instability of their aqueous solu-tions. In order to eliminate or drastically reduce the contamination by silver ions, theadrenaline - ilver oxide reaction mixture in methanol was filtered through a n anion-exchange resin bed in the chloride form prior to crystallizatioil (at -20) in orcler toremove ionic silver from solution as silver chloride. In this manner a highly crystallineproduct containing only traces of silver was obtained ( k0 .0 17 0) .

Th e paper chromatograpllic behavior of adrenochrome and rela ted compouncls hasrecently been studied by Fischer using n-butano1:aceticacid: water (8:2:2) andi-propanol:0.880 amm onia: water (8 :l :l ) solvent systems (12). The results reportecl were unsatis-factory, since some of the substances apparent ly decomposed in these solvents. Thi s is notaltogether surprising in view of the known sensitiv ity of these compounds toward aciclsand alltalies (7, 8, 9),aild furthermore Pavolini, Gambarin, ancl Godenigo in a study 011 theaction of ammonia on some 5,G-indolequinoiles, including adrenochrome, reportecl adefinite reaction in this case with the format ion of an unstable substance, exhibiting ayellow-green fluorescence, \vhich rapidly turns brown (13). We have repeated Fischer'sworlc and observed with the i-propanol-ammonia system tha t the bright red non-fluores-cent adrenochrome spot rapidly turned brownish-yellow in color and exhibited a yellow-green fluorescence, even on exposure to am rno~li a umes, and in contact with the solventthe spot soon became dark brown indicating that marlied decomposition had occurred.In the acid solvent system, a slower but definite decomposition of the adrenochrome spotwas observed during the chromatography. We have found th at dilute aqueous solutions ofadrenochrome prepared b y our method are relatively stable and tha t distilled water and2% acetic acid in water are the most suitable solvents for paper chromatography. Pureadrenochrome gave a single spot R f0. 8&0 .02 in water on previously washed Whatm anNo. 1 paper. Aclrenolutin had an R f of ca . 0.4 under these conclitions. Contamination ofadrenochrome samples with adrenolutin can easily be observed paper chroinatographi-cally, since the characteristic yellow adrenolutin spot showing a strong yellow-greenfluorescence is sooil visible behind the red adrenochrorne spot ; this can be observed veryquickly as it is only necessary to allo\\l the chromatogram to run a few inches in order tosee the two spots. The adrenolutin spot is quite distinct and not a diffuse "tail" to theadrenochrome spot, indicating that the ilnpurity was present initially and not formedby continuous isomerizatioil of the sta rti ng material. Polyineric melanin-type impuritiess t a r in the position of the original spo t. An interesting phenomenon was observed 011 clry-ing of the paper chromatograms; as the paper was allowed to d ry a t rooin temperature,the color of the adrenochrome spot gradually changed froin red to yello\v-bro\vn and thespot began t o exhibit the characteristic adrenolutin fluorescence. T he partial formationof adrenolutin was confirmed by two-dimensional chromatography, using water asrunning solvent in both di rections, although only one spo t KfO.S was observed in theinitial run ; after drying and rerunning a t 90' to the original direction two distinct spotswith the characteristic R,'s for adrenochrome and adrenolutin appeared .

Euler, in describing the paper c hro~n atogr aphy f adrenaline, states t hat after thechroinatograms have beell dried adre~~alinepots sho~va11 apple-green fluorescence( 14 , p. IG). A possible expla~ lat ion f t his observation n~oulclbe the air-oxidation of the

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I-IEACOCK ET AL.: AMINOCI-IROMES. I 855spot t o adrenochrome followed by isomerization to adrenolutin on the paper. Thereforeit would appear that a thin film of adrenochrome 011 paper will spontaneously rearrangeinto adrenolutin. Th is type of behavior has also been observed by Aust in, Chanley, andSobotka with the related compound epinochrome ( IV), which even in t he crystalline formundergoes a slow spontaneous rearrangement to 5,6-dihydroxy-N-methylindole (V)(15, 16). The paper chromatography of adrenochrome, adrenolutin, adrenoxyl (adreno-chrome monosemicarbazone) (VI) , and adrenochrome monoisonicotinic acid hydrazide

(VII) has been studied i l l six different solvent systems: (a) water, (b) 2% acetic acid inwater, (c) 75% methanol in water , (d) 75y0 ethanol in water, (e) n-butanol: acetic acid:water (8 :2: 2), and Cf) i-propanol :0.880 ammonia: water (8:l :l) . Although all solventsproved satisfactory for either oi the two derivatives, VI and VII, only ( a ) and (b ) weresuitab le for adrenochrome and possibly oilly (a ) for adreno lut in, since even in (b), (c),and (d) the fluoresceilce characterist ics of the com pou~l d eem to be irreversibly altered .

An at temp t to purify impure prepa rations of adrenochrome on cellulose columns usingwater as solve~ ltwas disappointiilg, since, although pure aqueous solutions of adreno-chrome were probably obtained, it was not possible to crystallize the adrenochrome fromwate r, and all samples prepared b y freeze-drying under high vacuum (a technique whichgave a very finely divided product) were shown chromatographically to be contaminatedto some extent with adreilolutiil (fortuitously formed dur ing the drying process).

In view of the very considerable interest th at ha s developed, in the pl~ysiologicalactivity of adrenochrome and related compounds, in recent years (see Baccl (17) andHoffer (18) for lists of references) the necessity for working with the compounds in apure and stab le form has become of paramount importance.

Prefinratiofz of AdrenochromeI.-Adrenaline (9.13 g.) \vas suspended in methanol (360 ml.) and '30% formic acid

added dropwise with stirring until a clear solution was obtained (ca. 3.5 nil.). Freshlyprepared silver oxide (36.3 8.)was added portioilwise during a period of 3 minutes , thereaction temperatu re being maintained between 18" and 23" dur ing the addition of theoxidant. The reaction mixture was filtered (with suction) through a Dourex-l(C1-)(200/400 mesh) resin bed (diam. = 6.5 cm. , height = 3.7 cm.)" and the deep red filtratewas allowed to crystallize slo~vly t -2.0". Pure adrenochrome (l.G g.) was obtained i l ldeep-violet needles, m.p. 112" with decomposition. F ound: C, 60.30; H, 4.69; S, .76.Calc. for CgHg03N:C, 60.33; H , 5.02; N , 7.81y0. Th e ultraviolet and visible absorptioilspectra were measured in aqueous solution (A,,,: 301, 487 mp; Amln: 62, 361 mp).

Samples of adrenochrome which we have prepared by repeating existing procedures con-tain , in ou r view, less well defined crystals than the product obtained as described above(rapid cooling to -80" tends to produce an ;~morph ous roduct) and the products are

" T I I E e s il t z ua s pr e pa r ed b y e s f c r ~ s k z a s l ~ i n g : 1)mi l h 3N ~ ~ y d 1 . 0 ~ ~ 2 ~ 0 r i cc i d , (2 )7i' ith ziratcr 1171lil 1e111r(12ol i t i ~ l z l s , n d (3 ) in al ly .ioillt ,irell1,a71,ol i~c lll he roasiz,i?~gs uere tra ?t sp a~ en to zll traviolet l ight.

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856 C.-\N.ADIAN JOURN:\L O F CH EMISTRY . VOL. 3Li, 1958invariably Inore or less contaminat ed with adrenolu tin and melanin. Th is has been demon-strate d paper chromatographically (using Wha tma n No. 1 paper with water as runningsolvent). No particular ad vanta ge either in yield, purity, or crystalline form, appearedto att end the use of absolutely d ry methanol. Th e silver conte nts of adrenochromesanlples prepared in this mallller and by the method of Sobotlta and Austin (5) weredetermined b y the dithizone method after complete oxidation of t he organic material inthe sample with a mixture of nitric an d perchloric acids (cf. 19, p. 644) and were observedto be very low (no t greater t han ca. 100 par ts per million) whereas preparations ob tainedby other rou tes had considerably higher silver conte nts (of the order of 1000 p.p .m.) .Sobotka and Austin filtered the m eth a~io l eaction mixture through a sodium sulphatebed prior to crystallization. This ma). have fortuitously acted as an anion-exchangeresin and removed silver ions from solutiorl as the sulph ate. I t is conceivable th at boththe resill and the sodiu~ll ulpha te beds would help to hold back colloidal as well as ionicsilver. One possible advantage of the resin over sodium su lphat e would be th at it \vouldenable the reaction to be carried out in aqueous solutioil if required.

Th e mother liquors were examined chromatographically on paper using water as solventand there mas evidence tha t th ey coiltai~ledadre~lochromeRJ0.82, a little adrenolutin11J0.4-0.45, a non-fluorescent da rk-vio let unidentified compound R 0.92, and a no~ lfluorescent colorless zone RJO.O-0.4. All these zones gave blue colors (i.e. reduced) withthe ferric chloride- potassium ferricyanide reagent (see next section). Th e low R j areaprobably contained some unchanged adrenaline, since it gave a red color when sprayedwith potassium ferricyanide or amm o~ii um ersulphate solution.Pape r Chromatogra$hy of Adrenochrome and Related Com$ounds

Whatman No. 1 paper was washed with distilled wat er for 12 hours and dried prior touse. I t was observed t ha t unwashed paper caused some decompositio~l f the adreno-chrome. The ascending method for paper chro matography was invariably employed, and10-20 pg. of sample were applied to the paper in each case. The resu lts obtaiiled are givenin Table I.

In all cases, the solvent was allowed to asceild a dis tance of ca. 9 to 12 inches. There wasa co~lsiderable ariability in the time required for this to occur; water and 2% aceticacid having the advantage of giving considerably quiclter results t han the othe r solventsystems. The approximate times taken by the various so lv e~ ~t sre given in Table 11.Detection 01$ots

All four compounds are colored and therefore are self-indicating. Adrenolutin (111)also exhib its a marked yellow-green fluoresceilce in ultraviolet light. I t has been previouslystated th at , on drying, the spots of I undergo a slow change to 111; this substance, beinga catechol derivative, has reducing properties a nd the chroma togram call be developedin the following manner (if a permanent record is required) . Th e developing solution wasprepared immediately before use, and coilsisted of 3% ferric chloride solutio~z(5ml.),3% potassium ferricya~iide olution (5 ml.), and water (90 rnl.). The papers were dippedin the reagent and the reducing cornpou~lds ormed p erm a~le nt lue spots. The excessreagent was washed off; the papers were dipped in 2 N hydrochloric acid and finallyexcess acid was washed off. Other chr on~ oge nic eagents for adre~lochronle nclude: ( a )Ehrlich's reagent (violet spot), ( b ) 3% ferric chloride solutioil (gray-brown spot), (6)diazotized p-nitroaniline (red-brown spot), and (d) zinc chloride solution (yellow-greenfluorescent spot in ultraviolet light).

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HBACOCK BT AL.: AMINOCHROMES. I

:2clrenochron1e (I) 0.8 f 0.02 0.82~k0.02 S. clec. (0.7-0.8) S. clec. (0.7-0.8) R. clec.7 I?. dec.i\drenolutin (III) :!' 0.45f 0.05 0.47+0.0'& S. tlec. (0.7-0.8) S. tlec. (0.7-0.8) R. dec. R. dec.:\clrenosyl (VI)t 0. 48 f 0.05 O.5Of 0.03 0. 65 f0.05 0.85+0.05! 0.57fO.05"~.58~0.05"'-4drenochrome~lionoisonicotinicacid hyclrazide(VII)$ 0.30NOTE.-.-l = IVater. B = 2% ace1.i~ocid i n zvater. C = d~ethano1:roater (3:i). D = Etltanol:zvater ( 3 : l ) .E = ?L-Butano1:c~cetic acid: water (8:2:2). F = i -Prop anol:0 .880 arnn$on.ia: water (8:1:1). S. dec. = slozv(lec0)1?positio71. . dec. = rapid decorisposi t iu?~." = Th e edreaolzl t i?~ oas prepared by t l~ erletl~od of Harley -i l fusoi t and Bu' Loc k (20).t = Szrppl ied by the Labas Co.t = Sz lpp l ied by t he P f i zer Co .5 = I n al l acidic solve?~ts drenolz ~t in zl l ibi ted a pink j ' lz~orescei~ce./ / = Occas io?~al l yirexpl icable lower R l 's were observed for adrenoxyl wit11 this solvent sy st e~ u.7 = Differing results cuere obtai7sed depettde?tt U I L t he t i ~ n ee k e tl t o r u n t l ~ e l ~ r o ~ n z l o g ra t i t . h e r c ~ l olor

of t l ~c pot slozoly faded wit h the for~, tntio ?t f a d irt y yellozo diffzise spot exhi bitin g a pink$zroresce?~ce.* C = Ti ~e sc esz~l ts re di f fere?t t f rom t l tose reborted breviozlslv (12): zue arc zrnnble to exblain these discre-pancies. TItc R, ualzles qtiot&l i n Fable I were n'btainci i,o?lsiste;ltlY ill O I L Y i ~ ~ v e s t ~ g a t i o ? t s .~ s c h e r ' sc la i r i ~ lm tc ldrenoxy l reso lves i n to adr en oc l~ ro ~~ ~end a f lzloresci?tg spot RI 0.88 zn the c~ ~~ z~ iz on ia ca lolve?st systent F (12)zuoz~ldap pe ar to be du e to a ~)zisi,tterpr etatiolt of tile evidence i n uiezo of k7tozon insta bil ity of adre?toclrrorne innm7nonia.'I'rIBLE I1

Solvent system .\ B C D E F.\pproximate running tillle (hours) 2.5-3.0 2.5-3.0 7.5 15.5-18.0 15.5-18.0 18.0-20.0

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